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P056 Soluble factors from the inflamed mucosa of patients with Crohn's disease (CD) or from cytokine-stimulated epithelial cells induce the expression of TL1A/TNFSF15 in human intestinal subepithelial myofibroblasts (ISMFs)

E. Filidou*1, G. Bamias2, D. Goukos3, V. Valatas4, K. Arvanitidis1, M. Panagopoulou1, G. Kouklakis5, G.L. Daikos3, S. Ladas2, G. Kolios1

1Democritus University of Thrace, Laboratory of Pharmacology, Faculty of Medicine, Alexandroupolis, Greece, 2Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, Academic Dpt. of Gastroenterology, Athens, Greece, 3Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, 1st. Dpt of Propaedeutic and Internal Medicine, Laboratory of Infectious Diseases, Athens, Greece, 4University of Crete, Laboratory of Gastroenterology, Faculty of Medicine, Heraklion, Greece, 5Democritus University of Thrace, Endoscopy Unit, Faculty of Medicine, University Hospital of Alexandroupolis, Alexandroupolis, Greece

Background

TL1A (TNFSF15) and DR3 (TNFRSF25) are a ligand/receptor pair of the TNF/TNFR superfamilies of proteins. Both proteins are highly upregulated and functionally involved in experimental intestinal inflammation and Inflammatory Bowel Disease. Recent studies in TL1A transgenic mice implicated TL1A/DR3 signalling in inflammation-induced intestinal fibrosis. Our aim was to examine whether the pro-inflammatory mucosal environment that exists in CD may provide stimulatory signals to ISMFs that lead to upregulation of TL1A.

Methods

ISMFs were isolated from endoscopically-obtained colonic biopsies from healthy controls (HC) and patients with CD. Cultured ISMFs were stimulated for 6h with supernatants from overnight cultures of colonic mucosal biopsies. Total RNA was extracted from cultured ISMF and HT-29 cells and TL1A mRNA expression was measured by real-time RT-PCR. HT-29 epithelial cells were cultured unstimulated or stimulated with rhIL-1α and/or rhTNF-α and/or rhIFN-γ and tested for upregulation of DR3 mRNA. Finally, the supernatants from cultured HT-29 epithelial cells were tested for their ability to induce the expression of TL1A in ISMFs.

Results

Supernatants from CD-derived colonic tissue cultures induced a significantly higher upregulation of TL1A expression in cultured ISMFs (>3-fold increase over HC, P<0.05). HT-29 epithelial cells significantly upregulated the expression of DR3 (the functional receptor for TL1A) when stimulated with the pro-inflammatory cytokines IL-1α, TNF-α and IFN-γ. Finally, supernatants from HT-29 epithelial cells cultures significantly induced the expression of TL1A in ISMFs when TNF-α was used for stimulation of the epithelial cells either alone or in combination with IL-1α and/or IFN-γ (P<0.05 for any combination vs. unstimulated HT-29 cell supernatant).

Conclusion

The pro-inflammatory mucosal milieu in CD contains soluble factors that induce the expression of TL1A in ISMFs and of its functional receptor, DR3, in intestinal epithelial cells. The latter further stimulate ISMFs to express TL1A. We conclude that interactions between ISMF derived TL1A and its receptor, DR3 on epithelial cells may contribute to the pathogenesis of chronic intestinal inflammation.