Fungal signature differentiates alcohol-associated liver disease from nonalcoholic fatty liver disease.

The fungal microbiota plays an important role in the pathogenesis of alcohol-associated liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). In this study, we aimed to compare changes of the fecal fungal microbiota between patients with ALD and NAFLD and to elucidate patterns in different disease stages between the two conditions. We analyzed fungal internal transcribed spacer 2 (ITS2) sequencing using fecal samples from a cohort of 48 patients with ALD, 78 patients with NAFLD, and 34 controls. The fungal microbiota differed significantly between ALD and NAFLD. The genera Saccharomyces, Kluyveromyces, Scopulariopsis, and the species Candida albicans (C. albicans), Malassezia restricta (M. restricta), Scopulariopsis cordiae (S. cordiae) were significantly increased in patients with ALD, whereas the genera Kazachstania and Mucor were significantly increased in the NAFLD cohort. We identified the fungal signature consisting of Scopulariopsis, Kluyveromyces, M. restricta, and Mucor to have the highest discriminative ability to detect ALD vs NAFLD with an area under the curve (AUC) of 0.93. When stratifying the ALD and NAFLD cohorts by fibrosis severity, the fungal signature with the highest AUC of 0.92 to distinguish ALD F0-F1 vs NAFLD F0-F1 comprised Scopulariopsis, Kluyveromyces, Mucor, M. restricta, and Kazachstania. For more advanced fibrosis stages (F2-F4), the fungal signature composed of Scopulariopsis, Kluyveromyces, Mucor, and M. restricta achieved the highest AUC of 0.99 to differentiate ALD from NAFLD. This is the first study to identify a fungal signature to differentiate two metabolic fatty liver diseases from each other, specifically ALD from NAFLD. This might have clinical utility in unclear cases and might hence help shape treatment approaches. However, larger studies are required to validate this fungal signature in other populations of ALD and NAFLD.

The role of the microbiome in liver disease.

PURPOSE OF REVIEW: The intestinal microbiome and the gut-liver axis play a major role in health and disease. The human gut harbors trillions of microbes and a disruption of the gut homeostasis can contribute to liver disease. In this review, the progress in the field within the last 3 years is summarized, focusing on metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), autoimmune liver disease (AILD), and hepatocellular carcinoma (HCC). RECENT FINDINGS: Changes in the fecal virome and fungal mycobiome have been described in patients with various liver diseases. Several microbial derived metabolites including endogenous ethanol produced by bacteria, have been mechanistically linked to liver disease such as MASLD. Virulence factors encoded by gut bacteria contribute to ALD, AILD and HCC. Novel therapeutic approaches focused on the microbiome including phages, pre- and postbiotics have been successfully used in preclinical models. Fecal microbiota transplantation has been effective in attenuating liver disease. Probiotics are safe in patients with alcohol-associated hepatitis and improve liver disease and alcohol addiction. SUMMARY: The gut-liver axis plays a key role in the pathophysiology of liver diseases. Understanding the microbiota in liver disease can help to develop precise microbiota centered therapies.

Serum aryl hydrocarbon receptor activity is associated with survival in patients with alcohol-associated hepatitis.

BACKGROUND AND AIMS: Patients with alcohol-associated hepatitis (AH) have an altered fecal metabolome, including reduced microbiota-derived tryptophan metabolites, which function as ligands for aryl hydrocarbon receptor (AhR). The aim of this study was to assess serum AhR ligand activity in patients with AH. APPROACH AND RESULTS: The study included 74 controls without AUD, 97 patients with AUD, and 330 patients with AH from 2 different multicenter cohorts (InTeam: 134, AlcHepNet: 196). Serum AhR activity was evaluated using an AhR reporter assay with HepG2-Lucia cells incubated with serum for 24 hours. Serum AhR activity was significantly higher in patients with AH compared with both controls (1.59 vs. 0.96-fold change, p < 0.001) and patients with AUD (1.59 vs. 0.93, p < 0.001). In both AH cohorts, patients with AhR activity ≥ 2.09 had significantly lower cumulative survival rates at 30, 60, 90, and 180 days compared to those with AhR activity < 2.09. When serum AhR activity was used to further stratify patients with severe AH, the cumulative 30, 60, 90, and 180-day survival rates for patients with severe AH and the AhR activity ≥ 2.09 group were all significantly lower than those with an AhR activity < 2.09 group. CONCLUSIONS: Serum AhR activity was significantly higher in patients with AH compared with controls and individuals with AUD, and this increased activity was associated with higher mortality. Consequently, serum AhR activity holds potential as a prognostic marker.

