Inflamm. Res. (2015) 64 (Suppl 2):S51–S248 DOI 10.1007/s00011-015-0839-4 Inflammation Research

The 12th World Congress on Inflammation

8–12 August 2015 Seaport Hotel and World Trade Center Boston, USA

The Abstracts

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List of Committees CONGRESS COMMITTEES New Investigators Award Committee Congress President John Somerville, Chair, USA Ian Adcock, UK Rod Flower, UK Brydon Bennett, USA Congress Co-Chairs Doug Morgan, USA Beverley Moore, USA Lisa Marshall, USA Ann Welton, USA Arpita Maiti, USA Outstanding Scientist Award Committee International Scientific Advisory Board John Hamilton, Co-Chair, Australia Anthony Coyle, USA Graham Wallace, Co-Chair, UK Leslie J. Crofford, USA Nathalie Vergnolle, France Eugen Faist, Germany Stephen A. Stimpson, USA Elisabeth Peen, Denmark Fiona Powrie, UK Women in Inflammation Science Award Committee Charles Serhan, USA Arlene H. Sharpe, USA Ellen Berg, Chair, USA Leslie J. Crofford, USA Local Scientific Committee Rod Flower, UK Graham Wallace, UK Lisa Schopf, Chair, USA Andrew Glasebrook, USA Career Development and Networking Committee Liwu Li, USA Alison O’Mahony, USA Caralee Schaefer, USA Caralee Schaefer, USA Larry Burgess, USA Stephen Stimpson, USA Tineke Meijers, Canada Joel Tocker, USA Sharlene Velichko, USA Thomas Wynn, USA Edward Yurkow, USA Social Activities Committee Abstracts and Poster Committee Doreen Gallagher, Chair, USA Edward Yurkow, Chair, USA Scientific Program Committee Lauren Aleksunes, USA Kent Barbay, USA Ian Adcock, UK Alison Budelsky, USA Ellen Berg, USA Subba Chintalacharuvu, USA Yasmine Belkaid, USA Heather Deutsch, USA Maria Belvisi, UK Karen Duffy, USA Kate Blease, USA Paul W. Fisher, USA Jason Brenchley, USA Catherine Healy, USA Larry Burgess, USA Brian Jones, USA Liudmila Buryachkovskaya, Russia Kevin Kreutter, USA Rachel Caspi, USA Erik Lubberts, Netherlands Ernest Choy, UK Ravi Malaviya, USA Leon Collis, USA Carl L. Manthey, USA Leslie Crofford, USA Christine McCauley, USA Salvatore Cuzzocrea, Italy Michael McQueney, USA James Ellis, USA Beverley Moore, USA Kate Fitzgerald, USA Lynne Murray, UK Rod Flower, UK Tatiana Ort, USA Marc Gavin, USA Holly Raymond, USA Claire Gelfman, USA Lani San Mateo, USA Andrew Glasebrook, USA Pitchumani Sivakumar, USA David Glass, USA Mary Ryan, USA Emma Guttman-Yassky, USA Joshua Wertheimer, USA Jenny Gumperz, USA Jennifer Hamilton, USA Advertising Committee Mark Hogarth, Australia Stephen Holdsworth, Australia Alison O’Mahony, Chair, USA David Howat, UK Heather Jones, USA David Huss, USA Doug Morgan, USA Heather Jones, USA Miranda Hanson-Baseler, USA Raj Kamath, USA

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Jeff Karp, USA Lisa Marshall, Chair, Boston 2015 World Congress on Inflammation Iain Kilty, USA Ian Adcock, Chair, London 2017 World Congress on Inflammation Paul Kirkham, UK Ian Ahnfelt-Rønne, Chair, Corporate Advisory Group Liwu Li, USA Peter Libby, USA IAIS Steering Committee (Executive Committee plus two Nikita Lomakin, Russia representatives from each member society) Alberto Mantovani, Italy Brazilian Inflammation Society (BIS, Brazil) Anthony Marotta, Canada Jason McDougall, Canada Mauro Teixeira Jane Mitchell, UK Fernando de Queir¢z Cunha James Mobley, USA Kathryn Moore, USA British Inflammation Research Association (BIRA, UK) Ikuo Morita, Japan Neville Punchard Lynne Murray, UK Matt Barnes Lisa Olson, USA Alison O’Mahony, USA Groupe de Recherche et d’Etude des Me´diateurs de Luke O’Neill, Ireland l’Inflammation (GREMI, France) Tatiana Ort, USA John Parkinson, USA Michel Chignard Robert Philibert, USA Vincent Lagente Vijay Rathinam, USA Simeon Ramsey, USA Inflammation Research Association (IRA, USA) Carlo Riccardi, Italy Arpita Maiti Eric Sasso, USA Joel E. Tocker Caralee Schaefer, USA Robert Schaub, USA Inflammation Research Network (IRN, Canada) Lisa Schopf, USA John L. Wallace Charles Serhan, USA Jason J. McDougall Arlene Sharpe, USA Gary Sims, UK Italian Inflammation Group (IIG, Italy) Mustapha Si-Tahar, France Anthony Slavin, USA Salvatore Cuzzocrea Pejman Soroosh, USA Carlo Riccardi Avi Spira, USA Japanese Society of Inflammation and Regeneration (JSIR, Japan) Hergen Spits, Netherlands Thaddeus Stappenbeck, USA Ikuo Morita Chris Stevenson, UK Tetsuya Taga Stephen Stimpson, USA Russian Inflammation Society (RIS, Russia) Tetsuya Taga, Japan Mauro Teixeira, Brazil Nikita V. Lomakin Nathalie Vergnolle, France Ludmila I. Buryachkovskaya John Wallace, Canada Society for Cytokines, Inflammation and Leukocytes Cara Williams, USA (SCIL, Australia) Li Chun Wang, USA Thomas Wynn, USA Stephen Holdsworth Tim Zheng, USA Samuel Breit

WCI 2015 Management IRA Officers FASEB Office of Scientific Meetings and Conferences Joel E. Tocker, President 9650 Rockville Pike Lisa Schopf, Vice-President Bethesda, MD 20814 Liwu Li, Treasurer Tel: 301-634-7010 Caralee Schaefer, Secretary Email: Infl[email protected] IRA Board Members

IAIS EXECUTIVE COMMITTEE & IRA OFFICERS Alison Budelsky AND BOARD MEMBERS Larry Burgess Anne M. Fourie IAIS Executive Committee Howard Kartstein John Hamilton, President Erik Lubberts Stephen A. Stimpson, Vice-President Arpita Maiti Graham R. Wallace, Treasurer Michael S. McQueney Nathalie Vergnolle, Secretary Alison O’Mahony Kouji Matsushima, Past-President Matthew A. Sleeman Boris Vargaftig, Chair, Brazil 2013 World Congress on Inflammation Edward Yurkow

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Table of Contents

Sunday, August 9, 2015 Patient Perspectives: Unmet Medical Need – Osteoarthritis ------S56 Symposium 1: Innate Memory and Programming in Acute and Chronic Inflammation ------S56 Symposium 2: Mechanisms Underlying Microbiome-Mediated Inflammation ------S57 Symposium 3: Disease Modification in Osteoarthritis: Myth or Reality? ------S58 Symposium 4: The ‘‘Eicosanoid Storm’’ ------S59 Symposium 5: Cytokines and Inflammation – Sponsored by ICIS ------S60 Symposium 6: Novel Biologics Therapies for Immune and Inflammatory Disorders – Sponsored by SCIL-AUS ------S61 Symposium 7: New Therapeutic Strategies for Respiratory Diseases – Sponsored by ERS ------S63 Symposium 8: The Neutrophil: An Old Dog with New Tricks – Sponsored by GREMI ------S64 Symposium 9: Ocular Inflammation, Translation Focused – Sponsored by Ora, Inc. ------S65 Symposium 10: Glucocorticoids in the Regulation of Inflammatory Response – Sponsored by Italian Inflammation Group ------S67 Symposium 11: A Role for Epigenetics in Inflammatory Disease: Spotlight on Monocytes and Macrophages ------S68 Symposium 12: Next Generation Kinase Inhibitors – Sponsored by BIRA-UK ------S69 Mini-Symposium 1: Novel Models of Inflammation ------S70

Monday, August 10, 2015 Patient Perspectives: Unmet Medical Need – Inflammatory Bowel Disease------S70 Symposium 13: MicroRNAs (miRs) in Inflammation ------S70 Symposium 14: Mucosal Immunity: Hot Topics in IBD ------S71 Symposium 15: Inhibitory Receptor/Immune Checkpoints/Immunotherapeutics – Sponsored by IRA-USA ------S73 Symposium 16: Mechanisms of Fibrosis ------S73 Symposium 17: Therapeutics with Localized Actions – Optimal Ways to Treat Tissue Inflammation ------S74 Symposium 18: Ion Channels and Inflammation – Sponsored by IRN-Canada ------S75 Symposium 19: Harnessing Novel Resolution Mechanisms to Control Inflammation ------S76 Symposium 20: Skeletal Muscle Inflammation ------S77 Symposium 21: Regulation of Inflammation in Tissues by Co-Stimulatory and Co-Inhibitory Pathways ------S78 Symposium 22: Atopic Dermatitis, Psoriasis and IL-4/IL-17 Biology ------S79 Symposium 23: Defining Sub-Phenotypes in Chronic Respiratory Diseases to Improve Drug Development and Disease Management - - S80 Symposium 24: Biological and Non-Biological Therapies of Autoimmune Diseases – Sponsored by JSIR-Japan ------S81 Mini-Symposium 2: Cellular and Molecular Mechanisms of Fibrosis ------S82 New Investigator Award Winner Presentations ------S82

Tuesday, August 11, 2015 Patient Perspectives: Unmet Medical Need – Lupus------S83 Symposium 25: Complement Targeted Therapies – Is There Light at the End of the Tunnel? ------S83 Symposium 26: Regenerative Medicine and Stem Cells in Inflammation ------S84 Symposium 27: iNKT Cells – Mediators at the Interface of Inflammation and Immunity ------S85 Symposium 28: Infection and Inflammation – Sponsored by BIS-Brazil------S87 Symposium 29: Biomarkers in Inflammation: Focus on Rheumatoid Arthritis ------S88 Symposium 30: SLE: Current Therapeutic Landscape and Future Promises ------S90 Mini-Symposium 3: Mechanisms Driving Mucosal Inflammation in the Gut ------S90 Symposium 31: New Visions in Ocular Therapy ------S91 Symposium 32: Tregs in Human Autoimmunity ------S91 Symposium 33: Gaseous Mediators as the Basis for Novel Anti-Inflammatory Drugs – Sponsored by IRN-Canada ------S92 Symposium 34: Therapeutic Pathways in Inflammation: New Faces of Old Friends – Sponsored by RIS------S93 Symposium 35: Clinical Developments in Fibrosis ------S94 Symposium 36: Novel Therapies in RA ------S95 Mini-Symposium 4: Signaling in Inflammation ------S97

Wednesday, August 12, 2015 Symposium 37: Novel Targets and/or Novel Therapies – Sponsored by Celgene ------S98 Symposium 38: Cardiovascular Inflammation------S99 Symposium 39: Predictive Toxicology ------S100 Symposium 40: Translational Medicine ------S101 Symposium 41: Innate Lymphoid Cells ------S101

Posters ------S102 Angiogenesis ------S102 Apoptosis ------S104 Autoimmune Diseases—RA, Psoriasis, SLE, IBD, MS ------S105

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Biologic Therapies for Targeting Inflammatory and Immune Mechanisms ------S117 Cartilage and Bone Remodeling ------S122 Cell Adhesion and Leukocyte Migration ------S123 Chemokines and Chemokine Receptors ------S126 Co-Stimulatory and Co-Inhibitory Pathways ------S128 Cytokines in Inflammatory Disease ------S128 Epigenetics and Regulation of the Immune Response ------S137 Fibrosis and Tissue Remodeling ------S138 GPCRs in Inflammation ------S141 Immunotherapeutics ------S143 Inflammasomes in Health and Disease ------S147 Inflammation and Aging ------S149 Inflammation and Cancer ------S150 Inflammation and Metabolic Disorders ------S153 Inflammatory Cell Signaling ------S156 Inflammatory Mediators ------S159 Inflammatory Pain and Analgesia ------S162 Inflammatory Processes in Cardiovascular Diseases ------S166 Inflammatory Processes in Central Nervous System Diseases ------S171 Inflammatory Processes in Shock and Trauma ------S174 Inflammatory Responses, Stem Cells and Tissue Regeneration ------S177 Inflammatory Skin Disorders ------S178 Inhibitory Receptors and Immune Checkpoints ------S181 Innate Immunity – Macrophages, Dendritic Cells, Neutrophills, Basophils, Mast Cells, Eosinophils ------S181 Innate Lymphoid Cells ------S189 Lipids, Their Enzymes and Inflammation ------S189 Microbiome in Health and Disease ------S190 microRNAs and lncRNAs in Inflammation (microRNAs=miRs; long Non-Coding RNAs=lncRNAs) ------S191 Molecular Patterns and Acute Inflammation ------S192 Mucosal Immunity ------S193 Neutrophils, NETs and PADs ------S193 New Models of Inflammatory Mechanisms and Diseases ------S195 Novel and Innovative Platforms for Drug Discovery ------S197 Novel Targets and New Drugs in Inflammation ------S198 Nuclear Hormone Receptors ------S207 Ocular Inflammation ------S208 Proteases ------S208 Resolution of Inflammation and Tissue Repair ------S211 Respiratory Disease and Inflammation ------S214 Small Molecule Therapeutics for Inflammatory Diseases ------S226 T Regulatory Cells ------S228 Tissue Damage/Repair------S229 Toll Receptors ------S231 Translational Medicine and Biomarkers ------S232 Author Index ------S238

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PATIENT PERSPECTIVES: UNMET PTX3 plays a role in the interplay between the cellular and the humoral arm of innate immunity. PTX3 deficiency was associated with increased MEDICAL NEED – OSTEOARTHRITIS susceptibility to mesenchymal and epithelial carcinogenesis. PTX3 expression was epigenetically regulated in selected human 001 tumors (e.g., leiomyosarcomas and colorectal cancer) by methylation of PATIENT PERSPECTIVES: UNMET MEDICAL the promoter region and of a putative enhancer. Thus, PTX3, an effector molecule belonging to the humoral arm of innate immunity, acts as an NEED–OSTEOARTHRITIS extrinsic oncosuppressor gene in mouse and man by regulating Comple- ment-dependent, macrophage-sustained, tumor promoting inflammation. Heather E. Jones References Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo Pfizer, Inc., Collegeville, PA, USA veritas. J Clin Invest. 2012, 122:787–95. Jaillon S, Moalli F, Ragnarsdottir B, Bonavita E, Riva F, et al. The Objective: To increase awareness of the true unmet medical need left humoral pattern recognition molecule PTX3 is a key component of innate by current therapeutic options for osteoarthritis and understand what immunity against urinary tract infection. Immunity, 40: 621–632, 2014. this means to the patient. Bonavita E, Gentile S, Rubino M, Maina V. et al., PTX3 is an The importance of recognizing the true unmet medical need in extrinsic oncosuppressor regulating complement-dependent inflam- osteoarthritis, the most common form of arthritis affecting millions of mation in cancer. Cell 160: 700–714, 2015. people worldwide, is higher than ever. No drug has so far been able to cure osteoarthritis, only to can help reduce pain and maintain joint movement. To progress science and clinical development there must be a focus on understanding the pathogenesis of the disease. From a 003 medical perspective we need to consider the patient as a whole human THE MACROPHAGE EPIGENOME AND THE being and not only based on the organ(s) involved. Osteoarthritis is a CONTROL OF THE INFLAMMATORY GENE degenerative disease that worsens over time and may become severe EXPRESSION PROGRAM enough to make daily tasks difficult. The morning session will begin by emphasizing the unmet medical need followed by an interview with a patient suffering from Gioacchino Natoli osteoarthritis, with an emphasis on what they see as key objectives for further development. At the end of the session the co-chairs will European Institute of Oncology, Milan, Italy summarize the signs and symptoms and the treatment algorithm for Induction of an inflammatory response requires the activation of a osteoarthritis, as well as the need for biomarkers to define the disease, complex gene expression program in which hundreds of genes are ensure safety, guide treatment and predict treatment response. activated or repressed in a kinetically complex fashion that reflects the specific functional role of their products. The activity of the transcrip- tion factors responsive to inflammatory stimuli, such as NF-kB, STAT SYMPOSIUM 1: INNATE MEMORY AND and IRF family members, is critically influenced by the pre-existing chromatin organisation (epigenome) of the cells in which they are PROGRAMMING IN ACUTE AND activated. This way inflammatory gene expression is qualitatively and CHRONIC INFLAMMATION quantitatively different depending on the cell type in which it is elicited. In turn, chromatin organisation in differentiated cells is controlled by 002 lineage-determining transcription factors, such as the essential myeloid master regulator PU.1 and its binding partners (RUNX1, IRF8 and PTX3 AS A PARADIGM FOR THE INTERPLAY others). A wealth of genomic, biochemical and functional data accu- BETWEEN CELLULAR AND HUMORAL INNATE mulated in the last years has demonstrated that an important role of PU.1 IMMUNITY IN INFLAMMATION AND CANCER is to make binding sites for inflammatory transcription factors accessi- ble, thus enabling their recruitment to chromatin and the activation of a macrophage-specific inflammatory gene expression program. Alberto Mantovani

Istituto Clinico Humanitas, Humanitas University, Rozzano, Italy Innate immunity consists of a cellular and a humoral arm. The long 004 pentraxin PTX3 as originally cloned (cDNA and genomics, mouse and EPIGENETIC REGULATION OF MACROPHAGE human) as an IL-1 inducible gene. We have used the long pentraxin ACTIVATION AND FUNCTION PTX3 as a paradigm for the humoral arm of innate immunity and its interplay with cells. PTX3 is a multifunctional soluble pattern recog- Lionel Ivashkiv nition receptor characterized by a C-terminal domain highly homologous to C-reactive protein and serum amyloid P component, Hospital for Special Surgery, Weill Cornell Medical College, New associated to a N-terminal domain unrelated to other known proteins. York, NY, USA PTX3 is produced upon stimulation with proinflammatory cytokines and Toll-like receptor engagement most prominently by mono- Macrophage activation phenotypes are determined by the interplay cytes/macrophages. The molecule binds complement components and between cytokines such as IFN-gamma or TNF and acute inflammatory microbial moieties. It mediates effector function via Fcg receptor and stimuli such as TLR ligands. Synergistic activation of inflammatory complement. Recent results suggest a function at mucosal surfaces. cytokine genes by IFN-gamma and TLR signaling is important for PTX3 plays non-redundant functions including innate immunity against innate immunity and inflammatory disease pathogenesis. We have selected microorganisms and regulation of inflammation. In addition identified a synergy mechanism whereby IFN-gamma creates a primed

123 Inflamm. Res. S57 chromatin environment to augment TLR-induced transcription of Infection of gut-resident CD4+ memory T-cells during acute HIV inflammatory genes such as TNF, IL6 and IL12B in human macro- and SIV infection is associated with rapid loss of these cells and phages. IFN-gamma primes enhancers by inducing sustained occupancy damage to the epithelial barrier. Damage to the epithelial barrier of STAT1, IRF-1 and associated histone acetylation. In contrast to allows translocation of microbial products from the intestinal lumen pervasive epigenomic remodeling for gene activation, IFN-gamma into the body. Immune activation caused by these microbial prod- stably silenced only a small number of genes via chromatin regulation. ucts has been associated with disease progression. Although These silenced genes are enriched in transcription factors that regulate microbial translocation has been demonstrated in SIV-infected aspects of alternative activation. These results suggest that M1 polar- nonhuman primates, the identity of translocating bacteria has not ization has a stable epigenetic component that blocks reprogramming of been determined. In this study we examined the community makeup select transcription modules by M2 stimuli, whereas the remaining of bacteria both within the GI tract and systemic tissues of both (majority) of M1 genes are dynamically regulated by environmental healthy and experimentally SIV-infected Asian macaques. While cues. In contrast to IFN-gamma, TNF tolerizes macrophages such that there were only modest changes in the GI tract-associated micro- inflammatory genes are not activated by TLR stimulation; this toler- biome resulting from infection, there is substantial dysbiosis after ization can be reversed by IFNs. Combined genome-wide analysis using administration of antiretrovirals. Analysis of bacterial DNA isolated RNAseq, ChIPseq and ATACseq identified chromatin-based mecha- from tissues of infected animals revealed a preference for the nisms for reversal of tolerance under conditions where signaling is phylum Proteobacteria, suggesting that Proteobacter preferentially strongly attenuated. Overall the results highlight the importance of translocate. Consistent with this finding, we observed increased chromatin-based mechanisms in regulating transcriptional responses to metabolic activity of Proteobacterial species within the colonic acute stimuli that activate canonical inflammatory signaling pathways. lumen of SIV-infected animals. Overall these data provide insights Interplay between epigenetic mechanisms and signaling pathways will into disease progression and suggest that therapies aimed at altering determine macrophage phenotypes in response to environmental cues. the composition and metabolic activity of the GI tract microbiome could benefit chronically-HIV infected individuals particularly those on antiretroviral therapies. 005 DYNAMIC PROGRAMMING OF INNATE IMMUNITY IN HEALTH AND DISEASE 007 SYMBIOTIC SPHINGOLIPIDS SHAPE THE HOST Liwu Li IMMUNE DEVELOPMENT AND HOMEOSTASIS

Virginia Tech, Blacksburg, VA, USA Dingding An1,2 Host innate leukocytes such as monocytes and neutrophils can be pre- 1Boston Children’s Hospital, Boston, MA, USA; 2Harvard Medical programmed into distinct states depending upon the nature and quantities School, Boston, MA, USA of external stimulants. The programming and rudimentary memory of innate immunity have significant implications in the pathogenesis both It has been increasingly appreciated that the development of the acute and chronic diseases such as sepsis and atherosclerosis. However, the mammalian immune system is profoundly dependent on the thou- responsible mechanisms are not well understood. We observed that super sands of microbial species that the host is associated with. However, low dose bacterial endotoxin lipopolysaccharide (LPS) skews and pro- the molecular details of how microbes modulate the immune system grams innate leukocytes into distinct functional states, as reflected by the are largely lacking. Here we report that sphingolipid-producing selective expression of inflammatory mediators in monocytes and unique symbiotic bacteria regulate the host mucosal homeostasis and dis- modulation of neutrophil extra-cellular traps (NET). At the pathological ease susceptibility. Many of the ubiquitous intestinal Bacteroides level, we observed that mice pre-conditioned with super-low dose LPS species possess sphingolipids, which are rare in bacteria and without experienced severe tissue damage, inflammation, and increased bacterial much known functions. We discovered that Bacteroides fragilis load in circulation when they were subjected to cecal-ligation and puncture produces immunomodulatory a-galactosylceramide molecules. (CLP). In contrast, CLP mice pre-conditioned with low dose LPS exhib- These molecules modulate the colonic invariant killer T (iNKT) cell ited reduced tissue damage, inflammation, and reduced bacterial load in proliferation by competing for the limited agonist-binding space on blood. In summary, our studies suggest that innate leukocytes can be the antigen-presenting CD1d protein. As a result, if the host is dynamically programmed by varying signal strength of innate challenges, exposed to the wild-type B. fragilis or its glycosphingolipid mole- and may have far-reaching patho-physiological consequences. cules early in life when iNKT cells actively proliferate, the expansion of colonic iNKT cells in response to endogenous antigens is modulated, resulting in a lower homeostatic colonic iNKT cell level in adult life. Consequently, the host becomes more resistant to SYMPOSIUM 2: MECHANISMS the iNKT cell-dependent colitis challenge. These results suggest an UNDERLYING MICROBIOME-MEDIATED unexpected mechanism by which symbionts can help the host attain immune balance by supplementing the endogenous lipid antigen INFLAMMATION milieu with unique inhibitory glycosphingolipids. These Bacteroides glycosphingolipids provide the second known example of 006 immunomodulatory molecules produced by a symbiont (the first DYSBIOTIC MICROBES TRANSLOCATE FROM being a zwitterionic polysaccharide also produced by B. fragilis). In addition, we have further discovered that bacterial sphingolipids also THE GI TRACT IN PROGRESSIVE SIV INFECTION mediate the homeostasis of the epithelial layer in the colon and are important for the host resistance to dextran sodium sulfate-induced Jason Brenchley, Zachary Klase colitis. Our work starts to reveal the profound impacts of these unique molecules produced by bacterial symbionts on host devel- LMM/NIAID/NIH, Bethesda, MD, USA opment and homeostasis.

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SYMPOSIUM 3: DISEASE MODIFICATION resurfacing of the entire joint surface. However, the presence of pro- inflammatory cytokines such as interleukin 1 or tumor necrosis factor IN OSTEOARTHRITIS: MYTH OR alpha has been shown to prevent chondrogenesis and induce degra- REALITY? dation of engineered tissues. In recent years, the advent of synthetic biology has led to the development of technologies for precisely modifying gene networks that control cell behavior. Here, we present 008 a combination of principles from these fields to rewire cellular gene NEW TARGETS FOR JOINT DAMAGE AND circuits in a manner that allows us to create a unique, custom-de- ASSOCIATED PAIN IN OSTEOARTHRITIS signed cell type that can sense and respond to its biochemical environment in a pre-programmed way to provide long-term signals Anne-Marie Malfait for controlling cell differentiation or immunomodulatory response. This strategy uses recently developed methods in genome editing Division of Rheumatology, Rush University Medical Center, Chicago, (CRISPR/Cas9) to reprogram cells to detect dynamic cues from the IL, USA environment and to actively regulate their response with customized decision-making capabilities. By combining techniques in synthetic Osteoarthritis (OA) is the most common form of arthritis. Pain is the biology, gene therapy, and functional tissue engineering, we have defining symptom of osteoarthritis (OA), yet available analgesic developed engineered tissues with the ability for tunable, inducible, or treatment options, of which NSAIDs are the most common, provide feedback-controlled, auto-regulated immunomodulatory properties. In inadequate pain relief and are associated with serious health risks. addition to recapitulating the biochemical and biomechanical prop- Disease-modifying OA drugs (DMOADs) are not yet available for erties of the tissue, these ‘‘smart’’ constructs can provide controlled clinical use. It is unknown to which extent efficacious DMOADs may drug delivery to the joint to enhance the success of engineered tissue also have a beneficial effect on pain associated with OA. Generally, the replacements. mechanisms that generate and maintain OA-associated pain are poorly understood, and so is the relationship between joint damage and pain Acknowledgments: Supported by grants from the NIH, NSF, Nancy pathways. This gap in knowledge exists partly because modeling OA Taylor Foundation for Chronic Diseases, Arthritis Foundation, and pain in laboratory animals is challenging. We employ the murine AO Foundation. destabilization of the medial meniscus (DMM) model for the longitu- dinal study of OA-related pain behaviors and their molecular/neuronal pathways. Following DMM surgery, in association with slowly pro- gressive joint pathology, mice develop pain-related behaviors in two 010 phases: firstly, secondary mechanical allodynia in the ipsilateral ADVANCES IN BIOMARKER QUALIFICATION TO hindpaw develops early on, when joint damage is minor (weeks 4–8 FACILITATE OSTEOARTHRITIS CLINICAL after surgery). The second phase (weeks 8–16) marks more severe joint TRIALS pathology and is characterized by persistent mechanical allodynia and the onset of movement-provoked pain, thus likely representing a stage of chronic pain. The temporal effects of novel analgesics will be dis- Virginia B. Kraus cussed. In addition, examples will be shown of how pharmaceutical modulation of joint damage affects pain pathways. Duke Molecular Physiology Institute and Department of Medicine, Duke University, Durham, NC, USA Objectives: Our current reference standard for disease diagnosis and 009 severity of osteoarthritis (OA) is often the radiograph (Hunter 2014). Moreover, the draft regulatory (FDA) guidance and current gold CELL-BASED THERAPIES FOR standard for measuring clinical efficacy in disease modifying therapy OSTEOARTHRITIS: FROM SYNTHETIC development in OA is radiographic which has a low responsiveness to MATERIALS TO SYNTHETIC BIOLOGY change and at most moderately correlates with clinical endpoints (Hunter 2014). Biomarkers are needed to provide a method for earlier diagnosis of OA, and to inform the prognosis, monitoring and ther- Farshid Guilak, Jonathan M. Brunger, Ananya Zutshi, Vincent P. apeutic strategies for OA (Kraus 2011 and 2015). The goal of our Willard, Katherine A. Glass, Alison K. Ross, Franklin T. Moutos, work has been to evaluate the ability of a set of soluble biomarkers to Bradley T. Estes, Charles A. Gersbach predict knee OA progression to provide new drug tools and surrogate outcomes in OA trials. Duke University Medical Center, Durham, NC, USA Methods: We undertook a nested case–control study within the The repair of articular cartilage following joint injury or degeneration Osteoarthritis Initiative in knees (one knee per subject) with a Kell- remains an important challenge for the field of tissue engineering. gren and Lawrence grade (KLG) of 1–3. Cases were defined as knees While a number of techniques have been developed for the treatment having the combination of radiographic progression (a decrease of of focal cartilage defects, there have been few attempts at tissue- C0.7 mm in medial tibiofemoral joint space) and pain progression (a engineered therapies for end-stage osteoarthritis. Some of the major persistent increase in WOMAC pain score of C9 on a 0–100 scale) at considerations for this approach include the design of mechanically the 24, 36 or 48 months follow-up compared to baseline. Controls functional scaffolds that can withstand joint loading, the identification (n = 406) were eligible knees that did not meet both endpoint cri- of an abundant and accessible stem cell source, and the reprogram- teria, and included 200 with neither radiographic nor pain ming of these cells for appropriate tissue differentiation within an progression, 103 with radiographic progression only and 103 with osteoarthritic environment. Using principles of ‘‘functional tissue pain progression only. A total of twelve OA-related soluble engineering’’, we have used techniques for three-dimensional weav- biomarkers were measured in serum and/or urine. ing to develop large, anatomically-shaped scaffolds that can be Results: Longitudinal burden of biomarker concentrations over time engineered with biomimetic mechanical properties that reproduce (area of concentration vs time curve) was a more robust predictor of those of native cartilage, providing the potential for complete case status than change scores over time.

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Conclusions: For dynamic markers such as soluble systemic single agent alone. Collectively, these findings uncover a previously biomarkers, strategies accounting for ‘biomarker burden’ over time unrecognized role for PGE2 in the promotion of tumor formation and appear to be more appropriate for identifying progressors than progression. strategies based on change over time. Acknowledgments: The FNIH OA Biomarkers Consortium 012 References Hunter DJ, Nevitt M, Losina E, Kraus V. Biomarkers for INDUCTION OF EICOSANOIDS BY THE osteoarthritis: current position and steps towards further validation. INFLAMMASOME Best Pract Res Clin Rheumatol 2014;28(1):61–71. Kraus VB, Burnett B, Coindreau J, Cottrell S, Eyre D, Gendreau M, Karsten Gronert Gardiner J, Garnero P, Hardin J, Henrotin Y, Heinegard D, Ko A, Lohmander LS, Matthews G, Menetski J, Moskowitz R, Persiani S, University of California Berkeley, Berkeley, CA, USA Poole AR, Rousseau JC, Todman M. Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis. Inflammasomes have remerged as key players in the innate immune Osteoarthritis Cartilage 2011;19(5):515–542. system and have been implicated in pathogenesis of inflammatory Kraus V, Blanco F, Englund M, Henrotin Y, Lohmander L, Losina E, diseases, autoimmune disorders and host defense. The inflammasome O¨ nnerfjord P, S P. Guidelines for soluble biomarker assessments in complex acts as a ‘‘last resort’’ intracellular sensor that detects osteoarthritis clinical trials. Osteoarthritis and Cartilage 2015;in press. intracellular microbial products or stress factors to mount a critical innate immune response. A common mechanism of most inflamma- somes is activation of caspase 1 and pyroptosis. Despite the fact that this system is designed to send an immediate inflammatory signal primary effector function has been relegated to cleavage and forma- SYMPOSIUM 4: THE ‘‘EICOSANOID tion of interleukin 1b (IL-1b) and IL-18, which takes hours. We STORM’’ recently discovered a novel early and critical effector function of inflammasome activation, namely the rapid formation of an eicosa- 011 noid storm by tissue macrophages. In view of emerging evidence that places eicosanoids as key regulators of innate as well as adaptive INFLAMMATION, INFLAMMATORY MEDIATORS immune responses this novel inflammasome effector function has far AND CANCER PROGRESSION reaching implications in health and disease.

Raymond N. DuBois1,2 013 1Arizona State University, Tempe, AZ, USA; 2Mayo Clinic Arizona, Phoenix, AZ, USA PGE2 AND THE INNATE IMMUNE RESPONSE Although epidemiologic and experimental observations support the Mariano Sanchez Crespo hypothesis that chronic inflammation and diet are risk factors for colorectal cancer, the mechanisms by which chronic inflammation and IBGM, CSIC, Valladolid, Spain diet contribute to the development of cancer are poorly understood. Evidence for the link between inflammation and cancer comes from Eicosanoids support local inflammation and exhibit immunomodula- epidemiologic and clinical studies showing that use of nonsteroidal tory properties. Current views have focused on prostaglandin E2 anti-inflammatory drugs (NSAIDs) reduce the relative risk for devel- (PGE2), because it is the most abundant and lasting eisosanoid in the oping colorectal cancer (CRC) by 40–50 %. NSAIDs exert some of inflammatory milieu due to the robust production elicited after chal- their anti-inflammatory and anti-tumor effects by targeting lenge with pathogen-associated molecular patterns by the constitutive cyclooxygenase enzymes (COX1 and COX2). Metabolism of arachi- and the inducible COX isoforms. The different functions and cell donic acid, a major ingredient in animal fats, by cyclooxygenase distribution of E prostanoid (EP) receptors explain the difficulty so far enzymes provides one mechanism for the contribution of dietary fats encountered to delineate the actual role of PGE2 in the immune and chronic inflammation to carcinogenesis. Prostaglandin E2 (PGE2) response. It is widely accepted that by acting in an autocrine/paracrine is a pro-inflammatory mediator that promotes tumor progression. manner PGE2 induces a regulatory phenotype including the expres- We found that PGE2 exerts its effects on chemokine and cytokine sion of IL-10, the inhibition of the release of IL-12 p70, and distinct expression by upregulation of the CXCR2 pathway, which increases effects on IL-23 production due to differential effects on the the level of myeloid derived suppressor cells (MDSC’s) in the tumor expression of the p40 and p19 (encoded by the gene il23) chains of microenvironment. MDSCs have been shown to contribute to cancer this cytokine. Given that PGE2 may be released concomitantly with immune evasion by suppressing T cell activation, proliferation, traf- other lipid mediators, the definite assignment of a role for each ficking, and viability. MDSC’s also can inhibit natural killer (NK) mediator is not an easy task. Our studies on the regulation of the gene cells and promote activation/expansion of Foxp3 positive Treg cells. il23a have disclosed the cooperation of leukotriene B4, cysteinyl- We also provided the first evidence demonstrating that MDSCs pro- leukotrienes, and the phospholipid mediator platelet-activating factor mote chronic colonic inflammation and colitis-associated in the response to fungal patterns. These mediators, by acting con- carcinogenesis via suppression of colonic CD8+ T cell cytotoxicity comitantly on their cognate G-protein coupled receptors, activate against tumor cells. Our recent findings not only provide a rationale phospholipase Cß and enhance the Ca2+- and kinase-dependent routes for developing effective therapeutic strategies to subvert inflamma- initiated by fungal patterns involving Syk kinase and phospholipase tion- and tumor-induced immunosuppression, but also support the Cc. The autocrine production of these mediators has a robust effect on hypothesis that combined treatment of anti-PD-1 and anti-CXCR2 the activation of transcription factors such as CREB and ATF2 that agents may provide more effective therapeutic effects than either cooperate with NF-jB to establish the cytokine signature.

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B153 015 COLONY-STIMULATING FACTOR-1 (CSF-1) AND TAM RECEPTOR TYROSINE KINASES TUMOR NECROSIS FACTOR-A (TNF-A) IN REGULATE THE MAGNITUDE OF THE IMMUNE ARTHRITIC PAIN AND DISEASE RESPONSE

See poster section for abstract. Carla Rothlin

Department of Immunobiology, School of Medicine, Yale University, New Haven, CT, USA SYMPOSIUM 5: CYTOKINES AND The innate immune response of dendritic cells (DCs) and other sen- INFLAMMATION—SPONSORED tinel cells functions as both the first line of defense against pathogens BY ICIS and also as the initiating trigger for T cell-mediated adaptive immunity. These fundamental activities notwithstanding, DC activa- tion must be tightly regulated. While reduced DC function leads to 014 increased susceptibility to infections, unrestrained, overactive DC RESOLVINS AND PRO-RESOLVING LIPID responses can lead to allergy, autoimmunity, chronic inflammatory MEDIATORS: RESOLVING THE STORM disease, and other pathological conditions. We have found that the TAM receptor tyrosine kinases, Tyro3, Axl and Mertk, are potent Charles N. Serhan, Nan Chiang, Jesmond Dalli negative regulators of the immune response in DCs. I will present new findings that show that the TAM signaling in DCs is triggered by cells Center for Experimental Therapeutics and Reperfusion Injury of the adaptive response with which DCs interact with. I will also Department of Anesthesia, Perioperative and Pain Medicine Harvard discuss the specificity of different TAM receptors in the regulation of Institutes of Medicine, BWH and Harvard Medical School, Boston, the immune response. MA, USA Local control of the acute inflammatory response by the host is critical for an ideal outcome and return of tissue function. Using a 016 systems approach with self-limited inflammatory infectious exudates FRENEMIES IN THE GUT: INFECTIOUS AGENTS to map tissue events, cell traffic and identification of protein and THAT DEFY CLASSIFICATION chemical mediators, we identified three structurally separate fami- lies of potent novel mediators, coined resolvins, protectins and Ken Cadwell maresins. Complete structural elucidation of these new molecules demonstrated their functions in vivo in the resolution of acute New York University School of Medicine, New York, NY, USA inflammation. Each family member is chemically distinct func- tioning as pro-resolving local mediators that control both the The gut microbiome includes trillions of commensal bacteria that provide duration and magnitude of acute inflammatory responses with key benefits to the host including aiding digestion, promoting the devel- actions in pico- to nano-gram concentration range. Mapping of opment of the immune system, and protecting the epithelial barrier. these resolution circuits provides new avenues to probe the However, an inappropriate immune response directed at members of the molecular basis of many widely occurring diseases and implicate microbiome can also lead to diseases such as inflammatory bowel disease the failure of resolution mechanism as a component of chronic (IBD). For these reasons, the bacterial microbiome has received much inflammatory diseases (CN Serhan Nature 2014). This presentation attention as critical regulators of mammalian health and disease. Less is shall focus on our recent advances on the biosynthesis and functions known about the impact of other intestinal inhabitants, such as viruses that of specialized pro-resolving mediators (SPM) and their actions in are part of the enteric virome. We present evidence that an intestinal counter-regulation of pro-inflammatory cytokines (IL-1, TNF, IL-6, animal virus can influence host physiology in a manner similar to com- etc.) that initiate leukocyte infiltration and inflammation. SPM mensal bacteria. Although murine norovirus (MNV) can persistently possess potent multi-pronged anti-inflammatory, pro-resolving, and infect mice without causing obvious signs of disease, we found that this anti-microbial actions in animal models. We use LC–MS-MS lipi- virus induces intestinal pathologies in a mouse model of IBD. Therefore, domics to profile SPM in human tissues and uncovered new like colonization by commensal bacteria, MNV infection is typically pathways that stimulate tissue regeneration. Several SPM are in harmless but can induce disease in a genetically susceptible host. clinical development and clinical trial in humans. Identification of Remarkably, we found that MNV infection reproduces beneficial func- SPM during inflammation-resolution indicates that resolution is an tions of bacteria as well. Viral infection of germ-free mice or antibiotics- active programmed process challenging the concept that resolution treated mice reversed abnormalities in intestinal and immune develop- is a passive process where chemotactic molecules dilute and wane mentthat were due to the depletion of bacteria. Moreover, MNV protected to resolve tissue inflammation. Together these findings indicate that bacterially-deficient mice from chemical and infectious damage to the endogenous resolution pathways may underlie prevalent diseases intestine. These finding indicate that viruses can perform functions that associated with uncontrolled inflammation and open the potential have been attributed to commensal bacteria, and has implications for how for resolution-based pharmacology. we view the role of non-bacterial members of the microbiome. The The authors acknowledges support of NIH GM038765 and GM relationship between MNV, cytokine signaling, and other infectious P01 GM095467. entities will be discussed in the context of disease susceptibility.

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017 019 INNATE IMMUNE SENSING OF LPS IN THE GM-CSF AND INFLAMMATORY PAIN CYTOSOL Andrew D. Cook Vijay Rathinam The University of Melbourne, Parkville, VIC, Australia UConn Health, Farmington, CT, USA Better therapies are needed for inflammatory pain. In arthritis the Innate immune system is central to the sensing of invading patho- relationship between joint pain, inflammation and damage is unclear. gens and the activation of the host immune response. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is Inflammasomes are multi-protein scaffolds in the cytosol containing important for the progression of a number of inflammatory conditions, a NLR receptor, an adapter ASC, and an effector, caspase-1. including arthritis, and there is encouraging data that its blockade may Inflammasome is an integral part of the immunosurveillance of the have clinical relevance. However, its contribution to inflammatory cytosol. Inflammasomes directly detect various ‘‘signature’’ micro- and arthritic pain is unknown. The aims of this study were to deter- bial products or indirectly sense signs associated with an infection. mine whether GM-CSF controls inflammatory and/or arthritic pain. Although lipopolysaccharide (LPS) of Gram-negative bacteria was A model of inflammatory pain (CFA footpad), as well as three -/- believed to be exclusively detected at the cell surface by Toll-like arthritis models, were induced in either GM-CSF mice or wild- receptor-4 (TLR4), it has very recently been described that the LPS type mice treated prophylactically or therapeutically with a mono- is sensed in the cytosol in a TLR4-independent manner by caspase- clonal antibody to GM-CSF. Development of pain (assessment of 11, an inflammatory caspase. Activation of caspase-11 by intracel- weight distribution) and arthritic disease (histology) were assessed. lular LPS leads to the proteolytic activation of caspase-1, which Pain was further measured in a GM-CSF-driven arthritis (mBSA/GM- then executes the activation of IL-1b and IL-18. Importantly, active CSF) model and the cyclooxygenase-dependence determined using caspase-11 triggers an inflammatory form of cell death (pyroptosis) indomethacin. and the release of endogenous alarmin or danger molecules that GM-CSF was absolutely required for pain development in both the perpetuate the inflammatory reactions. The mechanistic details of inflammatory pain and arthritis models. Therapeutic neutralization of this pathway regarding the molecular basis of caspase-11 activation GM-CSF not only abolished the pain within 3 days but also led to will be presented. significantly reduced cartilage damage in the collagenase-induced instability model of osteoarthritis. Pain in a GM-CSF-driven arthritis model, but not the disease itself, was abolished by the cyclooxygenase inhibitor, indomethacin, indicating separate pathways downstream of GM-CSF for pain and arthritis control. SYMPOSIUM 6: NOVEL BIOLOGICS GM-CSF is thus key to the development of inflammatory and THERAPIES FOR IMMUNE AND arthritic pain. Importantly, GM-CSF neutralization by a therapeutic monoclonal antibody-based protocol rapidly and completely abol- INFLAMMATORY DISORDERS— ished existing arthritic pain and suppressed the degree of arthritis SPONSORED BY SCIL-AUS development. These results suggest that it would be worth exploring the importance clinically of GM-CSF for pain and disease in inflammatory conditions in general. 018 TARGETING GM-CSF IN INFLAMMATORY DISEASES 020 Ian P. Wicks1,2,3 IL-3RA AS A THERAPEUTIC TARGET FOR LUPUS

1 2,3,4 5 2,3,4 1Inflammation Division, Walter and Eliza Hall Institute of Medical Nicholas J. Wilson , Shereen Oon , Eric Morand , Ian Wicks , 1 Research, Parkville, VIC, Australia; 2Rheumatology Department, Katherine Monaghan Royal Melbourne Hospital, Parkville, VIC, Australia; 3University of 1 2 Melbourne, Parkville, VIC, Australia CSL Limited, Broadmeadows, VIC, Australia; The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; 3The Granulocyte macrophage colony stimulating factor (GM-CSF) was Royal Melbourne Hospital, Parkville, VIC Australia; 4The University originally discovered by virtue of its effects as a haemopoietic growth of Melbourne, Parkville, VIC, Australia; 5Monash University, factor. However, GM-CSF turns out to have a variety of effects in Malvern East, VIC, Australia addition to those well recognised for bone marrow progenitor cells, including on cells of the immune and inflammatory systems. As such, Systemic lupus erythematosus (SLE) is a multi-system autoimmune GM-CSF can also be considered as a pro-inflammatory cytokine. Pre- disease characterized by auto-reactive antibodies and elevated pro- clinical studies established a strong rationale for targeting GM-CSF in duction of interferon-a (IFNa). SLE autoantibodies, directed against diseases such as rheumatoid arthritis (RA). Therapeutic monoclonal DNA and RNA components, form immune complexes that can acti- antibodies that antagonise GM-CSF signalling have been developed vate toll-like receptors (TLR)-7 and -9 on plasmacytoid dendritic cells and are being evaluated in human clinical trials. To date, the results in (pDCs), which produce large amounts of IFNa in response. PDCs also RA have been very promising and raise the prospect of extending this express high levels of interleukin-3 receptor alpha (IL-3Ra; CD123). approach to other inflammatory diseases. We have developed a novel, neutralizing anti-CD123 mAb (CSL362)

123 S62 Inflamm. Res. with enhanced antibody-dependent cell-mediated cytotoxicity kappaB and MAPKs). However, there are numerous aspects of (ADCC) against CD123-expressing cells. initiation and perpetuation of pro-inflammatory signalling responses To study the effects of CSL362 in SLE, we recruited a cohort of downstream of TLRs that are still poorly understood, some of which SLE patients (n = 34) and matched healthy donors (n = 34). We may be amenable to targeting. Here we report a novel cell-surface enumerated various cell subsets and determined their CD123 protein that is required for agonist-induced TLR4 phosphorylation, expression. In addition, the in vitro effect of CSL362 on pDC pro-inflammatory signalling and the TLR4-inducible production of a depletion and subsequent IFNa production and IFNa-inducible gene sub-set of pro-inflammatory cytokines (IL-6, IL-12). These studies expression was determined. The effect of CSL362 in vivo was also have revealed that proximal events involved in initiating TLR4 examined after subcutaneous (s.c.) administration to non-human pri- signalling impart specificity to downstream inflammatory responses, mates (NHPs). thus offering new avenues for selective manipulation of TLR-in- This study shows that in healthy controls and SLE donors, pDCs ducible cytokine production. We also describe the role of a specific have the highest average expression of CD123 (44,152 and 49,325 histone deacetylase (HDAC7) in driving HIF-1alpha-dependent receptors/cell respectively), followed by basophils (28,530 and 34,094 TLR4-inducible cytokine production in macrophages. HDAC7 is receptors/cell respectively) compared with other cell types that have expressed at elevated levels in inflammatory macrophages, thus \2000 receptors/cell. SLE donors had reduced cell numbers for most providing a mechanism for perpetuation of pathological inflamma- cell types studied, including pDCs, basophils and NK cells; however, tory responses. Broad-spectrum HDAC inhibitors are efficacious in they had normal numbers of eosinophils, neutrophils and plasma animal models of many inflammation-mediated diseases, but are blasts. CD123 expression was not statistically different between SLE also associated with a number of adverse effects. Thus, selective and healthy control donors for all cell types, with the exception of targeting of HDAC7 may enable the generation of new HDAC pDCs, which had higher CD123 expression in SLE than healthy inhibitors with anti-inflammatory properties, but reduced side donors. CSL362 potently depleted pDCs in vitro in both SLE and effects. healthy donors (EC90 = *0.01 lg/ml). CSL362 also partially depleted basophils and mDCs in vitro but did not significantly deplete other cell types. In addition, CSL362 abrogated ex vivo TLR-7 and - 9-induced IFNa production in SLE and healthy donors and selectively 022 inhibited an IFN gene signature induced by TLR-7 and -9 agonists, A NEW CANCER THERAPEUTIC THAT TARGETS but not other TLR agonists. In vivo, a dose-dependent depletion of ANGIOGENIC VESSELS pDCs and basophils was observed in response to s.c. administration of CSL362 to cynomolgus macaques. Depletion of pDCs and basophils Jennifer R. Gamble1,2, Yang Zhao1,2, Kaka Ting1,2, Jia Li1,2, by CSL362 was associated with activation of NK cells both in vitro Thorleif Moller3, Mathew A. Vadas1,2 and in vivo. Subcutaneous administration of CSL362 lead to a similar inhibition of IFN-induced genes in blood stimulated with TLR-9 1Centenary Institute, Sydney, NSW, Australia; 2University of Sydney, agonists ex vivo. Sydney, NSW, Australia; 3Mirrx Therapeutics, Vejle, Denmark This study shows that CSL362 potently and selectively depletes pDCs in vitro, in both SLE and healthy donors, and in vivo in NHPs. Tumour angiogenic vessels are considered an attractive target for the PDC-depletion by CSL362 was associated with decreased TLR-7/9- development of therapeutics. One of the prime targets is VEGF, the induced IFNa production and IFNa-inducible gene expression in vitro major growth factor for blood vessels. However, the anti-VEGF and in vivo accompanied by increased NK cell activation. Cytore- therapies have shown limited efficacy resulting in only a transient ductive therapy with CSL362 may therefore represent a novel effect on tumour growth but also resulting in an increase in tissue treatment strategy in SLE. Supported by Janssen Biotech hypoxia that ultimately promotes tumour growth and limits radio- and immune-therapy. Tumour angiogenic blood vessels are characterised by both structural and functional changes. Thus, they are tortuous, lack ade- 021 quate pericyte coverage and the matrix shows changes in density and TARGETING TLR-INDUCIBLE INFLAMMATORY composition. These changes result in increased permeability, reduced PATHWAYS IN MACROPHAGES perfusion and the tissue environment is hypoxic. The concept of vessel normalisation has been proposed as an alternative to ablation of tumour angiogenic vessels. In normalisation, the vessels are converted Matthew J. Sweet back to a structurally more normal architecture and function, with the potential to enhance radio-, chemo- and immune-therapy. IMB, The University of Queensland, St. Lucia, QLD, Australia We have developed a first-in-class drug (CD5-2) that with a single Toll-like receptors (TLRs) respond to both pathogen- and host- intravenous injection significantly enhances, in mice, tumour vessel derived danger signals to drive pro-inflammatory signalling perfusion and pericyte coverage, decreases vascular leak and tissue responses in innate immune cells such as macrophages. Inappro- hypoxia and inhibits tumour growth. CD5-2 increases the expression priate or dysregulated TLR activation propagates pathological of the major structural endothelial cell specific junctional adhesion inflammation in animal models of numerous inflammation-driven molecule VE-cadherin and has downstream effects on other associ- diseases including autoimmune diseases, atherosclerosis, neurode- ated pathways involved in vessel stabilisation namely the TIE-2 and generation, liver damage and cancer metastasis. Consequently, TLR the tight junction pathways. complexes and downstream signalling pathways represent attractive CD5-2 is a proprietary single stranded modified oligonucleotide. targets for inflammation-driven diseases. The textbook view of TLR The profound effects of CD5-2 are being currently examined in signalling is that receptor activation results in the recruitment of metastatic, cytotoxic and radiotherapy models. In an age where toll/interleukin-1 receptor (TIR) domain-containing proteins, which ‘personalised’ therapy of cancers is of great interest, drugs that have in turn recruit specific serine/threonine kinases (IRAKs) to relay potential utility across all solid cancers, and are thus ‘non-person- downstream pro-inflammatory signalling (e.g. activation of NF- alised’ offer a cost-effective advance.

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023 asthma patients do not achieve total control. The commonest reason GLUCOCORTICOID-INDUCED LEUCINE ZIPPER for this is poor adherence with asthma treatment. Some patients, however, will not achieve asthma control, even with maximal doses AS A NOVEL THERAPEUTIC IN INFLAMMATORY of currently available therapy, perhaps as many as 10 %. These DISEASE patients are considered to have severe refractory asthma. It has become evident that severe refractory asthma consists of a very Michael J. Hickey, Qiang Cheng, Eric F. Morand heterogeneous population of patients. In addition, many diseases can masquerade as severe asthma. An accurate diagnosis and careful Department of Medicine, Centre for Inflammatory Diseases, School of phenotyping is needed to identify which newer treatments may benefit Clinical Sciences at Monash Health, Monash University, Malvern an individual patient. East VIC, Australia A number of experimental treatments are being developed for severe refractory asthma. An example of the necessity to phenotype Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory patients with severe refractory asthma has been the development of protein first identified in T lymphocytes, being highly inducible in monoclonal antibodies (hMab) directed against interleukin (IL)-5, a response to dexamethasone. In previous work, we have observed anti- cytokine produced by Th2 cells in innate lymphocytes type 2 (ILC2). inflammatory effects of GILZ both in vitro and in vivo. Transfection of Two antibodies have been developed (mepolizumab and relizumab), GILZ into endothelial cells reduced their capacity to support interac- neither of which showed benefit in a non-selected cohort of patients tions with leukocytes, via effects on expression of adhesion molecules with difficult-to-treat asthma; however, when studied in patients with and chemokines. Similarly, in vivo delivery of GILZ via a viral vector a persisting airway eosinophilia, these treatments have been shown to reduced inflammation in a mouse model of collagen-induced arthritis. reduce asthma exacerbations and improve lung function. The use of As a result of these observations, we hypothesized that exogenously- induced sputum was essential to identify these patients with a per- delivered GILZ has potential as a novel anti-inflammatory agent, and sisting airway eosinophilia, despite optimal treatment. Similarly, a that it may provide a more specific anti-inflammatory effect than hMab directed against another type-2 cytokine, IL-13 significantly glucocorticoids by avoiding their well-known adverse metabolic improved lung function in patients with difficult-to-control asthma, effects. To address this, we developed a cell-permeable form of GILZ but only in those with an elevated serum periostin (a protein produced protein, termed HHph1-GILZ, via inclusion of a protein transduction by airway epithelial cells after stimulation with IL-13). Also, a hMab domain. HHph1-GILZ, but not native GILZ, was able to enter various directed against the IL-4Ra, which is the common component of the cell types including microvascular endothelial cells, macrophages and receptor for IL-4 and IL-13, is showing promise in patients with primary peripheral blood mononuclear cells, in vitro. Moreover, elevated blood eosinophil counts. HHph1-GILZ reduced activation of endothelial cells and macrophages Thymic stromal lymphopoietin (TSLP) is produced by airway in response to TNF, as determined using cell lines incorporating an epithelial cells in response to viruses and environmental allergens. It NFkB reporter construct. We are also assessing whether GILZ exerts is up-stream to the production of the type-2 cytokines, IL4, Il-5 and harmful metabolic effects similar to those induced by glucocorticoids, IL-13. Treatment with a hMab against TSLP also shows promise in using biochemical assays with hepatocyte cell lines and, to this point, asthma. there is no evidence that this is the case. In ongoing work, we are It is likely that all new treatments for severe refractory asthma will undertaking further characterisation of the molecular targets of require efforts at phenotyping to target therapy at the populations of HHph1-GILZ, with a view to assessing its anti-inflammatory capaci- patients likely to benefit, as this group of patients have such hetero- ties in in vivo models of inflammation. In conclusion, these findings geneous mechanisms causing their severe disease. support further investigation of the utility of cell-permeable GILZ as a novel therapeutic for inflammatory conditions.

025 THERAPEUTIC STRATEGIES FOR THE SYMPOSIUM 7: NEW THERAPEUTIC TREATMENT OF CYSTIC FIBROSIS STRATEGIES FOR RESPIRATORY Stuart Elborn DISEASES—SPONSORED BY ERS Queens University Belfast, Belfast, UK 024 Cystic fibrosis is the most common life-limiting genetic disorder ANTI-CYTOKINE THERAPY IN ASTHMA: FOCUS affecting North Western Europeans and it occurs in all populations ON TSLP across the world. It is caused by reduced or abnormal function of the cystic fibrosis transmembrane regulator protein (CFTR) which is a cyclic AMP regulated chloride channel. CFTR also transports other Paul M. O’Byrne ions particularly bicarbonate and regulates an epithelial sodium channel and interacts with inflammation related pathways within the McMaster University, Hamilton, ON, Canada cell. CFTR regulates hydration, pH and electrolyte concentration in Asthma management is focused on achieving total asthma control in epithelial lining fluid and in the lung this is mucus hydration, two domains. First is current control, where the patient is asymp- mucocilary clearance and innate immunity. tomatic all of the time, has normal lung function and no limitations in A range of therapeutic approaches have been utilised in an attempt activities. The second in minimizing future risk of severe exacerba- to restore CFTR function in people with cystic fibrosis. The aim is to tions, accelerated decline in lung function and avoiding side effects restore the airways surface liquid and associated micro environment from medications. to improve mucociliary clearance and innate immune function. In a Currently available treatment, particularly with inhaled corticos- ground-breaking small molecule programme ivacaftor was identified teroids (ICS) with or without long-acting inhaled b2-agonists (LABA) through high throughput screening to be a potentiator of mutant can achieve good asthma control in many patients. However, most CFTR. In patients with a particular mutation (G551D) where the

123 S64 Inflamm. Res. channel is in the cell membrane, clinical trials have demonstrated composed of dsDNA, histones and various proteins from the intra- efficacy, with improvements in lung function, quality of life and body cellular medium. The most recent reports emphasize the implication weight and a reduction in pulmonary exacerbations. More recently of NOX2 and rac, but also the role of SK3 channel, the mitochondrial- combination therapy with a second corrector which increases traf- derived reactive oxygen species, the mTOR pathway of autophagy or ficking of mutated F508del CFTR to the cell membrane with ivacaftor the complex mechanism of citrullination and its main importance in as a secondary potentiator have also demonstrated improvements in autoimmunity. Neutrophils might even retain some of their functional lung function and a reduction in exacerbations. These studies capacities while expressing NETs. demonstrate that CFTR is a drugable target and can be corrected. Netosis is one of the defense mechanisms against microbes and Other innovative approaches to specific mutations include read- neutrophils are able to choose to undergo netosis or to prefer through strategies and gene editing and gene therapy approaches. phagocytosis depending on the size of the pathogen; moreover some Correcting CFTR in people with CF is a very fruitful area for drug of them have developed strategies to overcome netosis. development. Ion channel therapy may also be a benefit in other Beside their important antimicrobial activity, NETs have been airway diseases and have effects on improving mucociliary clearance implicated in the pathophysiology of an increasing number of human and potentially restoring innate immunity. diseases. NETs components such as DNA, proteinase 3, myeloper- oxidase or citrullinated proteins are considered as auto-antigens by the immune system and can thus induce autoimmunity; systemic lupus erythematosus is one of the best documented example. 026 NETs also play a role in tissue damage in various clinical situa- NEW TREATMENT OPTIONS FOR PULMONARY tions. During severe infections, in particular in the liver and in the FIBROSIS lung, the majority of the injury might be induced by proteases-asso- ciated NETs rather by the microbe itself. Netosis, coagulation Toby M. Maher1,2 activation and platelets functions are linked and several lines of evi- dence implicate NETs in thrombosis-related diseases. Another 1Imperial College, London, London, UK; 2Royal Brompton Hospital, important challenge is to better understand how NETs are involved in London, UK cancer development. Among the new clinical studies documenting the deleterious effects of NETs, the description of the NET-related pri- Idiopathic pulmonary fibrosis (IPF) is an inexorably progressive mary graft dysfunction after lung transplantation is original. disease of unknown aetiology that conveys a dismal prognosis. Until Interestingly, NETs can participate to the regulation of the 12 months ago there were no licensed therapies in the United States immune response. For instance, they can downregulate dendritic cell for the treatment of IPF. However, a growth in pharmaceutical maturation, leading to Th2 polarization. They also express some interest in the treatment of fibrosis coupled with the development of inhibitors of the inflammatory response like SLPI. effective IPF clinical trials networks has led to a number of key Finally, given the deleterious effects of NETs on cells and tissues, developments in the treatment of IPF over the last decade. Both several therapeutic approaches have been suggested, via the use of pirfenidone and nintedanib have shown efficacy signals in multi- DNAse, the inhibition of their formation or their shedding in the centre phase 3 trials and have now both been approved in the US and circulation. Europe for the treatment of IPF. Furthermore, advances in the understanding of the pathogenetic processes involved in the devel- opment of pulmonary fibrosis have led to the identification of a multitude of novel therapeutic targets. This in turn has contributed to 028 a rapid expansion in the number of early phase trials in IPF with UNEXPECTED NEUTROPHIL CONTRIBUTIONS current targets including; protein kinases, cytokines, growth factors, TO ANTIBODY-INDUCED REACTIONS oxidative stress and matrix turnover. These developments offer gen- uine hope for individuals with IPF, and for their treating physicians, Pierre Bruhns1,2 that there may, in the relatively near future, be a range of therapeutic options available for the treatment of this devastating disease. 1Antibodies in Therapy and Pathology, Institut Pasteur, Paris, France; 2INSERM U760, Paris, France IgG antibodies are potent activators of myeloid cells that express IgG SYMPOSIUM 8: THE NEUTROPHIL: AN receptors (FccRs). Targeting of antibodies onto tissue-expressed OLD DOG WITH NEW TRICKS— antigens or onto circulating antigens can induce local or systemic SPONSORED BY GREMI inflammatory reactions, respectively. We aimed at understanding which myeloid cells are responsible for IgG antibody-induced inflammation in a local inflammatory conditions, i.e. a subcutaneous 027 tumor targeted by a therapeutic antibody, or in a systemic inflam- NEWS ABOUT NETS matory condition, i.e. circulating allergens opsonized by anti-allergen antibodies that results in allergic shock (anaphylaxis). Sylvie Chollet-Martin We show that neutrophils are responsible for antibody-induced therapy of subcutaneous tumors (a syngeneic melanoma or a human Bichat Hospital and INSERM Paris-Sud University, Orsay, France breast cancer xenograft) and for the induction of allergic shock. Both antibody-induced reactions were abolished in neutropenic mice or In 2004, A Zychlinsky evidenced that neutrophils were able to release the mice lacking FccRs. But both reactions could be restored upon so-called neutrophil extracellular traps (NETs) in order to kill pathogens transfer of FccR-expressing mouse or human neutrophils. In addition, via both microbe ensnaring and protease release. The present review aims transgenic expression of human IgG receptors expressed on activated to present some of the most important new findings on NETs. neutrophils, FccRI (CD64) or FccRIIA (CD32A), enabled both Since 2004, a large number of studies have described the mech- antibody-induced anti-tumor and anaphylactic reactions. Importantly, anisms leading to the formation of these web-like structures conditional knockout mice unable to perform FccR-mediated

123 Inflamm. Res. S65 activation and phagocytosis specifically in neutrophils were resistant regulation of the pro-survival PCNA scaffold and may have important to antibody-induced cancer therapy. therapeutic applications in chronic inflammatory diseases. Our work suggests that neutrophils are necessary and sufficient for (1) mAb-induced therapy of subcutaneous tumors, and (2) anaphy- lactic reactions to circulating allergens that mimics drug-induced anaphylaxis. These unexpected roles of neutrophils suggest novel B232 therapeutic avenues to improve antibody-induced anti-tumor therapy INTRAVITAL IMAGING OF VASCULATURE and to reduce drug-induced allergic shock. REVEALS THAT NEUTROPHIL EXTRACELLULAR TRAP (NET) COMPONENTS ATTACH TO VON WILLEBRAND FACTOR IN A DNASE-INDEPENDENT MANNER 029 DECIPHERING THE CYTOSOLIC SCAFFOLD OF See poster section for abstract. THE PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) TO CONTROL NEUTROPHIL SURVIVAL IN INFLAMMATORY DISEASES B204 RECOGNITION OF PRODUCTS FROM Veronique Witko-Sarsat1,2,3, Cle´mence Martin2,4, Delphine Ohayon1,2,3, Pierre-Regis Burgel2,4 NEUTROPHIL DEGRANULATION BY TOLL LIKE RECEPTORS MEDIATE KILLING OF 1INSERM U1016, Institut Cochin, Paris, France; 2Universite´ Paris LEISHMANIA AMAZONENSIS IN MACROPHAGES Descartes, Paris, France; 3Laboratoire d’Excellence INFLAMEX, 4 Paris, France; Cochin Hospital, Paris, France See poster section for abstract. The life span of a neutrophil must be tightly regulated, as extended survival is essential for the effective elimination of pathogens and cell death necessary to prevent the release of the highly cytotoxic contents of activated neutrophils and subsequent tissue damage. We have B282 previously shown that proliferating cell nuclear antigen (PCNA), a NEUTROPHIL PROTEASES ALTER THE nuclear factor involved in DNA replication and repair of proliferating INTERLEUKIN-22-RECEPTOR-DEPENDENT cells, is localized exclusively in the cytoplasm of neutrophils where it LUNG ANTIMICROBIAL DEFENCE regulates their survival. Nuclear PCNA functions are tightly linked to its ring-shaped structure, which allows PCNA to bind to numerous partner proteins to orchestrate DNA-related processes. We have See poster section for abstract. shown that only monomeric PCNA can expose its nuclear export sequence to be relocalized from nucleus to cytosol during granulocyte differentiation. When localized into the cytosol, PCNA is able to bind SYMPOSIUM 9: OCULAR procaspase-3, 8, 9 and 10, which in turn precludes their activation. Another well-characterised binding partner is the cyclin dependent INFLAMMATION, TRANSLATION kinase inhibitor p21/waf1, which binds PCNA at the interdomain- FOCUSED—SPONSORED BY ORA, INC. connecting loop which is the preferred site of interaction for numerous PCNA partners. A small peptide corresponding to the residues 141–160 of p21/waf1 known as carboxyp21 was found to 030 interfere with PCNA-protein interactions and in turn had a strong NK-DC CROSSTALK TRIGGERS AN INNATE antiproliferative effect. Treating neutrophils with carboxyp21 peptide IFN-c/IL-27 LOOP THAT CONTROLS THE triggered PCNA degradation and apoptosis, while treatment with a AUTOPATHOGENIC TH17 RESPONSE mutated version of this peptide incapable of binding to PCNA, had no effect on apoptosis. Moreover, carboxyp21 peptide significantly 1 1 2 inhibited G-CSF-induced neutrophil survival. We next examined if Rachel R. Caspi , Wai Po Chong , Nicholas van Panhuys , Jun Chen1, Phyllis B. Silver1, Yingyos (Ed) Jittayasothorn1, p21/waf1 expression in neutrophils can enhance their apoptosis and 2 potentiate the resolution of inflammation. The role of p21/waf1 in Ronald N. Germain Pseu- neutrophil survival was also examined in a murine model of 1 domonas aeruginosa Immunoregulation Section, Laboratory of Immunology, National Eye as well as in a model of peritonitis. After 7 days 2 of lung infection with P. aeruginosa, neutrophilic inflammation was Institute, NIH, Bethesda, MD, USA; Lymphocyte Biology Section, more prominent in p21-/- mice compared to WT controls. In vitro, Laboratory of Systems Biology, National Institute of Allergy and neutrophils isolated from p21-/- displayed enhanced survival in Infectious Diseases, NIH, Bethesda, MD, USA response to TNF-alpha and G-CSF associated with an increased in IFN-c is a proinflammatory cytokine. Paradoxically, its deficiency PCNA expression. Our data reveals a novel role for p21/waf1 in the exacerbates experimental autoimmune encephalomyelitis, uveitis and resolution of inflammation after P. aeruginosa infection via its ability arthritis. Here we demonstrate that innate production of IFN-c from to promote neutrophil apoptosis via the destabilization of cytosolic NK cells is necessary and sufficient to trigger a novel endogenous scaffold of PCNA. We concluded that p21/waf1 functions as an regulatory circuit that limits autoimmunity. Following autoantigen inducible and endogenous break in the PCNA pro-survival platform, challenge, DCs recruited IFN-c-producing NK cells to the draining which has been previously described (Witko-Sarsat et al. J Exp Med, lymph node and interacted with them in a CXCR3-dependent manner. 2010). To our knowledge, this is the first report showing negative DCs then produced IL-27, enhancing IFN-c production by NK cells

123 S66 Inflamm. Res. and forming a self-amplifying positive feedback loop that was ulti- mately controlled by NK/DC-intrinsic IL-10. IL-27 dampened the autopathogenic Th17 response and induced a regulatory Tr1 response, which ameliorated disease in an IL-10-dependent fashion. Thus, early NK-DC crosstalk controls adaptive immunity and limits autoimmune disease though an innate IFN-c/IL-27 axis.

031 COMPLEMENT: MORE THAN A ‘‘GUARD’’ AGAINST PATHOGENS

John D. Lambris

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Nearly a century after the significance of the human complement system was recognized, we have come to realize that its functions extend far beyond the elimination of microbes. It is increasingly perceived as an intricate network of effectors, regulators and regu- lators that not only drives core cascade functions in health and disease but also extensively communicates with associated physiological pathways ranging from immunity and inflammation to homeostasis and development. A steady stream of experimental data reveals new fascinating connections at a rapid pace; while opening unique opportunities for research discoveries, and therapeutic interventions 033 against various diseases and clinical conditions. I will discuss our DALAZATIDE (SHK-186), A KV1.3 POTASSIUM updated view of the function, structure and dynamics of the com- CHANNEL BLOCKER, SIGNIFICANTLY REDUCES plement network, highlight its interconnection with immunity at large INFLAMMATORY CELL INFILTRATES AND and with other endogenous pathways, and illustrate its multiple roles in homeostasis and disease emphasizing its role in ocular disease. DISEASE IN AN EXPERIMENTAL AUTOIMMUNE Finally, I will discuss the current status of complement therapeutics ANTERIOR UVEITIS MODEL and various approaches for complement based therapies. Ernesto J. Munoz, David W. Peckham, Kayla Norton, Shawn P. Iadonato 032 Kineta, Inc, Seattle, WA, USA ROCK-ISOFORM SPECIFIC POLARIZATION OF MACROPHAGES ASSOCIATED WITH AGE- Autoreactive effector memory T cell subsets play a major role in the etiology and chronicity of non-infectious and dry eye ocular RELATED MACULAR DEGENERATION disease by secretion of multiple inflammatory mediators. TH1 and TH17 effector memory cells infiltrate the anterior chamber during Ali Hafezi-Moghadam1,2 Sjogren’s syndrome and are key mediators in chronic uveitis models. Standard of care therapies for refractory uveitis and dry 1Harvard Medical School, Department of Radiology; 2Brigham and eye diseases are problematic, as they are broadly immunosup- Women’s Hospital, Boston, MA, USA pressive, cause significant side effects, or exhibit poor eye penetration. Age is a major risk factor in age-related macular degeneration Dalazatide (ShK-186) is a potent, highly specific blocker of Kv1.3, (AMD), but the underlying cause is unknown. We find increased Rho- a potassium channel required for sustained intracellular calcium associated kinase (ROCK) signaling and M2 characteristics in eyes of influx during activation of effector memory T cells. It is effective in aged mice, revealing immune changes in aging. ROCK isoforms preventing disease in preclinical models of diverse autoimmune dis- determine macrophage polarization into M1 and M2 subtypes. M2- eases. It was well tolerated with no serious adverse events reported in like macrophages accumulated in AMD, but not in normal eyes, phase 1 trials in healthy volunteers, and is currently being evaluated suggesting these macrophages may be linked to macular degenera- in a POC trial in psoriasis. tion. M2 macrophages injected into the mouse eye exacerbated In the current studies, topically-dosed dalazatide was evaluated for choroidal neovascular lesions, while M1 macrophages ameliorated its ability to penetrate the anterior chamber, and reduce disease and them, supporting a causal role for macrophage subtypes in AMD. Kv1.3+ and CD3+ cell infiltrates when given prophylactically in an Selective ROCK2 inhibition with a small molecule decreased M2-like EAAU disease model. macrophages and choroidal neovascularization. ROCK2 inhibition Dose-dependent dalazatide penetration of the anterior chamber upregulated M1 markers without affecting macrophage recruitment, was demonstrated, with concentrations in the aqueous fluid ranging underlining the plasticity of these macrophages. These results reveal from 5 to 62 and 275 to 949 ng/mL (0.1 and 1 % dalazatide dose, age-induced innate immune imbalance as underlying AMD patho- respectively). No histopathology was observed in H&E-stained sec- genesis. Targeting macrophage plasticity opens up new possibilities tions of 1 % dalazatide-treated naı¨ve eyes. In the EAAU model, for more effective AMD treatment.

123 Inflamm. Res. S67 dalazatide reduced disease penetration from 13 of 16 eyes (vehicle) to development. Notably, GILZ is an essential mediator for GC-induced 3 of 16 (dalazatide). Dalazatide also reduced the mean composite development of T regulatory cells (Treg), a T cell subpopulation clinical score for all eyes from 7.5 (vehicle) to 1.8 (dalazatide). responsible of anti-inflammatory effect. Consistent with these T cell Cellular infiltrates were reduced in anterior chamber fluid, as well. subpopulations changes there is an increased severity of colitis, Histopathology confirmed that dalazatide also reduced or eliminated arthritis and other inflammatory disease models in GILZ-KO, while a dilated iris vessels and cellular infiltrates. IHC demonstrated that reduction in GILZ-TG. dalazatide reduced or eliminated Kv1.3+ and CD3+ infiltrates of the Results indicate GILZ as a mediator of GC activity, provide new anterior chamber, ciliary body, and iris. means to predict sensitivity to treatment with GC and to outline new These results demonstrate that topical ocular dosing of dalazatide therapeutic approaches. has potential for treating autoimmune-mediated uveitis by targeting CD3+ and Kv1.3+ inflammatory cells which mediate disease, while limiting side effects. 036 GLUCOCORTICOID AND B CELL SYMPOSIUM 10: GLUCOCORTICOIDS IN DEVELOPMENT: ROLE OF GLUCOCORTICOID- THE REGULATION OF INFLAMMATORY INDUCED LEUCINE ZIPPER (GILZ) RESPONSE—SPONSORED BY ITALIAN Stefano Bruscoli, Michele Biagioli, Oxana Bereshchenko, Tiziana INFLAMMATION GROUP Frammartino, Daniele Sorcini, Monica Cimino, Carlo Riccardi

034 Department of Medicine, Section of Pharmacology, University of INTENSE CROSS–TALK BETWEEN TNF AND Perugia, Perugia, Italy GLUCOCORTICOID RECEPTOR Glucocorticoids (GC) are widely used as immunosuppressive drugs and antitumor agents in some acute leukemia and multiple myeloma. Claude Libert Therapeutic doses of GC induce growth suppressive and cytotoxic effects on various leukocyte types including B cells. Molecular Inflammation Research Center, VIB, Ghent, Belgium mechanisms of GC action include induction of GC target genes. Glucocorticoid-induced leucine zipper (GILZ) is a gene rapidly, GC protect extremely well against acute inflammatory conditions and potently and invariably up-regulated by GC treatment. It mediates a the cross talk between GC and cytokines is important. However, the number of GCs effects, such as control of cell proliferation, apoptosis exact mechanism of the anti-inflammatory function of GC is still and differentiation. GILZ suppresses Ras/MAPK/Erk and NFkB unclear. Notably, large groups of patients respond insufficiently to pathways and promotes TGF-b signaling in T cells. It belongs to GC, a condition called GC resistance. This problem occurs in some TSC22d family, members of which were recently found mutated in 5 % of asthma patients and in most of the sepsis patients. Sepsis is a diffuse large B cell lymphoma patients. form of systemic inflammatory response syndrome (SIRS) and a huge Here we address the physiologic role of GILZ in normal hema- unmet medical need. Study of the mechanism of loss of GC protec- topoiesis, and evaluate its role in mediation of GC effects on various tion, when GC aregiven after the onset of SIRS, indicate an intense blood cells, using genetic approach. cross talk between TNF and GC. Mice deleted for gilz gene were recently generated. We have monitored white blood cell counts in wild type (wt) and in gilz knock- out (KO) mice overtime. Development of lymphoid and myeloid lineages was evaluated both by peripheral blood (PB) cell counts 035 (Hematocrit), and by flow cytometry analysis of bone marrow (BM), ROLE OF GILZ IN MEDIATING THE ANTI- spleen and PB using Mac-1, B220, CD43, IgM and IgD staining. INFLAMMATORY ACTIONS OF Young gilz KO mice showed normal body and lymphoid tissues GLUCOCORTICOIDS weights and cell counts in PB, thymus, spleen, peripheral lymph nodes and BM. However, overtime gilz KO mice showed a 1.5- to 2-fold increase in white blood cell counts in PB. Increase in lym- Carlo Riccardi, Monica Cimino, Tiziana Frammartino, Daniele phocyte counts was due to accumulation of B220+ cells, while the Sorcini, Michele Biagioli, Oxana Bereshchenko, Stefano Bruscoli number of Mac-1+ cells did not differ between wt and gilz KO mice. Flow cytometry analyis of B220+ cell compartment in BM revealed Department of Medicine, University of Perugia, Perugia, Italy an increase in the frequency and number of pre-B cells (IgM loIgDlo), Glucocorticoids (GC) are of extraordinary therapeutic value in a wide immature (IgMhiIgDlo) and recirculating B cells (IgMloIgDhi) range of inflammatory and autoimmune diseases. Most of GC effects already in 8-week old mice. Preliminary data suggest that the defect are receptor (GR) mediated and relates to regulation of gene tran- starts as early as at common-lymphoid progenitor (CLP) stage. scription. Their therapeutic activity is due to effects on activation, Treatment of purified B220 + cell with GC in vitro resulted in dif- growth and differentiation in a number of cells and tissues, including ferent degree of apoptosis in wt and gilz KO cells, suggesting that the cells of the immune/inflammatory system such as T lymphocytes. increase in B cells in vivo may results from decreased sensitivity to We describe the GC-induced gene glucocorticoid-induced leucine the death induced by endogenous GC. zipper (GILZ), a protein rapidly induced by GC treatment. Notably, Our results show that lack of GILZ results in specific defect in B our results indicate GILZ is a mediator of GC effects. In particular, cell development, leading to the expansion of B220 + cells com- using GILZ-TG and GILZ-KO mice we demonstrate, in various partment, associated with the expansion of early B cell progenitor disease models, GILZ is an anti-inflammatory molecule. Interestingly, cells and suggest that deregulation of GILZ expression may con- GILZ regulates B and T cell activation, differentiation and survival. tribute to cell survival or differentiation of early B cells and Moreover, it regulates cytokine production and inflammatory process pathologies of B cell lineage.

123 S68 Inflamm. Res.

SYMPOSIUM 11: A ROLE FOR terminal domain (BET) family. Mice treated with a BET protein inhibitor were protected from the development of diabetes. Concor- EPIGENETICS IN INFLAMMATORY dantly, insulitis was either prevented or inhibited, depending on the DISEASE: SPOTLIGHT ON MONOCYTES treatment protocol. Mechanistically, BET inhibition induced an anti- AND MACROPHAGES inflammatory phenotype in pancreatic macrophages through the sup- pression of an NF-kappa-B-mediated inflammatory pathway in these cells. Strikingly, I-BET was also beneficial to pancreatic beta cells by 037 promoting beta cell turnover in vivo. Thus, small molecule inhibition THE EPIGENETIC SIGNATURE OF of BET protein bromodomains irreversibly blocks T1D in NOD mice INFLAMMATION IN WHITE BLOOD CELLS AND via combined effects of fostering anti-inflammatory macrophages and promoting beta cell function. Together, these data provide a rationale PULMONARY MACROPHAGES FROM SMOKERS: for T1D treatment by targeting epigenetic modifications. WHERE THERE IS SMOKE, THERE IS FIRE

Robert A. Philibert 039 EPIGENETIC DYSREGULATION IN IMMUNE Universtity of Iowa, Iowa City, IA, USA TOLERANCE: TWO SIDES TO THE SAME COIN- Smoking is the largest preventable cause of morbidity and mortality in CANCER AND AUTOIMMUNITY the United States. Each year, over a half million people die prema- turely from smoking related illnesses including coronary artery S A. Litherland, PhD1,2,3, Michael J. Clare-Salzler, MD2, Juan Pablo disease, stroke and chronic obstructive pulmonary disease. Peripheral 1 white blood cells and their derivatives, most notably, alveoloar mac- Arnoletti, MD rophages, are known to play important roles in mediating the 1Florida Hospital Cancer Institute, Orlando, FL, USA; 2University of pathogenesis of some of these smoking-related illnesses. In this pre- 3 sentation, we compare and contrast the whole genome DNA Florida College of Medicine, Gainesville, FL, USA; Sanford- methylation of DNA from peripheral white blood cells and pulmonary Burnham Medical Research Institute, La Jolla, CA, USA macrophages. We show that smoking is associated with dose-depen- Immune tolerance, the ability of the immune system to distinguish dent alteration of DNA methylation with preference for those genes self from non-self on the molecular level, varies throughout the body. mapping to immune system and inflammatory pathways in both A range of immune sensitive to immune privileged compartments are peripheral white blood cell and alveolar macrophage DNA. These found, dependent on the need to balance recognition of foreign changes in DNA methylation are mirrored by corresponding changes antigens mimicking self moieties with the ability to recognize and in the expression of genes mapping to inflammatory and immune destroy altered-self antigens. Maintenance of immune tolerance is a system pathways, and prior data demonstrating the role of smoking in double edged sword in disease: too little, and self-reactivity leads to driving the conversion of alveolar macrophages from the M1 to the M2 autoimmune pathology; too strong and altered-self cells go unde- phenotype. Network analyses of peripheral methylation data demon- tected or are actively protected from immune responses. A pro- strate that smoking alters the regulation of protein networks important inflammatory microenvironment aides both immune-related not only to inflammation, but to smoking associated illnesses such as pathologies. stroke, heart disease (notably F2RL3) and cancer. Finally, and perhaps In autoimmunity, epigenetic dysregulation promotes the inflam- most importantly, the sensitivity and reversibility of DNA methylation matory milieu conducive to the development of immunopathology. In at key loci, such as AHRR, may allow the derivation of new clinical this pro-inflammatory environment, antigen presenting cell regulation tools for improving smoking prevention and cessation. of self-reactive T cells becomes defective, self tolerance can be lost, and auto-reactivity ensues. In autoimmune type 1 diabetes (T1D), we found that epigenetic dysregulation affects the expression of GM-CSF 038 and its subsequent activation of COX2 expression and PGE2 pro- SUPPRESSION OF TYPE 1 DIABETES BY duction. The dysregulation is mediated by aberrant histone acetylation EPIGENETIC MODULATION though dysfunctional STAT5 activation and binding to enhancer elements in both gene loci. This dysfunction seen phenotypically in T1D human monocytes, can be genetically linked in NOD bicongenic Wenxian Fu1,2, Julia Farache1, Rab Prinjha3, Christophe Benoist1, mice myeloid cells through genetic variations found in enhancer Diane Mathis1 sequences affecting expression of CSF2 and PTGS2 gene loci. In cancer, promotion of immune self-tolerance can be lethal, 1Harvard Medical School, Boston, MA, USA; 2University of allowing relapse and metastatic growth of treatment resistant tumors. California, San Diego, Jolla, CA, USA; 3GlaxoSmithKline, Brentford, Epigenetic dysregulation of oncogenes in tumors is emerging as a USA common biomarker for tumor activation and development. Pro-in- Type 1 diabetes (T1D) is an organ-specific autoimmune disease flammatory products of myeloid derived suppressor cell (MDSC) characterized by selective destruction of the insulin-producing beta promote immunosuppression of tumor recognition. These functions cells of the islets of Langerhans of the pancreas. Like many complex appear to be enhanced by the interactions of MDSC with tumor cells. diseases, the pathogenesis of T1D is believed to be affected by both In pancreatic ductal carcinoma (PDAC), we have reproducibly genetic associations and environmental cues. Epigenetic regulation is detected histone acetylation of chromatin promoting the activation critical to integrate environmental signals for the cell to modulate the and gene expression of mutant K-RAS gene. We use this epigenetic functional output of its genome. However, the impact of epigenetic biomarker to identify circulating tumor cells (CTC) concentrated in mechanisms and regulations in T1D remains largely unexplored. Here the portal venous blood circulatory compartment between pancreas we report the modulation of T1D in non-obese diabetic (NOD) mice by and liver. The level of histone acetylation-mediated activation and K- an inhibitor that specifically interferes with the recognition of acety- RAS mutant mRNA expression is not seen in peripheral blood from lated lysine residues by proteins of the bromodomain and extra- the same patients or in the portal venous blood of patients without

123 Inflamm. Res. S69 cancer, suggesting the immune microenvironment of the portal cir- Toll-like receptors (TLR) and Interleukin-1 (IL-1) family of recep- culation may act as a potential metastatic cell reservoir for PDAC. tors. TLRs represent a first line of defense against pathogens such as Epigenetic dysregulation of inflammation and of critical gene bacteria, viruses and yeast with the IL-1 family of receptors also expression in immune targets represent two faces of the same playing important roles in the immediate inflammatory response to pathological coin, allowing for the development of pro-inflammatory invading organisms. In addition IRAK4 is expressed in T and B microenvironments and promoting aberrations of self-tolerance. lymphocytes and has been reported to play an important role in cross talk between the innate and adaptive immune system. IRAK4 has both a kinase dependent signaling role as well as a scaffolding role in a larger signaling complex including proteins such as MYD88 and 040 IRAK1. Interestingly, individuals who lack IRAK4 show impaired THE EPIGENETIC-ASSOCIATED FAMILY OF activation of the innate immune response but no increased suscepti- BROMODOMAIN ‘‘READER’’ PROTEINS AS DRUG bility to viral or fungal infection and only increased liability to TARGETS TO REGULATE ACTIONS OF THE infection by a narrow range of pyogenic bacteria prior to adolescence. Therefore IRAK4 has been recognized as an interesting pharmaco- IMMUNE SYSTEM logical target for the treatment of chronic inflammatory diseases. Using potent selective IRAK4 inhibitors we have characterized Eleonore Beurel the role of IRAK4 kinase activity in mediating inflammatory sig- naling in primary human cells from both healthy donors and patients University of Miami, Coral Gables, FL, USA suffering chronic inflammatory diseases, such chronic obstructive pulmonary disease, systemic lupus erythematosus and gout. More- Orchestration of the inflammatory response is crucial for clearing over, in vivo activity of compounds has also been investigated in a pathogens. Although the production of multiple inflammatory range of chronic inflammatory models. The emerging rationale for cytokines has been thought to be regulated by common mechanisms, IRAK4 inhibition across chronic inflammatory indications will be recent evidence indicates that the expression of some cytokines is presented. differentially regulated by epigenetic regulatory mechanisms. Inhi- bitors of bromodomain reader (BRD) proteins, including BRD2, BRD3 and BRD4, have provided promising results in a wide spec- trum of therapeutic applications, especially the regulation of the 042 immune system. BRD proteins are involved in translating histone A NEW GENERATION OF INHALED KINASES modifications, which dramatically impacts transcription. Thus, over- expressing BRD2 leads to the development of splenic B lymphomas. INHIBITORS FOR INFLAMMATORY AIRWAY BRD4 promotes NF-jB dependent gene responses after endotoxin DISEASES shock. BET bromodomain protein inhibitor (I-BET) protects from otherwise lethal septic shock and blocks lipopolysaccharide (LPS) Christopher S. Stevenson induced cytokine production. We found that IL-6 production is selectively inhibited by a low dose of I-BET151 in RAW264.7 cells Respivert Ltd, London, UK stimulated with LPS, whereas I-BET151 did not alter the production of several other cytokines (TNFa, IL-1b and IL-10) at the same Chronic obstructive pulmonary disease (COPD) and uncontrolled concentration of IBET151. I-BET151 prevented the binding of CBP asthma are obstructive airway diseases associated with pronounced to the promoter of IL-6, but I-BET151 did not affect acetylation, lung inflammation that cannot be attenuated with inhaled corticos- phosphorylation, nuclear translocation, or DNA binding of p65-NF- teroids. Several alternative anti-inflammatory approaches are jB. In vivo, I-BET151 treatment in the experimental autoimmune currently under clinical evaluation including orally administered encephalomyelitis mouse model of multiple sclerosis decreased the molecules that target protein kinases, which transduce inflammatory early clinical symptoms, which are thought to be dependent on signals triggered by cytokines, pathogens, and environmental stimuli cytokine production. Altogether, these data suggest that targeting (e.g., cigarette smoke). Unfortunately, the systemic adverse effects of epigenetic-related proteins, such as BET proteins, may provide a these kinase inhibitors have limited the clinical dose range that can be strategy to reduce inflammation and the severity of inflammatory assessed. In addition, the anti-inflammatory efficacy of these mole- diseases, such as multiple sclerosis. cules in other inflammatory indications (e.g., rheumatoid arthritis) have been limited due to physiological escape and/or the molecular redundancy that exists for these signaling pathways. To mitigate these potential risks, Respivert have developed two novel classes of inhaled SYMPOSIUM 12: NEXT GENERATION kinase inhibitors: (1) narrow spectrum kinase inhibitors (NSKIs) and (2) inhibitors of phosphatidylinositide 3-kinase isoforms. The mole- KINASE INHIBITORS—SPONSORED BY cules were designed for inhalation delivery to limit the systemic BIRA-UK exposure of these molecules (in order to maximize safety margins) and to specifically target multiple kinase isoforms involved in 041 mediating steroid resistant inflammation (to mitigate the potential for escape/redundancy). Each class of inhibitors was optimized using TARGETING IRAK4 AS A CENTRAL REGULATOR primary human cell phenotypic assays (including cells from patients) IN INNATE IMMUNITY to assess their potential for inhibiting steroid resistant inflammatory processes. In vivo efficacy and duration of action were assessed Iain Kilty across a number of preclinical models of steroid sensitive and insensitive inflammation. Early clinical data indicate that these Pfizer Inc, New York, NY, USA molecules are well tolerated and provide an anti-inflammatory benefit. Taken together, these data indicate that inhaled NSKIs and PI3 K Interleukin 1 receptor associated kinase 4 (IRAK4) represents a key inhibitors hold therapeutic potential by attenuating the steroid-resis- node in innate inflammatory signaling, directly downstream of the tant inflammation associated with COPD and uncontrolled asthma. 123 S70 Inflamm. Res.

MINI-SYMPOSIUM 1: NOVEL MODELS B179 OF INFLAMMATION MATERNAL IL17 PATHWAY PROMOTES AUTISM–LIKE PHENOTYPES IN OFFSPRING B071 See poster section for abstract. CIGARETTE SMOKE ENHANCES ADAPTIVE IMMUNE RESPONSE IN MURINE MODEL OF ALLERGIC AIRWAY INFLAMMATION PATIENT PERSPECTIVES: UNMET See poster section for abstract. MEDICAL NEED–INFLAMMATORY BOWEL DISEASE B092 043 DISEASE RELEVANT IN VITRO AND IN VIVO PATIENT PERSPECTIVES: UNMET MEDICAL MODELS FOR LUNG FIBROSIS NEED–INFLAMMATORY BOWEL DISEASE See poster section for abstract. Heather E. Jones

B002 Pfizer, Inc., Collegeville, PA, USA SYNCHROTRON MICROBEAM IRRADIATION Objective: To increase awareness of the true unmet medical need left INDUCES INFLAMMATION AND SELECTIVE by current therapeutic options for Crohn’s disease and understand what this means to the patient. VASCULAR DAMAGES IN ADULT ZEBRAFISH The importance of recognizing the true unmet medical need in autoimmune diseases is higher than ever. No drug has so far been able See poster section for abstract. to cure Crohn’s disease, only to induce remission. To progress science and clinical development there must be a focus on understanding the pathogenesis of the disease. From a medical perspective we need to B191 consider the patient as a whole human being and not only based on the DEVELOPMENT OF A MODEL OF DERMAL organ(s) involved. Autoimmune diseases in most cases are systemic disorders that affect more than one organ. INFLAMMATION AND IRRITATION (URTICARIA) The morning session will begin by emphasizing the unmet medical IN THE MINIATURE SWINE need followed by an interview with a patient with Crohn’s disease, with an emphasis on what they see as key objectives for further See poster section for abstract. development. At the end of the session the co-chairs will summarize the signs and symptoms and the treatment algorithm for Crohn’s disease, as B237 well as the need for biomarkers to define the disease, ensure safety, IN VITRO MODELLING ACUTE AND CHRONIC guide treatment and predict treatment response. INFLAMMATION BASED ON HUMAN MONOCYTES. ANALYSIS OF THE MODULATION SYMPOSIUM 13: microRNAs (MIRS) IN OF IL-1 FAMILY MEMBERS INFLAMMATION See poster section for abstract. 044 METABOLIC REGULATION BY MIR-33 IN B239 MACROPHAGES CONTROLS IMMUNE DYNAMIC EQUILIBRIUM OF NEUTROPHIL EFFECTOR RESPONSES TRAFFICKING AT SITES OF INFLAMMATION AND INFECTIONS Kathryn J. Moore1, Hasini Ediriweera1, U. Mahesh Gundra1, Katey J. Rayner3, P’ng Loke1, Philip Zamore2, Gregory Steinberg4, 1 See poster section for abstract. Mireille Ouimet

1New York University School of Medicine, New York, NY, USA; 2 B235 University of Massachusetts Medical School, Worcester, MA, USA; 3University of Ottawa Heart Institute, Ottawa, ON, Canada; SPONTANEOUS MUTATION IN ZFP-BINDING 4McMaster University, Hamilton, ON, Canada REGION OF TNF GENE CAUSES CHRONIC Cellular metabolism is increasingly recognized to control immune POLYARTHRITIS AND HEART VALVE DISEASE cell fate and functions. miR-33 is a regulator of cellular lipid meta- bolism that represses genes involved in cholesterol efflux, HDL See poster section for abstract. biogenesis and fatty acid oxidation. We demonstrate that by altering

123 Inflamm. Res. S71 the balance of aerobic glycolysis and mitochondrial oxidative phos- Sepsis is a systemic inflammatory response to infection and mediated via phorylation, miR-33 instructs macrophage inflammatory polarization activation of the innate immune system. Sepsis is the leading cause of and shapes innate and adaptive immune responses. Targeted deletion of death in patients in the intensive care units and opioids are the preferred miR-33 in macrophages increases oxidative respiration, enhances spare analgesic in this setting. The adverse effects of chronic morphine on the respiratory capacity, and induces the expression of genes that define M2 immune system has been well documented over the years. Higher levels macrophage polarization. We show that these changes are independent of morphine in systemic circulation reduces pathogen clearance, of effects on cholesterol efflux, but instead require miR-33 targeting of specifically in case of opportunistic infection, and also induces translo- the energy sensor AMP-activated protein kinase. Notably, inhibition of cation of gut microbes. Interestingly, in healthy individuals, both sepsis miR-33 also increases macrophage expression of the retinoic acid-pro- and consequent exposure to bacterial products is characterized by an ducing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro initial hyper-production of cytokines, followed by a ‘‘silencing’’ phase, and in vivo. Consistent with the ability of retinoic acid to foster inducible where Toll Like Receptor mediated production of pro-inflammatory regulatory T cells, anti-miR33-treated macrophages have an enhanced cytokines is suppressed. This has been variously referred to as ‘‘TLR capacity to induce FoxP3 expression in naı¨ve CD4+ T cells. Finally, reprogramming’’ or ‘‘endotoxin/LPS tolerance’’. The mechanisms pro- treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for posed for the endotoxin tolerance range from silencing of key mediators 8 weeks (conditions that do not alter HDL cholesterol levels) promoted of TLR signaling to impaired interaction between different signaling the accumulation of inflammation suppressing M2 macrophages and mediators. Within the past decade, while the ‘‘silencing’’ mechanism is FoxP3+ T regulatory cells in plaques, and reduced atherosclerosis pro- increasingly being implicated in describing drug/endotoxin tolerance, a gression by 40 %. Collectively, these results identify a novel role for new class of molecules, namely the micro-RNAs (miRNA) have miR-33 in the regulation of macrophage inflammation and show that emerged as key players in selectively silencing the intermediaries of antagonism of miR-33 reduces atherosclerotic inflammation by pro- TLR signaling between the surface receptor and eventual NF-jBacti- moting M2 macrophage polarization and regulatory T cell induction. vation. Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. We show that chronic mor- phine treatment tempers endotoxin tolerance resulting in persistent 045 inflammation, septicemia and septic shock. Morphine was found to microRNA REGULATION OF ENDOTHELIAL down-regulate endotoxin/LPS induced miR-146a and 155 in macro- INFLAMMATION phages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antag-

1,2 onizing miR-146a (but not miR-155) heightened the severity of Mark W. Feinberg morphine-mediated hyper-inflammation. These results suggest that

1 2 miR-146a acts as a molecular switch controlling hyper-inflammation in Brigham and Women’s Hospital, Boston, MA, USA; Harvard clinical and/or recreational use of morphine. Medical School, Boston, MA, USA Endothelial cell (EC) activation and vascular inflammation occur when the endothelium is exposed to various biochemical insults such as pro- SYMPOSIUM 14: MUCOSAL IMMUNITY: inflammatory cytokines, oxidative stress, hypertension, hyperglycemia, aging, and biomechanical stimuli such as shear stress. These insults lead HOT TOPICS IN IBD to the pathogenesis of a range of disease states, including atherosclerosis, insulin resistance, and obesity. Several signaling pathways, especially 047 nuclear factor jB mediated signaling, play crucial roles in these MONGERSEN, AN ORAL SMAD7 ANTISENSE pathophysiological processes. Recently, microRNAs (miRNAs) have emerged as important regulators of EC function by fine-tuning gene OLIGONUCLEOTIDE, IN ACTIVE CROHN’S expression. In this seminar, the audience will gain insights of how DISEASE miRNAs regulate EC function and vascular inflammation in response to a variety of pathophysiologic stimuli. Recent studies in mice and human Gerald Horan1, Giovanni Monteleone2, Markus F. Neurath3, subjects highlight an important role for miR-181b as a suppressor of Sandro Ardizzone4, Antonio Di Sabatino5, Massimo Claudio Fantini2, endothelial inflammatory responses in both acute (e.g., sepsis) and Fabiana Castiglione15, Maria Lia Scribano6, Alessandro Armuzzi7, chronic vascular disease states (e.g., atherosclerosis, insulin resistance, Flavio Caprioli8, Giacomo Carlo Sturniolo9, Francesca Rogai10, and obesity). These studies have uncovered emerging roles for novel Maurizio Vecchi8, Raja Atreya3, Fabrizio Bossa11, Sara Onali2, miRNA targets in a cell-specific manner. An understanding of the role of Maria Fichera4, Gino Roberto Corazza5, Livia Biancone2, miRNAs in EC activation and dysfunction may provide novel thera- Vincenzo Savarino12, Roberta Pica13, Ambrogio Orlando14, peutic opportunities for controlling a range inflammatory disease states. Francesco Pallone2

1Celgene Corp, Summit, NJ, USA; 2University of Tor Vergata, Rome, 3 4 046 Italy; University of Erlangen-Nu¨rnberg, Erlangen, Germany; ‘‘L. Sacco’’ University Hospital, Milan, Italy; 5University of Pavia, Pavia, MORPHINE INDUCED EXACERBATION OF Italy; 6Hospital San Camillo-Forlanini, Rome, Italy; 7Catholic SEPSIS IS MEDIATED BY TEMPERING University, Rome, Italy; 8University of Milan, Milano, Italy; ENDOTOXIN TOLERANCE THROUGH 9University of Padova, Padova, Italy; 10AOU Careggi University 11 MODULATION OF MIR-146A Hospital, Florence, Italy; ‘‘Casa Sollievo Sofferenza’’ Hospital, IRCCS, San Giovanni Rotondo, Italy; 12University of Genoa, Genoa, 13 14 1,2 1 1 Italy; Sandro Pertini Hospital, Rome, Italy; University of Sabita Roy , Santanu Banerjee , Jingjing Meng , Palermo, Palermo, Italy; 15University ‘‘Federico II’’ of Naples, Italy Sundaram Ramakrishnan2 Introduction: Crohn’s disease (CD)-related inflammation is charac- 1Department of Surgery; 2Department of Pharmacology, University of terized by defective activity of the immunosuppressive cytokine Minnesota, Minneapolis, MN, USA transforming growth factor (TGF)-b1, due to high Smad7 (an

123 S72 Inflamm. Res. inhibitor of TGF-b1) signalling. The effects of an oral, topically UPR target genes, in part by co-operating with XBP1s. XBP1 and active Smad7 antisense oligonucleotide, Mongersen, were evaluated ATF6 transactivate genes that are involved in protein translation, in a phase II study in patients with active CD. folding and quality control, but also in ER associated degradation Aims and methods: In a double-blind, placebo-controlled trial, the (ERAD), rendering the removal of misfolded proteins via the protea- efficacy of Mongersen as induction therapy was evaluated in steroid- some. Finally, PERK activation results in phosphorylation and hence dependent or steroid-resistant patients (utilizing ECCO consensus inactivation of elongation and initiation factor 2a (eIF2a) and conse- definition) with active CD [CD activity index (CDAI) score quent inhibition of mRNA transcription decreasing the global flux of 220–400]. Patients were randomized to Mongersen 10, 40 or proteins entering the ER. However, certain mRNAs that contain short 160 mg/day or placebo for 2 weeks. The primary outcomes were open reading frames in their 50 untranslated regions are preferentially clinical remission (CDAI score \150 at Day 15 and maintained for transcribed under conditions of limiting eIF2a. Amongst those is C2 weeks) and safety. Secondary endpoints included clinical activating transcription factor 4 (ATF4), a further UPR transcription response (CDAI score reduction of 100 points) at Day 28. factor that, among other target genes, transactivates CHOP (tran- Results: Clinical remission was achieved by significantly greater scription factor C/EBP homologous protein). CHOP in turn proportions of patients receiving Mongersen 40 (55.0 %) and transactivates genes involved in apoptosis induction and thereby 160 mg/day (65.1 %) compared with placebo (9.5 %; p \ 0.0001 for connects ER stress that has become unsustainable with cell death. A both). No significant difference in clinical remission was seen for critical cell type that is affected by abnormalities of the UPR is the 10 mg/day (12.2 %) vs. placebo. The rate of clinical response was intestinal epithelial cell (IEC) and especially Paneth cells. Many significantly greater among patients receiving 10 (36.6 %), 40 genetic and environmental defects which act upon the elements of the (57.5 %) or 160 mg/day (72.1 %) of Mongersen vs. placebo (16.7 %; UPR result in increased susceptibility to intestinal inflammation. One p = 0.039, p = 0.0001 and p \ 0.0001, respectively). The rates of such factor is XBP1, which when deleted in the IEC results in spon- adverse events (AEs) and serious AEs (SAEs) were similar across taneous enteritis. This presentation will focus on the role played by the groups. Nine SAEs occurred in 6 patients (placebo, n = 1; Mon- UPR in IECs and how these pathways interact with autophagy gersen 10 mg, n = 3; 40 mg, n = 1; 160 mg, n = 1). Most SAEs consisted of hospital admissions for CD-associated complications or symptoms, and included: pyrexia and cough (placebo); abdominal pain (n = 2), CD worsening and pyrexia (Mongersen 10 mg); seton 049 placement for perianal fistula and surgery for hemorrhoid thrombosis EMERGING UTILITY OF CULTURED (Mongersen 40 mg); and thermal burn (Mongersen 160 mg). INTESTINAL EPITHELIAL CELLS IN Conclusions: Induction therapy with orally administered, topically PERSONALIZED MEDICINE active Mongersen for CD was well tolerated; toxicities previously reported with systemically active antisense agents were not observed. Mongersen treatment resulted in significant improvements in clinical Thaddeus Stappenbeck remission and response rates within 4 weeks of initiation of treatment. Washington University School of Medicine, St. Louis, MO, USA One mode of personalized medicine is to utilize cells from patients to 048 perform in vitro assays that will in turn inform upon the treatment regimens of specific patients. Intestinal epithelial cells have the THE UNFOLDED PROTEIN RESPONSE AND potential to be a critical cell type in such assays. In the last several INTESTINAL INFLAMMATION years, as a field, we have developed the ability to efficiently isolate and propagate these cells from primary sources (animal models and Richard Blumberg humans). The lines creates have been the nidus for many new exciting experimental systems developed in the last several years that I will Harvard Medical School, Boston, MA, USA review here. I will also describe recent advances in the utility of human intestinal epithelial cell lines to show differences in the Protein function is fundamentally dependent on correct protein folding response to inflammatory stimuli that could direct treatments. and post-translational modification. For proteins entering the secretory pathway, this occurs in the endoplasmic reticulum (ER) which imposes stress on the ER when misfolding occurs. The unfolded protein response (UPR) has evolved to counter this stress by adapting the cell’s B209 protein folding capacity to the level of demand. Abnormalities in the ABSENCE OF DIETARY FIBRE OR THE UPR are increasingly recognized as an important contributor to disease METABOLITE SENSOR GPR43 EXACERBATES pathogenesis. Three major branches of the UPR have evolved, each consisting of an ER transmembrane protein that serves as a proximal NEUTROPHIL RECRUITMENT DURING ACUTE sensor of ER stress in conjunction with an ER resident chaperone, INFLAMMATORY RESPONSES grp78. These three ER transmembrane proteins are inositol-requiring enzyme 1 (IRE1), with its isoforms a and ß, activating transcription See poster section for abstract. factor 6 (ATF6; a and ß isoforms) and PKR (double-stranded RNA- dependent protein kinase)-like ER kinase (PERK), which activate specific transcriptional programs. IRE1 acts as an endoribonuclease, excising a 26nt stretch from the mRNA encoding X-box binding B032 protein 1 (XBP1), which results in a frame-shift and translation of the A NOVEL IRON-MEDIATED MECHANISM FOR active transcription factor (XBP1s, s for spliced) that transactivates DEVELOPMENT OF INFLAMMATORY BOWEL UPR target genes. ATF6 is packaged into vesicles, which are released from the ER and traverse to the Golgi, where ATF6 is cleaved by site-1 DISEASE and site-2 proteases (S1P, S2P), releasing the active transcription factor fragment that enters the nucleus and also transactivates a set of See poster section for abstract.

123 Inflamm. Res. S73

SYMPOSIUM 15: INHIBITORY demonstrated that ITAM can also initiate inhibitory signaling toward heterologous receptors. This inhibitory signaling by ITAM-bearing RECEPTOR/IMMUNE CHECKPOINTS/ receptors is coined inhibitory ITAM (ITAMi). Until now one human IgA IMMUNOTHERAPEUTICS—SPONSORED receptor, the FcaRI (CD89) and two human low-affinity IgG receptors, BY IRA-USA the FccRIIIA (CD16A) and the FccRIIA (CD32A), that are associated with the FcRc subunit or carrying an ITAM motif on their cytoplasmic tail, have been found to act as bifunctional receptors which, depending on 050 the type of interaction with their ligand, induce either activating or PREVENTION OF COLLATERAL DAMAGE BY inhibitory cell signaling. While multivalent cross-linking of these FcR by IMMUNE INHIBITORY RECEPTORS immune complexes induced proinflammatory signaling, monovalent or divalent targeting of these FcR with either IgA or IgG (or with Fab/ F(ab0)2 of anti-FcR) could trigger inhibitory signals towards a whole Linde Meyaard array of cellular functions such as phagocytosis, IgE-dependent degranulation and TLR- or cytokine-mediated responses. The ITAMi Laboratory of Translational Immunology, UMC, Utrecht, configuration is defined by weak phosphorylation of the ITAM, transient The Netherlands recruitment of Syk followed by stable recruitment of the tyrosine phos- The proper functioning of the immune system is based on a fine phatase SHP-1, whereas multivalent aggregation of these receptors balance between activation and inhibition. Immune suppression leads promotes strong ITAMa phosphorylation with stable Syk recruitment. to the threat of infection by pathogens and immunodeficiency, while The ITAMi configuration promotes the actin depolymerization-depen- insufficient inhibition can lead to damage to self or autoimmunity. dent ‘‘trapping’’of these FcR with the targeted activating receptors within Inhibitory immune receptors play a crucial role in this balance. the same lipid rafts, which is followed by the appearance of intracellular 0 Neutrophils are useful but also dangerous cells. They are essential inhibisome clusters. Monomeric IgA, IgG, IVIg or anti-FcR Fab/F(ab )2 to protect the host against invading pathogens and have the ability to were able to either prevent or reverse inflammation in asthma, nephritis or quickly enter the tissues. However, their activity also causes collateral arthritis mouse models through ITAMi signaling. These anti-inflamma- tissue damage that in ultimo can result in destruction of the tissue tory effects were independent of ITIM-bearing FccRIIB. These results architecture and the formation of pus, also in non-infectious inflam- demonstrate that circulating IgA or IgG are not functionally inert but act matory conditions. Balancing neutrophil influx and function is through continuous interaction with FcR inducing ITAMi signaling to essential to prevent excessive disease. Immune inhibitory receptors maintain immune homeostasis.These data alsosupport a new mechanism are potential regulators of this balance. of action for IVIg and demonstrate the therapeutic potential of FccRIIIA Next to phagocytosis and degranulation, neutrophils fight bacteria targeting in inflammation. Recently, we found a new function for 0 through neutrophil extracellular traps (NETs), which arise from the FccRIIA following receptor engagement by anti-FccRII F(ab )2 or by release of the neutrophil’s nuclear content into the extracellular space IVIg that reverses arthritis in mice. This mechanism has the ITAMi and consist of decondensed DNA decorated with antimicrobial pro- signaling molecular signature described for FcaRI and FccRIIIA but 0 teins. Despite the importance of NETs in host defense, NETs differs by involvement of a single tyrosine. Anti-FccRII F(ab )2 treat- contribute to pathology by several mechanisms. It is suggested that ment of inflammatory synovial cells from rheumatoid arthritis patients the DNA and antimicrobial peptides of NETs can be recognized by B induced inhibition of ROS production by switching their inflammatory cells which leads to the production of auto-antibodies, such as in SLE. activation state from an activated-ITAMa configuration to an ITAMi Furthermore, the antimicrobial peptides attached to the NETs have signaling. Shifting constitutive FccRIIA-mediated activation in poor target specificity, which leads to the damage of the tissues sur- rheumatoid arthritis to an ITAMi signaling could thus reverse inflam- rounding the NET. Finally, NETs have recently been shown to form a mation, providing ground for novel treatment options in this disease. platform for platelets and form a central role in thrombosis. There- fore, NET formation contributes to pathology in several diseases that involve tissue infiltration by neutrophils. We recently identified signal inhibitory receptor on leukocytes-1 SYMPOSIUM 16: MECHANISMS OF (SIRL-1) as a negative regulator of human neutrophil function. SIRL- FIBROSIS 1 ligation inhibits the oxidative burst and prevents the pathogenic release of NETs in SLE. SIRL-1 engagement can dampen sponta- neous and anti-neutrophil antibody-induced NET formation in SLE. 052 This opens up the interesting therapeutic opportunity of inhibiting BASIC MECHANISMS OF KIDNEY FIBROSIS collateral damage by engagement of inhibitory receptors. Jeremy S. Duffield1,2

051 1Biogen, Cambridge, MA, USA; 2Renal Division, University of ITIM AND ITAM BEARING INHIBITORY Washington, Seattle, WA, USA RECEPTORS ON TREATMENT OF Chronic kidney diseases, represent a major burden to society, INFLAMMATORY DISEASES affecting up to 10 % of the adult population, and are characterized by progressive fibrosis of the glomerulus and interstitial space. Currently organ failure is managed by intervention with dialysis and for some Renato C. Monteiro1,2,3 organ transplantation. Proof of concept experiments indicate that fibrosis per se con- 1Center for Research on Inflamation, Paris, France; 2Inflamex tributes to loss of function and organ demise, and that fibrosis Consortium, Paris, France; 3Paris Diderot University, Paris, France represents an aberrant wound-repair response. Recent studies in ani- Immunoregulation by Fc receptors (FcR) is controlled by ITAM and mals have delineated cellular mechanisms by which fibrosis occurs, ITIM motifs. This classic concept of the functional polarity of ITIM and and the challenge now is to identify pathways that drive this process ITAM motifs has been recently reevaluated. Several studies have where therapeutic interventions can be safely introduced.

123 S74 Inflamm. Res.

Fibrogenic cells undergo profound molecular, cellular and phe- mAb during priming, however, displayed decreased inflammation, notypic changes from the healthy state to the pathological state, and mucus production, and lung remodeling in the chronic phase. Toge- these require changes in inflammatory signaling pathways, growth ther, these studies reveal redundant roles for TSLP, IL-25, and IL-33 in factor signaling pathways and metabolic changes, all of which con- the maintenance of type 2 cytokine-driven pathologies and suggest that tribute to the maintenance of pathological matrix forming cells in some settings, early combined targeting of these mediators is nec- This presentation will highlight several novel pathways in the area essary to ameliorate progressive type 2-driven disease. of innate immunity that play roles in the pathological process of fibrogenesis, where intervention may be safe and tractable. SYMPOSIUM 17: THERAPEUTICS WITH 053 LOCALIZED ACTIONS—OPTIMAL WAYS TARGETING NOX ENZYMES IN PULMONARY TO TREAT TISSUE INFLAMMATION FIBROSIS 055 Victor J. Thannickal TARGETED POLYMERIC NANOPARTICLES: FROM DISCOVERY TO CLINICAL TRIALS University of Alabama at Birmingham, Birmingham, AL, USA The NADPH oxidases (Nox) family of enzymes are known to mediate Omid C. Farokhzad1,2 host defense functions in mammalian organisms. We have identified Nox4 as a highly inducible TGFb-regulated gene. Nox4 is essential to 1Brigham and Women’s Hospital, Boston, MA, USA; 2Harvard mediate TGFb-induced myofibroblast differentiation and to protect Medical School, Boston, MA, USA against injury-provoked pulmonary fibrosis. Recent studies support Controlled-release polymeric nanoparticles can deliver drugs in the the concept that Nox4-Nrf2 imbalance regulates cellular senescence optimum dosage over time, thus increasing the efficacy of the drug, and promotes persistent fibrosis associated with aging. maximizing patient compliance and enhancing the ability to use highly toxic, poorly soluble, or relatively unstable drugs, and can also be used to co-deliver two or more drugs for synergistic combination therapy. 054 Moreover, the surface engineering of these nanoparticles may yield COMBINATORIAL TARGETING OF TSLP, IL-25, them ‘‘stealth’’ to prolong their residence in blood, and the function- alization of these particles with targeting ligands can differentially AND IL-33 AND TYPE-2 CYTOKINE SIGNALING IN target their delivery or uptake by a subset of cells, further increasing CHRONIC INFLAMMATION AND FIBROSIS their specificity and efficacy. Nevertheless, the successful clinical translation of targeted polymeric nanoparticles for drug delivery Kevin M. Vannella1, Thirumalai R. Ramalingam1, Allen W. requires optimization of many distinct parameters including: variation Cheever1,2, Lee A. Borthwick1,3, Luke Barron1, Kevin M. Hart1, in the composition of the carrier system, drug loading efficiency and Robert W. Thompson1, Kristen N. Kindrachuk1, Sandra White1, Alison release kinetics, surface hydrophilicity, surface charge, particle size, L. Budelsky4, Michael R. Comeau4, Dirk E. Smith4, Thomas A. Wynn1 density of possible ligands for targeting, etc., resulting in potential variables for optimization which is impractical to achieve using a low 1Program in Tissue Immunity and Repair, Laboratory of Parasitic throughput approach. Combinatorial approaches precisely engineer Diseases, National Institute of Allergy and Infectious Diseases, nanoparticles and screen multiple nanoparticle characteristics simulta- National Institutes of Health; Bethesda, MD, USA; 2Biomedical neously with the goal of identifying formulations with the desired Research Institute; Rockville, MD, USA; 3Tissue Fibrosis and Repair physical and biochemical properties for each specific application. In Group, Institute of Cellular Medicine, Newcastle University; this talk, I will present our efforts in the design and optimization of Newcastle upon Tyne, NE2 4HH UK; 4Department of Inflammation targeted polymeric nanoparticles for medical applications, which Research, Amgen, Amgen Court West, Seattle, WA, USA formed the foundation for the clinical translation of the first-in-human targeted and controlled-release nanoparticles, BIND-014 and SEL-068. Thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 are impor- tant initiators of type 2-associated mucosal inflammation and immunity. However, their role in the maintenance of progressive type 056 2 inflammation and fibrosis is much less clear. Here, using chronic models of helminth infection and allergic lung inflammation, we show IMMUNE TARGETING NANOPARTICLES that collective disruption of TSLP, IL-25, and IL-33 signaling sup- CONTAINING siRNAs TO CURTAIL LEUKOCYTE- presses chronic and progressive type 2 cytokine driven inflammation IMPLICATED DISEASES and fibrosis. In a schistosome lung granuloma model or during chronic S. mansoni infection in the liver, individual ablation of TSLP, IL-25, or 1 Dan Peer IL-33/ST2 had no impact on the development of IL-4/IL-13-dependent inflammation or fibrosis. However, significant reductions in granu- 1 Tel Aviv University, Tel Aviv, Israel loma-associated eosinophils, hepatic fibrosis, and IL-13-producing group 2 innate lymphoid cells (ILC2s) were observed when signaling RNA interference (RNAi)-based approaches have greatly contributed of all three mediators was simultaneously disrupted. Combined to better understanding of gene expression and function in vitro. The blockade via mAb treatment also reduced IL-5 and IL-13 expression capability to apply these strategies in vivo in order to validate the role during primary and secondary granuloma formation in the lung. In a of specific genes in normal or pathological conditions, and to induce model of chronic house dust mite-induced allergic lung inflammation, therapeutic gene silencing, opened new avenues for utilizing RNAi as combined mAb treatment did not decrease established inflammation or a novel therapeutic modality. However, the translation of RNAi from fibrosis. TSLP/IL-33 double-knockout mice treated with anti-IL-25 an effective genomic tool into a novel therapeutic modality has been 123 Inflamm. Res. S75 hindered by the difficulty to deliver RNAi molecules into their target Primary somatosensory neurons densely innervate barrier tissues that tissues by systemic administration, especially to hematopoietic cells. are often exposed to pathogens, including the skin, respiratory, and In this presentation, I will describe some of the challenges and gastrointestinal tract. Pain, which is a hallmark of many infectious opportunities in modulating leukocytes response using RNAi and diseases, is mediated by nociceptor sensory neurons as a protective discuss adverse effects such as immuno-toxicity. Several examples mechanism against damaging stimuli. However, the molecular will be discussed among them the discovery of cyclin D1 as a potential mechanisms leading to nociceptor neuron activation during bacterial anti inflammatory targeted in Inflammatory bowel disease (IBD). infection and the role of the nervous system in host defense are not Cyclin D1 (CyD1) is a pivotal cell cycle–regulatory molecule and well understood. We find that nociceptor neurons directly detect two a well-studied therapeutic target for cancer. Although CyD1 is also up classes of bacterial ligands: N-formulated peptides and pore-forming regulated at sites of inflammation, its exact roles in this context toxins. Detection of these bacterial signals occurs through distinct remain less understood. To address this question, we developed a mechanisms, producing differential mechanical and thermal hyper- strategy to target gut mononuclear leukocytes and selectively silence algesia. Using a mouse model of skin infection, we find that CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) Staphylococcus aureus-induced pain does not depend on critical host were loaded with CyD1-small interfering RNA (siRNA). Antibodies defense pathways or immune cell-types. Hyperalgesia directly cor- to b7 integrin (b7 I) were then used to target specific leukocyte subsets relates with live bacterial load rather than tissue swelling or immune involved in gut inflammation. Using PET/CT scan we were able to activation. Targeted ablation of nociceptor neurons leads to signifi- 64 detect Cu-labeled -b7 I-tsNPs accumulation in the gut of mice with cant increases in immune cell recruitment and lymph node DSS-induced colitis. Intravenous administration of b7 I-tsNPs hypertrophy during infection. Therefore, sensory neurons directly silenced CyD1 in leukocytes and reversed colitis by suppressing detect bacterial ligands to produce pain and to modulate inflamma- leukocyte proliferation and TH1 cytokine expression. This study tion. We and others are working to further elucidate the molecular reveals CyD1 to be a potential anti-inflammatory target, and suggests interactions between bacterial pathogens and the somatosensory that the application of similar modes of targeting by siRNA may be nervous system that could be key mediators of pain, inflammation, feasible in other therapeutic settings. Examples of our current and host defense. research in blood cancer (mantle cell lymphoma) and from naı¨ve CD4 T cells will also be discussed. 059 057 TRPA1 CHANNELS ARE EARLY SENSORS OF AN INFLAMMATION RESPONSIVE PLATFORM GRAM-NEGATIVE BACTERIAL ENDOTOXINS FOR CONTROLLED DRUG DELIVERY Felix Viana1, Victor Meseguer1, Yeranddy A. Alpizar2, Enoch Luis1, 3 1 1 1,2,3 Sendoa Tajada , Bristol Denlinger , Jan-Albert Manenschijn , Otto Jeffrey M. Karp Fajardo1, Carlos Ferna´ndez-Pen˜a1, Arturo Talavera2, Tatiana 4 5 2 5 4 1 2 Kichko , Bele´n Navia , Alicia Sa´nchez , Rosa Sen˜arı´s , Peter Reeh , Brigham and Women’s Hospital, Boston, MA, USA; Harvard 3 3 3 Marı´a Teresa Pe´rez-Garcı´a , Jose´ Ramo´n Lo´pez-Lo´pez , Thomas Medical School, Boston, MA, USA; Harvard-MIT Division of Health 2 1 4 Voets , Carlos Belmonte Sciences and Technology, Cambridge, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA 1Instituto de Neurociencias de Alicante, UMH-CSIC, Alicante, Spain; We have developed an autonomous delivery system that titrates the 2Laboratory of Ion Channel Research, KULeuven, Leuven, Belgium; amount of drug released in response to the level of inflammation, 3Instituto de Biologı´a y Gene´tica Molecular, UV-CSIC, Valladolid, ensuring the drug is released only when needed at a therapeutically Spain; 4Institute of Physiology and Pathophysiology, University of relevant concentration. Translation of such technology would repre- Erlangen-Nuremberg, Erlangen, Germany; 5Department of sent a paradigm shift in the treatment of inflammatory diseases such Physiology, University of Santiago de Compostela, Santiago de as rheumatoid arthritis and inflammatory bowel disease which has Compostela, Spain characteristic flares followed by periods of lower disease activity. We TRPA1 are calcium permeable, non-selective cation channels have evaluated its ability to release drugs in response to enzymes that expressed in sensory endings of somatic and visceral nociceptors and are abundant in human synovial fluid and ulcerated gut tissue using a many non-neuronal cells, including fibroblasts, endothelial and glial series of in vitro and in vivo experiments. cells. These ion channels are critically involved in the biological response to physical stimuli (temperature and pressure) and natural and synthetic environmental irritants, including many electrophiles, SYMPOSIUM 18: ION CHANNELS AND reactive oxygen species and endogenous inflammatory mediators INFLAMMATION—SPONSORED BY IRN- produced following tissue damage. Abnormal activation of TRPA1 CANADA has been linked to the pathogenesis of neuropathic pain and itch, and a number of systemic inflammatory diseases, including atopic der- matitis, irritable bowel syndrome and asthma. 058 Gram-negative bacterial infections are accompanied by inflam- BACTERIAL SIGNALS TO SENSORY NEURONS mation and somatic or visceral pain. These symptoms are generally THAT MODULATE INFLAMMATION AND PAIN attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflam- mation by lipopolysaccharide (LPS), a toxic byproduct of gram- 1 2 Isaac M. Chiu , Clifford J. Woolf negative bacterial lysis, is thought to occur through activation of pattern-recognition receptors, in particular the Toll-like-receptor 4 1 Harvard Medical School, Department of Microbiology and (TLR4) signaling pathway. 2 Immunobiology, Boston, MA, USA; Boston Children’s Hospital, Using a combination of experimental techniques, including elec- Kirby Neurobiology Center, Boston, MA, USA trophysiological recordings and cellular calcium imaging, we found

123 S76 Inflamm. Res. that LPS exerts fast (seconds) activation of mouse somatic and vis- Inflammatory pain associated with tissue damage, infection or auto ceral nociceptors. These actions were mediated by opening of TRPA1 immune disease, results from sequential events initiated during channels leading to neuronal depolarization, firing of action potentials immune responses. Our lab investigates the plasticity of specialized and rapid elevation of intracellular calcium levels. Human TRPA1 pain-sensing dorsal root ganglion (DRG) neurons that participate in channels showed a similar sensitivity to LPS. LPS, injected intra- peripheral sensitization of the nociceptive system during inflamma- dermally, produced a rapid (minutes) inflammatory response that was tion. These neurons (nociceptors) exposed to inflammatory molecules accompanied by pain and acute vascular reactions, including neuro- undergo molecular changes that drive their hyperexcitability by genic inflammation and CGRP release (measured by enzyme lowering their threshold of activation. The hyperexcitability of immunoassay). These responses were severely blunted in TRPA1 KO nociceptors is mediated by altered expression, activity and trafficking mice and developed independently of TLR4 activation. Moreover, the of ion channels. Among a large variety of ion channels, DRG neurons capacity of various forms of LPS, purified from different pathogens, express TRP channels—a family of cationic ion channels that par- to activate TRPA1 in vitro correlated with their ability to induce ticipate in the regulation of Ca2+ homeostasis. TRP channels inflammatory responses in vivo. contribute to changes in cytosolic Ca2+ concentration [Ca2+]i by In summary, we identified TRPA1 channels as key molecular regulating Ca2+ influx at the membrane or Ca2+ release from internal determinants of rapid LPS effects on sensory neurons and their ter- Ca2+ stores. The Transient receptor potential vanilloid 1 (TRPV1) minals, leading to acute neurogenic inflammation and pain. These channel is a non-selective cation channel that transduces inflamma- results suggest that TRPA1 channels may play a previously unrecog- tory signals into electrical signals leading to action potential nized role in the first line of immune defense against microbial propagation in sensory neurons. In this presentation I will summarize pathogens. Pharmacological targeting of TPA1 channels could repre- our recent work that pertains to targeting TRPV1 subunit association sent a novel therapeutic avenue for treatment of complications derived to reduce inflammatory pain. In this study, we have identified the from gram negative bacterial infections, including septic shock. molecular determinants of TRPV1 channel subunit assembly and were able to abolish TRPV1 function and thus inflammatory pain by Acknowledgements: Financial support provided by project SAF2013- targeting this specific motif. In a second part of my talk, I will present 45608-R of MINECO. our findings on the regulation of TRPV1 signaling in the context of inflammation. Here, using the dextran sulfate sodium (DSS)-induced colitis model, we demonstrated a critical role of substance P-mediated sensitization of TRPV1 channel in the development of post-inflam- 060 matory pain. A ROLE OF THE CATION CHANNEL TRPV4 IN LUNG INFLAMMATION

Mustapha Si-Tahar SYMPOSIUM 19: HARNESSING NOVEL RESOLUTION MECHANISMS TO INSERM U1100, Centre d’e´tude des pathologies respiratoires CONTROL INFLAMMATION (CEPR), Tours, France Cystic fibrosis (CF) is an inherited disease associated to a severe and 062 chronic lung inflammation, causing an early death. To improve patients’ outcome, there is an urgent need to develop safe and effi- RESOLVINS AND SPM IN RESOLUTION AND cient anti-inflammatory treatments. THEIR RELATIONSHIP TO INFECTION AND To do so, we need to characterize new cellular and molecular TISSUE REGENERATION components that could contribute to mechanisms of lung inflammation. Here, we focused on the potential role of ‘‘Transient Receptor 00 Charles N. Serhan, Jesmond Dalli, Nan Chiang, Paul Norris, Sesquile Potential Vanilloid-4 (TRPV4), a non-selective, calcium channel in Ramon, Romain Colas respiratory epithelial cells-mediated inflammatory signaling. We first showed that 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids, Center for Experimental Therapeutics and Reperfusion Injury i.e. four natural lipid-based TRPV4 agonists are present in sputum of Department of Anesthesia, Perioperative and Pain Medicine Harvard CF patients. Then, we used in vitro and in vivo approaches to Institutes of Medicine, BWH and Harvard Medical School, Boston, demonstrate that epithelial TRPV4 triggers a secretion of pro-in- MA, USA flammatory mediators (cytokines, lipids) as well as a neutrophil recruitment. We also found an alteration of TRPV4-dependent sig- In the resolution of inflammation novel chemical mediators are pro- naling in the CF context, suggesting that TRPV4 could constitute a duced in resolving exudates that limit inflammation, stimulate promising target for the development of new anti-inflammatory resolution and tissue regeneration. Identification of novel signals that treatments in diseases such as CF. regulate these processes and their pathways is of general interest. This presentation shall review recent results from regenerating planaria, infectious murine exudates, human milk and macrophages, where we identified novel potent molecules that stimulate tissue regeneration in 061 planaria and promote tissue repair in mice. These molecules proved to SENSITIZATION OF THE TRANSIENT RECEPTOR be peptide-conjugated maresins and are coined maresins conjugates in POTENTIAL VANILLOID 1 (TRPV1) CHANNEL IN tissue regeneration (MCTR). They also displayed potent anti-inflam- NEUROGENIC INFLAMMATION AND PAIN matory and proresolving actions i.e. limiting neutrophil infiltration and promoting the containment and clearance of infections in vivo. At the cellular level, they stimulate bacterial phagocytosis by macrophages Christophe Altier and efferocytosis of apoptotic cells both with mice in vivo and human cells in vitro. MCTR1 and MCTR2 are each biosynthesized from University of Calgary, Calgary, AB, Canada docosahexaneoic acid and both carried sulfido-conjugated triene

123 Inflamm. Res. S77 double bond systems. These are joined by two other recently uncov- ‘‘inflammaging’’ and that rectifying these defects may improve vaccine ered novel series of bioactive conjugates from the resolvin and efficacy in the elderly. To understand these processes better as they protectin families denoted RCTR, PCTR, respectively. Together, these pertain to humans and human aging, we have developed models of results identify new, conserved resolution moduli present in planaria, acute, self-limiting inflammation in healthy volunteers. As a result of mice and humans that each regulate host responses to control infec- these studies we are mapping pathways of inflammation with emphasis tion, tissue regeneration and organ protection. on mononuclear phagocytes and lipid mediators and are finding clear differences between young and older individuals with regard to pro- resolution processes. In my presentation, I will present these new data and speculate on their potential contribution to the aetiology of chronic 063 inflammation and the role aging plays in this process. THERAPEUTIC INNOVATION IN INFLAMMATION: TARGETING PRO-RESOLVING RECEPTOR SIGNALING SYMPOSIUM 20: SKELETAL MUSCLE Mauro Perretti INFLAMMATION

Queen Mary University of London, London, UK 065 The resolution of acute inflammation is enabled by counter-regulatory DIFFERENT MACROPHAGE SUBSETS CONTROL checkpoints to terminate the host reaction, therefore, in line with the MATRIX REMODELING AND FIBROSIS DURING onset phase of inflammation, pro-resolving mediators encompass bioac- tive lipids, proteins and peptides, autacoids and gases, that act on target MUSCLE REGENERATION VERSUS CHRONIC cells to extinguish this response. When fully operative, the inflammatory DEGENERATIVE MYOPATHIES reaction is controlled in time and space, ensuring tissue repair and regain of function. Importantly, disruption of the key processes involved in Be´ne´dicte Chazaud1,2, Marielle Saclier2, Gae¨tan Juban1, Lin Yang3, resolution phase of inflammation could result in delayed restoration of Basil J. Petrof4,Re´mi Mounier1 tissue homeostasis, fibrosis and persistent inflammation. The large majority of current therapeutics for the clinical manage- 1Universite´ Claude Bernard Lyon 1, Villeurbanne, France; 2Institut ment of inflammatory diseases act by blocking specific enzymes or Cochin, INSERM, Paris, France; 3University of Florida, Gainesville, antagonising receptor targets. Using the endogenous protective path- FL, USA; 4McGill University, Montrel, QC, Canada way centred on a receptor termed FPR2/ALX, which is activated by Macrophages play beneficial roles during skeletal muscle regenera- Annexin A1, Lipoxin A4 and Resolvin D1, strategies that could lead to innovation for next generation anti-inflammatory medicines will be tion. Ly6Cpos pro-inflammatory macrophages entry the damaged discussed. In particular, we step-change derives from the notion that it is muscle where that sustain myoblast proliferation, then switch into not only important to select the appropriate ‘receptor target’ for the drug Ly6Cneg anti-inflammatory macrophages that stimulate myogenesis. discovery programmes, but also identify the pro-resolving signalling We characterized phenotypes and functions of macrophage subsets signature as guidance for predicting the wanted biological properties. in myopathies characterized by chronic inflammation and fibrosis. We Pro-resolving based medicines—identified and developed on the showed that pro-inflammatory marker-expressing macrophages were rationale of resolution, using targets and signalling validated in associated with fibrosis in both mouse and human Duchenne muscle. physio-pathological settings—will be modulatory in their function Functional experiments showed that in regenerating muscle, sorted and devoid of major side effects, affording a therapeutic approach Ly6Cneg macrophages were involved in matrix remodeling while based on the enhancement of the patient’s own mechanisms of repair. Ly6Cpos macrophages induced fibroblast apoptosis. On the contrary, in dystrophic muscle, Ly6Cpos stimulated fibroblasts to produce collagen, and sustained their persistence, triggering fibrosis, through the action of TGFb. 064 In vivo, prevention of Ly6Cpos macrophage entry into mdx MECHANISMS OF INFLAMMATORY muscle, as well as anti-inflammatory (NaHS) or Metformin treat- ments, improved the dystrophic phenotype, i.e. induced a reduction of RESOLUTION IN HUMANS necrosis and fibrosis, which was associated with a decrease of pro- inflammatory macrophages. Derek W. Gilroy

University College London, London, UK 066 Investigate the cells, soluble mediators and receptors that collectively help switch inflammation off, so-called inflammatory resolution. My CYTOKINE-MODULATED CELLULAR overall hypothesis is that understanding how acute inflammation RESPONSES IN SKELETAL MUSCLE DAMAGE resolves will provide insight into the aetiology of chronic inflammatory AND REPAIR diseases. In addition, identifying mediators and receptors essential for resolution will help develop drugs that will drive ongoing inflammation Andrew N. Billin down a pro-resolution pathway. However, recent work suggests that resolution of acute inflammation is not the end of immune responses to GlaxoSmithKline, Brentford, UK infection/injury but, that through cells of the mononuclear phagocyte system expressing arachidonic acid, docosahexaenoic acid and eicos- Regeneration of damaged skeletal muscle throughout life depends on a apentaenoic acid metabolising enzymes resolution acts as a bridge population of adult stem cells (satellite cells) that both self-renew and between innate and adaptive immunity. We believe that defects in these expand as myogenic precursors in response to injury. Satellite cell pathways may contribute to the aetiology of, for instance, regenerative functions are modulated by extracellular signals and these 123 S78 Inflamm. Res. signals are likely to play a pivotal role in the process of muscle repair. In Co-inhibitory or immune checkpoint receptors play a key role in order to identify key extracellular regulators of satellite cell activity we resolving tissue inflammation and restoring immune homeostasis. performed a high-throughput in vitro screen using murine satellite cells This reflects their function in terminating effector T cell responses. In and a library containing a comprehensive set of recombinant secreted cancer, the heightened expression and function of co-inhibitory proteins and the extracellular domains of transmembrane proteins. We receptors on tumor-infiltrating lymphocytes results in dampened anti- identified proteins that increased or decreased satellite cell proliferation tumor immunity. Currently, therapies that block the co-inhibitory and a significant fraction of the identified proteins are inflammatory receptors CTLA-4 and PD-1 are demonstrating unprecedented effi- cytokines. The enrichment of this class of proteins as active hits in the cacy in restoring productive anti-tumor immunity in certain cancers. screen suggests that inflammatory cytokines as a class play a major role in While these therapies continue to be very promising in the clinic, a determining the activity of satellite cells during muscle repair. The significant fraction of treated patients remain unresponsive to these implications of these findings will be discussed in the context of normal therapies and some cancers have proven refractory. This has spurred muscle repair and in the background of disease or aging. investigation into targeting other co-inhibitory receptor pathways. The growing landscape of co-inhibitory receptors raises new oppor- tunities but also important questions regarding mechanisms of action and therapeutic application. Data on emerging co-inhibitory receptor 067 targets will be discussed in the context of current therapies. MODULATION OF FIBROSIS BY INFLAMMATORY CELLS DISRUPTED IN CHRONIC MUSCLE INJURY 069

1 1 1 CONTROL OF FIBROTIC ACTIVITY IN THE Fabio Rossi , Dario Lemos , Farshad Babaeijandaghi , LUNG AND SKIN BY LIGHT (TNFSF14) Marcela Low1, Chih–Kai Chang1, Daniela Fiore2, Regan–Heng Zhang1, Sergei Nedospasov3 A. Nedospasov3,4 Michael Croft 1The Biomedical Research Centre, University of British Columbia, Vancouver BC, Canada; 2Department of Experimental Medicine, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Section of Medical Pathophysiology Food Science and The tumor necrosis factor receptor (TNFR) superfamily consists of 29 Endocrinology, Sapienza University of Rome, Rome, Italy; membrane and soluble proteins with similar structural features. Many 3 Engelhardt Institute of Molecular Biology and Lomonosov Moscow of these molecules are expressed on cells of the immune system and 4 State University, Moscow, Russia; German Rheumatism Research are potential targets of therapeutic intervention in a number of immune Center, Berlin, Germany based diseases. They can play strong roles as stimulatory receptors for Depending on the inflammatory milieu, injury can either result in a effector CD4 and CD8 T cells, as well as regulating activity of den- tissue’s complete regeneration or in its degeneration and fibrosis, dritic cells and B cells and functioning to organize lymphoid potentially leading to permanent organ failure. Our data indicates that structures. Recent studies have suggested that certain molecules in this in acutely damaged skeletal muscle, sequential interactions between family also may be important in regulating non-lymphoid cells con- multipotent mesenchymal progenitors and infiltrating inflammatory trolling the cross-talk that results from innate and adaptive immunity. cells determine the outcome of the reparative process. Infiltrating One consequence of this cross-talk between the immune system and inflammatory macrophages, through their expression of Tnf, directly non-lymphoid cells that occurs in chronic diseases is fibrosis and tissue induce apoptosis of fibro/adipogenic progenitors (FAPs). In states of remodeling. Fibrosis is a common feature of asthma, GvHD, IPF, and chronic damage, however, such as in mdx mice macrophages lost their several autoimmune diseases such as systemic sclerosis and RA. It ability to induce apoptosis and FAPs differentiate into fiborgenic cells. largely involves the deposition of extracellular matrix proteins such as Treatment with nilotinib, a kinase inhibitor with proposed anti-fibrotic collagen in the lungs, skin, and other tissues, and often accompanying activity, can restore FAP apoptosis and reduce muscle fibrosis in mdx this is differentiation of epithelial cells or fibroblasts such that they gain mice. Our findings suggest that disruption of the precisely timed characteristics of smooth muscle, which further impairs normal tissue inflammatory phases that characterize the response to acute damage function. Current treatment involves global immunosuppression and favors fibrotic degeneration of the muscle during chronic injury. therefore defining specific molecules that promote fibrosis may be important therapeutically. In a model of severe asthma, we found that LIGHT (CD258/TNFSF14) blockade reduced lung collagen produc- tion and smooth muscle hyperplasia, associated with impaired TGF- SYMPOSIUM 21: REGULATION OF beta and IL-13 expression, two cytokines previously implicated in driving fibrosis. We have extended this data to different inflammatory INFLAMMATION IN TISSUES BY conditions assessing fibrosis driven by rhinovirus infection, and by the CO-STIMULATORY AND CO- antibiotic bleomycin that induces a response similar to that in human systemic sclerosis and idiopathic pulmonary fibrosis. Data will be INHIBITORY PATHWAYS shown on the importance of LIGHT to these syndromes in studies of mice lacking LIGHT that show decreased collagen deposition and 068 smooth muscle mass in their lungs and skin and overall less fibrosis. TARGETING CO-INHIBITORY RECEPTORS IN LIGHT has two receptors in the TNFR superfamily, namely the herpes virus entry mediator (HVEM) and the lymphotoxin beta receptor CANCER (LTbetaR). Both receptors are broadly expressed on cells implicated in driving fibrotic activity, including epithelial cells in the lung, ker- 1,2 Ana C. Anderson atinocytes in the skin, and fibroblasts. Results will also be discussed assessing the range of activities of LIGHT on these cell types and how 1 2 Harvard Medical School, Boston, MA, USA; Brigham and Women’s this may contribute to the inflammatory milieu that drives both early Hospital, Boston, MA, USA and late stages of fibrotic disease.

