Membrane-bound O-acyltransferase 7 (MBOAT7) shapes lysosomal lipid homeostasis and function to control alcohol-associated liver injury.

Recent genome-wide association studies (GWAS) have identified a link between single-nucleotide polymorphisms (SNPs) near the MBOAT7 gene and advanced liver diseases. Specifically, the common MBOAT7 variant (rs641738) associated with reduced MBOAT7 expression is implicated in non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis. However, the precise mechanism underlying MBOAT7-driven liver disease progression remains elusive. Previously, we identified MBOAT7-driven acylation of lysophosphatidylinositol lipids as key mechanism suppressing the progression of NAFLD (Gwag et al., 2019). Here, we show that MBOAT7 loss of function promotes ALD via reorganization of lysosomal lipid homeostasis. Circulating levels of MBOAT7 metabolic products are significantly reduced in heavy drinkers compared to healthy controls. Hepatocyte- (Mboat7-HSKO), but not myeloid-specific (Mboat7-MSKO), deletion of Mboat7 exacerbates ethanol-induced liver injury. Lipidomic profiling reveals a reorganization of the hepatic lipidome in Mboat7-HSKO mice, characterized by increased endosomal/lysosomal lipids. Ethanol-exposed Mboat7-HSKO mice exhibit dysregulated autophagic flux and lysosomal biogenesis, associated with impaired transcription factor EB-mediated lysosomal biogenesis and autophagosome accumulation. This study provides mechanistic insights into how MBOAT7 influences ALD progression through dysregulation of lysosomal biogenesis and autophagic flux, highlighting hepatocyte-specific MBOAT7 loss as a key driver of ethanol-induced liver injury.

Membrane Bound O-Acyltransferase 7 (MBOAT7) Shapes Lysosomal Lipid Homeostasis and Function to Control Alcohol-Associated Liver Injury.

Several recent genome-wide association studies (GWAS) have identified single nucleotide polymorphism (SNPs) near the gene encoding membrane-bound O -acyltransferase 7 ( MBOAT7 ) that is associated with advanced liver diseases. In fact, a common MBOAT7 variant (rs641738), which is associated with reduced MBOAT7 expression, confers increased susceptibility to non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis in those chronically infected with hepatitis viruses B and C. The MBOAT7 gene encodes a lysophosphatidylinositol (LPI) acyltransferase enzyme that produces the most abundant form of phosphatidylinositol 38:4 (PI 18:0/20:4). Although these recent genetic studies clearly implicate MBOAT7 function in liver disease progression, the mechanism(s) by which MBOAT7-driven LPI acylation regulates liver disease is currently unknown. Previously we showed that antisense oligonucleotide (ASO)-mediated knockdown of Mboat7 promoted non-alcoholic fatty liver disease (NAFLD) in mice (Helsley et al., 2019). Here, we provide mechanistic insights into how MBOAT7 loss of function promotes alcohol-associated liver disease (ALD). In agreement with GWAS studies, we find that circulating levels of metabolic product of MBOAT7 (PI 38:4) are significantly reduced in heavy drinkers compared to age-matched healthy controls. Hepatocyte specific genetic deletion ( Mboat7 HSKO ), but not myeloid-specific deletion ( Mboat7 MSKO ), of Mboat7 in mice results in enhanced ethanol-induced hepatic steatosis and high concentrations of plasma alanine aminotransferase (ALT). Given MBOAT7 is a lipid metabolic enzyme, we performed comprehensive lipidomic profiling of the liver and identified a striking reorganization of the hepatic lipidome upon ethanol feeding in Mboat7 HSKO mice. Specifically, we observed large increases in the levels of endosomal/lysosomal lipids including bis(monoacylglycero)phosphates (BMP) and phosphatidylglycerols (PGs) in ethanol-exposed Mboat7 HSKO mice. In parallel, ethanol-fed Mboat7 HSKO mice exhibited marked dysregulation of autophagic flux and lysosomal biogenesis when exposed to ethanol. This was associated with impaired transcription factor EB (TFEB)-mediated lysosomal biogenesis and accumulation of autophagosomes. Collectively, this works provides new molecular insights into how genetic variation in MBOAT7 impacts ALD progression in humans and mice. This work is the first to causally link MBOAT7 loss of function in hepatocytes, but not myeloid cells, to ethanol-induced liver injury via dysregulation of lysosomal biogenesis and autophagic flux.

Altered anti-viral immune responses in monocytes in overweight heavy drinkers.

Alcohol abuse causes increased susceptibility to respiratory syndromes like bacterial pneumonia and viral infections like SARS-CoV-2. Heavy drinkers (HD) are at higher risk of severe COVID-19 if they are also overweight, yet the molecular mechanisms are unexplored. Single-cell RNA-sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells from lean or overweight HD and healthy controls (HC) after challenge with a dsRNA homopolymer (PolyI:C) to mimic a viral infection and/or with lipopolysaccharide (LPS). All monocyte populations responded to both PolyI:C and LPS with pro-inflammatory gene expression. However, the expression of interferon-stimulated genes, essential for inhibiting viral pathogenesis, was greatly reduced in overweight patients. Interestingly, the number of upregulated genes in response to the PolyI:C challenge was far greater in monocytes from HD compared to HC, including much stronger pro-inflammatory cytokine and interferon-γ signaling responses. These results suggest that increased body weight reduced anti-viral responses while heavy drinking increased pro-inflammatory cytokines.

Mincle-GSDMD-mediated release of IL-1ß small extracellular vesicles from hepatic macrophages in ethanol-induced liver injury.

BACKGROUND: Macrophage-inducible C-type lectin (Mincle) is expressed on hepatic macrophages and senses ethanol (EtOH)-induced danger signals released from dying hepatocytes and promotes IL-1ß production. However, it remains unclear what and how EtOH-induced Mincle ligands activate downstream signaling events to mediate IL-1ß release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating ß-glucosylceramide (ß-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which ß-GluCer engages Mincle on hepatic macrophages to release IL-1ß in the absence of cell death and exacerbates ALD. METHOD AND RESULTS: Concentrations of ß-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of hepatic macrophages with lipopolysaccharide and ß-GluCer induced formation of a Mincle and Gsdmd-dependent secretory complex containing chaperoned full-length gasdermin D (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1ß and components of the Caspase 8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEVs). Gao-binge EtOH exposure to wild-type, but not Mincle-/- and Gsdmd-/-, mice increased release of IL-1ß-containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from EtOH-induced liver injury. sEVs collected from EtOH-fed wild-type, but not Gsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge EtOH-induced liver injury. CONCLUSION: ß-GluCer functions as a danger-associated molecular pattern activating Mincle-dependent gasdermin D-mediated formation and release of IL-1ß-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

Myeloid-cell-specific role of Gasdermin D in promoting lung cancer progression in mice.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD) are implicated in lung cancer pathophysiology but it's not clear if their contributions promote or retard lung cancer progression. Using a metastatic Lewis lung carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lungs, exhibit markedly decreased lung cancer metastasis, and show a significant ∼50% increase in median survival rate. The cleaved forms of GsdmD and IL-1ß 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. Using bone marrow transplantations, we show a myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that GsdmD plays a myeloid-specific role in lung cancer progression.

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis.

BACKGROUND AND AIMS: 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)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. APPROACH AND RESULTS: Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice. CONCLUSION: Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

35kDa hyaluronan ameliorates ethanol driven loss of anti-microbial defense and intestinal barrier integrity in a TLR4-dependent manner.

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 35 kDa 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. Reg3ß 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.

Receptor-interacting protein 1 and 3 kinase activity are required for high-fat diet induced liver injury in mice.

