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Abstract The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D: IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer’s disease, notably apolipoprotein-e. Using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 (ZBP1) as a target of IRF3 that is relevant across various neuroinflammatory disorders. Lastly, we show IRF3 phosphorylation and IRF3-dependent ZBP1 induction in response to Aβ in primary microglia cultures. Together, our results identify IRF3 as an important regulator of LPS and Aβ -mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

Most chronic liver diseases progress to liver fibrosis, which, when left untreated, can lead to cirrhosis and hepatocellular carcinoma. MicroRNA (miRNA)-targeted therapeutics have become attractive approaches to treat diseases. In this study, we investigated the therapeutic effect of miR-155 inhibition in the bile duct ligation (BDL) mouse model of liver fibrosis and evaluated the role of miR-155 in chronic liver fibrosis using miR-155-deficient (miR-155 knockout [KO]) mice. We found increased hepatic miR-155 expression in patients with cirrhosis and in the BDL- and CCl4-induced mouse models of liver fibrosis. Liver fibrosis was significantly reduced in miR-155 KO mice after CCl4 administration or BDL. To assess the therapeutic potential of miR-155 inhibition, we administered an rAAV8-anti-miR-155 tough decoy in vivo that significantly reduced liver damage and fibrosis in BDL. BDL-induced protein levels of transforming growth factor β (TGF-β), p-SMAD2/3, and p-STAT3 were attenuated in anti-miR-155-treated compared with control mice. Hepatic stellate cells from miR-155 KO mice showed attenuation in activation and mesenchymal marker expression. In vitro, miR-155 gain- and loss-of-function studies revealed that miR-155 regulates activation of stellate cells partly via STAT3 signaling. Our study suggests that miR-155 is the key regulator of liver fibrosis and might be a potential therapeutic target to attenuate fibrosis progression.

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF.Methods: ACLF was triggered by a single alcohol binge (5 g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200 μg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed.Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1β was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicle-treated ACLF mice.Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment.

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1β via NLRP3 activation and that IL-1β acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Abstract Massive inflammation and liver failure are main contributors to the high mortality in alcohol‐associated hepatitis (AH). In recent clinical trials, granulocyte colony‐stimulating factor (G‐CSF) therapy improved liver function and survival in patients with AH. However, the mechanisms of G‐CSF‐mediated beneficial effects in AH remain elusive. In this study, we evaluated effects of in vivo G‐CSF administration, using a mouse model of AH. G‐CSF treatment significantly reduced liver damage in alcohol‐fed mice even though it increased the numbers of liver‐infiltrating immune cells, including neutrophils and inflammatory monocytes. Moreover, G‐CSF promoted macrophage polarization toward an M2‐like phenotype and increased hepatocyte proliferation, which was indicated by an increased Ki67‐positive signal colocalized with hepatocyte nuclear factor 4 alpha (HNF‐4α) and cyclin D1 expression in hepatocytes. We found that G‐CSF increased G‐CSF receptor expression and resulted in reduced levels of phosphorylated β‐catenin in hepatocytes. In the presence of an additional pathogen‐associated molecule, lipopolysaccharide (LPS), which is significantly increased in the circulation and liver of patients with AH, the G‐CSF‐induced hepatoprotective effects were abolished in alcohol‐fed mice. We still observed increased Ki67‐positive signals in alcohol‐fed mice following G‐CSF treatment; however, Ki67 and HNF‐4α did not colocalize in LPS‐challenged mice. Conclusion: G‐CSF treatment increases immune cell populations, particularly neutrophil counts, and promotes M2‐like macrophage differentiation in the liver. More importantly, G‐CSF treatment reduces alcohol‐induced liver injury and promotes hepatocyte proliferation in alcohol‐fed mice. These data provide new insights into the understanding of mechanisms mediated by G‐CSF and its therapeutic effects in AH.

