Human Alcoholic Liver Disease Research Articles

Establishment and bioinformatics evaluation of the ethanol combined with palmitic acid-induced mouse hepatocyte AFLD model (the Hu-Qiu Model)

Chronic alcoholic liver disease has brought great harm to human health. Alcoholic fatty liver disease is the first stage in the progression of all chronic alcoholic liver diseases. At present, there is no cell model that fully matches the etiology (high-fat diet + alcohol) of human alcoholic fatty liver disease. We used 100 mM ethanol +6.25 μM PA to establish the ethanol combined with PA-induced mouse hepatocyte AFLD model (EP-AFLD hepatocyte model) and performed the RNA-seq transcriptome sequencing. Through bioinformatics analysis and comparison, we discovered that the EP-AFLD hepatocyte model was more suitable for studying the pathological mechanism of AFLD than the mouse AFLD hepatocyte model induced by ethanol alone. And through bioinformatics analysis, we further discovered that 77 genes from the differential expression gene set of EP-AFLD hepatocyte model were engaged in the pathological process of mouse AFLD and 40 genes were involved in the pathogenesis of both mouse AFLD and human AFLD. In this study, a novel mouse hepatocyte AFLD model was successfully established by combining ethanol and PA, which can be used to study the molecular mechanism of the pathogenesis of AFLD in mice or humans. This study will provide a brand-new in vitro experimental platform for the in-depth study of AFLD pathogenesis and the screening of AFLD therapeutic drugs.

Targetable Brg1-CXCL14 axis contributes to alcoholic liver injury by driving neutrophil trafficking.

Alcoholic liver disease (ALD) accounts for a large fraction of patients with cirrhosis and hepatocellular carcinoma. In the present study we investigated the involvement of Brahma-related gene 1 (Brg1) in ALD pathogenesis and implication in ALD intervention. We report that Brg1 expression was elevated in mouse models of ALD, in hepatocyte exposed to alcohol, and in human ALD specimens. Manipulation of Brg1 expression in hepatocytes influenced the development of ALD in mice. Flow cytometry showed that Brg1 deficiency specifically attenuated hepatic infiltration of Ly6G+ neutrophils in the ALD mice. RNA-seq identified C-X-C motif chemokine ligand 14 (CXCL14) as a potential target for Brg1. CXCL14 knockdown alleviated whereas CXCL14 over-expression enhanced ALD pathogenesis in mice. Importantly, pharmaceutical inhibition of Brg1 with a small-molecule compound PFI-3 or administration of an antagonist to the CXCL14 receptor ameliorated ALD pathogenesis in mice. Finally, a positive correlation between Brg1 expression, CXCL14 expression, and neutrophil infiltration was detected in ALD patients. In conclusion, our data provide proof-of-concept for targeting the Brg1-CXCL14 axis in ALD intervention.

THU012 - Antibody response to gut microbiome bacteria in human alcoholic liver disease

THU012 - Antibody response to gut microbiome bacteria in human alcoholic liver disease

Abstract 785: Multi-omics profiling identifies genetic and epigenetic regulation underlying alcohol induced liver fibrosis in a porcine hepatocellular carcinoma model

