AGA Abstracts Table2: Rare missense variants in XIAP gene 390 Epigenetic and Post-Translational Modification of STAT3 Regulates Its Spatiotemporal Trafficking and Transcriptional Activity in Mesenchymal Cells of Crohn's Disease and Animal Models of Crohn's Disease Chao Li, John F. Kuemmerle STAT3 activity is governed by phosphorylation and acetylation. Post-translational modifica- tions determine localization to the nucleus for transcriptional activity, or to signaling endo- somes for regulation of signaling pathways. We recently identified a pattern of high pSTAT3(S727) and low pSTAT3(Y705) levels due to autocrine IL-6 production in mesenchy- mal cells of strictures in fibrostenotic Crohn's disease. The opposite is seen in the normal intestine in the same patient. This pattern characterizes B2 from other Crohn's phenotypes. We recently showed that STAT3 regulates transcription of TGF- β1 and collagen I. The AIM of this study is to determine the post-translational and epigenetic modifications that determine the subcellular trafficking and function of STAT3. METHODS: Subepithelial myofibroblasts (SEMF) and smooth muscle cells (SMC) were isolated from strictured intestine and normal intestine of patients with fibrostenotic Crohn's disease and from intestine of TNBS treated C57Bl/6J mice and RAG(-/-) mice following transfer of CD4+CD45RB hi T-cells both after 8 weeks. Isolated cells were used to prepare RNA and cell lysates. Histologic sections were also prepared for immunofluorescence. Cell lysates and immunofluorescent staining of primary cultured cells were used to determine STAT3 subcellular localization of phosphory- lated Y705 and S727 and acetylated K685. RESULTS: In both animal models expression of TGF-β1 and Collagen I increased in mesenchymal cells after 8 weeks similar to Crohn's disease. Similar patterns of S727 and Y705 phosphorylation and K685 acetylation and subcellular localization were observed in SEMF and SMC in B2 Crohn's disease (Figure 1), TNBS colitis and T-cell transfer. Phospho S727 and Acetyl K685 were increased and phospho Y705 was decreased in strictures in mice. Phospho S727 and acetyl K685 were localized to the nucleus, phospho Y705 was localized to endosomes in humans (Figrue 2) and mice. The opposite pattern was seen in normal intestine and control mice. IL-6-induced STAT3 nuclear localization of phospho S727 and acetyl K685 was enhanced by inhibition of histone deacetylation with Trichostatin A (500nM) and blocked by the inhibition of acetylation with the p300/CBP acetylation inhibitor, C646 (2.5 μM). IL-6-induced Erk1/2 activation and mesenchymal cell proliferation was inhibited by transfection of a dominant negative STA- T3(Y705F) mutant or by pharmacologic inhibition of STAT3 phosphorylation. CONCLU- SIONS: Activity of STAT3 is regulated by post-transcriptional modification of phosphoryla- tion state and by epigenetic modification of acetylation of lysine residues that determine its subcellular localization and activity. Distinct subcellular localization to nucleus or signaling endosome differentially regulate transcription of TGF- β1 and collagen I αI, and proliferation of mesenchymal cells, respectively. Distinct patterns of S727 and Y705 phosphorylation, and K685 acetylation in strictures and normal intestine of Crohn's disease S-82 AGA Abstracts Distinct subcellular localization of p-Y705 and p-S727 in Crohn's disease strictures. 391 A Crohn's Disease Susceptibility Gene A20 Regulates Autophagy Dynamics in CD4 T Cells Yu Matsuzawa, Shigeru Oshima, Masanori Kobayashi, Yoichi Nibe, Chiaki Maeyashiki, Averil I. Ma, Mamoru Watanabe BACKGROUND & AIMS: Genome wide association studies have identified autophagy related genes as the Crohn's disease susceptibility genes. Meanwhile, we have reported that TNFAIP3, which encodes the A20 protein, is also a Crohn's disease susceptibility gene. A20 is a ubiquitin-modifying enzyme and is critical for preventing inflammation in vivo. A20 is expressed in T cells and may play important roles in the human autoimmune diseases and Crohn's disease. However, the physiological role of A20 in T cells is not fully understood. In this research, we analyze the function of A20 in T cells, and demonstrate how important A20 is for autophagy regulation and inflammation. METHODS: To analyze the function of A20 and autophagy in vivo, we generated mice lacking A20 and ATG5 specifically in T cells by breeding A20FL and ATG5FL mice with CD4-Cre transgenic mice. Single-cell suspensions were prepared from thymus, spleen, and peripheral lymph nodes of each mice. Cell activation and signaling were analyzed by flow cytometry and immunoblotting. Next, to determine whether A20 regulates autophagy in T cells, A20 deficient naïve CD4 T cells were stimulated with anti-CD3 plus anti-CD28 in vitro. Live cells were analyzed by immunohistochemistry using LC3 antibody (LC3 is generally considered as a marker of autophagosome). Images were acquired on a confocal laser microscope. RESULTS: A20 and ATG5 double knockout (DKO) mice were obtained in Mendelian numbers and developed normally. Enlarged spleen and lymph nodes were observed in DKO mice. Surprisingly, the absolute number of peripheral naïve CD4 T cells was significantly reduced in DKO mice as compared to the control. Moreover, both B cells and myeloid cells were expanded in DKO mice. These data indicate that DKO mice have immune phenotype. The immunohisto- chemistry analysis using naïve CD4 T cells revealed that the number of LC-3 puncta was reduced in A20 deficient cells after stimulation. To further investigate the biochemical mechanism by which A20 regulates autophagy, we studied CD4 T cells in vitro. Finally, we found that mTOR (mammalian target of rapamycin) activity was enhanced in A20 deficient CD4 T cells. To confirm the function of enhanced mTOR activity, we treated CD4 T cells with mTOR inhibitor. Control CD4 T cells did not show altered LC3 puncta formation, but LC3 puncta formation increased with mTOR inhibitor in A20 deficient CD4 T cells. Thus, A20 regulates the induction of autophagy in CD4 T cells. CONCLUSIONS: Our studies demonstrate that A20 regulates autophagy dynamics, and provide new insights into how A20 regulates inflammation in T cells. 392 Short Bowel Syndrome: A Case Study to Illustrate How Gut Remodeling Can Impact Fecal Microbiota That in Turn Becomes Central to Understanding Clinical Outcome Laura Gillard, Camille Mayeur, Patricia Lepage, Sylvie Miquel, Véronique Robert, Johanne Le Beyec - Le Bihan, Andre Bado, Muriel Thomas, Francisca Joly Introduction: Short bowel syndrome (SBS) results from an extensive resection of small intestine. Clinical hallmarks of SBS are severe malabsorption and hyperphagia. We previously described an increase in colonic crypt depth of the remaining colon and in plasma GLP-1 and GLP-2 levels, secreted by L cells. SBS patients have a dysbiotic microbiota (referred to as lactobiota): preponderance of aero-tolerant ( Lactobacillus) and depletion of aero-sensitive (Bacteroides, C. leptum) bacteria. This lactobiota had a specific fermentative activity with high capacities to produce lactates. Our aim was to determine whether transfer of the dysbiotic microbiota from SBS patients to GF rats, affect crypt depth, cell proliferation and endocrine L cells number in colonic mucosa . Materials and methods: Germ-free (GF) rats