Coordinated action of a gut-liver pathway drives alcohol detoxification and consumption.

Alcohol use disorder (AUD) affects millions of people worldwide, causing extensive morbidity and mortality with limited pharmacological treatments. The liver is considered as the principal site for the detoxification of ethanol metabolite, acetaldehyde (AcH), by aldehyde dehydrogenase 2 (ALDH2) and as a target for AUD treatment, however, our recent data indicate that the liver only plays a partial role in clearing systemic AcH. Here we show that a liver-gut axis, rather than liver alone, synergistically drives systemic AcH clearance and voluntary alcohol drinking. Mechanistically, we find that after ethanol intake, a substantial proportion of AcH generated in the liver is excreted via the bile into the gastrointestinal tract where AcH is further metabolized by gut ALDH2. Modulating bile flow significantly affects serum AcH level and drinking behaviour. Thus, combined targeting of liver and gut ALDH2, and manipulation of bile flow and secretion are potential therapeutic strategies to treat AUD.

Inflammation in Alcohol-Associated Hepatitis: Pathogenesis and Therapeutic Targets.

Alcohol-associated hepatitis (AH) is an acute-on-chronic liver injury that occurs in patients with chronic alcohol-associated liver disease (ALD). Patients with severe AH have high short-term mortality and lack effective pharmacologic therapies. Inflammation is believed to be one of the key factors promoting AH progression and has been actively investigated as therapeutic targets over the last several decades, but no effective inflammatory targets have been identified so far. In this review, we discuss how inflammatory cells and the inflammatory mediators produced by these cells contribute to the development and progression of AH, with focus on neutrophils and macrophages. The crosstalk between inflammatory cells and liver nonparenchymal cells in the pathogenesis of AH is elaborated. We also deliberate the application of recent cutting-edge technologies in characterizing liver inflammation in AH. Finally, the potential therapeutic targets of inflammatory mediators for AH are briefly summarized.

Alcohol-associated liver cancer.

Heavy alcohol intake induces a wide spectrum of liver diseases ranging from steatosis, steatohepatitis, cirrhosis, and HCC. Although alcohol consumption is a well-known risk factor for the development, morbidity, and mortality of HCC globally, alcohol-associated hepatocellular carcinoma (A-HCC) is poorly characterized compared to viral hepatitis-associated HCC. Most A-HCCs develop after alcohol-associated cirrhosis (AC), but the direct carcinogenesis from ethanol and its metabolites to A-HCC remains obscure. The differences between A-HCC and HCCs caused by other etiologies have not been well investigated in terms of clinical prognosis, genetic or epigenetic landscape, molecular mechanisms, and heterogeneity. Moreover, there is a huge gap between basic research and clinical practice due to the lack of preclinical models of A-HCC. In the current review, we discuss the pathogenesis, heterogeneity, preclinical approaches, epigenetic, and genetic profiles of A-HCC, and discuss the current insights into and the prospects for future research on A-HCC. The potential effect of alcohol on cholangiocarcinoma and liver metastasis is also discussed.

Macrophages in necrotic liver lesion repair: opportunities for therapeutical applications.

Healthy livers contain 80% of body resident macrophages known as Kupffer cells. In diseased livers, the number of Kupffer cells usually drops but is compensated by infiltration of monocyte-derived macrophages, some of which can differentiate into Kupffer-like cells. Early studies suggest that Kupffer cells play important roles in both promoting liver injury and liver regeneration. Yet, the distinction between the functionalities of resident and infiltrating macrophages is not always made. By using more specific macrophage markers and targeted cell depletion and single-cell RNA sequencing, recent studies revealed several subsets of monocyte-derived macrophages that play important functions in inducing liver damage and inflammation as well as in liver repair and regeneration. In this review, we discuss the different roles that hepatic macrophages play in promoting necrotic liver lesion resolution and dead cell clearance, as well as the targeting of these cells as potential tools for the development of novel therapies for acute liver failure and acute-on-chronic liver failure.

Alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is a major cause of chronic liver disease worldwide, and comprises a spectrum of several different disorders, including simple steatosis, steatohepatitis, cirrhosis, and superimposed hepatocellular carcinoma. Although tremendous progress has been made in the field of ALD over the last 20 years, the pathogenesis of ALD remains obscure, and there are currently no FDA-approved drugs for the treatment of ALD. In this Review, we discuss new insights into the pathogenesis and therapeutic targets of ALD, utilizing the study of multiomics and other cutting-edge approaches. The potential translation of these studies into clinical practice and therapy is deliberated. We also discuss preclinical models of ALD, interplay of ALD and metabolic dysfunction, alcohol-associated liver cancer, the heterogeneity of ALD, and some potential translational research prospects for ALD.

