Tu1920 a Farnesoid-X-Receptor (FXR)-Glucocorticoid Receptor (GR) Cascade Regulates Intestinal Innate Immunity in Response to FXR Activation

AGA Abstracts

using Student's T test. Results: RQ is derived from deltaCT and demonstrates the fold change of gene expression between the two genotypes. RQ and p values were as follows: ERAP2 (1.92, <0.0001), CARD9 (2.51, 0.0006), FASLG (1.11, 0.69), CDKAL1 (1.44, 0.079), RANKL (1.48, 0.37). Conclusions: We have identified two IBD-associated SNP's (rs2549794, rs4077515) where variation correlates with expression of adjacent candidate genes ERAP2 and CARD9 in ileal biopsies. These results link GWAS risk SNPs with a potential mechanism in immunologically relevant genes, and help to explain their impact on IBD pathogenesis.

colitis. Endogenous and exogenous cathelicidin ameliorate intestinal inflammation by inhibiting NF-kappaB-dependent TNFalpha synthesis, suggesting a novel therapy for colitis. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, a Career Development Award grant from Crohn's and Colitis Foundation of America, and NIH K01DK084256 Award (HWK). Tu1920 a Farnesoid-X-Receptor (FXR)- Glucocorticoid Receptor (GR) Cascade Regulates Intestinal Innate Immunity in Response to FXR Activation Barbara Renga, Andrea Mencarelli, Claudio D'Amore, Angela Zampella, Eleonora Distrutti, Stefano Fiorucci Background. Nuclear receptors (NRs) are transcription factors that exert homeostatic functions at the interface between nutrient metabolism and innate immunity. The GR is prototypic of a subset of ligand-dependent NRs that integrate host immune responses with steroid metabolism. Glucocorticoids are widely used for the treatment of inflammatory, autoimmune and neoplastic diseases, and remain the first-line treatment for inducing moderate to severe active Crohn's disease and ulcerative colitis. Anti-inflammatory activities have been documented for other NRs, including the FXR, a bile acid sensor that regulates intestinal innate immunity and homeostasis. Aim. To investigate whether FXR regulation of intestinal innate immunity is mediated by GR gene activation. Methods. Colitis was induced in mice by intrarectal administration of TNBS. Mice were administered 6-ECDCA, a semi-synthetic FXR ligand (5 mg/kg) and the GR antagonist mifepristone (5 mg/kg) starting 3 days before TNBS. Relative mRNA expression of GR and pro-inflammatory genes was assayed by RT-PCR. The promoter analysis of human GR gene was performed with the Nubiscan program. Electrophoretic Mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) were carried out in THP1 monocytic cell line. Results. In Vivo activation of FXR protects against the development of colitis by down-regulating the expression of pro-inflammatory cytokines (TNFα, IL1β, IFNγ) and by increasing the expression of GR mRNA. Mifepristone reversed the protection exerted by the FXR agonist indicating a role for GR in the effects of FXR. Exposure of THP-1 cells to natural or synthetic FXR ligands increased GR mRNA expression. Analysis of both human and mouse GR promoters revealed the presence of a conserved ER-8 sequence (an FXRE). Co-transfection of FXR/RXR significantly enhanced FXR-induced luciferase activity while the transfection with an heterologous promoter construct containing the mutated FXRE failed to be activated by FXR ligand. ChIP experiment demonstrated that FXR is recruited on the GR promoter following FXR activation. EMSA experiment showed that FXR was able to bind to the ER-8 motif and failed to bind to an oligonucleotide bearing the mutation in the ER-8 FXRE. Conclusions. Our results demonstrated that regulation of intestinal innate immunity by FXR involves the GR.

