INVESTIGATING THE RELATIONSHIP BETWEEN MUCOSAL INFLAMMATION AND PROTEOLYTIC ACTIVITY FROM MUCOSA-ASSOCIATED MICROBIOTA IN CROHN’S DISEASE

Abstract

Background: The pathogenesis of inflammatory bowel disease, comprising of Crohn’s disease (CD) and ulcerative colitis (UC) involves host, genetic and environmental interactions, that include microbial factors. Proteolytic imbalance, characterized by increased inflammatory proteases, such as neutrophil elastase, and decreased epithelial antiproteases, such as elafin, has been described in IBD. Our lab has shown that high fecal proteolytic activity from IBD can be transferred to germ free mice via microbial colonization, leading to more severe colitis. This suggests the contribution of microbial proteases to inflammation in IBD. CD is characterized by areas of discrete inflammation often coexisting with nearby areas of spared mucosa. However, the mechanisms leading to this patchy pattern of inflammation is unknown. Building on our previous findings that inflamed CD mucosa is associated with bacteria “predicted” to exhibit proteolytic activity, I hypothesized that inflamed biopsies from CD patients display increased proteolytic activity and permeability. Aim: To investigate whether inflamed mucosal areas in CD are associated with higher proteolytic activity from microbial and/or host origin, and increased permeability. Methods: CD patients in flare (n=23) were recruited at McMaster University Medical Centre and matched (or paired) biopsies were collected at colonoscopy from inflamed and adjacent (within 5 cm) non-inflamed sites. Biopsy mucosal-to-serosal 51Cr-EDTA flux rate and tissue conductance were measured with Ussing chambers. Total proteolytic (trypsin), elastase-like (FITC-elastin), and mucolytic (0.05% mucin plates) activities were also assessed in biopsies from both sites and in situ zymography was used to measure and localize host derived elastase-like activity in biopsy samples. Mucosal brushing samples from the same participants were also collected and total RNA purified for metatranscriptomic analysis to characterize active microbial pathways with a focus on identifying highly expressed microbial proteases. Results: Paracellular permeability and tissue conductance were higher in biopsies from inflamed areas than in biopsies from matched non-inflamed areas (p<0.001). Elastase-like and mucolytic activities were higher in inflamed areas compared with matched non-inflamed areas (p<0.05 and p=0.04, respectively), no significance was detected in total proteolytic activity. In situ zymography revealed higher elastase-like activity in inflamed areas compared with non-inflamed areas (p=0.04), localized to both the epithelial and submucosa layers in inflamed tissues but only to the epithelial layer in matched non-inflamed tissue. Metatranscriptomics data analyze thus far suggests that specific microbial proteases are increased at inflamed sites in CD. Conclusions: The results support the hypothesis that localized variations in tissue inflammation, proteolytic activity and mucosal permeability in CD are causally linked, providing a rationale for the patchy distribution of mucosal injury. We propose a model where microbial elastase-like activity promotes inflammation and host proteolytic activity through barrier disruption. This mechanism could be targeted to modulate microbial proteolytic activity with the aim of decreasing host inflammation and tissue damage in CD.