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Diet-Induced Obesity Enhances Progression of Hepatocellular Carcinoma through Tenascin-C/Toll-Like Receptor 4 Signaling

2015; Elsevier BV; Volume: 186; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2015.09.015

ISSN

1525-2191

Autores

Jennifer H. Benbow, Kyle J. Thompson, Heidi L. Cope, Elizabeth Brandon‐Warner, Catherine R. Culberson, Krista Bossi, Ting Li, Mark W. Russo, Keith S. Gersin, Iain H. McKillop, Andrew S. deLemos, Laura W. Schrum,

Tópico(s)

Hippo pathway signaling and YAP/TAZ

Resumo

Obesity is an independent risk factor for the development of liver fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Tenascin-C (TnC), an extracellular matrix protein, is transiently expressed during tissue injury and plays a role in fibrogenesis and tumorigenesis. However, the mechanistic role of TnC signaling in the development of HCC remains unknown. We developed a diet-induced obesity HCC mouse model and examined TnC expression and liver injury. To determine the cellular mechanism of TnC signaling in promoting inflammation and hepatocyte epithelial-mesenchymal transition and migration, we used primary hepatocytes and hepatoma and macrophage cell lines. Further, to determine whether elevated TnC expression correlated with obesity-associated HCC, we measured plasma TnC in obese patients with various levels of liver injury. Increased tissue inflammation accompanied with elevated hepatic stellate cell-derived TnC and Toll-like receptor 4 expression was observed in the diet-induced obesity HCC animal model. In vitro studies found enhanced Toll-like receptor 4 signaling activated by TnC, promoting an increased inflammatory response, hepatocyte transformation, and migration. Further, obese patients with cirrhosis alone and in combination with HCC showed significant increases in plasma TnC compared with healthy volunteers and patients with less severe liver injury. Overall, these studies suggest TnC/Toll-like receptor 4 signaling as an important regulator in HCC; inhibiting this signaling axis may be a viable therapeutic target for impeding HCC. Obesity is an independent risk factor for the development of liver fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Tenascin-C (TnC), an extracellular matrix protein, is transiently expressed during tissue injury and plays a role in fibrogenesis and tumorigenesis. However, the mechanistic role of TnC signaling in the development of HCC remains unknown. We developed a diet-induced obesity HCC mouse model and examined TnC expression and liver injury. To determine the cellular mechanism of TnC signaling in promoting inflammation and hepatocyte epithelial-mesenchymal transition and migration, we used primary hepatocytes and hepatoma and macrophage cell lines. Further, to determine whether elevated TnC expression correlated with obesity-associated HCC, we measured plasma TnC in obese patients with various levels of liver injury. Increased tissue inflammation accompanied with elevated hepatic stellate cell-derived TnC and Toll-like receptor 4 expression was observed in the diet-induced obesity HCC animal model. In vitro studies found enhanced Toll-like receptor 4 signaling activated by TnC, promoting an increased inflammatory response, hepatocyte transformation, and migration. Further, obese patients with cirrhosis alone and in combination with HCC showed significant increases in plasma TnC compared with healthy volunteers and patients with less severe liver injury. Overall, these studies suggest TnC/Toll-like receptor 4 signaling as an important regulator in HCC; inhibiting this signaling axis may be a viable therapeutic target for impeding HCC. Nonalcoholic fatty liver disease (NAFLD) is estimated to affect nearly 30% of the US population.1Bohinc B.N. Diehl A.M. Mechanisms of disease progression in NASH: new paradigms.Clin Liver Dis. 2012; 16: 549-565Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar A subset of patients with NAFLD develop nonalcoholic steatohepatitis (NASH), characterized pathologically by hepatic steatosis, lobular inflammation, and ballooning. Although genetic variation, such as with patatin-like phospholipid domain containing protein 3,2Singal A.G. Manjunath H. Yopp A.C. Beg M.S. Marrero J.A. Gopal P. Waljee A.K. The effect of PNPLA3 on fibrosis progression and development of hepatocellular carcinoma: a meta-analysis.Am J Gastroenterol. 