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Cytokines 2020 Virtual Meeting – Abstract Book November 2020

ABSTRACT

BOOK

Local Organizing Committee Michael Gale, Jr., Ph.D., University of Washington, Seattle, USA, meeting chair Ram Savan, Ph.D., University of Washington, Seattle, USA, meeting co-chair Sarah Gaffen, Ph.D., University of Pittsburgh, Pittsburgh, USA Naeha Subramanian, Ph.D., Institute for Systems Biology, Seattle, USA Steve Ziegler, Ph.D., Benaroya Research Institute, Seattle, USA Yueh-Ming Loo, Ph.D., AstraZeneca, Inc., Gaithersburg, USA Renee Ireton, Ph.D., University of Washington, Seattle, USA

International Advisory Committee Simon Jones, Ph.D., Cardiff University, Cardiff, UK Eleanor Fish, Ph.D., FAAS, University of Toronto, Toronto, Canada Kate Fitzgerald, Ph.D., University of Massachusetts Medical School, Worcester, USA Georg Schett, M.D., Friedrich-Alexander-University, Erlangen, Germany Akinori Takaoka, M.D., Ph.D., Hokkaido University, Sapporo, Japan You-Me Kim, Ph.D., Pohang University of Science and Technology, Pohang, South Korea

1 2020 Virtual Meeting – Abstract Book November 2020

Contents Plenary 1: Cytokines and COVID19 ...... 3 Plenary 2: Cytokines in bacterial diseases ...... 3 Symposium 1: Cytokines in microbiome dynamics ...... 4 Symposium 2: ...... 4 Symposium 3: stimulated ...... 4 Symposium 4: Cytokines in cancer ...... 5 Symposium 5: Innate immunity ...... 6 Symposium 6: regulation and non-coding RNA...... 7 Symposium 7: Inflammation and barrier integrity ...... 8 Symposium 8: Regulators of - Philip I. Marcus Symposium ...... 9 Symposium 9: Host response to SARS-CoV-2 ...... 10 Symposium 10: Cytokines in neurobiology ...... 11 Symposium 11: Cytokines in neuroimmunology ...... 11 Symposium 12: Immune fate and function...... 12 Symposium 13: Cytokines in autoimmunity...... 14 Symposium 14: Japanese Interferon Society symposium ...... 15 Symposium 15: Late breaking talks in innate immunity, infection and cancer ...... 16 Lightning Talk Session 1: Adaptive Immunity...... 19 Lightning Talk Session 1: Cytokine regulation I ...... 29 Lightning Talk Session 1: Innate Immunity I...... 41 Lightning Talk Session 2: Autoinflammation and autoimmunity ...... 50 Lightning Talk Session 2: Cytokine regulation II ...... 62 Lightning Talk Session 2: Innate Immunity II...... 73 Lightning Talk Session 3: Cancer ...... 83 Lightning Talk Session 3: Innate Immunity III...... 91 Lightning Talk Session 3: Mucosal immunity ...... 102 Lightning Talk Session 3: SARS-CoV-2 or COVID-19 ...... 111

2 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Plenary 1: Cytokines and COVID19 established that perturbations in enteric bacteria combined O01 with genetic defects in the host immune responses to them DISTINCT SYSTEMIC AND MUCOSAL IMMUNE determine phenotypes of inflammatory bowel disease (IBD). RESPONSES TO SARS-COV2 However, the contributions of gut-resident eukaryotic and N. Smith1,*, P. Goncalves1, B. Charbit1, J. Hadjadj2, N. prokaryotic (collectively, the virome) that also Yatim1, T. Bruel1, L. Grzelak1, V. Rouilly3, V. Bondet1, S. fluctuate in IBD to host immunity and disease outcomes are Merkling1, H. Péré4, C. Blanc5, P.-L. Tharaux6, F. unknown. Here we show that viromes derived from healthy, Rozenberg4, A. Fischer2, O. Schwartz1, H. Mouquet1, F. ulcerative colitis (UC) or Crohn’s disease (CD) patient Rieux-Leucat2, S. Kernéis4, B. Terrier4, J. Di Santo1, D. intestine regulate host immunity differently and can Duffy1 independently modify disease state. Viromes from non-IBD 1Insitut Pasteur, 2Institut Imagine, 3DATACTIX, 4APHP- individuals directly elicited anti-inflammatory programs in CUP, 5Sorbonne Université, 6PARCC, Paris, France and intestinal epithelial cells. Conversely, viromes from both UC and CD patients promoted an Abstract Content: Coronavirus disease 2019 (Covid-19) is inflammatory response. Notably, healthy viromes characterized by distinct patterns of disease progression successfully dampened the IBD-virome inflammatory implying diverse host immune responses. We performed an response. Furthermore, mice with a “humanized” healthy integrated immune analysis on a cohort of fifty Covid19 virome had attenuated intestinal inflammation in patients with various disease severity, in both plasma and vivo while “humanized” IBD virome mice exhibited intestinal the nasopharynx, which reflects the initial site of infection. inflammation at steady state and exacerbated induction of Using state-of-the-art technologies including ultra-sensitive colitis. Finally, IBD-associated genetic digital Elisa (Simoa) to measure low levels of interferons variationin IFIH1 encoding the receptor MDA5 (IFN), mass cytometry (CyTOF), ultrasensitive droplet abrogated responses to the healthy enteric virome and based digital PCR (ddPCR), Nanostring nCounter disrupted intestinal epithelial integrity. Together, these technology and an ultrasensitive innovative antibody studies demonstrate that changes in the human virome, and detection technic (S-Flow), we identified a unique host immune responses to it, autonomously drive divergent phenotype in severe and critical patients. It consisted of a disease phenotypes. Manipulation of the human enteric profoundly impaired type I IFN response characterized by virome may be beneficial in IBD. low IFN-α production and activity, associated with a Disclosure of Interest: None Declared persistent blood viral load, an exacerbated inflammatory response, and a strong SARS-CoV2-specific antibody O03 response. Inflammation was partially driven by the STAT2 FACILITATES INTESTINAL INFLAMMATION BY transcriptional factor NFκB and characterized by increased INTERFERING WITH ANTIMICROBIAL IL-22 SIGNALING (TNF)-α and (IL)-6 A. M. Gamero1,*, T. Cremers1 production and signaling. These inflammatory cytokines in 1Medical Genetics and Molecular Biochemistry, Temple all patient groups were significantly correlated with Univ Sch of Med, Philadelphia, United States circulating viral levels, which was not associated with systemic type I interferon responses. Furthermore, we Abstract Content: Ulcerative colitis (UC) is one of two determine that while plasma levels major forms of inflammatory bowel disease. This debilitating were a hallmark of COVID-19 regardless of disease severity, chronic condition causes inflammation in the colon and cytokines present in the nasopharynx allowed a better heightens the risk of colorectal cancer. What causes UC categorization of disease severity. In addition to expected and its pathogenesis remains largely unclear. Type I and inflammatory markers this also included locally regulated type III interferons (IFN) are cytokines known to protect growth factors and suggesting viral induced against colitis. The STAT2 is a key mucosal tissue dysregulation. We propose that type-I IFN component of the ISGF3 complex that drives downstream deficiency in the blood could be a hallmark of severe Covid- the transcriptional responses of both cytokines. The aim of 19 and provide a rationale for combined therapeutic our study was to determine the role of STAT2 in the approaches which may be further optimized with a better development of intestinal inflammation by employing the understanding of infected tissue immune responses. murine dextran sodium sulfate (DSS) model of human UC- Disclosure of Interest: None Declared like colitis. We found that STAT2 deficiency caused less body weight loss and mild intestinal inflammation when Plenary 2: Cytokines in bacterial diseases compared to wild type mice. Pathology scoring of Stat2KO O02 colon sections showed reduced damage to the epithelial gut HUMAN ENTERIC VIRUSES SHAPE DISEASE barrier and attenuated immune cell infiltration. Co-housing PHENOTYPE THROUGH DIVERGENT of wild type with Stat2KO mice did not transfer colitis IMMUNOMODULATION susceptibility. Furthermore, colons from DSS-treated K. Jeffrey1,*, F. Adiliaghdam1 Stat2KO mice showed delayed and reduced IL6 induction 1Medicine, MASSACHUSETTS GENERAL HOSPITAL, accompanied by early and progressive increased induction HARVARD MEDICAL SCHOOL, Boston, United States of the antimicrobial cytokine IL-22; known to be protective against colitis. IL-22 treatment of intestinal cells with STAT2 Abstract Content: Environmental signals determine knockdown revealed increased STAT3 activation. Thus, our phenotype of disease in the context of genetics. It is well findings reveal that STAT2 is damaging and may promote 3 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 colitis by impairing IL-22 signaling by an as yet unknown Symposium 2: Inflammation mechanism that we are currently investigating. Collectively, O05 this bring to the forefront an unrecognized and damaging CYTOSOLIC DNTP CATABOLISM PREVENTS NLRP3 feature of STAT2 in the pathogenesis of UC, that when INFLAMMASOME OVERACTIVATION deregulated may progress CRC. Z. Zhong1,* Disclosure of Interest: None Declared 1Immunology, University of Texas Southwestern Medical Center, Dallas, United States Symposium 1: Cytokines in microbiome dynamics Abstract Content: Aberrant activation of NLRP3 O04 inflammasome drives the development of many diseases, COMMENSAL BACTERIA LIKELY CONTRIBUTE TO including atherosclerosis, gout, osteoarthritis, Alzheimer’s OCULAR MANIFESTATIONS AND EXCESSIVE IL-1 disease, macular degeneration, type 2 diabetes and cancer. BETA RESPONSE IN AUTOINFLAMMATORY DISEASES However, how the NLRP3 inflammasome activity is DUE TO NLRP3 GENE regulated at the molecular level remains incompletely M. Mattapallil1,*, J. Siak1, A. St. Leger2, T. Magone De understood. Our previous work demonstrated that new Quadros Costa3, W. Zein4, S. Alehashemi5, K. mitochondrial DNA (mtDNA) synthesis, induced at the Raychaudhuri1, G. Meng6, I. Fuss7, W. Strober7, R. inflammasome priming step, is a prerequisite for producing Goldbach-Mansky5, R. Caspi1 oxidized mtDNA (ox-mtDNA)—an activating of 1Lab. Immunology, NEI/NIH, Bethesda, 2Ophthalmology, NLRP3. Here we show that SAMHD1, a cytosolic dNTP Univ. Pittsburgh, Pittsburgh, 3Office of the Clinical Director, hydrolase, functions as a -intrinsic gatekeeper 4Ophthalmic Genetics & Visual Branch, NEI/NIH, that prevents NLRP3 inflammasome overactivation. 5Translational Autoinflammatory Disease studies unit, Mechanistically, inflammasome priming activates SAMHD1 NIAID/NIH, Bethesda, United States, 6Institut Pasteur of to inhibit cytosolic dNTP buildup, thereby preventing their Shanghai, University of Chinese Academy of Sciences, transport to mitochondria. This in turn protects mitochondria Shanghai, China, 7Lab. Clinical Immunology & Microbiology, from uncontrolled new mtDNA synthesis and ox-mtDNA NIAID/NIH, Bethesda, United States production, resulting in the attenuation of NLRP3 inflammasome activation. Consistent with these in vitro Abstract Content: We reported previously (PMID: observations, relative to their wild-type counterparts, 28709803) that ocular surface of healthy wild-type mice is SAMHD1-deficient mice produced substantially more IL-1b colonized by a commensal shared between mice and in vivo and were more sensitive to developing NLRP3 humans, Corynebacterium mastitidis (C. mast). On the inflammasome-dependent immunopathology. Together, murine ocular surface, C. mast elicits IL-17 from γδ T cells these results not only identify SAMHD1 as a novel cell- and promotes local host defense. Based on this we intrinsic inhibitor of NLRP3 inflammasome, but also hypothesized that ocular inflammation in Autoinflammatory demonstrate that cytosolic dNTP metabolism plays an disease patients, with NLRP3-associated mutations, may be essential role in orchestrating inflammation. triggered by an overactive response to their own ocular Disclosure of Interest: None Declared surface bacteria. We found that, unlike wild-type mice, knock-in mice bearing an NLRP3 gene found in Symposium 3: Interferon stimulated genes patients, developed conjunctivitis, conjunctival neutrophilia, O06 and increased IL-1β and IL-17 responses following ocular IMMUNITY-RELATED GTPASE IRGM1 GUARDS exposure to C. mast. scRNA-seq of murine conjunctival AGAINST SPONTANEOUS INTERFERON-DRIVEN cells confirmed that γδ T cells were a major immune cell AUTOIMMUNITY THROUGH MITOCHONDRIAL population and a source of Il- MAINTENANCE 17, Ccr2 and S100a8 transcripts, possibly explaining the P. Rai1,*, K. Janardhan1, J. Meacham1, J. Madenspacher1, neutrophilia. Importantly, patient PBMCs stimulated withC. W.-C. Lin1, P. Karmaus1, Q.-Z. Li2, M. Yan2, M. Grinstaff3, O. mast lysate overproduced IL-17A compared to healthy Shirihai4, G. Taylor5, M. Fessler1 controls by ELISA. However, in contrast to mice, scRNA-seq 1National Institutes of Environmental Health Sciences / NIH, of patient conjunctival cells and PBMCs showed Durham, 2University of Texas Southwestern Medical Center, upregulation of Th1 and Th17 signaling moleculesin ▫▫β T Dallas, 3Boston University, Boston, 4David Geffen School of cells rather than in γδ T cells. This suggests that response Medicine at UCLA, Los Angeles, 5Duke University Medical to C. mast in patients may be mediated by different immune Center, Durham, United States cells, and/or that other commensals may drive their ocular phenotype. Our study shows that commensals can elicit an Abstract Content: Type I interferon (IFN) plays a central exaggerated immune response in humans and mice role in the pathogenesis of common autoimmune disorders. with NLRP3-related autoinflammatory disease conditions. If Recently, we reported that mice deficient in the interferon-γ- indeed conjunctivitis in these patients reflects a response to inducible immunity-related GTPase - Irgm1, which regulates their own commensals, adjunct antimicrobial therapy could autophagy and also localizes to mitochondria, have an be an option. autoimmune exocrinopathy accompanied by increased Disclosure of Interest: None Declared autoantibodies and spontaneous induction of IFN-stimulated genes in several organs. We hypothesized that the pathogenic IFN response observed with Irgm1 deficiency is 4 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 driven by inappropriate sensing of cleared mitochondrial generate the second messenger 2-5A, which DNA (mtDNA). Here, we show that genetic deletion of the activates the latent ribonuclease RNase L. Activated RNase type I interferon receptor rescues the autoimmune tissue L potently restricts viral replication by degrading cellular pathology and excess autoantibody levels in Irgm1-/- RNAs to block host and induce . animals, demonstrating the pathogenicity of type I interferon Human OAS1 is C-terminally spliced into several isoforms, in these animals. Abnormal induction of type I interferon in and previous genetic studies have implicated a splice Irgm1-/- is associated with simultaneous acceptor site SNP (A/G, rs10774671) in the OAS1 gene with dysfunction in mitochondria and lysosomes, resulting in West Nile virus resistance. The susceptibility allele (A) soiling of the cytosol with mtDNA. The interferon response controls p42 expression and the resistance allele (G) is normalized upon depletion of cellular mtDNA and partially controls p46 expression, which suggests these isoforms corrected by restoration of lysosomal function. Further, we have distinct antiviral activity. We tested the antiviral activity show that mtDNA activates the double stranded DNA sensor of the p42 and p46 isoforms against encephalomyocarditis – cGAMP synthase (cGAS) and its adaptor, stimulator of IFN virus (EMCV), Coxsackie virus B (CVB), and West Nile Virus genes (STING), to drive type I interferon production in (WNV) and found p46, compared with p42, displays fibroblasts. In vivo, we found that the cGAS-STING pathway significantly stronger antiviral activity against EMCV, CVB3, underlies the pathology of salivary glands, autoantibodies as and WNV. Although both OAS1 isoforms require catalytic well as inflammatory cytokine production in Irgm1-null mice. activity and RNase L for antiviral activity, we discovered a Interestingly, the type I interferon response in Irgm1-/- C-terminal CaaX motif in p46. Proteins containing CaaX macrophages is dependent on endo-lysosomal signaling motifs at their C-termini are prenylated and targeted to and is associated with increased delivery of damaged membranes. Using confocal microscopy, we found p46 mitochondria to functional lysosomal compartments. Taken localized to the Golgi while p42 is cytosolic. Since EMCV, together, our findings support a model wherein Irgm1- CVB3, and WNV are positive-strand RNA viruses and mediated mitochondrial maintenance and turnover replicate their vRNA on intracellular organelle membranes, represses inappropriate mtDNA-dependent activation of we hypothesized membrane-targeting positions p46 in autoinflammatory interferon responses, possible in a tissue- proximity to vRNA replicating on organelle surfaces. In dependent manner. We propose that this fundamental support of this hypothesis, EMCV vRNA was mechanism may play a key role in suppression of type I immunoprecipitated with p46, but not p42, and membrane- interferon-dependent autoimmune syndromes. targeting was required for p46 to access v RNA. This was Disclosure of Interest: None Declared also confirmed by confocal microscopy, as p46, but not p42, localizes to sites of viral RNA accumulation during infection. O07 These data support a model in which membrane-targeting A WEST NILE VIRUS RESISTANCE SNP GENERATES A of OAS1 p46 enhances its antiviral activity by placing this MEMBRANE-TARGETED ISOFORM OF OAS1 WITH sensor in proximity to sites of positive-strand RNA virus ENHANCED ANTIVIRAL ACTIVITY replication at organelle surfaces. Collectively, our study F. W. Soveg1,*, J. Schwerk 1, A. Kell1, A. Forero1, K. Esser- shows how OAS1 specificity can be diversified through Nobis 1, J. R. Smith 1, J. Roby 1, T.-Y. Hsiang 1, S. Ozarkar alternative splicing and suggests differential subcellular 1, J. M. Clingan1, E. T. McAnarney2, A. E. L. Stone1, J. Perez targeting of OAS proteins may be important for the broad 3, C. Balu1, S. A. Zaver 4, J. J. Woodward4, V. D. antiviral specificity of this pathway. Menachery2, S. N. Sarkar3, D. B. Stetson1, M. Gale Jr1, R. Disclosure of Interest: None Declared Savan 1 1Immunology, The University of Washington, Seattle, Symposium 4: Cytokines in cancer 2Microbiology and Immunology, University of Texas Medical O08 Center , Galveston, 3Cancer Virology Program , University THE ACTIVATING STAT5BN642H DRIVER MUTATION of Pittsburgh Cancer Institute, Pittsburgh, 4Microbiology, DISRUPTS T-CELL DEVELOPMENT PROGRESSING TO The University of Washington, Seattle, United States T-CELL NEOPLASIA T. Suske1,*, F. Ruge1, H. Sorger1, B. Maurer1, H. T. T. Abstract Content: The antiviral activity of interferons, at Pham1, A. Boersma2, T. Rülicke2, H. A. Neubauer1, R. least in part, is mediated by the upregulation of interferon Moriggl1 stimulated genes (ISGs) whose products interfere 1Institute of Animal Breeding and Genetics, 2Institute of with virus replication. Collectively, these ISGs confer at least Laboratory Animal Science, Unitversity of Veterinary some protection against virtually all human viruses. Medicine Vienna, Wien, Austria However, mechanistic explanations for the antiviral activity and specificity of many ISGs is lacking. This is further Abstract Content: The activating STAT5BN642H mutation complicated by the fact that many antiviral ISGs are was found in over 120 patients suffering from various T-cell alternatively spliced, which could allow for fine tuning of the neoplasia subtypes. Studies have revealed that up to 8% of antiviral specificity or activity of an ISG. In this study, we T-cell acute lymphoblastic (T-ALL) patients have show a membrane-targeted isoform of oligoadenylate STAT5BN642H, correlating with poor prognosis and a higher synthetase 1 (OAS1) has enhanced antiviral activity against risk of relapse. Confirming the transforming capacity of this Flaviviruses and Picornaviruses. OAS proteins are an mutation, we used a STAT5BN642H vav-transgenic mouse antiviral system critical for restricting many different viruses. model, which rapidly develops CD8+ T-cell lymphoma that is Upon sensing viral RNA (vRNA), the catalytically active OAS transplantable and aggressively infiltrates into non- 5 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 hematopoietic organs. These transformed T-cells are infection at bay while mobilizing the adaptive immune mature and display a cytotoxic phenotype. Therefore, we response. Dendritic cells (DCs) and macrophages are key questioned biological and transforming consequences innate immune cell types that secret a cytokine milieu to governed by STAT5BN642H during T-cell development. establish a favorable immune environment for the Comparing thymocytes of STAT5BN642H to WT mice, we recruitment and activation of other immune cells. Within the found that cells double negative stage 1 (DN1) were highly tumor microenvironment (TME) of various cancers, enriched, whereas cells in DN2-4 were decreased. however, DCs and macrophages have been found to exert Importantly, double positive (DP) cell percentages were immunosuppressive effects. How the TME facilitates drastically reduced and CD8+ and CD4+ single positive tolerance or immune evasion has not been fully defined. We (SP8/SP4) cells were expanded in STAT5BN642H thymi, dissected the specific mechanisms by which the suggesting that STAT5 promotes T-cell maturation. This hepatocellular carcinoma TME modulates DC activity. We disruption in T-cell development caused by the mutation was also investigated the role of tumor-associated macrophages accompanied by an expansion of the thymic medulla, where in promoting in breast cancer. Mechanistic more mature T-cells reside. Comparing transcriptomes of insights from these studies will inform the design of novel DN, DP and SP8 cells by RNA-sequencing (seq), we found tumor vaccine strategies. We are currently developing a that STAT5BN642H triggers a highly activated and cytokine- vaccine platform that utilizes extracellular vesicles to load releasing and -responsive T-cell signature. To investigate tumor antigens into DCs, which are then delivered to whether STAT5BN642H can still exert this function patients transdermally by microneedle patches. Successful independently of the T-cell receptor (TCR), we crossed our implementation of this strategy will allow efficient delivery of transgenic mice to a RAG2-/- background. Strikingly, these DC vaccines that can overcome the immunosuppressive mice developed thymic neoplasia that retained immature T- TME to orchestrate an effective anti-tumor immune cell features with high TdT and reduced surface CD3 response. expression, resembling a T-ALL-like phenotype. Disclosure of Interest: None Declared Furthermore, high expression of TdT and oncogenes PIM1 and BCL-2 in diseased T-cells mirrors the situation in Symposium 5: Innate immunity STAT5BN642H-positive T-ALL patients. The mutation sufficed O10 to overcome RAG2-/--mediated DN stage arrest as most TYPE I AND III INTERFERONS DISRUPT LUNG STAT5BN642H RAG2-/- thymocytes were DP and SP8 T-cells. EPITHELIAL REPAIR DURING RECOVERY FROM VIRAL Comparing STAT5BN642H RAG2-/- to RAG2-/- thymocytes by INFECTION. RNA-seq showed upregulation of several TCR signaling J. Major1,*, A. Wack1, S. Crotta1, M. Llorian1, T. McCabe2, H. genes despite TCR deficiency, and enrichment of MYC H. Gad3, S. Priestnall4, R. Hartmann3 targets and mTOR signaling in these cells. We conclude that 1Francis Crick Institute, London, 2Galaxosmithkline, STAT5 hyperactivation induces an activated T-cell Stevenage, United Kingdom, 3Aarhus University, Aarhus, phenotype and thus drives proliferation in aggressive T-cell Denmark, 4The Royal Vetinary College, Hatfield, United neoplasia. Indeed, human T-ALL cell lines with highly Kingdom activated STAT5, caused by STAT5BN642H or IL-7R mutations, were sensitive to a specific STAT5 inhibitor, that Abstract Content: Excessive cytokine signaling frequently decreases proliferation of these cells, whereas cell lines with exacerbates lung tissue damage during respiratory viral low STAT5 activation respond less. In summary, infection. Disease severity is linked to lung epithelial hyperactive STAT5B disrupts thymic development, pushing destruction, due to both cytopathic viral effects and immune- T-cells into the CD8+ cytotoxic lineage and driving cytokine mediated damage. Type I (IFN-α/β) and III (IFN-λ) releasing and responsive factors. A RAG2-/- background interferons are host-produced antiviral cytokines. Due to demonstrated the mutation’s potency to overcome DN stage widespread expression of the type I IFN receptor (IFNAR) in arrest and drive immature T-cell neoplasia, which can be immune cells, IFN-α/β responses can result in explained by a partially restored TCR activation signature. immunopathology during viral infections. The IFN-λ receptor STAT5B is a clear target in many T-cell neoplasias and a (IFNLR) is mainly expressed at epithelial barriers, and beast in patients carrying the STAT5BN642H driver mutation. responses are therefore often characterized by their ability Disclosure of Interest: None Declared to confer localized antiviral protection at the site of infection, without driving damaging proinflammatory responses O09 associated with IFN-α/β. MECHANISMS OF IMMUNE MODULATION IN THE In our study, we show that IFN signaling interferes with lung TUMOR MICROENVIRONMENT repair during influenza recovery (1). Treatment with K. T. Chow1,* exogenous type I or III IFN late during infection reduces lung 1Biomedical Sciences, City University of Hong Kong, epithelial proliferation. Using IFNAR and IFNLR deficient Kowloon, Hong Kong mice, we show host-produced IFN-λ predominantly drives lung pathology. Additionally, comparison of IFN subtypes at Abstract Content: The ability to modulate the immune equal biological potency in primary airway epithelial cell system forms the basis of immunotherapy, which promises cultures revealed IFN-λ most potently induced new interventions for myriad clinical conditions including antiproliferative pathways. cancer. We focus on the modulation of the innate immune By studying specific effects in the respiratory epithelium, we system, which constitutes the first line of defense to keep identify a mechanism by which IFN exacerbates respiratory 6 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 virus disease, independent of immunomodulation. IFN- Gokhale3, S. M. Horner 3, M. J. McGeachy 1, A. C. Poholek4, induced p53 directly reduces lung epithelial proliferation and P. S. Biswas 1, S. L. Gaffen 1 differentiation during recovery from influenza virus infection, 1Division of Rheumatology & Clinical Immunology, therefore increasing disease severity, and susceptibility to University Of Pittsburgh, Pittsburgh, 2Department of bacterial superinfections. Thus, excessive or prolonged IFN- Medicine Endocrinology, Robert Wood Johnson Medical production aggravates viral infection by impairing lung School, Rutgers, 3Department of Molecular Genetics and epithelial regeneration. Our data indicate the need for Microbiology, Duke University Medical Center, Durham, effective regulation of host IFN responses, and the 4Division of Pediatrics, University of Pittsburgh, Pittsburgh, importance of timing and duration when considering IFNs as United States therapeutic strategies to treat respiratory virus infections, including influenza and 2019 coronavirus disease (COVID- Abstract Content: IL-17 is the founding member of a 19). unique and poorly understood cytokine subclass. Dysregulated activity of IL-17 and Th17 cells underlies many (1) J. Major et al., Type I and III interferons disrupt lung autoimmune conditions, but the molecular mechanisms by epithelial repair during recovery from viral infection. Science which IL-17 mediates pathogenic inflammation remain 10.1126/science.abc2061 (2020) poorly understood. IL-17 regulates pathogenic inflammatory Disclosure of Interest: None Declared genes by two key transcription factor classes, NF-ΚB and CCAAT/Enhancer Binding (C/EBP) proteins, specifically O11 C/EBPδ and C/EBPβ. Compared to activation of NF-ΚB, REACTIVE OXYGEN SPECIES OXIDIZE STING AND surprisingly little is known about mechanisms that activate SUPPRESS INTERFERON PRODUCTION C/EBPs, either by IL-17 or indeed most other inflammatory T. Reese1,*, L. Tao2, A. Lemoff3, G. Wang3, C. Zarek3, A. stimuli. In seeking to understand how IL-17 upregulates Lowe3 C/EBPs, we found that IL-17 signaling enhanced Cebpd 1Immunology, UT SOUTHWESTERN MEDICAL CENTER, mRNA stability, concomitant with increased levels of 2Immunology, 3UT Southwestern, Dallas, United States C/EBPδ translation. In contrast, IL-17 had only a marginal inductive effect on Cebpb mRNA, yet C/EBPβ protein was Abstract Content: Reactive oxygen species are by- nonetheless strongly upregulated. Examination of Cebpb products of cellular respiration that can promote oxidative and Cebpd noncoding sequences identified consensus sites stress and damage cellular proteins and lipids. One for N6-methyladenosine (m6A) modification, an canonical role of ROS is to defend the cell against invading epitranscriptomic mark that influences mRNA fate. The m6A bacterial and viral pathogens. Curiously, some viruses, pathway is still very understudied in the , so including herpesviruses, thrive despite the induction of to determine if this pathway was indeed involved in IL-17 reactive oxygen species, suggesting that reactive oxygen signaling, we knocked down the m6A ‘writer’ METTL3 and species are beneficial for the virus. However, the underlying the ‘eraser’ demethylase FTO. Indeed, METTL3 enhanced mechanisms remain unclear. Here, we found that reactive C/EBP expression, which was reversed by FTO. Moreover, oxygen species impaired interferon response during we found that loss of an unusual m6A ‘reader’ IGF2BP2 herpesvirus infection and the inhibition occurred (IMP2), an RNA binding protein known to control mRNA downstream of cytoplasmic DNA sensing. We further stability and translation, impaired IL-17 induction of C/EBPs demonstrated that reactive oxygen species suppressed the and consequently C/EBP-dependent genes. IMP2 bound type I interferon response by oxidizing Cysteine 147 on directly to Cebpb and Cebpd transcripts, leading to murine stimulator of interferon genes (STING), an ER- enhanced Cebpd half-life and enhanced translation of both associated protein that mediates interferon response after C/EBPδ and C/EBPβ. Mutation of putative IMP2 binding cytoplasmic DNA sensing. This inhibited STING sites within the Cebpd 3’ UTR impaired IMP2 binding. polymerization and activation of downstream signaling Transcriptomic analysis revealed that IMP2 regulates events. These data indicate that redox regulation of C/EBP-dependent genes, including IL-6 and Lcn2 Cysteine 147 of mouse STING, which is equivalent to (Lipocalin 2). Consistent with this, Imp2-/- mice were Cysteine 148 of human STING, controls interferon refractory to experimental autoimmune encephalomyelitis production. Together, our findings reveal that reactive (EAE, driven by IL-6) and autoantibody-induced oxygen species orchestrate anti-viral immune responses, glomerulonephritis (AGN, driven by Lipocalin 2). Thus, IL- which can be exploited by viruses to evade cellular 17-induced autoimmunity is mediated through m6A- defenses. dependent post-transcriptional regulation of C/EBP Disclosure of Interest: None Declared transcription factors, illustrating cross-talk between transcriptional and post-transcriptional programs driven by Symposium 6: Cytokine gene regulation and IL-17. This is one of the few studies demonstrating that the non-coding RNA m6A pathways is operative in cytokine signaling, and the first O12 to show that this pathway regulates C/EPBs and RNA M6A METHYLATION GUIDES IL-17-DRIVEN autoimmune pathogenesis. AUTOIMMUNITY THROUGH THE RNA-BINDING PROTEIN IMP2 Disclosure of Interest: None Declared R. Bechara1,*, N. Amatya1, R. Bailey1, S. Majumder 1, F. E. Aggor1, Y. Li1, C. V. Jawale 1, B. Coleman1, N. Dai2, N. S. 7 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

O13 produced during helminth induced type 2 inflammation and HOST CXCL10 IS REPRESSED BY THE MALARIA that PGD2 and its receptor CRTH2, known for their pro- PARASITE AND SERVES AS A DECISION MAKING inflammatory role during chronic Type 2 inflammation in the FACILITATOR FOR PARASITIC GROWTH AND SEXUAL lung, have a previously unappreciated anti-inflammatory DEVELOPMENT role during helminth-induced Type 2 intestinal inflammation. Y. Ofir-Birin1,*, N. Regev Rudzki1 CRTH2 deficient mice infected with Nippostrongylus 1Biomolecular sciences, Weizmann institute for science, brasiliensis cleared their worms more efficiently and had Rehovot, Israel increased intestinal mucin responses compared to wild type mice. Single cell RNA sequencing revealed an enrichment Abstract Content: The pathogenesis of infectious diseases for secretory lineage epithelial cells in the small intestine of is dominated by dynamic interactions between pathogen CRTH2 deficient compared to wild type mice. Critically, and host, as in the devastating parasitic disease malaria small intestinal epithelial cells and specifically goblet and tuft caused by Plasmodium falciparum.These interactions rely cells expressed the gene that encodes for CRTH2, Gpr44, on a network of secreted proteins, including and murine small intestinal organoids stimulated with Type CXCL10, which is found at high levels in cerebral malaria 2 cytokines downregulated expression of goblet cell- and tuft patients, but is reduced in non-complicated patients. We cell-associated genes following culture with PGD2. Finally, showed that Plasmodium falciparum actively inhibits the bone marrow chimeric mice that had CRTH2 deficiency synthesis of CXCL10 in using a sophisticated isolated to the non-hematopoietic system cleared worms mechanism involving blocking the association of the more efficiently and had elevated goblet cell responses immune host’s ribosomes to the CXCL10 RNA transcript. compared to mice with a wild type non-hematopoietic The underlying inhibition cascade involves delivery of RNA compartment, confirming a role for CRTH2 in regulating cargo via Plasmodium falciparum derived EVs into host epithelial responses and resulting worm clearance in vivo. monocytes. The EV-derived RNA triggers host RIG-I, which These findings highlight a novel regulatory effect of the then leads to the binding of HUR1 to an AU-rich domain in PGD2-CRTH2 pathway on helminth-induced Type 2 the 3’UTR of the CXCL10 transcript. Remarkably, in intestinal inflammation and may inform the development and response to the presence of CXCL10, the parasite starts use of therapies for treatment of Type 2 inflammatory accelerating growth and stimulating its sexual development. diseases. Thus, we identified a novel decision-sensing mechanism the Disclosure of Interest: None Declared deadly malaria parasite Plasmodium falciparum employs during the most severe form of malaria infection, cerebral O15 malaria. We reveal a ‘decision sensing system’: high CYTOKINES AND SKIN BARRIER INTEGRITY CXCL10 expression serves as an ‘alert facilitator’, B. J. Nickoloff1,* prompting Plasmodium falciparum to shift tactics and 1Eli Lilly & Co., Indianapolis, United States initiate escape actions. Disclosure of Interest: None Declared Abstract Content: When it comes to the skin functioning as a protective coat, the two essential components have Symposium 7: Inflammation and barrier traditionally been considered as the epidermal integrity producing a physical barrier, complemented by neuro- O14 immunocytic cells generating innate and adaptive immune THE PROSTAGLANDIN D2 RECEPTOR CRTH2 reactions to maintain homeostasis in an ever-changing SUPPRESSES EPITHELIAL RESPONSES DURING ecosystem. As skin is our outermost organ, its accessibility INTESTINAL HELMINTH INFECTION to biopsies and visual assessments of clinical changes has O. O. Oyesola1,*, M. Shanahan2, M. Kanke2, L. M. Webb1, P. made it an ideal target for translational studies and precision Campioli3, D. Pham3, S. Fruh3, M. Matheson1, E. Kamynina3, medicine yielding important lessons and insights into many S. A. Peng3, R. L. Cubitt2, M. S. Nadjsombati1, J. von inflammatory reactions beyond the integument. Almost 30 Moltke1, J. D. Lord4, P. Sethupathy2, E. D. Tait Wojno1 years ago, the myth that keratinocytes were only passive 1Immunology, University of Washington, Seattle, targets of immune responses, was dispelled by evidence 2Biomedical Sciences, 3Baker Institute for Animal Health indicating these cells were active producers of cytokines, as and Department of Microbiology and Immunology, Cornell well as adhesion molecules capable of initiating cutaneous University, Ithaca, 4Benaroya Research Institute and inflammation (Lancet 337:211, 1991). Indeed, we now Virginia Mason Medical Center, Division of recognize that barrier perturbation in skin activates cytokine Gastroenterology, University of Washington, Seattle, United production, as well as the reciprocal ability of cytokines to States alter barrier function (both tight junctions and filaggrin levels). It also has now evident that both bacterial and Abstract Content: Type 2 inflammation that is required for fungal commensal microbials contribute to cutaneous tissue repair and expulsion of helminth parasites must be immunobiology across the epidermal surface. Greater regulated to prevent chronic inflammation. While previous insights into the immunopathogenesis of two common studies show that cytokines play key roles in promoting and chronic inflammatory skin diseases characterized by altered resolving Type 2 inflammation, the role of bioactive lipid barrier function-psoriasis and atopic dermatitis have revealed complex, highly choreographed interactions mediators such as prostaglandin D2 in regulating these between cytokines and resident skin cells leading to processes is less clear. Here we show that PGD2 is 8 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 significant therapeutic progress for these immune-mediated promotes T2 pulmonary inflammation indirectly via IL-4Ra disorders. In this presentation we will review the importance signaling, but that IL-4Ra signaling can drive T2 of cytokines contributing to skin barrier integrity, and roles inflammation in the lung independently of TSLP. for commensal organisms and dysregulated cytokine Consequently, these data re-iterate that IL4Ra signaling is networks perturbing barrier function and contributing to the central cytokine node in promoting and maintaining T2 chronic skin inflammation. Clearly there has been inflammation. meaningful progress in our appreciation of the role for cytokines and interferons in skin health and disease far Research sponsored by Regeneron Pharmaceuticals, Inc. beyond the initial discovery that recombinant IFNg could Disclosure of Interest: None Declared induce MHC class II HLA-DR expression by human epidermal keratinocytes in 1984 (J Invest Dermatol 83:84, Symposium 8: Regulators of interferons - 1984). Philip I. Marcus Symposium Disclosure of Interest: B. Nickoloff Employee of: Eli Lilly & O17 Co. THE UNIQUE TYPE-I INTERFERON-EPSILON (IFNΕ) CONSTITUTIVELY PROTECTS THE FEMALE O16 REPRODUCTIVE TRACT FROM ZIKA VIRUS THYMIC STROMAL LYMPHOPOIETIN (TSLP) INFECTION. PRIMARILY DRIVES TYPE 2 INFLAMMATION THROUGH R. C. Coldbeck-Shackley1,*, M. Tate2, S. Rosli2, J. Gearing2, IL-4RA-DEPENDENT SIGNALING J. Gould2, S. Lim2, K. Van der Hoek1, N. Eyre3, S. 1,* 1 1 1 S. Srivatsan , J. Allinne , G. Scott , D. Birchard , L.-H. 4 5 2 1 1 1 1 1 Robertson , S. Best , P. Hertzog , M. Beard Ben , A. J. Murphy , M. A. Sleeman , J. M. Orengo 1 1 Molecular and Biomedical Science, The University of Regeneron Pharmaceuticals, Inc, Tarrytown, United States Adelaide, Adelaide, 2Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Abstract Content: , a fully human monoclonal Clayton, 3Biological Sciences, Flinders University , 4Health antibody that binds IL-4Ra and inhibits signaling of both IL- and Medical Sciences, The University of Adelaide, Adelaide, 4 and IL-13, is approved for the treatment of multiple Type 2 Australia, 5Rocky Mountain Laboratories, National Institutes (T2) inflammatory diseases, including asthma, atopic of Health, Montana, United States dermatitis (AD) and chronic sinusitis with nasal polyps, and has shown efficacy in eosinophilic esophagitis. Recently Abstract Content: Zika virus (ZIKV) can be transmitted studies with tezepelumab, a TSLP-neutralizing antibody, sexually with infection of the female reproductive tract (FRT) have also demonstrated positive effects in both eosinophilic and the developing foetus in utero. Like many RNA viruses and non-eosinophilic asthma, but interestingly not in ZIKV abrogates type-I and type-III IFN antiviral responses. AD. Given these observations, these studies highlight the This immune evasion is mediated by the actions of multiple need for a mechanistic comparison of these two pathways viral non-structural (NS) proteins. However, the unique type- on T2 inflammation to better understand the relative I IFN-epsilon (IFNε), is constitutively expressed by the contribution of each pathway to T2 immunity. mucosal epithelium of the FRT. Therefore, we hypothesised Here, we employ cytokine overexpression studies IFNε circumvents ZIKV mediated IFN evasion by (intranasal cytokine administration and hydrodynamic DNA constitutively protecting the FRT from infection. delivery) and multiple preclinical models of lung To explore the role of IFNε in vivo we intravaginally inflammation to evaluate the functional contributions of the inoculated IFNε KO (IFNε -/-), type-I IFN receptor KO TSLP and the IL-4/IL-13 signaling pathways to T2 airway (IFNAR1-/-) and wildtype (WT) mice with ZIKV (PRVABC59). inflammation. We demonstrate that induction of T2 immunity IFNε-/- mice had increased viral burden in the uterus and by TSLP requires IL-4Ra signaling. Several features of T2 ovary (qRT-PCR) and greater levels of ZIKV in vaginal airway inflammation induced by TSLP overexpression, washes when compared to WT mice (plaque assay). Next, including goblet cell metaplasia (GCM), lung tissue we intravaginally administered 4 μg of recombinant IFNε to eosinophilia and serum IgE are abrogated in Il4ra KO mice. IFNε-/- mice that resulted in the restoration of antiviral activity TSLP overexpression also dramatically alters the lung in the FRT compared to buffer treated controls. Furthermore, myeloid landscape, by inducing changes in alveolar application of 100 μg IFNε neutralising antibody to WT mice macrophages and subsets consistent with increased their susceptibility to infection compared to coordinating a T2 immune response, and requires intact IL- isotype controls. 4Ra signaling to mediate these changes. Conversely, IL-4 Next, we demonstrated that in primary transformed FRT cell or IL-13 overexpression promotes several of these T2 lines IFNε induced the expression of hundreds of interferon- hallmarks independently of TSLP signaling. Next, using stimulated genes similar to the antiviral profile induced by antibody-mediated pathway blockade in two distinct models IFNλ-III, not IFNα as expected being a type-I IFN of airway inflammation (the experimental ova-alum model (NextSeq550 V2.5, qRT-PCR). Interestingly, IFNε did not and the clinically relevant house dust mite model), we show induce expression of IRF1 and displayed limited induction of that blunting IL-4Ra signaling is significantly more effective pro-inflammatory cytokines compared to IFNα and IFNλ-III. than blocking TSLP at ameliorating key measures of T2 Pre-treatment with IFNε in FRT cell lines inhibited ZIKV inflammation, including GCM, lung tissue eosinophilia and replication like IFNα and IFNλ-III (qRT-PCR, plaque assay). associated chemokines. Thus, collectively across numerous However, post-infection these antiviral responses were endpoints in multiple models, we demonstrate that TSLP blocked via NS5 mediated STAT2 degradation. Conversely, 9 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 the expression of IFNε was impervious to ZIKV NS4A, NS1 that there was no IFN-λ antiviral effect early in gestation and NS5 mediated inhibition downstream of activated RIG-I (infection at E7 compared to E9), indicating that IFN-λ that delayed the production of other type-I and III IFNs (qRT- antiviral activity is dependent on gestational stage. Using PCR). Furthermore, we demonstrated by immunoblot that mice also lacking the type I IFN receptor (Ifnlr1-/ Ifnar1-/-), we IFNε signals constitutively despite IFN induced receptor studied the effect of IFN-λ signaling in pregnancies with desensitisation that limits IFNα mediated signal enhanced ZIKV replication and again found an antiviral transduction. effect of maternal IFN-λ signaling. Interestingly, early- Collectively these findings highlight the unique biological gestation pregnancies (infection at E7) with maternal IFN-λ role of constitutively expressed IFNε in prevention of ZIKV signaling also exhibited enhanced fetal pathology, infections in the FRT. Importantly, this shifts our suggesting that IFN-λ signaling can contribute to fetal understanding of FRT innate immunity from reliance on pathology in high-viremia models. Together these data show reactionary responses to pre-emptive protection against that IFN-λ signals to maternal tissues and has discrete viral infections. Combined with continued studies of IFNε functions at different gestational stages. expression in humans, this knowledge will help to inform Disclosure of Interest: None Declared sexual health recommendations for ZIKV and other infections of the FRT. Symposium 9: Host response to SARS-CoV-2 Disclosure of Interest: None Declared O19 LY6E IMPAIRS CORONAVIRUS FUSION AND CONFERS O18 IMMUNE CONTROL OF VIRAL DISEASE MATERNAL INTERFERON LAMBDA SIGNALING LIMITS K. B. Mar1,*, S. Pfaender2, E. Michailidis3, A. Kratzel4, I. TRANSPLACENTAL TRANSMISSION AND MEDIATES Boys1, P. V'kovski4, W. Fan1, J. Kelly4, D. Hirt4, N. Ebert4, H. FETAL PATHOLOGY IN A GESTATIONAL STAGE- Stalder4, H. Kleine-Weber5, M. Hoffman5, H. H. Hoffmann3, DEPENDENT MANNER DURING CONGENITAL ZIKA M. Saeed6, R. Dijkman4, E. Steinmann2, M. Wight-Carter1, VIRUS INFECTION IN MICE M. McDougal7, N. Hanners1, S. Pöhlmann5, T. Gallagher8, R. L. Casazza1,*, H. M. Lazear1 D. Todt2, G. Zimmer4, C. Rice3, J. Schoggins1, V. Thiel4 1Microbiology and Immunology, UNC Chapel Hill, Chapel 1UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL Hill, United States CENTER, Dallas, United States, 2Ruhr Universitat Bochum, Abstract Content: Interferons have a key role in the Bochum, Germany, 3Rockefeller University, New York City, development of the placenta and surrounding tissues United States, 4University of Bern, Bern, Switzerland, throughout pregnancy, and are regulated carefully to ensure 5Deutsches Primatenzentrum GmbH, Leibniz-Institut für fetal development as well as protection from maternal Primatenforschung, Göttingen, Germany, 6Boston pathogens. Unlike most viruses that cause congenital University, Boston, 7Ruhr Universitat Bochum, Dallas, infections in humans, Zika virus (ZIKV) pathogenesis can be 8Loyola University Chicago, Chicago, United States modeled in mice. Mouse models of congenital ZIKV infection provide a genetically tractable tool to distinguish maternal Abstract Content: Zoonotic coronaviruses (CoVs) are and fetal immune responses to viral infection, and their significant threats to global health, as exemplified by the distinct impacts on fetal outcomes. IFN-λ is secreted emergence of two severe acute respiratory syndrome CoVs constitutively from human placental trophoblasts, and limits (SARS-CoV and SARS-CoV-2) and Middle East respiratory ZIKV transplacental transmission in mouse congenital syndrome CoV (MERS-CoV) within two decades. Host infection models, but it has yet to be determined if IFN-λ is immune responses to CoVs are complex and regulated in secreted constitutively from the mouse placenta and what part through antiviral interferons. However, interferon- cell types respond to IFN-λ to restrict ZIKV congenital stimulated gene products that inhibit CoVs are not well infection. characterized. Here, we show that lymphocyte antigen 6 To determine the patterns of IFN-λ secretion from the complex, locus E (LY6E) potently restricts infection by mouse placenta, we evaluated IFN-λ activity in infected and multiple CoVs, including SARS-CoV, SARS-CoV-2, and uninfected dams at varied time points in gestation. In MERS-CoV. Mechanistic studies revealed that LY6E inhibits uninfected dams, IFN-λ activity was present in placentas CoV entry into cells by interfering with spike protein- from laboring dams, but was undetectable in mid-gestation mediated membrane fusion. Importantly, mice lacking Ly6e placentas. However mid-gestation placentas from ZIKV- in hematopoietic cells were highly susceptible to a murine infected dams had the highest levels of IFN-λ, indicating that CoV, mouse hepatitis virus. Exacerbated viral pathogenesis it is secreted in the mouse placenta under inflammatory in Ly6e knockout mice was accompanied by loss of hepatic conditions such as infection or labor, but not at baseline mid- immune cells, higher splenic viral burden, and reduction in gestation. global antiviral gene pathways. Accordingly, we found that To define the tissues that respond to IFN-λ at the maternal- constitutive Ly6e directly protects primary B cells from fetal interface, we assessed ZIKV transplacental murine CoV infection. Our results demonstrate that LY6E is transmission in mid-gestation (E9-E15) mouse pregnancies a critical antiviral immune effector that controls CoV infection in which either fetal or maternal tissues lacked the IFN-λ and pathogenesis. These findings advance our receptor (Ifnlr1-/-). Unexpectedly, the antiviral effects of IFN- understanding of immune-mediated control of CoV in vitro λ resulted exclusively from signaling in maternal tissues, as and in vivo, knowledge that could help inform strategies to fetal viral loads depended only on maternal Ifnlr1 genotype, combat infection by emerging CoVs. not the fetal/placental genotype. Furthermore, we observed Disclosure of Interest: None Declared 10 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Symposium 10: Cytokines in neurobiology O21 O20 CD28 AND B7 RECEPTOR HOMODIMER INTERFACES THE ROAD TO EPITHELIAL BARRIER HEALTH: CONTROL FORMATION OF THE B7/CD28 TARGETING THE IL-36R PATHWAY COSTIMULATORY AXIS AND LETHAL PRO- J. Fine1,*, E. Raymond1, G. Caviness1, R. Ganesan1, M. INFLAMMATORY SIGNALING Ramanujam1, A. Klimowicz1, J. Zheng1, C. Thoma2, K. R. Kaempfer1,*, A. Popugailo1, M. Levy1, O. Turgeman1, Z. Scheibe3, C. Neufert3, M. Neurath3 Rotfogel1 1Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, 1Biochemistry and Molecular Biology, Faculty of Medicine, United States, 2Boehringer Ingelheim Pharmaceuticals, Inc., The Hebrew University, Jerusalem, Israel Biberach, 3Universitatsklinikum Erlangen, Erlangen, Germany Abstract Content: The inflammatory response is indispensable for protective immunity, yet pathogens, Abstract Content: The Road to Epithelial Barrier Health: including coronavirus, often elicit a vastly excessive Targeting the IL-36R Pathway response harmful to the host. Full T-cell activation requires Ernest L. Raymond1, Gary Caviness1, Raj Ganesan2, Meera interaction between costimulatory receptors B7 and CD28. Ramanujam1, Alexander C. Klimowicz1, Jie Zheng1, In the extracellular domains of the CD28 as well as the B7 Christian Thoma3, Kristina Scheibe4, Clemens Neufert4, receptors, the self-adhesive homodimer interfaces are Markus F. Neurath4 and Jay S. Fine1 remote from the coligand binding sites. Yet, our finding is 1 Immunology and Respiratory Diseases Research, that short mimetic peptides derived from distinct regions Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, within the composite CD28 homodimer interface attenuate USA B7-2(CD86)/CD28 engagement as well as the tighter B7- 2 Biotherapeutics Discovery, Boehringer Ingelheim 1(CD80)/CD28 interaction, and thereby attenuate signaling Pharmaceuticals Inc., Ridgefield, CT, USA through the human B7/CD28 costimulatory axis, to dampen 3 Therapeutic Area Immunology Medicine, Boehringer induction of inflammatory cytokines. Conversely, short Ingelheim Pharma GmbH & Co. KG, Germany peptide mimetics derived from distinct regions within either 4 First Department of Medicine, Universitätsklinikum the composite B7-1 or B7-2 dimer interfaces selectively Erlangen, Friedrich-Alexander-Universität Erlangen- attenuate engagement of CD28 by the cognate B7 Nürnberg, Erlangen, Germany coreceptor, thereby attenuating B7/CD28 signaling and dampening the induction of inflammatory cytokines. Even Increasing evidence points to an important role for the when administered at exceedingly low concentrations, such interleukin (IL)-36 pathway in mediating a variety of peptides are capable of protecting mice from lethal bacterial inflammatory responses. The three IL-36 agonist cytokines and viral infections. Our results reveal that the CD28 and B7 (IL-36a, IL-36b and IL36g) are expressed by epithelial and homodimer interfaces each control formation of the immune cells and mediate signal transduction on epithelial B7/CD28 costimulatory axis and highlight the protective cells, immune cells and fibroblasts by binding to a common potential against severe infections of attenuating pro- receptor composed of IL-36R and IL1RAcP, in a manner inflammatory signaling through these receptor domains. Our analogous to other IL-1/R family members. IL-36Ra is a approach, development of host-oriented therapeutics that naturally occurring antagonist that binds to IL-36R but is target the human inflammatory response to control and unable to recruit IL1RAcP and induce downstream prevent the cytokine storm, is unlikely to be circumvented by signaling. Hyperactivation of the IL-36 pathway due to the pathogen mutations. absence of IL36Ra in generalized pustular psoriasis (GPP) Disclosure of Interest: None Declared suggests its fundamental role in the pathogenesis of pustular skin diseases. Symposium 11: Cytokines in In this presentation, we will describe novel IL-36 pathway neuroimmunology biology suggesting key regulatory roles in a) the loss of O22 epithelial barrier integrity in the inflamed skin and intestine, INTERFERON SIGNALLING IN ENDOTHELIAL CELLS and b) driving intestinal aberrant tissue remodeling MEDIATES PATHOGENESIS IN CEREBRAL TYPE I associated with Crohn’s disease and ulcerative colitis. INTERFERONOPATHIES Furthermore, we will discuss the rapid and sustained clinical B. Viengkhou1,*, E. Hayashida1, L. Loo2, K. Emelianova3, S. improvement achieved by selective IL-36R blockade with McGlasson3, G. Neely2, I. L. Campbell1, D. Hunt3, M. J. spesolimab in GPP patients presenting with an acute Hofer1 moderate to severe disease flare. Evidence for specific 1School of Life and Environmental Sciences and Charles cellular and molecular changes associated with treatment Perkins Centre, 2The Dr. John and Anne Chong Laboratory response will be described. for Functional Genomics, School of Life and Environmental Based on accumulating preclinical and clinical data, Sciences and Charles Perkins Centre, The University of spesolimab combines potent anti-inflammatory and anti- Sydney, Sydney, Australia, 3The UK Dementia Research fibrotic effects, demonstrates differentiation from standard of Institute, University of Edinburgh, Edinburgh, United care treatments, and may offer significant therapeutic Kingdom benefits in a range of inflammatory and fibrotic disorders. Disclosure of Interest: None Declared 11 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Abstract Content: Cerebral type I interferonopathies are a Abstract Content: Zika virus (ZIKV) infection during group of diseases that stem from chronic and elevated type pregnancy causes congenital Zika syndrome (CZS) I interferon (IFN-I) signalling, which is usually associated characterized by fetal abnormalities at birth and neurological with antiviral and immunoregulatory functions. These defects that manifest into early childhood. At present, there diseases include genetic disorders like Aicardi-Goutières is no approved vaccine or therapeutic for clinical use to syndrome, autoimmune diseases such as CNS-lupus and prevent ZIKV vertical transmission, making ZIKV an chronic and congenital viral infections. IFN-Is induce enduring worldwide public health concern for pregnant pathology, but the contribution of different cell types is not women. Nonhuman primate (NHP) models of ZIKV infection well understood. A hallmark of these diseases centres in pregnancy have demonstrated vertical transmission and around abnormal blood vessels in the brain. We thus fetal neuropathology; however, the impact of ZIKV infection hypothesised that endothelial cells play a critical role in on fetal brain development is underexplored. We performed mediating interferonopathic brain disease. To address our a comprehensive characterization of NHP fetal brain hypothesis, we used transgenic mice with CNS-targeted following ZIKV exposure in utero. Healthy pregnant pigtail chronic production of IFN-α, termed GIFN mice. GIFN mice macaques (PTM) received subcutaneous inoculations of are the only mouse model that recapitulates key clinical ZIKV (n=5) or media (n=3) in the forearms between 60-119 symptoms and pathologies of patients with cerebral type I days gestation (first-second trimesters). At birth, the PTM interferonopathies including a pronounced vasculopathy fetal brain was serially sectioned and samples collected for with aneurysms, vessel stenoses and perivascular histologic, transmission electron microscopy (TEM) and calcification. We analysed the transcriptome of single cells RNA-seq analyses. In ZIKV-infected fetal brains, from the CNS of GIFN mice to explore endothelial cell quantitative IHC analysis revealed decreased responsiveness to IFN-I in vivo. We also genetically ablated immunoreactivity of myelin basic protein (MBP) compared to IFN-I signalling in endothelial cells in GIFN mice (termed GIT control brains. MBP alterations were not associated with mice) to gauge the contribution of endothelial cells to significant changes in the percent positive staining for disease. In the CNS, endothelial cells had the highest Olig2+ nuclei in the white matter. Decreased MBP protein expression of the IFN-I receptor genes and in response to was also observed in NHP fetal brainstem by Western blot IFN-I showed a major change in their transcriptome that analysis and confirmed using a human oligodendrocytic reflected disease-relevant processes including endothelial M03.13 cell culture model. H&E analysis of NHP fetal brain dysfunction. Loss of IFN-I in endothelial cells in the presence found no obvious signs of immune cell infiltration or of elevated IFN-α in GIT mice resulted in a significant inflammation in post-ZIKV fetal brain, while EM analysis improvement of many disease features. This included indicated enhanced multifocal decompaction to myelin reduced mortality and motor impairment, the absence of sheaths. Differentially expressed (DE) genes related to calcification, aneurysms and neurodegeneration and myelination were upregulated in most post-ZIKV fetal brains minimal leukocyte infiltration and glial cell reactivity. and associated with gliogenesis genes in the recovery of Importantly, vascular morphology and blood brain barrier ZIKV infection. Our data sets and analyses define fetal integrity was rescued. These findings demonstrate that neurological profiles linked with ZIKV-induced brain injury vasculopathy is a major contributing factor to cerebral type I patterns. We hypothesize loss of MBP leads to instability of interferonopathies. Moreover, our findings indicate that myelin sheaths. ZIKV may directly target MBP for targeting the blood vessels offer a highly promising and degradation through an NS5-mediated mechanism that is novel approach to treat patients as in contrast to the brain similar to ZIKV interference of type I interferon signaling. The parenchyma the brain's blood vessels are more easily impact of ZIKV infection on the initiation of myelination accessible for therapeutic interventions. during prenatal development may have long term Disclosure of Interest: None Declared consequences into early childhood neurodevelopment. Disclosure of Interest: None Declared O23 ZIKA VIRUS INFECTION IS ASSOCIATED WITH Symposium 12: Immune cell fate and function ALTERATIONS IN MYELINATION DURING PRENATAL O24 DEVELOPMENT STRUCTURAL CELLS ARE KEY REGULATORS OF J. T. Go1,*, C. Stokes1, D. Newhouse1, K. Voss1, E. Smith1, ORGAN-SPECIFIC IMMUNE RESPONSES A. Baldessari2, M. Coleman3, A. Orvis4, J. Ogle2, A. T. Krausgruber1,*, N. Fortelny1, V. Fife-Gernedl1, M. Germond2, C. English2, M. Dacanay5, E. Parker6, B. W. Senekowitsch1, L. C. Schuster1, A. Lercher1, A. Nemc1, C. Johnson5, J. M. Snyder5, L. Rajagopal7, R. P. Kapur8, K. Schmidl1, A. F. Rendeiro1, A. Bergthaler1, C. Bock1,2 Adams Waldorf9, M. Gale1 1CeMM Research Center for Molecular Medicine of the 1Immunology, University of Washington, 2Washington Austrian Academy of Sciences, 2Department of Laboratory National Primate Research Center, 3Pediatric Infectious Medicine, Medical University of Vienna, Vienna, Austria Diseases, Seattle Children's Research Institute, 4Pediatric Infectious Diseases, Seattle Children’s Research Institute, Abstract Content: 5Comparative Medicine, 6Ophthalmology, 7Pediatrics, BACKGROUND: The mammalian immune system University of Washington, 8Pathology, Seattle Children’s implements a remarkably effective set of mechanisms for Hospital, 9Obstetrics & Gynecology, University of fighting pathogens. Its main components are Washington, Seattle, United States haematopoietic immune cells, including myeloid cells that control innate immunity and lymphoid cells that constitute 12 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 adaptive immunity. However, immune functions are not knockdown, overexpression plasmids, site-directed unique to haematopoietic cells, and many other cell types mutagenesis, and genetic knockout mice in metabolic flux display basic mechanisms of pathogen defence. assays, we aimed to understand the impact of Arg2 on IL- METHODS: To advance our understanding of immunology 10’s role as a metabolic rheostat in macrophages. We outside the haematopoietic system, we systematically demonstrate that IL-10 specifically enhances Arg2 investigate the regulation of immune genes in the three localization at the mitochondria in inflammatory major types of structural cells: epithelium, endothelium and macrophages, where it radically promotes a state of fibroblasts. We performed multi-omics profiling of these cell ‘fusion’, an effect that is dependent on both Arg2 catalytic types across twelve mouse organs - brain, caecum, heart, activity and its physical presence at the mitochondria. We kidney, large intestine, liver, lung, lymph node, skin, small demonstrate that Arg2 is critical for IL-10 induced oxidative intestine, spleen and thymus. Single-cell suspensions were respiration and ATP production in these cells. analysed by flow cytometry, and sort-purified cell Mechanistically, we illustrate that Arg2 distinctly influences populations were profiled with three genome-wide assays: complex II (also known as succinate dehydrogenase (i) profiling by low-input RNA-seq; (ii) (SDH)), a bi-functional enzyme that links the chromatin accessibility profiling by ATAC-seq; and (iii) mitochondrial electron transport chain (ETC) and the epigenome profiling by ChIPmentation with an antibody Tricarboxylic acid (TCA) cycle. Via its regulation of SDH, against promoter/enhancer-linked histone H3K4me2. Arg2 is essential for IL-10 mediated downregulation of RESULTS: We observed widespread expression of immune inflammatory mediators mitochondrial reactive oxygen regulators and cytokine/chemokine signalling molecules in species (mtROS) and interleukin 1β (IL-1β). Moreover, in an structural cells, organ-specific adaptation to the tissue acute model of inflammation when IL-10 expression is environment, and unexpectedly diverse capabilities for compromised, Arg2 expression is suppressed, suggesting interacting with hematopoietic cells. These cell-type-specific that Arg2 may be critical for IL-10 mediated protection and and organ-specific differences in immune gene activity were homeostasis. Altogether, these findings demonstrate a reflected by characteristic patterns of chromatin regulation. novel IL-10 polarization model where macrophages Most notably, we found evidence of an epigenetically leverage their metabolic enzymes as a mechanism to encoded immune potential under homeostatic conditions, maintain an anti-inflammatory state. and the affected genes were preferentially upregulated in Disclosure of Interest: None Declared response to an immunological challenge induced by systemic viral infection. We validated and functionally O26 dissected this epigenetic potential of structural cells by THE IL-10-STAT3 AXIS IS A CRITICALLY IMPORTANT further in vivo experiments with recombinant cytokines. LUNG-SPECIFIC PATHWAY DRIVING BLIMP-1 CONCLUSION: Our study highlights the prevalence and MEDIATED TH2 CELL DIFFERENTIATION TO PROMOTE organ-specific complexity of immune gene activity in non- ALLERGIC ASTHMA haematopoietic structural cells, and it provides a high- K. He1,2,*, A. Hettinga1,2, S. L. Kale2,3, S. Hu2,3, M. M. Xie4, A. resolution, multi-omics atlas of the epigenetic and L. Dent4, A. Ray2,3, A. C. Poholek1,2 on behalf of transcriptional networks that regulate structural cells in the Correspondence mouse. 1Division of Pediatric Rheumatology, Department of Disclosure of Interest: None Declared Pediatrics, 2Department of Immunology, 3Department of Pulmonary, Allergy, and Critical Care Medicine, Department O25 of Medicine, University of Pittsburgh School of Medicine, ARGINASE 2 IS ESSENTIAL FOR IL-10 METABOLIC Pittsburgh, 4Department of Microbiology and Immunology, REPROGRAMMING OF INFLAMMATORY Indiana University School of Medicine, Indianapolis, United MACROPHAGES AT THE MITOCHONDRIA States J. Dowling1, R. Afzal1, L. Gearing2, M. Cervantes-Silva1, C. De Santi1, N. Assmann3, D. Gough4, G. Bantug3, C. Abstract Content: Allergic asthma is a chronic McCoy5,* inflammatory disease of the lung that is growing worldwide 1school of Pharmacy and Biomolecular Science, RCSI, and has limited new therapeutic options. Lung inflammation Dublin, Ireland, 2Centre for Innate Immunity and Infectious is mediated by T cells responding to allergens that secrete Diseases, Hudson Institute of Medical Research, type 2 cytokines such as IL-4, IL-5 and IL-13 that drive Melbourne, Australia, 3Department of Biomedicine, airway hyperresponsiveness, increased mucus, and recruit University Hospital Basel, Basel, Switzerland, 4Centre for inflammatory cells such as eosinophils to the lung tissue Cancer Research, Hudson Institute of Medical Research, causing widespread damage. Th2 cell differentiation Melbourne, Australia, 5School of Pharmacy and classically requires signaling via the IL-4/STAT6 pathway, Biomolecular Science, RCSI, Dublin 2, Ireland which activates the master transcription factor GATA3. However, evidence of IL-4-independent Th2 cell Abstract Content: Recent evidence highlights the differentiation has demonstrated roles of IL-2/STAT5 and importance of mitochondrial bioenergetics and dynamics in STAT3 pathways. Blimp-1 is a transcriptional repressor regulating macrophage polarisation. We demonstrate that pleiotropically expressed by most effector T cells that Arginase-2 (Arg2), the mitochondrial isoform of arginase, is constrains -mediated autoimmunity. Using a house an interleukin-10 (IL-10) regulated gene in inflammatory dust mite induced murine model of allergic asthma, we have macrophages. Using approaches such as siRNA-induced revealed that Blimp-1 is required for Th2 cell development 13 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 in the lung to inhaled allergens. T cell specific deficiency of cytokine production, but did not exhibit cytotoxic impairment Blimp-1 intrinsically led to a significant decrease of Th2 cells or persistent antigen presentation. They retained KLRG1+ in the lung and a subsequent reduction in lung inflammation terminal effector differentiation and their transcriptional -/- and pulmonary function. In contrast, IgE and TFH cells were program was more comparable to WT than Prf1 mice in intact, suggesting Blimp-1 molecularly dissects Th2 and TFH their absence of an exhaustion signature. differentiation to allergens. We found STAT3 was required Mounting evidence suggests heterozygous mutations in to drive Blimp-1 expression in CD4 T cells and for Th2 cell cytotoxicity-related genes like PRF1 may promote development in response to allergens. Importantly, we show hyperinflammatory responses in MAS patients, who also that Blimp-1 is required for Th2 cells responding to inhaled have highly elevated IL-18 levels. Though neither excess IL- but not skin-injected allergens, suggesting there are lung- 18 nor perforin-deficiency individually cause specific pathways driving Th2 cells to allergens. Here, we immunopathology without inflammatory challenge, we sought to determine the cytokines responsible for observed lethal spontaneous hyperinflammation in expression of Blimp-1 through STAT3 to promote Th2 cells Il18tg;Prf1-/- mice. These mice showed expansion of a in allergen induced lung inflammation. We found than splenic PD-1+, TIGIT+, and Tim-3+ CD8 T-cell population, neither IL-6 nor IL-21 was responsible for Blimp-1 yet show increased IFNg production. Additionally, expression in Th2 cells in the lung. Counterintuitively, we spontaneous immunopathology was partially abrogated by found that the anti-inflammatory cytokine IL-10 was critical CD8 depletion or IFNg neutralization. Together, these data for Blimp-1 expression in Th2 cells and subsequent Th2 cell suggest that IL-18 and cytotoxicity can independently and development in response to allergens. Mechanistically, we synergistically drive pathologic CD8 T-cell activation and found that Blimp-1 acts through Bcl6, which is necessary to life-threatening immunopathology in HLH and MAS. drive GATA3 expression and subsequent Th2 formation in Disclosure of Interest: None Declared the lung in response to inhaled allergens. These data have uncovered a previously unrecognized IL-10-STAT3-Blimp-1 axis that is critically important for lung-specific allergen- Symposium 13: Cytokines in autoimmunity induced Th2 cell differentiation driving allergic asthma. O28 Disclosure of Interest: None Declared EARLY-ONSET AUTOINFLAMMATORY DISEASE CAUSED BY ETS FACTOR MUTATION IN HUMANS AND O27 MICE EXCESS IL-18 AND PERFORIN DEFICIENCY P. Tyler1, M. Bucklin1, T. Maher1, A. Rice1, W. Ji2, N. DISTINCTLY AND SYNERGISTICALLY PROMOTE Warner3, J. Pan3, R. Morotti4, I. Hollink5, V. Dalm5, J. PATHOLOGIC CD8 T-CELL ACTIVATION AND Catanzaro4, S. Lakhani2, A. Muise3, C. L. Lucas1,* EXPERIMENTAL HEMOPHAGOCYTIC 1Immunobiology, 2Yale University, New Haven, United LYMPHOHISTIOCYTOSIS States, 3Hospital for Sick Children, Toronto, Canada, 4Yale E. Rapp1,*, S. Canna1 New Haven Hospital, New Haven, United States, 5Erasmus 1University of Pittsburgh, Pittsburgh, United States University Medical Center, Rotterdam, Netherlands

Abstract Content: CD8 T-cell activation is necessary for Abstract Content: Unbiased disease gene discovery in host defense, but overactivation can also drive life- monogenic inflammatory disorders illuminates molecules threatening immunopathology as seen in Hemophagocytic essential for controlling the destructive potential of the lymphohistiocytosis (HLH). HLH is associated with genetic immune system directly in humans. Here, we describe three impairment of cytotoxic function (e.g. perforin deficiency), unrelated male patients with early-onset mucosal and Prf1-/- mice succumb to typically-mild LCMV infection via inflammation, oral ulcers, and fever caused by loss-of- CD8 T-cell/IFNγ-mediated immunopathology. Macrophage function variants in the gene encoding the ELF4 ETS family Activation Syndrome (MAS) is clinically similar to HLH, but transcription factor. The mutations disrupt transcriptional occurs in certain rheumatic/autoinflammatory diseases and activity, and genetically engineered mice with patient- has been associated with extraordinarily and chronically derived mutations similarly develop more inflammatory elevated serum IL-18. Both HLH and MAS are life- responses. Deficiency in ELF4 results in elevated serum IL- threatening cytokine storm disorders, and an improved 12p40, IL-18, and CXCL1, hyperinflammatory myeloid cells, understanding of their pathogenesis could significantly alter altered interferon-stimulated gene (ISG) induction, and patient screening, diagnosis, and management. exaggerated IL-17 responses from lymphocytes. Kinetic Using mice with excess IL-18 (Il18tg) to model the serum transcriptomic analysis revealed that mutant cells have findings in MAS, we found a baseline increase in PD-1+ CD8 relatively normal early responses (0-4 hrs) but fail to sustain T-cells over WT littermates. Il18tg mice develop enhanced expression of anti-inflammatory genes, including IL-1R immunopathology upon repeated systemic challenge with a antagonist (IL-1Ra), over time (16-24 hrs). Moreover, clinical TLR9-agonist, whereas TLR9-induced immunopathology treatment with recombinant IL-1Ra or neutralizing anti-IL- was comparable between Prf1-/- and WT mice. Additionally, 12p40 reversed features of disease. Thus, we identify a Il18tg mice developed features of MAS-like novel human autoinflammatory disease and define ELF4 as immunopathology upon infection with LCMV (Armstrong), a disease-relevant transcription factor with specialized which was largely mediated through CD8 T cells and IFNγ. function in orchestrating anti-inflammatory gene programs. As with LCMV-infected Prf1-/- mice, CD8 T cells from Disclosure of Interest: None Declared LCMV-infected Il18tg mice showed increased activation and 14 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Symposium 14: Japanese Interferon Society D. Newhouse1,*, J. Greene2, J. Chang1, E. Smith1, L. Law1, L. Picker2, J. Sacha2, M. Gale, Jr.1,3 symposium 1 O29 Deparment of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA, SYSTEMATIC DISSECTION OF CYTOKINE RESPONSES 2 IN LYMPH NODES AT SINGLE-CELL RESOLUTION Vaccine and Gene Therapy Institute, Oregon National 1,* 2 2 1 1 2 Primate Research Center, Oregon Health & Science A. Cui , T. Huang , S. Li , A. Ma , J. Perez , D. Keskin , C. 3 Wu2, N. Hacohen1 University, Portland, OR, Washington National Primate 1Broad Institute of MIT and Harvard, Cambridge, 2Dana- Research Center, University of Washington, Seattle, WA, Farber Cancer Institute, Boston, United States United States

Abstract Content: Cytokines mediate a highly complex Abstract Content: A pre-clinical HIV vaccine based on intercellular signaling network in health and diseases. While rhesus cytomegalovirus (RhCMV) vector, strain 68-1, many individual cytokines have been studied in-depth, we directs protective immunity against SIV infection in the do not have a systems-level view of cell-type-specific rhesus macaque (RM) model. This vaccine elicits a responses to each cytokine and do not yet understand how population of CD8+ T cells that are restricted by MHC-E cytokines orchestrate cell-cell communication networks in a presentation. As RMs show extensive MHC genetic complex immune response. To address these two diversity, we explored vaccine efficacy in Mauritian knowledge gaps in cytokine biology, we performed single- cynomolgus macaques (MCM), which exhibit limited MHC- cell RNA sequencing to measure gene expression profiles E genetic diversity that is similar to humans. Vaccination of over 17 mouse lymph node cell populations including with a MCM derived CMV vector (CyCMV) provided 50% lymphocytes, myeloid cells, and stromal cells in response to protection from SIV infection, similar to protection levels in over 80 cytokines in vivo. Using these data, we derived a the RM model. To uncover the molecular mechanisms compendium of cell-type-specific cytokine signatures, with underlying protection following CyCMV vaccination, we the goal of using this compendium to systematically performed longitudinal whole blood transcriptional profiling compare cytokine responses across cell types and to of protected and non-protected MCMs across 10 vaccination deconvolve complex immune responses. We first time points prior to challenge. Bioinformatic analyses and investigated how distinct cell types respond to the same computational modeling revealed a robust transcriptional cytokine, and found that while some pathways are activated response to vaccination among the protected animals. This across multiple cell types (e.g., anti-viral programs in protection signature was largely driven by the induction of response to interferons), many cytokines induced cell-type- genes involved in Rig-I-Like Receptor Signaling, Death specific gene expression signatures. Next, we performed a Receptor Signaling, and Interferon Signaling in protected global analysis to compare across cytokines and found that relative to non-protected animals. To further evaluate this cytokines induced both unique and shared biological outcome, we compared the CyCMV transcriptomic processes. For example, we found that IL- protection signature to the RhCMV transcriptomic signature 2/7/12/15/27/33/36a, IFNa1/b/g/e/k, LIF, cardiotrophin-1, linked with 68-1 vaccine protection against SIV in RM. We and neuropoietin each induced a cytolytic program in NK found that nearly 400 genes are shared between the two cells, but also cytokine-specific gene expression programs signatures and that the shared genes are enriched for innate involved in antigen presentation, NF-kB activation, and antiviral immunity pathways. Interestingly, the CyCMV mRNA splicing. Finally, we used our compendium of protection signature also showed substantial overlap with cytokine signatures to study the dynamic cytokine networks the interleukin (IL)-15 signaling component of the RhCMV that become active in mouse lymph nodes after protection signature. Since IL-15 signaling is integral to SIV immunization with vaccine adjuvants. By deconvolving protection in RMs, we treated an independent cohort of single-cell RNA-sequencing data from lymph nodes of MCMs with IL-15 and performed longitudinal whole blood immunized mice, we predict that more than 10 cytokines, transcriptional profiling to further interrogate the contribution including type I interferons and IL-15, act on and potentially of IL-15 to SIV vaccine protection. Our analyses identify activate cross-presenting dendritic cells after immunization many genes and specific functional pathways shared with a STING ligand-based adjuvant. Our study provides a between the RM and MCM vaccine protection, each global view of cell-type-specific cytokine responses in vivo, underscored by an IL-15 signature linked with vaccine providing new hypotheses for cytokine functions. protection. Thus, conservation of whole blood signatures of Furthermore, the framework can be readily applied to other immune programming and protection link IL-15 response transcriptomic datasets for assessing the roles of cytokines and innate immune programs with CMV vector-mediated and cell-cell communication networks in any immune SIV vaccine protection. Further, our studies reveal specific response. genes as blood biomarkers of vaccine protection that will inform human studies of CMV-based vaccine vectors for Disclosure of Interest: None Declared protection against HIV infection. Disclosure of Interest: None Declared O30 TRANSCRIPTOMIC RESPONSES TO CYCMV/SIV VACCINATION IN MAURITIAN CYNOMOLGUS MACAQUES

