Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway.

The gut microbiota has been found to play an important role in the progression of metabolic dysfunction-associated steatohepatitis (MASH), but the mechanisms have not been established. Here, by developing a click-chemistry-based enrichment strategy, we identified several microbial-derived bile acids, including the previously uncharacterized 3-succinylated cholic acid (3-sucCA), which is negatively correlated with liver damage in patients with liver-tissue-biopsy-proven metabolic dysfunction-associated fatty liver disease (MAFLD). By screening human bacterial isolates, we identified Bacteroides uniformis strains as effective producers of 3-sucCA both in vitro and in vivo. By activity-based protein purification and identification, we identified an enzyme annotated as ß-lactamase in B. uniformis responsible for 3-sucCA biosynthesis. Furthermore, we found that 3-sucCA is a lumen-restricted metabolite and alleviates MASH by promoting the growth of Akkermansia muciniphila. Together, our data offer new insights into the gut microbiota-liver axis that may be leveraged to augment the management of MASH.

The underappreciated diversity of bile acid modifications.

The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

Infection trains the host for microbiota-enhanced resistance to pathogens.

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.

The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

Interpersonal Gut Microbiome Variation Drives Susceptibility and Resistance to Cholera Infection.

The gut microbiome is the resident microbial community of the gastrointestinal tract. This community is highly diverse, but how microbial diversity confers resistance or susceptibility to intestinal pathogens is poorly understood. Using transplantation of human microbiomes into several animal models of infection, we show that key microbiome species shape the chemical environment of the gut through the activity of the enzyme bile salt hydrolase. The activity of this enzyme reduced colonization by the major human diarrheal pathogen Vibrio cholerae by degrading the bile salt taurocholate that activates the expression of virulence genes. The absence of these functions and species permits increased infection loads on a personal microbiome-specific basis. These findings suggest new targets for individualized preventative strategies of V. cholerae infection through modulating the structure and function of the gut microbiome.

Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.

Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.

Dysregulated Microbial Fermentation of Soluble Fiber Induces Cholestatic Liver Cancer.

Dietary soluble fibers are fermented by gut bacteria into short-chain fatty acids (SCFA), which are considered broadly health-promoting. Accordingly, consumption of such fibers ameliorates metabolic syndrome. However, incorporating soluble fiber inulin, but not insoluble fiber, into a compositionally defined diet, induced icteric hepatocellular carcinoma (HCC). Such HCC was microbiota-dependent and observed in multiple strains of dysbiotic mice but not in germ-free nor antibiotics-treated mice. Furthermore, consumption of an inulin-enriched high-fat diet induced both dysbiosis and HCC in wild-type (WT) mice. Inulin-induced HCC progressed via early onset of cholestasis, hepatocyte death, followed by neutrophilic inflammation in liver. Pharmacologic inhibition of fermentation or depletion of fermenting bacteria markedly reduced intestinal SCFA and prevented HCC. Intervening with cholestyramine to prevent reabsorption of bile acids also conferred protection against such HCC. Thus, its benefits notwithstanding, enrichment of foods with fermentable fiber should be approached with great caution as it may increase risk of HCC.

The hepatitis B virus receptor.

Hepatitis B virus (HBV) infection affects 240 million people worldwide. A liver-specific bile acid transporter named the sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for HBV and its satellite, the hepatitis D virus (HDV). NTCP likely acts as a major determinant for the liver tropism and species specificity of HBV and HDV at the entry level. NTCP-mediated HBV entry interferes with bile acid transport in cell cultures and has been linked with alterations in bile acid and cholesterol metabolism in vivo. The human liver carcinoma cell line HepG2, complemented with NTCP, now provides a valuable platform for studying the basic biology of the viruses and developing treatments for HBV infection. This review summarizes critical findings regarding NTCP's role as a viral receptor for HBV and HDV and discusses important questions that remain unanswered.

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi.

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi (BC). Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity characteristic of epithelial cells. Whether cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Time-course RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feedback to hepatoblast-to-hepatocyte differentiation.

Even Cancer Cells Watch Their Cholesterol!

Deregulated cell proliferation is an established feature of cancer, and altered tumor metabolism has witnessed renewed interest over the past decade, including the study of how cancer cells rewire metabolic pathways to renew energy sources and "building blocks" that sustain cell division. Microenvironmental oxygen, glucose, and glutamine are regarded as principal nutrients fueling tumor growth. However, hostile tumor microenvironments render O2/nutrient supplies chronically insufficient for increased proliferation rates, forcing cancer cells to develop strategies for opportunistic modes of nutrient acquisition. Recent work shows that cancer cells overcome this nutrient scarcity by scavenging other substrates, such as proteins and lipids, or utilizing adaptive metabolic pathways. As such, reprogramming lipid metabolism plays important roles in providing energy, macromolecules for membrane synthesis, and lipid-mediated signaling during cancer progression. In this review, we highlight more recently appreciated roles for lipids, particularly cholesterol and its derivatives, in cancer cell metabolism within intrinsically harsh tumor microenvironments.