Tu1918 Intestinal Dendritic Cells Survey Circulatory Antigens Prior to Induction of Regulatory CD8α/β T Cells Joo Hye Song, Sun Young Chang, Bayasi Guleng, Carmen Alonso, Seiji Arihiro, Christpher Karp, Atul K. Bhan, Cox Terhorst, Hans-Christian Reinecker Background: Antigen processing by DCs in the intestinal immune system has been linked to the development of tolerance to self-antigens, food and commensal microbiota. It remains controversial which intestinal DC subsets control the generation of regulatory CD4+ or CD8+ T cells to govern mucosal and systemic immune responses. Moreover, antigens in the intestine transition from fenestrated capillaries through the lamina propria into the draining lymphatics, but whether or how this process controls mucosal immune responses is unclear. CX3CR1+ DCs are uniquely positioned for the recognition of environmental and circulatory antigens in the lamina propria as they interact with both the epithelium and with the capillary vessel system in the lamina propria.

Methods: To investigate the uptake of circulatory antigen by CX3CR1+ DCs, we utilized In Vivo imaging system and electron microscopy after intravenous injection of Alexa 647 labeled OVA. OT-I CD8 T cells were cultured with CX3CR1+ DCs isolated from OVA injected CX3CR1-GFP mice and tested for their capacity to inhibit T cell activation In Vitro and to prevent IBD induced by injection of CD4+ CD45RBhigh cells into syngeneic immunodeficient mice.

Results: We demonstrate that small intestinal CX3CR1+ dendritic cells (DCs) acquire and process both circulatory and luminal antigens in the lamina propria. Through cross-presentation of antigen, CX3CR1+ DCs induce differentiation of IL-10 secreting CD8α/β T regulatory cells, that is dependent upon Programmed Death ligand 1/2 and its counter-structure PD1 of CD8 T cells. Differentiated CD8+ T regulatory cells represent a subset of intraepithelial lymphocytes (IELs) which express the specific adhesion molecule CD103. The CD8+ Tregs inhibit antigen specific CD4+ T cell activation through IL-10 secretion and prevent CD45RBhigh CD4 T cell-mediated small intestinal inflammation. Conclusion: Our finding shows that the lamina propria CX3CR1+ DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of selfand intestinally- absorbed antigens leading to the induction of CD8+ T regulatory cells, which govern tolerance in the mucosal immune system.

Tu1921 Selective Inhibitors of RIP2 Kinase Activity Reduce PRO-Inflammatory Cytokine Production in IBD Mucosal Biopsy Cultures and Inflammation in Murine TNBS-Induced Colitis Peter Gough, Bart Votta, Linda Casillas, Pamela Haile, David Lipshutz, Biva Desai, Barbara Swift, Carol Ann Capriotti, Michael Reilly, Kevin P. Foley, Paolo Biancheri, Anna Vossenkämper, Thomas T. MacDonald, Robert W. Marquis, John Bertin A breakdown in intestinal immune homeostasis, associated with epithelial barrier disruption, is a common feature among various forms of human inflammatory bowel disease. The resulting interactions between commensal bacteria and mucosal immune cells trigger inflammation via activation of pattern recognition receptors (PRRs). The PRRs NOD1 and NOD2, and their associated effector kinase RIP2, have the potential to be key drivers of intestinal inflammation through their ability to detect intracellular bacterial products such as peptidoglycan derivatives. In order to elucidate the role of NOD1 and NOD2 in mediating this PRRdriven inflammatory response, we have generated and characterized potent and highly selective small molecule inhibitors of RIP2 kinase. One such inhibitor, GSK'214, inhibited RIP2 kinase autophosphorylation (IC50 = 3.2 nM), and showed a high degree of selectivity (>2000-fold) when profiled against a panel of 300 kinases. GSK'214 blocked NOD1 and NOD2-dependent inflammatory cytokine production In Vitro and In Vivo, but did not inhibit TLR-2, -4, -7, -9 or TNFR-mediated signaling. To explore the role of RIP2 kinase in human inflammatory bowel disease we applied our specific inhibitors to cultured intestinal biopsies obtained from Crohn's Disease and Ulcerative Colitis patients. Strikingly, inhibition of RIP2 kinase activity in these cultures attenuated the spontaneous release of pro-inflammatory cytokines in a manner comparable to the steroid prednisilone. Furthermore, oral dosing of a RIP2 kinase inhibitor was able to reduce intestinal inflammation in a murine TNBS-induced colitis model. These results demonstrate the importance of RIP2 kinase activity in modulating intestinal inflammation and suggest that RIP2 kinase inhibition may be a novel therapeutic approach for the treatment of IBD.