Oral magnesium prevents acetaminophen-induced acute liver injury by modulating microbial metabolism.

Acetaminophen overuse is a common cause of acute liver failure (ALF). During ALF, toxins are metabolized by enzymes such as CYP2E1 and transformed into reactive species, leading to oxidative damage and liver failure. Here, we found that oral magnesium (Mg) alleviated acetaminophen-induced ALF through metabolic changes in gut microbiota that inhibit CYP2E1. The gut microbiota from Mg-supplemented humans prevented acetaminophen-induced ALF in mice. Mg exposure modulated Bifidobacterium metabolism and enriched indole-3-carboxylic acid (I3C) levels. Formate C-acetyltransferase (pflB) was identified as a key Bifidobacterium enzyme involved in I3C generation. Accordingly, a Bifidobacterium pflB knockout showed diminished I3C generation and reduced the beneficial effects of Mg. Conversely, treatment with I3C or an engineered bacteria overexpressing Bifidobacterium pflB protected against ALF. Mechanistically, I3C bound and inactivated CYP2E1, thus suppressing formation of harmful reactive intermediates and diminishing hepatocyte oxidative damage. These findings highlight how interactions between Mg and gut microbiota may help combat ALF.

Microbial Players in Primary Sclerosing Cholangitis: Current Evidence and Concepts.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with progressive biliary inflammation, destruction of the biliary tract, and fibrosis, resulting in liver cirrhosis and end-stage liver disease. To date, liver transplantation is the only definitive treatment option for PSC. The precise etiology of PSC remains elusive, but it is widely accepted to involve a complex interplay between genetic predisposition, immunologic dysfunction, and environmental influence. In recent years, the gut-liver axis has emerged as a crucial pathway contributing to the pathogenesis of PSC, with particular focus on the role of gut microbiota. However, the role of the fungal microbiome or mycobiome has been overlooked for years, resulting in a lack of comprehensive studies on its involvement in PSC. In this review, we clarify the present clinical and mechanistic data and concepts concerning the gut bacterial and fungal microbiota in the context of PSC. This review sheds light on the role of specific microbes and elucidates the dynamics of bacterial and fungal populations. Moreover, we discuss the latest insights into microbe-altering therapeutic approaches involving the gut-liver axis and bile acid metabolism.

Phage therapy: Targeting intestinal bacterial microbiota for the treatment of liver diseases.

Phage therapy has been overshadowed by antibiotics for decades. However, it is being revisited as a powerful approach against antimicrobial-resistant bacteria. As bacterial microbiota have been mechanistically linked to gastrointestinal and liver diseases, precise editing of the gut microbiota via the selective bactericidal action of phages has prompted renewed interest in phage therapy. In this review, we summarise the basic virological properties of phages and the latest findings on the composition of the intestinal phageome and the changes associated with liver diseases. We also review preclinical and clinical studies assessing phage therapy for the treatment of gastrointestinal and liver diseases, as well as future prospects and challenges.

Development of a Quantitative PCR Method for Detecting Enterococcus faecalis Cytolysin in Human Stool Samples.

Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials.

The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine.

