Search

Acute and chronic alcohol exposure compromise intestinal epithelial integrity, due to reduced expression of anti-microbial peptides (AMP) and loss of tight junction integrity. Ameliorating gut damage is beneficial in preventing associated distant organ pathologies. Orally administered purified hyaluronan (HA) polymers with an average size of 35kDa have multiple protective effects in the gut and are well-tolerated in humans. Therefore, we tested the hypothesis that HA35 ameliorates ethanol-induced gut damage. Specifically, mechanisms that restore epithelial barrier integrity and normalize expression of the Reg3 class of C-type lectin AMPs (i.e. Reg3β and Reg3γ) were investigated. Chronic ethanol feeding to mice reduced expression of C-type lectin AMPs in the proximal small intestine (jejunum), reduced expression of tight junction proteins and increased bacterial translocation to the mesenteric lymph node. Oral consumption of HA35 during the last 6 days of ethanol exposure ameliorated the effects of chronic ethanol. Similarly, in vitro challenge of isolated intestinal organoids from murine jejunum with ethanol reduced the expression of C-type lectin AMPs and impaired barrier integrity; these ethanol-induced responses were prevented by pre-treatment with HA35. Importantly, HA receptor null jejunum-derived organoids demonstrated that the HA receptor Tlr4, but not Cd44 nor Tlr2, was required for the protective effect of HA35. Consistent with the data from organoids, HA35 did not protect Tlr4-deficient mice from chronic ethanol-induced intestinal injury. Together, these data suggest therapeutic administration of HA35 is beneficial in restoring gut epithelial integrity and defense during the early stages of ethanol-driven intestinal damage.

Interferon (IFN) regulatory factor 3 (IRF3) is a transcription factor activated by phosphorylation in the cytoplasm of a virus-infected cell; by translocating to the nucleus, it induces transcription of IFN-β and other antiviral genes. We have previously reported IRF3 can also be activated, as a proapoptotic factor, by its linear polyubiquitination mediated by the RIG-I pathway. Both transcriptional and apoptotic functions of IRF3 contribute to its antiviral effect. Here, we report a nontranscriptional function of IRF3, namely, the repression of IRF3-mediated NF-κB activity (RIKA), which attenuated viral activation of NF-κB and the resultant inflammatory gene induction. In Irf3 −/− mice, consequently, Sendai virus infection caused enhanced inflammation in the lungs. Mechanistically, RIKA was mediated by the direct binding of IRF3 to the p65 subunit of NF-κB in the cytoplasm, which prevented its nuclear import. A mutant IRF3 defective in both the transcriptional and the apoptotic activities was active in RIKA and inhibited virus replication. Our results demonstrated IRF3 deployed a three-pronged attack on virus replication and the accompanying inflammation.

Resolution of pathways that converge to induce deleterious effects in hepatic diseases, such as in the later stages, have potential antifibrotic effects that may improve outcomes. We aimed to explore whether humans and rodents display similar fibrotic signaling networks.We assiduously mapped kinase pathways using 340 substrate targets, upstream bioinformatic analysis of kinase pathways, and over 2000 random sampling iterations using the PamGene PamStation kinome microarray chip technology. Using this technology, we characterized a large number of kinases with altered activity in liver fibrosis of both species. Gene expression and immunostaining analyses validated many of these kinases as bona fide signaling events. Surprisingly, the insulin receptor emerged as a considerable protein tyrosine kinase that is hyperactive in fibrotic liver disease in humans and rodents. Discoidin domain receptor tyrosine kinase, activated by collagen that increases during fibrosis, was another hyperactive protein tyrosine kinase in humans and rodents with fibrosis. The serine/threonine kinases found to be the most active in fibrosis were dystrophy type 1 protein kinase and members of the protein kinase family of kinases. We compared the fibrotic events over four models: humans with cirrhosis and three murine models with differing levels of fibrosis, including two models of fatty liver disease with emerging fibrosis. The data demonstrate a high concordance between human and rodent hepatic kinome signaling that focalizes, as shown by our network analysis of detrimental pathways.Our findings establish a comprehensive kinase atlas for liver fibrosis, which identifies analogous signaling events conserved among humans and rodents.