123 Inflamm. Res. S79

070 responses) in 80–90 % of patients using a single drug. This success IMMUNOREGULATORY MECHANISMS THAT has been enabled by a ‘‘fine mapping’’ of disease-related genes and pathways using genomic methods to identify disease-associated genes LIMIT AUTOIMMUNITY AND CANCER and associated responses to progressively more ‘‘narrow’’ immune or cytokine inhibitors. While psoriasis displays activation of Th1, Th17, Dario AA Vignali and Th22 T-cells (as well as corresponding CD8+ subsets, cd subsets, and innate lymphoid subsets), the central axis of disease pathogenesis University of Pittsburgh, Pittsburgh, PA, USA is now understood as the IL-23/T17 Pathway. This axis begins with excess IL-23 production by a group of myeloid (CD11c+) dentritic Regulatory T cells (Tregs) inhibit beneficial anti-tumor responses. Treg depletion enhances tumor rejection in animal models and the clinic but cells after these cells are stimulated in vivo by TNF, possibly from an also leads to substantial adverse events. Thus approaches have been autocrine activation loop (these DCs have been defined as TNF- and iNOS-producing or TIP-DCs). The excess IL-23 drives T-cell acti- sought to target Tregs in tumors while limiting systemic autoimmune vation as Th17, and more broadly as T17, with apparent co-activation and inflammatory manifestations. First, the signals that maintain Treg stability and potentiate their function remain obscure. We have shown of Th22 T-cells. T17 T-cells are activated within skin lesions to that the immune cell surface ligand semaphorin-4a (Sema4a) on produce excess IL-17A and IL-17F. In turn, IL-17 has major actions to alter gene transcription within epidermal keratinocytes and other conventional T cells and DCs, and the Treg-restricted receptor neu- skin-resident cells, leading to altered production of several-hundred ropilin-1 (Nrp1) interact to potentiate Treg function. Mice with a Treg- restricted deletion of Nrp1 exhibit limited tumor-induced tolerance gene products, many of which define the psoriasis vulgaris disease and thus substantial resistance to tumors, yet do not develop any phenotype. Induced genes include chemokines, cytokines, antimi- crobial peptides, and S100 proteins that create ‘‘feed forward’’ autoimmune manifestations. Thus, Nrp1 ligation maintains Treg sta- bility and function in highly inflammatory sites but is dispensable for activation loops for dendritic cells, T17 T-cells, and epidermal per- the maintenance of immune homeostasis, highlighting Nrp1 as a plasia/altered differentiation. Th22 T-cells produce high levels of IL- potential immunotherapeutic target in cancer. Second, the relative 22, which in combination with other ‘‘IL-20-family’’ cytokines (IL- 19, IL-20, IL-24), directly induce hyperplasia in epidermal ker- importance of different Treg suppressive mechanisms remains con- atinocytes. Clinical studies have shown that targeting the Th1 axis tentious. Interleukin-35 (IL35) is a Treg-secreted cytokine known to inhibit effector T cell proliferation and mediate infectious tolerance with antibodies to gamma-interferon is highly ineffective, whereas targeting the IL-23/T17 axis with antibodies to IL-23, IL-17A, or the via induction of suppressive IL35-producing induced Tregs, iTr35. IL-17 receptor induces PASI75 responses ([75 % disease improve- Using antibody-mediated neutralization, Treg-restricted deletion of Ebi3 and novel reporter mice, we have shown that IL35 facilitated ment) in 80–90 % + of treated patients and virtual disease remission tumor growth by limiting anti-tumor immunity in transplantable and (PASI100) in a high fraction of treated patients. Molecular profiling genetically-induced murine models of melanoma and lung carcinoma. of the treatment response to these antagonists has shown ability to These findings reveal the previously unappreciated importance of IL35 restore pathologic and disease-defining gene transcription changes to in limiting anti-tumor immunity and present IL35 as a potential ther- levels that are close to background skin. apeutic target in cancer. The inhibitory receptor LAG-3 regulates T cell function and is particularly evident in inflammatory sites. Indeed, we have observed extensive expression of LAG-3 on tumor-infiltrating 072 CD4+ and CD8+ T cells in three distinct transplantable tumors. We will THE TRANSLATIONAL REVOLUTION IN ATOPIC discuss the function of LAG3 on effector T cells versus Tregs in DERMATITIS CENTERED ON TH2/TH22 autoimmunity and cancer. Our recent observations suggest that LAG3 may be differentially utilized by different cell types. CYTOKINES

Emma Guttman-Yassky

SYMPOSIUM 22: ATOPIC DERMATITIS, Center for Excellence in Eczema and Laboratory for Inflammatory PSORIASIS AND IL-4/IL-17 BIOLOGY Skin Diseases, Icahn School of Medicine at Mount Sinai Medical Center, New York, NY, USA 071 Although originally two competing hypotheses were considered for atopic dermatitis (AD), the disease is emerging as a T-cell mediated PSORIASIS: AN IL-23/TH17-DRIVEN DISEASE disease, with cytokine-driven epidermal responses. The predominant inflammatory pathways in AD are T helper type 2 (Th2) and Th22, with James Krueger input from other T cell subsets such as Th1 and Th17. The cytokines interleukin (IL)-4 and IL-13 are key drivers of Th2 inflammation, and The Rockefeller University, New York, NY, USA their expression is associated with features of skin barrier dysfunction Psoriasis vulgaris is one of the most common T-cell mediated related to decreases in expression of filaggrin, increases in epidermal inflammatory diseases in humans, affecting 2–3 % of the population. hyperplasia, and spongiosis. The pathogenic role of IL-4/IL-13 sig- Psoriasis vulgaris is primarily a skin disease in which growth and naling and reversibility of the epidermal pathology in AD have been differentiation of resident skin cells is altered in response to infiltra- confirmed in studies of dupilumab in patients suffering from moderate- tion of skin by T-cells and CD11c+ dendritic cells, with attendant to-severe disease. Dupilumab is a fully-human monoclonal antibody production of inflammatory cytokines. Psoriasis is similar to other directed against the IL-4 receptor alpha subunit that blocks both IL-4 inflammatory diseases of peripheral organs in that it displays complex and IL-13 signaling. In these early phase clinical trials, dupilumab significantly improved clinical signs and symptoms of AD, and was genetics and genomics with [30 gene variants contributing disease 1–3 risk and with alterations in expression of [4000 mRNAs in skin well-tolerated with a favorable safety profile . Four weeks of dupi- lesions. However, psoriasis vulgaris is now the most successfully lumab treatment resulted in a dose-dependent shift from lesional to treated human inflammatory disease with a series of immune-targeted more non-lesional transcriptional phenotypes. Modulated genes therapeutics able to produce high grade improvement (PASI75 included those related to epidermal proliferation, keratin, and

123 S80 Inflamm. Res. inflammatory mediators4. Of note, dupilumab did not reduce mRNA The development of drugs targeting key modulators of immune expression of IL-22 in the skin; however, expression of IL-22-modu- function has brought about a revolution in the treatment of many lated genes was significantly suppressed, suggesting a modulation of diseases. However, such drugs pose unique and novel challenges for Th22 activity. Translational medicine offers insight into Th2- and drug development: as they target specific human immune components Th22-related mechanisms underlying AD pathogenesis. (Clinicaltri- it is often harder to extrapolate from in vitro or animal models what als.gov identifiers: NCT01259323, NCT01385657, NCT01548404, their clinical efficacy may be, and the clinical response can require NCT01639040, and NCT01859988) months to manifest. Thus longer trials with human patients are nec- essary, adding considerable cost, complexity and development time to References: the current cost of four billion dollars and 8 years in development for 1. Beck LA, Thac¸i D, Hamilton JD, et al. Dupilumab treatment in each successful drug brought to market. adults with moderate-to-severe atopic dermatitis. N Engl J Med. In particular, Psoriasis has become the ‘‘go to’’ immune disease for 2014;37:130–139. proof-of-concept studies of new immune targeted compounds, with 2. Thac¸i D, Simpson E, Bieber T, et al. Efficacy and Safety of more than 34 compounds in different phases of development spanning Dupilumab in adults with moderate-to-severe atopic dermatitis (AD) from biological agents to small molecules. As a result, there is inadequately controlled by topical therapies: final results of a phase abundant genomic data for model development, much of which has 2b study. American Academy of Dermatology—73rd Annual Meet- been generated by our laboratory over the past decade. Although ing. 2015; Abstract F037. https://www.aad.org/Symposium/LBAM psoriasis is highly responsive to pathway-specific immune antagonists 2015/. Accessed on April 30, 2015. it often takes 12–16 weeks for clinical response measures to be 3. Simpson E, Worm M, Soong W, et al. Dupilumab Improves meaningful, and efficacy ranges from 30 to 80 % success rate. Patient-Reported Outcomes (PROs) in a Phase 2 Study in Adults with We used longitudinal skin gene expression profiles to build a Moderate-to-Severe Atopic Dermatitis. J Allergy Clin Immunol. genomic classifier that use early time-points (baseline, 1st, 2nd and 2015;135:AB167. 4th weeks) to predict clinical response in psoriatic patients after 4. Hamilton JD, Sua´rez-Farin˜as M, Dhingra N, et al. Dupilumab 12 weeks of treatment. Clinical response was defined as PASI75 at improves the molecular signature in skin of patients with moderate-to- 12 weeks: i.e. the percentage of patients with at least 75 % reduction severe atopic dermatitis. J Allergy Clin Immunol. 2014;134:1293–1300. of the Psoriasis Area and Severity Index (PASI) after 12 weeks of treatment. Two classifiers are developed that predicts response 073 regardless of the agent the universal classifier, and a treatment- specific classifier. For the treatment specific classifier, baseline and FROM PATHWAY TO TARGETED THERAPY: IL-4 week 1 after treatment can predict treatment response with [97 % AND IL-13 IN ALLERGIC DISEASES accuracy for etanercept, metrotexate, adalimumab and ustekinumab. The universal classifier achieved 95 % accuracy with data up to Jennifer D. Hamilton 2 weeks and [97 % accuracy when data from week 4 is added. The universal classifier was evaluated in unseen data from 24 Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USA patients, 16 of whom were treated with ustekinumab/45 mg with profiles available for baseline and week 1 only and 8 who were treated Interleukin (IL)-4 and IL-13 are key cytokines driving allergic and T with placebos with profiles at baseline and week 2. helper cell type 2 (Th2)-polarized inflammatory processes. These The exciting results observed in predicting treatment response cytokines are now known to originate from several cell types, after a short clinical trial in psoriasis would allow us to use this including Th2 cells, innate lymphoid Type 2 cells (ILC2), mast cells, methodology in other diseases and conditions, like Atopic Dermatitis. eosinophils and basophils. IL-4 and IL-13 are upstream drivers of Th2 inflammatory processes, such as recruitment of T cells, mast cells, eosinophils and monocytes to tissues; mucus production; B cell acti- vation; and immunoglobulin E production. Different disease SYMPOSIUM 23: DEFINING endotypes may exist among patient subpopulations. Biologics target- SUBPHENOTYPES IN CHRONIC ing Th2-associated cytokine pathways are in development for several allergic diseases, including atopic dermatitis, asthma, chronic sinusitis RESPIRATORY DISEASES TO IMPROVE with nasal polyposis, and eosinophilic esophagitis. Data from early DRUG DEVELOPMENT AND DISEASE phase trials of these targeted therapies provide insight into Th2-me- MANAGEMENT diated inflammatory processes and provide further insight into the role of IL-4 and IL-13 in human allergic diseases. In addition to the potential for providing new treatment options for patients, targeted 075 therapeutics also provide a unique opportunity to advance the under- BIOMARKER DISCOVERY AND COMPANION standing of disease pathogenesis and endotypes in atopic disorders. DIAGNOSTIC DEVELOPMENT IN ASTHMA

074 Joseph R. Arron PREDICTION OF CLINICAL THERAPEUTIC Genentech, Inc, South San Francisco, CA, USA RESPONSES IN INFLAMMATORY SKIN DISEASES Asthma has been regarded as an allergic disorder characterized by type BASED ON EARLY TIME POINT GENE 2 cytokine expression and eosinophilic inflammation in the airways. EXPRESSION DATA However, it is clearly heterogeneous on molecular, pathological, and clinical levels. Genomic approaches have identified heterogeneous Mayte Suarez-Farinas1,2, Joel Correa da Rosa2 gene expression patterns in asthmatic airways corresponding to the degree of type 2 cytokine expression and eosinophilic inflammation. 1Mount Sinai School of Medicine, New York, NY, USA; 2Rockefeller These gene expression patterns have led to the identification of candi- University, New York, NY, USA date biomarkers of eosinophilic airway inflammation that do not require

123 Inflamm. Res. S81 bronchoscopy or sputum induction. Candidate biologic therapies tar- SYMPOSIUM 24: BIOLOGICAL AND geting mediators of type 2 airway inflammation have progressed through clinical studies in moderate-severe asthma patients in recent NONBIOLOGICAL THERAPIES OF years. Serum periostin, fractional exhaled nitric oxide (FeNO), and AUTOIMMUNE DISEASES—SPONSORED blood eosinophil counts have emerged as predictive and pharmacody- BY JSIR-JAPAN namic biomarkers that may enrich for clinical benefit in clinical studies of biologic therapies targeting IL13, IL5, and IgE. These clinical studies have shown that asthma heterogeneity impacts target, patient, and 077 outcome selection, and biomarker-guided clinical development will MOLECULAR UNDERSTANDING OF TARGETED help to: (1) deliver targeted therapeutics to patients most likely to THERAPIES IN PATIENTS WITH RHEUMATOID benefit, (2) redefine the unmet medical need in asthma to inform future target discovery research efforts. ARTHRITIS

Tsutomu Takeuchi 076 Division of rheumatology, Department of Internal Medicine, INTEGRATING CLINICAL AND MOLECULAR Keio University School of Medicine, Tokyo, Japan FEATURES TO IDENTIFY DISTINCT ASTHMA Recent understanding of pathogenesis of the autoimmune diseases has SUB-PHENOTYPES been enormously facilitated by clinical studies and careful observation with targeted therapies on the molecules involved in the disease pro- Frederic Baribaud cess. One example can be rheumatoid arthritis (RA), which is characterized by persistent destructive polyarthritis with autoantibod- Janssen R&D, Los Angeles, CA, USA ies against citrullinated proteins. The appropriate targets in this Rational: Asthma is a heterogeneous disease that includes clinically autoimmune disease, RA, are TNFa and IL-6, while the potential targets relevant subgroups. proposed by animal models such as IL-1ß and IL-17 did not exhibit the comparable clinical efficacy in human RA. In addition, it has been Objectives: To identify subgroups of asthma in the airways disease shown the rapid and excellent clinical response in placebo-controlled endotyping for personalized therapeutics (ADEPT) study using clus- ter analyses. dose finding study for fully human monoclonal antibody against GM- CSF receptor. Given such robust evidences of the appropriate targets, Materials and Methods: Clinical features and accessible biomarkers we further need to fully understand the role of these targets in the with molecular characteristics were collected by profiling asthmatic pathogenesis of the autoimmune disease. In this symposium, I will subjects and healthy non-atopic volunteers. Assessments included questionnaires, pulmonary function tests, airway hyper responsive- focus on the in vivo change of a variety of biomarkers including cytokines during anti-TNF or anti-IL-6 receptor therapies in patients ness, FENO, and biomarkers from induced sputum, endobronchial with RA in order to explore the relationship between TNFa and IL-6 as biopsy and brushings. Partition-around-medoid clustering was per- the appropriate targets. Then, we examined the in vivo change of formed on the ADEPT dataset using pre-specified clinical variables or using the gene set variation analysis (GSVA) enrichment scores (ES) immune cells during the targeted therapies by modern standardized immune-phenotyping to test their potential effects on B cells, T cell using an in vitro generated glucocorticoid (GC) signature. Models for subsets and monocyte subsets. Finally, I will discuss the molecular cluster classification probabilities were derived and applied to the U-BIOPRED adult asthma dataset. CCL26 (eotaxin-3) expression or distinction among the targeted therapies with comparable clinical GSVA ES of an IL13 IVS were used for evaluation of Th2high versus efficacy and the basis for personalized strategy of the targeted therapies. Th2low classification within clusters. Results: Four clinical clusters were identified in the ADEPT-asthma study population with unique clinical and biomarker profiles. Group 1 078 represents well-controlled asthma. Group 2 represents controlled, albeit LOW MOLECULAR WEIGHT highly hyper-reactive patients mostly with a Th2high phenotype. In con- trast, Group 3 has a less controlled, restrictive, Th2low phenotype. Group IMMUNOSUPPRESSANT THERAPY OF 4 consists of the most poorly controlled patients, a mixed Th2high/low AUTOIMMUNE DISEASES phenotype. U-BIOPRED asthma patients were classified to the ADEPT clusters with similar probability distributions as compared to ADEPT. Shinichi Kawai Importantly, the distributions for most clustering and non-clustering (e.g., demographics and biomarkers) variables were similar to those in Toho University School of Medicine, Tokyo, Japan ADEPT, rather than being biased by only a minority of variables. Three GC molecular response clusters were identified in ADEPT, a GC Autoimmune diseases are chronic inflammatory diseases characterized by responsive (R), a GC non-responsive (NR) and a GC partially-responsive immunological abnormalities and also by the specific organ disturbances. (PR) cluster. In clinical clusters 2 and 4 about 30 % of patients were GC Various low molecular weight immunosuppressants such as azathioprine, NR, 25 % were GC R and about 20 and 38 % were GC PR in cluster 2 cyclophosphamide, methotrexate, mycophenolate mofetil, cyclosporine and 4 respectively. In clinical cluster 3 however, about 10 % were GC A, and tacrolimus are widely used for treatment of autoimmune diseases. NR and PR respectively and about 70 % were GC R. The primary lesion of rheumatoid arthritis (RA), an autoimmune disease is Conclusions: Four discrete clinical clusters were identified, and considered to be in synovial membranes. Synovial cell proliferation confirmed, that have unique clinical and biomarker profiles. Clusters gradually affects surrounding cartilage and bones, frequently leading to 2 and 4 represent well-controlled, albeit highly hyper-reactive and the destruction and deformation of joints. Methotrexate and some other poorly controlled patient groups with a mixed GC responsive profile, immunosuppressants are used in patients with RA for not only improve- respectively, that may benefit from Th2high-targeted intervention. In ment of inflammation and immunological abnormalities but also contrast, cluster 3 has a Th2low, restrictive phenotype with a largely prevention of joint destruction. I will introduce the intracellular mecha- GC NR profile that would likely require different interventions. nisms of action of methotrexate in RA patients. I also introduce the basic

123 S82 Inflamm. Res. and clinical data of tacrolimus which is approved in Japan for treatment of B095 RA. Systemic lupus erythematosus (SLE) is an autoimmune disease that POLYCYSTIC LIVER DISEASE IS SUPPRESSED BY causes various symptoms and immunological abnormalities. The standard treatment is corticosteroid therapy, but a combination of corticosteroid IL-4/IL-13 AND TGF-ß SIGNALING with a low molecular weight immunosuppressant is used for SLE espe- cially with severe organ involvement. In Japan, tacrolimus is approved for See poster section for abstract. treatment of lupus nephritis as maintenance therapy. I will then introduce the data of some clinical studies of tacrolimus for SLE patients. In this presentation, some basic studies and clinical data of efficacy and safety of B096 several low molecular weight immunosuppressants especially for the EVALUATING IL-13RA2 AS A THERAPEUTIC Japanese patients with RA and SLE will be discussed. TARGET FOR PULMONARY FIBROSIS

See poster section for abstract. B034 WONDERBUMIN: A FULLY SYNTHETIC ALTERNATIVE TO IGT FOR AUTOIMMUNE B091 DISEASE IDENTIFICATION OF BOTH IMMUNOMODULATORY AND ANTI-FIBROTIC See poster section for abstract. ACTIONS OF NINTEDANIB AND PIRFENIDONE USING BIOMAPÒ HUMAN PRIMARY CELL- BASED DISEASE MODELS B047 NEUTRALIZING THE PRO-METASTATIC See poster section for abstract. IMMUNE RESPONSES OF MDSC AND T CELLS THROUGH B CELL TARGETING B025 THE SYK INHIBITOR FOSTAMATINIB LIMITS See poster section for abstract. TISSUE DAMAGE AND FIBROSIS IN A BLEOMYCIN-INDUCED SCLERODERMA MOUSE MODEL: EFFICACY IN SYK INHIBITION MINI-SYMPOSIUM 2: CELLULAR AND IN ANIMAL SCLERODERMA MOLECULAR MECHANISMS OF FIBROSIS See poster section for abstract. B093 LINEAGE TRACING OF RESIDENT FIBROBLASTS BY THE INTRATRACHEAL CELL TRANSFER NEW INVESTIGATOR AWARD WINNER ELUCIDATES THE CELLULAR ORIGIN OF PRESENTATIONS ACTIVATED FIBROBLASTS IN PULMONARY FIBROSIS B061 COMBATING SKIN INFLAMMATION WITH See poster section for abstract. CXCL12 CHEMOKINE NEUTRALIGANDS

See poster section for abstract. B097 CROSSTALK BETWEEN MACROPHAGES AND HUMAN HEPATIC FIBROBLASTS SHOW THAT B050 INFLAMMATORY RESPONSE ATTENUATE THE THE ROLE OF RESOLVIN D1 IN THE ACTIVATION OF FIBROBLASTS IN THE REGULATION OF INFLAMMATORY AND FIBROSIS RESPONSE CATABOLIC FACTORS IN OSTEOARTHRITIS

See poster section for abstract. See poster section for abstract.

B094 B099 PERICYTE MYD88 CONTROLS INFLAMMATORY ACETATE PROTECTS THE ISCHEMIC INTESTINE AND FIBROTIC RESPONSES TO TISSUE INJURY FROM REPERFUSION INJURY

See poster section for abstract. See poster section for abstract.

123 Inflamm. Res. S83

B246 There is increasing evidence that there is a linkage between devel- IDENTIFICATION OF CD44BRIGHT, A NOVEL opment of acute lung injury (ALI), complement and histones, both in humans and in mice with ALI. In C57BL/6 young male mice, ALI has SUBSET OF UTERINE NK CELLS CORRELATED been induced by airway instillation of LPS or recombinant murine TO ABORTION IN RECURRENT PREGNANCY C5a. This induces an intense neutrophil (PMN)-rich inflammatory LOSS MODEL MICE response in alveoli, accompanied by edema, fibrin deposition and extracellular histones. The requirement for complement (C5a) has See poster section for abstract. been demonstrated by the greatly reduced intensity of ALI as quan- titated by albumin leak and buildup of cytokines, chemokines and histones in bronchoalveolar fluid (BALF). The absence of receptors for C5a greatly reduces this inflammatory response including sharply B266 reduced BALF levels of histones. PMN depletion also is protective and A NOVEL ROLE FOR BK CHANNEL IN greatly reduces all parameters of injury (including BALF histones). It REGULATING METALLOPROTEASE ACTIVITY appears that generation of C5a with its reactions with C5a receptors on PMNs results in formation of neutrophil extracellular traps (NETs), which are associated with appearance of histones. Histones are See poster section for abstract. extremely phlogistic and prothrombotic. ALI can be sharply reduced by the presence of neutralizing antibodies to extracellular histones. These studies provide evidence for close interactions between com- PATIENT PERSPECTIVES: UNMET plement (C5a and its receptors) and histones in the setting of ALI. MEDICAL NEED–LUPUS 081 079 FINE-TUNING INFLAMMATION: ‘DESIGNER’ PATIENT PERSPECTIVES: UNMET MEDICAL LIGANDS FOR THE COMPLEMENT PEPTIDE NEED–LUPUS RECEPTORS Heather E. Jones Peter N. Monk Pfizer, Inc., Collegeville, PA, USA University of Sheffield, South Yorkshire, UK Objective: To increase awareness of the true unmet medical need left by current therapeutic options for SLE and understand what this Complement activation is a factor in most inflammatory diseases and means to the patient. The importance of recognizing the true unmet is also known to occur in many conditions not usually thought of as medical need in autoimmune diseases is higher than ever. No drug has having an inflammatory element, such as cancer. Small, biologically so far been able to cure the SLE, only to induce remission. To pro- active protein fragments, the complement peptides C5a and C3a, are gress science and clinical development there must be focus on generated which can act on many aspects of the immune response. understanding the pathogenesis of the disease. From a medical per- The receptors for these peptides are widely expressed on immune and spective we need to consider the patient as a whole human being and non-immune cells and have long been important drug targets. not only based on the organ(s) involved. Autoimmune diseases are C5a is generally pro-inflammatory and excessive production can systemic, in most cases affecting more than one organ. lead to tissue damage in rheumatoid arthritis, for example. In contrast, The morning session will begin by emphasizing the unmet medical C3a often has an anti-inflammatory role, sometimes working in need followed up by an interview of a patient with SLE, telling their opposition to C5a. However, in asthma, C5a has been shown to be story, with an emphasis on what they see as key objectives for further protective in the early sensitisation phase whereas both C5a and C3a development. are destructive in the late, effector, phase. Both C5a and C3a can also At the end of the session the co-chairs will summarize the signs be generated without activation of the complement cascade, through and symptoms and the treatment algorithm for SLE, as well as the the extrinsic pathway. Extra- and intra-cellular extrinsic pathways need for biomarkers to define the disease, ensure safety, guide have been described that enable local or autocrine production; such treatment and predict treatment response. pathways are now thought to be involved in T cell differentiation. In addition, some pathogens can modulate extrinsic generation of C5a and C3a to produce a less hostile microenvironment. This complex biology might limit the possibilities for therapeutic intervention in SYMPOSIUM 25: COMPLEMENT complement peptide-mediated inflammation. Our understanding of how the complement peptide receptors TARGETED THERAPIES—IS THERE (C5aR1, C5aR2 and C3aR) function is now leading to the generation of LIGHT AT THE END OF THE TUNNEL? new ligands which modulate receptor activity rather than acting simply as antagonists. Biased agonists that can select the downstream sig- 080 nalling pathways, partial agonists that diminish maximum responses, and strategies to control of receptor homo- and hetero-dimerisation will LINKAGES BETWEEN COMPLEMENT, HISTONES be discussed as promising strategies to control pathology associated AND ACUTE LUNG INJURY with complement peptide production. The first selective ligand for C5aR2 has recently been discovered and its ability to modify a subset Peter A. Ward of C5a responses will be described. The potential of this novel ligand for a complement peptide receptor will be illustrated by a newly-dis- University of Michigan Medical School, Ann Arbor, MI, USA covered pathway for the control of human T cell differentiation.

123 S84 Inflamm. Res.

082 Princeton, NJ, USA; 4Renal Section, Boston University Medical 5 COMPSTATIN CP40: AN IMMUNE MODULATOR School, Boston, MA, USA; Division of Rheumatology, University of Colorado Medical School, Aurora, CO, USA; 6Departments of ON A JOURNEY FROM DISEASE MODELS TO Neurology and Pharmacology and Physiology, George Washington CLINICAL DEVELOPMENT School of Medicine, Washington, DC, USA Introduction: Uncontrolled complement activation plays a pivotal role John D. Lambris1, Alexandra Primikyri1, Sophia Koutsogiannaki1, 1 1 1 in a variety of disorders such as PNH and aHUS. Malvina Papanastasiou , Jihyoung Seong , HongBin Wang , Gang Aims: ALN-CC5 is a subcutaneous (S.C.) investigational RNAi Chen2, Markus Huber-Lang3, Martijn van Griensven4, Bo Nilsson5, 5 5 6 therapeutic targeting complement C5 (C5) with the purpose of Kristina Nilsson-Ekdahl , Alireza Biglarnia , Richard J. Smith , decreasing terminal complement activity and thereby protects against George Hajishengallis7, Despina Yancopoulou8, Antonio M. 9 1 intravascular hemolysis and complement mediated tissue damage. Risitano , Daniel Ricklin Materials and methods: Preclinical studies in mouse, rat and non-

1 human primate (NHP) models were used to investigate the ability of University of Pennsylvania, Perelman School of Medicine, ALN-CC5 to reduce complement C5, inhibit complement-mediated Philadelphia, USA; 2Huazhong University of Science and 3 hemolytic activity and complement alternative- and complement Technology, Wuhan, China; Ulm University, Ulm, Germany; classic-pathway (CAP and CCP). Furthermore, ALN-CC5 was 4Technical University Munich, Munich, Germany; 5Uppsala 6 7 investigated in a rat membranous nephropathy model, the mouse anti- University, Uppsala, Sweden; University of Iowa, USA; University collagen antibody induced arthritis model and in a rat passive of Pennsylvania, School of Dentistry, Philadelphia, USA; 8Amyndas 9 myasthenia gravis model. Pharmaceuticals, S.A., Glyfada, Greece; Federico II University, A placebo controlled double blind phase 1 clinical study in healthy Naples, Italy volunteers and patients with PNH is ongoing. Several cohorts in part As a prime sensor of inflamed and damaged tissue, cellular debris and A, a single ascending dose study have been completed and Part B, a foreign cells/materials, the complement system critically contributes multiple ascending dose study is currently ongoing. Part C will be a to host defense and homeostasis, yet its formidable immune surveil- multiple dose study in patients with PNH. Primary endpoints are lance capabilities can turn awry and inflict damage to self-cells. safety and tolerability. Secondary endpoints are pharmacokinetics, Therapeutic complement inhibition has therefore been emerging as a reduction of circulating C5, reduction in hemolytic and CAP as well promising strategy in broad spectrum of immune, inflammatory, and as CCP activity. biomaterial-induced disorders. By blocking the central complement Results: Pre-clinical studies demonstrated that ALN-CC5 resulted component C3, the peptidic inhibitor compstatin and its analogs have in mean 98.4 ± 0.7 % reduction of C5 levels in NHPs, mean early shown promise in many disease models. Recent years have 88 ± 6.1 % reduction in hemolysis and mean 95.1 ± 0.93 % reduction provided insight into the mode of action and structural determinants in CAP with every-other week or monthly S.C. dosing. Furthermore, C5 of this inhibitor class and allowed for the design of compstatin silencing significantly reduced proteinuria in a rat membranous derivatives with largely improved pharmacodynamic and kinetic nephropathy model, reduced disease activity in the mouse arthritis properties. Among them, analog Cp40 has shown particular promise model to a level comparable to anti-C5 monoclonal antibody; and and has been successfully evaluated in a variety of clinically relevant reduced clinical disease severity in the myasthenia gravis model. models, ranging from C3 glomerulopathies, paroxysmal nocturnal In Part A of the phase 1 study, human volunteer subjects were hemoglobinuria and periodontal disease to hemodialysis-induced randomized (3:1) to placebo or a starting single subcutaneous dose of inflammation, hemorrhagic shock and solid organ transplantation. 50 mg ALN-CC5 and followed for a t least 70 days. Following safety Building on these strong preclinical results, a Cp40-based drug review, additional single ascending dose cohorts were authorized. Part (AMY-101, Amyndas) has meanwhile entered clinical development. B multiple ascending cohorts are planned to start in parallel to Part A. The combined preclinical and clinical studies already revealed a Up-to-date results on safety, tolerability and C5 knockdown, changes unique pharmacokinetic behavior that is expected to facilitate long- in CAP, CCP and hemolytic activity from study will be presented. term administration, and indicated a beneficial safety profile. This Conclusion: Collectively, these data suggest that the use of a novel presentation highlights milestones in the recent development of RNAi therapeutic targeting C5 is a promising approach for inhibiting compstatin Cp40, showcases data from disease-related studies, and complement in PNH, aHUS and other complement mediated diseases. discusses general implications on therapeutic complement inhibition The subcutaneous route of administration and infrequent dosing make at the level of C3. this a particularly encouraging potential therapy.

083 SYMPOSIUM 26: REGENERATIVE A SUBCUTANEOUSLY ADMINISTERED RNAI MEDICINE AND STEM CELLS IN THERAPEUTIC (ALN-CC5) TARGETING INFLAMMATION COMPLEMENT C5 FOR TREATMENT OF PNH AND COMPLEMENT MEDIATED DISEASES 084 THE ROLE OF STEM CELL EXHAUSTION IN 1 1 2 3 Anna Borodovsky , Benny Sorensen , Jorg Taubel , James Bush , AGING AND DISEASE Noriyuki Kawahata1, Lauren Melton1, Jeffrey Cehelsky1, Christine Powell1, Kristina Yucius1, Prasoon Chaturvedi1, Garvin Warner1, 4 5 5 6 Johnny Huard David Salant , V. Michael Holers , Nirmal Banda , Linda Kusner , Henry Kaminski6, Akshay Vaishnaw1, Pushkal Garg1 University of Pittsburgh, Pittsburgh, PA, USA 1Alnylam Pharmaceuticals, Cambridge, MA, USA; 2Richmond Aging is characterized by the progressive erosion of tissue home- Pharmacology, London, UK; 3Covance Clinical Research Unit, ostasis and functional reserve in all organ systems. There is evidence

123 Inflamm. Res. S85 that the accumulation of damage in stem cells renders them defective muscle satellite cells. Surprisingly, in vivo studies revealed that for self-renewing and regenerating damaged tissues. We have activation of NF-kB activity in muscle fibers, but not in satellite cells, demonstrated that a population of muscle progenitor cells (MPCs) drives muscle dysfunction and that life-long inhibition of NF-kB isolated from the ERCC1-deficient mouse model of accelerated aging, activity in myofibers preserves muscle regenerative potential with are defective in their proliferation abilities, differentiation capacity aging via cell-non-autonomous effects on satellite cell function. and resistance to oxidative stress. We have observed that intraperi- Building on this unexpected discovery, we analyzed differential gene toneal (IP) injections of wild-type (WT)-MPCs into Ercc1 knockout expression in muscle with varying NF-kB activity, and identified a (Ercc1-/-) mice resulted in an improvement in age related patholo- secreted phospholipase (PLA2G5) as a myofiber-expressed NF-kB gies. In an attempt to determine whether the defect observed in ERCC regulated gene that governs muscle regenerative capacity with age. deficient MPCs was not exclusive to this progeria model, we have Together, our data are consistent with a model in which NF-kB isolated and characterized MPCs from another progeroid mouse activation in muscle fibers increases Phospholipase 2 expression and models, the zinc metalloproteinase (Zmpste24) knock-out mouse, an subsequently drives the impairment in regenerative function charac- animal model of the Hutchinson-Gilford progeria syndrome (HGPS). teristic of aged muscle. Importantly, inhibition of NF-kB function Similar to ERCC deficient MPCs, we have observed that reverses this deficit in repair, suggesting that FDA-approved drugs, Zmpste24-/- MPCs have proliferation and differentiation defects, like salsalate, a prodrug form of sodium salicylate, may provide new characteristics also observed in MPCs isolated from naturally aged therapeutic avenues for elderly patients with reduced capacity to mice. These results suggest that the defect in MPCs is not specific to a recover effectively from muscle injury. Together, these studies thus particular model of progeria and can also be observed in naturally identify a novel NF-kB regulated, non-cell autonomous mechanism aged animals. Finally, we have investigated whether a defect in MPCs by which stem cell function is linked to lipid signaling and home- can also be observed in skeletal muscle disease such as Duchenne ostasis, and provide important new targets for therapeutic intervention muscular dystrophy (DMD), which is a degenerative muscle disorder to stimulate muscle repair in aged individuals. characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. Interestingly, DMD patients lack dystrophin from the time of birth; however, the onset of muscle weakness only becomes apparent at 4–7 years of age, which happens to coincide B121 with the exhaustion of the MPC pool. There are several lines of DYSREGULATION OF RESOLUTION OF ACUTE evidence that support this concept including the gradual impairment INFLAMMATION IN AGED HUMANS of the myogenic potential of MPCs isolated from patients during aging, which results in a reduction of muscle regeneration in older See poster section for abstract. patients. In addition to muscle weakness, patients acquire osteopenia, fragility fractures, and scoliosis indicating that DMD may represent a model of premature musculoskeletal aging with a potential dysfunc- tion in MPCs. Here, we report that dystrophin–utrophin double B190 knockout (dko) mice exhibit a spectrum of degenerative changes in PROTECTIVE EFFECT OF HEMATOPOIETIC various musculoskeletal tissues including skeletal muscle and bone. In contrast to that observed with MPCs isolated from the mdx mice STEM CELLS IN STROKE (dystrophin deficient and mild phenotype), we have recently shown a defect in the MPCs isolated from dKO mouse. We have observed that See poster section for abstract. the MPC defect from the dKO mouse model appears to be age dependent and not specific to MPC since other stem cell population also appears to be affected. SYMPOSIUM 27: INKT CELLS— MEDIATORS AT THE INTERFACE OF INFLAMMATION AND IMMUNITY 085 INHIBITING CHRONIC SKELETAL MUSCLE 086 INFLAMMATION IN AGING RESTORES THE HUMAN INVARIANT NATURAL KILLER T CELLS SATELLITE CELL NICHE AND IMPROVES ACTIVATE MONOCYTE-DERIVED DCS TO MUSCLE REGENERATION INITIATE A PATHWAY OF STERILE INFLAMMATION THAT PROMOTES HOST Amy J. Wagers DEFENSE Harvard University, Cambridge, MA, USA Jenny E. Gumperz1,2 It has been known for some time that muscle repair potential becomes increasingly compromised with advancing age, and that these age- 1 University of Wisconsin School of Medicine and Public Health; related defects are associated with reduced activity of muscle satellite 2 Department of Medical Microbiology and Immunology, Madison, cells and with the presence of chronic, low grade inflammation in the WI, USA muscle. Working from the hypothesis that a heightened inflammatory tone could contribute to poor regenerative capacity in aged muscle, Invariant natural killer T (iNKT) cells are innate T lymphocytes that we developed genetic systems to inducibly modulate inflammatory recognize lipids as antigens presented by CD1d glycoproteins. CD1d gene expression in muscle satellite cells or muscle fibers by modu- molecules are non-classical antigen presenting molecules expressed lation of the activity of nuclear factor kB (NF-kB), a master by most myeloid cell types (monocytes, macrophages, and DCs), as transcriptional regulator of inflammation in many tissues whose well as by B lymphocytes. iNKT cells are known to be able to activity is dramatically upregulated in aged skeletal muscle and potently affect the outcome of immune responses through cytokine

123 S86 Inflamm. Res. production and by interacting with and affecting the functions of 1Division of Oncology, Children’s Hospital of Philadelphia, CD1d+ antigen presenting cells. iNKT cells and CD1d+ myeloid cell Philadelphia PA, USA; 2Vaccinex, Inc. Rochester, NY, USA; types have been found in a variety of inflamed human epithelial and 3Research and Development, NKT Therapeutics, Waltham, MA, USA; endothelial tissues, and studies in murine model systems have sug- 4Department of Oncology, St. Jude Children’s Research Hospital, gested that both of these subsets are often early recruits to sites of Memphis, TN, USA tissue inflammation. To gain a better understanding of how iNKT Invariant natural killer T (iNKT) cells comprise a unique lineage of cells contribute during acute inflammation, we have investigated how CD1d-restricted lipid-reactive T lymphocytes that kill tumor cells and they influence the functions of human monocyte-derived DCs (a type exhibit robust capacity to transactivate the tumor-directed functions of DC that is often recruited to sites of inflammation). We show that of dendritic (DC), NK, T and B cells. In most cases, optimal iNKT iNKT cells induce sustained cytoplasmic calcium signaling in DCs cell killing of tumors is T cell receptor (TCR)-dependent, requires that is associated with the induction of eicosanoid lipid mediator tumor cell expression of the antigen-presenting molecule CD1d and biosynthesis, and that does not require stimulation by microbial the pre-loading of tumor cells with stimulatory lipid antigens that compounds. This sterile inflammatory pathway induces vascular induce iNKT cell activation. To capitalize on the anti-tumor proper- permeabilization and neutrophil recruitment and activation in vivo, ties of iNKT cells, our laboratory is developing novel iNKT cell based and appeared to facilitate host defense against challenge by the cancer therapies. As our first approach, we have engineered a opportunistic pathogen Candida albicans. recombinant fusion protein in which the human CD1d molecule is joined to a single chain antibody fragment (scFV) specific for human CD19, a tumor antigen expressed on most B cell cancers. We have produced and purified the fusion and demonstrated that it binds with 087 specificity to human CD19+ targets. Once loaded with a stimulatory IMAGING iNKT CELLS IN VIVO-DIFFERENT lipid antigen such as the prototypical iNKT cell agonist a-galactosyl ceramide (aGC), the fusion induces robust in vitro iNKT cell acti- FUNCTIONS IN DIFFERENT ORGANS vation, cytokine production and lysis of immortalized CD19+ CD1d- B cell lines. Currently, we are testing whether the fusion will activate Paul Kubes iNKT cells in situ and induce iNKT cell-dependent mechanisms of tumor clearance. As our second approach, we are characterizing the Snyder Institute for Chronic Diseases University of Calgary, Calgary, iNKT cell stimulatory properties of a recombinant humanized mon- AB, Canada oclonal antibody NKT 320, which binds selectively and with high affinity to the human invariant TCR. Soluble and immobilized NKT Using spinning disk microscopy allows us to track iNKT cells in 320 induces a dose-dependent iNKT cell activation, proliferation, various organs including the liver, joints, spleen and lung. In the liver degranulation and activation of bystander immune cells. iNKT cells iNKT cells crawl randomly in the sinusoids of the liver. During an stimulated by plate-bound NKT320 also robustly secrete Th1 and infection Kupffer cells catch bacteria from the vasculature and call Th2-type cytokines and chemokines. Consistent with these in vitro iNKT cells to their surface where they present pathogen-derived data, the treatment of Va24 transgenic mice, which express the human glycolipids on CD1d to the iNKT cells prompting them to make iTCR alpha chain, leads to up-regulation of activation markers and interferons. During non-infectious insults, the iNKT cells can make intracellular IFN-g production by iNKT cells, and incorporation of cytokines to help in repair. iNKT cells can even respond to neuro- BRDU, indicating iNKT cell proliferation. We are now developing transmitters that help to immunosuppress the host and reduce animal models with which to test the anti-tumor effects of NKT 320 inflammation. It appears that each organ has its own particular iNKT or the related antibody NKT 14 m, which binds to murine iNKT cells cells with organ specific localization. It is well known that iNKT cell and induces their activation in vitro and in vivo. deficient mice get a huge burden of Borreliosis into joints and when joints were visualized it became apparent that iNKT cells surround the vasculature of joints and prevent entry of Borrelia into this tissue. They do this independent of CD1d and via direct granzyme killing. They may also limit exit from joints of bacteria such as S. aureus. 089 iNKT cells are also found in the vasculature of the lungs where they iNKT CELLS: MEDIATORS AT THE INTERFACE reside in capillaries but remain stationary. Once a pathogen is OF INFLAMMATION AND IMMUNITY detected, they begin to crawl slowly migrating out of the vasculature where they receive stimulation via glycolipids found on dendritic cell Richard S. Blumberg1, Torsten Olszak1, Dingding An1, CD1d. However, their ability to emigrate appears to be closely Joana F. Neves1, Marie Dowds1, Sungwhan Oh1, Arthur Kaser1, associated with emigration of neutrophils. Clearly each organ has its Dennis L. Kasper1, Sebastian Zeissig1 own characteristic iNKT cell localized either within the vasculature, surrounding the vasculature or in the case of lung in the vasculature 1Brigham and Women’s Hospital, Harvard Medical School, Boston, with the capacity to emigrate out upon infection. MA, USA; 2Department of Internal Medicine I, University Medical Center Schleswig–Holstein, Kiel, Germany The mechanisms by which homeostasis at mucosal surfaces is 088 maintained is of central importance to the pathogenesis of inflam- HARNESSING THE IMMUNOSTIMULATORY matory bowel disease (IBD) and intestinal inflammation in general. PROPERTIES OF INVARIANT NATURAL KILLER Critical to these processes is the intestinal epithelial cell (IEC), which T (iNKT) CELLS IN NOVEL iNKT CELL-BASED regulates the responses of immune cells to environmental factors. CD1d has been demonstrated to function in IECs based upon studies THERAPIES FOR CANCER in in vitro models. These studies have shown that IEC lines can express CD1d and present alpha-galactosylceramide to invariant Rupali Das1, Peng Guan1, Elizabeth Evans2, Felix Scheuplein3, Natural Killer T (iNKT) cells. CD1d-iNKT restricted pathways play Maurice Zauderer2, Robert Schaub3, Kim E. Nichols4 an important role in antimicrobial responses in mucosal tissues for

123 Inflamm. Res. S87 both pathogens and management of microbial commensalism. CD1d The CCR5-/- mice have a more pronounced reduction of neutrophil has also been implicated in the pathogenesis of IBD using oxazolone- migration into the infection focus, compared with WT mice. Both induced colitis which is dependent upon CD1d and iNKT cells and events (CXCR1/2 down-regulation and CCR5 expression) are due to evidence of increased NKT cell activity in the human condition activation of Toll-like receptors (TLRs). Neutrophils harvested from based upon evidence for increased production of IL-13 upon stim- TLR-2 or -4 deficient mice submitted to polymicrobial (CLP) sepsis ulation with CD1d agonists. CD1d-restricted iNKT cells are also do not present chemotactic response to CXCR2 agonists, but regulated by the microbiota. Under GF conditions, oxazolone colitis become responsive to CCR5 agonists. Although TLR activation is increased and can only be normalized if recolonization of the GF induces both events, the signaling mechanisms are different. The state occurs during the first 2 weeks of life; indicating an important CXCR1/2 internalization in neutrophils harvested form septic mice window of opportunity in immune education of the iNKT cells in or patients or from naı¨ve mice and stimulated in vitro with LPS the colon. This is due to microbial induced signals that are yet to be associates with the increase of the expression and the activity of defined which regulate CXCL16 production of the intestinal G-protein-coupled receptor kinases (GRK-2/-5). On the other hand, epithelium and bacterial derived sphingolipids from specific the CCR2 expression in the same cells depends on TNF-a produc- microorganisms such as Bacteroides fragilis which impede the tion. The expression of GRKs and down-regulation of CXCR1/2 proliferation of the infiltrating colonic iNKT cells. In addition, were prevented by pharmacological inhibition of iNOS CD1d expression on IECs is decreased in human IBD suggesting a (1400 W)/soluble guanilate cyclase (ODQ)/PKG (KT5823) pathway. protective function for CD1d in the locale consistent with CD1d In conclusion, these results, by a side, emphasize the harmful role of cross-linking studies on IEC lines which induces IL-10 production. TLRs/iNOS/NO-GC-cGMP-PKG pathway on down-regulation of The production of IL-10 is due to a novel pathway of IEC-depen- CXCR1/2 receptors expression in neutrophils via induction of GRKs dent regulation of mucosal homeostasis that critically depends upon expression, and by other side, highlight a compensatory pathway epithelial cell expression of microsomal triglyceride transfer protein (TLRs/TNF-a production) in an attempt to reestablish the neutrophil (MTP), which we have previously shown is responsible for CD1d migration. lipidation in the secretory pathway, CD1d itself and production of Financial support CNPq, FAPESP and Timer-EC. HSP110 and IL-10 in a pathway that depends upon STAT3 sig- naling. Further, our studies implicate the hematopoietic system as the source of CD1d-mediated intestinal inflammation when acti- 091 vated. These studies reveal a critical role for CD1d-iNKT cells in SPECIALIZED PRO-RESOLVING MEDIATORS IN regulating the IEC-microbial interface. LUNG INFLAMMATION AND INJURY

Bruce D. Levy1,2 SYMPOSIUM 28: INFECTION AND INFLAMMATION – SPONSORED BY BIS- 1Brigham and Women’s Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA BRAZIL Acute lung inflammation is fundamentally important to host defense, but chronic or excessive inflammation can lead to several 090 important diseases. The resolution of inflammation is an active MECHANISMS INVOLVED IN THE IMMUNE- process that is directed, in part, by specialized pro-resolving DYSFUNCTIONS INDUCED BY SEVERE SEPSIS mediators that are enzymatically derived from polyunsaturated fatty acids. In health, cell–cell interactions at the onset of acute inflam- mation establish biosynthetic circuits for these pro-resolving Fernando Q. Cunha mediators, including the omega-3 fatty acid-derived resolvins, pro- tectins and maresins, which serve as agonists to orchestrate a return School of Medicine Ribeirao Preto, University of Sao Paulo, of the inflamed tissue to homeostasis. Understanding the cellular Sao Paulo, Brazil and molecular mechanisms for pro-resolving mediators in catabasis Mechanisms involved in the immune-dysfunctions induced by severe is providing new insights into lung tissue responses for resolution of sepsis FERNANDO Q. CUNHA, Department of Pharmacology of inflammation in health and the pathophysiology of disease, includ- School of Medicine of Ribeira˜o Preto, USP ing the host response to infections; as well as opportunities for Clearance of pathogens during infections depends on efficient therapeutic intervention. neutrophil migration. Using animal models and neutrophil from E-series and D-series resolvins are enzymatically derived from the patients, we are showing a defect in neutrophil recruitment into essential omega-3 fatty acids eicosapentaenoic acid and docosahex- infectious focus during severe sepsis, which is followed by a aenoic acid, respectively. Protectin D1 and maresin 1 (MaR1) are also reduced bacterial clearance. In fact, neutrophil from septic animals derived from DHA. Relevant to lung inflammation, lipoxin A4 or patients present reduced chemotactic activity to CXCR1/2 ago- (LXA4), Resolvin E1 (RvE1), resolvin D1 (RvD1) and protectin D1 nists. This impairment of neutrophil migration associated with the (PD1) are generated in murine lung. LXA4 and PD1 are generated in down-regulation of CXCR1/2 in this cell type. Investigating the human lung. Receptors for LXA4 and RvD1 (ALX/FPR2) and for mechanism involved in the neutrophil migration to infection focus RvE1 (CMKLR1) are expressed in lung and are dynamically regu- during sepsis, we are demonstrating that in parallel with CXCR1/2 lated with lung inflammation. Evidence will be presented for cellular internalization, the CCR5 is expressed in the neutrophil surface. It and molecular mechanisms for representative pro-resolving mediators seems that the CCR5 expression is a compensatory mechanism in an in their protective actions in the regulation of airway inflammation attempt to reestablish the neutrophil migration to infection focus. during innate and adaptive immune responses.

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092 deal with infection. In the context of Influenza A/WSN/33 H1N1 CHIKUNGUNYA VIRUS-INDUCED infection, there is much more neutrophil influx and damage in the lungs of PI3Kc-deficient mice. This is secondary to the decreased INFLAMMATORY DISEASE transmigration of T CD8+ lymphocytes, NK cells and resolving macrophages to the airways of PI3Kc deficient mice. Type I IFN Suresh Mahalingam responses induced by flu infection were abolished in lungs of KO mice leading to increased viral loads. Therefore, PI3Kc activation Griffith University, Nathan, QLD, Australia during Influenza A infection is necessary for an early antiviral Chikungunya virus (CHIKV)-induced inflammatory disease is a response that promotes recruitment of effector cells against the significant cause of human morbidity worldwide. CHIKV has caused infected cells and contributes to viral clearance. Therefore, our explosive outbreaks in Africa, Indian Ocean island territories, Asia, studies suggest that the role of PI3Kc during sterile inflammation Europe and now the Americas. The recent CHIKV outbreak in the and infection is context-dependent. Overall, inhibition or absence of Caribbean and subsequent spread within the Americas highlights the PI3Kc tend to decrease sterile inflammation by preventing excessive potential for a pandemic in neighboring North and South American leukocyte activation and tissue damage. During infection, these countries. The total number of cases in the Americas has now strategies tend to decrease infections with a strong systemic com- approached more than 1.2 million cases. There is a clear need to ponent but may affect negatively the capacity of the host to deal improve our understanding of the pathogenesis of this disease. In with focal infection, as in the model of pulmonary infection with recent years my laboratory has unravelled the mechanisms of how influenza A. The concept of anti-inflammatory drugs for infectious CHIKV cause musculoskeletal disease (arthritis, arthralgia, myosi- disease is a valid principle but needs to be tested in individual tis). We have carried out extensive work in understanding the infections to avoid unwanted increase in infection load and tissue pathogenesis of CHIKV disease and identifying new mechanisms, damage. which has led to the identification of new candidate therapies for viral arthritides. These mechanisms and recent human studies will be discussed. SYMPOSIUM 29: BIOMARKERS IN INFLAMMATION: FOCUS ON RHEUMATOID ARTHRITIS 093 MECHANISMS OF DISEASE AND PROTECTION 094 DURING VIRAL INFECTIONS TOWARDS A MOLECULAR TAXONOMY IN Mauro M. Teixeira1, Luciana Padua1, Cristiana C. Garcia2, RHEUMATOID ARTHRITIS: CAN WE DERIVE Daniele G. Souza1 CLINICALLY USEFUL PROGNOSTICS AND THERAPEUTIC PREDICTORS? 1Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2Instituto de Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil Iain B. McInnes The inflammatory response that accompanies infectious diseases is central for the host ability to contain the growth of the pathogen and University of Glasgow, Glasgow, UK prime an adaptive immune response. However, excessive, misplaced Rheumatoid arthritis (RA) treatment has been transformed by the or altered inflammatory responses may cause disease and even death advent of biologic therapeutics and by the application of formal of the host. It is possible that drugs which prevent the unwanted strategies to align and enhance the use of such therapeutics. Current inflammatory response to infection may be useful as co-adjuvant unmet clinical needs include the predominance of partial responses to treatment for infectious disease. We will discuss the potential effects most monotherapies, and the relatively rare occurrence of remission of inhibitors of PI3Kc in models of sterile inflammation and in the context of general treatment. Much work is ongoing to eluci- infection. PI3Ks are central signaling enzymes, involved in cell date mechanisms that underlie the emergence of autoimmunity in RA growth, proliferation, survival and migration. Class IB PI3K or and factors that mediate transition to, and maintain chronicity of PI3Kc is mainly expressed on leukocytes and activated by GPCR synovial inflammation thereafter, representing failure of homeostatic coupling, and involved in cell migration under inflammatory con- inflammation resolution. Complex interplay exists between ‘profes- ditions. Blockade or absence of PI3Kc is in general associated with sional’ immune cells and stromal cellular pathways that together decreased neutrophil activation but only partial inhibition of the contrive to perpetuate, or resolve articular inflammation. In this pre- influx of these cells in vivo. Decrease of the accumulation of neu- sentation I shall discuss the recent advances made in defining the trophils occurs only when PI3kc and PI3Kdelta are inhibited biosignatures that are proposed to predict natural history and thera- together. In the context of sepsis, absence or inhibition of PI3Kc is peutic response characteristics in RA. Unresolved issues surround the accompanied by an unexpected enhancement of neutrophil influx appropriate modality to define a molecular profile e.g. transcrip- and consequent greater ability to deal with the septic insult. This is tomics, proteomics and metabolomics, and even the utility of secondary to the decreased activation of the systemic activation of peripheral blood derived signals versus those form synovial tissues neutrophils. In the context of dengue infection, absence of PI3Kc obtained at biopsy. decreases systemic disease without altering the ability of the host to

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095 3. Explain how and why 14-3-3g serves as a personalized medicine 14-3-3 g: A JOINT-DERIVED MARKER WITH target. AUTOIMMUNE AND PERSONALIZED MEDICINE APPLICATIONS 096 Anthony Marotta VECTRA DA, A MULTI-BIOMARKER DISEASE Augurex Life Sciences Corp, Vancouver, BC, Canada ACTIVITY BLOOD TEST THAT PREDICTS RISK FOR JOINT DAMAGE IN PATIENTS WITH Background: RA is a chronic autoimmune disease that if left untreated results in severe joint destruction. Comorbid joint mani- RHEUMATOID ARTHRITIS festation occur in approximately 15–20 % of patients with other autoimmune conditions, such as SLE, psoriasis and inflammatory Eric H. Sasso bowel disease. It is estimated that 30–50 % of RA patients end up on workplace disability within 5 years of diagnosis. Early identification Crescendo Bioscience, South San Francisco, CA, USA of RA, assessment of disease severity at presentation, together with a Rheumatoid arthritis (RA) is a chronic disabling disease that prompt and effective treatment strategy can significantly improve requires ongoing monitoring of disease activity and adjustment of clinical outcomes and reduce the personal and economic burden of therapy to prevent irreversible damage to joints. The Vectra DA this disease. blood test measures 12 serum protein biomarkers and uses a vali- Need for New Markers: To enable this, new markers are needed to (1) dated algorithm to provide a score, on a scale of 1–100, that improve the diagnostic capture rate of early RA; (2) inform joint represents the level of RA disease activity. The Vectra DA score damage prognosis to optimize treat-to-target strategies; and (3) monitor was developed to correlate with DAS28-CRP, a conventional, the disease process along the uncoupling of inflammation and joint clinically based composite tool for assessing RA disease activity. damage aligning clinical response measures with damage progression Changes in Vectra DA score correlate with changes in DAS28-CRP. risk. The goal of personalized medicine for RA is an ambitious but In addition, the Vectra DA score has been shown to be associated worthy one because of the heterogeneity of disease amongst patients with risk for new joint damage, or radiographic progression, and is a and within individuals along the disease course. In that regard, if a better predictor of joint damage risk than clinically based measures biomarker target also serves as a drug target, companion biomarker or the CRP blood test. As such, Vectra DA can detect potentially levels can directly guide the selection of likely responders and allow for damaging disease activity in patients with low levels of disease tailored dosing and administration schedules. according to conventional measures and, conversely, it can identify 14-3-3g Markers and Personalized Medicine Potential: 14-3-3 patients with limited risk for progression despite a clinical appear- proteins are an evolutionary conserved family of molecular chap- ance of active disease. This presentation will review studies erones that play a critical role in the regulation of intracellular demonstrating that, as a predictor of joint damage risk, Vectra DA functions. In RA, externalization of 14-3-3g is believed to couple measures pathologically meaningful disease activity in patients with with the release of lipid microvesicles or exosomes in a similar RA and can supplement conventional assessment tools for thera- manner to other endokines. In vitro studies demonstrate that stim- peutic decision-making. ulation of cells with 14-3-3g lead to the induction of inflammatory transcripts, such as TNFa, and factors linked to the joint damage process, such as receptor activation of nuclear factor jB ligand (RANKL) and MMP-1. Since 14-3-3g is not normally present 097 extracellularly, the immune system sees soluble 14-3-3g as foreign MRI BIOMARKERS FOR DISEASE ACTIVITY AND resulting in a specific auto-antibody response. Serum 14-3-3g and its auto-antibodies together identify more than 90 % of early RA STRUCTURAL DAMAGE IN RHEUMATOID patients. Those who are 14-3-3g protein positive have worse dis- ARTHRITIS ease outcomes over 5 years, and in those patients who achieve DAS remission, a positive 14-3-3g test predicts a higher risk of joint Charles Peterfy damage progression, despite a good clinical response. 14-3-3g mechanistic findings converge strongly with in vivo data, wherein Spire Sciences, Inc, Boca Raton, FL, USA treatment of CIA mice with anti-14-3-3g monoclonal antibodies delays the disease onset and reduces its severity, providing a The past two decades have seen unprecedented advances in therapy for rational approach to personalized medicine with this novel factor. rheumatoid arthritis (RA), with several effective biologics introduced since 1998. Imaging played a central role in validating these novel Learning Objectives treatments; however, a number of factors have recently made using 1. List the unmet needs for biomarkers based on RA clinical radiography in clinical trials increasingly impractical if not infeasible. management objectives. Ironically, the most important of these has been the success of the 2. Describe 14-3-3g’s mechanistic role in RA pathogenesis and how biologics revolution itself, as the availability of effective therapy has its serological measurements assist with patient management. made extended placebo control unethical. Current guidelines call for

123 S90 Inflamm. Res. withdrawal or rescue therapy for non-responders within only MINI-SYMPOSIUM 3: MECHANISMS 12–14 weeks. Unfortunately, traditional radiographic endpoints, such as Sharp scoring, are unable to discriminate treatment effects that DRIVING MUCOSAL INFLAMMATION IN quickly, even with large numbers of patients per arm. Magnetic res- THE GUT onance imaging (MRI) has been shown to resolve structural joint damage more sensitively than radiography can, while also assessing the upstream inflammatory drivers of erosions and cartilage loss, B031 namely synovitis and osteitis. An increasing number of randomized MULTIPLE MESENTERIC LYMPHATIC clinical trials using MRI markers of these RA features have demon- ANOMALIES IN MURINE CROHN’S DISEASE strated that MRI can discriminate progression and treatment effect with respect to structural damage in only 12 weeks and with respect to See poster section for abstract. inflammation in as little as 2 weeks, using fewer than 50 patients per arm. Further, composite clinical measures of disease activity, such as DAS28, SDAI and CDAI are responsive to treatment, but often fail to B028 identify patients who continue to progress structurally despite satis- fying clinical criteria for remission. MRI can thus serve as a useful ENHANCEMENTS TO THE PREDICTABILITY adjunct to clinical measures in treat-to-target patient management as AND CLINICAL RELEVANCE OF TWO MURINE well. This presentation will review the performance of MRI as an MODELS OF CHRONIC INFLAMMATION: imaging biomarker for assessing inflammation and structural damage ADOPTIVE TRANSFER-INDUCED COLITIS AND in patients with RA from the perspectives of validity, discrimination and feasibility, and provide recommendations for its use. GVHD

See poster section for abstract.

SYMPOSIUM 30: SLE: CURRENT B215 THERAPEUTIC LANDSCAPE AND CRITICAL ROLE OF PLASMACYTOID FUTURE PROMISES DENDRITIC CELLS IN IMMUNOREGULATION INITIATED BY A COMMENSAL MICROBIAL 098 POLYSACCHARIDE MODULATION OF PLASMACYTOID DENDRITIC CELLS FOR TREATING AUTOIMMUNE DISEASES See poster section for abstract. LIKE LUPUS B013 Jo Viney SUPPRESSING INFLAMMATORY BOWEL Biogen, Cambridge, MA, USA DISEASE BY MODULATING PRO- Systemic lupus erythematosus (SLE) is a debilitating autoimmune INFLAMMATORY GENE EXPRESSION AND disease with potentially devastating consequences. Gene expression IMMUNE CELL POPULATIONS studies have revealed characteristic Type I interferon (IFN-I) signa- tures in SLE. The immune complexes that are prevalent in SLE bind See poster section for abstract. to FcgammaRIIa on plasmacytoid dendritic cells (pDC), driving the production of IFN-I (as well as other proinflammatory cytokines and chemokines). B020 Plasmacytoid DC exclusively express BDCA2, a receptor that AMELIORATION OF CHRONIC COLITIS IN MICE functions to inhibit the production of IFN-I. We have developed 24F4A, a humanized monoclonal antibody that binds to human and BY GENETIC DELETION OF THE IL-13 DECOY cynomolgus monkey BDCA2 with similar potency. In vitro human RECEPTOR whole blood and PBMC assays demonstrate the ability of 24F4A to potently inhibit IFN-I production and other proinflammatory medi- See poster section for abstract. ators following stimulation with TLR agonists or SLE-derived immune complexes. The inhibition of IFN-I is comparable in healthy and SLE donor assays. In vivo studies in cynomolgus B252 monkeys established a PK/PD correlation with strong concordance between drug levels, BDCA2 receptor internalization, and inhibition FILGOTINIB, JAK1-SELECTIVE INHIBITOR, of IFN-I production. REPRESSES SIMILARLY JAK1/STAT3 PATHWAY Mechanism of action studies have revealed the inhibitory effect of IN THE COLON OF MICE WITH DSS-INDUCED 24F4A on immune-complex-induced IFN-I production by pDC is COLITIS AND IN CULTURES OF COLON mediated via more than one mechanism. This presentation will highlight the novel therapeutic potential of an effector competent anti- BIOPSIES FROM INFLAMMATORY BOWEL BDCA2 mAb that has a dual mechanism to dampen pDC responses in DISEASE PATIENTS SLE and other autoimmune diseases. The author is an employee of Biogen. See poster section for abstract.

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SYMPOSIUM 31: NEW VISIONS IN SYMPOSIUM 32: TREGS IN HUMAN OCULAR THERAPY AUTOIMMUNITY

099 100 TOPICAL DELIVERY OF NOVEL MUCUS- ARE miRNA RESPONSIBLE FOR THE TREG PENETRATING RTKIS FOR SUCCESSFUL DEFECTS OBSERVED IN MULTIPLE SCLEROSIS? ANGIOGENESIS BLOCKADE Amy Lovett-Racke, Mary Severin, Mireia Guerau-de-Arellano, Lisa R. Schopf2, Elizabeth M. Enlow2, Pawel Nowak2, Winston Ong2, Michael Racke Jim Bourassa2, Kim Brazzell2, Hongming Chen2 Ohio State University, Columbus, OH, USA 1 Topical Delivery of Novel Mucus-penetrating RTKis for Successful Multiple sclerosis (MS) is an immune-mediated demyelinating dis- 2 Angiogenesis Blockade; Kala Pharmaceuticals, Waltham, USA ease of the central nervous system with an unknown etiology. Inflammation leading to neovascularization at the back of the eye is a Several studies have demonstrated a defect in CD4 regulatory T key contributor to vision loss in diseases such as age-related macular cells (Tregs) in MS patients but the cause of this defect lacks degeneration (AMD). Vascular endothelial growth factor (VEGF) explanation. We performed a comprehensive miRNA profiling study ligand blockade is a clinically proven treatment for AMD currently on CD4 T cells of untreated MS patients and controls. Numerous achieved through repeated intravitreal (IVT) injections of a biologic miRNA were differentially expressed in subpopulations of CD4 T agent. A significant challenge in ophthalmology continues to be cells of MS patients. Notably, naı¨ve CD4 T cells had altered delivering sufficient levels of therapeutic agent to the back of the eye expression of several miRNAs that were predicted to target com- by non-invasive means. The objective of this work was to develop a ponents of the TGFbeta, a pathway critical to the development and novel RTKi that selectively blocks the VEGF pathway and can be function of Tregs. Analysis of expression levels of various compo- delivered topically. While nanoparticles may have the potential to nents of the TGFbeta signaling pathway found that TGFbeta improve ocular tissue exposure from topical administration, this receptor 1 and Smad4 expression was reduced in CD4 T cells of MS potential has been undermined by the adhesive nature of the ocular patients. When these miRNAs were over-expressed in naı¨ve T cells mucus layer, which serves to protect the eye from allergens, patho- from control subjects, Treg development was impaired. Adminis- gens, and debris by effectively trapping and rapidly clearing foreign tration of these miRNAs to neonatal mice at the time when Treg particles from the ocular surface. The mucus-penetrating particle development is peaking in the thymus resulted in suppressed Treg (MPP) technology is a novel drug delivery platform that can be used development. Furthermore, adult-onset EAE was exacerbated in to design nanoparticles that effectively penetrate mucus secretions. mice which receive these miRNAs during early life. This study MPPs of certain compounds tested in the vaginal tract, lung, and suggests that miRNA dysregulation in naı¨ve CD4 T cells may gastrointestinal tracts of animals showed even distribution, and pro- underlie the Treg deficit observed in MS patients and may be a risk vided prolonged duration on these mucosal surfaces. Recently we factor for the development of MS. have demonstrated that MPPs of certain compounds can provide a prolonged residence time at the ocular surface, which in turn trans- lated into enhanced drug delivery to ocular tissues, superior 101 pharmacokinetics (PK) and improved efficacy in animals. We have designed a next generation RTKi with nanomolar DISENTANGLING THE ROLES OF IL-2 IN potency against VEGFR (KDR) and improved selectively over IMMUNE ACTIVATION AND SUPPRESSION undesirable targets such as EGFR, FGFR, CDK and RET. Formulated as an MPP the inhibitor has the ability to penetrate mucus, residing Jason D. Fontenot longer at the ocular surface in animals and allowing for biologically active concentrations to be delivered to the back of the eye. Tissue Biogen, Cambridge, MA, USA distribution studies in rabbits and mini-pigs revealed retinal and choroid levels well above the cellular IC50 and higher compared to IL-2 is a key nodal regulator of immune activation and suppression. systemic compartments (i.e., plasma, heart, kidney and brain). In a There is significant interest in therapeutic modulation of the IL-2 rabbit model of vascular leakage, the topically applied RTKi was pathway to potentiate cancer immunotherapy, facilitate transplant statistically equivalent to treatment with AvastinÒ. tolerance and treat autoimmune and inflammatory diseases. Given the In summary, there is a clear unmet medical need for non-invasive, ability of IL-2 to both promote effector T cell responses and limit topical drug delivery to both the anterior and especially to the pos- immune activation through maintenance of regulatory T cells, terior segment of the eye. We have demonstrated here that in animal extrapolating known effects on cell-type specific biologies to an models, an RTKi formulated as an MPP provided enhanced drug aggregate therapeutic impact is difficult to predict, but critical to penetration not only to the ocular surface but also to the posterior understand. Focusing on the role of CD25, the obligate component of segment. These data support the potential for a non-invasive therapy the high affinity IL-2 receptor, we examine the impact of targeting IL- for VEGF pathway blockade which would be a breakthrough in the 2 biology during immune homeostasis and in the context of autoim- treatment of neovascular ophthalmic diseases. mune pathology.

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102 proinflammatory mediators and elevated body temperature were not IN VIVO MAINTENANCE OF HUMAN induced with Fc.muteins. Unexpectedly, certain Fc.muteins were more effective than Fc.WT or Proleukin at increasing Treg frequency REGULATORY T CELLS DURING CD25 and upregulating FOXP3. This property was found to correlate with BLOCKADE an ability to stably associate with cell surface CD25 and stimulate low levels of IL-2R signaling for extended periods of time. Thus, optimal David J. Huss1, Devangi S. Mehta1, Akanksha Sharma1, Treg-selectivity resulted from a combination of reduced activation of Xiaojun You1, Katherine A. Riester1, James P. Sheridan2, effector cells and better agonism of Treg. Our results demonstrate that Lakshmi S. Amaravadi1, Jacob S. Elkins1, Jason D. Fontenot1 a high degree of Treg-selectivity can be achieved through subtle changes in IL-2/IL-2R interactions. 1Biogen, Cambridge, MA, USA; 2AbbVie Biotherapeutics, Redwood City, CA, USA Regulatory T (Treg) cells mediate immune tolerance to self and SYMPOSIUM 33: GASEOUS MEDIATORS depend on IL-2 for homeostasis. Treg deficiency, dysfunction and AS THE BASIS FOR NOVEL ANTI- instability are implicated in the pathogenesis of numerous autoimmune diseases, including relapsing-remitting multiple sclerosis (RRMS). INFLAMMATORY DRUGS—SPONSORED Daclizumab high yield process (DAC HYP) is a humanized mono- BY IRN-CANADA clonal antibody that binds the IL-2 receptor alpha subunit (IL2Ra or CD25) and prevents IL-2 binding. DAC HYP has demonstrated clin- 104 ical efficacy in patients with RRMS, despite causing a reduction in circulating Treg cell numbers. Here we investigate the impact of DAC IT’S JUST THE BEGINNING: NO, CO, H2S AND HYP-mediated CD25 blockade on Treg cell homeostasis in RRMS OTHER GASOTRANSMITTERS patients. Based on analysis of a large clinical sample set, we report that DAC HYP treatment caused an *50 % decrease in Treg cells by week Rui Wang 8 of treatment that was sustained over a 52-week period. Remaining Treg cells retained a demethylated TSDRin the FOXP3 promoter, Laurentian University, Sudbury, Ontario, Canada maintained active cell cycling and had minimal production of IL-2, IFN-gamma, and IL-17. In the presence of DAC HYP, IL-2 serum The revolution in our understanding of the cellular signaling mechanisms concentrations increased and CD25-independent intermediate affinity started from the discovery of nitric oxide (NO) as a novel signaling IL-2R-signaling induced STAT5 phosphorylation and sustained molecule of gas. Distinctive from conventional neurotransmitters, NO FoxP3 expression. Our results demonstrate that Treg cell phenotype transmits and amplifies biological signals intracellularly and intercellu- and lineage stability can be maintained in the face of CD25 blockade. larly without the requirement and restrictions of the existence of secretory vesicles, specific membrane receptors, or intracellular second messen- gers. Not alone in utilizing this unique signal transduction strategy, NO has been joined by carbon monoxide (CO) and hydrogen sulfide (H2S) in 103 the family of gasotransmitters. Being endogenously generated molecules MODULATING IL-2/IL-2R INTERACTIONS TO of gas, gasotransmitters are crucially important for the regulation of a THERAPEUTICALLY SHIFT THE BALANCE wide spectrum of cellular and molecular events. Altered bioavailability of gasotransmitters and their abnormal interactions with the target BETWEEN REGULATORY T CELLS AND molecules have direct correlation with numerous diseases. Among the EFFECTOR LYMPHOCYTES mostly studied molecular mechanisms for biological effects of gaso- transmitters are the post-translational modifications of proteins. While 2,1 1 1 1 Marc A. Gavin ,LiLi, Dina Alcorn , Margaret Karow , NO can induce S-nitrosylation of a protein to decrease its function, H2S- Jacqueline Kirchner1, Kevin Gorski1, David Martin1, Katsu Ishida1, induced protein S-sulfhydration usually increases the protein function. Guna Kannan1, Joshua Pearson1 Gasotransmitters can act on the same molecular target with different outcomes. They can also act on different molecular targets to achieve the 1Amgen, Inc., Thousand Oaks, CA, USA; 2Benaroya Research same functional outcome. The interactions among gasotransmitters may Institute, Seattle, WA, USA potentiate or cancel each other’s function. If two’s company and three’s a crowd (Wang, FASEB J, 16: 1792–1798, 2002), what is four? It is IL-2-based therapeutics that enhance CD4+ regulatory T cells (Treg) ammonium (NH3) (Wang, Trends Biochem Sci, 39 (5):227–232, 2014). represent a promising new modality for the treatment of inflammatory Against all criteria for gasotransmitters, ammonia certainly is qualified disease. IL-2-mediated Treg enrichment inhibits disease progression and its biological importance has been gaining more recognition. Gaso- in multiple mouse models of autoimmunity, and in initial clinical transmitter research has flourished in recent decades but it is still just the studies, low dose IL-2 (Proleukin) enriches Treg and alleviates beginning of the revolution. (Supported by Canadian Institutes of Health symptoms in patients suffering from various inflammatory conditions. Research). One concern with this approach, however, is that efficacy will be compromised in certain diseases or patients due to IL-2 activity on autoreactive T cells, NK cells, or innate lymphoid cells, or that IL-2- mediated lymphocyte activation will exacerbate pathology. To 105 explore ways to increase Treg-selectivity, we have generated human HYDROGEN SULFIDE-BASED ANTI- IL-2 muteins with reduced potency and increased dependence on high INFLAMMATORY THERAPEUTICS IL-2Ra/CD25 expression. In humanized mice and cynomolgus monkeys, Fc-fused IL-2 muteins (Fc.muteins) were highly effective at stimulating Treg growth, but were poor agonists of conventional T John L. Wallace cells and NK cells relative to Fc-fused wild-type IL-2 (Fc.WT) or Proleukin. Furthermore, unlike with Fc.WT or Proleukin, University of Calgary, Calgary, AB, Canada

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Hydrogen sulfide is a gaseous mediator that is produced throughout SYMPOSIUM 34: THERAPEUTIC the body, as well as by some bacteria residing in the gastroin- PATHWAYS IN INFLAMMATION: NEW testinal tract. In recent years, it has become clear that H2S mediates many physiological processes, including regulation of FACES OF OLD FRIENDS—SPONSORED blood flow, adherence of leukocytes to the vascular endothelium, BY RIS mitochondrial respiration, wound healing and regulation of blood pressure. The ability of H2S to increase the resistance of the gastrointestinal (GI) tract to injury, to accelerate the healing of pre- 107 existing damage, and to reduce inflammation, led us to design a THE NEW METHOD OF EVALUATION series of drugs that release H2S. Specifically, we focused on OF ß-ADRENORECEPTOR’S ACTIVITY existing anti-inflammatory drugs such as nonsteroidal anti-inflam- matory drugs (NSAIDs) and mesalamine (for treatment of 1 1 2 inflammatory bowel disease). Kirill A. Zykov , Olga Y. Agapova , Yuri S. Skoblov , Valery P. Masenko3, Irina E. Chazova3 Several H2S-releasing NSAIDs were developed and tested in a range of animals models. One of these, ATB-346, is a derivative of 1 naproxen, one of the most commonly used NSAIDs for treatment of Laboratory of Pulmonology, Moscow State University of Medicine and Dentistry named after A.I. Evdokimov, Moscow, Russian arthritis. In animal studies, ATB-346 exhibited comparable anti-in- 2 flammatory activity to the parent drug, but produced markedly less GI Federation; Laboratory of Isotope Analysis, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS (IBCh RAS), Moscow, Russian damage. Even when given to animals with severely compromised 3 mucosal defence, ATB-346 was found to be safe. Indeed, this com- Federation; Department of Arterial Hypertension, Russian pound significantly accelerated healing of pre-existing ulcers in the Cardiology Research And Production Complex, Moscow, Moscow, GI tract. In human trials, ATB-346 exhibited enhanced anti-inflam- Russian Federation matory activity as compared to the parent drug, presumably Body: ß-adrenoreceptor active drugs are often used in therapy of attributable to the anti-inflammatory effects of the H2S-releasing bronchoobstructive and cardiovascular diseases. These drugs can moiety, but also likely due to altered pharmacokinetics (including a change the expression and affinity of ß-receptors. Receptor’s affinity much longer half-life than that of naproxen). Clinical trials of this and expression can be assessed by a radioligand method on lym- compound are ongoing. phocytes and extrapolated to bronchial smooth muscle, but this H2S is a promising candidate for enhancing the activity of existing method is not suitable for clinic because it needs more than 20 million drugs and for a wide range of indications. cells (approximately 100 mL of blood). Objective: To evaluate the change of ß-adrenoreceptors relative activity in patients treated with ß-agonists using a new optimized radioligand method. 106 Methods: Evaluation of relative activity of ß-adrenoreceptor on SWITCHING ‘‘OFF’’ CHRONIC NEUROPATHIC T-lymphocytes was performed using an optimized method based on PAIN BY SWITCHING ‘‘ON’’ THE A3 ADENOSINE the radioligand assay and detection of the change of the amount of 125I-cyanopindolol in presence of specific ß2 ligand. We defined the RECEPTOR SUBTYPE (A3AR) relative receptor activity as the availability of the receptor on the cell surface for specific ligand under the given conditions. Optimal con- Daniela Salvemini ditions for the specific binding to assess ß2-adrenergic 0.5 fmol per 1 million T-lymphocytes using (125I)-cyanopindolol were found. Saint Louis University School of Medicine, St. Louis, MO, USA Healthy volunteers and patients with bronchial asthma (BA) and arterial hypertension (AH) were enrolled. Patients with BA had Chronic pain is a global burden that promotes disability and monotherapy with inhaled corticosteroids. unnecessary suffering. To date, efficacious treatment of chronic pain Results: 6 healthy persons aged 35.0 ± 7.8 initially had specific ß2- has not been achieved. Thus, new therapeutic targets are needed. receptor binding 4.2 ± 2.3 fmol/109; after 30 min of inhalation of a Our studies demonstrate that increasing endogenous adenosine short-ß2-agonist specific ß2-receptor binding decreased (3.6 ± 2.3). 3 levels through selective adenosine kinase inhibition produces pow- patients examined after 2 h. Specific ß2-receptor binding returns to erful analgesic effects in rodent models of experimental neuropathic the initial level (3.5 ± 1.6; 3.0 ± 1.9; 3.3 ± 1.7 fmol/109). pain through the A adenosine receptor (A AR) signalling pathway. 3 3 Five patients with AH and BA aged 59.6 ± 11.7 initially had Similar results were obtained by the administration of novel and specific ß2-receptor binding 2.5 ± 2.2, than 4.0 ± 2.1 and 1.9 ± 1.3 highly selective A AR agonists. These effects were prevented by 3 fmol/109 after 2 h. blockade of spinal and supraspinal A AR, lost in A AR knock-out 3 3 Conclusion: This method allows to assess relative activity of T-cells’ mice, and independent of opioid and endocannabinoid mechanisms. ß-adrenoreceptors. Different type of reaction of specific ß2- A AR activation also relieved non-evoked spontaneous pain beha- 3 adrenoreceptor binding were detected in healthy volunteers and viours without promoting analgesic tolerance or inherent reward. patients with BA and AH. Further research is needed to establish Further examination revealed that A AR activation reduced spinal 3 clinical relevance of different dynamics of relative activity of ß2- cord pain processing by decreasing the excitability of spinal wide adrenoreceptors of blood cells. dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and nora- drenergic bulbospinal circuits. Critically, engaging the A3AR 108 mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent CELLULAR SOURCES OF PROINFLAMMATORY neuropathic pain states. These studies reveal A3AR activation by CYTOKINES IN DISEASE AND CELL TYPE- adenosine as an endogenous anti-nociceptive pathway and support RESTRICTED CYTOKINE TARGETING the development of A3AR agonists as novel therapeutics to treat chronic pain. Sergei A. Nedospasov1,2,3

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1Engelhardt Institute of Molecular Biology, and Lomonosov Moscow (2.5 ± 0.55 vs. 2.0 ± 0.43, p = 0,036). Only 0.2–6.4 % platelets State University, Moscow, Russia; 2Lobachevsky University, Nizhni were CAR positive in healthy subjects. CAR was redistributed among Novgorod, Russia; 3German Rheumatism Research Center, a Leibniz all the platelet membrane. In DCMi pts the number of CAR positive Institute, Berlin, Germany platelets varied from 1 to 57 % with mean level of 8.0 percent that was significantly higher than in HS. In all DCMi patients CAR was Proinflammatory cytokines TNF, IL-6 and IL-1 may contribute to localized at the cites of intercellular communications in the small pathogenesis of autoimmune diseases and therefore systemic inhibi- platelet aggregates. The level of CAR expression correlated with tion of these cytokines became an essential part of the therapy. Our increased level of IL6 and TNF alpha (r = 0.51, 0.48). In pts spon- recent studies in experimental arthritis suggest that TNF from one taneous aggregation was 3.7 ± 1.8 % at the first 5 min, but to the particular cellular source may play an anti-inflammatory role. If so, 9–14 min its level grew up to 37 ± 17.3 % similar to 1.0-ADP systemic cytokine inhibition may be like a double-edged sword dis- induced aggregation as a result of platelet release reaction that was rupting both pathogenic and protective signaling. never observed in HS (p = 0.0001). In whole blood pts were detected Based on these findings we are developing an approach to cell circulating platelet microaggregates leukocyte-platelet and erythro- type-restricted cytokine neutralization by utilizing bispecific anti- cyte-platelet aggregates that are typical for inflammation bodies that would attach to the cell surface of a particular type of Summary: The level of CAR expression on cardiomyocytes is related immune cells, and capture the cytokine released by these cells, pre- to increased level of IL6. There is no relation between CAR venting its dissemination. Our constructs are based on single domain expression on cardiomyocyte and platelets in DCMi pts. Increased antibodies (VHH) specific for human or mouse TNF, human IL6 and CAR expression on platelets of pts is associated with increased level for cell type-specific markers, in particular, for F4/80 surface mole- of IL6 and TNF alpha. The high level of CAR expression, its cule expressed on macrophages. We find that such antibodies can appearing in the sites of intracellular connection and relation to effectively attach to the cell surface, capture and retain released increased platelet aggregation may indicate the role of this receptor in cytokines. Using mice humanized for the TNF system and with platelets changes during inflammation. macrophages isolated from such mice we assessed activity of these Financial support We acknowledge for financial support Rfbr: grant constructs in vitro and in vivo. Our findings may serve as a basis for 14-04-01754 bioengineering of new type of cytokine inhibitors. Supported by Russian Scientific Fund grant 14-25-00160 (for IL-6 related studies) and Russian Ministry of Science and Education grant 14.Z50.31.0008. B117 P38DELTA MAPK: A NOVEL EFFECTOR IN NLRP3 INFLAMMASOME ACTIVATION 109 UPREGULATED IN HUMAN CORONARY COXSACKIE: ADENOVIRUS RECEPTOR ATHEROSCLEROTIC LESIONS EXPRESSION ON CARDIOMYCYTES AND PLATELETS IN PATIENTS WITH See poster section for abstract. INFLAMMATORY CARDIAC PATHOLOGY

Elena M. Gupalo, Liudmila I. Buryachkovskaya, Irina A. Uchitel, B125 Peter V. Chumachenko, Natalia A. Mironova CONTROL OF LUNG CANCER WITH ASPIRIN- Russian Cardiology Research Complex, Moscow, Russian Federation TRIGGERED STIMULATION OF RESOLUTION Background: Coxsackie-adenovirus receptor is a protein of tight See poster section for abstract. junction, which persists of the different cell types. Recently it was shown that CAR can be expressed on the platelets surface, the cells which are known to play a crucial role in inflammation processes and which can directly reflect systemic inflammation. It remains unknown SYMPOSIUM 35: CLINICAL how CAR distributed in heart tissue and peripheral blood platelets in DEVELOPMENTS IN FIBROSIS patient with inflammatory cardiac pathology Methods: There were included 23 patients with inflammatory dilated cardiomyopathy (DCMi) and 12 healthy subjects (HS). Diagnosis of 110 DCMi was confirmed according endomyocardial biopsy (EMB) data. TARGETING IL13 IN IDIOPATHIC PULMONARY CAR persistence on cardiomyocytes was analyzed by immunohisto- FIBROSIS chemistry in EMB samples. Platelet morphology of pts and HS was assessed in whole blood by scanning electron microscopy. CAR persistence on platelets was analyzed using immunofluorescent Joseph R. Arron method. Spontaneous and 1.0 lM ADP-induced aggregation was estimated using light transmission aggregation analyzer BIOLA Genentech, Inc, South San Francisco, CA, USA (Russia). In all pts additionally the level of IL-6 and TNF-alpha was Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal analyzed be ELISA. parenchymal lung disease of unknown etiology. Preclinical models of Results: According to EMB evaluation analyses there was three fibrotic diseases have implicated interleukin-13 (IL13) activity on grades of CAR expression: (1) intensive staining of intercalated disc multiple cell types, including macrophages and fibroblasts, in initi- and whole membrane, (2) spread along all the cells surface, (3) weak ating and perpetuating pathological fibrosis, which can be ameliorated staining of lateral cell membranes. There was no relation between the by therapeutic inhibition of IL13 with monoclonal antibodies. IL13 level of CAR expression on platelets and cardiomyocytes. In patients and IL13-inducible target genes are expressed at significantly ele- with high IL-6 level the persistence of CAR on platelets was maximal vated levels in lung tissue from patients with IPF compared to control

123 Inflamm. Res. S95 lung tissue. Systemic biomarkers related to IL13 activity are elevated has been a growing appreciation of common mechanisms in their in subsets of IPF patients and have been associated with disease pathogenesis. The discovery of core fibrotic pathways shared by these severity and prognosis. Monoclonal antibody therapies targeting IL13 diseases suggests that therapeutics developed to target such pathways have demonstrated clinical efficacy and are generally well tolerated could have broad clinical applicability. We have identified important and safe in asthma patients, and are currently under investigation in roles for lysophosphatidic acid (LPA) in multiple mouse models of clinical studies of IPF patients. Given the molecular, pathological, and fibrotic diseases, each affecting a different organ, including the lung, clinical heterogeneity of IPF, biomarker-guided clinical development skin, peritoneum and kidney. Although LPA signals through at least strategies may be important in determining the success of targeted six different receptors, identified as LPA1–6, we have found important therapeutic approaches. roles for LPA signaling specifically through LPA1 in all organs we have investigated. In the lung, we have found that LPA-LPA1 sig- naling promotes epithelial cell apoptosis, vascular leak and fibroblast recruitment and persistence. In the skin, we have found that LPA- 111 LPA1 signaling is required for fibroblast differentiation into myofi- SMALL MOLECULE INHIBITORS OF LOXL2 broblasts. In the peritoneum and the kidney, we have identified a FOR FIBROSIS pathway through which LPA-LPA1 signaling promotes expression of pro-fibrotic genes, such as CTGF, which drive fibroblast proliferation. Wolfgang Jarolimek This pathway involves Ga12/13, RhoA and ROCK activation, actin polymerization, the transcriptional co-activators MRTF-A and Pharmaxis, Ltd, Frenchs Forest, NSW, Australia MRTF-B, and the transcription factor SRF. In contrast to similarities we have found in pro-fibrotic LPA signaling being mediated by LPA1 Fibrosis is the result of excessive accumulation of matrix proteins like across organs, we have found important cross-organ differences in the collagen and elastin. Degradation of these proteins is hindered by generation of LPA during the development of fibrosis. Two major increased activity of lysyl oxidases which cross link matrix protein to enzymatic pathways have been described for LPA production. LPA form fibrotic areas. Therefore, inhibition of lysyl oxidase activity can be produced from phosphatidic acid by phospholipase A1 (PLA1) seems to be a promising mechanism to treat fibrosis. Lysyl oxidases are and phospholipase A2 (PLA2) family members, including Lipase H. a five membered family (Lox, Loxl1, Loxl2, Loxl3, Loxl4) of which Alternatively, PLA1 and PLA2 can first convert phospholipids to Loxl2 seemed to be particularly important in diseases and a functional lysophospholipids such as lysophosphatidylcholine (LPC), which can antibody diminished fibrosis is various animal models (Nat Med then be converted to LPA by autotaxin (ATX). We have found that 16:1009–1017, 2010). However, similar results can be obtained with a ATX expression is elevated in, and its activity is required for, the functional Lox antibody (Cancer Res 73:1721–1732, 2013) suggesting development of dermal fibrosis. In contrast, we have found that ATX that the role of lysyl oxidases can be complimentary in diseases. activity is not required for the development of lung fibrosis. In To discover small molecules the only known small molecule keeping with LPA production not being ATX-dependent in lung inhibitor, b-aminoproprionitrile (BAPN), is not a good template as it fibrosis, mass spectroscopy analyses of lysophospholipid species is non-selective and does not have good developability properties. during the development of lung fibrosis do not support a substrate- Based on the successful development of a mechanism-based semi- product relationship between LPC and LPA. It consequently appears carbazide-sensitive amine oxidase inhibitor (SSAO/VAP-1) we that the LPA pathway can be inhibited more broadly across organs by attempted to develop Loxl2 selective small molecules. Through targeting the LPA1 receptor, rather than by targeting the enzymes that iterative medicinal chemistry efforts we could identify low molecular are involved in LPA generation. The strategy of targeting LPA1 is weight mechanism-based inhibitors that have various selectivity ratios currently being investigated in two fibrotic diseases, with antagonists and some are very selective for Loxl2 over Lox in biochemical of this receptor being evaluated in clinical trials in idiopathic pul- assays. These compounds show all characteristics of mechanism- monary fibrosis and scleroderma. based inhibition and have good developability properties. The CCl4-induced liver fibrosis model has been used to assess the efficacy of our small molecule Loxl2 inhibitors to reduce fibrosis. Consistently, the area of fibrosis and a-smooth muscle actin were significantly reduced by therapeutic applications of Loxl2 inhibitors. SYMPOSIUM 36: NOVEL THERAPIES IN In summary, small molecule, selective inhibitors of Loxl2 can be RA developed. These compounds are very efficacious anti-fibrotic drugs and may be useful for the treatment of various diseases. 113 RHEUMATOID ARTHRITIS: WHERE WE ARE AND WHAT IS THE FUTURE 112 TARGETING LPA AS A CORE FIBROTIC Ernest H. Choy PATHWAY: SIMILARITIES AND DIFFERENCES ACROSS ORGANS Cardiff University, Cardiff, UK Rheumatoid arthritis (RA) is a chronic inflammatory arthritis, which 1,2,3 Andrew M. Tager result in pain, disability and reduced quality of life. Chronic synovial inflammation leads to irreversible joint damage. Biologics have 1 2 Division of Pulmonary and Critical Care Medicine; Massachusetts transformed the management of RA, reducing disability and 3 General Hospital, Boston, MA, USA; Harvard Medical School, improving prognosis. In RA, ‘‘treat-to-target’’ to maintain suppression Boston, MA, USA of the disease activity is essential to halt joint destruction and is the Fibrosis—the pathologic accumulation of fibroblasts and extracellular current standard of care. However, \30 % of patients in routine matrix—can affect essentially any tissue or organ. Although fibrotic clinical practice. Therefore, there is a continued need to develop diseases present clinically with organ-specific manifestations, there novel treatments that achieve higher rate of disease remission. 123 S96 Inflamm. Res.

Over the two decades, five classes of biologic agents have been 2014) are protected from disease. Mavrilimumab is an IgG4 human licensed for the treatment of RA: tumor necrosis factor alpha (TNFa) monoclonal antibody that binds to the GMCSFR chain and prevents inhibitors; anti-interleukin-6 (IL-6) receptor monoclonal antibody GM-CSF binding to its receptor. Recently this antibody has demon- (mAb); the anti-CD20 B-cell–depleting mAb; T-lymphocyte co- strated efficacy in a 24 week Phase IIb double blind randomised stimulation inhibitor (abatacept); and the IL-1 receptor antagonist clinical trial in RA patients that have had an inadequate response to anakinra. More recently, a selective small molecule inhibitor of traditional disease modifying anti-rheumatic drugs (DMARDS). The Janus-activated kinase (JAK), tofacitinib, has been approved in the role of the GM-CSF pathway in RA will be introduced and the results US and other countries in the world for the treatment of RA1. of the clinical trials in rheumatoid arthritis will be presented. Optimizing treatment and predicting response to treatment are References important to maximise health economic value and minimise the risk Bell et al (1995). Measurement of colony-stimulating factors in of side effects. Currently, there are many research programmes trying synovial fluid: potential clinical value. Rheumatol Int. 14(5):177–82. to develop biomarkers that can predict response to biologic treatment, Greven et al (2014). Preclinical characterisation of the GM-CSF in particular, one study suggested that synovial histology obtained by receptor as a therapeutic target in rheumatoid arthritis. Ann Rheum biopsies before treatment may predict response to adalimuamb and 2 Dis. (Epub 16 June) tocilizumab . It found that patients with high ICAM expression and Hazenberg et al (1989). Correction of granulocytopenia in feltys low CXCL13 were more likely to respond to adalimumab conversely syndrome by GM-CSF. Simultaneous induction of IL-6 release and patients with low ICAM-1 and high CXCL13 were more likely to flare-up of the arthritis. Blood. 74(8): 2769–70. respond to tocilizumab. IL-6 differs from TNFa in that it has a major Campbell et al (1998) Protection from collagen-induced arthritis in role in the adaptive immune response through effects on T and B GM-CSF deficient mice. J. Immunol. 161(7): 3639–44. cells. CXCL13 is a B cell chemoattractant. High level of CXCL13 Cook et al. (2001). Blockade of collagen induced arthritis post-onset suggest active adaptive immune response. Recent studies using Bio- by antibody to GM-CSF: requirement for GM-CSF in the effector Map have lent further support this hypothesis by demonstrating that phase of the disease. Arthritis Res. 3(5): 293–8. methotrexate, adalimumab and tocilizumab have distinct immuno- logic phenotypic profiles consistent with their mode of action3. Interestingly methotrexate has a significant inhibitory effect on B cell function which is reflected in its ability to reduce immunogenicity of 115 therapeutic mAb. Methotrexate and adalimumab when added together had more pharmacodynamics interaction than combining methotrex- A NEW THERAPEUTIC APPROACH FOR RA ate to tocilizumab4. This may explain why the magnitude of benefit TREATMENT USING AN ANTI-TLR4 when methotrexate is combined with tocilizumab is less than those 5–7 MONOCLONAL ANTIBODY INHIBITING TLR4 observed with methotrexate and TNFa inhibitors . AND FCGAMMAR SIGNALING AND EXPLOITING References CITRULLINATED PROTEIN IMMUNE 1. Choy EH et al. Nat Rev Rheumatol. 2013 Mar;9(3):154–63 COMPLEXES AS POTENTIAL BIOMARKERS FOR 2. Dennis et al. Arthritis Research & Therapy 2014, 16:R90 3. Tan SL et al. ACR 2013. PERSONALIZED MEDICINE 4. O’Mahony A et al. EULAR 2014 Abs no THU0526. 5. Dougados et al. Ann Rheum Dis 2013;72:43–50. Emmanuel Monnet1, Limin Shang1, Genevie`ve Lapeyre1, 6. Breedveld FC, et al. Arthritis Rheum 2006; 54:26–37. Eric Hatterer1, Kathy deGraaf1, Venassa Buatois1, Greg Elson1, 7. Klareskog L et al. Lancet 2004; 363(9410): 675–681. Walter Ferlin1, Cem Gabay2, Jeremy Sokolove3, Simon Jones4, Ernest H. Choy4, Iain B. McInnes5, Marie Kosco-Vilbois1, Cristina de Min1 114 1 2 GM-CSF: A NEW TARGET FOR INTERVENTION Novimmune SA; Geneva University Hospitals,Geneva, Switzerland; 3Stanford University, Stanford, CA, USA; 4Cardiff Institute for Infection IN RHEUMATOID ARTHRITIS and Immunity, Cardiff , UK; 5Glasgow Biomedical Research Center, Institute of Infection, Immunity and Inflammation, Glasgow, UK Michael E. Weinblatt Background: Innate immunity is implicated in rheumatoid arthritis Brigham and Women’s Hospital, Boston, MA, USA (RA) pathogenesis and is likely initiated via TLR pathways. NI-0101 is the first humanized monoclonal antibody (mAb) that blocks TLR4 Granulocyte macrophage colony stimulating factor (GM-CSF) is a signaling independently of ligand type (i.e., exogenous/endogenous) pro-inflammatory cytokine involved in the activation, differentiation and concentration. and survival of cells of the myeloid compartment, notably neutrophils, Objectives: To determine preliminary tolerability, safety, pharmacoki- macrophages and dendritic cells. Over the last two decades significant netic (PK)/pharmacodynamic (PD) profiles after single administrations evidence points to this cytokine as playing a key role in rheumatoid of different NI-0101 doses to healthy volunteers (HV), as well as to arthritis (RA). Synovial fluids isolated from patients with RA have investigate NI-0101 ability to block TLR4-mediated inflammatory elevated GM-CSF levels (Bell et al. 1995), GM-CSF receptor positive cytokine production induced by endogenous TLR4 ligands. cells are increased in the synovial tissue (Greven et al. 2014) and Methods: A PK/PD guided single ascending dose Phase 1 study was chromosome 5q31, in the region of IL-3 and GM-CSF, was recently conducted in 73 HV, in the presence of in vivo and ex vivo challenges identified as a risk loci for RA (Okada et al. 2014). In addition the use with the exogenous TLR4 ligand, lipopolysaccharide (LPS). In parallel, of recombinant GM-CSF to treat neutropenia has been shown to monocytes obtained from RA patients were stimulated either with promote arthritis flares in patients with felty syndrome (Hazenberg endogenous TLR4 ligands or synovial fluids (SF) of RA patients in et al. 1989). These clinical observations are supported by arthritis presence and absence of NI-0101. The correlation of TLR4 blockade studies in vivo demonstrating that either mice deficient in GM-CSF with level of endogenous TLR4 ligands in SF was assessed. Finally, the (Campbell et al. 1998) or treated with neutralising monoclonal anti- anti-mouse TLR4 surrogate mAb, 5E3, was tested in murine models of bodies to either the ligand (Cook et al. 2001) or receptor (Greven et al. RA (IL-1Rn-/- mice and collagen induced arthritis).

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Results: NI-0101 was administered up to a single dose of 15 mg/kg in B259 the Phase 1 study in HV and showed no safety concerns. The pre- 14-3-3 ETA AS A NOVEL RA DRUG TARGET: dictable PK profile was biphasic, similar to other therapeutic IgG targeting cell surface receptors. NI-0101 administration completely ANTI-14-3-3 ETA MONOCLONAL ANTIBODY inhibited ex vivo and in vivo LPS-induced cytokine release from a DELAYS THE ONSET AND MITIGATES THE dose of 1 mg/kg, as well as prevented all laboratory and clinical SEVERITY OF ARTHRITIS IN CIA MICE changes expected following LPS administration to HV. NI-0101 PK/ PD profiles allowed, through modeling and simulation of multiple See poster section for abstract. administration of NI-0101, to identify an appropriate dose range to be tested in Phase 2. In vitro interaction of NI-0101 with TLR4 and Fcc Receptor efficiently blocked TLR4 activation by citrullinated protein immune complexes. NI-0101 blocked the activation of monocytes stimulated with SF from a sub-population of RA patients. This inhi- B316 bition correlated with the presence of anti citrullinated protein RESTORATION OF TREG CELL FUNCTION antibodies in synovial fluids and their matching sera. Therapeutic WITH PD-L1 FC PROTEIN IN RHEUMATOID administration of 5E3 ameliorated disease progression in both mouse ARTHRITIS models of arthritis. Conclusions: Taken together, these data strongly support TLR4 as a See poster section for abstract. valid therapeutic target in RA, and provide an opportunity to evaluate antibodies against citrullinated proteins as biomarkers predictive of treatment response in Phase 2. MINI-SYMPOSIUM 4: SIGNALING IN INFLAMMATION 116 CELL THERAPIES IN RHEUMATOID ARTHRITIS B139 SALT-INDUCIBLE KINASES (SIK) INHIBITION IN John D. Isaacs HUMAN MYELOID CELLS MODULATES TLR AND Newcastle University, Newcastle upon Tyne, UK IL-1R SIGNALING AND INDUCES AN ANTI- Rheumatoid arthritis (RA) is an autoimmune disease characterised by INFLAMMATORY PHENOTYPE joint inflammation as well as by extra-articular features such as interstitial pneumonitis, and comorbidities such as cardiovascular See poster section for abstract. disease. Current therapies for RA are anti-inflammatory and/or immunosuppressive, and the vast majority of patients require chronic treatment with the risk of adverse effects such as severe infections and possibly malignancy. Furthermore, even initially effective therapies B197 often lose their benefit with time. RIP KINASE SIGNALING IN KERATINOCYTE The ideal therapy for RA would switch off the autoimmune process and restore self-tolerance, providing long-term benefit from a short- CONTROLS NECROPTOSIS: MEDIATED SKIN term intervention. So-called therapeutic tolerance has been achieved in INFLAMMATION a number of animal models of autoimmunity and transplantation but translation to the clinic has been slow. Trials of tolerogenic antibodies See poster section for abstract. such as anti-CD4 have recently given way to tolerogenic cellular therapies. These include regulatory T-cells, tolerogenic dendritic cells and mesenchymal stem cells. Each of these is based around a natural immunoregulatory cell type and each has its own relative advantages B281 and disadvantages. Treatment generally involves the isolation of an BLOCKING LTßR SIGNALING IN VIVO appropriate cellular subset; its ex vivo manipulation, usually involving SUPPRESSES THE DEVELOPMENT OF SMOOTH expansion and/or differentiation, in a laboratory that conforms to cur- rent Good Manufacturing Practice regulations; and its subsequent MUSCLE HYPERTROPHY IN A CHRONIC administration following assessment against specific quality control or MURINE MODEL OF ASTHMA ‘release’ criteria. Some cellular therapies are autologous, others allo- geneic; and they can be administered fresh or following See poster section for abstract. cryopresevation. Most work in RA has focussed on tolerogenic den- dritic cells although only three trials have been reported to date, and only in abstract form at the time of writing. My own research group has recently completed the first ever trial of intra-articular tolerogenic B069 dendritic cells in rheumatoid and inflammatory arthritis patients (the OPPOSITE ROLE OF SOLUBLE FORMS OF IL-6 AuToDeCRA trial). As with any phase I study it was designed pri- RECEPTOR, SIL-6R AND SGP130, ON THE marily to study safety but potential efficacy was also explored. My lecture will use AuToDeCRA to illustrate the points to consider REGULATION OF INFLAMMATORY STATUS IN and understand before embarking on a tolerogenic therapy in humans, RHEUMATOID ARTHRITIS including how to measure ‘success’. The other cellular therapies that are in development for RA will also be discussed briefly. See poster section for abstract.

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B082 Oklahoma City, OK, USA; 5Allergy and Asthma Assoc. Santa Clara 6 COMBINATORIAL TARGETING OF TSLP, IL-25, County Research Center, San Jose, CA, USA; California Allergy and Asthma Medical Group, Los Angeles, CA, USA; 7Pennisula Research AND IL-33 IN TYPE 2 CYTOKINE-DRIVEN Assoc., Rolling Hills Estate, CA, USA; 8Clinical Research Institute of INFLAMMATION AND FIBROSIS Southern Oregon, Medford, OR, USA; 9University of Pittsburgh Asthma Institute, Pittsburgh, PA, USA See poster section for abstract. Asthma and other allergic diseases are associated with mast cell activation and prostaglandin D2 (PGD2) generation. PGD2 exerts pro-inflammatory activity via chemoattractant receptor-homologous B083 molecule expressed on Th2 cells (CRTh2). ARRY-502 is a potent DECIPHERING THE DELETERIOUS EFFECT OF and selective CRTh2 antagonist which was studied in mild atopic IFN-C IN A NEW RODENT MODEL FOR asthmatic adults in a double-blind, placebo-controlled 4 week phase 2a study. Enrolled patients were free of inhaled corticosteroids with EXPERIMENTAL SEVERE MALARIA an FEV1percent predicted of 60–85 %. Potential participants were screened and randomized to receive 200 mg BID ARRY-502 See poster section for abstract. (n = 93) or matching placebo (n = 91). The primary endpoint was change from baseline FEV1 compared to placebo. Secondary end- points included additional spirometry evaluations, measures of B325 asthma control and two quality of life (QOL) assessments. Safety NEURONAL TLR SIGNALING CONTROLS was evaluated by incidence and severity of adverse events, vital signs, hematology and EKG parameters. FEV1 improved 3.9 % NEURONAL EXCITABILITY, compared to placebo (Day 29, p = 0.02). Asthma Control Ques- NEUROINFLAMMATION, PAIN, AND ITCH tionnaire-7, ß-agonist use and symptom free days improved compared to placebo (p \ 0.001, p \ 0.001 and p = 0.07 respec- See poster section for abstract. tively). Asthma and Rhinitis QOL improved compared to placebo (p = 0.012, p = 0.007). ARRY-502 was well tolerated with less adverse events than placebo. In patients with elevated Th2 associ- SYMPOSIUM 37: NOVEL TARGETS AND/ ated biomarkers at baseline, activity outcomes between ARRY-502 and placebo were numerically and statistically greater in Th2 high OR NOVEL THERAPIES—SPONSORED (baseline) participants (for example: DFEV1 = 6.7 %; p = 0.008). BY CELGENE In addition, there was a significant reduction in markers of Th2 driven inflammation in these patients. These results support the activity of ARRY-502 in mild asthma patients, suggest that patients 117 that suffer from other Th2-driven allergic diseases like atopic DEVELOPING CAR T CELLS FOR CANCER dermatitis could benefit and warrant further development of ARRY- 502 Marcela Maus

University of Pennsylvania, Philadelphia, PA, USA 119 Chimeric antigen receptor (CAR) T cell immunotherapy has achieved MECHANISMS OF DUAL INHIBITION OF TNF unprecedented responses in hematologic B cell malignancies, and has AND IL-17: TRANSLATION TO CLINICAL been granted Breakthrough Therapy designation by the FDA. CAR T cells for solid tumors have shown great promise in pre-clinical STUDIES WITH ABT-122 models, and is in the early phases of clinical development. Despite having been developed in the academic setting, many T cell therapies Carolyn Cuff, Melanie Ruzek, Robert Padley, Heikki Mansikka, are now entering an industry setting to be developed into commercial Jeffrey Voss, Margaret Hugunin, Alexander Ivanov, therapies to treat cancer. We will discuss the components of chimeric Chung-Ming Hsieh antigen receptors, the technologies used in making a T cell product, some of the factors considered to be important for efficacy, and recent AbbVie, North Chicago, IL, USA results in hematologic and solid tumors. ABT-122 is an anti-TNF/IL-17 dual variable domain immunoglobulin (DVD-IgTM) currently in Phase II clinical trials for Rheumatoid Arthritis (RA) and Psoriatic Arthritis (PsA). This 118 molecule is capable of simultaneously binding to both TNF and IL- ARRY-502: A POTENT, SELECTIVE CRTH2 17. ABT-122 neutralized both TNF and IL-17 induced IL-6 pro- ANTAGONIST FOR ASTHMA duction by fibroblast-like synoviocytes in vitro and similar results were demonstrated with the sera from ABT-122-dosed healthy Laurence Burgess1, David Chantry1, Christine Eberhardt1, volunteers. Single dose Phase I studies have shown acceptable Robert Hopkins1, Michael Saunders1, Lisa Anderson1, safety and tolerability of ABT-122 that have enabled progression of Roger Aitchison1, Stacie Bell1, Kenji Izuhara2, Junya Ono3, clinical testing to Phase 2. In an effort to understand the mechanism Jeremy Cole4, James D. Wolfe5, Sheldon Spector6, Lawrence Sher7, of action of ABT-122 as well as identify potential biomarkers for Edward Kerwin8, Sally Wenzel9 clinical studies, gene arrays were evaluated in the collagen-induced arthritis mouse model, which demonstrated greater efficacy with 1Array BioPharma, Boulder, CO, USA; 2Saga Medical School, Saga, blockade of TNF and IL-17 compared to blockade of either cyto- Japan; 3Shino Test Corp., Tokyo, Japan; 4IPS Research Co., kine alone. This analysis revealed several genes and pathways

123 Inflamm. Res. S99 affected only under conditions of dual cytokine neutralization, SYMPOSIUM 38: CARDIOVASCULAR which identified multiple chemokines as potential soluble biomarkers that were robustly modulated. Consistent with modula- INFLAMMATION tion of chemokine pathways, we have also found certain chemokine receptors, CXCR4 and CXCR5, modulated on leukocyte subsets in 121 healthy volunteers following a single dose of ABT-122. In addition, MYELOID CELLS IN ISCHEMIC HEART DISEASE increased IL-10 and decreased GM-CSF responses were observed in PBMC with LPS stimulation ex vivo. As these chemokine receptors and cytokine responses have been suggested to play a role in dis- Matthias Nahrendorf ease pathology or its resolution, these data suggest that dual blockade of TNF and IL-17 by ABT-122 could provide a new Massachusetts General Hospital, Boston, MA, USA therapeutic approach to patients with RA and immune mediated Monocytes and macrophages are innate immune cells that reside and inflammatory diseases. accumulate in atherosclerotic lesions but also in the healthy and injured heart and brain. The cells and their subsets pursue distinct functions in steady state and disease, and their tenure may range between hours to months. Some subsets are highly inflammatory, 120 while others support tissue repair. The talk discusses current concepts RESULTS OF A SINGLE DOSE ASCENDING FIRST- of cell supply by the hematopoietic system, lineage relationships and IN-MAN TRIAL OF A NOVEL BIOLOGIC, HUMAN systems’ cross talk, highlights open questions, and describes imaging tools for studying monocytes, macrophages and their progenitors. STRESS PROTEIN RASOLVIR (BIP), IN RHEUMATOID ARTHRITIS (RA): THE RAGULO TRIAL 122 NETs FUELING DISEASE AND VICE VERSA Gabriel S. Panayi2,1, Bruce Kirkham1, Khaldoun Chaabo1, Christopher Hall2, Angela Vincent1, Joana Vasconcelos3, Denisa D. Wagner Toby Prevost3, Toby Garrood1, Valerie Corrigall2 Boston Children’s Hospital, Harvard Medical School, Boston, 1Department of Rheumatology, Guys & St Thomas’s NHS Hospital MA, USA 2 Trust (GSTT), London UK; Academic Department of Rheumatology, For many years, my lab has been studying the interplay between Centre for Molecular and Cellular Biology of Inflammation, Kings 3 inflammation and thrombosis. These processes occur together, stim- College London (KCL), London, UK; Department of Primary Care ulate each other and share cellular and molecular components. The and Public Health Sciences, KCL, London UK latest example of a common functional component is neutrophil Background: RasolvirTM (BiP) is a human endoplasmic reticulum- extracellular traps (NETs). NETs are chromatin released together with resident stress protein. In pre-clinical studies it has prolonged anti- toxic granular components from highly stimulated neutrophils. inflammatory properties by the induction of regulatory cells. A Originally found to trap/sequester invading pathogens, they were soon single intravenous infusion suppresses ongoing collagen-induced also seen to be part of sterile inflammatory and thrombotic processes. arthritis in the mouse; ameliorates arthritis and inhibits bone loss in They interact with VWF, which is also involved in platelet and the TNFa transgenic mouse; and suppresses inflammation in the RA leukocyte recruitment and is crucial for venous thrombus develop- synovial membrane/SCID transplantation model. It is thus a poten- ment after inferior vena cava stenosis. This will be discussed together tial new therapy for RA acting by a novel mode of action. The with the role of NETs and the enzyme that generates them (PAD4) in objectives of this randomised placebo-controlled, dose ascending animal models of deep vein thrombosis and myocardial infarction. double blind Phase I/IIA trial of RasolvirTM were safety (Primary We observed that various cancers in mice prime neutrophils for Objective) and efficacy as measured by the DAS28-ESR (Secondary NETosis. This causes cancer-associated thrombosis, and the produc- Objective). tion of NETs may affect tumor biology. Diabetes both in mice and Methods: 24 patients with active RA who had failed one or more man also promotes NETosis. NETs hinder wound healing and this is DMARDs were sequentially assigned to three cohorts each of eight exacerbated in diabetes. Healing of skin wound in PAD4-/- mice is patients randomly allocated to receive placebo (two patients) or faster than in wild type and it is not affected by diabetes. Surprisingly RasolvirTM (six patients). Patients received a single intravenous deficiency of PAD4 does not make mice immunodeficient in a sepsis infusion over 1 h and were observed as inpatients overnight. No model of cecal ligation and puncture. Thus inhibition of PAD4 to patients were re-treated. Clinical, rheumatological and laboratory prevent NETosis or NET destruction by DNase could be beneficial in assessments for safety and efficacy (DAS28-ESR) were carried out at diabetic wound healing and in many inflammatory and thrombotic stated intervals for 12 weeks. conditions where NETs contribute to the pathology. Findings: No infusion reactions or serious adverse reactions were noted. Adverse events were evenly distributed between placebo and RasolvirTM groups. There were no RasolvirTM-related toxicities. 123 Haematological, renal and metabolic parameters remained within the DEFECTIVE INFLAMMATION RESOLUTION IN normal range. No placebo patient and no patient who had received ATHEROSCLEROSIS: MECHANISMS AND 1 mg Rasolvir TM went into remission. At 12 weeks, one patient who had received 5 mg and two patients who had received 15 mg THERAPEUTIC OPPORTUNITIES RasolvirTM were in remission (DAS28 = \2.6). Interpretation: RasolvirTM is safe when administered up to a dose of Gabrielle Fredman1, Nazila Kamaly2, Omid Farokhzad2, Ira Tabas1 15 mg in patients with active RA. RasolvirTM induces remission after a single dose. It merits further study as a treatment for RA and other 1Columbia University Medical Center, New York, NY, USA; 2Harvard immuno-inflammatory diseases. Medical School, Boston, MA, USA 123 S100 Inflamm. Res.

The inflammatory response to acute and reversible infection or tissue Access BIO, Boyce, VA, USA damage, mediated by PAMPs and DAMPs, respectively, triggers an The purpose of preclinical safety assessment is to understand the essential resolution response that curtails inflammation and restores potential risks of a new drug or biologic in order to aid clinical tissue homeostasis. This resolution response is mediated by a panoply of decision-making. No drug or biologic is 100 % safe. A drug or bio- lipid and protein mediators that activate specific cell surface receptors logic is less safe if it is used in a way that decreases foreseeable and signal transduction pathways to trigger resolution endpoints. How- benefit, or increases the risk, or if the actual risks are greater than the ever, when the inflammatory response is indolent and persistent, the predicted risks. In vitro and ex vivo studies using cells or tissues from resolution phase is often not engaged, leading to a chronic, low-grade patients have been used to screen for compounds, understand mech- inflammatory response that causes clinically serious tissue damage. We anism of action and establish proof of concept. Animal models of have proposed that the maladaptive inflammatory response in human disease are also commonly utilized to gain insight into the atherosclerosis is, in essence, a classic example of failed resolution. The pathogenesis of disease and evaluate the efficacy but they are less initial inflammatory response is triggered by the subendothelial retention frequently utilized in safety assessment. While animal models of of apoB-containing lipoproteins, and resolution fails because this trigger human disease may not mimic all aspects of disease, understanding is not only persistent but is actually amplified. This scenario leads to a the limitation of the animal models together with understanding the vicious cycle of failed resolution, tissue damage-mediated DAMP for- human disease and the potential mechanisms of toxicity should allow mation, and amplified DAMP-mediated inflammation. This talk will for a better prediction of risk in the intended disease populations. review the features of atherosclerosis progression that are consistent with Importantly, regulatory authorities are a becoming more willing to failed resolution; reveal new molecular-cellular mechanisms of how accept and even recommend data from experimental animal disease certain resolution mediators activate signaling pathways in macro- models that combine efficacy and safety. phages, a key inflammatory cell type in atherosclerosis; and discuss and show data supporting the contention that inflammation resolution mediator therapy may be an ideal way to prevent atherosclerosis pro- gression in a manner that would not compromise in host defense. 126 TRANSLATIONAL SAFETY IN DRUG DEVELOPMENT: THE ROLE OF SAFETY SYMPOSIUM 39: PREDICTIVE BIOMARKERS IN PREDICTIVE TOXICOLOGY TOXICOLOGY John Michael Sauer 124 TRANSLATIONAL IMPACT OF SECONDARY Critical Path Institute, San Diego, CA, USA PHARMACOLOGY IN DRUG DISCOVERY In nonclinical safety studies applied during drug development, drug induced organ injury can be identified by histopathological changes. Laszlo A. Urban However, histopathological analysis is rarely an option in clinical drug development studies. Therefore, fluid based biomarkers are used to Preclinical Safety Profiling (PSP) at the Novartis Institutes for predict drug induced tissue injury in humans. Although many of these Biomedical Research (NIBR), Cambridge, MA, USA traditional safety biomarkers have acceptable predictive accuracy under controlled conditions, many also have deficits in their selectivity and/or The safety bar for medicines has been raised by regulatory authorities sensitivity. Furthermore, many of these biomarkers have not undergone and became a determinant factor in successful product launch. programmatic evaluation, but instead have been deemed acceptable Accordingly, the pharmaceutical industry introduced broader and based on clinical experience. The Predictive Safety Testing Consortium earlier risk assessment for better understanding of molecular bases of (PSTC) is a unique public private partnership that brings together toxic reactions which are often shared between drugs. pharmaceutical companies to share and validate each other’s safety Safety conscious product development requires parallel, integrated testing methods in cooperation with the U.S. Food and Drug Admin- optimization of efficacy and safety. For early risk assessment a pow- istration (FDA), the European Medicines Agency (EMA), and the erhouse of predictive in vitro and in silico tools have been developed Japanese Pharmaceuticals and Medical Devices Agency (PMDA). The which enable the inexpensive de-prioritization of chemical structures PSTC is currently involved in the rigorous evaluation of novel safety associated with ADRs, and prevents compounds with ‘‘toxic’’ off-target biomarkers for use in clinical trials with the ultimate goal of obtaining effects entering clinical trials. Learning from clinical experience led to regulatory endorsement and qualification of these biomarkers. PSTC is the development of the concept of ‘‘reverse pharmacology’’ which also working towards a translational safety strategy for use in drug further improved the predictive value of early risk assessment. These development that would integrate human biology-based in vitro mod- should result in diminished attrition, better side effect profiles, els, novel safety biomarkers, computational tools, and non-safety data improved compliance and significant savings for pharmaceutical drugs. under the umbrella of systems toxicology. The objective of this pre- In this presentation, I will review the strategy of early safety risk sentation will be to describe the ongoing research across the PSTC assessment/mitigation of drug candidates based on identification of working groups, and outline how these applied regulatory science molecular targets, pathways associated with pathophysiological clinical outcomes fit into our vision of translational safety. consequences.

125 127 OPTIMIZING CLINICAL DEVELOPMENT: LARGE MOLECULE PREDICTIVE SAFETY: LEVERAGING PRECLINICAL DISEASE MODELS EXPERIENCES AND POINTS TO CONSIDER FOR ASSESSING SAFETY AND EFFICACY Michelle J. Horner

Joy Cavagnaro Amgen, Thousand Oaks, CA, USA

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Drug induced toxicities with biologic therapeutics (i.e., protein-based Innate lymphoid cells (ILCs) have emerged recently as an important products) are generally a result of exaggerated pharmacology as these component of the immune system and the cell type that regulates products are specific for a particular target and are seldom associated mucosal immune responses and tissue homeostasis. Group 2 ILCs with off target toxicity. As a result, attrition of biotherapeutics due to (ILC2s) are a subset of ILCs, are resident in various tissues, and are safety issues has traditionally been uncommon, especially in comparison characterized by their capacity to produce type 2 cytokines and tissue to that of small molecule therapeutics (i.e., traditional pharmaceuticals). growth factors. These ILC2s play an important role in allergic However, innovation in therapeutic design has necessitated some immune response by linking the signals in the atmospheric environ- changes in de-risking strategies and to reduce concerns of potential ment to the immune system. Fungi are one of the major allergens liabilities (e.g. immune reactions, infections, cytokine storm, immuno- associated with human asthma, and animal and in vitro models using genicity). Industry is now challenged to provide diligence to discharge the fungal allergens have provided significant information to under- liabilities and adverse events that may impede progression of novel stand the mechanisms of allergic disease. In mouse models of fungus- biologics through the course of drug development. Using past experi- induced allergic airway inflammation, IL-33, IL-25, and TSLP are ences as guidance, evaluation and application of new approaches may released by airway epithelial cells. Lung ILC2s that respond to these help to understand mechanistic processes involved in unintended effects cytokines quickly produce a large quantity of type 2 cytokines, in in vitro models and non-clinical species, and their relevance to resulting in airway eosinophilia, mucus production, and airway humans. Implementation of these new approaches in the early stages of hyperreactivity even in the absence of adaptive immune cells. Evi- development for a biotherapeutic can minimize delays or terminations, dence also suggests that ILC2s interact with conventional immune and can also identify human hazards that cannot be efficiently screened cells, such as CD4+ T cells, and facilitate development of adaptive in pre-clinical species. Overall, a comprehensive predictive safety immune response and persistent airway inflammation. ILC2s are also strategy for novel biotherapeutics should include considerations for both present in peripheral blood and respiratory mucosa in humans. Further target/unintended target- and modality-driven putative liabilities. investigations into biology of ILC2s and their roles in the patho- physiology of allergic diseases will provide major conceptual advances in the field and may provide useful information toward SYMPOSIUM 40: TRANSLATIONAL development of new therapeutic strategy for patients. MEDICINE

Abstracts not available at time of printing. 130 LIPID MEDIATOR REGULATION OF ILC2 TH2 SYMPOSIUM 41: INNATE LYMPHOID CYTOKINE RESPONSE CELLS David Broide

128 University of California San Diego, San Diego, USA FUNCTION AND PLASTICITY OF HUMAN ILC Th2 cytokines (IL-4, IL-5, IL-9, IL-13) play an important role in the SUBSETS pathogenesis of allergic inflammation and asthma. Although allergen activated antigen specific CD4+ T cells are a significant source of Th2 Hergen Spits cytokines in asthma, recent studies suggest that innate lymphoid cell type 2 (ILC2) may also be a significant source of Th2 cytokines in AMC University of Amsterdam, Amsterdam, The Netherlands allergy and asthma. In contrast to allergen activation of CD4+ T cells to express Th2 cytokines, ILC2 do not respond to allergen as they do Innate lymphoid cells are emerging as important regulators and not express antigen receptors and are activated by cytokines including effectors in innate immunity and tissue homeostasis. Three groups of IL-33, TSLP, and IL-25 to express IL-5 and IL-13 but not IL-4. In ILC, ILC1, ILC2 and ILC3 have now been identified on the basis of addition to these cytokines, lipid mediators such as prostaglandin D2 cytokine production profiles, dependence on signature transcription (PGD2) and leukotrienes are able to regulate ILC2 responses factors and developmental pathways. ILC1 are defined by their including chemotaxis and expression of Th2 cytokines. PGD2 binds dependence on Tbet and capacity to produce Interferon gamma but to CRTH2 expressed on human ILC2 and induces their chemotaxis. are less well defined than ILC2 that depend on GATA3 and produce As PGD2 is released from allergen/IgE activated mast cells, the initial IL-4, IL-5 and IL-13 and ILC3 that are RORgt dependent and produce PGD2 release within minutes from mast cells following exposure to IL-17 and/or IL-22. I will give an overview of our present knowledge allergen may play an important role in chemotaxis of ILC2 at sites of of human ILC subsets and discuss data indicating that ILC highly allergic inflammation. PGD2 also potentiates the production of IL-13 plastic and that ILC subsets can shuttle between different functional by peripheral blood ILC2, whereas lipoxin A4 reduces levels of IL-13 states dependent on environmental cues. I will also discuss the produced by ILC2. Levels of cysteinyl leukotrienes (LTC4, LTD4, functional interactions of ILC with other hematopoietic cells in par- and LTE4) are increased in asthma and chronic rhinosinusitis. Mouse ticular between class II MHC expressing ILC3 and T cells lung ILC2 express the CysLT1receptor, which when activated mediates ILC2 calcium influx and Th2 cytokine production. Inter- 129 estingly, in contrast to IL-33 which does not induce ILC2 to express FUNCTIONS OF IL-33 AND GROUP 2 INNATE IL-4, LTD4 induces ILC2 to express high levels of IL-4 as well as other Th2 cytokines such as IL-5 and IL-13. As IL-4 is an important LYMPHOID CELLS IN CONTROLLING ALLERGIC switch factor for IgE synthesis, ILC2 activation by LTD4 may con- AIRWAY INFLAMMATION AND ASTHMA tribute to IgE production at sites of allergic inflammation. In addition, leukotriene induced ILC2 production of IL-4 may play an important + Hirohito Kita role in differentiation of CD4 cells to Th2 cells to amplify Th2 responses. In an innate mouse model of asthma induced by the mold Mayo Clinic, Rochester, MN, USA Alternaria (epidemiologically associated with severe human asthma),

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LTD4 enhances ILC2 proliferation and eosinophilia. Thus, overall febrile serositis, peritonitis, arthritis and pleuritis. Many studies have lipid mediators such as PGD2 and leukotrienes are highly expressed been performed as an attempt to understand the basis of the inflam- at sites of allergic inflammation including the lower airway in asthma. matory attacts in FMF. During the acute attacks, elevations in acute In addition to the effect of lipid mediators on structural cells such as phase reactant levels and increased several proinflammatory cytokines smooth muscle to induce bronchial constriction in asthma, lipid and anti-inflammatory peptides have been described. mediators such as PGD2 and leukotrienes may also serve an impor- Angiostatin is a physiologic angiostatic factor derived from the tant role in chemotaxis and activation of ILC2 to express Th2 proteolytic cleavage of plasminogen. Angiostatin is also known as a cytokines including IL-4 (in response to LTD4) which may amplify potent antiangiogenic mediator that can be found in increased levels IgE and Th2 responses. in the patients during various states of inflammation. It has been reported that angiostatin directly inhibite neutrophil migration and neutrophil mediated angiogenesis and also might inhibit inflamma- tion. The purpose of this study was to determine serum levels of 131 angiostatin in patients with FMF PATHWAYS THAT GOVERN ILC2S FUNCTION Material and method: In this study, 58 patients with FMF (38 female, AND HOMEOSTASIS 20 male, mean age 21.3 ± 4.1 years, mean disease duration 5.6 ± 2.8 years) and 22 healthy controls (14 female, 8 male, mean Omid Akbari age 20.8 ± 2.9 years) were included. Serum angiostatin levels were measured by ELISA. University of Southern California, Los Angeles, CA, USA Results: The mean serum angiostatin levels were 188.4 ± 33.7 ng/ml in patients with FMF and55.6 ± 19.5 ng/ml in the healthy controls. Type 2 Innate lymphoid cells (ILC2s) are a newly identified subset of The mean levels of serum angiostatin were 274.9 ± 36.1 ng/ml in immune cells that play important roles in the pathogenesis of allergic active stage and 104.4 ± 30.5 ng/ml in inactive stage. According to asthma by producing large amounts of IL-5 and IL-13. ILC2 lack these results; serum angiostatin levels were significantly higher in lineage markers but express CD45, IL-2R, IL-33R and IL-7R. We patients with FMF compared with healthy controls (p \ 0.001). In will discuss few recently identified pathways that modulate ILC2 addition, serum angiostatin levels were significantly higher in active function and homestasis. All murine ILC2s and a significant portion stage compared to in inactive stages (p \ 0.001). In the inactive of human ILC2s express Inducible T-cell COStimulator (ICOS) that is patients with FMF, serum angiostatin concentrations were found to be essential for T cell activation and function, however, the role of ICOS higher compared to healthy controls s (p \ 0.01). in ILC2s remains unknown. We investigated the role of ICOS in the In active patients, the mean ESR was 56.4 ± 3.7 mm/h, the mean function and survival of murine and human ILC2s using ICOS-/- and serum CRP level was 38.4 ± 3.9 mg/L and the mean serum fibrino- RAG2-/- and humanized mice in different experimental setups. gen level was 504.2 ± 96.1 mg/dl. In active FMF patients, the mean We found that ICOS-/- mice show lower IL-33-induced airway serum angiostatin level was correlated with the mean serum fibrino- hyperreactivity (AHR) and eosinophilia than wild type (WT) mice. gen level (r = 0.592 p = 0.012), serum CRP level (r = 0.534, Lack of ICOS decreased survival and impaired the production of IL-5 p = 0.022) and ESR (r = 0.522, p = 0.028). and IL-13 by ILC2s. We also for the first time introduced ILC2 Conclusion: The high levels of serum angiostatin, in active and humanized mice and showed that blocking ICOS:ICOS-Ligand inactive patients with FMF suggest that angiostatin may play a sig- interactions impair the production of IL-5 and IL-13, reduced IL-33 nificant role of in the pathogenesis of FMF. induced AHR and eosinophilia. Finally our data suggest that both human and murine ILC2s express ICOS-Ligand and cis or trans interactions of ICOS/ICOS-L, provide STAT5 dependant survival for the cells. These results indicate that ICOS is required for the ILC2- B002 mediated AHR and thus, therapeutic manipulation of the ICOS pathway is an attractive target for future strategies of treatment and SYNCHROTRON MICROBEAM IRRADIATION prevention of ILC2 dependent asthma. INDUCES INFLAMMATION AND SELECTIVE VASCULAR DAMAGES IN ADULT ZEBRAFISH

Daniel Bro¨nnimann1, Audrey Bouchet1, Werner Graber1, POSTERS Jean A. Laissue1, Valentin Djonov1, Raphael Serduc2, Elke Bra¨uer2 Angiogenesis 1University of Bern, Institute of Anatomy, Bern, Switzerland; 2European Synchrotron Radiation Facility, Grenoble, France B001 Background: Microbeam radiation therapy (MRT) is a new promising tumor treatment strategy used at preclinical stage. MRT is based on the SERUM CIRCULATING ANGIOGENESIS spatial fractionation into arrays of parallel microbeams, which are INHIBITOR ANGIOSTATIN LEVELS IN PATIENTS typically separated by few hundred micrometers. We have used the WITH FAMILIAL MEDITERRANEAN FEVER zebrafish caudal fin model to study the effects of synchrotron based microbeam irradiation on the mature and immature vasculature in vivo. Goksal Keskin1, Ali Inal2, Lale Ozısık3, Mehmet Yildiz3 Method: We used transgenic fli1:eGFP zebrafish to visualize endothelial cells in vivo. The ventral part of the caudal fin was par- 1Medical School of Ankara, Department of Immunology, Ankara, tially amputated to trigger regeneration and outgrowth of immature Turkey; 2Baskent University, Department of Immunology, Ankara, blood vessels. At 6 days post amputation, the caudal fin was irradiated Turkey; 3DYBEAH, Department of Internal Medicine, Turkey with three parallel microbeams of 50 lm widths and 400 lm spacing. Results: Three hours after the irradiation, the immature vasculature Background: Familial Mediterranean Fever (FMF) is an autosomal was characterized by severe blood flow disturbances and fragmented recessive genetic disorder characterized by recurrent episodes of endothelial cells in vivo. By the use of correlative microscopy

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(in vivo and electron microscopy), we revealed the presence of vac- in vitro. The number of LYVE-1 positive lymphatic vessels were uolated endothelial cells and a tremendous inflammatory response reduced in LLC/sVEGFR-2 inoculated on comparison with control inside the beam path. At 6 h post irradiation, the blood vessels were group in vivo. In addition, the expression of VEGFR-2, VEGFR-3 and characterized by the presence of activated macrophages phagocytiz- MMP-9 genes were also suppressed in primary lesion of LLC/ ing damaged endothelial cells. At this time point the capillary plexus sVEGFR-2 group, compared to that in control group. Furthermore, adopted a striated metronomic pattern, with alternating destroyed and the number of micro-metastatic colonies in lymph node were reduced intact zones, corresponding to the beam paths. At later time points, in LLC/sVEGFR-2 group inoculated mice in comparison with those activated macrophages were as well found in the loose connective control mice, when these cells directly inoculated in lung. tissue surrounding the vasculature. In contrast, the mature vasculature Conclusion: The present study indicated that intervention of remained intact in terms of blood perfusion, endothelial cell coverage sVEGFR-2 suppressed lymphangiogenesis in primary lesion and and inflammatory response. lymphogenic metastasis of lung tumor through depriving VEGF-C, Conclusions: Vascular toxicity and physiological effects of microbeam followed by down regulation VEGFR-2, VEGFR-3 and MMP-9. irradiation depend on the stage of capillary maturation and appear in the sVEGFR-2 might be one of the prominent target for the treatment of first hours after irradiation. Newly formed, immature capillaries are cancer by regulating lymphangiogenesis and lymphogenic metastasis. highly sensitive to microbeam irradiation, whereas mature vessels are barely affected. The selective vascular damage could serve in the future to create new and promising anti-angiogenic treatment strategies. B004 MLN4924 INHIBITION OF CULLIN-RING LIGASES ACTIVATES A HYPOXIC RESPONSE AND B003 SECRETION OF ANGIOGENESIS-PROMOTING SUPPRESSIVE EFFECTS OF SOLUBLE VASCULAR CYTOKINES IN HUMAN MCF-7 BREAST CANCER ENDOTHELIAL GROWTH FACTOR RECEPTOR-2 CELLS ON LYMPHOGENIC METASTASIS David Finkel, James Rivard, Amy James, Erin Eliria, Neal Lee, Shotaro Maehana1, Rimika Imai2, Rei Murakami2, Kathy Brumbaugh, Greta Wegner Masaki Nakamura2, Fumihiro Ogawa3, Fumiaki Kojima4, Masataka Majima3, Hidero Kitasato2 R&D Systems Inc, Minneapolis, MN, USA Hypoxia-inducible factors (HIFs) are the key transcription factors that 1Department of Environmental Microbiology, Kitasato University mediate the cellular response to low oxygen tension. During nor- Graduate School of Medical Sciences, Minato, Japan; 2Department of moxia, HIF activity is tightly controlled by its oxygen-dependent Microbiology, Kitasato University School of Allied Health Sciences, prolyl hydroxylation which allows polyubiquitination by von Hippel- Minato, Japan; 3Department of Pharmacology, Kitasato University Landau tumor suppressor (VHL), leading to HIF degradation by the School of Medicine, Minato, Japan; 4Department of Pharmacology, ubiquitin–proteasome pathway. During hypoxia, low oxygen tension Kitasato University School of Allied Health Sciences, Minato, Japan prevents HIF prolyl hydroxylation resulting in HIF accumulation and Objective: Vascular endothelial growth factor (VEGF) family is known subsequent transcription of HIF target genes. VHL is a known Cullin- to key factors of the vascular and lymph vessel formation. Among VEGF RING E3 ubiquitin ligase (CRL) that is dependent on Neddylation of families, VEGF-C is one of most important factor for lymphangiogenesis its Cullin-2 subunit by NEDD8 Activating Enzyme (NAE) for via binding with those receptors VEGF-R2 and -R3. At the same time, enzymatic activity. NEDD8, NEural precursor cell expressed Devel- soluble VEGFR-2 (sVEGFR-2) is known to have a potential activity for opmentally Down-regulated-8, is an ubiquitin-like molecule that is corneal alymphaticity in association with binding with VEGF-C and attached to its substrates in a manner similar to ubiquitin. The small selectively inhibit lymphangiogenesis but not angiogenesis. In this study, molecule inhibitor MLN4924 inhibits NAE Neddylation of Cullins by we transduced sVEGFR-2 into lung cancer cells and evaluated the effects covalently binding the nucleotide-binding site of NAE. HIF-1a is a on cancer progression including suppressor of genes regulating for lymph well-known CRL substrate that increases in abundance upon vessel formation and metastasis in vivo. MLN4924 treatment. This report presents evidence that MLN4924 Methods: VEGF-C and sVEGF-R2 genes were individually trans- activates a hypoxic (1 % O2, 48 h) response in MCF-7 human breast duced into Lewis lung carcinoma cells (LLC) using defective cancer cells with secretion of cytokines known to promote angio- retrovirus vector, then designed as LLC/VEGF-C and LLC/sVEGFR- genesis and inflammation. Cell lysates tested using the Human 2. The secreted proteins in supernatant of cultured these cells were Oncology Antibody Array and Western blot showed that both hypoxia detected by western blotting using specific antibodies (rabbit poly- and MLN4924 induced accumulation of HIF-1a protein, with higher clonal anti-mouse VEGFR-2 and mouse polyclonal anti-mouse protein levels in MLN4924 treated cells. This correlated with the VEGF-C), respectively. To examine the lymphangiogenesis in pri- expected attenuation of HIF-1a ubiquitination during hypoxia and mary lesion of tumor in vivo, LLC/sVEGFR-2 and LLC/VEGF-C cells MLN4924 treatment, as detected by the Human Ubiquitin Antibody were subcutaneously injected to C57/BL6 mice, respectively. After Array. Western blots showed that MLN4924 specifically inhibited the 14 days of the injection, immunohistochemical analysis was carried Neddylation of Cullin-2 and Cullin-1. Western blots also revealed that out by using antibody against lymphatic vessel endothelial hyaluronan MLN4924 indirectly inhibited mTORC1 complex-mediated Thr389 receptor 1 (LYVE-1), a lymphatic vessel marker. And then, the mRNA phosphorylation of p70 S6K, known to induce autophagy. Analysis of expression of VEGFR-2, VEGFR-3 and matrix metalloproteinase 9 cell culture supernates from normoxia, hypoxia, and MLN4924- (MMP-9) were also determined by real-time PCR. In addition, LLC/ treated cells using the Human XL Cytokine Antibody Array revealed sVEGFR-2 and LLC/VEGF-C were directly inoculated into left lung protein expression of several known hypoxia-activated targets. The of C57/BL6 mice, respectively, in order to determine the number of relative levels of Angiogenin, MIF, PDGF-AA, and VEGF were the micro-metastatic colonies in lung lymph node. found to be highest in MLN4924 treated cells. Subsequent ELISA Results: sVEGFR-2 and VEGF-C proteins were detected in cultured analysis confirmed these array data. The observations reported here supernatant of LLC/sVEGFR-2 and LLC/VEGF-C cells, respectively, are consistent with previous reports showing the mechanism of action

123 S104 Inflamm. Res. for MLN4924 mediated HIF-1a activation. In contrast, recent reports the endoplasmic reticulum, reactive oxygen species (ROS) genera- have found MLN4924 to inhibit tumor angiogenesis in mouse models, tion, mitochondria dysfunction with enhanced expression of Bax suggesting that the effects of MLN4924 on secretion of angiogenic protein levels, caspase-3 and caspase-7 activation, suggesting that factors by MCF-7 cells may be context dependent. LAAO-Bl causes nephrotoxicity by acting in multiple cell death pathways. Taken together our results suggest that LAAO-Bl is responsible for the nephrotoxicity observed in the envenomation by Apoptosis snakebites.

B005 L-AMINOACID OXIDASE, THE MAJOR B006 COMPONENT OF B LEUCURUS VENOM, BOTHROPOIDES PAULOENSIS VENOM INDUCES DIRECT NEPHROTOXICITY THROUGH CYTOTOXICITY IN RENAL TUBULAR ROS, MITOCHONDRIAL DYSFUNCTION AND EPITHELIA CELLS: CELL DEATH SIGNALING CASPASES PATHWAY PATHWAYS

Isabel Cristina Oliveira de Morais1, Gustavo Jose´ da Silva Pereira2, Aline Diogo Marinho, Isabel Cristina Oliveira Morais, Roberta Jeane Mar Orza´ez3, Marcos Hikari Toyama4, Soraya Soubhi Smaili2, bezerra Jorge, Ramon roseo Paula Pessoa bezerra Menezes, Enrique Pe´rez Paya´3, Alice Maria Costa Martins5 Joao alison moraes Silveira, Danya Bandeira Lima, Alice Maria Costa Martins, Helena Serra Azul Monteiro 1Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara´, Fortaleza, Ceara´, Brazil; 2Department Federal University of Ceara´, Fortaleza, Brazil of Pharmacology, Federal University of Sa˜o Paulo (UNIFESP), Snakebite envenomation constitutes a serious medical condition Brazil; 3Department of Medicinal Chemistry, Centro de Investigacio´n 4 common in tropical countries. In Brazil, snakes of the genus Bothrops Prı´ncipe Felipe, Valencia, Spain; Sa˜o Vicente Unit, Paulista Coastal are the main cause of venomous snake accidents. Acute renal failure Campus, Sa˜o Paulo State University (UNESP), Sa˜o Paulo, Brazil; 5 is a common complication caused by Bothrops snakebite with rele- Department of Clinical and Toxicological Analysis, Federal vant morbidity and mortality. The aim of the present study was to University of Ceara´, Fortaleza, Ceara´, Brazil investigate the effects of the Bothropoides pauloensis venom (BpV) in The pit viper Bothrops leucurus (White-tailed-jararaca) is a poisonous cultured renal tubular cells of the type Epithelial Madin–Darby snake habituating area in the northeast of Brazil. The biological Canine Kidney (MDCK). The determination of the cytotoxic potential effects due envenomation have similar profile than those observed was conducted by MTT reduction assay. Lactate dehydrogenase with other Bothrops, such as coagulant activity, hemorrhagic, fibri- (LDH) activity was determined in culture supernatants from experi- nolytic, and acute renal failure (ARF). ARF is a common mental groups for investigation of cell lysis induced by BpV. The complication caused by Bothrops snakebite with relevant morbidity mechanism of cell death induced by BpV was assessed by flow and mortality. Pathogenesis of ARF in snakebite envenomation may cytometry, by staining with propidium iodide (PI) and annexin be related to hypovolemia and hypoperfusion secondary to cardio- V-FITC. To verify the BpV effects on mitochondrial transmembrane vascular disturbances, deposit of fibrin in the glomerular capillaries potential (DWm) cells were stained with the mitochondrial specific leading thrombotic microangiopathy and high venom concentration at probe, tetramethylrhodamineethyl ester (TMRE) and analyzed by the renal tissue, direct venom action on the tubular cells and oxidative flow cytometry. Cytosolic ROS was measured using 2,7-dichlorodi- stress. Recently, we observed that Bothrops leucurus venom induces hydrofluorescein diacetate (DCFH-DA) and analyzed by flow nephrotoxicity in the isolated perfused kidney of rats associated with cytometry. The expressions of Caspase-3 and -7 were analyzed by cytotoxicity against renal tubular epithelia cells. In this study, it was Western blotting. Treatment with BpV caused decrease in cell via- evaluated the direct nefrotoxicity of a main component of B. leucurus bility until 6.25 lg/mL concentration with an IC50 of 7.5 lg/mL. The venom called L-aminoacid oxidase (LAAO-Bl) by using tubular results indicated an apparent membrane rupture in MDCK cells at the epithelial cell lines MDCK and HK-2. In these cells treated with highest concentrations studied [7.5 lg/mL (25.71 + 4.66); 15 lg/mL LAAO-Bl, 1.56–100 lg/mL for 12 h, there was a decrease in their (39.10 + 13.01); 25 lg/mL (49.36 + 14.67); 50 lg/mL (65.62 + viability in a concentration-dependent manner. We next evaluated if 6.28), control negative (0.0001 + 0.001); control positive necrosis was implicated in the cellular viability decrease observed by (100 + 0.001)], showing a increasing of LDH release. Annexin-PI + analyzing lactate dehydrogenase (LDH) release. In MDCK cells LDH loading cell analysis demonstrated an increase mainly in AX /PI- + + release was not observed after 12 h of LAAO-Bl exposure while cells at the concentrations of 7.5 lg/mL, as well as AX /PI cells in LAAO-Bl induced an apparent membrane rupture in HK-2 cells at the both concentrations (15 and 7.5 lg/mL), suggesting the participation highest concentrations studied. Annexin V/PI staining was applied to of apoptosis and late apoptosis on BpV-induced cell death. BpV detect apoptotic/necrotic cells after LAAO-Bl treatment. In MDCK 7.5 lg/mL (IC50) produced ROS overproduction and mitochondrial cells, LAAO-Bl significantly increased the percentage of early membrane potential decrease. The BpV (15 and 25 lg/mL) treat- apoptotic (Annexin-V+, PI-), necrotic (Annexin-V-,PI+) and sec- ment induced activation of caspase 3 and caspase 7 [7.5 lg/mL ondary necrotic cells (Annexin-V+ ,PI+) when compared with control (1.08 + 0.06); 15 lg/mL (3.04 + 0.12); 25 lg/mL (4.61 + 0.53); untreated cells. In HK-2 cells, in accordance with data obtained in the 50 lg/mL (5.17 + 0.36); control negative (0.87 + 0.24)]. In sum- LDH-release assay, the Annexin-V-PI loading cell analysis demon- mary, we have reported that BpV induced MDCK death, which strated an increase in necrotic (PI+ cells) and secondary necrotic cells involves an increase in ROS production and mitochondrial dys- (Annexin-V+ ,PI+) in a concentration-dependent manner. MDCK and function, with participation of apoptosis and late apoptosis HK-2 apoptosis induction was accompanied with Ca2+ release from mechanisms.

123 Inflamm. Res. S105

B007 neurovascular unit where brain microvascular endothelial cells HYPERGLYCEMIA FAVORS TH17 (BMVEC) form the morphological basis of the blood–brain barrier (BBB). Under inflammatory conditions peripheral leukocytes can DIFFERENTIATION AFTER PHAGOCYTOSIS OF exacerbate neurovascular dysfunction by the release of cytotoxic MRSA-INFECTED APOPTOTIC CELLS mediators that induce BBB breakdown. One of these mediators is hypochlorous acid (HOCl), which is formed via the myeloperoxidase 1,2,3 3 3 Naiara N. Dejani , Stephanie Brandt , Nicole L. Glosson-Byers , (MPO)/H2O2/chloride system of activated leukocytes. In vitro treat- 3 1,2 3 Soujuan Wang , Alexandra I. Medeiros , C. Henrique Serezani ment of BMVEC with the MPO/H2O2/chloride system or activated neutrophils results in oxidative modification of the endogenous cel- 1University of Sa˜o Paulo, USP, Ribeira˜o Preto, SP, Brazil; lular ether-phospholipid (plasmalogen) pool. This reaction generates 2Department of Biological Sciences, School of Pharmaceutical chlorinated fatty aldehydes (including 2-chlorohexadecanal; Sciences, UNESP, Universidade Estadual Paulista, Araraquara, SP, 2-ClHDA) and the corresponding remnant lysophospholipid. During Brazil; 3Indiana University School of Medicine, Indianapolis, IN, the present study we aimed to explore the hypothesis that 2-ClHDA USA and its metabolite 2-chlorohexadecanoic acid (2-ClHA) induce sus- tained and unresolved activation of the endoplasmic reticulum (ER) Clearance of infected apoptotic cells is an essential component of the stress response ultimately culminating in barrier dysfunction and host defense. Efferocytosis containing microbial products triggers the BMVEC apoptosis. production of cytokines crucial to T cells differentiation. Staphylo- Using the human BMVEC hCMEC/D3 line as in vitro BBB model coccus aureus infection leads to neutrophil abscess formation with we investigated whether 2-ClHDA can initiate the unfolded protein increased necrosis and apoptosis. In addition, S. aureus is frequently response (UPR)-mediated signal transduction pathway during ER isolated in the skin lesions of diabetic patients with chronic uncon- stress. Using Western blot analysis we analysed whether 2-ClHDA trolled hyperglycemia. It has been shown that IL-17 production in the and its redox metabolite 2-chlorohexadecanoic acid (2-ClHA) can skin is crucial for neutrophil migration and bacterial clearance. initiate the UPR via PERK mediated signalling. Treatment of Therefore, we hypothesized that during active S. aureus infection in hCMEC/D3 cells with 2-ClHDA and 2-ClHA induced phosphoryla- diabetics, the clearance of infected apoptotic cells drives dendritic tion of eukaryotic elongation factor 2a (eIF2-a) and elevated protein cells-mediated Th17 generation, favoring overwhelming inflamma- expression of activating transcription factor 4 (ATF4) and repre- tory response and increased lesion development. Bone marrow- senting crucial events in the PKR-ER like kinase (PERK) -mediated derived dendritic cells (BMDCs) from C57BL/6 mice were differ- UPR pathway activation. Furthermore 2-ClHDA and 2-ClHA stimu- entiated in the presence of GMCSF with high glucose (25 mM) or low lation induced the expression of pro-apoptotic transcription factor glucose (5 mM). After differentiation, BMDCs were co-culture with C/EBP homologous protein (CHOP) and mediated caspase-3 cleav- MRSA-infected apoptotic neutrophil cell line (HL-60) for 18 h and age, demonstrating 2-ClHDA and 2-ClHA induced apoptosis in the supernatants were collected to detect cytokines by ELISA and to hCMEC/D3. To further examine ER function in 2-ClHDA/2-ClHA evaluate the differentiation of naı¨ve CD4+ T cells. Our results show stimulated cells we investigated lipid composition in these cells as that high glucose does not influence BMDC efferocytosis (*40 % in many of the enzymes involved in lipid homeostasis reside in the ER. both low and high glucose). The abundance of Th17-driven cytokines These analyses revealed decreased cellular concentrations of C16 and TGF-ß, IL-1ß and IL-6 were slightly enhanced in high glucose. When C18 triglyceride and cholesterylester species. In addition, 2-ClHDA naı¨ve CD4+ T cells were differentiated in the presence of supernatants treatment resulted potently increased the concentration of several from co-culture of DC with MRSA-infected apoptotic cells, the per- ceramide species, findings accompanied by a concomitant decrease in centage of IL-17A-producing lymphocytes was 2-folder higher in the corresponding sphingomyelin classes. In conclusion the current high glucose. Indeed, we isolated naı¨ve CD4+ T cells from strepto- data provide evidence that a-chloro fatty aldehydes and a-chloro fatty zotocin-induced diabetic mice and observed increased Th17 acids that are generated under inflammatory conditions in vivo are differentiation using supernatants from co-culture or Th17-polarizing able to induce BBB dysfunction via induction of ER stress by the conditions. These data suggest that hyperglycemia favors Th17 PERK-mediated signalling cascade. responses and may increase inflammation during recognition of MRSA-infected apoptotic cells by DCs. Besides, overwhelming IL- 17A production may contribute to neutrophil recruitment, abscess formation and pathology in skin lesions in diabetics. Autoimmune Diseases: RA, Psoriasis, Financial support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7; SLE, IBD, MS NIH HL-103777; R01HL124159-01; T32AI060519; T32DK007519. B010 THE TYROSINE KINASE INHIBITOR PREVENTS B008 CYTOKINE INDUCTION IN THE PROGRESSION MPO-DERIVED CHLORINATED LIPIDS INDUCE OF ADJUVANT INDUCED ARTHRITIS ENDOPLASMIC RETICULUM STRESS IN BRAIN MICROVASCULAR ENDOTHELIAL CELLS Sheikh Fayaz Ahmad, Mushtaq Ahmad Ansari, Khairy M A Zoheir, Saleh A. Bakheet, Sabry M. Attia Nora Kogelnik1, Christoph Nusshold1, Christine Rossmann1, Gerald 2 1 3 1 Rechberger , Helga Reicher , Pierre-Oliver Courad , Ernst Malle , King Saud University, Riyadh, Saudi Arabia Wolfgang Sattler1 Rheumatoid arthritis (RA) is one of the major autoimmune diseases 1Medical University of Graz, Graz, Austria; 2University of Graz, of global prevalence. Irrespective of much research in RA disease, Graz, Austria; 3University Paris Descartes, Paris, France no drugs with capable safety profiles are yet available. Protein tyrosine kinases help to regulate the expression of many genes that Most regions of the brain operate within a well-controlled environ- play important roles in arthritis. We investigated the possible anti- ment separated from the milieu of the peripheral circulation by the arthritic effects of the tyrosine kinase inhibitor in a mouse model of

123 S106 Inflamm. Res. adjuvant induced arthritis (AIA). We report here that tyrosine kinase In summary, these data show that 19, similar to the original inhibitor exerts potent anti-arthritic effects in animal model of AIA tripeptide KdPT, is able to effectively ameliorate ongoing inflam- in vivo. In this study, we examined the effects of tyrosine kinase mation in skin and gut. Because of its improved phys.-chem. inhibitor on the key mediators of arthritic inflammation. Tyrosine properties 19 may be formulated as topical drug. Moreover, its oral kinase inhibitor treatment significantly attenuated the severity of bioavailability is likely to be improved compared to KdPT. AIA, reduced the arthritis scores, a substantial reduction in the levels of cell surface receptors, chemokines, and as well as the pro- inflammatory mediators. However, tyrosine kinase inhibitor signifi- cantly up-regulated the number of anti-inflammatory mediators B012 levels. Our results suggest that treatment with tyrosine kinase CYTOKINE PRODUCTION AND CALCIUM inhibitor attenuated AIA in mice might offer a promising alterna- tive/adjunct treatment for RA. INFLUX OF T LYMPHOCYTES UPON KV1.3 AND IKCA1 CHANNEL INHIBITION IN RHEUMATOID ARTHRITIS AND ANKYLOSING SPONDYLITIS B011 Gergely Toldi1, Luis Munoz2, Martin Herrmann2, Georg Schett2, ANTI-INFLAMMATORY EFFECTS OF Attila Balog3 TRIPEPTIDE WOL074-019 IN MURINE MODELS OF PSORIASIS AND COLITIS 1Semmelweis University, Budapest, Hungary; 2University of Erlangen-Nu¨rnberg, Erlangen, Germany; 3University of Szeged, Michael Soeberdt1, Carlo Sternemann2, Christoph Abels1, Szeged, Hungary 2 2 Thomas A. Luger , Karin Loser Voltage-sensitive Kv1.3 and calcium-dependent IKCa1 lymphocyte potassium channels have been implicated as important targets of 1 Dr. August Wolff GmbH & Co. KG Arzneimittel, Bielefeld, Germany; selective immunomodulation in autoimmune disorders. The transient 2 Department of Dermatology, University of Mu¨nster, Mu¨nster, increase of the cytoplasmic free calcium level, which is a prerequisite Germany of the downstream events of lymphocyte activation, is maintained by KdPT is a tripeptide with broad anti-inflammatory activity. It was the function of potassium channels. They conserve the electrochem- shown to be effective in different murine models of intestinal ical potential gradient via the efflux of potassium from cytoplasm. inflammation and psoriasis. Oral KdPT proved to be safe and well The relationship between the influx of calcium through the cell tolerated in a single and two multiple ascending dose clinical membrane and the efflux of potassium makes proliferation and acti- studies. Moreover, in a phase II trial it was shown to significantly vation of lymphocytes sensitive to pharmacological inhibition of accelerate disease remission in patients with mild to moderate active Kv1.3 and IKCa1 channels, and provides a potential opportunity for ulcerative colitis. However, KdPT’s phys.-chem. properties limit its targeted intervention. oral bioavailability and are not suitable for development of a topical We compared the alterations in cytokine production (IL-1b, IL1- formulation. Thus, we have designed and synthesized numerous RA, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A/F, IFN-g, analogues of KdPT with optimized phys.-chem. properties. Effects TNF-a) upon selective inhibition of Kv1.3 or IKCa1 channels (by determined on protein and gene level in vitro (murine and human T MGTX and TRAM, respectively) in 8 healthy donors (HD), 15 cells, HaCaT) showed anti-inflammatory activity of WOL074-019 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) (19) comparable to KdPT and thus 19 was selected for in vivo patients. We also determined calcium influx kinetics and its sensi- studies. tivity to Kv1.3 and IKCa1 channel inhibition following PHA The anti-inflammatory and immunomodulatory potential of 19 activation in Th1, Th2 and CD8 cells. in vivo was investigated in the mouse model of imiquimod-induced The application of TRAM resulted in a lower production of psoriasis-like skin inflammation (topical application for 8 days). At d 5 TNF-a and IL1-RA in all three study groups. Inhibition by TRAM and 6 after the start of treatment mice were injected with either PBS, had contrary effects on the production of IL-1b and IL-5: while their betamethasone dipropionate (BMDP), KdPT or 19 (5 lg, i.v.). Inter- production was increased by PBMCs of RA patients, this effect was estingly, similar to KdPT, treatment with 19 ameliorated ongoing skin not observed in HD and AS PBMCs. While treatment with MGTX inflammation as shown by the reduced thickness of epidermal rete resulted in a similar decrease of calcium influx in the CD4 and Th2 ridges and the decreased levels of pathogenic Th1 as well as Th17 subsets across all study groups, TRAM treatment had opposite cells in regional lymph nodes and lesional skin quantified by flow effects on RA and HD samples: it decreased calcium influx in the cytometry, real-time PCR and immunofluorescence staining. This Th2 and CD8 subsets in RA, while only Th1 cells were affected in effect was mediated by the reduction of pro-inflammatory cytokines HDs. like IL-1b,IL-6orTNF-a and the expansion of immunosuppressive PBMCs isolated from RA and AS patients react with a different Treg in 19-treated mice versus controls. In addition, 19 was tested in pattern of alterations in cytokine production and calcium influx the dextrane sodium sulphate (DSS)-induced colitis model in mice. kinetics compared to HDs. The application of lymphocyte potassium Mice that were injected with 19 (5 lg, i.p.) at d 4–7 were protected channel inhibitors has controversial effects on cytokine production from weight loss and moreover, did not show any signs of diarrhea or and calcium influx patterns in patients with RA and AS, questioning rectal bleeding. At day 8 (end of experiment) 19-treated mice looked its therapeutic relevance. TRAM decreases the secretion of TNF-a like BMDP-treated controls. Quantitative real-time PCR as well as and increases the secretion of the Th2 cytokine IL-5 in patients with immunofluorescence staining of colonic tissue revealed decreased RA. This would contribute to the shift of the immune balance levels of IL-6, IL-1b,TNF-a and IFN-c. Moreover, the numbers of towards less tissue damage and cellular paucity in RA. Nevertheless, neutrophils and macrophages were significantly reduced in mesenteric TRAM increases the secretion of IL-1b and at the same time lymph nodes and the colon from 19-treated mice versus PBS-injected decreases that of IL1-RA which is unfavorable, since it supports an controls. IL-1b mediated pro-inflammatory response. Additionally, TRAM

123 Inflamm. Res. S107 also inhibits the short-term activation of Th2 lymphocytes in RA, Methods:We retrospectively analyzed the data from the medical leaving that of Th1 cells unaffected, probably further contributing to records of 53 patients with TA (male 11, female 42) who received a shift of the inflammatory balance to a pro-inflammatory profile. treatment at our hospital From January, 2000 to September, 2014. The differences observed in cytokine response upon inhibition of Results:The average age at onset of disease was 28.6 ± 1.4 Kv1.3 and IKCa1 channels support the differential pathomechanisms (mean ± SE) years old, period from onset to diagnosis 14.8 ± of RA and AS. 7.43 months, and observation period was 181.8 ± 17.1 months, respectively. Disease classifications were type I (13), type II a (5), type II b (10), type III (n = 1), type IV (n = 1), and type V (n = 21). In adults, left subclavian artery involvement was common B013 (86.4 %), however, was less common in child (37.5 %). Celiac, SUPPRESSING INFLAMMATORY BOWEL mesentric and renal artery lesions were remarkably present in chil- dren compared with those of in adults (75.0 and 18.2 %, DISEASE BY MODULATING PRO- respectively). Stenosis was found in 43 cases, and in 37 patients INFLAMMATORY GENE EXPRESSION AND stenotic lesions were found at time of diagnosis. Aneurysm was IMMUNE CELL POPULATIONS found in 8 cases, of which 1 burst. Cerebral infarction was observed in 5 patients, 3 of which were on the first visit. Aortic regurgitation (AR) was found in 13 cases on the first visit, and 2 were found after Anh T. Do, Praveer Gupta, Abishek Iyer, David P. Fairlie treatment. Revascularization or valve replacement was performed 30 times in 13 patients. Combination therapy with immunosuppressant University of Queensland, St Lucia, QLD, Australia and glucocorticoid was administered in 12 patients with 3 biologic Inflammatory bowel diseases (IBDs) are a group of complex agents. Relapse was revealed 23 times in 11 cases. Duration from inflammatory conditions affecting colon and rectum. Different subsets first induction therapy to relapse was 26.6 ± 23.1 months. New of T-lymphocytes (Th1, Th2, Th17 and Treg) have been implicated in vascular involvement or progression of stenosis under the negative inflammatory bowel diseases (IBDs), with various inflammatory CRP or ESR was occurred in 7 cases. The survival was 97.5 % at cytokines and chemokines (IL-1, IL-6 and TNFa) involved in the 5 years, 96.9 % at 10 years, and 81.3 % at 20 years. Five patients disease pathogenesis. Anti-TNF therapy (e.g. Infliximab, Enteracep) died at 19.1 years of age after a diagnosis (3 infections, 1 cerebral is currently used as last resort treatment for refractory IBD patients. infarction, and 1 myocardial infarction). However, these drugs are expensive, not effective in everyone and Conclusion: Our findings suggested that the long-term outcome was have inherent serious side-effects. To treat IBD more effectively, we good, and TA patients with cerebral infarction and AR were infre- must first understand the nature of immune responses occuring from quent after immunosuppressive treatment. It is necessary to the acute stages through progression to chronic disease when commence treatment before severe stenosis, ischemia and AR occur. inflammation can trigger tumor formation. Using RT-PCR arrays and flow cytometry techniques we have serially characterized inflamma- tory changes in the progression of early to late stage of IBD and tumorigenesis in a dextran sulfate sodium (DSS)-induced IBD model B015 in mice. As disease progressed, cytokine expression pattern with more C-REACTIVE PROTEIN COULD PROMOTE involvement of lymphocyte cytokines, and progressively reduced OSTEOCLASTOGENESIS monocyte cytokines involvement. At the most chronic stage, 20 % of animals developed dysplastic lesions, associated with increases in Sang-Heon Lee1, Chong-Hyeon Yoon2 Il13, Il10 and Il4 mRNA, along with Cxcl13, Cxcl16, Ccl20 and G- Csf 1 2 . These are chemoattractants for T- and B-lymphocytes, tumor- Konkuk University School of Medicine, Seoul, South Korea; The associated neutrophils and macrophages, suggesting involvement of Catholic University of Korea, Colleg of Medicine, Seoul, South Korea different immune cells in dysplastic stages of disease. Furthermore, a lysine deacetylase inhibitor, N-Hydroxy-7-[5-(4-tertbutoxycarbony- Background: C-reactive protein (CRP) is one of the biomarkers for laminophenyl)-3-isoxazolecarboxamido]heptanamide (BML-281) the diagnosis and assessment of disease activity in rheumatoid altered these immune responses with suppression of multiple arthritis (RA). CRP is not only the by-product of inflammatory inflammatory cytokines/chemokines and reduced infiltrated B-lym- response, but also plays pro-inflammatory and pro-thrombotic roles. phocytes by 40 % in both colon and lymph node, leading to delay and Objectives: This study aims to determine the role of CRP on bone reduction of the dissease symptoms. destruction in RA. Methods: CRP levels in RA synovial fluid (SF) and serum were measured using the immunoturbidimetric method. The expression of CRP in RA synovium was assessed using immunohistochemical B014 staining. CD14 + monocytes from peripheral blood were cultured THE CLINICAL FEATURES OF TAKAYASU’S with CRP, and RANKL expression and osteoclast differentiation were ARTERITIS IN OUR HOSPITAL (53 CASES) evaluated by using real-time PCR, counting TRAP-positive multin- ucleated cells and assessing bone resorbing function. CRP-induced osteoclast differentiation was also examined after inhibition of Takumi Nagamoto1,2, Kenta Misaki1,2, Rintaro Saito1,2, Fcgamma receptors. Yuri Nakamura1,2, Yoshie Gon1,2, Toshihiko Yokota1,2 Results: There was a significant correlation between CRP levels serum and SF in RA patients. The SF CRP level was correlated with 1Kurashiki Central Hospital, Kurashiki, Japan; 2Department of interleukin (IL)-6 levels, but not with RANKL levels. Immunohis- Endocrinology and Rheumatology tochemical staining revealed that CRP was more abundantly Objectives:To reveal the clinical findings, and the long-term prog- expressed in the lining and sublining areas of the RA synovium, nosis of Takayasu’s arteritis (TA). compared with the osteoarthritis synovium. CRP promoted RANKL

123 S108 Inflamm. Res. production in monocytes, which in turn, induced osteoclast differ- Disclosures: All authors are employees of AbbVie. The design, study entiation from monocytes and increased bone resorption in the conduct, and financial support for this research were provided by absence of RANKL. AbbVie. AbbVie participated in the interpretation of data, review, and Conclusions: CRP could play an important role in the bony destruc- approval of the publication. tive process in RA through the induction of RANKL expression and promote direct differentiation of osteoclast precursors into mature osteoclasts. In the treatment of RA, lowering CRP levels is a sig- nificant parameter not only for improving disease activity but also for B017 preventing bone destruction. THE OPPOSING EFFECTS OF CERS2 AND CERS6 IN THE DEVELOPMENT OF EAE

Susanne Schiffmann4,2, Julia Barthelmes2, Max Eberle2, B016 1 1 3 A NOVEL APPROACH FOR TREATMENT OF Yael Pewzner-Jung , Anthony Futerman , Philipp Ebel , Klaus Willecke3, Gerd Geisslinger2, Sabine Gro¨sch2 RHEUMATOID ARTHRITIS: COMBINED TNF-A AND SCLEROSTIN INHIBITION BLOCKS 1Weizmann Institute of Science, Rehovot, Israel; 2University of Frankfurt, Frankfurt, Germnay; 3University of Bonn, Bonn, Germany; INFLAMMATION AND RESTORES BONE IN 4 A MOUSE COLLAGEN INDUCED ARTHRITIS Fraunhofer Institute IME-TMP, Frankfurt am Main, Germnay MODEL Ceramide synthases (CerS) synthesize ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune Wendy O. Waegell, Alexander V. Ivanov, Regina N. Mario, encephalomyelitis (EAE), an animal model of multiple sclerosis Shaughn Bryant, Trudi M. Veldman, Lisa M. Olson (MS), we observed a significant elevation of CerS2 and CerS6 and its products, C24-/16-ceramides, in CD11b+ cells (monocytes and neu- AbbVie Bioresearch Center, Worcester, MA, USA trophils) isolated from blood. This result correlates with the clinical One of the hallmarks of RA is progressive bone erosion that is finding that CerS2 and CerS6 mRNA expression and C24-/C16-ce- clinically managed by TNF blockade. In the majority of RA ramide levels were significantly increased in white blood cells of MS patients, anti-TNF therapy halts bone loss but little healing of bone patients. The increased CerS2 mRNA/C24-ceramide expression in erosions is observed. Sclerostin acts as an inhibitor of Wnt signal- neutrophils/monocytes seems to mediate pro-inflammatory effects, ing, inhibiting osteoblast differentiation and function. Two genetic while the increased CerS6 mRNA/C16-ceramide expression in neu- diseases, Van Buchem’s disease and sclerosteosis, result from loss trophils/monocytes seems to mediate anti-inflammatory effects. The of function mutations in the SOST gene encoding sclerostin, and genetic deletion of CerS2 or Cer6 significantly ameliorates or worsens result in overproduction of bone. Recent reports suggest that scle- the clinical symptoms, respectively, due to a reduced or increased rostin is up-regulated in the synovium of RA patients as well as in infiltration of immune cells, in particular neutrophils, into the central the HuTNF transgenic mouse model of RA. Blocking the inhibitory nervous system. CXCR2 chemokine receptors, expressed on neu- action of sclerostin and restoring Wnt signaling will therefore lead trophils, promote the migration of neutrophils into the central nervous to new bone formation. This has been demonstrated clinically in a system, which is a prerequisite for the recruitment of further immune phase I trial in which a single dose of anti-sclerostin mAb increased cells and the inflammatory process that leads to the development of BMD in the lumbar spine and hip. We propose a hypothesis that MS. Interestingly, neutrophils isolated from CerS2 or CerS6 null EAE combined blockade of TNFa and sclerostin may provide greater mice, as opposed to WT EAE mice, were characterized by signifi- efficacy and DMARD activity by combining the anti-inflammatory/ cantly lower or higher CXCR2 receptor mRNA expression resulting bone sparing of TNF inhibition with bone anabolic activity induced in their reduced or increased, respectively, migratory capacity by sclerostin inhibition, leading to bone healing, cartilage protection towards CXCL2. Most importantly, G-CSF-induced CXCR2 expres- and improved joint function. To test this hypothesis we have eval- sion was significantly reduced or increased in CerS2 null and CerS6 uated the efficacy of single agent versus dual agent inhibition of null neutrophils, respectively, and their migratory capacity was sig- TNF and sclerostin in collagen induced arthritis, a mouse model of nificantly impaired or improved, respectively. In conclusion, our data RA. Anti-TNF treatment initiated at first clinical signs of arthritis, a strongly indicate that CerS2 promotes inflammatory processes, while time-point with no bone loss, was effective in reducing inflamma- CerS6 inhibits them presumably via a feedback mechanism. Thus, tion either alone or in combination with anti-sclerostin mAb, CerS2 and CerS6 have opposing effects during the development of whereas sclerostin inhibition alone did not impact inflammation. In EAE and MS. contrast, when treatment was started 5 days after onset of clinical signs, a time-point with moderate bone loss, neither TNFa nor sclerostin inhibition had an effect on inflammation. However, scle- rostin blockade prevented additional bone loss after treatment B018 initiation based on micro-computed tomography evaluation. In an ADA2 DEFICIENCY ATTENUATES M2 additional study, we observed that sclerostin blockade in combina- MACROPHAGES DIFFERENTIATION tion with IL-a and IL-b blockade, which is able to block inflammation when treatment is initiated at the peak of inflamma- Dan Yang1, Guibin Chen1, Qing Zhou2, Yuting Huang1, Zhen Yu1, tion, led to bone restoration in the arthritic joint. Hence, blocking Avram Walts1, Alejandra Negro1, Cynthia St. Hilaire1, inflammation and bone resorption by inhibiting TNF or IL-1 and Ivona Aksentijevich2, Manfred Boehm1 promoting bone formation by inhibiting sclerostin could lead to healing of bone erosions and offer a promising new approach as a 1NHLBI, Bethesda, MD, USA; 2NHGRI, Bethesda, MD, USA novel treatment for RA.

