Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations.

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-1ß, 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.

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.

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.

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.

Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression.

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-1ß by ELISA; expression of cytokines Tnfα, Mcp1, Hmgb1, Il-17, Il-23, Il-6, and Cox2; and inflammasome components Il-1ß, 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-1ß. 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.

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.

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.

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.

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.

Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease.

AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD). METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1ß/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA). RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice. CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC.

Alcoholic hepatitis accelerates early hepatobiliary cancer by increasing stemness and miR-122-mediated HIF-1α activation.

Alcohol-related hepatocellular carcinoma (HCC) develops with advanced alcoholic liver disease and liver fibrosis. Using adult mice, we evaluate the effect of alcoholic steatohepatitis on early hepatobiliary carcinoma after initiation by diethyl-nitrosamine (DEN). Here we show that alcohol-fed DEN-injected mice have higher ALT and liver-to-body weight ratio compared to pair-fed DEN-injected mice. Alcohol feeding results in steatohepatitis indicated by increased pro-inflammatory cytokines and fibrotic genes. MRI and liver histology of alcohol+DEN mice shows hepatobiliary cysts, early hepatic neoplasia and increase in serum alpha-fetoprotein. Proliferation makers (BrdU, cyclin D1, p53) and cancer stem cell markers (CD133 and nanog) are significantly up-regulated in livers of alcohol-fed DEN-injected mice compared to controls. In livers with tumors, loss of miR-122 expression with a significant up-regulation of miR-122 target HIF-1α is seen. We conclude that alcoholic steatohepatitis accelerates hepatobiliary tumors with characteristic molecular features of HCC by up-regulating inflammation, cell proliferation, stemness, and miR-122 loss.

Progression of non-alcoholic steatosis to steatohepatitis and fibrosis parallels cumulative accumulation of danger signals that promote inflammation and liver tumors in a high fat-cholesterol-sugar diet model in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is becoming a pandemic. While multiple 'hits' have been reported to contribute to NAFLD progression to non-alcoholic steatohepatitis (NASH), fibrosis and liver cancer, understanding the natural history of the specific molecular signals leading to hepatocyte damage, inflammation and fibrosis, is hampered by the lack of suitable animal models that reproduce disease progression in humans. The purpose of this study was first, to develop a mouse model that closely mimics progressive NAFLD covering the spectrum of immune, metabolic and histopathologic abnormalities present in human disease; and second, to characterize the temporal relationship between sterile/exogenous danger signals, inflammation, inflammasome activation and NAFLD progression. METHODS: Male C57Bl/6 mice were fed a high fat diet with high cholesterol and a high sugar supplement (HF-HC-HSD) for 8, 27, and 49 weeks and the extent of steatosis, liver inflammation, fibrosis and tumor development were evaluated at each time point. RESULTS: The HF-HC-HSD resulted in liver steatosis at 8 weeks, progressing to steatohepatitis and early fibrosis at 27 weeks, and steatohepatitis, fibrosis, and tumor development at 49 weeks compared to chow diet. Steatohepatitis was characterized by increased levels of MCP-1, TNFα, IL-1ß and increased liver NASH histological score. We found increased serum levels of sterile danger signals, uric acid and HMGB1, as early as 8 weeks, while endotoxin and ATP levels increased only after 49 weeks. Increased levels of these sterile and microbial danger signals paralleled upregulation and activation of the multiprotein complex inflammasome. At 27, 49 weeks of HF-HC-HSD, activation of M1 macrophages and loss of M2 macrophages as well as liver fibrosis were present. Finally, similar to human NASH, liver tumors occurred in 41% of mice in the absence of cirrhosis and livers expressed increased p53 and detectable AFP. CONCLUSIONS: HF-HC-HSD over 49 weeks induces the full spectrum of liver pathophysiologic changes that characterizes the progression of NAFLD in humans. NAFLD progression to NASH, fibrosis and liver tumor follows progressive accumulation of sterile and microbial danger signals, inflammasome activation, altered M1/M2 cell ratios that likely contribute to NASH progression and hepatic tumor formation.

Moderate alcohol induces stress proteins HSF1 and hsp70 and inhibits proinflammatory cytokines resulting in endotoxin tolerance.

