Inflammation, Immunity and Cancer: and Other Leukocytes

The Society For Leukocyte Biology’s 49th Annual Meeting and “ 2016”

September 15-17, 2016 • University of Verona Congress Center • Verona, Italy Abstract Book

www.leukocytebiology.org 1 1. Cytoskeletal adapter proteins Kindlin-3 and Talin- 1 anchor clustered b2-integrins to the cytoskeleton Micro Scale in Vitro Models to Study Neutrophil and facilitate the transition from arrest to diapedesis. Behavior: Past, Present and Future This process represents a gatekeeper mechanism that David J. Beebe, University of Wisconsin regulates the rate and number of neutrophils that across endothelium and gain access to inflamed Microfluidics is now a mature field. The basic tissue. We recently reported that tensile force acts on physics of microfluidics are now well described and LFA-1 (CD11a/CD18) bonds inducing their a plethora of components, devices and systems have colocalization with Orai1, the predominant been demonstrated. The study of cell migration membrane store operated Ca2+ channel that (specifically neutrophils) was one of the first uses of cooperates with the endoplasmic reticulum to elicit microfluidics in cell biology research. I will review cytosolic flux. Employing custom fabricated our history of developing and applying micro scale microfluidic flow channels combined with total phenomena and technology to enable studies of internal reflection fluorescence microscopy, we neutrophil function. While our original focus was to applied defined shear stress to low- or high affinity enable and improve basic studies of cell migration, LFA-1 and imaged the spatiotemporal regulation of our current focus is multifold. First, we strive to bond formation with Kindlin-3 recruitment and make the systems simple and, thus, more accessible Ca2+ influx. We will present a model by which and useable by a broader community. Second, we neutrophils use focal adhesions as mechanosensors have begun to apply the systems in studies involving that convert shear stress–mediated tensile force into human subjects to, for example, understand immune local bursts of Ca2+ influx that catalyze cytoskeletal system regulation in response to various lifestyle engagement and protrusive force to drive a factors (e.g. caffeine, exercise, alcohol, meditation). migratory phenotype. Third, we have begun to build organotypic models that more accurately recapitulate structure/function 3 relationships to study neutrophil trafficking. These models include engineered blood vessels within Microfluidic Chambers to Screen Neutrophil complex 3D microenvironments to study neutrophil Killing of Aspergillus Fumigatus in the Presence migration (and reverse migration) in various of Novel Bifunctional Small Molecules contexts. While, the use of micro scale devices to Caroline N. Jones1,2, Benjamin P. Heithoff1, Kevin study leukocyte biology has a long history (actually Forrest3, Kevin Judice3, Mark C. Poznansky2, James predating the field of microfluidics), the next decade F. Markmann2, Jatin M. Vyas2, Daniel Irimia2 appears to be primed to begin to reap the full 1Virginia Polytechnic Institute and State University; benefits of these now well understood technologies 2Massachusetts General Hospital and Harvard in furthering our understanding of leukocyte Medical School; 3Cidara Therapeutics biology. The contribution of human neutrophils to the 2 protection against fungal infections by Aspergillus fumigatus is essential but not fully understood. On-Chip Phenotypic Analysis of Leukocyte Whereas healthy people can inhale spores of A. Inflammatory Recruitment in Acute and Chronic fumigatus without developing disease, neutropenic Diseases patients and those receiving immunosuppressive Scott I. Simon, University of California, Davis drugs have a higher incidence of invasive aspergillosis. We present a novel microfluidic Neutrophil arrest and migration on inflamed platform in which the interactions between human endothelium involves tethering via selectins and neutrophils and A. fumigatus were observed in real ligation of G-protein coupled receptors, both time, at single-cell resolution, in precisely controlled signaling mechanisms can elicit a conformational microenvironments. The design of the microfluidic shift in the b2-integrins (CD11/CD18) to a high- platform requires neutrophils to migrate along a affinity and clustered state that determines the migration channel to reach nanoliter-sized chambers, strength and lifetime of bond formation with ICAM- where they subsequently interact with A. fumigatus. Time-lapse imaging allows the recording of have implications for the faster diagnosis and start of neutrophil phenotypes including chemotaxis, NET treatment during infections. release, swarming, and , while simultaneously monitoring A. fumigatus growth 4 rates. The platform technology enabled the robust screening of neutrophil trafficking towards and Rapid Microfluidic Immune Cell Migration killing of A. fumigatus in the presence of novel Analysis for Biologically Oriented and Clinically bifunctional small molecules (Cloudbreak Oriented Research technology, C-001 and C-016). The Cloudbreak Francis Lin, University of Manitoba technology (C-001 and C-016) is a bifunctional small molecule utilizing a targeting moiety Directed cell migration is a complex cellular (antifungal agent) that binds to the cell wall of A. function that critically mediates a broad range of fumigatus and is conjugated to an effector moiety physiological and pathological processes such as (chemotactic peptide fMLP) that attracts and immune response and cancer metastasis. Chemical activates neutrophils, directing them to the site of concentration gradient is an important guidance cue infection and priming them for killing. We found for many cell types, particularly the diverse immune that neutrophils alone are weakly attracted to and cell types. Microfluidic devices can precisely modestly control the growth of A. fumigatus hyphae configure cellular microenvironments and therefore (49%). C-001 [100 nM] produced a high influx of have been increasingly employed to investigate the neutrophils and reduced hyphal growth to 5%. mechanism of immune cell migration and Chambers containing C-016 [10 nM] plus chemotaxis. Recently, microfluidics-based immune neutrophils were reduced to <1% hyphal growth. C- cell migration studies have also shown promise for 016 is significantly more effective at priming enabling clinical diagnostic applications. In this talk, neutrophils for effective fungal killing than controls I will discuss our recent work in the development of – fMLP or anti-fungal agent. Neutrophils from new microfluidic devices to study the biological patients receiving immunosuppressive treatment mechanisms of human blood neutrophil migration after kidney transplantation were less effective and chemotaxis. Some of these studies are connected against the fungus than those from healthy donors to disease models such as chronic kidney disease and and broader heterogeneity exists between patients, fibrosis. In addition, I will discuss the development compared with healthy individuals. The fungal of an integrated microfluidic cell migration system killing capacity of neutrophils isolated from to enable rapid all-on-chip human neutrophil immunosuppressed patients was significantly migration analysis for assessing chronic lung increased in the presence of C-001 and C-016 disease. (P<0.01). Bifunctional small molecules represent promising immunotherapies for the treatment of 5 aspergillosis or bacterial and viral infections and further study of these agents is warranted. The Application of Machine Learning Algorithms to microfluidic chamber device will accelerate the Identify Sepsis-Specific Neutrophil Migration study of dynamic host-pathogen interactions and will Signatures facilitate the development of immunomodulating Felix Ellett1, Julianne Jorgensen1, Yuk Ming Liu2, therapies. Currently, we are working on developing Myriam Martinez2, Kathryn Butler2, Daniel Irimia1 integrated multi-sensing platforms to measure 1BioMEMS Resource Center, Department of cytokine secretion rates and to detect levels of ROS Surgery, Massachusetts General Hospital, Harvard at the single cell level in the context of infection and Medical School, Shriners Burns Hospital, Boston, inflammation. The microfluidic platform developed MA 02129 ; 2Department of Surgery, Massachusetts in this study may eventually become a useful tool for General Hospital, Harvard Medical School, measuring the ability of neutrophils from patients to Shriners Burns Hospital, Boston, MA 02129 mount effective immune responses against fungi or other pathogens in vitro. Such tools may be able Sepsis affects more patients than breast, lung, and estimate the risk for infections for each patient, and prostate cancer combined, and is responsible for up to half of US hospital mortalities. Treatment of sepsis is the single largest expenditure in US during sepsis can lead to tissue damage, organ hospitals, but is misdiagnosed in one third of cases. dysfunction, and death. Enterotoxins such as S. Early diagnosis of sepsis is crucial, with mortality aureus enterotoxin A (SEA) are thought to play rates increasing by 10% for every 6 hours that major causative roles in the mechanism of injury. treatment is delayed. Current diagnostic approaches SEA directly crosslinks MHC II and specific T cell are complicated by clinical overlap with systemic receptor Vβ chains, triggering oligoclonal T cell inflammatory responses and long culture periods, activation and excessive immune response. We and provide low predictive power (AUROC = 0.72). showed previously in a mouse model that SEA Dysregulation of neutrophil biology, including an inhalation induced a rapid, systemic immune increase in circulating numbers, is a key response followed by local inflammation in lung characteristic of sepsis that has long provided tissue. Exposed mice presented with alveolitis diagnostic value. Recently, more detailed in vitro resembling acute lung injury, which often occurs analyses have also identified sepsis-specific changes septic patients. The purpose of this study was to to neutrophil function and behavior, including further characterize the resulting pulmonary altered phagocytic activity and chemotaxis. inflammation and to determine the mechanism However, most traditional in vitro assays of leading to increased lung permeability. Following neutrophil function only provide endpoint readouts SEA inhalation, mouse lung tissue was examined by and do not facilitate detailed measurement of confocal microscopy. We first inspected the lung for complex migration phenotypes. SEA binding and found that SEA was bound In this study, we designed a microfluidic device to predominantly to alveolar ; however, assess complex behavioral phenotypes of neutrophils SEA could no longer be detected 16 h after migrating spontaneously from a drop of whole inhalation. Clusters of SEA-specific T cells, blood. The device consisted of a simple maze that granulocytes, and active caspase 3-positive apoptotic allowed measurement of multiple aspects of cells were apparent by 48 h after inhalation. The neutrophil migration, better mirroring what can be mechanism by which SEA induces increased lung observed in vivo. permeability was investigated using mass cytometry Application of machine-learning approaches to (CyTOF) to detect subtle changes in cell populations complex neutrophil migration datasets identified key present in the lung. Pulmonary tissue was stained behaviors that segregated with septic and pre-septic with panels of 16 surface and 8 intracellular markers patients groups. Integration of these characteristics and analyzed using a mass cytometer. ViSNE maps into a scoring strategy allowed diagnostic and were generated characterizing discrete cell predictive identification of sepsis in patients with populations, which were then examined for changes AUROC values greater than 0.95. This represents an in marker expression patterns. T cells from SEA- enormous improvement upon current approaches, exposed mice showed greater expression of and provides a new diagnostic tool that could pSTAT3, pSTAT5 and pS6 but lesser expression of significantly impact patient outcomes. IκB. CD11c+SIGLEC F+ alveolar macrophages and CD11c+MHC II+ dendritic cells obtained from SEA- 6 treated mice had greater expression of MHC II, pSTAT1 and pSTAT3 compared to vehicle control. Staphylococcus Aureus Enterotoxin A Induced Finally, CD45−CD31+ endothelial cells had greater Pulmonary Inflammation is Accompanied by levels of pSTAT1 and MHC II following SEA Distinct Changes in Cell Populations within the inhalation compared to vehicle. Flow cytometric Lung analysis showed that total endothelial cell numbers Julia Svedova, Antoine M. Menoret, James Buturla, were significantly reduced 48 h after SEA Anthony T. Vella, UCONN Health inhalation. Isolation of RNA from sorted endothelial cells revealed reduced expression levels of Nos3 Staphylococcus aureus is a part of the normal human (iNOS) and Cdh5 (VE-cadherin) in SEA-exposed flora colonizing the skin and nasopharyngeal mice relative to vehicle control. These findings regions, but under pathological conditions it can also demonstrated that SEA inhalation leads to dynamic represent one of the most common etiologies of changes in cell populations within the lung and in sepsis. The abnormal immune response occurring particular, transcriptional and signaling changes in endothelial cells. Future studies will focus on flow cytometry. NETs formation was stimulated defining how the adaptive immune system integrates with PMA (phorbol 12-myristate 13-acetate; signals with the innate immune system to give rise to 100nM) or CI (calcium ionophore A23187; 4μM) for endothelial cell injury. Understanding SEA-induced 3h. NETs release was measured with a fluorometer pulmonary inflammation may unravel new using extracellular DNA binding dye Sytox Green. therapeutic options and help improve outcomes for NETs formation was also assessed qualitatively with patients with sepsis and acute lung injury related to fluorescent microscope after staining for DNA and sepsis. myeloperoxidase. The processes important for NETs release: reactive oxygen species (ROS) production 7 and citrullination of histone 3 were assessed respectively: fluorometrically after loading the cells Agents Differentiating Hl-60 Cells toward with 4 ug/ml DHR and by Western blotting after Granulocyte-Like Cells Determine Their Ability incubation with anti-CitH3 antibody. to Release Neutrophil Extracellular Traps Flow cytometry revealed, that ATRA differentiated Aneta Manda-Handzlik1,3, Sandra Sieczkowska2, HL60- cells most effectively toward granulocyte-like Weronika Bystrzycka2, Anna Stelmaszczyk-Emmel1, cells (% of CD11b+ cells: ATRA-97.8%±1.3%, Urszula Demkow1, Olga Ciepiela, 1Department of DMSO-91.3%±2.0%, DMF-90.8±2.9%), lack of Laboratory Diagnostics and Clinical Immunology of CD14 expression indicated that no treatment induced Developmental Age, Medical University of Warsaw; monocytic differentiation. Regardless to 2Students’ Scientific Group at Department of differentiating compound used, GLC were Laboratory Diagnostics and Clinical Immunology of morphologically similar to peripheral blood Developmental Age, Medical University of Warsaw; neutrophils. We found poor release of NETs in 3Postgraduate School of Molecular Medicine, ATRA-differentiated(d) HL-60 cells, moderate in Medical University of Warsaw DMSO-dHL-60 cells and the best in DMF-dHL-60 cells. Moreover, we found higher NETs release in Release of neutrophil extracellular traps (NETs) by CI-stimulated than in PMA-stimulated DMSO- and granulocytes, although firstly described over a DMF-dHL60 cells. Similarly to peripheral blood decade ago, remains a poorly understood neutrophils, both PMA and CI induced oxidative phenomenon. Notably, studying the functions of burst in DMSO- and DMF-dHL-60 cells. neutrophils have been constrained by the facts that Conversely, ATRA-dHL-60 cells were unable to these cells live shortly, can easily become release ROS after stimulation with CI. We observed preactivated during the isolation and are a difficult an increase in the level of CitH3 in DMSO- and object to genetic modifications using current DMF-, but not in ATRA-dHL-60 cells in an techniques. Thus, the investigations into neutrophil unstimulated state. Citrullination of histone 3 in biology could benefit from the availability of a cell these cells further increased after stimulation with CI line model well resembling the functions of (significant increase) and PMA (slight increase). peripheral blood neutrophils. The purpose of our Interestingly, the level of citH3 in ATRA-dHL60 study was to find a compound most effectively cells after stimulation with PMA and CI did not differentiating HL-60 (human promyelocytic increase and was even lower than in undifferentiated ) cells toward GLC able to release NETs. HL-60 cells stimulated with these compounds. HL-60 cells purchased from Sigma Aldrich were We concluded, that when studying NETs biology , cultured in RPMI 1640 + 10% fetal bovine serum DMF is the best stimulus for HL-60 cells and antibiotic/antimycotic in 5% CO2 humidified air differentiation. Conversely, ATRA-dHL-60 cells are at 37ºC. Human peripheral blood neutrophils were not recommended for these studies. obtained by density gradient centrifugation from buffy coats of healthy blood donors. HL-60 cells were differentiated to GLC by treatment with ATRA (1µM), DMSO (1,25%) or DMF (70 mM) for 5 days. Cell differentiation was assessed morphologically by May-Grünwald-Giemsa staining and by evaluating CD11b and CD14 expression by 8 From the FRET-FLIM results we were able to Imaging the Interaction of the Cytosolic Subunits extract structural information about the interactions of the NADPH Oxidase in Live Cells – from and to compare them with available structural data Quantitative Fret Imaging to 3D Model obtained from in vitro studies using small angle X- Oliver Nüβe, Cornelia Ziegler, Leila Bouchab, ray scattering (SAXS). The information from FRET- Fabienne Mérola, Sophie Dupré, Maria Erard, FLIM was coherent with the SAXS data. We then Laboratoire de Chimie Physique, Univ. Paris-Sud, aligned the available structures leading to the first CNRS UMR 8000, Université Paris Saclay Bat 349, 3D‑model of the cytosolic complex of the NADPH Orsay, 91405, France oxidase in the resting state in live cells. In conclusion, we developed a quantitative FRET- The phagocyte NADPH oxidase (NOX2) is a key FLIM approach that is not only able to distinguish enzyme of the immune system generating between specific and unspecific protein-protein superoxide anions, which are precursors for other interactions in live cells, but gives also information reactive oxygen species. Dysfunctions of NOX2 are about the structural organisation of the interacting associated with numerous diseases and thus detailed proteins. We can propose for the first time a 3D- knowledge about its regulation is needed. This model of the cytosolic complex of the NADPH oxidase is composed of five subunits, the membrane- oxidase covering the in vitro as well as the live cell bound gp91phox and p22phox and the cytosolic situation. p47phox, p67phox, and p40phox. The latter are assumed to be in a ternary complex that translocates 9 together with the small GTPase Rac to the membranous subunits during activation. Chlorinated Flavonoids Are Effective Modulators Our aim was to discover and to characterize specific of Hypochlorous Acid Production, Both in interactions of the cytosolic subunits of NOX2 in Isolated Human Neutrophils and Whole Blood live cells using a Förster Resonance Energy Transfer Carina Proença1, Daniela Ribeiro1, Sara Tomé2, (FRET) based approach. Since FRET depends on the Artur Silva2, Eduarda Fernandes1, Marisa Freitas1 distance between two fluorophores, it can be used to 1UCIBIO, REQUIMTE, Laboratório de Química reveal protein-protein interactions non-invasively by Aplicada, Departamento de Ciências Químicas, studying fluorescent protein (FP) tagged subunits. Faculdade de Farmácia, Universidade do Porto, The cytosolic subunits of NOX2, p40phox, p47phox Portuga; 2Departamento de Química & QOPNA, and p67phox were tagged with a FRET pair of FPs Universidade de Aveiro, 3810-193 Aveiro, Portugal on the N-terminus or the C-terminus and expressed in COS7 cells. FRET was measured by fluorescence Flavonoids are bioactive molecules found in a wide lifetime imaging microscopy (FLIM), because it variety of plants and fruits and are important allows a direct determination of the apparent and components of the human diet. These compounds molecular FRET efficiency, which contains both display many biological activities and have been qualitative and quantitative information about the suggested as effective antioxidant and anti- interaction and the structure of the interacting inflammatory agents. proteins. Fluorescence cross correlation During the complex and orchestrated inflammatory spectroscopy (FCCS) is a completely independent cascade, neutrophils play a crucial role and are method that is not based on distances like FRET but responsible for the production of various on the observation of the co‑diffusion of the FP- inflammatory mediators and reactive species, labelled subunits when they move across a small namely hypochlorous acid (HOCl), generated by confocal observation volume inside the cells. myeloperoxidase. HOCl is known to be the major Together, our FRET-FLIM and FCCS data allowed oxidant agent produced by activated neutrophils, us in a first step to discover heterodimeric with a long lifetime and potent microbicidal and interactions between all cytosolic subunits in live cytotoxic properties. It was previously shown that cells. They also indicate that each cytosolic subunit flavonoids are able to react with HOCl, forming is fully bound to its partners provided that the stable mono and dichlorinated products. Taking into partners are present in sufficient quantity. account the lack of information about the biological activities of chlorinated metabolites of flavonoids, ambit of “QREN e POPH e Tipologia 4.1 e our aim was to investigate the effect of synthetic Formação Avançada”, co-sponsored by FSE and by chlorinated flavonoids and their parent compounds national funds of MCTES. (Figure 1), quercetin and luteolin, in the modulation of HOCl production. For that purpose, this study was 10 performed in two different cellular models: in isolated human neutrophils and in human whole The Importance of Being "Pure" Neutrophils blood, to resemble, as close as possible, the in vivo Fabio Arruda-Silva, Federica Calzetti, Nicola physiologic state. Tamassia, Sara Gasperini, Marco A. Cassatella, The oxidative burst measurement was undertaken by University of Verona applying a fluorescent technique, in which the HOCl - induced oxidation of aminophenyl fluorescein In the last years, we have developed a simple (APF) was monitored in phorbol myristate acetate procedure to isolate neutrophils at > 99.6 % purity (PMA) - activated cells. It was observed that, in from either whole blood or buffy coats1 (herein general, chlorinated flavonoids were efficient defined as Neu). Neu incubated with 5 µM R848 (a modulators of HOCl production in the two cellular TLR8 ligand) were then shown to produce IL-6 and models tested. In human neutrophils, 3-chloro- TNFα, at maximal levels after an overnight 3’,4’,5,7-tetrahydroxyflavone and 6-chloro-3’,4’,5,7- incubation. Under the same experimental conditions, tetrahydroxyflavone were the most active however, Neu neither express IL-10 or interferon- compounds. In whole blood, quercetin, luteolin and stimulated genes (ISG) mRNA, nor produce IL-102. 6-chloro-3’,4’,5,7-tetrahydroxyflavone had the Recently, one-step isolation kits designed to highest activities. Even though the tested flavonoids guarantee a rapid isolation of highly pure neutrophils were more efficient in the study with isolated from the blood have become commercially available. neutrophils, the whole blood model should be We thus isolated neutrophils by using the considered as a preferential tool in this kind of ”MACSexpress® Neutrophil isolation kit” and the assays since it mimics the physiologic environment, “EasySep™ direct human neutrophil isolation kit” not excluding important blood elements that may (herein defined as, respectively, wbNeuM and take part in the immune response. wbNeuE) to check their capacity to express genes and produce cytokines in comparison to Neu. As a result, we found that both wbNeuM and wbNeuE expressed notable levels of IL-10, IFIT1 and ISG15 mRNA, as well as released detectable amounts of IL-10, when incubated with R848 for 20 h. Similarly, wbNeuM and wbNeuE, but not Neu,

treated with R848 produced elevated levels of TNFα Acknowledgements and IL-6 already after 4 h of incubation. This work received financial support from the Furthermore, both wbNeuM and wbNeuE expressed European Union (FEDER funds IFIT1 and ISG15 mRNAs upon incubation for 20 h POCI/01/0145/FEDER/007728) and National Funds with 50 µg/mL poly(I:C) (a TLR3 ligand), while (FCT/MEC, Fundação para a Ciência e Tecnologia Neu did not, in the latter case consistent with the and Ministério da Educação e Ciência) under the notion that human neutrophils lack TLR3. Partnership Agreement PT2020 Hence, we investigated the precise purity of UID/MULTI/04378/2013; and from the University wbNeuM, wbNeuE and Neu by flow cytometric of Aveiro and FCT/MEC for the financial support to analysis focusing on thirteen different cell markers. the QOPNA research Unit (FCT While Neu confirmed to be 99.7 % pure, both UID/QUI/00062/2013), through national founds and wbNeuM and wbNeuE, although 98.9 ± 0.5 % and where applicable co-financed by the FEDER, within 97.5 ± 1 % pure, respectively, were found to be the PT2020 Partnership Agreement. Marisa Freitas reproducibly contaminated by eosinophils (0.29 ± 0.37 % for wbNeuM and 0.53 ± 0.61 % for also acknowledges FCT the financial support for + + Pos-doctoral grant (SFRH/BPD/76909/2011) in the wbNeuE) and slan CD16 -monocytes (0.33 ± 0.16 % for wbNeuM and 0.22 ± 0.19 % for wbNeuE). Because slan+CD16+-monocytes display a well- the human cathelicidin LL-37 mediates an antiviral known capacity to produce elevated amounts of effect on RSV via direct damage to the viral cytokines when stimulated by TLR ligands, we set envelope, disrupting viral particles and decreasing up a protocol to remove them from wbNeuM and virus binding to, and infection of, epithelial cells. wbNeuE. Notably, wbNeuM completely lost the Delivery of exogenous LL-37 is protective in vivo in capacity to produce IL-10, as well as to express a murine model of pulmonary RSV infection, IFIT1 and ISG15 mRNA in response to either R848 demonstrating maximal efficacy when applied or poly(I:C) after slan+CD16+-monocyte removal. In concomitantly with virus. Furthermore, endogenous addition, both wbNeuM and wbNeuE lost the murine cathelicidin, induced by infection, has a capacity to express or produce IL-6 when stimulated fundamental role in protection against disease with R848 for 4 h, again after slan+CD16+-monocyte following infection with RSV. Finally, higher nasal depletion. On the other hand, slan+CD16+-monocyte- levels of LL-37 are associated with protection in a depletion was less effective for wbNeuE, particularly healthy human adult RSV infection model. for IFIT1 and ISG15 transcription, indicating that These data lead us to propose that cathelicidins are a other contaminating cells are responsible for the key, non-redundant component of host defence mRNA expression of IFN stimulated genes. Our against airway infection with RSV; functioning as a findings support the notion that we have been always first point of contact “antiviral shield”, and having recommending, namely that is absolutely mandatory additional later phase roles in minimising the to use highly purified populations of neutrophils severity of disease outcome. Consequently, when gene expression and/or neutrophil-derived cathelicidins represent an inducible target for cytokines are investigated. preventative strategies against RSV infection and may inform the design of novel therapeutic 1 Davey, M. S. et al. (2011) Nat. Immunol.; analogues for use in established infection. 11:1017-8 2 Zimmermann, M. et al. (2015) Nat. Commun.; 12 6:6061 YKL-39 is Expressed in Tumour-Associated 11 Macrophages, Stimulates Monocyte Migration and Reversely Correlates with Hematogenous Cathelicidins Have Direct Antiviral Activity and Lymphatic Metastasis in Human Breast against Respiratory Syncytial Virus in Vitro and Cancer Protective Function in Vivo in Mice and Humans. Julia Kzhyshkowska5,2,3, Tengfei Liu5, Irina Donald J. Davidson1, Silke M. Currie1, Emily Gwyer Mitrofanova2, Bin Song5, Michail Buldakov2, Findlay1, Amanda J. McFarlane1, Paul M. Fitch1, Marina Zavjalova2, Nikolaj Litviakov2, Nadezhda Bettina Bottcher1, Nick Colegrave1, Allan Paras2, Cherdyntseva2, Harald Klüter5,3, 1Institute of Agnieszka Jozwik2, Christopher Chiu2, Jurgen Transfusion Medicine and Immunology, Medical Schwarze1, Donald J. Davidson1, 1University of Faculty Mannheim, Heidelberg University; Edinburgh; 2Imperial College London 2Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, Respiratory syncytial virus (RSV) is a leading cause Russia; 3German Red Cross Blood Service Baden- of respiratory tract infection in infants, causing Württemberg – Hessen, Mannheim, Germany; significant morbidity and mortality. No vaccine or 5Institute of Transfusion Medicine and Immunology, specific, effective treatment is currently available. A Medical Faculty Mannheim, Heidelberg University, more complete understanding of the key components Germany of effective host response to RSV, and novel preventative and therapeutic interventions, are Introduction. Human chitinase-like proteins are urgently required. considered as а novel biomarker of cancer, Cathelicidins are host defence peptides, primarily inflammation and tissue remodeling. The role of expressed by neutrophils and epithelial cells, with YKL-39 and its associations with tumor progression key microbicidal and modulatory roles in innate host has not been studied until now. The aim of study defence against infection. Here we demonstrate that was to examine the intracellular trafficking pathways, and secretion mode of YKL-39 in M2 of the four conserved cysteine residues. Therefore, it macrophages, to explore the effect of YKL-39 on cannot adopt a canonical chemokine fold suggesting monocytes migration; and correlation of YKL39 a unique structure and so its function must also be levels with breast cancer progression. Methods. unique. It is constitutively expressed in various CD14+ monocytes were isolated from buffy coats, mucosal tissues suggesting a role in homeostasis. In cultured with IL-4, TGF-beta, dexamethasone and order to characterize CXCL17 function, we M-CSF to obtain M2 macrophages. YKL-39 generated a homozygous CXCL17 knockout (KO) expression level was measured by RT-PCR. The mice. Interestingly, these mice show no overt intracellular distribution of YKL-39 was checked by phenotype, which is in sharp contrast to knocking IF staining and confocal microscopy. YKL-39 out other homeostatic chemokines that are secretion was measured by ELISA assay. Monocyte embryonically lethal. Our studies to date indicate migration was performed with a trans-well system. that the KO mice have reduced basal levels of large The correlations of YKL-39 with metastasis in peritoneal macrophages suggesting that CXCL17 human breast cancer were verified by RT-PCR, IHC could play an important role in the strategic and IF staining. Results. YKL-39 gene level was positioning of immune cells for surveillance. In a strongly up-regulated with IL4/TGF-beta in human model of acute bacterial infection, we observed a macrophages: on day 6 (13.4 fold, p<0.05) and day decrease in macrophages but no change in neutrophil 12 (62.2 fold, p<0.05) (n=6). YKL-39 was detected levels. Surprisingly, we observed a significant in the TGN, p62lck positive late endosomes, Lamp-1 decrease in several inflammatory cytokines and positive lysosomes and CD63 positive secretory chemokines. Our results suggest that CXCL17 plays lysosomes. The extracellular secretion of YKL-39 a highly specialized pro-inflammatory role in (1.51 ng/ml) was detected on day 12. Recombinant bacterial infections. Our planned studies on YKL-39 significantly enhanced the migration of characterizing the signaling pathways linking and monocytes by 1.9 fold (p<0.01) in 1 hour, and establishing the crosstalk and the spatiotemporal reached to 4.9 fold (p<0.01) in 3 hours. In patients relationship between CXCL17 and leukocyte with breast cancer, low levels of YKL39 correlated trafficking should result in describing why CXCL17 with a higher frequency of lymphatic metastasis is one-of-a-kind chemokine. (p=0.036, n=74) and hematogenous metastasis (p=0.0337, n=74). The pre-dominant TAM 14 phenotypes were YKL-39 +/ stabilin-1+ (73%, n=10) and CD68+/YKL39+ (25%, Natural Nitration of CXCL12 Reduces Its n=10).Conclusions. YKL-39 is expressed in tumor- Signaling Capacity and Chemotactic Activity in associated macrophages in human breast cancer, is Vitro and Abrogates Intra-Articular Lymphocyte secreted via lysosomal pathway, stimulates Recruitment in Vivo migration of monocytes and its expression levels Rik Janssens1,2, Anneleen Mortier1, Daiane Boff2,1, have a reverse correlation with hematogenous and Vincent Vanheule1, Mieke Gouwy1, Charlotte lymphatic metastasis in human breast cancer. Franck1, Olav Larsen3, Mette M. Rosenkilde3, Jo Van Damme1, Flavio A. Amaral2, Mauro M. 13 Teixeira2, Sofie Struyf1, Paul Proost1, 1Laboratory of Molecular Immunology, Department of Role of Chemokine CXCL17 in Microbiology and Immunology, Rega Institute for Homeostasis and Infection Medical Research, KU Leuven; 2Departamento de Kirti V. Sawant, Roberto P. Garofalo, Krishna Bioquimica e Imunologia, Instituto de Ciencias Rajarathnam, University of Texas Medical Branch, Biologicas, Universidade Federal de Minas Gerais, Galveston, TX, USA. Brazil; 3Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Chemokines belong to a superfamily of secreted The Panum Institute, University of Copenhagen, proteins which function in the trafficking and Denmark activation of different types of leukocytes. CXCL17, one of the last chemokines to be identified, is a novel The chemokine CXCL12/stromal cell-derived one-of-a-kind chemokine since it contains only three factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and is acute inflammation and are characterized by their involved in tumor development. In vitro, CXCL12 ability to direct migration to the site of infection or activity is strongly regulated by proteolytic inflammation; a process called chemotaxis. Because processing. However, limited information is neutrophil chemotaxis is critical to many available on in vivo posttranslationally modified inflammatory diseases in humans and cattle, CXCL12. Therefore, natural CXCL12 from stromal chemoattractant receptors are subjects of exhaustive cells stimulated with leukocytes and inflammatory study. HCA2 expression and the response of specific agents was purified. CXCL12 with a nitration on ligands on bovine immune cells have not been Tyr7, designated [3-NT7]CXCL12, was detected. previously studied. In this study, we show HCA2 CXCL12 and [3-NT7]CXCL12 were chemically receptor mRNA expression in bovine neutrophils. synthesized to evaluate the biological effects of this Besides treatment with MK-1903 and nicotinic acid, modification. [3-NT7]CXCL12 recruited b-arrestin two HCA2 selective full agonists elicited a transient 2, phosphorylated the kinases Akt and ERK1/2 and rise of intracellular Ca2+ levels that were bound to glycosaminoglycans and the G protein- concentration dependent, suggesting that bovine coupled chemokine receptor CXCR4 similar to neutrophils express a functional HCA2 receptor. On CXCL12. However, it showed a reduced ability to the contrary, the structurally related compound enhance intracellular calcium concentrations, to Nicotinamide, which does not bind to HCA2 generate inositol triphosphate and to induce receptor had no effect on Ca2+ levels. We also monocyte and lymphocyte chemotaxis in vitro. observed that BHB, MK-1903 and NA but not Moreover, nitrated CXCL12 failed to induce in vivo nicotinamide increased bovine neutrophil extravasation of lymphocytes to the joint. In chemotaxis. Also, HCA2 agonists activate crucial summary, nitration on Tyr7 is a novel natural intracellular pathways in neutrophil chemotaxis such posttranslational regulatory mechanism on CXCL12 as PLC, AKT, p38 and AMPKα. In summary, these under inflammatory conditions which may affect the results contribute to our knowledge about novel CXCR4-mediated inflammatory and tumor- modulatory HCA2 mechanisms in the innate promoting activities of CXCL12. immune system that could be involved in various pathologies in cattle. 15 Acknowledgement Fondecyt 1151035 Agonists of the ß-Hydroxybutyrate Receptor (HCA2) Increase the Chemotactic Response of 16 Bovine Neutrophils Maria D. Carretta, Maria A. Hidalgo, Rafael A. Staphylococcus Aureus Proteases Inactivate Burgos, Institute of Pharmacology and Galectin-3 Immunoregulatory Functions and Morphophysiology, Universidad Austral de Chile Increase the Severity of Skin Infection Jonas Elmwall1, Jakub Kwiecinski1,2, Manli Na1, An elevated concentration of the ketone body β- Abukar Ali1, Veronica Osla1, Lindsey Shaw3, hydroxybutyrate (BHB) in dairy cattle during Wanzhong Wang4, Karin Sävman5, Elisabet lactation is associated with an increased incidence of Josefsson1, Johan Bylund6, Tao Jin1, Amanda inflammatory diseases, such as metritis and mastitis. Welin1, Anna Karlsson1, 1Department of However it remains unclear how BHB affects the Rheumatology and Inflammation Research, inflammatory response in dairy cows. BHB was Sahlgrenska Academy at the University of identified as endogenous ligand of the Gothenburg, Sweden; 2Department of Microbiology, Hydroxycarboxylic acid receptor 2 (HCA2 or Roy J. and Lucille A. Carver College of Medicine, GPR109A), a G protein-coupled receptor. HCA2 University of Iowa, USA; 3Department of Cell also is activated by nicotinic acid (NA), a lipid Biology, Microbiology and Molecular Biology, lowering drug. However, recent studies suggest that University of South Florida, USA; 4Department of HCA2 activation modulates the inflammatory Medical Biosciences, Umeå University, Sweden; response in human macrophages, monocytes and 5Perinatal Center, Departments of Physiology and neutrophils, where this receptor is highly expressed. Neuroscience and of Paediatrics, Sahlgrenska Neutrophils are undoubtedly the major effectors of Academy at the University of Gothenburg; 6Department of Oral Microbiology and Immunology, galectin-3-cleaving protease SspB increases the Sahlgrenska Academy at the University of severity of S. aureus skin infection, which suggests Gothenburg, Sweden that galectin-3-modification is an active function within S. aureus virulence. BACKGROUND: Galectin-3, a β-galactoside- binding human lectin, has significant impact on the 17 outcome of inflammation and infection, and is able to activate antimicrobial functions of neutrophils. Gro-α Induce Neutrophils Chemotaxis via the Galectin-3 carries a proteolytically sensitive domain Pure Chemotactic Receptor CXCR2 which, when cleaved by bacterial proteases, Karin Christenson1,3, Lena Björkman2, Johan generates a carbohydrate-binding fragment that is Bylund3, 1TIMM Laboratory, Sahlgrenska Cancer inert with regard to cellular activation. Center, Institution of Biomedicine ; 2Department of Staphylococcus aureus expresses four major Rheumatology and Inflammation Research, Institute proteases that are emerging as virulence factors: of Medicine; 3Department of Oral Microbiology and SspA, SspB, ScpA, and Aur. We hypothesized that Immunology, Institute of Odontology these proteases can cleave galectin-3 during infection, and thereby affect disease outcome. We Neutrophil chemotaxis is induced by a variety of thus investigated the ability of S. aureus proteases to stimuli, either from endogenous sources or from process galectin-3 and the impact of such activity on invading pathogens, via G-protein coupled receptors neutrophil activation and in vivo skin infection. (GPCRs) on the neutrophil surface. Typically, METHODS: Proteolytic cleavage of galectin-3 in ligation to the GPCRs also leads to neutrophil culture supernatants from S. aureus strain 8325-4 activation, e.g., granule mobilization and production and protease-lacking mutants was characterized by of reactive oxygen species from the NADPH- immunoblotting, and the impact of galectin-3 oxidase. The chemokine IL8 has been shown to cleavage on neutrophil NADPH-oxidase activation induce signaling in neutrophils via the GPCRs was assessed by isoluminol-enhanced CXCR1 and CXCR2. CXCR2 is also a receptor for chemiluminescence. The importance of galectin-3 several other chemoattractants and in this study we and bacterial galectin-3-cleaving proteases in vivo have investigated the CXCR2 agonist Gro-ɑ, a was investigated in a murine skin infection model, chemokine known to attract neutrophils, but less is by measurement of lesion sizes and bacterial load. described about how it affects other neutrophil Also, the localization of galectin-3 within healthy or functions. We evaluated chemotaxis and expression S. aureus-infected skin was characterized by of surface markers on human neutrophils, and as the immunohistochemistry. RESULTS: The transmigration process is calcium dependent, we also staphylococcal protease SspB was potently cleaving monitored intracellular calcium levels. We found galectin-3, abolishing the neutrophil-activating that similar to IL8, Gro-ɑ induced in vitro capacity of the lectin. In healthy murine skin, chemotaxis and intracellular calcium transients in galectin-3 was strongly associated to the skin neutrophils, but the Gro-ɑ induced signal was squamous and adnexa epithelial cells, as well as to completely blocked by a CXCR2 antagonist which tissue macrophages and dendritic cells. Upon was not the case for IL8.This suggests that in infection with SspB-expressing S. aureus, opposite to IL8, Gro-ɑ only induce signaling via infiltrating macrophages were strongly positive for CXCR2. Furthermore, stimulation with IL8 lead to galectin-3, as were the bacteria, while necrotic tissue granule mobilization with increased expression of had lost all galectin-3. When comparing the lesion complement receptor (CR) 3 on the neutrophil sizes induced by protease-expressing bacteria with surface while Gro-ɑ treated cells did not upregulate that of protease-deficient bacteria, SspB-expressing surface CR3 . The neutrophil NADPH-oxidase was S. aureus generated significantly greater lesion sizes, activated by stimulation of IL8, but Gro-ɑ was but only in galectin-3-expressing mice. incapable of inducing superoxide production. CONCLUSIONS: S. aureus is able to cleave In conclusion, we describe clear differences in galectin-3 by the action of secreted proteases, and activation patterns between IL8 and GRO-ɑ, and thereby inactivate the immunoregulatory functions found that the latter triggered chemotaxis without of the lectin. The combination of galectin-3 and the any further activation of the neutrophils. We suggest that CXCR2 is a pure chemotactic receptor that ROC AUC=0.828). In contrast, NGAL and elastase induces chemotaxis but no additional cell activation. showed no significant difference between AAA patients and healthy controls. Free DNA/histone 18 complexes were significantly higher in AAA patients with 45.8 vs. 28.9 relative units (P=0.036; Neutrophil Extracellular Traps in Abdominal ROC AUC=0.689). Neutrophils with citrullinated Aortic Aneurysm histones were readily detectable in the ILT Wolf Eilenberg, Branislav Zagrapan, Sarah Hetzer, associated with AAA. Katharina Muench, Suriya Prausmueller, Paimann Conclusion: Nawrozi, Vanessa Elleder, Christoph Neumayer, This is the first report on the association of NET Ihor Huk, Christine Brostjan, Medical University of markers with AAA. While free DNA/histone Vienna complexes in plasma seem to be less sensitive as diagnostic tool than the standard neutrophil Objective: activation marker MPO, NET parameters like Neutrophils are known to play an essential role in circulating citrullinated histones are currently under the formation of abdominal aortic aneurysms investigation and may be of particular interest in the (AAAs) since neutrophil depletion leads to prediction of AAA progression and rupture. inhibition of experimental AAA development in rodents. Furthermore, markers of neutrophil 19 activation are elevated in patients with AAA. The formation of so-called neutrophil extracellular traps Multifactorial Effects of Nitro-Oleic Acid on (NETs) is a process of extreme neutrophil activation Macrophage Mediated Cardiovascular and death involving histone modification Inflammation (citrullination) and DNA expulsion to entrap Gabriela Ambrozova1,2, Lukas Kubala1,2,3, Tana pathogens. Recently, NETs have been implicated in Fidlerova3, Hana Martiskova3, Volker Rudolph4, thrombotic processes. We hypothesized that NETs Bruce Freeman5, Michaela Pekarova1,2, 1Institute of may be associated with AAA development and NET Biophysics, Academy of Sciences of the Czech markers are increased in circulation of AAA Republic, Brno, Czech Republic; 2International patients. We further investigated whether NET Clinical Research Center - Center of Biomolecular parameters are more sensitive than regular and Cellular Engineering, St. Anne's University neutrophil activation markers in AAA detection. Hospital, Brno, Czech Republic; 3Faculty of Science, Methods: Masaryk University, Brno, Czech Republic; 4Heart Aortic tissue and peripheral venous blood were Centre, University Hospital of Cologne, Cologne, collected from 20 AAA patients scheduled for Germany; 5Department of Pharmacology and surgical repair. 21 age and sex matched healthy Chemical Biology, University of Pittsburgh, individuals served as controls. Markers of neutrophil Pittsburgh, PA, USA activation like elastase, myeloperoxidase (MPO) and neutrophil gelatinase associated lipocalin (NGAL) Chronic inflammation can lead to severe pathologies were measured in plasma by ELISA. In comparison, and tissue dysfunction and possibilities of its parameters of NET formation, free DNA-histone regulation are nowadays intensively studied. Nitro- complexes and citrullinated histone H3, were fatty acids are endogenous molecules with immuno- evaluated. Differences between groups were regulatory potential generated in the adaptive calculated by Mann-Whitney-U-test and diagnostic response of organisms to oxidative stress. Currently, marker potential was assessed by ROC analysis they are suggested as highly promising compounds (AUC=area under the curve). Furthermore, for treatment of diseases associated with immune neutrophils and NETs were detected in AAA tissue homeostasis deregulation. The purpose of our study and associated intraluminal thrombus (ILT) by was to characterize the effects of nitro-oleic acid immunofluorescence microscopy. (OA-NO2) in regulation of macrophage action Results: responsible for initiation of chronic inflammatory AAA patients had significantly elevated plasma responses in endothelium and point to a novel MPO levels of median 13.0 vs. 6.7 ng/ml (P<0.001; therapeutic strategy to treat distinct macrophage- cardiovascular inflammation. Following main induced inflammatory diseases. signalling pathways, we helped to clarify molecular The effect of OA-NO2 was tested in vitro employing mechanism of nitro-lipids action. OA-NO2 plays an RAW 264.7 macrophages, bone marrow-derived important role in regulation of chronic inflammation macrophages, and endothelial cells (MS-1, HUVEC) and pathologies of vascular endothelium. Therefore, activated or differentiated with various stimuli. In we can conclude that nitro-fatty acids are highly vitro experiments were further expanded by in vivo promising drugs for treatment of chronic observations using mouse model of angiotensin-II inflammation-accompanied diseases. induced cardiomyopathy. Our results showed that physiologically-relevant 20 concentrations of OA-NO2 significantly regulated macrophage action in different inflammation-related S100A9 Potentiates the Activation of Neutrophils pathologic processes. Firstly, we characterized the by the Etiological Agent of Gout, Monosodium immunomodulatory effect of OA-NO2 on Urate Crystals. macrophage polarization to pro-inflammatory and Louis-Simon Rousseau, Guillaume Paré, Asmaa immuno-regulatory subsets. We demonstrated that Lachhab, Paul Naccache, Philippe Tessier, Martin OA-NO2 significantly blocked the LPS-induced Pelletier, Maria Fernandes activation of transcription factors STAT1, NF-kB Université Laval and MAPKs as well as production of pro- inflammatory mediators, including reactive oxygen Introduction and rationale: Gout is one of the most and nitrogen species and cytokines TNF-α, IL-6, IL- painful types of arthritis, and its prevalence is 1β and TGF-β in pro-inflammatory macrophages. increasing worldwide. It arises when the body On the other hand, in IL-4-stimulated macrophages, mounts an acute inflammatory reaction against OA-NO2 inhibited STAT6 activation and arginase-1 monosodium urate crystals (MSU).During the expression and regulatory macrophage phenotype symptomatic phase, a large number of neutrophils development. Moreover, we demonstrated that OA- are recruited to the affected joint where they are NO2 effectively influences the process of activated by MSU to release a panoply of macrophage differentiation induced by growth inflammatory molecules (eg: IL-1beta, S100 factors GM-CSF/M-CSF and thus regulates their proteins), degradative enzymes and oxygen radicals. activation phenotype during inflammation. These activated neutrophils perpetuate the Further, we characterized OA-NO2 effects on inflammatory reaction and cause damage to the endothelium response to macrophage derived surrounding tissues. Neutrophil-derived mediators cytokines with consequent fibrosis development and neutrophils themselves, are thus attractive arising from vessel inflammation in different tissues. therapeutic targets to dampen inflammation during OA-NO2 prevented pathological activation of gout attacks. endothelial cells by reduction of pro-inflammatory Several lines of evidence strongly suggest that the cytokines and chemokines (RANTES, IL-6, GM- protein S100A9 plays a role in the pathogenesis of CSF and MCP5) production and adhesive molecules gout. The silencing of its expression in vivo reduces (ICAM-1) expression. Accordingly, OA-NO2 can MSU-induced inflammation. Moreover, S100A9 is prevent transformation of endothelial cells to the expressed throughout the synovium of gout patients. pro-fibrotic phenotype (α-SMA and fibroblast- Its expression is particularly enriched around blood specific protein 1) through blocking of endothelial- vessels. Moreover, synovium and sera of gout mesenchymal transition triggered by TGF-β patients contain elevated levels of S100A9 produced by macrophages. heterodimerized to S100A8. Regulation of macrophage polarisation and anti- Although extracellular S100A9 induces several fibrotic effects of OA-NO2 were verified by in vivo functional responses in human neutrophils, it experiments. remains unknown how neutrophils respond to MSU Our study provided the unique results showing the in the presence of extracellular S100A9. S100A9 protective effects of OA-NO2 in over-activation of alone, induces the shedding of L-selectin, an macrophages and ability to alternate pro- increase in the expression and activation of cell- inflammatory feedback of endothelial cells in during surface Mac-1, degranulation, adhesion to fibrinogen and chemotaxis of neutrophils. In contrast, it is Niels Heemskerk1,6, Lilian Schimmel1, Chantal unable to directly induce cytokine production and its Oort1, Jos van Rijssel1, Taofei Yin2, Bin Ma3, ability to produce radical oxygen species (ROS), Jakobus van Unen4, Bettina Pitter5, Stephan remains to be confirmed. S100A9 also enhances Huveneers1, Joachim Goedhart4, Yi Wu2, Eloi neutrophil phagocytosis and bactericidal activity. Montanez5, Abigail Woodfin3, Jaap D. van Buul1 Hypothesis: Since S100A9 is highly expressed 1Department of Molecular Cell Biology, Sanquin around blood vessels, we hypothesized that S100A9 Research and Landsteiner Laboratory, Academic primes human neutrophils for enhanced Medical Centre, University of Amsterdam, 1066CX responsiveness to MSU. Amsterdam, The Netherlands.; 2Genetics and Objective: In this study, we investigated the priming Developmental Biology, Center for Cell Analyses effect of S100A9 on various well characterized, and Modelling, University of Connecticut Health MSU-induced neutrophil effector functions. We also Centre, Farmington, Connecticut 06032, USA.; explored less well-known response of neutrophils to 3Experimental Medicine and Pharmacology, Centre MSU, namely, changes in metabolism. for Microvascular Research, William Harvey Methods: Human neutrophils were incubated with Research Institute, Barts and The London School of S100A9 prior to activation with MSU. The Medicine and Dentistry, Queen Mary, University of mobilization of intracellular calcium was determined London, Charterhouse Square, London, EC1M 6BQ, with Fura-2AM. Glycolysis and the oxygen UK.; 4Molecular Cytology, Swammerdam Institute consumption rate were determined in real-time with for Life Sciences, University of Amsterdam, the XF96 extracellular analyzer. The former was Amsterdam 1098XH, The Netherlands.; 5Department determined by measuring the extracellular of Angiogenesis, Walter-Brendel-Center of acidification rate (ECAR), and the latter by Experimental Medicine Ludwig-Maximilians measuring the oxygen consumption rate (OCR).IL-1 University Marchioninistr. 27 81377 Munich, and IL-8 production were determined by ELISA and Germany.; 6VU University medical center, signaling pathways were studied by Western blot Department of Molecular Cell Biology and analysis. Immunology, OI2 building, room 11 E 53, PO Box Results: S100A9 enhances several MSU-induced 7057, 1007 MB Amsterdam, The Netherlands. neutrophil effector functions including calcium mobilization, glycolysis, oxygen consumption as During immune surveillance and inflammation, well as IL-1 and IL-8 production. We also show that leukocytes exit the vasculature through transient S100A9 enhances the activation of signalling openings in the endothelium without causing plasma pathways known to underlie the response of leakage. However, the exact mechanisms behind this neutrophils to MSU. Of note, is the ability of intriguing phenomenon are still unknown. Here we S100A9 to induce glycolysis in human neutrophils. report that maintenance of endothelial barrier Conclusion: Our observations suggest that S100A9 integrity during leukocyte diapedesis requires local promotes the inflammatory reaction in gout by endothelial RhoA cycling. Endothelial RhoA enhancing the MSU-induced activation of depletionin vitroor Rho inhibitionin vivoprovokes neutrophils. The priming effect of S100A9 may be neutrophil-induced vascular leakage that manifests explained, in part, by its ability to enhance known during the physical movement of neutrophils MSU associated signalling pathways and by through the endothelial layer. Local RhoA activation inducing glycolysis. The latter may also underlie the initiates the formation of contractile F-actin priming of neutrophils by S100A9 for functional structures that surround emigrating neutrophils. responses towards other stimuli. The impact of this These structures that surround neutrophil-induced discovery on other auto-inflammatory and endothelial pores prevent plasma leakage through autoimmune pathologies remains to be investigated. actomyosin-based pore confinement. Mechanistically, we found that the initiation of 21 RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of F-Actin-Rich Contractile Endothelial Pores actomyosin-based endothelial pore confinement Prevent Vascular Leakage during Leukocyte involves ROCK2b, but not ROCK1. Thus, Diapedesis through Local RhoA Signalling endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial (LFA-1), suggesting that TIM-1 may directly pores that prevent vascular leakage during leukocyte interact with both PSGL-1 and LFA-1 on extravasation. neutrophils, or that TIM-1-PSGL-1 interaction transactivates integrins promoting subsequent 22 integrin-dependent neutrophil activation and arrest. Immunofluorescence studies showed constitutive Endothelial TIM-1 Triggers Neutrophil Adhesion TIM-1 expression on the surface of both brain- and under Inflammatory Conditions and Contributes cardiac-derived endothelial cells, and we also to the Induction of Autoimmunity detected TIM-1 on the vascular endothelium of Stefano Angiari1, Silvia Dusi1, Barbara Rossi1, Lara inflamed spinal cord (SC) pial venules during EAE, Toffali1, Tommaso Carlucci1, Carlo Laudanna1,2, in particular at disease pre-onset and onset phases. Gabriela Constantin1, 1Department of Medicine, Importantly, anti-TIM-1 blocking antibody inhibited Section of General Pathology, University of Verona neutrophil adhesion on endothelial cells in vitro as (Italy)University of Verona; 2The Center for well as neutrophil attachment in CNS vessels in Biomedical Computing (CBMC), University of intravital microscopy studies on exposed SC during Verona (Italy) EAE. Finally, our results demonstrate that anti-TIM- 1 treatment at disease pre-onset phase characterized BACKGROUND. Neutrophils are the first line of by high neutrophil infiltration of the CNS defense against invading pathogens and they have significantly inhibited EAE development. long been viewed as the final effector cells of an CONCLUSIONS. Our data suggest a pivotal role for acute inflammatory response. However, recent endothelial TIM-1 in controlling neutrophil evidence has demonstrated a role for neutrophils in trafficking under inflammatory conditions and that chronic inflammation, particularly in autoimmune targeting TIM-1 may represent an attractive diseases such as experimental autoimmune therapeutic approach in autoimmune diseases such encephalomyelitis (EAE), the animal model of as MS. multiple sclerosis (MS). Importantly, during EAE, neutrophils represent the first leukocyte subset 23 adhering to the blood-brain barrier and favoring immune cell invasion of the central nervous system X-Linked Neutropenia Caused by Overactive (CNS), but the molecular mechanisms controlling Mutations in the Wiskott-Aldrich Syndrome neutrophil trafficking in the CNS during EAE are Protein Renders Neutrophils Hyperactive largely unknown. The aim of this project was to Marton Keszei1, Paul Drescher1, Hannah Wurzer1, investigate a potential role for endothelial T cell Jaime James1, Laura Köcher1, Wenxia Song2, Scott immunoglobulin and mucin domain-containing Snapper3, Lisa Westerberg1, 1Karolinska Institutet, molecule-1 (TIM-1) in neutrophils trafficking under Department of Microbiology Tumour and Cell inflammatory conditions. biology, Stockholm, Sweden; 2University of RESULTS. Using under flow adhesion assays, we Maryland, Department of Cell biology and found that immobilized TIM-1 in capillary tubes Molecular Genetics, College Park, MD; 3Boston captured neutrophils under physiological flow Children's Hospital, Division of Gastroenterology, conditions in vitro, with most cells undergoing Boston, MA immediate firm arrest. Strikingly, adhered neutrophils rapidly displayed spreading after arrest, The gene encoding the Wiskott-Aldrich syndrome suggesting a prompt neutrophil activation following protein (WASp) is highly expressed and upregulated interaction with immobilized TIM-1. To identify during neutrophil maturation. Gain-of-function TIM-1 ligands on neutrophil surface, we performed mutations in WASp that destroys the auto-inhibited under flow experiments using blocking antibodies or conformation cause a rare form of X-linked neutrophils deficient for several adhesion molecules. neutropenia (XLN). Although neutrophil Surprisingly, our data showed that neutrophils are granulocytes are clearly affected in XLN patients, it captured on TIM-1 under flow through engagement remains largely unknown what role WASp plays in of mucin P-selectin glycoprotein ligand-1 (PSGL-1) neutrophil development and function. Here, we and integrin leukocyte function-associated antigen-1 generated two new mouse models that express the XLN patient mutations WASp-L272P and WASp- monosodium urate (MSU), calcium pyrophosphate I296T and compared neutrophils from these mice to dihydrate (CPPD) and silica. Live cell and fixed WASp-deficient neutrophils. We found that several fluorescence microscopy using DNA binding dyes neutrophil functions, such as chemotaxis, and cell vitality markers enabled dissection of the phagocytosis, adhesion, degranulation, and reactive kinetics of cell death and decondensation. oxygen species (ROS) production were regulated by Results: NETosis induced by crystal stimuli WASp. Surprisingly, while WASP-deficient occurred more rapidly than that induced by PMA neutrophils exhibited defective actin polymerization, and Candida, the latter two stimuli demonstrating a intracellular ROS production, phagocytosis, and characteristic latency prior to NETosis. Chromatin chemotaxis, XLN neutrophils showed increased decondensation occurs post-mortem in crystal polymerized actin, intracellular ROS, phagocytosis stimulated neutrophils but prior to cell death in PMA rate,and chemotaxis. In the competitive setting of and Candida treated neutrophils. NADPH oxidase bone marrow chimeric mice, WASp-deficient function assessed chemically and genetically was neutrophils had a selective disadvantage when required for PMA- and Candida-induced NETosis competing with wildtype cells. In contrast, XLN and cell death. MSU and CPPD induced robust neutrophils had an advantage over wildtype neutrophil reactive oxygen species generation but neutrophils in entering peripheral tissues in naïve this was not required for crystal-induced NETosis. mice and under sterile inflammation. These data Histone degradation during NETosis was evident indicate that there are unique requirements for the with all stimuli tested and was inhibited by presence and activation status of WASp in neutrophil elastase inhibitor (311616A). Neutrophil neutrophils and that activating mutations in WASp elastase inhibition reduced DNA decondensation render neutrophils hyperactive. with crystal and PMA-induced NETosis but did not alter the kinetics of cell death. 24 Conclusion: Pathologically relevant crystals trigger neutrophil cell death and chromatin decondensation Live Cell Time-Lapse Imaging Allows using distinct upstream biochemical pathways with Quantification of Netosis and Dissection of the neutrophil elastase as the final common mediator of Pathways Involved in Cell Death and Chromatin chromatin decondensation. Decondensation Simon M. Chatfield1,2,3, James M. Murphy1,2, Ian P. 25 1,2,3 1 Wicks , Walter and Eliza Hall Institute of Medical Research; 2University of Melbourne; 3Royal Cracking the Neutrophil Transcription Paradox: Melbourne Hospital Transcriptional Firing Drives Netosis Nades Palaniyar1,2, Meraj A. Khan1,2, 1The Hospital Objective: Neutrophil extracellular traps (NETs) and for Sick Children; 2University of Toronto the cell death process associated with their release (NETosis) have been studied using a variety of Background and rationale: The relevance of stimuli. It remains unclear whether different NET- transcription in neutrophils has remained a paradox. inducing stimuli operate through a common It is well established that neutrophils respond pathway. Furthermore, it remains unclear which quickly to stimuli and die within minutes to hours. pathways are important for cell death versus DNA Why would a short-lived cell such as neutrophil decondensation, the two hallmarks of NETosis. transcribe its geneome? We hypothesized that Methods: We used a novel quantitative live cell genome-wide transcription decondenses chromatin imaging strategy to explore biochemical pathways necessary to drive neutrophil extracellular trap involved in NETosis. The live cell platform allowed (NET) formation or NETosis. investigation of both the death and chromatin Methods: Human neutrophils from healthy donors decondensation components of NETosis. Healthy were purified and induced to undergo Nox- control neutrophils and neutrophils from a Chronic dependent (by PMA) or Nox-independent (by Granulomatous Disease patient were stimulated with calcium ionophore A23187) NETosis. phorbol myristate acetate (PMA), Candidal hyphae Transcriptomic analysis was conducted for and pathologically relevant crystals including unstimulated or NETotic neutrophils at different time points (30, 60 min; 3 conditions, 2 time points, Pathogen-triggered neutrophil (PMN) recruitment is 3 dornors; 18 transcriptomes). Kinase specific critical for innate immunity, but aberrant PMN genome-wide transcription was analysed by Human influx is also implicated in the pathogenesis of Affymetric array, and GeneSpring, Ingenuity and numerous inflammatory diseases of the gut and GeneGo Metacore softwares. Nuclear morphology, lungs. Fucosylated terminal glycans from the Lewis citrulination of histone and NETs were analyzed by glycan family, such as Lewis-x (Lex) and Sialyl flourescence microscopy. Effects of transcription Lewis-x, have previously been implicated in the and translation inhibitors on NETosis was regulation of important PMN functions, including determined by Sytox Green plate reader assays and Selectin-mediated PMN trafficking across the flourescence microscopy. vascular endothelium. While such glycans based on Results: We have identified that the transcriptional the type 2 sequence (Galb1-4GlcNAc-R) are activity reflects the degree of DNA decondensation abundant on PMNs, the presence of type 1 Galb1- occurring in both Nox-dependent and Nox- 3GlcNAc-R glycans required for the expression of independent NETosis. Genome-wide transcription Lewis-a (Lea) have not yet been reported. Here, we starts earlier in the rapid Nox-independent NETosis conclusively demonstrate by immunoblotting and than Nox-dependent NETosis. Transcriptomics immunofluorescence analyses that Lea is in fact analyses show that NETosis-specific kinase cascades expressed in human PMNs. Specific Lea glycan differentially activate transcriptional firing in these recognition by anti-Lea mAbs was verified using two types of NETosis. Inhibitors of transcription, but glycan array technology. In addition PCR and not translation, suppress both types of NETosis. In immunoblotting analyses revealed robust PMN particular, promoter melting and DNA unwinding expression of both a ß1-3 Galactosyltransferase and are important transcription steps necessary to drive an a1/4 Fucosyltransferase, glycosyltransferase NETosis. Immunofluorescence microscopy indicates enzymes required for Lea synthesis. We further that extensive chromatin-wide citrullination of report that antibody mediated ligation of PMN histones occurs only in Nox-independent NETosis. expressed Lea increases both PMN chemotaxis Hence, citrullination of histone could contribute to across collagen and migration across model the rapid transcription-mediated DNA intestinal epithelia. As would be expected treatment decondensation observed in Nox-independent of PMN from individuals deficient in a1/4 NETosis. However, blocking transcription fucosylation with anti-Lea mAbs did not result in suppresses both types of NETosis, without affecting changes in neutrophil trafficking. These results the reactive oxygen species production that is identify, for the first time, expression of Lea by necessary for anti-microbial functions of neutrophils. human PMN and demonstrate its relevance to PMN Conclusions: Therefore, we assign a novel function trafficking within the intestine. We propose that for transcription in neutrophils: Transcriptional PMN Lea represents a new target for regulating firing, regulated by NETosis-specific kinases, drives intestinal innate immunity and modulating NETosis. inflammation in diseases where dysregulated PMN Funded by: Canadian Institutes of Health Research influx is associated with bystander tissue damage. (CIHR) and Cystic Fibrosis Canada (CFC) research Furthermore, differential responses of human PMN grants to N.P. M.A.K receives a Mitacs postotoral to physiological ligands of Lea (based on an fellowship. individuals Lewis phenotype) may provide insights into the mechanisms linking specific Lewis 26 phenotypes with differential susceptibilities to bacterial infections, viral infections and coronary Expression of Lewis-A Glycans on PMN heart disease. Augments Function by Increasing Transmigration Jennifer C. Brazil1, Ronen Sumagin2, Richard D. Cummings3, Nancy A. Louis4, Charles A. Parkos1, 1University of Michigan; 2Northwestern Unversity; 3Harvard University; 4Emory University