Background: Mortality is high for severe alcohol-associated hepatitis (AH). Corticosteroids are the standard of care for patients without contraindications. Recent data showed that interleukin-1β receptor antagonist anakinra attenuated inflammation and liver damage. We designed a multicenter, double-blind, randomized controlled trial to assess the safety and efficacy of anakinra compared to prednisone. Methods: Patients meeting the clinical and biochemical criteria for severe AH with MELD scores between 20 and 35 were recruited at eight clinical sites. Eligible patients enrolled in the study were randomized to anakinra, 100 mg subcutaneous injection for 14 days, plus zinc sulfate 220 mg for 90 days, vs. prednisone 40 mg PO daily for 30 days. Matching placebos for anakinra, zinc, and prednisone were provided to mask the treatment. Participants were followed for 180 days. The primary outcome was overall survival at 90 days. An unadjusted log-rank test was used to compare the survival of the two treatments in the first 90 days. Between July 10, 2020, and March 4, 2022, we screened 1082 patients with severe AH, and 147 eligible patients were enrolled and randomized. The average baseline MELD score was 25 [range 20–35], Maddrey discriminant function (MDF) was 59.4 [range 20.2–197.5]. The mean aspartate transaminase (AST)-to-alanine transaminase (ALT) ratio was 3.5. The baseline characteristics were not statistically different between the two treatment groups. Conclusions: The study provided a direct comparison of the survival benefits and safety profiles of anakinra plus zinc vs. prednisone in patients with severe AH.

microRNAs (miRs) are small regulatory RNAs that are frequently deregulated in liver disease. Liver fibrosis is characterized by excessive scarring caused by chronic inflammatory processes. In this study, we determined the functional role of miR-132 using a locked nucleic acid (LNA)-anti-miR approach in liver fibrosis. A significant induction in miR-132 levels was found in mice treated with CCl4 and in patients with fibrosis/cirrhosis. Inhibition of miR-132 in mice with LNA-anti-miR-132 caused decreases in CCl4-induced fibrogenesis and inflammatory phenotype. An attenuation in collagen fibers, α SMA, MCP1, IL-1β, and Cox2 was found in LNA-anti-miR-132-treated mice. CCl4 treatment increased caspase 3 activity and extracellular vesicles (EVs) in control but not in anti-miR-132-treated mice. Inhibition of miR-132 was associated with augmentation of MMP12 in the liver and Kupffer cells. In vivo and in vitro studies suggest miR-132 targets SIRT1 and inflammatory genes. Using tumor cancer genome atlas data, an increase in miR-132 was found in hepatocellular carcinoma (HCC). Increased miR-132 levels were associated with fibrogenic genes, higher tumor grade and stage, and unfavorable survival in HCC patients. Therapeutic inhibition of miR-132 might be a new approach to alleviate liver fibrosis, and treatment efficacy can be monitored by observing EV shedding.

Alcohol use disorder is associated with systemic inflammation and organ dysfunction especially in the liver and the brain. For more than a decade, studies have highlighted alcohol abuse-mediated impairment of brain function and acceleration of neurodegeneration through inflammatory mechanisms that directly involve innate immune cells. Furthermore, recent studies indicate overlapping genetic risk factors between alcohol use and neurodegenerative disorders, specifically regarding the role of innate immunity in the pathomechanisms of both areas. Considering the pressing need for a better understanding of the relevance of alcohol abuse in dementia progression, here we summarize the molecular mechanisms of neuroinflammation observed in alcohol abuse and Alzheimer’s disease, the most common cause of dementia. In addition, we highlight mechanisms that are already established in the field of Alzheimer’s disease that may be relevant to explore in alcoholism to better understand alcohol mediated neurodegeneration and dementia, including the relevance of the liver-brain axis.

Acute liver injury (ALI) is associated with multiple cellular events such as necrosis, apoptosis, oxidative stress and inflammation, which can lead to liver failure. In this study, we demonstrate a new role of microRNA (miR)‐208a in ALI. ALI was induced in wild‐type (WT) and miR‐208a knockout (KO) mice by CCl4 administration. Increased alanine aminotransferase and decreased hepatic miR‐208a levels were found in WT mice after acute CCl4 treatment. Histopathological evaluations revealed increased necrosis and decreased inflammation in miR‐208a KO compared with WT mice after CCl4 treatment. CCl4 treatment induced a higher alanine aminotransferase elevation and increased numbers of circulating extracellular vesicles (exosomes and microvesicles) in miR‐208a KO compared with WT mice. We found increased CCl4‐induced nuclear factor kappa B activation and tumor necrosis factor‐α induction and decreased monocyte chemoattractant protein 1 levels in miR‐208a KO compared with WT mice. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling assay indicated aggravated hepatic apoptosis and necrosis in CCl4‐treated miR‐208a KO compared with WT mice. CCl4 treatment induced a greater increase in cleaved caspase‐8, p18, and caspase‐3 in miR‐208a KO compared with WT mice. p53 is involved in various cell death pathways, including necrosis and apoptosis. Our in silico analysis revealed p53 as a predicted miR‐208a target, and we found enhanced p53 and cyclophilin D protein expressions in miR‐208a KO mice after CCl4 treatment. Increased liver injury in miR‐208a KO mice was further associated with increased Bax (B cell lymphoma 2–associated X protein) and p21 expression. Our in vitro results indicated a role of miR‐208a in cell death. We found that CCl4‐induced cytotoxicity was partially rescued by miR‐208a overexpression in RAW macrophages. Altogether, our results revealed a role of miR‐208a in ALI in mice and suggest a role for miR‐208a in regulating cell death.

