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P079 Increased faecal proteolytic activity in pouchitis patients mediates epithelial barrier disruption through activation of protease-activated receptor-2

S. Hoffman*1, I. M. Carrol2, H. Tulchinski3,4, I. Borovok5, I. Dotan6

1Tel Aviv medical Center, Digestive and liver Disease Research Center, Tel Aviv, Israel, 2University of North Carolina, Center for Gastrointestinal Biology and Disease, School of Medicine, Chapel Hill, USA, 3Tel Aviv Medical Center, Division of Surgery Colorectal Unit, Tel Aviv, Israel, 4Tel-Aviv University, Sackler Faculty of Medicine, Tel Aviv, Israel, 5Tel-Aviv University, Department of Molecular and Microbiology and Biotechnology, Tel Aviv, Israel, 6Rabin Medical Center, Division of Gastroenterology, Petah Tikva, Israel

Background

Pouchitis in ulcerative colitis (UC) patients is thought to occur due to disruption of the epithelial barrier resulting in an abnormal immune response to a dysbiotic microbiota. We aimed to examine whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction and explore the pathways involved.

Methods

Faecal protease activity was measured using a FITC-casein florescence assay. Caco-2 cells monolayers were exposed to faecal supernatants of participants. Trans-epithelial electrical resistance and FITC-Dextran were used to determine monolayers' maturity and permeability. Tight junction (TJ) proteins integrity and protease-activated receptors (PARs) activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 expression/activation was examined in human pouch biopsies using antibodies directed to the N-terminus of the protein.

Results

Twenty-five patients, including 10 pouchitis, 6 normal pouch (NP) and 9 healthy (HC) participants, were recruited. Pouchitis patients exhibited a 5.19- and 5.35-fold increase in proteolytic activity (p ≤ 0.05) compared with NP and HC participants,, respectively. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers and disrupted TJ proteins, resulting in increased epithelial permeability. Truncation of PAR2 in Caco-2 monolayers, leading to its inactivation, abrogated increased faecal protease-mediated permeability. Human pouch biopsies displayed PAR2 activation in pouchitis but not in NP specimens.

Conclusion

Increased luminal proteolytic activity in pouchitis patients leads to disruption of tight junction proteins and increased epithelial cells permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.