Project Summary Humoral immunodeficiency is the most frequently diagnosed form of primary immunodeficiency in humans. Common variable immunodeficiency (CVID) is the most clinically severe form, and chronic gastrointestinal complications are both common in CVID patients and refractory to treatment by intravenous Ig therapy. A severe GI disorder is termed 'CVID enteropathy'; a small intestine (SI)-specific enteropathy that presents clinically as chronic diarrhea and malabsorption. Bile acids (BAs) are emulsifiers produced by the liver and secreted into the gut that promote the solubilization and absorption of dietary lipids and lipid-soluble vitamins. They may also serve as signaling molecules capable of inducing tolerogenic responses in tissue-resident immune cells. Mucosal antibody deficiency alters microbiota composition. Given that the microbiota profoundly influence luminal BA biochemistry, the overarching hypothesis this R01 proposal will address is that mucosal antibody- deficiency results in aberrant bacterial BA metabolism that abolishes BA-induced immunological tolerance in the SI. In this proposal, we provide evidence supporting that mucosal antibody-deficiency results in aberrant inflammatory immune responses that are associated with the development of SI enteropathy; potentially driven by elevated bacterial bile salt hydrolase (bsh) activity that causes BA malabsorption (BAM). We also identify that bsh ablation or oral BA supplementation alleviates BAM and suppresses inflammatory disease. The objective of Specific Aim #1 is to define how humoral immunity influences BA homeostasis, and how this coordinates establishment of a tolerogenic immune phenotype in the SI. Several complementary models will be utilized to address this fundamental question and will incorporate the advanced techniques of ultra-purity liquid chromatography mass spectrometry (UPLC-MS) to comprehensively characterize BA pool composition, and paired high-parameter flow cytometry and single cell RNA sequencing to characterize SI-resident immune cell phenotypes. The objective of Specific Aim #2 is to demonstrate how mucosal antibody deficiency influences the composition and BA-metabolizing capacity of the SI-resident microbial community. Several novel and complementary microbial colonization models, adoptive transfer models, and next-gen sequencing approaches will be utilized to address this. The objective of Specific Aim #3 is to test the efficacy of two candidate approaches to alleviate inflammatory SI enteropathies and involve oral BA supplementation or pharmacological knockdown of bacterial bsh activity. Innovations from this research include the first analysis of the role of humoral immunity plays in regulating BA metabolism, a comprehensive assessment of the role mucosal IgA and IgM antibody responses play in SI homeostasis, characterization of a novel mechanism by which dysbiosis drives disease, and characterization of the role bsh ablation and oral BA supplementa...