15 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Symposium 15: Late breaking talks in innate domain, which includes the caspase activation and immunity, infection and cancer recruitment domain (CARD). However, a natural pathogen- O31 derived effector that can activate human NLRP1 remains SARS-COV-2 ORF6 HIJACKS NUP98 TO BLOCK STAT unknown. Here, we use evolution-guided approach to NUCLEAR IMPORT AND ANTAGONIZE INTERFERON identify a sequence within human NLRP1 that mimics the SIGNALING consensus protease cleavage site of proteases from human L. Miorin1,*, T. Kehrer1, M. T. Sanchez-Aparicio1, K. Zhang2, enteroviruses. Proteolytic cleavage at this primate-specific P. Cohen1, R. S. Patel1, A. Cupic1, T. Makio3, M. Mei4, O. sequence is necessary and sufficient to activate pro- Danziger1, E. Moreno1, K. M. Khite1, R. Rathnasinghe1, M. inflammatory cytokine release from cells in which the NLRP1 Uccellini1, S. Gao2, T. Aydillo-Gomez1, N. Mena1, X. Yin5, L. inflammasome has been reconstituted. Moreover, we find Martin-Sancho5, N. J. Krogan6, S. K. Chanda5, M. that proteases from several other human picornaviruses Schotsaert1, R. W. Wozniak3, Y. Ren4, B. R. Rosenberg1, B. cleave NLRP1 within a rapidly evolving region of the protein, M. A. Fontoura2, A. Garcia-Sastre1 leading to host-specific and virus-specific activation of the 1Microbiology, Icahn School of Medicine at Mount Sinai, NLRP1 inflammasome. Our work demonstrates that NLRP1 New York, 2Cell Biology, UT Southwestern , Dallas, United acts as a “tripwire” to recognize the enzymatic function of a States, 3Cell Biology, University of Alberta, Edmonton, wide range of viral proteases, and suggests that host Canada, 4Biochemistry, Vanderbilt Univerity School of mimicry of viral polyprotein cleavage sites can be an Medicine, Nashville, 5Immunity and Pathogenesis Program, effective evolutionary strategy to activate a robust Sanford Burnham Prebys Medical Discovery Institute, La inflammatory immune response in the ongoing arms races Jolla, 6Department of Cellular and Molecular Pharmacology, between hosts and their viruses. University of California San Francisco, San Francisco, Disclosure of Interest: None Declared United States O32 Abstract Content: Severe acute respiratory syndrome DIETARY PALMITIC ACID INDUCES LPS TOLERANCE coronavirus 2 (SARS-CoV-2) is the causative agent of the AND INCREASED SEPSIS-ASSOCIATED ongoing coronavirus disease 2019 (COVID-19) pandemic IMMUNOPARALYSIS AND MORTALITY B. A. Napier1,*, A. L. Seufert 2, S. Traxler2, J. Hickman2 that is a serious global health problem. Evasion of interferon- 1 2 mediated antiviral signaling is a common defense strategy Portland State University, Portland, United States, Biology, that pathogenic viruses use to replicate and propagate in Portland State University, Portland, United States their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation Abstract Content: Sepsis is a deleterious immune response to infection that leads to organ failure and is the in order to impair transcriptional induction of interferon (IFN) th stimulated genes (ISGs). Our results demonstrate that the 11 leading cause of death worldwide. During sepsis, viral accessory protein Orf6 exerts this anti-IFN activity. We detection of microbial ligands by monocytes and found that SARS-CoV-2 Orf6 localizes at the nuclear pore macrophages leads to an early acute inflammatory immune complex (NPC) and directly interacts with Nup98-Rae1 via response followed by a robust anti-inflammatory immune its C-terminal domain to impair docking of cargo-receptor response. The early acute inflammatory response is (karyopherin/importin) complex and disrupt nuclear import. orchestrated by activated circulating monocytes and tissue- In addition, we show that a methionine-to-arginine resident macrophages that produce pro-inflammatory substitution at residue 58 impairs Orf6 binding to the Nup98- cytokines (ex: IL-6 and TNF). Inhibition of this inflammatory Rae1 complex and abolishes its IFN antagonistic function. response is termed sepsis-associated “immunoparalysis” All together our data unravel a new mechanism of viral and is correlated with increased systemic IL-10 and higher antagonism in which a virus hijacks the Nup98-Rae1 mortality rates in septic patients. Our recent publication complex to overcome the antiviral action of IFN. defined a role of Western Diet (WD), a diet high in SFA and Disclosure of Interest: None Declared sugar, in driving decreased activated circulating monocytes, increased immunoparalysis, and increased mortality during O31b (LPS)-induced sepsis in mice, compared DIVERSE VIRAL PROTEASES ACTIVATE THE NLRP1 to mice fed a Standard low-fat low-sugar Diet (SC). We INFLAMMASOME found that this phenotype was independent of microbiome B. V. Tsu1, C. Beierschmitt1, A. P. Ryan1, R. Agarwal2, P. S. and diet-associated obesity, suggesting the nutritional Mitchell2, M. D. Daugherty1,* components within the WD were regulating 1Division of Biological Sciences, University of California, San immunoparalysis. Here we use this same model of sepsis Diego, La Jolla, 2Division of Immunology & Pathogenesis , and find that a diet enriched only in SFAs and not sugar University of California, Berkeley , Berkeley, United States (Ketogenic Diet; KD), leads to the same induction of immunoparalysis and increased mortality, compared to mice Abstract Content: The NLRP1 inflammasome is a SC-fed mice. These data suggest the enriched SFAs within multiprotein complex that is a potent activator of the diet enhance immunoparalysis and worsen disease inflammation. Mouse NLRP1B can be activated through outcomes. Next, we wanted to identify specific dietary SFAs proteolytic cleavage by the bacterial Lethal Toxin (LeTx) responsible for enhanced immunoparalysis and worsened protease, resulting in degradation of the N-terminal domains disease outcome. It is well documented that circulating of NLRP1B and liberation of the bioactive C-terminal SFAs are dependent on the SFAs consumed in the diet; 16 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 thus, we applied liquid chromatography with tandem mass Ng infection at the same rate as IFNAR KOs. We spectrometry to serum of SC- and KD-fed mice and found discovered that IFN-e was critical for intravaginal Ng the SFA palmitic acid (PA) was significantly higher in the infection. IFN-e KOs phenocopy the IFNAR KOs with blood of KD-fed mice, compared to SC-fed mice. PA has attenuated Ng infection compared to WT mice. The rapid been historically described as an inflammatory lipid and, like clearance of Ng from the mouse genital tract of IFN-e KOs LPS, induces expression of pro-inflammatory cytokines in a was not due to neutrophils. In fact, Ng clearance was not TLR4-dependent manner in monocytes and macrophages. impeded when neutrophils, T lymphocytes or NK cells (or all Importantly, prolonged exposure to LPS results in a three cell types together) were systemically and locally decreased TLR4-dependent pro-inflammatory response in eliminated using depleting mAbs. Importantly, recombinant monocytes and macrophages, termed LPS tolerance. Here IFN-e delivered topically into the vaginal tract was sufficient we found that, similarly to prolonged exposure to LPS, 12- to entirely restore susceptibility to Ng infection in IFN-e KO. 24h exposure of a physiologically relevant level of the PA (Interestingly, IFN-e is suppressed by progesterone and induces LPS tolerance in macrophages by inhibiting progesterone treatment also prevents Ng infection in mice.) expression of il-1β, and not tnf or il-6, compared to We suggest that estrogen-dependent enhancement of Ng untreated macrophages. We show this induction of il-1β- infection is due to estrogen-induced IFN-e expression in the specific LPS tolerance was unique to PA, and not other vaginal tract and that IFN-e engagement of IFNAR promotes enriched SFAs found in the KD. This is the first observation Ng infection both within the genital tract and within myeloid of dietary SFAs inducing LPS tolerance. Further, we found cells. Thus, estrogen, via the IFN-e/IFNAR axis, provides a that monocytes from KD-fed mice showed increased permissive niche for Ng. The cellular and molecular expression of anti-inflammatory cytokine IL-10 when mechanisms of IFN-e/IFNAR enhanced Ng infection are exposed to LPS, compared to SC-fed monocytes, currently under investigation suggesting KD-associated PA is driving LPS tolerance in Disclosure of Interest: None Declared vivo. Together, these data implicate dietary SFAs as inducers of sepsis-associated immunoparalysis and O34 describe PA as a regulator of LPS tolerance in monocytes TDP-43 TRIGGERS MITOCHONDRIAL DNA RELEASE and macrophages. Importantly, diet-induced alterations to TO ACTIVATE CGAS/STING IN ALS monocytes and macrophages may be critical to consider in C.-H. Yu1,* on behalf of TDP-43 Neuroinflammation Working the treatment of septic patients, and dietary manipulation Group may serve as an essential therapeutic tool, especially for 1Inflammation Division, Walter and Eliza Hall Institute of those that consume excessive amounts of SFAs. Medical Research , Parkville, Australia Disclosure of Interest: None Declared Abstract Content: O33 Chien-Hsiung Yu1,3, Sophia Davidson1,3, Cassandra R. ESTROGEN-DRIVES EXPRESSION OF A UNIQUE TYPE Harapas1,3, James B. Hilton4,5, Michael J. Mlodzianoski2,3, I INTERFERON, IFN-EPSILON, AND PROMOTES Pawat Laohamonthonkul1,3, Cynthia Louis1,3, Dominic De NEISSERIA GONORRHOEAE INFECTION Nardo1,3,7, Katherine R. Balka1,3,7, Catriona A. E. Kurt-Jones1,*, S. Ram1, S. Gulati1, B. Zheng1, J. McLean6, Kelly L. Rogers2,3, Kate McArthur7, Peter J. Shaughnessy1, N. de Weerd1, A. Matthews1, R. de Oliveira1, Crouch4,5 and Seth L. Masters1,3,8. P. Hertzog1, D. T. Golenbock1 1Inflammation Division; 2Centre for Dynamic Imaging 1Division of Infectious Diseases and Immunology, The Walter and Eliza Hall Institute of Medical Research, Department of Medicine, University of Massachusetts Parkville 3052, Australia. Medical School, Worcester, United States 3Department of Medical Biology; 4Department of Pharmacology and Therapeutics; 5Florey Institute of Abstract Content: We explored the role of type I IFNs, Neuroscience and Mental Health particularly IFN-e, in controlling Neisseria gonorrhoeae (Ng) University of Melbourne, Parkville 3010, Australia. infections of the genital tract. In previous studies, we 6Anatomical Pathology, The Alfred Hospital, Victoria 3004, established that type I IFNs significantly enhance Ng Australia. replication in Mø and PMNs. Epithelial cells (EC) in the 7Department of Anatomy and Developmental Biology, human as well as mouse genital tract produce a unique type Monash Biomedicine Discovery Institute, Monash I IFN, IFN-epsilon (IFN-e), in response to estrogen. Clinical University, Clayton, Victoria 3168, Australia. studies indicate that estrogen play an important role in 8Immunology Laboratory, Guangzhou Institute of susceptibility of women to Ng infection. In mice, estrogen is Paediatrics, Guangzhou Women and Children’s Medical essential for Ng infection in the female genital tract while Centre, Guangzhou, Guangdong, 510623, China. progesterone inhibits Ng infection. Background: Cytoplasmic neuronal accumulation of the Our studies demonstrate that Type I IFN and IFN-e in normally nuclear protein TDP-43 is a disease hallmark for particular promote intravaginal Ng infection in estrogen many cases of Amyotrophic Lateral Sclerosis (ALS) and treated mice. Thus, estrogen-treated WT mice were Frontotemporal Lobar Degeneration (FTLD), with some susceptible to Ng infection with prolonged colonization (7-12 familial cases caused by mutant forms of the protein. Both days p.i.). In contrast, estrogen-treated IFNAR KO mice diseases are associated with a neuroinflammatory cytokine remained Ng infected for only 2-3 days. Similarly, WT mice profile related to upregulation of NF-kB and type I IFN treated systemically with anti-IFNAR Ab cleared intravaginal pathways. 17 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Objectives: In this project we sought to identify the innate isopeptidase USP18 deconjugates ISG15 and this requires immune sensor responsible for generating engagement of USP18’s catalytic triad with the ISG15 di-Gly neuroinflammation in response to TDP-43. motif. However, independently of its protease activity, Methods: We employed in vitro models with overexpression USP18 is critical for down-regulating IFN-I signalling. of TDP-43 in cell lines, and ALS patient derived iPSC cells Likewise, it was recently shown that ISG15 is critical for differentiated into motor . These were subjected to negative regulation of IFN-I signalling; in IFN-treated ISG15- CRIPSR mediated deletion or pharmacological inhibition of deficient cells, USP18 protein is rapidly degraded leading to various innate immune pathways. For in vivo studies a a model suggesting that ISG15’s essential role is to stabilise mouse model with human TDP-43(A315T) expressed under USP18, thus allowing regulated signalling. the prion promoter was used, crossed to mice that were To investigate this further, we generated lentiviral vectors genetically deficient for STING. that express wild-type (wt) or mutant forms of ISG15 under Results: Here we show that the cytoplasmic DNA sensor, the control of the endogenous, IFN-regulated, ISG15 cyclic GMP-AMP synthase (cGAS), is activated by an promoter and reconstituted ISG15-deficient cells. accumulation of wild-type TDP-43 and further exacerbated Reconstituted wt ISG15 retained its ability to bind USP18, by mutant TDP-43. Mislocalized TDP-43 invades whereas mutants could either bind with intermediate affinity mitochondria via TIM22 and causes the mitochondrial (C-term ΔGG) or were unable to bind USP18 (C-term di-Ala Permeability Transition Pore (mPTP) to release or ISG15-AA). Importantly, we show that following IFN mitochondrial DNA (mtDNA) into the , which treatment the capacity of ISG15 mutants to regulate IFN directly activates cGAS. Pharmacological inhibition or signalling faithfully mirrored their ability to bind USP18. For genetic deletion of cGAS and its downstream signalling example, the expression of ISGs in IFN-treated cells partner STING, prevents upregulation of NF-kB and type I expressing ISG15-AA were significantly higher than controls IFN induced by TDP-43 in human cell lines. Furthermore, and similar to that in ISG15-KO cells. Expression was, to genetic deletion of one allele of Sting in ALS-mutant TDP- some extent, regulated in ISG15-ΔGG-expressing cells with 43 transgenic mice is sufficient to increase lifespan, improve restoration of normal, regulated expression in wt ISG15- motor control and prevent loss, demonstrating that expessing cells. As expected, these data were corroborated partial inhibition of the pathway is sufficient to impact on when we investigated the levels of phospho-STAT1 (a disease. Finally, we document elevated levels of the specific marker of activated signalling) and following viral infection of cGAS signalling metabolite cGAMP in iPSC-derived motor IFN-treated cells: ISG15-deficient and ISG15-AA- neurons and post-mortem spinal cord samples of patients expressing cells were resistant to infection, ISG15-ΔGG with ALS. were partially permissive while wt ISG15-expressing cells Discussion and Conclusions: Our results identify mtDNA were fully permissive to infection. To stabilise the release and cGAS/STING activation as critical determinants ISG15:USP18 interaction, USP18 interacts with a of TDP-43 driven neurodegeneration and demonstrate the hydrophobic patch in ISG15 (coordinated by Trp123 or potential for targeting this pathway in neurodegenerative W123) that leads to a structural rearrangement of USP18 disease. and access of ISG15’s di-Gly motif to the catalytic groove in Disclosure of Interest: None Declared USP18. Importantly, expression of mutant ISG15-W123R (with di-Gly motif) unable to bind USP18 did not restore O35 regulated IFN-I signalling and phenocopied ISG15-deficient A CO-OPTED BINDING MECHANISM NORMALLY and ISG15-AA-expressing cells. Significantly, all ISG15 RESERVED FOR DE-ISGYLATION IS ESSENTIAL FOR mutants were able to stabilise USP18 showing that, in THE CONTROL OF TYPE I IFN SIGNALLING contract to existing models, the ISG15-dependent A. Vasou1,*, C. G. Bamford2, J. Andrejeva1, R. E. Randall1, stabilisation of USP18 is necessary but not sufficient for J. McLauchlan3, D. J. Hughes1 regulated IFN-I signalling. 1Biomedical Sciences Research Complex, University of St Therefore, we hypothesise that non-covalent binding of Andrews, St Andrews, 2Wellcome-Wolfson Institute for ISG15 and USP18 is necessary to facilitate USP18’s Experimental Medicine, Queen's University Belfast, Belfast, inhibitory function. Intriguingly, this is achieved through co- 3MRC-University of Glasgow Centre for Virus Research, option of a binding mechanism normally reserved for de- University of Glasgow, Glasgow, United Kingdom ISGylation. Disclosure of Interest: None Declared Abstract Content: The innate immune response is underpinned by the conserved type I interferon (IFN-I) O36 system and is of fundamental importance for our response TUMOR-SPECIFIC CD8 T CELL-MEDIATED ANTITUMOR to infection, cancer and immunotherapy. Importantly, IFN ACTIVITY INTRODUCED BY INTRATUMORALLY signalling must be moderated to prevent autoinflammatory INJECTED ANTI-CD40 ANTIBODY TOGETHER WITH IL- disease and tissue damage. The ubiquitin-like protein ISG15 15 IS CRITICAL IN TRAMP-C2 TUMORS IN MICE is strongly induced by IFN and is critical for regulating how W. Chen1,* on behalf of TA Waldmann's group cells respond to IFN. As part of the antiviral response, ISG15 1NCI, Bethesda, United States can modify proteins (incl. viral proteins) in a process known as ISGylation, where the C-terminal di-glycine motif of Abstract Content: Intratumoral immunotherapy that aims at ISG15 covalently attaches to lysine residues of target generating a potent priming of antitumor immunity, uses the proteins. As a reversible process, the ISG15-specific tumor as its own vaccine, for a systemic and durable clinical 18 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 benefit. Recently, Dr. Jeffrey Ravetch’s Group found that expression profiling using highly multiplexed targeted RT- use of the optimized anti-CD40 monoclonal antibody (2141- qPCR (Fluidigm Biomark). We observed robust CD4+ T cell V11) not only increases its antitumor efficacy but also infection in the CSF during Fiebig III-IV HIV-1 infection, with reduces its side effect through intratumoral injection (i.t.) 0.04-0.84% (median 0.14%) expressing spiced viral RNA ( 2016, 29(6), 820-831). Previously, we indicative of productive infection. A similar frequency was demonstrated that the combination regimen of the agonistic present in PBMC. As expected, markers of cellular anti-CD40 monoclonal antibody (FGK4.5) with IL-15 showed activation were increased, including CD38 on CD4+ and synergistic therapeutic efficacy when compared with either CD8+ T cells, and CD38 and CD169 on monocytes in both monotherapy alone in mouse tumor models (J Immunol PB and CSF (p<0.05). Parallel studies in a SHIV rhesus 2012, 188(12): 6156-64). Furthermore, IL-15 alone or macaque model indicated upregulation of CXCL10 in CSF agonistic CD40 did not yield tetramer positive tumor specific CD4+ T cells and monocytes compared to uninfected CD8 T-cells in the TRAMP-C2 model, whereas the controls, indicating a CNS inflammatory response during combination showed an at least 10-fold increase in the acute infection. These studies provide direct evidence of number of such tumor specific CD8 T-cells. viral replication in CD4+ T cells and broad immune activation Here, we investigated the combination of IL-15 with the in both the periphery and CNS during acute HIV-1 infection, intratumoral anti-CD40 therapy using a dosing schedule likely contributing to early CSF inflammation and based on our previous study in the TRAMP-C2 model. We neuropathology. demonstrated that given intratumorally of anti-CD40 mAb Disclosure of Interest: None Declared had the abscopal efficacy and shrunk the left, untreated site of tumor size. Also, the combination of IL-15 and anti-CD40 Lightning Talk Session 1: Adaptive Immunity mAb robust increased the numbers of the tumor specific LT001 tetramer+CD8+ T cells, which associate the boost of the SINGLE-CHAIN SOLUBLE RECEPTOR FUSION efficacy compare to the single treatment. These findings PROTEINS AS VERSATILE CYTOKINE INHIBITORS support the view that anti-CD40 given intratumorally has I. S. Afonina1,*, A. Holgado1, H. Braun1, K. Verstraete1, S. N. abscopal efficacy and IL-15 combined administration Savvides1, R. Beyaert1 increases this effect in Tramp-C2 tumor-bearing mouse 1VIB/UGent, Ghent/Zwijnaarde, Belgium model. Disclosure of Interest: None Declared Abstract Content: Cytokines are small secreted proteins that among many functions also play key roles in the O37 orchestration of inflammation in host defence and disease. NEUROIMMUNE CELLULAR AND CYTOKINE PROFILE Over the past years, a large number of biologics have been IN HIV-1 ACUTELY INFECTED PATIENTS developed to target cytokines in disease, amongst which K. McGuckin Wuertz1,2,*, V. Sharma1,3, M. Creegan1,3, P. soluble receptor fusion proteins have shown some promise Chan4, C. Sacdalan4, N. Phanuphak Pungpapong4, M. in pre-clinical studies. Eller1,3, S. Vasan1,3, D. Bolton1,3 We have previously reported the development of a new 1Military HIV Research Program (MHRP), Walter Reed Army biologic, termed IL-33trap, comprising the ectodomains of Institute of Research, Silver Spring, 2Center for Innate the cognate receptor ST2 and the co-receptor IL-1RAcP Immunity and Immune Disease, University of Washington, fused into a single-chain recombinant fusion protein. We Seattle, 3Military HIV Research Program (MHRP), Henry M. showed that IL-33trap is able to neutralize IL-33 released in Jackson Foundation for the Advancement of Military response to a fungal aeroallergen Alternaria alternata and to Medicine, Silver Spring, United States, 4SEARCH, Institute reduce lung inflammatory responses in a murine model of of HIV Research and Innovation (IHRI), Bangkok, Thailand airway inflammation. Here we extend the biophysical and biological characterisation of IL-33trap variants, and we Abstract Content: Neurological conditions associated with show that our design allows the formation of a single-chain HIV-1 remain a significant cause of morbidity and mortality fusion protein with high IL-33 antagonistic activity. globally with manifestations occurring throughout the central Furthermore, IL-33trap is a stable protein with a monomeric nervous system (CNS). Despite effective treatment with profile both at physiological temperatures and during liquid antiretroviral therapy, HIV-1 and chronic neuroinflammation storage at 4 °C. We also report that IL-33trap specifically can persist in the brain. The extent of viral replication in the targets biologically active IL-33 splice variants and is not CNS and origin of neuropathology associated with HIV-1 are affected by inactive variants. Finally, we document the poorly understood. In this study, we assessed the cellular generation and antagonistic activity of a single-chain IL- and innate immune profiles in both the CNS and peripheral 4/13trap, which inhibits both IL-4 and IL-13 signalling. compartments of patients (n = 6) during early acute HIV-1 Collectively, our results illustrate that single-chain soluble infection, when viral replication is maximal. Cell-associated receptor fusion proteins against IL-4, IL-13, and IL-33 are viral RNA and associated host cytokine profiles were novel biologics that might not only be of interest for research assessed in matched peripheral blood (PB) mononuclear purposes and further interrogation of the role of their target cell and cerebral spinal fluid (CSF) samples and compared cytokines in physiology and disease, but may also to uninfected donors (n = 3). PB and CSF CD4+ and CD8+ complement monoclonal antibodies for the treatment of T cells and macrophages were sorted and phenotyped for allergic and other inflammatory diseases. activation markers by flow cytometry directly ex vivo, Disclosure of Interest: None Declared followed by cytokine, chemokine and viral RNA gene 19 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT003 and lungs. A putative Ets binding site is found the promoter NOVEL REGULATORS OF IFNG-DEPENDENT MHCII region of the X. tropicalis IL-7R α gene, demonstrated by EXPRESSION others to be important in regulating transcription of the IL-7R L. Ankley1,*, M. Kiritsy2, J. Trombley1, G. Huizinga1, P. α gene in B and T cells. Comparison of the predicted amino Orning3, R. Elling4, K. Fitzgerald3, A. Olive1 acid sequence of the Xenopus IL-7R α indicates closest 1Microbiology and Molecular Genetics, Michigan State homology with the zebrafish IL-7R α, and significant University, East Lansing, 2Microbiology and Physiological homology with other characterized IL-7R α including those Systems, 3Division of Infectious Disease and Immunology, from humans, mice, chickens, alligators, primitive University of Massachusetts Medical School, Worcester, caecilians, green see turtles, and anole lizards. Analysis of United States, 4University Medical Center Freiburg, the predicted sequence indicates an intact Freiburg, Germany WSXWS motif (common to all IL-7R family members). The Box 1 motif, normally required for JAK interaction, has an Abstract Content: The IFNγ-dependent induction of MHCII asparagine replacing aspartic acid (similar variations are expression is critical for CD4+ T cell function in peripheral found in the zebrafish and anole lizard IL-7R α tissues. Once activated CD4+ T cells induce cytokine sequences). There are at least five potential N glycosylation production, macrophage activation, B cell responses and sites. There is a 16 amino acid insert unique to the Xenopus immune cell recruitment needed to control infection and sequence that is not found in the reported IL-7R α sequence disease. Dysregulation of MHCII is associated with of any other animal that we have examined. The protein autoimmunity, graft versus host disease, and increased structure model of IL-7R has the typical L-shape of the susceptibility to specific cancers and chronic infections. human and mouse receptors and conserved trio of disulfide Despite its central role in host immunity, the complex and bonds. Despite sharing only 20% identity with mouse IL-7R dynamic regulation of IFNγ-induced MHCII is poorly α, the RMSD between the model and the mouse structure understood. We hypothesize that targeting regulatory (4NN5:B) is only 1.775 Å. The 16 amino acid Xenopus insert networks of MHCII expression will allow the development of breaks up the C’2 β-strand in the fibronectin type-3 D2 new therapies to treat or prevent MHCII-expression related domain and could potentially affect ligand binding. Synteny disease states. Using a genome-wide CRISPR screen in analysis failed to identify a gene with homology to IL-7 at the macrophages we identified over 200 genes that control expected position, or anywhere else, in the Xenopus IFNγ-mediated MHCII surface expression. We validated genome. Interestingly, we have tentatively identified a these candidates using flow cytometry and qPCR transposon in place of the IL-7 gene at the expected uncovering a range of important MHCII regulators that were position. Signaling through a modified IL-7R using ligands previously unknown. We found two parallel pathways that other than IL-7 in Xenopus are being examined. require either the multifunctional kinase GSK3 or the Disclosure of Interest: None Declared mediator complex subunit Med16. Both pathways were required for IFNγ-mediated Ciita induction, MHCII expression, and CD4+ T cell activation. Using RNAseq LT005 analysis we defined the regulons of these distinct CONTINUED BCL6 EXPRESSION PREVENTS THE transcriptional networks and are using these data to define TRANSDIFFERENTIATION OF ESTABLISHED TFH the mechanisms of MHCII control in macrophages. Our CELLS INTO TH1 CELLS DURING ACUTE VIRAL results defining IFNγ-mediated MHCII regulation could INFECTION provide new therapeutic targets to modify MHCII expression D. Alterauge1, J. W. Bagnoli2, F. Dahlström1, B. M. and alleviate disease and infection. Bradford3, N. A. Mabbott3, T. Buch4, W. Enard2, D. Disclosure of Interest: None Declared Baumjohann1,5,* 1Institute for Immunology, 2Anthropology & Human LT004 Genomics, Department of Biology II, LMU Munich, Planegg- CHARACTERIZATION OF IL7R ALPHA IN XENOPUS Martinsried, Germany, 3The Roslin Institute and the Royal TROPICALIS: IMPLICATIONS FOR EVOLUTION OF (Dick) School of Veterinary Sciences, University of CYTOKINE SIGNALING SYSTEMS Edinburgh, Easter Bush, Midlothian, United Kingdom, P. M. Appasamy1,*, L. A. Lambert1, J. C. Sammons1, K. G. 4Institute of Laboratory Animal Science, University of Zurich, Daskalova1, M. Kostan1 Schlieren, Switzerland, 5Medical Clinic III for Oncology, 1Chatham University, Pittsburgh PA, Pittsburgh, United Hematology, Immuno-Oncology and Rheumatology, States University Hospital Bonn, Bonn, Germany

Abstract Content: Signaling through the IL-7 receptor is Abstract Content: T follicular helper (Tfh) cells are crucial essential for development of B and T cells, as evidenced by for the establishment of germinal centers (GCs) and potent the SCID-like phenotype seen in the absence of IL-7/IL-7 antibody responses that are elicited during infection and receptor signaling. The IL-7R is a heterodimer comprised of vaccination. In contrast, dysregulated Tfh cells can cause the IL-7R α chain and a γ chain that is shared by other IL-2 autoimmunity. Despite their importance for humoral family cytokines that utilize the JAK-STAT pathway. We immunity, the T cell-intrinsic factors that are required for the recently cloned the IL-7R α cDNA from Xenopus maintenance of already established Tfh cells and GCs tropicalis. IL-7R α is found in lymphoid (thymus, spleen) and remain largely unknown. Here, we used temporally guided non-lymphoid organs of X. tropicalis including intestine, eye, gene ablation in CD4+ T cells to dissect the contributions of 20 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 the Tfh-associated chemokine receptor CXCR5 and the H. K. Dewald1,*, R. I. Martínez-Zamudio2, T. Vasilopoulos 2, transcription factor Bcl6. Ablation of Cxcr5 in CD4+ T cells L. Gittens-Williams3, P. Fitzgerald-Bocarsly4, U. Herbig2 had only minor effects on the function of established Tfh 1Pathology, Immunology, and Laboratory Medicine, cells. Phenotypical and transcriptional analyses revealed 2Microbiology, Biochemistry, and Molecular Genetics, that Cxcr5-ablated cells still exhibited most features of 3Obstetrics, Gynecology, and Women's Health, 4Pathology, CXCR5-positive Tfh cells, including Il4 and Il21 expression. Immunology and Laboratory Medicine, Rutgers New Jersey In contrast, continued Bcl6 expression was essential to Medical School, Newark, United States maintain the GC Tfh cell phenotype and also the GC reaction. Importantly, CD4+ T cell-specific Bcl6 ablation Abstract Content: Aging is associated with a decline in the during acute viral infection resulted in transdifferentiation of immune system, termed “immunosenescence”. Cellular established Tfh cells into Th1 cells, e.g. characterized by senescence is defined by persistent cellular growth arrest upregulation of Tbet and concomitant downregulation and overall loss of function. If immune cells undergo cellular of Il6raand Il6st expression, thus highlighting the plasticity of senescence in vivo and whether cellular senescence has a Tfh cells. These findings have implications for strategies that role in immunosenesence remains controversial due to the boost or restrain Tfh cells and GCs in health and disease. lack of specific markers to reliably identify these cells in Disclosure of Interest: None Declared blood. Freshly-isolated human PBMC from younger and older donors were fluorescently labeled for senescence- LT006 associated b-galactosidase (SA-bGal) activity. CD8+ T cells IL-27 ENHANCES CYTOKINE SECRETION BY TIGIT+ were sorted based on SA-bGal and analyzed by qRT-PCR, HIVGAG-SPECIFIC T CELLS RNA sequencing, and microscopy. We found a higher J. Cheng1,*, T. G. Myers2, V. Shankavaram3, J. Kumar3, P. percentage of cells with high SA-bGal in the older vs. Kumar3, M. Catalfamo1 younger cohort. This increase was apparent in NK cells, 1Department of Microbiology and Immunology, Georgetown CD4+ and CD8+ T cells. CD8+ T cells with high SA-bGal University School of Medicine, Washington DC, 2Genomic displayed increased transcripts of p21, p16INK4a, and Technologies Section, Research Technologies Branch, inflammatory cytokines. They also had greater DNA damage National Institute of Allergy and Infectious Diseases, response foci and p16INK4a protein. SA-bGal increased as T National Institutes of Health, Bethesda, 3Division of cells differentiated into effector memory cells, which was Infectious Diseases and Travel Medicine, Georgetown consistent regardless of age. Intriguingly, a portion of T University School of Medicine, Washington DC, United effector cells did not exhibit a senescent phenotype and States maintained low SA-bGal; conversely, a portion of naïve T cells expressed high SA-bGal. RNA sequencing of CD8+ T Abstract Content: Although HIV infection can be effectively cells revealed alterations in gene expression were treated by lifelong administration of combination associated with SA-bGal and cells with low, medium, and antiretroviral therapy (cART), novel strategies are required high SA-bGal had distinct profiles. Cells with the same level for cART-free HIV control. Recent studies had shown that of SA-bGal shared similar gene expression, independent of IL-27, a member of IL-12 family of cytokines, can induce pro- age. Functional analysis of the sorted CD8+ T cells showed and anti-inflammatory effects and inhibits HIV replication in that cells with high SA-bGal proliferated significantly less monocytes, -derived dendritic cells, and T cells. In than SA-bGal low cells. CD8+ T cells expressing SA-bGal this study, we hypothesized that T- cell immune activation appear to be distinct from exhausted T cells as they do not driven by HIV will promote IL-27 dependent pro- uniformly express markers of T cell exhaustion such as inflammatory properties enhancing HIV-specific T-cell CD57, PD-1 and KLRG-1. The presence of p16INK4a, DNA immunity. We found that overnight in vitro stimulation with damage, inflammatory cytokines, and deceased IL-27 upregulated expression of CD69 and T-bet in CD4 and proliferation demonstrate that SA-bGal is a strong indicator CD8 T cells from both healthy and HIV infected donors. of cellular senescence. SA-bGal can be leveraged to further Accordingly, IL-27 increased IFN-ɣ and TNF-⍺ secretion by identify senescent immune cells and elucidate cellular TIGIT+ HIVGag specific CD4 and CD8 T cells. Moreover, the senescence’s role in immunosenesence. Understanding this RNA-Seq analysis showed that IL-27 induced global connection in healthy aging will further our understanding of transcriptional changes associated with signal transducer premature aging seen in some chronic diseases. and activator of transcription 1 (STAT1)/interferon inducible Disclosure of Interest: None Declared genes that were enriched in memory CD4 and CD8 T cells from HIV infected patients compared with healthy controls. LT008 This study provides new evidence about the effects of IL-27 DENDRITIC CELLS PRIMED BY MYCOBACTERIUM in enhancing HIVGag-specific T cell responses. LEPRAE ARE WEAK STIMULATORS OF T CELLS Disclosure of Interest: None Declared ACTIVATION G. V. Germano1,*, B. B. G. Carra1, V. C. Dos Santos2, D. LT007 Bertoluci3, A. S. A. A. Barbosa3, B. C. Sartori3, P. Rosa3, A. SENESCENCE-ASSOCIATED -GALACTOSIDASE P. Campanelli4, P. K. Das5, V. N. Brito de Souza3 ACTIVITY AND OTHER MARKERS OF SENESCENCE 1Botucatu School of Medicine, São Paulo State University, ARE PRESENT IN HUMAN PERIPHERAL BLOOD Botucatu, 2School of Sciences, São Paulo State University, MONONUCLEAR CELLS DURING HEALTHY AGING 3Lauro de Souza Lima Institute, 4Bauru School of Dentistry, São Paulo University, Bauru, Brazil, 5College of Medical and 21 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Dental Sciences, University of Birmingham, Birmingham, United Kingdom Abstract Content: The type I interferons (IFN-Is) are well recognised for their protective role during viral infection. Abstract Content: Leprosy is a public health problem in They induce an antiviral state, promote maturation of endemic areas with 208.641 news cases around the world leukocytes and thus aid in mediating viral clearance. in 2018. Mycobacterium leprae, the causative agent of However, IFN-Is can also provoke an immunosuppressive leprosy, is an obligate intracellular pathogen. The bacillus environment that promotes chronic infection by facilitating infects skin macrophages and Schwann cells of peripheral functional T cell exhaustion. However, the mechanisms that nerves where they proliferate causing neural injury. Several contribute to this dual role of IFN-Is in balancing clinical forms of leprosy can arise according to individual inflammatory vs. inhibitory signals are only partially immune response since M. leprae presents low genetic understood. variability. Tuberculoid patients show a predominance of Using the LCMV-Armstrong (LCMV-ARM) model of acute Th1 cellular immune response against M. leprae so the virus infection, we have recently shown that IFN-Is are disease is localized. On the other hand, lepromatous required for accumulation and activation of virus-specific patients demonstrate a predominant humoral response that CD8+ T cells. Moreover, LCMV-specific CD8+ T cells in is not efficient resulting in disseminated disease. Dendritic LCMV-ARM-infected mice lacking the IFN-I receptor IFNAR cells (DCs) are crucial for the activation of T cells during the (IFNAR KO) were functionally exhausted and unable to clear development of adaptive immunity. Although live M. leprae the virus, while wild type mice mounted a robust CD8+ T cell seems to be a weak promotor of DCs activation, the role of response and eliminated the virus. Given its importance in these cells in the polarization of immune response in leprosy regulating T cell responses during LCMV infection, we patients is not understood. This study aimed to evaluate the hypothesised that the PD1/PDL1 pathway contributed to profile of cytokines produced by autologous lymphocytes CD8+ T cell dysfunction in the IFNAR KO mice and that loss (LTs) co-cultured with DCs previously infected by live M. of PDL1 in IFNAR-deficient mice would prevent T cell leprae. Peripheral mononuclear blood cells were obtained exhaustion thereby permitting viral clearance. from healthy controls (HC) and leprosy patients through a To address this, mice lacking IFNAR were crossed with density gradient. Monocytes-CD14+ were positively PDL1-deficient mice to generate double-deficient IFNAR x selected by microbeads and differentiated in DCs using GM- PDL1 DKO mice. In contrast to infection of WT and PDL1 CSF and IL-14 for 6 days. DCs were stimulated by (i) live M. KO mice, which remain asymptomatic, and IFNAR KO mice, leprae at a MOI of 10:1 (bacilli:DC, MLDCs) or (ii) a standard which developed minor signs of disease, infection of IFNAR maturation cocktail (MC) composed by IL-1β, IL-6, TNF, x PDL1 DKO mice with LCMV-ARM resulted in a lethal PGE2 (MCDCs) or (iii) remained unstimulated (USDCs) for wasting disease. Whilst disease was accompanied by virus 48 hours. Then, DCs were co-cultivated with LTs for five spread and increased expression of cytokines this was not days. Intracellular and released cytokines were evaluated by dissimilar to IFNAR KO mice. Importantly, only DKO mice flow cytometry. The production of IFN-y, TNF, IL-9, and IL- displayed leukocyte infiltration into lungs, which led to 22 was higher in MCDCs+LTs cultures compared to LT necrotising pneumonia. Depletion of CD8+ cells in infected cultures with MLDCs and USDCs while IL-4 levels were IFNAR x PDL1 DKO mice prevented disease development lower in MLDCs+LTs cultures both in leprosy patients and indicating that a dysregulated CD8+ T cell response healthy controls. Lepromatous patients showed low IL-2 mediated lethality. However, phenotypical and functional levels when LTs were primed by MLDCs. In contrast, the analysis of CD8+ T cells showed that PDL1-deficiency did frequency of IL-4+ LTs was higher in these patients that not restore numbers or increase effector function of virus presented a lower frequency of IFN-y+ and IL-17A+ LTs in specific CD8+ T cells, suggesting that other mechanisms co-cultures with MLDCs. We conclude that DCs primed by were responsible for mediating lethality. M. leprae are not able to stimulate the full activation of LTs. Together, these findings underpin our limited understanding Besides, lepromatous patients presented a lower frequency of the relationship between IFN-Is and the PD1/PDL1 of Th1 and Th17 LTs. This suggests that these patients pathway and the impact on T cell responses. This has could present some impairment to develop the profiles of broader implications for our understanding and improving of immune response effective against M. leprae, favoring its immune check point therapy, where the underlying permanence in the host for long periods. Supported by: molecular mechanisms presently remain largely unknown. Fundação de Amparo à Pesquisa do Estado de São Paulo Disclosure of Interest: None Declared – FAPESP (Grant: #2015/23256-6). Disclosure of Interest: None Declared LT011 MIR-15/16 DEFICIENCY IN T CELLS LEADS TO LT009 ACCUMULATION OF CD25-LOW TREGS IN VIVO PDL1 PROTECTS LCMV-INFECTED TYPE I K. Johansson1,*, J. D. Gagnon1, S. Zhou1, K. M. Ansel1 INTERFERON RECEPTOR DEFICIENT MICE FROM 1Microbiology and Immunology, University of California San LETHAL IMMUNE MEDIATED DISEASE Francisco, San Francisco, United States M. J. Hofer1,*, T. Suprunenko1, E. J. Cutts1 1School of Life and Environmental Sciences, the Marie Abstract Content: Introduction: The transcription factor Bashir Institute for Infectious Diseases and Biosecurity, the FOXP3 defines the gene expression program of regulatory Charles Perkins Centre, and the Bosch Institute, The T cells (Tregs). High expression of the IL-2 receptor plays a University of Sydney, Sydney, Australia key role in Treg suppressor function as it allows Tregs to 22 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 consume IL-2 to limit T effector cell activation. Regulation of Abstract Content: CD4+ T lymphocytes consist of naïve, gene expression by microRNAs (miRNAs) is critical for Treg antigen-specific memory, and memory-phenotype (MP) cell differentiation and function. Here, we report that mice with compartments in steady state. We recently showed that MP miR-15/16 deficiency in T cells accumulate Tregs in multiple cells exert innate-like effector function in host defense. tissues with abnormal expression of FOXP3, IL2Rα/CD25 However, it remained to be defined whether MP CD4+ T cells and IL7Rα/CD127. are functionally heterogenous, and if so, what factors Methods: Flow cytometry was used for analysis of Tregs determine the selective differentiation of MP subpopulations (CD3+CD8-CD4+FOXP3+) and T effector cells in thymus, under homeostatic conditions. Here we characterize MP spleen and inguinal lymph nodes isolated from lymphocytes as consisting of T-bethigh, T-betlow, and T-bet- unchallenged miR-15/16fl/fl CD4cre or miR-15/16fl/fl FOXP3cre subsets, with innate Th1-like host-protecting activity mice (lacking both miR-15a/16-1/Mirc30 and miR-15b/16- exclusively associated with T-bethigh cells. We further show 2/Mirc10 clusters) and CD4wt or FOXP3wt miR-15/16fl/fl that steady-state differentiation of T-bethigh MP cells is controls. promoted by IL-12-producing type 1 dendritic cells (DC1). Results: Mice with miR-15/16 deficiency in all T cells Finally, we present evidence arguing that this tonic IL-12 (CD4cre) demonstrated an increased number of FOXP3+ production by DC1 requires TLR-MyD88 signaling triggered Tregs, but not FOXP3- conventional T cells, in thymus, independently of foreign agonists and is further enhanced spleen and lymph nodes compared to control mice. by CD40-CD40L interactions with CD4+ T lymphocytes. Interestingly, the expression level of FOXP3 (measured by Together our results argue that optimal differentiation of T- MFI) was significantly lower in miR-15/16 deficient Tregs, bethigh MP lymphocytes in steady state requires IL-12 indicating accumulation of functionally defective cells. tonically produced by DC1 in an MyD88 and CD40 signaling- Indeed, miR-15/16 deficient Tregs expressed low CD25 dependent fashion and thus reveal a previously levels. Additionally, the IL-7 receptor CD127, a direct target unappreciated homeostatic function of IL-12. of miR-15/16 and positive regulator of Treg survival, was This work was supported by the Intramural Research significantly increased in miR-15/16 deficient Tregs, Program of the NIAID and NCI, NIH. potentially contributing to expanded Treg pool. miR-15/16 Disclosure of Interest: None Declared deficient Tregs were capable of restricting T effector in vitro and in vivo by outnumbering T effector LT014 cells. The suppressive capacity of miR-15/16 deficient Treg RAPID DETERMINATION OF CYTOKINE RELEASE was significantly reduced on a per cell basis compared to FROM T CELLS WITH NO-TRANSFER, NO-WASH wild type Tregs. Spontaneous accumulation of LUMIT™ IMMUNOASSAYS FOXP3loCD25loCD127hi Tregs similarly developed in miR- D. Lazar1,*, K. Hsiao1, J. Gilden1, C. Sondgeroth1, K. 15/16fl/fl FOXP3cre mice which had larger spleen volume and Kupcho1, D. Thompson1, M. O'Brien1, J. Cali1 increased numbers of CD44hiCD62Llo T effector cells, 1R&D, Promega, Madison, United States suggesting insufficient Treg suppression. Conclusion: miR-15/16 deficient Tregs compensate for Abstract Content: We have utilized NanoLuc® Binary lower suppressive function by enhanced ability to expand. Technology (NanoBiT®) to develop a homogeneous and Disclosure of Interest: None Declared rapid assay method (< 70 min completion time) to measure cytokines released from cells in culture without the need for LT013 sample transfer and requiring only a standard, plate-reading IL-12 DERIVED FROM TYPE 1 DENDRITIC CELLS luminometer for signal acquisition. In this Lumit™ TONICALLY PROMOTES THE DIFFERENTIATION OF immunoassay approach, separate antibodies to a specific INNATE T-BET(HIGH) MEMORY-PHENOTYPE CD4(+) T cytokine are labeled with the small, 11-amino acid subunit of LYMPHOCYTES IN STEADY STATE NanoBiT luciferase (SmBiT) or its 17.6 kDa complementary T. Kawabe1,2,*, J. Yi3,4, A. Kawajiri2, K. Hilligan1, D. Fang5, N. subunit (LgBiT). When SmBiT- and LgBiT-labeled Ishii2, H. Yamane6, J. Zhu5, D. Jankovic1, K. S. Kim3,4, G. antibodies converge on the target cytokine, the resultant Trinchieri7, A. Sher1 proximity of SmBiT and LgBiT reconstitutes a bright 1Laboratory of Parasitic Diseases, National Institute of luciferase that produces light proportional to analyte Allergy and Infectious Diseases, National Institutes of levels. In this manner, Lumit immunoassays have been Health, Bethesda, United States, 2Department of developed for several cytokines, including human IL-1β, IL- Microbiology and Immunology, Tohoku University Graduate 2, IL-4, IL-6, IFN-γ, and TNF-α. These assays typically School of Medicine, Sendai, Japan, 3Academy of exhibit a lower limit of detection (LLOD) less than 10 pg/ml Immunology and Microbiology, Institute for Basic Science, and linearity over more than three logs of analyte 4Department of Integrative Biosciences and Biotechnology, concentration, with maximal signal-to-background ratios Pohang University of Science and Technology, Pohang, (S/B) greater than 1000. Korea, Republic Of, 5Laboratory of Immune System Biology, 6Vaccine Research Center, National Institute of Allergy and Following treatment of human peripheral blood mononuclear Infectious Diseases, National Institutes of Health, 7Center cells (PBMC) in 96-well plates with vehicle, LPS, R848, or a for Cancer Research, National Cancer Institute, National combination of PMA and ionomycin, the non-lytic cytokine Institutes of Health, Bethesda, United States detection reagents were added directly to the culture wells containing cells and medium. Dose- and time-dependent release of cytokines were observed over a wide range of 23 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 response levels without the need for sample administration at different time points. We assessed the dilution. Alternatively, replicate aliquots of medium from development of pain-like behaviour and joint pathology each cell treatment well were transferred to a separate using an incapacitance meter (a measurement of weight assay plate for simultaneous determination of multiple distribution between two hind limbs) and histological cytokines released from individual cell wells. In a cell model analysis, respectively. In comparison to wild-type (WT) comprised of purified CD8+ T cells and target Raji B cells mice, Ccl17E/E mice were protected from pain-like behaviour treated with increasing bispecific T-cell engager Blincyto®, and joint cartilage damage across all the arthritis models release of IL-2 and IFN-γ was observed with an EC50 of ~0.2 studied, while the degree of joint inflammation was ng/ml and maximal S/B for IL-2 and IFN-γ of 82- and 168- comparable to that seen in WT mice. For the inflammatory fold, respectively. In addition, differentiation of purified arthritis models, neutralizing CCL17 therapeutically CD4+ T cells into the Th2 phenotype was confirmed by (following the onset of pain) rapidly reversed established marked increases in IL-4 vs. IFN-γ release. Following pain-like behaviour and halted disease progression, as reagent addition, assay signal exhibited glow kinetics with a measured by structural changes; for the osteoarthritis half-life of approximately two hours. In addition, 384-well, model, we used treatment regimens neutralizing CCL17 at assay performance for determination of cytokine release different stages of disease (i.e. early vs. late) and from treated human PBMC resulted in Z’ factors greater than consistently found that CCL17 neutralization effectively 0.7, indicating the assay format is amenable to screening ameliorates pain-like behaviour. In contrast to the well- applications. recognized chemotactic role of CCL17 (e.g. preferential Th2 cell chemotaxis), these data reveal a novel, non- The implementation of this novel detection chemistry will chemotactic and algesic role of CCL17 in arthritis- enable rapid “add-and-read” determinations of cytokine associated pain and suggest that it could be a potential release from cells for both low- and high-throughput therapeutic target in arthritis patients. applications, including quantitative assessments of T cell Disclosure of Interest: None Declared activation and differentiation. Disclosure of Interest: D. Lazar Employee of: Promega LT017 Corporation, K. Hsiao Employee of: Promega Corporation, LIPID METABOLISM FUELS IL-17-PRODUCING J. Gilden Employee of: Promega Corporation, C. GAMMADELTA T CELLS AND DRIVES THEIR Sondgeroth Employee of: Promega Corporation, K. Kupcho EXPANSION IN OBESITY AND THE TUMOR Employee of: Promega Corporation, D. Thompson MICROENVIRONMENT Employee of: Promega Corporation, M. O'Brien Employee C. McIntyre1,*, M. Raverdeau2, A. Kohlgruber1, L. Agudelo3, of: Promega Corporation, J. Cali Employee of: Promega L. Dyck2, A. Douglas2, S. Cunningham2, H. Prendeville2, H. Corporation Kane2, C. Carmody1, M. Kellis3, M. Brenner1, L. Lynch1,2 1Brigham and Women's Hospital, Boston, United States, 2Trinity Biomedical Sciences Institute, Dublin, Ireland, LT015 3Massachusetts Institute of Technology, Boston, United A NOVEL THERAPEUTIC TARGET FOR ARTHRITIS States PAIN K. M.-C. Lee1,*, A. Jarnicki2, A. Achuthan1, A. Fleetwood1, G. Abstract Content: Metabolic reprogramming is required for Anderson2, J. Hamilton1, A. Cook1 T cell activation and effector cytokine production. Recent 1Medicine, 2Pharmacology, The University of Melbourne, studies have revealed the metabolic programs that Parkville, Australia govern αβ T cell subsets, NK cells and myeloid lineages, but little of known about gd T cell metabolism. γδ T cells hold Abstract Content: A GM-CSF→CCL17 pathway was significant promise for immunotherapy in cancer, however, originally identified in vitro using monocyte/macrophage they can be either anti-tumor or pro-tumor, depending on populations. We firstly explored the cellular source of the their production of IFNγ (γδIFN) or IL-17 (γδ17). Therefore, chemokine CCL17 and its GM-CSF dependence in various understanding the metabolic pathways that govern these inflammation models (zymosan- and antigen-induced distinct functions is essential. We found that γδ T cell peritonitis, and LPS-induced lung inflammation), using subsets had distinct metabolic requirements. γδ17 cells had Ccl17E/+ reporter mice and flow cytometry. We present enhanced oxidative metabolism, increased mitochondrial evidence that in these models CCL17 expression is mass and potential. This was accompanied by enrichment predominately expressed in macrophage-lineage of lipid-related metabolic pathways and intracellular lipid populations, namely MHCII+ macrophages and monocyte- droplets. γδ17, but not γδIFN cells, had high lipid uptake in derived dendritic cells and is suppressed upon GM-CSF vitro and administration of lipids in vivo by feeding a high fat blockade, which is consistent with our in vitro findings. We diet expanded the number of γδ17 cells and enhanced their next investigated the involvement of CCL17 in controlling ability to produce IL-17. In the tumor microenvironment, the pain and disease in various arthritis models, namely known to be lipid-rich, γδ17 cells were enriched, and were models of inflammatory arthritis (zymosan-, antigen- and further expanded in tumours of obese mice. This study cytokine-driven monoarticular arthritis) and osteoarthritis highlights the different metabolic requirements of γδ T cell (collagenase-induced osteoarthritis). For these particular subsets and suggests an important link between lipids and studies, we utilized CCL17 gene-deficient (Ccl17E/E) mice IL-17 production by γδ T cells. Lipid-driven IL-17 may and therapeutic protocols using monoclonal antibody influence the outcome of γδ T cell-based immunotherapy in 24 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 obesity and could be linked with IL-17 related diseases like new IL-7 delivery regimen, alone or in combination with psoriasis and multiple sclerosis that are increased in other cytokines, may help to reconstitute the T cell pool in obesity. lymphopenic conditions. Disclosure of Interest: None Declared Disclosure of Interest: None Declared

LT019 LT020 STEP-DOSE ESCALATION OF IL-7 TREATMENT LEADS SERTOLI CELLS SUPRESS ACTIVATION AND TO SYSTEMIC EXPANSION AND SURVIVAL OF CD4 INFLAMMATORY STATE OF T-CELLS IN VITRO AND CD8 T LYMPHOCYTES IN RHESUS MACAQUES B. Porubská1,2,*, M. Krulová 1,2, D. Vašek1 on behalf of H. Pandit1,*, A. Valentin1, C. Bergamaschi2, J. Bear2, M. Laboratory of Immune Regulations , M. Hájková1,2 Rosati1, B. Felber2, G. Pavlakis1 1Laboratory of Immunoregulation, Charles University, 1Human Retrovirus Section, 2Human Retrovirus Faculty of Science, 2Department of Transplantation Pathogenesis Section, NCI/NIH, Frederick, United States Immunology, Academy of Science, Institute of Experimental Medicine , Prague , Czech Republic Abstract Content: Background: (IL-7) is a homeostatic cytokine playing a central role in the growth and Abstract Content: Introduction: Sertoli cells (SCs) are survival of T lymphocytes. In several clinical studies, well known as nurse cells for maturating germ cells, their including HIV infection and cancer, IL-7 was able to immunological importance has become more and more efficiently reconstitute the T cell pool. With the rationale of pronounced last couple of years. They modulate immune using IL-7 as a vaccine enhancer or in combination with system and thus create immunologically privileged site other cytokines in immunotherapy protocols, We have within testis. Mesenchymal stem cells (MSCs) are even produced human and macaque IL-7 from human human better known for their immune-regulatory phenotype and are cells. This cytokines have the advantage of being properly also studied as a potential therapeutic tool in number of glycosylated and, therefore, will not be immunogenic in clinical studies. Due to the similarity of actions of these two humans. In this study, we address pharmacokinetics and cell types it was proposed that SCs are kind of MSCs. We systemic effects of recombinant macaque IL-7 (rmIL-7) aim to compare their ability to regulate the immune system produced from human HEK293 cells. and suppress the inflammatory response in vitro. Methods: Spleen cells were isolated from BALB/c mice, Study Design: Six rhesus macaques were treated with unstimulated or stimulated with Concanavalin A and rmIL-7 delivered subcutaneously using a protocol that cultivated in the presence of SCs or MSCs. After 48 included 3 injections of increasing cytokine doses (50, 100 (detection of cytokines) or 72 (detection of transcription and 200 mg/kg) every three days. Two days after the last factors) hours of incubation cells were analyzed using flow injection, animals were sacrificed and the systemic rmIL-7 cytometry. Cytokine production and expression of effects were analyzed on lymphocytes and myeloid cells transcription factors was determined intracellularly within from blood, lymph nodes (LN) and effector tissues by multi- spleen cells. Down-regulation of apoptosis and suppression parametric flow cytometry. of splenocyte proliferation in the presence of SCs or MSCs Results and Conclusions: Administration of rmIL-7 was was measured using Annexin V and Ki67, respectively. well-tolerated and resulted in increased plasma rmIL-7 Results: Percentage of IL-2 and TNFα positive splenocytes levels 4 hrs after each injection. Plasma rmIL-7 remained decreased after treatment with SCs, whereas co-culture with higher 48 hrs after each injection compared to the pre- MSCs reduced the number of IL-17 positive cells. timepoint. The increased systemic levels of rmIL-7 were Interestingly, a lower percentage of Th17 cells was evident by the reduced detection of CD127 (IL-7R) in PBMC observed in the presence of SCs when compared to and LN mononuclear cells at 48 hrs. rmIL-7 induced rapid presence of MSCs in the culture. Both cell types suppressed movement of central memory T cells from the blood at 4 hr. the proliferation of CD4+ T cells in a similar manner. Repeated rmIL-7 administration induced a gradual and Apoptosis of splenocytes decreased in the presence of systemic expansion of both CD4+ and CD8+ T cells, MSCs, in the presence of SCs, the percentage of apoptotic particularly in the memory compartment. rmIL-7 led to cells within CD4+ population remained similar to controls. increased proliferation (Ki67+) and expression of the Conclusion: SCs have suppressive effect on T helper cells survival factor (Bcl-2) on T cells from blood, LN and effector after activation. In some aspects, this effect was even sites, such as liver and gastrointestinal tract (duodenum, greater than that of MSCs. Therefore, they may be jejunum, ileum, colon and rectum). Compared to CD4 T interesting and important tool in therapies, for example in cells, higher proliferation among memory CD8 T cells was cases of testicular inflammation accompanied by infiltration found. Systemic administration of IL-7 signficantly increased of immune cells, which often impairs male fertility. plasma levels of CC chemokines such as CCL2, CCL4, Disclosure of Interest: None Declared CCL19 and CCL20 and CXC chemokines, CXCL10 and CXCL11. Cytokines, such as IL-6, IL-15 and TPO were also LT021 significantly increased in plasma of IL-7 treated macaques. T CELL RESPONSES TO PROTEOGLYCAN AGGRECAN Interestingly, within the lymph nodes, rmIL-7 treatment PEPTIDES IN KNEE OA PATIENTS increased the frequency of plasmacytoid dendritic cells T. Sae-jung1, N. Leearamwat2, N. Chaiseema2, P. (pDC) that may impact induction of adaptive immune Sengprasert1, S. Ngarmukos3, P. Yuktananda3, A. responses in vaccine and immunotherapy protocols. This Tanavalee3,4, N. Hirankarn2,5, R. Reantragoon2,5,* 25 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

1Medical Microbiology Interdisciplinary Program, Graduate broadly active antiviral. Tyrosine kinase 2 (TYK2), a JAK School, 2Immunology Division, Department of Microbiology, family member, is required for type I interferon (IFN-α/β) Faculty of Medicine, 3Department of Orthopedics, Faculty of signaling, but its role in type III IFN (IFN-λ) signaling is still Medicine, 4Osteoarthritis and Musculoskeletal Research unclear. We report here that the selective TYK2 inhibitor Unit, Faculty of Medicine, 5Center of Excellence in BMS-986165 blocks potentially detrimental type I IFN Immunology and Immune-mediated Diseases, Faculty of signaling without altering protective IFN-λ-mediated gene Medicine, Chulalongkorn University, Bangkok, Thailand expression. In contrast, the clinically used JAK1/2 inhibitor baricitinib was found to be equally potent in blocking Abstract Content: Knee osteoarthritis is accompanied by responses to both types of IFN. Mechanistically, we show inflammation and infiltration of immune cells eg. T cells and that epithelial cells do not require TYK2 for IFN-λ-mediated macrophages within the joint-surrounding tissues, such as, signaling or antiviral protection. Lack of TYK2 in vivo the infrapatellar fat pad and synovial linings. Many studies diminished IFN-α-induced protection against a lethal suggest the role of an antigen-driven response in influenza virus challenge in mice, but did not impair IFN-λ- osteoarthritic patients, eg. the presence of an oligoclonal T mediated antiviral protection. Our findings suggest that cell pattern and Ig-producing B cells in the synovium. selective TYK2 inhibitors likely represent a superior Previous studies have shown that OA patients were treatment option for type I interferonopathies than broadly responsive to proteoglycan aggrecan peptides especially acting JAK1/2 inhibitors, as selective TYK2 inhibitors may the p16-31 and p263-280 peptide fragment. Here, we show counteract inflammatory responses while preserving the that healthy individuals were capable of eliciting T cell beneficial antiviral effects of IFN-λ and its potential responses to the p16-31 and p263-280 peptide fragments, therapeutic use against COVID-19. suggesting primed T cells specific to these peptides. Disclosure of Interest: None Declared Interestingly, these T cells produced the pro-inflammatory cytokine, IL-6. T cells within the infrapatellar fat pad also LT027 elicited responses towards both peptide fragments. We also CRYSTAL STRUCTURE OF IL-27 IN COMPLEX WITH investigated the cytokines that were released from T cell SRF388, A FIRST-IN-CLASS IL-27 BLOCKING responses in response to these peptide stimulations. In the ANTIBODY UNDER EVALUATION IN A PHASE I synovial fluid of knee OA patients, many cytotoxic CLINICAL TRIAL IN PATIENTS WITH ADVANCED SOLID mediators, including IL-6, were detected at high levels. TUMORS Disclosure of Interest: None Declared J. Strand1,*, D. T. Logan2, M. Welin2, J. Hua1, R. Gilligan1, M. Rausch1, G. Tan1, P. M. Holland1, J. A. Hill1, D. Moodley1 LT023 1Surface Oncology, Cambridge, MA, United States, SELECTIVE INHIBITION PRESERVES 2SARomics Biostructures AB, Lund, Sweden INTERFERON-Λ-MEDIATED BARRIER PROTECTION D. Schnepf1,*, S. Crotta2, T. Thamamongood3, M. Stanifer4, Abstract Content: Introduction: IL-27 is a heterodimeric L. Polcik3, A. Ohnemus3, J. Vier5, M. Llorian6, H. H. Gad7, R. immunoregulatory cytokine composed of EBI3 and p28 Hartmann7, B. Strobl8, S. Kirschnek5, S. Boulant4, M. subunits. It is produced by activated macrophages and Schwemmle3, A. Wack2, P. Staeheli1 dendritic cells and binds with high affinity to IL-27RA 1Institute of Virology, Medical Center University of Freiburg, expressed on innate and adaptive immune cells. This Freiburg, Germany, 2Immunoregulation Laboratory, The interaction also relies on gp130 binding to mediate signaling Francis Crick Institute, London, United Kingdom, 3Institute through phosphorylated STAT1. This results in the of Virology, Medical Center Freiburg, Freiburg, 4Department downstream transcription of several immunoregulatory of Infectious Diseases, Heidelberg University, Heidelberg, receptors (eg, PD-L1) and inhibition of inflammatory 5Institute of Medical Microbiology and Hygiene, Medical cytokines (eg, IFNγ, IL-17, TNFα) that are important in Center Freiburg, Freiburg, Germany, 6Bioinformatics and dampening immune responses in certain tumor Biostatistics, The Francis Crick Institute, London, United microenvironments. Here, cytokine/receptor affinity studies Kingdom, 7Department of Molecular Biology and Genetics, and the X-ray crystal structure of the human IL-27 Aarhus University, Aarhus, Denmark, 8Institute of Animal heterodimer are reported. The structure was solved in Breeding and Genetics, University of Veterinary Medicine complex with the Fab of an IL-27 blocking antibody, Vienna, Vienna, Austria SRF388, currently being evaluated in a Phase I clinical trial in patients with advanced solid tumors (NCT04374877). Abstract Content: The ongoing SARS-CoV-2 pandemic Methods: IL-27 receptor binding and blocking studies were clearly illustrates that immune suppressive regimens must conducted by surface plasmon resonance. Additional be as targeted as possible to reduce the infection risk for binding studies using IL-27 cytokine and receptor subunits patients under treatment to the achievable minimum. were conducted by biolayer interferometry. Biological Current treatments of patients suffering from inflammatory activity studies using SRF388, recombinant IL-27, or the and other diseases with /2 (JAK1/2) inhibitors EBI3 or p28 subunits were tested using the human U937 cell can lead to inadvertent immune suppression and to line or human PBMCs. The SRF388 Fab fragment was increased risks for infections. Here we addressed whether complexed with the IL-27 heterodimer in a 1:1 stoichiometric selective JAK inhibition could facilitate a well-tailored ratio, purified, and crystallized. immune suppression preserving interferon-λ-mediated Results: The binding interactions between the cytokine and barrier protection and its potential therapeutic use as receptor subunits showed that IL-27 bound with strong 26 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 affinity to IL-27RA (21 pM). IL-27 binding to gp130 was Mechanistically, STAT5 localizes to enhancers and weaker (4 nM) but was still required for signaling. SRF388 promoters of a 50+ gene module encoding essential, often blocked the association of IL-27 with IL-27RA and inhibited rate- limiting enzymes transporters and associated proteins, all downstream signaling events. The EBI3/p28 heterodimer where it instructs transcriptional activity through p300 was required for high-affinity binding to IL-27RA and recruitment and epigenetic landscaping. Beyond downstream signaling, as each subunit on its own failed to autonomous transcriptional programing, systems level induce STAT1 phosphorylation. While p28 did not bind to analysis also revealed a STAT5-driven signaling network either receptor subunit, binding between EBI3 and IL-27RA, with broad influence on T cell metabolism and involving but not gp130, was detectable (3.8 nM). The IL-27 subunits three prominent metabolic agents, the PI3K-AKT and mToR had nanomolar affinity for each other (5 nM) and associated pathways, and the transcription factor MYC. Taken together, into a biologically active heterodimer extracellularly. The our findings provide a molecular roadmap for transcriptional structure of the IL-27/Fab complex was solved at 2.02 Å programing of T cell metabolism downstream of cg resolution. SRF388 Fab bound exclusively to p28 making cytokines and emphasize the Jak/STAT pathway in contacts with the αA and αC helices of the p28 four-helix powering T cell growth and proliferation. bundle and a portion of the poly-Glu sequence. EBI3 bound Disclosure of Interest: None Declared p28 at the hinge with contacts to the AB loop and the αB and αD helices of p28. Analysis of the p28 binding interface with LT029 EBI3 revealed multiple hydrophobic and salt-bridge WHO IS THE WORST JAK/STAT PROTEIN OF THEM interactions between the 2 halves. ALL? MODEL SYSTEMS TO EVALUATE THE Conclusion: These data highlight the structural interface CONSEQUENCES OF JAK/STAT DRIVER MUTATIONS responsible for the interactions between EBI3 and p28 that C. Wagner1,*, K. Sommer1, D. Pölöske1, H. Neubauer1, R. lead to a biologically active heterodimer that has high affinity Moriggl1 for IL-27RA. The combination of blocking and structural data 1Institute of Animal Breeding and Genetics, University of suggests that SRF388 inhibits binding of IL-27 to IL-27RA Veterinary Medicine, Vienna, Vienna, Austria through steric hindrance at Site 2 of p28. Further studies are underway to resolve the structure of the IL-27RA in complex Abstract Content: We know that distinct driver mutations of with IL-27. the Janus kinase (JAK)/signal transducer and activator of Disclosure of Interest: J. Strand Shareholder of: Surface transcription (STAT) signaling pathway can lead to Oncology, Employee of: Surface Oncology, D. Logan malignant cell transformation. However, their oncogenicity Shareholder of: SARomics Biostructures AB, Employee of: and lineage-specific transforming capacities have not yet SARomics Biostructures AB, M. Welin Employee of: been directly compared. Persistent activation of JAK/STAT SARomics Biostructures AB, J. Hua: None Declared, R. signaling proteins promotes aberrant DNA binding, gene Gilligan: None Declared, M. Rausch Shareholder of: Surface expression and chromatin structure. This is especially Oncology, Employee of: Surface Oncology, G. Tan: None dominant in the hematopoietic system and in acute Declared, P. Holland Shareholder of: Surface Oncology, leukemias or lymphomas. We demonstrated in mouse Employee of: Surface Oncology, J. Hill Shareholder of: models that gain of function JAK or STAT mutations can Surface Oncology, Employee of: Surface Oncology, D. cause leukemias. Enhanced function of JAKs can lead to Moodley: None Declared uncontrolled phosphorylation of diverse downstream proteins including STATs, whereas activating STAT LT028 mutations can increase DNA binding and gene expression. THE IL-2-STAT5 AXIS PROGRAMS HELPER T CELL Especially STAT5B gain of function mutations can also METABOLISM UPSTREAM OF AKT, MTOR AND MYC promote oligomer formation via the N-terminus. Compared A. Villarino1,*, A. Laurence2, Y. Kanno3, J. O"Shea3 with dimers, STAT5 oligomers are able to bind to different 1Microbiology & Immunology, University of Miami, Miami, DNA motifs and might be involved in chromatin looping. United States, 2University of Oxford, Oxford, United STAT3 and STAT5 were also shown to regulate the Kingdom, 3National Institute of Arthritis and Musculoskeletal chromatin landscape by recruiting chromatin remodeling and Skin Diseases (NIAMS), National Institutes of Health, enzymes to the DNA. A detailed understanding of these Bethesda, United States interactions and their consequences will be important for therapeutic intervention strategies. Abstract Content: Similar to cancer cells, activated To gain deeper insights into how different JAK/STAT lymphocytes adapt their metabolism to meet energetic and mutations influence gene expression, chromatin biosynthetic demands imposed by rapid growth and accessibility, oligomerization and chromatin formation in an proliferation. It has long been known that common gamma isogenic background, we established two model systems. (cg) chain cytokines are pivotal in this process but, while We introduced a broad panel of human JAK3, STAT3, numerous downstream mechanisms have been proposed, STAT5A and STAT5B variants into Ba/F3 cells, a their relationship is unclear and involvement of the lymphoid/myeloid progenitor, or mouse embryonic Jak/STAT pathway remains loosely defined. By integrating fibroblasts lacking Stat5a/b. To investigate the role of the genome-, transcriptome- and metabolome-wide analyses, STAT5 N-terminus in oligomerization and oncogene we demonstrate that the IL-2-STAT5 axis directly controls expression, we also introduced oligomerization-deficient key aspects of carbon metabolism, amino acid synthesis mutants. Overexpression of the different JAK/STAT mutants and synthesis in CD4+ ‘helper’ T cells. causes vast differences in cell proliferation, cytokine 27 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 independent growth and gene expression. We hypothesize binding sites in mRNAs of human Jurkat T cells and mouse that JAK/STAT driver mutations affect chromatin structure primary T cells that corresponded to known cis-regulatory differently, through differential STAT oligomerization, DNA elements. From our data, we identified CD69 as an immune binding and interaction with chromatin remodeling enzymes, transcript with multiple RBP binding sites in the 3’UTR, resulting in distinct oncogene activation and silencing of suggesting that the gene may be regulated on the post- tumor suppressor genes. transcriptional level. Disclosure of Interest: None Declared CD69 is an inducible protein that is rapidly up-regulated on LT030 the cell surface of T cells during stimulation and swiftly E3 UBIQUITIN LIGASE VON HIPPEL-LINDAU (VHL) down-regulated after activation. Interestingly, while effector PROTEIN PROMOTES TH17 DIFFERENTIATION memory cells remain CD69- in circulation, resident memory A. Chitrakar1, S. A. Budda1, J. G. Henderson1, R. C. Axtell2, T cells constitutively express CD69 despite absence of L. A. Zenewicz1,* antigen. How CD69 is regulated and the mechanism for the 1Department of Microbiology and Immunology, University of observed differential expression is unknown. Using our Oklahoma Health Sciences Center, 2Arthritis & Clinical GCLiPP dataset, we dissected CD69 3’UTR using CRISPR- Immunology Research Program, Oklahoma Medical Cas9 in human and mouse T cells to identify post- Research Foundation, Oklahoma City, United States transcriptional cis-elements which may be regulating the transcript and protein expression of CD69. We identified an Abstract Content: VHL is an E3 ubiquitin ligase that targets RBP occupied region in the human and mouse 3’UTR that proteins, including HIF-1a, for proteasomal degradation. may regulate transcript stability. VHL and HIF regulate the balance between glycolysis and Disclosure of Interest: None Declared oxidative phosphorylation, which is critical in highly dynamic T cells. HIF-1a positively regulates Th17 differentiation, a LT031b complex process in which quiescent naïve CD4 T cells CD4+ T CELL RECOGNITION OF HAEMAGGLUTININ undergo transcriptional changes to effector cells, which are EPITOPES ACROSS DIFFERENT INFLUENZA STRAINS commonly dysregulated in chronic inflammatory diseases. H. Sloane1,*, E. Grant1, D. Jayasinghe1, C. Szeto1, A. The role of VHL in Th17 cells is not known. In this study, we Nguyen1, S. Gras1 hypothesized VHL negatively regulates Th17 differentiation 1Biochemistry and Molecular Biology, Monash University, and deletion of VHL in CD4 T cells would elevate HIF-1a and Clayton, Australia increase Th17 differentiation. Unexpectedly, we found that VHL promotes Th17 differentiation. Mice deficient in VHL in Abstract Content: The 2019/20 SARS-Cov2 pandemic is a their T cells were resistant to an autoimmune disease, modern-day reminder of the impact that novel viral strains experimental autoimmune encephalomyelitis (EAE), often can have on a population without pre-existing immunity. The mediated by Th17 cells. In vitro Th17 differentiation was influenza A virus (IAV) has caused five pandemics in the last impaired in VHL deficient T cells. In the absence of VHL, 150 years and is particularly prone to mutations in the Th17 cells had decreased activation of STAT3 and SMAD2, surface haemagglutinin (HA) which is the suggesting that VHL indirectly or directly regulates these primary target of the CD4+ T cell and antibody responses. critical signaling molecules. Gene expression analysis revealed that in Th17 cells VHL regulates many cellular CD4+ T cells recognise peptide presented by human pathways, including genes encoding proteins involved leukocyte antigen class II (HLA-II) molecules with their T cell indirectly or directly in the glycolysis pathway. Compared to receptor (TCR). Although the pHLA-II-TCR interaction is wild-type, VHL deficient Th17 cells had elevated glycolysis extremely specific, it may cross-recognise similar peptides and glycolytic capacity. Our finding has implications on the leading to a cross-reactive response, providing broad design of therapeutics targeting the distinct metabolic needs protection against different IAV strains. The ability of CD4+ of T cells to combat chronic inflammatory diseases. T cells to cross-react with variants of a HA peptide from Disclosure of Interest: None Declared different IAV strains, especially those not in circulation, is not well understood. Furthermore, individuals expressing LT031 certain HLA-II molecules are known to be more susceptible MAPPING AND DISSECTING THE POST- to severe viral infections. In the context of IAV, HLA- TRANSCRIPTIONAL LANDSCAPE OF CD69 DRB1*07:01 has been associated with poor response to the D. Zhu1,*, A. Litterman1, R. Kageyama1, M. Ansel1 influenza vaccine whereas HLA-DRB1*11:01 has been 1Immunology and Microbiology, UCSF, San Francisco, associated with protection from viruses such as HIV and United States HCV. Investigation into the molecular basis underlying this association is warranted. Abstract Content: Untranslated regions (UTRs) of mRNA transcripts are bound by RNA binding proteins (RBPs) and Using intracellular cytokine staining and HLA-II tetramer microRNAs during constitutive RNA metabolism and gene staining, we found that HLA-DRB1*11:01+ individuals have specific regulatory interactions. To study these RNA-protein more cross-reactive responses towards HA peptide derived interactions, we developed a protocol for Global Cross- from different IAV strains than HLA-DRB1*07:01+ Linking Protein Purification (GCLiPP) to map RBP individuals, and that the cross-reacting CD4+ T cells occupancy transcriptome-wide. Our method identified RBP generally have lower polyfunctionality than specific CD4+ T 28 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 cells. Moreover, we provide the first insight into the Abstract Content: The activity of metabolic pathways, such molecular and functional basis of IAV epitope presentation as glycolysis, the tricarboxylic acid (TCA) cycle and the by different HLA-II molecules. electron transport chain (ETC) is regulated by cells via various signals, such as nutrient levels and antigen Disclosure of Interest: None Declared presentation. When it comes to the immune system, specific alterations in these metabolic pathways couple directly with Lightning Talk Session 1: Cytokine regulation their respective functions. For instance, it has been shown I that LPS-stimulated inflammatory macrophages have LT032 downregulated oxidative phosphorylation (OxPhos) and EPIGENETIC AND TRANSCRIPTIONAL REGULATION upregulated aerobic glycolysis, whereas such cells have OF CCL17 PRODUCTION BY DEXAMETHASONE been shown to restore their oxidative capacity in the A. Achuthan1,*, T. Lupancu1 presence of the anti-inflammatory cytokine interleukin-10 1The University of Melbourne, Parkville, Australia (IL-10). We demonstrate here that Arginase-2 (Arg2), the mitochondrial isoform of arginase, is an IL-10 regulated Abstract Content: Clinical trials in rheumatoid arthritis (RA) gene in LPS-stimulated macrophages. Our aim was to targeting the cytokine, granulocyte macrophage-colony understand the impact of Arg2 on IL-10’s role as a metabolic stimulating factor (GM-CSF) are showing promise although rheostat in macrophages. We firstly showed that Arg2 is its mode of action remains largely unknown. We have critical for IL-10 induced restoration of oxidative respiration previously shown that that GM-CSF drives CCL17 in LPS-stimulated macrophages. This effect was lost in the production via a new interferon regulatory factor 4 (IRF4)- absence of Arg2 as evidenced by the use of siRNA and Arg2 dependent pathway in human monocytes and mouse knock-out cells. Additionally, overexpression of Arg2 was macrophages. sufficient to significantly enhance OxPhos compared to Glucocorticoids, such as dexamethasone (Dex), are potent overexpression of an empty vector or the cytoplasmic anti-inflammatory and immunosuppressive agents broadly isoform, Arg1. We further established that the catalytic used in anti-inflammatory therapy, albeit with adverse side activity of Arg2 was crucial for its regulation of oxidative effects associated with long-term usage. The negative respiration by using a chemical inhibitor of arginase, nor- consequences of GC therapy provide an impetus for NOHA, as well as by utilizing a catalytic-dead mutant. research into gaining insights into the molecular Mechanistically, we showed that both Arg2’s presence and mechanisms of Dex action on immune cells. its enzymatic function is crucial for the activity of Complex II We report here that GM-CSF-induced CCL17 expression is (succinate dehydrogenase (SDH)), a bi-functional enzyme inhibited by Dex in human monocytes and mouse that links the mitochondrial ETC and the TCA cycle. macrophages. Moreover, we provide evidence for the first Additionally, an experiment coupling blue-native PAGE to time that Dex suppresses GM-CSF-induced IRF4 liquid chromatography tandem mass spectrometry (LC- expression via regulating the expression and activity of MS/MS) to characterize prospective protein-protein JMJD3, which demethylases trimethylated-H3K27. Further, interactions, suggested that Arg2 binds to SDH complexes we demonstrate that Dex suppresses the expression of A and B, providing potential insight into how it regulates SDH JMJD3. Significantly, we measured elevated levels of activity. Finally, we delineated that Arg2's impact on CCL17 in synovial fluid from patients with RA compared to oxidative metabolism is dependent on its own regulation by healthy controls. Using synovial fluid mononuclear cells from ERK1/2 and mTORC1 phosphorylation via site-directed RA patients, we provide molecular evidence for the anti- mutagenesis studies. These findings shed light on the inflammatory actions of Dex through epigenetic regulation of importance of a specific arm of metabolic regulation by Arg2, IRF4 expression and downstream inhibition of CCL17 that governs the IL-10 induced oxidative state in production. The delineated pathway potentially provides inflammatory macrophages. new therapeutic options for the treatment of inflammatory Disclosure of Interest: None Declared diseases and their associated pain. Disclosure of Interest: None Declared LT034 HIGH EXPRESSION OF TNF TYPE 1 RECEPTORS LT033 INCREASED PRO-APOPTOTIC DOSE-DEPENDENT ARGINASE-2 REGULATES IL-10 MEDIATED EFFECTS OF CYTOKINE IN PRESENCE OF TNF TYPE 2 METABOLIC REPROGRAMMING IN INFLAMMATORY RECEPTORS SIMULTANEOUS EXPRESSION, BUT NOT ALONE MACROPHAGES. 1,* 2 2 R. Afzal1,*, J. K. Dowling1, N. Assmann2, C. E. McCoy1 on A. A. Alshevskaya , J. Lopatnikova , J. Zhukova , M. Kuznetsova2, O. Koneva2, S. Sennikov 2 behalf of Immunobiology Laboratory, Department of 1 Biomedicine, University Hospital Basel; Biozentrum, Core Federal State Budgetary Scientific Institution "Research Institute of Fundamental and Clinical Immunology", Proteomics Facility, University of Basel; microRNA and 2 inflammation research group, RCSI; Curtis Clock lab, RCSI Novosibirsk, Russian Federation, Laboratory of Molecular 1School of Pharmacy and Biomolecular Sciences, Royal Immunology, Federal State Budgetary Scientific Institution College of Surgeons in Ireland (RCSI), Dublin, Ireland, "Research Institute of Fundamental and Clinical 2Department of Biomedicine, University Hospital Basel, Immunology", Novosibirsk, Russian Federation Basel, Switzerland Abstract Content: Background: 29 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Certain effect of cytokines on cell is mediated by number of THERAPEUTIC RESPONSE IN HTLV-1 ASSOCIATED parameters including change in receptor expression level or NEUROINFLAMMATION cytokine content. However, the possibility of modulating T. Assone1,*, F. de Toledo Gonçalves1, V. Angelo Folgosi1, specific biological effects through the change in expression G. da Silva Prates1, M. Braz2, A. C. Penalva de Oliveira3, D. level remains poorly understood. Since two types of highly Daelemans2, J. Casseb1, J. Van Weyenbergh4 specific receptors for TNF (TNFR1 and TNFR2) have the 1University of Sao Paulo, Sao Paulo, Brazil, 2KU LEUVEN, ability to activate fundamentally different effects in cells, this Leuven, Belgium, 3Instituto Emilio Ribas, Sao Paulo, Brazil, system is a convenient object for studying possible 4Rega Institute, KU LEUVEN, Leuven, Belgium regulation mechanisms. This study aimed to investigate the influence of TNF dose-dependent effect on the balance Abstract Content: Background: Human T-cell leukemia between pro-apoptotic and proliferation reactions depending virus type 1 (HTLV-1) is the causative agent of Adult T-cell on TNFR1/2 expression density parameters. Leukemia, but also causes HTLV-1-Associated Methods: Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Tumor cell lines (HEp-2, K-562, MCF-7, ZR-75/1, MOLT-4, Although a majority remains asymptomatic, 2-5% of people A-549, Raji) cell lines after characterisation for TNFR1/2 co- living with HTLV-1 (PLWH) can develop HAM/TSP, an expression using flow cytometry were studied for . incapacitating neuroinflammatory disorder with similarity to The dose-dependent effects of rhTNF on proportion of cells primary progressive multiple sclerosis. No disease- in apoptosis as well as in G1/G0, S, G2/M phases were modifying therapy is available for HAM/TSP but studied. Associations among studied parameters were corticosteroids and other immunomodulators provide some estimated using correlation and regression analyses. clinical benefit. Thus, biomarkers to predict and monitor Results: disease progression and therapeutic outcome are direly For ZR-75/1 cells (cell line with hith expression of both needed in HAM/TSP. We aimed to investigate systemic types) it was found that a dose-dependent increase in the cytokine levels as possible biomarkers of expression of both types of TNFα receptors on cells leads to immunopathogenesis in PLWH and of therapeutic response a decrease in the proliferative activity of cells. For MOLT-4 to corticosteroid pulse therapy in HAM/TSP. Patients and cell (with lower expression) increase in proliferative Methods: PLWH (n=92, of which 49 asymptomatic and 43 response of cells was positively associated with the HAM/TSP) were recruited at the HTLV outpatient clinic of percentage of both TNFR1 + and TNFR2 + cells. However, the "Emílio Ribas" Institute of Infectious Diseases (Sao for two other linesimilar expression profile was shown to be Paulo, Brazil). For 33 HAM/TSP patients, follow-up samples associated with opposite effect on cells. Both K-562 and were obtained after a mean of 4.6 (± 0.5) years of pulse MCF-7 expressed mainly 1 type of receptor (the total therapy with methylprednisolone, with a total follow-up of average percentage of TNFR1 + cells for the MCF-7 line is 181 person-years. Osame neurological motor scale was 66.8%±3.1%, for the K-562 line - 71.5% ±7.2%) and showed measured in all PLWH at diagnosis and at clinical follow-up. a statistically significant change in the relative number of Plasma cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN- cells in different phases of the cell cycle when different gamma and TNF were quantified by Cytometric Bead Array concentrations of TNFα are added to the culture. In both (BD Biosciences). Statistical analysis was performed using lines, the dose-dependent effect of the cytokine on the non-parametric tests (Wilcoxon and Spearman correlation) increase in TNFR2 + cells with an increase in the dose of using Prism 8.0. Results: With respect to demographics, TNFα was observed. However, MCF-7 cells responded to gender did not influence systemic cytokine levels, whereas the mediator with an increase in proliferative activity, and K- IL-6 (r=0.36; p= 0.00018) was positively correlated with age 562 cells, on the contrary, with a decrease. However, in PLWH. Regarding clinical status, we observed a between lines with similar effects (K-562 and ZR-75/1), a significant increase in systemic IFN-gamma (p=0.007) and similarity was observed in a large percentage of double- IL-17A (p=0.0001) in HAM/TSP patients, as compared to positive cells. No statistically significant dose-dependent asymptomatics. After pulse therapy, patients were classified effect of TNFα on the functional response of cells was found as responders (n=6) and non-responders (n=26) based on for the HEp-2, Raji, and A-549 cell lines. changes in Osame motor scale. All patients significantly Conclusions: decreased IL-17A levels after treatment (p=0.013) but only High expression of type 1 receptor for TNF is not always responders significantly decreased IFN-gamma levels after associated with predominant activation of pro-apoptotic treatment (p=0.008). In addition, pre-treatment TNF levels pathways. However, with high simultaneous expression of allowed significant prediction (p=0.037) of therapeutic both types of receptors, the proportion of double-positive outcome. Although none of the cytokines were correlated cells is crucial for activation either pro-apoptotic or with CD4, CD8 and NK cell levels quantified by flow proliferation pathways. cytometry at baseline, lower CD8 levels at diagnosis Study was supported by Grant of President of Russian significantly predicted a stronger IL-17A decrease after Federation for young scientists (МК-2433.2020.4) treatment. Conclusions: 1. An age-related increase in Disclosure of Interest: None Declared circulating IL-6 levels indicates possible inflammaging in PLWH. 2. Both IFN-gamma and IL-17A are increased in LT035 untreated HAM/TSP. 3. Pretreatment TNF predicts SYSTEMIC IL-6, IFN-GAMMA AND TNF AS CANDIDATE therapeutic response to corticosteroid pulse therapy, BIOMARKERS FOR INFLAMMAGING AND whereas post-treatment IFN-gamma reliably reflects therapeutic outcome in responders. 4. Inflammatory 30 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 cytokines IL-6, TNF and IFN-gamma are promising 1Biology Technician Team, 2Immunology Technician Team, candidate biomarkers for immunomonitoring of both PLWH Lauro of Souza Lima Institute, Bauru, Brazil and HAM/TSP patients, to be confirmed in independent cohort studies. Abstract Content: Introduction: Leprosy is a chronic Disclosure of Interest: None Declared infectious disease caused by Mycobacterium leprae, an obligate intracellular pathogen, thus, the resistance of the LT036 host to this pathogen depends on cellular immunity. In UNIQUE EVOLUTION OF TYPE 3 INTERFERON GENES experimental leprosy, BALB/c mouse has limited growth of IN PLACENTAL MAMMALS REVEALS NOVEL acid-fast bacilli (AFB) without dissemination to organs while REGULATORY MECHANISMS nude mouse present bacillary multiplication and C. G. G. Bamford1,*, J. McLauchlan2, R. J. Gifford2 dissemination. Objective: Considering the scarcity of 1Wellcome-Wolfson Institute for Experimental Medicine, studies evaluating the immune response in these two strains Queen's University Belfast, Belfast, 2MRC-University of of mice, this study aimed to evaluate the serum cytokines Glasgow Centre for Virus Research, University of Glasgow, profile and nitric oxide (NO), at different stages of the Glasgow, United Kingdom infection. Methods: BALB/c and nude mice were inoculated in the footpads with M. leprae and blood was collected at 3, Abstract Content: The type 3 interferons (IFNs) – 5 and 8 months post-inoculation. The cytokines profile (IL-2, otherwise known as IFN lambdas (λ) - are an important IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF) and NO were family of antimicrobial cytokines that defend against evaluated. The total number of AFB in the footpads was infection, particularly at barrier tissues such as the gut, lung determined. Results: Infection was progressive in the nude and placenta. Furthermore, recombinant IFNλs are being mice with bacillary multiplication in the footpads (2.5 x 107, trialled against hepatitis D and SARS-CoV-2 infections. 6.9 x 107 and 1.9 x 108 at 3, 5 and 8 months). In BALB/c However, the development of IFNλ-targeted therapies with mice, bacillary multiplication was more intense at 5 months improved safety and efficacy is hampered by an incomplete (8.4 x 105) and there was no significant difference in the understanding of fundamental IFNλ biology. To facilitate the number of bacilli at 3 and 8 months (2.6 x 105 and 2.5 x 105). exploration of IFNλs we apply a novel approach for the BALB/c mice showed increased levels of IL-2, IL-17, IFN- unbiased screening of vertebrate genomes (n=360) for γ and TNF at 8 months when compared to the control group IFNλ-like sequences, incorporating their genetic and decreased levels of IL-6 and NO, at the same times. relationships and chromosomal location. Finally, we query Nude mouse showed increased levels of TNF at 8 months this dataset to identify specific amino acid positions of compared to the control group. No differences in NO levels evolutionary significance. We identify IFNλs in all major were observed between these two leprosy experimental classes of vertebrates, showing that there are two major models. Conclusion: The results suggest the development IFNλ lineages in tetrapods: IFNλ4-like (herein IFNλA) and of an active immune response in BALB/c mice able to IFNλ3-like (IFNλB). The IFNλ locus underwent a unique control M. leprae multiplication in comparison to a expansion in the ancestor of placental mammals generating progressive and disseminate infection in nude mice. the IFNλA and B lineages. These lineages display distinct Disclosure of Interest: None Declared evolutionary dynamics, with only the IFNλBs undergoing rapid diversification. While nearly all mammalian genomes LT038 were found to contain identifiable IFNλ-like sequences, IFNλ SERUM LEVELS OF ADIPOKINES AND PRO- gene content differed by mammalian clade with most INFLAMMATORY CYTOKINES IN MALNOURISHED AND containing four IFNλs consisting of a single IFNλA and three NOURISHED BALB/C MICE INFECTED BY LACAZIA IFNλBs. Although their early evolution appears to have been LOBOI shaped by pressure to expand and diverge, it has since A. S. A. A. Barbosa1,*, B. G. C. Sartori1, T. J. Dionísio2, V. N. evolved by a combination of convergent episodic loss of B. Souza3, M. R. S. Nogueira1, F. R. Vilani-Moreno1, P. C. genes across the entire IFNλ system and pervasive gene M. Pereira4 conversion at the major IFNλ locus. At a molecular level, we 1Biology Technician Team, Lauro of Souza Lima Institute, identify amino acid positions in structurally-relevant sites 2Farmacology Laboratory, São Paulo University, undergoing recurrent diversification indicative of functional 3Immunology Technician Team, Lauro of Souza Lima importance. This work provides the first systematic Institute, Bauru, 4Tropical diseases, Botucatu Medical understanding of IFNλ evolution across placental mammals, School - Paulista State University, Botucatu, Brazil describing the diversity in these critical cytokines to facilitate the development of safe and effective IFNλ-targeted Abstract Content: Introduction: Adipose tissue presents interventions. neuroendocrine and immune functions, producing Disclosure of Interest: None Declared adipokines (adiponectin, leptin and resistin), as well as pro- inflammatory mediators such as TNF and IL-6. There is no LT037 study regarding the role of adipokines and its interaction EVALUATION OF THE SERUM CYTOKINES PROFILE with immune response in Jorge Lobo's disease (JLD), a AND NITRIC OXIDE IN MURINE EXPERIMENTAL chronic granulomatous LEPROSY cutaneous-subcutaneous fungal infection caused by A. S. A. A. Barbosa1,*, B. G. C. Sartori1, T. S. Lima1, V. N. B. Lacazia loboi. Aim: To evaluate the serum levels of Souza2, M. R. S. Nogueira1, F. R. Vilani-Moreno1 adipokines and cytokines in malnourished and nourished 31 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