2014; 109: 325-334Crossref PubMed Scopus (227) Google Scholar may explain some individual susceptibility to NASH, fibrosis progression, and, ultimately, hepatocellular carcinoma (HCC), the exact links in this pathway are largely unresolved. HCC is the fifth most common cancer in the world and the second leading cause of cancer-related mortality.3Jemal A. Bray F. Center M.M. Ferlay J. Ward E. Forman D. Global cancer statistics.CA Cancer J Clin. 2011; 61: 69-90Crossref PubMed Scopus (30317) Google Scholar Although incidence of HCC arising in the context of NASH is currently lower than that of chronic viral hepatitis,4El-Serag H.B. 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Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults.N Engl J Med. 2003; 348: 1625-1638Crossref PubMed Scopus (5935) Google Scholar Thus, the mechanisms underlying the development of HCC, both in obesity and NASH, are an attractive and clinically relevant area of investigation. One of the key events in the promotion of HCC development is activation of hepatic stellate cells (HSCs).7Amann T. Bataille F. Spruss T. Muhlbauer M. Gabele E. Scholmerich J. Kiefer P. Bosserhoff A.K. Hellerbrand C. Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma.Cancer Sci. 2009; 100: 646-653Crossref PubMed Scopus (221) Google Scholar, 8Geng Z.M. Li Q.H. Li W.Z. Zheng J.B. Shah V. Activated human hepatic stellate cells promote growth of human hepatocellular carcinoma in a subcutaneous xenograft nude mouse model.Cell Biochem Biophys. 2014; 70: 337-347Crossref PubMed Scopus (18) Google Scholar, 9Zhao W. Zhang L. Xu Y. Zhang Z. Ren G. 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Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice.J Pathol. 2007; 211: 86-94Crossref PubMed Scopus (98) Google Scholar, 18Carey W.A. Taylor G.D. Dean W.B. Bristow J.D. Tenascin-C deficiency attenuates TGF-beta-mediated fibrosis following murine lung injury.Am J Physiol Lung Cell Mol Physiol. 2010; 299: L785-L793Crossref PubMed Scopus (81) Google Scholar Moreover, TnC concentrations are increased in visceral adipose tissue of obese individuals with NAFLD, contributing to the sustained inflammatory response and matrix remodeling.19Catalan V. Gomez-Ambrosi J. Rodriguez A. Ramirez B. Rotellar F. Valenti V. Silva C. Gil M.J. Salvador J. Fruhbeck G. Increased tenascin C and Toll-like receptor 4 levels in visceral adipose tissue as a link between inflammation and extracellular matrix remodeling in obesity.J Clin Endocrinol Metab. 2012; 97: E1880-E1889Crossref PubMed Scopus (60) Google Scholar Elevation of TnC expression is associated with an invasive phenotype of primary cultured HCC cells20Lin Z.Y. Chuang W.L. Genes responsible for the characteristics of primary cultured invasive phenotype hepatocellular carcinoma cells.Biomed Pharmacother. 2012; 66: 454-458Crossref PubMed Scopus (66) Google Scholar and is shown to promote epithelial-mesenchymal transition (EMT) in colorectal cancer cells.21Takahashi Y. Sawada G. Kurashige J. Matsumura T. Uchi R. Ueo H. Ishibashi M. Takano Y. Akiyoshi S. Iwaya T. Eguchi H. Sudo T. Sugimachi K. Yamamoto H. Doki Y. Mori M. Mimori K. Tumor-derived tenascin-C promotes the epithelial-mesenchymal transition in colorectal cancer cells.Anticancer Res. 2013; 33: 1927-1934PubMed Google Scholar Further, EMT was found to play a pivotal role in migration of malignant hepatocytes during HCC progression.22van Zijl F. Zulehner G. Petz M. Schneller D. Kornauth C. Hau M. Machat G. Grubinger M. Huber H. Mikulits W. Epithelial-mesenchymal transition in hepatocellular carcinoma.Future Oncol. 