Host circadian signaling, feeding, and the gut microbiome are tightly interconnected. Changes in the gut microbial community can affect the expression of core clock genes, but the specific metabolites and molecular mechanisms that mediate this relationship remain largely unknown. Here, we sought to identify gut microbial metabolites that impact circadian signaling. Through a phenotypic screen of a focused library of gut microbial metabolites, we identified a bile acid metabolite, lithocholic acid (LCA), as a circadian modulator. LCA lengthened the circadian period of core clock gene hPer2 transcription in a dose-responsive manner in human colonic cells. We found evidence that LCA modulates the casein kinase 1 δ/ε (CK1δ/ε)-protein phosphatase 1 (PP1) feedback loop and stabilizes core clock protein cryptochrome 2 (CRY2). Furthermore, we showed that LCA feeding alters circadian transcription in mouse distal ileum and colon. Taken together, our work identifies LCA as a molecular link between host circadian biology and the microbiome. Because bile acids are secreted in response to feeding, our work provides potential mechanistic insight into the molecular nature of the food-entrainable oscillator by which peripheral clocks adapt to the timing of food intake. Given the association between circadian rhythm, feeding, and metabolic disease, our insights may offer a new avenue for modulating host health.

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammation and progressive fibrosis of the biliary tree. PSC pathogenesis remains poorly understood, and there are no effective therapies. Previous studies have observed associations between colonic and biliary microbiome alterations and PSC. We aimed to determine whether bacterial isolates cultured from PSC patient bile induce disease-associated phenotypes in cells, specifically cell death, epithelial permeability, inflammation, and changes in host-protective pathways. Bile was collected from PSC patients by endoscopic retrograde cholangiography and from non-PSC controls undergoing cholecystectomies. Biliary bacteria were cultured anaerobically, and 50 colonies per sample were identified by 16S sequencing. No bacteria were isolated from non-PSC controls, while bacteria were cultured from most PSC patients. The PSC bile microbiomes exhibited reduced diversity compared to the gut or oral cavity, with one or two species predominating. The effects of supernatants from seven PSC-associated bacterial isolates on cellular phenotypes were characterized using human colonic (Caco-2), hepatic (HepG2), and biliary (EGI-1) cells. Overall, PSC-associated bacteria produced factors cytotoxic to hepatic and biliary cells. An Enterococcus faecalis isolate, and to a lesser extent a Veillonella parvula isolate, induced epithelial permeability, while Escherichia coli, Fusobacterium necrophorum, and Klebsiella pneumoniae isolates induced inflammatory cytokines in biliary cells. Our data suggest that bacteria cultured from PSC bile induce cellular changes characteristic of PSC pathogenesis, with different isolates inducing distinct cellular responses. Our work provides a starting point for future research into bacterial contributions to PSC with the eventual goal of developing therapies for this disease.IMPORTANCEPrimary sclerosing cholangitis (PSC) is a chronic liver disease in which inflammation and scarring of the bile ducts cause bile to build up in the liver, leading to liver damage and eventually liver failure. The causes of this disease are poorly understood, and the only current treatment is a liver transplant. To develop new treatments, we must first better understand what leads to this disease. We examined whether bacteria isolated from PSC patient bile can cause disease-related responses in human biliary, liver, and intestinal cells. We observed that different PSC-associated bacteria can induce distinct disease-related cellular changes, including inflammation and cell death. These data suggest that the microbial community in PSC patients may indeed be linked to disease development. Our findings provide new starting points for further exploration into the poorly understood origins of PSC.

T cells, a cornerstone of the adaptive immune system, have pivotal roles at the host–microorganism interface. The gut microbiome profoundly influences T cell biology by producing a diverse repertoire of small molecules that are sensed by host cells. These microbial metabolites regulate all aspects of the T cell lifecycle, from cell development to differentiation and activation to exhaustion. Recent studies have uncovered microbially derived molecules, including short-chain fatty acids, secondary bile acids and tryptophan metabolites, as potent regulators of T cell function. However, the full scope of microbial metabolite–T cell interactions remains largely unexplored. This Review presents a mechanistic framework linking gut microbial metabolites to discrete stages of T cell fate and function. Expanding our understanding of these intricate host–microbiome interactions will reveal new aspects of immune regulation and inspire microbiome-guided therapeutic strategies for infections, autoimmune diseases and cancer immunotherapy. Tran et al. discuss the role of the gut microbiome and gut microbiome-derived metabolites in T cell biology.

