Hepatocellular carcinoma (HCC) is a major global health problem, ranking as the sixth most frequently diagnosed cancer and the third leading cause of cancer-related mortality worldwide. Although the incidence of viral infection-mediated HCC has decreased in recent years, the incidence of alcohol- and metabolic dysfunction-associated HCC has increased, driven by changes in lifestyle and diet. Excessive alcohol consumption contributes to advanced liver diseases, including liver fibrosis, cirrhosis and HCC. Despite the clinical relevance of alcohol-associated HCC, there are no suitable animal models that adequately reflect the pathophysiological features of alcohol-associated HCC in humans. Here, to address this limitation, we established a mouse model of alcohol-associated HCC through the combined administration of N-diethylnitrosamine and carbon tetrachloride (CCl4), followed by administration of an alcohol-containing Lieber-DeCarli diet. The results indicated that chronic alcohol exposure in the presence of N-diethylnitrosamine and CCl4 substantially accelerated HCC development, which was characterized by increased oxidative stress, inflammation and severe fibrosis. Furthermore, we found that chronic ethanol consumption disrupted hepatic immunity, characterized by natural killer/natural killer T cell depletion, increased PD1+CD8+ cells, reduced cytotoxicity and elevated inflammation. We also observed marked alterations in the gut microbiome following chronic alcohol administration. These immunological and microbiome alterations fostered an immunosuppressive microenvironment that accelerated HCC progression. Our newly developed mouse model induced liver tumorigenesis within a relatively short timeframe and recapitulated the clinical and pathological features of alcohol-associated HCC. The model therefore represents a valuable tool for studying the mechanisms underlying alcohol-associated HCC and related chronic liver diseases.

Previous work showed that rats that were exposed to a high-fat, low-carbohydrate/protein ketogenic diet (KD) exhibited elevated blood alcohol levels following alcohol exposure compared with rats fed regular chow. Additionally, the administration of a KD prior to alcohol exposure (i.e., a history of KD) reduced alcohol consumption in alcohol-dependent rats that were no longer on the diet. In the present study, we investigated the mechanisms by which a KD alters alcohol metabolism and tested whether ongoing KD exposure reduces alcohol consumption in rats. We hypothesized that chronic KD exposure alters hepatic alcohol-metabolizing enzymes, slows alcohol metabolism, and reduces alcohol self-administration in alcohol-dependent rats. We found that male and female rats maintained on a KD had higher blood alcohol levels, lower hepatic alcohol dehydrogenase 1 protein levels, and a higher nicotinamide adenine dinucleotide [NAD+]/[NADH] ratio in the liver cytoplasm compared with chow-fed control rats. Furthermore, KD-fed rats demonstrated lower brain glucose uptake relative to chow-fed control rats. In a model of alcohol dependence, the KD reduced alcohol consumption in male, but not female, rats compared with chow-fed rats. These findings suggest that a KD alters brain energetics and alcohol metabolism, which may contribute to reduced alcohol consumption in male rats.

BACKGROUND. In chronic alcohol consumers, immune cells may drive the progression from mild liver injury to more severe alcohol-associated liver disease (ALD), including alcohol-associated hepatitis (AAH) and cancer. Liver macrophages, both resident and infiltrating, express allograft inflammatory factor 1 (AIF1), which is upregulated during inflammation and enhances immune activation. METHODS. Using serum and urine samples from 868 individuals classified as having alcohol use disorder or not, based on DSM-IV/V criteria, along with serum and liver biopsy tissue from a second cohort of 27 patients diagnosed with AAH, we evaluated the impact of the AIF1 promoter single-nucleotide polymorphism (SNP) (rs3132451; C/C, C/G, G/G) on liver function markers and immune cell profiles. RESULTS. AIF1 transcript levels were genotype dependent: C/C homozygotes expressed 5.2% of the levels observed in G/G individuals, while C/G heterozygotes expressed 46%. Unlike most SNPs associated with harmful effects, the G/G genotype is highly prevalent, present in about 70% of patients. Among chronic alcohol users, G/G individuals exhibited elevated markers of liver injury and a more than 3-fold increase in hepatic immune cells, including infiltrating AIF1+ macrophages and neutrophils. Despite similar durations of alcohol misuse, G/G individuals had higher Model for End-Stage Liver Disease scores compared with C/G individuals, indicating a significantly greater 90-day mortality risk. Notably, some immune abnormalities, such as elevated neutrophils, persisted in G/G males even after alcohol abstinence. CONCLUSION. These findings suggest that functional genetic variation in AIF1 may contribute to the severity and persistence of ALD. TRIAL REGISTRATION. ClinicalTrials.gov NCT02231840. FUNDING. Research support was provided from the National Institute on Alcohol Abuse and Alcoholism of the NIH under grants 1ZIAAA000440-02 and R24AA025017.

