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

Abstract

Objective: The intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS.

Design: 39 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.

Results: Patients 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.

Conclusion: A 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.

Keywords: Campylobacter; gastroenteritis; germ-free mice; microbiome; trypsin.

Figure 1:

Characterisation of faecal proteolytic activity (PA). (A) Faecal PA in IBS and healthy subjects. PA of faecal supernatants (FSNs) was significantly higher in patients with IBS (n=39) compared with healthy volunteers (n=25) (p<0.05, one-way analysis of variance [ANOVA]). Eleven of 39 patients with IBS had PA >1429 BAEE units/mg protein (dotted line, 95th percentile PA for healthy volunteers), which was used to classify patients with IBS as ‘high PA’ and ‘low PA’. (B) Effect of protease inhibitors on faecal PA. PA from high-PA FSNs was significantly inhibited by serine protease inhibitor (AEBSF) and cysteine protease inhibitor (E64) (n=6/group, p<0.05, one-way ANOVA with repeated measures). No significant effect of inhibitors was seen with low-PA FSNs. (C) Serine protease characterisation using specific probes. High-PA IBS FSNs have greater trypsin-like, chymotrypsin-like, neutrophilic elastase-like activities (n=5/group, p<0.05, Mann-Whitney U test) but not pancreatic elastase-like or kallikrenin-like activities when compared with low PA IBS FSNs. AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; IBS-C, constipation-predominant IBS; PI-IBS, postinfection IBS.

Figure 2:

Effects of faecal supernatants on in vitro barrier function and effects of PAR-2 inhibition. (A) Effect of faecal PA on in vitro macromolecular flux. High proteolytic activity (PA) FSNs caused greater apical to basolateral flux of 3 KDa Texas Red Dextran across Caco-2 monolayers compared with low-PA FSNs (p<0.01) which was significantly inhibited by AEBSF (n=6/group, p<0.05, one-way analysis of variance [ANOVA] with repeated measures) but not by E64. Low-PA FSNs had no significant effect on flux. Similarly, AEBSF and E64 alone had no significant effect on flux. (B and C) Effect of faecal PA on in vitro transepithelial resistance (TER). High-PA FSNs caused significantly higher TER drop compared with low PA FSNs (n=6/group, p<0.01). TER drop caused by high-PA application was significantly inhibited by AEBSF and ENMD1068, a selective PAR-2 antagonist but not by E64 (n=6/group, p<0.05, one-way ANOVA with repeated measures for AEBSF and ENMD1068). Averaged 24-hour TER tracing: inhibitor added 30 min prior to FSN addition. (D) PAR-2 inhibition using siRNA. At day 7 following transfection of Caco-2 cells with RNAi, 40% inhibition of PAR-2 protein was observed. (E and F) siPAR-2-mediated inhibition of effects on barrier function. The TER drop after addition of high-PA FSN from day 6 to day 7 was significantly lower in siPAR-2 Caco-2 cells compared with negative controls (n=6/group, p<0.05, Mann-Whitney U test). FSNs, faecal supernatants.

Figure 3:

Effect of faecal supernatants on myosin light chain phosphorylation and occludin (A) pMLC/MLC protein expression following FSN incubation. High-PA FSNs caused significantly increased pMLC (20kDa) in Caco-2 cells compared to low PA FSNs. Red: target band; green: β-actin. (B) Quantitative changes in pMLC/MLC ratio. pMLC expression was significantly higher after high-PA FSNs application (n=5–6/group, p<0.01, Mann-Whitney U test). The protein bands were measured using densitometric quantitation with ImageJ software, normalised to β-actin. (C) pMLC localisation following FSN incubation. Immunofluorescence showing pMLC protein following high-PA and low-PA FSNs. pMLC colocalised with phalloidin staining. (D) Occludin protein expression following FSN incubation. High PA FSNs caused significantly decreased occludin protein (63kDa) in Caco-2 cells compared with low-PA FSNs. Red: target band; green: β-actin. (E) Quantitative changes in occludin protein. Occludin protein was significantly lower after high-PA FSNs application (n=5–6/group, p<0.05, Mann-Whitney U test). The protein bands were measured using densitometric quantitation with ImageJ software, normalised to β-actin. (F) Occludin localisation following FSN incubation. Immunofluorescence showing occludin following high-PA and low-PA FSNs. Occludin was internalised into cytoplasm from tight junction after high PA FSNs application. Scale bar=20μm. FSN, faecal supernatant; PA, proteolytic activity.

Figure 4:

Occludin protein and tight junction structure in sigmoid colonic biopsies from patients with high-PA and low-PA IBS. (A) Occludin protein expression. Biopsies of high-PA patients showed decreased expression of occludin (63 kDa) compared with low-PA patients. Red: target band; green: β-actin. (B) Quantitative changes in occludin expression. Occludin expression was significantly lower in high-PA patients (n=4–5/group, p<.05, Mann-Whitney U test). The protein bands were measured using densitometric quantitation with ImageJ software, normalised to β-actin. (C) Occludin localisation. Immunofluorescence showing occludin in sigmoid colonic biopsies of high-PA and low-PA IBS patients. (D and E) Apical junction complex ultrastructure. Representative scanning electron microscopy images from a high-PA and low-PA IBS patient, respectively. (F) Quantitative comparison of tight junction intercellular distance. High-PA patients revealed greater intercellular space at TJ space than low-PA IBS (n=6/group, p<.05, Mann-Whitney U test). (G) In situ zymography showing trypsin-like PA in sigmoid colonic biopsies. No differences were seen between a pilot set of high and low PA IBS patients (n=3–4/group, p>0.05, Mann-Whitney U test). PA, proteolytic activity.

Figure 5:

Microbiome composition of patients with high-PA and low-PA IBS. (A–C) Alpha diversity indices. Patients with high-PA IBS have lower microbial diversity than low-PA patients on observed operational taxonomic units (OTU), Shannon and Inverse Simpson tests (p<0.05). (D) Beta diversity index. Patients with high-PA IBS have different community structure than patients with low-PA IBS as shown by unweighted UniFrac analysis of beta diversity (p<0.05). PA, proteolytic activity.

Figure 6:

Proteolytic activity and in vivo permeability changes in humanised mice. (A) Schematic showing humanisation of germ-free mice. Faecal slurry from healthy, patients with high-PA IBS and low-PA IBS were administered in 4-week-old germfree mice. PA assessment was done just at baseline (just prior to administering faecal slurry) and after 6 weeks (humanised state). In vivo permeability assessment was done in humanised state. (B) PA changes after humanisation. Lower inhibition from baseline (germ free) or increased production were seen in mice humanised with high PA as compared with healthy and low PA human stool (n=2129 mice/group, 3–4 donors/group, p<0.05, one-way analysis of variance [ANOVA]). Hollow symbols represent mice that were only used for PA assessment and not for in vivo permeability assessment. (C) In vivo permeability assessment in humanised mice. Greater permeability for creatinine in high-PA humanised mice was seen as compared with low-PA and healthy humanised mice (n=9–13 mice/group, 3–4 donors/group, p<0.05 healthy vs high PA and p=0.05 low vs high PA, one-way ANOVA with post hoc calculations). FITC-Dextran 4kDa and Rhodamine-Dextran 70 kDa permeability were similar in the three groups. (D) Correlations between PA changes and in vivo permeability. Creatinine permeability correlates with % PA change following humanisation (Spearman r=0.4, p<0.05). FITC-Dextran 4kDa and Rhodamine-Dextran 70 kDa permeability do not correlate with % PA change. PA, proteolytic activity.