Adeline Bertola

Mouse model of chronic and binge ethanol feeding (the NIAAA model)

Chronic alcohol consumption is a leading cause of chronic liver disease worldwide, leading to cirrhosis and hepatocellular carcinoma. Currently, the most widely used model for alcoholic liver injury is ad libitum feeding with the Lieber-DeCarli liquid diet containing ethanol for 4–6 weeks; however, this model, without the addition of a secondary insult, only induces mild steatosis, slight elevation of serum alanine transaminase (ALT) and little or no inflammation. Here we describe a simple mouse model of alcoholic liver injury by chronic ethanol feeding (10-d ad libitum oral feeding with the Lieber-DeCarli ethanol liquid diet) plus a single binge ethanol feeding. This protocol for chronic-plus-single-binge ethanol feeding synergistically induces liver injury, inflammation and fatty liver, which mimics acute-on-chronic alcoholic liver injury in patients. This feeding protocol can also be extended to chronic feeding for longer periods of time up to 8 weeks plus single or multiple binges. Chronic-binge ethanol feeding leads to high blood alcohol levels; thus, this simple model will be very useful for the study of alcoholic liver disease (ALD) and of other organs damaged by alcohol consumption.

Interleukin‐22 induces hepatic stellate cell senescence and restricts liver fibrosis in mice

Interleukin (IL)‐22 is known to play a key role in promoting antimicrobial immunity, inflammation, and tissue repair at barrier surfaces by binding to the receptors, IL‐10R2 and IL‐22R1. IL‐22R1 is generally thought to be expressed exclusively in epithelial cells. In this study, we identified high levels of IL‐10R2 and IL‐22R1 expression on hepatic stellate cells (HSCs), the predominant cell type involved in liver fibrogenesis in response to liver damage. In vitro treatment with IL‐22 induced the activation of signal transducer and activator of transcription (STAT) 3 in primary mouse and human HSCs. IL‐22 administration prevented HSC apoptosis in vitro and in vivo, but surprisingly, the overexpression of IL‐22 by either gene targeting (e.g., IL‐22 transgenic mice) or exogenous administration of adenovirus expressing IL‐22 reduced liver fibrosis and accelerated the resolution of liver fibrosis during recovery. Furthermore, IL‐22 overexpression or treatment increased the number of senescence‐associated beta‐galactosidase‐positive HSCs and decreased alpha‐smooth muscle actin expression in fibrotic livers in vivo and cultured HSCs in vitro. Deletion of STAT3 prevented IL‐22‐induced HSC senescence in vitro, whereas the overexpression of a constitutively activated form of STAT3 promoted HSC senescence through p53‐ and p21‐dependent pathways. Finally, IL‐22 treatment up‐regulated the suppressor of cytokine signaling (SOCS) 3 expression in HSCs. Immunoprecipitation analyses revealed that SOCS3 bound p53 and subsequently increased the expression of p53 and its target genes, contributing to IL‐22‐mediated HSC senescence. Conclusion: IL‐22 induces the senescence of HSCs, which express both IL‐10R2 and IL‐22R1, thereby ameliorating liver fibrogenesis. The antifibrotic effect of IL‐22 is likely mediated by the induction of HSC senescence, in addition to the previously discovered hepatoprotective functions of IL‐22. (HEPATOLOGY 2012;56:1150–1159)

Chronic plus binge ethanol feeding synergistically induces neutrophil infiltration and liver injury in mice: A critical role for E‐selectin