123 Inflamm. Res. S109

Background: CECR1/ADA2 (ADA2) belongs to the adenosine Paulo, School of Pharmaceutical Sciences of Ribeira˜o Preto, Sa˜o deaminase family. Loss-of-function mutations in the CECR1 gene Paulo, Brazil; 5University of Sa˜o Paulo, Ribeira˜o Preto Medical (Deficiency of ADA2; DADA2) have been linked to early-onset School, Sa˜o Paulo, Brazil; 6University of Sa˜o Paulo, School of stroke, vasculopathy and inflammation. Mutant ADA2 protein and Pharmaceutical Sciences of Ribeira˜o Preto, Sa˜o Paulo, Brazil ADA2 knockdown in U937 cells have been shown to inhibit Introduction and objective: Rheumatoid arthritis (RA) is a chronic monocyte to macrophage differentiation. The mechanism of how disease characterized by synovial tissue inflammation, where neu- ADA2 affects monocyte/macrophage differentiation is largely trophils are the most abundant cells and are important mediators of unknown. Methods: We utilized human induced pluripotent stem tissue destruction. Tissue damage in AR involves excessive produc- cell (hiPSC)-derived monocytes, together with human primary tion of reactive oxygen species (ROS) triggered by immune monocytes and human myeloid U937 cell line, to study mono- complexes (IC) and neutrophils interactions via receptors FccR and cyte/macrophage differentiation. Flow cytometry, cytokine profiling, complement receptors (CR). Modulation potential of ROS generation phagocytosis assay, and immunofluorescence staining were used to may be relevant to the maintenance of body homeostasis. Compounds characterize cell markers and functions. Results: In DADA2 as flavonols have been considered helpful to inhibit and/or modulate patients’ monocytes, expression of M-CSF-induced M2 macrophage the neutrophils functions. In this study, we evaluated the oxidative markers CD163 and CD206 were decreased. GM-CSF-induced burst from peripheral blood neutrophils (PBN) from AR patients, patients’ M1 macrophages had higher expression of pro-inflamma- stimulated via FccR and FccR/CR receptors, the chemotactic activity tory cytokines upon LPS stimulation compared to control cells. This this cells and the ability of flavonols (quercetin and galangin) to suggests an M1/M2 differentiation imbalance in patients’ cells. inhibit these two functions. Consistent with data from the primary cells, hiPSC-derived patients’ Methods: Human peripheral blood was collected from the healthy macrophages expressed lower levels of the M2 markers CD206, volunteers and RA patients treated with methotrexate (MTX). The CD163, CD204, and CD209 compared to control hiPSC-derived measurement total ROS production of neutrophils was taken with cells. Besides, the expression of monocyte maturation markers luminol (CL-lum) chemiluminescence assays. We evaluated the HLA-DR, CD11c and CD64 also decreased in patients’ cells. This neutrophils chemotactic activity in vitro using acrylic migration indicates that the ADA2 deficiency leads to a disrupted M-CSF chambers and zymosan (Zy), a polysaccharide responsible for cell signaling. Furthermore, we observed that the addition of adenosine activation, was employed as complement activator for chemotactic leads to a down regulation in the expression of M2 markers, while fragments generation from NHS (normal human serum) and recombinant human ADA2 partially rescued this phenotype. We rheumatoid arthritis human serum (RAHS). Results: Comparison of found that the A3 adenosine receptor (A3AR) and A2bAR agonists the oxidative burst mediated by FccR (IC alone) and FccR/CR (IC partially mimic the effect of adenosine. Inversely, the addition of opsonized with serum) in neutrophils from RA patients and healthy A3AR antagonist rescued expression of the M2 marker in patient’s volunteers resulted in increased responses only in neutrophils from hiPSC-derived monocytes. This suggests that the adenosine-medi- patients with RA (p \ 0.001). Galangin and quercetin (mean ated pathways at least partially contribute to the impaired IC50 = 2.5 lM; 1.75 lM respectively) suppressed nearly 65 % CL- differentiation of M2 macrophages in patients with DADA2. Simi- lum of RA patients’ neutrophils. The chemotactic activity of neu- larly, ADA2 knockdown in U937 cells decreased M2-like trophils also was investigated. When neutrophils were incubated with macrophage markers. Meanwhile, reduced activation of PKC-delta NHS without the complement activator, the cells did not display and p38 suggesting a role for PKC/p38 signaling. Conclusion: increased migration, whereas enhanced migration was observed when deficiency of ADA2 attenuates M2 macrophage differentiation. Data cells were incubated with RAHS. These results indicate that RAHS from ADA2-knockdown in U937 cells suggest the mechanism by contains chemoattractant factors even before Zy activation occurs. which ADA2 regulates M2 macrophage differentiation via PKC/p38 The galangin diminished chemotactic activity when cells were incu- signaling. Data from primary monocytes suggests a role for ade- bated with RASH or Zy (p \ 0.01), indicated the possible modulation nosine signaling in this process. Together this data demonstrate a in complement activation. role for adenosine and PKC/p38 signaling pathways in the ADA2- Conclusion: Taken together the results showed thatthe flavonols galangin mediated macrophage differentiation. The altered ratio of tissue M2/ and quercetin partially inhibited the oxidative metabolism neutrophils M1 macrophages likely contributes to inflammation and the vascu- and only the galangin inhibited chemotactic activity as consequence of lopathic/vasculitis phenotype in patients with DADA2. complement activation. The use these compounds to modulate neutrophil functions can be a promising safe therapeutic strategy to control inflammation in RA patients. Support: FAPESP, CNPq. B019 FLAVONOLS MODULATE THE PRODUCTION OF REACTIVE OXYGEN SPECIES AND THE B020 CHEMOTACTIC ACTIVITY OF PERIPHERAL AMELIORATION OF CHRONIC COLITIS IN MICE BLOOD NEUTROPHILS FROM RHEUMATOID BY GENETIC DELETION OF THE IL-13 DECOY ARTHRITIS PATIENTS RECEPTOR

Trisha Pasricha, Thirumalai Ramalingam, Thomas Wynn Larissa F. Marchi1, Adriana P. Paschoalato2, Reneˆ R. Oliveira3, Ana Elisa C. Azzolini4, Eduardo A. Donadi5, Yara L. Valim6 National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD, USA 1University of Sa˜o Paulo, School of Pharmaceutical Sciences of Ribeira˜o Preto, Sa˜o Paulo, Brazil; 2University of Sa˜o Paulo, Ribeira˜o Introduction: The mainstays of therapy in inflammatory bowel dis- Preto Medical School, Sa˜o Paulo, Brazil; 3University of Sa˜o Paulo, ease, anti-TNF-alpha agents, are ineffective in a significant proportion Ribeira˜o Preto Medical School, Sa˜o Paulo, Brazil; 4University of Sa˜o of patients, underlying a great need to explore broader therapeutic

123 S110 Inflamm. Res. options within the inflammatory signaling pathways that govern collagen-induced arthritis (CIA) is widely accepted as a valid RA immune dysregulation. We hypothesized that IL-13Ra2 knock-out animal model for mimicking human RA. Thus, we aimed to inves- (KO) mice would demonstrate restored homeostatic activity of IL-13 tigate the role of HQ exposure on CIA in Wistar rats and the involved in a chronic inflammatory disease model and lead to improved mechanisms. physiological outcomes. Methods: Animals were immunized subcutaneously at the tail base by Objective: To assess the effects of genetic deletion of the IL-13 decoy using 200 lg of bovine type-II collagen emulsified 1:1 with complete receptor, IL-13Ra2, in a chronic model of inflammatory bowel Freund’s adjuvant (CFA). A booster injection of 100 lg of bovine disease. type-II collagen emulsified in CFA was administered 7 days later. Methods: Chronic colitis was induced in wild type (WT) C57B/6 The rats were divided into three groups according to exposition with mice and IL-13Ra2 KO C57B/6 mice via three 7-day cycles of 3 % saline, HQ vehicle (saline ethanol solution 1:20) or HQ 25 ppm. All dextran sodium sulfate (DSS) over the course of 5 weeks. Control animals were exposed for 35 consecutive days, 1 h/day, using a mice of both strains were administered normal water throughout the nebulization chamber. CIA was induced on day 7th and booster on course of the experiment. At day 35, colon tissue was harvested to day 14th. Animals were submitted to clinical evaluation (score scale analyze the severity of chronic colitis. Parameters assessed included of 0–4: 0 = no arthritis; 1–2 = weak arthritis, with inflamed digits; weight loss, qPCR analysis of inflammatory markers, colon collagen 3 = medium arthritis, with more than 2 digits and an inflamed foot- content measured by hydroxyproline assay. Two independent blin- pad; 4 = strong arthritis, with all inflamed digits and paws), ded reviewers performed histological scoring. Colon sections were hematological parameters and histological analysis of synovial graded on a 0–12 scale of increasing severity, assessing mucosal membranes. Data were obtained in nine animals per group. architecture, lymphocytic infiltration, epithelial defects, and goblet Results: Data obtained showed that HQ exposure caused a score 3–4 cell loss. of CIA; HQ vehicle 2–3 and saline 1–2. HQ exposure caused weight Results: All animals survived the study. On day 35 of the experiment, body loss (D day 35-day 1: saline = +14.92 %; vehicle = -3.53 %; IL-13Ra2 KO mice on DSS demonstrated greater weight than WT HQ =-9,66 %), no altered the numbers of circulating cells, and mice on DSS (p = 0.04). In qPCR analysis of distal colonic tissue, marked increased leukocyte numbers into the synovial fluid and IL-13Ra2 KO mice on DSS compared to WT mice demonstrated a neutrophils influx into the synovial membrane. Moreover, HQ expo- significantly reduced induction of TNF-alpha (p \ 0.0001), IFN- sure caused pannus formation and hyperplasia of the synovial cells. gamma (p = 0.02), IL-1B (p = 0.0009), and TGF-beta (p = 0.0008). Conclusion:: Our data clearly show that HQ exposure aggravates the For all qPCR studies, there were no significant differences between development of RA, suggesting that HQ may be a determinant WT mice on normal water, IL13Ra2 KO mice on normal water, and component of cigarette on RA induction. IL-13Ra2 mice on DSS. Additionally, IL-13Ra2 KO mice on DSS Supported by Capes. had a significantly less hydroxyproline content within equal length segments of colon tissue compared to WT mice on DSS (p = 0.004). Lastly, histological scores of IL-13Ra2 KO mice on DSS trended lower than WT mice on DSS. B022 Conclusions: We have shown that genetic deletion of the IL-13Ra2 MULTI-MODALITY PATHWAY receptor in mice can serve a protective role in a chronic inflammatory CHARACTERIZATION AND DISEASE bowel disease model across several physiological parameters. This data suggests that the IL-13 decoy receptor may be an important EVALUATION IN LUPUS MODEL therapeutic target for patients by restoring the normal homeostatic function of IL-13. Larger additional studies to confirm this regulatory Jie Zhang-Hoover, Alan Byford, Mark Zielstorff, Robert Faltus, pathway are currently underway. Joseph Eckman, Kimberly Bettano, Gain Robinson, Raquel Sevilla, Adam Hartigan, Weisheng Zhang, Milenko Cicmil

B021 Merck Research Laboratories, Merck & Co., Inc, Rahway, NJ, USA HYDROQUINONE EXPOSURE AGGRAVATES MRL/lpr model mimics some aspects of genetics and phenotypes of COLLAGEN INDUCED ARTHRITIS (CIA) human lupus disease in various tissues and has been used extensively DEVELOPMENT IN RATS for pharmacological evaluation of new therapies. We sought to val- idate utility of the model for potential pathways by measuring immune inflammation related gene expression levels in kidneys and Cintia S. Heluany1, Leoanrd k. Kupa1, Mariana N. Viana2, 2 1 characterizing immune cell phenotypes in peripheral blood of mice at Cristina M. Fernandes , Sandra P. Farsky various stages of disease development. In addition to routine in life biochemistry and terminal histology analyses, we monitored disease 1Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, 2 phenotypes longitudinally in vivo using multi-modality imaging: (1) Sa˜o Paulo, Brazil; Pharmacology Laboratory, Butantan Institute, tissue reactive oxygen species (ROS) level by luminol-biolumines- Sa˜o Paulo, Brazil cence imaging (luminol-BLI); (2) glomerular filtration rate (GFR) Background: Rheumatoid arthritis (RA) is a chronic autoimmune tracked by contrast agent Omnipaque washout using micro-computed disease and smoking is assumed to be the main etiological factor in tomography (micro-CT); (3) cerebral cortical thickness by brain the pathogenesis of RA. Studies have shown that smoking is a trigger magnetic resonance imaging (MRI). + factor in the development of RA and also it also worsens pre-existing MRL/lpr mice showed a disease associated change of CD3 + disease. However, the importance of cigarette components on the B220 cells (potential pathogenic immune cells) in peripheral blood overall effects of RA remains unknown. Hydroquinone (HQ) is a and up-regulation of PD1/PD-L2, BTLA/HVEM, GITR/GITR ligand, phenolic compound of natural or anthropogenic source, also found in and TIGIT gene expression in kidneys. Flow cytometry analysis + + high concentrations in cigarette (is the major oxidative component showed that CD3 B220 cells increased from around 2 % at 8 weeks found in cigarette), as well as it is a benzene metabolite. Type II of age to over 30 % of peripheral blood leukocytes at 19 weeks of age

123 Inflamm. Res. S111 in MRL/lpr mice. ROS production in the skin and hind limbs visu- During a median follow-up of 64 months (range 3–120 months), alized by luminol-BLI was detectable at 12 weeks of age and there were 12 flares according to the SLE flare index. SLE group with preceded gross skin lesion observation at 14 weeks of age. MicroCT serum chemerin levels above the cut-off value had significantly detected a GFR decrease as early as 14 weeks of age, compared to the shorter relapse-free survival than the other group (p = 0.05). blood urea nitrogen level increase at 16 weeks of age. Furthermore, brain MRI detected cerebral cortical thinning as early as 10 weeks of age. Cyclophosphamide treatment reduced disease progression sig- Table 1. The comparison of biochemical parameters of SLE and nificantly across multiple readouts. controls. For the first time we report a comprehensive approach by profiling SLE Controls p potential disease modulating gene signatures and immune cell phe- notypes and using clinically relevant PD markers to enhance the Glypican-4 (ng/ml) 3.76 ± 0.65 2.73 ± 0.52 \0.001 pharmacological translatability of compound evaluation in a lupus pre-clinical model. Chemerin (lg/L) 138.2 ± 115.2 58 ± 53.3 \0.001 RBP-4 (ng/ml) 54.39 ± 54.8 48.16 ± 81.3 0.72

B023 Conclusions: Serum chemerin and glypican-4 levels were increased in INCREASED SERUM CHEMERIN LEVELS MIGHT SLE patients; and they might be related to disease pathogenesis. PREDICT FLARES OF SYSTEMIC LUPUS Increased chemerin levels in SLE might be predictive for future flares. ERYTHEMATOSUS

Omer N. Pamuk, Mehmet S. Uyanik, Gulsum E. Pamuk B024 Trakya University Medical Faculty, Edirne, Turkey THE EFFECTS OF THE SPLEEN TYROSINE Background and Objectives: Recent studies reported that the receptor KINASE INHIBITOR, FOSTAMATINIB, ON AN for chemerin, Chem R23 was expressed intensively on plasmocytoid IMMUNE THROMBOCYTOPENIA MOUSE dendritic cells (DC); that chemerin was a chemoattractant factor for MODEL DC; and chemerin/Chem23R axis was responsible for especially the renal recruitment of DC in systemic lupus erythematosus (SLE). Gulsum E. Pamuk, Mehmet S. Uyanik, Turan Karaca, Selim Demirtas, Retinol-binding protein-4 (RBP-4) is also secreted by adipocytes; and Ahmet M. Demir, Omer N. Pamuk it is related to atherogenesis and insulin resistance. It was observed that anti-TNF therapy reduced RBP-4 levels significantly in anky- Trakya University Medical Faculty, Edirne, Turkey losing spondylitis (AS). In RA, the increased expression of glypican-4 was found to be related to angiogenesis in inflamed synovitis. Until Background: Immune thrombocytopenia (ITP) is the most common now, these cytokines have not been evaluated in systemic lupus autoimmune bleeding disorder. The major mechanism involves erythematosus (SLE). We determined serum chemerin, RBP-4, and the phagocytosis of antibody-coated platelets by Fc-gamma glypican-4 levels in SLE; and investigated clinical features associated receptor (FccR)-bearing macrophages. Many of the FccR-medi- with them. ated activities are dependent on the spleen tyrosine kinase (Syk) Methods: The study includes age-and-sex matched 46 patients with pathway. SLE (45 F, 1 M, mean age: 40.5 ± 12) and 26 apparently healthy Objectives: The aims of the present study were to test the hypotheses subjects (25 F, 1 M, mean age: 40.4 ± 10 years). The demographic of whether (1) The Syk pathway is involved in both the pathogenesis and clinical features of SLE patients were recorded from medical of ITP and in the treatment response to ITP, (2) Fostamatinib (Fos, charts. Serum chemerin, RBP-4 and glypican-4 levels were deter- R788), a novel Syk inhibitor drug, has any effects on the complement mined with the ELISA method. SLE disease activity index (SLEDAI) system, lymphocytes, and macrophages in ITP, and whether (3) Fos score was calculated at the time of inclusion into the study. SLE flare decreases the migration of T lymphocytes to the bone marrow and the was assessed by the SELENA-SLEDAI flare index. spleen in ITP. Results: Serum chemerin and glypican-4 levels in SLE were signifi- Methods: Six to eight week-old BALB/c mice were divided into cantly higher in SLE patients than in healthy controls (p eight groups: (1) Sham-operated; (2) isolated ITP; (3) Fos (3 g/kg, values \ 0.001). RBP-4 level was similar in SLE patients and in p.o.); (4) Fos + Liposomal clodronate (LC) (0.2 ml/U/mouse, i.p.); controls (p [ 0.05) (Table 1). In the SLE group which was active (5) Fos + cobra venom factor (CVF) (25 U/mouse, i.p.); (6) Intra- according to SLEDAI score ([4), serum chemerin level was signifi- venous immunoglobulin (IVIG) (2 g/kg, i.m.); (7) IVIG + LC; and cantly higher when compared to the inactive group (191.4 ± 159 vs. (8) IVIG + CVF. ITP model was formed by the i.v. injection of 4 lg 120.5 ± 72.5, p = 0.048). Serum glypican-4 level was significantly of anti-CD41/Integrin alpha-2b antibody. Spleen tissue sections were lower in SLE patients with thrombocytopenia when compared to cut and stained with Syk, phospho (p)-Syk, PECAM, and CX3CR1. nonthrombocytopenic SLE patients (3.34 ± 2.84 vs. 3.86 ± 0.63, Results: Administration of IVIG (p \ 0.001) and Fos (p \ 0.001) p = 0.041). prevented the fall in platelet counts in an ITP mouse model. Mac- ROC curve analysis revealed that the area under the curve values rophage depletion by LC, and complement depletion by CVF did not for glypican-4 and chemerin levels in SLE were, respectively, 0.903 provide any extra increments in the platelet counts. The intensity of (95 % CI 0.823–0.984) and 0.774 (95 % CI 0.656–0.893) (p val- Syk (p = 0.0001) and p-Syk (p = 0.0001) stainings were signifi- ues \ 0.001). Glypican-4 level of 3.1 had 88 % sensitivity and 83 % cantly higher in the isolated-ITP group than in the sham-operated specificity for SLE. For chemerin, a level of 62.6 had 74 % sensitivity group. Groups treated with Fos demonstrated less Syk and p-Syk and 72 % specificity. staining scores than ITP group (p \ 0.001 for Fos and Fos + LC;

123 S112 Inflamm. Res. p = 0.028 for Fos + CVF). Syk staining was significantly less intense Interestingly, treatment of mice with the the Syk inhibitor Fos in the IVIG group than in the ITP group (p = 0.0001). P-Syk levels in decreased significantly Syk expression in both skin and lung. The all IVIG groups were significantly higher than in other groups active form of Syk is phospho-Syk, and its expression is suppressed (p \ 0.001). The intensity of PECAM staining was significantly by Syk inhibitor therapy. Bleomycin induced the expression of higher in the ITP group than in the sham group (p = 0.0001). In ITP phospho-Syk but in the presence of the Syk inhibitor its expression and all treatment groups, PECAM staining score was higher than in was limited sginifcantly. Mice treated with bleomycin displayed more healthy controls (p \ 0.001). PECAM scores of all Fos groups (Fos, prominent TGF-b staining of skin and lung tissues. The administra- Fos + LC, and Fos + CVF) were similar to that of the ITP group. tion of Syk inhibitor to the bleomycin-treated mice abolished TGF-b CX3CR1 staining in spleens of the ITP group was more intense than staining. The number of mast cells were higher in the group of mice the sham group (p = 0.0001). CX3CR1 staining in Fos and Fos + LC that were treated with bleomycin alone (p \ 0.001) and the admin- groups was less intense than in ITP group (p \ 0.001). Fos + CVF istration of Fos reduced significantly the number mast cells. and ITP groups had similar CX3CR1 expression. CX3CR1 staining in Conclusions: The Syk inhibitor Fos prevented bleomycin-induced the Fos group was less intense than in the IVIG group (p \ 0.001). fibrosis and inflammation in the skin and the lung. The anti-fibrotic Conclusions: Our study showed that the Syk pathway played a role in effect of Fos is linked to reduced Syk phosphorylation and TGF-b the pathogenesis of ITP. The Syk inhibitor, fostamatinib, improved expression. The Syk pathway appears as a potential molecular target the thrombocytopenia in ITP by acting on the Syk pathway. In for therapeutic intervention in SSc. addition, fostamatinib decreased the migration of T lymphocytes to the spleen, but not to the bone marrow.

B026 B025 SERUM AND SYNOVIAL FLUID C5A LEVELS IN THE SYK INHIBITOR FOSTAMATINIB LIMITS PATIENTS WITH RHEUMATOID ARTHRITIS AND TISSUE DAMAGE AND FIBROSIS IN OSTEARTHRITIS A BLEOMYCIN-INDUCED SCLERODERMA 1 2 3 MOUSE MODEL: EFFICACY IN SYK INHIBITION Dilek Keskin ,Go¨ksal Keskin , Ali Inal IN ANIMAL SCLERODERMA 1University of Kırıkkale, Kırıkkale, Turkey; 2Ankara Univeristy, Ankara, Turkey; 3University of GATA, Turkey Omer N. Pamuk1, Guray Can1, Sulayman Can1, Turan Karaca1, Gulsum E. Pamuk1, Selim Demirtas1, George C. Tsokos2 Rheumatoid arthritis (RA) is a chronic inflammatory disease, with unknown etiology. Compleman 5a (C5a) which is generated during 1Trakya University Medical Faculty, Edirne, Turkey; 2Harvard acute flare up of Rheumatoid Arthritis (RA) the has a n important role University Medical Faculty, Boston, MA, USA in the pathogenesis of RA. Purpose: The aim of this study was to determine C5a levels in the Background/Objectives: The pathogenesis of fibrosis in scleroderma plasma and synovial fluid (SF) samples. (SSc) is unknown. TGF-b and platelet-derived growth factor are Patients and methods: Thirty patients with RA and Thirty-five important in the development of fibrosis and tyrosine kinases are patients with Osteoarthritis (OA) were enrolled to the study. The involved in these pathways. Spleen tyrosine kinase (Syk) is a protein mean duration of the disease was 6.7 ± 3.42 years in patients with tyrosine kinase which activates intracellular signal transduction RA and 8.3 ± 4.7 years in and patients with OA. All patients with pathways and has been claimed to be involved in the pathogenesis of RA were in active period. Synovial fluid was aspirated from knee systemic autoimmune diseases. We investigated the ability of a small joint and plasma samples were also taken at the same time and C5a drug Syk inhibitor, fostamatinib (Fos), to protect mice from bleo- levels were determined by ELISA. mycin-induced SSc. Results: The mean plasma C5a levels were in patients with active RA Methods: Four study groups of Balb/c mice were included in this and OA were 107.3 ± 18.2 lg/L and 88.5 ± 23.1 lg/L respectively. study: control, bleomycin (administered subcutaneously to BALB/c The mean SF C5a levels were 168.4 ± 41.7 lg/L in patients with mice for 21 days), bleomycin and Fos (mice fed with chow containing active RA and 103.8 ± 33.1 lg/L in patients with OA. There was no a Syk inhibitor for 21 days) and Fos alone groups. Skin and lung statistically significant difference in plasma C5a levels in both of the tissue specimens were obtained and evaluated histologically. groups. The SF C5a levels were significantly high in patients active Results: Mice treated with bleomycin alone had significantly more RA compared to patients with OA. skin thickness (416.1 ± 6.1) compared to control (260.1 ± 10.1) and Conclusion: The high levels C5a in SF suggests that C5a is inten- Fos (254.3 ± 7.9) treated mice (p \ 0.001). Mice subjected to bleo- sively synthesized in the region of inflammation. mycin and fed with Fos-containing chow generated more (312.3 ± 4.4) dermal thickness than control and Fos-treated mice (p values \ 0.001) but, significantly less when compared to mice treated with bleomycin alone (p \ 0.001). Alveolar hemorrhage, B027 edema, damage and leukocyte scores in the lungs of mice treated with bleomycin were significantly higher v compared to control or Fos THE ROLE OF NEUTROPHILS AND G-CSF IN THE alone-treated mice (p values \ 0.001). Mice though which were K/BxN SERUM-TRANSFER ARTHRITIS MODEL exposed to bleomycin and treated with Fos had significantly less alveolar damage and leucocyte infiltration compared to mice exposed Anne Deen Christensen1,2, Claus Haase2, Andrew D. Cook1, John A. to bleomycin and fed with regular chow. At the end of the 21-day Hamilton1 bleomycin administration, there was apparent prominent fibrosis which was reduced significantly in the group of mice which received 1Department of Medicine, University of Melbourne, Royal Melbourne in parallel Fos. Syk expression in both the lung and the skin was noted Hospital, Royal Parade, Parkville, Victoria, Australia; 2Novo Nordisk to be intense in the bleomycin-treated mice compared to control mice. A/S, Novo Nordisk Park, Ma˚løv, Denmark

123 Inflamm. Res. S113

Neutrophils are an important cell type in many autoimmune diseases (WL) and histopathology can be ambiguous as inflammation may such as rheumatoid arthritis. Furthermore, mobilization of neutrophils occur independent of WL, and WL and colitis may respond differ- from the bone marrow to the circulation during an inflammatory ently to treatment. To overcome these limitations, we used serial response is controlled by granulocyte-colony stimulating factor (G- FACS analyses of peripheral blood (PB) to assess engraftment, and CSF). In the K/BxN serum-transfer arthritis (STA) model, mimicking performed serial endoscopy (SE) to track onset and severity of CC. + the effector phase of rheumatoid arthritis, neutrophils have been Naive TH cells from C57Bl/6 donors, sorted as either CD4 , shown to play a key role. In this model neutrophils have been CD45RBHIGH, or CD4+, CD44-, CD62L+ were injected i.p. into depleted using either a rat anti-mouse LyG/C monoclonal antibody RAG2-/- recipients. Onset of colitis, determined by endoscopy (E), (mAb) (clone RB6.8C5) or a rat anti-mouse Ly6G mAb (clone 1A8). corresponded with onset of WL (d21-28). PB CD4+ cell counts However, the two antibodies have recently been shown to deplete assessed prior to appearance of observable disease (d14) were highly with different specificity as well as efficacy. In contrast, the impact of correlative with final (d56) WL (R2 = 0.74) and final E score G-CSF on arthritis in this model has not been investigated. The (R2 = 0.79). Positive control therapy (anti-TNFa mAb) had little objective of this study was to compare depletion of neutrophils with effect on terminal WL but significantly reduced E score (2.0 ± 0.15 the two different neutrophil-depleting antibodies, as well as to vs. 1.1 ± 0.14 at d49). These results and the method’s clinical rele- investigate the role of G-CSF in the K/BxN STA model. vance confirm that SE is a useful addition to WL as an enhanced For induction of arthritis, K/BxN serum was injected intraperi- readout for in-life disease severity and indicate that d14 CD4+ count toneally into C57BL/6 mice on day 0 and day 2. On day 0–10 after the is a reliable early predictor of disease severity. first serum transfer, arthritis was assessed by a clinical score as well as In the standard murine GVHD model, endpoints of WL and by measurement of paw swelling in the rear paws. On day 1, 4 and 7 GVHD score often fail to respond to positive control therapies levels of G-CSF, IL-1b, IL-10, CXCL10, CXCL2 and CXCL1 were (FK506 and anti-p40 mAb), highlighting the need for improved analysed in inflamed paws. Additionally, levels of G-CSF in serum translatability. We evaluated SE as an additional method to directly were measured on day 1, 2, 3, 4, 7 and 10. Neutrophils were depleted assess onset and progress of intestinal GVHD. GVHD was induced by by injecting 1 mg/mouse of either rat anti-mouse Ly6G/C mAb or rat harvesting bone marrow of male C57Bl/6 mice, depleting it of CD3+ anti-mouse Ly6G mAb intraperitoneally on day-1 and -2. On day 1 cells and transferring it along with splenocytes into lethally irradiated and 7 depletion was confirmed with flow cytometry. G-CSF was (8 Gy) Balb/c hosts. Subsequent SE showed disease levels that blocked by injection of a rat anti-mouse G-CSF mAb (clone 67604) strongly correlated with WL and GVHD score (R2 =-0.84 and one day prior to the first serum injection. On day 3 and 7, a blood 0.79). We next evaluated the utility of using serial peripheral blood analysis was performed by flow cytometry to quantify the absolute (SPB) FACS analysis of human leukocyte count (HLC) as a correlate number of neutrophils in peripheral blood. of WL and GVHD score in a humanized model in which GVHD was First, it was found that the neutrophil chemoattractants, CXCL1 induced by the adoptive transfer of human PBMCs into NSG mice. and CXCL2, were significantly increased after induction of arthritis SPB FACS analyses demonstrated a strong correlation between HLC compared to naı¨ve controls. We also found increased levels of G-CSF and WL and GVHD score (R2 =-0.85 and 0.67 on d42) indicating both in serum and in inflamed tissue during progression of arthritis. that HLC may be applicable to the evaluation of therapies directly After injection of the two neutrophil depleting antibodies, flow targeting these cell types. Early (d7) HLC predicted final WL and cytometric analysis showed that anti-Ly6G mAb only partially GVHD score with high correlation (R2 =-0.86 and 0.90). We depletes neutrophils in C57BL/6 mice, while anti-Ly6G/C mAb, in conclude that SE and FACS analyses are valuable additions to addition to completely depleting neutrophils, also depletes a fraction enhance the translatability, clinical relevance, and predictability of of monocytes. Treatment with anti-Ly6G mAb reduced arthritis sig- these adoptive transfer-based inflammatory disease models. nificantly, while anti-LyG/C mAb completely blocked arthritis. Interestingly, blockade of G-CSF led to an almost complete absence of arthritis as well as a reduced level of neutrophils in peripheral blood. This study emphasizes the importance of neutrophils in the B029 K/BxN STA model and demonstrates a crucial role for G-CSF in this IL-7 RECEPTOR POLYMORPHISMS IN model with possible significance for human disease. MULTIPLE SCLEROSIS: MECHANISM AND THERAPEUTIC IMPLICATION B028 Wenqing Li1, Julie A. Hixon1, Julia Tritapoe1, Joao T. Barata2, Scott ENHANCEMENTS TO THE PREDICTABILITY K. Durum1 AND CLINICAL RELEVANCE OF TWO MURINE MODELS OF CHRONIC INFLAMMATION: 1Laboratory of Molecular Immunoregulation, Cancer and 2 ADOPTIVE TRANSFER-INDUCED COLITIS AND Inflammation Program, CCR, NCI; Cancer Biology Unit, Instituto de Medicina Molecular, Lisbon University Medical School, Lisbon, GVHD Portugal

Dominic R. Beal, Sean M. Graham, Brett Van Dam, Gregory D. Lyng, IL-7 is required for survival of most T cell subsets and generally Stephen T. Sonis promotes T cell immune responses. IL-7 acts on lymphocytes by binding with high affinity to IL-7 receptor a chain (IL-7R) and Biomodels LLC, Watertown, MA, USA recruiting c, and then initiating the signaling cascade. Loss-of- functions of IL-7R in human has, for some time, been known to cause Mouse models are widely used to assess new therapies for autoim- severe combined immunodeficiency. We find that 9 % of childhood mune pathologies such as colitis and GVHD. We now report T-cell acute lymphoblastic leukemia (T-ALL) has somatic gain-of- enhancements which increase the translational value of 3 such mod- function mutations of IL-7R. These mutations, usually involved els. Adoptive transfer of naı¨ve THcells into immunodeficient mice is insertions of three or more amino acids immediately before or after one of the best characterized immunological models of chronic colitis residue 244 in the exon 6, create oncogenes driving T-ALL. More (CC). However, traditional disease severity endpoints of weight loss recently, polymorphisms in IL-7R have been shown be a risk factor

123 S114 Inflamm. Res. for multiple sclerosis (MS) and a number of diseases that are autoimmune or involve excess immune and inflammatory responses. The polymorphysims that affects risk to most of these immunopathologies is T244/I in exon 6, the same region we show to harbor gain-of-function mutations in T-ALL. To investigate the mechanism of IL-7R pathway association with MS and seek new therapeutic approaches directed to IL-7R and its downstream sig- naling component, we hypothesize that the functional significance of the 244T/I polymorphism in IL-7R is related to increased signaling. To test our hypothesis, the T244 and I244 alleles of IL7R are trans- fected into BaF3 cells. The high risk allele T244 induces stronger Stat5 activation and promotes better survival of BaF3 cells. We fur- ther examine the strength of signaling in vivo in mice. IL-7R deficient bone marrow hematopoietic progenitors are transduced by retroviral infection with T244 or I244 alleles of human IL-7R and transferred to Rag1 deficient mice. Human IL-7R rescues T cell development in this model. Consistent with the in vitro study, the CD8 cells harboring the high risk allele T244 display stronger Stat5 activation compared with cells with I244. Our preliminary study suggests that high risk allele T244 of IL-7R signals stronger than allele I244, and this may promote activation of autoimmune T calls in MS and other autoimmune Fig. 1 Serum 14-3-3g expression by disease. diseases. Conclusions: Serum 14-3-3g is a highly specific RA biomarker. As a novel mechanistic disease factor, 14-3-3g is expected to provide new insights and approaches to RA management and clinical studies. B030 SERUM 14-3-3 ETA IS AN RA SPECIFIC MECHANISTIC MARKER B031 MULTIPLE MESENTERIC LYMPHATIC Anthony Marotta1, Yauheniya Cherkas2, Bidisha Dasgupta2, Sarah Lamberth2, Karen Hayden2, Carrie Brodmerkel2, Mark Curran2 ANOMALIES IN MURINE CROHN’S DISEASE

1Augurex Life Sciences Corp, Vancouver, BC, Canada; 2Janssen Pierre-Yves von der Weid, Sonia Rehal R&D LLC, Raritan, NJ, USA University of Calgary, Calgary, AB, Canada Background: 14-3-3g is an emerging soluble rheumatoid arthritis (RA) biomarker that activates intracellular pathways that lead to the Background: Lymphatic pumping is the main mechanism used for upregulation of inflammatory and joint damage factors. It is reported propelling lymph and immune cells from peripheral tissues to lymph to be highly specific and sensitive for RA as a diagnostic marker, nodes and back to the blood stream. Decreased lymphatic pumping higher levels are associated with greater joint damage progression causes poor lymph drainage and accumulation of fluid, immune cells risk1,2 and 14-3-3g’s modulation with treatment suggests a role in and macromolecules in the tissues, ultimately leading to edema that disease monitoring. may contribute to the perpetuation of inflammation. Importantly, we Objectives: In this study, we examine the specificity of 14-3-3g in an have demonstrated that TNBS-induced inflammation of the ileum independent, clinically well-characterized cohort of moderate to alters pumping in the mesenteric lymphatic vessels via increased severe RA and disease control subjects. production of nitric oxide (NO) and prostaglandins caused by the Methods: Serum 14-3-3g levels were measured in a total of 147 upregulation of the enzymes inducible nitric oxide synthase (iNOS), patients using the Augurex 14-3-3g ELISA. The patient set com- and cyclooxygenases (COX1 and COX2). Our aim here was to further prised 36 with RA and 111 controls consisting of 20 with asthma (A), assess how inflammation impairs lymphatic contractile function, and 20 with Crohn’s disease (CD), 12 presumed healthy (H), 16 with to investigate whether TNF-a, a potent pro-inflammatory cytokine psoriasis (PsO), 20 with sarcoid arthritis (S), and 23 with spondy- upregulated in intestinal inflammation such as Crohn’s disease, alters larthropathies (SpA). Sample testing was done independently of lymphatic pumping and what are the mechanisms involved. Augurex. Mann–Whitney testing together with Kruskal–Wallis anal- Methods: Rats were euthanized and the small intestine with its ysis with the post hoc Dunn’s multiple comparison test was performed attached mesentery rapidly isolated. Mesenteric lymphatic vessels to assess group differences. Receiver operator characteristic curve were dissected out, and incubated in physiological and sterile con- (ROC) analysis was performed to assess the specificity of 14-3-3g for ditions for 24 h with TNF-a or vehicle (sham vessels) with or without RA. pharmacological inhibitors. Vessels were then either mounted on a Results: Median (IQR) serum 14-3-3g levels were significantly pressure myograph to measure contractile activity induced by the higher in RA [2.35 ng/ml (0.28-19.41)] than all controls [0 (0–0)], luminal pressure, or processed for protein and mRNA analysis by p \ 0.0001. ROC curve analysis further underscored this differential western blot and quantitative real-time PCR, respectively. Data expression yielding a significant area under the curve (AUC) of 0.86, obtained from sham and TNF-a-treated vessels were compared. p \ 0.0001. At the diagnostic positivity cut-off of C0.19 ng/ml, the Results: TNF-a significantly decreased lymphatic contraction fre- ROC curve delivered a sensitivity of 81 % with a corresponding quency in a concentration-dependent manner. Contractile activity was specificity of 84 %. Kruskal–Wallis testing revealed that serum 14-3- restored following administration of pyrrolidine dithiocarbamate 3g levels were significantly higher in RA in comparison to all other (PDTC), an inhibitor of the master regulator of inflammation NF-jB diseases, p \ 0.0001. This differential expression is illustrated in acting downstream of TNF-a. The involvement of NF-jB activation Figure 1. in lymphatic dysfunction was supported by western blot data, which

123 Inflamm. Res. S115 revealed an increase in phospho-IjB in TNF-a-treated vessels. Conclusion: The finding that targeted deletion of IRP1 but not IRP2 Quantitative real-time PCR showed upregulation of iNOS mRNA in completely abolished the inflammation in the TNFDARE/+ mouse TNF-a-treated vessels that was minimized in the presence of PDTC. suggests that IRP1 may be a target for specific inhibition to treat Furthermore, pumping was restored by pharmacological inhibition of inflammatory bowel diseases. the NO pathway with the iNOS inhibitor 1400 W or the soluble guanylate cyclase inhibitor ODQ, but not with the cyclooxygenase inhibitor indomethacin. Lymphatic pumping was also reestablished in B033 the presence of glibenclamide, a KATP channel blocker, suggesting activation of these channels and hyperpolarization of the lymphatic C-KIT IS EXPRESSED ON A SUBSET OF TH17 muscle as a likely cause of the contractile dysfunction. CELLS AND REGULATES THEIR IL-22 Conclusions: The NF-jB–iNOS–NO-KATP channel pathway is PRODUCTION involved in lymphatic pumping decrease via caused by TNF-a. Lym- phatic dysfunction may contribute to the perpetuation of inflammation. Kalil A. de Lima, Jhimmy Talbot, Paula Barbim, Thiago Cunha, Jose´ Carlos Alves-Filho, Paulo Louzada, Fernando Q. Cunha

B032 Ribeirao Preto Medical School, Ribeirao Preto, Brazil A NOVEL IRON-MEDIATED MECHANISM FOR The aryl hydrocarbon receptor (AHR) is a ligand-dependent tran- DEVELOPMENT OF INFLAMMATORY BOWEL scription factor. It is well know its involvement in TH17 cells DISEASE polarization, but the AHR downstream signaling remains unclear. Previous data in RORct-dependent group 3 innate lymphoid cells Shirly M. Belizowski1, Abraham Nyska2, Avi Zuckerman3, suggest the receptor tyrosine kinase c-kit as a possible downstream Fabio Cominelli4, Orly Savion1, Esther Meyron-Holtz1 target of AHR. Recently, c-kit expression was described in a pro- inflammatory human TH17 cells, however the c-kit/AHR interac- 1Technion Israel Institute of Technology, Haifa, Israel; 2Tel Aviv tion remains unclear in these cells. Thus, the aim of this study was University and Consultant in Toxicologic Pathology, Timrat, Israel; to investigate the role of c-kit expression in TH17 cells and cor- 3 4 relate it with the AHR pathway. We found that naive Aviv Projects, Ness Ziona, Israel; Case Western Reserve University, + + lo Cleveland, Ohio, USA CD4 CD62L CD44 T cells differentiated in vitro into TH17 cells originated around 7–8 % of c-kit+ cells. Investigating the kinetics Background: Iron accumulation in inflammatory lesions has been of c-kit expression under TH17-polarizing conditions we observed described in many settings but a molecular mechanism for this that c-kit expression was substantially upregulated 12 h after accumulation was not completely elucidated. We hypothesized that activation in the presence of TGF-b1 and IL-6, and its expression an iron independent activation of RNA-binding activity of the Iron remained throughout the TH17 differentiation. The real time anal- Regulatory Protein (IRP)1, via reactive oxygen and nitrogen species ysis to kit gene expression corroborate with protein analysis. To is the driving force for this iron accumulation and for the exacerbation characterize the relevance of c-kit to TH17 differentiation during of inflammation. the course of an immune response, we studied the in vivo popu- Methods: To test this hypothesis, we examined a mouse-model of lation expansion of TH17 cells after immunization. We immunized inflammatory bowel disease, the TNFa overexpressing mouse wild-type mice with MOG (35–55) and assessed the c-kit+ cells (TNFDARE/+) and an epithelial cell model for changes in iron home- frequencies in the lymph node and central nervous system-infil- ostasis during inflammation and analyzed the effect of IRP1 and 2 trating CD4+ T cells. Interestingly we also detected an expansion deletions on the course of the inflammation. Differential analysis of of c-kit-expressing TH17 cells. Additionally, we investigated the the different cell-types within the inflamed tissue was performed. effect of c-kit signaling on TH17 differentiation using c-kit ligand Results: We found that the terminal ileitis, which is developed in the and inhibitor, SCF and ISCK03 respectively. We did not observe TNFDARE/+ mouse, was accompanied not only by a local iron accu- any difference considering IL-17 production or expression. How- mulation but also by profound iron redistribution within the inflamed ever, cells treated with c-kit inhibitor showed significant tissue. While the local and infiltrating macrophages accumulated iron, improvement in their IL-22 expression, as shown by their higher the inflamed epithelial cells showed a state of significant iron defi- expression of Il22 and secretion of IL-22 into culture supernatants. ciency and elevated iron flux, which was mimicked in the epithelial cell Finally, to determine if AHR pathway has a role in c-kit expression model of inflamed Caco-2 cells. The significant iron redistribution was during TH17 cells differentiation, we expose naive T cells to the accompanied by elevated levels of the transcription factor Hypoxia AHR ligand FICZ and we observed that AHR activation enhanced inducible factor (HIF)2a, by distinctive altered RNA-binding activity c-kit expression as well as the frequency of c-kit+ cells. These of both IRP1 and IRP2 in the different local cell types, and by altered results suggest that c-kit signaling can be important to negatively levels of the proteins involved in the cellular iron homeostasis. These regulate IL-22 production on TH17 whereas AHR activation can new dynamics of iron homeostasis were completely reversed by the modulate c-kit expression in these cells. deletion of IRP1 in the TNFDARE/+ mouse and the Crohn’s like picture of inflammation of the TNFDARE/+ mouse was not detectable in the TNFDARE/+ xIRP1-/- mice. In contrast, TNFDARE/+ xIRP2-/- mice showed severe transmural ileitis. B034 Discussion: The non-iron mediated activation of IRP1 during WONDERBUMIN: A FULLY SYNTHETIC inflammation triggered an impaired iron homeostasis within the ALTERNATIVE TO IgT FOR AUTOIMMUNE inflammatory lesion that led to the accumulation of iron in the local DISEASE immune cells. The iron accumulation in these cells and the iron depletion of the epithelial cells both support the recruitment of a 1,2 3 1 systemic immune response and the propagation of an initially local Anne S. De Groot , Filipa Antunes , Eduardo Guillen , 1 1 1 1 and close to physiologic inflammation of the gut to a severe inflam- Sandra Lelias , Ryan Harvey , Christine Boyle , William Martin , 3 3 mation with massive infiltration of systemic immune cells. Paul Barrett , Darrell Sleep

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1EpiVax, Inc., Providence, USA; 2University of Rhode Island, Rhode that murine memory CD4+ T lymphocytes migrate specifically in Island, USA; 3Novozymes Biopharma Ltd, Nottingham, USA response to 7 a25-OHC via EBI2. Indeed, IL-17 producing EBI2-/- memory CD4+ T cells depicted delayed migration to the central Purpose: Immunoglobulin G Therapy (IgT) is the standard of care for nervous system compared to their wild type counterparts. However, several autoimmune diseases but it is not well tolerated by patients the expression and the role of EBI2 in human T lymphocytes, which due to systemic affects associated with the injection of large amounts are crucial cells in driving the adaptive immune response, have not of Ig. The use of IgT is expanding because it activates and expands been studied. Tregs, which are critical to the control of autoimmune disease (AID). Methods: the expression of EBI2 on healthy human peripheral blood We identified Treg epitopes, now called Tregitopes, contained in mononuclear cells was measured by flow cytometry using a specific conserved framework regions of IgG Fab and Fc. Tregitopes exhibit anti-human EBI2 antibody. The function of EBI2 in cell migration high affinity binding to multiple human HLA Class II DR and are was assessed using a transwell assay. conserved across mammalian species. Tregitopes activate CD4+/ Results: We observed maximal EBI2 expression on memory CD4+ T CD25hi/FoxP3+ Tregs and expand antigen-specific iTregs. We report cells; memory subsets of B and CD8+ T cells also depicted a modestly here on the development of a Tregitope-albumin fusion as a potential increased EBI2 expression compared to naı¨ve populations. Transwell alternative to IgT. migration assay experiments showed maximal migration of memory Methods: EpiVax applied in silico immunoinformatics tools to CD4+ T cells in response to 7a25-OHC. Even if globally less develop a polypeptide concatamer of Tregitopes for incorporation responsive to 7a25-OHC than the latter, memory subsets of B and into Novozymes’ VeltisTM platform—a drug delivery platform that CD8+ T cells were also found to migrate more strenuously than their combines therapeutically active molecules with the long half-life of naı¨ve counterparts. This chemotaxis was specific to EBI2 as selective human serum albumin. This Tregitope-Albumin fusion product was EBI2 inhibition unequivocally abrogated migration. evaluated for efficacy in a mouse model of OVA immunogenicity Conclusion:: These data are in line with our murine results and where OVA elicits a robust T cell response. T cell proliferation suggest an important role for EBI2 in human T cells migration. (CFSE) and phenotyping assays were performed following treatment Selective targeting of immune cells trafficking has become an of OVA-immunized mice with Tregitopes. important tool in the clinical setting to dampen autoimmunity, for Results: The mechanism of Tregitope-mediated tolerance induction example in the context of neuroinflammation such as multiple scle- appears to be as follows: (1) Tregitopes are delivered to antigen rosis. Uncovering the role of EBI2 in T cells trafficking, in particular presenting cells, possibly through their FcRN, which then present its ability to direct human CD4+ T cells chemotaxis, may open new Tregitopes on MHC Class II, (2) Treg recognizes them through their avenues for understanding the pathogenesis of autoimmunity and may T cell receptors and are activated, proliferate and produce IL-10, (3) promote novel therapeutic approaches. Treg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and (4) Treg and tolerogenic APC work together to suppress antigen-specific T cell responses. Conclusion: Tregitopes have been shown to be equivalent to IVIG in a number of murine models, and to induce bystander tolerance in B036 OVA immunization studies in mice. In addition to these pre-clinical HEALTHCARE RESOURCE USE AND COSTS OF studies, one clinical study has been performed using a single Tregi- ADRENOCORTICOTROPIC HORMONE IN tope, demonstrating 30 % efficacy (Sthoeger et al. Journal of RELAPSES OF MULTIPLE SCLEROSIS Autoimm. 2009). Tregitopes formulated as an albumin fusion may represent an important advance toward a fully synthetic alternative to Laurie S. Gold1, Patricia Schepman2, John Niewoehner2, IgT. 2 1 Supported by NIH SBIR Phase II R44 DK081261 Michael Philbin , Ryan Hansen 1University of Washington, Seattle, WA, USA; 2Mallinckrodt Pharmaceuticals, Ireland, Republic of Ireland B035 Background: Multiple Sclerosis (MS) is an autoimmune, neuro-in- OXYSTEROLS AND HUMAN MEMORY flammatory disorder characterized by acute exacerbations LYMPHOCYTES: WHEN INTERACTION MEANS (‘relapses’), interspersed between remissions. MS relapses impose a ATTRACTION heavy economic burden on society and are commonly treated with intravenous methylprednisolone (IVMP), and for some difficult-to- manage relapses, other hormonal therapies such as, adrenocorti- Aure´lie Clottu1, Fanny Chalmin1, Caroline Pot1,2 cotropic hormone (ACTH, H.P. Acthar GelÒ), intravenous immunoglobulin (IVIG; off label) and plasmapheresis (PMP). 1University of Geneva, Geneva, Switzerland; 2Geneva University Objective: We explored the economic burden accrued among patients Hospitals, Geneva, Switzerland with difficult to treat relapses, comparing ACTH versus IVIG or PMP Objectives: to study the expression and function of the oxysterols in this study over a 12- and 24-month follow-up period. receptor Epstein-Barr virus induced gene 2 (EBI2) in human Method: A retrospective analysis of commercial health insurance lymphocytes. claims of MS relapses was conducted using Truven Health Analytics Background: Oxysterols, hydroxylated cholesterol metabolites, have MarketScanÒ database. Patients with C2 MS relapse episodes recently been ascribed a role in promoting inflammation and in between 2007 and 2012 were identified; the first relapse was treated modulating the immune response. In this line we are interested in with IVMP and following relapses were treated with ACTH, IVIG, or studying the role of oxysterols, in particular 7a25-hydroxycholesterol PMP. The first calendar date of the second treated relapse was the (7a25-OHC), which is the strongest ligand of the G-coupled receptor index date. Patients with continuous health plan enrollment for EBI2. EBI2 is functionally expressed on human macrophages, 6-months prior and 12-months post index date were included, with a astrocytes and B cells, and participates to the latter positioning in the subset who were able to be followed for 24 months. We estimated the lymph nodes during an immune response. In the animal model healthcare resource use and costs (inpatient, outpatient, and phar- experimental autoimmune encephalomyelitis, we recently showed macy) separately for patients whose second relapse was treated with

123 Inflamm. Res. S117 each treatment option and then compared ACTH to patients who Flavocoxid and Zileuton improved weight loss, reduced colonic received either IVIG or PMP. Multivariate linear regression models myeloperoxydase activity, macroscopic and microscopic damage, were constructed to adjust for measured baseline patient character- LTB-4, and TNF-a serum levels. Flavocoxid was the only treatment istics and prior resource use. effective in reducing also malondialdheyde, PGE-2 levels, and Results: A total of 445 MS patients had 12 months of continuous apoptosis. In addition, histological features were strongly improved enrollment after their second MS relapse. 219 (49 %) patients were with Flavocoxid, as compared to Zileuton. treated with ACTH and 226 (51 %) patients with IVIG/PMP for their Our research demonstrate the protective effect of Flavocoxid in second relapse. Of those, 231 had 24 months of continuous follow-up IBDs, suggesting that this dual balanced inhibitor of COX and 5-LOX data, 99 (43 %) Acthar and 132 (57 %) IVIG/PMP. Patients who were may represent a future treatment for inflammatory bowel diseases. treated with ACTH had a significantly lower number hospitalizations (-0.4, 95 % CI -0.6 to -0.2) and outpatient visits (-17, 95 % CI -22 to -11) in the first 12 months, along with accompanying lower costs for those resources, with similar total costs at 1 year. ACTH Biologic Therapies for Targeting patients also had significantly fewer relapses (-0.9, 95 % CI -1.2 to Inflammatory and Immune Mechanisms -0.6). In multivariate linear regression, the significant differences in reduced inpatient costs, relapses, outpatient encounters, and outpa- tient costs remained. Additionally, total costs were still similar B038 between the two groups in adjusted analyses. The findings of reduced MYRIANTHUS ARBOREUS EXHIBITED ANTI- outpatient services and costs and comparable total costs were con- INFLAMMATORY PROPERTIES IN sistent among the subgroups with 24 months of continuous follow-up EXPERIMENTAL MODELS data. Conclusion: Difficult to manage MS relapses pose a challenge to 1 2 2 clinicians and patients. We found that treating these relapses with Oluwafemi G. Oluwole , Akinyinka Alabi , Olatunde Ganiyu ACTH is associated with decreased resource use and similar costs 1 compared to IVIG or PMP, thereby supporting the value of ACTH in Department of Pharmacology and Therapeutics, Faculty of Basic MS relapse. Medical Science, College Of Medicine, University Of Ibadan, Ibadan, Nigeria; 2Department of Pharmacology and Therapeutic, College of Medicine, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria B037 This study investigated the anti-inflammatory effects of ethanolic USE OF A BALANCED DUAL extracts of Myrianthus arboreus leaves in carrageenin-induced paw CYCLOOXYGENASE-1/2 AND 5-LYPOXYGENASE edema acute inflammation, formaldehyde- induced sub-acute INHIBITOR IN EXPERIMENTAL COLITIS inflammation, and lipopolysacharide (LPS) induced granuloma air pouch chronic inflammation in rats. Its membrane stabilizing activity 1 1 1 was also evaluated. Alessandra Bitto , Giovanni Pallio , Gabriele Pizzino , Rats (n = 5) were treated orally with M. arboreus (125, 250 Domenica Altavilla2, Francesco Squadrito1 and 500 mg/kg), Indomethacin (10 mg/kg) or distilled water (3 ml/ kg). Thirty minutes after treatment, acute inflammation was 1Department of Clinical and Experimental Medicine, Section of induced with sub-plantar injection of 0.1 ml of 1 % carrageenin Pharmacology, Medical School, University of Messina, Messina, into the right hind paw. The paw edema sizes were measured with Italy; 2Department of Paediatric, Gynaecological, Microbiological the aid of plethymometer Ugo basile over a period of 3 h. Using and Biomedical Sciences, University of Messina, Messina, Italy similar technique, 0.1 ml of 2.5 % formaldehyde was induced for Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), and their 3 days in sub-acute inflammation. Thereafter, anti-inflammatory products PGE-2 and LTB-4, play an important role in inflammatory effects of Marboreus was assessed based on neutrophil migration, bowel diseases (IBDs). Therefore we investigated the effects of volume of fluid exudates, also by free radicals scavenging activities Flavocoxid, a dual COX/LOX inhibitor, in experimental colitis based on the levels of MDA, GSH, Catalase, Nitric oxides and induced with either dinitrobenzenesulfonic acid (DNBS), or dextrane Prostaglandin released in the inflammatory fluids derived from sulphate sodium. Flavocoxid effect was compared with the 5-LOX 5 day LPS induced air pouch granuloma chronic inflammation. inhibitor, Zileuton. Membrane stabilizing activity was evaluated spectrophotometeri- In the DNBS model, colitis was induced in 21 rats by a single cally using RBC lysis induced phenlhydazine, heat, and then intra-colonic instillation (25 mg in 0.8 mL 50 % ethanol); after 24 h hypotonic medium, Histology of pouch tissues and stomach mucosa animals were randomized to receive, by gavage twice a day, either sections were also examined. Flavocoxid (10 mg/kg), or Zileuton (50 mg/kg), or vehicle. Sham M. arboreus (250, 500 mg/kg) produced a significant inhibition of (n = 7) or DNBS-vehicle (n = 7) animals received 0.8 ml of saline acute and sub-acute inflammation when compared with distilled water or 50 % ethanol, respectively. Rats were sacrificed 4 days after control; it also significantly stabilized membrane by preventing RBC induction and samples were collected for analysis. lysis, as well having antioxidant properties and modulates markers of In the DSS model, colitis was induced in 21 rats by the admin- inflammation significantly. istration of 8 % dextran sulfate sodium dissolved in drinking water; The results of this investigations suggested that M. arboreus certainly after 24 h animals were randomized to the same above reported contain active compounds therapeutically useful as anti-inflammatory treatments. Sham animals (n = 7) received standard drinking water. agents. Rats were sacrificed 5 days after induction and samples were col- Keywords: M. arboreus, anti-inflammatory, antioxidant, membrane lected for analysis. stabilizing.

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B039 with saline or 30 mg/kg of GM-0111 once daily (Days 2–7). On Day A MODIFIED GLYCOSAMINOGLYCAN, GM-0111 0, mice were irradiated with a single dose of x-rays at 20 Gy. On Day 8, we harvested tissues and determined the degree of inflammation by INHIBITS INFLAMMATORY MOLECULES gross and histological examinations, as well as detection of RELATED TO PERIODONTITIS myeloperoxidase (MPO). GM-0111 at 100 lg/mL significantly reduced x-ray-induced cell death in both HOEPs and macrophage Won Yong Lee1, Justin R. Savage1, Abigail Pulsipher1,2, cells. Mice treated with GM-0111 showed markedly reduced signs of Narayanam Rao1, Thomas P. Kennedy3,1, Maria E. Ryan4, inflammation in the tongue, with decreased infiltration of polymor- Glenn D. Prestwich2,1 phonuclear leukocytes in the mucosa, thicker epithelial layer, and normal mucous glandular morphology than the saline-treated group. 1GlycoMira Therapeutics, Inc., San Diego, USA; 2University of Utah, MPO analysis confirmed our histological observations, with signifi- Salt Lake City, Utah; 3Tulane University, New Orleans, Louisiana; cantly (p \ 0.01) lower concentrations of MPO in GM-0111 4Stony Brook University, New York, USA treatment groups compared to vehicle-treated groups (saline/20 Gy 206 ± 77 vs. GM-0111/20 Gy 104 ± 27 mU/mg protein; normal While approximately 30 % US adults are suffer from moderate to 62 ± 23 mU/mg protein). We suggest that the cytoprotective effects severe form of periodontitis, the available treatment options are far of GM-0111 potentially reduce clinical signs of oral mucositis that too limited. This chronic disease occurs due to a disruption of frequently occur during cancer therapy. homeostasis between the microorganisms residing in the oral cavity and the host immune system. GM-0111 is a modified glycosamino- glycan and it has demonstrated anti-inflammatory effects on various inflammatory diesease models molecular targets and possibly offer B041 alternative therapeutic choices for treating periodontitis. We investi- THE EFFECT OF DUPILUMAB ON BIOMARKERS gated whether GM-0111, a modified GAG, could block molecular events important for the development of periodontitis and the IN THE PERIPHERAL BLOOD AND NASAL resulting bone loss. First, we tested GM-0111 on RANKL-induced SECRETIONS IN THE TREATMENT OF CHRONIC osteoclast formation and bone resorption in cultured mouse pre-os- SINUSITIS WITH NASAL POLYPOSIS teoclasts. Next, we tested whether GM-0111 could block pro- inflammatory TLR2 and TLR4 signaling pathways in mouse macro- Brian N. Swanson1, Leda Mannent2, Jennifer D. Hamilton3, phage RAW 264.7 cells and heterologously expressed HEK293 cells. Donghui Zhang1, Nian Tian1, Ying Wang1, Gabriele Holtappels4, Lastly, we investigated the antibacterial effects of GM-0111 on Lars-Olaf Cardell5, Jeffrey E. Ming1, Neil Graham3, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomi- Gianluca Pirozzi1, Claus Bachert4 tans. GM-0111 blocked RANKL-induced osteoclast formation even at 300 ng/mL and it also reduced the resulting bone resorption. The anti- 1Sanofi, Bridgewater, NJ, USA; 2Sanofi, Chilly Mazarin, France; osteoclastic effects of GM-0111 were independent of RANKL to 3Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA; 4Ghent RANK signaling. We also found that GM-0111 selectively targets University Hospital, Ghent, Belgium; 5Karolinska Institute, TLR2 (IC50 of 1–10 ng/mL) than TLR4 (IC50 [ 100 lg/mL). By Stockholm, Sweden blocking TLR-mediated inflammatory pathways and inhibiting key process leading bone loss, we suggest that GM-0111 can provide a Background: Dupilumab, a fully human monoclonal antibody against therapeutic potential to treat periodontitis. interleukin-4 receptor alpha (IL-4Ra), potently inhibits both IL-4 and IL-13 signaling, and has shown efficacy in asthma and atopic der- matitis clinical trials. In patients with chronic sinusitis with nasal polyposis (CSwNP), dupilumab significantly reduced polyp scores, B040 sinus CT scans (Lund-Mackay scores), and suppressed symptoms GM-0111, A MODIFIED GLYCOSAMINOGLYCAN, (ClinicalTrials.gov: NCT01920893). We now report the effect of PREVENTS RADIATION-INDUCED MUCOSITIS dupilumab treatment on biomarkers related to type 2 helper (Th2) cell cytokine activity and eosinophil (Eos) inflammation in patients with CSwNP. Abigail Pulsipher1,2, Justin R. Savage1, Thomas P. Kennedy1,3, Methods: Adults with CSwNP were treated with dupilumab (600 mg Glenn D. Prestwich1,2, Won Yong Lee1 loading dose, then 300 mg subcutaneously weekly for 15 weeks) or placebo (PBO) for 16 weeks; each was added to daily treatment with 1GlycoMira Therapeutics, Inc., Salt Lake City, UT, USA; intranasal mometasone furoate. Nasal secretions (NS) were collected 2Department of Pharmacy, University of Utah, Salt Lake City, UT, using bilateral absorbent packings and then eluted into 3 mL of saline. USA; 3School of Medicine, Tulane University, New Orleans, LA, USA Biomarkers in peripheral blood and NS were assayed using com- Mucositis is an inflammatory disease that frequently develops during mercially available methodologies. radiation and chemotherapy, posing serious challenges to patients Results: Addition of dupilumab treatment (N = 30), versus PBO undergoing cancer treatments. GM-0111 is a modified glycosamino- (N = 30), significantly suppressed (least squares mean % change glycan that blocks various innate immune molecules such as TLRs from baseline): serum thymus and activation regulated chemokine and selectins. GM-0111 also broadly suppresses bacterial growth (TARC) from Week (W) 2 (-26.1 vs. +6.2, respectively; p \ 0.0001) present in the oral cavity. We investigated whether these anti-in- to W12 (-23.1 vs. +6.4; p = 0.0001); plasma eotaxin-3 from W2 flammatory and anti-bacterial effects of GM-0111 could be exploited (-41.9 vs. -5.8; p \ 0.0001) to W16 (-35.5 vs. +10.0; p \ 0.0001) to prevent radiation-induced oral mucositis by testing in vitro and and serum total immunoglobulin (Ig)E from W8 (-29.1 vs. -2.5; in vivo radiation-induced mucositis models (1). In vitro studies: p \ 0.0001) to W16 (-48.4 vs. +7.9; p \ 0.0001). In NS (saline human oral epithelial cells (HOEPs) and mouse macrophage cells eluates), Th2 biomarkers were lower after dupilumab versus PBO were treated with GM-0111 and irradiated with X-rays (10–15 Gy), (mean % change from baseline): eotaxin-3 from W8 (-32.4 vs. and cell viability was determined by staining cells with Annexin V/7- +74.4, respectively; p = 0.017) to W16 (-38.5 vs. +183.0; AAD. (2) In vivo studies: BDF1 mice were subcutaneously treated p = 0.040); eosinophil cationic protein (ECP) at W12 (-2.5 vs.

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+102.6; p = 0.055) and W16 (+7.2 vs. +120.2; p = 0.072); and total B043 IgE from W8 (-15.7 vs. +60.1; p = 0.043) to W16 (-15.9 vs. +43.8; NOVEL ANTI-INFLAMMATORY EFFECT OF p = 0.025). The mean serum ECP, unlike the mean NS ECP, did not show a decrease during dupilumab treatment. After dupilumab CONIFERYL ALDEHYDE BY SELECTIVE treatment, there were transient increases in blood Eos counts which INHIBITION OF STAT1-INOS SIGNALING resolved during treatment (mean % change from baseline: W4, +48.9 dupilumab vs. +0.2 PBO; W16, -5.8 vs, +3.8). Injection site reac- Muhammad Akram, Kim Kyeong-A, Ok-Nam Bae tions (ISRs), headache, and nasopharyngitis were the most frequently reported adverse events, with ISRs more frequent with dupilumab. Hanyang University, Seoul, South Korea Conclusions: Dupilumab suppressed IL-4Ra-dependent biomarkers in Coniferyl aldehyde (CA), a phenolic compound, can be found in the peripheral blood and NS. Reductions in TARC and eotaxin-3 several medicinal plants. Although the anti-oxidant and anti-radical suggested that dupilumab may reduce homing of Th2 cells and Eos. activities of CA have been previously reported, its therapeutic Declines in IgE indicate that dupilumab modulates Ig switching away potential remains to be elucidated. In this study, we tried to determine from an allergic phenotype. Relative decreases in eotaxin-3, ECP, and the anti-inflammatory activity of CA using in vivo and in vitro sys- IgE in NS versus PBO demonstrated a local effect of dupilumab on tems and identify the underlying molecular mechanisms. CA nasal inflammation. significantly reduced nitrite production, iNOS enzyme activity and iNOS expressions in LPS-stimulated murine macrophages, RAW 264.7 cells. While NF-kB and MAPKs (ERK1/2, JNK 1/2 and p38) pathways, the representative cellular pathways for iNOS induction, B042 were not affected by CA, LPS-induced STAT1 phosphorylation and TSPO GENE EXPRESSION ON 3T3-L1 p-STAT1 nuclear translocation were significantly inhibited by CA. In DIFFERENTIATION PROCESS IS MODULATED in vivo mouse models, topical application of CA produced significant BY DIAZEPAM TREATMENT AND LPS protective effects against TPA-induced ear edema. Systemic admin- istration of CA also significantly reduced carrageenan-induced paw edema in rats, confirming its potent anti-inflammatory activity. CA Eric D. Barioni, Rodrigo A. Loiola, Edson M. Oliveira, Ana Campa, significantly reduced carrageenan-induced iNOS induction and Sandra HP Farsky STAT1 phosphorylation in a dose dependent manner in paw edema samples. Taken together, we demonstrated a novel anti-inflammatory University of Sa˜o Paulo, Sa˜o Paulo, Brazil activity of CA and its selective inhibition of STAT1-iNOS signaling Backgound: Obesity is a chronic disease associated to low intensity using in vivo and in vitro models, suggesting its potential therapeutic chronic inflammation, characterized by influx of macrophages, application in various chronic diseases associated with excessive insulin resistance, hyperglycemia, dyslipidemia, hypertension, and production of nitric oxide. others associated diseases. The 18 kDa translocator protein (TSPO), Keywords: Coniferyl aldehyde, anti-inflammatory activity, STAT1, previously known as the peripheral benzodiazepine receptor, has iNOS been implicated in many important physiological functions, Funding Source: None including cholesterol transport and steroidogenesis, cellular respi- Disclosure Statement: None ration and immunomodulation. TSPO is expressed in immune cells, and considered a marker of inflammation. Moreover, TSPO is expressed on adipocytes, nevertheless its role in adipose tissue B044 differentiation is not established. Hence, we here proposed to investigate the TSPO expression on adipocytes in different AN INVESTIGATION OF ANTI-INFLAMMATORY conditions. ACTIVITIES PRODUCED BY SCHISANDRIN Methods: TSPO gene expression on the differentiation process of A AND SCHISANDRIN B IN VITRO AND IN VIVO 3T3-L1 lineage was quantified by Real Time-PCR. The growth curve of 3T3-L1 pre-adipocytes treated with TSPO benzodiazepine agonist Pou Kuan Leong, Kam Ming Ko (diazepam, 1, 10, 100 and 1000 nM) and dimethylformamide (DMF, diazepam vehicle, 0.01 %) was evaluated by optical microscopy. The Division of Life Science, The Hong Kong University of Science and viability of 3T3-L1 pre-adipocytes treated with diazepam and vehicle Technology, Hong Kong, China was quantified by flow cytometer. TSPO gene and protein expression on the differentiation process of 3T3-L1 lineage treated with diaze- The dried fruits of Schisandra chinensis and Schisandra sphenan- pam, vehicle and E. coli lipopolysaccharide (LPS, 10 ng/mL) was thera are commonly used Chinese herbs for the treatment of quantified by Real Time-PCR and western blot, respectively, and inflammatory diseases. Schisandrin A (Sch A) and schisandrin B 3T3-L1 triacylglycerol storage was quantified by Oil Red staining and (Sch B) are the active ingredients isolated from the fruits of S. colorimetric assay. sphenanthera and S. chinensis, respectively. In the present study, we Results: Our results showed the positive regulation of TSPO com- compared the mechanism underlying the anti-inflammatory activities plementary tapes on 3T3-L1 differentiation process. Furthermore, produced by Sch A and Sch B, using cultured lipopolysaccharide diazepam treatment induced TSPO gene expression (diazepam (LPS)-stimulated RAW264.7 macrophages. The exposure to LPS 10 nM) and did not evoke significantly changes in the growth curve (1 lg/mL) activated the c-Jun N-terminal kinase 1/2 (JNK1/2), p38 and viability of 3T3-L1 pre-adipocytes. Finally, 3T3-L1 triacylglyc- kinase and the nuclear factor-jB (NF-jB), induced the releases of erol storage was not modified by diazepam or LPS treatment. tumor necrosis factor alpha (TNFa) and interleukin-6 (IL-6), with Conclusion: Our data show that TSPO is expressed on 3T3-L1 cells the resultant induction of inducible nitric oxide synthase (iNOS) as during the differentiation process, and its modulated by TSPO agonist well as cyclooxygenase 2 (COX2) and increased production of nitric and LPS. Further studies are being carried out to investigate the role of oxide (NO) in RAW264.7 cells, of which are indicative of an TSPO activation by different agonist on the adipogenesis process. inflammatory response. Pre-incubation with Sch A and Sch B Supported by FAPESP (2013/11027-7). (50 lM, alone) for 6 h produced an anti-inflammatory action in

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LPS-stimulated RAW264.7 cells, as evidenced by significant inhi- The search results yielded 733 applications, with nearly 90 % of bitions on the release of TNFa and IL-6, the expression of iNOS them filed by biotech and pharmaceutical companies. Even if the and COX2 as well as the production of NO. Sch A and Sch B also applications were widely dispersed, clearly the leader in the field is suppressed the LPS-activated pro-inflammatory signaling cascade, Amgen with about 3 % of the applications. Besides inflammatory including JNK1/2, p38 kinase as well as NF-jB in LPS-stimulated diseases, the claims included the treatment of cancer (11.2 %), RAW264.7 cells, with the extent of inhibition afforded by Sch A infections (3.4 %), cardiovascular disorders (2.9 %) and allergies being more prominent. The effect of Sch A and Sch B on the (1.9 %), in addition to diagnostics (2.8 %). Biologicals can be cate- antioxidant response was also investigated. The result showed that gorized by the way they work in the body, thus in a large amount only Sch B but not Sch A activated the nuclear factor (erythroid- (29 %) of the applications the main matter found was antibodies, derived 2)-like 2, the master regulator of antioxidant system, in followed by therapeutic and fusion proteins (14 %). The methods of RAW264.7 cells. At 16 h after the Sch B exposure, the expression use together with dosage forms and compositions were claimed in of thioredoxin 1 (TRX) was increased by 30 %. The ability of Sch 30 % of the applications. Around 25 % of the applications include B to induce TRX expression was associated with significant sup- signal transduction modulators as the core mechanism of action pressions on the releases of TNFa and IL-6 as well as the inductions influencing the efficacy of biologic agents. Given that these drugs of iNOS, COX2 and NO in LPS-stimulated RAW264.7 cells. To mainly inhibit specific components of the immune system that play confirm the results obtained from the cell-based study, the effect of pivotal roles in diseases, mechanisms for neutralizing cytokines were single bolus dose (1 mmol/kg, p.o.) or long-term low dose found in almost 30 % of the documents, including antibodies, soluble (0.25 nmol/kg p.o. for five consecutive days) of Sch A/Sch B on k- receptors, receptor blockade, and activation of anti-inflammatory carrageenan-induced paw edema in female ICR mice was examined. pathways by bioengineered versions of several cytokines. The long-term low dose treatment with Sch A and Sch B reduced Over the past decade, biologics have accounted for just about one- the k-carrageenan-induced paw edema to a similar extent in mice. third of new drug approvals and many studies are under way inves- However, the treatment with a single bolus dose of Sch A (but not tigating targets in diseases with inflammatory etiologies. The findings Sch B) significantly reduced the extent of paw edema in k-car- reported here can have important implications for both policy makers rageenan-injected mice, suggesting the anti-inflammatory response and executives involved in making strategic decisions on the phar- induced by Sch A being more instantaneous. In conclusion, both of maceutical companies, in order to find better therapeutic options for Sch A and Sch B were found to produce a direct anti-inflammatory patients, with enhanced clinical efficiency and reduced side effects. action by the suppression of pro-inflammatory signaling cascade. In addition, Sch B may also reduce the extent of inflammation in an indirect manner, presumably via the induction of an antioxidant response. B046 ADP TREATMENT IMPROVES WOUND HEALING IN DIABETIC MICE B045 OVERVIEW OF NEW STRATEGIES FOR Claudia F. Benjamim, Paula B. Alvarenga, Janaı´na L. Georgii, Ariane R. Brogliato, Jose´ Roberto Meyer, Robson Coutinho, PATENTING BIOLOGIC AGENTS TARGETING Josiane S. Neves INFLAMMATORY DISEASES Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Iolanda M. Fierro2,1, Rodrigo A. Oliveria1, Fernando Tibau1, 2 2,3 Objectives and Backgrounds: Chronics wounds are a health problem Natalia L. Von Ranke , Adelaide Maria S. Antunes worldwide, which affect 6.5 millions patients in USA. Such problem

1 in association with high global prevalence of diabetes, reflects the Universidade do Estado do Rio de Janeiro; Rio de Janeiro; Brasil; increase in diabetic ulcers. Considering the absence of an effective 2Instituto Nacional da Propriedade Industrial; Rio de Janeiro; 3 treatment for chronic wound, we investigated the possible beneficial Brasil; Universidade Federal do Rio de Janeiro, Rio de Janeiro, effects of purinergic agonists in tissue repair. In the present work we Brasil explore the role of ADP in healing process of skin chronic wounds in In 2014 five of the 10 top-selling drugs were biologics, a number diabetic mice. expected to grow in the next coming years. Biologic agents (also Methods and results: In this study, Diabetes Mellitus was induced by a termed biologicals or biologics) are therapeutics synthesized by living single intravenous Alloxan injection (65 mg/kg) 8 h after fast in Swiss organisms and targeting distinct immune cell receptors or cytokines. male mice (20–24 g). Mice were then separated in four groups and Biologicals have changed the way to treat several inflammatory and treated once a day per 14 days as following: (a) control group: saline immune-mediated disorders, including rheumatoid arthritis, inflam- was topically applied on wound beds; (b) ADP group, 30 lM of ADP matory bowel disease and psoriasis. Patent data can be used as a was topically applied on wound beds; (c) clopidogrel + saline: in source of information to measure science and technology activities. which clopidogrel was given by gavage and saline was topically This work analyzes patent applications of biologics for the treatment applied on wound beds; and (d) clopidogrel + ADP, in which clopi- of inflammatory diseases, in order to identify technology trends and to dogrel was given by gavage and ADP was topically applied on wound better understand the strategies adopted by the main actors in the bed. Wound contraction was measured at days 3, 7, 10, and 14 days development of new formulations of these compounds. after skin lesions. Wounds were collected at day 7, formalin fixed and The technological forecasting focused on patent applications paraffin-embedded. Skin sections were stained with HE (for granula- between 2010 and 2015, considering that most of these medicines tion tissue), Sirius Red (for collagen), modified Sirius Red (for have gained approval only within the last few years. The search eosinophil), Alcian Blue (for mast cell), or immunostained for a-actin, strategy to recover these documents was based on predefined key- laminin, macrophages, VEGF and TGF-a. Wounds were also collected words using the database Thomson Reuters Integrity SM that indexes at day 7 for MPO activity and ELISA. Our data showed that ADP was patent applications in the World Intellectual Property Office in able to accelerate the wound healing of diabetic mice, which resembles addition to other six patent offices, covering the main world markets the healing of non-diabetic mice. Clopidogrel treatment, a P2Y12 in the pharmaceutical field. receptor antagonist, prevented the lesion closure in diabetic mice

123 Inflamm. Res. S121 treated with ADP. Interestingly, clopidogrel worsened the lesion clo- that control the chain of suppressive and pro-metastatic events, sure even in non-diabetic mice. Others nucleotides as adenosine, ATP, including the activation of MDSCs. AMP50 and AMP30 at 30 lM did not accelerate the lesion healing in This work was supported entirely by the Intramural Research Pro- diabetic mice, as observed for ADP. It was observed by histological gram of the NIH, National Institute on Aging analysis that ADP treatment improved the granulation tissue forma- 1. Olkhanud, P. B. et al. Tumor-evoked regulatory B cells promote tion, collagen synthesis and increased the recruitment of eosinophils breast cancer metastasis by converting resting CD4 T cells to and neutrophils, and the population of mast cells on the seventh day T-regulatory cells. Cancer research. 71, 3505–3515. doi:10.1158/ after the beginning of the lesion. ADP stimulated the release of the 0008-5472.CAN-10-4316 (2011). cytokine IFN-c on the third day and the IL-10 and IL-13 on the seventh 2. Bodogai, M. et al. Anti-CD20 antibody promotes cancer escape via day. In addition, at day 7 ADP was effective in increasing the differ- enrichment of tumor-evoked regulatory B cells expressing low levels entiation of myofibroblasts and the expression of laminin, VEGF and of CD20 and CD137L. Cancer research. doi:10.1158/ TGF-a. Still in this time point, ADP seemed to increase the argina- 0008-5472.CAN-12-4184 (2013). se + cells and to reduce iNOS + cells, which implies in the increase of 3. Olkhanud, P. B. et al. Breast cancer lung metastasis requires M2 macrophages in the wound after ADP. expression of chemokine receptor CCR4 and regulatory T cells. Conclusion: Our results suggest that ADP accelerates the wound Cancer Res.69, 5996–6004 (2009). healing in diabetic mice and the mechanism seems to be the recruitment of inflammatory cells to the lesion and by positive modulation of the wound repair. Financial support CAPES, CNPq and FAPERJ. B048 ROLE OF COLLAGEN VI TO IMPROVE TISSUE INTEGRATION OF DENTAL IMPLANTS B047 NEUTRALIZING THE PRO-METASTATIC Ramesh Tati1, Veronika Dill1, Melissa N. Langer1, Suado M. 1 1 1 1 IMMUNE RESPONSES OF MDSC AND T CELLS Abdillahi , Sara Nordin , Maria Baumgarten , Matthias Mo¨rgelin , Eric Gerner2, Christina Gretzer2 THROUGH B CELL TARGETING 1Lund University, Lund, Sweden; 2Dentsply Implants, Sweden Monica Bodogai1, Kanako Moritoh1, Catalina Lee Chang1, Background and objective: Followed by the surgical application of Christine M. Hollander3, Li Yang3, Robert Wersto2, Arya Biragyn1 dental implants, oral pathogens are able to infiltrate the damaged tissue leading to infection in the oral cavity. Collagen type VI, a sub- 1NIH, National Institute on Aging, Laboratory of Molecular Biology epithelial extracellular matrix component has been shown to have and Immunology, Bethesda, MD, USA; 2NIH, National Institute on adhesive properties as well as antimicrobial activity against patho- Aging, Flow Cytometry Unit, Bethesda, MD, USA; 3NIH, National gens. As a part of recent developments in the bioactive surfaces Cancer Institute, Laboratory of Cancer Biology and Genetics, antimicrobial treated implants, made of titanium, may be beneficial in Bethesda, MD, USA this case. The aims of the present study were to investigate the Although cancer metastasizes via expanding myeloid-derived sup- antimicrobial activity of collagen VI against invading pathogens and pressive cells (MDSCs) that suppress antitumor effector immune also its role in enhancement of tissue integration of dental implants. responses, our recent results questioned this simplistic view. The loss of Methods: Human keratinocytes were cultured on collagen coated cancer-induced B cells alone is sufficient to abrogate metastasis titanium discs followed by bacterial inoculation. Scanning electron without affecting the expansion of MDSCs. We report that metastasis microscopy was performed to check the keratinocyte morphology on of a highly aggressive 4T1 breast cancer requires the help from regu- titanium discs and also to detect bacterial killing in order to conform latory B cells (tBregs) converted from resting B2 cells in response to the antimicrobial activity of collagen VI. cancer-produced metabolites of 5-lipoxygenase. The tBregs produce Results: Results showed that the keratinocytes had shown better high levels of TGFb and convert CD4+ T cells into CCR4+ FoxP3+ - attachment on modified titanium surface. In case of infection, ker- Tregs that inactivate NK cells and CD8+ T cells protecting the lungs atinocyte survival was increased on collagen VI coated surfaces from metastasizing tumor cells. Here, using modelling studies with thereby confirming the antimicrobial activity of collagen VI against ex vivo generated MDSC and mice with different tumors, we report that oral pathogens. tBregs play a previously unknown, but essential role in metastasis— Conclusions: Together, these data suggest that extracellular matrix they activate the regulatory function of cancer-primed MDSCs. To do components are able to protect the tissues from invading pathogens this, cancer-induced B cells/tBregs directly activate the regulatory thereby improving the tissue integration onto the implant leading to function of both Mo-MDSCs and PMN-MDSCs, requiring at least in improved performance. part TgfbR signaling. The educated MDSCs increase production of ROS and NO and more efficiently suppress anti-tumor CD4+ and CD8+ T cells and thereby promote metastasis. As such, the loss of B cells or the TgfbR deficiency in MDSCs is sufficient to disable the B049 education of MDSCs and concurrently block metastasis. Thus, cancer APPLICATION OF TREGITOPES FOR TREATMENT induced B cells need to be removed to improve antitumor immune OF TYPE 1 DIABETES (T1D): OPTIMIZATION OF responses. However, the B-cell neutralizing strategy should be THE TREGITOPE DELIVERY VEHICLE approached cautiously, as depletion of B cells with anti-CD20 Ab/ rituximab caused enrichment of CD20Low 4-1BBLLow tBregs and Ann S. De Groot1,2, Leslie Cousens1, Christine Boyle1, Guilhem thereby further enhanced lung metastasis by exacerbating tBreg-me- 1 1 diated immunosuppression. In contrast, we show that BLC-arp-coupled Richard , William Martin

CpG-ODN can efficiently abrogate lung metastasis by inactivating 1 2 tBregs and activating immunostimulatory B cells. Overall, these results EpiVax, Inc, Providence, USA; University of Rhode Island, further underscore the importance of B cells/tBregs as important factors Rhode Island, USA

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HLA class II-restricted regulatory T cell (Trig) epitopes in IgG western blot. To induce osteoclasts (OC) recruitment, RAW264.7 (‘‘Tregitopes’’) have been reported to suppress immune responses to cells were incubated with 50 ng/ml LPS with or without RvD1 (1-10 co-administered antigens by stimulating expansion of natural Tregs uM) for 24 h. OC phenotype markers, namely, TRAP and (nTregs). In our previously published work we show that co-admin- cathepsin-K were assessed by western blot, enzymatic staining and istration of Tregitopes and T1D antigens delayed development of immunocytochemistry. hyperglycemia and reduced T1D incidence in NOD mice. T1D sup- Results: Our results show that RvD1 inhibits COX-2 and iNOS pression was observed even following disease onset.1 Current studies expression as well as PGE2, MMP-13 and NO generation. These aim to identify a suitable delivery vehicle and dosing regimen for effects are likely to be attributed to the blocking of the activation of Tregitope therapy. Using both an OVA-mouse model and NOD T1D p38 MAPK, SAPK/JNK1/2 and NF-kB/p65. Besides, RvD1 strongly mouse model, different delivery vehicles were used to co-deliver reduces OC recruitment as indicated by the inhibition of TRAP and Tregitopes and antigen. The effects of these in vivo regimes on cathepsin K expression. antigen-specific immune responses was measured in vitro. From these Conclusion: Our in vitro results clearly show that RvD1 presents very studies, a human serum albumin (HSA) Tregitope fusion emerged as interesting anti-inflammatory, anti-catabolic and anti-resorptive our lead candidate. To examine the therapeutic potential of this properties in OA. Additionally, our recent data that are not presented delivery vehicle, HSA-Tregitope fusion was administered to NOD showed a remarkable anti-apoptotic and antioxidant potential of mice with blood glucose level between 200-350 mg/dL on two con- RvD1 in OA chondrocytes. Taken together, these findings suggest secutive days. Mice that received the HSA-Tregitope fusion exhibit that RvD1 may offer a novel and original perspective to make a real prolonged adjusted survival compared to other treatment groups. contribution to OA therapy. These results show promise for implementation of these defined Acknowledgement Centre of excellence for arthroplasty research regulatory T cell epitopes for therapy of T1D, and the HSA-Tregitope (RACE). fusion as a potential treatment platform for the application of Tregi- tope therapy to other autoimmune diseases. 1. Cousens LP, Su Y, McClaine E, et al. Application of IgG-Derived Natural Treg Epitopes (IgG Tregitopes) to Antigen-Specific Toler- B051 ance Induction in a Murine Model of Type 1 Diabetes. Journal of EFFECT OF ATORVASTATIN ON Diabetes Research. 2013;2013:621693. doi:10.1155/2013/621693. INFLAMMATORY BONE LOSS IN RATS WITH GLUCOCORTICOID-INDUCED OSTEOPOROSIS: PARTICIPATION OF WNT/ß-CATENIN PATHWAY Cartilage and Bone Remodeling Karuza MA Pereira1, Karianne M. Mendonc¸a1, Luzia HT Sousa1, 1 1 1 B050 Ana L. Franc¸a , Eveline Linhares , Pedro H. Isaias , Ana GF Linhares1, Danielle RD Val2, Raul S. Freitas1, THE ROLE OF RESOLVIN D1 IN THE Mirna M. Bezerra1, Hellı´ada V. Chaves1, Gerly Anne DC Brito1, REGULATION OF INFLAMMATORY AND Conceic¸a˜o DS Martins1, Joanna T. Magalha˜es1, Paula Goes1 CATABOLIC FACTORS IN OSTEOARTHRITIS 1Federal University of Ceara´, Fortaleza, Brazil; 2Federal University Houda Benabdoune1,2, Shi Qin2, Julio Fernandes2, Pierre Ranger3, of Pernambuco, Recife, Brazil Hassan Fahmi1,4, Mohammed Benderdour1,2 Periodontal disease (PD) is a chronic infectious-inflammatory disease that causes loss of connective tissue and alveolar bone loss. Gluco- 1Pharmacology department, Universite´ de Montre´al, Montreal, QC, cortioid-induced osteoporosis (GIOP) is the main cause of secondary Canada; 2Orthopedic research laboratory-Hoˆ pital du Sacre´-Cœur de osteoporosis. Atorvastatin (ATV) is a hypolipemiant drug that has Montre´al, Montreal, QC, Canada; 3Hoˆ pital du Sacre´-Cœur de also present anti-inflammatory activity and bone anabolic effects. It Montre´al, Montreal, QC, Canada; 4Research Center-Centre has been recently reported the WNT/b-catenin signaling might have a hospitalier de l’universite´ de Montre´al, Montreal, QC, Canada role on osteoblastic differentiation. Therefore the aim of this work Introduction: Osteoarthritis (OA) is a degenerative joint disease, was to evaluate the effect of Atorvastatin on inflammatory bone loss characterized by cartilage and bone damage and accompanied by in rats with glucocorticoid-induced osteoporosis and the participation increased production of inflammatory and catabolic mediators such as of WNT/b-catenin pathway on this condition. Experiments were prostaglandin-E2 (PGE2), nitric oxide (NO), metalloproteinase-13 approved by the Institutional Animal Care and Use Committee of the ´ (MMP-13), tartrate-resistant acid phosphatase (TRAP) and cathepsin- Federal University of Ceara, Fortaleza, Brazil (49/2012). The animals K. Despite its important consequences, therapeutic modalities for OA were divided in four groups: PD receiving 0.9 % saline solution are limited to symptomatic treatments. Resolvin-D1 (RvD1) is a (2 mg kg-orally); GIOP: (7 mg/kg of dexamethasone once/wk for derivative of the omega-3 fatty acids and a potent anti-inflammatory 5 weeks – IM); GIOP + PD; ATV: GIOP + PD (27 mg/kg of ATV- agent synthesized during the resolution phase of inflammation. The orally). Parameters evaluated: (1) Alveolar bone loss (ABL), through overall objective of this study is to investigate the anti-inflammatory, macroscopic and radiographic analysis; (2) Radiographic density of anti-catabolic and anti-resorptive role of RvD1 in OA. femur; (3) Leukogram; (4) Bone-Specific Alkaline Phosphatase Methods: Post-surgery discarded human OA articular cartilage was (BALP); (5) Transaminases (TGO/TGP); (6) Histological analysis; obtained from OA patients who underwent total knee arthroplasty. (7) Immunohistochemistry for WNT 10b, b-catenin, DKK1; (8) First passage human OA chondrocytes, were treated with IL-1B with Gingival cytokine level IL-1b and TNF-a; (9) Myeloperoxidase levels or without RvD1 (1-10 uM) for 24 h. The expression of cyclooxy- in gingival tissue. The animals from GIOP + PD group showed greater ABL (7.53 ± 0.75 mm2) compared to PD (5.24 ± 0.39 mm2) genase-2 (Cox-2) and inducible NO synthase (iNOS) was 2 determined by western blot and real-time PCR. Levels of PGE2, and ATV prevented ABL (4.68 ± 0.39 mm ) compared to MMP-13 and NO were measured by EIA, ELISA and Greiss reac- GIOP + PD (p \ 0.05). The radiographic density analysis of maxillae tion, respectively. To investigate the signaling pathways, p38 corroborated the macroscopic analysis. ATV prevented the reduction MAPK, SAPK/JNK1/2, NF-kB/p65 activation was determined by of radiographic density of femur (173.91 ± 4.87 grey tones) when

123 Inflamm. Res. S123 compared to GIOP + PD (158.48 ± 5.38). ATV prevented the transporters renders monocytes/macrophages no longer sensitive to leukocytosis/neutrophilia (L = 8.5 ± 0.75 cell/mm3/N = 3.1 ± 0.53) chemoattractants. Our data suggests that acute infusion of apoA1 seen in GIOP + PD group (L = 15.89 ± 1.78/N = 6.3 ± 0.46). or apoA1 mimetics could significantly reduce inflammatory myeloid ATV treatment did not alter transaminases serum levels. ATV pre- cell recruitment via effects on responsiveness to multiple vented BALP reduction (25.56 ± 1.73 U/L) when compared baseline chemoattractants. (29.52 ± 0.93 U/L). GIOP + DP showed intense inflammatory infil- trate and bone loss (3 [3–3]) and ATV prevented these microscopic findings (1 [1–2]) (p \ 0.05). It was seen greater immunolabeling for WNT and beta-catenin and reduced immunolabeling for DKK on B053 ATV group when compared to GIOP + PD group. ATV prevented the TRPV4 MEDIATES MATRIX STIFFNESS- increase on TNF (2.53 ± 0.09 pg/ml) and IL-1 (4.50 ± 0.17) levels on gingival tissue, when compared to GIOP + PD (TNF = DEPENDENT ENDOTHELIAL ACTIVATION 4.61 ± 0.53; IL-1 = 6.33 ± 0.31) (p \ 0.05). ATV prevented the increase on MPO levels in gingiva. In this way we can conclude that Harry A. Scott, Xiao Yang, Soroush Ardekani, Andrea Cabrera, ATV has an anti-inflammatory effect and prevents alveolar bone loss Kaustabh Ghosh in GIOP + PD conditions through WNT/b-catenin signaling pathway. Funding Sources FUNCAP, CNPq, CAPES. University of California Riverside, Riverside, CA, USA Endothelial activation during inflammation, resulting from impaired activity of eNOS, is a key determinant of various conditions such as Cell Adhesion and Leukocyte Migration emphysema, diabetes and atherosclerosis. Past efforts to understand impaired NO-dependent endothelial activation have focused on genetic factors, soluble cues, and shear stress. However, these B052 inflammatory conditions are also marked by abnormal subendothelial ACUTE EXPOSURE TO APOLIPOPROTEIN A1 matrix. To determine whether matrix stiffness alone regulates INHIBITS MACROPHAGE CHEMOTAXIS endothelial activation, ECs were plated on fibronectin-coated poly- acrylamide (PA) gels to normal (1000 Pa), softer (200 Pa), and IN VITRO AND MONOCYTE RECRUITMENT stiffer sub-endothelial matrices (4000 Pa). Here we found, abnormal IN VIVO matrix stiffness induces increased monocyte-EC adhesion which correlates strongly with decreased phospho-eNOS and NO produc- Asif J. Iqbal1, Tessa J. Barrett3, Lewis Taylor1, Eileen McNeill1, tion. Importantly, this adhesion, at least in part, is mediated through Carlota Reico1, Gemma E. White1, Maximillian H. Brodermann1, stiffness-dependent biphasic ICAM-1 clustering with increased Dianne Cooper2, Keith M. Channon1, David R Greaves1 clustering on soft and stiff matrices. Notably, we show for the first time, stiffness-dependent variations in TRPV4 activity. This differ- 1University of Oxford, Oxford, UK; 2Queen Mary’s University of ential activity influences NO production, as determined by TRPV4 London, London, UK; 3NYU School of Medicine, New York City, NY, inhibition. Further, we show that stiffness-dependent endothelial USA activation can be reversed by TRPV4 modulation. These findings indicate TRPV4 as a key mediator in matrix stiffness-dependent Apolipoprotein A1 (apoA1) is the major protein component of high endothelial activation and implicate it as a therapeutic target for anti- density lipoprotein (HDL) and has been reported to have anti-in- inflammatory strategies. flammatory properties in addition to its role in reverse cholesterol transport. To better understand the anti-inflammatory activity of apoA1 we explored the effects on monocyte and macrophage chemotaxis in vitro and monocyte trafficking in vivo. B054 Acute apoA1 treatment (20–60 min) induced a substantial DOCK8 EXPRESSION IN DENDRITIC CELLS (50–90 %) decrease in murine macrophage chemotaxis in real-time assays to a range of pathophysiologically relevant chemoattractants DIFFERENTIALLY REGULATES CYTOTOXIC T (CCL2, CCL5, chemerin and complement C5a). In addition, human LYMPHOCYTE RESPONSES DURING monocyte chemotaxis to CCL2 was abrogated by short-term INNOCUOUS AND PATHOGENIC CHALLENGES treatment with apoA1, and human PBMC exposed to apoA1 for 45 min showed reduced rolling, adhesion and transmigration in Manuela Sales Lima Nascimento1,2, Dong Liu1, Pei Chen1, Lan Xu1, experiments with activated HUVEC monolayers under physiologi- Jayendra Kumar Krishnaswamy1, Samuele Calabro1, cal flow conditions. Remarkably, acute pre-treatment of mouse Gowthaman Uthaman1, Antonia Gallman1, Joa˜o Santana da Silva2, GFP+ monocytes with apoA1 ex vivo reduced recruitment by 65 % Stephanie C. Eisenbarth1 (p \ 0.01) following adoptive transfer into an on-going peritonitis model. 1Yale University School of Medicine, New Haven, Connecticut; Macrophages exposed to acute pre-treatment with the non-specific 2Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Sa˜o Paulo, cholesterol depleting agent methyl-ß-cyclodextrin or macrophages Brazil from ATP-binding cassette transporter A1 (ABCA1) null mice dis- play a similar reduction in macrophage migration, indicating that the DOCK8 is a guanine nucleotide exchange factor (GEF) that activates mechanism for A10s effects is independent of its role in reverse Rho GTPase CDC42, playing an important role in controlling cell cholesterol transport. We also demonstrate that the rapid effects of cytoskeletal function and migration capacity. DOCK8 deficiency apoA1 on monocyte/macrophage recruitment occur via reducing the leads to an immunodeficiency syndrome characterized by severe activity of PI3kinase, a signalling pathway important for actin-cy- atopy, early malignancy and recurrent viral infections in humans. We toskeleton reorganisation. have previously shown that the absence of Dock8 results in defective + Collectively our data support a model in which rapid depletion/re- DC migration, which also abrogates CD4 T cell activation. The role + organisation of membrane cholesterol independent of ABCA1 of DOCK8 on DC migration and the subsequent impact on CD8 T

123 S124 Inflamm. Res. cells responses, however, remain unclear. To study this, we took this pivotal enzyme in mediating some of Met’s anti-inflammatory advantage of protein antigen immunization and a bacterial infection effects. murine model. By adoptively transferring OVA-specific TCR Conclusion: Protection of the glycocalyx by Met via activation of transgenic OT-1 T cells into both WT and Dock8-/- mice, we AMPK may contribute to its anti-inflammatory activities and help demonstrated that CD8+ T cell priming is not impaired in contrast to explaining, in part, its cardiovascular benefits. a highly attenuated CD4+ OT2 T cell response in Dock8-/- mice Financial support Canadian Institutes of Health Research (CIHR), after OVA immunization. Moreover, ovalbumin coated splenocytes -/- Canadian Foundation for Innovation (CFI), Fonds de la Recherche du (OCS) immunized Dock8 mice generated comparable amount of Que´bec-Sante´ (FRQS) and the Wilbrod-Bherer Foundation. cytotoxic T lymphocytes (CTLs), which produce normal levels of IFN-c and granzyme-B, and are equally capable killing target cells in vivo. Surprisingly, our preliminary results suggest that Dock8-/- mice infected with Listeria monocytogenes (L. monocytogenes) B056 + showed a defect in CD8 T cell proliferation. Altogether, these data CHEMERIN: INFLAMMATORY BIOMARKER OR suggest that although DOCK8 is dispensable for CD8+ T cell priming during a noninvasive antigen challenge, it might be required ANTI-INFLAMMATORY MEDIATOR? for CTL priming to bacterial-derived antigens. Therefore, our data highlight the critical role of DOCK8 for an effective adaptive Daniel Regan-Komito, Asif J. Iqbal, David R. Greaves immune response. University of Oxford, Oxford, UK Background: Human pro chemerin (E21-S163) is a 142 amino acid protein that circulates in plasma in an inactive form. During inflam- B055 mation, the carboxyl terminus is cleaved by serine proteases to METFORMIN MODULATES NEUTROPHIL generate shorter bioactive forms of the protein which act as chemoattractants for macrophages, immature DCs, pDCs, and NK MIGRATION: IMPLICATION OF ENDOTHELIAL cells. Chemerin has been reported to play a role in the recruitment CELL AMPK, ADHESION PROTEIN EXPRESSION DCs, pDCs and NK cells to secondary lymphoid organs as well as AND GLYCOCALYX INTEGRITY sites of inflammation. A number of studies have reported elevated chemerin levels in patients with inflammatory and metabolic diseases Genevie`ve Bertheau Mailhot, Giambelluca S. Miriam, while others have reported anti-inflammatory effects of chemerin. We Cynthia Laflamme, Nathalie Bourcier, Aline Dumas, Benoıˆt Mailhot, have analysed endogenous chemerin levels during murine inflam- Luc Vallie`res, Steve Lacroix, Marc Pouliot matory disease models as well as the effects of modulating endogenous chemerin levels therapeutically. We hypothesised that during inflammation active murine chemerin T17-S156(corresponding Centre de recherche du CHU de Que´bec-Universite´ Laval, Quebec 21 157 City, Que´bec, Canada to Chemerin E -S in humans) is further cleaved by inflammatory proteases to generate shorter bioactive peptides with anti-inflamma- Introduction: Metformin (Met), a drug of the biguanide class, is a tory properties. first-line treatment for diabetes mellitus type 2. Beside its blood- Materials and methods: C57Bl6/J mice were injected intraperitoneally glucose controlling effect, Met ameliorates a number of associated with 100 lg of zymosan. Mice were treated with chemerin, dexam- complications such as cardiovascular diseases, suggesting anti-in- ethasone or PBS at different time points. The peritoneal cavity was flammatory properties. In the present study, we investigated the lavaged with ice cold PBS supplemented with 2 mM EDTA and impact of metformin on acute inflammatory responses. peritoneal exudate cells were quantified using flow cytometry. Methods: Air pouch, and dextran sulfate sodium (DSS) murine Chemokines, cytokines and chemerin levels were measured using models were used to measure neutrophils migration, viability and ELISA/Luminex. Plasma was also analysed for chemerin levels. activation. Mice received DSS (3.5 %) in drinking water, with or Results: Injection of 100 lg zymosan elicited a sterile inflammatory without Met 50 mg kg-1. Myeloperoxidase assays and immunohis- response in which neutrophils peaked at 4 h and mono- tochemistry were performed on colon tissues. In adhesion cytes/macrophages peaked at 8 h. T and B cells peaked in the experiments, human microvascular cells (HMEC-1) were stimulated peritoneum at later stages (9 and 13 days). Endogenous chemerin with LPS (0.01–5 lgmL-1), TNF (1–30 ggmL-1) or IL-1b levels increase both locally and systemically during the onset of (3–100-ggmL-1) with or without Met (0.001–3 mM). Human inflammation while dexamethasone significantly decreased chemerin neutrophils were obtained from healthy donors and incubated with levels. Pre-treatment with recombinant murine chemerin had no effect Calcein-AM. ELISAs quantified the surface expression of adhesion on monocyte recruitment. However, when administered as an inter- proteins and glycocalyx components. vention into an inflammatory environment chemerin decreased Results: In the air pouch model, Met reduced neutrophils migration monocyte recruitment by *27 % (P B 0.001) as well as CCL4 levels by up to 50 % to the site of injection. We also observed a reduction by *30 % (P B 0.05). Using mass spectrometry we have demon- in the DSS model, with significant decreases of myeloperoxidase strated that murine chemerin can indeed be further processed to activity and neutrophil numbers in colon tissues, in presence of Met. shorter bioactive peptides by the inflammatory neutrophil derived In vitro, Met reduced adhesion of neutrophils to HMEC-1 in a proteases elastase and cathepsin-G. concentration-dependent manner, by up to 50 %, without signifi- Conclusions: When chemerin was administered as a pre-treatment, cantly affecting the expression of adhesion proteins. Instead, Met there were no significant effects on monocyte recruitment. However, appeared to prevent degradation of the endothelial cell glycocalyx when administered into an inflammatory environment, we observed induced by inflammatory agonists, as measured by a reduction in the significant decreases in monocyte numbers and chemokine levels. We release of glycocalyx components. Incubation of HMEC-1 with an have also demonstrated the ability of neutrophil derived proteases to activator of 50 adenosine monophosphate-activated protein kinase cleave active chemerin into shorter bioactive chemerin peptides. (AMPK), 5-amino-4-imidazole carboxamide riboside (AICAR), Taken together our experimental data are consistent with further reproduced the effects of Met in that system, supporting a role for processing of chemerin (T17-S156) by proteases present at sites of

123 Inflamm. Res. S125 inflammation generating shorter anti-inflammatory peptides, which quantified by measuring the binding of fluorescently labelled leuko- may prove to have therapeutic benefit. cytes to immobilized intercellular adhesion molecule-1 (ICAM-1), the major ligand of LFA-1. The activation status of LFA-1 was monitored by quantifying the binding of the conformation-sensitive mAb MEM148. The impact of the inhibitors on the expression of LFA-1 B057 and other immune receptors was investigated by flow cytometry and SPIN90 DEFICIENCY INCREASES CXCL13- ImageStreamxtechnology. MEDIATED B CELL MIGRATION Results and conclusion: All inhibitors had in common that they blocked LFA-1-mediated leukocyte adhesion to ICAM-1, as expec- Sang-Heon Park ted. However, the ligand mimetic induced the LFA-1 activation epitope MEM148 in contrast to the allosteric inhibitor and the anti- Gwangju Institute of Science and Technology (GIST), Gwangju, LFA-1 antibody. Moreover, the antibody and the ligand mimetic Korea triggered internalization of LFA-1 while the allosteric inhibitor did not affect the surface expression of LFA-1. Changes in LFA-1 surface Cell migration is important wound healing and immune Responses. T expression were found to be associated with changes in T-cell cells and B cells migrate to regions of the lymph nodes where they receptor expression. In conclusion, the differential effects of these can interact with each other and facilitate T cell-dependent antibody LFA-1 inhibitors representing different modes of action may need to production. SPIN90 is a ubiquitously expressed. SPIN90 binds to be taken into account during their further pre-clinical and clinical actin related proteins, such as WASP, which mediates the effect of development. CDC42 on actin cytoskeleton reorganization, and SPIN90-C-termius interacts with Arp2/3 complex, and G- and F-actin, and participates in the regulation of actin dynamics. CXCL13 is one of the lymphoid chemokines. CXCR5 is the receptor for CXCL13, required for lym- B059 phoid follicle formation, follicular helper T cell (Tfh) and T cell- THE INFLUENCE OF MELANOCORTINS ON dependent B cell activation. In addition, the chemokine-receptor- PLATELET LEUKOCYTE INTERACTIONS mediated signal attracts B cells and T cells into lymph nodes or spleen, towards the B cell or T cell zone of lymphoid follicles. Felicity NE Gavins, Paul M. Holloway, Helen K. Smith, CXCR5 deficiency in T cells leads to failure of plasma cell later stage D.Neil Granger and germinal centre reponses. CXCL13-mediated B cell migration into lymphoid follicles is important during B cell-mediated immune LSUHSC-S, Shreveport, LA, USA reponses. In this study, we investigated the role of SPIN90 in CXCL13- Aberrant inflammatory responses play a significant role in the initi- mediated B cell migration using Spin90 gene-deficient mice. Our ation, maintenance and progression of a wide variety of both chronic chemokinesis analysis and transwell cell migration assay showed that and acute diseases. Further to their crucial role in haemostasis, it has SPIN90 is involved in CXCL13-mediated B cell migration without become evident that platelets display a variety of inflammatory affecting B cell development. Moreover, the level of CXCR5, which functions, acting both as target and effector cells, allowing cross talk is CXCL13 receptor, was increased in SPIN90-deficient B cells between inflammatory and thrombotic processes. compared with wild-type B cells. Overall, our data suggest that The melanocortin receptor system has demonstrated impressive SPIN90 plays an important role in B cell immune responses through anti-inflammatory properties and has been proposed as an endogenous the regulation of CXCL13-mediated B cell migration. system for the resolution of inflammation. Activation of melanocortin receptors can modulate a variety of inflammatory molecules also implicated in platelet functioning, however despite the intimate association between inflammation and thrombosis, the influence of B058 melanocortin signaling on platelet functioning has not yet been DIFFERENT MODES OF LFA-1 INHIBITION investigated. As such we have utilized both in vivo and in vitro models of platelet-leukocyte cross talk to evaluate the potential TRANSLATE INTO DIFFERENT DOWNSTREAM influence of melanocortins on platelet inflammatory and thrombotic EFFECTS IN VITRO functioning. Melanocortin receptor dependant and independent effects were investigated using the super potent melanocortin receptor Riccardo V. Mancuso1, Stephan Kra¨henbu¨hl1, agonist, NDP-a-MSH and the melanocortin peptide terminal sequence Gabriele Weitz-Schmidt2 KPV, which has previously been suggested to act independently of melanocortin receptor binding, instead acting as an IL-1b receptor 1University Hospital Basel, Division of Clinical Pharmacology and antagonist. Toxicology, Hebelstrasse 20, Basel, Switzerland; 2AlloCyte In a static leukocyte-platelet adhesion assay, treating murine Pharmaceuticals AG, Hochbergerstrasse 60C, Basel, Switzerland neutrophils with either the melanocortin receptor agonist NDP-a- MSH, or the terminal melanocrtin sequence KPV, was found to sig- Objectives: The integrin lymphocyte function-associated antigen-1 nificantly reduce murine neutrophil adherence to IL-1B stimulated (LFA-1) mediates the recruitment of leukocytes to sites of inflam- platelet monolayers. Furthermore platelets treated with KPV prior to mation and is a promising therapeutic target. Three classes of LFA-1 IL-1B stimulation showed significantly reduced affinity to untreated inhibitors with different modes of action have been identified: (1) neutrophils, further supporting KPV as acting as an IL-1b antagonist. Monoclonal antibodies (mAbs), (2) ligand mimetics, and (3) allosteric However, neither treatment influenced thrombin stimulated or vehicle inhibitors. The objective of this study was to compare these different treated platelets. types of LFA-1 inhibitors for downstream effects in vitro. To further investigate leukocyte platelet interactions in vivo, Methods: The effects of the LFA-1 inhibitors on (1) cell adhesion, (2) intravital microscopy was performed in the inflamed cerebral the LFA-1 conformational status and (3) the surface expression of microvasculature. C57BL/6 mice were treated with lipopolysaccha- LFA-1 and other immune receptors were analysed. Cell adhesion was ride (LPS) 4 h prior to visualizing the cerebral vessels and leukocytes

123 S126 Inflamm. Res. were visualized by i.v infusion of rhodamine 6G. Donor platelets molecules shaped by pathogens to escape the immune system, we were labeled with carboxylfluorescein succinimidyl ester (CSFE) and previously identified a small compound, the chalcone 4. By analogy infused 5 min prior to imaging. LPS treatment was found to signifi- to the effect of neutralizing antibodies, this molecule is called neu- cantly increase both leukocyte and platelet adherence to cerebral traligand, as it binds to the chemokine CXCL12 but not to the cognate microvessels. Treatment with KPV was found to significantly reduce receptor CXCR4. Besides the regulation of homeostatic processes, LPS induced platelet adherence whilst also reducing both leukocyte CXCL12 is implicated in skin inflammation, in particular the Th2- rolling and adherence on the inflamed vasculature. mediated atopic dermatitis (AD) disease. Given that this chemokine is This data suggests that melanocortin peptides may suppress pla- involved in AD on one hand, and the number of pharmacological telet leukocyte interactions and that the terminal melanocortin tools to investigate its function or to correct for defects is limited, it sequence KPV may directly influence platelet function. The mecha- seems very interesting to use specific chemokine inhibitors like the nisms by which melanocortins influence these events and the chalcone 4 neutralizing CXCL12. So, we evaluated the in vivo consequence on platelet thrombotic function are the subject of our on activity of this compound in a MC903-induced AD model, and we going investigations. showed that the topical application of chalcone 4 (350 lmol/kg) significantly reduced ear redness and thickness, as well as the infil- tration of inflammatory cells into the skin. In addition, the expression Chemokines and Chemokine Receptors of the Th2-related cytokines (L-4 and IL-5) in the skin, as well as plasma IgE levels were decreased in chalcone 4-treated mice. Alto- gether, our results showed that Th2 skin inflammation could be B060 pharmacologically controlled with anti-inflammatory CXCL12 neu- CCL3 DELETION PROTECTS MICE AGAINST traligands. Chemical probes with such neutralizing activity could ORAL SQUAMOUS CARCINOGENESIS therefore be useful tools to understand and/or treat chronic inflammation. Tarcilia A. Silva1, Janine M. Silva1, Aline C. Batista2, Mauro M. Teixeira1, Milene A. Rachid1, Remo C. Russo1 B062 ROLE OF INFLAMMATORY CHEMOKINE, CCL3, 1UFMG, Belo Horizonte, Brazil; 2UFG, Goiaˆnia, Brazil IN THE DOMINANT PROLIFERATION OF LEUKEMIA INITIATING CELLS IN CML BM The chemokine CCL3 displays a diversity of roles that may contribute to worse prognosis in oral carcinogenesis. The aim of this study was Tomohisa Baba, Naofumi Mukaida to evaluate the role of CCL3 in 4-nitroquinoline-1-oxide (4NQO)- induced oral carcinogenesis. C57BL/6 (WT) and CCL3 deficient mice Cancer Research Institute, Kanazawa University, Kanazawa, Japan (CCL3-/-) male mice were treated with 4NQO during 20 or 28 weeks. The tongues were collected for macroscopic, histopatho- Accumulating evidence suggests that a macrophage-derived inflam- logical and immunohistochemical analysis. Cytokine levels in tongue matory chemokine, CCL3, has multiple functions. Besides its pro- lesions were evaluated by ELISA. Our results showed a significantly inflammatory activities, CCL3 can in vitro inhibit the proliferation of higher incidence of tumors in tongue of WT- in comparison with hematopoietic stem/progenitor cells (HSPCs). Based on this unique CCL3-/–treated mice. Consistently, microscopic analysis demon- function, CCL3 is alternatively called as ‘‘stem cell inhibitor (SCI)’’. strated a pronounced cytological atypia in the WT-treated group. The Moreover, CCL3 can in vivo rapidly induce the mobilization of immunoexpression of PCNA and Ki67 revealed a significant increase HSPCs from bone marrow (BM) to peripheral blood (PB). Thus, of the proliferative index in WT compared with CCL3-/- treated CCL3 can potentially influence the homeostasis of HSPCs. In sharp group. The concentration of CCL3, TNF-a, CCL5 and CCL11 was contrast, CCL3 has few effects on leukemia initiating cells (LICs) in significantly enhanced after 4NQO treatment, but CCL3-/- treated chronic myeloid leukemia (CML), which share several characteristic mice showed lower TNF-a level when compared with WT. In con- capabilities including self-renewal and cellular quiescence, with clusion, these data suggest a protective effect of CCL3 deletion in oral normal HSCs. It was reported that BCR-ABL-mediated tyrosine tongue carcinogenesis which may be associated with local changes in kinase activity can directly desensitize LICs to the SCI activity of TNF-a production. CCL3. Thus, CCL3 can be a potent mediator to induce the dominant proliferation of LICs in the CML BM. However, it still remains to be investigated on the precise functions of endogenously-produced B061 CCL3 in the CML pathophysiology and normal hematopoiesis. COMBATING SKIN INFLAMMATION WITH In the present study, we at first determined the dynamics of the BM HSPCs and PB cell population in the CCL3-deficient mice or BM CXCL12 CHEMOKINE NEUTRALIGANDS chimeric mice arising from the transplantation with CCL3-deficient BM cells, in order to examine the role of endogenously-produced 1,2 Dayana Abboud CCL3 in physiological hematopoiesis and hematological reconstitu- tion. Of interest is that PB cell populations were excessively 1 Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/ reconstituted in the BM chimeric mice transplanted with CCL3-de- Universite´ de Strasbourg, and Labex Medalis, ESBS, Illkirch, France; ficient BM cells, despite of similar PB cell numbers in the 2 Laboratoire d’Innovation The´rapeutique, UMR 7200 CNRS/ physiological conditions between CCL3-deficient and wild-type (WT) Universite´ de Strasbourg, and Labex Medalis, Faculte´ de Pharmacie, mice. The HSPCs in BM transiently proliferated after BM trans- Illkirch, France plantation with WT BM cells and this proliferation was further Excessive signalling by chemokines has been associated with chronic augmented when CCL3-defcient BM cells was used for BM trans- inflammation or cancer, thus attracting substantial attention as plantation. Hence, we assume that endogenous CCL3 has SCI activity promising therapeutic targets. Inspired by chemokine-clearing only when the proliferation status of HSPCs is enhanced under the

123 Inflamm. Res. S127 stress conditions such as hematopoietic reconstitution after sub-lethal cell-transplanted mice, when 4T1.3 cells was injected into tibial irradiation. osseous tissue. In order to delineate the role of CCL3 in the dominant prolifera- Given the lack of CCR5 expression by 4T1.3 clone, tumor cell- tion of leukemia cells among normal hematopoietic cells, we derived CCL4 can act on CCR5-expressing bone marrow-derived established the non-irradiated mouse CML model, in which BCR-ABL normal cells to promote survival of 4T1 cells in bone marrow, and to gene-transduced LICs were directly transplanted into the BM cavity accelerate bone metastasis. of non-irradiated mice. In this model, an initial CML-like leukocy- tosis was accompanied with a transient increase in serum CCL3 concentration. Moreover, the ablation of the CCL3 gene in LICs dramatically inhibited the development of CML. Furthermore, the B064 genetic loss of the gene of either CCL3-specific receptor, CCR1 or PROTECTIVE ROLE OF THE ATYPICAL CCR5, induced normal HSPCs to directly impede the maintenance of CHEMOKINE RECEPTOR D6 DURING LICs in BM. Thus, these observations strongly suggest the crucial role EXPERIMENTAL SEPSIS of CCL3-mediated SCI activity in the competitive interaction between normal HSPCs and LICs in the CML BM. Fernanda VS Castanheira1, Fabiane Sonego1, Alexandre Kanashiro1, Vanessa F. Borges1, Paulo H. Melo2, Remo C. Russo3, Flavio A. Amaral4, Mauro M. Teixeira4, Gerard J. Graham5, B063 Massimo Locati6, Thiago M. Cunha1, Jose´ C. Alves Filho1, 1 CCL4-CCR5 INTERACTIONS HAVE PIVOTAL Fernando Q. Cunha ROLES IN SURVIVAL OF A MURINE BREAST 1Department of Pharmacologym, School of Medicine of Ribeira˜o CANCER CELL LINE IN OSSEOUS Preto, University of Sa˜o Paulo-Brazil, Sa˜o Paulo, Brazil; ENVIRONMENT 2Department of Immunology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo-Brazil, Sa˜o Paulo, Brazil; 3Department of Soichiro Sasaki Physiology and Biophysics, University of Minas Gerais-Brazil, Gerais, Brazil; 4Department of Biochemistry and Immunology, 5 Divison Molecular Bioregulation, Cancer Research Institute, University of Minas Gerais-Brazil, Gerais, Brazil; Institute of Kanazawa University, Ishikawa, Japan Infection, Immunity and Inflammation, University of Glasgow- Scotland, Glasgow, Scotland; 6Department of Medical Triple negative breast cancer (TNBC) is a subtype of breast cancer, Biotechnologies and Translational Medicine, Humanitas Clinical and which lacks the expression of estrogen receptor, progesterone Research Center, Milan, Italy receptor, and HER2. TNBC has poor prognostic outcome compared with other types of breast cancer, and frequently metastasizes to bone, Introduction: Sepsis is a systemic inflammatory response resulted thereby deteriorating the patients’ quality of life. However, the lack of from the inability of the immune system to control infections. During curative therapy for bone metastasis necessitates understanding on the an infection, neutrophils are the first cell line to reach the primary precise mechanisms controlling bone metastasis of breast cancer. focus, and chemokines have a fundamental role in this process. Intravenous administration or intracadiac injection of breast cancer However, in sepsis, these chemokines also contribute to the neutrophil cells is widely used as bone metastasis model. However, with these infiltration to remote organs and to multiple organ failure. Under maneuvers, a large number of tumor cells enter into bone even in the normal physiological conditions neutrophils do not express the CC early phase of metastasis, whereas only a small number of tumor cells chemokine receptor subfamily and, as consequence, do not respond to are presumed to be present in bone in the early phase. Thus, it is the CC chemokines. On the other hand, our group has shown that necessary to establish a breast cancer cell line, which can metastasize during sepsis neutrophils become responsive to these chemokines and to bone upon its orthotopic injection into mammary fat pad, in order express CC chemokine receptors, as CCR2 and CCR5. Recently, a to elucidate the molecular and cellular mechanisms of bone metas- new chemokine receptor named D6 has been described. It is an tasis, particularly at its early phase. atypical receptor due to its involvement in the removal and degra- From a murine TNBC breast cancer cell line, 4T1, we established dation of CC inflammatory chemokines. However, to date, there are a subclone which can metastasize to bone with a high probability no studies showing the involvement of D6 in sepsis physiopathology. upon its orthotopic injection. The resultant clone, 4T1.3, proliferated In this way, the aim of this study is to show the role of D6 during in vitro and in vivo at the primarily injected site, mammary fat pad, experimental sepsis. Methods:All experiments were performed with similar rates as the parental clone did. However 4T1.3 cells according to our institution’s ethical guidelines (169/2011). C57BL/6 -/- exhibited resistance to anoikis and an increase in CD44+ CD24-/low and D6 deficient mice (D6 ) were used to induce sepsis using cecal cell population, which can represent cancer stem cells (CSCs). ligation and puncture (CLP) model. Neutrophil migration to the Moreover, compared with the parental clone, 4T1.3 cells exhibited a peritoneal cavity, bacteremia, markers of organ damage, neutrophil higher survival rate when injected directly into tibial osseous tissue, infiltration and chemokine levels on remote organs were determined but no enhanced short-time migration ability, compared with the 24 h after sepsis induction. The survival rate of animals was assessed parental clone. twice a day, until the 100day after sepsis induction. The means of the Microarray analysis on in vitro cultured cells revealed that mRNA parameters evaluated in WT and D6 mice submitted to CLP were expression of a chemokines, CCL4, was enhanced in 4T1.3 clone, compared by ANOVA, followed by Bonferroni test, or by t test and compared with the parental clone. CCL4 shRNA transfection reduced the survival rate by the Mantel-Cox log rank test. Results and dis- -/- its tumor formation compared with control shRNA transfection, when cussion: it was observed that the D6 mice under sub-letal CLP- injected into osseous tissues, whereas the same treatment failed to surgery exhibited a significant reduction in the survival rate, as modulate CSC phenotypes of 4T1.3 cells. Tumor growth was reduced compared to WT animals. However, neutrophil migration to the in mice deficient in CCR5, a specific receptor for CCL4, or wild-type peritoneal cavity, bacterial load in the peritoneal exudate, blood and -/- (WT) mice transplanted with CCR5-deficient mouse-derived bone vital organs were similar between WT and D6 mice, 24 h after marrow cells, compared with WT mice or WT-derived bone marrow CLP. On the other hand, we showed increased levels of CC

123 S128 Inflamm. Res. chemokines in the lung, heart and kidney of the D6-/- mice and, as Conclusion: DICAM potently reduces differentiation and function of consequence, increased neutrophil infiltration in these organs, asses- THP-1 macrophage and skews a THP-1 polarization into M2-like sed by MPO, and increased levels of organs damage markers, when macrophage via suppression of integrin aVb3-dependent Akt- compared to WT animals. In conclusion, our data indicate that D6 has FoxO3a-IRF7 pathway. a protective role during sepsis, mediating the reduction of chemokine levels in remote organs and, consequently, the reduction of organ damage. Financial support CNPq, FAPESP, CAPES, FAEPA. B066 ABROGATION OF OX40 AND CD30 SIGNALING REDUCES HOUSE DUST MITE DRIVEN Co-Stimulatory and Co-Inhibitory Pathways ALLERGIC LUNG INFLAMMATION

Donald T. Gracias1, Hideo Yagita2, Michael Croft1 B065 DICAM ATTENUATES INFLAMMATORY 1La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA; MACROPHAGE DIFFERENTIATION VIA 2Juntendo University, Tokyo, Japan SUPPRESSION OF INTEGRIN AVß3–DEPENDENT Allergic asthma is an inflammatory disease that is induced by immune TYPE I INTERFERON SYSTEM responses to airborne allergens in the lungs. Several studies have identified T cells as being important players in asthma, with Type 2 Seungwoo Han1,2, Youn-Kwan Jung1, Min-Su Han1, Eun-Ju Lee1, helper T cells (Th2) being a critical subset. The selective removal of Hye-Ri Park1, Jiae Jang1, Gun-Woo Kim1,2 pre-existing pathogenic memory Th2 cells could potentially be a key process resulting in enhanced tolerance. We have attempted to identify 1Laboratory for Arthritis and Bone Biology, Fatima Research signaling molecules, in the TNF superfamily that regulate persistence Institute, Daegu, Republic of Korea; 2Department of Internal or activity of memory Th2 cells in a house dust mite (HDM) model of Medicine, Daegu Fatima hospital, Daegu, Republic of Korea allergic inflammation. C57BL/6J mice given a single intranasal instillation of HDM showed increased expression of several TNF Objective: DICAM, a dual Ig domain containing adhesion molecule, superfamily and cytokine genes in the lungs. Among these, up-regu- is involved in cell–cell adhesion through a direct interaction with lation of OX40L was significantly higher than other TNF family aVb3 integrin. In our previous study showing the inhibitory role in members measured. We then tested whether OX40-OX40L interac- osteoclastogenesis, we found a clue that DICAM also has a sup- tions were required for memory Th2 responses and lung inflammation pressive role in macrophage differentiation. However, it remains still driven by HDM. While OX40-deficient mice displayed strongly obscure the role of DICAM in macrophage differentiation and M1/M2 reduced overall lung inflammation, eosinophilia, and Th2 develop- polarization. ment in recall responses to HDM challenge, therapeutic blocking of Method: To induce differentiation into resting M0 macrophage, THP- OX40L at the time of memory T cell reactivation did not result in 1 cells were cultured with 100 nM PMA for 24 h, and then rested for similar inhibition of allergic responses, implying OX40L may have 6 days. For M1/M2 polarization, resting M0 THP-1 macrophages been important for initiation of the naı¨ve response but was not needed were treated with IFN-c or IL-4 for 24 h. To investigate the role of for the memory response. Similar negative results were gained by DICAM during THP-1 macrophage differentiation, THP-1 cells were targeting another TNF superfamily protein CD30L that was also infected with 50 moi of control LacZ adenovirus or with DICAM upregulated by HDM exposure. However, we observed significant adenovirus. reduction in airway inflammation and the Th2 response when OX40L Results: The expression of DICAM was increased during PMA-in- was simultaneously neutralized with CD30L. This suggests that duced THP-1 differentiation, and DICAM was slightly decreased by memory responses to complex allergens may be controlled by several IFN-c for M1 polarization and increased by IL-4 for M2 polarization. costimulatory interactions, and only abrogation of signaling from The overexpression of DICAM in THP-1 cells suppressed PMA-me- multiple TNF superfamily members may promote airway tolerance. diated macrophage differentiation in the number of activated branched macrophage and macrophage marker expression, CD14 and CD68. However, DICAM does not affect the viability and proliferation of PMA-stimulated THP-1 macrophage. Functionally, DICAM attenu- Cytokines in Inflammatory Disease ated the TNF-a secretion of differentiated THP-1 cells and their phagocytic activity as well. To investigate the molecular mechanisms B067 for DICAM-mediated suppression of macrophage differentiation, we conducted microarray analyses, which revealed that DICAM overex- TH17 CYTOKINES REGULATE pression significantly suppressed type 1 interferon system. Among OSTEOCLASTOGENESIS IN RHEUMATOID interferon regulatory factors (IRFs) family, IRF7 was most signifi- ARTHRITIS cantly reduced by DICAM. DICAM also attenuated Akt phosphorylation and increased a nuclear translocation of FoxO3a that Sang-Heon Lee1, Kyoung Woon Kim2, Hae-Rim Kim1 is known to be a critical negative regulator of IRF7. Consistently, b 1 2 DICAM also decreased total integrin 3 level and integrin-linked Konkuk University School of Medicine, Seoul, Korea; The Catholic kinase (ILK) phosphorylation, the major adaptor molecule of integrin University of Korea, Seoul, South Korea b3. Based on the fact that type 1 interferon is important in M1 mac- rophage polarization, we investigated the role of DICAM in M1/M2 Background: Th17 cells, which are defined by a selective interleukin polarization of macrophage. Overexpression of DICAM induced (IL)-17 secretion, are considered as a distinct lineage of CD4+ helper downregulation of M1-associated genes such as IL-12b p40, IL12 p19, T cells which are regulated by the other Th1 and Th2 cytokines. IL- TNFa, IL-6, and IL-1b but did not affect M2 genes, Arg1 and Fizz1. In 17A is a dominant proinflammatory cytokine produced by the Th17 addition, DICAM increased IL-10, but decreased TNFa and INF-b. cells. IL-21, IL-22 and IL-26 are also produced by the Th17 cells. In

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RA, IL-17A induces the production of proinflammatory mediators, Immunohistochemical approach revealed the prominent expression of such as IL-1 and tumor necrosis factor (TNF)-a from synovial IL-38 protein in the synovial lining of RA synovium. In contrast, IL- fibroblasts, macrophages, and chondrocytes. 38 was barely expressed in the synovial lining of OA synovium. Objectives: This study aimed to determine the regulatory effect of Moreover, we found that IL-38-deficient mice exhibited siginificant Th17 cytokines on the osteoclastogenesis in rheumatoid arthritis exacerevation of their arthritis scores, compared with their wild-type (RA). littermates. Correspomdingly, histological measures of arthritis were Methods: The expression of IL-17 and RANKL was determined in also exacerbated, and accompanied by increasedIL-1b and IL-6 gene synovial tissue, fibroblast-like synoviocytes (FLS) and synovial fluids expression in the ankles. of RA patients using immunohistochemical staining, ELISA and real- Conclusion: Our presentstudy is the first to identify the presence of time PCR. The Th17 cytokines inducedRANKL expression was soluble OL-38 protein in the serum andits expression in the synovium studied in RA FLS using real-time PCR, luciferase activity, and of RA patients. IL-38 may play a role as an inhibitor in the patho- western blot. Human peripheral blood monocytes were cultured with genesis of RA. M-CSF and Th17 cytokines, and then osteoclastogenesis was deter- minedby counting the number TRAP-positive multinucleated cells. The osteoclastogenesis was also determined after humanmonocytes were co-cultured with IL-17-prestimulated FLS. B069 Results: There was a significant correlation between RANKL and IL- OPPOSITE ROLE OF SOLUBLE FORMS OF IL-6 17 levels in RA synovial fluid. After RA FLS werestimulated with IL- 17, IL-21 and IL-22, the expression of RANKL mRNA increased and RECEPTOR, SIL-6R AND SGP130, ON THE the IL-17-induced RANKL expression was decreased by the inhibi- REGULATION OF INFLAMMATORY STATUS IN tion of Act1, TRAF6, NF-jB and the AP-1. Th17 cytokines and IL- RHEUMATOID ARTHRITIS 17-prestimulated FLS induced osteoclastogenesis from monocytes in the absence of osteoblasts or RANKL. Kazuyuki Yoshizaki, J Song Soken-Nakazawa, Kazuko Uno Conclusions: Th17 cytokines have a dual effect on osteooclastogen- esis in RA; direct induction of osteoclastogenesis from monocytes and Osaka University, Suita, Japan upregulation of RANKL production in RA FLS. Th17 cytokines/ RANKL axis could be a potential therapeutic target for bone The contribution of Interleukin 6 (IL-6) is well known in inflamma- destruction in RA. tion, especially in chronic inflammatory status. IL-6 signal is mediated into cells through two receptors, an IL-6 binding receptor (IL-6R) and an IL-6 signal transducer, gp130, which binds the com- plex of IL-6/IL-6R. Soluble forms of IL-6R (sIL-6R) and gp130 B068 (sgp130) are presented in serum. sIL-6R is produced by the proteinase IL-38: A NEW FACTOR IN RHEUMATOID cleavage of membrane IL-6R and the other sgp130 is secreted by ARTHRITIS splicing gp130 gene. Our previous in vitro study showed that sIL-6R with IL-6 induced and augmented the production of CRP, SAA, and Shinjiro Kaieda, Shin-ichi Takenaka, Tomoaki Hoshino hepcidin from hepatocyte, and that sgp130 inhibited the IL-6 signal and suppressed the production of acute phase proteins. Kurume University School of Medicine, Kurume, Japan Norwell et al. previously reported that sIL-6R enhanced IL-6 activity in the RA synovial, but that the development of arthritis could Background: IL-38 (IL-1F10) was originally described as an IL-1 be blocked by Fc coupled sgp130 in mouse model. However, less is family cytokine, and named IL-1HY2. The IL-38 gene is located in the known about the function of soluble receptors in inflammatory con- IL-1 family cluster on chromosome 2, next to the genes encoding the dition in human. Then, in order to know the significance of the soluble IL-1 receptor antagonist (IL-1Ra) and the IL-36 receptor antagonist forms of receptors in chronic inflammatory disease, we analyzed these (IL-36Ra). IL-38 shares 37 % homology with IL-1Ra, 43 % homology soluble receptors in the patients with rheumatoid arthritis (RA) who with Il-36 Ra, and has a three-dimensional structure similar to IL-1Ra. were treated with an anti-IL-6 receptor antibody, Tocilizumab. Il-38 was recently shown to inhibit Candida albicans-indeuced IL-17 Biomarkers including 4 soluble receptors with 19 cytokines and 8 and Il-22 production by human memory T cells. However, the role of chemokines were measured in serum before starting Tocilizumab IL-38 in inflammatory diseases such as RA remain unclear. therapy by Bio-Plex200 and Milliplex MAP. Simple and multiple Methods: We established several clones of mouse anti-human IL-38 linear regression analysis were used to determine if any relationship mAb. One anti-human IL-38 mAb can be used for sandwich ELISAs existed between pretreatment biomarkers and patient’s week 16 and immunohistochemistry. To determine the expression pattern of DAS28-CRP score. These values and clinical variables underwent a the IL-38 gene, a panel of cDNA derived from normal lung, pancreas, stepwise multiple linear regression analysis. The resulting parameters spleen, muscle and synoviocytes was analyzed by qRT-PCR. The with P \ 0.05 were considered significant. serum levels of IL-38 in 137 RA and 26 OA patients, and in 56 To find biomarkers that may contribute to 16 week DAS28 score, we healthy donors, were determined by ELISA. Synovial tissue samples performed single linear regression analysis of 16 week DAS28 score. were also obtained from7 RA and 3 OA patients. Arthritis was also We found that sgp130, sTNFR-1 and sIL-6R significantly coincided initiated in mice lacking IL-38, as well as their wild-type littermates, with the 16 week DAS28 score. Then multi linear regression analysis of via intraperitoneal administration of K/BxN serum. Total RNA was biomarker levels was performed to determine the best equation of isolated from mouse ankle joints and IL-1b and IL-6 mRNA were DAS28 improvement. We found that eight biomarkers including quantified by qRT-PCR. sgp130 values were significantly expressed in biological naı¨ve Tocili- Results: The IL-38 gene were strongly expressed in the lung, spleen zumab treated patients (R2 = 0.646, P \ 0.0001). In these markers, and synoviocytes. The serum levels of IL-38 were 5.7 ± 0.4, sgp130 level was a key biomarker in naı¨ve and non-naı¨ve patients. In 2.8 ± 0.8, and 2.8 ± 0.7 pg/ml in RA patients, OA patients, and fact, the distribution for remission and non-remission patients accord- healthy donors, respectively. Twenty-one of the 137 RA patients, 1 of ing to their pretreatment serum sgp130 level showed that among naı¨ve 26 OA patients, and 5 of the 56 controls had IL-38 levels that was patients 59.2 % of those who experienced remission and 19.0 % of non- above the detection limit our ELISa system (10 pg/ml). remission patients showed sgp130 level over 0.2 lg/ml. On the other

123 S130 Inflamm. Res. hand, among non-naı¨ve patients 66.6 % of remission and 19.3 % of These results suggest that IL-6 signaling pathway can differ in each non-remission patients had sgp130 levels exceeding 0.2 lg/ml. These RA patients with similar disease activity and further suggesting the results suggest that sgp130 is an important predictor of RA patient’s applicability of pStat3 as a biomarker to adjust TCZ interval in RA clinical outcome of Tocilizumab therapy, indicating that sgp130 may be patients. Measurement of pSTAT3 in RA patients treated with TCZ a natural IL-6 inhibitor inflammatory status. could be a promising strategy to optimize treatment.