PRMT5 is a type II arginine methyltransferase abundantly expressed in the colonic epithelium. It is up-regulated in inflammatory bowel disease and colorectal cancer. However, its role in mucosal defense against enteric infection has not been studied. Here, we report that Prmt5 in the murine colon is up-regulated in response to Citrobacter rodentium infection. Pathogen clearance in mice with haploinsufficient expression of Prmt5 is significantly delayed compared with wildtype littermate controls. Transcriptomic analyses further reveal that PRMT5 regulates the expression of canonical crypt goblet cell genes involved in mucus production, assembly, and anti-microbial responses via methyltransferase activity-dependent and -independent mechanisms. Together, these findings uncover PRMT5 as a novel regulator of mucosal defense and a potential therapeutic target for treating intestinal diseases.

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes.

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

Gut Microbiome-Centered Therapies for Alcohol-Associated Liver Disease.

Globally, liver disease caused by alcohol is becoming more prevalent each year. Misuse of alcohol causes a spectrum of liver diseases, such as liver steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The cornerstone of treatment is abstinence from alcohol. In spite of this, available treatment for alcohol-associated liver disease (ALD) shows limited effectiveness currently. There are numerous ways in which alcohol disrupts the gut-liver axis, including dysbiosis of the gut microbiome, disruption of mucus and epithelial cell barriers, impaired production of antimicrobial molecules, and dysfunction of the immune system, causing translocation of viable microbes and microbial products to the liver and systemic circulation. Microbial exposure results in not only inflammation and progression of liver disease but also infections in late-stage ALD. This led scientists to focus their therapeutic strategies and targets for ALD on the gut microbiome. Throughout this review, we address the role of gut microbiome-centered therapeutic approaches for ALD focusing predominantly on randomized controlled trials. We will summarize the latest clinical trials using probiotics, antibiotics, and fecal microbial transplants in modulating the gut-liver axis and for improvement of ALD.

A metabolite from commensal Candida albicans enhances the bactericidal activity of macrophages and protects against sepsis.

The gut microbiome is recognized as a key modulator of sepsis development. However, the contribution of the gut mycobiome to sepsis development is still not fully understood. Here, we demonstrated that the level of Candida albicans was markedly decreased in patients with bacterial sepsis, and the supernatant of Candida albicans culture significantly decreased the bacterial load and improved sepsis symptoms in both cecum ligation and puncture (CLP)-challenged mice and Escherichia coli-challenged pigs. Integrative metabolomics and the genetic engineering of fungi revealed that Candida albicans-derived phenylpyruvate (PPA) enhanced the bactericidal activity of macrophages and reduced organ damage during sepsis. Mechanistically, PPA directly binds to sirtuin 2 (SIRT2) and increases reactive oxygen species (ROS) production for eventual bacterial clearance. Importantly, PPA enhanced the bacterial clearance capacity of macrophages in sepsis patients and was inversely correlated with the severity of sepsis in patients. Our findings highlight the crucial contribution of commensal fungi to bacterial disease modulation and expand our understanding of the host-mycobiome interaction during sepsis development.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

The gastrointestinal microbiome plays a significant role in modulating numerous host processes, including metabolism. Prior studies show that when mice receive fecal transplants from obese donors on high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes, demonstrating the prominent role that gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, the impact of gut viruses on these phenotypes is understudied. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow over a 4-week study. By characterizing the gut bacterial biota via 16S rRNA amplicon sequencing and measuring mouse weights over time, we demonstrate that transplanted viruses affect the gut bacterial community, as well as weight gain/loss. Notably, mice fed chow but gavaged with HFD-derived viromes gained more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained less weight than their counterparts receiving HFD-derived viromes. Results were replicated in two independent experiments and phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Due to methodological limitations, we were unable to identify the specific bacterial strains responsible for respective phenotypic changes. This study confirms that virome-mediated perturbations can alter the fecal microbiome in vivo and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

The gut-liver axis and gut microbiota in health and liver disease.

The trillions of microorganisms in the human intestine are important regulators of health, and disruptions in the gut microbial communities can cause disease. The gut, liver and immune system have a symbiotic relationship with these microorganisms. Environmental factors, such as high-fat diets and alcohol consumption, can disrupt and alter microbial communities. This dysbiosis can lead to dysfunction of the intestinal barrier, translocation of microbial components to the liver and development or progression of liver disease. Changes in metabolites produced by gut microorganisms can also contribute to liver disease. In this Review, we discuss the importance of the gut microbiota in maintenance of health and the alterations in microbial mediators that contribute to liver disease. We present strategies for modulation of the intestinal microbiota and/or their metabolites as potential treatments for liver disease.