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Neutrophil Deposition of Microparticles onto Anti-Mouse Collagen XVII Human Inflamed Epithelium; A New Mechanism to Immunoglobulin a Antibodies Induce FcαRI - Disrupt Epithelial Cell-To-Cell Adhesions and Dependent Neutrophil Migration in a Novel Mice Promote Transepithelial Migration Model of LABD Veronika Butin-Israeli1, Madelyn C. Houser 3, Esil Aleyd1, Lydia P.E. van der Steen1, Sylvia J.P. Mingli Feng2, Edward B. Thorp1, Asma Nusrat2, Bogaards2, Stephan M. Pouw1, René J.P. Musters2, Charles A. Parkos2, Ronen Sumagin1, 1Northwestern Michel Cogné3, Cassian Sitaru4, Jantine E. Bakema5, University, Feinberg School of Medicine; Marjolein van Egmond1,6 2University of Michigan Medical School; 3Emory 1Department of Molecular Cell Biology and University School of Medicine Immunology, VUmc; 2Department of Physiology, VUmc; 3Department of Immunology, University of Neutrophil (PMN) migration across epithelial Limoges; 4Department of Dermatology, University monolayers is a hallmark of many inflammatory of Freiburg; 5Department of Otolaryngology/Head- mucosal disorders. While it is well established that Neck Surgery, VUmc; 6Department of Sugery, VUmc en-masse tissue infiltration by PMNs can cause tissue damage, mechanisms involved in this process Introduction are not yet well defined. The current work describes Immunoglobulin A (IgA) is the prevalent antibody a new mechanism whereby deposition of membrane- class at mucosal surfaces and plays an important role derived microparticles (PMN-MPs) by PMNs onto in mucosal defense. IgA can activate neutrophils, as intestinal epithelial cells (IECs) during these cells express a receptor for IgA, the FcαRI. We transepithelial migration (TEM) results in loss of have demonstrated that cross-linking of FcαRI by epithelial cadherins, leading to epithelial damage and IgA complexes induced neutrophil recruitment and increased tissue infiltration by PMNs. We release of the chemoattractant leukotriene B4 demonstrate that PMN-MPs secreted by activated, (LTB4) leading to amplification of neutrophil transmigrating PMNs display high levels of matrix migration. Our hypothesis is that excessive metalloproteinase 9 (MMP-9). Importantly, MMP-9 neutrophil activation by IgA–FcαRI interactions play associated with the PMN-MP surface was confirmed a harmful role in autoimmune diseases and lead to to have high enzymatic activity and to mediate tissue damage, such as in Linear IgA Bullous profound effects on the integrity of epithelial Disease (LABD). LABD is a chronic autoimmune monolayers. Isolated PMN-MPs efficiently bound to skin blistering disease characterized by the presence IECs, leading to cleavage of desmoglien-2 (Dsg-2) of anti-collagen XVII IgA autoantibodies and high and disruption of epithelial cell-to-cell adhesions. neutrophil influx. Furthermore, PMN-MP binding to intestinal We have now developed a novel mouse model to epithelium in-vitro and in-vivo significantly investigate the role of FcαRI and IgA-induced tissue enhanced PMN TEM. In both settings these effects damage in vivo. were specific to MMP-9 and were reversed in the Materials and Methods presence of specific pharmacological inhibitors. A hybridoma, producing human IgA (hIgA) directed Finally we demonstrate that while Dsg-2 negatively against mouse collagen XVII (anti-mCOL17 hIgA) regulates PMN TEM, activity of PMN-derived, but was generated. To determine the in vivo role of not IEC-derived MMP-9 was essential for proper FcαRI in IgA-induced neutrophil migration, FcαRI trafficking of PMNs across epithelial layers in-vitro transgenic mice (expressing human FcαRI on in a transwell assays and in-vivo in ligated intestinal neutrophils) were crossbred with LysEGFP mice loop preparations. Thus, our findings implicate (that have green fluorescent neutrophils). Anti- PMN-MPs in exacerbated, PMN-mediated mCOL17 hIgA mAbs were injected in the ears of inflammation and epithelial damage as observed in these mice, and intravital imaging was performed 48 inflammatory disorders of mucosal surfaces. hours later. Furthermore, FcαRI transgenic mice were crossbred with hIgA knock-in mice to investigate tissue damage after injection of anti- mCOL17 hIgA antibodies in the ears of these mice. Results Sepsis is a severe systemic inflammatory response to Anti-mCOL17 hIgA were able to bind to the bacterial infection. Due to prolonged stays in the basement membrane of mouse skin cryosections and ICU, complex therapies, and concerns of antibiotic- these antibodies were able to activate neutrophils in resistant microbes, sepsis remains a serious medical vitro. After injection of anti-mCOL17 hIgA in the issue. The cause of sepsis is heterogeneous and its ears of these mice, intravital imaging showed clinical features are diverse, and so determining activation and high numbers of neutrophils in the central regulators for effective host-directed blood vessels, which was absent when the vehicle therapies has been a considerable challenge. It is was injected as well as in control non-transgenic well established that sepsis impairs leukocyte and mice. Staining cryosections with GR-1 confirmed endothelial function. A rapid and robust recruitment the presence of a high neutrophil influx in response of circulating neutrophils at sites of infection is to anti-mCOL17 hIgA, which was not seen in non- critical for preventing bacterial spread, and it is well transgenic mice. established that this process is greatly impaired Importantly, minimal activation of neutrophils was during sepsis. The proteolytic activity of a observed when mice were injected with a FcαRI disintegrin and metalloprotease-17 (ADAM17) is blocking antibody. induced in the cell membrane of leukocytes upon We further analyzed whether IgA-induced neutrophil their activation, and this regulates the release of migration results in tissue damage and blister various pro-inflammatory factors as well as the formation after frequent injection of anti-mCOL17 surface density of an assortment of receptors hIgA antibodies in our FcαRI/hIgA mice model. We important for neutrophil effector functions, including were able to see differences in recruitment of their recruitment. We show that conditional neutrophils and tissue damage/blister formation in knockout mice lacking ADAM17 in all leukocytes the ears. Low numbers of neutrophils were seen in have a survival advantage when subjected to the ears injected with vehicle in hIgA and FcαRI/IgA polymicrobial sepsis. Bacteremia and circulating mice. Importantly, minimal neutrophil influx was pro-inflammatory cytokines, key determinants of seen in the ears injected with anti-mCOL17 hIgA sepsis severity, were significantly reduced in antibodies in hIgA mice, compared to the massive conditional ADAM17 knockout mice during sepsis. recruitment of neutrophils in the ears of FcαRI/hIgA The cecal bacterial microbiota and load were similar mice. in unmanipulated conditional ADAM17 knockout Conclusions and control mice, yet the peritoneal spread of Anti-mCOL17 hIgA antibodies induce neutrophil bacteria was significantly reduced in conditional activation and migration in vivo. We were able to ADAM17 knockout mice following sepsis induction. prevent neutrophil recruitment by blocking the IgA This was associated with an amplified recruitment of receptor FcαRI. Currently we are investigating neutrophils at the infectious locus. Taken together, whether we can reduce inflammation induced by our findings suggest that systemic ADAM17 activated neutrophils by blocking FcαRI on activation is a pivotal mechanism during sepsis that neutrophils. These results indicate that IgA-induced tips the balance between efficient and impaired neutrophil activation and migration is dependent on neutrophil recruitment. We are currently examining FcαRI in vitro and in vivo. Blocking IgA-FcαRI the therapeutic efficacy of ADAM17 targeting after interactions may be a therapy for IgA-induced sepsis onset in preclinical studies. blistering diseases. 30 29 Phosphoinositol-3-Phosphate Acts as a Timer for Targeting ADAM17 Increases Neutrophil ROS Production in the Phagosome by Recruitment and Reduces Bacterial Spread Controlling P67phox Accumulation during Sepsis ZhiMin Song1,2, Leila Bouchab1,2, Elodie Hudik1,2, Bruce Walcheck, Hemant K. Mishra, University of Romain Le Bars3, Oliver Nüβe1,2, Sophie Dupré1,2 Minnesota 1Université Paris-Sud, Orsay, France; 2CNRS U8000, LCP, Orsay, France; 3CNRS I2BC, Gif-sur- Yvette, France 1Instituto de Investigaciones Biomédicas- Neutrophils participate in the host defense via Universidad Nacional Autónoma de México; pathogen phagocytosis and microbial killing by 2Facultad de Odontología, Universidad Nacional producing reactive oxygen species (ROS) in the Autónoma de México phagosome. The ROS are produced by the NADPH oxidase (NOX2). The NADPH oxidase is activated Neutrophils (PMN) are the most abundant when the cytosolic subunits of NOX2 (p67phox, leukocytes in the blood. PMN migrate from the p47phox, p40phox) and Rac assemble with the circulation to sites of infection, where they are membrane subunits (gp91phox and p22phox) at the responsible for antimicrobial functions. PMN use phagosomal membrane. P67phox triggers the electron phagocytosis, degranulation, and formation of flow from NADPH to O2-. P40phox has a neutrophil extracellular traps (NETs) to kill phosphatidylinsositol-3phosphate (PI3P) binding microbes. Several stimuli, including bacteria, fungi, domain. The role of p40phox in the oxidase complex and parasites, and some pharmacological compounds is still unclear. Recent studies have revealed that the such as PMA are efficient inducers of NETs. PI3P may play an important role in locally boosting Antigen-antibody complexes are also capable of phagosomal NADPH oxidase activity through its inducing NET formation. Recently, it was reported binding to the p40phox NADPH oxidase subunit. We that FcγRIIIb crosslinking induced NET formation aim at investigating the importance of PI3P in similarly to PMA stimulation. Direct crosslinking of phagosomal ROS production and the recruitment of FcγRIIA or integrins did not promote NET the p67phox and p40phox subunits. formation. FcγRIIIb-induced NET formation We found that VPS34 IN1, a specific inhibitor of the presented a different kinetics from PMA-induced PI3Kinase, decreases the ROS production both in NET formation, suggesting differences in signaling. differentiated PLB-985 cells and human neutrophils. Because FcγRIIIb also indeces a strong activation of The general PI3Kinase inhibitor wortmannin ERK and nuclear factor Elk-1, and the transforming drastically decreases the time of presence of PI(3)P, growth factor-β-activated kinase 1 (TAK1) has p40phox and p67phox at the phagosome, which all left recently been implicated in the ERK signaling, in the the phagosome at the same time. An increase in present report, we explored the involvement of PI(3)P at the phagosome, triggered by siRNA TAK1 in the signaling pathway activated by against the PI3Kinase associated protein Rubicon FcγRIIIb leading to NET formation. FcγRIIIb was and/or the PI3phosphatase MTM1, increases ROS stimulated by specific monoclonal antibodies and production inside the phagosome and extends the NET formation was evaluated in the presence or accumulation of p67phox at the phagosome. absence of pharmacological inhibitors. The Furthermore, the down-regulation of PI(3)P at the antibiotic LL Z1640-2, a selective inhibitor of TAK1 phagosomal membrane, by overexpression of prevented FcγRIIIb-induced, but not PMA-induced MTM1, prevents the ROS production and the NET formation. Both PMA and FcγRIIIb accumulation of p67phox. crosslinkng induced phosphorylation of ERK. But, In conclusion, PI(3)P sustains ROS production in the LL Z1640-2 only inhibited the FcγRIIIb-mediated phagosome by allowing p40phox and p67phox to stay at activation of ERK. Also, only FcγRIIIb, similarly to the phagosomal membrane. Our data suggest that transforming growth factor-β-induced TAK1 p40phox works as a late adaptor controlled by PI(3)P phosphorylation. A MEK (ERK kinase) specific to maintain p67phox in the NADPH oxidase complex, inhibitor was able to prevent ERK phosphorylation PI(3)P acts as a timer for NADPH oxidase assembly. induced by both PMA and FcγRIIIB. These data show for the first time that FcγRIIIB crosslinking 31 activates TAK1 and that this kinase is required for triggering the MEK/ERK signaling pathway to Transforming Growth Factor-ß-Activated Kinase NETosis. 1 (TAK1) is Required for Human FcγRIIIb - Induced Neutrophil Extracellular Trap (NET) Formation Carlos Rosales1, Omar Rafael Alemán1, Eileen Uribe-Querol2, Nancy Mora1 32 could show that the Src/Syk-pathway downstream regulates the PI3K/Akt-, p38MAPK- and ERK 1/2- Immobilized Immune Complexes Induce the pathways upon iIC stimulation. ROS do not mediate Formation of Neutrophil Extracellular Traps the activation of Akt, p38MAPK and ERK 1/2 since (NETS) treatment with NOX inhibitor DPI, in contrast to the Martina Behnen, Christoph Leschczyk, Sonja Src-inhibitor PP2, prior to iIC stimulation did not Möller, Tobi Batel, Werner Solbach, Tamás Laskay affect their phosphorylation. University of Lübeck, Department of Infectious Diseases and Microbiology, Lübeck, Germany

Background: One important antimicrobial effector mechanism of neutrophils is the release of neutrophil extracellular traps (NETS), which are composed of chromatin, histones and antimicrobial proteins and

contribute to pathogen containment. However, NETs Figure1: Immobilized immune complexes induce the have also been suggested to play a formation of NETs. pathophysiological role in autoimmune diseases, which are characterized by the presence of Fluorescence microscopy (FM) and scanning autoimmune complexes. In several autoimmune electron mircroscopy (SEM) images of neutrophils diseases pathogenic immune complexes are formed incubated on HSA-coated and iIC-coated surfaces. on the extracellular matrix and thus are immobilized. For FM cells were stained with SYTOXgreen and We report, that immobilized immune complexes overlay of brightfield and fluorescence is shown. induce the release of NETs from primary human Conclusion: Our data show for the first time that iIC neutrophils. induces ROS-dependent NET formation in human Methods: Human blood neutrophils were incubated neutrophils, which is enhanced in the presence of the with immobilized HSA/anti HSA immune pro-inflammatory mediator TNF-α. This indicates complexes (iIC) and the ability to generate reactive that an (auto-) inflammatory milieu, characterized by oxygen species (ROS) and NETs was investigated. iIC and pro-inflammatory cytokines is highly potent The contribution of various ROS was assessed by regarding the induction of NETs in the absence of using inhibitors of ROS-generating pathways and the microbial stimuli. Thus we conclude that NETs antioxidants L-ascorbic acid and 5-ASA. Roles of contribute to pathology in autoimmune Fc-gamma receptors (FcγR) and the integrin Mac-1 inflammatory disorders associated with surface- were determined by incubation with blocking bound immune complexes. antibodies. Intracellular signaling pathways engaged downstream of the receptors were investigated by Data published in: use of specific inhibitors and western blotting. Behnen M, Leschczyk C, Möller S, Batel T, Klinger Results: Treatment of human neutrophils with iIC M, Solbach W, Laskay T: Immobilized Immune induced the release of NETs. Pre-incubation of Complexes Induce Neutrophil Extracelluar Trap neutrophils with TNF-α significantly increased iIC- Release by Human Neutrophil Granulocytes via induced NET production. We could show that iIC FcγRIIIB and Mac-1. Journal of Immunology 2014; induce NETs in a ROS-dependent manner via 193:1954-1965 activation of NADPH-oxidase (NOX) and

myeloperoxidase. The activation of iIC induced 33 oxidative burst was shown to depend on stimulation of both FcγRIIa and FcγRIIIb, whereas only HS1 Deficiency Impairs Neutrophil Activation FcγRIIIb is sufficient for iIC-induced NET release. and Extravasation Mac-1 blocking also abolished NET formation. This 1,2 2 2 Michael Schnoor , Joanna Latasiewicz , Ding Jing , suggests that FcγRIIIb, that lacks an intracellular 2 3 Silke M. Currie , Inés M. Antón , Dietmar domain, can signal in association with Mac-1. As 2 1 Vestweber , Department of Molecular Biomedicine, intracellular signaling pathways involved we 2 Cinvestav-IPN, Mexico-City, Mexico; Department identified the tyrosine kinases Src/Syk pathway. We of Vascular Biology, Max-Planck-Institute for 34 3 Molecular Biomedicine, Münster, Germany; Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Group V Secreted Phospholipase A2 Mediates the Spain Production of Angiogenic and Anti-Angiogenic Factors from Human Neutrophils Neutrophil extravasation is a critical step in innate Stefania Loffredo1, Francesco Borriello1, Raffaella immunity in response to tissue injury or invading Iannone1, Maria Rosaria Galdiero1, Anne Lisa pathogens. The interaction of neutrophils with the Ferrara1, Francescopaolo Granata1, Marco A. endothelium requires activation of adhesion Cassatella2, Gianni Marone1,3, 1Department of molecules such as the β2-integrin LFA-1 leading to Translational Medical Sciences and Center for Basic firm adhesion, polarization and intraluminal and Clinical Immunology Research (CISI), crawling to the actual site of diapedesis. These steps University of Naples Federico II, Naples, Italy; alsorequire coordinated cytoskeletal remodeling. The 2Department of Medicine, Section of General cortactin homologue in leukocytes, hematopoietic Pathology, University of Verona, Italy; 3CNR cell-specific lyn substrate (HS1) is an actin-binding Institute of Experimental Endocrinology and protein (ABP) that regulates actin dynamics and may Oncology “G. Salvatore”, Naples, Italy thus be relevant in the regulation of . Analyzing the CXCL1-stimulated Background cremaster by intravital microscopy, we found that Angiogenesis, the formation of new blood vessels leukocyte adhesion and extravasation was strongly from preexisting ones, plays a prominent role in inhibited. This was accompanied by an increased chronic inflammatory disorders and tumors. This rolling velocity pointing to a disturbed transition process is sustained by the coordinated production of from rolling to firm adhesion in the absence of HS1. several angiogenic factors including Vascular Moreover, CXCL1-induced activation of the small Endothelial Growth Factors (VEGFs) and GTPases Rac1 and Rap1 was strongly inhibited Angiopoietins (Angs). Secreted phospholipases A2 leading to disturbed actin remodelling and aberrant (sPLA2) are multivalent mediators involved in pseudopod formation. In response to inflammation, inflammatory diseases and tumors. Human HS1 interacted with the ABPs Wiskott-Aldrich neutrophils (PMNs) are both a source and a target of syndrome protein (WASP), WASP-interacting sPLA2s. These cells release group V sPLA2 and can protein (WIP) and vasodilator-stimulated be activated by sPLA2s to release CXCL8. We have phosphoprotein (VASP). Interestingly, these investigated the role of group V sPLA2 in the processes were dependent on PKA since PKA production of angiogenic factors from PMNs. inhibition blocked chemokine-induced Rap1 Methods activation and interaction with the mentioned ABPs. VEGF-A, -B, -C, -D and Angs (Ang1 and Ang2) Absence of HS1 likely prevents the formation of this expression was evaluated by RT-PCR in highly signalling complex thus explaining aberrant actin purified (>99%) PMNs. Release of VEGF-A, polymerization and impaired neutrophil VEGF-A165b, Ang1 and CXCL8 was evaluated by extravasation. The importance of PKA for HS1- ELISA. sPLA2 activity was measured by EnzChek® mediated support of extravasation was corroborated PLA2 Assay Kit. by the fact that PKA activation increased whereas Results and Conclusions inhibition reduced transmigration of WT neutrophils PMNs constitutively express mRNAs for the but not of HS1-KO neutrophils. Our results are the proangiogenic molecules VEGF-A165, VEGF-B167, first in vivo evidence that HS1 is crucial for VEGF-B186, and Ang1. mRNA for VEGF-A121, orchestrating the molecular events in neutrophils VEGF-A189, VEGF-C, VEGF-D, and Ang2 was not during inflammation that mediate extravasation. detected. PMNs also expressed mRNA for the anti- angiogenic factor VEGF-A165b. In vitro stimulation of PMNs with increasing concentrations (0.1 to 5 mg/ml) of human recombinant group V sPLA2 (hGV) caused the release of VEGF-A, Ang1 and CXCL8. hGV also induced the release of VEGF- A165b. hGV-induced release of VEGF-A was significant after 15 min (p<0.01) and progressively a reduced ability to turn in response to a change in increased up to 6 hours. Preincubation (30 min, direction of fMLP stimulation. ROS formation to a 37°C) of hGV with Me-Indoxam (1 mM), which variety of stimuli in unprimed and primed Tiam1 -/- blocks M-type receptor-mediated effects of sPLA2s, neutrophils is unaffected. Finally, a significant abolished the release of VEGF-A, Ang1 and CXCL8 defect in neutrophil recruitment to the peritoneum but not that of VEGF-A165b. The release of VEGF- upon thioglycollate challenge is observed in Tiam1 - A165b was reduced by preincubation (37°C, 30 min) /- mice. of neutrophils with P11 (100 nM) and/or TCS 2314 In conclusion, Tiam1 has an important role in (100 nM), antagonists of integrin receptors (anb3 neutrophil function by regulating a specific subset of and a4b1, respectively). These results indicate that Rac-dependent responses, and further hGV induced the production of both angiogenic and characterisation of these responses in progress. anti-angiogenic factors from PMNs by different receptor-mediated mechanisms. Activation of PMNs 36 by fMLF induced the release of hGV as well as of VEGF-A and CXCL8. Preincubation (10 min, 37°C) Regulation of Neutrophil Responses by P-Rex of PMNs with Me-Indoxam (1 mM) before and Norbin stimulation with fMLF (1 mM) inhibited (≈ 55%) Chiara Pantarelli, Dingxin Pan, Heidi C. Welch, the release of VEGF-A and CXCL8. These results Babraham Institute are compatible with the hypothesis that endogenous hGV may be involved in fMLF induced release of Phosphatidylinositol 3, 4, 5-trisphosphate-dependent VEGF-A and CXCL8. Rac exchanger 1 (P-Rex1) is a guanine nucleotide exchange factor (GEF) for the RAC family of small 35 GTP-binding proteins (GTPases). It catalyses the active conformation of Rac GTPases, thus regulating The Role of T-Cell Lymphoma Invasion and many different cell responses, including morphology Metastasis-Inducing Protein 1 (Tiam1) in and motility. P-Rex1 activation is controlled Neutrophils. synergistically by PIP3, the lipid product of PI3K Kirsti Hornigold1, Martin Baker1, Anna-Karin activity, and by the Gbg, subunits of heterotrimeric Johnsson1, Angeliki Malliri2, Heidi Welch1, G proteins. 1Signalling Programme, The Babraham Institute, A screen carried out recently by the Welch lab Babraham Research Campus, Cambridge, CB22 identified a new binding partner of P-Rex1, the 4AT, UK; 2Cell Signalling Group, Cancer Research GPCR adaptor protein Norbin. This study revealed a UK Manchester Institute, The University of novel mechanism of regulation of P-Rex1 by Manchester, Manchester MB20 4BX, UK Norbin, where Norbin is an important regulator of P- Rex1 subcellular localization and a direct stimulator Rac GTPases have an important role in regulating of the P-Rex1 Rac-GEF activity (Pan D. et al, 2016, the cytoskeleton and are involved in many neutrophil JBC). The study showed furthermore that Norbin is functions such as adhesion, spreading, chemotaxis, expressed in neutrophils. In order to assess the phagocytosis, reactive oxygen species (ROS) functional importance of the P-Rex1/Norbin formation and degranulation. interaction in neutrophils, we generated two new We have investigated the functional importance of genetically-modified mouse strains: a strain with a the Rac-activator Tiam1 in a variety of neutrophil conditional Norbin deletion in myeloid cells and a functions. We have shown that Tiam1 is expressed strain with combined Norbin and P-Rex1 deficiency. in mouse neutrophils and is absent in neutrophils Using these new mouse strains, we found that from Tiam1 -/- mice. Neutrophils from Tiam1 -/- isolated Norbin-deficient neutrophils show increased mice primed with GM-CSF and TNF-alpha show an ROS production, adhesion, spreading and polarity increase in adhesion in response to f-Met-Leu-Phe upon stimulation of the GPCR fPR1 (fMLP (fMLP) stimulation, however neutrophil spreading is receptor). Under some conditions, the Norbin unaffected. fMLP-stimulated chemotaxis is impaired deficiency was found to override functional in neutrophils from Tiam1 -/- mice primed with GM- impairments caused by the P-Rex1 deficiency. These CSF and TNF-alpha, and Tiam1 -/- neutrophils have data indicate that Norbin plays an important functional role in neutrophils. Ongoing experiments discrete subcellular pools of Rac. Further work aims are aimed at investigating whether Norbin acts by to determine the dependence of these Rac-GEF modulating P-Rex1 dependent Rac activity and mediated pools of Rac activity on specific adhesion whether it affects P-Rex1 dependent neutrophil molecules and signalling pathways. recruitment during inflammation in vivo. The characterization of neutrophil responses through 38 the analysis of mouse strains deficient in Norbin, P- Rex1 or both, in vitro as well as in vivo, will enable RIPK1-Dependent NET Formation Requires us to fully understand the importance of Norbin in MLKL and PAD4 neutrophils and its interaction with P-Rex1 for the Ben A. Croker1,2, Meghan Bliss-Moreau1,2, Arshed ability of neutrophils to fight bacterial and fungal Al-Obeidi1,2, Mathilde Gavillet1,2, David A. infections. Williams1,2, Maria Ericsson3, Akshay D'Cruz1,2, 1Division of Hematology/Oncology, Boston 37 Children’s Hospital, Boston MA; 2Department of Pediatrics, Harvard Medical School, Boston MA; Rac-GEFS Activate Discrete Spatiotemporal 3Department of Cell Biology, Harvard Medical Pools of the Small GTPase Rac to Elicit Specific School, Boston MA Neutrophil Responses Heidi CE Welch, Anna-Karin Johnsson, Signalling Neutrophil extracellular trap (NET) formation can Programme, The Babraham Institute, Cambridge, generate short-term functional anuclear cytoplasts United Kingdom. and trigger the loss of cell viability. Whether regulated non-apoptotic cell death pathways Rac is a small GTPase that can be activated by stimulate NETosis is poorly studied. By developing numerous guanine-nucleotide exchange factors methods to engage receptor-interacting protein (Rac-GEFs) to control a wide range neutrophil kinase-3 (RIPK3) and mixed lineage kinase domain- functions, including adhesion, migration, like (MLKL) in mouse and human neutrophils, we degranulation, phagocytosis, ROS and NET demonstrate that the kinase activity of receptor- formation. An important question in this field is how interacting protein kinase-1 (RIPK1) induces MLKL the many different types of Rac-GEFs can activate clusters at the membrane and peptidylarginine Rac to elicit specific neutrophil responses. deiminase 4 (PAD4)-dependent hypercitrullination Intramolecular fluorescence resonance energy of histone H3 leading to NET formation. NETs transfer (FRET) probes are a useful tool for studying prevented S.aureus replication ex vivo and mice Rac signalling. However, use of these probes in lacking RIPK3 or MLKL were sensitive to S.aureus. neutrophils has been limited because these cells are Loss of RIPK3 and MLKL prevented NET terminally differentiated, short-lived and difficult to formation but stimulated Caspase-8-dependent cell manipulate to express exogenous proteins. We death, suggesting that the activation of Caspase-8 or recently developed a reporter mouse strain that RIPK3/MLKL by the kinase activity of RIPK1 was expresses a FRET reporter for Rac activity, Raichu- dependent on substrate availability. Rac, and used it to define spatiotemporal patterns of Rac activity during the adhesion and migration of 39 primary mouse neutrophils (Johnsson A-K et al, 2014, Cell Rep). To determine if different neutrophil Blockade of Neutrophil Adhesion Has Rac-GEFs can activate specific spatiotemporal pools Therapeutic Effect in Animal Models of of Rac, we crossed this Rac-activity reporter strain Alzheimer’S Disease with mouse strains deficient in neutrophil Rac-GEFs Elena Zenaro, Enrica Pietronigro, Vittorina Della such as Prex1, Vav1 and Dock2. By correlating the Bianca, Genny Piacentino, Marco Bonani, Tanaz Rac-GEF dependence of spatiotemporal Rac activity Saatochi, Gabriela Constantin, University of Verona pools with the ability of neutrophils to undergo adhesion, spreading, chemotaxis and phagocytosis, Alzheimer's disease (AD) is the most common we were able to identify how different Rac-GEFs neurodegenerative disorder and is characterized by a elicit specific neutrophil responses by activating progressive decline of cognitive functions. The neuropathological features of AD include amyloid activation and restoration of synaptic proteins beta (Aβ) deposition, intracellular neurofibrillary compared to animals treated with a control antibody. tangles derived from the cytoskeletal Collectively, our results suggest that hyperphosphorylated tau protein, amyloid pharmacological targeting of integrins controlling angiopathy, the loss of synapses, and neuronal leukocyte trafficking may represent a new degeneration. In the last decade, inflammation has therapeutic strategy to address AD. emerged as a key feature of AD, but most studies have focused on the role of microglia-driven 40 neuroinflammation mechanisms. Vascular inflammation and a dysfunctional blood-brain- Impaired Neutrophil Extracellular Traps (NETs) barrier (BBB) have been implicated in the Release in Children with Acute pathogenesis of AD. However, the role of leukocyte Magdalena Ostafin1, Edyta Ulińska2, Olga Ciepiela1, trafficking mechanisms in the induction of Urszula Demkow1, 1Department of Laboratory neuropathological changes and memory deficit in Diagnostics and Clinical Immunology of AD is unclear. We have recently demonstrated that Developmental Age, Medical University of Warsaw; neutrophils infiltrate the AD brain and contribute to 2Department of Pediatrics, Hematology and the induction of cognitive deficit and Oncology, Medical University of Warsaw neuropathological changes in animal models of AD. The aim of our study was to investigate the role of Neutrophils have been recognised as the front-line neutrophil integrins in the pathogenesis of disease in fighters of the innate immune system. One of their animal models of AD. Two-photon laser-scanning weapon is mechanism named NETosis, created to microscopy (TPLSM) experiments in the brain of destroy bacteria, fungi, parasites and even some of mice with AD-like disease showed that LFA-1 the viruses. In this process neutrophilic granulocytes integrin blockade prevents neutrophil adhesion in release a web-like structure outside the cells, brain vessels, extravasation, and inhibits composed mainly of deoxyribonucleic acid, histones intraparenchymal motility, suggesting a key role for and enzymes derived from their granules. Emergent this integrin in neutrophil recruitment in AD. construction is known as neutrophils extracellular Notably, the blockade of neutrophil trafficking by an traps (NETs), and in some cases is equivalent of the anti-LFA-1 antibody rescued cognitive deficits in cell death. Insufficient releasing of NETs or AD-like mice. 3xTg-ADxItgal-/-mice lacking LFA-1 formation them as a defective structure, leads to integrin showed improved memory in cognitive tests increases susceptibility to contamination with compared to wild-type animals, suggesting that different pathogens. In children with acute leukemias LFA-1 integrin contribute to the induction of serious, life-threatening infections remain a major cognitive deficit in AD mice. These findings were cause of morbidity and mortality. Therefore the goal supported by neuropathological studies showing a of our study was to verify of the contribution of lower density and activation state of microglia and a NETs to infectious complications of acute leukemias reduction of amyloid beta deposition and tau in children. hyperphosphorylation in 3xTg-AD mice deficient of Blood for analysis was collected from 30 leukemic LFA-1 integrin compared to aged-matched controls. children at the time of the diagnosis and from 10 Moreover, the blockade of alpha4 integrins, which healthy donors. Neutrophils after isolation, were represent an alternative mechanism for neutrophil seeded on 96-well plate or Lab-Tek slides, and migration during inflammatory conditions, clearly activated with well-known NETs stimulator - PMA improved memory function in AD mice and the (phorbol-12-myristate-13-acetate) or treated only by restoration of cognition was maintained also at later cell medium – RPMI (negative control). time points, suggesting that therapeutic blockade of Extracellular DNA, as a marker of NETs formation, leukocyte adhesion during early stages of disease was quantitated by fluorometry using a fluorescent provides a long-term beneficial effect on cognition dye. Furthermore, construction of this structure was in older mice. In addition, neuropathological studies assessed by using fluorescent microscopy. performed in mice treated with an anti-alpha4 Averages of extracellular DNA release after 3 – antibody, showed reduction of amyloid beta hours incubation with PMA were 9317 ± 182 RFU deposition, tau hyperphosphorylation, microglial (relative fluorescent units) for children with acute leukemia and 15382 ± 499 RFU for healthy children. DF2156A presented decreased number of total cells Differences between these two groups were and neutrophils into the inflamed joint when statistically significant. Comparison of unstimulated compared to non-treated arthritic mice. This reduced samples between these groups resulted in averages cellular migration in DF2156A-treated mice was 3001 ± 57 RFU for children with acute leukemias associated to a lower TNF-α and IL-1β in inflamed and 2658 ± 86 RFU for healthy children. The tissue. Furthermore, DF2156A-treated mice had differences observed between negative controls were decreased articular damage and reduced statistically unsignificant. hypernociception compared to vehicle-treated mice. In our investigation we presented that neutrophils On the other hand, DF2156A-treated group showed isolated from blood derived from children with acute slightly increased in bacterial load at the joint when leukemia (ALL/AML) demonstrated lower compared with non-treated mice. In a similar expression of extracellular traps formation than manner, the local treatment with DF2156A increased neutrophils derived from blood of healthy children. the bacterial load compared to non-treated mice. We hope that this observation will be the first step to Purified human neutrophils stimulated with CXCL8 explain the marked susceptibility of these patients to increased the killing of S. aureus compared to non- recurrent life-threatening bacterial and fungal stimulated neutrophils. Conclusions: The blockage infections, and contribute to find solution of this of CXCR2 prevents the accumulation of neutrophils problem. in the joint and decreases the articular inflammation, tissue damage and dysfunction following S. aureus 41 infection. However, CXCR2-binding chemokines are very important to control the bacterial load by Role of Receptor CXCR2 in the Pathogenesis of neutrophils. Thus, the use of CXCR2 antagonists in Experimental Septic Arthritis the context of S. aureus-induced septic arthritis must Daiane Boff1,2, Vívian LS Oliveira2, Paul Proost1, be carefully investigated to avoid the loss of Mauro M. Teixeira2, Flávio A. Amaral2, bacterial control. Financial support: CAPES, 1KULEUVEN; 2UFMG CNPq, FAPEMIG and FWO

Introduction: Septic Arthritis is the joint disease 42 which occurs when pathogens invade the joint causing infection. The main microorganism involved Nuclear Segmentation of Neutrophils and in that pathology is Staphylococcus aureus. The Migration of Neutrophils through Narrow Pores; disease results in high mortality and morbidity, Cause or Consequence? about 50% of patients have irreversible loss of joint Erinke van Grinsven1,2, Nienke Vrisekoop1,2, Peter function. The most prominant cell involved in Pickkers3, Leo Koenderman2,1, 1Laboratory for immune response to bacterial infections is the Translational Immunology, University Medical neutrophil. This cell is the first to arrive at the site of Centre Utrecht; 2Department of Respiratory inflammation, thereby helping to combat infection Medicine, University Medical Centre Utrecht; by means of several enzymes and mediators. The 3Radboud University Nijmegen Medical Centre CXC chemokines that signals via CXCR2 activate neutrophils and promote their adhesion to the Neutrophils have to pass narrow pores when they endothelium. The objective of this work was to extravasate and patrol for pathogens in the investigate the role of chemokine receptor CXCR2 extracellular matrix. The current dogma states that in inflammation caused by S. aureus in an migration through narrow pores is favored by the experimental model of septic arthritis. Methods and characteristic segmented nucleus of neutrophils. A Results: Experimental septic arthritis was induced bulky nucleus is thought to be a limiting factor in by intra-articular injection of S. aureus (107 CFU; 10 cell migration (Wolf et al., 2013). μL) in C57/Bl6 mice (5-6 mice/group). Mice were Therefore, we tested the hypothesis that immature treated orally with an alosteric inhibitor (DF2156A) neutrophils, characterized by a band-shaped nucleus, of CXCR2 receptor 1 h after the injection of bacteria would migrate less efficiently than mature and daily for 7 days or treated intra-articularily every neutrophils, recognized by a segmented nucleus (2-3 two days for 7 days. The oral treatment with lobes). Hypersegmented neutrophils (≥4 lobes) would in turn migrate most efficiently. These three common Gram-negative bacteria to cause subtypes of neutrophils with different nuclear nosocomial pneumonia in immunocompromised segmentation appear in the blood during acute patients. Evidenced through neutropenic patients inflammation (Pillay et al., 2012). being predisposed to respiratory infection, We evoked acute inflammation by systemic neutrophils are critical for lung immunity and challenge of healthy volunteers with bacterial promote bacterial clearance. To carry out these lipopolysaccharide (LPS) and FACS-sorted the three effector functions neutrophils are first required to neutrophil subtypes. We imaged migration of traffic from the blood to tissues, and, for lung subtypes in dense collagen matrices in custom glass immunity, to the airspaces. Neutrophil recruitment chambers (as described in Wolf et al.,2013). from the systemic circulation occurs predominantly Additionally, transendothelial migration was studied in post-capillary venules, which involves a well- in a transwell system, as a model for extravasation. studied neutrophil signaling cascade for β2 integrins In Fluoroblok™ transwells, the kinetics of the transferring from a low to a high affinity state. With transmigrating neutrophil subtypes were recorded in respiratory infections, neutrophils are sequestered real-time. Before and after transendothelial within the alveolar capillary bed independent ofβ2 migration, the expression of cell surface markers and integrins, and then pass into the interstitium and nuclear segmentation were determined. airspaces. In response to certain infectious stimuli, In 3D collagen matrices as well as during β2 integrins retain sequestered neutrophils within the transendothelial migration, the type of nuclear pulmonary capillary and play a role in migration into morphology was not associated with a difference in the airspaces. This study expands this knowledge migration capacity or speed. In parallel, we tested using a technique to quantify the intravascular, the hypothesis that nuclear morphology could also interstitial and airspace fractions of the murine lung be a consequence of migration through narrow in order to look at the role of β2 integrin activation pores. However, nuclear segmentation was not in neutrophil trafficking during acute P. aeruginosa- altered after transendothelial migration or during two induced pneumonia. Talin-1 and Kindlin-3 are hours of migration in collagen. involved in activating β2 integrins to their In contrast to the current dogma, nuclear intermediate and high affinity states. Tissue analysis segmentation does not facilitate neutrophil migration of neutrophil trafficking in mixed chimeric mice either in a matrix or through an endothelial suggests that talin-1 and Kindlin-3 knockout monolayer. Nuclear (hyper)segmentation may be neutrophils exit the pulmonary capillaries and enter important for other functions of the neutrophils, or the interstitial spaces more efficiently than their may be a consequence of separate differentiation of wild-type counterpart. Pharmacological inhibition of neutrophil subtypes. high affinityβ2 integrins through the small molecule References XVA143 increases the number of neutrophils Wolf et al. J Cell Biol. 2013;201(7):1069–1084 entering the pulmonary interstitial spaces and Pillay et al. J Clin Invest. 2012;122(1):327–336 airspaces during acute pseudomonal pneumonia. Bacterial clearance within the airspaces in vivo was not significantly impacted by XVA143 treatment, 43 despite enhanced neutrophil recruitment. XVA143 attenuated neutrophil phagocytosis of P. aeruginosa, High Affinity ß2 Integrins Inhibit Neutrophil possibly contributing to unimproved clearance in Recruitment during Acute Murine Pseudomonas vivo. This study highlights the role of high aeruginosa Pneumonia affinityβ2 integrins in restricting neutrophils from Zachary S. Wilson1,2, Lawrence Ahn2, William S. exiting the pulmonary vasculature during acute P. Serratelli2, Mehmet Sen3, Craig T. Lefort2, 1Brown aeruginosa respiratory infection, which may provide University; 2Rhode Island Hospital; 3University of a target to augment host defense against pneumonia Houston in immunocompromised patients.

Pneumonia is a significant burden to healthcare as the second leading cause of mortality worldwide. Pseudomonas aeruginosa is one of the most 44 peptides prevent chemokines from properly performing their chemotactic function. Such high Competition with High Affinity affinity GAG-binding peptides may serve as a lead Glycosaminoglycan Binding Peptides Inhibits molecules for further development of inhibitors of Neutrophil Dependent Inflammation inflammation based on their interference with Paul Proost1, Vincent Vanheule1, Daiane Boff2,1, Rik interactions between multiple chemoattractants and Janssens1, Pedro E. Marques2, Nikola Kitic3, Nele GAGs. Berghmans1, Andreas J. Kungl3, Flavio A. Amaral2, Jo Van Damme1, Mauro M. Teixeira2, Anneleen 45 1 1 Mortier , KU Leuven, Leuven, Belgium; 2Universidade Federal de Minas Gerais, Belo The Small Molecule CR3 (CD11B/CD18) Agonist Horizonte, Brazil; 3Karl-Franzes Universitaet, Graz, Leukadherin-1 is Protective of the Endothelial Austria Barrier Disruption When Challenged by Neutrophils Obtained from Critically Ill Patients. The CXC chemokines CXCL9 and CXCL12gamma Jonathan S. Reichner1, Catherine M. Dickinson1, are characterized by a long, highly positively Xian M. O'Brien1, Daithi Heffernan1, William G. charged COOH-terminal region, absent in most other Cioffi1, Mohd H. Faridi2, Vineet Gupta2, 1Rhode chemokines. To investigate the role of the COOH- Island Hospital/Brown University, Department of terminal region of CXCL9 and CXCL12gamma, Surgery, Providence, Rhode Island, USA; several COOH-terminal peptides were chemically 2Department of Internal Medicine, Rush University synthesized. These peptides display high affinity for Medical Center, Chicago, IL, USA glycosaminoglycans (GAGs) with Kd’s in the low nM range and compete with functional intact During critical illness, whether from trauma, sepsis, chemokines for GAG binding. The COOH-terminal or invasive surgery, the vascular integrity is peptides don’t signal through the classical G protein- disturbed leading to edema, hypotension and, in coupled chemokine receptors (GPCR) nor act as severe cases, multisystem organ failure. This GPCR antagonists. Moreover, they do not influence endothelial dysfunction is mediated in part by the neutrophil chemotactic activity of CXCL8 in neutrophil-to-endothelial interactions driven by vitro. Based on the GAG binding data, an anti- endothelial activation and expression of ICAM, inflammatory role for these peptides may be neutrophil activation by pathogen- and damage- expected in vivo. Intravenous injection of site- associated molecular patterns, and subsequent specifically labeled peptides showed staining on the neutrophil transmigration through the endothelium. endothelial surface of blood vessels. Simultaneous The β2 integrin receptor, CR3 (Complement intravenous injection of the GAG-binding peptides Receptor 3, CD11b/CD18, αMβ2), on the neutrophil with CXCL8 injection in the peritoneum or joint surface plays a key role in both adhesion and diminished CXCL8-induced neutrophil diapedesis. Leukadherin-1 (LA-1) is a small extravasation. Analogously, monosodium urate molecule CR3 agonist that binds to the alpha I crystal-induced neutrophil migration to the domain of CR3 and allosterically stabilizes the tibiofemural articulation, a murine model of gout, is receptor in a high affinity conformation with open highly reduced by intravenous injection of headpiece, while decoupling the receptor from some CXCL9(74–103). In addition, the peptides of its intracellular signaling. LA-1 has been found to significantly inhibited neutrophil extravasation in increase neutrophil cell adhesion, decrease response to locally administrated bacterial toll-like chemotaxis, and decrease transendothelial migration. receptor agonists LPS and peptidoglycan. These data While animal models have suggested an anti- show that chemokine-derived peptides with high inflammatory effect of this molecule, the impact of affinity for GAGs may be used as anti-inflammatory increased neutrophil adherence to an activated peptides. By competing with several active endothelium on endothelial integrity and barrier neutrophil attractants for binding and immobilization function has not been investigated. on GAGs, these peptides may lower local chemokine Human umbilical vein endothelial cell (HUVEC) presentation on the endothelium and disrupt the monolayers were generated and Electrical Cell- generation of a chemokine gradient. Thereby the substrate Impedance sensing (ECIS) was used to quantify barrier disruption over time after neutrophil Neutrophils are the first responders in bacterial adhesion. Blood was obtained from healthy infections. They are also the primary target of volunteers and patients in the surgical or trauma injection by the T3SS in early stages of P. intensive care unit at Rhode Island Hospital. aeruginosa infections. Injection of two effectors, Neutrophils were isolated by dextran sedimentation ExoS and ExoT, promotes survival of P. aeruginosa and allowed adhere to TNFα-activated endothelial during establishment of the infection. These monolayers before additional fMLF stimulation. effectors prevent killing of the invading bacteria by Results from a combined ICU population including neutrophils recruited to the site of the infection. trauma patients, sepsis patients, and surgical patients ExoS and ExoT are bifunctional effectors that found neutrophils from critically ill patients caused contain distinct GTPase activating protein (GAP) significantly more endothelial damage than those and ADP-ribosyltransferase (ADPRT) activities. The from healthy donors. Neutrophils obtained from increased survival of P. aeruginosa in murine models healthy donors and activated by fMLF ex vivo had of pneumonia and keratitis, as well as survival in significantly less endothelial disruption when treated neutrophil in vitro, can be attributed entirely to with LA-1 than untreated neutrophils. Patient ADPRT activities of these two proteins. Given that neutrophils treated with LA-1 showed significantly neutrophils are the first and predominant immune less damage that untreated neutrophils from the same cell present in the lungs, blood stream, and cornea population. LA-1 treated patient neutrophils had a during in vivo infection, investigating the effect of reduction in endothelial damage to levels ExoS and ExoT on primary neutrophils is critical to indistinguishable from neutrophils from healthy understanding P. aeruginosa pathogenesis. donors. Using peripheral blood human neutrophils from LA-1 treatment attenuated loss of endothelial barrier healthy volunteers, we found that the injection of function in response to activated neutrophils from ExoS or ExoT into the cytoplasm of PMNs result in both critically ill patients and from healthy inhibition of reactive oxygen species (ROS) volunteers. This result identifies CR3 as a strong production. Here, we show for the first time that P. therapeutic target in treatment of endothelial aeruginosa targets the Ras-mediated PI3K signaling dysfunction and warrants further investigation into cascade that is responsible for the assembly of applications for the anti-inflammatory effects of LA- NADPH oxidase complex which leads to ROS 1 in situations of critical illness. (Supported by NIH production. Specifically, in human neutrophils, ExoS HL25265 and T32GM065085). and ExoT, prevent the phosphorylation of the PI3K associated regulatory kinase Akt and the cytosolic 46 NADPH oxidase component p40phox thereby rendering both inactive and preventing ROS The ADP-Ribosyl Transferase Activity of Exos production. Importantly, preventing ROS production from P. aeruginosa Targets Ras to Inhibit by neutrophils leads to increased survival of P. Reactice Oxygen Species Production in Human aeruginosa in vivo and in vitro. Our in vitro studies Neutrophils revealed that ExoS targets Ras for ADP-ribosylation Chairut vareechon1, Stephanie Zmina1, Mausita in human neutrophils. ExoS had been shown Karmaker1, Eric Pearlman 2,1, Arne Rietsch1, 1Case previously to ADP-ribosylate Ras in epithelial cells Western Reserve University; 2University of at either Arg41 or Arg128. Intracellular delivery of a California Irvine mutated Ras (R41Kh), which is unable to be Pseudomonas aeruginosa is a major cause of acute ribosylated at Arg41, rescued ROS production in infections, such as hospital-acquired pneumonia, neutrophils infected with P. aeruginosa. This blood stream infections, and microbial keratitis, as increase in ROS production was accompanied by a well as chronic lung infections in cystic fibrosis decrease in intracellular survival of P. aeruginosa in patients. Like many Gram-negative bacterial human neutrophils harboring Ras (R41K). pathogens, P. aeruginosa relies on a type III Together, our data indicate that P. aeruginosa utilizes secretion system (T3SS) to directly inject effector its T3SS to inject ExoS into the neutrophil proteins into the cytoplasm of host cells. These cytoplasm which directly targets Ras. Riboyslation effectors paralyze normal cellular functions, thereby of Ras at Arg41 leads to the inhibition of ROS enabling successful establishment of infection. production and, therefore, increased intracellular neutrophils. The activation of neutrophils by urate survival within the neutrophil. crystals require the Src kinase-dependent phagocytosis of the crystals. Identification of these 47 novel players in urate crystal-induced intracellular signaling pathways in neutrophils leads to a better Src Family Kinases in Monosodium-Urate understanding of the pathogenesis of gout and may Crystal-Induced Inflammatory Responses help to develop novel therapeutic strategies in MSU Krisztina Futosi, Miklós Vértes, Tamás Németh and crystal- associated inflammatory diseases. Attila Mócsai, Department of Physiology, Semmelweis University School of Medicine, 48 Budapest, Hungary and MTA-SE “Lendület” Inflammation Physiology Research Group of the Effect of Ly6G Ligation on Neutrophil Hungarian Academy of Sciences and Semmelweis Extravasation Varies with the Dependence of University, Budapest, Hungary Migration on Beta2 Integrins Pierre Cunin1, Pui Y. Lee1,2, Edy Kim3, Roy Background: Deposition of monosodium urate Soberman4, Peter A. Nigrovic1,2, 1Department of (MSU) crystals in the joints or other tissues is a Medicine, Division of Rheumatology, Immunology hallmark in the pathogenesis of gout, which is and Allergy, Brigham and Women’s Hospital, known to be mediated by neutrophils besides Harvard Medical School, Boston, MA, USA.; monocytes and macrophages. Although MSU 2Department of Medicine, Division of Immunology, crystal-mediated signal transduction is in the focus Boston Children’s Hospital, Harvard Medical of recent investigations, the molecular mechanism is School, Boston, MA, USA.; 3Department of only partially characterized. In this study, we Medicine, Division of Pulmonary and Critical Care investigated the role of Src family kinases in MSU Medicine, Harvard Medical School, Boston, MA, crystal induced myeloid cell activation. USA.; 4Department of Medicine, Division of Methods: Bone marrow isolated neutrophils from Nephrology, Massachusetts General Hospital, wild type and triple Src family kinases-deficient Harvard Medical School, Boston, MA, USA. (Hck–/–Fgr–/–Lyn–/–) mice were stimulated by different concentrations of MSU crystals followed BACKGROUND: Ly6G is a GPI-linked murine by the analysis of superoxide production and neutrophil surface protein of unknown function that cytokine release. The process of crystal phagocytosis is frequently targeted for depletion and labeling was investigated by videomicroscopy and flow studies. Ligation via specific antibody abrogates cytometry in neutrophils derived from wild type and neutrophil migration in IgG-dependent experimental Src family kinases-deficient mice or in human K/BxN arthritis but not in some other systems, and neutrophils treated by the Src-inhibitor dasatinib or migration in Ly6G-null mice is intact. We sought to vehicle control. understand whether the association of Ly6G with Results: The MSU crystal-induced superoxide beta2 integrins could account for these apparently release and cytokine production were dramatically discordant findings. impaired in Src family kinases-deficient or in METHODS: We employed CD18-null mice to dasatinib-treated neutrophils. Cytochalasin D, which define the contribution of integrins to neutrophil is an actin polimerization blocker that can effectively migration in peritonitis and lung inflammation. To inhibit phagocytosis, strongly reduced the isolate the role of neutrophil beta2 integrins, we superoxide release of neutrophils activated by urate employed simultaneous adoptive transfer of WT and crystals. Both in the presence of cytochalasin D or CD18-null neutrophils to animals subjected to dasatinib, the crystal phagocytosis of human inflammation in lung or peritoneum using either neutrophils was abrogated. Neutrophils with genetic sterile (IL-1beta) or infection-like (LPS or E. coli) deficiency of Src family kinases also failed to stimuli. We then administered the anti-Ly6G phagocytose the MSU crystals. antibody 1A8 to test the impact of Ly6G ligation as Conclusions: The Src family kinases Hck, Fgr and a function of neutrophil surface integrins. Lyn play an indispensable role in MSU crystal- RESULTS: Unlike K/BxN arthritis, which is strictly induced superoxide and cytokine production in dependent on CD18 expression, beta2 integrins play a reduced role in experimental inflammation in lung partially understood. Likewise, current or peritoneum. Co-engrafted WT and CD18-/- methodological approaches to quantify NETs suffer neutrophils migrated equally to bronchial irritation from significant drawbacks, not the least being the induced by either LPS or IL-1beta, and inclusion of sometimes abundant nonspecific administration of 1A8 had no effect on neutrophil fluorescent signal. In this study, we developed a recruitment. In peritoneum, WT and CD18-/- quantification method based on a novel, cell- neutrophils migrated equally to E. coli, but integrin- impermeable fluorescent polymer that only binds sufficient neutrophils demonstrate a distinct if partial extruded NETs – not intracellular DNA or advantage when inflammation was induced via IL- membrane-bound proteins. This new approach 1beta. Correspondingly, in mice subjected to allows for a straightforward, reliable, standardized peritoneal inflammation after co-engraftment with quantification of NETosis, and was applied to WT and CD18-/- neutrophils, Ly6G ligation had no decipher the upstream signaling pathways effect on the migration of either cell population in E. controlling the phenomenon. In neutrophils activated coli peritonitis but selectively impaired entry of with various physiological stimuli, we confirmed integrin-sufficient neutrophils in IL-1beta peritonitis. that inhibition of the Syk or PI3K pathways blocks CONCLUSIONS: Neutrophil migration exhibits NETosis, and found that it does so by preventing variable dependence on beta2 integrins, depending chromatin extrusion from activated neutrophils. We not only on site but also on inflammatory stimulus. also observed that inhibition of the TAK1, p38 Impairment of migration through Ly6G ligation is MAPK or MEK pathways partially prevent selective for beta2 integrin-expressing neutrophils NETosis, albeit without preventing chromatin and occurs only under conditions where beta2 extrusion – only filament generation seems to be integrins contribute to neutrophil extravasation. The affected. This indicates that extrusion does not mechanism by which Ly6G ligation alters integrin- necessarily lead to filament formation, and that the dependent migration, and the implications of this latter step can be regulated separately. By contrast, mechanism for the endogenous function of Ly6G, inhibiting PKC or Src family kinases failed to remain to be determined. prevent NETosis in activated neutrophils. Finally, we revisited the issue of whether NET generation is 49 ROS-dependent. Inhibition of the NADPH oxidase expectedly prevented NETosis in response to PMA, Physiological Stimuli Induce ROS-Independent but was found to be largely ROS-independent in NETosis Using PAD4 and Discrete Signaling response to several soluble physiological agonists. Pathways. Conversely, inhibition of PAD4 potently prevented Patrick P. McDonald, Pulmonary Division, Faculty NETosis by all stimuli tested. In summary, we of Medicine, Université de Sherbrooke and CR- describe a reliable method to quantify NETosis, that CHUS, Sherbrooke, Québec (Canada). can be used to identify new molecular targets for therapeutic intervention in pathologies known to In addition to being professional phagocytes, feature NET involvement. neutrophils produce a wide spectrum of inflammatory cytokines, which shape both the innate 50 and adaptive immune responses. More recently, neutrophils were shown to extrude decondensed Temporal Dynamics of Hepatic CD4+FoxP3+ chromatin, thus forming NETs (neutrophil Tregs following Chronic CCL4-Induced Liver extracellular traps). These structures immobilize Fibrosis pathogens, thus preventing their spreading, but also Rebecca L. McCullough1, Megan R. McMullen1, feature antimicrobial molecules that efficiently kill Laura E. Nagy1,2,3, 1Department of various microorganisms. NETs were additionally Pathobiology,Center for Liver Disease Research, shown to participate in the pathogenesis of Cleveland Clinic, Cleveland, OH; 2Department of autoimmune and inflammatory disorders. Gastroenterology, Cleveland Clinic, Cleveland, OH; Despite the importance of NETs, the molecular 3Department of Molecular Medicine, Case Western mechanisms underlying their formation, as well as Reserve University, Cleveland, OH the upstream signaling pathways involved, are only Objective: Wound healing is a naturally occurring displayed a biphasic response after CCl4 injection, physiologic response; however, upon excessive with a modest increase at 18hrs, a decrease at 24hrs, exposure to toxicants (chronic alcohol abuse, CCl4) a modest increase at 48hrs and a dramatic increase at or persistent injury (viral infections, nonalcoholic 72hrs. The expression of FoxP3 mRNA in liver also steatohepatitis), fibrosis occurs. During fibrosis, followed this biphasic response. Interestingly, hepatic stellate cells (HSCs) become activated and circulating CD4+FoxP3+Tregs increased at 18hrs, deposit extracellular matrix, hepatocytes are decreased at 24hrs, increased at 48hrs, and damaged, and hepatic nonparenchymal cells (NPCs) ultimately decreased again 72hrs post-CCl4. promote tissue injury. Both the progression and Conclusions: These data demonstrate the dynamic resolution of fibrosis depends on the interactions of response of the liver to the final challenge with CCl4 NPCs, including HSCs, resident leukocytes, and after chronic CCl4 exposure. Even after chronic infiltrating leukocytes. Regulatory T cells (Tregs) injury, this additional insult led to leukocyte are key contributors to the resolution phase of infiltration to the liver and induction of fibrogenic inflammation and maintain immunological tolerance responses within 18-24hrs. By 48-72hrs after the via secretion of a myriad of immunosuppressive and final challenge, resolution of inflammation and pro-resolving cytokines, including IL-10 and TGF-b. extracellular matrix remodeling transpires. Here, we Here, we evaluated the dynamic regulation of demonstrate that hepatic CD4+FoxP3+Tregs , major inflammation and resolution in liver following pro-resolution effector T cells, are also dynamically chronic CCl4-induced liver fibrosis. regulated, with increased recruitment to the liver Methods: Wild-type C57BL/6J female mice were during the wound healing/resolution phase of the treated twice weekly with an injection of carbon response, likely serving to promote tissue repair tetrachloride (CCl4) for five weeks; control mice following CCl4-induced liver fibrosis. This work was received olive oil (OO) (J Leuk Biology 2015 supported by NIH/NIAAA R01AA011975 and 97(1):161-9). Final doses were timed so that mice R37AA011876 (LEN) and NIH/NIDDK were euthanized 18, 24, 48, or 72 h post-injection so 5T32DK007319 (RLM). that dynamic responses to the final challenge with CCl4 could be assessed. 51 Results: CCl4-treated mice displayed elevated indices of hepatocyte injury, including plasma Neutrophil Subsets and Activation Markers Are alanine and aspartate aminotransferase (ALT, AST) Sensitive Diagnostic Indicators of the Abdominal activities; peak injury occurred 18 and 24hrs post- Aortic Aneurysm CCl4 injection, which almost resolved by 72hrs. Branislav Zagrapan, Wolf Eilenberg, Katharina Markers of liver fibrosis were elevated in CCl4- Muench, Sarah Hetzer, Vanessa Elleder, Edith treated animals, with bridging deposition of collagen Lackner, Christa Drucker, Bernd Jilma, Christoph 1a1 (COL1A1) at 24hrs. Expression of pro-fibrotic Neumayer, Ihor Huk, Christine Brostjan, Medical genes alpha-smooth muscle actin (a-SMA) and University of Vienna COL1A1 were increased at early time points (18 and 24hrs), which was maintained at 72hrs. Similarly, Objective CCl4 treatment increased the expression of pro- Neutrophils and monocytes are prominent cells in inflammatory mediators TNFa, MCP-1, and IL-6 in the intraluminal thrombus (ILT) associated with liver, with peak elevations at 18 and 24hrs. FACS abdominal aortic aneurysm (AAA). Their analysis of liver NPCs revealed infiltration of recruitment and subsequent secretion of proteolytic CD11c+ dendritic cells, Ly6G+CD11b+ neutrophils enzymes may substantially contribute to the overall and CD11b+Ly6Chi inflammatory scar-associated pathological role of the ILT in aortic wall macrophages (SAM) at 24hrs following CCl4 weakening, thus increasing aneurysm growth and the treatment. Interestingly, CD11b+Ly6Clow tissue risk of rupture. Recently, neutrophil subsets have restorative SAMs persisted throughout the recovery been identified with distinct inflammatory activity. period. The mRNA expression of pro-resolution We hypothesised that the distribution of the mediators TGF-b and IL-10 robustly increased at 18 quiescent (CD66b bright CD62L bright CD16 and 24hr, decreased at 48hr, and moderately bright), activated (CD66b bright CD62L dim CD16 increased at 72hrs. Liver CD4+FoxP3+Tregs bright) and newly released (CD66b bright CD62L bright CD16 dim) neutrophil populations as well as Nitrogen mustard (NM, mechlorethamine) is a the level of neutrophil activation markers in plasma cytotoxic alkylating agent known to induce is altered in AAA patients compared to healthy extensive lung injury, which progresses to fibrosis. controls. Comparably, the frequency of monocyte This is associated with a persistent macrophage subsets (based on CD14 and CD16 surface dominant inflammatory response. Recent evidence expression) was investigated. suggests that lipid laden foamy macrophages play a Methods role in fibrogenesis. In these studies we used high 22 AAA patients with advanced disease (prior to definition magnetic resonance imaging (MRI) and surgical intervention) and 21 age, sex, BMI and computed tomography (CT) to characterize the smoker status-matched healthy individuals were progression of lung injury and fibrosis induced by assessed. Monocytes and neutrophils were stained in NM. We also determined if NM caused hirudinised, fixed whole blood with fluorescent derangements in lung lipids resulting in the antibodies and quantified by flow cytometry. Patient formation of foamy macrophages. Male Wistar rats plasma was analysed for neutrophil elastase, NGAL were treated i.t. with 0.125 mg/kg NM or PBS and MPO by ELISA. SDF-1α, RANTES, MIP-1α, control. Gradient (GRE) and fast spin (FSE) echo MIP-1β, MCP-1, IL-8, IP-10, GRO-α, and eotaxin MRI, as well as CT scans were performed on each were quantified by Procarta Multiplex animal 1, 3, 7, 14, 21 and 28 d later. Image analysis Immunoassay. and high definition 3D lung reconstruction showed Results that NM exposure resulted in a significant increase Among the secreted factors only MPO (mean+-SD in injury volume within 1 d, which persisted up to 16.1+-11.2 vs. 7.3+-3.0 ng/ml, p<0.001) and MCP-1 28d; this was accompanied by reduced volumes of (95.8+-29.4 vs. 62.7+-24.3 pg/ml, p=0.002) were air and increased consolidated lung tissue. NM significantly increased in AAA patients compared to exposure also resulted in phospholipid accumulation healthy individuals. Correspondingly, a significant in bronchoalveolar lavage fluid (BAL). This was increase in the frequency of activated as well as associated with a time-related accumulation of newly released neutrophils was identified in AAA oxidized phospholipids in lung macrophages. By 28 patient blood (2.95+-0.66 vs. 1.92+-0.36 and 3.20+- d, these cells were enlarged, vacuolated, laden with 0.87 vs. 1.67+-0.39%, p<0.001). A significant neutral lipid, and clustered in areas of fibrosis. correlation was identified between MPO level and Liver-X-receptor (LXR) and Farnesoid-X-receptor the proportion of activated neutrophils (r=0.407, (FXR) are transcription factors involved in lipid p<0.001). The distribution of monocyte subsets did homeostasis. Three d post NM, LXR and its target not differ significantly between AAA patients and genes, cholesterol efflux transporters ABCA1 and the control group. ABCG1, were decreased in lung macrophages; Conclusion conversely, FXR was increased, along with An increased frequency of newly released and scavenger receptor CD36. Treatment of alveolar activated neutrophil subsets is significantly macrophages with lipid enriched fractions of BAL associated with AAA, as further reflected in a higher collected from rats 3 d after NM resulted in plasma level of MPO which is secreted by activated upregulation of proinflammatory genes, NOS2, neutrophils. The potential of neutrophil subsets and ApoE and PTGS2, while BAL collected from rats 28 activation markers to predict AAA progression is d post NM induced the formation of foamy currently under investigation. macrophages and upregulated anti-inflammatory IL- 10, CD163, and CX3CR1 gene expression. These 52 data demonstrate that MRI and CT imaging are effective tools to visualize the progression of NM- Nitrogen Mustard-Induced Pulmonary Injury induced lung injury and fibrosis in rats. Moreover, and Fibrosis Visualized by MRI and CT Imaging altered lipid homeostasis induced by NM results in is Accompanied by an Accumulation of Foamy macrophage foam cell formation and differential cell Macrophages in the Lung activation, responses that likely contribute to Alessandro Venosa, Derek Adler, Edward Yurkow, fibrogenesis. Jeffrey Laskin, Andrew Gow, Debra Laskin, Rutgers Supported by NIH AR055073, HL096426, University ES004738, and ES005022. 53 release of NETs was detected by LPS-stimulated neutrophils from healthy DIO mice. However, when Impact of Obesity on Formation of Neutrophil neutrophils where collected from LPS-challenged Extracellular Traps by Murine Neutrophils septic mice, the above effect was abolished. We during Sepsis further studied NET formation by neutrophils from Elzbieta Kolaczkowska, Iwona Cichon, Michal DIO and lean mice upon incubation with different Santocki, Jagiellonian University, Krakow, Poland concentrations of glucose and again detected differential responses between the two groups. Our Neutrophils combat pathogens by phagocytosis, studies indicate that formation of NETs is altered is release of antimicrobial agents and proteases, and obese mice and this is at least partially connected to release of neutrophil extracellular traps (NETs) increased glucose levels present in obese individuals. (Kolaczkowska & Kubes, Nat Rev Immunol. Detailed evaluation of mechanisms responsible for 2013;13:159). NETs are structures composed of the higher chances of surviving sepsis in obesity, in DNA decorated with histones and granular proteins, connection to NETs, might also help understand including proteases such as neutrophil elastase (NE) mechanisms of NET formation in relation to and antimicrobials. NETs play both beneficial and immunometabolism in general. disadvantageous role since they facilitate capturing of bacteria during infectious inflammation 54 (Brinkmann et al. Science. 2004;303:1532) and bystander damage to host tissues due to their Neutrophils Extracellular Traps (NETs) persistent presence in vasculature, respectively Formation in the Synovial Fluid of Heifers with (Kolaczkowska et al. Nat Commun. 2015;6:6673). Acute Ruminal Acidosis Both of these aspects of NET functioning have to be Alejandra I. Hidalgo, Maria D. Carretta, Pablo taken into consideration during inflammatory Alarcón, Stefanie E. Teuber, Maria A. Hidalgo, responses and their treatment. This is of importance Rafael A. Burgos, Institute of Pharmacology, especially during sepsis in which NETs are formed Facultad de Ciencias Veterinarias, Universidad and present in blood vessels. Sepsis is a systemic, Austral de Chile, Valdivia, Chile deleterious host response to infection with a high mortality rate. Surprisingly, however, obese patients Neutrophil extracellular traps (NETs) were have higher chances of surviving sepsis (e.g. previously described as a defense mechanism against Wacharasint et al. Crit Care 2013;17:R122). This harmful stimuli in tissues, however also are involved phenomenon might be connected to differential in the pathogenesis of several joint disease including immune responses in lean and obese individuals as it gout and rheumatoid arthritis in humans. NETs is well know that obesity is accompanied by low- structures are composed by DNA strands, grade chronic inflammation. Most importantly, antimicrobial and cytotoxic peptides, such as obese individuals have elevated levels of NE and, as myeloperoxidase, elastase neutrophilic, Matrix shown in obese mice, it contributes to inflammation- Metalloproteinase-9 (MMP-9) and histones. Aseptic induced metabolic disease, namely, neutrophil NE polysynovitis is a pathological condition described mediates insulin resistance (Talukdar et al. Nat Med. in bovine with acute ruminal acidosis, a metabolic 2012;18:1407). We hypothesized that NE present in disease produced by high intake of fermentable higher amounts in obese individuals might at least carbohydrates. The acidosis is characterized by the partially originate from NETs, and that some NETs presence of high levels of D-lactic acid in ruminal might be already present in adipose tissue and/or fluid. Until now the pathogenesis of aseptic vasculature of obese individuals prior to sepsis. For polysynovitis in bovine with ruminal acidosis is this reason we studied NET formation by non- unknown. activated and liposaccharide (LPS)- or phorbol-12- We assessed the synovial fluid of four heifers with myristate-13-acetate (PMA)-activated neutrophils acute ruminal acidosis induced by oligofructose from mice with diet-induced obesity (DIO) and age- overload (13 g/kg BW). pH of ruminal fluid was matched lean mice. C57Bl/6J mice were maintained recorded with a portable pH meter after on high fat diet (DIO, 60% kcal from fat) or chow ruminocentesis. Arthrocenthesis of tarso-crural joint diet (lean mice) for at least 12 weeks. Increased was performed to assess in the synovial fluid; total protein, DNA, ATP, prostaglandin E2and IL-1β, 55 using a microplate reader, MMP-9 by zymography, NETs formation was visualized by Assessment of Arl4c Expression in T-Cell Acute immunofluorescence microscopy using anti- Lymphoblastic Leukemia and Acute Myeloid citrunillated histone 3 antibody (Alexa 594) and Leukemia Cell Lines Picogreen as DNA probe. Also a cytological analysis Sylvain G. Bourgoin1,2, Lynn Davis2, 1University was done. The level of D-lactic acid in the synovial Laval; 2CHU de Québec fluid was detected using HPLC, equipped with a column for chiral separation. Additionally, we Background: Arl4c is a small GTPase that belongs assessed the role of D-lactic in the formation of to the Arf family. Liver X-receptor (LXR) and NETs using blood neutrophils isolated from healthy retinoid X-receptor (RXR) agonists or cholesterol bovines. All experiments were conducted in loading induces Arl4c expression. Arl4c is known to accordance with institutional review board-approved regulate cholesterol efflux and vesicular trafficking protocols and the National Guidelines on the Use of from early endosomes to recycling endosomes via Experimental Animals of the Comision Nacional de interaction with microtubules. Wnt3a and EGF Ciencia y Tecnologia de Chile. signaling synergistically induce Arl4c, which Ruminal fluid of animals with acute ruminal acidosis regulates cell migration and the actin cytoskeleton. after oligofructose overload, showed a decrease of As cancer cells often express high levels of Arl4c pH below 5.0. The synovial fluid appears cloudy and mRNA, we evaluated the expression of Arl4c in T- with abundant presence of neutrophils and high total cell acute lymphoblastic leukemia (T-ALL) and protein, DNA and ATP level. The formation of myeloid leukemia (AML) cell lines. NETs structures in the synovial fluid were confirmed Materials and methods: Arl4c monoclonal by the visualization of DNA decorated with antibodies (mAb) were generated and characterized. citrunillated histone 3. Additionally, MMP-9, Arl4c expression in T-ALL (MOLT-3, JJHAN, prostaglandin E2and IL-1β were increased in the HSB2) and AML (PLB-985) cell lines was synovial fluid of heifer with acute ruminal acidosis. monitored using qPCR and Western blotting. PLB- An increase of D-lactic acid was also detected in the 985 cells were cultured with or without LXR synovial fluid. In vitro experiments revealed that D- (GW3965, T0901317) and RXR (LG268) agonists, lactic acid induced NETs formation with the either alone or in combination. Granulocytic presence of DNA, citrunillated histone H4 and differentiation was induced using db-cAMP, DMSO, CD11b. We demonstrated that Cl-amidine reduced or DMF. the NET formation induced by D-lactic acid, Results: Arl4c mAb 21F12 was capable of detecting suggesting a possible role of PAD4 in this response. 2 ng of Arl4c, with no significant cross-reactivity to We concluded that animals with acute ruminal Arf proteins, Arl4a, and Arl4d. Higher levels of acidosis develop an aseptic neutrophilic Arl4c protein were measured in MOLT-3 and HSB2 polysynovitis, characterized by the presence of lymphoblastic T-cells compared to JJHAN cells. NETs structures, D-lactic acid, ATP, prostaglandin Higher Arl4c mRNA but lower Arl4c protein levels E2 and IL-1β in the joint. Also, our results suggest were detected in neutrophils compared to that D-lactic acid could contribute to NETs undifferentiated PLB-985 cells. Granulocytic formation in the initial steps of aseptic polysynovitis. differentiation of PLB-985 cells markedly enhanced Acknowledgments: This work was supported by Arl4c mRNA copy numbers. Differentiation with FONDECYT 1151035, Beca Conicyt Doctorado db-cAMP (3 days) increased Arl4c mRNA 500 to Nacional, MECESUP AUS1203. Programa de 1000-fold, while slightly decreasing Arl4c protein Doctorado en Ciencias Veterinarias, UACh. (15-40%). Combinations of LXR and RXR agonists during db-cAMP differentiation further decreased endogenous Arl4c protein. Conclusions: Leukemia cell lines express Arl4c. Although granulocytic differentiation markedly increases Arl4c mRNA levels, those of Arl4c protein were decreased and down regulated by combinations of LXR and RXR agonists. 56 there is a connection between obesity and inflammation, inflammation can have a role in Functional Role of Free Fatty Acid Receptor 4 T2DM. The melanocortin 5 receptor (MC5r) has a (FFAR4/GPR120) Agonists in Bovine Neutrophils role in lipolysis in adipocytes and in the induction of Stefanie Teuber, Ivan Olmo, Matias Muñoz, Rafael antigen-specific regulatory immunity, so had dual A. Burgos, Maria A. Hidalgo, Institute of roles in both metabolism and inflammation. Ocular Pharmacology, Universidad Austral de Chile, Chile immune privilege exists to protect the eye from damaging and potentially blinding inflammation, Omega-3 fatty acids are recognized ligands of the G- and one aspect of ocular immune privilege is the protein coupled receptor Free Fatty Acid Receptor 4 induction of antigen-specific systemic regulatory (FFAR4), and a role on inflammatory response has immunity. In some cases, inflammation of the eye been suggested. Previously, we suggested the (uveitis) does occur and can be autoimmune in expression of FFAR4 in bovine neutrophils; nature. Autoimmune uveitis is studied with a mouse however the role of FFAR4 agonists has not been model, experimental autoimmune uveitis (EAU). In demonstrated yet. The aim of this study was to mice, EAU spontaneously resolves and a determine the functional role of FFAR4 agonists in consequence of the resolution is the induction of bovine neutrophils. First, we demonstrated the systemic regulatory immunity. This regulatory presence of FFAR4 in bovine neutrophils by RT- immunity requires MC5r expression on a PCR, immunoblot and immunofluorescence. Then, CD11b+F4/80+Ly6G+Ly6Clo (Ly6G+) suppressor the natural and synthetic FFAR4 agonist macrophage. We therefore asked if MC5r has a role docosahexaenoic acid (DHA) and TUG891, in obesity induced hyperglycemia and if EAU- respectively, were used to analyse the associated regulatory immunity can protect from metalloproteinase-9 (MMP-9) granules release by obesity induced hyperglycemia. zymography, and MAPK and Akt phosphorylation Body weight, blood glucose, and the macrophage by immunoblot. We observed that DHA and composition in white adipose tissue (WAT) and TUG891, between 50 and 200 μM, significantly brown adipose tissue (BAT) was compared between increased MMP-9 granules release. The analysis of wild-type (WT) and MC5r(-/-) mice on a normal fat protein phosphorylation showed that DHA (50 μM), diet (NFD) or high fat diet (HFD), and compared between 15-30 min, induced the ERK1/2 and Akt with EAU mice. At 8 weeks on the HFD, MC5r(-/-) phosphorylation. Similarly, TUG891 increased the mice showed a significant increase in body weight phosphorylation of ERK1/2, p38 MAPK and Akt. In compared to HFD WT mice. Despite the increase in conclusion, our results showed that FFAR4 agonists weight, the HFD MC5r(-/-) mice showed significantly increased granules release and phosphorylation of lower blood glucose compared to HFD WT mice. intracellular signaling pathways in bovine When immunized for EAU both HFD and NFD WT neutrophils, thus suggesting that bovine neutrophil mice showed significantly lower blood glucose function could be modulated in the presence of compared to HFD WT mice. However, MC5r(-/-) omega-3 fatty acids. mice immunized for EAU showed significantly Supported by Grant FONDEF ID14I10050 and higher blood glucose on either NFD and HFD. The Fondecyt 1151047 percentage of Ly6G+ macrophages was 10 fold greater in BAT compared to WAT, and Ly6G+ 57 macrophages in BAT from HFD mice was reduced by half. This population showed a similar increased Dual Immunometabolic Roles of the accumulation in BAT from mice at resolution of Melanocortin 5 Receptor in Obesity Induced EAU on both NFD and HFD. These observations Diabetes indicate that a deficiency in MC5r provides 1,2 1 1 Darren J. Lee , Dawei Wang , University of protection from HFD induced hyperglycemia. More 2 Oklahoma Health Sciences Center; Dean McGee interestingly, these observations suggest that as Eye Institute inflammation in the eye resolves systemic regulatory immunity emerges that is protective from HFD Type 2 diabetes mellitus (T2DM) is a serious global induced hyperglycemia and may be due to a MC5r- problem with obesity as a major risk factor. Since dependent accumulation of Ly6G+ macrophages in also causing an overall unbalanced cytokine BAT. production and immunity. Batf3-deficient mice have impaired IL-12 production, which results in 58 defective Th1 immunity, but also leads to an exacerbated Th2 immunity during helminthes Dendritic Cells: Holding the Balance of Immunity infection such as Schistosoma mansoni. Increased and Tissue Integrity Th2 polarization and cytokine secretion had major Roxane Tussiwand1, Bart Everts 2, Gary E. Grajales- histo-pathological consequences with sever liver Reyes3, Edward J. Pearce4, Kenneth M. Murphy3, damage. However, following H. Polygyrus infection 1University of Basel, Switzerland ; 2Leiden increased Th2 cytokines in Batf3 deficient mice, University Medical Center, Leiden Netherlands; improved parasite burden, ensuing reduced worm 3Washington University in St. Louis, MO, USA ; and egg counts. 4Department of Immunometabolism, Max Planck Collectively, the outcome of an immune response is Institute of Immunobiology and Epigenetics, highly dependent on the site of infection, the Freiburg, Germany pathogen and the generation of the appropriate inflammatory microenvironment. A balanced Dendritic cells (DCs) can be subdivided in different cytokine production by DCs is instrumental to subsets: conventional DC type-1 (cDC1), cDC2 and generate a pathogen-specific immune response. plasmacytoid DCs (pDCs). Each subset has Altered DC development in the absence of one of the specialized functions and is required for protection key TFs, will result in an improper innate cell against selected pathogens. pDCs are major source activation as well as Th-cell polarization generating of type-1 IFNs during viral infections. cDC1 are an unbalanced immune response. DCs are therefore essential producers of IL-12 leading to IFN-g not only professional antigen presenting cells, but responses. cDC2 are a more heterogeneous subset; also essential gatekeepers of innate immunity and on one hand responsible for the production of IL-23, tissue integrity. required for the induction of Th17 immunity, and on the other hand responsible for mediating Th2 59 immunity. This DC subset specialization is obtained and maintained through a complex network of T Cells Undergoing Homeostatic Expansion transcription factors (TFs). We could establish the Induce Acute Lymphoblastic Leukemia in IL-15- requirement for specific TF during DC development Deficient NOD.Scid Mice through the generation of KO mouse models. Subburaj Ilangumaran, Diwakar Bobbala, Sheela Further, we could correlate the expression of these Ramanathan, Department of Pediatrics, Immunology TF with a specific function linked to a DC subset. In Division, Faculty of Medicine and Health Sciences, the absence of Irf8 or Batf3 cDC1 commitment is Université de Sherbrooke, Sherbrooke J1H 5N4, compromised, and their development abrogated. Québec, Canada This results in impaired generation of Th1 and CTL immunity. Mice defective for either TF are therefore Non-obese diabetes mice with severe combined highly susceptible to intracellular pathogens such as immunodeficiency (NOD/SCID) are widely used in parasites i.e Toxoplasma gondii or viral infections, leukemia research. We made a serendipitous finding i.e. Herpes simplex virus, and also show defective that NOD/SCID.Il15-/- mice, but not NOD/SCID anti-tumor response. The other branch of cDCs controls, became overtly ill following adoptive depends on the TFs Irf4 and Klf4. In the absence of transfer of splenocytes from 8.3-NOD mice these TFs, Th17 and Th2 immune responses are expressing an MHC class I-restricted, transgenic T defective. In particular, mice lacking Klf4 have cell antigen receptor (TCR). These NOD/SCID.Il15- impaired Th2 immunity against helminthes infection /- recipients developed acute lymphoblastic leukemia such as Schistosoma mansoni and are resistant to the that infiltrated the liver and kidney. This development of house dust mite mediated asthma. leukemogenic activity was restricted to donor CD4+ Altered immunity in the absence of one of these TFs T cells and also occurred when non-TCR transgenic is however, not only rendering mice susceptible to donor cells were used. The leukemic cells showed an specific pathogens, for which the TF is required, but immature T cell phenotype (CD4+CD8intTCR- CD5hiB220-), readily grew in vitro, showed multiple essential signals for HRS cell survival. cytogenetic lesions and rapidly transferred disease to Interleukin 31 (IL-31) is related to the IL-6 family immune-competent NOD mice. Further investigation and secreted mainly by activated Th2 cells. Thymic revealed that the leukemic cells did not arise from Stromal Lymphopoietin (TSLP) is highly expressed donor cells, but originated from recipient cells and in cutaneous and bronchial epithelial cells from expressed terminal deoxynucleotidyl transferase. In atopic dermatitis and asthma patients. Both agreement, NOD/SCID mice lacking IL-15 or IL-15 cytokines are mediators of chronic pruritus in receptor alpha developed spontaneous leukemia various skin diseases. Pruritus is observed in about within 8 months of age with 100% penetrance. These 30% of patients with cHL, more often in the nodular findings indicate that IL-15 deficiency or impaired sclerosis type with mediastinal mass. Aim of this IL-15 signaling in a lymphopenic environment study was to investigate the possible role of IL-31 promotes leukemogenesis from T cell precursors and TSLP in pruritus and the potential contribution during homeostatic expansion of CD4+ T cells. of these cytokines to disease pathogenesis in patients These findings highlight the importance of IL-15 with cHL. signaling in conferring protection against potentially Plasma samples from 109 HL patients were collected neoplastic T cell precursors from developing into at diagnosis and tested for IL-31/TSLP secretion by fatal leukemia. ELISA. IL-31 was detected in 65 patients out of 109 tested and ranged from 5 to 7937 pg/ml with a median of 245 pg/ml; TSLP was detected in 52 out 60 of 75 patients with a range from 9 to 4209 pg/ml, and a median of 171 pg/ml. IL-31/TSLP levels were Interleukin-31 and Thymic Stromal found not to be associated with presence or degree of Lymphopoietin Expression and Clinical itching in our patient cohort. However, we identified Relevance in Classical a subgroup of patients showing elevated plasma Patients levels of both IL-31 and TSLP who had an Vito Pistoia1, Elisa Ferretti1, Stefan Hohaus2, International Prognostic Score (IPS) >2, indicative Arianna Di Napoli3, Beatrice Belmonte4, Elisa of high risk disease (P=0.002 for IL-31, =0.03 for Cupelli2, Eugenio Galli2, Gino Tripodi5, Claudio TSLP). Among the clinical parameters included in Tripodo4, Giulio Fraternali-Orcioni6, Jean Louis IPS, white blood cells count >15x103/ml was found Ravetti6, Anna Corcione1, 1Laboratory of Oncology, to be significantly associated with high IL-31 and Istituto Giannina Gaslini, Genova, Italy; 2Institute of TSLP plasma levels (P=0.01, n=19/89 for IL-31; Hematology, Catholic University of the Sacred P=0.02, n=15/59 for TSLP). In addition, cHL Heart, Roma, Italy; 3Department of Clinical and patients with a positive interim PET-scan (indicative Molecular Medicine, Pathology Unit, Sant'Andrea of insufficient response to chemotherapy) had higher Hospital, Sapienza University, Roma, Italy ; 4Tumor IL-31/TSLP levels at diagnosis (P=0.01 for IL-31, Immunology Unit, Department of Health Science, =0.05 for TSLP) compared with patients with a Human Pathology Section, University of Palermo, negative interim PET-scan.. Palermo, Italy; 5Immunohaematology and Then, we investigated by flow cytometry the Transfusion Centre, Istituto Giannina Gaslini, expression of IL-31/TSLP and their heterodimeric Genova, Italy; 6Unit of Pathology, IRCCS Azienda receptors (IL-31R alpha/OSMR for IL-31 and Ospedaliera Universitaria San Martino – IST – TSLR/IL-7R alpha for TSLP) in lymph node Istituto Nazionale per la Ricerca sul Cancro, biopsies form cHL patients. HRS cells were found to Genova, Italy express intracytoplasmic and surface IL-31 and TSLP, as well as their receptors on the cell surface. Classical Hodgkin Lymphoma (cHL) is Among the major immune cells infiltrating cHL characterized by a low percentage of neoplastic lymph nodes, B cells and macrophages displayed Hodgkin/Reed-Stenberg (HRS) cells, that are surface and cytoplasmic expression of IL-31 and embedded in a reactive microenvironment including TSLP and expressed the corresponding receptors. CD4 T cells, B cells, macrophages, dendritic cells, CD4 T cells also expressed both cytokines (in the eosinophils, fibroblasts, and basophils/mast cells. intracellular compartment only) and their surface This inflammatory microenvironment provides receptors. This study demonstrates for the first time that IL- found that levels of IL-39 in serum and peritoneal 31/TSLP are present in the plasma of HL patients cavity significantly increase in response to infection. but do not appear to be the mediators of itching. Surprisingly, IL-39 deficient mice were more However, the cytokines are associated with HL resistant to the T.gondii infection as indicated by clinical characteristics of prognostic relevance, reduced pathogen load and increased survival. suggesting a role of these two cytokines as potential Analysis of various cytokines and chemokines in biomarkers of chemoresistance and surrogate serum from infected WT and IL-39 deficient mice biomarkers of early-PET. Moreover, the expression revealed that IL-39-/- mice had lower levels of of IL-31/TSLP and their receptors on both malignant several pro-inflammatory mediators including and immune cells suggests that numerous paracrine CXCL1 and CCL2. High levels of these chemokines and/or autocrine interactions involving these pairs of have strong correlation with increased mortality cytokines and receptors may take place in vivo. suggesting that, with sublethal doses of T.gondii infection, mice die from sepsis rather than from the 61 infection itself. Therefore, our results indicate that reduced inflammation levels in IL-39-/- mice protect Functional Characterization of Meteorin- them from immune-mediated tissue damage during Like/IL-39, a Novel Cytokine Produced by T.gondii infection. Together, our studies indicate Macrophages and Barrier Tissues that IL-39 is a pro-inflammatory cytokine that plays Irina Ushach, Christine Schneider, Melissa B. important roles in both innate and/or adaptive Lodoen, Albert Zlotnik, UC Irvine immune responses.