B070 B071 ALTERATION OF IL-6 SIGNALING IS USEFUL IN CIGARETTE SMOKE ENHANCES ADAPTIVE RHEUMATOID ARTHRITIS TREATMENT: IMMUNE RESPONSE IN MURINE MODEL OF ADJUSTMENT OF DOSING INTERVAL OF ALLERGIC AIRWAY INFLAMMATION TOCILIZUMAB, A IL-6 RECEPTOR-INIHIBITOR Thayse R. Bruggemann, Paula Fernandes, Jessica M. Oliveira, Beatriz M. Saraiva-Romanholo, Mı´lton A. Martins, Shuntaro Saito, Keisuke Izumi, Yuko Kaneko, Katsuya Suzuki, Fernanda M. Arantes-Costa Kunihiro Yamaoka, Tsutomu Takeuchi Laboratory of Experimental Therapeutics, LIM20, School of Medicine Division of Rheumatology, Department of Internal Medicine, Keio of Sa˜o Paulo, Sa˜o Paulo, Brazil University School of Medicine, Tokyo, Japan Introduction: Several studies have shown that cigarette smoke interacts Introduction: Interleukin-6 (IL-6) is a key cytokine in the pathogen- with the asthma phenotype and has the potential to interfere in the esis of rheumatoid arthritis (RA). Tocilizumab (TCZ) binds to physical, chemical and immunological barrier. Moreover, asthmatics membrane and soluble forms of human IL-6 receptor, efficiently subjects who have smoke habits have more severe symptoms and inhibiting IL-6-STAT3 signaling with standard every 4 weeks intra- higher number of hospitalizations. Methods: Male balb/c mice venous administration. However, subset of patient show poor (n = 8/group) were sensitized with two intraperitoneal injections of response or become tolerant to TCZ, reminiscent of the strength of ovalbumin (OVA, 20 lg) and alum (3 mg) on days 0 and 14 and were IL-6 signaling could differs in each individual. Therefore, we mea- challenged with an aerosol of OVA (1 %, 30 min) on days 21, 23, 25 sured phosphorylated STAT3 (pSTAT3) in RA patients treated with and 27 for OVA and OVA + CS groups. The CS and OVA + CS TCZ, with those whose administration interval was adjusted. groups were exposed to cigarette smoke once a day (7 cigarettes/ses- Methods: Three hundred fifty-five patients were treated with TCZ sion) for twelve consecutive days (from 16 to 27). CS and Control (8 mg/kg) at our institution between 2008 and 2014. Patients treated (SAL) groups received saline and alum intraperitoneal injection and with dosing interval of 3 weeks (shortened group, n = 25), 4 weeks were challenged with saline 0.9 %. 24 h after the last challenge the (normal group, n = 266) and C5 weeks (prolonged group, n = 64). levels of IL-5, IL-10, IL-13 and IFN-c were measured in bron- Whole blood from 10 patients in each group and methotrexate treated choalveolar lavage fluid (BALF) by cytometric bead array (CBA). patients (control group, n = 10) with low disease activity ELISA measured TSLP levels in lung homogenate and edema area and (CDAI \ 10, CDAI = Clinical Disease Activity Index) was stimu- number of eosinophils were measured by histological lung stained lated with different concentration of recombinant human (rh) IL-6 (0, slides with Luna. Results: OVA-sensitization caused rich pulmonary 0.1, 1, 10, 100 ng/ml). Proportion of pSTAT3 positive CD4+ T cells inflammatory infiltrate of eosinophils, an increase in inflammatory was measured by phosflow cytometric analysis. cytokines such as IL-5 and IL-13 and an increase in peribronchoalve- Results: olar edema that were all even enhanced when was co-exposition to A. Clinical efficacy of adjusting administration interval of TCZ cigarette smoke. We showed a remodeling process been initiated by Twenty-five patients (7.0 %) were in shortened group due to increased edema on peribronchoalveolar space on OVA + CS group. insufficient response to 4-week dosing for mean of 33.8 weeks. TSLP levels showed increased in animals exposed to cigarette smoke Disease activity improved after two administrations with CDAI but decreased when OVA-sensitized. The regulatory cytokine IL-10 changing from 20.8 to 7.8 (p \ 0.05). Sixty-four patients (18.0 %) increased only on CS group. Levels of the Th1 cytokine IFN-c, that were in prolonged group, after achieving remission for mean of acts in a allergic inflammation with a regulatory manner, showed 70 weeks. Among them, 51 patients (79.7 %) continued prolonged increased in OVA and OVA + CS groups. Conclusion: In summary, interval, while 13 (20.3 %) were placed back to 4-week interval our study showed that smoking acts as an aggressive agent that worsens due to exacerbation of disease activity after a mean of 41.9 weeks. the precondition of asthma. Furthermore, we could see that neither IL- B. Assessment of IL-6 induced pSTAT3 in CD4+ T cell Proportion 10 nor IFN-c managed the inflammation based on increased levels of of pSTAT3 positive CD4+ T cells increased in a dose dependent this cytokines and the inflammatory mediators in same groups. manner in each treatment group. Although, all patients were in low disease activity, pSTAT3 was significantly increased in control group from the lowest concentration (0.1 ng/mL) of rhIL- B072 6 compared to TCZ treated patients. Among the TCZ treated patients, pSTAT3 was significantly suppressed in shortened THE ANTI-INFLAMMATORY ROLE OF group with low detectable pSTAT3 even when stimulated with ADIPONECTIN IN PATIENTS WITH CHRONIC the highest concentration (100 ng/mL) of rhIL-6. On the other CHAGAS DISEASE hand, pSTAT3 was increased from low concentration (1 ng/mL) of rhIL-6 in prolonged group and normal group resulted in in- Ka´rita CF Lidani, Thaisa L. Sandri, Marcia H. Beltrame, Renato M. between of prolonged and shortened group. Nisihara, Iara JT Messias-Reason Conclusion: Our study demonstrated that the strength of IL-6-STAT3 inhibition was altered depending on the adjustment of TCZ admin- Laboratory of Molecular Immunopathology, Clinical Hospital, istration interval, resulting in low disease activity in RA patients. Federal University of Parana´ (HC-UFPR), Curitiba, PR, Brazil

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Chagas disease (CD) is a tropical neglected disease caused by Try- important role in immunity and inflammation, and its expression has panosoma cruzi, which affects more than eight million people in Latin been reported in most barrier tissues of the body, such as lung, gut and America and represents an emerging problem in North America and skin as well as in the placenta. Natural killer (NK) cells express AhR Europe due to international migrations. Although most individuals and AhR activation stimulates antitumor activity as well as resistance remain asymptomatic in the indeterminate form all lifelong, around to infections. However, the exact mechanism by which NK cells 2–5 % of them progress each year, to one of the symptomatic forms of contribute to obesity with severe sepsis remains unknown. The pur- the disease, developing cardiomyopathy, digestive megasyndromes, or pose of this study was to investigate the role of NK cells in the both. In chronic CD, adipose tissue and adipocytes have been related inflammatory response and also evaluate the potential protective as a long-term storage site of T. cruzi, where the adipokines have a effect of AhR activation by6-formylindolo[3,2-b]carbazole (FICZ) critical role in inflammatory process. Thus, this study investigated the during cecal ligation and puncture (CLP) induced sepsis on high-fat levels of adiponectin in patients with indeterminate or cardiac clinical diet-fed mice. forms of chronic CD compared to controls, besides the contribution of Methods: Male C57BL/6 mice at 6 weeks of age were randomized to the adipokine in chronic chagasic cardiomyopathy (CCC) progression. a high-fat diet (HFD) or standard chowdiet (SD) for 10 weeks. After Adiponectin plasma levels were determined in chronic CD women 10 weeks of feeding, polymicrobial sepsis was induced by CLP fol- (n = 64) from Southern Brazil using an ELISA commercial kit. The lowed by a peritoneal injection of FICZ or saline. Twenty-four hours cardiac patients were graded according to the cardiac insufficiency after CLP, AhR expression of lung, spleen tissues and splenic NK classification of the American Heart Association, adapted for Chagas cells were analysed. The effect of FICZ administration on early disease (2005) in A, B1, B2, C, and D groups. Demographical, labo- immune response and survival rate was investigated as well. ratorial, and clinical findings of the specific CD forms were obtained Results: Lung and spleen tissue from HFD mice with sepsis expressed from medical records. Moreover, 80 Southern Brazilian women with significantly less AhR than SD controls (P \ 0.05). We also found negative T. cruzi serology and without clinical complaints were used that purified splenic NK cells from HFD mice expressed far lower as control subjects. All parameters were analyzed with adjustment for level AhR, displaying a significant decrease of their capacity to age and Body Mass Index (BMI) using logistic regression analysis. produce IFN-c compared with that of SD mice after CLP (P \ 0.01). Formal written consent was obtained from each individual and this IFN-c synthesis of HFD NK cells was partly restored by addition of study was approved by local ethics committee. Our results demon- IL-15 and FICZ in vitro. Accordingly, HFD mice had a higher mor- strated higher levels of adiponectin in controls than chronic CD tality than SD mice. Treatment of HFD mice with FICZ augmented patients (p \ 0.0001, means 15316.5 vs. 2217.48 ng/lL, respectively) NK cell function with increased IFN-c production and improved and higher in controls when compared to indeterminate form sepsis survival. Either AhR antagonist or NK cells depletion reversed (p \ 0.0001, means 15316.5 vs. 2208 ng/lL, respectively). Besides, the beneficial effects of AhR activation on immune defense to CLP higher levels of adiponectin were observed in controls than in patients induced sepsis. with cardiac form (p = 0.0001, means 15316.5 vs. 2113.24 ng/lL, Conclusions: These data demonstrate that HFD induced obesity have respectively). Although there was no difference between indetermi- detrimental effects on experimental polymicrobial sepsis due to nate and cardiac clinical forms (p = 0.32, means 2356.5 vs. impairedIFN-cproducing NK cells. AhRactivation by FICZ could 2113.24 ng/lL, respectively) the adiponectin levels were negatively increases survival of obesity with sepsis by improving NK cells correlated with cardiometabolic parameters, such as triglycerides function with increased IFN-c production during early inflammation (q =-0.41 and p = 0.034) and cholesterol (q =-0.45 and response. p = 0.018). Patients with arterial hypertension presented higher adi- ponectin levels than those non hypertensive (p = 0.03, medians 3060 vs. 2062 ng/lL, respectively). In addition, comparing adiponectin levels in patients with cardiac insufficiency (C + D forms) and without B074 it (A + B), no significance difference was observed (p = 0.94, means CHRONIC LEVELS OF IFN GAMMA DECREASED 2202 vs. 2155 ng/lL, respectively). As the chronic Chagas disease is associated with low levels of adiponectin, its anti-inflammatory role CHEMOTAXIS OF BONE MARROW DERIVED inT. cruzi infection might contribute to CCC or risk of metabolic MACROPHAGES AT SITES OF INFLAMMATION syndrome in all clinical forms. Julio C. Valencia

B073 LEI, NCI-Frederick, Frederick, MD, USA ARYL HYDROCARBON RECEPTOR ACTIVATION Interferonc (IFNc, also called Type II interferon or immune inter- BY 6-FORMYLINDOLO[3,2-B]CARBAZOLE (FICZ) feron, is a critical factor in several autoimmune disorders. IFNc is a modulator of inflammation capable of influencing the clinical out- INCREASES SURVIVAL IN HIGH-FAT DIET-FED come of autoimmune disorders with both pro- and anti-inflammatory MICE WITH SEPSIS: CRITICAL ROLE FOR NK functions. IFNc signaling activates both innate and adaptive immune CELLS cells such as macrophages and T-cells. Recent evidence indicates that immune activated macrophages are first recruited and later repro- Min Zhu1, Peng Xiong1, Wei Zhang2, Hai-Wei Sang1, Xiang-Ning Fu1 grammed by the microenvironment at sites of inflammation. Given that chronic inflammatory responses are associated with a macro- 1Department of Thoracic Surgery, Tongji Hospital, Tongji Medical phage-mediated inflammatory response, we examined the role of c College, Huazhong University of Science and Technology, Wuhan, chronic IFN exposure on the activation and trafficking of macro- China; 2Wuhan Institute of Burn, Wuhan No. 3 Hospital, Wuhan, phages. Bone marrow (BM) macrophages were isolated from a mouse c c China model of chronic IFN expression generated by deletion of the IFN 30 UTR AU-Rich element (ARE-DEL). Isolated BM macrophages Background: Sepsis is a worldwide life-threatening disease, espe- from ARE-DEL+/+ wild type (WT) or ARE-DEL-/- (KO) C57B6/L6 cially in criticallyillobesepatients. The aryl hydrocarbon receptor mice were immortalized using an avian retrovirus (J2) carrying the (AhR) is a ligand-activated transcription factor which plays an Myc and Raf oncogenes. FACS analysis confirmed that immortalized

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WT and KO macrophages expressed the markers CD11b/MAC1 and protective haplotype in patients with CP (OR = 0.159, 95 % F4/80 consistent with their BM origin, while the cells were negative CI = 0.035–0.728, p = 0.0054) and in patients with T2DM for CD11c and GR-1, markers for dendritic cells and neutrophils, (OR = 0.179, 95 % CI = 0.049–0.654, p = 0.0039). On the con- respectively. Combined FACS and immunoblotting analysis revealed trary, the GGG haplotype is associated with an increased risk for that KO macrophages had higher basal STAT1 protein levels and T1DM (OR = 1.870, 95 % CI = 1.035–3.379, p = 0.0399). produced higher levels of phosphorylated STAT1 (Y701) after short Conclusions: Our results indicate that different IL6 promoter haplo- (10 min) or long (24 h) stimulation with mouse recombinant IFNc types can affect the risk for CP and DM development. However, the (rIFNc), compared to WT macrophages. These results suggest that individual IL6 gene promoter polymorphisms do not act indepen- chronic exposure to IFNc primed KO macrophages for enhanced dently and may affect each other. responsiveness to further stimulation. To examine the consequences This study was supported by thegrantsGACR GB14-37368G, IGA of IFNc-mediated priming on macrophage motility, transwell NT11405-6 and by the project MUNI/A/1359/2014. migration assays were carried out over a period of 18 h in the pres- ence or absence of mouse rIFNc. Interestingly, although in a primed state, KO macrophages had a significantly lower motility than WT B076 macrophages (p \ 0.05, ANOVA). Furthermore, pre-treatment with 50, 100 and 200 U/mL of mouse rIFNc for 72 h decreased signifi- PLATYMISCIUM FLORIBUNDUM VOG, cantly the motility of WT macrophages (p \ 0.05, ANOVA) A MEDICINAL PLANT, DECREASES THE compared to untreated controls. Collectively, these data indicate that PRODUCTION OF PRO-INFLAMMATORY TNF-a macrophage chemotaxis is severely decreased after long term expo- AND IL-1ß IN A RAT MODEL OF LIGATURE- sure to IFNc at the site of inflammation. As a result, this mechanism has potential therapeutics implications to limit further macrophage INDUCED PERIODONTITIS recruitment at sites of chronic inflammation. Karuza MA Pereira1, Luzia H. Teixeira1, Danielle RD Val2, Hellı´ada V. Chaves1, Gerly Anne DC Brito1, Conceic¸a˜o DS Martins1, Paula Goes1, Antonia TA Pimenta1, Mary Anne S. Lima1, B075 Alrieta H. Teixeira1, Jordaˆnia MDO Freire1, Alice RD Freitas1, HAPLOTYPE ANALYSIS OF INTERLEUKIN-6 Janine M. Bastos1, Antoˆ nia TDPP Parente1, Mirna M. Bezerra1 GENE PROMOTER POLYMORPHISMS IN 1 2 PATIENTS WITH CHRONIC PERIODONTITIS AND Federal University of Ceara´, Fortaleza, Brazil; Federal University of Pernambuco, Recife, Brazil DIABETES MELLITUS Periodontitis is known to be one of most prevalent worldwide chronic Simona Valova1, Petra Borilova Linhartova1,2, Katerina Kankova1, inflammatory diseases. During periodontitis course, it has been shown Hana Poskerova2, Jan Vokurka2, Antonin Fassmann2, that proinflammatory cytokines such as TNF-a and IL-1b modulate Lydie Izakovicova Holla1,2 bone loss. Platymiscium floribundum Vog. is a tree that occurs in Northeast Brazil, where it is known as ‘‘sacambu’’ and ‘‘jacaranda-do- 1Department of Pathophysiology, Faculty of Medicine, Masaryk litoral’’, that has been used in traditional medicine as anti-inflam- University, Brno, Czech Republic; 2Clinic of Stomatology, Institutions matory. Some authors have reported that this species are a source of Shared with St. Anne’s Faculty Hospital, Faculty of Medicine, isoflavonoids that possess various biological effects such as antifun- Masaryk University, Brno, Czech Republic gal, antimitotic, and anticancer properties. Since a few of studies have been developed in the search of others chemotherapeutic values from Objectives: Strong evidence confirms a bidirectional relationship this plant, the aim of this study was to evaluate the effects of between chronic periodontitis (CP) and diabetes mellitus (DM). Platymiscium floribundum Vog. on alveolar bone loss in a rat model Pathogenesis of both diseases has been associated with high levels of of ligature-induced periodontitis, investigating the involvement of interleukin-6 (IL-6), pleiotropic cytokine involved in immune regu- TNF-a and IL-1b. Methods: This study was conducted with the lationand control ofinflammatory processes. The IL-6 expression is approval of the Ethics Committee of the UFC, Fortaleza, Brazil influenced by polymorphisms in the IL6 gene promoter region. The (CEPA no. 08/13). Periodontitis was induced by placing a nylon aim of our study was to investigate the association of single thread (3.0) in the upper molars of female Wistar rats (180-200 g). nucleotide polymorphisms at positions -174G/C (rs1800795), Rats (6 per group) were treated (per os) daily with Platymiscium -572G/C (rs1800796) and -597G/A (rs1800797), and their combi- floribundum Vog (0.1, 1 or 10 mg/kg) or vehicle (saline) for 11 days. nations (haplotypes) in patients with CP, type 1 DM (T1DM) and type The rats were killed on day 11 after ligature placement, and the 2 DM (T2DM) in comparison with healthy controls. maxillae were removed and processed for macroscopic analyses of Methods: 900 Czech subjects were enrolled in this case–control study: alveolar bone loss (mm2) using the ImageJÒ software. Gingival 381 diabetic patients without periodontitis (84 T1DM and 297 T2DM samples were collected to evaluate TNF-a and IL-1b levels (pg/ml) patients), 115 subjects suffered both from diabetes and periodontitis by ELISA. Rats were weighed daily. Results: Platymiscium flori- (37 T1DM + CP and 78 T2DM + CP), 223 patients had only CP and bundum Vog (1 or 10 mg/kg) reduced (P \ 0.0001) alveolar bone 181 subjects were healthy controls. The individual polymorphisms in loss (2.33 ± 0.59 and 1.99 ± 0.34 mm2, respectively), compared to IL6 gene promoter (-174G/C, -572G/C and -597G/A) were the vehicle group (4.17 ± 0.19). Further, Platymiscium floribundum determined by polymerase chain reaction-restriction fragment length Vog decreased (P \ 0.05) both TNF-a and IL-1b levels in gingival polymorphism (PCR–RFLP) assay. tissues (3.88 ± 0.13 and 4.44 ± 0.29 pg/ml, respectively), compared Results: The allele and genotype frequencies of the studied poly- to the vehicle group (6.83 ± 0.36 and 9.43 ± 0.42 pg/ml, respec- morphisms between groups of patients and healthy controls did not tively). Conclusion: These findings reveal that Platymiscium differ statistically significantly. However, we proved significant dif- floribundum Vog decreases bone loss in ligature-induced periodontitis ferences in haplotype frequencies between groups of patients with CP, in rats by reducing, at least in part, TNFa and IL-1b levels. T1DM, T2DM and healthy controls. The GGA haplotype acts as a Funding Sources RENORBIO, CNPq, CAPES, and INCT-IBISAB.

123 Inflamm. Res. S133

B077 Francisca RLD Silva1, Francisco EA Rodrigues1, Jair Mafezoli1, 1 1 1 GLYCOGEN SYNTHASE KINASE-3 SIGNALLING Vicente de Paulo T. Pinto , Gerardo C. Filho , Virgı´nia CC Gira˜o , Ba´rbara M. Bastos1, Angela MC ARRIAGA1, Aline CD Medeiros1 AND REGULATION OF TNF MRNA TRANSLATION IN HUMAN NEUTROPHILS 1Federal University of Ceara´, Fortaleza, Brazil; 2Federal University of Pernambuco, Recife, Brazil Miriam S. Giambelluca1, Genevie`ve Bertheau Mailhot1, Emmanuelle 1 1 2 1 Periodontitis is an inflammatory condition that result in the Rollet-Labelle , Cynthia Laflamme , Marc J. Servant , Marc Pouliot destruction of the supporting structures of the dentition. Though the etiology of periodontitis is bacterial, some evidences strongly suggest 1Centre de Recherche du CHU de Que´bec-Univesite´ Laval, Quebec 2 that the pathogenesis of the disease is mediated by the host immune- City, QC, Canada; Universite´ de Montre´al, Faculty of Pharmacy, inflammatory response. Stemodia maritima Linn. (Scrophulariaceae) Montreal, QC, Canada is a very common shrub that grows wild in northeastern Brazil near Introduction: Glycogen synthase kinase-3 (GSK-3) is a serine/thre- the sea, where it is known as ‘‘melosa’’. It is used to treat stom- onine kinase involved in the regulation of multiple cellular processes. achache and swelling by the local population. This study aimed to Its two known isoforms, a and b, are differentially expressed in tis- evaluate the effects of a hydro-alcoholic extract of Stemodia mar- sues throughout the body and exert overlapping, but sometimes itima Linn. (Sm) on alveolar bone loss in a rat model of ligature- distinct functions. GSK-3 is typically active in resting cells, inhibition induced periodontitis, investigating the involvement of TNF-a and by phosphorylation of Ser21 (GSK-3a) or Ser9 (GSK-3b) being the IL-1b. Methods: The experimental protocol was conducted with the most common regulatory mechanism. GSK-3 activity has been linked approval of the Ethics Committee of the UFC, Fortaleza, Brazil recently with immune system functions, yet little is known about the (CEPA no. 08/13). Periodontitis was induced by placing a nylon role of this enzyme in neutrophils, the most abundant type of thread (3.0) in the upper molars of female Wistar rats (180–200 g). leukocytes. In this study, we examined GSK-3 expression, signaling Rats (n = 6 per group) were treated (per os) daily with Sm (0.1, 1 or and impact of GSK-3 inhibition by lithium or specific GSK-3 inhi- 10 mg/kg) or vehicle (non-treated group) for 11 days. The rats were bitors, on the production of TNF by neutrophils. killed on day 11 after ligature placement, and the maxillae were Methods: Freshly-isolated human neutrophils and the murine air- removed and processed for macroscopic analyses of alveolar bone pouch model of inflammation were used for this study. GSK-3 loss (mm2) using the ImageJÒ software. Gingival samples were isoform identification and activation were monitored by western collected to evaluate TNF-a and IL-1b levels (pg/ml) by ELISA. blots, whereas the effects of GSK-3 inhibition on mRNA, cytokine Rats were weighed daily. Results: Sm (10 mg/kg) reduced production, transcriptional and translational pathways were studied (P \ 0.05) alveolar bone loss (1.93 ± 0.54 pg/ml), when compared by RT-PCR, ELISAs, Western Blots and quantitative mass to non-treated group (4.39 ± 0.31 pg/ml). Further, Sm (10 mg/kg) spectrometry. decreased (P \ 0.05) both TNF-a and IL-1b levels in gingival tissues Results: GSK-3a was found to be the predominant isoform. It was (3.53 ± 0.15 and 5.90 ± 0.36 pg/ml, respectively), compared to the constitutively expressed and cell stimulation with different agonists non-treated group (6.83 ± 0.36 and 9.43 ± 0.42 pg/ml, respec- did not alter its expression. The main pathways contributing to the tively). Conclusion: These findings suggest that Stemodia maritima control of GSK-3 activity were: Src kinase, PKC, PI3K/AKT and Linn. might have a protective potential on gingival tissue inflam- ERK/RSK in lipopolysaccharides (LPS)-stimulated neutrophils, mation and alveolar bone loss during the process of periodontitis by PI3 K/AKT, ERK/RSK and PKC in GM-CSF-stimulated neutrophils. inhibiting pro-inflammatory cytokines, resulting in improved peri- In the murine air pouch model of inflammation, injection of odontal tissue in experimental periodontitis, which is an attractive lithium chloride potentiated TNF production induced by LPS. In model to evaluate inflammatory bone loss. human neutrophils, GSK-3 inhibitors mimicked the impact of lithium Funding Sources RENORBIO, CNPq, CAPES, and INCT-IBISAB. chloride, also potentiating TNF release. When cellular mechanisms were investigated, no alteration in the NF-Jbor CREB pathways activation, or in the levels of TNF mRNA, was observed but an association with the p38/MNK1/eIF4E translation pathway and RNA- B079 binding proteins activity was revealed. GSK-3 inhibition potentiated HIGH DOSE OF LEPTIN INDUCES FIBROTIC de novo TNF synthesis. CONVERSION OF ENDOTHELIAL CELLS INTO Conclusions: GSK-3a emerges as a central kinase in human neu- FIBROBLAST trophil physiology, being regulated by a number of different stimuli and playing a central role in regulating TNF mRNA translation. Alvaro Becerra1, Macarena Rojas1, Alejandro Vallejos1, Financial support Canadian Institutes of Health Research (CIHR), Felipe Simon1,2 Canadian Foundation for Innovation (CFI), Fonds de la Recherche en Sante´ du Que´bec (FRSQ) and the Wilbrod-Bherer Foundation. 1Departamento de Ciencias Biolo´gicas, Facultad de Ciencias Biolo´gicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; 2Millennium Institute on Immunology and B078 Immunotherapy, Santiago, Chile EFFECTS OF A STEMODIA MARITIMA LINN. Introduction: During sepsis syndrome progression, a number of EXTRACT ON BONE RESORPTION AND TNF-A inflammatory mediators are released into the blood vessels, expo- AND IL-1ß LEVELS IN RATS WITH LIGATURE- suring a wide range of inflammatory mediators to endothelial cells (ECs). Several evidences have been shown that inflammatory medi- INDUCED PERIODONTITIS ators induce a fibrotic conversion to become ECs into activated fibroblasts through a process known as endothelial-to-mesenchymal 1 1 2 Karuza MA Pereira , Luzia H. Teixeira , Danielle RD Val , transition (EndMT). It has been demonstrated that during the course 1 1 1 Raul S. Freitas , Hellı´ada V. Chaves , Paula Goes , of sepsis the adipokine leptin is increased in serum. Despite that it has 1 1 Alrieta H. Teixeira , Jordaˆnia MDO Freire , been described that leptin exert a modulatory function on immune

123 S134 Inflamm. Res. system, the role played by leptin during sepsis is poorly understood. B081 Thus, our aim was to investigate whether leptin, similarly to CYTOKINES AND CHEMOKINES ANALYSIS IN inflammatory mediators, induces conversion of ECs into activated fibroblasts. PREGNANT WOMEN WITH ERYTHROCYTE Methods and Results: Using primary cultures of rat mesenteric ALLOIMMUNIZATION endothelial cells (RMEC), we demonstrated that RMEC exposed to high doses of leptin (50–100 ng/ml) for 72 h exhibits a conversion of Juliana AC Schettini1,2, Thoma´s V. Gomes1, Alexandra Karla S. ECs in activated fibroblasts. Leptin-induced fibrotic conversion was Barreto1, Claudeir D. Silva Ju´nior1, Marina C. da Matta1, determined by changes in morphology and protein expression pattern. Isabela C. Coutinho2, Maria do Carmo VC Oliveira3, Results showed a leptin-induced fibrotic-like ECs morphology com- Leuridan C. Torres1 pared to cells in absence of leptin. It was found a decreasing of the endothelial markers VE-Cadherin and CD31/PECAM and a increas- 1Translational Research Laboratory, Instituto de Medicina Integral ing in the expression of the fibroblast-specific proteins, a smooth Prof. Fernando Figueira (IMIP), Recife, Brazil; 2Department of muscle actin (a-SMA) and fibroblast-specific protein-1 (FSP-1). Obstetrics and Gynecology of the Instituto de Medicina Integral Prof. Furthermore, extracellular matrix (ECM) proteins, fibronectin (FN) Fernando Figueira (IMIP), Recife, Brazil; 3Fundac¸a˜o de and collagen type III (Col III), were also increased upon exposition to Hematologia e Hemoterapia de Pernambuco (HEMOPE), Recife, high doses of leptin. In addition, changes on intracellular distribution Brazil of the endothelial, fibrotic and MEC protein in ECs exposed to high Cytokines and chemokines analysis in pregnant women with ery- doses of leptin were studied. ECs exposed to high doses of leptin throcyte alloimmunization. increased the secretion of TGFß-1, suggesting an autocrine/paracrine Introduction: It is not clear if there is any difference in chemokine and effect to induce conversion of ECs. Using an inhibitor of the ALK5 cytokine profile according to the presence or absence of maternal activation, we observed that ALK5 is also involved in the sepsis- RhD alloimmunization. induced EC fibrosis. Objective: Evaluate the levels of inflammatory cytokines and Conclusion: Our data demonstrated that ECs exposed to high doses of chemokynes in RhD negative pregnant women with erythrocyte leptin acquire fibroblasts-like features mediating the conversion of alloimmunization. ECs in activated fibroblasts through a TGF-ß1/ALK5-dependent Methods: The study was carried out the Outpatient Clinic for Preg- pathway. Results provided here contribute to understand the patho- nancies (OCP) and Translational Research Laboratory of the Instituto physiology of sepsis revealing a novel photogenic mechanism de Medicina Integral Professor Fernando Figueira (IMIP) between involved in sepsis and other inflammatory diseases. August 2013 to December 2014. Cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, TNF-a and IFN-c) and chemokines (RANTES, MIG, MCP1, IP10) were measured in culture supernantants. Maternal plasma RHD genotyping was performed for RhD negative sensitized mothers. About 103 women were divided in groups of pregnants: 16 B080 RhD (-) alloimmunized [indirect antiglobulin test = IAT (+)] and 43 MEASURING RECEPTOR DIMERIZATION TO RhD (-) non-sensitized; and groups of controls: 24 RhD (+) IAT (-) CREATE FUNCTIONAL CELL-BASED ASSAYS pregnant and 20 non-pregnant women. Analyses of groups was used FOR [ 85 % OF THE INTERLEUKIN RECEPTOR the Mann–Whitney test two-tailed. The correlation between variables was determined by calculating the Spearman correlation coefficient r. FAMILY A p value \ 0.05 was considered significant. Results: IL12p70 concentration was higher in RhD(-) IAT(-) and Scott Gridley, Hyna Dotimas, Sangeetha Gunthuri, Hanako Daino- RhD(+) IAT(-) compared with those in women control (p** = 0.005 Laizure, Albert Doan, Phil Achacoso, Jane Lamerdin and p* = 0.02, respectively). MCP1 level was increased in the RhD (-) IAT (+) (median = 20648 pg/mL) when compared to RhD(-) DiscoveRx Corporation, San Francisco, CA, USA IAT(-) (median = 16136 0 g/mL, p \ 0.0001) and women control (median = 476 pg/mL, p \ 0.0001). In the RhD(-) IAT(+) group Ligand-induced receptor dimerization is the first functional step in IL-8 was positively correlated with IL-10 (r = 0.51, p = 0.04) and receptor activation, representing the most proximal, functional read- b out for receptor activation. It is well understood that the family of IL-6 (r = 0.32; p = 0.01). IL1- was positively correlated with IL-6 (r = 0.57, p = 0.02) and IL-10 (r = 0.57, p = 0.02). Anti-D Interleukin receptors will dimerize with the other members of its alloantibody was present in 10 mothers and anti-D and C in 6 family leading to a complicated signaling cascade that is critically mothers. RHD genotyping showed that 12 fetus were RHD positive involved in a variety of auto-immune, inflammatory and oncogenic RHD diseases. Surprisingly, existing cellular assays have been unable to and 4 negative. There were two neonatal death and 1 stillbirth secondary to fetal hydrops. faithfully monitor these interactions in a proximal manner to the Conclusions: There is a increase in proinflammatory cytokines in receptor. Here we present a novel application of the Enzyme Frag- sensitized pregnant women. ment Complementation system to monitor receptor-receptor interactions at the surface of intact live cells, applicable to diverse receptor types such as Interleukin receptors, BMP receptors, receptor tyrosine kinases and cytokine receptors, with a specific focus on the B082 interleukin family of receptors. The high specificity, simplicity of the COMBINATORIAL TARGETING OF TSLP, IL-25, assay protocol, large signal to noise ratio, serum tolerance and reproducibility of these assays has enabled their use in cell-based AND IL-33 IN TYPE 2 CYTOKINE-DRIVEN screening, functional characterization, QC lot release assays and INFLAMMATION AND FIBROSIS neutralizing antibody studies. Examples discussed here include assays for the IL-1, IL-2, IL-6, IL-10 receptor families, with assays devel- Kevin M. Vannella1, Thirumalai R. Ramalingam1, Allen W. Cheever2, oped for over 85 % of the Interleukins and their receptors. Lee A. Borthwick3, Luke Barron1, Kevin M. Hart1, Robert W.

123 Inflamm. Res. S135

Thompson1, Kristen N. Kindrachuk1, Sandra White1, Alison L. stromal radio-resistant cells can impact myeloid progenitors, we are Budelsky4, Michael R. Comeau4, Dirk E. Smith4, Thomas A. Wynn1 currently testing whether IFN-c can shape the development and/or the mobilization of aberrant inflammatory monocytes during N67C 1National Institute of Allergy and Infectious Disease, Bethesda, USA; infection, which may lead to immune-mediated splenic pathology and 2Biomedical Research Institute; 3Newcastle University, Newcastle rapid mortality, in the absence of parasite clearance. The elucidation upon Tyne, England; 4Amgen Inc., Thousand Oaks, USA of this IFN-c signaling mechanism may enable further development of interventions to decrease human morbidity and mortality associ- Thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 are impor- ated with severe malaria. tant initiators of type 2-associated mucosal inflammation and immunity. However, their role in the maintenance of progressive type 2 inflammation and fibrosis is much less clear. Here, using chronic models of helminth infection and allergic lung inflammation, we show B084 that collective disruption of TSLP, IL-25, and IL-33 signaling sup- IMMUNE RESPONSE TO LATENCY ASSOCIATED presses chronic and progressive type 2 cytokine driven inflammation and fibrosis. In a schistosome lung granuloma model or during chronic MYCOBACTERIUM TUBERCULOSIS ANTIGEN S. mansoni infection in the liver, individual ablation of TSLP, IL-25, or AMONG LATENT AND ACTIVE TB PATIENTS IL-33/ST2 had no impact on the development of IL-4/IL-13-dependent inflammation or fibrosis. However, significant reductions in granu- Manju Namdeo1, Dipendra Kumar Mitra1, Deepshi Thakral1, loma-associated eosinophils, hepatic fibrosis, and IL-13-producing Steven G. Reed2, Rhea Coler2 group 2 innate lymphoid cells (ILC2s) were observed when signaling of all three mediators was simultaneously disrupted. Combined 1All India Institute of Medical Sciences, New Delhi, India; 2Infectious blockade via mAb treatment also reduced IL-5 and IL-13 expression Disease Research Institute, Seattle, WA, USA during primary and secondary granuloma formation in the lung. In a model of chronic house dust mite-induced allergic lung inflammation, Purpose: Polyfunctional T cells are believed to be critical for the combined mAb treatment did not decrease established inflammation or immune containment of Mycobacterium tuberculosis (Mtb) infection. To address the role of latency associated Mtb antigen (Rv1813) in breach fibrosis. TSLP/IL-33 double-knockout mice treated with anti-IL-25 + mAb during priming, however, displayed decreased inflammation, of latency, we intended to delineate the host polyfunctional CD4 Tcell response in latent TB infection (LTBI) versus active TB patients. mucus production, and lung remodeling in the chronic phase. Toge- + ther, these studies reveal partially redundant roles for TSLP, IL-25, Methods: We evaluated polyfunctional CD4 T cell response in LTBI and active TB patients. PBMCs isolated from Mtb infected subjects and IL-33 in the maintenance of type 2 pathology and suggest that in + some settings, early combined targeting of these mediators is neces- were stimulated in vitro with Mtb antigen Rv1813 and CD4 T cell sary to ameliorate progressive type 2-driven disease. response was assessed for the production of IFN-c, IL-2, TNF-a at single cell level. Using flow cytometry, we identified single (IFN-c+ , IL-2+ , TNF-a+), dual (IFN-c+ TNF-a+ , IFN-c+ IL-2+ , and TNF- a+ IL-2+) and triple (IFN-c+ TNF-a+ IL-2+) producer T cells specific to Rv1813 among LTBI versus active TB patients. B083 + DECIPHERING THE DELETERIOUS EFFECT OF Results: We observed a decrease in polyfunctional CD4 T cell response (Dual and Triple cytokine producers) in active TB patients IFN-C IN A NEW RODENT MODEL FOR as compared to LTBI. Among the dual cytokine producers, IFN- EXPERIMENTAL SEVERE MALARIA c+ TNF-a+ phenotype was significantly reduced in active TB patients. This could imply that polyfunctional CD4+ T cells response Norinne Lacerda-Queiroz, Nicolas D. Riteau, Richard Eastman, in TB patients is impaired, presumably due to immune evasion Alan Sher, Dragana Jankovic, Xin-Zhuan Su strategies by Mtb. We have previously shown that Programmed death- 1 receptor (PD-1) plays a critical role in inhibition of effector T cell NIAID/NIH, Rockville, MD, USA response. Therefore, we envisage that blocking PD-1 could rescue the polyfunctional T cell response. This strategy may be used for rescuing Immune response plays an important role in controlling malaria host polyfunctional T cell response against Mycobacterium tubercu- infection; however, excessive inflammatory response can lead to losis, thereby preventing the breach of latency. severe disease. Here we have investigated the mechanism of patho- Conclusion: Our results suggest that polyfunctional CD4+ T cell genesis in mice after infection with a virulent rodent malaria parasite, response among infected subjects against Rv1813 may serve as Plasmodium yoelii nigeriensisstrain N67C. Previous studies showed immunological correlates of protection, preventing disruption of that C57BL/6-infected mice display high parasitemia and a strong latency. Rescuing polyfunctional T cell response may have implica- pro-inflammatory response, culminating in extensive splenic damage tions for vaccination strategies. and host lethality at around 7 days post-infection. The present work aims to characterize the cellular and molecular events associated with host immune-mediated pathology following intravenous infection with 106 N67C infected red blood cells. We confirmed that infected B085 mice develop a progressive inflammation in the spleen, characterized PORPHYROMONAS GINGIVALIS’ NUCLEOSIDE by an intense cellular death and an up-regulation of pro-inflammatory DIPHOSPHATE KINASE CONTRIBUTES TO IL-1ß cytokines (notably IFNc, IL-6, CCL2 and CXCL1). Interestingly, we identified splenic CD4+ and CD8+ T cells as major sources of IFN-c PRODUCTION AND SECRETION IN as early as day 4 post-infection. Additionally, we demonstrated that MACROPHAGES IFN-c is a major trigger of tissue pathology since its absence or blockade prolonged host survival and prevented excessive host Ca´ssio L C Almeida-da-Silva1, Gabrielle C. Rocha1, inflammatory response. Moreover, by employing chimeric mice, we Erivan S. Ramos-Ju´nior1, Ana Carolina F. Morandini1, showed that the absence of IFN-c receptor in the non-hematopoietic Ju´lio Scharfstein1,O¨ zlem Yilmaz2, David M. Ojcius3, compartment protect mice from early death. Since IFN-c signaling in Robson Coutinho-Silva1 123 S136 Inflamm. Res.

1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; tissue degradation, actually worsening the disease. Current therapies 2University of Florida, Gainesville, FL , USA; 3University of the target cytokines upstream of NF-jB, rendering the patient immuno- Pacific, Stockton, CA, USA compromised and susceptible to reactivation of infection. Therefore, a therapeutic strategy to combat pro-inflammatory manifestations of Porphyromonas gingivalis (Pg) are Gram-negative bacteria associ- cytokines, without affecting the immune response, could be effective ated with periodontitis. One of Pg’s virulence factors is a homolog of in preventing vascular destabilization. nucleoside disphosphate kinase (NDK) enzyme, which is secreted by By dissecting pathways that trigger degradation of barrier func- Pg. NDK cleaves extracellular ATP (eATP), suppressing ATP-in- tion, we have identified that the small GTPase ADP-ribosylation duced P2 9 7-dependent apoptosis and ATP-induced reactive oxygen factor 6 (ARF6) is a convergence point of several destabilization species (ROS) production via P2X7-NADPH-oxidase, and contributes cues: the cytokine interleukin-1b (IL-1b), the endotoxin LPS, and to Pgpersistence in gingival epithelial cells. Our group showed that proliferation signals such as vascular endothelial growth factor ATP via P2X7 controls infections with different kind of intracellular (VEGF). We report that IL-1b and ARF6 control the permeability of parasites such as Leishmania amazonensis and Toxoplasma gondii via cultured human dermal microvascular endothelial cells, and that IL-1b production. We also showed that IL-1b secretion induced by ARF6 is a critical regulator of VE-cadherin localization on the cell- P2X7 signaling in macrophages is impaired by Pg’s fimbriae. Here, surface. More importantly, inhibition of the NF-jB canonical path- we investigated the role of Pg’s NDK in infection in vitro. To analyze way by siRNAs does not affect ARF6 activation or vascular whether IL-1b secretion is modulated by Pg’s NDK, we infected bone permeability, implying that cytokine-induced ARF6 activation and marrow derived macrophages (BMDM) with wild-type Pg or NDK- downstream signaling are independent of NF-jB-mediated immune deficient (Pg D ndk) (MOI of 100) for 6, 18 or 24 h, and treated the responses. cells or not with 5 mM ATP during the last 30 min. BMDM infected Collectively, these results suggest that cytokine signaling bifur- with Pg D ndk showed less IL-1b secretion by ELISA than wild-type cates into NF-jB-mediated immune activation and ARF6-mediated Pg at all times tested. IL-1b secretion was dependent on P2X7 sig- VE-cadherin endocytosis and membrane hyperpermeability. We have naling since there was no secretion in BMDM from P2X7-/- mice verified this hypothesis by blocking ARF6 activation in animal infected with Pg. To verify immature pro-IL-1b production, we models of inflammation. ARF6 inhibition after the onset of collagen- analyzed IL1-b in BMDM infected with Pg by Western blotting. By induced arthritis in mice reduced vascular permeability in the joints, densitometry, we quantified 46 % less pro-IL-1b in cellular extracts but had no effect on cytokine levels at 24 h after treatment. We also and 46 % less IL-1b in supernatants of BMDM infected with Pg D confirmed the efficacy of ARF6 inhibition in animal models of other ndk than wild-type Pg. Next, we tested the invasion capacity of Pg conditions that result from compromised vasculature. Blocking ARF6 strains using the antibiotic protection assay. Pg strains infected at was sufficient to reduce vascular leak and enhance survival during similar levels after 2 h of infection. Further, to verify whether Pg’s endotoxic shock in mice, without inhibiting the host cytokine NDK hydrolyzes eATP, we quantified this nucleotide in BMDM response. In addition to reducing cytokine- and endotoxin-mediated supernatants after Pg infection. We detected more ATP in BMDM endothelial permeability, we report that ARF6 also blunts the inter- supernatants infected with Pg D ndk than wild-type Pg. Recently, nalization of VEGFR, dampens signaling to multiple downstream adenosine (Ado) was shown to potentiate IL-1b secretion in macro- cascades including MARCKS and ERK, and mutes pathologic phages treated with LPS/ATP. Therefore, we evaluated IL-1b release endothelial hyperpermeability in mouse models of diabetic retinopa- in BMDM supernatants after infection with Pg and treatment with thy. Collectively, these data identify ARF6 activation to be a ATP, Ado or its pan-antagonist receptor (CGS15943). Ado potenti- promising target to modulate the inflammatory process by stabilizing ated IL-1b secretion induced by Pg/ATP, and this enhancement was vasculature in conditions such as arthritis, acute respiratory distress abolished by CGS15943. Together, these results indicate that Pg’s syndrome, and vascular eye diseases. NDK contributes to IL-1b production and secretion in BMDM, and that the mechanism might be due to Ado production and signaling via Ado receptor after ATP cleavage by Pg’s NDK and other BMDM ectonucleotidases. B087 Financial support CAPES, CNPq, FAPERJ, National Institutes of DIFFERENT PHENOTYPES OF LUNG Health. PARENCHYMAL CD4+ T CELLS ACTIVATION DURING AGGRESSIVE FORMS OF B086 TUBERCULOSIS ARF6 INHIBITION STABILIZES THE Eduardo P. Amaral1, Simone C.M. Ribeiro2, Caio Cesar Bomfim1, VASCULATURE WITHOUT COMPROMISING THE Rafael M. Salgado1, Veronica R. Lanes2, Mario Hirata3, IMMUNE RESPONSE OF CYTOKINES Jose M.M. Alvarez1, Elena Lassounskaia2, M. Regina D’Imperio-Lima1 Dean Li1,2, Weiquan Zhu1, Kirill Ostanin2, Alan Mueller2 1Departament of Immunology, Biomedical Science Institute, 2 1University of Utah, Salt Lake City, UT, USA; 2Navigen Pharma, Salt University of Sa˜o Paulo, Sa˜o Paulo, Brazil; Laboratory of Biology of Lake City, UT, USA Recognition, State University of North Fluminense, Campos dos Goytacazes, Brazil; 3Departament of Clinical Chemistry and The innate immune system is the first line of defense that facilitates Toxicology, Faculty of Pharmaceutical Sciences, University of Sa˜o recognition and clearance of invading microbes and ensures tissue Paulo, Sa˜o Paulo, Brazil repair. During infection, macrophages release cytokines to activate the nuclear factor-jB (NF-jB)-dependent signaling pathways, leading Severe tuberculosis (TB) is characterized by lung necrosis, pneumo- to recruitment of inflammatory cells. Cytokines also activate path- nia, atelectasis and bacillus dissemination. However, the mechanism ways that disrupt the endothelial barrier, facilitating clearance of by which hypervirulent mycobacteria induce severe disease and how infection by leukocytes. However, dysregulated cytokine release in the host immune response is activated during that process is not so conditions like chronic inflammation and sepsis causes excessive clear. In this study, we sought to determine the major features of

123 Inflamm. Res. S137 bacillus-host interaction responsible for immune response activation cellular and mitochondrial ROS were attenuated by either RNA induced by hypervirulent strains (Beijing 1471, MP287/03 and B2 knockdown or SETD7 inhibitor under different stress conditions. strains). C57BL/6 mice were infected with a low-dose of bacteria Conclusions: In this study we identify novel roles of SETD7 in (*100 bacilli) and evaluated bacteria burden, cytokine production oxidative stress response. Our data suggest that SETD7 is important and CD4+ T cells activation in the lung parenchyma and pulmonary in fine-tuning cellular oxygen signaling and protecting cells against vasculature by using intravenous CD45 staining. We found that dis- excessive production of ROS. ease severity correlated directly with the intensity of bacillus multiplication in vivo and in vitro. C57BL/6 mice suffering from severe TB showed extensive necrotic areas in the lungs, pneumonia and bacillus dissemination. Either extremely low or high productions B089 of pro-inflammatory mediators. In contrast, during mild disease (in- EFFECT OF HYDROGEN SULFIDE (H2S) ON duced by H37Rv strain), the production of intermediate levels of pro- inflammatory cytokines was associated with low bacillus burden. HISTONE METHYLATION OF LPS-STIMULATED Moreover, mice infected with hypervirulent mycobacteria (Beijing MACROPHAGES 1471 and MP287/03) showed similar frequencies of CD4+ CD44+ T + cells. All CD4 T subsets that presented expression of CD69 were Ricardo C. Petroni, Denise F. Barbeiro, Thais M. de Lima, found inside the lung parenchyma. Mice infected with Beijing 1471 Suely K. Ariga, Francisco G. Soriano showed increased frequency of lung parenchymal CD4+CD44+CD69+ T cell subset, whereas reduced frequencies of that subset were found University of Sa˜o Paulo, Medical School, Sa˜o Paulo, Brazil in mice infected with MP287/03. These phenotypes of lung par- enchyma CD4+ T cells reflected in the production of IFN-c and Hydrogen sulfide (H2S) is an endogenously produced gaseous mes- number of Mtb-ESAT-6 tetramer+ CD4+T cells in the tissue. This senger that has gained increasing recognition as an important player study shows that several pathological manifestations of severe TB can in modulating inflammatory mediator production. Recent studies have be reproduced in mice infected with hypervirulent mycobacteria. shown the role of H2S in chromatin modulation by histone acetyla- Furthermore, the lung parenchyma millieu can impact the CD4+ T cell tion. However, its role in histone methylation (associated with gene activation, contributing to the outcome of aggressive TB. repression and hallmark of condensed chromatin at silent loci) and its effects on inflammatory cytokine production is currently unknown. In this study, we investigated the role of H2S on histone methylation in an invitro model of lipopolysaccharide (LPS)-induced inflammation Epigenetics and Regulation of the and evaluated its effects on inflammatory cytokine production. Macrophage-differentiated THP-1 cells were pre-treated with Immune Response propargylglycine (PAG) (inhibitor of enzyme CSE) at 1 mM for 90 min or sodium hydrosulfide (NaHS) (an H2S donor) at 1 mM for B088 30 min. To stimulate cytokine production, the cells were challenged LYSINE METHYLTRANSFERASE SETD7 with bacterial LPS (1 lg/ml) for 24 h. Histone H3 and H4 methyla- tion was analyzed by western blotting and cytokine production was REGULATES OXYGEN HOMEOSTASIS measured by ELISA. H2S inhibition increased the production of THROUGH OXIDATIVE STRESS interleukin-6—IL6(692.2 ± 1.6) and tumor necrosis factor-a—TNF- DETOXIFICATION AND MITOCHONDRIA a(9790 ± 1318) compared to NaHS group (4.1 ± 1) and FUNCTIONS (163.5 ± 61), respectively. This effect was associated with ade- creased H3 methylation at lysines 9 (26,5035.9 ± 17880) and 27 (73,1460 ± 73004.7) compared to NaSH treated cells Shuying He, Dafydd Owen, Scott Jelinsky, Lih-Ling Lin (40,1700 ± 5032) and (1,735,050 ± 5783.2) respectively and H4 Lys 20 methylation (75,161 ± 2473.7) in H2S inhibited cells compared to Pfizer, New York City, NY, USA NaSH cells (312,985.5 ± 3923.7). Thus, the findings of the present Introduction: Lysine methylation is critical for signaling regulation. study suggest that H2S inhibition suppresses histone methylation, Lysine methyltransferase SETD7 has been found to methylate lysine which, in turn, induce chromatin openness, leading to an increase in residues in both histone and non-histone proteins. Reactive oxygen the gene transcription of pro-inflammatory cytokines. species (ROS) have profound disease implication in neurodegenera- tion, cardiovascular defects, metabolic dysfunction, respiratory diseases and cancer. Our hypothesis is that SETD7 is required for cellular oxidative stress defense. Understanding the functions of B090 SETD7 in ROS homeostasis will provide new insight into the DUAL TRANSCRIPTOME SEQUENCING REVEALS mechanisms of lysine methylation and oxidative stress response, and RESISTANCE OF TLR4 LIGAND-ACTIVATED identify novel therapeutic targets against ROS-associated diseases. Methods: In order to study the functions of SETD7, siRNA knock- BONE MARROW-DERIVED MACROPHAGES TO down was performed in human lung bronchial epithelial cells (Beas- INFLAMMATION MEDIATED BY THE BET 2B) and primary lung fibroblasts. Cells were stimulated by cigarette INHIBITOR JQ1 smoke extract, hydrogen peroxide or TGFb1 to induce oxidative stress. Gene expression was quantified by western blot and qPCR. Amitabh Das1, Jin Choul Chai2, Chul-su Yang2, Young Seek Lee2, Cellular ROS and mitochondrial ROS levels were measured after Kyoung Hwa Jung3, Young Gyu Chai1,2 stimulation. Similar experiments were performed in the presence of SETD7 inhibitor to evaluate compound potency. 1Department of Bionanotechnology, Hanyang University, Seoul, Results: Expression of NRF2, PGC1a, MnSOD and Catalase was 133-791, Republic of Korea; 2Department of Molecular and Life elevated in both siSETD7- and compound- treated cells. Inflammatory Sciences, Hanyang University, Ansan, 426-791, Republic of Korea; cytokine production was inhibited in the knockdown cells. Both

123 S138 Inflamm. Res.

3Institute of Natural Science and Technology, Hanyang University, models called BioMAP systems. Each system consists of complex co- Ansan, 426-791, Republic of Korea cultures of human primary cell types, stimulated in a manner designed to recapitulate the complex signaling and multi-component biology The persistence of macrophage activation is associated with the pro- contributing to inflammation, fibrosis, and wound healing. Screening duction and secretion of various pro-inflammatory genes, cytokines and of nintedanib and pirfenidone across a wide panel of BioMAP sys- chemokines, which may initiate or amplify inflammatory disorders. A tems revealed, as expected, potent anti-fibrotic activities in systems novel synthetic BET inhibitor, JQ1, was proven to exert immunosup- modeling fibroblast biology. Interestingly, both compounds also dis- pressive activities in macrophages. However, a genome-wide search for played potent immunomodulatory activities on both adaptive and JQ1 molecular targets is largely unexplored in macrophages. The pre- innate immune cell types, including T cells, B cells and mono- sent study aimed at evaluating the anti-inflammatory function and cytes/macrophages. Activities include: suppression of pro- underlying genes that are targeted by JQ1 in lipopolysaccharide (LPS)- inflammatory chemokine and cytokine production by immune cells as stimulated primary bone marrow-derived macrophages (BMDMs) well as anti-proliferative activity, which could result in significant using two transcriptomic techniques:global transcriptomic RNA immune cell skewing. These data indicate the efficacy of these drugs sequencing and quantitative real-time PCR. Among annotated genes, is due to combinatorial effects impacting both anti-fibrotic and anti- transcriptional sequencing of BMDMs treated with JQ1 revealed inflammatory aspects involved in IPF. The effectiveness of these selectively reduced expression of cytokines/chemokines, interferon- agents supports either further development of drugs with this desired stimulated genes, and prominent (transcription factors) TFs. Addi- polypharmacology, or novel combination approaches designed to tionally, we found that JQ1 reduced the expression of previously target both the fibrotic and inflammatory components driving IPF unidentified genes that are important in immune regulation. Impor- pathogenesis. tantly, these inflammatory genes were not affected by JQ1 treatment alone. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1 treatment in BMDMs. These B092 unprecedented results suggest that the BET inhibitor JQ1 is a candidate DISEASE RELEVANT IN VITRO AND IN VIVO for the prevention or therapeutic treatment of inflammatory disorders. MODELS FOR LUNG FIBROSIS

Jeroen DeGroot1, Krista Ouwehand1, Blandine Mille-Baker1, Alan Young2, Mary McElroy3, Joe Cornicelli4, David Bonnel5, Fibrosis and Tissue Remodeling Jonathan Stauber5, David F. Fischer1

1 2 B091 Charles River Laboratories Leiden, the Netherlands; Charles River Laboratories Oxford, UK; 3Charles River Laboratories Edinburgh, IDENTIFICATION OF BOTH UK; 4Charles River Laboratories, Wilmington, MA, USA; IMMUNOMODULATORY AND ANTI-FIBROTIC 5ImaBiotech Lille, France ACTIONS OF NINTEDANIB AND PIRFENIDONE Objectives: Fibrosis is characterized by excessive deposition of Ò USING BIOMAP HUMAN PRIMARY CELL- extracellular matrix due to exaggerated repair in response to damage. BASED DISEASE MODELS While the initiating event(s) and the underlying pathophysiological processes may vary between organs and diseases, common features Sharlene Velichko, Sylvie Privat, Dat Nguyen, Hannah Cho, include the involvement of inflammation, appearance of myofibrob- Alison O’Mahony lasts, and changes in tissue architecture and function. In lung fibrosis, persistent and non-resolving injury to the alveolar epithelium is BioSeek, a division of DiscoveRx, South San Francisco, CA, USA thought to drive the disease. Because of the complex interactions between various cell types require state-of-the art in vitro and in vivo Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease models are needed that capture disease relevant processes to enable that is characterized by pleural scarring of the lung, a result of an the discovery and development of new drugs for fibrotic diseases. aberrant wound healing response to acute lung injury. Given the Methods: Primary human bronchial epithelial cells and fibroblasts, unfavorable response of IPF patients to both corticosteroid treatment isolated from IPF donors and control donors were cultured in 96-well and other anti-inflammatory agents in clinical trials, the impact of format. Trans-differentiation was induced with TGF-b and assessed inflammation on IPF, particularly at end-stage disease, remains by high content imaging (INCell 2200) following immunostaining for unclear. However, recent reports on the presence of inflammatory cell fibronectin (EMT in the epithelial cells) and aSMA (FMT in the types in IPF lung tissue as well as in the periphery support the con- fibroblasts). The effect of SB525334 (ALK5 inhibitor; assay positive tribution of inflammation in disease pathogenesis. Agents targeting the control), perfenidone, nintedanib, imatinib, MB06322, thalidomide, anti-fibrotic effects, such as TGFbR signaling inhibitors, have also N-acetyl cysteine, tofacitinib, and GSK2126458 was evaluated. failed in IPF trials and are actually pro-inflammatory. Collectively, In vivo, lung fibrosis was induced in rats by seven daily bleomycin these data suggest that inhibition of both fibrotic and inflammatory doses (at 1 mg/kg) that were delivered by oropharyngeal aspiration. processes may have a greater impact on IPF pathology than targeting Readout parameters (day 7 and 22) included body weight, clinical either aspect alone. Recently, nintedanib (OfevÒ), a multi-angiokinase signs, respiratory parameters, histopathology and molecular MALDI inhibitor targeting downstream mediators of VEGF, PDGF and bFGF imaging. signaling, and pirfenidone (EsbrietÒ), an anti-fibrotic small molecule Results: Both EMT and FMT assays were compatible with high with an unknown mechanism of action, were approved by the FDA and throughput screening of small molecules and RNAi vectors. Signal/ the EMA for the treatment of IPF. We used the BioMAP technology background ratio, vehicle tolerance, assay window and intra and inter- platform to predict how these agents work both mechanistically and assay variability all passed pre-set QC criteria. Nintenadib and biologically to improve therapeutic responses in IPF patients. GSK2126458 inhibited EMT and FMT. Imatinib and MB06322 The BioMAP technology platform enables target agnostic phe- inhibited FMT but not EMT. The other compounds did not modulate notypic screening of agents in human primary cell-based disease trans-differentiation (up to 10 lM). In vivo, lung function (mean tidal

123 Inflamm. Res. S139 volumes) decreased and respiratory rate increased statistically sig- Sterile inflammation is increasingly recognized as a central problem nificantly following repeated bleomycin administration. Inflammatory in many acute and chronic diseases but the mechanisms controlling and fibrotic changes at necropsy were consistent with progressive responses to sterile inflammation are poorly understood. Injured or fibrosis. A combination of MALDI imaging and microscopy allowed dying cells release products, or Danger Associated Molecular Patterns monitoring the distribution of bleomycin in the lung, as well as the (DAMPs), recognized by immune receptors contributing to disease dynamics of (novel) fibrosis markers. progression. Here we show that DAMPs isolated from injured kidney Conclusions: The primary human cell-based assays allow evaluation activate TLR2/4 and MyD88 dependent transcription of immune of compounds targeting various molecular mechanisms. In vivo, the genes in non-immune kidney resident cells, pericytes. When treated combination of mass spectrometry imaging and histological staining with extracellular DAMPs pericytes form an active NLRP3 inflam- allowed detailed assessment of disease processes by identifying dis- masome, process pro-IL1 and pro-IL18 to their active secreted forms ease markers and studying their co-distribution at sites of active and undergo pyroptosis, thereby contributing to inflammation and disease. leukocyte trafficking. Recent studies have indicated that kidney pericytes represent a major source of interstitial myofibroblasts in renal fibrogenesis. We B093 show that treatment with DAMPs in vitro activate pericyte to LINEAGE TRACING OF RESIDENT FIBROBLASTS myofibroblast transition indicated by upregulation of transcription of fibrotic genes Col1a1 and Acta2 and production of SMA. MyD88-/- BY THE INTRATRACHEAL CELL TRANSFER pericytes showed significant reduction in fibrogenesis markers. In ELUCIDATES THE CELLULAR ORIGIN OF wound healing assays DAMPs caused pericyte migration, which was ACTIVATED FIBROBLASTS IN PULMONARY significantly reduced in the absence of MyD88. FIBROSIS Cell-specific ablation of MyD88 in perivascular and stromal cells in models of ischemic acute kidney injury in mice significantly attenuates innate immune activation, injury and fibrogenic responses. Tatsuya Tsukui1,3, Satoshi Ueha1,3, Shigeyuki Shichino1,3, 2,3 1,3 In addition, we show that human pericytes respond to DAMPs in a Yutaka Inagaki , Kouji Matsushima similar manner by activating immune genes, secreting pro-inflam- matory cytokines and inducing pyroptotic cell death. 1Department of Molecular Preventive Medicine, Graduate School of 2 In conclusion, pericytes respond to sterile inflammation via two Medicine, The University of Tokyo, Tokyo, Japan; Center for Matrix divergent mechanisms both of which are controlled by MyD88: Biology and Medicine, Graduate School of Medicine, Tokai 3 activation of immune signaling which enables detection and ampli- University, Kanagawa, Japan; Japan Science and Technology fication of the inflammatory signal; and activation of fibrogenesis Agency-CREST Program, Tokyo, Japan contributing to pathology. Therefore, this regulation could be an Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease important new therapeutic target for tissue injury. with few therapeutic options, characterized by architectural destruc- tion of alveoli and formation of fibroblastic foci. Activated fibroblasts, such as myofibroblasts that express a-smooth muscle actin (a-SMA), accumulate in fibroblastic foci and produce excessive B095 amounts of extracellular matrix components. To develop specific POLYCYSTIC LIVER DISEASE IS SUPPRESSED BY treatments for IPF, the pathologic nature and origin of activated IL-4/IL-13 AND TGF-ß SIGNALING fibroblasts need to be clarified. One problem in recent lineage tracing studies is that the rigorous lineage tracing of resident fibroblasts, David A. Cantu1, Kristen Kindrachuk1, Lee Borthwick1, Doug which are classically suggested as progenitors of activated fibroblasts, Morris2, Danielle Donahue2, David Dorwood3, Brenda Klaunberg2, is still lacking. Here we report lineage tracing of resident fibroblasts Thirumalai Ramalingam1, Sandy White1, Robert Thompson1, Thomas by using the intratracheal adoptive cell transfer method in bleomycin- Wynn1 induced lung injury. Histological analysis of GFP reporter mice of type 1 collagen showed that activated fibroblasts migrated into 1Immunopathogenesis Section, Laboratory of Parasitic Diseases, epithelium-denuded alveolar airspaces in the early phase of bleomy- National Institute of Allergy and Infectious Diseases, National cin-induced pulmonary fibrosis. Purified resident fibroblasts delivered Institutes of Health (NIH), Bethesda, MD, USA; 2Magnetic Resonance into injured alveoli through an intratracheal route displayed similar Imaging Research Facility/Mouse Imaging Facility, National activated signatures and comprised fibroblastic foci. Neither pericytes Institutes of Health (NIH), Bethesda, MD, USA; 3Microscopy Unit, nor epithelial cells had the same potential. Transferred resident Research Technologies Branch, NIAID/Rocky Mountain Laboratories fibroblasts proliferated and highly up-regulated pro-fibrotic genes Hamilton, MT, USA including a-SMA. These data may suggest resident fibroblasts as the major origin of activated fibroblasts in pulmonary fibrosis. Introduction: Polycystic liver disease (PLD) is a relatively rare but devastating condition that causes cysts—fluid filled sacs—to grow throughout the liver. The immunological mechanisms controlling B094 liver cyst formation, however, remain unclear. PERICYTE MYD88 CONTROLS INFLAMMATORY Methods: Briefly, IL-17A knockout (KO), IL-4/IL-13 double KO (dKO) mice, and IL-4/IL-13/IL-17A triple KO (tKO) were infected AND FIBROTIC RESPONSES TO TISSUE INJURY with 35 cercariae of the Puerto Rican (NMRI) strain of Schistosoma mansoni (S. Mansoni) harvested from biomphalaria glabrata snails Irina Leaf1,3, Ivan Gomez1,2, Shunsaku Nakagawa2, Bryce Johnson1,2, (Biomedical Research Institute Rockville, MD). Mouse livers were Jin Joo Cha2, Julia Lichtnekert2, Kristen Mittelsteadt2, Alan Aderem3, harvested 7-12 weeks post-infection for hydroxyproline content, flow Bill Altemeier2, Jeremy Duffield1,2 cytometry, gene expression, and histological analysis. Results: We show that during infection with the helminth parasite S. 1Biogen, Cambridge, MA, USA; 2University of Washington, Seattle, Mansoni, severe PLD-like disease develops when infected mice are WA, USA; 3Seattle BioMed, Seattle, WA, USA deficient in both IL-4 and IL-13, suggesting that Stat6-mediated

123 S140 Inflamm. Res. signaling is critically required to prevent the development of PLD. of IL-10, suggesting that Th2 cytokines may help regulate Th17- We hypothesized that the parasite eggs might be contributing to cyst mediated inflammation. To investigate the interplay between IL- formation by promoting damage to microvessels in the liver, which 13Ra2 and Th17/Th2 immunity, wild-type (WT) C57BL/6, IL-13Ra2 are not efficiently repaired when mice are deficient in both IL-4 and KO, and IL-10 KO mice were subjected to intratracheal bleomycin IL-13. Because IL-4 and IL-13 are linked with wound repair and are injections and were subsequently evaluated for inflammation and potent inducers of transforming growth factor-beta (TGF-b) expres- fibrosis over a 30-day period. Temporal gene expression analysis in sion, we examined whether cyst formation in IL-4/IL-13-deficient WT mice showed a very early increase in IL-13Ra2 expression that mice was linked with uncontrolled type 1-associated inflammation was rapidly accompanied by increased expression of IL-17A, TNF-a, and/or maladaptive repair. Although IL-17A was up-regulated in IL- and IP-10, in the absence of a prominent early Th2 signature. IL- 4/IL-13 double knockout (dKO) mice, supporting the emergence of a 13Ra2 isolated from the bronchoalveolar lavage fluid of bleomycin- Th17-driven inflammatory response, IL-17A KO and IL-4/IL-13/IL- treated animals remained unsaturated throughout the 30-day period, 17A triple KO (tKO) were equally susceptible to the development of suggesting that IL-13Ra2 could be contributing to the development of PLD, although the tKO displayed slightly improved survival. Histo- pulmonary fibrosis by mitigating the potential dampening effects of logical analysis of liver tissues from IL-4/IL-13 dKO mice revealed IL-13 on Th1/Th17-mediated inflammation during the early inflam- an elevated infiltration of F4/80+ macrophages that co-localized with matory stage following injury. Though our studies with IL-13Ra2KO TGF-b-inducible protein along the cyst border, suggesting TGF-b mice did not show statistically significant changes in fibrosis, gene signaling was elevated in the dKO mice. To elucidate the role of expression analysis in these mice suggested increased IL-13 effector TGF-b, infected IL-4/IL-13 dKO mice were treated with a pan-neu- functions and IL-10 expression compared to WT mice, supporting the tralizing TGF-b monoclonal antibody (mAb). Strikingly, the number notion that IL-13Ra2 suppresses both IL-13 and IL-10 bioactivity. and size of cysts was dramatically increased when type-2 cytokines Furthermore, our findings contradict earlier observations showing that and TGF-b signaling were simultaneously blocked, suggesting that IL-13Ra2, through its induction of TGFb, is required in bleomycin- IL-4, IL-13, and TGF-b collaborate to prevent PLD during chronic S. induced lung fibrosis, as we observed no impairment in the expression mansoni infection. of TGF-bi, a surrogate marker for TGFb activity, in the lungs of IL- Conclusion: These studies suggest that therapeutic strategies that 13Ra2 KO mice. Future studies in a more chronic model of pul- increased IL-4/IL-13 and/or TGF-b signaling may provide a novel monary inflammation and fibrosis may better address the role of IL-13 approach to treat patients with progressive PLD. and IL-13Ra2 in established and progressive pulmonary fibrosis. References [1] Lee et al. IL-13 Induced Tissue Fibrosis by Selectively Stimu- lating and Activating Transforming Growth Factor b1. J. Exp. Med. 2001; 194(6): 809–821 B097 [2] Chiaramonte et al. An IL-13 inhibitor blocks the development of CROSSTALK BETWEEN MACROPHAGES AND hepatic fibrosis during a T-helper type 2-dominated inflammatory response. J. Clin. Invest. 1999; 104: 777–785. HUMAN HEPATIC FIBROBLASTS SHOW THAT INFLAMMATORY RESPONSE ATTENUATE THE ACTIVATION OF FIBROBLASTS IN THE B096 FIBROSIS RESPONSE EVALUATING IL-13RA2 AS A THERAPEUTIC Sacha Robert1, Thomas Gicquel1, Aude Bodin1, Alain Fautrel2, TARGET FOR PULMONARY FIBROSIS Vincent Lagente1, Elisabeth Boichot1

Nikhil Jiwrajka1, Thirumalai Ramalingam1, Josh Sciurba1, Sandy 1UMR991 INSERM, Universite´ de Rennes 1, Rennes, France; 2H2P2, Oland1, Rafael Prado1, Kevin Hart1, Trisha Pasricha1, Kevin Histopathological Platform, Universite´ de Rennes 1, Rennes, France Vannella1, Bernadette Gochuico2, Marion T. Kasaian3, Thomas A. Wynn1 Liver fibrosis is a physiological response due to acute injury and resulting in an inflammatory process, imbalance between matrix 1Immunopathogenesis Section, Laboratory of Parasitic Diseases, metalloproteinases (MMPs) and the tissue inhibitors of metallopro- National Institute of Allergy and Infectious Diseases, National teinases (TIMPs), and the production of scar tissue (collagen). When Institutes of Health, Bethesda, MD, USA; 2Undiagnosed Diseases the injuries persist, excessive collagen deposition could lead to seri- Program, National Human Genome Research Institute, National ous pathologies. At cellular level, collagen production is mediated by Institutes of Health, Bethesda, MD, USA; 3Department of activated fibroblasts, also known as hepatic stellate cells. This acti- Inflammation and Immunology, Pfizer Research, Cambridge, MA, vation occurs after crosstalk between fibroblasts and liver USA macrophages which express pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß) cleaved into active form by caspase-1 Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease associated with NLRP3-inflammasome pathway. Driving a coculture that remains incurable despite recent therapeutic advances. Gene model of macrophages and hepatic fibroblasts, we put on light that the expression profiling studies in IPF patients have repeatedly revealed inflammatory response and especially IL-1ß seems to attenuate the increased expression of IL-13Ra2, a high-affinity receptor that binds activation of fibroblasts in the fibrosis response. IL-13 and sequesters it from the surrounding milieu. We sought to THP-1 cell line or human peripheral blood monocyte cells were examine the role of IL-13Ra2 in pulmonary fibrosis by interrogating differentiated into macrophages with respectively phorbol-myristate- the murine bleomycin-induced lung injury model. Previous work has acetate at 10 ng/mL for 3 days and GM-CSF at 50 ng/mL for 7 days, indicated that fibrosis in this model is driven by the inflammatory then treated with lipopolysaccharide at 0,1 lg/mL and followed after cytokine IL-17A. Recently, several groups have shown that Th17 18 h by monosodium urate crystals at 300 lg/mL for 6 h. Two cells express a functional IL-13 receptor, IL-13Ra1, that serves to coculture models were chosen with two types of liver fibroblasts. We downregulate the production of IL-17A and upregulate the production used the LX-2 cell line, which are human immortalized liver myofi-

123 Inflamm. Res. S141 broblasts, or human hepatic stellate cells from liver biopsies. Firstly, GPCRs in Inflammation macrophages were placed in coculture at the top of inserts with liver fibroblasts in the bottom of the multi-well dish. Secondly, the supernatant of macrophages was removed and put on fibroblasts. B099 After 24 h, mRNA expression was evaluated by RT-qPCR and after ACETATE PROTECTS THE ISCHEMIC INTESTINE 48 h, proteins secretion was measured in the supernatant using ELISA FROM REPERFUSION INJURY method and MMPs activities by zymography. Cocultures of liver fibroblasts with macrophages result in Zoe V. Schofield1, Reena Halai1, Matthew A. Cooper1, Trent M. increased mRNA levels of MMP-1, MMP-3 and MMP-9 and also Woodruff2 their collagenase activity whereas there is a decrease in a-SMA, type I and IV collagens, endothelin-1 and PDGF-BB. IL-1ß from the mac- 1Institute for Molecular Bioscience, St Lucia, QLD, Australia; rophages also exacerbates the pro-inflammatory environment by 2School of Biomedical Sciences, The University of Queensland, St stimulating IL-1ß, IL-6, IL-8 and chemokines such as GRO-a and Lucia, QLD, Australia MCP1 expression and/or release. These effects were partially reduced by pre-treatment of macrophages with an inhibitor of inflammasome It has long been advocated that acetate has therapeutic benefits activation (by targeting caspase-1), the Z-YVAD-FMK, or pre-treat- however, the mechanism of action for this has not been determined. ment of fibroblasts with an antagonist of IL-1ß receptor, Anakinra. SCFA’s can transiently modulate cell function or alter cell function IL-8 and TNF-a seems also to participate in the process as they show through the free fatty acid 2 receptor (FFA2) [1]. In the past decade similar effects. FFA2 has been shown to be involved in several diseases including Taken together, these results demonstrate that the inflammatory cancer, obesity, diabetes mellitus, asthma and inflammatory bowel response, mostly involving IL-1ß, trigger an anti-fibrogenic envi- disease [2, 3]. ronment on liver fibroblasts and that regulation of the differentiation FFA2 is abundantly expressed on neutrophils [3] which are the state of macrophages is important for the production of collagen in the primary responders in ischemia reperfusion injury (IRI) and represent control of chronic liver fibrosis. important components in the prolonged inflammatory response and severity associated with these conditions [4]. IRI is a common, damaging and untreatable condition that is common during surgery, trauma and stroke [4] with a 50 % mortality rate. B098 In this study, we investigated the role of acetate in a mouse model PERICYTE AND MYOFIBROBLAST ACTIVATION of mesenteric IRI to address the potential anti-inflammatory effects of REGULATED BY THE TWEAK/FN14 PATHWAY acetate in IRI. Untreated mice showed significant damage of the villi in the small intestine, whereas protection was afforded to the villi by Ivan G. Gomez1,2, Naoki Nakagawa2, Timothy S. Zheng1, Jeremy S. acetate. Interestingly despite this protective effect, neutrophil infil- Duffield1,2, Linda C. Burkly2 tration was significantly increased in the intestine after dosing with acetate. Our data suggests that acetate may modulate neutrophil 1Biogen, Cambridge, MA, USA; 2Renal Division, University of function and equally protect cells against ischemic damage. Washington, Seattle, WA, USA References Background: Pericytes, mesenchymal cells that partially wrap capil- 1. Vinolo M, Hosana AR, Rodrigues G, Hatanaka E, Sato FT, Sam- lary endothelium and are critical for supporting their integrity, have paio SC, Curi R. Suppressive effect of short-chain fatty acids on been shown by genetic fate-mapping to be a major progenitor pool for production of proinflammatory mediators by neutrophils. J Nutr myofibroblasts in a number of fibrotic contexts. Pericytes provide Biochem 2011. directional cues that route the innate immune cells extravasating into 2. Ulven T.Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/ injured tissue. Previously it was shown that TWEAK, a TNF family GPR41 as new potential therapeutic targets. Front endocrinol. 2012. cytokine produced largely by leukocytes, promotes macrophage 3. Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, infiltration and fibrosis in the UUO model of renal injury. We Schilter HC, et al. Regulation of inflammatory responses by gut hypothesized that TWEAK mediates its effect through its injury-in- microbiota and chemoattractant receptor GPR43. Nature. 2009. ducible receptor, Fn14. Since Fn14 is expressed by mesenchymal 4. Schofield ZV, Woodruff TM, Halai R, Wu MC-L, Cooper MA. lineage cells and regulates their fate, we hypothesized that TWEAK/ Neutrophils-a key component of ischemia–reperfusion injury. Shock. Fn14 might be a key element for pericyte activation in response to 2013. tissue injury. Results: We show that Fn14 deficiency ameliorates myofibroblast appearance and renal fibrosis in the UUO model, phenocopying B0100 TWEAK deficient mice. TWEAK activates primary murine cultured renal pericytes through Fn14, inducing low level proliferation, PRIMARY MICROGLIA ADOPT migration and production of proinflammatory mediators, as well as A PROINFLAMMATORY PHENOTYPE AFTER pericyte to myofibroblast transition measured by induction of EXPOSURE TO INCREASED LPA LEVELS a-smooth muscle actin expression and stress fiber formation. Fur- thermore, myofibroblasts are activated by TWEAK/Fn14 signaling, Joanna Plastira1, Eva Bernhart1, Astrid Hammer2, Wolfgang Sattler1 exhibiting higher-level proliferation, migration and cytokine pro- duction as well as cytoskeletal changes. Transcriptional profiling of 1Institute of Molecular Biology and Biochemistry, Medical University TWEAK-stimulated myofibroblasts supports its multifaceted role and Graz, Graz, Austria; 2Institute of Cell Biology, Histology and identified TWEAK-response genes. Embryology, Medical University Graz, Graz, Austria Conclusions: These findings suggest that targeting the TWEAK/Fn14 pathway is an approach to modulate pericyte and myofibroblast Microglia, the immunocompetent cells of the CNS, are rapidly acti- activation and a novel way to target both inflammatory and fibrotic vated in response to brain injury and disease. Microglial migration aspects of fibrotic disease. towards and homing at damaged tissue plays a key role in CNS

123 S142 Inflamm. Res. regeneration. Classically activated microglia (M1) are able to syn- Methods: PAFR deficient (PAFRKO) and wild type animals (WT) thesize pro-inflammatory factors that inflict neuronal damage, while were fed standard and high fat diet (HFD) for 16 weeks or aged in the production of trophic and anti-inflammatory factors by M2 standard diet for 40 weeks. Glucose and insulin tolerance tests were microglia can support neuronal survival and regeneration. Under performed by blood monitoring. Macrophages were isolated from chronic inflammation as observed in many neurodegenerative dis- epididymal withe adipose tissue (WAT) and evaluated by FACS. eases, microglia are characterized by over activation and secretion of Gene and protein expression was investigated by PCR and western- proinflammatory and neurotoxic factors that can induce neuronal blot, respectively. damage. Results: Macrophages infiltrated in WAT showed a pro-inflammatory Lysophosphatidic acid (LPA) is produced via the autotaxin path- M1-phenotype, characterized by increased population of F4/ way or by phospholipase A-mediated pathways. LPA has diverse 80+CD11c+ cells (twofold) and increased gene expression of Ccr7 biological functions mediated by downstream signaling through dif- (twofold), Nos2 (3-fold), Il6 (threefold), and Il12 (twofold); whereas ferent receptors. These receptors play prominent role in the central IL10 gene was less expressed (p \ 0.05 vs. WT animals). This was nervous system, and signaling is amplified at sites of inflammation observed in animals fed standard or high fat diet and in aged mice. where LPA concentrations are increased. During earlier work we WAT from PAFRKO and WT showed similar levels of adiponectin could show that LPA regulates microglia protein expression and and chemerin, anti- and pro-inflammatory adipokines, respectively. affects their migrational response in a PKD dependent pathway. Compared to WT, PAFRKO presented significant (p \ 0.01), Western blotting for MAPKs and PKDs revealed that lysophos- increased weight (25 %) and adipocyte size (30 %), which are char- phatidic acid (LPA) can potently activate these pathways and acteristics of obesity; higher fasting serum glucose levels, followed by confocal microscopy analysis depicted LPA induced changes in decreased glucose tolerance and insulin resistance; impaired insulin- microglia cytoskeleton. Using flow cytometry, we obtained evidence induced AKT phosphorylation in the liver and hepatic damage. When that microglia (both BV-2 and primary) polarized towards an M1 fed with HFD, PAFR deficient mice presented increased weight gain, proinflammatory phenotype. LPA treatment induced high CD40 (M1 insulin resistance that affected the liver and skeletal muscle and marker) expression while the levels of CD206 (M2 marker) dramat- developed liver steatosis. ically decreased. Western blot analysis demonstrated that LPA Conclusion: Our results indicate that PAFR expressed by adipose increased protein expression of iNOS and COX-2 (both M1). On the tissue macrophages have an important role in maintaining their anti- contrary, the basal expression of arginase I and RELMa (FIZZ) was inflammatory profile and this process is essential to preserve adipose gradually suppressed (both M2). Immunofluorescence for iNOS, tissue homeostasis and balanced glucose metabolism. COX2, Arginase I and RELMa confirmed these results. These find- ings were accompanied by increased IL-6 secretion and nitric oxide (NO) production further supporting LPA-mediated polarization towards M1. B102 A better understanding of the role of bioactive lipid mediators in GUT MICROBIAL METABOLITES PROTECT microglia physiology and pathophysiology and the characterization of AGAINST AUTOIMMUNE DIABETES BY signaling cascades that drive microglia polarization could open new PRESERVING GUT HOMEOSTASIS AND possibilities to modulate microglia function. PROMOTING PERIPHERAL TREG BIOLOGY

Eliana Marino1, James L. Richards1, Keiran H. McLeod1, B101 Dragana Stanley2, Yu Anne Yap1, Hoey Yein Goh1, Jose M. Polo1, PLATELET ACTIVATING FACTOR RECEPTOR Jan Kranich3, Ana Carolina Oliveira4, Alan G. Baxter5, Trevor J. Lockett6, Julie M. Clarke7, David L. Topping7, CONTROLS ADIPOSE TISSUE MACROPHAGES 8 1,9 PHENOTYPE AND GLUCOSE METABOLISM Leonard C. Harrison , Charles R. Mackay 1School of Biomedical Sciences, Faculty of Medicine, Nursing and 1 1,2 1 Luciano R. Filgueiras , Francisco J. Rios , Marianna M. Koga , Health Sciences, Monash University, Wellington Road, Clayton, 3 1 1 Paula G. Quaresma , Edson K. Ishizuka , Marlise BA Montes , Victoria, Australia; 2Central Queensland University, School of 3,4 3 1 Patricia O. Prada , Mario S. Saad , Sonia Jancar Medical and Applied Sciences, Rockhampton, Queensland, Australia; 3Institute for Immunology, Ludwig Maximilians University, Munich, 1 Department of Immunology, Institute of Biomedical Sciences, Munich, Germany; 4Instituto de Biofı´sica Carlos Chagas Filho, 2 University of Sa˜o Paulo, Sa˜o Paulo, Brazil; Institute of Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Cardiovascular and Medical Sciences, British Heart Foundation Brazil; 5Comparative Genomics Centre, Molecular Sciences Bldg 21, Glasgow Cardiovascular Research Centre, University of Glasgow, James Cook University, Townsville, Queensland, Australia; 6CSIRO 3 Glasgow, United Kingdom; Department of Internal Medicine, State Preventative Health National Research Flagship, North Ryde, NSW, 4 University of Campinas (UNICAMP), Campinas, SP, Brazil; School Australia; 7CSIRO Preventative Health National Research Flagship, of Applied Sciences, State University of Campinas (UNICAMP), Adelaide, SA, Australia; 8Walter & Eliza Hall Institute of Medical Limeira, SP, Brazil Research, Parkville, Victoria, Australia; 9Charles Perkins Centre, Luciano R Filgueiras and Francisco J Rios contributed equally The University of Sydney, Camperdown NSW, Australia Introduction: Metabolic dysfunction is associated with inflammatory Diet and its effects on the composition of the gut microbiota may response in adipose tissue mediated by activated macrophages. We underlie the increased incidence of certain inflammatory diseases in previously showed that the interaction between oxidized lipids and western countries. Type 1 diabetes (T1D) in humans is an autoim- platelet activating factor receptor (PAFR) shifted macrophages mune disease with a genetic basis, but incidence around the world is towards anti-inflammatory phenotype. We thus questioned whether influenced by as yet unknown environmental factors. Here we used in vivo deficiency of PAFR changes the activation profile in adipose the non-obese diabetic (NOD) mouse model, to demonstrate that tissue macrophages and influences metabolic parameters in these pathogenesis T1D is critically dependent on bacterial metabolites, animals. particularly the short chain fatty acids (SCFA) acetate and butyrate.

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We found high concentrations of bacterial metabolites acetate and In summary, the chemotaxis and phagocytosis data suggest that butyrate in blood and faeces of male but not female mice, and in activation of GPR40 by GW9508 may have subtle and selective NOD.MyD88-/- mice, which correlated with protection from disease. effects on neutrophil biology, and possibly, the resolution of We employed specialised high acetate- and butyrate-yielding diets, inflammation. which significantly reduced the high diabetes incidence in female Supported by Science Without Borders/CNPq/Brazil and William NOD mice, through changes in intestinal microbial composition and Harvey Research Institute Founding. improved gut epithelial integrity. This protection was associated with both acetate and butyrate promoting the expansion of colonic as well as peripheral Treg cells, and unresponsiveness of islet-reactive (IGRP-reactive TCR transgenic) CD8+ T cells in NOD mice. Acetate B104 and butyrate reduced inflammatory cytokines IL-21 and TNFa, and IMPORTANCE OF THE S1P1 PHOSPHORYLATION increased IL-22 and TGFb. Protection relied on the metabolite sensor DOMAINS AND RECEPTOR INTERNALIZATION GPR43, a receptor for both acetate and butyrate. Hence diet and IN REGULATING INFLAMMATORY RESPONSES metabolites can dramatically influence disease pathogenesis, and autoantigen reactivity, in the NOD model of autoimmune diabetes. These results fit with the notion that altered dietary habits in humans Manisha Menon, Leigh Maher, Kamal M. Khanna from western countries may also lead to an altered gut microbial ecology and reduced SCFAs, important for immune regulation University of Connecticut Health Center, Farmington, CT, USA and tolerance. We propose these factors favour the development of Identification of novel mediators of inflammation is important for T1D in genetically susceptible individuals. Thus diet and bacterial development of new therapeutics. The sphingosine 1 phosphate metabolites may represent an effective, non-pharmacological means (S1P)—sphingosine 1 phosphate receptor-1(S1P1) signaling axis has to modulate gut and immune homeostasis, to treat autoimmune been implicated in regulating inflammatory responses. Previous diabetes. reports have shown that the phosphorylation-deficient mutant S1P1- S5A exacerbates experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (MS). Here, we B103 demonstrate a potential anti-inflammatory role for the phosphory- EXPRESSION AND ACTIVATION OF THE LONG- lation domain of S1P1. S1P1-S5A mice exhibit inflammatory phenotypes characterized by enlarged spleen and intestinal tissue CHAIN FATTY ACID RECEPTOR GPR40 IN damage. H&E stained images of the small bowel and the colon HUMAN NEUTROPHILS show significant damage to the villi architecture. By using intravital two-photon dynamic imaging of live mouse gut we demonstrate that Patricia RS Souza, Hefin R. Jones, Lucy V. Norling, Mauro Perretti S1P1-S5A mice show compromised barrier function that likely contributes to intestinal tissue damage and heightened inflammation. William Harvey Research Institute/QMUL, London, UK Interestingly, the tissue damage appears to be exasperated in S1P1- S5A mice with age. Furthermore, we used bone marrow chimeras to Omega-3 fatty acids are essential polyunsaturated fatty acids with a isolate the receptor internalization deficiency specifically in the double bond between the third and fourth carbon atoms from the stromal versus hematopoietic compartment to determine the mech- methyl end of the carbon chain. Nutritionally important omega-3 fatty anism responsible for the aforementioned observations. These results acids include eicosapentaenoic acid (EPA) and docosahexaenoic acid indicate that proper regulation of S1P1 receptor internalization even (DHA), which are correlated with lower incidence of chronic dis- under steady state conditions is essential to maintain immune and eases. Beside recent ground-breaking work on resolvins and intestinal homeostasis. protectins as bioactive derivatives of omega-3 fatty acids, there are still molecular mechanisms to be unveiled. DHA and EPA can acti- vate the long-chain free fatty acid receptors GPR40 and GPR120, two GPCRs with a poorly investigated biology. In this work, we focused Immunotherapeutics on GPR40 by using selective agonist GW9508. Using real-time PCR analysis, we detected GPR40 transcript in human neutrophils, a result confirmed at the protein level by flow cytometry and image stream B105 analysis. Expression of GPR40 was up-regulated after 10 min cell DECIPHERING THE HUMAN ANTI- stimulation with platelet-activating factor (PAF, 10 nM) or leuko- CARBOHYDRATE REPERTOIRE OF IGG, triene B4 (LTB4, 10 nM). To establish the affinity of GW9508 for IGA AND IGM GPR40, coupled to Gaq/11 protein, intracellular calcium fluxes were assessed. Tested on human neutrophils, GW9508 elevated intracel- 1 1 2 lular calcium when applied within the 0.1–10 lM range. Christoph Schneider , Marc Wehrli , David F. Smith , Richard D. Cummings2, Alex Straumann3, Adrian Zu¨rcher4, The up-regulation of GPR40 expression by pro-inflammatory 1 stimuli suggested to us potential counter-regulatory roles for this Stephan von Gunten receptor during inflammation. We therefore investigated whether the 1Institute of Pharmacology, University of Bern, Bern, Switzerland; GPR40 agonist GW9508 could counter-regulate neutrophil adhesion 2 molecules expression induced by TNF-a (10 ng/ml, 15 min). How- Protein-Carbohydrate Interaction Core H, Consortium for ever, GW9508 did not modulate the expression of CD11b and Functional Glycomics in the Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA; 3Department of L-selectin. According, GW9508 was inactive when tested neutrophil- 4 endothelial interaction under flow. However, 1 and 10 lM GW9508 Gastroenterology, Kantonsspital, Olten, Switzerland; Research and increased neutrophil chemotaxis in response to the cytokine IL-8 Development, CSL Behring AG, Bern, Switzerland (30 ng/ml). In addition, GPR40 activation by GW9508 modulated Despite the paradigm that carbohydrates are T cell-independent phagocytosis of E. coli by human neutrophils by approximately 50 % antigens, isotype-switched glycan-specific IgG antibodies and when tested at 0.1 and 1 lM. polysaccharide-specific T cells are found in humans. We employed a

123 S144 Inflamm. Res. systems level approach combined with glycan array technology to B107 decipher the repertoire of carbohydrate-specific antibodies. A strik- INTERFERON BETA CELL THERAPY SHOWED ingly universal architecture of this repertoire with modular organization among different donor populations revealed an associ- ANTI-TUMOR EFFECTS ASSOCIATED WITH ation between immunogenicity or tolerance and particular structural INHIBITION OF TUMOR CELL PROLIFERATION features of glycans. Antibodies were identified with specificity not AND ACTIVATION OF IMMUNE CELLS only for microbial antigens, but for a broad spectrum of host glycans that serve as attachment sites for viral and bacterial pathogens and/or Masaki Nakamura1, Lisa Kagawa1, Norihiro Nakada2, Masashi exotoxins. The comparison of IgG with IgA and IgM further reveals Satoh3, Shotaro Maehana6, Fumiaki Kojima5, Hideki Amano4, Yoshiki novel insights into the isotype-specific glycan-recognition in human Murakumo2, Kazuya Iwabuchi3, Masataka Majima4, Hidero Kitasato1 sera, breast milk, saliva and in the gastrointestinal tract. Our study highlights the power of systems biology approaches to analyze 1Department of Microbiology, Kitasato University School of Allied immune responses and reveals potential glycan antigen determinants Health Sciences, Sagamihara, Japan; 2Department of Pathology, that are relevant to vaccine design, diagnostic assays, and antibody- Kitasato University School of Medicine, Sagamihara, Japan; based therapies. 3Department of Immunology, Kitasato University School of Medicine, Sagamihara, Japan; 4Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Japan; 5Department of B106 Pharmacology, Kitasato University School of Allied Health Sciences, Sagamihara, Japan; 6Department of Environmental Microbiology, SITE-TARGETING NITROGLYCERIN Kitasato University Graduate School of Medical Sciences, NANOTHERAPEUTIC FOR LOCAL Sagamihara, Japan IMMUNOSUPPRESSION WITHOUT INDUCTION Objectives: Interferon beta (IFNb) is known to have anti-tumor OF TOLERANCE effects via suppression of tumor cell proliferation, activation of immune cells, and inhibition of tumor angiogenesis. IFNb has been Soroush Ardekani1, Shane Eum1, Sharad Gupta3, Harry A. Scott1, clinically used for the treatment of several types of cancers including Xiao Yang1, Alexander R. Brunelle2, Sean M. Wilson2, Umar melanoma and brain tumor. However, the clinical benefit by the Mohideen1, Kaustabh Ghosh1 systemic administration of IFNb itself is limited because of its short half-life and sever systematic side effects such as hepatic dysfunction 1University of California, Riverside CA, USA; 2Loma Linda and thrombocytopenia. In this study, we showed a novel IFNb cell University School of Medicine, Loma Linda CA, USA; 3Indian therapy, in which therapeutic cells highly producing IFNb were Institute of Technology, Indore, India injected around the tumor as cancer therapy. We demonstrated the efficacy and safety of the IFNb cell therapy using a murine melanoma Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavail- model, and histologically investigated the mechanism of anti-tumor ability. However, its ability to mimic the anti-inflammatory properties effects in this therapy. of NO remains unknown. Here, we examined whether NTG can Methods: To establish IFNb therapeutic cells, murine fibroblasts were suppress endothelial cell (EC) activation during inflammation and transfected with murine IFN-b gene by using a retrovirus vector. To developed NTG nanoformulation to simultaneously amplify its anti- evaluate directly suppression of tumor cell proliferation, the super- inflammatory effects and ameliorate adverse effects associated with natant of therapeutic cell was added into B16 murine melanoma cells, high-dose NTG administration. then the proliferation of B16 cells was evaluated with MTT assay Our findings reveal that NTG significantly inhibits human mono- in vitro. As a in vivo study of murine tumor model, C57BL/6 mice cyte adhesion to NO-deficient human microvascular ECs in vitro were subcutaneously inoculated with B16 murine melanoma cells. (EC50 = 0.64 lM) through an increase in endothelial NO and Then IFNb therapeutic cells were injected locally around the B16 decrease in endothelial ICAM-1 clustering, as determined by NO tumor as cell therapy. After a week, the tumor tissue was excised and analyzer, microfluorimetry, and immunofluorescence staining. analyzed histologically. Subsets of lymphocyte infiltrating surround Nanoliposomal NTG (NTG-NL) was formulated by encapsulating the B16 tumor were also analyzed by flow cytometry. In addition, the NTG within unilamelar lipid nanoparticles that were * 150 nm in concentration of IFN-b in blood and tumor tissues were determined to diameter and readily uptaken by ECs, as determined by dynamic light estimate the efficacy and side effects of the IFNb cell therapy. Sys- scattering and quantitative fluorescence microscopy, respectively. tematic side effects including hepatic dysfunction and More importantly, NTG-NL produced an approximately two orders of thrombocytopenia were also examined. magnitude greater anti-inflammatory effect than free NTG while Results: Interferon beta cell therapy significantly suppressed the preventing excessive mitochondrial superoxide production and loss of proliferation of the B16 cells in vitro, and the progression of the B16 arterial vasorelaxation associated with high NTG doses. Finally, to tumors in a murine tumor model in vivo. By histological analysis, facilitate targeting of NTG-NL to inflamed ICAM-1-expressing ves- proliferation of tumor cells was suppressed and cell degeneration was sels, we have tethered a non-immunogenic fragment of ICAM-1 also observed after the IFNb cell therapy. Interestingly, significant antibody to the surface of NTG-NL. Our preliminary in vitro studies lymphocyte infiltration was observed around the tumor in respones to show that NTG-NL modified with anti-ICAM-1 fragment exhibits the IFNb cell therapy. Flow cytometry analysis further revealed that sixfold greater binding to inflamed (ICAM-1-expressing) ECs than to NK cells was the most dominant population among the infiltrating normal ECs. Thus, by identifying the superior therapeutic effects of lymphocytes. The level of IFN-b in the tumor tissue was high enough NTG nanoformulation and conferring potent site-targeting capability to suppress tumor growth, while blood level of IFNb was not ele- to it, this study provides the rationale for detailed investigation of vated. Systemic side effects including hepatic dysfunction and NTG nanotherapeutic as a potentially superior anti-inflammatory thrombocytopenia were not detected after the IFNb cell therapy, therapy. suggesting safety and efficacy of this therapy.

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Conclusion: The present study indicates that novel IFNb cell therapy Indiana University School of Medicine, Indianapolis, IN, USA suppresses the tumor growth both in vitro and in vivo. In addition, the Methicillin-resistant Staphylococcus aureus (MRSA) is the major activation of NK cells by IFNb cell therapy partly plays an important cause of skin and soft tissue infections. Due to antibiotic resistance, role for anti-tumor effects. IFNb cell therapy might be one of the MRSA infections are challenging to treat which demonstrates a need prominent strategy for the treatment of cancer. for novel therapeutic strategies. Leukotriene B4 (LTB4) is a bioactive lipid mediator produced by the 5-lipoxygenase (5-LO) metabolism of arachidonic acid. LTB4 signals through its high affinity receptor LTB4 B108 receptor 1 (BLT1) and induces leukocyte recruitment and enhances EVALUATION OF MOLECULAR MARKERS AND macrophage antimicrobial effector functions. We hypothesize that LTB4 is a homeostatic regulator of skin host defense by increasing THERAPEUTIC TARGETS IN TNBS-MODEL OF different arms of the innate immune response. INTESTINAL INFLAMMATION MRSA skin infection leads to macrophage-dependent enhanced -/- 5-LO and BLT1 expression and LTB4 abundance. 5-LO and -/- Aline Witaicenis Fantinati, Alexandre S. Chagas, Alexandre BLT1 mice infected subcutaneously with MRSA had larger Tanimoto, Luiz C. Di Stasi abscesses and higher bacterial burden than wild type mice at day nine post infection, suggesting an impairment in immune response. -/- Sao Paulo State University, UNESP, Araraquara, Brazil BLT1 mice had less neutrophil recruitment and more apoptotic cells compared to wild type mice at day two post infection. Mice Introduction: Ulcerative colitis and Crohn’s disease are major forms treated with a pharmacological BLT1 inhibitor had more apoptotic of Inflammatory Bowel Diseases (IBD) in humans; both diseases are cells in the skin, suggesting that LTB4 has a protective role in both characterized by excessive immune response against normal con- neutrophil recruitment and survival during MRSA skin infection. stituents of the gastrointestinal tract. Recent studies show that Endogenously produced LTB4 enhanced MRSA ingestion and killing, different genes are involved in the initiation, amplification and per- which correlated with enhanced generation of reactive oxygen species petuation of intestinal inflammation. Based on these, the present study and antimicrobial molecules in response to MRSA. Additionally, aims to identify new molecular markers and/or future therapeutically BLT1-/- mice produced similar TNFa levels but less IL1b than wild target in animal models of intestinal inflammation. type mice in response to in vivo MRSA infection. IL1b is an Methods: Anaesthetised rats (n = 8) were daily treated (p.o.) with important inflammatory cytokine involved in controlling MRSA coumarin derivatives with proven intestinal anti-inflammatory activity infections. IL1b requires maturation through caspase-1 activity. in rats, such as 4-methylesculetin (4-M, 5 mg/Kg), esculetin (5 mg/ Inhibiting BLT1 reduced MRSA-induced caspase-1 activity, sug- Kg), esculin (25 mg/Kg), or with reference drugs: sulphasalazine gesting that LTB4 promotes IL1b production and processing. Topical (50 mg/Kg), azathioprine (2 mg/Kg) or prednisolone (2 mg/Kg) for treatment with an ointment containing LTB4 greatly improved bac- 4 days and 2 h prior to TNBS administration into the colon (preven- terial clearance and accelerated healing in both 5-LO-/- mice and tive protocol) or 2 h after TNBS instillation and daily until the 7th day wild type mice. (curative protocol). After the animals were killed, colon segments These findings show that LTB4 is vital for skin host defense (100 mg) were collected and regulation of mRNA expression of mechanisms and suggests that topical LTB4 treatment is a likely can- HSP70, HPRT, NF-kB p50 and p65, JAK1, 2, 3; TYR2, STAT1, 2, 3, didate for an immunotherapeutic agent to control MRSA infections. 4, 6; MAPK1, 3, 6, 9; and TIMP1 were analyzed by qRT-PCR. Financial support NIH Immunology and Infectious Diseases T32 Results: In preventive protocol, TNBS-control group showed AI060519, R00HL103777, and R03 AI110990-01A1 increased expression of HSP70 and TIMP1 and decreased NF-kB p65, JAK1, JAK3, STAT3 and MAPK9 expressions in comparison to non- colitic animals; all treatments reduced HSP70 mRNA levels; esculetin increased JAK3 and MAPK1 expressions; esculin, azathioprine and B110 prednisolone increased the JAK3 and MAPK6; and sulphasalazine TARGETING NORMAL AND NEOPLASTIC reduced STAT6 and increased MAPK1 and TIMP1 mRNA levels. In HUMAN B CELLS IN VITRO AND IN VIVO USING curative protocol, only expression of MAPK6 and MAPK9 were decreased and TIMP1 was increased in TNBS-control group. Treat- ANTIBODY-MEDIATED DELIVERY OF SIRNA ments with esculetin, 4-M and prednisolone decreased TIMP1 gene expression and 4-M increased MAPK9 expression. Gary P. Sims, Fred Karnell III, Lena Shirinian, Vladimir Voynov, Conclusion: Gene expression alterations occur mostly in the early Daniel C. Rowe, Marlon C. Rebelatto, Elizabeth Ward, Bo Chen, phase of inflammatory process. The genes HSP70, JAK3, MAPK6 and Nazzareno Dimasi, Changshou Gao, Ronald Herbst TIMP1 are altered after TNBS administration; therefore these genes might be new molecular markers and/or future therapeutical targets in Research and Development, MedImmune, Gaithersburg, MD, USA IBD. The treatment with the coumarins derivatives regulated somehow The use of synthetic siRNAs to specifically silence gene expression in the altered gene expressions in intestinal inflammation. key pathological pathways provides appealing therapeutic opportu- Financial Support FAPESP (11/50824-4; 11/50512-2). nities. However, without an efficient cell-specific delivery system, the clinical use of siRNA therapeutics has been restricted. Herein, we combined the precise cell recognition and internalization properties of B109 anti-CD22 antibody with the nucleic acid-binding capacity of pro- LEUKOTRIENE B4 IS A HOMEOSTATIC tamine, to specifically deliver siRNA to B cells. Blimp-1 siRNA delivered to primary B cells, directed the knockdown of the target COMPONENT OF METHICILLIN-RESISTANT mRNA, and inhibited plasma cell differentiation and immunoglobulin STAPHYLOCOCCUS AUREUS SKIN INFECTION secretion. Increasing the siRNA payload by conjugating additional protamine peptides to the antibody increased the potency 20-fold. Stephanie Brandt, Soujuan Wang, Sebastian Carrasco, Stacy Blank, Delivery of siRNAs targeting the oncogenes Bcl-2 and c-myc Nathan Delafield, C. Henrique Serezani inhibited growth of acute lymphoblastic leukemia cells in vitro, and

123 S146 Inflamm. Res. systemic administration resulted in target knockdown and the The objective of this animal study and laboratory investigation were remarkable regression of established subcutaneous tumors in vivo. to investigate whether the decrease in the rate of mortality caused by This study demonstrates the therapeutic potential of cell-specific lipopolysaccharide (LPS) tolerance may be associated with an antibody-mediated delivery of siRNA for the targeted, functional increased population of CD4+ T regulatory lymphocytes and Th17. inhibition of normal and neoplastic B cells. Male black C57/6 mice received subcutaneous (s.c.) injections of LPS (1 mg/kg) for 5 days, followed by cecal ligation and puncture (CLP). Cytokines and marked lymphocytes were measured after tolerance and CLP challenge. Both of T reg subpopulation (induced and natu- B111 ral) and Th17 lymphocytes, showed increase in cells in the spleen and A NONHUMAN PRIMATE MODEL OF ALLERGIC plasma after tolerance. Mortality reduced in tolerant animals. This RHINITIS AS A MECHANISTIC MODEL FOR study demonstrated that reduced mortality after tolerance may be FOOD ALLERGY associated with increasing population of Treg and Th17 cells due to immunoregulation of the hyperinflammatory response and neutrophil recruitment. Joanne Schiding, Nick Colletti, Mike Shaw, Neil Fitch

Sanofi US Inc, BioInnovation, Cambridge, MA, USA B113 Introduction: Food allergy affects approximately 8 % of US children and is thus a major public health concern. The most common food THE EFFECT OF TRANSCUTANEOUS CERVICAL allergens induce an IgE-mediated response which can sometimes be ELECTRICAL VAGAL NERVE STIMULATION ON severe enough to cause fatalities. While children will outgrow their AUTONOMIC INDICES IN HEALTHY HUMANS: allergies to some foods, other food allergies persist over a lifetime. A POTENTIAL ANTI-INFLAMMATORY Current strategies to treat food allergy involve specific immunotherapy to desensitize the patient to the food allergen. We THERAPY? are looking at a novel strategy involving the use of immunomodu- lators to divert the immune response away from the IgE-mediated JP Errico3, Christina Brock1, Bruce Simon3, Qasim Aziz2, Th2 phenotype. Asbjorn M. Drewes1, Adam D Farmer2,1 Hypothesis: A nonhuman primate natural allergic rhinitis model, (which is IgE mediated and driven by a Th2 cytokine phenotype), can 1University of Aalborg, Aalborg, Denmark; 2Barts and the London act as both a surrogate as well as a mechanistic model for food allergy School of Medicine, London, UK; 3ElectroCore Medical LLC, in assessing therapeutic benefits of an immunomodulator. Basking Ridge, NJ, USA Methods: A clinical acoustic rhinometer was adapted for use with the Background: The autonomic nervous system is a bidirectional hier- nonhuman primate. The rhinometer transmits a sound wave into the archically controlled brain body nexus that integrates the external nasal cavity and measures the reflected signal. Algorithms are used to environment with the internal milieu. The parasympathetic nervous calculate nasal volume and the minimum cross-sectional area of the system (PNS), whose main neural substrate is the vagus nerve, nasal cavity. Intranasal delivery of allergen induces nasal congestion influences inflammation through the cholinergic anti-inflammatory which is evidenced by a reduction in both volume and area. Animals pathway. A non-invasive transcutaneous cervical vagus nerve stim- were treated with the pro-Th1 TLR-4 agonist Glucopyranosyl lipid A ulator (t-VNS), gammaCore (Basking Ridge, NJ), has been recently (GLA) in an attempt to ameliorate the allergic response to antigen. developed for the treatment of epilepsy and migraine. If this new Results: Treatment with 10ug GLA co-administered with 10ug anti- technology has the potential to be used as a modulator of the immune gen intramuscularly once weekly for 4 weeks blocked the response to system, it is important to first establish that it can modulate cardiac intranasal antigen challenge when measured 24 h after the final vagal tone in a measurable manner. treatment. Efficacy was maintained 2–4 weeks after the final treat- Aim: To investigate the effect of t-VNS on cardiac vagal tone (CVT) ment. If the antigen was administered at a distal site relative to the in healthy subjects. GLA, there was no significant effect. FACS analysis indicated that Methods: 20 healthy subjects (13 female, median age 34 years, range GLA treatment reduced (1) basophil responsiveness (demonstrated by 23–56) had heart rate (HR), blood pressure (BP) and CVT, a validated reduced histamine release on stimulation) and (2) antigen-specific real time non-invasive parameter of brain stem mediated efferent PNS CD4+ effector T-cells. Cytokine analysis of serum suggested that tone (1), measured at baseline, during 4 min of bilateral t-VNS and C-reactive protein (CRP) may be a valuable biomarker for GLA dose post t-VNS at 90 min and 24 h. Venous blood was also sampled at monitoring. these time points to assess the effect of t-VNS on inflammatory Conclusion: This nonhuman primate allergic rhinitis model was cytokine expression. immunomodulated by a Th1-inducing TLR4 agonist. This suggests Results: All subjects tolerate t-VNS well except one subject (1 that this mechanism may translate into efficacy in animal models of female, 30 years old) who felt light headed during the stimulation. A food allergy and in patients with food allergy. repeated measures analysis of variance (ANOVA) was conducted to ascertain the effect of t-VNS on HR, systolic BP and diastolic BP. T-VNS had no effect on HR (F (3,76) = 2.5, p = 0.07) or diastolic B112 BP (F (2.6, 49.1) = 2.2, p = 0.1). T-VNS had a small effect in THE LPS TOLERANCE AFFECTS ON CD4, TREG reducing systolic BP (F (2.7, 52.4) = 4.5, p = 0.01). A non-para- metric Friedman test was conducted to assess the effect of t-VNS on AND TH17 LYMPHOCYTES CVT, yielding a Chi square of 28.7, p \ 0.0001, see figure 1. Conclusions: t-VNS raises CVT, suggesting that it is sufficient to Mariana M. Andrade, Andre Botega, Denise F. Barbeiro, potentially stimulate the cholinergic anti-inflammatory pathway. Should Hermes Barbeiro, Francisco G. Soriano the cytokine expression data from this study demonstrate an anti-in- flammatory effect of t-VNS, further work will be warranted to translate Medicine Faculty of Sa˜o Paulo Univerity, Sa˜o Paulo, Brazil theses findings in clinical disorders such inflammatory bowel disease.

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Figure 1 The effect of t-VNS on cardiac vagal tone. The maximal showed that a 24-h treatment with MSU crystals induced the effect of t-VNS on CVT was seen 90 min post stimulation, with an expression of P2X7R. Furthermore, addition of the P2X7 purinergic effect still present at 24 h, **** p \ 0.0001, *p \ 0.05. receptor antagonist A-740003 significantly impeded IL-1b production after treatment with LPS + MSU or LPS + BzATP. Contrarily, addition of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro- 2H-benzofuro-3,2-e]-1,4-diazepin-2-one (5-BDBD) did not affected IL-1b release after treatment with LPS + MSU whereas 5-BDBD significantly reduced production of IL-1b after treatment with LPS + BzATP. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited significantly the release of IL-1b and other M1 macrophage cytokines (such as IL-1a, IL-6 and TNF-a) from MDMs stimulated with LPS + MSU. Our findings confirm the quantitative difference between NLRP3-inflammasome activation by MSU crys- tals and activation by exogenous ATP, and suggest that blockade of the NLRP3-inflammasome pathway or the P2 purinergic P2X7R receptors is a novel potential therapeutic approach to control the inflammatory process in several associated pathologies.

B115 INFLAMMASOME ACTIVATION BY LANTHANIDE UPCONVERSION Reference 1. Borokova et al. Nature 2000. 2. Farmer et al. Ann Gastro 2014. NANOPARTICLES AND ITS ABROGATION BY A SURFACE COATING PEPTIDE

Inflammasomes in Health and Disease Longping Wen, Yunjiao Zhang, Han Yao School of Life Science, University of Science and Technology of B114 China, Hefei, Anhui, China INVOLVEMENT OF P2X4 AND P2X7 PURINERGIC Lanthanide upconversion nanoparticles (UCNs) hold great promise RECEPTORS IN IL-1ß PRODUCTION AND NLRP3- for in vivo theranostic applications. It is known that a variety of INFLAMMASOME PATHWAY ACTIVATION IN nanocrystals, including rare earth oxide nanoparticles, elicits potent inflammatory response through activation of NLRP3 inflammasomes, HUMAN MACROPHAGES but whether UCNs have the similar effect remains to be shown. Here we have assessed the ability of UCNs to activate inflammasomes and 1,2 1 1 1 Thomas Gicquel , Sacha Robert , Pascal Loyer , Tatiana Victoni , investigated in detail the underlying mechanism. UCNs triggered 1 1 1 Aude Bodin , Catherine Ribault , Gleonnec Florence , inflammasome activation, characterized by the activation of caspase 1 3 1 1 Isabelle Couillin , Elisabeth Boichot , Vincent Lagente and release of IL-1 and IL-18, in a dose- and time-dependent fashion in macrophages, including human THP-1 cells, mouse bone marrow 1 UMR991 INSERM, Universite´ de Rennes 1, Rennes, France; derived macrophage (BMDM) cells, and mouse peritoneal micro- 2 Laboratoire de toxicologie biologique et me´dico-le´gale, CHU phage cells. Cellular internalization, reactive oxygen species (ROS) 3 Rennes, France; UMR-IEM CNRS6218, Universite´ d’Orle´ans, and lysosomal damage were all critical, while potassium efflux only France played a minimal role, for UCN-induced inflammasome activation. The Nod-like receptor family protein 3 (NLRP3)-inflammasome Consistent with published reports, NLRP3 inflammasome were mainly responsible for UCN-elicited inflammation, as the IL-1 release pathway is known to be activated by danger signals such as mono- -/- sodium urate (MSU) or adenosine triphosphate (ATP). Here, we was largely reduced in nlrp3 mouse BMDM cells treated with investigated the role of P2X4R and P2X7R, two P2 purinergic UCN. However, significant IL-1 release, corresponding to approxi- mately 30 % level to that seen in the wild-type cells, was still receptors in the activation of NLRP3-inflammasome pathway in pri- -/- mary human monocyte-derived macrophages (MDMs). After initial observed in the UCN-treated nlrp3 BMDM cells, indicating the stimulation with a low concentration of LPS (0.1 lg/mL), a 6-h involvement of non-NLRP3 inflammasomes during UCN-elcited treatment with ATP agonists, 20,30-O-(4-benzoylbenzoyl) adenosine inflammatory response. RE-1, a lanthanide-specific surface coating 50-triphosphate (BzATP), Adenosine 50-[c-thio]triphosphate tetra- peptide we discovered previously, significantly abrogated the lithium salt (ATPcS) or MSU crystals induced the MDMs to release inflammasome-activating activity of UCN. RE-1 coating did not IL-1b in a dose-dependent manner. Moreover, the caspase-1 inhibitor affect the ability of the nanocrystals to enter cells through phagocy- Z-YVAD-FMK reduced production of IL-1b in a dose-dependent tosis, but led to significant reduction in UCN-induced ROS generation manner after LPS + MSU treatment. We used RT-qPCR to show that and lysosomal damage. In conclusion, we demonstrated that UCNs treatment with MSU (500 lg/mL) or BzATP (250 lM) induced sig- were potent inflammasome activators in macrophages, and the ability nificantly the expression of NLRP3 after LPS priming or not. We also of RE-1 peptide to abrogate this effect may be of great value for found that MSU treatment, but not BzATP, induced P2X7R mRNA in vivo applications of UCNs and other rare earth-based expression. Using flow cytometry and immunoblotting analysis, we nanomaterials.

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B116 expression in human coronary artery specimens obtained from 10 WHOLE EXOME ANALYSIS OF INDIVIDUALS explanted hearts. In advanced atherosclerotic plaques (AHA IV-VI), a significant upregulation of 12 target genes was found compared to AND FAMILIES WITH CHRONIC RECURRENT early lesions (AHA I-III), among them several core components of the MULTIFOCAL OSTEOMYELITIS (CRMO) inflammasome pathway. Immunohistochemical stainings of the advanced plaques revealed the presence around necrotic cores of Allison J. Cox1,2, Benjamin W. Darbro1, Xinyu Bing1, macrophage foam cells positive for NLRP3 inflammasome compo- Alexander G. Bassuk1, Polly J. Ferguson1 nents. The PCR array target p38delta mitogen-activated protein kinase (MAPK) was upregulated in advanced coronary plaques and 1Department of Pediatrics, University of Iowa, Iowa City, IA USA; expressed in macrophage foam cells of the plaques. In cultured human 2Interdisciplinary Graduate Program in Genetics, University of Iowa, monocyte-derived macrophages, activators of the NLRP3 inflamma- Iowa City, IA, USA some, cholesterol crystals and ATP, triggered strong p38delta MAPK activation. The activation of p38delta was required for inflamma- Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, some-mediated IL-1beta secretion and depended on intracellular autoinflammatory bone disease presenting in infancy and childhood. stress signals previously linked to NLRP3 inflammasome activation. While CRMO is characterized by painful bone lesions, it is often Conclusions: Our data revealed upregulation of several core comorbid with psoriasis or Crohn disease and many patients have components of the inflammasome pathway in advanced coronary close relatives with either of the more common disorders. Some plaques compared to early lesions from the same patients, implying a syndromic cases of CRMO are caused by truncation mutations in the role for the inflammasome pathway in disease progression. Among interleukin-1 receptor agonist (IL-1RA) gene and these children the upregulated genes, p38delta MAPK was identified as a novel respond very well to treatment with recombinant IL-1RA. However, mediator of NLRP3 inflammasome activation, and thus represents a most cases of CRMO are non-syndromic with an unknown genetic potential target for modulation of atherosclerotic inflammation. cause, although many patients respond to TNF-a blocking agents, implicating the pathway in the disease. Using whole exome sequencing paired with genetic analyses based on inheritance patterns in affected families, we have determined that the genetic basis of CRMO is heterogeneous and complex. However, for several families, B118 we have determined the most likely causative mutations, in both NLRC4 IS INVOLVED IN ACTIVATION OF THE novel and previously characterized genes affecting a shared pathway. NLRP3 INFLAMMASOME IN RESPONSE TO Our research demonstrates how multiple approaches to candidate gene selection may be merged to help determine the underlying cause LEISHMANIA AMAZONENSIS INFECTION of a rare and complex genetic disease. Alexandre L.N Silva1, Djalma S. Lima-Junior1, Larissa D. Cunha1,2, Dario S. Zamboni1

B117 1University of Sa˜o Paulo, Medical School Ribeira˜o Preto, Sa˜o Paulo, P38DELTA MAPK – A NOVEL EFFECTOR IN Brazil ; 2St Jude Children’s Research Hospital, Memphis, TN, USA NLRP3 INFLAMMASOME ACTIVATION NLRs are a group of intracellular receptors that recognize endogenous UPREGULATED IN HUMAN CORONARY and exogenous danger signals. Some members of this family partic- ATHEROSCLEROTIC LESIONS ipate of the inflammasome formation. The inflammasome is able to activate inflammatory caspases, such as caspase-1 that is responsible for processing and secretion of IL-1b and IL-18, as well to induce an Kristiina Rajamaki1, Mikko I. Mayranpaa2, Jarno Tuimala4, inflammatory form of cell death called pyroptosis. Leishmaniasis is a Katariina Nurmi1, Kari K. Eklund3, Katariina Oorni1, group of diseases caused by protozoa of Leishmania genus. These Petri T. Kovanen1 parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome of host macrophages. Macrophages are 1Wihuri Research Institute, Helsinki, Finland; 2Department of critical for effective and protective immune responses to different Pathology, Haartman Institute, University of Helsinki and HUSLAB diseases, including Leishmaniasis. Macrophages express different Division of Pathology, Meilahti Laboratories of Pathology, Helsinki pattern recognition receptors (PRR) responsible for recognition of University Central Hospital, Helsinki, Finland; 3Division of molecular patterns associated with microbes (PAMPs). While many Rheumatology, Department of Medicine, Helsinki University Central publications emphasize the importance of TLRs in the susceptibility Hospital, Helsinki, Finland; 4RS Training, Helsinki, Finland and control of Leishmania infection, the role of NLRs is still unclear. Background: Inflammation is recognized as a major driving force in We have recently demonstrated that the NLRP3 inflammasome is atherogenesis, yet the mechanisms involved remain elusive. Recently, activated in response to L. amazonensis infection in a process that is cholesterol crystals commonly found in atherosclerotic plaques were required for restriction of parasite infection. However, the molecules shown to trigger a strong proinflammatory response via the NLR involved in activation of NLRP3 in response to L. amazonensis family pyrin domain containing 3 (NLRP3) inflammasome in mac- infection are unknown. In this study, we found that NLRC4 is rophages, the key immune cells in atherosclerotic plaques. The involved in activation of the NLRP3 inflammasome in response to NLRP3 inflammasome regulates the proteolytic maturation and Leishmania infection. By using mice deficient in NLRC4, we found secretion of potent proinflammatory and proatherogenic cytokines, that NLRC4 is activated in response to L. amazonensis infection and interleukin (IL)-1beta and IL-18. plays an important role in controlling infection in macrophages and Aims: The aim of the current study was to comprehensively charac- in vivo. We also demonstrate that the NLRC4 receptor is phospho- terize, for the first time, the expression of inflammasome pathway rylated after infection with L. amazonensis and found that the NLRC4 components and regulators in human atherosclerotic lesions. inflammasome is assembled in response to L. amazonensis infection Methods and Results: A quantitative PCR array targeting 88 inflam- in macrophages when macrophages are transduced with retrovirus masome pathway-related molecules was employed to analyze mRNA encoding NLRC4-GFP. Collectively, these studies identify NLRC4 as

123 Inflamm. Res. S149 an important molecule participating on NLRP3 activation in response Inflammation and Aging to L. amazonensis infection. B120 MEASUREMENT OF NITRIC OXIDE B119 METABOLITES AS POTENTIAL MARKERS OF L. AMAZONENSIS ELIMINATION BY P2X7 INFLAMMATION IN DEMENTIA RECEPTOR AND LEUKOTRIENE B4 REQUIRES NLRP3 INFLAMMASOME ACTIVATION AND IL- Annie Knight1, Miranda J. Smallwood1, Emma Taylor1, David 1R SIGNALING Llewellyn1, Patrick G. Kehoe2, Paul G. Winyard1

1 Mariana M. Chaves1,De´bora A. Sinflo´rio1, Maria Bellio1, Dario University of Exeter Medical School, Exeter, EX2 4TE, UK; 2 Zamboni2, Cla´udio Canetti1, Robson Coutinho-Silva1 University of Bristol, School of Clinical Sciences, Learning and Research Building, Southmead Hospital, Bristol, UK 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2 Inflammation is present in the Alzheimer’s disease (AD) brain, with University of Sa˜o Paulo, Sa˜o Paulo, Brazil activation of complement, cytokine and chemokine pathways. Acti- Objectives: P2X7 receptors activation via extracellular ATP (eATP) vated microglia and astrocytes release reactive oxygen species, such Á Á¯ has been reported as an important molecule signal to intracellular as nitric oxide ( NO) and superoxide (O2 ). These species react to parasites elimination. After activation, the P2X7 receptor can lead to form peroxynitrite (ONOO¯ ) leading to nitration of tyrosine residues apoptosis, and cytokines release such as IL-1b, among others mech- (i.e. 3-nitrotyrosine; Tyr-NO2) within brain proteins. Tyrosine resi- anisms. Recently, it was demonstrated that IL-1b release, after dues are also nitrated by the myeloperoxidase (MPO)–H2O2-nitrite - NLRP3 inflammasome activation, participates of the resistance to (NO2 ) system. Immunohistochemical studies have suggested Leishmania amazonensis. Furthermore, our group showed that L. increased Tyr-NO2 in AD brain tissue. However, there is limited amazonensis elimination through P2X7 receptor activation depends of quantitative data on Tyr-NO2 levels in AD brain, compared with leukotriene B4 (LTB4) production. Thus, we investigated whether L. normal brain tissue. If Tyr-NO2 is elevated in the AD brain, this amazonesis elimination by P2X7 receptor and LTB4 involves NLRP3 would suggest that serum Tyr-NO2 should be tested as a biomarker in inflammasome activation and IL-1 receptor (IL-1R) signaling. future studies. Methods and Results: Peritoneal macrophages from C57Bl/6 (C57Bl/ In order to assess if Tyr-NO2 is a potential marker of inflammation -/- -/- -/- 6), P2X7 receptor (P2X7 ), NLRP3 (NLRP3 ), ASC (ASC ) in AD, Tyr-NO2 was measured along with the stable oxidation end- -/- • - - and IL-1R (IL-1R ) deficient mice, 2–7 months, were infected or products of NO, NO2 and NO3 (nitrate). Frontal lobe homogenates not with L. amazonensis at 10:1 MOI ratio. These were tested for from people with AD, vascular dementia (VaD) and non-demented - parasitic load presence or absence of 500 lM ATP, 100 nM LTB4, or elderly controls (n = 15 for each group) were analysed. NO2 and - 100 pg/mL IL-1b for 30 min at 37 °C. After 24 h, infected macro- NO3 were measured by ozone based chemiluminescence and Tyr- phages were fixed and stained with Panotic kit and analysed by optical NO2 was measured using a novel electrochemiluminescence based microscope. C57Bl/6 and P2X7-/- mice were infected in the footpad ELISA. 6 -/- - with 10 L. amazonensis promastigotes for 7 days. After, P2X7 Brain NO2 levels were significantly different between the mice were treated in infect footpad twice a week with 5 ng of LTB4 for three groups (P = 0.03, Kruskal–Wallis test) with AD and VaD 3 weeks and thickness paw and parasitic load were determined. Paw’s significantly higher than non-demented controls, P \ 0.05 (median thickness was accompanied with thickness gauge and the parasitic load (IQR): non-demented 0.08 (0.07–0.09); AD 0.1 (0.08–0.18) and VaD was established with limiting dilution assay (LDA). Nitric oxide (NO) 0.1 (0.08–0.12) lmol/g protein). There were no statistically signifi- - levels were measured by Griess assay. The graphs were generated and cant differences between groups for NO3 levels: controls 2.1 analyzed using the GraphPad Prism 5.0. (1.8–2.3); AD 2.6 (1.8–4.9); VaD 2.4 (1.9––3.3) lmol/g protein. -/- Our results showed that infected macrophages from NLRP3 mice Levels of Tyr-NO2 in brain tissue showed no statistically significant treated with ATP and LTB4 did not decrease parasitic load (difference differences between the three groups: normal volunteers 0.296 between means 4 %, n = 4; 5 %, n = 3, respectively). However, when (0.224–0.548); AD 0.292 (0.185–0.572) and VaD 0.357 -/- NLRP3 infeceted macrophages were treated with IL-1b, a decrease in (0.182–0.402) pmol BSA-Tyr-NO2 equiv/mg protein. There were no -/- - ¯- parasitic load was noted (20 %; n = 3). Furthermore, ASC infected statistically significant correlations between NO2 ,NO3 or Tyr-NO2 macrophages treated both ATP and LTB4 also did not diminish parasitic and subject age or post-mortem delay. - load (13 %, n = 3; 7 %, n = 3, respectively). The same happened with The increased concentration of NO2 in AD and VaD brain tissue Á IL-1R-/-infected macrophages (3 %, n = 3; 3 %, n = 3, respectively). suggests increased production of NO. Yet, our data set challenges the -/- Similarly, infected macrophages from P2X7 mice treated with IL-1b current consensus that Tyr-NO2 is increased in AD. A potential - decreased parasitic load (difference between means 39 %, n = 3). Fur- mechanism of Tyr-NO2 generation is via the MPO-H2O2-NO2 sys- -/- - thermore, P2X7 mice infected with L. amazonensis in the footpad tem. Our results demonstrate that NO2 is available in the AD/VaD treated with exogenous LTB4 showed more resistance to infection, brain as a substrate for MPO. However, the known loss of superoxide because their footpad had lower parasite load (difference between means dismutase activity in the AD brain, may limit the rate of H2O2 gen- 1.1x109 ± 4.7 9 108 parasites; n = 8) and lower lesion eration. This may, in turn, limit MPO catalysed nitration, thereby -/- (37.94 ± 11.45 mm, n = 8) when compared to untreated P2X7 mice. explaining the lack of increased Tyr-NO2 in AD brain tissue in the This resistance does not seem to be via NO. current study. Conclusion: Thereby, these data suggest the involvement of LTB4 in Acknowledgement We would like to thank the South West Dementia resistance of WT mice when compared with P2X7-/- mice, and that L. Brain Bank (SWDBB) for providing the brain tissue homogenates for amazonensis elimination by P2X7 receptor mediated by LTB4 this study. The SWDBB is supported by BRACE (Bristol Research depends of NLRP3 activation and IL-1R signaling. into Alzheimer’s and Care of the Elderly), Brains for Dementia Financial Support CNPq, CAPES, FAPERJ Research and the Medical Research Council.

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B121 Design of investigation includes four groups of 107 female DYSREGULATION OF RESOLUTION OF ACUTE patients: control group with normal endometrium, simple hyperplasia, complex hyperplasia with and without atypia. In tissue samples of INFLAMMATION IN AGED HUMANS endometrium we performed a quantitative analysis of estrogen and progesterone receptors and CD45+ (common leukocyte antigen) Roel P. H. De Maeyer, Madhur Motwani, Justine S. Newson, expression by immunohistochemical method. The level of estrogens, Derek W. Gilroy progesterone and cytokines IL-1ß, IL-6 and TNF-a investigated in uterine flushing’s by ELISA methods. The elastase-like and trypsine- Division of Medicine, Centre for Clinical Pharmacology and like activities and level of acid-stable inhibitors and antitrypsine Therapeutics, University College London, London, UK activity were measured by enzyme methods. Age-related pathophysiologies are becoming an increasing global The obtained results showed that more intensive imbalance with health concern as the population reaches newfound heights of long- prevalence of estrogens and estrogen receptor expression it was evity. Susceptibility of the elderly to severe and lasting infections showed in simple EH and we have not found the progression of coincides with a chronic inflammatory state termed inflamm-aging. hormonal changes in complex EH. But we found increase of mor- phological characteristics of chronic inflammatory process in the Thus far, few data are available on whether resolution, or the ability to + switch off innate immune-mediated responses, is dysregulated in aged endometrium and increase common leukocyte antigen CD45 individuals. Therefore, we investigated the inflammatory response in expression during the transition from normal endometrium healthy young and aged volunteers using the cantharidin skin blister (4.2 ± 1.2 %) to complex hyperplasia (57.8 ± 2.4 %). Study of local model of self-resolving, tissue injury driven inflammation. A novel cytokine levels showed that they are also increased in 3–5 times in and extensive multi-colour fluorescence staining protocol was simple EH and 15–20 times in both types of complex EH. The activity designed to identify all cell populations in the blister using flow of elastolytic enzymes significantly increased in 7.5 times in simple cytometry. We observed classic neutrophilia associated with inflam- EH and in 8–9 times in both forms of complex hyperplasia. Intensity matory onset in both age groups, but found a significantly increased of change depended from presence of infectious and inflammatory number of HLA-DR+/CD14+/CD16lo monocytes/macrophages (Mo/ processes in the urogenital system and not dependent from the Mu) present in the blisters of aged volunteers. By resolution, neu- menstrual cycle. trophils cleared from the blisters of young volunteers in a manner that This results showed that in simple EH more important progressive was proportional to Mo/Mu influx and that was associated with robust factor can be hormonal imbalance, but in complex and atypical EH increase role of inflammatory process. This inflammatory process Mo/Mu p38a/MAPK14 expression as well as prostaglandin E2 which follow EH, interpreted in our investigation such as ‘‘endome- (PGE2) synthesis, established molecular consequences of effective Mu phagocytosis of apoptotic neutrophils. However, in blisters of trial hyperplasia associated’’ or ‘‘endometrial hyperplasia related’’ aged volunteers, correlations between neutrophil clearance and Mo/ inflammation. Formation of this inflammatory changes in endometrial Mu numbers was lost being associated with a significant (threefold) hyperplasia can be considered factor of promotion and progression of pathology, as well as attributed risk factor of malignancy in reduction in PGE2. qPCR on isolated blister Mo/Mu also revealed that, in the aged, these cells fail to upregulate both cyclooxygenase-2 endometrial hyperplasia. (COX-2) and p38a/MAPK14. These data implicate a dysregulation of efferocytosis, a process which ultimately leads to a pro-resolution phenotypic switch of the Mo/Mu. We therefore speculate that a B123 dysregulated pro-resolution cascade may contribute, at least in part, to IL-1 RECEPTOR ANTAGONIST (IL-1RA) AND the syndrome of inflamm-aging. DEXAMETHASONE (DEX) THERAPY IN PATIENTS WITH SMOLDERING/INDOLENT Inflammation and Cancer MYELOMA (SMM/IMM) SHOWS IMPROVED SURVIVAL IN PATIENTS WITH A DECREASED B122 C-REACTIVE PROTEIN (CRP): INTERPLAY ROLE OF INFLAMMATION IN PROMOTION AND BETWEEN IL-1, IL-6 AND IL-17 PROGRESSION OF ENDOMETRIAL John A. Lust, Kathleen A. Donovan HYPERPLASIA Division of Hematology, Mayo Clinic, Rochester, MN, USA Anatoliy V. Kubyshkin, Evgeniia Kovalenko, Leonid Aliev, Vladimir Kubyshkin In early stage myeloma, IL-6 is a central myeloma growth factor and we have shown that abnormal production of IL-1 in the myeloma V.I.Vernadsky Crimea Federal University, Simferopol, Republic of microenvironment stimulates the generation of IL-6 in a paracrine Crimea fashion. IL-1 has also been shown to be a crucial factor in the induction of IL-17 producing T-cells in vivo. We have completed a In many non-inflammatory pathologies after some time begin devel- Phase II trial using IL-1Ra and dexamethasone, in patients with opment associated inflammatory processes. This type of inflammation smoldering/indolent MM (SMM/IMM), showing that IL-1Ra targets had secondary origin and depended from primary non-inflammatory the myeloma proliferative component which parallels a decrease in metabolic or tissue rearrangements in organs and systems. This the C-reactive protein (CRP), a surrogate for IL-6 production. Patients mechanism takes part in the development of cancer-related inflam- that had [10 % bone marrow plasma cells and/or an IgG or IgA mation (Colotta et al. 2009; Hanahan 2011). In some investigations M-spike [3 g/dL were eligible. All patients received 100 mg of described inflammation which also associated with atherosclerosis, Anakinra (IL-1Ra) SQ qd for 6 months. Patients with evidence of obesity, allergic processes, chronic kidney disease, angiopathies and reduction in M-protein levels continued receiving IL-1Ra alone. others. In our investigation role of inflammation in the development Patients with stable disease at 6 months or those with a rising of endometrial hyperplasia (EH) it was studied. M-protein before 6 months received low dose dexamethasone (20 mg

123 Inflamm. Res. S151 qweek) in addition. Data were available on 47 patients based on intent from the blood samples and the genotypes were analyzed by Real- to treat, and patients were classified as smoldering (72 %) vs. indolent Time PCR and confirmed by DNA sequencing. Allele and genotype (28 %). All 47 patients received IL-1Ra initially and 25/47 subse- frequencies were calculated by the gene counting method and the quently received IL-1Ra/Dex. Median follow-up was 7.7 years. Hardy–Weinberg equilibrium was estimated using GENEPOP 4.2. Myeloma cell growth rate (PCLI), C-reactive protein (an in vivo Results: Out of the 600 healthy individuals investigated, 228 (38 %) marker of IL-6 levels) and IL-17 were measured in patients on trial. were homozygote for the GG, 109 (18.2 %) for the AA and 263 Seven patients had a decrease in the plasma cell labeling index (PCLI) (43.8 %) were heterozygote. Out of the 145 NHL patients investigated on IL-1Ra alone which paralleled a decrease in the C-reactive protein 57 (39.3 %) were homozygote for the GG allele, 18 (12.4 %) were in all cases. Three patients achieved a minor response to IL-1Ra alone homozygote for the AA allele and 70 (48.3 %) were heterozygote. and nine patients achieved a PR/MR after addition of dexamethasone. The minor allele frequency for the A allele was 0.4008 for controls When patients were grouped into whether they exhibited a reduction and 0.365 for patients. The Hardy–Weinberg equilibrium was found to in the C-reactive protein from baseline after 6 months of therapy, the be significantly deviated (P = 0.0292) in the healthy controls whereas median PFS for patients without (22 patients) or with (25 patients) a in the patient samples was found within equilibrium (P = 0.72). greater than 40 % reduction in baseline CRP was 10.8 months vs. Conclusion: Our data shows that the allelic and genotypic frequencies 8.6 years (p \ 0.0001). Similarly, the median OS for patients without of the investigated SNP are different between the healthy and patient or with a 40 % reduction in baseline CRP was 7.9 years vs. median population. This might be an indicator that the polymorphism in this not reached (p = 0.001). Analyses of biomarkers suggested that SNP might be associated with a change in the risk to develop lym- patients with elevated IL-17 levels may be less likely to respond to phoma. Future work investigating other Ghrelin SNPs and additional IL-1Ra treatment. Sixty percent of patients without a CRP decrease statistical analysis is needed to establish a relationship between these had IL-17 levels [10 pg/mL versus 25 % of patients with a CRP SNPs and the risk to develop NHL. decrease. The median TTP in the IL-17 \10 pg/mL group was 2047 vs. 1367 days in the IL-17[10 pg/ml group. In conclusion, the above results suggest that agents such as IL-1Ra that specifically inhibit IL-1 induced paracrine IL-6 production are effective at targeting the pro- B125 liferative myeloma component and warrant further investigation in CONTROL OF LUNG CANCER WITH ASPIRIN- combination with standard myeloma therapies. Elevated IL-17 levels TRIGGERED STIMULATION OF RESOLUTION may suggest that the inflammatory process is too far advanced in some individuals to respond to IL-1 blockade, and biomarkers such as Molly M. Gilligan1,2, Megan L. Sulciner1,2, Sesquile Ramon3, Romain CRP and IL-17 may be useful to predict those patients that are most A. Colas3, Sui Huang4, Charles N. Serhan3, Dipak Panigrahy1,2 likely to benefit from anti-IL-1 therapies. 1Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Department of Pathology, Beth Israel Deaconess Medical Center, B124 3 Harvard Medical School, Boston, MA, USA; Center for ANALYSIS OF ALLELIC AND GENOTYPIC Experimental Therapeutics and Reperfusion Injury, Brigham and FREQUENCY OF GHRL-4427G [ A Women’s Hospital, Harvard Medical School, Boston, MA; 4Institute POLYMORPHISM IN A SAMPLE OF THE for Systems Biology, Seattle, WA, USA KUWAITI POPULATION AND LYMPHOMA Objective: To establish aspirin-triggered resolvins as a novel treat- PATIENTS ment to enhance current cancer therapies through the resolution of inflammation and clearance of tumor cell debris inevitably induced by chemotherapy and targeted therapy. Maryam H. Alrashid, Suzanne A. Al-Bustan, Jeethu Anu Geo Background: Inflammation in the tumor microenvironment is now recognized as a strong promoter of tumor growth. Substantial epi- Kuwait University, Kuwait City, Kuwait demiological evidence suggests that aspirin, which suppresses Analysis of allelic and genotypic frequency of GHRL-4427G [ A inflammation, also reduces the risk of cancer. However, the mecha- polymorphism in a sample of the Kuwaiti population and Lymphoma nism by which aspirin inhibits cancer remains unclear and toxicity has patients limited its clinical use. Aspirin is not only anti-thrombotic and anti- Maryam H. Alrashid, Suzanne A. Al-Bustan, Jeethu Anu Geo inflammatory, but, as more recently discovered, low-dose aspirin also Introduction: The candidate gene approach is a well-established stimulates the production of pro-resolving mediators, such as aspirin- method to study disease-associated polymorphisms in a population triggered resolvins (AT-RvDs). Resolvins are novel pro-resolution and to investigate the relationship between certain genes underlying lipid mediators derived from omega-3 polyunsaturated fatty acids and complex diseases. Ghrelin is an anti-inflammatory peptide hormone are being evaluated as effective anti-inflammatory agents in phase III that is produced and released mainly during caloric restriction and ocular human clinical trials. We recently discovered that resolvins weight loss. It has been shown that ghrelin exerts protective effects in have potent anti-tumor activity, making resolvins potential candidates various inflammatory disease models. Ghrelin SNP polymorphisms to mediate the anti-cancer activity of aspirin. Thus, we hypothesize have been reported to be associated with a change in cancer risk, that aspirin’s tumor-inhibitory effect is mediated in part by aspirin- specifically the GHRL-4427G [ A (rs1629816) with non-Hodgkins triggered (AT) resolvins via novel anti-inflammatory and pro-reso- Lymphoma (NHL). Since association of certain polymorphisms with lution mechanisms. specific conditions can be population specific, we aimed to determine Results: Aspirin-triggered resolvins inhibit cancer progression by the genotypic and allelic frequencies for rs1629816 SNP in a sample enhancing endogenous clearance through macrophage phagocytosis of Kuwaiti healthy population and lymphoma patients. of tumor debris at nanomolar concentrations. In addition, AT-re- Methods: Blood samples and relevant biostatistics and background solvins counter-regulated macrophage secretion of cytokines/ data were collected from healthy Kuwaiti volunteers from public chemokine exposed to tumor debris, including PAI-1 and IL1-ra. To Polyclinics and patients from Sheikha Badriya Al Sabah Medical abrogate AT-resolvin receptor activity, we utilized a pharmacological Oncology Center (18–75 years old, both sexes). DNA was extracted antagonist of the AT-resolvin D1 receptor (ALX/FPR-2) termed

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WRW4. WRW4 neutralized both aspirin’s anti-tumor activity and Objective: As tumor cells undergo cell death, the resulting cell debris low-dose aspirin’s stimulation of macrophage phagocytosis. contributes to an underappreciated component of the tumor Conclusions: Resolvins may provide a novel pathway and molecular microenvironment that promotes tumor progression. To stimulate the mechanism to explain how aspirin reduces cancer risk so broadly. natural debris-clearing process in breast cancer, we utilized endoge- These anti-inflammatory and pro-resolving lipid mediators may nous specialized pro-resolving mediators (SPMs), specifically complement current therapies for lung cancer with minimal resolvins and maresins, which are natural tissue products that nor- toxicity. mally function to terminate inflammation through a process called resolution. Background/methods: Current cancer therapies, including chemotherapy and targeted therapy, reduce tumor burden by killing B126 tumor cells, but as a result, create debris. Breast cancer debris may LIPOXIN A4 SELECTIVELY SHIFTS, IN VITRO stimulate tumor growth via associated inflammation and by increasing AND IN VIVO, THE PROFILE OF TUMOR- cancer stem cells. Thus chemotherapy and targeted therapy may be a ASSOCIATED MACROPHAGES, IMPAIRING THE doubled-edged sword. Therefore, enhancing clearance of tumor debris with SPMs may target novel and endogenous mechanisms that drive TUMOR GROWTH AND ANGIOGENESIS tumor growth, resistance, and dormancy escape. Maresin 1 (MaR1), a key mediator of inflammation resolution, is biosynthesized by human Christina Barja-Fidalgo, Rafael L. Simo˜es, Natalia M. Brito, macrophages from endogenous docosahexaenoic acid. We hypothe- Hayandra C. Costa, Veronica Morandi, Iolanda M. Fierro size that maresins represent a novel modality in breast cancer treatment by inhibiting tumor growth through enhancing endogenous Department of Cell Biology, IBRAG, Universidade do Estado do Rio clearance of tumor debris by macrophage phagocytosis. de Janeiro, Rio de Janeiro, Brazil Results: Flow cytometry confirmed tumor cell debris generated by chemotherapy and anti-hormone therapy. Co-injection of breast car- In tumor microenvironments, macrophages acquire anti-inflammatory cinoma debris with a sub-inoculum of viable tumor cells promotes and pro-tumor characteristics. Tumor-associated macrophages primary breast tumor growth via breast cancer stem cells and (TAMs) exhibit an M2-like profile, with low cytotoxicity and defi- inflammation. Specialized proresolving mediators (SPMs) inhibit cient production of NO and ROS. Lipoxins (LX) are lipid mediators breast cancer tumors stimulated by cell debris. Systemic administra- with anti-inflammatory and pro-resolution activities in mononuclear tion of anti-hormone therapy can stimulate tumor dormancy escape. cells. We investigated the effects of 15-epi-LX A4 on modulation of SPMs inhibit spontaneous mammary tumor growth (MMTV-PyT) in TAM in vivo and in vitro. TAMs, obtained by incubation of human a genetically engineered model of breast cancer. SPMs promote the macrophages with conditioned medium of metastatic melanoma cells, + clearance of tumor debris by stimulating macrophage phagocytosis of exhibited increasing M2 surface markers: CD206 ; high Arginase apoptotic tumor cells, and by counter-regulating critical pro-inflam- (Arg); and low iNOS expression. Treatment with LX, which shifted matory cytokines, as well as stimulating natural endogenous anti- cells from M1 (induced by LPS/IFN) to an M2-like profile, have inflammatory cytokines such as IL-1 RA. selectively decreased M2 markers in TAM, but not in M2 cells (in- Conclusions: The maresin pathway or the enhancement of endoge- duced by IL-4). LX also stimulated in vitro NO production, increasing nous resolution processes, may offer an entirely novel approach for the iNOS/arginase ratio, and activated NADPH-oxidase activity, clearing tumor cell debris inevitably induced by conventional cancer triggering ROS production. Alterations in TAM profile in vitro therapy. Our results suggest that resolution-based therapies may reduced their antiapoptotic effects and increased cytotoxicity on complement current breast cancer treatments, which inevitably pro- melanoma cells. Additionally, in a murine melanoma model, LX duce tumor cell debris. induces in vivo a significant reduction in tumor weight and volume. This effect was accompanied by a decrease in CD206 expression on the macrophages associated in vivo to the tumor. Furthermore, LX inhibited the angiogenic process activated by TAM in vitro (tubulo- genesis assay), and also diminishing, in vivo, CD105 expression in B128 tumor mass. Together, the results revealed a novel data on the effect PERITONEAL LEUKOCYTE BIOLOGY of lipoxins in cancer, showing that the pro-resolutive lipid derivatives down-modulate the tumor progression stimulated by TAM, induce the DISRUPTION DURING INFLAMMATORY shift of cells from M2- to an M1-like profile, restoring the tumoricidal OVARIAN CANCER activity of macrophages. (FAPERJ; Capes; CNPq/Brazil) James E. Riggs, Kelley DePierri, Naomi Goldman, Gretel Torres, John Somerville

B127 Biology Department, Rider University, Lawrenceville, NJ, USA CONTROLLING BREAST CANCER THROUGH The i.p. transfer of ID8 cells (mouse ovarian surface epithelial cell THE STIMULATION OF RESOLUTION carcinoma) into intact, syngeneic, C57BL/6 J (wild type, WT) mice leads to hemorrhagic ascites within 5 weeks. CD11b+Gr-1+ (granu- 1 1 2 3 Kristen A. Lehner , Megan Sulciner , Sesquile Ramon , Sui Huang , locytic) myeloid-derived suppressor cells (MDSCs) arise concomitant 2 1 Charles Serhan , Dipak Panigrahy with ascites appearance in both the spleen and peritoneal cavity. To determine if pro- (IFNc) or anti- (IL4, IL10) inflammatory cytokines 1 Center for Vascular Biology Research, Department of Pathology, are drivers of disease in this model we transferred ID8 into C57BL/6J Beth Israel Deaconess Medical Center, Harvard Medical School, mice lacking either these molecules or their receptors. Relative to WT 2 Boston, MA, USA; Center for Experimental Therapeutics and mice, the development of hemorrhagic ascites was accelerated in Reperfusion Injury, Brigham and Women’s Hospital and Harvard IL10-/-, IL4-/-, and IFNcR-/- mice. MDSC and ID8 expansion 3 Medical School, Boston, MA, USA; Institute for Systems Biology, were greatest in IL10-/- mice. ID8 also triggered a marked increase Seattle, WA, USA in PerC macrophages in the IL10-/- mice and lymphocytes in the

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IFNcR-/- and IL4-/- mice. However, the percent representation of B have antitumor activity against breast cancer also by modulating cells in the total PerC cell pool declined markedly for all mice. B cell tumor microenvironment inflammatory response. subset analysis revealed initial depletion of B-1 (CD11b+IgMhiIgDlo) Support: CNPq B cells and persistence of B-2 B cells (CD11b-IgMloIgDhi). Immu- nization with thymus-independent type 1 (TI-1, FITC-LPS) and type 2 (TI-2, FITC-Dextran) antigens revealed initial loss of the TI-2 response. Beyond the peritoneal locus of disease, both B and T Inflammation and Metabolic Disorders lymphocyte percentages dropped in the spleens of all mice. These observations reveal that inflammation disrupts leukocyte biology B130 during ovarian cancer emergence and might serve to inform strategies to develop biomarkers for this disease. Supported by NIH AREA ADIPOSE TISSUE INFLAMMATION AND R15CA173688. REMODELING DURING WEIGHT CYCLING IN MICE

Cı´ntia RP Caria, Caroline C. Oliveira, E´ rica MF Gotardo, B129 Tanila W. Dos Santos, Alessandra Gambero MODULATION OF INFLAMMATORY RESPONSE INDUCED BY INHIBITOR MOLECULES OF THE Sa˜o Francisco University, Sa˜o Paulo, Brazil KINESIN EG5 WITH PRONOUNCED ANTITUMOR Expansion of adipose tissue (AT) during obesity induces accumula- ACTIVITY AGAINST BREAST CANCER tion of macrophages and other immune cells which produce proinflammatory mediators and contribute to a local and systemic Luis Felipe F. Silva1, Rafael Correa1, Breno AD Neto2, inflammation. AT expansion also triggers events known as AT Jose´ Raimundo Correa3, Kelly G. Magalha˜es1 remodeling that involves adipocytes, immune cells infiltrated and extracellular matrix proteins. Dysfunctional remodeling events are a 1Laboratory of Immunology and Inflammation, University of Brası´lia, hallmark of obesity associated with metabolic disease. During weight Brazil; 2Laboratory of Medicinal and Technological Chemistry, loss, macrophage infiltration and local inflammation decrease, but University of Brası´lia, Brazil; 3Laboratory of Electron Microscopy, several anti-weight loss mechanisms probably acts and weight regain University of Brası´lia, Brazil occurs easily in the vast majority of individuals. We studied the inflammation in AT after weight loss and regain focusing adipokine Breast cancer is the second most common type of cancer and release and remodeling for a better understanding the anti-weight loss responsible for the death of millions of women around the world. In mechanisms. Eight-week-old male Swiss mice were fed a high-fat order to find new solutions for the treatment of this disease, our group diet (HFD; 60 % of the calories derived from fat) during 8 weeks. has previously synthesized and selected 5 new derivatives (4M, 4BT, Obese mice were subjected to moderate caloric restriction for sub- 4P, 4Bc and 4X) from 3,4-dihydropryrimididone, which is a class of sequent 8 weeks (weight loss group, WL). WL was reintroduced to inhibitory molecules of the motor spindle protein Eg5. These mole- HFD for additional 8 weeks (weight regain group, WR). Group of cules have been previously described as potent antitumor compounds age-matched obese mice was used as control (Ob16 and Ob24). against different breast cancer cells lines. To provide a wider Glucose homeostasis was evaluated by glucose blood level and understanding on the effects of these drugs, the present study aimed to insulin tolerance test (ITT). Mice were sacrificed and AT collected. characterize the modulation of inflammatory response induced by our Adipokine and matrix metalloproteinases (MMP) were quantified by newly synthesized Eg5 inhibitory compounds (EG5ICs). To address multiplex kit. Moderated caloric restriction in mice reduced final this goal, weinvestigated the effects of EG5ICs on the: (I) biogenesis body weight (63 ± 2 and 40 ± 1 g for Ob16 and WL, respectively; of cellular lipid droplets, (II) release of nitric oxide (NO), (III) p \ 0.05) and adiposity (2.5 ± 0.2 and 1.1 ± 0.1 g of epididymal AT secretion of inflammatory cytokines (IL-6 and TNF-a) and (IV) for- for Ob16 and WL, respectively; p \ 0.05). HFD reintroduction mation of Reactive Oxygen Species (ROS). RAW 264.7, mice resulted in a final body weight of 71 ± 6 and 70 ± 3 for WR and peritoneal macrophages and human monocytes cells were stimulated Ob24, respectively) and adiposity gain. Weight loss improved local or not with the lipopolysaccharide (LPS) and treated with EG5ICs adipokine production reducing leptin, monocyte chemoattractant (4M, 4BT, 4P, 4Bc and 4X) at different concentrations and time protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and periods. Monastrol, Taxol and LPS were used as assay controls. IL-6, interleukin-6 (IL-6). Although, MMP-3 expression was also reduced, TNF-a and NO were quantified in cells supernatant by ELISA and MMP-2 (4.1 ± 0.5 and 4.0 ± 1.1 ng/mL for Ob16 and WL, respec- NO assay, respectively. Lipid droplet biogenesis was assessed by flow tively) and MMP-12 (27.1 ± 5.6 and 23.6 ± 11.0 ng/mL for Ob16 cytometry and confocal microscopy. ROS generation was detected by and WL, respectively) remain elevated in AT after weight loss. Weigh flow cytometry. Our results showed that EG5ICs 4P, 4BC, 4X and regain resulted in higher levels of IL-6 (641 ± 96 and 351 ± 38 pg/ Monastrol, but not 4M and 4BT, were capable of inhibit the LPS- mL for WR and Ob24, respectively; p \ 0.05) and PAI-1 (3.6 ± 0.9 induced TNF-a secretion after 24 h of stimulation. All EG5ICs, and 1.7 ± 0.3 ng/mL for WR and Ob24, respectively;p \ 0.05). In except 4BT, decreased significantly LPS-induced IL-6 secretion after WR group, MMPs levels were not different when compared with 24 h of stimulation. Only Monastrol significantly reduced NO levels obese-matched group, but glucose blood levels (234 ± 28 and induced in cells. Lipid droplet biogenesis was decreased only in cells 195 ± 17 mg/dL of glucose for WR and Ob24, respectively, treated with EG5ICs 4X and Monastrol. All EG5ICs increased ROS p = 0.10) and insulin resistance (2.0 ± 0.3 and 3.3 ± 0.4 kITT for generation in cells after 3 h of stimulation. Taken together, our results WR and Ob24, respectively, p = 0.10) were worsened in WR group. show that most of the EG5ICs tested tend to modulate macrophages In conclusion, inflammation in AT is resolved during weight loss, but and monocytes to an anti-inflammatory profile where the most remodeling process is remained. We hypothesized that a new cycle of prominent results were seen by Monastrol, 4BC and 4X. On the other expansion when AT is in a remodeling process could worsened hand, the EG5ICs 4BT has shown a tendency to modulate the mac- inflammation establishment and metabolic alteration, resulting that rophages to the pro-inflammatory profile. Therefore, EG5ICs can weight cycles are more deleterious that obesity maintenance.