Bile acid and nonalcoholic steatohepatitis: Molecular insights and therapeutic targets.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) has been the second most common cause of liver transplantation in the United States. To date, NASH pathogenesis has not been fully elucidated but is multifactorial, involving insulin resistance, obesity, metabolic disorders, diet, dysbiosis, and gene polymorphism. An effective and approved therapy for NASH has also not been established. Bile acid is long known to have physiological detergent function in emulsifying and absorbing lipids and lipid-soluble molecules within the intestinal lumen. With more and more in-depth understandings of bile acid, it has been deemed to be a pivotal signaling molecule, which is capable of regulating lipid and glucose metabolism, liver inflammation, and fibrosis. In recent years, a plethora of studies have delineated that disrupted bile acid homeostasis is intimately correlated with NASH disease severity. AIMS: The review aims to clarify the role of bile acid in hepatic lipid and glucose metabolism, liver inflammation, as well as liver fibrosis, and discusses the safety and efficacy of some pharmacological agents targeting bile acid and its associated pathways for NASH. KEY SCIENTIFIC CONCEPTS OF REVIEW: Bile acid has a salutary effect on hepatic metabolic disorders, which can ameliorate liver fat accumulation and insulin resistance mainly through activating Takeda G-protein coupled receptor 5 and farnesoid X receptor. Moreover, bile acid also exerts anti-inflammation and anti-fibrosis properties. Furthermore, bile acid has great potential in nonalcoholic liver disease stratification and treatment of NASH.

Fecal cytolysin does not predict disease severity in acutely decompensated cirrhosis and acute-on-chronic liver failure.

BACKGROUND: Cirrhosis with acute decompensation (AD) and acute-on-chronic liver failure (ACLF) are characterized by high morbidity and mortality. Cytolysin, a toxin from Enterococcus faecalis (E. faecalis), is associated with mortality in alcohol-associated hepatitis (AH). It is unclear whether cytolysin also contributes to disease severity in AD and ACLF. METHODS: We studied the role of fecal cytolysin in 78 cirrhotic patients with AD/ACLF. Bacterial DNA from fecal samples was extracted and real-time quantitative polymerase chain reaction (PCR) was performed. The association between fecal cytolysin and liver disease severity in cirrhosis with AD or ACLF was analyzed. RESULTS: Fecal cytolysin and E. faecalis abundance did not predict chronic liver failure (CLIF-C) AD and ACLF scores. Presence of fecal cytolysin was not associated with other liver disease markers, including Fibrosis-4 (FIB-4) index, 'Age, serum Bilirubin, INR, and serum Creatinine (ABIC)' score, Child-Pugh score, model for end-stage liver disease (MELD) nor MELD-Na scores in AD or ACLF patients. CONCLUSIONS: Fecal cytolysin does not predict disease severity in AD and ACLF patients. The predictive value of fecal cytolysin positivity for mortality appears to be restricted to AH.

Acute alcohol-associated hepatitis: Latest findings in non-invasive biomarkers and treatment.

Acute alcohol-associated hepatitis (AH) is a syndrome that occurs in heavy and long-term drinkers and results in severe jaundice and liver failure. The mortality rate in severe cases is 20%-50% at 28 days, and in cases that do not improve despite appropriately timed corticosteroid therapy, the mortality rate reaches 70% at 6 months. The only curative treatment is early liver transplantation, but less than 2% of patients with severe AH are eligible. In order to improve the prognosis, diagnostic tools are needed to detect appropriate cases at risk of severe conditions, and new therapies need to be developed that can replace corticosteroids. Recent research has revealed that the pathogenesis of AH involves a complex of factors, including changes in the gut microbiota, inflammatory and cytokine signalling, oxidative stress and mitochondrial dysfunction, and abnormalities in the hepatic regenerative capacity. Non-invasive diagnostic tools focusing on these specific pathologies have been reported in recent years. In addition, several novel agents targeting specific pathways are currently being developed and tested in clinical trials. This review will provide an overview of alcohol-associated hepatitis and focus on the latest diagnostic tools, particularly non-invasive biomarkers, and novel therapies.