Cytokines play a key role in nearly all biological 62 processes including hematopoiesis, wound healing, inflammation, cell differentiation, lymphoid The Gut Microbiome is Essential for the trafficking, proliferation and survival. The cytokine Development of IL-33-Responsive ILC2s That superfamily includes various cytokines, chemokines, Mediate Chronic Inflammation in Ileitis-Prone interleukins, tumor necrosis and growth factor Mice families. Thirty eight interleukins have been Theresa T. Pizarro, K-Ann Buela, Case Western described. Recently, we reported the identification of Reserve University a novel cytokine, Meteorin-like (Metrnl), for which we have proposed the name IL-39to reflect its It is well established that IL-33 and its receptor, association with the immune system. ST2, are increased in several autoimmune and IL-39 is produced by macrophages in response to inflammatory disorders, including inflammatory various stimulating agents including TNFα, IL-17α, bowel disease (IBD). In fact, IL-33 has been IL-4 and IL-12 whereas TGFβ and IFNƴ inhibit IL- reported by several groups to be potently 39 production. Moreover, levels of IL-39 in upregulated in patients with both ulcerative colitis circulation rise in response to inflammation induced and Crohn’s disease, the two main etiopathogenic by thioglycollate injection into the peritoneal cavity. forms of IBD, compared to healthy controls. To elucidate the function of IL-39, we have However, uncovering its precise role utilizing produced an IL-39-/- mouse. Although IL-39 (primarily acute, chemically-induced) models of deficient mice have comparable numbers of major colitis has produced ambiguous results in the immune cell populations in their immune organs, the literature, indicating both pathogenic and protective effector functions of some of these cells are altered. functions of IL-33. Using a relevant, spontaneous For example, splenocytes isolated from IL-39-/-mice model of Crohn’s-like ileitis, i.e., SAMP1/YitFc produce abnormal levels of several cytokines and (SAMP) mouse strain, we recently reported that chemokines following activation (with anti- blockade of IL-33 signaling ameliorates chronic CD3/anti-CD28) when compared to splenocytes intestinal inflammation and that the gut microbiome isolated from their WT littermates. is essential for the induction of IL-33 in these mice. To elucidate the role of IL-39 in innate and/or Since IL-33 is also known to be important in the adaptive immune responses, we tested the IL-39-/- development of group 2 innate lymphoid cells mice in a model of acute T.gondii infection. We (ILC2s), the aim of this study was to determine the contribution of IL-33-responsive ILC2s to chronic overgrowth in the small intestine one day following intestinal inflammation in ileitis-prone SAMP mice. injury. We have demonstrated that interleukin-22 By flow cytometry, our results showed a dramatic (IL-22) administration helps maintain gut barrier increase in both the frequency and absolute numbers integrity and prevents bacterial overgrowth of ILC2s within the draining mesenteric lymph following the combined injury. We also observed nodes (MLNs) (P<0.05 for both frequency and that IL-22 restores proliferation and reduces absolute numbers) as well as the ileal lamina propria apoptosis in epithelial cells after ethanol and burn (both P<0.05) of SAMP vs. AKR (parental control) injury, which was partially dependent on signal mice, and increased with age as disease became transducer and activator of transcription factor 3 more severe in 20- vs. 4-wk-old SAMP (P<0.007), (STAT3). In this study, we wanted to elucidate which also demonstrated increased MLN-derived IL- whether IL-22 limits acute intestinal inflammation, 5-expressing ILC2s (P<0.05). In fact, MLN-derived and if STAT3 and downstream suppressor of ST2+ ILC2s potently expanded in normal AKR mice cytokine signaling factor 3 (SOCS3) signaling is after exogenous IL-33 administration vs. vehicle- necessary for IL-22 mediated protection. To study treated controls (P<0.0001). Interestingly, germ-free this, male C57BL/6 or intestine epithelial cell SAMP raised under gnotobiotic conditions exhibited specific VillinCre STAT3-/- knockout mice were a dramatic decrease in MLN-derived ILC2s gavaged with ~2.9g/kg ethanol or vehicle (water). compared to specific pathogen-free raised SAMP When blood alcohol content was ~100mg/dL, mice (P<0.01), which corresponded with a decrease in were anesthetized and given a ~12.5% total body MLN-derived ILC2s in SAMP lacking the bacterial surface area scald burn by immersion in 85-90 C sensor for muramyl dipeptide (SAMPxNOD2-/- vs. water for 7 seconds. Mice in sham groups were SAMPxNOD2+/+, P<0.01), suggesting that immersed in 37 C water for 7 seconds. Mice were⁰ components of the gut microbiome may be necessary immediately given 1mL normal saline resuscitation for ILC development and expansion during chronic with or without ⁰1mg/kg recombinant mouse IL-22. gut inflammation. Together, our data indicate that One day following injury total small intestine tissue, the IL-33/ST2 axis may mechanistically contribute small intestine epithelial cells (IECs), and blood to chronic intestinal inflammation, such as that were collected. Small intestine tissue was analyzed observed in IBD, through the development of for inflammatory cytokine expression (IL-6, IL-18, pathogenic ILC2s. KC) by ELISA, small IEC SOCS3 gene expression was measured by qRT-PCR, and IL-22 levels in 63 circulation were quantified by ELISA. We found increased levels of IL-22 in both sham IL-22 treated IL-22 and STAT3 Signaling Protect Intestinal (70 pg/mL) and burn ethanol (38 pg/mL) animals Barrier Integrity despite Persisting Intestinal compared to sham vehicle controls (undetectable). Inflammation following Alcohol and Burn Injury Burn ethanol mice receiving IL-22 treatment had Adam M. Hammer, Niya L. Morris, Abigail R. significant increases in serum IL-22 (352 pg/mL) Cannon, Omair M. Khan, Robin C. Gagnon, Nellie compared to all other groups. There was an increase V. Movtchan, Ilse van Langeveld, Suhail Akhtar, in IL-6 (1.5-fold), IL-18 (3.75-fold), and KC (2.2- Xiaoling Li, Mashkoor A. Choudhry, Alcohol fold) in the small intestine following ethanol burn Research Program, Burn & Shock Trauma Research injury compared to shams, and a 6-fold increase in Institute, Loyola University Chicago Health Science SOCS3 gene expression in small IECs. Burn ethanol Division, Maywood, Illinois, USA 60153 animals treated with IL-22 displayed even higher SOCS3 gene expression (9-fold increase) in small Burn injury has been shown to result in intestinal IECs, but not reduced IL-6, IL-18, or KC levels. inflammation and leakiness, which can contribute to SOCS3 expression was completely diminished in sepsis and multiple organ failure in burn-injured small IECs of STAT3-/- mice, however, we patients. Co-morbidities, such as ethanol observed similar increases in IL-6 (2.4-fold) and IL- intoxication at the time of trauma, negatively impact 18 (1.8-fold) in STAT3-/- burn ethanol injured patient prognosis. We have previously shown that animals compared to sham vehicle controls. ethanol intoxication prior to burn results in gut Together along with our previous findings, these barrier leakiness, inflammation, and bacterial data suggest that IL-22 signaling does not directly regulate gut inflammation, rather, IL-22 and STAT3 endogenous IL-21 in restraining CD8+ T cell protect the intestinal barrier by promoting epithelial reactivity towards low affinity TCR ligands. barrier regeneration following ethanol and burn injury. 65

64 Role of PR3 in IL-1b Cleavage and Release in Human Neutrophils A Novel Role for IL-21 in Modulating T Cell Hiroto Kambara1,2, YuanYuan Chen1,2, Hongbo R. Antigen Receptor Responsiveness towards Low Luo1,2, 1Department of Lab Medicine, Boston Affinity Ligands in CD8+ T Lymphocytes Children’s Hospital; 2Department of Pathology, Subburaj Ilangumaran, Diwakar Bobbala, Sheela Harvard Medical School Ramanathan, Department of Pediatrics, Immunology Division, Faculty of Medicine and Health Sciences, INTRODUCTION: Université de Sherbrooke, Sherbrooke, Québec, Interleukin 1b (IL-1b) is a key proinflammatory Canada cytokine in innate immunity and host response to microbial invasion and tissue injury. IL-1b is first IL-21 promotes the development of spontaneous and expressed as a biologically inactive precursor and virus-induced autoimmune type-1 diabetes (T1D) by the proIL-1b is processed into mature IL-1b by increasing the efficiency of CD4+ T cell help. Caspase 1. It is known that not only Caspase 1 but However, it is unclear whether IL-21 is also directly also serine proteases such as proteinase 3 (PR3), involved in the activation of autoreactive CD8+ T neutrophil elastase and Cathepsin G cleave the cells, which play a key role in T1D pathogenesis. proIL-1b in neutrophils. However, the roles of serine Here, we assessed the requirement of IL-21 in a proteases, especially PR3, on IL-1b cleavage remain virus-induced, CD8+ T cell-mediated T1D model poorly understood. For example, there is no clear where disease occurs independently of CD4 help. In evidence suggesting mouse proIL-1b is cleaved by this model expressing the glycoprotein (GP) antigen mouse PR3 even though previous studies have of lymphocytic choriomeningitis virus (LCMV) shown that human proIL-1b is cleaved by human under the rat insulin promoter (RIP-GP), LCMV PR3. Also, the exact PR3 cleavage site of IL-1b is infection activates CD8+ T cells reactive to the GP- unknown. In this study, we will analyze the role of derived antigenic peptide GP33 that attack the islets PR3 on IL-1b cleavage both in human and mouse. and cause T1D. IL-21 deficiency in RIP-GP mice METHODS: did not affect T1D induction by LCMV, indicating Recombinant human and mouse PR3 were incubated that IL-21 is dispensable for eliciting the pathogenic with recombinant human and mouse proIL-1b in functions of autoreactive CD8+ T cells. Previously vitro. The exact cleavage site of IL-1b was we have shown that IL-21 enables autoreactive determined by ESI-TOF mass spectrometry. The CD8+ T cells to respond to weak TCR ligands and PR3 cleaved mature IL-1b was expressed in E. coli induce T1D in RIP-GP mice. Therefore, we assessed and purified by Ni-NTA affinity column followed by IL-21 requirement for T1D induction by variant SUMO protease cleavage. HeLa cells were LCMV expressing altered GP33 epitopes that are incubated with the purified recombinant IL-1b and less efficient in inducing T1D in RIP-GP mice. its biological activity was measured by quantifying Surprisingly, an LCMV variant expressing the the amount of IkB. The release of IL-1b was altered peptide ligand induced T1D more efficiently measured by ELISA using both human PR3 in Il21-/-RIP-GP mice than in control mice. knockout HL-60 cells and mouse PR3 knockout Moreover, a LCMV variant expressing a very weak neutrophils. The PR3 cleaved IL-1b specific peptide mimic of the GP33 epitope activated antibody is generated. diabetogenic T cells that caused disease in a RESULTS: lymphopenic environment. Using Nur77GFP reporter Human PR3, but not mouse PR3, generates a long mice, we show that CD8+ T cells from Il21-/- mice form of mature IL-1b in vitro. The long mature IL- expressing the GP33-specific transgenic P14 TCR 1b cleavage site is 9 amino acid upstream of Caspase showed increased reactivity towards low affinity 1 cleavage site. The IkB was rapidly degraded after TCR ligands. Our findings suggest a novel role for treatment of the HeLa cells with the recombinant long mature IL-1b, suggesting that the long mature BW) by intraperitoneal injectionat time of injury. IL-1b is biologically active. PR3 knockout human One day after injury, mice were sacrificed and cells release significantly less IL-1b compared to spleen and small intestine were collected. Splenic T wild-type cells, whereas there was no significant cells were isolated and cultured with plate-bound difference in IL-1b release between PR3 knockout anti-CD3 and soluble anti-CD28 for 48 hours. and wild-type mouse neutrophils. The PR3 cleaved Supernatants were collected for the measurement of long mature IL-1b is specifically recognized by anti- IL-17 and IL-22 by ELISA, and cells were collected long mature IL-1b, but not commercially available for determination of the AHR downstream molecule, anti-mature IL-1b. CYP1A1, via mRNA expression by PCR. Lamina CONCLUSIONS: propria (LP) cells were also isolated from small PR3 cleaves proIL-1b in a species-specific manner. intestine and stimulated with CD3 and CD28 for 16 The long mature IL-1b may be a potential hours to determine IL-17 and IL-22 by FACS. In therapeutic target or biomarker for neutrophil- addition, inflammatory cytokines were measured in dominated inflammation in human. small intestinal homogenates by ELISA. We observed significant decreases in IL-17 and IL-22 in 66 both splenic T cells and LP cells as well as a significant decrease in CYP1A1 expression in Aryl Hydrocarbon Receptor FICZ Protects, splenic T cells harvested from mice receiving Intestinal Barrier by Upregulating Th17 Cell ethanol and burn injury compared to sham vehicle Responses in a Mouse Model of Alcohol and Burn mice. Furthermore, significant increases in IL-6, KC, Injury IL-18, and apoptosis in small intestine and a Xiaoling Li, Adam M. Hammer, Abigail R. Cannon, significant increase in intestinal permeability were Niya L. Morris, Robin C. Gagnon, Mashkoor A. observed in mice receiving ethanol and burn injury Choudhry, Alcohol Research Program, Burn & compared to sham vehicle mice. These data were Shock Trauma Research Institute, Loyola University similar to our previous findings. However, in this Chicago Health Sciences Division, Maywood, IL study we found that treatment of mice with FICZ 60153, USA prevented the decrease in IL-17 and IL-22, which was accompanied by a significant increase in Aryl Hydrocarbon Receptor (AHR) is a ligand- CYP1A1 mRNA expression in T cells isolated from dependent transcription factor and regulates Th17 ethanol and burn mice. Treatment of mice with FICZ cell differentiation and functions. IL-22 produced by also prevented increases in IL-6, KC, IL-18, and Th17 cells plays a critical role in maintaining apoptosis in the small intestine, as well as intestinal intestinal barrier function. In a previous study, we permeability following ethanol and burn injury. have shown that acute alcohol (ethanol) intoxication Together, these data suggest that the AHR agonist, combined with burn injury suppresses T cell release FICZ, normalizes Th17 effector functions, prevents of IL-17 and IL-22. This accompanied an increase in intestinal inflammation, and maintains intestine intestinal inflammatory cytokines, such as IL-6, KC, barrier after injury. (Support: R01 AA015731, R01 and IL-18, as well as an increase in intestinal AA015731-08S1 and T32 AA013527) permeability. In this study, we treated mice withan AHR agonist 6-Formylindolo (3,2-b) Carbazole 67 (FICZ) to determine whether AHR activation modulates the Th17 cell response and protects gut IL-33 Increases Cysteinyl-Leukotriene Receptor barrier after ethanol and burn injury. To test this, Expression in Human Peripheral Lymphocytes male C57BL/6 mice were divided into four groups: Marie Boudaud1,2, Sylvie Turcotte1,2, Jana sham vehicle, sham vehicle treated with FICZ, burn Stankova1,2, Marek Rola-Pleszczynski1,2, 1Université ethanol and burn ethanol treated with FICZ. In the de Sherbrooke; 2Centre de recherche du CHUS ethanol and burn group, mice were gavaged with ethanol (~ 3.0 g/Kg BW) to achieve a blood ethanol During allergic inflammation, interleukin-33 (IL-33) level of ~100 mg/dL prior to receiving a ~12.5% is released as an alarmin by damaged airway total body surface area burn. In the FICZ treated epithelial cells and fibroblasts. Following a group, mice were treated with FICZ (200 µg/Kg proteolytic cleavage by inflammatory proteases, the cytokine is recognized by its specific receptor ST2 dehydration. Alcohol consumption is known to be expressed on leukocyte cells, contributing to cell both pro-inflammatory and directly harmful to gut activation and Th2 immune response. Cysteinyl- barrier function, and it was recently reported to leukotrienes (CysLTs) are well characterized as induce flare periods in IBD patients. Our preliminary contributing to the Th2 phenotype. CysLTs are lipid findings demonstrate increased weight loss, colon mediators derived from acid arachidonic metabolism shortening, and large intestine inflammation in mice and include leukotriene (LT) C4, D4 and E4. They exposed to binge alcohol paradigm following DSS have initially been described as slow-reacting induced colitis. During UC remission, IL-22 substance of anaphylaxis for their strong broncho- expression is upregulated, acting as a hallmark of constrictive activity, but they also target leukocyte entrance into a UC remission period as it stimulates populations and stimulate chemotaxis and cytokine proliferation, mucous protection, and AMP secretion production. The CysLTs act via, at least, two within the intestine. In this study, we examined the receptors, CysLT1 and CysLT2, which are expressed effect of alcohol on both IL-22 expression during on the majority of leukocytes in different UC remission and mortality from C. rodentium proportions. infection in DSS induced colitis.To accomplish this, In this study, we have explored the regulation of male C57BL/6 were divided into two groups: DSS CysLT1 and CysLT2 expression by IL-33 in human and sham. In DSS group, mice received 2% DSS ad peripheral lymphocytes isolated from healthy libitum in their drinking water for 5 days to induce donors. UC. Mice in sham/control group received water. On CysLT1 and CysLT2 mRNA expression levels were day 5, DSS and sham/control group mice were not affected by IL-33 treatment. However, cytometry further subdivided into two subgroups: mice analysis showed that IL-33 increased CysLT1 and gavaged with alcohol (~3g/kg) or mice gavaged with CysLT2 protein levels, up to 120% and 60% water days 5, 6, and 7. This resulted in four overall respectively, in T and B lymphocytes, suggesting a experimental groups - sham vehicle, sham alcohol, translational or post-translational regulation of these DSS vehicle, and DSS alcohol. Three hours after the receptors. In addition, calcium influx and last gavage on day 7, mice were humanely chemotaxis in response to LTD4 were also increased euthanized and large intestines were harvested and in IL-33-stimulated T lymphocytes. processed for quantification of IL-22 and IL-17 These results reveal a potential regulation of protein levels by ELISA. A separate group of DSS peripheral lymphocyte responses to cysteinyl- vehicle and DSS alcohol mice were gavaged with 1 leukotrienes by IL-33, and reinforce the x105 CFUs of Citrobacter rodentium 3 hours after inflammatory capacity of this alarmin in allergic last gavage day 7. On day 11, mice were euthanized diseases. and large intestines were harvested and processed for quantification of IL-22, IL-6, and KC. Body weight 68 and mortality were monitored over the 11 days. Protein levels of large intestine IL-22 were Alcohol Suppresses IL-22 Expression during UC significantly decreased (6.9 fold) in DSS alcohol Remission and Enhances Mortality to the Mouse compared to DSS alone. In contrast, levels of the Enteropathogen C. Rodentium pro-inflammatory cytokine, IL-17, remained Abigail R. Cannon, Adam M. Hammer, Niya L. elevated in DSS alcohol mice compared to DSS Morris, Robin C Gagnon, Suhail Akhtar, Xiaoling vehicle. Upon gavage with C. rodentium, DSS Li, Mashkoor A. Choudhry, Loyola University alcohol mice experienced 25% mortality by day 9 Chicago Biomedical Sciences and 50% mortality by day 11 compared to no mortality in DSS vehicle. This accompanied Over 1.4 million Americans present with increases in weight loss in DSS alcohol plus C. Inflammatory Bowel Disease (IBD), and ulcerative rodentium mice compared to DSS vehicle C. colitis (UC) accounts for half of those diagnoses. UC rodentium treated mice. Furthermore, IL-22 levels in is a cyclical, life-long illness characterized by the large intestine trended toward a decrease in DSS disease remission and active disease flares causing alcohol receiving C. rodentium compared to DSS symptoms of abdominal pain, increased weight loss, vehicle plus C. rodentium, while levels of IL-6 and intestinal inflammation, rectal bleeding, and KC were elevated. However, neither of these were statistically significant. Along with our previous alcohol (~3 mg/kg) 4 hours prior to receiving a findings, these data suggest alcohol exacerbates an ~12.5% total body surface area full thickness burn UC flare period by preventing upregulation of the injury. Mice were given 5 mg/kg of PX-478 via IL-22 mediated repair response needed for entrance intraperitoneal injection immediately after burn into remission. Additionally, alcohol further impairs injury. Mice were euthanized one day after the DSS-colitis intestinal defense mechanisms against injury, small intestine was harvested and processed invading pathogens resulting in increased mortality, for isolation of epithelial cells. IECs were used for weight loss, and inflammation. (Support: R21 the measurement of HIF-1α, components of AA022324, T32AA013527) microRNA biogenesis (Drosha and Argonaute-2) and miR-150 expression. In addition, we measured 69 IL-6 and IL-18 levels in the small intestine total tissue. As shown previously, alcohol and burn injury HIF-1α Inhibitor PX-478 Modulates Microrna reduced Drosha (1.25 fold) and Argonaute-2 (1.5 Biogenesis and Inflammation in the Small fold) expression as well as miR-150 (2 fold) Intestine following Alcohol and Burn Injury expression in IECs compared to sham vehicle. Niya L. Morris1, Adam M. Hammer1, Abigail R. Treatment of mice with PX-478 at the time of injury Cannon1, Robin C. Gagnon1, Xiaoling Li1, 1Loyola normalized the expression of Drosha and miR-150 to University Chicago Health Sciences Division; sham values. PX-478 did not affect the expression of 2Integrated Cell Biology Program; 3Burn & Shock Argonaute-2 in IECs following alcohol and burn Trauma Research Institute, Department of Surgery; injury. The PX-478 restoration of miR-150 was 4Alcohol Research Program accompanied by reduced inflammation as assessed by the measurement of IL-6 and IL-18 levels in the Alcohol intoxication at the time of burn injury is a intestine following alcohol and burn injury. major contributing factor in post burn pathogenesis. Together, these data suggest that the HIF-1α Numerous adverse effects after alcohol and burn inhibitor PX-478 restores the expression of Drosha injury are linked to gut barrier disruption. The gut is and miR-150 while reducing intestine inflammation the largest bacterial reservoir within the body and a after alcohol and burn injury. Supported by compromised barrier could result in bacterial R01AA015731, R01AA015731-08S1, and translocation from the gut to extra-intestinal sites T32AA013527-12. leading to systemic inflammatory response, sepsis and multiple organ failure. Earlier studies from our 70 laboratory have shown that oxygen delivery is significantly reduced to the gut following alcohol Macrophage Migration Inhibitory Factor is and burn injury. Furthermore, we observed a Hepatoprotective following Chronic-Binge significant increase in hypoxia inducible factor Ethanol Feeding in Mice through Suppression of (HIF)-1α, a marker of hypoxia in small intestinal Neutrophil Accumulation. epithelial cells (IECs) after alcohol and burn injury. Kyle L. Poulsen1, Megan R. McMullen1, Adam R. Hypoxia has been demonstrated to negatively affect Morris1, Laura E. Nagy1,2, 1Pathobiology, Center for microRNA biogenesis. MicroRNAs are major Liver Disease Research - Cleveland Clinic, regulators of cellular homeostasis and play a critical Cleveland, OH, United States ; 2Gastroenterology role in gut inflammation and barrier maintenance. In and Hepatology - Cleveland Clinic, Cleveland, OH, a recent study, we observed a decrease in miR-150 United States expression in IECs following alcohol and burn injury. We further found that alcohol and burn injury Background: Chronic alcohol abuse is a leading reduced the expression of microRNA biogenesis cause of preventable morbidity and mortality components Drosha and Argonaute-2. To test worldwide. Alcoholic liver disease (ALD) is whether altered microRNA biogenesis and reduced characterized by a chronic progression from miR-150 following alcohol and burn injury are steatosis, inflammation, to fibrosis and cirrhosis. In linked to increased HIF-1α, we treated a group of addition, acute alcoholic hepatitis, characterized by mice with HIF-1α inhibitor PX-478 immediately massive neutrophilic infiltration, is a severe form of after injury. In short, male mice were gavaged with ALD with a high mortality rate. In order to better understand the mechanisms underlying different Conclusions: Chronic-Binge ethanol feeding induced stages of ALD, we make use of different animal marked chemokine synthesis in adipose tissue, models that model chronic progression (chronic contributing to neutrophil mobilization and ethanol feeding) or alcoholic hepatitis (Chronic- recruitment into the liver in WT mice. MIF-/- mice Binge/Gao-Binge). Macrophage Migration showed greater chemokine expression and neutrophil Inhibitory Factor (MIF) is a regulator of innate recruitment, suggesting that MIF serves as a brake to immunity with chemokine- and cytokine-like neutrophil mobilization. These data suggest that, in activities. MIF contributes to the progression of addition to MIF’s well-established chemokine several diseases, including alcohol-induced liver activity, MIF also plays a robust upstream role in the injury in response to chronic ethanol feeding in regulation of chemokine expression in response to an mice. However, the role of MIF in models of acutely severe episode of alcoholic hepatitis in mice. alcoholic hepatitis is not known. Here, making use Supported by R01-AA011876 and U01-AA020821 of MIF-/- mice, we have investigated the role of MIF (L.E.N.) and F32-AA024955 (K.L.P.) in neutrophil recruitment in response to the Gao- binge model of alcoholic hepatitis 71 Experimental Design: Wild-type C57BL6/J and MIF-/- female mice were fed a liquid diet of ethanol Butyrate Protects Intestinal Immune Response for 10 days (5% v/v) then acutely binged with during Acute on Chronic Ethanol Exposure ethanol (5 g/kg) on day 11 (Gao-binge model). Gail A. Cresci, Bryan Glueck, Laura E. Nagy, Results: In wild-type mice, Chronic-Binge feeding Departments of Gastroenterology and Pathobiology, resulted in liver damage characterized by increased Cleveland Clinic, Cleveland, Ohio, USA ALT/AST and neutrophil accumulation. Chronic- binge ethanol exposure also resulted in a dynamic Background: Excessive alcohol consumption is regulation of chemokines in circulation, including a pathogenic contributing to increased morbidity and robust increase in the C-X-C chemokine LIX, a mortality. Following a physiologic insult, an potent chemotactic factor for neutrophils in mice, immune response needs to be adequately mounted that preceded neutrophil appearance in the liver and and resolved for proper organ function homeostasis. injury. Interestingly, in contrast to the protection of The immune response to EtOH exposure differs MIF-/- mice from progression of chronic ethanol- based on multiple factors including the duration of induced liver injury, ALT/AST activities were ethanol exposure. While chronic EtOH exposure is exacerbated in MIF-/- mice in response to Chronic- proinflammatory, acute EtOH can actually dampen Binge ethanol exposure compared to WT mice. MIF- immune responses. Butyrate, a fermentation /- mice had a more robust and rapid accumulation of byproduct of the gut microbiota, influences gut neutrophils in the liver. Hepatic neutrophils from inflammation and immunity. Our prior work reveals MIF-/- mice were also more activated, as measured butyrate protects against EtOH-induced intestinal by Ly6G+CD11b+ cells in FACS analysis. and hepatic injury. This study aimed to determine Peripheral chemokines in the plasma were largely butyrate’s impact on innate immune responses in the blunted in MIF-/- mice, with the exception of LIX, intestine following acute binge-on-chronic EtOH which was present at higher concentrations in MIF-/- exposure. compared to WT mice. Interestingly, hepatic Methods: Female C57BL/6J age-matched mice were expression of LIX mRNA was actually suppressed adapted to a control liquid diet for 5 days, then following Chronic-Binge ethanol feeding in both randomly assigned to a 5% v/v (27% total kcal) WT and MIF-/- mice, suggesting an extrahepatic ethanol-containing diet or an isocalorically- source of ethanol-induced LIX. Adipose tissue is an substituted maltose dextrin containing diet for EtOH important source of cytokines and chemokines in for 10 days. Ethanol-fed groups were allowed free response to ethanol. Indeed, expression of cytokines access to the diet and control mice were pair-fed. On TNF and IL-1β, as well as C-X-C chemokines MIP2 day 11, mice were orally gavaged a 5 g/kg dose of and LIX mRNA was increased in adipose tissue EtOH or isocaloric maltose. Mice were euthanized 9 following Chronic-Binge ethanol feeding in WT hours post-binge. Mice were provided equimolar mice; this response was further exacerbated in MIF- glycerol or tributyrin (5mM) as part of the liquid diet /- mice. over the 10 days of EtOH exposure as well as added into the EtOH or maltose gavage at a dose of 2.5 1Immunology Division, Pediatrics, Faculty of mM. Tributyrin is a structured lipid consisting of a Medicine and Health Sciences, Clinical Research glycerol backbone and 3 esterified butyrate Center, Université de Sherbrooke, Sherbrooke, molecules which are released following digestion by Quebec, Canada; 2Medicine, Faculty of Medicine gastric and pancreatic amylase and lipase. Intestinal and Health Sciences, Clinical Research Center, expression of markers of inflammation was Université de Sherbrooke, Sherbrooke, Quebec, evaluated. Additionally an in vitro co-culture model Canada was developed using a transwell system. Caco2 (intestinal epithelial) and RAW 264.7 (macrophage) During the past decade, obesity has become a global cells were seeded in the apical and basolateral health problem and is associated with chronic chamber, respectively. Differentiated Caco2 cells diseases such as diabetes, hypertension and were pretreated with sodium butyrate (5 mM) for 24 cardiovascular diseases, referred to as metabolic hours followed by EtOH (40 mM) for 3 hrs. Cell syndrome. Obesity and metabolic syndrome are culture supernatant and cells were collected and associated with chronic inflammation. In this study processed for protein and mRNA expression of we characterized the expression of chemokines and cytokines, a G-protein coupled receptor for butyrate cytokines in subcutaneous and visceral adipose and chemokine’s via ELISA and real time PCR. All tissues obtained from obese patients undergoing animal procedures were approved by Cleveland bariatric surgery. Obese patients were classified into Clinic Institutional Animal Care and Use non-diabetic, pre-diabetic and diabetic groups. Committee. Lysates prepared from frozen tissues were analyzed Results: As predicted, in response to acute on for 42 analytes using commercial services. We chronic EtOH exposure, genes for immune observed that certain chemokines are expressed at mediators exhibited decreased expression in the higher levels in visceral adipose tissues when proximal colon including IL12p40, IFNγ, IL1β, compared to subcutaneous adipose tissues. Ly6C, CD68 and NOD2. However, mice co-treated Furthermore we did detect significant expression of with tributyrin during EtOH exposure had genes and cytokine signatures associated with T comparable mRNA expression of these immune lymphocytes in the visceral adipose tissues from parameters as pair-fed control mice. Similarly, in these patients. Our results show that the pattern of response to EtOH exposure, supernatant from the expression of chemokines and cytokines differ apical co-culture system exhibited decreased protein between the visceral and subcutaneous adipose expression of the chemokine IL-8 compared to tissues. control, butyrate and EtOH-butyrate treated cells. Interestingly, mRNA expression of a butyrate 73 receptor (GPR109A) was depleted with EtOH treatment, in both in vivo and in vitro experimental Peroxisome Proliferator-Activated Receptor- models, and this depletion was mitigated with Gamma (PPARγ) Plays a Critical Role in butyrate co-treatment Pulmonary Immunity by Altering Macrophage Conclusion: These findings show butyrate protects Cell Surface Receptor Expression in a Mouse against the blunting of the immune response caused Model of Pseudomonas aeruginosa Infection by acute binge ethanol exposure in the intestine and Brenda J. Curtis1, Jill A. Shults2, Devin M. Boe1, suggests that this response may be linked with a G- Elizabeth J. Kovacs1, 1University of Colorado protein coupled receptor, GPR109A, for which Denver; 2Loyola University Chicago butyrate is the main ligand. Alveolar macrophages (AMs) play a pivotal role in 72 regulating the pulmonary response to infection and maintaining tissue homeostasis. They initiate Regulation of Inflammation and Thermogenesis inflammatory responses, through pathogen in Adipose Tissues by Pro-Inflammatory phagocytosis and pro-inflammatory cytokine Cytokines production, and then switch phenotype for Sheela Ramanathan1, Volatiana Rakotoarivelo1, inflammation resolution, through neutrophil Marian Mayhue1, Marie-France Langlois2, efferocytosis and anti-inflammatory cytokine release. PPARγ is a ligand-activated nuclear receptor 74 involved in promoting resolution. Previous work in our laboratory showed that in comparison to Microfluidics-Based Side View Flow Chamber wildtype mice, intratracheal P. aeruginosa infection Reveals Tether-To-Sling Transition in Rolling caused impaired respiratory function and Neutrophils dramatically elevated levels of lung pro- Alex Marki1, Edgar Gutierrez2, Zbigniew Mikulski1, inflammatory cytokines in macrophage-specific Alex Groisman2, Klaus Ley1,3, 1La Jolla Institute for PPARγ knockout mice (Mac PPARγ KO). Allergy and Immunology; 2Department of Physics Histologically, lungs of Mac PPARγ KO mice also UCSD; 3Department of Bioengineering UCSD had increased cellularity, interstitial thickening, and alveolar collapse following intratracheal P. Neutrophils rolling at high shear stress (above 6 aeruginosa infection. Since AMs play a critical role dyn/cm2) form tethers in the rear and slings in the in regulating lung inflammation following infection, front. Here, we developed a novel photo- we chose to examine AMs in this model. Wildtype lithographically fabricated, silicone(PDMS)-based (C57BL/6) or Mac PPARγ KO mice were given P. side-view flow chamber to dynamically visualize aeruginosa or PBS control (intratracheally, 40,000 tether and sling formation. Fluorescently membrane- CFU). 24 hours later, bronchoalveolar lavage cells labeled mouse neutrophils rolled on P-selectin were analyzed by flow cytometry for absolute substrate at 10 dyn/cm2. Most rolling cells formed 5 numbers of AMs and cell surface receptor tethers that were 2-30 μm long. Breaking of a single expression, as determined by mean fluorescence tether caused a reproducible forward microjump of intensity (MFI). Markers for efferocytosis (CD11b the cell, showing that the tether was load-bearing. and MARCO), antigen presentation (MHC Class II About 15% of all tether-breaking events resulted in and Toll-like receptor-4 (TLR4)/MD-2), and slings. The tether-to-sling transition was fast (<100 macrophage phenotype (CD206) were assessed. ms) with no visible material extending above the When compared to vehicle-treated controls, CD11c+ rolling cell, suggesting a very low bending modulus CD11b-SiglecF+ AM were decreased by 64.7% of the tether. The sling downstream of the rolling following P. aeruginosa infection in wildtype mice cell aligned according to the streamlines before and 96.9% in Mac PPARγ KO mice (p<0.05). landing on the flow chamber. These new Levels of CD11b were reduced by 26.4% and observations explain how slings form from tethers 50.14% in infected wildtype and Mac PPARγ KO and provide insight into their biomechanical mice, respectively (p<0.05). There was also a trend properties. towards heightened MARCO expression, as levels were increased by 14.3% and 28.3% in infected 75 wildtype and Mac PPARγ KO mice, respectively. No differences in TLR4/MD-2 were observed Morpho-Rheological Fingerprint of Neutrophils between groups. When comparing vehicle treated in fMLP Activated Blood and RSV Infections animals, Mac PPARγ KO mice had 38.8% higher Nicole Toepfner1,2, Christoph Herold2, Neda Farahi1, expression of MHC Class II (p<0.05) than wildtype, Jochen Guck2, Edwin R. Chilvers1, 1Department of which was not changed in the presence of infection. Medicine, University of Cambridge, School of Lastly, we measured levels of the anti-inflammatory Clinical Medicine, Cambridge, UK ; 2Biotechnology marker (M2), CD206. CD206 was reduced by 82% Centre, Technische Universität Dresden, Dresden, in AM from wildtype mice (p<0.05) and by 97.9 % Germany in Mac PPARγ KO (p<0.05). These findings suggest that AM deficient in PPARγ upregulate cell surface Background: Neutrophil activation induces changes markers important for efferocytosis and antigen in a number of biophysical characteristics including presentation more robustly than wildtype AM do in cell size, shape and mechanical properties. By a response to P. aeruginosa infection and that the microfluidic technology named RealTime switch to a M2, anti-inflammatory phenotype may Deformability Cytometry (RTDC) we have be impaired in Mac PPARγ KO AMs. Supported by advanced mechanical cell analysis from rates of 1 R01AA012034 (EJK) and R01AG018859 (EJK). cell/min up to 3000 cells/s (Otto et al., Nature Methods, 2015). To enable label-free RTDC identification of neutrophils in whole blood, we sensitive way to detect even minimal shape change developed a cell distinction algorithm to allow novel (priming). Additionally, AR realized the label-free morpho-rheological blood cell characterization. This distinction of neutrophil subsets during activation. morpho-rheological fingerprint of blood neutrophils Priming starts rapidly following fMLP stimulation, was assessed in RSV infection and healthy volunteer in which even neutrophils of AR < 1.05 decreased in neutrophils activated in-vitro in whole blood by the size and were less deformable, and shape changed bacterial tri-peptide fMLP. neutrophils (AR > 1.05) were detected. This was Method: Morpho-rheological neutrophil typing was followed by a wave of de-priming characterized by assessed in five patients with RSV+ lower increased neutrophil size and deformability as seen respiratory tract infections. All patients were in clinical RSV infection. The mechanism and recruited on the day of hospitalization with a core physiological meaning of this latter response is yet temperature > 38.5°C and the need for supplemental to be established. oxygen. The blood of 20 healthy donors was used as control. Blood samples were analyzed by RTDC 76 within 30 minutes of venipuncture. For in-vitro fMLP activation, blood samples of five healthy Long-Term Intensive Endurance Exercise Leads donors were stimulated with 100nM fMLP. Samples to an Innate Immune Response Characterised by were incubated at 37 °C, 450 rpm, analyzed at RT Mobilisation of CD16+CD62L- Neutrophils after 15, 30, 45 and 60 min and compared to Selma van Staveren1, Twan ten Haaf2, Margot unstimulated controls. In brief, RTDC blood analysis Klöpping1, Gerjen Tinnevelt3, Jeroen Jansen3, Jos de involved 50 µl blood diluted in 950 µl PBS Koning2, Nienke Vrisekoop1, Leo Koenderman1, methylcellulose at 15mPas and was carried out in 20 1UMC Utrecht; 2VU Amsterdam; 3Radboud µm PDMD channels at a flow rate of 0.12 µl/s. University Nijmegen Morpho-rheological phenotyping was processed via a series of in-house python scripts. Area and Regular physical exercise is correlated with fewer deformability were calculated for neutrophils with upper respiratory tract infections (URTI), when an area ratio of 1.0 to 1.05 (AR; reflecting concavity compared to a sedentary lifestyle. In contrast, of shape). intensive endurance training is associated with an Results: The cross sectional area of blood above average risk to URTI. Different studies have neutrophils of patients with RSV infections was shown that peripheral blood neutrophil counts increased in comparison to controls (p=0.0014). increase upon exercise and recover within 6-24 Additionally, blood neutrophils from RSV patients hours. This applies both for anaerobic as well as for were more deformable in comparison to controls aerobic exercise bouts. We have previously (p=0.0374). A change of AR was only detected in described the mobilisation of 3 neutrophil subsets in one patient with RSV infection, whereas the others response to acute inflammation such as evoked by did not differ from the control cohort (p=0.3624). systemic LPS challenge in healthy volunteers. These fMLP activation of blood neutrophils caused an cells can be identified by differential expression of initial size reduction and the expected stiffening of FcgammaRIII (CD16) and of L-selectin (CD62L) cells (the ‘priming’ response). However, this was (Pillay et al, 2012). The aim of this study was to test followed by an increase in cell size and an the hypothesis that these 3 subsets also arise upon unexpected increase in deformability 30 min post long term endurance exercise. stimulation (corresponding to the ‘de-priming’ To test this hypothesis we studied the leukocyte phase); AR peaked at 15 min with consecutive kinetics in the peripheral blood of thirty (11 female, decreases thereafter (all p < 0.01). 19 male) contestants of an 8-day intensive cycling Discussion: Neutrophil activation can be detected tour (on average 160 km and 2500 altimeters per label-free in whole blood of patients with RSV day). Next to this, we measured the expression of infection. The morpho-rheological fingerprint CD16, CD62L and an additional 8 markers on their provides a novel view on mechanical neutrophil peripheral blood neutrophils by flow cytometry. changes under ‘near to’ physiological conditions. Blood was sampled on three mornings and three Correlation to the in-vitro kinetics of fMLP activated afternoons purified neutrophis revealed that AR provides a During the second part of the tour overall neutrophil numbers did not normalise overnight and an Background: CARD9 is a CARMA-like enhanced neutrophil count was found in the morning intracellular protein that is highly expressed in of day 8 (1.32 fold-increase; 95%CI 1.04-1.58 myeloid cells. We previously presented that CARD9 p<0.05). When analysing the 3 neutrophil subsets plays an important role in the development of separately we found that “mature” CD16+CD62L+ experimental arthritis and autoimmune blistering neutrophils normalised to baseline overnight, skin disease in mice, likely by linking the Syk whereas CD16+CD62L- neutrophils were tyrosine kinase to chemokine production in innate significantly increased at the mornings of day 5 immune cells.Here, we investigated whether CARD9 (1.66 fold-increase; 95% CI 1.10-2.22) to day 8 expression in neutrophils is required for the in vivo (2.65 fold-increase; 95% CI 1.9-3.4 p<0.0001) when pathogenesis. compared to the morning of day 1. Also a significant Methods: Neutrophil-specific CARD9-deficiency increase in CD16-CD62L+ neutrophils was found in was achieved by crossing MRP8 promoter-driven the morning of day 8 (2.78 fold-increase; 95% CI Cre recombinase transgenic (MRP8-Cre) animals 1.33-4.23 p<0.05). Using FLow cytometric with Card9flox/flox mice (MRP8-Cre Orthogonal Orientation for Diagnosis (FLOOD), a Card9flox/flox animals). The efficacy and computational analysis method for multidimensional specificity of lineage-specific deletion was analyzed flow cytometry data (Jansen, Hilvering et al 2016), by Western-blot in neutrophil and macrophage cell we analysed our data in a response specific space. lysates. Experimental arthritis was induced by a We identified a new CD62L- population within the single intraperitoneal injection of K/BxN serum and total CD16+CD62L- population which has was assessed by clinical scoring, ankle thickness differential marker expression when compared to the measurements and by testing articular function. classical CD16+CD62L- cells. Epidermolysis bullosa acquisita was triggered by In conclusion, long-term intensive endurance anti-collagen type VII antibodies. Global neutrophil exercise induces a systemic innate immune response gene expression changes were detected by characterised by a change in neutrophil phenotypes. Affymetrix Microarrays. CXCL2 levels in the This response does not recover to baseline overnight synovium were measured by ELISA. after 5-7 days of exercise. Multidimensional Results:In contrast to wild type animals, neutrophils FLOOD analysis of peripheral blood neutrophils of the MRP8-Cre Card9flox/flox mice did not allows the analysis of multiple markers and the express CARD9, while their macrophages had definition of a new neutrophil phenotype mobilised normal levels of the protein. Neutrophil-specific during extreme exercise. CARD9-deficiency caused a partial, but significant decrease in the clinical arthritis score, the ankle 77 thickening and the articular dysfunction, which did not differ significantly from that seen in Card9–/– Neutrophil CARD9 Mediates Autoantibody- animals. A similar phenomenon was observed in induced Autoimmune Diseases by Controlling connection with the autoimmune blistering skin Gene Expression Changes in Vivo disease as MRP8-Cre Card9flox/flox animals Tamás Németh1, Krisztina Futosi1, Cassian Sitaru2, showed a substantial but incomplete reduction in Jürgen Ruland3 and Attila Mócsai1 inflammation, which was indistinguishable from 1Department of Physiology, Semmelweis University theCard9–/– phenotype. Furthermore, CARD9- School of Medicine, Budapest, Hungary and MTA- deficiency resulted in a 78 % reduction in immune SE “Lendület” Inflammation Physiology Research complex-induced global gene expression changes in Group of the Hungarian Academy of Sciences and neutrophils in vitro. In line with this, synovial Semmelweis University, Budapest, Hungary CXCL2 levels were significantly lower in mice with 2Department of Dermatology, University Hospital neutrophil-specific CARD9-deficiency compared to Freiburg and BIOSS Centre for Biological wild type animals. Signalling Studies, Freiburg, Germany Conclusions: CARD9 in neutrophils plays an 3Department of Clinical Chemistry and important role in the development and progression Pathobiochemistry, Technical University of Munich, of autoimmune arthritis and autoimmune blistering Munich, Germany skin disease in mice, likely by controlling gene expression changes and chemokine release. These are the first data in the literature showing a direct separation, proteins were extracted, trypsin digested role for neutrophil gene expression changes in vivo. and the resulting peptides were iTRAQ labelled This project was supported by the János Bolyai followed by HILIC fractionation and nLC-MS/MS Research Scholarship of the Hungarian Academy of analysis. Database searches, normalization and Sciences. statistical analysis of our proteomic analysis resulted in the identification of 2437 protein groups that were 78 assigned to five different clusters (Fig.1) based on the relative abundance profiles among the Neutrophil Proteomics Reveals Pathways experimental groups. Statistical analysis of the Involved in Preconditioning Prevention of proteins within each cluster led to the identification Ischemia/Reperfusion Injury of significantly up and downregulated proteins in IR Samina Arshid3,1, Muhammad Tahir1,2, Belchor and IPC. Cluster based analysis revealed that after Fontes3, Edna F.S. Montero3, Mariana S. Castro1, intestinal ischemic preconditioning some KEGG Simone Sidoli2,4, Veit Schwämmle2, Peter pathways are regulated. The most relevant Roepstorff2, Wagner Fontes1, 1Laboratory of upregulated pathways were actin cytoskeleton, Biochemistry and Protein Chemistry, Department of metabolism, phagocytosis, chemokine signaling, Cell Biology, Institute of Biology, University of focal adhesion and leukocyte transendothelial Brasilia, Brasília-DF, Brazil.; 2Department of migration whereas downregulation was detected in Biochemistry and Molecular Biology, University of ribosome, spliceosome, RNA transport, protein Southern Denmark, 5230 Odense M, Denmark.; processing in endoplasmic reticulum and 3Laboratory of Surgical Physiopathology (LIM-62), proteasome. Furthermore, enzyme prediction Faculty of Medicine, University of São Paulo, analysis revealed the regulation of some important Brazil.; 4Epigenetics Program, Department of antioxidant enzymes and having their role in reactive Biochemistry and Biophysics, Perelman School of oxygen species production. To our knowledge, this Medicine, University of Pennsylvania, Philadelphia, work describes the most comprehensive and detailed PA 19104, USA quantitative proteomic study of the neutrophil showing the beneficial role of ischemic Intestinal ischemia and reperfusion injury often preconditioning and its effects on the neutrophil results into lung injury and multiple organ failure proteome. This data will be helpful to understand the also observed after trauma and surgery. Ischemic effect of underlying protective mechanisms preconditioning preceding ischemia and reperfusion modulating the role of PMNs after IPC and provide was shown to attenuate this injury and has a a trustworthy basis for future studies. potential therapeutic application; however the exact underlying mechanism is not clear. Neutrophils are key participants in the mechanism of ischemia and reperfusion injury while the ischemic preconditioning led to a decrease in neutrophil stimulation and activation. Proteomic analysis is widely used as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding a longer ischemia and reperfusion on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia/reperfusion and Figure 1: Expression profile of regulated proteins ischemic preconditioning. Ischemia was performed found in the four experimental conditions (quiescent by clamping the superior mesenteric artery for 45 control, laparotomy, ischemia / reperfusion and min, reperfusion was allowed for 120 min. The preconditioning). All identified proteins were preconditioning was obtained by a short (10 min) grouped in 5 clusters. ischemia/reperfusion event preceding the longer ischemic event. At the end of the surgical procedure, central venous blood was collected. After neutrophil 79 were functional as shown by ROS production upon activation with PMA. This rapid method to isolate Direct One-Step Isolation of Untouched Human untouched neutrophils will facilitate studies of Neutrophils from Whole Blood without Lysis neutrophil function in inflammatory disease. Carrie E. Peters, Nooshin Tabatabaei-Zavareh, Tim A. LeFevre, Steven M. Woodside, Terry E. Thomas, 80 STEMCELL Technologies Inc, Vancouver, BC, Canada Multiple Signals Coordinate Human-Neutrophil Migration during Swarming in a Novel Ex vivo Neutrophils are the first line of defense against Assays microbial infections, sensing pathogen-associated Daniel Irimia1,2,3, Eduardo Reategui1,2, Aimal molecular patterns through a wide range of Khankhel1, Fatameh Jalali1, Elisabeth Wong1, receptors. Once activated, neutrophils eliminate Hansang Cho1,2, Charles Serhan2, Jesmond Dalli2, infection via phagocytosis of pathogens, releasing Hunter Elliot2, Daniel Irimia1,2,3, 1Massachusetts pro-inflammatory cytokines and antimicrobial General Hospital; 2Harvard Medical School; peptides, and producing reactive oxygen species 3Shriners Burns Hospital, Boston (ROS). Current procedures to enrich neutrophils from human whole blood involve at least two steps; Neutrophils display a highly coordinated behavior one to remove red blood cells (RBC) and one to against large microbial targets, a process known as select neutrophils or to remove unwanted cells. For swarming. Swarming is important for containing example, RBCs may be first depleted using fungal infection but also key to the pathology of ammonium chloride lysis or sedimentation, followed various inflammatory diseases, including rheumatoid by fluorescence-activated cell sorting or magnetic arthritis or gout. Better understanding the molecular cell separation. Alternatively, neutrophils may be players that control the swarming of human separated from other leukocytes using density neutrophils may help develop new treatments against gradient centrifugation. However, in many cases infections and inflammatory diseases. However, mononuclear cells (MNC), eosinophils, and despite some recent advances from the in vivo basophils may still contaminate the pelleted cells, studies of swarming in mice, progress has been slow. and they are still mixed with RBCs which then need Animal experiments are low throughput, have poor to be lysed. Finally, neutrophils may be lost or their control over local microenvironment, and their viability compromised at every stage of a multi-step relevance to human immune responses is untested. procedure, thus isolation in a single step from the Here, we circumvent the limitations of current starting sample is preferable. techniques by designing a novel ex vivo assay that We have developed a one-step immunomagnetic, enables us to control the size of swarming targets column-free negative selection method to enrich and space between arrays of targets (see figure). neutrophils from whole blood which does NOT With this assay, we monitored the recruitment of require RBC lysis, sedimentation or density gradient neutrophils towards targets at highest resolution centrifugation. RBC, MNC, platelets, eosinophils observation to date, we synchronized the starting and basophils were targeted for removal with time for thousands of swarms at once and analyzed antibody complexes which crosslinked them to the proteins and lipids in the supernatant, and magnetic particles. The sample was then placed in an verified the role of some of these mediators by EasySep™ magnet, and the labeled, unwanted cells targeted inhibition of receptors or neutralization of were retained in the magnet while the neutrophils mediator. We uncovered a highly redundant network were simply poured off. The sample was diluted of chemotactic cytokines that coordinate neutrophil with buffer and the magnetic incubation and pour-off swarming, consisting of more than two dozen steps repeated once more, for a total protocol time of cytokines and lipid inflammation mediators that are 25 minutes. differentially secreted by neutrophils between The purity of neutrophils isolated directly from swarming vs. simple phagocytosis, and measured whole blood was 97.3 ± 1.4%, with an average their temporal dynamics. Interestingly, some of the recovery of 0.6 – 2.9 x 10^6neutrophils / mL of cytokines stimulate receptors present exclusively on blood (means ± SD, n=14). The isolated neutrophils other immune cells besides the neutrophils and also non-immune cells, suggesting that the human lactoferrin. These data indicate that two other types neutrophils participating in swarms are at the center of sugar residue, distinct from CD15, characterize of a constellation of coordinated cellular responses specific granules. One sugar type is present in of the innate and adaptive immune systems, blood specific granules and myeloperoxidase-positive vessels, adipose tissue, tissue fibroblasts etc. azurophilic granules, thereby defining an alternative Overall, new knowledge regarding the nature and category of granules and supporting our initial temporal dynamics of the cytokines coordinating hypothesis. swarming will advance our understanding of the physiologic and pathological processes associated 82 with inflammation, during health and disease. Assessing the Production of Reactive Oxygen and 81 Nitrogen Species by Human Neutrophils Marisa Freitas, Carina Proença, Daniela Ribeiro, Mapping of Glycoepitopes in Neutrophil Eduarda Fernandes, UCIBIO, REQUIMTE, Granules Laboratório de Química Aplicada, Departamento de Régis Dieckmann, Vignesh Venkatakrishnan, Niclas Ciências Químicas, Faculdade de Farmácia, Karlsson, Johan Bylund, Anna Karlsson, University Universidade do Porto, Portugal of Gothenburg, Sweden Neutrophils are the first immune cells to arrive at the The time of protein synthesis during neutrophil site of inflammation, especially as a result of differentiation is the main identified mechanism bacterial infections, where they recognize and driving the targeting of proteins to granules. Unlike phagocyte the microorganisms. One of the most for the delivery of proteins to the endocytic pathway, important defense systems of neutrophils against the no identified sugar residue participate in the sorting invaders corresponds to their ability to trigger a of proteins to neutrophils granules. Thus, granule strong oxidative burst through the formation of subsets are characterized not only by a distinct reactive oxygen species (ROS) and reactive nitrogen protein content. Indeed, pauci-mannose residues species (RNS). While oxidative burst is important have been identified by mass spectrometry on for the elimination of invading microorganisms, the azurophilic proteins. Galectins bind to poly-N- overproduction of reactive species or the impairment acetyllactosaminyl backbones attached to proteins of endogenous antioxidant defenses may result in mobilized from specific granules. We also showed, detrimental effects to the host. The nature and the by immunofluorescence, that the selectin ligands, extent of reactive species production by neutrophils CD15 and CD15S, localize to different intracellular in response to different stimuli is, consequently, a compartments. Therefore, proteins carrying distinct matter of extensive research, with scientific reports sugars are targeted to distinct granules. showing an enormous variability on the detection These observations lead us to hypothesize that sugar methodologies employed. This presentation attempts residues might define other granule subsets than to provide a critical assessment of the most effective those identified by proteins and that they might also approaches to detect the reactive species formed play a role in the genesis and structure of granules. during the neutrophils’ oxidative burst. In that sense, Therefore, we started mapping glyocoepitopes on some practical examples will be given arising from neutrophil granules using a combination of our experience in the modulation of human immunofluorescence, subcellular fractionation and neutrophils’ oxidative burst by bioactive glycomics. In an initial screen, we selected six compounds, as flavonoids. The detection lectins for their specific binding to myeloid cells and mechanisms and performance, as well as advantages their ability to stain intracellular vesicles in and limitations of the different methodologies, will neutrophils. The binding of two lectins to granules be presented, focusing on the use of luminescent was strongly reduced in neutrophils lacking specific probes. granules, due to a specific mutation in neutrophil Acknowledgements differentiation. Interestingly, one of the two lectins This work received financial support from the co-localized partially with myeloperoxidase whereas European Union (FEDER funds none of the two lectins co-localized with CD15 or POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia Morphological analyses show that, whereas HDN and Ministério da Educação e Ciência) under the appear to be a homogenous population of mature, Partnership Agreement PT2020 segmented neutrophils, LDN are heterogeneous and UID/MULTI/04378/2013. Marisa Freitas also contain segmented as well as morphologically acknowledges FCT the financial support for Pos-doc immature (banded and ring-shaped) neutrophils. grant (SFRH/BPD/76909/2011) in the ambit of Furthermore, LDN had higher forward scatter (FSC) “QREN e POPH e Tipologia 4.1 e Formação than HDN with similar side scatter (SSC), Avançada”, co-sponsored by FSE and by national suggesting that they are larger than HDN while funds of MCTES. maintaining similar granularity. LDN display a reduced inflammatory profile, impaired neutrophil 83 function and immunosuppressive properties, characteristics that are in stark contrast to those of Diversity of Neutrophil Subpopulations in Cancer mature HDN. Importantly, we show that the mature Jitka Y. Sagiv1, Janna Michaeli2, Siman Assi1, Inbal subset of LDN is derived from HDN, in a TGFß Mishalian2, Hen Kisos1, Paola Damti2, D Lumbroso3, dependent mechanism. Lola Polyansky1, RV Sionov1, A Ariel3, AH Hovav1, Our findings challenge the concept that mature Zvi Fridlender2, Zvi Granot1, 1The Hebrew neutrophils are a homogenous group of cells. HDN University of Jerusalem; 2Hadassah-Hebrew possess anti-tumor properties, whereas LDN have University Medical Center, Jerusalem; 3University features associated with pro-tumor activity. During of Haifa early tumor development, HDN are the predominant neutrophil subpopulation leading to an overall Neutrophils, the predominant circulating leukocyte antitumor response. With tumor progression, LDN population, play a well-established role in host become dominant resulting in an overall neutrophil defense and are usually associated with phenotype switch toward tumor-promoting inflammation and with counteracting infection. functions. The functional differences between HDN Neutrophils are largely viewed as a homogeneous and LDN together with the continuous increase in population of terminally differentiated white blood the proportion of LDN with tumor progression cells lacking significant plasticity. Interest in these provide a solid explanation for the controversy that cells has increased during the last decade, because of has surrounded neutrophil function in cancer. the accumulating data that suggest important and significant roles for neutrophils in tumor biology. Controversy, however, surrounds their role as 84 neutrophils were shown to provide both pro- and anti-tumor functions. Neutrophils and Lymphocytes Express We hypothesized that the controversy regarding Vomeronasal Type-1 Receptor (VN1R) Genes neutrophil function in cancer stem from the Patrick Marcinek, Dietmar Krautwurst, Deutsche existence of distinct neutrophil subsets which may Forschungsanstalt für Lebensmittelchemie – Leibniz posses different and even conflicting functional Institut, 85354 Freisinig, Germany properties. Along these lines, the ratio between these subpopulations and the potency of their activity Blood leukocytes express a variety of G protein- should determine the overall contribution of coupled receptors (GPCR) to distinguish non-self neutrophils. We now show that two circulating from self, and to orchestrate immune responses in neutrophils subpopulations may be detected in adaptive and innate immunity alike. For example, cancer based on their different densities. Using some 40 GPCR are highly expressed in T-cells, B- mouse models of cancer, and samples from breast cells, and polymorphonuclear granulocytes and lung cancer, we identified a heterogeneous (neutrophils) [1, 2]. Recently, and in addition to the subset of low-density neutrophils (LDN) that appear canonical immunologically relevant GPCR, we and continuously accumulate with cancer demonstrated the expression of some 80 progression. These LDN are phenotypically different chemosensory odorant, taste, and trace amine- from the normal high-density neutrophils (HDN) associated GPCR in five different types of blood that are the predominant population in health. leukocytes [3, 4]. Here we show the expression in neutrophils, T- and B-cells of yet another family of acesulfam-K and cyclamate concentrations on the chemonsensory GPCR, comprising the four regulation of sweet and bitter taste chemosensory vomeronasal type-1 receptors (VN1R) 1, 2, 4, and 5. receptor genes and genes of innate immunity in VN1R are receptors that are canonically expressed in isolated PMN, in vitro and in a human intervention a specialized chemosensory epithelium, the study. In summary, we suggest that artificial vomeronasal organ (VNO), enabling the detection of sweeteners beyond their role as dietary hedonic species-specific chemical cues pertaining to social supplements are bioactives on our cellular immune interaction – humans, however, supposedly have no system. functional VNO. An ectopic expression of VN1R in 1. Futosi, K., S. Fodor, and A. Mocsai, Reprint of blood leukocytes opens up new venues for research Neutrophil cell surface receptors and their on the functional relevance of VN1R expression for intracellular signal transduction pathways. Int our cellular immune system. Immunopharmacol, 2013. 17(4): p. 1185-97. 1. Futosi, K., S. Fodor, and A. Mocsai, Reprint of 2. Malki, A., et al., Class I odorant receptors, Neutrophil cell surface receptors and their TAS1R and TAS2R taste receptors, are markers intracellular signal transduction pathways. Int for subpopulations of circulating leukocytes. J Immunopharmacol, 2013. 17(4): p. 1185-97. Leukoc Biol, 2015. 97(3): p. 533-45. 2. Kehrl, J.H., G-protein-coupled receptor signaling, RGS proteins, and lymphocyte 86 function. Crit Rev Immunol, 2004. 24(6): p. 409- 23. Plasma Membrane Profiling of the Neutrophil by 3. Malki, A., et al., Class I odorant receptors, Amino-Oxybiotinylation and Tandem Mass Tag TAS1R and TAS2R taste receptors, are markers Mass Spectrometry for subpopulations of circulating leukocytes. J Joanna M. Pocock1, Francois H. Demay1, James C. Leukoc Biol, 2015. 97(3): p. 533-45. Williamson2, Paul J. Lehner2, Edwin R. Chilvers1 4. Babusyte, A., et al., Biogenic amines activate 1Department of Medicine, University of Cambridge, blood leukocytes via trace amine-associated United Kingdom; 2Cambridge Institute for Medical receptors TAAR1 and TAAR2. Journal of Research, University of Cambridge, United Leukocyte Biology, 2013. 93: p. 387-394. Kingdom

