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Oral Presentations

Oral Presentations

Cytokine 87 (2016) 46–57 Contents lists available at ScienceDirect Cytokine journal homepage: Oral Presentations...

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Cytokine 87 (2016) 46–57

Contents lists available at ScienceDirect

Cytokine journal homepage:

Oral Presentations

O001 DISCOVERY OF A NOVEL TRANSCRIPTIONAL REGULATOR OF SUSTAINING TLR-MEDIATED INFLAMMATORY RESPONSES B. Lin1,*, B. Dutta1, K.-S. Oh1, I. Fraser1 1 Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institues of Health, Bethesda, United States Introduction: Inflammatory genes induced by Toll-like receptor (TLR) activation show varying induction kinetics, with some genes strongly induced early after activation (like Nfkbiz), while others (like Il6) show delayed induction that is sustained for several hours [1]. This phenomenon is unexpected, considering the fact that many of the induced genes share common activating transcription factors such as NF-kB and AP-1 [2]. Methods: To investigate the underlying mechanisms, we carried out an RNAi screen using IL-6 as output, aiming to identify regulators involved in sustaining TLR-activated inflammatory gene induction. Results: A novel regulator (Hit4) was identified and found to be specifically required to sustain the late inflammatory genes induced by TLR activation. RNAi knockdown of Hit4 in mouse bone-marrow derived macrophages led to decreased expression of the secondary response genes Il6, Edn1 and Lcn2, and the late primary response genes Il1a, Il1b, and Csf2, but did not affect induction of the primary response genes Nfkbiz, Egr1, and Egr2. Most interestingly, Hit4 functioned specifically in sustaining rather than initiating the transcription, as Hit4 showed no effect on the early mRNA transcription before 2 h. We also find that Hit4 had no effect on interferon response genes, either induced by Lipid A (TLR4) or poly(I:C) (TLR3). In contrast, late inflammatory response genes induced by R848 (TLR7), Pam3CSK4 (TLR2), and Lipid A (TLR4) were all enhanced by Hit4. These data suggest that Hit4 is primarily involved in the regulation of MyD88- but not TRIF-mediated gene induction. Further mechanistic investigation revealed that Hit4 functioned at the transcriptional level rather than post-transcriptional level, as Hit4 enhanced the level of nascent transcripts at later time points. This was further supported by the finding that this protein was required for recruitment of RNA pol II to the regulated gene loci at these times. Moreover, Hit4 KO RAW264.7 macrophage cells failed to discriminate persistent and transient LPS stimulation. The KO cells failed to 1043-4666/Ó 2016 Published by Elsevier Ltd.

sustain the response by persistent LPS stimulation, which led to only low level of response similar to that in transient LPS stimulation. This suggests a role for Hit4 in discriminating brief spurious pulses of infectious signal from a more sustained real infection signal. Conclusion: In summary, we have identified a novel transcriptional regulator functioning to sustain the transcription of late inflammatory genes. This protein could provide important new insight to the transcriptional mechanisms involved in sustaining the inflammatory response to persistent microbial signals. (Supported by the Intramural Research Program of NIAID, NIH) References [1] M. Rabani, R. Raychowdhury, M. Jovanovic, M. Rooney, D.J. Stumpo, A. Pauli, N. Hacohen, A.F. Schier, P.J. Blackshear, N. Friedman, I. Amit, A. Regev, High-resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies, Cell 159(7) (2014) 1698–1710. [2] O. Takeuchi, S. Akira, Pattern recognition receptors and inflammation, Cell 140(6) (2010) 805–820. Disclosure of Interest: B. Lin Grant/research support from: Intramural Research Program of NIAID, NIH, B. Dutta Grant/research support from: Intramural Research Program of NIAID, NIH, K.-S. Oh Grant/research support from: Intramural Research Program of NIAID, NIH, I. Fraser Grant/research support from: Intramural Research Program of NIAID, NIH.

O002 TRANSGENE EXPRESSION OF INTERLEUKIN-37 IN IL-10KO MICE INHIBITS COLON CARCINOGENSIS A. Ringleb1, R. Schwaiger1, S. Mountford1, D. Mayr2, C.A. Dinarello3, P. Bufler1,* 1 Pediatric Gastroenterology and Hepatology, Dr. von Hauner Children’s Hospital Ludwig-Maximilians-University Munich, Germany; 2Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany; 3 Div. Infectious Diseases, University of Colorado Denver, Aurora, United States Introduction: Inflammatory bowel disease is associated with an increased risk of developing colon cancer. Interleukin (IL-) 37 is a fundamental inhibitor of innate immunity by reducing systemic and local inflammation. IL-37 exhibits intra- and extracellular functionality and we recently identified the IL-37 receptor composed of IL-18 receptor 1 and single Ig IL-1R-related molecule (SIGIRR). We showed that IL-37 reduces the LPS- or IL-1b-induced inflammatory response

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of immune cells and protects transgene mice against LPS-induced sepsis as well as in a model of acute colitis. IL-37 protein is expressed in healthy and diseased human bowel tissue and shows a similar expression pattern as IL-18. In the present study, we tested whether transgene IL-37 expression protects colon carcinogenesis secondary to chronic colitis in IL-10ko mice. Methods: IL-37tg mice (C57Bl6) were crossbred with IL-10ko (C57Bl6) mice. Homozygous IL-10ko/IL-37tg mice were selected by genotyping. At 6 weeks of age mild colitis was induced by 2% dextran sulphate sodium (DSS) in drinking water for 5 days (IL-10ko n = 10, IL-10ko/IL-37tg n = 5). Subsequently, cyclooxygenase 2 inhibitor celecoxib (500 ìg/mouse) was applied by gastric gavage on day 7, 10 and 13 to trigger colon carcinogenesis as described (Glauben et al. Gut 2008). Mice were sacrified on day 171. Endpoints were clinical parameters as weight, rectal bleeding, stool consistency, colon length as well as cytokine response in LPS-stimulated whole blood and explanted colon cultures. Cytokine measurements were performed by Elisa (IL-6) and Bioplex assay (IL-1b, IL-10, IL-17, IFNc, TNFa). Colon inflammation, number of adenoma/carcinoma and colitis-associated liver inflammation were analyzed by histology (HE staining). Results: During the DSS-induction phase IL-10ko and IL-10ko/IL37tg mice had a similar weight loss due to mild acute colitis. From day 115 there was a significantly improved weight gain in IL-10ko/ IL-37tg mice. Colon length was similar in both groups. Whole blood assays showed similar basal cytokine levels. However, after LPS response, IL-10ko/IL-37tg compared to IL-10ko mice released less IL6 (p = 0.03), IL-1b (p = 0.03), IFNc (p < 0.0001), TNFa (p < 0.003), but more IL-10 (p < 0.0001) in supernatants of ex vivo stimulated whole blood. Ex vivo colon cultures showed a trend towards lower levels of pro-inflammatory cytokine production in IL-10ko/IL-37tg mice. Hemoglobin levels were higher in IL-10ko/IL-37tg mice (p = 0.013). All IL-10ko mice developed colon adenoma and carcinoma (5–10 adenoma/carcinoma per mouse). No adenoma or carcinoma were detected in the colon of IL-10ko/IL-37tg mice. Colitis-induced liver inflammation was markedly less in IL-10ko/IL37tg mice. Detailed histological and immunohistochemistical analysis will be presented. Conclusion: In conclusion, IL-37 transgene expression protects IL10ko mice from colon carcinogenesis secondary to chronic colitis. Reduced pro-inflammatory immune responses and higher levels of anti-inflammatory IL-10 by IL-37 overexpression are central protective mechanisms. It remains unclear whether IL-37 has direct tumor suppressing properties. Disclosure of Interest: None declared.

O003 EFFECTS OF MYCOBIOTA DYSBIOSIS DURING INTESTINAL INFLAMMATION I. Leonardi1,*, A.S. Bar1, I. Iliev1 1 Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York City, United States Introduction: Although a complex community of fungi (so called mycobiota) resides in the gastrointestinal tract of various mammals, its contribution to the maintenance of intestinal homeostasis is poorly understood. This study examines how fungi can modulate intestinal inflammation. Methods: We used a custom-developed database for the fungal ‘‘internal transcribed spacer” (ITS1) and a sequencing-based analysis