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B131 measured in culture supernatants using the AmplexRedÒ assay. Pre- SERUM ANTIBODIES TO ENTEROBACTERIAL adipocytes were differentiated into mature adipocytes with rosiglita- zone and lipid droplet accumulation, adiponectin production and LIPOPOLYSACCHARIDES AND THEIR browning (UCP1 mRNA levels) were assessed. The effect of RELATIONSHIP WITH INFLAMMATORY MB06322 (CS-917; FBP1 inhibitor; hepatocyte assay positive con- MARKERS IN DIABETES MELLITUS trol), BADGE (PPARc antagonist; adipocyte assay positive control), pioglitazone, sitagliptin, salsalate, acetylsalicylic acid, metformin, Anatoliy V. Kubyshkin, Andrey Gordienko, Vladimir Beloglazov, geldanamycin and rimonabant were evaluated. Natalia Khimich, Yuliana Shramko Results: Both hepatocyte and adipocyte assays were compatible with high throughput screening of small molecules and adenoviral RNAi V.I.Vernadsky Crimea Federal University, Simferopol, vectors. Signal/background ratio, vehicle tolerance, assay window Republic of Crimea and intra- and inter-assay variability all passed pre-set QC criteria. MB06322 and metformin inhibited gluconeogenesis with IC50 values Cardiovascular complications conditioned by the chronic intima’s that approximate literature values. Geldanamycin, rimonabant and inflammation of low intensity are considered the main reason of dis- MB06322 inhibited adiponectin production, with geldanamycin ability and mortality in diabetes mellitus (DM). Enterobacterial showing signs of cytotoxicity. Rimonabant, MB06322 and pioglita- lipopolysaccharides are regarded as the potential factor in maintenance zone modulated lipid droplet formation. Other compounds did not of low intensity chronic inflammation in DM. The laboratory study of inhibit the readouts at concentrations [ 10-fold higher than the IC50 51 patient with type 1 diabetes mellitus (DM-1) and 60 ones with type on their primary target(s). 2 diabetes mellitus (DM-2) was performed. The concentration of serum Conclusions: A panel of primary human cell-based assays allows C-reactive protein (CRP) and levels of serum antibodies to enter- evaluation of compounds targeting various molecular mechanisms, as obacterial lipopolysaccharides (anti-LPS-Ab) were determined with the potential lead molecules to treat metabolic syndrome. In the current enzyme- linked immunosorbent assay (ELISA) in both groups of the study, non-steroidal anti-inflammatory compounds such as salsalate patients. Claster analysis determined that in 40.8 % of patient with and acetylsalicylic acid that have been reported to potentially mod- DM-1 increased concentration of CRP was associated with significant ulate aspects of metabolic syndrome (insulin resistance, decrease of anti-LPS-IgA, anti-LPS-IgM and anti-LPS-IgG. In 56.7 % cardiovascular events) did not modulate the disease phenotype in of patient with DM-2 accompanied by the high concentration of CRP patient-derived primary human cell-based assays, suggesting a dif- the rise of anti-LPS-IgA and anti-LPS-IgM was not reliable, whereas ferent mode of action in vivo. the levels of anti-LPS-IgG were reliably increased. At the same time, activization of the inflammatory response with the subsequent rise of serum CRP in the patients with DM-2 was followed by the substantial B133 increase of anti-LPS-IgA and anti-LPS-IgG levels and the tendency of INFLAMMATORY MARKERS IN PATIENTS WITH anti-LPS-IgM levels decrease. Consequently, the obtained data indicate the existence of the close interactions between inflammation of low STABLE ANGINA AND DIABETES MELLITUS OF intensity and the immune response to enterobacterial lipopolysaccha- TYPE 2 rides in diabetes mellitus type 1 and 2. Ludmila I. Gapon, Tatiana I. Petelina, Natalia A. Musikhina, Irina V. Osipova, Vadim A. Kuznetsov B132 Tyumen Cardiology Center, Tyumen, Russian Federation PRIMARY HUMAN CELL-BASED MODELS FOR METABOLIC SYNDROME Objectives: To study inflammatory markers and lipid profile in group of Stable Angina (SA) patients with and without (type 2 diabetes mellitus) DM. Jeroen DeGroot1, Annelieke Strijbosch1, Folkert Verkaar1, 1 2 1 Methods: 86 patients aged 64.3 ± 6.5 years with SA and insignificant Blandine Mille-Baker , Joe Cornicelli , David F. Fischer coronary stenosis (\75 %) were examined.

1 2 Group I included 33 patients with SA and DM, group II consisted Charles River Laboratories. Leiden, the Netherlands; Charles River of 53 patients with SA without DM. All patients received statins, Laboratories. Wilmington, MA, USA ACE inhibitors, beta blockers, l antiplatelet therapy. In group I all Objectives: Metabolic syndrome is a disorder of energy utilization patients received antihyperglycemic therapy. Lipid profile parameters and storage, characterized by obesity, elevated blood pressure, plasma (total cholesterol, triglycerides, LDL cholesterol, VLDL cholesterol, glucose, triglycerides, and decreased HDL cholesterol. Metabolic lipoprotein (a), Apo-A, Apo-B), inflammatory markers (hs-CRP, syndrome increases the risk of developing diabetes and cardiovascular TNF-alpha, homocysteine, interleukine 1 b, 6, 8, sCD40 L, MMP-9, disease. Adipocytes play a pivotal role in the development of meta- TIMP-1), endothelial dysfunction markers (endothelin-1, nitrites) bolic syndrome, since they contribute to the production of circulating were measured. proinflammatory cytokines and adipokines (such as TNFa, adipo- Results: There were high levels of hs-CRP, TNF-alpha, lipoprotein nectin, resistin, and PAI-1). Hepatocytes play a key role in energy (a), MMP-9, triglycerides, and endothelin-1 in both groups. The level metabolism (storage and emergency glucose release). Because of the of TIMP-1 was significantly reduced in both groups. Patients in group complex interactions between various cell types, state-of-the art 1 had significantly elevated levels of total cholesterol, LDL choles- in vitro and in vivo models are needed that capture the disease rele- terol, homocysteine, Apo-B, Apo-B/Apo A-1 ratio, IL-1 b. In group 1 vant processes to enable the discovery and development of new the following positive correlations were found: between glycohe- disease-modifying drugs. moglobin and Apo-B, Apo-B/Apo A-1 ratio, homocysteine, IL-1 b, Methods: Human primary hepatocytes (50,000 cell/well) and pre- sCD40 L; IL-6 and hs-CRP; homocysteine and LDL cholesterol, adipocytes (4000 cells/well), both from T2D donors were cultured in MMP-9, duration of CAD; endothelin-1 and sCD40L, TNF-alpha. 96-well plates. Hepatocytes were glucose-starved and subsequently In group I the following positive correlations were found: between incubated with a gluconeogenic substrate. Glucose levels were scale of Syntax Score and hs-CRP and homocysteine level.

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Conclusion: In patients with SA and DM there was a significant Introduction: Uric acid is the main antioxidant that accumulates in increase in the levels of atherogenic lipid fractions as well as plasma (200–500 lM). Its mono-anion form, urate (pKa 5.4), chelates homocysteine, hs-CRP and IL-1 b which may indicate a higher risk of transition metals ions, reacts with hydroxyl radical, singlet oxygen coronary events even in the absence of significant coronary stenosis. and repairs protein radicals. Despite its antioxidant potential, the release of urate from dying cells initiates inflammatory response and neutrophils recruitment. Urate primes immune cells and induces cytokine production through Toll-like receptors. Here, we investi- B134 gated the effects of urate in superoxide (O2Á-), hypochlorous acid IRON SUPPLEMENTATION EFFECTS ON (HOCl) and cytokines release to understand the redox mechanisms by SYSTEMIC AND ADIPOSE TISSUE which urate modulate inflammatory response. INFLAMMATION DURING OBESITY IN MICE Methods and Results: HL-60 cells were differentiated into neutrophils (dHL-60; 5 days, 1.3 % dimethylsulfoxide) and activated with phor- bol myristate acetate (PMA; 100 ng/mL). Superoxide production was Alessandra Gambero, Cintia RP Caria, Erica MF Gotardo detected using dihydroethidium probe (DHE). The products of DHE oxidation, 2-hydroethidium (2-OH) and ethidium were separated in a Sa˜o Francisco University, Sa˜o Paulo, Brazil HPLC (column Phenomenex Synergi, 4 lm Polar-RP 80A, The conception of obesity as a chronic low systemic inflammatory 150 9 60 mm) and quantified by fluorescence (kexc 480 nm and kem condition has been recently linked to the reduction in plasma iron 580 nm). The consumption of O2 was evaluated by polarography observed in obese subjects. Visceral adipose tissue (AT) releases using Clark electrode. Hypochlorous acid was measured by taurine several pro-inflammatory cytokines, as interleukin (IL)-6 when this chloroamine-DTNB assay. Tumor necrosis factor alpha (TNF-a) was local is infiltrated by macrophages. IL-6 induces hepcidin mRNA in quantified by Enzyme-Linked Immunosorbent Assay (ELISA). At macrophages and adipocytes and hepcidin decreases iron release from low concentration (50 lM) urate decreased in 15 % the levels of •- these cells and reduces iron absorption by intestinal cells resulting in O2 . However, at a concentration of 500 lM, urate increased in 51 % Á- hypoferremia of inflammation in obese patients and experimental the levels of O2 . Urate slightly increased O2 consumption in cells models. Iron is important micronutrients but, it also is able to generate activated with PMA. The incubation with PMA increased five times dangerous hydroxyl radical in the presence of reactive oxygen species the levels of HOCl by dHL-60. Urate (0.5–2 mM) significantly (ROS) and contribute to inflammation exacerbation. In this work, we decreased HOCl concentration. The PMA also induced a hundred fold studied the effects of systemic iron supplementation to obese mice increase in TNF-a release (98.8 ± 1.2 pg/mL). Urate (2 mM) caused with hypoferremia upon adipose tissue and systemic inflammation. a slightly but significant decreased in TNF-a release (88.9 ± 3.2 pg/ Eight-week-old male Swiss mice were fed a high-fat diet (HFD; 60 % mL). of the calories derived from fat) during 24 weeks. In the last 2 weeks, Conclusion: These results demonstrate that urate could have a dual obese mice received iron citrate (IC; 50 mg/kg/week, i.m.). Age- effect in the redox balance during the inflammatory oxidative burst. If matched lean mice were used as control. Glucose homeostasis was in one hand it increased oxygen consumption and superoxide pro- evaluated by glucose blood level and insulin tolerance test (ITT). duction, on the other hand it decreased HOCl. In our model, urate Mice were sacrificed and AT collected. Serum and AT cytokines were slightly decreased TNF-a release. Additional studies are being per- quantified by multiplex kit. Iron supplementation increased iron levels formed to understand the modulatory mechanisms of urate upon improving hypoferremia (376 ± 34, 172 ± 13 and 217 ± 16 lg/dL immune cells. for IC, non-treated obese group (NT) and lean control, respectively; Financial support FAPESP (2011/18106-4, 2013/07937-8, p \ 0.05) but it did not promote alterations in insulin tolerance or 2013/02195-3), CNPq and USP. basal glucose levels altered by obesity. Iron supplementation did not modify the amount of hepcidin, adiponectin, leptin, resistin, tumor necrosis factor (TNF)-a, IL-6 or monocyte chemoattractant protein-1 B136 (MCP-1) in adipose tissue altered by obesity, but we observed a tendency to decrease local production of plasminogen activator PHYSIOLOGICALLY DIGESTED CARRAGEENAN inhibitor-1 (PAI-1; 104 ± 21 and 220 ± 76 ng/ml of PAI-1 for IC or AFFECTS INTESTINAL BARRIER FUNCTION NT, respectively). Serum levels of cytokine, including PAI-1 were not altered by iron supplementation. In conclusion, iron supplementation Lulu Fahoum, Uri Lesmes, Esther G. Meyron did not worsen adipose tissue or systemic inflammation in a model of hypoferremia associated to obesity in mice. Increased PAI-1 levels Technion-Israel Institute of Technologh, Haifa, Isreal were previously described for iron scarce or hypoxic conditions in cancer cells. PAI-1 was increased in AT during obesity and iron Background: Intestinal inflammation is becoming more frequent in supplementation had a tendency to reduced it, suggesting that intra- the western world. The dramatic increase within all ethnic groups cellular iron level or hypoxia in AT could be improved by iron supports the theory that environmental, including nutritional factors supplementation, but without modify AT inflammation. contribute to the disease development. One food additive that has been associated with the induction of gut epithelial inflammation is carrageenan (E407) (CGN), a sulfated marine polysaccharide that is widely incorporated in processed foods as a thickener, stabilizer, and B135 texturizer. The use of CGN in processed foods has been limited to high molecular weight CGN in contrast to degraded low molecular EVALUATION OF THE URIC ACID EFFECT IN weight CGN that is recognized to mediate intestinal inflammation and INFLAMMATORY RESPONSE is designated as a Group 2B carcinogen. We hypothesized that poly- disperse high molecular weight CGN permitted in foods may undergo Larissa AC Carvalho, Eliziane S. Patricio, Joa˜o PP Bonifacio, partial physiological digestion (based on a semi-dynamic in vitro Flavia C. Meotti digestion system recreating human digestion) and consequently affect the epithelium-integrity and possibly impair the physiologic intestinal Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil homeostasis.

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Objectives: Our study addresses both the functional and the mor- IL-1b but not TNF-a expression. Moreover, we found that HMW phological impacts of physiologically digested CGN on an intestinal adiponectin suppressed LPS-induced C/EBPb expression and epithelium model in order to elucidate its effects on intestinal nuclear translocation. Interestingly, contrary to expectation, inhibi- homeostasis and its potential role in the progression of inflammatory tion of NF-jB was not necessary for reduced IL-1b expression, bowel diseases. because HMW adiponectin did not lead to the significant alteration Methods and results: A Caco-2 cell model for intestinal epithelium of NF-jB reporter activity. These results suggest that HMW adi- was incubated with physiologically relevant concentrations of CGN ponectin has potent anti-inflammatory activities, as suppressor of IL- digesta. Barrier-function was analyzed by immunofluorescence and 1b expression, by inhibition of Akt-C/EBPb signaling pathway in immunoblots of subcellular fractionations of proteins relevant to the macrophages. tight-junction functionality and by evaluation of apical to basolateral transport of fluorescent dextran molecules in a transwell-system. We detected increased paracellular permeability to macromolecules and disruption of the monolayer structure. Specifically, changes in tight Inflammatory Cell Signaling junction protein zonula occludens 1 (Zo1) morphology we detected along with redistribution of the protein in the sub-cellular compart- B138 ments. The apical actin filaments were also affected by CGN showing phenotypic characteristics typically associated with stress fibers. ERYTHROPIETIN PROTECTS ENDOTHELIAL Conclusion: This study provides new evidence revisiting the safety CELL FROM HIGH GLUCOSE INDUCED INJURY evaluation of CGN by demonstrating deleterious effects of food-grade CGN after physiologic digestion, on epithelial barrier-function Yasunori Iwata, Yasuyuki Shinozaki, Haruka Yasuda, Kengo implying a possible deleterious effect of this food additive on con- Furuichi, Norihiko Sakai, Takashi Wada sumer health. Kanazawa University, Kanazawa, Japan Diabetic nephropathy (DN) is a major cause of end stage kidney B137 disease and a strong risk factor for cardiovascular diseases. High HMW ADIPONECTIN SUPPRESS LPS-INDUCED glucose induces endothelial injury in vasculature, resulting in tissue injury in diabetic condition. Chronic inflammation has been reported IL-1ß EXPRESSION THROUGH INHIBITING AKT- to play an important role for the progression of high glucose induced C/EBPß INFLAMMATORY SIGNALING IN cell injury. Growing data showed that erythropoietin (EPO) protect RAW264.7 MACROPHAGE the tissues from some kind of injury, such as hypoxia and mechanical stress. However, the contribution of EPO to high glucose induced Toshihiro Tanioka, Yoshinao Tainaga, Michiyo Yamada, Yuuri tissue injury remains to be explored. Kamimura, Mariko Suzuki, Saori Takatori, Yasuko Nakano Therefore, we hypothesized that EPO protects endothelial cells from high glucose (HG) induced injury via the regulation of inflam- School of Pharmacy, Showa University, Tokyo, Japan matory and anti-inflammatory balance. To explore this possibility, we performed genome-wide transcriptome profiling in human umbilical Adiponectin is abundant adipocytokine secreted from adipocytes, vein endothelial cells (HUVEC), which were stimulated by high which exists as trimer, hexamer, high molecular weight (HMW) glucose (HG) with/without EPO treatment and detected the expres- adiponectin and proteolytic product, globular adiponectin (gAd) in sion of inflammation associated genes. plasma. Several groups have reported that adiponectin has anti-in- Hieralchial clustering analysis showed the different pattern of flammatory effects on macrophages, however, anti-inflammatory mRNA expression in HG stimulated HUVEC with/without EPO. action of adiponectin are still largely controversial, because of the While inflammatory cytokines/chemokines mRNA expression were different sources of recombinant adipoenctin were used. Recently, increased by the HG stimulation in HUVEC, Th2 related cytokine we established a very simple and effective purification method for receptors and intracellular signaling molecules showed the reduced HMW adiponectin from human plasma. In this study, we compared mRNA expression levels. EPO treatment reduced inflammatory purified native HMW with various recombinant adiponectin to cytokines/chemokines mRNA expression and increased Th2 related investigate whether adiponectin induces anti-inflammatory or pro- cytokine mRNA expression levels. Real-time PCR analysis confirmed inflammatory action on macrophages. Pretreatment with 10 lg/mL the increased expression of inflammatory related genes, those were HMW for 16 h inhibited LPS-induced IL-1bexpression but not decreased in HG stimulated HUVEC with EPO treatment. Moreover, TNF-a expression, whereas treatment with HMW and LPS at the EPO stimulation increased mRNA expression of EPO receptor and same time showed no inhibitory effects on IL-1b expression. As b-common receptor. expected, exposure of macrophages to 10 lg/mL recombinant Taken together, EPO signaling protects high glucose induced cell human adiponectin (rhAd) expressed in CHO cells, which contains injury by the regulation of immune balance. mainly hexamer, attenuated LPS-induced IL-1b expression but not TNF-a. Unexpectedly, 10 lg/mL recombinant mouse adiponectin (rmAd) expressed in CHO cells, which contains mainly HMW, markedly inhibited bothIL-1bandTNF-a expression. Recombinant B139 human and mouse globular adiponectin (rhgAd and rmgAd), SALT-INDUCIBLE KINASES (SIK) INHIBITION IN expressed in E. Coli also significantly suppressed IL-1b expression. HUMAN MYELOID CELLS MODULATES TLR AND In contrast, these molecules showed no effect on TNF-a expression. Further analysis of the underlying molecular mechanisms revealed IL-1R SIGNALING AND INDUCES AN ANTI- that HMW adiponectin abrogated the ability of LPS to induce INFLAMMATORY PHENOTYPE phosphorylation of Akt (Ser 473). Importantly, inhibition of PI3K, as upstream molecule of Akt, by pharmacological inhibitor in Maria Stella Lombardi1,2, Corine Gillie´ron1,2, Damien Dietrich1,2, combination with adiponectin additively diminished LPS-induced Cem Gabay1,2

123 Inflamm. Res. S157

1Division of Rheumatology, Department of Internal Medicine production also modifying the expression of genes directly involved Specialties, University Hospitals of Geneva, 26 Avenue de Beau- in immune response. Macrophages play key roles in innate immune Se´jour, 1211 Geneva 14, Switzerland; 2Department of Pathology and response and have high utilization rate of amino acid glutamine Immunology, University of Geneva School of Medicine, 1 Rue Michel- (GLN), essential for energy and nitrogen supply. In addition, acti- Servet, 1211 Geneva 4, Switzerland vation of macrophages in vitro increases the transcription process and secretion of proteins, such as pro-inflammatory cytokines. This type Background: Experiments performed primarily in mouse cells showed of activation leads to an increased synthesis of mRNA, in which GLN that salt-inducible kinases (SIKs) synergize with Toll-like receptor has an important role, acting as a precursor of nitrogenous bases. In (TLR) signaling to restrict the formation of regulatory macrophages this context, GLN metabolism in macrophages is essential for the involved in the resolution of inflammation via the production of high cytokines synthesis and it is dependent on the extracellular levels of anti-inflammatory IL-10 and low levels of proinflammatory concentration. IL-12 and tumor necrosis factor (TNF-a) cytokines. Mechanistically, Considering the modulatory effects of GLN and DR, we proposed pharmacological inhibition of SIK leads to de-phosphorylation and addressing some complex aspects of immune regulation of the NF-kB nuclear translocation of CREB transcriptional co-activator (CRTC3) transcription factors, and the influence of GLN supplementation and class II histone deacetylase HDAC4, respectively. CRTC3 modulating these processes in peritoneal cells from DR mice. interacts with p-CREB to promote a gene expression program Materials and methods: We evaluated the effects of different con- including strong up-regulation of IL-10, whereas HDAC4 deacety- centration of GLN (0, 0.6, 2 or 10 mM) in vitro on TNF-a, IL-12 and lates p65-NF-kB leading to repression of pro-inflammatory cytokines. IL-10 production by peritoneal cells. The expression of NF-jB, IkB Objectives: To examine the regulation of expression and function of and their respective phosphorylated portion were evaluated by Wes- SIKs in human monocytes and monocyte-derived macrophages tern Blotting (WB), under in vitro stimulation of LPS (1.25 lg), the (MDM) and dendritic cells (MDC) in culture. source of cells were DR BALB/c mice, subjected to a DR for 10 days, Methods: We used two structurally unrelated small molecule SIK reducing their ration consumption in 30 % when compared to the inhibitors: HG-9-91-01 and ARN-3236 (Arrien Pharmaceuticals, Salt control group. Lake City, UT). Cells were pretreated 1 h with SIK inhibitors before Results: We observed that animal’s weight, Lee index, cholesterol, challenge with TLR4 (LPS) or TLR2 (Pam3CSK4) agonist or IL-1b triglycerides and glucoses serum concentrations were reduced in DR for 3–24 h. Total mRNA was isolated and gene expression deter- group. Anemia, leucopenia, peritoneal and spleen cellularity reduc- mined by RT-qPCR. Cell lysates were analysed by immunoblotting tion in animals submitted to DR was also observed. for the expression of SIK kinases family members (SIK 1-3) or using In peritoneal cells, reduction of IL-12 and TNF-a production were specific phospho-antibodies for CRTC3 and HDAC4. Cytokines observed when cultivated without GLN or supplemented with 2 or secreted in the supernatants were determined by ELISA. 10 mM of GLN in the DR group. Results: Our results demonstrated that the differentiation from periph- Peritoneal cells from DR group supplemented with 10 mM of eral blood monocytes to MDM or MDC cells induced a marked up- GLN showed increased production of IL-10 when compared with regulation of SIK protein expression. We showed that SIK inhibition cells that did not received GLN, we also observed that cells from significantly decreased proinflammatory cytokines (TNF-a, IL-6, IL-1b control group produced less IL-10 in comparison to cells from DR and IL-12p40) and increased IL-10 secretion by human myeloid cells group when both were cultivated in medium with 10 mM of GLN. stimulated with TLR2 and-4 agonists. Differently than in mouse cells, WB results showed reduced pIKBa/IKBa ratio expression in cells SIK inhibition did not enhance IL-1Ra production in human MDM. from both groups cultivated with 10 mM of GLN and compared to the Interestingly, SIK inhibition impaired proinflammatory (M1) polariza- others GLN concentrations studied. In addition, the pNFkB/NFkB tion of MDMs as assessed by the downregulation of different validated expression in cells from both groups cultivated with 2 or 10 mM markers (including CD80 and CCXL9), and induced a regulatory-like showed reduced expression when compared to cells cultivated with- M2b/c (IL-10high/IL-12low) phenotype. More importantly, we showed out GLN. Comparison between control and DR groups did not show for the first time that SIK inhibition decreases pro-inflammatory differences. cytokines secretion (TNF-a and IL-6) in human MDMs and MDCs upon In conclusion, we can infer that GLN can modulate the IKBa and IL-1R stimulation. The downstream effects observed with SIK inhibi- NF-kB expression in peritoneal cells interfering in cytokines pro- tors on cytokine modulation correlated with direct SIK targets (CRTC3 duction and increasing IL-10 production in DR mice. and HDAC4) dephosphorylation. Conclusion: Altogether our results show that SIK inhibition exerts anti-inflammatory effects in human myeloid cells and expand the therapeutic potential of SIK inhibitors for the treatment of immune- B141 mediated inflammatory diseases. EFFECT OF URIC ACID IN THE BACTERICIDAL ACTIVITY OF INNATE IMMUNE CELLS

B140 Joa˜o P. P. Bonifacio, Larissa A. C. Carvalho, Flavia C. Meotti EVALUATION OF GLUTAMINE AS MODULATOR OF THE TRANSCRIPTION FACTOR NF-KB IN University of Sa˜o Paulo, Sa˜o Paulo, Brazil MACROPHAGES FROM MICE SUBJECTED TO Introduction: Uric acid (UA) is considered an important antioxidant in DIETARY RESTRICTION human plasma and is also a pro-inflammatory agent. Urate activates inflammatory cytokine production by priming Toll-like receptors. The oxidation of urate in the inflammatory burst generates a pro-oxidant Dalila C. Oliveira, Ed W, Santos, Jackeline S. Beltran, Primavera intermediate that could modulate the killing activity of innate immune Borelli, Ricardo A. Fock cells. We have found that urate increases Pseudomonas aeruginosa 14 (PA14) survival by decreasing tumor necrosis factor alpha (TNF-a) University of Sa˜o Paulo, Sa˜o Paulo, Brazil and interleukin 1b (IL-1b) release in neutrophils and macrophages. In Dietary restriction (DR) modify the innate and adaptive immune this study, we investigate the mechanisms by which urate increases response, exerting effects on inflammatory and regulatory cytokine bacterial survival.

123 S158 Inflamm. Res.

Methods and Results: Human THP-1 and HL-60 cells were differ- composition of LD with particularly attention for presence of phos- entiated into macrophages and neutrophils by incubation with phorbol phorylated kinases. myristate acetate or 1.3 % dimethylsulfoxide, respectively. Effect of Methods and Results: For this study, HEK 293T cells were transfected urate on immune cells viability and phagocytosis was evaluated by with different constructs containing TLRs, co-receptors and PPARc flow cytometry. The interference in hypochlorous acid (HOCl) pro- genes. BCG infection was able to induce NF-kB activation, as duction was measured by taurine chloroamine-DTNB assay. Effect of observed by luciferase activity, and IL-8 production in TLR2-trans- urate (0.5 mM) in the microbicidal activity of a cell free system fected cells in the presence or absence of PPARc; whereas only in containing myeloperoxidase (MPO, 100 nM), hydrogen peroxide PPARc-transfected HEK 293T LD biogenesis was induced by BCG. (H2O2, 0.5 mM) and chloride (0.9 %) was evaluated against PA14 By western blot analysis, there was an increase expression of PPARc (107 cells). Activation of NF-jB was measured by Western blot. and FABP4, a target gene of this nuclear receptor, in response to BCG Urate did not affect immune cells viability or phagocytosis. Incuba- infection. Moreover, BCG activated the mTOR pathway in TLR2- tion of PA14 with neutrophils did not induce a significant increase in transfected HEK 293T cells, inducing the phosphorylation of mTOR, HOCl formation (3.8 ± 0.6 in neutrophils and 4.8 ± 1.9 lMin S6 kinase and 4E-BP1. BCG infection-induced mTOR pathway acti- neutrophils plus PA14). No difference was found in cells incubated vation was also observed in mouse bone marrow-derived macrophages with urate. Urate did not interfere in the bacterial killing capability of (BMDM). Treatment with rapamycin inhibited BCG-induced PPARc - the MPO/H2O2/Cl system since no growth was observed in this expression. Our next goal was to study the LD composition following system against 0.2 9 105 ± 0.0 CFU in controls. Urate significantly BCG infection in BMDM. LDs were purified by ultracentrifugation in increased HOCl formation in this cell free system from 13.11 ± 6.1 sucrose gradient; non-stimulated cells or oleic acid, a well-known LD to 38 ± 14 lM in absence or presence of urate, respectively. Incu- inducer, were used respectively as negative and positive control. bation of immune cells with PA14 activated p65 NF-jB Phosphokinases were identified by a membrane-based phosphokinase phosphorylation. The ratios for phospho-p65/b-actin and for the antibody array, followed by confirmation by western blot. BCG was inhibitor IjB-a/b-actin were 0.207, 0.38 and 0.056 O.D. and 0.408, able to trigger the compartmentalization within LD of activated 0.315 and 0.591 O.D in macrophages alone, macrophages plus PA14 members of MAPK, mTOR and Src signaling pathways. and macrophages plus PA14 plus urate. Conclusion: The above set of results shows that the Mycobacterium Conclusion:: These results present a new role for uric acid in bovis BCG can play a key role in manipulating the intracellular immune cell response in bacterial killing. The inhibitory effect of signaling system of host cells, stimulating the PPARc expression/ urate upon NF-jB would explain the decrease in TNF-a and IL-1b. activation and LD formation in a mTOR-dependent mechanism and The anti-microbicidal effect of urate is not related to a decrease in leading to the compartmentalization of important cell signaling immune cell viability, phagocytosis and oxidants production. The pathways within host LD. These effects may contribute to the escape modulation NF-jB pathway by urate might be the responsible by the mechanism of the parasites on host responses. alteration in the bactericidal activity of the immune cells. Financial Support CNPq, FAPERJ and CAPES. Financial support FAPESP (2011/18106-4, 2014/01936-2, 2013/07937-8), CNPq and USP. B143 PLATELETS MEDIATE MONOCYTE AND B142 ENDOTHELIAL CELL ACTIVATION IN DENGUE MYCOBACTERIUM BOVIS BCG ENHANCES THROUGH PLATELET-CELL INTERACTION AND PPAR EXPRESSION/ACTIVATION THROUGH c CYTOKINE SIGNALING MTOR DEPENDENT PATHWAYS AND COMPARTMENTALIZES IMPORTANT CELL Eugenio D. Hottz1,2, Juliana F. Lopes1, Gisele Barbosa-Lima1, SIGNALING ENZYMES WITHIN LIPID DROPLETS Isabel Medeiros-de-Moraes1, Andrew S. Weyrich3, Guy A. Zimmerman3, Fernando A. Bozza2, Patrı´cia T. Bozza1 Alan B. Carneiro1, Henrique NS Carvalho1, Tamyres S. Salles1, 1 2 Giovana H. Carvalho1, Miriam B. Werneck2, Joa˜o P. Viola2, Laborato´rio de Imunofarmacologia, IOC, Fiocruz, Brazil; Instituto 3 Heloı´sa D’A´ vila3, Patrı´cia T. Bozza1 Nacional de Infectologia, Fiocruz, Brazil; University of Utah, Salt Lake City, USA 1 Laborato´rio de Imunofarmacologia, Instituto Oswaldo Cruz, Dengue is the most prevalent human arbovirus disease worldwide. 2 FIOCRUZ-RJ, Brazil.; Instituto Nacional do Caˆncer, RJ, Brazil.; Dengue virus (DENV) infection may be asymptomatic or cause dis- 3 Universidade Federal de Juiz de Fora, MG, Brazil ease which intensity may vary from self-limiting febrile illness (mild Introduction: Lipid droplet (LD) is considered a dynamic cellular dengue) to life-threatening disease of bleeding and shock (severe organelle that can play several functions on cells. LD biogenesis, dengue). The pathogenesis of severe dengue is not completely composition and function may vary according to the cell and stimuli. understood, however it is known that progression to severe dengue is Recent studies indicate a remarkable action of this organelle during associated to increased immune activation with overproduction of host responses against pathogens infection. Our group already inflammatory mediators. Although thrombocytopenia and increased demonstrated that Mycobacterium bovis BCG infection induces LD vascular permeability are hallmarks of severe dengue, the role played biogenesis in macrophages through mechanisms dependent on TLR2 by platelets in dengue pathogenesis is not completely understood. and induction of PPARc expression (Almeida et al. 2009). PPARc is a Platelets, classically known as essential effectors of hemostatic nuclear receptor that plays a substantial role in the regulation of responses, are now increasingly recognized by their role in inflam- cellular differentiation, metabolism and inflammation. Furthermore, mation. Platelets can mediate immune and inflammatory responses the PPARc inhibition leads to LD biogenesis reduction together to through different mechanisms including secretion of immune medi- increased mycobacterium killing. So, the objectives of this work are ators and interaction with leucocytes. Here we investigate the to evaluate the signaling pathways induced by M. bovis BCG involved potential contribution of platelet activation in dengue pathogenesis. in PPARc expression/activation and to analyze the protein We evaluate the levels of inflammatory mediators secreted by DENV-

123 Inflamm. Res. S159 activated platelets and the ability of activated platelets to modulate Conclusion: Here, we demonstrate that insularin has an important monocyte and endothelial responses through cytokine signaling and/ effect on ischemia and reperfusion, with improvement of renal or platelet-cell interaction. We observed that platelets from dengue- function, reducing inflammation and tissue injury. infected patients and platelets exposed to DENV in vitro became Financial support Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o activated. Platelets activated by DENV in vitro secreted pro-inflam- Paulo (FAPESP 2013/11712-1), CAPES, CNPq. matory mediators including IL-1b, RANTES, VEGF and MIF. During dengue infection in patients, activated platelets formed circulating platelet-monocyte aggregates. Using a model of platelet-monocyte interaction ex vivo, we demonstrated that dengue-activated platelets B145 lead to monocyte activation with secretion of IL-1b, IL-8 and IL-10, EARLY IDENTIFICATION OF SECONDARY expression of CD80, and accumulation of lipid droplets in monocytes. BACTERIAL INFECTIONS We showed that cytokine secretion by platelet-monocyte aggregates depended on P-selectin on activated platelet. In addition, IL-10 was Praveen Papareddy, Gopinath Kasetty, Erik Malmstro¨m, specifically secreted in response to phosphatidylserine on apoptotic Johan Malmstro¨m, Heiko Herwald platelets. Blocking of MIF with the MIF antagonist Iso-1 prevented platelet-induced lipid accumulation in monocytes. We also observed Lund University, Lund, Sweden that MIF and IL-1b in conditioned medium from DENV- or thrombin- stimulated platelets were able to increase endothelial cell perme- Background: Bacterial infections are major causes of sepsis, leading ability in vitro. Our data provide new evidence that platelet driven to millions of deaths worldwide. Particularly secondary infections are cytokine and cell–cell signaling contribute to inflammatory responses emerging clinical problem that patients surviving the early phase of in dengue. sepsis are not able to eradicate the secondary infection. This group of Financial Support CNPq, FAPERJ, CAPES, PRONEX patients is at great risk to die from subsequent secondary nosocomial infections because their hyper-inflammatory state may lead to an immunosuppressive condition and a subsequent impaired immune response. The host response involves hundreds of mediators and they Inflammatory Mediators keep changing in various stages of sepsis. It is therefore unlikely that one single biomarker is able to satisfy all the needs and expectations for sepsis research and management. B144 Aim: To identify a new sepsis biomarker that can aid in early thera- INSULARIN REDUCES INFLAMMATION IN AN peutic decision-making and add information about screening, EXPERIMENTAL MODEL OF RENAL ISCHEMIA diagnosis, risk stratification, and monitoring of the response to therapy. AND REPERFUSION Methods: For better understanding the molecular mechanisms behind secondary infection processes, mice were infected intraperitoneally

1 2 2 with S. aureus, followed by an intranasal challenge with P. aerugi- Reinaldo C. Silva , Raphael J.F Felizardo , Marcos A. Cenedeze , nosa. Plasma samples were analyzed for cytokine profiling and lung ´ 2 3 Alvaro Pacheo-Silva , Maisa Della-Casa morphology was analyzed by histology and electron microscopy (SEM). 1University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Federal University of 3 Results: The primary infection in mice caused either by bacteria S. Sa˜o Paulo, Sa˜o Paulo, Brazil; Institute Butantan, Sa˜o Paulo, Brazil aureus or P. aeruginosa had no significant effect on survival. Introduction: Platelets are circulating cells that respond to vascular Whereas in mice with established primary S. aureus infection fol- disorders, adhering to endothelial substructures and also physically lowed by secondary P. aeruginosa challenge, dramatically decrease interact with other platelet, leukocytes and endothelial cells by adhe- their survival rate. Analysis of cytokines in plasma samples of pri- sion receptors expressed on their surfaces. Platelets secrete chemotactic mary infected mice displayed high levels of pro-inflammatory agents, growth factors and fibrinogen, stimulating tissue remodeling cytokines IL-6, TNF-a, MCP-1 and anti-inflammatory cytokine IL-10. after injury through mechanisms involving cell migration, proliferation SEM and histology of lung tissues of primary and secondary infected and matrix synthesis. In renal pathologies, the role of platelets is still mice demonstrated more pulmonary leakage of proteins and damage restricted to observations and associations without exposing its real of lung morphology in mice with secondary infection compared to mechanism of action. Therefore, this study aims to evaluate that pla- primary infected mice. telets participate in the process of tissue remodeling after injury, Conclusions: Here we show the host immune responses at various because of its role in adhesion and cell signaling mechanisms. stages in severe bacterial infectious. Advances gained by this Methods and Results: For this purpose, C57BL/6 mice were subjected approach, will lead to a better understanding of the molecular to ischemia and reperfusion (I/R) and treated or not with insularin mechanisms and help in developing novel antimicrobial treatments 50 lg/kg, one via the bifunctional inhibitor of integrin. The animals and tools for diagnosis. were sacrificed 24 h after ischemia, corresponding to the stabilization of renal injury. Renal dysfunction was analyzed by biochemical testing using blood samples. Serum creatinine, urea, renal tissue B146 histology and gene expression of cytokines were evaluated. Animals subjected to I/R showed an increase in renal dysfunction when DUAL FUNCTIONALITY OF ALPHA1- compared to control group, while the I/R group treated with insularin ANTITRYPSIN: AN INFLAMMATORY shows a significant reduction in serum creatinine and urea. Increased S-NITROSYLATED FORM STIMULATES gene expression of proinflammatory cytokines was also observed, as MACROPHAGES DURING BACTERIAL IL1b, TNF and IL-6 in the I/R group, with significant reduction in animals treated with insularina. Histological analysis showed less INFECTIONS tissue damage in I/R group treated with insularina and lower platelet adhesion in renal tissue. Ziv Kaner1, Galit Shahaf1, Shahar Dotan1, Rotem Engelman2, Yaffa Mizrachi Nebenzahl1, Moran Benhar2, Eli C. Lewis1 123 S160 Inflamm. Res.

1Ben-Gurion University of the Negev, Beer Sheva, Israel; 2Technion Objectives: Recent evidence suggests an excessive inflammatory Israel Institute of Technology, Haifa, Israel response in maternal obesity during pregnancy and it is associated with adverse pregnancy outcome. Whether this imbalance can be Background: Human alpha1-antitrypsin (hAAT) is a circulating ser- transferred from mother to breast milk remains to be established. ine-protease inhibitor that rises during acute phase responses and Methods: 15 lean, 15 overweight and 15 obese pregnant woman were possesses immune-modulating and tissue-protective activities. hAAT recruited in this study. Maternal blood was collected from each reduces the levels of inflammatory cytokines (e.g., TNFa, IL-1b and woman before delivery and colostrum samples were collected post- IL-6) and chemokines (KC and MCP-1) while increasing anti-in- partum in the first 48 h. Samples were analyzed for interleukin (IL)-2, flammatory proteins, such as IL-10 and IL-1Ra; nonetheless, although IL-6 and tumor necrosis factor (TNF)-a by flow cytometer and the highly consistent, these outcomes are observed primarily during sterile data were analyzed using the software FCAP Array 1.0. Leptin was immune responses. We recently reported that hAAT is responsible for measured using a high-sensitivity enzyme-linked immunosorbent an unexpected early and temporary inflammatory peak in responding assay method and C-reactive protein (CRP) were determined by innate cells and actually reduces bacterial load during peritonitis. turbidimetric method. While hAAT has no direct anti-bacterial properties, it has been shown Results: No significant variations were detected in the IL-2 concen- to be S-nitrosylated (S-NO-hAAT) upon excess nitric oxide and then trations between the different groups studied (p [ 0.05). Maternal to directly block bacterial growth. Yet there is no data regarding the serum levels of IL-6, TNF-a, leptin and CRP were significantly dif- effect of S-NO-hAAT on innate cell responses to live bacteria. ferent between the groups and obese mothers presented the highest Aim: Investigate innate cell activation in the presence of S-NO- levels (p \ 0.05). Colostrum leptin levels were higher in the obese hAAT. group versus lean group (p \ 0.05). No differences in colostrum Methods: Mice transgenic for lung-specific hAAT or strain-matched cytokines and CRP concentration were detected among the groups wild-type mice were inoculated intranasally with lethal and sublethal (p [ 0.05). doses of Streptococcus pneumonia (strain WU2) and animal survival Conclusions: Maternal obesity in pregnancy is associated with and bacterial burden were determined, respectively. In vitro, clinical- changes in cytokines and acute phase proteins of maternal blood in grade hAAT underwent chemical S-nitrosylation and then introduced the third trimester, with an increase in IL-6, TNF-a and CRP in the to cultures of peritoneal macrophages, either alone or in the presence obese category, but these changes were not observed in breast of LPS (10 ng/ml); for nitric oxide control, equimolar concentrations milk. Obese woman show elevations in serum leptin relative to of the nitric oxide donor, S-nitroso-glutathione (GSNO), were used. normal weight woman during pregnancy and in human milk in Subsequently, macrophage activation was evaluated. To assess whe- postpartum. Further investigation is needed to determine the extent ther general physical properties had changed between hAAT and to which obesity-induced changes affects maternal and infant S-NO-hAAT, their thermal stability was measured. health. Results: hAAT-transgenic mice displayed significantly reduced mortality rates and lower bacterial load. In vitro, in the absence of added LPS, hAAT reduced TNFa levels 1.9-fold, but S-NO-hAAT Table 1 Laboratory measurements according to Body Mass Index increased TNFa levels 4.3-fold, compared to untreated cells. In the Category presence of LPS, hAAT and GSNO significantly reduced TNFa levels while S-NO-hAAT did not. After 6 h of incubation, S-NO-hAAT Cytokines Samples Concentration induced transcript levels of IL-1b, TNFa, KC and IL-6, as well as iNOS, TLR2 and CD14 (ranging 1.3- to 3.6-fold relative to untreated Lean Overweight Obese cells); no significant changes were observed in IL-10 and IL-1Ra. IL-2 Colostrum 3.35 ± 1.25 1.59 ± 0.23 1.65 ± 0.14 Structurally, S-NO-hAAT was found to be significantly more heat- (pg/mL) labile than hAAT. Maternal 1.97 ± 0.71 2.0 ± 0.35 2.05 ± 0.25 Conclusions: hAAT has a context-specific dual function upon S-ni- Blood trosylation that may play a role in enhancing bacterial clearance, in IL-6 Colostrum 34.73 ± 7.68# 38.20 ± 7.07# 44.04 ± 10.28# part by acting as a macrophage-stimulating agent. Its anti-inflam- (pg/mL) Maternal 1.73 ± 0.18 2.87 ± 0.67 4.03 ± 0.76* matory properties are thus postulated to occur distal to the site of Blood infection. The mechanism behind this profound duality is unknown; it # # # is possible that nitrosylation-induced structural changes affect its TNF-a Colostrum 10.58 ± 1.77 11.67 ± 2.1 12.91 ± 2.44 interaction with binding partners, modulating immune signaling to, (pg/mL) Maternal 5.49 ± 0.70 5.86 ± 0.64 8.63 ± 0.61* ideally, minimize tissue vulnerability and maximize the performance Blood of innate cells towards a more effective host defense. Leptin Colostrum 1.70 ± 0.32# 1.94 ± 0.33# 3.24 ± 0.56*# (ng/mL) Maternal 21.60 ± 3.10 32.81 ± 3.77* 36.84 ± 1.92 Blood B147 CRP Colostrum 0.38 ± 0.12# 0.47 ± 0.13# 0.56 ± 0.18# MATERNAL OBESITY AND MARKERS OF (mg/dl) Maternal 0.91 ± 0.33 1.50 ± 0.37 8.50 ± 2.01* INFLAMMATION IN BLOOD AND HUMAN MILK Blood

Mahmi Fujimori1, Vanessa Fiorin2, Tassiane C. Morais1, Eduardo L. Franc¸a3, Adenilda C. Hono´rio-Franc¸a3, Luiz C. Abreu1,2 The results reepresent the mean and standard error of cytokines concentration of the 15 samples of colostrum and maternal blood. # 1University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2School of Medicine of * Indicates intergroup differences within each sample; and ABC, Spokane, USA; 3Federal University of Mato Grosso, Cuiaba´, indicates differences between sample (colostrum and blood) consid- Brazil ering the same cytokines.

123 Inflamm. Res. S161

B148 Thus, the cellular recruitment and its kinetic is a process to calibrate MAS RECEPTOR DEFICIENCY EXACERBATES before evaluating any drugs. In this work, we characterized the effect of different doses of zymosan and of TNF-a on the kinetic of LIPOPOLYSACCHARIDE-INDUCED recruitment of cells in the peritoneal cavity. We determined the INFLAMMATION IN THE MOUSE BRAIN functional lipidomic signature of TNF-a-induced peritonitis. Methods: Experiments were carried out by injecting 0.3; 1 and 3 mg of One´sia Cristina Oliveira Lima1, Mauro Cunha Xavier Pinto1, Johan zymosan or 0.001 mg of TNF-a in the peritoneal cavity. Inflammatory Duchene2, Michael Bader2, Natalia Alenina2, Robson Augusto Souza parameters were assessed by counting the cells in the peritoneal lavage Santos1, Juliana Carvalho Tavares1 and by identification of the leukocyte subpopulations. SPMs and other mediators issued from fatty acids were analyzed using a liquid chro- 1Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Max matography-tandem mass spectrometry (LC–MS/MS) methodology to Delbru¨ck Center for Molecular Medicine, Berlin, Germany quantitatively evaluate their production. Results: In these experiments, we initially studied the inflammatory The G protein-coupled receptor Mas is a functional binding site for kinetics by counting cells and identifying leukocyte subpopulations the angiotensin-(1–7). In the brain, the Mas receptor is expressed on over time in the peritoneal lavage after zymosan or TNF-a injection in vascular endothelium, neurons and microglia. Several evidences the peritoneal cavity. Kinetic, inflammatory peaks and the resolution supports the involvement of Mas receptor in inflammatory response, intervals were calculated and have been shown to be dependent on the however it’s role in neuroimmunological mechanisms remains to be dose of zymosan. These data may vary according to the batch of elucidated. The aim of this work was to evaluate the role of Mas zymosan. Concerning the TNF-a-induced peritonitis, we have shown receptor on brain inflammation induced by Lipopolysaccharide. a lower and sustainable recruitment from 2 to 18 h at around 2 mil- C57Bl/6 wild type (WT) and Mas deficient mice (Mas-/-), lions of leukocytes compared to the zymosan-induced peritonitis. We 8–12 weeks-old, were challenged by intra-peritoneal (i.p.) injection evaluated also the SPMs signature in the TNF-a-induced peritonitis. of Lipopolysaccharide (LPS Escherichia coli O111:B4, 5 mg/Kg). We have shown that TNF-a is able to induce production of The experiments were performed at 3 and 24 h after LPS injection. 14-HDOHE and 17-HDOHE as well as 7-Mar1. The leukocyte endothelial interaction in the brain microvasculature Conclusions: Taken together those results suggest that the kinetic and was evaluated by intravital microscopy. The phenotype of leukocyte the cellular events controlling peritonitis are key components to recruited to the brain microvasculature, as well the microglial acti- evaluate before doing any experiments on inflammation. Zymosan- or vation were evaluated by immunofluorescence. Brain expression of TNF-a-induced peritonitis are two different models of cell recruit- MCP-1; CXCL-1; CXCL2 and IL-1b were evaluated by ELISA. The ment in kinetic and intensity that allows evaluation of different type CD11b expression on bone marrow neutrophils was evaluated by of compounds with different mechanisms of action. Flow cytometry. Mas-/- mice presented significant increase in leukocyte adhesion to the brain microvasculature compared to WT, as well as, a higher number of monocytes and neutrophils recruited to the pia-mater. The higher number of adherent leukocytes to the brain B150 in Mas-/- mice was associated with increased brain expression of CXCL1, CXCL2 and MCP-1 24 h after LPS injection, as well as, DECIPHERING THE SIGNALS REQUIRED FOR increased expression of CD11b in bone marrow neutrophils from FOLLICULAR HELPER T CELL GENERATION IN -/- Mas mice. The leukocyte adhesion to brain microvasculature 3 h THE ABSENCE OF EXOGENOUS MICROBIAL after LPS injection was not associated with microglial activation, STIMULI which occurred 24 h after LPS in both groups. In conclusion, this study suggest a potential role for the Mas receptor as regulator of brain inflammatory response induced by LPS in mice, once Mas Nicolas Riteau, Sandy D. Oland, Dragana Jankovic, Alan Sher receptor deficiency seems to be related to exacerbated inflammation in LPS-challenged mice and delay the cerebral process of resolution National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA of inflammation. Follicular helper T (Tfh) cells are specialized providers of T cell help to B cells by delivering essential signals for germinal center formation and affinity maturation. Therefore, a more detailed understanding of B149 Tfh biology could aid in the design of improved vaccines based on the induction of humoral immunity. While the triggers required for Th1, PERITONITIS AS A KEY TOOL FOR Th2 or Th17 polarization are well defined, those responsible for Tfh EVALUATION OF RESOLUTION differentiation are still only partially delineated. In order to identify PHARMACOLOGY signals that could be used to promote Tfh responses, we have utilized an experimental model in which C57BL/6 or gene KO mice are Vincent Baillif, Ge´rald Cheˆne, Charlotte Guigne´, Estelle Wanecq, immunized with either peptide or protein antigen emulsified in the oil Emeline Van Goethem, Marc Dubourdeau and surfactant-only containing adjuvant IFA (Incomplete Freund’s Adjuvant). Using this immunization protocol in wild-type mice we Ambiotis SAS, Toulouse, France find that at the peak of the response in the draining lymph nodes, half of the antigen specific CD4+ T cells have differentiated into Tfh based Introduction: Inflammation is a natural process for host defense. It on their expression of the chemokine receptor CXCR5 and the lineage starts, increases and stops. The end signaling is a key and active defining transcription factor Bcl6 while little or no increase in Th1 or process, which allows the come back to homeostasis. Return to tissue Th17 cells is observed. Similar results were observed with other oil equilibrium involves many cellular and molecular factors. Immuno- and surfactant-only adjuvants. Interestingly, both the overall number inflammatory cells issued and recruited from the blood stream are key and the frequency of antigen specific Tfh were substantially decreased components for the control of inflammation. They are notably in IFA immunized MyD88-/- mice, but not in animals lacking TRIF, involved in the secretion of bioactive mediators derived from TLR4 or IL-1R signaling. In addition, while gut microbiota-derived polyunsaturated fatty acids that participate and orchestrate resolution. TLR5 triggering has recently been reported to be required for the Tfh

123 S162 Inflamm. Res. response to unadjuvanted vaccines, our results show that TLR5-/- 1Konyang University, Nonsan, South Korea; 2Chonnam National mice or orally antibiotic treated immunized mice have no impairment University, Gwangju, South Korea in Tfh differentiation. Furthermore, although STAT3 signaling is Cathelicidin-related antimicrobial peptide (CRAMP) is a murine known to be critical for the Tfh response to infections, we found only antimicrobial peptide that has various biological functions such as a minor effect on IFA triggered Tfh differentiation of conditional immune regulation, chemotaxis, and direct bacteria killing effect. In STAT3 deficiency in CD4+ T cells. Importantly, a much larger defect the present study, we sought to determine whether CRAMP has in Tfh polarization was observed in the absence of type I IFN sig- antimicrobial effect against multidrug resistant Acinetobacter bau- naling. The nature of this type I IFN requirement is currently under mannii. The gene and protein expression of CRAMP was increased in investigation but does not appear to reflect TLR3, MDA5 or STING the lung of mice infected with A. baumannii at 6 h after infection. dependent production of the cytokine. Together, our results show that Such CRAMP expression was impaired in the lungs of TLR2/4 Tfh responses can be promoted by adjuvants that lack exogenous double-deficient mice, as compared with WT mice. In vitro assay PAMPs through a pathway involving type I IFN and, paradoxically, revealed that neutrophils are a major source of CRAMP expression. In the microbial sensing related adaptor MyD88. addition, the bacterial growth was inhibited by the addition of recombinant CRAMP in a dose-dependent manner. In vivo study showed that bacterial clearance was impaired in the lungs of B151 CRAMP-deficient mice at 1 day after infection, as compared with CTX-4430, A POTENTIAL FIRST-IN-CLASS WT mice. These results suggest that CRAMP has antimicrobial effect against A. baumannii and can be a candidate for therapeutic agents. LEUKOTRIENE A4 HYDROLASE INHIBITOR FOR TREATMENT OF INFLAMMATORY DISEASES

E. Springman, R. Grosswald, L. Bhatt, T. Van, S. Ahuja Inflammatory Pain and Analgesia

Celtaxsys, Inc., Atlanta, United States B153 The Leukotriene B4 (LTB4) pathway has seen a resurgence of interest COLONY-STIMULATING FACTOR- 1 (CSF-1) AND for pharmaceutical development stemming from recent discoveries TUMOR NECROSIS FACTOR- a (TNF- a)IN linking LTB4 to a diversity of inflammatory processes, including ARTHRITIC PAIN AND DISEASE neutrophil swarming behavior, NALP3 inflammasome activation, and adipose-related inflammation. Additionally, the role of LTB4 in stimulating immune response is no longer thought to be limited to Reem A. Saleh, Derek C. Lacey, John Hamilton, Andrew Cook innate immunity, but rather to also stimulate certain facets of adaptive response. The balance between LTB4 and its anti-inflammatory Department of Medicine, Royal Melbourne Hospital, Melbourne, counterpart Lipoxin A4 (LXA4) appears critical for restoring immune Victoria, The University of Melbourne, Parkville, Victoria, Australia homeostasis during bouts of infection or inflammation. The LTB4 and Colony-stimulating factor-1 (CSF-1) is a key cytokine that has been LXA4 pathways share a common precursor in Leukotriene A4 associated with the development of arthritis in animal models. Its role (LTA4) and their production is, thus, linked in an opposing manner. in neuropathic pain has been studied by several laboratories; however, For example, when LTB4 is overproduced, LXA4 production is its potential involvement in arthritic pain has not received attention. reduced. LTAA4 Hydrolase (LTA4H) is the enzyme responsible for Tumor necrosis factor-alpha (TNF-a) is a pro-inflammatory cytokine catalyzing the rate limiting step in formation of LTB4 and, therefore, which has been implicated in the pathogenesis and progression of RA, serves as an interesting target for both reducing LTB4 production and as well as the development of arthritic pain. Thus, the objectives of potentially restoring LXA4 balance. the current study were as follows: (1) to examine the role of CSF-1 in Here we report the mechanism of action and pharmacodynamics arthritic pain using an acute mono-articular methylated bovine serum of CTX-4430, a potent and orally active inhibitor of LTA4H that is albumin (mBSA)-induced arthritis model; (2) to compare the TNF- currently undergoing clinical trials. Orally administered CTX-4430 mediated pain to the CSF-1-induced pain; and (3) to elucidate the rapidly reduces production of LTB4 in the blood and airways in mechanisms by which CSF-1 can mediate arthritic pain. animals and humans and has demonstrated therapeutic effects in Results from this study showed that systemic administration of animal models of skin, lung, vascular and CNS inflammation. Its CSF-1 can induce pain in the mBSA model and exacerbate arthritis. pharmacodynamic and pharmacokinetic profile in humans suggests an Data showed that early CSF-1-induced pain was not reversed fol- optimal dose range of 50–200 mg for once daily administration. The lowing indomethacin administration, suggesting that CSF-1-mediated rationale for ongoing and planned proof-of-concept clinical trials will pain is cyclooxygenase-independent. Systemic administration of be briefly discussed. TNF-a was also able to induce pain in the novel mBSA model and exacerbate arthritis severity. Unlike CSF-1, early TNF-mediated pain was abolished following the administration of indomethacin. This indicated that the TNF-induced pain is mediated through the pro- B152 duction of eicosanoids. In addition, indomethacin treatment had no CATHELICIDIN RELATED ANTIMICROBIAL anti-inflammatory effects on the CSF-1-mediated arthritis, whereas, PEPTIDE CONTRIBUTES TO BACTERIAL TNF-mediated arthritis was relatively reduced following indometha- CLEARANCE AT EARLY STAGE OF PULMONARY cin administration. In conclusion, both cytokines were able to induce pain; however, INFECTION WITH ACINETOBACTER each cytokine induced arthritic pain via a different pathway. Further BAUMANNII insight into the mechanisms by which CSF-1 mediates pain could determine its contribution to arthritic pain and may provide novel Seong Gak Jeon1, Jun-Young Lee2, Yu-Jin Jeong1, Min-Jung Kang2, therapeutic targets or strategies for joint pain in inflammatory diseases Jong-Hwan Park2 such as rheumatoid arthritis (RA).

123 Inflamm. Res. S163

B154 and Traditional Medicine, National Institute for Pharmaceutical LIMONENE REDUCES HYPERALGESIA INDUCED Research and Development, P.M.B. 21, Abuja, Nigeria BY GP120 AND CYTOKINES BY MODULATION OF Introduction: Arthritis is a major inflammatory disorder usually NF-JB, SOD, IL-1 b IN MICE affecting the joints and characterized by inflammation of the synovial membrane, pain and restricted joint movement. Ocimum gratissimum leaves contain polyphenolics that may be responsible for protection Candida A. L. Kassuya1, Ana Claudia Piccinelli1, Priscila N. 1 1 2 3,1 against chronic disease due to their anti-inflammatory and anti-oxi- Morato , Elisabete C. Konkiewitz , Edward B. Ziff , Julio Croda dant activities. In this study, the anti-inflammatory and anti-oxidant effects of standardized methanol extract of O gratissimum leaves was 1Federal University of Grande Dourados, College of Health Science, 2 tested using a model of carrageenan/kaolin—induced monoarthritis. Dourados, MS, Brazil; Department of Biochemistry and Molecular Methods and results: The methanol extract (ME) was obtained by Pharmacology, New York University School of Medicine, New York, 3 successive maceration of O gratissimum dried leaves first with USA; Oswaldo Cruz Foundation, Campo Grande, Brazil n-hexane and chloroform and then methanol to obtain a polyphenolic The envelope glycoprotein gp120 of HIV can play an important role enriched extract. Fingerprint of the extract was obtained using a High in the generation of pain. Intrathecal administration gp120 induces Performance Liquid Chromatographic method. Female Wistar rats the production of cytokines, including IL-1, IL-6 and TNF, which in (180–250 g) were subjected to 3 % carrageenan/3 % kaolin induced turn induces neuroinflammation and hyperalgesia. A previous study monoarthritis model. Groups were divided into vehicle, ME (100, 200 from our group demonstrated antihyperalgesic and antidepressive and 400 mg/kg, p.o for 3 days) and Indomethacin (2 mg/kg, p.o for actions of limonene administered orally in a neuropathic pain model. 3 days). One h after the last treatment, 0.1 mL of a mix of 3 % The present work has investigated the antihyperalgesic effects of carrageenan and 3 % kaolin was injected into the knee joint cavity (R)-(+)-limonene in mice that received intrathecal gp120 by ana- and the leg was flexed and extended for about 5 min. The following lyzing the roles of cytokines involved in these processes as well as parameters were then assessed; Paw volume and knee circumference, the mechanisms. Male Swiss mice (n = 6) received gp120 mechanical hyperalgesia withdrawal threshold, locomotor activity, (50–500 ng) intrathecally or sterile saline as a control. Intrathecal joint histopathology and plasma anti-oxidant levels (reduced glu- administration of gp120 increased mechanical sensitivity measured tathione, superoxide dismutase and thiobarbituric reacting with an electronic Von Frey apparatus, but not cold hypersensitivity substances). (measured by the acetone test), at 2 and 3 h after the injections. O gratissimum extract dose-dependently and significantly Limonene significantly decreased this mechanical sensitivity at 3 h (p \ 0.05) reduced swelling (as assessed by paw volume and knee after of the injection. In addition, intrathecal injection of gp120 thickness), and inflammatory pain (as assessed by mechanical increased IL-1b (measured by the ELISA test) in the serum of mice, hyperalgesia and locomotor activity). Rats injected with KC and and limonene prevented the ability of gp120 to increase this cyto- treated with vehicle alone had higher TBARS and reduced GSH and kine. Limonene also inhibited TNF and IL-1b-induced mechanical SOD levels as compared to groups treated with ME (100, 200 and hyperalgesia and IL-1b-induced cold hypersensitivity. Western blot 400 mg/kg) and Indomethacin (2 mg/kg). Similarly, the extract and assay demonstrated limonene was capable of significantly decreasing Indomethacin reduced histological signs of inflammation in the knee the expression of NF-kB while limonene increased SOD expression joint cavity. in cytoplasm of cells from spinal cord at 4 h after intrathecal IL-1b Conclusion: The standardized extract of O gratissimum leaves injection. These results demonstrate that gp120 administered exerted a beneficial effect in carrageenan/kaolin—induced intrathecally causes mechanical hyperalgesia and a peripheral monoarthritis model in rats and is probably related to its anti-in- increase in IL-1b, and that limonene inhibits this change. Also flammatory and antioxidant properties, which may be due to its limonene modulates the activation of NF-kB and SOD expression in polyphenolic content. the spinal cord after spinal IL-1b application. The ability of limonene to inhibit the mechanical hyperalgesia induced by TNF and IL-1b emphasizes the anti-inflammatory action of limonene, specifically its ability to inhibit cytokine production. B156 ANTI-INFLAMMATORY AND ANTINOCICEPTIVE EFFECTS OF NEW N-PHENYL-PYRAZOLE B155 COMPOUND ANTI-INFLAMMATORY AND ANTI-OXIDANT Daiany P. B. Silva1, Iziara F. Florentino1, Daniela C. Vinhal2, Luiz EFFECTS OF STANDARDIZED EXTRACT OF C. Cunha3, Ricardo Menegatti2, Elson A. Costa1 OCIMUM GRATISSIMUM L LEAVES IN CARRAGEENAN/KAOLIN—INDUCED 1Laboratory of Pharmacology of Natural Products, Institute of MONOARTHRITIS IN RATS Biological Sciences, Federal University of Goias, Goiaˆnia, GO, Brazil; 2Laboratory of Medicinal Pharmaceutical Chemistry, Faculty

1 1 3 of Pharmacy, Federal University of Goias, Goiaˆnia, GO, Brazil; Abayomi M. Ajayi , Benneth Ben-Azu , Samuel E. Okhale , Olusegun 3 1 Nucleus of Studies and Research Toxic Pharmacological, Faculty of G. Ademowo Pharmacy, Federal University of Goias, Goiaˆnia, GO, Brazil 1Department of Pharmacology and Therapeutics, Faculty of Basic Introduction: LQFM-102 was projected from the molecular Medical Sciences, College of Medicine, University of Ibadan, Ibadan, hybridization strategy using the analgesic acetaminophen and anti- Nigeria; 2Department of Pharmacology and Therapeutics, Faculty of inflammatory Clopirac, aims to maintain the therapeutic activities Basic Medical Sciences, College of Medicine, University of Ibadan, profile of both in the new compound created and to promote the Ibadan, Nigeria; 2Department of Pharmacology and Therapeutics, reduction of its side effects. The aim of this study is to evaluate the Faculty of Basic Medical Sciences, College of Medicine, University of anti-inflammatory and antinociceptive effects of LQFM-102, as well Ibadan, Ibadan, Nigeria; 3Department of Medicinal Plant Research as clarify the mechanisms involved.

123 S164 Inflamm. Res.

Methods: Experiments were performed using male Swiss albino mice articular injection of zymosan (Zy) (2 mg, 40 lL) in the left TMJ. In [35–40 g (n = 9). The anti-inflammatory and antinociceptive effects another series of experiments rats were treated with ZnPP-IX (3 mg/ of LQFM-102 were evaluated by methods of formalin-induced pain, kg), a specific HO-1 inhibitor, or with aminoguanidine (30 mg/kg) hot plate test, paw edema and pleurisy induced by carrageenan. All (i.p.),a selective inhibitor of nitric oxide synthase (iNOS), before AEL the experimental protocols were approved by the Research Ethics (1 mg/kg). Von Frey test was used to evaluate hypernociception (g) at Committee of the UFG (Protocol No. 182/10]. 4 h after Zy. 6 h after Zy injection it was collected synovial lavage for Results and discussion: In the first phase of formalin-induced pain test leukocyte counting and myeloperoxidase (MPO) measurement, and (0–5 min.), the treatment with LQFM-102 (75, 150 or 300 mg/kg TMJ tissue for histopathological analysis (H&E) and immunohisto- p.o.) reduced the time of reactivity to pain by 36, 48 or 43 % as chemistry for TNF-a, IL-1b, HO-1. Also TMJ periarticular tissue and compared to the control group treated with vehicle 10 mL/kg (81 s). trigeminal ganglion were removed for TNF-a and IL-1b dosage In the second phase (15–30 min.), the treatments with LQFM-102 (ELISA). Vascular permeability was evaluated by Evans Blue (75, 150 or 300 mg/kg) reduced the time of reactivity to pain by 30, extravasation measurement. Results: AEL (0.01, 0.1 or 1 mg/kg) 35 or 70 %, respectively, as compared to the control group (207 s). In increased (p \ 0.05) the nociceptive threshold (56.7 ± 1.1; the test of paw edema induced by carrageenan, treatments with 69.1 ± 2.1 or 81 ± 1.7, respectively), when compared to Zy group LQFM-102 75, 150 or 300 mg/kg (p.o.) reduced the edema by 12, 21 (43.8 ± 2.2). AEL (1 mg/kg) reduced (p \ 0.05) cell influx or 22 %, respectively in the first hour, 20, 28 or 33 %, respectively in (1183 ± 219.3), MPO activity (22.1 ± 10.7), inflammatory cell the second hour, 16, 35 or 32 % respectively in the third hour and influx in the synovial membrane (0.5 ± 0.2), and Evans Blue finally in the fourth hour treatments with LQFM-102 150 or 300 mg/ extravasation measurement (131.7 ± 3.1), compared to Zy group kg reduced the edema by 29 or 31 % as compared to the control group (37844 ± 6203; 127.5 ± 27.6; 3 ± 0.4; 166.3 ± 6.7, respectively). (Difference between the paws 136, 130, 123 and 108 lL, first, second, AEL (1 mg/kg) also reduced TNF-a and IL-1b levels in both TMJ third and fourth respectively). In the test of pleurisy induced by tissue (2.52 ± 0.07) (3.11 ± 0.49) and trigeminal ganglion carrageenan, treatment with LQFM-102 150 mg/kg p.o. reduced cell (2.52 ± 0.09) (1.55 ± 0.34), when compared to Zy group migration by 39 % compared to the control group (8.8 9 106 (7.94 ± 0.49, 11.01 ± 0.83 and 7.12 ± 0.40, 4.72 ± 0.47, respec- Leukocytes/mL) and also reduced the proteic exudation by 77 % as tively). TMJ immunohistochemical analyses showed that AEL (1 mg/ compared to the control group (Blue Evan’s concentration 18 lg/ kg) increased HO-1 and decreased TNF-a and IL-1b expression was mL). In the hot plate test, treatment with LQFM-102 150 mg/kg p.o. observed in the AEL-treated group compared to the zymosan. These did not increase the latency to thermal stimulus. The results obtained effects of AEL were not observed in the presence of ZnPP-IX, but in the formalin test suggest an antinociceptive activity that might be they were maintained with aminoguanidine. Conclusions: AEL is dependent on an anti-inflammatory activity. The edematogenic effect effective in zymosan-induced TMJ inflammatory hypernociception in in the paw edema and reduced cell migration and proteic exudation rats, and its efficacy, at least in part, depends on TNF-a and IL-1b suggest that LQFM-102 has anti-inflammatory effect. The result in inhibition and the HO-1 pathway integrity. AEL may represent a the hot plate test suggest that this compound doesn’t has a central potential therapeutic to ameliorate the inflammatory TMJ painful analgesic action. condition. Financial support CAPES ans CNPq. Funding sources FUNCAP, CNPq, CAPES, and INCT-IBISAB.

B157 B158 LECTIN FROM ABELMOSCHUS ESCULENTUS A SEMI-SYNTHETIC DERIVED FROM DECREASES LEVELS OF TNF-a AND IL-1b AND NATURALLY OCCURRING TEPHROSIA DOWN-REGULATES HEME OXYGENASE-1 ON TOXICARIA PERS. REDUCES TNFa AND IL-1b ZYMOSAN-INDUCED TEMPOROMANDIBULAR LEVELS AND ACTIVATES L, D, AND KAPPA JOINT INFLAMMATORY HYPERNOCICEPTION RECEPTORS IN THE MODEL OF FORMALIN- IN RATS INDUCED TEMPOROMANDIBULAR JOINT INFLAMMATORY HYPERNOCICEPTION IN RATS Mirna MB Brayner1, Raul S. Freitas1, Jose´ Thalles JGD Lacerda3, 3 3 Tatiane Santi-Gadelha , Carlos Alberto DA Gadelha , Vicente de Mirna MB Brayner1, Danielle RD Val2, Alice RD Freitas1, Rodrigo 1 1 1 Paulo T. Pinto , Gerardo C. Filho , Karuza MA Pereira , Gerly Anne DS Santos1, Fernanda MLDS Lopes1, Raul S. Freitas1, Maria VS 4 1 4 DC Brito , Hermany C. Freitas , Renata FDC Leita˜o , Ellen LD Teixiera1, Aldeˆnia RD Santos1, Francisca RLD Silva1, Angela MC 1 2 1 1 Assis , Danielle RD Val , Lorena V. Vieira , Hellı´ada V. Chaves Arriaga1, Cristina GD Macedo3, Simone MS Lamana3, Juliana TC Napimoga3, Maria BDS Maia2, Hellı´ada V. Chaves1 1Federal University of Ceara´, Sobral, Brazil; 2Federal University of 3 Pernambuco, Recife, Brazil; Federal University of Paraı´ba, 1Federal University of Ceara´, Ceara´, Brazil; 2Federal University of 4 Paraı´ba, Brazil; Federal University of Ceara´, Fortaleza, Brazil Pernambuco, Recife, Brazil; 3State University of Campinas, The antinociceptive and anti-inflammatory effects of a newly-dis- Campinas, Brazil covered lectin named AEL, isolated from organic Okra seeds Temporomandibular joint (TMJ) disorders are a group of conditions (Abelmoschus esculentus L Moench) (Okra), were investigated in the associated with high levels of inflammatory pain-related disability. zymosan-induced temporomandibular joint (TMJ) inflammatory The activation of opioid receptors (l, d, and j) may suppress the hypernociception in rats. Methods: Experiments were approved by the production of inflammatory neurotransmitters. Tephrosia toxicaria Institutional Animal Care and Use Committee of the Federal Pers (Tt). is a shrub that has been used in Amazonian countries tra- University of Ceara´, Fortaleza, Brazil (74/2013). Rats were pretreated ditional medicine to alleviate inflammatory pain. Recently our group i.v. with AEL (0.01, 0.1 or 1 mg/kg) or saline 30 min before the intra- demonstrated the effect of Tt in a model of inflammatory hyperalgesia

123 Inflamm. Res. S165 induced by zymosan in rats. Considering these results we developed a and inflammation induced by CGN when injected into the TMJ of semi-synthetic derived from naturally occurring Tt identified by the rats, and to pharmacologically characterize the mechanisms involved. acronym PHO. Thus, the present study was aimed at investigating the The protocol was approved by the local Ethics Committee for antinociceptive and anti-inflammatory efficacy of PHO in the model Animal Experimentation (CEUA-ICB 46, book 2/85, 2010). Under of formalin-induced TMJ inflammatory hypernociception in rats. anesthesia with inhalatory isofluorane (3 % in O2), male Wistar rats Additionally, we also investigated whether PHO efficacy involves (7 week old) received an intra-articular (i.art.) injection of 500 lgof activation of the central opioid system. Experiments were approved CGN. Four hours later, mechanical allodynia was evaluated by by the Institutional Animal Care and Use Committee of the Federal measuring the force threshold necessary forhead withdrawal with the University of Ceara´, Fortaleza, Brazil (57/2010) Rats were pretreated aid of an electronic analgesimeter based on the Von Frey filaments (per os) with PHO (0.01, 0.1 or 1 mg/kg) 60 min before formalin principle. Myeloperoxidase (MPO) activity was measured in the TMJ (1.5 %) or serotonin (225 lg) injection (intra-TMJ). Non-treated capsule tissue as a marker of neutrophil infiltration. GYY-4137 group received formalin (1.5 %) or serotonin (225 lg) injection (in- (1.25–20 lg/joint), glibenclamide (a KATP channel blocker, at 10 and tra-TMJ) and saline (per os). Behavioral nociception response was 30 lg/joint) and ODQ [1H-(1,2,4) oxadiazolo [4,3-a] quinoxalin-1- evaluated for a 45 min observation period. Periarticular tissues were one, a specific soluble guanylate cyclase inhibitor, at 0.8 and 8 lg/ collected 45 min after formalin injection to TNFa and IL-1b assays joint] were co-injected with CGN. The results were analysed by (ELISA). In other series of experiments rats were pretreated with an unpaired Student t-test or ANOVA followed by the Dunnett’s test, intrathecal injection of naloxone or CTOP [antagonist of Mu (l) when applicable. opioid receptor], naltrindole hydrochloride [antagonist of Delta (d) In comparison with saline (control group), the intra-articular opioid receptor], or Nor-Binaltorphimine [antagonist of Kappa (j) injection of CGN into the rat TMJ evoked mechanical allodynia, as opioid receptor], before PHO (0.01 mg/kg; per os) injection, and evidenced by the significant decrease in the force threshold followed by formalin (1.5 %) intra-TMJ. PHO (0.01, 0.1 or 1 mg/kg) (-31.6 ± 3.5 vs. 0.3 ± 4.0 g; p \ 0.001) and increased MPO reduced (p \ 0.05) formalin-induced hypernociception (82.4 ± 5.3; activity (26.8 ± 5.2 vs. 1.3 ± 0.3 U/joint; p \ 0.001). GYY-4137 89.7 ± 6.6; and 110.8 ± 8.4, respectively), when compared to non- significantly reduced CGN-induced mechanical allodynia in a dose- treated group (245.0 ± 14.2). PHO (0.01 mg/kg) also reduced sero- dependent manner (between 2.5 and 20 lg/joint, p \ 0.01) as well as tonin-induced hypernociception (22.2 ± 7.2), when compared to non- MPO activity (12.9 ± 4.5 vs. 26.8 ± 5.2 U/joint, p \ 0.05) at the treated group (99.0 ± 5.1). PHO (0.01 mg/kg) decreased (p \ 0.05) 2.5 lg/joint dose. Glibenclamide (30 lg/joint) prevented the both TNFa (3.3 ± 0.1) and IL-1b (5.2 ± 0.2) levels in the periar- antinociceptive effect of GYY-4137 (22.3 ± 2.9 vs. 8.8 ± 0.8 g; ticular tissue, when compared to non-treated group (6.3 ± 0.3 and p \ 0.05) while ODQ did not alter these effects. 15.1 ± 2.2, respectively). PHO was not able to reduce formalin-in- These data provide evidence on the anti-inflammatory and duced hypernociception when opioid receptors antagonists were antinociceptive effects of GYY-4137 on the CGN-induced TMJ administered. PHO effectively decreased formalin-induced TMJ synovitis in rats. Pathways involving KATP channels but not cGMP inflammatory hypernociception dependent, at least in part, upon seem to be involved in the antinociceptive effects of GYY-4137. TNFa and IL-1b inhibition and opioid receptors activation. Taking Financial support FAPESP (Grant #2014/24518-1), CNPq and into account the well-demonstrated antinociceptive and anti-inflam- CAPES. matory efficacy of PHO, the designing of alternative compounds to classical anti-inflammatory and analgesic agents is very encouraged to define new pharmacological targets for the inflammatory TMJ painful condition treatment. B160 Funding sources FACEPE, RENORBIO, CNPq, CAPES, and INCT- THERAPEUTIC EFFICACY OF FISH OIL IBISAB. CONCENTRATE ON EXPERIMENTAL MODEL OF NEUROPATHIC PAIN

B159 Ana Luisa P. Miranda, Rafaela V. Silva, Cleverton Kleiton F. Lima, ANTI-INFLAMMATORY AND ANTI- Ewerton P A Santos, Bianca W. Lobo NOCICEPTIVE EFFECTS OF GYY-4137, A SLOW- RELEASING HYDROGEN SULFIDE (H2S) DONOR, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil ON TEMPOROMANDIBULAR JOINT SYNOVITIS Introduction: Neuropathic pain (NP) is a multifactorial condition INDUCED BY CARRAGEENAN IN RATS arising from injury or malfunction of peripheral or central nervous system. The pathophysiology is characterized by strong neuro-im- mune interaction. Omega 3 polyunsaturated fatty acids such as Flavia B. Lira1, Marcos A. de Paula1, Simone A. Teixeira1, Mark eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are Wood2, Matt Whiteman2, Soraia K. Costa1, Marcelo N. Muscara1 well known for their immunomodulatory activity displayed by their endogenous conversion to resolvins and protectins as lipid mediators 1Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, (Serhan 2002). Brazil; 2University of Exeter Medical School, Exeter, USA Aim: To evaluate therapeutic efficacy of fish oil concentrate (FOC), Temporomandibular joint (TMJ) disorders are usually associated with rich in EPA and DHA, for the treatment of partial sciatic nerve inflammation and pain. We have previously demonstrated that ligation- induced (PSNL) NP in mice. hydrogen sulfide (H2S) exerts beneficial effects on nociception and Methodology: NP was induced by PSNL on the left paw (Seltzer inflammation secondary to carrageenan (CGN)-induced knee joint 1990). Two treatment protocols were employed: daily oral treatment synovitis in rats. GYY-4137 is a slow H2S-releasing compound that initiated at 5th day after surgery (therapeutic protocol) and daily oral has shown promising results as anti-inflammatory agent, although the treatment initiated prior to surgery (preventive protocol). Animals mechanisms involved have not been yet completely defined. We thus were treated with vehicle, FOC (4.6 and 2.3 g/kg) or Gabapentin decided to investigate the effects of GYY-4137 on the nociception (100 mg/kg). Thermal hypernociception and mechanical allodynia

123 S166 Inflamm. Res. were assed up to 24 h following first administration on 5th day and B162 also at 7th and 9th days after surgery. Ethics committee protocol PERSISTENCE OF INFLAMMATION ACCORDING number FARMACIA04 (CEUA-UFRJ). Results: The highest dose of FOC increased mechanical and thermal TO ENDOMYOCARDIAL BIOPSY AND CARDIAC withdrawal threshold in animals receiving daily oral administration in MAGNETIC RESONANCE AND THE LEVEL OF a therapeutic protocol initiated on 5th day after NP induction. In the CIRCULATING AUTOANTIBODIES AND first 24 h after FOC administration it was observed a decrease in CYTOKINES IN PATIENTS WITH DILATED mechanical allodynia, but not in thermal hypernociception. A statis- tically significant reduction in mechanical allodynia was observed at CARDIOMYOPATHY AND CONDUCTION 7th and 9th days post-surgery whereas thermal hypernociception was DISTURBANCES totally reversed at 9th day after PSNL, showing the therapeutic effi- cacy of FOC. In a preventive treatment protocol, beginning at the day Elena M. Gupalo, Natalia A. Mironova, Liudmila I. Buryachkovkaya, of the surgery, the highest dose of FOC prevented mechanical allo- Olga Stukalova, Tatiana Malkina, Tatiana Sharf, Evgeniy Efremov, dynia behavior induced by PSNL. Petr V. Chumachenko, Sergei Golitsyn Conclusions: Our results indicate that FOC oral treatment reverses mechanical and thermal hypersensitivity after peripheral nerve injury Russian Cardiology Research and Production Complex, Moscow, in mice in preventive and therapeutic treatment protocol. Therefore, it Russia might arise as an alternative treatment for neuropathic pain. Purpose: To evaluate the level of circulating autoantibodies and Financial support CAPES, CNPq, FAPERJ. cardiovascular magnetic resonance (CMR) data in dilated cardiomy- opathy (DCM) patients (pts) and pts with conduction disturbances and Inflammatory Processes structurally normal heart (CD). Methods: 30 pts with CD and structurally normal heart (11 male, in Cardiovascular Diseases mean age 39.3 ± 11.0) and 40 pts with DCM (18 male, mean age 40.5 ± 10.5) underwent Holter ECG monitoring with subsequent B161 CMR with assessment of myocardial edema (ME), early gadolinium EVALUATION OF THE EFFECTS ON enhancement (EGE) and late gadolinium enhancement (LGE) All DCM pts underwent EMB with PCR analyses for virus persistence. In CARDIOVASCULAR PARAMETERS BY CHRONIC serum of all pts persistence of autoantibodies to beta1-adrenergic TREATMENT WITH SELECTIVE receptor (b1-AAbs) and M2 muscarinic receptor (M2-AAbs) as well CYCLOOXYGENASE 2 INHIBITORS IN NORMAL as cytokines TNFa, IL6, TGFb1 was evaluated by ELISA. RATS Results: EMB revealed active inflammation 24 (60 %) DCM patients resolved myocarditis with huge areas of fibrosis in 8 (20 %) pts and no inflammation in 8 cases (20 %). In 16 (40 %) samples parvovirus Janetti N. Francischi, Regina MM Turchetti-Maia, Ligia F. Brenck, B19 was detected. Virus persistence in EMB samples was not Taissa IC Frade, Geovanne D. Cassali, Andrea S. Haibara associated with inflammatory cell infiltration. CMR found EGE in 10 (33.3 %) pts and ME in 6 (20 %) pts CD pts. None of CD pts Federal University of Minas Gerais, Belo Horizonte, MG, Brazil revealed LGE at CMR. 4 (13.3 %) pts had both EGE and ME which Introduction: Use of selective cyclooxgenase (COX) 2 inhibitors matched topographically in ventricular septum. The most frequent (ICOX-2) to treat inflammation and pain may be associated with an finding in DCM pts was LGE—in 19 pts (48 %), ME was found in increase in blood pressure following chronic administration. The aim 11 pts (40 %), sighs of EGE in 16 pts (40 %). Totally C2 positive of the present study was to evaluate whether celecoxib or nimesulide MRI criteria indicating myocarditis [4] were detected in 13 (32.5 %) would affect the blood pressure and the heart rate of normotensive DCM pts. TNFa was detected in 62.5 % DCM and 23.3 % CD pts rats following chronic administration. (p = 0.024), IL6 in 27.5 % DCM and 5.3 % CD pts (p = 0.016). Materials and methods: Holtzman male rats weighing (165–200 g) The increased level of TNFa and IL6 didn’t correlate with the were randomly assigned to one of the three groups: (1) control activeness of inflammation according to EMB in DCM pts. In some (n = 5), (2) nimesulide (NS; n = 8), (3) Celecoxib (CX; n = 8). CD pts increased level of these cytokines can indicate a systemic After 5 days of adaptation, all the animals were anesthetized and inflammation. CD pts demonstrated increased level of TGF-b1 (in implanted with a probe in the abdominal aorta, for collection of 44.7 % pts compared to 17.5 % in DCM group, p = 0.012). continuous blood pressure and heart rate data by telemetry. Within 10 Increased level of the main profibrotic cytokine did not correlated days of recovery, control and drug-treated animals received saline, a with fibrosis volume in DCM pts (r = 0.15). 31 (77.5 %) of DCM dose of 30 mg/kg (CX) or 5 mg/kg (NS) by gavage (in 0.1 mL/ pts have shown elevated level of b1-AAbs IgG. 18 (60 %) CD pts 100 g), divided in two halves at 8:00 and 17:00 h of the same day, revealed enhanced level of IgM to M2-AAbs, while in DCM group starting at the day zero. Experiments were developed during 30 days it was positive only in 14 (35 %) pts. The titer of M2-AAbs was in two turns (April and August 2014). Data were plotted and analyzed higher in CD group than in DCM pts (0.68 [0.49; 1.24] vs 0.55 using SigmaPlot and Sigma Stat software, respectively. [0.43; 0.86], p = 0.04). Thelevel of IgM to M2-AAbs correlated + + + + Results: Animals didn’t present any variation in blood pressure or the with the number of CD3 , CD4 , CD8 and CD68 cells in EMB heart rate all through the study. However, two animals in each drug- samples of DCM pts (r = 0.54, 0.72, 0,67 and 0,75). The amount of group died during the experiments. b1-AAbs in DCM pts correlated with number of non-sustained Conclusions: Chronic treatment with selective COX-2 inhibitors is ventricular tachycardia (VT), and volume of myocardium with LGE not, by itself, sufficient to alter the blood pressure and the heart rate in (r = 0.71, 0.61, respectively). LGE positive pts were characterized normal rats. However, death may happen in a parcel of the individuals by higher number of VPC (3643 [255; 8500] vs 659 [64; 2403] (25 %) in the chronically treated ICOX-2 groups. p = 0.04) and higher level of b1-AAbs IgG (1.16 [0.71; 2.05] vs Support: CNPq and Capes. 0.69 [0.54; 0.93], p = 0.019).

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B163 B164 A COMPLEX ATHEROSCLEROTIC MACROPHAGE LIPID LOADING LEADS TO TYPE PHENOTYPE OF MIF GENE-DEFICIENT I INTERFERON HYPORESPONSIVENESS MICE IN AN ATHEROGENIC APOE2/2 BACKGROUND UNCOVERS A NOVEL Marieke C.S. Boshuizen, Marten A. Hoeksema, Annette E. Neele, CONNECTION BETWEEN MIF Saskia van der Velden, Jan Van den Bossche, Menno P.J. de Winther AND B CELLS Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Corinna Schmitz, Omar El Bounkari, Christina Klasen, Heidi Noels, Macrophage-derived foam cells are critical components of Ju¨rgen Bernhagen atherosclerotic lesions and the ways in which the inflammatory response of foam cells influences atherogenesis is of great interest. RWTH Aachen University, Aachen, Germany Previously we demonstrated that interferon-beta (IFN-b) promotes Macrophage migration inhibitory factor (MIF) is an evolution- atherogenesis. But how IFN-b influences foam cell inflammatory arily conserved pleiotropic cytokine. Originally identified as an responses is not understood yet. IPA analysis indicated a downregu- inhibitor of random macrophage migration, MIF was recently lated interferon signaling upon monocyte and macrophage lipid found to act as a major regulator of inflammation and athero- loading. Hence we wanted to assess whether macrophage lipid genesis by displaying chemokine-like functions. This was shown loading also results in a decreased interferon response. in various experimental disease models including Mif gene To do so, we loaded bone marrow-derived macrophages overnight knockout in Ldlr-KO mice, antibody blockade of MIF in ApoE- with acLDL followed by 6 h IFN-b treatment. Surprisingly, lipid KO mice, and more recently by adenoviral knock-down of Mif in loading impaired the induction of IFN-b target genes like CCL5 and ApoE-KO mice. CXCL10. To validate these findings in vivo, LDLR-/- mice were put Pro-atherogenic effects of MIF involve amplification of plaque on normal chow (NC) or a high cholesterol diet (HCD) for 10 weeks. inflammation and destabilization, but most important are effects on Peritoneal macrophages (PEMs) were collected 4 days after intraperi- leukocyte recruitment. Upon engaging the non-cognate chemokine toneal thioglycollate administration, combined with IFN-b,IFN-c (both receptors CXCR2 and CXCR4, MIF mediates monocyte and T lym- 5000 U/mL) or PBS administration 24 and 8 h before sacrifice. This phocyte recruitment, respectively, into atherosclerotic plaques. Its diet-induced lipid loading also resulted in PEM IFN-b-hyporespon- blockade in the atherosclerotic prone ApoE-KO mouse was associated siveness, since several IFN-b target genes were again less expressed with diminished amounts of intimal immune cells and inflammatory compared to NC PEMs. In addition, ex vivo culturing of PEMs from mediators. IFN-b-treated animals on HCD versus NC showed an overall decreased Thus its broadly implication in inflammatory diseases is kind of inflammatory activity, as gene expression of inflammatory markers was clear-cut, however the role of MIF in cardiovascular diseases (CVD) reduced and secretion of IL-6, TNF and NO was decreased. This seems to be more complex as MIF-effects in acute phases of hyporesponsiveness was specific for IFN-b,asIFN-c target genes were myocardial infarctions even include cardioprotective mechanisms still induced upon IFN-c treatment, and an adequate inflammatory like metabolic activation, apoptosis suppression and antioxidative response could also still be observed ex vivo. Interestingly, similar stress. In addition peripheral neutralization of MIF by local appli- effects were observed in human primary macrophages. The mechanism cations of a blocking monoclonal antibody in ApoE-KO mice behind this hypercholesterolemia-induced hyporesponsive state showed marked impact on inflammatory markers but did not reduce remains to be discovered. Currently we assess whether the IFN-b- lesion size. induced activation of STAT2 and IRF9 is affected under lipid condi- Here we studied the functional role of MIF in experimental tions, as we found STAT1 protein expression to be unaffected. atherosclerosis in the ApoE-KO mouse model by comparing mice Altogether, we observed that macrophage lipid loading results in genetically depleted in Mif (Mif-/- ApoE-/-) with MIF-expressing hyporesponsiveness to IFN-b stimulation, in different models of foam ApoE-/- animals. cell formation. Future research will clarify whether this hypore- As expected, Mif-/- ApoE-/- mice fed a western type diet sponsiveness also affects IFN-b-mediated antiviral activity, which showed atheroprotective properties towards equally treated MIF- might have implications for obesity related disorders. expressing mice. Interestingly, significant reductions in plaque sizes This work was supported by the Dutch Heart Foundation, Grant were observed in brachiocephalic arteries and in abdominal aortas of #2010B022. Mif-/- ApoE-/- mice whereas different atherosclerotic prone regions like aortic roots or aortic arch were not affected. Analysis on plaque composition revealed no differences in infiltrated cell B165 quantities but changes in systemic immune responses could be HIGHER MYOCARDIAL INFLAMMATION IN observed. Considering the more recent recognition on the role of B cells and DCM PATIENTS SUBMITTED TO HEART adaptive immune responses by artery tertiary lymphoid organ forma- TRANSPLANT WAS ASSOCIATED WITH GOOD tion in atherosclerosis, we assume that a previously unrecognized effect PULSE THERAPY RESPONSE WITH DECREASE of MIF on B cells may account for the complex atherosclerotic phe- IN BACTERIA ANTIGEN LEVELS IN THE notype seen upon genetic Mif deletion in the ApoE-/-atherosclerosis model. MYOCARDIUM At the conference, we will present data, suggesting effects of MIF on B cell maturation and subset differentiation that may underlie this Joyce T. Kawakami, Maria de Lourdes Higuchi, Renata n. Ikegami, phenotype and that are suggestive of a novel role for MIF-mediated B Marcia M. Reis, Pablo Pomerantzeff, Sandrigo Mangini, Fernando cell regulation in atherogenesis. Bacal, Edmar Bocchi

123 S168 Inflamm. Res.

Heart Institute (InCor), University of Sa˜o Paulo Clinics Hospital, Sa˜o found in archaea. Now we searched if circulating MPS are associated Paulo, Brazil with archaeal elements in the serum of HF chagasic pts. Methods: 2 groups of sera from chagasic pts, IF (n = 8) and HF Purpose: Dilated Cardiomyopathy (DCM) has been related to bacte- (n = 7), were studied by electron microscopy (EM). Sera were cen- rial and viral infection accompanied or not by myocardial trifuged obtaining a pellet, which was processed for electron inflammation. In this paper we studied if Borrelia burgdorferi (Bb), microscopy exam embedding in EPON/Araldite resin. Immunolectron Mycoplasma pneumoniae (Mp) and myocarditis are present in DCM technique was used with primary anti-archaemetzincin-1 antibody receptor hearts (RH), and related to outcome after heart (AMZ1, Novus Biologicals) and in situ hybridization (ARCH 915 transplantation. probe), both linked with 10 nm colloidal gold. The mean number of Methods: Endomyocardial biopsies (EMBs) were studied regarding MPs and positive dots/photo inside and outside MP, of 50,0009 Bb and Mp bacterial antigens in RH, donor heart (zero time); mod- magnification of each case was obtained. erate rejection (MR) and persistent rejection (PR) by Results: The mean numbers of MPs in HF versus IF groups did not immunohistochemistry. GI (n = 5)—patients having one episode of differ. HF had higher numbers of AMZ1 immunogold positive dots MR and GII (n = 5)—patients presenting persistent MR. In RHs, we outside MPs than IF group (Table 1). The mean numbers of MPs also evaluated the CD3 Tcells/mm2. exhibited a positive correlation with numbers of archaeal DNA out- Results: The mean % area of Bb and Mp antigens in RH of GI were: side MPs in HF sera but not in IF. 7.5 and 26.6, and in GII were 9.3 and 22.6 without significant dif- Conclusion: The increased amount of AMZ1 collagenase in the sera ference. There was significant increased numbers of CD3T cells/mm2 differentiated HF chagasic patients from IF pts. The correlation in GI (18.6 ± 9.8) than in GII (4.3 ± 1.2), p \ 0.01. In GI, a positive between number of MPs vs archaea DNA extracellular in HF group correlation was observed between Bb vs Mp (r = 0.76, p = 0.23) suggests that these MPs are pathogenic archaea, releasing collage- without statistical significance (meaning a symbiotic association) and nases. Further studies may reveal if the increased amount of archaeal absence of correlation in GII (r = 0.07, p = 0.93). The mean % area AMZ1 in the serum may be biomarkers of HF. of Bb and MP in EMBs of zero time—donor, MR and after PT in GI and GII are shown in the figure. In zero time of both groups there were a large amount of Mp and few Bb, which increased during MR Table 1 Mean number/EM photo of Microparticles (MP), AMZ1 episodes. In group GII, Bb remained increasing after pulse therapy positive dots extra MPs, and Archaeal DNA positive dots (Arch (PT), and decreased in GI. DNA) extra MPs, in the pellet of sera from Heart Failure (HF) and Conclusion: Higher myocardial inflammation in DCM pts submitted Indetermiante Form (IF) of chagasic patients to HT was associated with MR regression after PT and low myocardial inflammation with persistent MR. Bb in symbiotic asso- HF IF p ciation with Mp may be related to a good PT response during episodes of MR in GI. MPs 7.5 7.3 0.48 AMZ1 79.2 15.2 0.01 Arch DNA 15.4 11.3 0.35 Correl no MPs 9 Arch DNA-r (P) 0.95 -0.370 (0.001) (0.76)

B167 INFLAMMATORY MARKERS IN PATIENTS WITH UNSTABLE ANGINA AFTER CORONARY ANGIOPLASTY AND STENTING

B166 Ludmila I. Gapon1, Natalia A. Musikhina1, Tatiana I. Petelina1, HEART FAILURE IN CHAGAS’ DISEASE MAY BE Irina V. Osipova1, Vadim A. Kuznetsov1 RELATED TO MICROPARTICLES AND 1Tyumen Cardiology Center, Tyumen, Russia ARCHAEAL ELEMENTS IN THE SERUM Aim: To study inflammatory markers and lipid profile in patients with Joyce T. Kawakami, Maria de Lourdes Higuchi, Jaqueline J. Pereira, unstable angina (UA) after coronary stenting. Barbara Ianni, Sandrigo Mangini, Renata N. Ikegami, Methods: A total of 95 patients (mean age 60.5 ± 9.5 years) with UA Marcia M. Reis, Edmar Bocchi and significant coronary artery stenosis [75 % undergoing percuta- neous coronary intervention (PCI) with drug-eluting stent placement Heart Institute (InCor), University of Sa˜o Paulo Clinics Hospital, Sa˜o were examined. The parameters were evaluated at baseline and 3, 6 and Paulo, Brazil 12 months after PCI. All patients received optimal medical treatment that included statins and dual antiplatelet therapy. Lipid profile Introduction: In Chagas’ disease, approximately 30 % of infected parameters, inflammatory markers (hs-CRP, TNF-alpha, homocysteine, individuals develop myocarditis and heart failure (HF). Antigens and interleukin 1b, 6, 8, 16; sCD40 L, MMP-9, TIMP-1); endothelial T. cruzi DNA are scarce, not explaining the intensity of myocarditis. dysfunction markers (endothelin-1, nitrites) were measured. The individuals remaining asymptomatic, are called indeterminate Results: High levels of atherogenic index, hs-CRP, TNF-alpha, MMP- form (IF). Circulating microparticles (MPs) in the serum have been 9, CD40, sCD40L, homocysteine, endothelin-1 were found initially as associated with heart failure in dilated cardiomyopathy patients (pts). well as the following positive correlations were detected: between Previously we found archaeal-like MPs in the myocardium and serum homocysteine and APO B/A-1, CD 40, MMP-9. After 3 months of HF chagasic pts. Collagenases are activated in HF, and are widely significantly elevated levels of homocysteine, MMP-9 and TIMP-1

123 Inflamm. Res. S169 and valid reduction of TNF-alpha, CD40, sCD40L, endothelin-1 were of Nuclear Medicine, University Hospital of Wu¨rzburg, Wu¨rzburg, revealed. In 6 month MMP-9, TNF-alpha, hs-CRP remained high, Germany; 4Research Center Magnetic Resonance Bavaria (MRB), however level of sCD40L was elevated. After 12 months of follow-up Wu¨rzburg; 5Department of Molecular Pathology, University Hospital we detected reduction in atherogenic index, total cholesterol, of Tu¨bingen, Tu¨bingen, Germany; 6Interdisciplinary Bank of triglycerides, MMP-9, TNF-alpha, and significant increase in the Biomaterials and Data Wu¨rzburg (ibdw), University Hospital concentration CD40. Endothelial dysfunction was also observed: the Wu¨rzburg, Germany level of endothelin-1 remained high. Objective: To non-invasively detect and characterize myocardial Conclusions: The study showed that prolonged endothelial inflam- inflammation in an experimental rat model of myocarditis. matory process is an initiating factor of atherosclerotic process Background: Myocarditis is characterized by inflammation, myocyte destabilization in patients with UA after coronary angioplasty and necrosis/apoptosis and subsequent fibrotic replacement of heart stenting. muscle. In the human, about 30 % of myocarditis-patients develop DCM. Because the clinical picture of myocarditis is multi-faceted, its diagnosis is difficult. B168 Methods: n = 150 female Lewis rats were immunized with (pig) cardiac SMALL INTESTINE MODULATES SYSTEMIC myosin (CM) emulsified in CFA, injected with heat killed B. Pertussis on days 0 and 3, and were antigen-boosted on days 7, 14, and 28. Develop- INFLAMMATION AND CARDIOVASCULAR ment of anti-myosin-antibodies was followed by ELISA and cardiac FUNCTION INDUCED BY DIETARY OXIDIZED function was surveyed by echocardiography and cardiac magnetic reso- FATTY ACIDS THROUGH nance imaging (cMRI). The development of myocardial effusion (day LYSOPHOSPHATIDYLCHOLINE PATHWAY 18–21) was visualized by cMRI. On day 21, the rat hearts were removed and consecutive 3 lm heart sections were stained with Hematoxylin/ Eosine (HE), Masson Goldner trichrome (MG), or with anti-CD68 (im- Azadeh Esmaeili mune-staining of mononuclear cells), and then compared with MRI findings (T2 and T2*/Flash sequences). On top, we then assayed nuclear David Geffen School of Medicine, University of California Los tracers such as gallium 67 citrate, gallium 68 linked to somatostatin, and Angeles, Los Angeles, CA, USA gallium 68 linked to integrin peptide (RGD) to detect cellular infiltrates in Background: Systemic inflammation induced by oxidized lipids affects ourmodel. Inparallel, apicalcardiactissues were analyzedin-depth forthe cardiovascular function. Our group has demonstrated that dietary lipids expression of pro-inflammatory and pro-fibrotic markers. can undergo oxidative modification in the GI tract and result in the Results: Sera from immunized rats strongly reacted with cardiac myosin. elevation of circulating factors including serum amyloid A (SAA). In immunized rats, echocardiography and subsequent MRI revealed large Objective: In the current study we sought to determine the role of the pericardial effusion (days 18–21). Analysis of the kinetics of myocardial small intestine in modulating systemic inflammation stimulated by infiltrates revealed maximal macrophage invasion between day 14 and dietary lipids. Methods, Male LDL receptor deficient mice (groups with 28. Histological findings correlated well with MRI-findings but strikingly n = 23) were treated with (a) Laboratory rodent chow, or (b) high fat better with autoradiograms showing an high uptake of gallium 67 citrate high cholesterol diet (Western diet) for 2 weeks. At this time the mice and gallium 68 linked to somatostatin in strongly infiltrated myocardial were fasted overnight, blood was removed from the retroorbital sinus, areas. Disappearance of macrophages resulted in replacement-fibrosis in the mice were perfused with cold saline, the intestine was removed, formerly invaded myocardial areas. This finding was confirmed by the washed with cold saline and kept at -80 °C. Lipids were extracted time-dependent differential expression of corresponding cytokines in the from plasma and from jejunum prepared for liquid chromatography- myocardium. Immunized animals reacted either with an early or a late electrospray ionization mass spectrometry to determine the level of patternofpost-inflammationfibrosis. lysophosphatidylcholine (LPC) and its oxidative derivatives. Conclusion: By applying a broad panel of biochemical, histological, Results: the level s of LPC with stearic acid at the sn-1 position did not molecular and imaging methods here we revealed that different patterns differ significantly in the plasma or in jejunum between the two groups. of reactivity may occur upon induction of myocarditis using one and the Plasma and intestinal levels of LPC with oleic, linoleic or arachidonic same rat strain. In particular, immunized Lewis rats may react either acid at the sn-1 position showed significantly higher level in the Wes- with an early or a late pattern of macrophage invasion and subsequent tern diet treated group (p \ 0.01, 0.02 and 0.01 respectively). post-inflammation fibrosis. As compared to cMRI, promising results Conclusion: Dietary fat can convert to the potent growth promoter achieved in the acute inflammatory phase of the rat hearts with gallium LPC in the intestine resulting in elevated levels of SAA that readily 67 citrate and gallium 68 linked to somatostatin will, this will stimulate has deleterious inflammatory effects on cardiovascular function. further development of nuclear strategies for the non-invasive detection of acute myocarditis, in the near future perhaps in patients.

B169 TO NON-INVASIVELY DETECT AND B170 CHARACTERIZE MYOCARDIAL CP-3(IV), A NOVEL AZAPEPTIDE THAT BINDS INFLAMMATION IN AN EXPERIMENTAL RAT SCAVENGER RECEPTOR CD36, ATTENUATES MODEL OF MYOCARDITIS INFARCT SIZE AND OXIDATIVE STRESS AND PRESERVES CARDIAC FUNCTION IN A MURINE 1 1,2 4 Priyadarshini A. Panjwani , Vale´rie Boivin-Jahns , Xavier Helluy , MODEL OF TRANSIENT MYOCARDIAL Takahiro Higuchi2,3, Yu-Xiang Ye4, Franz Kaiser3,2, Karin Klingel5, Karl-Heinz Hiller4, Peter Jakob4, Martin Lohse1, Roland Jahns1,2,6 ISCHEMIA

1 1 1 1Institute of Pharmacology and Toxicology, University of Wu¨rzburg, David N. Huynh , Vale´rie L. Bessi , Liliane Me´nard ,Je´roˆ me 1 1 2 1 Wu¨rzburg, Germany; 2Comprehensive Heart Failure Center (CHFC), Piquereau , Caroline Proulx , Maria Febbraio , William D. Lubell , 3 1 1 1 University Hospital of Wu¨rzburg, Wu¨rzburg, Germany; 3Department Andre´ C. Carpentier , Yan Burelle , Huy Ong , Sylvie Marleau

123 S170 Inflamm. Res.

1Universite de Montreal, Montreal, Canada; 2University of Alberta, thrombotic complications, but their effect on the subpopulation of Edmonton, Alberta; 3Universite de Sherbrooke, Sherbrooke, Que´bec large platelets is not as yet well-known. Methods: Sixty patients (pts) with acute coronary syndrome treated Aims: The CD36 receptor plays a pivotal role in the regulation of with primary percutaneous intervention (PCI) and dual antiplatelet energy metabolism in the heart, where it facilitates fatty acid transport therapy with clopidogrel were tested for platelet reactivity using light to the myocardium. The present study aimed to investigate the car- transmission aggregometry (spontaneous and 1.0 lM ADP aggrega- dioprotective effect of a novel azapeptide (azaPhe4) derivative, CP- tion). Platelet morphology and leukocyte-platelet aggregates (LPA) 3(iv) (Ala-D-Trp-Ala-AzaPhe-D-Phe-Lys-NH2), as a potent and were assessed by scanning electron microscopy. Mean platelet vol- selective CD36 ligand in a model of transient occlusion of the left ume (MPV) was estimated by thrombocytocrit method, serum high anterior descending coronary artery in mice. sensitive CRP (hs-CRP) level was analysed by nephelometric method. Methods: C57BL/6 mice (CD36+/+) and CD36-deficient (CD36-/-) Tests were performed 30 days and then 1 year post-PCI. mice were pretreated with a daily subcutaneous injection of CP-3(iv) Results: In all pts 30 days after PCI the levels of LPA and LRP were (289 nmol/kg) or 0.9 % NaCl vehicle for 14 days before undergoing 62.7 ± 12.4 and 6.8 ± 1.7 %, respectively, which are normally not myocardial ischaemia (30 min) and reperfusion (MI/R) (6 or 48 h) observed in healthy subjects (HS). The presence of LPA and LRP is elicited by the temporary ligation of the left anterior descending usual in pts with inflammation. In 52 % of pts the number of LRP was coronary artery, or underwent sham surgery. Myocardial infarction higher than 4 %, and that correlated with the level of hs-CRP size was determined by the Evans blue/TTC double staining method (1.3 ± 0.2 lg/l in pts with \4 % of LRP and 3.1 ± 0.4 lg/l in pts and planimetry of digital photographs. Left ventricular (LV) function with LRP [4 %). MPV was 12.6 ± 2.7 vs 8.4 ± 0.7 fl in HS and was assessed using pressure–volume conductance catheter technique. correlated with the LRP level. Spontaneous and 1.0 lM ADP LV and mitochondrial reactive oxygen species (ROS) were assessed aggregation increased. At 1 year post-PCI all parameters went back to using lucigenin-enhanced chemiluminescence and saponin-perme- normal with 91.1 % of pts. Spontaneous aggregation was retained in 4 abilized ghost muscle fibers with the fluorescent probe Amplex Red, patients, LPA went down to 12.4 ± 1.6 %, the level of LRP did not respectively. exceed 1.2 %, MPV was 8.9 ± 0.8 fl. hs-CRP level returned to nor- Results: MI/R was associated with a consistently large mean AAR to mal and constituted 0.6 ± 0.1 lg/l. total LV area of 53 ± 3 and 47 ± 2 % in vehicle-treated CD36+/ Conclusion: An inflammatory process in pts with acute coronary + and CD36-/- mice, respectively. Whereas CP-3(iv) reduced infarct syndrome treated with PCI is related to an increased level of super- area (IA) by 54 % (p \ 0.001) in CD36+/+ mice, no effect of the active platelet production. Dual antiplatelet therapy, in addition to its azapeptide was observed in CD36-/- mice. CP-3(iv) treatment antithrombotic action, may also have anti-inflammatory potential. improved myocardial haemodynamics as shown by a significant increase in stroke volume (SV), cardiac output (CO), and of the load- independent contractility parameter, PRSW, by 36 % (p \ 0.05), 40 % (p \ 0.01), and 47 % (p \ 0.001), respectively, while B172 decreasing total peripheral resistance (TPR) by 26 % (p \ 0.05) when MESENTERIC ARTERY RELAXATION TO compared to vehicle-treated MI/R group. Stimulated ROS production levels in heart and mitochondrial LV were decreased by 51 % EXOGENOUS NITRIC OXIDE ‘‘IN VITRO’’ IS (p \ 0.01) and 20 % (p \ 0.05), respectively, whereas aconitase IMPAIRED IN RATS WITH LIGATURE-INDUCED activity was increased by 33 % (p \ 0.05) at 6 h of reperfusion of PERIODONTITIS ischaemic hearts in CP-3(iv)-treated CD36+/+ mice. Conclusion: Our results show that a pretreatment with the CP-3(iv) Flavia N. de Jesus1, Elly S. do Amaral Neto1, Camila F. Wenceslau2, azapeptide analog exerts a CD36-dependent cardioprotective effect in Simone A. Teixeira1, Aline Maia-Dantas1, Gisele K. Couto2, part through attenuating myocardial oxidative stress. Our results Luciana V. Rossoni2, Luis C. Spolidorio3, Soraia K. Costa1, support the potential application of azapeptide derivatives targeting Marcelo N. Muscara1 CD36 for cardioprotection. 1Department of Pharmacology, ICB University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2University of Sa˜o Paulo, Sa˜o Paulo, Brazil; B171 3State University of Sa˜o Paulo, Sa˜o Paulo, Brazil THE LEVEL OF CIRCULATING LARGE We have previously shown that periodontal disease has some nitric RETICULATED PLATELETS AND oxide (NO)-mediated remote effects on rat heart and kidney, as well INFLAMMATION IN PATIENTS WITH ACUTE as aorta endothelial dysfunction and decreased adrenergic contrac- tililty. We thus decided to study the effects of periodontitis on the CORONARY SYNDROME TREATED WITH mesenteric resistance artery. PRIMARY PERCUTANEOUS INTERVENTION The protocol was approved by the Ethics Committee for Animal Experimentation (CEUA-ICB 170, book 2/113, 2011). Cotton liga- Nikita V. Lomakin1, Ludmila I. Buryachkovskaya2, tures were subgingivally placed around both lower first molars of Alexander B. Sumarokov2, Irina A. Uchitel2 male adult Wistar rats; sham animals had the ligatures immediately removed. Seven days later, the animals were euthanized and the 1Central Clinical Hospital of the Presidential Administration of vessels (3rd. order mesenteric artery branches) were isolated and Russian Federation, Moscow, Russia; 2Russian Cardiology Research mounted on a myograph for evaluation of the in vitro responses to Complex, Moscow, Russia acetylcholine (Ach), phenylephrine (Phe), sodium nitroprusside (SNP) and sildenafil (Sil). From the concentration–response curves, Background: Platelets represent an important link between inflam- potency (pD2) and maximal response (Emax) values were calculated. mation, thrombosis and atherogenesis. Mediators of inflammation The vessels were also analysed for NOS isoenzyme gene expression affect megakaryocytes, thereby causing generation and release into and activity of the antioxidant enzymes superoxide dismutase (SOD) blood of large reticulated platelets (LRPs) with high thrombogenic and catalase (CAT). The results were analysed by unpaired Student activity. Antiplatelet drugs are widely used to reduce the risk of t-test.

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Alveolar bone loss was observed in the animals with ligature, as to Nfkbiz+/+ hearts at 4 weeks after TAC. Western blot analysis bilaterally measured at the first and second molar levels. No differ- showed that the phosphorylation levels of Akt, which can be activated ences were observed between the groups in terms of vascular by IL-1b and contributes to cardiac adaptive mechanisms, were responses to Phe or ACh. However, SNP-induced relaxation was less higher in Nfkbiz+/- hearts than in Nfkbiz+/+ hearts at 4 weeks after potent in the ligature group (pD2: 6.4 ± 0.2 vs. 7.1 ± 0.1; p \ 0.01), TAC. Consistently, mRNA expression levels of suppressor of cyto- similarly to Sil (pD2: 8.6 ± 0.2 vs. 10.3 ± 0.2; p \ 0.001), but kine signaling 3 (SOCS3), that is induced by chronic gp130 signaling efficacies (Emax) were unaltered. Decreased SOD activity and suppresses Akt phosphorylation, were higher in Nfkbiz+/+ hearts (0.7 ± 0.05 vs. 0.4 ± 0.05; p \ 0.01) and increased iNOS mRNA that in Nfkbiz+/- hearts. In vitro experiments demonstrated that (1.0 ± 0.1 vs. 20.9 ± 5.9; p \ 0.01) and CAT activity (2.3 ± 0.2 pharmacological activation of the NF-jB system induced apoptosis of vs.3.2 ± 0.3; p \ 0.05) were found in the arteries from rats with cardiomyocytes and proliferation of cardiac fibroblasts, which were periodontitis. suppressed by IjBf knockdown. With basis on the results above, we conclude that during the early Conclusions: Our results suggest that IjBf regulates the transition phase of ligature-induced periodontitis in rats, functional changes from adaptive cardiac hypertrophy to heart failure during pressure related to the NO-cyclic GMP pathway occur in the mesenteric artery overload, possibly in part, through the modulation of proinflammatory smooth muscle, which may be secondary to the excessive production cytokine expression pattern in the heart, which may regulate the of iNOS-derived NO, superoxide anion or their combination. balance between adaptive and maladaptive signaling pathways. Thus, Financial support FAPESP (Grant #2011/17800-4), CAPES, IjBf may be a novel therapeutic target for treating heart failure. CNPQ.

B174 B173 DRUG A SUPPRESSES INFLAMMATORY THE INDUCIBLE NUCLEAR PROTEIN IjBf RESPONSES INDUCED BY REGULATES THE TRANSITION FROM ADAPTIVE 27-HYDROXYCHOLESTREOL CARDIAC HYPERTROPHY TO HEART FAILURE DURING PRESSURE OVERLOAD Bo Young Kim, Koanhoi Kim

Yasutomi Higashikuni1, Daiju Fukuda2, Masataka Sata3, Pusan National University, Busan, South Korea 1 Issei Komuro Atherosclerosis is an inflammatory disease that is characterized by the infiltration of immune cells into the artery wall, followed by the 1 Department of Cardiovascular Medicine, The University of Tokyo, development of atherosclerotic plaques. We reported that 27-hy- 2 Tokyo, Japan; Department of Cardio Metabolic Medicine, The droxycholesterol (27OHChol) detected in abundance in 3 University of Tokushima, Tokushima, Japan; Department of atherosclerotic lesions could induce inflammation by activating Cardiovascular Medicine, The University of Tokushima, Tokushima, monocytic cells. In the current study, we sought to find drugs or Japan chemicals that influence inflammatory responses induced by Background: Inflammation has been implicated in the pathophysiol- 27OHChol. Treatment of THP-1 cells with 27OHChol resulted in ogy of heart disease. However, the roles and the regulatory significant increase in expression of inflammatory chemokines like mechanisms of inflammation in the heart remain largely unknown. It CCL2, CCL3, and CCL4, and the increase was does-dependently has been reported that the NF-jB system contributes to inflammatory inhibited in the presence of drug A, but not of aspirin. Also, drug A responses in the heart and pathological cardiac hypertrophy during attenuated the expression of MMP-9 induced by 27OHChol. Condi- pressure overload. The inducible nuclear protein IjBf, encoded by the tioned media isolated from 27OHChol-treated THP-1 cells, which NFKBIZ gene, regulates proinflammatory cytokine expression pattern contained a high amount of the ligands, enhanced migration of by modulating the NF-jB system. This study was performed to clarify monocytic cells and Jurkat T cells expressing CCR5, a characteristic the role of IjBf in heart disease. chemokine receptor of Th1 subtype. Addition of drug A resulted in Methods and results: Pressure overload was induced in mice by complete inhibition of the migration of both types of cells. We pro- transverse aortic constriction (TAC). TAC-operated Nfkbiz+/+ hearts pose that drug A can be used for the treatment of inflammatory showed adaptive cardiac hypertrophy until 2 weeks after the opera- responses in atherosclerotic lesions. tion, whereas at 4 weeks after TAC, Nfkbiz+/+ mice fell into heart failure with impaired systolic function and left ventricular dilatation. The failing hearts showed higher mRNA expression levels of IjBf with higher interleukin-6 (IL-6) to IL-1b expression ratios compared Inflammatory Processes in Central to the hearts with adaptive hypertrophy. Nfkbiz+/- mice showed similar adaptive cardiac hypertrophy to Nfkbiz+/+ mice until 2 weeks Nervous System Diseases after TAC. However, at 4 weeks after TAC, Nfkbiz+/- hearts showed preserved systolic function with attenuated LV dilatation compared B175 +/+ with Nfkbiz hearts. The bone marrow transplantation experiment MODULATION OF IMMUNE-INDUCED AND revealed that IjBf expressed in the heart, but not in bone marrow- derived cells, contributes to cardiac phenotype during pressure TRAUMATIC CNS INJURY BY DENDRITIC overload. Histological assessment showed attenuated interstitial CELLS’ EXPRESSION OF CEBPD, A NOVEL fibrosis and macrophage infiltration and higher capillary density in THERAPEUTIC TARGET Nfkbiz+/- hearts compared with those in Nfkbiz+/+ hearts at 4 weeks +/+ after TAC, whereas cardiomyocyte hypertrophy in Nfkbiz and 1 1 +/-- Masoud Hassanpour Golakani , Mohammad G. Mohammad , Nfkbiz mice was comparable. Real-time PCR revealed that IL-6 to 1 1 2 1 +/- Vicky W. Tsai , Hui Li , Marc Ruitenberg , Samuel N. Breit , IL-1b expression ratios remained lower in Nfkbiz hearts compared Paul E. Sawchenko3, David A. Brown1

123 S172 Inflamm. Res.

1Laboratory of Neuroinflammation, St Vincent’s Centre for Applied FACS, and cytokines (IL1b, TGF) secreted in the culture media was Medical Research and University of New South Wales, Sydney, NSW, measured by ELISA. Control cells were incubated with cultured Australia; 2Queensland Brain Institute, The University of media. Queensland, Brisbane, Queensland, Australia; 3Laboratory of Results: Incubation of BV2 cells with LPS increased TSPO expres- Neuronal Structure and Function, The Salk Institute for Biological sion (70 % vs control); ANXA1 per se did not alter the TSPO Studies, La Jolla, CA, USA expression. Co-incubation with ANXA1 (10 or 100 ng/mL) inhibited the LPS-induced (10 ng/mL) expression of TSPO after 4 and 12 h CCAAT/enhancer binding protein delta (CEBPD) is a transcription (53.5 % or 50.42 %); effect was not detected with 100 ng/mL LPS. factor that modulates many biological processes including central Moreover, ANXA1 treatment reduced the LPS-induced IL-1b nervous system (CNS) injuries. However, the cellular machanisms (86,9 %) and inhibited reduction of TGF secretion evoked by LPS. remain largely unknown. We aimed to determine the mechanism of Discussion: TSPO is expressed by LPS stimulation in BV2 cells and action of CEBPD in immune-mediated and traumatic spinal cord this effect is modulated by ANXA1. In addition, ANXA1 seems to injury (SCI). For immune-induced SCI we used a mouse model of inhibit the BV2 LPS-induced polarization. The role of TSPO in this multiple sclerosis (MS), experimental autoimmune encephalomylitis latter effect has been investigated. (EAE), which is actively induced with myelin oligodendrocyte gly- Agency: FAPESP (2013/25903-3 fellowship grant; Financial support coprotein (MOG). Our findings indicate that mice deficient in CEBPD 2014/07328-4) had less sever EAE. This was associated with a significant reduction of ratios of pro-inflammatory T helper (Th) 17 cells to anti-inflam- matory regulatory T-cells (Tregs) in an interleukin-10 (IL-10)- dependant manner. We have showed that this is mediated by CD11c+ B177 dendritic cells (DCs). Bone marrow chimeric mouse experiments EFFECTS OF GP120 GLYCOPROTEIN INJECTION showed that peripherally-derived immune cells mediated the benefi- AT NUCLEUS ACCUMBENS IN RODENT cial effects of reduced CEBPD expression in EAE. Examination of CD11c+ DCs mediated Th-cell development in vitro and in vivo Elisabete C. Konkiewitz1, Ana C. Piccinelli1, Priscila N. Morato1, suggested that CEBPD modulated interleukin-10 (IL-10) expression Jose´ A. Pochapski2, Edmar Miyoshi2, Edward B. Ziff2, in DCs. Direct examination confirmed that the changes mediated by Caˆndida L. Kassuya1 reduced CEBPD expression was mediated by IL-10 secretion both in vitro and in vivo. The inhibition of IL-10 actions by a specific anti- 1Universidade Federal da Grande Dourados (UFGD), Dourados, IL-10 receptor antibody treatment reversed the effect of absent DC- Brazil; 2New York University, New York City, NY, USA; CEBPD both in vitro and in vivo. Extending these findings to a model 2Universidade Estadual de Ponta Grossa (UEPG), Ponta Grossa, of traumatic spinal cord injury, we found that CEBPD knockout mice Brazil display significantly augmented locomotor recovery after contusive spinal cord injury compared to wild-type mice. These findings iden- HIV infection affects the central nervous system causing depression tify CEBPD as an important DC transcription factor modulating CNS and behavioral and motor disorders. The gp120 protein plays a role in injury from a variety of causes and suggest it as a novel potential these effects of HIV infection with its ability to induce neuroin- therapeutic target in CNS injuries. flammation, which may lead to apoptotic cell death. The Nucleus accumbens is a brain region that functions in behavioral and emo- tional regulation. Lesions to this brain region may result in prominent symptoms of apathy as well as behavioral changes. Therefore, the B176 present work has investigated histological parameters and neuronal apoptosis following stereotaxic injection of gp120 into the Nucleus ANNEXIN A1 MODULATES THE LPS INDUCED accumbens in rats. The behavioral effects were also evaluated. Male TSPO EXPRESSION IN BV2 CELLS Wistar rats (n = 7) were divided into groups: control, gp120-injected (400 ng) and TNF-alpha-injected (100 ng). Cannulas were implanted Lorena N. Pantaleao1, Rodrigo Azevedo1, Erick Barioni1, in rats through stereotaxic surgery, and after 5 days the rats received a Mauro Perretti2, Egle Solito2, Sandra P. Farsky1 bilateral injection in the nucleus accumbens of saline, gp120 or TNF- alpha, as noted. After 2 days, the open field and sucrose preference 1FCF-USP, Department de Ana´lises Clı´:nicas e Toxicolo´gicas (Av. tests were conducted to evaluate locomotor activity and depression. Prof. Lineu Prestes, 580 Bloco 13B, Cid. Universita´ria, SP CEP: After sacrifice, brains were collected, fixed in 4 % of paraformalde- 05508-000); 2William Harvey Research Institute, QMUL, London, hyde and then embedded in paraffin. Sections were stained for UK immunohistochemistry analysis. NeuN primary antibody (Millipore MAB377, 1:200) was used to stain for neuronal apoptosis. The Introduction: The translocator protein (18 kDa) (TSPO) is a benzo- immunohistochemistry results showed that TNFa decreased NeuN diazepine receptor localized in the outer mitochondrial membrane. It levels 18.70 %, and gp120 decrease the NeuN expression 16.01 % is as a biomarker in brain inflammation and modulates the develop- compared the control group. Gp120 and TNFa however did not ment of inflammatory diseases. Annexin A1 (ANXA1) is 37 kDa induce significant changes in depression in sucrose preference test, or protein produced by different cell types and inhibits and induces the in rearing and locomotor activity in the open field test when compared development and resolution of innate inflammation, respectively. to the control group. The results suggest that although gp120 and Gene and protein expression of ANXA1 is induced by glucocorticoids TNF-alpha did not cause behavioral changes, injection into the and TSPO modulation on inflammation is dependent on Nucleus accumbens resulted in loss of neurons. glucocorticoids. Objective: To investigate the connection of ANXA1 and TSPO in References BV2 in basal or inflammatory conditions. Nasirinezhad F, Jergova S, Pearson JP, Sagen J. Attenuation of per- Method: Murine BV2 microglial cells were seed in culture plates, sistent pain-related behavior by fatty acid amide hydrolase (FAAH) exposed to different concentration of an inflammatory stimuli (LPS, inhibitors in a rat model of HIV sensory neuropathy. Neuropharma- 10 or 100 ng/mL), and co-incubated or not with ANXA1 (10 cology. 2014 Dec 5. S0028-3908(14) 00440-7. doi:10.1016/ or100 ng/mL) for 4 or 12 h. TSPO expression was evaluated by j.neuropharm.2014.11.024.

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Reddy PV, Gandhi N, Samikkannu T, Saiyed Z, Agudelo M, Yndart suggest a role for DCs and macrophages in the resolution of SCI and A, Khatavkar P, Nair MP. HIV-1 gp120 induces antioxidant response identify them as a potential therapeutic target. element-mediated expression in primary astrocytes: role in HIV associated neurocognitive disorder. Neurochem Int. 2012 Oct; 61(5):807–14. doi:10.1016/j.neuint.2011.06.011. B179 MATERNAL IL17 PATHWAY PROMOTES AUTISM–LIKE PHENOTYPES IN OFFSPRING B178 DYNAMICS OF INFLAMMATORY IMMUNE Jun Huh RESPONSE IN A MOUSE MODEL OF TRAUMATIC SPINAL CORD INJURY University of Massachusetts Medical School, Worcester, USA Accumulating evidence points to a central role for immune dysreg- Masoud Hassanpour Golakani1, Mohammad G. Mohammad1, ulation in utero as a risk factor in Autism Spectrum Disorder (ASD). Hui Li1, Marc Ruitenberg2, Samuel N. Breit1, David A. Brown1 Human studies suggest that maternal viral infections early in preg- nancy correlate with an increased frequency of ASD in their 1Laboratory of Neuroinflammation, St Vincent’s Centre for Applied offspring. This observation, coined maternal immune activation Medical Research and University of New South Wales, Sydney, NSW, (MIA), has been modeled in rodents by inducing inflammation in Australia; 2Queensland Brain Institute, The University of pregnant dams. MIA requires the pro-inflammatory effector cytokine, Queensland, Brisbane, Queensland, Australia interleukin 6 (IL-6), to produce ASD-like phenotypes in the offspring. However, the specific immune cell population(s) that induces MIA Aim: Inflammatory responses post spinal cord injury (SCI) may be phenotypes has not been identified. We have found that pro–inflam- detrimental or beneficial. However, the dynamics of this inflamma- matory CD4+ T helper cells expressing RORat are required in tory response are largely unknown. The aim of this study was, using a mothers for MIA to induce ASD–like phenotypes in affected off- mouse model of traumatic SCI, to quantify and characterise immune spring. These data suggest that the Th17 cell lineage, a major cells in and around the spinal cord injury site and their relationship contributor to autoimmune inflammation, may serve as a therapeutic with locomotor functional recovery after SCI. target in susceptible pregnant mothers to reduce the likelihood of Methods: Mice had a severe, 70 kilo dyne force, contusive SCI bearing children with inflammation–induced ASD phenotypes. induced using the Horizon impactor after laminectomy. Locomotor function was assessed, using the Baso Mouse Scale (BMS) scoring system. At days 7, 14, 21, and 28 post injury, the entire spinal cord was removed and mononuclear cells associated with SCI were iso- B180 lated, prior to fluorescent labeling for multiparameter flow cytometry IL-33 SIGNALLING IS ESSENTIAL TO to characterize and quantify 15 subtypes of immune cells. Results ATTENUATE THE DEVELOPMENT OF VIRAL were validated using both immunohistochemistry (IHC) and immunofluorescence (IF) staining and correlated with histological INDUCED ENCEPHALITIS BY assessment of injury parameters and injury course. DOWNREGULATING INOS EXPRESSION IN THE Results and discussion: Post SCI mice had complete paralysis, with CENTRAL NERVOUS SYSTEM functional recovery to score 3–4 on BMS at 28 days post injury (dpi). Smaller mice had significantly less recovery. Peripherally Rafael FO Franca1, Renata S. Costa2, Jaqueline R. Silva1, derived immune cells progressively increased over the course of Raphael S. Peres2, David F. Colon2, Fernando Q. Cunha2 recovery from SCI, most of which were T-cells. Both CD4 and CD8 T-cells increased by four- to five-fold at day 28 compared to 1Oswaldo Cruz Foundation FIOCRUZ, Rio de Janeiro, Brazil; day 7 dpi. The CD4 T-cell population were predominantly INFg 2University of Sa˜o Paulo, USP, Sa˜o Paulo, Brazil (Th1) expressing, with FoxP3+ (T-regulatory) and IL17+ (Th17) cells being less frequent. Myeloid DCs (mDCs) were the pre- Interleukin-33 (IL-33) is an immunomodulatory cytokine, member of dominant subtype of DCs present and increased significantly from the IL-1 family that binds to IL-1 receptor ST2, leading to Th2- day 21 to 28 dpi. On the other hand, CD8a DCs were the least associated cytokines production. Despite a few studies, the role of IL- frequent DC subtypes and significantly decreased by about three- 33 signaling on viral infections is still poorly explored. Viral fold at day 21 and then increased at 28 dpi. Plasmacytoid DCs encephalitis are a common cause of lethal infections in humans and (pDCs) were mostly unchanged. There were also no significant several different viruses are documented to be responsible. However, changes in macrophages and B cells over days 7–28 dpi, indicating the immunopathogenic process of these different infections is still early recruitment. poorly understood. Rocio virus (ROCV) is a flavivirus that causes a Immunohistology revealed CD11c+ cells and CD3+ T-cells were severe lethal encephalitis syndrome in humans and also mice, pro- predominantly confined to the injury core. While GFAP+ astrocytes viding an interesting model to study the central nervous system (CNS) surrounded the core. Iba-1+ microglia were dispersed throughout the compartmentalized immune response. Therefore, we aimed to explore entire spinal cord. White mater surrounding the injury core was how the IL-33/ST2 axis regulates the local immune response during substantially demyelinated and myelin was observed in injury core. ROCV infection. We have shown that ST2 receptor is expressed in Functional recovery was significantly correlated with the total the CNS of ROCV infected mice and that T1/ST2(-/-) mice pre- numbers of injury associated macrophages and their percentage of sented increased susceptibility to infection. ST2 deficiency were total CD45+ cells at 28 dpi. There was also a significant correlation correlated with increased tissue pathology, higher viral load, cellular with CD11c+ DCs’ percentage of total CD45+ cells, which were infiltration and increased CNS levels of TNF-a and IFN- c transcripts, mostly mDCs. On the other hand, pDC numbers were negatively compared to with wild-type (WT) mice. As consequence, increased correlated with functional recovery. The changes for T-cells and their CNS Th1 cytokines level acted on infiltrating macrophages inducing subtypes were not significantly related to improvement. These data the expression of inducible nitric oxide synthase (iNOS), contributing

123 S174 Inflamm. Res. to brain injury. Moreover, iNOS-/- mice were more resistant to critical states is still one of the most debatable problems. It is known, ROCV encephalitis, presenting a lower clinical score and reduced that massive injuries of tissues are accompanied by discharge of big mortality rate, despite the increased tissue pathology. In conclusion, amount of biologically active substances, which are supposed to be we provide evidences of a specific role for IL-33 receptor signaling in the key factor for formation of systemic inflammatory response NO induction through local IFN-c modulation, suggesting that NO syndrome. However, the question, in what measure can the mentioned overproduction might have an important role in progression of changes be the starting factor for development of complications and experimental viral encephalitis. organs pathology formation, is still unstudied. We studied the organs pathology development by using the model of reperfusion injury by applying the tourniquets on both posterior Inflammatory Processes in Shock limbs of rats at the level of inguinal fold for 6 h. Revascularization was provided 6 h after applying the tourniquets. The activity of and Trauma proteinase-inhibitory system’s components was determined using enzymatic methods with spectrophotometer Biomat 5. The concen- B181 tration of cytokines IL-1b, IL-6 and TNF-a was determined by ROLE OF HMGB1 IN NUCLEIC ACID-MEDIATED ELISA method (RayBio). The morphological changes in the lungs, liver, kidneys, heart and intestine were studied using immunohisto- INNATE IMMUNE RESPONSES AND chemical and electron-microscopic methods. INFLAMMATORY DISEASES The provided experimental researches have shown, that under reperfusion injury the activation of nonspecific proteinases occurs Hideyuki Yanai within 24 h on the local (injured muscular tissue), systemic (blood serum) and organ levels (bronchoalveolar lavage, peritoneal fluid, Department of Molecular Immunology, Institute of Industrial Science, tissues of kidney and liver). At the same time, the content of pro- University of Tokyo, Japan inflammatory cytokines considerably grows. We have found more than tenfold grow of IL-1b and more than forty-fold grow of IL-6 and High-mobility group box protein 1 (HMGB1) is a nuclear protein TNF-a during 12–24 h after the reperfusion. At the same time we conserved among almost all eukaryotic cells. HMGB1 is thought to be have found the signs of inflammatory changes in the target organs; a danger signal mediator when it is released both actively from these signs manifested by activation of the inflammation’s and activated immune cells and passively from dead cells into extracel- necrosis’ markers and by ultrastructural changes on the cellular level. lular milieu upon pathogen infections or sterile inflammations. The obtained results allow to conclude that excessive systemic Released HMGB1 act as an inflammatory cytokine and promote activation of pro-inflammatory cytokines and nonspecific proteinases inflammatory disorders. In addition to released HMGB1, recently we under decrease of inhibitor control may play an important role in have found that the role of cytosolic HMGB1 in innate immune activation of injury processes in the target organs by formation of responses. We found that HMGB1, 2 and 3 bind to immunogenic numerous inflammatory foci and all these changes can leads to nucleic acids and are involved in nucleic acid-mediated innate development of multiple organ dysfunction syndrome. immune responses. We hypothesized that selective activation of nucleic acid-sensing cytosolic and Toll-like receptors is contingent on the promiscuous sensing of nucleic acids by HMGB proteins. Con- sistent with this notion, we also found that non-immunogenic B183 nucleotides with high affinity for HMGB proteins can strongly sup- PHOSPHATASE AND TENSIN HOMOLOG press the activation of innate immune responses induced by cytosolic CONTROLS MACROPHAGE ENDOTOXIN nucleic acid receptors including RIG-I-like receptors and Toll-like TOLERANCE receptors. In addition, in vivo function of HMGB1 is still elusive since Hmgb1 gene-deficient mice are lethal and we established HMGB1 conditional knockout (cKO) mice recently. In this poster Nicole L. Glosson-Byers, Soujuan Wang, C. Henrique Serezani presentation, we want to discuss our findings in relation to the critical role of HMGBs in initiating immune responses and the possible use of Indiana University School of Medicine, Indianapolis, IN, USA these non-immunogenic nucleotides in therapeutic interventions. The host immune response to sepsis is characterized by an initial Further, we would like to discuss our findings in HMGB1 cKO mice. dominant hyper-inflammatory phase followed by a persistent immunosuppressive phase. A signature of sepsis-induced immuno- suppression is macrophage tolerance, which might contribute to B182 increased susceptibility to secondary infections and mortality in sepsis. Phosphatase and tensin homolog (PTEN) is a dual phosphatase rec- THE ROLE OF INFLAMMATION IN FORMATION ognized as a tumor suppressor, but we and others have also shown that OF MULTIPLE ORGAN DYSFUNCTION PTEN inhibits many macrophage antimicrobial effector functions. We SYNDROME AT CRITICAL STATES hypothesize that PTEN regulates macrophage tolerance and sepsis outcome. Using the cecal ligation and puncture (CLP) mouse model of polymicrobial sepsis, we found that post-septic mice are more sus- Anatoliy V. Kubyshkin, Ludmila Anisimova, Michail Fedosov, Irina ceptible to secondary pulmonary infection with methicillin-resistant Fomochkina, Vladimir Kharchenko, Viacheslav Mikchailichenko, Staphylococcus aureus (MRSA), a pathogen commonly found in ICUs, Olga Malchenko in comparison to sham-treated mice. Interestingly, Pten expression is significantly increased in both the peritoneal cavity and the lung of V.I. Vernadsky Crimea Federal University, Simferopol, Republic of mice 3–4 days after CLP. Enhanced Pten expression is accompanied Crimea by increased expression of immunosuppressive mediators such as Il10, One of the most dangerous complications of critical states, which lead Socs3 and Irak3. Using an in vitro endotoxin tolerance model, we to the death of patients, is the multiple organ dysfunction syndrome found that Pten expression is increased in tolerant macrophages and (MODS). At present, the role of inflammation in pathogenesis of that pharmacologic inhibition of PTEN impairs tolerance in both

123 Inflamm. Res. S175 murine macrophages and human monocytes. In addition, peritoneal B185 cells isolated from CLP-treated mice display PTEN-mediated toler- ESTRADIOL AFFECTS THE LEUKOCYTE ance upon in vitro stimulation with endotoxin. Together, these data suggest that PTEN could be an important mediator of the immuno- MOBILIZATION CAUSED BY INTESTINAL suppressive phase of sepsis by controlling macrophage endotoxin ISCHEMIA AND REPERFUSION IN MALE RATS tolerance. Thus, PTEN might be a therapeutic target to control sepsis- induced immunosuppression and susceptibility to secondary infec- Evelyn T. Fantozzi1, Fernanda Y Ricardo da Silva1, Sara Rodrigues tions, resulting in increased survival of septic patients. Garbin1, Ricardo M. Oliveira Filho1, Bernardo B. Vargaftig1, Ana Cristina Breithaupt Faloppa2, Wothan Tavares de Lima1

B184 1Department of Pharmacology, ICB/USP, Sa˜o Paulo, Brazil; ESTRADIOL MODULATES LEUCOCYTES 2Cardiovascular Surgery and Circulation Pathophysiology MOBILIZATION AFTER INTESTINAL ISCHEMIA/ Laboratory, Heart Institute (Incor), Sa˜o Paulo University Medical REPERFUSION IN FEMALE RATS School, Sa˜o Paulo, Brazil Objects: Intestinal ischemia and reperfusion (i-IR) induces local and Evelyn T. Fantozzi1, Fernanda Y. Ricardo da Silva1, Sara Rodrigues remote organ injury characterized by leukocyte mobilization, Garbin1, Ricardo M. Oliveira Filho1, Bernardo B. Vargaftig1, Ana increased microvascular permeability and systemic inflammation. Cristina Breithaupt Faloppa2, Wothan Tavares de Lima1 Studies show female resistance to local repercussions of i-IR over male, which is attributed to sex hormones mechanisms, notably 1Department of Pharmacology, ICB/USP, Sa˜o Paulo, Brazil; estradiol. However the mechanisms underlying the effects on male are 2Cardiovascular Surgery and Circulation Pathophysiology not totally explored. Laboratory, Heart Institute (Incor), Sa˜o Paulo University Medical Methods: The studies were performed in accordance to IACUC from School, Sa˜o Paulo, Brazil the Institute of Biomedical Sciences, University of Sao Paulo. Anesthetized male rats (Wistar, 60 days old) were submitted to Objectives: Experimental evidence shows that female sex hormones superior mesenteric artery occlusion (45 min), followed by reperfu- may exert protective effect on organ injury caused by trauma-hem- sion (2 h), during this period the rats had the intestine packed in a orrhagic shock (T-HS). The intestinal ischemia/reperfusion (i-I/R) plastic bag. As control, sham operated animals (Sham) were used. causes local and remote injuries similar to those found in the T-HS. Estradiol (17b) was given (280 mg/kg, s.c.) 24 h before induction of Since estradiol appears as a mediator of protection against the organ i-IR (E24). White blood cell (WBC) and bone marrow cell (BMC) injury after T-HS, in the present study we sought to analyze the role count were assessed. Also, the intestinal fluid collected from the of estradiol on the control of leucocytes migration into gut after i-I/R. intestinal bag was analyzed for total and differential leucocyte counts In parallel, the involvement of estradiol on the magnitude of bone (optical microscopy) and chemokines were quantified in the serum marrow cells and blood leukocytes was also studied. (Multiplex). Methods: The studies were performed in accordance to IACUC from the Results: Comparisons between groups (n = 6–11), were made by Institute of Biomedical Sciences, Universityof Sao Paulo.After 7 days of one-way ANOVA followed by Bonferroni post test (p \ 0.05). WBC ovaries removal (OVx), Wistar rats (60 days old) were submitted to count did not differ between the groups. However, after i-IR BMC 45 min occlusion of the superior mesenteric artery, followed by 2 h count decreased in comparison to Sham and E24 treatment increased reperfusion. A group of OVx rats received one single dose (280 mg/kg, the number of cells to similar levels found in Sham group s.c.) of estradiol 24 h before induction of i-I/R (i-I/R + E). OVx-sham i-I/ (Sham = 51.83 ± 2.35; i-IR = 31.19 ± 4.08; E24 = 47.72 ± 4.91 R rats were used as controls. Circulating leukocytes were quantified in 9 106 cells/mL). The intestinal fluid analysis showed increased leu- blood samples using a hematological analyzer. Neutrophil recruitment to cocytes count in i-IR group and E24 group decreased the cells the gut was evaluated by myeloperoxidase (MPO) activity assay. During (Sham = 30.31 ± 7.9; i-IR = 143.1 ± 24.34; E24 = 46.74 ± 12.97 i-I/R the intestine of rats was packed in a plastic bag in order to collect the 9 104 cells/mL). Intestinal fluid in Sham group was predominantly intestinal fluid to quantify the total and differential leucocytes by optical neutrophilic, whereas in i-IR the number of mononuclear cells was microscopy. Comparisons between groups were made by one-way predominant. Yet, E24 group increased the neutrophils (Mononuclear ANOVA followed by Bonferroni post test. cells: Sham = 13.5 ± 1.55 vs i-IR = 72.6 ± 7.27 %; E24 = Results: Estradiol treatment reduced the percentage of increase (before 58.83 ± 8.006; Neutrophils: Sham i-I/R = 87.4 ± 2.82; i-IR = and after i-I/R) of total white blood cell relative to non-treated i-I/R. In 24.4 ± 6.4 %; E24 = 54.50 ± 9.021). Chemokines increased after contrast, estradiol did not alter the neutrophil number in blood, whereas i-IR, and were decreased in E24 group: IP-10 (Sham = 370.1 ± that of monocytes was increased compared to non-treated i-I/R group (i- 19.43; i-IR = 988.3 ± 125.7; E24 = 598.1 ± 59.80 pg/mL), GM- 3 I/R = 522.7 ± 70.23; i-I/R + E=864.3 ± 102.5 monocytes/mm , CSF (Sham = 0.89 ± 0.09; i-IR = 3.22 ± 0.75; E24 = 1.25 ± p = 0.0027). Bone marrow cell number was not altered by i-I/R. 0.18 pg/mL), GRO/KC (Sham = 203.9 ± 37.74; i-IR = 2248 ± Interestingly, estradiol increased MPO activity in the intestine after i-I/R 422.8; E24 = 855.6 ± 137.9 pg/mL), MIP-1a (Sham = (i-I/R = 0.23 ± 0.024; i-I/R + E=0.4 ± 0.09; p = 0.0024). In com- 16.55 ± 1.3; i-IR = 234.7 ± 52.17; E24 = 97.20 ± 31.69 pg/mL), parison to non treated i-I/R group, estradiol reduced the number of VEGF (Sham = 63.37 ± 12.45; i-IR = 119.6 ± 17.61; E24 = mononuclear cells (i-I/R = 72 ± 4.9; i-I/R + E=55.56 ± 2.84 %, 68.96 ± 6.97 pg/mL). p = 0.0062) and increased those of neutrophils in intestinal fluid Conclusion: Our data suggest that i-IR mobilizes leukocytes from the (OVx = 22.86 ± 2.95; E = 35.75 ± 2.33 %, p = 0,012). bone marrow and increases the levels of chemokines in serum by a Conclusion: Our data suggest that during i-I/R, estradiol modulates mechanism under control of estradiol. Thus, the previous treatment differently the traffic of circulating leukocytes into gut and to with estradiol may be of interest to control the local and systemic intestinal cavity, likely controlling the local inflammatory response effects of ischemic events. and, as consequence the remote organ injury observed after i-I/R. Ethics Committee number: 111/10/03—CEUA—ICB/USP. Ethics Committee number: 111/10/03—CEUA—ICB/USP. Supported by CAPES, CNPq and FAPESP 2013/15291-0. Financial support CAPES, CNPq and Fapesp (2013/15291-0).

