Fecal supernatants from constipated IBS patients increase colonic permeability in mice by occludin degradation linked to cysteine-protease activity

Background and aims: Increased gut permeability has been described in all IBS subgroups. We have previously shown that fecal cysteine-protease (CP) activity is elevated only in constipated IBS (IBS-C) patients among IBS subtypes. Since CPs are involved in the increase of airway permeability by digesting tight junction proteins, we evaluated the ability of CP in the IBS-C fecal supernatant (FSN) to increase permeability and degrade tight junction proteins in mice and T84 human cell cultures.

Methods: CP activity was assayed in fecal samples from healthy subjects (n=20) and IBS-C patients (n=25) using a selective substrate and controlled by a selective CP inhibitor, E64. FSN from IBS-C and healthy controls pre-incubated or not with E64, or papain solution (as a control CP) or vehicle were (i) infused intracolonically (IC) in C57/BL6 mice (twice at 12h interval) or (ii) added to the apical site of T84 cells seeded in a 24-well Fluoblock® plate system. Intestinal permeability to oral FITC-4kD dextran was assessed 4h after the end of the second IC infusion. Permeability to FITC-4kD dextran was measured every hour over 24 hours in T84 cells. Expression of occludin in mice and T84 cells was evaluated by Western blotting and immunostaining.

Results: CP activity in IBS-C FSN was significantly increased (p<0.05) vs. healthy controls (2420±718 and 319±91 Δfluo/mg prot). IBS-C FSN and papain IC infusion increased colonic permeability in mice compared to healthy control FSN or saline (403±53 vs. 333±43; 562±73 vs. 341±40 Δfluo/µl respectively) as well as in T84 cells (34% increase vs. control FSN, starting at 15h after application). This effect was markedly reduced by E64 both in mice and T84 cells. Occludin protein levels were decreased by IBS-C FSN or papain infusion in mice colon by 53 and 50% vs. healthy FSN or vehicle, respectively, and this effect was prevented by E64. Occludin degradation was observed in T84 cells incubated with IBS-C FSN or papain, prevented by E64.

Conclusion: This study indicates both in vivo and in vitro that elevated cysteine-protease activity found in IBS-C fecal supernatant disrupts tight junction integrity through occludin enzymatic degradation. Our results suggest the potential role of elevated fecal cysteine-protease activity in the pathomechanism of IBS-C.

TÁMOP-4.2.2–08/01–2008–0002