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Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis

2013; Elsevier BV; Volume: 54; Issue: 5 Linguagem: Inglês

10.1194/jlr.m034876

1539-7262

George N. Ioannou, W. Geoffrey Haigh, David Thorning, Christopher E. Savard,

Diet, Metabolism, and Disease

We sought to determine whether hepatic cholesterol crystals are present in patients or mice with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH), and whether their presence or distribution correlates with the presence of NASH as compared with simple steatosis. We identified, by filipin staining, free cholesterol within hepatocyte lipid droplets in patients with NASH and in C57BL/6J mice that developed NASH following a high-fat high-cholesterol diet. Under polarized light these lipid droplets exhibited strong birefringence suggesting that some of the cholesterol was present in the form of crystals. Activated Kupffer cells aggregated around dead hepatocytes that included strongly birefringent cholesterol crystals, forming "crown-like structures" similar to those recently described in inflamed visceral adipose tissue. These Kupffer cells appeared to process the lipid of dead hepatocytes turning it into activated lipid-laden "foam cells" with numerous small cholesterol-containing droplets. In contrast, hepatocyte lipid droplets in patients and mice with simple steatosis did not exhibit cholesterol crystals and their Kupffer cells did not form crown-like structures or transform into foam cells. Our results suggest that cholesterol crystallization within hepatocyte lipid droplets and aggregation and activation of Kupffer cells in crown-like structures around such droplets represent an important, novel mechanism for progression of simple steatosis to NASH. We sought to determine whether hepatic cholesterol crystals are present in patients or mice with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH), and whether their presence or distribution correlates with the presence of NASH as compared with simple steatosis. We identified, by filipin staining, free cholesterol within hepatocyte lipid droplets in patients with NASH and in C57BL/6J mice that developed NASH following a high-fat high-cholesterol diet. Under polarized light these lipid droplets exhibited strong birefringence suggesting that some of the cholesterol was present in the form of crystals. Activated Kupffer cells aggregated around dead hepatocytes that included strongly birefringent cholesterol crystals, forming "crown-like structures" similar to those recently described in inflamed visceral adipose tissue. These Kupffer cells appeared to process the lipid of dead hepatocytes turning it into activated lipid-laden "foam cells" with numerous small cholesterol-containing droplets. In contrast, hepatocyte lipid droplets in patients and mice with simple steatosis did not exhibit cholesterol crystals and their Kupffer cells did not form crown-like structures or transform into foam cells. Our results suggest that cholesterol crystallization within hepatocyte lipid droplets and aggregation and activation of Kupffer cells in crown-like structures around such droplets represent an important, novel mechanism for progression of simple steatosis to NASH. Nonalcoholic fatty liver disease (NAFLD), defined as increased lipid deposition within hepatocytes in the absence of viral hepatitis or excessive alcohol consumption, is extremely common affecting 15–46% of adults in the United States (1Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2927) Google Scholar, 2Bellentani S. Saccoccio G. Masutti F. Croce L.S. Brandi G. Sasso F. Cristanini G. Tiribelli C. Prevalence of and risk factors for hepatic steatosis in Northern Italy.Ann. Intern. Med. 2000; 132: 112-117Crossref PubMed Scopus (1065) Google Scholar, 3Williams C.D. Stengel J. Asike M.I. Torres D.M. Shaw J. Contreras M. Landt C.L. Harrison S.A. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study.Gastroenterology. 