Abstract Background Chronic alcohol consumption is associated with neuroinflammation, neuronal damage, and behavioral alterations including addiction. Alcohol-induced neuroinflammation is characterized by increased expression of proinflammatory cytokines (including TNFα, IL-1β, and CCL2) and microglial activation. We hypothesized chronic alcohol consumption results in peripheral immune cell infiltration to the CNS. Since chemotaxis through the CCL2-CCR2 signaling axis is critical for macrophage recruitment peripherally and centrally, we further hypothesized that blockade of CCL2 signaling using the dual CCR2/5 inhibitor cenicriviroc (CVC) would prevent alcohol-induced CNS infiltration of peripheral macrophages and alter the neuroinflammatory state in the brain after chronic alcohol consumption. Methods C57BL/6J female mice were fed an isocaloric or 5% (v/v) ethanol Lieber DeCarli diet for 6 weeks. Some mice received daily injections of CVC. Microglia and infiltrating macrophages were characterized and quantified by flow cytometry and visualized using CX3CR1eGFP/+ CCR2RFP/+ reporter mice. The effect of ethanol and CVC treatment on the expression of inflammatory genes was evaluated in various regions of the brain, using a Nanostring nCounter inflammation panel. Microglia activation was analyzed by immunofluorescence. CVC-treated and untreated mice were presented with the two-bottle choice test. Results Chronic alcohol consumption induced microglia activation and peripheral macrophage infiltration in the CNS, particularly in the hippocampus. Treatment with CVC abrogated ethanol-induced recruitment of peripheral macrophages and partially reversed microglia activation. Furthermore, the expression of proinflammatory markers was upregulated by chronic alcohol consumption in various regions of the brain, including the cortex, hippocampus, and cerebellum. Inhibition of CCR2/5 decreased alcohol-mediated expression of inflammatory markers. Finally, microglia function was impaired by chronic alcohol consumption and restored by CVC treatment. CVC treatment did not change the ethanol consumption or preference of mice in the two-bottle choice test. Conclusions Together, our data establish that chronic alcohol consumption promotes the recruitment of peripheral macrophages into the CNS and microglia alterations through the CCR2/5 axis. Therefore, further exploration of the CCR2/5 axis as a modulator of neuroinflammation may offer a potential therapeutic approach for the treatment of alcohol-associated neuroinflammation.

Severe alcoholic hepatitis (AH) is an acute and often devastating form of alcohol-associated liver disease. Clinically, AH is characterized by elevated bilirubin, model for end stage liver disease scores >20, and nonspecific symptoms that are caused by underlying inflammation, hepatocyte injury, and impaired intestinal barrier function. Compromised immune defense in AH contributes to infections, sepsis and organ failure. To date, corticosteroids are the only recommended treatment for severe AH, however it does not provide survival benefits beyond 1 month. Recent preclinical and early clinical studies in AH aided understanding of the disease and presented opportunities for new therapeutic options targeting inflammation, oxidative stress, liver regeneration and modification of intestinal microbiota. In this comprehensive review, we discuss promising preclinical results and ongoing clinical trials evaluating novel therapeutic agents for the treatment of severe AH.