Chronic plus binge ethanol feeding acts synergistically to induce liver injury in mice, but the mechanisms underlying this phenomenon remain unclear. Here, we show that chronic plus binge ethanol feeding synergistically up‐regulated the hepatic expression of interleukin‐1β and tumor necrosis factor alpha and induced neutrophil accumulation in the liver, compared with chronic or binge feeding alone. In vivo depletion of neutrophils through administration of an anti‐Ly6G antibody markedly reduced chronic‐binge ethanol feeding‐induced liver injury. Real‐time polymerase chain reaction analyses revealed that hepatic E‐selectin expression was up‐regulated 10‐fold, whereas expression of other neutrophil infiltration‐related adhesion molecules (e.g., P‐selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1) was slightly up‐ or down‐regulated in this chronic‐binge model. The genetic deletion of E‐selectin prevented chronic‐binge ethanol‐induced hepatic neutrophil infiltration as well as elevation of serum transaminases without affecting ethanol‐induced steatosis. In addition, E‐selectin‐deficient mice showed reduced hepatic expression of several proinflammatory cytokines, chemokines, and adhesion molecules, compared to wild‐type mice, after chronic‐binge ethanol feeding. Finally, the expression of E‐selectin was highly up‐regulated in human alcoholic fatty livers, but not in alcoholic cirrhosis. Conclusions: Chronic‐binge ethanol feeding up‐regulates expression of proinflammatory cytokines, followed by the induction of E‐selectin. Elevated E‐selectin plays an important role in hepatic neutrophil infiltration and injury induced by chronic‐binge feeding in mice and may also contribute to the pathogenesis of early stages of human alcoholic liver disease. (Hepatology 2013;58:1814–1823)

Inflammation-associated interleukin-6/signal transducer and activator of transcription 3 activation ameliorates alcoholic and nonalcoholic fatty liver diseases in interleukin-10–deficient mice†‡

Alcoholic and nonalcoholic steatohepatitis are characterized by fatty liver plus inflammation. It is generally believed that steatosis promotes inflammation, whereas inflammation in turn aggregates steatosis. Thus, we hypothesized the deletion of interleukin (IL)‐10, a key anti‐inflammatory cytokine, exacerbates liver inflammation, steatosis, and hepatocellular damage in alcoholic and nonalcoholic fatty liver disease models that were achieved via feeding mice with a liquid diet containing 5% ethanol for 4 weeks or a high‐fat diet (HFD) for 12 weeks, respectively. IL‐10 knockout (IL‐10−/−) mice and several other strains of genetically modified mice were generated and used. Compared with wild‐type mice, IL‐10−/− mice had greater liver inflammatory response with higher levels of IL‐6 and hepatic signal transducer and activator of transcription 3 (STAT3) activation, but less steatosis and hepatocellular damage after alcohol or HFD feeding. An additional deletion of IL‐6 or hepatic STAT3 restored steatosis and hepatocellular damage but further enhanced liver inflammatory response in IL‐10−/− mice. In addition, the hepatic expression of sterol regulatory element‐binding protein 1 and key downstream lipogenic proteins and enzymes in fatty acid synthesis were down‐regulated in IL‐10−/− mice. Conversely, IL‐10−/− mice displayed enhanced levels of phosphorylated adenosine monophosphate‐activated protein kinase and its downstream targets including phosphorylated acetyl‐coenzyme A carboxylase and carnitine palmitoyltransferase 1 in the liver. Such dysregulations were corrected in IL‐10−/−IL‐6−/− or IL‐10−/−STAT3Hep−/− double knockout mice. Conclusion: IL‐10−/− mice are prone to liver inflammatory response but are resistant to steatosis and hepatocellular damage induced by ethanol or HFD feeding. Resistance to steatosis in these mice is attributable to elevation of inflammation‐associated hepatic IL‐6/STAT3 activation that subsequently down‐regulates lipogenic genes but up‐regulates fatty acid oxidation‐associated genes in the liver. (HEPATOLOGY 2011; 54:846–856)

Animals models of gastrointestinal and liver diseases. Animal models of alcohol-induced liver disease: pathophysiology, translational relevance, and challenges.

Over the last four decades, chronic ethanol feeding studies in rodents using either ad libitum feeding or intragastric infusion models have significantly enhanced our understanding of the pathogenesis of alcoholic liver disease (ALD). Recently, we developed a chronic plus binge alcohol feeding model in mice that is similar to the drinking patterns of many alcoholic hepatitis patients: a history of chronic drinking and recent excessive alcohol consumption. Chronic+binge ethanol feeding synergistically induced steatosis, liver injury, and neutrophil infiltration in mice, which may be useful for the study of early alcoholic liver injury and inflammation. Using this chronic+binge model, researchers have begun to identify novel mechanisms that participate in the pathogenesis of alcoholic liver injury, thereby revealing novel therapeutic targets. In this review article, we briefly discuss several mouse models of ALD with a focus on the chronic+binge ethanol feeding model.