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B186 1Cardiovascular Surgery and Circulation Pathophysiology EXTRACELLULAR MICRORNA PROMOTES Laboratory, Heart Institute, Sao Paulo University Medical School (HC-FMUSP), Sao Paulo, Brazil; 2Department of Pharmacology, COMPLEMENT FACTOR B EXPRESSION VIA ICB/USP, Sao Paulo, Brazil TLR7-MYD88 SIGNALING IN BACTERIAL SEPSIS Objectives: Lung transplantation depends on heartbeating donors after brain death. Brain death (BD) is associated with inflammation, release Lin Zou, Ganqiong Xu, Yan Feng, Wenling Jian, Wei Chao of mediators and generalized ischemia–reperfusion injury, which is accompanied by upregulation of inducible nitric oxide synthase Massachusetts General Hospital, Boston, MA, USA, Harvard Medical (iNOS) expression. Female sex hormones influence the lung inflam- School, Boston, MA, USA matory and immune responses after trauma and this raises questions Objective: Bacterial sepsis induces massive activation of the com- relative to their influence on donor lung state after BD. In this study, plement system including the alternative pathway (AP). Complement we investigated the differences between male and female rats on the factor B (cfB) is an essential component of AP activation. We have lung inflammation after brain death in rats. recently demonstrated that cfB is the downstream effector of TLRs Methods: Groups of male (M), proestrus female (PF) (high and plays a pivotal role in the pathogenesis of sepsis. However, the estradiol secretion period) and ovariectomized (OVx) Wistar rats upstream mediator leading to cfB production in sepsis remains largely were submitted to BD by intracranial balloon catheter sudden infla- unclear. microRNA (miRNA) is a group of small non-coding RNAs tion. BD was confirmed by maximally dilated and fixed pupils, apnea, that negatively regulate target gene translation. Host miRNAs are absence of reflexes, and a drop in mean arterial pressure (MAP). After released into the extracellular space during bacterial sepsis. Whether 6 h, lung samples were collected and the iNOS gene expression was or not miRNAs induce cfB production is unknown. analysed. Lung sections were analysed by histology and the iNOS Methods: Bacterial sepsis was created by cecum ligation and puncture expression by immunohistochemistry. Lung myeloperoxidase activity (CLP) in mice. Plasma miRNAs were analyzed using microRNA array (MPO) was determined and vascular permeability (VP) assessed 24 h after sham or CLP surgery. Bone marrow-derived macrophages using the Evans blue dye extravasation method. Estradiol (E) and (M/) from WT or TLR3-/-, TLR7-/-, MyD88-/- mice were treated progesterone (P) serum levels were quantified. with 50 nM of miRNA mimics in the presence of lipofectamine for Results: After 6 h of BD, E and P concentrations in serum of 18 h. Medium cfB, phospho-ERK1/2 and P38 were tested with Wes- proestrus female rats were significantly reduced (E initial: tern blot. Three mg RNase was injected i.p. 1 h before and 12 h after 60.51 ± 1.74, 6 h: 0.89 ± 0.5 pg/mL, p \ 0.0001; P initial: surgery. Cardiac complement mRNA was tested by qRT-PCR. 1362 ± 379, 6 h: 231 ± 104 ng/mL, p = 0.0017). Proestrus female Results: Plasma miRNA array demonstrated that among the 68 rats presented augmented MPO (PF = 0.837 ± 0.029 versus miRNAs tested, six miRNAs had more than twofold increase in septic M = 0.724 ± 0.017; p = 0.028) and VP (PF = 171.9 ± 10.27 ver- mice as compared with sham mice with fluorescence counts [100, sus M = 104.9 ± 8.35; p = 0.0017). iNOS relative gene expression namely miR-145, miR-146a, miR-122, miR-34a, miR-192 and miR- was significantly higher in the proestrus female in comparison to 210. Treatment with miRNA mimics miR-145, -146a, -34a, -122, but other groups (PF = 701.2 ± 223.3, OVx = 37.68 ± 5.05, not miR-192 or -210, induced significant increase in cfB production in M = 191.4 ± 23.77; p = 0.0046). In relation to leukocyte infiltration M/. This effect appeared to be specific because their UÒA mutants to the lung, we did not find significant differences among the groups, did not induce any increase in cfB expression. In M/ deficient of but the iNOS protein expression was higher in the lungs of the TLR7 or MyD88, these miRNA mimics failed to induce cfB pro- proestrus female group compared with other groups. duction. In contrast, TLR3-deficiency had no impact on the effect of Conclusion: The data evidenced important differences between gen- these miRNAs. In addition, miR-146a treatment led to phosphoryla- ders after BD with higher lung inflammatory compromise in proestrus tion of ERK1/2 and P38. TLR7-deficiency completely blocked this female rats, which have the hormone levels acutely reduced. There is activation. To test the role of extracellular RNA in cfB production in evidence that estradiol limits the induction of iNOS, thus we suggest animal model of sepsis, we tested cfB gene expression in the heart that the estradiol reduction might lead to higher lung iNOS expression following sepsis. CLP led to a significant increase in cfB gene and inflammation. The results are also consistent with the notion that expression in the heart but had no impact on other complements female sex hormones could influence the result of lung transplant and tested, such as C3 and C5. Eliminating extracellular and circulating should be considered as important elements for the maintenance of RNA by RNase administration led to a 46 % reduction in cfB gene donor lung status. expression, suggesting that extracellular RNA including miRNA may Financial support Sao Paulo Research Foundation (FAPESP in part mediate cardiac cfB production during sepsis. 2013/20282-0). Conclusion: We demonstrate that miRNAs induce cfB production in M/ via TLR7-MyD88-dependent mechanism. Polymicrobial infec- tion leads to specific cardiac cfB gene expression and extracellular RNA in part mediates the cfB production in vivo. B188 EFFECTS OF Co-ultraPEALut ON INFLAMMATORY EVENTS ASSOCIATED WITH B187 TRAUMATIC BRAIN INJURY LUNG INFLAMMATION AND INDUCIBLE NITRIC Rosalia Crupi1, Giuseppe Bruschetta1, Irene Paterniti1, OXIDE SYNTHASE AFTER BRAIN DEATH: Rosalba Siracusa1, Marika Cordaro1, Emanuela Esposito1, PROESTRUS FEMALE VERSUS MALE DONORS Salvatore Cuzzocrea1,2

Ana Cristina Breithaupt-Faloppa1, Sueli G. Ferreira1, 1Department of Biological and Environmental Sciences, University of Guilherme K. Kudo1, Roberto Armstrong Junior1, Messina, Viale Ferdinando Stagno D’Alcontres 31-98166 Messina, Wothan Tavares de Lima2, Paulina Sannomiya1, Italy; 2Manchester Biomedical Research Centre, Manchester Royal Luiz Felipe P. Moreira1 Infirmary, School of Medicine, University of Manchester, UK

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Introduction: Traumatic brain injury (TBI) is considered important Background and objectives: Acute respiratory distress syndrome burdens to society and remain challenging to diagnose, manage, and (ARDS) is a severe inflammatory disorder characterized by acute treat. His diagnosis includes a broad range of short- and long-term respiratory failure, resulting from severe, destructive lung inflam- physical, cognitive, and emotional impairments. It is characterized by mation and irreversible lung fibrosis. Here, we evaluated the use of neurological dysfunction, due to the progressive destruction of local stem cells derived from human exfoliated deciduous teeth (SHEDs) or and distal neuronal networks, resulting from injury-induced tissue SHED-derived serum-free conditioned medium (SHED-CM) as damage and subsequent local, cellular, and biochemical reactions. The treatments for bleomycin (BLM)-induced mice acute lung injury neuroinflammatory cascade contributes to neuronal damage and (ALI), exhibiting several pathogenic features associated with the behavioral impairment. N-palmitoylethanolamide (PEA) is an human disease ARDS. endogenous fatty acid amide belonging to the family of the Methods: Mice with BLM-induced ALI with or without SHED or N-acylethanolamines (NAEs). PEA is an important analgesic, anti- SHED-CM treatment were examined for weight loss and survival. inflammatory and neuroprotective mediator, acting at several molec- The lung tissue was characterized by histological and real-time ular targets in both central and sensory nervous systems as well as quantitative PCR analysis. The effects of SHED-CM on macrophage immune cells. However, PEA lacks a direct antioxidant capacity to differentiation in vitro were also assessed. prevent the formation of free radicals, and to counteract the damage of Results: A single intravenous administration of either SHEDs or DNA, lipids and proteins. Luteolin (Lut), a common flavonoid present SHED-CM attenuated the lung injury and weight loss in BLM-treated in many plants, has strong antioxidant and pharmacological activities, mice, and improved their survival rate. Similar recovery levels were including a memory-improving effect. It displays excellent radical seen in the SHEDs- and SHED-CM-treatment groups, suggesting that scavenging and cytoprotective properties, particularly when tested in SHED improves ALI by paracrine mechanisms. SHED-CM contained complex biological systems where it can interact with other antioxi- multiple therapeutic factors involved in lung-regenerative mecha- dants, such as vitamins. Lut displays specific anti-inflammatory nisms. Importantly, SHED-CM attenuated the BLM-induced pro- effects, which are only partly explained by its antioxidant capacities. inflammatory response and generated an anti-inflammatory/tissue- Aim: In the present study, we performed a widely-used model of TBI regenerating environment, accompanied by the induction of anti-in- to determine the neuroprotective propriety of palmitoylethanolamide flammatory M2-like lung macrophages. Furthermore, SHED-CM (PEA) and the antioxidant effect of a flavonoid luteolin (Lut), given promoted the in vitro differentiation of bone marrow-derived mac- as a co-ultramicronized compound Co-ultraPEALut. rophages into M2-like cells, which expressed high levels of Methods: TBI was induced in mice by controlled cortical impactor. Arginase1, CD206, and Ym-1. Co-ultraPEALut (1 mg/kg, soluble 10 % ethanol, i.p.) were admin- Discussion and conclusions: Taken together, our results suggest that istered 1 h after craniotomy. At 24 h after TBI, the brains were SHED-secreted factors provide multifaceted therapeutic effects, collected. including astrong M2-inducing activity, for treating BLM-induced Results: We demonstrated that the treatment with Co-ultraPEALut ALI. This work may open new avenues for research on stem-cell- resulted in a significant improvement of motor and cognitive based ARDS therapies. recovery after controlled cortical impact (CCI), as well as markedly reducing lesion volumes. Moreover, our results revealed the ability of Co-ultraPEALut, to reduce brain trauma through modulation of NF-jB activation. In addition, treatment with Co-ultraPEALut B190 significantly enhanced the post-TBI expression of the neuropro- PROTECTIVE EFFECT OF HEMATOPOIETIC tective neurotrophins GDNF compared to vehicle. Co-ultraPEALut STEM CELLS IN STROKE at the dose of 1 mg/kg, also modulated apoptosis, the release of cytokine and ROS, the activation of chymase, tryptase and Felicity NE Gavins, Helen K. Smith, Shantel Vital, D.Neil Granger nitrotyrosine. Conclusions: Thus, our data demonstrated that Co-ultraPEALut at a LSUHSC-S, Shreveport, LA, USA lower dose compared to PEA alone, can exert neuroprotective effects and the combination of both could improve their ability to Increasing evidence suggests that stem cells may be beneficial as a counteract the neurodegeneration and neuroinflammation induced treatment for stroke. We investigated the effects of hematopoietic by TBI. stem cells (precursors to cells of the blood) in a mouse model of cerebral ischemia and reperfusion (I/R). Mice underwent surgical middle cerebral artery occlusion (MCAo) for 30 min using a 6-0 nylon filament, which was then removed to Inflammatory Responses, Stem Cells induce cerebral I/R. 24 h later, mice were injected with 1 million lineage negative bone marrow cells (Lin- BMCs, hematopoietic cell and Tissue Regeneration precursors which expressed no markers of differentiation), derived from donor mice. B189 After the following 24 h (i.e. 48 h post cerebral I/R) and up to 2 FACTORS SECRETED FROM DENTAL PULP weeks, various improvements in Lin- BMC-treated mice versus vehi- cle-treated mice were assessed through outcome parameters including: STEM CELLS SHOW MULTIFACETED BENEFITS mortality rates, infarct volume, stroke score (behavioral assessment) FOR TREATING ACUTE LUNG INJURY IN MICE and levels of inflammation (as indicated by leukocyte-endothelial interactions). All of these were significantly reduced in Lin- BMC Hirotaka Wakayama1, Naozumi Hashimoto2, Yoshihiro Matsushita1, versus vehicle-treated mice. In addition to a large drop in mortality Noriyuki Yamamoto1, Masaya Nishikawa1, Yoshinori Hasegawa2, (from 50 to 7 % in Lin- BMC and vehicle-treated mice, respectively), Akihito Yamamoto1 results showed a clear improvement in infarct volume, leukocyte activity and stroke score, which were all reduced by reduced by at least 1Department of Oral and Maxillofacial Surgery of Nagoya University, 50 % at 48 h reperfusion and \95 % by 1 week (p \ 0.05). Nagoya, Japan; 2Department of Respiratory Medicine of Nagoya After establishing their efficacy, it is important to investigate the University, Nagoya, Japan differentiation of transplanted cells and/or their release of protective

123 S178 Inflamm. Res. substances after stroke; we therefore aimed to identify the mechanism appeared to be at 25 mg/mL. When urticaria was induced with 10 mg/ by which the cells were of benefit. The observed reduction in mL histamine, Allegra slightly inhibited both dermal irritation and leukocyte activity indicated a possible immunomodulatory mecha- wheal and flare, whereas Cortaid inhibited the wheal and flare only. nism. As such, brain slices were stained for Iba-1 to indicate the When urticaria was induced with 25 and 50 mg/mL histamine, presence of activated microglia—previously identified to be of sig- Allegra as well as Cortaid inhibited both dermal irritation and wheal nificance in mediating damage after stroke. These images showed a and flare. The inhibitory effects of Allegra and Cortaid were observed 50 % reduction in activated microglia, both contra- and ipsilaterally, at 25 and 45 min post dose. Histamine at 50 mg/mL was shown to in mice treated with Lin- BMCs versus vehicle. Microglia activation induce urticaria with sustained dermal irritation and wheal and flare in and an excessive inflammatory response have consistently been Hanford miniature swine. associated with poor outcome following stroke, therefore there results Conclusion: A histamine induced urticaria model has been established suggest a way in which hematopoietic stem cells may be beneficial as with the female Hanford miniature swine and can be used for the a treatment. testing of topical treatments for dermal irritation and inflammation. In conclusion, not only have we provided significant and broad evidence of the therapeutic effects of hematopoietic stem cells in stroke (including improved mortality, infarct volume, levels of inflammation and functional recovery), but we have given a primary B192 insight into their immunomodulatory potential by demonstrating a DOES SUBSTANCE P PLAY A ROLE IN ALLERGIC down-regulation of microglial activation. We believe our data will CONTACT DERMATITIS? contribute to the development of an optimized stroke therapy based on the use of stem cells. Francis FY Lam, Ethel SK Ng, Nick H. Ng

The Chinese University of Hong Kong, Hong Kong, China Inflammatory Skin Disorders Objective: Allergic contact dermatitis (ACD) is a common inflam- matory skin disease. It is a hypersensitive reaction to allergens that B191 causes rash or skin lesion at the site of exposure. Substance P(SP) is a sensory neuropeptide known to be involved in neurogenic inflam- DEVELOPMENT OF A MODEL OF DERMAL mation. In this study, the importance of SP in the development of INFLAMMATION AND IRRITATION (URTICARIA) ACD was investigated. IN THE MINIATURE SWINE Methods: Male 6-week-old Balb/c mice were used in the present studies. Test drugs were applied topically on to one ear of each- Miao Zhong, Alain Stricker-Krongrad, Jason Liu, Guy Bouchard mouse for 6 times over a course of 10 days to induce ACD. The contralateral ear received parallel vehicle application to serve as an Sinclair Research Center, LLC, Auxvasse, USA internal control. Agents tested as inducers of ACD include the standard ACD inducer dinitrofluorobenzene (DNFB), SP, and cap- Background: Urticaria is dermal edema that results from vascular saicin (anagent that can deplete sensory neuropeptides). Agents dilation and leakage of fluid into the skin in response to molecules tested as inhibitors of ACD include capsaicin, RP67590 (a NK1 released from mast cells. Histamine, the major mediator of mast cells, receptor antagonist), and dexamethasone (an anti-inflammatory is known to induce a short lived urticaria when applied intra-dermally glucocorticoid). in human. Unlike other animals, the swine has a fixed skin tightly Results: Topical application of DNFB increased the thickness and attached to the subcutaneous tissues similar to that in humans, which weight of the mice ears, indicating successful induction of ACD. makes it a preferable model for dermal studies. The effect of his- Microscopic examination confirmed spongiosis and inflammatory tamine to induce urticaria in pig has not been investigated. cells in the affected ears. The same treatment with SPor capsaicin did Objectives: To develop a reproducible dermal urticaria model in not produce ACD. The symptoms of ACD induced by DNFB were miniature swine. suppressed by dexamethasone and capsaicin, but not by SP or the Methods: Three female Hanford (3–18 months old) were used in the NK1 receptor antagonist RP67580. study. The animals were pricked on their back skins with a skin test Conclusions: The present findings do not support a role for SP in the device (Lincoln Diagnostics Inc, Decatur, IN) that were loaded with development of ACD in mice. However, similar to dexamethasone, vehicle or histamine in vehicle. The irritation and wheal and flare capsaicin produced marked inhibition on the ACD symptoms induced responses of the dermis were evaluated with Draize scoring and with by DNFB. This is unexpected since SP is not involved in the devel- wheal size measurement. To investigate the time and dose responses opment of ACD. The mechanisms of the inhibitory effects of of histamine, dermal reactions were evaluated at 10 and 20 min post capsaicin on ACD remain to be elucidated. prick. To evaluate the abilities of cortaid (1 % hydrocortisone) and allegra (2 % diphenhydramine) creams to block the histamine induced dermal urticaria, animals were challenged with histamine for 15 min before being treated with test formulations. The biological reactions of skin were assessed at 10, 25, and 45 min after test for- B193 mulation application. AGGREGATION OF THROMBIN C-TERMINAL Results: Histamine dose-dependently induced skin irritation at both FRAGMENTS—A NOVEL HOST DEFENSE 10 and 20 min after treatment. The most prominent erythema and MECHANISM edema (Draize score) responses were observed at 10 min after treatment, which were slightly diminished at 20 min after treatment. Histamine also caused skin wheal that ranged between 4 and 7 mm in Jitka Petrlova, Finja Hansen diameter. Although wheal sizes increased over time following treat- ment, this change in wheal size appeared not related to histamine Division of Dermatology and Venereology, Departments of Clinical effect. The optimal concentration of histamine to induce urticaria Sciences, Lund University, Lund, Sweden

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Host defense peptides (HDPs) have important roles in the first line of Results: In the RAW cells, KJ extract inhibited LPS-induced defense against invading microorganisms in blood and at epithelial inflammatory cytokines (TNF-a, IL-6 and IL-1b) production. Fur- surfaces. Thrombin is a key enzyme in the coagulation cascade, a thermore, KJ extract reduced skin hyperplasia and expressions of IL- fundamental host defense system, activated during injury or infection. 6, IL-1b and MCP-1 on dorsal skin of mice models. We have previously reported that human neutrophil elastase (HNE) Conclusion: It was proved that the KJ extract inhibited the secretions cleaves thrombin, generating an 11 kDa major fragment, denoted of pro-inflammatory cytokines in LPS-stimulated RAW cells. More- thrombin-derived C-terminal peptide (TCP), with antibacterial over, KJ extract applications on the dorsal skins of the DNCB- effects. Here, we generated recombinant TCP (rTCP) and applied induced CD mice reduced the thickness of the epidermis and dermis biophysical and microbiological techniques to determine its mode of by decreasing the secretions of inflammatory cytokines and chemo- action and interaction with lipopolysaccharide (LPS). Our novel data kines. These findings not only indicate that applications of KJ extract demonstrate that LPS induces aggregation of rTCP. Amorphous rTCP may lessen skin thickening of CD by inhibiting the expressions of containing aggregates were detected by negative stain electron inflammatory mediators, but also suggest that it is a natural option for microscopy, and an increase of b-sheet structure was identified by the treatment of skin inflammation. circular dichroism spectroscopy and a thioflavin T1 assay. Similar results were obtained using intact thrombin digested by HNE. Fur- thermore, antimicrobial effects of rTCP against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa were demon- B195 strated, and found to be mediated by rTCP-induced bacterial BEYOND THE ADRENAL GLAND: THE aggregation. In vivo, a colocalization of C-terminal thrombin frag- ments and LPS was detected in aggregates present in fibrin sloughs, ESSENTIAL ROLE OF LOCALLY SYNTHESISED obtained from patients with infected wounds, indicating the presence GLUCOCORTICOIDS IN REGULATING of LPS-TCP-aggregates at physiological conditions. Furthermore, INFLAMMATION IN HEALTHY SKIN AND generation of C-terminal thrombin fragments of similar molecular PSORIASIS size as TCP, were detected in acute wound fluid. Taken together, our in vitro and in vivo data disclose previously unknown host defense 1 2 3 mechanism of TCPs, involving LPS induced aggregation/scavenging Rosalind F. Hannen , Chinedu Udeh-Momoh , Mike Wright , 3 1 4 2 and microbial killing, thus providing a protection against endotoxins David Halsall , Rod Flower , Lisa Sevilla , Stafford Lightman , 4 1 and pathogens at the wound site. Paloma Perez , Mike Philpott

1Queen Mary University of London, London, UK; 2University of Bristol, Bristol, UK; 3Addenbrookes Hospital, Cambridge, UK; B194 4Instituto de Biomedicina de Valencia, Valencia, Spain KORTHALSELLA JAPONICA ALLEVIATES THE Endogenous glucocorticoids (GCs) are essential for maintaining the DERMAL AND EPIDERMAL HYPERPLASIA IN epidermal skin barrier and their powerful anti-inflammatory effects A CONTACT DERMATITIS MICE MODEL are exploited therapeutically to treat inflammatory skin conditions such as psoriasis. Skin contains the enzymatic machinery required for Jinju Kim synthesising GCs from cholesterol and local cortisol production has been observed by healthy human skin (1–4). However, little is known Department of Korean Physiology, College of Pharmacy, Kyung Hee about the interplay between local and systemic GC production and University, Seoul, South Korea how this impacts on skin pathologies. Here we show that both de novo cortisol synthesis and GC Aim of the study: Contact dermatitis (CD) is skin inflammation that is receptor expression are severely compromised in psoriatic skin. characterized by redness, swelling, heat, and itching. During the Specifically, de novo cortisol synthesis was reduced to less than 10 % occurrence of skin inflammation, macrophages accumulate at the production in both non-lesional and lesional psoriatic tissue compared specific skin sites and secrete many inflammatory mediators, includ- to healthy skin controls (healthy skin 809.6 ± 120.4 ng/mL, psoriatic ing cytokines and chemokines. These inflammatory mediators non-lesional 63.1 ± 7.9 ng/mL, psoriatic lesional 67.7 ± 11.8 ng/ promote a wide range of inflammatory responses, such as itchiness mL, n = 8, p = 0.001 as measured by LC–MS/MS). In addition, and skin thickening. Korthalsella japonica (KJ) is a traditional radiometric assay demonstrated that cultured primary psoriatic ker- medicinal plant, which has various biological and pharmacological atinocytes also exhibited dysfunctional cortisol synthesis (healthy activities. In this study, we investigated anti-inflammatory effect of keratinocytes, 38.8 ± 6.1 %, uninvolved keratinocytes 8.7 ± 0.4 %, (KJ) ethanol extract in lipopolysaccharide (LPS)-stimulated RAW lesional keratinocytes 8.6 ± 0.2 % of cortisol formed from 264.7 macrophages and 2, 4-dinitrochlorobenzene (DNCB) induced [3H]pregnenolone after 24 h, n = 4, p = 0.01). This suggests that skin inflammation mice models. defective GC synthesis is an inherent defect of psoriatic skin. Materials and methods: RAW 264.7 cells were pretreated with KJ Expression of the GC receptor was reduced in non-lesional psoriatic extract for 1 h, and then stimulated with LPS for 24 h. The anti- tissue and was further down-regulated in lesional psoriatic skin. inflammatory activity of KJ extract was determined by measuring Experiments using adrenalectomised wild type (WT) and GR epi- production of tumor necrosis factor-a (TNF-a), interleukin (IL)-6 and dermal knockout (GREKO) mice highlighted the significance of IL-1b. In the in vivo study, BALB/c mice were sensitized by topically systemic vs local GC production in experimental model of skin applying 100 ll of 1 % DNCB (in acetone: olive oil = 4:1) on inflammation induced by topical PMA. Remarkably, adult GREKO shaved dorsal skin on days 1–3. Four days later, the mice were re- mice compensated for loss of GR by up-regulating localized GC sensitized by applying dorsal skin with 100 ll of 0.5 % DNCB for production and this protected the animals from PMA induced IL-1b/ 2 weeks. KJ extract was treated on before 2 h of DNCB application. IL-6/TNF-a generation, even post-adrenalectomy. Thickness of the epidermis and dermis were determined by skin These studies demonstrate that skin-derived GC production is histological examination. IL-6, IL-1b and monocyte chemoattractant capable of protecting the tissue from inflammation in the absence of protein-1 (MCP-1) synthesis were analyzed using ELISA. systemic GCs. Crucially this pathway is defective in psoriatic skin

123 S180 Inflamm. Res. and therefore presents a critical pathway to target in this inflammatory Conclusions: Overall, 5 % IMQ was found to consistently cause skin condition. psoriasis-like skin lesions in mice as evaluated using both in-life and histopathological methods. Steroidal agents clobetasol propionate, References which is the most commonly used drug for skin disorders including 1. Hannen, R.F., Michael, A.E., Jaulim, A., Bhogal, R., Burrin, J.M., psoriasis, and dexamethasone significantly reduced IMQ-induced skin and Philpott, M.P. 2011. Steroid synthesis by primary human ker- changes in mice. Antibodies against TNF-alpha and IL17A were atinocytes; implications for skin disease. Biochem Biophys Res barely effective although only a single dose of each was tested and Commun 404:62–67. thus further studies with higher doses of these clinically used anti- 2. Vukelic, S., Stojadinovic, O., Pastar, I., Rabach, M., Krzyza- psoriatic agents are warranted. nowska, A., Lebrun, E., Davis, S.C., Resnik, S., Brem, H., and Tomic- Canic, M. 2011. Cortisol Synthesis in Epidermis Is Induced by IL-1 and Tissue Injury. J Biol Chem 286:10265–10275. 3. Cirillo, N., and Prime, S.S. 2011. Keratinocytes synthesize and B197 activate cortisol. J Cell Biochem 112:1499–1505. RIP KINASE SIGNALING IN KERATINOCYTE 4. Slominski, A., Wortsman, J., Tuckey, R.C., and Paus, R. 2007. Differential expression of HPA axis homolog in the skin. Mol Cell CONTROLS NECROPTOSIS—MEDIATED SKIN Endocrinol 265–266:143–149. INFLAMMATION

Snehlata Kumari, Manolis Pasparakis B196 PHARMACOLOGICAL VALIDATION OF THE CECAD Research Centre, Institute for Genetics, University of MOUSE IMIQUIMOD (IMQ)-INDUCED PSORIASIS Cologne, Germany MODEL Receptor interacting kinase 1 (RIPK1) is involved in cell death and inflammation. We used conditional targeting to investigate the role of Harunor Rashid, Fraser McIntosh, Agathe Bedard, Luc Chouinard, RIPK1 in epidermal keratinocytes. Mice with epidermis-specific E-KO Joe Cornicelli, Rana Samadfam RIPK1 deletion (RIPK1 ) develop a progressive inflammatory skin disease starting after six to seven postnatal days resulting in Charles River Labs, Wilmington, USA severe cutaneous inflammation by the age of 3–4 weeks. The skin lesions of RIPK1E-KO mice are characterized by non-cell autonomous Background: Psoriasis is an immune-mediated inflammatory disease epidermal hyperproliferation, infiltration of immune cells and that mainly affects skin and is characterized by thickening of epi- upregulation of pro-inflammatory cytokines such as IL-1b, IL-33 and dermis and red scaly patches. A recent article (van der Fits et. al. TNF. We identified increased numbers of apoptotic and necrotic J Immunology 2009; 182:5836–5845) suggested development of keratinocytes in the epidermis of RIPK1E-KO mice and reasoned that psoriasis-like skin lesions in mice by application of TLR7/8 ligand, keratinocyte death could trigger inflammation. To specifically inhibit imiquimod (IMQ). necroptosis, we generated RIPK1E-KO mice lacking Receptor inter- Objective: The aim of the present study was to further characterize acting kinase 1 (RIPK3) or Mixed lineage kinase domain-like the IMQ-induced psoriasis model in mice using both pharmacological (MLKL) gene, which are essential for necroptosis. We found that the and histological approaches. RIPKE-KO; Ripk3-/- or RIPKE-KO; Mlkl-/- double deficient mice, did Methods: Male BALB/c mice were purchased from Charles River not show keratinocyte necrosis and did not develop inflammatory skin Canada and acclimated for 7–8 days. On the Day 1 of the study, skin lesions, demonstrating that inhibition of necroptosis prevented skin was shaved and baseline thickness measurements were taken before inflammation. Furthermore, deficiency of TNFR1, partially protected randomizing animals into different treatment groups. IMQ cream RIPK1E-KO mice from keratinocyte death and inflammation showing (5 %, 62.5 or 47 mg) or control Vaseline cream was then applied on that TNFR1 signaling contributes to the induction of necroptosis. Our back skin and/or ear from Day 1 to Day 5. From Day 2 to Day 6, back study identified a unique role of epithelial RIPK1 signaling in the skin and/or ear thickness was measured using an engineering caliper maintenance of skin homeostasis and the prevention of skin inflam- and visual scoring was done for extent of erythema and scales. On mation. Importantly, knock-in mice expressing a kinase inactive Day 6, animals were euthanized and back skin and/or ear tissues were RIPK1 allele did not develop skin lesions showing that kinase inde- collected for histopathological evaluation. For assessing pharmacol- pendent RIPK1 functions regulate keratinocyte survival and skin ogy with commonly used anti-psoriatic agents, effects of the inflammation. Collectively, these results identified RIPK1-medited following drugs were examined: dexamethasone (0.3 and 3 mg/kg, inhibition of necroptosis as a key mechanism for the maintenance of oral gavage, Day 1–6, QD), clobetasol (0.05 % cream, topical, Day skin homeostasis and the prevention of inflammation, suggesting that 1–6, QD), etanercept (10 mg/kg, SC, Day 1 and 3) and IL17A anti- keratinocyte necroptosis could be relevant for the pathogenesis of body (100 ug per mouse, SC, Day -1, 2 and 4). human inflammatory skin diseases. Results: Application of 5 % IMQ cream caused erythema, scaling and thickening of skin starting from Day 2 and these effects peaked at Day 6. Effects on back skin seemed more pronounced than on the ear, B198 and thus only back skin was chosen for IMQ cream application for IL-1b PRODUCING MONOCYTE DERIVED subsequent studies. In histopathological evaluation of skin samples collected terminally, epidermal thickness was measured using Aperio DENDRITIC CELLS ARE CRITICAL FOR IL-23- or Image-Pro system, and IMQ cream was found to increase epider- INDUCED SKIN INFLAMMATION mal thickness (acanthosis) very similar to the in-life results. 5 % IMQ cream also caused parakeratosis and inflammatory infiltration in the Tej Pratap Singh1, Howard H. Zhang1, Michael N. Hedrick1, skin tissues. Among the reference drugs, 0.05 % clobetasol cream Brian L. Kelsall1, Bjorn E. Clausen2, Joshua M. Farber1 almost completely blocked IMQ-induced skin lesions while dexam- ethasone had moderate effects at 3 mg/kg dose. Etanercept and anti- 1Laboratory of Molecular Immunology, National Institute of Allergy mouse IL17 antibody had no effects at the doses tested. and Infectious Diseases, National Institutes of Health, Bethesda, MD,

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USA; 2Institute for Molecular Medicine, University Medical Center of an atypical association with regulatory proteins to repress gene the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany expression. This mechanism is termed transrepression and is thought to be the consequence of the post-translational modification (PTM) of Human studies have strongly implicated IL-23 in psoriasis, and PXR1 by SUMO (Small Ubiquitin-Like Modifier). This work sought injecting IL-23 in mouse skin produces psoriasis-like inflammation. to investigate the link between SUMO and the anti-inflammatory role IL-23 injection leads to accumulation of dendritic cells (DCs), which of the PXRs. Additionally, the possible anti-inflammatory function of are also abundant in psoriatic skin. In analyzing myeloid cells, we PXR3 was examined. This is important since PXR gene mutations are found monocyte-derived langerin-positive cells (moLCs) appearing in linked to the onset of inflammatory bowel disease. the epidermis, and increased numbers of other monocyte-derived Materials and methods: Site-directed mutagenesis was used to create cells, including monocyte-derived dermal DCs (moDDCs) and mac- PXR1/PXR3 Lys to Arg mutants at predicted sumoylation sites. The rophages, plus non-monocyte derived DCs (cDCs) in the dermis after human Il-8 and mouse iNOS reporters, possessing NF-jB response IL-23 injection. Depletion of all CD11c+ cells in diphtheria toxin- elements were induced by lipopolysaccharide (LPS) in RAW264 treated CD11c-DTR mice blocked IL-23-induced IL-22, IL-19, IL-17, macrophages. The activity of these reporters in the presence of the IL-36, IL-1b and s100A, as well as the psoriasis-like changes. IL-22 PXR1/PXR3 mutants was then measured using luciferase reporter and IL-17 are known to be important mediators of pathology in this assays. Next, each wild type PXR1/PXR3 or putative SUMO site model. Using Il1r1-/- mice, we also identified a major role for IL-1, mutant was transiently co-transfected into HELA cells with His-tagged which we found was produced by moLCs and moDDCs but not cDCs. SUMO1, SUMO2 or SUMO3. Nickel pull down assays and Despite the dramatic effects of depleting CD11c+ cells, Batf3-/-, immunoblotting were then performed to probe for the presence of PXR. diphtheria toxin-treated Bdca2-DTR, and Flt3l-/- mice, which lack Results and discussion: In the presence of wild type PXR1/PXR3, LPS- CD103+ cDCs, pDCs, and all cDCs and pDCs, respectively, each mediated induction of the human Il-8 (Interleukin-8) and mouse iNOS showed little protection against IL-23-induced changes. Although (Inducible nitric oxide synthase) reporters were repressed close to basal depleting conventional LCs alone had no effect, inflammation was levels of promoter activity. PXR1 SUMO mutants repressed LPS-in- much diminished in langerin-DTR mice also depleted of moLCs. duced IL-8 activity but to a lesser extent than wt PXR1. However, Furthermore, depleting Ly6C+ monocytes but not neutrophils signif- repression of this activity by the PXR3 SUMO mutants was compa- icantly reduced the moLC and moDDC accumulation in skin, IL-22, rable to that of wt PXR3. This may indicate the occurrence of IL-17A/F and IL-1b production, and skin inflammation. Together, sumoylation events at multiple sites within PXR and that the mutation these data suggest that CD11c+ moDDCs and moLCs contribute of one single site is insufficient to prevent sumoylation. Nickel pull significantly to pathology. Previously, we have reported that Ccr6-/- down assays confirmed previous findings that PXR1 was conjugated by mice are resistant to IL-23-induced skin inflammation, and the skin of each of the SUMO proteins. This was also true for PXR3. Conjugation Ccr6-/- mice showed no changes in numbers of CD11c+ cells in of proteins with SUMO like other PTMs, alters the functions of pro- dermis or epidermis after IL-23 injection. Repeated and selective teins. The sumoylation of both PXR1 and PXR3 could help elucidate depletion of Ccr6+/+ CD11c+ cells using mice reconstituted with a how PXR acquires this anti-inflammatory ability in addition to its mixture of bone marrows from Ccr6-/- and (Ccr6+/+) CD11c-DTR transactivation function. Importantly, the repressive activity and mice revealed that CCR6 was required for the recruitment and/or sumoylation of PXR3 are novel findings and may specify a role for activity of the pathologic CD11c+ cells or their precursors. Using PXR3 in inflammation. In fact, PXR1 and PXR3 are expressed in many Ccr6-EGFP mice, we identified expression of Ccr6 in blood mono- of the same tissues, therefore the anti-inflammatory activity attributed cytes and we demonstrated that monocytes migrate to the CCR6 to PXR1 may actually be attributed to both PXR1 and PXR3. ligand, CCL20. Importantly, we found that although both Ccr6+/+ and Ccr6-/- monocytes restored inflammation when injected, together with IL-23, into ears of Ccr6-/- mice, only the Ccr6+/+ monocytes were effective if injected intravenously. Together, our data suggest Innate Immunity—Macrophages, that blood monocytes are recruited to inflamed skin using CCR6 and Dendritic Cells, Neutrophills, Basophils, give rise to moDDCs and moLCs, which produce IL-1b and are critical for IL-23-induced psoriasis-like pathology. Mast Cells, Eosinophils

B200 Inhibitory Receptors and Immune ROLE OF PROSTAGLANDIN D2 IN EOSINOPHIL Checkpoints ACTIVATION INDUCED BY LEPTIN Nata´lia Tasmo-Amorim1, Ludmilla Dellatorre-Teixeira1, B199 Tatiana Luna-Gomes1, Patricia Bozza2, Clarissa Maya-Monteiro2, THE ANTI-INFLAMMATORY ACTIVITY OF THE Christianne Bandeira-Melo1 NUCLEAR RECEPTOR PXR AND THE MINOR 1Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; ISOFORM PXR3 2Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil

Jerusalem Alleyne, Andrew Bennett Eosinophils are classically associated with allergic diseases and hel- minth infections. Recently, immunomodulatory roles of eosinophils FRAME laboratory, School of Biomedical Sciences, University of have been described, such as the ability of adipose tissue-resident Nottingham, UK eosinophils to regulate adipose macrophage functions and tissue hemostasis. Leptin, a hormone/cytokine produced by adipocytes, is a Objective and design: The major isoform of the Pregnane X receptor survival factor for eosinophils, which are known to express its (PXR1) controls the expression of genes involved in liver detoxifi- receptor. Inasmuch as eosinophils are known to be cell sources of an cation. Recent findings have revealed that PXR1 represses autocrine/paracrine functionally active prostaglandin D2 (PGD2), the inflammatory pathways. The minor isoform PXR3 is transcriptionally aim of this study was to evaluate the role of eosinophil-derived PGD2 inactive and its role is unknown. It is believed that PXR1 undertakes in leptin-induced eosinophil activation. For in vivo assays, mouse 123 S182 Inflamm. Res. pleurisy was triggered by intrapleural injection of leptin. Within 24 h, B202 in parallel to eosinophil influx and activation (characterized by PLATELET ACTIVATING ACETYLHYDROLASE eosinophil lipid body biogenesis and increased levels of pleural INVOLVED IN INFLAMMATION RESOLUTION IN LTC4), we found increased levels of PGD2 in the pleural cavity of leptin-stimulated mice. PGD2 appeared to be a key mediator of MACROPHAGES AND NEUTROPHILS WHICH leptin-induced eosinophil activation in vivo, since the pre-treat- HAVE BEEN STIMULATED WITH URATE ment with HQL-79, an inhibitor of hematopoetic PGD synthase, CRYSTALS inhibited both lipid body biogenesis within eosinophils and LTC4 synthesis. Accordinlgy, using either human eosinophils purified Darshna Yagnik from healthy donors or bone marrow-differentiated mouse eosi- nophils, leptin was also able to trigger PGD synthesis in vitro as 2 Middlesex University, Department of Natural Sciences, London, UK detected in eosinophil supernatants within 1 h of stimulation. In vitro HQL-79 pre-treatment inhibited both eosinophil lipid body Background: A model of gout which consists of human blood derived biogenesis and LTC4 synthesis elicited by leptin. Our data clearly in vitro differentiated monocytes/macrophages and neutrophils are uncover the ability of leptin to activate PGD2 synthesizing immune cells which have been investigated over the years to deter- machinery in both mouse and human eosinophils. Moreover, such mine the role these cells play in the resolution phase of gout. leptin-driven eosinophil-derived PGD2 displays autocrine/para- Macrophages and neutrophils are able to phagocytose monosodium crine regulatory role in leptin-induced eosinophil activation. urate monohydrate (MSU) crystals without releasing inflammatory Inasmuch as PGD2 is now emerging as a immunomodulatory factors. This study analysed platelet activating acetylhydrolase (PAF- molecule, our findings may indicate potential functions on obesity AH) secretion by human macrophages and neutrophils derived from and other inflammatory disorders. whole blood upon phagocytosis of MSU crystals. Method: Monocytes and neutrophils were isolated from whole blood taken from human blood donations collected from the National Blood NHS Service using standard isolation procedures using percoll and B201 dextran sedimentation. Isolated monocytes were differentiated for a INTERNALIZED CRYPTOCOCCUS NEOFORMANS period of 7 days in vitro into macrophages. Day 1 monocytes or day 7 ACTIVATES NOT ONLY THE CANONICAL in vitro differentiated macrophages were then stimulated with MSU CASPASE-1 BUT ALSO THE NONCANONICAL crystals (0.5 mg/mL), or LPS (10 lg/mL) and signalling inhibitors for a period of 17 h. Cultured supernatants were then collected and CASPASE-8 INFLAMMASOMES assayed for Tumour necrosis (TNF) alpha, PAF acetyl hydrolase (PAF-AH) secretion by ELISA. 1 1 1 1 Guangxun Meng , Mingkuan Chen , Yue Xing , Ailing Lu , Results: Analysis of supernatants from in vitro differentiated mac- 2 2 Wei Fang , Wanqing Liao rophages stimulated with MSU crystals revealedPAF-AH secretion whereas monocytes did not secreted PAF-AH. Release of macrophage 1 Key Laboratory of Molecular Virology and Immunology, Institut PAF-AH (0.6 pg/mL ± 0.01) was inhibited by a protein kinase Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, inhibitor (SB203580 at a range of 50–300 nM) when added to the 2 Chinese Academy of Sciences, Shanghai, China; Shanghai Key assay at the same time as MSU crystals. Similar results were obtained Laboratory of Molecular Medical Mycology, Changzheng Hospital, with neutrophils. Shanghai, China Conclusion: This study identifies a role for neutrophil and macro- Cryptococcus neoformans (C. neoformans) is an opportunistic fungal phage derived PAF-AH in inflammation resolution through PKC pathogen which causes Cryptococccosis in immunocompromised signalling in the pathway by which immune cells ingest MSU crystals patients as well as immunocompetent individuals. Host cell surface and resolve the concomitant inflammation. The PAF-AH enzyme receptors that recognize C. neoformans have been widely studied. could be used therapeutically for treatment of patients with recurrent However, intracellular sensing of this pathogen is still poorly under- or treatment resistant gout. stood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NLRP3 inflammasome. In the current study, it was found that opsonization B203 mediated internalization of encapular C. neoformans also activated the canonical NLRP3-ASC-caspase-1 inflammasome. In addition, the BACTERIAL METABOLITES, SHORT CHAIN internalized C. neoformans activated the noncanonical NLRP3-ASC- FATTY ACIDS, ATTENUATE THE IMMUNE caspase-8 inflammasome as well, which resulted in robust IL-1b RESPONSE TO AGGREGATIBACTER secretion and cell death from caspase-1 deficient dendritic cells. ACTINOMYCETEMCOMITANS Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further Renan Oliveira Correa, Aline Vieira, Erica Moraes Sernaglia, mechanistic studies showed that both the phagolysosome membrane Marco Aurelio Ramirez Vinolo permeabilization and potassium efflux were responsible for C. neo- formans induced activation of either the canonical NLRP3-ASC- State University of Campinas (UNICAMP), Campinas, Brazil caspase-1 inflammasome or the noncanonical NLRP3-ASC-caspase-8 inflammasome. Moreover, infection with Candida albicans or challenge Short chain fatty acids (SCFAs) including butyrate, propionate, and with the fungal PAMP zymosan also led to the activation of the non- acetate are products of bacterial fermentation. These metabolites canonical NLRP3-ASC-caspase-8 inflammasome in cells absent for attenuate inflammation and may be useful in the treatment of condi- caspase-1. Collectively, these findings uncover a number of novel tions such as inflammatory bowel disease and obesity. However, their signaling pathways for the innate immune response of host cells to C. role in the immune response during infectious diseases is still unknown. neoformans infection, and suggest that manipulating NLRP3 signaling The aim of this study was to test the effect of SCFAs in leukocytes may help to control fungal challenge. recruitment and effector function in response to Aggregatibacter

123 Inflamm. Res. S183 actinomycetemcomitans (Aa), a bacteria implicated in periodontal B205 disease. For this, we have used the in vivo subcutaneous chamber PGD2 MEDIATES EOSINOPHILIC model in C57BL/6 mice. Ten days after subcutaneous implantation of a stainless coil chamber, a solution containing Aa with or without dif- INFLAMMATION DURING S. MANSONI ferent concentrations of SCFAs was inoculated. After 4 or 24 h, the INFECTION animals were sacrificed and the inflammatory exsudates were collected for measurement of cytokines (TNF-a,IL-1b, IL-6, IL-10, IL-12) and Laı´s C. Agra1, Camila R.R. Pa˜o1, Isaac Bellas1, Tatiana Luna- chemokines (Cxcl1, Cxcl2), evaluation of leukocyte recruitment, bac- Gomes1, Valdirene S. Muniz2, Ludmilla Dellatorre-Teixeira1, terial killing capacity and phagocytic activity. The presence of the Natalia R. Amorim1, Ligia A. Paiva3, Christianne Bandeira-Melo1 SCFAs did not modulate leukocyte recruitment in response to bacteria. However, the production of TNF-a, IL-10, and Cxcl2 decreased in the 1Instituto de Biofı´sica Carlos Chagas Filho, UFRJ, Rio de Janeiro, group inoculated with SCFAs at 24-h. In addition, the killing of Aa by Brazil; 2Instituto de Cieˆncias Biome´dicas, UFRJ, Rio de Janeiro, the immune cells was 10 times less effective for the SCFAs-treated Brazil; 3Instituto Oswaldo Cruz, Fiocruz/RJ, Instituto Oswaldo Cruz, group. Furthermore, the phagocytic activity of the immune cells was Fiocruz/RJ, Rio de Janeiro, Brazil also reduced when treated with SCFAs for 4 h. In vitro experiments Background: Prostaglandin D2 (PGD2) is a lipid mediator mainly have confirmed these in vivo observed patterns. By incubating neu- produced by mast cells in chronic allergic asthma and other inflam- trophils and Aa (or LPS) together with non-cytotoxic concentration of matory disorders. Recently, it has been demonstrated that eosinophils SCFAs separately for 6 h, the production of TNF-a and IL-10 are also cell sources of PGD2, which in turn is recognized as a potent decreased when treated with some concentrations of propionate, stimulus of eosinophil activation. During S. mansoni infection, butyrate, and acetate (this last one only for IL-10 in the LPS group). schistosoma-derived PGD2 has emerged as a key parasite regulator of However, an increase in the production of IL-1b was observed for immune defense evasion, controlling cutaneous immune response similar conditions. Moreover, the SCFAs also reduced the phagocytic through inhibition of Langherhans cells migration. However, the role activity of neutrophils in vitro after 2 h of incubation. The results of of host PGD2 during S. mansoni infection is not established. this study are still preliminary, but they suggest that SCFAs reduce the Aim: In this work, we investigated the role of host-derived PGD2 immune response to Aa and may play act as a bacterial mechanism of during the progression of experimental S. mansoni infection. evasion. This may be relevant for periodontitis development but also Methods: C57/Bl6 mice were infected by transcutaneous penetration for other types of infections caused by anaerobic bacteria. of 60–70 cercariae of Schistosoma mansoni (BH strain, Institute Oswaldo Cruz, RJ, Brazil). At 24th day, osmotic pumps (AlzetÒ pump; 100 ll) containing HQL-79 (deliver rate: 24 lg/day), a B204 specific inhibitor of hematopoeitic PGD synthase (H-PGDS), were RECOGNITION OF PRODUCTS FROM implanted subcutaneously in infected and non-infected mice for 4 weeks, when animals were sacrificed. Besides, infection parame- NEUTROPHIL DEGRANULATION BY TOLL LIKE ters, eosinophilia, lipid mediator production and fibrosis were RECEPTORS MEDIATE KILLING OF analyzed in bone marrow, blood, peritoneal fluid, and livers. LEISHMANIA AMAZONENSIS IN MACROPHAGES Results: First, we learned that increased levels of PGD2 are found in liver and peritoneal cavity of S. mansoni-infected mice and that such Natalia M. Tavares1, Lilian Afonso1, Martha Suarez1, systemic production of PGD2 was impaired by HQL-79 treatment. Mariana R. Ampuero1,3, Stella d´Areˆde1,3, Aldina Barral1,3, HQL-79 decreased eosinophilia in peripheral blood, peritoneal fluid and George dos Reis2, Vale´ria M. Borges1, Cla´udia Brodskyn1,3 in liver granulomas of S. mansoni-infected mice, while no alteration was observed in bone marrow eosinophilia, indicating that inhibition of 1CPqGM/FIOCRUZ, Salvador, Brazil; 2Universidade Federal do Rio PGD2 synthesis affects only peripheral eosinophilia. Whereas the size de Janeiro, Rio de Janeiro, Brazil; 3Universidade Federal da Bahia, of hepatic granulomas was not modified by HQL-79, inhibition of Salvador, Brazil PGD2 synthesis reduced the number of schistossomal granulomas in the liver compared to non-treated mice. Moreover, HQL-79 treatment Neutrophils mediate early responses against pathogens and they become decreased hepatic amounts of leukotriene C4, indicating that infection- activated during endothelial transmigration towards the inflammatory related endogenous PGD2 induces lipoxygenase activity during infec- site. In the present study, human neutrophils were activated in vitro with tion. However, we did not observe any difference in infection-induced immobilized extracellular matrix (ECM) proteins. Neutrophil activation collagen deposition between HQL-79-treated and non-treated mice. by fibronectin (FN), but not other ECM proteins, induces the release of Conclusion: Our results indicate that hematopoeitic cells-derived granules content, measured by matrix metalloproteinase 9 (MMP-9) and endogenous PGD2 is a key lipid mediator of schistosomiasis, con- neutrophil elastase (NE) activity in culture supernatant, as well as trolling granuloma formation and systemic eosinophilia installation reactive oxygen species (ROS) production. Upon contact with L. ama- during infection. zonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granules content (NE, MMP-9) and production of B206 inflammatory mediators, TNF-a and leukotriene B4 (LTB4), participate INTERFERON REGULATORY FACTOR 8 in parasite killing by infected macrophages. Besides, inhibition of degranulation from FN-activated neutrophilsabrogatedLTB4 production REGULATES THE MICROGLIAL CELL and reverted parasite killing. Moreover, the addition of exogenous RESPONSE TO STERILE INJURY IN THE BRAIN granules proteases increased leishmanicidal activity of infected mac- rophages and this effect was dependent on Toll like receptor (TLR). Iain L. Campbell1, Rue Dan Xie1,Na`dia Villacampa2, Together, these results points out the role of degranulation from FN- Beatriz Almolda2, Berta Gonza´lez2 activated neutrophils in the induction of protective mechanisms of macrophages that may contribute to the control of Leishmania infection, 1University of Sydney, Sydney, Australia; 2Autonomous University of but also to the local inflammatory response. Barcelona, Barcelona, Spain

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The transcription factor interferon regulatory factor (IRF) 8 regulates by both agonists. The IL-6 and TNF-a peak responses were reduced myeloid cell differentiation and function. We identified IRF8 to be a only in LPS-stimulated macrophages. Addition of cPAF did not affect constitutive nuclear factor that regulates the homeostatic properties of macrophages response to the MyD88-independent Poly(I:C). Adi- microglia—the tissue-resident macrophages of the brain. Here we tionally, cPAF reduced the LPS-induced COX-2 expression and asked if IRF8 modulates the microglial response to sterile neuronal PGE2 production, but did not affect the iNOS expression and nitrite injury. Facial nerve axotomy (FNA) was performed in wild type (WT) formation. The down regulation of cytokine production induced by and IRF8-/-(KO) mice and the brains removed at different times cPAF was independent on the adaptor molecules, once it did not post-lesion. A subset of mice was injected with bromodeoxyuridine influence MyD88 and TRIF mRNA expression. However, cPAF (BrdU) prior to retrieval of the brain. Changes in the facial nucleus decreased the phosphorylation of NF-jB p65 subunit induced by LPS (FN) were examined by immunohistochemistry and histochemistry. In and Pam3Cys in 15 min, without altering IjB-a phosphorylation and brains from IRF8 KO mice, nucleoside diphosphatase (NDPase) p65 translocation to the nucleus. histochemistry and lectin staining revealed gross alterations in the Conclusion: These findings indicate that the down regulation induced morphology of microglia, which were stunted and hypertrophied. by PAF in TLR-triggered responses was caused by impaired tran- After FNA in WT mice, a progressive increase in microglial activa- scriptional activity of the p65 subunit. These data unveil a heretofore tion was observed in the lesioned FN peaking at day 7 and was unrecognized role for the PAF-R in MyD88-dependent activation of accompanied by dense staining for Iba1, lectin, NDPase and CD11b. NF-jB in macrophages. By contrast, in IRF8 KO mice, the microglial response to FNA was Financial support FAPESP, CAPES and CNPq markedly attenuated with little staining for Iba1, while the density of staining for lectin, NDPase and CD11b was reduced significantly. The attenuated microglial response in IRF8 KO mice was paralleled by a significant decrease at day 3 post-lesion in proliferation of these cells B208 when compared with WT. Furthermore, a decrease in the number of CD39/ENTPD1 EXPRESSION ON MACROPHAGES PU.1-positive cells was observed in the FN of IRF8 KO mice com- LIMITS ATP-P2X7 RECEPTOR PRO- pared with WT. The wrapping of individual motor neuron cell bodies INFLAMMATORY SIGNALING RESPONSES IN in the axotomised FN by microglia involved in synaptic stripping and phagocytosis was impaired in the absence of IRF8. Finally, in IRF8 SEPSIS KO mice, the degeneration of axotomised motor neurons was sig- nificantly increased. These studies demonstrate a crucial requirement Luiz Eduardo B. Savio1,2, Paola A. Mello2,4, Vanessa R. Figliuolo 1, for IRF8 in regulating multiple functions of microglia in the innate Thiago Fernandes A. Almeida1, Patrı´cia T. Santana1, Suellen D’arc response of these cells to neuronal injury. S. Oliveira1,3, Claudia Lucia M. Silva3, Linda Feldbru¨gge 2, Yan Wu2, Simon C. Robson2, Robson Coutinho-Silva1

1Laboratory of Immunophysiology, Biophysics Institute Carlos B207 Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, 2 PAF IMPAIRS MYD88-ENHANCED NF-jB P65 Brazil; Department Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Harvard University, TRANSCRIPTIONAL ACTIVITY IN MURINE 3 Boston, MA, USA; Laboratory of Molecular and Biochemical MACROPHAGES Pharmacology, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 4Faculty of Pharmacy, Edson K. Ishizuka1, Carlos H. Serezani2, Luciano R. Filgueiras1, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Sonia Jancar1 Background: Sepsis is a leading cause of death worldwide. Mortality is related to onset of shock with inflammatory responses, which are 1University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Indiana University dependent upon Toll-like receptor (TLR) activation in monocyte- School of Medicine, Indianapolis, IN, USA macrophages. Upon TLR activation, adenosine triphosphate (ATP) is Introduction: Platelet-activating factor receptor (PAF-R) and Toll-like released into the extracellular milieu. Extracellular ATP (eATP) receptors (TLRs) are receptors highly expressed in macrophages and serves as a novel danger signal to heighten inflammatory responses are important components of homeostatics processes and host defense via activation of the type 2-purinergic receptor P2X7 (P2X7R). We against invading pathogens. Macrophage responses usually derive propose that the proinflammatory properties of eATP-P2X7R sig- from integration of signals transduced by more than one receptor. In naling will be countered by ectonucleotidases, such as CD39 and this study, we investigated the effect of PAF-R activation in the associated with lipid rafts in plasma membranes. responsiveness of thioglycollate (TG)-elicited macrophages to Aim: To investigate the modulatory role of CD39 during ATP-P2X7 MyD88-dependent and independent agonists. signaling in sepsis. Methods: C57BL/6 male mice were intraperitoneally injected with Methods: For in vitro assays, peritoneal macrophages (MU)orbone 2 mL of 3 % thioglycollate. After 4 days, peritoneal macrophages marrow derived MU were primed with 1 lg/mL LPS for 4 h, followed (PMs) were collected and stimulated simultaneously with the stable by stimulation with ATP (500 lM) or Bz-ATP (100 lM; a P2X7 PAF (1-hexadecyl-2-N-methylcarbamyl-glycero-3-phosphocholine; agonist) for additional 3 h. Before priming with LPS, cells were also cPAF) 100 nM and the TLR agonists (LPS 100 ng/mL, Pam3Cys pretreated with select P2X7R antagonists (oxidized ATP (oATP): 100 ng/mL or [Poly(I:C)] 50 lg/mL). Pro-inflammatory cytokines 300 lM, 2 h; or A740003: 100 nM, 30 min), or with imipramine (acid (IL-6, TNF-a and IL-12p40) and PGE2 were measured by ELISA and sphingomyelinase inhibitor; 30 lM, 1 h). Ecto-enzymatic activities the expression of adaptor molecules MyD88 and TRIF by qPCR. were assayed by TLC, protein localization determined by immunos- Protein samples were subjected to Western blot analysis to investigate taining, intracellular signaling pathways by Western blot and cytokine the NF-jB pathway. release assayed by ELISA. Sepsis was induced by cecal ligation and Results: We found that in macrophages stimulated with MyD88-de- puncture (CLP) in wild type (WT) and CD39-/- mice. P2X7R function pendent agonists (LPS and Pam3Cys), addition of exogenous cPAF was inhibited pharmacologically in vivo, using the receptor antagonist drastically reduced the peak response of IL-12p40 production induced brilliant blue G (45.5 mg/kg via i.p. 24 h before CLP).

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Results: ATP or Bz-ATP boosted CD39 activity in LPS-primed MU. B210 Furthermore, CD39 co-localized with the lipid raft marker Flotillin-2 NFAT SIGNALING PATHWAY IN INNATE in the LPS- and ATP-treated cells. Drugs that disrupt cholesterol- enriched domains—such as nystatin and methyl-b-ciclodextrin—de- IMMUNE CELLS PRIMES THE INFLAMMATORY creased CD39 enzymatic activity, irrespective of prior stimulation PROCESS DURING SKIN FUNGAL INFECTIONS with LPS and ATP. Both oATP and imipramine attenuated ATP- induced increases in CD39 expression and activity as well as con- Francesca Granucci1,2, Simona Barresi2, Francesca Mingozzi2, current STAT3 activation in LPS-primed cells. LPS- and ATP- William Santus2, Marina Vai2, Ivan Orlandi2, Ivan Zanoni2,1,3 stimulated CD39-/- cells produced more IL-1b and less IL-10 than WT cells. P2X7 inhibition also decreased LPS-induced cytokine 1Humanitas Clinical and Research Center, Milan, Italy; 2University production, as well as ATP-elicited activation of NF-kappaB and of Milano-Bicocca, Milan, Italy; 3Harvard Medical School, Boston, AKT/mTOR pathways. CD39-/- septic mice exhibited increased IL- MA, USA 1b levels and a reduced production of IL-10 in blood and peritoneal Candida (C.) albicansinfections occur frequently in hospitalized lavage fluid (PLF), when compared to matched WT counterparts. patients, especially if they are debilitated or immunocompromized. P2X7 blockade decreased blood and PLF levels of IL-1b, IL-6 and -/- Candida (C.) albicans infections can develop in different anatomic IL-10 in both WT and CD39 septic mice. sites, including skin and subcutaneous tissues. Fungal ligands are Conclusion: This study confirms that eATP signaling through P2X7R among the most potent stimuli able to induce the activation of the contributes to inflammatory responses in sepsis. Further, our findings NFAT signaling pathway in innate immune cells. The NFAT pathway highlight the crucial role of CD39 in the control of sepsis-related functions have been best characterized in adaptive immunity while responses and suggest modulatory roles of lipids. CD39 comprises an very little is know in innate immune cells. In the present work we important purinergic regulatory mechanism crucial in limiting have investigated the consequences of NFAT activation in innate inflammation and restoring homeostasis during sepsis. immune cells in models of Candida induced inflammation. Financial support NIH, CNPq, CAPES. We found that in vitro NFAT activation in BMDCs in response to curdlanor Candida (C.) albicans stimulation potentiates PGE2 pro- duction. When we infected mice intradermally with C. albicans we B209 found NFATc2 nuclear translocation in dermal DCs 1 h post infec- ABSENCE OF DIETARY FIBRE OR THE tion. Moreover, inflammatory cell infiltration and ulcer formation were observed in wild type animals infected with Candida. Ulcers METABOLITE SENSOR GPR43 EXACERBATES normally resolved in 1 week. Diversely, in CD11c+ cells-depleted or NEUTROPHIL RECRUITMENT DURING ACUTE NFATc2-deficient mice we observed the formation of a necrotic cyst INFLAMMATORY RESPONSES due to fibroblasts proliferation with no recruitment of inflammatory cells and no ulceration. The necrotic cyst persisted for the entire Connie H. Wong1, Marjon Kamp1, Raymond Shim1, Ana Carolina duration of the experiment. Fibroblast proliferation and cyst forma- Oliveira2, Linda J. Mason1, Lauren Binge1, Charles R. Mackay1 tion were induced by the release of active TGFbeta. In wt animals, TGFbeta functions were counteracted by NFAT-dependent PGE2 1Monash University, Melbourne, Victoria; 2Universidade Federal do production. No PGE2 production was, indeed, observed in NFATc2- Rio de Janeiro, Rio de Janeiro, Brazil deficient animals and the injection of PGE2 or TGFbeta inhibitors blocked cyst formation and reversed the phenotype. We also observed Fermentation of dietary fibre in the gut yields high amounts of short that both CD11c+ cells and NFATc2 were required to induce C. chain fatty acids (SCFAs). SCFAs can impart immediate biological albicans transport to the draining lymph node and the activation of responses on cells through their engagement of ‘metabolite-sensing’ G adaptive T cell responses. In the absence of DCs or NFAT activation protein-coupled receptors (GPCRs). One of the main SCFA receptors, C. albicans remained confined to the dermis inside the necrotic cyst. GPR43, is highly expressed by neutrophils, a cell type central to many Altogether, our data support the hypothesis that NFAT activation inflammatory reactions. This suggests that dietary fibre intake and the in innate immune cells, particularly in CD11c+ cells, regulates the actions of GPR43 may affect neutrophil recruitment during inflam- earliest events of the innate immune response elicited against fungi. matory responses in vivo. We examined the inflammatory response of Our data demonstrate that NFAT-dependent production of PGE2 -/- wildtype and Gpr43 mice, fed on normal chow, or diets consisting orchestrates inflammatory cell recruitment at the infection site and the of no fibre or high fibre, in models of acute intestinal inflammation. efficient transport of the pathogen to the draining lymph node thereby Under basal conditions, bone marrow neutrophils of no fibre-fed controlling adaptive immune system activation. wildtype mice exhibited elevated migratory behaviour towards CXC chemoattractants compared to normal chow-fed wildtype mice. Inter- estingly, this hyper-migratory behaviour of neutrophils could also be reproduced through simple transfer of a no fibre microbiota into germ- B211 free mice, suggesting that the composition and function of microbiota DUAL ACTION OF PLATELETS AND stemming from a no fibre diet mediated the changes in neutrophil GLYCOPROTEIN VI IN IMMUNE COMPLEX- migration. Using intravital imaging of the small intestine, we found accelerated intravascular neutrophil rolling and adhesion in Gpr43-/- MEDIATED INFLAMMATION mice in response to LPS at 1 h, with increased numbers of neutrophils 1 1 1 found in the lamina propria at 4 h. Similarly, GPR43-deficient leuko- Angele Gros , Varouna Syvannarath , Lamia Lamrani ,Ve´ronique 1 1 2,3 4 cytes demonstrated exacerbated migration into the peritoneal cavity Ollivier , Ste´phane Loyau , Tobias Goerge , Bernhard Nieswandt , 1 1 following cecal ligation and puncture, or after fMLP injection. More- Martine Jandrot-Perrus , Benoıˆt Ho-Tin-Noe´ over, fMLP-induced leukocyte migration was reduced in wildtype, but 1 not Gpr43-/- mice fed a high fibre diet, or acetate. Therefore, GPR43 UMRS-1148 Inserm-University Paris Diderot, Laboratory for and a microbiota composition that allows for SCFA production regulate Vascular Translational Science, Bichat Hospital, Paris, France; 2 proper neutrophil recruitment during inflammatory responses. Department of Dermatology, University Hospital of Mu¨nster,

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Mu¨nster, Germany; 3Interdisciplinary Center for Clinical Research Methods: We firstly detected the expression and the activity of the (IZKF), University of Mu¨nster, Mu¨nster, Germany; 4Chair of indoleamine 2,3-dioxygenase-1(IDO-1) in the human monocyte- Vascular Medicine, University Hospital Wu¨rzburg & Rudolf Virchow derived dendritic cells (moDC) infected with N. gonorrhoeae; Then Center, University of Wu¨rzburg, Germany we tested the effect of the IDO-1 inhibitor, L-1-MT, on the prolif- eration of the T cells stimulated by the N. gonorrhoeae treated moDC; Background: Beyond their role in thrombus formation and primary Finally, neutralizing antibodies against the IFN-c and the TLR4 were hemostasis, platelets are crucial actors of innate and adaptive immune added to the coculture system of the N. gonorrhoeae and the moDC, responses. They maintain vascular integrity during inflammation. separately, to determined the involvement of the IFN-c or TLR4 in Evidence suggests that this protective function is independent of the induction of the active IDO-1. thrombus formation and involves platelet immunoreceptor tyrosine Results: N. gonorrhoeae could induce the active IDO-1 in human activation motif signalling. However, it remains unclear how platelets moDC, and L-1-MT can enhance the proliferation of the T cells prevent inflammatory bleeding. Our hypothesis is that platelets could stimulated by the N. gonorrhoeae treated moDC. The induction of dampen or repair neutrophil-inflicted vascular damage. IDO-1 by N. gonorrhoeae does not depend on the viability and the Aim: Our objective was to determine how platelets and glycoprotein intactness of the bacterial cells, but partially mediates by TLR4 and VI (GPVI) contribute to maintain vascular integrity during partially depends on the production of IFN-c. inflammation. Conclusions: N. gonorrhoeae induces the production of IDO-1 in Methods: In models of immune complex (IC)-mediated dermatitis human moDC to down regulate the T cell proliferation, which could and peritonitis combined with immunodepletion of platelets and/or be one of the immune regulation mechanisms of the N. gonorrhoeae. neutrophils in wild-type and/or GPVI deficient mice, we investigated the contribution of platelets to the regulation of neutrophil recruit- ment, infiltration, and injurious activities. Using intravital microscopy, we analyzed the contribution of GPVI to platelet B213 recruitment and the interactions between platelets and neutrophils at THE ROLE OF MerTK AND ITS CLEAVAGE IN the reaction site. Through a transfusion-based experiment of platelets treated with tyrosine kinase inhibitors in GPVI-/- mice, we assessed THE RESOLUTION OF ACUTE STERILE the consequences of GPVI signaling inhibition on the prevention of INFLAMMATION neutrophil inflicted vascular damage. Results: Depletion of neutrophils prevented skin bleeding observed Bishuang Cai, Gabby Fredman, Ira Tabas in thrombocytopenic and GPVI-deficient mice subjected to der- matitis, indicating that platelets counter the deleterious effect of Columbia University, New York City, NY, USA neutrophils. However, during dermatitis and peritonitis, neutrophil infiltration was reduced in thrombocytopenic mice whereas degran- Efficient clearance of apoptotic cells (ACs), or efferocytosis, and the ulation, and oxidative stress were reduced in both GPVI-deficient generation specialized pro-resolving mediators (SPMs) are required and thrombocytopenic mice as compared to wild-type mice. Intrav- for inflammation resolution. Mer tyrosine kinase (MerTK) is a key ital microscopy revealed that in inflamed vessels, platelets interacted efferocytosis receptor in macrophages. Our lab and others have shown directly with both neutrophils and the vascular wall. Furthermore, that MerTK plays a critical role in clearing ACs in advanced during IC-mediated dermatitis, intravascular binding sites for GPVI atherosclerosis. The ectodomain of MerTK can be cleaved by the were exposed by neutrophils, and GPVI supported the recruitment of metalloproteinase ADAM17 by pro-inflammatory stimuli, resulting in platelets to these spots. The platelet secretory response accompany- a decrease of surface MerTK on macrophages. While the anti-in- ing IC-mediated inflammation was partly mediated by GPVI and flammatory actions of MerTK have been described, little is currently blocking of GPVI signalling impaired the vasculoprotective action of known about MerTK’s pro-resolving roles. We hypothesize that platelets. preventing MerTK cleavage and thus retaining surface MerTK leads Conclusion: Our results indicate that platelets and GPVI play a dual to a pro-resolution circuit that involves enhanced efferocytosis and role in inflammation by enhancing neutrophil recruitment and dam- SPM generation. To test this hypothesis, we used a sterile peritonitis model initiated by zymosan A in wild type (WT), Mertk-/- or newly aging activities while ensuring sealing of neutrophil-inflicted vascular cr injury through GPVI-dependent adhesion and activation of single engineered MerTK cleavage resistant (Mertk ) mice and assessed platelets. Our study suggests that platelets should be considered as resolution intervals (Ri), in vivo efferocytosis and SPMs in peritoneal exudates. We found a significant delay in resolution measured by Ri integral players of immune complex-mediated inflammation that -/- intervene at all phases of the inflammatory response. of 13 h in WT vs 23 h in Mertk . Importantly we observed a sig- nificant decrease in both in vivo efferocytosis and SPM (i.e. lipoxin -/- A4 and Resolvin D1) synthesis in Mertk mice compared with the control mice, indicating that inflammation resolution was delayed in -/- B212 Mertk mice. In contrast, PMN clearance was enhanced and both in vivo efferocytosis and pro-resolving mediators were increased in cr THE IMMUNE REGLLATION OF THE NEISSERIA Mertk mice with a decrease of Ri by 54 %, indicating that blocking GONORRHOEAE INVOLVED INDOLEAMINE 2,3- MerTK cleavage improved inflammation resolution. To further elu- DIOXYGENASE-1 cidate the mechanism we observed that stimulating macrophages with a MerTK activating antibody resulted in significantly enhanced LXA4 production compared to the IgG control. Importantly, the MerTK Sai Li, Wenjing Le, Xiaohong Su -/- activating antibody was unable to stimulate LXA4 in Mertk macrophages. To understand these results in a more physiologic Institute of Dermatology, Chinese Academy of Medical Sciences, context, we also observed that Mertk-/- macrophages produced less Chaoyang, China LXA4 during efferocytosis than WT controls. Based on these studies, Objective: This study plans to prove the role of the immune regulation we conclude that MerTK signaling directly promotes inflammation of the indoleamine 2,3-dioxygenase-1 in N. gonorrhoeae infection. resolution by enhancing efferocytosis and SPM production.

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B214 The microbiota is critical in shaping the mammalian host’s MECHANISMS AND CONSEQUENCES OF LIPID immune system. Polysaccharide A (PSA), the archetypical immunomodulatory microbial molecule of the gut commensal BODY-PHAGOSOME INTERACTION IN Bacteroides fragilis, induces regulatory T cells to secrete the anti- MYCOBACTERIUM BOVIS, BCG INFECTION inflammatory cytokine interleukin 10. We show, in a model of colitis, that PSA requires both innate and adaptive immunity to Natalia R. Roque1, Silvia L. Lage1, Roberta Navarro1, generate protection. Dendritic cells mediate PSA’s effect on IL-10 Clarissa M. Maya-Monteiro1, Jens Rietdorf3, Rossana Melo2, production. Unlike conventional DCs, plasmacytoid DCs exposed Heloisa D’Avila2, Patricia T. Bozza1 to PSA do not produce the proinflammatory cytokines tumor necrosis factor-a and IL-12 but PDCs do specifically stimulate IL- 1Oswaldo Cruz Foundation, Fiocruz, Rio de Janeiro, Brazil; 2Federal 10 secretion by CD4+ T cells and efficiently mediate PSA-medi- University of Juiz de Fora, Juiz de Fora, Brazil; 3Center of ated immunoprotection. PSA induces and preferentially ligates Technological Development in Health, Fiocruz, Rio de Janeiro, Toll-like receptor 2 on PDCs but not on CDCs. Compared with Brazil other TLR2 ligands, PSA better enhances PDC expression of co- stimulatory molecules required for protection against colitis. PDCs Lipid bodies (LB) also named lipid droplets, are lipid rich organelles orchestrate beneficial immunoregulatory interaction of commensal that have been often associated with inflammatory and infectious microbial molecules with CD4+ T cells through both innate and conditions. The increase in number and size of these organelles is a adaptive immunity. well-regulated phenomenon that seems to be involved with bacterial persistence. Here we investigate the mechanisms by which mycobac- teria induced—lipid bodies may act in favor of infection. We observed that M. bovis bacillus Calmette-Gue´rin (BCG) and Lipoarabinoman- nan (LAM), but not nonpathogenic mycobacteria M. smegmatis or non- B216 coated latex beads, was able to induce LB formation in macrophages STRESS CONCENTRATIONS OF in vitro. By Transmission electron microscopy (TEM), we showed GLUCOCORTICOIDS CAN ENHANCE IMMUNE ADRP-marked LB interacting with phagosomes during BCG infection in vivo. Also, we showed LB in close apposition with phagosomes AND INFLAMMATORY PATHWAYS containing beads coated with LAM from M. tb, but not with non-coated beads. LAM coated beads interacts with LB 30 % more than non- Paul M. Guyre, Jane E. Collins, Nick M. Jensen, Patricia A. Pioli, coated beads in macrophages loaded with oleic acid. Moreover, we Fiona Barr, Sara Metzler, Brian D. Sites, Mark P. Yeager observed that Rab7, an important endocytic GTPase and late endosome marker, as well as its effector RILP but not Rab5 was co-localized in Geisel School of Medicine at Dartmouth, Hanover, NH, USA LB induced by BCG infection at 24 h. By TEM, we observed that Rab7 Glucocorticoids (GCs) are widely understood to suppress inflam- was co-localized with LB in the site of interaction with an infected mation through regulation of signaling pathways in leukocytes and phagosome during the experimental BCG infection in vivo. Interest- other effector cells. Until recently, these potent and clinically ingly, we observed a decrease of LAM coated bead-LB apposition important effects of GCs have obscured results from early research when macrophages were treated with CID1067700, a competitive documenting that GCs can stimulate in vivo defense mechanisms. inhibitor of Rab7. Next, we evaluated the role of LB modulation on We and others have shown that a 6-h in vivo exposure of healthy mycobacterial infection. Treatment with C75 (fatty acid synthase humans to stress-associated concentrations of cortisol induces a inhibitor) down-regulated LB formation and PGE2 synthesis induced substantial increase in the pro-inflammatory response to a subse- by BCG. Also, these treatments were able to enhance TNF-a while quent challenge with bacterial endotoxin. In those studies, stress inhibit the IL-10 production and down-modulation of mycobacterial cortisol pretreatment resulted in increased serum TNF-a IL-6, and survival and growth assessed by CFU count. These results suggest that reduced serum IL-10 during subsequent endotoxemia (Crit Care LB-phagosome interaction are well-regulated phenomena modulated Med. 2009; 37:2727–32). We have now used gene expression by mycobacteria cell wall components and dependent on Rab7. The microarray analysis of CD14-selected human monocytes, and hijack of active Rab7 to LB may enable the interaction with phago- additional in vivo studies to begin to identify signaling pathways some, and may favour the exchange of nutrients to mycobacterial that are differentially regulated by stress cortisol versus pharma- survival. In addition, LB modulate pro- and anti- inflammatory medi- cological concentrations of hydrocortisone. Human monocytes ators in favor of BCG survival and replication. Thus suggesting that were cultured for 24 h with either 50 nM or cortisol followed by inhibition of LB formation and/or function as a promising target for mRNA isolation and analysis using Agilent Technologies 44,000 therapeutic interventions in mycobacterial infection. element DNA microarrays. Several genes exhibiting differential Financial support CNPq, FAPERJ, Fapemig, PAPES-FIOCRUZ, up- or down-regulation by stress versus pharm cortisol were con- CAPES, CDTS. firmed by qPCR. Our results suggest that time-dependent changes Animal ethical approval number: # LW32/12. in expression of mRNA for IL1R2, ATF3, DUSP1, DUSP10, TLR2, NFKBIA, CCR2, CX3CR1 and SOCS3 may make impor- tant contributions to immune enhancement by stress cortisol. B215 Furthermore, when subjects were pretreated with cortisol or saline CRITICAL ROLE OF PLASMACYTOID and monocyte migration into sterile blisters was assessed 24 h DENDRITIC CELLS IN IMMUNOREGULATION later, mean monocyte density was 3.0 ± 0.6 following control INITIATED BY A COMMENSAL MICROBIAL treatment and 7.0 ± 1.9 following cortisol treatment (mean ± S.D.). Flow cytometric studies of cortisol-treated mono- POLYSACCHARIDE cytes showed upregulation of TLR2, CCR2 and CD163. These studies have begun to identify permissive and preparatory molec- Suryasarathi Dasgupta, Dennis L. Kasper ular changes induced by stress cortisol in human monocytes that can enable a more robust response to subsequent challenge by an Harvard Medical School, Boston, MA, USA infectious agent.

123 S188 Inflamm. Res.

B217 receptor (AHR), which is activated by a range of ligands of environ- ROLE OF INFLAMMASOME-DEPENDENT mental or dietary origin, has emerged recently as an important regulator of innate immunity. We have been interested in character- AND—INDEPENDENT MECHANISMS IN THE izing in detail primary host macrophage transcriptional responses to ADJUVANT EFFECT OF FLAGELLIN M.tb. infection. Transcriptome profiling of host macrophages revealed that M. tuberculosis (Mtb) infection induced expression of several Silvia L. Lage2,1,3, Carina B. Lima4, Eduardo P. Amaral3, Kelly C. enzymes controlling tryptophan (TRP) catabolism. This included Matteucci1,2,3, Lilian M. Massis4, Marcelo Y. Icimoto5, Adriana K. indole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase Carmona5, M. Regina D’Imperio-Lima3, Mauricio M. Rodrigues1,2, (TDO2), which catalyze the rate-limiting step in the kynurenine Luis C.S. Ferreira4, Gustavo P. Amarante-Mendes3, pathway, producing ligands for the aryl hydrocarbon receptor (AHR). Karina R. Bortoluci1,2 The AHR and heterodimeric partners AHR nuclear translocator (ARNT) and RelB are robustly expressed in macrophages, and AHR 1Departamentos de Cieˆncias Biolo´gicas e Microbiologia, Imunologia, and RelB levels further increased during infection. Infection enhanced e Parasitologia da Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, AHR/ARNT and AHR/RelB DNA binding, and stimulated expression Brazil; 2Centro de Terapia Celular e Molecular da Universidade of AHR target genes, including that encoding the inflammatory Federal de Sa˜o Paul, Sa˜o Paulo, Brazil; 3Departamentos de cytokine IL1B. AHR target gene expression was further enhanced by Imunologia, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; exogenous kynurenine, and exogenous TRP, kynurenine or synthetic 4Departamento de Microbiologia, Universidade de Sa˜o Paulo, Sa˜o agonist indirubin reduced mycobacterial viability. Comparative Paulo, Brazil; 5eDepartamento de Biofı´sica, Universidade Federal de expression profiling revealed that AHR ablation in infected macro- Sa˜o Paulo, Sa˜o Paulo, Brazil phages diminished expression of numerous genes implicated in innate immune responses, including cytokines, antimicrobial peptides and Flagellin is a natural agonist of TLR and NLR receptors that has been enzymes regulating intracellular signaling. Notably, infected cells extensively investigated as an adjuvant, although the mechanisms depleted for AHR exhibited reduced expression of IL23A and IL12B involved in its immunomodulatory properties remains controversial. transcripts, which encode subunits of interleukin 23 (IL23), a macro- Innate immune recognition of flagellin is shared by transmembranic phage cytokine that stimulates production of IL22 by innate lymphoid TLR5 and cytosolic Naip5/NLRC4 inflammasome complex. TLR5 cells. The AHR directly induced IL23A transcription in human and signaling activates a number of pro-inflammatory genes through MyD88 mouse macrophages through near-upstream enhancer regions con- pathway, while inflammasome is responsible for the induction of the pro- taining extended-consensus AHR binding sites. Taken together, these inflammatory cytokines IL-1b,IL-1a and IL-18 and necrotic cell death findings show that AHR signaling is strongly engaged in Mtb-infected named pyroptosis through caspase-1 activation. However, the molecular macrophages, and has widespread effects on innate immune responses regulation of inflammasome activation in response to flagellin, as well as to infection. Moreover, they reveal a cascade of AHR-driven innate the mechanisms involved in its imunomodulatory effect are still poorly immune signaling, as IL1b and IL23 induce expression of the gene understood. In this study, we evaluated macrophage activation in encoding IL22, another direct target of AHR transactivation. response to flagellin from inserted into lipid vesicles (Dotap), which allows flagellin delivery into cell cytosol, as well as, the impact of that stimuli in the adaptative immune response generation. By using this approach, atypical cell death induction was found in peritoneal macro- B219 phages deficient in inflammasome components (NLRC4, ASC and RECOMBINANT PENTRAXIN-2 PROTECTS caspase-1), besides the absence of IL-1b production. In this context, PROXIMAL TUBULAR EPITHELIAL CELLS IN macrophage death maintains its inflammatory and antimicrobial out- come, being accompanied by IL-1a secretion and early loss of THE KIDNEY DURING CELL STRESS membrane integrity. We also demonstrated that this process is regulated by a lysosomal pathway with a redundant role for cathepsins B and D. Ivan G. Gomez1,2, Naoki Nakagawa2, Mark Lupher3, Richard Jack3, Also, cathepsin B regulates the IL-1a and IL-1b secretion, suggesting a Jeremy S. Duffield1,2 cooperation between the inflammasome and lysosomal pathway in response to flagellin associated with Dotap. Furthermore, preliminary 1Biogen, Cambridge, MA, USA; 2Renal Division, University of results demonstrated that dendritic cells stimulated with flagellin from Washington, Seattle, WA, USA; 3Promedior Inc., Lexington, MA, USA Salmonella typhimurium inserted into Dotap was able to induce IFN-c Background: Pentraxin-2 is a naturally produced circulating plasma and IL-17 production by CD4+ T cells, being dependent of IL-1 sig- protein involved in innate immunity, whose level is decreased in naling. Thus, our data open new perspectives in the generation of vaccine chronic human fibrotic diseases. Recent studies indicate that systemic and therapeutic strategies based on the inflammasome activation. delivery of recombinant PTX-2 inhibits inflammatory diseases asso- ciated with fibrosis by blocking pro-fibrotic macrophage activation and promoting anti-inflammatory and regulatory macrophages. B218 Methods: Human PTECs were isolated from discarded human kidneys ENGAGEMENT OF THE ARYL HYDROCARBON that were digested using a collagenase-based method followed by magnetic immune affinity separation. To induce cell-stress, PTECs RECEPTOR IN M. TUBERCULOSIS-INFECTED were treated with TGFb (5 ng/mL) or 10 % human plasma with or MACROPHAGES HAS PLEIOTROPIC EFFECTS without rhPTX-2 (25 mg/mL) for 16 h or 24 h and evaluated by ON INNATE IMMUNE SIGNALING immune staining for a mesenchymal marker (Vimentin) and an epithelial marker (E-cadherin), mitochondrial reactive oxygen species (ROS) production and with qPCR for epithelial-mesenchymal-transi- Babak Memari, Manuella Bouttier, Vassil Dimitrov, John H. White tion (EMT) markers. Additionally, Col4a3 deficient mice were treated with Recombinant human pentraxin-2 (rhPTX-2) twice per week for McGill University, Montreal, Que´bec, Canada 5 weeks and functional and histological endpoints were measured. The innate arm of the immune system detects environmental patho- Results: Recombinant human pentraxin-2 (rhPTX-2) therapy attenu- gens and mounts a primary immune response. The aryl hydrocarbon ates the progression of Alport nephropathy in Col4a3 deficient mice

123 Inflamm. Res. S189 and rhPTX-2 is distributed not only to macrophages but also to Dietary fatty acid balance is recognized as an important factor in proximal tubular epithelial cells (PTECs). We hypothesized that inflammation regulation and disease controls. Especially omega-3 rhPTX-2 would directly prevent tubular injury in human PTECs. polyunsaturated fatty acid (PUFA) including eicosapentaenoic acid Immunostaining for vimentin was increased and epithelial marker (EPA) and docosahexaenoic acid (DHA) are widely held to be ben- E-cadherin expression was decreased in response to cell stress whereas eficial in many inflammatory disorders. Also, elevation in tissue those changes were dramatically attenuated by rhPTX-2. The mRNA omega-3 PUFA levels in omega-3 desaturase (fat-1) transgenic mice expression levels of vimentin, a-smooth muscle actin and Twist1 were that endogenously biosynthesize omega-3 PUFA from omega-6 increased by cell stress but significantly suppressed by rhPTX-2 PUFA exhibits resistance to inflammatory disease models. To eluci- compared to vehicle. In response to cell stress, PTECs also rapidly date the molecular mechanisms underlying the beneficial effects of generated ROS, and lost mitochondrial function but PTECs pretreated omega-3 PUFA, we developed a comprehensive LC–MS/MS-based with rhPTX-2 produced far less ROS, which was accompanied by lipidomics method that can detect and quantify more that 500 of fatty decreased caspase-3 activity. rhPTX-2 has a specific binding activity acid metabolites simultaneously. Using a genetic model, namely fat-1 for proximal tubule cells and is internalized via a clathrin-mediated transgenic mice, we examined the impact of enhanced omega-3/ pathway, suggesting an epithelial cell receptor. omega-6 ratio in inflammation and tissue homeostasis. Also we Conclusions: rhPTX-2 protects proximal tubular epithelium against demonstrated LC–MS/MS-based lipidomic analyses, and identified plasma- or TGFb-mediated EMT changes, mitochondrial dysfunction potent anti-inflammatory metabolites and key metabolic pathways for and cell death. rhPTX-2 is a potential new therapy for human chronic omega-3 PUFAs. These metabolites may underlie some of the ben- kidney diseases. eficial actions of omega-3 PUFAs in controlling inflammation and related diseases. References: Innate Lymphoid Cells 1. Endo J et al. 18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload-induced maladaptive car- B220 diac remodeling. J Exp Med 211, 1673–1687 (2014) 2. Kubota T et al. Eicosapentaenoic acid is converted via omega-3 PROFILING OF INFLAMMATORY BIOMARKERS epoxygenation to anti-inflammatory metabolite 12-hydroxy-17,18- FROM STIMULATED NATURAL KILLER CELLS epoxyeicosatetraenoic acid. FASEB J 28, 586–593 (2014) 3. Kunisawa J et al. Dietary omega-3 fatty acids exerts anti-allergic Richard K. Fuerstenberg, Shaya Anderson, Kathy Brumbaugh effect through the conversion to 17,18-epoxyeicosatetraenoic acid in the gut. Sci Rep in press R&D Systems, Inc, Minneapolis, USA NK (natural killer) cells are immune cells of lymphoid origin but unlike their brothers the T and B cells, they are a component of the innate B222 immune system and thus do convey long-lasting or protective immu- ELUCIDATING THE MOLECULAR MECHANISMS nity to the host. They instead destroy compromised host cells, such as OF INTERACTION BETWEEN MASLINIC ACID tumor cells or virus-infected cells, recognizing such cells through a missing self mechanism. Over the past decade, NK cells have come AND HUMAN GROUP IIA-SECRETED under greater scrutiny as major influencers of the host’s immune PHOSPHOLIPASE A2 (HGIIA-SPLA2) AND ITS response via secretion of cytokines, chemokines and other factors. In EFFECT IN REGULATING INFLAMMATORY addition, studies of the mechanisms of NK cells as antitumor effector RESPONSE cells have advanced our understanding of possible cancer cures. IL-12 is a well-known and potent stimulator of NK cells but is also a key 1 2 3 regulator of T cell responses, thereby bridging the gap between innate Wei Hsum Yap , Nafees Ahemad , Yang Mooi Lim response and adaptive immunity. In this study, NK cells were enriched 1Taylor’s University, Subang Jaya, Malaysia; 2Monash University from peripheral blood using a negative selection method, grown 3 overnight in tissue culture flasks both with and without IL-12. The cell Malaysia, Bandar Sunway, Malaysia; Universiti Tunku Abdul supernates from both conditions were sampled and assayed for secreted Rahman, Petaling Jaya, Malaysia biomarkers as large multiplexes using the R&D Systems, Luminex Maslinic acid (2-a,3-b-dihydroxyolean-12-en-28-oic acid), a natural Screening Assay. The markers that showed a positive response were pentacyclic triterpenoid found in pomace olive oil has various phar- identified for further analysis, thereby establishing a secretory profile macological properties which include anti-inflammatory activity. A for the study of the actions of NK cells. recent study showed that secretory phospholipase A2 (sPLA2) may be a potential binding target of maslinic acid. Considering sPLA2s contribute to the biosynthesis and release of pro-inflammatory Lipids, Their Enzymes and Inflammation mediators and cytokines in inflammatory cells, it represents a novel target for treating inflammatory diseases. The present study examined the interaction between maslinic acid and human Group IIA (hGIIA)- B221 sPLA2 and its effect in monocyte differentiation and migration. In this EMERGING ROLES OF OMEGA-3 FATTY ACID study, it is demonstrated that maslinic acid inhibit hGIIA-sPLA2 METABOLITES IN CONTROLLING enzyme activity in a concentration-dependent manner, with 75 % INFLAMMATION AND TISSUE HOMEOSTASIS inhibition at 100 lM concentration. Molecular docking study using Studio Suite 4.0 software further showed that maslinic acid binds to the calcium binding site and interfacial phospholipid binding site via Makoto Arita1,2,3 hydrogen bonding and hydrophobic interactions. These results sug- gest that maslinic acid inhibit the access of catalytic calcium ion 1RIKEN-IMS, Yokohama, Japan; 2Yokohama City University, required for enzymatic reaction and substrate binding to membrane Yokohama, Japan; 3PRESTO, JST, New York City, NY, USA

123 S190 Inflamm. Res. phospholipid, thereby inhibiting the enzymatic activity of hGIIA- myeloperoxydase activity to 37.8 ± 1.6 mU/mL, when compared to sPLA2. We further elucidate the role of maslinic acid in regulating control group (101.1 ± 12.7 mU/mL). In conclusion, the results hGIIA-sPLA2-mediated cellular responses inmonocyte THP-1 cells. presented in this study revealed that the new compound exhibited Our results showed thathGIIA-sPLA2is capable of inducing THP-1 anti-inflammatory activity, by presenting inhibitory effects on dif- cell differentiation and migration. Incubation of hGIIA-sPLA2-in- ferent enzymes involved in the inflammatory process. duced THP-1 cells with maslinic acid significantly inhibited cell Sources of research support CNPq, FAPEG, UEG and CAPES. adhesion at a concentration of 10, 20 and 50 lM and cell migration at a concentration of 20 and 50 lM. In conclusion, these findings pro- vide insight into the interaction between maslinic acid and hGIIA- B224 sPLA2 and its effect towards hGIIA-sPLA2-induced THP-1 cell adhesion and migration, an important immune-inflammatory pro- A HOST-MICROBIOME INTERACTION cesses occurring in atherosclerosis. MEDIATES THE OPPOSING EFFECTS OF OMEGA-6 AND OMEGA-3 FATTY ACIDS ON CHRONIC LOW-GRADE INFLAMMATION B223 EVALUATION OF ANTI-NOCICEPTIVE AND Jing X. Kang, Kanakaraju Kaliannan ANTI-INFLAMMATORY EFFECT OF NEW Massachusetts General Hospital and Harvard Medical School, BUTYLATED HYDROXYTOLUENE COMPOUND Boston, USA Metabolic endotoxemia, commonly derived from gut dysbiosis, is a Roberta Campos Lino1,2, Larissa de Oliveira Gonc¸alves3, 1 3 1 primary cause of chronic low grade inflammation that underlies many Iziara Ferreira Florentino , Ricardo Menegatti , Elson Alves Costa chronic diseases. Here we show that mice fed a diet high in omega-6 fatty acids exhibit higher levels of metabolic endotoxemia and systemic 1Laboratory of Pharmacology of Natural Products ICB-UFG. 2 low-grade inflammation, while transgenic conversion of tissue omega-6 Goiaˆnia-Go, Goiaˆnia, Brazil; Department of Pharmacy–State to omega-3 fatty acids dramatically reduces endotoxemic and inflam- University of Goia´s, Caˆmpus Itumbiara-Go, Itumbiara, Brazil; 3 matory status. These opposing effects of tissue omega-6 and omega-3 Laboratory of Pharmaceutical Medicinal Chemistry, FF-UFG. fatty acids can be eliminated by antibiotic treatment and animal co- Goiaˆnia-Go, Itumbiara, Brazil housing, suggesting the involvement of the gut microbiota. Analysis of Research seeks the development of new analgesic agents with better gut microbiota and fecal transfer revealed that elevated tissue omega-3 pharmacological activity. LQFM-091 or BHT, new butylated fatty acids enhance intestinal production and secretion of intestinal hydroxytoluene compound, derivative of nimesulide and BF-389, alkaline phosphatase (IAP), which induces changes in the gut bacteria inhibitors of COX-2 and 15-LIPOX respectively. This worked aimed composition resulting in decreased lipopolysaccharide production and to evaluate the anti-nociceptive and anti-inflammatory effects of the gut permeability, and ultimately, reduced metabolic endotoxemia and new compound obtained by hybridizationtechnique. inflammation. Our findings uncover an interaction between host tissue The experiments were conducted as approved by the Animal fatty acid composition and gut microbiota as a novel mechanism for the Ethics Committee of the UFG (no. 37/14). Female albino Swiss mice anti-inflammatory effect of omega-3 fatty acids. Given the excess of weighing approximately 30 g were used in this study. Pharmacology: omega-6 and deficiency of omega-3 in the modern Western diet, the in vitro cyclooxygenases activity and lipoxigenase activity. In vivo: differential effects of tissue omega-6 and omega-3 fatty acids on gut acetic acid-induced abdominal writhing, formalin-induced pain, car- microbiota and metabolic endotoxemia provide insight into the etiology rageenan-induced pleurisy, the TNF-a level and myeloperoxidase and management of today’s health epidemics. activity were dosage. LQFM-091 inhibited the activity of lipoxygenase enzyme (IC50 = 38.9 lmol/mL), COX-1 (IC50 = 89.0 lmol/mL) and COX-2 Microbiome in Health and Disease (IC50 = 89.0 lmol/mL). LQFM-091 (70, 140 or 280 lmol/kg, p.o) reduced the number of writhing to 57.1 ± 2.4; 51.5 ± 2.9; 50.5 ± 3.8, respectively when compared to control group (20 % B225 DMSO, p.o.) 84.7 ± 3.2 writhes. The treatment with nimesulide MESALAMINE PREVENTS TRANSLOCATION OF (162 lmol/kg, p.o.) also reduced the writhes to 64.3 ± 5.0. In the first phase of the formalin test, the treatments with LQFM-091 or nime- PLANKTONIC PATHOBIONTS RELEASED IN GUT sulide not reduced the licking time, but morphine (13.7 lmol/kg s.c.) MICROBIOTA BIOFILMS FROM reduced the licking time to 3.0 ± 2.5 s when compared to control INFLAMMATORY BOWEL DISEASE PATIENTS, group: 41.0 ± 5.8 s. In the second phase of the formalin test, the BUT NOT FROM HEALTHY INDIVIDUALS treatments with LQFM-091(140 or 280 lmol/kg, p.o); nimesulide

(162 lmol/kg, p.o.) or morphine reduced the licking time to 1 2 1 131.7 ± 15.9; 93.5 ± 10.4; 73.2 ± 14.6 e 8.4 ± 8.4 s, respectively Jean-Paul Motta , John L. Wallace , Andre G. Buret when compared to control group: 181.0 ± 18.9 s. The treatment with 1University of Calgary, Department of Biological Sciences, Calgary, LQFM-091 (140, 280 or 560 lmol/kg, p.o.), or dexamethasone 2 reduced the leukocytes migrated to the pleural cavity to 4.4 ± 0.3; Alberta, Canada; University of Calgary, Department of Physiology 3.1 ± 0.4; 2.8 ± 0.5 9 106/mL (5 lmol/kg, p.o), 2.7 ± 0.2 9 106/ and Pharmacology, Calgary, Alberta, Canada 6 mL when compared to control group: 6.6 ± 0.6 9 10 /mL. LQFM- Introduction: Inflammatory bowel disease (IBD) is commonly asso- 091 (280 lmoL/kg, p.o) reduced the levels of: TNF-a to 21.3 ± 9.0 ciated with alterations of gut microbiota composition and structure. In qg/mL, when compared to control group (62.3 ± 5.6 qg/mL), Evan’s rodents, commensal bacteria live as multispecies biofilms in the blue concentration in inflammatory exudate to 11.3 ± 3.8 e lg/mL, healthy gut. We observed that during experimental colitis, these when compared to control group (26.9 ± 3.1 lg/mL), and biofilms were fragmented, closely apposed to the epithelial surface,

123 Inflamm. Res. S191 and associated with severe bacterial translocation. Mesalamine is the ncRNA in plasma of b-thalassemia major patients. Identification of first line of treatment for patients with IBD. It has been found to affect exosomes by dynamic light scattering (DLS), flow cytometry and bacterial gene expression and reduce the concentration of mucosa- western blots. TRAP staining and bone resorption assay for osteo- adherent bacteria. We hypothesized that (1) human IBD is associated clasts number and function. Lentiviral infection, ncRNAs transfection with biofilms that release planktonic bacteria able to break the and in vitro knockdown or overexpression obviously increased or epithelial barrier, (2) and that mesalamine is able to prevent this ameliorated exosome-induced osteoclasts differentiation. Bioinfor- abnormality. matics analysis, luciferase assay and in vitro studies revealed that Methods: Human colon biopsies from healthy donors and Crohn’s ncRNAs functioned as a repressing mediator and formed a feedback Disease (CD) patients (active or inactive sites) were seeded anaero- loop with ncRNAs and target gene to mediate osteoclast bically into the Calgary Biofilm DeviceTM (CBD) to grow ex vivo differentiation. into their multispecies biofilm and planktonic counterparts. Live Results: We observed that APOC3 and ncRNA is differential biofilm bacteria were quantified using XTT assay. Biofilms were expressed in the exosomes of b-thalassemia major. We analyzed the exposed for 24 h to various concentrations of mesalamine (from 0 to effects of ex vivo-derived exosomes for osteoclast differentiation by 2 mg/mL). Planktonic bacteria were collected from treated biofilms osteoclast precursor model. In vitro model showed that exosomes of and were exposed apically to human epithelial monolayers on tran- b-thalassemia major were internalized by THP-1/RAW264.7 cells, swells (Caco-2, 18 h). Translocation of bacteria was quantified by and increase osteoclasts number and differentiation via up-regulated plating aerobically or anaerobically, and was visualized by fluorescent the p38/pAKT/NF-kB/TRAF6/NFATc1 pathway. In addition, Exo- in situ hybridization. somal APOC3-transferred ncRNA mediates inflammatory cytokine Results: At concentrations of 0.1, 1 and 2 mg/mL, mesalamine expression. reduced survival of biofilm bacteria originating from active CD but Conclusions: This study reveals the role of extracellular Exosomal had no effect biofilms from healthy and inactive CD patients. Aerobic APOC3-transferred ncRNA in the regulation of osteoclast differen- planktonic bacteria from healthy biofilms did not cross the epithelial tiation, which may provide the molecular mechanism for a new monolayer, and translocation of anaerobic bacteria was minimal. In therapeutic strategy in variety of osteoclast-related disorders. contrast, both aerobic and anaerobic planktonic bacteria from active and inactive CD biofilms crossed the epithelial monolayers. Planktonic bacteria collected from active CD translocated significantly more than planktonic bacteria from inactive CD sites. 3D images confirmed B227 translocation through paracellular route. At concentration of 2 mg/mL, EXTRACELLULAR RNA INDUCES CELLULAR mesalamine reduced translocation of aerobic bacteria from active and INFLAMMATION VIA TLR7-MYD88 SIGNALING inactive CD patients, and anaerobic bacteria from active CD. Conclusions: Our results demonstrate that host-microbiota biofilm Yan Feng, Hongliang Chen, Lin Zou, Dan Yan, Ganqiong Xu, interactions differ between the healthy and the IBD intestine. Wei Chao Planktonic bacteria released from IBD microbiota biofilms are able to cross the epithelial barrier, increasing the likelihood to trigger Massachusetts General Hospital, Boston, MA, USA inflammatory flares. The findings also demonstrate that mesalamine targets pathobionts evading from IBD microbiota biofilms, paving the Introduction: We have recently reported that extracellular RNA way towards a novel therapeutic paradigm using this drug. (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes (CMs) and immune cells, and may play a role in a mouse model of myocardial ischemia/reperfusion (I/R) injury. How- ever, the identities of these exRNAs and the molecular mechanism by which exRNAs exhibit the proinflammatory effect are unknown. microRNAs and lncRNAs Methods: I/R model mice were subjected to sham procedure or in Inflammation (microRNAs 5 miRs; coronary occlusion for 45 min followed by reperfusion. exRNA RNA Long Non-Coding RNAs 5 lncRNAs) in media and sera was extracted using Trizol LS and quantified using Quant-iTTM RNA assay kit. miRNA array 68 miRNAs in plasma were quantified using miRNA assay kit (Firefly, Cambridge, MA). Cell B226 treatment and cytokine measurement CMs, neutrophils, and bone EXOSOMAL APOC3-TRANSFERRED NCRNA marrow-derived macrophages (BMDMs) were treated with exRNA PROMOTE OSTEOCLASTS DIFFERENTIATION mixed with lipofectamine for 18 h. Cytokines in media were mea- sured by ELISA. miRNA uptake Uptake of fluorescent FAM-labeled miRNA in BMDMs was measured by fluorescent microscopy and Chi-Yuan Li1, Kuo-Ting Sun1 flow cytometry . Results: Twenty-four h after hypoxia/serum-deprivation, RNA in CM 1China Medical University Hospital, Taiwan, China; 2China Medical culture medium rose from 12 ng/mL to approx. 600 ng/mL. In vivo, University Hospital, Taiwan, China 24 h after I/R, serum RNA level was increased from 247 ± 46 to Background: Bone homeostasis requires an orchestrated balance 464 ± 92 ng/mL. Unbiased plasma miRNA array data indicate that between osteoblasts and osteoclasts during bone formation and 31 out of 68 miRNAs tested were significantly increased by [ two- resorption. Exosomes containing bioactive proteins and genetic mate- fold at 4 h following I/R compared to the sham mice. To test the rials that may be regulates cell development and differentiation. ability of exRNA to induce inflammatory response, CMs and immune ncRNA occurs in response to the cellular program to mediate differ- cells (neutrophils and BMDMs) were treated with cardiac RNA entiation. Exosomal APOC3-transferred ncRNA may be a crucial (2.5–10 lg/mL) or 8 miRNA mimics (10–1500 nM) selected from the function in osteoclasts differentiation. However, the mechanism is miRNA array data. Both RNA and 6 miRNA mimics (miR-34a, -122, remains unclear. The aim of this study was to investigate how Exo- -133a, 142a, -146a, -208a) were found to induce MIP-2 production in some-transferred APOC3-ncRNA adapts to osteoclasts differentiation. a dose-dependent manner. The effects were abolished by pre-treat- Methods: Using Proteomics, RNA-sequencing and lncRNA Q-PCR ment of RNase, but not DNase. Moreover, RNA-induced cytokine array, we demonstrated expression of exosomal APOC3-transferred production was significantly inhibited by a specific TLR7 inhibitor in

123 S192 Inflamm. Res. both CMs and immune cells, or diminished in TLR7-/- or Antigen assay. In the BD CBA cytokine assay, IL-2 and IL-4 were MyD88-/-, but not TLR3-/- or Trif-/- BMDMs. Similarly, miRNA- below the lower limit of detection (LLOD) and IL-17A was below the induced cytokine response was completely blocked in TLR7-/- or LLOQ. While TNF-a and IL-6 were the cytokines that were quanti- MyD88-/-, but not TLR3-/- or Trif-/- BMDMs. To test whether fied at similar plasma level to those detected with the Myriad-RBM TLR7 mediates miRNA trans-membrane uptake, WT and TLR7-/- assays, IFN-c and IL-10 were quantified at lower plasma levels. BMDMs were incubated with FAM-miRNA-133a for 4 h. There was Relatively higher level of IFN-c was detected by Myriad-RBM in the no difference in cellular fluorescent miRNA-133a between WT and 2-h plasma samples, whereas relatively higher level of IFN-c was TLR7-/- BMDMs as determined by fluorescent microscopy and flow detected with BD CBA cytokine assay in the 4-h plasma samples. cytometry. Finally, i.p. administration of cardiac RNA or miRNA Conclusion: Reproducible circulating TNF-a and IL-6 levels were mimics induced acute peritoneal inflammation as evidenced by IL-6 obtained for plasma samples of the LPS treated mice with the assays production and neutrophil activation. from both Myriad-RBM and BD Biosciences. The BD CBA cytokine Conclusions: Our data demonstrate that (1) RNA, including miRNA, assay was not as sensitive as the Myriad-RBM assays in detecting and is released from hypoxic CMs in vitro and ischemic myocardium quantitating circulating IL-2 and IL-4 and IL-17A levels in the LPS in vivo; (2) exRNA, both cardiac RNA and miRNA mimics, induce treated mice, but was more sensitive and reliable in measuring cir- cytokine production via TLR7-MyD88; (3) exRNA induces acute culating IFN-c levels. Reliable circulating IL-4 measurements were peritonitis after i.p. injection. These data suggest that exRNA is a not achieved by either assay. potent proinflammatory ligand that signals through TLR7-MyD88.

B229 Molecular Patterns and Acute COPPER-INDUCED INFLAMMATION LEADS TO Inflammation CHANGES IN SIRTUIN GENE EXPRESSION AND ALTERED BEHAVIOR IN ZEBRAFISH B228 COMPARATIVE SENSITIVITY AND RELIABILITY Talita C B Pereira1, Carlos Eduardo Leite2, Laura R. Nery3, 1,2 1,3 OF A MULTI-ANALYTES PLATFORM AND Maria Martha Campos , Mauricio R. Bogo A MULTIPLEX FLOW CYTOMETRY IN THE 1Programa de Po´s-Graduac¸a˜o em Medicina e Cieˆncias da Sau´de, QUANTITATION OF CIRCULATING CYTOKINES PUCRS, Brazil; 2Instituto de Toxicologia e Farmacologia, PUCRS, LEVEL IN A MOUSE MODEL OF ENDOTOXEMIA Brazil; 3Programa de Po´s-Graduac¸a˜o em Biologia Celular e Molecular, PUCRS, Brazil Alain Stricker-Krongrad, Jason Liu, Guy Bouchard, Miao Zhong Copper is an essential micronutrient and key catalytic cofactor in a wide range of enzymes. Under inflammatory conditions, serum cop- Sinclair Research Center, LLC, Auxvasse, MO, USA per levels are increased, and when in excess it triggers oxidative stress Background: A number of cytokines, such as interleukin 2 (IL-2), responses that can finally lead to apoptosis. Copper exposure can be interferon c (IFN-c), tumor necrosis factor a (TNF-a), IL-4, IL-6, IL- easily modeled in zebrafish, a consolidated model in many research 10 and IL-17A, are elevated in response to inflammation and are also areas. Due to developmental, economical and genetic advantages, it is key regulators of immune responses. IL-2, IFN-c, and TNF-a are suitable for large-scale screenings, representing a powerful experi- associated with the Th1 response. IL-2, IL-4, IL-6 and IL-10 are mental tool to mechanism-related studies. Toxicological effects of associated with the Th2 response. IL-6 and IL-17A are associated copper have been extensively investigated in zebrafish, and its use as with the Th17 response. Hence, measuring expression profiles of an inflammatory agent has increased. Sirtuins comprises a unique these cytokines is important in monitoring the polarization of the class of evolutionary conserved NAD+ -dependent deacetylases. immune response and therefore the results should be independent of They play important roles regulating metabolic and enzymatic the quantitation methods. To evaluate this effect, the 7 circulating activity, stress, DNA repair and apoptosis targeting not only histones Th1/Th2/Th17 cytokines were quantified in plasma of lipopolysac- but also transcriptional factors and structural proteins. Under patho- charide (LPS ± dexamethasone versus vehicle) treated mice with two logical conditions, sirtuins have also been associated to inflammation, different multiplex platforms (Myriad-RBM and BD Biosciences). with both pro- and anti-inflammatory effects. Here, we aimed to Objectives: To compare two different multiplex assay formats to evaluate the effects of copper-induced inflammation on locomotor quantify the release of the 7 circulating Th1/Th2/Th17 cytokines in behavior in 7dpf zebrafish larva, and mRNA expression levels of all plasma of LPS challenged mice. sirtuin family members (SIRT1-7). IL-1b, IL-10, TNF-a and COX-2 Methods: Female C57BL6 mice (6–9 months old) were treated with mRNA expression was evaluated as indicative of inflammation. The vehicle (0.5 % methyl cellulose in water) or dexamethasone (5 mg/ larvae (n = 20/group) were treated for 4 h or 24 h with copper sulfate kg), followed by LPS (0.2 mg/kg) via intravenous injection (IV). At as inflammatory agent, and immediately submitted to a recorded 0.5, 1, 2, 4, and 6 h after LPS challenge, blood samples were analyzed locomotors behavior assay. Relative mRNA expression levels were -DDCT with the Rodent MAP V3.0 Antigens assay, with the Mouse Cytokine determined by qPCR analysis (2 method), using EF1-a and Panels A & B assay (Myriad-RBM; Luminex platform) and compared Rpl13-a as reference genes. Results were statistically compared by to the Mouse Th1/Th2/Th17 cytokines (Becton–Dickinson: BD CBA) T-test, or one-way ANOVA followed by Tukey test, considering assay on a BD Accuri C6 flow cytometer platform. p B 0.05 as significant. Larval swimming performance showed a Results: TNF-a, IL-2, IL-4, IFN-c, IL-6 and IL-10 were shown to be significant decrease on traveled distance, mean speed and number of the early phase responders, and IL-17A was shown to be a sustained rotations for both 4 and 24 h exposures. The inflammation mediators responder, which was up-regulated through LPS treatment. Pre- IL-1b, IL-10, TNF-a related genes showed a significant activation in treatment with dexamethasone reduced expressions of the 7 circu- all treatments, as well as COX-2 gene for the 24 h treatment, when lating Th1/Th2/Th17 cytokines. Plasma levels of IL-4 were detected compared to the control group. SIRTs 2, 3, 4, 6 did not present altered with the Mouse Cytokine Panels A and B assay, but were below the expression; however, we found a decrease on SIRT1 gene expression Lower limit of quantification (LLOQ) of the Rodent MAP V3.0 after 4 h-treatment and an activation of SIRT7 after 24 h exposure.

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SIRT3.2, a SIRT3 paralogue, showed increased expression on both Federal University of Rio de Janeiro, Rio de Janeiro, Brazil treatments. These findings show potential involvement of, at least, Introduction: Eosinophils mediate the immune response in a number SIRT1, SIRT3.2 and SIRT7 in inflammatory events. A tissue-specific of infectious diseases, including parasitic helminth, bacterial, fungal approach seems promising to evaluate inflammation effect on sirtuins and viral infections. The process of extracellular release of DNA nets family members, due their differentiated expression pattern. (traps) by leukocytes has been described as an important mechanism of the innate immune response in different infectious diseases including fungal infections. Purified human eosinophils release Mucosal Immunity extracellular DNA traps (EETs) by different stimuli including bac- teria and cytokines. Aspergillus fumigatus (AF) is an opportunistic filamentous fungus that may cause invasive aspergillosis, a patho- B230 logical condition of high morbidity and mortality in ROLE OF NK CELLS IN IFN-E-MEDIATED immunocompromised patients. In vivo, eosinophils are recruited to PROTECTION AGAINST FEMALE the lung after exposure to AF and release cationic proteins, which demonstrate an important role in the elimination of this pathogen. REPRODUCTIVE TRACT INFECTION In vitro, eosinophils present potent fungicidal activity against AF. However, the mechanisms that lead to recognition as well as the death 1 2 1 Jemma R. Mayall , Niamh E. Mangan , Malcolm R. Starkey , of AF by eosinophils remain unknown. In this work we investigated 1 1 2 Richard Y. Kim , Alexandra C. Brown , Paul J. Hertzog , whether eosinophils release EETs in response to AF and the mech- 1 1 Jay C. Horvat , Philip M. Hansbro anisms involved. Methods and Results: We isolated eosinophils from blood of healthy 1 2 University of Newcastle, Newcastle, Australia; Monash University, donors by negative immunomagnetic selection. Cells were then Melbourne, Victoria, Australia stimulated with AF in the ratios (fungus:cell) 1:1, 10:1, 50:1 and Chlamydia trachomatis is the most common bacterial sexually 100:1 being the release of EETs evaluated at different incubation transmitted infection (STI) and infection frequently results in repro- times by a quantitative fluorimetric method and by confocal fluores- ductive tract (RT) sequalae such as pelvic inflammatory disease and cence microscopy. We observed that EETs were significantly released infertility. However, the immune factors and processes involved in after 6, 9 and 12 h of incubation. The incubation time of 6 h and the the clearance and immunopathology of Chlamydia infection are not ratio (fungus:cell) 10:1 were then selected for further studies (con- well understood. In previous studies we showed that interferon (IFN)- trol = 84.08 ± 10.71 FU, AF = 281.6 ± 33.82 FU, n = 8, student’s e, a novel type I IFN that is exclusively and constitutively expressed t test p \ 0.05, FU = fluorescence unit). Pretreatment of eosinophils in the female RT, plays an important role in protecting against Ch- for 30 min with DPI (20 lmol/mL) (AF = 307.2 ± 50.21 FU, lamydia infections at the earliest stages of infection. Here, we DPI = 232.3 ± 45.95 FU, n = 7) or apocynin (100 lmol/mL) examined the effects of IFN-e on innate cellular responses in the (AF = 248.50 ± 38.47 FU, Apo = 229.0 ± 40.57 FU n = 4), both female RT in order to elucidate the potential mechanisms that inhibitors of reactive oxygen species (ROS), did not inhibit the AF- underpin how IFN-e protects against Chlamydia infections. Female induced EETs release. However, the pretreatment with piceatannol wild type and IFN-e deficient (-/-) C57BL/6 mice were pre-treated (40 lmol/mL) (AF = 214.5 ± 32.47 FU, PCT = 97.08 ± 13.12 FU, with progesterone to synchronise their oestrous cycles and prime for n = 6, student’s t test p \ 0.05) or OXSI (2 lmol/mL) infection. Seven days later, mice were infected intra-vaginally with (AF = 303.5 ± 59.26 FU, OXSI = 146.9 ± 25.34 FU, n = 6, stu- Chlamydia muridarum or sham-infected. Uterine horns were har- dent’s t test p \ 0.05), both inhibitors of Syk tyrosine kinases, vested at 3 days post infection and the effects of IFN-e deficiency on significantly inhibited the AF-induced EETs release. Chlamydia infection, immune factor expression and cellular infiltra- Conclusion: Our results indicate that human eosinophils release EETs tion were assessed using real-time qPCR and flow cytometry. We in response to AF through a mechanism which involves the Syk show that IFN-e-/- mice have increased Chlamydia 16S expression tyrosine kinases pathway, but independent of ROS. in the upper RT. This corresponded with fewer NK cells, which are Financial support FAPERJ, CNPq and Capes. known to play a role in protecting against Chlamydia through the production of IFN-c. IFN-c producing CD45+ cells were decreased in -/- the infected IFN-e mice, of which over 60 % were NK cells. B232 Tissue-resident uterine (u) NKs were also decreased in these mice. The changes in IFN-c+ and NK cells were associated with reduced INTRAVITAL IMAGING OF VASCULATURE IL-15, CXCL10, iNOS and STAT1 expression. These findings sug- REVEALS THAT NEUTROPHIL gest that IFN-e may protect against Chlamydia RT infections EXTRACELLULAR TRAP (NET) COMPONENTS by potentiating the recruitment of protective IFN-c-producing NK ATTACH TO VON WILLEBRAND FACTOR IN cells. A DNASE-INDEPENDENT MANNER

Elzbieta Kolaczkowska2,1, Craig N. Jenne1, Bas G. Surewaard1, Neutrophils, NETs and PADs Ajitha Thanabalasuriar1, Woo-Yong Lee1, Maria-Jesus Sanz1, Kerri Mowen4, Ghislain Opdenakker3, Paul Kubes1 B231 1University of Calgary, Canada; 2Jagiellonian University, Krakow, HUMAN EOSINOPHILS RELEASE Poland; 3University of Leuven, Belgium; 4Scripps Research Institute EXTRACELLULAR DNA TRAPS IN RESPONSE TO La Jolla, San Diego, CA, USA ASPERGILLUS FUMIGATUS Neutrophils form neutrophil extracellular traps (NETs) in response to pathogens and/or during severe inflammation, including sepsis. NETs Josiane S. Neves, Valdirene S. Muniz, Mariana SR Bica, Yasmim AV are composed of extracellular DNA, histones and granular proteins, Braga, Rodrigo T. Figueiredo including proteases such as neutrophil elastase (NE). The structures 123 S194 Inflamm. Res. function to trap and immobilize pathogens and subsequently con- demonstrated that RSV Fusion protein induce NET formation through tribute either directly, or indirectly to pathogen elimination. In vivo activation of TLR-4, NADPH Oxidase-derived reactive oxygen spe- characteristics of NET formation and removal remain unclear. cies generation and ERK and p38 MAPK phosphorylation. Thus, we Therefore we studied NET release, their life-span and consequences asked whether an active RSV infection would induce NET release. of their presence in vasculature by means of intravital imaging, using This study aims to investigate the mechanisms underlying the pro- spinning-disk confocal microscopy, during systemic infection with duction of NETs induced by alveolar epithelial cells infected with Staphylococcus aureus. Intravascular S. aureus injection is lethal for RSV. Human neutrophils were stimulated with different concentra- Kupffer cell- or platelet-depleted mice, but not those lacking neu- tions of RSV (102–104 PFU/mL) for 3 h at 37oC under 5 % CO2. trophils suggesting NETs are dispensable. In wild-type mice, Alternatively, alveolar epithelial cells (A549 cell line -1 9 105/mL) neutrophils rapidly infiltrated the liver and formed NETs that per- were infected with active RSV or UV-inactivated RSV in RPMI sisted in the vasculature for several hours following infection, far medium without FCS for 2 h at 37 °C under 5 % CO2. Afterwards, beyond the time needed for S. aureus blood clearance. The prolonged medium was replaced by RPMI 10 % FCS and the culture was presence of NETs led to profound damage to the liver and this maintained for 48 h. After this, medium was replaced to remove virus damage was associated with proteolytic activity of neutrophil elastase particles and neutrophils (5 9 105/mL) were added to the culture. The attached to NETs. The prevention of NET formation, as observed in co-culture was incubated for 150 min. At the end of incubation, NETs NE- and peptidyl arginine deiminase type IV (PAD4)-deficient mice, were quantified in culture supernatants, using Quant-iT dsDNA HS kit. eliminated the liver injury. Surprisingly, attempts to remove NETs by Here we show that RSV was able to directly induce NET release by DNase were only partially successful. While this treatment did human neutrophils in a concentration-dependent fashion. We per- remove all of the extDNA, only some histones and NE detached upon formed a MTT assay to evaluate the dose- and time-dependent effect the treatment. NET components were determined to be attached to the of RSV infection on alveolar epithelial cells viability. We found that glycocalyx via von Willebrand factor (VWF): blockade of VWF the RSV concentrations used were not able to affect A549 cell viability before sepsis induction, or VWF shedding during the ongoing sepsis until 48 h. In 72 h, RSV (103 and 104 PFU/mL) induced A549 cell was sufficient to significantly decrease the attachment of NET com- death; leading us to infect the cells for 48 h. RSV infection of alveolar ponents to the endothelium. Most recently we observed that although epithelial cells was able to induce NET formation in a concentration- platelets were not involved in NET formation during S. aureus-in- dependent manner. This effect was due to an active RSV infection, duced sepsis, platelets were important for the primary interactions since UV-inactivated RSV did not stimulate NET release nor pro- between the pathogen and Kupffer cells. In summary, we show that in moted a cytopathic effect in A549 cells, as visualized by optical the vasculature, unlike what is observed under in vitro conditions, microscopy. The excessive production of NETs induced by RSV could NET components attach to a substratum independently of extDNA fill the lungs and impair lung function and consequently aggravate the and this attachment hinders their removal by DNase. Albeit removal inflammatory symptoms of the infection in young children and babies. of VWF might be a therapeutic option, the consequences on platelet We propose that the associated use of DNase could potentially lead to functioning must be better understood before this approach can be novel therapeutic approaches to help control RSV-induced inflam- used. Due to the difficulty in removing NET components once matory consequences and pathology of viral bronchiolitis. released, prevention of NET formation proved to be the most effec- tive therapeutic intervention. B234 AUTOPHAGY MEDIATES INTERLEUKIN-1 BETA B233 EXPORTATION FROM HUMAN NEUTROPHILS RESPIRATORY SYNCYTIAL VIRUS INFECTION OF BOTH ALVEOLAR EPITHELIAL CELLS AND Analia S. Trevani1,2, Irene Keitelman1, Florencia Sabbione1, 1,2 1 NEUTROPHILS INDUCES THE FORMATION OF Carolina C. Jancic , Leonardo Iula NEUTROPHIL EXTRACELLULAR TRAPS 1IMEX-CONICET- Academia Nacional de Medicina, Buenos Aires, Argentina; 2Facultad de Medicina, Universidad de Buenos Aires, Ba´rbara N. Porto1, Ste´fanie P. Muraro1, Giselle A. Funchal1, Argentina Nata´lia Jaeger1, Rafael S. Czepielewski1, Gabriela F. Souza1, 2 b b Renato T. Stein Introduction: Interleukin-1 (IL-1 ) is a pro-inflammatory cytokine synthesized in the cytoplasm as a precursor, pro-IL-1b, which has to 1Laboratory of Clinical and Experimental Immunology, Pontifical be proteolytically processed to acquire biological activity. We have b Catholic University of Rio Grande do Sul, Porto Alegre, Brazil; previously demonstrated that human neutrophil IL-1 processing is 2Laboratory of Pediatric Respirology, Pontifical Catholic University dependent of caspase-1 and elastase and/or proteinase-3. The release b of Rio Grande do Sul, Porto Alegre, Brazil of IL-1 does not follow the conventional ER-Golgi route of secre- tion, being secreted by poorly-defined non-conventional secretory Respiratory Syncytial Virus (RSV)-induced acute bronchiolitis is the pathways among which autophagy has been proposed to be involved. most prevalent disease in children under 2 years old, which causes a However, controversial findings have been reported regarding the role huge impact in hospitalizations and costs to the health system. The of autophagy in controlling IL-1b secretion in other myeloid cells; alveolar epithelial cells are the initial targets for RSV infection, as well with studies indicating either that autophagy targets IL-1b for as the first site for activation of the innate immune response. Epithelial degradation or that it is involved in its unconventional secretion. Here cells infected by RSV secrete pro-inflammatory cytokines and neu- we tested the hypothesis that an unconventional autophagy mecha- trophil chemokines, which attract these granulocytes to the site of nism is required for neutrophil IL-1b secretion. infection. The excessive neutrophil activation by epithelial cells Methods and results: We found that autophagy inhibitors like infected with RSV can be harmful to the host, as neutrophils release 3-methyadenine markedly reduced IL-1b secretion induced by 5 h neutrophil extracellular traps (NETs), which kill microorganisms but stimulation of human neutrophils with LPS or LPS+ ATP evaluated also damage host cells and tissues. NETs are composed by decon- by ELISA, without affecting cell viability. By contrast, these inhi- densed chromatin and antimicrobial proteins. We have recently bitors did not modulate IL-8 secretion, a cytokine whose release

123 Inflamm. Res. S195 follows the canonical ER-Golgi pathway. In neutrophil-differentiated Introduction: High sugar diet (HSD) induces insulin resistance (IR) in PLB985 cells, siRNA knockdown of ATG5, an essential component larvae and hyperglycemia in adults Drosophila flies. HSD-induced IR of the autophagic pathway, abolished IL-1b secretion. In agreement is characterized by delayed eclosion and decreased body weight of with these findings, stimulation of autophagy by cell starvation pro- imago [1]. Chronic low grade inflammation is one of the major risk moted IL-1b secretion induced by both agonists. At 4 h post- factors for the development of IR and diabetes. Molecular mecha- stimulation with LPS+ ATP, intracellular IL-1b showed a vesicular nisms mediating the impact of chronic inflammation on the distribution co-localizing with LC3B, a marker of autophagy vesicles, development of IR have not been finalized. We suggested that dys- by confocal microscopy. Furthermore, neutrophil starvation increased regulation of kynurenine (KYN) pathway of tryptophan (TRP) IL-1b and LC3B colocalization. metabolism is one of the mechanisms of development of inflamma- Conclusion: Taken together, our studies reveal that autophagy is part tion-induced IR. KYN formation from TRP is catalyzed by rate- of the pathway involved in IL-1b exportation from human PMN. limiting enzymes: indoleamine 2,3-dioxygenase (IDO) and TRP 2,3- dioxygenase (TDO-2) [2]. IDO is activated by pro-inflammatory cytokines while TDO in induced by stress hormones. Rate-limiting enzyme of TRP—KYN metabolism in Drosophila is an evolutionary New Models of Inflammatory conserved ortholog of human IDO and TDO that is encoded by ver- Mechanisms and Diseases milion gene. Formation of KYN from TRP is deficient in vermilion mutants of Drosophila melanogaster. B235 Hypothesis: Vermilion flies will be more resistant to inducement of IR by HSD. SPONTANEOUS MUTATION IN ZFP-BINDING Aim: To evaluate the effect of HSD on development of IR in ver- REGION OF TNF GENE CAUSES CHRONIC milion mutants of Drosophila melanogaster. POLYARTHRITIS AND HEART VALVE DISEASE Method: The effect of HSD on eclosion time and body weight of imago was compared in vermilion mutants and wild type (Oregon) 1,2 1,2 3 flies. Derek C. Lacey , Peter Hickey , Benedicta D. Arhatari , Lorraine 1,2 1,2 4 4 Results: HSD delayed eclosion time of Oregon flies by 54 % O’Reilly , Leona Rohrbeck , Helen Kiriazis , Xiao-Jun Du , 1,2 (151.2 ± 19.2 and 232.5 ± 23.1 h, resp., n = 400, p \ 0.003, Philippe Bouillet Mann–Whitney two tailed test) and decreased body weight of imago 1 by 37 % (0.99 ± 0.004 and 0.62 ± 0.003 mg, resp). In vermilion The Walter and Eliza Hall Institute of Medical Research, Parkville, 2 mutants HSD delayed eclosion time by 18 % (176.5 ± 27.6 and Victoria, Australia; Department of Medical Biology, The University 208.9 ± 22.3 h, resp.) and did not affect body weight of imago of Melbourne, Melbourne, Victoria, Australia; 3ARC Centre of (0.93 ± 0.004 and 0.92 ± 0.003 mg, resp). Excellence for Advanced Molecular Imaging, Department of Physics, 4 Discussion: Although HSD delayed eclosion in both vermilion La Trobe University, Victoria, Australia; Baker IDI Heart and mutants and Oregon flies, delay of eclosion was threefold shorter in Diabetes Institute, and Central Clinical School, Monash University, vermilion mutants (18 vs 54 %) than in Oregon flies suggesting that Melbourne,Victoria, Australia deficient TRP conversion in KYN attenuates development of IR We have studied a new spontaneous mutation that causes severe RA induced by HSD. Our results are in line with our working hypothesis symptoms and heart valve disease in BPSM1 (bone phenotype that deficient formation of KYN from TRP attenuates inducement of spontaneous mutation 1) mice. The insertion of a retrotransposon into IR by HSD. Our data suggest that modulation of inflammation-in- the 30 untranslated region (UTR) of TNF causes its strong overex- duced activation of TRP—KYN metabolism might be utilized for pression in myeloid cells. We identified several members of a family prevention and treatment of IR and diabetes. of RNA–binding CCCH-containing zinc finger proteins, which can Supported by NIH Grant MH104810 (GO). positively or negatively control TNF expression through in the TNF 1. Pasco MY and P Le´opold. (2012) High Sugar-Induced Insulin 30UTR. We have also identified a new regulatory element in TNF Resistance in Drosophila Relies on the Lipocalin Neural Lazarillo 30UTR, which is essential for the newly identified zinc finger protein’s PLoS One. 2012; 7(5): e36583. doi:10.1371/journal.pone.0036583 control of TNF but not Zfp36. The disease in BPSM1 mice is inde- 2. Oxenkrug G. (2013) Insulin Resistance and Dysregulation of pendent of the adaptive immune system, and does not appear to Tryptophan-Kynurenine and Kynurenine Nicotinamide Adenine involve inflammatory cytokines other than TNF. In addition to the Dinucleotide Metabolic Pathways. Mol Neurobiol. 48: 294–301. doi: severe joint destruction, mutant mice also develop aortic root 10.1007/s12035-013-8497-4 aneurism and aorto-mitral valve disease that can be fatal depending on the genetic background. This is the first animal model showing both RA and heart disease as a direct result of TNF deregulation. B237 IN VITRO MODELLING ACUTE AND CHRONIC B236 INFLAMMATION BASED ON HUMAN DOWN-REGULATION OF KYNURENINE MONOCYTES. ANALYSIS OF THE MODULATION FORMATION FROM TRYPTOPHAN OF IL-1 FAMILY MEMBERS ATTENUATES INDUCTION OF INSULIN Paola Italiani1, Ettore Mosca 2, Luciano Milanesi2, Diana Boraschi1 RESISTANCE IN DROSOPHILA MELANOGASTER 1Institute of Protein Biochemistry, National Research Council of Valeriya Navrotska1, Lyudmila Vorobyova1, Paul Summergrad2, Italy, Naples, Italy; 2Institute of Biomedical Technologies, National Gregory Oxenkrug2 Research Council of Italy, Naples, Italy

1 2 The course of an acute physiological inflammatory response can be Karazin National University, Kharkiv, Ukraine; Tufts University, modelled in vitro by exposing human primary monocytes in culture to Boston, MA, USA

123 S196 Inflamm. Res. a sequence of conditions simulating the recruitment from blood into cause of waterborne diarrheal disease. Giardia infections have been an inflamed tissue, the encounter with inflammatory agents, and associated with decreased incidence of diarrheal disease and fever, eventually the conditions promoting resolution and tissue repair. and lower serum inflammatory scores via unknown mechanisms. CD14+ monocytes were exposed sequentially to CCL2, Furthermore, Giardia infections have been concurrently observed Gram + bacterial vesicles, TNF-a, IFN-c, IL-10, and TGF-b, with with other pro-inflammatory gastrointestinal pathogens, such as concomitant changes in temperature and oxygen levels. A parallel enterohemorrhagic Escherichia coli. model of chronic inflammation was obtained with a different Objective: The purpose of this study was to assess whether in vivo sequence of stimuli (CCL2, a cocktail of bacterial and viral stimuli Giardia infections modulate host responses or susceptibility to co- plus IFN-c immune complexes, survivin, M/GM-CSF) and main- infection with intestinal pathogenic bacteria and to identify the taining the culture at 39 °C in hypoxic conditions. mechanisms involved. We have profiled by RNAseq the transcriptome of monocytes and Methods: Male C57BL/6 mice were co-infected with Giardia muris analysed the variations of expression of the IL-1 family members. and Citrobacter rodentium and sacrificed at 14 days post-infection. Cytokines and receptors of the IL-1 family members are major Animal weights were recorded and stool samples collected to assess players in an inflammatory reaction, taking part in all its phases both for bacterial colony forming units (CFUs). Colonic tissues were in normal defence and in pathological situations. IL-1 family stained by hematoxylin-eosin (H&E) and scored for macroscopic cytokines encompass inflammatory and anti-inflammatory factors, damage. Granulocyte infiltration was assessed by myeloperoxidase including natural antagonists. IL-1 family receptors can be either (MPO) assay. Gut bacteria were stained using fluorescent in situ activating, accessory or non-signalling ‘‘decoy’’ receptors. Alteration hybridization (FISH—probe against all bacteria), and Citrobacter of the qualitative, quantitative and spatio-temporal regulation of the rodentium was stained by immunofluorescence (anti-C. rodentium IL-1 family network, as observed during a physiological resolving lipopolysaccharide, LPS). The capacity of colonic crypts to kill E.coli inflammatory defensive response, may lead to pathological was performed by crypt killing assays. inflammation. Results: At 7 and 14 days post-infection,weight loss was reduced in In the early phase of both acute and chronic inflammation, the G. muris + C. rodentium co-infected mice as compared to mice expression of six cytokines (IL1B, IL1A, IL1RN, IL18, IL18BP, infected with just C. rodentium. Overall bacteria and C. rodentium IL36G) and four receptors (IL1R1, IL1R2, IL1RAP, SIGIRR) is sim- CFUs in the stool were reduced in G. muris + C. rodentium animals at ilarly regulated, peaking between during the inflammatory phase and 2 and 7 days post-infection, compared to C. rodentium animals. returning to the low basal level during the resolution phase in the Macroscopic and microscopic damage scores and MPO activity were acute model (except IL18 that was lower than in basal conditions), also reduced in co-infected animals compared to C. rodentium infected while remaining higher than in fresh monocytes in the chronic model. mice at 14 days post-infection. In the colon of C. rodentium infected Exceptions are the genes of the inhibitors IL18BP and SIGIRR. mice, we observed translocation of bacteria into the lamina propria SIGIRR decreased during the entire course of the reaction, whereas (FISH staining), and mucosal attachment and translocation of C. IL18BP significantly increased in the late phases of chronic inflam- rodentium via LPS staining. In G. muris + C. rodentium animals, we mation. By GSEA analysis, the expression of all the IL-1 family observed less bacterial translocation, and less mucosal attachment of members is mainly modulated during the first 14 h of stimulation in C. rodentium. Accordingly, aerobic and anaerobic CFUs in liver and both models, i.e. in the timeframe in which the largest variation of spleen homogenates were significantly reduced in co-infected mice as expression occurs. The analysis of differentially expressed genes compared to the C. rodentium group. Crypt killing assays demon- between two consecutive time points shows that IL-1 family cytoki- strated higher antimicrobial capacity against E. coli,inG. muris and G. nes and receptors are modulated during the entire course of the muris + C. rodentium groups as compared to C. rodentium group. inflammatory reactions, except in the late phases of chronic inflam- Conclusion: Giardia muris infection reduces the severity of C. mation (in which they remain practically unchanged). rodentium-induced colitis in mice. These results suggest that Giardia These results underline the strong modulation of IL-1 family infections are protective against the development of severe gastroin- genes, in particular in the initial phases of inflammation, suggesting testinal disease induced by a co-infecting gastrointestinal bacterial their involvement in regulating the inflammation outcomes. pathogen, potentially via their ability to enhance the anti-microbial Work supported by the EU FP7 project BioCog (GA 602461) and capacity of the intestinal epithelium. by the Cluster project Medintech of the Italian Ministry of University and Research. B239 DYNAMIC EQUILIBRIUM OF NEUTROPHIL B238 TRAFFICKING AT SITES OF INFLAMMATION GIARDIA MURIS INFECTION REDUCES THE AND INFECTIONS SEVERITY OF CITROBACTER RODENTIUM- INDUCED COLITIS Daniel Irimia1,2,3

Jean-Paul Motta1, Anna Manko2,1, James A. Cotton1, 1Massachusetts General Hospital, Boston, MA, USA; 2Harvard Bruce A. Vallance3 Medical School, Boston, MA, USA; 3Shriners Burns Hospital, Boston, MA, USA 1University of Calgary, Biological Sciences, Calgary, Alberta; Appropriate inflammatory responses to wounds and infections require 2University of Calgary, Department of Physiology and the presence of adequate numbers of neutrophils at injury sites. Both Pharmacology, Calgary, Alberta; 3University of British Columbia, insufficient and excessive neutrophil recruitment can be detrimental, Division of Gastroenterology, Vancouver, Canada favoring the spread of infection or triggering severe tissue damage, Background: Our understanding of polymicrobial gastrointestinal respectively. However, the fundamental rules that regulate the traf- infections and their effects to host biology remains incomplete. ficking of neutrophils at sites of inflammation/infection are complex Giardia is a protozoan parasite that infects humans, and is a prevalent and consequently difficult to study using traditional in vivo models.

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To elucidate the neutrophil trafficking rules,we designed devices further investigate in an intact living organism the mechanisms that in which human neutrophils emerge directly from a droplet-size promote hematopoiesis development and related diseases. samples of whole blood and migrate towards chambers with Funding FONDECYT 1140702/FONDAP 15090007. chemoattractants and microbe-like particles. Inside these devices, human neutrophils could be monitored in detail, under precise control of the mechanical, biochemical, and microbe interactions conditions. We found that the number of neutrophils recruited by chemotaxis Novel and Innovative Platforms for Drug and departing by retrotaxis increases and stabilizes to dynamic Discovery equilibrium in the presence of chemoattractants alone. The migration of individual neutrophils ceases immediately after phagocytosis, altering the balance between chemotaxis and retrotaxis B241 and increasing the number of neutrophils accumulating to the site. ASSESSING NK CELL KILLING ACTIVITY TO This number is proportional to the number of microbe-like parti- INFORM DRUG DISCOVERY PROGRAMS ON cles in the chambers. CLINICAL INFECTION RISK Overall, autonomous neutrophil traffic regulation assures that a continuous supply of fresh neutrophils is available to infection sites, that the number of neutrophils accumulating is appropriate to the Cristina A. St. Pierre, Laura Engstrom, Alexandra Hicks, Ravisankar number of microbes, and that overpopulation is avoided. A. Ramadas

Merck Research Labs, Rahway, NJ, USA B240 Natural killer (NK) cells are critical for the clearance of pathogen- infected cells and tumor cells that lack class I MHC expression. We IN VIVO IMAGING OF MAMMALIAN HSCS developed a non-radioactive flow cytometry based assay to evaluate ENGRAFTMENT AND COLONIZATION INTO THE NK cell killing activity in human and mouse tissues. In brief, human CHT OF ZEBRAFISH EMBRYOS PBMC or mouse splenocytes were co-incubated with target cells (MHC class I negative tumor cell lines) under optimized conditions Margarita M. Parada Kusz1,2, Anne Clatworthy2, Humberto Jijon3, and the percentage of target cell killing was used to evaluate NK cell Eduardo J. Villablanca4 activity. We found that SYK/ZAP70 antagonists inhibited NK cell killing function in human, but not mouse tissues, suggesting that 1Centro FONDAP de Regulacio´n del Genoma, Universidad de Chile, certain pathways or pathway inhibitors can have species specific Santiago, Chile; 2Department of Molecular and Cellular Biology, effects. We used this assay to compare the impact of clinical Harvard University, Cambridge, MA, USA; 3Center for Computational immunosuppressants (cyclosporine A, leflunomide, methotrexate, and Integrative Biology, Massachusetts General Hospital, Boston, MA, mycophenolate mofetil, prednisolone, rapamycin, and tofacitinib) and USA, Harvard Medical School, Boston, MA, USA; 4Translational pathway specific inhibitors (BTK, IRAK4, JAK, PI3Kd, RORcT, and Immunology Unit, Department of Medicine, Solna, Karolinska SYK/ZAP70) on NK cell activity, which has the potential to help Institutet and University Hospital, Stockholm, Sweden inform on dose selection and the relationship between efficacy and safety. This assay could also serve as a phenotypic screen to identify Hematopoietic stem cells (HSCs) generate the full repertoire of blood immunostimulators and is amenable to the evaluation of antagonists and immune cell types throughout an organism’s life. They are pro- as well as agonist small molecules and biologics in NK cell responses. duced during ontogeny, and actively respond to niche-specific environmental cues to mobilize, expand or differentiate. These nourishing niches dynamically change locations throughout devel- opment, beginning in the aorta–gonad–mesonephros (AGM) region B242 and the yolk sac, followed by the placenta, fetal liver, spleen and bone marrow. Similar to mammals, HSCs in zebrafish originate early in the ETANERCEPT, ABATACEPT AND ANAKINRA aorta, move transiently to the caudal hematopoietic tissue (CHT) and TREATMENT AMELIORATES INFLAMMATION ultimately mobilize to the thymus and kidney (equivalent to the AND PAIN IN A NOVEL MONO-ARTHRITIC mammalian bone marrow). Capitalizing on these similarities, we are MULTI-FLARE MODEL OF STREPTOCOCCAL developing a xenograft model to study HSCs homing and niche-in- teractions in vivo and in real time using mammalian bone marrow CELL WALL INDUCED ARTHRITIS: FURTHER derived HSCs transplanted into fish larvae. To this aim, CD117+ cells CHARACTERIZATION IN A RODENT MODEL OF where isolated from mice bone marrows by means of negative COLLAGEN INDUCED ARTHRITIS selection, fluorescently stained, and transplanted into the blastoderm of 5 hpf zebrafish embryos. Live imaging of transplanted embryos Kalyan Chakravarthy, Robert Faltus, Anwar Murtaza, Milenko throughout development revealed that CD117+ cells in general Cicmil colocalized with the intermediate cell mass (ICM) at 24 hpf, with the AGM region at 48 hpf, and CHT at 72 hpf. CD117+ cells would Merck Research Laboratories, Rahway, NJ, USA evidence in situ proliferation, and in some cases, moved into circu- lation. The engraftments do not suggest mayor alteration of To delineate the role of TNF, T cells, and IL-1 in pathogenesis of endogenous hematopoiesis as zebrafish neutrophil development and SCW-induced arthritis, we investigated the activity of clinical agents, function is not impaired. Using this approach, murine HSCs could be Etanercept, Abatacept and Anakinra ina novel mono-arthritic multi- followed for at least 7 dpf. Engraftment with human promyelocytic flare Rat Streptococcal Cell Wall (SCW) model. Comparative eval- HL-60 cells also shows that they are able to colonize the CHT. uation of these targeted therapies was also performed in the rat Collectively, these data reveal the extent of conservation and Collagen Induced Arthritis (CIA) model. SCW arthritis was induced homology shared on protein cues among vertebrates during hemato- in female Lewis rats with an intra-articular injection in the hind ankle poiesis development, positioning the zebrafish as a good model to joint on day 1 (flare 1) followed by two intravenous challenges on

123 S198 Inflamm. Res. days 21 (flare 2) and 42 (flare 3) of SCW extract PG-PS 100p. CIA readout platform. Optimal adenoviral shRNA delivery conditions was induced using methods previously described. Inflammation and were established using reporter constructs in two adenoviral back- pain were monitored by measuring paw swelling and withdrawal bones (C01 and C20) and quantified by FACS. Cytotoxicity was threshold respectively. In addition, cytokine profiling, cell pheno- assessed using Cell titer blue assay. Negative control (ffluc and eGFP) typing, bioluminescence/lCT imaging and histopathology were also and putative positive control (IL17RA, NFjB, C/EBPb, CXCL1, performed in the local joint. In the SCW model late prophylactic IL17RC and MAPK14) shRNAs were used to establish optimal administration of Etanercept, Abatacept and Anakinra significantly screening conditions and assay window. A set 320 shRNA viruses inhibited paw swelling by C60 % (p \ 0.001), C60 % (p \ 0.001), from the SilenceSelectTM shRNA library was screened at three MOIs 88 % (p \ 0.001) and pain by 37 % (p \ 0.05), C28 % (p \ 0.05) to establish final screening characteristics. and 64 % respectively in flare 2. Etanercept in flare 3 inhibited paw Results: Recombinant human IL-17A induced a dose-dependent swelling by 60 % (p \ 0.001) and partially inhibited pain by 27 %. increase in GROa production in all three donors, in both 96- and Interestingly, prior treatment with Etanercept in flare 2 followed by a 384-well format. The signal to background ratio was comparable for wash out period of 14 days and re-administration in flare 3 led to a AlphaLisa and MSD both in the presence (C259) and absence (C69) loss in efficacy, potentially due to immunogenicity. Abatacept of 5 ng/mL TNFa stimulation. The AlphaLisa assay generated more administration in flare 3 had no effect on either paw swelling or pain reproducible data. Optimal (100 %) transduction was observed with in rats that were treated in flare 3 alone or in rats that were treated C20 backbone at MOI *20 in 96 well format. The effect of negative previously in flare 2. In the CIA model, both late prophylactic and control viruses was \25 % compared to non-transduced cells. therapeutic treatment with Etanercept inhibited paw swelling by 50 % Knockdown by shRNA of IL17RA, NFjB, C/EBPb,CXCL1,IL17RC (p \ 0.001). A loss of efficacy with Etanercept was also observed in and MAPK14 consistently reduced IL-17A induced GROa production. the CIA model when administered prophylactically possibly due to The assay was successfully automated and screened in duplicate, on immunogenicity. Prophylactic, late prophylactic and therapeutic two separate occasions, with a pilot screen set. No plate effects administration of Abatacept in the CIA model significantly inhibited observed based on heat maps. Pearson correlation coefficient and paw swelling by 100 % (p \ 0.001), 42 % (p \ 0.001) and 34 % Spearman correlation coefficient were [0.4. Kappa statistics [0.2. (p \ 0.001) respectively. The additional biomarkers corroborated Conclusions: A reproducible and robust assay was developed in pri- with efficacy in both models. We developed a novel multi-flare SCW mary human foreskin fibroblasts to identify novel therapeutic targets model that can be used to evaluate clinically relevant parameters of modulating the IL17 pathway. inflammation and pain simultaneously. Using clinical agents Etaner- cept, Abatacept and Anakinra targeting TNF, T cells and IL-1 respectively we have delineated distinct pathogenic mechanisms of inflammation and pain at different stages of disease in the SCW Novel Targets and New Drugs model. We also show similar profiles of efficacy in late prophylactic in Inflammation and therapeutic regimens in the CIA model. The flaring mechanism in the SCW model allows for drug washout periods in between com- pound administration. This might provide useful pre-clinical insights B244 on potential immunogenicity mechanisms that may be relevant in a THYMOQUINONE INHIBITS INFLAMMATION IN clinical setting. Our novel model can facilitate innovative assessment IL-1BETA-STIMULATED SK-N-SH CELLS of anti-rheumatic agents in multiple flares and offers a powerful tool for drug discovery. Olumayokun Olajide1, Ravikanth Velagapudi1, Asit Kumar2, Simon Schindler2, Bernd L. Fiebich2

1University of Huddersfield, Huddersfield, England; 2University of B243 Freiburg Medical School, Freiburg, Germany A NOVEL ASSAY FOR HIGH THROUGHPUT Thymoquinone is a phytochemical antioxidant compound in the oil shRNA-BASED TARGET DISCOVERY IN THE obtained from the seeds of Nigella sativa (black cumin seed oil). IL-17 PATHWAY Studies have suggested that thymoquinone produces anti-inflamma- tory property. Previously, we have shown that thymoquinone inhibited neuroinflammation in LPS-activated rat priamry microglia. Jeroen DeGroot, Blandine Mille-Baker, David F. Fischer However, nothing is known about its direct effect on neurons. In this study, we have investigated the effects of thymoquinone on inflam- Charles River Laboratories, Leiden, The Netherlands mation induced in SK–N–SH neuroblastoma cells stimulated with Objectives: The IL-17 pathway is associated with pathology in interleukin-1beta (IL-1b). Cultured SK–N–SH cells were treated with numerous autoimmune and inflammatory conditions, such as psoria- thymoquinone (0.5, 1 and 2.5 lM) prior to stimulation with IL-1b. sis, rheumatoid arthritis, multiple sclerosis, Crohn’s Disease and SLE. Levels of prostaglandin E2(PGE2) production was measuredusing Consequently, IL-17 and its downstream signaling routes emerge as enzyme immunoassay (EIA), while ELISAs were used to detect levels potential targets for therapy. Monoclonal antibodies against IL-17 of pro-inflammatory cytokines tumour necrosis factor-alpha (TNFa) (such as secukinumab; recently approved for psoriasis) and IL-17R and interleukin-6 (IL-6). Levels of cyclooxygenase-2 (COX-2, (e.g. Brodalumab; in phase 2) have been developed. The objective of microsomal prostaglandin E synthase-1 (mPGES-1) were measured the current study was to develop and validate a high throughput assay with western blot. Further experiments were carried out on I&B for the identification novel drugable target genes downstream of IL- phosphorylation and degradation using western blots. Results showed 17 receptor A (IL-17RA) using an arrayed adenoviral shRNA library that thymoquinone (0.5, 1 and 2.5 lM) produced concentration-de- (SilenceSelect). pendent and significant inhibition of PGE2, TNFa and IL-6. These Methods: Human foreskin fibroblasts (3 donors) were cultured in concentrations of the compound also reduced protein levels of COX-2 96 well plates and stimulated with IL-17 in the presence or absence of and mPGES-1. At 1 and 2.5 lM, there was marked inhibition of I&B TNFa. GROa production was assessed by MesoScale Discovery phosphorylation and degradation by this compound. Taken together, (MSD) or AlphaLisa to confirm cellular functionality and select the these results demonstrate that thymoquinone suppressed inflammation

123 Inflamm. Res. S199 in neurons, suggesting its therapeutic potential in inflammation-me- inflammatory effect of LQFM-021, shown that the nitric oxide is diated neuronal damage found in neurodegenerative disorders. important to this effect. Sources of research support: CNPq and CAPES.

B245 B246 MECHANISMS INVOLVED IN THE ANTI- IDENTIFICATION OF CD44BRIGHT, A NOVEL INFLAMMATORY ACTIVITY OF THE NEW SUBSET OF UTERINE NK CELLS CORRELATED PYRAZOLE COMPOUND 5-(1-(3- TO ABORTION IN RECURRENT PREGNANCY FLUOROPHENYL)-1H-PYRAZOL-4-YL)-2H- LOSS MODEL MICE TETRAZOLE (LQFM-021) Jun Tanaka, Yuki Fukunaga Iziara F. Florentino1, Daiany Priscilla B. Silva1, Roberta C. Lino1, Taciane S. Silva2, Carlos Roge´rio Tonussi2, Ricardo Menegatti1, Central Reseaech Laboratory, Japan Blood Products Organization, Elson A. Costa1 Izumisawa, China

1 2 Introduction: Women undergoing three or more consecutive sponta- Federal University of Goia´s, Goiaˆnia, Brazil; Federal University of neous abortions are believed to suffer from recurrent pregnancy loss Santa Catarina, Floriano´polis, Brazil (RPL). Women with RPL display a higher rate of and cytotoxic activity Introduction: Inflammation is an adaptive response that is triggered by of NK cells in the periphery and endometrium. Conversely, normal noxious stimuli and conditions, such as infection and tissue injury. uterine NK (uNK) cells play an important role in the establishment and The pyrazole compounds are known to possess anti-inflammatory, outcome of pregnancy. The triggering mechanism that induces NK cells analgesic and antipyretic activities. The aim of this work was to to attack the fetus remains unexplained; however, most RPL cases have evaluate anti-inflammatory effect of the new pyrazole derivative 5-(1- been explained by autoimmune abnormalities. Intravenous (3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM-021) in acute immunoglobulin (IVIg) has been utilized in the treatment of several and chronic models of inflammation and investigate mechanisms inflammatory and autoimmune disorders. The efficacy of IVIg in the involved in this effect. treatment of RPL has been confirmed in several clinical trials; however, Methods: The LQFM-021, pyrazole derivative, was synthesized at the its precise mechanism remains unknown, mainly because the mecha- Laboratory of Medicinal Pharmaceutical Chemistry/UFG. In this nism of abortion remains to be elucidated. Therefore, the mechanism of study were used Swiss albino mice and Wistar rats females. The IVIg action, as well as that of abortion in RPL must be explained. experimental protocol was approved by the Research Ethics Com- Methods: All animal procedures were approved by the Animal Care mittee of UFG (number 017/13). The pharmacological tests: and Use Committee of our organization. In this study, the CBA/ Carrageenan-induced paw edema and pleurisy tests and CFA-induced J 9 DBA/2 N mating mouse model was employed to analyze RPL. chronic arthritis model. The abortion rate was increased by activating maternal immunity via Results: In the paw edema test, the treatments with LQFM-021 30 or an intraperitoneal LPS injection; the preventive effect of IVIg 60 mg/kg p.o., began to reduce the edema starting from the 2nd hour by administration on immune reproductive failure was also examined. 25.3 and 33.7 %, respectively, at 3rd hour by 25.5 and 33.0 %, The peripheral and uterine NK cells were defined as CD45 + CD3- respectively, and at 4th hour by 30.4 and 29.0 %, respectively. In the CD49b+ . The percentage of activated peripheral NK (CD69+) cells carrageenan-induced pleurisy test, the treatment with LQFM-021 or the specific uNK cell subset was determined by flow cytometry. (30 mg/kg p.o.) reduced the leukocyte migration into pleural cavity by Results: IVIg did not affect peripheral immune cell activation; 38.1 %, with reduction of polymorphonuclear recruitment, also was however, it attenuated the rate of abortion in RPL model mice in a observed a reduction of the Evan’s blue concentration in the pleural dose-dependent manner. Additionally, we confirmed a significant exudates by 30.9 %, decrease myeloperoxidase activity by 43.0 % and increase in the number of uNK cells in RPL model mice, which decreased the TNF-a level by 47.5 % and IL-1b levels by 26.8 %. was suppressed by IVIg injection. We also demonstrated a novel Moreover, was also seen which the pre-treatment with L-name was flow cytometry-based approach for the characterization of uNK capable of reversed the effect of the LQFM-021 on leukocytes subsets by CD44 expression. Specifically, we discovered the migration, TNF-a and IL-1b levels. In the CFA-induced chronic presence of two distinct subsets: CD44bright and CD44mid. We arthritis model, the treatment with LQFM-021 (30 mg/kg once/day) observed an increase in the CD44bright uNK subset in RPL model promoted a progressive reduction of Paw Lifting Time (PLT) from the mice, while the CD44mid uNK subset remained unchanged. Fur- 2nd until 6nd day, by 26.3; 31.4; 31.3; 41.8 and 47.0 %, respectively. thermore, the CD44bright uNK subset number remained The formation of edema was reduced from the 1st day of the treatment unchanged when the abortion was reduced by IVIg administration, by 36.9; 23.6; 20.7; 21.7; 24.6 and 18.0 %, respectively. Also, there facilitating the differentiation between pathological uNK cells and was a reduction in the number of leukocytes by 34.2 % and poly- normal uNK cells, based on CD44 expression. We observed a low morphonuclear by 54.3 % to synovial fluid. The treatment with LQFM- expression of the CD94 inhibitory receptor on the CD44bright 021 (15 mg/kg twice/day), reduced the PLT from the 2nd until 6nd uNK subset. Moreover, we also confirmed the presence of acti- day, by 29.6; 15.6; 21.2; 29.2 and 29.5 %, respectively. vated peripheral NK cells belonging to the CD44bright subset in Conclusions: The pyrazole compound LQFM-021 presented anti-in- RPL model mice with or without IVIg injection. flammatory activity in acute and chronic models of inflammation. In Conclusion: Based on these results, we proposed CD44 to be the marker this anti-inflammatory effect involve the reduction leukocyte migra- of pathological uNK cells, with the CD44bright uNK subset acting as a tion, the TNF-a and IL-1b levels and decreased of the novel RPL target. Elucidation of the mechanism underlying IVIg myeloperoxidase activity. In addition, we also found that the inhibi- activity against RPL may help explain the optimal design of IVIg tion of nitric oxide synthase promoted by L-NAME reduced the anti- injection, as well as the mechanisms underlying abortion in RPL.