85 Background Following stimulation, a multitude of processes are Food Supplements Regulate the Expression of instigated which require the neutrophil to modulate Chemosensory Receptor Genes in Neutrophils the expression of cell surface molecules. Patrick Marcinek, Dietmar Krautwurst, Deutsche Comprehensive tracking of these changes by mass Forschungsanstalt für Lebensmittelchemie - Leibniz spectrometry against a background plasma Institut, D-85354 Freising, Germany membrane proteome has been limited by

conventional cell and membrane purification Neutrophils express a variety of G protein-coupled techniques, which result in cell priming and receptors (GPCR) to identify tissue damage, organelle membrane contamination. In addition, the bacterial infection, and to orchestrate innate immune short neutrophil half-life ex-vivo precludes the use responses. For example, about 10 canonical, non- of stable isotype labelling by amino acids in cell chemosensory GPCR are known to highly express in culture (SILAC). neutrophils [1]. There is, however, increasing The use of amino-oxybiotin to label plasma evidence of an ectopic expression of both sweet and membrane proteins prior to MS has been bitter taste receptors in a variety of non- demonstrated in fibroblasts, resulting in high chemosensory tissues and cells, including numbers of protein identifications with good leukocytes. Recently, we demonstrated saccharin to membrane purity (Weekes 2010). Furthermore, it is be capable of activating chemotaxis in neutrophils now possible to multiplex this procedure, combining via both sweet and bitter taste GPCR, which are multiple culture conditions for comparison on a functionally expressed in these and other leukocytes single MS run using Tandem Mass Tags (TMT) [2]. Here, we investigated the role of diet lemonade (Matheson 2015). This abstract describes the first consumption-typical, postprandial saccharin, use of this technique in human neutrophils. first time in a primary cell. It has the potential to Methods allow investigation of how the neutrophil alters Pilot plasma membrane profiling was carried out on surface signalling in response to a wide range of neutrophils isolated from 3 healthy volunteers. stimuli and pathogens, using a candidate-free Plasma-Percoll gradient separated neutrophils were hypothesis, and ultimately to identify novel drug incubated with amino-oxybiotin, enriched with targets. streptavidin, TMT labelled and subjected to high performance liquid chromatography MS. The 87 resulting plasma membrane proteins were assigned corresponding gene ontology terms to determine A Genome-Scale Screen for New Players in subcellular localisation, and input cell numbers were Neutrophil Biology increased to improve the number of proteins Craig T. Lefort1,2, William S. Serratelli1, 1Rhode identified. Island Hospital; 2Warren Alpert Medical School of To validate the technique, neutrophils were Brown University incubated with the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) for up to 4 Genetic modification of neutrophils is frustrated by hours prior to biotinylation, and expression of the their short lifespan and sensitivity to manipulation. adhesion molecules CD11b and CD62L was We circumvented these limitations by deriving compared to untreated neutrophils using both TMT conditionally immortalized murine myeloid MS and flow cytometry. progenitors that are receptive to gene knockout via Results CRISPR/Cas9. We expressed Cas9 endonuclease Plasma membrane profiling of 2x10^7 neutrophils and the GeCKOv2 small guide RNA library in by amino-oxybiotinylation followed by MS resulted myeloid progenitors to achieve knockout of a single in identification of 263 unique proteins, with high gene in each cell. The GeCKOv2 library targets enrichment for membrane proteins (92%) as judged more than 20,000 different genes. We performed by gene ontology annotation. Increasing the cell proof-of-concept experiments using single cell PCR number to 1x10^8 neutrophils led to an increase in amplification and sequencing of the sgRNA barcode. identified proteins to 819, of which 77% were The library-expressing myeloid progenitors were membrane-associated. Biotinylation efficiency was then differentiated into neutrophils to establish a high, with a 3-log increase in streptavidin binding in range of functional assays. The tools developed in biotinyated neutrophils compared to unbiotinylated, this study will allow us to identify novel genes that and the protein set was comparable across replicates regulate neutrophil activation, recruitment and from 3 donors, with the most variable proteins being effector function. predominantly HLA molecules. Plasma membrane profiling of GM-CSF-treated 88 neutrophils over a 4 hour time course showed similar temporal changes across donors, and TMT MS NADPH Oxidase Activation Regulates Apoptotic temporal expression profiles of CD11b and CD62L Cell Clearance and Antigenicity correlated with that demonstrated by flow cytometry Juhi Bagaitkar1, Jing Huang1, Melody Y. Zeng2, (r2=0.978 for CD11b, r2=0.594 for CD62L). Nancy K. Pech1, Mary C. Dinauer1, 1Department of Conclusions Pediatrics, Washington University School of Here we describe an unbiased quantitative proteomic Medicine in St. Louis, St. Louis, MO; 2Department of approach to characterizing the neutrophil plasma Pathology and Comprehensive Cancer Center, membrane by amino-oxybiotinylation and MS. The University of Michigan Medical School, Ann Arbor, additional use of Tandem Mass Tags allowed MI comparison of multiple culture conditions on the same MS run, and we validated our findings by The phagocyte NADPH oxidase generates tracking cell surface molecule changes known to superoxide in response to multiple agonists. occur following stimulation with GM-CSF. This Superoxide and derivative reactive oxygen species new proteomic approach has been described in other (ROS) are important in microbial killing and many cell types but here is successfully adapted for the immunological processes including antigen presentation, LC3-associated phagocytosis (LAP) 89 and modulation of inflammatory responses. Chronic granulomatous disease (CGD) patients with Activated Protein C (APC) Binds Leukocytes and inactivating NADPH oxidase subunit mutations are Signals to Inhibit Neutrophil Extracellular Trap susceptible to recurrent microbial infections and Formation and Apoptosis; Possible Additional chronic inflammatory conditions. Moreover, variant Mechanism for Anti-Inflammatory and NADPH oxidase subunit alleles causing only partial Cytoprotective Effects of APC loss of enzyme activity are also associated with Laura D. Healy1, Cristina Puy2, Jose A. Fernández3, increased susceptibility to autoimmune diseases. Tiffany Chu2, András Gruber2, John H. Griffin3, However specific molecular mechanisms by which Owen JT McCarty1,2, 1Cell, Developmental & NADPH oxidase and ROS prevent autoimmunity are Cancer Biology Department, Oregon Health & poorly defined. Here, we demonstrate a mechanistic Science University, Portland, OR, USA; role of the NADPH oxidase in regulating apoptotic 2Department of Biomedical Engineering, Oregon cell clearance by macrophages, dendritic cells, and Health & Science University, Portland, OR, USA; prevention of autoimmune responses in vivo. 3Department of Molecular and Experimental Efficient clearance of apoptotic cells is essential to Medicine, The Scripps Research Institute, La Jolla, prevent secondary necrosis, inflammation and CA, USA aberrant immune responses to apoptotic cell antigens. Our studies show that ingestion of Background: At sites of microbial infections, apoptotic cells in vitro by wild type mouse activated neutrophils can expel their nuclear content macrophages and dendritic cells activated NADPH to form microbicidal protein-exposing neutrophil oxidase in a CD11b-TLR4-MyD88-dependent extracellular traps (NETs). This process of NETosis manner. Furthermore, apoptotic cell-derived ligands provides a structural framework for pathogen (MSU crystals, ATP) were strong activators of the clearance. The exact mechanism of NETosis, oxidative burst, while complement deposition (iC3b) including its regulation, if any, is unclear. Activated on apoptotic cells further augmented superoxide protein C (APC) is a natural antithrombotic, anti- generation. After uptake, NADPH oxidase activity inflammatory and cytoprotective blood enzyme that directly regulated the rate of proteolysis, and inhibits the migration of neutrophils. We subsequent cross-presentation of apoptotic cell- hypothesized that the anti-inflammatory function of derived peptides. Macrophages from CGD mice APC is due in part to the inhibition of NETosis and lacking NADPH oxidase activity due to inactivation cell death. of Cybb (encodes gp91phox- the main catalytic Aim: To determine the effect of APC on phorbol subunit of NADPH oxidase), were delayed in myristate acetate (PMA)-induced NETs formation digestion of ingested apoptotic cargo in vitro and and tumor necrosis factor-α (TNFα)-induced strongly activated CD8 T cell responses that apoptosis. correlated with enhanced cross-presentation of Methods: Human neutrophils were purified from model antigen - ovalbumin in vitro and in vivo. peripheral blood and allowed to adhere to a Long-term infusion of apoptotic cells in CGD mice fibronectin-coated surface. Neutrophils were then resulted in anti-nuclear antibody generation and pretreated with increasing concentrations (30-300 glomerulonephritis. Thus our studies identify nM) of human APC prior to stimulation with PMA receptors, ligands and molecular pathways leading to to induce NETosis. NETosis was quantified using activation of NADPH oxidase following apoptotic the DNA-binding dye, Hoescht 33342, as well as cell ingestion. Furthermore, this study for the first neutrophil elastase and citrullinated histone. The time explains the direct role that NADPH oxidase- area of DNA was quantified via fluorescence derived plays in the digestion of apoptotic cells, and microscopy and MATLAB-based image analysis. is most likely to be the underlying mechanism For apoptosis studies, neutrophils were treated with leading to hyperinflammation and autoimmune increasing concentrations of APC prior to complications observed in the of context NADPH stimulation with TNFα in the presence or absence of oxidase deficiency. cycloheximide (CHX) to induce apoptosis. Cells were stained with the apoptosis molecule Yo-Pro1 and Hoescht 33342 and quantified via fluorescence microscopy as the percent of Yo-Pro1 cells over control mice, splenic macrophages from Bxsb/Yaa total cells. mice had increased basal and TLR4/7-induced Results: APC prevented NETosis induced by the phosphorylation of IRAK1, ERK and p38 MAPKs, protein kinase C activator, PMA. The ability of APC p65 NF-κB, and enhanced TNF-a and CCL5 gene to prevent NETosis was reversed after pretreatment expression. In contrast, lupus development led to of APC with the protease inhibitor, Phe-Pro-Arg decreased levels of Toll-interacting protein and chloromethylketone (PPACK). Function blocking IRAK-M in splenic macrophages, but did not affect antibodies to endothelial protein C receptor (EPCR), IRAK4 or IRAK1 mRNA expression. Mice protease activated receptor-3 (PAR-3) and Mac-1 harboring kinase-inactive IRAK4 on the lupus-prone and pharmacological inhibitors of the PDK and PLC Yaa background had reduced glomerulonephritis, signaling pathways prevented APC from inhibiting splenomegaly, and levels of serum anti-nuclear Abs PMA-induced NETosis. Recombinant APC with compared to lupus-prone mice expressing kinase- several mutations in and near the protease domain sufficient IRAK4. A loss of IRAK4 kinase activity failed to prevent the ability of PMA to induce led to decreased numbers of splenic F4/80+ NETosis. APC reduced the ability of TNFα to macrophages, CD11c+ dendritic cells, IFN-?+ T- induce neutrophil apoptosis in a dose-dependent lymphocytes and B220+CD138+ B-lymphocytes and manner. Function blocking antibodies to EPCR, reduced TNF-a expression in macrophages. Thus, PAR-3 and Mac-1 abrogated the anti-apoptotic IRAK4 kinase activity positively regulates the effect of APC. development of lupus in mice and could represent a Conclusions: Our data suggest that ex vivo, new target for therapeutic interventions in lupus via exogenous APC can inhibit PMA-induced NETosis applications of peptide- or small molecule-based and TNFα-induced apoptosis in a EPCR/Mac- IRAK4 inhibitors. 1/PAR-3 dependent manner. Our data identify an Supported bythe National Institute of Health Grant additional possible link to the anti-inflammatory and R21 AI099430 (to AEM) antithrombotic activities of APC in immunothrombosis. 91

90 Cullin-5 Promotes TRAF6 Polyubiquitination and LPS Signaling Deficiency in IRAK-4 Activity Attenuates Richard D. Ye1, Ziyan Zhu2, Lili Wang2, Rui Hao2, Manifestations of Murine Lupus Bo Zhao2, Lei Sun2, 1University of Macau; Andrei Medvedev, Michael Murphy, Tissa 2Shanghai Jiao Tong University Manavalan, Goutham Pattabiraman, University of Connecticut Health Center TNF receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase that integrates signals from multiple IL-1R-associated kinase (IRAK) 4 initiates TLR cell surface receptors for the activation of nuclear signaling and plays an important role in host defense factor κB (NF-κB). However, the mechanism against infections. As an active kinase, IRAK4 underlying lipopolysaccharide (LPS)-induced elicits full spectra of MyD88-dependent responses, TRAF6 signaling remains largely unknown, nor is it while kinase-inactive IRAK4 adapter induces a clear how TRAF6 responds to various types of subset of cytokines and negative TLR regulators receptor signals. We report that cullin-5 (Cul-5), a whose expression is not regulated by mRNA cullin family scaffold protein that resides in the stability. While IRAK4 activity is critical for host cytoplasm of unstimulated cells, forms a complex resistance against Streptococcus pneumonia, its role with TRAF6 and promotes TRAF6 in autoimmunity is incompletely understood. In this polyubiquitination at Lys63 in LPS-stimulated study, we determined the role of IRAK4 activity in macrophages. A direct interaction is established systemic lupus erythematosus, using 4 month-old between the C-terminal domain of Cul-5 and the lupus-prone Bxsb male mice expressing the Y TRAF-C domain of TRAF6 that facilitates chromosome-linked autoimmunity accelerator (Yaa) dimerization and polyubiquitination of TRAF6. and age-matched control Bxsb female mice without Targeted deletion ofCul-5(cul-5+/-) abrogates LPS lupus manifestations. Compared to cells from response and significantly improves the survival rate of mice receiving systemic LPS challenge. Cul- pediatric intensive care unit length of stay. Based on 5deficiency also attenuates acute lung injury our finding of an alteration in the density of TLR1 resulting from intratracheal injection of LPS to on the cell surface in the setting of this SNP, we thecul-5+/-mice. Macrophages isolated from the cul- hypothesized that TLR1 biosynthesis and trafficking 5 hemizygous mice display significantly delayed was impacted by this SNP. Moreover, we postulated phosphorylation of p65/RelA, ERK, JNK and p38 that PMN responses to a “natural” bacterial ligand MAP kinases, with a marked decrease in NF-κB for TLR2/1 from Mycobacterium tuberculosis would activation and significant reduction in inflammatory be similarly affected by this SNP. Using confocal cytokine expression. The effect of Cul-5 to promote microscopy for quantitative colocalization analysis TRAF6 binding and polyubiquitination is abrogated for TLR1 and TLR2 proteins, we found a Pearson’s by inhibition of NEDD8 conjugation, suggesting the correlation coefficient of 0.9337 in a donor with the involvement of neddylation in LPS signaling leading 1805T SNP. PMNs displayed colocalization of to TRAF6 polyubiquitination. Taken together, these TLR1 with TLR2 both on the cell surface and in findings identify Cul-5 as a novel link between the intracellular compartments. Immunoprecipitation LPS-activated TLR4-MyD88 complex and TRAF6, studies demonstrated enhanced association of TLR1 an immune cell check-point that governsactivation with both TLR2 and with gp96, an ER chaperone of NF-κB. protein, in PMNs from 1805T vs. 1805G donors. Unexpectedly, cell activation in 1805 G vs. T PMNs 92 in response to purified lipoarabinomannan from M. tuberculosis (a TLR2/1 ligand) demonstrated a Toll-Like Receptor Trafficking and Cellular completely different phenotypic signature from that Responses are Determined by Common Single seen in response to Pam3CSK4. 1805T PMNs Nucleotide Polymorphisms displayed no differences in priming of NADPH Jessica G. Moreland, Jessica S. Hook, Mou Cao, Lin oxidase activity in response to fMLF as compared Jordan, Renee M. Potera, University of Texas with 1805G PMNs, but there was significantly Southwestern Medical School greater cytokine production by both monocytes and PMNs and enhanced phosphorylation of ERK 1/2. Despite advances in personalized medicine for many We conclude that leukocyte responses to TLR disease processes, our therapies for sepsis remain stimulation are significantly impacted by SNPs in algorithmic rather than patient specific, with these receptors and that may result from impaired morbidity and mortality still unacceptably high. trafficking within the cell. Moreover, cellular However, it is well recognized that the host innate responses are highly ligand-specific with unique immune response has pronounced patient-specific phenotypes that could be very relevant to disease variability and often determines the patient outcome. pathogenesis. We speculate that these ligand-specific Neutrophil (PMN) activation, although required for differences may result from modification of TLR2/1 pathogen killing, can be a mediator of significant responses by other pattern recognition receptors, host tissue damage during acute inflammatory including the inhibitory actions of TLR10. events, via release of reactive oxygen species, granule exocytosis, and cytokine production. 93 Bacterial ligands elicit priming or pre-activation of neutrophils through interaction with pattern- HHV-8 Replication in Subtypes of Myeloid recognition receptors including Toll-like receptors. Dendritic Cells is Regulated by Differential We recently demonstrated that stimulation of PMN Expression of Virus Receptors with the synthetic TLR2/1 ligand Pam3CSK4 Giovanna Rappocciolo, Diana Campbell, Paolo elicited a priming phenotype in approximately 50% Piazza, Mariel Jais, Frank J. Jenkins, Charles R. of donors, with the remainder displaying minimal/no Rinaldo, University of Pittsburgh response. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was determined by a Background: HHV-8 (KSHV) infection of myeloid common SNP in TLR1 (1805 G>T). We dendritic cells (DC) is considered important in host subsequently analyzed a pediatric sepsis database control of the virus and prevention of Kaposi’s and found that patients with this SNP had prolonged sarcoma (KS). There is, however, limited information of HHV-8-DC interaction. Monocyte- Langerin and EphA2 as receptors. EphA2 is known derived dendritic cells (MDDC) only support to synergize with CIB1 (calcium and integrin abortive replication of HHV-8 in vitro, with binding protein 1) to facilitate HHV-8 entry and expression of viral lytic proteins but minimal virion establish successful productive primary infection in production. HHV-8 infected MDDCs have impaired endothelial cells. Ligand binding to EphA2 influence cytokine production and antigen processing, with a activation and maturation of DC by affecting significant decrease in endocytosis and lower integrin affinity and the cell cytoskeleton. Thus, capacity to activate antigen-specific CD8+ T cells. HHV-8 binding to LC and DDC through EpHA2 Because DC are actually a family of functionally and receptor could render these cells less efficient in the regionally diverse antigen presenting cells (APC), ability to stimulate T cell responses. Taken together, we assessed Langerhans cells (LC) and our data show for the first time that HHV-8 interstitial/dermal DCs (IDDC) compared to MDDC differentially infects and replicates in several types to better define virus receptor usage and their ability of DC and supports a role of these cells in HHV-8 to support virus replication. infection and pathogenesis. Methods: The 3 DC populations were derived from neonatal cord blood and infected with BAC16, a 94 recombinant virus expressing green fluorescent protein (GFP). The percentage of HHV-8-infected ICOS Signaling Contributes to Staphylococcus cells expressing GFP was enumerated by flow Aureus Pneumonia cytometry. To measure virus replication, viral DNA Dane Parker, Alice Prince, Rudy Jacquet, Columbia in cell pellets and cell cultures was quantitated by University RT-PCR and the amount of infectious virus in cell culture supernatants was measured by a flow Staphylococcus aureus is a major cause of both cytometry-based TCID50 assay. Virus receptor community and healthcare acquired pneumonias. We usage was determined by blocking infection of DC have shown previously that CD4+ T cells contribute with mAbs directed against either DC-SIGN, to pneumonia through the production of Langerin or Ephrin type-A receptor 2 (EphA2). proinflammatory cytokines and the CD28 co- Results: Phenotyping studies confirmed that LC receptor plays a role in this signaling. The ICOS express Langerin and EphA2 but not DC-SIGN, pathway is part of the CD28 family of proteins and IDDC express DC-SIGN and low levels of EphA2 is a target for immune checkpoint therapy. To and MDDC express DC-SIGN but not Langerin or determine the contribution of ICOS to the EphA2. Our data indicate that full cycle, HHV-8 pathogenesis of S. aureus pneumonia we intranasally lytic replication occurs in LC and IDDC as measured infected WT and Icos-/- mice with S. aureus by at least one log10 increase in viral DNA copies USA300, the current epidemic MRSA isolate. Icos-/- and infectious virus over 72 hours of infection. Only mice had improved survival in a mortality model and abortive HHV-8 infection was detected in MDDC, in significant reductions in bacterial burden in the agreement with our previous results. Anti-DC-SIGN airway (65% reduction, P<0.01) and in lung tissue mAb blocked viral infection of both IDDC and (92% reduction, P<0.01) in a non-lethal acute MDDC by 75%. By contrast, HHV-8 appeared to pneumonia model. We then examined the cytokine use alternate receptors to infect LC, as blocking with differences in bronchoalveolar lavage fluid between mAb against either Langerin or EphA2 abrogated WT and Icos-/- mice. Icos-/- mice had major infection by 42% and 59%, respectively, and 83% reductions in most cytokines analyzed including: IL- when cells were treated with both mAbs prior to 6, KC, TNF, GM-CSF, CCL2, Eotaxin (CCL11), IL- infection, with no detectable infectious virus 1β, IL-17, IL-5, IL-7 and IL-13. To better production. Infected LC and DDC were poor MLR understand the mechanisms behind the improvement stimulators. in the Icos-/- mice we examined the cellular response Discussion: We expand on our previous results by to infection by antibody staining and flow showing that HHV-8 utilizes DC-SIGN as a major cytometry. Examination of recruitment to the airway receptor for infection of both DC and IDDC, but identified significant reductions in two cell types, the only fully replicates in IDDC. HHV-8 infection of Ly6C+ inflammatory monocyte (82%, P<0.05; LC is more complex in that the virus utilizes both CD45+Ly6C+Cd11b+CD11c-MHCII-) and eosinophils (73%, p<0.01; CD45+Ly6C- mediated by myosin IIA which was also present on CD11b+SiglecF+CD11c-). It has also been recently the phagosome. Together this work indicates that shown that ICOS is expressed on group 2 innate opsonins direct a different fate for coated pathogens lymphoid cells (ILC2) and we observed a 4-fold and CR3-mediated phagocytosis promotes reduction in ILC2 cells in the Icos-/- mice. We are mechanical over enzymatic destruction of target currently investigating the role of ICOS in other particles. airway pathogens and delineating the respective roles of inflammatory monocytes, eosinophils and 96 ILC2 cells in the pathogenesis of S. aureus pneumonia. Our results indicate that ICOS plays a Differential Activation of Formyl-Peptide significant role in orchestrating the innate immune Receptors by Staphylococcus Aureus and response to S. aureus and could be potential Consequences for Inflammation immunomodulatory target to attenuate S. aureus- Elisabeth Weiß1, Dennis Hanzelmann1, Friederike related immunopathology. von Loewenich2, Katja Schlatterer1, Jan Liese3, Andreas Klos4, Andreas Peschel1, Dorothee 95 Kretschmer1, 1Interfaculty Institute for Microbiology and Infection Medicine, University of Tübingen, Phagocytosis by Opsonic Receptors in Tübingen, Germany; 2Institute for Medical Macrophages Microbiology and Hygiene, Universitätsmedizin Rene E. Harrison, Mathieu B. Poirier, Urja Naik, Mainz, Mainz, Germany; 3Institute for Medical University of Toronto Microbiology and Hygiene, UKT Tübingen, Tübingen, Germany; 4Department of Medical Our lab studies the activation of macrophages and Microbiology and Hospital Epidemiology, Hannover cellular mechanisms behind the targeted destruction Medical School, Hannover, Germany of pathogens by phagocytosis. We and others have shown that the type of receptor engaged on the Formyl peptide receptors (FPR1-3) are crucial macrophages influences the uptake of the phagocytic pattern recognition receptors governing leukocyte target into macrophages. We have done a chemotaxis and cytokine release in response to comprehensive analysis of kinase and cytoskeletal microbe-associated molecular patterns (MAMPs). requirements for Fcgamma and CR3-mediated FPR1 senses formylated peptides produced by all phagocytosis when challenged with single or dual- kinds of bacteria, while FPR2 and 3 respond to opsonized particles. Scanning electron microscopy certain endogenous peptides. In addition, we have as well as fluorescent imaging of F-actin in recently demonstrated that phenol-soluble modulin macrophages has revealed that very different surface (PSM) peptides from highly pathogenic structures are induced by opsonic receptors and the Staphylococcus aureus are not only important timing of particle internalization varies accordingly. cytolytic toxins but also highly efficient ligands for Interestingly, coating particles with both antibody the human FPR2. Mouse neutrophils also respond to and complement (to stimulate Fcgamma and CR3, PSMs, but it has remained unclear, which of the respectively) has an additive effect on particle mouse FPR paralogs senses staphylococcal PSMs. binding. The prominent membrane ruffles that To analyze the role of mouse FPRs, stably capture complement-opsonized particles do not need transfected RBL cells were generated, which either Src activity but have a strong requirement for express mFpr1 or mFpr2. After stimulation with microtubules, while the opposite is observed during PSMs or culture filtrates of PSMs-secreting S. Fcgamma receptor engagement. The activation of aureus strain USA300, we noticed strong calcium the receptor also influences the maturation of the influx and degranulation in mFpr2-transfected cells, phagosome once inside. Phagosomes of but no response in mFpr1-transfected cells and complement-opsonized particles show delayed control cells. Moreover, by using HoxB8 acidification and striking F-actin recruitment to the neutrophils, a primary neutrophil cell line prepared phagosome in waves or flashes. The presence of the from wild-type (WT) and mFpr2 knockout mice F-actin flash correlates with deformation of the (Fpr2-/-), we observed strong calcium influx, internalized particle, the contraction which is likely chemotaxis, MIP2 release and CD11b upregulation in wild-type HoxB8 but not in Fpr2-/- HoxB8 after 4 and IFNγ of monocytes. However, stimulation by stimulation with PSMs or culture filtrates of IL-4 and IFNγ, when polarizing monocyte-derived USA300. macrophages, resulted in a decreased expression of These data indicate that the mouse Fpr2 is cyt-PTPε among M1 and an increase of RPTPε in specifically activated by PSMs. Therefore, PSMs M2. Activation of Src is also different in M1 and M2 represent the first secreted MAMPs for the mouse macrophages subtypes, after stimulation with LTD4, Fpr2. Our data support the hypothesis that the mouse as a decrease in the phosphorylation of the Src Fpr2 is the functional orthologue of the human FPR2 inhibitory residue (Y530) was observed in M1 and and that a mouse infection model may be a suitable an increase in M2. The opposite tendency was model for analyzing the role of PSMs and FPRs observed at the ROCKII inhibitor residue (Y722) during infection. where M1 had an increase in phosphorylation of the residue and M2, a decrease. 97 Using Boyden chambers for directional migration experiments, our results demonstrate a correlation LTD4-Induced Monocyte/Macrophage between PTPε expression, ROCK activity and Migration: a Role for PTPε? monocyte chemotaxis. Indeed, monocytes treated Fanny Lapointe, Marie Boudaud, Sylvie Turcotte, with siRNA against PTPε (siPTPε) or with a ROCK Marek Rola-Pleszczynski, Jana Stankova, Université inhibitor, Y27632, display a significantly reduced de Sherbrooke LTD4-induced chemotaxis. However, the combination of siPTPε and Y27632, results in a Cysteinyl-leukotrienes (cys-LTs), which include restoration of migration. leukotriene (LT) C4 (LTC4), LTD4 and LTE4, have Our results demonstrate a possible interaction of well-characterized physiopathological roles in the PTPε and ROCK in LTD4-induced monocytes development of several inflammatory diseases, chemotaxis. In addition, the differential modulation including asthma. Cys-LTs lead to the production of of cyt-PTPε and RPTPε expression in polarized various inflammatory mediators, to the recruitment macrophages M1 and M2 may result in differential of multiple cellular subtypes and to an exacerbation CysLT1R signalling and/or migration. of asthma symptoms. Thus, the function and signalling of CysLT1 receptors (CysLT1R), highly 98 expressed on leukocytes, have been the focus of research in our laboratory. In our attempt to discover The Role of IL-15 Trans-Presentation in Antigen potential CysLT1R-interacting proteins, we Cross-Presentation identified protein tyrosine phosphatase epsilon Sheela Ramanathan1, Sakina Orkhis1, Subburaj (PTPε). Interestingly, a polymorphism in the PTPRε Ilangumaran1, Alfredo Menendez2, 1Immunology gene, PTPRε_rs7081735A>G, has been associated Division, Departments of Pediatrics, Faculty of with allergic asthma, potentially making its Medicine and Health Sciences, Université de interaction with CysLT1R even more significant. Sherbrooke, Sherbrooke, Quebec, Canada; Furthermore, as PTPε has been found to be involved 2Departments of Microbiology, Faculty of Medicine in the formation and function of podosomes in and Health Sciences, Université de Sherbrooke, osteoclasts, through Src activation, we were Sherbrooke, Quebec, Canada interested in its role in monocyte migration, as well as, in LTD4-induced CysLT1R signalling in Interleukin-15 (IL-15) is a pro-inflammatory polarized monocyte-derived macrophages (M1 and cytokine that is required for the survival and M2). activation of memory CD8+ T cells, NK cells, innate There are two major protein isoforms of PTPε, the lymphoid cells, macrophages and dendritic cells. IL- receptor-like (RPTPε) and the cytoplasmic (cyt-) 15 is implicated in the pathogenesis of various PTPε. In most cells, only one of the isoforms is autoimmune diseases such as rheumatoid arthritis, expressed, however in human monocytes both are inflammatory bowel disease, psoriasis and present, although the cyt-PTPε predominates. In this autoimmune type 1 diabetes (T1D). IL-15 receptor regard, we demonstrate a positive modulation of cyt- (IL-15R) consists of a specific a chain, the b chain PTPε expression following stimulation by LTD4, IL- that is shared with IL-2R and the common g chain. IL-15 is unique in the manner in which it binds and (left ventricular assist device). The outcome of signals through its receptor subunits. IL-15 that is LVAD therapy differs significantly between patients complexed with IL-15Ra binds to the bg receptor with over 50% mortality after LVAD explantation complex present on the responding cell to mediate and successful myocardial recovery in others. its biological effects, through a process referred to as Identifying the immunopathological mechanisms trans-presentation. The trans-presented IL-15 is and biomarkers of patients’ reaction to LVAD are essential to mediate the biological effects on T urgently needed. We used stablin-1 as a bimarker to lymphocytes and NK cells. We show that in vitro, idensitfy M2 subsets in healing heart. Heart sections neither IL-15 nor IL-15Ra is required for cross- were obtained directly before LVAD installation and presentation of bacterial antigens. However IL-15, out the explained hearts at the moment of donor but not IL-15Ra is required for cross-presentation of heart transplantation. Analysis of macrophages antigens derived from intracellular pathogens in phenotypes in paraffin sections of 21 patients with vivo. Our findings provide insight into the HF was performed using IHC, IF/confocal complexities of IL-15 signaling in the initiation and microscopy. Three types of macrophage subtypes maintenance of CD8+ T cell-mediated immune have been identified: CD68+stabilin-1; responses. CD68+Stabilin-1+; CD68-stabilin-1+. In 10 patients the percentage of CD68-Stabilin1+ was decreased or 99 not changed during LVAD therapy. In 8 out of these 10 cases ejection fraction (EF) of the heart was Stabilin-1 is a Functional Biomarker for Pro- improved after LVAD therapy. In 11 cases the Fibrotic Alternative Macrophages Predicting percentage of CD68-Stab1+ was increased after Pathological Heart Remodelling in Patients with LVAD therapy. In 10 out of these 11 cases EF was Heart Failure during the Left Ventricular Assist not improved. Pro-fibrotic cytokine TGFbeta Device Therapy strongly activated functionally active stabilin-1 in Julia Kzhyshkowska1,2,4, Kondaiah Moganti1, human macrophages in vitro. We concluded that Katarina Wassilew3, Bin Song 1, Evgenij Potapov3, CD68-Stabilin-1+ macrophages represent new Thomas Krabatsch3, Michael Dandel 3, Amanda subtype of pro-fibrotic M2 in healing heart, and Mickley 1, Harald Klueter1,2, Roland Hetzer3, dynamic accumulation of CD68-Stabilin-1+ 1Institute of Transfusion Medicine and Immunology, macrophages is predictive for the pathological heart Medical Faculty Mannheim, Heidelberg University, remodelling during LVAD therapy. Germany; 2German Red Cross Blood Service Baden- Württemberg–Hessen, Mannheim, Germany; 100 3 German Heart Centre Berlin, Germany; 4Laboratory for translational cellular and molecular Loss of MEKK2 and MEKK3 Results in biomedicine, Tomsk State University, Tomsk Russia Increased ROS and is Protective in LPS-Induced Lung Injury Alternatively activated macrophages (M2) control Abdul Basit, Qianying Yuan, Wenwen Tang, immunological tolerance and healing processes. Dianqing Wu, Yale School of Medicine Stabilin-1 expressed on M2 and acts as a multifunctional scavenger/sorting receptor that Introduction: mediates clearance of unwanted-self components Patients with chronic granulomatous disorder (CGD) and delivery into secretory pathways of soluble have defective NOX2 and impaired release of ROS. mediators, including chitinase-like proteins. Stabilin- These patients not only suffer from recurrent levels can be also elevated in chronic inflammatory infections but also develop sterile inflammatory conditions. Chronic inflammation in myocardium lesions. This latter observation suggests that ROS results in development of caridomyopathy and heart have anti-inflammatory potential. failure (HF). Advanced HF is a life-threatening MEKK3 is highly expressed in neutrophils while, disorder affecting 6-10% of people above 65 y.o. MEKK2,its homolog is expressed at lower level. Less than 50% of HF patients survive 5 year after However, the two homologs share over 95% the first symptoms are identified. Advanced HF is sequence identity in their kinase domains suggesting treated with mechanical circulatory support LVAD that MEKK2 can compensate for the loss of 101 MEKK3. Hypothesis: Distinct Role for MyD88 Signaling in Myeloid MEKK2 and MEKK3 regulate NOX2 in neutrophils. Cells and Stromal Cells in an Imiquimod-Induced Methods: Mouse Model of Psoriasis 1. Generation of double knock out (DKO) mice Sara Costa1, Dalila Bevilacqua1, Anthony L. with loss of MEKK3 in myeloid cells and DeFranco2, Clifford A. Lowell2, Marco Cassatella1, global loss of MEKK2. Giampiero Girolomoni3, 1Department of Medicine 2. Generation of bone marrow (BM) chimeric Section of General Pathology University of Verona; mice with transplantation of DKO BM into 2Departments of Laboratory Medicine and wild type (WT) mice and WT BM into WT Microbiology/Immunology, University of California, mice. San Francisco, CA, United States; 3Department of 3. Development of LPS-induced lung injury Medicine-Section of Dermatology and Venereology, via intranasal delivery of 50 ul of LPS University of Verona, Italy (1mg/ml). 4. Expression of MEKK3 , kinase domain Psoriasis is a chronic skin disease associated with mutant MEKK3 (KD), PB1 domain deregulated interplays between immune cells and deficient MEKK3 (delta PB1 MEEK3) in keratinocytes. The contribution of T cells and DCs neutrophils and cos-7 cells. to psoriasis pathogenesis is well established, 5. Expression of NOX2 components and whereas the role of innate myeloid cells is not well p47phox mutant S208E (47phoxSE) incos-7 understood. Aim of this study was to investigate the cells. contribution of MyD88 signaling in neutrophils and 6. In vitro kinase assay. monocytes/macrophages during psoriatic 7. GST pulldown assay. inflammation. Results: Materials & Methods. We induced imiquimod- 1. DKO neutrophils release significantlymore induced psoriasis-like skin model in conditional ROS compared to WT neutrophils. knockout mice lacking MyD88 in 2. DKO mice have significantly less monocytes/macrophages and neutrophils (Myd88fl/fl permeability to FITC albumin as compared LySM-Cre mice) or in the total hematopoietic to WT mice in an LPS- induced model of compartment (Myd88fl/fl Vav-Cre mice). Total lung injury. MyD88 knockout mice (MyD88 -/-), Myd88fl/fl and 3. This permeability change is sensitive to anti- C57BL/6 mice were also included as controls. The oxidant treatment (BHA) suggesting that the development of psoriasis was induced by topical protective effect is being mediated by ROS. application of AldaraTM (5% IMQ cream) on the 4. Overexpressed MEKK3GFP inhibited ROS shaved back for 6 consecutive days. release in WT and DKO neutrophils Results. Myd88fl/fl LySM-Cre mice manifested a suggesting that MEKK3 regulates NOX2 significant reduction in the activation and negatively. recruitment of innate immune cells and gd T cells in 5. MEKK3 mediated NOX2 inhibition is the draining lymph nodes and in the skin. This dependent upon its kinase activity. correlates with a reduction of the expression of 6. MEKK3 phosphorylates p47phox on S208. several inflammatory mediators in these tissues, 7. MEKK3 mediated phosphorylation of including a specific proliferative and inflammatory p47phox on S208 disengages p47phox from skin markers, however keratinocyte proliferation and p22phox. epidermal acanthosis were not significantly reduced Conclusion: in Myd88fl/fl LySM-Cre mice. Interestingly, we Increased ROS can be protective under certain observed that significant levels of skin inflammation circumstances, and the regulation of NOX2 by were still present, at similar levels, also in both MEKK3 opens a new direction for the development Myd88fl/fl Vav-Cre and MyD88 -/- mice, probably due of novel anti inflammatory therapies. to the reported TLR7-indipendent signaling activation induced by IMQ in keratinocytes. Conclusions. These data demonstrate that MyD88 signaling in innate myeloid cells plays an important periods of time (0, 1, 4, 8, 12 and 20 hrs). RNA was role on disease propagation and exacerbation in the isolated from these cells and induced expression of IMQ-induced model of psoriasis. However, MyD88 the TNF-α, IL-6, IL-1β, IFN-β, RANTES (CCL-5), signaling is dispensable for the initiation of IL-10 and IP-10 genes was determined by keratinocyte-mediated inflammatory responses. SybrGreen RT-qPCR assays, with 18S rRNA used as housekeeping control. 102 Our preliminary data shows that donkey monocytes are highly sensitive to LPS (EC50 = ~27 pg/ml) and Responses of Donkey Monocytes to Toll-Like are less sensitive to the TLR2 agonist PAM3CSK4 Receptor Agonists (EC50 = ~8 ng/ml), similar to what is observed in Michel L. Vandenplas, Kathleen Yvorchuk -StJean, equine monocytes. LPS and PAM3CSK4 induced Lisa Ebner, Erika Little, Sean Callanan, Bernard higher expression of TNF-α, IL-6, IL-1β than poly Grevemeyer, Dept. Clinical & Biomedical Sciences, I:C , with weaker expression of IL-10 only at early Ross University School of Veterinary Medicine, time intervals. Poly I:C induced higher expression of Island Main Rd, Basseterre, St. Kitts and Nevis IL-10 (late time intervals), RANTES and IFN-β. These findings are similar to those observed in As opposed to other animal species, stimulation of equine monocytes indicating that LPS activation of equine monocytes with lipopolysaccharide (LPS) these cells leads to a largely pro-inflammatory gene through Toll-like receptor 4 (TLR4) leads to expression profile. induction of pro-inflammatory gene expression profiles showing engagement of only the MyD88 103 pathway. In contrast, activation of TLR2 or TLR3 shows the expected engagement of the pro- Differential Role of the Transcriptional and Non- inflammatory MyD88 and the anti-inflammatory Transcriptional Functions of IRF-3 in High Fat TRIF pathways in these cells. These unique Diet-Induced Liver Injury responses of equine monocytes to LPS, coupled with Carlos Sanz Garcia1, Sanjoy Roychowdhury1, the low EC50 of LPS in these cells, may be Saurabh Chattopadhyay2, Katherine Pollard1, Megan responsible for the finding that endotoxemia is McMullen1, Ganes Sen2, Laura Nagy1, associated with the leading causes of death in horses. 1Pathobiology, Center for Liver Disease Research, In contrast colic events in the university donkey herd Lerner Research Institute, Cleveland Clinic, are typically not associated to clinical endotoxemia Cleveland, Ohio, USA; 2Immunology, Center for as seen in the equine population. In addition there Liver Disease Research, Lerner Research Institute, appear to be no reports on the response of donkey Cleveland Clinic, Cleveland, Ohio, USA monocytes to Toll-like receipt agonists in the literature. To determine whether closely related Background and Aims. Metabolic diseases, like non- animal species have similar responses to TLR alcoholic fatty liver disease (NAFLD), are agonists we have examined the responses of isolated characterized by hepatic steatosis and increased donkey peripheral blood monocytes to the respective expression of inflammatory cytokines and TLR4, TLR3 and TLR2 specific agonists LPS, Poly chemokines. These responses in turn contribute to I:C and PAM3CSK4 hepatocyte injury characterized by endoplasmic Donkey monocytes were isolated from peripheral reticulum (ER) stress and mitochondrial dysfunction blood using density gradient centrifugation over resulting in hepatocyte injury. While the Histopaque gradients and adherence of the pathogenesis of NAFLD is complex and not monocytes to plastic. In the first series of completely understood, activation of Toll-like experiments the EC50 of E. coli O55:B5 LPS and receptor (TLRs), through increased exposure to fatty PAM3CSK4 was determined by stimulating the acids, as well as gut derived microbial products, monocytes (>95% purity) with a range of agonist contribute to disease progression. Interferon concentrations and measuring secreted TNF-α by regulatory factor 3 (IRF3) is a master regulator of ELISA assays. In the second series of experiments host response to viral infection, but is also activated donkey monocytes were stimulated with diluent only via the TLR4-MyD88-independent pathway. Upon (control), LPS, PAM3CSK4, or poly I:C for fixed activation, IRF3 is phosphorylated and translocates to the nucleus where it initiates a specific of Immunology, Faculty of Biochemistry, Biophysics transcriptional program. Recently, two non- and Biotechnology, Jagiellonian University, transcriptional functions for IRF3 have been Cracow, Poland identified: 1) a novel pro-apoptotic function termed RIG-I-induced IRF3-mediated pathway of apoptosis Professional phagocytes are equipped with complex (RIPA) and 2) an interaction with the p65 subunit of mechanisms of pattern recognition, allowing them NFκB that prevents p65 translocation to the nucleus. not only to identify different pathogens (PAMPs), While IRF3 has been implicated in the progression but also to recognize the self-derived molecular of high fat diet-induced steatosis and insulin patterns of dying, damaged or stressed cells resistance, the contribution of the transcriptional vs (DAMPs and ACAMPs). A term: engulfment non-transcriptional functions of IRF3 are not well synapse, has been proposed to depict the complex understood. Here we made use of IRF3-deficient nature of the phagocyte-target contact site which mice (IRF3 KO), as well as a novel knock-in of a comprises hundreds of proteins that play a key role mutated IRF3 that cannot translocate to the nucleus in deciphering the target surface and in defining host and only retains the non-transcriptional functions of response. HSP90, a stress-induced chaperone IRF3 (nt-IRF3 KI). protein, has been demonstrated as a regular Methods: Wild-type C57BL/6, IRF3-KO and nt- component of LPS-signalling complexes on IRF3 KI mice were allowed free access to a high fat macrophages but its role has not been elucidated. As diet or chow-fed control diets for 12 weeks. LPS is a prototypical PAMPs it is tempting to Measures of liver injury were assessed. speculate that HSP90 also assists recognition of Results: High fat diet (HFD) increased plasma ALT different molecular patterns by professional and AST activities indicators for hepatic injury and phagocytes. expression of inflammatory cytokines and In this study, we detected the cell surface HSP90 on chemokines in liver of wild-type mice. These human monocyte-derived macrophages by staining responses were exacerbated in IRF3 KO mice, but with a cell-impermeable specific inhibitor. Confocal completely ameliorated in nt-IRF3 KI mice. In analysis of live hMDMs revealed that the HSP90- contrast, hepatic triglycerides were elevated after inhibitor complexes were rapidly clustered on the HFD in wild-type mice, with a modestly reduced macrophages surface and, when incubation was steatosis in IRF3 KO mice and further reduced in nt- prolonged, recycled from cell surface through IRF3 KI mice. Fibrosis, assessed by expression of endosomal compartment. This finding, supported by collagen 1α mRNA and Sirius red staining, was detection of N-terminal domain of surface HSP90 by increased in wild-type mice after HFD; the fibrotic specific mAbs suggests that N-terminal (ATP-ase) response was even higher in IRF3 KO mice, but nt- domain of HSP90 is exposed to and accessible from IRF3 KI mice were protected from HFD-induced the outside of the cell. To study the role of surface fibrosis. HSP90 on phagocytic cells we used two different Conclusion: IRF3 has previously been implicated in classes of molecular patterns: pathogen- or apoptotic the development of HFD-induced steatosis and cell-associated. We have shown that blocking the insulin resistance. Here we have identified a cell-surface HSP90 pool leads to the dramatic differential contribution of the transcriptional vs decrease of TNF production by primary human non-transcriptional actions of IRF3, finding that the monocytes and hMDMs exposed to wide range of expression of only the nt-IRF3 can protect from soluble (TLRs-specific ligands) and particulate HFD-induced inflammation and fibrosis in the liver. (bacteria S. aureus and P. gingivalis) PAMPs. Surprisingly, in human macrophages the functional 104 cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or Activity of Cell Surface 90kDa Heat Shock bacteria cells. The presented data suggest that the Protein (HSP90) is Necessary for Cytokine surface HSP90 is a ‘signaling complex chaperone’ Production but Not for Target Engulfment which activity is essential for cytokine response but following Pattern Recognition by Macrophages not for target engulfment following pattern Malgorzata Bzowska, Anna Nogieć, Krystian Bania, recognition by macrophages. Magdalena Zygmunt, Krzysztof Guzik, Department 105 while silencing Tie1 did not alter lung tissue Ang- 2/Ang-1 ratio, we found inflammatory cytokines, Silencing Endothelial Cell Receptor, Tie1, vessel permeability and lung injury to be decreased Reduces Inflammation and Vascular in Tie1 siRNA treated mice. These data suggest that Permeability following Hemorrhage Priming for Tie1 contributes to EC activation by inhibiting Ang- the Development of Acute Respiratory Distress 1/Tie2 interaction, potentially leading to EC Syndrome in Mice dysfunction and the development of iARDS. Joanne L. Lomas-Neira1,2, Chun-Shiang Chung1,2, (Funded by NIH GM103652) Jiali Zhu1, 1Rhode Island Hospital; 2Brown University 106

Loss of endothelial cell barrier function plays a Role of CK2 in Neutrophil Signaling and significant role in the development of acute Functional Responses respiratory distress syndrome (ARDS). Thornin Ear, Patrick P. McDonald, Pulmonary Angiopoietins 1 & 2 mediate endothelial cell (EC) Division, Faculty of Medicine, Université de activation through competitive binding to their Sherbrooke and CR-CHUS, Sherbrooke, Québec shared receptor, Tie2, expressed on ECs. Ang-1 (Canada). binding induces Tie2 phosphorylation and signaling for downstream anti-inflammatory and anti- Casien kinase 2 (CK2) is a highly conserved, apoptotic protein synthesis and vessel barrier ubiquitously expressed, and pleiotropic Ser/Thr integrity. Ang-2/Tie2 binding, alternatively kinase, endowed with constitutive activity, that can promotes increased pro-inflammatory signaling be modulated by phosphorylation. The CK2 (increased ICAM-1 expression) and decreased holoenzyme consists of two regulatory (beta) and barrier function (lung leak). two catalytic (alpha or alpha’) subunits, the latter of Ang-2 is significantly elevated in plasma from which can also function independently of the patients with ARDS and in our murine model of tetramer. CK2 has hundreds of substrates hemorrhagic shock priming for the development of participating in the regulation of numerous cellular indirect (i)ARDS followed a subsequent septic processes including oncogenesis, cell cycle challenge. We have demonstrated that EC interaction progression, proliferation, apoptosis, transcription, with hemorrhage-primed neutrophils contributes to inflammation, and immune responses. Neutrophils EC activation and Ang-2 release, and that depletion and their products profoundly influence innate and of peripheral blood neutrophils prior to adaptive immunity, notably through the generation hemorrhage/sepsis increases Tie2 phosphorylation. of inflammatory cytokines. Several signaling Recent publications as well as our preliminary data cascades are known to control neutrophil responses, suggest that the orphan receptor, Tie1, may also play including the TAK1, IKK/NF-kB, p38 MAPK, a role in modulating EC activation by forming a MEK/ERK, and PI3K/Akt pathways. Whether CK2 complex with Tie2. This complex inhibits Ang- affects neutrophil functional responses or signaling, 1/Tie2 interactions promoting an activated/pro- however, remains unknown. inflammatory EC phenotype. To better understand In this study, we report that neutrophils the significance of Tie1/Tie2 interaction in constitutively express CK2 subunits. Following cell mediating EC activation leading to loss of barrier stimulation by TNFalpha or LPS, CK2alpha function and the development of ARDS in our becomes rapidly phosphorylated. Similarly, the model, we delivered liposomal-encapsulated short phosphorylation of several phospho-(Ser/Thr) CK2 interference (si)RNA, targeting Tie1, via tail vein substrates is observed in resting neutrophils, and injection 1 hour post hemorrhage. Mice were made increases following TNF or LPS stimulation. septic [cecal ligation and puncture (CLP)] 24 hour Inhibitors of MAPKs, PI3K, or TAK1 failed to following hemorrhage and euthanized 24 hours after affect the pattern of phospho-(Ser/Thr) CK2 CLP. Targeted siRNA treatment decreased Tie1 substrates. This suggests that these various kinases expression in lung tissue homogenates by 65% +/- do not act upstream of CK2. Conversely, neutrophil 7% compared to mice that received non- pretreatment with a CK2 inhibitor (CX-4945) led to targeting/scrambled siRNA (control). Importantly, a dose-dependent inhibition of phospho-(Ser/Thr) CK2 substrates. The kinase, Akt, appeared to inhibitor SP-600125 was used for inhibiting the JNK represent one such substrate, as its phosphorylation pathway. NETosis kinetics was measured by the was attenuated by CX-4945 in activated neutrophils. Sytox-Green assay and ROS production was By contrast, the phosphorylation of other signaling measured by the DHR123 assay. Immunoblots and intermediates, including p38 MAPK, JNK, or IKK confocal microscopy was used for determining the was unaffected by CK2 inhibition. We next pattern of NET inhibition, JNK activation, and investigated whether CK2 might affect neutrophil apoptosis. functional responses. The inducible gene expression Results: Our immunoblot results suggest that JNK is and secretion of several inflammatory cytokines activated in response to LPS, but not PMA or media were profoundly impaired following CK2 inhibition. control. Further, JNK inhibition by SP-600125 Similarly, the delayed apoptosis observed in significantly reduces LPS-induced ROS production response to various neutrophil stimuli was mostly while PMA-induced ROS production was reversed by CK2 inhibition. Our data unveil a role unchanged. This shows that JNK is essential for for CK2 in controlling the Akt cascade and LPS-mediated ROS production. In addition, JNK downstream functional responses in primary human inhibition suppresses LPS-induced NETosis but does neutrophils, making it a promising target for not affect PMA-induced NETosis. Our microscopy therapeutic intervention in view of the foremost role images show that JNK inhibition suppresses LPS- of neutrophils in several chronic inflammatory mediated NETosis such that the cells resemble the conditions. media control. However, JNK inhibition did not inhibit PMA-mediated NETosis such that no 107 difference was observed compared to the PMA group. Additionally, our cleaved caspase-3 A Novel Role for JNK in Neutrophils: JNK immunoblots and confocal images indicate that JNK Uniquely Regulates ROS Production and suppression does not induce apoptosis in neutrophils NADPH-Oxidase-Dependent Netosis but prolongs the lifespan of neutrophils in the LPS Meraj A. Khan1,2, Armin Farahvash1,2, Johann group. To further confirm the biological significance Christoph-Licht1,2, Neil Sweezey1,2, Nades of LPS-induced NETosis, we tested the effects of Palaniyar1,2, 1The Hospital for Sick Children; JNK inhibition in E. coli- and PA-induced NETosis. 2University of Toronto We found that pretreating neutrophils with SP- 600125 significantly suppresses E. coli and PA- Introduction & Objectives: JNK is a stress-activated induced NETosis. kinase that regulates cell survival and death under Conclusion: We show that JNK is essential in LPS-, different conditions. However, the roles of JNK in E. coli- and PA-mediated, but not PMA-mediated neutrophils are not clearly established. We NETosis. JNK is involved in ROS production in considered that JNK plays an important role in LPS-mediated NETosis pathway such that its neutrophil Extracellular Trap (NET) formation or inhibition reduces LPS-induced ROS production and NETosis. NETs are decondensed DNA decorated subsequent NETosis. Additionally, JNK inhibition with antimicrobial peptides that are capable of does not lead to apoptosis but increases the life span trapping bacteria. The molecular pathway of of the LPS treated neutrophils. NETosis is not fully understood. Although the Funded by: This study was supported by the CIHR participation of mitogen-activated protein kinases (to N.P) and CFC (to N.P and N.S). M.A.K is a (MAPKs) ERK and p38 in NETosis has been studied Mitacs postdoctoral fellow. A.F is a recipient of in considerable detail, the role of c-Jun N-terminal LMP summer studentship. Kinase (JNK), another MAPK, in NETosis is not clearly established. Methods: NETosis was activated in human neutrophils by a diacylglycerol mimic phorbol 12- myristate 13-acetate (PMA), gram-negative bacteria cell wall component lipopolysaccharide (LPS), and gram-negative bacteria such as E.coli and Pseudomonas aeruginosa (PA). JNK specific 108 by their localization to the epithelium, which was governed by the presence of neutrophils during early Neutrophils and Influenza-Specific CD8 T Cells infection. Both in vitro and in vivo imaging showed in the Flu-Infected Airways that migrating neutrophils leave behind long-lasting Kihong Lim, Minsoo Kim, University of Rochester trails from their elongated uropods that are

prominently enriched in the chemokine CXCL12. Influenza virus infects the epithelial cells that line We observed that CXCL12 derived from the the respiratory tract. Therefore, cytotoxic CD8+ T epithelial cells remained close to the epithelium, cells must traffic to the mucosal epithelium to while CXCL12 derived from neutrophils was the mediate elimination of infected cells. Neutrophils are main source of CXCL12 in the tissue interstitium key players that help organs initiate and maintain during infection. Experiments with granulocyte- immune reactions and shape the overall immune specific CXCL12 conditional knock-out mice and a response by signaling to DCs, monocytes, and T CXCR4 antagonist revealed that CXCL12 derived cells. Under most inflammatory conditions, from neutrophil trails is critical for virus-specific neutrophils are the first cell type that crosses the + CD8 T cell recruitment and anti-viral effector blood vessel endothelium into the tissue, often functions. preceding a subsequent wave of effector T cells. The data presented here demonstrate that migrating Although neutrophil-mediated recruitment of T cells neutrophils leave behind chemoattractant-containing into infected sites has been documented in both trails, resulting in the local accumulation of bacterial and viral infections and in chronic neutrophil-derived chemoattractant signals in inflammatory diseases, the molecular mechanisms inflamed tissues. As chemokines are small, that link neutrophil and T cell migration remain diffusible molecules, perhaps these trails serve to unknown. package the chemoattractant so that it can be The chemokine receptor family is known to be the preserved and survive severe mechanical most potent and subset-selective, tissue-specific perturbation during inflammation, lest it be present homing receptors for T cell. Therefore, it is widely only transiently or immediately diffuse away from assumed that the differential expression of the the site. chemokines and their receptors is responsible for the distinct migratory properties and distribution 109 patterns of different subsets of specialized T cells. However, although this idea has been verified Role of Neutrophils at the Site of Unhealing experimentally in some settings, multiple chemokine Leishmania mexicana Induced Lesion receptors expressed on the effector T cells and the 1 1 Benjamin P. Hurrell , Manon Beaumann , Sandrina redundancy in their signaling pathways suggest the 2 2 Heyde , Andreas J. Müller , Fabienne Tacchini- presence of a more complex mechanism that can 1 1 2 Cottier , University of Lausanne; Otto-von- confer the specificity and selectivity of T cell Guericke University Magdeburg recruitment. Furthermore, less is known about how chemokines released from newly recruited The protozoan Leishmania mexicana parasites cause leukocytes act together with the local chemokines chronic non-healing cutaneous lesions in humans produced within the inflamed tissue. To address this, and mice with poor parasite control. Promastigote we performed intravital multi-photon microscopy forms of the parasites are injected by sand flies and (IV-MPM) imaging of the influenza-infected mouse transform into intracellular amastigote stage forms in trachea and explored how neutrophil-derived the vertebrate hosts. We have previously shown that chemokines cooperates with the tissue-specific within hours of infection, L. mexicana promastigotes inflammatory cues to finely control the recruitment induce the local recruitment of neutrophils that of CD8+ T cells to the influenza-infected trachea. sequester the parasites, shaping the Here, we show that the early recruitment of microenvironment, eventually favoring the neutrophils into influenza-infected trachea is development of a chronic lesion. Here, we essential for CD8+ T cell-mediated immune investigated interactions between neutrophils and the protection. Especially, the relative motility of virus- intracellular amastigote form of the parasite. We first specific CD8+ T cells in the trachea was determined show that amastigotes are 2-3 times less internalized by neutrophils than promastigotes in vitro, however deficient in both receptors for TNFα. Furthermore, opsonization with immune serum led to comparable the directional crawling of neutrophils onto the internalization. In line with these data, injection of lumen of lymphatic endothelial cells is altered in mice with amastigote forms of L. mexicana only mice pre-treated with an anti-TNFα blocking poorly recruits neutrophils. This is in contrast to antibody, a response associated with a reduction in what is observed following injection of the infective ICAM-1 expression by lymphatic endothelial cells in promastigotes. Of note, intralesional neutrophils WT animals. Collectively, our results demonstrate represent 6-10% of the local cells during chronic the critical role of TNFα in promoting neutrophil infection. Strikingly,these neutrophils are heavily recruitment into the lymphatic vasculature in vivo infected with over 70% of them harboring two or during the inflammatory response of antigen more intact amastigotes, as determined by the use of challenge. fluorescently labelled parasites and ImageStream This work is supported by Arthritis Research UK analysis. Recent reports reveal increased lifespan of (19913). tissue neutrophils upon inflammation, suggesting that neutrophils could not only harbor live parasites 111 but that parasites may replicate within these cells. To explore this, we have generated transgenic L. Effects of Antibody Opsonization on Early mexicana parasites expressing the photoswitchable Yersinia Pestis-Host Cell Interactions in the Skin kikume protein, allowing visualization of and Draining Lymph Node in a Mouse Model of intracellular parasite replication. New data will be Bubonic Plague presented which focus on a better understanding of Jeffrey G. Shannon, B. Joseph Hinnebusch, the role of neutrophils at the site of unhealing Laboratory of Zoonotic Pathogens, Rocky Mountain parasite infection. Laboratories, NIAID, NIH, Hamilton, MT, USA

110 Plague is a zoonosis caused by the bacterial pathogen Yersinia pestis. Bubonic plague, the most TNFα Drives the Migration and Crawling of prevalent form in humans, results when Y. pestis is Neutrophils into Afferent Lymphatic Vessels deposited in the skin via the bite of an infected flea. during Antigen Challenge In vivo. Bacteria subsequently traffic from the dermis to the Mathieu-Benoit Voisin, Samantha Arokiasamy, draining lymph node (dLN) where they replicate to Sussan Nourshargh, Queen Mary University of large numbers forming a severely enlarged lymph London node called a bubo. The bacteria can escape from the bubo and spread systemically, which if left Neutrophils are now viewed as key effectors of both untreated, is highly fatal. Though several plague innate and adaptive immunities in many vaccine candidates are currently at various stages of physiological and pathological conditions. Whilst development, no licensed vaccine is available in the the trafficking of neutrophils through blood vessels United States. Polyclonal and several monoclonal has been extensively studied, the mechanism that antibodies (Ab) can provide complete protection control their entry into the lymphatic system is against plague in animal models. The mechanisms poorly understood. For this purpose, we have involved in this antibody-mediated immunity (AMI) developed a murine model of cremasteric to Y. pestis remain poorly understood. Here we inflammation to visualise by intravital confocal examine the effect of opsonizing Ab on Y. pestis microscopy the migration of neutrophils into tissue- interactions with macrophages and neutrophils in associated afferent lymphatic vessels in vivo. In the vitro and in vivo. We generated mouse anti-Y. pestis present study, we report that neutrophils migrate immune serum by vaccinating C57Bl/6 mice with an rapidly into the lymphatic vessels of inflamed attenuated pCD1+ strain of Y. pestis and confirmed cremaster muscles of WT animals upon antigen the presence of high Ab titers by western blot and challenge in a CCR7-dependent manner. immunofluorescence assays. Immune serum- Interestingly, neutrophil intravasation into opsonized Y. pestis showed modest but significant lymphatics (but not extravasation through blood increases in phagocytosis by mouse macrophages vessels) is inhibited (~70%) in mice genetically and neutrophils in vitro compared to naïve serum controls. Ab opsonization had little effect on versus IgG-induced neutrophil effector functions and intracellular survival of bacteria within these cells or to elucidate the specific signaling pathways of FcαRI on neutrophil oxidative burst. Previous intravital in neutrophils, responsible for IgA-induced microscopy studies showed a rapid and robust neutrophil migration and activation. neutrophil response to Y. pestis in the dermis of Methods naïve mice. We used this system to determine if Ab The quantitative number of Fc receptors expressed opsonization alters this response. We observed was determined with a Qifikit. Furthermore, increased association of immune serum-opsonized Y. neutrophils were stimulated with complexed serum pestis with neutrophils in the dermis of LysGFP IgA or IgG to determine the differences in mice compared to naïve serum-treated controls. phagocytic index, lactoferrin release, induction of Similar results were obtained with passively and reactive oxygen species (ROS), NETosis, cytokine actively immunized mice. We also imaged the release, metabolite release, calcium flux and popliteal LNs after i.d. injection of bacteria in the induction of signaling. footpad and observed increased Y. pestis-neutrophil Results interactions and increased neutrophil clustering in Neutrophils express a higher number of Fcγ response to Ab-opsonized bacteria. Thus, despite receptors (FcγRII and FcγRIII) compared to FcαRI. only having a modest effect in in vitro assays, No differences were observed in phagocytosis of opsonizing Ab has a dramatic in vivo effect on Y. IgA- versus IgG-coated beads, but triggering the Fc pestis-neutrophil interactions in the dermis and dLN alpha receptor with IgA led to enhanced release of very early after infection. Additionally, anti-Y. pestis cytokines, chemokines and pro-inflammatory Ab may alter neutrophil recruitment and localization metabolites. Additionally, IgA stimulation resulted within infected dLNs. These data shed new light on in a higher induction of ROS, NETosis and calcium the importance of neutrophils in AMI to Y. pestis flux. Furthermore, crosslinking the FcαRI led to a and may provide a new correlate of protection for stronger and more sustained phosphorylation of evaluation of plague vaccine candidates. different signaling molecules. Conclusion 112 Activating neutrophils with IgA leads to increased pro-inflammatory effector functions and enhanced Fc Receptors on Neutrophils: Two Sides of the signaling, which could not be explained by a higher Same Coin? expression of FcαRI on neutrophils. This suggests Marieke H. Heineke1, Esil Aleyd1, Sanae Ben that the signaling routes after IgA and IgG triggering Mkaddem2, Renato C. Monteiro2, Jantine E. are different, which is against the current dogma Bakema3, Marjolein van Egmond1,4, 1Department of which dictates that both FcαRI and Fcγ receptors use Molecular Cell Biology and Immunology, VU similar signaling pathways via immunoreceptor University Medical Center, Amsterdam, The tyrosine-based associated motifs (ITAMs). The Netherlands; 2National French Institute of Health delineation of the exact signaling pathways of FcαRI and Medical Research (INSERM) Unit 1149, Center and Fcγ receptors in neutrophils is the subject of of Research on Inflammation, Paris, France; 3Tumor further studies, as specifically targeting IgA Biology Section, Department of signaling pathways may represent a novel Otolaryngology/Head-Neck Surgery, VU University therapeutic strategy to prevent tissue damage in IgA- Medical Center, Amsterdam, The Netherlands; mediated autoimmune diseases. 4Department of Surgery, VU University Medical Center, Amsterdam, The Netherlands 113