Background & AimsMacrophage inducible C-type lectin (Mincle) is expressed on Kupffer cells and senses ethanol-induced danger signals released from dying hepatocytes and promotes IL-1β production. However, it remains unclear what and how ethanol-induced Mincle ligands activate downstream signaling events to mediate IL-1β release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating β-glucosylceramide (β-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which β-GluCer engages Mincle on Kupffer cells to release IL-1β in the absence of cell death and exacerbates ALD.Approach and ResultsConcentrations of β-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of Kupffer cells with LPS and β-GluCer induced formation of aMincleandGsdmd-dependent secretory complex containing chaperoned full-length GSDMD (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1β and components of the Casp8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEV). Gao-binge ethanol exposure to wild-type, but notMincle-/-andGsdmd-/-, mice increased release of IL-1β containing sEVs from liver explant cultures. Myeloid-specific deletion ofGsdmdsimilarly decreased the formation of sEVs by liver explant cultures and protected mice from ethanol-induced liver injury. sEVs collected from ethanol-fed wild-type, but notGsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge ethanol-induced liver injury.Conclusionβ-GluCer functions as a DAMP activating Mincle-dependent GSDMD-mediated formation and release of IL-1β-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.

Liver fibrosis is a disease with characteristics of an aberrant wound healing response. Fibrosis is commonly the end-stage for chronic liver diseases like alcohol-associated liver disease (ALD), metabolic-associated liver disease, viral hepatitis, and hepatic autoimmune disease. Innate immunity contributes to the progression of many diseases through multiple mechanisms including production of pro-inflammatory mediators, leukocyte infiltration and tissue injury. Chemokines and their receptors orchestrate accumulation and activation of immune cells in tissues and are associated with multiple liver diseases; however, much less is known about their potential roles in liver fibrosis. This is a narrative review of current knowledge of the relationship of chemokine biology to liver fibrosis with insights into potential future therapeutic opportunities that can be explored in the future.A comprehensive literature review was performed searching PubMed for relevant English studies and texts regarding chemokine biology, chronic liver disease and liver fibrosis published between 1993 and 2021. The review was written and constructed to detail the intriguing chemokine biology, the relation of chemokines to tissue injury and resolution, and identify areas of discovery for fibrosis treatment.Chemokines are implicated in many chronic liver diseases, regardless of etiology. Most of these diseases will progress to fibrosis without appropriate treatment. The contributions of chemokines to liver disease and fibrosis are diverse and include canonical roles of modulating hepatic inflammation as well as directly contributing to fibrosis via activation of hepatic stellate cells (HSCs). Limited clinical evidence suggests that targeting chemokines in certain liver diseases might provide a therapeutic benefit to patients with hepatic fibrosis.The chemokine system of ligands and receptors is a complex network of inflammatory signals in nearly all diseases. The specific sources of chemokines and cellular targets lend unique pathophysiological consequences to chronic liver diseases and established fibrosis. Although most chemokines are pro-inflammatory and contribute to tissue injury, others likely aid in the resolution of established fibrosis. To date, very few targeted therapies exist for the chemokine system and liver disease and/or fibrosis, and further study could identify viable treatment options to improve outcomes in patients with end-stage liver disease.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD), the final executor of inflammasome activity, are implicated in lung cancer pathophysiology but it’s not clear if their contributions promote or retard lung cancer progression. GsdmD plays a role in release of interleukin-1beta (IL-1 β), and the CANTOS trial and recent studies have highlighted a crucial role of IL-1β in promoting lung cancer. Expression of GsdmD was shown to be upregulated in human non-small cell lung cancer (NSCLC) tissue, but its contribution to in vivo lung cancer metastasis is not known. Using a metastatic Lewis Lung Carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lung, and exhibit markedly decreased lung cancer metastasis. Furthermore, GsdmD-/- mice show a significant ~ 50% increase in median survival rate vs. isogenic WT C57BL6J mice. The cleaved forms of GsdmD and IL-1 β were detected in lung tumor tissue, indicating inflammasome activity in lung tumor microenvironment (TME). Increased migration and growth of LLC cells was observed upon exposure to the conditioned media derived from inflammasome-induced wild type, but not the GsdmD-/-, macrophages. Exposure of human A549 lung cancer cells to the conditioned media derived from inflammasome-induced THP-1 macrophages also resulted in increased cell migration. Using bone marrow transplantation, we show the myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that inflammasome activation in macrophages promotes lung cancer growth and migration, and GsdmD plays a myeloid-specific role in lung cancer progression in mice.

