DNMT3b-mediated methylation of ZSWIM3 enhances inflammation in alcohol-induced liver injury via regulating TRAF2-mediated NF-κB pathway.

The regulation of macrophages during inflammatory responses is a crucial process in alcoholic liver disease (ALD) and aberrant macrophage DNA methylation is associated with inflammation. Our preliminary screening results of macrophage methylation in the present study demonstrated the zinc finger SWI2/SNF2 and MuDR (SWIM)-domain containing 3 (ZSWIM3) were hypermethylated in the 5' untranslated region (5'-UTR) region. ZSWIM3, a novel zinc finger-chelate domain of SWIM, is predicted to function in DNA-binding and protein-binding interactions. Its expression was found to be consistently decreased in macrophages isolated from livers of ethyl alcohol (EtOH)-fed mice and in EtOH+lipopolysaccharide (LPS)-induced RAW264.7 cells. Over-expression of ZSWIM3 was found to attenuate chronic+binge ethanol feeding-induced liver injury and inhibit inflammatory responses in vivo. Enforced expression of ZSWIM3 in vitro was also found to have anti-inflammatory effects. Aberrant expression of ZSWIM3 in alcohol-induced liver injury (ALI) was found to be associated with hypermethylation. Analysis of CpG prediction indicated the presence of two methylated sites in the ZSWIM3 promoter region and methylation inhibitor and DNA methyltransferases (DNMTs)-siRNA transfection were found to restore down-regulated ZSWIM3. Chromatin immunoprecipitation (ChIP) assay and molecular docking affirmed the role of DNMT 3b (DNMT3b) as a principal regulator of ZSWIM3 expression. Mechanistically, ZSWIM3 might affect inflammation by binding with tumor necrosis factor receptor-associated factor 2 (TRAF2), which further mediates the activation of the nuclear transcription factor κB (NF-κB) pathway. The present study, therefore, provides detailed insights into the possible structure and function of ZSWIM3 and thus, contributes new substantial research in the elucidation of the pathogenesis of ALI.

Differential Activation of Unconventional T Cells, Including iNKT Cells, in Alcohol-Related Liver Disease.

BACKGROUND: Liver is enriched in several innate-like unconventional T cells, but their role in alcohol-related liver disease (ALD) is not fully understood. Studies in several acute alcohol feeding models but not in chronic alcoholic steatohepatitis (ASH) model have shown that invariant natural killer T (iNKT) cells play a pathogenic role in ALD. Here, we investigated the activation of iNKT cells in an intragastric (iG) infusion model of chronic ASH as well as the frequency and cytokine phenotype of 3 different unconventional T cells: iNKT, mucosal-associated invariant T (MAIT), and CD8+ CD161hi Vα7.2- cells in peripheral blood of ALD patients. METHODS: Hepatic iNKT cells were investigated using the iG model of chronic ASH that combines feeding of high-cholesterol/high-fat diet (HCFD) with intragastric feeding of ethanol diet (HCFD + iG Alc). Human iNKT, MAIT, and CD8+ CD161hi Vα7.2- cells were examined by flow cytometry in peripheral blood of patients with severe alcoholic hepatitis (SAH) and chronic alcoholics (ChA) and compared with healthy controls. RESULTS: In the iG model of chronic ASH, IFNγ+ iNKT cells accumulate in their livers compared with pair-fed control mice and activated hepatic iNKT cells show high expression of Fas and FasL. Notably, IFNγ+ iNKT cells are also significantly increased in peripheral blood of ChA patients compared with SAH patients. MAIT cells are significantly reduced in all ALD patients, but CD8+ CD161hi Vα7.2- cells are increased in SAH patients. Although MAIT and CD8+ CD161hi Vα7.2- cells displayed a similar cytokine production profile, the production of IFNγ and TNFα is significantly increased in SAH patients, while significant IL-17A production is found in ChA patients. CONCLUSIONS: We found that the 3 unconventional T cells are activated in ALD patients showing interesting differences in their frequency and cytokine production profile between SAH and ChA patients. In the iG murine model of chronic ASH, iNKT cells are also activated secreting proinflammatory cytokines suggesting their involvement in liver disease.

TMEM88 modulates the secretion of inflammatory factors by regulating YAP signaling pathway in alcoholic liver disease.

