ECM1 attenuates hepatic fibrosis by interfering with mediators of latent TGF-ß1 activation.

OBJECTIVE: Extracellular matrix protein 1 (ECM1) serves as a gatekeeper of hepatic fibrosis by maintaining transforming growth factor-ß1 (TGF-ß1) in its latent form. ECM1 knockout (KO) causes latent (L) TGF-ß1 activation, resulting in hepatic fibrosis with rapid mortality. In chronic liver disease (CLD), ECM1 decreases with increasing CLD severity. We investigate the regulatory role of ECM1 in TGF-ß1 bioavailability and its impact on CLD progression. DESIGN: RNAseq was performed to analyse hepatic gene expression. Functional assays were performed using hepatic stellate cells (HSCs), Ecm1-KO and Fxr-KO mice, patient liver tissue and computer simulations. RESULTS: Expression of LTGF-ß1 activators, including thrombospondins (TSPs), ADAMTS proteases and matrix metalloproteinases (MMPs), increased along with profibrotic gene expression in liver tissue of Ecm1-KO mice. In HSCs, overexpression of ECM1 prevented LTGF-ß1 activation mediated by TSP-1, ADAMTS1, and MMP-2/9. In vitro interaction assays demonstrated that ECM1 inhibited LTGF-ß1 activation by interacting with TSP-1 and ADAMTS1 via their respective, intrinsic KRFK or KTFR amino acid sequences and by suppressing MMP-2/9 proteolytic activity. In mice, ECM1 overexpression attenuated KRFK-induced LTGF-ß1 activation while KTFR treatment reversed Ecm1-KO-mediated and Fxr-KO-mediated liver injury. In patients with CLD, ECM1 expression was inversely correlated with TSP-1, ADAMTS1, MMP-2/9 expression and LTGF-ß1 activation. And, these results were complemented by a computational compartment model representing the key network of cellular phenotypes and predicted interactions in liver fibrogenesis. CONCLUSION: Our findings underscore the hepatoprotective effect of ECM1, which interferes with mediators of LTGF-ß1 activation, suggesting ECM1 or its representative peptide as potential antifibrotic therapies in CLD.

Metabolic dysfunction-associated liver disease and diabetes: Matrix remodeling, fibrosis, and therapeutic implications.

Metabolic dysfunction-associated liver disease (MASLD) and steatohepatitis (MASH) are becoming the most common causes of chronic liver disease in the United States and worldwide due to the obesity and diabetes epidemics. It is estimated that by 2030 close to 100 million people might be affected and patients with type 2 diabetes are especially at high risk. Twenty to 30% of patients with MASLD can progress to MASH, which is characterized by steatosis, necroinflammation, hepatocyte ballooning, and in advanced cases, fibrosis progressing to cirrhosis. Clinically, it is recognized that disease progression in diabetic patients is accelerated and the role of various genetic and epigenetic factors, as well as cell-matrix interactions in fibrosis and stromal remodeling, have recently been recognized. While there has been great progress in drug development and clinical trials for MASLD/MASH, the complexity of these pathways highlights the need to improve diagnosis/early detection and develop more successful antifibrotic therapies that not only prevent but reverse fibrosis.

Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Pre-clinical evidence of a dual NADPH oxidase 1/4 inhibitor (setanaxib) in liver, kidney and lung fibrosis.

Fibrosis describes a dysregulated tissue remodelling response to persistent cellular injury and is the final pathological consequence of many chronic diseases that affect the liver, kidney and lung. Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) enzymes produce reactive oxygen species (ROS) as their primary function. ROS derived from NOX1 and NOX4 are key mediators of liver, kidney and lung fibrosis. Setanaxib (GKT137831) is a first-in-class, dual inhibitor of NOX1/4 and is the first NOX inhibitor to progress to clinical trial investigation. The anti-fibrotic effects of setanaxib in liver, kidney and lung fibrosis are supported by multiple lines of pre-clinical evidence. However, despite advances in our understanding, the precise roles of NOX1/4 in fibrosis require further investigation. Additionally, there is a translational gap between the pre-clinical observations of setanaxib to date and the applicability of these to human patients within a clinical setting. This narrative review critically examines the role of NOX1/4 in liver, kidney and lung fibrosis, alongside the available evidence investigating setanaxib as a therapeutic agent in pre-clinical models of disease. We discuss the potential clinical translatability of this pre-clinical evidence, which provides rationale to explore NOX1/4 inhibition by setanaxib across various fibrotic pathologies in clinical trials involving human patients.

Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

BACKGROUND & AIMS: Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. METHODS: Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed in vivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. RESULTS: Shc was significantly induced in patients with AH (P < .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1ß, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. In vivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. CONCLUSIONS: Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

Non-alcoholic Fatty Liver Disease and Liver Fibrosis during Aging.

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) have emerged as the leading causes of chronic liver disease-related mortality. The prevalence of NAFLD/NASH is expected to increase given the epidemics of obesity and type 2 diabetes mellitus. Older patients are disproportionally affected by NASH and related complications such as progressive fibrosis, cirrhosis and hepatocellular carcinoma; however, they are often ineligible for liver transplantation due to their frailty and comorbidities, and effective medical treatments are still lacking. In this review we focused on pathways that are key to the aging process in the liver and perpetuate NAFLD/NASH, leading to fibrosis. In addition, we highlighted recent findings and cross-talks of normal and/or senescent liver cells, dysregulated nutrient sensing, proteostasis and mitochondrial dysfunction in the framework of changing metabolic milieu. Better understanding these pathways during preclinical and clinical studies will be essential to design novel and specific therapeutic strategies to treat NASH in the elderly.

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called "chicken wire," which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson's trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis. The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

Shc inhibitor idebenone ameliorates liver injury and fibrosis in dietary NASH in mice.

Shc expression rises in human nonalcoholic steatohepatitis (NASH) livers, and Shc-deficient mice are protected from NASH-thus Shc inhibition could be a novel therapeutic strategy for NASH. Idebenone was recently identified as the first small-molecule Shc inhibitor drug. We tested idebenone in the fibrotic methionine-choline deficient (MCD) diet and the metabolic fast food diet (FFD) mouse models of NASH. In the fibrotic MCD NASH model, idebenone reduced Shc expression and phosphorylation in peripheral blood mononuclear cells and Shc expression in the liver; decreased serum alanine aminotransferase and aspartate aminotransferase; and attenuated liver fibrosis as observed by quantitative polymerase chain reaction (qPCR) and hydroxyproline quantification. In the metabolic FFD model, idebenone administration improved insulin resistance, and reduced inflammation and fibrosis shown with qPCR, hydroxyproline measurement, and histology. Thus, idebenone ameliorates NASH in two mouse models. As an approved drug with a benign safety profile, Idebenone could be a reasonable human NASH therapy.

Editorial: Noninvasive Fibrosis Biomarkers in Patients With NASH With Diabetes.

This is an editorial about non-invasive fibrosis markers in patients with NASH and type 2 diabetes mellitus.

Soluble Epoxide Hydrolase Hepatic Deficiency Ameliorates Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored. METHODS: To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model. RESULTS: We observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding. CONCLUSIONS: These findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.

AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging.

BACKGROUND AND AIMS: Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. APPROACH AND RESULTS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. CONCLUSIONS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

Leptin/adiponectin ratio correlates with hepatic steatosis but not arterial stiffness in nonalcoholic fatty liver disease in Japanese population.

