Deficiency of miR-208a Exacerbates CCl4-Induced Acute Liver Injury in Mice by Activating Cell Death Pathways.

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.

Dysregulated Autophagy and Lysosome Function Are Linked to Exosome Production by Micro-RNA 155 in Alcoholic Liver Disease.

Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3-II levels. Alcohol reduced autophagy flux in vivo in chloroquine-treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol-related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy-related protein 7 (Atg7), proteins involved in phagophore-autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal-associated membrane protein 1 (LAMP1) and lysosomal-associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro-RNA 155 (miR-155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR-155-deficient mice were protected from alcohol-induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down-regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol-induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR-155 as a mediator of alcohol-related regulation of autophagy and exosome production in hepatocytes and macrophages.

Biomarkers of Macrophage Activation and Immune Danger Signals Predict Clinical Outcomes in Alcoholic Hepatitis.

Although mortality due to acute alcoholic hepatitis (AH) correlates with Model for End-Stage Liver Disease (MELD) scores, biomarkers are critically needed to manage this disease. Increases in inflammatory markers and macrophage activation are associated with acute AH and could be potential biomarkers of clinical events and/or mortality. We enrolled 89 clinically diagnosed AH patients in four US academic medical centers. Plasma from AH patients had a significant increase in gut microbial translocation indicators (endotoxin, bacterial 16S ribosomal DNA) and host response indicators (soluble cluster of differentiation 14 [sCD14] and lipopolysaccharide binding protein [LBP]) compared to controls. Patient MELD score and Glasgow Alcoholic Hepatitis score (GAHS) correlated with endotoxin levels. AH patients also had a significant increase in high mobility group protein 1 (HMGB1), a sterile danger signal molecule, and osteopontin (OPN), a multifunctional phosphoprotein involved in neutrophil activation, compared to controls. Increased levels of OPN positively correlated with increasing MELD score, GAHS, and LBP levels. Consistent with these results, AH patients had significantly increased circulating levels of macrophage activation (sCD163 and sCD206) markers compared to healthy controls, and sCD163 and sCD206 significantly and positively correlated with OPN, HMGB1, and LBP levels as well as with MELD score and GAHS. These findings indicate a connection between microbial translocation, immune cell activation, and AH severity. Plasma sCD14, OPN, sCD163, and sCD206 levels were significantly higher in nonsurvivors than survivors. In multivariate regression models, we identified sCD14, sCD163, and OPN as independent predictors of 90-day mortality, infection, and organ failure development, respectively. Conclusion: Our study suggests that sCD14, LBP, OPN, sCD163, and sCD206 are biomarkers to indicate severity and predict clinical outcomes in AH.

Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen-1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol-related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi ) and reduced proinflammatory Ly6Chi MØ in livers of alcohol-fed mice. CVC increased liver T-cell numbers and attenuated Il-2 expression without an effect on CD69+ or CD25+ T-cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.

Macrophage-Specific Hypoxia-Inducible Factor-1α Contributes to Impaired Autophagic Flux in Nonalcoholic Steatohepatitis.

Inflammatory cell activation drives diverse cellular programming during hepatic diseases. Hypoxia-inducible factors (HIFs) have recently been identified as important regulators of immunity and inflammation. In nonalcoholic steatohepatitis (NASH), HIF-1α is upregulated in hepatocytes, where it induces steatosis; however, the role of HIF-1α in macrophages under metabolic stress has not been explored. In this study, we found increased HIF-1α levels in hepatic macrophages in methionine-choline-deficient (MCD) diet-fed mice and in macrophages of patients with NASH compared with controls. The HIF-1α increase was concomitant with elevated levels of autophagy markers BNIP3, Beclin-1, LC3-II, and p62 in both mouse and human macrophages. LysMCre HIFdPA fl/fl mice, which have HIF-1α levels stabilized in macrophages, showed higher steatosis and liver inflammation compared with HIFdPA fl/fl mice on MCD diet. In vitro and ex vivo experiments reveal that saturated fatty acid, palmitic acid (PA), both induces HIF-1α and impairs autophagic flux in macrophages. Using small interfering RNA-mediated knock-down and overexpression of HIF-1α in macrophages, we demonstrated that PA impairs autophagy via HIF-1α. We found that HIF-1α mediates NF-κB activation and MCP-1 production and that HIF-1α-mediated impairment of macrophage autophagy increases IL-1ß production, contributing to MCD diet-induced NASH. Conclusion: Palmitic acid impairs autophagy via HIF-1α activation in macrophages. HIF-1α and impaired autophagy are present in NASH in vivo in mouse macrophages and in human blood monocytes. We identified that HIF-1α activation and decreased autophagic flux stimulate inflammation in macrophages through upregulation of NF-κB activation. These results suggest that macrophage activation in NASH involves a complex interplay between HIF-1α and autophagy as these pathways promote proinflammatory overactivation in MCD diet-induced NASH.