Global and national prevalence of nonalcoholic fatty liver disease in adolescents: An analysis of the global burden of disease study 2019.

BACKGROUND AND AIMS: NAFLD in adolescents is an increasing health crisis worldwide, but its exact global, continental, and national prevalence, its relationship with other metabolic conditions, and the human development index (HDI) globally are not known. APPROACH AND RESULTS: We analyzed data from the Global Burden of Disease Study 2019 to compare global, continental, and national prevalence rates of adolescent NAFLD and associations with other metabolic conditions and HDI. The global NAFLD prevalence in adolescents increased from 3.73% in 1990 to 4.71% in 2019 (a relative increase of 26.27%). The prevalence for the male and female populations was 5.84% and 3.52% in 2019, respectively. The Oceanian and North American continents had the highest adolescent NAFLD prevalence (median: 6.54% and 5.64%, respectively), whereas Europe had the lowest prevalence (median: 3.98%). South America and North America had the highest relative increase in adolescent NAFLD prevalence from 1990 to 2019 (median: 39.25% and 36.87%, respectively). High body mass index and type 2 diabetes mellitus increased significantly in adolescents worldwide. However, only high body mass index and not type 2 diabetes mellitus correlated with NAFLD prevalence in adolescents globally. Countries with a higher HDI had larger increases in adolescent NAFLD prevalence from 1990 to 2019 although countries with the highest HDI (HDI: > 0.9) had the lowest NAFLD prevalence in 2019. CONCLUSIONS: NAFLD in adolescents is an increasing health problem on all continents. Improving environmental factors, including lifestyle but also healthcare policies, can help to prevent NAFLD from developing in children and adolescents and help to improve outcomes in children and adolescents with NAFLD.

Toll-like receptor 2 activation in monocytes contributes to systemic inflammation and alcohol-associated liver disease in humans.

BACKGROUND AND RATIONALE: In the context of gut leakiness and translocation of microbial products in alcohol-associated liver disease (ALD), it is possible that systemic and liver inflammation involve the activation of circulating monocyte through gut-derived factors. We explored the association between monocytes, microbial translocation, systemic inflammation, and ALD. METHODS: Patients with alcohol use disorder following a rehabilitation program were compared with healthy controls. We determined the circulating number and proportion of monocyte subsets by FACS. The activation of signaling pathways by gut-derived microbes was analyzed by quantitative PCR in isolated monocytes. Cytokines secretion by monocytes and phagocytosis were assessed in vitro. Serum microbial translocation markers and cytokines were measured by ELISA and multiplex assay, respectively. ALD severity and liver inflammatory responses were analyzed in liver biopsies by various methods. RESULTS: In patients with alcohol use disorder, the number of blood monocytes increased compared with controls. Monocytes from patients with alcohol use disorder upregulated IL-1ß and IL-8 together with toll-like receptor 2 and downstream AP-1, while fungal sensor CARD9 was downregulated. IL-1ß and IL-8 were actively secreted upon stimulation in vitro with the toll-like receptor 2 ligand peptidoglycan. Exposure with Escherichia coli confirmed preserved bacterial phagocytic activity. In contrast, Candida albicans stimulation leads to downregulation of IL-1ß and TNFα compared with controls. Systemic cytokines and monocyte changes correlated with microbial translocation. Hepatic IL-1ß and IL-8 increased with ALD severity together with liver macrophage activation and upregulation of chemokines involved in monocyte attraction. CONCLUSIONS: Our results point to the contribution of activated monocytes to systemic inflammation and ALD. Monocytes likely infiltrate the liver, transform into monocyte-derived macrophages and release IL-1ß and IL-8 in response to peptidoglycan and toll-like receptor 2 activation.