2011; 140: 124-131Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar). However, the majority of patients with NAFLD have "simple steatosis" defined by hepatic steatosis in the absence of substantial inflammation or fibrosis. Such patients generally have a benign clinical course with a very low probability of developing progressive liver dysfunction and cirrhosis (4Matteoni 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 (2798) Google Scholar). In contrast, 10–30% of patients with NAFLD develop a more aggressive condition known as nonalcoholic steatohepatitis (NASH) (3Williams C.D. Stengel J. Asike M.I. Torres D.M. Shaw J. Contreras M. Landt C.L. Harrison S.A. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study.Gastroenterology. 2011; 140: 124-131Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar), characterized by varying degrees of hepatic inflammation, balloon hepatocytes, and fibrosis in addition to hepatic steatosis. Steatohepatitis can progress to cirrhosis, liver failure, and hepatocellular carcinoma in a variable proportion of patients (4Matteoni 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 (2798) Google Scholar, 5Bugianesi 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 (1262) Google Scholar). It is clearly established that central obesity and insulin resistance are important risk factors for the development of hepatic steatosis. However, the factors responsible for the development of progressive steatohepatitis remain unclear. Determining these factors would: i) clarify the pathogenesis of progressive steatohepatitis; ii) help to distinguish the subgroup of patients with NAFLD who are likely to develop progressive NASH and cirrhosis; and iii) potentially point to targeted treatments. Recent reports by our group and others suggest that dietary and hepatic cholesterol are critical factors in the development of steatohepatitis in animal models (6Matsuzawa 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 (410) Google Scholar, 7Zheng 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 (142) Google Scholar, 8Subramanian S. Goodspeed L. Wang S.A. Kim J. Zeng L. Ioannou G.N. Haigh W.G. Yeh M.M. Kowdley K.V. O舗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 (174) Google Scholar, 9Van 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: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Human studies also support the hypothesis that dietary cholesterol plays a role in the development of steatohepatitis. In a large nationally representative epidemiological study, we reported that dietary cholesterol consumption was independently associated with the development of cirrhosis (10Ioannou 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 (130) Google Scholar). Finally, inhibition of intestinal cholesterol absorption by administration of ezetimibe to patients with NASH (11Yoneda 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: 613-621Crossref PubMed Scopus (112) Google Scholar, 12Park 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 (158) Google Scholar) has been reported to improve hepatic inflammation and steatosis; these studies were not, however, randomized or controlled. Cholesterol, a naturally occurring molecule abundant in most tissues, has traditionally been viewed as inert. Recently, however, its crystalline form has been shown to induce inflammation by stimulating the NLRP3 inflammasome in animal models of atherosclerosis (13Duewell 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 (2586) Google Scholar). We therefore hypothesized that cholesterol crystals may form in fatty livers, which are characterized by high concentrations of cholesterol as well as other lipids, and may be the hitherto unrecognized signal that leads to the progression from simple steatosis to progressive steatohepatitis. Cholesterol crystals have been described before in the livers of patients with cholesteryl ester storage disease (14Di Bisceglie A.M. Ishak K.G. Rabin L. Hoeg J.M. Cholesteryl ester storage disease: hepatopathology and effects of therapy with lovastatin.Hepatology. 