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P061 Succinate promotes EMT in intestinal epithelial cells through SUCNR1: Relevance in fistula development

J. Cosin-Roger*1,2, D. Ortiz-Masia3, M. Aragón-Borrego1, L. Gisbert-Ferrándiz1, S. Calatayud1, M. D. Barrachina1

1University of Valencia, Pharmacology, Valencia, Spain, 2Hospital Dr Peset, Valencia, Spain, 3University of Valencia, Medicine, Valencia, Spain

Background

Intestinal fistula is a common complication in CD patients whose aetiology is unknown. It is associated with an exacerbated inflammation and epithelial-to-mesenquimal transition (EMT), a process which allows a switch from epithelial towards a fibrotic phenotype. Under inflammatory conditions, succinate is accumulated and activates its receptor, SUCNR1, which has recently been related to intestinal fibrosis. We aim to analyse the role of succinate and SUCNR1 in EMT.

Methods

HT-29 cells were treated with succinate (0, 0.1, 0.5, 1.5 mM) or TGF-β (5 ng/ml) during 48 h and transfected with SUCNR1 siRNA. Expression of EMT markers was analysed by qPCR and western blot. Intestinal fibrosis was induced in vivo using the heterotopic transplant model in WT and Sucnr1−/− mice and expression of EMT markers was analysed by qPCR and by confocal microscopy. Intestinal resections were obtained from CD and non-IBD patients. The expression of SUCNR1, Snail1, Snail2 and E-Cadherin was analysed by qPCR and succinate levels were quantified with a Succinate Assay Kit. Results are expressed as fold induction (mean ± SEM, n ≥5). Statistical analysis was performed with one-way ANOVA followed by Newman–Keuls test. Correlations were analysed with the Spearman coefficient.

Results

Succinate induces, in HT-29 cells a significant increase in Vimentin, Snail1, and Snail2 expression and a significant reduction in E-Cadherin expression compared with vehicle-treated cells and these changes were significantly prevented in cells transfected with SUCNR1 siRNA, (1.87 ± 0.09 vs. 1.12 ± 0.12, 1.85 ± 0.18 vs. 0.90 ± 0.09 and 2.57 ± 0.43 vs. 1.07 ± 0.26 vs.,, respectively). WT-grafts at Day 7 showed a significant increase in Vimentin expression (3.50 ± 0.48), Snail1 (4.87 ± 0.79) and Snail2 (2.45 ± 0.25) and a significant reduction in E-Cadherin expression (0.52 ± 0.07) vs. WT-grafts at day 0. KO-grafts at Day 7 showed a significant reduction in Vimentin expression (1.84 ± 0.14), Snail1 (1.91 ± 0.28) and Snail2 (1.07 ± 0.26) and an increase in E-Cadherin (0.94 ± 0.05) compared with WT-grafts at Day 7. Finally, in intestinal resections from B3-CD patients: (a) levels of succinate were higher than in that from B2-CD patients or non-IBD patients (244.90 ± 26.03 μM, 142.00 ± 21.66 μM and 99.73 ± 11.12 µM,, respectively); (b) SUCNR1 mRNA expression was significantly increased when compared with B2-CD or non-IBD controls. SUCNR1 mRNA expression correlates positively with Snail1 (r = 0.560) and Snail2 (r = 0.588) and negatively with E-cadherin (r = −0.714).

Conclusion

Succinate activates EMT in intestinal epithelial cells through SUCNR1. Both succinate levels and SUCNR1 expression are increased in intestine from B3-CD patients and correlates with EMT markers, which points to a new possible target for fistula treatment.