Gut Bacteria in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) poses a significant global public health challenge, with high patient mortality rates and economic burden. The gut microbiome plays an important role in the onset and progression of alcohol-associated liver disease. Excessive alcohol consumption disrupts the intestinal barrier, facilitating the entry of harmful microbes and their products into the liver, exacerbating liver damage. Dysbiosis, marked by imbalance in gut bacteria, correlates with ALD severity. Promising microbiota-centered therapies include probiotics, phages, and fecal microbiota transplantation. Clinical trials demonstrate the potential of these interventions to improve liver function and patient outcomes, offering a new frontier in ALD treatment.

Hepatic immune regulation and sex disparities.

Chronic liver disease is a major cause of morbidity and mortality worldwide. Epidemiology, clinical phenotype and response to therapies for gastrointestinal and liver diseases are commonly different between women and men due to sex-specific hormonal, genetic and immune-related factors. The hepatic immune system has unique regulatory functions that promote the induction of intrahepatic tolerance, which is key for maintaining liver health and homeostasis. In liver diseases, hepatic immune alterations are increasingly recognized as a main cofactor responsible for the development and progression of chronic liver injury and fibrosis. In this Review, we discuss the basic mechanisms of sex disparity in hepatic immune regulation and how these mechanisms influence and modify the development of autoimmune liver diseases, genetic liver diseases, portal hypertension and inflammation in chronic liver disease. Alterations in gut microbiota and their crosstalk with the hepatic immune system might affect the progression of liver disease in a sex-specific manner, creating potential opportunities for novel diagnostic and therapeutic approaches to be evaluated in clinical trials. Finally, we identify and propose areas for future basic, translational and clinical research that will advance our understanding of sex disparities in hepatic immunity and liver disease.

IL-22 resolves MASLD via enterocyte STAT3 restoration of diet-perturbed intestinal homeostasis.

The exponential rise in metabolic dysfunction-associated steatotic liver disease (MASLD) parallels the ever-increasing consumption of energy-dense diets, underscoring the need for effective MASLD-resolving drugs. MASLD pathogenesis is linked to obesity, diabetes, "gut-liver axis" alterations, and defective interleukin-22 (IL-22) signaling. Although barrier-protective IL-22 blunts diet-induced metabolic alterations, inhibits lipid intake, and reverses microbial dysbiosis, obesogenic diets rapidly suppress its production by small intestine-localized innate lymphocytes. This results in STAT3 inhibition in intestinal epithelial cells (IECs) and expansion of the absorptive enterocyte compartment. These MASLD-sustaining aberrations were reversed by administration of recombinant IL-22, which resolved hepatosteatosis, inflammation, fibrosis, and insulin resistance. Exogenous IL-22 exerted its therapeutic effects through its IEC receptor, rather than hepatocytes, activating STAT3 and inhibiting WNT-ß-catenin signaling to shrink the absorptive enterocyte compartment. By reversing diet-reinforced macronutrient absorption, the main source of liver lipids, IL-22 signaling restoration represents a potentially effective interception of dietary obesity and MASLD.

Viral antibody response predicts morbidity and mortality in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Alcohol-associated hepatitis (AH) is associated with very high mortality despite abstinence from alcohol; up to 40% of patients die within 6 months of diagnosis. Patients with AH are especially prone to infections, which can lead to multiorgan dysfunction and poorer prognosis. APPROACH AND RESULTS: We performed comprehensive serological profiling of the viral and bacterial infection history of 36 healthy controls, 48 patients with alcohol use disorder, and 224 patients with AH from 2 multicenter observational studies. We used systematic viral and bacterial epitope scanning by VirScan, a phage-display immunoprecipitation and sequencing technology that detects the peptides recognized by antibodies in patient sera, to comprehensively analyze antiviral and antibacterial antibodies and identify serologic biomarkers to predict patient outcomes. We found significant differences in the serological profiles of the 3 populations. The number of serum antibody epitopes in patients with alcohol use disorder during abstinence was increased compared with during active alcohol use. A decreased number and diversity of viral and bacterial antibody targets were detected in the sera of patients with AH, particularly those with a higher Child-Pugh score. In patients with AH, a decrease in the serum antiviral, but not antibacterial, antibody repertoire was associated with decompensation and mortality. Ninety-day mortality in AH could be predicted using a serum viral epitope signature. CONCLUSIONS: Abstinence from alcohol is associated with a significant increase in serum viral and bacterial antibody response. Decreased serum antiviral antibody repertoire is predictive of decompensation of liver disease and mortality in patients with AH.

Gut mycobiome alterations and implications for liver diseases.

Chronic liver disease and its complications are a significant global health burden. Changes in fungal communities (mycobiome), an integral component of the gut microbiome, are associated with and contribute to the development of liver disease. Fungal dysbiosis can induce intestinal barrier dysfunction and allow fungal products to translocate to the liver causing progression of disease. This review explores recent progress in understanding the compositional and functional diversity of gut mycobiome signatures across different liver diseases. It delves into causative connections between gut fungi and liver diseases. We emphasize the significance of fungal translocation, with a particular focus on fungal-derived metabolites and immune cells induced by fungi, as key contributors to liver disease. Furthermore, we review the potential impact of the intrahepatic mycobiome on the progression of liver diseases.