Background and aims Acute liver damage causes hepatocyte stress and death, but in chronic liver disease impaired hepatocyte regeneration and immune cell infiltration prevents recovery. While the roles of both impaired liver regeneration and immune infiltration have been studied extensively in chronic liver diseases, the differential contribution of these factors is difficult to assess. Approach and results We combined single-cell RNA-sequencing (RNA-seq) data from healthy livers and peripheral immune cells to measure cell proportions in chronic liver diseases. Using bulk RNA-seq data from patients with early alcohol-associated hepatitis, severe AH (sAH), HCV, HCV with cirrhosis, and NAFLD, we performed gene deconvolution to predict the contribution of different cell types in each disease. Patients with sAH had the greatest change in cell composition, with increases in both periportal hepatocytes and cholangiocyte populations. Interestingly, while central vein hepatocytes were decreased, central vein endothelial cells were expanded. Endothelial cells are thought to regulate liver regeneration through WNT signaling. WNT2, important in central vein hepatocyte development, was down in sAH, while multiple other WNTs and WNT receptors were up-regulated. Immunohistochemistry revealed up-regulation of FZD6, a noncanonical WNT receptor, in hepatocytes in sAH. Immune cell populations also differed in disease. In sAH, a specific group of inflammatory macrophages was increased and distinct from the macrophage population in patients with HCV. Network and correlation analyses revealed that changes in the cell types in the liver were highly correlated with clinical liver function tests. Conclusions These results identify distinct changes in the liver cell populations in chronic liver disease and illustrate the power of using single-cell RNA-seq data from a limited number of samples in understanding multiple different diseases.

We aimed to identify a microRNA (miRNA)‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways and investigate the underlying molecular mechanisms in alcohol‐associated hepatitis (AH). An miRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways was constructed using integrated bioinformatics analysis. Differentially expressed hub miRNAs (GSE59492) and their validated miRNA target genes (GSE28619) were identified in the liver of patients with AH compared with healthy controls. Liver samples from patients with AH and healthy individuals and mice exposed to Gao‐binge (acute on chronic) ethanol were used for experimental validation. Using hub miRNAs identified by weighted correlation network analysis, a miRNA‐E3 ubiquitin ligase regulatory network was established based on 17 miRNAs and 7 E3 ligase genes targeted by these miRNAs that were down‐regulated in AH. Among the miRNAs in this regulatory network, miR‐150‐5p was the only miRNA regulating the E3 ligase cytokine‐inducible SH2 containing protein (CISH), the E3 ligase that regulates the largest number of substrates among all E3 ligase family members. Therefore, the CISH regulatory pathway for ubiquitinated substrates was selected for subsequent experimental validation. Consistent with the bioinformatics analysis results, expression of miR‐150‐5p was markedly increased, while CISH was decreased, in the livers of patients with AH and mice exposed to Gao‐binge ethanol. Moreover, ubiquitination of Fas‐associated protein with death domain, a predicted CISH substrate involved in the regulation of programmed cell death, was reduced in livers from mice after Gao‐binge ethanol. Conclusion: Identification of the miRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in the cell death pathways provides insights into the molecular mechanisms contributing to hepatocyte death in AH., This study identifies a microRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways. We report that expression of miR‐150‐5p was elevated in AH and experimentally verified the changes in the key components in this miR‐150‐5p‐CISH‐FADD regulatory network in the liver from both patients with AH and a murine model of acute‐on‐chronic ethanol exposure.

Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)Bone marrow transplants between MlklTogether, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

The C-type lectin receptor Mincle is known for its important role in innate immune cells in recognizing pathogen and damage associated molecular patterns. Here we report a T cell–intrinsic role for Mincle in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Genomic deletion of Mincle in T cells impairs TH17, but not TH1 cell-mediated EAE, in alignment with significantly higher expression of Mincle in TH17 cells than in TH1 cells. Mechanistically, dying cells release β-glucosylceramide during inflammation, which serves as natural ligand for Mincle. Ligand engagement induces activation of the ASC-NLRP3 inflammasome, which leads to Caspase8-dependent IL-1β production and consequentially TH17 cell proliferation via an autocrine regulatory loop. Chemical inhibition of β-glucosylceramide synthesis greatly reduces inflammatory CD4+ T cells in the central nervous system and inhibits EAE progression in mice. Taken together, this study indicates that sensing of danger signals by Mincle on TH17 cells plays a critical role in promoting CNS inflammation.