BACKGROUND:Acetaldehyde (AcH), a highly reactive metabolite of ethanol, plays a pivotal role in the pathogenesis of alcohol-associated liver disease (ALD) and alcohol use disorder (AUD). Post alcohol consumption, AcH generated in hepatocytes is further metabolized into acetate by aldehyde dehydrogenase 2 (ALDH2) or excreted into circulation and bile via the basolateral and apical membranes, respectively. Our previous studies have demonstrated that aquaporin 8 (AQP8), which is a water channel and mainly expressed on the apical membrane, facilitates AcH excretion into bile. AQP9 is predominantly expressed on the basolateral membrane of hepatocytes; however, the roles of AQP9 in AcH relocation and pathogenesis of ALD and AUD remain unknown. METHODS:AQP9 expression was examined in human ALD liver samples. The role of AQP9 was investigated by using the NIAAA mouse model of ALD, voluntary and binge-drinking behavior paradigms, global Aqp9 knockout (KO) mice, in situ liver perfusion, and primary hepatocyte culture assays. RESULTS:Our data demonstrated that hepatic AQP9 expression was markedly downregulated in ALD patients and correlated with liver injury markers and metabolic gene expression. In mice, Aqp9 KO ameliorated early-stage ALD by reducing hepatic lipogenesis, lipid peroxidation, and inflammation. In vivo and in vitro experiments revealed that AQP9 promotes AcH influx into hepatocytes. By using drinking in the dark experiments, we found that Aqp9 KO mice had reduced binge-like alcohol consumption compared with wild-type mice, while two-bottle choice experiments revealed that Aqp9 KO mice had slightly higher alcohol preference compared with wild-type mice. CONCLUSIONS:Our findings suggest that AQP9 promotes hepatocytes to take up AcH, thereby exacerbating ALD progression and regulating alcohol-drinking behavior.

Acetaldehyde (AcH), the first metabolite of ethanol, is an aversive and bioactive compound that plays a key role in modulating alcohol consumption and liver injury. The traditional notion is that AcH is primarily metabolized in the liver by aldehyde dehydrogenase 2 (ALDH2). However, our recent study suggests that the gut-liver ALDH2 axis, rather than the liver alone, plays a key role in metabolizing and clearing AcH partially via bile secretion. Aquaporin 8 (AQP8) is a membrane channel that localizes at the canalicular membrane of hepatocytes and is known to increase bile flow. Here, we identify hepatic AQP8 as an important channel of AcH excretion, mediating its efflux from hepatocytes into bile both with and without altering bile flow. We demonstrated that acute alcohol exposure enhanced AQP8-mediated bile flow and AQP8 promoted hepatic AcH clearance and increased alcohol consumption in both male and female mice. Furthermore, chronic alcohol exposure downregulated hepatic Aqp8 expression, whereas overexpression of hepatic Aqp8 alleviated dysregulated lipid metabolism and liver inflammation in a murine model of alcohol-associated liver disease (ALD). Collectively, our study uncovers a novel role for AQP8 in AcH secretion, demonstrating how this pathway influences both alcohol consumption and liver injury. These findings provide a foundation for exploring AcH excretion as a therapeutic target in alcohol use disorder and ALD.

Background:Ethanol metabolism is intimately linked with the physiological and behavioral aspects of ethanol consumption. Ethanol is mainly oxidized by alcohol dehydrogenase (ADH) to acetaldehyde and further to acetate via aldehyde dehydrogenases (ALDHs). Understanding how ethanol and its metabolites work together to initiate and drive continued ethanol consumption is crucial for identifying interventions for alcohol use disorder (AUD). Therefore, the goal of our study was to determine how ADH1, which is mainly peripherally-expressed and metabolizes >90% of ingested ethanol, modulates ethanol metabolite distribution and downstream behaviors. Methods:Ethanol consumption in drinking-in-the-dark (DID) and two-bottle choice (2BC) drinking paradigms, ethanol metabolite concentrations, and lickometry were assessed after ADH1 inhibition and/or in Adh1-knockout (Adh1 KO) mice. Results:We found that Adh1 KO mice of both sexes exhibited decreased ethanol consumption and preference compared to wild-type (WT) mice in DID and 2BC. ADH1 inhibitor fomepizole (4-MP) also significantly decreased normal and sweetened ethanol consumption in DID studies. Measurement of ethanol and its metabolites revealed that ethanol was increased at 1h but not 15 min, peripheral acetaldehyde was slightly decreased at both time points, and ethanol-induced increases in acetate were abolished after ethanol administration in Adh1 KO mice compared to controls. Similarly, ethanol accumulation as a function of consumption was 2-fold higher in Adh1 KO or 4-MP treated mice compared to controls. We then used lickometry to determine how this perturbation in ethanol metabolism affects drinking microstructure. Adh1 KO mice consume most of their ethanol in the first 30 min like WT mice but display altered temporal shifts in drinking behaviors and do not form normal bout structures, resulting in lower ethanol consumption. Conclusions:Our study demonstrates that ADH1-mediated ethanol metabolism is a key determinant of ethanol consumption, highlighting a fundamental knowledge gap around how ethanol and its metabolites drive ethanol consumption.

