Liver and biliary diseases affect more than a billion people worldwide, with high associated morbidity and mortality. The impact of the intestinal bacterial microbiome on liver diseases has been well established. However, the fungal microbiome, or mycobiome, has been overlooked for a long time. Recently, several studies have shed light on the role of the mycobiome in the development and progression of hepatobiliary diseases. In particular, the fungal genus Candida has been found to be involved in the pathogenesis of multiple hepatobiliary conditions. Herein, we compare colonisation and infection, describe mycobiome findings in the healthy state and across the various hepatobiliary conditions, and point toward communalities. We detail how quantitation of immune responses to fungal antigens can be employed to predict disease severity, e.g. using antibodies to Saccharomyces cerevisiae or specific anti-Candida albicans antibodies. We also show how fungal products (e.g. beta-glucans, candidalysin) activate the host's immune system to exacerbate liver and biliary diseases. Finally, we describe how the gut mycobiome can be modulated to ameliorate hepatobiliary conditions.

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.

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.

Alcohol use is a leading cause of chronic liver disease worldwide, and changes in the microbiome associated with alcohol use contribute to patients' risk for liver disease progression. Less is known about the effects of alcohol use on the intestinal viral microbiome (virome) and interactions between bacteriophages and their target bacteria. We studied changes in the intestinal virome of 62 clinically well-characterized patients with alcohol use disorder (AUD) during active alcohol use and after 2 weeks of alcohol abstinence, by extracting virus-like particles and performing metagenomic sequencing. We observed decreased abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages in patients with active AUD when compared with controls, whereas after 2 weeks of alcohol abstinence, patients with AUD demonstrated an increase in the abundance of Propionibacterium, Lactobacillus, and Leuconostoc phages. The intestinal virome signature was also significantly different in patients with AUD with progressive liver disease, with increased abundance of phages targeting Enterobacteria and Lactococcus species phages compared with patients with AUD with nonprogressive liver disease. By performing moderation analyses, we found that progressive liver disease is associated with changes in interactions between some bacteriophages and their respective target bacteria. In summary, active alcohol use and alcohol-associated progressive liver disease are associated with changes in the fecal virome, some of which are partially reversible after a short period of abstinence. Progression of alcohol-associated liver disease is associated with changes in bacteriophage-bacteria interactions.

Approximately half of all deaths from liver cirrhosis, the tenth leading cause of mortality in the United States, are related to alcohol use. Chronic alcohol consumption is accompanied by intestinal dysbiosis and bacterial overgrowth, yet little is known about the factors that alter the microbial composition or their contribution to liver disease. We previously associated chronic alcohol consumption with lower intestinal levels of the antimicrobial-regenerating islet-derived (REG)-3 lectins. Here, we demonstrate that intestinal deficiency in REG3B or REG3G increases numbers of mucosa-associated bacteria and enhances bacterial translocation to the mesenteric lymph nodes and liver, promoting the progression of ethanol-induced fatty liver disease toward steatohepatitis. Overexpression of Reg3g in intestinal epithelial cells restricts bacterial colonization of mucosal surfaces, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. Thus, alcohol appears to impair control of the mucosa-associated microbiota, and subsequent breach of the mucosal barrier facilitates progression of alcoholic liver disease.

Background

Obesity, non-alcoholic fatty liver disease (NAFLD) and its more advanced form non-alcoholic steatohepatitis (NASH) are important causes of morbidity and mortality worldwide. Bile acid dysregulation is a pivotal part in their pathogenesis. The aim of this study was to evaluate the bile acid sequestrant colesevelam in a microbiome-humanized mouse model of diet-induced obesity and steatohepatitis.

Methods

Germ-free C57BL/6 mice were associated with stool from patients with NASH and subjected to 20 weeks of Western diet feeding with and without colesevelam.

Results

Colesevelam reduced Western diet-induced body and liver weight gain in microbiome-humanized mice compared with controls. It ameliorated Western diet-induced hepatic inflammation, steatosis, fibrosis and insulin resistance. Colesevelam increased de novo bile acid synthesis and decreased hepatic cholesterol content in microbiome-humanized mice fed a Western diet. It further induced the gene expression of the antimicrobials Reg3g and Reg3b in the distal small intestine and decreased plasma levels of LPS.

Conclusions

Colesevelam ameliorates Western diet-induced steatohepatitis and obesity in microbiome-humanized mice.

Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol-associated dysbiosis are largely unknown. The aim of this study was to identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over-representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)-15 protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)-7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine-restricted FXR agonist fexaramine, which protected mice from ethanol-induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF15, a nontumorigenic FGF19 variant-a human FGF15 ortholog-was overexpressed in mice using adeno-associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis.

Conclusion

Taken together, alcohol-associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid-FXR-FGF15 signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol-induced liver disease in mice. (Hepatology 2018;67:2150-2166).

Background & aims

Studies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis.

Methods

We performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B.

Results

The presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis.

Conclusions

Non-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NASH.

Lay summary

Non-alcoholic fatty liver disease is one of the most common chronic liver diseases and is associated with changes in the fecal bacterial microbiome. We show that patients with non-alcoholic fatty liver disease and more severe disease stages have a specific composition of fecal fungi and an increased systemic immune response to Candida albicans. In a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis, we show that treatment with antifungals reduces liver damage.

Background & aims

The degree of cholestasis is an important disease driver in alcoholic hepatitis, a severe clinical condition that needs new biomarkers and targeted therapies. We aimed to identify the largely unknown mechanisms and biomarkers linked to cholestasis in alcoholic hepatitis.

Methods

Herein, we analyzed a well characterized cohort of patients with alcoholic hepatitis and correlated clinical and histological parameters and outcomes with serum bile acids and fibroblast growth factor 19 (FGF19), a major regulator of bile acid synthesis.

Results

We found that total and conjugated bile acids were significantly increased in patients with alcoholic hepatitis compared with controls. Serum FGF19 levels were strongly increased and gene expression of FGF19 was induced in biliary epithelial cells and ductular cells of patients with alcoholic hepatitis. De novo bile acid synthesis (CYP7A1 gene expression and C4 serum levels) was significantly decreased in patients with alcoholic hepatitis. Importantly, total and conjugated bile acids correlated positively with FGF19 and with disease severity (model for end-stage liver disease score). FGF19 correlated best with conjugated cholic acid, and model for end-stage liver disease score best with taurine-conjugated chenodeoxycholic acid. Univariate analysis demonstrated significant associations between FGF19 and bilirubin as well as gamma glutamyl transferase, and negative correlations between FGF19 and fibrosis stage as well as polymorphonuclear leukocyte infiltration, in all patients with alcoholic hepatitis.

Conclusion

Serum FGF19 and bile acids are significantly increased in patients with alcoholic hepatitis, while de novo bile acid synthesis is suppressed. Modulation of bile acid metabolism or signaling could represent a promising target for treatment of alcoholic hepatitis in humans.

Lay summary

Understanding the underlying mechanisms that drive alcoholic hepatitis is important for the development of new biomarkers and targeted therapies. Herein, we describe a molecule that is increased in patients with alcoholic hepatitis. Modulating the molecular pathway of this molecule might lead to promising targets for the treatment of alcoholic hepatitis.