Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier

Sarit Hoffman, Nathaniel Aviv Cohen, Ian M. Carroll, Hagit Tulchinsky, Ilya Borovok, Iris Dotan, Nitsan Maharshak*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Background and Aims: The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. Methods: Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR 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 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. Results: Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Conclusions: Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Original languageEnglish
Pages (from-to)1558-1568
Number of pages11
JournalJournal of Crohn's and Colitis
Volume13
Issue number12
DOIs
StatePublished - 10 Dec 2019

Funding

FundersFunder number
Alexander Germanis Research Fund in Gastroenterology and the Research Fund of the Sackler Faculty of Medicine of Tel Aviv University
Leona M. and Harry B. Helmsley Charitable Trust

    Keywords

    • Pouch
    • gut mucosal barrier function
    • microbiome
    • proteolytic activity
    • tight junctions

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