Abstract Background: Cirrhosis is a form of end-stage liver disease which manifests with a host of metabolic and hematologic derangements, and is commonly the result of long-term ethanol exposure. Large animal models of cirrhosis, and of one of its common long-term sequelae, hepatocellular carcinoma (HCC), are needed to study novel and emerging therapeutic interventions. Methods: Liver fibrosis was induced in the Oncopig cancer model, a large animal model of HCC, via intrahepatic, intra-arterial ethanol infusion. Post-mortem liver sections from five fibrosis induction and five age-matched controls were harvested for RNA-seq (mRNA and lncRNA), small RNA-seq (miRNA), and reduced representation bisulfite sequencing (RRBS; DNA methylation). Supervised, multi-omic analysis of the 5,000 genes and methylated regions demonstrating the largest inter-sample variance with non-zero counts along with all sequenced miRNA were analyzed using the DIABLO method within the mixOmics 6.8.5 R library. Results: Single component DIABLO latent variable analysis of mRNAs, lncRNAs, miRNAs, and methylated regions was performed to identify the genetic elements (variables) which most reliably discriminated between experimental arms. Analysis of the top 100 discriminatory variables from each ‘omics data set was performed to provide sufficient data for pathway analysis while maintaining separation of the experimental arms within the latent space. IPA pathway analysis of these genes revealed differential regulation of pathways related to hepatic fibrosis, adipogenesis, ketogenesis as well as several other liver-specific metabolic pathways. Identification of the five most discriminatory variables was also performed. Single component, five variable analysis revealed ELOVL2, LIPK, and LAMA3 most reliably differentiated control and fibrosis groups, along with two lncRNA transcripts, XLOC035153 and XLOC036409 which were predicted to target S1PR1 and UHRF1BP1L, respectively. LAMA3, XLOC036409, and its target UHRF1BP1L were overexpressed in Oncopig fibrotic liver samples. UHRF1, a potential binding target for UHRF1BP1L, has previously been implicated as an oncogene for hepatocellular carcinoma. S1PR1 is involved in vasculogenesis and cancer cell motility via the RAC-CDC42 and ERK pathways. Conclusions The differentially regulated genetic elements identified by the multi-omic analyses were largely concordant with known alterations in human alcoholic liver disease. This agreement lends validity to the Oncopig model as a model organism for human alcoholic liver disease. In addition, novel genes, UHRF1BP1L and S1PR1, were associated with porcine liver fibrosis and further work to characterize their precise mechanistic roles in the natural history of this disease is required. Citation Format: Mark Hieromnimon, Lawrence Schook, Ron Gaba, Kyle Schachtschneider. Multi-omics profiling identifies genetic and epigenetic regulation underlying alcohol induced liver fibrosis in a porcine hepatocellular carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 785.

Interleukin-11 drives human and mouse alcohol-related liver disease

ObjectiveAlcoholic hepatitis (AH) reflects acute exacerbation of alcoholic liver disease (ALD) and is a growing healthcare burden worldwide. Interleukin-11 (IL-11) is a profibrotic, proinflammatory cytokine with increasingly recognised toxicities in...

NLRP6 Inflammasome Modulates Disease Progression in a Chronic-Plus-Binge Mouse Model of Alcoholic Liver Disease.

A considerable percentage of the population is affected by alcoholic liver disease (ALD). It is characterized by inflammatory signals from the liver and other organs, such as the intestine. The NLR family pyrin domain containing 6 (NLRP6) inflammasome complex is one of the most important inflammatory mediators. The aim of this study was to evaluate a novel mouse model for ALD characterized by 8-week chronic-plus-binge ethanol administration and to investigate the role of NLRP6 inflammasome for intestinal homeostasis and ALD progression using Nlrp6-/- mice. We showed that chronic-plus-binge ethanol administration triggers hepatic steatosis, injury, and neutrophil infiltration. Furthermore, we discovered significant changes of intestinal microbial communities, including increased relative abundances of bacteria within the phyla Bacteroidota and Campilobacterota, as well as reduced Firmicutes. In this ALD model, inhibiting NLRP6 signaling had no effect on liver steatosis or damage, but had a minor impact on intestinal homeostasis via affecting intestinal epithelium function and gut microbiota. Surprisingly, Nlrp6 loss resulted in significantly decreased hepatic immune cell infiltration. As a result, our novel mouse model encompasses several aspects of human ALD, such as intestinal dysbiosis. Interfering with NLRP6 inflammasome activity reduced hepatic immune cell recruitment, indicating a disease-aggravating role of NLRP6 during ALD.

ZFP36L1 Regulates Fgf21 mRNA Turnover and Modulates Alcoholic Hepatic Steatosis and Inflammation in Mice