BACKGROUND AND AIMS Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown). APPROACH AND RESULTS Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations. CONCLUSIONS Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.

BACKGROUND/AIMS: Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. METHODS: Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. RESULTS: Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment. CONCLUSION: An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are the two major types of chronic liver disease worldwide. Inflammatory processes play key roles in the pathogeneses of fatty liver diseases, and continuous inflammation promotes the progression of alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH). Although both ALD and NAFLD are closely related to inflammation, their respective developmental mechanisms differ to some extent. Here, we review the roles of multiple immunological mechanisms and therapeutic targets related to the inflammation associated with fatty liver diseases and the differences in the progression of ASH and NASH. Multiple cell types in the liver, including macrophages, neutrophils, other immune cell types and hepatocytes, are involved in fatty liver disease inflammation. In addition, microRNAs (miRNAs), extracellular vesicles (EVs), and complement also contribute to the inflammatory process, as does intertissue crosstalk between the liver and the intestine, adipose tissue, and the nervous system. We point out that inflammation also plays important roles in promoting liver repair and controlling bacterial infections. Understanding the complex regulatory process of disrupted homeostasis during the development of fatty liver diseases may lead to the development of improved targeted therapeutic intervention strategies.

Background and aims Given the lack of effective therapies and high mortality in acute alcohol-associated hepatitis (AH), it is important to develop rationally designed biomarkers for effective disease management. Complement, a critical component of the innate immune system, contributes to uncontrolled inflammatory responses leading to liver injury, but is also involved in hepatic regeneration. Here, we investigated whether a panel of complement proteins and activation products would provide useful biomarkers for severity of AH and aid in predicting 90-day mortality. Approach and results Plasma samples collected at time of diagnosis from 254 patients with moderate and severe AH recruited from four medical centers and 31 healthy persons were used to quantify complement proteins by enzyme-linked immunosorbent assay and Luminex arrays. Components of the classical and lectin pathways, including complement factors C2, C4b, and C4d, as well as complement factor I (CFI) and C5, were reduced in AH patients compared to healthy persons. In contrast, components of the alternative pathway, including complement factor Ba (CFBa) and factor D (CFD), were increased. Markers of complement activation were also differentially evident, with C5a increased and the soluble terminal complement complex (sC5b9) decreased in AH. Mannose-binding lectin, C4b, CFI, C5, and sC5b9 were negatively correlated with Model for End-Stage Liver Disease score, whereas CFBa and CFD were positively associated with disease severity. Lower CFI and sC5b9 were associated with increased 90-day mortality in AH. Conclusions Taken together, these data indicate that AH is associated with a profound disruption of complement. Inclusion of complement, especially CFI and sC5b9, along with other laboratory indicators, could improve diagnostic and prognostic indications of disease severity and risk of mortality for AH patients.

Patients with severe alcohol-associated hepatitis (AH) have high mortality. Corticosteroids improve survival only for 30 days. We targeted inflammation, cellular injury, and gut leakiness in a randomized clinical trial comparing combination therapy to corticosteroids on 180-day survival.Subjects with a clinical diagnosis of severe AH (Model for End-Stage Liver Disease [MELD]20, Maddrey discriminant function [MDF]32) were randomized to receive methylprednisolone (PRED; 28 days) or a combination of anakinra (14 days) plus pentoxifylline (28 days) plus zinc (COMB; 180 days). The primary endpoint was survival at 180 days. The study was designed in 2013, initiated in October 2014, and completed in March 2018. Five hundred patients were screened to randomize 104 subjects with a clinical diagnosis of AH with a MELD score20. Fifty-three patients were randomized into the COMB and 50 to the PRED treatment; 1 dropped out of the study before randomization. Mean age was 45.3 ± 10.4 years; 60.6% were males, 92.3% White, and mean MELD 25.7 ± 3.9. Kaplan-Meier survival estimate at 180 days was 67.9% in COMB and 56% in PRED (HR = 0.69; p = 0.3001). Survival curves separated by 90 days (COMB, 69.8%; PRED, 58.0%; HR = 0.69; p = 0.28). Survival at 28 days was similar between the COMB (83.4%) and PRED groups (81.2%; HR = 0.91; p = 0.85). There were no unexpected serious adverse events, and incidence of infection was comparable between groups. MELD 20-25 and MELD26 strata showed nonsignificant treatment effects in favor of COMB.A combination of anakinra, pentoxifylline plus zinc provides similar survival benefits compared to corticosteroid therapy in severe AH.

Alcohol-associated hepatitis (AH) is an acute inflammatory disease in which gut-microbial byproducts enter circulation and peripheral immune cells infiltrate the liver, leading to nonresolving inflammation and injury. Single-cell RNA sequencing of peripheral blood mononuclear cells isolated from patients with AH and healthy controls paired with lipopolysaccharide (LPS) challenge revealed how diverse monocyte responses are divided among individual cells and change in disease. After LPS challenge, one monocyte subtype expressed pro-inflammatory genes in both disease and healthy controls, while another monocyte subtype was anti-inflammatory in healthy controls but switched to pro-inflammatory in AH. Numerous immune genes are clustered within genomic cassettes, including chemokines and C-type lectin receptors (CTRs). CTRs sense byproducts of diverse microbial and host origin. Single-cell data revealed correlated expression of genes within cassettes, thus further diversifying different monocyte responses to individual cells. Monocyte up-regulation of CTRs in response to LPS caused hypersensitivity to diverse microbial and host-derived byproducts, indicating a secondary immune surveillance pathway up-regulated in a subset of cells by a closely associated genomic cassette. Finally, expression of CTR genes was higher in livers of patients with severe AH, but not other chronic liver diseases, implicating secondary immune surveillance in nonresolving inflammation in severe AH.

Alcohol use and consequent liver disease are major burdens that have worsened during the COVID-19 pandemic. There are several facets to the pathophysiology and clinical consequences of alcohol-use disorder (AUD) and progression to alcohol-associated liver disease (ALD) that require a concerted effort by clinicians and translational and basic science investigators. Several recent advances from bedside to bench and bench to bedside have been made in ALD. We focused this review on a case-based approach that provides a human context to these important advances across the spectrum of ALD.

BACKGROUNDThere is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested.METHODSWe used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing.RESULTSWe show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome.CONCLUSIONSThe microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

Protein synthesis and autophagy are regulated by cellular ATP content. We tested the hypothesis that mitochondrial dysfunction, including generation of reactive oxygen species (ROS), contributes to impaired protein synthesis and increased proteolysis resulting in tissue atrophy in a comprehensive array of models. In myotubes treated with ethanol, using unbiased approaches, we identified defects in mitochondrial electron transport chain components, endogenous antioxidants, and enzymes regulating the tricarboxylic acid (TCA) cycle. Using high sensitivity respirometry, we observed impaired cellular respiration, decreased function of complexes I, II, and IV, and a reduction in oxidative phosphorylation in ethanol-treated myotubes and muscle from ethanol-fed mice. These perturbations resulted in lower skeletal muscle ATP content and redox ratio (NAD+/NADH). Ethanol also caused a leak of electrons, primarily from complex III, with generation of mitochondrial ROS and reverse electron transport. Oxidant stress with lipid peroxidation (thiobarbituric acid reactive substances) and protein oxidation (carbonylated proteins) were increased in myotubes and skeletal muscle from mice and humans with alcoholic liver disease. Ethanol also impaired succinate oxidation in the TCA cycle with decreased metabolic intermediates. MitoTEMPO, a mitochondrial specific antioxidant, reversed ethanol-induced mitochondrial perturbations (including reduced oxygen consumption, generation of ROS and oxidative stress), increased TCA cycle intermediates, and reversed impaired protein synthesis and the sarcopenic phenotype. We show that ethanol causes skeletal muscle mitochondrial dysfunction, decreased protein synthesis, and increased autophagy, and that these perturbations are reversed by targeting mitochondrial ROS.