to characterize the mycobiota of C57Bl6 mice along the digestive tract. The abundance of selected fungal species was validated by qPCR. Profiling of the bacterial microbiota was performed by 16S sequencing. The fungal and bacterial community were analyzed for composition, diversity (Shannon and Inverse Simpson indexes) using the phyloseq package in R. Beta diversity was calculated with the Bray–Curtis index using the vegan package. LDA Effect Size (LEfSe) was used to identify genera characterizing the different regions. The generalized linear models implemented in MaAsLin was used to find associations between the topology of the sample and microbiota composition. Network analysis was performed to detect co-occurrence and mutual exclusion at the genus level. Colitis was induced by oral administration of DSS (in the drinking water for 7 days). The development of colitis was monitored by the weight loss and mortality rate. Immunophenotyping of lamina propria (LP) and mesenteric lymph node (mLN) and histology were performed at the end day (3 days after the withdrawal of DSS). Results: We show that in contrast to bacterial community, where colonic and caecal bacteria microbiota clustered apart from other region, fungal communities clustered closely to one another. Interestingly, analysis at the genus levels revealed differences in the relative abundance among different regions, with the abundance of Candida spp. being higher in the murine colon. LefSE analysis of the murine esophagus and colon, showed that OTUs belonging to the Candida and Saccharomyces genus were significantly more abundant in the latter whereas Nectariaceae and Sordariomycetes were decreased. We therefore explored the effect of Candida spp. and Saccharomycopsis fibuligera on mucosal immunity and inflammation in the colon. Our results show that, in the colon, administration of some fungal species, such as Candida, exacerbate the outcome of DSS colitis leading to a more pronounced weight loss and increased mortality rates. In contrast, other species, such as S. fibuligera, exert a protective effect. Conclusion: Our results show that the composition of the intestinal mycobiota varies across the length of the intestine, with the Candida genus being more prevalent in the lower gastrointestinal tract. Our data suggest that specific fungi might be associated with site specific immune responses in the colon and might promote inflammatory conditions as a result of aberrant immunity to fungi. Disclosure of Interest: None declared. O004 MICROBIOME-DEPENDENT MODULATION OF MUCOSAL IMMUNITY AT THE OCULAR SURFACE A.J.St. Leger1,*, J. Desai2, R. Drummond2, F. Almaghrabi1, P. Silver1, M. Lionakis2, R. Caspi1 1 Laboratory of Immunology, National Eye Institute, NIH, United States; 2 Fungal Pathogenesis Unit, NIAID, NIH, Bethesda, United States Introduction: Mucosal sites that interface with the environment and provide barrier function include the intestine, nasopharynx, lung, female reproductive tract and the ocular surface. Disruption of immune homeostasis at the ocular surface is associated with discomfort, inflammation and potential loss of vision. Immune cells are present within the conjunctiva and can be affected by environmental factors, potentially including microorganisms. However, proof that a resident ocular microbiome exists and influences local immunity has been elusive. We used a mouse model of ocular surface disease to study whether commensal microbes are present in ocular mucosa and modulate immunity.


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Methods: Mice were either treated with PBS, topical antibiotics, or were ocularly inoculated with a Corynebacterium sp. that we show influences the immune signature within the conjunctiva. Tears were assessed for anti-microbial components and functionality. Conjunctivae were isolated and assessed for neutrophilic infiltration and IL17 production. We used an ocular model of Candida albicans to assess the functional implications of commensal bacteria colonization at the ocular surface. Results: We found that IL-17 is constitutively produced within the conjunctiva-associated lymphoid tissue (CALT) and is necessary to recruit neutrophils to the ocular surface in the steady state and after a bacterial challenge. IL-17 sources in CALT include cd T cells, ab T cells and innate lymphoid cells (ILCs), in that order. Notably, a strain of Corynebacterium isolated from ocular tissue of mice, and known to also colonize the ocular surface of humans, induced the conjunctival cd T cells to secrete IL-17, which modified the local inflammatory signature. This interaction appears necessary to regulate local immunity at the ocular surface, since elimination of these bacteria by antibiotic treatment, or their introduction into non-colonized mice, correlated inversely with severity of an experimental Candida albicans infection. Conclusion: Our results show for the first time that a relationship exists between commensals and immune cells at the ocular surface, which is critical for maintenance of homeostasis and host defense within the ocular mucosa. Disclosure of Interest: None declared.

O005 LKB1 HAPLOINSUFFICIENCY IN T CELLS IS SUFFICIENT TO DRIVE THE DEVELOPMENT OF PEUTZ-JEGHERS SYNDROME M.C. Poenberger1,*, E. Ma1, B. Samborska1, S. Izreig1, J. Blagih1, G. Boily1, R.G. Jones1 1 Goodman Cancer Research Centre, McGill University, Montreal, Canada Introduction: Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by gastrointestinal (GI) polyps and a 93% cumulative risk of cancer development by the age of 65. PJS development has been linked with heterozygous expression of LKB1, a multi-faceted protein with roles in cellular proliferation, cell polarity and cellular metabolism. Methods: Mice heterozygous for LKB1 (LKB1+/-) aged to =52 weeks develop GI polyps similar to those found in PJS patients. While heterozygous expression of LKB1 has clearly been implicated in disease development, the mechanism by which LKB1 mutation leads to GI polyp and cancer development has not yet been fully elucidated. Our group has previously determined that LKB1 is involved in T cell development, activation and metabolism. To determine if LKB1 deletion in T cells plays a role in PJS development, we aged mice with homozygous or heterozygous deletion of LKB1 in T cells alone (LTko, LThet). Results: We have observed that aged LTko and LThet mice developed GI polyps histologically identical to those in LKB1+/- mice indicating that altered LKB1 expression in T cells alone is sufficient to drive the development of PJS. T cells isolated from LTko and LThet mice were further analyzed following anti-CD3/anti-CD28 activation to determine the effect of LKB1 loss on cytokine production. We observed that LKB1-/- and LKB1+/- CD4+ and CD8+ T cells express increased levels of a number of inflammatory cytokines including IFNc, TNFa, IL-17 and IL-6 upon activation. Furthermore, GI polyps and adjacent healthy tissue in aged LKB1+/- mice had increased mRNA expression of inflammatory cytokines as well as phosphorylated STAT3.

Conclusion: A causative link between immune dysfunction and tumor development has been observed in many cancer models. As our findings suggest that PJS may be another such disease, identification of LKB1 as a regulator of inflammation-induced cancer development could indicate a role for metabolic regulatory genes in other inflammation-associated cancers. References The liver kinase B1 is a central regulator of T cell development, activation, and metabolism. (2011) N.J. MacIver, J. Blagih, D.C. Saucillo, L. Tonelli, T. Griss, J.C. Rathmell, R.G. Jones. J Immunol. 187 (8):4187–4198. Disclosure of Interest: None declared.

O006 COMPLEX REGULATION OF TNF EXPRESSION CONTROLLED BY THE 3’ UNTRANSLATED REGION P. Bouillet1,*, D. Lacey1 1 Molecular Genetics of Cancer, The Walter and Eliza Hall Institute, Parkville, Australia Introduction: High levels of the pro-inflammatory cytokine tumour necrosis factor (TNF) have been associated with many diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), inflammatory bowel disease (IBD) and psoriasis. We have recently studied a spontaneous dominant mouse mutation that causes severe polyarthritis and heart valve disease in BPSM1 (Bone Phenotype Spontaneous mutant 1) mice. In these animals, very high Tnf overexpression is caused by the insertion of a retrotransposon in the 3’ untranslated region (UTR) of the Tnf gene (1). Methods: We have used a reporter system to study the posttranscriptional regulation of Tnf by a family of CCCH zinc fingercontaining proteins. On another hand, we have used the CRISPR/Cas9 technology to create mouse mutants that harbour subtle mutations in the 3’UTR of the Tnf gene. Results: The CCCH zinc finger-containing proteins Zfp36 and Roquin regulate TNF expression via the AU-rich elements (ARE) and the constitutive decay element (CDE) located in the 3’UTR, respectively. We have investigated the regulatory potential of 50 other CCCHcontaining ZPF on the 3’UTR of TNF and identified new regulators of TNF mRNA stability, as well as a new regulatory element (NRE) within the 3’UTR. The regulatory elements contained within TNF 3’UTR appear to act synergistically and in a cell type-specific fashion. We have engineered mutant mice lacking individual TNF regulatory elements (ARE, CDE or NRE), or combinations of these elements. Remarkably, all these mutants develop different diseases. Conclusion: Our results indicate that the regulation of TNF expression is mostly regulated at a post-transcriptional level, through a complex mechanism involving several regulatory elements and multiple proteins that control the degradation of TNF mRNA. References [1] Lacey D, et al. (2015) Spontaneous retrotransposon insertion into TNF 3’UTR causes heart valve disease and chronic polyarthritis. Proceedings of the National Academy of Sciences of the United States of America 112(31):9698–9703. Disclosure of Interest: None declared.

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these mutations consist of insertions containing cysteine into the juxtamembrane region, creating homodimers. These signal independently of IL-7 or the gamma-c chain of the IL-7 receptor, and constitutively activate Jak1.

D. Yu1,*, J. He2, X. Zhang2, Y. Wei3, X. Sun2, Y. Chen1, N. Shen4, E. Morand1, Z. Li2 1 Monash University, Clayton, Australia; 2Peking University People’s Hospital, Beijing, China; 3Shandong Academy of Sciences, Jinan, China; 4 Renji Hospital, Shanghai, China

Methods: Two approaches have been undertaken to target the IL-7R pathway in T-ALL: inhibition of Jak1, and development of monoclonal antibodies (MAbs) against IL-7R alpha.

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with multi-system involvement, and associated with an imbalance between effector and regulatory CD4+ T cells. The development and activation of regulatory and effect CD4+ T cell subsets are critically regulated by interleukin-2 (IL-2), but its production is impaired in SLE patients. We hypothesized that lowdose IL-2 can be used to treat SLE by selectively expanding regulatory CD4+ T (Treg) cells and suppressing follicular helper T (Tfh) cells and interleukin 17 (IL-17)-producing helper T (Th17) cells.

