Title: Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH
Abstract: Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation. Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation. Nonalcoholic fatty liver disease (NAFLD) is characterized by increased lipid deposition within hepatocytes attributable to metabolic causes in the absence of viral hepatitis or liver disorders caused by excessive alcohol or toxic drug consumption or other liver disorders. In the majority of patients, NAFLD manifests histologically as "simple steatosis" defined as hepatic steatosis without substantial inflammation or fibrosis. Simple steatosis carries a very low risk of progression to cirrhosis and liver dysfunction (1Matteoni C.A. Younossi Z.M. Gramlich T. Boparai N. Liu Y.C. McCullough A.J. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.Gastroenterology. 1999; 116: 1413-1419Abstract Full Text Full Text PDF PubMed Scopus (2791) Google Scholar). However, 10–30% of patients with NAFLD have or develop nonalcoholic steatohepatitis (NASH), characterized by hepatic lobular inflammation and fibrosis in addition to steatosis. Progression to cirrhosis occurs in a proportion of patients (1Matteoni C.A. Younossi Z.M. Gramlich T. Boparai N. Liu Y.C. McCullough A.J. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.Gastroenterology. 1999; 116: 1413-1419Abstract Full Text Full Text PDF PubMed Scopus (2791) Google Scholar, 2Bugianesi E. Leone N. Vanni E. Marchesini G. Brunello F. Carucci P. Musso A. De Paolis P. Capussotti L. Salizzoni M. et al.Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma.Gastroenterology. 2002; 123: 134-140Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar). The factor(s) responsible for the development of progressive NASH, as opposed to simple steatosis, remain unclear. A prominent concept that has been invoked to explain the development of NASH is that of lipotoxicity. Hepatic lipotoxicity implies that exposure to, or accumulation of, certain lipid species within hepatic cells may directly cause cellular toxicity or act in a proinflammatory or profibrotic manner. According to the hypothesis of hepatic lipotoxicity, NASH develops when the liver is exposed to lipotoxic lipid species, whereas simple steatosis develops in response to over-nutrition when the liver is not significantly exposed to lipotoxic lipid species. It is generally accepted that triglycerides, which constitute the majority of hepatic lipids in NASH and simple steatosis, are a "safe" storage lipid with little or no lipotoxic potential. Relatively small quantities of other lipotoxic lipid species may exert a disproportionate impact in the development of NASH. Lipidomic analyses of human livers with NAFLD reported that levels of free (unesterified) cholesterol (FC) were increased in NASH but not in simple steatosis, whereas levels of free fatty acids were no different (3Puri P. Baillie R.A. Wiest M.M. Mirshahi F. Choudhury J. Cheung O. Sargeant C. Contos M.J. Sanyal A.J. A lipidomic analysis of nonalcoholic fatty liver disease.Hepatology. 2007; 46: 1081-1090Crossref PubMed Scopus (908) Google Scholar). Recent experimental studies suggest that FC is an important lipotoxic molecule that promotes the development of NASH (3Puri P. Baillie R.A. Wiest M.M. Mirshahi F. Choudhury J. Cheung O. Sargeant C. Contos M.J. Sanyal A.J. A lipidomic analysis of nonalcoholic fatty liver disease.Hepatology. 2007; 46: 1081-1090Crossref PubMed Scopus (908) Google Scholar, 4Matsuzawa N. Takamura T. Kurita S. Misu H. Ota T. Ando H. Yokoyama M. Honda M. Zen Y. Nakanuma Y. et al.Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet.Hepatology. 2007; 46: 1392-1403Crossref PubMed Scopus (407) Google Scholar, 5Zheng S. Hoos L. Cook J. Tetzloff G. Davis Jr, H. van Heek M. Hwa J.J. Ezetimibe improves high fat and cholesterol diet-induced non-alcoholic fatty liver disease in mice.Eur. J. Pharmacol. 