Patients with alcohol-associated cirrhosis (AC) may develop severe alcohol-associated hepatitis (sAH), a disease with high short-term mortality. Our previous studies demonstrated that sAH, but not AC livers, are infiltrated with a high number of self-sustaining IL-8+ neutrophils that likely drive the transition from AC to sAH. Monocyte-derived macrophages (MoMFs) also infiltrate the liver in sAH, but their roles remain largely obscure. In the present study, we characterized liver macrophages in human liver explants from sAH and AC patients. Our data revealed a marked reduction in Kupffer cells, whereas MoMFs were increased in sAH and AC. Single-cell RNA-Seq analyses revealed several populations in both AC and sAH, including C1Q+, S100A8+, APOE+, TNF+ and VSIG4+ macrophages, with sAH containing unique C1Q+ macrophages potentially playing a role in removing apoptotic neutrophils in sAH. C1Q+ macrophages also express many genes involved in phagocytosis and proinflammatory and anti-inflammatory functions, suggesting that C1Q+ macrophages have diverse functions in sAH. The roles of C1Q, S100A8, and APOE were further examined in experimental models of alcohol-induced liver injury. Our data revealed that C1q KO mice and macrophage-specific S100a8 KO mice presented similar alcohol-induced liver injury and hepatic neutrophil infiltration, while Apoe KO mice developed much more severe liver injury than did WT mice following chronic-plus-binge ethanol challenge. Taken together, sAH and AC are infiltrated with multiple populations of macrophages that perform diverse functions to drive chronic disease progression. Unique C1Q+ macrophages in sAH play a compensatory role in removing dead cells but may also promote inflammation in sAH.

Background:Healthy livers contain a large number of resident macrophages named Kupffer cells (KCs), which are partially replaced by infiltrating monocyte-derived macrophages (MoMFs) during acute or chronic liver injury. Despite extensive research, understanding macrophage heterogeneity, spatial distribution and interactions with other cells within the liver remains challenging. Methods:This study employs sequential multiplex immunofluorescence staining, advanced image analysis and single-cell RNA sequencing (scRNA-seq) analysis to characterise macrophages in both healthy and diseased livers in mice. Results:Our data revealed that liver KCs made up more than 80% of total immune cells in healthy mouse livers, while massive amounts of MoMFs infiltrated into the livers after acute and chronic liver injury. KCs were more abundant and larger in Zones 1 and 2 compared with Zone 3 in healthy livers. Zone 1 KCs exhibited higher phagocytic activity than Zone 2/3 KCs and MoMFs. We simultaneously evaluated cell proliferation and apoptosis on one slide and found that proliferation and apoptosis of KCs and MoMFs significantly increased in acutely injured livers. We also performed scRNA-seq to investigate liver macrophage gene expression in naïve and concanavalin A (ConA)-treated mice. MoMF clusters expanded following ConA treatment, while KCs remained stable. Macrophages were divided into distinct subtypes, including C1q+ MoMFs, with differential expression of genes like Trem2, Spp1, Fabp5 and Gpnmb. Newly recruited C1q- MoMFs expressed high levels of Lyz and Ccr2, while Itgax (Cd11c)+ MoMFs expressed endothelin converting enzyme 1 (Ece1), a gene encoding ECE1 enzyme that activates endothelin to promote hepatic stellate cell contraction and necrotic lesion resolution. By immunostaining analysis of the proteins encoded by these signature genes, we identified several populations of MoMFs that were mainly located surrounding the necrotic lesion area and expressed various proteins that are involved in dead cell debris clearance. Conclusion:We developed a robust framework for studying liver macrophages in vivo, offering insights into their roles in host defence and liver injury/repair. We identified several populations of MoMFs that surround necrotic lesion areas and express proteins that promote dead cell debris clearance. These necrotic lesion-associated macrophages likely play key roles in promoting necrotic lesion resolution.