FXR and TGR5 Agonists Ameliorate Liver Injury, Steatosis, and Inflammation After Binge or Prolonged Alcohol Feeding in Mice.

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-1ß 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.

Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use.

BACKGROUND & AIMS: Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking. METHODS: Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion. RESULTS: Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice. CONCLUSIONS: Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis. LAY SUMMARY: Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

Extracellular vesicles from mice with alcoholic liver disease carry a distinct protein cargo and induce macrophage activation through heat shock protein 90.

A salient feature of alcoholic liver disease (ALD) is Kupffer cell (KC) activation and recruitment of inflammatory monocytes and macrophages (MØs). These key cellular events of ALD pathogenesis may be mediated by extracellular vesicles (EVs). EVs transfer biomaterials, including proteins and microRNAs, and have recently emerged as important effectors of intercellular communication. We hypothesized that circulating EVs from mice with ALD have a protein cargo characteristic of the disease and mediate biological effects by activating immune cells. The total number of circulating EVs was increased in mice with ALD compared to pair-fed controls. Mass spectrometric analysis of circulating EVs revealed a distinct signature for proteins involved in inflammatory responses, cellular development, and cellular movement between ALD EVs and control EVs. We also identified uniquely important proteins in ALD EVs that were not present in control EVs. When ALD EVs were injected intravenously into alcohol-naive mice, we found evidence of uptake of ALD EVs in recipient livers in hepatocytes and MØs. Hepatocytes isolated from mice after transfer of ALD EVs, but not control EVs, showed increased monocyte chemoattractant protein 1 mRNA and protein expression, suggesting a biological effect of ALD EVs. Compared to control EV recipient mice, ALD EV recipient mice had increased numbers of F4/80hi cluster of differentiation 11b (CD11b)lo KCs and increased percentages of tumor necrosis factor alpha-positive/interleukin 12/23-positive (inflammatory/M1) KCs and infiltrating monocytes (F4/80int CD11bhi ), while the percentage of CD206+ CD163+ (anti-inflammatory/M2) KCs was decreased. In vitro, ALD EVs increased tumor necrosis factor alpha and interleukin-1ß production in MØs and reduced CD163 and CD206 expression. We identified heat shock protein 90 in ALD EVs as the mediator of ALD-EV-induced MØ activation. CONCLUSION: Our study indicates a specific protein signature of ALD EVs and demonstrates a functional role of circulating EVs containing heat shock protein 90 in mediating KC/MØ activation in the liver. (Hepatology 2018;67:1986-2000).

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease.

BACKGROUND & AIMS: Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. METHODS: We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells. RESULTS: Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with mice given a control vector. Levels of hypoxia-inducible factor 1 alpha (HIF1α) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1α developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. CONCLUSIONS: Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.

Correction: Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice.

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

Alcohol-induced miR-155 and HDAC11 inhibit negative regulators of the TLR4 pathway and lead to increased LPS responsiveness of Kupffer cells in alcoholic liver disease.

Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut-derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR-155, an inflammatory miRNA in isolated KCs. We hypothesized that miR-155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol-fed mice showed a decrease in IRAK-M, SHIP1, and PU.1, and an increase in TNF-α levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR-155 deficiency on LPS responsiveness, as KCs that were isolated from miR-155 KO mice showed a greater induction of IRAK-M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPß, a validated miR-155 target, stimulates IL-10 transcription. We found a higher induction of C/EBPß and IL-10 in KCs that were isolated from miR-155 KO mice after LPS treatment. Gain- and loss-of-function studies affirmed that alcohol-induced miR-155 directly regulates IRAK-M, SHIP1, suppressor of cytokine signaling 1, and C/EBPß, as miR-155 inhibition increased and miR-155 overexpression decreased these genes in LPS or alcohol-pretreated wild-type KCs. HDAC11, a regulator of IL-10, was significantly increased and IL-10 was decreased in KCs that were isolated from alcohol-fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL-10 increase in LPS or alcohol-pretreated Mϕ. We found that acetaldehyde and NF-κB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol-induced responsiveness of KCs to LPS, in part, is governed by miR-155 and HDAC11.

Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice.

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.

Interleukin-1 inhibition facilitates recovery from liver injury and promotes regeneration of hepatocytes in alcoholic hepatitis in mice.

BACKGROUND & AIMS: Inflammation and impaired hepatocyte regeneration contribute to liver failure in alcoholic hepatitis (AH). Interleukin (IL)-1 is a key inflammatory cytokine in the pathobiology of AH. The role of IL-1 in liver regeneration in the recovery phase of alcohol-induced liver injury is unknown. METHODS: In this study, we tested IL-1 receptor antagonist to block IL-1 signalling in a mouse model of acute-on-chronic liver injury on liver inflammation and hepatocyte regeneration in AH. RESULTS: We observed that inhibition of IL-1 signalling decreased liver inflammation and neutrophil infiltration, and resulted in enhanced regeneration of hepatocytes and increased rate of recovery from liver injury in AH. CONCLUSION: Our novel findings suggest that IL-1 drives sustained liver inflammation and impaired hepatocyte regeneration even after cessation of ethanol exposure.

Circulating and Exosome-Packaged Hepatitis C Single-Stranded RNA Induce Monocyte Differentiation via TLR7/8 to Polarized Macrophages and Fibrocytes.

Monocytes and macrophages (MΦs) play a central role in the pathogenesis of chronic hepatitis C virus (HCV) infection. The tissue microenvironment triggers monocyte differentiation into MΦs, with polarization ranging within the spectrum of M1 (classical) to M2 (alternative) activation. Recently, we demonstrated that HCV infection leads to monocyte differentiation into polarized MΦs that mediate stellate cell activation via TGF-ß. In this study, we aimed to identify the viral factor(s) that mediate monocyte-to-MΦ differentiation. We performed coculture experiments using healthy monocytes with exosome-packaged HCV, cell-free HCV, or HCV ssRNA. Coculture of monocytes with exosome-packaged HCV, cell-free HCV, or HCV ssRNA induced differentiation into MΦs with high M2 surface marker expression and production of pro- and anti-inflammatory cytokines. The HCV ssRNA-induced monocyte activation and differentiation into MΦs could be prevented by TLR7 or TLR8 knockdown. Furthermore, TLR7 or TLR8 stimulation, independent of HCV, caused monocyte differentiation and M2 MΦ polarization. In vivo, in chronic HCV-infected patients, we found increased expression of TLR7/8 in circulating monocytes that was associated with increased intracellular expression of procollagen. Furthermore, knockdown of TLR8 completely attenuated collagen expression in monocytes exposed to HCV, and knockdown of TLR7 partially attenuated this expression, suggesting roles for TLR7/8 in induction of fibrocytes in HCV infection. We identified TLR7/8 as mediators of monocyte differentiation and M2 MΦ polarization during HCV infection. Further, we demonstrated that HCV ssRNA and other TLR7/8 ligands promote MΦ polarization and generation of circulating fibrocytes.

Hepatocellular carcinoma is accelerated by NASH involving M2 macrophage polarization mediated by hif-1αinduced IL-10.