2008; 584: 118-124Crossref PubMed Scopus (141) Google Scholar, 6Van Rooyen D.M. Larter C.Z. Haigh W.G. Yeh M.M. Ioannou G. Kuver R. Lee S.P. Teoh N.C. Farrell G.C. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis.Gastroenterology. 2011; 141,1403: 1393,e1-1403,5Google Scholar, 7Savard C. Tartaglione E.V. Kuver R. Haigh W.G. Farrell G.C. Subramanian S. 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Thus, using diverse animal models [dietary with cholesterol-containing atherogenic (Ath) diets (4Matsuzawa N. Takamura T. Kurita S. Misu H. Ota T. Ando H. Yokoyama M. Honda M. Zen Y. Nakanuma Y. et al.Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet.Hepatology. 2007; 46: 1392-1403Crossref PubMed Scopus (407) Google Scholar, 5Zheng S. Hoos L. Cook J. Tetzloff G. Davis Jr, H. van Heek M. Hwa J.J. Ezetimibe improves high fat and cholesterol diet-induced non-alcoholic fatty liver disease in mice.Eur. J. Pharmacol. 2008; 584: 118-124Crossref PubMed Scopus (141) Google Scholar, 7Savard C. Tartaglione E.V. Kuver R. Haigh W.G. Farrell G.C. Subramanian S. Chait A. Yeh M.M. Quinn L.S. Ioannou G.N. Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis.Hepatology. 2013; 57: 81-92Crossref PubMed Scopus (191) Google Scholar), LDLR knock-out and ApoE knock-in mice (9Wouters K. van Bilsen M. van Gorp P.J. Bieghs V. Lutjohann D. Kerksiek A. Staels B. Hofker M.H. Shiri-Sverdlov R. Intrahepatic cholesterol influences progression, inhibition and reversal of non-alcoholic steatohepatitis in hyperlipidemic mice.FEBS Lett. 2010; 584: 1001-1005Crossref PubMed Scopus (82) Google Scholar, 10Wouters K. van Gorp P.J. Bieghs V. Gijbels M.J. Duimel H. Lutjohann D. Kerksiek A. van Kruchten R. Maeda N. Staels B. et al.Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammation in hyperlipidemic mouse models of nonalcoholic steatohepatitis.Hepatology. 2008; 48: 474-486Crossref PubMed Scopus (381) Google Scholar, 11Bieghs V. Van Gorp P.J. Wouters K. Hendrikx T. Gijbels M.J. van Bilsen M. Bakker J. Binder C.J. Lutjohann D. Staels B. et al.LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease.PLoS ONE. 2012; 7: e30668Crossref PubMed Scopus (122) Google Scholar, 12Subramanian S. Goodspeed L. Wang S. Kim J. Zeng L. Ioannou G.N. Haigh W.G. Yeh M.M. Kowdley K.V. O'Brien K.D. et al.Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice.J. Lipid Res. 2011; 52: 1626-1635Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), appetite-defective Alms1 mutant (foz/foz) (6Van Rooyen D.M. Larter C.Z. Haigh W.G. Yeh M.M. Ioannou G. Kuver R. Lee S.P. Teoh N.C. Farrell G.C. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis.Gastroenterology. 2011; 141,1403: 1393,e1-1403,5Google Scholar, 8Van Rooyen D.M. Gan L.T. Yeh M.M. Haigh W.G. Larter C.Z. Ioannou G. Teoh N.C. Farrell G.C. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.J. Hepatol. 2013; 59: 144-152Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), or melanocortin 4 receptor-deficient (Mc4r-ko) (13Itoh M. Suganami T. Nakagawa N. Tanaka M. Yamamoto Y. Kamei Y. Terai S. Sakaida I. Ogawa Y. Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis.Am. J. Pathol. 2011; 179: 2454-2463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) mice fed an Ath diet, and ABCB4 mutant opossums with defective hepatic cholesterol excretion in bile (14Chan J. Sharkey F.E. Kushwaha R.S. VandeBerg J.F. VandeBerg J.L. Steatohepatitis in laboratory opossums exhibiting a high lipemic response to dietary cholesterol and fat.Am. J. Physiol. 2012; 303 (Gastrointest. Liver Physiol.): G12-G19Crossref PubMed Scopus (19) Google Scholar)], there is support for a consistent association between hepatic FC content and NASH pathology. Human epidemiological studies and nonrandomized clinical trials of cholesterol-lowering drugs also appear to support a role of cholesterol in the development of NASH (15Ioannou G.N. Morrow O.B. Connole M.L. Lee S.P. Association between dietary nutrient composition and the incidence of cirrhosis or liver cancer in the United States population.Hepatology. 