Zinc finger protein 36 like 1 (ZFP36L1) enhances the turnover of mRNAs containing AU-rich elements (AREs) in their 3'-untranslated regions (3'UTR). The physiological and pathological functions of ZFP36L1 in liver, however, remain largely unknown. Liver-specific ZFP36L1-deficient (Zfp36l1flox/flox/Cre+; L1LKO) mice were generated to investigate the role of ZFP36L1 in liver physiology and pathology. Under normal conditions, the L1LKO mice and their littermate controls (Zfp36l1flox/flox/Cre-; L1FLX) appeared normal. When fed a Lieber-DeCarli liquid diet containing alcohol, L1LKO mice were significantly protected from developing alcohol-induced hepatic steatosis, injury, and inflammation compared with L1FLX mice. Most importantly, fibroblast growth factor 21 (Fgf21) mRNA was significantly increased in the livers of alcohol diet-fed L1LKO mice compared with the alcohol diet-fed L1FLX group. The Fgf21 mRNA contains three AREs in its 3'UTR, and Fgf21 3'UTR was directly regulated by ZFP36L1 in luciferase reporter assays. Steady-state levels of Fgf21 mRNA were significantly decreased by wild-type ZFP36L1, but not by a non-binding zinc finger ZFP36L1 mutant. Finally, wild-type ZFP36L1, but not the ZFP36L1 mutant, bound to the Fgf21 3'UTR ARE RNA probe. These results demonstrate that ZFP36L1 inactivation protects against alcohol-induced hepatic steatosis and liver injury and inflammation, possibly by stabilizing Fgf21 mRNA. These findings suggest that the modulation of ZFP36L1 may be beneficial in the prevention or treatment of human alcoholic liver disease.

Pathophysiological Aspects of Alcohol Metabolism in the Liver.

Alcoholic liver disease (ALD) is a globally prevalent chronic liver disease caused by chronic or binge consumption of alcohol. The liver is the major organ that metabolizes alcohol; therefore, it is particularly sensitive to alcohol intake. Metabolites and byproducts generated during alcohol metabolism cause liver damage, leading to ALD via several mechanisms, such as impairing lipid metabolism, intensifying inflammatory reactions, and inducing fibrosis. Despite the severity of ALD, the development of novel treatments has been hampered by the lack of animal models that fully mimic human ALD. To overcome the current limitations of ALD studies and therapy development, it is necessary to understand the molecular mechanisms underlying alcohol-induced liver injury. Hence, to provide insights into the progression of ALD, this review examines previous studies conducted on alcohol metabolism in the liver. There is a particular focus on the occurrence of ALD caused by hepatotoxicity originating from alcohol metabolism.

A mini-review of the rodent models for alcoholic liver disease: shortcomings, application, and future prospects.

Rodents are the most common models in studies of alcoholic liver disease (ALD). Although several rodents ALD models have been established and multiple mechanisms have been elucidated based on them, these models have some non-negligible shortcomings, specifically only inducing early stage (mainly steatosis, slight to moderate steatohepatitis) but not the whole spectrum of human ALD. The resistance of rodents to advanced ALD has been suggested to be due to the physiological differences between rodents and human beings. Previous studies have reported significant interstrain differences in the susceptibility to ethanol-induced liver injury and in the manifestation of ALD (such as different alteration of lipid profiles). Therefore, it would be interesting to characterize the manifestation of ethanol-induced liver damage in various rodents, which may provide a recommendation to investigators of ALD. Furthermore, more severe ALD models need to be established for the study of serious ALD forms, which may be achieved by using genetic modified rodents.

Modeling Alcoholic Liver Disease in a Human Liver-Chip

Fatty liver disease (FLD) is a major public health burden that affects up to 30% of people in Western countries and leads to progressive liver injury, comorbidities, and increased mortality. One risk factor for developing FLD is alcohol consumption, which is growing in prevalence worldwide. There is an urgent need for human-relevant preclinical models to improve our understanding of FLD progression to steatohepatitis in alcoholic liver disease (ALD) and to develop sensitive noninvasive diagnostics and therapies of FLD in general. Here, by leveraging a tri-culture human Liver-Chip with biomimetic hepatic sinusoids and bile canaliculi, we achieve the first in vitro modeling of ALD that uses human relevant blood alcohol concentrations (BAC) and affords multimodal profiling of clinically relevant endpoints. Our ALD Liver-Chip recapitulates established FLD markers in response to ethanol in a concentration-dependent manner, including lipid accumulation and oxidative stress. The ALD Liver-Chip also supports the study of secondary insults common in patients with advanced ALD, such as high blood endotoxin levels due to alcohol-associated increased intestinal permeability and barrier dysfunction. Moreover, the ALD Liver-Chip enables the measurement of structural changes of the hepatic bile canaliculi network as a novel in vitro quantitative readout of alcoholic liver toxicity, and the co-culture of human stellate cells to study their role in FLD progression and liver fibrosis. In summary, we report the development of a human ALD Liver-Chip as a new platform for modeling the progression of alcohol-induced liver injury and fatty liver disease with direct translation to clinical research.

Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

Alcohol’s impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol’s inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD.

Effects of Dihydroxylated Fatty Acid Mediators on Ethanol‐Induced Liver Injury in a Mouse Model of Alcoholic Liver Disease

BackgroundAlcoholic liver disease (ALD) is the most common chronic liver disease and a major contributor to liver disease burden worldwide, and a disease for which there is no FDA‐approved treatment. The mechanisms of ALD pathogenesis are not completely understood. Data from our group indicate that dihydroxylated fatty acids (dihydroxy‐FAs) are increased in the livers of human alcoholic hepatitis patients. Dihydroxy‐FA lipid mediators are produced via cytochrome P450/epoxide hydrolase‐mediated metabolism of polyunsaturated fatty acids and are generally pro‐inflammatory. Despite their increase in human ALD, the role of dihydroxy‐FAs in ALD has not been explored.HypothesisExogenous administration of dihydroxy‐FAs will exacerbate liver injury in a mouse model of ALD.MethodsMale C57BL/6 mice were provided ethanol (EtOH) using a chronic‐binge Lieber DeCarli ad libitum EtOH feeding model. Mice were administered either 8,9‐DiHETE (arachidonic acid metabolite), a combination of 9,10‐DiHOME and 12,13‐DiHOME (linoleic acid metabolites), or a vehicle control by i.p. injection once daily for two days prior to sacrifice. Liver injury, inflammation, and steatosis were measured. To further elucidate the effects of dihydroxy‐FAs on liver cells, primary mouse hepatocytes or HepG2 cells were treated with 8,9‐DiHETE, 9,10‐DiHOME, 12,13‐DiHOME, or a vehicle control. Effects on baseline or palmitic acid (PA)‐induced cell death were measured by MTT assay and apoptotic cell death was identified by Annexin‐V staining (cellomics).ResultsIn vivo, 8,9‐DiHETE treatment modestly while 9,10/12,13‐DiHOME significantly increased liver injury as determined by elevated plasma ALT levels (1.3‐fold and 2.2‐fold p=0.04, respectively). While 8,9‐DiHETE had no effect on steatosis, 9,10/12,13‐DiHOME decreased liver triglyceride/free fatty acid levels compared to vehicle control. Dihydroxy‐FA treatment decreased the hepatic expression of several pro‐inflammatory chemokines at the mRNA level, including Mip2a and Mcp1. In vitro, 8,9‐DiHETE treatment of HepG2 cells or primary mouse hepatocytes significantly exacerbated PA‐induced cell death as determined by MTT assay, whereas 9,10‐DiHOME, and 12,13‐DiHOME had no effect on PA‐induced or baseline cell death or apoptosis.ConclusionsDifferent dihydroxy‐FAs have diverse effects in vivo, in a mouse model of ALD, and in vitro. In vivo, dihydroxy‐FAs exacerbated liver injury and was associated with down‐regulated several pro‐inflammatory cytokines. In vitro, 8,9‐DiHETE increased hepatocyte cell death, while 9,10/12,13‐DiHOME showed no effects. Future studies are warranted to dissect the effects of individual dihydroxy‐FAs in ALD.Support or Funding InformationThis work was funded by grants from the National Institutes of Health and the Department of Veterans Affairs.

