Human gut bacteria produce T_H17-modulating bile acid metabolites

Abstract

The microbiota plays a pivotal role in gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17a (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. While it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown. Furthermore, it was not clear whether 3-oxoLCA and other immunomodulatory bile acids are associated with gut inflammatory pathologies in humans. Using a high-throughput screen, we identified human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Like 3-oxoLCA, isoLCA suppressed TH17 differentiation by inhibiting RORγt (retinoic acid receptor-related orphan nuclear receptor γt), a key TH17 cell-promoting transcription factor. Levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase (3α-HSDH) genes required for their biosynthesis were significantly reduced in patients with inflammatory bowel diseases (IBD). Moreover, levels of these bile acids were inversely correlated with expression of TH17 cell-associated genes. Overall, our data suggest that bacterially produced TH17 cell-inhibitory bile acids may reduce the risk of autoimmune and inflammatory disorders such as IBD.

A bacterial bile acid metabolite modulates Treg activity through the nuclear hormone receptor NR4A1

“…Bacteroidetes species produce isoalloLCA from the gut bacterial metabolite 3-oxoLCA To identify individual gut bacterial strains that produce isoal-loLCA, we performed a screen of 990 gut bacterial isolates obtained from human stool samples (Paik et al, 2021) (Figure S1A). Single colonies were incubated for up to 96 h with either LCA or 3-oxolithocholic acid (3-oxoLCA) (100 mM), two bile acids found in the human gut (Hamilton et al, 2007;Hang et al, 2019) that we hypothesized could be precursors to isoalloLCA (Figure 1A).…”

“…Human gut bacteria, specifically Clostridium scindens and its close genetic relatives, convert the primary bile acid chenodeoxycholic acid (CDCA) (1) to the secondary bile acid LCA (2) through enzymes encoded by the bile-acid-inducible (bai) operon (Figure 1A) (Funabashi et al, 2020;White et al, 1980). Furthermore, in recent work, we showed that a variety of gut bacteria from the phyla Actinobacteria and Firmicutes convert LCA (2) to 3-oxoLCA (3) through the action of 3a-hydroxysteroid dehydrogenase (3a-HSDH) enzymes (Paik et al, 2021). Given our finding that gut bacteria convert 3-oxoLCA (3) to isoalloLCA (4), we hypothesized that multiple species of gut bacteria could be acting together to convert the host-produced bile acid CDCA to isoalloLCA.…”

“…Secondary bile acids are produced exclusively by gut bacteria and can be further transformed by the resident microbiota into a variety of metabolites with distinct physiological activities (Paik et al, 2021;Ridlon et al, 2006;Russell, 2003;Wahlströ m et al, 2016). In many cases, the bacteria and the biosynthetic pathways responsible for the production of these metabolites are unknown.…”

“…To screen human isolates for isoalloLCA production, isolates were retrieved from stock plates from our human fecal screen library (Paik et al, 2021) and cultured in 600 mL Cullen-Haiser Gut (CHG) media (Hall et al, 2017), which consists of BHI supplemented with 1 % BBL vitamin K1-hemin solution (BD Biosciences, 212354), 1% trace minerals solution (ATCC, MD-TMS), 1 % trace vitamins solution (ATCC, MD-VS), 5% fetal heat-inactivated bovine serum (FBS) (Genesee Scientific, 25-514), 1 g/L cellobiose (Sigma-Aldrich, C7252), 1 g/L maltose (Sigma-Aldrich, M5895) and 1 g/L fructose (Sigma-Aldrich, F0127), containing 0.5% (w/v) arginine (Sigma-Aldrich, A5006) for 48 hours at 37 C in 96-well plates. Each isolate, as well as the negative controls, was then diluted 1:10 in new media containing 100 mM LCA (Sigma-Aldrich) or 100 mM 3-oxoLCA (Steraloids) for an additional 48 hours.…”

“…We identified isoalloLCA in the HMP2 metabolomics dataset by running a pure standard of isoalloLCA in negative mode under the same LC-MS conditions used to collect HMP2 data and matching the m/z and chromatographic retention time of this known compound to a compound in HMP2: C18n_QI13593, m/z = 375.2905 at 11.04 min (Paik et al, 2021). Differential abundance of isoalloLCA with respect to IBD diagnosis and dysbiosis state in the longitudinally sampled HMP2 cohort were determined using linear-mixed model models following our recent work (Paik et al, 2021).…”