2009; 50: 175-184Crossref PubMed Scopus (129) Google Scholar, 16Yoneda M. Fujita K. Nozaki Y. Endo H. Takahashi H. Hosono K. Suzuki K. Mawatari H. Kirikoshi H. Inamori M. et al.Efficacy of ezetimibe for the treatment of non-alcoholic steatohepatitis: An open-label, pilot study.Hepatol. Res. 2010; 40: 566-573Crossref PubMed Scopus (112) Google Scholar, 17Park H. Shima T. Yamaguchi K. Mitsuyoshi H. Minami M. Yasui K. Itoh Y. Yoshikawa T. Fukui M. Hasegawa G. et al.Efficacy of long-term ezetimibe therapy in patients with nonalcoholic fatty liver disease.J. Gastroenterol. 2011; 46: 101-107Crossref PubMed Scopus (157) Google Scholar). The mechanisms by which cholesterol, a naturally occurring molecule abundant in most tissues, might exert lipotoxicity and promote the development of NASH remain unclear. We recently reported that cholesterol crystals were present within the lipid droplets of steatotic hepatocytes in patients with NASH and in a mouse model of NASH induced by a high-fat, high-cholesterol diet but not in patients or mice with simple steatosis (18Ioannou G.N. Haigh W.G. Thorning D. Savard C. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.J. Lipid Res. 2013; 54: 1326-1334Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). We also demonstrated that enlarged Kupffer cells (KCs) surrounded steatotic, dead hepatocytes containing cholesterol crystals. Such KCs appeared to process the remnant lipid droplets within these hepatocytes to form characteristic crown-like structures (CLSs) similar to those recently described in inflamed visceral adipose tissue (19Cinti S. Mitchell G. Barbatelli G. Murano I. Ceresi E. Faloia E. Wang S. Fortier M. Greenberg A.S. Obin M.S. Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.J. Lipid Res. 2005; 46: 2347-2355Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar, 20Strissel K.J. Stancheva Z. Miyoshi H. Perfield 2nd, J.W. DeFuria J. Jick Z. Greenberg A.S. Obin M.S. Adipocyte death, adipose tissue remodeling, and obesity complications.Diabetes. 2007; 56: 2910-2918Crossref PubMed Scopus (688) Google Scholar). This lipid scavenging resulted in profound accumulation of cholesterol within small droplets in markedly enlarged, activated KCs that took the appearance of the lipid-laden "foam cells" found in atheroma. These findings are particularly relevant because cholesterol crystals have recently been shown to activate the NLRP3 inflammasome in animal models of atherosclerosis (21Duewell P. Kono H. Rayner K.J. Sirois C.M. Vladimer G. Bauernfeind F.G. Abela G.S. Franchi L. Nunez G. Schnurr M. et al.NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.Nature. 2010; 464: 1357-1361Crossref PubMed Scopus (2570) Google Scholar, 22Rajamaki K. Lappalainen J. Oorni K. Valimaki E. Matikainen S. Kovanen P.T. Eklund K.K. Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link between cholesterol metabolism and inflammation.PLoS ONE. 2010; 5: e11765Crossref PubMed Scopus (688) Google Scholar), thus providing a mechanism by which exposure of KCs to cholesterol crystals could lead to chronic inflammation and resultant fibrosis in NASH. In the current study, we used a combination of approaches to test this concept. We explored a metabolic syndrome murine model of NASH [Alms1 mutant (foz/foz) mice], in which an appetite defect leads to obesity, diabetes, and metabolic syndrome (as in Mc4r-ko mice), to determine whether a) the links between hepatic cholesterol crystals, CLSs, and NASH are reproducible in a completely different animal model from the one used initially to describe these findings and b) pharmacological treatment aimed at reducing hepatic cholesterol content also removed hepatic cholesterol crystals, leading to resolution of crown-like structures in association with reversal of NASH fibrosis. These experiments were performed on mice used previously to analyze the therapeutic effects of cholesterol-lowering drugs on NASH pathology (8Van Rooyen D.M. Gan L.T. Yeh M.M. Haigh W.G. Larter C.Z. Ioannou G. Teoh N.C. Farrell G.C. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.J. Hepatol. 2013; 59: 144-152Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In brief, female Alms1 mutant (foz/foz) mice were fed a high-fat (23%), high-sucrose diet containing 0.2% cholesterol (Specialty Feeds, Australia), hereafter referred to as an Ath diet, for 16 weeks, then assigned to one of four pharmacological groups for an additional 8 weeks while continuing the Ath diet. Agents were administered orally with the diet as previously reported (8Van Rooyen D.M. Gan L.T. Yeh M.M. Haigh W.G. Larter C.Z. Ioannou G. Teoh N.C. Farrell G.C. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.J. Hepatol. 2013; 59: 144-152Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar): a) vehicle control (n = 8); b) atorvastatin (n = 10), 20 mg/kg body weight/day (Lipitor® Pfizer West Ryde, NSW, Australia); c) ezetimibe (n = 10), 5 mg/kg/day (Ezetrol® Schering-Plough, Whitehouse Station, NJ); and d) ezetimibe, 5 mg/kg/day and atorvastatin, 20 mg/kg/day (n = 9). The foz/foz mice have a spontaneous mutation of the Alström syndrome gene murine equivalent, Alms1, which is associated with loss of hypothalamic neuronal cilia causing severely defective central appetite regulation (23Heydet D. Chen L.X. Larter C.Z. Inglis C. Silverman M.A. Farrell G.C. Leroux M.R. A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice.Dev. Neurobiol. 2013; 73: 1-13Crossref PubMed Scopus (46) Google Scholar). This results in hyperphagia, rapid weight gain with inactivity, obesity, insulin resistance, hypercholesterolemia, and NAFLD. We previously reported that in foz/foz mice intake of an Ath diet accelerates the development of obesity, insulin resistance, hyperinsulinemia, hyperglycemia, hypercholesterolemia, hypoadiponectinemia, hypertension (metabolic syndrome), and transformation of simple steatosis to NASH (6Van Rooyen D.M. Larter C.Z. Haigh W.G. Yeh M.M. Ioannou G. Kuver R. Lee S.P. Teoh N.C. Farrell G.C. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis.Gastroenterology. 2011; 141,1403: 1393,e1-1403,5Google Scholar). Thus, foz/foz mice on the Ath diet mirror the metabolic and histological features of human NASH. Similar findings have been noted in mice with other hypothalamic appetite defects, such as Mc4r-ko mice (13Itoh M. Suganami T. Nakagawa N. Tanaka M. Yamamoto Y. Kamei Y. Terai S. Sakaida I. Ogawa Y. Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis.Am. J. Pathol. 2011; 179: 2454-2463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), reflecting the importance of Alms1 as an appetite disorder with no direct implications for liver biology (hepatocytes do not express a primary cilium). Experiments were approved by the Australian National University Experimentation Ethics Committee (Canberra, Australia) and by the Research and Development Committee of the Veterans Affairs Puget Sound Health Care System (Seattle, WA). Formalin-fixed, paraffin-embedded liver sections were stained with hematoxylin and eosin and Sirius red (for collagen). Histological steatosis, inflammation, ballooning degeneration, and fibrosis were assessed semiquantitatively by a "blinded" liver pathologist to determine the NAFLD Activity Score and the overall impression of NASH according to the scoring system proposed by Kleiner et al. (24Kleiner 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. et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7112) Google Scholar). Frozen mouse liver tissue embedded in Optimal Cutting Temperature compound was sectioned at 10 µM in thickness. Sections were allowed to come to room temperature and were immediately cover-slipped using pure glycerol as the mounting medium without applying stain. They were examined using a Nikon Eclipse microscope with or without a polarizing filter to evaluate for the presence of birefringent crystals that we have previously shown to represent cholesterol crystals (18Ioannou G.N. Haigh W.G. Thorning D. Savard C. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.J. Lipid Res. 2013; 54: 1326-1334Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). With the polarizing filter in place, images of 10 random fields were taken (200× original magnification; 0.26 mm2 total area/image) in each liver, avoiding major blood vessels. Image J density software (National Institutes of Health, Bethesda, MD) was used to calculate the percent area of each image that was birefringent, and the average of 10 images per liver was calculated. Frozen liver sections were also stained with filipin to visualize FC (18Ioannou G.N. Haigh W.G. Thorning D. Savard C. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.J. Lipid Res. 2013; 54: 1326-1334Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Filipin interacts with the 3β-hydroxy group of cholesterol to fluoresce blue (25Rudolf M. Curcio C.A. Esterified cholesterol is highly localized to Bruch's membrane, as revealed by lipid histochemistry in wholemounts of human choroid.J. Histochem. Cytochem. 2009; 57: 731-739Crossref PubMed Scopus (62) Google Scholar). When attempting to demonstrate cholesterol crystals and simultaneously stain for KCs or for FC, the procedures used for these stains (i.e., long incubation times and repeated washing) cause partial disruption of the cholesterol crystals and somewhat diminished birefringence so that they may not appear as prominent as when they are looked at in unprocessed, frozen liver sections. Frozen liver sections were stained with anti-CD68 and anti-F4/80 antibodies, which identify macrophages (including hepatic KCs) and with anti-TNF-α antibodies, which identify activated M1 macrophages, as described previously (8Van Rooyen D.M. Gan L.T. Yeh M.M. Haigh W.G. Larter C.Z. Ioannou G. Teoh N.C. Farrell G.C. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.J. Hepatol. 2013; 59: 144-152Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 18Ioannou G.N. Haigh W.G. Thorning D. Savard C. Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.J. Lipid Res. 2013; 54: 1326-1334Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). These stains were used to identify activated macrophages surrounding and processing steatotic hepatocytes containing cholesterol crystals to form CLSs. CLSs were counted in 10 random fields (200× original magnification) per liver. The percent area that stained with anti-TNFα in 10 random fields per liver was averaged as an assessment of the number of activated macrophages. We also stained liver sections with anti-CD3e (Life Technologies, Grand Island, NY) and with antimyeloperoxidase (Thermo Scientific, Fremont, CA) to evaluate for involvement of T-cells or neutrophils, respectively, in CLSs. Liver sections were stained with anti-NLRP3 antibodies (R&D Systems, Minneapolis, MN) to look for expression of this component of the NLRP3 inflammasome in the KCs of CLSs. Liver sections were also stained for activated (cleaved) caspase 1 using the FAM-FLICA caspase 1 assay kit (Immunochemistry Technologies, Bloomington, MN). Detection of cleaved caspase 1 demonstrates the presence of an activated NLRP3 inflammasome: cleaved caspase-1 can mediate the downstream effects of the NLRP3 inflammasome by cleaving and activating pro-interleukin (IL)-1 and pro-IL-18. All primary antibodies were identified using secondary antibodies labeled with AlexaFluor 488 (Invitrogen, Camarillo, CA) examined with the FITC filters on a Nikon fluorescent microscope. We used real-time PCR to quantify mRNA gene expression levels as previously described (8Van Rooyen D.M. Gan L.T. Yeh M.M. Haigh W.G. Larter C.Z. Ioannou G. Teoh N.C. Farrell G.C. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.J. Hepatol. 