While Roux-en-Y gastric bypass is an effective treatment for obesity and type 2 diabetes, up to one-third of patients develop post-bariatric hypoglycaemia (PBH). Individuals with PBH exhibit increased postprandial secretion of the intestinal hormone fibroblast growth factor 19 (FGF19, Fgf15 in mice). However, the underlying mechanisms contributing to PBH remain uncertain. Here we demonstrate that faecal and plasma bile acid (BA) profiles are significantly altered in postoperative individuals with PBH versus those without hypoglycaemia. Furthermore, altered BAs in PBH induce FGF19 secretion in intestinal cells in a manner dependent on the apical sodium-dependent BA transporter (ASBT). We demonstrate that ASBT inhibition reduces Fgf15 expression and increases postprandial glucose in hypoglycaemic mice. Our data suggest that dysregulation of luminal BA profiles and transport may contribute to PBH and provide proof of concept that ASBT inhibition could be developed as a new therapeutic strategy for PBH. FGF19 increase in post-bariatric hypoglycaemia is mediated by bile acid dysregulation, and can be countered by inhibiting bile acid transport.

Recent studies have demonstrated that metabolites produced by commensal bacteria causally influence health and disease. The sulfated metabolome is one class of molecules that has recently come to the forefront due to efforts to understand the role of these metabolites in host–microbiome interactions. Sulfated compounds have canonically been classified as waste products; however, studies have revealed a variety of physiological roles for these metabolites, including effects on host metabolism, immune response and neurological function. Moreover, recent research has revealed that commensal bacteria either chemically modify or synthesize a variety of sulfated compounds. In this Review, we explore how host–microbiome collaborative metabolism transforms the sulfated metabolome. We describe bacterial and mammalian enzymes that sulfonate and desulfate biologically relevant carbohydrates, amino acid derivatives and cholesterol-derived metabolites. We then discuss outstanding questions and future directions in the field, including potential roles of sulfated metabolites in disease detection, prevention and treatment. We hope that this Review inspires future research into sulfated compounds and their effects on physiology.

Abstract Disclosure: S.O. Moser: None. M.D. McCurry: None. J. Walsh: None. D.V. Winter: None. M. Smieško: None. D.J. Morris: None. A.S. Devlin: None. A. Odermatt: None. In sodium-transporting epithelial cells, 11β-hydroxysteroid dehydrogenase 2 (11βHSD2) protects the mineralocorticoid receptor (MR) from activation by glucocorticoids. MR activation results in sodium and water retention and potassium excretion. Apparent mineralocorticoid excess (AME) results from HSD11B2 mutations, leading to cortisol-driven MR activation characterized by low renin and aldosterone levels. 11βHSD2 inhibition contributes to acquired AME, associated with essential hypertension. Licorice-derived glycyrrhetinic acid inhibits 11βHSD2, allowing cortisol accumulation and subsequent MR activation. A "cloud" of steroids and steroid-like factors is proposed to influence the activities of 11βHSD isoforms and modulate MR and GR activity in vivo. These endogenous steroidal compounds, termed glycyrrhetinic acid-like factors (GALFs), are produced by peripheral tissues and gut microbiota. GALFs exhibit potent inhibitory effects towards 11βHSD2. The significance of these GALFs in the microenvironment is emphasized when investigating essential hypertension. Experimental interventions, such as antibiotic treatment and fecal transplant, demonstrated the microbiota's influence on blood pressure regulation. 3α,11β-Dihydroxy-5β-tetrahydroprogesterone (3α,11β-diOH-5β-THP) and 3β,11β-dihydroxy-5α-tetrahydroprogesterone (3β,11β-diOH-5α-THP) are metabolites that may be formed from tetrahydrocorticosterone by the gut microbiota. In this study, we assessed the potential of 3α,11β-diOH-5β-THP and 3β,11β-diOH-5α-THP to inhibit 11βHSD2 activity as well as their capability to modulate MR transcriptional activity. Comparison of 3α,11β-diOH-5β-THP and 3β,11β-diOH-5α-THP showed that 3β,11β-diOH-5α-THP is a potent inhibitor of human 11βHSD2, with an IC50 of 47 nM whilst 3α,11β-diOH-5β-THP had an IC50 of approximately 10 µM. Both metabolites were less potent inhibitors against mouse 11βHSD2, with an IC50 of 1.3 µM for 3β,11β-diOH-5α-THP and about 25% inhibition at 20 µM 3α,11β-diOH-5β-THP. 3α,11β-diOH-5α-THP was previously shown to inhibit 11βHSD2 with an IC50 of 0.12 µM using sheep microsomes. This data suggests that a 5α-conformation of the steroid core is important for the inhibitory capacity towards 11βHSD2. 3β,11β-diOH-5α-THP was able to activate human MR, although with an EC50 two or three orders of magnitude higher than those for cortisol or aldosterone. Similarly, 3β,11β-diOH-5α-THP was able to activate mouse MR, with an one or two orders in magnitude higher EC50 than those for cortisol and aldosterone. 3α,11β-diOH-5β-THP did not activate human and mouse MR at 1 µM. In conclusion, these results highlight an endogenous metabolite that influences the functional interaction of 11βHSD2 and MR, with relevance for essential hypertension. Presentation: 6/2/2024

