Divergent immue responses to commensal Clostridia classified based on motility gene arrangement and flagellin diversity

Flagella-dependent motility is a defining feature of several species of Clostridia that colonize the intestine. The genomes of these bacteria encode varying numbers of flagellins – the basic subunits of flagella - and the machinery necessary for synthesis, assembly, and function of flagella. Although adaptive immune responses to commensal flagellins are hallmarks of Crohn's disease, it is unclear whether flagellins themselves drive inflammation, or what defines pro-inflammatory flagellated commensals. Here, we show that the arrangement of motility loci and the diversity of encoded flagellins can separate flagellated Clostridia into at least 2 functionally distinct subgroups. Both subgroups induce tolerogenic responses at homeostasis but possess divergent capacities for TLR5 activation and induction of inflammation. We identify a hypervariable region of the flagellin D0 domain that modulates the capacity for TLR5 activation and demonstrate enrichment of bacteria in the pro-inflammatory subgroup in Crohn's disease biopsies. Collectively, our study identified a subset of colitogenic commensals and revealed one mechanism whereby these organisms can directly initiate intestinal inflammation. Overall design: Three groups of untreated mice: 1) gnotobiotic with three select G1 bacteria, 2) gnotobiotic with three select G2 bacteria, and 3) germ-free; three replicates of each group for a total of nine samples.