2009; 5: 1169-1179Crossref PubMed Scopus (261) Google Scholar TnC interacts with several extracellular matrix molecules and cell receptors, including Toll-like receptor 4 (TLR4), increasing proinflammatory cytokine expression [eg, tumor necrosis factor (TNF)-α, IL-6, IL-8].23Midwood K. Sacre S. Piccinini A.M. Inglis J. Trebaul A. Chan E. Drexler S. Sofat N. Kashiwagi M. Orend G. Brennan F. Foxwell B. Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease.Nat Med. 2009; 15: 774-780Crossref PubMed Scopus (537) Google Scholar Liver cells (eg, HSCs, Kupffer cells, endothelial cells, and hepatocytes), benign tumors, and malignant cancer cells express TLR4.24Yu L.X. Yan H.X. Liu Q. Yang W. Wu H.P. Dong W. Tang L. Lin Y. He Y.Q. Zou S.S. Wang C. Zhang H.L. Cao G.W. Wu M.C. Wang H.Y. Endotoxin accumulation prevents carcinogen-induced apoptosis and promotes liver tumorigenesis in rodents.Hepatology. 2010; 52: 1322-1333Crossref PubMed Scopus (226) Google Scholar, 25Mai C.W. Kang Y.B. Pichika M.R. Should a Toll-like receptor 4 (TLR-4) agonist or antagonist be designed to treat cancer? TLR-4: its expression and effects in the ten most common cancers.Onco Targets Ther. 2013; 6: 1573-1587Crossref PubMed Scopus (91) Google Scholar In normal immune cells, TLR4 was shown to enhance antitumor immunity; however, with chronic inflammation, TLR4 signaling augments cancer development.25Mai C.W. Kang Y.B. Pichika M.R. Should a Toll-like receptor 4 (TLR-4) agonist or antagonist be designed to treat cancer? TLR-4: its expression and effects in the ten most common cancers.Onco Targets Ther. 2013; 6: 1573-1587Crossref PubMed Scopus (91) Google Scholar TLR4 expression is elevated in murine livers after diet-induced obesity.26Gabele E. Dostert K. Dorn C. Patsenker E. Stickel F. Hellerbrand C. A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis.Alcohol Clin Exp Res. 2011; 35: 1361-1367Crossref PubMed Scopus (70) Google Scholar Further, TLR4 and TLR2 expression increases in livers from patients with hepatitis, cirrhosis, or HCC.27Soares J.B. Pimentel-Nunes P. Afonso L. Rolanda C. Lopes P. Roncon-Albuquerque Jr., R. Goncalves N. Boal-Carvalho I. Pardal F. Lopes S. Macedo G. Lara-Santos L. Henrique R. Moreira-Dias L. Goncalves R. Dinis-Ribeiro M. Leite-Moreira A.F. Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence.Innate Immun. 2012; 18: 700-708Crossref PubMed Scopus (55) Google Scholar Conversely, mice with impaired TLR4 signaling are less susceptible to liver fibrosis.28Cong M. Iwaisako K. Jiang C. Kisseleva T. Cell signals influencing hepatic fibrosis.Int J Hepatol. 2012; 2012: 158547Crossref PubMed Google Scholar In a metastatic model, silencing TLR4 resulted in a lowering of tumor burden24Yu L.X. Yan H.X. Liu Q. Yang W. Wu H.P. Dong W. Tang L. Lin Y. He Y.Q. Zou S.S. Wang C. Zhang H.L. Cao G.W. Wu M.C. Wang H.Y. Endotoxin accumulation prevents carcinogen-induced apoptosis and promotes liver tumorigenesis in rodents.Hepatology. 2010; 52: 1322-1333Crossref PubMed Scopus (226) Google Scholar; conversely, TLR4 activation by lipopolysaccharide (LPS) contributed to inflammation-driven tumor promotion in HCC.29Dapito D.H. Mencin A. Gwak G.Y. Pradere J.P. Jang M.K. Mederacke I. Caviglia J.M. Khiabanian H. Adeyemi A. Bataller R. Lefkowitch J.H. Bower M. Friedman R. Sartor R.B. Rabadan R. Schwabe R.F. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4.Cancer Cell. 2012; 21: 504-516Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar Herein, we present data showing that elevated expression of TnC by HSCs promotes enhanced TLR4 signaling, leading to inflammation, hepatocyte transformation, and migration, which is correlated with development of obesity-associated HCC in a mouse model. Furthermore, TnC concentrations are also increased in plasma from patients with NASH cirrhosis, and this concentration is further increased in patients with HCC. Interfering with and/or blocking TnC/TLR4 signaling may prove to be a therapeutic target for preventing HCC. Human hepatoma cell line Huh 7.0 and mouse macrophage cell line Raw 264.7 were cultured in Dulbecco's modified Eagle's medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2.0 mmol/L glutamine, and 10% fetal bovine serum. HepG2 and Hep3B human hepatoma cell lines were cultured in minimal essential