OBJECTIVE: Transmembrane protein 88 (TMEM88), a new protein of increasing concern existed in cell membrane, inhibits the typical Wnt/ß-catenin signaling pathway to play a regulatory role on cell proliferation by binding to Dishevelled-1. Until recently, the connection between TMEM88 and alcoholic liver disease is unknown. In this research, we explored the effect of TMEM88 on the secretion of inflammatory cytokines in ethanol (EtOH)-induced RAW264.7 cells, moreover, the function of YAP signaling pathway in EtOH-induced RAW264.7 cells were investigated. METHODS: We administered TMEM88 adenovirus (ADV-TMEM88) by tail vein injection into C57BL/6J mice in vivo. In vitro, RAW264.7 murine macrophages were stimulated with EtOH and were transfected with pEGFP-C1-TMEM88 and TMEM88 siRNA, respectively, protein expression and mRNA expression of IL-6 and IL-1ß were assessed by Western Blotting and RT-qPCR, respectively. RESULTS: Our group found that the overexpression of TMEM88 led to an up-regulation of IL-6 and IL-1ß secretion, hinting that it had the possibility of linking with the initiation, the development, and the end of inflammation. In addition to that, TMEM88 silencing reduced the secretion of IL-6 and IL-1ß in RAW264.7 cells. Moreover, we demonstrated that the YAP signaling pathway under the action of EtOH was activated by TMEM88. CONCLUSIONS: All in all, these experimental outcomes indicated that TMEM88 had an indispensable impact on EtOH-induced secretion of inflammatory cytokines (IL-6 and IL-1ß) in RAW264.7 cells through YAP signaling pathway.

Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice.

OBJECTIVE: Antimicrobial C-type lectin regenerating islet-derived 3 gamma (REG3G) is suppressed in the small intestine during chronic ethanol feeding. Our aim was to determine the mechanism that underlies REG3G suppression during experimental alcoholic liver disease. DESIGN: Interleukin 22 (IL-22) regulates expression of REG3G. Therefore, we investigated the role of IL-22 in mice subjected to chronic-binge ethanol feeding (NIAAA model). RESULTS: In a mouse model of alcoholic liver disease, we found that type 3 innate lymphoid cells produce lower levels of IL-22. Reduced IL-22 production was the result of ethanol-induced dysbiosis and lower intestinal levels of indole-3-acetic acid (IAA), a microbiota-derived ligand of the aryl hydrocarbon receptor (AHR), which regulates expression of IL-22. Importantly, faecal levels of IAA were also found to be lower in patients with alcoholic hepatitis compared with healthy controls. Supplementation to restore intestinal levels of IAA protected mice from ethanol-induced steatohepatitis by inducing intestinal expression of IL-22 and REG3G, which prevented translocation of bacteria to liver. We engineered Lactobacillus reuteri to produce IL-22 (L. reuteri/IL-22) and fed them to mice along with the ethanol diet; these mice had reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain and upregulated expression of REG3G in intestine. However, L. reuteri/IL-22 did not reduce ethanol-induced liver disease in Reg3g-/- mice. CONCLUSION: Ethanol-associated dysbiosis reduces levels of IAA and activation of the AHR to decrease expression of IL-22 in the intestine, leading to reduced expression of REG3G; this results in bacterial translocation to the liver and steatohepatitis. Bacteria engineered to produce IL-22 induce expression of REG3G to reduce ethanol-induced steatohepatitis.

Protective Effects of Fucoxanthin against Alcoholic Liver Injury by Activation of Nrf2-Mediated Antioxidant Defense and Inhibition of TLR4-Mediated Inflammation.

Fucoxanthin (Fx) is a natural extract from marine seaweed that has strong antioxidant activity and a variety of other bioactive effects. This study elucidated the protective mechanism of Fx on alcoholic liver injury. Administration of Fx was associated with lower pathological effects in liver tissue and lower serum marker concentrations for liver damage induced by alcohol. Fx also alleviated oxidative stress, and lowered the level of oxides and inflammation in liver tissue. Results indicate that Fx attenuated alcohol-induced oxidative lesions and inflammatory responses by activating the nuclear factor erythrocyte-2-related factor 2 (Nrf2)-mediated signaling pathway and down-regulating the expression of the toll-like receptor 4 (TLR4)-mediated nuclear factor-kappa B (NF-κB) signaling pathway, respectively. Our findings suggest that Fx can be developed as a potential nutraceutical for preventing alcohol-induced liver injury in the future.

Alcohol or Gut Microbiota: Who Is the Guilty?