Alcohol-associated bowel disease: new insights into pathogenesis.

Excessive alcohol drinking can cause pathological changes including carcinogenesis in the digestive tract from mouth to large intestine, but the underlying mechanisms are not fully understood. In this review, we discuss the effects of alcohol on small and large intestinal functions, such as leaky gut, dysbiosis and alterations of intestinal epithelium and gut immune dysfunctions, commonly referred to as alcohol-associated bowel disease (ABD). To date, detailed mechanistic insights into ABD are lacking. Accumulating evidence suggests a pathogenic role of ethanol metabolism in dysfunctions of the intestinal tract. Ethanol metabolism generates acetaldehyde and acetate, which could potentially promote functional disruptions of microbial and host components of the intestinal barrier along the gastrointestinal tract. The potential involvement of acetaldehyde and acetate in the pathogenesis of the underlying ABD, including cancer, is discussed. We also highlight some gaps in knowledge existing in the field of ABD. Finally, we discuss future directions in exploring the role of acetaldehyde and acetate generated during chronic alcohol intake in various pathologies affecting different sites of the intestinal tract.

Toll-like receptor 2 activation in monocytes contributes to systemic inflammation and alcohol-associated liver disease in humans.

BACKGROUND AND RATIONALE: In the context of gut leakiness and translocation of microbial products in alcohol-associated liver disease (ALD), it is possible that systemic and liver inflammation involve the activation of circulating monocyte through gut-derived factors. We explored the association between monocytes, microbial translocation, systemic inflammation, and ALD. METHODS: Patients with alcohol use disorder following a rehabilitation program were compared with healthy controls. We determined the circulating number and proportion of monocyte subsets by FACS. The activation of signaling pathways by gut-derived microbes was analyzed by quantitative PCR in isolated monocytes. Cytokines secretion by monocytes and phagocytosis were assessed in vitro. Serum microbial translocation markers and cytokines were measured by ELISA and multiplex assay, respectively. ALD severity and liver inflammatory responses were analyzed in liver biopsies by various methods. RESULTS: In patients with alcohol use disorder, the number of blood monocytes increased compared with controls. Monocytes from patients with alcohol use disorder upregulated IL-1ß and IL-8 together with toll-like receptor 2 and downstream AP-1, while fungal sensor CARD9 was downregulated. IL-1ß and IL-8 were actively secreted upon stimulation in vitro with the toll-like receptor 2 ligand peptidoglycan. Exposure with Escherichia coli confirmed preserved bacterial phagocytic activity. In contrast, Candida albicans stimulation leads to downregulation of IL-1ß and TNFα compared with controls. Systemic cytokines and monocyte changes correlated with microbial translocation. Hepatic IL-1ß and IL-8 increased with ALD severity together with liver macrophage activation and upregulation of chemokines involved in monocyte attraction. CONCLUSIONS: Our results point to the contribution of activated monocytes to systemic inflammation and ALD. Monocytes likely infiltrate the liver, transform into monocyte-derived macrophages and release IL-1ß and IL-8 in response to peptidoglycan and toll-like receptor 2 activation.

Serum keratin 18-M65 levels detect progressive forms of alcohol-associated liver disease in early noncirrhotic stages.

BACKGROUND AND AIMS: The progression of alcohol-associated liver disease (ALD) in its early precirrhotic stages can be a silent process. Serum keratin 18 levels (K18-M65) predict severe events and mortality in advanced stages of ALD, but data on this biomarker in early stages are scarce. We evaluated the diagnostic accuracy of K18-M65 levels in identifying early forms of ALD. METHODS: We prospectively evaluated two cohorts of actively drinking patients with alcohol use disorder (AUD) following a rehabilitation program (training (n = 162) and validation (n = 78)) and matched healthy controls (n = 21). Clinical, laboratory, and imaging data were used to distinguish AUD patients with simple steatosis (minimal ALD) and steatohepatitis/fibrosis (early ALD). We measured serum K18-M65 levels and assessed their ability to predict early ALD. RESULTS: High levels of K18-M65 characterized AUD patients with early ALD, while levels in the minimal ALD group were similar to those in healthy controls. K18-M65 levels distinguished minimal liver disease from early ALD (AUROC = 0.8704; p < 0.0001) with an optimal cutoff at 265.9 U/L. K18-M65 levels strongly correlated with transaminases and predicted early ALD (OR 25.81; 95% CI 3.166-336.1; p < 0.0001), controlled attenuation parameter, and liver stiffness independently from transaminases and other potential confounders. K18-M65 levels did not discriminate between fibrosis and steatohepatitis but correlated with histological signs of hepatocellular injury and inflammation (all p < 0.05). The K18-M65 cutoff detected early ALD in the validation cohort with high accuracy (sensitivity 86.67%, specificity 96.67%) and a very high positive likelihood ratio (28.6; 95% CI 4.14-197.73). CONCLUSIONS: Serum K18-M65 levels can be used as a biomarker to detect early ALD stages with excellent predictive value.