2013; 59: 144-152Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) of the following components of the NLRP3 inflammasome in liver tissue: Caspase-1, Nalp3, and Asc (apoptosis-associated speck-like, caspase recruitment domain containing protein) (26Ganz M. Csak T. Nath B. Szabo G. Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver.World J. Gastroenterol. 2011; 17: 4772-4778Crossref PubMed Scopus (95) Google Scholar). Continuous data are presented as mean (±SD) and analyzed by ANOVA with Tukey post hoc testing. Histological assessments were done using Kruskal-Wallis test and group comparisons with Mann-Whitney U test. A P value <0.05 was considered statistically significant. As previously reported (6Van Rooyen D.M. Larter C.Z. Haigh W.G. Yeh M.M. Ioannou G. Kuver R. Lee S.P. Teoh N.C. Farrell G.C. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis.Gastroenterology. 2011; 141,1403: 1393,e1-1403,5Google Scholar, 8Van Rooyen D.M. Gan L.T. Yeh M.M. Haigh W.G. Larter C.Z. Ioannou G. Teoh N.C. Farrell G.C. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome.J. Hepatol. 2013; 59: 144-152Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), Ath-fed foz/foz mice develop fibrotic NASH after 24 weeks in association with obesity, hyperinsulinemia, hyperglycemia, hypoadiponectinemia, and increased hepatic FC content (Table 1). Administration of atorvastatin or ezetimibe, and particularly combined treatment with both, agents lowered hepatic FC content in Ath-fed foz/foz mice (Table 1). This was associated with amelioration of NASH, which was present in 22% of mice treated with both ezetimibe and atorvastatin, as compared with 100% of vehicle-treated controls, and with fibrosis (fibrosis stage ≥1 present in 33% of mice treated with both ezetimibe and atorvastatin mice as compared with 100% of vehicle-treated controls). Administration of ezetimibe and atorvastatin also resulted in a significant reduction in hepatic histological inflammation scores, hepatic levels of the proinflammatory transcription factor NF-κB, and its downstream proinflammatory markers vascular cell adhesion molecule 1 and ICAM-1, whereas hepatic expression of the NF-κB inhibitor B-α (IκB α) increased (Table 1).TABLE 1.Characteristics (mean ± SD) of foz/foz mice after 24 weeks on a high-fat diet with 0.2% cholesterol, categorized by receipt of pharmacological treatment during the last 8 weeks of the experimentPharmacological TreatmentVehicle n = 8Atorvastatin n = 10Ezetimibe n = 10Ezetimibe + Atorvastatin n = 9Body weight (g)52.2 ± 2.349.1 ± 1.650.0 ± 3.352.4 ± 1.4Liver weight/body weight (%)10.9 ± 0.58.4 ± 0.4aP < 0.05 compared with vehicle-treated mice.7.3 ± 0.5aP < 0.05 compared with vehicle-treated mice.6.7 ± 0.4aP < 0.05 compared with vehicle-treated mice.Hepatic lipids (mg/g)Triglyceride379 ± 48238 ± 39283 ± 40161 ± 28aP < 0.05 compared with vehicle-treated mice.Cholesterol ester39.2 ± 415 ± 4aP < 0.05 compared with vehicle-treated mice.11 ± 3aP < 0.05 compared with vehicle-treated mice.4 ± 2aP < 0.05 compared with vehicle-treated mice.Cholesterol1.3 ± 0.50.7 ± 0.20.7 ± 0.30.4 ± 0.1aP < 0.05 compared with vehicle-treated mice.Serum levels, fastingALT (U/l)358 ± 27206 ± 20aP < 0.05 compared with vehicle-treated mice.151 ± 41aP < 0.05 compared with vehicle-treated mice.88 ± 19aP < 0.05 compared with vehicle-treated mice.Cholesterol (mmol/l)5.4 ± 0.44.2 ± 0.33.3 ± 0.43.2 ± 0.4aP < 0.05 compared with vehicle-treated mice.HDL cholesterol (mmol/l)2.2 ± 0.11.8 ± 0.11.6 ± 0.1aP < 0.05 compared with vehicle-treated mice.1.5 ± 0.2aP < 0.05 compared with vehicle-treated mice.Triglyceride (mmol/l)1.17 ± 0.10.84 ± 0.11.22 ± 0.191.46 ± 0.3Glucose (mmol/l)8.35 ± 0.39.26 ± 0.79.46 ± 0.57.93 ± 0.2Insulin (ng/ml)7.80 ± 2.22.29 ± 1.09aP < 0.05 compared with vehicle-treated mice.3.20 ± 1.13aP < 0.05 compared with vehicle-treated mice.2.62 ± 0.3aP < 0.05 compared with vehicle-treated mice.Adiponectin (ng/ml)3.38 ± 0.25.9 ± 0.5aP < 0.05 compared with vehicle-treated mice.6.1 ±