medium with the same supplements as described for Huh 7.0 cells. Adult male Sprague-Dawley rats (200 to 250 g for hepatocytes and >500 g for HSCs; Charles River Laboratories, Durham, NC) were used for these studies. All experiments were reviewed and approved by the Carolinas Medical Center (Charlotte, NC) Institutional Animal Care and Use Committee (protocols 09-12-04A and 09-13-01A). Primary rat hepatocytes were isolated and plated with a modified two-step collagenase perfusion as previously reported.30Samson C.M. Schrum L.W. Bird M.A. Lange P.A. Brenner D.A. Rippe R.A. Behrns K.E. Transforming growth factor-beta1 induces hepatocyte apoptosis by a c-Jun independent mechanism.Surgery. 2002; 132: 441-449Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Primary rat HSCs were isolated by pronase/collagenase perfusion digestion, followed by subsequent density-gradient centrifugation, as previously reported.31Li T. Eheim A.L. Klein S. Uschner F.E. Smith A.C. Brandon-Warner E. Ghosh S. Bonkovsky H.L. Trebicka J. Schrum L.W. Novel role of nuclear receptor rev-erbalpha in hepatic stellate cell activation: potential therapeutic target for liver injury.Hepatology. 2014; 59: 2383-2396Crossref PubMed Scopus (40) Google Scholar Huh 7.0 cells, primary rat hepatocytes, and Raw 264.7 cells were treated with 1× phosphate-buffered saline (control), 1 μg/mL LPS (diluted in 1× phosphate-buffered saline), or 50 nmol/L TnC ± 1 μg/mL LPS for 6, 15, or 24 hours. To confirm the involvement of the TLR4 signaling pathway, Huh 7.0 cells and Raw 264.7 cells were preincubated with either 25 μmol/L TLR4-specific antagonist (VIPER; Imgenex, San Diego, CA) or control peptide (CP7; Imgenex) 90 minutes before treatment with TnC ± LPS. Primary rat HSCs were treated on day 7 for 72 hours with dimethyl sulfoxide control or 10 μmol/L SR9009 (EMD Millipore, Billerica, MA). Male C57BL/6 mice (aged 21 to 25 days; Charles River Laboratories) were randomized, weighed, and injected intraperitoneally one time with diethylnitrosamine (DEN; Sigma-Aldrich, St. Louis, MO) or olive oil vehicle before mice were randomly assigned to a 10% kcal% fat, control diet (CD; Research Diets, Inc., New Brunswick, NJ; D12450B) or a 60% kcal% fat (40% unsaturated: 60% saturated fat lard), high-fat diet (HFD; Research Diets; D12492) at 5 weeks as previously reported.32Thompson K.J. Swan R.Z. Walling T.L. Iannitti D.A. McKillop I.H. Sindram D. Obesity, but not ethanol, promotes tumor incidence and progression in a mouse model of hepatocellular carcinoma in vivo.Surg Endosc. 2013; 27: 2782-2791Crossref PubMed Scopus (12) Google Scholar At 42 weeks, mice were euthanized, and the livers were resected and stored (−80°C). All work with mice performed at Carolinas Medical Center was reviewed and approved by the Institutional Animal Care and Use Committee at this institution (protocol no. 12-07-01A). Plasma, snap-frozen liver, and formalin-fixed, paraffin-embedded liver from male C57BL/6 mice fed the CD or HFD for 10 and 20 weeks were obtained from The Jackson Laboratories (Bar Harbor, ME). Whole blood was collected and submitted to the Liver, Biliary and Pancreatic Repository according to an institutional review board-approved protocol for Carolinas HealthCare System. Obese patients [body mass index (BMI; calculated as kg/m2) > 30] undergoing bariatric surgery, hepatobiliary surgery, or with chronic liver disease were eligible for enrollment. Obese patients were negative for hepatitis B and hepatitis C viruses. Plasma samples were obtained from healthy volunteers (BMI < 30) as a control group. Concentrations of TnC in plasma were determined with the use of the human TnC (FN III-C) Assay enzyme-linked immunosorbent assay (ELISA) kit (IBL, Minneapolis, MN). For statistical analysis, TnC results were stratified by fibrosis stage and NAFLD activity score,33Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Yeh M. McCullough A.J. Sanyal A.J. Nonalcoholic Steatohepatitis Clinical Research NetworkDesign and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7151) Google Scholar determined by liver biopsy at the time of bariatric surgery. Patients with cirrhosis and HCC were diagnosed by cross-sectional imaging34Wald C. Russo M.W. Heimbach J.K. Hussain H.K. Pomfret E.A. Bruix J. New OPTN/UNOS policy for liver transplant allocation: standardization of liver imaging, diagnosis, classification, and reporting of hepatocellular carcinoma.Radiology. 