Mice with Endogenously Increased ωPUFAs are Resistant to Alcohol‐Mediated Down‐Regulation of Wnt Signaling

Background/ObjectivesChronic alcohol consumption can lead to a plethora of detrimental health effects, including alcoholic liver disease (ALD), which is characterized by steatosis and steatohepatitis with varying degrees of fibrosis and cirrhosis. Unresolved inflammation and impaired mechanisms of liver tissue repair and regeneration are among major contributors to disease progression. Canonical Wnt signaling is important for tissue regeneration following hepatocellular damage and regulation of inflammation. It has been shown that hepatic Wnt signaling is down‐regulated in rodent models of ALD and that restoration of this pathway can ameliorate this disease. Our group is focused on nutritional interventions as a treatment/prevention paradigm for ALD, specifically with ω3 polyunsaturated fatty acids (PUFAs), which have numerous beneficial effects, including metabolism to specialize pro‐resolving mediators (SPM), a class of lipid molecules that are important for limiting the inflammatory response and a return to homeostasis. The objective of the current study was to test the hypothesis that ω3 PUFAs are beneficial in ALD via improving EtOH‐induced alterations in Wnt signaling.MethodsMale WT and fat‐1+/− mice (that endogenously convert ω6 PUFAs to ω3) were provided a liquid diet that was supplemented with ethanol (EtOH) as follows: 0–3% (v/v) over the course of 1 week, 4% for 1 week, 5% for 1 week, and 6% for 3 weeks. To simulate human ALD associated with endotoxemia, LPS was administered to mice 24h before euthanasia (5 mg/kg, i.p.). Liver injury was assessed by plasma ALT activity, histopathology, and the expression of pro‐inflammatory cytokines. Liver RNA was extracted and the expression of 84 genes in the Wnt signaling pathway was determined by PCR array.ResultsWe found that EtOH‐fed, LPS‐challenged fat‐1 mice had attenuated liver injury (reduced ALT levels), with reduced hepatic neutrophil accumulation, significant reduction in the expression of proinflammatory Pai‐1 and an increase in anti‐inflammatory Il‐10 levels, compared to WT mice. We also found that EtOH+LPS treated WT mice had a greater decrease in the expression of Axin2 (an endogenous readout of canonical Wnt signaling) than fat‐1 mice (8‐fold vs. 3‐fold, respectively, vs. control, pair‐fed mice). EtOH+LPS treated WT mice also had a significant increase in hepatic expression of the Wnt inhibitor, Dkk3, whereas fat‐1 mice had decreased expression of this gene when compared to pair‐fed controls. Finally, the expression of the repressor protein, Nfatc1, was increased to a greater extent in EtOH‐LPS treated WT vs. fat‐1 mice. Taken together, these data suggest that EtOH+LPS‐mediated downregulation of Wnt signaling was attenuated in fat‐1 mice.Conclusionsfat‐1 mice are partially protected from alcohol‐induced liver disease. Canonical Wnt signaling was reduced to a greater extent in WT than in fat‐1 mice and was associated with increased liver damage and inflammation. These data provide evidence that restoration of the Wnt signaling pathway through dietary means may be a viable strategy to treat or prevent ALD.Support or Funding InformationThe work presented in this study was supported by the National Institutes of Health and the Department of Veterans Affairs.

Perturbations in Mitochondrial Dynamics Are Closely Involved in the Progression of Alcoholic Liver Disease.

Mitochondria play a fundamental role in the pathogenesis of alcoholic liver disease (ALD). The preservation of functional mitochondria during toxic alcohol insults is essential for cell survival and is maintained by key processes known as mitochondrial dynamics, including fragmentation and fusion, which are regulated by mitochondria-shaping proteins (MSP). We have shown mitochondrial dynamics to be distorted by alcohol in cellular and animal models, but the effect in humans remains unknown. Hepatic gene expression of the main MSP involved in the mitochondrial fusion and fragmentation pathways was evaluated in patients with alcoholic hepatitis (AH) by DNA microarray (n=15) and Reverse Transcription Polymerase Chain Reaction (n=32). The activation of dynamin-1-like protein (Drp1) was also investigated in mitochondria isolated from liver biopsies of ALD patients (n=8). The effects of alcohol on mitochondrial dynamics and on MSP protein expression were studied in human precision-cut liver slices (PCLS) exposed for 24hours to increasing doses of ethanol (EtOH; 50 to 250mM). A profound hyperactivation of the fragmentation pathway was observed in AH patients, with a significant increase in the expression of Drp1 and its adapters/receptors. The translocation of Drp1 to the mitochondria was also induced in patients with severe ALD and was affected in the PCLS with short-term exposure to EtOH but only mildly. The fusion pathway was not altered in ALD, and this was confirmed in the PCLS model. The present study reveals the role of mitochondrial dynamics in human ALD, confirming our previous observations in animal and cell culture models of ALD. Taken together, we show that alcohol has a significant impact on the fragmentation pathway, and we confirm Drp1 as a potential therapeutic target in severe ALD.