Background: The RIP1-RIP3-MLKL-mediated cell death pathway is associated with progression of non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH). Previous work identified a critical role for MLKL, the key effector regulating necroptosis, but not RIP3, in mediating high fat diet-induced liver injury in mice. RIP1 and RIP3 have active N-terminus kinase domains essential for activation of MLKL and subsequent necroptosis. However, little is known regarding domain-specific roles of RIP1/RIP3 kinase in liver diseases. Here, we hypothesized that RIP1/RIP3 kinase activity are required for the development of high fat diet-induced liver injury. Methods: Rip1K45A/K45A and Rip3K51A/K51A kinase-dead mice on a C57BL/6J background and their littermate controls (WT) were allowed free access to a diet high in fat, fructose and cholesterol (FFC diet) or chow diet. Results: Both Rip1K45A/K45A and Rip3K51A/K51A mice were protected against FFC diet-induced steatosis, hepatocyte injury and expression of hepatic inflammatory cytokines and chemokines. FFC diet increased phosphorylation and oligomerization of MLKL and hepatocyte death in livers of WT, but not in Rip3K51A/K51A, mice. Consistent with in vivo data, RIP3 kinase deficiency in primary hepatocytes prevented palmitic acid-induced translocation of MLKL to the cell surface and cytotoxicity. Additionally, loss of Rip1 or Rip3 kinase suppressed FFC diet-mediated formation of crown-like structures (indicators of dead adipocytes) and expression of mRNA for inflammatory response genes in epididymal adipose tissue. Moreover, FFC diet increased expression of multiple adipokines, including leptin and plasminogen activator inhibitor 1, in WT mice, which was abrogated by Rip3 kinase deficiency. Discussion: The current data indicate that both RIP1 and RIP3 kinase activity contribute to FFC diet-induced liver injury. This effect of RIP1 and RIP3 kinase deficiency on injury is consistent with the protection of Mlkl-/- mice from high fat diet-induced liver injury, but not the reported lack of protection in Rip3-/- mice. Taken together with previous reports, our data suggest that other domains of RIP3 likely counteract the effect of RIP3 kinase in response to high fat diets.

Hepatic kinome atlas: An in-depth identification of kinase pathways in liver fibrosis of humans and rodents.

BACKGROUND AND AIMS: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: Our findings establish a comprehensive kinase atlas for liver fibrosis, which identifies analogous signaling events conserved among humans and rodents.

Gut microbial trimethylamine is elevated in alcohol-associated hepatitis and contributes to ethanol-induced liver injury in mice.

There is mounting evidence that microbes residing 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. We 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. We show the gut microbial choline metabolite 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. The 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.

Role of MIF in coordinated expression of hepatic chemokines in patients with alcohol-associated hepatitis.

The chemokine system of ligands and receptors is implicated in the progression of alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. This study involved coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in 2 distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific KO were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH. Selected C-X-C chemokine members of the IL-8 chemokine family and C-C chemokine CCL20 were highly associated with AH compared with HC but not in patients with liver diseases of other etiologies (nonalcoholic fatty liver disease [NAFLD] and hepatitis C virus [HCV]). Our previous studies implicate macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of WT mice; this was prevented in hepatocyte-specific Mif-KO (MifΔHep) mice. This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and that hepatocyte-derived MIF might drive this inflammatory response.

Multiomics-Identified Intervention to Restore Ethanol-Induced Dysregulated Proteostasis and Secondary Sarcopenia in Alcoholic Liver Disease.

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 ß-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 ß-hydroxymethyl-butyrate treatment. CONCLUSION: An innovative multiomics approach followed by experimental validation showed that ß-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

Differential role of MLKL in alcohol-associated and non-alcohol-associated fatty liver diseases in mice and humans.