Recent studies suggest that human-associated bacteria interact with host-produced steroids, but the mechanisms and physiological impact of such interactions remain unclear. Here, we show that the human gut bacteria Gordonibacter pamelaeae and Eggerthella lenta convert abundant biliary corticoids into progestins through 21-dehydroxylation, thereby transforming a class of immuno- and metabo-regulatory steroids into a class of sex hormones and neurosteroids. Using comparative genomics, homologous expression, and heterologous expression, we identify a bacterial gene cluster that performs 21-dehydroxylation. We also uncover an unexpected role for hydrogen gas production by gut commensals in promoting 21-dehydroxylation, suggesting that hydrogen modulates secondary metabolism in the gut. Levels of certain bacterial progestins, including allopregnanolone, better known as brexanolone, an FDA-approved drug for postpartum depression, are substantially increased in feces from pregnant humans. Thus, bacterial conversion of corticoids into progestins may affect host physiology, particularly in the context of pregnancy and women's health.

Bariatric surgical procedures such as sleeve gastrectomy (SG) provide effective type 2 diabetes (T2D) remission in human patients. Previous work demonstrated that gastrointestinal levels of the bacterial metabolite lithocholic acid (LCA) are decreased after SG in mice and humans. Here, we show that LCA worsens glucose tolerance and impairs whole-body metabolism. We also show that taurodeoxycholic acid (TDCA), which is the only bile acid whose concentration increases in the murine small intestine post-SG, suppresses the bacterial bile acid-inducible (bai) operon and production of LCA both in vitro and in vivo. Treatment of diet-induced obese mice with TDCA reduces LCA levels and leads to microbiome-dependent improvements in glucose handling. Moreover, TDCA abundance is decreased in small intestinal tissue from T2D patients. This work reveals that TDCA is an endogenous inhibitor of LCA production and suggests that TDCA may contribute to the glucoregulatory effects of bariatric surgery.

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 (T H 17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits T H 17 cell differentiation 1 . 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 T H 17 differentiation by inhibiting RORγt (retinoic acid receptor-related orphan nuclear receptor γt), a key T H 17 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 T H 17 cell-associated genes. Overall, our data suggest that bacterially produced T H 17 cell-inhibitory bile acids may reduce the risk of autoimmune and inflammatory disorders such as IBD.

Here, I reflect on my trajectory from a graduate student in organic chemistry to an early-career scientist in the microbiome field. I discuss strategies for discoveringmicrobiome-derived molecules and their activities, and I contemplate how we will uncover which of the molecules we identify are responsible for driving host phenotypes.

Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3′-phosphate-5′-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414–BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host. Characterization of a biosynthetic pathway for the sulfonation of steroidal metabolites, such as cholesterol, by gut bacteria may have implications for immune cell trafficking and inflammatory bowel disease.

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.

