Project Summary The gut is a major immunological organ where host-microbe interactions shape both local and systemic immune tolerance. However, current views of intestinal immune regulation ignore fundamental differences in the function and metabolite composition of the two distinct organs that comprise the gut—the small and large intestine (or SI and LI). This impedes a more detailed understanding of immune regulatory mechanisms along the intestinal tract, and limits efforts to develop safer, more targeted treatments for the two major forms of human inflammatory bowel diseases (IBDs), ulcerative colitis and small bowel Crohn’s disease. We hypothesize that mucosal CD4 T cells use different sets of ligand-regulated nuclear receptors (NRs) in the SI and LI to control key regulatory functions, including IL-10 expression, to local concentration gradients of bile- and microbe-derived metabolites. On one hand, we have discovered that Foxp3– effector (Teff) subsets in the SI—including Th1 and Th17 cells— utilize the nuclear xenobiotic receptor, constitutive androstane receptor (CAR/Nr1i3), to direct a ‘hepatocyte-like’ transcriptional response to contend with high local bile acid (BA) concentrations, which are far greater in SI than in LI (millimolar vs micromolar) due to ‘enterohepatic’ circulation—where primary BAs synthesized in the liver, stored in the gallbladder, and secreted into the duodenum are actively reabsorbed by specialized enterocytes in the ileum for portal recirculation to the liver. Because BAs are lipophilic, they can be toxic and pro-inflammatory, and several nuclear receptors—including CAR—have evolved to suppress BA toxicity. These studies suggest that enterohepatic circulation establishes a unique SI microenvironment that is distinct from that in the LI and requires unique transcriptional machinery to protect T cells in the SI. Conversely, the LI harbors 103-107 times more bacteria than SI, and ~1000-fold lower BA concentrations. Accordingly, microbes and their metabolites— short chain fatty acids (SCFAs; e.g., butyrate), secondary BAs (produced via microbial metabolism of residual primary BAs)—are central to immune regulation in the LI. SCFAs inhibit histone deacetylase enzymes (HDACs) and stabilize Foxp3 gene expression in peripherally-induced T regulatory cells (iTregs), whereas secondary BAs appear to promote regulatory functions of RORgt+ Tregs in the LI through another NR, vitamin D receptor (VDR). Thus, while antigens from the enteric flora prime both pro- and anti-inflammatory T cell responses throughout the gut, marked concentration gradients of bile- and bacteria-derived metabolites in the SI vs. LI are sensed by different NRs to execute compartmentalized T cell regulatory functions. In testing this hypothesis, we will apply cutting-edge genomics and computational approaches to comprehensively map the contributions of each of the 49 mouse NRs to specialized regulatory functions in the SI and LI in vivo, using IL-10 expression as the primary screening target. We will also interrogate the regulation and molecular functions of two key NRs, CAR/Nr1i3 and VDR/Nr1i1, in SI type 1 regulatory (Tr1) and LI iTreg cells, respectively. These studies will advance understanding of lymphocyte specialization in the gut, and inform new approaches to treat IBDs.

Project Narrative The gut is a key immunological organ, where host-microbe interactions shape both local and systemic immune tolerance. Prevailing immunological views conflate the two organs that comprise the gut—the small and large intestines (or SI and LI)—yet recent evidence from our labs, and others, suggest that T cells infiltrating the SI and LI utilize unique sets of ligand-regulated nuclear receptors (NRs) to tether key regulatory functions, including IL-10 expression, to discrete bile- and microbe-derived metabolite classes. In this proposal, we use both agnostic and targeted approaches to interrogate the mechanisms by which NRs execute T cell regulatory functions in the SI vs. LI, thus laying a foundation for understanding compartmentalized immune regulation in the gut.