Restricted immunological and cellular pathways are shared by murine models of chronic alcohol consumption

Murine models of chronic alcohol consumption are frequently used to investigate alcoholic liver injury and define new therapeutic targets. Lieber-DeCarli diet (LD) and Meadows-Cook diet (MC) are the most accepted models of chronic alcohol consumption. It is unclear how similar these models are at the cellular, immunologic, and transcriptome levels. We investigated the common and specific pathways of LD and MC models. Livers from LD and MC mice were subjected to histologic changes, hepatic leukocyte population, hepatic transcripts level related to leukocyte recruitment, and hepatic RNA-seq analysis. Cross-species comparison was performed using the alcoholic liver disease (ALD) transcriptomic public dataset. Despite LD mice have increased liver injury and steatosis by alcohol exposure, the number of CD45+ cells were reduced. Opposite, MC mice have an increased number of monocytes/liver by alcohol. The pattern of chemokine gradient, adhesion molecules, and cytokine transcripts is highly specific for each model, not shared with advanced human alcoholic liver disease. Moreover, hepatic RNA-seq revealed a limited and restricted number of shared genes differentially changed by alcohol exposure in these 2 models. Thus, mechanisms involved in alcohol tissue injury are model-dependent at multiple levels and raise the consideration of significant pathophysiological diversity of human alcoholic liver injury.

Intestinal SIRT1 Deficiency Protects Mice from Ethanol-Induced Liver Injury by Mitigating Ferroptosis

Aberrant liver sirtuin 1 (SIRT1), a mammalian NAD+-dependent protein deacetylase, is implicated in the pathogenesis of alcoholic liver disease (ALD). However, the role of intestinal SIRT1 in ALD is presently unknown. This study investigated the involvement of intestine-specific SIRT1 in ethanol-induced liver dysfunction in mice. Ethanol feeding studies were performed on knockout mice with intestinal-specific SIRT1 deletion [SIRT1i knockout (KO)] and flox control [wild-type (WT)] mice with a chronic-plus-binge ethanol feeding protocol. After ethanol administration, hepatic inflammation and liver injury were substantially attenuated in the SIRT1iKO mice compared with the WT mice, suggesting that intestinal SIRT1 played a detrimental role in the ethanol-induced liver injury. Mechanistically, the hepatic protective effect of intestinal SIRT1 deficiency was attributable to ameliorated dysfunctional iron metabolism, increased hepatic glutathione contents, and attenuated lipid peroxidation, along with inhibition of a panel of genes implicated in the ferroptosis process in the livers of ethanol-fed mice. This study demonstrates that ablation of intestinal SIRT1 protected mice from the ethanol-induced inflammation and liver damage. The protective effects of intestinal SIRT1 deficiency are mediated, at least partially, by mitigating hepatic ferroptosis. Targeting intestinal SIRT1 or dampening hepatic ferroptosis signaling may have therapeutic potential for ALD in humans.

Deficient IL-6/Stat3 Signaling, High TLR7, and Type I Interferons in Early Human Alcoholic Liver Disease: A Triad for Liver Damage and Fibrosis.