Progression of Chronic Liver Inflammation and Fibrosis Driven by Activation of c-JUN Signaling in Sirt6 Mutant Mice

Background: Sirt6 plays important roles in metabolism and lifespan; however, its role in inflammation is unknown. Results: Sirt6 deficiency in the immune cells of mice results in liver inflammation and fibrosis through activating the c-JUN signaling. Conclusion: Sirt6 has anti-inflammatory function in mice. Significance: Small chemical compounds that activate Sirt6 might be useful in therapeutic treatment of chronic liver inflammation. The human body has a remarkable ability to regulate inflammation, a biophysical response triggered by virus infection and tissue damage. Sirt6 is critical for metabolism and lifespan; however, its role in inflammation is unknown. Here we show that Sirt6-null (Sirt6−/−) mice developed chronic liver inflammation starting at ∼2 months of age, and all animals were affected by 7–8 months of age. Deletion of Sirt6 in T cells or myeloid-derived cells was sufficient to induce liver inflammation and fibrosis, albeit to a lesser degree than that in the global Sirt6−/− mice, suggesting that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6−/− mice showed increased MCP-1, IL-6, and TNFα expression levels and were hypersensitive to LPS stimulation. Mechanistically, SIRT6 interacts with c-JUN and deacetylates histone H3 lysine 9 (H3K9) at the promoter of proinflammatory genes whose expression involves the c-JUN signaling pathway. Sirt6-deficient macrophages displayed hyperacetylation of H3K9 and increased occupancy of c-JUN in the promoter of these genes, leading to their elevated expression. These data suggest that Sirt6 plays an anti-inflammatory role in mice by inhibiting c-JUN-dependent expression of proinflammatory genes.

Fat-Specific Protein 27/CIDEC Promotes Development of Alcoholic Steatohepatitis in Mice and Humans

BACKGROUND & AIMS Alcoholic steatohepatitis (ASH) is the progressive form of alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma. We studied mouse models and human tissues to identify molecules associated with ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in white adipose tissues and promotes formation of fat droplets. METHODS C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27 (Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5% ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5 or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor (GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral vectors were used to express transgenes or small hairpin (sh) RNAs in cultured hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH and analyzed histologically and immunohistochemically and by transcriptome, immunoblotting, and real-time PCR analyses. RESULTS Chronic-plus-binge ethanol feeding of mice, which mimics the drinking pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray analyses revealed similar alterations in expression of many hepatic genes in ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given injections of adenovirus-Fsp27shRNA had markedly reduced ASH following chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic AMP-responsive element binding protein H (CREBH) prevented the increases in Fsp27α and FSP27β mRNAs, respectively, and reduced liver injury in this chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol exposure had synergistic effects in inducing production of mitochondrial reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA expression was increased in patients with AH and correlated with the degree of hepatic steatosis and disease severity including mortality. CONCLUSIONS In mice, chronic-plus-binge ethanol feeding induces ASH that mimics some histological and molecular features observed in patients with AH. Hepatic expression of FSP27/CIDEC is highly up-regulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation contributes to alcohol-induced liver damage.

Short- or long-term high-fat diet feeding plus acute ethanol binge synergistically induce acute liver injury in mice: an important role for CXCL1.