Hepatocellular death contributes to progression of alcohol-associated (ALD-associated) and non-alcohol-associated (NAFL/NASH) liver diseases. However, receptor-interaction protein kinase 3 (RIP3), an intermediate in necroptotic cell death, contributes to injury in murine models of ALD but not NAFL/NASH. We show here that a differential role for mixed-lineage kinase domain-like protein (MLKL), the downstream effector of RIP3, in murine models of ALD versus NAFL/NASH and that RIP1-RIP3-MLKL can be used as biomarkers to distinguish alcohol-associated hepatitis (AH) from NASH. Phospho-MLKL was higher in livers of patients with NASH compared with AH or healthy controls (HCs). MLKL expression, phosphorylation, oligomerization, and translocation to plasma membrane were induced in WT mice fed diets high in fat, fructose, and cholesterol but not in response to Gao-binge (acute on chronic) ethanol exposure. Mlkl-/- mice were not protected from ethanol-induced hepatocellular injury, which was associated with increased expression of chemokines and neutrophil recruitment. Circulating concentrations of RIP1 and RIP3, but not MLKL, distinguished patients with AH from HCs or patients with NASH. Taken together, these data indicate that MLKL is differentially activated in ALD/AH compared with NAFL/NASH in both murine models and patients. Furthermore, plasma RIP1 and RIP3 may be promising biomarkers for distinguishing AH and NASH.

Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease.

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.

Functionally Diverse Inflammatory Responses in Peripheral and Liver Monocytes in Alcohol-Associated Hepatitis.

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.

Inhibition of IRAK4 kinase activity improves ethanol-induced liver injury in mice.

BACKGROUNDS & AIMS: Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Interleukin-1 receptor associated kinase 4 (IRAK4), the master kinase of Toll-like receptor (TLR)/IL-1R-mediated signalling activation, is considered a novel therapeutic target in inflammatory diseases, but has not been investigated in the context of ALD. METHODS: IRAK4 phosphorylation and IRAK1 protein were analysed in liver from alcohol-related hepatitis patients and healthy controls. IRAK4 kinase activity-inactive knock-in (Irak4 KI) mice and bone marrow chimeric mice were exposed to chronic ethanol-induced liver injury. IL-1ß-induced IRAK4-mediated signalling and acute phase response were investigated in cultured hepatocytes. IRAK1/4 inhibitor was used to test the therapeutic potential for ethanol-induced liver injury in mice. RESULTS: Increased IRAK4 phosphorylation and reduced IRAK1 protein were found in livers of patients with alcoholic hepatitis. In the chronic ethanol-induced liver injury mouse model, hepatic inflammation and hepatocellular damage were attenuated in Irak4 KI mice. IRAK4 kinase activity promotes expression of acute phase proteins in response to ethanol exposure, including C-reactive protein and serum amyloid A1 (SAA1). SAA1 and IL-1ß synergistically exacerbate ethanol-induced cell death ex vivo. Pharmacological blockage of IRAK4 kinase abrogated ethanol-induced liver injury, inflammation, steatosis, as well as acute phase gene expression and protein production in mice. CONCLUSIONS: Our data elucidate the critical role of IRAK4 kinase activity in the pathogenesis of ethanol-induced liver injury in mice and provide preclinical validation for use of an IRAK1/4 inhibitor as a new potential therapeutic strategy for the treatment of ALD. LAY SUMMARY: Herein, we have identified the role of IRAK4 kinase activity in the development of alcohol-induced liver injury in mice. Hepatocyte-specific IRAK4 is associated with an acute phase response and release of proinflammatory cytokines/chemokines, which synergistically exacerbate alcohol-induced hepatocyte cell death ex vivo. Pharmacological inhibition of IRAK4 kinase activity effectively attenuates alcohol-induced liver injury in mice and could have therapeutic implications.

MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis.

BACKGROUND & AIMS: Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. METHODS: Rip3+/+, Rip3-/-, Mlkl+/+ and Mlkl-/- mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). RESULTS: The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl-/- mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes. CONCLUSIONS: Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy. LAY SUMMARY: Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.

Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High-Fat Diet-Induced Liver Injury.

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 (IKKß) 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.