title: Luminal cysteine-proteases degrade colonic tight junction structure and are responsible for abdominal pain in constipation-predominant IBS.
abstract: OBJECTIVE
  Luminal serine-proteases lead to increased colonic paracellular permeability and visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Other proteases, namely cysteine-proteases (CPs), increase airway permeability by digesting epithelial tight junction proteins. In this study, we focused on constipation-predominant IBS (IBS-C) and we aimed to (i) evaluate CP levels in two cohorts of IBS patients, (ii) test if IBS-C fecal supernatant (FSN) affects permeability, and visceral sensitivity after repeated administrations in mice, and (iii) evaluate occludin expression in IBS-C colonic biopsies.
  
  
  METHODS
  Fecal CP activity was determined using selective substrate and inhibitor (E64). The effect of papain, as positive control, and IBS-C FSN administrations were evaluated on colonic paracellular permeability and mucosal occludin levels in mice and T84 monolayers. Occludin protein levels were evaluated in IBS-C colonic biopsies. Sensitivity to colorectal distension (CRD) was measured after repeated administrations of IBS-C FSN.
  
  
  RESULTS
  We found in a subset of IBS-C patients an enhanced fecal CP activity, in comparison with healthy controls and IBS-D patients. CP activity levels positively correlated with disease severity and abdominal pain scoring. This association was confirmed by receiver operating characteristic curve analysis. In mice, repeated application of IBS-C FSN into colon triggered increased permeability, linked to the enzymatic degradation of occludin, and was associated with enhanced visceral sensitivity to CRD. Finally, occludin levels were found decreased in colonic biopsies from IBS-C patients, and IBS-C FSNs were able to degrade recombinant human occludin in vitro. All these effects were abolished by preincubation of IBS-C FSN with a CP inhibitor, E64.
  
  
  CONCLUSIONS
  These data suggest that luminal CPs may represent a new factor contributing to the genesis of symptoms in IBS.
authors.affiliation: INRA, UMR1331, Neuro-Gastroenterology and Nutrition Group, Toulouse, France.
authors.forename: Anita
authors.initials: A
authors.lastname: Annaházi
authors.forename: Laurent
authors.initials: L
authors.lastname: Ferrier
authors.forename: Valerie
authors.initials: V
authors.lastname: Bézirard
authors.forename: Mathilde
authors.initials: M
authors.lastname: Lévêque
authors.forename: Helene
authors.initials: H
authors.lastname: Eutamène
authors.forename: Afifa
authors.initials: A
authors.lastname: Ait-Belgnaoui
authors.forename: Moïse
authors.initials: M
authors.lastname: Coëffier
authors.forename: Philippe
authors.initials: P
authors.lastname: Ducrotté
authors.forename: Richárd
authors.initials: R
authors.lastname: Róka
authors.forename: Orsolya
authors.initials: O
authors.lastname: Inczefi
authors.forename: Krisztina
authors.initials: K
authors.lastname: Gecse
authors.forename: András
authors.initials: A
authors.lastname: Rosztóczy
authors.forename: Tamás
authors.initials: T
authors.lastname: Molnár
authors.forename: Tamar
authors.initials: T
authors.lastname: Ringel-Kulka
authors.forename: Yehuda
authors.initials: Y
authors.lastname: Ringel
authors.forename: Thierry
authors.initials: T
authors.lastname: Piche
authors.forename: Vassilia
authors.initials: V
authors.lastname: Theodorou
authors.forename: Tibor
authors.initials: T
authors.lastname: Wittmann
authors.forename: Lionel
authors.initials: L
authors.lastname: Bueno
mesh_terms: D015746:Abdominal Pain / Q000201:enzymology / Q000473:pathology; D000328:Adult; D000704:Analysis of Variance; D000818:Animals; D001706:Biopsy; D015153:Blotting, Western; D016022:Case-Control Studies; D002478:Cells, Cultured; D003248:Constipation / Q000201:enzymology / Q000473:pathology; D057056:Cysteine Proteases / Q000378:metabolism*; D004576:Electromyography; D005243:Feces / Q000201:enzymology; D005260:Female; D006801:Humans; D007408:Intestinal Absorption; D043183:Irritable Bowel Syndrome / Q000201:enzymology* / Q000473:pathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D062793:Occludin / Q000378:metabolism; D010147:Pain Measurement; D016133:Polymerase Chain Reaction; D012372:ROC Curve; D011795:Surveys and Questionnaires; D019108:Tight Junctions / Q000201:enzymology* / Q000473:pathology*
chemical_list: D062793:Occludin; D057056:Cysteine Proteases