BALB/c mice infected by Lacazia loboi. Methods: Mice were mediating role for pro-inflammatory cytokines as measured divided into four groups: G1: infected with restricted diet, by an ELISA-based technique. In a first step, monocytes G2: not infected with restricted diet, G3: infected with regular derived from a healthy donor were treated with serum from diet, AA participants of a community-based cohort study in G4: not infected with regular diet. Mice from G1 and G2 were Washington DC without SARS-CoV-2 (n=84 AA (95% submitted to malnutrition for 20 days before infection and female), mean age 59.7 yr, BMI 33.5). Subsequently, TLR2 kept with restricted diet during the experiment. After 4 SME was determined by flow cytometry. Multivariable months, mice were euthanized for the collection of regression was used to examine the association between serum and footpads. Serum levels of adipokines and NDI and TLR2 SME. Sera-treated monocytes revealed that cytokines was evaluated. The number of fungi and viability higher NDI was associated with a downregulation of TLR2 were SME (ß=-0.34, p=0.03) after adjusting for atherosclerotic evaluated in the inoculated footpads. Results: Viability and cardiovascular disease (ASCVD) 10-year risk score, BMI, number of fungi were higher in nourished than and individual-level income. In a second step, monocyte in malnourished mice. No differences in adiponectin levels TLR2 SME was determined in vivo from freshly drawn blood. were found. Leptin production was decreased in Again, higher NDI was associated with lower monocyte the malnourished groups (G1 and G2) while resistin levels TLR2 SME (ß=-0.34, p=0.02) after adjustments verifying our was lower in infected groups (G1 and G3). Besides, IL-6 previous ex vivo results. In a third step, we determined that and TNF levels were lower in malnourished groups (G1 and monocyte TLR2 SME displayed a significant negative G2). Conclusion: The process of malnutrition promoted association with serum-level tumor necrosis factor-alpha a decrease in the production of leptin and pro-inflammatory (TNF-α) (ß=-0.46, p=0.01) after adjustments. Structural cytokines in spite of infection, suggesting some equation modeling showed that TNF-α significantly interaction among these mediators, while the infection by L. mediated the relationship between NDI and in vivo loboi caused a decrease in resistin release. In an monocyte TLR2 SME by 67.5% when using ASCVD 10-year unexpected way the malnutrition status seems to favor the risk score and BMI as covariates. In summary we control of L. loboi infection by the host. Altogether, the determined that increasing NDI, potentially via increasing results reveal a complex scenario in JLD involving nutrition TNF-α levels, was associated with decreased monocyte and immunity. Supported by: Fundação de Amparo à TLR2 expression, a phenomenon which has been Pesquisa do Estado de São Paulo – FAPESP (nº associated with decreased survival in SARS-CoV-1. Further 2017/15664-2). studies are needed to identify the connection of monocyte Disclosure of Interest: None Declared TLR2 levels and SARS-CoV-2 severity or mortality as well as potential underlying mechanisms. However, LT039 understanding immunomodulating features of NDI may NEIGHBORHOOD DEPRIVATION ASSOCIATES WITH provide therapeutic insights in the fight against SARS-CoV- DECREASED MONOCYTE TLR2 EXPRESSION VIA TNF- 2, especially for individuals living in deprived ALPHA POTENTIALLY CONTRIBUTING TO SARS-COV- neighborhoods. 2 DISPARITIES IN AFRICAN AMERICANS Disclosure of Interest: None Declared Y. Baumer1,*, C. A. Gutierrez-Huerta1, B. S. Turner1, M. P. Playford2, S. E. Claudel1, R. Islam1, V. M. Mitchell1, B. S. LT041 Collins1, K. Tamura1, N. N. Mehta2, T. M. Powell-Wiley1 PLASMACYTOID DENDRITIC CELLS ENHANCE TLR- 1Social Determinants of Obesity and Cardiovascular Risk MEDIATED B-LYMPHOCYTE ACTIVATION AND Laboratory, 2Section of Inflammation and Cardiometabolic DIFFERENTIATION IN AN STAT1 AND TYPE I IFN- Diseases, NHLBI, Bethesda, United States DEPENDENT MANNER H.-H. Chen1,*, C.-K. Lee1 Abstract Content: Neighborhood deprivation (ND) as a 1Graduate Institute of Immunology, National Taiwan measure of neighborhood-level socioeconomic status (SES) University College of Medicine, Taipei, Taiwan has been implicated as an important social determinant of SARS-CoV-2. SARS-CoV-2 disproportionally affects African Abstract Content: B cells function to secret antibodies Americans (AA) as well as individuals residing in more upon activation and differentiation, which play an important deprived neighborhoods. An upregulation of monocyte toll- role in adaptive immunity. Plasmocytoid dendritic cells like receptor (TLR2) surface marker expression (SME) has (pDCs), on the other hand, are a subset of DCs known for been shown in individuals infected with SARS-CoV-1, robust type I IFN (IFN-I) production upon TLR stimulation or suggesting its importance for an adequate immune viral infection and are crucial for antivirus response of innate response to coronaviruses. In animal models of social immunity. B cells and pDCs can cooperate to boost the defeat TLR2 and TLR4 SME were modulated, indicating humoral immunity. However, the detailed mechanisms their relevance in the body’s response to social factors. But remain elusive. Here, we set up an in vitro coculture system little is known about the relationship between ND and of splenic pDCs and B cells and stimulated with, R848, a TLR2/4 SME modulation. Therefore, we utilized ex vivo and TLR7 agonist. pDCs enhanced TLR7-mediated B cell in vivo approaches to test the hypothesis that increasing ND activation with increased expression of MHCII, CD86 and is associated with monocyte TLR2 SME. NDI is defined by CD69. Moreover, increased proliferation and plasma cell a U.S. Census-derived ND index (NDI; higher NDI=lower differentiation but decreased survival and germinal center B NSES). Furthermore, we aimed to identify a potential cell (GC B) formation were also found through both soluble 32 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 factor- and cell-to-cell contact-dependent manner. In LT043 addition, follicular B cells (FO B) are more sensitive to THE ROLE OF DNMT1 AND G9A pDC/R848 stimulation than marginal zone B cells (MZ B). METHYLTRANSFERASE IN VIRUS-ACTIVATED PDC Among soluble factors secreted in the coculture system, IFN-ALPHA PRODUCTION IN HEALTHY DONORS AND IFN-I was found to be critical for the enhanced response. B COVID-19 PATIENTS cells lacking IFNAR1 displayed impaired responses. A. Codrington1,*, H. Dewald1, Z. Yin2, S. Singh2, P. Interestingly, IFN-I receptor signaling in pDCs contributed to Fitzgerald-Bocarsly1 the enhanced responses as well. A similar phenotype was 1Rutgers University- RBHS, 2University Hospital-NJMS, also observed for cell-intrinsic requirement of STAT1 Newark, United States signaling for both B cells and pDCs. STAT1 was activated by serine 727 phosphorylation (pS727) in B cells directly by Abstract Content: In chronic viral infections such as HIV, R848 stimulation. S727 to A mutation in STAT1 of B cells pDC initially produce a robust amount of IFN-alpha but then attenuated the synergism between pDC and B cells. lose their ability to produce IFNs, with the cells themselves Adoptive transfer of B cells into Rag1-/- mice that had been being rapidly turned-over. Our lab is investigating the role of depleted of pDCs also reduced IgM production in response DNA and histone methylation in virus-activated pDC to R848, suggesting an essential role of pDC in T- cytokine response from healthy donors and individuals with independent antibody response in vivo. Collectively, we COVID-19. DNMT1 proteins are known for their dual have identified that murine splenic pDCs directly enhance B functions including 1) methylation at cytosine phosphate cell activation, proliferation and plasma cell differentiation in guanine (CpG) sites and, 2) recruiting histone deacetylases vitro and in vivo, and define a crucial role of IFN-I and STAT1 (HDACs), promoting histone methylation both of which are signaling in both pDCs and B cells to positively modulate the known to silence cytokine gene expression. To investigate humoral immunity. the effects of virus activation on pDC, we isolated fresh Disclosure of Interest: None Declared PBMC from healthy donors and COVID-19 patients and stimulated with RNA viruses IAV or HIV-1, and DNA virus, LT042 HSV-1, which signal in pDC through TLR7 and -9, REVEALING THE HIDDEN HELP FROM IL-21: respectively, for 8 and 24 hrs. Flow cytometry was used to FOLLICULAR HELPER T CELLS RE-SENSITISE GC B quantify the expression of DNA methyltransferase-1 CELLS TO IL-21 FOR PLASMA CELL DIFFERENTIATION (DNMT1) in pDC. We also treated pDC with the G9a Z. Chen1,2,*, Y. Yao2, H. Wang1, J. Yunis2, R. Wang1, X. methyltransferase inhibitor, BIX01294, and measured IFN- Gao1, P. Zhou1, D. Yu1,2 alpha and DNMT1 6-hr post stimulation with HSV and IAV. 1John Curtin School of Medical Research, The Australian RNA from purified pDC was analyzed for expression of National University, Acton, 2Diamantina Institute, University DNMT1 and G9a methyltransferase via qRT-PCR. More of Queensland, Brisbane, Australia recently, pDC from hospitalized COVID-19 patients were stimulated with virus, and expression of DNMT1 and IFN- Abstract Content: The differentiation of plasma cells from alpha was measured by flow cytometry. IAV and HSV- germinal centre (GC) B cells requires the selection signals stimulated pDC, DNMT1 was significantly upregulated at 24- from follicular helper T (Tfh) cells. However, how the hrs compared to 8-hr stimulated and unstimulated controls. competent GC B cells commit the plasma cell differentiation IFN-alpha production and DNMT1 expression in IAV- remains unclear. In this study, we demonstrate that the activated pDC, but not in HSV, was significantly inhibited by magnitude of GC B cells to plasma cell differentiation is in the G9a methyltransferase inhibitor, BIX01294. G9a proportion to the signalling potency of Interleukin-21 (IL-21), methyltransferase mRNA was elevated in IAV and HIV-1- a key effector cytokine produced by Tfh cells. Importantly, stimulated pDC, but minimally expressed in HSV-stimulated we revealed that the sensitivity of GC B cells to IL-21 is not cells. Furthermore, hospitalized COVID-19 patients had determined by IL-21 receptor (IL-21R), but by the decreased numbers of pDC as well as decreased IFN-alpha expression level of a novel coreceptor, which enhances IL- production, while DNMT1 proteins were elevated compared 21 signalling strength by increasing the cellular to healthy controls. Our studies indicate that DNMT1 is bioavailability to IL-21. In GC B cells, the expression of this increased in virus-activated pDC at 24-hrs post-stimulation, putative coreceptor is stringently regulated and a time when IFN-a production is turning off, possibly progressively increased upon receiving the help signals suggesting a role for DNMT1 in controlling excessive from Tfh cells. GC B cells with the deficiency of this immune activation. Further, the reduction of DNMT1 in IAV, coreceptor showed prolonged retention in GCs and impaired but not HSV-activated pDC upon treatment with BIX01294 GC B cells to-plasma cell differentiation. We propose a implies a virus or TLR pathway (TLR7 vs -9)-specific role of model whereby Tfh cell-mediated upregulation of IL-21 DNMT1. In addition, decreased IFN-alpha production and coreceptor enhances the sensitivity of GC B cells to IL-21 elevated DNMT1 expression by COVID-19 patient pDC and thus promotes plasma cell differentiation. This model is suggests that DNMT1 may play a vital role in silencing complementary to antigen affinity-based selection of plasma genes associated with IFN-alpha production, resulting in cells and provide new insights in the formation of broad pDC dysfunction. neutralising antibodies in infection and vaccination, and also Disclosure of Interest: None Declared for the generation of autoantibodies in autoimmune diseases. Disclosure of Interest: None Declared 33 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT044 structures, through control of Th17 cell expansion. This may IL-27 REGULATES THE MAGNITUDE OF ECTOPIC provide a viable therapeutic option for treating patients with LYMPHOID STRUCTURES IN EXPERIMENTAL autoimmune diseases. SIALADENITIS VIA CONTROL OF TH17 CELLS Disclosure of Interest: None Declared R. Coleby1,*, D. Lucchesi1, E. Pontarini1, D. Hill2, A. D. Soria3, I. Humphreys3, C. Pitzalis1, G. Jones2, M. LT045 Bombardieri1 TISSUE SOURCE OF ISOLATION INFLUENCES 1Experimental Medicine and Rheumatology, William Harvey MESENCHYMAL STROMAL CELLS CHEMOKINE Research Institute, London, 2School of Cellular and SECRETION AND THEIR IMMUNOMODULATORY Molecular Medicine, University of Bristol, Bristol, 3Division of PROPERTIES Infection and Immunity, Cardiff University, Cardiff, United N. Cuesta Gomez1,2,*, G. Graham1, J. Campbell2 Kingdom 1University of Glasgow, Glasgow, 2Scottish National Blood Transfusion Service, Edinburgh, United Kingdom Abstract Content: Introduction: IL-27 is a key regulator of adaptive immunity and has both pro- and anti-inflammatory Abstract Content: Since the discovery of mesenchymal roles. One of its main functions is to exert stromal cells (MSCs) ability to repair craniofacial defects and immunomodulatory effects on CD4 T cells. For example, IL- immunomodulatory properties, they became focus of 27 inhibits the expansion of Th17 cells which secrete the research due to their cellular therapeutic potential. The pro-inflammatory cytokine IL-17. CD4 T cells, and in Advanced Therapeutics Department within the Scottish particular Th17 cells have been implicated as a driver of National Blood Transfusion Service (SNBTS) has developed many autoimmune diseases and were previously shown to standardised methodologies for the isolation of MSCs from contribute to ectopic lymphoid structure (ELS) development. pancreatic islets (Is MSCs), adipose tissue (Ad MSCs) and These organised aggregates of immune cells can harbour a bone marrow (BM MSCs). However, studying and functional germinal centre and are often present in comparing their in vivo function and the immunomodulatory autoimmune diseases where they correlate with worse potential is essential prior to their use within a clinical setting. disease progression. Here, we aim to elucidate the role of The immunomodulatory abilities of human MSCs have been IL-27 in ELS formation in a murine model of inducible studied using mouse models that lack a control for sialadenitis, using transcriptomic data. mismatched major histocompatibility complex molecule Methods: We administered a replication deficient expression. Thus, this study aimed to objectively compare adenovirus (AdV) to the salivary glands (SG) of C57BL6 the immune cell recruitment and potential (WT) and IL27Rα-deficient (KO) mice via retrograde immunomodulatory functions in vivo of murine BM, Is and cannulation. IL-17A signalling was blocked using a Ad MSCs in a stringent, standardised manner, without neutralising antibody. Salivary gland samples were species or gender mismatch that could lead to both cell– analysed using immunofluorescence, flow cytometry and mediated and humoral immune responses. RNAseq. Results: In experimental sialadenitis in WT mice, the Under resting conditions, MSCs from the three tissue immune cell infiltration in the SG, was organised in sources were able to secrete chemokines at similar levels, aggregates by 12 days post cannulation (dpc) and fully were CCL2 was secreted the most; however, this secretion formed ELS by 19 dpc. Temporally tracking the top did not produce a recruitment of leukocytes above control upregulated genes showed that at early time points (1 and 5 levels. Inflammatory stimulation of MSCs with a cytokine dpc) genes involved in inflammation (Cxcl11, Acod1, cocktail (TNF-α, IL-1β, IFN-γ) led to the upregulation of the S100a8/a9 and Saa2/3) were the most upregulated, secretion of chemokines where Is MSCs secreted the whereas at 12 and 19 dpc T and B cell signature genes highest levels of CCL2, CCL5, CXCL1 and CXCL10 while (Icos, Cd19, Cxcr5 and Pax5) were highly upregulated. IL- CXCL12 was secreted at higher levels by BM MSCs. CCL2, 27 producing cells and IL-27 family gene expression were CCL5 and CXCL1 are strong chemoattractans, but despite upregulated in the SG of WT mice. KO mice had larger and the higher secretion by Is MSCs under inflammatory more numerous SG ELS and presented an expansion of conditions, Ad MSCs were able to recruit significantly more Th17 cells compared to WT. Transcriptomic data leukocytes in vivo than BM and Is MSCs. More importantly, corroborated this showing a strong and sustained ELS gene Ad MSCs were the only MSCs able to produce the signature in the KO mice, between 12 and 19 dpc. IL-17 recruitment of T cells. Recipient cytotoxic cells are blocking in KO mice resulted in the reduction of ELS size considered detrimental in clinical settings but they are and activity to levels comparable to WT, without affecting the essential to initiate MSC–mediated immunosuppression; ELS number. IL-17 blockade in KO mice also resulted in a thus, we could hypothesize that Ad MSCs have a greater gene signature more similar to WT with a reduction in immunosuppression potential than BM and Is MSCs. expression of the ELS-related genes including Aicda, Ccl19, Ltb and Cxcr5. Furthermore, we Chemokines not only have leukocyte recruitment properties detected an upregulation of Tfh cell and T regulatory cell- as examples, CXCL1, CXCL2 and CXCL12 have angiogenic related genes at 19dpc in the KO compared to WT, that was properties while CXCL10 has angiostatic potential. only partially reduced upon IL-17A blockade. Chemokines are not the only molecules secreted by MSCs Conclusion: Our data shows that IL-27 plays a critical role with the potential to regulate angiogenesis. Ad MSCs in regulating the magnitude and function of ectopic lymphoid secreted the most IL–6, which can promote VEGF secretion, 34 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 but Is MSCs secreted the most VEGF under resting and hormone (GH), and leukaemia inhibitory factor (LIF) stimulatory conditions, which combined with the increased signalling. Ongoing work will assess cytokine induction of secretion of CXCL1, CXCL2 and CXCL12, we hypothesize SOCS mRNA levels in these cell lines and determine that Is MSCs could have greater re–vascularisation whether the SOCS proteins themselves are direct potential. substrates for CRL4AMBRA1 mediated ubiquitination and proteasomal degradation. Altogether, this study highlights that MSCs from different sources differ in their ability to recruit and immunomodulate Conclusion: In this study AMBRA1 was validated as a surrounding immune cells in vivo. These differences have prominent interactor of the CRL5 SOCS box complex and the potential to influence their clinical performance. shown to modulate SOCS1 and SOCS3 activity, two Disclosure of Interest: None Declared important negative regulators of inflammatory cytokine signalling. This further suggests that AMBRA1 will have a LT046 key role in regulating multiple members of the greater SOCS AMBRA1 REGULATES CYTOKINE SIGNALLING BY box family, most likely by ubiquitinating and directing them TARGETING THE SUPPRESSORS OF CYTOKINE to proteasomal degradation. SIGNALLING FAMILY FOR PROTEASOMAL DEGRADATION Disclosure of interest: None declared F. Dehkhoda1,2,*, H. Wong1, T. Kolesnik1, L. Dagley1,2, J. Babon1,2, S. Nicholson1,2 References 1The Walter and Eliza Hall Institute of Medical Research, 1. Chen, S.H., et al., CRL4(AMBRA1) targets Elongin 2Department of Medical Biology, University of Melbourne , C for ubiquitination and degradation to modulate CRL5 Parkville, Australia signaling. EMBO J, 2018. 37(18). Disclosure of Interest: None Declared Abstract Content: Introduction: The canonical suppressors of cytokine signalling (SOCS) family proteins LT047 include CIS and SOCS1-7, with CIS and SOCS1-3 acting as IL-6 TYPE CYTOKINE SIGNALLING FROM AN classical negative feedback inhibitors of cytokine signalling INFORMATION THEORETIC POINT OF VIEW – pathways. The SOCS proteins possess an N-terminal MECHANISMS TO REDUCE UNCERTAINTY OF domain of varying length, a central SH2 domain, and a C- SIGNALLING terminal SOCS box motif. In response to cytokine activation U. Billing1,2, J. Fiebelkow1, B. Guendel1,3, T. Jetka4, M. of downstream signalling pathways, SOCS proteins bind to Komorowski4, S. Waldherr5, C. Garbers6, F. Schaper1, A. phosphorylated target proteins via their SH2 domain and Dittrich1,* facilitate proteasomal degradation of the target by recruiting 1Department of Systems Biology, Otto-von-Guericke an E3 Cullin 5-RING ligase (CRL5) complex through their University Magdeburg, 2Experimental Paediatrics and SOCS box motif. The CRL5 complex is utilised by more than Neonatology, University Hospital Magdeburg, Magdeburg, 80 human SOCS box-containing proteins and consists of the Germany, 3Pancreas Cancer Research Lab, Department of adaptors Elongins B and C, the RING protein Rbx2 and Clinical Intervention and Technology (CLINTEC), Karolinska Cullin 5. Recently, the E3 Cullin4-RING ligase (CRL4) Institute, Stockholm, Sweden, 4Division of Modelling in AMBRA1 was shown to regulate CRL5SOCS3 activity by Biology and Medicine, Polish Academy of Sciences, targeting Elongin C for proteasomal degradation [1]. Warsaw, Poland, 5Bio- & Chemical Systems Technology, However, the exact mechanism/s by which SOCS3 and Reactor Engineering and Safety Section, KU Leuven, other SOCS family members are regulated, remains to be Leuven, Belgium, 6Institute for Pathology, University elucidated. Hospital Magdeburg, Magdeburg, Germany

Methods: An affinity purification-mass spectrometry (AP- Abstract Content: Cytokines of the IL-6-type cytokine MS) approach was utilised to identify proteins which might family, which are characterised by the shared use of the regulate SOCS levels. Co-immunoprecipitation and signal transducing β-receptor gp130, are involved in a immunoblotting were used to validate AP-MS findings and variety of processes in both health and disease. The investigate cytokine signalling responses. CRISPR/Cas9 orchestration of these processes critically depends on gene editing was used to fully or partially delete AMBRA1 in reliable intracellular signalling. It is remarkable that the HEK293T and A549 cell lines. Q-PCR was used to assess expression and activation of signalling proteins differ SOCS transcriptional profiles. markedly even between isogenic cells of the same cell type within a living organism. Obviously, this cell-to-cell variability Results: AMBRA1 was identified by AP-MS in CIS protein does not prevent reliable IL-6-type cytokine signalling. complexes, together with known CRL5CIS complex The aim of this study is to elucidate molecular mechanisms, members, Elongin B, Elongin C, Cullin-5, and Rbx2. Co-IP that allow reliable IL-6-type cytokine signal transmission studies confirmed that, in addition to CIS, SOCS1-7 were despite cell-to-cell variability. Despite the common activation able to interact with AMBRA1 and this interaction occurred of the signal transducing β-receptor gp130 and subsequent via their respective SOCS box motifs. Reduction of AMBRA1 JAK/STAT signalling, individual IL-6-type cytokines execute levels in HEK293T and A549 cell lines resulted in enhanced distinct functions. Whether these distinct functions are SOCS inhibition of (IFNγ), growth accompanied by different molecular mechanisms governing 35 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 reliable signalling is unclear. To address these questions we modified von Frey method. The distribution of NLRP apply multiplexed single cell analyses and information receptors was investigated with immunoperoxidase method, theoretic measures to investigate robustness and Channel and single immunofluorescent labelings. After CFA injection Capacity of IL-6-type cytokine-induced JAK/STAT expressional changes of NLRP1, NLRP2 and NLRP3 signalling. proteins were investigated by double immunofluorescent Interestingly, different mechanisms that enable robustness labelings and Western blotting. The colocalisational values of IL-6-induced JAK/STAT signalling against varying STAT3 with GFAP marker were obtained by confocal microscopy expression complement each other at different time scales and IMARIS software. and cytokine doses. Early IL-6-induced STAT3 activation is Results robust as long as cytokine-concentrations are low to After CFA injection the mechanical withdrawal threshold of intermediate. Robustness at high cytokine concentrations is the treated animals was significantly reduced. Studying the ensured by high STAT3 protein copy-number or by STAT3 distribution of NLRP receptors, immunoperoxidase serine phosphorylation. At late time-points the kinase- reactions confirmed the presence of NLRP proteins in the inhibitor SOCS3 increases robustness. Time- pain-processing Rexed lamina I-II of the spinal gray complementary to the late-acting SOCS3, the protein matter. We found significant increase of the NLRP2 but not tyrosine phosphatase SHP2 increases robustness of IL-6- NLRP1 or NLRP3 protein levels within the spinal dorsal horn induced STAT3 signalling at early time-points. three days following CFA injection. According to the findings The amount of information transmitted by JAK/STAT of double fluorescent labelings only NLRP2 protein was signalling is quantified by Channel Capacity. Channel abundantly expressed by astrocytes in chronic pain Capacity of IL-6-induced JAK/STAT signalling is limited by conditions. cell-to-cell variability in STAT3 expression. Additionally, Conclusion Channel Capacity is affected by the same mechanisms that Here we provide evidence that the spinal astrocytes are the govern robustness. An increase in STAT3 copy number major source of IL-1β in CFA-induced inflammatory pain and increases both Channel Capacity and robustness, whereas the production of mature IL-1β is induced by the NLRP2 an increase in STAT3 tyrosine phosphorylation reduces inflammasome at the peak of mechanical pain sensitivity. robustness but increases Channel Capacity. The currently available literature on NLRP2 protein in CNS In summary, the application of information theory is an is scanty, and these works mainly focus on its effects in elegant and intuitive way to elucidate the mechanisms that reproductive system and embryonic development. prevent disease-associated dysregulated signalling by enabling reliable JAK/STAT signalling despite cell-to-cell Keywords: interleukin-1β, NLRP2, inflammatory pain, spinal heterogeneity. cord, astrocyte, Disclosure of Interest: None Declared Disclosure of Interest: None Declared

LT048 LT049 CHRONIC PAIN INDUCED UPREGULATION OF NLRP 2 REVERSE-SIGNALING OF SOLUBLE TNF RECEPTOR I INFLAMMASOME IN THE RAT SPINAL DORSAL HORN BINDING TRANSMEMBRANE TNFΑ INDUCES A WAKE- L. Ducza1,*, A. Gajtko1, K. Hollo1 LIKE STATE IN VITRO 1Faculty of Medicine, Department of Anatomy, Histology and C. J. Dykstra-Aiello1,*, J. M. Krueger1, K. M. S. Koh1, J. Embryology, University of Debrecen, Debrecen, Hungary Nguyen1 1Integrative Physiology and Neuroscience, Washington Abstract Content: Background State University, Spokane, United States Long-term noxious stimulation related to chronic inflammation lead to central sensitization of neural circuit Abstract Content: Introduction within the spinal dorsal horn. It is widely accepted that Tumor necrosis factor alpha (TNF) is involved in brain inflammasomes play a cardinal role in this process. development and regulating physiological sleep and sleep Inflammasomes are regarded as key signaling platforms responses to pathological insult. Cell activity affects that detect pathogenic microorganisms and activate the neuronal TNF expression; increased action potentials (APs) proinflammatory cytokines such as IL-1β. Precursor of the enhance expression. Neuron/glia co-cultures treated with IL-1β is cleaved by the protease caspase-1 converting it to TNF exhibit a sleep-like state. Binding of soluble TNF a mature biologically active form. In our previous results we receptors (sTNFR) to transmembrane (tm) TNF induces demonstrated a significant enhancement in the IL-1β reverse , contrary to conventional signaling expression in astrocytes, therefore our next aim was to induced by sTNF binding tmTNFRs. We previously inhibited identify the potential inflammasomal type(s), which may sleep in vivo by administering sTNFR. Thus, we orchestrate the formation of IL-1β. hypothesized tmTNF-sTNFR binding would generate Materials and methods reverse signaling in vitro and induce a wake-like state. Male Wistar rats (n=12) were used for animal experiments. Methods Inflammatory pain was evoked by unilateral intraplantar Wildtype (WT) mice and mice lacking either TNF (TNFKO) injection of complete Freund adjuvant (CFA). The or both TNFRs (TNFRKO) provided somatosensory cortical mechanical nociceptive threshold of control and CFA- neurons/glia for co-cultures on six-well multi-electrode treated animals was measured daily for withdrawal arrays (MEAs). Cells were allowed three days of responses before and after CFA administration with undisturbed, incubated development. Developmental 36 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 analyses used daily one-hour MEA recordings taken on cytokine responses, monolayers were treated apically with days four through 14. A one-hour baseline was recorded on microbial stimuli (lipopolysaccharide (LPS), lipoteichoic acid day 14 immediately prior to sTNFR1 treatment (0.0 ng/μL- (LTA), and flagellin), host stimuli (IL-1α, and TNF), or 120 ng/μL). Additional one-hour recordings were made microbial- and host-produced histamine in either complete immediately after treatment and 24 hours later. Measures organoid media or an optimized minimal media. Secreted for in vitro development rates and treatment-induced cytokines were examined by Luminex Magpix in the changes were identical to those defining in vivo sleep: supernatant after 16 hours of incubation. Minimal cytokine synchronization of electrical activity (SYN), APs per second production was observed in organoids incubated in the (APs/s), slow wave delta power (SWP; 0.25-3.75 Hz), and commonly used complete human intestinal organoid burstiness index (BI). medium. In contrast, our optimized organoid media resulted Results in the production ROS, as assessed by H2DCFDA Developmental rates were highest in TNFKO cells, lowest in fluorescence, and robust cytokines in response to select WT cells. sTNFR1 treatment response was decreased in microbial- and host-derived stimuli. Of the host-derived TNFKO cells compared to TNFRKO and WT cells. Dose- stimuli, 1 µg/ml TNF stimulated the largest number of dependent response varied across time and between epithelial cytokines (13 cytokines; >2 fold change to media TNFKO and TNFRKO cells. TNF-expressing TNFRKO cells control); TNF stimulated IL-8, IL-1α, IL-1α, IL-1Rα, IL-7, IL- showed decreased SYN and SWP indicating a wake-like 15, EGF, GRO, GM-CSF, IP-10, MCP-1, MCP-3, MDC, and state that was abolished 24 hours post-treatment. TGF-α. IL-1α (1 µg/ml) only stimulated 9 cytokines: IL-8, Conclusion TNF, IL-1Rα, EGF, G-CSF, IL-15, IL-7, GRO, and MDC. We provide the first evidence of reverse TNF signaling in While LPS has been shown in a number of cancer-derived sleep/wake states and offer a new perception of sleep state cell lines to stimulate IL-8 and other cytokines, in human regulation for potential clinical applications. organoids LPS (1 µg/ml) only stimulated TNF. Moreover, Disclosure of Interest: None Declared LTA (1 µg/ml) only stimulated GRO. In contrast, flagellin (100 ng/ml) stimulated several cytokines: IL-8, TNF, GRO, MCP-1, and MCP-3. The functionality of organoid secreted cytokines was confirmed using Boyden chambers with LT050 human neutrophils. Interestingly, although histamine MICROBIAL- AND HOST-DERIVED STIMULI ACTIVATE receptor 1 was identified by RNAseq, no concentration of REACTIVE OXYGEN SPECIES AND GENERATE histamine (0.1-100 µg/ml) stimulated any cytokine ROBUST CYTOKINE RESPONSES IN HUMAN production. Conclusions: This work is among the first to INTESTINAL ORGANOIDS identify the optimal media conditions to generate human M. A. Engevik1,2,*, W. Ruan3, A. Chang-Graham4, J. Hyser4, organoids that are amenable to microbial and host-derived J. Versalovic2 stimuli. These findings pave the way for studying host and 1Pathology & Immunology, Baylor College of Medicine, microbial interactions in a physiologically relevant model. 2Pathology, 3Pediatric Gastroenterology, Texas Children's Disclosure of Interest: None Declared Hospital, 4Molecular Virology & Microbiology, Baylor College of Medicine, Houston, United States LT051 PBMC AND LYMPH NODE TRANSCRIPTIONAL Abstract Content: Background: The epithelium of the RESPONSES TO ZIKA VIRUS INFECTION IN PIGTAIL gastrointestinal tract represents a key interface between the MACAQUES luminal environment of the gut and the host immune system. J. T. Go1,*, D. Newhouse1, K. Voss1, M. A. O’Connor2, T. B. Intestinal organoids provide a unique system to explore this Lewis2, C. J. Miller3, E. Smith1, N. R. Klatt4, D. Fuller2, M. epithelial-cross-talk. Organoids can be derived from human Gale1 intestinal stem cells and differentiated into all the intestinal 1Immunology, 2Microbiology, 3Pharmaceutics, University of cell types: enterocytes, goblet cells, paneth cells, Washington, Seattle, 4Surgery, University of Minnesota, enteroendocrine cells, etc. Moreover, they are non- Minneapolis, United States transformed, non-cancer-derived and propagatable. Based on their similarity to normal intestine, many researchers Abstract Content: have begun to use this tool to examine epithelial signaling. However, current human organoid systems have been Background: Zika virus (ZIKV) is a pathogenic flavivirus refractory to stimulation of cytokine production due to the primarily transmitted by Aedes mosquitoes that causes an presence of antioxidants in culture media. We have made acute febrile illness. Monocytes are a major cellular target several technical refinements to create human organoids during acute ZIKV infection, with innate immunity shaping capable of producing measurable amounts of cytokines. ZIKV tropism and persistence. Here, we used a nonhuman Using this new system, we hypothesize that human primate model to investigate innate immune responses in intestinal organoids will respond to pro-inflammatory stimuli whole blood and lymphoid tissue during acute ZIKV derived from host and microbes in unique patterns. infection. We performed whole transcriptome sequencing on Methods & Results: Human intestinal organoids derived longitudinal PBMC samples and lymph node, a known from the jejunum of healthy volunteers were grown as two- anatomical site of ZIKV persistence. dimensional monolayers. RNAseq revealed expression of TLRs, cytokine receptors and NOD receptors. To assess 37 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Materials & Methods: Four adult male PTM were inoculated physiological, and immunological alterations described in with ZIKV subcutaneously at multiple sites in the forearms. PTSD patients. However, developing PTSD requires a Whole blood and lymph node biopsies were collected traumatic event that is actively avoided. The social fear throughout the 30-day experimental time course. We conditioning paradigm (SFC) is a mouse model that induces performed total RNA-seq on PBMC samples at baseline (5 social fear and social avoidance by pairing social days before inoculation), 1-4, 7, 14 and 23 days post- investigation with electric foot shocks. Here, we tested the inoculation (dpi), and lymph node tissue biopsied at hypothesis that (i) prior CSC exposure potentiates the baseline, 7, 14, and 21 dpi. Gene Set Enrichment Analysis effects of SFC, and (ii) this is accompanied by central and (GSEA) of the whole transcriptome was performed for each peripheral changes in immune activation. Therefore, male time-point to identify functional categories responding to mice were exposed to 19 days of CSC before they were ZIKV infection. A ZIKV qRT-PCR assay was used to detect tested in the SFC paradigm. During social fear acquisition viral RNA copy numbers in samples. (day1), fewer shocks were needed to reliably evoke social fear in CSC mice compared to single housed controls. Results: Viremia peaked 2-3 dpi and was completely Moreover, CSC mice displayed a delayed extinction of resolved in the blood in all animals by day 7 p.i. In contrast, social fear (day2), which was still visible during recall (day3). ZIKV RNA persisted in the peripheral lymph node (PLN) to Regarding the immune system, in vitro spleen and lymph 21 dpi. Differential expression analyses of PBMC samples node responsiveness, plasma cytokine levels as well as revealed gene enrichment related to interferon signaling and central immune markers including microglia and cytokine RIG-I-like receptor signaling pathways, with increased expression were differentially regulated following CSC, differential gene expression between day 2-4 p.i. There was SFC, or their combination. Supported by DFG Ne465/33-1 a prominent innate immune and cytokine signaling gene and BMBF (OptiMD). signature at 4 dpi. Differentially expressed genes involved in Disclosure of Interest: None Declared the PBMC antiviral innate immune response included DDX58 (encoding RIG-I) and TRIM25. Several interferon- LT053 stimulated genes were strongly upregulated, including ZIKA VIRUS DYSREGULATES TROPHOBLAST IFIT1/3/5, OAS1-3 and STAT1/2. By 7 dpi an inflammatory INVASION VIA INHIBITION OF LIF-MEDIATED STAT3 gene signature had evolved, with gene enrichment related SIGNALING to IL-1 production, granulocyte activation, neutrophil- N. Hajari1,2,*, E. Verstelle1,3, M. Gale.Jr1,3 mediated immunity, and chemokine production. In contrast 1University of Washington, 2School of Public Health, to PBMCs, there was little change in gene expression in PLN Pathobiology Program , 3Immunology, Center for Innate samples, despite the persistence of ZIKV RNA in the Immunity and Immune Disease , Seattle, United States lymphoid tissue. Abstract Content: Zika virus (ZIKV) is a flavivirus that Conclusions: Our data sets and analyses define causes fetal infection and disease including fetal demise. immunologic profiles linked with acute ZIKV infection. While During pregnancy ZIKV infects the placenta leading to PBMCs mount a strong antiviral innate immune response alteration of the maternal-fetal interface and placental early in ZIKV infection, we hypothesize that the muted damage but the virus/host interactions that underlie response in the PLN contributes, in part, to the persistence placental infection and disease are not defined. Human of viral RNA in this anatomic site. This may have placenta has three layers, with distinct cell types in each; consequences on the transmission of ZIKV after clinical trophoblasts, mesenchymal cells, macrophages (Hofbauer symptoms resolve. cells) and fibroblasts. Trophoblasts are epithelial cells that Disclosure of Interest: None Declared form the outer layer of the blastocyst and are major cell types of the placenta that are responsible for invading LT052 maternal decidua to facilitate implantation. Trophoblast CHRONIC PSYCHOSOCIAL STRESS AGGRAVATES differentiation and invasion are critical for implantation and SOCIAL FEAR CONDITIONING: THE ROLE OF THE proper exchange of oxygen and nutrients during the IMMUNE SYSTEM development. Trophoblast invasion is mediated by specific K. Gryksa1,*, A. K. Schmidtner1, S. O. Reber2, I. D. trophoblast type and is controlled through JAK/STAT Neumann1 signaling by developmental cytokines. During development 1Behavioural and Molecular Neurobiology, University of Leukemia inhibitory factor (LIF) binds to LIF receptor and Regensburg, Regensburg, 2Psychosomatic Medicine and gp130 on trophoblasts to induce JAK/STAT signaling to Psychotherapy, University of Ulm, Ulm, Germany mediate the phosphorylation and activation of STAT3. Active STAT3 then directs the expression of genes that Abstract Content: Chronic psychosocial stress is a risk mediate trophoblast invasion and implantation. Our previous factor for somatic and affective disorders, including studies show that ZIKV imparts a broad blockade to posttraumatic stress disorder (PTSD). Human and animal JAK/STAT signaling that includes a block to STAT3 studies suggest that an over-reactive immune system activation. During infection of first trimester trophoblast cell accompanies stress-associated disorders and is involved in lines we hypothesize that ZIKV blocks STAT3 activation to their pathogenesis. The mouse model of chronic control LIF-regulated JAK/STAT signaling. We used an in subordinate colony housing (CSC) mimics this type of vitro invasion/migration model, immunoblotting and health-compromising stress and induces behavioral, immunofluorescence (IF) staining to test this hypothesis. We 38 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 show that ZIKV infection disrupts LIF-induced trophoblast- production is strongly increased in liver, lung, and spleen in matrix interactions concomitant with STAT3 suppression. this model. In vitro studies have shown that IL-18BP mRNA Our findings suggest that ZIKV infection of trophoblasts and is expressed by a variety of cell types (myeloid, endothelial suppression of LIF dependent STAT3 signaling can impact and epithelial). However, IL-18BP-producing cells have critical steps of implantation to disrupt placental been poorly described in vivo. This study aimed to identify development. Il-18bp-producing cells at steady state and following CpG Disclosure of Interest: None Declared injections in blood, spleen, liver and lung. Methods: As Il-18bp is a secreted protein, the identification LT054 of Il-18bp-producing cells by immunostaining is bound to be ADARZA’S ZIVA MULTIPLEX PROTEIN DETECTION unreliable. Thus, we generated an Il18bpTomato knock-in PLATFORM OFFERS A NEW ULTRA-SENSITIVE ASSAY (KI) mouse line carrying a nuclear tdTomato reporter FORMAT FOR FEMTOGRAM LEVELS OF SENSITIVITY inserted upstream of the first coding of the Il18bp gene J. Haley1,*, E. Mueller1, T. Wente-Roth1, T. Hodge1, J. to visualize Il-18bp-producing cells by flow cytometry and Mitchell1, J. Schmuke1 immunofluorescence (IF). The tdTomato reporter and Il- 1Adarza Biosystems, St. Louis, United States 18bp coding sequences are separated by an A2 self- cleavable peptide, allowing independent production of the Abstract Content: We have used Arrayed Imaging reporter and the Il-18bp protein. Reflectometry (AIR™), a label-free protein microarray Results: In comparison to WT littermate controls, Il-18bp technology that relies on the target binding-induced expression was impaired in Il18bpTomato KI/KI mice perturbation of an antireflective coating on the surface of a suggesting that insertion of the targeting construct partially silicon chip, to create a multiplex antibody array for the interferes with basal and CpG-induced Il-18bp expression. detection of both low and highly abundant targets. ZIVA is Nevertheless, tdTomato mRNA levels reliably mirrored a new multiplex protein detection platform that leverages relative Il-18bp expression in different organs of naïve and AIR™ technology and can multiplex up to 100 unique protein CpG-treated mice and the reporter protein production targets.We have previously detailed our Human Cytokine allowed for identification of Il-18bp producing cells. In naïve Screening Array, an assay that footprints a large number of mice, we identified three Tomato (Tom)+ cell populations high and low abundant proteins with sensitivity ranging from based on the intensity of fluorescence of the reporter: picogram to nanogram/ml (analyte dependent and Tomlow, Tomint and Tomhigh by flow cytometry. The Tomlow optimized).In this presentation, we demonstrate a newly cells likely correspond to F4/80+ CD11b+ monocytes, developed mass enhancement option that increases macrophages, and/or granulocytes. A large population of sensitivity down to low picogram and femtogram/ml Ly6G+ Tomint neutrophils was identified in spleen and in the levels. Using this mass enhancer, we have developed an circulation. Interestingly, all neutrophils analyzed were 18-plex Human Ultra-Sensitivity Cytokine Array and have Tomint. In liver and lung, Tomint cells corresponded validated the array to include linearity, spike-recovery, and respectively to CD146+ Liver Sinusoidal Endothelial cells reproducibility. With array capabilities offering affordable (LSEC) and CD31+ endothelial cells. In addition, 44% of assays from one to hundreds of plex combined with a F4/80+ CD11b- red pulp macrophages (RPM) and 34% of hands-free, automation-friendly workflow enabled by the Kupffer cells (KC) were Tom+ in spleen and liver, ZIVA instrument, the technology has been designed to respectively. Finally, we observed red fluorescence in liver achieve “tunable” levels of sensitivity empowered by AIR™ F4/80+ macrophages and in stabilin-2+ LSEC by IF. technology and novel mass enhancers. Conclusion: We identified neutrophils, endothelial cells and Disclosure of Interest: None Declared resident macrophages as major Il-18bp-producing cells in the circulation, spleen, liver and lung of naïve mice. This LT055 project is currently pursued for a precise description of the IDENTIFICATION OF IL-18BP-PRODUCING CELLS different cell types involved in Il-18bp production in CpG- USING NEWLY DEVELOPED TDTOMATO REPORTER induced MAS. MICE Disclosure of Interest: None Declared M. Harel1,2,*, E. Rodriguez1,2, G. Palmer1,2, C. Gabay1,2 1Department of Pathology and Immunology, University of LT056 Geneva, 2Division of Rheumatology, Department of MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) Medicine, Geneva, Switzerland IS RELEASED BY NECROTIC MONOCYTES/MACROPHAGES Abstract Content: J. Harris1,* on behalf of Monash Rheumatology Research Introduction: Interleukin (IL)-18 is a pro-inflammatory Group cytokine, the activity of which is regulated by its natural 1Centre for Inflammatory Diseases, Monash University, inhibitor IL-18 binding protein (IL-18BP). If the balance Clayton, Australia between IL-18 and IL-18BP is dysregulated, abnormal levels of free bioactive IL-18 (fIL-18) are present in the circulation, Abstract Content: Macrophage migration inhibitory factor as is the case in sera of patients with macrophage activation (MIF) is a pleiotropic inflammatory molecule with both syndrome (MAS). We showed that endogenous Il-18bp cytokine and non-cytokine activity. MIF is constitutively exerts a protective role in a murine model of MAS induced released from multiple cell types via an unconventional by repeated injections of the TLR9 agonist CpG. Il-18bp secretory pathway that is not well defined. Here, we show 39 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 that both mouse immortalised bone marrow-derived as expected. The relations between IL-1a and SBP, macrophages (iBMM) and human THP-1 monocytic cells between IL-6 and SBP, and between IL-18 and SBP, were constitutivelt release relatively high levels of MIF in culture all loosely directly correlated in these healthy SEA subjects. and rapidly replenish extracellular MIF following a change of Since BP is affected by gender differences, we repeated the culture medium. Previous studies have also shown that MIF analysis by separating male and female groups. The direct can be released in response to different inflammatory correlations between SBP and each of the inflammatory stimuli, including the TLR4 agonist lipopolysaccharide cytokines were more pronounced in female than in male (LPS). Here, we looked at MIF release from human and subjects. But the healthy male subjects showed significantly mouse monocytes/macrophages in response to different higher levels of plasma IL-1a, IL-6, and IL-18 than their stimuli. In iBMM and THP-1 cells, MIF release was not female counterparts (p* < 0.0001 for each cytokine significantly altered in response to LPS, the TLR2 agonist comparison). PAM3CYSK3, the TLR7/8 agonist resiquimod (R848) or Conclusion: From analyzing the SEA data, the blood heat-killed Escherichia coli. However, cytotoxic stimuli pressure setting of healthy individuals could be directly strongly promoted release of MIF. In particular, MIF release correlated with the plasma levels of inflammatory cytokines was highly upregulated in cells undergoing necrosis, IL-1a, IL-6, and IL-18. Yet the levels of these cytokines likely necroptosis and NLRP3 inflammasome dependent were also affected by sexual hormones, as demonstrated pyroptosis. Our data suggest that cell death represents a here that the levels were generally much higher for similarly major route for MIF release from myeloid cells. The healthy male subjects than female subjects. Our findings functional significance of these findings and their potential suggest that the gender factor should be taken into importance in the context of autoimmune and inflammatory consideration for inflammation-related hypertension. diseases, particularly those in which cell death and defective Disclosure of Interest: None Declared clearance of apoptotic cells are drivers of pathology, warrants further investigation. LT057b Disclosure of Interest: None Declared CYTOKINE RESPONSES AND TISSUE PATHOLOGY IN NLRP12-DEFICIENT MICE IN A MODEL OF DIET- LT057 INDUCED OBESITY (DIO) GENDER DIFFERENCES IN INFLAMMATION-RELATED K. Berman1,*, E. Lien2, A. Stanton3 BLOOD PRESSURE SETTING 1Biology, Westfield State University, Westfield, 2Medicine, H.-J. Lin1, C.-C. Liao2, Y.-C. Huang3, J. C.-Y. Lai4, K. Hsu1,* UMASS Medical School, Worcester, 3Westfield State 1Medical Research, MacKay Memorial Hospital, Tamsui, University, Westfield, United States 2Department of Laboratory Medicine, 3Health Evaluation Center, 4Department of Medical Research, Taitung MacKay Abstract Content: Metabolic syndrome is named for a Memorial Hospital, Taitung, Taiwan group of risk factors that predisposes patients to cardiovascular disease and type 2 diabetes (T2DM). These Abstract Content: The relationships between inflammation risk factors include obesity, elevated serum cholesterol, and hypertension have long been observed. Although a triglycerides, blood pressure and elevated fasting glucose. major trigger to inflammation-related hypertension is obesity Significantly increased visceral adipose tissue depots (VAT) and/or unhealthy lifestyle, many mechanistic details remain can cause central obesity, hallmarked by a chronic, low unclear. This study aimed to examine possible relationships grade inflammation. Obese, diabetic patients have between blood pressure (BP) and levels of inflammatory increased levels of circulating pro-inflammatory cytokines, cytokines among healthy eastern Taiwanese (a Southeast such as TNF, IL-6, MCP-1, and the IL-1 family members, IL- Asian [SEA] population). 1β and IL-18. These pro-inflammatory cytokines originate Methods: Adults subjects were recruited at the Health from multiple sites, including both the liver and from Checkup Center of Taitung MacKay Memorial Hospital (TT- infiltrating of the white VAT. Though our MMH), with approval of the MMH Institutional Review Board. understanding of the pathways that trigger insulin resistance Besides the general physical assessment, recruited has grown, and includes activation of several innate immune subjects were asked to fill out a questionnaire that assessed signaling pathways, including Toll-like receptors (TLR) and their lifestyle. Their plasma samples were tested for IL-1a, the Nod-like receptor (NLR), NLRP3, the complex IL-6, and IL-18. interaction between innate immune signaling and Results: The data from age-matched, healthy subjects (116 metabolism is only starting to be appreciated. We tested the male & 110 female) were selected for analyses. The role of NLRP12 in a model of diet-induced obesity (DIO) and averaged body-mass indices (BMI) for both gender groups metabolic disease. Compared to wildtype mice, NLRP12- were similar (male: 25.7+/-3.9; female: 25.2+/-5.7). The deficient mice gained more weight, were more insulin averaged BPs for male subjects were 126.4+/-15.9 mmHg resistant and glucose intolerant. NLRP12-deficient mice (systolic) and 84.0+/-12.7 mmHg (diastolic), while the had higher levels of serum cholesterol, leptin, and insulin as averaged BPs for female were 115.8+/-13.9 mmHg well as the appearance of fatty liver not observed in WT (systolic) and 73.5+/-9.9 mmHg (diastolic). Both gender mice. When we performed both co-housing experiments groups reported similarly scores of sleep quality. The male and littermate control experiments between NLRP12 and group in average exercised twice more often than the female WT mice, we found the phenotype to be transferable. group per week. By correlation analyses, we observed a Pro-inflammatory cytokine levels, specifically IL-18, were roughly direct relation between BMI and systolic BP (SBP) generally higher in NLRP12-deficient mice serum. Analysis 40 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 of tissues revealed that NLRP12-deficient mice had events in cancer. Yet virtually nothing is known about how significantly higher levels of pro-inflammatory cytokines in altered epigenetic regulators directly contribute to the VAT and pancreas. Histological analysis of tissues immunological disease. Speckled Protein 140 (SP140) is a revealed that NLRP12-deficient mice had significantly chromatin “reader” protein, with high homology to increased area of pancreatic islets as compared to wildtype autoimmune regulator AIRE, that is restricted to the immune mice. These studies further identify NLRP12 as a novel system. Loss-of-function mutations within SP140 associate suppressor of metabolic disease. with Crohn’s disease (CD) (1, 2) multiple sclerosis (MS) and Disclosure of Interest: None Declared chronic lymphocytic leukemia (CLL) (3). We previously identified SP140 as a key orchestrator of LT057c macrophage identity and function through repression of CHARACTERIZATION AND PREDICTION OF ISRE lineage-inappropriate genes such as HOX but the BINDING PATTERNS ACROSS CELL TYPES UNDER mechanisms by which this novel epigenetic reader functions TYPE I INTERFERON STIMULATION are unknown. Here we reveal SP140 as a repressor of S. Leviyang1,* topoisomerase activity and DNA accessibility via its PHD 1Georgetown University, Washington, United States and Bromodomain. We employed a global proteomic strategy to find SP140 in complex with DNA unwinding and Abstract Content: Many studies have noted cell-type chromatin remodeling machinery, including topoisomerases specificity in the ISGs differentially expressed in response to (TOP1, 2A), FACT and SMARCA complexes. We find that type I IFN stimulation. Non-canonical IFN signaling has SP140 acts to repress topoisomerases to maintain gene been clearly associated with variation in the ISGs across cell silencing. Loss of SP140 in CD patients bearing SP140 types, for example in cases where STAT3 is activated and mutations, led to disruption of the SP140-TOP complex, shifts signaling away from ISGF3 associated regulation. The increased topoisomerase activity, increased DNA double- existence of variation associated with binding to ISREs by stranded breaks and irregular gene expression programs in ISGF3 and its impact on ISGs is less clear. We examined leukocytes that could be successfully rescued with ISRE binding patterns under IFN stimulation across six cell topoisomerase inhibitors. Our results illustrate how types using existing ChIPseq datasets available on the GEO epigenetic and transcriptional alterations induced by SP140 and ENCODE databases. We find that ISRE binding is cell mutations disrupt immune cell identity and function. This specific, particularly for ISREs distal to transcription start comprehensive understanding of how SP140 exerts sites, potentially associated with enhancer elements, while transcriptional repression for macrophage identity, ISRE binding in promoter regions is more conserved. Given encourages development of precision medicine strategies variation of ISRE binding across cell types, we investigated for manipulation of the aberrant immune transcriptome due associations between the cell type, homeostatic state and to loss of SP140 in immunological disease. ISRE binding patterns. Taking a machine learning approach and using existing ATACseq and ChIPseq datasets Disclosure of Interest: None Declared available on GEO and ENCODE, we show that the epigenetic state of an ISRE locus at homeostasis and the LT061 DNA sequence of the ISRE locus are predictive of the NOVEL MECHANISMS OF NUCLEIC ACID SENSING: ISRE's binding under IFN stimulation in a cell type, specific IMPLICATIONS FOR ANTIVIRAL RESPONSES manner, particularly for ISRE distal to transcription start K. Baid1,*, K. Mossman2 sites. 1Biochemistry and Biomedical Sciences, 2Pathology and Molecular Medicine, McMaster University, Hamilton, Reference: Canada

Leviyang S. (2020) Characterization and Prediction of ISRE Abstract Content: Intracellular detection of viral nucleic Binding Patterns Across Cell Types Under Type I Interferon acids leads to the production of type I interferons (IFNs) and Stimulation. BioRxiv. https://t.co/5ILY5rKUT3 subsequent establishment of an antiviral state in infected Disclosure of Interest: None Declared and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN responses in infected cells, however, viral nucleic acids released from dying cells can Lightning Talk Session 1: Innate Immunity I stimulate IFN production in surrounding or distal uninfected LT059 cells. We have previously shown that class A scavenger SP140 EPIGENETIC READER REPRESSES receptors (SR-As) internalize extracellular double-stranded TOPOISOMERASE ACTIVITY TO MAINTAIN RNA (dsRNA) to mediate an IFN response. Cytosolic MACROPHAGE IDENTITY sensors are essential in mediating an antiviral response H. Amatullah1,*, S. Digumarthi1, G. Bonilla2, R. Sadreyev2, K. against the endocytosed dsRNA, but the mechanism of L. Jeffrey1 endoplasmic release and cytoplasmic entry of dsRNA 1Gastroenterology, 2Department of Molecular Biology, remains an enigma. In this study, we hypothesized that a Massachusetts General Hospital, Boston, United States dsRNA-channel protein facilitates entry of dsRNA into the cytoplasm after endocytosis. To test our hypothesis, we Abstract Content: Dysregulated epigenetic enzymes and studied the role of SIDT2, a mammalian ortholog of aberrant chromatin landscapes and architecture are sentinel Caenorhabditis elegans SID-1 dsRNA transporter in 41 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 facilitating dsRNA entry into cytoplasm and subsequent changes where observed with rhIL-4 and rhIL-10 treatment. activation of antiviral responses. We generated and Moreover, rhIFN-g increased TNF-α secretion in Mtb- assessed antiviral responses to dsRNA in SIDT2 deleted stimulated THP-1 cells. However, SLAM did not regulate human cells. Wildtype and SIDT2 deleted cells did not TNF-α nor IL-1β production as measured by ELISA. respond differently to dsRNA. Preliminary observations Rhodamine-stained Mtb (Mtb-R) or live M. tuberculosis-RFP suggest that SIDT2 might not be necessary in facilitating were used to evaluate SLAM role on bacterial uptake by FC. extracellular dsRNA recognition in mammalian cells. We are Costimulation trough SLAM with an agonistic antibody currently using high-throughput assays to identify cellular further induced Mtb-R and M. tuberculosis-RFP endocytosis molecules that may be involved in the antiviral response to by macrophages and THP-1 cells (p<0.05). extracellular dsRNA. Data from our studies will allow us to To elucidate SLAM role as a microbial sensor, we first identify mechanisms of antiviral immune response that are demonstrate a direct interaction between SLAM and Mtb by mediated by extracellular nucleic acids. We shall harness FC and fluorescence microscopy. Furthermore, we found this knowledge to identify mechanisms to boost innate that SLAM colocalized with Mtb (Manders’ index (MI) of immune responses against viruses and develop nucleic 0.74) and with early (EEA1) and late (LAMP2) acid-based therapies that can bypass endosomal /lysosomes markers by confocal microscopy (MI entrapment and lysosomal degradation. of 0.6 and 0.7 respectively). These results indicate that Disclosure of Interest: None Declared SLAM could recognize Mtb and participate in the endolysosomal maturation process. LT062 Notably, higher SLAM levels were detected in CD14 positive SLAM MEDIATES MYCOBACTERIUM TUBERCULOSIS cells from pleural effusions compared to peripheral blood RECOGNITION AND ENDOLYSOSOMAL MATURATION CD14 positive cells from Tuberculosis patients, indicating a IN HUMAN MONOCYTE-DERIVED MACROPHAGES possible active function of SLAM at the site of infection. A. M. Barbero1,2,*, R. E. Hernández Del Pino1,2, A. Trotta3, Taken together, our results provide evidence of SLAM as a M. Genoula3, M. A. Estermann2,4, J. Celano2, F. Fuentes3, potential microbiological sensor that improves M. V. E. Garcia5,6, L. Balboa3, P. Barrionuevo3, V. Pasquinelli1,2 tuberculosis uptake in human monocyte-derived 1Centro de Investigaciones y Transferencias del Noroeste macrophages. de la Provincia de Buenos Aires (CIT NOBA), UNNOBA- Disclosure of Interest: None Declared Universidad Nacional de San Antonio de Areco (UNSAdA)- Consejo Nacional de Investigaciones Científicas y Tecnicas LT063 (CONICET)., Buenos Aires, 2Centro de Investigaciones CHARACTERIZATION OF INNATE AND ADAPTIVE Básicas y Aplicadas (CIBA), Universidad Nacional del IMMUNE IMPACTS OF CHEMICALLY DISSIMILAR Noroeste de la Provincia de Buenos Aires (UNNOBA), STING ADJUVANTS Junin, 3Instituto de Medicina Experimental (CONICET- D. Boehm1,*, N. Mizuno1, K. Pryke1, V. R. DeFilippis1 Academia Nacional de Medicina), Buenos Aires, Argentina, 1Vaccine and Gene Therapy Institute, Oregon Health and 4Department of Anatomy and Developmental Biology, Science University, Portland,OR, United States Monash University, Victoria, Australia, 5Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Abstract Content: Vaccination represents the most Departamento de Química Biológica, 6CONICET- effective and adaptable strategy for combating viral Universidad de Buenos Aires, Instituto de Química Biológica infections. Live-attenuated vaccines generate a potent and de la Facultad de Ciencias Exactas y Naturales long-lived immune response; however, they can be (IQUIBICEN), Buenos Aires, Argentina associated with contraindications, especially in immune- insufficient individuals. Protein subunit vaccines offer a safer Abstract Content: Tuberculosis is the leading cause of solution, yet these do not activate all necessary facets of an death from a single infectious agent in the world. immune response and in some cases confer shorter Mycobacterium tuberculosis has smartly manipulated the protection than their replication-competent counterparts. immune system to survive within host macrophages over These limitations can be mitigated through the co- ages. The Signaling Lymphocytic Activation Molecule administration of adjuvants, non-antigenic molecules (SLAM) is a self-ligand receptor that internalizes Gram- capable of activating innate immune cells through the bacteria and regulates macrophages’ phagosomal induction of PRR-mediated, cytokine-terminal innate functions. In Tuberculosis, SLAM promotes Th1 protective signaling pathways. Unfortunately, few adjuvants are immune responses. In this work, we studied SLAM role on licensed in the U.S. and identification of new ones macrophages’ functions against M. tuberculosis infection. represents an unmet clinical need. These may additionally Human monocyte-derived macrophages were obtained from facilitate the development of novel vaccines through precise healthy donors by CD14 positive selection. After 2h of T helper polarization of the immune response. The PRR adherence, cells were cultured in complete media overnight STING recognizes cyclic dinucleotides (CDN) synthesized before stimulation with sonicated-M. tuberculosis (Mtb). by the cytosolic enzyme cGAS in response to microbial or THP-1 cells were differentiated with PMA for 24h before Mtb cellular DNA. The activation of STING stimulates innate stimulation. antiviral activity characterized by a localized type I IFN and We observed that both Mtb and rhIFN-g stimulation induce proinflammatory cytokine response and, as such, exhibits SLAM expression in monocyte-derived macrophages and characteristics of an effective adjuvant target. However, the THP-1 cells, as determined by flow cytometry (FC). No activation of STING also drives transcription-independent 42 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 processes such as apoptosis and autophagy. As a result, platform for caspase-4 recruitment and activation at LPS- the general phenotype elicited in response to STING containing membranes as the first step of non-canonical activation can vary substantially between STING-inducing inflammasome signalling. stimuli such as CDN species and small molecule agonists. Disclosure of Interest: None Declared While pharmacologic STING activation enhances immunogenicity of vaccine antigens, the impact of LT065 differences in STING-associated intracellular responses on THE ANTIVIRAL EFFECTOR RTP4 IS ENGAGED IN A processes important for establishment of adaptive immunity HOST-VIRUS ARMS RACE IN BATS AND OTHER has not been explored. To address this, we utilize a mouse MAMMALS model of vaccination against Chikungunya virus. This is I. Boys1,*, E. Xu1, P. De La Cruz-Rivera1, J. Eitson1, K. Mar1, done in combination with chemically diverse STING J. Schoggins1 agonists including CDNs and small molecules. While all 1UT Southwestern Medical Center, Dallas, United States CDNs and small molecules directly activate STING, the downstream immune reactivity to the antigen varied Abstract Content: Viruses and their hosts continuously significantly. Furthermore, our exploration of STING- impose evolutionary pressure on one another. Host antiviral mediated innate correlates of these differences led to genes frequently underlie these evolutionary conflicts, with unexpected observations regarding agonist-specific examples in primates, but fewer in other mammals. cytokine induction, APC maturation, follicular helper T cell Mammalian cells deploy a range of antiviral effectors as part enhancement, and antigen trafficking and presentation. This of the interferon (IFN) response, a critical component of has important implications in at least two areas: 1) It is antiviral immunity. While the IFN pathway is conserved crucial to filling the numerous gaps that exist with regard to across vertebrates, many antiviral IFN-stimulated genes understanding mechanisms that drive STING-associated (ISGs) exhibit species-specific properties. In screens to immune enhancement; 2) It will reveal properties desirable identify antiviral effectors in bats, which harbor many for STING-based adjuvants to help direct future zoonotic viruses, we identified black flying fox Receptor optimization. Transporter Protein 4 (RTP4) as a potent interferon-induced Disclosure of Interest: None Declared inhibitor of human pathogens in the Flaviviridae family, including Zika, West Nile, and hepatitis C viruses. LT064 Mechanistically, RTP4 associates with the flavivirus GUANYLATE-BINDING PROTEINS AND CASPASE-4 replicase and disrupts interactions required for replication. COOPERATE FOR NON-CANONICAL INFLAMMASOME Comparative approaches demonstrate that RTP4 is ACTIVATION undergoing positive selection, that a flavivirus can mutate to D. Boucher1,2,*, J. Santos 2, L. Kapinos-Schneider3, M. escape RTP4-imposed restriction, and that diverse Dilucca2, B. Demarco 2, K. W. Chen2, R. Heilig 2, K. mammalian RTP4 orthologs exhibit striking patterns of Shkarina2, R. Lim 4, P. Broz 2 specificity against distinct Flaviviridae members. Our 1Biology, University of York, York Biomedical Research findings reveal an antiviral mechanism that has likely Institute, Heslington, York, United Kingdom, 2Biochemistry, adapted over 100 million years of mammalian evolution to University of Lausanne , Epalinges, Lausanne , 3Biozentrum accommodate unique host-virus genetic conflicts. , University of Basel, 4Biozentrum , Universty of Basel, Disclosure of Interest: None Declared Basel, Switzerland LT066 Abstract Content: The human non-canonical METFORMIN EFFECT ON HUMAN MACROPHAGE inflammasome enables caspase-4 activation and INFLAMMATORY RESPONSE AND PHAGOCYTOSIS OF Gasdermin-D-dependent pyroptosis in response to cytosolic MYCOBACTERIUM TUBERCULOSIS bacterial lipopolysaccharide (LPS). LPS has been reported J. L. Cervantes1,*, A. Sanca2, J. Barragan1 to bind and oligomerizes caspase-4 and the pathway is 1Texas Tech University Health Sciences Center, 2UTEP, El thought to proceed without dedicated LPS sensors or an Paso, United States activation platform. Here we report that interferon-inducible guanylate-binding proteins (GBPs) are required for non- Abstract Content: Metformin (MTF) has a well- canonical inflammasome activation following documented ability to control hyperglycemia, which has cytosolic Salmonella infection or upon cytosolic delivery of been shown to have effects on macrophage and lymphocyte LPS. In epithelial cells, GBP1 associates with the surface of functions that are key to controlling tuberculosis (TB) cytosolic Salmonella seconds after bacterial escape from infection. their vacuole and initiates the recruitment of GBP2-4 to Here, we aimed to better understand the effects of MTF on assemble a GBP coat on the bacteria. The GBP coat recruits the phagocytosis of Mycobacterium tuberculosis (Mtb) by caspase-4 to the bacterial surface and allow caspase human macrophages. PMA-differentiated THP-1 cells with dimerization and activation, without lysing the bacteria. two reporters for nuclear factor-ᴋB (NF-ᴋB), and interferon- Mechanistically, GBP1 binds LPS directly with high affinity regulatory factors (IRFs) were treated with 2mM of MTF for through electrostatic interactions. LPS recognition increases 4 hours, and then inoculated with Mtb from various lineages. GBP1 activity and promote oligomerisation on the Since MTF can also directly inhibit key metabolic processes bacteria. Our findings indicate that in human epithelial cells of Mtb, we controlled this variable by using of gamma- GBP1 acts as a cytosolic LPS sensor and assembles a 43 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 irradiated mycobacteria. Phagocytosis was assessed by LT068 immunofluorescent assay. NEURONAL HYPEREXCITABILITY IS A DLK- Phagocytosis of Mtb increased in MTF-treated DEPENDENT TRIGGER OF HSV-1 REACTIVATION THAT macrophages. NF-ᴋB activation after Mtb stimulation was CAN BE INDUCED BY IL-1B lower in MTF-treated macrophages. The effect on IRF S. R. Cuddy1,*, A. Schinlever1, S. Dochnal1, J. Suzich1, P. activation was minimal. Kundu1, T. Downs1, M. Farah1, B. Desai1, C. Boutell2, A. Our results indicate that MTF improves phagocytosis of Mtb Cliffe1 by macrophages, while it at the same time modulating their 1University of Virginia, Charlottesville, United States, 2MRC- inflammatory response. Downregulation of type I IFN University of Glasgow Centre for Virus Research, Glasgow, pathways, associated with active TB infection, could allow United Kingdom for improved activation of macrophages in the presence of TB infection. These results support the effects of MTF in Abstract Content: -1 (HSV-1) keys steps TB infection control, and support its use as an establishes a latent infection in neurons wherein viral additional treatment for TB. transcription is restricted and the viral promoters are Disclosure of Interest: None Declared associated with heterochromatin. Periodically, HSV-1 can reactivate in response to certain stimuli to induce lytic gene LT067 expression and permit transmission. The physiological DISSECTION OF NLRP3 INFLAMMASOME ACTIVATION triggers of reactivation and the intrinsic neuronal signaling BY INFLUENZA A VIRUS SC35M (H7N7) USING A pathways that mediate reactivation are not well-understood. NOVEL VIRUS-ENCODED REPORTER TOOL Previously, we found that dual leucine zipper kinase (DLK)- M. H. Christensen1,*, M. Uchima1, T. Zillinger2, F. I. Schmidt1 mediated activation of c-Jun N-terminal kinase (JNK) was 1Institute of innate immunity, 2Institute of Clinical Chemistry essential for reactivation in response to inhibition of PI3K & Clinical Pharmacology, University of Bonn, Bonn, signaling, which mimics loss of nerve (NGF) Germany support. Moreover, reactivation was associated with a DLK/JNK-dependent histone phospho/methyl switch on lytic Abstract Content: Inflammasome sensors are an important gene promoters. Given that the same histone part of the mediating ASC- phospho/methyl switch occurs in cortical neurons following dependent cytokine secretion and pyroptotic cell death. hyperexcitability (triggered by forskolin), we examined Influenza A virus is known to activate the NLRP3 whether HSV reactivation was linked to neuronal inflammasome, however the exact mechanism, the hyperexcitability and the contribution of DLK/JNK activity importance of the ASC speck assembly and the existence of and histone phosphorylation. Using our primary neuronal additional inflammasome sensors are still debated. model of HSV-1 reactivation, we demonstrate that HSV reactivation is triggered by stimuli that induce neuronal Inflammasome activation is often evaluated by the secretion hyperexcitability. Neuronal hyperexcitability-induced of IL-1b and IL-18 from infected cells while the quantification reactivation was dependent on voltage-gated ion and of ASC speck assembly often is neglected. We have hyperpolarization-activated cyclic nucleotide-gated (HCN) developed a novel, fluorescent inflammasome reporter, channels, demonstrating that neuronal excitability is which allows the visualization and quantification by flow required for HSV-1 reactivation. Additionally, activation of cytometry of ASC nucleation, upstream of cytokine DLK/JNK was essential for HSV-1 reactivation triggered by secretion. neuronal hyperexcitability. The initial burst of HSV-1 lytic gene expression in response to neuronal hyperexcitability We detected high inflammasome activity in primary occurred independently of histone demethylase activity and macrophages and macrophage-like THP-1 reporter cells was accompanied with a histone phospho/methyl switch. upon infection with the SC35M (H7N7) strain, which was not Neuronal hyperexcitability can also be induced by IL-1β, an observed upon infection with lab-adapted WSN (H1N1) inflammatory cytokine released during chronic psychological strain. The SC35M-induced inflammasome was dependent stress and fever; both are triggers of clinical HSV-1 on a late stage of the virus replication cycle but independent reactivation. Notably, in mature sympathetic neurons, IL-1β of virus release or activation of the type I IFN pathway. To triggered HSV-1 reactivation that was dependent on DLK dissect the inflammasome response further, we introduced activation and neuronal excitability. Thus, HSV co-opts an our inflammasome reporter into the genome of the SC35M innate immune pathway resulting from IL-1 stimulation of strain, allowing expression of the reporter tool at early time neurons to induce reactivation. points of infection. By screening a range of THP-1 KO cells Disclosure of Interest: None Declared we confirmed the existence of IAV-induced NLRP3 inflammasomes in myeloid cells. However, reporter virus LT069 infection of primary human airway epithelial cells activated CLASS IIA HISTONE DEACETYLASES DRIVE THE an NLRP3-independent inflammasome response, indicating TOLL-LIKE RECEPTOR-INDUCIBLE GLYCOLYTIC that multiple inflammasome sensors may detect influenza SWITCH AND MACROPHAGE INFLAMMATORY viruses. RESPONSES VIA PYRUVATE KINASE M2 Disclosure of Interest: None Declared K. Das Gupta1,*, M. R. Shakespear1, J. E. B. Curson1, A. Iyer1, D. Ramnath1, R. C. Reid1, T. Kobayashi2, J. H. Gunter3, D. P. Fairlie1, M. J. Sweet1 44 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