Neutrophils express Fc receptors, which recognize Neutrophil Dysfunction in the Pre-Hospital the antibodies IgA (FcαRI) or IgG (Fcγ receptors). Setting following Traumatic Injury: Results from We previously demonstrated that neutrophil the “Golden Hour” Study migration is only induced after IgA activation, which Jon Hazeldine1,2, Emma Toman2, David Davies2, may play a role in IgA-autoantibody mediated Zhangjie Su2, Peter Hampson1,3, Robert J. autoimmune diseases. The aim of this study is to Dinsdale1,3, Nicholas Crombie4, Paul Harrison1,3, investigate in depth the differences between IgA- Antonio Belli1,2, Janet M. Lord1,2, 1Institute of Inflammation and Ageing, University of CD11b and shedding of L-selectin. The phagocytic Birmingham, Birmingham, UK.; 2NIHR Surgical activity of neutrophils in pre-hospital samples was Reconstruction and Microbiology Research Centre, negatively associated with the absolute number of Queen Elizabeth Hospital Birmingham, UK.; circulating IGs and CD16BRIGHT CD62LDIM 3Healing Foundation Centre for Burns Research, neutrophils. As a readout of neutrophil function in Queen Elizabeth Hospital, Birmingham, UK.; vivo, plasma samples from patients were screened 4Midlands Air Ambulance, Unit 16 Enterprise for citrullinated histone H3 (CitH3). A marker of Trading Estate, Brierley Hill, West Midlands, UK. neutrophil extracellular trap (NET) formation, we detected CitH3 in pre-hospital samples, but not Providing immediate frontline protection against those acquired 4-12 hours or 48-72 hours post rapidly dividing bacteria, fungi and yeast, injury, thereby demonstrating that NET formation is neutrophils are critical effector cells of the innate a feature of the ultra-early immune response to immune system. In the acute phase (4-12 hours) sterile trauma. following major traumatic injury, significant Taken together, we have shown for the first time that alterations have been described in neutrophil neutrophil dysfunction is evident within minutes of phenotype and function, changes that may contribute major traumatic injury and attribute this to the increased susceptibility of trauma patients to phenomenon to a trauma-induced creation of a nosocomial infection. However, no study to date has heterogeneous neutrophil pool, consisting of IGs, examined neutrophil behaviour in the immediate activated neutrophils and CD16BRIGHT CD62LDIM aftermath of major injury. Thus, it remains to be suppressive neutrophils. established how quickly following a traumatic insult neutrophil biology is altered. Here, by analysing 114 blood samples acquired in the pre-hospital setting from 75 adult trauma patients (mean time to blood The Type of Monosaccharides Influences the sample 43.69±1.37 minutes post injury) we have Metabolic and Functional Responses of performed for the first time a detailed assessment of Neutrophils Activated with Pro-Inflammatory the composition, function and phenotype of the Cytokines circulating neutrophil pool in the ultra-acute phase of Martin Pelletier1,2, Asmaa Lachhab1,2, Maude major trauma. Leclerc1, Benjamin Brisebois1, 1Infectious Diseases We observed within minutes of injury a significant and Immunity Research Division, CHU de Québec- neutrophilia, which was accompanied by a marked Université Laval Research Center; 2Department of elevation in the percentage and number of Microbiology, Infectious Disease and Immunology, circulating immature granulocytes (IGs), suggesting Faculty of Medicine, Université Laval enhanced bone marrow haemopoietic activity in the immediate post injury phase. Relative to a cohort of Pro-inflammatory cytokines IL-6, TNF and IL-1β age and sex-matched healthy controls, freshly are involved in chronic inflammatory diseases. They isolated neutrophils from patients exhibited modulate the responses of immune cells like significantly reduced surface expression of CD62L, polymorphonuclear neutrophils (PMNs), but their CD88, CXCR1 and CXCR2 as well as increased impact on their bioenergetics is not well defined. expression of CD11b, phenotypical changes that are Using the extracellular flux analyzer, we observed a indicative of neutrophil activation. Furthermore, quick and robust glycolytic response in TNF- multi-colour flow cytometry revealed a significant activated PMNs compared to IL-1β- or IL-6- trauma-induced increase in the frequency and activated cells. TNF also induced rapid oxygen absolute number of circulating CD16BRIGHT consumption, an effect enhanced with IL-1β. IL-6 or CD62LDIM suppressive neutrophils. In ex vivo IL-1β alone had no effect on PMNs’ oxygen functional analysis, neutrophils from trauma patients consumption. At the molecular level, we observed exhibited impaired phagocytosis of the gram that TNF- and IL-1β-activated PMNs had enhanced negative bacteria Escherichia Coli and were gene transcripts coding for pro-inflammatory refractory to stimulation with the bacterial peptide cytokines and for the metabolic adaptor HIF-1A. The formyl-methionine-leucine-phenylalanine as expression of the glucose transporter GLUT-3 and evidenced by significantly reduced up-regulation of the amino acid transporters CD98 and ASCT2 transcripts were only modulated by TNF. Pre- differences in swelling directly correlate with incubation of PMNs with a competitive inhibitor of neutrophil/macrophage accumulation in the synovial glucose revealed that the cytokine-activated cavity of these mice. These findings are also oxidative burst depended on glycolysis. Since supported by data obtained in a model of PMA- monosaccharides regulate this process, we examined induced ear inflammation. Currently, our research is the effect of sugars on TNF and IL-1β-activated focused on the potential cross-talk between the PMNs. The cytokine-induced oxidative burst involved PHD2-deficient myeloid populations and occurred in the presence of sugar-free medium, or in its effect on chemotaxis in both in vivo models. media containing glucose and galactose. This effect was completely inhibited in the presence of 116 mannose, even when glucose and galactose were added. Moreover, the induction of pro-inflammatory Secondary Necrotic Neutrophils Release cytokine transcripts in response to TNF and IL-1β Interleukin-16C and MIF from Stores in the was significantly increased in PMNs when Cytosol mannose was used compared to glucose-, galactose- Tamás Laskay, Sara Roth, University of Lübeck or sugar-free medium. These results indicate that pro-inflammatory cytokines exert different effects on Background: Neutrophils harbor a number of PMNs’ energy metabolism and that the type of preformed effector proteins that allow for immediate monosaccharides can influence their cytokine antimicrobial functions without the need for time- expression and functional responses. consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, 115 including IL-1ra, CXCL8. The present study aimed to identify additional preformed cytokines in PHD2 as a Regulator of Myeloid Inflammatory primary human neutrophils and investigate the Response conditions of their release/secretion. Sundary Sormendi-Gómez, Ben Wielockx, Klinische Materials and methods: In search for additional Pathobiochemie, Medizinische Fakultaet, preformed cytokines, a cytokine array analysis was Technische Universitaet Dresde, Germany performed with lysates from human primary neutrophils. Confocal immunofluorescence Hypoxia-inducible factors (HIF) are master microscopy as well as western blot analysis of regulators of the cellular response to oxygen subcellular fractions was carried out to investigate deprivation and coordinate a transcriptional program the subcellular localization of cytokines. Neutrophil that ensures optimal functional, metabolic, and apoptosis was induced by overnight incubation and vascular adaptation to oxygen levels. Regulation of by UV irradiation. these transcription factors relies on the oxygen- Results: IL-16 and macrophage migration inhibitory dependent enzymes prolyl-hydroxylase domain factor (MIF) were identified as preformed cytokines (PHD1-3) proteins. Although several studies have in human primary neutrophils. Both IL-16 and MIF shown how the hypoxia pathway controls innate and are unconventional cytokines because they lack a adaptive immunity response in different signal sequence. IL-16 and MIF were found to be inflammatory disorders, the role of these oxygen- stored in the cytosol rather than in the granules of sensors during inflammation is still largely human neutrophils, which implies an unconventional unknown. secretion mechanism for both cytokines. IL-16 is Using an antibody-induced arthritis model, we synthesized and stored as a precursor (pre-IL-16). observed that loss of PHD2 in hematopoietic cells We present evidence that the processing of pre-IL-16 (Vav:cre-PHD2f/f) leads to an augmented to the biologically active IL-16C is mediated by inflammatory response, revealed by an increase in caspase-3 and occurs during both spontaneous and paw thickness in conditional deficient mice. UV-induced apoptosis of human neutrophils. Therefore, we hypothesize that PHD2 (main Although IL-16 processing occurs during apoptosis, regulator of the HIFα proteins) in hematopoietic IL-16C and MIF release was observed only during cells might have a protective role during the onset of secondary necrosis of neutrophils. Screening a panel inflammatory arthritis. Indeed, the observed of microbial substances and pro-inflammatory cytokines did not identify any stimulus that induced produced by PMN were assessed by use of the the release of IL-16C and MIF independent of redox-sensitive dye C-DCDHF-DA coupled to secondary necrosis. spectrofluorimetry. Cell morphology was assessed Conclusions: The data presented here suggest that by transmission electron microscopy (TEM) and IL-16 and MIF are neutrophil-derived inflammatory scanning electron microscopy (SEM). D1-like (D1 mediators released under conditions of insufficient and D5) and D2-like (D2, D3 and D4) dopaminergic clearance of apoptotic neutrophils, as typically receptor (DR) expression was analyzed by flow occurs at sites of infection and autoimmunity. cytometry. In PMN from HC, migration induced by fMLP 0.1 117 µM was (mean±SEM) 9.4±1.9 µm. DA 1 µM reduced migration to 26.2±18.0% of fMLP-induced Morpho-Functional Changes of Human values (n = 5, P<0.01 vs fMLP alone). The effect Neutrophils Treated with Dopaminergic Agents: was mimicked by the D1-like DR agonist SKF- Relevance for Multiple Sclerosis 38393 0.1 µM (22.3±6.4% of fMLP-induced values, Monica Pinoli1, Emanuela Rasini1, Michela Gaiazzi1, n = 5, P<0.01 vs fMLP alone) and reverted by the Magda De Eguileor2, Laura Pulze2, Terenzio D1-like DR antagonist SCH-23390 1 µM Congiu3, Mauro Zaffaroni4, Marco Cosentino1, (22.7±13.9%, n = 3, P>0.05 vs fMLP alone, and Franca Marino1, 1Center for Research in Medical P<0.05 vs fMLP+DA). The D2-like DR agonist Pharmacology, University of Insubria, Varese, Italy; pramipexole 1 µM did not affect migration (not 2Department of Biotechnology and Life Sciences, shown). ROS generation induced by fMLP was University of Insubria, Varese, Italy; 3Department of 302.8±124.1 arbitrary units. DA 1 µM reduced Surgical and Morphological Sciences, University of fMLP-induced ROS generation to 63.0±18.0% of Insubria, Varese, Italy; 4Multiple Sclerosis Center, fMLP-induced values (n = 11, P<0.01 vs fMLP S. Antonio Abate Hospital, Gallarate, Varese, Italy alone). The effect was mimicked by SKF-38393 0.1 µM (56.7±44.3%, n = 13, P<0.01 vs fMLP alone) Polymorphonuclear leukocytes (PMN) are innate but not by pramipexole 1 µM (not shown). DA 1 µM immune cells that during inflammatory or infective prevented fMLP-induced morphological changes of conditions leave the bloodstream and enter the PMN and was antagonized by SCH-23390 1 µM. tissues, promoting repair or contributing to the Flow cytometric assay of DR showed that PMN amplification of chronic damage depending on local were 79-80% and 48-63% positive for D1-like and and systemic factors. D2-like DR in HS (n = 9) and 65-72% and 37-63% Multiple sclerosis (MS) is the main autoimmune positive in PTS (n = 7). Functional experiments on disease of the central nervous system (CNS), and PMN from PTS are ongoing. emerging evidence suggest novel roles for PMN in DA exerts inhibitory effects in human PMN, which this disease. In MS patients PMN show an activated might be beneficial in MS. Dopaminergic profile and, in response to pro-inflammatory stimuli, modulation of PMN function must be however they migrate into the CNS through the compromised assessed also in cells from PTS. blood-brain-barrier, producing proteases and reactive oxygen species (ROS) thus resulting in increased 118 inflammation and disease progression. Dysregulation of dopaminergic pathways in the Zebrafish as Animal Model to Study Intestinal immune system play a key role in multiple sclerosis Inflammation (MS), however few information exists on Carmen G. Feijoo, Pilar Ulloa, Camila Solis, dopaminergic regulation of PMN function in healthy Universidad Andres Bello conditions and during MS. Our aim was to investigate the morpho-functional changes induced Inflammatory Bowel Disease (IBD) is an by dopaminergic agents in PMN from healthy immunological mediated disorder of the controls (HC) and from MS patients (PTS). gastrointestinal tract, characterized by uncontrolled Cell migration was measured by optic microscopy inflammation resulting from inappropriate and and quantified as the difference (Δ) between resting persistent activation of the mucosal immune system. values and values induced by treatments. ROS Although it is widely accepted that IBD results from a deregulated mucosal immune response to neutrophils and macrophages. The binding of environmental factors in genetically susceptible PAMPs to PRRs on phagocytes initiates pathogen- hosts, the precise cause of the disease has not yet specific innate immune responses to eliminate the been fully elucidated. The hallmark of active IBD is invading microorganisms. To evade host immune an aberrant mucosal infiltration by innate immune systems, pathogenic mycobacteria express effector cells (primarily neutrophils) and adaptive immune molecules, including lipoarabinomannan (LAM), on cells. their cell surface. ManLAM has been associated In our laboratory, we developed a novel approach with several PRRs and binding molecules on using the zebrafish model to study the intestinal phagocytes, including αMβ2-integrin, macrophage inflammation triggered by the intake of a soybean- mannose receptor, and DC-SIGN. Moreover, meal based diet (50SBM). It is well accepted that Mycobacteria have been shown to target lipid rafts soybean is a potent allergen and that produce (membrane microdomains) to enter into host cells. intestinal inflammation in fish. By the use of However, little is known about the molecular Tg(bacmpx:gfp)i114 zebrafish larvae line, that labels mechanisms by which LAM associates with lipid specifically neutrophils fluorescently, we evaluated rafts. Previously, we found that a neutral GSL, the presence of this granulocyte in the intestine, as a lactosylceramide (LacCer, CDw17), forms lipid rafts marker of an inflammatory process. Likewise, we on cell membranes. Moreover, we showed that also determined the effect of the soybean meal on LacCer-enriched lipid rafts mediate the phagocytosis the intestinal barrier integrity by of non-opsonized mycobacteria by human immunofluorescence on intestinal section. Also, neutrophils. Here, we investigated the binding qPCR assays were performed to analyse the specificity of mycobacterial LAM to LacCer and transcription level of mucosal immunity markers. To LAM-induced phagocytosis. determine if the susceptibility to pathogen was We first examined the binding specificity of LAM to increased in larvae with intestinal inflammation, we LacCer using surface plasmon resonance (SPR). developed a challenge assay with the enteropathogen SPR analysis showed that LacCer liposomes bind Edwardsiella tarda. specifically to Mycobacterium tuberculosis-derived Our results indicate that after as early as 4 day of mannose-capped LAM (ManLAM). In contrast, feeding, larvae fed with 50SBM showed an increase ganglioside GM3 liposomes failed to bind to in the number of neutrophils present in the intestine ManLAM. We also tested whether LAM binds to compared to control ones. Also, experimental larvae glycosphingolipids-coated plastic plates, finding that were more susceptible to infection. At the moment, both ManLAM and non-pathogenic M. smegmatis- we are working on the epithelial integrity assays. derived phosphatidylinositol-capped LAM (PILAM) Founding: Fondecyt 1140297; Fondap 1510027; bind specifically to LacCer- but not GM1-coated Unab DI-1229-16/R plastic plates, In contrast, PILAM from an M. smegmatis α1,2-mannosyltransferase deletion mutant 119 (ΔMSMEG_4247), which lacks α1,2-monomannose side branches of the LAM mannan core, did not bind Human Neutrophils Phagocytose Mycobacteria to LacCer. Next, we examined the phagocytosis of through Interactions between Laccer and α1,2- LAM-coated polystyrene beads by human Monomannose Side Branches of LAM neutrophils. Neutrophils phagocytosed M. Hitoshi Nakayama1,2, Noriko Yokoyama2, Kazuhisa tuberculosis-derived ManLAM- and M. smegmatis- Iwabuchi1,2, 1Juntendo University Faculty of Health derived PILAM-coated beads, with the phagocytic Care and Nursing; 2Institute for Environmental and indices of these ManLAM- and PILAM-coated Gender-Specific Medicine, Juntendo University beads being similar. Moreover, as observed in the Faculty of Medicine binding experiments, neutrophils did not phagocytose beads coated with PILAM derived from Microorganisms express highly conserved ΔMSMEG_4247. Taken together, these findings pathogen-associated molecular patterns (PAMPs). suggest that, regardless of the pathogenicity of These molecules are able to bind to a diverse array Mycobacteria, α(1→2)-monomannose side branches of pattern-recognition receptors (PRRs), such as of LAM mannan core are essential for LacCer αMβ2-integrin, expressed on the surfaces of binding and for neutrophil phagocytosis of Nursing, Juntendo University; 3Laboratory for Mycobacteria in humans. Biochemistry, School of Health Care and Nursing

120 Neutrophils play a central role in the innate immune system, and express several specific pattern Cytoskeleton Re-Organization during NET recognition receptors (PRRs) on their cell surface Formation that recognize pathogen- associated molecular Darko Stojkov, Poorya Amini, Hans-Uwe Simon, patterns (PAMPs) on microorganisms. Shida Yousefi, University of Bern, Institute of Microorganisms possess binding avidities to several Pharmacology types of glycosphingolipids (GSLs), suggesting that GSLs are involved in host-pathogen interactions. Neutrophils are most prominent cells in blood GSLs cluster to form lipid rafts with several circulation and their anti-microbial defence transducer molecules on plasma membranes, which mechanism has been defined by their ability to have been implicated in a number of important phagocytise microbes as well as forming Neutrophil membrane events. A neutral GSL, lactosylceramide Extracellular Trap (NET). For the last decade efforts (LacCer, CDwl7) has been shown to bind has been done to elucidate the molecular mechanism specifically to several types of pathogenic of NET formation. In this study we demonstrate that microorganisms, including Escherichia coli, Bacillus physiological activation of neutrophils generate dysenteriae, Candida albicans and Mycobacteria. reactive oxygen species (ROS), specifically LacCer is highly expressed on human neutrophils, hydrogen peroxide (H2O2), which then negatively and forms lipid rafts coupled with Src family kinase regulate actin polymerization by inducing actin Lyn and trimeric G proteins. LacCer-enriched lipid glutathionylation. In absence of NADPH oxidase rafts directly mediate neutrophil chemotaxis, activity, CGD patients’ neutrophils have defect in phagocytosis and superoxide generation. Moreover, actin reorganization, and NET formation. Exogenous LacCer-enriched lipid rafts serves as signal source of H2O2 however, restores actin transduction platforms for αMβ2 integrin-mediated glutathionylation, and ability of neutrophils to trap phagocytosis. and kill bacteria. Consistent with above Here we introduce how LacCer organizes the lipid observations, Grx1-deficient mouse neutrophils rafts with several transducer molecules and mediate upon activation accumulated high level of actin signaling. Two different anti-LacCer monoclonal glutathionylation, which attenuated actin antibodies T5A7 and Huly-ml3 showed the different polymerization, and impaired NET formation despite binding behaviors and specificities. Although Huly- presence of high level of ROS. ml3, but not T5A7, immunoprecipitated LacCer- Taken together these data support our hypothesis enriched domains, the biological activity of T5A7 on that cytoskeleton rearrangements are essential for neutrophil functions was much higher than that of physiological functions of neutrophils such as NET Huly-ml3. T5A7 bound to human and mouse formation. Pre-requisite for F-actin recycling is ROS neutrophils, while Huly-ml3 only bound to human activity; which is reversibly regulated by Grx1 neutrophils. SPR analysis revealed that the binding enzyme activity. These findings enlighten our affinity of Huly-ml3 but not T5A7 to LacCer understanding of regulated molecular mechanism depended on the amount of LacCer in the domains. involved in NET formation. The ultra-high resolution STED microscope observation revealed that Huly-ml3 and T5A7 bound 121 to distinct LacCer domains/clusters. The binding experiments revealed that T5A7 recognizes the Organization and Immunological Functions of phosphatidylcholine (PC)-enhanced three- Lactosylceramide-Enriched Lipid Rafts dimensional structure of LacCer clusters. In contrast, Kazuhisa Iwabuchi1,2,3, Hitoshi Nakayama1,3, Noriko PC did not affect the binding of Huly-m13 to LacCer Yokoyama1, Kumiko Ishii1, 1Institute for cluster. It seems, therefore, that Huly-ml3 binds to Environmental and Gender-Specific Medicine, the core region of lactose clusters in LacCer- Graduate School of Medicine; 2Infection Control enriched domains, while T5A7 binds to dispersed Nursing, Graduate School of Health Care and LacCer clusters in the phase boundary regions of the lipid rafts. Immune-electron microscopic mutations cause autosomal dominant optic atrophy observations revealed that Lyn molecules were (ADOA), a condition characterized by the located at the phase boundary regions of the rafts. preferential loss of retinal ganglion cells and Photo labeling experiments revealed that LacCer subsequent progressive optic nerve degeneration in directly associated with Lyn and Gαi proteins ADOA patients. through the direct connection between C24 fatty acid We studied the role of OPA1 in mtDNA release, chains of LacCer and palmitic chains of these NET formation and antibacterial activity of signaling molecules. A Src family kinase Hck was neutrophils. We generated mice lacking Opa1 gene not associated with LacCer-enriched lipid rafts in specifically in myeloid population, by cross breeding resting neutrophils. During phagocytosis, Hck was Opa1flox/flox with Lyz2Cre/Cre mice. These mice associated with LacCer-enriched lipid rafts on the have Opa1 knockout restricted to myeloid lineage membrane of non-pathogenic but not pathogenic (Opa1∆neut). In addition, we used isolated mycobacterial phagosomes. M. tuberculosis-derived neutrophils from blood of autosomal dominant optic lipoarabinomannan (ManLAM) directly bound to atrophy (ADOA) patients. LacCer clusters. ManLAM inhibited the association Freshly isolated neutrophils from Opa1∆ mice are of LacCer-enriched lipid rafts with Hck on unable to form functional NETs while the phagosomal membranes, and prevented phagosome neutrophils from genetically matched mice maturation. Thus, ManLAM may interfere the (Lyz2Cre/Cre) retain this ability. NETs are known to formation of LacCer clusters to make direct contribute to antibacterial defence mechanisms. association of LacCer and Hck. Taken together, it Intranasal inoculation with the nonmucoid seems that reorganization of LacCer-enriched lipid Pseudomonas aeruginosa revealed failure of Opa1∆ raft with signal transducer molecules is essential for mice to clear the bacteria from lung despite immunological functions of neutrophils. increased neutrophil infiltration in their lung and intact phagocytic activity compare to control 122 (Lyz2Cre/Cre) mice. We also confirmed the absence of NETs formation by staining of extracellular DNA Optic Atrophy 1 (OPA1) Controls Energy fibres associated with MPO and infiltrated Metabolism and Contributes to Antibacterial neutrophils in lung sections of infected Opa1∆mice. Functions of Neutrophils Moreover, inability of NETs formation and Poorya Amini1, Darko Stojkov1, Kevin Oberson1, disruption of microtubule was also observed in Luca Scorrano2, Charaf Benarafa3, Shida Yousefi1, ADOA patient’s neutrophils. We conclude that, the Hans-Uwe Simon1, 1Pharmacology University of difference in antibacterial killing ability is mainly Bern; 2Institute of Molecular Medicine (VIMM), due to the fact that Opa1∆neut are not able to form Padua; 3Theodor Kocher Institute, University of NETs. Bern 123 Neutrophil extracellular traps (NETs) are implicated in killing extracellular pathogens. Our laboratory Activation of Neutrophils by Immunoglobulin a demonstrated that activated neutrophils form (IgA) Exacerbates Pathogenesis of Inflammatory extracellular DNA traps, whereby the DNA Bowel Disease (IBD) originates from mitochondria. Marijn Bögels1, Lydia PE van der Steen1, Gerd Content exchange between mitochondria, Bouma2, Marjolein van Egmond1,3, 1Department of mitochondrial shape control, and mitochondrial Molecular Cell Biology and Immunology, VU communication with the cytosol are processes that University Medical Center Amsterdam; 2Department require mitochondrial fusion and fission. OPA1 in of Gastroenterology, VU University Medical Center inner membrane, and MFN1 and MFN2 in outer Amsterdam; 3Department of Surgery, VU University membrane of mitochondria are three proteins, which Medical Center Amsterdam orchestrate fusion of mitochondria in mammalian cells. OPA1, a large GTPase protein, is required for Background fusion of phospholipid bilayer and tightness of Mucosal lesions in IBD, and particularly ulcerative cristae junction in mitochondria. Pathogenic OPA1 colitis, are characterised by massive neutrophil infiltration and aggregation. Earlier we showed that Background: The normal, healthy, closed eye is binding of IgA, the prominent antibody in the gut remarkably different than the open eye, and is mucosa, to its receptor FcalphaRI on neutrophils characterized by many pro-inflammatory cytokines, initiates chemotaxis of these cells. As such, we complement activation products, matrix hypothesize that abnormal activation of neutrophils metalloproteinases, and has a characteristic by mucosal IgA might explain enhanced neutrophil accumulation of hundreds of thousands of infiltration in IBD, which results in undesirable neutrophils–500,000 on average. Previous work has tissue damage. demonstrated that these neutrophils have a refractory Materials and methods phenotype, whereby they do not upregulate surface Fresh blood and snapfrozen colon biopsies of IBD membrane receptors in response to inflammatory patients were stained for different immunological stimuli. However, these neutrophils have an markers and analysed with FACS or activated phenotype as compared to blood-isolated immunofluorescence, respectively. Neutrophils were neutrophils with increased expression of CD66b, activated by IgA and co-cultured with CaCo2 CD11b, and CD54. The closed eye leukocyte milieu epithelial cells. A humanised mouse model, also contains a small percentage (2%) of T cells. expressing both human IgA and FcalphaRI, was Preliminary investigations have shown that roughly used to study the role of IgA/FcalphaRI interactions 20% of all CD4+ T cells in the closed eye are Th17 in pathogenesis in a DSS-induced colitis model. cells. Dry eye disease is a complex multifactorial Results disease of the ocular surface, which results in Phagocytosis of IgA opsonised particles led to symptoms of pain, irritation, and discomfort and activation of neutrophils, leading to extensive may ultimately lead to a significant reduction in reactive oxygen production and release of quality of life. Dry eye disease is commonly Neutrophil Extracelluar Traps (NETs), which associated with inflammation and Th17 influx, and consisted of aggregated DNA strands covered by in severe cases, neutrophil extracellular traps have reactive enzymes like myeloperoxidase (MPO) and also shown to be present. The purpose of this pilot neutrophil Elastase (NE). Culturing ‘NETosing’ investigation was to determine if additional neutrophils with epithelial cells led to enhanced neutrophil hyperactivity or increased T cell apoptosis and cell death of epithelial cells. In human infiltration could be observed as part of the closed IBD biopsies enhanced neutrophil infiltration eye leukocyte population in dry eye disease. correlated with presence of NETs positive for MPO Methods: Six normals and six dry eye subjects were and NE. FcalphaRI on neutrophils furthermore, recruited and were trained to wash the ocular surface resulted in enhanced DSS induced colitis in with phosphate buffered saline for at-home self- IgA/FcalphaRI mice, compared to mice that only collection of tear film leukocytes following a full expressed human IgA. night of sleep. Cells were isolated and counted, and Conclusions were incubated with fluorescently-labeled antibodies Aberrant activation of neutrophils in the gut mucosa against CD45, CD14, CD15, CD16, CD11b, and via IgA/Fcalpha interactions may lead to extensive CD66b to identify neutrophils. Antibodies against NETs release, resulting in epithelial cell death and CD45, CD3, CD4, CD8, CD196, and CD161 were tissue damage. Inhibiting migration and activation of used to identify T cells. A Becton Dickinson (BD; neutrophils by restraining IgA/FcalphaRI San Jose, CA) proprietary fixable viability stain was interactions can be a valuable new approach to used to exclude dead cells from analysis. A BD LSR dampen the disease burden. II flow cytometer was used for all analyses. Results: Neutrophils isolated from dry eye subjects 124 demonstrated increased expression of CD11b, CD66b, and CD16 as compared to normals, Refractory Neutrophils in the Closed Eye Are indicating a hyperactive neutrophil phenotype. Associated with Ocular Surface Dysregulation Similarly, there were more CD4+ T cells and more Cameron K. Postnikoff, Kelly K. Nichols, University Th17 cells (as identified as being CD196+CD161+) of Alabama at Birmingham in the closed eye leukocyte populations as compared to normals. Combined, the results demonstrate increased inflammation in the dry eye closed eye compared to the normal closed eye. haploinsufficient mice. Interestingly, we found that Conclusions: In a small cohort, the closed eye both female NZM.NCF2-/- and NZM.NCF2+/- mice leukocyte population in dry eye disease appeared to showed accelerated onset of lupus nephritis. have increased presence of Th17 cells and an Proteinuria and renal pathologies including associated neutrophil hyperactivity. The normal, glomerular hypercellularity, glomerular crescents, homeostatic, closed eye is in an inflammatory state, mesangial matrix deposition and interstitial and this investigation suggests that local inflammation, were comparable in 4 month old inflammatory markers may be upregulated in dry eye NZM.NCF2-/-, ~5.5 month old NZM.NCF2+/- and disease. This allows for novel biomarkers of study ~7.5 months old wild type NZM females. and may offer a potential site for intervention. Correspondingly, survival was shortened in NCF2 The authors thank Dr. Karen Ersland for her null mice (4~6 months), followed by the assistance with flow cytometry. haploinsufficient strain (6~10 months), as compared to NZM.NCF2+/+ mice (8~12 months). We 125 evaluated splenic immune cell populations and found that the appearance of hyperactive B cells Deficiency of NADPH Oxidase NCF2 Synergizes including GC-B cells and antibody producing with Other Lupus-Predisposing Genes to plasma cells, and T cells including activated T and Accelerate Lupus in NZM.2328 Mice CD69+ T cells was significantly accelerated in Dae-goon Yoo1, Ning Yu2, Lizet Perez-Zapata1, NZM.NCF2-/- mice. Moreover, increased type I IFN Emilia A. Barbu1, Jeanette T. Pingel1, Monica M. activity is spotlighted as a mediator in autoimmune Purmalek3, Mariana J. Kaplan3, Chaim O. Jacob2, disorders such as SLE. We demonstrated that a type Mary C. Dinauer1, 1Washington University in St. I IFN signature gene expression was significantly Louis; 2University of Southern California; 3National increased in NZM.NCF2-/- mice even in the pre- Institutes of Health disease state (7 wks age old females). Finally, neutrophil extracellular traps (NETs) have been Systemic Lupus Erythematosus (SLE) is an associated with SLE pathogenesis. Given the autoimmune disease characterized by extensive hypothesized role of NETosis as a potential mediator immune dysregulation and loss of tolerance to self to induce type I IFN signaling and inflammation nucleic acids, inducing chronic inflammation in and/or a role as autoantigens in SLE, we measured multiple organs. Variants in multiple genes involved NET formation. As expected, NCF2-null PMNs did in immune regulation can influence the development not release NETs following phorbol myristate of lupus. Recent genetic studies revealed that acetate stimulation. However, lupus serum missense variants in Neutrophil Cytosolic Factor 2 stimulated NETosis by NCF2-null neutrophils to a (NCF2) are associated with SLE. NCF2 is an similar extent as wild type NZM neutrophils, as essential subunit of the leukocyte NADPH oxidase studied in 7 wk old mice. In conclusion, deficiency that generates superoxide and derivative reactive of NCF2 and NADPH oxidase activity synergizes oxygen species (ROS), which have both with other lupus-predisposing genes in NZM mice to antimicrobial and immunomodulatory functions. accelerate immune dysregulation and the emergence Human SLE shows substantial variability in clinical, of lupus. This study complements the genetic studies genetic, disease status and medications, limiting the in human SLE that identified NCF2 variants as mechanistic studies on the impact of NCF2 linked to SLE, and establishes a new experimental mutations in human SLE leukocyte samples. Thus, system to dissect mechanisms for the role of to investigate the role of NCF2 in SLE, we generated NADPH oxidase-derived ROS in regulating NCF2 null mice on the C57BL/6 background and autoimmunity. backcrossed the mutation into the New Zealand Mixed (NZM).2328 spontaneous lupus model. 126 NZM.2328 mice are an inbred strain derived from NZB x NZW mice, and females develop SLE with Neutrophil Activation in Systemic Anaphylaxis: lupus nephritis by 7 – 8 months of age. NZM.NCF2- Results from the Multicentric NASA Study /- knockout mice lacked leukocyte NADPH oxidase Luc de Chaisemartin2,1, Friederike Jönsson3,4, activity whereas this was reduced in NZM.NCF2+/- Aurélie Gouel-Chéron3,4,9, Vanessa Granger2,1, Caitlin Gillis3,4, Neukirch Catherine5,10, Nicaise- was associated with anaphylaxis severity. Finally, Roland Pascale1, Fadia Dib6,7, Florence Tubach6,7, we show a significant decrease of neutrophil FcγR Dan Longrois9,8, Sylvie Chollet-Martin2,1, Pierre expression, pointing towards their engagement by Bruhns3,4, 1APHP, Hôpital Bichat, Immunology, immune complexes. This decrease correlated Paris, France; 2UMR996 - Inflammation, significantly with NET release and with the severity Chemokines and Immunopathology -, INSERM, Univ of the anaphylactic reaction. Together, our results Paris-Sud, Université Paris-Saclay, Chatenay- strongly suggest an activation of neutrophils by Malabry, France; 3Institut Pasteur, Département NBMA-IgG complexes during anaphylaxis. d'Immunologie, Unité des Anticorps en Thérapie et Conclusion Pathologie, Paris, France; 4INSERM, U 1222, Paris, We provide for the first time evidence for of an IgG- France ; 5APHP, Hôpital Bichat, Service dependent neutrophil activation pathway during Pneumologie A, Paris, France; 6APHP, Hôpital anaphylaxis in human. This additional mechanism Bichat, Département d'épidémiologie et de opens potential applications in anaphylaxis recherche clinique, Paris, France; 7UMR1123, diagnostics and treatment. ECEVE, INSERM, Université Paris Diderot, Sorbonne Paris Cité, Paris, France; 8Université 127 Paris Diderot, Sorbonne Paris Cité, Paris, France; 9APHP, Hôpital Bichat, Département d'Anesthésie- Neutrophils and Monocytes Induce a Synergistic Réanimation, Paris, France; 10UMR1152, INSERM, Increase of Blister Formation in a Human Skin Université Paris Diderot Paris 7, Paris, France Cryosection Model of Bullous Pemphigoid Elisabeth de Graauw1, Cassian Sitaru2, Luca Background Borradori3, Shida Yousefi1, Dagmar Simon1, 3 and Anaphylaxis is a severe systemic allergic reaction Hans-Uwe Simon1 , 1Institute of Pharmacology, that can be life-threatening. Evidence of the classical University of Bern, Switzerland, 2Department of anaphylaxis pathway, which involves IgE and IgE Dermatology, University Medical Center Freiburg, receptors, can be detected in most (85%) but not all Germany, 3Department of Dermatology, Inselspital cases. An alternative pathway involving IgG and Bern, Switzerland. neutrohil IgG receptors (FcγRs) has recently been suggested by animal models. We hypothesized that Background: Bullous pemphigoid (BP) is an such a mechanism may also exist in humans and autoimmune subepidermal blistering disease studied this possibility in a multicentric prospective characterized by tissue bound and circulating cohort of patients suspected of perioperative autoantibodies to the hemidesmosomal antigens anaphylaxis to neuromuscular blocking agents BP180 (BPAG2, type XVII collagen) and BP230 (NMBA). (BPAG1). Although neutrophils have been described Materials and Methods to mediate blister formation, the contribution of Consecutive patients suspected of perioperative other immune cells to neutrophil-dependent tissue anaphylaxis (n=86 cases) were recruited and paired damage in BP is unknown. with 86 control patients. Blood samples were Objective: To investigate whether neutrophils collected for cases and controls promptly after collaborate with other immune cells in the induction anesthesia induction. For cases, an extensive of BP autoantibody-dependent blister formation and allergological evaluation was performed 6-8 weeks the mechanisms involved. after the reaction. Circulating elastase, neutrophil Methods: In an ex vivo skin model, cryosections of extracellular traps (NETs), tryptase, histamine, and normal skin were incubated with purified human IgG and IgE anti-NMBA were measured by ELISA. neutrophils and peripheral blood mononuclear cells FcγR expression on the major blood cell populations (PBMCs) in the presence or absence of BP was analyzed by flow cytometry. autoantibodies. Reactive oxygen species (ROS) and Results granule protein release was inhibited by using We found higher circulating NETs and elastase chemical inhibitors of NADPH oxidase or serine levels during an anaphylactic reaction as compared proteases, myeloperoxidase or MMP-9 respectively. to controls. IgG anti-NMBA were found in both The dermal-epidermal separation (DES) was cases and controls, however for cases the IgG titer assessed by light microscopy and quantified by Fiji software. utilizing blood neutrophils obtained from WT and Results: Neutrophils synergised with monocytes to PAD4-/- mice we determined that the only induce enhanced DES in a BP autoantibody- neutrophil functional deficit in the PAD4-/- mice dependent manner. This synergistic effect on DES was the inability to make NETS; since neutrophils was inhibited by the NADPH-oxidase inhibitor from both groups were comparable in ex vivo diphenyleneiodonium (DPI). Blockage of MMP-9 motility, ROS production, and phagocytosis killing. resulted in decreased DES To examine the role of NETs in iALI we subjected Conclusion: Our study revealed a partnership PAD4-/- mice and WT mice to a “2 hit” model of between neutrophils and monocytes, resulting in traumatic shock (fixed-pressure hemorrhage; Hem) increased MMP-9 release, leading to an followed by septic (CLP) insult (Hem/CLP). Mice enhancement of BPS-dependent DES in a human BP were hemorrhaged, resuscitated, 24 hours post-Hem model. More studies are needed to elucidate the role mice were then subjected to CLP, and 24 h later lung of an immunological synapse between neutrophils tissue, blood, and bronchial lavage fluid (BAL) and monocytes, and the molecular pathways and samples were collected. To evaluate PMN targeting mechanisms involved, in their synergistic induction to the lung we looked at MIP-2, KC, and MPO of skin blister formation in BP. levels in lung tissue after Hem/CLP. PAD4-/- mice displayed an increase in MIP-2 levels compared to 128 WT mice while KC levels were comparably elevated in both WT and PAD4-/-. However, there was a Do NETs Matter? Establishing a Role for marked decrease in MPO in PAD4-/- lung tissue. To Neutrophil Extracellular Traps in Patho-Biology the extent that these changes were associated with of Indirect Acute Lung Injury local lung as opposed to systemic cytokine response Bethany Biron Girard1,2, Chun-Shiang Chung1,2, to iALI we noted that while, there were no Yaping Chen2, Jonathan Reichner1,2, Alfred Ayala1,2, differences in BAL IL-10 or TNF-α levels between 1Brown University; 2Rhode Island Hospital the two groups, PAD4-/- mice displayed a marked decrease in IL-6 levels in BAL after Hem/CLP. Acute lung injury (ALI) and acute respiratory PAD4-/- appears to have no significant effect on distress syndrome (ARDS) represent clinical indices of lung injury, as increased vascular syndromes of acute respiratory failure, with an permeability was noted in the lungs of both negative incidence rate between 80 and 60 per 100,000 per control and experimental mice. Conclusion: These year, respectively. Indirect ALI (iALI) is caused by a data taken together suggest that NET formation non pulmonary inflammatory process as a result appears to contribute to neutrophil influx as well as from insults such as non-pulmonary sepsis, the pro-inflammatory response within the lung, in hypotensive shock, etc. Neutrophils are thought to response to pathological processes regulating iALI have a significant role in mediating ALI, with the here development of iALI being characterized by dysregulation and recruitment of activated 129 neutrophils to the lung. Recently a novel mechanism of microbial killing by neutrophils was identified Neutrophils Contribute to Pathology but not through the formation of neutrophil extracellular Neovascularization following Ocular Herpes traps (NETs). NETs are comprised of large webs of Simplex Virus Type 1 Infection decondensed chromatin coated with granule proteins Daniel J. Carr, Christopher D. Conrady, Derek J. that are released from activated neutrophils into the Royer, Hem R. Gurung, University of Oklahoma extracellular space which is regulated by the enzyme Health Sciences Center PAD4 through mediating chromatin decondensation via citrullination of target histones. Components of Neutrophils - one of the “first responders” to ocular NETs including neutrophil derived circulating free herpes simplex virus type 1 (HSV-1) infection - DNA as well as circulating histones has been traffic to the cornea within the first 24 hr following implicated in ALI. However, it is unknown if there infection. Previous studies indicated depletion of Gr- is any pathological significance of NET formation in 1+ ‘neutrophils’ exacerbated virus replication and ALI caused by non-pulmonary insult. Firstly, spread. In addition, earlier studies reported neutrophils contributed to HSV-1-induced play a significant role in the neovascularization of neovascularization of the normally avascular cornea the mouse cornea following HSV-1 infection. through the production of pro-angiogenic factors and enzymes. We proposed to follow-up these reports by 130 other groups and more closely evaluate the role of neutrophils in ocular pathology in response to acute Tumor-Associated Neutrophils from a Bird’s Eye corneal HSV-1 infection. Initially using IFNAR1- View deficient (CD118-/-) and CCL2-deficient mice along Simon Sody1, Katrin Hahnel1, Matthias Gunzer2, with neutrophil-specific depleting antibody—Ly6G Sven Brandau1, 1Department of (1A8), we found inflammatory monocytes Otorhinolaryngology, University Hospital Essen, (F4/80+Gr-1+) but not neutrophils (F4/80– Gr-1+) University of Duisburg-Essen, Germany; 2Institute controlled HSV-1 infection within the first 48-72 hr for Experimental Immunology and Imaging, post ocular infection - as measured by flow University Hospital Essen, University Duisburg- cytometry and viral plaque assay. These results were Essen consistent with our findings that showed an increase in infectious virus recovered in the cornea of mast Tumor-associated neutrophils (TAN) were recently cell-deficient KitW-sh mice, which was associated identified as the most important prognostic leucocyte with a specific increase in neutrophil infiltration but subset associated with an adverse outcome over a not that of other myeloid populations. Enrichment of broad range of tumor diseases. However, the neutrophils from the corneas of infected mast cell- underlying pathophysiological mechanisms still deficient KitW-sh mice using immunomagnetic remain unclear and a dichotomy of pro- versus separation showed they possessed an elevation in the antitumoral functions is described in the literature. HSV-1 lytic gene, thymidine kinase, in comparison Insight into TAN function is yet restricted to to enriched neutrophils from wild type mice which classical histology or in vitro models. In this project we interpreted to suggest the cells were possibly a we combine the advances of intravital two-photon source of virus replication. Indeed, upon transfer of imaging with a novel transgenic mouse model with neutrophils from the cornea of infected mice into red fluorescent neutrophils (CatchupIVM) to naive CD118-/- mice resulted in significant mortality longitudinally explore the interaction of TAN and reinforcing the notion that neutrophils were a source tumors in the living mouse. Additionally the of HSV-1 replication and likely contributed to virus establishment of a tumor clearing protocol allows for dissemination. We further investigated the role of three-dimensional reconstruction of whole tumor neutrophils in corneal pathology by assessment of samples. With this approach we were able to monitor corneal neovascularization following HSV-1 neutrophil infiltration in newly established tumors infection of mice. Depletion of neutrophils as well as and characterize the migratory behavior of TAN in other leukocyte populations in the cornea using anti- the tumor environment. Ultimately, this project will Gr-1 (RB6-8C5) antibody had no significant provide novel insight into the complex biology of consequence on the genesis of corneal blood or TAN leading to innovative approaches for neutrophil lymphatic vessels in response to HSV-1 infection as targeting in the tumor host. measured by confocal microscopy. In contrast, the This work is supported by the Else Kröner- intraperitoneal administration of the potent anti- Fresenius-Stiftung. inflammatory steroid, dexamethasone, as a single bolus following ocular clearance of virus 131 dramatically reduced neovascularization without compromising the number of leukocytes, including Hypoxia Upregulates PI3Kinase-Dependent neutrophils, residing in the cornea. In summary, our Neutrophil Degranulation and Neutrophil- results show neutrophils are a detriment to the host Mediated Tissue Injury in response to ocular HSV-1 infection as they serve Katharine M. Lodge1, Kim Hoenderdos1, Wei Li1, as an additional source for virus replication and do Alison M. Condliffe2, Edwin R. Chilvers1, not contribute to viral surveillance during acute 1University of Cambridge, UK; 2University of infection of mice. However, in contrast to previously Sheffield, UK published data, our results suggest neutrophils do not Introduction: combination with GM-CSF/fMLP. Hypoxic Neutrophils are the first line of defence against neutrophil supernatants induced extensive HPAEC invading pathogens. However, damage to host tissue detachment and death, which was prevented by co- from persistent neutrophilic inflammation has been incubation with the anti-protease alpha-1 antitrypsin. implicated in the pathogenesis of many diseases, Neither normoxic nor hypoxic neutrophil including chronic obstructive pulmonary disease supernatants changed HPAEC expression of (COPD). Infected and inflamed tissues can be adhesion molecules ICAM-1, VCAM-1 or E- profoundly hypoxic; hypoxia promotes a destructive selectin. Silver stained protein bands from neutrophil phenotype with prolonged neutrophil precipitated neutrophil supernatants separated by survival and augmented degranulation of histotoxic SDS-PAGE, identified by mass spectrometry, proteases. Hence, hypoxic neutrophils have the suggested a hypoxic increase in damage associated potential for enhanced tissue damage. molecular pattern (DAMP) proteins S100A8 and Methods: S100A9 (cytoplasmic rather than granule- Neutrophils from healthy volunteers were isolated associated). However, when this was interrogated by by centrifugation over discontinuous plasma- ELISA, there was no significant difference between Percoll® gradients. Neutrophils were re-suspended the amount of S100A8, S100A9 or A8/A9 in IMDM under normoxia (21% O2) or hypoxia heterodimer in normoxic versus hypoxic (0.8% O2; Ruskinn SCI-tive dual hypoxia supernatants. workstation) for 4 hours, and subsequently treated Conclusion: with granulocyte-macrophage colony-stimulating Hypoxia augments neutrophil azurophilic granule factor (GM-CSF), platelet activating factor (PAF) or exocytosis in a priming agonist- and PI3Kinase-γ- tumour necrosis factor-α (TNFα), followed by dependent manner but NET production is not formylated peptide (fMLP). Neutrophil elastase increased. Hypoxic neutrophil supernatants have (NE) activity was measured by Enzchek® assay. enhanced capacity to damage endothelial cells in Western blotting for total and phosphorylated Akt vitro but do not affect endothelial adhesion molecule was performed on cell lysates. Neutrophil expression. Endothelial cell damage is likely due to extracellular trap (NET) production was assessed by increased release of neutrophil elastase and can be incubation of neutrophils with SYTOX Green and prevented by the addition of alpha-1 antitrypsin. The measurement of extracellular DNA by fluorescence contribution of S100A8 and S100A9 proteins to this absorbance. Neutrophil supernatants were incubated damage is currently unclear. Hence, hypoxia with primary human pulmonary artery endothelial promotes a destructive neutrophil phenotype with cells (HPAEC, Lonza) for 4, 24 or 48 hours. HPAEC enhanced capacity to damage pulmonary endothelial death and detachment were measured by MTT assay cells in vitro, with relevance to chronic and confocal microscopy; HPAEC activation was inflammatory diseases such as COPD. assessed by flow cytometric analysis of ICAM-1, VCAM-1 and E-selectin. TCA-precipitated 132 neutrophil supernatants were separated by SDS polyacrylamide gel electrophoresis (PAGE) and Rhesus Monkey Neutrophils are Hyper Reactive silver stained. S100A8, S100A9 and A8/A9 as Compared to Human and Marmoset Monkey heterodimer content of neutrophil supernatants was Neutrophils; the Rhesus Monkey Experimental assessed by ELISA. Autoimmune Encephalomyelitis (EAE) as a Results: Model for Acute Disseminated Encephalomyelitis Following exposure to hypoxia, neutrophils treated (ADEM). with GM-CSF or PAF, and fMLP displayed Krista G. Haanstra1, Nicole Heijmans1, Bert A. 't significantly greater NE release. TNFα treatment did Hart1,2, 1Department Immunobiology, Biomedical not increase NE release under hypoxia. Hypoxia also Primate Research Centre, Rijswijk, The augmented resting and cytokine-stimulated Akt Netherlands.; 2University of Groningen, Department phosphorylation; PI3Kinase-γ inhibition abrogated Neuroscience University Medical Center, Akt phosphorylation and prevented the hypoxic Groningen, The Netherlands uplift of NE release. There was no increase NET release in response to hypoxia alone or in The rhesus monkey experimental autoimmune 133 encephalomyelitis (EAE) model is characterized by an early onset after disease induction and rapid Functional and Metabolic Reprogramming deterioration. Attempts to alter the disease course in Drives the Development of a Pathogenic Subset of this species have been largely unsuccessful. EAE Neutrophils in Inflammatory Airway Diseases models in the other non-human primates, Osric A. Forrest1, Sarah Ingersoll1,2, Marcela cynomolgous and marmoset monkeys, have a less Preininger2, Julie Laval1,2, Dominique Limoli1,2, acute and destructive disease course. Milton Brown1,2, Frances Lee3, Branchetna Bedi3, In rhesus monkeys, EAE onset is preceded by a Ruxana Sadikot3, Joanna Goldberg1,2, Vin peripheral neutrophilia and lesions are characterized Tangpricha3, Rabindra Tirouvanziam1,2, 1Center for by perivenular lesions with a prominent influx of Cystic Fibrosis and Airways Disease Research, macrophages and neutrophils. This is in contrast to Children's Healthcare of Atlanta; 2Department of the lesions in MS patients and the EAE model in Pediatrics, Emory University School of Medicine; marmosets in particular, which are characterized by 3Department of Medicine, Emory University School T-cell infiltrates and macrophages. The pathology of Medicine and clinical presentation observed in the rhesus monkey EAE model is much more reminiscent of Airway inflammation in Cystic Fibrosis (CF), human acute disseminated encephalomyelitis Chronic Obstructive Pulmonary Disease (COPD), (ADEM), or even its most severe variants acute and severe asthma is in part characterized by the haemorrhagic massive recruitment of neutrophils (PMNs) into the leucoencephalitis/leukoencephalomyelitis lumen of the airways. In CF in particular, neutrophil (AHL/AHEM), two forms of acquired demyelinating elastase (NE) that is released from the primary syndromes mostly observed in children that have granules of PMNs is a strong predictor of lung been linked to viral infections and vaccination. function and survival. However, it is still not clear The involvement of neutrophils in the rhesus why and how long PMNs are able to survive in the monkey EAE model with the subsequent differences airways. Methods: Blood and sputum were in pathology and in contrast to EAE in marmosets or collected from CF, COPD, severe asthma, and HC cynomolgous monkeys points to a particular role of patients, and analyzed for viability, degranulation, neutrophils in this species. The prominent role of and other functional changes. In a transepithelial neutrophils in rhesus monkeys has also been migration assay, airway fluid from patients with CF, observed in other disease models, such as collagen- COPD, and severe asthma were used to recruit induced arthritis (CIA). PMNs and analyzed for survival, degranulation, Characterization of rhesus monkey neutrophils in metabolic activity, and bacterial killing capacity. vitro indicates that they have a higher tendency to Results: From these analyses we observed that (i) produce reactive oxygen species (ROS) as compared CF, COPD, and severe asthma airway fluid induces to humans or marmosets. rapid migration and survival of neutrophils, (ii) live The EAE and CIA models in rhesus monkeys have a PMNs release their primary granules and (iii) clear dependency on autoantibody induction, which migration towards CF airway fluid metabolic correlates with ADEM and the childhood version of reprograms PMNs to become increasingly glycolytic arthritis, juvenile idiopathic arthritis (JIA). Similar to while at the same time decreasing their ability to kill what has been described for systemic lupus P. aeruginosa. Finally, (iv) In our model system we erythematosus (SLE), we are currently studying the can utilize inhibitors of neutrophil migration role of Fc receptor mediated activation of (LTA4H inhibitor) that significantly reduces the neutrophils by disease specific autoimmune chronic influx of PMNs. Conclusions: These data antibodies. taken together suggest a primary role for the The rhesus monkey EAE model presents a unique inflammatory airway microenvironment in inducing opportunity to study neutrophil mediated increased survival, phenotypic and metabolic inflammation and may be useful for evaluation of reprogramming of PMN and identifies a pathogenic treatments aimed at the modulation of neutrophils in subset of neutrophils that can targeted general, and more specifically for ADEM or AHL. therapeutically in various inflammatory airway diseases. 134 aforementioned oxidant species in RA patients’ and healthy subjects’ neutrophils: it inhibited nearly 60% Galangin as Modulator of NADPH Oxidase and of total ROS production at 2.5 µM, 30% of MPO Myeloperoxidase Activity in Peripheral Blood activity / taurine chloramine production at 20 µM, Neutrophils from Patients with Rheumatoid and ~50% of NADPH oxidase activity / superoxide Arthritis anion production at 5 µM. Conclusion: Galangin Larissa F. Marchi1, Adriana B. Paoliello- acts as an effective inhibitor of IC-stimulated Paschoalato2, Lucineia R. Albiero2, Micassio F. neutrophil ROS generation by modulating the Andrade2, Rene D. Oliveira2, Ana Elisa S. Azzolini1, activities of NADPH oxidase and MPO, in RA Eduardo A. Donadi2, Yara M. Lucisano-Valim1, patients who are resistant to methotrexate therapy. 1School of Pharmaceutical Sciences of Ribeirao The use of this flavonol to modulate neutrophil Preto, University of Sao Paulo; 2Ribeirao Preto functions can be a promising therapeutic strategy to Medical School, University of Sao Paulo control inflammation in RA patients. Support: FAPESP, CNPq. Introduction and Objective: Rheumatoid arthritis (RA) is a chronic disease characterized by synovial 135 tissue inflammation, where neutrophils are the most abundant cells. These cells are important players in Neutrophils’ Influx and Propensity to Form tissue damage in RA, which involves the excessive NETs is Regulated by SLPI in Experimental production of reactive oxygen species (ROS) Model of Psoriasis triggered by immune complexes (IC) via Fcg and Monika Majchrzak-Górecka, Beata Grygier, Joanna complement receptors (FcgR and CR, respectively). Cichy, Dept. of Immunology, Faculty of NADPH oxidase (NADPHox) and myeloperoxidase Biochemistry, Biophysics and Biotechnology, (MPO) are the key enzymes that generate ROS in Jagiellonian University, Krakow, Poland activated neutrophils. In this sense, modulation of neutrophil ROS generation can help to maintain the Introduction tissue homeostasis in RA. Flavonols are natural Psoriasis is a chronic autoinflammatory skin compounds with promising anti-inflammatory and condition. As skin infiltrating neutrophils that often immunomodulating activity that have emerged as form epidermal microabscesses are a hallmark of selective modulators of the effector functions of psoriasis, it is likely that these cells play a role in the neutrophils. In this study, we examined whether disease. Neutrophils have been implicated in variety galangin modulates the neutrophil NADPH oxidase of autoimmune-like conditions, largely due to their and MPO activities in methotrexate-treated RA ability to form neutrophil extracellular traps (NETs). patients with active disease who do not respond very These structures can serve as a source of effectively to this drug therapy. Methods: Peripheral autoantigens as well as stimulate production of type blood was collected from healthy volunteers and RA I interferon (IFNI) in plasmacytoid dendritic cells patients, and neutrophils were isolated by the gelatin (pDC). The production of IFNI by pDCs is method. The total ROS production was measured by considered pivotal for the development of psoriasis, the luminol-enhanced chemiluminescence assay. The indicating that NETs may play a role at early stages NADPH oxidase activity was assessed by the of the disease. Neutrophil elastase (NE) – enzyme lucigenin-enhanced chemiluminescence assay, crucial for NET formation (NETosis) – is inhibited which detects the reaction product superoxide anion. by secretory leukocyte protease inhibitor (SLPI). The MPO activity was measured by a colorimetric Together, these data suggest that SLPI can impact assay using 3,3′,5,5′-tetramethylbenzidine, which the severity of this disease possibly by regulating detects the end-product taurine chloramine formed NETosis. by the reaction of HOCl with taurine. Results: The Methods levels of total ROS, superoxide anion, and taurine To investigate if/how neutrophils and SLPI chloramine production in RA patients’ neutrophils contribute to psoriasis we took advantage of were significantly higher than those detected in experimental model that closely resembles human healthy subjects’ neutrophils (p< 0.001). Galangin disease. The model is based on multiple applications equally suppressed the production of all the of imiquimod (TLR7-ligand)-containing cream (Aldara) on murine skin. C57Bl/6xSvj129 SLPI Neutrophil-derived microvesicles induces knock-out and wild type mice were treated twice a aggregation of bacteria and, hence, arrest the growth day for up to 6 days and analyzed daily for psoriasis- of bacteria. Neutrophils also deposit chemokine- like changes by macroscopic and microscopic containing extracellular vesicles known as trail, observation. Next SLPI+/+ and -/- animals were which can guide the migration of virus-specific injected intracuteneously with SLPI in PBS or PBS CD8+ T cells. Although these neutrophil-derived only and subsequently treated with Aldara for 2 extracellular vesicles have diverse functions ranging days. Samples of treated skin were analyzed by from the immune modulation to antimicrobial histology and immunofluorescence. activity, their specific characterization has not been Results fully understood. Here, we studied the differences in SLPI deficient mice exhibited increase in scaling and compositions and functions between neutrophil- expanded skin lesions which are indicative of more derived microvesicles and trails. We investigated the severe psoriasis. These changes were accompanied effects of different stimulants on microvesicles and by enhanced skin inflammation measured by the trails formation. Chemoattractants, inflammatory amount of infiltrating leucocytes in lesions. These cytokines, and bacteria induced the generation of included Ly6G+ granulocytes that were predominant microvesicles and trails from neutrophils. immune cells observed at early stages of skin Microvesicles and trails showed similar patterns of alteration. Interestingly, neutrophils from the early surface marker expression including influx showed increased propensity for NET- phosphatidylserine and MCP-1. Both mircovesicles formation in SLPI KO mice as compared to control and trails have direct bactericidal activity and mice. The ectopic delivery of recombinant SLPI induced chemotaxis of monocytes. However, alleviated skin inflammation and significantly microvesicle and trails have different effects on the reduced frequency of neutrophils producing NETs in phenotype polarization of macrophages. SLPI -/- animals. Additionally, neutrophil-derived extracellular Conclusions vesicles were also detected in the serum of healthy The differences in abundance and function of donors, and their number was significantly increased neutrophils and corresponding severity of psoriatic in the serum of septic patients. Together, our study symptoms are dependent on the presence of SLPI suggests the important insights into the which suggests that regulation of NETosis may play understanding the neutrophil-derived microvesicles. a role in the development of psoriasis. Together, these findings establish a protective role for SLPI 137 during the pathogenesis of experimental psoriasis. Acknowledgements Targeting Specific Phosphoinositide 3-Kinase This research was supported by NCN Maestro grant Isoforms Reduces Damaging Neutrophil 2011/02/A/NZ5/00337. Functions without Impairing Bacterial The Jagiellonian University is a partner of the Phagocytosis in Chronic Obstructive Pulmonary Leading National Research Center (KNOW) Disease supported by the Polish Ministry of Science and Georgia M. Walton1, Adam JA McGuinness1, Higher Education. Malcolm Begg2, Augustin J. Amour2, Edith M. Hessel2, Adam Usher1, James A. Stockley1, 136 Christopher Chadwick1, Matthew Pottle1, Hannah 1 1 1 Greenwood , Robert A. Stockley , Elizabeth Sapey Different Functions of Neutrophil-Derived 1University of Birmingham, UK; 2GlaxoSmithKline, Microvesicles and Trails UK Sanjeeb Shrestha1, Young-Jin Youn1,2, Jun-Kyu Kim2, Shin Kim3, Chang-Won Hong2, 1Hallym INTRODUCTION: Chronic Obstructive University; 2Kyungpook National University; Pulmonary Disease (COPD) is a chronic 3Keimyong University inflammatory lung condition, with systemic manifestations, and is the fourth leading cause of Neutrophils release different types of extracellular death worldwide. Neutrophils are centrally vesicles with diverse biological activities. implicated in COPD pathogenesis and their numbers, and products, correlate with clinical concentration, these compounds did not impair decline. Inaccurate neutrophil migration, increased opsonised or non-opsonised phagocytosis of degranulation and excess superoxide release have all Staphylococcus aureus. previously been described and may contribute to CONCLUSIONS: COPD neutrophils display a disease development and lung tissue damage. Less is functional phenotype favouring host damage that known about phagocytosis. There are currently no may contribute to disease pathogenesis. The disease-modifying treatments available for patients PI3Kgi/PI3di/SHIP1a restore these functions to with COPD and generic anti-inflammatory “healthy” levels, without impairing bacterial preparations do not prevent COPD progression. phagocytosis. Their use in COPD may reduce the There is great interest in modifying disease-specific potential for neutrophil-related host damage while targets to improve outcomes and phosphoinositide 3 maintaining bacterial clearance. This further kinase (PI3K) has been identified as a potential supports their development as potential therapies. target. Delta and Gamma isoforms of class I PI3K are of particular interest, as these are central to 138 accurate migration. We wished to assess if a PI3Kdelta selective inhibitor (PI3Kdi), PI3Kgamma Peptidomimetics as novel formyl peptide receptor selective inhibitor (PI3Kgi) or SHIP1 activator 2 (FPR2) modulators in human and mouse (SHIP1a) could improve neutrophil migration in neutrophils COPD without inhibiting other cell functions André Holdfeldt1, Claes Dahlgren1, Huamei important for bacterial clearance. Forsman1,Sarah Line Skovbakke2, Henrik Franzyk2 METHODS: Peripheral blood neutrophils were 1Department of Rheumatology and Inflammation isolated from 70 COPD patients and 70 age matched Research, Sahlgrenska Academy at the University of controls (aged 39 – 80 years). Functional assays Gothenburg, Sweden, 2Department of Drug Design were also conducted with PI3Kgi, PI3Kdi, SHIPa or and Pharmacology, Faculty of Health and Medical vehicle control (DMSO). Neutrophil migration was Sciences, University of Copenhagen. assessed using an Insall chamber towards CXCL8, CXCL1, LTB4 and fMLP. Surface expression of Formyl peptide receptor 2 (FPR2) is a CD63 (a measure of primary granule release) and chemoattractant receptor belonging to the family of phagocytosis of opsonised and non-opsonised G-protein coupled receptors (GPCRs). FPR2 is pHrodo-conjugated Staphylococcus aureus, or alexa- abundantly expressed in neutrophils and have a fluor488 labelled Streptococcus pneumoniae and major regulatory role in the inflammatory processes Haemophilus influnzae was assessed by flow involving these cells, indicating that modulators of cytometry. Extracellular reactive oxygen species FPR2 have therapeutic potential. However, most (ROS) production was studied using an isoluminol molecules that interact with the receptor are peptides assay in basal conditions and following stimulation that display low bioavailability and low affinity for with fMLP. the mouse counterpart (Fpr2), which render them RESULTS: Neutrophil migration in COPD inadequate for animal studies. Peptidomimetics are remained faster but less accurate than healthy peptide-like molecules that have unnatural residues controls across all age groups. COPD neutrophils incorporated in their backbones, thus making them demonstrated systemic cell activation (evidenced by proteolytically stable and attractive as novel increased basal cell polarization), and increased modulators for both FPR2 and Fpr2. Isolated human degranulation (evidenced by increased CD63 peripheral blood neutrophils and bone marrow- expression). This cell phenotype could not be derived mouse neutrophils were used in this study. A replicated by exposing “healthy” neutrophils to library of lipidated α-peptide/β-peptoid hybrid COPD plasma. Phagocytosis of all bacterial species peptidomimetics was screened for the ability to: (i) was preserved in COPD but total extracellular activate/inhibit the NADPH-oxidase in human and reactive oxygen species production was increased. mouse neutrophils, (ii) induce an intracellular The PI3K delta and gamma isoform selective calcium transient in HL-60 cells transfected to inhibitors and SHIP1-activator restored neutrophil overexpress FPR2 or FPR1. Through the screening migratory accuracy and reduced total extracellular process, peptidomimetics that activates NADPH- reactive oxygen species production. At the same oxidase activity in both human and mouse neutrophils were identified. The lipid headgroup was loop of FPR2 specifically activate this receptor. In found to be critical for the agonistic activity. The this study, we have determined the effects (rise in activities of the compounds were sensitive to FPR2 intracellular Ca2+ and NADPH-oxidase activity) of antagonists and the receptor specificity was pepducins generated from some other GPCRs confirmed in the cells overexpressing either of the expressed in neutrophils. two FPRs. Results: A pepducin with an amino acid sequence In conclusion, a novel class of lipidated α-peptide/β- derived from the third intracellular loop of FPR2 peptoid hybrid peptidomimetics was found to (F2Pal) specifically activates this receptor and the activate both human and mouse neutrophils. The signaling cascade initiated was similar to the one preferred receptor was human FPR2 and the mouse triggered by a classical FPR2 agonist. A pepducin orthologue Fpr2. This novel class of with a peptide sequence identical to the third peptidomimetics is expected be an excellent tool in intracellular loop of FPR1 (F1Pal), however, mouse models of human diseases as they enable inhibited neutrophil function and unexpectedly the experiments that will increase our understanding of inhibition was found to be selective for FPR2 but not the role of FPR2 in the complex processes of FPR1. Also pepducins with amino acid sequences inflammation, and they might constitute the basis for corresponding to the second and third intracellular the development of therapeutic drugs that loops of P2Y2R activated neutrophils. But the specifically target FPR2. receptor desensitization profile and the effects of receptor specific antagonists suggest the 139 involvement of FPR2 rather than P2Y2R. Similarly to FPR1 and P2Y2R pepducins, a pepducin derived Pepducins Designed to Target Their Cognate G- from the first intracellular loop of CXCR4 (ATI- Protein Coupled Receptors FPR1, P2Y2R And, 2341) targeted FPR2 to activate neutrophils. CXCR4 Allosterically Modulate the Function of Conclusions: In summary, our data demonstrate that FPR2 pepducins designed to target FPR1, P2Y2R, and Michael Gabl, Andre Holdfeldt, Malene Winther, CXCR4 constitute a novel class of modulators for Claes Dahlgren, Huamei Forsman, The Phagocyte FPR2. These findings are in contrast to the generally Research Laboratory, Department of Rheumatology accepted dogma for the mode of action of pepducins & Inflammation Research, University of and raise the possibilities of FPR2 being a pattern Gothenburg, Sweden recognition receptor for lipopeptide structures, as well as a possible “off-target” receptor for pepducins Background: Pepducins are a class of lipopeptides designed to allosterically modulate other receptors. with a short peptide sequence identical to one of the intracellular domains of a G-protein coupled 140 receptor (GPCR) that act as allosteric GPCR activators or inhibitors. Their activities are An Emerging Oral Pathogen, Peptoanaerobacter transduced following interaction with a receptor Stomatis, Promotes Degranulation and Primes distinct from the orthosteric binding site used by the Respiratory Burst Response in Human conventional ligands. The physio-chemical Neutrophils. properties of a pepducin (i.e. fatty acid chain, charge Silvia M. Uriarte1, Emeri Jimenez-Flores1, Maria V. and hydrophobicity) suggest a “flipping” of the Sizova2, Slava S. Epstein3, Richard J. Lamont1, molecule through the cell membrane and an 1University of Louisville; 2Evelo Biosciences; interaction directly with the cytosolic parts of the 3Northeastern University cognate receptor. Human neutrophils express many GPCRs important in immune regulation including Peptoanaerobacter stomatis is a newly appreciated P2Y2R, the receptor for the “danger signal” ATP, the species associated with chronic inflammatory chemoattractant receptors CXCR4, as well as the periodontal disease. However, little is known about pattern recognition receptor FPR1 and the closely this organism’s potential virulence and its interaction related receptor FPR2. Using the pepducin approach, with the host immune system. Therefore, to better we have earlier shown that pepducins with amino understand the role of P. stomatis in periodontitis, acid sequences derived from the third intracellular we studied its interactions with human neutrophils. P. stomatis strain CM2 was used to challenge School of Medicine, Kyungpook National University, neutrophils at a multiplicity of infection of 10. Daegu, 41944, Republic of Korea.; 4Lung Research Bacterial internalization was examined by imaging Institute of Hallym University, College of Medicine, flow cytometry and bacterial viability was assessed Hallym University, Seoul, 05355, Republic of Korea; by colony-forming unit assays. Phagocytosis- 5Division of Pulmonary, Allergy and Critical Care stimulated respiratory burst response was measured Medicine, Department of Internal Medicine, Hallym by flow cytometry, and superoxide release by the University Sacred Heart Hospital, Anyang, 14068, reduction of ferricytochrome c. Exocytosis of Republic of Korea.; 6Division of Pulmonary, Allergy secretory vesicles, specific granules and azurophil and Critical Care Medicine, Department of Internal granules was determined using flow cytometry to Medicine, Chuncheon Sacred Heart Hospital, measure the expression of CD35, CD66b and CD63, Chuncheon, 24252, Republic of Korea.; 7Division of respectively. Exocytosis of gelatinase granules was Pulmonary, Allergy and Critical Care Medicine, determined by ELISA for gelatinase (MMP9) Department of Internal Medicine, Kangdong Sacred release. After 30 min of incubation, only 25% of the Heart Hospital, Seoul, 05355, Republic of Korea.; P. stomatis associated with neutrophils in suspension 8Department of Pulmonary and Critical Care were internalized. Approximately 43% of the Medicine, Asan Medical Center, University of Ulsan bacterial inoculum was killed within a 30 min College of Medicine, Seoul, Republic of Korea. interaction with neutrophils. P. stomatis induced a robust intracellular respiratory burst response Excessive or dysregulated functions of neutrophils compared to S. aureus. Minimal superoxide release are considered responsible for the pathogenesis of was observed by the bacterial challenge after 5-30 sepsis. To understand the function of neutrophil min, however P. stomatis significantly increased during sepsis, we hypothesized that neutrophils from fMLF-stimulated superoxide release to a similar septic patients are either primed or easily stimulated, extent as cells primed with TNFα. P. stomatis and autophagy is responsible for the activation of induced significant exocytosis of secretory vesicles, neutrophils. To investigate this, we isolated gelatinase granules, specific granules, and azurophil neutrophils from community acquired pneumonia- granules. These results suggest that although induced septic patients, and investigated the neutrophils have a low phagocytic efficiency for P. functions of neutrophils. Neutrophils were isolated stomatis; robust intracellular ROS production from septic patients who have admitted to the nonetheless occurs, and exocytosis of the four intensive care units, and the morphology, the neutrophil granules is induced. The release of expression of surface phenotypic markers, the reactive oxygen intermediates and matrix generation of reactive oxygen species (ROS), metalloproteinases such as MMP9 from neutrophil neutrophil extracellular traps (NETs) formation, granules could contribute to tissue damage in granule release, and autophagy were examined. periodontal disease. Neutrophils isolated from septic patients showed increased vacuolization with decreased mean lobe 141 counts and showed several changes in phenotypic markers. Further, sepsis neutrophils have increased Autophagy Primes Neutrophils for Neutrophil NETs formation and degranulation markers in Extracellular Trap Formation during Sepsis response to stimulation. We also found that So Young Park1, Sanjeeb Shrestha2, Shin-Yeong autophagy is responsible for the priming effect of Kim2, Young-Jin Youn2, Jun-Kyu Kim3, Ki-Suck sepsis neutrophils. Moreover, sepsis neutrophils Jung4,5, Myung Goo Lee4,6, Yong Bum Park4,7, Eun from non-survivor showed impairment in autophagy Kyung Mo4,7, Yousang Ko4,7, Suh-Young Lee4,6, with decreased NETs formation. In murine sepsis Younsuck Koh8, Myung Jae Park2, Dong-Keun model, the enhancement of neutrophil autophagy Song2, Chang-Won Hong3, 1Department of improved survival via increased NETs formation. Pulmonary and Critical Care Medicine, KyungHee Together, our study suggests an important insights University Medical Center, Seoul, 20447, Republic into role of autophagy in neutrophils during sepsis. of Korea.; 2Department of Pharmacology, College of Medicine, Hallym University, Chuncheon, 24252, Republic of Korea.; 3Department of Physiology, 142 143