Tu1919 Anti-Inflammatory Effects of Anti-Microbial Peptide Cathelicidin in Mice With Acute Colitis Hon Wai Koon, Jeremy Chen, Ryan Ichikawa, David Q. Shih, Samantha Ho, Michelle Cheng, Tressia Hing, Michelle Vu, Ivy Law, Dezheng Zhao, Richard Gallo, Stephan R. Targan, Charalabos Pothoulakis Background and Aims: Cathelicidins (LL-37 in human and mCRAMP in mice) represent a family of endogenous peptides with well-established anti-microbial activity. We previously reported an anti-inflammatory role for endogenous cathelicidin in the dextran sulfate (DSS) mouse model, resembling ulcerative colitis (UC) (Gastroenterology 2011 141:1852-63). Here we examined the role of endogenous and exogenous cathelicidin in the trinitrobenzene sulphonic acid (TNBS) mouse model, resembling Crohn's disease (CD), and determined the anti-inflammatory mechanism of this response. Methods: Wild-type (WT) and mCRAMP deficient (Camp-/-) mice were injected with TNBS intracolonically (100mg/kg, 5 mice/ group). Some mice were treated intracolonically with mCRAMP (5 mg/kg). After 7 days mouse colons were collected for analysis. Results: In colonic biopsies from UC and CD patients (n=10/group), high TNFalpha was statistically associated with elevated LL-37 protein level, suggesting colitis-mediated cathelicidin expression. Following TNBS administration, colonic mCRAMP mRNA levels in WT mice were significantly increased (~15 fold, p= 0.0001), compared to ethanol-treated groups. TNBS exposed Camp-/- mice had significantly higher colitis histology score (by 35%, p=0.04) and TNFalpha levels (by ~25%, p=0.03) compared to WT mice. On the other hand, intracolonic mCRAMP ameliorated acute colitis evidenced by significantly reduced colonic histology score (~40%, p=0.0006) and tissue TNFalpha protein level (~40%, p=0.03). Exposure of human primary colonic epithelial cells to a proinflammatory cytokine cocktail (TNFalpha, IL-1beta and IFNgamma, 10 ng/ml each) increased TNFalpha mRNA and protein expression (by ~10 fold, p=0.0001) which was reduced (by ~20%, p=0.02) in the presence of LL-37 (1 microM). Similarly, incubation (46 hrs) of NCM460 human colonocytes with LL-37 (5-10 microM) completely abolished proinflammatory cytokine cocktail induced TNFalpha with inhibition of NF-kappaB phosphorylation (by ~40%, p=0.04) and transcriptional activity (by ~50%, p=0.01). Inflamed colons from IBD patients (by ~10 fold, p=0.03) and TNBS exposed mice (~2 fold, p=0.0008) had increased expression of phosphorylated NF-kappaB, compared to controls. At day 7 of TNBS colitis, Camp-/- mice developed stronger NF-kappaB phosphorylation (by 50%, p= 0.04) than WT mice but all were significantly diminished by (>50%, p=0.004) intracolonic mCRAMP. Conclusions: Endogenous cathelicidin is induced in colons in response to acute

AGA Abstracts

Tu1922 Genome-Wide Epigenomic Analysis of Lamina Propria Mononuclear Cells of Inflammatory Bowel Disease Takeshi Otsubo, Yuki I. Kawamura, Kenshiro Oshima, Takaho A. Endo, Tetsuro Toyoda, Teruki Hagiwara, Yutaka J. Kawamura, Fumio Konishi, Hideaki Yano, Yukio Saito, Masahira Hattori, Taeko Dohi Background & Aim: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are caused by an inappropriate inflammatory response in a genetically susceptible host triggered by various environmental factors. We assume that epigenetic changes in the immune system may reflect the influence of these environmental factors. Accumulating evidences have demonstrated that chronic inflammation is associated with epigenetic changes, and it may contribute to IBD etiology; however, to our knowledge, epigenetic alterations of inflammatory immune cells in IBD had not been reported. To find out aberrant DNA methylation and chromatin modification in lamina propria mononuclear

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