Fecal gelatinase does not predict mortality in patients with alcohol-associated hepatitis.

Alcohol-associated liver disease is highly prevalent worldwide, with alcohol-associated hepatitis as a severe form characterized by substantial morbidity, mortality, and economic burden. Gut bacterial dysbiosis has been linked to progression of alcohol-associated hepatitis. Fecal cytolysin secreted by the pathobiont Enterococcus faecalis (E. faecalis) is associated with increased mortality in patients with alcohol-associated hepatitis. Although gelatinase is considered a virulence factor in E. faecalis, its prevalence and impact on alcohol-associated hepatitis patient outcomes remains unclear. In this study, 20 out of 65 (30.8%) patients with alcohol-associated hepatitis tested positive for gelatinase in their stool. There were no significant differences in 30-day and 90-day mortality between gelatinase-positive and gelatinase-negative patients (p=0.97 and p=0.48, respectively). Fecal gelatinase had a low discriminative ability for 30-day mortality (area under the curve [AUC] 0.50 vs fibrosis-4 Index (FIB-4) 0.75) and 90-day mortality compared with other established liver disease markers (AUC 0.57 vs FIB-4 0.79 or 'age, serum bilirubin, INR, and serum creatinine' (ABIC) score 0.78). Furthermore, fecal gelatinase was not an important feature for 30-day or 90-day mortality per random forest analysis. Finally, gelatinase-positive patients with alcohol-associated hepatitis did not exhibit more severe liver disease compared with gelatinase-negative patients. In conclusion, fecal gelatinase does not predict mortality or disease severity in patients with alcohol-associated hepatitis from our cohort.

Prevalence of steatotic liver disease, advanced fibrosis and cirrhosis among community-dwelling overweight and obese individuals in the USA.

BACKGROUND: There are limited prospective data among overweight and obese individuals on the prevalence of advanced fibrosis, and cirrhosis using advanced MRI-based methods in the USA. The aim of this study was to fill that gap in knowledge by prospectively determining the MRI-based prevalence of steatotic liver disease (SLD) and its subcategories, advanced fibrosis and cirrhosis among overweight and obese individuals residing in the USA. METHODS: This is a cross-sectional analysis of prospectively enrolled overweight or obese adults aged 40-75 years from primary care and community-based settings in Southern California. Participants were classified as having SLD if MRI proton density fat fraction ≥5%, and subclassified as metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction and alcohol-associated liver disease (MetALD) and alcohol-related liver disease (ALD) consistently with the new nomenclature guidance per AASLD-EASL-ALEH. Advanced fibrosis and cirrhosis were defined as magnetic resonance elastography (MRE) ≥3.63 kPa and MRE ≥4.67 kPa, respectively. RESULTS: The cohort included 539 participants with mean (±SD) age of 51.5 (±13.1) years and body mass index of 32.6 (±6.2) kg/m2, respectively. The prevalence of SLD, advanced fibrosis and cirrhosis was 75%, 10.8% and 4.5%, respectively. The prevalence of MASLD, MetALD and ALD was 67.3%, 4.8% and 2.6%, respectively. There was no difference in prevalence of advanced fibrosis and cirrhosis among subcategories. CONCLUSIONS: Using advanced MRI methods among community-dwelling overweight and obese adults, the prevalence of cirrhosis was 4.5%. Most common SLD subcategory was MASLD with 67% of individuals, whereas MetALD and ALD were less common. Systematic screening for advanced fibrosis among overweight/obese adults may be considered.

Relationship between updated MELD and prognosis in alcohol-associated hepatitis: Opportunities for more efficient trial design.

BACKGROUND: Alcohol-associated hepatitis (AH) is associated with significant mortality. Model for End-Stage Liver Disease (MELD) score is used to predict short-term mortality and aid in treatment decisions. MELD is frequently updated in the course of AH. However, once the most updated MELD is known, it is uncertain if previous ones still have prognostic value, which might be relevant for transplant allocation and trial design. We aimed to investigate the predictive performance of updated MELDs in a prospectively collected cohort of patients with AH by the InTeam consortium. METHODS: Three hundred seven patients (with 859 MELD values within 60 d of admission) fulfilled the inclusion criteria. The main endpoint was time to death or transplant up to 90 days. We used a joint model approach to assess the predictive value of updated MELDs. RESULTS: Updated MELD measurements had a strong prognostic value for death/transplant (HR: 1.20, 95% CI: 1.14-1.27) (p < 0.0001). Previous MELD values did not add predictive value to the most current MELD. We also showed that MELD at day 28 (MELD28) had a significant predictive value for subsequent mortality/transplant in a landmark analysis (HR: 1.18, 95% CI: 1.12-1.23). We show that the use of an ordinal scale including death, transplant, and MELD28 as a trial outcome could substantially reduce the sample size required to demonstrate short-term benefit of an intervention. CONCLUSION: We show that updated MELDs during the trajectory of AH predict subsequent mortality or the need for transplant. MELD28 inclusion in an ordinal outcome (together with death or transplant) could increase the efficiency of randomized controlled trials.

