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

Research paper by Sarit S Hoffman, Nathaniel Aviv NA Cohen, Ian M IM Carroll, Hagit H Tulchinsky, Ilya I Borovok, Iris I Dotan, Nitsan N Maharshak

Indexed on: 07 May '19Published on: 06 May '19Published in: Journal of Crohn's & colitis



Abstract

The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether fecal 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. Fecal samples were measured for protease activity using a fluorescein isothiocyanate (FITC)-casein florescence assay. Caco-2 cell monolayers were exposed to fecal 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 analyzed using Illumina MiSeq platform of the 16S rRNA gene. Ten pouchitis patients, 6 normal pouch (NP) patients and 9 healthy controls (HC) were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher fecal protease (FP) activity (p ≤ 0.05) compared to the NP and HC participants, respectively. The Haemophilus species was positively associated with FP activity (R=0.718, FDR<0.1).Fecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted TJ proteins, and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated fecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Protease-producing bacteria may increase fecal proteolytic activity that results in pouch inflammation through disruption of TJ proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation. Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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