CD4 + effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff actually adopt in frontline tissues in vivo , we applied single-cell transcriptome and chromatin analysis on colonic Teff cells, in germ-free or conventional mice, or after challenge with a range of phenotypically biasing microbes. Subsets were marked by expression of interferon-signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic T H subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T H markers distributed in a polarized continuum, which was also functionally validated. Clones derived from single progenitors gave rise to both IFN-γ and IL17-producing cells. Most transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activity of AP1 and IRF transcription factor families, not the canonical subset master regulators T-bet, GATA3, RORγ.

Host inflammatory responses against pathogenic organisms can be abrogated by commensals; however, the molecular mechanisms by which pathogenesis is prevented are still poorly understood. Previous studies demonstrated that administration of a single dose of Bacillus subtilis prevented disease and inflammation by the enteric mouse pathogen Citrobacter rodentium, which causes disease similar to the human pathogen enteropathogenic E. coli. No protection was observed when an exopolysaccharide (EPS)-deficient mutant of B. subtilis was used, suggesting that EPS are the protective factor. Here, we isolated and characterized EPS, and showed that they also prevent C. rodentium-associated intestinal disease after a single injection. Protection is TLR4-dependent, since EPS-treated TLR4 KO mice developed disease. Further, protection could be conveyed to wild-type (wt) mice by adoptive transfer of macrophage-rich peritoneal cells from EPS-treated mice. We found that EPS specifically bind peritoneal macrophages and because mice lacking MyD88 signaling in myeloid cells were not protected by EPS, we conclude that bacterial EPS prevent colitis in a TLR4-dependent manner that requires myeloid cells. These studies provide a simple means of preventing intestinal inflammation caused by enteric pathogens.

Commensal bacteria contribute to immune homeostasis in the gastrointestinal tract; however, the underlying mechanisms for this are not well understood. A single dose of exopolysaccharide (EPS) from the probiotic spore-forming bacterium Bacillus subtilis protects mice from acute colitis induced by the enteric pathogen Citrobacter rodentium. Adoptive transfer of macrophage-rich peritoneal cells from EPS-treated mice confers protection from disease to recipient mice. In vivo, EPS induces development of anti-inflammatory M2 macrophages in a TLR4-dependent manner, and these cells inhibit T cell activation both in vitro and in C. rodentium-infected mice. In vitro, the M2 macrophages inhibit both CD4+ and CD8+ T cells, and the inhibition of CD4+ T cells is dependent on TGF-β, whereas inhibition of CD8+ T cells is dependent on both TGF-β and PD-L1. We suggest that administration of B. subtilis EPS can be utilized to broadly inhibit T cell activation and thus control T cell-mediated immune responses in numerous inflammatory diseases.

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