This review covers some important new aspects of the alcohol-induced communications between liver parenchymal and non-parenchymal cells leading to liver injury development. The information exchange between various cell types may promote end-stage liver disease progression and involves multiple mechanisms, such as direct cell-to-cell interactions, extracellular vesicles (EVs) or chemokines, cytokines, and growth factors contained in extracellular fluids/cell culture supernatants. Here, we highlighted the role of EVs derived from alcohol-exposed hepatocytes (HCs) in activation of non-parenchymal cells, liver macrophages (LM), and hepatic stellate cells (HSC). The review also concentrates on EV-mediated crosstalk between liver parenchymal and non-parenchymal cells in the settings of HIV- and alcohol co-exposure. In addition, we overviewed the literature on the crosstalk between cell death pathways and inflammasome activation in alcohol-activated HCs and macrophages. Furthermore, we covered highly clinically relevant studies on the role of non-inflammatory factors, sinusoidal pressure (SP), and hepatic arterialization in alcohol-induced hepatic fibrogenesis. We strongly believe that the review will disclose major mechanisms of cell-to-cell communications pertained to alcohol-induced liver injury progression and will identify therapeutically important targets, which can be used for alcohol-associated liver disease (ALD) prevention.

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

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.

Acyl-CoA synthetase 1 (ACSL1) is an enzyme that converts fatty acids to acyl-CoA-derivatives for lipid catabolism and lipid synthesis in general and can provide substrates for the production of mediators of inflammation in monocytes and macrophages. Acsl1 expression is increased by hyperglycemia and inflammatory stimuli in monocytes and macrophages, and promotes the pro-atherosclerotic effects of diabetes in mice. Yet, surprisingly little is known about the mechanisms underlying Acsl1 transcriptional regulation. Here we demonstrate that the glucose-sensing transcription factor, Carbohydrate Response Element Binding Protein (CHREBP), is a regulator of the expression of Acsl1 mRNA by high glucose in mouse bone marrow-derived macrophages (BMDMs). In addition, we show that inflammatory stimulation of BMDMs with lipopolysaccharide (LPS) increases Acsl1 mRNA via the transcription factor, NF-kappa B. LPS treatment also increases ACSL1 protein abundance and localization to membranes where it can exert its activity. Using an Acsl1 reporter gene containing the promoter and an upstream regulatory region, which has multiple conserved CHREBP and NF-kappa B (p65/RELA) binding sites, we found increased Acsl1 promoter activity upon CHREBP and p65/RELA expression. We also show that CHREBP and p65/RELA occupy the Acsl1 promoter in BMDMs. In primary human monocytes cultured in high glucose versus normal glucose, ACSL1 mRNA expression was elevated by high glucose and further enhanced by LPS treatment. Our findings demonstrate that CHREBP and NF-kappa B control Acsl1 expression under hyperglycemic and inflammatory conditions.

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.

Bile acids (BAs) activate various dedicated receptors, including the farnesoid X receptor (FXR) and the Takeda G protein‐coupled receptor 5 (TGR5). The FXR agonist obeticholic acid (OCA) is licensed for the treatment of primary biliary cholangitis and has shown promising results in NASH patients, whereas TGR5 agonists target inflammation and metabolism. We hypothesized that FXR and/or TGR5 agonists may be therapeutic in early alcoholic liver disease (ALD) in mice, in which hepatic inflammation plays a major role. OCA, INT‐777, and INT‐767 are BA derivatives with selective agonist properties for FXR, TGR5, or both, respectively. These compounds were tested in two mouse models (3‐day binge model and prolonged Lieber DeCarli diet for 12 days) of early ALD. Serum alanine aminotransferase and liver histology were used to assess liver injury, Oil Red O staining of liver sections to assess steatosis, and real‐time polymerase chain reaction to assess changes in gene expression. In the ethanol binge model, treatment with OCA and INT‐777 decreased hepatic macrovesicular steatosis and protected from ethanol‐induced liver injury. After prolonged ethanol administration, mice treated with OCA, INT‐767, or INT‐777 showed decreased hepatic steatosis, associated with reduced liver fatty acid synthase protein expression, and protection from liver injury. Treatment with BA receptor agonists in both models of ethanol administration modulated lipogenic gene expression, and decreased liver interleukin‐1β mRNA expression associated with increased ubiquitination of NLRP3 inflammasome through cyclic adenosine monophosphate–induced activation of protein kinase A. Conclusion: OCA, INT‐767, or INT‐777 administration is effective in reducing acute and chronic ethanol‐induced steatosis and inflammation in mice, with varying degrees of efficacy depending on the duration of ethanol administration, indicating that both FXR and TGR5 activation can protect from liver injury in ALD models.