Obesity-related inflammation promotes cancer development. Tissue resident macrophages affect tumor progression and the tumor micro-environment favors polarization into alternatively activated macrophages (M2) that facilitate tumor invasiveness. Here, we dissected the role of western diet-induced NASH in inducing macrophage polarization in a carcinogen initiated model of hepatocellular carcinoma (HCC). Adult C57BL/6 male mice received diethyl nitrosamine (DEN) followed by 24 weeks of high fat-high cholesterol-high sugar diet (HF-HC-HSD). We assessed liver MRI and histology, serum ALT, AFP, liver triglycerides, and cytokines. Macrophage polarization was determined by IL-12/TNFα (M1) and CD163/CD206 (M2) expression using flow cytometry. Role of hif-1α-induced IL-10 was dissected in hepatocyte specific hif-1αKO and hif-1αdPA (over-expression) mice. The western diet-induced features of NASH and accelerated HCC development after carcinogen exposure. Liver fibrosis and serum AFP were significantly increased in DEN + HF-HC-HSD mice compared to controls. Western diet resulted in macrophage (F4/80+CD11b+) infiltration to liver and DEN + HF-HC-HSD mice showed preferential increase in M2 macrophages. Isolated hepatocytes from western diet fed mice showed significant upregulation of the hypoxia-inducible transcription factor, hif-1α, and livers from hif-1α over-expressing mice had increased proportion of M2 macrophages. Primary hepatocytes from wild-type mice treated with DEN and palmitic acid in vitro showed activation of hif-1α and induction of IL-10, a M2 polarizing cytokine. IL-10 neutralization in hepatocyte-derived culture supernatant prevented M2 macrophage polarization and silencing hif-1α in macrophages blocked their M2 polarization. Therefore, our data demonstrate that NASH accelerates HCC progression via upregulation of hif-1α mediated IL-10 polarizing M2 macrophages.

Endoplasmic Reticulum Stress-induced Hepatocellular Death Pathways Mediate Liver Injury and Fibrosis via Stimulator of Interferon Genes.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-ß (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4 Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4 In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease.

UNLABELLED: The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol-induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal-regulated kinase 1 and 2-mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol-induced hepatic steatosis and interferon regulatory factor 3-mediated apoptosis. CONCLUSION: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell-driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis. (Hepatology 2016;64:1057-1071).

Therapeutic Benefits of Spleen Tyrosine Kinase Inhibitor Administration on Binge Drinking-Induced Alcoholic Liver Injury, Steatosis, and Inflammation in Mice.

BACKGROUND: Binge drinking is increasingly recognized as an important cause of liver disease with limited therapeutic options for patients. Binge alcohol use, similar to chronic alcohol consumption, induces numerous deregulated signaling events that drive liver damage, steatosis, and inflammation. In this article, we evaluated the role of spleen tyrosine kinase (SYK), which modulates numerous signaling events previously identified linked in the development alcohol-induced liver pathology. METHODS: A 3-day alcohol binge was administered to C57BL/6 female mice, and features of alcoholic liver disease were assessed. Some mice were treated daily with intraperitoneal injections of a SYK inhibitor (R406; 5 to 10 mg/kg body weight) or drug vehicle control. Liver and serum samples were collected and were assessed by Western blotting, biochemical, ELISA, electrophoretic mobility shift assays, real-time quantitative polymerase chain reaction, and histopathological analysis. RESULTS: We found that binge drinking induced significant SYK activation (SYK(Y525/526) ) with no change in total SYK expression in the liver. Functional inhibition of SYK activation using a potent SYK inhibitor, R406, was associated with a significant decrease in alcohol-induced hepatic inflammation as demonstrated by decreased phospho-nuclear factor kappa beta (NF-κB) p65, NF-κB nuclear binding, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 mRNA in the liver. Compared to vehicle controls, SYK inhibitor treatment decreased alcohol binge-induced hepatocyte injury indicated by histology and serum alanine aminotransferase. Strikingly, SYK inhibitor treatment also resulted in a significant reduction in alcohol-induced liver steatosis. CONCLUSIONS: Our novel observations demonstrate the role of SYK, activation in the pathomechanism of binge drinking-induced liver disease highlighting SYK a potential multifaceted therapeutic target.

The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD. METHODS: Wild-type (WT) (C57/BL6J) or miR-155 knockout (KO) and TLR4 KO mice received Lieber DeCarli diet for 5weeks. Some mice received corn oil or CCl4 for 2 or 9weeks. RESULTS: We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased peroxisome proliferator-activated receptor response element (PPRE) and peroxisome proliferator-activated receptors (PPAR)α (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARγ expression in naïve and alcohol treated RAW macrophages. Alcohol increased lipid metabolism gene expression (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet caused an increase in the number of CD163(+) CD206(+) infiltrating macrophages and neutrophils in WT mice, which was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPß. Pro-fibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT mice, attenuation in CCl4 induced hydroxyproline and α-SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet. CONCLUSIONS: Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages.

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFß and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages.