and invasiveness of intestinal epithelial cells (IEC). The underlying extracellular matrix isbroken down to enable IEC to invade. Here, we investigated the impact of pathogen-associated molecular patters (PAMPs) such as muramyldipeptide (MDP) on the inductionof EMT in a three-dimensional multicellular model of HT-29 IEC and matrix destructionin human fistula tissue. Methods: Multicellular three-dimensional HT-29 IEC constructswere exposed to MDP (100 ng/ml) for seven days and observed microscopically over atimeline of seven days as well as on a molecular level. Human perianal fistula tissue wasassessed for matrix metalloproteinases (MMP) using immunohistochemical procedures.Results: After exposure to MDP for seven days, the spheroids lost the well-defined globularshape and the epithelial cells separated indicating the onset of EMT. This was not seen inuntreated control spheroids. In accordance with these microscopic findings, MDP treatedspheroids up-regulated mRNA levels of EMT-related genes, including SNAIL1 (p < 0.05),TGF-β (p < 0.05) and IL-13 (p < 0.05) after treatment for seven days. In contrast to MDP,interleukin (IL)-22 did not induce EMT in spheroids. Furthermore, we found that MMP-3was highly expressed around fistula tracts in human CD fistula tissue while only few stainedcells were found in the surrounding inflamed tissue. Conclusions: Here, we demonstratethat PAMPs might contribute to the onset of EMT and therefore to the development of CDfistulae. The excessive expression of MMP-3 around the fistula tracts might contribute tothe invasive growth of the EMT cells into deeper tissue layers. In summary, our data suggestthat bacterial pathogens likely impact the pathogenesis of CD-associated fistulae.

Sa1729

ER Stress Leads to Epithelial to Mesenchymal Transition (EMT) in IntestinalEpithelial Cells in a JNK- and SRC-Dependent MannerClaudia Stanzel, Anne Terhalle, Michael Scharl, Gerhard Rogler

Background: Fistulae are a frequent complication in Crohn's disease patients. Epithelial tomesenchymal transition (EMT) has been identified as an important driving force of theirformation. On the other hand, the importance of ER stress and related pathways in intestinalinflammation has been demonstrated by recent evidence from animal models, the phenotypeof human IBD, as well as genetic data. We showed previously, that ER stress leads to theonset of EMT in intestinal epithelial cells. Here, we investigated candidate signaling pathwaysand therapeutic substances. Methods: HT-29 cells were stimulated with the ER stress inducertunicamycin (TM). In order to identify candidate pathways, we used specific inhibitorstargeting JNK, Src, Smad or siRNA targeting Ets1 transcription factor prior to stimulationwith TM. Polyamines, butyrates and bile acids were tested for their potential therapeuticeffect by pre-treating the cells with the substances followed by addition of TM. The extentof ER stress and EMT was determined by measuring typical markers by Western blot andquantitative real time PCR. Results: Upon treatment with TM induction of the transcriptionfactor Ets1 was found, which is known to be induced upon ER stress and to play a role ininduction of EMT. However, knockdown of Ets1 by siRNA did not influence mRNA levelsof the typical fibroblast markers vimentin, beta-6-integrin or the ER stress marker gp96.Similar, inhibiting Smad only lead to slightly reduced levels of beta-6-integrin. On the otherhand, stimulation with JNK inhibitor II revealed that induction of Ets, beta-6-integrin andgp96 was JNK-dependent. In addition, the Src kinase inhibitor PP2 reduced the expressionof Ets1 and gp96, while induction of beta-6-integrin was found to be Src-independent.Analysing potential therapeutic substances, neither spermidine nor sodium phenylbutyratecould counteract the effect of TM. However, pre-incubation of the cells with 10 mM of thebile acid tauroursodeoxycholic acid decreased significantly the levels of vimentin, beta-6-integrin and gp96. Conclusion: Our data suggest that the JNK and Src pathway are involvedin the regulation of ER stress-mediated EMT during fistula formation in Crohn's disease.The bile acid tauroursodeoxycholic acid represents a therapeutic treatment option.

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Aryl Hydrocarbon Receptor-Driven Signals Down-Regulate Intestinal FibrosisIvan Monteleone, Francesca Zorzi, Irene Marafini, Roberta Caruso, Alfredo Colantoni,Francesco Pallone, Giovanni Monteleone

BACKGROUND & AIM : Strictures are a major complication of Crohn's disease (CD) andrepresent one of the main indications for surgery. The pathogenesis of strictures remainsunknown even though fibroblast activation and excessive deposition of collagen are keyhallmarks of this condition. Compounds which interfere with such a process could thus beuseful for preventing the development of strictures in CD. AhR, best known for mediatingthe dioxin-toxicity, modulates human myofibroblast differentiation. In this study we investi-gated the role of AhR in the prevention/treatment of intestinal fibrosis. METHODS: AhRexpression was evaluated in fibroblasts isolated from normal controls, patients with CD andpatients with ulcerative colitis by RTR-PCR and flow cytometry. Fibroblasts isolated fromintestinal samples of patients with fibrostenosing CD were stimulated with TGF-beta1 orTNF-alpha with or without Ficz, an activator of AhR, or an AhR antagonist. Collagen,cytokines and matrix metalloproteinases (MMP) were evaluated by colorimetric assay orReal-Time PCR (RT-PCR). To assess the in vivo role of AhR in the development of intestinalfibrosis, chronic intestinal inflammation-mediated fibrosis was induced in mice by weeklyintrarectal administration of trinitrobenzene-sulfonic-acid (TNBS) and animals were giventherapeutically Ficz or the AhR antagonist. RESULTS: Intestinal fibroblasts constitutivelyexpressed AhR with no apparent difference between CD and controls. Treatment of fibroblastswith Ficz inhibited TGF-beta and TNF-alpha driven myofibroblast differentiation and colla-gen production, while the AhR antagonist increased collagen synthesis. Ficz treated micewere largely protected from TNBS-induced fibrosis and showed a marked down-regulationof collagen, fibrogenetic cytokines and MMP. Differently, abrogation of AhR-signaling by aspecific antagonist increased intestinal collagen accumulation, thus leading to the develop-ment of a more severe intestinal fibrosis. CONCLUSIONS: AhR-driven signals inhibit pro-fibrogenetic stimuli in the gut and suggest that AhR-related compounds may be useful forpreventing human intestinal fibrosis.

S-283 AGA Abstracts

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HMGB1 Controls the Intestinal Epithelial Autophagy/Apoptosis CheckpointDuring Mucosal Inflammation and IBDXiaorong Zhu, Jeannette S. Messer, Yunwei Wang, Fanfei Lin, Candace M. Cham,Jonathan Chang, Timothy Billiar, Michael T. Lotze, David L. Boone, Eugene B. Chang

HMGB1 is chromosome-binding protein that is released from cells to act as a damage-associated molecular pattern molecule (DAMP) during many inflammatory diseases, includinginflammatory bowel disease (IBD). While the extracellular, pro-inflammatory functions ofthis protein are well-characterized, its intracellular functions are only poorly understood.In this study we investigated the role of intracellular HMGB1 in colitis using an intestinalepithelial (IEC) conditional HMGB1 knockout mouse (HMGB1f/f, Vil-CRE) and samplesfrom humans with IBD. Loss of HMGB1 in IEC exacerbated colitis in the dextran sodiumsulfate (DSS) and IL10-/- murine colitis models. This suggested that, in addition to serving asan immune response molecule, HMGB1 may also have a cytoprotective function. Autophagy isa cellular response that promotes survival during stress and HMGB1 is reported to bind tothe autophagy protein beclin-1. In the DSS colitis model, loss of HMGB1 was associatedwith decreased autophagy and increased IEC death. Autophagy failure is linked to cell deaththrough accumulation of damaged organelles and protein aggregates. However, the pro-autophagic proteins beclin-1 and Atg5 can also be cleaved by proteases to generate pro-apoptotic protein fragments. In HMGB1f/f, Vil-CRE mice, beclin-1 and Atg5 were cleavedin a manner consistent with calpain cleavage after DSS administration and in IL10-/- mice.We confirmed that HMGB1 protects beclin-1 and Atg5 from calpain-mediated cleavage invitro and in enteroids derived from HMGB1f/f and HMGB1f/f, Vil-CRE mice stimulated withmuramyl dipeptide, a bacterial cell wall component. Treatment of mice with calpeptin, acalpain inhibitor, ameliorated DSS colitis in HMGB1f/f, Vil-CRE mice and prevented beclin-1 and Atg5 cleavage. These data suggest that HMGB1 protects cells from death by preventingcalpain-mediated cleavage of beclin-1 and Atg5 during inflammation. Human IBD is alsocharacterized by IEC death so we investigated HMGB1 expression, calpain activity, andbeclin-1 and Atg5 cleavage in endoscopic intestinal biopsies from patients with controlledor active IBD. HMGB1 expression was increased at the level of mRNA, but decreased at theprotein level in patients with active colitis versus controls. Patients with active colitis alsohad increased calpain activity and cleavage of beclin-1 and Atg5. Thus, humans with activeIBD have decreased HMGB1 and a switch between the pro-autophagic and pro-apoptoticfunctions of beclin-1 and Atg5 to favor apoptosis. This study represents a major advancein understanding both the function of HMGB1 and the process of inflammation in IBD.Beclin-1 and Atg5 cleavage in IBD patients suggests that the autophagy/apoptosis checkpointis not functioning properly with the result that the equilibrium in the gastrointestinal mucosais tipped away from autophagy and toward cell death.