Faecal proteomics links neutrophil degranulation with mortality in patients with alcohol-associated hepatitis.

OBJECTIVE: Patients with alcohol-associated hepatitis (AH) have a high mortality. Alcohol exacerbates liver damage by inducing gut dysbiosis, bacterial translocation and inflammation, which is characterised by increased numbers of circulating and hepatic neutrophils. DESIGN: In this study, we performed tandem mass tag (TMT) proteomics to analyse proteins in the faeces of controls (n=19), patients with alcohol-use disorder (AUD; n=20) and AH (n=80) from a multicentre cohort (InTeam). To identify protein groups that are disproportionately represented, we conducted over-representation analysis using Reactome pathway analysis and Gene Ontology to determine the proteins with the most significant impact. A faecal biomarker and its prognostic effect were validated by ELISA in faecal samples from patients with AH (n=70), who were recruited in a second and independent multicentre cohort (AlcHepNet). RESULT: Faecal proteomic profiles were overall significantly different between controls, patients with AUD and AH (principal component analysis p=0.001, dissimilarity index calculated by the method of Bray-Curtis). Proteins that showed notable differences across all three groups and displayed a progressive increase in accordance with the severity of alcohol-associated liver disease were predominantly those located in neutrophil granules. Over-representation and Reactome analyses confirmed that differentially regulated proteins are part of granules in neutrophils and the neutrophil degranulation pathway. Myeloperoxidase (MPO), the marker protein of neutrophil granules, correlates with disease severity and predicts 60-day mortality. Using an independent validation cohort, we confirmed that faecal MPO levels can predict short-term survival at 60 days. CONCLUSIONS: We found an increased abundance of faecal proteins linked to neutrophil degranulation in patients with AH, which is predictive of short-term survival and could serve as a prognostic non-invasive marker.

Endogenous ethanol production in health and disease.

The gut microbiome exerts metabolic actions on distal tissues and organs outside the intestine, partly through microbial metabolites that diffuse into the circulation. The disruption of gut homeostasis results in changes to microbial metabolites, and more than half of the variance in the plasma metabolome can be explained by the gut microbiome. Ethanol is a major microbial metabolite that is produced in the intestine of nearly all individuals; however, elevated ethanol production is associated with pathological conditions such as metabolic dysfunction-associated steatotic liver disease and auto-brewery syndrome, in which the liver's capacity to metabolize ethanol is surpassed. In this Review, we describe the mechanisms underlying excessive ethanol production in the gut and the role of ethanol catabolism in mediating pathogenic effects of ethanol on the liver and host metabolism. We conclude by discussing approaches to target excessive ethanol production by gut bacteria.

Fecal Carriage of Multidrug-Resistant Organisms Increases the Risk of Hepatic Encephalopathy in Cirrhotic Patients: Insights from Gut Microbiota and Metabolite Features.

Background: Impact of fecal colonization by multidrug-resistant organisms (MDROs) on changes in gut microbiota and associated metabolites, as well as its role in cirrhosis-associated outcomes, has not been thoroughly investigated. Methods: Eighty-eight cirrhotic patients and 22 healthy volunteers were prospectively enrolled with analysis conducted on plasma metabolites, fecal MDROs, and microbiota. Patients were followed for a minimum of one year. Predictive factors for cirrhosis-associated outcomes were identified using Cox proportional hazards regression models, and risk factors for fecal MDRO carriage were assessed using logistic regression model. Correlations between microbiota and metabolic profiles were evaluated through Spearman's rank test. Results: Twenty-nine (33%) cirrhotic patients exhibited MDRO carriage, with a notably higher rate of hepatic encephalopathy (HE) in MDRO carriers (20.7% vs. 3.2%, p = 0.008). Cox regression analysis identified higher serum lipopolysaccharide levels and fecal MDRO carriage as predictors for HE development. Logistic regression analysis showed that MDRO carriage is an independent risk factor for developing HE. Microbiota analysis showed a significant dissimilarity of fecal microbiota between cirrhotic patients with and without MDRO carriage (p = 0.033). Thirty-two metabolites exhibiting significantly different expression levels among healthy controls, cirrhotic patients with and without MDRO carriage were identified. Six of the metabolites showed correlation with specific bacterial taxa expression in MDRO carriers, with isoaustin showing significantly higher levels in MDRO carriers experiencing HE compared to those who did not. Conclusion: Fecal MDRO carriage is associated with altered gut microbiota, metabolite modulation, and an elevated risk of HE occurrence within a year.

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.

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.

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.

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.

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.