Duodenal CD8+ T resident memory cell apoptosis contributes to gut barrier dysfunction and microbial translocation in early alcohol-associated liver disease in humans.

BACKGROUND: Intestinal T cells are key in gut barrier function. Their role in early stages of alcohol-associated liver disease (ALD) remain unknown. AIM: To explore the links between intestinal T cells, microbial translocation and ALD METHODS: Patients with alcohol use disorder (AUD) following a rehabilitation programme were compared to subjects with non-alcoholic fatty liver disease (NAFLD) and healthy controls. Clinical and laboratory data (liver stiffness, controlled attenuation parameter, AST, ALT, K18-M65) served to identify AUD patients with isolated steatosis (minimal liver disease) or steatohepatitis/fibrosis (ALD). Serum microbial translocation markers were measured by ELISA, duodenal and plasma levels of sphingolipids by targeted LC-MS. T lymphocytes in duodenal biopsies were characterised by immunohistochemistry, flow cytometry and RNA sequencing on FACS-sorted cells. Mechanisms for T-cell alterations were assessed in vitro. RESULTS: Patients with ALD, but not those with minimal liver disease, showed reduced numbers of duodenal CD8+ T resident memory (TRM) cells compared to controls or patients with NAFLD. TRM transcriptomic analysis, in vitro analyses and pharmacological inhibition of cathepsin B confirmed TRM apoptosis driven by lysosomal membrane permeabilisation and cathepsin B release into the cytosol. Altered lipid metabolism and increased duodenal and plasma sphingolipids correlated with apoptosis. Dihydroceramide dose-dependently reduced viability of TRM. Duodenal TRM phenotypic changes, apoptosis and transcriptomic alterations correlated with increased levels of microbial translocation markers. Short-term abstinence did not reverse TRM cell death in patients with ALD. CONCLUSIONS: Duodenal CD8+ TRM apoptosis related to functional changes in lysosomes and lipid metabolism points to impaired gut adaptive immunity specifically in patients with AUD who developed early ALD.

Lipidomics for the Prediction of Progressive Liver Disease in Patients with Alcohol Use Disorder.

Alcohol-related liver disease is a public health care burden globally. Only 10-20% of patients with alcohol use disorder have progressive liver disease. This study aimed to identify lipid biomarkers for the early identification of progressive alcohol-related liver disease, which is a key step for early intervention. We performed untargeted lipidomics analysis in serum and fecal samples for a cohort of 49 subjects, including 17 non-alcoholic controls, 16 patients with non-progressive alcohol-related liver disease, and 16 patients with progressive alcohol-related liver disease. The serum and fecal lipidome profiles in the two patient groups were different from that in the controls. Nine lipid biomarkers were identified that were significantly different between patients with progressive liver disease and patients with non-progressive liver disease in both serum and fecal samples. We further built a random forest model to predict progressive alcohol-related liver disease using nine lipid biomarkers. Fecal lipids performed better (Area Under the Curve, AUC = 0.90) than serum lipids (AUC = 0.79). The lipid biomarkers identified are promising candidates for the early identification of progressive alcohol-related liver disease.

Liver alterations are not improved by inulin supplementation in alcohol use disorder patients during alcohol withdrawal: A pilot randomized, double-blind, placebo-controlled study.