2013; 266: 376-382Crossref PubMed Scopus (279) Google Scholar and compatible clinical symptoms/signs of cirrhosis.35Chalasani N. Younossi Z. Lavine J.E. Diehl A.M. Brunt E.M. Cusi K. Charlton M. Sanyal A.J. American Gastroenterological Association, American Association for the Study of Liver Diseases, American College of GastroenterologyThe diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology.Gastroenterology. 2012; 142: 1592-1609Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar Patient characteristics are described in Table 1.Table 1Demographic and Clinical Characteristics of Study SubjectsVariableMetavir scoreF0 (n = 36)F1 (n = 31)F2–F3 (n = 22)F4 (n = 26)F4 + HCC (n = 13)F4 + HCC nonobese (n = 15)Sex, n (%) Male5 (13.9)5 (16.1)4 (18.2)12 (46.2)10 (76.9)13 (86.7) Female31 (86.1)26 (83.9)18 (81.8)14 (53.9)3 (23.1)2 (13.3)Age, year43.4 ± 1.547.1 ± 1.449.0 ± 2.255.4 ± 1.867.5 ± 2.363.4 ± 2.4BMI, kg/m243.6 ± 1.144.2 ± 1.041.5 ± 1.837.5 ± 1.531.9 ± 1.726.8 ± 0.8AST (IU/L)21.6 ± 1.432.6 ± 4.141.2 ± 6.151.9 ± 3.687.9 ± 18.374.6 ± 11.0ALT (IU/L)21.9 ± 2.041.7 ± 7.047.2 ± 11.336.9 ± 3.059.2 ± 13.457.7 ± 1.2AFP (IU/L) Median0 (0)0 (0)1.15 (2/22)∗Liver biopsies/AFP not available for all patients.3.65 (20/26)∗Liver biopsies/AFP not available for all patients.7.6 (12/13)∗Liver biopsies/AFP not available for all patients.17.0 (13/15)∗Liver biopsies/AFP not available for all patients. Range0 (0)0 (0)1.1–1.21.4–12.70.9–1,032,4002.8–2399Tumor size, n (%) ≤3 cmNANANANA8 (61.5)9 (60.0) >3 cmNANANANA5 (38.5)6 (40.0)Steatosis, n (%) 021 (58.3)2 (6.5)0/21 (0)∗Liver biopsies/AFP not available for all patients.0/9 (0)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 111 (30.6)12 (38.7)14/21 (66.7)∗Liver biopsies/AFP not available for all patients.5/9 (55.6)∗Liver biopsies/AFP not available for all patients.3/5 (60.0)∗Liver biopsies/AFP not available for all patients.3/3 (100)∗Liver biopsies/AFP not available for all patients. 24 (11.1)14 (45.2)4/21 (19.0)∗Liver biopsies/AFP not available for all patients.3/9 (33.3)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 30 (0)3 (9.7)3/21 (14.3)∗Liver biopsies/AFP not available for all patients.1/9 (11.1)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients.Lobular inflammation, n (%) 028 (77.8)8 (25.8)2/20 (10.0)∗Liver biopsies/AFP not available for all patients.2/9 (22.2)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 18 (22.2)18 (58.1)7/20 (35.0)∗Liver biopsies/AFP not available for all patients.4/9 (44.4)∗Liver biopsies/AFP not available for all patients.4/5 (80.0)∗Liver biopsies/AFP not available for all patients.2/3 (66.7)∗Liver biopsies/AFP not available for all patients. 20 (0)5 (16.1)9/20 (45.0)∗Liver biopsies/AFP not available for all patients.3/9 (33.3)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.1/3 (33.3)∗Liver biopsies/AFP not available for all patients. 30 (0)0 (0)2/20 (10.0)∗Liver biopsies/AFP not available for all patients.0/9 (0)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients.Ballooning, n (%) 032 (88.9)17 (54.8)4/20 (20.0)∗Liver biopsies/AFP not available for all patients.3/9 (33.3)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.1/3 (33.3)∗Liver biopsies/AFP not available for all patients. 14 (11.1)12 (38.7)12/20 (60.0)∗Liver biopsies/AFP not available for all patients.5/9 (55.6)∗Liver biopsies/AFP not available for all patients.4/5 (80.0)∗Liver biopsies/AFP not available for all patients.2/3 (66.7)∗Liver biopsies/AFP not available for all patients. 