16S rRNA sequencing of human stool

Abstract

<h4>Background & aims</h4>Bacterial flagellin is an important antigen in inflammatory bowel disease, but the role of flagellin-specific CD4⁺ T cells in disease pathogenesis remains unclear. Also unknown is how changes in intestinal microbiome intersect with those in microbiota-specific CD4⁺ T cells. We aimed to quantify and characterize flagellin-specific CD4⁺ T cells in Crohn's disease (CD) and ulcerative colitis (UC) patients and study their relationship with intestinal microbiome diversity.<h4>Methods</h4>Blood was collected from 3 cohorts that included CD patients, UC patients, and healthy controls. Flow cytometry analyzed CD4⁺ T cells specific for Lachnospiraceae-derived A4-Fla2 and Escherichia coli H18 FliC flagellins, or control vaccine antigens. Serum antiflagellin IgG and IgA antibodies were detected by enzyme-linked immunosorbent assay and stool samples were collected and subjected to 16S ribosomal DNA sequencing.<h4>Results</h4>Compared with healthy controls, CD and UC patients had lower frequencies of vaccine-antigen-specific CD4⁺ T cells and, as a proportion of vaccine-specific cells, higher frequencies of flagellin-specific CD4⁺ T cells. The proportion of flagellin-specific CD4⁺ T cells that were CXCR3^negCCR4⁺CCR6⁺ Th17 cells was reduced in CD and UC patients, with increased proportions of CD39⁺, PD-1⁺, and integrin β7⁺ cells. Microbiome analysis showed differentially abundant bacterial species in patient groups that correlated with immune responses to flagellin.<h4>Conclusions</h4>Both CD and UC patients have relative increases in the proportion of circulating Fla2-specific CD4⁺ T cells, which may be associated with changes in the intestinal microbiome. Evidence that the phenotype of these cells strongly correlate with disease severity provides insight into the potential roles of flagellin-specific CD4⁺ T cells in inflammatory bowel disease.