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.

Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta ceil failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insuiin resistance through activation of peripheral CB1 receptors (CB1Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB1R signaling in beta ceils, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB1R-deficient [Cnr1−/−) or Nlrp3−/− mice, with the endocannabinoid anandamide. Peripheral CB1R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB1R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB1R as a therapeutic target in T2DM.

Interleukin-22 (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 3 (STAT3) in primary mouse and human HSCs. IL-22 administration prevented HSC apoptosis in vitro and in vivo, but surprisingly, the overexpression of IL-22 via either gene targeting (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 β-galactosidase-positive HSCs and decreased α-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 via p53- and p21-dependent pathways. Finally, IL-22 treatment upregulated suppressor of cytokine signaling 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 anti-fibrotic effect of IL-22 is likely mediated via the induction of HSC senescence in addition to the previously discovered hepatoprotective functions of IL-22.

T-cell regulation in adipose tissue provides a link between inflammation and insulin resistance. Because of alterations in adipose tissue T-cell composition in obesity, we aimed to identify the antigen-presenting cells in adipose tissue of obese mice and patients with insulin resistance. Dendritic cells (DCs) and T cells were studied in mice and in two cohorts of obese patients. In lean mice, only CD11c+ DCs were detected in adipose tissue. Adoptive transfer of naive CD4+ T cells in Rag1−/− mice led to a predominant Th1 response in adipose tissue. In contrast, during obesity DCs (human CD11c+CD1c+ and mouse CD11chighF4/80low) accumulated in adipose tissue. CD11chighF4/80low DCs from obese mice induced Th17 differentiation. In patients, the presence of CD11c+CD1c+ DCs correlated with the BMI and with an elevation in Th17 cells. In addition, these DCs led to ex vivo Th17 differentiation. CD1c gene expression further correlated with homeostatic model assessment-insulin resistance in the subcutaneous adipose tissue of obese patients. We show for the first time the presence and accumulation of specific DCs in adipose tissue in mouse and human obesity. These DCs were functional and could be important regulators of adipose tissue inflammation by regulating the switch toward Th17 cell responses in obesity-associated insulin resistance.

We have previously demonstrated that chronic plus binge ethanol feeding acts synergistically to induce liver injury in mice; however, the mechanisms underlying this phenomenon remain unclear. In the current study, we show that chronic plus binge ethanol feeding synergistically upregulated the hepatic expression of IL-1β and TNF-α and induced neutrophil accumulation in the liver compared to chronic or binge feeding alone. The in vivo depletion of neutrophils through the administration of an anti-Ly6G antibody markedly reduced chronic-binge ethanol feeding-induced liver injury. Real-time PCR analyses revealed that hepatic E-selectin expression was upregulated 10-fold, whereas the expression of other neutrophil infiltration-related adhesion molecules (such as P-selectin, ICAM-1, and VCAM-1) was slightly upregulated or downregulated in this chronic-binge model. The genetic deletion of E-selectin prevented chronic-binge ethanol-induced hepatic neutrophil infiltration and the 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 upregulated in human alcoholic fatty livers but not in alcoholic cirrhosis. Conclusions Chronic-binge ethanol feeding upregulates the expression of proinflammatory cytokines, followed by the induction of E-selectin. Elevated E-selectin plays an important role in hepatic neutrophil infiltration and injury in mice induced by chronic-binge feeding, and may also contribute to the pathogenesis of early stages of human alcoholic liver disease.

Obesity and alcohol consumption often coexist and work synergistically to promote steatohepatitis; however, the underlying mechanisms remain obscure. Here, we demonstrated 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 a 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 upregulated 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 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 the Cxcl1 exacerbated steatohepatitis in HFD-fed mice. Furthermore, the expression of Cxcl1 mRNA was upregulated in hepatocytes, hepatic stellate cells and endothelial cells isolated from HFD+ethanol-fed mice compared to mice that were only given a HFD, with the highest fold induction observed in hepatocytes. In vitro stimulation of hepatocytes with palmitic acid upregulated the expression of Cxcl1 mRNA, and this upregulation was attenuated after treatment with ERK1/2, JNK, or NF-κB inhibitors. In addition, hepatic or serum levels of free fatty acids (FFAs) were higher in HFD+ethanol-fed mice than in the control groups. In conclusion, a HFD combined with acute ethanol consumption synergistically induces acute liver inflammation and injury via the elevation of hepatic or serum FFAs and subsequent upregulation of hepatic CXCL1 expression and promotion of hepatic neutrophil infiltration.

BackgroundObesity modulates inflammation and activation of immune pathways which can lead to liver complications. We aimed at identifying expression patterns of inflammatory and immune response genes specifically associated with obesity and NASH in the liver of morbidly obese patients.Methodology/Principal FindingsExpression of 222 genes was evaluated by quantitative RT-PCR in the liver of morbidly obese patients with histologically normal liver (n = 6), or with severe steatosis without (n = 6) or with NASH (n = 6), and in lean controls (n = 5). Hepatic expression of 58 out of 222 inflammatory and immune response genes was upregulated in NASH patients. The most notable changes occurred in genes encoding chemokines and chemokine receptors involved in leukocyte recruitment, CD and cytokines involved in the T cell activation towards a Th1 phenotype, and immune semaphorins. This regulation seems to be specific for the liver since visceral adipose tissue expression and serum levels of MCP1, IP10, TNFα and IL6 were not modified. Importantly, 47 other genes were already upregulated in histologically normal liver (e.g. CRP, Toll-like receptor (TLR) pathway). Interestingly, serum palmitate, known to activate the TLR pathway, was increased with steatosis.Conclusion/SignificanceThe liver of obese patients without histological abnormalities already displayed a low-grade inflammation and could be more responsive to activators of the TLR pathway. NASH was then characterized by a specific gene signature. These findings help to identify new potential actors of the pathogenesis of NAFLD.

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 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 (Fsp27Hep−/− 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 of the peroxisome proliferator-activated receptor-γ (PPARG) (GW9662). 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 by histologic, immunohistochemical, transcriptome, immunoblot, 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 upregulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27Hep−/− 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. Conclusion 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 upregulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this upregulation contributes to alcohol-induced liver damage.