Cristina Llorente

Intestinal REG3 Lectins Protect against Alcoholic Steatohepatitis by Reducing Mucosa-Associated Microbiota and Preventing Bacterial Translocation.

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.

The Gut Microbiota and Liver Disease

The leaky gut hypothesis links translocating microbial products with the onset and progression of liver disease, and for a long time they were considered one of its major contributors. However, a more detailed picture of the intestinal microbiota contributing to liver disease started to evolve. The gut is colonized by trillions of microbes that aid in digestion, modulate immune response, and generate a variety of products that result from microbial metabolic activities. These products together with host-bacteria interactions influence both normal physiology and disease susceptibility. A disruption of the symbiosis between microbiota and host is known as dysbiosis and can have profound effects on health. Qualitative changes such as increased proportions of harmful bacteria and reduced levels of beneficial bacteria, and also quantitative changes in the total amount of bacteria (overgrowth) have been associated with liver disease. Understanding the link between the pathophysiology of liver diseases and compositional and functional changes of the microbiota will help in the design of innovative therapies. In this review, we focus on factors resulting in dysbiosis, and discuss how dysbiosis can disrupt intestinal homeostasis and contribute to liver disease.

Methods to determine intestinal permeability and bacterial translocation during liver disease

Liver disease is often times associated with increased intestinal permeability. A disruption of the gut barrier allows microbial products and viable bacteria to translocate from the intestinal lumen to extraintestinal organs. The majority of the venous blood from the intestinal tract is drained into the portal circulation, which is part of the dual hepatic blood supply. The liver is therefore the first organ in the body to encounter not only absorbed nutrients, but also gut-derived bacteria and pathogen associated molecular patterns (PAMPs). Chronic exposure to increased levels of PAMPs has been linked to disease progression during early stages and to infectious complications during late stages of liver disease (cirrhosis). It is therefore important to assess and monitor gut barrier dysfunction during hepatic disease. We review methods to assess intestinal barrier disruption and discuss advantages and disadvantages. We will in particular focus on methods that we have used to measure increased intestinal permeability and bacterial translocation in experimental liver disease models.

Intestinal fungi contribute to development of alcoholic liver disease

Chronic liver disease with cirrhosis is the 12th leading cause of death in the United States, and alcoholic liver disease accounts for approximately half of all cirrhosis deaths. Chronic alcohol consumption is associated with intestinal bacterial dysbiosis, yet we understand little about the contribution of intestinal fungi, or mycobiota, to alcoholic liver disease. Here we have demonstrated that chronic alcohol administration increases mycobiota populations and translocation of fungal &bgr;-glucan into systemic circulation in mice. Treating mice with antifungal agents reduced intestinal fungal overgrowth, decreased &bgr;-glucan translocation, and ameliorated ethanol-induced liver disease. Using bone marrow chimeric mice, we found that &bgr;-glucan induces liver inflammation via the C-type lectin–like receptor CLEC7A on Kupffer cells and possibly other bone marrow–derived cells. Subsequent increases in IL-1&bgr; expression and secretion contributed to hepatocyte damage and promoted development of ethanol-induced liver disease. We observed that alcohol-dependent patients displayed reduced intestinal fungal diversity and Candida overgrowth. Compared with healthy individuals and patients with non–alcohol-related cirrhosis, alcoholic cirrhosis patients had increased systemic exposure and immune response to mycobiota. Moreover, the levels of extraintestinal exposure and immune response correlated with mortality. Thus, chronic alcohol consumption is associated with an altered mycobiota and translocation of fungal products. Manipulating the intestinal mycobiome might be an effective strategy for attenuating alcohol-related liver disease.

Gastric acid suppression promotes alcoholic liver disease by inducing overgrowth of intestinal Enterococcus

Chronic liver disease is rising in western countries and liver cirrhosis is the 12th leading cause of death worldwide. Simultaneously, use of gastric acid suppressive medications is increasing. Here, we show that proton pump inhibitors promote progression of alcoholic liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis in mice by increasing numbers of intestinal Enterococcus spp. Translocating enterococci lead to hepatic inflammation and hepatocyte death. Expansion of intestinal Enterococcus faecalis is sufficient to exacerbate ethanol-induced liver disease in mice. Proton pump inhibitor use increases the risk of developing alcoholic liver disease among alcohol-dependent patients. Reduction of gastric acid secretion therefore appears to promote overgrowth of intestinal Enterococcus, which promotes liver disease, based on data from mouse models and humans. Recent increases in the use of gastric acid-suppressive medications might contribute to the increasing incidence of chronic liver disease.Proton pump inhibitors (PPIs) reduce gastric acid secretion and modulate gut microbiota composition. Here Llorente et al. show that PPIs induce bacterial overgrowth of enterococci, which, in turn, exacerbate ethanol-induced liver disease both in mice and humans.

Modulation of the intestinal bile acid/farnesoid X receptor/fibroblast growth factor 15 axis improves alcoholic liver disease in 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).

Pyroptosis by caspase11/4‐gasdermin‐D pathway in alcoholic hepatitis in mice and patients

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross‐analysis has identified Casp11 (CASP4 in humans) as a commonly up‐regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin‐D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin‐18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte‐specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation. Conclusion: These results implicate pyroptosis induced by the CASP11/4‐GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737‐1753).

Deficiency of intestinal mucin-2 protects mice from diet-induced fatty liver disease and obesity

Nonalcoholic fatty liver disease (NAFLD) and obesity are characterized by altered gut microbiota, inflammation, and gut barrier dysfunction. Here, we investigated the role of mucin-2 (Muc2) as the major component of the intestinal mucus layer in the development of fatty liver disease and obesity. We studied experimental fatty liver disease and obesity induced by feeding wild-type and Muc2-knockout mice a high-fat diet (HFD) for 16 wk. Muc2 deficiency protected mice from HFD-induced fatty liver disease and obesity. Compared with wild-type mice, after a 16-wk HFD, Muc2-knockout mice exhibited better glucose homeostasis, reduced inflammation, and upregulated expression of genes involved in lipolysis and fatty acid β-oxidation in white adipose tissue. Compared with wild-type mice that were fed the HFD as well, Muc2-knockout mice also displayed higher intestinal and plasma levels of IL-22 and higher intestinal levels of the IL-22 target genes Reg3b and Reg3g. Our findings indicate that absence of the intestinal mucus layer activates the mucosal immune system. Higher IL-22 levels protect mice from diet-induced features of the metabolic syndrome.

Publisher Correction: Gastric acid suppression promotes alcoholic liver disease by inducing overgrowth of intestinal Enterococcus

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 10 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.