INTRODUCTION: Bariatric surgery is the most effective therapy for type 2 diabetes (T2D). We previously discovered cholic acid 7-sulfate (CA7S), a gut-restricted metabolite that is upregulated by sleeve gastrectomy in both mice and humans. CA7S lowers blood glucose by inducing secretion of glucagon-like peptide-1 (GLP-1) in the gut. Thus, CA7S is a potential oral GLP-1–based T2D therapy, unlike most GLP-1 analogs today. Sitagliptin is an approved T2D drug that prolongs GLP-1 half-life by inhibiting its degradation. Here, we investigate the antidiabetic efficacy of CA7S alone or in combination with sitagliptin. METHODS: Sixteen-week-old male, diet-induced obese, insulin-resistant C57BL/6J mice, were weight and fasted-glucose matched and received PBS, CA7S (100 mg/kg), sitagliptin, or CA7S (100 mg/kg) + sitagliptin. An incremental dose of sitagliptin was used: 0.1, 1, and 100 mg/kg. Fasted mice were orally gavaged with the indicated compound(s), and 3 hours later, were subjected to an oral glucose tolerance test (OGTT). Active serum GLP-1 levels were also measured 15 minutes after glucose administration. RESULTS: CA7S alone reduces peak blood glucose levels (p-BGL) by 26.6% (p < 0.01), decreasing the total area under the curve (t-AUC) by 30.5% (p < 0.05) when compared with control. A similar effect is seen after 1 mg/kg sitagliptin (p < 0.01). Co-administration of CA7S and sitagliptin drops p-BGL by 63.7% and t-AUC by 60.1% (p < 0.001, Figs. 1A–1D). GLP-1 increases by 233% after CA7S, p < 0.01; 352% after 1 mg/kg Sitagliptin (p < 0.001) and 600% after the combination of the 2 (p < 0.0001) when compared with control (Fig. 1C)Figure 1CONCLUSION: Single and combination therapy of CA7S, a surgically induced, gut-restricted metabolite and sitagliptin improves glucose tolerance and augments GLP-1 secretion.

Bile acids play crucial roles in host physiology by acting as both detergents that aid in digestion and as signaling molecules that bind to host receptors. Gut bacterial bile salt hydrolase (BSH) enzymes perform the gateway reaction leading to the conversion of host-produced primary bile acids into bacterially modified secondary bile acids. Small molecule probes that target BSHs will help elucidate the causal roles of these metabolites in host physiology. We previously reported the development of a covalent BSH inhibitor with low gut permeability. Here, we build on our previous findings and describe the development of a second-generation gut-restricted BSH inhibitor with enhanced potency, reduced off-target effects, and durable in vivo efficacy. SAR studies focused on the bile acid core identified a compound, AAA-10, containing a C3-sulfonated lithocholic acid scaffold and an alpha-fluoromethyl ketone warhead as a potent pan-BSH inhibitor. This compound inhibits BSH activity in conventional mouse fecal slurries, bacterial cultures, and purified BSH proteins and displays reduced toxicity against mammalian cells compared to first generation compounds. Oral administration of AAA-10 to wild-type mice for 5 days resulted in a decrease in the abundance of the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse GI tract with low systemic exposure of AAA-10, demonstrating that AAA-10 is an effective tool for inhibiting BSH activity and modulating bile acid pool composition in vivo.

The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5 agonists as type 2 diabetes therapeutics. However, the clinical application of TGR5 agonists has been hampered by side effects of these compounds that primarily result from their absorption into circulation. Here we describe an in vitro screening protocol to evaluate the TGR5 agonism, GLP-1 secretion, and gut-restricted properties of small molecules. The protocol involves differentiating gut epithelial and endocrine cells together in transwells to assess both the pharmacodynamics of TGR5 agonists and the toxicity of compounds to the intestinal monolayer. As a proof of concept, we demonstrate the use of the protocol in evaluating properties of naturally occurring bile acid metabolites that are potent TGR5 agonists. This protocol is adapted from Chaudhari et al. (2021).

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology for which there are no approved therapeutic options. Patients with PSC display changes in gut microbiota and in bile acid (BA) composition; however, the contribution of these alterations to disease pathogenesis remains controversial. Here we identify a role for microbiota-dependent changes in BA synthesis that modulates PSC pathophysiology. In a genetic mouse model of PSC, we show that loss of microbiota-mediated negative feedback control of BA synthesis results in increased hepatic BA concentrations, disruption of bile duct barrier function and, consequently, fatal liver injury. We further show that these changes are dependent on decreased BA signalling to the farnesoid X receptor, which modulates the activity of the rate-limiting enzyme in BA synthesis, CYP7A1. Moreover, patients with advanced stages of PSC show suppressed BA synthesis as measured by serum C4 levels, which is associated with poor disease prognosis. Our preclinical data highlight the microbiota-dependent dynamics of BA metabolism in cholestatic liver disease, which could be important for future therapies targeting BA and gut microbiome interactions, and identify C4 as a potential biomarker to functionally stratify patients with PSC and predict disease outcomes.