International audience; BACKGROUND and AIMS: Only a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk. METHODS: Three cohorts (GenomALC-1: n=1690, GenomALC-2: n=3037, UK Biobank: relevant n=6898) with a history of heavy alcohol consumption (≥80 g/day (men), ≥50 g/day (women), for ≥10 years) were included. Cases were participants with alcohol-related cirrhosis. Controls had a history of similar alcohol consumption but no evidence of liver disease. Risk scores were computed from up to eight genetic loci identified previously as associated with alcohol-related cirrhosis and three clinical risk factors. Score performance for the stratification of alcohol-related cirrhosis risk was assessed and compared across the alcohol-related liver disease spectrum, including hepatocellular carcinoma (HCC). RESULTS: A combination of three single nucleotide polymorphisms (SNPs) (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) and diabetes status best discriminated for cirrhosis risk. The odds ratio (OR) and 95% confidence intervals (CI) for the extreme score quintiles (Q1-Q5) of the 3-SNP score, based on independent allelic effect size estimates, were 5.99 (4.18;8.60) (GenomALC-1); 2.81 (2.03;3.89) (GenomALC-2); and 3.10 (2.32;4.14) (UK Biobank). Patients with diabetes and high-risk score, compared to those without diabetes and a low-risk score, had ORs increased to 14.7 (7.69;28.1) (GenomALC-1) and 17.1 (11.3;25.7) (UK Biobank). Patients with cirrhosis and HCC had significantly higher mean risk scores than patients with cirrhosis alone (0.76±0.06 versus 0.61±0.02, p=0.007). Score performance was not significantly enhanced by information on additional genetic risk variants, body mass index or coffee consumption. CONCLUSIONS: A risk score based on three genetic risk variants and diabetes status can provide meaningful risk stratification for cirrhosis in excess drinkers, allowing earlier prevention planning including intensive intervention. LAY SUMMARY: Excessive chronic drinking leads to liver cirrhosis in some people, but so far there is no way to identify those at high risk of developing this debilitating disease. Our study has developed a genetic risk score (GRS) test that can identify patients at high risk and shows that the risk of cirrhosis is increased >10-fold with just two risk factors - diabetes and high GRS. Risk assessment using this test has potential for early and personalised management of this disease in high-risk patients.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption actually hinge on gut microbial metabolism. We show that a single gut microbial flavonoid catabolite is sufficient to reduce diet-induced cardiometabolic disease burden in mice. Dietary supplementation with elderberry extract attenuated obesity and continuous delivery of the catabolite 4-hydroxphenylacetic acid was sufficient to reverse hepatic steatosis. Analysis of human gut metagenomes revealed that under one percent contains a flavonol catabolic pathway, underscoring the rarity of this process. Our study will impact the design of dietary and probiotic interventions to complement traditional cardiometabolic treatment strategies.One-Sentence SummarySelect gut microbes can metabolize flavonoids from a fruit and vegetable diet to monophenolic acids, which improve fatty liver disease.Graphical abstract

Interferon regulatory factor 3 (IRF3) has both transcriptional and nontranscriptional functions. Transcriptional activity is dependent on serine phosphorylation of IRF3, while transcription-independent IRF3-mediated apoptosis requires ubiquitination. IRF3 also binds to inhibitor of nuclear factor kappa B kinase (IKKβ) in the cytosol, restricting nuclear translocation of p65. IRF3-deficient mice are highly sensitive to high-fat diet (HFD)-induced liver injury; however, it is not known if transcriptional and/or nontranscriptional activity of IRF3 confers protection. Using a mouse model only expressing nontranscriptional functions of IRF3 (Irf3 S1/S1), we tested the hypothesis that nontranscriptional activity of IRF3 protects mice from HFD-induced liver injury. C57BL/6, Irf3 -/-, and Irf3 S1/S1 mice were fed an HFD for 12 weeks. In C57BL/6 mice, the HFD increased expression of interferon (IFN)-dependent genes, despite a decrease in IRF3 protein in the liver. The HFD had no impact on IFN-dependent gene expression Irf3 -/- or Irf3 S1/S1 mice, both lacking IRF3 transcriptional activity. Liver injury, apoptosis, and fibrosis were exacerbated in Irf3 -/- compared to C57BL/6 mice following the HFD; this increase was ameliorated in Irf3 S1/S1 mice. Similarly, expression of inflammatory cytokines as well as numbers of neutrophils and infiltrating monocytes was increased in Irf3 -/- mice compared to C57BL/6 and Irf3 S1/S1 mice. While the HFD increased the ubiquitination of IRF3, a response associated with IRF3-mediated apoptosis, in Irf3 S1/S1 mice, protection from liver injury was not due to differences in apoptosis of hepatocytes or immune cells. Instead, protection from HFD-induced liver injury in Irf3 S1/S1 mice was primarily associated with retardation of nuclear translocation of p65 and decreased expression of nuclear factor kappa B (NFκB)-dependent inflammatory cytokines. Conclusion: Taken together, these data identify important contributions of the nontranscriptional function of IRF3, likely by reducing NFκB signaling, in dampening the hepatic inflammatory environment in response to an HFD.

Background Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. Methods Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. Results While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. Conclusions Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.