123 S200 Inflamm. Res.

B247 B248 RESVERATROL IMPROVES THE PATHOGENESIS NEW ANALOGUES FROM IKK-b INHIBITOR OF NONALCOHOLIC STEATOHEPATITIS LASSBIO-1524 AS POTENT ANTI- THROUGH INHIBITION OF ENDOTOXIN- INFLAMMATORY CANDIDATES INDUCED LIVER INFLAMATION AND FIBROSIS Nata´lia M. Cordeiro, Rosana H.C. Freitas, Carlos Alberto M. Fraga, Takaomi Kessoku1, Koichiro Wada2, Yasushi Honda1, Yuji Ogawa1, Patricia D. Fernandes Kento Imajo1, Atsushi Nakajima1 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 1 2 Yokohama city university, Yokohama, Japan; Shimane University LASSBio-1524, a new ikB kinase b inhibitor, participates in the Faculty of Medicine, Matsue, Japan activation of NF-kB canonical pathway and demonstrated anti-in- Introduction: Nonalcoholic fatty liver disease (NAFL) morbidity rate flammatory efficacy in vivo (Eur J Med Chem., 46, 1245, 2011). in Asia Pacific region is close to 12–24 %, while in Western countries Our objective is to evaluate the anti-inflammatory potential of new is about 20–30 %. and NAFLD can progress to nonalcoholic steato- substances synthesized from LASSBio-1524: LASSBio-1760, hepatitis (NASH), cirrhosis and hepatocellular carcinoma. In spite of LASSBio-1763, LASSBio-1764. its high prevalence, up till now here is no proven effective treatment Cell migration was induced by carrageenan injection in the for NAFLD. Although gut-derived endotoxin (ET), such as subcutaneous air pouch (SAP), mice (Swiss webster, 22–25 g, lipopolysaccharide (LPS), plays a key role in the pathogenesis of n = 6–8) received 1 h before carrageenan injection, treatment nonalcoholic steatohepatitis (NASH), detailed mechanisms of this with LASSBios (3, 10, 30, 100 lmol/kg, p.o.), IKK-b inhibitor pathogenesis becomes clear. We previously reported that overex- SC-514 (30 lmol/kg p.o.) or dexamethasone (1.5 lmol/kg, i.p.). pression of CD14 via activation of leptin-STAT3 signaling in Kupffer Air pouch exudate was collected for quantification of TNF-a. cells induced hyper-inflammatory response to low-dose ET, resulting Leukocytes from the SAP were pretreated ex vivo with LASSBios in progression from simple steatosis to steatohepatitis with liver (1, 10 and 30 lM) to assess reactive oxygen species (ROS). fibrosis. Therefore, we hypothesized that inhibition of leptin-STAT3 Resultsareexpressedasmean± SD and statistical analysis were signaling in Kupffer cells may lead to attenuate the progression of performed by ANOVA followed by Bonferroni test (*p \ 0.05). steatohepatitis via inhibition of CD14 expression. The protocol for animal use was approved by CEUA/UFRJ Aim: The aim of this study was to investigate whether the resveratrol #DFBCICB015-04/16. which is known to inhibit activation of STAT3, improves the All substances reduced dose-dependent and significantly leukocyte pathogenesis of steatosis or steatohepatitis in murine model. migration. Animals pretreated orally with vehicle (Polysorbate 80) Methods: 8-week-old male C57BL/6 J mice were randomly dis- injected with carrageenan in the SAP presented increase in leukocyte 3 tributed into 3 groups of 10 animals each: a high fat diet group (HF), number (78.3 ± 13.5 9 10 cells/lL) when compared to the group 3 HF supplemented with 2 mg/kg resveratrol daily (HFR2), and HF injected with PBS in SAP (1.1 ± 0.5 9 10 cells/lL). Treatments 3 supplemented with 20 mg/kg resveratrol daily (HFR20). After with SC-514 (36.2 ± 19.4 9 10 cells/lL), dexamethasone (19.4 ± 3 3 12 weeks of dietary treatment, the rats were euthanized and relevant 8.6 9 10 cells/lL); LASSBio-1524 (3 lmol/kg: 51.8 ± 3.6 9 10 3 tissues were prepared for subsequent analysis. In this study, E. coli- cells/lL, 10 lmol/kg: 42.2* ± 3.9 9 10 cells/lL, 30 lmol/kg: 3 3 derived LPS (0.25 mg/kg) was used. 22* ± 4.1 9 10 cells/lL, 100 lmol/kg: 12.7* ± 4.2 9 10 cells/ lL) or its three analogues LASSBio-1760 (3 lmol/kg: 51.9 ± A. We investigated whether the resveratrol attenuates HFD-induced 32.2* 9 103 cells/lL, 10 lmol/kg: 40.2* ± 22.3 9 103 cells/lL, steatosis. 30 lmol/kg: 35.7* ± 12.1 9 103 cells/lL, 100 lmol/kg: 11.0* ± B. We investigated whether the resveratrol attenuates ET-induced 4.2 9 103 cells/lL), LASSBio-1763 (3 lmol/kg: 47.0* ± 18.3 9 103 liver damage via inhibition of response to ET. cells/lL, 10 lmol/kg: 39.3* ± 4.7 9 103 cells/lL, 30 lmol/kg: C. We investigated whether the resveratrol improves the pathogen- 27.5* ± 10.0 9 103 cells/lL, 100 lmol/kg: 3.0* ± 1.7 9 103 cells/ esis of long-term exposed ET-induced steatohepatitis with liver lL) and LASSBio-1764 (3 lmol/kg: 40.9* ± 8.8 9 103 cells/lL, fibrosis. 10 lmol/kg: 34.0 * ± 9.6 9 103 cells/lL, 30 lmol/kg: 26.9* ± 10.2 9 103 cells/lL, 100 lmol/kg: 1.7* ± 1.1 9 103 cells/lL) Results: Resveratrol prevented the high fat–induced steatosis assessed reduced leukocyte migration. All doses also inhibited TNF-a pro- by semiquantitative grading, which furthermore corresponded with a duction: LASSBio-1524 reduced in 79, 79, 87 and 94 %; LASSBio- complete normalization of the hepatic triglyceride content 1760 reduced in 99, 97, 95 and 93 %; LASSBio-1763 reduced in 92, (p \ 0.001), despite no change in total body fat, and hepatic 96, 93 and 99 % and LASSBio-1764 reduced in 96, 97, 95 and 96 %, SREBP1c expression was significantly decreased as compared with respectively. All substances significantly inhibited ROS production: HF. HFR showed significant inhibition of hepatic CD14 expression LASSBio-1524 reduced in 30, 41 and 45 %; LASSBio-1760 reduced through suppression of STAT3 activity in Kupffer cells, following in 42, 33, 29 %; LASSBio-1763 reduced in 61, 97, 98 % and inhibition of a single low-dose LPS-induced liver damage. Moreover, LASSBio-1764 reduced in 83, 84, 98 %. long-term low-dose LPS-induced liver fibrosis in HFR is significantly The new analogues of LASSBio-1524 showed anti-inflammatory decreased as compared with HF. potential even better than the original molecule, placing them as new Conclusion: These data indicated that the resveratrol improves not candidates for the development of new anti-inflammatory drugs. only the pathogenesis of steatosis thorough inhibition of lipogenesis Financial support CNPq, CAPES, FAPERJ and Instituto Vital Brazil but also steatohepatitis through inhibition of endotoxin-induced liver (donation of animals). damage via suppression of STAT3-CD14 signaling in Kupffer cells. Technical support Alan Minho. The resveratrol may have application for the treatment of NAFLD.

123 Inflamm. Res. S201

B249 Inflammation is a beneficial host response to injury that has as goal LIPOSOMES LOADED WITH the restoration of tissue structure and function. The LASSBio-1828 is a new n-acilhydrazone derived from LASSBio-1524, an IKK-b 3-PHENYLCOUMARIN DERIVATIVES AND inhibitor (Eur J Med Chem., 46, 1245, 2011). BACCHARIS DRACUNCULIFOLIA EXTRACT Our objective was to evaluate the anti-inflammatory potential of REDUCE THE INFILTRATE OF NEUTROPHILS IN LASSBio-1828 through formalin-induced licking and carrageenan- SYNOVIAL FLUID OF ARTICULAR induced inflammation in the subcutaneous air pouch (SAP). Male Swiss webster mice (22–25 g, n = 4–6) were pre-treated with INFLAMMATION INDUCED BY ZYMOSAN LASSBio-1828 (10, 30, 100 lmol/kg, p.o.), LASSBio-1524 (30 lmol/ kg, p.o.), SC-514 (IKK-b inhibitor, 30 lmol/kg, p.o.) or vehicle 1 h 1 2 Mica´ssio F. Andrade , Andre´a S.G. Figueiredo-Rinhel , Ana Elisa before formalin injection in mice paws or carrageenan injection in the 2 3 3 3 C.S. Azzolini , Jairo K. Bastos , Fla´vio S. Emery ,Moˆ nica T. Pupo , SAP. Formalin-induced licking response was evaluated during 30 2 Yara M. Lucisano-valim subsequent minutes and 24 h after carrageenan injection in the SAP the exsudate was collected for leukocyte counting, TNF-a and nitric oxide 1 Department of Biochemistry and Immunology, School of Medicine, (NO) measurements. Reactive oxygen species (ROS) were measured in University of Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil; leukocytes obtained from exsudate of the carrageenan-induced 2 Department of Physics and Chemistry, School of Pharmaceutical inflammation in the SAP. Cells were ex vivo incubated with LASSBio- Sciences, University of Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil; 1524 (30 lM) or LASSBio-1828 (1, 10, 30 lM) before treatment with 3 Department of Pharmaceutical Sciences, School of Pharmaceutical phorbol myristate acetate (PMA). Results are expressed as mean ± SD Sciences, University of Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil and statistical analysis were performed by ANOVA followed by Introduction: Neutrophils have an important part in acute inflamma- Bonferroni test (p \ 0,05 %). Protocols for animal use received num- tory responses. When they find a stimulus, these cells produce ber #DFBCICB015-04/16. reactive oxygen species (ROS), release lytic compounds of their LASSBio-1828 showed any antinociceptive effect in the 1st phase granules, as well as release of their DNA content. Therefore, these of formalin-induced licking, but all doses inhibited the 2nd phase: processes can disorders provoked. Natural and synthetic compounds 10 lmol/kg = 128 ± 41* s; 30 lmol/kg = 76 ± 26* s; 100 lmol/ are able to reduce the deleterious effects of neutrophils activation. In kg = 83 ± 32* s when compared to vehicle = 193 ± 32 s. Car- 0 0 rageenan-induced leukocyte migration to SAP was significantly this context, 6,7-dihydroxy-3-[3 ,4 -methylenedioxyphenyl]-coumarin 6 (3-PD), a synthetic 3-phenylcoumarin derivative and Baccharis dra- reduced by 100 lmol/kg dose (42 ± 12*cells 9 10 /mL, when compared to PBS in SAP = 3±2cells 9 106/mL or carrageenan in cunculifolia D.C. (Asteraceae) leaf extracts (BdE) showed, in vitro 6 assays, a negative modulation of neutrophils oxidative metabolism. SAP = 65 ± 9cells 9 10 /mL). All doses of LASSBio-1828 signif- Thus, the aim of this work is investigate the effect of these two icantly reduced TNF-a level in exudate: 10 lmol/kg = 321 ± substances, incorporated in liposomes (LPM), in animal model of 210*ng/mL; 30 lmol/kg = 371 ± 272*ng/mL; 100 lmol/kg = articular inflammation induced for zymosan. 390 ± 113*ng/mL, when compared with PBS in SAP (20 ± 41 ng/ Methods and results: Wistar rats (200 g) were treated i.p., with lipo- mL) or carrageenan in SAP (755 ± 84 ng/mL). Results of LASSBio- somes loaded with 3-PD (LPM 3PD 1.5 mg/Kg), BdE (LPM BdE 9 mg/ 1524 and SC-514 were 99 ± 49*ng/mL and SC-514 = 527 ± Kg), or with control substances [Quercetin (LPM Quer 1.5 mg/Kg)— 270 ng/mL, respectively. Concentration of NO in the exudate phenolic compound pattern, or with dexamethasone (Dexa 4 mg/Kg)— decreased approximately 50 % at all doses of LASSBio- drug pattern, or with dimethyl sulfoxide (LPM DMSO)—positive 1828: 10 lmol/kg = 66 ± 65*lM; 30 lmol/kg = 90 ± 62*lM; control]. Liposomes were prepared by ethanol injection method. Vesi- 100 lmol/kg = 100 ± 49*lM, when compared to PBS in SAP cles composed of soya phosphatidylcholine and cholesterol (26 ± 2 lM) or carrageenan in SAP (279 ± 86 lM). The results of (SPC:CHOL 5:1). One h late of treatment, the animals were injected LASSBio-1524 and SC-514 were 65 ± 50 and 64 ± 44*lM, intra-articular with 300 lg of zymosan. Six h late, the animals were respectively. Reduction in ROS production was: 1 lM = 4 9 105 ± 3 9 104 arbitrary units; 10 lM = 2 9 105 ± 1 9 104*au; sacrificed and back the synovial fluid for total leucocytes and neu- 5 4 trophils recruitment analyze. The diameter of articulation was verified 30 lM = 1 9 10 ± 1 9 10 *au, when compared to control group (6 9 105 ± 1 9 105au). The result of LASSBio-1524 was before and after of induction of inflammation. The animals treated with 5 4 LPM 3-PD and LPM BdE showed reduction significantly in diameter of 3 9 10 ± 1 9 10 au. articulation in relation of control animals (LPM DMSO). Therefore, the These results indicate that the new n-acylhydrazone, LASSBio- treatments reduce significantly the infiltrate of total leucocytes and 1828, seems to be, at least in part, better than the original molecule neutrophils in articulation. This reduction was similar for all treatments. LASSBio-1524 in tested models suggesting this new substance to be Conclusion: The treatment of animals with LPM 3-PD and LPM BdE evaluated in other models of inflammation. reduced all the inflammatory parameters analyzed. Taken together, Financial support CAPES, CNPq, FAPERJ, Instituto Vital Brazil the dates show these substances, incorporate in this drug carrier (donation of animals). system, with potential therapeutic application in the treatment of Technical support Alan Minho. inflammatory diseases. Support FAPESP, CNPq and CAPES. B251 ANTI-INFLAMMATORY ACTIVITY OF B250 FRACTIONS OBTAINED FROM CHOISYA AZTEC- LASSBIO-1828: A NEW IKK-b POTENTIAL PEARL, A NEW HYBRID OF CHOISYA TERNATA INHIBITOR Patrı´cia Ribeiro de Carvalho1, Denise Ropero2,Fa´bio Boylan 2, Tayna´ S. Valerio, Nata´lia M. Cordeiro, Rosana H.C. Freitas, Patricia Dias Fernandes1 Carlos Alberto M. Fraga, Patricia D. Fernandes 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Trinity Federal University of Rio de Janeiro, Rio de Janeiro, Brazil College Dublin, Dublin, Republic of Ireland

123 S202 Inflamm. Res.

C. Aztec-Pearl (Rutaceae) is a hybrid of C. Ternata and C. Dumosa Marie-Christine Ceccotti1, Christelle David1, Laetitia Perret1, var. arizonica. In traditional medicine C. ternata has been used in Martine De Vos3, Reginald Brys2, Rene´ Galien1 Mexico. Infusions prepared with the leaves of the latter species are claimed to have antispasmodic and stimulant properties. 1GALAPAGOS SASU, Romainville, France; 2GALAPAGOS NV, Our objective was to investigate a possible anti-inflammatory Mechelen, Belgium; 3Ghent University Hospital, Ghent, Belgium activity from C. Aztec-Pearl’s leaves crude ethanol extract and its Objective: Filgotinib (known as GLPG0634) is a JAK inhibitor that fractions. has been shown to be selective for JAK1 over the 3 other family Leaves of C. Aztec-Pearl were collected in Dublin and a voucher members (JAK2, JAK3 and TYK2) in biochemical and human whole specimen (#TCD2,895) was deposited at TCD Herbarium. The crude blood assays. It has been shown to be efficacious in arthritis and ethanol extract (E) was submitted to a liquid–liquid extraction yielding colitis mouse models. Filgotinib is currently being assessed as a the fractions: hexane (H) and ethyl acetate (EA). Anti-inflammatory treatment for Crohn’s disease (CD) in a Phase 2 study. Here, we activities were evaluated by the formalin-induced licking and car- compare signaling mechanisms, in particular JAK1 inhibition-related rageenan-induced inflammation into the subcutaneous air pouch (SAP) effects, regulated by filgotinib in mouse and human colon in vivo or models. Mice (Swiss Webster, 22–25 g, n = 4–6) were orally treated ex vivo, respectively. with 10, 30 or 100 mg/kg of each fraction 1 h before formalin or car- Methods: In vivo efficacy of 30 mg/kg filgotinib QD was evaluated in rageenan injection. After 24 h of carrageenan injection into the SAP, a mouse chronic DSS (dextran sodium sulphate, 3 cycles)-induced animals were euthanized and exsudate was collected for subsequent colitis model using macroscopic and histopathological scorings, dosages of nitric oxide (NO) and protein. The results are presented as immunohistochemistry and gene expression analysis by qPCR. mean ± SD and statistical analysis were performed by ANOVA fol- Inflamed colon biopsies from IBD patients were cultured for 18 or lowed by Bonferroni test (*p \ 0.05). Protocols for animal use received 24 h in the presence of 5 lM GLPG0634 and gene expression as well number #DFBCICB015-04/16 (COBEA/UFRJ/Brazil). as STAT phosphorylation were analyzed using qPCR and 5-Plex None of the fractions inhibited the first phase (nociceptive) of the STAT kit Luminex, respectively. response to formalin, although all of them significantly reduced the Results: Filgotinib strongly prevents experimental colitis as demon- 2ndphase (inflammatory). The results were: 10 mg/kg: strated by reduction of DAI, lower histological disease severity, E = 75 ± 18*s; H = 82 ± 10*s; EA = 123 ± 15*s; 30 mg/kg: reduced inflammatory cell infiltration and lower expression of E = 32 ± 43*s; H = 32 ± 10*s; EA = 68 ± 39*s; 100 mg/kg: inflammatory genes in colon tissue from filgotinib-treated mice E = 70 ± 29*s; H = 13 ± 9*s; EA = 44 ± 13*s, compared to compared to controls. Colons from DSS-treated mice displayed vehicle = 212 ± 22 s. E and EA significantly reduced leukocyte higher STAT3 phosphorylation levels, which were prevented by migration into the SAP at doses of 30 mg/kg: E = 13 ± 6*- treatment with filgotinib. Of interest, the expression of genes that cells 9 106/mL; EA = 27 ± 17*cells 9 106/mL and 100 mg/kg: activate (IL-6) or are induced by the JAK1 pathway (MX1, MX2) E = 15 ± 10*cells 9 106/mL; EA = 24 ± 16*cells 9 106/mL, followed the same regulation as pSTAT3, suggesting a relationship when compared to vehicle-treated group:1.1 ± 0.6 cells 9 106/mL between these readouts and a role of the pathway in the disease. and carrageenan-injected group:70 ± 21 cells 9 106/mL. Remarkably, the same regulation of IL-6 and MX1 expression and NO concentration in the exsudate was significantly reduced by E relationship with STAT3 phosphorylation were observed in cultured and H fractions at all doses, 10 mg/kg: E = 238 ± 117 lM*; colon biopsies from IBD patients in presence of filgotinib. H = 151 ± 35 lM*, 30 mg/kg: E = 413 ± 141 lM*; H = 167 ± Conclusions: These new data provide further mechanistic under- 42 lM*; 100 mg/kg: E = 237 ± 142 lM*; H = 246 ± 146 lM*, standing for the efficacy of JAK1 inhibition in the pre-clinical mouse the EA reduced NO production in more than 60 % only in two doses: colitis model, highlighting the importance of JAK1/STAT3 pathway. 30 mg/kg: 20 ± 14 lM* and 100 mg/kg: 81 ± 44 lM*, when Similarly a human colon biopsy model further supports the impor- comparing to vehicle-treated: 32 ± 19 lM and carrageenan-treated tance of the JAK1 pathway in the etiology of IBD. These data suggest group: 796 ± 117 lM. All fractions also reduced protein extravasa- that filgotinib may be beneficial in treating CD patients and support its tion at doses: 10 mg/kg: E = 165 ± 64 lM*; H = 130 ± 72 lM*; evaluation in a clinical study. EA = 135 ± 39 lM*; 30 mg/kg: E = 147 ± 112 lM*; H = 33 ± 17 lM*; EA = 51 ± 21 lM*; 100 mg/kg: E = 161 ± 84 lM*; H = 154 ± 60 lM*; EA = 146 ± 97 lM*, when comparing to vehicle-treated: 24 ± 15 lM and carrageenan-treated group: B253 215 ± 76 lM. GPR84 INHIBITION AS A NOVEL THERAPEUTIC Our results indicate a significant anti-inflammatory activity of this plant, and contribute to the studies with the Rutaceae family. APPROACH IN INFLAMMATORY BOWEL Financial support Capes, Cnpq, FAPERJ and Instituto Vital Brazil DISEASE (IBD): MECHANISTIC AND (donation of animals). TRANSLATIONAL STUDIES Technical support Alan Minho. Sonia Dupont1, Ingrid Arijs3, Roland Blanque´1, Debby Laukens4, Kris Nys3, Marie-Christine Ceccotti1, Didier Merciris1, Steve De Vos2, B252 Mate´ Ongenaert2, Isabelle Parent1, Veerle De Vriendt2, Fre´de´ric FILGOTINIB, JAK1-SELECTIVE INHIBITOR, Labe´gue`re1, Rene´ Galien1, Martine De Vos4, Paul Rutgeerts3, REPRESSES SIMILARLY JAK1/STAT3 PATHWAY Nick Vandeghinste2,Se´verine Vermeire3, Reginald Brys2 IN THE COLON OF MICE WITH DSS-INDUCED 1Galapagos SASU, Romainville, France; 2Galapagos NV, Mechelen, COLITIS AND IN CULTURES OF COLON Belgium; 3Translational Research Center for Gastrointestinal BIOPSIES FROM INFLAMMATORY BOWEL Disorders (TARGID), KU Leuven, Leuven, Belgium; 4Department of DISEASE PATIENTS Gastroenterology, University Hospital, Ghent, Belgium Objective: GPR84 is a GPCR activated by medium chain fatty acids, Sonia Dupont1, Carole Delachaume1, Veerle De Vriendt2, Debby mainly expressed in white blood cells and regulating cell migration. Laukens3,Be´atrice Vayssie`re1, Didier Merciris1, Steve De Vos2, Recently, we reported that pharmacological GPR84 inhibition reduces

123 Inflamm. Res. S203 disease activity in experimental colitis (Dupont et al. UEGW 2014). Purpose: In this study, we examined the therapeutic benefits of To gain mechanistic insights, the impact of GPR84 inhibition on SHED-CM, Bone marrow mesenchymal stem cells (BMMSC)-CM colon gene expression was investigated in experimental colitis. In and skin fibroblast (Fibro)-CM for treatment of the Ab peptide addition, translational studies were engaged, measuring GPR84 injected mouse AD-like model. expression levels in patient samples. Materials and methods: Mouse AD-like model was generated by the Methods: In vivo efficacy of GLPG1205, a GPR84 antagonist, was i.c.v. injection of oligomerized 5 lgofAb1-40, or Ab40-1 was into evaluated in a mouse chronic DSS (dextran sodium sulphate, 3 the 9-week-old ICR mice brain. Twenty-four h after the i.c.v. injec- cycles)-induced colitis model using macroscopic and histopathologi- tion, a total of 50 ll of various type CM, were administered to each cal scorings and microarray gene expression analysis. Blood and mice via the olfactory pathway. The therapeutic effects were evalu- intestinal samples from ulcerative colitis (UC) and Crohn’s disease ated by the behavioral, biochemical and histological examination. (CD) patients were collected (before and after first Infliximab treat- In vitro, effects of SHED-CM on microglia and neurons were ment) as well as from healthy controls. GPR84 expression was investigated by biochemical examination and TUNEL staining. All confirmed by QrtPCR and immunohistochemistry (IHC). experiments were performed in accordance with theGuidelines for Results: Disease activity in a DSS model was strongly reduced by Animal Experimentsof Nagoya University Graduate School of GLPG1205 (10 mg/kg, q.d.) and sulfasalazine (SSZ, 20 mg/kg, q.d.). Medicine. Microarray expression profiles in DSS-treated mice showed a strong Result: SHED-CM suppressed the expressions of pro-inflammatory negative correlation between GLPG1205 treatment and disease cytokines, iNOS and 3-nitrotyrosine and improved cognitive function (R2 = 0.8), comparable to the impact of SSZ (R2 = 0.75). Compar- of the mouse AD-like model. SHED-CM shifted the M1-type pro- ison to human expression datasets showed an impact of GLPG1205 inflammatory microenvironment associated with mouse AD-like on several gene sets associated with IBD. Microarray and QrtPCR model toward the M2-type anti-inflammatory/neuroprotective one. data indicated an increase in GPR84 expression in inflamed UC colon Conclusion:: Our data demonstrate that SHED-CM exerted multi- and CD colon/ileum which was more pronounced in patients not faceted neuro-repairing activities for the treatment of mouse AD-like responding to infliximab. Moreover IHC showed a positive GPR84 model without adverse effects. SHED-CM may provide a novel cell- staining in mucosal inflammatory cells which was increased in active free neuro-reparative therapy for AD. IBD mucosa and very pronounced in granuloma and pouchitis sam- ples. Additionally, patient white blood cell samples displayed increased GPR84 expression levels as well. Conclusions: GLPG1205 has profound effects on experimental colitis B255 that were confirmed at gene expression level and were comparable to NOVEL CARTILAGE-PROTECTIVE MEDIATORS SSZ. In patient biopsies, an increased GPR84 expression specifically IN PRO-RESOLVING EXUDATES in active disease conditions was observed, which represents an advantage with regard to the safety profile of GPR84 inhibitors. As Magdalena K. Kaneva1, Sarah E. Headland1, Prashant Mori2, GPR84 expression is increased in patient blood, GPR84 inhibition Adrian Moore2, Mauro Perretti1 may impact disease through systemic effects as well. 1William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK; 2UCB Pharma, Slough, UK B254 Introduction: The end-point of the resolution of inflammation is FUNCTIONAL RECOVERY OF ALZHEIMER’S healing, repair and restoring tissue physiology. We set out to search DISEASE USING STEM CELLS FROM HUMAN for novel tissue-protective activities produced within the resolution EXFOLIATED DECIDUOUS TOOTH-DERIVED phase of acute inflammation and applied an integrated approach with CONDITIONED MEDIUM experimental exudates, proteomic analyses and bio-assays with human chondrocytes and experimental arthritis. Methods: Rat pleural exudates (24 h post-carrageenan injection) were Tsuneyuki Mita1, Yoko Hibi2, Chiaki Shimojima1, Hisasi Hattori1, subjected to gel filtration chromatography and run for mass spec- Kiyofumi Yamada1, Akihito Yamamoto1 trometry using ion-trap mass analyzer (MS/MS LTQ Orbitrap XL). Human C28/I2 chondrocytes were grown in 3D high-density micro- 1Department of Oral and Maxillofacial Surgery, Nagoya University mass (PMID: 21946086), incubated with the fractions with or without Graduate School of Medicine, Nagoya, Japan; 2Department of catabolic stimuli like interleukin (IL)-1b (20 ng/mL) or osteoarthritic Neuropsychopharmacology and Hospital Pharmacy, Nagoya synovial fluids (OASF) for 48 h. a1-Antitrypsin (AAT) and Gelsolin University, Graduate School of Medicine, Nagoya, Japan (GSN) were tested against catabolic stimulation. Sulphated gly- Introduction: Alzheimer’s disease (AD), a progressive neurodegen- cosaminoglycans (GAGs) deposition was assessed using Alcian Blue erative disease, is characterized by deterioration of cognitive function (AB) staining. Gene expression was quantified by qPCR. Data are accompanying with deposition of b-amyloid (Ab) peptides. Ab pep- expressed as 2-DDCt equation–relative amount of target genes, nor- tides are liberated from larger transmembrane amyloid precursor malized to GAPDH and vehicle, with expression set to 1.0. proteins (APP) and generate Ab42/Ab40 peptides. These aberrant Ab Experiments were run 3 times. Inflammatory arthritis was induced peptides form amyloid plaques induce M1-type pro-inflammatory though K/BxN serum transfer model (PMID: 22562975). Knee joint microglia, releasing high levels of cytokines, glutamate, reactive structure and cartilage integrity were determined 48 h after i.a. oxygen species, and nitric oxide (NO). This oxidative stress accel- injection of AAT or GSN (n = 8) by the GAG-sensitive toluidine erates generation of 3-nitrotyrosine in neuron and consequently blue staining of knee sections. One-way ANOVA was used for sta- suppresses the catecholamine synthesis and depressesneuro-synaptic tistical analyses. transmission. Inhibition of the neurotoxic factor production may be a Results: Whilst high molecular weight (HMW) fractions of the exu- novel AD treatment. We have reported strong immunosuppressive dates inhibited (B60 %) GAG deposition from 3D chondrocyte activities of serum free conditioned medium derived from Stem Cells cultures, low MW fractions (LMW) were anabolic both on their own from Human Exfoliated Deciduous Tooth (SHED-CM). and even more effective in the presence of IL-1b, causing [60 %

123 S204 Inflamm. Res. increase in GAG deposition mirrored by *30 % rise in COL2A1 and significant thinning. Finally, we applied AN5322 topically to the ears ACAN gene expression (p \ 0.05). Proteome analysis identified 66 of mice stimulated with PMA and examined the ears for the levels of exudate proteins, including GSN and AAT. several cytokines; IL-22 mRNA was suppressed more than fifty-fold Exogenous AAT and GSN abrogated the effect of IL-1b on by AN5322. Similarly AN5322 suppressed calcipotriol-induced pro- MMP13 (eightfold and threefold increase over vehicle; p \ 0.05). duction of TSLP protein by more than eighty-fold. Incubation of chondrocyte micromasses with OASF augmented IL6 New topical therapeutics with novel mechanisms are needed for and MMP13 mRNA, with concomitant down-regulation of COL2A1 mild to moderate Ps and AD due to the significant side effects and and ACAN gene products (p \ 0.01): tested at 3–10 lg/mL, both modest activity of corticosteroids and calcineurin inhibitors. These AAT and GSN attenuated these effects (p \ 0.01). GAG deposition studies provide evidence that topical PDE4Is may suppress cytokines confirmed the chondroprotection afforded by these proteins, that promote inflammation, itch and keratinocyte hyperproliferation in with *80 % reduction by OASF and reversal to control levels Ps and AD. observed following AAT and GSN addition (p \ 0.01). In arthritic mice, knee joint cartilage was eroded by *60 % (GAG content; p \ 0.05). Intra-articular injection of AAT (100 ng) or GSN (30 ng) recovered cartilage integrity by 52 and 34 %, respec- B257 tively, compared to vehicle-injected contralateral joints (p \ 0.05). EVALUATION OF ANTI-INFLAMMATORY Conclusion:: Using resolving exudates as a bio-source, we identified and characterized two novel chondroprotective proteins AAT and ACTIVITY OF ISATIN, N-METHYL ISATIN AND GSN, paving the way for new strategies to repair/restore compro- OXOPROPYL ISATIN mised cartilage functions. Thais B.S. Giorno, Ba´rbara V. Silva, Angelo C. Pinto, Patricia D. Fernandes B256 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil TOPICALLY ACTIVE PHOSPHODIEASTERASE INHIBITORS SUPPRESS IL-17, IL-23, IL-22 AND Isatin is found in plants of the genus Isatis. Due to the great synthetic versatility there has been growing interest in the synthesis and eval- TSLP PRODUCTION, INDICATING POTENTIAL uation of the pharmacological effects of new derivatives. APPLICATION IN PSORIASIS AND ATOPIC Our objective was to characterize the anti-inflammatory activity of DERMATITIS isatin, N-methyl-isatin (MI) and N-methyl-3-(2-oxopropyl)-3-hy- droxy-2-oxindole (NMO). Substances (0.1–10 mg/kg, p.o.) were evaluated in the car- Kurt Jarnagin, Chen Dong, Olga Zemska, Charlotte Frates, Shannon rageenan-induced cell migration into the subcutaneous air pouch Jonesiatauro, Grober Baltazar, Tsutomu Akama (SAP) to assess cell migration and dosages of nitric oxide (NO), TNF- a and IL-1b (by ELISA). To evaluate reactive oxygen species (ROS) Anacor Pharmaceuticals, Palo Alto, CA, USA production induced by phorbolmyristate acetate (PMA) in leukocytes Psoriasis (Ps) and Atopic Dermatitis (AD) are serious skin diseases collected from the SAP after carrageenan injection, the cells were affecting the lives of millions of patients. We examined three topical pretreated ex vivo with substances (1–30 lM). The fluorescence was benzoxaborole PDE4 inhibitors (PDE4Is), AN3889, AN5322, and captured in the FL-1 channel of a flow cytometer and expressed as AN6415, for their affinity, selectivity, and cytokine suppression fluorescence intensity. The protocol for the use of animals was toward: TNFa, IL-23, IL-2, IFNc, IL-17, IL-4, IL-5 and IL-13. approved by CAUAP/UFRJ and received the number DFBCICB015- Activity was compared to known Ps or AD therapeutics. The crystal 04/16. Statistical analysis was performed by ANOVA and Bonfer- structure of AN6415 bound to PDE4 was determined and provides an roni’s test (*p \ 0.05). understanding for the origin of its 0.3 nM affinity and selectivity. A Pretreatment of animals with different doses of all substances dose-limiting side effect of glucocorticoids is skin thinning; we significantly reduced cell migration to the SAP: PBS in SAP = examined topical AN5322 for its ability to thin the skin of hairless 1.1 ± 0.5 9 106 cells/mL; carrageenan in SAP = 80 ± 8.2 9 106 mice. Aberrant growth of keratinocytes mediated by IL-22 is a feature cells/mL; 0.1 mg/kg: Isatin = 66 ± 2.3 9 106 cells/mL; MI = of both diseases. Itch is intense in AD and can be severe in Ps 69.6 ± 8.5 9 106 cells/mL; NMO = 49.6 ± 1.4 9 106* cells/mL; patients, and TSLP is an itch mediator. We also examined the ability 1 mg/kg: Isatin = 59 ± 2.5 9 106* cells/mL; MI = 47.5 ± of these inhibitors to suppress IL-22 secretion in mouse skin treated 3.2 9 106* cells/mL; NMO = 45 ± 2.8 9 106* cells/mL; 10 mg/ with PMA, and TSLP skin treated with calcipotriol. kg: Isatin = 52.5 ± 7.5 9 106* cells/mL; MI = 48.4 ± 7.2 9 106* AN3889, AN5322 and AN6415 are potent PDE4Is, with IC50 of cells/mL; NMO = 46.8 ± 7.8 9 106* cells/mL. Isatin (1 mg/ 1.8, 3.0 and 0.31 nM, respectively. All three are equally active on the kg = 124.5 ± 9.6* lM; 10 mg/kg = 97.7 ± 21.7* lM) and NMO catalytic domain and full length PDE4 enzymes, and thus do not (0.1 mg/kg = 96.2 ± 10.4* lM; 1 mg/kg = 75.5 ± 13.9* lM; interact with UCR1/2. They are not highly selective among the PDE4 10 mg/kg = 33.6 ± 20.5* lM) significantly decrease the amount of A, B, C or D isoforms. The three candidates are more potent on PDE4 NO. With respect to quantification of TNF-a and IL-1b, MI (0.1 mg/ than 15 other cyclic-nucleotide specific phosphodiesterases, exhibit- kg = 377.1 ± 93,6/1009 ± 48.3 pg/mL; 1 mg/kg = 80.3 ± 15.5*/ ing selectivity ratios from 15 to 300,000-fold. In cultured human 719 ± 73.1* pg/mL; 10 mg/kg = 55.9 ± 28.4*/571.4 ± 124.3* pg/ leukocytes these compounds are potent suppressors of monocyte mL and NMO (0.1 mg/kg = 55.3 ± 13.3*/831 ± 114.6 pg/mL; cytokine secretion, with IC50’s for TNFa and IL-23 \ 100 nM; they 1 mg/kg = 35.9 ± 6.5*/576.9 ± 84.9* pg/mL; 10 mg/kg = 32.0 ± suppress T-cell Th1 cytokines potently with IC50’s \ 50 nM; they 18.5*/539.6 ± 25* pg/mL, respectively) were able to reduce the suppress IL-17 secretion with IC50’s \1 lM, and Th2 cytokines with values when compared with carrageenan in SAP group (529.1 ± 13.3 IC50’s B 3 lM. These patterns are similar to other PDE4Is and and 1226.5 ± 139.8 pg/mL, respectively). highly distinct from glucocorticoids, or calcineurin inhibitors. Isatin, MI and NMO also reduced ROS production (control = AN5322 did not cause skin thinning when applied topically for 0.04 9 105 ± 0.5 9 104, PMA = 5.6 9 105 ± 3.2 9 104; Isatin: 16 days, by comparison, dexamethasone and clobetasol caused 1 lM = 1.7 9 105 ± 1.5 9 104; 10 lM = 1.9 9 105 ± 1.9 9

123 Inflamm. Res. S205

104; 30 lM = 0,4 9 105 ± 0.4 9 104; MI: 1 lM = 2.3 9 105 ± generation in an ethanol-induced gastropathy model in mice, the 2.4 9 104; 10 lM = 1,5 9 105 ± 1.0 9 104; 30 lM = 0.83 9 designing of alternative compounds to classical antisecretory drugs is 105 ± 1.0 9 104; NMO: 1 lM = 2.2 9 105 ± 2.5 9 104; 10 lM = very encouraged to define new pharmacological targets for preventing 1.2 9 105 ± 2.6 9 104; 30 lM = 0.8 9 105 ± 1.2 9 104). mucosal cell damages in clinical trials. With these results we can conclude that: (1) Isatin, MI and NMO Funding sources FUNCAP, CNPq, CAPES, and INCT-IBISAB. inhibited leukocyte migration; (2) Isatin and NMO inhibit NO pro- duction; (3) MI and NMO inhibited the production of TNF-a and IL- 1b; (4) Isatin, MI and NMO decreased ROS production. Data suggest that these substances could be used as new prototype forthe devel- B259 opment of new anti-inflammatory drugs. 14-3-3 ETA AS A NOVEL RA DRUG TARGET: Acknowledgements Alan Minho for technical assistance, Instituto ANTI-14-3-3 ETA MONOCLONAL ANTIBODY Vital Brazil (Nitero´i, Brazil) for donation of mice. DELAYS THE ONSET AND MITIGATES THE Financial support CAPES, CNPq and FAPERJ. SEVERITY OF ARTHRITIS IN CIA MICE

Anthony Marotta1, Abedelnasser Abulrob2, Mario Mercier2, Slavisa B258 Corluka2, Roger MacKenzie2, Shalini Raphael2, Sara Michienzi1, 1 1 3 ANTIULCER AND ANTIOXIDANT ACTIVITY OF Jane Savill , Yuan Gui , Walter P. Maksymowych

LECTIN FROM MUCUNA PRURIENS SEEDS ON 1 2 Augurex Life Sciences Corp., Vancouver, Canada; National ETHANOL-INDUCED GASTROPATHY IN MICE Research Council, New York City, NY, USA; 3University of Alberta, Edmonton, Alberta Vicente de Paulo T. Pinto1, Isabela R. Pinto1, Danielle RD Val2, Background: 14-3-3g is a joint-derived soluble biomarker that is Katia A. Ribeiro4, Raul S. Freitas1, Samuel MP Filho1, Auriana S. available as a diagnostic test in the US, Canada and Europe. As an Vasconcelos1, Francisca CFD Sousa1, Tatiane Santi-Gadelha3, Jose´ extracellular ligand, 14-3-3g potently and concentration-dependently Thalles JGD Lacerda3, Carlos ADA Gadelha3, Gerardo C. Filho1, upregulates the expression of multiple factors including TNFa, IL-6, Karuza MA Pereira1, Ellen LD Assis1, Hellı´ada V. Chaves1, Antonio and RANKL and its clinical detection is associated with joint damage AR Silva1, Mirna M. Bezerra1 progression risk. Several disease modifying agents are available for the treatment of RA with remission efficacy rates around 30 %. No 1Federal University of Ceara´, Ceara´, Brazil; 2Federal University of agent has direct companion biomarkers for patient selection and Pernambuco, Recife, Brazil; 3Federal University of Paraı´bam, therapy response monitoring. Since RA is driven by multiple factors Paraı´ba, Brazil; 4Federal University of Rio de Janeiro, Rio de to varying degrees, within and between patients along the disease Janeiro, Brazil course, personalized medicine that enables the specific targeting and Antiulcer and antioxidant activity of lectin from Mucuna pruriens measurement of disease potentiators, such as 14-3-3g, is highly (MpLec) seeds on ethanol-induced gastropathy in mice were studied. desirable. After treatment with ethanol 99.9 %, mice were pre-treated with Objectives: To evaluate the in vivo feasibility of targeting 14-3-3g MpLec (0.001; 0.01 or 0.1 mg/kg, i.v.), ranitidine (80 mg/kg, p.o.), or with a monoclonal antibody drug candidate to mitigate the onset and saline. Mice were sacrificed 30 min after ethanol challenge and severity of collagen-induced arthritis (CIA) in mice. hemorrhagic/ulcerative lesions were measured using ImageJO` . His- Methods: 27 DBA/1 mice were randomized to four study groups: non- tological assessment (H&E), iron-induced lipid peroxidation, GSH induced mice (negative control, n = 5), 0.5 mg/kg of dexamethasone content, SOD activity and mucosal PGE2 were measured. Yohimbine, group (positive control, n = 6), vehicle (saline) injected mice (pla- indomethacin, naloxone or L-NAME was added in order to clarify cebo group, n = 10), and the treatment arm that was administered MpLec action mechanisms. Mice received MpLec (5 or 10 mg/kg; 10 mg/kg of 14-3-3g mAb (n = 6). Treatments were initiated 2 days i.v.) and were observed for toxicity signs and mortality. Ethanol prior to immunization with collagen and administered daily for induced gastric damage, edema, and hemorrhagic patch formation, 6 weeks. A collagen booster was injected on day 18 of the study for which were reduced by MpLec (2.44 ± 0.8 % versus all immunized mice. Arthritis scores were determined daily by an 21.95 ± 2.96 % injured area %; p \ 0.05). Histopathological established and standardized chart evaluating inflammation and assessment show induced a high degree of epithelial cell loss and swelling of each paw (0 to 4). All animals were sacrificed 42 days edema as well as hemorrhagic patch formation on the gastric tissue after the beginning of the treatments. Paws were further analysed by were significantly reduced (p \ 0.05) in animals administered MpLec x-ray. Student t-test was performed to examine differences (onset (0.1 mg/kg). Also, MpLec decreased lipid peroxidation (163 ± 30.73 CIA, maximum, and end scores, and paw scores) amongst the two versus 341.6 ± 78.83 lg/g of tissue, p \ 0.05), SOD activity groups. Kruskal–Wallis test was used to compare group daily score (1.55 ± 0.32 versus 0.46 ± 0.09 lSOD/lg protein, p \ 0.05) and differences over the course of disease. increased GSH content (1271 ± 126.8 versus 837.8 ± 37.23 lg/g of Results: Non-induced and dexamethasone mice did not develop vis- tissue, p \ 0.05), Mucosal PGE2 level (2387 ± 133.1 versus ible signs of arthritis over the course of disease while 100 % of the 1683 ± 143.6 pg/mL protein, p \ 0.05). MpLec effect was reversed mice within the saline arm did. 17 % of the mice in the 14-3-3g mAb by yohimbine (33.54 versus 2.44 injured area %, p \ 0.05) or indo- group did not develop any signs of arthritis. The CIA score for the methacin (36.51 versus 2.44 injured area %, p \ 0.05), suggesting the 14-3-3g mAb arm had significantly lower CIA onset scores alpha-2 adrenoceptors and prostaglandin involvement in MpLec (0.83 ± 0.41 vs 2.7 ± 1.57, p = 0.0119), maximum CIA scores efficacy. Nevertheless, MpLec was able to protect mucosa against (2.33 ± 1.75 vs 5.3 ± 1.83, p = 0.0052), and end CIA scores ethanol gastropathy even in presence of both naloxone (8.809 versus (1.83 ± 1.84 vs 4.3 ± 1.7, p = 0.0197) than the saline treated 2.438 %, p [ 0.05) and L-NAME (9.88 versus 2.44 %, p [ 0.05). No groups. Figure 1 further demonstrates that 14-3-3g mAb treated mice symptoms of toxicity or death were observed during the acute toxicity have significantly lower disease over the disease course than animals tests. MpLec possesses antiulcer and antioxidant activity through in the saline group, p \ 0.01, with x-ray paw analysis also demon- involvement of alpha-2 adrenoceptors activation and prostaglandin strating significance (p = 0.0041).

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LASSBio-1524 = 120 ± 49 s. In SAP, the 30 lmol/kg dose = 24 ± 14* cells 9 106/mL reduced significantly the migration cell compared to carrageenan group = 70 ± 6 cells 9 106/mL and PBS group = 2.4 ± 1.0 cells 9 106/mL. The results of dexametha- sone = 19 ± 7.7 cells 9 106/mL; SC-514 = 36 ± 18 cells 9 106/ mL; LASSBio-1524 = 22 ± 4 cells 9 106/mL. All concentrations of LASSBio-1829 significantly reduced production of ROS: 1 lM = 3 9 105 ± 2 9 104*; 10 lM = 1 9 105 ± 1 9 104*; 30 lM = 1 9 105 ± 3 9 104* compared to group that was stimulated with PMA = 5 9 105 ± 8 9 104. LASSBio-1524: 30 lM = 4 9 104 ± 1 9 104. Only the dose of 10 lmol/kg = 34 ± 26 lM was able to significantly inhibit the production of nitrite compared to group that received SC-514 = 74 ± 43 lM and carrageenan group = 279 ± 17 lM. PBS: 26 ± 5 lM; LASSBio-1829 (30 lmol/kg) = 249 ± 55 lM; dose of 100 lmol/kg = 221 ± 77 lM. The results suggest that LASSBio-1829 has an anti-inflammatory Fig. 1 CIA scores by treatment arm over the course of disease. effect due to reduced licking time, cell migration and inflammatory mediators. Financial support Capes, CNPQ, FAPERJ, Instituto Vital Brazil. Conclusions: 14-3-3g is a mechanistic joint damage factor involved in Technical support Alan Minho. the pathogenesis of RA. A research program to exploit modifying the 14-3-3g pathway is underway to develop improved antibody thera- peutics for delaying the onset and reducing the severity of this disease. B261 A PHASE 1 CLINICAL STUDY OF CTX-4430 IN CYSTIC FIBROSIS PATIENTS B260 LASSBIO-1829, A NOVEL ANTI-INFLAMMATORY E. Springman2, R. Grosswald2, E. Philpot2, G. MacGregor3, MOLECULE DERIVED FROM LASSBIO-1524 A. Horsley4, D. Bilton5, J. Stewart6, S. Elborn1

Tayna´ S. Vale´rio, Thaı´s S. Nascimento, Nata´lia M. Cordeiro, Rosana 1Centre for Infection and Immunity, Queens University School of H.C.N Freitas, Carlos Alberto M. Fraga, Patricia D. Fernandes Medicine, Dentistry and Biomedical Sciences, Belfast, UK; 2Celtaxsys, Inc., Atlanta, GA, USA; 3West of Scotland CF Centre, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Gartnavel General Hospital, Glasgow, UK; 4Manchester Adult Cystic Fibrosis Centre, University Hospital of South Manchester, Inflammation is the organism response to infection or tissue injury Manchester, UK; 5Royal Brompton Hospital, Department of that leads to restoration of tissue structure and function (LAWRENCE Respiratory Medicine, London, UK; 6Celerion, Belfast, UK et al. 2002). LASSBio-1829 was developed based on the structure of LASSBio-1524, an IKK-b inhibitor (AVILA et al. 2011). Introduction: Safe and effective anti-inflammatory treatment (AIT) Our aim is to assess the prospective anti-inflammatory effect of remains a significant unmet need for CF. Inflammation plays a central LASSBio-1829. role in CF and is responsible for lung injury. Along with infection, Formalin model is characterized by two phases: nociceptive (1st inflammation drives acute pulmonary exacerbations and chronic phase) and inflammatory (2nd phase). Formalin was injected in hind decline in lung function. Therefore, AIT for CF must balance sup- paw 1 h after treatment with LASSBio-1829 (30 lmol/kg) and the pression of immune responses leading to inflammation against time that the animal remained licking its paw was recorded. The preservation of immune responses required for microbial control. This subcutaneous air pouch model (SAP) was conducted to evaluate cell need for balance is illustrated by two AIT drugs having divergent migration. The animals received 10 mL of sterile air into the outcomes for CF. High-dose ibuprofen has been shown to reduce the intrascapular area and the reinforcement was made after 3 days. At rate of FEV1 decline and confer a survival benefit, particularly when the 6th day, a carrageenan solution (1 %) was injected in the cavity started at a young age. Whereas, amelubant (BIIL 284) failed in 1 h after treatment with LASSBio-1829 (10, 30, 100 lmol/kg, p.o.) Phase2 due to an increase in pulmonary exacerbations. Both act via after 24 h the animals were euthanized and the exudate was collected the leukotriene B4 (LTB4) pathway, with ibuprofen acting as a weak for total leukocyte counting and mediators measurement. To deter- inhibitor of LTB4 production and amelubant, a strong receptor mine reactive species of oxygen production (ROS), cells were treated antagonist, blocking binding of LTB4 to the receptors BLT1/2. While with LASSBio-1829 (1, 10 and 30 lM) 1 h before stimulation with the reasons for this divergence in outcomes is unclear, it could be due PMA. Finally, we added the probe DCF-DA. ROS production was to the different degrees of pathway inhibition achieved between these assessed by measuring the fluorescence intensity, FL1 of each cell drugs in the clinic. CTX-4430 is a once daily oral Leukotriene A4 expressed through the flow cytometer. Determination of nitric oxide Hydrolase inhibitor that modulates the LTB4 pathway more potently was performed by nitrite measurement. Controls: LASSBio-1524 than ibuprofen but less potently than amelubant. It is currently in (30 lmol/kg, p.o.), SC-514, (an iKK-b inhibitor, 30 lmol/kg, p.o.), clinical development as a once-daily oral AIT for CF. dexamethasone (0.5 lmol/kg, i.p.) and acetylsalicylic acid (ASA Methods: This ascending dose study was conducted at four clinical 20 mg/kg). Mice (n = 6–8) were used. Statistical analysis was per- sites in the United Kingdom (NCT01944735). It included 17 patients formed by ANOVA followed by Bonferroni’s test. Results are with mild to moderate CF. Doses of 50 or 100 mg CTX-4430 or expressed as mean ± SD and (p \ 0.05*). placebo were studied during 15 days of treatment. Safety measures In formalin test, treatment with LASSBio-1829 was significant in were taken on days 1, 8 and 15, including assessments of adverse the inflammatory phase, reducing licking time: 101 ± 35 s compared events, hematology, blood chemistries and pulmonary function. to vehicle = 197 ± 27 s. The result of ASA was 129 ± 55 s and Experimental measures included serum CRP, sputum DNA, elastase

123 Inflamm. Res. S207 and microbial counts. Additional assessments of pharmacokinetics study in all late-stage cancer patients, those receiving SSI as part of and pharmacodynamics in blood are reported separately. their treatment had a median survival advantage of 12 months com- Results: Here we report the results of this phase 1 study in CF patients pared to those not receiving SSI (n = 43 per group). While this including assessments of safety and tolerability, pulmonary function experience comprises uncontrolled, unblinded observations, the data and inflammatory markers in blood and sputum. In brief, CTX-4430 suggest that SSI may induce productive anti-tumour immunity. Qu’s was safe and well tolerated at both dose levels. No deleterious QBKPN SSI product, designed to induce a lung site-specific response, changes in pulmonary function, sputum microbiology, or circulating is currently being studied in a Phase 2a clinical trial in patients with neutrophil counts were observed. Positive trends were observed in non-small cell lung cancer, in collaboration with the BC Cancer blood and sputum inflammatory markers (CRP and elastase, respec- Agency (Trial NCT02256852). tively) and sputum WBC and neutrophil counts demonstrating potential use of CTX-4430 to preserve CF lung function over time. Conclusion:: These results support further clinical development of CTX-4430 for treatment of CF. Nuclear Hormone Receptors

B263 B262 NORETHISTERONE, A PROGESTIN USED IN SITE-SPECIFIC IMMUNOMODULATOR (SSI): CONTRACEPTION AND HRT, PROMOTES A NOVEL IMMUNOTHERAPY FOR CANCER INFLAMMATION IN THE ECTOCERVICAL ENVIRONMENT VIA THE GLUCOCORTICOID David W. Mullins1,2, Shirin Kalyan1, Momir Bosiljcic1, Ashley Westerbeck1, Rebecca Anderson1, Daniel Patton1, Ralf Kleef4, RECEPTOR Gauthier Bouche23, Nina Ludwig3, Simon Sutcliffe1, Hal Gunn1 Donita J. Africander1, Renate Louw-du Toit1, Janet P. Hapgood2 1Qu Biologics, Inc., Vancouver, Canada; 2Geisel School of Medicine at Dartmouth, Hanover, USA; 3Reliable Cancer Therapies, Belgium; 1University of Stellenbosch, Stellenbosch, South Africa; 2University of 4Institute for Immunotherapy and Integrative Oncology, Vienna, Cape Town, Cape Town, South Africa Austria Medroxyprogesterone acetate (MPA) and norethindrone enanthate The immune system is armed with the intrinsic capacity of recog- (NET-EN) are widely used as injectable contraceptives in Southern nizing and eliminating cells that have undergone malignant Africa. In addition, for hormone replacement therapy (HRT), MPA is transformation. The observation that an intricate relationship exists the most commonly used progestin in the U.S.A., while norethin- between immune activation and cancer dates back to the 1700s, when drone-acetate (NET-A) is generally used in Europe. Both MPA and spontaneous tumor remission was observed in some patients experi- NET are synthetic progestins designed to mimic the actions of the encing acute microbial infections. Building upon this history, Qu endogenous hormone progesterone (Prog) by binding to the proges- Biologics has discovered that repeated subcutaneous injection of an terone receptor. However, these progestins also bind to other immunotherapy derived from specific species of killed bacteria members of the steroid receptor family such as the glucocorticoid known to commonly cause infection in a particular organ or tissue receptor (GR). Studies have indicated that MPA disrupts normal may provide an effective method for the treatment of cancers growing immune responses in the female genital tract which could increase in that organ/body site. We hypothesize Qu’s proprietary platform of inflammation, and likely affect susceptibility to sexually transmitted immunotherapies, called Site-Specific Immunomodulators (SSI), infections. Furthermore, we have recently shown that MPA favours a stimulate the body’s immune system to reverse the immune sup- pro-inflammatory milieu in human ectocervical epithelial cells via a pression and dysfunction in the tumor microenvironment, enabling GR dependent mechanism. However, not much is known about the effective anti-cancer immune responses. To test this hypothesis, we effects of NET on local immune function in the female genital tract. evaluated tumor growth and survival in preclinical lung cancer This study thus investigated whether NET-A can also regulate the models following repeated subcutaneous administration of Qu’s lung expression of endogenous cytokine genes in a human ectocervical specific SSI, QBKPN (derived from Klebsiella pneumoniae). SSI epithelial (Ect1/E6E7) cell line. Using real-time quantitative PCR, we significantly reduced tumor burden at day 16 post tumor inoculation show upregulation of the pro-inflammatory cytokine IL-12p40 gene (p \ 0.0001) and improved median survival by 10 days (p \ 0.005). and downregulation of the anti-inflammatory cytokine gene IL-10 by Similar results were obtained using the B16 model, an aggressive and NET-A. An in-depth investigation into the underlying mechanism of poorly-immunogenic melanoma cell line growing as metastatic-like IL-12p40 and IL-10 regulation by NET-A, using a combination of lesions in the lungs, demonstrating the site-specific anticancer effi- whole cell binding assays, Western blotting, siRNA technology, cacy is independent of cancer type. Anticancer efficacy was chromatin immunoprecipitations (ChIP) and re-ChIP assays, show associated with site-specific infiltration of newly recruited monocytes that the GR is mediating these effects. Moreover, the transcription and neutrophils to the lung and mobilization of antigen presenting factors CCAAT-enhancer-binding protein (C/EBP)-b and nuclear cells in the lung-draining lymph node. These data provide strong factor kappa B (NFjB) are needed for the recruitment of NET-A- evidence that Qu’s SSI platform may reconstitute effective bound GR to the promoter of IL-12p40, while recruitment to the IL- immunosurveillance in the tumor microenvironment. Therefore, we 10 promoter requires signal transducer and activator of transcription evaluated SSIs (including QBKPN, QBSAU [derived from Staphy- (STAT)-3. Taken together, our biochemical study clearly indicates lococcus aureus], or QBECO [derived from Escherichia coli]) in that NET-A exerts similar GR-mediated pro-inflammatory effects in compassionate use protocols. 254 patients with advanced cancer were the human ectocervical epithelial cell line as previously observed with treated with one or more SSIs for up to 3.5 years. In retrospective MPA. These results suggest that the use of NET-A in hormonal analysis, patients with metastatic breast cancer receiving SSIs as part therapy may modulate inflammation and immune function in the of their treatment (the largest patient group) had a 20 months longer ectocervical environment, possibly leading to enhanced susceptibility median survival than those not treated with SSIs. In a case-matched to infections such as HIV-1.

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B264 death of retinal pigment epithelial cells. In the present study, we THE BONE MARROW MICROENVIRONMENT investigated the detailed action mechanism by which 4-HNE induces dysfunction of adult retinal pigmented epithelial cells (ARPE-19) PROTECTS PLASMA CELLS FROM THE in vitro. Treatment of ARPE-19 cells with 4-HNE decreased pro- LYMPHOLYTIC EFFECTS OF duction of inflammatory cytokine and vascular endothelial growth GLUCOCORTICOIDS factor (VEGF) in a time-dependent manner. Also, 4-HNE exerted prominent cytotoxic effects in cultured ARPE-19, manifested by Derek W. Cain, Johnny A. Cidlowski morphological changes and diminished cellular viability. At the same time, 4-HNE enhanced NOX4 expression and activity, National Institute of Environmental Health Sciences, Research resulting in aggravated ROS production in addition to mitochondrial Triangle Park, NC, USA ROS. The 4-HNE-inudced ROS production and cell death were inhibited by apocynin, diphenyleneiodonium (DPI) and VAS2870, Glucocorticoids exert wide-ranging effects on the immune system, while 4-HNE-inudced IL-6 and VEGF decrease was not blocked. acting primarily through the glucocorticoid receptor (GR). The anti- The results suggest that 4-HNE-induced NOX4 expression and ROS inflammatory actions of glucocorticoids have been attributed to the production plays a major role in cell death but not in cytokine capacity of liganded GR to inhibit expression of pro-inflammatory production of ARPE-19 cells. genes and to induce lymphocyte apoptosis. Glucocorticoid therapy is associated with reduced serum immunoglobulin concentrations, but it is unclear if glucocorticoids act directly or indirectly on antibody-secreting plasma cells. We have previously shown that B Proteases cells in mice express GR from early stages of bone marrow development through maturation in the periphery, and that imma- ture and mature B cells undergo apoptosis in response to the B266 synthetic glucocorticoid dexamethasone. In this study, we investi- A NOVEL ROLE FOR BK CHANNEL IN gated GR signaling and effects of dexamethasone on murine plasma REGULATING METALLOPROTEASE ACTIVITY cell survival. Splenic and bone marrow plasma cells express GR and are sensitive to dexamethasone-induced cell death in vitro. Minae Yoshida, Dean Willis Dexamethasone treatment of mice reduces plasma cell numbers in the spleen but not in bone marrow, suggesting that the bone mar- Department of Neuroscience, Physiology and Pharmacology, row microenvironment protects plasma cells from glucocorticoid- University College London, London, UK induced death. Provision of IL-6, but not IL-5, APRIL, BAFF, or CXCL12, mitigates plasma cell death by glucocorticoid treatment Objective: The cells of the immune system expresses an array of ex vivo. We conclude that peripheral plasma cells are susceptible to different ion channels, yet their roles in these cells are not fully glucocorticoid-induced lympholysis, but that specialized niches understood. To address the shortfall in this area of inflammation within bone marrow insulate plasma cells from the apoptotic effects research, we investigated the role of BK channels in LPS mediated of glucocorticoids. activation using a mouse macrophage cell line, RAW264.7 cells. Method: Functional cell membrane expressed BK channel was investigated by whole-cell voltage clamp recordings. A ramp protocol (25 s ranges from -40 to 100 mV) was applied in the presence/ Ocular Inflammation absence of a BK channel selective blocker, 20 nM iberiotoxin (IbTX). The IbTX sensitive current was obtained by subtraction. Released B265 TNF-a was assessed by ELISA and cellular TNF-a protein by Wes- a ROLE OF NOX4-DERIVED ROS PRODUCTION IN tern-blot. mRNA were extracted and TNF- mRNA levels were determined by qRT-PCR. TACE activity was assessed by incubating 4-HNE-INDUCED DYSFUNCTION AND DEATH OF cells with FAM fluorescence quenched TACE substrate II, a substrate ARPE-19 ADULT RETINAL PIGMENT for ADAM17 and ADAM10. Activity is assessed by fluorescence EPITHELIAL CELLS resulting from the cleavage of substrate. Data are presented as mean ± S.E.M and analysed student t-test. Results: Whole cell voltage clamp recordings indicated the almost Sajita Shah2, Jung-Ae Kim1 absence of BK channel current on the cell membrane in non-stimu- lated cells. However the BK channel current was present after 24 h 1Yeungnam University, Gyeongsan, South Korea; 2Yeungnam LPS (10 ng/mL) stimulation. Subsequently, LPS pre-stimulated University, Gyeongsan, South Korea (10 ng/mL for 24 h) cells were used in the following experiments to Role of Nox4-derived ROS production in 4-HNE-induced dysfunction investigate the role for membrane active BK channels in TNF-a and death of ARPE-19 adult retinal pigment epithelial cells. synthesis/release. Pre-stimulated RAWs were treated with LPS Sajita Shah, Jung-Ae Kim* (150 ng/mL) for 4 h. Application of IbTX (20 nM) during the LPS College of pharmacy, Yeungnam University, Gyeongsan 712-749, stimulation significantly enhanced the release of TNF-a. We then South Korea investigated whether the TNF-a regulation occurred at pre/post- Defects of the retinal pigment epithelium (RPE) which consti- transcriptional level. Despite the significant difference in TNF-a tutes the blood-retina barrier result in photoreceptor damage leading release, TNF-a mRNA was almost equivalent in both control and to age-related macular degeneration (AMD). RPE produces large IbTX treated group. In comparison, the IbTX treated cells contained amounts of ROS during phagocytosis of shed outer segments of significantly less TNF-a within the cells. These results indicated that photoreceptors. During phagocytosis, oxidation of polyunsaturated the BK channel may be involved in the release of TNF-a molecule. fatty acids (PUFA), rich components in the outer segments, results TNF-a is known to exist as transmembrane protein and is subse- in oxidative stress by generating lipid aldehyde radicals including quently released by the action of metalloproteases, e.g. ADAM17 and 4-hydroxy-2-nonenal (4-HNE) which contributes to dysfunction and ADAM10. To investigate the direct link between BK channel and

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TNF-a release, TACE activity assay was performed. TACE activity medium. In addition, ELA hyperactivity induced an unbalance of was increased to 2.5 fold by IbTX treatment. cytokines expression favouring the increase of pro-inflammatory Conclusion: In this study, we have shown that the membrane active cytokines. BK channel controls TNF-a release in RAW264.7 cells. We found Ad-ELA administration in murine colon allowed epithelial ELA evidence that BK channel directly regulates proteolytic activity of expression in tissues and increased elastase activity in epithelial cells. ADAMs. This study provides a novel insight into the role of ion Ad-ELA treated mice presented an increase of macroscopic inflam- channel in regulating metalloprotease activity and macrophage matory damage score compared to control Ad-null mice. reponses. Conclusion:: Our study demonstrates that epithelial cells constitute a major source of elastolytic activity and secretes a form of elastase in the extracellular space. In IBD epithelium, elastase is hyperactive disrupting the epithelial barrier function and maintaining the inflam- B267 matory response. INTESTINAL EPITHELIAL CELLS SECRETE AN ELASTASE WHICH PARTICIPATES TO MUCOSAL INFLAMMATION IN IBD B268

1,2 1 EPITHELIAL CELLS RELEASE TRYPSIN Ce´line Deraison , Alexandre Denadai-Souza , Anna Carolina ACTIVITY IN INFLAMMATORY CONDITION: Florence1, Anissa Edir1, Corinne Roland1, Jean-Paul Motta3, Chrystelle Bonnart1, Elena Verdu4, Derek McKay2, Laurent Alric5, ROLE IN IRRITABLE BOWEL SYNDROME Nathalie Vergnolle1 Claire Rolland Fourcade1, Alexandre Denadai-Souza1, Jean-Paul 1Inserm, U1043, Toulouse, France; 2University of Calgary, Faculty of Motta2, Tereza Bautzova1, Nicolas Cenac1, Ian Spreadbury3, Stephen Science, Department Physiology and Pharmacology, Calgary, J. Vanners3,Ce´line Deraison1, Nathalie Vergnolle1 Canada; 3University of Calgary, Faculty of Science, Department Biological Sciences, Calgary, Canada; 4Farncombe Family 1Inserm CPTP, Toulouse, France; 2University of Calgary, Calgary, Digestive Health Research Institute, McMaster University, Hamilton, Alberta, Canada; 3Queen’s University, Kingston, Kingston, Canada Canada; 5CHU Purpan, Gastroenterology department, Toulouse, Trypsin activity is released by human colonic biopsies from IBS France patients compared to control patients (Cenac et al. 2007). However, Proteases have a crucial role in the persistence of chronic inflam- the cellular source of this activity remains controversial. Mesotrypsin matory responses of the gastro-intestinal tract. We have previously or trypsin IV is a form of trypsin, which is expressed in the small demonstrated that expression of ELAFIN, an elastase inhibitor, is intestine of IBS patients (Kerckhoffs et al. 2008), but the cellular drastically down-regulated in IBD epithelium. The imbalance origin of this protease, and its functions are not clear. between proteases and their inhibitors appears to be crucial to the This study aims at determining the cellular source of trypsin development of Inflammatory Bowel Diseases (IBD). activity, its release under inflammatory condition, its effect on neu- This study aims at identifying the source of hyperactive elastase ronal signalling and in inflammatory pain. The general objective of released by IBD tissue biopsy and to decipher the consequences of its this study was to understand the role of trypsin activity in the context production on colonic barrier function and inflammatory response. of IBS. Methods: In situ zymography with FITC-elastin was performed on Cryosections of biopsies from IBS and control patients were used cryosection of colonic biopsies from healthy (n = 7) and IBD patients to perform an in situ zymography of trypsin activity. Intestinal (n = 10) taken from inflamed and non-inflamed area. Immunostain- epithelial cells (Caco2) were cultured on transwell, and stimulated ing was performed on cryosections from human biopsies. Caco2 with LPS as an inflammatory stimulus. Conditioned media from epithelial cell line overexpressing elastase (Tg-ELA) was constructed basolateral and apical sides of epithelial monolayers were used to and the potential of elastase hyperactivity to modulate the release of stimulate primary afferent isolated from mouse Dorsal Root Gan- cytokines and permeability changes was evaluated. ELA cDNA-re- glia. Mesotrypsin protein was detected by western blot and combinant adenovirus (Ad-ELA) was construct to assess the effect immunohistochemistry and trypsin activity was measured using ELA overexpression in vivo. Mice were pre-treated by 2 % DSS for Tosyl GPR as substrate. Mesotrypsin was administered in mouse 3 days and then Ad-ELA was administrated intracolonically. At day colon and nociceptive responses to colorectal distension were 7, colons were harvested for measurement of inflammation recorded. parameters. In situ zymography revealed that trypsin activity was more Results: Using in situ zymography, we showed a strong elastolytic intense in tissues from IBS patients compared to healthy controls, activity in the enterocytes in healthy human colonic tissue, which and the strongest trypsin activity was detected in epithelial cells. In was greatly enhanced in inflamed biopsies as well as in non-in- culture conditions, epithelial cell monolayers released more trypsin flamed biopsies from IBD patients. An elastase’s cDNA was cloned activity in the basolateral compartment after LPS stimulation. This from human enterocytes. Immunostaining of colonic tissues showed polarised secretion could signal to neuronal terminations. Culture that this form of elastase was only expressed in epithelial cells. In media harvested from basolateral but not apical side of LPS- IBD, epithelial elastase expression was enhanced. Analysis of stimulated intestinal epithelial cells induced calcium signalling in elastase expression shows also an up-regulation in colonocytes from cultured sensory neurons, which was inhibited by trypsin inhibitor, DSS-treated mice but absence of protein in colons from germ-free suggesting that trypsin activity is responsible for such activation. mice. Expression of cationic, anioc and mesotrypsin was quantified in Tg-ELA epithelial cells showed defective barrier function: inflamed epithelial monolayer. Only mesotrypsin expression is increase of Dextran-FITC permeability and of bacterial translocation. increased after LPS stimulation and preferentially released at the Western blot analysis revealed that ZO-1 and occluding amounts were basolateral side. Mesotrypsin administration in mouse colonic decreased in Tg-ELA compared to Caco-2 cells. Thus these proteins lumen induces visceral hypersensitivity in a dose dependant of tight junction are targeted by elastase hyperactivity secreted in the manner.

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This study reveals that in IBS patients, trypsin activity is strongly Table 1 detected in epithelial cells, which are able to produce this activity specifically from the basolateral side. Sensory neurons can be acti- Control- Control- Smoke- Smoke- vated by basolateral media of stimulated epithelia, in a trypsin VE rBmTIA VE rBmTIA activity-dependent manner. Mesotrypsin was identified as a possible candidate released by Protocol 1 intestinal epithelial cells in response to inflammation, which can Lm (lm) 50.06 ± 1.10 53.69 ± 2.53 61.57 ± 2.49* 58.89 ± 1.83* signal to neurons and further induce visceral hypersensitivity. These data highlight epithelial proteases as important signalling molecules Protocol 2 to enteric neurons that might be heavily involved in IBS-associated Lm (lm) 42.75 ± 1.51 46.11 ± 2.78 59.68 ± 2.61* 47.18 ± 3.07*,# hypersensitivity symptoms. Elastic 17.43 ± 0.79 14.25 ± 1.15 23.17 ± 0.39* 19.72 ± 0.87*,# fibers (%) Collagen 14.01 ± 0.71 11.36 ± 0.94 19.47 ± 1.29* 13.88 ± 0.85*,# fibers (%) B269 MAC-2 1.65 ± 0.32 1.32 ± 0.14 2.99 ± 0.48* 2.68 ± 0.29* EFFECTS OF A PROTEASE INHIBITOR FROM (cells/lm2) THE TICK RHIPICEPHALUS BOOPHILUS MMP-12 1.17 ± 0.15 1.17 ± 0.11 3.04 ± 0.43* 2.01 ± 0.17*,# (cells/lm2) MICROPLUS IN A CIGARETTE SMOKE-INDUCED MMP-9 1.02 ± 0.09 1.01 ± 0.07 1.29 ± 0.06* 1.52 ± 0.11* EMPHYSEMA (cells/lm2)

Juliana D. Lourenc¸o1, Daniela A.B. Cervilha1, Juliana T. Ito1, Davi S. Sales1, Alyne Riani1, Clarice R. Olivo1, Adriana Duran2, Sergio D. Data were expressed as mean ± SE;* p \ 0.05 compared to Control Sasaki2,Mı´lton A. Martins1, Fernanda D.T.Q.S. Lopes1 groups; # p \ 0.05 compared to Smoke-VE group. Supported by FAPESP. 1University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2UFABC, Santo Andre´, Brazil Introduction: Considering that tabagism is the main risk factor for the B270 Chronic Obstructive Pulmonary Disease (COPD) development, and CPM ACTIVITY MODULATION BY KININ B1 the cigarette smoke (CS) exposure models appear to best represent RECEPTOR IN ENDOTHELIAL CELL MODELS human COPD, it is important to test new therapies to avoid the impairment in parenchymal destruction in such model. Paola B. Guimara˜es1, Carolina C. Hoff5,1, Liliam Fernandes2, Objectives: To verify the effects of rBmTI-A treatment, a protease Jair R. Chagas4,1, Thaysa Paschoalin1, Adriana K. Carmona1, inhibitor, on the emphysema pathogenesis. Michael Bader3, Joa˜o B. Pesquero1 Methods: Protocol 1: Mice were exposed to CS (twice daily/30 min/ 5 days a week/12 weeks), and the control animals were exposed to 1 room air. These animals received two doses of rBmTI-A inhibitor or Federal University of Sa˜o Paulo, Sa˜o Paulo Campus, Sa˜o Paulo, 2 its vehicle (Saline Solution 0.9 %) by nasal instillation after the CS Brazil; Federal University of Sa˜o Paulo, Diadema Campus, Sa˜o 3 end exposure (Dose 1: 24 h after the end of CS exposure; Dose 2: Paulo, Brazil; Max Delbru¨ck Center for Molecular Medicine, Sa˜o 4 7 days after the first dose). Fifteen days after the final of CS exposure, Paulo, Brazil; Federal University of Sa˜o Paulo, Santos Campus, Sa˜o 5 animals were submitted to procedures for respiratory mechanics and Paulo, Brazil; Paulista University, Santos-Rangel Campus, Sa˜o the mean linear intercept (Lm) evaluations. Protocol 2: We used the Paulo, Brazil same protocol for CS exposure, but the rBmTI-A treatment was performed during the exposure time (Dose 1: 24 h before the start of Carboxypeptidase M (CPM) is a glycophosphatidylinositol anchored CS exposure; Dose 2: 1 month after the exposure start, Dose 3: enzyme that plays a role in the kallikrein-kinin system (KKS). CPM 2 months after the start). After 24 h of exposure end, we obtained the catalytic domain hydrolyzes Lys or Arg from C-terminal peptides lung mechanical measurements and the Lm analysis. We also mea- (i.e., bradykinin and kallidin), generating B1 receptor (B1R) agonist, sured the volume proportion of collagen and elastic fibers, and the as des-Arg9-BK. Consequently, both CPM and B1R are important in density of macrophages, MMP-12 and MMP-9 positive cells in cardiovascular homeostasis and inflammation. It is known that CPM parenchyma. and kinin B1R are co-localized in plasma membrane microdomains, Results: For both protocols, there was no difference in respiratory where they interact with each other, facilitating the receptor signaling. mechanics between the groups. Although Smoke-rBmTIA and Enzyme-receptor interactions in membrane microdomains are not Smoke-VE groups showed an increase of Lm compared to Control well understood, especially from the enzymatic point of view. In this groups in both protocols, there were a decrease in the Smoke-rBmTIA context, this study becomes crucial, considering the role of ectoen- group compared to Smoke-VE group only in the second one. The zymes, like CPM in KKS. The hormonal function of this system same behavior was observed for the volume proportion analysis of depends on circulating levels of its agonists, whose local availability elastic and collagen fibers in parenchyma. We also observed an is controlled by the enzymes which are close to their receptors. increase in the density of macrophages, MMP-12 e MMP-9 positive Therefore, we hypothesized that CPM-kinin B1R interaction might cells in the Smoke groups, and the treatment with rBmTI-A only also affect the enzyme activity. Thus, the aim this work was to decreased the density of MMP-12 positive cells (Table 1). evaluate the CPM activity and expression in the presence of B1R in Conclusion:: These results suggest that rBmTI-A inhibitor attenuated endothelial cell models. For this purpose, we studied two different the development of emphysema when administered during the disease kind of endothelial cell system: (1) a B1R ablation system, namely -/- induction, probably to a decrease in MMP-12 levels, but there is no primary culture of endothelial cells from B1 mice; (2) endothelial treatment effect after the established disease. cells from transgenic rats TGR (Tie2B1) overexpressing the B1R

123 Inflamm. Res. S211 exclusively in the endothelium. Primary culture was characterized by B272 both immunohistochemistry using anti-von Willebrand factor (vWf) RESOLUTION FACTORS: WILL THEY BE GOOD antibody and angiogenesis in vitro on Matrigel. CPM mRNA levels was assayed by real time PCR; CPM protein expression was analyzed CONTROLLERS OF THE INFLAMMATORY by Western blot using anti-CPM primary antibody; and CPM activity RESPONSE? was assayed with 200 lM Dansyl-Ala-Arg-OH fluorescent substrate. CPM activity was performed in the presence or absence of 1 lMkinin Fla´via CS Fonseca, Janetti N. Francischi B1R antagonist R715. Primary culture of endothelial cells presents positive label for vWf and it forms tubular structures on Matrigel, some Department of Pharmacology, Federal University of Minas Gerais, characteristics of endothelial cell. Results show that cells from B1-/- Brazil group, in which B1R receptors are absent, have decreased CPM Introduction: Detection of resolution factors such as Resolvins, Pro- activity, along with reduction in both CPM mRNA levels and protein tectins and Maresins (SERHAN et al. 2015) demarcated a turning point in content. However, when control endothelial cells were pre-treated with the understanding of the pathophysiology of the inflammatory process. B1R antagonist R715, CPM activity did not change. On the other hand, The aim of the present work was to verify the profile of biological activity CPM activity is increased in cells with kinin B1R overexpression. In presented by Resolvin E1 in the rat paw edema and hyperalgesia test. The summary, our data show that kinin B1R positively modulates both used phlogogenic stimuli were: carrageenan (CG), prostaglandin E2 CPM activity and expression, suggesting that CPM-kinin B1R inter- (PGE2), histamine (H), 5-hydroxytriptamine (5-HT) and substance P action in membrane microdomains might affect enzyme activity, (SP) in doses known to induce paw edema and/or hyperalgesia (nano- beyond interfering in receptors signaling. This work highlights the gram to microgram range). Paw edema and hyperalgesia were measured interactions among the different components of the KKS and con- at 0, 5, 15, 30 min, 1, 2, 3, 4, 6 and 24 h with an Ugo Basile plethys- tributes to a better understanding of its patho-physiological role. mometer and the Randall-Selitto analgesimeter (mechanical Financial support CAPES, CNPq and FAPESP. hyperalgesia), respectively, using male Holtzman rats (weight = 150–180 g). Resolvin E1 (RvE1; in 0.1 mL) diluted in 5 % ethanol was administered intraplantarly by subcutaneous route, 10 min before the Resolution of Inflammation and Tissue phlogogenic stimuli, also given in 0.1 mL at zero time. Initial dose of Repair RvE1 was based on the work of XU et al. 2010. The experiments were approved by UFMG Ethics’s Committee for Animal Use (no. 199/2014). Results: RvE1 reduced edema induced by CG but did not affect SP- B271 and PGE2-induced edema. Surprisingly, RvE1 increased H and 5-HT RESOLVIN D4 STRUCTURAL CONFIRMATION edema. RvE1 also reduced CG-, PGE2-, 5-HT- and SP-induced AND POTENT PRO-RESOLVING ACTIONS IN hyperalgesia as resumed on Table 1 below. Conclusions: Given the predictability of the carrageenan paw edema INFLAMMATION AND INFECTION model in rats to detect potential (non-steroidal) antiinflammatory drugs (MUKHERJEE et al. 1996), RvE1 may represent a good analgesic 1 1 1 Jeremy W. Winkler , Sarah K. Orr , Romain A. Colas , Jesmond candidate, but its prolonged use might be associated with adverse 1 1 2 1 Dalli , Nan Chiang , Nicos A. Petasis , Charles A. Serhan effects related with its proinflammatory activities. Support CNPQ and CAPES. 1Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 2University of Southern California, Los Angeles, CA, USA Table 1: Profile of anti-inflammatory and analgesic activity of Resolvin E1 in rats Objective: Resolvins are a family of structurally distinct pro-resolving mediators biosynthesized from polyunsaturated fatty acids that are Phlogogen stimulus/Dose EDEMA Hyperalgesia critical to control acute inflammation. This study presents evidence for the first total organic synthesis and complete stereochemical Control – – assignment and confirmation of novel Resolvin D4 (RvD4). + RvE1 – – Materials and methods: Synthetic compound and endogenous Resolvin D4 were examined by MS–MS metabolo-lipidomic profil- CG -100 lg +++ +++ ing. For in vivo actions and endogenous production, mice received S. + RvE1 + + aureus infection in dermal pouch. Concomitant with a bacterial PGE2 -200 ng ++ + challenge, synthetic compound was administered and leukocytic + RvE1 ++ – response measured overtime. For in vitro RvD4 was administered to macrophages and phagocytosis carried out. SP -1 lg+++ Results: The absolute stereochemical confirmation was established as + RvE1 + – 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic 5-HT -1 lg++++ acid by matching authentic endogenous RvD4 with synthetic pre- cursor from total organic synthesis. Our results demonstrate that + RvE1 +++ – RvD4 is produced endogenously in a self-limited skin infection model H–20 lg+– at levels commensurate with its bioactions. We confirmed the potent + RvE1 ++ (hypoalgesia) actions of RvD4 in vivoin reducing neutrophils both in murine peri- tonitis and dermal infection by 40–60 %. Resolvin D4 promoted the + presence or - absence of the response. Dose of RvE1: clearance of cellular debris and apoptotic PMN by human macro- 100 ng/paw phage in doses as low as 0.1–10 nM. Conclusion: Total synthesis and matching of RvD4 provides a new tool References to explore RvD4 bioactions and opens opportunities for control of 1. SERHAN, C. N.; DALLI, J.; COLAS, R. A.; WINKLER, J. W.; unwanted inflammation and infection via Resolution Pharmacology. CHIANG, N. Review: Protectins and maresins: New pro-resolving Keywords: Anti-inflammatory, pro-resolving, infection, mediator families of mediators in acute inflammation and resolution bioactive

123 S212 Inflamm. Res. metabolome. Biochimica et Biophysica Acta 1851, p. 397–413, 17-HDHA, RvE1 and VEGF equivalent to that of WT MEFs. Collec- 2015. tively, these data support the hypothesis that oxygenated n-3 PUFA 2. XU, Z-Z.; ZHANG, L.; LIU, T.; PARK, J. Y.; BERTA, T.; YANG, metabolites directly target endothelial cells to enhance pro-angiogenic R.; SERHAN, C. N.; JI, R-R. Resolvins RvE1 and RvD1attenuate potential in a PPARb/d-dependent manner and demonstrate a key role inflammatory pain via central and peripheral actions. Nature Medi- for FPR2 in mediating the endothelial-directed pro-repair actions of cine, v.16, n.5, p. 592–597, 2010. RvD1. These mechanisms likely contribute to explaining the purported 3. MUKHERJEE, A.; HALE, V. G.; BORGA, O.; STEIN, R. Pre- beneficial roles of dietary n-3 PUFA as regulators of vascular home- dictability of the clinical potency of NSAIDS from the preclinical ostasis and effective tissue repair. pharmacodynamics in rats. Inflammation Research, v. 45, p. 531– 540, 1996. B274 OLD DRUGS WITH NEW SKILLS: BIASING THE B273 MELANOCORTIN RECEPTORS TO RESOLVE OXYGENATED N-3 PUFA METABOLITES INFLAMMATORY ARTHRITIS PRESENT IN THE CIRCULATION AFTER ACUTE CONSUMPTION OF N-3-PUFA CONTAINING OILS Trinidad Montero-Melendez1, Thomas Gobbetti1, Sadani N. Cooray1, REGULATE THE ANGIOGENIC POTENTIAL OF Thomas EN Jonassen2, Perretti Mauro1

HUMAN ENDOTHELIAL CELLS THROUGH 1 PPARb/D- AND GPCR-DEPENDENT PATHWAYS The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, UK, Charterhouse Square, London, UK; 2Department of Biomedical 1 1 2 Caroline PD Wheeler-Jones , Sally H. Latham , Anna Nicolaou , Sciences, University of Copenhagen, Copenhagen, Denmark Alexandra Kendall2, Ashton R. Faulkner1, David Bishop-Bailey1, Mauro Perretti3, Thomas Gobbetti3, Kathleen M. Botham1, Melanocortin receptors (MC1–MC5) are druggable GPCRs that upon Wendy L. Hall4, Robert Purcell1 activation elicit pro-resolving properties, dampening inflammatory processes. The MC peptide ACTH has been used for over 60 years to 1Royal Veterinary College, London, UK; 2University of Manchester, treat rheumatoid arthritis, gout and other inflammatory conditions. Manchester, UK; 3Queen Mary, University of London, London, UK; However, lack of selectivity of ACTH and the synthetic MC drugs 4King’s College London, London, UK developed so far limits the translational delivery of novel MC drugs into patients. A new concept of significant therapeutic interest is the Mechanistic understanding of how the nutritionally important n-3 one of biased agonism, which implies ligand-dependent selectivity for polyunsaturated fatty acids (PUFA) EPA (eicosapentaenoic acid) and certain signal transduction pathways. Here we characterize the DHA (docosahexaenoic acid) may preserve vascular homeostasis and molecule AP1189 ((E)-N-[trans-3-{1-(2-nitrophenyl)-1H-pyrrol-2- tissue repair is lacking. In a randomized trial in 16 healthy males we yl} allylidenamino] guanidium acetate), which acts as a biased ago- demonstrated divergent postprandial responses to high fat meals con- 2+ nist at MC1 and MC3, since it activates ERK1/2 and Ca signaling taining EPA + DHA-rich oil versus DHA-rich oil, suggestive of but not the canonical cAMP pathway, of relevance as MC1 pro-me- differential effects on FA metabolism. Here, we used targeted lipi- lanogenic side effects depend on cAMP (see figure below). AP1189 domics to profile the oxygenated fatty acid metabolites present in reduces cytokine release by macrophages in vitro, an effect driven by baseline and postprandial (6 h) plasma from study participants, and MC1 and MC3, evidenced with knockout cells. It also promotes investigated the direct actions of selected mediators and their down- efferocytosis in an ERK1/2 dependent manner. No melanogenesis stream metabolites on human endothelial cells (ECs). Plasma was induced by AP1189 in B16-F10 melanocytes. In vivo, oral lipidomics revealed patterns of EPA- and/or DHA-derived metabolites AP1189 reduces leukocyte trafficking in acute peritonitis, activating that were largely consistent with the composition of the oils ingested. the resolution of inflammation three times faster as compared to Significant increases in a range of COX- and LOX-derived mediators vehicle-treated mice when the drug was administered at peak of were evident following meals containing EPA and/or DHA, with par- inflammation. Finally, when tested in models of inflammatory ticularly high concentrations of 18-HEPE and 17-HDHA, pathway arthritis, AP1189 reduced clinical score, paw swelling, disease precursors for the pro-resolving RvE1 and RvD1, respectively. severity and leukocyte infiltration. 18-HEPE, 17-HDHA, RvE1 and RvD1, but not the n-6 PUFA-derived In summary, biased agonists at MC receptors offer therapeutic metabolites 9- and 13-HODE, increased capillary-like tube formation innovation for the development of drugs with improved profile by by HUVEC and adult microvascular ECs in vitro, and these effects were activating those pathways that are therapeutically relevant and inhibited by the PPARb/d antagonist GSK0660. Responses to 18-HEPE evading those associated with side effects. and RvE1 were reduced by treatment with CCX832, a selective ChemR23 antagonist. Quantitative RT-PCR and immunofluorescence in actively tubing cells confirmed expression of FPR2 in ECs. RvD1 B275 2+ elevated intracellular Ca , enhanced ERK1/2 phosphorylation and HUMAN MILK SPECIALIZED PRORESOLVING reduced caspase3/7 activity in ECs, responses that were blocked by WRW4 (an FPR2 antagonist) and mimicked by MMK1 (FPR2 agonist). LIPID MEDIATORS STIMULATE RESOLUTION The pro-angiogenic activities of RvD1 and MMK1, but not 17-HDHA, OF ACUTE INFLAMMATION were attenuated by WRW4. VEGF-induced tube formation was abro- gated by PPARb/d blockade but was unaffected by antagonists of FPR2 Hildur H. Arnardottir, Sarah K. Orr, Jesmond Dalli, or ChemR32. MMK1, RvD1, 17-HDHA, RvE1 and VEGF increased Charles N. Serhan the tube forming capacity of wild type (WT) embryonic fibroblasts (MEFs) whereas FPR2-/- MEFs failed to undergo tubulogenesis in Center for Experimental Therapeutics and Reperfusion Injury, response to either MMK1 or RvD1 but retained sensitivity to Department of Anesthesiology, Perioperative and Pain Medicine,

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Harvard Institutes of Medicine, Brigham and Women’s Hospital and resolution of inflammation seem to play an essential role in the ter- Harvard Medical School, Boston, MA, USA mination of tissue repair and the promotion of its return to homeostatic structure and function. Human milk contains nutrients and bioactive products relevant to infant immune development and protection. Here we investigated the pro-resolving properties of human milk using human milk lipid mediator isolates (HLMI) and determined their impact on resolution B277 programs In vivo and with human macrophages. In murine acute SATIATED MACROPHAGES UPREGULATE peritonitis, HLMI reduced maximum neutrophil numbers from 14.6 ± 1.2 9 106 cells/exudate to 11.0 ± 1.0 9 106 cells/exudate CD24 EXPRESSION THROUGH UNCOUPLING and shortened the resolution interval (Ri) by 54 % (12 h vs. 26 h) PROTEIN 2 compared to peritonitis plus vehicle mice. Using liquid chromatog- raphy tandem mass spectrometry (LC–MS–MS)-based lipid mediator Janan Saadi, Senthil K. Satyanarayanan, Nawras Abboud, (LM) metabololipidomics, we found that human milk possesses a Sagie Schif-Zuck, Amiram Ariel proresolving LM signature profile, containing specialized proresolv- ing lipid mediators (SPM) at concentrations within their bioactive University of Haifa, Department of Human Biology, Haifa, Israel range (pico-nanomolar concentrations) that enhanced human macro- phage efferocytosis and bacterial (E. coli) containment. The SPM The engulfment of apoptotic leukocytes (efferocytosis) by macro- identified in human milk included D-series resolvins, [e.g. Resolvin phages during the resolution of inflammation is essential for tissue (Rv) D1, RvD2 and RvD3], Protectin D1, Maresin 1, E-series resol- homeostasis and results in macrophage reprogramming/immune-si- lencing. However, a distinct subset of resolution phase macrophages vins (e.g. RvE1, RvE2 and RvE3) and lipoxins (LXA4 and LXB4). Milk from inflamed human mammary glands (mastitis) had altered loss their phagocytic potential, and hence were termed satiated LM profile, with lower SPM and higher proinflammatory LM com- macrophages. Here, we used Illumina-based RNA-Seq analysis and pared to milk from non-inflamed (healthy) mammary glands. Hence, Western blotting to identify proteins that are upregulated upon mac- these results demonstrate that human milk contains proresolving rophage satiation. We found that CD24 expression is highly chemical signals that simulate resolution of acute inflammation and upregulated in satiated macrophages, while the expression of infection. Taken together, these findings provide a novel mechanism Uncoupling Protein 2 (UCP2), a mitochondrial protein that prolongs in maternal-infant biochemical imprinting. apoptotic cell clearance, was down-regulated. Moreover, pharmaco- logic inhibition of UCP2 in resolution phase macrophages resulted in elevated CD24 expression as well as increased TNFa production. Thus, macrophage satiation during the resolution of inflammation B276 seems to be associated with increased CD24 expression and dimin- THE SATIATED MACROPHAGE: A KEY PLAYER ished TNFa production through UCP2 down-regulation. IN THE RESOLUTION OF INFLAMMATION

Amiram Ariel1, Senthil K. Satyanarayanan1, Noa Sher2,1, B278 Sagie Schif-Zuck1 NATURAL KILLER CELLS ARE INDISPENSABLE FOR RESOLUTION OF ANTIGEN-INDUCED 1Department of Human Biology, University of Haifa, Haifa, Israel; 2Tauber Bioinformatics Research Center, University of Haifa, Haifa, INFLAMMATION IN MICE Israel Ingibjorg Hardardottir1, Osk Anuforo1,3,4, Hulda S. Jonasdottir5, The engulfment of apoptotic leukocytes (efferocytosis) by macro- Martin Giera5, Jona Freysdottir2,3,4 phages during the resolution of inflammation is essential for tissue homeostasis and results in macrophage reprogramming/immune-si- 1Biochemistry and Molecular Biology, Faculty of Medicine, lencing. However, a distinct subset of resolution phase macrophages Biomedical Center, University of Iceland, Reykjavik, Iceland; loss their phagocytic potential, and hence were termed satiated 2Department of Immunology, Faculty of Medicine, Biomedical macrophages. To characterize the satiation of resolution phase mac- Center, University of Iceland, Reykjavik, Iceland; 3Centre for rophages, apoptotic cells, latex beads (LB) or opsonized LB were Rheumatology Research, Landspitali, The National University injected alongsides the fluorescent dye PKH2-PCL during zymosan Hospital of Iceland, Reykjavik, Iceland; 4Department of Immunology, A-induced peritonitis, and phagocytosis of the dye as well as the Landspitali, The National University Hospital of Iceland, Reykjavik, expression of efferocytic receptors, such as CD36, CD206, and Mer, Iceland; 5Center for Proteomics and Metabolomics, Leiden was evaluated. In addition, phagocytic or satiated macrophages were University Medical Center, Leiden, The Netherlands sorted and differences in their gene expression profile were deter- mined by Illumina-based RNA-Seq analysis. Here, we show the Resolution of inflammation is important in preventing chronic engulfment of apoptotic cells, but not other particles, In vivo leads to inflammation. Natural killer (NK) cells are known to act as the first line macrophage satiation and loss of phagocytic capacity. Moreover, of defense during an infection, but have recently been implicated in satiated macrophages were generated during the resolution of resolution of allergic airway inflammation. This study examined the inflammation and maintained proliferative potential exhibited by role of NK cells in resolution of inflammation using an antigen-induced phagocytic/M2-like macrophages. In addition, we found satiation to peritonitis model, resembling a flare-up in autoimmune diseases. be irreversible and associated with reduced surface expression of Mice were immunized twice subcutaneously with methylated BSA efferocytic receptors. Furthermore, a differential gene expression (mBSA) and peritonitis induced by injecting mBSA into their peri- analysis revealed that macrophage satiation results in a comprehen- toneum. Mice were injected intravenously with the NK cell depleting sive molecular switch that conforms, for the most part, with transition antibody, anti-asialo GM1, or a control antibody 24 h prior to peritonitis from a reparative/pro-fibrotic phenotype to a pro-resolving/anti-fi- induction. Prior to and at several time-points following peritonitis brotic one. Thus, satiated macrophages generated during the induction, peritoneal exudates were collected, cells counted and

123 S214 Inflamm. Res. expression of surface molecules determined by flow cytometry. Con- B280 centration of cytokines, soluble cytokine receptors and growth factors SPECIALIZED-PRORESOLVING MEDIATORS was determined by ELISA and lipid mediators using LC–MS/MS. The NK cell depleting antibody led to a 50 % decrease in the AND TRANCRIPTOMIC SIGNATURES IN number of NK cells in the peritoneum 36 h after injection (12 h after BLEOMYCIN-INDUCED LUNG FIBROSIS induction of the inflammation). Most of the NK cells were mature, CD11b+CD27+ cells. Immature CD11b-CD27+ NK cells increased Ge´rald Cheˆne, Vincent Baillif, Guigne´ Charlotte, Belloy Marcy, after induction of inflammation in the control group but not in the NK Wanecq Estelle, Emeline Van Goethem, Marc Dubourdeau cell depleted group. The number of peritoneal neutrophils increased after induction of inflammation and peaked in the control group at Ambiotis SAS, Toulouse, France 6 h, whereas their number in the depleted group continued to increase Introduction: Idiopathic pulmonary fibrosis (IPF) is a disease with and peaked at 12 h, being at that time-point twice the number in the unknown origin. IPF is characterized by the apparition of collagen control group. By 48 h the number of neutrophils in the control group fibers into the lung parenchyma, leading to an excessive and irre- had almost returned back to baseline but in the depleted group their versible healing of the tissue, associated with a loss of its function. numbers remained high. NK cell depletion had little effect on other The clinical course of IPF is dramatic since it is estimated that the cell types. Peritoneal concentrations of 15-HEPE and PGE were 2 5-year survival is between 20 and 40 %, a higher mortality rate than lower in the depleted group than in the control group 12 h following many cancer types, including colon cancer, myeloma multiple and induction of inflammation and the concentration of LXA had a 4 bladder cancer. Following the description of specialized mediators of tendency to be lower. NK cell depletion prevented timely reduction in inflammation resolution (SPMs) and their potential involvement in IL-6, IL-12p40 and G-CSF and increased peritoneal concentrations of the control of fibrosis, their study in animal models of pulmonary IL-1ra and TGF-b at late time-points. fibrosis might help deciphering mechanisms involved in this pathol- These results indicate that NK cells may be important for halting ogy and thus open new potential therapeutic directions. neutrophil recruitment and/or inducing neutrophil apoptosis and are Methods: For this purpose, mice were inoculated by intranasal chal- indispensable for resolution of antigen-induced inflammation. lenge of bleomycin. After 2 weeks, mice were euthanized and lungs were collected and prepared for analysis. SPMs and other mediators issued from fatty acids were analyzed using a liquid chromatography- tandem mass spectrometry (LC–MS/MS) methodology to quantita- B279 tively evaluate their production. RNA expression was evaluated EXUDATE SUCTION BLISTER: A NOVEL IN VIVO thanks to a Fluidigm dynamic array. MODEL OF ACUTE INFLAMMATION AND Results: During these experiments, we have shown an increase of RESOLUTION IN HUMANS PGE2, TXB2, PGD2 and 15-HETE, mediators depending on the arachidonic acid pathways. For metabolites linked to EPA, we were also able to demonstrate an increase of PGE3, 15-HEPE and 18-HEPE Madhur Motwani1, Roel De Maeyer1, James Fullerton1, Daniel 2 2 1 the precursor of resolvins of type E. Finally, we also observed DHA Marks , Andrew Smith , Derek W. Gilroy metabolites with an increase of 13-HDoHE and 17-HDoHE but also of RvD2. Concerning trancriptomic experiments, we evaluated over 90 1Centre for Clinical Pharmacology, Division of Medicine, University 2 genes and classified them according to their systemic overexpression, College London, London, UK; Centre for Molecular Medicine, inhibition or no effect over 3 independent experiments that we per- Division of Medicine, University College London, London, UK formed. Interestingly and as an example, we notably noticed an Resolution of acute inflammation is an active process and its dysreg- overexpression of inflammatory genes (such as COX1, IL6, Ccl1, ulation may contribute to chronic inflammatory disorders. While MCP-1) and genes involved in the remodeling of the extracellular animal models have been used to study resolution their appropriateness matrix (Col1, Fn1, Mmp2, Timp1). We also measured an inhibition of has been questioned. We describe a novel model of resolving inflam- Alox12 and no effect on Alox5 mRNAs expression. mation in humans triggered by intradermal injection of UV killed Conclusions: Taken together those results suggest that fibrosis context E. coli into the forearm of healthy volunteers (n = 30). Inflammatory is associated with an inflammatory status that did not seem to be exudate was drawn into a skin blister induced by negative pressure counterbalanced by production of SPMs. Indeed, even if RvD2 is applied over the injection site. Blister cells were analyzed by flow increased and the enzymes COX and 15-LOX activated (as seen with cytometry while the cell free exudates were assayed for cytokines. To production of PGE2 and 15-HETE), none of the other SPMs were study the different phases of inflammation, blisters were raised at detected. These results might thus draw the first evidence of a defect multiple time points post-injection: 4, 8, 14, 24 h, Day 3, Day 5, Day 7, during fibrosis to produce a complete network of SPMs. Day 14. Vascular response was monitored by laser Doppler. Onset (4 h) was characterised by peak erythema and neutrophilia (HLA-DR-/ CD16++). By 24 h, neutrophil numbers had fallen by 70 % while Respiratory Disease and Inflammation monocytes/macrophages (HLA-DR+/CD14+) increased, indicating cellular resolution. From 48 h blood flow declined alongside mono- B281 cytes/macrophages. Lymphocytes (CD4+, CD8+) dominated the site at BLOCKING LTbR SIGNALING IN VIVO day 3 and persisted up to day 14. Levels of TNF-a,IL-1b were SUPPRESSES THE DEVELOPMENT OF SMOOTH maximal during onset phase, IFN-c and IL-8 during resolution, whilst post-resolution phase showed higher levels IL-16 and IL-7. Exudate MUSCLE HYPERTROPHY IN A CHRONIC Suction Blister is safe and reproducible model that allows, for the first MURINE MODEL OF ASTHMA time, exploration of cells and soluble mediators in parallel with the clinical signs of resolution in humans. Potential applications include Silke NK Hobbie, Peter Maier, Erb J. Klaus testing efficacy of novel anti-inflammatory and pro-resolving therapies and investigate whether resolution defects underlie pathogenesis of Boehringer Ingelheim Pharma GmbH & Co KG, Rhineland- chronic inflammatory disorders. Palatinate, Germany

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LTbR receptors are expressed on many different cell types and bind to life-threatening nature of these exacerbations, the underlying mech- LIGHT (TNFSF14), which is expressed on activated T cells, and anisms remain unclear although a high number of neutrophils in the lymphotoxin a and b, expressed on activated T, B and NK cells. lungs of COPD patients is known to correlate with poor prognosis. Signaling via LTbR receptor has been suggested to play a role in We and others previously demonstrated that interleukin (IL)-22 pro- remodeling in chronic airway diseases (Dohertey et al. 2011; Herro tects and regenerates respiratory epithelial cells upon virus infections et al. 2015). We investigated the effects of blocking LTbR signaling and also limits secondary bacterial infections. Indeed, IL-22, through on the development of fibrosis and smooth muscle thickening of the its receptor (IL-22R), is a cytokine that plays a pivotal role in lung airways in a chronic asthma model in mice. Mice were subjected to an antimicrobial defence and tissue protection. Regarding the IL-22/ immunization/challenge protocol using a combination of house dust IL22R antimicrobial pathway, we hypothesized that any alteration of mite extract, cockroach extract and ovalbumin, which leads to a IL-22R in the lungs of COPD patients may compromise innate severe asthma remodeling phenotype (Duechs et al. 2014). These defence mechanisms and enhance susceptibility to infections. More animals were either treated with dexamethasone or murLTbR-Fc specifically, we examined whether IL-22R expressed on lung (which blocks LIGHT and LTab signaling). We found that the epithelial cells is targeted by the neutrophil proteases present at the treatment of the mice with the murLTbR-Fc but not dexamethasone surface of lung mucosa. significantly suppressed the development of smooth muscle thicken- Using in vitro and in vivo approaches as well as RT-qPCR, flow ing and reduced fibrosis without affecting inflammation or lung cytometry and/or western-blotting techniques, we first showed that function. This supports a role of LTbR signaling in remodeling. Since pathogens such as influenza virus promote IL-22R expression in soluble LIGHT but not LTa and LTb protein levels were increased in human bronchial epithelial cells whereas Pseudomonas aeruginosa, broncho-alveolar lavage in this model, the LIGHT-induced signaling bacterial LPS do not. We also investigated whether IL-22R lung seems to be mediating these processes. To further investigate the expression was impaired by cigarette smoke in epithelial cells or in direct effect of LIGHT on lung target cells we performed several mice chronically exposed to cigarette smoke. Finally, we examined in vitro experiments. In normal human lung fibroblasts (NHLF) lung tissue from 129 patients (11 % non-smokers, 41 % ‘‘healthy’’ LIGHT increased the proliferation rate twofold after 24 h stimulation. smokers, 48 % COPD patients). IL-22R1 RNA expression was nei- LIGHT alone had no effect on cytokine release in NHLF, however, in ther associated with the cigarette smoke exposure nor the COPD the presence of IFNg, LIGHT increased IL-8 and IP-10 release, 2- and status. sevenfold respectively. In the broncho-epithelial cell line A549 In view of the foregoing data and the evidence that neutrophilia is LIGHT induced EMT (epithelial-mesenchymal fibroblast transition). a pathological hallmark of COPD, we hypothesized that neutrophils LIGHT was as efficacious as TGF-b in this assay with a later on-set of induce post translational modifications of IL-22R expression and action. In human bronchial-smooth muscle cells LIGHT together with function. We first exposed human epithelial cells to supernatant from IFNg (but neither alone) also induced a significant increase in the activated neutrophils and observed that IL-22R1 protein expression proliferation of cells. These data indicate that LIGHT could be was strongly decreased under these conditions, whereas a-1 pro- involved in remodeling processes in various cell types in vitro also teinase inhibitor prevented the disappearance of the receptor. Most conferring profibrotic properties. Taken together our results show that importantly, neutrophil proteases impaired IL-22-dependent immune blocking LTbR signaling reduces remodeling in a severe asthma signalling and expression of antimicrobial effectors such as b-de- model in mice. The presence of soluble LIGHT and not LTa and LTb fensin-2. This proteolysis resulted in the release of a soluble fragment in the lung of the mice together with the stimulating effects on human of IL-22R, which was detectable both in cellular and animal models fibroblasts and smooth muscle cells in vitro point at LIGHT mediating as well as in sputa from COPD patients with acute exacerbations. these effects. Hence, our study reveals an unsuspected regulation by the prote- olytic action of neutrophil enzymes of IL-22-dependent lung host response. This process likely enhances pathogen replication and ultimately COPD exacerbations. B282 NEUTROPHIL PROTEASES ALTER THE INTERLEUKIN-22-RECEPTOR-DEPENDENT B283 LUNG ANTIMICROBIAL DEFENCE STEM CELL FACTOR AND INTERLEUKIN 31 EXPRESSION: ASSOCIATION WITH SERUM IgE 1,3,2 1,3,2 1,2 Antoine Guillon , Youenn Jouan , Deborah Brea , LEVELS AND DISEASE SEVERITY IN ATOPIC Fabien Gueugnon 1,2, Emilie Dalloneau 1,2, Thomas Baranek 1,2, Cle´mence Henry 1,2, Eric Morello 1,2, Jean-Christophe Renauld 4,5, AND NON-ATOPIC BRONCHIAL ASTHMA Muriel Pichavant 7,8,9, Philippe Gosset 7,8,9, Yves Courty 1,2, 1,2 1,2 Patrice Diot1,2, Mustapha Si-Tahar 1,2 Mai Moaaz , Salma Aboelnazar

1 2 1INSERM, Centre d’Etude des Pathologies Respiratoires, U1100, Medical Research Institute, Colombo, Sri Lanka; University of Tours, France; 2Universite´ Franc¸ois Rabelais de Tours, Tours, Alexandria, Alexandria, Egypt 3 France; CHRU de Tours, Service de Re´animation Polyvalente, Bronchial asthma is a chronic inflammatory disorder, its prevalence Tours, France; 4Ludwig Institute for Cancer Research, Brussels 5 continues to increase over the world and it remains a significant cause branch, Brussels, Belgium; De Duve Institute, Universite catholique of morbidity. The origins of the disease remain elusive. Recently, it de Louvain, Brussels, Belgium; 6Universite´ Lille Nord de France, 7 has been reported that stem cell factor (SCF) and interleukin-31(IL- Lille, France; Institut Pasteur de Lille, Center for Infection and 31) may play a major role in bronchial asthma. The aim of the current Immunity of Lille, Lille, France; 8CNRS, Unite´ Mixte de Recherche 9 study was to validate the obscure associations of these two markers 8204, Lille, France; INSERM, U1019, Team 8, Lille, France with the susceptibility to asthma and correlate them with serum Acute episodes of exacerbations triggered by respiratory pathogens immunoglobulin E (IgE) level and disease severity. The present study mark the progression of chronic obstructive pulmonary disease included 160 Egyptian subjects who were divided into 75 atopic (COPD), leading to substantial morbidity and mortality. Despite the asthmatic patients, 35 non-atopic asthmatic patients and 50 normal

123 S216 Inflamm. Res. subjects. After measuring disease severity, pulmonary function tests B285 and serum IgE; RNA was isolated from peripheral blood mononuclear OSTEOPONTIN MODULATES CYTOTOXIC cells (PBMCs) to determine gene expression of SCF and IL-31 by real-time PCR. The levels of SCF mRNAs in atopic asthmatic patients EFFECTS OF EXTRACELLULAR HISTONES IN PBMCs were significantly higher than those in control subjects COPD (p = 0.0001) and non-atopic asthmatics (p = 0.0001). There was a high statistical significant difference also as regards IL-31 between Gopinath Kasetty, Ravi Kiran Varma Bhongir, Matthias Morgelin, Atopic asthmatics and controls (p = 0.0001) and between them and Arne Egesten non-atopics (p = 0.014). There was a strong significant direct cor- relation between SCF, IL-31 (r = 0.827 and p = 0.0001**) and Lund University, Lund, Sweden between both of them and IgE in asthmatics (r = 0.543 and Background: Lung inflammation, cell death and extensive lung tissue p = 0.0001**) (r = 0.443 and p = 0.0001**) respectively. A direct remodeling characterize COPD. Regulation of the activity of poten- correlation between SCF, IL-31 and forced expiratory volumes (FEV- tially harmful extracellular histones released from necrotic cells is 1/FVC %),c- reactive proteins (CRP), clinical severity scores and important for the prevention of excessive tissue damage and disease wheezing existed. These findings suggest that both SCF and IL-31 progression in COPD. Osteopontin (OPN) is an anionic glycoprotein enhances IgE-mediated inflammation in bronchial asthma proposing upregulated during a number of physiological and pathological pro- their role in mediating inflammation and enhancing severity of cesses has been involved in the regulation of inflammation. In recent allergic asthma studies high levels of OPN have been detected in the airways of patients with COPD. However contribution of OPN to the host response during COPD has not been well characterized. Objective: Investigate if OPN binds to extracellular histones and B284 modulate their cytotoxic effects. FUNCTIONAL DEFECT OF MAIT CELLS IN Methods: The binding affinity and kinetics of histones with OPN was PATIENTS WITH ACTIVE TUBERCULOSIS IS measured utilizing SPR BIAcore. The cytotoxic effects of extracel- lular histones in presence and absence of OPN was determined by MEDIATED BY PD-1 SIGNALING measuring lysis of erythrocytes, epithelial and endothelial cells and antimicrobial activities of histones in presence of OPN were Xiaoxing Cheng, Jing Jiang, Hongjuan An determined. Results: OPN binds to all subclasses of histones with varying affini- Institute of Tuberculosis, Beijing, China ties. Functional analysis has revealed that OPN neutralizes the cytotoxic effects of extracellular histones, which is evidenced by low Mucosal-associated invariant (MAIT) cells are innate-like T cells that levels of LDH release from epithelial and endothelial cells and are restricted by the evolutionarily conserved major histocompati- complete inhibition of erythrocyte lysis. In addition the antimicrobial bility complex-like molecule MR1. Human MAIT cells display a T activities of histones was suppressed in presence of OPN. cell receptor alpha chain that consists of TRA1-2 gene paired with Conclusions: These findings suggest a possible protective mechanism different TRAJ genes including TRAJ33, TRAJ12 and TRAJ20. of OPN in modulating host immune response by neutralizing extra- MAIT cells are activated by cells infected with bacteria and yeast, cellular histones, thereby presenting a novel treatment strategy to such as Mycobacterium tuberculosis, Escherichia coli, Shigella flex- prevent excessive tissue damage and disease progression in COPD. neri. Vitamin B metabolites synthesized by microbes can bind MR1 and activate MAIT cells. MAIT cells are abundant in humans, including peripheral blood, liver, gut lamina propria and lungs, and they not only produce cytokines, such as IFN-gamma, TNF-alpha, IL- B286 17, but also have cytotoxic effect. Tuberculosis (TB) is the second THE POLYPHENOL RESVERATROL IMPROVES most common cause of death from infectious disease. Despite high rate of M. tuberculosis infection in humans, especially in developing THE PULMONARY OXIDATIVE STRESS IN countries, only 5–10 % of infected people develop activeTB in their ASTHMATIC AND OBESE MICE BY SIRT1 life time. Interactions between M. tuberculosis and host largely PATHWAY ACTIVATION determine the development and outcome of TB infection. In this study, we compared IFN-gamma and TNF-alpha production of MAIT Diana M. Andre, Marina C. Calixto, Eduardo C. Alexandre, Carolina cells from healthy controls and patients with active TB. To prevent S. Sollon, Gabriel F. Anhe, Edson Antunes influence of previous antigenexposure and make it comparable in the two groups, we used PMA/ionomycin and E. coli as stimulator. In State University of Campinas, Campinas, Brazil patients with active TB, defect in IFN-gamma and TNF-alpha pro- duction was observed in MAIT cells when E. coli was used for Objectives: To evaluate the antioxidant effects of resveratrol via stimulation, while MAIT cell response to nonspecific stimulation with activation of SIRT1 in the lungs of asthmatic and obese mice PMA/ionomycin appeared to be normal. To determine whether there Methodology: The experimental protocols have been approved by the is any association between inhibitory receptors and functional defect Ethics Committee of UNICAMP (No. 2709-1). Male C57Bl/6/JUnib of MAIT cells, we compared expression of PD-1, PD-L1, PD-L2, mice received high-fat or standard-chow diet for 12 weeks. Lean and Tim-3, CD244, LAG-3, BTLA, CD160 on MAIT cells from patients obese mice received daily administration of resveratrol (100 mg/kg, with active TB and healthy controls. Among the inhibitory receptor gavage, 2 weeks). Mice were sensitized with OVA (100 lg, s.c) and examined, PD-1 was the only receptor that had much higher intranasally challenged with this antigen (10 lg) 2 weeks later. At expression on MAIT cellsfrom patients with active TB as compared 48 h after OVA challenge, SIRT1, p47 phox and FOXO1 expressions with healthy controls. Blockade of PD-1 signaling significantly were examined in the lung tissues. improved IFN-gamma production in MAIT cells. It is concluded that Results: SIRT1 expression in lung tissue of obese OVA-challenged MAIT cells from patients with active TB exhibited functional defect mice was reduced by about of 50 % compared to OVA-challenged that is mediated by PD-1. lean group (p \ 0.05), which was fully restored by resveratrol

123 Inflamm. Res. S217 treatment. Resveratrol treatment increased by 20 % the p-FOXO1 Introduction: Heparin exhibits anti-inflammatory activity independent expression in obese compared with lean mice (p \ 0.05). The p47 of its anti-coagulant action. However, the precise mechanism(s) un- phox expression in lung tissue showed no statistical differences derlying this anti-inflammatory activity is not well established. We between obese and lean groups, but resveratrol reduced by 56 % this have investigated the effect of ex-vivo treatment of platelets with a protein expression in obese group (p \ 0.05), with no alterations in non-anti coagulant fraction of heparin, N-acetyl-O-Sulfated Heparin lean group. (NSH), on neutrophil migration to the lung induced by lipopoly- Conclusions: Our data show that activation of SIRT1 by resveratrol sacharide (LPS, E. coli), and expression of P-selectin and PSGL-1 on may lead to increased FOXO1 expression and decreased of p47 phox the surface of platelets. expression, possibly reducing the pulmonary oxidative stress, thus Methods: Platelets were collected from anaesthetized LPS-primed improving asthma. mice (10 lg i.p.) by cardiac puncture and treated ex-vivo with NSH Financial support CNPq. (0.5 mg/mL) for 15 min. Platelets were washed and transfused back into recipient mice rendered thrombocytopenic with Busulfan (20 mg/ kg, i.p.). Lung inflammation was induced by intranasal instillation of 10 lg of LPS and bronchoalveolar lavage and samples of blood B287 undertaken 4 h later. In an independent experiment, mice were treated LUNG EPITHELIAL TRPA1 TRANSDUCES THE orally with 20 mg/Kg of NSH just before intra-nasal instillation of EXTRACELLULAR ROS INTO LPS. Four h later, blood samples were stained with anti-CD41 FITC TRANSCRIPTIONAL REGULATION OF LUNG and anti-CD62P (P-selectin) or anti-CD162 (PSGL-1) and after 30 min incubation, lysed and prepared for flow cytometry. INFLAMMATION INDUCED BY CIGARETTE Results: Mice rendered thrombocytopenic presented a significantly SMOKE: THE ROLE OF INFLUXED CA2+ lower neutrophil recruitment in response to LPS (LPS: 29.1 ± 1.3 vs LPS/Busulfan: 3.1 ± 0.9 cells 9 104/mL). Replenishment with LPS- An-Hsuan Lin1, Meng-Han Liu1, Hsin-Kuo Ko1,2, activated platelets, but not platelets pre-treated with NSH, signifi- Diahn-Warng Perng2, Tzong-Shyuan Lee1, Yu Ru Kou1 cantly re-established the neutrophilic response to LPS in the lung (LPS/Busulfan/plat.:16.0 ± 1.3 vs LPS/Busulfan/NSH: 6.80 ± 2.3 4 1Department of Physiology, School of Medicine, National Yang-Ming 9 10 /mL, p \ 0.0001). LPS significantly increases the expression of University, Taipei, Taiwan; 2Department of Chest Medicine, Taipei P-selectin, but not PSGL-1 in the surface of platelets, in comparison Veterans General Hospital, Taipei City, Taiwan to saline treated mice (saline: 6.6 ± 3.2 vs LPS: 22 ± 1.4 % increase in expression). Pre-treatment with 20 mg/Kg of NSH significantly The mechanism underlying the inflammatory role of TRPA1 in lung inhibited the expression of P-selectin, on the surface of platelets in epithelial cells (LECs) remains unclear. Here, we show that cigarette comparison to LPS (NSH: 13.2 ± 1.3 %). smoke extract (CSE) sequentially induced several events in LECs. 2+ Conclusion:: The non-anti coagulant form of heparin, N-acetyl-de-o- The Ca influx was prevented by decreasing extracellular reactive Sulfated heparin, inhibits cell recruitment into the lung by a platelet oxygen species (ROS) with the scavenger N-acetyl-cysteine, remov- dependent mechanism. ing extracellular Ca2+ with the chelator EGTA or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cys- B289 teine or HC030031. The activation of the MAPKs/NF-kB signaling was suppressed by EGTA, N-acetyl-cysteine or HC030031. IL-8 COMPREHENSIVE ANALYSIS OF RICIN BINDING induction was inhibited by HC030031 or TRPA1 siRNA. Addition- IN VIVO AND CELLULAR DAMAGE UPON ally, chronic cigarette smoke (CS) exposure in wild-type mice PULMONARY EXPOSURE induced TRPA1 expression in LECs and lung tissues. In CS-exposure -/- trpa1 mice, the increased BALF level of ROS was similar to that Anita Sapoznikov, Reut Falach, Ron Alcalay, Nehama Seliger, of CS-exposure wild-type mice, yet lung inflammation was lessened. Yoav Gal, Tamar Sabo, Chanoch Kronman Thus, in LECs, CSE may initially increased extracellular ROS, which 2+ activates TRPA1 leading to an increase in Ca influx. The increased Israel Institute for Biological Research, Ness Ziona, Israel intracellular Ca2+ contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/ The Departments of Biochemistry and Molecular Genetics, Israel NF-kB signaling leading to IL-8 induction. This mechanism may Institute for Biological Research, Ness Ziona, Israel possibly be at work in mice chronically exposed to CS. Ricin is one of the most poisonous natural toxins from plants; it belongs to a group of ribosome-inactivating protein type II (RIP II) family, which inhibits protein synthesis. Pulmonary exposure to ricin results in the generation of an acute edematous inflammation, fol- B288 lowed by respiratory insufficiency and death. In order to develop a N-ACETYL-DE-O-SULFATED HEPARIN INHIBITS potent therapeutic countermeasure, better understanding of the nature LYPOPOLYSACCHARIDE-INDUCED of the interaction between the target host cell and the toxin, and its LEUKOCYTE MIGRATION BY A PLATELET direct consequences on the whole lung tissue is required. In this study we examined the kinetics of ricin binding and DEPENDENT MECHANISM internalization to lung cells in vivo and its effect on the cellular milieu. Interactions between ricin and lung cells were examined Yanira Riffo Vasquez1,2, Richard Amison1,2, Simon Pitchford1,2, concomitantly by fluorescently-labeled-toxin and by specific anti- Clive P. Page1,2 ricin antibody binding. We show that although ricin could potentially bind to any of the lung cells, as demonstrated in ex-vivo labeling 1King’s College London, London, England; 2Sackler Institute of experiments; In vivo, it bound preferentially to alveolar macrophages Pulmonary Pharmacology, London, England (MU), dendritic cells (DCs), pneumocytes type II (PTII) and

123 S218 Inflamm. Res. endothelia. Furthermore, the binding to lung cells following pul- expression of caspase 3 and Fas-L, but not Bax and caspase 9. OVA- monary exposure of mice to ricin occurred at two different kinetics, challenge or NaHS-treatment was unable to modulate the apoptosis of displaying peak levels at early and late time points. The differential BAL eosinophils. However, the NaHS avoid the apoptosis increase in binding pattern could be explained by interactions with cells bearing bronchial epithelial cells promoted by OVA challenge. An increase in different classes of receptors, the galactose-containing glycoprotein/ the expression of CSE enzyme in the bronchial epithelium and in the glycolipid receptors or the cellular mannose receptor. MU, DCs and vascular endothelium was observed in the lungs of allergic mice and PTII cells, which express the mannose receptor at their surface, dis- was amplified by NaHS-treatment. No changes were observed for played early binding, as opposed to endothelial cells which displayed CBS enzyme. In conclusion our results suggest that NaHS-treatment late binding. Although MU, DCs and PTII cells all bound ricin prevented apoptosis and, consequently, the bronchial epithelium rapidly, elimination of PTII cells from the lungs occurred at a con- damage, which contributes to the pulmonary inflammation decrease. siderably later time-point, in comparison to MU and DCs, which The CSE enzyme may be involved in this process. Therefore, the H2S population had been reduced already few hours after intoxication. can have a protective effect against lung damage caused by allergic Interestingly, pneumocytes type I that are known to be very sensitive reaction, representing a potential therapeutic agent for allergic pul- cells, were not affected by the toxin. Endothelial cells, lacking the monary disorders, such as asthma. mannose receptor, displayed a late binding profile, and their elimi- nation from the lungs occurred after several days. In addition, we could observe, an early reduction in endothelial and epithelial tight- junction protein (VE-Cadherin, Occludin and Claudin5) expression. B291 In contrast to the toxin-binding cells, neutrophils, which were mas- MODERATE PHYSICAL EXERCISE DECREASES sively recruited to the lungs, did not interact with ricin. Taken AIRWAY INFLAMMATION IN A MURINE MODEL together, these results show that differential ricin binding and inter- OF ASTHMA nalization directly contribute to the loss of maintenance of tissue integrity, which results in functional lung damage. Paula Fernandes1, Thayse R. Bru¨ggemann1, Clarice R. Olivo1, Fernanda M. Bruni2,3,Mı´lton A. Martins1, Fernanda M. Arantes-Costa1 B290 1Laboratory of Experimental Therapeutics, LIM20 School of DOES HYDROGEN SULFIDE INFLUENCE 2 APOPTOSIS PROCESS IN LUNGS FROM Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil; Institute for Investigation in Immunology (iii), INCT, Sa˜o Paulo, Brazil; ALLERGIC MICE? 3Laboratory of Clinical Immunology and Allergy, LIM60 Scholl of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil Heloisa Helena A. Ferreira1, Jackeline A. Mendes2, Matheus C. Introduction: The benefits of moderate physical exercise in allergic Ribeiro3, Gislaine Cristina P. Moreira3, Mateus S. Silva3, Natalia H. asthma are well known, indicating that the physical exercise has a Dias3, Bruna T. Albaladejo3, Jose´ A. Pereira3, Thalita Rocha3 anti-inflammatory effect by reducing the Th2 responses and the pul- monary remodeling, although the mechanisms of this 1Sa˜o Leopoldo Mandic Institute and Research Center, Campinas, immunoregulation are not very known yet. Therefore, we made this Brazil; 2State University of Campinas/Unicamp, Campinas, Brazil; study to investigate the mechanisms of the immunoregulation by 3Sa˜o Francisco University, Campinas, Brazil moderate physical exercise on asthma inflammation. Many studies have shown that hydrogen sulfide (H2S) has a relevant Methods: Male Balb/c mice (n = 8/group) were sensitized with five role in the pathophysiology of lung diseases. This study aimed to intraperitoneal injections of ovalbumin (OVA, 20 lg) and alum investigate the effect of H2S in modulating apoptosis in lungs from (3 mg) on days 0 and 14, 21, 28 and 42 and were challenged with an allergic mice. BALB-C mice were sensitized and challenged with aerosol of OVA (1 %, 30 min) three times per week, starting on day ovalbumin (OVA group). Some sensitized mice received sterile saline 21 until day 51 [OVA and OVA+ exercise (OVA + EX) groups]. A without OVA at the time of challenge (saline group). Others mice maximal exercise capacity test was performed on days 21 and 51. The were sensitized but were treated with H2S donor—sodium hydrosul- EX (exercise) and OVA + EX groups practiced moderated exercise fide (NaHS) 30 min before OVA-challenge (OVA/NaHS group). The on days 21 until 51. EX and Control (SAL) groups received saline and euthanasia was performed 48 h after allergen challenge. Bron- alum intraperitoneal injection and were challenged with saline 0.9 %. choalveolar lavage (BAL) was collected for eosinophils isolation by Twenty-four h after the last challenge, we evaluated the inflammatory immunomagnetic method. The right lung lobe was removed and cells influx on the bronchoalveolar lavage fluid, and also the pro- homogenized to study the expression of caspase 3, caspase 9, Bax and duction of immunoglobulin IgG1 and IgG2a in plasma. Fas-L by western blotting. The left lung lobe was fixed in formalin for Results: Physical capacity was increased only in trained groups 1) histological analysis of inflammatory cell infiltrate using hema- (p = 0.036). OVA-sensitized mice showed increased influx of mac- toxylin/eosin staining (HE); 2) in situ apoptosis analyses by TUNEL rophages (p = 0.016) and eosinophils (p = 0.05) to the airway and assay and 3) verification of expression of cistationia-c-lyase enzymes the training reduced this inflammatory infiltrate (OVA + EX) (CSE) and cystathionine-b-synthase (CBS) by immunohistochem- (p = 0.05 and p \ 0.01, respectively). We noticed that IgG1 and istry. The histological results showed an inflammatory infiltrate IgG2a levels increases in both OVA-sensitized groups (OVA and around the bronchi and bronchioles in the OVA group, with a OVA + EX; p = 0.02 and p \ 0.01, respectively). prevalence of eosinophils, which was prevented by NaHS-treatment. Conclusion: We conclude that exercise reduces the lung inflammation The treatment of allergic mice with NaHS also decreased the in asthmatic mice.

123 Inflamm. Res. S219

B292 site suggesting an involvement of natural PAR2 ligands such as mast PROTEINASE-ACTIVATED RECEPTOR 2 PLAYS cell tryptase on this process. Financial support CNPq and CAPES. A PIVOTAL ROLE IN ALLERGEN-INDUCED LEUKOCYTE RECRUITMENT IN EXPERIMENTAL LUNG INFLAMMATION B293 Natalia A. Matos1, Raphael G. Ferreira2, Annie R P Alvarez2, Karine PROTECTIVE ROLE OF SURFACTANT PROTEIN- A. Damasceno3, Geovanni D. Cassali3, Jose´ C. Alves-Filho2, Andre´ D IN NITROGEN MUSTARD INDUCED LUNG Klein1 TOXICITY

1 Department of Pharmacology, Federal University of Minas Gerais, 1 1 1 2 Vasanthi R. Sunil , Kinal P. Vayas , Jessica Cervelli , Michael Belo Horizonte, Brazil; Department of Pharmacology Ribeira˜o Preto Goedken1, Rama Malaviya1, Jeffrey D. Laskin2, Debra L. Laskin1 Medical School, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3 Department of Pathology, Federal University of Minas Gerais, Belo 1Rutgers University, Brunswick, New Jersey; 2Robert Wood Johnson Horizonte, Brazil Medical School, Brunswick, New Jersey Introduction: Proteinase-activated receptors (PARs) are G-protein- Nitrogen mustard (NM) and related alkylating agents are potent coupled receptors comprising a family of four members. We have vesicants that target the lung, causing inflammation and fibrosis. demonstrated that PAR2 plays a role in eosinophil (E/) recruitment Evidence suggests that alveolar macrophages contribute to the in experimental allergen-induced pleurisy supporting additional evi- pathogenic response. Surfactant Protein (SP)-D is a pulmonary col- dences for a role for PAR2 in allergic diseases. However, the role of lectin known to suppress alveolar macrophage activity. In the present PAR2 in lung inflammation has not been fully understood. In this studies, we analyzed the effects of loss of SP-D on NM-induced lung work, we evaluated the effects of PAR2 activation in allergen-induced toxicity. Wild type (WT) and SP-D-/- mice were treated with PBS or lung inflammation. NM (0.08 mg/kg) intratracheally. Bronchoalveolar lavage (BAL) and Methods: BALB/c mice (20–25 g) were sensitized intraperitoneally lung tissue were collected 14 days later. Treatment of WT mice with (i.p.) with 100 lg of ovalbumin (OVA) in 2 % Al(OH)3 gel adjuvant NM resulted in perivascular inflammation and increases in BAL cell on day 0, then challenged intranasally (i.n.) on days 8–10 with 10 lg and protein content, indicating alveolar epithelial injury. Loss of SP- of OVA or PBS. Bronchoalveolar lavage (BAL) were obtained 4, 24, D resulted in a significant increase in the sensitivity of the mice to 48 and 72 h after challenging. In some experiments PAR2 activating NM; thus, perivascular inflammation was more severe and there was peptide SLIGRL-NH2 (SLIG, 30 lg) was coadministered i.n. with evidence of foamy macrophages, perivascular edema, bronchiolar OVA challenging on days 8–10 and the BAL was evaluated 72 h epithelial hypertrophy and hyperplasia, bronchiolar alveolar hyper- after. PAR2 detection on surface of BAL cells was performed through plasia, bronchiectasis, fibrosis and emphysema. NM-induced immunohistochemistry on cytospin preparations. Furthermore, mice oxidative stress, as assessed by hemeoxygenase-1 expression by were treated i.p. with the PAR2 antagonist ENMD1068 (ENMD, alveolar macrophages, was also increased. Enlarged and highly vac- 11 lg) 1 h before OVA i.n. administration on days 8-10 and the uolated profibrotic Ym-1+ and mannose receptor-1+ alveolar number of infiltrating leukocytes and cytokine levels present on BAL macrophages were also present; in contrast, cyclooxygenase-2+ were analysed by ELISA. Statistical analyses: One-Way ANOVA proinflammatory macrophages were decreased. These data indicate followed by Newman-Keuls post-test. The experiments were that SP-D plays an important role in protecting against NM-induced approved by UFMG Ethics’s Committee for Animal Use (certificated pulmonary toxicity. Elucidating mechanisms regulating macrophage number 348/2014). activity following NM exposure may be important in developing Results: OVA challenging in sensitized mice induces E/ recruitment therapeutics to treat vesicant-induced lung injury. 4–72 h peaking 48 and 72 h when compared to PBS-treated mice Support: NIH AR055073 and ES005022. (48 h: PBS, 0.2 ± 0.2; OVA, 9.7 ± 3.7*; 72 h: PBS, 0.0 ± 0.0; OVA, 8.3 ± 3.5* E/s 9 104,*p\ 0.01). SLIG + OVA induced E/ recruitment in sensitized mice 72 h after when compared to PBS- treated mice (PBS, 0.0 ± 0.0; OVA + SLIG 10.4 ± 2.6* E/ B294 4 s 9 10 ,*p \ 0.001), and PAR2 is localized in both E/s and TRANSCRIPTOMIC AND PROTEOMIC mononuclear cells 48 h after the OVA challenging. ENMD abolished PROFILING OF SPUTUM REVEALS the neutrophil (N/) recruitment induced by OVA 2, 4, 8, 12 h after challenging (2 h: PBS + OVA, 9.0 ± 1.5; ENMD + OVA, INFLAMMASOME-ASSOCIATED SIGNATURES IN 4.1 ± 0.3*, N/s 9 104), (4 h: PBS + OVA, 1.7 ± 0.1; SEVERE ASTHMA ENMD + OVA, 0.3 ± 0.04**), (8 h: PBS + OVA, 0.8 ± 0.04; ENMD + OVA, 0.46 ± 0.04**), (12 h: PBS + OVA, 0.7 ± 0.06; Ian M. Adcock1, Christos Rossios1,2, Stelios Pavlides2, Matthew 5 ENMD + OVA, 0.27 ± 0.02**) N/s 9 10 ,*p\ 0.01 **p \ 0.001. Loza2, Fred Baribaud2, Scott Kuo1,2, Anthony Rowe2, Uraj Hoda1,2, ENMD also inhibited E/ recruitment induced by OVA 8, 12, 24 h Ratko Djukanovic2, Peter J. Sterk2 after (8 h: PBS + OVA, 2.1 ± 0.17; ENMD + OVA 1.2 ± 0.2*, E/ 4 s 9 10 ) (12 h: PBS + OVA, 0.2 ± 0.03; ENMD + OVA, 1NHLI, Imperial College London, London, UK; 2U-BIOPRED 0.06 ± 0.01**), (24 h: PBS + OVA, 0.4 ± 0.05; ENMD + OVA, Consortia 0.07 ± 0.01**) E/s 9 105 *p \ 0.01 **p \ 0.001. ENMD reduces KC, IL-6, RANTES, AREG levels 2 h after OVA challenging when Background: Asthma is a chronic inflammatory disease of the airway compared to OVA-treated mice and increased IL-10 4 h after OVA driven by Th2-driven eosinophilia. Severe asthma is characterised by challenging. poor therapeutic response to corticosteroid therapy and a diverse Conclusion:: This study demonstrates a pivotal role for PAR2 on airway inflammation profile. leukocyte recruitment in allergen-induced lung inflammation, at least Aims and objectives: To investigate the relationship between in part through the cytokine modulation at the allergic inflammatory sputum eosinophils and proteomic and transcriptomic profiles in

123 S220 Inflamm. Res. sputum from the U-BIOPRED cohort of severe non-smoking asthma up-regulated (FDR\0.01) on days 1, 3, 4 and 7 respectively. In total (A), severe smoking asthma (B), non-severe asthma (C) and controls 26 genes were up-regulated at all time points including genes such as (D). CCL26, POSTN and ITGB2 which are associated with cytokine, Methods and results: Median sputum eosinophil and neutrophil counts chemokine and integrin signalling. were respectively: A: 2.75, 54.0 % (n = 128); B: 4.13, 55.0 % These 26 genes were used as a GSVA gene signature to map the (n = 53); C: 1.05, 44.5 % (n = 47); and D: 0.0, 39.6 % (n = 41). CFA/HDM severe asthma model to the U-BIOPRED cohort. Defining an eosinophil cut-off [1.32 % and neutrophil count Enrichment of this signature in samples collected from blood, biopsy, [85.7 % (2 SD above mean in cohort D), cohort A had eosinophilic bronchial brushing, sputum and nasal brushing from all cohorts were (55.5 %), neutrophilic (9.4 %), mixed granulocytic (7.8 %) and nor- analysed. There was a significant enrichment of the signature in mal granulocytic (27.3 %) patterns of inflammation. severe non-smoking asthma compared to healthy subjects in biopsies, Gene expression profiling demonstrated 496 differentially-ex- bronchial brushings and sputum samples. This was not significantly pressed genes between severe asthma and healthy subjects (FDR different in non-severe asthma. \0.05, twofold/healthy cohort), with the most differentially expres- Conclusion: The signature of up-regulated genes in the CFA/HDM sed genes associated with the inflammasome: IL18R1, IL1RL1, model of severe asthma was elevated in human severe non-smoking IL1R2, IL18RAP, IRAK3. Genes for Charcot-Leyden crystal (CLC) asthma but not in non-severe asthma. This approach may be used to protein and IL-33R (IL1RL1) were highly expressed (5.04- and 5.52- investigate how other murine models of asthma align with human fold) and were most highly correlated with sputum eosinophil counts asthma allowing the selection of distinct mouse models to reflect the (r = 0.77 and 0.76; p \ 10-8). Gene set variation analysis for specific characteristics of the asthma phenotype to be tested in man. microarray data (GSVA) of Th2 signatures in Cohorts A and B Funded by the Innovative Medicines Initiative. compared to cohorts C and D. Somalogic SOMAscanTM V3 analysis of sputum showed that 54 analytes were differentially expressed between SA non-smokers (n = 43) and healthy subjects (n = 18) (FDR \0.05 [ twofold/ B296 healthy cohort). Pregnancy-associated plasma protein A (PAPP-A), ENDOGENOUS CHOLINERGIC SYSTEM IS Serum amyloid P (SAP) and C-reactive protein (CRP) were highly INVOLVED IN SEXUAL DIMORPHISM ON up-regulated in both smoking and non-smoking SA (14.7-, 6.27- and PULMONARY INFLAMMATION IN AN ASTHMA 3.21-fold). PAPP-A (r = 087; p \ 10-10), SAP (r = 0.6; p \ 10-10) and periostin (r = 0.55; p \ 10-10) correlated with sputum eosino- EXPERIMENTAL MODEL phils, while b-endorphin (r = 0.67; p \ 10-10), azurocidin (r = 0.63; p \ 10-10), IL-5 (r = 0.6; p \ 10-10) and IL-8 (r = 0.51; p \ 10-8) Nathalia M. Pinheiro1, Fernanda P.R. Santana2, Francine M. Almeida1, with sputum neutrophils. Adenir Perini1, Iolanda F.L.C Tibe´rio1, Milton A. Martins1, Conclusion:: Severe asthma is associated with acute phase and eosi- Marco Antoˆnio M. Prado3,Vaˆnia F. Prado3, Ana Cristina B. Faloppa1, nophil- and neutrophil- associated analytes in sputum. Expression Wothan T. Lima1, Carla M. Prado2 profiling and proteomics in induced sputum of severe asthma provides inflammasome and Th2-high gene signatures that are associated with 1University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2University Federal of eosinophilia. Inflammasome activation appears strongest in eosino- Sa˜o Paulo, Sa˜o Paulo, Brazil; 3University of Western Ontario, philic asthma. The significance of the inflammasome activation in London, Ontario stable disease remains to be determined. Introduction: Asthma is an immune disorder characterized by chronic Funded by Innovative Medicine Initiative. airway inflammation and hyperresponsiveness. Cholinergic anti-in- flammatory pathway modulates immune systemic inflammatory responses in different models by release its main neurotransmissor acetylcholine (ACh). The vesicular acetylcholine transporter B295 (VAChT) mediates ACh storage in synaptic vesicles, which is A GENE SIGNATURE FROM THE CFA/HDM essential for ACh release. It is known that estradiol and progesterone MODEL OF ASTHMA PREFERENTIALLY MAPS drive cellular recruitment and affect inflammation in asthma. TO HUMAN SEVERE ASTHMA Aim: We tested the relationship between cholinergic deficiency and females hormones. First, male and female VAChT-deficiency (VAChTKD-HOM) or wild type (WT) mice were submitted to Ian M. Adcock1,2, Kirsty Russell1,2, Stelios Pavlides2, 1,2 2 1,2 ovalbumin (OVA) subcutaneous protocol on days 0, 7, and 14 and Christos Rossios , Navin Rao , Kian F. Chung then inhaled with OVA or saline protocol. Then, ovariectomized

1 2 VAChTKD-HOM mice (7 days before the beginning of OVA pro- NHLI, Imperial College London, London, UK; U-BIOPRED tocol) and those treated with estradiol, progesterone or a7nicotinic Consortia receptor (a7 nACHR) agonist (PNU, 10 mg/kg i.p) were submitted to Background and aim: A major concern in drug discovery is that the same OVA protocol. On day 29th, BAL was collected and animal models of disease do not always translate to human disease. inflammatory cells were counted. VAChTKD-HOM male mice To improve translation, we have used transcriptomic profiles and gene exposed to OVA presented an increase in total cells, lymphocyte and set variation analysis (GSVA) obtained from a novel mouse model of eosinophil in BALF compared to WT male mice exposed to OVA. severe asthma to map onto expression profiles of various compart- This effect was not observed in female VAChTKD-HOM. The OVX ments in human severe asthma. reduced the inflammatory cells, macrophage and eosinophils in Methods and Results: Male Balb/c mice were exposed to house dust female WT but not in VAChTKD-HOM exposed to OVA. Estradiol, mite (HDM) in Complete Freunds Adjuvant (CFA) for 2 weeks treatment maintained the values of inflammatory cells reduced similar before being challenged with either saline or HDM intranasally. Gene to those observed in OVX female, but the progesterone increased the expression analysis was conducted using Affymetrix HT_MG- total cells compared to OVX female mice. The stimulus of a7nAChR 430_PM arrays on lung tissue collected at 1, 3, 4 and 7 days post with PNU in VAChTKDHOM OVX mice reduced the total cells to challenge. A total of 167, 798, 385 and 129 genes were differentially similar values obtained in WT OVX female mice.

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Conclusions: In this model of asthma experimental, cholinergic Rationale: Resistance to inhaled corticosteroid therapy in conjunction deficiency worsens lung inflammation only in male mice suggesting with concomitant neutrophilic and eosinophilic inflammation are that gender dimorphism influence the effects of cholinergic defi- hallmarks of patients with severe asthma. Treatment options in this ciency. However, our data also suggest that the anti-inflammatory patient population are limited and drug development has been ham- effects of ovariectomy in allergic female, which seems to be modu- pered by limited pre-clinical animal models. Here we present a lated more by progesterone, depend, at least in part, by intact clinically relevant corticosteroid-resistant allergic mouse model of cholinergic anti-inflammatory system. severe asthma characterized by the generation of neutrophilic and eosiniophilic inflammation and Th1/Th2 cytokines which correlate with airway hyperresponsiveness and abnormal changes in lung B297 mechanics. SODIUM METABISULFITE CHALLENGE; LITTLE Methods: Balb/c mice were sensitized s.c. on day 0 with House Dust Mite (HDM) combined with Complete Freund’s Adjuvant (CFA) fol- EVIDENCE OF INFLAMMATORY POTENTIAL lowed by i.n. challenge of HDM on day 14 and methacholine challenge on day 15. A comparator set of animals was sensitized and challenged Daniel E. Maddox, Sharon M. Flynn i.n. with HDM using a classical model to develop an eosinophilic Th2 inflammatory response. In both models, a group of animals were treated Mayo Clinic, Phoenix, USA with fluticasone propionate (FP). Inflammatory responses were char- Since the original 1976 report by Prenner and Stevens of metabisulfite acterized by total and differential cell counts in bronchoalveolar lavage sensitivity, there has been controversy surrounding the mechanisms by fluid (BALF), analysis of Th1/Th2 panels of cytokines and chemokines which patients respond to the compound. The asthmatic responders have by Luminex, lung histopathology and assessment of lung mechanics and airway hyperresponsiveness to methacholine challenge using the been postulated to be converting the metabisulfite in the lung to sulfur Ò dioxide, which is known to activate irritant receptors in the hyperreac- flexiVent system (SCIREQ ). tive airways of asthmatics. Save for the report by Sokol and Hydick, Results: Mice exposed to HDM/CFA developed an inflammatory postulating IgE-mediated sensitivity to the compound being implicated profile characterized by Th1 and Th2 responses with the presence of in urticaria and angioedema, other investigators have not been able to pulmonary neutrophils, eosinophils and Th1 (IL-2) and Th2 (IL-4) demonstrate positive evidence supporting this mechanism. Over the past cytokines and chemokines. FP failed to inhibit neutrophilic inflam- 24 years, since computerization of the Allergy Clinical Laboratory at mation and moderately inhibited eosinophils in the HDM/CFA Mayo Clinic, we have performed 103 challenges to sodium animals, but did inhibit eosinophilic inflammation in the classically metabisulfite. The patient population was 29 males and 74 females. All challenged HDM animals. Airway hyperresponsiveness and lung but two of the challenges were orally administered metabisulfite, with mechanics were enhanced in the HDM/CFA challenged animals as two patients (whose reactions being investigated had occurred to compared to responses observed in the classically challenged HDM injectable drugs preserved with metabisulfite) undergoing skin testing. animals. FP- treatment reduced these responses more dramatically in The symptoms being investigated were anaphylaxis (12 cases), urti- the classically challenged HDM animals with minimal effects in the caria/angioedema (44 cases), food-associated gastrointestinal symptoms severe asthmatic mice. (12 cases), food/drink-associated asthma flares (19 cases), and there was Conclusions: We have successfully established a model of severe one case each of flushing only associated with food and drink, and one asthma with matching inflammatory profiles to human severe asthma case of food-associated laryngospasm. Shortness of breath with no and for which concurrent clinically relevant functional parameters of history of asthma was reported by 20 cases. There were a total of only 3 airway hyperresponsiveness and lung mechanics measurements were positive challenges, 2 in urticaria/angioedema patients, and 1 in an seen. This model will be a useful tool in assessing novel therapies in asthma patient. One case that was pulled out of the database was pulled the treatment of ICS-resistant severe asthma. because there was 0.5 mg/mL metabisulfite in a specific local anesthetic product used in dentistry, to which the patient was being tested, but that test was incomplete because of emergence of symptoms thought related to the included epinephrine in the solution. There were 18 placebo- B299 responders in this series, or 6 times the number of active material EFFECTS OF CHOLINERGIC HYPOFUNCTION IN responders. Overall, it appears that there is very low or no pro-inflam- LUNG MECHANICS AND HISTOPATHOLOGY IN matory potential for metabisulfite to trigger these kinds of symptoms in patients with recurrent underlying inflammatory disorders. Our data AN EXPERIMENTAL MODEL OF LUNG raises the question of whether this particular investigation is helpful in INFLAMMATION INDUCED BY AIR POLLUTION the clinical evaluation of patients reporting food or drink-associated IN MICE allergic or pseudoallergic symptoms. Fernanda PR Santana1,3, Nathalia M. Pinheiro1,Ma´rcis MB Mernak3, Perini Adenir1, Kelly Yoshizaki2, Mariaˆngela Macchione2, B298 Paulo H. Saldiva2, Luciana C. Caperuto3, Milton A. Martins1, A CORTICOSTEROID-RESISTANT ALLERGIC Iolanda FLC Tibe´rio1, Marco Antoˆ nio M. Prado4,5, 4,5 3,1 MOUSE MODEL OF SEVERE ASTHMA WITH Vaˆnia F. Prado , Carla M. Prado CLINICALLY RELEVANT ENDPOINTS: 1Department of Medicine School of Medicine, Universidade de Sa˜o CORRELATING INFLAMMATORY RESPONSES Paulo, Sa˜o Paulo, Brazil; 2Department of Pathology School of TO ALTERATIONS IN LUNG MECHANICS Medicine, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; 3Department of Biological Science, Universidade Federal de Sa˜o 4 Paulette W. Andreotta, Dominic R. Beal, Martina Rosado, Paulo, Sa˜o Paulo, Brazil; Department of Physiology and Samantha Rogers, Stephen T. Sonis, Gregory D. Lyng Pharmacology, University of Western Ontario, London, Ontario; 5Department of Anatomy and Cell Biology, University of Western Biomodels, LLC, Boise, Idaho Ontario, London, Ontario

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Introduction: Diesel automotive engines are widely used in urban (PI3K) pathways and histone deacetylase (HDAC)2, and to investi- centers and their exhausts is considered a major environmental and gate new treatment approaches. toxic pollutant to human health, mainly because diesel exhaust par- Methods: novel mouse models of chlamydia, and haemophilus, ticulate (DEP) reaches more distal airways and lung parenchyma, respiratory infection and ovalbumin-induced, severe, neutrophilic, inducing an inflammatory response. ACh has been recently described steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice as the main mediator of cholinergic anti-inflammatory pathway since were developed. The roles and potential for targeting of infection- it suppresses cytokine generation which controls the innate immune induced miR-21 expression and PI3K-dependent signaling in the lung response. were examined using therapeutic treatments with a specific miR-21 Aim: In the present study, the authors evaluated whether cholinergic inhibitor (antagomir, Ant-21) and the pan-PI3K inhibitor LY294002. dysfunction is involved in DEP-induced lung inflammation. In the Results: Respiratory infection induced a miR-21-dependent, PI3K- present study, the authors evaluated whether cholinergic dysfunction mediated cell-signaling pathway that promoted steroid-insensitive, is involved in DEP-induced lung inflammation. neutrophilic inflammation and airway hyper-responsiveness (AHR) in Methods: Male mice with VAChT reduction were used, divided AAD. This involved the suppression of nuclear HDAC2 levels. according to genotyping for wild-type (WT) and knock-down for Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and VAChT (KD), and submitted to DEP exposure protocol (10 lLof restored HDAC2 levels. Treatment also restored sensitivity to steroid DEP, 3 mg/mL for 30 days) or saline. Pulmonary mechanics, lung administration. inflammation, cytokine expression, extracellular matrix fiber deposi- Conclusions: We have identified a previously unrecognised patho- tion, and mucus production in the airway and nasal epithelial were genic role for a miR-21/PI3K/pAkt/HDAC2 signaling axis in evaluated. Both in WT and KD mice, DEP instillation induced lung severe, neutrophilic, steroid-insensitive AAD. Our data highlights parenchyma responsiveness; increased mononuclear cells in periph- miR-21 as a novel therapeutic target for the treatment of this form eral blood and macrophages in bronchoalveolar lavage fluid (BALF); of asthma. increased positive cells to TNF-a, IL-4, IL-6 and IL-13 in lung tissue; collagen fiber deposition in alveolar septa; increased mucus produc- tion in airway epithelial associated with an increase in secretory cells; and a decrease in ciliary cells. Additionally, DEP exposure in mutant B301 mice also induced an increase in granulocytes in peripheral blood; in TUMOUR NECROSIS FACTOR-RELATED neutrophils and lymphocytes in BALF; in elastic fibers content in APOPTOSIS INDUCING LIGAND PROMOTES THE alveolar septa; and in acid mucus in nasal epithelial. These alterations DEVELOPMENT OF EXPERIMENTAL CHRONIC were not observed in wild-type mice. Moreover, the levels of IL-4 and TNF-a were also higher in mutant mice exposed to DEP compared OBSTRUCTIVE PULMONARY DISEASE with wild-type mice exposed to DEP. Conclusion: In conclusion, VAChT-deficient mice seem to be more Philip M. Hansbro1, Tatt J. Haw1,2, Malcolm R. Starkey1,2, Prema M. vulnerable to DEP-induced lung alterations, suggesting that air pol- Nair1, Irwan Hanish3, Duc H. Nguyen1, Gang Liu1, Mark D. Inman4, lution effects in the respiratory system are likely to be influenced by Richard Y. Kim1, Adam M. Collison1, Darryl A. Knight1, the cholinergic anti-inflammatory system. Hideo Yagita5, Joerg Mattes1, Jay C Horvat1 Financial support FAPESP, CNPq, LIM-20 HCFMUSP. 1Department of Health and Medicine, Faculty of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, Priority Research Centre for Asthma and Respiratory Diseases, University of B300 Newcastle, New South Wales, Australia; 2Authors contributed microRNA-21 DRIVES SEVERE, STEROID- equally; 3Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, INSENSITIVE EXPERIMENTAL ASTHMA BY 4 AMPLIFYING PI3K-MEDIATED SUPPRESSION Selangor, Malaysia; Department of Medicine, McMaster Immunology Research Centre, Firestone Institute for Respiratory OF HDAC2 Health, St. Joseph’s Healthcare, McMaster University, Hamilton, Ontario, Canada; 5Department of Immunology, Juntendo University Philip M. Hansbro1, Richard Y. Kim1, James W. Pinkerton1, Malcolm School of Medicine, Bunkyo-ku, Tokyo, Japan R. Starkey1, Ama T. Essilfie1, Jemma R. Mayall1, Bernadette Jones1, Background: Chronic obstructive pulmonary disease (COPD) is the Tatt Jhong Haw1, Simon Keely1, Joerg Mattes1, Ian M. Adcock2, third leading cause of death worldwide and causes significant Paul S. Foster1, Jay C. Horvat1 healthcare and economic burden. Cigarette smoking is a major risk factor. There is a lack of effective treatments for COPD due to poor 1The University of Newcastle, Callaghan, Australia; 2National Heart understanding of the underlining mechanisms. Tumour necrosis fac- and Lung Institute, Imperial College London, London, UK tor-related apoptosis-inducing ligand (TRAIL) is implicated in Background: Severe, steroid-insensitive asthma is a substantial clin- respiratory diseases, namely asthma and pulmonary fibrosis. How- ical problem accounting for [50 % of asthma-associated health-care- ever, the role of TRAIL in the pathogenesis of COPD is unknown. costs. Effective treatments are urgently required, however, their Objectives: To determine the effect of cigarette smoke-induced development is hampered by a lack of understanding of the mecha- experimental COPD on production of TRAIL. To elucidate whether nisms that promote the pathogenesis of disease. Steroid-insensitive genetic deletion or antibody-mediated neutralization of TRAIL asthma is associated with respiratory infections, and non-eosinophilic influences the pathogenesis of COPD. endotypes of disease, including neutrophilic asthma. The mechanisms Methods: Wild-type or TRAIL-deficient mice were exposed to nose- that underpin infection-induced, severe, neutrophilic, steroid-insen- only cigarette smoke inhalation for 8 weeks and key pathological sitive asthma may be elucidated using mouse models of disease. features of disease were assessed. To determine therapeutic potential Objectives: To develop representative mouse models of this endotype of targeting TRAIL, wild-type mice were exposed to cigarette smoke of asthma. To use these models to identify novel pathogenic mech- inhalation for 12 weeks and given neutralising anti-TRAIL antibody anisms involving microRNA (miR)-21, phosphoinositide-3-kinase or isotype control intraperitoneally from week 7 to week 12.

