Gut Microbes · 15 authors, 7 centres
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This study demonstrates that gut microbiota from Crohn's disease patients with high proteolytic activity (CD-HPA) increases colitis severity in mice compared to microbiota with low proteolytic activity. The proinflammatory effect requires intact PAR2 signaling, as PAR2 cleavage-resistant mice were protected from worsened colitis. These findings identify microbial proteolytic activity as a potential therapeutic target in Crohn's disease.
**Background:** Emerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but its role in Crohn's disease (CD) remains unclear. Proteases from gut bacteria can promote inflammation through multiple mechanisms including matrix remodeling, mucus degradation, activation of protease-activated receptors (PARs), and modulation of cytokines. This study investigated whether high microbial proteolytic activity from CD patients is colitogenic and explored the underlying signaling pathways.
**Methods:** Fecal samples were selected from CD patients with high (CD-HPA, n=2) or low (CD-LPA, n=1) proteolytic activity, and from healthy controls with high (HC-HPA, n=1) or low (HC-LPA, n=1) activity. Adult and neonatal germ-free C57BL/6 mice were colonized with these microbiota. Additional experiments used Nod2−/− mice and R38E-PAR2 mice (PAR2 cleavage-resistant). After 3 weeks of colonization, fecal proteolytic, elastolytic, and mucolytic activities were measured. Microbial composition was assessed by 16S rRNA gene sequencing and PICRUSt2 functional predictions. Inflammatory gene expression was analyzed by NanoString nCounter. Acute colitis was induced using 2% DSS in drinking water for 5 days followed by 2 days of water, and histological scores were determined.
**Key Results:** Germ-free mice had high baseline proteolytic activity. Colonization with HC-LPA or CD-LPA decreased this activity, while CD-HPA increased overall proteolytic, elastolytic, and mucolytic activities. In vitro PAR2 cleavage was high in germ-free mice and decreased after colonization with HC-LPA, HC-HPA, or CD-LPA, but not CD-HPA. NanoString analysis identified 172 differentially expressed genes between groups; 18 genes differed between CD-LPA and CD-HPA, including upregulation of Map2k6, Rps6ka5, Ccl11, Ccl21, NFκB1, Il1rap, Il22ra2, and Claudin 1 in CD-HPA mice. PMN cell counts were higher in CD-HPA versus CD-LPA mice. CD-HPA mice had lower alpha diversity (Chao1 and Shannon indexes) and distinct beta diversity compared to CD-LPA. Beneficial bacteria including Akkermansia muciniphila, Alistipes putredinis, and Ruminococcus bromii were decreased, while Hungatella hathewayi and Romboutsia ilealis were increased in CD-HPA mice. The serine protease K04772 from H. hathewayi was actively transcribed at higher levels in CD-HPA mice. In DSS colitis experiments, C57BL/6 mice colonized with CD-HPA had higher microscopic scores than CD-LPA mice. Nod2−/− mice colonized with CD-HPA also had higher colitis severity. However, R38E-PAR2 mice colonized with CD-HPA had milder colitis compared to CD-LPA, with blunted Pi3k and Mapk3k responses.
**Clinical Implications:** This study provides evidence that high microbial proteolytic activity in CD is colitogenic and requires intact PAR2 signaling. The identification of H. hathewayi serine protease K04772 as a potential contributor to the proinflammatory phenotype suggests a specific microbial target. These findings support the investigation of microbial proteolytic signatures in IBD to identify patients who may benefit from anti-proteolytic therapies. The results also suggest that PAR2 could be a therapeutic target for mitigating microbiota-driven inflammation in CD.