1Institute for Molecular Bioscience, The Univeristy of that loss of intermediate filaments was driven by the Queensland, Australia, 2Diamantina Institute, The University calcium-dependent protease, calpain. However, as in of Queensland, 3Translational Research Institute, apoptosis, the mechanism driving pyroptotic microtubule Queensland University of Technology, Brisbane, Australia loss is unknown. Here we show that pyroptotic microtubules dissociate from their connections at the cell periphery, Abstract Content: The metabolic state of innate immune suggesting that microtubules detach from tethers that link cells is intrinsically linked to their functions. Toll-like them to the periphery and prevent Receptor (TLR) signaling enhances aerobic glycolysis depolymerization. Consistent with this idea, we during macrophage activation, resulting in skewing of demonstrate that the microtubule-actin tether protein, cytokine production towards proinflammatory responses. IQGAP1, is cleaved during both pyroptotic and apoptotic cell Here, we use metabolomic, pharmacological, genetic and death of both primary and tumor derived cells from mice and proteomic approaches to demonstrate that class IIa histone humans, and we show that cleavage of IQGAP1 is deacetylases (HDACs) play key roles in fuelling metabolism- modulated by calpain. Together our data support a model mediated inflammation. Inhibition of these enzymes in whereby apoptotic and pyroptotic calcium fluxes activate macrophages attenuates lipopolysaccharide (LPS)- calpain, to regulate cleavage of IQGAP1 which dissociate inducible production of lactate and other glycolytic microtubules from stabilizing connections at the periphery intermediates, reduces proinflammatory IL-1b and CCL2, and drive microtubule depolymerization. We hypothesize and amplifies immunoregulatory IL-10. We have previously that this process may enhance the release of IL-1β and other reported a role for Hdac7 in driving macrophage damage associated molecular pattern molecules for inflammatory responses. Myeloid-specific overexpression of triggering the inflammatory response. Hdac7 in transgenic mice amplifies LPS-inducible lactate, Disclosure of Interest: None Declared and promotes glycolysis-associated inflammatory cytokine release. Conversely, pharmacological or genetic targeting of LT072 Hdac7 and other class IIa HDACs attenuates LPS-inducible WHEN VIRUSES AND THE INNATE IMMUNE SYSTEM glycolysis and associated inflammatory responses in MEET: COMPARISON OF DIFFERENT PESTIVIRAL E- macrophages. A proteomic screen identified the glycolytic RNS IN EVASION OF INNATE IMMUNITY enzyme, pyruvate kinase M isoform 2 (Pkm2) as an E. De Martin1,*, C. Lussi1, M. Schweizer1 interacting partner of proinflammatory Hdac7 in murine 1Institute of Virology and Immunology, Bern, Switzerland macrophages. We show that an Hdac7-Pkm2 complex is a novel immunometabolism signaling hub, where both Pkm2 Abstract Content: The genus Pestivirus, family and Hdac7 are required for optimal macrophage Flaviviridae, includes four approved species named bovine inflammatory functions. Screening for potential lysine viral diarrhea virus (BVDV)-1 and -2, classical swine fever residues that might be a target for Hdac7, we report that virus (CSFV), and Border disease virus (BDV). In addition to Pkm2 deacetylation at lysine 433 is crucial for its these species, currently named as pestivirus A-D, some of proinflammatory functions. The interaction of Hdac7 with the related viruses previously grouped as “unassigned Pkm2 was increased when using an enzyme-dead variant of pestiviruses” are now classified as pestivirus E-K. A Hdac7, consistent with an enzyme-substrate interaction. common feature to all the members is the presence of two Pharmacological targeting of this complex also suppresses unique proteins that pestiviruses evolved to regulate the inflammatory responses in vivo. Our findings therefore host’s innate immune response: the nonstructural protein highlight a novel role of class IIa HDACs (HDAC4/5/7/9) as Npro (only missing in the virus isolated from P. phocoena), key molecular links between toll-like receptor (TLR)- and the envelope glycoprotein Erns. inducible aerobic glycolysis and macrophage inflammatory Particularly, the Erns is also released in the extracellular responses. space in a soluble form, where it is taken up by endocytosis Disclosure of Interest: None Declared into neighboring cells to exert its function. Erns is an RNase able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the immune response by this potent pathogen- LT071 associated molecular pattern (PAMP). This activity is at the IQGAP1 CLEAVAGE AND MICROTUBULE LOSS basis of the ability of BVDV to establish persistent infections DURING PYROPTOTIC CELL DEATH. and immunotolerance, with relevant implications for the M. A. Davis1,*, M. R. Fairgrieve1, B. Turnbull1, M. Gale, Jr.1 epidemiology of the infection. Therefore, this study aims to 1Immunology, University of Washington, Seattle, United characterize the basic features of the Erns proteins of States different classified and unassigned pestiviruses that have not been described yet. To that end, we purified a wide Abstract Content: Pyroptosis and apoptosis are two forms variety of Erns proteins and characterized them in vitro and of programmed cell death that eliminate unnecessary, in cell cultures. The ability to dimerize, the enzymatic activity dysfunctional, infected, or damaged cells; and whereas on different RNA substrates and the intracellular localization apoptosis is considered immunologically silent, pyroptosis is of Erns and its ability to inhibit dsRNA-induced interferon inflammatory to release interleukin-1 beta (IL-1β) to (IFN) synthesis, were the main features we analyzed in this stimulate a downstream inflammatory response. We project. recently discovered that as in apoptosis, the cytoskeleton is Overall, we found large differences between the various Erns destroyed during pyroptosis. Moreover we demonstrated proteins that cannot be predicted solely based on their 45 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 primary amino acid sequences. This provides valuable laboratory mice. This ‘dirty’ mouse model offers a platform information to delineate the structure-function relationship of for studying the acute transmission of viruses between hosts pestiviral endoribonucleases, and on their potential to via natural mechanisms—through direct contact, air, and regulate the host’s IFN response. saliva and other fluids. We co-housed pet store mice with Disclosure of Interest: None Declared wild type laboratory mice and mice that are deficient in interferon-I or interferon-III receptors and harvested various LT073 organs for RNA sequencing analysis. After identifying TBK1 AND IKK-EPSILON ACT REDUNDANTLY TO viruses present in both the reservoir and new host, we used MEDIATE STING-INDUCED NF-KAPPA-B RESPONSES an amplicon-based strategy to quantify variance in viral IN MYELOID CELLS populations and assessed the constrains the interferon K. R. Balka1, C. Louis2, T. L. Saunders1, A. M. Smith3, D. J. response places on transmission and dissemination Calleja2, D. B. D'Silva2, F. Moghaddas2, M. Tailler1, K. E. bottlenecks. We have also co-housed our laboratory mice Lawlor4, Y. Zhan5, C. J. Burns6, I. P. Wicks2, J. J. Miner3, B. with the bedding of pet store rats to analyze species-specific T. Kile1, S. L. Masters2, D. De Nardo1,* immune and non-immune barriers to transmission. We have 1Monash Biomedicine Discovery Institute, Monash identified viruses present in the pet store rats that do and do University, Clayton, 2Inflammation Division, Walter&Eliza not transmit to mice. Overall, this mouse model allows for Hall Institute, Parkville, Australia, 3Department of Medicine, the analysis of barriers to transmission of natural rodent Department of Molecular Microbiology, and Department of viruses. Pathology and Immunology, Washington University School Disclosure of Interest: None Declared of Medicine, St. Louis, MO, United States, 4CiiiD, Hudson Institute, Clayton, 5Immunology Division, 6Chemical Biology LT075 Division, Walter&Eliza Hall Institute, Parkville, Australia ANALYSIS OF THE UNIQUE BIOLOGICAL FUNCTIONS ELICITED BY TYPE III INTERFERONS IN THE Abstract Content: STING is a critical component of host EPITHELIUM innate immune defense, but can contribute to chronic A. Forero1,*, S. Ozarkar1, H. Li2, C. H. Lee1, E. A. Hemann1, autoimmune or autoinflammatory disease. Once activated, M. S. Nadjsombati1, M. R. Hendricks1, L. So1, R. Green1, C. the cGAS-STING pathway induces both type I IFN N. Roy3, S. N. Sarkar3, J. von Moltke1, S. K. Anderson2, M. expression and NF-kB-mediated cytokine production. Gale Jr1, R. Savan1 Currently these two signaling arms are thought to be 1University of Washington, Seattle, 2NCI, Fredrick, mediated by a single upstream kinase, TBK1. 3University of Pittsburgh, Pittsburgh, United States Here, using genetic and pharmacological approaches we show that TBK1 alone is dispensable for STING-induced Abstract Content: The epithelium is a highly proliferative NF-kB responses in human and mouse immune cells, as tissue under constant exposure to pathogenic and well as in vivo. We further demonstrate that TBK1 acts commensal microbes where type I and III interferons (IFNs) redundantly with IKKe to drive NF-kB upon STING activate similar downstream signaling cascades that result activation. Interestingly, we show that activation of IRF3 is in kinetic differences in the expression of antiviral IFN- highly dependent on TBK1 kinase activity, while NF-kB is stimulated genes (ISG). Our study addresses the significantly less sensitive to TBK1/IKKe kinase inhibition. fundamental question of why these two IFN families are Our work redefines signaling events downstream of cGAS- required to prevent viral dissemination at barrier sites. While STING. Our findings further suggest cGAS-STING will need previous observations had suggested type I IFNs (IFNα/β), to be targeted directly to effectively ameliorate the type III IFNs (IFNλ) do not elicit strong inflammatory inflammation underpinning disorders associated with STING responses in vivo, this study provides some of the molecular hyperactivity. mechanisms underlying this disparity. IFNα/β uniquely Disclosure of Interest: None Declared induce expression of the transcription factor IRF1, with little to no IRF1 induction observed after IFNλ treatment. Through LT074 genome-wide expression analysis, we demonstrate that INNATE IMMUNE BARRIERS TO INTRA- AND INTER- IRF1 expression is dispensable for the antiviral activity of SPECIES TRANSMISSION OF VIRUSES IFNα/β, but necessary for the induction of inflammatory E. Fay1,*, K. Balla2, S. Roach1, D. Putri1, A. Tucker1, M. chemokines and immune cell infiltration. The muted Pierson1, S. Kotenko3, D. Masopust1, N. Elde2, R. Langlois1 induction of IRF1 by IFNλ is due to insufficient STAT1 1University of Minnesota, Minneapolis, 2University of Utah, activation and homodimerization given the limited IFNλ Salt Lake City, 3Rutgers, Newark, United States receptor 1 subunit (IFNLR1) expression. We show that in the epithelium, the expression of IFNLR1 is not affected by Abstract Content: Viruses emerging from zoonotic inflammatory stimuli or viral challenge, although exogenous reservoirs represent a major public health concern. While overexpression of IFNLR1 can enhance both antiviral and identifying potential zoonoses is paramount to pandemic chemokine gene expression through IRF1 induction. This preparedness, there are few experimental models that are suggests that sustaining low IFNLR1 expression in the able to characterize virus and host factors necessary for epithelium is crucial for preventing deleterious inflammation. cross-species transmission. Here, we leverage a model On the other hand, our gene expression profiling identified whereby pet store mice—which harbor a myriad of mouse genes that were expressed uniquely in IFNλ stimulated pathogens—are co-housed with specific pathogen-free cells. Gene set enrichment analyses suggested that many 46 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 of these genes are involved in pathways consistent with the Johnson, M., Jessberger, N., Robertson, A., Burgio, G., resolution of inflammation and the maintenance of barrier Tscharke, D., Fox, E., Leyton, D., Kaakoush, N., Märtlbauer, function. Many of these pathways, likely regulated E., Leppla, S. and Man, S.M., 2020. Bacillus cereus non- independently from canonical JAK/STAT signaling. Overall, haemolytic enterotoxin activates the NLRP3 inflammasome. we show that IFNα/β and IFNλ work in concert to sustain Nature Communications, 11(1). antiviral immunity and limit tissue damage at sites of Disclosure of Interest: None Declared pathogen exposure. The transient inflammatory responses to IFNα/β help recruit immune effectors to promote LT078 protective immunity while IFNλ act as potent antiviral RATIONAL DESIGN OF ANTISENSE effectors and remodelers of the epithelial barrier. OLIGONUCLEOTIDES MODULATING THE ACTIVITY OF Disclosure of Interest: None Declared TLR7/8 AGONISTS M. P. Gantier1,*, A. Alharbi1 LT076 1Centre for Innate Immunity and Infectious Diseases, NON-HAEMOLYTIC ENTEROTOXIN INDUCES PORE- Hudson Institute of Medical Research, Clayton, Australia FORMATION AND ACTIVATION OF THE INFLAMMASOME AND PYROPTOSIS Abstract Content: Oligonucleotide-based therapeutics D. R. Fox1,*, A. Mathur1, S. M. Man1 have become a reality, and are set to transform 1Department of Immunology and Infectious Disease, The management of many diseases. Nevertheless, the John Curtin School of Medical Research, The Australian modulatory activities of these molecules on immune National University, Canberra, ACT, Australia responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense Abstract Content: Non-haemolytic enterotoxin induces oligonucleotides (ASOs) can have opposing activities on pore-formation and activation of the inflammasome Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent and pyroptosis suppression of TLR7 and activation of TLR8, in a sequence- dependent manner. Through a screen of 192 2′OMe ASOs Daniel Fox, Anukriti Mathur, Si Ming Man and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we Department of Immunology and Infectious Disease, The identified core motifs preventing the immunosuppressive John Curtin School of Medical Research, The Australian activities of 2′OMe ASOs on TLR7. Based on these National University, Australia. observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve Inflammasomes are central to innate immunity mediating TLR7 function, and promote strong activation of phagocytes host defence against pathogens and maintaining and immune cells. We also provide proof-of-principle data homeostasis with commensal microbes. The sensing of that gene-targeting ASOs can be selected to synergize with pathogens, danger, or homeostasis-altering signals by TLR8 agonists currently under investigation as inflammasome sensor proteins is the molecular basis driving immunotherapies, and show that rational ASO selection can assembly of the inflammasome complex. Here, we identify be used to prevent unintended immune suppression of non-haemolytic enterotoxin (NHE) as an activator of the TLR7. Accordingly, we propose that rational selection of NLRP3 inflammasome. We show that NHE, found in the TLR8-potentiating ASOs could present new opportunities in neglected human foodborne pathogen Bacillus cereus, is a the therapeutic development of bifunctional ASOs with non-redundant toxin to haemolysin BL (HBL) despite gene-targeting and immunostimulatory activities. Taken exhibiting a similar mechanism of action. NHE-induced together, our work characterizes the immumodulatory activation of NLRP3 has a delayed kinetic to that induced by effects of ASOs to advance their therapeutic HBL. Subunit C of NHE, via its putative transmembrane development. doi: 10.1093/nar/gkaa523 region, initiates binding to the plasma membrane, leading to Disclosure of Interest: None Declared the recruitment of subunit B and subunit A, forming a tripartite lytic pore permissive to efflux of potassium. NHE LT079 mediates killing of cells from multiple lineages and hosts, MONOCYTE-DERIVED DENDRITIC CELLS FROM highlighting its versatile functional repertoire in different host LEPROSY PATIENTS PRODUCED LOW LEVELS IL- species. Moreover, our data suggest that both NHE and HBL 12P70 AFTER STIMULUS WITH VIABLE operate synergistically to induce inflammation in the host. MYCOBACTERIUM LEPRAE Overall, our results highlight that multiple virulence factors G. V. Germano1,*, B. B. G. Carra1, V. C. Dos Santos2, D. from the same pathogen exhibiting conserved function and Bertoluci3, A. S. A. A. Barbosa3, B. C. Sartori3, P. Rosa3, A. mechanism of action can be exploited for sensing by a single P. Campanelli4, P. K. Das5, V. N. Brito de Souza3 inflammasome, ultimately leading to increased capacity of 1São Paulo State University, Botucatu, 2São Paulo State the host to detect and defend against naturally-occurring University, 3Lauro de Souza Lima Institute, 4Bauru School of genetic variants. Dentistry , São Paulo University, Bauru, Brazil, 5College of Associated Reference: Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom Fox, D*., Mathur, A*., Xue, Y., Liu, Y., Tan, W., Feng, S., Pandey, A., Ngo, C., Hayward, J., Atmosukarto, I., Price, J., 47 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Abstract Content: Leprosy is a chronic infectious disease 1frederick national laboratory for cancer research, Frederick, caused by Mycobacterium leprae, an obligate intracellular United States, 2LHRI, frederick national laboratory for pathogen, with a predilection for Schwann cells in peripheral cancer research, Frederick, United States nerves. In the absence of treatment, nerve injuries bring significant physical disabilities that affect the quality of life. Abstract Content: Interleukin-27 (IL-27) is known as an Tuberculoid leprosy patients present a strong Th1 immune anti-HIV cytokine. We have recently demonstrated that IL- response that controls bacilli multiplication resulting in 27-pretreatment promotes phytohaemagglutinin (PHA)- localized disease while lepromatous patients show a stimulated CD4(+) T cells into HIV-1-resistent cells. In the predominant and ineffective humoral immune response that current study, we investigated profiles of known and favors disease dissemination. Dendritic cells (DCs) play a unknown microRNA in the IL-27-treated CD4(+) T cells and crucial role in the activation of specific immune responses characterized anti-viral effect of microRNAs. by antigen presentation, expression of costimulatory PHA-stimulated CD4(+) T cells were treated with or without molecules and production of cytokines during immunological IL-27 for three days. MicroRNA profiles were analyzed using synapses. Accordingly, the cytokines released by DCs MicroRNA sequencing. To assess anti-HIV effect, T cells or during infection by M. leprae can prevent or favor its survival macrophages were transfected with synthesized microRNA and spread. The aim of this study was to analyze the mimics and then infected with HIVNL4.3 (T cell-tropic HIV) cytokine profile produced by DCs from healthy subjects and or HIVAd8 (Macrophage-tropic HIV). Anti-HIV effect was leprosy patients after infection with viable M. leprae. monitored by a p24 antigen ELISA kit. IFN-a, -b, or -l Peripheral blood mononuclear cells were obtained by production was quantified using each subtype-specific gradient density and submitted to positive selection ELISA kit. employing anti-CD14 microbeads and magnetic columns to A comparative analysis of microRNA profiles indicated that monocytes isolation. Monocytes were cultivated with IL-4 expression of known miRNAs was not significantly changed and GM-CSF for differentiation of DCs. After 6 days, in IL-27-treated cells compared to untreated T cells, immature DCs were infected by live M. leprae obtained from however, a total of 15 novel microRNAs athymic mice at a MOI of 10:1 (bacilli:DC) and cultivated for (miRTC1~miRTC15) were identified .The over-expression 48 hours. A standard maturation cocktail (MC) composed by of each novel microRNA revealed that 10 nM IL-1β, IL-6, TNF, PGE2 was used as a positive control for miRTC14 remarkably suppressed HIV replication by DC maturation. In order to evaluate the capability of IL- 99.9+0.04% (n=5) in macrophages but not in T cells. The 12p70 production, M. leprae-primed DCs were co-cultured inhibition was associated with induction of >1000 pg/mL of with J588 transfected cells expressing CD40L. The multiple subtypes of IFN-a, ~600pg/ml of IFNl1, and ~50 cytokines profile was determined in the supernatant by pg/ml of IFN-b. cytometric bead array. Our results showed that M. leprae As a conclusion, we discovered a total of 15 novel stimulus did not induce an increase in the production of IL- microRNAs in T cells and characterized that miRTC14, one 1β, IL-6, TNF and IL-23, while MC elicit high production of of the novel microRNAs, was a potent IFN-inducing anti-HIV these cytokines, proving the effective potential of activation miRNA, implicating that regulation of the expression of of DCs from both leprosy patients and healthy controls. DCs miRTC14 may be a potent therapeutic tool for not only HIV from lepromatous patients produced a higher amount of IL- but also other IFN-sensitive virus infection. 1β in response to M. leprae in comparison to tuberculoid ones and larger amounts of IL-23 and IL-10 if compared to Disclosure of Interest: None Declared healthy controls. Of special interest, DCs from leprosy patients produced lower levels of IL-12p70 in response to M. LT081b leprae than healthy controls. In conclusion, our results show IL-25 EXACERBATES CHRONIC AIRWAY to M. leprae as a weak inducer of DCs activation and leprosy INFLAMMATION BY PROMOTING PATHOGENIC TH2 patients as unable to produce the cytokine essential to CELLS induce the development of Th1 immune response, the most N. Amatya1,*, G. Qian1, P. J. Bryce1, Y.-H. Wang1 effective against intracellular pathogens. Altogether these 1Immunology and Inflammation, Sanofi US, Cambridge, findings highlight a complex pathogen-host interaction, United States pointing a role for human and bacillary factors in the outcome of leprosy. Supported by: Fundação de Amparo à Abstract Content: Chronic allergic disorders are difficult to Pesquisa do Estado de São Paulo – FAPESP (Grant: treat due to the complex nature of heterogenous cells in the #2015/23256-6). inflammatory tissues after repetitive re-exposures to Disclosure of Interest: None Declared allergens. Pathogenic TH2 cells (peTH2) are a key driver of allergen recall-induced exacerbations of chronic atopic LT080 diseases such as allergic asthma. peTH2 cells are a subset A COMPARATIVE STUDY OF NOVEL MICRORNAS of antigen-specific memory T helper cells that are IDENTIFIED IN INTERLEUKIN-27-INDUCED HIV- phenotypically distinct from conventional TH2 cells. RESISTANT T CELLS Furthermore, peTH2 cells show enhanced Type 2 effector S. Goswami1,2,*, X. HU1, Q. CHEN1, J. Qiu1, J. YANG1, D. functions compared to conventional TH2 cells. However, the POUDYAL1, B. T. SHERMAN1, W. CHANG1, T. Imamichi1 mechanisms underlying the pathogenicity of peTH2 cells are poorly understood. Using mouse model of chronic airway inflammation, we show that IL-25 amplified the generation of 48 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 allergen-induced peTH2 cells and exacerbated pulmonary asthma. ILC2s are able to produce Th2-cytokines rapidly in inflammation. IL-25-induced peTH2 cells were antigen- early immune responses to allergens and non-specific specific and expressed higher levels of IL-17RB (IL-25 stimuli. ILC2s are important for initiation of adaptive 2 receptor) and ST2 (IL-33 receptor) than non-peTH2 cells. In immune responses and regulation of persistent airway normal human PBMCs, the frequency of peTH2 positively inflammation. It was also shown that ILC2s dynamically correlated with the ability of IL-25 to enhance allergen- express the checkpoint inhibitor molecules PD-1 and PD-L1. induced IL-13 production. Furthermore, IL-25 amplified However, it is still largely unexplored how the stronger allergic immune responses in normal human immunophenotypic characteristics of ILC2s change in PBMCs than either IL-33 or TSLP. Together, these findings patients with asthma and if they are clinically relevant. The indicate IL-25 as a critical inducer of peTH2 cells during role of PD-1 and PD-L1 expression on ILC2s seems to be chronic airway inflammation, and may be a therapeutic controversial. According to recent studies PD-1 can be an target for chronic comorbid allergic disorders. important negative regulator of iILC2s, whereas activating Disclosure of Interest: None Declared function of PD-L1 in type 2 immune responses was described . Therefore, it is of the utmost importance to LT081c explore PD-1 and PD-L1 expression on ILC2s in different CCL2-MEDIATED REVERSAL OF IMPAIRED SKIN diseases, particularly T2-driven, e.g. bronchial asthma, WOUND HEALING IN DIABETIC MICE BY allergic rhinitis etc. NORMALIZATION OF NEOVASCULARIZATION Materials and methods: The study included 9 patients (51.1 Y. Ishida1,*, Y. Kuninaka1, M. Nosaka1, M. Akiyama2, N. ± 4.7 years) with a T2-high asthma endotype, whose blood Mukaida2, M. Kawaguchi1, T. Kondo1 samples were obtained from the Clinic of Immunopathology 1WAKAYAMA MEDICAL UNIVERSITY, Wakayama, of RIFCI, Novosibirsk, and 11 healthy donors (44.3 ± 4.7 2Kanazawa University, Kanazawa, Japan years). ILC2s were identified as a Lin-FceRI- CD127+CRTH2+ cell population distinct from human Abstract Content: Patients with diabetes frequently peripheral blood mononuclear cells. We investigated present complications such as impaired skin wound healing. phenotypic characteristics of ILC2s, i.e. expression of PD-1 Skin wound sites display a markedly enhanced expression and PD-L1 on inflammatory (KLRG1+) and natural (KLRG1- of CCL2, a potent macrophage chemoattractant, together ) ILC2 cells . with macrophage infiltration during the early inflammatory Results: Frequencies of inflammatory ILC2s were phase in skin wound sites of healthy normal individuals, but significantly increased in patients with asthma (median it remains elusive on the association of CCL2 with delayed 91,1% (IQR 84,6-93,5)) compared with healthy donors- skin wound healing in diabetic patients. Compared to control 83,3% (71,1-88,7), p=0,04. The percentages of PD- mice, streptozotocin-induced diabetic mice displayed 1+ iILC2s were three times higher in patients- 17,9 % (15,4- impaired healing after excisional skin injury, with decreased 18,0) than in healthy donors- 4,3% (2,2-6,6, neovascularization, CCL2 expression, and macrophage p=0,008. There also was significant decrease of PD-1- infiltration, as compared with control mice. Compromised nILC2s and of PD-L1-nILC2s in patients (4,9% (4,0-7,4); skin wound healing in diabetic mice was reversed by the 6,0% (2,9-7,7) resp.) in comparison with healthy donors administration of topical CCL2 immediately after the injury, (11,5% (9,1-19,5); 13,45% (9,4-22,0) resp.), p=0,006 and as evidenced by normalization of wound closure rates, p=0,003 respectively. neovascularization, and infiltration of macrophages Conclusion: With the present study we observed significant expressing vascular endothelial growth factor, a potent changes of ILC2s phenotypic characteristics in patients with angiogenic factor. Eventually, CCL2 treatment increased the T2-driven bronchial asthma: increase in inflammatory ILC2 accumulation of endothelial progenitor cell at the wound cells and frequencies of PD-1+ iILC2s, significant decrease sites of diabetic mice and eventually accelerated in frequencies of PD1- and PD-L1-nILC2s. It is yet to be neovascularization. Thus, the tropical application of CCL2 clarified if such changes may play a role in therapy success can be an effective therapeutic option for the treatment of and can be used as a diagnostic marker. Further studies are diabetic patients with defective wound repair, promoting required to assess functional characteristics of ILC2s and to neovascularization at skin wound sites. establish the role of PD-1 and PD-L1 expression on Disclosure of Interest: None Declared ILC2s in patients with bronchial asthma and allergy. The reported study was funded by RFBR and Novosibirsk LT081e oblast according to the research project № 19-415-543004. PHENOTYPIC CHARACTERISTICS OF TYPE 2 INNATE Disclosure of Interest: None Declared LYMPHOID CELLS IN PERIPHERAL BLOOD OF PATIENTS WITH T2-DRIVEN BRONCHIAL ASTHMA LT081f COMPARED WITH HEALTHY DONORS NEUTROPHILS PROMOTE ANTIBODY ISOTYPE A. O. Makeeva1, E. A. Pashkina1,*, E. A. Blinova1, D. V. SWITCHING IN RESPONSE TO THE PNEUMOCOCCAL Demina1, V. A. Kozlov1 CONJUGATE VACCINE IN AN INTERFERON GAMMA- 1RIFCI, Novosibirsk, Russian Federation INDEPENDENT MANNER

Abstract Content: Background: Group 2 innate lymphoid E. Y. I. Tchalla1,*, M. Bhalla1, E. A. Wohlfert 1, E. N. Bou cells (ILC2s) are considered to be the most significant Ghanem1 mediators during the orchestration of immune responses in 49 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

1Microbiology and Immunology, University at BUffalo, signals is influenced by the activation state of the APC, Buffalo, United States which is determined by an interplay between signal transduction and metabolic pathways. While TLR ligation Abstract Content: Streptococcus pneumoniae relies on glycolytic metabolism for the expression of (pneumococcus) cause serious infections including inflammatory mediators, less is known about the metabolic pneumonia and invasive pneumococcal disease. dependencies of other signals, such as IFNγ. Using Neutrophils or polymorphonuclear cells (PMNs) are innate CRISPR-Cas9, we performed a series of genome-wide immune cells that are important for initial control of knockout screens in macrophages to identify the regulators pneumococcal infection. However, we recently showed that IFNγ-inducible T cell-stimulatory/inhibitory proteins MHCII, PMNs are also required during immunization with the CD40, and PD-L1. Our multi-screen approach identified new pneumococcal conjugate vaccine (PCV) for subsequent pathways that uniquely associate with each marker. In host protection against infection. Depletion of PMNs in mice addition, meta-analysis implicated complex I of the at the time of vaccination impaired protective antibody mitochondrial respiratory chain in the expression of all three responses, resulting in a decrease in anti-pneumococcal markers, and by extension IFNγ signaling. We report that antibody class switching to IgG2c and IgG3 and reduced the IFNγ response requires mitochondrial respiration and antibody opsonophagocytic activity. However, the APCs are unable to activate T cells upon genetic or chemical mechanism by which PMNs mediate production of inhibition of complex I. Further characterization found that protective antibodies during vaccination is unknown. As complex I loss alters T-cell mediated control of M. interferon gamma (IFN-g) promotes switching to IgG3 and tuberculosis but did not inhibit TLR-mediated inflammation IgG2, we explored its role during vaccination. We first tested or type I IFN responses. These findings suggest a whether PMNs produced IFN-g in response to PCV. We dichotomous metabolic dependency between IFNγ and toll- found no increase in IFN-g production in vaccinated hosts like receptor signaling; implicating mitochondrial function as as compared to naïve controls by mature PMNs in the a fulcrum of innate immunity and central to critical spleen and vaccine draining lymph nodes of mice 18- or 48- interactions between APCs and T cells. hours following vaccination. We also used antibodies to Disclosure of Interest: None Declared block IFN-g at the time of vaccination and assessed antibody production as well as host protection following Lightning Talk Session 2: Autoinflammation pneumococcal lung infection. IFN-g-blocked vaccinated and autoimmunity mice had comparable anti-pneumococcal antibody LT082 production, bacterial burden in the lung and blood as well as INCREASED METFORMIN DOSAGE SUPPRESSES survival as compared to isotype-treated vaccinated controls. PRO-INFLAMMATORY CYTOKINE LEVELS IN These data suggest that IFN-g does not play an important SYSTEMIC CIRCULATION AND MIGHT CONTRIBUTE role in the ability of PMNs to induce protection during TO ITS BENEFICIAL EFFECTS vaccination. However, when we investigated the B. Amoani1,*, S. A. Sakyi2, R. Mantey2, E. F. Laing2, R. E. phenotypes of PMNs, we found that upon vaccination, Dadzie3, O. S. kantanka4, S. Koffie2, E. Obese5,5 PMNs expressed molecules involved in T cell interaction 1Biomedical Sciences, University of Cape Coast, Cape and antigen presentation. As compared to naïve controls, Coast, 2Molecular Medicine, KNUST, Kumasi, 3 Medical the phenotype of circulating PMNs remained unchanged, Laboratory Sciences, University of Cape Coast, Cape however, PMN in the spleen and vaccine draining lymph Coast, 4Department of Medicine, Diabetes Clinic, School of nodes upregulated expression of CD11b, MHC-II, CD80 and Medical Sciences, KNUST, Kumasi, 5Department of CD86 following vaccination. These findings suggest that Pharmacology, School of Medical Sciences, University of PMNs contribute to PCV responses by potentially acting on Cape Coast, Cape Coast, Ghana T cells. Exploring the mechanisms by which PMNs induce protective anti-pneumococcal antibodies is important for Abstract Content: Background: Type 2 diabetes Mellitus designing more effective vaccines against Streptococcus (T2DM) is a metabolic disorder, characterized by persistent pneumoniae. elevation of blood glucose either due to insulin resistance or Disclosure of Interest: None Declared insulin insufficiency. Inflammatory responses plays significant role in the pathogenesis and progression of LT081g T2DM. Cytokines play a pivotal role in modulation of immune FUNCTIONAL GENETIC APPROACHES UNCOVER A reactions and disease pathogenesis. T-helper type 1 (Th1) CRITICAL REQUIREMENT FOR MITOCHONDRIAL and type 17 (Th17) cells, are important pro-inflammatory RESPIRATION FOR ANTIGEN-PRESENTING CELLS TO CD4+ T cell subsets secreting TNF-α, and INF-γ (Th1), and RESPOND TO IFN-GAMMA 1 1 2,* (Th17). These cytokines has been shown to M. Kiritsy , C. Sassetti , A. Olive play a crucial role in inflammation, insulin resistance, and 1University of Massachusetts Medical School, Worcester, 2 development of T2DM. Metformin is the recommended first Michigan State University, East Lansing, United States line of medication for the management of T2DM and is known to improve insulin sensitivity and prevents Abstract Content: The immunological synapse allows hyperglycemia by reducing chronic inflammatory responses. antigen presenting cells (APC) to convey a wide array of Here, we provide the first report on the therapeutic effect of distinct signals to the T cell, which ultimately shape the metformin dosage on pro-inflammatory cytokine levels immune response. The strength of stimulatory/inhibitory among T2DM patients. 50 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Methodology: Two hundred and ninety eligible male and inhibitors from three distinct chemotypes to identify potent female participants admitted to or receiving treatment at the suppressors of this critical cytokine (IC50 < 3μM). diabetic unit of the Komfo Anokye Teaching Hospital (KATH) Compounds were counter-screened for cytotoxicity in the in the Ashanti region of Ghana were recruited in a hospital- myeloma cell line, H929, known to be sensitive to Sec61 based cross-sectional study design. Serum samples inhibition. Activity of potent TNFα translocation inhibitors collected from the participants were analyzed for the with a range of H929 cytotoxicity levels was confirmed in concentrations of TNF-α, INF-γ, GM-CSF and IL-17 cytokine primary cell assays. Following LPS or anti-CD3/anti-CD28 levels by solid phase sandwich ELISA stimulation of isolated human PBMCs or mouse Results: We found that participants on 3000 mg/day dose splenocytes, inhibition of multiple pro-inflammatory of metformin had significantly lower levels of TNF-α cytokines was observed, including GM-CSF, IFNγ, IL-2, IL- (p<0.001) and IFN-γ (p=0.014) compared to the other 6, IL-23, and TNFα, but not IL-1β, which is known to be dosages (1000 mg and 2000 mg/day). However, GM-CSF secreted in a Sec61-independent manner. Cytokine and IL-17 levels were not affected by increased metformin inhibition potencies in primary cells were comparable to or dosages. After adjusting for age, gender, dose and duration higher than those observed for transfected luciferase of metformin use, we observed that participants who took reporter lines of the same cytokines. Of these confirmed higher doses of metformin had significantly reduced levels inhibitors, representatives from each chemotype were of TNF-α (β= -0.0297, 95% CI = (-0.005 to -0.002) p<0.001. selected for inflammatory mouse model testing based on Metformin dosage independently predicted TNF-α levels anti-cytokine potency (IC50 < 100-250nM), low toxicity which explained 14.4% of the variations in the dosage against unstimulated primary immune cells (IC50 > 25μM), levels. and in vitro metabolic stability. Conclusion: Increased metformin dosage suppresses pro- Dose-dependent decreases in serum pro-inflammatory inflammatory cytokine levels in systemic circulation and cytokines were observed following injection of compounds hence might contribute to its beneficial effects. two hours prior to administration of a mitogen to mice. Key words: Type 2 diabetes mellitus, pro-inflammatory Furthermore, dosing in a mouse model of rheumatoid cytokine, metformin dosage arthritis revealed dose-dependent amelioration of disease Disclosure of Interest: None Declared activity at doses <1/8th of the compound MTDs. Paw lysates obtained at the peak of clinical scoring (observed for vehicle- LT083 treated animals) exhibited significant reductions in pro- BLOCKADE OF CYTOKINE PRODUCTION AND inflammatory cytokines and chemokines subsequent to ATTENUATION OF EXPERIMENTAL ARTHRITIS Sec61 inhibitor treatment, including IL-6, KC/GRO, and PROGRESSION BY NOVEL SMALL MOLECULE TNFα. INHIBITORS OF SEC61-DEPENDENT PROTEIN These data support Sec61 as a novel therapeutic target for SECRETION autoimmune disorders via simultaneous suppression of J. L. Anderl1,*, R. A. Fan1, J. Jiang1, H. W. B. Johnson1, A. multiple pro-inflammatory cytokines. We continue to Kanicki1, C. J. Kirk1, E. R. Lewis1, E. Lowe1, D. L. McMinn1, optimize Sec61 inhibitors for development as oral therapies B. Millare1, T. Muchamuel1, M. Rao1, J. Taunton2, C. Tun1, for the treatment of chronic inflammatory conditions. J. Wang1, J. A. Whang1, J. Zhang1, P. Zuno1 Disclosure of Interest: J. Anderl Shareholder of: Kezar Life 1Kezar Life Sciences, South San Francisco, CA, 2University Sciences, Employee of: Kezar Life Sciences, R. A. Fan of California, San Francisco, San Francisco, CA, United Shareholder of: Kezar Life Sciences, Employee of: Kezar States Life Sciences, J. Jiang Shareholder of: Kezar Life Sciences, Employee of: Kezar Life Sciences, H. Johnson Shareholder Abstract Content: The Sec61 membrane protein complex of: Kezar Life Sciences, Employee of: Kezar Life Sciences, gates translocation into the (ER) and A. Kanicki Shareholder of: Kezar Life Sciences, Employee subsequent targeting of nearly all proteins destined for of: Kezar Life Sciences, C. Kirk Shareholder of: Kezar Life secretion or membrane incorporation. Entrance to the ER Sciences, Employee of: Kezar Life Sciences, E. Lewis via Sec61 is negotiated by signal sequences or Shareholder of: Kezar Life Sciences, Employee of: Kezar transmembrane anchors unique to each translating protein. Life Sciences, E. Lowe Shareholder of: Kezar Life Sciences, This interaction can be impeded by small molecule Employee of: Kezar Life Sciences, D. McMinn Shareholder translocation inhibitors. Although biologics targeting of: Kezar Life Sciences, Employee of: Kezar Life Sciences, individual cytokines and their receptors have been highly B. Millare Shareholder of: Kezar Life Sciences, Employee successful in the treatment of inflammatory and autoimmune of: Kezar Life Sciences, T. Muchamuel Shareholder of: diseases, there is increasing interest in the simultaneous Kezar Life Sciences, Employee of: Kezar Life Sciences, M. suppression of multiple targets for improved efficacy. While Rao Shareholder of: Kezar Life Sciences, Employee of: Sec61 inhibitors such as mycolactone reduce cytokine Kezar Life Sciences, J. Taunton Shareholder of: Kezar Life secretion, cytotoxicity associated with their broad Sec61 Sciences, Employee of: University of California, San client specificity has been observed. Here we describe Francisco, C. Tun Employee of: Kezar Life Sciences, J. subset-selective protein secretion inhibitors capable of Wang Shareholder of: Kezar Life Sciences, Employee of: targeting multiple pro-inflammatory cytokines, yet with Kezar Life Sciences, J. Whang Shareholder of: Kezar Life limited cytotoxicity. Sciences, Employee of: Kezar Life Sciences, J. Zhang HEK293 cells stably overexpressing TNFα fused to a Shareholder of: Kezar Life Sciences, Employee of: Kezar luciferase reporter were utilized to screen over 1000 Sec61 51 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Life Sciences, P. Zuno Shareholder of: Kezar Life Sciences, LT087 Employee of: Kezar Life Sciences EBI3 AND IL-1BETA PLAY A MAJOR ROLE IN THROMBOSIS DURING BEHÇET DISEASE BY LT086 REGULATING ACTIVATED PROTEIN C RESISTANCE CHARACTERIZATION OF AUTOIMMUNE OVARIAN H. Belguendouz1,*, N. E. H. Ghozali1,2, Z. Abdesselam3, D. FAILURE UPON REMOVAL OF THE IFN-GAMMA 3’ UTR Messaoudene2, Z. Hadjimi1, M. Terahi4, I. M. Kediha5, K. AU-RICH ELEMENT – A NOVEL MOUSE MODEL. Lahmar1, M. Tazir5, H. Amroun Ait Belkacem3, C. Touil- E. Bafor1,*, M. Hess1, J. Fenimore1, R. Erwin-Cohen1, M. Boukoffa1 Sanford1, J. Valencia1, H. Young1 1LBCM, FSB, USTHB,, algiers, 2Department of biology, 1Laboratory of Cancer Immunometabolism, National Cancer Faculty of sciences, UMBB, Boumerdes, 3Central Institute, Frederick, United States laboratory, 4Department of ophtalmology, CHU Nafissa Hammoud, 5department of neurology, CHU Mustapha Abstract Content: Premature ovarian failure (POF), which Bacha, algiers, Algeria is when the ovaries cease to function, affects approximately Abstract Content: Behcet disease (BD) is a chronic 1-10% of women less than 40 years of age and is associated systemic inflammatory disorder that is considered as a with increased risks of osteoporosis, cardiovascular vasculitis touching both veins and arteries of all sizes. The pathologies, and diminished quality of life. Hence, laboratory underlying mechanism is under investigation with emphasis studies using animal models for the development and on the role of inflammation. In this study, we intended to testing of effective POF therapies are therefore necessary. explore the role of IL-1b with two other proinflammatory Existing drug-induced POF animal models lack some clinical cytokines, IL-32 and EBI3, in the regulation of coagulation features and exhibit variable POF. In this regard, the poor during BD. reproductive capacity observed in mice with homozygous Eighteen patients with BD (6 active and 12 inactive stage) loss of the 3’ untranslated region (3’UTR) AU-rich element and 10 control subjects were enrolled in this study. Freshly (ARE) of the interferon gamma (IFNγ) gene and clinical collected blood on citrated tubes was used in the study. characteristics of systemic lupus erythematosus led us to Hemostasis parameters and factors were assessed in investigate the possibility of autoimmune POF. freshly collected plasma. IL-1b, IL-32 and EBI3 were A day before experiments, mice were estrus synchronized measured by ELISA (Biotechne). Statistical analyses were using urine-infused spent male bedding. On the day of the measured by Mann Witney U test for group comparison experiment, the ovaries, uteri, and spleen (controls) were while Spearman test was used for correlation analyses. collected, weighed, and dissociated. Vaginal epithelial cells We observed a significant increase in EBI3 and IL-32 levels were also collected. Using flow cytometry and during BD in comparison to controls (p<0.05). IL-1b and IL- immunohistochemistry (IHC), the distribution of immune 32 were increased during active stage (p<0.05) but not EBI3 cells in the ovaries, uteri, vaginal epithelial cells (VECs), and (p=0.123). All Hemostasis parameters didn’t show any spleen (controls) were assessed. Histopathology was used differences between patients and control subjects (p>0.05). to assess the morphological presentation of the organs. However, correlation studies showed significant Estrus cycle stages were determined, and organ weights relationships between cytokines and some hemostasis assessed. From the results obtained thus far, flow parameters in patients and not in control subjects. cytometric analysis showed increased CD8+-, CD4+ -T cells, Especially, EBI3 was positively correlated with fibrinogen (r= CD11c+- and F4/80+ cells in the ovaries and uteri of ARE-/- 0.583, p=0.018) and Activated protein C resistance compared to the wildtype (WT) and heterozygous (ARE+/-) (r=0.626, p=0.01). IL-1b was positively associated with mice. MHCII+ increased in the ARE-/- uteri. VECs showed Activated protein C resistance (r=0.782, p=0.013) but decreased neutrophils with a simultaneous increase in negatively correlated with FIX (r= - 0.846, p=0.008). IL-32 eosinophils, macrophages, and MHCII populations in ARE-/- showed no significant correlation with the tested parameters . IHC revealed a dominance of CD8+ and CD4+ T cells in the (p>0.05). ovaries and uteri with infiltration of the zona pellucida and Our results showed that EBI3 and IL-32 were increased in granulosa cells of maturing follicles and corpora lutea while BD in comparison to control subjects while IL-1b and IL-32 sparing the primordial and primary follicles. Histopathology were more associated to disease activity. Even if we didn’t showed atrophied ovaries and uteri in the ARE-/- mice while observe any alteration in the hemostasis in patients with BD, ovary and uterine weights were also decreased, indicative we observed that IL-1b increase is associated the intrinsic of atrophy. The ARE-/- and ARE+/- also showed reduced coagulation pathway suggesting its implication in the susceptibility to estrus synchronization. alteration of endothelial cells. EBI3 was more associated This study has shown that the female ARE-/- mice exhibit with fibrinogen genesis. The two cytokines were associated features consistent with autoimmune ovarian failure with Activated protein C resistance reflecting a state of phenotype with clinical POF features. While further hypercoagulability during Behçet disease. These findings characterization of POF continues, the ARE mouse model highlight the role of inflammatory cytokines, especially IL-1b holds the potential to advance research in the management and EBI3, in the prothrombotic state during BD. of autoimmune POF. Disclosure of Interest: None Declared Disclosure of Interest: None Declared

52 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT088 which type I IFN suppress the effectivity of GC, which could TYPE I INTERFERON INHIBITS GLUCOCORTICOID- be targeted to improve therapeutic efficacy in SLE. INDUCED LEUCINE ZIPPER (GILZ) EXPRESSION AND Disclosure of Interest: None Declared UPREGULATION BY GLUCOCORTICOIDS W. Dankers1,*, M. Northcott1, T. Bennett2, B. Russ2, J. LT089 Flynn1, S. Jones1, E. Morand1 IL-10 PROMOTES REMYELINATION IN AN EX-VIVO 1Rheumatology Research Group, School of Clinical MODEL OF MYELIN DAMAGE Sciences at Monash Health, 2Department of Microbiology, C. Duffy1,*, C. McCoy1 Monash University, Clayton, Australia 1School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin 2, Ireland Abstract Content: Background Glucocorticoids (GC) are broadly used in the treatment of Abstract Content: Multiple sclerosis (MS) is an inflammatory diseases, including systemic lupus autoimmune disease characterized by peripheral immune erythematosus (SLE). Despite their widespread use, most infiltration of the CNS and subsequent demyelination of SLE patients do not reach a state of low disease activity on axons, leading in turn to neuron damage and symptoms in GC treatment. Currently it is not completely understood what patients. This damage can be repaired via a process of factors play a role in the response to this treatment. It is remyelination of previous damaged neurons. The cytokine thought that many anti-inflammatory effects of GC are IL-10 has been shown to promote a micro-environment mediated through upregulation of glucocorticoid-induced conducive to myelin repair in MS, however the underlying leucine zipper (GILZ). GILZ expression is decreased in the mechanism remains poorly understood. Remyelination can blood of SLE patients compared to healthy controls. be modelled ex vivo by application of the demyelinating drug Interestingly, we have previously shown that this is inversely lysolecithin to cultured organotypic cerebellum and brain correlated with the interferon (IFN) signature induced by slice cultures, then applying factors and assessing the type I IFN, including IFNα and IFNβ. Given the important role extent of remyelination via Immunofluorescent microscopy. of type I IFN in SLE pathogenesis, we studied whether IFNα We show brain slice cultures were successfully could suppress GILZ and thereby reduce the effectiveness demyelinated using lysolecithin and subsequently of GC. remyelination was observed ex vivo. Demyelinated slices Methods were then treated LPS, IL-10 and a combination of both, and Human peripheral blood mononuclear cells (PBMC) were we observe a trend towards greater remyelination in brain isolated from healthy individuals and treated with 1000 IU slices treated with the combination of both stimuli. Our lab IFNα2a, 100 nM dexamethasone (DEX) or both, with or has previously identified Arginase 2 as a downstream without the Jak1/Tyk2 inhibitor tosylate salt (TS). GILZ immunometabolic target of IL-10. In brain slices obtained expression in these cultures was analysed using RT-PCR. from an Arg2 knockout strain, this observed LPS+IL-10 STAT1 binding sites were analyzed from public datasets effect is lost. Arg2 is also a target of miR-155, and in brain and using chromatin immunoprecipitation followed by RT- slices lacking miR-155, remyelination is enhanced PCR. Public datasets were also used to study the effect of compared with controls, suggesting a mechanism via which IFNα and the interplay between GC and IFNα on GILZ miR-155 may mediate the observed LPS + IL-10 effect via expression in SLE patients. Arg2. Results Disclosure of Interest: None Declared IFNα treatment reduces the expression of GILZ in human PBMC in a dose- and time-dependent manner. Interestingly, LT090 it also reduces the DEX-induced upregulation of GILZ. This THE ASSOCIATION BETWEEN TMAO AND corresponds to data in SLE patients, where GC treatment is NEIGHBORHOOD DEPRIVATION IS MEDIATED BY less effective at inducing GILZ in patients with a high IFN PRO-INFLAMMATORY CYTOKINES AMONG AFRICAN- score than in patients with a low IFN score. Mechanistically, AMERICANS AT RISK FOR CARDIOVASCULAR we found that IFNα2a reduces GILZ expression via the DISEASE. Jak1/Tyk2 signalling pathway, as treatment with the specific N. Farmer1,*, C. Gutierrez-Huerta2, Y. Baumer2, G. R. inhibitor TS reversed the effects of IFNα2a. In public Wallen1, T. Powell-Wiley2 datasets, and then confirmed via ChIP, we subsequently 1National Institutes of Health Clinical Center, 2National Heart found that the transcription factor STAT1, downstream of Lung and Blood Institute , Bethesda, United States Jak1/Tyk2, has multiple DNA binding sites surrounding the GILZ locus. These STAT1 binding sites coincide with Abstract Content: Trimethylamine N-oxide (TMAO), is binding sites of the glucocorticoid receptor (GR), which may identified as a biomarker for atherosclerosis. Likely explain the mechanism by which IFNα2a reduces the DEX- mechanisms for TMAO and atherosclerosis include the induced GILZ upregulation. promotion of pro-inflammatory cytokines such as Il-1β and Conclusion TNF-α from in vitro studies. The role of TMAO in In human PBMC, IFNα2a reduces GILZ expression and the cardiovascular (CVD) risk among resource limited DEX-induced upregulation of GILZ via the Jak1/Tyk2 communities that are most impacted by neighborhood risk signalling pathway and direct DNA binding of STAT1 to the factors, such as limited food access or exposure to GILZ locus. These data reveal a potential mechanism by pollutants has not been fully investigated. We therefore sought to examine the relationship of TMAO at both an 53 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 individual and neighborhood level and to evaluate a ARE mice had increased cellular infiltrates into heart tissue mediating role for pro-inflammatory cytokines. and mitochondria linked metabolic changes that were Plasma samples were utilized from African-American study concomitantly observed following a stress- exacerbated participants who lived in neighborhoods surrounding and decrease in heart function. Our data also demonstrate an including Washington, D.C. TMAO plasma levels were increase in glucose expenditure and lower general mobility determined by ELISA (Biohippo). Cytokine levels were of ARE mice as well as structural and ultrastructural determined by the ELISA-based technique, U-Plex changes in the heart. We also observe a decrease in the (MesoScale). Clinical CVD risk factor data included BMI, transcripts and function of aerobic respiratory components cholesterol levels and ASCVD risk score calculation. in the muscle of male mice. Notable, we observe a decrease Demographic data collected included addresses, which in X linked pyruvate dehydrogenase, a key allowed for the calculation of the census linked variable, component in pyruvate’s entry to the TCA cycle, and other neighborhood deprivation index (NDI). Associations downstream mitochondrial genes. Furthermore, the in vivo between variables were determined by bivariate and data revealed an increase in factors associated with fatigue, multivariate ANOVA using STATA. Mediation analysis was such as lactic acid production and changes in expression of also conducted using STATA. genes associated with an increase in anaerobic respiration Results from 60 African-American individuals (female 93%, in cardiac musculature. These results indicate that the median age 60.8, median BMI = 33.0) showed a mean chronic expression of IFN-g results in a model for male TMAO level of 37.54 ng/ml. Bivariate analysis showed a biased heart failure under stress in an autoimmune significant association between NDI and TMAO, after environment, similar to the idiopathic cardiomyopathy found adjusting for BMI and ASCVD risk score (β= 0.314, in male Systemic Lupus Erythematosus patients. p=0.017). There were significant associations between NDI Considering that fatigue is a symptom often associated with and pro-inflammatory cytokines TNF-α (β=0.485, p<0.001) autoimmune diseases, we hypothesize that cytokine-driven and IL-1β (β=0.434, p<0.001). As well as with TMAO and changes in cardiac musculature may play a critical role in TNF-α (β=0.368, p=0.004) and IL-1 β (β=0.222, fatigue-related disease associated with autoimmune p=0.053). Together both TNF-α and IL-1β explained 61.7% disorders. This work provides new insights into the diverse of the relationship between NDI and TMAO. Interestingly, actions of IFN-g in fatigue and cardiomyopathy. other pro-inflammatory cytokines, such as IL-6 did not Disclosure of Interest: None Declared contribute to the mediation model (p=0.895). In conclusion, by using cross-sectional data available from LT092 African-American adults with CVD risk factors living in PATHOGENIC, GLYCOLYTIC PD-1+ B CELLS variable neighborhood environments, we found an ACCUMULATE IN THE HYPOXIC RA JOINT. association between plasma TMAO levels and A. Floudas1,*, N. Neto2, M. Vivianna1, K. Murray3, B. Moran4, neighborhood deprivation and that the relationship was M. Monaghan2, L. Candice3, R. H. Mullan5, V. Krishna6, S. mediated by cytokines, IL-1β and TNF-α. Findings from this Nagpal6, D. J. Veale3, U. Fearon7 study may serve to identify biological mechanisms related to 1Molecular Rheumatology, Trinity Biomedical Sciences neighborhood deprivation and CVD risk, but also serve to Institu, 2Department of Mechanical and Manufacturing provide insight on TMAO, a previously unidentified Engineering, Trinity Biomedical Sciences Institute, 3EULAR biomarker for adverse health outcomes at the neighborhood Centre of Excellence, Centre for Arthritis and Rheumatic level. Diseases, UCD, 4Trinity Biomedical Sciences Institute, Disclosure of Interest: None Declared Dublin, Ireland, 5Dept. of Rheumatology, Trinity Biomedical Sciences Institute, Dublin, Ireland, 6Janssen, Spring House, LT091 United States, 7Molecular Rheumatology, Trinity Biomedical METABOLIC AND STRUCTURAL MUSCULAR Sciences Institute, Dublin, Ireland CHANGES DRIVEN BY CHRONIC DISEASE-LIKE IFN-G EXPOSURE Abstract Content: Background: J. Fenimore1,*, D. Springer2, J. Valencia1, E. Edmonston1, R. Rheumatoid arthritis (RA) often has a progressive and Erwin-Cohen1, H. Young1 debilitating course, with significant impact on the patient’s 1LCIM, NIH, Frederick, 2NIH, Bethesda, United States quality of life. Despite the long-known association with autoantibodies, knowledge of the role of B cells and their Abstract Content: The effect of cytokines on non-traditional potential direct contribution to RA pathogenesis remains immunological targets under conditions of chronic elusive. inflammation have been fruitful at explaining many Methods: comorbidities in disease. Cardiovascular myopathies and Synovial tissue biopsies from RA patients were obtained fatigue have been associated with chronic inflammatory through key-hole arthroscopy followed by single cell flow responses and activated cell-mediated immunity. We report cytometric analysis of B cell subpopulations and chemokine aberrant cardiovascular activity in the form of reduced receptor expression. SPICE analysis was performed for the ejection fraction in vivo in male mice with persistent low-level chemokine receptor expression of synovial B cells and B cell expression of IFN-g (ARE mice). ARE mice have a deletion invasion assays. Functional characterization of sorted RA B and replacement with random in the 3’ UTR of cells, cultured in vitro under hypoxia, simulating the inflamed the IFN-g gene that serves to remove a regulatory element joint environment. Characterization of B cell metabolism and that is integral at controlling the stability of IFN-g mRNA. transcriptional regulation of pSTAT3 by flow cytometry. 54 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Novel, non-invasive Fluorescent Lifetime Imaging development of lymphocyte rich BALTs in the lung as early Microscopy (FLIM) assay for the characterisation of the as 6 weeks of age and develops progressive fibrotic disease metabolic status of sorted RA patient PD-1 B cells coupled by 16 weeks. with RNAseq analysis was performed. To test the requirement of lymphocytes in SAVI disease, we Results: bred VM Rag1-/- mice and found they were protected from Extensive flow-cytometric characterisation and SPICE lung and systemic inflammation, as indicated by histology, algorithm analyses of single-cell synovial tissue from RA- splenomegaly, and body weight. To define which patients revealed the accumulation of switched and double lymphocyte subsets were required for disease, we negative memory PD-1 expressing B cells at the site of genetically and pharmacologically ablated T and B cells inflammation. Accumulation of memory B cells is mediated respectively using VM TCRβ-/- mice or VM mice treated with by CXCR3, evident by the observed increase in CXCR3 αCD20 antibodies. Systemic inflammatory disease expressing synovial B cells compared to the periphery, persisted in both cohorts, suggesting neither αβ T cells nor differential regulation by key synovial cytokines, and B cells were required for disease. Additionally, lung disease restricted B cell invasion demonstrated in response to persisted in VM TCRβ-/- mice, and it has been reported prior -/- CXCR3 blockade. Notably, under 3% O2 hypoxic conditions that lung disease persists in B cell deficient µMT STING that mimic the joint-microenvironment, RA B cells maintain N153S SAVI mice. These results support the notion that T marked expression of MMP-9,-TNF and IL-6, with PD-1+ B- and B cells could independently contribute to SAVI lung cells demonstrating higher expression of CXCR3, CD80, disease. Moreover, we found that VM splenic αβ T and B CD86, IL-1β and GMCSF than their PD-1- counterparts. cells depended on each other for maximal hyperactivation, Following functional analysis and flow cell sorting of RA PD- suggesting a potential synergistic interaction as well. 1+ vs PD-1- B-cells, we demonstrate using RNAseq and Next, we generated chimeric mice by transferring donor VM novel FLIM visualisation of cellular NADH, a significant shift bone marrow (BM) into irradiated WT mice to test if immune in metabolism of RA PD-1+ B-cells towards glycolysis, abnormalities were due to hematopoietic intrinsic STING associated with an increased transcriptional signature of key Gain-of-Function (GOF). Although BM intrinsic STING GOF cytokines and chemokines that are strongly implicated in RA was sufficient to drive lymphopenia, it was not sufficient for pathogenesis. lymphocyte activation. To test if lymphocyte activation could Conclusions: instead be driven by extrinsic STING GOF in radioresistant The accumulation of pro-inflammatory PD-1 B cells in the cells, we transferred WT BM into irradiated VM recipients synovium of RA patients, is CXCR3 mediated and offers an and found WT splenic T cells developed hyperactivation that opportunity for early therapeutic intervention. Once in the phenocopied VM mice. WT B cells in these chimeras also hypoxic environment of the inflamed RA joint, PD-1 B cells became activated, although to a lesser degree. Importantly, show altered activation, pro-inflammatory cytokine progressive lung disease persisted in these chimeric mice, production and metabolism that could prove important for indicating radio-resistant cell STING GOF was sufficient to understanding the role of B cells in RA pathogenesis. perpetuate SAVI inflammatory lung fibrosis. Disclosure of Interest: None Declared Together, these findings suggest that STING GOF in radioresistant cells organizes and activates a profibrotic LT093 inflammatory lymphocyte response in the lung. We are STING GAIN-OF-FUNCTION IN RADIORESISTANT currently investigating candidate radioresistant cell CELLS SUPPORTS A LYMPHOCYTE DEPENDENT populations as initiators of this process including AUTO-INFLAMMATORY LUNG DISEASE endothelium and lung epithelium, as well as mechanisms by K. M. Gao1,2,*, M. Motwani1, A. Marshak-Rothstein1,2, K. A. which lymphocytes drive SAVI lung pathology. These Fitzgerald1 findings are clinically relevant for SAVI patients, as well as 1Program in Innate Immunity, Division of Infectious more broadly for patients suffering from interstitial lung Diseases, 2Division of Rheumatology, Department of disease associated with rheumatic disease and suggest that Medicine, University Of Massachusetts Medical School, lymphocyte depleting therapies may mitigate further lung Worcester, United States injury and worsening of fibrosis. Disclosure of Interest: None Declared Abstract Content: cGAS-STING is a cytosolic dsDNA sensing pathway whose regulation is vital to immune LT094 homeostasis. Pediatric patients with constitutively active ASTROCYTE, BUT NOT MICROGLIA STING mutations develop an autoinflammatory syndrome UNRESPONSIVENESS TO TYPE I INTERFERON IS known as STING Associated Vasculopathy with onset in PROTECTIVE IN A MOUSE MODEL OF CEREBRAL Infancy (SAVI) and suffer from treatment resistant lung TYPE I INTERFERONOPATHIES fibrosis. Interestingly, SAVI patients develop immune E. Hayashida1,*, B. Viengkhou1, M. J. Hofer1 abnormalities including lymphopenia concomitant with 1School of Life and Environmental Sciences, Marie Bashir hyperactivation, and lung biopsies from SAVI patients show Institute for Infectious Diseases and Biosecurity, Charles lymphocyte predominant immune aggregates which strongly Perkins Centre and Bosch Institute, The University of resemble bronchus associated lymphoid tissues (BALT), Sydney, Sydney, Australia suggesting a role for lymphocytes in SAVI lung disease. We have genetically engineered a mouse model of SAVI Abstract Content: Cerebral type I interferonopathies are an (STING V154M or VM mice) that recapitulates the expanding group of neuroinflammatory and 55 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 neurodegenerative disorders associated with the signaling. IL-38 polymorphisms are associated with overproduction of interferon-α (IFN-α) in the central nervous increased susceptibility for auto-inflammatory diseases such system (CNS). Aicardi-Goutières Syndrome (AGS) is a as psoriasis, spondyloarthritis and rheumatoid arthritis, but monogenic disorder that is considered the prototypical its role in IL-17-driven inflammation in vivo remained largely cerebral type I interferonopathy. Clinically, patients with obscure. AGS suffer cognitive deficits and motor dysfunction, as well Methods: The experimental autoimmune encephalomyelitis as other pathological features such as leukocytosis, (EAE) mouse model was used as an IL-17-dependent model intracerebral calcifications and chronic encephalitis. Other of autoimmune disease of the central nervous system, which cerebral type I interferonopathies include autoimmune closely resembles human multiple sclerosis. EAE was disorders such as CNS-lupus and congenital and chronic induced in WT and IL-38 KO mice by immunization with viral infections. Currently, there is no cure for these MOG35-55 peptide emulsified with Complete Freund’s disorders. Adjuvant (CFA), followed by intraperitoneal administration of pertussis toxin (PTX) 2 h and 22 h after immunization. Mice In the CNS, astrocytes and microglia are key effectors of were examined daily for body weight and clinical signs of innate immune defenses against infection and injury and EAE using clinical scores that follow different degrees of play important roles in the maintenance of a normal, paralysis. Immune cell composition and cytokine profiles of homeostatic environment. However, recent evidence has the spinal cord and blood were analyzed by flow cytometry. implicated both astrocytes and microglia in the pathogenesis IL-38 and pro-inflammatory mRNA expression levels were of neuroinflammation and neurodegeneration. Transgenic evaluated by quantitative PCR and RNAScope. Spinal cord mice with CNS-restricted chronic production of IFN-α histology was performed by PhenOptics. Statistical (termed GIFN mice) recapitulate many of the hallmark comparisons between two groups were performed using features seen in human patients with cerebral type I Mann Whitney test, unpaired two-tailed Student’s t test. interferonopathies. To determine the contribution of One- or two-way analysis of variance (ANOVA) followed by astrocytes and microglia in the pathogenesis of this disease, appropriate post-tests. we generated novel triple transgenic GIFN mouse lines in Results: IL-38 was shown to be expressed in the spinal cord which either astrocytes or microglia are unresponsive to and its absence in IL-38 KO mice, against expectations IFN-I, using Cre/lox-mediated recombination of the Ifnar1 strongly reduced clinical scores in the EAE model indicating gene. attenuated disease development. This finding was substantiated by a better spinal cord structure in IL-38 KO Astrocyte-specific deletion of IFNAR in GIFN mice delayed mice, including increased number of neurons and reduced onset and progression of disease and improved survival myelin deposition, as well as less immune cell infiltration. compared to GIFN mice. However, no improvement was Indeed, we observed less macrophages, monocytes, seen in GIFN mice with microglia-specific deletion of IFNAR. neutrophils and T helper cells in the spinal cord of IL-38 KO Despite these differences, both triple transgenic mice mice compared to WT mice, while the percentage of these showed protection from CNS pathology including immune cell populations tended to be higher in the blood. neurodegeneration, calcifications, aneurysms and Moreover, IL-38 KO mice showed lower mRNA expression microgliosis observed in GIFN mice. These findings indicate of inflammatory markers in the spinal cord, as well as lower that while microglia responses to IFN-α appear not to concentration of TNFα at protein level. Alteration of the contribute to disease pathogenesis in cerebral type I macrophage number and macrophage-related gene interferonopathies, astrocytes may play a greater role in the expression in the spinal cord of IL-38 KO mice was related pathogenesis of disease. This is a key step for the to the absence of IL-38 in a cell-intrinsic manner. Indeed, development of cell-specific, targeted therapies. classically activated IL-38 KO macrophages expressed Disclosure of Interest: None Declared lower levels of inflammatory genes such as TNFα or IL-23, compared to WT macrophages. LT095 Conclusion: Unlike other auto-inflammation models, IL-38 ABLATION REDUCES INFLAMMATION AND ablation of IL-38 dramatically reduced disease severity in DISEASE SEVERITY IN EXPERIMENTAL AUTOIMMUNE the EAE model. This appeared to be due to IL-38 expression ENCEPHALOMYELITIS by macrophage themselves, as opposed to IL-38 by other A. Huard1,*, H. N. Do1, B. Brüne1, N. Debruin2, A. Weigert1 sources acting on macrophage, which usually dampens 1Institute of Biochemistry I, 2Branch for Translational inflammation. suggest IL-38 as a potential target for CNS Medicine and Pharmacology TMP, Fraunhofer Institute for autoimmune diseases such as multiple sclerosis or Molecular Biology and Applied Ecology IME, Frankfurt am autoimmune encephalopathies. Main, Germany Disclosure of Interest: None Declared

Abstract Content: Introduction: Interleukin-38 (IL-38) is a LT096 IL-1 family member that shares homology with both IL-1Ra B LYMPHOCYTE-DERIVED CCL7 AUGMENTS and IL-36Ra, and is therefore proposed as a negative NEUTROPHIL AND MONOCYTE RECRUITMENT, regulator of inflammation. IL-38 is known to be released from EXACERBATING ACUTE KIDNEY INJURY apoptotic cells to limit inflammatory macrophage and A. Inaba1,*, Z. K. Tuong1, A. M. Riding1, R. J. Mathews1, J. downstream T lymphocyte IL-17 production by blocking X- L. Martin2, K. Saeb-Parsy2, M. R. Clatworthy1 linked IL-1 receptor accessory protein-like 1 (IL1RAPL1) 56 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

1Medicine, 2Surgery, University of Cambridge, Cambridge, SA-IL-4 decreased integrin expression in antigen-reactive United Kingdom CD4+ T cells, indicating impaired cell migration capability. SA-IL-4 increased the number of and programmed death Abstract Content: Acute kidney injury (AKI) is a serious ligand-1 expression on granulocyte-like myeloid-derived condition affecting a fifth of hospital in-patients. B suppressor cells, a key EAE disease suppressor, in the lymphocytes have immunological functions beyond antibody spinal cord-draining LN (dLN). SA-IL-4 decreased the production and may produce cytokines and chemokines number of Th17 cells, a pathogenic cell population for EAE which modulate inflammation. Here we investigated disease. In the chronic phase of EAE, SA-IL-4 also showed leucocyte responses in a mouse models of AKI and marked therapeutic effects, accompanied by inhibition of observed an increase in circulating and kidney B cells, immune cell infiltration into the spinal cord and complete particularly a B220low subset, following AKI. We found that B abrogation of immune response to the myelin antigen in the cells produce the chemokine CCL7, with the potential to spleen. Engineered SA-IL-4 demonstrate translational facilitate neutrophil and monocyte recruitment to the injured promise for MS as both a preventative and therapeutic kidney. Siglec-G-deficient mice, which have increased treatment via accumulation in SLOs. numbers of B220low innate B cells and a lower B cell To extend our study, we also made SA fusion to IL-10, an activation threshold, had increased Ccl7 transcripts, immunosuppressive cytokine. Rheumatoid arthritis (RA) is a neutrophil and monocyte numbers in the kidney and more major autoimmune disease that causes synovitis and joint severe AKI. CCL7 blockade in mice reduced myeloid cell damage. Although clinical trials using IL-10 have been infiltration into the kidney and ameliorated AKI. In two performed as a potential treatment of RA, its therapeutic independent cohorts of human patients with AKI, we effects have been limited. After intravenous injection to observed significantly higher CCL7 transcripts compared to mice, SA fusion to IL-10 led to enhanced LN accumulation controls, and in a third cohort, an increase in urinary CCL7 compared with unmodified IL-10. Intravenous SA-IL-10 levels in AKI, supporting the clinical importance of this treatment restored immune cell composition in the paws to pathway. Together our data suggest that B cells contribute a normal status, elevated the frequency of suppressive M2 to early sterile inflammation in AKI via the production of macrophages, reduced IL-17A amount in the paw- draining leucocyte-recruiting chemokines. LN, and protected joint morphology. Intravenous SA-IL-10 Disclosure of Interest: None Declared treatment showed similar efficacy as treatment with an anti- TNF-a antibody. SA-IL-10 was equally effective when LT097 administered intravenously, locally or subcutaneously, PROLONGED RESIDENCE OF ALBUMIN-FUSED ANTI- which benefits clinical translation of this molecule. SA fusion INFLAMMATORY CYTOKINES IN THE SECONDARY to IL-10 is a simple but effective engineering strategy for RA LYMPHOID ORGANS AMELIORATES EXPERIMENTAL therapy and holds clinical translational potential. Overall, we AUTOIMMUNE ENCEPHALOMYELITIS AND are the first to show that SA fusion to the RHEUMATOID ARTHRITIS immunosuppressive cytokines would increase the residence J. Ishihara1,*, A. Ishihara1, E. Yuba1, E. Budina1, A. in the SLO, and strongly suppresses autoimmune diseases Mansurov1, A. T. Alpar1, J. Hubbell1 symptoms through decreasing pathogenic cell population. 1Pritzker school of molecular engineering, University of Disclosure of Interest: None Declared Chicago, chicago, United States LT098 Abstract Content: Multiple sclerosis (MS) is a common and LUPUS-LIKE DISEASE IN B6.NBA2 MICE SHOWS A severe demyelinating autoimmune disease of the central BIPHASIC PROGRESSION nervous system. Although interleukin (IL)-4, an E. J. Keller1,2,*, T. Jorgensen1,2, N. Patel1,3, N. Campbell1, L. immunosuppressive cytokine, suppresses development of Davison1,2,4 pathology in a murine MS model, experimental autoimmune 1Inflammation and Immunity, Lerner Research Institute, encephalomyelitis (EAE), IL-4 has not been clinically 2Molecular Medicine, 3Case Western Reserve University, translated to the clinic. We hypothesized that the poor Cleveland, 4Teneobio, Newark, United States efficacy of recombinant IL-4 administration is due to insufficient residence of IL-4 in the secondary lymphoid Abstract Content: Lupus is an autoimmune disease with a organs (SLOs), because antigen-specific T cell priming and diverse pattern of symptoms affecting approximately 1.5 Th17 cell development primarily occur in the SLO. We have million Americans. Over the past decade, new biologics engineered a fusion protein of serum albumin (SA) and IL-4 (f.ex. Belimumab, Anifrolumab) have emerged targeting (SA-IL-4) to increase the persistence of IL-4 in the SLOs. cytokines and/or cells known to be pathogenic in lupus, SA-IL-4 showed greater accumulation and residence time in however, response-rates among varied patient populations lymph nodes (LNs) and spleen compared to wild-type (wt) remain, low supporting the heterogeneity of the disease. IL-4, which was dependent on neonatal Fc receptor (FcRn) B6.Nba2 mice exhibit a spontaneously occurring constitutive binding. Administration of SA-IL-4 prevented EAE disease progressive lupus-like disease pattern driven by type I development in all mice and demonstrated higher interferons (IFNα). Using this model, ablation of therapeutic efficacy compared to FTY720, a clinically used plasmacytoid dendritic cells (pDCs) is effective if initiated drug (fingolimod), and wt IL-4. SA-IL-4 prevented immune before, but not after, 4 months of age, suggesting a biphasic cell infiltration into the spinal cord, facilitating maintenance disease pattern with pDCs/IFNα playing a role during the of spinal cord structure and resulting neurological function. early phase only. To investigate the second phase, we 57 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 compared disease manifestations in 4 and 9 month old higher BCL6, IL21 and IL6 expression. In conclusion, B6.Nba2 mice. As expected, IFNα-induced gene transcripts perturbed signalling pathways in GPA naïve CD4 T cells in diminished after 4 months of age as did levels of IL-6; response to T cell activation result in decreased STAT5 another product of pDCs, while we saw no changes in IL-21 activation versus STAT3 activation, and lead to aberrant levels. In contrast, splenic BAFF levels increased over time BCL6 expression, promoting enhanced TFH differentiation. both at the transcriptional and protein level as did levels of Strategies targeting BCL6 expression and/or IL6/STAT3 Irf4 and Bcl6 transcripts, supporting an upregulation in activation may have therapeutic benefit in the treatment of specific B cell subsets including germinal center B cells and GPA/ANCA associated vasculitis. plasma cells in aging mice. These data indicates that pDCs Disclosure of Interest: None Declared and their products, IFNα and IL-6, play a critical role in early lupus like disease progression, while BAFF and B cells are LT100 involved in the later stage of the disease progression. Thus, TYPE III INTERFERON LIMITS RECOVERY AND treatment with different biologics may be needed at different PROMOTES AXONAL INJURY DURING AUTOIMMUNE time points of disease. We suggest that investigating NEUROINFLAMMATION patterns of gene expression combined with obtaining an S. Manivasagam1,*, J. L. Williams2, L. L. Vollmer1, A. inclusive patient history is necessary to determine the Archambault1, G. F. Wu1, R. S. Klein1 treatment approach needed for each individual lupus 1Washington University in St. Louis, St. Louis, 2Cleveland patient. Clinic, Cleveland, United States Disclosure of Interest: None Declared Abstract Content: Multiple sclerosis (MS) is a chronic LT099 autoimmune disease characterized by pathologic infiltration IMPAIRED IL2/STAT5 SIGNALLING IN NAÏVE CD4 T of lymphocytes and macrophages into the central nervous CELLS PROMOTES TFH DIFFERENTIATION IN system (CNS) that leads to demyelination and axonal injury. GRANULOMATOSIS WITH POLYANGIITIS PATIENTS Extent of axonal injury is strongly correlated with disease progression and permanent disability in MS patients. S. Kim1,*, L. Boehme1, L. Nel2, A. Casian2, E. Nova- Currently available treatments for MS have varying efficacy, Lamperti3, J. Spencer1, P. Lavender1, D. D’Cruz1, S. John1 do not impact transition to progressive disease, or promote 1King's College London, 2Guy’s and St. Thomas’ NHS Trust, axonal recovery. Here, we define a role for type III interferon london, United Kingdom, 3University of Concepcion, (interferon lambda or IFNλ) in the maintenance and Concepción, Chile progression of CNS autoimmune diseases. Interferons (IFN) are a class of cytokines that play a role in Abstract Content: Granulomatosis with Polyangiitis (GPA) antiviral immunity, as well as in immune-mediated diseases. is a systemic autoimmune disease, classified as an anti- In particular, type I IFN has been highly studied and neutrophil cytoplasmic antibodies (ANCA) associated implicated in CNS autoimmune diseases, such as MS; vasculitis. Accompanying elevated levels of ANCA in however, less is known about the closely related type III IFN patient’s blood, an imbalance in circulating functional (IFNλ) in this context. IFNλ is most known for its antiviral effector CD4 T cell subsets is strongly associated with GPA properties at mucosal barriers, promotion of Th1 adaptive pathogenesis. Recent studies have observed a suppression immune responses, and ability to alter blood-brain barrier of regulatory T cell (Treg) function and an increase in permeability. Based on these characteristics, we follicular helper T (FH) / Th17 cells in GPA. However, the hypothesized that IFNλ’s immunomodulatory properties may mechanism(s) responsible for this imbalance in GPA remain alter immune-mediated inflammation during CNS unknown. In this study, we hypothesise that transcriptomic autoimmune disease. changes may occur in naïve CD4 T cells in GPA that lead to We found that IFNλ signaling impacts recovery in mice with an imbalanced effector CD4 T cell population. Bioinformatic experimental autoimmune encephalomyelitis (EAE), a well- analysis of microarray data revealed a dysregulation of established murine model of CNS autoimmunity. Genetic IL2Rβ/JAK-STAT signalling pathway, and higher expression deletion of the IFNλ receptor (Ifnrl1) and antibody mediated of BCL6 (a key TFH transcription factor) and BCL6-related neutralization of IFNλ both result in clinical recovery during genes in GPA naïve CD4 T cells compared to healthy EAE. Recovery was linked to resolution of CNS controls (HC). In naïve CD4 T cells pre-activated with anti- inflammation, with animals deficient in IFNλ signaling CD3 and anti-CD28 antibodies, IL2 induced STAT5 exhibiting fewer infiltrating, cytokine-producing lymphocytes activation (which suppresses BCL6 expression) was and decreased microglial activation compared to wildtype significantly reduced in GPA compared to HC as measured animals. Deletion of Ifnlr1 also protected animals from by western blot analysis. In contrast to STAT5 activation, no axonal injury of the spinal cord, while surprisingly having no differences in STAT3 activation (which upregulates BCL6 effect on demyelination or remyelination. Cell specific expression) by IL6 and IL2 were observed. In vitro CD4 T deletion of Ifnlr1 revealed CD11c+ antigen presenting cells, cell differentiation assays showed that BCL6 is highly critical for T cell reactivation, to be targets of IFNλ during expressed in response to T cell activation in GPA naïve CD4 EAE. Finally, in human tissue, we found increased levels of T cells. Moreover, under conditions designed to skew CD4 IFNλ in lesions of secondary progressive MS patients T cell differentiation towards a TFH phenotype, GPA naïve compared to relapsing remitting MS patients. These data CD4 T cells exhibited a more pronounced increase in TFH reveal a novel role for IFNλ in maintaining chronic characteristics; higher CXCR5+ PD1+ frequencies, and 58 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 neuroinflammation and as a potential therapeutic target in MS patients. Abstract Content: Multiple Sclerosis (MS) is a chronic Funded by: NIH grant F31 NS108629-01A1 (SM) and NMSS demyelinating autoimmune disease characterised by central Research Grant (RSK) nervous system (CNS) infiltration of peripherally derived Disclosure of Interest: None Declared immune cells, the largest fraction of which are macrophages. The macrophage role in MS is multifaceted, LT101 in a pro-inflammatory or ‘M1’ state instructing demyelination DETERMINING HNRNP-A2/B1’S ROLE IN and axonal loss, while the anti-inflammatory ‘M2’ state holds CONTRIBUTING TO INFLAMMATION IN RHEUMATOID a key role in promoting tissue repair and regeneration. There ARTHRITIS is considerable interest in elucidating and manipulating M. Mohammed Salih1,* factors that mediate switching between these phenotypes. 1Molecular and Cell Developmental Biology, UC Santa Cruz, Previously, we have identified that IL-10 inhibition of miR- Santa Cruz, United States 155 is a prominent mechanism utilised by macrophages to maintain an M2 state. Moreover, using a miR-155 floxed x Abstract Content: The innate immune system is the first LysMCre model, where miR-155 is specifically deleted from line of defense against pathogens; it functions through myeloid cells, there was reduced disease onset and less various pattern recognition receptors (PRRs) that recognize lesion burden in the experimental autoimmune microbial products or danger signals leading to the encephalomyelitis (EAE) animal model. activation of signaling pathways which initiate transcription Therefore, we hypothesise miR-155 inhibition may of inflammatory genes. Activation of the innate immune favourably modulate the macrophage population to an ‘M2’ response is essential to resolve infections, however, its or pro-repair phenotype, reducing inflammation, alleviating dysregulation can result in pathological inflammation, disease progression, mimicking an IL-10 mediated effect. contributing to an array of diseases, such as Thus, the objective of this study is to investigate the atherosclerosis, autoimmunity and cancer. HnRNP-A2/B1, a therapeutic potential of a miR-155 anti-miRNA ubiquitous RNA processing protein, is emerging as an oligonucleotide (AMO) packaged in nanoparticle-based important player in a wide variety of autoimmune diseases carriers to enhance uptake into macrophages. From a range such as Rheumatoid Arthritis (RA). Loss of tolerance to of anti-miRNA oligonucleotide (AMO), we have identified the native and citrullinated forms of hnRNP-A2/B1 is a hallmark best miR-155 AMO to inhibit mir-155 in Raw 264.7 and in of RA; abundance of autoantibodies against it in patients’ murine bone marrow derived macrophages (BMDMs). We sera as well as it’s abundance in patients’ synovial tissue are investigated a range of macrophage polarisation well documented. Despite its use as a biomarker for RA, the parameters, including pro-inflammatory cytokine degree of its involvement in disease pathogenesis is not yet expression, Nitric Oxide production, expression of M2 studied. The aim of our work is to determine if hnRNP-A2/B1 markers Arginase-1 and CD206, and identified that is playing a direct role in contributing to the inflammation Arginase-2 is the most robust miR-155 target that mimics an associated with RA. We performed siRNA mediated knock IL-10 M2 mediated phenotype. We also demonstrate the down of hnRNP-A2/B1 in -like synoviocytes (FLS) capacity of miR-155 AMO for nanoparticle encapsulation obtained from RA patients to investigate whether into PLGA and delivery using novel star shaped knockdown of hnRNP-A2/B1 leads to changes in expression polypeptides in vitro. Our future studies will examine the of ISGs and inflammatory genes associated with RA. Our capacity for in vivo delivery and directing macrophage gene expression analysis using RNA-seq and Nanostring specific uptake. technology uncovered a role for hnRNP-A2/B1 in driving Disclosure of Interest: None Declared inflammation, where loss of this protein dampened the inflammatory circuit associated with RA pathogenesis. We LT103 are currently working to determine the mechanism by which INTEGRIN ΑLPHA 3 PROMOTES THE EXTRAVASATION hnRNP-A2B1 contributes to controlling inflammation within OF PROINFLAMMATORY TH17 CELLS IN the human RA samples. In addition we will utilize our EXPERIMENTAL AUTOIMMUNE recently generated hnRNP-A2/B1 conditional knock out ENCEPHALOMYELITIS. mouse to further characterize this protein’s involvement in E. Park1,*, W. E. Barclay1, M. Shinohara1, M. Ciofani1 amplifying the inflammatory circuit in the arthritic joint in 1Immunology, Duke University, Durham, United States vivo. Disclosure of Interest: None Declared Abstract Content: The disruption of blood-brain barrier and the infiltration of leukocytes into the central nervous system LT102 (CNS) parenchyma are critical in the pathogenesis of NANOMODULATION OF MICRORNAS IN multiple sclerosis (MS). Because integrin α 4 mediates MACROPHAGES lymphocyte tethering and transendothelial migration, F. Nally1,*, V. Lyons2,3, C. De Santi1, S. A. Cryan1, N. Payne2, integrin α 4 has been therapeutically targeted by C. McCoy1 natalizumab to inhibit the infiltration of CD4+ T cells in MS. 1School of Pharmacy and Biomolecular Sciences, Royal However, natalizumab is less effective in inhibiting the College of Surgeons in Ireland, Dublin, Ireland, 2Australian migration of Th17 cells which play major pathogenic roles in Regenerative Medicine Institute, 3Hudson Institute of MS due to the low expression of integrin α 4 on Th17 cells. Medical Research, Monash University, Melbourne, Australia Moreover, long-term use of natalizumab down-regulates 59 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Th1-mediated immune surveillance and increases the risk of evaluate PTN as a possible biomarker. Written informed opportunistic JC-virus infection in the brain leading to consent was obtained from all participants. progressive multifocal leukoencephalopathy. Therefore, it is Results: PTN serum levels were significantly higher in necessary to understand Th17 cell trafficking during patients when compared to controls (329.49±68.94 pg/mL inflammation in order to identify new therapeutic targets in and 139.61±67.52 pg/mL, respectively) (p<0.05). In IFN-β MS treatment. In this study, we identified that integrin α 3 is treated patients, PTN levels were higher after 8 years of expressed by inflammatory Th17 cells that infiltrate into the disease duration, compared to 8 years duration or less CNS parenchyma in experimental autoimmune (p<0.05). These patients showed a positive correlation encephalomyelitis (EAE) through an IL-6-Stat3 signaling between time of evolution (r=0.484, p<0.05) and time of IFN- pathway. Moreover, the deletion of integrin α 3 in CD4+ T β use (r=0.423, p<0.05) with PTN levels. In addition, we cells or IL-17A-fate-mapped cells attenuated the disease found a negative correlation between PTN concentration severity of EAE by impeding CD4+ T cell infiltration into the and the MS Severity Score (r=-0.451, p<0.05). There were CNS parenchyma. Immunofluorescence staining of spinal no findings such as these in GA-treated and non-treated cord sections revealed an increased retention of integrin α patients. PTN expression was not detected by qPCR in 3-deficient CD4+ T cells in laminin-rich perivascular spaces. PBCM in any group. The ROC curve analysis had an AUC The significance of integrin α 3 in Th17 cell migration is of 0.678, with a sensibility of 64.9% and specificity of 72.2% further corroborated by the enhanced migration of Th17 to identify patients and controls. However, in males PTN cells in the presence of laminin-α5 in transwell migration concentration had a better predictive power, with an AUC of assay. In conclusion, these data collectively demonstrate 0.824, a sensibility of 70.8%, and a specificity of 77.8%. The that integrin α 3 promotes the extravasation of inflammatory AUC for females was considerably lower (0.564), meaning Th17 cells in the context of EAE and suggest integrin α 3 as that hormones could be regulating PTN. a therapeutic target in MS. Conclusion: Our study provides first evidence that PTN is Disclosure of Interest: None Declared increased in patients with RRMS compared to controls, with a differential expression and correlation according to LT105 treatment, being significantly for IFN-β treatment. Future PLEIOTROPHIN SERUM CONCENTRATIONS studies must be developed to confirm the role of PTN in CORRELATE WITH CLINICAL VARIABLES IN RRMS, its possible use as a biomarker, and its participation INTERFERON-BETA (IFN-Β) TREATED RELAPSING- in the connections between CNS and IS. REMITTING MULTIPLE SCLEROSIS (RRMS) PATIENTS. References: M. P. Reyes-Mata1,*, A. E. Rojas-Mayorquín2, L. Carrera- 1) Filippi, M. et al. Nat Rev Dis Primers 4, 43 (2018) Quintanar3, C. González-Castillo4, M. A. Mireles-Ramírez5, 2) González-Castillo, C. et al. Front Cell Neurosci 8, (2015) J. D. J. Guerrero-García6, D. Ortuño-Sahagún1 3) Sorrelle, N., et al. J. Leukoc. Biol. 102, 277–286 (2017) 1Instituto de Investigación en Ciencias Biomédicas, CUCS - 4) Wu, G.C. et al. Oncotarget 8, 40181–40189 (2016) Universidad de Guadalajara, 2Departamento de Ciencias 5) Pufe, T. et al. Arthritis Rheum 48, 660–667 (2003) Ambientales, CUCBA - Universidad de Guadalajara, 6) Kuboyama, K. et al. J. Neurosci. 35, 12162–12171 (2015) 3Laboratorio de Ciencias de los Alimentos, CUCS - Disclosure of Interest: None Declared Universidad de Guadalajara, Guadalajara, 4Escuela de Medicina y Ciencias de la Salud, Tecnológico de Monterrey, LT107 Zapopan, 5Unidad Médica de Alta Especialidad (UMAE), CCL18 INDUCES HEALTHY FIBROBLAST-LIKE Hospital de Especialidades (HE), Centro Médico Nacional SYNOVIOCYTES TO DEVELOP GENE EXPRESSION de Occidente (CMNO), IMSS, 6Banco de Sangre Central, PROFILING SIMILAR TO FIBROBLAST-LIKE Centro Médico Nacional de Occidente (CMNO), IMSS, SYNOVIOCYTES OF RHEUMATOID ARTHRITIS Guadalajara, Mexico Y. Tan1,*, Y. Mo2 on behalf of co-corresponding author, N. Z. Lai1 on behalf of co-corresponding authors Abstract Content: Introduction: RRMS is the most 1R&D division, Advanced Medigene, Rockville, 2 National common form of Multiple Sclerosis (MS). It is an Institute of Dental and Craniofacial Research, National autoimmune demyelinating disease mainly affecting the Institutes of Health, Bethesda, United States Central Nervous System (CNS) (1). Pleiotrophin (PTN), a neuromodulatory (2) and immunomodulatory cytokine (3), is Abstract Content: a good candidate for establishing a bidirectional Background: CC chemokine ligand 18 (CCL18) is a communication between the CNS and the Immune System secretion chemokine that is produced by tumor associated (IS). It is involved in other autoimmune diseases (4,5) and macrophages in a variety of solid cancers, or by activated has been studied in an experimental model for MS (6), monocytes/macrophages and dendritic cells in however, PTN concentration has not been explored in nonneoplastic diseases. Our previous study demonstrated RRMS patients. CCL18 significantly enriched in synovial fluid of patients with Methods: PTN serum levels were quantified by ELISA in 57 rheumatoid arthritis (RA), which may correlate with one-year patients with RRMS (treated with Glatiramer acetate (GA), radiographic progression through promoting migration of RA IFN-β, or non-treated) and in 18 controls. We also fibroblast-like synoviocytes (RA-FLS). Studies which reveal determined PTN mRNA expression in Peripheral Blood the biological regulation of CCL18 on FLS would increase Mononuclear Cells (PBMC) by qPCR. Non-parametric the understanding of how the chemokine involves in analyses were performed. ROC curves were constructed to autoimmune disease. 60 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Objectives: To explore whether CCL18 can induce healthy Abstract Content: Behcet disease (BD) is a chronic FLS (HFLS) to develop gene expression profiling similar to systemic inflammatory disorder that is considered as a RA-FLS, and determine the biological function of CCL18 on vasculitis touching both veins and arteries of all sizes. The HFLS. disease clinical expression varies among patients. However, aphtosis is a common feature. Oral microbiota has been Methods: Primary RA-FLS were obtained from Parker- incriminated in BD genesis. However, the implication of local Pearson needle biopsy of synovial tissues and were cultured immune response is still under investigation. In this study, in vitro. Primary HFLS cells were purchased from Cell we intended to explore the local versus systemic role of IL- Applications Inc. Total RNA was isolated from HFLS treated 32 and nitric oxide in the oral immune responses during BD. with recombinant CCL18 (500ng/mL) or vehicle, and five Sixty patients with BD (36 active and 24 inactive stage) and cases of primary RA-FLS. RNA-Seq were performed using 25 control subjects were enrolled in this study. Freshly Illumina SBS technology. DESeq2 was implemented to collected blood on heparinized tubes and spontaneously assess differentiation expression genes (DEGs). Ingenuity secreted saliva were used in the study. In the same time, pathway analysis (IPA) core analyses were conducted to oral health and hygiene were assessed through a previously identify the molecular activities of the DEGs along with prepared questionnaire. The samples were centrifuged and literature reviewed. filtered then supernatants were conserved at -80. IL-32 was measured by ELISA (Biotechne) while nitric oxide was Results: Compared with HFLS, 2698 genes were estimated by a modified Griess method in both samples upregulated and 2317 genes downregulated in RA-FLS, types. Statistical analyses were measured by Mann Witney while 249 upregulated genes and 311 downregulated gene U test for group comparison while Spearman test was used in HFLS treated with CCL18 were determined when for correlation analyses. comparing with the untreated cells. Compare the above two We observed a significant increase in IL-32 and nitric oxide datasets, it was found 117 upregulated DEGs and 148 levels during BD in comparison to controls (p<0.05). Both downregulated DEGs in both RA-FLS and HFLS treated markers were increased in plasma and saliva (p<0.05). In with CCL18, suggesting HFLS treated with CCL18 had plasma, both markers were increased during disease similar gene expression profiling to RA-FLS. IPA core activity while in saliva, only nitric oxide levels were increased analyses were conducted focusing on these 265 common (p<0.05). In addition, nitric oxide levels were correlated with DEGs. Except unclassified categories, the first functional the number of mouth ulcers (r=0.303; p=0.035). In contrast, category of proteins encoded by these genes was enzyme, salivary IL-32 was not associated with aphtosis (r=0.009, followed by transcription factor and transporter. Diseases & p=0.961) but was strongly correlated with the dental plaque functions module in IPA showed these genes enriched in index (r=0.819, p<0.0001). We did not observe any migration associated pathways including formation of correlation in control group (p>0.05). cellular protrusions, formation of filopodia, migration of Our results showed that Nitric oxide and IL-32 were connective tissue cells and disruption of focal adhesions. increased in BD in comparison to control subjects. We According to Ingenuity in IPA and publications, 19 migration- observed a difference between local and systemic associated genes were identified, including ANXA2, CDH11 responses. Although both markers were associated with and SUMO1 which were reported overexpression in RA-FLS disease activity in plasma, in saliva, nitric oxide was and to promote cell migration. Besides, 10 genes encoded associated with the genesis of oral mucosal lesions while IL- cytokines, growth factors, chemokines and pro-inflammatory 32 seems to play an important role in the excessive proteins in extracellular space, such as CCN2 and CCL7, inflammatory responses to oral flora especially, plaque flora. both of which were reported overexpression in RA patient’s Our results suggest different regulating mechanisms for the serum and synovial fluid. two considered molecules during BD. Disclosure of Interest: None Declared Conclusion: CCL18 induces HFLS to develop gene expression profiling similar to RA-FLS. CCL18 enriched in LT109 RA synovial fluid may promote migration of FLS though STAT4 REGULATES PATHOGENIC IL-21 AND IFN-G upregulation of migration-associated genes and maintain PRODUCTION IN T FOLLICULAR HELPER (TFH) CELLS inflammation through upregulation of certain pro- IN MURINE AND HUMAN LUPUS inflammatory proteins. X. Dong1, O. Antao2, G. Sanchez2, F. Koumpouras1, J. Disclosure of Interest: None Declared Craft1, J. Weinstein2,* 1Yale Medical School, New Haven, 2Center for Immunity and LT108 Inflammation, Rutgers New Jersey Medical School, Newark, SALIVARY IL-32 IS ASSOCIATED WITH ORAL HYGIENE United States LACK DURING BEHCET DISEASE Z. Hadjimi1, H. Belguendouz1, N. E. H. Ghozali1, K. Lahmar1, Abstract Content: Follicular helper T cells (Tfh) cells shape M. Terahi2, D. Hakem3, C. Touil-Boukoffa1,* the germinal center (GC) response by delivery of contact- 1Cytokines and NO synthases team, LBCM, FSB, USTHB, dependent signals and cytokines, including IFN-γ and IL-21. 2Department of ophtalmology, CHU Nafissa Hammoud, The contribution of these discrete extrinsic signals to the 3Department of internal medicine, CHU Bab El Oued, mal-regulation of GC B cells in lupus is unclear. We found Algiers, Algeria that in Tfh cells from young lupus-prone mice, the 61 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 transcription factors Bcl6 and T-bet are co-expressed, as pancreatic β cells. Lnc10 is located in the nucleus of β cells, they are in conventional Tfh cells following acute type 1 both in basal and stimulated conditions, suggesting a (viral) challenge, with robust IL-21 and IFN-γ production. In potential role in transcriptional regulation. Lnc10 lupus, by contrast to acute viral challenge, GCs persist as overexpression in combination with disease progresses, and we observed that Bcl6 and T-bet polyinosinic:polycytidylic acid (PIC) transfection to mimic a expression declined in Tfh cells from chronic GCs during viral infection, increased the expression of several antiviral such progression, as they continued to robustly co-produce genes of the iDIN pathway, including key genes in type I IFN both IL-21 and IFN-γ with ongoing genesis of IgG2 signaling and antiviral response (IRF7, STAT1, IFIT1, IFIT2 autoantibody production necessary for immune complex and ISG15). Interestingly, the type IFN signaling has been glomerulonephritis. Transcriptional analysis of lupus-Tfh shown to play a crucial role in the initial stages of T1D cells at different stages of disease revealed an evolving pathogenesis in pancreatic β cells. gene expression profile, with an increased STAT4 gene In conclusion, our results show that Lnc10 is upregulated signature coincident with STAT4 phosphorylation that after a viral infection in pancreatic β cells. Upregulation of corresponded to disease progression. In a like manner to Lnc10 in combination with a viral infection exacerbates the our murine findings, we found that in human lupus patients expression of several antiviral genes from the iDIN pathway. circulating Tfh cells secreted IL-21 and IFN-γ and that Further studies are required to elucidate the molecular STAT4 phosphorylation was enhanced upon IFN- or IL-12 mechanisms by which this lncRNA regulates the expression stimulation, coinciding with clinical disease activity. of iDIN genes and to determine its impact in virus-induced β Furthermore, IFN-I blockade in lupus mice resulted in a cell dysfunction and T1D development in genetically decrease in IL-21 and IFN-γ production by Tfh cells. These susceptible individuals. data indicate that in the chronic inflammatory state Disclosure of Interest: None Declared characterizing lupus, STAT4 guides pathogenic cytokine production, providing a potential therapeutic target in Lightning Talk Session 2: Cytokine regulation patients with active disease. II Disclosure of Interest: None Declared LT110 TRANSFORMING GROWTH FACTOR BETA 1 (TGF-B1) LT109b EXPRESSION UPREGULATED FOLLOWING THE T1D-ASSOCIATED LNCRNA LNC10 MODULATES PROLOTHERAPY TREATMENT FOR CARTILAGE THE TYPE I IFN SIGNALING AND ANTIVIRAL REGENERATION RESPONSE IN PANCREATIC BETA CELLS. 1,* 2 1,2,* 1,2,3 E. D. Johnston , J. Tyburski I. Gonzalez-Moro , I. Santin 1 1 Palos Verdes Peninsula High School, Rancho Palos Endocrinology and Diabetes Research Group, university of Verdes, 2Nelson Scientific Labs LLC , Ashburn, United the basque country, Leioa, 2Biocruces Bizkaia Health 3 States Research Institute, barakaldo, Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Abstract Content: Osteoarthritis is a highly prevalent Disorders (CIBERDEM), Madrid, Spain disease associated with a shift in cytokine regulation within joints. Current treatments, such as corticosteroids, aim to Abstract Content: Type 1 diabetes (T1D) is a complex reduce pain but do not slow disease progression. By autoimmune disease in which genetic factors interact with understanding and targeting the inflammatory component of environmental factors (e.g. viral infections) to trigger an the disease, future therapies can more effectively treat autoimmune assault against insulin producing pancreatic β patients. One promising therapy is proliferative therapy cells. The majority of T1D-associated SNPs lay into non- (prolotherapy), a complementary medical intervention with coding regions of the and many have been promising clinical evidence. Scientists hypothesize this predicted to affect the expression, secondary structure and treatment induces a localized inflammatory response function of long non-coding RNAs (lncRNAs). However, the leading to cartilage repair. However, the underlying molecular mechanisms by which these non-coding mechanisms are not well understood. This present study molecules contribute to T1D pathogenesis remain to be investigates the molecular basis of prolotherapy by studying clarified. whether a common prolotherapy treatment, P2G, Lnc10 is a lncRNA harboring a T1D-associated SNP that upregulates proliferation‐enhancing cytokines in has been described to control Ebi2 expression in immune undifferentiated MC3T3-E1 preosteoblast cells as a model cells. Ebi2 is a trans-eQTL that regulates the IRF7-driven for chondroprogenitors. In vitro stimulation of inflammatory network (iDIN), an antiviral gene network chondroprogenitor cells was carried out with phenol- which is enriched with T1D-associated genes. Taking into glucose-glycerin (P2G) prolotherapy treatment followed by account that the T1D-associated SNP is located in Lnc10, qRT‐PCR quantification to determine the mRNA expression our hypothesis is that Lnc10 might be dysregulated in T1D of the genes BMP2, TGF‐β1, AKT, and STAT1. We found patients, influencing the regulation of the iDIN network at the that cells treated with P2G did not exhibit significant changes pancreatic β cell level and provoking an exacerbated in AKT, STAT1, or BMP2 expression. However, cells antiviral response, and eventually, β cell destruction. increased expression of TGF-β1 approximately 2.3 fold by Preliminary results of our group have demonstrated that 30 hours after treatment initiation (p<0.001). Given the role diabetogenic stimuli, such as pro-inflammatory cytokines of TGF-β1 in proliferation and stimulating chondrogenic and viral infections, upregulate Lnc10 expression in collagen deposition, it follows that TGF-β1 may be involved 62 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 in P2G’s mechanism in the chondroprogenitor response. Disclosure of Interest: None Declared The involvement of TGF-β1 could potentially slow or reverse cartilage degradation as chondroprogenitor cells proliferate LT112 and rebalance catabolic/anabolic roles to repair and SYSTEMATIC ANALYSIS OF KINOME INHIBITION FOR maintain cartilage. INTERFERON AND CYTOKINE GENE EXPRESSION Disclosure of Interest: None Declared K. S. A. Khabar1,* on behalf of Molecular BioMedicine LT111 Program INTEGRATED SINGLE-CELL ANALYSIS OF 1King Faisal Specialist Hospital and Research Centre, MULTICELLULAR IMMUNE DYNAMICS DURING Riyadh, Saudi Arabia HYPERACUTE HIV INFECTION S. W. Kazer1,2,*, T. P. Aicher1, D. M. Muema3, J. Ordovas- Abstract Content: Montanes4, C. G. K. Ziegler5, K. L. Dong1, Z. M. Ndhlovu6, T. Ndung'u7, B. D. Walker1, A. K. Shalek2 Interferon and cytokine responses comprise complex 1Ragon Institute of MGH, MIT & Harvard, 2Institute for intertwined signaling and effector pathways, many of which Medical Engineering & Sciences, Massachusetts Institute of are driven by kinases' activities. Here, we employed Technology, Cambridge, United States, 3Africa Health chemical inhibitor (n=387) library targeting the kinome to Research Institute, Durban, South Africa, 4Boston Children's investigate their effects on two different responses; one is Hospital, Boston, 5Division of Health, Science, & transcriptional (IFN response), and the other is post- Technology, Harvard Medical School & Massachusetts transcriptional (cytokine expression). The ISRE-linked Institute of Technology, Cambridge, United States, 6African transcriptional reporter was used in Huh-7 cell stimulated Health Research Institute, Durban, 7African Health with IFN-α. The hierarchal clustering for multivariant Research Institute, Cambridge, South Africa analysis (Reduction and Selectivity) pointed to a cluster of 92 small molecules that selectively inhibit IFN response by Abstract Content: Development of effective vaccines and targeting 40 individual kinases, involved in more than 12 therapeutics is facilitated by understanding the earliest biological pathways. The most notable pathways were immune responses to infection. Here, we apply single-cell inferred as PI3K/mTOR, cell cycle, JAK-STAT, protein RNA-sequencing (scRNA-seq) and a novel gene module tyrosine kinase, and several signaling pathways, previously discovery-based framework to longitudinally profile unknown for involvement in IFN response. Representative peripheral multicellular immune responses in untreated kinase inhibitors from each group were selected, and it was hyperacute HIV infection. found that the majority were able to reduce IFN-stimulated gene (ISG) expression at the mRNA level. Whereas, few We profiled >59,000 single cells from four individuals across inhibitors acted at the protein level without affecting mRNA multiple acute infection timepoints (including pre-infection), expression. utilizing scRNA-seq data to identify transient immune The cytokine expression model employed a reporter system responses with cell subset resolution. Onset of viremia selective for 3'-untranslated region-AU-rich elements (ARE) induced interferon stimulated gene (ISG) responses derived from the TNF-α gene. The ARE sequence elements integrated across multiple cell types, wherein monocytes are important determinants of mRNA decay and translation notably contributed to the cytokine milieu, producing CXCL9 and exist in many pro-inflammatory cytokine genes. Fifteen and CXCL10. Otherwise obscured in bulk analyses, we kinase inhibitors were found to reduce the TNF-ARE describe a second set of responses following ISG reporter activity. These inhibitors targeted fewer pathways upregulation that align in time: pro-inflammatory T cell largely MAPK pathway and cell cycle, indicating restricted differentiation, prolonged monocyte MHC-II upregulation, kinase activity at the post-transcriptional level. and persistent NK cytolytic killing. Leveraging our temporal, cell-type resolved data, we nominate putative cell-cell Among those novel players that affect ISG and ARE- signaling networks and their key cytokines during infection: dependent cytokine expression, is polo-like kinase 1 (PLK1). IL-6/IL-8/IL-17 restrict CD8 T cell killing of HIV infected cells; Accordingly, RNAseq experiments were performed with the TNF and IL-1β affect only CD4+ T cells, and IL-2/IL-15/IL- PLK1 inhibitor, volasertib, showing reduction of many 18 mediate persistent killing by NK cells and CD8 T cytokine and chemokine ARE-mRNAs. PLK1 inhibition cells. Two participants who later develop viremic control caused accelerated mRNA decay of cytokine mRNAs, and associated with elevated frequencies of proliferating was associated with reduced phosphorylation of the mRNA cytotoxic cells, inclusive of a previously unappreciated NK decay-promoting protein, tristetraprolin (TTP/ZFP36). In cell subset, immediately following HIV detection. contrast, ectopic expression of PLK1 caused an increased abundance of the phosphorylated TTP in normal cells. PLK1 Our study reveals both cooperative and cell subset specific effect was associated with the MAPK-MK2 pathway, a major immune responses during untreated hyperacute HIV regulator of ARE-mRNA stability, as evident from MK2 infection with temporal resolution, nominating monocytes, inhibition, in vitro phosphorylation, and knockout NK cells, and pro-inflammatory signaling as perturbation experiments. Moreover, mutational analysis demonstrated targets for future vaccine studies. Moreover, our gene that TTP serine 186 is a target for the PLK1 effect. module discovery framework can be readily applied to other Treatment of mice with the PLK1 inhibitor reduced both longitudinal studies in humans and other model organisms. 63 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

TTP/ZFP36 phosphorylation and cytokine expression in LT115 xenograft tissues. THE CYTOKINE RESPONSE TO TOTAL KNEE ARTHROPLASTY IS ASSOCIATED WITH RISK OF To our knowledge, this is the first large and detailed screen PERSISTENT POSTOPERATIVE PAIN of the kinome inhibitors with IFN and cytokine responses M. Kirksey1,2,*, G. Birch1, H. Zhong1, A. Sideris1, V. and a resource for the assessment of the kinome for Rotundo1, A. Gonzalez Della Valle3, M. Parks3, M. Otero4, potential therapeutic effects and off-target effects. The data P. Sculco3 also showed that PLK1 is an important regulator of 1Department of Anesthesiology, Critical Care, and Pain TTP/ZFP36 phosphorylation in cytokine expression, and Management , Hospital for Special Surgery, 2Department of that kinase-mediated post-transcriptional process could be Anesthesiology, Weill Cornell Medical College, 3The Stavros a target for therapeutic purposes. Niarchos Foundation Complex Joint Reconstruction Center, Disclosure of Interest: None Declared 4Hospital for Special Surgery, New York, United States

LT113 Abstract Content: Introduction: Persistent postoperative SEVERE FEVER WITH THROMBOCYTOPENIA pain following total knee arthroplasty (TKA) can occur in as SYNDROME VIRUS NON-STRUCTURAL PROTEIN many as 20-40% of TKA patients1. There is growing (SFTSV-NSS) ACTIVATES NFΚB-DEPENDENT evidence that cytokines and adipokines are associated with CYTOKINE STORM IN VITRO osteoarthritis (OA) severity, progression, and associated J. A. Khalil1,*, T. Yamada2, H. Kato2, T. Fujita1 pain severity2. Specifically, tumor necrotic factor alpha 1Department of Molecular and Cellular Immunology, Kyoto (TNFα) and (IL6) have been identified as University, Kyoto, Japan, 2Medical Faculty, University of correlating with increased pain in osteoarthritis patients3,4. In Bonn, Bonn, Germany this study, we sought to determine if there are associations between blood and synovial fluid cytokine levels in end- Abstract Content: Severe Fever with thrombocytopenia stage OA patients during the TKA perioperative period and syndrome (SFTS) is an emerging infectious disease caused development of persistent postoperative pain. by a tick-borne bunyangvirus of the family phenuiviridae, the Methods: 162 patients undergoing primary unilateral TKA SFTS virus (SFTSV). The syndrome is characterized by were prospectively enrolled. Exclusion criteria included severe inflammatory process affecting the integrity of host autoimmune/inflammatory disease, systemic steroid use tissues, such as liver, spleen, and lymph nodes and often within 6 months of surgery, active infection, a history of associated with high morbidity and mortality rates (reaching opioid use and/or chronic pain. Synovial fluid was collected 30%). Unfortunately, very little is known about the causative pre-arthrotomy and plasma was collected prior to incision, in virus and its pathogenesis. As a result, there is currently no the recovery room, and on postoperative days (POD) 1 and vaccine or efficient treatment for the syndrome. 2. Numerical Rating Scale pain levels were recorded at rest Using different cell lines, DNA plasmids expressing various and with movement preoperatively, on POD0, 1, 2 and 6 SFTSV viral proteins, and real virus infection, we identified months post-operatively. Pain Catastrophizing Score (PCS) both the viral protein responsible for the cytokine storm was recorded on POD2. Postoperative persistent pain was induction, and the transcription factor mediating the defined as ambulatory NRS ≥ 4 at 6 months. Cytokine levels inflammatory process in the host. were measured using the V-Plex Human Cytokine 30-Plex In summary, the viral non-structural protein (SFTSV-NSs) is Panel (Mesoscale - Rockville, Maryland, USA). a potent activator of NFκB pathway, which induces the Results: 15/162 (9.3%) of patients met criteria for expression of cytokines at the transcriptional level, resulting postoperative persistent pain at 6 months following TKA for in overly enhanced interlukin-8 (IL-8), interlukin-6 (IL-6), OA. Postoperative plasma levels of 4/31 cytokines studied tumor necrosis factor α (TNF-α), and C-C motif chemokine were significantly different between persistent pain and non- ligand 2 (CCL2). Interestingly, interfering with and inhibiting persistent pain patients. IL10 levels were significantly lower NFκB resulted in significant decrease in the cytokines (p=0.033) immediately postoperatively in the pain group. activation and alleviated the cytokine storm fundamentally. IL1Β was significantly higher (p=0.0005) among persistent Since cytokine storm has been described clinically in SFTS postoperative pain patients on POD1. VEGF was patients, we propose that NFκB could be a therapeutic target significantly lower (p=0.0239) on POD1. IL12_23p40 in the future for treating SFTS. differentiated at both POD1 and POD2 (p=0.0199, Taken together, these findings present a new insight into the p=0.0439) between groups. IL10 preoperative synovial fluid pathogenesis of lethal SFTS, and may promote not only the cytokine level was associated with development of understanding of virus-host interactions but also the postoperative persistent pain (p=0.0482). development of antiviral therapeutics. Conclusion: This prospective cohort study describes Keywords: Cytokine storm, NFκB, SFTSV, Non-structural variations in 4 cytokines in the perioperative period in protein. patients who developed persistent pain after TKA, suggesting a distinct inflammatory profile in at risk patients. Disclosure of Interest: None Declared While baseline IL10 levels were lower in the synovial fluid of patients who developed persistent postoperative pain, differences in serum cytokine levels were only detected postoperatively in response to surgery. These results

64 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 support the hypothesis that pre- and post-operative cytokine receptor families, including Toll Like Receptors (TLRs), pathways may influence long term postsurgical outcomes. cytokine receptors (TNF-alpha and IL-1 receptors), and Fc References: receptors, leading to the expression of inflammatory 1. Searle R et al. Continuing Education in Anaesthesia, mediators. Our lab has previously shown that the Tpl2- Critical Care & Pain 2010;10:12-14 /- mice are more susceptible to influenza infection, and the 2. Orita S et al. BMC Musculoskelet Disord 2011;12:134 purpose of this study is to delineate the mechanisms 3. Shimura Y et al. Osteoarthritis Cartilage 2013;21(9):1179- responsible. Tpl2-/- mice ultimately clear the virus albeit with 84 delayed kinetics. Despite eventual viral control, Tpl2-/- mice 4. Stannus, OP et al. Ann Rheum Dis. 2013;72(4):535-40 begin to exhibit severe clinical signs and require euthanasia. Disclosure of Interest: None Declared Therefore, we hypothesize that an overexuberant immune response rather than impaired viral control leads to severe LT116 pathology in Tpl2-/- mice in response to influenza infection. STAT3 REDUCES THE EXPRESSION OF THE MTOR Histological examination of the lungs of influenza- INHIBITOR REDD1 IN A NON-CANONICAL FASHION infected Tpl2-/- mice showed increased alveolar septal N. Köhler1,*, A. Dittrich1, F. Schaper1 necrosis, pleuritis, and more widespread lesions, which are 1System Biology, Otto von Guericke University, Magdeburg, signs of epithelial-endothelial barrier damage and Germany inflammation. In order to assess potential cellular mediators of inflammation, the cellular profile of the lung was analyzed Abstract Content: Interleukin 6 (IL-6) is a pleiotropic at 7 days post infection (dpi), when significant morbidity was cytokine and an activator of Mammalian Target of observed in Tpl2-/- mice. An excess influx of inflammatory Rapamycin (mTOR). In contrast, mTOR activity is negatively monocytes was observed in the lungs of Tpl2-/- mice, along regulated by Regulated in Development and DNA Damage with a corresponding increase in serum levels of MCP-1, the Responses 1 (REDD1). Expression of REDD1 is induced by chemokine required for inflammatory monocyte recruitment. cellular stressors such as glucocorticoids and DNA Because type I interferons promote inflammatory monocyte damaging agents. We show that the expression of basal as recruitment via the induction of MCP-1, ongoing studies are well as stress-induced REDD1 is reduced by IL-6. The examining whether Tpl2 paradoxically limits inflammation reduction of REDD1 expression by IL-6 is independent of during influenza infection by constraining the production of proteasomal or caspase-mediated degradation of REDD1 type I IFNs. This information will enhance our understanding protein. Instead, induction of REDD1 mRNA is reduced by of Tpl2 signaling and interferon regulation, which could IL-6. The regulation of REDD1 expression by IL-6 is provide insights into host-targeted therapies for treating independent of Phosphatidylinositide-3-Kinase (PI3K) and highly pathogenic influenza which is typically associated Mitogen-Activated Protein Kinase (MAPK) signalling but with excess type I IFNs and robust inflammatory monocyte depends on the expression and activation of Signal recruitment. Transducer and Activator of Transcription 3 (STAT3). Disclosure of Interest: None Declared Furthermore, the reduction of basal REDD1 expression by IL-6 correlates with IL-6-induced activation of mTOR LT118 signalling. Inhibition of STAT3 activation blocks IL-6-induced IMMUNE REGULATOR LGP2 INHIBITS K63 mTOR activation. POLYUBIQUITINATION The gene-repressory action of STAT3 is contrary to the J. Lenoir1,*, C. M. Horvath1 canonical transcription-inducing function of STAT3, possibly 1Department of Molecular Biosciences, Northwestern reflecting a new non-canonical function of STAT3. We University, Evanston, United States demonstrate an IL-6-dependent recruitment of STAT3 to the REDD1 promoter. Moreover, we determine the position of Abstract Content: During RNA virus infection, type I the STAT3 binding site within the REDD1 promoter. In interferon (IFN) production allows for the cell to achieve a summary, we present a novel STAT3-dependent robust antiviral response and it must be tightly regulated to mechanism of both IL-6-induced activation of mTOR and IL- prevent cytotoxic effects. IFN production is initiated when 6-dependent reversion of stress-induced inhibition of mTOR viral sensor proteins RIG-I, MDA5, and LGP2 bind to viral activity. RNA, activating downstream E3 ubiquitin ligases TNF Disclosure of Interest: None Declared receptor-associated factors (TRAFs). Through the synthesis of K63-linked polyubiquitin chains, TRAFs recruit kinases LT117 which drive transcription of IFN. Overexpression of LGP2 TUMOR PROGRESSION LOCUS 2 (TPL2) PREVENTS has been shown to negatively regulate IFN production. The IMMUNOPATHOLOGY DURING INFLUENZA Horvath lab identified that LGP2 inhibits the production of INFECTIONS K63 polyubiquitin chains on TRAF proteins, however the mechanism was not well understood. Experiments K. Latha1,*, K. Sakamoto1, W. Watford1 presented here demonstrate that LGP2 inhibits K63 1Department of Infectious Diseases, University of Georgia, polyubiquitination and downstream signaling without the Athens, United States need of a binding site on TRAF6. TRAF6 was unable to interact with its E2 conjugating enzyme Ubc13/Uev1a in the Abstract Content: Tpl2 (Tumor Progression Locus 2) is a presence of LGP2. Using a different E3 ubiquitin ligase, serine/threonine kinase that transmits signals via several LGP2 prevents K63 polyubiquitination by preventing 65 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 interaction between the E3 ligase and Ubc13/Uev1a. Using strategy for engineering single-cell variability in response co-immunoprecipitation experiments, we determined that due to family-wide heterogeneity in receptor abundance. Ubc13/Uev1a interacts with LGP2 and this interaction is Disclosure of Interest: A. Farhat: None Declared, A. important in the inhibition of polyubiquitin chain formation. Weiner: None Declared, C. Posner Employee of: Visterra, These findings establish a mechanism that LGP2 uses in Inc., Z. Kim: None Declared, B. Orcutt-Jahns: None order to inhibit K63 polyubiquitination of E3 ubiquitin ligases Declared, S. Carlson Employee of: Visterra, Inc., A. Meyer involved in the antiviral response. Grant / Research support from: Visterra, Inc. Disclosure of Interest: None Declared LT121 LT120 ABSENCE OF CCL5/CCR5 AXIS EXAGGERATES MODELING CELL-SPECIFIC DYNAMICS AND THROMBUS FORMATION THROUGH REDUCED UPA, REGULATION OF THE TPA AND VEGF EXPRESSION IN MURINE DVT MODEL CYTOKINES M. Nosaka1,*, Y. Ishida1, Y. Kuninaka1, A. Ishigami1, H. A. M. Farhat1, A. C. Weiner2, C. Posner3, Z. S. Kim4, B. Yamamoto1, A. Kimura1, N. Mukaida2, T. Kondo1 Orcutt-Jahns4, S. M. Carlson5, A. S. Meyer4,* 1Department of Forensic Medicine, Wakayama Medical 1College of Medicine-Chicago, University of Illinois, University, Wakayama, 2Division of Molecular Bioregulation, Chicago, 2Computational Biology & Medicine, Tri-Institute Cancer Research Institute, Kanazawa University, Program, New York, 3Visterra, Inc, Waltham, MA, Kanazawa, Japan 4Bioengineering, University of California, Los Angeles, Los Angeles, 5Visterra, Inc., Waltham, MA, United States Abstract Content: Introduction Abstract Content: Many receptor families exhibit both Deep vein thrombosis (DVT) is multifactorial and often pleiotropy and redundancy in their regulation, with multiple results from a combination of risk factors such as genetic ligands, receptors, and responding cell populations. Any conditions, obesity, drugs, pregnancy, aging, trauma, intervention, therefore, has multiple effects, confounding inflammation and malignancy. And DVT is a complex intuition about how to precisely manipulate signaling for biological event, with endothelial injury, venous stasis and therapeutic purposes. The common γ-chain cytokine blood hypercoagulability. In this study, we examined the receptor dimerizes with complexes of the cytokines pathophysiological role of CCL5/CCR5 axis in the resolution interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 and their of DVT on Ccr5 knockout (KO) mice. corresponding "private" receptors. These cytokines have Methods existing uses and future potential as immune therapies due Eight-week-old C57BL/6 (WT) and KO mice underwent to their ability to regulate the abundance and function of anesthesia and, via a laparotomy the femoral, the inferior specific immune cell populations. However, engineering cell vena cava (IVC) was exposed and ligated with 3-0 silk specificity into a therapy is confounded by the complexity of suture. At 1, 3, 5, 7, 10, 14 and 21 days after the IVC ligation, the family across responsive cell types. the blood flow in thrombosed IVCs were measured and mice were euthanized. After that thrombosed IVCs were Here, we build a binding-reaction model for the ligand- harvested. The morphological study, immunohistochemical receptor interactions of the common γ-chain receptor analyses, and double-color immunofluorescence analyses cytokines enabling quantitative predictions of response. We were performed. At 1, 3, 5, 10 and 14 days, the thrombus show that accounting for receptor-ligand trafficking is was separated from the vein wall and the intrathrombotic essential to accurately model cell response. This model gene expressions were obtained by real-time RT-PCR. accurately predicts ligand response across a wide panel of Results cell types under diverse experimental designs. Further, we Immunohistochemically, in WT mice intrathrombotic CCR5- can predict the effect and specificity of natural or engineered positive cells were detected whole of the observation period ligands across cell types. Using a panel of engineered, Fc- and increased gradually with time after IVC ligation. conjugated cytokines, we identify that reducing CD122/γ- Concomitantly, the gene expression of Ccr5 was detected chain affinity cannot overcome reductions in CD25 affinity in the thrombi. When KO mice were treated in the same when engineering regulatory T cell specificity. The manner, thrombus size was much larger than WT mice. orientation of Fc conjugation can also strongly affect affinity Moreover, the blood flow of the IVC was more recovered in to each receptor and thus the signaling specificity. Finally, WT than in KO mice. And intrathrombotic Plat, Plau and we show that tensor factorization is a uniquely powerful tool Vegf mRNA expressions were significantly reduced in KO to visualize changes in the input-output behavior of the mice than WT mice. We explored the contribution of BM- family across time, cell types, ligands, and concentration. derived CCR5+ cells to thrombolysis by using BM chimeric mice generated from WT and KO mice. Both WT and KO In total, these results present a more accurate model of mice transplanted with WT mouse-derived BM cells ligand response validated across a panel of immune cell exhibited a higher thrombolytic activity than the recipients of types and demonstrate an approach for generating KO mouse ones. Supportingly, recombinant murine (rm) interpretable guidelines to manipulate the cell type-specific CCL5 treatment enhanced gene expression of Plau, Plat targeting of engineered ligands. These techniques will in and Vegf in WT-derived macrophages but not KO-derived turn help to study and therapeutically manipulate many other ones. These observations indicated that CCR5-deficiency complex receptor-ligand families. Lastly, we will outline a 66 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 impaired fibrinolytic activity and collagen production, both LT123 indispensable steps for thrombus resolution. INDUCES RNA METHYLATION CHANGES Conclusion THAT REGULATE INTESTINAL INFLAMMATION VIA The absence of CCL5/CCR5 axis can have a detrimental ALLELE-SPECIFIC XPO1 TRANSLATION role in the thrombus resolution by suppressing uPA, tPA and A. Olazagoitia-Garmendia1,2,*, L. Zhang3, P. Mera4,5, M. VEGF expression respectively. The CCL5/CCR5 axis can Sebastian-delaCruz1,2, I. Garcia-Santisteban1,2, A. Huerta6, be a good molecular target for the DVT treatment. I. Irastorza2,7, G. Bhagat8, P. H. Green8, L. Herrero4,5, D. Disclosure of Interest: None Declared Serra4,5, J. A. Rodriguez1, C. He3, J. R. Bilbao1,2, A. Castellanos-Rubio1,2,9 LT122 1Genetis, Physical Anthropology and Animal Physiology, TRANSCRIPTIONAL REGULATION OF IL-21 UPV/EHU, Leioa, 2Biocruces Bizkaia Health Research EXPRESSION IN T FOLLICULAR HELPER CELLS Institute, Barakaldo, Spain, 3Howard Hughes Medical R. Nurieva1,*, L. Qin1, A. Sahoo1, M. Divenko1, A. Alekseev1 Institute, University of Chicago, Chicago, United States, 1Immunology, The University of Texas MD Anderson Cancer 4Department of Biochemistry and Physiology, School of Center, Houston, United States Pharmacy and Food Sciences,, Institut de Biomedicina de la Universitat de Barcelona , Barcelona, 5CIBEROBN, Abstract Content: Introduction: T follicular helper (Tfh) Instituto de Salud Carlos III, Madrid, 6Enfermedades cells play a key role in providing help to B cells during Digestivas, Hospital de Galdakao-Usansolo, Galdakao, germinal center (GC) reactions. The generation and function 7Pediatrics, UPV/EHU, Leioa, Spain, 8Center for Celiac of Tfh cells is regulated by multiple checkpoints including Disease, Department of Medicine, Columbia University, their early priming stage in T zones and throughout the New York, United States, 9Ikerbasque, Basque Foundation effector stage of differentiation in GCs. Signaling pathways for Science, Bilbao, Spain activated downstream of cytokine and costimulatory receptors as well as consequent activation of subset- Abstract Content: Celiac disease (CD) is an inflammatory specific transcriptional factors are essential steps for Tfh cell intestinal disorder that develops in genetically susceptible generation. While to date early signaling events for fate individuals upon gluten ingestion. It is known that gluten committed differentiation of Tfh cells are sufficiently studied, triggers a proinflammatory response, but the underlying mechanisms allowing Tfh cells to maintain their mechanisms remain unclear. CD-associated SNPs map commitment/programming are still unclear. mainly to noncoding regions and their effects are difficult to Methods: The regulation of IL-21 expression by assess. Additionally, post-transcriptional RNA modifications, STAT1/STAT3/Batf/IRF4 transcriptional complex in Tfh cells including m6A, play crucial roles in the regulation of different was assessed by FACS staining, quantitative Real-Time autoimmune diseases. PCR, Chromatin Immunoprecipitation (ChIP) assay, Our hypothesis is that noncoding CD-associated SNPs can luciferase reporter assay and retroviral transduction. For alter m6A levels in adjacent motifs, affecting different cell these assays FACS-sorted CD4+CD44hiCXCR5hiPD1hi Tfh processes and increasing CD predisposition. Along this line, cells were utilized from wild-type, STAT1-, STAT3-, Batf-, SNP rs3087898, in the 5’ UTR of the XPO1 gene, is located and IRF4-deficient mice subjected to Ova immunization. close to an m6A motif. We confirmed the methylation in this Immunoblot analysis was performed to explore the region, and observed that the RNA carrying the CD-risk mechanism whereby STAT1 along with STAT3, Batf, and allele shows higher methylation and binds the YTHDF1 IRF4 contributes to IL-21 production in Tfh cells. reader more efficiently, promoting higher XPO1 translation. Results: Here we report that signal transducer and activator Knocking down the m6A-related machinery resulted in lower of transcription 1 (STAT1) deficiency in CD4+ T cells leads XPO1 protein levels; while gluten stimulations induced to a diminished number of Tfh cells at day 7 and 14 days XPO1, METTL3 and YTHDF1 levels, both in vitro and in after antigen immunization, while at day 3-4, number of Tfh vivo. We also observed that gluten-induced activation of cells was comparable between STAT1-sufficient and STAT1 NFkB pathway, a hallmark of CD, could be reversed by deficient mice, suggesting the role of STAT1 in Tfh lineage inhibition of XPO1 or m6A machinery and that the NFkB maintenance rather than in Tfh cell priming. In fact, in both target IL8 cytokine and its murine homologs Cxcl1 and mouse and human Tfh cells interleukin (IL)-21 mRNA and Mip2a increased upon gliadin stimulations via this m6A- protein expression is dependent on the presence of STAT1. XPO1 pathway. Finally, we confirmed that rs3087898 SNP Functionally, STAT1 in cooperation with STAT3, Batf, and genotype influences XPO1 protein levels in human intestinal IRF4 triggers IL-21 production in Tfh cells by directly binding biopsy samples. In addition, CD patients show increased, to and activation of the IL-21 gene locus. Moreover, utilizing gluten-dependent, XPO1, METTL3 and YTHDF1 expression gene knockout approach, we have determined that Batf is a and downstream IL8 inflammatory cytokine. crucial connector to form STAT1/STAT3/Batf/IRF4 active Altogether, we have demonstrated that intergenic SNPs complex for triggering IL-21 transcription. associated with autoimmune disorders can alter m6A levels Conclusion: Our results thus indicate functional and contribute to disease pathogenesis. Moreover, this is contribution of STAT1 towards regulation of IL-21 the first study linking gluten with alterations in m6A-related expression and Tfh lineage maturation/maintenance. mechanisms. We have observed that gluten induces the Disclosure of Interest: None Declared m6A machinery resulting in an allele-specific increase of m6A levels in the 5’UTR of XPO1, which in turn leads to augmented XPO1, NFkB activation and IL8 secretion in 67 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 patients harboring the risk allele, thus opening the door to overexpression) is strongly suppressed in mouse lungs in new therapeutic approaches. vivo by PR8 infection, and this may be due to direct effects Disclosure of Interest: None Declared of Type 1 Interferons and/or IFNγ on IL-33 expressing cells. Thus, although influenza infection in lungs may LT124 induce OSM, influenza may also suppress OSM effects INFLUENZA A/PR8 VIRUS INFECTION ATTENUATES later in infection such as its regulation of IL-33. (Supported OSM-INDUCED IL-33 CYTOKINE LEVELS AND TH2/M2 by CIHR). SKEWED LUNG INFLAMMATION IN MICE IN VIVO Disclosure of Interest: None Declared C. D. Richards1,*, A. Dubey1, F. Botelho1, A. Yip1, K. MacDonald1, K. Ask1, M. Miller1 LT125 1McMaster Immunology Research Centre, MCMASTER IL-6 PROMOTES TH17 CELL RESPONSES IN PATIENTS UNIVERSITY, Hamilton, Canada WITH T1R LEPROSY REACTIONS. C. Saini1,*, R. K. Srivastava1, V. Ramesh2, A. Sharma3 Abstract Content: Influenza A (Flu) infections typically 1Biotechnology, All India Institute of Medical Sciences, induce innate immune mechanisms (including type 1 2Dermatology, SJH Hospital, New Delhi, 3Biochemistry, All Interferons) and adaptive immunity with both B-cell and T- India Institute of Medical Sciences, Delhi, India cell (typically Th1) responses. IL-33, a nuclear- localized "alarmin" cytokine with pro-inflammatory/immune Abstract Content: Background: effects generated by IL-33Receptor positive cells, has been Type 1 reactions (T1R), an inflammatory condition, are suggested to be elevated in Flu models. IL-33 can be represented as inflammation of local skin patches in leprosy induced by the gp130 cytokine (OSM), which patients. Approximately, 30-40% leprosy patients have we have previously shown to induce a Th2/M2-skewed undergone T1R during the course of MDT. Previously, we inflammatory response (eosinopils, Arg1+Macrophages, IL- have reported that IL-23 is involved in Th17 cells 33, IL-4, eotaxin-2) in mouse lungs. Since IL-33 can differentiation and IL-6 in non-reactions (NR) leprosy influence both pathways of immune responses, and patients. Subsequently, recent finding by our group on mediates the Th2/M2-skewed inflammation induced by immunopathology of leprosy reactions showed that IL-6 OSM, we examined both OSM and IL-33 regulation by induces Th17 cell differentiation together with TGF-β in Influenza A (PR8 strain) lung infection of Naïve mice or of leprosy reactions. Here, we thus asked the next question mice primed with Advector to overexpress OSM whether IL-6 or IL-23 induced Th17 cells are different in (AdOSM). C57Bl/6 female mice (n=5/group) infected nature? intranasally with PR8 (1000pfu) showed time- Methods & Materials dependent elevation of OSM protein (ELISA) detected A total of 56 newly diagnosed and untreated non reaction in Broncoalveolar Lavage (BAL) (day 2, peaking day 4, still and T1R reactions patients were recruited. 48 hours PBMCs elevated at Day 7). Day 7 of mice infected with 250 or cultures were established with different combination of 1000pfu PR8 showed dose-dependent elevated OSM recombinant IL-6, IL-23 and TGF-β with or without mRNA in total-lung-homogenates (TLhomogenates). Mycobacterium leprae sonicated antigen (MLSA). However, mRNA levels of IL-33 in TLhomogenates were Subsequently PBMCs cultures were blocked with either reduced by >50% in PR8-infected mice, and total IL-33 antagonized sIL6R or sIL23R antibodies. Real Time PCR protein was also reduced by >50% as assessed was further used for gene expression analysis of IL-17A, IL- by Western blots in PR8 infected mice. Although we 17F, IL-6R and IL-23R. Different phenotypes of Th17 cells detected small increases in IL-33 by ELISA in BAL (day 2 were studied by flowcytometry and culture supernatant was and 4) the total amount of IL-33 in lavage was a small estimated for cytokine ELISA. fraction of IL-33 estimated in TLhomogenates (ELISA, Results: corrected to mg/protein) which were reduced by >50% by In this study, leprosy reactions showed high percentage of PR8 infection, consistent with mRNA and Western blot data. IL-17A producing CD4+ IL6R+ T cells as compared to stable Since this suggested active suppression of global lung IL-33 leprosy patients (p<0.001). On the other hand T1R leprosy by PR8, we then assessed the effects of PR8 super-infection reactions showed significantly low (p<0.001) IL-17A in mice with ongoing Th2/M2-skewed inflammatory producing CD4+ IL23R+ Th17 cells as compared to NR environment induced by AdOSM administered 7 days leprosy patients in 48 hours MLSA stimulated cultures prior. The effect of PR8 (250pfu, n>5 per group) was . Furthermore, recombinant IL-6, IL-23 and TGF-β examined at day 2 and day 5 post-PR8, and showed, significantly (p<0.001) promoted IL-17A in CD4+IL6+ T cells. starting at Day 2 and most evident at day 5, that Subsequently, IL-6R and IL-23R blocking experiments PR8 markedly reduced eosinophil and Arg1+Macrophage showed significantly (p<0.01) down regulated IL-17A in T1R accumulation, IL-33, IL-4, and eotaxin-2 expression in reaction as compared to NR leprosy patients. lung. This was associated with increased levels of both IFNβ and IFNγ expression in total lungs of PR8/AdOSM Conclusion: treated animals. Both IFNβ and IFNγ suppressed OSM- This study for the first time establishes that pathogenic Th17 induced IL-33 levels in mouse lung epithelial cells or cells produce IL-17 via IL-6R pathway in leprosy T1R fibroblasts in vitro, as assessed by Western blots of cell reactions. Thus, present approaches that specifically target lysates. Collectively, the results show that IL-33 Th17 cells and/or the cytokines that promote their expression (either basal, or previously elevated by OSM- development, such as IL-6, TGF-β and IL-23A may provide 68 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 more focused treatment strategies for the management of Abstract Content: Interferons are rapidly induced to M. leprae and its reaction. Hence, therapeutic approaches combat the virus. After virus clearance, interferons need to that aim to re-establish homeostasis by decreasing the return to basal levels as they can cause tissue damage. We production of IL-17 by Th17 cells may prove effective in the identified zinc-finger antiviral protein (ZAP) as a negative control of leprosy reaction and during its emergency. regulator of interferon, that binds to the 3’UTR of both type I Disclosure of Interest: None Declared and III interferon mRNAs. Previous studies have shown that ZAP is a major antiviral restriction factor of alphaviruses and LT126 HIV. However, we have uncovered two distinct functions of INTERLEUKIN-7 IS A POTENT INDUCER OF HIV- ZAP which are determined by alternative splicing. Of the two SUPPRESSIVE CHEMOKINES major alternatively spliced ZAP isoforms, the constitutively H. Schmeisser1,*, S. Owusu1, O. I. Hansen1, Q. Liu1, H. expressed long isoform (ZAP-L) acts as the host’s major Miao1, R. Cimbro2, T. Myers3, A. Fauci4, P. Lusso1 alphavirus restriction factor through direct binding of vRNA 1Laboratory of Immunoregulation, Viral Pathogenesis and recruitment of the exosome machinery for vRNA decay. Section, National Institute of Allergy and Infectious Conversely, the short isoform (ZAP-S), which contains Diseases, Bethesda, MD, 2Division of Rheumatology, Johns identical RNA-binding domains but is only expressed late Hopkins University School of Medicine, Baltimore, MD, after virus and interferon sensing, binds to and mediates the 3Genomic Technologies Section, Research Technologies degradation of several interferon mRNAs, thus acting as a Branch, 4Laboratory of Immunoregulation, National Institute negative feedback regulator of the interferon response. of Allergy and Infectious Diseases, Bethesda, MD, United While ZAP-S and ZAP-L are expressed from States the ZC3HAV1 gene and harbor identical RNA-binding domains, we uncovered differences in their intracellular Abstract Content: Although interleukin-7 (IL-7) may localization and mRNA splicing are the major determinants promote T-cell reconstitution in HIV-1 infection, its effects on for their specificity toward host versus viral RNA. ZAP-S is HIV-1 replication are still uncertain. In this study, we diffusely cytoplasmic, whereas ZAP-L forms perinuclear foci investigated the ability of IL-7 to induce the production of and co-localizes with membranes of modified endosomes – chemokines that may play a role in the endogenous control used as replication organelles by the alphavirus Sindbis of HIV-1 replication in infected hosts. Treatment with IL-7 virus – which is mediated by the C-terminal prenylation motif induced peripheral blood mononuclear cells (PBMC) from in ZAP-L. Through this, ZAP-L restricts viral replication early healthy donors to secrete significant amounts of the CCR5 during infection, while ZAP-S curbs interferon responses at ligands CCL3/MIP-1α, CCL4/MIP-1β, and CCL5/RANTES, a later stage. Through mRNA splicing assays, we as well as other antiviral chemokines, both in the presence identified ZAP-S mRNA is produced by a weaker intronic and absence of concomitant T-cell receptor-mediated non-canonical polyadenylation signal (ncPAS). The stimulation. Dose-response experiments showed that selection of this ncPAS to produce ZAP-S mRNA is supra-homeostatic concentrations of IL-7 are required for interferon dependent. We also identified the cleavage induction of HIV-suppressive chemokines. Neutralization of stimulation factor subunit 2 (CSTF2) as the effector protein CCL3, CCL4, and CCL5 increased HIV-1 replication in IL-7- that preferentially induces alternative polyadenylation to treated cells starting on day 3 post infection. Although T cells produce ZAP-S mRNA. Together, our study provides the were the main chemokine source in IL-7-stimulated molecular mechanisms underlying mRNA splicing and PBMC, depletion of monocytes significantly reduced distinct subcellular localization of ZAP isoforms, which chemokine production levels, suggesting that cross-talk determine their distinct specificity to host and viral target between T cells and monocytes is critical for this effect. We RNA. also investigated the signaling pathways involved in the Disclosure of Interest: None Declared mechanism of IL-7-mediated chemokine induction, showing that STATs play an important role. These results delineate LT128 potential mechanisms whereby IL-7 may facilitate HIV-1 GUT DYSBIOSIS EXISTS IN PULMONARY control in infected individuals. NONTUBERCULOUS MYCOBACTERIA INFECTION Disclosure of Interest: None Declared C.-C. Shu1,* 1Internal Medicine, National Taiwan University Hospital, LT127 Taipei, Taiwan DISTINCT ANTIVIRAL AND IMMUNE RESOLUTION FUNCTIONS OF ZINC-FINGER ANTIVIRAL PROTEIN Abstract Content: Background: Pulmonary ISOFORMS ZAP-S AND ZAP-L nontuberculous mycobacteria infection increases in J. Schwerk1,*, F. Soveg1, A. Ryan2, K. Thomas1, L. Hatfield1, recent decades. However, the clinical relevance of positive S. Ozarkar1, A. Forero1, A. Kell1, J. Roby1, L. So1, J. Hyde3, sputum for nontuberculous mycobacteria is far around M. Gale jr.1, M. Daugherty2, R. Savan1 30-50 %. indicating its probable host susceptibility. The 1Immunology, University Of Washington, Seattle, 2Biological cytokine response of peripheral mononuclear cells has been Sciences, University of California San Diego, La Jolla, reportedly decreased but the nature remains unclear. The 3Microbiology, University of Washington, Seattle, United role of gut dysbiosis of microbiota could be responsible for it States but lacks of study in Pulmonary nontuberculous mycobacteria infection.

69 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Methods: Patients with Pulmonary nontuberculous characterised by an exaggerated IFN-α response and mycobacteria infection and healthy controls were enrolled. increased levels of STAT1 activation by tyrosine 701 and The cytokine response of peripheral mononuclear cells and serine 727 phosphorylation but not STAT3 activation in gut microbiota as well as stool toll like receptor (TLR) ligands comparison to single transgenic controls. To study further were checked and analyzed. the role of STAT1 transactivation in IFN-α and IL-6 signalling Results: We stimulated the participant’s PBMCs and found together, we interbred the GFAP-IFN-IL6 mice with mice that that cytokines of interferon-gamma (IFN-γ) and tumor express a S727A mutant STAT1. In the absence of pS- necrosis factor-alpha (TNF-α) were lower in patients that STAT1, mice were partially protected against the IFN-α and that in the controls. In regard to gut microbiota, there is IL-6 driven neurological disease, with decreased expression dysbiosis with decreasing Shannon index in the patients of inflammatory genes. This indicates that serine 727 with pNTM comparing with their controls. The TLR2 ligand phosphorylation regulates STAT1 transcriptional activity, was decreased in pNTM patients than the controls. and enhances the pathogenic actions of IFN-α and IL-6 Conclusions: We preliminarily found that the cytokines of together in the CNS. PBMC response, stool TLR2 ligand and microbiota diversity Overall, our findings suggest that serine phosphorylation of of gut were decreased in patients STAT1 is a potent modulator of neuroinflammation. withPulmonary nontuberculous mycobacteria infection. Consequently, defining its modulatory role in these diseases The gut dysbiosis might play a role in immunosuppression will advance current understandings of complex cytokine for pathogenesis of Pulmonary nontuberculous signaling and the pathogenesis of neuroinflammatory mycobacteria infection. The mechanism required further diseases. investigation. Disclosure of Interest: None Declared Disclosure of Interest: None Declared LT130 LT129 EFFECTS OF PROBIOTIC SUPPLEMENTATION ON SERINE 727 PHOSPHORYLATION OF STAT1 WORSENS CYTOKINES BALANCE AFTER A MARATHON RACE. A SEVERE NEUROLOGICAL DISEASE INDUCED BY E. Tavares-Silva1,*, G. S. Leite2, H. A. Batatinha3, A. S. COMBINED CHRONIC PRODUCTION OF IFN-Α AND IL- Resende2, A. H. Lancha Jr.2, J. C. Neto3, R. V. Thomatieli- 6 Santos1 on behalf of LAFISBE - Laboratório de Fisiologia e P. Songkhunawej1,*, M. J. Hofer1 Bioquímica do Exercício 1School of Life and Environmental Sciences, The University 1Psychobiology, UNIFESP, 2Biodynamics of the Movement of Sydney, The University of Sydney, Australia of the Human Body, 3Cell and Tissue Biology, USP, São Paulo, Brazil Abstract Content: In the central nervous system (CNS), cellular production and signaling of cytokines is tightly Abstract Content: Background regulated. Not surprisingly, an increase in cytokine signature Previous studies have demonstrated several changes in the has been documented as a key feature of a number of cytokines production after strenuous exercise. Cytokines neurological disorders including Aicardi-Goutières greatly influence the immune response and metabolism of syndrome, multiple sclerosis and neuromyelitis optica. athletes who often suffer immunomodulation after prolonged Cytokine-induced disease features are closely modeled exercise. Probiotic supplementation has been used to through the use of transgenic mice with CNS-targeted mitigate the effects of strenuous exercise on the immune production of single cytokines such as IFN-α (GFAP-IFN system. Thus, the present study aimed to evaluate the effect mice) and IL-6 (GFAP-IL6 mice) where clinical and of daily probiotic supplementation on IL-2, IL-4, IL-10 and pathological phenotypes reflect the actions of the particular TNF-α concentration and IL2/IL-4 and TNF-α/IL-10 ratios of cytokine produced. A key finding in GFAP-IFN and GFAP- runners submitted to an official Marathon race. IL6 mouse models is the activation of transcription factor Methods STAT1 by serine 727 phosphorylation (pS-STAT1). Runners (n:30) were supplemented for thirty days with However, GFAP-IFN or GFAP-IL6 mice that express S727A probiotics 10x109 CFU of Lactobacillus Acidophilus and mutant STAT1 are indistinguishable from GFAP-IFN and Bifidobacterium Lactis or placebo double-blinded. At GFAP-IL6 controls, suggesting a minimal biological impact baseline (30d before), 24 hours before (1d before), one hour for pS-STAT1 in response to either IFN-α or IL-6 in the living after (1h after), and five days after an official marathon (5d CNS. Here we studied the contribution of pS-STAT1 in a after), blood were collected for analyses. The plasma mouse model of combined IFN-α or IL-6 production in the concentration of IL-2, IL-4, IL-10, and TNF-α was analyzed CNS. using the Simultaneous analysis of multiple cytokines To study the actions of IFN-α and IL-6 in combination, we (Luminex technology with magnetic Beads). Data normality interbred GFAP-IFN mice and GFAP-IL6 mice to obtain verified using the Shapiro-Wilk's test, and the Anova Two- double transgenic GFAP-IFN-IL6 mice. Compared with Way with a significance level of p ≤ 5% was applied. single transgenic animals, GFAP-IFN-IL6 mice developed a Results more pronounced and accelerated disease that was In both groups, IL-2 decreased at all moments compared to characterised by severe ataxia and tremors. In the the baseline. At probiotic group, the IL-2 decreased 1h after cerebellum of these mice, gross leukocyte infiltrates, and 5d after compared to 24h before. Compared to the destruction of the Purkinje cell layer and increased baseline, IL-4 concentration decreased 1h after and 5d after microgliosis and astrogliosis was observed. This was on placebo group, while significantly decreased 5d after on 70 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 probiotic group. IL-10 increased 2.700% and 2.100% on differences in the microglia response to CSF1, we examined placebo and probiotic, respectively, 1h after the Marathon the potential contribution of the . race compared to 1d before and return to baseline levels 5d Although we never detected infiltration of lymphocytes in the after in both groups. TNF-α decreased at all moments spinal cord after i.t. CSF1, we did find a robust expansion of compare to 30d before in both groups. No statistical lymphocyte populations, in particular T-lymphocytes, in differences were found between groups for Th1/Th2 spinal cord meninges. Furthermore, i.t. CSF1 readily balance. However, 5d after the race, there was a change in provoked mechanical hypersensitivity in T-lymphocyte IL-2/IL-4 favoring the immune response in the probiotic depleted female mice. Ongoing work is identifying the role group compared to 30d before. The IL-10/TNF-α ratio was of specific T-lymphocyte subsets in CSF1 induced pain lower at 1d before and 5d after compared to 30d before in hypersensitivity. In conclusion, our data reveals that CSF1 both groups. After the marathon race, the IL-10/TNF-α activates microglia in a sex specific manner and that the balance was higher compared to all moments demonstrating adaptive immune system is involved in this process. higher IL-10 concentrations. Disclosure of Interest: None Declared Conclusions The supplementation of 10x109 CFU of Lactobacillus LT133 Acidophilus and Bifidobacterium Lactis for thirty days did not CHANGES IN CYTOKINE PRODUCTION BY IMMUNE modify the plasma concentration of IL-2, IL-4, IL-10 and CELLS AND ADIPOSE TISSUE DURING COLD TNF-α and Th1/Th2 balance compared to placebo. Specific ADAPTATION changes in the probiotic group were found 5d after the D. Vašek1,*, M. Hájková1,2, P. Vebr3, B. Porubská1,2, A. marathon race favoring the Th1 response. Strenuous Kratochvílová1, J. Žurmanová3, M. Krulová1,2 on behalf of . physical exercise has a potential to increase momently the 1Department of Cell Biology, Faculty of Science, Charles IL-10/TNF-α balance. Several studies demonstrate benefits University, 2Department of Transplantation Immunology, of probiotic supplementation. Further investigations are Institute of Experimental medicine CAS, 3Department of necessary to verify the efficacy of these specifics strains and Physiology, Faculty of Science, Charles University, Prague, dose to immune system. Czech Republic Acknowledgments - Financial Support: FAPESP #2016/25821-5: We declare that there is no conflict of Abstract Content: Maintaining energy homeostasis at interest in research. reduced temperatures is essential for the survival of the Disclosure of Interest: None Declared organisms. We determined the effect of cold stress and cold adaptation on the immune system in a rat model, as well as LT132 on the cytokine production in different types of adipose FEMALE T-LYMPHOCYTES INHIBIT CSF1-MICROGLIA tissue. INDUCED PAIN HYPERSENSITIVITY Various factors participate in the process of I. D. Vainchtein1,*, J. A. Kuhn2, J. Braz2, M. Bernstein2, J. thermoregulation, a crucial step involves the binding of Ortiz-Carpena3, A. B. Molofsky3, A. V. Molofsky1, A. I. norepinephrine (NE) to adrenergic receptors, and in long- Basbaum2 term adaptation, thyroid hormones play an important role. 1Department of Psychiatry, 2Department of Anatomy, To decipher neuro-immune interaction at the reduced 3Department of Laboratory Medicine, University of temperature, we monitored changes in the presence and California, San Francisco, San Francisco, United States activation of individual immune cell populations isolated from peripheral blood, spleen and peritoneal cavity at Abstract Content: Activation of spinal cord microglia is an various time points of cold adaptation and correlated them important contributor to peripheral nerve injury-induced with the expression of enzymes crucial for NE and thyroid mechanical hypersensitivity, a hallmark of neuropathic pain. hormone (triiodothyronine – T3) activity. Significant changes We previously reported that nerve injury induces de novo in immune response to various stimuli, including cytokine primary sensory neuron expression of colony-stimulating production, in particular IL-1β, IL-4, IL-6, IL-10 and TNF-α, factor 1 (CSF1), a cytokine that activates spinal cord indicated switch to anti-inflammatory environment and microglia, and that upregulation of CSF1 is required for the response skewed towards Th2 immunity in cold adapted associated mechanical hypersensitivity. Here we report that animals. intrathecal (i.t) injection of CSF1 is sufficient to induce Surprisingly, we observed change in IL-17 production that mechanical hypersensitivity in male but not female mice. was associated with γδT-cell population. Adaptation to the Interestingly, i.t. injection of CSF1 not only promotes reduced temperature was also accompanied by changes in proliferation, but also leads to microglia activation and loss cytokine production in brown and white adipose tissue. We of morphology complexity in males, but not females. suggest that cellular metabolism and activity of uncoupling Transcriptional profiling of spinal cord microglia after i.t. proteins in immune cells are responsible for observed CSF1 demonstrated that male, but not female microglia, changes. However, further studies of neuro-immune robustly upregulate immune activation genes involved in crosstalk that occur during cold adaptation are necessary. cytokine production, chemotaxis and cell adhesion and Understanding mechanisms of cold adaptation and overall acquire a phenotype previously identified as ‘disease subsequent metabolic changes can help in the development associated microglia’. In addition, male microglia of therapeutic approaches for obesity and related metabolic downregulate many of their tissue supportive functions. To disorders. identify the mechanisms that underlie these sex specific Disclosure of Interest: None Declared 71 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

role in neural precursor function; ii) be used for engagement LT134 of NPCs for enhanced oligodendrocyte regeneration; and iii) CHEMOKINE FRACTALKINE (CX3CL1) MEDIATES be involved in novel cell-to-cell interactions between NPCs NEURAL PRECURSOR CELL FUNCTION FOR and microglia, both of which express chemokine receptors, ENHANCED OLIGODENDROCYTE PRODUCTION such as CX3CR1. Our future studies will address the ability A. Voronova1,*, A. E. Watson1, Y. J. Li1, M. M. Alves de of exogenous fractalkine to enhance remyelination and Almeida1, N. Dittmann1, K. Goodkey1, T. Footz1 modify precursor-microglia crosstalk in mouse models of 1Medical Genetics, University of Alberta, Edmonton, Multiple Sclerosis. Canada Disclosure of Interest: None Declared

