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

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 score inflammation and patients with histological simple defined as score with M. C. Liu Y.C. A. et and of a histological for nonalcoholic fatty liver PubMed Scopus Google Scholar). We liver from these patients that been in liver were to liver as well as of and of and clinical C57BL/6J mice were fed for a high-fat with cholesterol or a high-fat high-cholesterol were by and their composition is described in were of the by following We reported that mice fed a developed increased hepatic fat deposition with inflammation and fibrosis C. Kuver R. Haigh Farrell G.C. S. A. Yeh M.M. L.S. Ioannou G.N. of dietary cholesterol and dietary fat in PubMed Scopus Google Scholar). a developed more hepatic substantial inflammation, and fibrosis associated with adipose inflammation and a in C. Kuver R. Haigh Farrell G.C. S. A. Yeh M.M. L.S. Ioannou G.N. of dietary cholesterol and dietary fat in PubMed Scopus Google Scholar). Human and mouse liver was and with and and and with or inflammation, and fibrosis were by a liver to the by et M. C. Liu Y.C. A. et and of a histological for nonalcoholic fatty liver PubMed Scopus Google Scholar). Human liver were in and in liver to the human livers were in and liver were in and in in were to to as the and a with or a to for the presence of birefringent crystals. liver were with which free cholesterol by with its group M. C.A. cholesterol is to as by lipid in of human 2009; PubMed Scopus Google Scholar), as for in liver were with and with in for was in a small of to in and to the for 1 at were with and were and a with an in mouse and human were with which hepatic Kupffer by with and a determine whether were mouse liver were also with known as or by a to or with by with We the cells” of the to that some of these cells may be from the of of mouse or human liver were in in the were in and were with a of by and viewed a of the that been in were with and viewed a light at the of fatty and therefore potentially distinguish lipid droplets that free cholesterol with from lipid droplets that or cholesterol were from mouse liver the J. M. simple for the and of from animal Full Text PDF PubMed Google Scholar). lipid were by and the cholesterol and free cholesterol were and by fatty were by and the were by a human liver was to hepatic lipid was from mouse liver and to was the with as the hepatic of 4 to the NLRP3 inflammasome were and M. T. G. and the inflammasome in the J. Gastroenterol. 2011; PubMed Scopus Google Scholar) 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). studies were mouse liver tissue. were with from the and the Care and of the Veterans Affairs Puget Sound Health Care Human for in our to the Health Care and of patients with NASH or simple steatosis were obese with and patients with NASH of three patients with simple steatosis of patients with simple steatosis or in a fed a developed steatohepatitis and and liver hepatic cholesterol and free cholesterol and insulin and mice fed a which developed simple steatosis of mice fed a mice fed a (NASH) for NASH fatty a between and with a of in a a between and with a of of human and mouse liver with and and the presence of inflammation, and fibrosis in with and the absence of substantial inflammation or fibrosis in those with simple steatosis polarized light of liver from and mice with steatohepatitis strongly birefringent crystals within a large proportion of hepatocyte lipid droplets. droplets with birefringence also with suggesting that the birefringence was to cholesterol crystals. In livers with simple steatosis birefringence filipin in cells around hepatocytes which clearly birefringent lipid droplets and most around mouse hepatocytes the most strongly birefringent lipid which appeared as polarized light the Kupffer cells that hepatocytes with birefringent lipid with filipin that free and for and suggesting that were These were in mice and with NASH, the Kupffer cells appeared more in the human livers and these suggest that in NASH, Kupffer cells around hepatocytes that cholesterol crystals, and of mice a (NASH) with and Kupffer cells or and activated Kupffer cells or and activated Kupffer cells and viewed with polarized light to birefringent crystals, or and are of the liver viewed with or polarized light Kupffer cells around birefringent lipid droplets cholesterol in form that a in and in suggest that the Kupffer cells are activated and very similar to described crown-like structures in inflamed visceral adipose tissue. We that cells in are are and