Abstract Background The end-organ effects of alcohol span throughout the entire body, from the gastrointestinal tract to the central nervous system (CNS). In the intestine, alcohol use changes the microbiome composition and increases gut permeability allowing translocation of microbial components into the circulation. Gut-derived pathogen-associated signals initiate inflammatory responses in the liver and possibly elsewhere in the body. Because previous studies showed that the gut microbiome contributes to alcohol-induced liver disease, we hypothesized that antibiotic administration to reduce the gut microbiome would attenuate alcohol-induced inflammation in the brain and small intestine (SI). Methods Six- to 8-week-old C57BL/6J female mice were fed alcohol in a liquid diet or a calorie-matched control diet for 10 days with an acute alcohol binge or sugar on the final day (acute-on-chronic alcohol administration). Some mice were treated with oral antibiotics daily to diminish the gut microbiome. We compared serum levels of TNFα, IL-6, and IL-1β by ELISA; expression of cytokines Tnfα, Mcp1, Hmgb1, Il-17, Il-23, Il-6, and Cox2; and inflammasome components Il-1β, Il-18, Casp1, Asc, and Nlrp3 in the CNS and SI by qRT-PCR. Microglial morphology was analyzed using immunohistochemical IBA1 staining in the cortex and hippocampus. Results Antibiotics dramatically reduced the gut microbiome load in both alcohol- and pair-fed mice. Alcohol-induced neuroinflammation and increase in SI cytokine expression were attenuated in mice with antibiotic treatment. Acute-on-chronic alcohol did not induce serum TNFα, IL-6, and IL-1β. Alcohol feeding significantly increased the expression of proinflammatory cytokines such as Tnfα, Mcp1, Hmgb1, Il-17, and Il-23 in the brain and intestine. Reduction in the gut bacterial load, as a result of antibiotic treatment, attenuated the expression of all of these alcohol-induced proinflammatory cytokines in both the brain and SI. Alcohol feeding resulted in microglia activation and morphologic changes in the cortex and hippocampus characterized by a reactive phenotype. These alcohol-induced changes were abrogated following an antibiotic-induced reduction in the gut microbiome. Unexpectedly, antibiotic treatment increased the mRNA expression of some inflammasome components in both the brain and intestine. Conclusions Our data show for the first time that the acute-on-chronic alcohol administration in mice induces both neuroinflammation and intestinal inflammation and that reduction in the intestinal bacterial load can attenuate alcohol-associated CNS and gut inflammation. Gut microbiome-derived signals contribute to neuroinflammation in acute-on-chronic alcohol exposure.

Introduction. Microvascular invasion (MVI) in hepatocellular carcinoma (HCC) patients is a poor prognostic factor after liver transplantation and/or resection. Any correlation between MVI and segmental location of HCC has yet to be studied. Our aim is to evaluate the segmental location of HCC and any correlation with the presence of MVI, portal vein thrombosis (PVT) in explanted livers, and the recurrence of HCC after transplantation. Another objective of the study is to assess the treatment history (ablation or transarterial chemoembolization (TACE)) and size of the tumor with respect to the risk of MVI. Methods. A single center, retrospective chart review, including 98 HCC patients, aged 18 years and older who had liver transplantation in our institute between 2012 and 2017. We reviewed the radiological images of the HCC tumors, the pathological findings of the explanted livers, and the follow-up imaging after transplantation. Results. 98 patients with the diagnosis of HCC underwent liver transplantation between 2012 and 2017. The mean age of the cohort was 63 ± 8.2. Males represented 75% and Caucasian race represented 75% of the cohort. The most common etiology of cirrhosis was chronic hepatitis C virus infection followed by alcohol abuse and nonalcoholic steatohepatitis (NASH) with percentages of 50%, 23%, and 10%, respectively. Microvascular invasion was found in 16% of the patients while PVT and the recurrence of HCC were found in 17% and 6 % of the cohort, respectively. MVI was found in 10 single HCC and 6 multifocal HCC. Right lobe HCC had more MVI when compared to the left and multilobar HCC, with percentages of 11%, 2%, and 3%, respectively. Localization of HCC in segment 8 was associated with the highest percentage of MVI when compared to all other segments. The risk of MVI in segment 8 HCC was 3.5 times higher than the risk from the other segments (p=0.002) while no vascular invasion was found in segments 1, 3, and 5. The risk of vascular invasion in untreated HCC is 3 times the risk in treated HCC (P=0.03). Conclusion. Our data indicate that the risk of microvascular invasion is highest in tumors localized to segment 8. The size and number of HCC tumors were not associated with an increased risk of microvascular invasion.

[This corrects the article DOI: 10.1371/journal.pone.0174544.].