BACKGROUND: Emerging evidence highlights that targeting the gut microbiota could be an interesting approach to improve alcohol liver disease due to its important plasticity. This study aimed to evaluate the effects of inulin supplementation on liver parameters in alcohol use disorder (AUD) patients (whole sample) and in a subpopulation with early alcohol-associated liver disease (eALD). METHODS: Fifty AUD patients, hospitalized for a 3-week detoxification program, were enrolled in a randomized, double-blind, placebo-controlled study and assigned to prebiotic (inulin) versus placebo for 17 days. Liver damage, microbial translocation, inflammatory markers and 16S rDNA sequencing were measured at the beginning (T1) and at the end of the study (T2). FINDINGS: Compared to placebo, AST (ß = 8.55, 95% CI [2.33:14.77]), ALT (ß = 6.01, 95% CI [2.02:10.00]) and IL-18 (ß = 113.86, 95% CI [23.02:204.71]) were statistically significantly higher in the inulin group in the whole sample at T2. In the eALD subgroup, inulin supplementation leads to specific changes in the gut microbiota, including an increase in Bifidobacterium and a decrease of Bacteroides. Despite those changes, AST (ß = 14.63, 95% CI [0.91:28.35]) and ALT (ß = 10.40, 95% CI [1.93:18.88]) at T2 were higher in the inulin group compared to placebo. Treatment was well tolerated without important adverse events or side effects. INTERPRETATION: This pilot study shows that 17 days of inulin supplementation versus placebo, even though it induces specific changes in the gut microbiota, did not alleviate liver damage in AUD patients. Further studies with a larger sample size and duration of supplementation with adequate monitoring of liver parameters are needed to confirm these results. Gut2Brain study: https://clinicaltrials.gov/ct2/show/NCT03803709 FUNDING: Fédération Wallonie-Bruxelles, FRS-FNRS, Fondation Saint-Luc.

Host Factors in Dysregulation of the Gut Barrier Function during Alcohol-Associated Liver Disease.

Chronic alcohol consumption and alcohol-associated liver disease (ALD) represent a major public health problem worldwide. Only a minority of patients with an alcohol-use disorder (AUD) develop severe forms of liver disease (e.g., steatohepatitis and fibrosis) and finally progress to the more advanced stages of ALD, such as severe alcohol-associated hepatitis and decompensated cirrhosis. Emerging evidence suggests that gut barrier dysfunction is multifactorial, implicating microbiota changes, alterations in the intestinal epithelium, and immune dysfunction. This failing gut barrier ultimately allows microbial antigens, microbes, and metabolites to translocate to the liver and into systemic circulation. Subsequent activation of immune and inflammatory responses contributes to liver disease progression. Here we review the literature about the disturbance of the different host defense mechanisms linked to gut barrier dysfunction, increased microbial translocation, and impairment of liver and systemic inflammatory responses in the different stages of ALD.

Intestinal permeability, microbial translocation, changes in duodenal and fecal microbiota, and their associations with alcoholic liver disease progression in humans.

BACKGROUND: Animal data suggest a role of the gut-liver axis in progression of alcoholic liver disease (ALD), but human data are scarce especially for early disease stages. METHODS: We included patients with alcohol use disorder (AUD) who follow a rehabilitation program and matched healthy controls. We determined intestinal epithelial and vascular permeability (IP) (using urinary excretion of 51Cr-EDTA, fecal albumin content, and immunohistochemistry in distal duodenal biopsies), epithelial damage (histology, serum iFABP, and intestinal gene expression), and microbial translocation (Gram - and Gram + serum markers by ELISA). Duodenal mucosa-associated microbiota and fecal microbiota were analyzed by 16 S rRNA sequencing. ALD was staged by Fibroscan® (liver stiffness, controlled attenuation parameter) in combination with serum AST, ALT, and CK18-M65. RESULTS: Only a subset of AUD patients had increased 51Cr-EDTA and fecal albumin together with disrupted tight junctions and vasculature expression of plasmalemma Vesicle-Associated Protein-1. The so-defined increased intestinal permeability was not related to changes of the duodenal microbiota or alterations of the intestinal epithelium but associated with compositional changes of the fecal microbiota. Leaky gut alone did not explain increased microbial translocation in AUD patients. By contrast, duodenal dysbiosis with a dominance shift toward specific potential pathogenic bacteria genera (Streptococcus, Shuttleworthia, Rothia), increased IP and elevated markers of microbial translocation characterized AUD patients with progressive ALD (steato-hepatitis, steato-fibrosis). CONCLUSION: Progressive ALD already at early disease stages is associated with duodenal mucosa-associated dysbiosis and elevated microbial translocation. Surprisingly, such modifications were not linked with increased IP. Rather, increased IP appears related to fecal microbiota dysbiosis.

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)-1ß, 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.