Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS

Abstract

ObjectiveThe intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS.Design39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states.ResultsPatients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota.ConclusionA subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.

Role of gut microbiota in regulating gastrointestinal dysfunction and motor symptoms in a mouse model of Parkinson’s disease

“… 32 Briefly, on the day of measurement mice were fasted for 2 h then given a 200 µl oral gavage containing 100 mg/mL creatinine, 60 mg/mL FITC-4 kDa Dextran, and 40 mg/mL of rhodamine B isothiocyanate-70 kDa dextran (Sigma) to allow assessment of pore (radius 2.6 Å), leak (radius 13 Å) and unrestricted (radius 64 Å) barrier pathways, respectively. 32 Mice were fasted for additional 2 h after which food was replaced. Five hours post-gavage, the intracardiac puncture was performed to harvest blood and collect serum samples.…”

Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation

“…Taken together, these data have lent some credence to host serine proteases as playing critical roles in controlling the homeostatic network at the mucosal surface of the intestine, while largely dismissing the effect of their bacterial counterparts. A tight regulation of such proteases is though needed since an increased proteolytic activity could be detrimental to the epithelium and impair the intestinal barrier 49,50 …”

Extracellular vesicles released by the human gut symbiont Bacteroides thetaiotaomicron in the mouse intestine are enriched in a selected range of proteins that influence host cell physiology and metabolism

“…Moreover, 7 additional serpins (protease inhibition activity, InterPro family IPR000215) were identified with similar abundance in fasted and fed mice. It has been reported that high protease activity measured in the feces of patients suffering from irritable bowel syndrome correlates with a decrease in microbial diversity [62]. It is therefore tempting to speculate that the various serine protease inhibitors detected and identified in EVs produced in mice mono-colonised with Bt (submitted to Vesiclepedia, number) is a consequence of the lack of microbial diversity, and is to counteract the detrimental effect of proteases present in high abundance in the gut lumen [62] of mono-colonised mice.…”