Obesity and alcohol consumption often coexist and work synergistically to promote steatohepatitis; however, the underlying mechanisms remain obscure. Here, we demonstrate that feeding mice a high‐fat diet (HFD) for as little as 3 days markedly exacerbated acute ethanol binge–induced liver neutrophil infiltration and injury. Feeding mice with an HFD for 3 months plus a single binge of ethanol induced much more severe steatohepatitis. Moreover, 3‐day or 3‐month HFD‐plus‐ethanol binge (3d‐HFD+ethanol or 3m‐HFD+ethanol) treatment markedly up‐regulated the hepatic expression of several chemokines, including chemokine (C‐X‐C motif) ligand 1 (Cxcl1), which showed the highest fold (approximately 20‐fold and 35‐fold, respectively) induction. Serum CXCL1 protein levels were also markedly elevated after the HFD+ethanol treatment. Blockade of CXCL1 with a CXCL1 neutralizing antibody or genetic deletion of the Cxcl1 gene reduced the HFD+ethanol‐induced hepatic neutrophil infiltration and injury, whereas overexpression of Cxcl1 exacerbated steatohepatitis in HFD‐fed mice. Furthermore, expression of Cxcl1 messenger RNA was up‐regulated in hepatocytes, hepatic stellate cells, and endothelial cells isolated from HFD+ethanol‐fed mice compared to mice that were only given the HFD, with the highest fold induction observed in hepatocytes. In vitro stimulation of hepatocytes with palmitic acid up‐regulated the expression of Cxcl1 messenger RNA, and this up‐regulation was attenuated after treatment with an inhibitor of extracellular signal–regulated kinase 1/2, c‐Jun N‐terminal kinase, or nuclear factor κB. In addition, hepatic or serum levels of free fatty acids were higher in HFD+ethanol‐fed mice than in the control groups. Conclusion: An HFD combined with acute ethanol consumption synergistically induces acute liver inflammation and injury through the elevation of hepatic or serum free fatty acids and subsequent up‐regulation of hepatic CXCL1 expression and promotion of hepatic neutrophil infiltration. (Hepatology 2015;62:1070‐1085)

Activation of invariant natural killer T cells impedes liver regeneration by way of both IFN-γ- and IL-4-dependent mechanisms

Invariant natural killer T (iNKT) cells are a major subset of lymphocytes found in the liver. These cells mediate various functions, including hepatic injury, fibrogenesis, and carcinogenesis. However, the function of iNKT cells in liver regeneration remains unclear. In the present study, partial hepatectomy (PHx) was used to study liver regeneration. α‐Galactosylceramide (α‐GalCer), a specific ligand for iNKT cells, was used to induce iNKT cell activation. After PHx, two strains of iNKT cell‐deficient mice, CD1d−/− and Jα281−/− mice, showed normal liver regeneration. Injection of α‐GalCer before or after PHx, which rapidly stimulated interferon‐gamma (IFN‐γ) and interleukin (IL)‐4 production by iNKT cells, markedly inhibited liver regeneration. In vitro treatment with IFN‐γ inhibited hepatocyte proliferation. In agreement with this in vitro finding, genetic disruption of IFN‐γ or its downstream signaling molecule signal transducer and activator of transcription (STAT)1 significantly abolished the α‐GalCer‐mediated inhibition of liver regeneration. In vitro exposure to IL‐4 did not affect hepatocyte proliferation, but surprisingly, genetic ablation of IL‐4 or its downstream signaling molecule STAT6 partially eliminated the inhibitory effect of α‐GalCer on liver regeneration. Further studies revealed that IL‐4 contributed to α‐GalCer‐induced iNKT cell expansion and IFN‐γ production, thereby inhibiting liver regeneration. Conclusion: iNKT cells play a minor role in controlling liver regeneration after PHx under healthy conditions. Activation of iNKT cells by α‐GalCer induces the production of IFN‐γ, which directly inhibits liver regeneration, and IL‐4, which indirectly attenuates liver regeneration by stimulating iNKT cell expansion and IFN‐γ production. (Hepatology 2014;60:1356–1366)

Animal models of alcoholic liver disease: Pathogenesis and clinical relevance

Alcoholic liver disease (ALD), a leading cause of chronic liver injury worldwide, comprises a range of disorders including simple steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Over the last five decades, many animal models for the study of ALD pathogenesis have been developed. Recently, a chronic-plus-binge ethanol feeding model was reported. This model induces significant steatosis, hepatic neutrophil infiltration, and liver injury. A clinically relevant model of high-fat diet feeding plus binge ethanol was also developed, which highlights the risk of excessive binge drinking in obese/overweight individuals. All of these models recapitulate some features of the different stages of ALD and have been widely used by many investigators to study the pathogenesis of ALD and to test for therapeutic drugs/components. However, these models are somewhat variable, depending on mouse genetic background, ethanol dose, and animal facility environment. This review focuses on these models and discusses these variations and some methods to improve the feeding protocol. The pathogenesis, clinical relevance, and translational studies of these models are also discussed.