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P032. Pro-inflammatory cytokines induce the expression of TL1A/TNFSF15 in primary intestinal subepithelial myofibroblasts

G. Bamias1, E. Filidou2, D. Goukos3, V. Valatas4, K. Arvanitidis2, M. Panagopoulou2, G. Kouklakis5, G. Daikos3, S. Ladas1, G. Kolios2, 1Laikon Hospital, Ethnikon & Kapodistriakon University of Athens, Academic Dpt. of Gastroenterology, Athens, Greece, 2Democritus University of Thrace, Laboratory of Pharmacology, Faculty of Medicine, Alexandroupolis, 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 belongs to the TNF superfamily of cytokines (TNFSF15). It provides co-stimulatory signals for activated lymphocytes that express the receptor DR3. TL1A and DR3 are highly upregulated in intestinal areas with active IBD-related inflammation. Recently it was reported that transgenic mice with lymphoid- or myeloid-specific TL1A overexpression develop colonic fibrosis. We aimed to determine whether primary human intestinal subepithelial myofibroblasts (ISMF) express TL1A under stimulation with IBD-related pro-inflammatory cytokines.

Methods

ISMF were isolated from endoscopically-obtained colonic biopsies from healthy controls (HC) and Crohn's disease patients (CD). ISMF were cultured unstimulated or stimulated under various conditions: (a) with rhTNF-a and/or rhIL-1a; (b) with supernatants from cultures of colonic biopsies obtained from HC and CD patients; and, c) with supernatants from cultured HT-29 epithelial cells which were unstimulated or stimulated with rhTNF-a, rhIL-1a and rhIFN-g, added alone or in combination. Total RNA was extracted from the cultured ISMF and the mRNA expression of TL1A and the receptor DR3 was measured by real-time RT-PCR.

Results

Stimulation of ISMF with either TNF-a or IL-1a resulted in significant upregulation of the relative expression for TL1A mRNA at 6h (unstimulated, 2.89±5.20 average±sdev; IL-1a, 93.58±17.44; TNF-a, 66.59±15.53; IL-1a+TNF-a, 127.04±19.40, P < 0.00001 for any condition vs. control). The average increase in TL1A expression was 32.8-fold for IL-1a, 23-fold for TNF-a, and 43.9-fold for their combination. No difference was seen at 1h whereas at 24h only stimulation with TNF-a was significantly higher than the control condition. We did not detect DR3 mRNA in ISMF under any of the above conditions. We also found that supernatants from cultured HT-29 epithelial cells were able to induce the expression of TL1A in ISMF when TNF-a was used for stimulation of the epithelial cells either alone or in combination with IL-1a and/or IFN-g (P < 0.05 for any combination vs. unstimulated HT-29 cell supernatant). Finally, supernatants from CD-derived colonic tissue cultures induced a significantly higher upregulation of TL1A mRNA expression in cultured ISMF (>5-fold increase over tissue cultures from healthy controls, P < 0.01).

Conclusion

Pro-inflammatory cytokines that are abundantly expressed in intestinal areas with active CD (TNF-a, IL-1a, IFN-g) induce the expression of the co-stimulatory cytokine TL1A in ISMF either directly or through the induction of epithelial-derived mediators. Our results raise the possibility that interactions between ISMF-derived TL1A and its receptor DR3 on epithelial cells or lymphocytes may participate in the pathogenesis of chronic intestinal inflammation.