Functional GI DisordersLuminal Cysteine-Proteases Degrade Colonic Tight Junction Structure and Are Responsible for Abdominal Pain in Constipation-Predominant IBSAnnaházi, Anita MD1, 2, 3, 9; Ferrier, Laurent PhD1, 2, 9; Bézirard, Valerie MSc1, 2, 9; Lévêque, Mathilde BSc1, 2; Eutamène, Helene PhD1, 2; Ait-Belgnaoui, Afifa PhD1, 2; Coëffier, Moïse MD, PhD4; Ducrotté, Philippe MD, PhD5; Róka, Richárd MD, PhD3; Inczefi, Orsolya MD3; Gecse, Krisztina MD, PhD3; Rosztóczy, András MD, PhD3; Molnár, Tamás MD, PhD3; Ringel-Kulka, Tamar MD, MPH6; Ringel, Yehuda MD7; Piche, Thierry MD, PhD8; Theodorou, Vassilia PhD1, 2; Wittmann, Tibor MD, PhD3; Bueno, Lionel PhD1, 2Author Information 1 INRA, UMR1331, Neuro-Gastroenterology and Nutrition Group, Toulouse, France 2 EI-Purpan, INP, UMR1331, Neuro-Gastroenterology and Nutrition Group, Toulouse, France 3 1st Department of Internal Medicine, University of Szeged, Szeged, Hungary 4 INSERM UMR1073, Rouen University and Nutrition Unit, Rouen University Hospital, Rouen, France 5 INSERM UMR1073, Rouen University and Department of Gastroenterology, Rouen University Hospital, Rouen, France 6 Department of Maternal and Child Health, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, USA 7 Department of Medicine, Division of Gastroenterology and Hepatology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA 8 Department of Gastroenterology, INSERM U576, Hôpital de l’Archet II, Nice, France 9 These authors contributed equally to this work Correspondence: Lionel Bueno, PhD, INRA, UMR1331, Neuro-Gastroenterology and Nutrition, 180 Chemin de Tournefeuille, 31027 Toulouse Cedex 3, France. E-mail: firstname.lastname@example.org SUPPLEMENTARY MATERIAL accompanies this paper at http://links.lww.com/AJG/A790 Received 22 January 2013; accepted 22 April 2013 published online 28 May 2013 American Journal of Gastroenterology: August 2013 - Volume 108 - Issue 8 - p 1322–1331 doi: 10.1038/ajg.2013.152 Buy SDC Metrics Abstract OBJECTIVES: 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 occludinin 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. © The American College of Gastroenterology 2013. All Rights Reserved.