BACKGROUND AND AIMS: Cardiovascular disease (CVD) is a leading cause of mortality in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship of leptin-to-adiponectin (L/A) ratio with hepatic steatosis and arterial stiffness in NAFLD. METHODS: The subjects were 871 Japanese adults who participated in a health survey. Dietary intake, body composition, lipid profile, serum interleukin-6 (IL-6), leptin, and adiponectin were analyzed. NAFLD was defined as fatty liver on ultrasonography in the absence of other causes of steatosis. Arterial stiffness was evaluated by the brachial-ankle pulse wave velocity (baPWV). RESULTS: The subjects with NAFLD had a greater body mass index (BMI) and body fat percentage (BFP); a higher intake of daily energy (kcal) and carbohydrates; and a higher prevalence of hypertension, diabetes, and hyperlipidemia. The subjects with NAFLD had higher serum leptin and lower serum adiponectin concentrations and a higher L/A ratio than subjects without NAFLD. The L/A ratio increased with increasing severity of steatosis. The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. Women had higher L/A ratio and BFP levels than men regardless of the presence or absence of NAFLD. There was a weak positive correlation between baPWV and severity of steatosis. BaPWV was strongly correlated with age, while no relation was found between baPWV and L/A ratio. IL-6 level was correlated with baPVW and age, while the correlation between Il and 6 level and L/A ratio was very weak. The L/A ratio was correlated with triglycerides and the ratio of total cholesterol to high-density lipoprotein-cholesterol. CONCLUSION: L/A ratio and arterial stiffness were associated with the severity of steatosis, whereas there was no correlation between L/A ratio and arterial stiffness in NAFLD. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the pathogenesis for atherosclerosis in NAFLD.

Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH.NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.

Patterns and co-occurrence of risk factors for hepatocellular carcinoma in four Asian American communities: a cross-sectional study.

OBJECTIVES: To investigate risk factor patterns and the simultaneous occurrence of multiple risk factors in the viral, metabolic and lifestyle domains among Asian Americans, who have had the highest mortality rates from hepatocellular carcinoma (HCC). SETTING: Sacramento County, California, USA. PARTICIPANTS: Eligible participants were county residents ages 18 and older who had not been screened for chronic hepatitis B virus (HBV) and were born in a CDC-defined endemic area or whose parent was born in that area. Of 1004 enrolled, 917 were foreign-born Chinese (130 women, 94 men), Hmong (133 women, 75 men), Korean (178 women, 90 men) or Vietnamese (136 women, 81 men) with complete risk factor data. PRIMARY AND SECONDARY OUTCOME MEASURES: We tested participants for HBV and chronic hepatitis C virus (HCV); measured haemoglobin A1c and waist circumference; and recorded self-reported history of diabetes, hypertension, alcohol use and smoking status. We identified risk factor patterns using cluster analysis and estimated gender-specific age-standardised prevalence rates. RESULTS: We identified four patterns: (1) viral (chronic HBV or HCV); (2) lifestyle (current smoker or alcohol user, no viral); (3) metabolic (≥2 metabolic, no lifestyle or viral); and (4) lower risk (≤1 metabolic, no lifestyle or viral). Vietnamese men (16.3%, 95% CI 7.4% to 25.3%) and Hmong women (15.1%, 95% CI 7.8% to 22.5%) had the highest viral pattern prevalence. Hmong women had the highest metabolic (37.8%, 95% CI 29.8% to 45.9%), and Vietnamese men the highest lifestyle (70.4%, 95% CI 59.1% to 81.7%) pattern prevalence. In multiple domains, Hmong men and women were most likely to have viral+metabolic risk factors (men: 14.4%, 95% CI 6.0% to 22.7%; women: 11.9%, 95% CI 5.6% to 18.3%); Vietnamese men were most likely to have lifestyle+viral (10.7%, 95% CI 2.7% to 18.8%), and lifestyle+metabolic but not viral (46.4%, 95% CI 34.4% to 58.5%) risk factors. CONCLUSIONS: Efforts to reduce HCC must comprehensively address multiple risk factors. TRIAL REGISTRATION NUMBER: NCT02596438.

Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis in Experimental Steatohepatitis.

BACKGROUND & AIMS: Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. METHODS: A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. RESULTS: HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B-induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. CONCLUSIONS: The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI's Gene Expression Omnibus (GEO accession number: GSE98153).