Alcoholic liver disease (ALD), a disorder caused by excessive alcohol intake represents a global health care burden. ALD encompasses a broad spectrum of hepatic injuries including asymptomatic steatosis, alcoholic steatohepatitis (ASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The susceptibility of alcoholic patients to develop ALD is highly variable and its progression to more advanced stages is strongly influenced by several hits (i.e., amount and duration of alcohol abuse). Among them, the intestinal microbiota and its metabolites have been recently identified as paramount in ALD pathophysiology. Ethanol abuse triggers qualitative and quantitative modifications in intestinal flora taxonomic composition, mucosal inflammation, and intestinal barrier derangement. Intestinal hypermeability results in the translocation of viable pathogenic bacteria, Gram-negative microbial products, and pro-inflammatory luminal metabolites into the bloodstream, further corroborating the alcohol-induced liver damage. Thus, the premise of this review is to discuss the beneficial effect of gut microbiota modulation as a novel therapeutic approach in ALD management.

Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease.

BACKGROUND/AIMS: Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown. METHODS/DESIGN: We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively. RESULTS: In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments. CONCLUSIONS: In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the 'leaky' gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

Liver tissue metabolically transformed by alcohol induces immune recognition of liver self-proteins but not in vivo inflammation.

Precision-cut liver slices (PCLSs) provide a novel model for studies of alcoholic liver disease (ALD). This is relevant, as in vivo ethanol exposure does not appear to generate significant liver damage in ethanol-fed mice, except in the National Institute on Alcohol Abuse and Alcoholism binge model of ALD. Previous studies have shown that the two metabolites of ethanol consumption, malondialdhyde (MDA) and acetaldehyde (AA), combine to form MDA-AA (MAA) adducts, which have been correlated with the development and progression of ALD. In this study, murine PCLSs were incubated with ethanol and examined for the production of MAA adducts. PCLSs were homogenized, and homogenates were injected into C57BL/6 mice. PCLSs from control-, pair-, and ethanol-fed animals served as targets in in situ cytotoxic assays using primed T cells from mice hyperimmunized with control or ethanol-exposed PCLS homogenates. A CD45.1/CD45.2 passive-transfer model was used to determine whether T cells from the spleens of mice hyperimmunized with PCLS ethanol-exposed homogenates trafficked to the liver. PCLSs incubated with ethanol generated MAA-modified proteins in situ. Cytotoxic (CD8+) T cells from immunized mice killed naïve PCLSs from control- and pair-fed mice in vitro, a response that was blunted in PCLSs from ethanol-fed mice. Furthermore, CD45.1 CD8+ T cells from hyperimmunized mice trafficked to the liver but did not initiate liver damage. This study demonstrates that exposure to liver tissue damaged by ethanol mediates robust immune responses to well-characterized alcohol metabolites and native liver proteins in vitro. Moreover, although these proinflammatory T cells traffic to the liver, these responses appear to be dampened in vivo by locally acting pathways. NEW & NOTEWORTHY This study shows that the metabolites of ethanol and lipid breakdown produce malondialdehyde-acetaldehyde adducts in the precision-cut liver slice model system. Additionally, precision-cut liver slices exposed to ethanol and harboring malondialdehyde-acetaldehyde adducts generate liver-specific antibody and T cell responses in the spleens of naïve mice that could traffic to the liver.

Galectin-9: Diverse roles in hepatic immune homeostasis and inflammation.

Glycan-binding proteins, which include galectins, are involved at all stages of immunity and inflammation, from initiation through resolution. Galectin-9 (Gal-9) is highly expressed in the liver and has a wide variety of biological functions in innate and adaptive immunity that are instrumental in the maintenance of hepatic homeostasis. In the setting of viral hepatitis, increased expression of Gal-9 drives the expansion of regulatory T cells and contraction of effector T cells, thereby favoring viral persistence. The dichotomous nature of Gal-9 is evident in hepatocellular carcinoma, where loss of expression in hepatocytes promotes tumor growth and metastasis, whereas overexpression by Kupffer cells and endothelial cells inhibits the antitumor immune response. In nonalcoholic fatty liver disease, Gal-9 is involved indirectly in the expansion of protective natural killer T-cell populations. In ischemic liver injury, hepatocyte-derived Gal-9 is both diagnostic and cytoprotective. In drug-induced acute liver failure, plasma levels correlate with outcome. Here, we offer a synthesis of recent and emerging findings on Gal-9 in the regulation of hepatic inflammation. Ongoing studies are warranted to better elucidate the pathophysiology of hepatic immune-mediated diseases and to develop new therapeutic interventions using glycan-binding proteins. (Hepatology 2017;66:271-279).

NLRP3 Inflammasome and IL-33: Novel Players in Sterile Liver Inflammation.