Results: Ruxolitinib is a recently FDA approved inhibitor of Jak1, and we show it is an effective inhibitor of cells driven by mutant IL7Ralpha in vitro and in immunodeficient mice. Two novel mouse MABs were developed that are directed against two different epitopes on human IL-7Ralpha, and they recognize both mutant and WT proteins. MAbs were chimerized with human IgG1 to optimize antibody-dependent cell mediated cytotoxicity (ADCC). These MAbs were highly effective in ADCC assays, mediating NK cell killing of T-ALL cells harboring mutant IL-7R alpha, and in xenografts of patient T-ALL cells with mutant or WT IL-7R.

Methods: Forty patients with active SLE were recruited; 38 patients completed the treatment with three cycles of recombinant human IL-2 (rhIL-2). In each cycle, 1 million IU rhIL-2 was administered subcutaneously every other day for 2 weeks, followed by a 2-week break. Both clinical and immunological responses were assessed. The primary end points were the SLE Responder Index (SRI) and safety at week 12. Secondary end points were the effects of the therapy on Treg, Tfh and Th17 cells. Results: An SRI response was seen in 34/38 patients (89.5%) at week 12. Resolution of clinical activity which had been present at baseline was observed in multiple manifestations, including rash (20/24 patients), alopecia (13/14), arthritis (10/11), fever (3/3), leukopenia (18/19) and thrombocytopenia (4/4) (all P < 0.05). No severe adverse events were observed. Laboratory parameters showed reductions of anti-dsDNA titres (p < 0.001) and proteinuria (p = 0.016), and increased levels of C3 (p = 0.028) and C4 (p = 0.001). Immunological analysis revealed that low-dose rhIL-2 administration was associated with selective expansion of Treg cells (p < 0.001) and conversely with reductions of Tfh and Th17 cells (p < 0.001), but not Th1 and Th2 cells. Conclusion: Low-dose IL-2 was effective and well tolerated in active SLE. The effect was associated with selective modulation of CD4+ T cell subsets. Disclosure of Interest: None declared.

Conclusion: These approaches are being developed as new therapeutics for acute lymphoblastic leukemia, most of use the IL-7R pathway either through normal ligand signaling or mutations in the pathway. Disclosure of Interest: None declared.

O009 INTERFERON-DRIVEN METABOLIC REPROGRAMMING OF HEPATOCYTES A. Bergthaler1,* 1 CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria Introduction: Tissue damage and inflammation caused by viral hepatitis is a major cause of morbidity and mortality worldwide. The liver is located at the interface of systemic metabolism and inflammation, with hepatocytes providing the stage for metabolic turnover and immunoregulation. A better understanding of the involved molecular crosstalk may open avenues to novel therapeutic strategies against infectious and inflammatory diseases. Methods: We used primary hepatocytes and mouse models of viral hepatitis to investigate the role of cytokines in metabolic-inflammatory crosstalk.

S. Durum1,*, J. hixon1, E. senkevitch1, S. cramer1, J. barata2, S. walsh3, W. li1 1 National Cancer Institute, Frederick, United States; 2University of Lisbon, Lisbon, Portugal; 3University of Maryland, College Park, United States

Results: This approach led to the identification of virus-induced early transcriptional changes in the redox pathways in the liver, including the downregulation of superoxide dismutase 1 (Sod1). Sod1-deficient mice exhibited increased inflammation and aggravated liver damage upon viral infection. Type I interferon (IFN-I) led to a downregulation of Sod1 and altered redox metabolism, and caused oxidative liver damage in Sod1-deficient and wild-type mice. Ablation of the IFN-I signaling pathway prevented the death of hepatocytes, delineating IFN-I mediated oxidative stress as a key mediator of virus-induced liver damage.

Introduction: Acute lymphoblastic leukemia, derived from immature T or B cells, is the most common cancer in children. Current chemotherapeutic regimen are effective in over 80% of T-ALL, but the failure rate and the harshness of current treatments makes new approaches desirable. We and others have recently identified gainof-function mutations in IL-7R alpha which serve as driver oncogenes in T cell acute lymphoblastic leukemia (T-ALL). Most of

Conclusion: This mechanism of innate immunity-driven pathology links IFN-I signaling with antioxidant host defense and infectionassociated tissue damage. Ongoing studies aim to extend these findings towards a comprehensive understanding of how cytokines contribute to metabolic reprogramming of hepatocytes. We thereby expect fundamental insights into the role of cytokines in systemic metabolism, inflammation and pathology.



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References Bhattacharya A, Hegazy AN, Deigendesch N, Kosack L, Cupovic J, Kandasamy RK, Hildebrandt A, Merkler D, Kühl AA, Vilagos B, Schliehe C, Panse I, Khamina K, Baazim H, Arnold I, Flatz L, Xu HC, Lang PA, Aderem A, Takaoka A, Superti-Furga G, Colinge J, Ludewig B, Löhning M, Bergthaler A. Superoxide Dismutase 1 Protects Hepatocytes from Type I Interferon-Driven Oxidative Damage. Immunity. 2015 Nov 17;43(5):974–986. doi: 10.1016/j.immuni.2015.10.013. Disclosure of Interest: None declared.

O010 IFNAR SIGNALING OF MYELOID CELLS MODULATES VIRAL HEPATITIS BUT NOT KUPFFER CELL REPLENISHMENT K. Borst1,*, T. Frenz1, J. Spanier1, P. Tegtmeyer1, C. Chhatbar1, J. Skerra1, S. Lienenklaus1, M. Köster2, S. Namineni3, M. Heikenwdlder4, G. Sutter5, U. Kalinke1 1 Experimental Infection Research, TWINCORE GMBH, Hannover, Germany; 2Department of Gene Regulation and Dierentiation, Helmholtz Center for Infection Research, Brunswick, Germany; 3Institute of Virology, Technical University Munich, Munich, Germany; 4Chronic Inflammation and Cancer, German Cancer Research Centre, Heidelberg, Germany; 5Institute for Infectious Diseases and Zoonoses, LudwigMaximilians University, Munich, Germany Introduction: Upon viral infection, type I interferons (IFN-I) are induced that play a crucial role for the induction of anti-viral immunity. During systemic infection most viral particles reach the liver where they are taken up by liver-resident macrophages (Kupffer cells, KC). KC have been shown to be important in order to control pathogenic insult within the liver, and on the other hand are essential in restoring injured liver tissue as well as stabilizing the immune-tolerogenic milieu. Vaccinia virus (VACV), a large DNAencoded poxvirus, encodes several IFN-I evasins, including the viral IFN-I receptor B18. Here we studied the significance of IFNARsignaling within the liver upon VACV infection for the induction of anti-viral immunity and myeloid cell function. Methods: In order to dissect type I IFN receptor dependent effects within the liver, mice with a hepatocyte specific IFNAR deletion (AlbCreIFNARfl/fl mice), or with a myeloid cell specific IFNAR deletion (LysM-CreIFNARfl/fl mice) were used. Histochemisty of infected livers was performed. For analysis of liver resident KC and infiltrating monocytes, leukocytes were isolated from liver and analysed by FACS. Results: In vivo imaging (IVIS) of IFN-b- and Mx2-luciferase reporter mice revealed that systemic VACV infection induces local IFN-b responses in secondary lymphoid organs, which are mainly sensed within the liver. VACV infected IFN-I receptor deficient (IFNAR-/-) mice develop fulminant disease: They show high virus load in the liver, aberrant cytokine responses, and succumb to the infection. Histological analysis indicated massive liver inflammation and infiltration of immune cells in the absence of IFNAR-signaling. This hepatitis was partially induced by the lack of IFNAR-signaling of myeloid cells (LysM-CreIFNARfl/fl mice). Furthermore, as analyzed by FACS, IFNAR-signaling was essential to mediate KC replenishment after infection induced deletion of this cell subset. By using CX3CR1GFP mice we could show that replenishment of the KC pool is mediated by liver-infiltrating monocytes, and not by proliferation of local cells. To analyze whether liver-infiltrating monocytes need direct IFNAR-signaling in order to develop into KC, we set up mixed bone marrow chimeras with WT and IFNAR-/- BM in WT mice and checked KC replenishment. Surprisingly, 4 dpi the replenished KC pool composed significantly more IFNAR-/- than WT cells, indicating that direct IFNAR-signaling of infiltrating monocytes is detrimental

for KC repopulation. In contrast, IFNAR-signaling of cells of the nonhematopoietic compartment was essential to control virus replication in the liver as well as replenishment of the KC pool. Interestingly, in Alb-CreIFNARflox/flox mice no liver inflammation was detected after VACV infection. Conclusion: Collectively these results indicate that although serum IFN-I is sequestered, VACV infection induces local IFN-I responses that play a central role in balancing cytokine responses to prevent liver damage during acute hepatitis. Moreover, local IFN-I responses within the liver critically contribute to protection from viral hepatitis, modulate KC replenishment after the infection, and thus assure survival. Disclosure of Interest: None declared.

O011 A ‘‘RHEOSTAT FUNCTION FOR THE GP130/STAT3 SIGNALING CASCADE FOR GASTROINTESTINAL WOUND-HEALING AND TUMORIGENESIS M. Ernst1,*, T. Putoczki2, M. Eissmann1, F. Masson1 1 Cancer and Inflammation, Olivia Newton-John Cancer Research Institute, Heidelberg (Melbourne), Australia; 2Inflammation, Walter and Eliza Hall Institute, Melbourne, Australia Introduction: The gastrointestinal epithelium undergoes continuous homeostatic renewal, which is controlled by tonic WNT signaling. However, the harsh and abrasive environment of the intestine requires an effective, but localized, epithelial wound healing response to avoid prolonged exposure of innate immune sentinels to intestinal microbes and associated expansion of neoplastically transformed epithelial (stem) cells. Methods: We use a comprehensive suite of mouse strains comprising an allelic series of gp130 signaling mutants, cell type-specific constitutive Stat3 activation or heterozygous Stat3 inactivation (Stat3flox/wt), as well as an inducible short hairpin (shStat3) allele, in combination with adoptive bone marrow transfers. We assess and molecularly characterize susceptibility of the resulting mice to acute and chronic DSS colitits as well as sporadic and colitis asscoaited colon, and gastric cancer. Results: We find that gp130/Stat3 signaling is largely dispesible for homeostatic renewal of the gastrointestinal mucosa, but limits the extent of WNT-driven wound-healing without affecting expression of WNT target genes. Likewise, the growth and progression of gastrointestinal tumors driven by mutations in componets of the canonical WNT cascade (i.e. Apc or beta-catenin), or of PIK3CA or KRAS/BRAF is determined by the extent of gp130/Stat3 signaling. Accordingly, the intestinal wound-healing response can be modified and the growth of primary tumors in these models can be suppressed by genetic restriction of interleukin (IL)-11 receptor-dependent signaling, gp130 or Stat3 expression. Importantly, these observations can be replicated by therapeutic induction of systemic short-hairpin Stat3 expression or administration of small molecular Jak inhibitors, most likely through an epithelial-cell intrinsic mechanism that does not impact homeostatic turnover of the gastrointestinal mucosa. Conclusion: Our observations suggest that neoplastic gastrointestinal cells exploit a mechanism whereby epithelial gp130/Stat3 signaling links the extent of the wound healing response of their non-transformed counterparts to the presence of inflammatory IL11 family cytokines. Disclosure of Interest: None declared.

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aggravates the risk of several extrahepatic diseases such as myocardial infarction, particularly in those with the inflammatory stage of steatohepatitis (NASH).

S. Gaen1,*, H. Conti2, E. Childs1, S. Sinha3, S. Filler4, V. Bruno5, J. Kolls6 1 University of Pittsburgh, Pittsburgh, United States; 2University of Toledo, Toledo, United States; 3SUNY Bualo, Bualo, United States; 4UCLA, Los Angeles, United States; 5University of Maryland, Baltimore, United States; 6Children’s Hospital of Pittsburgh, Pittsburgh, United States

Methods: Here, we investigated the impact of diet-induced NASH on the severity of imiquimod-induced psoriasiform dermatitis.

Introduction: Candida albicans is a commensal fungus responsible for opportunistic oropharyngeal candidiasis (OPC, thrush) in settings of immunodeficiency such as HIV/AIDS and chemotherapy. IL-17 is essential for protective immunity to Candida, as impairments in IL17 receptor (IL-17R) signaling components (IL-17RA, IL-17RC, Act1) cause mucosal candidiasis in mice and humans. The IL-17R complex is ubiquitously expressed, and antifungal signaling by IL-17 has been documented in cells of mesenchymal and epithelial origin but also in hematopoietic cells. In this study we sought to delimit the essential IL-17-responsive cells necessary to mediate protection from OPC. Methods: We used a murine OPC model to evaluate sensitivity of various mice to oral C. albicans infections. To test the hypothesis that OECs are the essential IL-17-responsive cell type in the context of OPC, we created and validated a new conditional knockout mouse in which CRE expression is driven by the murine Keratin-13 (Krt13, K13) proximal promoter (K13CRE). We performed RNA-Seq analyses of human OECs infected with C. albicans and treated with TNF and IL17. Data were compared to RNA-Seq profiles of WT versus either Il17ra-/- mice or Il17raK13Cre mice. Results: Reciprocal radiation chimeras in WT and Il17ra-/- mice ruled out a requirement for IL-17R signaling in hematopoietic cells. Expression profiling comparing C. albicans-infected cultured human oral epithelial cells (OECs) and tongue tissue from mice with OPC revealed remarkable similarities between gene pathways induced in both settings. Consistent with the known expression profile of Krt13, mice in which CRE was driven by the K13 promoter exhibited specificity for suprabasal layers of oral and esophageal epithelial tissue. K13CRE mice were crossed to Il17rafl/fl mice (Il17raK13Cre) and subjected to OPC. Il17raK13Cre mice exhibited fungal loads and disease symptoms comparable to Il17ra-/- mice, with similar impairments in OPC-associated gene expression in the oral mucosa. Microarray analyses suggested that OEC-dependent IL-17 signaling impairs antimicrobial peptides (AMP), particularly Defb3 (b defensin 3). Conclusion: Oral epithelial cells, not hematopoietic cells, dominantly control the IL-17-dependent antifungal immune response to OPC, with particularly potent effects on AMP expression. Disclosure of Interest: None declared.

O013 IL-17A EXACERBATES PSORIASIFORM DERMATITIS IN A MOUSE MODEL OF HIGH-FAT DIET-INDUCED STEATOHEPATITIS J.-C. Lecron1,*, P. Vasseur1,2, L. Serres3, J.-F. Jegou1, M. Pohin1, A. Delwail1, I. Petit-Paris1, P. Levillain3, L. Favot1, M. Samson4, H. Yssel5, F. Morel1,3, C. Silvain1 1 Université de Poitiers, LITEC, Poitiers, France; 2Centre Hospitalier Nord Deux-Sèvres, Bressuire, France; 3CHU de Poitiers, Poitiers, France; 4 IRSET, Inserm, U1085, Université de Rennes 1, Rennes, France; 5 Sorbonne Universités, UPMC Univ Paris 06, CIMI, Inserm U1135, Paris, France Introduction: Recent studies suggest that psoriasis may be more severe in patients with nonalcoholic fatty liver disease, known to

Results: Mice fed with a high-fat diet enriched in cholate developed steatohepatitis reminiscent of human NASH with ballooning hepatocytes and significant liver fibrosis. Mice with steatohepatitis also displayed moderate cutaneous inflammation characterized by erythema, dermal infiltrates of CD45+ leukocytes and a local production of interleukin (IL)-17A. In the cutaneous draining lymph nodes, these mice showed an expansion of CD3+ CD4+ TCRcd- IL17A+ Th17 lymphocytes. Moreover, steatohepatitis was associated with an epidermal activation of caspase-1 and cutaneous overexpression of IL-1b. Intradermal injection of rIL-1b in standard dietfed mice was able to recapitulate some features observed in mice with steatohepatitis: it induced skin erythema along with a cutaneous overexpression of CCL20, a Th17 lymphocyte-recruiting chemokine, and finally an overexpression of IL-17A. Imiquimodinduced psoriasiform dermatitis was exacerbated in mice with steatohepatitis as compared to animals fed with a standard diet. Scale formation and acanthosis (62 ± 9 lm vs 90 ± 10 lm, p < 0.05) were aggravated, in correlation with increased IL-17A and IL-22 expression in inflamed skins. Finally, intradermal injection of IL-17A in standard diet-fed mice recapitulated the cutaneous pathology of mice with steatohepatitis. Conclusion: The results show that high-fat diet-induced steatohepatitis aggravates the inflammation in psoriasiform dermatitis, via the cutaneous production of IL-17A. The epidermal activation of caspase-1 observed in mice with steatohepatitis might be an upstream event for IL-17A production in the skin of these mice. In agreement with clinical data, this description of a novel extrahepatic manifestation of NASH should sensitize dermatologists to the screening and the management of fatty liver in psoriatic patients. Disclosure of Interest: None declared.

O014 CARDIOTROPHIN-LIKE CYTOKINE (CLCF1) PARTICIPATES IN LIPOPROTEIN COMPLEXES S. Pasquin1,*, S. Chehboun1, A. Dejda2, M. Sharma3, P. Sapieha2, C. Martel4, J.-F. Gauchat1 1 Pharmacology, Canada; 2Biochemistry, Université de Montréal, Montreal, Canada; 3KCVA Medical Center, Kansas City, United States; 4 Medicine, Université de Montréal, Montreal, Canada Introduction: CLCF1 is a CNTFR ligand. CNTF regulates metabolism and has been tested in clinic for the treatment of metabolic diseases [1]. In mice, systemic treatment with CLCF1 led to significant weight loss [2]. Moreover, mutations in the gene coding for CLCF1 or its secretion partner CRLF1 lead to syndromes with characteristics such as strong episode of aseptic fever early in life, altered response to environmental temperatures and loss of interest in food indicative of a metabolic dysregulation [3,4]. Recent studies have identified CLCF1 as a ligand for sortilin and SorLA [5,6]. Sortilin is a receptor for apolipoprotein E (ApoE), a major component of lipid metabolism [7]. The N-terminus domain of ApoE, composed of 4 helix a-chains, shares structural similarities with CLCF1 and was shown to interact with CNTF [8]. The aim of this study was therefore to evaluate


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whether ApoE or ApoE-containing lipoproteins interact with CLCF1, regulates its activity and thereby contribute to its role in metabolism regulation. Methods: The interactions between CLCF1 and ApoE or CLCF1 and lipoproteins were investigated using co-immunoprecipitation, sizeexclusion chromatography, aLISA proximity assays and ligand blots. The properties and biological activities of these complexes were studied by binding and phosflow assays on Ba/F3 transfected cells and cell lines constitutively expressing CNTFR. The therapeutic effects of CLCF1 and CLCF1-VLDL complexes were compared in vivo in a mouse model of oxygen-induced retinopathy. Results: We observed that CLCF1 forms complexes with the three major forms of apolipoprotein E. FPLC analysis of lipoprotein complexes in mouse and human sera mixed with CLCF1 indicated that the cytokine can directly interact with plasma VLDL, LDL and HDL. Unexpectedly, studies with sera from ApoE-/- mice indicate that ApoE is not required for CLCF1-lipoprotein interaction. LipoproteinCLCF1 binding was confirmed using proximity assays and ligand blots. In vitro assays have demonstrated that CLCF1-induced STAT3 phosphorylation was significantly reduced when the cytokine was complexed with VLDL. In a mouse model of oxygen-induced retinopathy, beneficial effect of CLCF1 on retinal pathological neovascularisation and vaso-obliteration was prevented by coadministration with VLDL. Conclusion: CLCF1 when mixed with purified lipoproteins or lipoprotein containing physiological liquids such as serum binds lipoprotein complexes and appears to behave as a ‘‘lipocytokine”. This interaction modulates CLCF1 biological activity. It remains to be determined whether it directly contributes to metabolic regulation and its putative role in diseases such as focal segmental glomerulosclerosis (FSGS). References [1] S. Pasquin, M. Sharma, J.F. Gauchat. Cytokine & growth factor reviews 26, 507–515 (2015). [2] G. Senaldi et al., Proc Natl Acad Sci U S A 96, 11458–11463 (1999). [3] P.M. Knappskog et al., Am J Hum Genet 72, 375–383 (2003). [4] L. Crisponi et al., Am J Hum Genet 80, 971–981 (2007). [5] J.V. Larsen et al., Mol Cell Biol 30, 4175–4187 (2010). [6] J.V. Larsen et al., Mol Cell Biol 36, 1272–1286 (2016). [7] A.S. Carlo et al., J Neurosci 33, 358–370 (2013). [8] C.R. Gutman, W.J. Strittmatter, K.H. Weisgraber, W.D. Matthew. J Neurosci 17, 6114–6121 (1997). Disclosure of Interest: None declared.

O015 AN ESSENTIAL ROLE FOR EPITHELIAL CELL HISTONE DEACETYLASE 3 IN INTESTINAL HOST DEFENSE T. Alenghat1,* 1 Division of Immunobiology, Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, OH, United States Introduction: Mucosal tissues represent initial sites of infection for numerous pathogens, however the mechanisms regulating how epithelial cells lining mucosal surfaces direct protective inflammatory responses to infectious agents are not well understood. Here, we aim to determine whether epithelial expression of the epigenomicmodifying enzyme HDAC3 regulates pathways that are critical for innate immune responses and clearance of bacterial pathogens from the intestine.

Methods: C57Bl/6 mice expressing Cre-recombinase (VilCre) or tamoxifen-dependent Cre-recombinase (VilCre-ER) expressed under the control of the villin promoter were bred to floxed HDAC3FF mice to generate HDAC3DIEC and HDAC3DIEC-IND mice, respectively. For infections, we employed Citrobacter rodentium, a mouse pathogen that causes colitis similar to attaching and effacing Escherichia coli in humans. Groups of 4–6 male or female HDAC3FF and HDAC3DIEC mice were infected with C. rodentium by oral gavage, monitored regularly for bacterial burdens, and sacrificed on days 6 or 12 postinfection. Immunologic, pathologic, and microbiologic assays were performed. Results: We found an essential role for histone deacetylase 3 (HDAC3) in intestinal epithelial cells (IECs) in host defense against bacterial infection. Despite sharply increased pathogen burden, IFNc production by intraepithelial lymphocytes in mice lacking expression of IEC-intrinsic HDAC3 (HDAC3DIEC mice) was significantly impaired in response to Citrobacter rodentium. Reciprocally, stimulating IFNc in the colon of HDAC3DIEC mice reversed susceptibility to C. rodentium in HDAC3DIEC mice, suggesting that IEC-intrinsic regulation by HDAC3 is required for protective immune cell function in the intestine. C. rodentium induced early IEC expression of the IFNc-inducing interleukin (IL)-18 in wildtype mice, however induction of IL-18 did not occur in IECs of HDAC3DIEC mice. To eliminate contributions of circulating cells, we employed an ex vivo culture model and found that infection-induced production of IFNc requires IEC-intrinsic HDAC3 expression and that IL-18 could restore effector function to tissue-resident lymphocytes in HDAC3DIEC mice. Conclusion: These results highlight the complex interactions between IECs and resident lymphocytes, uncovering a novel level of regulation by which epithelial HDAC3 coordinates protective inflammatory responses to mucosal infection. Disclosure of Interest: None declared.

O016 INTERFERON-LAMBDA CONTROLS THE SPREAD OF RESPIRATORY VIRUSES FROM THE UPPER RESPIRATORY TRACT TO THE LUNGS AND RESTRICTS VIRUS TRANSMISSION IN MICE P. Staeheli1,*, J. Klinkhammer2, D. Schnepf2, L. Ye2, M. Schwaderlapp2, D. Garcin3, T. Malakoiv2 1 Virology, Medical Center University of Freiburg, Germany; 2University of Freiburg, Germany, Freiburg, Germany; 3University of Geneva, Geneva, Switzerland Introduction: Infection studies in which influenza viruses were administered preferentially to the lower respiratory tract of mice suggested that interferon-k (IFN-k) plays only a minor role in the defense against respiratory viruses. However, it remained unclear whether this experimental setting mimicked a physiological scenario. Methods: To solve this issue, we selectively applied viruses to the upper respiratory tract of mice to mimic a natural infection. Results: Under such experimental conditions, we found that multiplication of influenza and Sendai viruses was largely restricted to the nasal cavity in wild-type mice. By contrast, in mice lacking functional receptors for IFN-k, these viruses frequently spread to the lungs. Virus spreading to lungs was observed significantly more frequently in mice lacking functional IFN-k receptors than in mice lacking IFN-a/b receptors, indicating that IFN-k is of particular importance for virus restriction in the upper respiratory tract.

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Sendai virus and H3N2 influenza A viruses are readily transmitted among mice lacking functional receptors for IFN-a/b and IFN-k. We observed that mice selectively lacking functional IFN-k receptors also transmitted these viruses fairly efficiently to IFN-deficient cage mates. By contrast, virus transmission was observed only infrequently when wild-type mice or animals lacking functional IFNa/b receptors were used as virus spreaders.

O018 LOSS OF AUTOPHAGY DRIVES INFLAMMATION VIA TYPE I INTERFERONS M. Samie1,*, J. Baughman2, I. Peng2, S. Park2, N. Chai2, M. Xu2, B. Mckenzie2, D. Kirkpatrick2, M. van Lookeren Campagne2, A. Murthy1 1 Cancer Immunology, United States; 2Genentech, South San Francisco, United States

Conclusion: By using infection models that closely mimic natural transmission of respiratory viruses, we demonstrate a previously unrecognized crucial role of IFN-k in preventing virus transmission among mice. Our data indicate that IFN-k plays a more prominent role in antiviral defense of mucosal surfaces than IFN-a/b, suggesting that a subset of epithelial cells in the nose is blind for IFN-a/b and relies on IFN-k for antiviral defense.

Introduction: Autophagy is a conserved intracellular catabolic pathway responsible for maintaining homeostasis by degrading damaged organelles or dysfunctional proteins. Growing body of evidence has shown that defects in autophagy is associated with increased risk of chronic inflammatory diseases such as inflammatory bowel disease, arthritis and lupus. However, the mechanisms describing how autophagy suppresses inflammatory signaling remain poorly understood.

Disclosure of Interest: None declared.

O017 NEGATIVE REGULATION OF INNATE IMMUNE SIGNALING BY THE RNA SENSOR, LGP2 C. Horvath1,* 1 Dept. of Molecular Biosciences, Northwestern University, Evanston, IL, United States Introduction: The detection of RNA virus infections triggers the production type I interferon (IFN), and is a major component of the powerful antiviral system that directly interferes with virus replication and contributes to both innate and adaptive immune responses. Virus replication intermediates such as double stranded RNA (dsRNA) or RNAs with distinct non-self modifications are recognized in the cytoplasm by pattern recognition receptors including RIG-I, MDA5, and LGP2. These innate immune sensors recognize non-self RNAs via their C-terminal domains, and RIG-I and MDA5 use Nterminal CARD regions to mediate interactions that induce the activation of the downstream signaling scaffold, MAVS. MAVS coordinates the assembly of signaling machinery including TRAFs and IKK-family kinases that result in the activation of latent IRF3 and NFkB transcription factors. LGP2 lacks an N-terminal CARD region, and uses distinct mechanisms to act as both a positive regulator of MDA5 and a negative regulator of MDA5 and RIG-I. Currently available evidence suggests LGP2 acts as a both a positive and negative regulator of antiviral signaling. For positive actions, LGP2 uses its unique catalytic activity to enhance MDA5 RNA recognition, dsRNA filament assembly, and enable more efficient activation of MAVS and downstream signal transduction. Methods: The mechanism of LGP2 negative regulation has been investigated using molecular and biochemical methods. Results: Results indicate that LGP2-mediated inhibition occurs downstream of RIG-I, MDA5, and MAVS. LGP2 inhibition does not require either enzymatic activity or RNA recognition capacity. Instead, results indicate that LGP2 targets TRAF signaling proteins, disengaging their ubiquitin ligase activity, and potently preventing NFkB activation. Conclusion: Identification of TRAF proteins as inhibitory targets not only resolves the dual roles of LGP2 in antiviral signaling regulation, but also potentially expands the impact of LGP2 negative regulation to inflammatory signaling. Disclosure of Interest: None declared.

Methods: ELISA, Western blot, QPCR, Confocal Microscopy, Tissue culture, Histology, Humn subjects, Immunoprecipitation. Results: Here we show that defective autophagy leads to elevated production of Interferon-beta (IFNb) by macrophages, both transcriptionally and at the protein level. Myeloid-cell specific ATG16L1 deficiency in mice led to elevated levels of IFNb in the serum and promoted morbidity following S. typhimurium-induced colitis or endotoxin-induced sepsis. Pre-treatment of mice with an IFNAR1 blocking antibody protected the mice lacking ATG16L1 from LPSinduced sepsis. On the other hand, ATG16L1 deficient mice were protected against Influenza-A pulmonary infection, which correlated with elevated IFNb levels in lung tissue and lavage fluid. Finally, human monocyte derived macrophages carrying the Atg16L1 T300A risk allele showed increased IFNb production versus non-risk carriers when stimulated with TLR3 and TLR4 ligands. Conclusion: Our data illustrates that autophagy negatively regulates Type I IFN production and provides a mechanistic explanation of how genetic variants affecting autophagy can predispose to chronic inflammation. Disclosure of Interest: None declared.

O019 INTERFERON-INDUCIBLE PROTEINS ARE ESSENTIAL FOR THE ACTIVATION OF THE INFLAMMASOMES S.M. Man1,*, R. Karki1, T.-D. Kanneganti1 1 Immunology, St. Jude Children’s Research Hospital, Memphis, United States Introduction: Recognition of pathogens by the cell is paramount for the initiation of an appropriate immune response. A subset of pattern-recognition receptor family, including NLRP3, NLRC4 and AIM2, are cytosolic sensors which induce formation of the inflammasome, a multi-protein complex which drives the release of the pro-inflammatory cytokines IL-1beta and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes, however, the mechanism by which concealed ligands are liberated in the cytoplasm have remained unclear. Methods: We used biochemical, molecular and imaging approaches to examine the role of guanylate-binding proteins and other novel interferon-inducible GTPases in the activation of the inflammasome. Results: We found that guanylate-binding proteins and other novel interferon-inducible GTPases were required for activation of the


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DNA-sensing AIM2 inflammasome by Francisella novicida, and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria. Interferon-inducible GTPases targeted the bacteria in a sequential manner and recruited to the bacterial cell membrane to mediate bacteriolysis. Lysis of the bacteria in the host cytoplasm liberates DNA and LPS for sensing by AIM2 and caspase-11, respectively. Conclusion: Localization of interferon-inducible GTPases to the bacteria compromised their structural integrity and mediated cytosolic release of ligands for sensing by inflammasomes. Overall, our results reveal a specific mechanistic link between cell-autonomous immunity and innate immune sensing pathways in response to bacterial infection. Disclosure of Interest: None declared.

O020 THE LAB ENVIRONMENT RECAPITULATES PRIMARY HUMAN IMMUNE RESPONSES IN MICE W. Elsegeiny1, T. Eddens2, M. Zheng3, J. Kolls4,* Pediatrics, Childrens Hospital of Pittsburgh, United States; 2Children’s Hospital of Pittsburgh, United States; 3CHP, United States; 4Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, United States 1

Introduction: Despite the discovery of key pattern recognition receptors and CD4+ T-cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Recently for CD8+ T-cell immunity, co-housing of laboratory mice with antigen-experienced feral or store bought mice was able to recapitulate many aspects of adult CD8+ T-cell immunity in exposed lab mice. Whether this is true for CD4+ T-cell immunity is unclear. A hallmark infection of functional CD4+ T-cell immunity is resistance to Pneumocystis pneumonia. Indeed this infection was the first AIDS-defining illness and the opportunistic infection most closely tied with peripheral CD4+ T-cell counts. Due to successful control of HIV, the epidemiology of Pneumocystis infection in children is now primarily due to primary human immunodeficiency. Methods: We evaluated murine models of human primary deficiency in terms of susceptibility to primary Pneumocystis infection including mutations in Rag1, STAT4, STAT6, STAT3, RORC, and IL-21R. Results: To this end, we found that nearly every human genetic immunodeficiency that results in susceptibility in humans is faithfully replicated in SPF mice including the recently described IL-21 receptor deficiency. IL-21R was essential for the generation of protective class-switched anti-fungal antibody responses. Additionally, CD4+ T-cell intrinsic expression of IL-21R and the downstream STAT3 signaling pathway was required for the generation of antifungal effector responses. RNAseq analysis of purified CD4+ T-cells during the effector response showed that IL-21R was essential for IL22 expression. Treatment of IL-21R-/- mice with IL-22:Fc resulted in a reduction of fungal burden;; specifically the replicative life from – the trophozoite which grows firmly attached to type I pneumocytes. IL-22:Fc treatment resulted in reduced fungal attachment and also induced cathelicidin anti-microbial protein which displayed in vitro anti-fungal activity against the trophozoite form of the organism. Conclusion: This study shows that laboratory mice faithfully replicate genetic susceptibility to the hallmark opportunistic infection, Pneumocystis. In conclusion, SPF laboratory mice faithfully

replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T-cell immunity. Disclosure of Interest: None declared.

O021 FAMILIAL AUTOINFLAMMATION WITH NEUTROPHILIC DERMATOSIS REVEALS A NOVEL REGULATORY MECHANISM OF PYRIN ACTIVATION P.J. Baker1,2,*, V. Lagou3,4,5, I. Jéru6,7,8, L. Van Eyck4,5, D.A. Parry9, D. Lawless10, D. De Nardo1,2, J.E. Garcia-Perez4,5, L.F. Dagley1,2,11, C. Holley12, J. Dooley4,5, F. Moghaddas1,2, E. Pasciuto4,5, P.-Y. Jeandel13, R. Sciot14,15, D. Lyras16, A.I. Webb2,11, S.E. Nicholson1,2, L. De Somer15, E. van Nieuwenhove4,5,15, J. Ruuth-Praz7,8, B. Copin8, E. Cochet8, M. Medlej-Hashim17, A. Megarbane18, K. Schroder12, S. Savic19,20, A. Goris3, S. Amselem6,7,8, C. Wouters4,15, A. Liston4,5, S.L. Masters1,2 1 Inflammation Division, Walter & Eliza Hall Institute of Medical Research, Australia; 2Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia; 3Department of Neurosciences, Belgium; 4Department of Microbiology and Immunology, KU Leuven, Belgium; 5Translational Immunology Laboratory, VIB, Leuven, Belgium; 6 INSERM, France; 7Université Pierre et Marie Curie–Paris, UMR S933, France; 8Assistance Publique Hôpitaux de Paris, Hôpital Trousseau, Service de Génétique et d’Embryologie médicales, Paris, France; 9Centre for Genomic and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom; 10Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Wellcome Trust Brenner Building, Saint James’s University Hospital, Leeds, United Kingdom; 11Systems Biology & Personalised Medicine Division, Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; 12Institute for Molecular Bioscience and IMB (Institute for Molecular Bioscience) Centre for Inflammation and Disease Research, University of Queensland, Brisbane, Queensland, Australia; 13Département de Médecine Interne, Hôpital Archet 1, Université Nice Sophia-Antipolis, Nice, France; 14Department of Pathology, KU Leuven, Belgium; 15University Hospitals Leuven, Leuven, Belgium; 16Department of Microbiology, Monash University, Clayton, Victoria, Australia; 17Department of Life and Earth Sciences, Faculty of Sciences II, Lebanese University, Beirut, Lebanon; 18AlJawhara Center, Arabian Gulf University, Manama, Bahrain; 19Department of Allergy and Clinical Immunology, Saint James’s University Hospital, United Kingdom; 20National Institute for Health Research– Leeds Musculoskeletal Biomedical Research Unit and Leeds Institute of Rheumatic and Musculoskeletal Medicine, Wellcome Trust Brenner Building, Saint James’s University Hospital, Leeds, United Kingdom Introduction: Pyrin, encoded by the MEFV gene, is implicated in the innate immune response to a number of bacterial pathogens. These bacteria secrete toxins that indirectly stimulate pyrin-dependent IL1b release by inhibiting Rho-GTPase signalling. MEFV is so called due to mutations in this gene being responsible for the recessively inherited autoinflammatory disease Familial Mediterranean Fever (FMF). FMF is characterised by short periodic attacks of fever and serositis. We have studied several families with a dominantly inherited autoinflammatory disease that is clinically distinct from FMF and is predominantly characterised by neutrophilic dermatosis, prolonged bouts of fever and an absence of serositis. This disease, which we have named Pyrin-Associated Autoinflammation with Neutrophillic Dermatosis (PAAND), is caused by a single amino acid substitution (S242R). Methods: We have used both a 293T overexpression model and CRISPR knockout THP-1 monocytes to study formation of the pyrin

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inflammasome in the presence of the S242R mutation. Findings in these systems have been directly translated to primary peripheral blood monocytes from PAAND patients and healthy controls.

molecular mechanisms underlining self-DNA-mediated autoinflammatory disease and provides a target for the concept of new therapeutics designed to alleviate such disorders.