Mechanisms underlying alcohol‐induced liver injury and its progression still remain incompletely understood. Animal models can only address some aspects of the pathophysiology that requires studies directly in humans, which are scarce. We assessed liver inflammatory and immune responses at early stages of alcoholic liver disease in a unique cohort of alcohol‐dependent patients undergoing a highly standardized alcohol withdrawal program. In active drinkers, quantitative real‐time polymerase chain reaction revealed alcohol‐induced activation of tumor necrosis factor alpha, interleukin (IL)‐1β, and nuclear factor kappa B in liver tissue already at early disease stages. Double immunofluorescence staining indicated that this proinflammatory response was restricted to activated, CD68‐positive macrophages. In parallel, down‐regulation of IL‐6, inhibition of the signal transducer and activator of transcription 3 (Stat3) pathway, as well as blunted cyclin D expression in hepatocytes, reduced proliferation and favored hepatocyte apoptosis. In addition, immunofluorescence and quantitative real‐time polymerase chain reaction of liver tissue showed that alcohol also activated the toll‐like receptor (TLR) 7–interferon (IFN) axis in hepatocytes, which was confirmed in alcohol‐stimulated primary human hepatocytes and precision‐cut liver slices in vitro. Activation of the TLR7–IFN axis strongly correlated with liver fibrosis markers and disease progression. Two weeks of abstinence attenuated the inflammatory response but did not allow recovery of the defective Stat3 pathway or effect on fibrosis‐associated factors. Conclusion: In humans, inflammation, activation of the TLR7–IFN axis, and inhibition of Stat3‐dependent repair mechanisms in early alcoholic liver disease pave the way for fibrosis development and ultimately disease progression.

Serum Metabolomic Profiling Identifies Key Metabolic Signatures Associated With Pathogenesis of Alcoholic Liver Disease in Humans.

Alcoholic liver disease (ALD) develops in a subset of heavy drinkers (HDs). The goals of our study were to (1) characterize the global serum metabolomic changes in well‐characterized cohorts of controls (Cs), HDs, and those with alcoholic cirrhosis (AC); (2) identify metabolomic signatures as potential diagnostic markers, and (3) determine the trajectory of serum metabolites in response to alcohol abstinence. Serum metabolic profiling was performed in 22 Cs, 147 HDs, and 33 patients with AC using ultraperformance liquid chromatography–tandem mass spectrometry. Hepatic gene expression was conducted in Cs (n = 16) and those with AC (n = 32). We found progressive changes in the quantities of metabolites from heavy drinking to AC. Taurine‐conjugated bile acids (taurocholic acid [TCA], 127‐fold; taurochenodeoxycholic acid [TCDCA], 131‐fold; and tauroursodeoxycholic acid, 56‐fold) showed more striking elevations than glycine‐conjugated forms (glycocholic acid [GCA], 22‐fold; glycochenodeoxycholic acid [GCDCA], 22‐fold; and glycoursodeoxycholic acid [GUDCA], 11‐fold). This was associated with increased liver cytochrome P450, family 7, subfamily B, member 1 and taurine content (more substrates); the latter was due to dysregulation of homocysteine metabolism. Increased levels of GCDCA, TCDCA, GCA, and TCA positively correlated with disease progression from Child‐Pugh A to C and Model for End‐Stage Liver Disease scores, whereas GCDCA, GCA, and GUDCA were better predictors of alcohol abstinence. The levels of glucagon‐like peptide 1 (GLP‐1) and fibroblast growth factor (FGF) 21 but not FGF19 were increased in HDs, and all three were further increased in those with AC. Conclusion: Serum taurine/glycine‐conjugated bile acids could serve as noninvasive markers to predict the severity of AC, whereas GLP‐1 and FGF21 may indicate a progression from heavy drinking to AC.

Alcoholic liver disease: Utility of animal models.

Alcoholic liver disease (ALD) is a major cause of acute and chronic liver injury. Extensive evidence has been accumulated on the pathological process of ALD during the past decades. However, effective treatment options for ALD are very limited due to the lack of suitable in vivo models that recapitulate the full spectrum of ALD. Experimental animal models of ALD, particularly rodents, have been used extensively to mimic human ALD. An ideal animal model should recapitulate all aspects of the ALD process, including significant steatosis, hepatic neutrophil infiltration, and liver injury. A better strategy against ALD depends on clear diagnostic biomarkers, accurate predictor(s) of its progression and new therapeutic approaches to modulate stop or even reverse the disease. Numerous models employing rodent animals have been established in the last decades to investigate the effects of acute and chronic alcohol exposure on the initiation and progression of ALD. Although significant progress has been made in gaining better knowledge on the mechanisms and pathology of ALD, many features of ALD are unknown, and require further investigation, ideally with improved animal models that more effectively mimic human ALD. Although differences in the degree and stages of alcoholic liver injury inevitably exist between animal models and human ALD, the acquisition and translational relevance will be greatly enhanced with the development of new and improved animal models of ALD.

Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte-like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.-Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.