Binge or moderate alcohol exposure impairs host defense and increases susceptibility to infection because of compromised innate immune responses. However, there is a lack of consensus on the molecular mechanism by which alcohol mediates this immunosuppression. In this study, we show that cellular stress proteins HSF1 and hsp70 play a mechanistic role in alcohol-mediated inhibition of the TLR4/MyD88 pathway. Alcohol exposure induced transcription factor HSF1 mRNA expression and DNA binding activity in primary human monocytes and murine macrophages. Furthermore, HSF1 target gene hsp70 mRNA and protein are upregulated by alcohol in monocytes. In vitro pre-exposure to moderate alcohol reduced subsequent LPS-induced NF-κB promoter activity and downstream TNF-α, IL-6 and IL-1ß production in monocytes and macrophages, exhibiting endotoxin tolerance. Mechanistic analysis demonstrates that alcohol-induced HSF1 binds to the TNF-α promoter in macrophages at early time points, exerting transrepression and decreased TNF-α expression. Furthermore, association of hsp70 with NF-κB subunit p50 in alcohol-treated macrophages correlates with reduced NF-κB activation at later time points. Hsp70 overexpression in macrophages was sufficient to block LPS-induced NF-κB promoter activity, suggesting alcohol-mediated immunosuppression by hsp70. The direct crosstalk of hsp70 and HSF1 was further confirmed by the loss of alcohol-mediated endotoxin tolerance in hsp70- and HSF1-silenced macrophages. Our data suggest that alcohol-mediated activation of HSF1 and induction of hsp70 inhibit TLR4-MyD88 signaling and are required for alcohol-induced endotoxin tolerance. Using stress proteins as direct drug targets would be clinically relevant in alcohol abuse treatment and may serve to provide a better understanding of alcohol-mediated immunosuppression.

Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.

BACKGROUND & AIMS: Heat shock protein 90 (hsp90) is an emerging therapeutic target in chronic liver diseases. Hsp90 plays an important role in liver immune cell activation; however its role in alcoholic liver disease (ALD) remains elusive. Here we hypothesize that hsp90 is crucial in alcohol induced steatosis and pro-inflammatory cytokine production. To test this hypothesis, we employed a pharmacological inhibitor of hsp90, 17-DMAG (17-Dimethylamino-ethylamino-17-demethoxygeldanamycin) in an in vivo mouse model of acute and chronic alcoholic liver injury. METHODS: C57BL/6 mice were given either a single dose of ethanol via oral gavage (acute) or chronically fed alcohol for 2 weeks followed by oral gavage (chronic-binge). 17-DMAG was administered during or at the end of feeding. Liver injury parameters, inflammatory cytokines and lipid metabolism genes were analysed. RESULTS: Our results reveal increased expression of hsp90 in human and mouse alcoholic livers. In vivo inhibition of hsp90, using 17-DMAG, not only prevented but also alleviated alcoholic liver injury, determined by lower serum ALT, AST and reduced hepatic triglycerides. Mechanistic analysis showed that 17-DMAG decreased alcohol mediated oxidative stress, reduced serum endotoxin, decreased inflammatory cells, and diminished sensitization of liver macrophages to LPS, resulting in downregulation of CD14, NFκB inhibition, and decreased pro-inflammatory cytokine production. Hsp90 inhibition decreased fatty acid synthesis genes via reduced nuclear SREBP-1 and favoured fatty acid oxidation genes via PPARα. CONCLUSIONS: Inhibition of hsp90 decreased alcohol induced steatosis and pro-inflammatory cytokines and inhibited alcoholic liver injury. Hsp90 is therefore relevant in human alcoholic cirrhosis and a promising therapeutic target in ALD.

Immunity and inflammatory signaling in alcoholic liver disease.

The pathogenesis of alcoholic liver disease (ALD) is multifactorial and characterized by steatosis, steatohepatitis and cirrhosis. Several signaling pathways in different liver cell types that contribute to the development and progression of alcoholic liver injury have been identified. Among these, immune cells and signaling pathways are the most prominent and central to ALD. Both innate and adaptive immune responses contribute to ALD. The key features of inflammatory pathways in ALD including liver innate and adaptive immune cell types, signaling receptors/pathways, and pro- and antiinflammatory/protective responses are summarized here.

Oxidative stress and inflammation: essential partners in alcoholic liver disease.

Alcoholic liver disease (ALD) is a multifaceted disease that is characterized by hepatic steatosis or fat deposition and hepatitis or inflammation. Over the past decade, multiple lines of evidence have emerged on the mechanisms associated with ALD. The key mechanisms identified so far are sensitization to gut-derived endotoxin/lipopolysaccharide resulting in proinflammatory cytokine production and cellular stress due to oxidative processes, contributing to the development and progression of disease. While oxidative stress and inflammatory responses are studied independently in ALD, mechanisms linking these two processes play a major role in pathogenesis of disease. Here we review major players of oxidative stress and inflammation and highlight signaling intermediates regulated by oxidative stress that provokes proinflammatory responses in alcoholic liver disease.