20 (0)2 (6.5)4/20 (20.0)∗Liver biopsies/AFP not available for all patients.1/9 (11.1)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients.NAFLD Activity Score, n (%) 019 (52.8)1 (3.2)0/20 (0)∗Liver biopsies/AFP not available for all patients.0/9 (0)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 19 (25.0)5 (16.1)1/20 (5.0)∗Liver biopsies/AFP not available for all patients.1/9 (11.1)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 23 (8.3)2 (6.5)3/20 (15.0)∗Liver biopsies/AFP not available for all patients.1/9 (11.1)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.1/3 (33.3)∗Liver biopsies/AFP not available for all patients. 34 (11.1)12 (38.7)4/20 (20.0)∗Liver biopsies/AFP not available for all patients.3/9 (33.3)∗Liver biopsies/AFP not available for all patients.2/5 (40.0)∗Liver biopsies/AFP not available for all patients.2/3 (66.7)∗Liver biopsies/AFP not available for all patients. 41 (2.8)8 (25.8)5/20 (25.0)∗Liver biopsies/AFP not available for all patients.2/9 (22.2)∗Liver biopsies/AFP not available for all patients.1/5 (20.0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 50 (0)3 (9.7)4/20 (20.0)∗Liver biopsies/AFP not available for all patients.2/9 (22.2)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 60 (0)0 (0)1/20 (5.0)∗Liver biopsies/AFP not available for all patients.0/9 (0)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 70 (0)0 (0)0/20 (0)∗Liver biopsies/AFP not available for all patients.0/9 (0)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients. 80 (0)0 (0)2/20 (10.0)∗Liver biopsies/AFP not available for all patients.0/0 (0)∗Liver biopsies/AFP not available for all patients.0/5 (0)∗Liver biopsies/AFP not available for all patients.0/3 (0)∗Liver biopsies/AFP not available for all patients.AFP, α-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; HCC, hepatocellular carcinoma; NA, not applicable; NAFLD, nonalcoholic fatty liver disease.∗ Liver biopsies/AFP not available for all patients. Open table in a new tab AFP, α-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; HCC, hepatocellular carcinoma; NA, not applicable; NAFLD, nonalcoholic fatty liver disease. Sections (4 μm) of formalin-fixed, paraffin-embedded liver tissue were blocked with 2.5% normal horse serum and incubated with either anti-TLR4 antibody (dilution 1:75; Abcam, Cambridge, MA;) or anti-CD68 antibody (dilution 1:250; Abcam) for macrophage detection. Detection of bound antibody was visualized with an anti-rabbit IgG ImmPRESS polymerized reporter enzyme staining system (Vector Labs, Burlingame, CA) with diaminobenzidine (Vector Labs). The nuclei were counterstained with Mayer's hematoxylin. Representative liver sections were examined microscopically (×20) on an Olympus BX40 microscope (Olympus America, Inc., Center Valley, PA) with a DP72 camera and CellSense standard 2.0 software (Olympus America, Inc.) and scored by three blinded independent investigators with the use of the following scoring scale: 0, no staining to 40%. Individual scores were averaged to medians ± SEM, providing a numerical score for each liver section and then used for statistical analysis. Sections (4 μm) of formalin-fixed liver tissue were incubated in Naphthol AS-D-Chloroacetate Esterase working solution and then counterstained with Gill's hematoxylin, followed by permanent coverslipping with Permount. Representative liver sections were examined microscopically (×40) on an Olympus BX40 microscope (Olympus America, Inc.) with a DP72 camera and CellSense standard 2.0 software (Olympus America, Inc.), and neutrophils were counted and averaged from four randomly selected areas per slide by two independent investigators. Four representative sections (4 μm) of formalin-fixed, paraffin-embedded liver tissue were incubated with the following primary antibodies: rabbit anti-TnC (dilution 1:100; Abcam) and goat anti–α-smooth muscle actin (α-SMA; dilution 1:100; Abcam) overnight then detected with the following secondary antibodies: anti-rabbit cyanine 3 (dilution 1:250; Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor donkey anti-goat IgG 488 (dilution 1:2500; Life Technologies, Carlsbad, CA). The nuclei were stained with DAPI (Life Technologies). To suppress nonspecific background staining, slides were incubated with Sudan Black B (Sigma-Aldrich) before b

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