Results: Phosphorylation of the serine 242 residue creates a noncanonical 14-3-3 binding motif that we show is disrupted by either the S242R mutation or treatment with the Clostridium difficilederived Rho-GTPase-modifying toxin TcdB, leading to increased IL1b production. Mutations in pyrin commonly seen in patients with FMF did not affect the pyrin:14-3-3 interaction, suggesting alternate mechanisms of pyrin activation are implicated in these diseases.

Disclosure of Interest: None declared.

Conclusion: Our results suggest that dephosphorylation of serine 242 is a primary mechanism of pyrin activation in the innate immune response, which is deregulated in individuals presenting with PAAND. Targeting of IL-1b has proven to be a successful therapy for treating PAAND in at least eight patients. Disclosure of Interest: None declared.

O022 STING-DEPENDENT CYTOKINE SIGNALING MANIFESTS SELF DNA INDUCED AUTOINFLAMMATORY DISEASE. J. Ahn1,*, G. Barber1 1 Department of Cell Biology, University of Miami Miller School of Medicine, Miami, United States Introduction: Inflammatory autoimmune diseases such as systemic lupus erythematosus (SLE) and polyarthritis are characterized by chronic cytokine overproduction. Although the mechanisms are not well understood, defective clearance of self-DNA, perhaps from apoptotic or necrotic cells is thought to be a major cause of proinflammatory cytokine production such as TNF-a and type I interferons (IFNs). For example, defects in key Deoxynucleases (DNase), which are critically important for degrading self-DNA, can lead to auto inflammatory disease. Patients defective in the 3’–5’ DNA exonuclease TREX1 can suffer from severe SLE and AicardiGoutieres syndrome (AGS), which presents as a lethal neurological syndrome with high cytokine levels. STING (Stimulator of Interferon Genes) has been found to be essential for triggering the production of various cytokines including type I IFNs.

O023 IL-27 INDUCED BY SELECT CANDIDA SPP. VIA TLR7/NOD2 SIGNALING AND IFN-B PRODUCTION INHIBITS FUNGAL CLEARANCE S.J. Orr1,*, E.C. Patin1, A.V. Jones2, A. Thompson1, M. Clement1, G.W. Jones1 1 Infection & Immunity, Cardi University, United Kingdom; 2University Dental Hospital, Cardi, United Kingdom Introduction: Candida spp. elicit cytokine production downstream of various pathogen recognition receptors (PRRs) including C-type lectin-like receptors (CLRs), Toll-like receptors (TLRs) and nucleotide oligomerisation domain (NOD)-like receptors (NLRs). IL-12 family members, IL-12p70 and IL-23, are important for host immunity against Candida spp. Herein we show that IL-27, another IL-12 family member, is produced by myeloid cells in response to select Candida spp. Methods: Mice were matched by gender and age (8–12 weeks old) and 100 ll of C. parapsilosis or C. albicans in PBS was injected i.v. Mice were monitored and weighed daily. Experiments were continued for a maximum of 42 days for C. parapsilosis or 30 days for C. albicans. Results: We demonstrate a novel mechanism for C. parapsilosismediated induction of IL-27 in a TLR7-, MyD88- and NOD2dependent manner. Our data revealed that IFN-b is induced by C. parapsilosis, which in turn signals through the interferon-a/b receptor (IFNAR) and STAT1/2 to induce IL-27. Moreover, IL-27R (WSX-1) deficient mice systemically infected with C. parapsilosis displayed enhanced pathogen clearance compared to WT mice. This was associated with increased levels of pro-inflammatory cytokines in the serum and increased IFN-g and IL-17 responses in the spleens of IL-27R deficient mice.

Methods: To delineate the role of STING in Trex1-manifested disease, we examined the hearts and cardiac infiltrating leukocytes from wild-type (WT), STING-/-(SKO), TREX-/- (TKO) or STING-/-TREX1-/- (STKO) mice. To further evaluate the role of hematopoietic cells in Trex1-manifested disease, we performed retrieved bone marrow derived cells (BM) from WT or TKO mice and adoptively transplanted them into irradiated TKO mice.

Conclusion: Thus our data define a novel link between C. parapsilosis, TLR7, NOD2, IFN-b and IL-27 and we have identified an important role for IL-27 in the immune response against C. parapsilosis. Overall these findings demonstrate an important mechanism for the suppression of protective immune responses during infection with C. parapsilosis, which has potential relevance for infections with other fungal pathogens.

Results: Trex1 null mice similarly suffer from inflammatory myocarditis with a short life span. Our data indicates that STING (Stimulator of Interferon Genes) is responsible for lethal proinflammatory cytokine production in TREX1 (DNaseII) as well as DNAseIII defective mice. Loss of STING (Trex1-/- STING-/-) was observed to rescue the lethal phenotype lifespan of Trex1-/- and DNaseIII-/- mice due to the prevention enhanced pro-inflammatory gene induction. We found that Trex1 was responsible for eliminating erroneous DNA left over from hematopoetic cell division processes that would otherwise activate STING.

Disclosure of Interest: None declared.

Conclusion: Collectively, our data indicates that STING plays a key role of facilitating pro-inflammatory gene induction induced by intrinsic self-DNA. This study provides new insight into the

O024 HOMEOSTATIC STAT5 SIGNALING DRIVES ACCUMULATION AND FUNCTION OF INNATE LYMPHOCYTES A. Villarino1,*, G. Sciumè1, G. Robinson2, L. Hennighausen2, J. O’Shea 1 1 National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS); 2National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health (NIH), Bethesda, United States Introduction: Innate lymphoid cells (ILCs) patrol environmental interfaces to defend against infection and protect barrier integrity.


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Methods: Using mouse models, we demonstrate that the transcription factor STAT5 is critical for accumulation and function of all ILC subsets. Genome wide analysis of DNA occupancy and gene transcription were used to define molecular mechanisms for STAT5 activity in NK cells, the prototypical ILC subset. Results: Our studies reveal a hierarchy of STAT5 dependency among different ILC subsets, starting with NK cells and ILC1 and at the top of the scale, followed by NCR+ ILC3 and then ILC2 and LTi cells. We also report a similar trend for innate-like T cells, with CD8aa T cells, gamma/delta T cells and NKT cells exhibiting the greatest STAT5 dependencies. Integrated transcriptomic and DNA binding analysis indicates that STAT5 has pervasive effects in NK cells. Not only does it regulate genes involved with apoptosis and cellular homeostasis, as predicted by our in vivo findings, but also key genes involved with lineage identity and functional maturation. These studies also reveal surprising features of STAT5 dynamics, most notably, the striking, genome-wide re-distribution that occurs when NK cells shift from constitutive, steady-state signaling to acute, high dose signaling. Conclusion: Collectively, our data position STAT5 as a central node in ILC biology and provide mechanistic insights on how it operates at cellular, molecular and genomic levels. Disclosure of Interest: None declared.

O025 INNATE LYMPHOID CELL FUNCTION IN ALLERGIC INFLAMMATION: ARGINASE 1 IS A CRITICAL METABOLIC CHECKPOINT CONTROLLING TYPE 2 INFLAMMATION L.A. Monticelli1,*, M.D. Buck2, A.-L. Flamar1, S.A. Saenz1, E.D. Tait Wojno1, N.A. Yudanin1, L.C. Osborne1, M.R. Hepworth1, S.V. Tran1, H.-R. Rodewald3, H. Shah4, J.R. Cross4, J.M. Diamond5, E. Cantu5, J.D. Christie5, E.L. Pearce2, D. Artis1 1 Weill Cornell Medicine, New York, United States; 2Max Plank Institute of Immunobiology and Epigenetics, Freiburg, Germany; 3German Cancer Research Center, Heidelberg, Germany; 4Memorial Sloan Kettering Cancer Center, New York, United States; 5University of Pennsylvania, Perelman School of Medicine, Philadelphia, United States Introduction: Group 2 ILCs (ILC2s) are activated by cell-extrinsic factors including host-derived cytokines such as interleukin-33 (IL-33) and can promote inflammation and tissue repair. However, the cell-intrinsic metabolic pathways that control ILC2 effector function are unknown. Methods: In this study we utilized murine models of acute and chronic lung inflammation, as well as primary human lung tissue from patients with advanced chronic lung disease, to perform immuno-metabolic assays in order to interrogate the role of Arginase-L-arginine metabolism in ILC2 function and development of type 2 inflammation.

curtailing aerobic glycolysis. In humans, expression of ILC2-intrinsic Arg1 was associated with progression of chronic human lung disease. Conclusion: Collectively these studies are the first interrogation of the metabolic pathways that control ILC2 effector function and identify Arg1 as a previously unknown regulator of ILC2 bioenergetic programming that controls their proliferative capacity and proinflammatory function. Disclosure of Interest: None declared.