Cross-Talk of Dendritic Cells with Neutrophils Neutrophil Expression of B7-H6 and HLA – that have been Activated with Immunoglobulin A Implications on Interactions between NK Cells Annelot Breedveld, Reina Mebius, Marjolein van and Neutrophils in Inflammation Egmond Elin Bernson1, Karin Christenson1,2, Malin Pasanen1, VU Medical Center Amsterdam Rebecca Riise1, Firoozeh Amirbeagi3, Johan Bylund2, Fredrik Bergh Thorén1 Introduction 1TIMM Laboratory, Sahlgrenska Cancer Center, Immunoglobulin A (IgA) is the most prevalent University of Gothenburg, Sweden; 2Department of antibody at mucosal sites and a potent stimulus of Oral Microbiology and Immunology, Institute of neutrophils (PMN) via the IgA Fc receptor (FcαRI). Odontology, University of Gothenburg, Sweden; In patients with ulcerative colitis, mucosal 3Department of Rheumatology and Inflammation infiltration of PMNs as well as interaction with Research, University of Gothenburg, Sweden dendritic cells (DCs) is seen. We hypothesize that mucosal pathology and immune responses are As neutrophils contain toxic content and degrading influenced by FcαRI induced PMN activation. The enzymes, they constitute a potential danger to the aim of this study is to investigate crosstalk of IgA surrounding tissue. A silent neutrophil death, i.e., activated PMN with DCs. apoptosis, is therefore of importance for termination Methods of an inflammatory process. We have previously Retinoic acid stimulated DCs (RA-DCs) and demonstrated that Natural Killer (NK) cells can monocyte-derived DCs (MoDCs) were derived from induce apoptosis in healthy neutrophils via the monocytes after being cultured for 6 days in the activating NK cell receptor NKp46 and in a Fas- presence of IL-4 and GM-CSF, with or without dependent manner. retinoic acid (RA) respectively. Aldehyde NK cell cytotoxicity is regulated by array of dehydrogenase activity was measured using the activating and inhibitory receptors. The major ALDEFLUOR kit. Fresh isolated PMNs inhibitory receptors are the killer immunoglobulin- phagocytosed IgA coated latex beads (IgA PMN) like receptors (KIRs), which bind to corresponding after which they were co-cultured with DCs for 16h. HLA ligands, while the activating receptors include Subsequent, maturation marker expression on DCs the natural cytotoxicity receptors (NCRs). B7-H6 was assessed. Cell-cell interactions were analyzed has been identified as a ligand to the activating NCR with live cell microscopy and cytokine production in NKp30, and it was recently reported that neutrophils supernatants of co-cultures was measured. display B7-H6 on their surface after pro- Results inflammatory stimulation and that B7-H6 exists in a RA-DCs differed in morphology, expressed more soluble form in inflammatory conditions. CD103, and had higher aldehyde dehydrogenase In this study we investigated how the human activity compared to MoDCs. RA-DCs expressed neutrophil surface expression of B7-H6 and HLA less CD1c, CD80, CD83, CD86 and HLA-DR, and was regulated during inflammatory conditions and more CD141 and CD103 after co-culture with IgA how the relative expression of these ligands affected PMN compared to MoDCs. Cell interactions and the outcome of NK cell interactions with bead transfer between IgA PMN and DCs were seen neutrophils. in live cell microscopy. IL-12 was produced by We found B7-H6 to be expressed already on the MoDCs after co-culture with IgA PMN, but not by surface of resting, circulating neutrophils. RA-DCs. Furthermore, an additional pool of pre-formed B7- Conclusions H6 was localized in easily mobilized neutrophil Both MoDCs and RA-DCs interacted with IgA granules. When we monitored the expression of B7- PMN, and bead transfer from PMNs to DCs was H6 on the surface of neutrophils over time in observed. However, after co-culture with IgA PMN, response to inflammatory stimuli, we found the RA-DCs did not produce IL-12, suggesting expression of B7-H6 to be highly dynamic. Further, induction of immune tolerance. our in vitro studies demonstrated that the surface

expression of HLA was rapidly decreased upon Hodgkin lymphoma (NHL) remains unclear. neutrophil activation. Methods: The frequency and immunophenotype of These in vitro observations were confirmed in a CD66b+CD33dimHLA-DR- cells were analyzed in human in vivo skin chamber model where PBMCs from HL and NHL patients (n=124) and transmigrated neutrophils demonstrated decreased healthy donors (n=48). The immunosuppressive levels of HLA on their cell surface. Also the surface functions of these cells were tested in vitro. expression of B7-H6 was altered and we could Correlations between CD66b+CD33dimHLA-DR- detect soluble B7-H6 in exudates of the skin cells and patient clinicopathological features and chambers. In an ex vivo cytotoxicity assay, we outcome, were evaluated. observed that the transmigrated neutrophils from the Results: CD66b+CD33dimHLA-DR- cells were skin chambers were significantly more sensitive to increased in PBMCs from HL and NHL patients as NK cell-mediated cytotoxicity, supporting our compared to healthy donors. Their frequency hypothesis that altered expression of NK cell remained significantly higher even considering HL receptor ligands on inflammatory neutrophils (n=31), indolent (n=31) and aggressive (n=62) NHL determines their susceptibility to NK cell-induced patients separately. CD66b+CD33dimHLA-DR- death. cells in patient PBMCs were mostly composed of Collectively, our data shed light on the complex mature CD11b+CD16+ low-density neutrophils in regulation of interactions between NK cells and an activated status as compared to conventionally neutrophils during an inflammatory response and isolated autologous or healthy donor neutrophils. suggest a role for NK cells in the resolution phase of The in vitro depletion of CD66b+ cells from patient inflammation. PBMCs restored the proliferation of autologous T cells. Higher frequencies of CD66b+CD33dimHLA- 144 DR- G-MDSCs correlated with unfavorable prognostic index scores and a significantly shorter Identification of Granulocytic Myeloid-Derived freedom from disease progression. Suppressor Cells (G-MDSCs) in the Peripheral Discussion: Novel areas of research for lymphoma Blood of Hodgkin and Non-Hodgkin Lymphoma patients include improving the efficacy of adoptive Patients cellular therapies, modulating T regulatory cells, Olivia Marini1,2, Cecilia Spina1, Elda Mimiola1, developing novel lymphoma vaccine and enhancing Adriana Cassaro1, Giovanni Malerba3, Giuseppe tumor-specific innate immune response. In this Todeschini1, Omar Perbellini1, Maria Scupoli1,4, context, our study demonstrate that in HL and B-cell Giuseppe Carli1, Davide Facchinelli1, Marco A. NHL patients a population of functionally defined Cassatella2, Patrizia Scapini2, Cristina Tecchio1, G-MDSCs mainly composed of mature activated 1Department of Medicine, Section of Hematology LDNs is present and its generation seems to be more and Bone Marrow Transplant Unit, University of related to the biological aggressiveness of the Verona, Verona, Italy; 2Department of Medicine, disease than to its size. Section of General Pathology, University of Verona, Conclusions: Our findings disclose a previously Verona, Italy; 3Department of Neurological, unknown G-MDSC-mediated mechanism of Biomedical and Movement Sciences, Section of immune-escape in lymphomas, therefore anticipating Biology and Genetics, University of Verona, Verona, possible targets for therapeutic interventions. Further Italy; 4Department of Inderdepartmental Laboratory studies are awaited in order to establish whether for Medical Research (LURM), University of CD66b+CD33dimHLA-DR- G-MDSCs could be Verona, Verona, Italy inhibited or reprogrammed for therapeutic purposes. Bibliography: Marvel D., Gabrilovich D.I. Purpose: Human granulocytic myeloid-derived Myeloid-derived suppressor cells in the tumor suppressor cells (G-MDSCs) have been described as microenvironment: expect the unexpected. J Clin low-density immunosuppressive Invest. 2015;125(9):3356-3364. CD66b+CD33dimHLA-DR- granulocytes that co- doi:10.1172/JCI80005. purify with mononuclear cells after density gradient centrifugation of blood from cancer patients. The role of G-MDSCs in Hodgkin (HL) and non- 145 146

Role of Neutrophils in an Imiquimod-Induced Is There a Role for Neutrophils in the Mouse Model of Psoriasis Adjuvanticity of Alum? Dalila Bevilacqua1, Sara Costa1, Elena Caveggion1, Manuel Reithofer, Dominika Polak, Claudia Marco Cassatella1, Giampiero Girolomoni2, Patrizia Kitzmüller, Barbara Bohle, Beatrice Jahn-Schmid Scapini1, 1Università di Verona, Dipartimento di Medical University of Vienna Medicina; 2Università di Verona, Sezione di Dermatologia e Venerologia Insoluble aluminum salts (alum) are still the most widely used adjuvants in humans. The actual Psoriasis is a chronic immune-mediated skin disease mechanism of the adjuvanticity of alum is still not associated with deregulated interplays between fully understood. In vivo studies are limited to immune cells and keratinocytes. Despite the fact that animals and in the human system only its effect on a prominent skin infiltration of neutrophils is a monocytes and dendritic cells has been assessed. distinctive hallmark feature of psoriatic Recently, it has been shown in mice that host- inflammation, the role of neutrophils in psoriasis derived DNA, which could not be assigned to a pathogenesis remains unclear. Aim of this study was single cell type, is involved in the adjuvant effect of to investigate the specific contribution of neutrophils alum. Neutrophils are the most abundant circulating during psoriatic inflammation. white blood cells in humans, the first cell type to Methods. Psoriasis was induced by topical enter sites of tissue damage or inflammation and can application of AldaraTM, 5% Imiquimod (IMQ) produce so-called NETs, i.e. “neutrophil cream in B6 mice treated or not with anti-LY6G extra­cellular traps” upon different stimuli (e.g. (1A8) antibody to deplete neutrophils. Disease bacteria, fungi, immune complexes, C3a). These development was evaluated by flow cytometry and NETs consist of decondensed nuclear chromatin gene expression analysis of draining lymph nodes (DNA, histones) or mitochondrial DNA forming and skin biopsies, as well as by histological web-like filaments and are decorated with evaluation of skin inflammation. antimicrobial granular material such as Results: Epidermal thickening, together with the myeloperoxidase, elastase or LL37, which are expression of several inflammatory cytokines (i.e. expelled during an induced death process called IL-22, IL-17, CXCL1 and IL-23) and psoriatic “NETosis”. In addition to their obvious associated genes (i.e. Lipocalin 2 and S100A7), was antimicrobial activity, components of NETs are significantly increased in the skin of neutrophil meanwhile also discussed as host derived danger depleted mice in response to IMQ treatment. signals (DAMPs) in the initiation of innate immune Interestingly, IMQ-treated neutrophil depleted mice responses and may play an important role for the manifested also a significantly increased recruitment adjuvant effect of alum vaccines. of activated IL-17-producing ɣδT cells in the The aim of this study is to investigate, whether alum draining lymph nodes. In line with the latter induces NETosis in human neutrophils and hereby observation, we demonstrated that neutrophils may influence the initiation of immune responses to inhibited ɣδT cell proliferation and IL-17 production alum-containing vaccines. in vitro. First, freshly isolated human neutrophils were Conclusion:Overall, these data demonstrate that stimulated with alum to induce a possible NET- neutrophils may negatively contribute to disease response and PMA or ionomycin were used as propagation and exacerbation in the IMQ-induced positive controls. Strong NET-formation was mouse model of psoriasis by impairing ɣδT cell induced in neutrophils by all three stimuli when effector functions. visualized by co-staining of extracellular DNA and different granular proteins using fluorescence/confocal microscopy. In addition, the enzymatic activity of neutrophil elastase in culture supernatants indicated alum-induced NETosis. Regarding the mode of action, the production of cellular reactive oxygen species (ROS) were analysed by flow cytometry or plate reader assays. the cell wall of fungi. Co-ligation of CR3 with Fn at Prominent cytosolic (c)ROS production was only the I-domain and β-glucan at the lectin-like domain observed with PMA, as expected, whereas alum and is possible due to spatially distinct locations and ionomycin induced significant production of generates unique cellular responses not seen with mitochondrial (m)ROS. Using a plate-reader method ligation of either domain separately. In the setting of to quantify DNA-release over time, for alum and immobilized β-glucan and Fn, CR3 exhibits immune ionomycin a similar time course (namely much responses not seen when exposed to either substrate faster than with PMA) was observed and application alone correlating to in vivo fungal infection. Recent of different ROS-inhibitors or EDTA revealed a work in our lab demonstrates in response to Fn + β- significant dependence on mROS and extracellular glucan, as well as Candida hyphae in the context of Ca++ for both stimuli. Inhibition of NET-release Fn, neutrophils form homotypic aggregates and with cytochalasin D indicated phagocytosis as initial rapid NET release. step for alum-induced NETosis, which in aqueous In this study, we further characterized the solution is present in colloid particles, as also chemotactic signals underlying neutrophil evaluated by microscopy. Similarily, amine- or homotypic aggregation. Moreover, while the key carbonate-modified latex beads (3 µm) were surface receptors have been identified, less is known phagocytosed by neutrophils and induced NETs, about the intracellular signaling pathways whereas uncharged beads did not. First co-cultures downstream of these receptors in the context of of NET-forming neutrophils and monocyte-derived neutrophil aggregation. We additionally interrogated dendritic cells (mdDC) showed a decrease in LPS- the role of a variety of signaling proteins that were or alum-induced upregulation of CD83 and co- identified via mass-spectrometry as having stimulatory molecules of these mdDC. differential tyrosine phosphorylation in neutrophils In summary, alum is capable of potently inducing clustered on surfaces coated with Fn and β-glucan, NETosis in human neutrophils. This response is including extracellular signal-regulated kinase dependent on phagocytosis (most probably (ERK), mitogen-activated protein kinase kinase 1 facilitated by its charged nature), mROS production (MEK1), phosphatidylinositol-4,5-bisphosphate 3- and extracellular Ca++. While Alum directly kinase (PI3K), spleen tyrosine kinase (Syk), activates mdDC, co-cultured neutrophils producing glycogen synthase kinase 3 β (GSK3β), and protein NETs may rather inhibit mdDC activation. kinase C (PKC). The long term goal is to determine differential stimulation of intracellular signaling 147 pathways as a function of CR3 domain occupancy. Understanding the signaling mechanisms underlying Signaling Pathways Mediating the Human chemoataxis, aggregation, and NETosis in the Neutrophil Response to Fungal ß-Glucan neutrophil anti-fungal response may provide Hadley Witt1,2,3, Xian M. O'Brien1,2, Courtney M. therapeutic targets which may benefit to candidiasis Johnson1,2,3, Allan Huang2, Jonathan S. Reichner1,2,3, patients in the future by reducing harmful neutrophil 1Division of Surgical Research, Department of behaviors and/or stimulating behaviors crucial to Surgery, Rhode Island Hospital; 2Warren Alpert fungal clearance. Medical School, Brown University; 3Graduate Program in Pathobiology, Brown University 148

Neutrophils are the critical immune cell necessary STAT Factors Control the Expression of Certain for eliminating invasive fungal infections from the Genes in Human Neutrophils, Yet without Much tissue and recognize fungi via CR3, a β2 subfamily Impact on Functional Responses integrin involved in phagocytosis. CR3 is a unique Thomas Z. Mayer, Patrick P. McDonald, Pulmonary receptor with two spatially distinct binding domains, Division, Faculty of Medicine, Université de the I-domain and the lectin-like site. The I-domain Sherbrooke and CR-CHUS, Sherbrooke, Québec binds number of ligands including iC3b, ICAM, (Canada). fibrinogen, and fibronectin (Fn). The lectin-like site binds polysaccharides, including β-glucan, a Inflammation and immunity are crucially impacted pathogen associated molecular pattern found within by cytokines and chemokines produced by neutrophils. We have previously shown that this 149 response is tightly regulated by transcription factors NF-κB, C/EBP and CREB in response to The Role of Hemorrhage-Primed Neutrophils in physiological stimuli. The STAT family of Altering Endothelial Cell Tie1/Tie2 Expression in transcription factors is also thought to control the Development of Indirect Acute Respiratory cytokine production in neutrophils, as they do in Distress Syndrome other leucocytes. Supporting this idea is the fact that Jiali Zhu1,2, Alfred Ayala1, Joanne Lomas-Neira1 several STAT proteins are phosphorylated on both 1Rhode Island Hospital/Brown University, tyrosine residues in response to neutrophil stimuli Providence, RI; 2Changhai Hospital, Secondary such as IFNγ, GM-CSF, or G-CSF, that are known Military Medical University, Shanghai, China to induce responses such as cytokine production, delayed apoptosis, and expression of surface Severe hemorrhage/hemorrhagic shock(Hem) has receptors. However, a direct link between STAT been shown to be causal for the development of activation and these functional responses remains to extra-pulmonary/indirect (i)ARDS and is associated be demonstrated. with severe endothelial cell (EC) injury. EC growth Using pharmacological inhibitors selective for factors, Angiopoietin (Ang)-1 & 2, maintain STAT1/3 or for STAT3/5, and DNA microarray vascular homeostasis via tightly regulated analysis, we identified numerous genes that are competitive interaction for the tyrosine kinase controlled by STATs in response to IFNγ, GM-CSF, receptor, Tie2, expressed on ECs. Ang-1/Tie2 or G-CSF. Among them were genes encoding binding leads to Tie2 phosphorylation (pTie2) and cytokines or chemokines (IL-1b, CXCL1, CXCL10), signaling for downstream anti-inflammatory and surface receptors (CD64, PD-L1, TLR5, IL-18R), pro-survival phenotype, whereas Ang-2, stored pre- and SOCS proteins (CIS, SOCS1, SOCS3). From a formed and released for activated ECs, can act as an more functional standpoint, cytokine/chemokine apparent antagonist. We have shown Ang-2 secretion was largely unaffected by STAT inhibitors, significantly elevated in our murine acute respiratory or by siRNAs directed against STAT1 or STAT3. distress syndrome (ARDS) model of hemorrhage Likewise, the increase in CD64 membrane (priming) followed by secondary septic challenge, expression induced by IFNγ or G-CSF was not and additionally, that neutrophil (PMN) interaction impaired in presence of STAT inhibitors or siRNAs. with resident pulmonary vascular ECs contributes Finally, neutrophil apoptosis is strongly delayed by significantly to Ang-2 release and mediates Ang-2- IFNγ, G-CSF, or GM-CSF, but STAT inhibition associated pulmonary EC dysfunction central to the only hindered the survival effect of IFNγ, while that development of indirect ARDS. The orphan of G-CSF or GM-CSF was unaffected. Together, receptor, Tie1, has recently been described as these results indicate that STAT proteins play but a contributing additional level of regulation to marginal role in neutrophil responses, despite Ang/Tie2 signialing. While neither Ang-1 nor Ang-2 controlling the expression of several genes involved bind Tie1, Tie1 complexing with Tie2, has been in said responses. In the particular case of shown to inhibit Ang-1/Tie2 interactions, thus CIS/SOCS, while their inducible gene expression promoting an activated, pro-inflammatory EC was found to be under STAT control, the phenotype. However, Whether Hem alters Tie1/Tie2 corresponding proteins were not detected, in keeping expression and interactions leading to the with other studies. Thus, the actual outcome of primed/activated EC phenotype, and further, how the STAT-controlled SOCS expression is difficult to Hem primed PMN contribute to this altered assess. Studies are in progress to clarify the issue. In expression, is not known. To begin to investigate summary, while STAT factors definitely govern the this, we tested the hypothesis that Tie1/Tie2 expression of several target genes in neutrophils, this expression is altered following Hem, and this change seems to have little or no effect on functional is a result of EC interaction with activated (shock responses in these cells. primed) PMN. A murine model of hemorrhagic shock–induced priming for the development of iARDS after subsequent septic challenge was used in this study. To assess the contribution of Hem-primed PMN to changes in Tie1/Tie2 expression, mice were depleted intravital confocal microscopy, TNFα also promotes of peripheral blood PMN via intra-peritoneal (i.p.) the intraluminal crawling of all intravasated injection 500 ug of rat anti-mouse PMN antibody, neutrophils along the lymphatic endothelium. anti-Gr1(clone RB6–8C5, rat IgG2b)/mouse, 48 Additionally, TNFα-induced inflammation results in hours before Hem. Mice were euthanized at 6 and 24 ICAM-1 upregulation on lymphatic vessels. Taken hours following Hem. Tie1, Tie2 and together, our results demonstrate for the first time a phosphorylated Tie2 expression in lung critical role for TNFα in priming neutrophils to homogenates were assessed using commercial migrate into the lymphatic vessels in vivo, through ELISA kits and Western blot. the upregulation of ICAM-1 and CCR7 signalling. Our findings show that 1) p-Tie2 is decreased at 6h, but is restored by 24h after hemorrhagic shock; 2) This work is supported by Arthritis Research UK Tie1 expression is elevated at both 6h and 24h after (19913) and Queen Mary Principal's Research hemorrhagic shock; 3) Depletion of neutrophil Studentship. significantly increases p-Tie2 and decreases Tie1 expression. These findings support our hypothesis 151 and suggest 1) that Tie1 plays a role in mediating EC dysfunction potentially by prolonging EC activation Deficiency in Toll-Interacting Protein (Tollip) by decreasing Tie2 availability for Ang-1 binding Skews Inflamed yet Incompetent Innate and, 2) that Hem-primed PMN contributes to the Leukocytes in Vivo during DSS-Induced Septic altered Tie2/Tie1 expression. (Funded by Colitis NIH.5P20GM103652 [JLN] &R35GM118097[AA]) Yao Zhang1, Na Diao2, Liwu Li1, 1Department of Biological Sciences, Virginia Tech; 2Department of 150 Gastroenterology, Nanfang Hospital, Southern Medical University, China TNFα Promotes CCR7-Dependent Migration of Neutrophils and Their Subsequent Crawling into Functionally compromised neutrophils contribute to the Lymphatic Vessels in Vivo adverse clinical outcomes in patients with severe Samantha Arokiasamy, Wen Wang, Sussan inflammation and injury such as colitis and sepsis. Nourshargh, Mathieu-Benoit Voisin, Queen Mary However, the ontogeny of dysfunctional neutrophil University of London during septic colitis remain poorly understood. We herein report that the dysfunctional neutrophil may Neutrophils through their diverse immune functions be derived by the suppression of Toll-interacting- play an essential role in innate immunity against protein (Tollip). We found that Tollip deficient mice invading pathogens and during inflammatory were more susceptible to DSS induced acute colon processes. Interestingly, these leukocytes have been injury. Tollip deficient mice displayed serious rectal shown recently to be capable to shape the adaptive bleeding, quicker weight loss, higher mortality, and immune responses following immunisation and shorter colon length significantly as compared to during pathogen infections; however, the mechanism wild type mice. Further studies showed that Tollip of neutrophil trafficking to the lymphatic system is deficient neutrophils had compromised migratory still unclear. In the present study, we have used the capacity, as well as reduced potential to generate murine model of cremasteric inflammation to bacterial-killing neutrophil extra-cellular trap (NET), decipher some of the molecular mechanisms of and compromised bacterial killing activity. Together, neutrophil trafficking into the tissue-associated our data reveal a novel mechanism in Tollip lymphatic vessels. Here we show that in vivo alteration that underlies the inflamed and stimulation of the tissues with TNFα induces the incompetent polarization of neutrophils leading to migration of neutrophils into cremasteric lymphatic severe outcomes of colitis. capillaries in a time-dependent manner. This response is strictly CCR7-dependent as the neutrophil trafficking into lymphatic vessels is suppressed in CCR7KO animals (~97% inhibition as compared to WT mice). Furthermore, using 152 proteolytic processing of IL-1β. T. gondii inhibited LPS-induced caspase-1 cleavage by 83%. Together, Toxoplasma gondii down Regulates Il-1ß these results show that T. gondii limits the Production by Human Neutrophils in the production of LPS-induced IL-1β in human Presence of Lipopolysaccharide neutrophils and that this effect is associated with the Tatiane Soares de Lima, Lanny Gov, Melissa down regulation of IL-1β and NLRP3 at the Lodoen, University of California Irvine transcript level and a reduction in NF-κB activation. Moreover, T. gondii inhibited the cleavage and During acute infection with the intracellular parasite, activation of the inflammasome protease caspase-1. Toxoplasma gondii, monocytes and neutrophils are Collectively, these results describe a new strategy of among the first immune cells recruited to the site of immune evasion in which T. gondii down regulates infection. These cells can be infected by T. gondii, the immune response of human neutrophils, but, how neutrophils, in particular, respond to this potentially facilitating the survival and spread of this pathogen is poorly understood. Since IL-1β is a key parasite during acute infection. regulator of innate immunity, we investigated the production of this cytokine during T. gondii 153 infection. Human peripheral blood monocytes or neutrophils were infected with T. gondii (MOI 2) Long Noncoding RNA MEG3-4 Tissue and after 16 hours, the secretion of IL-1β was Specifically Regulates IL-1β during Pulmonary measured by ELISA. Unlike monocytes, which Bacterial Infection by Repressing MicoRNA-138 produce IL-1β in response to T. gondii infection, the Min Wu, Rongpeng Li, Department of Biomedical infection of neutrophils did not induce IL-1β release. Sciences, School of Medicine and Human Health, Interestingly, in neutrophils, T. gondii infection also University of North Dakota, USA inhibited LPS-induced IL-1β by 62%. This inhibition was observed when the stimulation with Long noncoding RNAs (lncRNAs) modulate various LPS and the infection were simultaneous or when biological processes; however, their function in host LPS was added 3 hours before infection. To test if immunity response against infection remains elusive. IL-1β inhibition required active parasite invasion, Here, we identify an intergenic lncRNA MEG3 neutrophils were cultured with parasites that were (linc-MEG3) as a tissue specific regulator for heat killed, treated with DMSO (vehicle control) or pulmonary immunity during Gram-negative bacterial pretreated with mycalolide B (5 µM), an irreversible infection. Among the 10 transcripts of linc-MEG3, actin polymerization inhibitor, which prevents transcripts 1 and 4 are main regulators as being parasite invasion. Heat killed and mycalolide B- discovered the most downregulated in mouse lungs treated parasites did not limit IL-1β production, after bacterial infection, but with insignificant indicating that this effect required active parasite alterations in other organs. Overexpression of linc- invasion. Since LPS activates NF-κB, which is MEG3-4 in mice led to exacerbated inflammatory involved in transcriptional activation of pro-IL-1β response, severe lung injury, systemic dissemination and the inflammasome sensor NLRP3, a possible and ultimately mouse death. Alveolar epithelial cells effect of T. gondii on the transcription of these two and alveolar macrophages (AM) were the major cell genes was investigated by qPCR. T. gondii reduced types that are targeted by linc-MEG3-4. Due to its 3’ the transcription of IL-1β by 95% and NLRP3 by sequences complementary to microRNA-138 (miR- 75% in the presence of LPS. Furthermore, since LPS 138), linc-MEG3-4 competitively binds miR-138 typically activates NF-κB through the canonical and thereby freeing its target IL-1b mRNA to pathway, we investigated if T. gondii inhibition of intensify inflammatory responses. Our results IL-1β and NLRP3 transcription was related to an characterize linc-MEG3 as a novel pulmonary effect on NF-κB activation. In T. gondii-infected inflammatory regulator of bacterial infection through neutrophils stimulated with LPS, phosphorylation of a miR-138/IL-1b/NF-kB pathway, indicating that p65 was reduced by 60%, compared to LPS lincRNAs may be critical for coordination of treatment alone, indicating that the parasite prevents complex immunity and inflammation in response to NF-κB activation. Finally, we investigated the effect infection. of T. gondii on caspase-1, a key mediator of *This project was supported by Flight Attendant for HIV-1 replication and persistence in Medical Research Institute (FAMRI, Grant macrophages. #103007), NIH AI101973-01, AI109317-01A1, and AI097532-01A1. 155

154 Signaling Pathways in Neutrophils are Regulated by Surgical Trauma - a Proteomics Study USP18 is Required for HIV-1 Infection in a Samina Arshid3,1, Muhammad Tahir1,2, Belchor Human iPSC-Derived Macrophage Infection Fontes3, Edna F.S. Montero3, Mariana S. Castro1, Model Simone Sidoli2,4, Peter Roepstorff2, Wagner Fontes1 Jared P. Taylor1,2, Daniel L. Aldridge1,2,3, Melanie N. 1Laboratory of Biochemistry and Protein Chemistry, Cash1,2, Katherine E. Santostefano1,2, Lucas H. Department of Cell Biology, Institute of Biology, Armitage1,2, Nao Terada1,2, Mark A. Wallet1,2, University of Brasilia, Brasília-DF, Brazil.; 1University of Florida; 2Department of Pathology, 2Department of Biochemistry and Molecular Immunology & Laboratory Medicine; 3HHMI Biology, University of Southern Denmark, 5230 Science for Life Odense M, Denmark.; 3Laboratory of Surgical Physiopathology (LIM-62), Faculty of Medicine, The type I interferon [T1-IFN] pathway plays a dual University of São Paulo, Brazil.; 4Epigenetics role in viral defense by inducing expression of Program, Department of Biochemistry and restriction factors to limit viral replication, while Biophysics, Perelman School of Medicine, simultaneously increasing expression of host factors University of Pennsylvania, Philadelphia, PA 19104, involved in antigen processing and presentation. USA Although the innate antiviral response can potently restrict virus replication in nearly all cell types, in Neutrophils are important players in the some antigen presenting cells like macrophages, it is development of the systemic inflammatory response important for viruses to replicate sufficiently that after trauma. To understand in more detail the antigen is present for priming cell-mediated adaptive neutrophil activation at early stages after trauma, in immunity. For HIV-1, however, this defensive this study neutrophils were isolated from rats strategy is not effective for eradicating the virus. divided in two groups: control and surgical trauma. Recruitment of CD4+ T cells actually provides Rats from the surgical trauma group were submitted additional target cells for the virus and macrophages to laparotomy, eviscerated and kept hydrated for themselves become persistent virus producing cells. 120min, after what the abdominal wall was closed CD4+ T cells are highly susceptible to the cytopathic and blood was collected. The control group was effects of HIV-1 while macrophages are relatively anesthetized and observed for the same period. resistant to virus-induced cell death pathways. To Isolated neutrophils were lysed and the extracted understand the mechanisms of survival of HIV-1- proteins were analyzed using nano liquid infected macrophages, we performed whole chromatography coupled to tandem mass transcriptome profiling of HIV-1-infected cells and spectrometry. We identified a total of 2924 rat found that ubiquitin specific proteinase 18 [USP18] neutrophil proteins in our analysis, of which 393 was highly upregulated in infected cells. were found differentially regulated between control USP18 is a negative regulator of T1-IFN signaling and surgical trauma groups. Functional pathways through the Jak/STAT pathway. We hypothesized analysis revealed transcription initiation and protein that USP18 expression inhibits the antiviral response biosynthesis to be enriched containing several of the normally initiated through the T1-IFN signaling 190 proteins up-regulated in surgical trauma. On the pathway allowing for macrophage survival and HIV- other hand, among the 203 proteins down-regulated 1 replication. Indeed knockout of USP18 in iPSC- in surgical trauma we found enrichment for proteins derived macrophages results in marked reduction of of the immune response, proteasome degradation HIV-1 replication. USP18 deficiency also makes the and actin cytoskeleton (Fig.1). Moreover, we cells hypersensitive to stimulation with T1-IFNs and performed enzyme prediction analysis, that revealed other pattern recognition receptor ligands. Together regulated enzymes involved in neutrophil apoptosis, our data suggest that USP18 expression is essential directional migration and chemotaxis. Our observations were confirmed by in silico protein- p38 MAPK, Erk-1/2, PI3K/Akt, Hck and Syk were protein interaction analysis. targets of A2AR activities, while Lyn, SHIP-1 or Collectively, our results reveal that neutrophils PTEN phosphorylation levels were spared. drastically regulate their biochemical pathways after Prostaglandin E2, or a mixture of the compounds RO the early stages of surgical trauma, showing lower 20-1724 (inhibitor of phosphodiesterase IV) and activity. This implies higher susceptibility of the forskolin (activator of adenylate cyclase) largely trauma patients to infection and bystander tissues mimicked the effect of CGS 21680. We used LC- damage. MS/MS for the global survey of the neutrophil phosphoproteome, mainly occurring on serine and threonine residues. Stimulation of the cells solicited the MAPK, PIP3/Akt, Rho GTPases, NF-kB, cytokine signaling, inflammasomes and mTOR pathways. Activated A2AR positively impacted proteins involved in functions of the Golgi, nitric oxide, and sumoylation of proteins. In contrast, ERK/MAPK targets, the AP-1 family of transcription factors and DNA replication processes, were down-regulated. In resting cells, A2AR engagement down-regulated phosphorylation events linked with receptor signaling pathways, transcription and glucose uptake processes. Results

unveal a number of intracellular signaling pathways Figure 1: Protein interaction diagram showing the targeted by the A R, several of which might be key proteinsdownregulatedin surgical trauma, 2A in the regulation of neutrophils functions during highlighting proteins involved in significantly inflammation.Proteomics data are available via enriched pathways. ProteomeXchange with identifier PXD004172.

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Early Tyrosine Phosphorylation and Identifying Novel Immune Modulating Factors in Comparative Proteomics Provide New Insights a Genome-Wide S. aureus Screen in Human into Adenosine A Receptor Activities in Human 2A Neutrophils Neutrophils Dingyi Yang1, John Connolly2, Simon Foster2, Marc Pouliot, Miriam S. Giambelluca, Florence Lynne Prince1, 1Department of Infection, Immunity Roux-Dalvai, Frédéric Fournier, Arnaud Droit, & Cardiovascular Disease, Medical school, the Benjamin Nehmé, Université Laval University of Sheffield, Sheffield, UK; ; 2Department

of Molecular Biology and Biotechnology, the Adenosine, through activation of the adenosine A 2A University of Sheffield, Sheffield, UK (A R) receptor, elevates intracellular levels of 2A cyclic AMP and in this way acts as a physiological Rationale: inhibitor of inflammatory neutrophil functions. In Staphylococcus aureus is a highly adaptive and the present study, we looked into the impact of A R 2A widespread bacterial pathogen causing numerous engagement on early phosphorylation events. clinical problems due to antibiotic resistance. Recent Neutrophils were stimulated for 2 min at 37˚C with human-pathogen interaction studies reveal S. aureus well-characterized, pro-inflammatory agonists, in uses multiple evasion mechanisms to survive or even absence or presence of the A R agonist, CGS 2A replicate within neutrophils, the primary cellular 21680. As assessed by immunoblotting, several defence against this pathogen. In addition, S. aureus protein bands were tyrosine phosphorylated; CGS induces rapid and profound neutrophil necrosis, 21680 markedly decreased tyrosine phosphorylation which further disables the immune response. Our levels of four regions (37-45 kDa, 50-55 kDa, 60 study aims to identify novel immune modulators kDa and 70 kDa) and key-signaling protein kinases, through the screening of a genome-wide S. aureus studied, mainly because neutrophils are seen as mutant library in neutrophil cell death assay. short-lived effectors cells. Multiple knockout models Methodology: have demonstrated the importance of the The 1,952 strain mutant library was constructed by transcription factor (TF) PU.1 in the differentiation transposon insertion in the clinically relevant of myeloid cells. Indeed, this pioneer TF, that acts as community acquired methicillin-resistant S. aureus a master transcriptional regulator, initiates (USA300) background. Individual S. aureus strains nucleosome remodelling followed by histone were co-incubated with primary human neutrophils modification depositions at large numbers of isolated from healthy volunteers, at an MOI of 10 for genomic regions. These changes in the chromatin 3 hours before ToPro-3 staining and assessment of landscape serve as beacons for the recruitment of cell loss via flow cytometry. additional factors, which ultimately drive both cell- Findings: specific gene expression and signal-dependent Wild type USA300 caused profound neutrophil cell responses. loss by 3 hours (≥75%). Quantification of ToPro-3 Herein, by performing chromatin negativity and viable cell counts identified 34 mutant immunoprecipitation (ChIP) assays followed by strains that showed attenuated neutrophil cell death. high-throughput sequencing in freshly isolated A number of internal controls with known pro-death human neutrophils and monocytes, we globally functions including lukAB, lukGH, agrA and saeS mapped the genomic distribution of the enhancer were among the identified attenuated strains. Four mark histone H3 lysine 4 monomethylation gene mutations not previously associated with cell (H3K4me1) and the master regulator PU.1. We death: purB, lspA, clpP and pfo were also among the found that the numbers of PU.1-bound genomic sites most highly-attenuated strains and have been further were 63461 in neutrophils and 49041 in monocytes, validated by transduction strategies, and current whereas the H3K4me1 peaks were 54269 in studies to verify them by gene complementation are neutrophils and 63661 in monocytes. While most of underway. These findings may identify novel the PU.1 peaks that are localized in promoter- mechanisms of S. aureus induced neutrophil cell proximal regions showed similar occupancy levels in death, which may aid in the design of future both neutrophils and monocytes, cell-specific PU.1 antibiotic-independent therapeutic strategies to recruitment was predominantly located at enhancer- restore failures of innate immunity. distal regions in both types of phagocytes. Analysis of the genomic distribution of H3K4me1 within a 158 definite region of approximately 3000 bp from the centre of enhancer-distal PU.1 peaks showed a Genome-Wide Localization of PU.1 and typical bimodal distribution. In addition, we found H3K4me1 in Human Neutrophils and Monocytes that PU.1 recruitment at cell-specific enhancer sites Francisco MA Bianchetto-Aguilera1, Nicola strictly correlates with distal cell-specific H3K4me1 Tamassia1, Maili Zimmermann1, Gioacchino Natoli2, also in neutrophils. Moreover, by performing Marco A. Cassatella1, 1Department of Medicine, microarray gene expression profiling, we identified University of Verona; 2Department of Experimental either common or cell-specific transcripts in freshly Oncology, European Institute of Oncology (IEO) isolated neutrophils and monocytes. Finally, by integrating the transcriptomic and epigenetic Neutrophils and monocytes are the two most signatures, we observed a stringent correlation represented population of phagocytes in the human between cell-specific transcript expression and bloodstream. Neutrophils are integrated in the H3K4me1 and PU.1 deposition at specific enhancer activation, regulation and effector mechanisms of the regions of the related genes. Altogether, these innate and adaptive immune systems. Beyond their findings suggest that histone modifications and PU.1 well-known role in acute inflammatory responses recruitment at cell-specific genomic regions are and resistance to extracellular pathogens, neutrophils likely responsible of the differences observed at the also function as major players of diverse pathologies transcriptional level between neutrophils and via the release of de novo formed mediators. monocytes. However, the mechanisms that in neutrophils control transcription of mediators are still very poorly 159 species was increased in these normal subjects. Funded by NCI Contract No. HHSN261200800001E Analysis of NCF1 in Patients with P47phox Deficient Chronic Granulomatous Disease and 160 Normal Subjects by Droplet Digital PCR Douglas B. Kuhns1, Xiaolin Wu2, Amy P. Hsu3, Neutrophil and Monocyte Survival in Vivo and in David Sun2, Paul Griffith2, Steven M. Holland3, Vitro Depends on an Additive Cytoprotective Harry L. Malech4, John I. Gallin4, 1Neutrophil Shield by Serpinb1a and Serpinb6a against Monitoring Laboratory, Applied/Development Granule Proteases Research Directorate, Leidos Biomedical Research, Sabrina Sofia Burgener1,2, Melanie Brügger1, Phillip Inc., Frederick National Laboratory for Cancer Ian Bird3, Charaf Benarafa1, 1Theodor Kocher Research, Frederick, MD; 2Genomics Laboratory, Institute, University of Bern, Bern, Switzerland; Cancer Research Technology Program, Leidos 2Graduate School for Cellular and Biomedical Biomedical Research, Inc., Frederick National Science, University of Bern, Bern, Switzerland; Laboratory for Cancer Research, Frederick, MD; 3Department of Biochemistry & Molecular Biology, 33Laboratory of Clinical Infections, NIAID, NIH, Monash University, Clayton, Australia Bethesda, MD ; 44Laboratory of Host Defenses, NIAID, NIH, Bethesda, MD Background: Serine protease inhibitors, serpins, are highly conserved proteins regulating many Mutations in gene NCF1 (encoding protein p47phox of physiological processes. While most serpins are the NADPH oxidase) result in an autosomal secreted into the plasma, the clade B serpins control recessive form of chronic granulomatous disease proteases intracellularly in the cytoplasm. We have (CGD), a rare genetic disease with impaired previously shown that Serpinb1a is essential in phagocyte production of reactive oxygen species and regulating a cell intrinsic cathepsin G-dependent recurrent infections, granulomatous complications, neutrophil death in steady state and in infection and premature death. Identification of the specific studies leading to impaired bacterial clearance. Mice genetic defect in patients with p47phox CGD is lacking Serpinb1a were previously shown to have complicated by the presence of two highly conserved 50% less neutrophils in the bone marrow. SerpinB6, (>98%) pseudogenes. The NCF1 gene has a GTGT a related clade B serpin expressed in myeloid cells, at the start of exon 2 while the pseudogenes (NCF1B also inhibits cathepsin G. Yet, lack of SerpinB6 and NCF1C) delete one GT (ΔGT). Unequal leads to a recessive inherited stimulus-induced crossing-over events between the wild-type gene and deafness in humans and mice but the role of the pseudogenes have been suggested to account for SerpinB6 in monocytes and neutrophils remains the majority of mutations in p47phox deficient CGD. unknown. Due to the sequence identity between the wild type Results: First, we generated double knock-out mice gene and pseudogenes, standard Sanger sequencing by crossing Serpinb1a-/- mice with mice lacking has proven to be inadequate to assign a specific Serpinb6a, the mouse homolog of SerpinB6. genetic mutation in these patients. Using droplet Serpinb1a.Serpinb6a-/-mice showed a more severe digital polymerase chain reaction (ddPCR), we neutropenia in the bone marrow compared to the developed an assay to differentiate between ΔGT Serpinb1a-/- mice. Strikingly, Serpinb1a.Serpinb6a-/- and other mutations in NCF1. Using the ratio of mice also presented reduced blood neutrophil as well intact, GTGT alleles to total number of NCF1 as blood and bone marrow monocyte percentages, alleles, this assay can accurately determine carrier which were not observed in Serpinb1a-/- mice. status of relatives within the p47phoxCGD families. Accordingly, Serpinb1a.Serpinb6a-/- neutrophils and Unexpectedly, analysis of normal subjects revealed monocytes had a considerably higher kinetic of cell that a significant proportion (14%) exhibited >2 death induced by granule permeabilization in vitro. alleles containing the GTGT, while 17 subjects Caspase inhibition did not rescue neutrophil death in exhibited 3 alleles and 4 subjects (of 151 normal cells lacking serpins. In contrast, cathepsin G subjects tested) exhibited 4 alleles. Despite increased deletion fully rescued granule permeabilization- numbers of GTGT alleles, neither p47phox protein induced cell death in neutrophils, but not in expression nor the production of reactive oxygen monocytes. Conclusion: Our results demonstrate that Serpinb1a Tissue injury arising from trauma, surgery or and Serpinb6a cumulatively provide a cytoprotective ischemia elicits a prompt inflammatory response. A shield in neutrophils and monocytes in steady state primary goal of the inflammatory response is to and after granule leakage by inhibiting the serine eliminate exogenous pathogens and to promote the protease cathepsin G, which induces death clearance of dead cells. Timely resolution of the independently of caspases. inflammatory phase enables the transition from inflammation to tissue repair, whereas chronic 161 unresolved inflammation perturbs tissue repair. Resolvins are lipid mediators generated by Neutrophil Differentiation and Functions after leukocytes (neutrophils and macrophages) during the Modulation of DDR Pathway resolution phase of inflammation. Here, we show Oleg N. Demidov1,2, Anastasia R. Goloudina2, that resolvins promote the transition from Bogdan Grigorash1, Burhan Uyanik3, Frédéric inflammation to tissue repair and enhance tissue LIRUSSI3, Ettore Appella4, 1Institute of Cytology, revascularization following ischemia. In mice RAS; 2Inserm U866; 3University of Burgundy; 4NCI, undergoing hind limb ischemia (HLI) induced by NIH femoral artery ligation, we identified resolvin D2 (RvD2) in bone marrow and skeletal muscle by mass The main function of genes involved in DNA spectrometry. We also identified RvD2 in skeletal damage pathway is regulation of cellular response to muscle biopsies from humans with peripheral artery genotoxic stress. Modulation of expression and/or disease. Monocytes were recruited to skeletal muscle activity of DNA damage response (DDR) genes in during HLI and isolated monocytes produced RvD2 leukocytes and their progenitors affects not only in a lipoxygenase-dependent manner. Using laser DDR but also other functions of neutrophils such as Doppler perfusion imaging, we found that differentiation, proliferation and pro-inflammatory exogenous RvD2 enhanced perfusion recovery in functions. Deregulation of expression and activity of HLI and micro-computed tomography (microCT) of wild type p53-induced phosphatase in mouse and limb vasculature revealed greater volume, with human neutrophils led to their stabilization in in evidence of tortuous arterioles indicative of vitro culture, promoted neutrophil differentiation and arteriogenesis. Unlike other treatment strategies for proliferation and changed the repertoire of secreted therapeutic revascularization that exacerbate cytokines. Using genetic deletion or chemical inflammation, RvD2 did not increase vascular inhibition in deregulated neutrophils, we permeability, but reduced neutrophil accumulation manipulated p38MAPK, tp53 and Stats pathways to and the plasma levels of TNF-a and GM-CSF, while determine dependency of observed neutrophil promoting a tissue reparative monocyte/macrophage phenotypes from indicated pathways. The results phenotype. In mice treated with RvD2, obtained in in vitro experiments were confirmed by histopathological analysis of skeletal muscle of manipulation with indicated above pathways in in ischemic limbs showed more regenerating myocytes vivo mouse models of several human inflammatory with centrally located nuclei and mice deficient in and oncological pathologies. RvD2 receptor, Gpr18, had an endogenous defect in The novel strategies in treatment of human diseases perfusion recovery following HLI. Thus, RvD2 based on our findings were proposed. stimulates arteriogenic revascularization during HLI The work was supported by Grant #14-15-00636, suggesting that resolvins may be a novel class of Russian Scientific Fund mediators that both resolve inflammation and promote arteriogenesis. 162

Inflammation-Resolving Lipid Mediator, Resolvin D2, Promotes Revascularization and Tissue Regeneration during Limb Ischemia Matthew Spite, Harvard Medical School and Brigham and Women's Hospital, Boston, MA

163 compounds that robustly increased neutrophil apoptosis ≥2 fold and have greatest specificity for Protein Kinase Inhibitor Compound Screen their kinase targets. Epidermal Growth Factor Reveals ErbB Family Kinases as Regulators of Receptor family (ErbB) was identified as a frequent Neutrophil Apoptosis in the Context of target of the identified compounds. Inhibition of Inflammatory Diseases ErbB1 and ErbB2 with selective small molecule Atiqur Rahman1, Katherine Henry2, David Sammut1, inhibitors, Erbstatin analog and Tyrphostin AG825 A. A. Roger Thompson1, Bill Zuercher3, Moira respectively, accelerated spontaneous apoptosis in Whyte4, David Dockrell1, Stephen Renshaw1,2, neutrophils from healthy subjects and COPD Lynne Prince1, 1Department of Infection, Immunity patients, and also reversed GMCSF-, LPS-, hypoxia- and Cardiovascular Disease, University of Sheffield, and cAMP-mediated neutrophil survival. Tyrphostin Sheffield, UK; 2Bateson Centre, University of AG825 was also able to reduce GMCSF induced Sheffield, UK; 3SGC-UNC, Division of Chemical AKT-phosphorylation, suggesting that ErbB2- Biology and Medicinal Chemistry, UNC Eshelman mediated neutrophil survival signals may be School of Pharmacy, University of North Carolina at transduced via the PI3-AKT pathway. In an in vivo Chapel Hill, USA; 4MRC centre for Inflammation zebrafish tail transection model, Tyrphostin AG825 Research, University of Edinburgh, UK significantly reduced neutrophils at the site of inflammation at 4 and 8 hours post injury. Rational and Hypothesis: Tyrphostin AG825 also significantly increased BAL Persistent neutrophilic inflammation is observed in a neutrophil apoptosis and alveolar macrophage number of inflammatory conditions, including efferocytosis of apoptotic neutrophils in an acute chronic obstructive pulmonary disease (COPD). lung injury mouse model. Prolonged neutrophil survival in the lung is known Conclusion: to contribute to COPD pathophysiology. Uncovering We are hopeful that our findings may identify the mechanisms underpinning pro-survival clinically targetable protein kinase dependent phenotypes may allow development of new and pathways that will allow the development of novel effective therapies to treat the cause of inflammation therapeutic strategies for chronic inflammatory in COPD. Protein kinase cascades play pivotal roles diseases. in diverse cellular functions including cell proliferation and apoptosis, and kinase inhibitors are 164 used as pharmacologic drugs in multiple pathologies. Here, we screened a protein kinase Atypical Molecular Pattern Exposed by inhibitor library to identify therapeutically targetable Peripheral Blood Neutrophils Treated with novel neutrophil apoptosis pathway(s). Sphingolipid Analog Drug FTY720 Methods: Joanna Skrzeczynska-Moncznik, Malgorzata Neutrophils were isolated from peripheral blood of Bzowska, Krzysztof Guzik healthy subjects and COPD patients by Percoll Department of Immunology, Faculty of gradient centrifugation. A protein kinase inhibitor Biochemistry, Biophysics and Biotechnology, library consisting of 298 compounds that have been Jagiellonian University, Cracow, Poland fully profiled against 224 human protein kinases, was screened in neutrophil apoptosis assays. Recently we have described an atypical cell death Neutrophil apoptosis was assessed by light induced in human neutrophils by sphingolipid microscopy and flow cytometry. Inhibitors were analog drug FTY720 (fingolimod). The neutrophil tested in mpx:GFP zebrafish tail injury model of death is characterised by rapid translocation of inflammation by counting GFP positive neutrophils HSP27 to the cell surface and externalization of at the site of inflammation. LPS nebulized C57BL/6 phosphatidylserine (PS) and annexin I mice were injected intraperitoneally with the kinase (Skrzeczynska-Moncznik et all. 2015, J Leuc Biol). inhibitor Tyrphostin AG825, and bronchoalveolar In continuation we studied the engulfment of the lavage (BAL) was performed at 48 hours. FTY720-treated neutrophils by macrophages. The Results: FTY720-treated neutrophils were extensively Screening and validation experiments identified 12 phagocytized by monocyte-derived macrophages although the exposure of PS or annexin I did not macrophage receptors. This process is called correlate with the clearance. Interestingly, inhibitors “efferocytosis”. Macrophage engulfment of the of HSP90 (geldanamycin, 17-DMAG, radicicol) apoptotic material leads to the induction of a reduced the externalization of PS, moreover some downstream signaling in which efferocytic features of FTY720-induced neutrophil death were macrophages acquire a resolving phenotype by regulated by albumin or sphingolipids. releasing TGFb1 or IL-10 and by further Paradoxically, the decrease of the established 'eat upregulation of phagocytic receptors and bridging me' signals was accompanied by a substantial molecules. Resolving macrophages are central to increase in the neutrophils clearance. The resolution of inflammation (RoI), which promotes engulfment of the FTY720-treated neutrophils by restoration of tissue homeostasis. macrophages was mediated by scavenger receptors We have previously described Developmental and triggered cytokine production. Cumulatively, endothelial locus-1 (Del-1) as an endogenous our data strongly argue that the clearance of negative regulator of leukocyte adhesion by blocking FTY720-treated neutrophils by macrophages is not the interaction between the integrin LFA-1 and related to the typical changes associated with ICAM-1. Here we show that Del-1 also acts as an apoptosis, especially the PS redistribution to the cell eat-me signal thereby enhancing the efferocytic surface. In conclusion, we postulate the existence of ability of macrophages in the context of RoI. This a new molecular pattern (DAMP) on neutrophils action is facilitated by a high affinity binding of the which is independent of physiological apoptosis, discoidin I-like domains of Del-1 to PS on apoptotic regulated by sphingolipids and HSP90, results in cells, and the interaction of Del-1 with the rapid neutrophil clearance, and thus may importantly phagocytic receptor alphaVbeta3-integrin on contribute to the neutrophil turnover. macrophages, which is mediated by the RGD site in the second EGF-repeat of Del-1. The increased 165 efferocytosis in the presence of Del-1 was associated with the induction of the resolving macrophage The Dual Role of the Endogenous Homeostatic phenotype, as demonstrated by upregulation of Factor Del-1 in the Modulation of Acute Self- phagocytic receptors, bridging molecules, the Limited Inflammation nuclear receptor LXRa and TGFb1. These Del-1 Ioannis Kourtzelis1, Ioannis Mitroulis1, Janusz von effects were dependent on the alphaVbeta3-integrin, Renesse1, Daniel Grosser2, Christian Doreth1, Lan- as beta3-integrin deficient macrophages did not Sun Chen1, Anaisa Ferreira1, Kavita Hosur3, Pallavi display the Del-1-mediated upregulation of LXRa Subramanian1, Ünal Coskun2, George and TGFb1. Blockade of LXRa decreased Hajishengallis3, Triantafyllos Chavakis1 efferocytosis-dependent upregulation of TGFb1 1Technische Universität Dresden, Department of expression. The presence of the apoptotic Clinical Pathobiochemistry and Institute for Clinical neutrophils rather than solely PS was required to Chemistry and Laboratory Medicine, 01307 mediate the Del-1-mediated macrophage re- Dresden, Germany; 2Laboratory of Membrane programming, as phagocytosis of PS-coated beads in Biochemistry, Paul Langerhans Institute Dresden, the presence of Del-1 was not sufficient to induce a Faculty of Medicine Carl Gustav Carus at the TU resolving phenotype in macrophages, while Dresden, 01307 Dresden, Germany; 3University of inhibition of the degradation of phagocytosed Pennsylvania, Penn Dental Medicine, Department of apoptotic material resulted in decreased LXRa and Microbiology, Philadelphia, PA 19104 USA TGFb1 expression. In vivo, monosodium urate (MSU) crystal-induced peritonitis resulted in Acute inflammation is initiated by neutrophil and increased accumulation of neutrophils and monocyte recruitment to the inflamed site, followed monocytes/macrophages due to Del-1 deficiency, by release of pro-inflammatory mediators. After consistent with the effect of Del-1 to block LFA-1- their acute proinflammatory actions, recruited dependent neutrophil and Mac-1-dependent neutrophils undergo apoptosis and are phagocytosed monocyte recruitment, respectively. Conversely but by macrophages, a process that is mediated by the consistently, mice with endothelial over-expression recognition of phosphatidylserine (PS) on apoptotic of Del-1 displayed significantly decreased leukocyte neutrophils by “eat-me” signals and specific numbers 8h after MSU injection. Besides this acute anti-inflammatory effect of Del-1, Del-1 contributed flavonoids as modulators of human neutrophils’ to RoI in vivo, as Del-1-deficient mice displayed oxidative burst and as inhibitors of leukotriene B4 enhanced numbers of apoptotic neutrophils and (LTB4) production. As it is a comprehensive and impaired induction of the resolving macrophage systematic study, a series of 24 structurally related phenotype. Local administration of Del-1 resulted in flavonoids (fig. 1) was studied and the structure- enhanced efferocytosis of apoptotic neutrophils in relationship was stablished. For this purpose fresh MSU-induced peritonitis. human neutrophils were daily isolated. The RS Taken together, our data provide evidence that Del-1 produced were detected through the use of exerts a dual immunomodulatory role by both chemiluminescent and fluorescent probes and the blocking early inflammatory cell recruitment and by LTB4 produced was detected by applying an ELISA promoting macrophage efferocytic capacity, thus kit. The obtained results showed that the catechol identifying new mechanisms that can be targeted to group in B-ring is important for the activities modulate RoI. studied. Indeed, 3’,4’-dihydroxyflavone, 5,3’,4’- trihydroxyflavone, 7,3’,4’-trihydroxyflavone, and 166 luteolin prove to have the essential structural features to influence the inflammatory mediators Flavonoids Can Mediated Inflammation studied, constituting promising alternatives for the Resolution through Inhibition of Human resolution of inflammatory process. Neutrophils' Oxidative Burst and Leukotriene B4 Production Daniela Ribeiro1, Sara M. Tomé2, Carina Proença1, Artur M.S. Silva2, Marisa Freitas1, Eduarda Fernandes1, 1UCIBIO, REQUIMTE, Laboratório de Química Aplicada, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Portugal; 2Departamento de Química & Fig. 1 - Substitution patterns of the studied QOPNA, Universidade de Aveiro, Aveiro, Portugal flavonoids.

The first reports of the clinical features of Acknowledgements: inflammation date from 3000BC and this complex This work received financial support from the process and their modulation have been object of European Union (FEDER funds study for many years. Inflammation is a carefully POCI/01/0145/FEDER/007728) and National Funds orchestrated response of the organism, involving the (FCT/MEC, Fundação para a Ciência e Tecnologia recruitment of inflammatory cells, namely and Ministério da Educação e Ciência) under the neutrophils. Neutrophils are the major body’s first Partnership Agreement PT2020 line defence, but they are also found in some chronic UID/MULTI/04378/2013; and from the University inflammatory diseases. They are responsible for the of Aveiro and FCT/MEC for the financial support to production of various inflammatory mediators, such the QOPNA research Unit (FCT as reactive species (RS) by the rapid consumption of UID/QUI/00062/2013), through national founds and oxygen, prostaglandins, leukotrienes, cytokines, where applicable co-financed by the FEDER, within among others. Although inflammatory response is the PT2020 Partnership Agreement. Marisa Freitas generally self-limited, the balance between the pro- also acknowledges FCT the financial support for and anti-inflammatory signals may become Pos-doctoral grant (SFRH/BPD/76909/2011) in the dislocated, leading to deleterious widespread ambit of “QREN e POPH e Tipologia 4.1 e inflammatory responses. Therefore, the need for Formação Avançada”, co-sponsored by FSE and by efficient drugs with low side effects has become a national funds of MCTES. hot topic in the scientific research field. Flavonoids have been studied as possible alternatives for the anti-inflammatory therapy, as they already demonstrated antioxidant effects. In this sense, the main aim of this work was to study the potential of 167 by C3aR-dependent NETosis drives protumorigenic neutrophils during small intestinal tumorigenesis. Coagulation Induced by C3aR-Dependent Nat Commun. 2016 Mar 21;7:11037. PubMed NETosis Drives Protumorigenic Neutrophils PMID: 26996437. during Small Intestinal Tumorigenesis Carsten Krieg1, Silvia Guglietta1, Elena Zagato1, Lu Bao3, Simona Gandini4, Paola S. Ravenda5, Giuseppe Penna1, Maria Rescigno1, 1European Institute of Oncology, Department of Experimental Oncology; 1University Zurich, Institute of Experimental Immunology; 3Childrens’ Hospital, Harvard Medical School; 4Division of Epidemiology and Biostatistic, European Institute of Oncology; 5Gastrointestinal and Neuroendocrine Tumor Unit, European Institute of Oncology; 6Department of Oncology and Haemato-Oncology

Cancer patients show hypercoagulation and neutrophila but a link has up to now been unclear. We set out to study hereditary colorectal cancer (CRC) in the APCMin/+ model. Similar to familial 168 adenomatous polyposis (FAP) in humans in this model CRC development is driven by the truncation Myeloperoxidase in the Regulation of at codon 850 of the apc gene. Subsequently APCMin/+ Polymorphonuclear Neutrophil Cell Death 2,1,3 1 mice develop mainly in the small intestine (ileum) Lukas Kubala , Silvie Kremserova , Tomas 2,4 1,3 2,5 and sporadically in the large intestine (colon) polyps. Perecko , Anna Kocurkova , Anna Klinke , 1 Tumorigenesis is associated with the manifestation Institute of Biophysics, Academy of Sciences of the 2 of hypercoagulation and neutrophil accumulation. Czech Republic, Czech Republic; International All these effects on the haematopoietic system were Clinical Research Center - CBCE, St. Anne's 3 found to be driven by haematopoietic extrinsic University Hospital Brno, Czech Republic; Faculty factors. During tumoroginesis neutrophils showing a of Science, Masaryk University, Czech Republic; 4 low-density (LDN) phenotype associated with a Institute of Experimental Pharmacology and functional pro-tumorigenic N2 profile emerged that Toxicology, Slovak Academy of Sciences, Slovak 5 were pre-set to spontaneous NETosis and were Republic; Heart Center, University of Cologne, directly imprinted by blood clots. Finally, Germany tumorigenesis results in increased circulating lipopolysaccharide induced activation of the Polymorphonuclear neutrophils (PMNs) play a key complement cascade and we demonstrate the role in host defense. However, their massive existence of a feedback loop between neutrophils accumulation at the site of injury can initiate chronic and complement. We confirm these results in a inflammatory processes, thus the clearance of PMN cohort of patients with small bowel cancer. In our mediated by regulated cell death is key process. model enhanced thrombus formation induces a Myeloperoxidase (MPO), highly abundant enzyme protumorigenic phenotype in neutrophils which in in PMN granules, primarily connected with PMN addition to spontaneous NETosis, further strengthens defense machinery is suggested to be involved in hypercoagulation. This study expands our view on PMN regulated cell death. Nevertheless, the mechanisms underlying cancer-associated mechanisms how MPO affects PMN cell death hypercoagulation, which could be further exploited remain incompletely characterized. as a therapeutic intervention. Interestingly, MPO deficient PMNs revealed FURTHER READING: Guglietta S, Chiavelli A, significantly decreased rate of cell death Zagato E, Krieg C, Gandini S, Ravenda PS, Bazolli characterized by phosphatidylserine surface B, Lu B, Penna G, Rescigno M. Coagulation induced expression in response to activation of oxidative burst by12-myristate 13-acetate (PMA). An inhibitor sepsis. In these experiments hPR3Tg mice displayed of MPO activity 4-ABAH (50 μM) showed only decreased survival where 50% of WT mice survived limited effect on PMA induced cell death compared for 7 days post-surgery while 100% of hPR3Tg mice to MPO deficiency. Interestingly, PMA stimulated died prior to day 5. This decreased survival was also PMNs do not present activation of other markers associated with increased neutrophil infiltration. In characteristic for apoptotic cell death including vitro, neutrophils from hPR3Tg mice displayed activation of caspase 3 and 8 and DNA enhanced survival during spontaneous apoptosis fragmentation. In contrast, markers characterizing compare to WT controls, as well as decreased autophagy such as cleavage of LC3 protein and apoptosis induced by TNF-α treatment and this may increased expression of p62 were observed in PMA in part explain the increased neutrophil numbers stimulated PMNs. observed during the later stages of inflammation. The important role of MPO in the regulation of the Taken together, our data suggests that hPR3 plays a course of inflammation, independent of its putative proinflammatory role in vivo and increased levels of microbicidal functions, can be potentially linked to this protein during inflammation affects neutrophil MPO ability to modulate the life span of PMN accumulation and survival. accumulated at the site of inflammation. 170 169 Polarization of Innate Neutrophils Due to Tollip Mice Expressing Human Proteinase 3 Show Deficiency Modulates Colonic Tumorigenesis Sustained Neutrophil Infiltration and a Defect in Christina Lee, Virginia Polytechnic Institute and the Resolution of Inflammation State University Katherine R. Martin1,2,4, Magali Pederzoli-Ribeil1,2,3, Emeline Lebourdais 4,5,6, Adam Dahdah4,5,6, Celine Differential programming of innate neutrophils may Candalh1,2,3, Sabrina Burgener7, Charaf Benarafa7, bear critical relevance in host defense as well as Pierre Launay4,5,6, Veronique Witko-Sarsat1,2,4, tumorigenesis. However, molecular mechanisms 1INSERM U1016; 2CNRS-UMR 8104; 3Université responsible for neutrophil polarization are still not Paris Descartes; 4Labex Inflamex; 5INSERM U1149; well understood. Here, we aim to identify and 6Université Paris Diderot; 7Theodor Kocher characterize the neutrophil phenotypes in chronic Institute; 8Graduate School for Cellular and colitis (CC) mouse model. The study focuses at Biomedical Sciences clarifying the molecular and physiological signature of neutrophils in mice subjected to chronic colitis Proteinase 3 (PR3) is serine protease expressed with AOM and DSS challenges. We identify that exclusively in neutrophils, monocytes and Toll-interacting- protein (Tollip) may play a key role macrophages. This protein has a number of well- in modulating the long-term polarization and characterized pro-inflammatory activities including function of neutrophils as well as anti-tumor cleaving extracellular protein matrix, activating defense. We observed that Tollip deficient mice had chemokines as well as controlling cell survival and reduced generation of colon polyps when subjected proliferation. To better understand the function of to prolonged challenges with AOM and DSS. At the PR3 in vivo, a transgenic mouse was generated cellular level, Tollip deficient mice challenged with expressing human PR3 (hPR3Tg). hPR3 mRNA was AOM-DSS had distinct neutrophil activation profiles detected in myeloid cells isolated from the bone as compared to wild type mice, including the marrow of transgenic mice. During zymosan- differential expression of cell surface co-stimulatory induced peritonitis, hPR3Tg mice displayed an molecules, migratory behaviors, as well as cytokine increased accumulation of neutrophils compared to expression profiles. Based on the in vivo and in vitro WT controls 24 hours after the induction of studies, we conclude that Tollip may play a key role inflammation and this difference increased by 48h. during the polarization of innate neutrophils There was no difference in the recruitment of involved in the modulation of chronic colitis tumor macrophages, B or T lymphocytes at any time point environment. Further studies with regard to the examined. Mice were also subjected to cecum molecular mechanisms may enable a better ligation and puncture, a model used to mimic human understanding of neutrophil modulation in colon inflammation, we found that integrin-independent tumorigenesis. migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, 171 integrin-dependent transmigration into the alveolar space was completely impaired. Finally, signal Cystic Fibrosis is a New Type of Leukocyte transduction analysis showed that, in CF monocytes, Adhesion Deficiency Disease chemoattractant-triggered activation of RhoA and Alessio Montresor1, Claudio Sorio1, Matteo CDC42 rho small GTPases (controlling integrin Bolomini-Vittori 2, Sara Caldrer1,4, Barbara Rossi1, activation and chemotaxis, respectively) was Silvia Dusi1, Stefano Angiari1, Jan E. Johansson1, strongly deficient. Marzia Vezzalini1, Teresinha Leal3, Elisa CONCLUSIONS Calcaterra1, Baroukh M. Assail4, Paola Melotti4, Altogether, these data highlight the critical Carlo Laudanna1, 1University of Verona, Department regulatory role of CFTR in integrin activation by of Medicine; 2Radboud University Medical Center chemoattractants in monocytes and identify CF as a Geert Grooteplein ; 3Université Catholique de new, cell type-selective, leukocyte adhesion Louvain; 4Cystic Fibrosis Center, Azienda deficiency disease (LAD-IV), providing new Ospedaliera Universitaria Integrata di Verona insights into CF pathogenesis.