1990; 11: 764-772Crossref PubMed Scopus (45) Google Scholar), but not, to our knowledge, in patients with NAFLD/NASH. We therefore aimed to determine whether cholesterol crystals are present in the livers of patients with NAFLD/NASH or in a mouse model of NAFLD/NASH. Additionally, we wanted to determine whether the presence or distribution of hepatic cholesterol crystals correlates with the presence of NASH and distinguishes it from simple steatosis. From an existing human liver biorepository at Veterans Affairs Puget Sound Health Care System (VAPSHCS), we randomly selected 4 patients with histological NASH, defined as NAFLD activity score (NAS) of 5 or greater, with at least 1 point in each of the three components of the NAS score (steatosis 0–3, lobular inflammation 0–3, ballooning degeneration 0–2); and 3 patients with histological simple steatosis, defined as NAS score 2–3 with no ballooning degeneration (15Kleiner 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 (7151) Google Scholar). We retrieved liver tissue from these patients that had been flash frozen in liquid nitrogen immediately after liver biopsy. Fasting laboratory tests were prospectively performed just prior to liver biopsy as well as completion of questionnaires and collection of demographic and clinical information. Male C57BL/6J littermate mice were fed for 30 weeks either a high-fat (HF) (15%, w/w) diet with no cholesterol (n = 8) or a high-fat (15%) high-cholesterol (1%) (HFHC) diet (n = 8). The experimental diets were prepared by Bioserve (Frenchtown, NJ) and their composition is described in supplementary Table I. Mice were euthanized 30 weeks after initiation of the experimental diets by cervical dislocation following isoflurane anesthesia. We previously reported that mice fed a HF diet developed increased hepatic fat deposition with little inflammation and no fibrosis (simple steatosis) (16Savard C. Tartaglione E.V. Kuver R. Geoffrey Haigh W. 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 (192) Google Scholar). Mice on a HFHC diet developed significantly more profound hepatic steatosis, substantial inflammation, and perisinusoidal fibrosis (steatohepatitis), associated with adipose tissue inflammation and a reduction in plasma adiponectin levels (16Savard C. Tartaglione E.V. Kuver R. Geoffrey Haigh W. 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 (192) Google Scholar). Human and mouse formalin-fixed paraffin-embedded liver tissue was sectioned and stained with hematoxylin and eosin (H and E) and with Masson舗s trichrome or Sirius red (for collagen). Histological steatosis, inflammation, and fibrosis were assessed semiquantitatively by a "blinded" liver pathologist according to the scoring system proposed by Kleiner et al. (15Kleiner 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 (7151) Google Scholar). Human liver biopsies were placed in cryovials and frozen in liquid nitrogen immediately after liver biopsy. Just prior to cutting, the frozen human livers were embedded in optimal cutting temperature (OCT) compound and frozen on dry ice. Mouse liver portions were embedded in OCT and frozen in liquid nitrogen immediately after removal. Frozen sections (10 µM in thickness) were allowed to come to room temperature, immediately coverslipped using pure glycerol as the mounting medium without applying any stain, and examined using a Nikon Eclipse microscope with or without a polarizing filter, to evaluate for the presence of birefringent crystals. Frozen liver sections were stained with filipin, which identifies free cholesterol by interacting with its 3β-hydroxy group (17Rudolf M. Curcio C.A. Esterified cholesterol is highly localized to Bruch舗s membrane, as revealed by lipid histochemistry in wholemounts of human choroid.J. Histochem. Cytochem. 2009; 57: 731-739Crossref PubMed Scopus (62) Google Scholar), as follows. After fixing for 15 min in formalin, liver sections were washed with phosphate buffered saline (PBS), and then treated with 10% fetal bovine serum (FBS) in PBS for 30 min. Filipin (Sigma Chemical Co., St. Louis, MO) was dissolved in a small volume of dimethylsulfoxide then diluted to 0.25 mg/ml in 10% FBS/PBS and added to the tissue for 1 h at room temperature. Slides were washed with 10% FBS/PBS once and PBS twice. Slides were coverslipped using Aquamount (Lerner, Pittsburgh, PA) and examined using a fluorescent Nikon Eclipse microscope with an excitation 340-380/emission 435-485 filter in place. Frozen mouse and human sections were stained with anti-CD68 antibodies, which identify macrophages (including hepatic