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Results: TRAIL mRNA expression and/or protein levels in the lung B303 (airway and parenchyma) and serum were increased in a mouse model THE ROLE OF PD-1 IN VIRAL PREDISPOSITION of cigarette smoke-induced experimental COPD. Genetic deletion of TRAIL significantly reduced cigarette smoke-induced pulmonary TO SECONDARY BACTERIAL PNEUMONIA inflammation, expression of key pro-inflammatory mediators, 1 1 1 emphysema-like alveolar enlargement and improved lung function in Philip Hansbro , Alexandra Brown , Ama-Tawiah Essilfie , 1 3 2 2 experimental COPD. Interestingly, genetic deletion of TRAIL led to Emma Beckett , Alison Thorburn , Hideo Yagita , Hideo Yagita , 1 1 spontaneous small airway remodeling characterized by increased Paul Foster , Jay Horvat airway epithelial cell thickness and collagen deposition. Importantly, 1 2 antibody-mediated neutralization of TRAIL reduced cigarette smoke- University of Newcastle, Callaghan, NSW, Australia; Jutendo 3 induced pulmonary inflammation, emphysema-like alveolar enlarge- University, Tokyo, Japan; Monash University, Clayton, VIC, ment and small airway remodeling. Australia Conclusions: Our study is the first to show that TRAIL plays an Background: Secondary bacterial pneumonia is a common conse- important role in the pathogenesis of COPD and provides further quence of respiratory viral infections, and is a debilitating and life evidence for TRAIL being a pivotal inflammatory cytokine in respi- threatening disease. Studies have revealed many components of the ratory diseases. immune response to be dysfunctional, suggesting the presence of multi-factorial immune suppressants such as the PD-1/PD-L pathway. Aim: We aim to demonstrate that PD-1/PD-L signalling is up-regu- B302 lated during viral infections and their suppressive function predisposes to secondary bacterial pneumonia. RelB-DEFICIENT DENDRITIC CELLS PROMOTE Methods: We employed a mouse model of viral predisposition to THE DEVELOPMENT OF SPONTANEOUS bacterial pneumonia by infecting mice with pneumonia virus of mice ALLERGIC AIRWAY INFLAMMATION IN MICE (PVM), and at the time of viral clearance infected them with a mouse- adapted strain of Streptococcus pneumoniae (Spn, D39). PD-1/PD-L Philip M. Hansbro1,3, Prema M. Nair1, Malcolm R. Starkey1, Tatt J. expression and immune cell profiles of the lung were analysed by Haw1, Roland Ruscher2, Muralidhara R. Maradana2, Ranjeny flow cytometry. The role of PD-1 signalling was determined by Thomas2,3, Brendan O’Sullivan2,3 administration of a PD-1 blocking antibody. Results: Bacterial titres in the lungs of a secondary Spn infection were 1School of Biomedical Sciences and Pharmacy, Priority Research increased compared to a primary Spn infection alone. Cellular profiles Centre for Asthma and Respiratory Disease, Faculty of Health and of mice with a prior PVM infection demonstrated elevated PD-1 and Hunter Medical Research Institute, University of Newcastle, NSW, PD-L1 expressing cells in response to Spn infection. Blocking PD-1 Australia; 2University of Queensland Diamantina Institute, signalling reduced bacterial titres in the lung during secondary Spn Translational Research Institute, Princess Alexandra Hospital, infection. Woolloongabba, QLD, Australia; 3Authors contributed equally Conclusion:: A prior PVM infection induces an immunosuppressive environment in the lungs during Spn infection that may hamper the Background: RelB is a member of the NF-jB family that is essential hosts’ ability to clear bacterial infection. We have also demonstrated a for dendritic cell (DC) function and inflammatory responses. How- pivotal role of PD-1 signalling in the clearance of a secondary bac- ever, the contribution of RelB to the development of allergic airway terial infection. This study also highlights a potential role of PD-1 inflammation (AAI) is unknown. blocking antibody as a novel therapeutic approach for secondary Objectives: To determine the role of RelB in the spontaneous bacterial pneumonia. development of AAI that is independent of allergen exposure and to identify the importance of RelB in DCs. Methods: Two strains of naı¨ve RelB-deficient (-/-) mice were used to assess features of AAI (a targeted knockout and a mutant expressing an MHC transgene). To B304 identify the importance of RelB in DCs, splenic CD11c+ DCs from ANTIFIBROGENIC EFFECT OF DOMINANT- RelB-sufficient mice were adoptively transferred intravenously into NEGATIVE INHIBITOR OF SOLUBLE TNF XPRO RelB-/- recipients and features of AAI were assessed. Results: Both strains of naı¨ve RelB-/- mice had increased total 1595 ON EXPERIMENTAL SILICOSIS IN MICE leukocytes, eosinophils, lymphocytes and neutrophils in bron- 1 1 choalveolar lavage fluid (BALF) and more severe parenchymal Patricia MR Silva , Bianca T. Ciambarella , Ana Carolina S. 1 1 2 inflammation compared to their respective wild-type (RelB+/+) and Arantes , Yago AJ Sa , David E. Szymkowski , Marco Aurelio 1 heterozygous (RelB+/-) controls. RelB-/- mice also had increased Martins chemokines (CCL-2, -3, -4, -5, -11, -17, CXCL9, CXCL10, 1 2 CXCL13); Th2-associated cytokines (IL-4 and -5); serum IgE; mucus Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; Xencor, secreting cells (MSCs); collagen deposition around the airways; and Monrovia, USA airway epithelial thickening. Transfer of RelB-sufficient DCs to -/- Tumor necrosis factor (TNF) is a multifunctional cytokine known to RelB mice decreased total leukocytes and eosinophils in BALF; regulate inflammation, which is presented on the cell surface as parenchymal inflammation; chemokines (CCL-2, -3, -4, -5, -11, -17, transmembrane TNF (tmTNF), acting to promote juxtacrine signaling, CXCL9, CXCL10, CXCL13); Th2-associated cytokines (IL-4, -5, - and soluble (solTNF), acting in a paracrine fashion. We demonstrated 13, -25, -33, TSLP); serum IgE; numbers of type-2 innate lymphoid that mice depleted from TNFR1 gene and treatment with thalidomide cells, myeloid DCs and cd T cells; numbers of MSCs; collagen markedly inhibited lung function alterations and tissue fibrosis in deposition and airway epithelial thickening. mice stimulated with silica particles, indicating that TNF plays an Conclusions: These findings demonstrate the importance of RelB in important role in such dysfunction. A previous report described the DCs in controlling AAI and a strategy towards DC-targeted therapy invention of a novel class of anti-TNF biologics (DN-TNFs), which for AAI. selective inhibits the soluble form of TNF (Zalevsky J, J. Immunol,

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179, 1872, 2007). In this study, we aimed to investigate the effect of Animal weight was monitored weekly, inflammatory and anti-ox- the XPro 1595 on the experimental model of silicosis in mice. The idative markers as well as the mean linear intercept (Lm), a measure monoclonal antibody against TNF infliximab was used for compari- of alveolar space area, were assessed 24 h after the last exposure to son. Male Swiss-Webster mice were intranasally instilled with silica CS. Exposure to CS caused a reduction of about 2 g throughout the particles, and then treated therapeutically with XPro 1595 (1.25 and experiment in both WT and ACKR2-/- mice. Surprisingly, 10 mg/kg, i.p.) and infliximab (1.25 mg/kg) on days 7, 14 and 21 ACKR2-/- mice showed a significant increase in Lm values both in post-silica. The analyses were performed 24 h after the last dose and AA condition (from 31.6 ± 0.4 to 35.4 ± 0.7 lm; p \ 0.001) and CS included: (1) lung function and airways hyperreactivity to metha- condition (from 41.8 ± 0.5 to 44.5 ± 0.4 lm; mean ± SEM, choline (invasive plethysmography—Finepointe Buxco System), (2) p \ 0.05). ACKR2-/- mice also exhibited a substantial decrease in morphology and morphometry (H&E and Picrus sirius), (3) collagen catalase levels following AA exposure (from 9.9 ± 0.1 to 1.9 ± 2.1 deposition (Sircol method); iv) cytokine generation (ELISA) and U/mg ptn; p \ 0.001), suggesting the existence of a COPD predis- NFkB expression (western blot). We showed that stimulation with posing phenotype. However, no significant catalase changes were intranasal silica led to an increase in the basal levels of lung resistance seen in CS mice despite tendency (from 2.8 ± 0.2 in WT to 1.3 ± 0.1 and elastance as well to airways hyper-reactivity to aerosolized U/mg ptn in KO mice, mean ± SEM). Following CS exposure, lung methacholine. A marked tissue leukocyte infiltration accompanied by MPO levels showed a twofold increased (p \ 0.05) in ACKR2-/- fibrosis (collagen deposition and granuloma formation) was also mice compared to WT mice. Finally, though the levels of IL-17, TNF- noted in the lungs of silicotic mice. Increased levels of cytokines a and CCL3L1 were slightly higher in the lung of ACKR2-/- as (MIP-1alpha, MIP-2, KC, MCP-1, IL-1beta and TGF-beta) and NF- compared to WT mice grown in ambient air, levels of these inflam- kB activation were detected in lungs of silica-challenged mice. matory mediators were similarly increased in the lung of WT and Therapeutic administration of XPro 1595 and infliximab significantly ACKR2-/- mice exposed to CS. These findings suggest that the inhibited the decreased lung function and hyper-reactivity as well as atypical chemokine receptor ACKR2 prevents the development of fibrosis, including augmentation in collagen deposition and granu- pivotal COPD features, including chronic inflammation and loma formation in silica-stimulated mice. Cytokine generation and emphysema. NF-kB activation were also sensitive to treatment with XPro 1595 and Financial support UE FP7- 2007-2013—Grant agreement infliximab. Altogether our findings show that treatment with XPro HEALTH-F4-2011-281608. 1595 and infliximab effectively inhibited some critical features of silicosis, including alteration of lung function and fibrogenic response, reinforcing the idea that TNF is implicated in this disease. They also indicate that inflammation in mouse silicosis seems to be B306 primarily driven by sol TNF. COMPARATIVE PROTEOMIC ANALYSIS OF Financial support FIOCRUZ/CNPq/FAPERJ (Brazil). SURVIVAL AND NON-SURVIVAL SEPSIS PATIENTS USING iTRAQ-BASED ABSOLUTE QUANTITATION B305 1 1 1 ROLE OF THE ATYPICAL CHEMOKINE Narendra K. Sharma , Alexandre K. Tashima , Milena KC Brunialti , Flavia R. Machado2, Eliezer Silva3, Otelo Rigato4, Reinaldo RECEPTOR ACKR2 ON CIGARETTE SMOKING- Salomao1 INDUCED COPD IN MICE 1Escola Paulista de Medicina, Universidade Federal de Sao Paulo; Diego S. Coutinho1, Tatiana Paula T. Ferreira1, Davidson F. Dias1, Brazil; 2ICU, Hospital Sa˜o Paulo, Escola Paulista de Medicina, Ana Carolina S. Arantes1, Bianca CIAMBARELLA1, Magda F. Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; 3ICU, Serra1, Andrey J. Fernandes1, Antonio Gabriel S. Silva1, Patricia M R Hospital Israelita Albert Einstein, Sa˜o Paulo, Brazil; 4ICU, Hospital Silva1, Massimo Locati2, Marco Aure´lio Martins1 Sirio Libanes, Sa˜o Paulo, Brazil Sepsis is a systemic inflammatory response caused by infection whose 1Laboratory of Inflammation, Oswaldo Cruz Institute, FIOCRUZ, Rio biochemical mechanisms are poorly known. The early detection of de Janeiro, Brazil; 2Humanitas Clinical and Research Center and sepsis remains a great challenge for clinicians because no single University of Milan, Milan, Italy biomarker capable of its reliable prediction, hence, delayed diagnosis Active cigarette smoking is the main risk factor for chronic frequently undermines treatment efforts, thereby contributing to high obstructive pulmonary disease (COPD) development. ACKR2 (pre- mortality. There are several source of infection reported for devel- viously known as D6) is an atypical chemokine receptor implicated in opment of sepsis such as pneumonia, abdominal infection and urinary sequestering and internalization of chemokines and resolution of tract infection etc. In the present study, we compared the plasma inflammation. Remarkably, ACKR2 is highly expressed on human proteome profile of survival (N-20 at day 0 and N-17 after day 7) and alveolar macrophages of COPD patients. Although its non-redundant non-survival (N-13 at day 0 and N-9 after day 7) patients of severe role in chronic inflammatory conditions is well recognized, the bio- sepsis/septic shock secondary to community acquired pneumonia with logical role of ACKR2 in COPD remains elusive. This study was healthy volunteers (N-23), to understand molecular dynamics of undertaken in order to assess the impact of ACKR2 on main patho- disease. In brief, we depleted high abundant proteins from represen- logical changes of COPD triggered by cigarette smoke inhalation in tative groups and labelled with iTRAQ 8plex kit. Typically 40 mice. Thirty-two healthy adult female C57Bl/6 mice were equally fractions were collected by SCX fractionation and analysed by LC– distributed into 4 groups: (1) WT mice exposed to ambient air (AA), MS/MS (Synapt G2 HDMS, Waters). (2) WT mice exposed to cigarette smoke (CS), (3) ACKR2-/- mice In this study, we identified total 932 proteins (NCBI database) exposed to AA and (4) ACKR2-/- mice exposed to CS. Animals with 1 % FDR corresponding to several biological pathways such as were exposed to CS 7 days per week for 12 weeks. The exposure acute inflammatory response, inflammatory response reactive oxygen procedure was based on 3 cigarette-smoking cycles per day, 4 species and oxidative stress etc. which are altered at the protein level cigarettes per cycle and an inhalation time of 6 min per cigarette. when compared to healthy volunteers as well as between survival and

123 Inflamm. Res. S225 non-survival sepsis patients. We also identified some known marker several parameters of inflammation, ameliorated histological score, protein such as C-reactive protein (CRP) in corroborating previous without interfering with bacterial load or survival. Partial blockade of studies. We are reporting the first protein level comparison between pulmonary inflammation may be beneficial for the murine host, survival and non-survival sepsis patients in time interval progression mainly when combined to standard antibiotics treatment. Mechanis- of disease with single source of infection. This study may provide tically, combined treatment improved infection outcome by insight for diagnosis, prognosis, and novel therapeutic targets in balancing/reducing inflammation and increasing expression of the sepsis. pro-resolving and tissue protective protein AnxA1 with bacterial phagocytosis, restoring lung homeostasis. In addition, AnxA1 may play an important role during pneumococcal pneumonia revealing a new treatment strategy. B307 MODULATION OF INFLAMMATORY RESPONSE DURING STREPTOCOCCUS PNEUMONIAE B308 INFECTION PREVENTS LUNG INJURY AND PHOTOTHERAPY (PHT): ATTENUATION OF IMPROVES BACTERIA CLEARANCE IN MICE CHOLINERGIC HYPERREACTIVITY, b2- ADRENERGIC HYPORRESPONSIVENESS AND Luciana P. Tavares1,4,3, Cristiana C. Garcia2, Juliana P. Vago1,6, Celso M. Queiroz-Junior1,7, Bruna A. David1,7, Milene A. Rachid1,8, MITOCHONDRIAL mRNA EXPRESSION IN RAT Remo C. Russo1,5, Patrı´cia R. Martins2, Lirlaˆndia P. Sousa1,6,3, BRONCHI SEGMENTS AND PULMONARY TISSUE 1,4,3 Mauro M. Teixeira IN E. COLI LIPOPOLYSACCHARIDE-INDUCED AIRWAY INFLAMMATION BY A NF-kB, AP-1 AND 1Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2Instituto Oswaldo Cruz, RJ, Brazil; 3Laborato´rio de MITOCHONDRIAL DEPENDENT MECHANISM Imunofarmacologia; 4Departamento de Bioquimica e Imunologia; 5Departamento de Fisiologia; de Biologia Celular; 8Departamento de Fla´via Mafra de Lima1, Renata Kelly Palma2, Niels Olsen Saraiva Patologia Camara1 Introduction: Community-acquired pneumonia (CAP), caused mainly 1University of Sa˜o Paulo, Av Prof. Lineu Prestes 1730, Cidade by Streptococcus pneumonia, is a major global health problem and a Universita´ria, Sa˜o Paulo, Brazil; 2Rehabilitation Sciences leading cause of morbidity and mortality among children and the Departament, Centro Universita´rio Nove de Julho, Uninove. Rua elderly. Despite the use of antibiotics, the incidence of pneumococcal Vergueiro, SP, Brazil infections did not change. Inflammation is crucial to control dis- semination of bacteria; however, it could cause tissue damage and Background and objective: It is unknown if the decreased ability to high mortality during pneumoccocal infection. Therefore, our ques- relax airways smooth muscles in acute respiratory distress syndrome tion was whether modulating the inflammation by using the PDE4 (ARDS), can be influenced by low level laser therapy irradiation. In inhibitor rolipram (ROL) could improve the outcome of the host this context, the present work was developed in order to investigate if during infection. PhT could reduce dysfunction in inflamed bronchi smooth muscles Methods and Results: Male Balb/C mice were infected intranasally (BSM) in rats. with S. pneumoniae serotype 3 (ATCC 6303, 104 CFU) and pre- Methods: A controlled ex vivo study was developed where bronchi treated with ROL (6 mg/kg) or vehicle (V). ROL treatment decreased from Wistar rat were dissected and mounted in an organ bath neutrophil recruitment in lungs and airways, and decreased cytokines apparatus with or without a TNF-a. In the experimental assays with levels in bronchoalveolar lavage fluid (BALF). Number of bacteria in the participation of TNF-a, the reactivity of BSM to cholinergic BALF and lethality were similar in both groups. However, ROL- agonist or to b2-adrenergic agonist was analyzed. The cAMP level treated animals presented better lung pathology scores than V group. was also investigated in BSM bathed or not with TNF-a and Then, immunomodulatory actions of ROL were investigated in treated or not with PhT. In other series of experiment, the BSM combination with the antibiotic ceftriaxone (CFX). Bacterial counts from rat were incubated with LPS in order to investigate the PhT were diminished by CFX, but not by ROL treatment. Combined effect on TNF-a mRNA expression in BSM. Mechanical properties treatment reduced even more bacteria counts by enhancing macro- of lungs were measured (QTAS HORAS) after LPS injection. Lung phage phagocytosis. Delayed treatment with either CFX or ROL did resistance (RL) and elastance (EL) were computed by linear not prevent neutrophil infiltration into lungs or TNF-a, CXCL1 and regression fitting of recorded signals during mechanical ventilation CCL2 increased levels, whereas combined treatment strikingly (tracheal pressure, flow and volume). Static (Est) and dynamic reduced neutrophil numbers in the lungs and airways and cytokine (Edyn) elastances were obtained by the end-inspiratory occlusion levels. ROL or combined treatment markedly improved overall lung method. injury and dynamic compliance of infected mice. Annexin A1 Results: TNF-a caused cholinergic hyperreactivity and b2-adrenergic (AnxA1), is an anti-inflammatory and tissue protective protein bio- hyporesponsiveness when compared to respective controls. PhT logically active in the intact form. Whereas cleaved (inactive) AnxA1 administered perpendicularly to a point in the middle of the dissected band was more evident than intact AnxA1 band in V group at 84 h of bronchi with a wavelength of 655 nm and a fluence of 2.6 J/cm2, infection, ROL, but not CFX treatment partially prevented this partially decreased BSM hyperreactivity to cholinergic agonist, cleavage. Combined treatment greatly reduced AnxA1 cleavage. In restored BSM relaxation to isoproterenol and reduced the TNF-a addition, AnxA1 KO mice had a higher lethality than WT mice and a mRNA expression. The cAMP level diminished by TNF-a in com- single dose of an active portion of Annexin A1 (Ac2-26–10 mg/kg) parison with isoproterenol was restored by PhT. An NF-kB antagonist could partially rescue mice from lethality and decrease inflammation blocked the PhT effect on dysfunction and cAMP level in inflamed related to infection. BSM. The RL and EL were higher after LPS injection in compared to Conclusion: Inflammation was an important determinant of morbidity control and laser treatment reduces these values. Similar increases after infection with S. pneumoniae. Pretreatment with ROL decreased were induced by LPS injection in Est and Edyn.

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Conclusion:: We show that low level laser therapy (LLLT), can firmed by surface plasmon resonance. Our results reveal that, in decrease hyperreactivity in isolated organ and improve the pulmonary addition to Hsp90 modulation, gedunin acts as a competitive inhibitor ventilation (in vivo) after QTAS HORAS ventilation. Our results of LPS, blocking the formation of TLR4/MD-2/LPS complex. shown that this effect is mediated by MyD88, IRAK1, and this treatment is efficient to activate Cyt c, TFAM, NFR2 and increase mitochondrial membrane potential. B310 VENETOCLAX (ABT-199), A POTENT AND Small Molecule Therapeutics SELECTIVE BCL-2 INHIBITOR, IS EFFICACIOUS for Inflammatory Diseases IN MOUSE MODELS OF LUPUS NEPHRITIS AND REDUCES HUMAN LYMPHOCYTE LIFESPAN IN VITRO B309 GEDUNIN BINDS TO MD-2 AND IMPAIRS LPS- Stephen Clarke1, Stuart Perper1, Kristie Grebe1, Annette Schwartz1, INDUCED TLR4 SIGNALING IN MACROPHAGES Christian Goess1, Liz O’Connor1, Dawna Hartman1, Susan Westmorland1, Candace Graff1, Andrew Souers1, Joel Leverson1, Perla V. Borges1, Katelim H. Moret1, Clarissa M. Monteiro3, Steven Elmore1, Lisa Olson1, Li Chun Wang1 Franklin S. Silva4, Carlos R. Alves4, Ernesto Caffarena5, Paulo R. Batista5, Maria das Grac¸as Henriques1,6, Patrı´cia Pacheco1, Carmen 1AbbVie Bioresearch Center Worcester, MA, USA1; AbbVie, Inc. Penido1,6 North Chicago, IL, USA Venetoclax (ABT-199), a Potent and Selective BCL-2 Inhibitor, is 1Laborato´rio de Farmacologia Aplicada, Farmanguinhos, Fundac¸a˜o Efficacious in Mouse Models of Lupus Nephritis and Reduces Human Oswaldo Cruz, Rio de Janeiro, Brazil; 3Laborato´rio de Lymphocyte Lifespan in vitro Imunofarmacologia, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Stephen Clarke1, Stuart Perper1, Kristie Grebe1, Annette Cruz, Rio de Janeiro, Brazil; 4Laborato´rio de Biologia Molecular de Schwartz1, Christian Goess1, Liz O’Connor1, Dawna Hartman1, Susan Doenc¸as Endeˆmicas, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Westmoreland1, Candace Graff1, Andrew Souers2, Joel Leverson2, Cruz, Rio de Janeiro, Brazil; 5Programa de Computac¸a˜o Cientı´fica, Steven Elmore2, Lisa Olson1 and Li Chun Wang1 Grupo de Biofı´sica Computacional e Modelagem Molecular, 1Abbvie Bioresearch Center, Worcester, MA, United States; Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil; 6Centro de 2AbbVie, Inc. North Chicago, IL, United States Desenvolvimento Tecnolo´gico em Sau´de, CDTS/INCT, Fundac¸a˜o Proteins in the BCL-2 family are key regulators of apoptosis, or Oswaldo Cruz, Rio de Janeiro, Brazil programmed cell death. We have evaluated the effects of venetoclax Recognition of bacterial lipopolysaccharide (LPS) by innate immune (ABT-199), a highly potent and orally available BCL-2 selective system is mediated by the CD14/TLR4/MD-2 complex. In the present inhibitor, in two mouse models of lupus nephritis and in human cells study, we have investigated the modulatory effect of gedunin, a in vitro. In the mouse models (spontaneous and IFN alpha-induced limonoid from species of the Meliaceae family described as Hsp90 NZBW F1), venetoclax treatment dose-dependently reduced the inhibitor, on LPS-induced response in immortalized murine macro- incidence of severe proteinuria and prolonged survival in both phages. The pretreatment of wild type (WT) macrophages with models compared to vehicle controls and attenuated glomeru- gedunin (0.01–10 mM, non-cytotoxic concentrations) inhibited LPS lonephritis, tubular dilatation, immune cell infiltrates and IgG (50 ng/mL)-induced calcium influx, TNF-a and nitric oxide (NO) deposition in the kidney. Venetoclax mediated a significant reduc- production, in a concentration-dependent manner. The selective effect tion in the numbers of splenic T cells but conferred a preferential of gedunin onMAL/MyD88- and TRAM/TRIF-dependent signaling reduction in select B cell subsets. Interestingly, venetoclax did not pathways was further investigated. The pretreatment of WT, TRIF impair the number of CD138+ long-lived plasma cells in the bone KO and MAL KO macrophages with gedunin (10 lM) significantly marrow, which was consistent with unaltered circulating anti- inhibited LPS (50 ng/mL)-induced TNF-a, IL-1b and IL-6 produc- dsDNA titers in these animals. Venetoclax efficacy also correlated tion, within 6 h and 24 h, suggesting that gedunin modulates a with a dose-dependent reduction of lymphocytes in peripheral blood common event between both signaling pathways. Furthermore, of NZBWF1 mice. Consistent with these findings, human lympho- gedunin (10 lM) inhibited LPS-induced PGE2 production, cytes from both healthy donors and systemic lupus erythematosus cyclooxygenase-2 (COX-2) expression and NFjB translocation into (SLE) patients treated with venetoclax in vitro have reduced lifes- the nucleus of WT macrophages, demonstrating a wide range effect of pan. Taken together, these data underscore the essential role of this chemical compound. In addition to the ability to inhibit LPS- BCL-2 in the pathogenesis of lupus and support further exploration induced pro-inflammatory mediators, gedunin also triggered antiin- of selective BCL-2 inhibition in autoimmune diseases such as SLE/ flammatory factors. The pretreatment with gedunin induced the Lupus Nephritis. production of IL-10 and the expression of heme oxigenase-1 (HO-1) Disclosures: All authors are employees of AbbVie. The design, and heat shock protein (Hsp)70 in macrophages stimulated or not with study conduct, and financial support for this research were provided LPS. In silicomodeling studies revealed that gedunin efficiently by AbbVie. AbbVie participated in the interpretation of data, review, docked into the MD-2 LPS binding site, a phenomenon further con- and approval of the publication.

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B311 was collected. For pulmonary model of infection, mice were pre- CHARACTERIZATION OF A SMALL MOLECULE treated with BCP (p.o.; 50 mg/kg) 1 h before and 24 h after intranasal instillation (i.n.) of BCG (1x106 CFU/lung). The effects were eval- IRAK4 KINASE INHIBITOR FOR THE uated 48 h after infection in the bronchoalveolar lavage (BAL). The TREATMENT OF AUTOIMMUNE AND total and differential leukocyte migration were analyzed in both INFLAMMATORY DISEASES models (PW and BAL) using May Grunwald-Giemsa staining or by flow cytometry. Cytokine production was assessed by ELISA. All Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy protocols were approved by the Fundac¸a˜o Oswaldo Cruz Animal Frances, Stacey Siu, Sylvia Braselmann, Don Payan, Darren Welfare Committee (LW-43/14.). Adhesion and chemotaxis were McMurtrie, Ryan Kelley, Thilo Heckrodt, Rose Yen, Yan Chen, Hui performed in vitro. The stimulus with BCG induced an increase of Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor total leukocytes, predominantly neutrophil influx in both models. The oral pretreatment with BCP (50 mg/kg) decreased the numbers of Rigel Pharmaceuticals, South San Francisco, CA, USA total leukocyte in the pleural cavity and in the lungs, showing a remarkable effect on neutrophil migration, 45 and 97 % of inhibition, IRAK4 is a key protein kinase in immune cells. It is responsible for respectively. In pleurisy, BCP was able to reduce the levels of IL-12 initiating MyD88-dependent signaling from most Toll-Like Receptors (21 %) and NO (54 %). In the lungs, BCP inhibited IL-10 (99 %), (TLR) and Interleukin-1 Receptors (IL-1R), resulting in downstream MCP-1 (100 %), IFN-c (98 %) and IL-12 (97 %) release. GP1a and production of pro-inflammatory cytokines. Hence, inhibitors of Dex also reduced the neutrophil migration and NO production in PW. IRAK4 kinase activity represent valuable therapeutic tools to treat Flow cytometry analysis revealed that BCP reduced integrin CD11b/ cytokine-driven autoimmune and inflammatory diseases. Through CD18 expression on Ly6G positive cells in PW (neutrophils). In cell-based screening, we identified potent small molecule IRAK4 addition, neutrophils pretreated with BCP (10 lM) reduced adhesion kinase inhibitors that block TLR4- and IL-1R-induced cytokine pro- to endothelial cells previously stimulated with mTNF-a (10 ng/mL) duction with potencies \100 nM. Our inhibitors exhibit good and chemotaxis toward LTB4 (10–7 M). These data suggests that selectivity against a broad panel of kinases. In vivo, our lead com- BCP modulates early inflammatory events in BCG infection by pound decreases serum IL-6 in an acute mouse model of IL-1beta- inhibition of neutrophil’s migration to inflamed site. induced cytokine release, demonstrating excellent pharmacokinetics Financial support CAPES; Faperj; CNPq. properties. In addition, our IRAK4 inhibitor reduces the paw swelling in the chronic rat model of collagen-induced arthritis, as well as the clinical score and overall inflammatory phenotype of mice in the imiquimod-induced psoriasis model. The in vitro and in vivo char- B313 acterization of our lead candidate is promising and confirms IRAK4 DISCOVERY OF SELECTIVE RORc INVERSE as an attracting therapeutic target for the management of cytokine- AGONISTS AND DEMONSTRATION OF EFFICACY driven diseases. IN INFLAMMATORY DISEASE MODELS

Ravi K. Ujjinamatada, Ravi D. Krishna Babu, Kavitha Nellore, B312 Samiulla S. Dodheri, Charamanna K B, Subhendu Mukherjee, b-CARYOPHYLLENE MODULATES NEUTROPHIL Kancharla BabuRao, Wesley Roy Balasubramanian, Sanjay B. MIGRATION IN INFLAMMATORY REACTION Rathod, Anirudha Lakshminarasimhan, Narasimha K. Rao, Anuradha Ramanathan, Natarajan Mahalingam, Girish A R, Shekar Chelur, INDUCED BY MYCOBACTERIUM BOVIS-BCG Susanta Samajdar, Chetan Pandit, Murali Ramachandra

Magaiver Andrade-Silva1, Luana B. Correa1, Andre´ Cande´a1, Simone Aurigene Discovery Technologies Limited, Bengaluru, Karnataka C. Cavalher-Machado1, Elaine C. Rosas1, Maria das Grac¸as Objective: Th17 cells play a key pro-inflammatory role in a variety of Henriques1,2 autoimmune diseases including psoriasis, arthritis, lupus, multiple sclerosis and asthma. The nuclear hormone receptor RORc controls the differentiation of Th17 cells and expression of IL-17. Aurigene’s 1Laboratory of Applied Pharmacology, Farmanguinhos, Fundac¸a˜o objective is to develop RORc inverse agonists for treatment of Oswaldo Cruz, FIOCRUZ, RJ, Brazil; 2National Institute for Science autoimmune disorders. and Technology on Innovation on Neglected Diseases (INCT/IDN), Methods: RORc inverse agonists were designed using a combi- Center for Technological Development in Health (CDTS); Fundac¸a˜o nation of approaches including structure and knowledge based Oswaldo Cruz, FIOCRUZ, RJ, Brazil methods. Compounds were screened in a RORc radio-ligand binding assay using 3H 25-Hydroxycholesterol, as well as in a cell based b-caryophyllene (BCP) is a natural sesquiterpene found in essential reporter assay to demonstrate inverse agonism. Selected compounds oils from a variety of plant species and it was described as a natural were screened against RORa as well as other nuclear receptors to agonist of cannabinoid 2 (CB2) receptor. Previous studies have evaluate selectivity. Crystal structure of RORc in complex with demonstrated an anti-inflammatory effect of BCP in different known inverse agonists as well as novel compounds, were solved. inflammation models. However, there are no reports of its action on Th17 differentiation assay was developed using primary mouse/hu- inflammatory process induced by mycobacteria. The aim of this study man CD4+ ve T-cells to determine functional effect of the was to evaluate the effect of BCP on neutrophils migration in an compounds. DMPK profile of potent compounds was determined to inflammatory reaction induced by M. bovis-BCG. To evaluate the support evaluation in animal models. Efficacy of lead compounds was effects of BCP on pleurisy, mice received an intrathoracic injection evaluated in LPS induced IL-17 model as well as in collagen induced (i.t.) of BCG (4x105 CFU/cavity) 1 h after the oral pretreatment (p.o) arthritis model. with BCP (50 mg/kg), dexamethasone (by intraperitoneal injection, Results: Lead compounds have demonstrated good activity i.p.;Dex, 2 mg/kg) or synthetic selective CB2 agonist, GP1a (\100 nM) in reporter assay. Co-crystal structure of RORc with (i.p.;10 mg/kg). After 24 h of the injection, the pleural wash (PW) compounds clearly showed the mode of binding. Selected compounds

123 S228 Inflamm. Res. demonstrated [100 fold selectivity against RORa as well as other Conclusions: Our results suggest that the obstruction progression in nuclear receptors. Compounds from multiple series have shown sig- smokers was related to a decrease in inflammation regulatory activity nificant inhibition (\100 nM) of IL-17 release from differentiated on the small airways, mediated by T regulatory cells, leading to less Th17 cells. Lead compounds have shown optimal physicochemical expression of interleukin 10 (IL-10) and increased interleukin 17 (IL- profile including good solubility and high free fraction, leading to 17) production. Also, we verified that such inflammation regulatory excellent pharmacokinetic profile in mice. Efficacy of lead com- activity showed differences between BALTs and small airways. pounds has been demonstrated in LPS induced IL-17 model as well as Supported by FAPESP and Instituto de Laborato´rios de Investigac¸a˜o in collagen induced arthritis model. Me´dica-HC/FMUSP Conclusions: We have identified structurally diverse small molecule inverse agonists of RORc and have demonstrated efficacy in relevant disease models. B315 IMPACT OF REGULATORY T CELLS ON T Regulatory Cells CHEMOKINE RESPONSE IN TUBERCULOSIS

B314 Divya Kamboj, Dipendra Kumar Mitra, Anant Mohan THE FOXP3 T REGULATORY CELLS IN All India Institute of Medical Sciences, New Delhi, India OBSTRUCTIVE AND NON OBSTRUCTIVE SMOKERS: A DESCRIPTIVE STUDY IN AIRWAYS Purpose: Regulatory T cells dampen the inflammatory immune response in various diseases including tuberculosis. In tuberculosis, AND LYMPHOID FOLLICLES local immune response is an important determinant in shaping the disease manifestation. A robust response at the disease site, results in Davi S. Sales, Ivy A. Zanchetta, Juliana T. Ito, Raquel Annoni, effective granuloma formation containing the infection thus pre- Thais Mauad, Milton A. Martins, Fernanda DTQS Lopes venting its dissemination. Utilizing various mechanisms, regulatory T cells suppress protective effector T cell response. Granuloma for- University of Sa˜o Paulo, Sa˜o Paulo, Brazil mation requires local production of Th1 associated chemokines facilitating the effector T cells at the pathological site. We previously Background: The COPD progression has been associated with a reported higher levels of Treg cells and IL-10 at the local disease site persistent inflammatory process into the airways walls characterized in tuberculosis. Here, we envisage that locally enriched regulatory T by an innate and adaptive inflammatory immune cells infiltration cells may alter the tissue chemokine response thereby influencing the which can be organized into bronchus-associated lymphoid tissue effector T cell influx. This may be a mechanism underlying the local (BALT) or may be dispersed as lymphoid clusters lacking organiza- immune deficit observed in tuberculosis patients. tion (iBALT) in parenchyma. Smoking is configured as the main risk Materials and methods: In this study, we expanded regulatory T cells factor for COPD development; however, not all smokers develop in vitro and examined the effect of Mycobacterium tuberculosis clinically significant COPD, which suggests that there are individual specific T reg expansion on chemokines like MIP1-a and MIP1-b using intrinsic factors involved in such disease progression. Although polychromatic flow cytometry and in vitro cell culture techniques. lymphoid follicles are a feature of the adaptive immune response and Results: We observed that T reg cells from tuberculosis patients could have been described in COPD patients, there are few studies be expanded using All Trans Retenoic Acid (ATRA) in vitro. After T describing the inflammatory cells types in such structures and its reg cells expansion, it was found that there was decreased production differences between obstructive and non obstructive smokers. of MIP1-a and MIP1-b by both monocytes and lymphocytes. Pres- Aims: To compare the inflammatory profile associated to the regu- ence of ATRA also led to increased proliferation of T reg cells latory adaptive immune response in obstructive and non obstructive subsequently resulting in heightened IL-10 and TGF-b production. smokers. Blocking of IL-10 and TGF-b in culture could rescue these Methods: Small and large airways, BALTs and iBALTs were assessed chemokines which influence the Th1 cytokines. in surgically resected lung tissue from patients, divided into three Conclusion: Here, we show that T reg cells derived from tuberculosis groups: Control (n = 21), Obstructive Smokers (COPD) (n = 17) and patients can inhibit the production of Mycobacterium tuberculosis Non Obstructive Smokers (FNO) (n = 22). CD4+ and CD8+ T cells, specific chemokines required for the recruitment of Th1 effector T FOXP3 regulatory T (Treg) cells, and the expression of IL-10 and IL- cells. We propose that this may lead to failure of effector T cell homing 17 interleukins were evaluated by immunohistochemistry and mor- and elicitation of optimal local immunity in tuberculosis. Our results phometric analysis. indicate a novel role of in vitro expanded T reg cells in modulating the Results: We observed an inflammatory process characterized by an chemokine response resulting in weakened recruitment of effector T increase in CD4+ and CD8+ lymphocytes in small and large airways, cells. This impedes the granuloma formation leading to failure of BALT and iBALT in smokers; however the higher values were immune containment and dissemination of disease. detected in small airways of COPD individuals (p \ 0,001). In addition, it was observed a decrease in regulatory T cells density only in small airways of this group (p \ 0,001), with a consequent B316 decrease in IL-10 expression in small and large airways (p \ 0,001). RESTORATION OF TREG CELL FUNCTION WITH However in BALT we observed a different response, with an increase in Treg cells density in COPD group, and in iBALT we observed an PD-L1 FC PROTEIN IN RHEUMATOID ARTHRITIS increase in Treg cells only in FNO group without differences between the groups in IL-10 expression in both structures. Regarding the IL17 Kaustav Chowdhury1, Dipendra Kumar Mitra1, Uma Kumar1, expression, there was an increase in small and large airways Soumabha Das1, Prabin Kumar1, Uma Kanga1, Jaydeep Chaudhuri2, (p \ 0,001), and iBALTs (p \ 0,001) as the airflow obstruction Santu Bandyopadhaya2, Parasar Ghosh3, Ravi Kiran Basyal1, progressed. Maumita Kanjilal1

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1All India Institute of Medical Sciences, New Delhi, India; 2Indian excisional wound healing. 8 weeks-old C57Bl/6 mice (n = 5–7 ani- Institute of Chemical Biology, Kolkata, West Bengal; 3Rheumatology mals/group) received either IgG2 isotype control or anti-CXCR7 Center, IPGMER, Kolkata, West Bengal blocking antibodies (1.7 mg/mL, i.p.) 24 h before creation of dorsal excisional wounds and once a week till euthanasia. Wound area was Programmed death receptor -1 (PD-1) and its ligands (PD-L1, PD-L2) calculated after digital caliper measures and results were expressed as interaction is known to dampen effector T cell response. Rheumatoid closure percentage from original size. Treatment with anti-CXCR7 arthritis (RA) is a chronic autoimmune disease of joints resulting in impaired the wound closure when compared to isotype control. joint inflammation and bone erosion. Here, we investigated the Accordantly, wound epithelization, assessed in H&E-stained histo- importance of PD-1/PD-L1 interaction in modulating inflammatory logical sections, was reduced in anti-CXCR7-treated animals. pathogenic T cell response at the disease site of RA. We attempted to Interestingly, myeloperoxidase and N-acetyl-b-D-glucosaminidase study the cause of failure of locally enriched Treg cells in controlling activities, used to assess accumulation of neutrophils and macro- the inflammatory cytokine producing cells. We demonstrate that lack phages, respectively, were marked increased in wounds of anti- of PD-L1 on synovial fluid derived machorphages (CD14+ cells) CXCR7-treated mice. However, levels of the chemokines CCL2/ incapacitates functionality of Treg cells and upon engaging PD-L1 MCP-1, a macrophage recruiter, or CXCL1/KC, a neutrophil recrui- can functionally resuscitate the Treg cells. We recruited active RA ter, were lower in wounds of anti-CXCR7-treated mice. Instead, patients (n = 15) for the study and isolated mononuclear cells from levels of CXCL12 and TNF-a were higher in wounds of anti-CXCR7- peripheral blood (PBL) and synovial fluid (SF) of the same individual treated mice than in control group. Similarly, and offering insight on patients. PD-1 and its ligand expression (PD-L1) were seen on Treg the evolutionary conservation of CXCR7 pathway, we observed that cells and macrophages (CD14+) by the use of flowcytometry. Anti anti-CXCR7-treated 3-day-old zebrafish larvae (1.7 mg/mL in culture inflammatory cytokine IL-10, TGF-b in Treg (CD4+ FoxP3+) and medium) presented increased cell recruitment to wound site. proinflammatory cytokines IFN-c, IL-17A, TNF-a in effector T cells Regarding vascularization, anti-CXCR7-treated mice showed reduced (CD4+) were identified with FACS based intracellular staining after blood supply and angiogenesis in the wounded area, as evaluated by treatment with PD-L1 Fussion chimeric protein (PD-L1 Fc) in pres- laser Doppler perfusion imaging and histology. Conversely, wounds ence of TCR engagement with anti-CD3 & anti-CD28. Our result of anti-CXCR7-treated mice exhibited greater accumulation of col- shows that synovial Treg cells are compromised in their IL-10, TGF-b lagen (Sirius Red histological staining) and scar tissue area, which production in-spite of local enrichment of PD-1+ Treg were associated with increased levels of TGF-b1 in wounds when (CD4+ FOXP3+) cells. Engaging PD-1-PD-L1 with PD-L1 Fc, Treg compared to control group. Therefore, we may point the impaired cells could produce higher level of IL-10 & TGF-b. On the other hand wound healing is associated with impaired epithelization and persis- such engagement decreased the frequency of synovial effector T cells tent accumulation of inflammatory cells and CXCL12 and TNF-a in producing inflammatory cytokines (IFN-c+, TNF-a+, IL-17A+) and the injured area, suggesting CXCR7 absent activity may be linked to proliferation of these cells (Ki67+). Interestingly blocking IL-10, chronicity. This scenery is also characterized by reduced angiogenesis TGF-b with specific antibody in similar condition(s) failed to reduce and increased scarring. Overall, our findings suggest CXCR7 is the number of proinflammatory cytokine producing T cells. This essential for controlling skin wound healing by modulating critical suggests a critical role of PD-1-PD-L1 pathway in controlling features such as inflammation, angiogenesis, and fibrogenesis after inflammation in RA to get resolution. injury.

Tissue Damage/Repair B318 THE DEVELOPMENT OF NEW THERAPY FACIAL B317 NERVE INJURY USING THE M2 MACROPHAGE CRITICAL ROLE OF CXCR7 IN MODULATING INDUCTION FACTORS INFLAMMATION, ANGIOGENESIS AND FIBROGENESIS IN SKIN INJURY IN MICE Fumiya Kano, Kohki Matsubara, Akihito Yamamoto

Alan S. Barbosa1,2, Leandro C. Freitas-Limas1,2,CamilaP.Almeida1,2, Nagoya University Graduate School of Medicine, Nagoya, Japan Luiza D.C. Lima1,2, Maria C.C. Canesso1,2,Ange´lica T. Vieira1,3, 2 4 4 Backgrounds: The divergent activation states of mono- Robson A.S. Santos , Daniela V. Rosa , Erika K. Sacramento , cyte/macrophage linages play central roles in the inflammatory Marco A. Romano-Silva4, Mauro M. Teixeira1,3, Charles C. Mackay5, 1,2 responses of the traumatic nerve injury. M1-like cells initiate Lucı´ola S. Barcelos inflammation by releasing high levels of pro-inflammatory cytokines,

1 2 glutamate, reactive oxygen species, and nitric oxide (NO). These Immunopharmacology group; Department of Physiology neurotoxic factors accelerate glial scar formation, neuronal cell death and Biophysics, Federal University of Minas Gerais, Belo Horizonte, and the retraction of damaged dystrophic axons. In contrast, M2-like Brazil; 3Department of Biochemistry and Immunology, Federal 4 cells counteract the pro-inflammatory M1 conditions and promote University of Minas Gerais, Belo Horizonte, Brazil; School tissue repair by secreting anti-inflammatory cytokines, phagocytizing of Medicine, Federal University of Minas Gerais, Belo Horizonte, 5 cellular debris, enhancing axonal elongation, and promoting the Brazil; Faculty of Medicine, Nursing and Health Sciences, Monash proliferation and differentiation of oligodendrocyte progenitor cells. University, Melbourne, Victoria, Australia We have identified a novel set of set of inducers for anti-inflammatory CXCL12/SDF-1a, a chemokine expressed in both homeostatic and M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) pathological conditions, and its main known receptor, CXCR4, have and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 been implicated in skin wound healing, although with controversial (ED-Siglec-9). MCP-1 and ED-Siglec-9 administration into the roles. The disparity may be related to a recently identified second high injured spinal cord induced M2-like macrophages and led to a marked affinity receptor, CXCR7; however, its role in wound healing was recovery of hindlimb locomotor function after SCI (Matsubara et al. never evaluated. Our aim was to elucidate the role of CXCR7 on The Journal of neuroscience, 35, 2452–2464, 2015).

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Objective: We examined the therapeutic effects of MCP-1 and ED- Financial support FAPESP (2012/21603-2) and CNPq (308144/2014- Siglec-9 in the treatment of the peripheral facial nerve injury. 7). Method: The rat facial nerve was resected 5 mm. Collagen scaffolds infiltrated with MCP-1 and ED-Siglec-9 were placed in the resected site. The facial nerve recovery was evaluated by whiskers’ behavior, histological analysis with electron microscope and the tracer analysis B320 for the restoration of the nerve circuit. THE WORSENING ASTHMA PROCESS DRIVEN Results: MCP-1/ED-Siglec-9 treatment restored the whiskers’ behav- BY BRONCHOALVEOLAR EPITHELIUM WITH ior, regenerated resected facial nerve and restored the nerve circuit. CO-EXPOSITION TO CIGARETTE SMOKE Conclusion:: MCP-1/ED-Siglec-9 may provide a novel strategy to treat the peripheral nerve injury. Thayse R. Bru¨ggemann1, Paula Fernandes1, Fernanda M. Bruni2,3, Mı´lton A. Martins1, Fernanda M. Arantes-Costa1

B319 ASSESSMENT OF PORCINE DERMIS SCAFFOLDS 1Laboratory of Experimental Therapeutics, LIM20 School AND THEIR BIOCOMPATIBILITY USING of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2 A MODEL OF XENOGENEIC IMPLANTATION Laboratory of Clinical Immunology and Allergy, LIM60 School of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3Institute for Investigation in Immunology (iii), INCT, Sa˜o Paulo, Brazil Sonia M. Oliani1, Kallyne KO Mimura1, Andreia R. Moraes1, Tahera Ansari2, Karin V. Greco2 Introduction: Recent genetic, structural and functional studies have identified the airway and lung epithelium as a key orchestrator of the immune response. The inability of the airway epithelium of asth- 1University of Sa˜o Paulo State (UNESP), Sa˜o Paulo, Brazil; matics to effectively defend the lung against normally innocuous 2University College London (UCL), London, UK inhaled agents suggests that the airway epithelial barrier of asthmatics is compromised. We designed this study to investigate the role of Off-the-shelf availability of tissue-engineered skin constructs, tailored bronchial epithelium against the exposition to cigarette smoke in a by different combinations of reagents to produce a highly preserved model of allergic asthma. biological matrix is often the only means to help patients suffering Methods: Male Balb/c mice (n = 8/group) of the OVA and skin damage. The challenge facing tissue engineering, however, is to OVA+ cigarette smoke (CS) groups were sensitized with two combine different reagents to preserve the structure and function of a intraperitoneal injections of ovalbumin (OVA, 20 lg) and alum complex mixture of proteins that forms the extracellular matrix (3 mg) on days 0 and 14, and were challenged with an aerosol of (ECM) and keep their functions to promote tissue regeneration. OVA (1 %, 30 min) on days 21, 23, 25 and 27. The CS and Therefore, this study assessed the composition of porcine dermal OVA + CS groups were exposed to cigarette smoke once a day (7 scaffolds and their biocompatibility in rats. The two types of methods cigarettes/session) for twelve consecutive days (from 16 to 27). CS (1 and 2) used to produce dermal scaffolds showed to be efficient not and Control (SAL) groups received saline and alum intraperitoneal only in removing cellular material and debris from dermal scaffolds injection and were challenged with saline 0.9 %. Twenty-four h after but also in preserving the ECM components (glycosaminoglycans and the last challenge, we evaluated the production of cytokines (IFN-c, collagen). Histological examinations and analysis by SDS-PAGE IL-5 and IL-13) in bronchoalveolar lavage fluid by cytometric bead revealed that these scaffolds retained structural properties comparable array (CBA), GM-CSF, TSLP and TGF-b in lung homogenate by to a native dermal matrix. Biocompatibility (integration, cell density, ELISA. Epithelium remodeling was measured by lung stained slides mineralization, and collagen quantification), ED-1 macrophages and with Periodic acid-Schiff alcian blue (PAS-AB) for epithelium area blood vessels of the scaffolds were assessed in rats on the 3°,14° and and mucus production. 21° days after implantation. Blood was collected for leukocytes Results: OVA-sensitized mice showed increased production of quantification and plasma for TNF-a, IL-6 and IL-1b analysis. cytokines as IL-5 and IL-13 that was even enhanced when there was Histopathological analysis showed a gradual increase in the integra- co-exposition to cigarette smoke. We noticed that TSLP levels tion and cell density of both scaffolds (mainly on scaffold 1) in increases with exposition to cigarette smoke, but when combined to comparison to PermacolÒ (used as control). No mineralization was OVA-sensitization it reduces significantly but not until OVA levels. observed on the assessed time-points. Under polarized light a The levels of TGF-b increased on CS and OVA groups. Comparing decrease of collagen was observed on the 14° day in comparison to OVA + CS to OVA groups, we noted that the levels of TFG-b the 3°. However, on the 21° day we observed deposition of new showed maintained, but decreased when compared to CS group. The collagen fibers, suggesting initiation of the tissue remodeling process. OVA-sensitization process per se was capable to increase IFN-c that Our results showed an initial increase on leukocytes (day 3) with a was not observed when there was exposition to cigarette smoke significant reduction in the subsequent time-points. However, no combined or alone. We also observed a remodeling process of the significant difference was observed in the inflammatory mediators. epithelium on sensitized group by increased epithelium area and Immunohistochemical techniques revealed significant increase in ED- mucus production that was intensified after co-exposition to cigar- 1 positive macrophages inside the scaffolds after day 14, and blood ette smoke. We observed no changes in GM-CSF levels among all vessels positive to vWF after 21 days of implantation. Altogether our groups. findings suggest that both scaffolds were biologically compatible, Conclusion: Based on this data, we conclude that co-exposure of permitting cells recruitment, vasculogenesis and gradual substitution sensitized mice to cigarette smoke once a day worse the lung of the acellular matrix; and therefore supporting tissue remodeling inflammation induced by the antigen OVA. It is clear that epithelium and regeneration. in involved in this process by the remodeling presented on it as well The experimental procedure were approved by the Research Ethics as the enhanced production of cytokine such as TSLP that is mainly Committee (0182/12). produced by the epithelium cells.

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B321 1Rutgers Molecular Imaging Center, Rutgers University, Piscataway 2 NERVE REGENERATION IN INJURED TOOTH IS NJ 08854; Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854 MEDIATED BY THE INTERACTION BETWEEN THE COMPLEMENT FRAGMENT C5A AND PULP Nitrogen mustard (NM, mechlorethamine) is a cytotoxic alkylating agent that induces acute lung injury followed by delayed fibrosis. We FIBROBLASTS previously characterized the effects of NM on lung histology, inflammation and function. In this study we used molecular imaging 1 2 1 1 Fanny Chmilewsky , Imad About , Warda Ayaz , Seung H. Chung techniques to periodically track the development of NM-induced alterations in the lungs of live animals throughout a 28-day period. 1 2 University of Illinois at Chicago, USA; Aix-Marseille universite´, Male Wistar rats (225–250 g; n = 3/group) were treated with Marseille, France 0.125 mg/kg NM or PBS control by intratracheal instillation. Mag- Dentin-pulp regeneration is closely linked to the presence of nerve netic Resonance Imaging (MRI), consisting of gradient spin echo fibers in injured teeth, since there is an amplification of healing (GRE) and fast spin echo (FSE) sequences, as well as Computed Tomography (CT) were performed on each animal on days 1, 3, 7, 14, mechanism by the sprouting of the nerve fiber’s terminal branches TM in the pulp beneath injury (Kumazawa et al. 1996; Arai, 1991). and 28. VivoQuant image processing software was used to analyze Although this process seems to be associated with a local MRI and CT scans. Three-dimensional images of lungs were rendered enhancement of several growth factors such as Nerve Growth Factor from MRI scans and the volumes of the injured and normal lung (NGF) (Byers et al. 1992a), little is known about initial mechanisms tissue were quantified. Following NM exposure, the volume of injured that regulate the dental pulp nerve sprouting beneath the injured site. tissue increased significantly within 1 day, a response that persisted It has been recently demonstrated that the complement system for 28 days. Lung volumes of air, dynamic lung tissue, and consoli- activation, which occurs during carious decay (Chmilewsky et al. dated lung tissue were determined via segmentation of CT scans 2013), contributes to tissue regeneration through the local produc- based on voxel intensity. The dynamic lung volume was significantly tion of anaphylatoxin C5a (Chmilewsky et al. 2014). The aim of this decreased 3 days after NM administration. Conversely, the volume of study is to examine the roles of the active fragment C5a in dental consolidated lung tissue was significantly increased on both day 14 nerve regeneration following tooth damage and further identify the and day 28, when compared to day 1 and day 3. These data show that underlying mechanisms. MRI and CT imaging are useful methods to detect and track the Immunofluorescence staining was performed in vivo on intact and progression of NM-induced lung injury in rats. Since these molecular carious tooth sections to localize the C5a receptor (C5aR). The C5aR imaging techniques can be conducted on live animals, each animal expression was then examined in vitro using western blotting and can serve as its own control, fewer animals can be used, and paired double immunofluorescence staining with untreated or lipoteichoic data analysis can be conducted, thus reducing variability. Supported acid (LTA)–stimulated (carious condition) human pulp cells. The by NIH AR055073 and ES005022. neurotrophin production by untreated, or LTA–stimulated human pulp cells, treated or not with a C5aR specific antagonist (W54011), was quantified by ELISA assay. Finally, the neurite outgrowth from Toll Receptors human neurons toward untreated, or LTA–stimulated human pulp cells, treated with or without W54011, was examined with the AXIS Axon isolation device. B323 Our results show that pulp cells, localized beneath the carious A NOVEL CELL SURFACE PROTEIN THAT injury, express the C5aR. This observation is consistent with our IMPARTS CYTOKINE SPECIFICITY TO TLR4 in vitro results that showed an ectopic expression of C5aR in pulp fibroblasts stimulated with LTA, a complex component of Gram- SIGNALLING positive bacteria cell walls. Finally, the treatment with specific C5aR antagonist significantly inhibited both the NGF and Brain-Derived Nilesh J. Bokil1,2, Lin Luo1,2, Jennifer L. Stow1,2, Matthew J. Sweet1,2 Neurotrophic Factor (BDNF) expressions in LTA-stimulated pulp fibroblasts, and the neurite outgrowth from human neurons toward the 1Institute for Molecular Bioscience, Brisbane, Australia; 2University direction of LTA stimulated fibroblasts. of Queensland, Brisbane, Australia These findings suggest that upon bacterial infection, the C5a, Toll-like receptors (TLRs) are a family of pattern recognition through its interaction with pulp fibroblast C5aR, contributes to the receptors that sense both pathogen-associated molecular patterns as tissue regeneration by mediating nerve outgrowth on the injured site. well as endogenous host factors to drive pro-inflammatory responses. This effect seems to be due to the significant C5a involvement in For example, the archetypal TLR, TLR4, recognises both neurotrophin secretions by pulp fibroblasts. These data may provide a lipopolysaccharide (LPS) from Gram-negative bacteria, as well as a useful therapeutic tool targeting pulp fibroblasts in dentin-pulp range of endogenous host factors including amyloid-beta peptide and regeneration process. oxidized low-density lipoprotein. Upon ligand recognition, TLR4 dimerises and recruits the Toll/Interleukin-1 Receptor (TIR) domain- containing proteins MAL (MyD88 adaptor-like protein), MyD88 (Myeloid differentiation primary response gene 88), TRAM (TRIF- B322 related adapter molecule) and TRIF (TIR-domain-containing adapter- MOLECULAR IMAGING TECHNIQUES TO inducing interferon-b) through homotypic TIR-TIR domain interac- EVALUATE NITROGEN MUSTARD INDUCED tions, ultimately enabling the activation of specific serine/threonine LUNG INJURY kinases to relay downstream signalling. TLR4 is also tyrosine phos- phorylated upon activation, however the mechanisms controlling this process are much less understood. Here we report a novel cell surface 1 2 2 Edward J. Yurkow , Justin Schumacher , Alessandro Venosa , protein that is required for LPS-induced TLR4 phosphorylation. 1 2 2 Derek Adler , Jeffrey Laskin , Debra L. Laskin Using siRNA-mediated gene knock-down in primary mouse

123 S232 Inflamm. Res. macrophages we also show that this protein is required for primary B325 LPS signalling responses (activation of NF-kappaB, p38 and JNK), as NEURONAL TLR SIGNALING CONTROLS well as LPS-inducible secretion of IL-6 and IL-12. Interestingly, this pathway had no role in regulating TNF production. These differential NEURONAL EXCITABILITY, effects were also apparent at the level of gene expression. Conversely, NEUROINFLAMMATION, PAIN, AND ITCH gain-of-function studies by retroviral over-expression in primary murine macrophages revealed selective upregulation of IL-6 and IL- Ru-Rong Ji, Chul-Kyu Park, Tong Liu, Zhen-Zhong Xu, 12, with no effect on TNF production. Our findings thus reveal a new Qingjian Han, Yong Ho Kim, Temugin Berta level of specificity emanating from the cell surface to mediate the TLR4-inducible production of a sub-set of pro-inflammatory Duke University Medical Center, Durham, NC, USA cytokines. It is generally believed that Toll-like receptors (TLRs) are expressed by immune cells to regulate innate immunity. However, we found functional TLR3 and TLR7 are also expressed by small-sized primary B324 sensory neurons in dorsal root ganglion (DRG) that are known to ALTERED TOLL-LIKE RECEPTOR SIGNALING IN regulate pain and itch. Interestingly, TLR3 agonist PIC and TLR7 agonist immiquimod and loxoribine can cause immediate activation INDIVIDUALS WITH TYPE 1 BIPOLAR DISORDER (within seconds) of sensory neurons via a novel non-canonical sig- naling pathway that requires TLR and ion channel (e.g., TRPA1) but 1,2,3 1,2 1 Andrea Wieck , Carine Hartmann do Prado , Laura Petersen , not Myd88. Immiquimod induces robust scratching (itch-like behav- 4 2,1 Ba´rbara Nery Porto , Rodrigo Grassi-Oliveira , ior) in wild-type mice, which is reduced in Tlr7-deficient mice. 1,5,3 Moise´s Evandro Bauer Strikingly, we found that specific miRNAs containing GUUGUGU motif such as Let-7b can act as endogenous ligands of TLR7. 1 Laboratory of Immunosenescence, Institute of Biomedical Research, Extracellular application of let-7b to dissociated DRG neurons is Pontifical Catholic University of the Rio Grande do Sul, PUCR, Porto sufficient to induce rapid inward currents and action potentials in 2 Alegre, Brazil; Cognitive Neuroscience Research Group (GNCD), DRG neurons. These electrophysiological responses are abolished in Centre of Studies and Research in Traumatic Stress (NEPTE), mice lacking Tlr7 or Trpa1. Injection of let-7b into a hind paw of Postgraduate Program in Psychology, PUCRS, Porto Alegre, Brazil; mouse elicits rapid spontaneous pain via TLR7 and TRPA1 in wild- 3 Postgraduate Program in Biomedical Gerontolgy, PUCRS, Porto type mice. Although let-7b-induced acute pain does not require 4 Alegre, Brazil; Centro Infant, Pontifical Catholic University of Rio MyD88, Myd88-mediated canonical signaling is essential for let-7b- 5 Grande do Sul, PUCRS, Porto Alegre, Brazil; Faculty of induced chronic pain. Specific deletion of Myd88 in nociceptive Biosciences, PUCRS, Porto Alegre, Brazil sensory neurons (Nav1.8-expressing neurons) lead to a reduction in Bipolar disorder (BD) has been associated with immune imbalance, chemotherapy-induced neuropathic pain. Furthermore, chemother- characterized with chronic low-grade inflammation profile. Toll-like apy-induced innate immunity and adaptive immunity in the peripheral receptors (TLR) are pivotal receptors in microbial recognition, initi- nervous system (DRG) is impaired after conditional deletion of ating innate immune responses and providing a link between innate Myd88 in nociceptive neurons. Finally, we found that TLR7 is also and adaptive immune responses. Given the importance of TLR in co-expressed with TRPA1 in human DRG neurons from non-diseased coordinating inflammatory immune responses, the main objective of donors, and activation of TLR7 by let-7b is also sufficient to excitate this study is to immunophenotype circulating monocytes for TLR human DRG neurons via TRPA1. Together, our findings have expression (TLR1, TLR2, TLR4, TLR5 and TLR6) and signaling. demonstrated that TLR signaling in primary sensory neurons controls Thirteen euthymic participants with type 1 BD and 15 healthy con- acute pain, acute itch, chronic pain, and neuroinflammation via both trols (HC) took part in this study. Peripheral blood mononuclear cells non-conical and canonical signaling pathways. Our study also sug- (PBMCs) were isolated, differentiated into monocytes and stimulated gests that RNAs (such as total RNAs and miRNAs) can serve as in vitro with specific TLR agonists (flagelin, LPS, LTA, BLP and extracellular signaling molecules to activate TLR3 and TLR7 in PGN) to assess inflammatory cytokines (IL-8, IL1-b, IL-6, IL-10, sensory neurons. TNF-a and IL-12p70). TLRs and cytokines were assessed by multi- This work is supported by NIH RO1 Grants NS67686, DE17794, color flow cytometry. Increased percentages of TLR1+ and DE22743, and NS87988 to R.R.J. TLR2+ monocytes were observed in BD (p = 0.007 and p = 0.005 respectively). In contrast, reduced expression of TLR5 in monocytes from patients was also observed (p = 0.034). Following stimulation Translational Medicine and Biomarkers with specific TLR agonists, BD patients had higher percentages of TLR1 + and TLR2 + monocytes (p = 0.018 and p = 0.023 respec- tively), as well as TLR6 (p = 0.010) as compared with HC. Increased B326 levels of IL-8, IL12-p70 and TNF-a (p = 0.0001; p = 0.006 and MOLECULAR DETECTION OF INFLAMMATION p = 0.0001, respectively) were observed following stimulation with IN CELL AND ANIMAL MODELS USING specific TLR1, TLR2 and TLR6 ligands, further indicating increased signaling via those receptors in BD patients. Our data indicates HYPERPOLARIZED 13C-PYRUVATE altered signaling via TLR1 and TLR2 in BD patients, which was corroborated by increased levels of cytokines when stimulated with Julia Nguyen, Jeffrey T. Gu, Linda Nguyen, Renuka Sriram, specific agonists. Given the importance of TLR2/1 in triggering Dave Korenchan, John Kurhanewicz, John D. MacKenzie immune responses, our data suggests an important role for innate immunity in development/maintenance of the immune imbalance Department of Radiology and Biomedical Imaging at UCSF, observed in BD. San Francisco, CA, USA

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Purpose: To test and validate hyperpolarized 13C-pyruvate and mediators (SPMs) resolvins, protectins and maresins on phagocytosis, alterations in its conversion to 13C-lactate as an imaging biomarker inflammation and cognition. for disease severity and treatment response in inflammation. Methods: Phagocytosis of FITC- Abeta1-42 was measured by flow Methods: With IACUC approval, inflammation was induced with cytometry [expressed as mean fluorescence intensity (MFI)] and flu- Freund’s complete adjuvant (0.4 mL/kg) in healthy Balb/c mice. orescence microscopy; resolvin D1 (RvD1) by ELISA; inflammation At the peak of inflammation (7d post-induction), animals were by RNA-Seq of PBMCs; cognition by Minimental state examination anesthetized and positioned in the NMR scanner. Proton (1H) (MMSE) score; and macrophage phenotype by the M1 markers CD54 and carbon-13 (13C) imaging was performed on a 14.1-T Varian (ICAM-1) and CD80 (costimulatory protein) and the M2 markers 600WB animal NMR scanner through the area of inflammation CD163 (scavenger receptor) and CD206 (mannose receptor). (paw) and control paw. 13C spectra were obtained immediately Results: 12 MCI patients and 2 pre-MCI patients have been followed 13 after the intravenous injection of 300 ll of hyperpolarized C1- on daily nutritional supplementation with omega-3 fatty acids (DHA pyruvate using DNP (15–20 % liquid-state polarization). Tissues 1 gm and EPA 1 gm in the Smartfish drink, Oslo Norway) for were processed and inspected for histological changes of 5–18 months (the first visit was without supplementation and subse- inflammation. quent visits on supplementation). MFI increased from 530 on the first J774A.1 mouse macrophage cells were cultured, stimulated with visit to 1306 on the last visit (p \ 0.016). RvD1 (n = 6) increased in lipopolysaccharide (LPS) into their activated inflammatory state, and 3 and did not change in 3 patients. MMSE score (n = 8) improved in biomarkers were studied to evaluate the cells response to treatment 7 but later declined in 4 (related to disease or noncompliance) and (mRNA levels of lactate transporters MCT1/4 & lactate dehydroge- declined in one ApoE4 patient. Most patients had mixed macrophage nase (LDH) A/B using qRT-PCR and nitric oxide (NO) production phenotype with highest expression of CD54 and CD206; two had an with Griess assay). Cells were also pre-treated with indomethacin inflammatory type but one switched to a mixed type on a subsequent (IND) (10 lM) 4 days prior to stimulation and were also administered visit. On the initial visit, FITC- Abeta1-42 phagocytosis by macro- IND at the time of LPS stimulation. phages of most patients was improved more by in vitro treatment with To measure 13C-lactate production, J774A.1 cells were encapsu- resolvin D1, resolvin D2 or maresin D1 than by omega-3 emulsion lated into alginate beads and either left as control or activated with (Fig. 1a), whereas on later visits omega-3 was generally more 100 ng/mL lipopolysaccharide (LPS) for 24 h. The cells were then effective (Fig. 1b). RNA sequencing (n = 4) showed inflammatory placed in a bioreactor, and the production of 13C-lactate measured signature in 2 and non-inflammatory signature in 2. with NMR after incubation with hyperpolarized 13C-pyruvate. Conclusions: In MCI patients, omega-3 fatty acids have positive Imaging data were processed with VNMRJ and SIVIC, and ACD/ effects on immunity against Abeta1-42 through SPMs. NMR imaging software. Results: Elevated levels of lactate were detected in areas of inflam- mation in mice, using 13C-MRSI. LPS-stimulated cells had a substantial increase in the production of mRNA of MCT4 lactate transporter (4.7 ± 1.8 SD fold increase) whereas LDHA/B and MCT1 remained at control levels. Indomethacin inhibited NO production by LPS-stimulated cells. Nine-fold elevation in the production of hyperpolarized 13C-lactate was observed after stimulation in a mac- rophage cell model of inflammation. Discussion: We have identified a robust method of studying inflam- mation with 13C-MRSI. Our experiments indicate that lactate production is elevated in inflammation, which can be readily detected by 13C-MRSI with hyperpolarized 13C-pyruvate. Unlike other molecular imaging techniques that are still experimental, this tech- nique has IND approval and should translate rapidly to clinical usage for inflammatory disorders such as adult rheumatoid and juvenile idiopathic arthritis. B328 TRANSLATIONAL PROPERTIES OF A FOLATE- B327 AMINOPTERIN SMALL MOLECULE DRUG OMEGA-3 NUTRITIONAL SUPPLEMENTATION CONJUGATE WITH A METABOLICALLY IMPROVES AMYLOID-BETA IMMUNITY ACTIVATED LINKER SYSTEM THROUGH SPECIALIZED PRO-RESOLVING MEDIATORS Yingjuan Lu1, Satish Rao1, Paul Kleindl1, Fei You1, Michael Pugh1, Vicky Cross1, Elaine Westrick1, Alex Lloyd1, Leroy Wheeler II1, Milan Fiala, Sam Famenini, Rachel Rubattino, Patrick Klein1, Iontcho P. Vlahov1, Philip S. Low2, Bien Sagong, Jessica Leung, Ramesh Halder Christopher P. Leamon1

Department of Surgery, UCLA School of Medicine, Los Angeles, 1Endocyte, Inc., West Lafayette, IN, USA; 2Purdue University, West CA, USA Lafayette, IN, USA Introduction: Alzheimer disease and Minor cognitive impairment Introduction: Chronic human diseases and conditions are often caused (MCI) patients have a defect in the phagocytosis of amyloid-beta 1-42 or worsened by excessive activation of macrophages and their (Abeta1-42) and abnormally low or high activation of inflammation. monocyte precursors. During immune adaptation to the local envi- Objectives: We are studying the effects of the omega-3 drink ronment, activated macrophages of both M1 and M2 phenotypes (Smartfish, Oslo, Norway) and the specialized pro-resolving express a nanomolar-affinity folate receptor (FR)-b. This

123 S234 Inflamm. Res. phenomenon has allowed for a receptor-targeted approach for site- electrochemiluminescence (ECL). The reported ECL-ELISA method, specific delivery of both imaging and therapeutic agents. In patients which has a 96 well format, offers high sensitivity [limit of quanti- with rheumatoid arthritis and osteoarthritis, etarfolatide (99mTc- tation 0.04 nM nitrated bovine serum albumin (BSA-Tyr-NO2) EC20), a FR-specific radioimaging agent, was found to preferentially equivalents] and good linearity across four dilutions accumulate in the inflamed joints or ‘‘hot spots’’. In the therapeutic (R2 = 0.96 ± 0.04, n = 3). realm, we have previously shown that EC0746, a folic acid (FA)- To test the ability of the assay to detect Tyr-NO2 changes, in an aminopterin (AMT) small molecule drug conjugate, can modulate example of an acute inflammatory response, we analysed serum macrophage functionality and is effective in rodent models of adju- samples from patients undergoing a range of surgical procedures, vant arthritis, autoimmune uveitis and encephalomyelitis. In order to where serum had been collected both before and after surgery. A facilitate therapeutic translation, the current investigation focuses on statistically significant increase in nitration was seen post-surgery species differences in linker metabolism and potential implications in (n = 35, p \ 0.05, Wilcoxon Matched Pairs) compared to the levels extrapolating metabolism data from animal models to humans. before surgery; median (IQR): 0.59 (0–1.3) and 0.97 (0–1.7) Tyr-NO2 Methods: A series of FA-AMT conjugates bearing subtle or signifi- (fmol BSA-Tyr-NO2 equivalents/mg protein) for pre- and post-sur- cant structural variations next to or in the linker region were gery respectively. These results confirm that the new assay is indeed synthesized. In-vitro and in vivo similarities and differences in able to detect an increase in serum nitration following an acute pharmacodynamics, pharmacokinetics, and metabolism among dif- inflammatory response. ferent species were investigated using multiple relevant biological In conclusion we have developed and validated a new, sensitive systems. and robust ECL-ELISA for the measurement of Tyr-NO2 that offers Results: Release of free AMT (and its active analog) directly or high-throughput analysis of human samples. Our assay is able to indirectly from intact conjugates was largely enzymatic. Using whole- detect an increase in serum levels of Tyr-NO2 following an cell homogenates and liver S9 fractions from different species, it was inflammatory response induced by surgery. The present Tyr-NO2 found that the linker region influences the rate of free AMT release. In assay may be applicable in providing a pharmacodynamic endpoint preclinical models, activity and tolerability of the folate-AMT con- in pre-clinical and clinical studies in which therapeutic effects on jugates correlated well with differences seen in linker metabolism and oxidative stress are implicated. For example the assay may be useful pharmacokinetics. By fine-tuning the enzymatic substrate specificity in testing novel anti-inflammatory drugs that lower ROS production. in the linker region we could control the rate of free AMT release Several such drugs are currently in commercial development, in vitro and in vivo. including selective NADPH oxidase inhibitors and myeloperoxidase Conclusion: The main challenge in preclinical translation has always inhibitors. been to circumvent species differences in drug metabolism. The promiscuity of metabolic enzymes in rodents complicates the inter- pretation and prediction of linker metabolism in dogs and man. We have identified specific metabolic enzymes that are responsible for B330 AMT release from our folate-AMT series. We have been able to MEASUREMENT OF SERUM NITRATE utilize this knowledge and incorporate species-dependent and -inde- CONCENTRATION FOR THE DIAGNOSIS OF pendent structural variations to control the rate of linker metabolism and optimize conjugate efficacy and tolerability. Consequently, a lead INFECTIVE GASTROENTERITIS compound has emerged with superior translational properties for advancement into the clinic. Miranda J. Smallwood1, Kyle Stewart2, Thalia Groom1, Jessica Cross1, Nigel Benjamin2, Paul G. Winyard1

1University of Exeter Medical School, Exeter, UK; 2Torbay Hospital, B329 Torquay, UK NEW ELECTROCHEMILUMINESCENCE-BASED An increased serum concentration of nitrate, a metabolic end product ELISA FOR HIGH-THROUGHPUT ANALYSIS OF of nitric oxide (NO), has been reported in patients with infective SERUM OXIDATIVE DAMAGE ASSOCIATED gastroenteritis, and this increase in nitrate is much greater than is seen WITH INFLAMMATION in patients with sepsis or other causes of the inflammatory response. The observed extreme increase of serum nitrate concentration in infective gastroenteritis is thought to be due to a host inflammatory Annie Knight1, Emma Taylor1, Jane E. Carre´1, Yuktee Dogra1, ‘superinduction’ of inducible nitric oxide synthase activity, over and Jo Tarr1, Roman Lukaszewski2, Paul G. Winyard1 above that seen in many autoimmune inflammatory diseases. In serum samples collected from 149 patients upon their admission 1University of Exeter Medical School, Exeter, UK; 2Dstl, Porton to hospital with suspected infective gastroenteritis, the nitrate con- Down, Salisbury, UK centration was measured by both the established method of ozone- Activation of an inflammatory response leads to the generation of based chemiluminescence and by a new, validated, modification of a reactive oxygen species (ROS); these species cause oxidative damage rapid and cheap spectrophotometric plate-based method. such as nitration of tyrosine residues within proteins, thereby gener- As measured by chemiluminescence, the median serum nitrate ating 3-nitrotyrosine (Tyr-NO2). Previous studies have reported concentration was significantly increased (p \ 0.0001) in patients in elevated serum Tyr-NO2 in many human inflammatory diseases, e.g. whom a causative organism was identified (75.7 lM; IQR, sepsis and systemic lupus erythematosus, and it has been suggested 37.3–150.0 lM; n = 51) compared with those in whom no organism that Tyr-NO2 is a potential marker of inflammatory activity. Mea- was found (43.6, 21.8–66.0 lM, n = 98). Patients with a bacterial surement of oxidative stress markers in clinical studies has been cause of gastroenteritis (Campylobacter or Salmonella) had particu- hindered by methodological issues such as low sensitivity and low larly high concentrations of plasma nitrate (median: 115.3, IQR: throughput. 78.6–268.8, and median: 205.4, 65.0–2472.0 lM respectively), Therefore a high-throughput method, for Tyr-NO2 detection, has compared with serum nitrate concentrations from patients who did not been developed based on the highly sensitive platform, have a bacterial cause of gastroenteritis. Patients diagnosed with

123 Inflamm. Res. S235 norovirus had similar serum nitrate concentrations (51.5, at later times suggesting crosstalk between pathways; BIRB-796 and 25.1–145.0 lM) to patients with non-infective gastroenteritis (43.6, PD-184352 both decrease p-p38 at 24 h. Whereas PD-184352 and 21.8–66.0). In patients in whom a causative organism was identified, JNK IX decrease p-c-Jun, BIRB-796 unexpectedly increases p-c-Jun. the serum nitrate concentration correlated with the severity of disease Phosphorylation of HSP27, a cytoprotective protein implicated to have as measured by the amount of intravenous fluids required (R2 = 0.58; a role in skin injury, is decreased by p38i and JNKi but increased by p \ 0.02, n = 18). However serum nitrate concentration was not MEKi. Together, this systems biology approach reveals that MAPK correlated with CRP values (R2 = 0.02, n = 43). When measured by inhibitors do not act in a target-isolated manner but rather impact a the spectrophotometric 96-well plate-based assay, serum nitrate network of inflammatory, proliferative and stress response pathways. concentrations correlated well with nitrate measured by chemilumi- In conclusion, phenotypic screening and phosphorylation profiling nescence (R2 = 0.97), suggesting that the spectrophotometric method enables the identification of sentinel biomarkers that can be associated could serve as a high throughput assay. with efficacy and the potential for drug-related skin rash AE. The present study provides evidence that a serum nitrate con- centration of greater than 70 lM indicates bacterial gastroenteritis, particularly Campylobacter and Salmonella. We have also demon- strated the feasibility of screening hospital patients for serum nitrate, B332 using a convenient high-throughput nitrate assay. We therefore sug- CRP AND 14-3-3 ETA ARE EACH ASSOCIATED gest that serum nitrate measurement may be a useful diagnostic test in gastroenteritis patients. WITH JOINT DAMAGE PROGRESSION, THEIR TITRES DO NOT CORRELATE AND ARE BETTER PREDICTORS OF PROGRESSION TOGETHER B331 THAN ALONE

MECHANISMS OF DRUG-INDUCED SKIN RASH 1 4 2 Ò Anthony Marotta , Nathalie Carrier , Artur J. Fernandes , Patrick USING BIOMAP HUMAN PRIMARY CELL- Liang2, Ariel Masetto2, Yuan Gui1, Jane Savill1, Sara Michienzi1, BASED SYSTEMS Henri A. Menard6,3, Walter P. Maksymowych5, Gilles Boire2

Jason Ptacek, Karen Matta, Sylvie Privat, Dat Nguyen, Ellen Berg, 1Augurex Life Sciences Corp., British Columbia, Canada; 2University Alison O’Mahony of Sherbrooke, Sherbrooke, Que´bec, Canada; 3McGill University Health Center Research Institute, Montreal, Quebec; 4Centre BioSeek, a division of DiscoveRx Corp, South San Francisco, CA, USA hospitalier universitaire de Sherbrooke, Sherbrooke, Que´bec, Canada; 5University of Alberta, Edmonton, Alberta, Canada; 6MSK The relationship between drug efficacy and adverse events (AE) is Research Axis, Canada dependent on (1) target selectivity, (2) primary and secondary effects of target modulation and (3) the concentration at which these inter- Background: As a marker of inflammation and an acute phase reac- actions occur. BioMAP technology uses a systems biology approach tant, C-reactive protein (CRP) is routinely used in clinical practice to capture the complexity of physiologically relevant tissue and in RA classification criteria. Higher or sustained levels of CRP microenvironments and provide information about how compounds are associated with a worse prognosis. 14-3-3g is a joint derived behave in these settings with respect to efficacy and safety. BioMAP mechanistic marker that up-regulates factors that perpetuate disease. systems consist of cultures of human primary cells stimulated to Similar to CRP, higher or persistent levels of 14-3-3g are associated model disease biology and protein-based biomarker endpoints to with a worse prognosis, and a corresponding decrease in circulating detect phenotypic changes resulting from compound exposure. Here, levels is associated with better clinical outcomes. Inflammation and we use BioMAP technology to gain a phenotypic and mechanistic joint damage are now understood to be processes that uncouple along understanding of the differentiating properties between classes of the course of disease and treatment strategies have been tightened to MAPK inhibitors. We show these inhibitors have expected anti-in- achieve both clinical and joint damage remission. flammatory and anti-proliferative activities but differ in the induction Objectives: The aim of this study was to examine both the indepen- of a pro-inflammatory response associated with skin rash, an AE dent and combined effects of CRP and 14-3-3g on radiographic reported clinically for the MEK and p38 inhibitor classes. progression. We performed a phenotypic screen of three target-selective MAPK Methods: Baseline (BL) serum 14-3-3g titres were assessed in 331 inhibitors: PD-184352 (MEKi), BIRB-796 (p38i), and JNK IX (JNKi). recent onset polyarthritis patients from the Sherbrooke EUPA Cohort In BioMAP systems modeling inflammation, these compounds across a with 5 years of radiographic follow-up data. Patients were DMARD range of concentrations decreased several inflammatory biomarkers naı¨ve at BL, median age was 60 years, and 62 % were female. CRP (CD69, IL-1a,TNFa). Interestingly, at the same exposure levels, and 14-3-3g positivity were defined as [8 mg/L and C0.19 ng/mL, BIRB-796 and PD-184352 showed pro-inflammatory activities (in- respectively. Spearman correlation was performed to assess the creased VCAM-1, IP-10, MIG) in a BioMAP system modeling wound relationship between 14-3-3g and CRP titres. Radiographic changes healing. Multiple highly selective p38 inhibitors had similar activities, (change in total Sharp/van der Heijde (DSHS) score over 30 months indicating that this effect is target-class, not compound specific. Con- were assessed in relation to CRP and 14-3-3g co-expression using versely, JNK IX had the opposite profile in the wound healing model ANOVA analysis. Chi square was used to assess the relationship with anti-inflammatory activities over several concentrations. between CRP positivity with radiographic changes at 30 months To further elucidate the differences between the MAPK inhibitors based on DSHS C1, 3, and 5 unit cut-offs. in the wound healing system, we used phosphorylation profiling of Results: Of 331 patients, 207 (63 %) and 153 (46 %) were CRP and key signaling proteins to compare underlying network interactions at 14-3-3g positive at BL, respectively. Spearman correlation revealed early and late time points. Specifically, PD-184352, BIRB-796 and that titres of CRP and 14-3-3g did not correlate, r =-0.00025 JNK IX decreased the levels of p-ERK, p-p38, and p-c-Jun, respec- p = 0.996. As noted in Table, Chi square analysis returned both CRP tively, at early time points. A broader, network impact is manifested and 14-3-3g as significantly associated with radiographic changes.

123 S236 Inflamm. Res.

. relation to the established inflammatory marker C-reactive protein (CRP). Variable DSHS BL to DSHS BL to DSHS BL to Methods: Patients admitted to the Acute Medical Unit, Hvidovre year 3 C 1 year 3 C 3 year 3 C 5 Hospital, between 11/18/2013 and 03/31/2014 were included. The study population comprised 4,343 patients. suPAR was measured OR p- OR p- OR p- with suPARnosticÒ AUTO Flex ELISA. CRP was measured with value value value COBAS 6000 analyzer. Data on admissions and vital status were CRP positivity 1.73 0.034 2.47 \0.001 2.22 0.001 extracted from Danish patient administrative registries using the patients’ unique Danish civil registry number. Duration of follow-up 14-3-3g positivity 1.69 0.046 1.74 0.016 1.70 0.023 was 30 days. CRP and 14-3-3g 2.82 0.002 3.68 \0.001 3.16 \0.001 Results: Median suPAR was 3.2 ng/mL (95 % RI: 1.3–11.3 ng/mL), positivity and suPAR levels increased with admission time (p \ 0.0001). CRP and suPAR were significantly correlated (Spearman’s rank correlation coefficient: 0.43, p \ 0.0001), however, 20.6 % of the patients had The cumulative probability plot illustrates that patients who were low CRP (\10 mg/l) but high suPAR levels ([4.5 ng/mL). The positive for both CRP and 14-3-3g had the significantly greatest median suPAR level was higher in patients who were readmitted increase in radiographic progression (p \ 0.001) with over 50 % of during follow-up (3.9 ng/mL, IQR 2.7–5.6, n = 854) compared to patients having DSHS C5 over the 30 month period. patients who were not readmitted (3.0 ng/mL, IQR 2.2–4.5, p \ 0.0001). Similarly, the median suPAR level was higher in patients who died during follow-up (6.8 ng/mL, IQR 4.7–9.9, n = 224) compared to survivors (3.1 ng/mL, IQR 2.2–4.5, p \ 0.0001). Both suPAR and CRP predicted 30 days mortality, and the ROC AUCs for suPAR and CRP were 0.84 (95 % CI 0.81–0.86) and 0.77 (95 % CI 0.74–0.80), respectively. In Cox regression anal- ysis of log2-transformed suPAR values, suPAR was a strong independent predictor of readmission and mortality with a HR for 30 days mortality of 2.56 (95 % CI 2.13–3.08, p \ 0.0001) when adjusted for age, sex, Charlson score, and CRP. In patients with low CRP (\10 mg/l), suPAR remained an independent predictor of readmission and mortality. Conclusions: The new inflammatory biomarker suPAR is associ- ated with readmission and mortality in acute medical patients. suPAR adds information on high risk patients to the established inflammation marker CRP. Conclusions: CRP and 14-3-3g are both associated with joint damage progression at 3 years and titres do not correlate consistent with their distinct roles in RA disease processes. Interaction analysis further reveals that the combination of these two markers is a better predictor of future radiographic damage than either marker alone. Concomitant B334 serial testing of both these modifiable markers may assist with tight SERUM INTERLEUKIN-18 AND S100A8/A9 AS control RA treatment strategies. PREDICTION MARKERS FOR DISEASE ACTIVITY IN PATIENTS ADULT ONSET STILL’S DISEASE

B333 Dae Hyun Yoo, Jinju Kim, Seung Taek Song SUPAR: A NOVEL INFLAMMATORY BIOMARKER IN CLINICAL ROUTINE ASSOCIATED WITH Department of Rheumatology, Hanyang University Hospital for Rheumatic Diseases, Hanyang, Japan READMISSION AND MORTALITY Background: Adult-onset Still’s disease (AOSD) is a rare systemic inflammatory disease. Although the pathogenesis of AOSD is still Line JH Rasmussen, Steen Ladelund, Thomas H. Haupt, unknown, proinflammatory cytokines including interleukin (IL)-1, IL- Jørgen H. Poulsen, Gertrude Ellekilde, Ove Andersen 6, IL-18 and TNF-a contribute to clinical manifestations and labo- ratory abnormalities. Determination of disease activity is difficult Copenhagen University Hospital, Hvidovre, Denmark because there are lack of disease specific clinical findings and sero- Background: Soluble urokinase plasminogen activator receptor logic markers. (suPAR) is the circulating form of uPAR, which is expressed on Objectives: To investigate the significance of serum IL-18 and immune and endothelial cells. suPAR levels increase with inflam- S100A8/A9 protein in the assessment of disease activity among mation and immune activation. suPAR is an unspecific marker of AOSD patients. chronic low-grade inflammation associated with prognosis in both Methods: Forty patients satisfying Yamaguchi’s criteria for AOSD, infectious and chronic diseases. suPAR was recently introduced in 26 healthy controls and 23 patients with rheumatoid arthritis (RA) clinical routine as one of the standard blood tests measured on all were enrolled. We collected clinical data including demographic patients admitted to the Acute Medical Unit, Hvidovre Hospital, findings and laboratory findings in active state and inactive state. Denmark. Serum levels of IL-18 and S100A8/A9 proteins were measured by Objectives: We aimed to investigate the association between the new enzyme-linked immunosorbent assay (ELISA). Activity state was inflammatory biomarker suPAR, readmission, and mortality as well as divided by modified Pouchot’s (mPouchot’s) score. Inactive state was

123 Inflamm. Res. S237 defined mPouchot’s score less than 4 at least consecutive 2 months, 1Children’s Hospital of Philadelphia, Philadelphia, USA; after 6.2 ± 1.4 months of active state. 2University of Pennsylvania, Philadelphia, USA Results: There were significantly higher levels of serum IL-18 CAR T cells with anti-CD19 specificity have demonstrated consid- (107951.1 ± 94440.9 pg/mL) in active state than inactive state of erable promise against highly refractory hematologic malignancies. AOSD (14772.2 ± 22126.7 pg/mL), RA patients (333.8 ± 309.0 pg/ Dramatic clinical responses with complete remission rates as high as mL) and healthy controls (350.7 ± 160.4 pg/mL). Although, serum 90 % have been reported in children with relapsed/refractory ALL S100A8/A9 protein were significantly higher in active state of AOSD treated with CTL019 (Maude et al. NEJM 2014). Marked in vivo (33340.3 ± 22029.3 ng/mL) than inactive state CAR T cell proliferation (100 to 100,0009) leads to improved effi- (11826.5 ± 15368.4 ng/mL), healthy controls (1812.7 ± 2506.1 ng/ cacy but can be associated with adverse advents, including cytokine mL) and RA (13577.5 ± 12893.6 ng/mL), S100A8/A9 in inactive release syndrome (CRS). To better understand the manifestations of state was not showed different compared to RA patients. The levels of CRS, we studied 39 children (median age 11; range 9–16) treated with serum IL-18 and S100A8/A9 protein showed mild to moderate cor- CTL019 and present clinical, laboratory, biomarker data. relations with other serologic markers such as WBC, ESR, CRP and T cells were lentivirally transduced with a CAR composed of anti- ferritin. And they also showed moderate correlations with mPouchot’s CD19 scFv/4-1BB/CD3 as previously described (Porter, NEJM score. The cutoff points of differentiating activity in AOSD were 2011). 44 cytokines were measured, using Luminex bead array. Other determined by receiver operator characteristic (ROC) curve. When biomarkers were tested in a CLIA/CAP certified clinical lab. using a 24399.85 pg/mL of IL-18 as an AOSD activity marker, sen- 36/39 patients developed grade 1-4 CRS (CRS1-4) at peak T cell sitivity was 82.5 % and specificity was 82.5 %. And using a expansion. 11 pts developed grade 4 CRS (CRS4), defined as 12101.55 ng/mL of S100A8/A9 protein level showed lower sensi- requiring high dose vasoactive medications and/or mechanical ven- tivity (72.5 %) and specificity (72.5 %) than IL-18. The 25 % of tilation. These 11 pts and 2 of 7 pts with grade 3 CRS (CRS3, defined AOSD patients with clinically inactive state became active state and/ by less severe critical illness) were treated with the IL-6 inhibitor or elevated ESR and CRP within 2 months. AOSD patients showing tocilizumab, and all had rapid marked improvement in CRS. higher IL-18 and S100A8/A9 protein than activity cutoff points in We hypothesized and demonstrated patients with CRS4 develop inactive state, showed disease flare within 2 months [OR = 14.0 clinical and laboratory manifestations similar to macrophage activa- (2.1–93.2), OR = 9.8 (1.9–50.6), respectively]. tion syndrome (MAS)/hematophagocytic syndrome (HLH). 5 of 10 Conclusions: Serum IL-18 and S100A8/A9 protein were significantly CRS4 pts developed splenomegaly (1 subject too obese to evaluate), increased in active AOSD patients compared with inactive AOSD, and 7/10 developed hepatomegaly, whereas no CRS0-3 patients RA and healthy controls. However, IL-18 was more useful cytokine to developed organomegaly. Fibrinogen (not sent on all subjects) was distinguish from RA in inactive AOSD compared to S100A8/A9 decreased (\150 mg/dl) in 9/10 with CRS4 vs 2/15 with CRS0-3. protein. Serum level of IL-18 and S100A8/A9 protein may be useful Ferritin and LDH were also markedly clinically and statistically predictors of disease activity within several months in inactive state of significantly elevated in all pts with CRS4 compared to CRS1-3. Of AOSD patients. the 44 tested cytokines, 16 have been previously studied in children with primary HLH. We found an identical pattern of cytokines dif- ferentially elevated in HLH also in patients with CRS4 compared with B335 CRS0-3. There was a clinically, statistically, and biologically sig- CLINICAL AND BIOLOGIC CHARACTERIZATION nificant difference in IFN-g, IL10, sIL2Ra, IL6, IL8, MCP1, and MIP1B in CRS4 vs CRS0-3 and no clinically or biologically signif- OF CYTOKINE RELEASE SYNDROME AFTER icant difference in IL1B, IL2, IL4, IL5, IL7, IL12, IL13, IL17, and CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL TNF alpha. Both IL6 and IL6R were markedly elevated in patients THERAPY FOR ACUTE LYMPHOBLASTIC with CRS4; this IL6 cytokine pattern, along with the pronounced LEUKEMIA (ALL) response to tocilizumab, establish that IL6 trans-signaling is clinically relevant. These data represent the largest and most comprehensive profiling David T. Teachey1,2, Simon Lacey2, Pamela A. Shaw2, Jos 2 2 1,2 2 of the clinical and laboratory manifestations of CAR T cell related Melenhorst , Noelle Frey , David Barrett , Fang Chen , CRS and provide novel insights into the biology of CRS. They also Julie Fitzgerald1,2, Vanessa Gonzalez2, Shannon Maude1,2, 2 1,2 2 2 provide direct therapeutic relevance and may guide future cytokine Edward Pequignot , Scott Weiss , Carl June , David Porter , directed therapy. Stephan A. Grupp1,2

123 S238 Inflamm. Res.

Author Index

A An, H. B284 Banerjee, S. 046 Abboud, D. B061 Andersen, O. B333 Baranek, T. B282 Abboud, N. B277 Anderson, A.C. 068 Barata, J.T. B029 Abdillahi, S.M. B048 Anderson, L. 118 Barbeiro, D.F. B089, B112 Abels, C. B011 Anderson, R. B262 Barbeiro, H. B112 Aboelnazar, S. B283 Anderson, S. B220 Barbim, P. B033 About, I. B321 Andrade, M.M. B112 Barbosa, A.S. B317 Abreu, L.C. B147 Andrade, M.F. B249 Barbosa-Lima, G. B143 Abulrob, A. B259 Andrade-Silva, M. B312 Barcelos, L.S. B317 Achacoso, P. B080 Andre, D.M. B286 Baribaud, F. 076, B294 Adcock, I.M. B294, B295, B300 Andreotta, P.W. B298 Barioni, E.D. B042 Ademowo, O.G. B155 Anhe, G.F. B286 Barioni, E. B176 Adenir, P. B299 Anisimova, L. B182 Barja-Fidalgo, C. B126 Aderem, A. B094 Annoni, R. B314 Barr, F. B216 Adler, D. B322 Ansari, M.A. B010 Barral, A. B204 Afonso, L. B204 Ansari, T. B319 Barresi, S. B210 Africander, D.J. B263 Antunes, A.M.S. B045 Barreto, A.K.S. B081 Agapova, O.Y. 107 Antunes, E. B286 Barrett, D. B335 Agra, L.C. B205 Antunes, F. B034 Barrett, P. B034 Ahemad, N. B222 Anu Geo, J. B124 Barrett, T.J. B052 Ahuja, S. B151 Anuforo, O. B278 Barron, L. 054, B082 Aitchison, R. 118 AR, G. B313 Barthelmes, J. B017 Ajayi, A.M. B155 Arantes, A.C.S. B304, B305 Bassuk, A.G. B116 Akama, T. B256 Arantes-Costa, F.M. B071, B291, B320 Bastos, B.M. B078 Akbari, O. 131 Ardekani, S. B053, B106 Bastos, J.K. B249 Akram, M. B043 Ardizzone, S. 047 Bastos, J.M. B076 Aksentijevich, I. B018 Arhatari, B.D. B235 Basyal, R.K. B316 Alabi, A. B038 Ariel, A. B276, B277 Batista, A.C. B060 Albaladejo, B.T. B290 Ariga, S.K. B089 Batista, P.R. B309 Al-Bustan, S.A. B124 Arijs, I. B253 Bauer, M.E. B324 Alcalay, R. B289 Arita, M. B221 Baumgarten, M. B048 Alcorn, D. 103 Armstrong Junior, R. B187 Bautzova, T. B268 Alenina, N. B148 Armuzzi, A. 047 Baxter, A.G. B102 Alexandre, E.C. B286 Arnardottir, H.H. B275 Beal, D.R. B028, B298 Aliev, L. B122 Arnoletti, J.P. 039 Becerra, A. B079 Alleyne, J. B199 Arriaga, A.M.C. B078, B158 Beckett, E. B303 Almeida, C.P. B317 Arron, J.R. 075, 110 Bedard, A. B196 Almeida, F.M. B296 Assis, E.L.D. B157, B258 Belizowski, S.M. B032 Almeida, T.F.A. B208 Atreya, R. 047 Bell, S. 118 Almeida-da-Silva, C.L.C. B085 Attia, S.M. B010 Bellas, I. B205 Almolda, B. B206 Ayaz, W. B321 Bellio, M. B119 Alpizar, Y.A. 059 Azevedo, R. B176 Belmonte, C. 059 Alrashid, M.H. B124 Aziz, Q. B113 Beloglazov, V. B131 Alric, L. B267 Azzolini, A.E.C.S. B019, B249 Beltrame, M.H. B072 Altavilla, D. B037 Beltran, J.S. B140 Altemeier, B. B094 B Benabdoune, H. B050 Altier, C. 061 Baba, T. B062 Ben-Azu, B. B155 Alvarenga, P.B. B046 Babaeijandaghi, F. 067 Benderdour, M. B050 Alvarez, A.R.P. B292 BabuRao, K. B313 Benhar, M. B146 Alvarez, J.M.M. B087 Bacal, F. B165 Benjamim, C.F. B046 Alves, C.R. B309 Bachert, C. B041 Benjamin, N. B330 Alves-Filho, J.C. B033, B064, B292 Bader, M. B148, B270 Bennett, A. B199 Amano, H. B107 Bae, O.-N. B043 Benoist, C. 038 Amaral, E.P. B087, B217 Baillif, V. B149, B280 Bereshchenko, O. 035, 036 Amaral, F.A. B064 Bakheet, S.A. B010 Berg, E. B331 Amarante-Mendes, G.P. B217 Balasubramanian, W.R. B313 Bernhagen, J. B163 Amaravadi, L.S. 102 Balog, A. B012 Bernhart, E. B100 Amison, R. B288 Baltazar, G. B256 Berta, T. B325 Amorim, N.R. B205 Banda, N. 083 Bertheau Mailhot, G. B055, B077 Ampuero, M.R. B204 Bandeira-Melo, C. B200, B205 Bessi, V.L. B170 An, D. 007, 089 Bandyopadhaya, S. B316 Bettano, K. B022

123 Inflamm. Res. S239

Beurel, E. 040 Brenck, L.F. B161 Castanheira, F.V.S. B064 Bezerra, M.M. B051, B076, B258 Brito, Gerly A.D.C. B051, B076, B157 Castiglione, F. 047 Bhatt, L. B151 Brito, N.M. B126 Cavagnaro, J. 125 Bhongir, R.K.V. B285 Brock, C. B113 Cavalher-Machado, S.C. B312 Biagioli, M. 035, 036 Brodermann, M.H. B052 Ceccotti, M.-C. B252, B253 Biancone, L. 047 Brodmerkel, C. B030 Cehelsky, J. 083 Bica, M.S.R. B231 Brodskyn, C. B204 Cenac, N. B268 Biglarnia, A. 082 Brogliato, A.R. B046 Cenedeze, M.A. B144 Billin, A.N. 066 Broide, D. 130 Cervelli, J. B293 Bilton, D. B261 Bro¨nnimann, D. B002 Cervilha, D.A.B. B269 Bing, X. B116 Brown, A.C. B230, B303 Cha, J.J. B094 Binge, L. B209 Brown, D.A. B175, B178 Chaabo, K. 120 Biragyn, A. B047 Bru¨ggemann, T.R. B071, B291, B320 Chagas, A.S. B108 Bishop-Bailey, D. B273 Bruhns, P. 028 Chagas, J.R. B270 Bitto, A. B037 Brumbaugh, K. B004, B220 Chai, J.C. B090 Blank, S. B109 Brunelle, A.R. B106 Chai, Y.G. B090 Blanque´, R. B253 Brunger, J.M. 009 Chakravarthy, K. B242 Blumberg, R.S. 048, 089 Bruni, F.M. B291, B320 Chalmin, F. B035 Bocchi, E. B165, B166 Brunialti, M.K.C. B306 Chan, M. B311 Bodin, A. B097, B114 Bruschetta, G. B188 Chang, C.-K. 067 Bodogai, M. B047 Bruscoli, S. 035, 036 Channon, K.M. B052 Boehm, M. B018 Bryant, S. B016 Chantry, D. 118 Bogo, M.R. B229 Brys, R. B252, B253 Chao, W. B186, B227 Boichot, E. B097, B114 Buatois, V. 115 Charlotte, G. B280 Boire, G. B332 Budelsky, A.L. 054, B082 Chaturvedi, P. 083 Boivin-Jahns, V. B169 Burelle, Y. B170 Chaudhuri, J. B316 Bokil, N.J. B323 Buret, A.G. B225 Chaves, H.V. B051, B076, B078, B157, Bomfim, C.C. B087 Burgel, P.-R. 029 B158, B258 Bonifacio, J.P.P. B135, B141 Burgess, L. 118 Chaves, M.M. B119 Bonnart, C. B267 Burkly, L.C. B098 Chazaud, B. 065 Bonnel, D. B092 Buryachkovskaya, L.I. 109, B162, B171 Chazova, I.E. 107 Boraschi, D. B237 Bush, J. 083 Cheever, A.W. 054, B082 Borelli, P. B140 Byford, A. B022 Chelur, S. B313 Borges, P.V. B309 Chen, B. B110 Borges, V.M. B204 C Chen, F. B335 Borges, V.F. B064 Cabrera, A. B053 Chen, G. 082 Borilova Linhartova, P. B075 Cadwell, K. 016 Chen, G. B018 Borodovsky, A. 083 Caffarena, E. B309 Chen, H. B227 Borthwick, L.A. 054, B082, B095 Cai, B. B213 Chen, H. 099 Bortoluci, K.R. B217 Cain, D.W. B264 Chen, J. 030 Boshuizen, M.C.S. B164 Calabro, S. B054 Chen, M. B201 Bosiljcic, M. B262 Calixto, M.C. B286 Chen, P. B054 Bossa, F. 047 Campa, A. B042 Chen, Y. B311 Botega, A. B112 Campbell, I.L. B206 Cheˆne, G. B149, B280 Botham, K.M. B273 Campos, M.M. B229 Cheng, Q. 023 Bouchard, G. B191, B228 Can, G. B025 Cheng, X. B284 Bouche, G. B262 Can, S. B025 Cherkas, Y. B030 Bouchet, A. B002 Cande´a, A. B312 Chiang, N. 014, 062, B271 Bouillet, P. B235 Canesso, M.C.C. B317 Chiu, I.M. 058 Bourassa, J. 099 Canetti, C. B119 Chmilewsky, F. B321 Bourcier, N. B055 Cantu, D.A. B095 Cho, H. B091 Bouttier, M. B218 Caperuto, L.C. B299 Chollet-Martin, S. 027 Boylan, F. B251 Caprioli, F. 047 Chong, W.P. 030 Boyle, C. B034, B049 Cardell, L.-O. B041 Chouinard, L. B196 Bozza, F.A. B143 Caria, C.R.P. B130, B134 Chowdhury, K. B316 Bozza, P.T. B142, B143, B200, B214 Carmona, A.K. B217, B270 Choy, E.H. 113, 115 Braga, Y.A.V. B231 Carneiro, A.B. B142 Christensen, A.D. B027 Brandt, S. B007, B109 Carpentier, A.C. B170 Chumachenko, P.V. 109, B162 Braselmann, S. B311 Carrasco, S. B109 Chung, K.F.B295 Bra¨uer, E. B002 Carre´, J.E. B329 Chung, S.H. B321 Brayner, M.M.B. B157, B158 Carrier, N. B332 Ciambarella, B.T. B304, B305 Brazzell, K. 099 Carvalho, G.H. B142 Cicmil, M. B022, B242 Brea, D. B282 Carvalho, H.N.S. B142 Cidlowski, J.A. B264 Breit, S.N. B175, B178 Carvalho, L.A.C. B135, B141 Cimino, M. 035, 036 Breithaupt Faloppa, A.C. B184, B185, B187 Caspi, R.R. 030 Clare-Salzler, M.J. 039 Brenchley, J. 006 Cassali, G.D. B161, B292 Clarke, J.M. B102

123 S240 Inflamm. Res.

Clarke, S. B310 Das, R. 088 Dumas, A. B055 Clatworthy, A. B240 Das, S. B316 Dupont, S. B252, B253 Claudio Fantini, M. 047 Dasgupta, B. B030 Duran, A. B269 Clausen, B.E. B198 Dasgupta, S. B215 Durum, S.K. B029 Clottu, A. B035 David, B.A. B307 Colas, R.A. 062, B125, B271 David, C. B252 E Cole, J 118 D’A´ vila, H. B142, B214 Eastman, R. B083 Coler, R B084 deGraaf, K. 115 Ebel, P. B017 Colletti, N. B111 De Groot, A.S. B034, B049 Eberhardt, C. 118 Collins, J.E. B216 DeGroot, J. B092, B132, B243 Eberle, M. B017 Collison, A.M. B301 Dejani, N.N. B007 Eckman, J. B022 Colon, D.F. B180 de Jesus, F.N. B172 Edir, A. B267 Comeau, M.R. 054, B082 Delachaume, C. B252 Ediriweera, H. 044 Cominelli, F. B032 Delafield, N. B109 Efremov, E. B162 Cook, A.D 019, B027, B153 de Lima, K.A. B033 Egesten, A. B285 Cooper, D. B052 de Lima, T.M. B089 Eisenbarth, S.C. B054 Cooper, M.A. B099 Della-Casa, M. B144 Eklund, K.K. B117 Cooray, S.N. B274 Dellatorre-Teixeira, L. B200, B205 Elborn, S. 025, B261 Corazza, G.R. 047 De Maeyer, R.P.H. B121, B279 El Bounkari, O. B163 Cordaro, M. B188 de Min, C.115 Eliria, E. B004 Cordeiro, N.M. B248, B250, B260 Demir, A.M. B024 Elkins, J.S. 102 Corluka, S. B259 Demirtas, S. B024, B025 Ellekilde, G. B333 Cornicelli, J. B092, B132, B196 de Morais, I.C.O. B005 Elmore, S. B310 Correa da Rosa, J. 074 Denadai-Souza, A. B267, B268 Elson, G. 115 Correa, J.R. B129 Denlinger, B. 059 Emery, F.S. B249 Correa, L.B. B312 de Paula, M.A. B159 Engelman, R. B146 Correa, R. B129 DePierri, K. B128 Engstrom, L. B241 Corrigall, V. 120 Deraison, C. B267, B268 Enlow, E.M. 099 Costa, E.A. B156, B223, B245 De Vos, M. B252, B253 Errico, J.P. B113 Costa, H.C. B126 De Vos, S. B252, B253 Esmaeili, A. B168 Costa, R.S. B180 De Vriendt, V. B252, B253 Esposito, E. B188 Costa, S.K. B159, B172 de Winther, M.P.J. B164 Essilfie, A.T. B300, B303 Cotton, J.A. B238 Dias Fernandes, P. B251 Estelle, W. B280 Couillin, I. B114 Dias, D.F. B305 Estes, B.T. 009 Courad, P.O. B008 Dias, N.H. B290 Eum, S. B106 Courty, Y. B282 Dietrich, D. B139 Evans, E. 088 Cousens, L. B049 Dill, V. B048 Coutinho, D.S. B305 Dimasi, N. B110 F Coutinho, I.C. B081 Dimitrov, V. B218 Fahmi, H. B050 Coutinho-Silva, R. B046, B085, B119, B208 D’Imperio-Lima, M.R. B087, B217 Fahoum, L. B136 Couto, G.K. B172 Diot, P. B282 Fairlie, D.P. B013 Cox, A.J. B116 Di Sabatino, A. 047 Fajardo, O. 059 Croda, J. B154 Di Stasi, L.C. B108 Falach, R. B289 Croft, M. 069, B066 Djonov, V. B002 Faloppa, A.C.B. B296 Cross, J. B330 Djukanovic, R. B294 Faltus, R. B022, B242 Cross, V. B328 Do, A.T. B013 Famenini, S. B327 Crupi, R. B188 do Amaral Neto, E.S. B172 Fang, W. B201 Cuff, C. 119 Doan, A. B080 Fantozzi, E.T. B184, B185 Cummings, R.D. B105 Dodheri, S.S. B313 Farache, J. 038 Cunha, F.Q. 090, B033, B064, B180 Dogra, Y. B329 Farber, J.M. B198 Cunha, L.D. B118 Donadi, E.A. B019 Farmer, A.D. B113 Cunha, L.C. B156 Donahue, D. B095 Farokhzad, O.C. 055, 123 Cunha, T.M. B033, B064 Dong, C. B256 Farsky, S.P. B021, B042, B176 Curran, M. B030 Donovan, K.A. B123 Fassmann, A. B075 Cuzzocrea, S. B188 Dorwood, D. B095 Faulkner, A.R. B273 Czepielewski, R.S. B233 dos Reis, G. B204 Fautrel, A. B097 dos Santos, T.W. B130 Fayaz Ahmad, S. B010 D Dotan, S. B146 Febbraio, M. B170 Daino-Laizure, H. B080 Dotimas, H. B080 Fedosov, M. B182 Dalli, J. 014, 062, B271, B275 Dowds, M. 089 Feinberg, M.W. 045 Dalloneau, E. B282 Drewes, A.M. B113 Feldbru¨gge, L. B208 Damasceno, K.A. B292 Du, X.-J. B235 Felizardo, R.J.F. B144 da Matta, M.C. B081 DuBois, R.N. 011 Feng, Y. B186, B227 Darbro, B.W. B116 Dubourdeau, M. B149, B280 Ferguson, P.J. B116 d´Areˆde, S. B204 Duchene, J. B148 Ferlin, W. 115 Das, A. B090 Duffield, J.S. 052, B094, B098, B219 Fernandes, A.J. B305

123 Inflamm. Res. S241

Fernandes, A.J. B332 Futerman, A. B017 Greco, K.V. B319 Fernandes, C.M. B021 Gretzer, C. B048 Fernandes, J. B050 G Gridley, S.B080 Fernandes, L. B270 Gabay, C. 115, B139 Gronert, K. 012 Fernandes, P.D. B248, B250, B257, B260 Gadelha, C.A.D.A. B157, B258 Groom, T. B330 Fernandes, P. B071, B291, B320 Gal, Y. B289 Gros, A. B211 Ferna´ndez-Pen˜a, C. 059 Galien, R. B252, B253 Gro¨sch, S. B017 Ferreira, H.H.A. B290 Gallman, A. B054 Grosswald, R. B151, B261 Ferreira, L.C.S. B217 Gambero, A. B130, B134 Grupp, S.A. B335 Ferreira, R.G. B292 Gamble, J.R. 022 Gu, J.T. B326 Ferreira, S.G. B187 Ganiyu, O. B038 Guan, P. 088 Ferreira, T.P.T. B305 Gao, C. B110 Guerau-de-Arellano, M. 100 Fiala, M. B327 Gapon, L.I. B133, B167 Gueugnon, F. B282 Fichera, M. 047 Garcia, C.C. 093, B307 Gui, Y. B259, B332 Fiebich, B.L. B244 Garg, P. 083 Guigne´, C. B149 Fierro, I.M. B045, B126 Garrood, T. 120 Guilak, F. 009 Figliuolo, V.R. B208 Gavin, M.A. 103 Guillen, E. B034 Figueiredo, R.T. B231 Gavins, F.N.E. B059, B190 Guillon, A. B282 Figueiredo-Rinhel, A.S.G. B249 Geisslinger, G. B017 Guimara˜es, P.B. B270 Filgueiras, L.R. B101, B207 Georgii, J.L. B046 Gumperz, J.E. 086 Filho, G.C. B078, B157, B258 Germain, R.N. 030 Gundra, U.M. 044 Filho, S.M.P. B258 Gerner, E. B048 Gunn, H. B262 Finkel, D. B004 Gersbach, C.A. 009 Gunthuri, S. B080 Fiore, D. 067 Ghosh, K. B053, B106 Gupalo, E.M. 109, B162 Fiorin, V. B147 Ghosh, P. B316 Gupta, P. B013 Fischer, D.F. B092, B132, B243 Giambelluca, M.S. B077 Gupta, S. B106 Fitch, N. B111 Gicquel, T. B097, B114 Guttman-Yassky, E. 072 Fitzgerald, J. B335 Giera, M. B278 Guyre, P.M. B216 Florence, A.C. B267 Gillie´ron, C. B139 Florence, G. B114 Gilligan, M.M. B125 H Florentino, I.F. B156, B223, B245 Gilroy, D.W. 064, B121, B279 Haase, C. B027 Flower, R. B195 Giorno, T.B.S. B257 Hafezi-Moghadam, A. 032 Flynn, S.M. B297 Gira˜o, V.C.C. B078 Haibara, A.S. B161 Fock, R.A. B140 Glass, K.A. 009 Hajishengallis, G. 082 Fomochkina, I. B182 Glosson-Byers, N.L. B007, B183 Halai, R. B099 Fonseca, F.C.S. B272 Gobbetti, T. B273, B274 Halder, R. B327 Fontenot, J.D. 101, 102 Gochuico, B. B096 Hall, C. 120 Foster, P.S. B300, B303 Goedken, M. B293 Hall, W.L. B273 Frade, T.I.C. B161 Goerge, T. B211 Halsall, D. B195 Fraga, C.A.M. B248, B250, B260 Goes, P. B051, B076, B078 Hamilton, J.D. 073, B041 Frammartino, T. 035, 036 Goess, C. B310 Hamilton, J. B027, B153 Franc¸a, A.L. B051 Goh, H.Y. B102 Hammer, A. B100 Franc¸a, E.L. B147 Gold, L.S. B036 Han, M.-S. B065 Franca, R.F.O. B180 Goldman, N. B128 Han, Q. B325 Frances, R. B311 Golitsyn, S. B162 Han, S. B065 Francischi, J.N. B161, B272 Gomes, T.V. B081 Hanish, I. B301 Frates, C. B256 Gomez, I.G. B094, B098, B219 Hannen, R.F. B195 Fredman, G. B213 Gon, Y. B014 Hansbro, P.M. B230, B300, B301, B302, Fredman, G. 123 Gonc¸alves, L.O. B223 B303 Freire, J.M.D.O. B076, B078 Gonza´lez, B. B206 Hansen, F. B193 Freitas, A.R.D. B076, B158 Gonzalez, V. B335 Hansen, R. B036 Freitas, H.C. B157 Gordienko, A. B131 Hapgood, J.P. B263 Freitas, R.S. B051, B078, B157, B158, B258 Gorski, K. 103 Hardardottir, I. B278 Freitas, R.H.C.N. B248, B250, B260 Gosset, P. B282 Harrison, L.C. B102 Freitas-Limas, L.C. B317 Gotardo, E.M.F. B130, B134 Hart, K.M. 054, B082, B096 Frey, N. B335 Graber, W. B002 Hartigan, A. B022 Freysdottir, J. B278 Gracias, DT. B066 Hartman, D. B310 Fu, W. 038 Graff, C. B310 Hartmann do Prado, C. B324 Fu, X.-N. B073 Graham, G.J. B064 Harvey, R. B034 Fuerstenberg, R.K. B220 Graham, N. B041 Hasegawa, Y. B189 Fujimori, M. B147 Graham, S.M. B028 Hashimoto, N. B189 Fukuda, D. B173 Granger, D.N. B059, B190 Hassanpour Golakani, M. B175, B178 Fukunaga, Y. B246 Granucci, F. B210 Hatterer, E. 115 Fullerton, J. B279 Grassi-Oliveira, R. B324 Hattori, H. B254 Funchal, G.A. B233 Greaves, D.R. B052, B056 Haupt, T.H. B333 Furuichi, K. B138 Grebe, K. B310 Haw, T. B300, B301, B302

123 S242 Inflamm. Res.

Hayden, K. B030 Ishizuka, E.K. B101, B207 Kaneva, M.K. B255 He, S. B088 Italiani, P. B237 Kang, J.X. B224 Headland, S.E. B255 Ito, J.T. B269, B314 Kang, M.-J. B152 Heckrodt, T. B311 Iula, L. B234 Kanga, U. B316 Hedrick, M.N. B198 Ivanov, A.V. 119, B016 Kanjilal, M. B316 Helluy, X. B169 Ivashkiv, L. 004 Kankova, K. B075 Heluany, C.S. B021 Iwabuchi, K. B107 Kannan, G. 103 Henriques, M. B309, B312 Iwata, Y. B138 Kano, F. B318 Henry, C. B282 Iyer, A. B013 Karaca, T. B024, B025 Herbst, R. B110 Izakovicova Holla, L. B075 Karnell III, F. B110 Herrmann, M. B012 Izuhara, K. 118 Karow, M. 103 Hertzog, P.J. B230 Izumi, K. B070 Karp, J.M. 057 Herwald, H. B145 Kasaian, M.T. B096 Hibi, Y. B254 J Kaser, A. 089 Hickey, M.J. 023 Jack, R. B219 Kasetty, G. B145, B285 Hickey, P. B235 Jaeger, N. B233 Kasper, D.L. 089, B215 Hicks, A. B241 Jahns, R. B169 Kassuya, C.A.L. B154, B177 Higashikuni, Y. B173 Jakob, P. B169 Kawahata, N. 083 Higuchi, M. B165, B166 James, A. B004 Kawai, S. 078 Higuchi, T. B169 Jancar, S. B101, B207 Kawakami, J.T. B165, B166 Hiller, K.-H. B169 Jancic, C.C. B234 KB, Charamanna, B313 Hirata, M. B087 Jandrot-Perrus, M. B211 Keely, S. B300 Hixon, J.A. B029 Jang, J. B065 Kehoe, P.G. B120 Hobbie, S.N.K. B281 Jankovic, D. B083, B150 Keitelman, I. B234 Hoda, U. B294 Jarnagin, K. B256 Kelley, R. B311 Hoeksema, M.A. B164 Jarolimek, W. 111 Kelly Palma, R. B308 Hoff, C.C. B270 Jelinsky, S. B088 Kelsall, B.L. B198 Holers, V.M. 083 Jenne, C.N. B232 Kendall, A. B273 Hollander, C.M. B047 Jensen, N.M. B216 Kennedy, T.P. B039, B040 Holloway, P.M. B059 Jeon, S.G. B152 Kerwin, E. 118 Holtappels, G. B041 Jeong, Y.-J. B152 Keskin, D. B026 Honda, Y. B247 Ji, R.-R. B325 Keskin, G. B001, B026 Hono´rio-Franc¸a, A.C. B147 Jian, W. B186 Kessoku, T. B247 Hopkins, R. 118 Jiang, J. B284 Khanna, K.M. B104 Horan, G. 047 Jijon, H. B240 Kharchenko, V. B182 Horner, M.J. 127 Jittayasothorn, Y. (Ed), 030 Khimich, N. B131 Horsley, A. B261 Jiwrajka, N. B096 Kichko, T. 059 Horvat, J.C. B230, B300, B301, B303 Johnson, B. B094 Kilty, I. 041 Hoshino, T. B068 Jonasdottir, H.S. B278 Kim, B.Y. B174 Ho-Tin-Noe´, B. B211 Jonassen, T.E.N. B274 Kim, G.-W. B065 Hottz, E.D. B143 Jones, B. B300 Kim, H.-R. B067 Hsieh, C.-M. 119 Jones, H.E. 001, 043, 079 Kim, J. B194, B334 Huang, S. B125, B127 Jones, H.R. B103 Kim, J.-A. B265 Huang, Y. B018 Jones, S. 115 Kim, K. B174 Huard, J. 084 Jonesiatauro, S. B256 Kim, K.W. B067 Huber-Lang, M. 082 Jorge, R.J. B006 Kim, R.Y. B230, B300, B301 Hugunin, M. 119 Jouan, Y. B282 Kim, Y.H., B325 Huh, J. B179 Juban, G. 065 Kindrachuk, K.N. 054, B082, B095 Huss, D.J. 102 June, C. B335 Kirchner, J. 103 Huynh, D.N. B170 Jung, K.H. B090 Kiriazis, H. B235 Jung, Y.-K. B065 Kirkham, B. 120 I Kita, H. 129 Iadonato, S.P. 033 K Kitasato, H. B003, B107 Ianni, B. B166 Kagawa, L. B107 Klase, Z. 006 Icimoto, M.Y. B217 Kaieda, S. B068 Klasen, C. B163 Ikegami, R.N. B165, B166 Kaiser, F. B169 Klaunberg, B. B095 Imai, R. B003 Kaliannan, K. B224 Klaus, E.J. B281 Imajo, K. B247 Kalyan, S. B262 Kleef, R. B262 Inagaki, Y. B093 Kamaly, N. 123 Klein, A. B292 Inal, A. B001, B026 Kamboj, D. B315 Klein, P. B328 Inman, M.D. B301 Kamimura, Y. B137 Kleindl, P. B328 Iqbal, A.J. B052, B056 Kaminski, H. 083 Klingel, K. B169 Irimia, D. B239 Kamp, M. B209 Knight, A. B120, B329 Isaacs, J.D. 116 Kanashiro, A. B064 Knight, D.A. B301 Isaias, P.H. B051 Kaneko, Y. B070 Ko, H.-K. B287 Ishida, K. 103 Kaner, Z. B146 Ko, K.-M. B044

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Koga, M.M. B101 Lee, E.-J. B065 Lopes, J.F. B143 Kogelnik, N. B008 Lee, J.-Y. B152 Lo´pez-Lo´pez, J.R. 059 Kojima, F. B003, B107 Lee, N. B004 Loser, K. B011 Kolaczkowska, E. B232 Lee, S.-H. B015, B067 Lourenc¸o, J.D. B269 Komuro, I. B173 Lee, T.-S. B287 Louw-du Toit, R. B263 Konkiewitz, E.C. B154, B177 Lee, W.Y. B039, B040 Louzada, P. B033 Korenchan, D. B326 Lee, W.-Y. B232 Lovett-Racke, A. 100 Kosco-Vilbois, M. 115 Lee, Y.S. B090 Low, M. 067 Kou, Y.R., B287 Lehner, K.A. B127 Low, P.S. B328 Koutsogiannaki, S., 082 Leita˜o, R.F.D.C. B157 Loyau, S. B211 Kovalenko, E., B122 Leite, C.E. B229 Loyer, P. B114 Kovanen, P.T., B117 Lelias, S. B034 Loza, M. B294 Kra¨henbu¨hl, S. B058 Lemos, D. 067 Lu, A. B201 Kranich, J. B102 Leong, P.K. B044 Lu, Y. B328 Kraus, V. B010 Lesmes, U. B136 Lubell, W.D. B170 Krishna Babu, R.D. B313 Leung, J. B327 Lucisano-valim, Y.M. B249 Krishnaswamy, J.K. B054 Leverson, J. B310 Ludwig, N. B262 Kronman, C. B289 Levy, B.D. 091 Luger, T.A. B011 Krueger, J. 071 Lewis, E.C. B146 Luis, E. 059 Kubes, P. 087, B232 Li, C.-Y. B226 Lukaszewski, R. B329 Kubyshkin, A.V. B122, B131, B182 Li, D. B086 Luna-Gomes, T. B200, B205 Kubyshkin, V. B122 Li, H. B175, B178, B311 Luo, L. B323 Kudo, G.K. B187 Li, J. 022 Lupher, M. B219 Kumar, A. B244 Li, L. 103 Lust, J.A. B123 Kumar, P. B316 Li, L. 005 Lyng, G.D. B028, B298 Kumar, U. B316 Li, S. B212 Kumari, S. B197 Li, W. B029 M Kuo, S. B294 Liang, P. B332 Macchione, M. B299 Kupa, L.K. B021 Liao, W. B201 Macedo, C.G.D. B158 Kurhanewicz, J. B326 Libert, C. 034 MacGregor, G. B261 Kusner, L. 083 Lichtnekert, J. B094 Machado, F.R. B306 Kuznetsov, V.A. B133, B167 Lidani, K.C.F. B072 Mackay, C.C. B317 Kyeong-A, K. B043 Lightman, S. B195 Mackay, C.R. B102, B209 Lim, Y.M. B222 MacKenzie, J.D. B326 L Lima, C.B. B217 MacKenzie, R. B259 Labe´gue`re, F. B253 Lima, C.K.F. B160 Maddox, D.E. B297 Lacerda, J.T.J.G.D. B157, B258 Lima, D.B. B006 Maehana, S. B003, B107 Lacerda-Queiroz, N. B083 Lima, L.D.C. B317 Mafezoli, J. B078 Lacey, D.C. B153, B235 Lima, M.A.S. B076 Mafra de Lima, F. B308 Lacey, S. B335 Lima, O.C.O. B148 Magalha˜es, J.T. B051 Lacroix, S. B055 Lima, W.T. B296 Magalha˜es, K.G. B129 Ladelund, S. B333 Lima-Junior, D.S. B118 Mahalingam, N. B313 Laflamme, C. B055, B077 Lin, A.-H. B287 Mahalingam, S. 092 Lage, S.L. B214, B217 Lin, L.-L. B088 Maher, L. B104 Lagente, V. B097, B114 Linhares, A.G.F. B051 Maher, T.M. 026 Laissue, J.A. B002 Linhares, E. B051 Maia, M.B.D.S. B158 Lakshminarasimhan, A. B313 Lino, R.C. B223, B245 Maia-Dantas, A. B172 Lam, F.F.Y. B192 Lira, F.B. B159 Maier, P. B281 Lamagna, C. B311 Litherland, S.A. 039 Mailhot, B. B055 Lamana, S.M.S B158 Liu, D. B054 Majima, M. B003, B107 Lamberth, S. B030 Liu, G. B301 Maksymowych, W.P. B259, B332 Lambris, J.D. 031, 082 Liu, J. B191, B228 Malaviya, R. B293 Lamerdin, J. B080 Liu, M.-H. B287 Malchenko, O. B182 Lamrani, L. B211 Liu, T. B325 Malfait, A.-M. 008 Lanes, V.R. B087 Llewellyn, D. B120 Malkina, T. B162 Langer, M.N. B048 Lloyd, A. B328 Malle, E. B008 Lapeyre, G. 115 Lobo, B.W. B160 Malmstro¨m, E. B145 Laskin, D.L. B293, B322 Locati, M. B064, B305 Malmstro¨m, J. B145 Laskin, J.D. B293, B322 Lockett, T.J. B102 Mancuso, R.V. B058 Lassounskaia, E. B087 Lohse, M. B169 Manenschijn, J.-A. 059 Latham, S.H. B273 Loiola, R.A. B042 Mangan, N.E. B230 Laukens, D. B252, B253 Loke, P. 044 Mangini, S. B165, B166 Le, W. B212 Lomakin, N.V. B171 Manko, A. B238 Leaf, I. B094 Lombardi, M.S. B139 Mannent, L. B041 Leamon, C.P. B328 Lopes, F.D.T.Q.S. B269, B314 Mansikka, H. 119 Lee Chang, C. B047 Lopes, F.M.L.D.S. B158 Mantovani, A. 002

123 S244 Inflamm. Res.

Maradana, M.R. B302 Merciris, D. B252, B253 Mullins, D.W. B262 Marchi, L.F. B019 Mernak, M.M.B. B299 Muniz, V.S. B205, B231 Marcy, B. B280 Meseguer, V. 059 Munoz, E.J. 033 Marinho, A.D. B006 Messias-Reason, I.J.T. B072 Munoz, L. B012 Marino, E. B102 Metzler, S. B216 Murakami, R. B003 Mario, R.N. B016 Meyaard, L. 050 Murakumo, Y. B107 Marks, D. B279 Meyer, J.R. B046 Muraro, S.P. B233 Marleau, S. B170 Meyron, E.G. B136 Murtaza, A. B242 Marotta, A. 095, B030, B259, B332 Meyron-Holtz, E. B032 Muscara, M.N. B159, B172 Martin, C. 029 Michienzi, S. B259, B332 Musikhina, N.A. B133, B167 Martin, D. 103 Mikchailichenko, V. B182 Martin, W. B034, B049 Milanesi, L. B237 N Martins, A.M.C. B005, B006 Mille-Baker, B. B092, B132, B243 Nagamoto, T. B014 Martins, C.D.S. B051, B076 Mimura, K.K.O. B319 Nahrendorf, M. 121 Martins, M.A. B304, B305 Ming, J.E. B041 Nair, P.M. B301, B302 Martins, M.A. B071, B269, B291, B296, Mingozzi, F. B210 Nakada, N. B107 B299, B314, B320 Miranda, A.L.P. B160 Nakagawa, N. B098, B219 Martins, P.R. B307 Miriam, G.S. B055 Nakagawa, S. B094 Masenko, V.P. 107 Mironova, N.A. 109, B162 Nakajima, A. B247 Masetto, A. B332 Misaki, K. B014 Nakamura, M. B003, B107 Mason, L.J. B209 Mita, T. B254 Nakamura, Y. B014 Massis, L.M. B217 Mitra, D.K. B084, B315, B316 Nakano, Y. B137 Masuda, E. B311 Mittelsteadt, K. B094 Namdeo, M. B084 Mathis, D. 038 Miyoshi, E. B177 Napimoga, J.T.C. B158 Matos, N.A. B292 Mizrachi Nebenzahl, Y. B146 Nascimento, M.S.L. B054 Matsubara, K. B318 Moaaz, M. B283 Nascimento, T.S. B260 Matsushima, K. B093 Mohammad, M.G. B175, B178 Natoli, G. 003 Matsushita, Y. B189 Mohan, A. B315 Navarro, R. B214 Matta, K. B331 Mohideen, U. B106 Navia, B. 059 Mattes, J. B300, B301 Moller, T. 022 Navrotska, V. B236 Matteucci, K.C. B217 Monaghan, K. 020 Nedospasov, S.A. 067, 108 Mauad, T. B314 Monk, P.N. 081 Neele, A.E. B164 Maude, S. B335 Monnet, E. 115 Negro, A. B018 Mauro, P. B274 Monteiro, C.M. B309 Nellore, K. B313 Maus, M. 117 Monteiro, H.S.A. B006 Nery Porto, B. B324 Mayall, J.R. B230, B300 Monteiro, R.C. 051 Nery, L.R. B229 Maya-Monteiro, C.M. B200, B214 Monteleone, G. 047 Neto, B.A.D. B129 Mayranpaa, M.I. B117 Montero-Melendez, T. B274 Neurath, M.F. 047 McElroy, M. B092 Montes, M.B.A. B101 Neves, J.F. 089 McInnes, I.B. 094, 115 Moore, A. B255 Neves, J.S. B046, B231 McIntosh, F. B196 Moore, K.J. 044 Newson, J.S. B121 McKay, D. B267 Moraes Sernaglia, E. B203 Ng, E.S.K. B192 McLeod, K.H. B102 Moraes, A.R. B319 Ng, N.H. B192 McMurtrie, D. B311 Morais, I.C.O. B006 Nguyen, D. B091, B331 McNeill, E. B052 Morais, T.C. B147 Nguyen, D.H. B301 Medeiros, A.I. B007 Morand, E.F. 020, 023 Nguyen, J. B326 Medeiros, A.C.D. B078 Morandi, V. B126 Nguyen, L. B326 Medeiros-de-Moraes, I. B143 Morandini, A.C.F. B085 Nichols, K.E. 088 Mehta, D.S. 102 Morato, P.N. B154, B177 Nicolaou, A. B273 Melenhorst, J. B335 Moreira, G.C.P. B290 Nieswandt, B. B211 Mello, P.A. B208 Moreira, L.F.P. B187 Niewoehner, J. B036 Melo, P.H. B064 Morello, E. B282 Nilsson, B. 082 Melo, R. B214 Moret, K.H. B309 Nilsson-Ekdahl, K. 082 Melton, L. 083 Mo¨rgelin, M. B048, B285 Nishikawa, M. B189 Memari, B. B218 Mori, P. B255 Nisihara, R.M. B072 Menard, H.A. B332 Moritoh, K. B047 Noels, H. B163 Me´nard, L. B170 Morris, D. B095 Nordin, S. B048 Mendes, J.A. B290 Mosca, E. B237 Norling, L.V. B103 Mendonc¸a, K.M. B051 Motta, J.-P. B225, B238, B267, B268 Norris, P. 062 Menegatti, R. B156, B223, B245 Motwani, M. B121, B279 Norton, K. 033 Menezes, R.r.P.P.b. B006 Mounier, R. 065 Nowak, P. 099 Meng, G. B201 Moutos, F.T. 009 Nurmi, K. B117 Meng, J. 046 Mowen, K. B232 Nusshold, C. B008 Menon, M. B104 Mueller, A. B086 Nys, K. B253 Meotti, F.C. B135, B141 Mukaida, N. B062 Nyska, A. B032 Mercier, M. B259 Mukherjee, S. B313

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O Panjwani, P.A. B169 Piquereau, J. B170 O’Byrne, P.M. 024 Pantaleao, L.N. B176 Pirozzi, G. B041 O’Connor, L. B310 Pa˜o, C.R.R. B205 Pitchford, S. B288 Ogawa, F. B003 Papanastasiou, M. 082 Pizzino, G. B037 Ogawa, Y. B247 Papareddy, P. B145 Plastira, J. B100 Oh, S. 089 Parada Kusz, M.M. B240 Pochapski, J.A. B177 Ohayon, D. 029 Parent, I. B253 Polo, J.M. B102 Ojcius, D.M. B085 Parente, A.T.D.P.P. B076 Pomerantzeff, P. B165 Okhale, S.E. B155 Park, C.-K., B325 Porter, D. B335 Olajide, O. B244 Park, G. B311 Porto, B.N. B233 Oland, S.D. B096, B150 Park, H.-R. B065 Poskerova, H. B075 Oliani, S.M. B319 Park, J.-H. B152 Pot, C. B035 Oliveira Correa, R. B203 Park, S.-H. B057 Pouliot, M. B055, B077 Oliveira Filho, R.M. B184, B185 Paschoalato, A.P. B019 Poulsen, J.H. B333 Oliveira, A.C. B102, B209 Paschoalin, T. B270 Powell, C. 083 Oliveira, C.C. B130 Pasparakis, M. B197 Prada, P.O. B101 Oliveira, D.C. B140 Pasricha, T. B020, B096 Prado, C.M. B296, B299 Oliveira, E.M. B042 Paterniti, I. B188 Prado, M.A.M. B296, B299 Oliveira, J.M. B071 Patricio, E.S. B135 Prado, R. B096 Oliveira, M.C.V.C. B081 Patton, D. B262 Prado, V.F. B296, B299 Oliveira, R.R. B019 Pavlides, S. B294, B295 Prestwich, G.D. B039, B040 Oliveira, S.S. B208 Paya´, E.P. B005 Prevost, T. 120 Oliveria, R.A. B045 Payan, D. B311 Primikyri, A. 082 Olivo, C.R. B269, B291 Pearson, J. 103 Prinjha, R. 038 Ollivier, V. B211 Peckham, D.W. 033 Privat, S. B091, B331 Olsen Saraiva Camara, N. B308 Peer, D. 056 Proulx, C. B170 Olson, L.M. B016, B310 Penido, C. B309 Ptacek, J. B331 Olszak, T. 089 Pequignot, E. B335 Pugh, M. B328 Oluwole, O.G. B038 Pereira, G.J.S. B005 Pulsipher, A. B039, B040 O’Mahony, A. B091, B331 Pereira, J.J. B166 Pupo, M.T. B249 Onali, S. 047 Pereira, J.A. B290 Purcell, R. B273 Ong, H. B170 Pereira, K.M.A. B051, B076, B078, B157, Ong, W. 099 B258 Q Ongenaert, M. B253 Pereira, T.C.B. B229 Qin, S. B050 Ono, J. 118 Peres, R.S. B180 Quaresma, P.G. B101 Oon, S. 020 Perez, P. B195 Queiroz-Junior, C.M, B307 Oorni, K. B117 Pe´rez-Garcı´a, M.T. 059 Opdenakker, G. B232 Perini, A. B296 R O’Reilly, L. B235 Perng, D.-W. B287 Rachid, M.A. B060, B307 Orlandi, I. B210 Perper, S. B310 Racke, M. 100 Orlando, A. 047 Perret, L. B252 Rajamaki, K. B117 Orr, S.K. B271, B275 Perretti, M. 063, B103, B176, B255, B273 Ramachandra, M. B313 Orza´ez, M. B005 Pesquero, J.B. B270 Ramadas, R.A. B241 Osipova, I.V. B133, B167 Petasis, N.A. B271 Ramakrishnan, S. 046 Ostanin, K. B086 Petelina, T.I. B133, B167 Ramalingam, T. 054, B020, B082, B095, O’Sullivan, B. B302 Peterfy, C. 097 B096 Ouimet, M. 044 Petersen, L. B324 Ramanathan, A. B313 Ouwehand, K. B092 Petrlova, J. B193 Ramirez Vinolo, M.A. B203 Owen, D. B088 Petrof, B.J. 065 Ramon, S. 062, B125, B127 Oxenkrug, G. B236 Petroni, R.C. B089 Ramos-Ju´nior, E.S. B085 Ozısık, L. B001 Pewzner-Jung, Y. B017 Ranger, P. B050 Philbin, M. B036 Rao, N.K. B313 P Philibert, R.A. 037 Rao, N. B039 Pacheco, P. B309 Philpot, E. B261 Rao, N. B295 Pacheo-Silva, A´ . B144 Philpott, M. B195 Rao, S. B328 Padley, R. 119 Pica, R. 047 Raphael, S. B259 Padua, L. 093 Piccinelli, A.C. B154, B177 Rashid, H. B196 Page, C.P. B288 Pichavant, M. B282 Rasmussen, L.J.H. B333 Paiva, L.A. B205 Pimenta, A.T.A. B076 Rathinam, V. 017 Pallio, G. B037 Pinheiro, N.M. B296, B299 Rathod, S.B. B313 Pallone, F. 047 Pinkerton, J.W. B300 Rayner, K.J. 044 Pamuk, G.E. B023, B024, B025 Pinto, A.C. B257 Rebelatto, M.C. B110 Pamuk, O.N. B023, B024, B025 Pinto, I.R. B258 Rechberger, G. B008 Panayi, G.S. 120 Pinto, M.C.X. B148 Reed, S.G. B084 Pandit, C. B313 Pinto, V.P.T. B078, B157, B258 Reeh, P. 059 Panigrahy, D. B125, B127 Pioli, P.A. B216 Regan-Komito, D. B056

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Rehal, S. B031 Saadi, J. B277 Schumacher, J. B322 Reicher, H. B008 Sabbione, F. B234 Schwartz, A. B310 Reico, C. B052 Sabo, T. B289 Sciurba, J. B096 Reis, M.M. B165, B166 Saclier, M. 065 Scott, H.A. B053, B106 Renauld, J.-C. B282 Sacramento, E.K. B317 Scribano, M.L. 047 Riani, A. B269 Sagong, B. B327 Seliger, N. B289 Ribault, C. B114 Saito, R. B014 Sen˜arı´s, R. 059 Ribeiro de Carvalho, P. B251 Saito, S. B070 Seong, J. 082 Ribeiro, K.A. B258 Sakai, N. B138 Serduc, R. B002 Ribeiro, M.C. B290 Salant, D. 083 Serezani, C.H. B007, B109, B183, B207 Ribeiro, S.C.M.B087 Saldiva, P.H. B299 Serhan, C.A. B271 Ricardo da Silva, F.Y. B184, B185 Saleh, R.A. B153 Serhan, C.N. 014, 062, B125, B127, B275 Riccardi, C. 035, 036 Sales, D.S. B269, B314 Serra, M.F. B305 Richard, G. B049 Salgado, R.M. B087 Servant, M.J. B077 Richards, J.L. B102 Salles, T.S. B142 Severin, M. 100 Ricklin, D. 082 Salomao, R. B306 Sevilla, L. B195 Riester, K.A. 102 Salvemini, D. 106 Sevilla, R. B022 Rietdorf, J. B214 Samadfam, R. B196 Shah, S. B265 Rigato, O. B306 Samajdar, S. B313 Shahaf, G. B146 Riggs, J.E. B128 Sanchez Crespo, M. 013 Shang, L. 115 Rios, F.J. B101 Sa´nchez, A. 059 Sharf, T. B162 Risitano, A.M. 082 Sandri, T.L. B072 Sharma, A. 102 Riteau, N.D. B083, B150 Sang, H.-W. B073 Sharma, N.K. B306 Rivard, J. B004 Sannomiya, P. B187 Shaw, M. B111 Robert, S. B097, B114 Santana, F.P.R. B296, B299 Shaw, P.A. B335 Robinson, G. B022 Santana, P.T. B208 Sher, A. B083, B150 Robson, S.C. B208 Santi-Gadelha, T. B157, B258 Sher, L. 118 Rocha, G.C. B085 Santos, A.R.D. B158 Sher, N. B276 Rocha, T. B290 Santos, E.W. B140 Sheridan, J.P. 102 Rodrigues Garbin, S. B184, B185 Santos, E.P.A. B160 Shichino, S. B093 Rodrigues, F.E.A. B078 Santos, R.A.S. B317 Shim, R. B209 Rodrigues, M.M. B217 Santos, R.A.S. B148 Shimojima, C. B254 Rogai, F. 047 Santos, R.D.S. B158 Shinozaki, Y. B138 Rogers, S. B298 Santus, W. B210 Shirinian, L. B110 Rohrbeck, L., B235 Sanz, M.J. B232 Shramko, Y. B131 Rojas, M. B079 Sapoznikov, A. B289 Silva Jr., C.D. B081 Roland, C. B267 Saraiva-Romanholo, B.M. B071 Silva, A.L.N. B118 Rolland Fourcade, C. B268 Sasaki, S.D. B269 Silva, A.A.R. B258 Rollet-Labelle, E. B077 Sasaki, S. B063 Silva, A.G.S. B305 Romano-Silva, M.A. B317 Sasso, E.H. 096 Silva, B.V. B257 Ropero, D. B251 Sata, M. B173 Silva, C.L.M. B208 Roque, N.R. B214 Satoh, M. B107 Silva, D.P.B. B156 Rosa, D.V. B317 Sattler, W. B008, B100 Silva, D.P.B. B245 Rosado, M. B298 Satyanarayanan, S.K. B276, B277 Silva, E. B306 Rosas, E.C. B312 Sauer, J.M. 126 Silva, F.R.L.D. B078, B158 Ross, A.K. 009 Saunders, M. 118 Silva, F.S. B309 Rossi, F. 067 Savage, J.R. B039, B040 Silva, J.M. B060 Rossios, C. B294, B295 Savarino, V. 047 Silva, J.R. B180 Rossmann, C. B008 Savill, J. B259, B332 Silva, J.S. B054 Rossoni, L.V. B172 Savio, L.E.B. B208 Silva, L.F.F. B129 Rothlin, C. 015 Savion, O. B032 Silva, M.S. B290 Rowe, A. B294 Sawchenko, P.E. B175 Silva, P.M.R. B304, B305 Rowe, D.C. B110 Scharfstein, J. B085 Silva, R.V. B160 Roy, S. 046 Schaub, R. 088 Silva, R.C. B144 Rubattino, R. B327 Schepman, P. B036 Silva, T.S. B245 Ruitenberg, M. B175, B178 Schett, G. B012 Silva, T.A. B060 Ruscher, R. B302 Schettini, J.A.C. B081 Silveira, J.A.M. B006 Russell, K. B295 Scheuplein, F. 088 Silver, P.B. 030 Russo, R.C. B060, B064, B307 Schiding, J. B111 Simo˜es, R.L. B126 Rutgeerts, P. B253 Schiffmann, S. B017 Simon, B. B113 Ruzek, M. 119 Schif-Zuck, S. B276, B277 Simon, F. B079 Ryan, M.E. B039 Schindler, S. B244 Sims, G.P. B110 Schmitz, C. B163 Sinflo´rio, D.A. B119 S Schneider, C. B105 Singh, R. B311 Sa, Y.A.J. B304 Schofield, Z.V. B099 Singh, T.P. B198 Saad, M.S. B101 Schopf, L.R. 099 Siracusa, R. B188

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