Abstract Content: LT135 Introduction: Oligodendrocytes produce myelin, an INTERLEUKIN-10 DEFICIENCY EXACERBATES BRAIN essential component of the central nervous system. In the IL-6 AND LPS-INDUCED TAU PATHOLOGY adult brain, oligodendrocytes are generated via a 2-step L. Weston1,*, S. Jiang1, D. Chisholm1, K. Bhaskar1 process: neural precursor cells (NPCs) in the subventricular 1University Of New Mexico, Albuquerque, United States zone (SVZ) niche are committed to oligodendrocyte precursor cells (OPCs), which then differentiate into Abstract Content: Neurodegenerative diseases, oligodendrocytes. Formation of oligodendrocytes from their such as Alzheimer's disease, are associated with multipotent progenitors is regulated by signals present in the accumulation of microtubule associated protein tau (MAPT NPC niche. The chemokine fractalkine (CX3CL1), which is or tau). The presence of hyperphosphorylated tau (p-tau) secreted by neurons in the NPC niche and signals via its has been shown to strongly correlate with cognitive decline sole receptor (CX3CR1), increases oligodendrocyte and neuroinflammation. Though anti-inflammatory cytokines formation from cortical NPCs during embryonic are key players in regulating immune responses, the direct development (Voronova et al., Neuron, 2017). CX3CR1 is effects of certain prominent anti-inflammatory cytokines in also expressed in postnatal and adult NPCs and OPCs tau pathology is still unclear. One of the most established (reviewed in Watson et al., Neurosci Lett 2020). However, anti-inflammatory cytokines is interleukin-10 (IL-10) which the role of fractalkine signalling in postnatal NPCs and OPCs plays a role in downregulating inflammatory microglia is not currently known. responses. We hypothesized that IL-10 plays a role in Methods: We utilized single cell RNA fluorescent in situ regulating the pro-inflammatory pathway that induces tau hybridization (RNA scope) to detect CX3CR1 expression in phosphorylation within the brain. postnatal brain. We also injected fractalkine directly Using lipopolysaccharide (LPS, 3 mg/kg b.w.; i.p.; conjugated to fluorophore Alexa-647 (CX3CL1-647) directly single dose) to induce inflammation in C57BL/6J mice, we into the lateral ventricle of adult brain to identify which cell compared the effects of systemic inflammation on types bind fractalkine in vivo. We then infused fractalkine endogenous mouse tau in the brains of Il10-deficient (Il10-/- into the lateral ventricle of adult NPC lineage tracing mice ) mice versus wildtype controls (Il10+/+). First, basal levels of (NestinCreERT2;RosaYFPSTOP/+) to assess the role of tau phosphorylation measured through western blot with fractalkine signaling in de novo oligodendrocyte genesis. To AT8, AT180, and PHF1 antibodies were no different in Il10- test the direct effect of fractalkine on NPCs, we generated /- and Il10+/+ mice. Following LPS administration, tau NPCs or OPCs from murine postnatal SVZ neural stem cell phosphorylation increased in both groups with significant neurospheres, in which the only cells that propagate are multi-fold increases of p-tau in Il10-/- hippocampal (Hp) precursors while other cells such as microglia, neurons and lysates compared to Il10+/+ controls. Interestingly, total tau astrocytes do not survive. Microglia-free NPC or OPC levels were significantly reduced in Il10-/- mice after LPS monolayer cultures were incubated in the presence of administration indicating loss of neuronal tau. Quantification soluble fractalkine or CX3CL1 or CX3CR1 function blocking of AT8+ and AT180+ area through IHC confirmed antibodies and analyzed for differences in proliferation, significantly increased phosphorylated tau epitopes in the differentiation and survival. dentate gyrus of Il10-/- mice. Additionally, we observed a Results: Here we provide evidence that in addition to small but statistically significant decrease in NeuN+ area microglia, postnatal SVZ NPCs express CX3CR1 and bind with notable detection of TUNEL positive staining in the CA3 fractalkine in vitro and in vivo. When FKN is added to region of Il10-/- LPS mice compared to the control group. microglia-free NPC cultures, it enhances their differentiation Microglia cell morphology and inflammatory into OPCs and oligodendrocytes without affecting NPC cytokines within the perfused brain tissue was examined. proliferation or survival. Inhibition of fractalkine signalling in Quantitative comparison of microglia morphology showed microglia-free OPC cultures with function-blocking that Il10-/- microglia had significantly reduce branching antibodies inhibits oligodendrocyte differentiation. Finally, compared to Il10+/+ microglia suggesting an activated cell infusion of fractalkine into lateral ventricle of adult NPC type. In both groups, LPS administration elevated active p38 lineage tracing mice enhances OPC and oligodendrocyte MAPK, which is pro-inflammatory and shown to genesis from Nestin-positive SVZ NPCs in vivo. phosphorylate tau. In Il10-/- mice, phospho-p38 elevation Conclusions: In summary, we demonstrate fractalkine was nearly two-fold significantly higher than the Il10+/+ signalling directly mediates postnatal SVZ NPC and OPC group. Interestingly, statistically significant lower amounts of function. Our results raise the possibility that immunological Total p38 were observed in Il10-/- Veh group compared to chemokines, such as fractalkine, may i) play an important Il10+/+ Veh controls. After LPS, the Il10+/+ group had elevated 72 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 levels of IL-6, IL-1b, and TNFa with concomitant increase in Abstract Content: The cytokine IL-1β is an important driver IL-10 within cortical brain lysates. Furthermore, Il10-/- mice of fever and a key factor in the host response against had significantly enhanced levels of IL-6 and TNFa infection. Interestingly, IL-1 β differs from other common compared to Il10+/+ mice after 24 hours of LPS cytokines (TNF- , IL-8) as it is produced as a pro-form and administration. IL-6 was particularly untethered in the IL-10 requires catalytical processing by caspase-1 yielding its deficient mice compared to the IL-10 positive group. To mature form; but also since is released in an unconventional evaluate the potential role of this cytokine to promote tau manner, rather than being released over the conventional phosphorylation in neurons, we administered recombinant route via ER and Golgi. It was shown that caspase-1 also IL-6 directly to primary neuronal mouse cells, in vitro, and processes the protein gasdermin-D (GSDMD) and that the observed a pattern of tau phosphorylation and total tau loss N-terminal GSDMD fragment that is generated by this compatible with in vivo observations. These data support the cleavage forms pores in the plasma membrane, thereby significance of IL-10 in modulating the pro-inflammatory causing a caspase-1 dependent form of necrotic cell death, pathway that induces microglia activation and tau known as pyroptosis. Since IL-1β secretion also depends on phosphorylation. GSDMD we investigated the role of GSDMD in Disclosure of Interest: None Declared unconventional secretion of IL-1β. Here we show that gasdermin-D is required for IL-1 secretion by LT136 macrophages, dendritic cells and partially in neutrophils, SALT GENERATES ANTI-INFLAMMATORY TH17 CELLS and that secretion is a cell-lysis-independent event. BUT AMPLIFIES THEIR PATHOGENICITY IN PRO- Liposome transport assays in vitro further demonstrate that INFLAMMATORY CYTOKINE MICROENVIRONMENTS gasdermin-D pores are large enough to allow the direct J. Matthias1 on behalf of Zielinski, C. Zielinski2,* on behalf of release of IL-1 . Moreover, IL-18 and other small soluble Julia Matthias cytosolic proteins can also be released in a lysis- 1TUM, 2TranslaTUM, Technical University of Munich, independent but gasdermin-D-dependent mode, while Munich, Germany proteins larger than 25 kDa or artificially enlarged IL-1 (tagged with eGFP) remain trapped in the cytosol, Abstract Content: T helper cells integrate signals from their suggesting that the gasdermin-D pores allow passive the microenvironment to acquire distinct specialization release of cytosolic proteins in a size-dependent manner. programs for efficient clearance of diverse pathogens or for Disclosure of Interest: None Declared immunotolerance. Ionic signals have recently been demonstrated to affect T cell polarization and function. LT138 Sodium chloride (NaCl) was proposed to accumulate in TOLL-LIKE RECEPTOR 4-MEDIATED INFLAMMATION peripheral tissues upon dietary intake and to promote TRIGGERED BY EXTRACELLULAR IFI16 IS ENHANCED autoimmunity via the Th17 cell axis. Here we demonstrate BY LIPOPOLYSACCHARIDE BINDING that high NaCl conditions induced a stable, pathogen- A. Iannucci1,2,*, V. Caneparo1, S. Raviola1,2, G. Griffante2, S. specific, anti-inflammatory Th17 cell fate in human T cells in Landolfo3, M. Gariglio1,2, M. De Andrea1,3 vitro. The p38/MAPK pathway, involving NFAT5 and 1Center for Translational Research on Autoimmune and SGK1, regulated FoxP3 and interleukin (IL)-17A-expression Allergic Diseases, 2Department of Translational Medicine, in high-NaCl conditions. The NaCl-induced acquisition of an University of Eastern Piedmont, Novara, 3Department of anti-inflammatory Th17 cell fate was confirmed in vivo in an Public Health and Pediatric Sciences, University of Turin, experimental autoimmune encephalomyelitis (EAE) mouse Turin, Italy model, which demonstrated strongly reduced disease symptoms upon transfer of T cells polarized in high NaCl Abstract Content: conditions. However, NaCl was coopted to promote murine Background: Since its discovery in the early 90s, a and human Th17 cell pathogenicity, if T cell stimulation cornucopia of biological activities has been attributed to the occurred in a pro-inflammatory and TGF-b-low cytokine IFI16 protein, including cell cycle regulation, tumor microenvironment. Taken together, our findings reveal a suppression, apoptosis, DNA damage signaling, virus context-dependent, dichotomous role for NaCl in shaping sensing, and virus restriction. In addition, aberrant IFI16 Th17 cell pathogenicity. NaCl might therefore prove expression and release in the extracellular space has been beneficial for the treatment of chronic inflammatory diseases reported in a series of inflammatory conditions. The current in combination with cytokine-blocking drugs. hypothesis is that overexpression of the IFI16 protein occurs Disclosure of Interest: None Declared in tissue compartments where it is not physiologically expressed during inflammation. The ensuing release of the Lightning Talk Session 2: Innate Immunity II IFI16 protein into the extracellular space may allow it to LT137 behave like a damage-associated molecular pattern THE GASDERMIN-D PORE ACTS AS A CONDUIT FOR (DAMP) that signals through the Toll-like receptor 4 (TLR4) IL-1Β SECRETION triggering inflammation by itself or through interaction with R. Heilig1,*, M. S. Dick2, L. Sborgi2, S. Hiller2, P. Broz1 exogenous molecules, e.g. lipopolysaccharide (LPS). 1Department of Biochemistry, University of Lausanne, Methods: Pull down assays and ELISA were used to Lausanne, 2Biozentrum Basel, University of Basel, Basel, characterize IFI16 binding activity to LPS. The human Switzerland monocytic cell line THP-1 and the renal carcinoma cell line 786-O were used as target cells to define IFI16-induced 73 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 proinflammatory activity. Co-immunoprecipitation (co-IP), infection disease courses. Overall, the severity and number surface plasmon resonance (SPR), and silencing of lines exhibiting brain histopathology increased over the experiments were used to define IFI16 signaling. studied time course, with the greatest histopathological Results: We show that the IFI16 HINB domain binds to the disease severity in the largest number of lines occurring at lipid A moiety of either high or weak TLR4 agonist LPS day 12 post-infection. Meningitis, perivascular variants. Treatment of THP-1 or 786-O cells with IFI16 led inflammation, and parenchymal inflammation were the most to increased production of proinflammatory cytokines, which prevalent types of brain histopathological phenotypes. We was further enhanced when IFI16 was pre-complexed with found that brain histopathology associated with other WNV sub-toxic doses of high TLR4 agonist LPS but not low disease measures, such as weight loss and brain viral load, agonists. Silencing of TLR4/MD-2 or MyD88 abolished and may associate with gene expression of IFIT, IFN-beta, cytokine production. These findings alongside with other in and IL-12 in the brain. Using the histological phenotype vitro binding experiments indicate that IFI16 interacts and information, we performed genetic mapping of quantitative signals through TLR4. trait loci (QTL) and discovered genomic regions of interest Conclusions: Collectively, our data provide compelling that were associated with WNV-infection induced brain evidence that: i) IFI16 is a DAMP that triggers inflammation histopathologies. Together our findings show that the CC through the TLR4/MD2-MyD88 pathway; and ii) its activity is model of WNV is a relevant and powerful mouse model for strongly enhanced upon binding to LPS variants regarded mechanistic studies of WNV infection and neuropathology. as full TLR4 activators. These data strengthen the notion Disclosure of Interest: None Declared that extracellular IFI16 functions as DAMP and point to new pathogenic mechanisms involving the crosstalk between LT140 IFI16 and subtoxic doses of LPS. OBESITY-RELATED AND LDL-MEDIATED LOSS OF NK Disclosure of Interest: None Declared CELL DEGRANULATION AND CYTOTOXOCITY IS IN PART MEDIATED BY NFKB INHIBITION LT139 R. M. Islam1,* on behalf of Powell-Wiley , C. Gutierrez- BRAIN HISTOLOGY AND PATHOBIOLOGY OF WEST Huerta11, B. Turner1, V. M. Mitchell1, B. S. . Collins1, Y. NILE VIRUS INFECTION IN THE COLLABORATIVE Baumer1, T. M. Powell-Wiley1 CROSS MOUSE MODEL 1National Heart Lung and Blood Institute, National Institutes R. C. Ireton1,2,*, A. Sekine2, S. Thomas2, P. Treuting3, R. of Health, Ashburn, United States Green2, K. Voss2, M. Mooney4, L. Whitmore2, S. McWeeney4, M. Gale, Jr.1,2,5,6 Abstract Content: The global obesity epidemic increases 1Center for Innate Immunity and Immune Disease, cardiovascular disease (CVD) risk worldwide. In the U.S., 2Immunology, 3Comparative Medicine, University of obesity is most prevalent among African-American (AA) Washington, Seattle, 4Medical Informatics & Clinical women, contributing to CVD disparities. Natural Killer (NK) Epidemiology, Oregon Health & Science University, cells have been reported as important in CVD development Portland, 5Microbiology, 6Pathology, University of and display impaired function in obese individuals. However, Washington, Seattle, United States little is known about the potential drivers of obesity-related NK cell dysfunction especially in at-risk groups like AA Abstract Content: West Nile Virus (WNV) is an emerging women. Thus we explored links between NK cells and neuroinvasive flavivirus. During human infection, WNV obesity and aimed to identify potentially significant biological activates host innate antiviral immunity through the RIG-I- pathways for both chronic disease outcomes and like receptor pathway, but evades the actions of interferon prevention. First, we compared the NK cell profile of lean AA (IFN) and can cause serious encephalitis disease with a 6% women (BMI<25) and AA women with obesity (BMI>35) case lethality. WNV infection immunopathology has been (n=15 lean/14 obese, age-matched). While we saw no well characterized in C57BL/6J mice, but understanding the significant differences in overall CD3-/CD56+ NK cell full impact of host genetics on the variety of disease proportions we found a significant decrease in outcomes and susceptibility is limited with this inbred model CD56dim/CD16hi cytotoxic NK cells and a trending increase because of the lack of genetic variability. We used the in CD56hi/CD16low proliferating NK cells among those with Collaborative Cross (CC) mouse model to evaluate the obesity. CD3-/CD56+ isolated NK cells of 7 women in each pathobiology and innate immune regulation that underlies group were further characterized. NK cells isolated from AA WNV disease progression and neuroinvasion. By using a women with obesity displayed less degranulation towards standardized histology scoring system to evaluate viral K562 cells. Decreased intracellular levels of Granzyme B localization and immunopathology of WNV infection in brain were also found. In a second step, we used an ex vivo tissue isolates from CC lines, we evaluated correlates of approach to determine possible mediating variables. innate immune action and IFN actions in 101 genetically Primary naïve NK cells of a healthy donor were treated with diverse CC mouse lines. Our studies reveal that WNV study participants’ serum overnight (n=60, 93% female, age infection in the CC captures the diversity in disease 60.8 years, all AA). Degranulation and intracellular cytokine susceptibility and outcomes observed in humans, with 72% levels were detected after exposure to K562 cells and the of the evaluated lines demonstrating brain histopathologies results subjected to multivariable regression analysis during the course of infection. We also observed lines with against clinical parameters. Interestingly, after adjusting for unique disease-course histopathologies, which may provide ASCVD and BMI, serum LDL levels negatively associated a new modeling resource for the study of alternative WNV with CD107a (b=-0.23, p=0.07), IFNg (b=-0.35, p=0.005), 74 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

GM-CSF (b=-0.38, p=0.002), perforin (b=-0.30, p=0.02), and Collectively, CD86 appears to be an alternative useful Granzyme B (b=-0.39, p=0.002), indicating loss in function marker for Sca-1, which allows us to analyze “bona fide” and cytotoxicity with increasing LDL levels. To determine if hematopoietic responses under biological stresses such as LDL could be a driver of obesity-related NK cell function infection and inflammation. loss, naïve isolated NK cells were treated with LDL. In Disclosure of Interest: None Declared accordance with the ex vivo data significant decreases in CD107a, IFNg, and perforin were detected, with additional LT142 downregulation of TNFa. Analysis of the secreted cytokines A DISTINCTIVE THERAPEUTIC CANDIDATE TO of LDL-treated NK cells revealed a decrease in IFNg and RESTRICT INFLUENZA VIRUS-CAUSED LUNG TNFa. By using proteomics of LDL-treated naïve NK cells INFECTION: INTERFERON-LAMBDA-LOADED (n=3), we determined that NFkb was significantly decreased SURFACTANT NANOPARTICLE in its expression. It is noteworthy that NFkb pathway is H. J. Kim1,*, C. H. Gil2, A. Jo2, J. Won2, S. Kim2 important in NK cell cytotoxicity and therefore critical for NK 1Department of Otorhinolaryngology, 2SEOUL NATIONAL cell function. Together, our data suggest that obesity as a UNIVERSITY COLLEGE OF MEDICINE, Seoul, Korea, significant risk factor of CVD acts on NK cells function and Republic Of cytotoxicity via LDL by regulating NFkb signaling pathways. Our findings suggest crucial signaling mechanisms in NK Abstract Content: Rationale: We verified that interferon cell mediated cytotoxicity that may be most affected by (IFN)-λ gene expression and secreted protein were more obesity and hyperlipidemia as CVD risk factors. highly induced after influenza A virus (IAV) infection and also propose that IFN-λ is the primary interferon to mediate an Disclosure of Interest: None Declared anti-viral defense in vivo respiratory epithelium. We found that inhaled delivery of IFN-λs induced antiviral innate LT141 immune responses in respiratory tract and surfactant-based CD86-BASED ANALYSIS ENABLES IDENTIFICATION nanoparticle might be a useful material for IFN-λ delivery to OF HEMATOPOIETIC PROGENITORS UNDER in vivo lung. BIOLOGICAL STRESSES WHICH UPREGULATE Material and Methods: C57BL/6 mice (N=30) were INTERFERON exposed to whole body mainstream by the SCIREQ M. Kanayama1,*, T. Ohteki1 “InExpose” system and surfactant-based nanoparticle 1 Department of Biodefense Research, Medical Research (diameter 100nM) including recombinant IFN-λ2/3 (10 ug), Institute, Tokyo Medical & Dental University, Tokyo, Japan surfactant (1mg/ml), protamine (2.5ug/ml) was inoculated into mice by inhaled delivery. Abstract Content: Systemic infection and inflammation Results: IAV-infected mice were observed to have most skew hematopoiesis and break balances between blood cell significant loss of body weight, severe pathologic findings, populations. Examining the impact of infection/inflammation viral replication and highest viral titer in their lung at 7 days in hematopoiesis could be helpful to understand the after infection. IAV mRNA levels, nucleoprotein and viral titer pathology. Sca-1 has long been used as an essential cell were completely lower in the lung of IAV-infected mice which surface marker to distinguish hematopoietic stem progenitor were inoculated with IFN-λ-loaded nanoparticles at the cells (HSPCs; Sca-1+) from Sca-1- same time with IAV infection. In addition, the lung erythrocyte/megakaryocyte- or myeloid-progenitors such as inflammation was significantly improved and body weight MEPs, GMP and CMPs. It has been recently reported that was maintained until 7 days after infection in IAV-infected interferon (IFN), which could be upregulated during viral and mice with IFN-λ-loaded nanoparticles. IAV-infected mice bacterial infection, make the identification of hematopoietic with administration of with IFN-λ-loaded nanoparticles progenitors difficult by inducing Sca-1 expression in the Sca- exhibited an even greater increase of IFN-stimulated genes 1- downstream progenitors. (ISG) in their lung and more induction of ISG after In this study, we confirmed IFN-dependent Sca-1 inoculation of IFN-λ-loaded nanoparticles could last longer upregulation in downstream progenitors after compared the IAV-infected mice with single treatment with lipopolysaccharide administration or induction of systemic IFN-λ. infection with a cecal ligation puncture model. IFN-mediated Conclusion: The current data show that inhaled delivery of Sca-1 upregulation in downstream progenitors IFN-λs mediated more potent antiviral immune responses overestimated apparent number of HSPCs and and restrict IAV replication in the respiratory tract. underestimated the number of downstream progenitors. To Surfactant-based nanoparticle can deliver IFN-λ more solve this issue, we screened a panel of cell surface efficiently to in vivo lung and IFN-λ-loaded surfactant molecules and newly identified CD86 as an alternative nanoparticle might be effective therapeutics to limit IAV- marker for Sca-1. Under steady-states, the CD86- caused lung infection. expressing cells mostly overlapped with the cells expressing Disclosure of Interest: None Declared Sca-1. Contrary to Sca-1, however, CD86 expression was nearly unchanged under infection/inflammation. At an early LT144 phase of infection, CD86-based analysis demonstrated the DELETION OF TRANSFERRIN RECEPTOR 2 (TFR2) intact stemness of HSCs and activation of erythropoiesis, ENHANCES INFLAMMATION AND MACROPHAGE which were not observed by using Sca-1-based analysis. ACTIVATION

75 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

M. G. Ledesma Colunga1,*, U. Baschant1, L. C. Hofbauer1, 1Georgetown University, 2George Mason University, M. Rauner1 Washington DC, United States 1Department of Medicine III & Center for Healthy Aging, Technische Universität Dresden, Dresden, Germany Abstract Content: Introduction Interferon stimulated genes (ISGs) are a collection of genes Abstract Content: important in the early innate immune response. The Rheumatoid arthritis is an autoimmune disease regulation of ISGs has been extensively studied in cells characterized by disordered innate immunity, chronic exposed to significant interferon stimulation, but less is inflammation and progressive joint destruction with frequent known about ISG regulation in homeostatic regimes. Most synovial iron deposition. Transferrin receptor 2 (Tfr2) is a previous homeostatic ISG studies have focused on a small focal point in the regulation of systemic iron levels and bone number of ISGs in a single cell type. Our goal in this study mass. Loss of Tfr2 function results in low hepcidin was to characterize homeostatic ISG regulation across a expression and iron overload. As neither the role of iron nor broad array of ISGs and potential regulation factors and Tfr2 has been explored in arthritis, here, we aimed to across multiple cell types. investigate whether Tfr2 deletion and/or iron overload Methods impacts on the pathogenesis of inflammatory arthritis. We collected 33 expression datasets from the GEO We analyzed iron overloaded Tfr2-deficient mice (Tfr2-/- database across four cell types: CD4, pulmonary epithelial, ) and their wild-type (Tfr2+/+) littermate controls as well as fibroblast mice lacking Tfr2 only in macrophages that are not iron and macrophage cells. We sought to construct ISG- overloaded for their capacity to develop K/BxN serum regulator modules that identified subsets of ISGs with transfer arthritis (STA). Arthritis severity was assessed by expression levels that correlated with expression levels of clinical scores of paw swelling and hind paws were collected subsets of regulator genes. We used a linear regression for micro-CT, histomorphometry, quantitative PCR and flow model to construct such modules and to quantify the degree cytometry. to which ISG expression levels was predicted by regulator Tfr2-/- mice developed more pronounced joint swelling with expression levels. enhanced mRNA expression of inflammatory markers (Il1b, Results Inos and Ifng), synovial hypertrophy and bone erosion as Our results show regulation of 100s of ISGs at homeostasis compared to Tfr2+/+ mice. Furthermore, the infiltration of through the ISGF3 molecule and also suggest additional myeloid cells including GR1+ neutrophils and F4/80+ regulation through IRF7 and IRF8 associated pathways. We macrophages/monocytes at day 7 was increased in the find that at homeostasis roughly 50% of ISGs have joints of Tfr2-/- mice, suggesting a role of Tfr2 and/or iron expression levels significantly correlated with ISGF3 overload in arthritis progression. To elucidate whether Tfr2 expression. We find that ISG expression levels varied in has a direct role in the pathogenesis of arthritis or whether their correlation with ISGF3 across cell types, with epithelial the effects are merely mediated via the systemic iron and macrophage cells showing more correlation than CD4 overload, we induced STA in mice with a conditional deletion and fibroblast cells. Our analysis also provides a novel of Tfr2 in the myeloid lineage that have normal iron loading. approach for decomposing and quantifying ISG regulation. Tfr2fl/fl-LysMCre+ showed increased disease development Reference: with enhanced synovial hypertrophy and bone erosion in the Leviyang S., Strawn N., Griva I. (2020) Regulation of arthritic joints compared to Cre- control littermates. interferon stimulated gene expression levels at Since iron controls inflammatory responses and homeostasis. Cytokine. macrophages sense and regulate iron availability, we Disclosure of Interest: None Declared hypothesized that Tfr2-deficiency in macrophages could modulate their activation and polarization into a pro- LT146 inflammatory phenotype that contributes to arthritis TUBERCULOSIS IMPACTS IMMUNE-METABOLIC progression. Tfr2-/- macrophages showed a differential PATHWAYS RESULTING IN PERTURBED IL-1 transcriptional response to IFN-γ stimulation by increasing CYTOKINE RESPONSES the expression of Il1b, Il6, Inos, Cxcl10 compared to Tfr2+/+ A. Llibre1,*, N. Smith1, V. Rouilly2, M. Musvosvi3, E. Nemes3, macrophages. Furthermore, the deletion of Tfr2 in C. Posseme1, S. Mabwe3, B. Charbit1, S. Kimbung3, V. macrophages led to enhanced and prolonged IFN-γ-induced Saint-Andre1, V. Bondet1, P. Bost1, H. Mulenga3, N. Bilek3, STAT1 activation, while it downregulated STAT3 activation M. L. Albert4, T. J. Scriba3, D. Duffy1 likely via increasing Socs3 expression. 1Institut Pasteur, 2DATACTIX, Paris, France, 3University of Taken together, these findings suggest a protective role of Cape Town, Cape Town, South Africa, 4Insitro, San Tfr2 in the progression of arthritis and that it is involved in Francisco, United States repressing macrophage pro-inflammatory activation. Disclosure of Interest: None Declared Abstract Content: Tuberculosis (TB) remains a major public health problem. Host-directed therapeutics are proposed as novel treatment strategies, with TNF, IL1, and LT145 IFN cytokine pathways all strongly implicated in differential REGULATION OF INTERFERON STIMULATED GENES disease outcome. However, the successful development of AT HOMEOSTASIS host-directed therapeutics still requires a comprehensive S. Leviyang1,*, N. Strawn1, I. Griva2 76 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 understanding of how Mycobacterium tuberculosis (M.tb) examination of innate immune activation by infection impacts immune responses. ZIKV/Dakar/1984/ArD41519 and To address this challenge, we applied standardised ZIKV/Malaysia/1966/P6740 demonstrated differential and immunomonitoring tools to compare induced immune selective activation of the IRF3 and NF-κB signaling axes responses between individuals with latent M.tb infection within infected cells, revealing strain-specific innate immune (LTBI) and active TB disease. This revealed distinct polarization. Our observations show that viral amino acid responses between TB and LTBI groups at transcriptomic, sequence differences link with ZIKV replication kinetics and proteomic and metabolomic levels. Specifically we observed detection by host cells wherein early virus detection and dysregulated induced IL-1 responses in TB patients, which innate immune activation serve as critical restriction points could be explained through differences in upstream TNFα to control viral replication. Distinct ZIKV-induced IRF3 and and type I interferon, and downstream granzyme B NF-κB activation demonstrates the concept of virus-directed responses. At baseline, we identified pregnane steroids and innate immune polarization leading to antiviral versus the PPARγ pathway as novel immune-metabolic drivers of inflammatory signaling to drive distinct immune responses IL-1ra secretion. linked with ZIKV infection and disease. These findings improve our knowledge of how M.tb alters Disclosure of Interest: None Declared immune responses, and may support development of improved diagnostic, prophylactic and therapeutic tools. LT148 Disclosure of Interest: None Declared POST-TRANSCRIPTIONAL REGULATION OF ANTIVIRAL GENE EXPRESSION BY N6- LT147 METHYLADENOSINE COMPARISON OF GEOGRAPHICALLY DISTINCT ZIKA M. J. Mcfadden1,*, N. S. Abell2, A. B. McIntyre3, N. S. VIRUS STRAINS REVEALS DIFFERENTIAL Gokhale4, B. Xhemalce5, C. E. Mason3, S. M. Horner1 ACTIVATION OF HUMAN HOST INNATE IMMUNITY 1Molecular Genetics & Microbiology, Duke University, DIRECTING INNATE IMMUNE POLARIZATION Durham, 2Genetics and Pathology, Stanford University, A. Y. Lu1,2,*, D. J. Newhouse2, M. Gale, Jr.1,2 Stanford, 3Weill Cornell Medical College, New York, 1Global Health, 2Immunology, University of Washington, 4University of Washington, Seattle, 5Department of Seattle, WA, United States Molecular Biosciences, University of Texas at Austin, Austin, United States Abstract Content: Recently, Asian lineage-derived Zika virus (ZIKV) strains have emerged, causing outbreaks Abstract Content: Type I interferons (IFN) drive the across the Pacific Ocean resulting in new disease induction of hundreds of IFN-stimulated genes (ISGs). The manifestations, including fetal infection with congenital ZIKV expression of these genes must be carefully regulated to syndrome and adult infection presenting Guillain-Barré allow for both efficient production of antiviral effectors and syndrome. We hypothesize that changes in viral sequence controlled shut-off of inflammatory factors to avoid tissue contribute to phenotypic differences in virus replication and damage and autoimmunity. The RNA base modification N6- host cell interactions between geographically distinct ZIKV methyladenosine (m6A) has emerged as an important post- strains that underlie differential disease. We therefore transcriptional regulator of RNA expression. Indeed, m6A conducted de novo viral sequence and comparison has been shown to regulate both viral infection and the analyses to evaluate how viral sequences of several expression of IFN-beta, but its role in the type I IFN response geographically distinct ZIKV strains link with virologic has not been explored. To investigate the role of m6A in the differences and features of innate immune activation in type I IFN response, we mapped m6A in the IFN-induced human cells in vitro. Endemic African lineage-derived transcriptome using meRIP-seq, and found that the ZIKV/Dakar/1984/ArD41519, endemic Asian lineage- transcripts of many ISGs are m6A-modified. We then derived ZIKV/Malaysia/1966/P6740, and emerging Asian perturbed the expression of the m6A deposition enzymes, lineage-derived ZIKV/French Polynesia/2013/H-PF-2013, METTL3 and METTL14 (METTL3/14) and found that the ZIKV/Puerto Rico/2015/PRVABC59, and ZIKV/São protein expression of certain m6A-modifed ISGs is enhanced Paulo/2015/SPH2015 strains responsible for the Pacific and by METTL3/14, however their mRNA levels were South American ZIKV outbreaks were assessed. De novo unaffected. Polysome profiling determined that METTL3/14 viral sequence determination and alignment revealed regulates the translation of these m6A-modified ISGs. To several amino acid substitutions in each viral genome identify the full complement of METTL3/14-regulated ISGs, compared to pre-emergent ZIKV/Malaysia/1966/P6740. we performed ribosome profiling and quantitative mass One-step virus growth analysis revealed profound spectrometry. These data revealed a class of ISGs whose differences in replication kinetics between strains, whereas protein expression is enhanced by m6A, including many measurement of host cell IRF3 and NF-κB activation genes with known antiviral functions. Indeed, we found that identified distinct virus-specific innate immune activation METTL3/14 augments the antiviral effects of type I IFN profiles. Among these, ZIKV/French Polynesia/2013/H-PF- during vesicular stomatitis virus infection. Together, these 2013 exhibited faster replication kinetics and more robust studies identify m6A as a post-transcriptional control of ISG innate immune activation compared to ZIKV/São translation during the type I IFN response, which promotes Paulo/2015/SPH2015 and ZIKV/Puerto an antiviral cellular state. Rico/2015/PRVABC59 linked with slower replication kinetics Disclosure of Interest: None Declared and weaker innate immune activation. Moreover, 77 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT149 hydrophobic stretch in the N-terminus region of Siglec5 was SYSTEMATIC ANALYSIS OF CELL-INTRINSIC INNATE found to be required for efficient recognition of these IMMUNE ANTAGONISM BY RNA VIRUSES lipophilic ligands. Notably, this hydrophobic stretch is E. Mesev1,*, R. LeDesma1, C. Claudio1, E. Guare1, B. dispensable for sialic acid recognition. Siglec5 is an Heller1, A. Ploss1 inhibitory receptor, which negatively regulates cell activation 1Molecular Biology, Princeton University, Princeton, United upon ligand binding. The lipophilic ligands suppressed IL-8 States production in human monocytic cells expressing Siglec5. Siglec14 and 5 have high sequence identity in the Abstract Content: Emerging and re-emerging RNA viruses extracellular region and thus, Siglec14 also recognizes the such as Zika virus (ZIKV), yellow fever virus (YFV), SARS- lipophilic ligands. In contrast to Siglec5, Siglec14 transduces CoV-2, and endemic RNA viruses such as hepatitis E virus activating signals upon ligand recognition. Indeed, the (HEV) remain global health problems. Most of these viruses lipophilic ligands induced IL-8 production in human have devised mechanisms to evade antiviral defenses, monocytic immune cells expressing Siglec14. Collectively, thereby creating a host cellular environment that is more our study demonstrates that Siglec5 and 14 have the conducive to viral replication. In particular, proteases potential to recognize lipophilic ligands and modulate innate encoded by RNA viruses have been shown to inactivate immune responses. To our knowledge, this is the first study different components of antiviral signaling pathways, to report the binding of Siglecs to lipid ligands, which will blunting the cell’s ability to mount type I and III interferon expand our understanding of the biological function and (IFN) dependent defenses. Examples of this antagonism in importance of Siglecs. well-characterized RNA viruses include the hepatitis C virus Disclosure of Interest: None Declared NS3/4A protease and ZIKV NS2B3 protease, which cleave the innate immune adaptor proteins MAVS and STING LT151 respectively. In this work, we have developed a platform to REPORTER NANOPARTICLE FOR REAL-TIME interrogate the putative innate immune antagonizing INFLAMMASOME MONITORING DURING A DISEASE- activities of several poorly-characterized human viruses: PROGRESSION HEV, Usutu virus (USUV), and Powassan virus (POWV). D. Nandi1,*, A. Nguyen2, A. Kulkarni2,3 Utilizing a live-cell STAT1 reporter in cells expressing a viral 1Veterinary and Animal Sciences Department, 2Department protease of interest, we are able to determine the necessity of Chemical Engineering, 3Department of Biomedical of type I and type III IFN signaling in the host cells’ ability to Engineering, University of Massachusetts, Amherst, mount an antiviral defense. Subsequent biochemical Amherst, United States analyses allow us to quantify protein and transcript levels of innate immunity proteins before and during protease Abstract Content: Inflammasome activation is critical to expression to pinpoint the stage of innate immune signaling variety of chronic inflammatory diseases like Colitis, Non- that is antagonized by the virus. Preliminary data suggests Alcoholic-Steatohepatitis, Alzheimer’s. This innate immune that HEV can dampen multiple innate immunity pathways at response induced by microbial or sterile invasion activates an intermediate level not seen in other viral families, which caspase-1 enzyme, an important hallmark of inflammasome opens up exciting new avenues of investigation. activation. Active caspase-1 further cleaves pro-IL-1b and Collectively, our work provides mechanistic insights into how pro-IL18, converting them to their active forms which are these emerging and endemic human pathogens evade released outside the cell, resulting in enhanced immune innate immunity and may also reveal new targets for antiviral response and pyroptosis. Currently available techniques fail therapy. to provide temporal kinetics of inflammasome activation and Disclosure of Interest: None Declared cytokine release during disease progression, presenting a window of opportunity for the development of inflammasome LT150 monitoring tools. To address this gap, we have developed a IDENTIFICATION OF NOVEL LIPOPHILIC LIGANDS OF polymer-nanoparticle encapsulated caspase-1 activatable SIGLEC RECEPTORS THAT MODULATE INNATE probe that will emit signal specifically at inflamed sites upon IMMUNITY active-caspase-1 cleavage. This biocompatible platform Y. Miyake1,*, H. Yoshida1 enhances the probe’s residence time in circulation by 1Faculty of medicine, Saga University, Saga, Japan preventing its opsonization and allowing its sustained release over time. Preliminary observations show that the Abstract Content: Sialic acid-binding immunoglobulin-like probe is cleaved specifically by recombinant active- lectins (Siglecs) are a family of cell-surface receptors that caspase-1 and can be encapsulated stably inside polymer bind to sialic acid at terminal glycan residues. In this study, nanoparticle. We have also successfully demonstrated its we found that Siglec5 and 14 recognize Trichophyton, a in-vitro testing in inflammasome-activated-bone-marrow- pathogenic fungal species. Biochemical approaches derived-macrophages. We further plan to translate this revealed the chemical structures of the ligands, which were study in relevant animal models. This entire system will help identified as alkanes and triacylglycerols. This unexpected in the precise estimation of caspase-1 activation and finding prompted us to further search for novel endogenous cytokine release kinetics during disease progression, hence lipid ligands. Although Siglec5 weakly recognized several ensuring early detection of pathogenic inflammation and its endogenous lipids, cardiolipin and 5-palmitic acid-hydroxy timely treatment. stearic acid (5-PAHSA) showed potent ligand activity. The Disclosure of Interest: None Declared 78 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

1Immunology, 2Obstetrics & Gynecology, University of LT152 Washington, Seattle, United States CLEARANCE OF PEGYLATED INTERFERON BY KUPFFER CELLS DETERMINES Abstract Content: The persistence of latent cells that INNATE IMMUNE ACTIVATION AND RESPONSE TO silently harbor HIV proviruses despite antiretroviral therapy TREATMENT OF CHRONIC HEPATITIS B (ART) is a major barrier to an HIV cure. HIV infection triggers cell-intrinsic immune defenses which can block viral A. Nishio1,*, F. Bolte1, K. Takeda2, K. Valdez1, M. G. Ghany1, replication in acutely infected cells. These innate immune B. Rehermann1 defenses are mediated by pathogen recognition receptors 1Liver Diseases Branch, National Institute of Diabetes and that sense viral products and activate type 1 interferon (IFN) Digestive and Kidney Diseases, National Institutes of production, leading to induction of hundreds of interferon- Health, DHHS, 2Pathology Core, National Heart, Lung and stimulated genes (ISGs), many with antiviral activity. Blood Institute, National Institutes of Health, DHHS, Despite these defenses, some acutely infected cells Bethesda, United States become latent and silently persist for years in vivo, suggesting they have escaped IFN-mediated immunity. Abstract Content: Approximately 257 million people Using a variety of primary and cell line models of HIV worldwide are chronically infected with the hepatitis B virus latency, we evaluated innate immune activation and IFN (HBV). About 900,000 people die from HBV-related liver signaling in latently infected cells. We found that HIV latent failure and/or hepatocellular carcinoma each year, which T cell lines have functional viral RNA sensing pathways that makes HBV more deadly than HIV, malaria and tuberculosis drive IRF3 activation and IFN production. However, unlike combined. Pegylated interferon-alpha (PegIFNα) is the sole uninfected cells, latent cells respond poorly to type 1 IFN curative treatment for chronic hepatitis B virus (HBV) treatment and fail to induce specific antiviral ISGs. RNA infection, but it is unknown why only a minority of patients sequencing analysis of latent vs uninfected cells stimulated respond to PegIFNα. Cure of chronic HBV infection is with IFN revealed a network of dysregulated ISGs that we defined as HBsAg loss from the blood. hypothesize may be important for restricting latency. Here, we investigated the mechanisms that contribute to a Furthermore, HIV infected primary CD4+ T cells suppressed patient’s response to PegIFNα therapy. Using sequential with ART demonstrate dampened responses to IFN blood and liver samples, we found that patients with early stimulation compared to uninfected cells. These findings natural killer (NK) cell activation after PegIFNα injection suggest that reservoir cells have intrinsic defects in IFN experienced greater liver inflammation, lysis of HBV- signaling, which may be selected for during initial infection infected hepatocytes and HBsAg decline than those without. and/or actively maintained throughout the transition to The level of NK cell activation depended on PegIFNα latency. We are further investigating the mechanism of IFN pharmacokinetics, because serum levels of PEG and IFNα signaling dysregulation in latent cells. Our work reveals a were 80-fold and 125-fold higher in patients with NK cell previously undescribed role for type 1 IFN in regulating HIV responses than in patients without NK cell responses at 6 latency, which may be exploited to design curative therapies hours and 24 hours after the first PegIFNα injection, aimed at eradicating the reservoir. respectively. The PegIFNα level in the blood was Disclosure of Interest: None Declared determined by differential Kupffer cell-mediated clearance of PegIFNα/PEG-antibody complexes as revealed by immunohistochemistry of liver biopsies, taken prior to and 6 LT154 hours after PegIFNα injection. VANCOMYCIN INTERMEDIATE STRAINS OF These results indicate that NK cell activation and NK cell- STAPHYLOCOCCUS AUREUS HAVE DAMPENED mediated lysis of HBV-infected hepatocytes plays a pivotal INDUCTION OF TYPE I INTERFERON PRODUCTION role in the clinical response to PegIFNα therapy, and that A. Peignier1,*, D. Parker1 PEG clearance by Kupffer cells impairs therapeutic activity 1Pathology, Center for Immunity and Inflammation, Rutgers, during PegIFNα treatment. We postulate that repeated The State University of New Jersey, NEWARK, United exposure to PEG, which is widely used in dairy products States (such as shampoo, cosmetics, laxative) might induce the production of anti-PEG antibody prior to PegIFNα treatment Abstract Content: Staphylococcus aureus is a leading and accelerate PEG clearance by Kupffer cells. Accelerated cause of bacterial pneumonia and we have shown PEG clearance by Kupffer cells needs to be considered previously that type I interferon (IFN) contributes to beyond the HBV field to improve the efficacy of a wide pathogenesis of S. aureus pneumonia. In this study, we variety of pegylated drugs, that are now used and being screened 75 S. aureus strains for their ability to induce type developed for multiple chronic diseases and cancer. I & III IFN. Both cytokine pathways were differentially Disclosure of Interest: None Declared induced by various S. aureus strains independently of their isolation sites or methicillin resistance profiles. These LT153 induction patterns persisted over time and type I & III IFN HIV LATENT CELLS HAVE ACQUIRED RESISTANCE TO generation differentially correlated with TNF-α production. CELL-INTRINSIC INNATE IMMUNITY Investigation of one strain, strain 126, showed a significant R. Olson1,*, G. Gornalusse2, D. Newhouse1, F. Hladik2, M. defect in type I IFN induction that persisted over several time Gale Jr.1 points. The lack of induction was not due to differential 79 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 phagocytosis, subcellular location or changes in endosomal mechanisms may provide targets for preventing and/or acidification. Strain 126 had a decreased rate of autolysis treating VAERD. and increased resistances to lysostaphin degradation and Disclosure of Interest: None Declared host cell mediated killing. This strain displayed decreased virulence in a murine model of acute pneumonia compared LT157 to USA300 (current epidemic strain and commonly used in INNATE IMMUNE SIGNALING DRIVES PATHOGENIC research) and had reduced capacity to induce multiple EVENTS LEADING TO AUTOIMMUNE DIABETES cytokines. We observed this isolate to be a vancomycin N. Qaisar1,*, B. Satish2, R. Racicot,3, A. Kucukural4, A. Derr5, intermediate S. aureus (VISA) strain. The relationship S. Redick6, Z. Guo3, J. Mordes3, J. Wang1 between reduced type I IFN induction levels and decreased 1Medicine,Diabetes Center of Excellence, Program in autolysis was found to be shared by other VISA strains and Molecular Medicine, 2Medicine, Diabetes Center of studied using an isogenic pair of wild type and VISA mutant Excellence, Program in Molecular Medicine, 3Medicine, strains. We conclude that the reduction in type I IFN 4Program in Bioinformatics and Integrative Biology, induction was largely the result of inherent cellular 5Program in Bioinformatics and Integrative Biology, properties of VISA strains whose reduced autolysis prevents Diabetes Center of Excellence, Program in Molecular the release of pathogen-associated molecular patterns. Medicine, 6Diabetes Center of Excellence, Program in Overall, this study demonstrates the heterogeneity of IFN Molecular Medicine, University of Massachusetts Medical induction by S. aureus and uncovered a unique property of School, Worcester, United States VISA strains in their inability to induce type I IFN production. Disclosure of Interest: None Declared Abstract Content: Introduction: Type 1 diabetes (T1D) is a chronic autoimmune disease, characterized by the LT156 immune-mediated destruction of insulin-producing b cells of HOST CYTOKINE DYSREGULATION AND ENHANCED pancreatic islets. Essential components of the innate RESPIRATORY DISEASE ELICITED BY VACCINE- immune antiviral response, including type I interferon (IFN) INFLUENZA MISMATCH. and IFN receptor (IFNAR)-mediated signaling pathways, are J. Powell1,*, E. Abente1, B. Kimble1, D. Rajao1, D. Bayles1, candidates for determining susceptibility to human T1D. A. Vincent1 Methods: We previously generated Ifnar1-/- LEW.1WR1 1USDA, ARS- National Animal Disease Center, Ames, rats that have a significant reduction in diabetes frequency United States following Kilham rat virus (KRV) infection. To delineate the impact of IFNAR1 loss on adaptive immune responses in Abstract Content: Influenza A virus outbreaks in humans KRV-induced diabetes, we examined immune cell and pigs are mitigated through vaccination. Pigs vaccinated populations in the spleen during the preinsulitic stage. with adjuvanted whole-inactivated virus vaccine (WIV) that We used approaches such as flow cytometry, bulk RNA are later infected with a mismatched influenza strain of the sequencing, and single-cell RNA-sequencing to identify same HA subtype can develop vaccine associated major leukocyte subsets which are altered in spleen enhanced respiratory disease (VAERD). VAERD occurs in preceding insulitis in both WT and Ifnar1-/- rats after viral the presence of high titer non-neutralizing antibodies, with infection. similar viral loads between VAERD-affected and Results: Examination of splenic T cells and natural killer unvaccinated animals. To elucidate the mechanisms (NK) cells before the onset of insulitis and diabetes revealed underlying the severe pulmonary pathology, we examined a significant decrease in CD8+ T cells and NK cells in Ifnar1- host gene alterations in WIV vaccinated pigs affected by /- rats compared to WT rats. In addition, splenic regulatory T VAERD compared to naïve infected pigs. qPCR arrays and cells were diminished in WT but not Ifnar1-/- rats. whole-genome RNA-seq analyses from pulmonary alveolar Conversely, splenic neutrophils were increased in KRV- macrophages and lung tissue were utilized to assess the infected Ifnar1-/- rats compared to the infected WT rats. We transcriptomic response. ELISA and MAGPIX® Luminex™ also compared gene expression changes in spleens from platforms were used to analyze responses at the protein virus-infected WT and Ifnar1-/- rats using bulk RNA-seq at level. Pigs with VAERD had dysregulation in many anti-viral the prediabetic stage. RNA-seq revealed diminished or pro-inflammatory cytokine genes compared to naïve interferon-stimulated genes and inflammatory gene challenged pigs, including a diminished type-I interferon expression in spleens from Ifnar1-/- rats relative to WT rats. response (IFN-a). Genes associated with helper T-cell Single cell RNA-seq of spleens confirmed a decrease of NK driven inflammation (IL-17A, IL-22 and IL-23) were higher at cells in KRV-infected Ifnar1-/- rats compared to WT rats. the RNA and protein level in VAERD animals compared to Conclusions: Collectively, these findings identify novel naïve challenged pigs. Additionally, RNA-seq analysis innate immune players in the KRV-induced diabetes in rats identified genes involved with wound repair and tissue and firmly establish that disruption of IFNAR signaling remodeling were upregulated in VAERD pigs. These results dynamically alters immune cell populations in a manner revealed that cytokines associated with helper T-cell which is protective against the development of autoimmune responses were upregulated with VAERD and may provide diabetes. possible mechanisms that lead to the severe pulmonary Disclosure of Interest: None Declared immunopathology; whereas wound repair and tissue remodeling gene upregulation may be linked to the resolution of the VAERD lesions. These underlying 80 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT158 macrophage inflammation in vitro and were interested to ANTIVIRAL EFFECT OF THE RIG-I INDUCED PATHWAY know whether this effect was mediated via NLRP3 OF APOPTOSIS (RIPA) IS BOOSTED BY ITS ABILITY TO inflammasome complex. For this, bone marrow derived TRIGGER DEGRADATION OF THE DEUBIQUITINASE, macrophages (BMDM) from tibia and femurs of mice were OTULIN differentiated by granulocyte-macrophage colony- R. Raja1,*, G. C. Sen1 stimulating factor (GMCSF) to skew them towards M1 1Department of inflammation and immunity, Lerner phenotype. The rexinoid drug suppressed expression of Research Institute Cleveland clinic, Cleveland, United NLRP3 protein and hindered the processing of IL-1 beta and States Caspase-1 proteins in LPS+ATP-primed M1 macrophages. Secreted IL-1 beta and IL-18 mRNAs and proteins were also Abstract Content: Infection of mammalian cells by many significantly (p<0.05) decreased in LPS+ATP-stimulated M1 RNA viruses trigger the RIG-I pathway which activates IRF3. macrophages when treated with either the rexinoid drug or IRF3 exerts its antiviral effects by inducing IFN synthesis NLRP3 siRNA. and another antiviral pathway, called RIG-I induced pathway These data suggest that the rexinoid drug can reduce of apoptosis (RIPA). RIPA requires linear polyubiquitination NLRP3-mediated activations in M1 macrophages. Future of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 studies will explore models of macrophage mediated binds to Bax and translocates it to mitochondria causing the inflammatory diseases. release of Cytochrome C, activation of caspases and Disclosure of Interest: None Declared apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, LT160 inhibits RIPA by binding to LUBAC and deubiquitinating MURINE AND HUMAN MACROPHAGES MEDIATE IRF3. Ablation of Otulin expression enhanced RIPA and its RECOGNITION OF THE INTRAERYTHROCYTIC overexpression inhibited RIPA. We discovered that to APICOMPLEXAN PARASITE BABESIA DIVERGENS. overcome Otulin-mediated inhibition, RIPA actively S. Rius-Rocabert1,2,3,*, E. Montero4, L. M. Gonzalez4, E. degrades Otulin. This degradation required sequential Nistal-Villan5,6 actions of RIPA-activated Caspase 3 and proteasomes. 1Pharmaceutical and Health Science, University CEU San Caspase 3 cleaved Otulin first at D31 and then at D322; the Pablo, Boadilla del Monte, 2 Instituto de Medicina Molecular D31A mutant was not cleaved at all. The caspase-cleaved Aplicada (IMMA), Universidad San Pablo-CEU, 3CEMBIO fragment was totally degraded by proteasomes, which (Centre for Metabolomics and Bioanalysis), Facultad de required its K48-linked ubiquitination. The E3-ubiquitin Farmacia, Universidad San Pablo-CEU, Madrid, ligase, HOIP, a component of LUBAC, ubiquitinated Otulin 4Laboratorio de Referencia e Investigación en Parasitología, at K64 and K197 to trigger proteasome-mediated Centro Nacional de Microbiología, ISCIII , Majadahonda, degradation. To assess the impact of Otulin degradation on Madrid, 5Pharmaceutical and Health Science, University RIPA-mediated antiviral action, we expressed in Otulin- CEU San Pablo, 6Instituto de Medicina Molecular Aplicada ablated cells, a non-degradable mutant of Otulin, in which (IMMA), Universidad San Pablo-CEU, Boadilla del Monte, D31, K64 and K197 had been mutated. In these cells, RIPA Madrid, Spain was less active and replication of Sendai virus was more pronounced. Thus, our study has revealed an important Abstract Content: Babesia divergens, is a natural positive feedback loop of RIPA. pathogen of cattle, and one of the species responsible for Disclosure of Interest: None Declared human babesiosis, causing death unless treated promptly. This worldwide emerging disease, with many clinical LT159 features that are like those of malaria, is transmitted by ticks A SYNTHETIC REXINOID REDUCES LPS-ACTIVATED and transfusion and is increasing in frequency and NLRP3 ACTIVATION IN BONE MARROW DERIVED M1 geographic range. Thus, babesiosis has a significant impact MACROPHAGES. on human and animal health. N. Raychaudhuri1,*, S. Basak1 During its asexual life cycle, B. divergens infects and 1ATGC Group, Los Angeles, United States reproduces within red blood cells (RBCs) and builds a complex population of intraerythrocytic (IE) and free Abstract Content: The NLR family pyrin domain containing infective merozoites. The invasion by the free merozoites 3 (NLRP3) inflammasome is a critical mediator of and posterior destruction of the RBCs is a critical point in the inflammation in response to pathogens, tissue damage, and progress and prognosis of the disease. inflammatory diseases. These large multi-protein complexes Macrophages are innate immune cells responsible of form rapidly in response to danger and pathogen-associated recognition and elimination of pathogens at early stages of signals, serving as scaffolds to promote maturation of infection. Macrophage polarization is known to be important caspase-1, a cysteine protease that processes inactive pro- in parasitic infections. Cytokines and chemokines produced IL-1 beta and pro-IL-18 to their active pro-inflammatory by macrophages are crucial for establishment of immune cytokines, IL-1 beta and IL-18, respectively. response against pathogens and disease prognosis. Retinoid X receptors (RXRs) are nuclear receptors and play Thus, in this study we describe an in vitro response of both essential roles for macrophage biology. Rexinoids are human and murine bone-marrow differentiated synthetic molecules that bind and activate RXRs. We found macrophages (BMDM) exposed to mock treatment, that our rexinoid drug inhibited LPS-induced M1 uninfected human red blood cells (RBC), B. divergens- 81 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 infected RBCs or to free merozoites in order to characterize 2Department of Medical Genetics, University of British the possible reaction against the parasite during infection. Columbia, Vancouver, Canada, 3Departments of Pediatrics, Results show a strong expression of IFN-β mRNA and some Developmental Biology, Cell Biology and Physiology, proinflammatory cytokines such as IL-6, TNF-α, IL-1β in University of Pittsburgh School of Medicine, Pittsburgh, GM-CSF derived BMDM, in comparation with M-CSF United States BMDM, in response to the presence of free merozoite. Notably, as compared to the free merozoite, a weak Abstract Content: During bacterial infection, the host is proinflammatory response was induced in BMDM against B. confronted with multiple overlapping signals that are divergens-infected RBCs in both murine and human BMDM. integrated at the organismal level to produce defensive host This result suggests that macrophages cannot efficiently responses. How multiple infection signals are sensed by the recognize the B. divergens parasite when is within its host host and how they elicit the transcription of host defense cell. genes is much less understood at the whole-animal level Disclosure of Interest: None Declared than at the cellular level. The model organism Caenorhabditis elegans is known to mount transcriptional LT160c defense responses against intestinal bacterial infections that CONGENITAL DEFICIENCY IDENTIFIES CRITICAL elicit overlapping starvation and infection responses, but the ROLE OF ISG15 IN SKIN HOMEOSTASIS regulation of such responses is not well understood. Direct F. Pessler1 on behalf of Consortium on Skin Lesions in comparison of C. elegans that were starved or infected with ISG15 Deficiency , M. N. Malik2, F. Waqas1,* Staphylococcus aureus revealed a large infection-specific 1TWINCORE Centre for Experimental and Clinical Infection transcriptional signature. This signature was almost Research, Hannover, Germany, 2University of Lahore, completely abrogated by deletion of transcription factor hlh- Lahore, Pakistan 30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of Abstract Content: Ulcerating skin lesions are a newly fmo-2/FMO5 severely compromised infection survival, thus described manifestation of human ISG15 deficiency, a type identifying the first FMO with innate immunity functions in I interferonopathy. However, it is unclear whether their animals. Moreover, the mechanism of fmo-2/FMO5 etiology can be completely explained by chronic induction required the nuclear hormone receptor, NHR- inflammation in dermal cells. We describe two siblings with 49/PPAR-α, which induced fmo-2/FMO5 and host defense chronic recurrent skin ulcers that healed with scar formation cell non-autonomously. These findings for the first time upon corticosteroid treatment. Both had a homozygous reveal an infection-specific host response to S. aureus, nonsense mutation in ISG15, which led to a truncated ISG15 identify HLH-30/TFEB as its main regulator, reveal that protein that was unstable in vitro. We created immortalized FMOs are important innate immunity effectors in animals, dermal fibroblasts, HaCaT keratinocytes, and hiPSC- and identify the mechanism of FMO regulation through derived vascular endothelial cells with targeted deletions of NHR-49/PPAR-α in C. elegans, with important implications ISG15. Transcriptomic and proteomic analyses revealed the for innate host defense in higher organisms. expected hyperinflammatory phenotype, but also Disclosure of Interest: None Declared dysregulated expression of molecules critical for connective tissue and epidermis integrity, such as reduced collagens LT160e and adhesion molecules, but increased matrix CELL-INTRINSIC COMPLEMENT PATHWAY metalloproteases. ISG15-/- cells exhibited elevated ROS ENHANCES TLR4-DEPENDENT PRO-INFLAMMATORY production and reduced ROS scavenger expression. As CYTOKINE PRODUCTION AND TLR4 ENDOCYTOSIS opposed to hyperinflammation, defective collagen and VIA INHIBITION OF CAMP integrin synthesis could not be rescued by conjugation B. A. Napier1,*, J. Hickman1, J. Corcoran2 deficient ISG15. A 3D model of epidermis formation showed 1Biology, 2Portland State University, Portland, United States markedly reduced desmosome density, loose architecture, and reduced collagen synthesis, all of which could be Abstract Content: Hyper-production of inflammatory reversed by treatment with the Jak/Stat inhibitor ruxolitinib cytokines by cells of macrophage-monocyte lineage is plus doxycycline/TGF-β1. These results reveal regulation of associated with increased severity of disease and mortality connective tissue and epidermis homeostasis as a novel in sepsis patients. Sepsis is a deleterious immune response function of ISG15 that likely requires its conjugation to yet to infection that leads to organ failure and is the cause of unidentified targets. over 10 million deaths annually. Currently, septic patients Disclosure of Interest: None Declared are treated with palliative care due to the lack of effective immune therapeutics, thus there is an urgent need to LT160d understand the cellular pathways within human NHR-49/PPAR-ALPHA AND HLH-30/TFEB PROMOTE C. macrophages that contribute to this pro-inflammatory ELEGANS HOST DEFENSE VIA A FLAVIN-CONTAINING response. The complement pathway and toll-like receptor MONOOXYGENASE (TLR) signaling are two cell-intrinsic innate immune K. A. Wani1,*, D. Goswamy1, S. Taubert2, R. Ratnappan3, A. pathways within macrophages/monocytes that contribute to Ghazi3, J. E. Irazoqui1 the production of proinflammatory cytokines during sepsis. 1Microbiology and Physiological Systems, University Of We have recently reported that membrane-bound Massachusetts Medical School, Worcester, United States, complement receptor C3aR is required for expression of 82 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 pro-inflammatory cytokines downstream of toll-like receptor- activities by the surveillance of homeostatic processes 4 (TLR4) activation during exposure to Gram-negative within cells. In contrast to PRRs, which are well-known for bacterial lipid lipopolysaccharide (LPS), in primary murine their roles in many types of infections, the role of guard and human macrophages. Moreover, we have shown that proteins in most infectious contexts remains less well C3aR-dependent expression of pro-inflammatory mediators understood. In this study, we demonstrate that inhibition of is required for protection against multiple Gram-negative host-protein synthesis during viral infection is sensed as a bacterial pathogens, and that c3ar is over-expressed in the virulence strategy and initiates pyroptosis in primary human blood of septic patients, suggesting clinically relevant epithelial cells. We identify the highly labile Bcl-2 family implications for the crosstalk between C3aR and TLR4. member Mcl-1 as a sensor of translation shutdown. Mcl-1 Currently, it is not known how C3aR is enhancing TLR4 depletion during infections with multiple distinct viruses signaling and cytokine expression within macrophages. In stimulates caspase-dependent cleavage of gasdermin E, this study, we find that C3aR is functioning as an inhibitory resulting in pyroptosis and the release of pre-formed IL-1α. G protein-coupled receptor (Gi) within macrophages to Moreover, direct inhibition of Mcl-1, in the absence of inhibit production of cyclic-AMP (cAMP) during LPS infection, was sufficient to engage this pyroptotic pathway. stimulation. cAMP regulates intracellular signaling via Thus, Mcl-1 can act as a guard protein that senses the multiple target proteins, including two ubiquitously dysregulation of host-cell translation during viral infections expressed cAMP receptor enzymes: protein kinase A (PKA) of barrier epithelia. and exchange protein directly activated by cAMP (Epac). Disclosure of Interest: None Declared We found that C3aR inhibited activation of PKA, but not Epac, and this inactivation of PKA correlated with decreased Lightning Talk Session 3: Cancer expression of both NFκB (IL-6, IL-1β) and IRF3 (IFNβ and Rsad2) regulated genes during LPS stimulation. It has been LT162 previously shown that increased cAMP and PKA activation PLASMA CYTOKINES PREDICT LYMPHEDEMA results in dysregulated TLR4 trafficking and decreased DEVELOPMENT IN BREAST CANCER PATIENTS 12 inflammation in macrophages. Here we find that inhibition of MONTHS BEFORE CLINICAL PRESENTATION C3aR signaling results in altered TLR4 endocytosis at 60- M. B. Aldrich1,*, S. F. Shaitelman2, J. C. Rasmussen1, E. M. 120 minutes post-LPS stimulation, similar to what was Sevick-Muraca1, L. Isales2, S. M. DeSnyder3 previously seen with pharmacological activation of PKA. 1Center for Molecular Imaging, The Brown Institute for Together, these data suggest that C3aR activation is Molecular Medicine, UTHealth, 2Radiation Oncology, dampening intracellular cAMP production and PKA 3Breast Surgical Oncology, Nellie B. Connelly Breast Cancer activation, resulting in altered TLR4 endocytosis and Center, MD Anderson Cancer Center, Houston, United inhibition of NFκB- and IRF3-regulated pro-inflammatory States cytokine expression in macrophages during LPS stimulation. This study provides the first mechanistic insight Abstract Content: BACKGROUND: Lymphedema (LE) is underlying the molecular crosstalk between complement a dreaded complication of cancer treatment, afflicting at pathway and TLR signaling macrophages, and is pivotal in least 40% of breast cancer survivors. Arm, trunk, and breast understanding the relationship between microbe-sensing swelling, along with pain, depression, skin fibrosis, and TLRs and the complement pathway, two pro-inflammatory cellulitis susceptibility, severely reduce quality of signaling pathways that have been implicated in regulating life. Clinically, LE is diagnosed once limb swelling exceeds sepsis mortality and outcome. In future studies, it will be 5% over baseline volume, but there is growing evidence of imperative to understand if this pathway can be a “latent,” pre-swelling form of LE. LE-associated swelling therapeutically targeted in septic patients. can appear during chemotherapy, surgery, and/or radiation Disclosure of Interest: None Declared treatment, or suddenly appear years later. There is currently no cure for LE, but early palliative treatment has LT160f been shown to significantly improve outcomes, by stalling or VIRUS-MEDIATED LOSS OF MCL-1 PROMOTES reversing LE swelling. Standard-of-care, palliative LE GSDME-DEPENDENT INFLAMMATORY CELL DEATH IN treatment includes manual lymphatic drainage (specialized BARRIER EPITHELIAL CELLS massage) to remove excess fluid, compression bandaging M. H. Orzalli1,*, A. Prochera2,3, A. Smith4, J. A. Garlick4, J. C. to prevent fluid buildup, pneumatic compression therapy for Kagan2,3 home use, and meticulous skin care to prevent 1Department of Medicine , University of Massachusetts infections. Lymphatic microsurgeries are growing in Medical School, Worcester, 2Division of Gastroenterology, popularity to improve LE outcomes, but outcomes for these Boston Children's Hospital, 3Program in Immunology, procedures are optimal only for treatment of early-stage Harvard Medical School, 4Department of Diagnostic LE. Clearly, there is a need for a tool to detect LE at the Sciences, Tufts University School of Dental Medicine, earliest, possibly “latent,” stage possible. Boston, United States METHODS: Data were collected as part of an ongoing, prospective, longitudinal surveillance of 100 breast cancer Abstract Content: Pattern recognition receptors (PRRs) patients undergoing neoadjuvant chemotherapy, axillary and intracellular guard proteins are critical components of lymph node dissection (ALND), and radiation treatment (RT) the innate immune system. While PRRs recognize microbial (ClinicalTrials.gov, NCT02959726). Milliplex products, guard proteins often detect virulence factor cytokine/chemokine levels were determined from plasma 83 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 samples that were collected from patients before ALND, derived suppressor cells (MDSCs) expand in GBM patients after ALND but before RT, and at 6, 12, and 24 months after and are associated with poor prognosis. Using multiple RT. Perometry, a method to measure arm volumes using syngeneic mouse GBM models, we investigated whether an optoelectronic device, was performed at each of the time MDSC subsets promoted tumorigenesis in a sex-specific points, and LE was clinically diagnosed when cancer- manner. Our results demonstrated that monocytic MDSCs affected-side arm volume exceeded contralateral arm (mMDSCs) accumulate in tumors of male mice, while female volume by 5%. Data from the first 12 study subjects were tumor-bearing mice had a two-fold increase in circulating analyzed for correlation between median granulocytic MDSC (gMDSC) frequency. Consistently, male cytokine/chemokine levels for the groups with or without GBM patients had greater immunosuppressive myeloid cell clinical LE before ALND and at 12 months post-RT infiltration, and female patients with a high gMDSC gene (Wilicoxon nonparametric test), as well as linear regression signature had a worse outcome. In line with the differential analysis of perometer/% relative limb volume change (% MDSC subset association, targeting gMDSCs with anti- RVC) values and picograms/milliliter plasma Ly6G neutralizing antibodies significantly extended the cytokine/chemokine levels at 12 months post-RT. lifespan of female mice without affecting males. However, RESULTS: Levels of several cytokines/chemokines (G- mMDSCs were protected from the anti-Ly6C depletion CSF, GM-CSF, fractalkine, IL-12p40, IL-15, and IL-2) were strategy due to their systemic and local proliferation, as significantly elevated in plasma samples, taken at 12 indicated by ex vivo Ki-67 staining. Drug-prediction using the months post-RT, from study subjects who developed LE at differential gene expression profiles of MDSC subsets and 12 months post-RT. R-squared values for correlation subsequent pre-clinical testing established that mMDSCs between plasma levels of these six cytokines/chemokines can be targeted by chemotherapies in males. In contrast, IL- and % arm swelling were 0.53, 0.76, 0.67, 0.63, 0.50, and 1β, which was expressed at higher levels by female murine 0.60, respectively. Moreover, values of these gMDSCs and correlated with poor prognosis of female cytokines/chemokines were also significantly elevated patients, was identified as a drug candidate to target gMDSC before ALND—over 12 months before clinical diagnosis of activity. Neutralization of this cytokine provided a female- LE, in the group that developed LE by 12 months post-RT. specific survival advantage by reducing circulating gMDSC CONCLUSIONS: Plasma cytokine/chemokine levels of G- frequency. This effect was accompanied by a decline in the CSF, GM-CSF, fractalkine, IL-12p40, IL-15, and IL-2 could number of tumor-infiltrating microglia and tumor cell be used as a tool to identify breast cancer patients who are proliferation rate. In vitro assessment of IL-1β inhibition at high risk to develop LE within 12 months post-RT. This resulted in reduced viability and expression of activation screening tool could enable early initiation of LE treatment markers by primary microglia. These findings highlight a (“pre-hab”), and greatly improve patient outcomes. gMDSC-microglia communication axis mediated by IL-1β Disclosure of Interest: None Declared signaling as a driver of GBM in females and indicate that MDSC subset variation and the IL-1β signaling axis LT163 represent opportunities for improved immunotherapy IL-1B DRIVES GLIOBLASTOMA PROGRESSION IN A efficacy while accounting for sex as a biological variable. SEX-SPECIFIC MANNER Disclosure of Interest: None Declared D. Bayik1,*, Y. Zhou2, C. Park1, C. Hong1, D. Vail3, A. Lo1, D. Watson4, A. Lauko1, G. Roversi1, D. Silver1, T. Alban1, M. LT164 Sorensen5, B. Otvos1, M. Grabowski1, M. Vogelbaum6, C. HETERODIMERIC IL-15 THERAPY PROMOTES Horbinski7, B. Kristensen5, A. Khalil3, T. H. Hwang1, M. INTRATUMORAL LYMPHOCYTE AND DENDRITIC CELL Ahluwalia8, F. Cheng1, J. Lathia1 ACCUMULATION BY A CYTOKINE NETWORK 1Lerner Research Institute, Cleveland Clinic, Cleveland, INVOLVING XCL1, IFN-GAMMA, CXCL9 AND CXCL10, 2Lerner Research Institute, Cleveland Clinic, Clevelannd, RESULTING IN CONTROL OF PRIMARY AND 3Department of Genetics and Genome Sciences, Case METASTATIC TUMOR Western Reserve University, 4Hematology/Oncology, C. Bergamaschi1,*, H. Pandit2, B. Nagy1,2, D. Stellas2, S. University Hospitals Cleveland Medical Center, Cleveland, Karaliota3, S. M. Jensen4, K. Dimas2, B. A. Fox4, B. K. United States, 5Department of Pathology, University of Felber1, G. N. Pavlakis2 Southern Denmark, Odense, Denmark, 6Department of 1Human Retrovirus Pathogenesis Section, Vaccine Branch, Neuro-Oncology, Moffitt Cancer Center, Tampa, CCR, 2Human Retrovirus Section, Vaccine Branch, CCR, 7Department of Pathology and Neurosurgery, Northwestern National Cancer Institute at Frederick, 3Vaccine Branch, University, Chicago, 8Cleveland Clinic, Cleveland, United Basic Science Program, Frederick National Laboratory for States Cancer Research,, Leidos Biomedical Research, Frederick, 4Robert W Franz Cancer Research Center, Earle A Chiles Abstract Content: Research Institute, Providence Cancer Center, Providence Glioblastoma (GBM), the most common primary malignant Portland Medical Center, Portland, United States brain tumor, remains uniformly lethal due to therapeutic resistance by profound immunosuppression. Males Abstract Content: Immunotherapy has emerged as a constitute 60% of GBM patients and have a worse disease valuable strategy for the treatment of several type of outcome. However, the contribution of the differences in cancers. The presence of tumor-infiltrating lymphocytes is anti-tumor immunity to GBM sexual dimorphism has not considered an important predictive biomarker for clinical been elucidated. We previously demonstrated that myeloid- benefit in response to immunotherapies. Interleukin-15 (IL- 84 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