Kupffer cells), followed by secondary antibodies labeled with Alexa Fluor 488 (Invitrogen, Camarillo, CA) and then examined using a fluorescent microscope. To determine whether macrophages were activated, mouse liver sections were also stained with rat anti-mouse CD11b (also known as macrophage-1 antigen or Mac-1) antibodies identified by a goat anti-rat secondary antibody linked to horseradish peroxidase or with anti-tumor necrosis factor α (TNFα) antibodies identified by secondary antibodies labeled with Alexa Fluor 488. We will henceforth use the term "Kupffer cells" (resident tissue macrophages of the liver) to describe CD68-positive cells, while acknowledging that some of these cells may be macrophages recruited from the circulation. At time of tissue removal, pieces of mouse or human liver were fixed in Trump舗s fixative. After postfixation in osmium tetroxide, the samples were embedded in epoxy resin and thinly sliced (0.12 µM). The sections were stained with a solution of uranyl acetate followed by aqueous lead citrate and viewed using a JEOL (Tokyo, Japan) transmission electron microscope. Thicker sections (1 µM) of the embedded tissue that had been fixed in osmium tetroxide were counterstained with methylene blue and viewed under a regular light microscope. Osmium tetroxide binds at the carbon-carbon double bonds of unsaturated fatty acids, and therefore could potentially distinguish lipid droplets that contained only free cholesterol (no staining with osmium) from lipid droplets that contained triglycerides or cholesterol esters. Lipids were extracted from frozen mouse liver using the Folch method (18Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The neutral lipid fractions were prepared by solid phase extraction on Bond Elut Si cartridges (Varian Corp., Walnut Creek, CA) and the triglycerides, diglycerides, cholesterol esters, and free cholesterol were then separated and quantified by normal phase HPLC/ELSD. Free fatty acids were esterified by boron trifluoride/methanol and the methyl esters were then separated by GC, using a 60 m HP-Innowax capillary column (Agilent Technologies, Santa Clara, CA). Insufficient frozen human liver tissue was available to perform hepatic lipid analysis. Total RNA was isolated from mouse liver tissue using RNeasy minicolumns (Qiagen, Valencia, CA) and reverse transcribed to cDNA. Quantitative real-time RT-PCR was performed using the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA) with β-actin as the housekeeping gene. The hepatic gene expression levels of 4 genes related to the NLRP3 inflammasome were assessed [Nalp3, ASC (apoptosis-associated speck-like caspase recruitment domain containing protein), Caspase-1, and Pannexin-1] (19Ganz 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) because cholesterol crystals have been shown to induce inflammation by stimulating the NLRP3 inflammasome in animal models of atherosclerosis (13Duewell 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 (2586) Google Scholar). Gene expression studies were only performed on mouse liver tissue. All experimental procedures were undertaken with approval from the Institutional Review Board and the Institutional Animal Care and Use Committee of the Veterans Affairs Puget Sound Health Care System. Human subjects provided informed consent for participation in our biorepository. Animal investigations conformed to the Public Health Policy on Humane Care and Use of Laboratory Animals. All patients with NASH (n = 4) or simple steatosis (n = 3) were obese males with mildly elevated serum ALT level and normal serum bilirubin level (Table 1). All patients with NASH had diabetes mellitus while only one of three patients with simple steatosis had diabetes.TABLE 1Characteristics of patients with simple steatosis versus NASHSimple SteatosisNASHPatient number1234567Steatosis (0–3)1222321Inflammation (0–3)1112122Ballooning (0–2)0001222Fibrosis (0–4)0001A1A23Age37606244414561GenderMaleMaleMaleMaleMaleMaleMaleRaceWhiteWhiteWhiteBlackWhiteWhiteWhiteDiabetesNoYesNoYesYesYesYesBody mass index (Kg/m2)30323136443536Plasma or serum levels, fastingALT (U/L)746144788367104Bilirubin (mg/dl)0.50.20.30.30.60.60.4Insulin (µU/ml)161322405996133Glucose (mg/dl)85109102143127273104Hemoglobin A 1c (%)5.27.05.47.07.17.97.6 Open table in a new tab Mice fed a HFHC diet developed steatohepatitis and had higher body weight and liver weight; higher hepatic triglyceride, diglyceride, cholesterol ester, and free cholesterol concentration; higher plasma ALT, cholesterol, and insulin levels; and lower serum adiponectin levels than mice fed a HF diet which developed simple steatosis (Table 2).TABLE 2Characteristics (mean ± SD) of mice fed a HF diet (simple steatosis) versus mice fed a HFHC diet (NASH) for 30 weeksHF Diet Simple Steatosis (N = 8)HFHC Diet NASH (N = 8)Body weight (g)36.3 ± 4.939.9 ± 3.8*Body weight gain (%)13 ± 432 ± 7*Liver weight (g)1.7 ± 0.83.4 ± 0.1*Liver weight/body weight (%)4.7 ± 1.38.6 ± 1.7*Hepatic lipids (mg/g)Triglyceride97 ± 81235 ± 75*Diacylglyceride1.2 ± 0.23.0 ± 1.3*Cholesterol ester0.8 ± 0.6108 ± 38*Cholesterol0.48 ± 0.231.22 ± 0.6*Free fatty acid3.1 ± 0.63.7 ± 1.5Plasma levels, fastingALT (U/L)88 ± 83319 ± 111*Cholesterol (mg/dl)86 ± 49262 ± 66*Triglyceride (mg/dl)44 ± 1235 ± 14Glucose (mg/dl)239 ± 35290 ± 44*Insulin (ng/ml)2.78 ± 1.773.26 ± 2.49Adiponectin (µg/ml)5.2 ± 1.43.2 ± 0.8*Hepatic mRNA analysis (relative expression)Nalp31 ± 0.51.8 ± 1.3ASC1 ± 0.61.5 ± 0.9Caspase-11 ± 0.71.5 ± 1.5Pannexin-11 ± 0.51.2 ± 0.7*Indicates a comparison between HFHC and HF with a statistical significance of P < 0.05. Open table in a new tab *Indicates a comparison between HFHC and HF with a statistical significance of P < 0.05. Light microscopy of formalin-fixed human and mouse liver sections stained with H and E, Sirius red, and Masson舗s trichrome confirmed the presence of lobular inflammation, steatosis, and perisinusoidal fibrosis in specimens with steatohepatitis, and the absence of substantial inflammation or fibrosis in those with simple steatosis (Fig. 1). Examination under polarized light of frozen liver sections from humans and mice (Fig. 2) with steatohepatitis revealed strongly birefringent crystals within a large proportion of hepatocyte lipid droplets. Those droplets with birefringence also stained prominently with filipin, suggesting that the birefringence was due to cholesterol crystals. In both species, livers with only simple steatosis showed neither birefringence nor filipin staining in steatotic hepatocytes. CD68-positive cells (Kupffer cells) formed "crown-like" aggregates only around hepatocytes which contained clearly birefringent lipid droplets (Figs. 3, 4, and supplementary Fig. I), most notably around mouse hepatocytes containing the most strongly birefringent lipid droplets, which appeared as "Maltese crosses" under polarized light (Fig. 4B, D). Moreover, the Kupffer cells that surrounded hepatocytes with birefringent lipid droplets, stained intensely with filipin (Fig. 3), indicating that they contained free cholesterol, and for CD11b and TNFα (Fig. 4E, F) suggesting that they were activated. These findings were observed both in mice and humans with NASH, although the Kupffer cells appeared more slender in the human livers (Fig. 3A, B and supplementary Fig. III). Taken together, these findings suggest that in NASH, Kupffer cells aggregate around hepatocytes that contain cholesterol crystals, accumulate cholesterol, and become activated.Fig. 4Liver sections of mice on a HFHC diet (NASH) stained with anti-CD68 [stains green and identifies Kupffer cells (A–D)] or anti-CD11b [stains brown and identifies activated Kupffer cells (E)] or anti-TNFα [stains green and identifies activated Kupffer cells (F)] and viewed with fluorescent microscopy (A, C, F), polarized light (B, D) to identify birefringent crystals, or bright field microscopy (E). Pairs (A, B) and (C, D) are photomicrographs of the same liver section viewed with either fluorescent or polarized light showing Kupffer cells (green) aggregating around intensely birefringent lipid droplets containing cholesterol in crystallized form that creates a Maltese cross appearance. CD11b stain in panel (E) and TNFα staining in panel (F) suggest that the Kupffer cells are activated and show morphology very similar to previously described crown-like structures in inflamed visceral adipose tissue. We confirmed that TNFα colocalizes on