BACKGROUND:Alcohol-induced intestinal dysbiosis disrupts homeostatic gut-liver axis function and is essential in the development of alcoholic liver disease. Here, we investigate changes in enteric microbiome composition in a model of early alcoholic steatohepatitis and dissect the pathogenic role of intestinal microbes in alcohol-induced liver pathology. MATERIALS AND METHODS:Wild type mice received a 10-day diet that was either 5% alcohol-containing or an isocaloric control diet plus a single binge. 16S rDNA sequencing defined the bacterial communities in the cecum of alcohol- and pair-fed animals. Some mice were treated with an antibiotic cocktail prior to and throughout alcohol feeding. Liver neutrophils, cytokines and steatosis were evaluated. RESULTS:Acute-on-chronic alcohol administration induced shifts in various bacterial phyla in the cecum, including increased Actinobacteria and a reduction in Verrucomicrobia driven entirely by a reduction in the genus Akkermansia. Antibiotic treatment reduced the gut bacterial load and circulating bacterial wall component lipopolysaccharide (LPS). We found that bacterial load suppression prevented alcohol-related increases in the number of myeloperoxidase- (MPO) positive infiltrating neutrophils in the liver. Expression of liver mRNA tumor necrosis factor alpha (Tnfα), C-X-C motif chemokine ligand 1 (Cxcl1) and circulating protein monocyte chemoattractant protein-1 (MCP-1) were also reduced in antibiotic-treated alcohol-fed mice. Alcohol-induced hepatic steatosis measured by Oil-Red O staining was significantly reduced in antibiotic treated mice. Genes regulating lipid production and storage were also altered by alcohol and antibiotic treatment. Interestingly, antibiotic treatment did not protect from alcohol-induced increases in serum aminotransferases (ALT/AST). CONCLUSIONS:Our data indicate that acute-on-chronic alcohol feeding alters the microflora at multiple taxonomic levels and identifies loss of Akkermansia as an early marker of alcohol-induced gut dysbiosis. We conclude that gut microbes influence liver inflammation, neutrophil infiltration and liver steatosis following alcohol consumption and these data further emphasize the role of the gut-liver axis in early alcoholic liver disease.

Background & Aims: Monocyte and macrophage (MΦ) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MΦs and monocytes recruited as precursors of MΦs into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MΦs in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MΦs. Methods: Healthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MΦ markers and cytokine levels. A similar analysis was performed on circulating monocytes and liver MΦs from HCV-infected patients and controls. Results: Huh7.5/JFH-1 cells induced monocytes to differentiate into MΦs with increased expression of CD14 and CD68. HCV-MΦs showed M2 surface markers (CD206, CD163, and Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)) and produced both proinflammatory and anti-inflammatory cytokines. HCV-induced early interleukin 1β production promoted transforming growth factor (TGF)β production and MΦ polarization to an M2 phenotype. TGF-β secreted by M2-MΦ led to hepatic stellate cell activation indicated by increased expression of collagen, tissue inhibitor of metalloproteinase 1, and α-smooth muscle actin. In vivo, we observed a significant increase in M2 marker (CD206) expression on circulating monocytes and in the liver of chronic HCV-infected patients. Furthermore, we observed the presence of a unique collagen-expressing CD14+CD206+ monocyte population in HCV patients that correlated with liver fibrosis. Conclusions: We show an important role for HCV in induction of monocyte differentiation into MΦs with a mixed M1/M2 cytokine profile and M2 surface phenotype that promote stellate cell activation via TGF-β. We also identified circulating monocytes expressing M2 marker and collagen in chronic HCV infection that can be explored as a biomarker. Keywords: Collagen, Fibrocytes, CD206, Biomarkers

BACKGROUND & AIM:MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis. METHODS:Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed. RESULTS:MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice. CONCLUSIONS:Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis.

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.

Binge drinking, the most common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its biological consequences are poorly defined. Previous studies demonstrated that chronic alcohol use results in increased gut permeability and increased serum endotoxin levels that contribute to many of the biological effects of chronic alcohol, including alcoholic liver disease. In this study, we evaluated the effects of acute binge drinking in healthy adults on serum endotoxin levels. We found that acute alcohol binge resulted in a rapid increase in serum endotoxin and 16S rDNA, a marker of bacterial translocation from the gut. Compared to men, women had higher blood alcohol and circulating endotoxin levels. In addition, alcohol binge caused a prolonged increase in acute phase protein levels in the systemic circulation. The biological significance of the in vivo endotoxin elevation was underscored by increased levels of inflammatory cytokines, TNFα and IL-6, and chemokine, MCP-1, measured in total blood after in vitro lipopolysaccharide stimulation. Our findings indicate that even a single alcohol binge results in increased serum endotoxin levels likely due to translocation of gut bacterial products and disturbs innate immune responses that can contribute to the deleterious effects of binge drinking.

Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.

BACKGROUND & AIMS:Hepatitis C virus (HCV) is difficult to eradicate and type III interferons (IFN-λ, composed of IL-28A, IL-28B and IL-29) are novel therapeutic candidates. We hypothesized that IFN-λ have immunomodulatory effects in HCV- infected individuals. MATERIALS AND METHODS:We analyzed the expression of IFN-λ and its receptor (composed of IL-10R2 and IFN-λR subunits) in the blood and livers of patients with chronic (c)HCV infection compared to controls (those who cleared HCV by sustained virological response, SVR, and those with liver inflammation of non-viral origin, non-alcoholic steatohepatitis, NASH). We also compared the proliferative capacity of dendritic cells (DCs) obtained from healthy individuals and those with chronic HCV using a mixed leukocyte reaction combined with 3H-Td incorporation. In addition, the composition of the IFN-λ receptor (IFN-λR) on myeloid DCs, plasmacytoid DCs, PBMCs, and T cells was determined by FACS analysis. RESULTS:We report that the expression of IFN-λ protein in serum and mRNA in liver is increased in cHCV patients, but not in those with HCV SVR or NASH, compared to controls. Liver level of IFN-λR mirrored the expression of serum IFN-λ and was higher in cHCV, compared to controls and HCV-SVR patients, suggesting that elevation of IFN-λ and IFN-λR are HCV-dependent. We further identified that innate immune cell populations expressed complete IFN-λ receptor. In vitro, recombinant IFN-λ promoted differentiation of monocyte-derived dendritic cells (DCs) into a phenotype with low T cell stimulatory capacity and high PD-L1 expression, which further promoted expansion of existing regulatory T cells. IFN-λ-DCs failed to induce de novo generation of regulatory T cells. The inhibitory capacity of IFN-λ-DCs was counteracted by recombinant IL-12 and by neutralization of the PD-1/PD-L1 system. CONCLUSIONS:Our novel findings of the immunomodulatory effect of IFN-λ contribute to the understanding of the anti-inflammatory and/or anti-viral potential of IFN-λ in cHCV.

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

Alcoholic liver disease (ALD) is a major global health problem. Chronic alcohol use results in inflammation and fatty liver, and in some cases, it leads to fibrosis and cirrhosis or hepatocellular carcinoma. Increased proinflammatory cytokines, particularly TNF alpha, play a central role in the pathogenesis of ALD. TNF alpha is tightly regulated at transcriptional and posttranscriptional levels. Recently, microRNAs (miRNAs) have been shown to modulate gene functions. The role of miRNAs in ALD is getting attention, and recent studies suggest that alcohol modulates miRNAs. Recently, we showed that alcohol induces miR-155 expression both in vitro (RAW 264.7 macrophage) and in vivo (Kupffer cells, KCs of alcohol-fed mice). Induction of miR-155 contributed to increased TNF alpha production and to the sensitization of KCs to produce more TNF alpha in response to LPS. In this paper, we summarize the current knowledge of miRNAs in ALD and also report increased expression of miR-155 and miR-132 in the total liver as well as in isolated hepatocytes and KCs of alcohol-fed mice. Our novel finding of the alcohol-induced increase of miRNAs in hepatocytes and KCs after alcohol feeding provides further insight into the evolving knowledge regarding the role of miRNAs in ALD.

In the multifactorial pathophysiology of alcoholic liver disease (ALD), inflammatory cascade activation plays a central role. Recent studies demonstrated that Toll-like Receptors, the sensors of microbial and endogenous danger signals, are expressed and activated in innate immune cells as well as in parenchymal cells in the liver and thereby contribute to ALD. In this paper, we discuss the importance of gut-derived endotoxin and its recognition by TLR4. The significance of TLR-induced intracellular signaling pathways and cytokine production as well as the contribution of reactive oxygen radicals is evaluated. The contribution of TLR signaling to induction of liver fibrosis and hepatocellular cancer is reviewed in the context of alcohol-induced liver disease.