15) promotes growth and activation of cytotoxic CD8+T and elucidated in previous studies of ourselves and others, acute natural killer (NK) cells. Bioactive IL-15 is produced in the stimulation with high doses of IFNβ enhances therapeutic body as a heterodimeric cytokine, comprising the IL-15 and responses, whereas chronic stimulation with low doses of IL-15 receptor alpha chains that are together termed IFNβ increases resistance to DNA damage by up-regulating heterodimeric IL-15 (hetIL-15). Several preclinical models a set of IFN-related DNA damage resistance signature have supported the anti-tumor activity of hetIL-15, and (IRDS) genes. based on these results, it has advanced to clinical trials. The We find that the expression of PD-L1 and IRDS genes, objectives of this study were to explore how hetIL-15 including OAS1, OAS2, Mx1, IFIT1, and IFIT3, is promotes lymphocyte entry into the tumor and to concurrently elevated in a subset of cancer cells of various characterize the interactions between tumor-infiltrating origin, including breast, colon, lung, and kidney. Using an in lymphocytes (TILs) and other cell types through the analysis vitro model in which only cancer cells are present, we found of cytokines and chemokines affected by hetIL-15 treatment. that lung cancer cells expressing high levels of PD-L1 and hetIL-15 treatment was effective in controlling primary tumor IRDS proteins are highly resistant to cisplatin or ionizing growth in murine cancer models, including the MC38 colon radiation, and that knock-down of PD-L1 re-sensitizes these carcinoma, TC-1 cervical carcinoma, B16 cells. Since IRDS genes are induced by IFN-I, we examined models and orthotopic breast cancer models. In breast cancer-cell intrinsic IFN-I responses in the cancer cells, cancer models, hetIL-15 also reduced significantly finding that the cGAS-STING-IFNβ pathway is constitutively metastatic disease. hetIL-15 directly affects NK, CD8+ and activated. When we knocked down cGAS or the IFN-I CD4+ T cells within the tumor promoting their proliferation receptor (IFNAR1), the expression of PD-L1 as well as IRDS (Ki67), survival (Bcl-2) and cytotoxic commitment, with high proteins was substantially decreased, suggesting that levels of IFN-γ production and upregulation of GzmA, GzmB cancer cell-intrinsic chronic IFN-I responses are responsible and perforin. Activated lymphocytes secrete XCL1 in for high levels of PD-L1 and IRDS expression in the cancer response to hetIL-15 treatment, a chemokine responsible for cells. Interestingly, knock-down of PD-L1, which sensitizes the enhanced intratumoral influx of cross-presenting the cancer cells to DNA damage, substantially decreased dendritic cells (cDC1). Such cells expressing XCR-1, IRF-8 IRDS expression, indicating that PD-L1 makes cancer cells and CD103 were detected in tumors of hetIL-15-treated resistant to DNA damage by enhancing chronic IFN-I animals. In addition to macrophages and monocyte-derived responses, which induce IRDS expression. On the other DC, cDC1 produce CXCL9 and CXCL10 in response to hand, acute responses to high levels of IFN-I are themselves hetIL-15. Importantly, hetIL-15-driven CXCL9 and CXCL10 cytotoxic, and also a new synthesis of IFN-I in cancer cells secretion by myeloid cells required exposure to IFN-γ, increases the cytotoxicity of radiation or chemotherapeutic indicating a positive feedback loop whereby the hetIL-15- agents. We found that PD-L1 inhibits the acute IFN-I dependent increase in intratumoral XCL-1 and IFN-γ– responses, as shown by our observation that PD-L1 knock- producing lymphocytes enhances cDC1 recruitment and down dramatically increases the phosphorylation of STAT1 amplifies the CXCL9/10-directed intratumoral infiltration by and STAT2 in response to high doses of IFN-I. Taken CXCR3+ effector lymphocytes. The role of cDC1 in hetIL-15 together, this study reveals that the resistance of a subset of anti-cancer efficacy is currently being investigated in Batf3 cancer cells to DNA damage is due to the ability of PD-L1 to knock-out mice, lacking cDC1. regulate IFN-I responses by sustaining chronic IFN-I Overall, our results show that hetIL-15 orchestrates a responses that induce IRDS expression and by inhibiting multipronged immune response that includes leucocyte cytotoxic acute IFN-I responses. expansion and trafficking to the tumor, lymphoid-myeloid Immune checkpoint blockade, including anti-PD-L1, is a cell interactions and enhanced cytotoxic responses through landmark advance for treating advanced cancers but is not the induction of a favorable cytokine milieu. hetIL-15 therapy effective in patients who have poor pre-existing immunity. may be a general method to enhance T and cDC1 cell entry This study elucidating the immune-independent roles of PD- in tumors, increasing the success rate of immunotherapeutic L1 in cancer therapy will help to direct therapeutic strategies interventions. using PD-L1 inhibitors to improve their response rates, Disclosure of Interest: None Declared regardless of patients’ pre-existing immune status. Disclosure of Interest: None Declared LT166 PD-L1 ENHANCES RESISTANCE TO DNA DAMAGE BY LT167 REGULATING DOUBLE-EDGED RESPONSES TO TYPE THE AIM2 DNA SENSOR PROMOTES STAT3-DRIVEN I INTERFERON IN CANCER CELLS TUMORIGENESIS INDEPENDENT OF H. Cheon1,*, E. G. Hovey-Bates1, G. R. Stark1 INFLAMMASOMES VIA EPITHELIAL CELL MIGRATION 1Cancer Biology, Lerner Research Institute/ Cleveland R. E. Dawson1,2,*, V. Deswaerte1,2, K. Tang1,2, M. Oshima3, Clinic, Cleveland, United States P. Tan4,5,6, B. Jenkins1,2 1Hudson Insitute of Medical Reseach, 2Monash University, Abstract Content: Programmed death ligand 1 (PD-L1) on Melbourne, Australia, 3Kanazawa University, Kanazawa , cancer cells binds to PD-1 on activated T cells, suppressing Japan, 4Duke-NUS Graduate Medical School, 169857, anti-cancer immunity. In this study, we investigated immune- 5Genome Institute of Singapore, 138672, 6National independent functions of PD-L1, which inhibit the efficacy of University of Singapore, 117456, Singapore radiation or chemotherapy by regulating type I interferon (IFN-I) responses in a cancer cell-intrinsic manner. As 85 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Abstract Content: The link between chronic gastric Abstract Content: Invasive ductal carcinoma (IDC) is a inflammation – caused by over-activation of the immune serious problem for patients as it metastasizes, decreasing system – and gastric cancer (GC) is well defined, however 5-year patient survival from >95% to ~27%. The breast the identity of key regulators of the immune system that tumor microenvironment (TME) is often saturated with promote GC is not well understood. The pattern recognition proinflammatory cytokines, such as oncostatin M (OSM), receptor (PRR) absent in melanoma 2 (AIM2) is a cytosolic which are secreted from tumor-activated neutrophils and DNA sensor which contributes to the pathogenesis of macrophages and whose expression is correlated with numerous autoimmune and chronic inflammatory diseases increased breast cancer metastasis. Remodeling the in the context of inflammasome complexes. AIM2 is also extracellular matrix (ECM) of the TME plays an important linked with cancers, although its roles have been reported to role in promoting invasive and metastatic potential of IDC. be tissue-specific and often ill-defined. Considering the role Specifically, the reorganization and alignment of collagen of AIM2 in innate immune responses, we investigated fibers in stromal ECM leads to increased tumor cell motility, whether it contributed to the pathogenesis of GC. which promotes metastasis. Lysyl oxidase like-2 (LOXL2) is a protein that catalyzes ECM remodeling by crosslinking of AIM2 expression at the mRNA and protein levels was collagen I in the ECM. We propose a novel mechanism significantly upregulated in tumours of GC patients, whereby OSM induces LOXL2 expression, mediating specifically in the epithelium, and strongly correlated with stromal ECM remodeling of the breast TME. Our studies patient survival. Using the well-established gp130F/F mouse demonstrate that IDC patients with high LOXL2 and OSM model of GC, we also observed high expression of AIM2 in co-expression have a worse prognosis than those with high tumour-bearing mice compared to their wild-type levels of each, individually, and LOXL2 expression is counterparts. The coupling of the gp130F/F mouse model positively correlated to OSM/ OSM receptor (OSMR) with mice lacking AIM2 significantly decreased tumour expression in IDC patients. Furthermore, human IDC cells burden, thus supporting the pro-tumourigenic role of AIM2 treated with OSM results in a significant increase in in GC. Interestingly, our data showed that while gastric secreted, enzymatically active LOXL2. OSM-induced tumourigenesis was suppressed, inflammation or activation LOXL2 promotes significantly increased ECM collagen I of the inflammasome was unaffected, suggesting the fiber crosslinking and alignment. Taken together, these existence of a novel inflammasome-independent pro- results provide a new paradigm through which tumourigenic mechanism for AIM2 in GC. proinflammatory cytokine OSM promotes tumor progression, by promoting ECM remodeling in the TME. Specifically, we identified a decrease in the migration of Disclosure of Interest: None Declared AIM2-deficient primary mouse gastric epithelial cells and, conversely, an increase in migration of AIM2- LT169 overexpressing human GC MKN-1 cells, measured by in IL33 CYTOKINE SIGNALLING IN GASTROINTESTINAL vitro 3D migration transwell and spheroid assays. This which CANCERS - A THERAPY TARGET? was accompanied by cellular morphological differences, M. F. Eissmann1,*, C. Dijkstra1, M. Ernst1 alterations in actin cytoskeleton polymerisation, increased 1Olivia Newton-John Cancer Research Institute, Heidelberg, filopodia formation and differences in adhesion gene Australia expression. These data support changes in epithelial cell migration and motility as the underlying mechanism of the Abstract Content: Cytokine-mediated inflammation is a pro-tumourigenic role of AIM2 in GC. A strategy to identify driver of gastric and colonic tumorigenesis. IL1-family potential AIM2-interacting partners that mediate the pro- cytokine (IL33) regulates inflammatory migratory function of AIM2 will also be discussed. responses and antibodies targeting IL33 or its receptor ST2 are in clinical trials against various inflammatory and allergy Taken together, this work uncovers a novel, inflammasome- diseases. Only recently, a role of IL33 in cancer started to independent pathogenic mechanism for AIM2 in GC which emerge. Depending on the cancer stage or type IL33 can may pave the way for novel and improved strategies for GC provoke either pro- or anti-tumoral responses 1. patient outcomes through the use of AIM2 as a biomarker to To evaluate the potential of targeting IL33 signalling to identify patients which may benefit from the development of abrogate gastric and colonic cancer growth, we employ therapeutic targeting of AIM2. gp130Y757F/ Y757F (FF) gastric cancer mice and the 6xAOM Disclosure of Interest: None Declared sporadic colon cancer model as well subcutaneously engrafted MC38 colon cancer models. IL33 signalling LT168 inhibition was achieved via genetic deficiency (St2-/-), while A NEW MECHANISM FOR PROINFLAMMATORY recombinant IL33 was administered to hyper activate the CYTOKINE-PROMOTED EXTRACELLULAR MATRIX IL33-signalling pathway. REMODELING IN BREAST CANCER We found, that IL33 cytokine and ST2 receptor are S. Dinca1,*, D. Greiner2, K. Weidenfeld3, L. Bond1, D. overexpressed in human gastric cancers and high ST2 Barkan3, C. Jorcyk1 expression predicts poor survival for gastric cancer patients. 1Boise State University, Boise, 2University of Utah, Salt Lake In accordance, IL33 and ST2 are highly elevated in our FF City, United States, 3University of Haifa , Haifa, Israel mouse tumors. Deficiency of IL33 signalling (ST2-/-) diminishes gastric tumour growth, and is associated with a decrease in tumour-adjacent pro-tumoral mast cells and 86 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 tumour-infiltrating macrophages (TAM) as well as reduced members. Human breast tissue microarray analysis shows angiogenesis. Furthermore, ST2-deficiency potentiates the that OSM is expressed at highest levels in the precancerous anti-tumor effects of oxaliplatin chemotherapy in the FF epithelial cells of ductal carcinoma in situ (DCIS), mouse model. Mechanistically, we show that tumour- suggesting a role for autocrine-produced OSM in invasive produced IL33 can activate mast cells, which in turn recruit potential. Tumor-associated neutrophils (TANs) release pro-tumoral and pro-angiogenic macrophages to the tumour large amounts of OSM when they come into contact with through release of chemo-attractants like Ccl2, Ccl3 and breast cancer cells, and this paracrine-produced OSM Ccl7 2. induces invasive capacity. Employing human breast and In the colon cancer setting, ST2-deficiency led to increased mouse mammary orthotopic xenograft and syngeneic tumour burden in 6xAOM sporadic colorectal cancer. mouse models, OSM promotes metastasis to bone, lung, Reciprocal bone marrow chimera experiments indicated that and other organs. Furthermore, OSM increases circulating the radio-resistant non-hematopoietic cell compartment tumor cell (CTC) numbers, which are reduced in an OSM contributes to the increased tumour growth. Indeed, we knockout background, demonstrating the importance of both found St2 expression in the mesenchymal cells of the tumor paracrine- and autocrine-produced OSM in this process. microenvironment. Loss of IL33 signalling in the non- When supported by in vitro results, where OSM remains haematopoietic radio-resistant compartment coincided with active when it binds to extracellular matrix (ECM) proteins a strong reduction of an IFNy gene expression signature. and induces tumor cell-produced protease secretion and Importantly, IL33 cytokine administration reduced colon other ECM-modifying enzymes, our findings suggest a role cancer allograft growth associated with tumour-infiltrating for OSM in early stage metastasis. Finally, the importance IFNy-positive T cells 3. of OSM relative to IL-6 in these processes will be Our data demonstrates the opposing roles of IL33 signalling discussed, and as anti-IL-6 cancer therapies () in the growth of gastric and colonic cancers and identifies are failing in clinical trials, our results suggest that OSM may cellular mediators of these divergent tumor responses. be a more rational target. Collectively, these data suggest Further studies are warranted to stratify IL33-signalling that OSM, whether TME- or autocrine-produced, is a critical sensitive GI cancers and to verify the potential of anti- factor driving breast cancer invasion and tumor cell IL33/ST2 antibodies against gastric cancer and activating dissemination with subsequent metastasis. IL33 cytokine administration in colon cancers as novel Funding provided by NIH grants P20GM109095, therapy strategies. P20GM103408, and R25GM123927, NSF-BSF 2017237, the METAvivor Quinn Davis Northwest Arkansas 1 Eissmann MF, et al. IL33 and Mast Cells—The Key METSquerade Fund, and the Smylie Family Cancer Fund. Regulators of Immune Responses in Gastrointestinal We also acknowledge the support of The Biomolecular Cancers? Front. Immunol. 2020;11:1389. doi: Research Center at Boise State University. 10.3389/fimmu.2020.01389 Disclosure of Interest: None Declared 2 Eissmann MF, et al. IL-33-mediated activation promotes gastric cancer through macrophage mobilization. LT173 Nature Communications 2019 Jun; 21;10(1):2735 COMPLETE REGRESSION OF MURINE BREAST 3 Eissmann MF, et al. Interleukin 33 Signaling Restrains TUMORS AND LONG-TERM ANTI-TUMOR IMMUNITY BY Sporadic Colon Cancer in an Interferon-y-Dependent HETIL-15 MONOTHERAPY IS MEDIATED THROUGH Manner. Cancer Immunol Res 2018 April; 6(4):409-421. THE INTERACTION OF T, NK, CDC1 CELLS AND A Disclosure of Interest: None Declared NOVEL POPULATION OF DENDRITIC CELLS D. Stellas1, S. Karaliota2,*, V. Stravokefalou1,3, B. Nagy1,4, LT171 C. Bergamaschi4, B. K. Felber4, G. N. Pavlakis1 CURRENT UNDERSTANDINGS ON HOW AN 1Human Retrovirus Section, Vaccine Branch, Center for INFLAMMATORY TUMOR MICROENVIRONMENT Cancer Research, National Cancer Institute at Frederick, PROMOTES INVASION AND EARLY STAGE BREAST 2Vaccine Branch, Basic Science Program, Frederick CANCER METASTASIS National Laboratory for Cancer Research, Frederick, United S. Dinca1, D. Greiner2, K. Tawara1, C. L. Jorcyk1,* States, 3Department of Pharmacology, Faculty of Medicine, 1Biological Sciences/Biomolecular Sciences, Boise State University of Thessaly, Larissa, Greece, 4Human Retrovirus University, Boise, 2Biochemistry, University of Utha, Salt Pathogenesis Section, Vaccine Branch, Center for Cancer Lake City, United States Research, National Cancer Institute at Frederick, Frederick, United States Abstract Content: The propensity of primary breast cancer Abstract Content: Background and Hypotheses: IL-15 is to invade and metastasize has been partially explained by a cytokine which stimulates the proliferation and cytotoxic high levels of endogenous inflammation. However, it is not function of CD8+ T and NK cells. We have produced the fully understood how inflammatory mediators, expressed native heterodimeric IL-15 (hetIL-15) of human, macaque or either by the primary breast cancer cells or cells of the tumor mouse origin. The objectives of the study were to explore microenvironment (TME), specifically contribute to the mechanisms of anti-tumor effects of hetIL-15 after metastasis. Our lab has been studying how oncostatin M locoregional administration in orthotopic breast cancer (OSM), an interleukin-6 (IL-6)-family inflammatory cytokine, models and to characterize the interactions between tumor- promotes the development of early metastatic infiltrating lymphoid and myeloid cells. properties distinct from IL-6 and other family 87 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Study Design and Methods: We studied the therapeutic Correspondence: Yuliya Pylayeva-Gupta, Ph.D., Lineberger efficacy of hetIL-15 in the murine EO771 orthotopic breast Comprehensive Cancer Center, University of North cancer model in syngeneic C57BL/6 mice. The effects of Carolina, 450 West Drive, Chapel Hill, 27599. hetIL-15 on immune cells were analyzed by flow cytometry, immunohistochemistry (IHC) and gene expression profiling. Abstract We also assessed the metabolic profile of tumor infiltrating T cells. Transcription factors guided by interaction of signals through Results and Conclusions: hetIL-15 peritumoral cytokine and toll-like receptors are critical for B cell administration resulted in complete regression in 40% of the differentiation. We recently showed IL-35, a novel immune- treated animals and increased survival. Subsequent regulatory cytokine produced by B cells suppress anti-tumor rechallenges with the same cell line failed to generate T cell response and promotes pathogenesis of pancreatic tumors. Therefore, locoregional hetIL-15 administration cancer. Previous studies have shown that STAT3/STAT4 induced long term immunological memory. CD8+ T and NK heterodimer is linked to IL-35 downstream signaling in T cells were found increased in hetIL-15 treated tumors and cells. However, the exact role of IL-35 and its downstream showed enhanced activation and proliferation. signaling pathways in B cells that can induce immune Transcriptomic analysis confirmed the activated state of the suppressive Breg cell differentiation has not been fully T and NK cells, whereas metabolic flux analysis of the tumor understood. In this study, we showed that IL-35 induced infiltrated CD8+T cells from treated mice confirmed a rise in STAT1/STAT3 signaling is essential for generation of Breg oxygen consumption rate (OCR) with substantial increase of cells from naïve B precursors and their tumor accumulation. spare respiratory capacity. In addition, peritumoral hetIL-15 Increased IL-35 and IL-10 production and altered administration resulted in an increase of infiltrating, suppressive activity were found in pancreatic cancer conventional type 1 dendritic cells (cDC1s), suggesting educated B cells. IL-35 strongly induces p35, EBi3, IL-10 mechanisms of DC-lymphocyte interactions in the tumor. and CD1d gene expression in pancreatic cancer educated We also observed a new population of intratumoral DCs naïve B cells as well as promotes their tumor infiltration. found only in the hetIL-15 treated animals. The cDC1s and Chromatin immunoprecipitation confirms Breg cell the new DC population were inversely correlated with the suppressive genes p35, EBi3, IL-10 and CD1d are the direct tumor size. Phenotypic profile of the new DC population target of STAT1/STAT3 in IL-35 dependent manner. Our showed expression of several cDC1 specific markers, results demonstrate that IL-35, via STAT1/STAT3, induces including CD103 and IRF8. RNAseq analysis of the sorted generation of immunosuppressive Breg phenotype and DC populations derived from the treated tumors, verified maintenance in tumor microenvironment. In sum, these unique clustering of this new DC population. studies define new insight into the specific role of Therefore, locoregional administration of hetIL-15 results in STAT1/STAT3 signaling particularly in the Breg cell lineage complete eradication of EO771 primary breast cancer differentiation and identify STAT1/STAT3 axis as a novel tumors, prolonged survival and long-lasting specific anti- target for treatment of B cell dependent deadly cancers like tumor immunity. The anti-cancer activity of hetIL-15 in pancreatic cancer. primary EO771 tumors is orchestrated by the interplay of NK, CD8+T cells, cDC1 and a novel subset of antigen Disclosure of Interest: None Declared presenting cells with a distinct phenotypic profile. Disclosure of Interest: None Declared LT176 CHARACTERIZATION OF IL-15 DEFICIENT LT175 THYMOCYTES IN THE MODEL OF SPONTANEOUS IL-35 SIGNALING PROMOTES REGULATORY B CELL LEUKEMIA GENERATION AND TUMOR ACCUMULATION BY M. Nandi1,*, S. Ramanathan1 ENHANCING STAT1/STAT3 AXIS 1Universite de Sherbrooke, Sherbrooke, Quebec, Canada B. Mirlekar1,*, Y. Pylayeva-Gupta1 1Lineberger Comprehensive Cancer Center, University of Abstract Content: Leukemia is a diverse group of North Carolina at Chapel Hill, Chapel Hill, United States hematopoietic malignancies that cause significant morbidity and mortality in children and adults. T cell acute Abstract Content: IL-35 signaling promotes regulatory lymphoblastic leukemia (T-ALL) is an aggressive tumor that B cell generation and tumor accumulation by enhancing accounts for 10-15% of pediatric ALL. Even though intensive STAT1/STAT3 axis chemotherapy can cure 80% of pediatric cases, chemo- resistant and relapse cases have poor Bhalchandra Mirlekar1,2, Yuliya Pylayeva-Gupta1,2 prognosis. Treatment of such cases requires the development of new approaches through better 1The Lineberger Comprehensive Cancer Center, University understanding of the molecular mechanisms of of North Carolina School of Medicine, Chapel Hill, North leukemogenesis. Carolina 2Department of Genetics, University of North Carolina We reported that NOD.Scid (NS) mice lacking either School of Medicine, Chapel Hill, North Carolina, 27599. interleukin-15 NOD.Scid.Il15-/- or IL-15 receptor alpha chain NOD.Scid.Il15ra-/- spontaneously develop T cell leukemia with 100% penetrance by 30 weeks of age. Leukemic cell 88 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 lines established from NOD.Scid.Il15-/- mice express the T- deletions and 19% hemizygous detentions. Next to it, ALL marker TdT, display constitutive NOTCH1 activation complete loss of the type I Interferon (IFN) gene cluster, and are sensitive to NOTCH inhibition. Depletion of NK cells represent 1/3 of all the tumors that have lost CDKN2A. did not promote the development of leukemia in NOD.Scid Newcastle disease virus (NDV), an avian paramyxovirus, mice, indicating NK-independent role for endogenous IL-15 has been proposed for the treatment of different tumors, in preventing the survival and outgrowth of leukemic cell including GBM, due to its oncolytic and immunostimulatory precursors. Analysis of thymi from 4 wks-old NOD.Scid.Il15- properties. IFN cluster deletion implies the loss of an /- mice show discernible changes in the phenotype of DN efficient antiviral response. The secretion of type I IFNs that thymocytes compared to those of control mice. Whereas triggers an antiviral response blocking virus replication. NOD.Scid mice showed developmental arrest at the DN3 GBM susceptibility to NDV is dependent on the loss of the stage, substantial numbers of DN4, DP and SP cells were type I IFN. A recombinant NDV bearing the NS1 gene observed in NOD.Scid.Il15-/- thymi. (rNDV-NS1), a strong antagonist of the IFN response, could These findings strongly suggest that IL-15 signaling within exert higher oncolytic activity in the type I IFN-competent the thymus controls the emergence of leukemogenic CSCs. GBM patient stratification, evaluating type-I IFN precursors from developing thymocytes. competence, could predict NDV and other oncolytic Disclosure of Interest: None Declared therapies. Disclosure of Interest: None Declared LT177 NEWCASTLE DISEASE VIRUS (NDV) ONCOLYTIC LT178 ACTIVITY IN HUMAN GLIOMA TUMORS IS DEPENDENT INTRACELLULAR EXPRESSION OF IFN- 4 LEADS TO ON TYPE I IFN GENE CLUSTER CODELETION. ER STRESS, ENHANCED IRF1 SIGNALING AND E. Nistal-Villan1,2,*, I. Palacin-Aliana3,4, N. García-Romero DECREASED PROLIFERATION IN HEPATIC CELLS 5,6, R. Madurga5,7, S. Esteban-Rubio7, S. Rius-Rocabert2,8,9, THAT MIGHT PROTECT HCV+ PATIENTS FROM LIVER J. Carrión-Navarro5,10, J. Presa11, S. Cuadrado-Castaño12, CIRRHOSIS P. Sanchez-Gomez13, A. Garcia-Sastre14,15,16, Á. Ayuso- O. Onabajo1,*, F. Wang1, M.-H. Lee2, O. Florez-Vargas1, A. Sacido10,17 Obajemu1, M. A. Castro3, J. Vargas1, S.-F. Liao2, C.-Y. 1Pharmaceutical and Health Science, University CEU San Shen4, Y.-H. Huang2, R. Banday1, T. R. O'Brien5, A. G. Pablo, 2 Instituto de Medicina Molecular Aplicada (IMMA), Robertson6, L. Prokunina-Olsson1 Universidad San Pablo-CEU, 3Atrys Health, 4Fundación de 1Laboratory of Translational Genomics, Division of Cancer Investigación HM Hospitales, HM Hospitales, 5Faculty of Epidemiology and Genetics, National Cancer Institute, Experimental Sciences, Universidad Francisco de Vitoria, Bethesda, United States, 2Institute of Clinical Medicine, 6Brain tumour laboratory, Fundación Vithas, Grupo National Yang-Ming University, Taipei, Taiwan, Hospitales Vithas, 7Facultad de Medicina, Instituto de 3Bioinformatics and Systems Biology Lab, Federal Medicina Molecular Aplicada (IMMA), Universidad San University of Parana, Curitiba, Brazil, 4Institute of Biomedical Pablo-CEU, 8Microbiology Section, Dpto. CC, Sciences, Academia Sinica, Taipei, Taiwan, 5Infections and Farmacéuticas y de la Salud, Facultad de Farmacia, Immunoepidemiology Branch, Division of Cancer Universidad San Pablo-CEU, 9CEMBIO (Centre for Epidemiology and Genetics, National Cancer Institute, Metabolomics and Bioanalysis), Facultad de Farmacia, Rockville, United States, 6Canada’s Michael Smith Genome Universidad San Pablo-CEU, 10 Brain tumour laboratory, Sciences Center, BC Cancer Agency, Vancouver, Canada Fundación Vithas, Grupo Hospitales Vithas, 11Independent Researcher, 12Department of Microbiology, Global Health Abstract Content: Several genetic variants in the human and Emerging Pathogens Institute, Icahn School of IFNL3/IFNL4 locus have been associated with reduced Medicine at Mount Sinai, 13Neurooncology Unit, Instituto de hepatic fibrosis despite poor clearance of HCV infection. We Salud Carlos III-UFIEC, Madrid, Spain, 14Microbiology expanded this analysis to cirrhosis, a more advanced stage Department, Global Health and Emerging Pathogens of fibrosis. In 2931 individuals with chronic HCV, the IFNL4 Institute, Icahn School of Medicine at Mount Sinai, genotype that generates IFN-l4 was associated with 15Department of Medicine, Division of Infectious Disease, protection from cirrhosis (OR=0.65, p=0.012, adjusted for Icahn School of Medicine at Mount Sinai, New York, 16The age and sex). The IFNL4 genotype affects the production of Tisch Cancer Institute, Icahn School of Medicine at Mount IFN-l4 and, additionally, may affect expression levels of IFN- Sinai, New York, United States, 17 Faculty of Experimental l3, making it difficult to delineate the individual contribution Sciences, Universidad Francisco de Vitoria, Madrid, Spain of these IFNs. To address this, we established HepG2- based cell models engineered to inducibly express either Abstract Content: Glioblastoma (GBM) is the most IFN-l3 or IFN-l4. Using RNA-seq based expression profile devastating and least treatable primary brain tumor in adults generated in these hepatic cell lines, we explored the global with a median overall survival of 15 months. Some of the transcriptome of liver tumors (n=373) from The Cancer most common and relevant mutations in gliomas are the Genome Atlas to identify transcription factor networks deletions at Ch9p21, which involves the genetic loss of the affected by these IFNs in the liver. Several networks, CDKN2A. We have evaluated the genetic loss of the type I including of the IRF1, were upregulated by IFN-l4 more IFN locus in 1018 glioma tumors and the NDV oncolytic strongly than by IFN-l3. Intracellular expression of IFN-l4 but effect in six GBM cancer stem cells (CSCs). Human GBM not of IFN-l3 also led to potent IRF1-dependent CDKN2A (p16) deletion represents 57% homozygous antiproliferative effects. Live cell imaging revealed that IFN- 89 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 l4 was poorly secreted, mainly accumulated in lysosomes, Results: RANKL/PD-L1 expression profile on specimens and caused apoptosis, suggesting increased ER-stress via from each breast cancer subtypes showed that both the misfolded protein response. Knockdown of DNA immunosuppressive molecules are expressed by all breast damage-inducible transcript 3 (DDIT3), an ER-stress cancers with a significantly more intense immunoreactivity response effector, significantly attenuated the for triple negative breast cancers. Most of the cell lines antiproliferative effects of IFN-l4. This novel interplay of expressed both proteins. We found that RANKL is secreted enhanced IRF1 signaling coupled with intracellular in their extracellular environment. RANKL inhibitors are accumulation and induction of ER stress by IFN-l4 may have efficient in vitro and are currently been tested in vivo. First complex consequences on liver homeostasis during chronic results highlight a decreased tumor growth in the presence HCV infection but also mediate anti-cirrhotic phenotypes. of RANKL inhibitors. Moreover, an increase in TCD4+ and Disclosure of Interest: None Declared TCD8+ cells and a decrease in granulocytes and M2 macrophages have been observed in the different immune LT179 cell populations infiltrating the tumor. COMBINING PD-1/PD-L1 BLOCKADE AND RANKL Conclusions: Several other murine triple negative breast INHIBITORS TO TREAT BREAST CANCERS cancer cell lines will also be sub-cutaneously injected in UNRESPONSIVE TO STANDARD THERAPY mice and the efficacy of both RANKL and PD-L1 inhibitors C. Pilard1,*, P. Roncarati1, E. Hendrick1, D. Bruyère1, T. will be evaluated (separately or in combination). The Lerho1, M. Ancion1, C. Reynders1, P. Delvenne1, M. Herfs1, infiltration of tumor microenvironment by different immune P. Hubert1 cell populations, the presence of metastasis and the tumor 1Laboratory of Experimental Pathology, GIGA-Cancer, growth will be analyzed. University of Liege, Liege, Belgium, Liège, Belgium Disclosure of Interest: None Declared

Abstract Content: Background: In the past decade, LT180 immunotherapy using immune checkpoint inhibitors DONOR IFNL4 GENOTYPE PREDICTS NON-RELAPSE (especially targeting the PD-1/PD-L1 axis) has been MORTALITY AFTER UNRELATED DONOR demonstrated as a promising strategy for the treatment of MYELOABLATIVE HEMATOPOIETIC CELL cancers that do not respond to classical chemoradiotherapy. TRANSPLANTATION FOR ACUTE LEUKEMIA Given that cancer cells have the potential to express many L. Prokunina-Olsson1,*, S. Gadalla2, Y. Wang2, T. Wang3, immunosuppressive molecules other than PD-L1, the O. R. Onabajo1, R. Banday1, A. Obajemu1, E. Karaesman4, combination of immune checkpoint inhibitors with other L. Sucheston-Campbell4, T. Hahn5, J. A. Sees6, S. R. drugs thwarting tumor immunosuppressive Spellman6, S. J. Lee7, H. A. Katki8 microenvironment could represent a promising strategy. 1Laboratory of Translational Genomics, Division of Cancer Among these immunosuppressive molecules, RANKL, a Epidemiology and Genetics, 2Clinical Genetics Branch, member of the TNF superfamily, which mainly affects the Division of Cancer Epidemiology and Genetics, National immune system and bone remodeling, has been shown to Cancer Institute, Bethesda, 3Division of Biostatistics, be a key factor promoting the progression of breast cancer. Medical College of Wisconsin, Milwaukee, 4College of In addition, RANKL induces the formation of tolerogenic Pharmacy, The Ohio State University, Columbus, 5Roswell dendritic cells and Treg cells, which promotes Park Comprehensive Cancer Center, Buffalo, 6Center for immunotolerance to the tumor. International Blood and Marrow Transplant Research, The aim of this research project is to study the impact of Minneapolis, 7Clinical Research Division, Fred Hutchinson several RANKL inhibitors on triple negative breast cancer Cancer Research Center, Seattle, 8Biostatistics Branch, and to analyze the efficiency of their association with anti- Division of Cancer Epidemiology and Genetics, National PD-1/PD-L1 agents. Cancer Institute, Bethesda, United States Material and methods: We studied RANKL and PD-L1 expression in several murine and human breast cancer cell Abstract Content: Background. IFNL4 genotype regulates lines by immunohistochemistry. The secretion of RANKL the immune response by controlling the production of IFN- was analyzed by ELISA. Inhibitors of RANKL were then λ4, a type III interferon. We evaluated the association tested in vitro. We selected several RANKL inhibitors: anti- between donor and recipient IFNL4 genotype with survival RANKL antibody, RANK-Fc, Isoliquiritigenin and in patients with acute leukemia who received myeloablative Gallocatechin gallate. The efficacy of these inhibitors was 10/10 HLA-matched hematopoietic cell transplant (HCT). indirectly evaluated with the murine macrophage RAW264.7 Methods. We conducted a two-stage cohort study: in a cell line which undergoes, in the presence of RANKL, an discovery set of 404 patients we genotyped IFNL4 osteoclast differentiation (TRAP and Cathepsin K polymorphisms (rs368234815, rs12979860, and expression). The efficacy of RANKL inhibitors was then rs117648444) with TaqMan assays; and in an independent evaluated, in this model, by RT-qPCR. Apoptosis, validation of 1245 patients from the DISCOVeRY-BMT study proliferation and mitochondrial respiration of the cancer cell we used existing Illumina array genotype data. Pre-HCT lines in the presence of the inhibitors were also analyzed. blood samples and outcome data were provided by the RANKL inhibitors were then tested in vivo in a mice model. Center for International Blood and Marrow Transplant Tumor growth was followed and the infiltration of tumor Research. microenvironment by different immune cell populations were Findings. IFNL4-positive genotype of donor but not analyzed by flow cytometry. recipient was associated with increased risk of non-relapse 90 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 mortality (NRM; HR-per allele=1.60, 95% CI=1.23-2.10, L. N. Schulte1,2,* on behalf of SFB-TR84 "Innate Immunity of p=0.0005, and HR=1.22, 95% CI=1.05-1.40, p=0.007, in the the Lung" discovery and validation, respectively). The effect was 1Institute for Lung Research, Philipps-University Marburg, driven by excess risk of deaths from infections and graft- Marburg, 2German Center for Lung Research (DZL), versus-host disease (HR-combined sets=1.54, 95% Giessen, Germany CI=1.16-2.04, p=0.003, and 1.46, 95% CI=1.11-1.94, p=0.008), respectively. An association between donor Abstract Content: The activity of mammalian phagocytes IFNL4-positive genotype and patient post-HCT overall is controlled at several levels by regulatory RNAs, which survival (OS) was also noted (HR-combined sets=1.11, 95% ensure the correct timing of soluble inflammatory mediator CI=1.02-1.22, p=0.02). Single-cell RNA-sequencing production. While the fine-tuning activities of microRNAs in analysis identified IFNL4 as the main interferon transcript this process are well-understood, the mechanistic expressed in bone marrow cells, detected in 0.67% of all implications of long non-coding RNAs (lncRNAs) are only cells and 83.1% of all interferon-expressing cells. beginning to emerge. We previously determined the global Interpretation. Prioritizing HCT donors with IFNL4-Null association of TLR-inducible lncRNAs with macrophage genotype may decrease the risk of NRM and improve OS protein machines by combining subcellular fractionation with without significantly limiting the candidate donor pool. density gradient centrifugation and RNA-seq and found that Disclosure of Interest: None Declared lncRNAs in LPS-activated macrophages are often localized in the cytoplasm and interact with ribosomes. Here, we LT180b selected from the non-ribosome-associated lncRNAs the top ERK IS INVOLVED IN STEROID-LIKE CUCURBITACIN D- 10 LPS-inducible transcripts in blood-derived and tissue- INDUCED ANTITUMOR EFFECTS ON ATL CELLS resident human macrophages for a small-scale CRISPR D. Wang1, M. Shen1, Y. Yoshida1,* interference screen. Loss-of-function phenotypes of the 1Immunology and Parasitology, Univ. of Occupational and tested lncRNAs ranged from imbalances in the TLR-NFκB Environmental Health, Kitakyushu, Japan and JAK-STAT signalling pathways to changes in macrophage differentiation programs. Mechanistically, LPS- Abstract Content: Adult T cell leukemia (ATL) is an inducible lncRNAs interacted with cytoplasmic TLR aggressive andmalignant blood disease. We previously signalling components, alternative splice factors and reported that steroid-structured cucurbitacin D (CuD) membraneless nuclear organelles to regulate pro- induces apoptosis in ATL cells from patients and cell lines. inflammatory macrophage activity. Our data suggest that In this study, we investigated the effects of mitogen- alongside potentially translated novel LPS-responsive activated protein kinase (MAPK) signaling inhibitors on RNAs, true long non-coding RNAs constitute an important CuD-induced cell death in peripheral blood lymphocytes pillar of the cytoplasmic and nuclear networks involved in the (PBLs) isolated from ATL/ acute lymphoblastic leukemia adequate activation of human macrophage immunity. (ALL) patients and ATL cell lines (MT-1 and MT-4). Cell Disclosure of Interest: None Declared surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex. LT182 Cell proliferation was assessed using the Cell Titer-Glo GLUTEN CONSUMPTION MAINTAINS VIRAL INDUCED luminescent cell viability assay. Protein expression was TYPE I INTERFERON PATHWAY THROUGH AN determined by western blotting. PBLs from patients highly INCREASE OF M6A LEVELS expressed CD4 and CD5. Serum from the patient contained M. Sebastian-delaCruz1,2,*, A. Olazagoitia-Garmendia1,2, L. high levels of interleukin (IL)-8, IL-10, IL-18, and IFN-γ M. Mendoza1, M. Legarda3, C. Tutau3, J. R. Bilbao1,2,4, A. compared to the healthy donor. CuD-induced cell death was Castellanos-Rubio1,2,4,5 enhanced by the mitogen-activated protein kinase kinase 1Genetics, Universtity of the Basque Country, Leioa, (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal 2BioCruces Bizkaia Health Research Institute, 3Pediatric kinase (JNK) inhibitor prevented CuD-induced cell death. Gastroenterology Section, Cruces Hospital, Barakaldo, Immunoblot analyses revealed that CuD reduced the 4CIBERDEM, Madrid, 5IKERBASQUE, Basque Foundation phosphorylation of ERK, p38, and JNK, and co-treatment for Science, Bilbao, Spain with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced Abstract Content: cell death, and U0126 enhanced the CuD-induced de- Celiac disease (CeD) is a complex autoimmune disorder in phosphorylation of ERK in MT-1 and MT-4 cells. We which gliadin from gluten is the known triggering agent and conclude that CuD reduces ERK activation, resulting in where both the innate and the adaptive immune responses enhanced antitumor effects on ATL/ALL cells. are implicated. While adaptive immune mechanisms have Disclosure of Interest: None Declared been deeply studied, the role of innate immunity in the development of the disease remains mainly unclear. Among Lightning Talk Session 3: Innate Immunity III other environmental factors, enteroviral infections have LT181 been linked to the risk of developing the disease. The type I A NETWORK OF CYTOPLASMIC AND NUCLEAR LONG interferon (IFN-I) pathway, which is upregulated in CeD NON-CODING RNAS REGULATES HUMAN patients, is activated upon viral infections, being an MACROPHAGE IMMUNITY important innate immune response mechanism. It has been described that m6A methylated RNAs are able to control the 91 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 innate immune response to infections by targeting IFN-Is relevance of this phenomenon was confirmed by inhibiting and have also been involved in the development of the action of Erk1/2 and JNK kinases: in the presence of autoimmunity. MAPK inhibitors, mir-24-3p was not induced, STING level We hypothesized that gluten consumption can reactivate the was unaltered and HSV1 replicated less efficiently. In Vivo IFN-I pathway in patients that have been previously infected mouse studies revealed reduced HSV-1 replication and by enteric virus, and that this may be regulated by an m6A- alleviated disease symptoms upon silencing of miR-24-3p in dependent mechanism. To test this hypothesis, we used mice brain. Silencing of miR-24 enhanced IFN-β driven polyinosinic-polycytidylic acid (PIC) treatment as a viral antiviral response against HSV-1 infection in mice. Thus, our mimic in combination with gluten stimulations in HCT15 experimental findings identified a new pathway by which intestinal cells. As expected, PIC treatment induced the HSV-1 can evade antiviral immune responses by inhibiting IRF7-IFNB pathway, together with downregulation of m6A STING mRNA translation. eraser ALKBH5 and an overall increase in m6A levels after Disclosure of Interest: None Declared 24h. We then combined PIC and gliadin stimulations to evaluate the effect of gluten on the m6A machinery and IFN- LT184 I pathway after a mimic viral infection. We observed a ADAPTIVE CHANGES IN HIV-1 ENVELOPE RESULTING synergistic effect, since both ALKBH5 decrease and IRF7 FROM IN VIVO ADAPTATION OF SHIVS CONFERS induction were augmented in cells treated with both agents. RESISTANCE TO INTERFERON METTL3 expression was also increased by gliadin A. C. Smith1, H. Weight2, J. Overbaugh2, A. Sharma1,* stimulation after PIC treatment. In addition, ALKBH5 1Department of Veterinary Biosciences; Department of silencing and METTL3 overexpression increased IRF7 Microbial Infection & Immunity, Ohio State University, expression and downstream genes STAT1 and CXCL10. Columbus, 2Human Biology Division, Fred Hutchinson Methylation of IRF7 mRNA was confirmed by m6-RIP- Cancer Research Center, Seattle, United States qPCR, suggesting that the induction observed after the combination of viral mimic and gliadin stimulation might be, Abstract Content: HIV-1 does not persistently infect at least partially, mediated by an m6A increase. Likewise, macaques due to restriction by several type-I interferon we observed that total m6A levels together with some m6A (IFN)-induced host factors. To overcome these restrictions machinery genes are higher in biopsies from CeD patients. chimeric SIV/HIV-1 viruses (SHIVs), which encode the SIV Besides, IRF7, STAT1 and CXCL10 are constitutively antagonists of the known restriction factors and HIV-1 upregulated in celiac patients. Envelope glycoprotein (Env), are used to infect macaques In conclusion, our results suggest that in the context of viral to model HIV-1 infection. Existing SHIV/macaque models infection, gliadin consumption may lead to an autoimmune typically employ SHIVs that encode HIV-1 sequences from response by the alteration of m6A machinery and the viruses amplified in culture that were further adapted in induction of innate immunity pathways. macaques (adapted SHIVs). Development of SHIVs Disclosure of Interest: None Declared encoding HIV-1 variants derived directly from infected humans without adaptation (unadapted SHIVs) has been LT183 challenging as these SHIVs replicate poorly in macaque ROLE OF MICRORNA-24-3P IN THE REGULATION OF cells. Our prior studies demonstrated that in contrast to the HERPES SIMPLEX VIRUS-1 INDUCED STING adapted SHIVs, the unadapted SHIVs have lower replication SIGNALLING kinetics in macaque lymphocytes and are sensitive to IFN. N. Sharma1,*, G. C. Sen1 The HIV-1 envelope gene was defined as the major 1Inflammation and Immunity, Lerner research institute, determinant of both replication and sensitivity to the IFN Cleveland clinic, cleveland, United States response in macaque lymphocytes. However, these studies did not directly examine the determinants of IFN adaptation Abstract Content: The type I interferon system is the first in SHIVs that were adapted through serial macaque- line of cellular antiviral innate immune response; virus passage. infection is recognized by various pattern recognition In order to identify the viral determinant(s) of macaque- receptors in the infected cell and it activates the interferon passaged SHIVs that confer resistance to IFN in vivo, we system to inhibit virus replication. However, viruses have took advantage of our previous observations, which showed evolved various mechanisms to reduce the cellular immune that the serial macaque-passage of a subtype C SHIV response and enhance viral replication. For Herpes Simplex encoding a recently transmitted strain of HIV-1 led to a more Virus-1, the cGAS/STING pathway is the principal route of IFN-resistant virus population. We generated SHIV interferon induction; we report here a new mechanism by infectious molecular clones (IMCs) encoding the parental which HSV1 evades this process by inducing a microRNA subtype C env and representative env clones from the that inhibits STING expression. Bioinformatics analysis sequential macaque-passaged viruses. Importantly, these revealed that a cellular miRNA, miR-24-3p, can target the IMCs are isogenic in the SIV portion of the genome and 3’UTR of STING mRNA and experimental manipulation of differ only in HIV-1 env sequences. We found that the its cellular level confirmed that it regulates STING unpassaged, parental SHIV IMC is potently inhibited by IFN expression. HSV1 stimulated miR-24 synthesis through (mean IC50 range 1.9 to 54.6 U/ml), whereas the SHIV IMCs activation of the Erk1/2 and JNK MAP kinases; encoding macaque-passaged envs are resistant to IFN consequently, in the infected cell, miR-24-3p level was inhibition (mean IC50 >5000 U/ml), consistent with the higher and STING level was lower. The physiological studies of the adapted SHIV quasispecies. In addition, we 92 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 found that SHIV IMCs encoding macaque-passaged envs pyroptosis. This process drives a pro-inflammatory form of have high replication capacity and most, but not all, have cell death that serves to release the mature cytokines along more virion Env content. Next, we took a gain-of-function with other inflammatory mediators, thereby initiating the approach and generated chimeras that introduce portions of inflammatory response. While multiple regulators of the env gene from IFN-resistant SHIV IMC into the parental IFN- NLRP3 inflammasome have been described, specific sensitive SHIV IMC. Using this approach, we mapped the ligands of NLRP3 and its mechanism of activation remain determinant of IFN resistance and high replication capacity largely unknown. We performed a proteomics screen using to the gp120 subunit of HIV-1 Env. co-immunoprecipitation (IP) and mass spectrometry to In conclusion, our results indicate that the adaptive changes identify cellular proteins that bind NLRP3. Using this screen, in the gp120 subunit of HIV-1 env resulting from the in vivo we identified multiple NLRP3 interactors, including a family adaptation of SHIVs via serial macaque-passage are of solute carrier (SLC) proteins that localize to the inner sufficient to increase resistance to IFN replication capacity, mitochondrial membrane. Co-IP experiments verified that and virion Env content. four of these proteins, namely SLC25A1, a citrate Disclosure of Interest: None Declared transporter, SLC25A6, an ADP/ATP translocase, SLC25A11, a 2-oxoglutarate/malate carrier, and SLC25A13, LT185 a calcium-dependent glutamate transporter, specifically RAPID REMODELING OF POISED CHROMATIN interact with NLRP3. Because of its linkage with known LANDSCAPES AND TRANSCRIPTION FACTOR metabolic disease, we further assessed the role of REPURPOSING FACILITATE GENE INDUCTION IN SCL25A13 in inflammasome function. When ectopically NATURAL KILLER CELLS expressed, SLC25A13 induced IL-1β release in cells, while H.-Y. Shih1,*, G. Sciumè2, J. O'Shea2 CRISPR/Cas9 knockout of SLC25A13 from THP-1 1NEI, NIH, 2NIAMS, NIH, Bethesda, United States macrophages partially abrogated NLRP3-dependent pyroptosis, IL-1β release, and ASC speck formation. This Abstract Content: Innate immune responses rely on rapid regulatory activity of SLC25A13 was dependent on its and precise orchestration of gene regulation mediated by calcium-sensing N-terminal domain. Importantly, this accessibility of regulatory regions to key transcription factors regulation was specific to NLRP3 and did not affect the AIM2 (TFs). Enhancers, whose competence, but not potency, is inflammasome or other cell death pathways. Altogether, our established during cell lineage acquisition, are promptly studies reveal a novel role for SLC25A13 in regulation of the engaged to mount acute responses to pathogens. However, NLRP3 inflammasome and inflammatory cytokine release. the extent to which acute activation alters DNA-accessibility This material is based upon work supported by the National and the roles of distinct classes of TFs in this process are Science Foundation Graduate Research Fellowship unsatisfactorily understood. Here, we investigated Program under Grant No. DGE-1256082, as well as NIH epigenomic modifications and transcriptomic changes grants R01 AI118916, R01 AI104002, and R01 AI127463. occurring upon activation of natural killer (NK) cells. In Disclosure of Interest: None Declared addition to developmentally primed enhancers, rapid, high- level gene induction was regulated by de novo chromatin LT187 accessibility and enhancer remodeling. Activation resulted IMPAIRED TYPE I INTERFERON RESPONSE TO ZIKA in redeployment of a lineage-specific TF INFECTION IN HUMAN NEURAL PROGENITOR CELLS to de novo enhancers, independent of its DNA sequence- DUE TO LIMITED RIG-I SIGNALING specific motif recognition. Thus, in NK cells, acute signaling C. Stokes1,2,*, J. Go2, M. De La Riva2, N. Wren3, D. shapes enhancer function through the combinatorial usage Newhouse2, J. Young4, M. Gale, Jr.2 and repurposing of both lineage-determining and signal- 1Pediatrics, 2Immunology, 3School of Medicine, 4Pathology, regulated TFs to ensure a rapid innate immune response. University of Washington, Seattle, United States

Disclosure of Interest: None Declared Abstract Content: Intrapartum infection with Zika virus (ZIKV) has been linked to devastating fetal microcephaly LT186 and other abnormalities of brain development. Neural MITOCHONDRIAL SLC25A13 INTERACTS WITH NLRP3 progenitor cells (NPCs) appear to be the major tropic cell of AND REGULATES INFLAMMASOME ACTIVITY ZIKV in the central nervous system (CNS), and because A. Shuvarikov1,*, M. Davis1, K. Esser-Nobis1, M. Gale, Jr.1 persistence of ZIKV in brain tissue is a hallmark of 1Immunology, University of Washington, Seattle, WA, United congenital infection, we hypothesized that an impaired States innate immune response might underlie the susceptibility of NPCs to ZIKV. Consistent with this, we found that during in Abstract Content: The NLRP3 protein is a key initiator of vitro infection NPCs support higher ZIKV RNA copy number inflammation in humans. NLRP3 becomes activated by a than do differentiated cells but do not induce IFN-β. To multitude of danger signals, including microbial infection, explore the underlying mechanisms for this differential metabolic dysfunction, and cell-internalized particulates. response we first examined expression of several PRRs and Upon activation, NLRP3 nucleates formation of a found that compared to differentiated cells NPCs express multiprotein complex called the inflammasome, in which lower levels of retinoic acid-induced gene I (RIG-I) the major caspase-1 activity mediates processing of the pro- pathogen recognition receptor detecting ZIKV and other inflammatory cytokines IL-1β and IL-18 and induces flaviviruses. We performed fluorescence-activated cell 93 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 sorting and single-cell RNA sequencing to characterize the Disclosure of Interest: None Declared innate immune response of identified cell types including NPCs, post-mitotic neurons and astrocytes. We found that LT189 astrocytes are a primary source of IFN-β production and PML-DEPENDENT MEMORY OF TYPE I IFN response during ZIKV infection. In contrast, NPCs exhibit TREATMENT PROMOTES A REPRESSIVE FORM OF blunted response to IFN- with lower induction of interferon- HERPES SIMPLEX VIRUS LATENCY stimulated genes (ISGs), including RIG-I. These findings J. Suzich1,*, S. Cuddy 1, H. Baidas 1, A. Schinlever1, S. suggest that limited signaling via RIG-I in NPCs underlies an Dochnal1, A. Babnis1, C. Boutell2, A. Cliffe1 attenuated innate immune response and increased 1University of Virginia, Charlottesville, United States, 2MRC- permissiveness for ZIKV for impaired viral control in fetal University of Glasgow Centre for Virus Research (CVR), ZIKV infection. Moreover, our findings demonstrate that Glasgow, United Kingdom significant heterogeneity exists in the innate immune Abstract Content: Herpes simplex virus-1 (HSV-1) response to viral infection across cell types in the developing establishes a lifelong latent infection of neurons. HSV-1 brain. Given the importance of innate immunity and type I latency is characterized by the association of viral lytic interferon actions in processes ranging from viral infection to promoters with repressive heterochromatin. At the individual degenerative disease of the CNS, these findings have genome level, HSV-1 latency is heterogenous, which likely important implications for the development of targeted also results in populations of latent genomes with distinct therapeutic interventions for ZIKV and other neurologic types of heterochromatin. Although latent viral genomes diseases. have been shown to associate with different subnuclear Disclosure of Interest: None Declared structures, including promyelocytic leukemia-nuclear bodies (PML-NBs), how this nuclear distribution arises and its LT188 impact on the ability of a latent genome to reactivate MANGANESE ENHANCES KU70-STING MEDIATED IFN- following various neuronal stresses remain unknown. We LAMBDA 1 INDUCTION BY INCREASING IN hypothesized that inflammatory conditions during initial PHOSPHORYLATION OF TBK1 infection could impact the nature of HSV latency. We H. Sui1,*, Q. Chen1, J. Yang1, S. Srirattanapirom 1,2, T. specifically investigated the contribution of type I IFN to Imamichi1 regulating the nature of HSV-1 latency using a primary 1Frederick National Laboratory for Cancer Research, neuronal model system. Surprisingly, we found that Frederick, 2Biochemistry and Molecular Biology, University treatment of primary neurons with type I IFN solely at the of Maryland, Baltimore, United States time of infection resulted in a form of HSV latency that was more restricted for reactivation. We also show that primary Abstract Content: Transition metals are essential for a murine sympathetic and sensory neurons are largely devoid variety of physiological processes including reproduction, of PML-NBs. However, treatment with type I IFN infection neuronal function, and antioxidant defenses, but little is induces the formation of PML-NBs, which persist even known for their roles in innate immunity. Here, we found that, following cessation of ISG expression. A proportion of latent among the transition metals (Cr2+, Mn2+, Fe2+, Co2+, Ni2+, HSV-1 genomes are stably entrapped within PML-NBs Cu2+, and Zn2+), only Mn2+ significantly enhanced Ku70- throughout a latent infection of IFN pre-treated sympathetic STING DNA sensing pathway in HEK cells, THP-1 cells and neurons. Furthermore, depletion of PML prior to infection human primary macrophages. In the presence of Mn2+ at rescues the ability of the virus to reactivate only in IFN- the concentration of 25 mM, DNA-mediated induction of treated neurons, therefore indicating that IFN-induced PML- IFN-λ1, IFN-α, and IFN-β was increased by 7.1-, 15.8- and NBs directly contribute to a deeper form of latency that is 9.3-folds, respectively. To further characterize the gene resistant to reactivation. This study demonstrates how type regulation profile by Mn2+ treatment, a microarray analysis I IFN-treatment during de novo infection alters subnuclear was performed. The gene expression was compared positioning of viral genomes and ultimately the ability of a between presence and absence of Mn2+ treatment. We latent genome to reactivate. found that the expression of 351 genes was ≥2-fold greater Disclosure of Interest: None Declared with Mn2+ and DNA treatments than with DNA stimulation alone. Using DAVID, a functional annotation bioinformatics LT190 tool, we further identified that most of those genes were COULD A WORLD WAR II DRUG BE REPURPOSED TO involved in biological processes such as innate immune LIMIT VIRAL HYPERINFLAMMATION? response, inflammatory cytokines or cytokine-mediated M. D. Tate1,*, S. Rosli1, A. Bawazeer1, F. Kirby1, A. Mansell1 signaling pathway. Western blot analysis revealed that, in 1Centre for Innate Immunity and Infectious Diseases, the presence of DNA, Mn2+ treatment greatly enhanced the Hudson Institute of Medical Research, Clayton, Australia activation of STING-TBK1-IRFs signaling pathway. And Mn2+ treatment alone induces phosphorylation of TBK1. Abstract Content: Severe influenza A virus (IAV) infections Finally, we found that Mn2+ protected human primary are associated with damaging hyperinflammation that can macrophages from HSV-1 infection by increasing in IFN lead to mortality. The repurposing of drugs with existing and induction. Our finding sheds a light on a potential beneficial studied pharmacokinetic and safety profiles is an attractive role of Mn on the application of DNA-based therapeutic or strategy to develop new therapeutics for severe and preventive reagents. (Funded by NCI Contract No. pathogenic IAV infections. We have previously HHSN261200800001E) demonstrated that the NLRP3 inflammasome plays a 94 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 temporal role during severe IAV infection with early of OM85BV. We hypothesized that OM85BV may modulate protective responses, however, subsequent dysregulation innate immune functions central to anti-microbial defence. leads to excessive inflammation, contributing to disease severity. While therapeutic treatment with the potent NLRP3 Aims: To elucidate the effect of OM85BV treatment on inhibitor MCC950 provided protection later in infection, it innate immune function in a high-risk infant cohort during renders mice susceptible early in infection. their first winter using gene co-expression network analysis. We have identified two existing drugs which target P2X7 receptor signalling and dampen NLRP3 inflammasome Methods: OM85BV treatment (or placebo) was carried out responses during severe IAV infection. Our study across the 5-month winter period; PBMC were collected demonstrates that the anti-inflammatory drugs Probenecid post-treatment and stimulated in vitro with PolyI:C and LPS. and AZ11645373 are effective at dampening RNAseq profiling was performed to identify gene co- hyperinflammation and severe influenza disease providing expression networks underlying immune responses. potentially new therapeutic strategies for treating severe or Cytokines were measured via Luminex. pathogenic IAV infections. Intranasal therapeutic treatment Results: of mice displaying severe influenza disease reduced pro- The OM85BV-treatment and placebo groups mount robust inflammatory cytokine production, cellular infiltrates in the responses to PolyI:C (3468 differentially expressed genes lung and provided protection against disease. Importantly, [DEG] and 3191 DEG respectively (FC>1.5)); and LPS Probenecid and AZ11645737 could be administered at (1930 and 1982 DEG respectively (FC>1.5)). In both groups, either early or late stage of disease and provide therapeutic the PolyI:C DEG were enriched with type I/III interferons, efficacy. Probenecid has been used in humans since World and the LPS DEGs were enriched with cytokine signalling War II to extend the life of penicillin and is currently used to (IL23, IL27 and IL12) and IFNg. Network analysis revealed treat Gout. Furthermore, AZ11645373 has been clinically that both OM85BV-treatment and placebo groups activate a trialled for treating inflammatory diseases such as arthritis. large antiviral module containing type I and type III interferon We propose that drugs such as Probenecid and signalling in response to PolyI:C. In the LPS response, both AZ11645737 which are less potent than those that directly groups upregulated a cytokine signalling module containing target NLRP3 such as MCC950, are effective as they a number of proinflammatory genes (IL6, IL8, CCL3, IL1b). dampen NLRP3 responses to a level that is not detrimental This module was less complex and contained fewer genes or damaging, but sufficient to provide protection in the OM85BV group (n=263) compared to Placebo Understating the kinetics and mechanisms involved in this (n=881). TNF was contained within the large interferon ‘switch’ from early protective to late damaging NLRP3 module in Placebo. The OM85 group contained an responses during IAV infection is of therapeutic benefit. additional small innate immunity module that contained TNF Myeloid-derived cells, such as neutrophils and Ly6C+ and was downregulated in response to LPS. IL6 protein was inflammatory macrophages are present in very low numbers significantly lower in OM85BV (p<0.05) with a trend towards in the airways in the absence of infection and accumulate lower TNF protein (p=0.055). peaking on day 3 following severe HKx31 infection (mice require ethical euthanasia by day 4). We have identified that Conclusion: OM85BV treatment during infancy tunes following IAV infection, infiltrating myeloid-derived cells such maturation of mechanisms that regulate innate immune as neutrophils and Ly6C+ inflammatory macrophages responses to PolyI:C and LPS such that proinflammatory express significant NLRP3. We are currently examining the signals are selectively attenuated without constraining roles of epithelial cells and infiltrating myeloid cells, in interferon-associated networks central to first-line mediating excessive NLRP3 responses may differ and antimicrobial defence. hypothesise that cellular targeting with anti-P2X7 receptor 1PMID:31185221 drugs in vivo could potentially improve anti-NLRP3 efficacy. Disclosure of Interest: None Declared Disclosure of Interest: None Declared LT192 LT191 LYMPHOID-MYELOID CELL FATE DETERMINATION BY OM85BV PROTECTS AGAINST RESPIRATORY VIRAL ENDORIBONUCLEASES REGNASE-1 AND -3 INFECTION IN HIGH-RISK INFANTS BY MODULATING T. Uehata1,*, S. Yamada1, M. Miyazaki1, A. Giladi2, A. INNATE IMMUNITY Vandenbon1, I. Amit2, H. Kawamoto1, O. Takeuchi1 N. Troy1,*, S. Galbraith2, Z. Islam2, M. Serralha1, B. Holt3, D. 1Kyoto university, Kyoto, Japan, 2Weizmann Institute of Strickland1, P. Sly2, A. Bosco1, P. Holt 1 Science, Rehobot, Israel 1Telethon Kids Institute, Perth, 2University of Queensland, Brisbane, 3Telethon Kids Institute , Peth, Australia Abstract Content: Hematopoietic stem and progenitor cells (HSPCs) give rise to all blood lineage cells through complex Abstract Content: Background: Lower respiratory lineage-determining mechanisms. Recent work shows that infections (LRI) in infancy are a major cause of morbidity. In multipotent progenitor (MPP) and even hematopoietic stem a recent clinical trial, we demonstrated that treatment of cell (HSC) clones are heterogeneous and possess infants with the microbial-derived immunomodulator differentiation preferences toward either myeloid or OM85BV provided significant protection against LRI1. The lymphoid lineages, suggesting the existence of lineage bias. focus of this study is on the mechanisms-of-action Such lineage bias plays an important role in stressed hematopoiesis (e.g. inflammatory conditions), which 95 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 enhances myelopoiesis at the expense of lymphopoiesis. signaling, known as “oncolytic signaling.” Our lab has However, it is unclear how the HSPC lineage commitment is identified a viral pathogen associated molecular pattern regulated under native and stressed hematopoiesis. (PAMP) RNA motif from hepatitis C virus (HCV) that when Here we show that Regnase-1 (also known as Zc3h12a) and delivered to cells in a liposomal formulation engages RIG-I its family member, Regnase-3/Zc3h12c, govern early-stage to induce innate immune signaling. We found that PAMP lymphopoiesis by regulating mRNA decay in HSPCs. Lack RNA can direct RIG-I dependent signaling to drive cell death of Regnase-1 and Regnase-3 (DKO) in mice resulted in a in tumor cells, indicating that targeting the RIG-I pathway severe loss of B lymphocytes but not myeloid lineage cells. with PAMP has applications for oncolytic destruction of In contrast, a single deficiency of Regnase-1 or Regnase-3 tumors. Preliminary structure-function assays reveal that did not show impairment in lymphopoiesis. Competitive RIG-I binds to a novel protein TRIM16, and that TRIM16 is reconstitution of control and DKO fetal liver cells resulted in required for this oncolytic phenotype. Therefore, we a profound reduction of DKO lymphoid progenitors including hypothesize that TRIM16 mediates a non-canonical RIG-I lymphoid-primed multipotent progenitor (LMPP). However, dependent cell death pathway. In-vitro studies using DKO cells showed a mild increase in myeloid and CRISPR knockout cell lines confirmed that this cell death megakaryocyte/erythroid (Meg/E) lineages. Nevertheless, signaling pathway requires RIG-I and the downstream proliferative activity and cell viability of HSC and LMPP were adaptor protein MAVS. Overexpression assays show that comparable between control and DKO. Single cell RNA-seq TRIM16 does not affect IFN-beta expression nor does IFN- (scRNA-seq) analysis of Lineage–Sca1+c-Kit+ (LSK) fraction beta affect TRIM16 expression, suggesting that TRIM16 is revealed that DKO cells had a skewing of lineage biases not involved in innate immune signaling or response but toward myeloid and Meg/E cells, not only in lineage instead imparts a novel and non-canonical RIG-I mediated composition but in gene expression profiles. In addition, bulk pathway of cell death. We are currently working to identify RNA-seq analysis of LMPP showed that DKO cells showed the structural components of RIG-I and TRIM16 required for higher expression of sets of myeloid- as well as Meg/E- cell death signaling and are developing an in-vivo model to related genes, compared to the counterpart. From the data study PAMP oncolytic actions. In addition, we are of scRNA-seq and bulk RNA-seq, we identified Nfkbiz as a developing TRIM16 conditional knockout mice to interrogate novel promoter of myelopoiesis. Crosslinking and the actions of TRIM16 in vivo. This research will reveal the immunoprecipitation (CLIP)-seq analyses revealed that role of TRIM16 and the novel RIG-I mediated cell death Regnase-1 and Regnase-3 can interact shared stem loop signaling pathway toward design of anti-tumor therapeutics structures present in mRNA 3’ untranslated regions. Nfkbiz leveraging viral PAMP actions. was turned out to be a target mRNA both for Regnase-1 and Disclosure of Interest: None Declared Regnase-3 for degradation. Interestingly, ablation of Regnase-1 and Regnase-3 in cultured LMPPs resulted in a LT194 rapid increase of myeloid-related genes including Nfkbiz, TLRS 1/2, 2/6, 4 AND 7/8 ENGAGE THE ALTERNATIVE accompanied by subsequent myeloid differentiation. NLRP3 INFLAMMASOME PATHWAY TO INDUCE Overexpression of Nfkbiz in lymphoid progenitor cells INTERLEUKIN-1Β RELEASE FROM PRIMARY HUMAN showed a marked induction of myeloid lineage cells and a MONOCYTES. severe reduction in B cell differentiation , indicating that the S. Unterberger1,*, L. Mullen1, M. S. Flint2, S. Sacre1 Regnase-1/-3-Nfkbiz axis acts as a rheostat for regulating 1Brighton and Sussex Medical School, University of Sussex, myeloid- and lymphoid-lineage bias in LMPPs. Finally, Brighton, BN1 9PS, 2School of Pharmacy and Biomolecular inflammatory conditions by LPS challenge decreased the Sciences, University of Brighton, Centre for Stress and Age- expression of Regnase-1/3 in HSPCs, consistent with the related Disease, Brighton, BN2 4GJ, United Kingdom enhancement of myelopoiesis. Thus, Regnase-1/-3 may contribute to emergency myelopoiesis through the alteration Abstract Content: in their protein expressions. Taken together, the post- Introduction transcriptional mechanism mediated by Regnase-1 and Interleukin-1β (IL-1β) is expressed as a pro-form that Regnase-3 is a novel regulatory phase for the lymphoid- requires cleavage to an active form by caspase-1 as a part myeloid cell fate decision, acting at the interface between of the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) HSPCs and inflammation. inflammasome. This process usually requires two signals; Disclosure of Interest: None Declared toll-like receptor (TLR) activation to transcribe pro-IL-1β and NLRP3, alongside a second signal to activate the LT193 inflammasome, such as K+ efflux. In addition, caspase-1 VIRAL PAMP SIGNALING DIRECTS RIG-I-DEPENDENT cleaves gasdermin D (GSDMD) leading to the formation of DEATH OF TUMOR CELLS membrane pores that permit the release of IL-1β and induce B. Ulloa1,*, A. Tan1, E. Verstelle1, M. Gale Jr.1 pyroptosis1,2. However, an alternative pathway has been 1Immunology, University of Washington, Seattle, United described in BLaER1 cells, a human cell line which shares States some similarity to primary human monocytes. Upon TLR4 stimulation, IL-1β was released by a NLRP3 and caspase-8 Abstract Content: Most malignant cells are poorly dependent mechanism that was independent of pyroptosis immunogenic and fail to elicit an effective antitumor or K+ efflux3. Our study set out to investigate if this response. However, viral infection of cancer cells can trigger mechanism operates downstream of TLRs 1/2, 2/6, 4 and intracellular innate immune defenses that impart cell death 7/8 in primary human peripheral blood monocytes. 96 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Methods immunity. We defined phenotypic categories of dynamic CD14+ monocytes were isolated from peripheral blood by susceptibility to WNV and innate immune control of infection density centrifugation followed by iso-osmotic Percoll among CC strains. Our studies reveal that during WNV gradient centrifugation. Cells were then stimulated with a infection the dynamics of innate immune activation and range of TLR ligands for 6 or 24 hours in the presence or engagement of the innate/adaptive immune interface are absence of a NLRP3 inhibitor (MCC950), a potassium linked with gastrointestinal (GI) disease, and the partitioning ionophore (nigericin), caspase-1 or -8 inhibitors, a GSDMD of infection among peripheral tissue, and the central and inhibitor (NSA) or excess extracellular potassium chloride. enteric nervous systems. These outcomes reflect WNV IL-1β secretion was measured by ELISA, pyroptosis by infection dynamics in humans. Thus, the CC model is a measurement of release of lactate dehydrogenase from unique resource allowing researchers to study and define monocytes and GSDMD cleavage was assessed by host genetic features of innate immune regulation and WNV Western blot. disease phenotypes. Results Disclosure of Interest: None Declared TLR-induced IL-1β release was NLRP3, caspase-1 and caspase-8 dependent. It was also mediated by GSDMD but LT196 did not require K+ efflux or induce pyroptosis. Additional TYROSINE PHOSPHORYLATION BY EGFR IS activation of K+ efflux with nigericin amplified GSDMD REQUIRED FOR STING TO TRANSLOCATE TO THE dependent IL-1β secretion by all TLRs apart from TLR7/8. , ACTIVATE IRF3, INDUCE INTERFERON Conclusion AND INHIBIT HSV1 REPLICATION. TLRs 1/2, 2/6, 4 and 7/8 all use the alternative NLRP3 C. Wang1,* inflammasome pathway to release IL-1β from primary 1Inflammation and Immunity, Cleveland Clinic, Cleveland, human monocytes. TLR7/8 stimulation alone can induce United States maximal IL-1β release in the absence of pyroptosis. Abstract Content: Tyrosine phosphorylation by EGFR is References required for STING to translocate to the endosome, 1 Evavold, C.L., et al., The Pore-Forming Protein Gasdermin activate IRF3, induce interferon and inhibit HSV1 D Regulates Interleukin-1 Secretion from Living replication. Macrophages. Immunity, 2018. 48(1): p. 35-44.e6. Chenyao Wang and Ganes C Sen. Department of 2 He, W.-t., et al., Gasdermin D is an executor of pyroptosis Inflammation and Immunity, Cleveland Clinic, Cleveland, and required for interleukin-1β secretion. Cell research, Ohio, USA 2015. 25(12): p. 1285-1298. Stimulator of interferon genes (STING) mediates protective 3 Gaidt, Moritz M., et al., Human Monocytes Engage an cellular response to microbial infection and tissue damage; Alternative Inflammasome Pathway. Immunity, 2016. 44(4): however, its aberrant activation leads to autoinflammatory p. 833-846. diseases. Upon ligand-stimulation, STING, an endoplasmic Disclosure of Interest: None Declared reticulum (ER) protein, translocates to the endosomes to induce interferons; but an alternate route takes it directly to LT195 the autophagosomes. Here, we report that EGFR-mediated USING THE COLLABORATIVE CROSS MOUSE MODEL tyrosine phosphorylation of STING was required for its ability TO DETERMINE INNATE IMMUNE CORRELATES OF to activate IRF3, which drives the induction of IFN. Upon RESISTANCE TO WEST NILE VIRUS cGAMP stimulation, EGFR interacted with STING in the ER K. M. Voss1,*, S. Thomas1, C. Wilkins1, R. Green1, A. membrane and directly phosphorylated its Y245 residue. Tyr Sekine1, D. Hendrick1, R. Ireton1,2, M. Gale Jr.1,2,3,4 phosphorylation of STING was not required for binding 1Immunology, 2Center for Innate Immunity and Immune TBK1; STING-bound TBK1 was activated by Disease, 3Microbiology, 4Pathology, University of autophosphorylation and it phosphorylated Ser366 of Washington, Seattle, United States STING, a prerequisite for IRF3 binding. However, the Y245F mutant of STING rapidly transited to the autophagosome Abstract Content: Inbred mice are inadequate in capturing and did not translocate to the endosome, where, as we the diversity of human responses to infections. The demonstrated, IRF3 interacted with STING. In the absence Collaborative Cross (CC) mouse model is a unique resource of this critical post-translational modification of STING Y245, for modeling disease response in a genetically diverse IRF3 activation, interferon production and antiviral action population similar to human genetic diversity. To better were compromised in cell cultures and mice; in contrast, its understand how genetic diversity contributes to innate autophagic activity was enhanced. Our observations immune responses to West Nile infection, we examined the illuminate a new connection between the tyrosine kinase innate immune and cytokine gene expression following activity of EGFR and innate immune functions of STING; West Nile virus (WNV) infection in 114 CC mouse lines, they also suggest experimental and therapeutic approaches which encompass 90% of genetic variation in laboratory for selective regulation of STING functions. mice while mirroring human genetic diversity. We measured weight loss, clinical score, viral load, and innate immune Disclosure of Interest: None Declared responses, including IL-12 and IFN-beta, in each CC mouse line over an acute infection time course as the host response engaged innate immunity and transitioned to adaptive 97 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT197 LT198 MICROGLIA UNIQUELY REGULATE THEIR PROTECTIVE TRANSCRIPTOMIC SIGNATURE AND PHENOTYPE AND FUNCTION IN IL-6 AND IFN-ALPHA- BIOMARKERS OF THE RHCMV/SIV VACCINE MEDIATED NEUROLOGICAL DISEASE RESPONSE P. K. West1,*, A. N. McCorkindale1, B. Guennewig1, T. M. L. S. Whitmore1,*, F. Barrenas1,2, S. Hansen3, L. Law1, D. Ashhurst1, B. Viengkhou1, E. Hayashida1, S. R. Jung1, O. Newhouse1, E. Smith1, J. Chang1, I. Golez1, J. Butovsky2, M. J. Hofer1, I. L. Campbell1 Komorowski2,4,5, P. Edlefsen6, L. J. Picker3, M. Gale1 1University of Sydney, Sydney, Australia, 2Harvard Medical 1Department of Immunology, Center for Innate Immunity and School, Boston, United States Immune Disease, Washington National Primate Research Center, University of Washington, Seattle, United States, Abstract Content: Chronic production of interleukin (IL)-6 2Department of Cell and Molecular Biology, Uppsala or interferon (IFN)-alpha in the CNS is the cause of several University, Uppsala, Sweden, 3Vaccine and Gene Therapy neurological diseases including neuromyelitis optica and Institute, Oregon Health and Science University, Portland, Aicardi-Goutières syndrome respectively. Transgenic mice United States, 4Swedish Collegium for Advanced Study, that produce IL-6 (GFAP-IL6) or IFN-alpha (GFAP-IFN) in Uppsala University, Uppsala, Sweden, 5Institute of the CNS recapitulate many aspects of the respective human Computer Science, Polish Academy of Sciences, Warsaw, diseases. Microglial activation is a prominent feature in the Poland, 6Statistical Center for HIV/AIDS Research and CNS of GFAP-IL6 and GFAP-IFN mice and is implicated in Prevention, Fred Hutchinson Cancer Research Center, the pathogenesis of these cytokine-mediated neurological Seattle, United States diseases. To define the role of microglia in disease, we performed a detailed phenotypic comparison of microglia in Abstract Content: The 68-1 RhCMV vaccine, based on GFAP-IL6 versus GFAP-IFN mice. Rhesus Macaque (RM) modified cytomegalovirus (CMV), can direct a protective immune response against SIV GFAP-IL6 microglia were highly proliferative, pY-STAT3 infection in RMs and serves as a preclinical model for CMV- positive, and were smaller and had stunted processes, while based vaccine vectors in HIV vaccination. We longitudinally GFAP-IFN microglia were pY-STAT1 positive and evaluated the blood transcriptional signature of the 68-1 significantly larger with hyper-ramified processes. Purified vaccine after oral or subcutaneous vaccination of 15 RMs microglia ex vivo had altered transcriptomic and surface who underwent a prime and week 18 boost regimen before marker profiles in response to IL-6 and IFN-alpha. GFAP- challenged with the virus multiple time starting IL6 microglia had transcriptomic and surface marker approximately 90 weeks after prime vaccination. Using changes associated with proliferation and phagocytosis, differential gene analysis and linear modeling we identified while GFAP-IFN microglia had changes associated with viral a unique transcriptional gene response linked with vaccine immunity and antigen presentation. protection against multi-challenge SIV infection that includes genes within inflammasome, interleukin signaling, Toll-like In order to clarify the contribution of microglia to the receptor signaling, T cell receptor signaling functional development of cytokine-mediated neurological disease, pathways. We found that induction of the interleukin (IL)-15 microglia were ablated from the CNS of GFAP-IL6 and response also tracked with vaccine protection. Our studies GFAP-IFN mice using the CSF1R blocking drug, PLX5622. reveal gene correlates of 68-1 vaccine protection and The capacity of surviving microglia to repopulate the empty identify whole blood gene biomarkers linked with vaccine CNS niche was differentially altered in response to the efficacy. cytokine environment driven by IL-6 and IFN-alpha. GFAP- Disclosure of Interest: None Declared IL6 microglia repopulated the CNS more rapidly and robustly, while GFAP-IFN microglia were unable to LT199 repopulate the CNS. Microglia-depleted GFAP-IL6 and TWO DISTINCT INTERFACES MEDIATE THE GFAP-IFN but not WT mice had exaggerated disease, with ASSOCIATION BETWEEN CASPASE-1 AND pronounced tissue destruction and severe calcification of GASDERMIN D the cerebellum. Surprisingly, although cerebral calcification T. S. Xiao1,*, Z. Liu1, C. Wang1, J. Yang2, Y. Chen3, B. Zhou1, is a hallmark of IFN-mediated disease, this exaggerated D. Abbott1 disease pathology was not associated with increased IFN or 1Pathology, Case Western Reserve University, Cleveland, IFN-regulated gene expression. 2Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, 3Physiology and We conclude that the microglia response to each cytokine Biophysics, Case Western Reserve University, Cleveland, environment is wide-ranging and gives rise to divergent United States phenotypes. The acquisition of unique phenotypic features may impart microglia with distinct functions in the Abstract Content: The recognition and cleavage of neurological diseases mediated by IL-6 and IFN-alpha. gasdermin D (GSDMD) by inflammatory caspases-1/4/5/11 Additionally, microglia depletion in both GFAP-IL6 and is an essential step in initiating pyroptosis following GFAP-IFN mice, rather than being protective, is deleterious. inflammasome activation. Previous work has identified Disclosure of Interest: None Declared cleavage site signatures in substrates such as GSDMD and inflammatory cytokines, but it is unclear if these are the sole determinants for caspase engagement. Here we report the 98 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 crystal structure of a complex between human caspase-1 protein nuclear bodies (PML bodies) may provide (CASP1) and the full-length murine GSDMD. Our work interactions between SUMO and SUMO interaction motifs reveals that the GSDMD N-terminal domain does not bind (SIMs) for contribution ASC speck formation. We further CASP1, whereas the cleavage site-containing linker in showed that K21,22,24,26 on ASC is essential for GSDMD adopts a long loop structure that engages the SUMOylation and interact with PLZF, also required for ASC CASP1 active site. This active site interface resembles speck formation. Accordingly, macrophage-specific deletion previously reported structures of CASP1 in complex with a of ZBTB16 attenuates inflammation induced by Mono pan-caspase inhibitor z-VAD-FMK or a GSDMD-derived Sodium Urate Crystal-induce peritonitis through NLRP3 peptide inhibitor. In addition to GSDMD engagement of the activation. Remarkably, ablating ZBTB16 also relieves caspase-1 active site, an anti-parallel b sheet at the inflammatory pathogenesis in a murine model of Muckle- caspase-1 L2 and L2’ loops binds a hydrophobic pocket Wells syndrome caused by hyperactive mutant NLRP3. Our within the GSDMD C-terminal domain distal to its N-terminal findings reveal a novel role for SUMO-mediated regulation domain. This “exosite” interface is in agreement with the of inflammatory disease caused by ASC that is modulated recent publication by Wang and colleagues reporting the through ZBTB16. structures of caspases-1/4/11 in complex with the GSDMD Disclosure of Interest: None Declared C-terminal domains. The exosites in inflammatory caspases that bind a hydrophobic pocket formed by a5-a6 and a7’-a8 LT201 in GSDMD C-terminal domains endow a novel function for MG53/TRIM72 DAMPENS TYPE I INTERFERON the GSDMD C-terminal domain as a caspase-recruitment PRODUCTION AND PROTECTS FROM LETHAL module, in addition to its role in autoinhibition. Such dual INFLUENZA VIRUS INFECTION WHEN functionality of the C-terminal domain may allow stringent THERAPEUTICALLY ADMINISTERED substrate selectivity in the resting state while facilitating A. Kenney1, M. Sermersheim1, Z. Li1, Z. Bian1, P.-H. Lin1, X. cleavage and pyroptosis upon inflammasome activation. Zhou1, H. Li1, A. Zani1, J. Li1, K. Gumpper1, T. Adesanya1, The residues forming the hydrophobic pocket are conserved T. McMichael1, K.-H. Park1, B. Whitson1, N. Mokadam1, T. between human and murine GSDMD, but not in GSDME, Tan1, C. Cai1, J. Ma1, J. Yount1,* suggesting that the exosite interface may underlie the 1The Ohio State University, Columbus, United States specific recognition of GSDMD but not GSDME by inflammatory caspases. Our study thus reveals dual Abstract Content: Tripartite motif (TRIM) family proteins interface engagement of GSDMD by caspase-1, which may play integral roles in the innate immune response to virus be applicable to other physiological substrates of caspases. infections. Mitsugumin 53 (MG53), also known as TRIM72, Disclosure of Interest: None Declared is believed to be a muscle-specific TRIM protein that is essential for membrane repair and maintenance. LT200 We now show that human macrophages express MG53 and ZBTB16 CONTROLS SUMOYLATION OF ASC AND that its knockdown leads to increased production of type I INFLAMMASOME ACTIVITY interferon (IFN) upon virus infection. MG53 knockout mice D. Dong1, Y. Du1, X. Li1, A. Mansell2, G. Meng3, A. Sadler2, infected with influenza virus show comparable viral titers to D. Xu1,* WT mice, but experience increased morbidity accompanied 1Faculty of Medical Laboratory Science, Ruijin Hospital, by accumulation of inflammatory cells and elevation of IFNβ School of Medicine, Shanghai Jiao Tong University, in the lung. We found that MG53 knockdown results in Shanghai, China, 2Hudson Institute of Medical Research, activation of NF-ΚB signaling, which is linked to an increase Monash University, Vic, Australia, 3CAS Key Laboratory of in intracellular calcium oscillation mediated by ryanodine Molecular Virology & Immunology, Institut Pasteur of receptor (RyR). Further, we observed that MG53 inhibits Shanghai,, Chinese Academy of Sciences, Shanghai, China IFNβ induction in an RyR-dependent manner. Since MG53 is also secreted by muscle cells as a to provide Abstract Content: Appropriate control of Inflammasome regenerative benefits to multiple tissues, we sought to activity is critical to limit damaging immune responses determine the effects of enhancing systemic levels of MG53 through rapidly escalates the intensity of inflammation by during influenza virus infection. Intravenous administration activating interleukin (IL)-1β, IL-18 and cell death by of clinical grade recombinant human (rh) MG53 during lethal pyroptosis. Here we identify a novel control mechanism that influenza virus infection protected mice from severe modulates the central Caspase-1 and NLR (Nod-like disease, with a 92% survival rate, and with mice remaining receptor) adaptor ASC (apoptosis-associated speck-like ambulant and active throughout infection. rhMG53 treatment protein containing a CARD). We identify SUMO (small of infected mice preserved lung integrity, corresponding with ubiquitin-like modifier) modification of ASC represses the a decrease in lung inflammation, including decreases in nuclear to cytoplasmic translocation and its subsequent IFNβ and inflammasome activation, without directly oligomerization, ASC speck formation. We also discover affecting viral titers. Collectively, our studies reveal a new that nuclear ZBTB16 (Zinc finger and BTB domain- role for MG53 in limiting tissue-damaging inflammation containing protein 16) interacts with ASC to suppress the during virus infection, and identify rhMG53 as a promising conjugation of SUMO-1, thereby promoting inflammasome host-directed biologic therapy for infectious lung injury. activity. ZBTB16 (also known as PLZF , nuclear body protein Disclosure of Interest: A. Kenney: None Declared, M. promyelocytic leukemia zinc-finger protein) which Sermersheim: None Declared, Z. Li: None Declared, Z. colocalize with specialized Promyelocytic leukemia Bian: None Declared, P.-H. Lin: None Declared, X. Zhou: 99 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

