P079 Increased faecal proteolytic activity in pouchitis patients mediates epithelial barrier disruption through activation of protease-activated receptor-2. (25th January 2019)

• Record Type: Journal Article
• Authors: Hoffman, S; Carrol, I M; Tulchinski, H; Borovok, I; Dotan, I
• Is Part Of: Journal of Crohn's and colitis. Volume 13(2019)Supplement 1
• Page Start: S124
• Page End: S125
• Publication Date: 2019-01-25
• Subjects: Inflammatory bowel diseases -- Periodicals; 616.344005
• DOI: 10.1093/ecco-jcc/jjy222.203
• Languages: English
• ISSNs: 1873-9946

Abstract

Abstract: 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. Conclusions: 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.