Sa1732

TNF-α Inhibits Mucosal Protection Effects of Osteopontin in Crohn's Diseasevia IRF1Ruihan Tang, Guang Yang, Shenghong Zhang, Minhu Chen

Crohn's disease (CD) is characterized by damaged intestinal epithelium barrier. Variousendogenous protecting substances are dampamed under inflammed condition. Osteopontin(OPN) was reported to regulate cell survival and growth in many tissues. However, its rolein IBD injured intestinal epithelium has not been well defined yet. In our patient group,average opitical densites (AOD) of OPN were significantly lower than control group (n=156, AOD=0.151±0.042 vs n=60, AOD=0.243±0.013, P=0.001) . The hs-CRP correlatedclosest with OPN AOD (Spearman's rank correlation coefficient r = -0.415, P=0.001), followedby ESR ( r = 0.301, P=0.017), CDEIS( r = -0.237, P=0.011), SES-CD( r =- 0.227, P=0.017),and the CDAI ( r = -0.217, P=0.012), indicating that OPN decreases when disease recurrence.Intriguingly, we found that another factor interferon regulator factor 1 (IRF1) was upregulatedin CD patients, and was strongly correlated with hs-CRP (r = 0.439, P=0.001) and CDAI (r = 0.198, P=0.022). The IRF1 level showed a negative correlation with OPN expression(r = 0.207, P=0.010). To unveil its underlying mechanisms, we investigated their effects onproliferation and apoptosis in TNF-α treated HCT116 and Caco2 cell lines. TNF-α inducedIRF1 and decreased the OPN expressions in both 2 cell lines. OPN over-expression andrhOPN reduced the apoptosis induced by TNF-α while IRF1 over-expression aggravatedapoptosis, indicating the opposite functions of OPN and IRF1 in CD. Luciferase assay andChIP assay showed that IRF1 transcriptionally inhibited the expression of OPN. TNF-αinhibited the OPN induced up-regulation of p-ERK, p-P38 and p-Akt. In conclusion, OPNmight play a protective role for intestinal epithelial cell but its function was impaired in thepresence of TNF-α. IRF-1 is one of molecular targets for TNF-α against OPN in CD.

Sa1733

Tofacitinib Inhibits JAK1 and STAT3-Dependent Increases in Profibrotic TGF-β1 and Collagen IαI in Muscle Cells From Patients With Montreal B2Fibrostenotic Crohn's DiseaseChao Li, Audra Iness, Jennifer Yoon, Jay Kuemmerle

Background: We recently showed that IL-6 production is significantly increased in musclecells of affected segments of ileum in patients with Montreal B2 phenotype Crohn's diseasecompared to the normal proximal resection margin in the same patient or normal intestinein non-Crohn's patients. In muscle cells of strictured intestine STAT3(S727) is increasedand STAT3(Y705) is decreased. The opposite pattern is seen in the normal margin of thesame patient. IL-6 stimulates STAT3(S727) phosphorylation that increases STAT3(S727)DNA binding affinity and transcriptional activity of TGF-β. This results in increased expres-sion of TGF-β1 and collagen IαI: two characteristics of fibrosis in patients with MontrealB2 Crohn's disease and strictures. Aims: The determine whether IL-6 activates Janus kinase(Jak), and whether the Jak inhibitor, tofacitinib, can inhibit the IL-6-induced, STAT3-dependent pro-fibrotic effects on TGF-β1 and collagen I expression in intestinal musclefrom Montreal B2 phenotype Crohn's disease patients. Methods: Histologic sections wereprepared and smooth muscle cells were isolated from ileal strictures and the normal resection

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