E. Mingarelli

Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice

Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram‐negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high‐fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut‐sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL‐ or HFD‐treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram‐negative versus Gram‐postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram‐negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD‐treated mice; liver damage was further enhanced by transplantation of selected Gram‐negative bacteria obtained from cecum content of HFD+BDL‐treated mice. Conclusions: Dietary habits, by increasing the percentage of intestinal Gram‐negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD. (Hepatology 2014;59:1738–1749)

HCC Development Is Associated to Peripheral Insulin Resistance in a Mouse Model of NASH

NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. Aim: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. Methods: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. Results: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1–3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. Conclusions: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD.

Activation of the developmental pathway neurogenin‐3/microRNA‐7a regulates cholangiocyte proliferation in response to injury

The activation of the biliary stem‐cell signaling pathway hairy and enhancer of split 1/pancreatic duodenal homeobox‐1 (Hes‐1/PDX‐1) in mature cholangiocytes determines cell proliferation. Neurogenin‐3 (Ngn‐3) is required for pancreas development and ductal cell neogenesis. PDX‐1‐dependent activation of Ngn‐3 initiates the differentiation program by inducing microRNA (miR)−7 expression. Here we investigated the role Ngn‐3 on cholangiocyte proliferation. Expression levels of Ngn‐3 and miR‐7 isoforms were tested in cholangiocytes from normal and cholestatic human livers. Ngn‐3 was knocked‐down in vitro in normal rat cholangiocytes by short interfering RNA (siRNA). In vivo, wild‐type and Ngn‐3‐heterozygous (+/−) mice were subjected to 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) feeding (a model of sclerosing cholangitis) or bile duct ligation (BDL). In the liver, Ngn‐3 is expressed specifically in cholangiocytes of primary sclerosing cholangitis (PSC) patients and in mice subjected to DDC or BDL, but not in normal human and mouse livers. Expression of miR‐7a‐1 and miR‐7a‐2 isoforms, but not miR‐7b, was increased in DDC cholangiocytes compared to normal ones. In normal rat cholangiocytes, siRNA against Ngn‐3 blocked the proliferation stimulated by exendin‐4. In addition, Ngn‐3 knockdown neutralized the overexpression of insulin growth factor‐1 (IGF1; promitotic effector) observed after exposure to exendin‐4, but not that of PDX‐1 or VEGF‐A/C. Oligonucleotides anti‐miR‐7 inhibited the exendin‐4‐induced proliferation in normal rat cholangiocytes, but did not affect Ngn‐3 synthesis. Biliary hyperplasia and collagen deposition induced by DDC or BDL were significantly reduced in Ngn‐3+/− mice compared to wild‐type. Conclusion: Ngn‐3‐dependent activation of miR‐7a is a determinant of cholangiocyte proliferation. These findings indicate that the reacquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes. (Hepatology 2014;60:1324–1335)

Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD

Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A “multi-hit” theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3−/− and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3−/−-HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3−/−-HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy.

P353 ACTIVATION OF THE DEVELOPMENTAL PATHWAY Ngn-3/miR-7a REGULATES CHOLANGIOCYTES PROLIFERATION IN RESPONSE TO INJURY

Background and Aims: The activation of the biliary stemcell signalling pathway Hes-1/PDX-1 in mature cholangiocytes determines cell proliferation. Neurogenin-3 (Ngn-3) is required for pancreas development and for ductal cell neogenesis. PDX-1dependent activation of Ngn-3 initiates the differentiation program, by inducting microRNA (miR)-7 expression. We aimed to verify whether Ngn-3 regulates cholangiocyte proliferation. Methods: Expression levels of Ngn-3 and miR-7 isoforms were tested in cholangiocytes from normal and cholestatic livers. Ngn-3 was knocked down in vitro by siRNA. In vivo, wild type (WT) and Ngn-3-heterozygous (+/−) mice were subjected to Bile Duct Ligation (BDL) for 2 weeks. Results: In the liver, Ngn-3 is expressed in cholangiocytes of mice subjected to BDL and of patients affected by PSC, but not in normal conditions. Expression of miR-7a-1 and miR-7a-2 isoforms, but not miR-7b, was increased in BDL cholangiocytes as compared to normal ones. In vitro, Ngn-3 siRNA neutralized the increases in cell proliferation and in the expression of IGF-1 (a pro-proliferative effector) and miR-7a, but not of PDX-1 or VEGF, observed after exposure to FBS or exendin-4. Anti-sense miR-7 neutralized the FBS or exendin-4 induced increases in cell proliferation but not in PDX-1 and Ngn-3 synthesis. In vivo, increases in bile duct mass and collagen deposition induced by BDL were significantly reduced in Ngn-3 mice. Conclusions: Ngn-3-dependent activation of miR-7a is a determinant of cholangiocyte proliferation. These findings indicate that the re-acquisition of a molecular profile typical of organ development is essential for the biological response to injury by mature cholangiocytes.