Impact of dietary carbohydrate restriction on the pathobiology of Hepatocellular Carcinoma: The gut-liver axis and beyond.

Despite decades of fiercely competitive research and colossal financial investments, the majority of patients with advanced solid cancers cannot be treated with curative intent. To improve this situation, conceptually novel treatment approaches are urgently needed. Cancer is increasingly appreciated as a systemic disease and numerous organismal factors are functionally linked to neoplastic growth, e.g. systemic metabolic dysregulation, chronic inflammation, intestinal dysbiosis and disrupted circadian rhythms. It is tempting to hypothesize that interventions targeting these processes could be of significant account for cancer patients. One important driver of tumor-supporting systemic derangements is inordinate consumption of simple and highly processed carbohydrates. This dietary pattern is causally linked to hyperinsulinemia, insulin resistance, chronic inflammation and intestinal dysbiosis, begging the pertinent question whether the adoption of dietary carbohydrate restriction can be beneficial for patients with cancer. This review summarizes the published data on the role of dietary carbohydrate restriction in the pathogenesis of Hepatocellular Carcinoma (HCC), the most frequent type of primary liver cancer. In addition to outlining the functional interplay between diet, the intestinal microbiome and immunity, the review underscores the importance of bile acids as interconnectors between the intestinal microbiota and immune cells.

Bile acid signaling in metabolic and inflammatory diseases and drug development.

Bile acids are the end products of cholesterol catabolism. Hepatic bile acid synthesis accounts for a major fraction of daily cholesterol turnover in humans. Biliary secretion of bile acids generates bile flow and facilitates biliary secretion of lipids, endogenous metabolites and xenobiotics. In intestine, bile acids facilitate the digestion and absorption of dietary lipids and fat-soluble vitamins. Through activation of nuclear receptors and G protein-coupled receptors and interaction with gut microbiome, bile acids critically regulate host metabolism and innate and adaptive immunity, and are involved in the pathogenesis of cholestasis, metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), type-2 diabetes, and inflammatory bowel diseases (IBD). Bile acids and their derivatives have been developed as potential therapeutic agents for treating chronic metabolic and inflammatory liver diseases and gastrointestinal disorders. Significance Statement Bile acids facilitate biliary cholesterol solubilization and dietary lipid absorption, regulate host metabolism and immunity, and modulate gut microbiome. Targeting bile acid metabolism and signaling hold promise for treating metabolic and inflammatory diseases.

Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Identification of Proteome-Based Immune Subtypes of Early Hepatocellular Carcinoma and Analysis of Potential Metabolic Drivers.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, ranking fourth in frequency. The relationship between metabolic reprogramming and immune infiltration has been identified as having a crucial impact on HCC progression. However, a deeper understanding of the interplay between the immune system and metabolism in the HCC microenvironment is required. In this study, we used a proteomic dataset to identify three immune subtypes (IM1-IM3) in HCC, each of which has distinctive clinical, immune, and metabolic characteristics. Among these subtypes, IM3 was found to have the poorest prognosis, with the highest levels of immune infiltration and T-cell exhaustion. Furthermore, IM3 showed elevated glycolysis and reduced bile acid metabolism, which was strongly correlated with CD8 T cell exhaustion and regulatory T cell accumulation. Our study presents the proteomic immune stratification of HCC, revealing the possible link between immune cells and reprogramming of HCC glycolysis and bile acid metabolism, which may be a viable therapeutic strategy to improve HCC immunotherapy.

Intestinal bile acids provide a surmountable barrier against C. difficile TcdB-induced disease pathogenesis.

Intestinal bile acids play an essential role in the Clostridioides difficile lifecycle having been shown in vitro to modulate various aspects of pathogenesis, including spore germination, vegetative growth, and more recently the action of the primary virulence determinant, TcdB. Here, we investigated whether physiological levels of the total pool of intestinal bile acids in mice and humans protect against TcdB action. Small molecules extracted from the lumenal contents of the small intestine, cecum, colon, and feces were found to inhibit TcdB in accordance with the differential amounts of total bile acids in each compartment. Extracts from antibiotic-treated and germ-free mice, despite harboring dramatically altered bile acid profiles, unexpectedly also prevented TcdB-induced cell rounding to similar extents. We show that protection, however, is surmountable and can be overcome at higher doses of TcdB-typical to those seen during severe C. difficile infection-suggesting that the protective properties of intestinal bile acids are operant primarily under low to moderate toxin levels. Taken together, these findings demonstrate a role for intestinal bile acids in attenuating virulence, provide insights into asymptomatic carriage of toxigenic C. difficile, and inform strategies to manipulate bile acid levels for therapeutic benefit.