Aditya Ambade

An essential role for monocyte chemoattractant protein‐1 in alcoholic liver injury: Regulation of proinflammatory cytokines and hepatic steatosis in mice

The importance of chemokines in alcoholic liver injury has been implicated. The role of the chemokine, monocyte chemoattractant protein‐1 (MCP‐1), elevated in patients with alcoholic liver disease is not yet understood. Here, we evaluated the pathophysiological significance of MCP‐1 and its receptor, chemokine (C‐C motif) receptor 2 (CCR2), in alcoholic liver injury. The Leiber‐DeCarli diet containing alcohol or isocaloric control diets were fed to wild‐type (WT) and MCP‐1‐deficient knockout (KO) mice for 6 weeks. In vivo and in vitro assays were performed to study the role of MCP‐1 in alcoholic liver injury. MCP‐1 was increased in Kupffer cells (KCs) as well as hepatocytes of alcohol‐fed mice. Alcohol feeding increased serum alanine aminotransferase in WT and CCR2KO, but not MCP‐1KO, mice. Alcohol‐induced liver steatosis and triglyceride were attenuated in alcohol‐fed MCP‐1KO, but high in CCR2KO mice, compared to WT, whereas serum endotoxin was high in alcohol‐fed WT and MCP‐1KO mice. Expression of liver proinflammatory cytokines tumor necrosis factor alpha, interleukin (IL)‐1β, IL‐6, KC/IL‐8, intercellular adhesion molecule 1, and cluster of differentiation 68 was induced in alcohol‐fed WT, but inhibited in MCP‐1KO, mice independent of nuclear factor kappa light‐chain enhancer of activated B cell activation in KCs. Oxidative stress, but not cytochrome P450 2E1, was prevented in chronic alcohol‐fed MCP‐1KO mice, compared to WT. Increased expression of peroxisome proliferator‐activated receptor (PPAR)α and PPARγ was accompanied by nuclear translocation, DNA binding, and induction of fatty acid metabolism genes acyl coenzyme A oxidase and carnitine palmitoyltransferase 1A in livers of alcohol‐fed MCP‐1KO mice, compared to WT controls. In vitro assays uncovered an inhibitory effect of recombinant MCP‐1 on PPARα messenger RNA and peroxisome proliferator response element binding in hepatocytes independent of CCR2. Conclusion: Deficiency of MCP‐1 protects mice against alcoholic liver injury, independent of CCR2, by inhibition of proinflammatory cytokines and induction of genes related to fatty acid oxidation, linking chemokines to hepatic lipid metabolism. (HEPATOLOGY 2011)

Cutting Edge: Necrosis Activates the NLRP3 Inflammasome

Cells undergoing necrosis release endogenous danger signals that possess proinflammatory potential. In this study we show that mature IL-1β and IL-18 are released by necrotic cells but not by apoptotic cells. We identify 7-bromoindirubin-3′-oxime, an indirubin oxime derivative that induces necrosis, as a potent inducer of caspase-1 activation and release of mature IL-1β and IL-18. Inflammasome activation was triggered by other necrosis-inducing treatments but was not observed in response to apoptosis-inducing stimuli. Necrosis-induced inflammasome activation was mediated by the NLRP3 and ASC molecules. Release of IL-18 and IL-1β in response to necrosis-inducing stimuli was observed in THP-1 macrophages and the MSTO-211H human mesothelioma cell line independently of LPS priming. Using the in vivo model of naphthalene-induced airway epithelial cell injury, we showed that necrosis activates the ASC inflammasome in vivo. Our study identifies a new mechanism through which necrosis generates proinflammatory molecules that contributes to the sterile inflammatory response.

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.

Inhibition of heat shock protein (molecular weight 90 kDa) attenuates proinflammatory cytokines and prevents lipopolysaccharide‐induced liver injury in mice

Endotoxin‐mediated proinflammatory cytokines play a significant role in the pathogenesis of acute and chronic liver diseases. Heat shock protein 90 (molecular weight, 90 kDa) (hsp90) functions as an important chaperone of lipopolysaccharide (LPS) signaling and is required for the production of proinflammatory cytokines. We hypothesized that inhibition of hsp90 would prevent LPS‐induced liver injury by decreasing proinflammatory cytokines. C57BL/6 mice were injected intraperitoneally with an hsp90 inhibitor, 17‐dimethylamino‐ethylamino‐17‐demethoxygeldanamycin (17‐DMAG), and LPS. Parameters of liver injury, proinflammatory cytokines, and associated mechanisms were studied by in vivo and in vitro experiments. Inhibition of hsp90 by 17‐DMAG prevented LPS‐induced increases in serum alanine aminotransferase activity and significantly reduced serum tumor necrosis factor alpha (TNFα) and interleukin‐6 (IL‐6) protein as well as messenger RNA (mRNA) in liver. Enhanced DNA‐binding activity of heat shock transcription factor 1 (HSF1) and induction of target gene heat shock protein 70 (molecular weight, 70 kDa) confirmed hsp90 inhibition in liver. 17‐DMAG treatment decreased cluster of differentiation 14 mRNA and LPS‐induced nuclear factor kappa light‐chain enhancer of activated B cells (NFκB) DNA binding without affecting Toll‐like receptor 4 mRNA in liver. Mechanistic studies revealed that 17‐DMAG‐mediated inhibition of TNFα showed no effect on LPS‐induced NFκB promoter‐driven reporter activity, but significantly decreased TNFα promoter‐driven reporter activity. Chromatin immunoprecipitation assays showed that 17‐DMAG enhanced HSF1 binding to the TNFα promoter, but not the IL‐6 promoter, suggesting HSF1 mediated direct inhibition of TNFα, but not IL‐6. We show that HSF1 indirectly regulates IL‐6 by the induction of another transcription factor, activating transcription factor 3. Inhibition of HSF1, using small interfering RNA, prevented 17‐DMAG‐mediated down‐regulation of NFκB‐binding activity, TNFα, and IL‐6 induction, supporting a repressive role for HSF1 on proinflammatory cytokine genes during hsp90 inhibition. Conclusion: Hsp90 inhibition in vivo reduces proinflammatory cytokines and prevents LPS‐induced liver injury likely through repressive action of HSF1. Our results suggest a novel application for 17‐DMAG in alleviating LPS‐induced liver injury. (HEPATOLOGY 2011)

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-1β 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.

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.

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

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)

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

ABSTRACT 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.

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-1β/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.