RATIONALE Cystic fibrosis (CF) is a genetic 172 disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Human Blood Monocytes are Able to Form Persistent lung inflammation, characterized by Extracellular Traps increasing polymorphonuclear leukocyte Luc de Chaisemartin1,2, Vanessa Granger1,2, Benoit recruitment, is a major cause of the decline in Noel1, Yann Gallais1, Natacha Szely1, Marc respiratory function in patients with CF and is a Pallardy1, Sylvie Chollet-Martin1,2, 1UMR996 - leading cause of morbidity and mortality. CFTR is Inflammation, Chemokines and Immunopathology - expressed in various cell types, including leukocytes, ,INSERM, Univ Paris-Sud, Université Paris-Saclay, but its involvement in the regulation of leukocyte 92296, Châtenay-Malabry, France ; 2APHP, Bichat recruitment in inflammation is unknown. Hospital, Immunology Department, Paris, France OBJECTIVES We evaluated whether CF leukocytes might present alterations in integrin activation, cell Background adhesion and migration controlled by Neutrophil extracellular traps (NETs) are chemoattractants, key processes governing innate extracellular DNA filaments formed during and acquired immune responses. neutrophil activation, a process called netosis and METHODS originally associated with neutrophil antibacterial In this study we used integrin affinity assays, ex vivo properties. Several lines of evidence now suggest a adhesion and chemotaxis assays, flow cytometry, major role for netosis in thrombosis, auto-immune immunofluorescence staining, in vivo analysis and diseases and cancer. A similar mechanism, called rho small GTPase signaling activity assays. etosis, has been reported in other immune cells such MEASUREMENTS AND MAIN RESULTS as eosinophils. We investigated if such a mechanism We found that chemoattractant-induced activations could exist in human monocytes. of β1 and β2 integrins and of chemotaxis are Materiel and Methods defective in mononuclear cells isolated from patients Magnetically sorted CD14+ monocytes and with CF. In contrast polymorphonuclear leukocyte monocyte-derived dendritic cells were stimulated adhesion and chemotaxis were normal. The with phorbol-12-myristate-13-acetate, calcium functionality of β1 and β2 integrins was restored by ionophore (A23187), platelet-activating factor and treating CF monocytes with the CFTR-correcting zymosan A. Extracellular traps release was drugs VRT325 and VX809. Moreover, treatment of quantified by Sytox Green fluorescence. NETs were healthy monocytes with the CFTR inhibitor visualized by immunofluorescence with antibodies CFTR(inh)-172 blocked integrin activation by against myeloperoxidase, lactoferrin, citrullinated chemoattractants. In a murine model of lung histones, elastase, and tissue factor. DNA- myeloperoxidase complexes were measured by Tennessee State University, 1301 E. Main St., ELISA and histone citrullination was assessed by Murfreesboro, TN 37132, USA; 5Department of western blot. Medicine, Solna, Karolinska University Hospital, Results Stockholm S17176, Sweden; 6Division of Veterinary We demonstrate that human blood monocytes are Resources, Office of the Director, National Institutes capable of extracellular trap (ET) release in response of Health, Bethesda, MD 20892, USA; 7Department to several chemical and biological stimuli. In of Biochemistry and Molecular Pharmacology, contrast, monocyte-derived dendritic cells are not University of Massachusetts Medical School, capable of etosis but rather undergo necrotic cell Worcester, MA 01605, USA; 8Division of death upon stimulation. By microscopy, we show Rheumatology, Department of Internal Medicine, that monocyte ETs display a morphology analogous University of Michigan, Ann Arbor, MI 48109, USA to NETs, and are associated with myeloperoxidase, lactoferrin, citrullinated histones and elastase. Rheumatoid arthritis (RA) is a chronic systemic Monocyte etosis depends on oxidative burst via autoimmune disorder characterized by inflammation NADPH oxidase activation but not on and destruction of the synovial joints and by the myeloperoxidase activity, in contrast to neutrophils. development of humoral and cellular autoimmunity Finally, we provide evidence of tissue factor on to citrullinated proteins. Autoimmunity to monocyte ETs, a feature that could be relevant to citrullinated antigens appears years before the onset monocyte thrombogenic properties. of clinical symptoms of RA and is highly specific Conclusion for this disease. Previous studies implicate the We demonstrate that upon ex vivo stimulation, formation of neutrophil extracellular traps (NETs) as human blood monocytes can release extracellular a source of citrullinated autoantigens and as a traps bearing several active proteins. This new mechanism promoting the activation of RA synovial cellular mechanism is likely to improve our fibroblasts (FLS), cells with crucial roles in joint understanding of monocytes’ role in multiple damage. We investigated the molecular mechanism pathological contexts such as inflammatory by which NETs promote a proinflammatory disorders, infection or thrombosis. phenotype in FLS, and whether these interactions play a role in the generation of adaptive immune 173 responses to citrullinated autoantigens. Autoantibodies recognizing citrullinated Arthritogenic Citrullinated Peptides Present in antigens(ACPAs), isolated from RA patients, Neutrophil Extracellular Traps are Internalized canenhance NETosis and recognize multiple by Synovial Fibroblasts through a RAGE-TLR9 citrullinated peptides present in NETs. Proteomic Axis and Activate Adaptive Immunity in analysis of NETs demonstrates that myeloperoxidase Rheumatoid Arthritis (MPO) and neutrophil elastase, among other Carmelo Carmona-Rivera1, Erica Moore1, Nithya proteins, are citrullinated. Autoantibodies against Lingampalli2, Hannes Uchtenhagen3, Eddie James3, citrullinated MPO are present in synovial fluid from Kevin L. Bicker4, Heidi Wahamaa5, Victoria RA patients. Furthermore, NETs containing these Hoffmann6, Anca I. Catrina5, Paul R. Thompson7, citrullinated peptides are internalized by FLS Jane H. Buckner3, William H. Robinson2, David A. through a RAGE-TLR-9 pathway. NETs’ Fox8, Mariana J. Kaplan1, 1Systemic Autoimmunity internalizationis required for the upregulation of Branch, National Institute of Arthritis and proinflammatory cytokine synthesis by FLS. Musculoskeletal and Skin Diseases, National Interleukin-17B (IL-17B), externalized in NETs, Institutes of Health, Bethesda, MD 20892, USA; 2VA induces upregulation of major histocompatibility Palo Alto Health Care System, Palo Alto, CA 94304, complex class II (MHCII) in FLS. Once internalized, USA and the Division of Immunology and NET-peptides are loaded into MHCII intracellularly Rheumatology, Stanford University School of and trafficked to the FLS plasma membrane. Medicine, Stanford, CA 94305, USA; 3Benaroya Arthritogenic NET peptidesare then presented by Research Institute at Virginia Mason, The University FLS to Ag-specific T cells and induce their of Washington School of Medicine, Seattle, WA activation. Humanized HLA-DRB1*0401 transgenic 98101, USA ; 4Department of Chemistry, Middle mice immunized with mouse FLS loaded with NETs develop ACPAs and antigen-specific T cell 175 responses. Epitope mapping arrays demonstrate the enhanced presence of autoantibodies against Ifn-γ and IL-17A Establish the Balance between citrullinated forms of relevant autoantigens P. aeruginosa Clearance and Inflammatory implicated in RA pathogenesis in those animals Potential during Infection of Human immunized with FLS-NETs. These results suggest Macrophage-Neutrophil Co-Cultures that NETs are a source of arthritogenic peptides in Sirina Muntaka, Miguel Cámara , Luisa Martínez- the synovium and implicate FLS as mediators in RA Pomares, University of Nottingham pathogenesis, through the internalization and presentation ofcitrullinated peptides to the adaptive Neutrophils are essential for protection against immune system leading to ACPA formation and T extracellular bacteria but neutrophil-dominated cell activation. inflammation can also compromise organ function through tissue damage. Optimal anti-bacterial 174 immunity should harness the microbicidal activity of neutrophils while minimising their potential for PI3K Activity in the Tumor Microenvironment causing injury. Neutrophils do not act in isolation Promotes Mammary Tumor Growth and and during inflammation, they respond to cues Metastasis and Inhibits Anti-Tumor Immunity provided by other cells such as macrophages, which Philip Owens1, Jiqing Sai1, Sergey Novitskiy1, Anna influence their activation and life span.In E. Vilgelm1, Jinming Yang1, Tammy Sobolik1, vivomodels have dominated the study of anti- Nicole Lavender1, Andrew C. Johnson1, Colt bacterial immunity and there is a need of human McClain1, Gregory D. Ayers1, Mark C. Kelley1, models for the dissection of macrophage-neutrophil Melinda Sanders1, Harold L. Moses1, Mark communication during bacterial infection. This work Boothby1, Ann Richmond2,1, 1Vanderbilt University; describes the development of a human macrophage- 2Tennessee Valley Healthcare System, Department neutrophil infection assay and its use to model Th1 of Veteran Affairs, Nashville, TN (IFN-γ)- and Th17 (IL-17A)-driven microbicidal activity and inflammatory potential upon infection. Identification of targeted therapies that also activate Results show that IFN-γ and IL-17A have opposite anti-tumor immunity could offer significant hope for effects on the killing ability of macrophages and cancer patients. In this report, therapeutic effects of neutrophils co-cultures; bacterial killing was reduced the pan-PI3K inhibitor, BKM120, on growth and by IFN-γ and promoted by IL-17A. In addition metastasis of mammary tumor allografts in mice, macrophages and neutrophils specifically were observed to be associated with increased collaborated for the production of IL-1β and IL-1α infiltration of IFN-γ and TNFα expressing immune and IFN-γ-treated co-cultures generated significantly cells into tumors. Moreover, in a humanized mouse less IL-1β and IL-1α compared to those treated with model using patient derived xenografts growing in IL-17A. This effect was not observed with other immune compromised mice engrafted with the cytokines such as TNF-α, IL-6, MIP-1α and MCP-1. patient’s hematopoietic cells, BKM120 also Thus phagocyte co-cultures provide a suitable model inhibited tumor growth and enhanced anti-tumor of human anti-microbial immunity and unveil an immune responses. Surprisingly, similar effects unappreciated collaboration between macrophages occurred when PI3Kγ-/- mice were implanted with and neutrophils in the promotion of IL-1-mediated PI3KWT MMTV-PyMT tumor, without any inflammation which is quenched in the presence of exogenous inhibitor treatment. Effects of host PI3Kγ IFN-γ. loss on tumor growth were partially reversed by CD8+T depletion. Altogether our data indicate a key 176 mechanism for PI3K inhibition of tumor growth and metastasis involves enhancement of anti-tumor Advances on the Biology of 6-Sulfo LacNac immunity, particularly CD8+T effector cells, in (slan)/M-Dc8+ Cells addition to direct effects on tumor cells. Alessandra Micheletti1, Federica Calzetti1, Giulia Finotti1, Silvia Lonardi2, William Vermi2, Marco A. Cassatella1, 1Section of General Pathology, Department of Medicine, University of Verona, differentiation program occurring within tonsils. Our Verona ; 2Section of Pathology, Department of data uncover novel information on plasticity by Molecular and Translational Medicine, University of blood slanDCs/M-DC8+ cells and their ultimate Brescia, Brescia commitment within tissue microenvironments.

A population of human myeloid cells, sharing some 177 functional characteristics with classical myeloid dendritic cells (DCs), have been recently identified IFNα Enhances the Production of IL-6 by Human by using specific mAbs (M-DC8) recognizing the 6- Neutrophils Activated via TLR8 Sulfo LacNac (slan) carbohydrate modification of Nicola Tamassia1, Maili Zimmermann1, Fabio PSGL-1, whose acronym gave thus origin to their Arruda-Silva1, Francisco MA Bianchetto-Aguilera1, name (namely, “slanDCs”). However, on a two- Claudio Lunardi2, Marco A. Cassatella1, dimensional flow cytometry dot plot of CD14 and 1Department of Medicine, Section of General CD16 expression in peripheral blood mononuclear Pathology, University of Verona; 2Department of cells (PBMCs), slanDCs/M-DC8+ myeloid cells Medicine, Section of Internal Medicine, University overlap, in part, with the CD14dimCD16+ monocytes, of Verona suggesting that they might actually represent a subset of the so-called non-classical monocytes. PURPOSE Functionally, blood slanDCs/M-DC8+ cells have Recently, we reported that human neutrophils been described as potent pro-inflammatory cells, produce biologically active IL-6 when incubated based on their capacity to produce large amount of with agonists activating TLR81, a receptor TNFα and IL-12p70 upon stimulation with TLR recognizing viral single strand RNA. Herein, we ligands. In addition, blood slanDCs/M-DC8+ cells investigated the effect of IFNα, a cytokine known to are known to promote proliferation, cytotoxicity and modulate the early innate immune responses toward IFNγ production by natural killer (NK) cells, as well viral and bacterial infections, on the production of as strong antigen-specific T-cell responses. IL-6 by TLR8-activated neutrophils. Furthermore, it is well established that slanDCs/M- METHODS DC8+ cells locate in peripheral tissues, especially Human neutrophils isolated from healthy donors or under inflammatory conditions, and selectively in systemic lupus erythematosus (SLE) patients by carcinoma-draining lymph nodes, where they negative selection using immunomagnetic beads marginate metastatic cells. However, even though (99.7 ± 0.2 % purity), were incubated for up to 20 h blood slanDC/M-DC8+ cell function and phenotype with or without 5 μM R848 (a TLR8 agonist), in the have been broadly delineated, an extensive presence or the absence of 1000 U/ml IFNα. mRNA comparison between blood and tissue slanDCs/M- expression and cytokine production were then DC8+ cells, as well as between tissue slanDCs/M- measured by, respectively, RT-qPCR and ELISA, DC8+ cells and other tissue DC/macrophage while C/EBPβ transcription factor recruitment at the populations, has never been performed. For such a IL-6 genomic locus was investigated by chromatin purpose, we recently made an extensive phenotypic immunoprecipitation (ChIP) assays. and functional characterization of slanDCs/M-DC8+ RESULTS cells in human tonsils. We found that tonsil In this study2, we demonstrate that IFNα potently slanDCs/M-DC8+ cells represent a unique enhances the production of IL-6 in neutrophils population of dendritic cells, displaying a surface incubated with R848. Such an effect is not caused by marker repertoire distinct from their circulating an IFNα-dependent induction of TLR7, another counterpart and from other tonsil DC and receptor for R848, but, rather, by an increased macrophage subsets described to date. Functionally, release of TNFα, which in turn amplifies IL-6 tonsil slanDCs/M-DC8+ cells exhibit an efficient expression. Endogenous TNFα, in fact, was shown antigen presentation capacity and a constitutive to promote an augmented synthesis of the IkBζ co- secretion of TNFα. Notably, such DC phenotype and activator and an enhanced recruitment of C/EBPβ to function were substantially reproduced by culturing the IL-6 promoter. Moreover, our data uncover that blood slanDCs/M-DC8+ cells in tonsil-derived neutrophils from active SLE patients, displaying an medium, supporting the hypothesis of a full DC IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNFα in neutrophils, and characterized using response to R848 as compared to healthy donors. immunofluorescence microscopy, electron DISCUSSION microscopy and live-cell spinning disk imaging. The Altogether, data clarify the molecular bases of the mechanism of EP was interrogated using specific IFNα-dependent enhancement of IL-6 production in inhibitors as well as in mice with informative genetic TLR8-activated neutrophils. More in general, we deletions. The impact of EP was interrogated show that TLR8 ligands, IFNα and TNFα, three through engraftment of MKs co-cultured with players often coexisting in many diseases of viral or neutrophils back into recipient mice. autoimmune origin, promote a strong production of RESULTS: EP is evident in approximately 5% of IL-6 in human neutrophils, placing the same MKs in bone marrow, increasing 2-3-fold with LPS neutrophils among potential targets for and peripheral platelet depletion. Imaging discloses immunotherapeutic interventions. sequential phases, beginning with uptake into a BIBLIOGRAPHY distinct vacuole followed by release of the neutrophil 1 Zimmermann, M. et al. (2015) Nat directly into the MK cytoplasm (Figure 1). Uptake is Commun.;6:6061 mediated in part through neutrophil beta2 integrins 2 Zimmermann, M. et al. (2016) Sci Rep.;6:19674 and requires active cytoskeletal rearrangement by both neutrophils and MKs. Within the cytoplasm, 178 neutrophils develop direct membrane contiguity with the MK’s platelet-producing demarcation membrane Emperipolesis is a Novel Cell-In-Cell system (DMS), resulting in transfer of neutrophil Phenomenon that Mediates Transfer of proteins to the MK surface and to platelets produced Neutrophil Membrane to and by MKs engrafted into live mice. MK cell contents Platelets are reciprocally transferred to neutrophils prior to Pierre Cunin1, Eric Boilard2, Pui Y. Lee1,3, Allyn their release from MKs alive and intact. Compared Morris1, Joseph E. Italiano4, Peter A. Nigrovic1,3, with MKs cultured alone, MKs cultured together 1Department of Medicine, Division of Rheumatology, with neutrophils demonstrate more efficient platelet Immunology and Allergy, Brigham and Women’s generation in vivo, suggesting that one effect of EP Hospital, Harvard Medical School, Boston, MA, is to enhance the ability of MKs to respond to USA.; 2Centre de Recherche du Centre Hospitalier thrombocytopenic stress. Universitaire de Québec (CHUL), Faculté de CONCLUSIONS: Emperipolesis is a highly novel Médecine de l’Université Laval, Québec, QC, cell-in-cell interaction that is mediated in part by Canada.; 3Department of Medicine, Division of neutrophil beta2 integrins and results in the Immunology, Boston Children’s Hospital, Harvard reciprocal exchange of cell contents between Medical School, Boston, MA, USA.; 4Department of megakaryocytes and neutrophils, potentially Medicine, Hematology Division, Brigham and enhancing production of platelets. Additional Women's Hospital, Harvard Medical School, Boston, consequences of emperipolesis for MKs and MA, USA. neutrophils remain to be defined.

BACKGROUND: Emperipolesis (EP) is a poorly- understood phenomenon wherein bone marrow megakaryocytes (MKs) actively enclose intact neutrophils. Common even in healthy individuals, EP increases in frequency in myeloproliferative states and in states of accelerated platelet production. However, the mechanism and significance of EP are unknown. METHODS: We assessed the frequency of EP in healthy mice and in animals subjected to systemic LPS and to induced thrombocytopenia. A novel model of EP was developed through incubation of cultured bone marrow MKs together with 179 CSF3R the synthesis of Nicotinamide phosphoribosyltransferase (NAMPT), which is an Type I Interferon-Mediated Polarization of unique enzyme-cytokine molecule. It is a rate- Tumor-Associated Neutrophils Depends on limiting enzyme, converting nicotinamide (NA) into CSF3R Signaling Pathway NAD+ that in turn activates NAD+-dependent Jadwiga Jablonska1,2, Mozhgan D. Harati2, Ekaterina protein deacetylases sirtuins (SIRTs). Moreover, Pylaeva1, Sven Brandau1, Karl Welte2, Julia extracellularly, NAMPT exhibits also the cytokine- Skokowa2, Stephan Lang1, 1University Hospital like functions that lead to upregulation of pStat3. Essen; 2University Hospital Tuebingen Extracellular NAMPT levels are increased in various inflammatory conditions, e.g. tumors. That makes Neutrophils play strategic role during inflammatory this pleiotropic molecule an important player in responses of the host. Importantly, inflammation has tumor/host cross-talk. NAMPT serves as an inhibitor long been associated with enhanced susceptibility of neutrophil apoptosis and as neutrophil for cancer thus neutrophils, as a crucial component chemoattractant. It is a potent pro-inflammatory of this process, play essential role in tumorigenesis factor upregulating ROS release and pro-angiogenic and remain an independent prognostic marker in a factor (smooth muscle maturation). NAMPT is broad variety of neoplasias. strongly overexpressed in many cancers and its It is known that neutrophils are influenced by the elevated levels are correlated with tumorigenicity. tumor microenvironment and differentiate into anti- Analysis of CSF3R downstream signaling in tumor (N1) or pro-tumor (N2) cells. The most neutrophils from interferon deficient mice revealed plausible explanation for the different neutrophil highly elevated expression of NAMPT, several phenotypes in tumor is alteration of the local Sirtuins and pStat3, on gene and protein expression cytokine milieu. Notably, we could recently level. This was in line with enhanced tumorigenesis demonstrate that type I interferons polarize in these mice. Notably, treatment of such animals neutrophils into N1 phenotype. In mice that lack with Nampt or Sirtuin inhibitors leads to strongly endogenous type I IFNs elevated numbers of N2 inhibited tumor growth and altered neutrophil neutrophils could be observed, accumulating in differentiation towards N1 phenotype. tumors and pre-metastatic lungs. Such neutrophils Based on these observations, we identified a new efficiently support tumor growth by up-regulating mechanism of interferon-mediated polarization of pro-angiogenic molecules e.g. VEGF and MMP9. TANs that involves NAMPT inhibition. Importantly, Moreover, these cells display prolonged survival, both – enzymatic and cytokine-like activity of secrete higher amounts of neutrophil-attracting NAMPT seems to be influenced by interferons. chemokines and support metastatic processes by up- Since TANs represent a highly potent therapeutic regulation of pro-metastatic proteins, like Bv8, target, these data highlight the therapeutic potential MMP9, S100A8 and S100A9. Importantly, these of interferons as NAMPT inhibitors, suggesting neutrophils show also impaired tumor cell killing optimization of their clinical use as potent anti-tumor due to repressed ROS production and less effective agent. NETs release. Treatment with rmIFN-b reverses these effects, leading to anti-tumor N1 polarization. 180 Importantly, neutrophils isolated from melanoma patients undergoing type I IFN therapy show Tumor Associated Neutrophils (TAN) Impair augmented N1 anti-tumor characteristics, suggesting Cytotoxic T-Cells Anti-Tumor Effect by similar regulating mechanisms in human. Inducting CD8+ Cells Apoptosis and Inhibiting Here, we evaluated the molecular mechanisms Proliferation involved in interferon-mediated alterations of Merav E. Shaul, Janna Michaeli, Inbal Mishalian, neutrophil polarization. We observed that in the Zvi G. Fridlender, Institute of Pulmonary Medicine, absence of type I interferons the expression level of Hadassah-Hebrew University Medical Center G-CSF is markedly elevated in neutrophils from different anatomical compartments. This could be The tumor microenvironment includes a complex reversed by rmIFN-β treatment. Importantly, we network of immune cells comprising macrophages, could show that G-CSF induces through its receptor MDSCs, neutrophils, T-cells and more. The contribution of Tumor Associated Neutrophils Breast cancer is the most common cancer among (TAN) to tumor progression has been a matter of Canadian women over the age of 20, representing debate as both pro- and anti-tumor functions have 26% of all cancer cases in Canadian women. been reported. In recent years, there has been a Metastatic breast cancer is the most advanced stage growing interest in the relationship and co-regulation (stage IV) of the disease and it is largely incurable. between tumor-infiltrating myeloid cells and Breast cancer cells display preferences for specific cytotoxic T cells (CTLs). Infiltrating T lymphocytes metastatic sites including the bone, lung, and liver. have been attributed an anti-tumor function since the The liver represents a prominent site for breast presence of activated CD8+T cells both within the cancer metastasis, with 50-70% of women with tumor and in the peritumoral stroma provided a metastatic breast cancer developing hepatic positive prognosis for patients. In our current work, metastases. we evaluated the effects of TAN on CD8+ cytotoxic The steps involved in the metastatic cascade rely on T-cells (CTLs) survival, proliferation and anti-tumor reciprocal interactions between cancer cells and their effect. Using a modified Winn assay, we first microenvironment. Distinct immune infiltrates can demonstrated in vivo that while CD8+ T-cells have either impair the metastatic process or conversely, the ability to slow down tumor initial growth, TANs assist in the seeding, colonization and growth of revert this anti-tumor effect of CD8+ CTLs. We disseminated cancer cells. Within distal organs, found that neutrophils isolated from murine tumors immune cells and their mediators are known to of lung cancer and mesothelioma, induce significant facilitate metastasis formation. However, the apoptosis of CD8+ CTLs. This effect was found to contribution of tumor-induced systemic be contact-dependent, and mediated by iNOS. inflammation to metastasis and the mechanisms Furthermore, this effect was markedly reduced in regulating systemic inflammation are not well TNFα knock-out mice, suggesting that TNFα has an characterized. Using lung and liver-metastatic essential role in this process. Although TAN were variants of 4T1 breast cancer cells model, we have found to have a mild positive effect on the activation revealed that there are increased recruitment of of CD8+ T-cells, the use of activation markers such myeloid-derived/granulocytic (Gr-1+) and as CD69 and intracellular cytokines IFN-γ showed neutrophils (NE+) in the lungs and livers of mice that TAN primarily promote the apoptosis of non- bearing lung and liver metastasis respectively. activated CD8+ T-cells. Using the CFSE method, we However, based on the Gr-1+ depletion studies, it also demonstrated that TAN inhibit the proliferation was observed that infiltrating myeloid- of CD8+ CTLs. derived/granulocytic cells, including neutrophils, Our findings demonstrate the importance of TAN in were essential for the formation of liver metastases the regulation of the immune reaction to cancer, but not for lung metastases. Intriguingly, we have adding another layer on the understanding of the role found that in peripheral blood, lung and liver and effects of neutrophils in cancer. metastases have the ability to mobilize differently the two distinct populations of high-density (HDNs) 181 and low density neutrophils (LDNs) based on a density gradient centrifugation. In the peripheral Differential Mobilization of Circulating blood of mice bearing liver metastases, there is a Neutrophil Subpopulations in Breast Cancer dramatic increase in the mobilization of LDNs Metastasis compared to mice bearing lung metastases. While Brian E. Hsu1,2, Sébastien Tabariès1,2, Matthew G. HDNs are characterized as being more of an anti- Annis1,2, Claudia U. Duerr4, Jörg H. Fritz4,5, Peter M. tumor “N1” neutrophil with increased phagocytic Siegel1,2,3, 1Goodman Cancer Research Centre, activity, production of reactive oxygen species, and McGill University; 2Department of Medicine, McGill N1 associated genes (IFN-B and iNOS), LDNs University; 3Department of Biochemistry, McGill function as a pro-tumorigenic “N2” neutrophil with University; 4Department of Microbiology and increased expression of N2 associated genes (Arg1 Immunology, Complex Traits Group, McGill and MMP9). These two different neutrophil University; 5Department of Physiology, Complex populations are functionally important in liver Traits Group, McGill University metastases where HDNs have the ability to suppress metastases while LDNs cannot. Thus, we believe that liver metastatic breast cancer cells rely on 183 interactions with neutrophils within the liver microenvironment for colonization and growth. Our Elucidating the Mechanism of Neutrophil results demonstrate the importance of investigating Apoptosis Inhibition by Francisella Tularensis the role played by these two neutrophil Lauren C. Kinkead1,2, Justin T. Schwartz1, Brandi B. subpopulations which may represent a new potential Ketelsen1,2, Laura C. Whitmore1, Lee-Ann H. therapeutic strategy to inhibit metastatic diseases. Allen1,2 1University of Iowa; 2VA Medical Center 182 Francisella tularensis is a Gram-negative, Characterization of Neutrophil Sub-Populations facultative intracellular pathogen and the etiologic in Steady State and during Cancer Inflammation agent of the zoonotic disease tularemia. Inhalation of Using High Dimensional Data Analysis as few as ten organisms can result in severe, and Carsten Krieg1, Silvia Guglietta2, Sven Brandau3, potentially lethal, pneumonic disease. The Burkhard Becher1, 1University Zurich, Institute of pathogenicity of F. tularensis is dependent on its Experimental Immunology; 2European Institute of ability to modulate host immune responses, and to Onclogy, Department of Experimental Oncology; survive and replicate within several different types 3University Hospital Essen, Department of of cells, including neutrophils. It is well established Otorhinolaryngology that defects in neutrophil turnover are defining features of an abnormal and ineffective immune Experimental and clinical evidences of the past years response that exacerbates inflammation and results revealed an unexpected plasticity of neutrophils in in host tissue destruction. Notably, published data several pathologic conditions including cancer. indicate that neutrophils contribute to the pathology Similarly to macrophages, it has been proposed that of tularemia, as demonstrated by the fact that neutrophils show a pro-tumorigenic (N2) or an anti- blocking neutrophil migration into infected tissues tumorigenic (N1) phenotype. In addition, it has been decreases bacterial burden and tissue damage, and suggested that neutrophils are of key importance in enhances host survival. Previous data from our the metastatic process. Whether and how many laboratory demonstrated that F. tularensis prevents neutrophil sub-populations could potentially exist in NADPH oxidase assembly and activation, escapes cancer is still a matter of debate. Here we used a the phagosomes, and replicates in the neutrophil mass cytometry based high dimensional staining cytosol. Furthermore, we have demonstrated that F. panel to analyze blood neutrophils in healthy donors. tularensis inhibits spontaneous neutrophil apoptosis Mass cytometry uses metal-tagged instead of and significantly extends neutrophil lifespan. At the fluorochrome-tagged antibodies thus eliminating molecular level, F. tularensis impairs activation and spectral overlap and in combination with barcoding activity of the intrinsic and extrinsic caspases in enables the measurement of more than 30 molecular addition to modulating various anti- and pro- species on a single cell in multiple parallel samples. apoptotic proteins; however, the underlying Due to the complexity of the data set and in order to molecular mechanisms are incompletely defined and reduce analytical bias we use advanced bacterial factors required for this aspect of virulence bioinformatics in order to identify key immune are unknown. We now show that one or more factors populations. Using this approach we were able to secreted by F. tularensis can extend neutrophil identify multiple neutrophil populations in the lifespan, albeit to a lesser extent than direct peripheral blood of healthy donors and now you infection. Additional studies have identified a role want to explore their role and changes during cancer for bacterial lipoproteins (BLP) in delaying inflammation. neutrophil apoptosis, whereas two major Francisella virulence factors, lipopolysaccharide and capsule, are neither necessary nor sufficient for this delay. Preliminary data suggest BLP act via a Toll-like receptor (TLR) 2/1-dependent manner; this mechanism is influenced by a TLR1 single nucleotide polymorphism that affects TLR1 trafficking to the plasma membrane. Our data also nm (small) or 10 µm (large). First, GO flakes were indicate that phosphoinositide-3-kinase activity is confirmed to be endotoxin-free. Both small and large essential for F. tularensis-mediated neutrophil flakes decreased cell viability (about 30% loss of apoptosis inhibition, and whether this response is cell viability at 3 h versus control, as assessed on mechanistically linked to TLR2/1 is under ATP measurements). Using transmission electron investigation. Finally, we show that a related microscopy, we noted that GO flakes were aligned organism, F. novicida, also delays neutrophil with the cell membrane, possibly leading to apoptosis by mechanisms similar yet distinct from F. membrane stripping, which could be linked to the tularensis. Taken together, our data are significant as loss of cell viability. Furthermore, we observed GO- they provide fundamental insight into the triggered NET formation, as evidenced by scanning mechanisms of pathogen-mediated perturbation of electron microscopy and confocal microscopy. neutrophil lifespan during infection and may Using an enzymatic assay for neutrophil elastase contribute to the identification of targets for (NE), as an indirect readout for NET release, we therapeutic intervention or vaccines. showed that GO triggers NET formation in a size- dependent manner, with the large flakes inducing 184 stronger NET release. NETs were, in turn, able to degrade GO. Hence, using confocal Raman analysis, Graphene Oxide (GO) Triggers Neutrophil we observed degradation of GO in purified NETs, Extracellular Trap (NET) Formation in Human and this was blocked in the presence of a selective Neutrophils in a Size- Dependent Manner inhibitor of myeloperoxidase. Additionally, using Beatrice Lazzaretto1, Sourav P. Mukherjee1, Kunal confocal microscopy, we observed that small and Bhattacharya1, Matthew Fielden2, Birgit Brandner3, large GO flakes completely disrupted lipid rafts in a Kjell Hultenby4, Neus Lozano5, Kostas Kostarelos5, manner comparable to methyl-β-cyclodextrin. Bengt Fadeel1, 1Nanosafety & Nanomedicine Overall, our work has provided new insights Laboratory, Division of Molecular Toxicology, regarding the interaction of neutrophils with GO, a Institute of Environmental Medicine, Karolinska 2-D carbon-based material. Institutet, Stockholm, Sweden; 2Department of Applied Physics, Royal Institute of Technology, 185 3 Stockholm, Sweden; SP Technical Research Institute of Sweden, Stockholm, Sweden; Neisseria gonorrhoeae Co-Opts Complement 4Department of Laboratory Medicine, Karolinska Receptor 3 (CR3) for Silent Entry into Human Institutet, Stockholm, Sweden; 5Nanomedicine Neutrophils Laboratory, School of Medicine & National Asya Smirnov1, Kylene P. Daily1, Morgan B. Graphene Institute, University of Manchester, Johnson1, Joshua C. Eby2, Victor J. Torres3, Alison Manchester, United Kingdom K. Criss1, 1Department of Immunology, and Cancer Biology, University of Virginia, USA; 2Division of Neutrophils, as the first-line defense of the innate Infectious Diseases and International Health, immune system, are capable of detecting and Department of Medicine, University of Virginia, promptly eliminating a wide range of foreign USA; 3Department of Microbiology, NYU, USA intruders in order to maintain organismal homeostasis. Neutrophils can thus engulf and digest Neisseria gonorrhoeae (Gc) is a human-specific, microbes intracellularly or they may release gram-negative bacterium that causes the sexually- neutrophil extracellular traps (NETs) to capture and transmitted infection gonorrhea. Symptomatic destroy microbes extracellularly. In this study, we gonorrhea is characterized by the influx of investigated whether neutrophils are also capable of neutrophils to sites of infection. Notably, Gc can be ‘sensing’ and responding to graphene oxide (GO). cultured from neutrophil-rich secretions. The ability GO is a synthetic, carbon-based material envisioned of Gc to survive after exposure to human neutrophils for a range of different applications including in is influenced by bacterial expression of phase- medicine. To this end, we isolated primary human variable opacity-associated (Opa) outer membrane peripheral blood neutrophils and exposed them to proteins. Using adherent, chemokine-treated primary GO displaying average lateral dimensions of 80-100 human neutrophils, we found that Opa- Gc delays phagosome fusion with neutrophil primary granules opsonized Gc in their interactions with neutrophils to prevent exposure to granule degradative (e.g. lack of oxidative burst, delayed phagosome components and consequent bacterial killing maturation, actin-dependent phagocytosis). We posit (Johnson and Criss, Cellular Microbiology 2013). In that Opa- bacteria co-opt CR3 to “silently” infect addition, Opa- bacteria do not promote NADPH neutrophils and avoid cellular activation, to establish oxidase activation and do not induce oxidative burst a safe niche that promotes bacterial survival. in neutrophils. In contrast, Opa+ Gc that engage neutrophil CEACAMs induces a potent oxidative 186 burst in neutrophils and is phagocytosed into mature phagolysosomes that are bactericidal. These findings Epithelial Cells Directly Contribute to Attraction suggest that the route of bacterial phagocytosis and Activation of Neutrophils during Infection influences the extent of neutrophil activation and with the Intracellular Bacterium Chlamydia thus Gc survival. However, the mechanism by which trachomatis neutrophils phagocytose unopsonized, Opa- Gc is Susanne Kirschnek, Saskia Lehr, Juliane Vier, unknown. In primary cervical epithelial cells, Opa- Stephanie Dille, Georg Häcker, Institute of Medical Gc are internalized via a non-opsonic interaction Microbiology and Hygiene, Medical Centre, between pili and porin on the Gc surface and University of Freiburg, Germany epithelial Complement Receptor 3 (CR3; CD11b/CD18; αMβ2integrin; Mac-1) (Edwards et Genital infection with the obligate intracellular al, Cellular Microbiology 2002). In the current bacterium Chlamydia trachomatis can cause study, we tested the hypothesis that Opa- Gc uses inflammation of genital tract and pelvis with the CR3 for phagocytosis by primary human potential consequence of tubal scarring and female neutrophils. We found a dramatic decrease in the infertility. The mechanisms that lead to genital tract binding and internalization of Opa- Gc by tissue damage are still unclear but previous studies neutrophils following treatment with blocking suggest a contribution from neutrophil granulocytes. antibodies against either CD11b or CD18. Although infected epithelial cells may secrete Conversely, expression of CR3 enhanced uptake of various mediators it is not clear how these cells are Opa- Gc by HL-60 cells, and was dependent on the recognized by and stimulate neutrophils. We here I-domain of CD11b. We found that IL-8 primed, rebuild this situation in vitro with the goal of adherent neutrophils express more total and understanding the factors involved on the sides of C. activated CR3 on their surface than suspension trachomatis, infected epithelial cell and responding neutrophils. Treating unprimed neutrophils in neutrophil. Mouse neutrophils were generated from suspension with phorbol ester increased total and progenitors ‘conditionally immortalized’ with activated CR3 on the neutrophil surface and was regulable Hoxb8. Epithelial cells were infected with sufficient to increase Gc association with the cells. C. trachomatis; to test for the role of the chlamydial These findings indicate that activated CR3 is cryptic plasmid known to contribute to tissue required for Opa- Gc uptake and may in part explain inflammation we also used a plasmidless bacterial the discrepancy between our results and those in the strain. Supernatants of infected cells were collected field regarding phagocytosis of Opa- Gc. We found and coincubated with neutrophils. no evidence that human neutrophils released C3 to In transwell migration assays, neutrophils showed opsonize Gc and generate the iC3b ligand for CR3. considerably higher migration towards supernatants Interestingly, unlike in the epithelial cell model, the of Chlamydia infected HeLa cells than to those of CR3-mediated association of Opa- Gc with uninfected cells. When wt neutrophils were neutrophils was independent of pili or pilin incubated in supernatants of infected HeLa cells, glycosylation. However, pili were found to enhance neutrophil apoptosis was significantly reduced. This contact and Gc proximity to the neutrophil surface, activity was neutrophil MyD88/TRIF independent in a CR3-independent manner. We conclude that and was absent from supernatants of HeLa cells unopsonized, Opa- Gc uses activated CR3 as the infected with the plasmidless strain. Surprisingly, predominant route of entry into primary human supernatants from both strains induced similar levels neutrophils. Our findings help explain why of TNF in wt but not MyD88-/-TRIF-/- neutrophils. unopsonized, Opa- Gc phenocopy complement- These findings suggest that neutrophil-activation by infected epithelial cells occurs both TLR- Neutrophils were isolated from 8 buffy coats of dependently and –independently and at least partly healthy blood donors using density gradient depends on the chlamydial plasmid. We are centrigugation and sedimentation in 1% polyvinyl currently investigating the role of the cryptic alcohol. Bacteria were isolated from peripheral chlamydial plasmid with particular focus on the blood after culturing for 24 hours in BacT Alert PF virulence factor Pgp3 using a Pgp3-deficient media, kept at -80ºC and thawed directly before plasmid-reconstituted chlamydial strain. Pgp3 had experiments. Bacteria were allowed to grow on been shown by others to play a role in pathogenicity Columbia agar with 5% sheep blood, and were then and tissue damage formation during chlamydial suspended in 0.9% NaCl in OD600 of 0.5. infection in mice and thus is a promising candidate. Neutrophils were stimulated with 80 µl of bacteria Our system illustrates that the interaction of infected suspension or phorbol 12-myristate 13-acetate epithelial cells with neutrophils alone may entertain (PMA, 100 nM). NETs were quantified as amount of inflammation and will permit more in-depth study of extracellular DNA bound to Sytox Green and immune response and tissue damage during measured with a fluorometer and visualized by chlamydial infection. immunofluorescent microscopy with Sytox Orange and antibody anti-MPO conjugated with FITC. 187 Intracellular production of reactive oxygen species (ROS) was analyzed by flow cytometry after Release of Neutrophil Extracellular Traps dihydrorhodamine 123 (DHR) staining. Extracellular (NETs) and Production of Reactive Oxygen release of ROS was measured by Species (ROS) in Response to Acinetobacter chemiluminescence assay using luminol as a Baumanii Isolated from Septic Patients. substrate and PMA, bacteria or medium alone as Olga Ciepiela1, Sandra Sieczkowska2, Weronika stimulators. Degradation of NETs was evaluated by Bystrzycka2, Joanna Rogulska1, Aneta Moskalik2, fluorescent microscopy after adding bacteria to Katarzyna Lewandowska2, Aneta Manda- NETs already released by PMA stimulation, after Handzlik1,3, Urszula Demkow1, 1Department of 1,2, 3 and 4 hours of coincubation of NETs and Laboratory Diagnostics and Clinical Immunology of bacteria at 37ºC and 5% CO2. Developmental Age, Medical University of Warsaw; Acinetobacter baumani stimulated NETs release 2Students’ Scientific Group at Department of which was confirmed in qualitative and quantitative Laboratory Diagnostics and Clinical Immunology of experiments. Amount of NETs released from cells Developmental Age, Medical University of Warsaw; measured as fluorescence of Sytox Green bound to 3Postgraduate School of Molecular Medicine, extracellular DNA was 29428±8 713RFU in Medical University of Warsaw unstimulated samples and 35878±11809 RFU in bacteria stimulated samples, p=0.047. Intracellular Neutrophil extracellular traps (NETs) constitute an ROS production was slightly induced: DHR important part of innate immunity. These threads of fluorescence was 15.3±3.97 mcf for live cells and DNA in complex with bactericidal enzymes, which 23.2±4.7 mcf for cell incubated with bacteria, are primarily accumulated within cytoplasmatic p=0.03; however fluorescence for PMA stimulated granules of neutrophils, are actively released from samples was 123.3±31.7 mcf. A. baumanii did not cells to immobilize and entrap invading pathogens. caused release ROS to extracellular matrix, as found Several studies indicated that NETs are released in chemiluminescent assay (lack of increase in during sepsis, as confirmed by plasma freeDNA and luminol fluorescence comparing with non stimulated citrullinated histones presence in patients blood. cells, p=0.94). A. baumani did not degrade NETs. Acinetobacter baumanii is a Gram-negative bacteria We found that Acinetobacter baumanii isolated from that can cause opportunistic infections, including patients with sepsis stimulates innate immunity by sepsis in immunocomprised patients. induction of NETs release and intracellular ROS The aim of the study was to assess the ability of production. No NETs degradation proves lack of Acinetobacter baumanii isolated from blood of nucleases expression in the bacteria. septic child to induce NETs and production of ROS from healthy neutrophils.

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The Role of Neutrophils in Mice during Cytotoxic PSMα Peptides from S. aureus Induce Chlamydia Infection Rapid Cytotoxicity and NET Formation Tom Zortel, Juliane Vier, Georg Häcker, Susanne Halla Björnsdottir1, Agnes Dahlstrand Rudin1, Karin Kirschnek, Institute for Medical Microbiology and Christenson1, Huamei Forsman2, Marios Stylianou3, Hygiene, University Medical Center, Hermann- Constantin Urban3, Claes Dahlgren2, Anna Herder-Straße 11, 79104 Freiburg i. Br. Karlsson2, Johan Bylund1 , 1Department of Oral Microbiology and Immunology, University of The sexually transmitted Chlamydia trachomatis Gothenburg, Sweden; 2The Phagocyte Research genital infection is prevalent worldwide. This Laboratory, Department of Rheumatology & obligate intracellular bacterium can cause severe Inflammation Research, University of Gothenburg, tissue damage such as hydrosalpinx formation and Sweden; 3Antifungal Immunity Group, Laboratory subsequent infertility in women. It is still not fully for Molecular Infection Medicine Sweden (MIMS) understood how the immune reaction causes this Department of Clinical Microbiology, Umeå pathology. Neutrophil granulocytes are candidates University, Sweden because of their ability to induce tissue damage in other infections. We studied infiltration of immune Community-associated methicillin resistant cells and tissue damage in a model of genital Staphylococcus aureus (CA-MRSA) are MRSA infection with Chlamydia muridarum in mice. We strains that have emerged over the last few decades analyzed lymphoid and myeloid subpopulations in and are both antibiotic resistant and have enhanced the genital tract after various time points by flow virulence and fitness. Among the secreted virulence cytometry. To analyze the effect of neutrophils on factors are phenol-soluble modulin (PSM) peptides. chlamydial infection and inflammation we used A group of PSM peptides of the alpha type are conditional Mcl-1 knock out (Mcl-1 ko) mice capable of activating human neutrophils, e.g. to deficient in mature neutrophils. produce reactive oxygen species, but they also As expected, the chlamydial burden in the genital possess cytotoxic properties to neutrophils at higher tract increased in the first week after infection and concentrations. bacteria were cleared 3 weeks later. There was no We here corroborated that PSMα peptides were difference in chlamydial burden during the course of cytotoxic to neutrophils in a concentration dependent infection between wild type (wt) and Mcl-1 ko mice. manner and found that cell death was associated In the first two weeks, neutrophils, monocytic cells with the formation of structures indistinguishable and T cells infiltrated the genital tract of wild type from neutrophil extracellular traps (NETs). These mice. We could not detect mature neutrophils in PSMα-induced NETs contained DNA, histones, and Mcl-1 ko mice but identified a population of γδ T granule proteins. As for the function of the PSMα- cells recruited to the genital tract that appeared only induced NETs we found that both bacteria (S. in Mcl-1 ko mice. Compared to the wt mice, the aureus) and fungi (Candida albicans) were captured number of monocytic and NK cells was reduced in in NETs, however with prolonged incubation S. the Mcl-1 ko mice. Preliminary data indicate that all aureus could degrade the NET structures, which is of the infected mice developed hydrosalpinx but the in accordance with their ability to secrete DNase. C. dilatation of the hydrosalpinx seemed to be less albicans on the other hand could not degrade the prominent in the Mcl-1 ko mice. Our findings NET structures and the PSMα-induced NETs could suggest that neutrophils had an impact on the inhibit the growth of C. albicans at comparable recruitment of other infiltrating immune cells and levels to NETs induced by the classic inducer may have an influence on genital tract tissue phorbol myristate acetate (PMA). damage. Other immune cell populations may In contrast to NET formation induced by PMA, contribute to these events. PSMα-induced NETs formed very rapidly (within minutes) and the process was independent of ROS production and MPO activity. The nuclear structure of neutrophils treated with PSMα was rapidly disintegrated suggesting that the peptide-induced nuclear destabilization could result in NET BXD strains exhibited a more lymphocytic formation. alveolitis. For example, BALF recovered from mice To establish whether the effect of PSMα peptides of the BXD45 strain was 68% neutrophils and 7% T was specific for neutrophils we studied the effects on lymphocytes whereas; BALF recovered from mice non-myeloid cells treated with PSMα. A melanoma of the BXD73 strain was 92% neutrophils and 2% T cell line was also sensitive to the cytotoxicity of lymphocytes. In addition, there was considerable PSMα peptides, but this death was not associated variability in activated CD4+ T cells; BALF from with expulsion of DNA extracellularly. mice of the BXD24 strain had 33% CD4+/CD69+ T Our data reveal an unusual cytotoxic effect of cells and from the BXD74 strain had 72% PSMα peptides that lead to the formation of NETs CD4+/CD69+ T cells. To examine the extent of and show that rapid NET formation may be triggered inflammation in the lung tissue H&E staining was through membrane disturbance by the cytotoxic performed. The results demonstrate differences in peptides. the lung pathology; the C57Bl/6 mice exhibited typical foci of inflammation whereas mice from the 190 BXD45 strain exhibited a limited diffuse infiltration into the lungs. These data support the contention that Genetic Susceptibility to Hypersensitivity genetic differences critically influence SR Pneumonitis responsiveness given that environmental factors Tae-Won Yoon1, Ae-Kyung Yi1, Robert W. were held constant. Williams2, Elizabeth A. Fitzpatrick1, 1Dept.of Micro. Immun. & Biochem., University of Tennessee Health 191 2 Science Center; Dept. of Genetics, Genomics and Informatics, University of Tennessee Health Science Neutrophil Plasticity in Thyroid Cancer Center Maria R. Galdiero1, Francesco Borriello1, Stefania Loffredo1, Gilda Varricchi1, Raffaella Iannone1, Hypersensitivity Pneumonitis (HP) is a T cell- Tiziana Lansione1, Gianni Marone1,2, 1Department of mediated interstitial lung disease that develops Translational Medical Sciences (DiSMeT) and following repeated exposure to a wide variety of Center for Basic and and Clinical Immunology inhaled environmental antigens. The disease is Research (CISI), University of Naples Federico II, characterized by alveolitis, noncaseating granulomas Naples, Italy; 2Institute of Experimental and, in some patients, develops into a chronic form Endocrinology and Oncology “Gaetano Salvatore” which is associated with fibrosis and emphysema. (IEOS), National Research Council (CNR), Naples, Not all individuals exposed to HP causative antigens Italy develop disease suggesting that genetic factors play a role in susceptibility. The host co-factor(s) that Background: Neutrophil function has long been play a role in determining whether an individual is viewed limited to the acute phase of inflammation susceptible to disease are unknown. We used the and resistance against pathogens. The role(s) of innovative systems genetics tool, the BXD panel of neutrophil in tumor initiation and progression remain recombinant inbred mice with the poorly understood. Neutrophils are found within Saccharopolyspora rectivirgula (SR) model of HP to inflammatory cells infiltrating the tumors and recent identify genetic loci and candidate genes associated studies placed them as key effector cells in the with disease development. We exposed the parental orchestration of the immune and inflammatory strains, C57Bl/6 and DBA/2, and eight BXD strains responses. However, the association between to SR for 3 weeks and analyzed the lungs 18h after neutrophil infiltration, clinicopathological features the last exposure. Bronchoalveolar lavage (BAL) and outcome in cancer patients remain to be was performed and cells recovered from the BAL clarified. Thyroid cancer (TC) is the most frequent fluid were analyzed by flow cytometry to determine type of cancer of the endocrine system, accounting the frequency of infiltrating immune cells. The for 70% of deaths due to endocrine cancers. No results reveal wide variability in the cellular studies are so far available investigating the role of composition of the BALF; with some BXD strains neutrophils in TC. exhibiting a neutrophilic alveolitis whereas other Objective: The objective of this study was to investigate the role of tumor-infiltrating neutrophils vaccinate mice implanted with subcutaneous in TC. Method: Highly purified human neutrophils syngeneic colon (CT26), prostate (TRAMP-C2) or (>99%) from healthy donors were stimulated, in renal (RENCA) cell line carcinomas. Vaccination vitro, with conditioned media derived from thyroid inhibited, and in some of the cases regressed, tumor cancer cell lines (TC-CM). Neutrophil biology and growth in a dose-dependent manner (94%, 71% and functions (e.g. chemotaxis, activation, plasticity, 72%, respectively, p<0.01), and demonstrated survival, gene expression and protein release of immune memory that prevented tumor recurrence neutrophils) were evaluated. Results: We found that (p<0.001). When tumor cells were administered TC cell lines produced soluble factors able to through the tail vein to generate lung metastases, promote neutrophil chemotaxis and significantly vaccination effectively reduced the number of improved neutrophil survival. In addition, TC-CM metastatic foci (by 15- and 23-folds, p<0.001), and induced neutrophil morphological changes and increased mice survival (p<0.01) relative to the activation (CD11b and CD66b up-regulation, scrambled control peptide. In all experiments, no CD62L shedding). Finally, TC CM induced the significant adverse responses were observed. production of reactive oxygen species (ROS), the Mechanistically, these effects were achieved by expression of pro-inflammatory and angiogenic significantly immune-modulating the tumor stimuli (CXCL8/IL-8, VEGF-A) and the release of microenvironment (TME): vaccination induced matrix metalloproteinase-9 (MMP-9). Conclusions: production of EMMPRIN-specific antibodies, TC cell lines produce soluble factors able to increased CD8+ T cells infiltration, enhanced their ‘educate’ neutrophils towards an activated functional cytotoxicity, and alleviated immune suppression by state. Experiments are in progress to understand the decreasing TGFbeta and elevating TNFalpha and IL- role of these “tumor-educated neutrophils” in 1beta concentrations. Additionally, vaccination modifying TC behavior. These data will advance our reduced angiogenesis and cell proliferation, but understanding on the molecular and cellular enhanced apoptosis. Thus, our results establish mechanism of the innate immune system in cancer. EMMPRIN as an attractive target, and show that peptide vaccination could be considered an efficient 192 immune modulating approach for the treatment and prevention of some types of cancer. Octa-Branched Peptide (MAP) Vaccination against Emmprin/CD147 Inhibits Tumor Growth and Metastasis by Immune Modulating the Microenvironment Michal A. Rahat1,2, Elina Simanovich1,2, Maya M. Rahat1, Ella Drazdov1, Miriam Walter1, Vera Brod1 1Immunotherapy lab, Carmel Medical Center; 2Ruth and Bruce Rappaport Faculty of Medicine, Technion

The Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is a multifunctional protein that mediates tumor cell-macrophage interactions, leading to induction of MMPs and VEGF. EMMPRIN is weakly expressed on immune cells and normal glandular epithelial cells, but overexpressed in tumor cells of high grade and stage and metastasis, in correlation to poor prognosis. We previously identified a novel epitope in EMMPRIN that is responsible for both MMP-9 and VEGF induction, and developed a polyclonal antibody that efficiently inhibited tumor growth and metastases in vivo. Here, we used this epitope, synthesized as an octa-branched multiple antigenic peptide (MAP), to LB01 iodide/RNase solution, and apoptosis was determined by measuring the sub-G0 population by The Role of Scavenger Receptor a (CD204) in flow cytometry. We found that in untreated cells the Apoptosis of Septic Neutrophils percentage of apoptotic WT cells was lower than the Tammy Ozment, Nikolas Hopkins, David Williams, percentage of apoptotic SRA deficient cells. When Department of Surgery, Quillen College of treated with LPS, the percentage of apoptotic cells Medicine, East Tennessee State University increased in both groups; however, the percentage of apoptotic cells was greater in the WT cells. We can In the United States ~750,000 patients/year develop conclude that apoptosis of primed neutrophils is sepsis syndrome with an overall mortality rate of increased in the absence of SRA while apoptosis of 28.6% (~215,000 deaths/year). Those patients that activated neutrophils is increased in the presence of survive the initial event may ultimately succumb to SRA. Thus, the decreased inflammation seen in widespread organ dysfunction due to prolonged septic SRA deficient mice is unlikely to be immune dysfunction and infection. Scavenger attributable to increased neutrophil apoptosis. receptor A (SRA, aka CD204) is a multi-functional receptor which binds endogenous ligands and LB02 pathogen associated molecular patterns. We have shown that SRA plays a pivotal role in mediating the Source and Mechanisms of Recruitment of a New pathogenesis of sepsis. Specifically, SRA deficient Subset of Neutrophils, the Pro-Angiogenic mice exhibit improved survival and decreased Neutrophils bacterial burdens in cecal ligation and puncture Cédric Seignez, Evelina Vågesjö, Mia Phillipson, (CLP) induced sepsis. SRA has previously been University of Uppsala considered a macrophage specific receptor; however, we have discovered that neutrophils express SRA in Background: We have recently described that response to sepsis. Neutrophils play an integral role hypoxic tissues induce the recruitment of a distinct in host defense by killing pathogens either after population of neutrophils (CD49d+ VEGFR1high internalization or by releasing degradative enzymes, CXCR4high and MMP9high), called pro-angiogenic reactive oxygen species, and neutrophil extracellular neutrophils, crucial for restoring the oxygen supply traps into the extracellular milieu. However, of the tissue by inducing the development of new neutrophils can also inflict significant collateral and functional blood vessels. This study aims to damage to the tissues. One way in which this characterise the origin of the pro-angiogenic collateral damage is limited in the normal immune neutrophils and the signals driving their recruitment response is by apoptosis of activated neutrophils. In into the hypoxic tissue. fact, it has been shown that delayed neutrophil Materials and methods: The study is performed in apoptosis is associated with increased inflammation a mouse model of ischemic hind limb following and tissue damage. Our data have revealed high ligation of the femoral artery. Different organs levels of SRA expression on tissue infiltrating (muscles, spleen, lung and blood) are dissociated and neutrophils during CLP sepsis in WT mice. In cell populations are analysed by flow cytometry 3 contrast there is decreased inflammation in the lungs hours following ischemia induction. Different of SRA deficient mice, though neutrophil infiltration pharmacological inhibitors are used to identify the was similar to that of WT mice. It is possible that the molecular signals inducing the mobilisation of pro- decreased inflammation found in SRA deficient angiogenic neutrophils visualised by histological septic mice could be the result of a higher rate of staining and intravital confocal microscopy. neutrophil apoptosis compared to that seen in WT Results: Pro-angiogenic neutrophils are stored in septic mice. To determine if this is the case bone two different pools in lung and spleen where they marrow neutrophils were harvested 72 h after CLP. represent respectively 10 and 15% of the total Cells were cultured with media alone or with ultra- neutrophil population of these organs. Following pure LPS (100 ng/ml) for 16 h. Untreated cells were induction of muscle ischemia, the pro-angiogenic considered primed, while LPS treated cells were neutrophils are recruited into the hypoxic muscle and considered primed and activated. Cells were fixed increase in numbers more than 200%. These with ethanol and stained with a propidium neutrophils are mobilised into circulation mainly from the spleen. Moreover, the inhibition of the derived from gelsolin were without effects on the adrenergic receptors impairs the release of the pro- neutrophil activity induced in mouse cells by angiogenic neutrophils from the spleen. formylated agonistic peptides specific for Fpr1 or Furthermore, the inhibition of the chemokine Fpr2, respectively. receptor CXCR4, of the vascular endothelial growth We have earlier shown that a pepducin derived from factor receptor 1 (VEGFR1) or of the integrin the third intracellular domain of FPR2 activates CD49d blocks the intramuscular recruitment of the neutrophils through FPR2, whereas the pro-angiogenic neutrophils. corresponding pepducin derived from FPR1 inhibits Conclusions: The newly described pro-angiogenic human neutrophil activity. The inhibitory effect was neutrophil are recruited into hypoxic tissues from the not mediated through FPR1 as expected. Instead the spleen by a multistep process. The recruitment is closely related FPR2 was hijacked by the pepducin. driven first by the release from the spleen after the This FPR2 inhibiting pepducin activates mouse activation of the receptors of the catecholamines and neutrophils and it was a dual ligand that targeted then by the extravasation into the muscle involving both Fpr1 and Fpr2. the receptors CXCR4 and VEGFR1 and the integrin Conclusions: Our basic pharmacological CD49d. characterization of a number of human FPR ligands in mouse neutrophils reveals that the modulating LB03 effect and receptor preference by certain ligands differ significantly across species. Prominent Distinct Modulating Effect and Receptor examples are, i) that the most potent FPR Preference on the Formyl Peptide Receptors antagonists have no effects on the activities triggered Expressed in Mouse Neutrophils for Ligands with by the corresponding mouse receptors and, ii) the Known Specificities for the Receptors Expressed FPR1 derived pepducin that inhibits FPR2 is a dual in Human Cells agonist for both Fpr1 and Fpr2. Malene Winther1, Michael Gabl1, Johan Bylund1, Ji Ming Wang2, Claes Dahlgren1, Huamei Forsman1, LB04 1 The Phagocyte Research Laboratory, University of Gothenburg; 2Cancer and Inflammation Program, Immune Cell Response to Bacterial Infection Frederick National Laboratory for Cancer Research Shayda R. Maleki-Toyserkani, Christopher P. Thomas , Emma J. Kidd, Les Baillie, Cardiff Background: The neutrophil expressed formyl University peptide receptors (FPR1 and FPR2 in human; and Fpr1 and Fpr2 in mouse) are pattern recognition Sepsis arises when the body’s response to an receptor belonging to the family of G-protein infection damages its own tissues and organs; coupled receptors (GPCRs). The activities of the defined as life threatening organ dysfunction caused FPRs, that play pivotal roles in host defense and by a dysregulated host response to infection. The inflammatory reactivity, are regulated by a large body’s entire system is engulfed by a deleterious number of conventional peptide agonist/antagonists, inflammatory response as opposed to local small molecules, reversed agonists, and allosteric inflammation resulting from a local infection. This modulators which display binding preference for can lead to septic shock and death if not recognised FPR1 or FPR2, or both (dual ligands). It is well early and treated promptly. Eicosanoids, including known that the prototype high affinity FPR1 agonist prostaglandins and leukotrienes, are a family of fMLF is a very low affinity agonist for Fpr1, but lipids that play key roles in inflammation including very little is known about the activity induced in helping leukocytes fight infection. Cells of the innate mouse neutrophils by other FPR ligands. In this immune system are major contributors of local study, we have determined recognition profiles for eicosanoids. It is known that varying bacterial the mouse Fprs of a number of ligands with known components result in a different profile of lipids and specificities for FPR1/FPR2. cytokines, and by characterising this lipid signature Results: The very potent FPR1 antagonist it may be possible to define a biomarker fingerprint cyclosporine H and the most potent FPR2 inhibitor predictive for sepsis. This study will address the described, a rhodamine linked PIP2 binding peptide hypothesis that the profile of the eicosanoid and cytokine response varies depending on the nature of diagnostics, allowing clinicians to administer the the bacterial pathogen. The experimental aim is to correct antibiotics for sepsis patients at the earliest identify signatures of eicosanoids and cytokines that opportunity. may represent useful prognostic indicators of bacterial infection. LB05 The spectrum of the human innate immune response is influenced by a number of factors including host A Mouse Model of Neonatal Staphylococcus genetic background and physiology. A standardised Epidermidis Infection human monocyte cell line (THP-1) was employed to Jacqueline CY Lai1, Pernilla Svedin1, Duc Ninh characterise the eicosanoid and cytokine response of Nguyen2, Per T. Sangild2,3, Tobias Strunk4,5, Andrew different bacterial agonists. A culture of THP-1 Currie6, Ofer Levy7, Xiaoyang Wang1, Carina monocyte cells was differentiated into monocyte- Mallard1, 1Perinatal Medicine Center, Department derived macrophages and production of eicosanoids of Physiology, University of Gothenburg, and cytokines was analysed. Eicosanoids were Gothenburg, Sweden; 2Section of Comparative identified using a combination of high performance Pediatrics and Nutrition, University of Copenhagen, liquid chromatography and multiple-reaction Denmark; 3Department of Pediatrics and Adolescent monitoring mass spectrometry. A DuoSet ELISA Medicine, Rigshospitalet, Copenhagen, Denmark; was used to identify the production of inflammatory 4Centre for Neonatal Research and Education, The cytokines. Primary cell responses to bacterial University of Western Australia, Perth, Australia; components were analysed using neutrophils 5Neonatal Clinical Care Unit, King Edward isolated from healthy human donors. Isolated Memorial Hospital, Subiaco, Australia; 6School of neutrophils were activated under various conditions Veterinary & Life Sciences, Murdoch University, and lipid extracts were characterised as described Murdoch, Australia; 7Department of Medicine, above. Division of Infectious Diseases, Boston Children's Initial THP-1 cell optimisation found that monocyte- Hospital, Harvard Medical School, Boston, USA derived macrophages differentiated from THP-1 cells with 0.025nM, 2nM, 25nM and 100nM phorbol Background and objective: Neonatal sepsis is a 12-myristate 13-acetate allowed no generation of the serious problem in neonatal intensive care units, eicosanoid 15-hydroxyicosatetraenoic acid when causing increased hospitalization costs and stimulated with calcium ionophore. THP-1 cells prolonged hospitalization stays. The coagulase- differentiated for 24-hours with 0.025nM PMA negative staphylococci, Staphylococcus epidermidis, produced the inflammatory cytokine IL-1β following have emerged as the predominant pathogen of late- treatment with calcium ionophore, and TNF-α onset neonatal sepsis in premature infants, following treatment with lipopolysaccharide from accounting for up to 78% of neonatal late-onset Escherichia coli. Generation of the eicosanoid 5- sepsis. Our laboratory previously determined that hydroxyicosatetraenoic acid by isolated human intravenous injection of S. epidermidis in postnatal neutrophils was detected following exposure to day 1 mice induces systemic cytokine production human toll-like receptor agonists (InvivoGen). Once and affects brain development. As neonatal mice the ability to detect agonist specific eicosanoid and have an immature immune system and deficiencies cytokine profiles in THP-1 cells has been established in leukocyte populations, we tested the response of primary immune effector cells will be used for older neonatal mice to the bacteria, so as to further experimental validation. determine the vulnerability window for infection- In conclusion, preliminary results indicate that induced brain injury in neonates. isolated human neutrophils display suitable Methods: S. epidermidis grown to mid-log phase physiological characteristics and substantiate that was administered intraperitoneally into postnatal day THP-1-derived cells are indeed macrophages. In 4 mice. Animals were monitored over time for body future work these approaches will be utilised to weight and temperature changes and long-term characterise the innate immune responses to survival. Bacterial colony-forming units from the bacterial infection with a view to identifying peripheral blood, liver and spleen was determined by pathogen-specific profiles. Bacteria-specific bio- spot plating. Cytokine production was determined by signatures will underpin the development of rapid multiplex cytokine bead array analysis. Circulating cell-free DNA was detected using a Quant-iT functions; immune complexes are critical in driving PicoGreen dsDNA Assay Kit. Immuno- range of chronic autoinflammatory disorders that histochemistry of brain sections was performed to rely on neutrophilic inflammation. However, determine the effects of the infection and insoluble immune complexes are also known to inflammation in the brain. induce neutrophil apoptosis, an essential process in Results: Injection of S. epidermidis led to the resolution of inflammation. bacteremia within two hours in a dose-dependent Here we present a novel, non-canonical signaling manner. There was a dose-dependent decrease in pathway, FcgR – PI3Kb/d – Cdc42 – Pak – Mek – body weight gain after the infection. Pro- Erk, that promotes immune-complex induced inflammatory cytokines were significantly up- apoptosis in human neutrophils. This pathway regulated in the peripheral blood of infected animals represents an example of Ras and Raf-independent by 2 to 24 hours post infection and tapered down by Erk activation, where instead, an alternative small 48 hours, in association with the clearance of the GTPase (Cdc42) and an alternative MAP3K (Pak) bacteria from the blood. Neutrophil and monocyte are involved. The non-canonical pathway is chemotactic cytokines (CXCL1 and MCP-1), moreover a rare example of PI3K driven activation granulocyte-colony stimulating factor, and caspase-3 of Cdc42 rather than Rac. activity was also elevated in the brain by post Mechanistically, we show that the non-canonical infection. An elevated level of cell-free DNA was signaling pathway drives apoptosis of human observed in postnatal day 9 but not day 4 mice after neutrophils by regulating the ratio of the Bcl2 family S. epidermidis infection. No difference in the gray proteins Mcl1 and Bax1. We conclude that our and white matter volume in the brain was detected novel, non-canonical signaling pathway may be 10 days after the infection. important for the resolution of inflammation in Conclusions: Although intraperitoneal injection of chronic inflammatory diseases that rely on immune- S. epidermidis into postnatal day 4 animals led to a complexes driven neutrophil activation. similar infection pattern and response compared to In addition, we demonstrate that other effector intravenous infection of 1-day old animals, no gross functions induced by insoluble immune complexes morphological injury in the brain was detected. (ROS generation, selectin shedding, cytokine These data give support to the hypothesis that the release) are also PI3Kb/d dependent but employ response to infection, and the vulnerability of the separate pathways downstream of PI3K. immature brain to infection and inflammation is age Given that neutrophils are terminally differentiated dependent. Delineating the difference between cells that are not amenable to transfection or neonatal mice of different ages will provide insight transduction, we analysed mouse neutrophils with a into factors leading to brain injury upon infection view to making use of available knock-out models to and inflammation, and allow us to develop enable studying the novel pathway genetically. therapeutic treatments for infants affected by these Although insoluble immune complexes drive PI3K conditions. and Erk dependent apoptosis of mouse neutrophils, the two species use separate pathways. Control LB06 experiments carried out with fMLP stimulated neutrophils demonstrated that fMLP stimulated Erk Immune Complex Induced Apoptosis is activation is also controlled by separate pathways in Controlled by Non-Canonical PI3K – Cdc42 – human and mouse neutrophils. Our work therefore Pak – Erk Signaling Promotes in Human but not emphasizes that results obtained with mouse Mouse Neutrophils neutrophils need to be interpreted with caution, as Julia Chu, Sonja Vermeren, Ian Dransfield, Adriano they may or may not be conserved in human. G. Rossi, The University of Edinburgh LB07 Neutrophils are peripheral blood leukocytes that represent the first line of defense against bacterial Neutrophil Extracellular Trap Formation among and fungal infections and are also key to generating Neutrophil Subpopulations. inflammatory response. Immune complexes are Carmen Herrera-Hidalgo1, Cedric Seignez1, Gustaf important mediators of many neutrophil effector Christoffersson2,1, Håkan Wallén3, Charlotte Thålin3, Laszlo Szekely3, Mia Phillipson1, 1Department of pathogens. In contrast to NETs, chromatin within Medical Cell Biology, Uppsala University, Uppsala, intact nuclei is highly organized in three- Sweden.; 2La Jolla Institute for Allergy and dimensional space. Here we utilized genome-wide Immunology, La Jolla, CA.; 3Department of Clinical chromosome conformation capture to map the Sciences, Danderyd Hospital, Stockholm, Sweden organization of neutrophil chromatin during NETosis. We find that the lobed morphology of the Neutrophils have traditionally been considered to neutrophil nucleus enforces a unique organization of contribute to the non-specific first line of immune chromatin compared to that of non-lobed nuclei. We defence during acute inflammation, and as such further identify distinct changes in chromatin disregarded as rudimentary leukocytes. However, as organization that are dependent on the NET- their contributions to tumour homeostasis, chronic inducing stimulus. Together with epigenetic and inflammation and adaptive immunity were transcriptional profiling, we offer the first molecular- discovered, neutrophils have been more recently resolution view of neutrophil activation and NET acknowledged for offering a diverse set of formation. specialized functions. We recently identified a pro- angiogenic circulating neutrophil subset (CD49d+ LB09 VEGFR1high CXCR4high) that was specifically recruited by VEGF-A to sites of hypoxia, where they Role of the Atypical Chemokine Receptor were fundamental for angiogenesis and restitution of CCRL2 in Intestinal Inflammation blood flow at least partly due to their high Tiziana Schioppa1,2, Annalisa Del Prete1,2, Francesca expression levels of MMP-9. The current project Sozio1,2, Stefania Vetrano1, Silvano Sozzani1,2, investigates the ability of classic as well as 1University of Brescia, Dept of Molecular and proangiogenic neutrophils to form neutrophil Translational Medicine, Italy; 2Humanitas Clinical extracellular traps (NETs), and if this is altered in and Research Center, Rozzano, Italy persons with cancer. In addition, the ability of ascites fluid from cancer patients to induce NET Intestinal bowel disease (IBD), comprising Crohn's formation is studied in the different neutrophil disease and ulcerative colitis, is a chronic subsets. Interestingly, our results indicate that when inflammatory immune-mediated disease at the isolated from cancer patients, the proportion of pro- intersection of complex interactions between angiogenic neutrophils positive for NETs was higher genetics, environment and gut microbiota. The main than that of the pro-inflammatory subset. Further, pathological feature of IBD involves a massive ascites fluid from patients with cancer strongly infiltration of immune cells, in particular neutrophils induced basal neutrophil NET formation. into the intestinal tissue. However, the beneficial versus deleterious role of neutrophils during this LB08 pathological condition is still a matter of debate. Chemokine and their receptors play an important Neutrophil Chromatin Undergoes Dramatic role in the recruitment of leucocytes in the intestinal Stimulation-Specific Topological Changes during mucosa of IBD patients (1-2). CCRL2/ACKR5 is an Extracellular Trap Formation orphan chemokine receptor which apparently does Matthew Denholtz, Yina Zhu, Simon Dohrmann, not activate any chemokine conventional signaling. Takeshi Isoda, Victor Nizet, Corenlis Murre, For this reason, it has been included in the subset of University of California, San Diego “atypical chemokine receptors” (ACKRs; 3), which are also characterized by ligand scavenging Upon encountering activating stimuli neutrophils functions that dampen local inflammation. This undergo a unique form of lytic cell death termed receptor is rapidly induced in many activated cells, NETosis. During NETosis the neutrophil nucleus including macrophages, neutrophils, and dendritic undergoes a catastrophic loss of organization, as the cells (4) and in preliminary experiments is integrity of the nuclear envelope is compromised and upregulated in the mucosa of Crohn’s patients. chromatin is extruded into the extracellular space. The aim of our work was to define the role of The resulting cytotoxic granule-laced neutrophil CCRL2 during an experimental model of extracellular trap (NET) confines and kills inflammation-induced colitis (dextran sodium sulphate-DSS model) by using CCRL2 deficient dopamine receptors D1A, D2, and D4, GABA-A mice. CCRL2 KO mice treated with 2.5% DSS for 7 receptors a2, a3, and a4, serotonin receptor 3a, days show exacerbated phenotype, including higher glutamate receptor m5, glutamate receptor body loss, increased bleeding and shorter colon ionotropic NMDA 1, 2a, 2b, and 2c, and the length compared to WT animals. The pathogenic cholinergic receptor nicotinic beta 4 subunit. In mechanism underlying the observed phenotype in addition, we found decreased expression of immune- CCRL2 KO is under investigation. related IL-5, IL-12a, IL-16, and solute carrier family The understanding of the role of CCRL2 in the 6 (Slc6), members 2 and 4. We then used the Library recruitment of immune cells at colonic mucosa level of Integrated Network-Based Cellular Signatures might provide new insights in the treatment of (LINCS), a biological network-based strategy that pathological intestinal inflammation. utilizes gene profile signatures to suggest probability (1) Fegn, L., and Wang, Z., J. Laryngol. Otol. 2009. outcomes in terms of drug mimics and effects, to (2) Katoh H., et al., Cancer Cell 2013. further examine the altered gene profile during (3) Bachelerie et al, Pharmacol Rev 2004. endotoxin tolerance. LINCS analysis revealed that (4) Del Prete A. et al, European J. of Immunology the top three compounds having similar effects as 2013. those seen in the endotoxin tolerant state are RG108, an inhibitor of DNA methyltransferase; roscovitine, LB10 a potent and selective cyclin dependent kinase (CDK) inhibitor for Cdc2, CDK2, and CDK5; and Gene Expression of Neurotransmitter Receptors selumetinib, a highly selective mitogen-activated and Immune-Related Proteins in the Nucleus protein kinase 1 (MEK1) inhibitor. Our LINCS Accumbens during Endotoxin Tolerance analysis data suggests that, during endotoxin Sulie L. Chang, Wenfei Huang, Andrew M. tolerance, expression of DNA methyltransferase, Dieterich, Institute of NeuroImmune Pharmacology CDK, and MEK1 in the NAc may be inhibited. and Department of Biological Sciences, Seton Hall (Supported in part by NIH grant DA036175). University LB11 The nucleus accumbens (NAc) is the brain area involved in the reward system and the development Neutrophils are Recruited to the Lung in a CCR1 of addictive behavior. We previously reported that and Complement C5 Dependent Manner systemic disorders, such as HIV and bacterial Independent from Complement C3 and Type I infections, may trigger neuro-inflammation leading IFN Receptor during Infection with Modified to increased use of addictive substances. We also Vaccinia Virus Ankara found that, during endotoxin tolerance, Philip J.R. Price1, Lino E. Torres-Dominguez1, lipopolysaccride (LPS)-induced expression of TNF- Christine Brandmuller1, Bruno Luckow2, Zoltan alpha, IL-1beta, and IL-6 still occurs in the brain Banki3, Heribert Stoiber3, Admar Verschoor4, even when these three cytokines are no longer Carsten J. Kirschning5, Gerd Sutter1, Michael H. induced in the periphery. In the present study, we Lehmann1, 1Institute for Infectious Diseases and measured the gene expression of the dopamine (DA) Zoonoses, Ludwig-Maximilians-Universität receptors, GABA-A receptors, serotonin receptors München, Munich, Germany; 2Klinikum der (Htr), glutamate receptors (Gr), glutamate receptor Universität München, Medizinische Klinik und ionotropic NMDA (Grin), cholinergic (Ch) Poliklinik IV, Arbeitsgruppe Klinische Biochemie, receptors, and immune-related genes in the NAc of Munich, Germany; 3Division of Virology, Medical male F344 rats (7-8 wks) treated with LPS University of Innsbruck, Innsbruck, Austria; (progressively from 250 μg/kg, 500 μg/kg, 1 mg/kg, 4Institute for Medical Microbiology, Immunology 2 mg/kg, 4 mg/kg, 8 mg/kg, 16 mg/kg to 32 mg/kg) and Hygiene, Technische Universität München, using real-time PCR assay. Among the 68 genes Munich, Germany; 5Institute of Medical examined, we found increased gene expression for Microbiology, University of Duisburg-Essen, Essen, lL-1alpha, IL-1beta, as well as the Types 1 and 2 IL- Germany 1 receptors. There was decreased gene expression for key neurotransmitter receptors, including Innate immune responses influence the outcome of vaccination. Modified Vaccinia virus Ankara 1Department of Molecular Medicine, College of (MVA), which is used as a versatile vector for Medicine, Inha University, Incheon 22212, Republic antigen delivery, has been proven effective in of Korea; 2Hypoxia-related Disease Research preclinical and clinical studies. To understand why Center, College of Medicine, Inha University, vaccination with non-replicating MVA induces Incheon 22212, Republic of Korea; 3Department of immunity without need for an adjuvant, we study Radiology, University of Texas Health Science MVA-induced cytokine expression in macrophages Center at San Antonio; 4Department of Clinical and leukocyte recruitment to the site of inoculation. Laboratory Sciences, University of Texas Health We and others have shown that MVA, but not other Science Center at San Antonio ; 5Department of vaccinia virus strains, induces robust innate immune Biochemistry, University of Texas Health Science responses including recruitment of leukocytes to the Center at San Antonio lung within hours (Lehmann et al. 2009 J. Virol., Lehmann et al. 2015 Virol. J.). During the early Diabetes promotes the S-glutathionylation, stages of MVA infection neutrophils represent the inactivation and subsequent degradation of mitogen largest cell population of the infiltrating leukocytes, activated protein kinase phosphatase 1 (MKP-1) in which are recruited by a mechanism involving blood monocytes, and hematopoietic MKP-1- chemokine (C-C motif) receptor 1 (CCR1) (Price et deficiency in atherosclerosis-prone mice accelerates al. 2014 J. Virol.) but not type I interferon receptor atherosclerotic lesion formation, but the underlying (Lehmann et al. 2016 J. Leukoc. Biol.). mechanisms were not known. To determine the Vaccinia virus activates both the classical and mechanisms through which MKP-1 deficiency in alternative complement pathways and complement is monocytes and macrophages promotes known to play an important role in poxvirus atherogenesis. Transplantation of MKP-1-deficient immunity. Activation of the terminal C5 component bone marrow into LDL-R-/- (MKP-1LeuKO) mice leads to the release of the small peptide fragment accelerated high fat diet (HFD)-induced C5a, which is a potent immune activator and atherosclerotic lesion formation. After 12 weeks of chemoattractant, particularly for neutrophils. HFD feeding, MKP-1LeuKO mice showed increases Therefore, the role of complement component C5 in in lesion size in both the aortic root (1.2-fold) and MVA-triggered recruitment of leukocytes was the aorta (1.6-fold), despite reduced plasma investigated in C5 deficient mice as well as in wild cholesterol levels. Macrophage content was type mice treated with an antibody that blocks C5. increased in lesions of MKP-1LeuKO mice Neutrophil recruitment to the lungs of C5 deficient compared to mice that received wildtype bone mice and anti-C5 treated wild type mice was marrow. After only 6 weeks on a HFD, the in vivo dramatically reduced as compared to untreated chemotactic activity of monocytes was already control mice at 12 h post infection. Additionally, significantly increased in MKP-1LeuKO mice. using C3 deficient mice, we found that C5 functions MKP-1 deficiency in monocytes and macrophages independently of the central complement component promotes and accelerates atherosclerotic lesion C3 to support neutrophil recruitment during the formation by hyper-sensitizing monocytes to inflammatory response to MVA (Price et al. 2015 J. chemokine-induced recruitment, predisposing Immunol.). We conclude that CCR1 and macrophages to M1 polarization, decreased complement component C5 play non-redundant autophagy and oxysterol-induced cell death whereas roles during the process of neutrophil recruitment to overexpression of MKP-1 protects macrophages the site of viral infection. against metabolic stress-induced dysfunction. MKP- 1 serves as master-regulator of macrophage LB12 phenotype and function and its dysregulation by metabolic stress may be a major contributor to Monocytic MKP-1 is a Sensor of the Metabolic atherogenesis and the progression of atherosclerotic Environment and Regulates the Functional and plaques. Phenotypic Fate of Monocyte-Derived Macrophages in Atherosclerosis Hong Seok Kim1,2, Sina Tavakoli3, Leigh Ann Piefer4, Huynh Nga Nguyen5, Reto Asmis3,4,5 with two model pathogens, representing bacterial LB13 (Staphylococcus aureus) and fungal (Candida albicans) sepsis for a 4h time course, followed by QUANTIM- Quantification of Innate Immune FACS-based analyses of cell activation and Function in Whole Blood Infection Assays pathogen association with immune cells. Ines Leonhardt1,2, Kerstin Hünniger1,2, Daniel Preliminary analysis showed a strong post-operative Thomas-Rüddel3, Teresa Lehnert2, Marc-Thilo increase in neutrophil cell counts. Interestingly, the Figge2, Oliver Kurzai1,2, 1Septomics Research increase in cell number results in a faster association Center, Friedrich Schiller University Jena, Jena; of C. albicans and S.aureus, but does not correlate 2Leibniz Institute for Natural Product Research and with a stronger activation of neutrophils. In ongoing Infection Biology, Hans-Knöll-Institute, Jena; 3Jena experiments, differences in pathogen elimination University Hospital, Department of Anesthesiology before and after extracorporeal circulation will be and Intensive Care Medicine quantified and analyzed using biomathematical modeling. Additionally, cytokine levels will be Little progress has been made in recent years assessed and compared to known cytokine profiles towards bringing immunomodulatory therapies for in terms of immune activation. sepsis into clinical use. A major reason for this is the In conclusion, with QUANTIM we provide novel heterogeneity of sepsis as a clinical syndrome, methods for individualized quantification of resulting in highly diverse pathological conditions alterations in immune effector functions by using an and showing variable kinetics within patient ex vivo whole blood model of infection combined populations. Resultingly, classification of sepsis with advanced biomathematical quantification. patients in terms of their immune status is important and necessary for future studies (e.g. LB14 immunomodulatory therapy approaches). With QUANTIM we seek to address this using a human Recycling and Scavenging Properties of CCRL2 whole blood model of infection combined with Chiara Mazzotti1, Annalisa Del Prete1,2, Luisa biomathematical modeling to quantify the global Gazzurelli1, Marcus Thelen3, Daniela Bosisio1, status of the innate immune response to infection Silvano Sozzani1,2, 1Dept of Molecular and (Hünniger et al, PLOSCompBiol 2014). Translational Medicine, University of Brescia; In preliminary work, human peripheral blood was 2Humanitas Clinical and Research Center; 3Institute collected from healthy volunteers and infected with a for Research in Biomedicine, Bellinzona broad range of relevant pathogens. The resulting data provides time-resolved data on cell activation CCRL2 (C-C chemokine receptor-like 2) is a non- and allows a comparative analysis of regulatory signalling seven-transmembrane domain receptor networks governing inflammation and pathogen related to the atypical chemokine receptors (ACKRs) elimination. Before the whole blood model is ready family. CCRL2 is rapidly expressed by many to be examined with as heterogeneous of a activated leukocytes, including neutrophils[1]. At population as septic patients, it needs to be validated present, the best-characterized function of CCRL2 is with a more homogeneous population. For this its ability to act at the endothelial surfaces as a purpose, cardiac surgery with extracorporeal presenting molecule for chemerin[2], [3]. The aim of circulation is ideal, as it provides an inflammatory this study was to explore the biology of CCRL2, stimulus that is both time-defined and relatively focusing on the common ACKRs features, that homogeneous. With the ability to investigate the include constitutive internalization, recycling, ligand blood of the same patient at defined time points scavenging and transcytosis[3], [4]. before and after extracorporeal circulation, inter- To this goal and to avoid the steric hindrance of individual differences and effects of inflammation antibodies, HeLa cells were transfected with a can be clearly distinguished. construct carrying the Acyl Carrier Protein (ACP) For this study, blood was taken from patients before tag at the N-terminus of CCRL2. Receptors were cardiac surgery (pre-operative) immediately after then labeled using a phosphopantetheine transferase surgery (post-operative) and 1 day after admission to that binds a fluorophore to the ACP tag. Cell intensive care. Whole blood infection was performed analysis by fluorescence microscopy revealed constitutive internalization and recycling of CCRL2. We here use an inducible Hoxb8 system to generate Inside the cell, CCRL2 co-localized with EEA1- and differentiate mouse neutrophil progenitors to positive and Rab5-positive vesicles and with investigate the capacity of leukaemia-associated recycling compartments mainly characterized by mutations to affect differentiation and survival of Rab11-positive vesicles. Apparently, CCRL2 neutrophil granulocytes. Several C/EBPα mutations internalization was not regulated by the presence of were expressed in these neutrophil progenitors, chemerin or CCL19, a chemokine also suggested including an internal tandem duplication (K313 binding this receptor. However, the expression of duplication) in the leucine zipper region, the BRM2 CCRL2 conferred to transfected cells the ability to mutation (which carries two amino acid substitutions scavenge chemerin, but not CCL19, from the cell in the basic region) and the p30 N-terminal supernatant. truncation mutant, which is generated by using an To further investigate the mechanisms involved in alternative start codon. CCRL2 internalization, receptors carrying different Expression of p30 C/EBPα impaired neutrophil degree of truncations at the C-terminus were differentiation and had a negative effect on survival expressed in HeLa cells. FACS analysis of during differentiation. Expression of C/EBPα transfected cells showed reduced membrane carrying the K313 duplication exerted a strong expression of all the truncated forms, as described differentiation-retarding effect while the BRM2 for other ACKRs, such as ACKR2[5]. Nevertheless, mutant only led to mild differentiation defects. The preliminary experiments highlighted a lower rate of lack of differentiation was associated with internalization of the truncated receptor forms maintained high expression levels of anti-apoptotic suggesting the involvement of CCRL2 C-terminus in Bcl-2 and Mcl-1 and low levels of pro-apoptotic the internalization process. Bim. Future experiments will be performed to better The K313 mutation has been reported to have low characterize the mechanisms of CCRL2 transcriptional activity in other cellular systems. internalization. Also, it will be interesting to confirm However reporter assays showed strong C/EBPα the scavenging ability, to date observed in activity in neutrophil progenitors expressing the transfected cells, in a more physiological setting. K313 mutant. We show that C/EBPα expression is [1] A. Mantovani, et al. Nat. Rev. Immunol., vol. 6, upregulated during differentiation and subsequently no. 12, pp. 907–918, 2006. downregulated at later stages of maturity, but [2] B. a Zabel, Set al. J. Exp. Med., vol. 205, no. 10, interestingly, expression of the K313 mutant as well pp. 2207–2220, 2008. as the BRM2 mutant was much higher than wild [3] S. Gonzalvo-Feo, et al. J. Immunol., vol. 192, no. type despite similar levels of transcription. These 5, pp. 2366–73, 2014. expression levels remained high during [4] A. Vacchini, et al. J. Leukoc. Biol., pp. differentiation. These results suggest that the K313 jlb.2MR1015–477R–, Jan. 2016. and BRM2 mutations may contribute to AML by [5] C. V. McCulloch, et al. J. Biol. Chem., vol. 283, blocking the downregulation of C/EBPα at later no. 12, pp. 7972–82, Mar. 2008. stages of differentiation keeping the cells in an incompletely differentiated state. LB15 LB16 Leukaemia-Associated Mutations of C/Ebpa Inhibit Neutrophil Differentiation Platelets Enhance Antifungal Activity of Human Isabel Mölter, Ian Gentle, Georg Häcker, Neutrophils during Candida Infection Universitätsklinikum Freiburg Kerstin Hünniger1,2, Diana Nessel1,2, Oliver Kurzai1,2, 1Septomics Research Center, Friedrich CCAAT enhancer binding protein alpha (C/EBPα), a Schiller University Jena, Jena; 2Leibniz Institute for master regulator of granulopoiesis, is known to be Natural Product Research and Infection Biology, involved in cell proliferation and differentiation. Hans-Knöll-Institute, Jena C/EBPα has been reported to be mutated in 9 % of acute myeloid leukaemia (AML) cases. Besides their crucial importance in blood hemostasis, platelets have been identified as effector cells with diverse immune functions that participate LB17 in the host defense against pathogens. Using an ex vivo human whole blood infection model we have Swimming Motility Mediates NET Formation already shown that innate immune activation Stimulated by Flagellated Bacteria triggered by the two most prevalent Candida spp., Balazs Rada1, Madison Floyd1, Matthew Winn1, Candida albicans and Candida glabrata, differs Payel Sil1, Benoit Chassaing2, Linda L. McCarter3, considerably. However, if platelets contribute to Andrew T. Gewirtz2, Joanna B. Goldberg4, these differences needs to be elucidated. 1University of Georgia, Department of Infectious To compare platelet activation triggered by C. Diseases; 2Georgia State University, Institute for albicans and C. glabrata, we infected human whole Biomedical Sciences; 3The University of Iowa, blood and quantified platelet effector mechanisms Department of Microbiology; 4Emory University after confrontation with the two species at different School of Medicine time points. Both species induced increase in surface exposure of activation marker CD62P on platelets Pseudomonas aeruginosa is an opportunistic associated with immune cells over time. However, pathogen causing severe infections often infection with C. glabrata resulted in a less characterized by robust neutrophilic infiltration. pronounced response than C. albicans. In line with Neutrophils provide the first line of defense against this, secretion of platelet effector proteins, soluble P- P. aeruginosa. Aside from their phagocytic defense, Selectin (sCD62P), PDGF-BB and PF4, was more neutrophils also release neutrophil extracellular traps induced by C.albicans. Whereas platelets interact (NETs) to kill bacteria. Although NET formation is with various leukocytes during whole blood an important antimicrobial process, the details of its infection, no association to the pathogens has been mechanism are largely unknown. The identity of the observed. To get further insight in the interaction of main components of P. aeruginosa responsible for platelets with the two Candida spp. we performed triggering NET formation in neutrophils is unclear. confrontation assays with purified platelets. Using In this study, our focus was to identify the main autologous plasma with heat-inactivated bacterial component mediating NET formation and complement during confrontation, a time-dependent to gain insight into the underlying mechanism. We increase in platelet-C. albicans interaction could be found that bacterial flagellum is the primary observed. Binding of platelets to C.glabrata was component of P. aeruginosa responsible for inducing markedly lower than compared to C. albicans. NET extrusion as flagellum-deficient P. aeruginosa Beside direct interaction with pathogens, immune does not trigger NETs. Surprisingly, purified P. functions of stimulated platelets are mediated by aeruginosa flagellin, the monomeric component of crosstalk with immune cells. Therefore, we the flagellum, does not stimulate NET formation in investigated the contribution of platelets on human neutrophils. P. aeruginosa-induced NET Candida-induced activation of neutrophils, which formation is independent of the flagellum-sensing have been shown to play a predominant role during receptors TLR5 and NLRC4 in both human and C. albicans infection. When confrontation of mouse neutrophils. Flagellum-expressing but primary neutrophils and C.albicans was performed flagellar motor-deficient bacterial strains failed to in the presence of platelets, neutrophil activation induce NET release. On the contrary, when detected by increased surface levels of CD66b and flagellum- and flagellar motor-deficient P. down-regulation of CD16 was markedly higher than aeruginosa mutants were centrifuged on neutrophils, induced by C. albicans alone. This synergistic effect their abilities to trigger NET formation were entirely seems to be independent of the complement. In restored. Thus, we found that swimming motility, contrast, neutrophil activation in response to C. but not flagellum expression, mediates NET release glabrata was markedly lower than compared to C. induced by flagellated P. aeruginosa. The motAB albicans and only slightly enhanced by the presence operon of P. aeruginosa encoding essential flagellar of platelets. motor genes proved to be critical. Complementation Taken together, our data demonstrate a strong experiments showed that reintroduction of the activation of platelets by C. albicans that motAB genes into motAB-deficient P. aeruginosa synergistically influence neutrophil activation strains also restored their impaired ability to trigger induced by C. albicans. NET formation. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also of human neutrophils by A. fumigatus CEA10 essential for flagellum to mediate NET production. hyphae without prior separation of the two cell types This indicates that flagellated bacteria are first at a time point of NET formation (3.5h). phagocytosed prior to release NET formation. We developed a protocol for parallel extraction and Taken together, our study presents here for the first enrichment of both NETs covering hyphae and time flagellum as the main organelle of planktonic secreted proteins during NET formation. Besides a bacteria responsible for mediating NET release. lysis-based approach for the cellular fraction Furthermore, swimming motility propelled by the consisting of hyphae entrapped in NETs, secreted flagellum, rather than binding of the bacteria to proteins were enriched by C4 solid-phase extraction flagellum-sensing receptors on host cells, is required of the supernatant fraction. Using a multiplexed for P. aeruginosa to induce NETs. isobaric labelling approach and TiO2 phosphopeptide enrichment combined with nLC- LB18 MS/MS analysis, we were able to identify and quantify 273 differentially regulated proteins of 856 Simultaneous Dual Proteome Analysis towards proteins in total on the fungal side and 298 Understanding Neutrophil Interaction with differentially regulated proteins of 1950 proteins in Aspergillus Fumigatus total on the host side compared to NET controls Iordana A. Shopova1,2, Thomas Krüger1, Olaf induced with PMA and hyphae grown alone. Kniemeyer1, Axel A. Brakhage1, 11Leibniz Institute We showed that neutrophil activities induce higher for Natural Product and Infection Biology (Hans expression of fungal signal transduction proteins Knöll Institute); 2Friedrich Schiller University, Jena from the GPCR-cAMP and CalA axis as well as Ste- 20 tyrosine kinase compared to hyphae grown alone Systemic infections in immunocompromised hosts as control. Neutrophil stress caused repression of caused by the opportunistic filamentous fungus fungal proteins responsible for siderophore–and- Aspergillus fumigatus are a significant burden for ergosterol biosynthesis and of the 60S ribosomal the hospital community due to limited therapeutic translation. Fungal Afyap1 dependent and options and poor understanding of efficient immune independent heat-shock and ROS response was mediated clearance in healthy individuals. overall repressed. Neutrophils are indispensable in executing the Antifungal NETs contained in higher abundance the elimination of fungal spores and hyphal elements by calprotectin complex, lactotransferrin and PTX3 various intra-and-extracellular means: degranulation, versus control NETs and alternative and core phagocytosis, formation of reactive oxygen (ROS) - histones with changed stoichiometric ratios were and–nitrogen species (RNI) as well as neutrophil detected. We observed that neutrophil activation by extracellular trap (NET) formation. We the fungus and by PMA has distinct signatures by demonstrated that NETs occur both in vitro and in detecting unique 275 interactome-specific proteins vivo but exhibit only fungistatic effects. Dual and 50 PMA-specific proteins. The results of the transcriptome analysis resulted in high coverage of phosphopeptide enrichment were implemented in a the fungal transcriptome and low coverage of the model of the signal transduction cascade of NET human transcriptome. Stress-response proteomics formation. revealed that neither ROS, nor RNI mediate the direct killing of A. fumigatus, since the fungus LB19 expressed an arsenal of fungal ROS/RNI detoxifying systems (catalase, superoxide dismutase, Endogenously Produced Tnfa Contributes to the flavohaemoproteins) switched by the transcription Expression of CXCL10 in IFN?3- Activated factors (TFs) Afyap1, AfSkn7. These factors, were Plasmacytoid Dendritic Cells surprisingly dispensable for fungal virulence in Giulia Finotti, Nicola Tamassia, Federica Calzetti, infection mouse models. Marco A. Cassatella, University of Verona Thus, a key paradigm- how A. fumigatus hyphae activate neutrophils and how they ultimately kill the The interplay between plasmacytoid dendritic cells fungus, needs be resolved. We therefore conducted a (pDCs) and members of the IFNλfamily (IFNλ1, simultaneous dual proteome study of the activation IFNλ2 and IFNλ3) is becoming increasingly relevant, particularly at the light of their key role in fungus Paracoccidioides brasiliensis (Pb). induction of the antiviral state during HCV infection. Polymorphonuclear neutrophils (PMN) participate in Previous data showed that pDCs respond to IFNλ1 an active way of the innate immunity and directing (1). Hovewer, the immunomodulatory activities of the acquired immune response towards an effective IFNλ3 on pDCs are poorly defined. response. PMN of paracoccidioidomycosis patients We report (2) that pDCs incubated with IFNλ3: are less effective than those of healthy individuals, prolong their survival; alter their expression pattern suggesting that Pb exerts some deactivating effect on of surface HLA-DRα, CD123, CD86, and CD303; this cell population. In that way, we aimed to are induced to express typical ISG mRNAs, develop a treatment capable of stimulating PMN that including IFIT1, ISG15, and CXCL10; and time migrate to the site of injury through low-level laser dependently produce IFNα, CXCL10 and even therapy (LLLT). Methodology: We employed modest quantities of TNFα. Nevertheless, LLLT (50 mW of power; wavelength of 780 nm; endogenously produced TNFαwas found to be energy density of 37.5 J/cm2; 30 seconds per point) essential for driving the expression of CXCL10 in applied in vivo on alternate days at two points on IFNλ3-treated pDCs. each hind paw of mice with Zymosan (Z) in Our data report that human pDCs respond to IFNλ3. subcutaneous air pouches. Unirradiated animals Moreover, we demonstrate that IFNλ3 induces the were used as controls. PMN that migrated to the production of biologically active TNFα. In fact, inoculation site provided, 10 days later, a highly antibodies neutralizing the IFNαR pure population of cells which were subsequently orendogenoTNFαconfirmed that the production of cultured in the presence of either a virulent (Pb18) or CXCL10 is scarcely dependent on endogenous an avirulent (Pb265) isolate. Experiments to assess IFNαbut rather driven by TNFα, regardless of the the viability of PMN after culture, mitochondrial CXCL10 amounts in the cell supernatant. activity and reactive oxygen species (ROS) Our study suggest that IFNλ3, by triggering the production by these cells were performed. production of IFNα, CXCL10 and TNFαmay impact Results: PMN were more deactivated in the on the polarization of Th cells as well as the presence of Pb18 than those maintained in culture recruitment/activation of CXCR3+cells in HCV with Pb265. LLLT treatment of PMN had the ability patients. to keep them more active even when exposed to BIBLIOGRAPHY Pb18. This virulent isolate increased the (1) Megjugorac, NJ. et al. (2009).J Leukoc Biol. 86, mitochondrial activity of PMN compared with the 1359–1363 avirulent isolate. Treatment with LLLT increased (2) Finotti G. et al. (2016).J Leukoc Biol. 99(1):107- mitochondrial activity of PMN exposed to either Pb 19 isolate when compared to controls. PMN co-cultured with Pb265 produced more ROS than those LB20 maintained in culture with Pb18, which demonstrates that the virulent Pb isolate determines The Deactivator Effect Elicited by more marked deactivation of PMN. The in vivo Paracoccidioides Brasiliensis in Neutrophils Can exposure of immature PMN in the bone-marrow to Be Reversed with Low Level Laser Therapy LLLT was able to activate these cells , significantly Applied “In Vivo” increasing their ROS production when co-cultured Eva Burger1, Ana Cristina L. Pereira1, Julianne C. with the virulent Pb isolate. Curiously, PMN Grisolia1, Giulia Maria C. Bani1, Felipe F. exposed to Pb265 when treated with LLLT Sperandio1, Liana Maria C. Verinaud1,2, Zoilo P. decreased their ability to release ROS. Camargo1,3, Ana Carolina S.C. Mendes1, 1Federal Conclusion: Paracoccidioides brasiliensis exerts an University of Alfenas (UNIFAL-MG), Alfenas, MG, inactivating effect on neutrophils, which depends on Brazil ; 2State University of Campinas (UNICAMP), the virulence of the fungal isolate. In vivo LLLT Campinas, SP, Brazil ; 3Federal University of São treatment reverts this effect, rendering PMN more Paulo (UNIFESP), São Paulo, SP, Brazil active metabolically against the virulent isolate, probably due to increased production of ROS. Introduction: Paracoccidioidomycosis (PCM) is a Acknowledgements: The authors were recipient of systemic mycosis caused by the thermodimorphic the following Grants: CNPq #304827/2012-6 and FAPEMIG #CBB-PPM- associated with increased susceptibility to 00119-14. chemically-induced cancerogenesis. Mendes, ACSC and Grisolia, JC are recipients of Until recently neutrophil function was mostly related CAPES scholarship and Pereira, ACL is recipient of to acute inflammation and defense against CNPQ scholarship. pathogens. We (and others) have challenged this dogma and demonstrated that neutrophils represent LB21 an essential component in the control of tumor onset and development. Neutrophils are Protective in Cancerogenesis by Altering Tumor Microenvironment and LB22 Controlling Intestinal Microbiota Andrea Ponzetta1, Marialuisa Barbagallo1, Maria The Differential Metabolic Responses of Non- Rosaria Galdiero1, Martina Molgora1, Eduardo Reprogrammed Vs Reprogrammed Human Bonavita1, Elena Magrini1, Nadia Polentarutti1, Neutrophils Involved in Chronic Inflammation Marina Sironi1, Fabio Pasqualini1, Cecilia Martin Pelletier, Asmaa Lachhab, Maude Leclerc, Garlanda2,1, Alberto Mantovani2,1, Sebastien Jaillon2, Flavia Ribeiro de Vargas, Isabelle Allaeys, Patrice 1Humanitas Clinical and Research Center; E. Poubelle, CRCHU de Québec-Université Laval 2Humanitas University Neutrophil plasticity is characterized by the ability The view of neutrophil as a cell involved only in the of some neutrophils to be reprogrammed into other early phases of inflammation has recently been cell types. Reprogramming of neutrophils into challenged and neutrophils are now considered key dendritic-like cells (DC) is obtained by culturing players in the orchestration of the immune response. blood neutrophils with diverse combinations of GM- Though several studies relied on antibody-based CSF, IFN-γ, IL-4 and TNF. Importantly, neutrophils neutrophil depletion to determine their contribution with DC characteristics are present in rheumatoid to tumor development, rigorous in vivo genetic synovial fluids in which GM-CSF, TNF and IL-4 are evidence explaining the neutrophil role in present. Human neutrophilic skin inflammation is cancerogenesis is missing. locally regulated by GM-CSF, TNF, IFN-γ, IL-4 and We investigated this issue using key preclinical IL-5. GM-CSF, TNF and IL-4 are also factors models of chemically-induced cancer (3-MCA responsible for the presence of neutrophils in lungs induced sarcoma and AOM/DSS induced colorectal of a mouse model of asthma. These pathological cancer, CRC) and taking advantage of a genetic conditions lend support to study these reprogrammed model of neutrophil deficiency (i.e. csf3r-/- mice). neutrophils in vitro. We showed that normal human Neutrophil deficiency was associated with increased blood neutrophils cultured with GM-CSF, IL-4 and susceptibility to sarcoma and CRC, and tumor TNF lead to long-lived (LL) neutrophils with microenvironment displayed protumoral features expression of DC markers and modified functions (e.g. increased frequency of M2 macrophages and (i.e. elevated production of superoxide anions and reduced IFNg concentration). In addition, in WT leukotrienes in response to second signals, and mice increased neutrophil infiltrate significantly decreased degranulation). Compared to adequate correlated with reduced proliferation rate of tumor controls, do these LL neutrophils exhibit changes in cells and adoptive transfer of naïve neutrophils their bioenergetics? Using the extracellular flux reduced tumor growth in csf3r-/- mice. Importantly, analyzer, we observed that LL neutrophils maintain the increased susceptibility to CRC in csf3r-/- mice their glucose metabolism over time while only a was dependent on intestinal microflora, and was transient increase in glucose metabolism was abolished in cohousing experiments. In addition, the observed in control neutrophils. Moreover, upon dysbiosis displayed bycsf3r-/-mice was also stimulation, LL neutrophils increase their glucose responsible for the increased susceptibility to acute metabolism to the level of glycolysis of freshly colitis, which has a causal link with intestinal cancer purified neutrophils. The increase in glycolysis was development. associated with an increase in oxygen consumption, Collectively, our data support that genetic deficiency while adequate control neutrophils were of neutrophils affects the anti-tumor response and is unresponsive to stimulation in terms of their bioenergetics. These results suggest that Bultynck2, Jean-Baptiste Parys2, Maurice B. Hallett1 reprogramming of neutrophils leads to neutrophils 1Cardiff University; 2KU Leuven with major changes of their energy metabolism in parallel to their modified functions. Background: The spreading of neutrophils onto a surface involves LB23 a massive change in cell shape. As ezrin forms crucial crosslinks between the plasma membrane and An Investigation into the Pathways Involved in cortical F-actin, in neutrophils and other myeloid Neutrophil Extracellular Trap Induction cells, it is thought to maintain the structure of cell Elaine F. Kenny, Volker Brinkmann, Arturo surface microridges which act as a reservoir of Zychlinsky, Max Planck Institute for Infection plasma membrane for spreading. It has been Biology suggested that the effect of elevating cytosolic Ca2+ concentration, and thereby activation of the Ca2+- To date many of the investigations into the activated cysteine protease calpain, would be to mechanism of neutrophil extracellular trap (NET) break the ezrin link and permit cell spreading. In this formation have relied on the use of the ligand PMA. work, we have expressed fluorescent constructs of We aimed to examine if the ligands A23187 (a ezrin to investigate the dynamic relationship calcium ionophore), nigericin (a potassium between elevatedCa2+ concentration in the ionophore), group B streptococcus (GBS, a gram microdomain of ezrin and its release from the cell positive bacteria) and Candida albicans (a fungus) periphery. utilise a similar pathway as PMA to induce NET Materials and methods: formation. To carry out this study healthy Blood was taken from healthy donors and human neutrophils were examined for NET production in neutrophils were isolated by dextran sedimentation. response the chosen ligands in the presence of Immunocytochemistry was performed using FITC- inhibitors of proteins known to be central to PMA conjugated secondary antibodies. RAW 267.4 cells induced NET formation (ex: PKC, neutrophil were transfected with plasmids encoding ezrin- elastase, myeloperoxidase and reactive oxygen mEmerald, mCherry-ezrin, ezrin-mCherry, GFPi- species, ROS). Neutrophils from patients with ezrin and ezrin-CEPIA3 using the Cell Line mutations in the pathways involved in NET Nucleofector™ Device (Lonza). Ca2+ influx formation, such as chronic granulomatous disease wasachieved using a high Ca2+ cocktail with patients (CGD, patients with a mutation in proteins thapsigargin and ionomycin. of the NADPH oxidase complex resulting in a lack Results: of ROS generation by granulocytes) or MPO- Immunocytochemical detection showed that deficient patients were also use to verify the endogenous ezrin was exclusively at the cell pathways required for NET formation formed by the periphery in human neutrophils. Ezrin constructs ligands of interest. with available N-FERM domain (e.g. ezrin- Our findings reveal that PMA and the physiological mEmerald, ezrin-mCherry and GFPi-ezrin) were all ligands Candida albicans and GBS make use of a located at the cell membrane in transfected myeloid related pathway for NET induction whereas the cells, whereas ezrin constructs with a fluor linked to ionophores require very few of these signalling this domain (as with mCherry-ezrin) remained molecules for NET induction. This demonstrates that cytosolic. This suggests a dominant role of N-FERM NET induction does not occur through one binding in ezrin localisation to the cell periphery, signalling pathway but is a widely varied method of compared to C-ERMAD domain binding to F-actin. host cell defence against pathogen attack. Elevation of cytosolic Ca2+ concentration resulted in a rapid loss of ezrin from the cell cortex, and a LB24 simultaneous dramatic increase in cell size. By 2+ attaching a low affinity Ca -sensing fluor to ezrin Dynamics of Ezrin Location at the Plasma (ezrin-CEPIA3), it was found that in the membrane Membrane: Relevance to Neutrophil Spreading protrusions, or ezrin-containing microdomains, Ca2+ Rhiannon E. Roberts1, Tim Vervliet2, Geert concentration was elevated to approximately 60 µM before release of ezrin and cell expansion. Conclusions: role is the phagocytosis on the spot [3]. The surface It was concluded that elevation of cytosolic Ca2+ composition of the conidia plays a decisive role in concentration caused ezrin to be lost from the cell their recognition. Our further interest was to periphery. This may result in the reduction of investigate the importance of GPI-anchored proteins membrane surface microridges, and contribute to the on fungal spores. GPI observed increase in cell size. For the first time, Ca2+ (glycosylphosphatidylinositol)-anchors fix proteins concentration has been measured locally in the to the conidia surface and are highly abundant in microridges of myeloid cell membranes. Ca2+ fungi. By proteome analysis of the conidial surface 8 concentration in the microridges of myeloid cells GPI-anchored proteins were identified. Knock-out increased upon cell stimulation, to a sufficiently high mutants of the encoding genes were generated. Then, concentration to activate calpain (Kd 30 µM). The the phagocytosis rate in MHS-macrophages was dynamics of ezrin localisation and response to Ca2+ determined microscopically. The images were concentration at the cell periphery can be modelled analysed by an algorithm. As a result, for 4 fungal into the mechanism of neutrophil spreading upon mutants significantly reduced phagocytosis was extravasation, where localised elevated cytosolic calculated. From these, we chose 3 mutants for Ca2+ levels activate local calpain to cleave ezrin and further analysis. The respective genes code for a ß- liberate plasma membrane for neutrophil spreading. 1,3-glucanosyltransferase, a β-1,3-endoglucanase and a glutaminase. The mutant conidia were LB25 coincubated with neutrophils and the oxidative burst was measured. All 3 mutants initiated a stronger Deciphering the Recognition Process of the oxidative burst than wild-type conidia. Thus it is Human Pathogenic Fungus Aspergillus likely that the analysed GPI-anchored proteins affect Fumigatus by the Immune System with Live-Cell the recognition of conidia in macrophages and Imaging and Analysis of Fungal Mutant Strains neutrophils. Hanno Schoeler, Juliane Macheleidt, Vera Pähtz, [1] Erjavec et al., 2009, Clin Microbiol Infect Kaswara Kraibooj, Naim Al-Zaben, Thorsten [2] Heinekamp et al., 2015, Sem Immunpath Heinekamp, Marc Thilo Figge, Axel Brakhage [3] Behnsen et al., 2007, PLoS Pathog Leibniz-Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute LB26

Invasive aspergillosis caused by the fungus EFFECT of ADMINISTRATION of ANTI- Aspergillus fumigatus (Af) is a life-threatening INFLAMMATORY DRUG CELECOXIB infection of immunocompromised hosts with high SIMULTANEOUSLY with LOW POWER mortality rates and annually increasing incidences LASER THERAPY in MURINE [1]. The small-sized conidia of Af are inhaled and NEUTROPHILS easily reach the lung alveoli. Here, they encounter Eva Burger1, Ana Carolina S.C. Mendes1, Julianne the first line of defense, mainly neutrophilic C. Grisolia1, Giulia Maria A.C. Bani1, Felipe F. granulocytes and macrophages [2]. Neutrophils are Sperandio1, Zoilo P. Camargo1,2, Liana Maria C. fast-moving cells that are able to quickly invade Verinaud1,3, Ana Cristina L. Pereira1, 1Federal infected tissue. We studied their motility by live-cell University of Alfenas (UNIFAL-MG), Alfenas, MG, imaging. Neutrophils were coincubated with GFP- Brazil; 2Federal University of São Paulo labeled spores (conidia) and images were taken (UNIFESP), São Paulo, SP, Brazil; 3State University every 10 seconds. A bioinformatic algorithm tracked of Campinas (UNICAMP), Campinas, SP, Brazil the movement of every single neutrophil, which enabled us to perform an in-depth analysis of the Introduction: Celecoxib (CX) is a nonsteroidal anti- interaction. More sophisticated parameters were inflammatory drug, extensively employed in applied to determine reactions of neutrophils like patients’ therapy. Previously, we studied the effect touching events, phagocytic efficiency, the number of administration by the oral route of this drug in of phagocytosed spores per cell or the average mice infected ip with P. brasiliensis (Pb), and neutrophil granulocyte speed over time. By contrast, observed that the treatment increased the expression macrophages are slowly moving cells whose main of GM-CSF, responsible for the bone marrow maturation of polymorphonuclear neutrophils Conclusions: Our results suggest that administration (PMN). We also studied the effect of low level laser of CX increases mitochondrial activity by PMN therapy (LLLT) applied to the bone marrow of resulting, eventually in increased GM-CSF animals infected with Pb or inoculated with production. Laser therapy acts favoring these cellular Zymosan (Z) in subcutaneous air pouches, and processes, but with less efficiency than the anti- reported that this therapy was able to activate PMN inflammatory drug Celecoxib alone. that subsequently migrated to the site of the Acknowledgements: Burger, E. is recipient of inoculum. In the present study we evaluate the CNPq #304827/2012-6, FAPEMIG CBB-PPM- overall effect of LLLT application on bone-marrow 00119-14 grants. immature neutrophils followed by administration of Mendes, A.C.S.C. and Grisolia, J.C. are recipients of CX locally, at the site of subcutaneous air pouches in CAPES scholarship and Pereira, A.C.L. is recipient a sterile inflammation model (Z inoculation) in mice. of CNPQ scholarship. Methodology: The treatments employed were: A) laser radiation, employing LLLT (50 mW of power; LB27 wavelength of 780 nm; energy density of 37.5 J/cm2; 30 seconds per point) at two points on each The Effects of High-Dose Statins on Neutrophil hind paw of mice on alternate days in mice Functions in Healthy Elders and during previously inoculated with Z in subcutaneous air Respiratory Infections and Sepsis; in Vitro pouches, B) local treatment at the site of the air Studies and a Randomised, Placebo Controlled pouch with CX, in the last three days prior to Cross-Over Clinical Trial. collection of cells (at day +8), C) a sum of both Adam JA McGuinness, Hannah L. Greenwood, treatments and D) Untreated mice. Cells were Jaimin Patel, Georgia M. Walton, Charendeep collected and the number of total and viable PMN, Sadhra, Dhruv Parekh, Rachel CA Dancer, Peter mitochondrial activity, reactive oxygen species Nightingale, Janet M. Lord, David R. Thickett, (ROS) and proteins production were determined. Elizabeth Sapey, Institute of Inflammation and Results: The administration of CX significantly Aging, University of Birmingham decreased the influx of cells at the inoculation site, while treatment with LLLT managed to increase it. Background Submitted to both, CX and LLLT, the number of Life expectancy is increasing leading to cells was similar to that of controls, as these two unprecedented global health resource use. treatments had opposite effects. None of the Pneumonia a leading infectious cause of death in the treatments affected cell viability. CX treatment was elderly with mortality rates unchanged over the last able to strikingly increase mitochondrial activity, decade(1). Sepsis is a clinical syndrome associated when compared to both, the LLLT-treated group, the with systemic immune dysregulation produced by CX+LLLT treated group and the non-treated invasive microorganisms. Severe sepsis is seen in controls. The group of CX-LLLT treated mice also 48% of pneumonia patients and is more common had significantly increased the metabolic activity as (with worse outcomes) in the elderly(2). Innate compared to the LLLT-treated group. Regarding the immunosenescence (the decline in innate immunity release of ROS and production of proteins there with age) is thought to contribute to these poorer were no difference between treatments. When outcomes(3).Neutrophils are key effector cells analyzing GM-CSF-production, CX caused a during bacterial infections, and there is evidence of remarkable increase as compared with all groups sub-optimal neutrophil responses in the elderly. (non-treated, laser-treated and even LLLT+CX There is great interest in developing therapeutic treated group). LLLT treatment increased the strategies to enhance immune responses. expression this cytokine when compared to control, HMG CoA Reductase Inhibitors (“statins”) are used and the combined treatment (LLLT+CX treated to reduce cardiovascular risk. Observational studies group) has antagonistic effect on this production. of patients taking statins suggest improved survival This cytokine is known to stimulate the maturation during infections, in vitro studies describe enhanced of neutrophils in the bone marrow, which may have neutrophil function, yet interventional trials of caused the increased mitochondrial activity of the statins during severe sepsis have not shown benefit. CX group. However, clinical trials have recruited younger patients with severe sepsis for an acute intervention, the elderly in health and during infections, but not at observational studies included older patients and in low dose nor during sepsis. This supports pre- vitro studies used pharmacologically irrelevant emptive statin treatment in at risk populations but concentrations. To clarify the potential utility of not as an acute intervention. statins for bacterial infections we assessed in vitro and in vivo affects of pharmacologically relevant LB28 concentrations on neutrophil functions in relation to donor age, infection severity and sepsis. Secretion of the Phosphorylated Form of the Methods Alarmin S100A9 from Neutrophils is Essential Migratory dynamics (with simvastatin or control) for the Pro-Inflammatory Functions of were assessed for neutrophils from healthy subjects, Extracellular S100A8/A9, Véronique Schenten, patients with lower respiratory tract infections, Fabrice Tolle, Sabrina Bréchard, Sébastien Plançon, pneumonia or pneumonia-associated sepsis. Prior Eric J. Tschirhart, University of Luxembourg, Life statin use and outcomes in pneumonia were assessed Sciences Research Unit- Calcium Signalling and retrospectively from hospital records in an Inflammation Laboratory observational study of 2068 patients. Simvastatin effects on neutrophil functions were assessed in a S100A8 and S100A9 are members of the S100 randomised, double-blinded cross-over clinical trial family of cytoplasmic EF-hand Ca2+-binding in healthy elders. proteins and are abundantly expressed, mostly under Results heterodimeric form, in the cytosol of neutrophils. In In health, increasing age was associated with addition to various intracellular roles, reduced neutrophil migratory accuracy (R2=-0.48, S100A8/S100A9 can be secreted in the extracellular p<0.0001). Infection progressively worsened environment and be considered as alarmins neutrophil functions in the elderly, in accordance modifying the inflammatory response through the with insult severity (chemotaxis: mean difference to interaction with pattern recognition receptors such as aged healthy; Lower Respiratory Tract Infection TLR4 or RAGE. High concentrations of S100A8/A9 (LRTI) -0.7μm/min, p=0.04; Pneumonia - are found at local sites of inflammation or in the 1.1μm/min, p=0.02; Pneumonia with Sepsis - serum of patients with inflammatory diseases and are 1.6μm/min, p=0.01). thus used as inflammation biomarkers. Although Pharmacologically relevant doses of simvastatin S100A8/A9 secreted by neutrophils are of rescued the inaccurate neutrophil migration seen importance in the pathophysiology of many with age in a dose dependent manner. Simvastatin inflammatory diseases, the mechanisms by which (1uM) also restored “old” neutrophil migratory these proteins are released remain unclear. The accuracy during LRTI and pneumonia, but not in intracellular activity of S100A8/A9 was shown to be patients with sepsis and only at high dose. An regulated by S100A9 phosphorylation among other, observational pneumonia study confirmed improved however the phosphorylation state of S100A9 in the survival with prior statin use, demonstrating a dose extracellular environment and the importance of this response with better outcomes associated with putative phosphorylation on the extracellular activity higher doses. of S100A8/A9 has not yet been extensively studied. 2 weeks of 80mg simvastatin improved neutrophil Therefore, we focused our work on one hand on the chemotaxis in cells isolated from healthy elderly phosphorylation state of the secreted S100A9 and on subjects in response to interleukin-8 (IL8) (p=0.04) the other hand on the effects of the and fMLP (p=0.006) compared to placebo, without unphosphorylated and the phosphorylated complex impeding other crucial neutrophils functions. on the pro-inflammatory function of neutrophils. Simvastatin effects appear to be mediated through First of all, we characterized the secretion of normalisation of cell adherence. S100A8/A9 under different stimulatory conditions Conclusion and investigated the phosphorylation state of Age is associated with a reduction in neutrophil secreted S100A9. Our results on neutrophil-like migration accuracy, which worsens during infections differentiated HL-60 cells (dHL-60) and purified in accordance with the severity of the infectious human neutrophils show a time-dependent secretion insult. Simvastatin improves neutrophil function in of S100A8/A9 when induced by PMA and importantly, S100A9 was found in a phosphorylated health problem, representing the fourth cause of form in these supernatants. Then we studied the cancer death worldwide. Prediction of outcome in influence of S100A8/A9 or patients with CRC remains difficult because of the S100A8/PhosphoS100A9 stimulation on pro- complexity and heterogeneity of this pathology 1. inflammatory cytokine expression and secretion in Inflammation, including soluble and cellular dHL-60 cells. In this aim, time course experiments effectors is an essential component of the tumor with unphosphorylated or phosphorylated microenvironment 2. Tumor-associated neutrophils S100A8/A9 were performed and the expression and (TAN) are a component of the inflammatory secretion levels of IL1α, IL1ß, IL6, TNFα, CCL2, microenvironment of tumors but the significance of CCL3, CCL4 and CXCL8 were measured by real- TAN in CRC has been the subject of conflicting time PCR and cytometry bead array respectively. reports 3. The present study was designed to set up a Our results clearly show that only the reliable methodology to assess TAN infiltration in phosphorylated form of the complex induces pro- CRC and to evaluate their clinical significance. inflammatory cytokine expression and secretion. Methods: CD66b and myeloperoxidase (MPO) were Finally, we were able to show that assessed as candidate neutrophil markers in CRC S100A8/PhosphoS100A9 is inducing cytokine using immunohistochemistry. CRC patients (n=271) secretion through TLR4 signaling. (stageI-IV) were investigated retrospectively by As a conclusion, we were able to show for the first computer-assisted imaging on whole tumor sections. time that S100A8/A9 is released under a Results: CD66b was found to be a reliable marker to phosphorylated form from both dHL60 cells and identify TAN in CRC tissues, whereas MPO also purified human neutrophils and that this identified a subset of CD68+ cells. Higher TAN phosphorylation is essential for the pro- density in stage I-IV patients was associated with inflammatory activity of the S100 complex on better prognosis. Importantly, an interaction was neutrophil-like dHL-60 cells. observed between clinical stage, TAN density and 5- FU-based chemotherapy, and, in stage III patients, LB29 TAN density had a dual clinical significance depending on the use of chemotherapy. Occurrence and Significance of Tumor- Discussion: TAN are an important component of the Associated Neutrophils in Patients with immune cell infiltrate of CRC which can be reliably Colorectal Cancer quantified by CD66b staining. Unexpectedly, higher Maria Rosaria GALDIERO 1,2, Paolo BIANCHI 1, TAN density was associated with response to Fabio GRIZZI1, Giuseppe DI CARO 1, Gianluca chemotherapy. BASSO1, Andrea PONZETTA 1, Eduardo Conclusions: Assessment of TAN infiltration may BONAVITA 1, Marialuisa BARBAGALLO 1, Silvia help identify patients likely to benefit from 5-FU- TARTARI1, Nadia POLENTARUTTI 1, Alberto based chemotherapy. MALESCI 1,3, Gianni MARONE 2, Massimo Bibliography: RONCALLI1,4, Luigi LAGHI 1, Cecilia 1. Bindea, G., et al. Immunity 39, 782-795 (2013). GARLANDA 1,4, Alberto MANTOVANI 1,4, 2. Hanahan, D. & Weinberg, R.A. Cell 144, 646-674 Sébastien JAILLON1,4, 1Humanitas Clinical and (2011). Research Center, via Manzoni 56, 20089 Rozzano 3. Jaillon, S., et al. Semin Immunopathol 35, 377- (Mi), Italy; 2Department of Translational Medical 394 (2013). Sciences and Center for Basic and Clinical Immunology Research (CISI), University of Naples LB30 Federico II, via Pansini 5, Naples, Italy ; 3Department of Biotechnologies and Translational Neutrophil Infiltration Enhances the Favorable Medicine, University of Milan, via Manzoni 56, Prognostic Significance of CD8+ T Cell 20089 Rozzano (Mi), Italy; 4Department of Infiltration in Colorectal Cancer Biomedical Sciences, Humanitas University, Via Valeria Governa1,2, Valentina Mele1, Luigi Tornillo2, Manzoni 113, 20089 Rozzano – Milan, Italy Luigi Terracciano2, Giandomenica Iezzi1, Giulio C. Spagnoli1, 1Department of Biomedicine, Basel Purpose: Colorectal cancer (CRC) is a major public University Hospital and University of Basel, Basel, CH;; 22Molecular Pathology Division, Institute of Dendritic cell (DC) migration in peripheral tissues Pathology, Basel University Hospital serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration OBJECTIVE: Tumor infiltration by different T towards lymphatic vessels (LVs) upon maturation. lymphocyte subsets was repeatedly reported to be These migratory events determine the efficiency of associated with favorable prognosis in colorectal the adaptive immune response. cancer (CRC). Still debated is the role of innate In this seminar, I will discuss about the cellular immune system. We investigated clinical relevance, rearrangements of the actin cytoskeleton needed to phenotypes and functional features of CRC optimize migration of DCs in tissues. I will show infiltrating CD66b+ neutrophils and their crosstalk that migration of immature DCs depends on two with CD8+ T cells. major actin pools: (1) a RhoA-mDia1-dependent DESIGN: CD66b and CD8 immunohistochemical actin pool located at their back, which facilitates evaluation was performed on a tissue microarray forward locomotion and (2) a Cdc42-Arp2/3- including >650 evaluable CRC samples. Phenotypic dependent actin pool present at their front, which profiles of tissue infiltrating and peripheral blood limits migration but promotes antigen capture. CD66b+ cells were evaluated by flow cytometry. Following LPS-induced maturation, Arp2/3- CD66b+/ CD8+ cells crosstalk was investigated by dependent actin enrichment at the cell front is in vitro experiments. drastically reduced. Consequently, mature DCs RESULTS: CD66b+ cell infiltration in CRC is switch to a faster and more persistent locomotion significantly associated with increased survival. mode that facilitates chemotactic migration to LVs Interestingly, neutrophils frequently co-localize with and lymph nodes. I will further discuss on how these CD8+ T cells in CRC tissues. Functional studies cytoskeleton rearrangements are used by DCs to indicate that although neutrophils are devoid of move in irregular landscapes facilitating the direct antitumor potential, co-culture with peripheral migration of leukocytes in the complex geometry of blood and tumor associated neutrophils (TANs) tissues. enhances CD8+ T cell activation, proliferation and cytokine release induced by stimulation with LB32 suboptimal concentrations of anti-CD3 monoclonal antibody (mAb). Moreover, under optimal activation Ultra High Throughput Purification and conditions, CD8+ cells initially stimulated in the Concentration of Leukocytes and Progenitor presence of CD66b+ cells show decreased Cells from Peripheral Blood and Bone Marrow expression of PD-1 “exhaustion” marker and are Kyle Smith1, Thomas Barber1, Mehmet Toner2, Ron significantly less susceptible to apoptosis induced by Tompkins2, Ravi Kapur1,2 ,1 MicroMedicine, Boston, T- cell receptor triggered re-stimulation. MA , 2 Massachusetts General Hospital, Boston, MA Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with While tremendous advances have been made in significantly better prognosis, as compared to CD8+ extracting valuable information from specific T cell infiltration alone. cellular components in blood (and other bodily CONCLUSIONS: Neutrophils enhance the fluids), the bulk processing methods used to isolate responsiveness of CD8+ T cells to TCR triggering. these subpopulations have remained crude and Accordingly, infiltration by neutrophils enhances the stagnant for decades, thereby compromising prognostic significance of CRC infiltration by CD8+ precious patient samples and hindering downstream T cells, suggesting that they might modulate clinical assays. MicroMedicine’s innovative antitumor immunity. LeukoChip™ cell isolation platform addresses this issue by enabling enrichment with commensurate LB31 efficiency and elegance to the available backend cell and molecular interrogation techniques. The core of Mechanobiology of dendritic cell migration in the platform is a high-precision, injection-molded confined environments microfluidic disc that enables ultra high-throughput Pablo Vargas, Institut Curie purification and concentration of leukocytes from peripheral blood or bone marrow. and quality of information in Clinical Diagnostics, The LeukoChip™ cell isolation platform Cellular Therapy and Life Sciences. achieves this high level of performance by utilizing inertial focusing to sort and concentrate cells. In the LB33 first stage, the sample stream enters the microfluidic channel flowing alongside a stream of buffer CHARACTERIZATION OF A NOVEL solution. Target cells flowing near the elongated NEUTROPHIL-DEFICIENT MOUSE STRAIN island structures in the channel experience a fluidic J.Z. Csepregi*, O. Kása*, T. Németh*, E. Zajta†, K. lift force that induces them to gradually migrate Csonka†, A. Gácser†, Y.W. He‡ & A. Mócsai*, laterally from the sample stream into the buffer *Department of Physiology, Semmelweis University stream, leaving behind the contaminating cells. The School of Medicine and MTA-SE „Lendület” target cells then proceed to the second stage of the Inflammation Physiology Research Group, device to a series of curving channels in which the Budapest, Hungary; †Department of Microbiology, fluidic lift force induces them to migrate towards the University of Szeged, Szeged, Hungary; ‡Department center of channel. This creates a cell-free stream of of Immunology, Duke University Medical Center, fluid at the edge of the curving channel that is Durham, NC, USA siphoned into an adjacent channel. After a number of iterations, the volume in which the target cells are Background and objectives: Genetic deletion of suspended is significantly reduced. The platform specific leukocyte lineages strongly contributes to processes blood at a rate of 200 mL/hour with each understanding the role of various leukocyte subsets cell spending just a fraction of a second in the chip. in physiological and pathological conditions. There The LeukoChip™ platform maintains cells have been a number of attempts to generate in their endogenous state, with no contact with genetically neutrophil-deficient mouse strains. physical objects or antibodies, and with minimal However, all those strains suffer from substantial change in homeostasis. Minimal manipulation limitations such as the limited efficiency of enables recovery of target cells (rare or abundant) neutrophil deletion, the effect of the mutations on with high yield, high purity, and high functionality. other lineages or the limited survival of the mutant Preserving the functionality of the cells, and mice. We have previously shown that the integrity of the protein, DNA, and RNA sub- antiapoptotic Mcl-1 protein is essential for the components, enables ex-vivo, and in-vivo survival of neutrophils but not macrophages. therapeutic and diagnostic applications. Specific Therefore, we tested whether myeloid-specific examples include: (1) Retention, enrichment, and deletion of Mcl-1 could provide a novel and more concentration of abnormal cells (and their suitable genetic model of neutrophil deficiency in phenotype, genotype, proteomics, and experimental mice. metabolomics) from blood or bone marrow, enables Materials and methods: LysMCre mice expressing diagnosis of disease using traditional tools of Cre recombinase in the myeloid compartment were cytopathology, and recent advances in next crossed with animals carrying the Mcl-1flox mutation generation sequencing of RNA/DNA. (2) Retention to obtain LysMCre/CreMcl-1flox/flox (referred to as Mcl- of cellular motility, and cell signaling enables 1ΔMyelo) mice characterized by myeloid-specific isolated autologous radioisotope tagged neutrophils conditional deletion of Mcl-1. Leukocyte to migrate and detect infection in-vivo. (3) populations in the peripheral blood, spleen and bone Retention of cell division and cell signaling enables marrow of Mcl-1ΔMyelo mice were tested by flow use of isolated autologous progenitor cells (from cytometry. The viability and fertility of the mice was either bone marrow or mobilized peripheral stem monitored for 6 months under SPF conditions. The cells) for wound repair. (4) Retention of the susceptibility of the Mcl-1ΔMyelo mice to infection immunogenicity of circulating T cells enables was tested following intraperitoneal injection of isolation and ex-vivo conditioning for autologous 2×107 Staphilococcus aureus bacteria/mouse or immune therapy. intravenous injection of 105 Candida albicans By providing gentle, precise, high cells/mouse. K/BxN serum-transfer arthritis and the throughput and efficient cell enrichment, the mouse model of the epidermolysis bullosa acquisita LeukoChip™ platform will enhance the performance (EBA) were used as neutrophil-dependent in vivo inflammation models. Results: Mcl-1ΔMyelo mice were viable, though their long-term survival was somewhat reduced compared to wild type mice. Importantly, Mcl-1ΔMyelo mice were fertile even in homozygous form. The mice have a strong (>99%) reduction of circulating neutrophil counts and severe deficiency of splenic and bone marrow neutrophils. However, other leukocytes such as circulating eosinophils, monocytes, T and B cells, or splenic dendritic cells and macrophages are not affected. Following infection with S. aureus, all Mcl-1ΔMyelo mice died on the first day. The neutrophil-deficient mice were also significantly more sensitive compared to the control group in the candidemia model. Mcl-1ΔMyelo mice are completely protected from all signs of arthritis development in the K/BxN serum-transfer model. Following injection of mCVII antibody, the neutrophil-deficient mice, in contrast to control group, showed no skin lesions or blisters in the EBA model. Conclusions: The Mcl-1ΔMyelo mutation leads to dramatic reduction of the number of circulating and tissue neutrophils without affecting other blood and splenic leukocyte lineages. This reduced innate immune response makes these mice more susceptible for bacterial and fungal infections. However, Mcl-1ΔMyelo mice are viable and fertile in homozygous form and are completely protected in two known neutrophil-dependent in vivo disease models. Taken together, the Mcl-1ΔMyelo mice may provide a useful novel model of genetically determined neutrophil deficiency. Program Chairs Invited Speakers Lionel Ivashkiv, Hospital for Special Surgery Ido Amit Liwu Li, Virginia Tech Yasmine Belkaid Danica Chen Plenary Topics Mashkoor Choudhry Memory of Tissue Location and Micro-environment Hogler Eltzschig Epigenetic Mechanisms of Memory in Innate Immune Andrew Gewirtz Cell Activation Chris Glass Epigenetic Memory in Lymphocytes Ajanda Goldrath Leukocyte Memory in Chronic Disease Daniel Irimia Lionel Ivashkiv Leukocyte Memory in Infection and Injury Lewis Lanier Concurrent Topics Liwu Li Gioacchino Natoli Microbiome in leukocyte memory Mihai Netea Systems analyses of leukocyte memory John O'Shea Leukocyte Memory and Drug Development Virginia Pascual Systems biology of adaptive immune memory Gwendalyn Randolph Metabolism in leukocyte memory Ellen Rothenberg Leukocyte memory in autoimmune disease Carla Rothlin Engineering analyses of leukocyte memory Matthew Sleeman Best of Journal of Leukocyte Biology Ira Tabas