Altered host-microbe interactions and increased intestinal permeability have been implicated in disease pathogenesis. However, the mechanisms by which intestinal microbes affect epithelial barrier integrity remain unclear. Here, we investigate the impact of bacterial metabolism of host-produced bile acid (BA) metabolites on epithelial barrier integrity. We observe that rats fed a choline-deficient, l -amino acid–defined, high-fat diet (CDAHFD) exhibit reduced intestinal abundance of host-produced conjugated BAs at early time points, coinciding with increased gut permeability. We show that in vitro, conjugated BAs protect gut epithelial monolayers from damage caused by bacterially produced unconjugated BAs through micelle formation. We then demonstrate that inhibition of bacterial BA deconjugation with a small-molecule inhibitor prevents the development of pathologic intestinal permeability and hepatic inflammation in CDAHFD-fed rats. Our study identifies a signaling-independent, physicochemical mechanism for conjugated BA-mediated protection of epithelial barrier function and suggests that rational manipulation of microbial BA metabolism could be leveraged to regulate gut barrier integrity.

In this renaissance era of gene therapy, a new study published by the Susan Kaech lab in Science demonstrates the use of CRISPR-Cas9 technology to selectively deplete conjugated bile acids in the liver by targeting the bile acid–CoA:amino acid N-acyltransferase (Baat) gene to improve responsiveness to immunotherapy. This study highlights the role of conjugated bile acids in impairing intratumoral T cell function by directly accumulating in resident liver T cells and driving mitochondrial dysfunction. Knockout of Baat reduced hepatic conjugated bile acid production, thus improving immunotherapy potency and reducing tumor burden. Subsequently, Baat liver knockout reduced levels of microbially produced secondary bile acids such as lithocholic acid, a known carcinogen and T cell toxin. This study mechanistically links bile acids to liver cancer immunotherapy success, setting the stage for bile acid-based screening approaches and pharmacologic manipulations for improved patient outcomes.

Folates participate in the one-carbon metabolism (OCM) cycle, supporting many biochemical pathways. Existing methods to profile folate are limited in the diversity of vitamers they measure and the samples they profile. Here we present a metabolomics workflow for stable extraction, separation and measurement of folates, along with precursors and products of OCM-associated pathways. We profile these metabolites in 37 mouse tissues to chart an interactive 'OCM atlas' ( https://chaudharilab.com/folate-atlas/ ), revealing vast heterogeneity across organs and an uncharacterized folate derivative. We discover that, in adult mice, the gut microbiota is a consumer of folate and folate polyglutamylation in the host is not regulated by folate availability. Germ-free mice show tissue-specific shifts in methyl donor abundances relative to conventionally raised mice, indicative of altered DNA methylation. Correlation analyses uncover the central roles of folates in potentially modulating other biochemical pathways in tissues, thus linking microbial folate consumption directly to its global impacts on host metabolism.

Agonists of the G protein-coupled receptor TGR5 have long been sought-after for their metabolic benefits. Intestinal TGR5 activation induces secretion of the antidiabetic hormone GLP-1, which can systemically improve diabetes phenotypes in multiple organs. However, no TGR5 agonist drug candidate has succeeded in clinical trials due to their low potency and unwanted side effects. A challenge in the field has been the development of TGR5 agonists that are non-toxic, long-acting, and have functional selectivity for G protein-biased agonism. In this study, we propose a systematic pipeline for engineering optimal TGR5 agonists with antidiabetic properties. This pipeline is interdisciplinary, combining in silico, in vitro, and in vivo assays to design and validate drug candidates. We identify 2 lead compounds that outline the necessary beneficial properties for a successful TGR5 agonist against diabetes. We uncover the molecular mechanisms that allow TGR5 agonists to induce the transcription of their target, TGR5, in intestinal enteroendocrine cells. Lastly, we investigate the molecular interactions of our lead candidates in the TGR5 binding pocket to identify optimal parameters for stability and biological activity. Our strategy for TGR5 agonist design and evaluation has the potential to guide the discovery process for targeted TGR5 therapeutics for metabolic diseases.

SUMMARY: Methotrexate (MTX) therapy in inflammatory bowel disease (IBD) is often limited by inter-individual variability in clinical response and adverse effects. Gut microbiota contribute to MTX therapeutic response and toxicity by metabolizing MTX and altering its bioavailability. However, how inflammation alters microbial MTX metabolism and how its metabolites influence the host remain poorly understood. Here, we identify Clostridium asparagiforme as a potent and efficient metabolizer of methotrexate, producing deoxyaminopteroic acid (DAMPA) in the distal gastrointestinal tract. We demonstrate that DAMPA preserves mitochondrial integrity by promoting mitophagy in intestinal epithelial cells through mitochondrial STAT3 signaling. DAMPA administration attenuates intestinal inflammation in vivo , and improves metabolic dysfunction associated with IBD. Together, these findings reveal an unappreciated role for a gut microbial MTX metabolite in mediating epithelial homeostasis during intestinal inflammation, thus reframing microbial MTX metabolism from passive drug detoxification to active regulation of host mitochondrial and inflammatory homeostasis.