Crohn’s disease proteolytic microbiota enhances inflammation through PAR2 pathway in gnotobiotic mice

ABSTRACTEmerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but whether it also plays a role in Crohn’s disease (CD) remains unclear. We investigated the effects of colonizing adult and neonatal germ-free C57BL/6 mice with CD microbiota, selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), or microbiota from healthy controls with LPA (HC-LPA) or HPA (HC-HPA). We then investigated colitogenic mechanisms in gnotobiotic C57BL/6, and in mice with impaired Nucleotide-binding Oligomerization Domain-2 (NOD2) and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (Nod2−/−; R38E-PAR2 respectively). At sacrifice, total fecal proteolytic, elastolytic, and mucolytic activity were analyzed. Microbial community and predicted function were assessed by 16S rRNA gene sequencing and PICRUSt2. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity in germ-free mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with germ-free mice. CD-HPA mice had lower alpha diversity, distinct microbial profiles and higher fecal proteolytic activity compared with CD-LPA. C57BL/6 and Nod2−/− mice, but not R38E-PAR2, colonized with CD-HPA had higher colitis severity than those colonized with CD-LPA. Our results indicate that CD proteolytic microbiota is proinflammatory, increasing colitis severity through a PAR2 pathway

Acetylcholine-producing T cells in the intestine regulate antimicrobial peptide expression and microbial diversity

The cholinergic anti-inflammatory pathway reduces systemic tumor necrosis factor (TNF) via acetylcholine-producing memory T cells in the spleen. These choline acetyltransferase (ChAT)-expressing T cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-enhanced green fluorescent protein (eGFP) reporter mice. CD4(Cre) mice were crossed to ChAT(fl/fl) mice to achieve specific deletion of ChAT in CD4(+) T cells. We observed that the majority of ChAT-expressing T cells in the human and mouse intestine have characteristics of Th17 cells and coexpress IL17A, IL22, and RORC The generation of ChAT-expressing T cells was skewed by dendritic cells after activation of their adrenergic receptor β2 To evaluate ChAT T cell function, we generated CD4-specific ChAT-deficient mice. CD4ChAT(-/-) mice showed a reduced level of epithelial antimicrobial peptides lysozyme, defensin A, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT(-/-) mice. We conclude that ChAT-expressing T cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT-expressing T cells may function to mediate the host AMP secretion, microbial growth and expansio