O026 REGULATION AND FUNCTION OF THE TYPE 2 IMMUNE MODULE IN ADIPOSE TISSUE A.B. Molofsky1,* Laboratory Medicine, University San Francisco, United States






Introduction: Rates of obesity are rising across the world in both children and adults, with over one in three Americans classified as obese. Obesity promotes local inflammation in visceral adipose tissue (AT), ultimately contributing to systemic inflammation, insulin resistance, and the development of type 2 diabetes. However, the function and regulation of normal immune cells in healthy, lean adipose tissue is poorly understood. We have found that ‘allergic’ or ‘type 2’ immune cells, including group 2 innate lymphoid cells (ILC2) and regulatory T (Treg) cells, are surprisingly abundant in lean AT, maintaining metabolic health and limiting obesity induced inflammation and insulin resistance. Our findings suggest type 2 immunity, traditionally associated with protection from multicellular helminthic worms, also plays a central role in the physiologic regulation of adipose tissue metabolism. Methods: We have used cytokine reporters, genetically altered mice, RNA sequencing, and advanced microscopy and flow cytometry to delineate the regulation and metabolic contributions of AT immune cells and cytokines. Results: We have identified the nuclear cytokine Interleukin-33 (IL33) as a major activator of AT ILC2 and the type 2 immune module, protecting against obesity-induced insulin resistance. In contrast, with obesity and aging Interferon-g (IFN-g)-producing lymphocytes increase in AT, promoting insulin resistance and progression to type 2 diabetes. We have found that IFN-g directly represses ILC2s, suggesting IFN-g mediated ILC2 repression may be a central feature in the progression of insulin resistance and diabetes. We have identified relevant cellular sources of IL-33 and IFN-g that regulate AT ILC2. Conclusion: By focusing on the normal composition of the AT ILC2 tissue ‘‘niche”, we hope to understand how ILC2 can promote both beneficial and pathologic type 2 immune responses. Disclosure of Interest: None declared.

Results: Here we demonstrate that constitutive expression of the enzyme Arginase 1 (Arg1) is a conserved trait of mature ILC2s across diverse tissue sites and that expression is maintained independently of classical IL-33-IL-33R signaling. In a model of acute type 2 lung inflammation, deletion of murine ILC-intrinsic Arg-1, but not myeloid cell-intrinsic Arg1, abrogated disease and was associated with impaired ILC2 proliferation and reduced effector cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and

O027 ELUCIDATING THE EPIGENETIC REGULATION OF CD4+ T CELL DERIVED IL-10 DURING INFLUENZA INFECTION N. Fonseca1,*, A. Lorzadeh2, S.A. Redpath1, M. Hirst2, G. Perona Wright1 1 Department of Microbiology and Immunology, Canada; 2Centre for High-Throughput Biology, Department of Microbiology & Immunology, University of British Columbia, Vancouver, Canada

Oral Presentations / Cytokine 87 (2016) 46–57

Introduction: Surviving influenza requires a careful balance of proinflammatory signals that promote viral clearance and anti-inflammatory signals that prevent immunopathology. IL-10 is a potent immunosuppresive cytokine that is essential to this balance. Evidence suggests that in CD4+ T cells, IL-10 expression is critically dependent on IL-27 signaling. Our ex vivo analysis of CD4+ T cell IL10 expression shows that IL-10 is indeed dependent on IL-27 signaling in a primary response to influenza but in a recall response, CD4+ T cells express IL-10 in an IL-27 independent manner. Previous studies have shown that T cell gene expression can be epigenetically regulated through histone modifications; therefore we hypothesised that the il10 gene locus in memory CD4+ T cells could be remodelled to an open state following primary influenza infection. Methods: We tracked cytokine expression from CD4+ T cells during influenza with IL-10 eGFP reporter mice and flow cytometry. To assess the correlation between permissive histone modification patterns and secondary CD4+ T cell IL-10 expression, we performed native ChIP-Seq on aliquots of ~10,000 flow-sorted naïve and influenza-specific memory CD4+ T cells. Four histone modifications (H3K4me3, H3K4me1, H3K27me3, and H3K27ac) were profiled with the ChIP Seq analysis tool MACS2. We used Transcription Factor Affinity Production Web Tools (TRAP) to identify potential transcription factor binding motifs at the il10 gene locus. Results: We observed that memory CD4+ T cells exhibit a permissive H3K4me3, H3K4me1 and H3K27Ac signature and lose the repressive H3K27me3 mark at the il10 locus compared to naïve CD4+ T cells. We also found a strong H3K27Ac signal associated with active enhancers from within the il10 gene body in memory CD4+ T cells. At this enhancer site, we identified potential transcription factor binding motifs for STAT1 and STAT4. Conclusion: Together our data suggest that despite dependence on IL-27 in a primary response, memory CD4+ T cells acquire a permissive epigenetic signature at the il10 locus which positively correlates with their ability to express IL-10 independently of IL-27 signaling in a recall response to influenza. Disclosure of Interest: None declared.

O028 IMMUNE-RESTRICTED EPIGENETIC READER SP140 MAINTAINS TRANSCRIPTIONAL PROGRAMS REQUIRED FOR MACROPHAGE IDENTITY AND INTESTINAL HOMEOSTASIS S. Mehta1, D.A. Cronkite1, M. Basavappa1, C. Wijmenga2, R.J. Xavier1, K.L. Jerey1,* 1 Medicine, Massachusetts General Hospital, Harvard, Boston, United States; 2Department of Genetics, University of Groningen, Groningen, Netherlands Introduction: Chromatin regulators such as ‘‘readers” that recognize post-translational modifications on histones have become attractive therapeutic targets for cancer and inflammation. SP140 is one such


bromodomain/plant homeo domain (PHD)-containing reader with immune-restricted expression, and single nucleotide polymorphisms (SNPs) within SP140 associate with Crohn’s disease (CD). However, the function of SP140 and the consequences of diseaseassociated SP140 SNPs have remained unknown. Methods: To identify transcriptional programs controlled by SP140, we performed global gene-expression analysis of lipopolysaccharide (LPS)-stimulated mouse bone marrow-derived macrophages carrying a lentiviral short-hairpin (sh)RNA against Sp140. To determine the global occupancy of SP140, we performed chromatin immunoprecipitation (ChIP) sequencing of SP140 in primary human M(IFNc) and M(IFNc + LPS) macrophages. To understand the influence of SP140 SNPs on SP140 expression we assessed SP140 mRNA isoforms in peripheral blood mononuclear cells (PBMCs) from individuals carrying no, one or two alleles of CD-risk intronic SP140 SNPs rs7423615 and rs6716753 by RNAseq. We compared gene expression in PBMCs from CD patients harboring SP140 SNPs, CD patients without SP140 SNPs and healthy controls at steady state and following Toll-like receptor stimulation ex vivo. Finally, we examined SP140 mRNA levels in intestinal biopsies as a potential predictor of responsiveness to anti-TNF therapy. Results: Here we show that SP140 is critical for the maintenance of macrophage identity and activation states attained in response to cytokines and microbes, through repression of key gatekeepers of cellular de-differentiation. We found that SP140 was preferentially enriched at lineage-inappropriate genes bearing repressive histone 3 Lysine 27 tri-methylation (H3K27me3) in primary human macrophages exposed to IFNc and/or LPS. SP140 occupancy was highest at the HOX cluster of genes and was essential for silencing HOXA9 that promotes stem cell expansion and prevents myeloid differentiation. Thus, by contributing to the elaborate epigenetic regulation of HOX genes, SP140 ensures functional reprogramming of macrophages and preservation of that activation state. Furthermore, we establish lossof-function consequences of two highly significant intronic SNPs within SP140 that are associated with CD. Individuals carrying CDassociated SNPs within SP140 had defective SP140 mRNA splicing, diminished SP140 protein and suppressed innate immune gene signatures in PBMCs, that stratified them from other CD patients not harboring SP140 SNPs. Furthermore, lower expression of SP140 in intestinal biopsies correlated with improved response to anti-TNF therapy. Conclusion: Collectively, our data show that the immune-restricted epigenetic reader SP140 is an essential orchestrator of transcriptional programs that support macrophage differentiation and activation status. These programs are indispensable for intestinal and immune homeostasis, as a loss of SP140 by genetic variation contributes to a moleculary defined subset of CD that is driven by inadequate innate immunity. Moreover, SP140 levels require tight regulation, as it is both essential for, and predictive of, the inflammatory state. Disclosure of Interest: None declared.