In sterile liver inflammation, danger signals are released in response to tissue injury to alert the immune system; e.g., by activation of the NLRP3 inflammasome. Recently, IL-33 has been identified as a novel type of danger signal or "alarmin", which is released from damaged and necrotic cells. IL-33 is a pleiotropic cytokine that targets a broad range of immune cells and exhibits pro- and anti-inflammatory properties dependent on the disease. This review summarizes the immunomodulatory roles of the NLRP3 inflammasome and IL-33 in sterile liver inflammation and highlights potential therapeutic strategies targeting these pathways in liver disease.

Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients.

BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD. METHODS: MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif-/- bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). RESULTS: HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif-/-→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif-/-) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. CONCLUSIONS: Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.

High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

UNLABELLED: Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRß chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRß nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. CONCLUSION: We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease-relevant T cells in order to better understand and treat liver disease.

Biological implications of selenium in adolescent rats exposed to binge drinking: Oxidative, immunologic and apoptotic balance.

Alcohol intermittent binge drinking (BD) during adolescence decreases the levels of selenium (Se), a trace element that plays a key biological role against oxidative damage in hepatocytes through different selenoproteins such as the antioxidant enzymes glutathione peroxidases (GPx1 and Gpx4) and selenoprotein P (SelP). In this context, it has been found that GPx4 has an essential antioxidant role in mitochondria modulating the apoptosis and NF-kB activation (a factor intimately related to apoptosis and immune function). To further investigate the effectiveness of selenium supplementation in oxidative balance, inflammation and apoptosis, the present study examined the protective effects of 0.4ppm of dietary selenite administrated to adolescent rats exposed to BD. BD consumption depleted Se deposits in all the tissues studied. In liver, GPx1 activity and expression were decreased leading to protein and lipid hepatic oxidation. Moreover GPx4 and NF-kB expression were also decreased in liver, coinciding with an increase in caspase-3 expression. This hepatic profile caused general liver damage as shown the increased serum transaminases ratio AST/ALT. Proinflammatory serum citokines and chemocines were decreased. Se supplementation therapy used restored all these values, even AST levels. These findings suggest for first time that Se supplementation is a good strategy against BD liver damage during adolescence, since it increases GPx1 and GPx4 expression and avoids NF-kB downregulation and caspase-3 upregulation, leading to a better oxidative, inflammatory and apoptotic liver profile. The therapy proposed could be considered to have a great biological efficacy and to be suitable for BD exposed teenagers in order to avoid future hepatic complications.

Peptide hormones and lipopeptides: from self-assembly to therapeutic applications.

This review describes the properties and activities of lipopeptides and peptide hormones and how the lipidation of peptide hormones could potentially produce therapeutic agents combating some of the most prevalent diseases and conditions. The self-assembly of these types of molecules is outlined, and how this can impact on bioactivity. Peptide hormones specific to the uptake of food and produced in the gastrointestinal tract are discussed in detail. The advantages of lipidated peptide hormones over natural peptide hormones are summarised, in terms of stability and renal clearance, with potential application as therapeutic agents. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Genetic and Epigenetic Profile of Patients With Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a definition encompassing a spectrum of disorders ranging from simple steatosis to cirrhosis and hepatocellular carcinoma. Excessive alcohol consumption triggers a series of metabolic reactions that affect the liver by inducing lipogenesis, increasing oxidative stress, and causing abnormal inflammatory responses. The metabolic pathways regulating lipids, reactive oxygen species (ROS), and immune system are closely related and in some cases cross-regulate each other. Therefore, it must be taken into account that major genetic and epigenetic abnormalities affecting enzymes involved in one of such pathways can play a pivotal role in ALD pathogenesis. However, recent studies have pointed out how a significant predisposition can also be determined by minor variants, such as relatively common polymorphisms, epigenetic modifications, and microRNA abnormalities. Genetic and epigenetic factors can also affect the progression of liver diseases, promoting fibrogenesis, cirrhosis, and ultimately hepatocellular carcinoma. It is noteworthy that some of these factors, such as some of the cytokines involved in the abnormal inflammatory responses, are shared with non-alcoholic liver disease, while other factors are unique to ALD. The study of the genetic and epigenetic components involved in the liver damages caused by alcohol is crucial to identify individuals with high risk of developing ALD, design personalized protocols for prevention and/or treatment, and select the best molecular targets for new therapies.

Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease.

BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is characterised by neutrophil infiltration that contributes to hepatic injury and disease. Lipocalin-2 (LCN2) was originally identified as siderophore binding peptide in neutrophils, which exerted tissue protective effects in several disease models. Here we investigate the role of LCN2 in the pathogenesis of alcohol-induced liver injury. METHODS: We compared hepatic LCN2 expression in ASH patients, alcoholic cirrhosis patients without evidence of ASH and patients with non-alcoholic fatty liver disease (NAFLD; i.e. simple steatosis). To mechanistically dissect LCN2 function in alcohol-induced liver injury, we subjected wild-type (WT) and Lcn2-deficient (Lcn2(-/-)) mice to the Lieber-DeCarli diet containing 5% ethanol (EtOH) or isocaloric maltose. Adoptive transfer experiments were performed to track neutrophil migration. Furthermore, we tested the effect of antibody-mediated LCN2 neutralisation in an acute model of ethanol-induced hepatic injury. RESULTS: Patients with ASH exhibited increased hepatic LCN2 immunoreactivity compared to patients with alcoholic cirrhosis or simple steatosis, which mainly localised to neutrophils. Similarly, ethanol-fed mice exhibited increased LCN2 expression that mainly localised to leukocytes and especially neutrophils. Lcn2(-/-) mice were protected from alcoholic liver disease (ALD) as demonstrated by reduced neutrophil infiltration, liver injury and hepatic steatosis compared to WT controls. Adoptive transfers revealed that neutrophil-derived LCN2 critically determines hepatic neutrophil immigration and persistence during chronic alcohol exposure. Antibody-mediated neutralisation of LCN2 protected from hepatic injury and neutrophilic infiltration after acute alcohol challenge. CONCLUSIONS: LCN2 drives ethanol-induced neutrophilic inflammation and propagates the development of ALD. Despite a critical role for LCN2 in immunity and infection, pharmacological neutralisation of LCN2 might be of promise in ALD.

Gut-liver axis in alcoholic liver disease.

Alcoholic liver disease (ALD) has been among the leading causes of cirrhosis and liver-related death worldwide for decades. Early discoveries in alcoholic liver disease identified increased levels of bacterial endotoxin in the portal circulation, suggesting a role for gut-derived toxins in ALD. Indeed, alcohol consumption can disrupt the intestinal epithelial barrier and result in increased gut permeability that increasingly is recognized as a major factor in ALD. Bacterial endotoxin, lipopolysaccharide, is a prototypic microbe-derived inflammatory signal that contributes to inflammation in ALD through activation of the Toll-like receptor 4. Recent studies also have shown that alcohol consumption is associated with alterations in the gut microbiome, and the dysbalance of pathogenic and commensal organisms in the intestinal microbiome may contribute to the abnormal gut-liver axis in ALD. Indeed, bacterial decontamination improves ALD both in human and animal models. This short review summarizes recent findings and highlights emerging trends in the gut-liver axis relevant to ALD.

Inhibition of type I natural killer T cells by retinoids or following sulfatide-mediated activation of type II natural killer T cells attenuates alcoholic liver disease in mice.

UNLABELLED: Innate immune mechanisms leading to liver injury subsequent to chronic alcohol ingestion are poorly understood. Natural killer T (NKT) cells, enriched in the liver and comprised of at least two distinct subsets, type I and II, recognize different lipid antigens presented by CD1d molecules. We have investigated whether differential activation of NKT cell subsets orchestrates inflammatory events leading to alcoholic liver disease (ALD). We found that after chronic plus binge feeding of Lieber-DeCarli liquid diet in male C57BL/6 mice, type I, but not type II, NKT cells are activated, leading to recruitment of inflammatory Gr-1(high) CD11b(+) cells into the liver. A central finding is that liver injury after alcohol feeding is dependent upon type I NKT cells. Thus, liver injury is significantly inhibited in Jα18(-/-) mice deficient in type I NKT cells as well as after their inactivation by sulfatide-mediated activation of type II NKT cells. Furthermore, we have identified a novel pathway involving all-trans retinoic acid (ATRA) and its receptor (RARγ) signaling that inhibits type I NKT cells and, consequently, ALD. A semiquantitative polymerase chain reaction analysis of hepatic gene expression of some of the key proinflammatory molecules shared in human disease indicated that their up-regulation in ALD is dependent upon type I NKT cells. CONCLUSIONS: Type I, but not type II, NKT cells become activated after alcohol feeding. Type I NKT cell-induced inflammation and neutrophil recruitment results in liver tissue damage whereas type II NKT cells protect from injury in ALD. Inhibition of type I NKT cells by retinoids or by sulfatide prevents ALD. Given that the CD1d pathway is highly conserved between mice and humans, NKT cell subsets might be targeted for potential therapeutic intervention in ALD.

STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease.

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis.

TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration.

Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.