CD68-positive cells (Kupffer cells) in supplementary Fig. IV [(A, B, E, F) are ×200 magnification; (C, D) are ×800 magnification].View Large Image Figure ViewerDownload Hi-res image Download (PPT) The osmium and methylene blue stained sections as well as the electron micrographs demonstrated that the aggregates of Kupffer cells actually occurred around the remnant lipid droplets of dead hepatocytes (marked by asterisks in Fig. 5 and supplementary Fig. II). In many instances, the Kupffer cells directly abutted on lipid cores without evident hepatocyte cytoplasm (Fig. 5D and supplementary Fig. IID, E, H).This created crown-like structures identical to those recently described in inflamed visceral adipose tissue (20Cinti 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 (1770) Google Scholar, 21Strissel 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 (691) Google Scholar). While normal lipid droplets (marked LD in Fig. 5) in healthy hepatocytes stained gray/brown with osmium either on electron microscopy or light microscopy (Fig. 5), the remnant lipid droplets of dead hepatocytes (marked by asterisks) that were surrounded by Kupffer cells did not stain with osmium, suggesting that they no longer contained triglyceride or cholesterol esters. Instead, they contained a granular precipitate and corresponded to the intensely birefringent droplets, including those exhibiting Maltese crosses, suggesting that they contained cholesterol in crystallized form. Furthermore, the Kupffer cells that aggregated around remnant lipid droplets had enlarged and become foam cells filled with a large number of much smaller lipid droplets which did not stain with osmium (Fig. 5C, E and supplementary Fig. IIB, F). The intense staining of the Kupffer cells with filipin and the lack of osmium staining suggests that these droplets contained free cholesterol, but not cholesterol esters, triglycerides, or free fatty acids. Taken together these findings suggest that Kupffer cells aggregate around remnant lipid droplets of dead hepatocytes that contain cholesterol crystals forming crown-like structures. Triglycerides and cholesterol esters within lipid droplets are hydrolyzed and resultant free fatty acids oxidized, but free cholesterol accumulates in activated Kupffer cells which turn into foam cells. In contrast, mouse and human frozen liver sections with simple steatosis did not have any birefringent material and did not stain with filipin within hepatocyte lipid droplets (Fig. 2), suggesting absence of cholesterol crystals and unesterified cholesterol in simple steatosis. Although CD68-positive cells (Kupffer cells) were identified, they did not cluster around steatotic hepatocytes and did not stain with filipin. On electron microscopy, these Kupffer cells were not enlarged, did not contain lipid droplets, and did not aggregate around steatotic hepatocytes (supplementary Fig. II). Hepatic mRNA levels of 4 genes associated with the NLRP3 inflammasome (Nalp3, ASC, Caspase-1, and Pannexin-1) were all greater in mice with NASH than in mice with simple steatosis, but did not reach statistical significance (Table 2). Our results show that cholesterol crystals were present within steatotic hepatocytes in patients with NASH and in a mouse model of NASH induced by a HFHC diet, but not in patients or mice with simple steatosis. Enlarged Kupffer cells surrounded steatotic dead hepatocytes that included cholesterol crystals and appeared to process the remnant lipid droplets within these hepatocytes forming crown-like structures similar to those recently described in inflamed visceral adipose tissue (20Cinti 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 (1770) Google Scholar, 21Strissel 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 (691) Google Scholar). This lipid scavenging resulted in profound accumulation of cholesterol within small droplets in markedly enlarged activated Kupffer cells that took the appearance of lipid-laden foam cells. This process may represent an important pathogenetic mechanism in NASH because exposure of macrophages to excess free cholesterol and cholesterol crystals has been shown to lead to their activation (13Duewell P. Kono H. Rayner K.J. Sirois C.