None Declared, H. Li: None Declared, A. Zani: None Declared, J. Li: None Declared, K. Gumpper: None Abstract Content: Cell-intrinsic antiviral immunity is pivotal Declared, T. Adesanya: None Declared, T. McMichael: to combat virus infections. Sensing of conserved pathogen- None Declared, K.-H. Park: None Declared, B. Whitson: associated molecular patterns (PAMPS) by cellular None Declared, N. Mokadam: None Declared, T. Tan receptors such as retinoic acid-inducible gene I (RIG-I) Shareholder of: TRIM-edicine Inc., C. Cai: None Declared, results in the production and release of interferons and J. Ma Shareholder of: TRIM-edicine Inc., J. Yount: None massive expression of interferon-stimulated genes (ISGs), Declared conferring an antiviral state. Notably, this ISGs-driven state not only comprises antiviral effectors but also displays LT202 cytostatic and anti-tumorigenic properties, inducing STAT1 AND TYPE I IFN SIGNALING PATHWAYS apoptosis, and suppressing proliferation and migration. REGULATE CONVENTIONAL DENDRITIC CELL Surprisingly, recent work showed that this antiviral pathway DEVELOPMENT FROM COMMON LYMPHOID is not only relevant in viral infection, but also for proper PROGENITORS DURING INFLAMMATION tumor-cell death following treatment with DNA damage inducing chemotherapeutics [1, 2]. H. Yu-Ling1,*, L. Chien-Kuo1 Our study aims to identify and characterize the components 1Graduate Institute of Immunology/National Taiwan and the mechanism of RIG-I mediated cell-death upon DNA University, Taipei, Taiwan damage. We generated A549 CRISPR/Cas9 knockout cell lines targeting different components of innate immunity and Abstract Content: Dendritic cells (DCs), including determined rates of cell death upon treatment with conventional DCs (cDCs) and plasmacytoid DCs (pDCs), cytostatics or γ-irradiation by life cell imaging. We confirmed are key regulators of immune response. However, the that doxorubicin treatment induces cell death dependent on developmental origin of both subsets of DCs and the signals a functioning RIG-I/MAVS axis, while involvement of the orchestrating DC generation remain incompletely classical ISG response remains unclear. Moreover, we have understood. Here, we demonstrated that TLR-mediated identified proteins that contribute to cell death but are not inflammation drastically altered the developmental process related to the canonical RIG-I pathway. We are currently of progenitors of both lymphoid and myeloid lineages by investigating the mechanistic role of these proteins in DNA enhancing cDC production in vitro and in vivo. The ratio of damage-induced cell death. cDC1 to cDC2 also increased upon TLR stimulation. More 1. Sistigu, A., et al., Cancer cell-autonomous contribution importantly, ex vivo DC development from CLPs of mice of type I interferon signaling to the efficacy of chemotherapy. previously treated with R848, a TLR7 agonist, also favored Nature Medicine, 2014. 20: p. 1301. cDC generation even though R848 is omitted in the culture 2. Ranoa, D.R., et al., Cancer therapies activate RIG-I-like conditions, suggesting that TLR signaling reprograms DC receptor pathway through endogenous non-coding RNAs. development in the progenitors. Fate mapping using IL-7R- Oncotarget, 2016. 7(18): p. 26496-26515. driven reporter mice demonstrated that CLPs contributed to Disclosure of Interest: None Declared increased cDC population, cDC1 in particular, during inflammation. The TLR7 effect on cDC development was LT204 partially rescued in MyD88KO CLPs when cocultured with COINFECTION WITH HELIGMOSOMOIDES WT CLPs, suggesting that both primary and secondary POLYGYRUS INCREASES ACUTE MURINE signaling events downstream of TLR7 are involved. One of GAMMAHERPESVIRUS-68 INFECTION IN THE the secondary signaling events was dictated by IFN-I PERITONEUM induced by the stimulation, which partially controlled TLR- C. Zarek1,*, T. Reese2 dependent enhancement of cDC generation. While STAT1 1Immunology, 2UT Southwestern Medical Center, Dallas, is a canonical signal mediator of IFN-I, it was also directly United States activated by R848 through phosphorylation at S727. Either STAT1KO or S727A mutation in STAT1 impeded the effect. Abstract Content: In certain areas of the world, the human In sum, these findings reveal that developmental process of gammaherpesviruses, Epstein-Barr virus (EBV) and DCs from their progenitors is very dynamic in steady state Kaposi’s sarcoma-associated herpesvirus (KSHV), create a and inflammation. Moreover, we define a novel function of substantial health burden through lymphoproliferative STAT1 and IFN-I signaling pathway in TLR-mediated disorders. Gammaherpesviruses cause chronic infections in reprogramming of DC development from CLPs. B cells, macrophages, and dendritic cells. While single Disclosure of Interest: None Declared pathogen infections in laboratory settings are common, coinfections are not, despite the fact that most infections LT203 people experience are coinfections. For example, in the RELEVANCE OF THE CELL-INTRINSIC ANTIVIRAL places that have endemic EBV or KSHV, there is also high SIGNALING FOR THE INDUCTION OF CELL DEATH incidence of helminth infection. While some co-infecting UPON DNA DAMAGE pathogens induce similar immune responses, such as D. Y. Zander1,*, S. Burkart1, S. Wüst1, V. Goncalves bacteria and virus, other combinations of pathogens induce Magalhaes1, M. Binder1 diverse responses. Helminths induce a Th2 immune 1Virus-associated Carcinogenesis, German Cancer response and alternatively activate macrophages through Research Center (DKFZ), Heidelberg, Germany IL-4 and IL-13 signaling. Meanwhile, viruses primarily 100 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 induce interferon, Th1 responses, and classical activation in production in recently activated Th9 cells. In murine asthma macrophages. In order to examine how parasite coinfection models, inhaled IL-2 and IL-4 were sufficient to support a affects gammaherpesvirus infection, we infected mice with population of transferred lung-resident Th9 cells, and a helminth, Heligmosomoides polygyrus (HP), and then ablating TCR signaling did not prevent Th9-mediated infected with murine gammaherpesvirus-68 (MHV68) a allergic immunopathology in sensitized mice, demonstrating week later. Serum from coinfected mice had increased Th2- an innate, antigen-independent function for tissue Th9 associated cytokines, including IL-4 and IL-5, during the first cells in vivo. Taken together, these data suggest that Th9 week of MHV68 infection. Further, we observed increased cells respond to STAT-dependent cytokines in a unique viral titer in the coinfected mice at day 2 of MHV68 infection innate manner, independent of TCR, to promote allergic that was dependent on STAT6. The immune cell populations disease. of the peritoneum were altered in single and co-infection Disclosure of Interest: None Declared conditions. Mice infected only with MHV68 had increased numbers of monocyte-derived macrophages, while LT204c coinfected mice had increased numbers of resident T HELPER 9 CELLS ARE ASSOCIATED WITH AN peritoneal macrophages. In addition to an increased viral INCREASED RISK OF PSORIATIC CARDIOVASCULAR replication of MHV68, coinfected mice had increased DISEASE AND DIRECTLY PROMOTE HUMAN numbers of subsets of infected macrophages at day 2, day ARTERIAL ENDOTHELIAL CELL DYSFUNCTION 4 and day 7 of MHV68 infection. We aim to determine the D. Schwartz1,*, Y. Baumer2, A. M. Burma1, A. K. Dey2, M. contribution of expanded macrophage populations in co- Kitakule1, C. A. Gutierrez Huerta2, H. Teague2, M. Playford2, infected mice and the role of STAT6 signaling on MHV68 T. M. Powell-Wiley2, J. D. Milner3, P. F. Guerrerio1, N. N. infection in peritoneal macrophage populations. Mehta2 Disclosure of Interest: None Declared 1Laboratory of Allergic Diseases, NIAID, NIH, 2NHLBI, NIH, Bethesda, 3Division of Allergy, Immunology and LT204b Rheumatology, Columbia University Medical Center, New DYNAMIC ACCESSIBILITY UNDERLIES TH9 LINEAGE York, United States PLASTICITY AND CONFERS INNATE-LIKE FUNCTION IN VIVO TO PROMOTE ALLERGIC DISEASE Abstract Content: CD4+ T helper (Th) cells are undeniably D. M. Schwartz1,*, A. Son1, F. Meylan2, K. Manthiram3, J. D. central mediators of psoriasis, where they produce Milner4, P. F. Guerrerio1 cytokines like IL-17A and IL-23, which recruit inflammatory 1Laboratory of Allergic Diseases, NIAID, NIH, 2NIAMS, NIH, cells to the skin and directly promote 3NHGRI, NIH, Bethesda, 4Division of Allergy, Immunology proliferation. Yet psoriasis is a systemic disease with various and Rheumatology, Columbia University, New York, United extracutaneous manifestations including arthritis, States inflammatory bowel disease, and atherosclerotic cardiovascular disease. Although these extracutaneous Abstract Content: Interleukin-9 (IL-9), an inflammatory manifestations are a major source of morbidity and mortality cytokine derived primarily from T helper 9 (Th9) cells, is a in patients with psoriasis, the immunopathologic critical mediator of allergy, autoimmunity, and antitumor mechanisms driving extracutaneous psoriatic pathology are immunity. Yet Th9 cells are tissue-resident, and IL-9 incompletely characterized. To determine which T helper- expression can be transient. The mechanisms underlying derived cytokines might promote extracutaneous psoriatic Th9 lineage plasticity and the in vivo significance of disease, we immunophenotyped CD4+ T cells from psoriasis transient IL-9 production are not well characterized. We patients and looked for associations with cutaneous and found that human and murine Th9 cells produced IL-9 upon extracutaneous pathology. We tested 10 cytokines: IL-17A, exposure to paracrine IL-2 and IL-4, but not other cytokines. IL-4, IL-5, IL-13, IL-9, IL-21, IL-10, IL-2, interferon (IFN) -γ, Mechanistically, IL-2 and IL-4 induced active transcription of and tumor necrosis factor (TNF) -α. Unexpectedly, we found stable IL9 transcript via JAK-STAT signaling. Cytokine- a correlation between circulating IL-9+ T helper (Th9) cells induced IL-9 production was independent of T-cell receptor and non-calcified coronary atherosclerotic burden (TCR) stimulation, indicating that it did not require antigen (unadjusted p = 0.039, adjusted p = 0.002) that was not seen recognition. This mechanism was unique to IL-9, as for other cytokines. Analysis of public single cell RNA- interferon (IFN)-γ, IL-2, IL-4, IL-5, IL-13, IL-17A, and IL-10 sequencing data revealed increased numbers of CD4+ T were not induced under these conditions. IL-2 and IL-4 were cells expressing the Th9 master transcription factor (PU.1) sufficient to induce IL-9 production from recently activated in in human atherosclerotic plaque compared with peripheral vitro differentiated or expanded Th9 cells and from ex blood (FDR<0.01). This indicated that Th9 cells were vivo tonsillar Th9 cells, but not from circulating memory Th9 plaque-resident and suggested that they might directly cells. Examining the dynamic accessibility of the induce aortic endothelial cell dysfunction, which is critical to extended Il9 locus revealed three critical STAT- the pathogenesis of atherosclerotic cardiovascular disease. dependent cis-regulatory regions that were accessible in Accordingly, human aortic endothelial cells (HAoECs) recently activated Th9 cells, maintained accessibility for expressed both subunits of the heterodimeric IL-9 several days, and lost accessibility thereafter. Dynamic receptor. In vitro treatment of cultured HAoECs with IL-9 accessibility was not seen for other extended Type 2 caused STAT3 activation, induced STAT3 target genes, and cytokine loci. Taken together, these results indicate that promoted endothelial dysfunction. Taken together, these dynamic accessibility underlies STAT-dependent IL-9 results suggest that Th9 cells promote psoriatic 101 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 cardiovascular disease by directly inducing human arterial 1Division of Rheumatology & Clinical Immunology, endothelial cell dysfunction. University of Pittsburgh, Pittsburgh, 2Fungal Pathogenesis Disclosure of Interest: None Declared Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious LT204d Diseases, NIH, Bethesda, United States, 3Division of RED BLOOD CELLS ALLOIMMUNIZATION AND Infectious Diseases, University of Pittsburgh, Pittsburgh, -, CYTOKINES IN SCD PATIENTS IN AFRICA 4Department of Dermatology, University of Pittsburgh, L. Siransy1,*, A. Honore2, K. Sidonie3, K. Patricia1, D. Sery1 Pittsburgh, United States, 5Centre for Host-Microbiome 1Université Felix houphouet Boigny, 2UFR Sciences Interactions, Faculty of Dentistry, Oral & Craniofacial Médicales, 3Centre National de Transfusion Sanguine, Sciences, King’s College London, London, United Kingdom, Abidjan, Côte d'Ivoire 6Cytokines and Immunity Section, Cancer and Inflammation Program, National Cancer Institute, NIH, Frederick, Abstract Content: INTRODUCTION 7Institute for Genome Sciences, University of Maryland Numerous factors are involved in the process of School of Medicine, Baltimore, 8Center for Translational alloimmunization. Not all patients respond in the same way Research in Infection and Inflammation, Tulane University, to alloimmunization. Some are immunological responders New Orleans, United States and others not. Inflammation has been identified as a factor and would increase the risk of immunization. In sickle cell Abstract Content: Oropharyngeal candidiasis (OPC, oral disease the most prevalent genetic disease worldwide, thrush) is an opportunistic infection caused by the inflammation is a permanently phenomenon. This work commensal fungus . IL-17 and IL-22 are attempts to identify the pattern of few inflammatory and non cytokines produced by Type 17 lymphocytes. Both cytokines inflammatory cytokines and their relationship with mediate antifungal immunity yet activate quite distinct alloimmunization. downstream signaling pathways. While much is now PATIENTS AND METHODS understood about how IL-17 promotes immunity in OPC, the 50 subjects (4 to 18 years) were prospectively enrolled in activities of IL-22 are far less well delineated. We show that, the study after an informed consent. The patients were despite having similar requirements for induction from Type assigned in 2 groups, patients in steady state and patients 17 cells, IL-22 and IL-17 function non-redundantly during admitted for crisis. Pattern of 4 cytokines were analysed OPC. We find that the IL-22 and IL-17 receptors are required (IL10, IL4, IL17 and IFNγ) and serums were measured by in anatomically distinct locations within the oral mucosa; loss using LEGENDplexTM Human Inflammation Panel assays. of IL-22RA1 or STAT3 in the oral basal epithelial layer (BEL) RESULTS causes susceptibility to OPC, whereas IL-17RA is needed in We recruit 50 patients with a diagnosis of sickle cell disease the suprabasal epithelial layer (SEL). Transcriptional (SCD). Of these, only 31 have benefited from the research profiling of the tongue linked IL-22/STAT3 to oral epithelial of irregular antibodies (62%) comprising 14 males (45,16%) cell proliferation and survival, but also, unexpectedly, to and 17 females (54, 84%). The overall alloimmunization driving an IL-17-specific gene signature. We show that IL-22 prevalence was 16, 12%. We noted an increased level of mediates regenerative signals on the BEL that replenish the IL10 in nonalloimmunized patients with SCD when IL-17RA-expressing SEL, thereby restoring the ability of the compared with alloimmunized patients. IL4 was higher in oral epithelium to respond to IL-17 and thus to mediate alloimmunized patients compared to non alloimmunized. antifungal events. Consequently, IL-22 signaling in BEL Concerning IL17, we noted a small increase in patients ‘licenses’ IL-17 signaling in the oral mucosa, revealing without alloantibodies and lower levels of IFNγ spatially distinct yet cooperative activities of IL-22 and IL-17 CONCLUSION in oral candidiasis. The impact of biomarkers in the occurrence of Disclosure of Interest: None Declared alloimmunization is a constant preoccupation of researchers. Despite the small number of patients enrolled, LT206 our study shows the possible role of IL-10 as protecting from SHIGELLA DISRUPTS TYPE I AND III INTERFERON alloimmunization but this information must be explored in a SIGNALLING IN EPITHELIAL CELLS larger cohort. N. Alphonse1,2,* Disclosure of Interest: None Declared 1Infectious Diseases, King's College London, 2Immunoregulation Laboratory, Francis crick Institute, Lightning Talk Session 3: Mucosal immunity London, United Kingdom LT205 ORAL EPITHELIAL IL-22/STAT3 SIGNALING LICENSES Abstract Content: Type I and type III interferons (IFNs) are IL-17-MEDIATED IMMUNITY TO ORAL MUCOSAL immune cytokines produced in response to the detection of CANDIDIASIS microbes by epithelial cells (ECs) and innate immune cells. F. E. Y. Aggor1,*, T. J. Break2, G. Trevejo-Nuñez 3, N. Type I IFNs, predominantly composed of IFNα and IFNβ, are Whibley1, B. M. Coleman1, R. D. Bailey1, D. H. Kaplan4, J. secreted by almost every cell and signal through the R. Naglik5, W. Shan6, A. C. Shetty7, C. McCracken7, S. K. ubiquitously expressed IFN-α receptor 1 (IFNAR1)/IFNAR2 Durum6, P. S. Biswas1, V. M. Bruno7, J. K. Kolls8, M. S. complex receptor. In contrast, type III IFNs, comprise IFN Lionakis2, S. L. Gaffen1 λ1-4 and signal through the IFN-λ receptor 1 (IFNLR1)/IL- 10R2 complex receptor and are restricted to epithelia at 102 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 barrier sites, thereby playing an important role in mucosal superinfections. In this study, we analyzed how innate immunity. Upon binding with their respective receptor, they immune activation in response to viral RNA contributes to induce the expression of interferon-stimulated genes (ISGs) the pathogenesis of RNA-viruses. We demonstrate that type through janus kinase (Jak) – signal transducer and activator III interferons are produced by dendritic cells in the lung via of transcription (STAT) signalling. the activation of the TLR3-TRIF pathway in response to Although they have important roles for the clearance of synthetic double stranded RNA, and cause barrier damage, numerous viruses, their roles in the defence against compromising host tolerance and resistance. In particular, bacterial pathogens remain poorly understood. Our lab type III interferons act directly on epithelial cells and inhibit highlighted that type I and III interferons are secreted upon tissue repair by impairing lung epithelial cell proliferation, bacterial infection. The different roles of type I and III causing susceptibility to lethal bacterial superinfections. interferons as well as their mechanisms of action in the Overall, our data describe a previously context of bacterial infections are the focus of our lab. unappreciated pathophysiological role of this group of interferons which mandates a careful evaluation of their My project specifically investigates the functions of type I possible use in the clinical practice against endemic as well and III IFNs in immunity against Shigella sonnei (S. as emerging viral infections. sonnei). S. sonnei is a gram-negative bacterium responsible Disclosure of Interest: None Declared for Shigellosis, a disease particularly life-threatening for children under five, characterized by bloody diarrhea, fever LT208 and abdominal pain. Upon ingestion, Shigella reaches the LOC339803 LNCRNA REGULATES INTESTINAL colon where it activates a type-3-secretion system (T3SS), PROINFLAMMATORY CYTOKINE RESPONSE BY AN through which it secretes virulence factors or effectors ALLELE-SPECIFIC RNA METHYLATION-DEPENDENT required for its entry, replication and dissemination MECHANISM into/within ECs. T3SS-effectors also interfere with innate A. Castellanos-Rubio1,2,*, A. Olazagoitia-Garmendia2, M. sensing pathways and can modulate pro-inflammatory Sebastian-delaCruz2, I. Irastorza3, I. Santin2, J. R. Bilbao2 cytokine expression. However, little is known their impact on 1Ikerbasque, Basque Foundation for Science, Bilbao, signalling downstream of IFN receptors. Using a reporter 2University of the Basque Country, Leioa, 3Biocruces ISRE-Luc transfection system, our lab recently identified Bizkaia Health Institute, Barakaldo, Spain that some Shigella effectors are potent inhibitors of IFN signalling. These effectors were able to block IFNβ or IFNλ- Abstract Content: LOC339803 is a lncRNA with unknown mediated STAT1 phosphorylation and subsequent ISG function that is located on the intestinal inflammatory expression. Furthermore, I investigated the impact of IFNβ disease-associated region 2p15. One of the disease- on S. sonnei invasion of ECs by measuring intracellular associated SNPs is within a m6A methylated region, so we bacterial numbers in IFNβ-treated ECs infected with S. hypothesized that SNP-related methylation changes could sonnei for 1,3 or 5 hours. The results showed a decrease of affect lncRNA function. To test this hypothesis, we used an intracellular bacteria number at each time point, suggesting intestinal cell line heterozygous for the associated SNP that IFNβ impairs S. sonnei entry into ECs. The impact of together with RNA extracted from human small intestinal type III IFN on S. sonnei infection of ECs is currently under samples of controls and individuals with an intestinal assessment in the lab to complete these findings. inflammatory condition (i.e. celiac disease).

Overall, our studies greatly contribute to the understanding To asses whether the associated SNP genotype was of interferon regulation and functions which benefits our influencing m6A methylation of the lncRNA we performed an knowledge of host-microbial interactions at mucosal allele-specific meRIP and observed that the risk conferring surfaces. G allele was preferentially methylated. Stability analysis Disclosure of Interest: None Declared showed that the preferentially methylated G allele is less stable, and when the m6A levels were increased by LT207 overexpressing METTL3 methylase, its levels were further IFN-▫▫ INDUCED BY VIRAL RECOGNITION IN THE LUNG decreased, pointing to an involvement of the methylation of PREDISPOSES TO BACTERIAL SUPERINFECTIONS BY this lncRNA in its function. Assessment of the subcellular INHIBITING EPITHELIAL REPAIR. location of the lncRNA showed that LOC339803 is primary A. Broggi1,2,*, S. Ghosh1, B. Sposito1, I. Zanoni1 nuclear in intestinal cells suggesting a role in transcriptional 1Immunology, Boston Children's Hospital, 2Harvard medical regulation. Overexpression of LOC339803 induced allele- school, Boston, United States dependent downregulation of the adjacent COMMD1 gene and augmented downstream NFkB levels, while CRISPR- Abstract Content: RNA viruses such as influenza virus, Cas9 mediated deletion of the lncRNA respiratory syncytial virus and the new pandemic enhanced COMMD1 and decreased NFkB expression. coronavirus SARS-CoV-2 induce severe clinical disease in Additionally, overexpression of LOC339803 activated the the lower respiratory tract. Clinical and experimental expression of the cytokines IL1B and IL6, a hallmark of evidence indicate that most severe and lethal cases do not intestinal inflammation, which was counteracted by depend on the viral burden and are, instead, characterized overexpressing COMMD1. RNA immunoprecipitation by an aberrant immune response which induce tissue experiments followed by mass-spectrometry, using in vitro damage and predisposes to lethal microbial transcribed biotinylated lncRNA, showed that HNRNPC 103 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 protein, which has been shown to bind m6A-switches, is LT210 able to interact with LOC339803 in intestinal cells. INHIBITION OF SALMONELLA ENTERICA INVASION Interestingly, proteins that bind COMMD1 promoter were AND INTRACELLULAR REPLICATION BY also present within the immunoprecipitated complexes. INTERFERON-STIMULATED GENES. Moreover, expression analysis in human intestinal biopsies R. Dickenson1,* revealed that this lncRNA is increased in celiac disease 1Infection and immunity, King's College London, London, patients showing higher levels in those patients United Kingdom homozygous for the risk allele. The results so far suggest that lncRNA LOC339803 has an Abstract Content: Salmonella enterica serovar allele-specific effect in NFkB-mediated intestinal Typhimurium is an intracellular pathogen that causes food- inflammation by the induction of IL1B and IL6. The allele and water-borne gastroenteritis. Once consumed it invades specificity seems to be related to m6A methylation levels host cells, including epithelial cells, M cells and dendritic and to the ability of the lncRNA to regulate the expression of cells within the gastrointestinal tract. Recognition of the the nearby COMMD1 gene probably via its interaction with pathogen by the host immune system induces a pro- the HNRNPC protein. inflammatory state that usually results in clearance of the Disclosure of Interest: None Declared pathogen by neutrophils and other immune cells. Interferons (IFNs) are archetypal antiviral cytokines that act in an LT209 autocrine and paracrine manner to activate JAK/STAT REGULATORY T CELLS CONTROL THE DYNAMIC AND signalling and induce the expression overlapping sets of SITE-SPECIFIC POLARIZATION OF CD4 T CELLS hundreds of interferon-stimulated genes (ISGs). IFNs have FOLLOWING SALMONELLA INFECTION also been shown to be expressed during bacterial infections, S. Clay1,2,*, A. Bravo Blas2,3, D. Wall2, M. MacLeod2, S. although whether they help the host or the pathogen is Milling2 highly variable between bacterium, biological context, and 1Department of Immunology and Infectious Diseases, IFN type. As they are induced in concert, the individual roles Harvard School of Public Health, Boston, United States, of many ISGs are not well understood and there is a dearth 2Institute of Infection, Immunity and Inflammation, University of knowledge regarding their effects in bacterial infections. of Glasgow, 3Institute of Cancer Sciences, University of In this study, epithelial cell lines were incubated with IFNs Glasgow and Cancer Research UK Beatson Institute, prior to infection with S. Typhimurium. Data indicates that Glasgow, United Kingdom IFNb can inhibit the invasion of S. Typhimurium into epithelial cells and may also inhibit intracellular replication. Abstract Content: FoxP3+ regulatory T cells (Tregs) control The project aims to conduct a comprehensive screen of inflammation and maintain mucosal homeostasis, but their ISGs to better understand how they restrict bacterial potential to control CD4 T helper (Th) bias is poorly invasion into, and replication within, host epithelial cells. understood. Th1, Th2 and Th17 cells can be identified by Disclosure of Interest: None Declared expression of cytokines and the master transcription factors (TFs) T-bet, GATA3 and RORγT, respectively. Tregs also LT211 express these TFs, but it is unclear what role Tregs MODC'S-TARGETING TNF FUSION PROTEINS expressing Th-type TFs play during infection. To address ENHANCE CYCLIC DI-GMP MUCOSAL VACCINE this knowledge gap, we developed a model of non-lethal ADJUVANTICITY IN MIDDLE-AGED AND AGED MICE colitis using an attenuated strain of Salmonella enterica H. Gogoi1,*, S. Mansouri1, D. S. Katikaneni1, L. Jin1 serotype Typhimurium. Early after infection we observed 1Division of pulmonary, critical care, and sleep medicine, increased numbers of colonic IL17A+ Th17 cells, followed by University of Florida, Gainesville, United States a long-lasting Th1 bias with increased IFNγ production. Unexpectedly, the early Th17 response paralleled an Abstract Content: Since the discovery of alum in the late increase in T-bet+ Tregs, and the later Th1 bias occurred in 1920's, vaccine adjuvants have been the "immunological tandem with increased RORγT+ Tregs. This reciprocal bullets" for nearly a century. Cyclic dinucleotides (CDNs) are dynamic may indicate sub-populations of Tregs selectively promising mucosal vaccine adjuvants and have been suppress Th cells, shaping the overall immune response. To reported to induce a balanced, potent humoral and cellular determine if Tregs were required for the dynamic Th bias, immune responses. However, CDNs' efficacy in an aged depletion experiments were carried out. Treg depletion 1-2 environment is unclear. We examined the mucosal vaccine days post-infection shifted the early Th17 response to a Th1 efficacy of cyclic di-GMP (CDG), the founding member of bias; and depletion 6-7 days post-infection abrogated the CDNs, in 1-year-old (middle-aged), and 2-year-old (aged) Th1 bias. Thus, Tregs are necessary for the early Th17 C57BL/6J mice. We found that 1-year-old and 2-year-old response, and for a maximal Th1 response later. These data C57BL/6J mice are defective in CDG-induced memory Th1 show that Tregs shape the colonic CD4 T cell response in a and Th17 responses and high-affinity serum IgG, mucosal nuanced and fine-tuned way. This highlights the potential for IgA production. Next, we generated two novel TNF fusion subpopulations of Tregs to be targeted in novel proteins; soluble TNF and transmembrane TNF to target immunotherapies to restrain pathogenic T cell responses. TNF receptors in monocyte-derived DCs (moDCs) to Disclosure of Interest: None Declared enhance CDG vaccine efficacy in 1-year- old and 2-year-old mice. The moDCs -targeting TNF fusion proteins restored CDG-induced memory Th1, Th17, and high-affinity IgG, IgA 104 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 responses in the 1-year-old and 2-year-old mice. Together, inflammation, and the keratinization modules we identified the data suggested that aging negatively impacts CDG were associated with an upregulation of genes vaccine adjuvanticity. However, moDC- targeting TNF that regulate inflammatory signaling, apoptosis, and fusion proteins enhanced CDG adjuvanticity in the aging necroptosis. Ongoing single-cell RNAseq analysis aims to mice. It can be said that, age related immune impairment is discern the role of RIG-I-directed responses in epithelial not irreparable and can be recovered with precise targeting. cells of the vaginal mucosa and to determine the potential Disclosure of Interest: None Declared role of tissue-specific programmed cell death in controlling ZIKV infection. At this time, we hypothesize that LT212 accelerated keratinization serves as a host-directed effort to INTRAVAGINAL ZIKA VIRUS INFECTION INITIATES shed infected cells rapidly, and that ZIKV persistence in the TISSUE-SPECIFIC INNATE IMMUNE PROGRAMS IN THE FRT and dissemination to other sites may benefit from the FEMALE REPRODUCTIVE TRACT resultant anti-inflammatory state. A. Gustin1,2,*, K. Voss1, N. Klatt2, M. Diamond3, M. Gale1 Disclosure of Interest: None Declared 1Immunology, University of Washington, Seattle, 2Surgery, University of Minnesota, Minneapolis, 3Microbiology, LT213 Washington University, St. Louis, United States A SMALL-MOLECULE RIG-I AGONIST ENHANCES VACCINE PROTECTION AGAINST PANDEMIC AND Abstract Content: Introduction: Innate immunity plays a HIGHLY PATHOGENIC AVIAN INFLUENZA A VIRUS critical role in protection of the female reproductive tract INFECTION (FRT) to mediate tissue-specific defense against invading E. A. Hemann1,*, M. Knoll1, S. P. Iadonota2, R. Ireton1, Y.-M. pathogens while maintaining tolerance toward commensal Loo1, M. Gale, Jr1 bacteria, sperm and fetal products. We have shown that 1University of Washington, 2Kineta, Inc, Seattle, United innate immune defenses against ZIKV are triggered through States RIG-I. Importantly, ZIKV suppresses IFN signaling to facilitate infection, but how these processes direct innate Abstract Content: Despite the constant threat of pandemic immune actions, gene expression, and viral dissemination influenza virus emergence, vaccines against influenza from the FRT are not known. We found that ZIKV targets remain suboptimal in eliciting an effective adaptive immune and persistently replicates in the vaginal mucosa after response. Novel strategies to develop new adjuvants may subcutaneous challenge in nonhuman primates, thus aid in increasing the functionality of currently existing defining the FRT and as a relevant site of viral tropism and influenza virus vaccines. In order to address this need, we persistence. leveraged knowledge of host innate immune signaling Methods: To define the innate immune correlates that pathways to develop a novel small-molecule RIG-I agonist, respond to ZIKV and impart antiviral defense or KIN1148, to adjuvant vaccination against pandemic H1N1 susceptibility to infection, we established a small animal (pH1N1) or highly pathogenic avian influenza (H5N1). model of intravaginal ZIKV challenge with mouse-adapted C57Bl/6 mice were vaccinated intramuscularly (i.m.) with ZIKV/Dakar in C57xBL6/J mice featuring knock-in of the inactivated split vaccine against pH1N1 or H5N1 and human STAT2 gene (S-KI). This model is fully immune adjuvanted with formulated KIN1148 or vehicle. Thirty days competent and is susceptible to ZIKV infection and viral following vaccination mice were challenged with antagonism, displaying virus/host dynamics similar to NHP homologous virus for survival studies or the lungs were and human infection. harvested at day 5 p.i. for analyses of immune response. Results: We found that intravaginal infection in S-KI mice Alternatively, mice were administered two homologous resulted in virus dissemination to the upper reproductive vaccinations on days 0 and 14 with immune response tract and draining lymph nodes as assessed by qRT-PCR assessment of serum, dLN, and/or spleen performed on day as early as one day post-infection (dpi). Virus was 19. Our results demonstrate that KIN1148 binds to and subsequently detected in the spleen and gastrointestinal activates RIG-I leading to the activation of IRF3 and innate tract at 4-6 dpi, and spinal cord at 8-10 dpi. We next immune genes. Ex vivo treatment of dendritic cells with conducted bulk RNA sequencing on tissues of the lower and KIN1148 leads to cellular activation and maturation. upper reproductive tract, draining lymph nodes, and Administration of KIN1148 with pH1N1 or H5N1 i.m. spleen over the infection time course. Innate immune vaccination significantly increased IAV-specific antibody induction in the lower female reproductive tract - as responses compared to vaccination alone, and led to an measured by differential gene expression - followed a increase in HI titers against other H5 HAs. Intriguingly, biphasic pattern, with a rapid response via the RIG-I KIN1148 also induced IAV-specific CD4 and CD8 T cell pathway followed by a subsequent induction of genes responses following vaccination, a unique and cross- downstream of types I and III IFN. Complimentary co- protective cellular immune response not typically observed expression analysis of the entire LRT gene set identified a downstream of i.m. split vaccination. This broad humoral suite of keratinization and cornification genes upregulated in and cellular immune response induced following a single infected animals as early as 1 dpi that may suggest an vaccination with KIN1148 led to enhanced protection during acceleration in programmed epithelial cell death following high dose pH1N1 or H5N1 challenge and reduced virus ZIKV infection. titers and pathology in the lungs. In sum, KIN1148 Discussion: Homeostatic cell renewal in stratified administered with pH1N1 or H5N1 vaccination leads to the squamous epithelial tissues requires suppression of induction of a broad and enhanced anti-IAV immune 105 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 responses compared to vaccination alone. Overall, these intestinal microbiota can induce tonic type I interferon (IFN) results suggest targeting of the RIG-I pathway typically and interferon a/b receptor (IFNAR) signaling is a known activated during infection to confer immunity with a small regulator of lamina propria lymphocyte populations at molecule compound can enhance vaccine protection, steady-state, we hypothesized that microbiota-induced highlighting the potential of KIN1148 to enhance vaccine- IFNAR signaling also influences γδ IEL homeostasis. induced protection against RNA virus infection. Morphometric analysis of jejunum from 8-week-old GFP γδ Disclosure of Interest: None Declared T cell reporter mice (TcrdEGFP, WT) and TcrdEGFP; IFNAR KO mice showed that the number of GFP+ γδ IELs was LT214 increased 2-fold in IFNAR KO mice compared to WT. IFN-LAMBDA INDUCTION BY MURINE ASTROVIRUS Administration of 5-Ethynyl-2´-deoxyuridine (EdU) revealed REFLECTS GOBLET CELL TROPISM AND IS that γδ IELs isolated from IFNAR KO mice exhibit a 50% RECAPITULATED BY ENTEROID CULTIVATION increase in proliferation compared to WT. Surprisingly, the H. Ingle1,*, E. Hassan1, J. Gawron1, B. Mihi1, Y. Li1, H. relative proportion of γδ IELs was skewed toward Vγ7- Makimaa1, S. Lee1, M. Good1, M. Baldridge1 subsets in IFNAR KO mice. EdU incorporation within each 1Washington University School of Medicine, St. Louis, Vγ subset revealed that IFNAR KO mice exhibit a 66% and United States 22% increase in proliferation in Vγ7- and Vγ7+ IEL populations, respectively, relative to WT. Bulk TCR Abstract Content: Despite being a global cause of viral sequencing and clonotype analysis of γδ IELs isolated from gastroenteritis in children, astroviruses are understudied WT and IFNAR KO mice showed polyclonal TCRγδ due to the lack of a well-defined small animal model. Murine repertoires, indicating that the expanded γδ IEL population astroviruses (muAstVs) chronically infect immunodeficient was not due to clonal expansion. We observed a 35-fold mice, but an in vitro cultivation system and understanding of increase in γδ IELs in IFNAR KO mice one week after birth, their in vivo pathogenesis has been lacking. Here, we indicating that this phenotype begins early in life. describe a platform to cultivate muAstV using air-liquid Interestingly, IFNAR KO mice rederived into a murine interface (ALI) cultures derived from mouse enteroids. The norovirus (MNV)-free facility had normal γδ IEL ALI system supports apical infection of and viral release by compartment similar to WT mice, indicating that loss of type epithelial cells. MuAstV also stimulates IFN-λ production in I IFN signaling alone is not sufficient to induce the ALI, recapitulating our in vivo findings. We report that hyperproliferative phenotype, but that an environmental chronic muAstV infection in immunodeficient mice is factor may also be involved. To test this, MNV- IFNAR KO predominantly in the small intestine, is microbiota- breeding pairs were housed with dirty bedding from IFNAR independent, and correlates with higher IFN-λ expression. KO mice from our barrier facility. The mice born from these We demonstrate that goblet cells and presumptive pairs had increased numbers of γδ IELs, indicating that the enterocytes are the in vivo targets for chronic muAstV γδ IEL hyperproliferative phenotype is transmissible and infection. During chronic infection, muAstV stimulates IFN-λ may be independent of IFNAR signaling. To control for production in goblet cells, in turn inducing widespread ISG maternal effects on the microbiome, we crossed separately- expression in the intestinal epithelium. Collectively, these housed WT and IFNAR KO mice from our barrier facility to findings provide insights into the cellular tropism for and generate F2 littermates. Adult F2 WT mice had a 2-fold innate immune responses to muAstV, and demonstrate an increase in the number of γδ IELs, similar to parental SH enteroid-based culture system to readily propagate muAstV IFNAR KO mice. Further, F2 WT γδ IELs exhibited a 71% in vitro. increase in proliferation compared to SH WT. Fecal 16S Disclosure of Interest: None Declared rRNA sequencing analysis showed a greater diversity in SH IFNAR KO and F2 littermates compared to SH WT or MNV- LT215 controls (Shannon Index, p<0.01). While the precise IDENTIFICATION OF A TRANSMISSIBLE EARLY LIFE contribution of IFNAR signaling and the intestinal GAMMA DELTA INTRAEPITHELIAL LYMPHOCYTE microbiome remains to be determined, we have identified a HYPERPROLIFERATIVE PHENOTYPE THAT IS novel, vertically-transmitted γδ IEL hyperproliferative ASSOCIATED WITH CHANGES IN THE INTESTINAL phenotype that develops during early life prior to weaning. MICROBIOME Disclosure of Interest: None Declared L. Jia1,*, G. Wu2, Y. Lam2, A. Lemenze1, L. Zhao2, K. L. Edelblum1 LT216 1Center for Immunity and inflammation, Rutgers university- THE CYCLIC DINUCLEOTIDES-SENSING New Jersey Medical School, Newark, 2Department of INDEPENDENT FUNCTION OF HAQ STING IN BODY Biochemistry and Microbiology, New Jersey Institute for METABOLISM Food, Nutrition & Health, New Brunswick, United States D. S. Katikaneni1, S. Mansouri1, A. Singh2, S. Patel1, G. de Lartigue2, L. Jin1,* Abstract Content: Intestinal intraepithelial lymphocytes 1Medicine, 2Pharmacodynamics, University Of Florida, expressing the γδ T cell receptor (γδ IEL) function as a first Gainesville, United States line of defense against injury or microbial invasion. The majority of the γδ IELs express the Vγ7 T cell receptor Abstract Content: The human R71H-G230A-R293Q (TCR); however, the epithelial compartment also contains (HAQ) STING allele is common in non-Africans. The Vγ7- cells typically found in the periphery. Given that the population frequencies for HAQ ranges from ~23% in 106 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Caucasians to ~63% in East Asians. However, HAQ is one of the major IL-27-producing cell types in the gut- extremely rare in Africans (<1%). In contrast, ~40% of associated tissue. Whereas IL-27 expression in Africans have the AQ (G230A-R293Q) STING, whose conventional dendritic cells (DCs) or myeloid cells is population frequency in non-Africans is <1%. HAQ STING respectively required for the differentiation of a specialized evolved from the AQ STING. The modern anatomically regulatory T (Treg) cell subset and IL-10 production, gut humans migrated from Africans about ~40,000 years ago. epithelial IL-27 specifically promotes a distinct CD4CD8αα The rapid replacement of AQ-STING by the HAQ-STING intraepithelial lymphocyte (IEL) population that acquires allele in non-Africans suggests a strong natural selection for their functional differentiation upon arrival at the intestinal HAQ in early human migration out of Africa. STING senses epithelium. Either loss of IL-27 in IECs or disruption of its cyclic dinucleotides (CDNs) including bacterial cyclic di- receptor in T cells could lead to a selective defect in GMP, cyclic di-AMP and mammalian 2’3’-cyclic GMP-AMP. CD4CD8αα IELs over time. Consequently, mice with IEC- We previously showed that the HAQ STING is defective in specific IL-27 ablation exhibited uncontrolled pathogen responses to cyclic dinucleotides (CDNs) sensing in vivo burden during parasitic infection and this could be rescued and in vitro (Jin et al., 2011; Patel et al., 2017; Sebastian et by transfer of exogenous CD4CD8αα IELs. Collectively, our al., 2020). To address the question of why HAQ replaced data reveals that in addition to its known regulatory AQ, we generated an AQ knock-in mouse. We found that properties in preventing immune hyperactivity, gut epithelial AQ mice responded to CDNs similarly to the WT mice. IL-27 confers barrier immunity through inducing a specific Interestingly, we noticed that mature adult AQ mice had less IEL subset and further suggests that IL-27 derived from body weight than the HAQ or WT mice on a normal diet. different cellular sources plays distinct roles in maintaining STING-/- mice had similarly lean phenotypes as the AQ mice. intestinal homeostasis. Thus, the evolution of AQ to HAQ STING, while resulted in Disclosure of Interest: None Declared the loss of CDNs sensing ability, restored STING function in body metabolism, which appeared to be essential for early LT218 humans’ survival. We will present data further characterizing DEVELOPMENTAL PATHWAYS REGULATE this previously unrecognized, CDNs-sensing independent CYTOKINE-DRIVEN EFFECTOR AND FEEDBACK function of STING in calibrating body metabolism. We RESPONSES IN THE INTESTINAL EPITHELIUM propose that the de facto role of STING in humans is to H. T. Lindholm1,*, N. Parmar1, C. Drurey2, J. Ostrop1, A. control body metabolism. Díez-Sanchez1, R. Maizels2, M. Oudhoff1 1CEMIR – Centre of Molecular Inflammation Research, References Department of Clinical and Molecular Medicine, Norwegian Jin, L., Xu, L.G., Yang, I.V., Davidson, E.J., Schwartz, D.A., University of Science and Technology, Trondheim, Norway, Wurfel, M.M., and Cambier, J.C. (2011). Identification and 2Wellcome Centre for Integrative Parasitology, Institute of characterization of a loss-of-function human MPYS variant. Infection, University of Glasgow, Glasgow, United Kingdom Genes Immun 12, 263-269. Patel, S., Blaauboer, S.M., Tucker, H.R., Mansouri, S., Ruiz- Abstract Content: The intestinal tract is a common site for Moreno, J.S., Hamann, L., Schumann, R.R., Opitz, B., and infection, and it relies on an appropriate immune response Jin, L. (2017). The Common R71H-G230A-R293Q Human to defend against pathogens. The intestinal epithelium has TMEM173 Is a Null Allele. J Immunol 198, 776-787. an important role in effector responses, which is coordinated Sebastian, M., Hsiao, C.J., Futch, H.S., Eisinger, R.S., by immune-type specific cytokines. It is incompletely Dumeny, L., Patel, S., Gobena, M., Katikaneni, D.S., Cohen, understood how cytokines drive epithelial responses. Here, J., Carpenter, A.M., Spiryda, L., Heldermon, C.D., Jin, L., using organoid-cytokine co-cultures, we provide a and Brantly, M.L., (2020). Obesity and STING1 genotype comprehensive analysis of how key cytokines affect the associate with 23-valent pneumococcal vaccination efficacy. intestinal epithelium, and relate this to in vivo infection JCI Insight May 7; 5(9): e136141. 5. models. We use image analysis based on a convolutional Disclosure of Interest: None Declared neural network to automatically categorize organoids into "spheroid" and "budding". We show that these categories LT217 correlate to the maturity of organoids and that cytokines GUT EPITHELIAL IL-27 CONFERS INTESTINAL affect the relative percentage of these categories. IMMUNITY THROUGH THE INDUCTION OF Furthermore, we use transcriptomic analysis to reveal that INTRAEPITHELIAL LYMPHOCYTES cytokines use developmental pathways to define intestinal C.-H. Lin1,*, M.-C. Chen1, L.-L. Lin1, D. A. Christian2, C. A. epithelial effector responses. For example, we find that IL- Hunter2, L.-F. Lu1 22 and IL-13 dichotomously induce goblet cells, in which 1Biological Sciences, University of California San Diego, only IL-13 driven goblet cells are associated with NOTCH San Diego, 2Pathobiology, University of Pennsylvania, signaling. We further show that IL-13 induces BMP signaling Philadelphia, United States to act as a negative feedback loop in IL-13 induced tuft cell hyperplasia, an important aspect of type 2 immunity. Abstract Content: IL-27 is known to control a diverse range Together, we show that targeting developmental pathways of immune responses in many different disease settings. may be a useful tool to tailor epithelial effector responses Multiple cell types are capable of expressing IL-27 with that are necessary for immunity to infection. innate immune cells considered to be the dominant cellular Disclosure of Interest: None Declared sources. Here, we identify intestinal epithelial cells (IECs) as 107 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT219 5Cystic Fibrosis Regional Reference Center, Fondazione A NOVEL INTERFERON-BETA SIGNALING PATHWAY IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, CONTROLS THE TOLEROGENIC PROGRAM IN Italy PULMONARY TNFR2+ CDC2 SUBSET IN VIVO S. Mansouri1,*, D. S. Katikaneni1, H. Gogoi1, L. Jin1 Abstract Content: Introduction 1Division of Pulmonary, Critical Care and Sleep Medicine, Cystic fibrosis (CF) and chronic obstructive respiratory Department of Medicine, University of Florida, Gainesville, disease (COPD) are characterized by a pulmonary United States inflammatory status coordinated by a network of cytokines, often associated with bacterial colonization. Nontypeable Abstract Content: Introduction: Dendritic cells (DCs) play Haemophilus influenzae (NTHi) is a bacterium commonly a vital role in maintaining lung immune tolerance by isolated from the airways of patients that suffer from CF or generating regulatory T cells (Tregs). Lung tolerance must COPD. However, to what extent NTHi persistence be tightly controlled to avoid the development of chronic contributes to the lung inflammatory burden during chronic inflammatory lung diseases. We recently identified the airway disease is controversial. Here, we aimed at tolerogenic TNFR2+ cDC2 subset (R2D2) in the lung that determining the pro-inflammatory role of NTHi persistence induces regulatory T cells and maintains lung immune in a cohort of CF patients and in a newly generated mouse tolerance (Mansouri et al., 2020)(PMID: 31959883). R2D2 model of NTHi persistence. require type I interferon signaling to induce Tregs. Type I Methods IFNs, including IFNb and IFNa, signal through the shared Nasopharyngeal aspirates from 19 CF patients with variable IFANR1/IFNAR2 receptors, however their signaling genotypes were collected from the Regional CF Center at outcomes can have different outcomes. Here, we sought to Milan’s Ospedale Maggiore Policlinico during routine care delineate the tolerogenic IFNb signaling in R2D2. visits, and levels of CF pro-inflammatory cytokines were Methods: To identify the molecular mechanisms evaluated by ELISA. responsible for the tolerogenic IFNb signaling in vivo, Long-term chronic infection was established with NTHi- various DC-specific conditional knock-out mice were embedded agar beads by adapting a murine model intranasally treated with IFNb, IFNa, aIFNAR1 and previously described for other pathogens (Lorè NI et al, Sci. aIFNAR2. Characterization of lung DCs was examined by Rep. 2016). Cytokines levels and lung infiltrating cells were flow cytometry 24 hours after treatment. Induction of Tregs evaluated by ELISA, flow cytometry and histological was examined by flow cytometry on day 14. analysis during the development of chronic infection (2 and Results: We have delineated a non-canonical signaling 14 days). pathway in which IFNb activates the anti-inflammatory Results properties of the tolerogenic R2D2. Interestingly, inhaled In our study cohort, we found that CF patients chronically IFNb and IFNa had opposing effects on R2D2. IFNb colonized by NTHi had significantly higher levels of IL-8 and promoted the tolerogenic program in R2D2 and enhanced CXCL1 than those who were not colonized. To better define Treg generation. IFNa repressed the tolerogenic program the impact of NTHi persistence in fuelling the airway and inhibited Treg induction in the lung. Furthermore, IFNb inflammatory response, we developed a novel mouse model signaling acts through IFNAR1, not IFNAR2, to generate using both laboratory and CF clinical strains. NTHi Tregs in the lung. We will present data further characterizing persistence associated with a sustained inflammation of the the non-canonical IFNb-IFNAR1 signaling pathway in the lung, characterized by recruitment of neutrophils and by the tolerogenic R2D2 cells. release of related pro-inflammatory cytokines (KC, Mip-2, G- Conclusions: We delineated an IFNb-specific signaling CSF, IL-6) at 2 and 14 days post-infection. Moreover, an pathway in the lung tolerogenic DC subset that is essential increased burden of T cell mediated response (CD4+ and γδ for maintaining lung tolerance. These findings elucidate a T cells) was observed in the lungs at 14 days post-infection. fundamental mechanism controlling lung tolerance and We also found that both CD4+IL-17+ cells and levels of IL- identifies a strategy to restore lung tolerance in inflammatory 17A cytokine (IL-17A and IL-17F) were enriched in mice at lung diseases. advanced stage of NTHi chronic infection, in association Disclosure of Interest: None Declared with higher levels of matrix metalloproteinase 9 as a marker of tissue remodelling. LT221 In addition, we were able to demonstrate by THE PERSISTENCE OF NONTYPEABLE HAEMOPHILUS immunohistochemistry that that CD3, B220 and CXCL-13 INFLUENZAE FUELS TYPE 17 IMMUNITY IN THE LUNG expressing cells co-localized in bronchus-associated F. Saliu1,2,3,*, G. Rizzo 2,3, A. Bragonzi3, L. Cariani4, D. M. lymphoid tissues(BALT)-like structure in infected lungs at Cirillo1, C. Colombo5, V. Daccò5, D. Girelli4, S. Rizzetto4, B. day 14 post-infection. Sipione3, C. Cigana3, N. I. Lorè1,2,3 Conclusion 1Division of Immunology, Transplantation, and Infectious Our results demonstrate that NTHi persistence exerts a pro- Diseases, Emerging bacterial pathogens, IRCCS San inflammatory activity, mediated by type 17 immunity, and Raffaele Scientific Institute, 2Università Vita-Salute San could therefore contribute to the exaggerated burden of lung Raffaele, 3Division of Immunology, Transplantation, and inflammation in patients with chronic respiratory diseases. Infectious Diseases, Infections and cystic fibrosis unit, Disclosure of Interest: None Declared IRCCS San Raffaele Scientific Institute, 4Cystic Fibrosis Microbiology Laboratory, Fondazione IRCCS Ca' Granda, 108 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

LT222 1CHUNGNAM NATIONAL UNIVERSITY, TAEJON, Korea, INTERFERON-LAMBDA RECEPTOR 1 EXPRESSION IS Republic Of DECREASED IN THE SMALL INTESTINE OF PEDIATRIC INFLAMMATORY BOWEL DISEASE PATIENTS Abstract Content: Porcine epidemic diarrhea virus (PEDV) D. M. Santer1,*, H. Armstrong2, S. Lamb1, E. Wine2, M. infects pigs and causes enteric disease characterized by Houghton1, D. L. J. Tyrrell1 watery diarrhea and dehydration. Despite the extensive 1Medical Microbiology and Immunology, Li Ka Shing vaccination, PEDV strains with variations in the spike gene Institute of Virology, University of Alberta, 2Pediatrics, have been reported in several countries, including Korea, Centre of Excellence for Gastrointestinal Inflammation and China, and the United States. Variant PEDV belonging to Immunity Research, University of Alberta, Edmonton, genogroup G2 has higher pathogenicity and morbidity than Canada conventional PEDV belonging to genogroup G1. To understand the pathogenesis of the variant PEDV, we Abstract Content: Introduction: Type III interferons (IFN- examined the susceptibility of the variant PEDV to interferon lambdas, IFN-λs) are important antiviral cytokines that and PEDV specific neutralizing antibodies, which are known modulate immune responses by acting through a unique to important for the control of PEDV infection. We found that IFN-λR1/IL-10R2 heterodimeric receptor that is highly variant PEDV (WG strain) showed higher resistance to expressed on epithelial cells especially at mucosal surfaces. interferon than conventional PEDV (KPEDV-9 strain). Mouse colitis models clearly demonstrate Ifnlr1 deficiency Similarly, KPEDV-9 strain grew well in interferon-deficient leads to exacerbated inflammation, while Ifn-λ treatment Vero cells but did not grow in interferon-releasing porcine promotes gut barrier integrity and ameliorates colitis in alveolar macrophages, whereas WG strain showed similar wildtype mice. With little data on whether IFN-λR responses infectivity in both cells. We also found that WG strain is not are dysregulated in human inflammatory bowel disease blocked by the neutralizing antibodies against KPEDV-9, (IBD), we determined if IFN-λR1 expression and IFN-λ suggesting the differences in the antigenicity of two strains. responses were altered in pediatric IBD patients. To elucidate the possible mechanism of resistance to Methods: IFN-λR1 expression was assessed by interferon and neutralizing antibody in variant PEDV, immunohistochemistry of terminal ileum formalin-fixed sequencing analysis was performed. We found huge paraffin-embedded biopsies from healthy (n=8), Crohn’s variations in spike and ORF3 proteins. To test the impacts disease (n= 7), and ulcerative colitis (n=10) pediatric of these mutations on the acquisition of interferon and patients, along with healthy patient liver biopsies (antibody neutralizing antibody resistance, we are currently generating positive control). Fresh patient biopsies (terminal ileum) pseudotyped PEDV and recombinant PEDVs with were cultured ex vivo in media with or without IFN-λ3 mutations. Our results indicate that the recently introduced overnight and gene expression was quantified by RT-qPCR. variant PEDV exhibits higher resistance to the interferon Results: A novel monoclonal antibody was identified that than the existing PEDV and avoids the neutralizing antibody accurately stains IFN-λR1 in human mucosal tissue samples against the existing PEDV. These results also provide for the first time. We found distinct IFN-λR1 staining in understanding of the occurrence of variant PEDV and its intestinal epithelial cells (average 74% positive), but also in pathogenesis. specific immune cells, which was not previously described in mouse models. This was corroborated by examining Disclosure of Interest: None Declared single cell RNA sequencing datasets. There was a significantly lower percentage of epithelial and immune cells LT224 positive for IFN-λR1 in pediatric Crohn’s disease and INFLUENZA-INDUCED MONOCYTE-DERIVED ulcerative colitis patients compared to pediatric healthy ALVEOLAR MACROPHAGES CONFER PROLONGED controls (P<0.01). IFN stimulated gene (ISG) transcript ANTIBACTERIAL PROTECTION VIA INTERLEUKIN-6 levels also differed between healthy and IBD patients after A. Wack1,*, H. Aegerter2, J. Kulikauskaite1, S. Crotta1, H. IFN-λ3 stimulation of fresh biopsies. Patel3, G. Kelly3, E. M. Hessel4, S. Beinke4 Conclusion: Our novel finding of decreased expression of 1Immunoregulation Laboratory, The Francis Crick Institute, IFN-λR1 in pediatric IBD compared to healthy patients London, United Kingdom, 2Immunoregulation Unit, VIB indicates patients may be less able to induce critical IFN-λ- Center for Inflammation Research, Ghent, Belgium, mediated antiviral responses and protective anti- 3Bioinformatics and Biostatistics, The Francis Crick Institute, inflammatory pathways. This work supports the possibility London, 4Respiratory Therapy Area, GlaxoSmithKline, for new therapeutic approaches to promote IFN-λ signaling Stevenage, United Kingdom to improve regulation of inflammatory pathways in pediatric IBD patients. Abstract Content: Despite the prevalence and clinical Disclosure of Interest: None Declared importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral LT223 clearance and clinical recovery, at 1 month after infection NEWLY ISOLATED PORCINE EPIDEMIC DIARRHEA with influenza, mice are better protected fromStreptococcus VIRUS RESISTANCE TO INTERFERON AND pneumoniae infection due to a population of monocyte- NEUTRALIZING ANTIBODY derived alveolar macrophages (AMs) that produce H. Shin1,*, J. PARK1 increased interleukin-6 (IL-6). We show that IL-6 is dispensable for antibacterial protection in naïve mice, but 109 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 crucial for enhanced protection in influenza-experienced LT226 mice. Influenza-induced monocyte-derived AMs have a IL-17 DEPENDENT FIBROBLASTIC RETICULAR CELLS surface phenotype similar to resident AMs but display a SET THE THRESHOLD FOR PROTECTIVE ANTIBODY unique functional, transcriptional and epigenetic profile that PRODUCTION IN THE GUT is distinct from resident AMs. In contrast, influenza- D. Wu1,*, S. Majumder1, M. J. McGeachy1 experienced resident AMs remain largely similar to naive 1Division of Rheumatology & Clinical Immunology, AMs. Over longer time periods, recruited AMs become Pittsburgh, United States transcriptionally more similar to resident AMs. Thus, influenza changes the composition of the AM population to Abstract Content: Attaching/Effacing (A/E) bacterial provide prolonged antibacterial protection. Monocyte- infection of the intestine is a significant cause of mortality derived AMs persist over time but lose their protective and morbidity worldwide. Bacteria specific antibody profile. Our results help to understand how transient production are critical for clearing the murine A/E bacteria, respiratory infections, a common occurrence in human life Citrobacter rodentium. Interleukin 17 (IL-17) promotes B-cell and a feature of the current COVID-19 pandemic, can activation and T cell-dependent antibody responses in constantly alter lung immunity by contributing monocyte- autoimmune models, and is known to contribute to C. derived, recruited cells to the AM population. rodentium clearance. Here we report that IL-17 acts via Disclosure of Interest: None Declared lymph node fibroblastic reticular cells (FRC) to drive C. rodentium specific antibody production. Mice with FRC- LT225 restricted IL-17RA deletion (CCL19CreIL17RAfl/fl), had T HELPER 2 CELL RESPONSES ARE DEPENDENT ON reduced germinal center (GC) formation in gut-draining LN, TISSUE-RESIDENT BASOPHILS AND THE NOTCH and reduced C. rodentium-specific antibody in both serum SIGNALING PATHWAY IN BASOPHILS DURING and feces, along with higher C. rodentium burden and HELMINTH-INDUCED TYPE 2 INFLAMMATION increased colon inflammation. Besides, loss of IL-17RA L. Webb1,*, O. Oyesola2, M. Matheson2, E. Tait Wojno2 specifically in FRC causes defective protective immunity 1Department of Immunology, Uinversity of Washington, against N, and reduced C. rodentium-specific antibody in 2Department of Immunology, University of Washington, both serum and feces, along with higher C. rodentium re- Seattle, United States infection. On the other hand, previous IL-17 dependent activation of gut-draining LN FRC by inducing mild colitis Abstract Content: Type 2 inflammation is characterized by with dextran sodium sulfate enhanced anti-Citrobacter production of the cytokines IL-4, IL-5 and IL-13 and antibody production, leading to quicker C. rodentium promotes clearance of gastrointestinal helminths, which clearance and less colon inflammation. In conclusion, these infect over 2 billion people worldwide. Basophils are innate data demonstrate an important mechanism for IL-17 in immune cells that accumulate in the intestine during mucosal immunity, acting indirectly through LN stromal cell infection with the helminth Trichuris muris, and are potent activation to promote protective antibody responses. producers of inflammatory mediators. However, the Disclosure of Interest: None Declared molecular mechanisms that control basophil function and gene expression during helminth-induced type 2 LT227 inflammation have been unclear. We previously showed that MODULATING IFNG PRODUCTION AS A NEW during T. muris infection, basophils upregulated the Notch EFFECTIVE WAY IN COMBATING RECURRENT UTI signaling pathway, which regulates gene expression J. Wu1,*, B. W. Hayes2, C. Bao2, S. N. Abraham1,2 programs during development and inflammation. 1Immunology, 2Pathology, Duke University, Durham, United Transcriptional profiling of Notch-deficient basophils States revealed that Notch directs basophil responsiveness to inflammatory cues and effector gene expression. In vivo, Abstract Content: basophils displayed decreased interaction with CD4+ cells in Urinary tract infection (UTI) is one of the most common the cecum. Consequently, mice lacking basophil-intrinsic bacteria infections, mostly initiated by uropathogenic E. Notch signaling had reduced Gata3+ T helper 2 cells in the coli (UPEC) invading bladder. UTI is mainly affecting intestinal site and decreased type 2 inflammation women, young children and aging population, and it was following T. muris infection, resulting in impaired worm estimated that about 50% of women will get at least one UTI clearance. We are now investigating the role of productive during lifetime. Besides its high incidence rate, high basophil-T cell interactions in driving fulminant type 2 recurrence rate is another major problem of UTI. Various immunity in the intestine. These studies highlight that clinical research showed that 27% to 44% of UTI patients inhibition of Notch signaling in basophils inhibits infection- will have UTI recurrence. Actually, a previous history of UTI induced gene expression changes in CD4+ T cells, while in is widely accepted as a strong risk factor for another UTI in vitro coculture experiments demonstrate that basophils clinics. This is striking, considering that bacteria infections in drive Th2 responses in CD4+ T cells. These findings other body sites such as lung and gut have relatively low broaden our understanding of type 2 immunity, providing recurrence rate, as adaptive immunity activated during the important insights for development of effective therapeutics initial infection should establish memory preventing aimed at this arm of host defense. infections of the same pathogen. Due to incomplete Disclosure of Interest: None Declared understanding of the adaptive immunity of bladder, no effective treatment is available in preventing recurrent UTI. 110 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

To get a better understanding of bladder adaptive immunity Virology and Department of Medical Microbiology and and to develop an effective way preventing recurrent UTI, Immunology, University of Alberta, Edmonton, Alberta, we studied CD4 T cells, a pivotal regulator of adaptive Canada, 6Pritzker School of Molecular Engineering and immunity, in bladder. Previous studies on bladder CD4 T Department of Biochemistry and Molecular Biology, cells failed to determine their function in bladder due to lack University of Chicago, Chicago, United States, 7Division of of proper tools. With newly developed tools, such as Cellular Polarity and Viral Infection, German Cancer cytokine reporter mice, several knock-out mice and bladder Research Center (DKFZ), Heidelberg, Germany transplantation technique, we are able to study the dynamics of cytokine secretion from bladder CD4 T cells and Abstract Content: Severe acute respiratory syndrome determine their function. We found that both IFNg secreting coronavirus-2 (SARS-CoV-2), which causes COVID- Th1 cells and IL4 secreting Th2 cells are activated during 19, utilizes angiotensin-converting enzyme 2 (ACE2) for UTI, and their activation is potentially modulated by bladder entry into target cells. ACE2 has been proposed as an microbiota. However, the percentage of IL4 secreting Th2 interferon-stimulated gene (ISG). Thus, IFN- cells is significantly higher than the percentage of driven variability in ACE2 expression levels or functionality IFNg secreting Th1 cells. Those bladder Th2 cells are could be important for susceptibility to COVID-19 and its induced to repair bladder epithelium damaged during UTI, outcomes. Here, we report the discovery of a novel, N- but they are inhibiting bacterial clearance. The Th2 biased terminally truncated primate-specific isoform of ACE2, adaptive immunity was further reinforced during the second designated as deltaACE2 (dACE2), which we demonstrate and third infections. Consequently, the bladder obtains an is an ISG but not ACE2. In The Cancer Genome Atlas abnormal structure due to overwhelmed IL4 mediated (TCGA), dACE2 expression was enriched in squamous epithelium repair and an inhibited bacterial clearance epithelial tumors of the respiratory, upper gastrointestinal, capability due to reduced IFNg secretion. We further and urogenital tracts. In vitro, dACE2, which lacks 356 aa of identified that these cytokine secreting activities were the peptidase domain, neither interacted with the SARS- modulated by a novel bladder specific OX40L+CD301b+ CoV-2 spike protein nor affected binding between the spike dendritic cell. Knowing CD4 T cells didn’t contribute to protein and ACE2. In contrast with ACE2, the bladder clearance during recurrent UTI because of inhibited carboxypeptidase activity of dACE2 was undetectable. The IFNg secretion, we explored whether we can enhance the existence of two functionally distinct ACE2 isoforms IFNg production from CD4 T cells by immunization to reconciles several functional properties previously attributed combat recurrent UTI. By using UPEC antigens and different to ACE2, with dACE2 being an ISG, and ACE2 being the adjuvants prone to induce Th1 cells such as CpG and IL-12, SARS-CoV-2 entry receptor and carboxypeptidase. We we successfully increased IFNgproduction in bladder and propose that the ISG-type induction of dACE2 is unlikely to enhanced bacterial clearance during recurrent UTIs. affect the cellular entry of SARS-CoV-2 and would not Mechanism study on CpG induced IFNg mediated bacterial promote infection. Our results warrant further studies on clearance found out that it is not dependent on antibody other functions of dACE2 and their potential relevance for production. Considering that no effective UTI vaccine is COVID-19. available now and most of the attempts are focusing on Disclosure of Interest: None Declared inducing high antibody production, inducing local IFNg secretion by CD4 T cells is a new effective method for LT232 combating UPEC in patients with one or recurrent UTIs. SARS-COV-2 RECEPTOR ACE2 IS AN INTERFERON- Disclosure of Interest: None Declared STIMULATED GENE: IMPLICATIONS FOR CYTOKINE REGULATION OF DIVERGENT HOST OUTCOMES Lightning Talk Session 3: SARS-CoV-2 or DURING COVID-19 C. G. K. Ziegler1, V. N. Miao1, A. W. Navia1, S. K. Nyquist1, COVID-19 2 3 3 4 LT228 Y. Tang , F. Taliaferro , S. W. Kazer , H. R. Laird , A. Owings4, M. Senitko4, T. O. Robinson4, S. C. Glover4, B. H. ΔACE2: A NOVEL PRIMATE-SPECIFIC INTERFERON- 2 1 3,* STIMULATED GENE AND ITS POTENTIAL ROLE IN Horwitz , A. K. Shalek , J. Ordovas-Montanes on behalf of HCA Lung Biological Network COVID-19 1 A. R. Banday1,*, O. Onabajo1, W. Yan1, A. Obajemu1, M. Institute for Medical Engineering and Science (IMES), MIT; Stanifer2, D. M. Santer3, O. Florez-Vargas1, H. Piontkivska4, Department of Chemistry, MIT; Koch Institute for Integrative D. L. J. Tyrrell5, J. Mendoza 6, S. Boulant7, L. Prokunina- Cancer Research, MIT; Ragon Institute of MGH, MIT and 1 Harvard; Broad Institute of MIT and Harvard, Cambridge, Olsson 2 1Laboratory of Translational Genomics, Division of Cancer MA, USA, Cambridge, Division of Gastroenterology, Boston Children’s Hospital, Department of Pathology, Brigham and Epidemiology and Genetics , National Cancer Institute, 3 National Institutes of Health, Bethesda, United States, Women’s Hospital, Boston, MA, USA , Division of 2Department of Infectious Disease, Molecular Virology, Gastroenterology, Boston Children’s Hospital; Program in University Hospital Heidelberg, Heidelberg, Germany, 3Li Ka Immunology, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard; Harvard Stem Cell Shing Institute of Virology and Department of Medical 4 Microbiology and Immunology, University of Alberta, Institute, Cambridge, MA, USA, Boston, Department of Edmonton, Alberta, Canada, 4Department of Biological Medicine, University of Mississippi Medical Center, Jackson, Sciences and Brain Health Research Institute, Kent State MS, USA, Jackson, United States University, Kent, United States, 5Li Ka Shing Institute of 111 Cytokines 2020 Virtual Meeting – Abstract Book November 2020

Abstract Content: There is pressing urgency to understand targeting TRAF3, a critical switch point for the induction of the pathogenesis of the severe acute respiratory syndrome IFN-I by TNF receptors, innate sensors (TLRs, RLRs) and coronavirus clade 2 (SARS-CoV-2) which causes the inflammatory cytokines (IL1β, IL18). Viral inactivation of disease COVID-19. SARS-CoV-2 spike (S)-protein binds TRAF3 can limit IFN-I secretion, leading to dysregulated ACE2, and in concert with host proteases, principally cytokine production and a hyper-inflammatory state. Using TMPRSS2, promotes cellular entry. The cell subsets a multiomic screen we discovered a novel set of antiviral targeted by SARS-CoV-2 in host tissues and how local proteins within the LTβR-TRAF3 pathway that are potentially cytokine activity influences divergent host outcomes, remain involved in controlling LTβR-TRAF3->IFN-I response unknown. Importantly, the COVID-19 pandemic has (Virgen-Slane et al., 2020 J. Immunol). At least two disproportionately affected Black, Indigenous, and Latinx conserved SARS-CoV-2 proteins (Nsp3/PLpro and Nsp9) communities which are significantly underrepresented in target the novel components in the TRAF3 interactome. biomedical research. Here, we leverage pre-COVID-19 Nsp3/PLpro deubiquitinates TRAF3 suppressing activation human, non-human primate, and mouse single-cell RNA- of NIK and IFN-I. Nsp9 targets DCAF7 (DDB1 and CUL4 E3 sequencing (scRNA-seq) datasets across health and Ub ligase associated factor 7) that forms a complex with disease to uncover the putative targets of SARS-CoV-2 DYRK1A (Dual-specificity tyrosine phosphorylation amongst tissue-resident cell subsets and guide our ongoing regulated kinase 1A). DYRK1A has been shown to be collection and analysis of COVID-19 samples. We identify involved in the development of B and T cells, however ACE2 and TMPRSS2 co-expressing cells within lung type II specific pathway contribution and the functional role remain pneumocytes, ileal absorptive enterocytes, and nasal goblet elusive. We show that DYRK1A specifically binds to TRAF3 secretory cells. Strikingly, we discover that ACE2 is a human protein and regulates the stability of TRAF3. In the absence interferon-stimulated gene (ISG) in vitro using airway of Dyrk1A, Traf3 protein is abnormally degraded and epithelial cells, and extend our findings to in vivo viral induces the survival of germinal center B cells. Further, infections. Our data suggest that SARS-CoV-2 could exploit Dyrk1A-deficient B cells have higher non-canonical NF-κB species-specific interferon-driven upregulation of ACE2, a signaling. Together, we show a crucial regulatory role for tissue-protective mediator during lung injury, to enhance DYRK1A in TRAF3 and TNFR-mediated signaling infection. pathways. These pathways may play important roles during In ongoing work, our group is using scRNA-seq to the infection with SARS-CoV-2. characterize the direct cellular targets of viral infection, and Disclosure of Interest: None Declared bystander responding cells, from the nasal mucosa of a predominantly Black cohort (enrollment n=15 non-infected LT234 controls and n=60 SARS-CoV-2+) of COVID-19 patients. COVID19 AND ENDOCRINOLOGY We have successfully started to profile this cohort, P. Rapti1,*, T. Palouka 1 illustrating technical feasibility, and will report on all of our 1Endocrinology, Endocrinologist, Athens, Greece collected data through November 1st, as well as making all Abstract Content: The unexpected appearance of the viral cell x gene matrices publicly available on a rolling basis infection COVID-19, with tremendous global consequences, before and beyond Cytokines 2020. We hypothesize that the creates thoughts and concerns to doctors and researchers, intrinsic response of ACE2+TMPRSS2+ goblet secretory regarding the possible impacts on other systems of the cells to infection, as well as their interactions with innate and organism.Various researches, show that an interesting adaptive immune cells in the nasal mucosa, play a crucial correlation seems to exist between the COVID-19 infection role in limiting COVID-19 disease severity. Identifying and particular endocrine and metabolic pathways. Those whether and how upper airway host-virus interactions pathways are related to the existence of the ACE2 receptor, specify divergent outcomes is essential to understand Additionally, a correlation seems to exist between COVID- SARS-CoV-2 pathogenesis and identify treatments and 19 and production of proinflammatory factors or cytokines. vaccine strategies uniquely suited to individuals suffering We know that cytokines and especially Interleukin 6 from COVID-19. (pleiotropic), are produced in various endocrine systems Disclosure of Interest: None Declared and glands throughout the organism, as well as in other organs. A further examination of those endocrine and metabolic LT233 pathways might be important in understanding, dealing and THE KINASE DYRK1A CONTROLS TRAF3 TURNOVER finding the disease severity markers, as well as in studying AND IS POTENTIALLY MODULATED BY SARS-COV-2 the genetic differences between the two sexes or between P. Ramezani-Rad1,*, J. J. Grist1, R. Virgen-Slane1, R. C. different populations. Rickert1, C. F. Ware1 the- 2 (ACE2 / the equivalent of the known ACE, which 1Sanford Burnham Prebys Medical Discovery Institute, La converts Ang I to Ang II) .Normally, ACE2 cleaves Jolla, United States Angiotensin II (Ang II) to Angiotensin (1-7) and consequently Abstract Content: Progression of SARS-CoV-2 infected converts Ang I to Ang (1-9). ACE2 receptors play a role in patients to life threatening disease may result from a virus- the pathogenesis of hypertension and diabetes mellitus, and mediated, dysregulated immune response associated with they are expressed in the epithelial cells of many organs. excessive production of inflammatory cytokines, cytokine Angiotensin II stimulates the release of the antidiuretic release syndrome. Coronaviruses are known to suppress hormone (ADH), the adrenocorticotropic hormone (ACTH), the innate, type I interferon (IFN-I, IFN⍺β) response by the prolactin, the luteinizing hormone (LH), the oxytocin, the 112 Cytokines 2020 Virtual Meeting – Abstract Book November 2020 aldosterone and others. However, it is important that drugs, scans. Yet, chronic stress-related neural activity and its such as diuretics, mineralocorticoids, glucocorticoids, underlying mechanism has not been explored in the context estrogens, oral contraceptives, adrenocorticotropic of SARS-CoV-2 disparities. In this study, 60 African hormone, sodium, potassium, as well as the body posture American individuals without SARS-CoV-2 were recruited to during blood collection, influence Angiotensin II levels. In the NIH Clinical Center (n=60 , 93% female, median COVID-19, the levels of Angiotensin II appear to increase. age=60.8, median BMI=33.0). Amygdala activity (AmygA) Ιf there is an involvement, the question is what is the was measured by standard uptake value of 18FDG-PET/CT importance of the neuroendocrine system and the endocrine and plasma cytokines (IFNγ, TNFα) were measured using glands, (e.g. thyroid, adrenal, hypothalamus, pituitary, fatty an ELISA-based multiplex technique. Coronavirus mortality tissue of the pancreas), and hormones, (such as prolactin, risk scores (CMRS), a score indicative of potential infection- estrogens, testosterone, pituitary hormones, thyroxin, related death if the participants were to contract SARS- insulin, various prohormones, etc.), and the genital systems COV-2, were calculated based on U.S. available data. of the two sexes, (ovaries, testes), as well as the Linear regression modeling was performed to identify involvement in the pathophysiology of various diseases (e.g. associations between each individual’s amygdala activity, metabolic syndrome, type 2 diabetes mellitus, insulin cytokine serum level, and CMRS while adjusting for body resistance, thyroid disorders, Addison, hypertension, mass index (BMI). Increasing AmygA was associated with obesity, etc.) lThe Hypothalamic – Pituitary – Adrenal axis is increasing TNFα and IFNγ (ß=0.43, p=<0.01 and ß=0.31, a particularly intricate complex, with their interaction as p=0.02, respectively). In addition, IFNγ associated with feedback. This axis and the interaction of the three, CMRS (ß=0.39, p=<0.01 ) but TNFα only trended towards compose the neuroendocrine system, defining and significance (ß=0.24, p=0.06). After adjusting for both regulating the responses of many biological procedures of neighborhood- and individual-level SES, the association the organism, including stress mechanism, metabolism, with CMRS holds true for IFNγ (ß=0.32, p=0.04), but is defensive system, affect, emotions, sexuality and energy eliminated in TNFα (ß=0.22, p=0.16), suggesting that the storage. association between TNFα and CMRS is dependent on The Hypothalamic – Pituitary – Adrenal (HPA) axis, the SES. Structural equation modeling identified cytokine Hypothalamic – Pituitary – Gonadal (HPG) axis, the mediators between AmygA and CMRS. IFNγ accounted for Hypothalamic – Pituitary – Thyroid (HPT) axis and the 52.6% of the AmygA-CMRS relationship, suggesting that Hypothalamic – Neurohypophyseal system, form the four IFNγ may be an important mediator between chronic stress- neuroendocrine systems, through which the hypothalamus related neural activity and SARS-CoV-2 mortality risk. We and the pituitary, guide and perform the neuroendocrine therefore present that chronic stress-related neural activity functions. may relate to SARS-CoV-2 mortality risk by way of Hormones Potentially, these neuroendocrine axes are increased IFNγ levels in circulation in an African American, involved, through the cytokines and other proinflammatory community-based cohort. Interestingly, IFNγ overproduction factors, and play an important role in the pathophysiology by immune cells has recently been connected with SARS- and the development of a hyperreaction of the immune CoV-2-related death. The mechanism linking circulatory system and the evolution of the disease, triggered by IFNγ levels and SARS-CoV-2 mortality remains to be coronavirus infection, and this should be further elucidated, but our data show that IFNγ may potentially be investigated. used as an early biomarker to identify individuals at greater Disclosure of Interest: None Declared risk for SARS-CoV-2 mortality, especially in the most vulnerable populations. Identifying these individuals is LT236 crucial to allow for earlier therapeutic intervention to INTERFERON GAMMA MAY BE A CRITICAL TARGET decrease SARS-COV-2 mortality in disadvantaged FOR CHRONIC STRESS-ASSOCIATED SARS-COV-2 populations. DISPARITIES Disclosure of Interest: None Declared B. Turner1,*, Y. Baumer1, C. A. Guiterrez-Huerta1, A. Dey2, M. Playford2, R. Islam1, V. M. Mitchell1, B. S. Collins1, N. N. Mehta2, T. M. Powell-Wiley1 1Social Determinants of Obesity and Cardiovascular Risk, 2Inflammation and Cardiometabolic Diseases Laboratory, National Institutes of Health, Bethesda, United States

Abstract Content: COVID-19, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global pandemic that has exacerbated existing cardiometabolic disease disparities in the US and across the globe. SARS-CoV-2 mortality has been seen to disproportionately affect individuals who live in lower resourced neighborhoods, are of lower socioeconomic status (SES), and are of minority background; these factors are associated with increased chronic stress-related neural activity measurable by amygdala activity in 18FDG-PET/CT 113