as well as the that the of Kupffer cells around the lipid droplets of dead hepatocytes by in 5 and In the Kupffer cells lipid hepatocyte and crown-like structures to those recently described in inflamed visceral adipose S. G. G. E. E. Wang S. M. and in adipose of obese mice and Lipid Res. 46: Full Text Full Text PDF PubMed Scopus Google Scholar, K.J. H. J. adipose and obesity 2007; PubMed Scopus Google Scholar). lipid droplets in in hepatocytes with or light the lipid droplets of dead hepatocytes by that were by Kupffer cells did not with suggesting that or cholesterol a and to the birefringent those suggesting that cholesterol in the Kupffer cells that aggregated around lipid droplets and foam cells with a large of lipid droplets which did not with and of the Kupffer cells with filipin and the of that these droplets free but not cholesterol or free fatty these suggest that Kupffer cells around lipid droplets of dead hepatocytes that cholesterol crystals forming crown-like and cholesterol within lipid droplets are and free fatty but free cholesterol accumulates in activated Kupffer cells which into foam cells. In contrast, mouse and human liver with simple steatosis did not have birefringent and did not with filipin within hepatocyte lipid droplets suggesting absence of cholesterol crystals and cholesterol in simple steatosis. cells were identified, did not around hepatocytes and did not with these Kupffer cells were not did not lipid and did not around hepatocytes Hepatic of 4 associated with the NLRP3 inflammasome and were in mice with NASH in mice with simple but did not Our results that cholesterol crystals were present within hepatocytes in patients with NASH and in a mouse model of NASH by a but not in patients or mice with simple steatosis. Kupffer cells dead hepatocytes that included cholesterol crystals and appeared to process the lipid droplets within these hepatocytes forming crown-like structures similar to those recently described in inflamed visceral adipose S. G. G. E. E. Wang S. M. and in adipose of obese mice and Lipid Res. 46: Full Text Full Text PDF PubMed Scopus Google Scholar, K.J. H. J. adipose and obesity 2007; PubMed Scopus Google Scholar). lipid in of cholesterol within small droplets in activated Kupffer cells that the of lipid-laden foam cells. process may represent an important mechanism in NASH of to free cholesterol and cholesterol crystals has been shown to to their activation (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). of suggest that dietary cholesterol plays an important role in the pathogenesis of NASH. We reported that addition of dietary cholesterol to a high-fat causes progression from simple steatosis to steatohepatitis in C57BL/6J mice C. Kuver R. Haigh Farrell G.C. S. A. Yeh M.M. L.S. Ioannou G.N. of dietary cholesterol and dietary fat in PubMed Scopus Google Scholar). of dietary cholesterol to a high-fat to increased hepatic inflammation, and fibrosis in LDL receptor-deficient mice 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). of cholesterol to the of which are obese and insulin to of hepatic free hepatocyte and liver fibrosis 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). of a liver to mice with NASH hepatic of free cholesterol and hepatic of by a in hepatic inflammation K. van M. van A. R. cholesterol inhibition and of non-alcoholic steatohepatitis in 2010; 584: PubMed Scopus Google Scholar). in that the effects of for most of the were from the effects of free cholesterol responsible for the K. van M. van A. R. cholesterol inhibition and of non-alcoholic steatohepatitis in 2010; 584: PubMed Scopus Google Scholar). of the liver to cholesterol the progression of simple steatosis to steatohepatitis unclear. Our results suggest that a critical be crystallization of cholesterol within fatty We a between the presence of cholesterol crystals within hepatocyte lipid droplets and the presence of NASH simple steatosis in and hepatocyte lipid droplets were in NASH and simple cholesterol crystals were present in the lipid droplets of livers with NASH but not in the lipid droplets of livers with simple steatosis. the hepatocytes with strong birefringence were associated with in Kupffer suggesting that the cholesterol crystals were more dead hepatocytes that cholesterol crystals were by Kupffer which were into activated foam cells lipid droplets of free cholesterol but fatty or cholesterol of foam cells lipid-laden hepatocyte crown-like structures similar to those recently described in inflamed visceral adipose S. G. G. E. E. Wang S. M. and in adipose of obese mice and Lipid Res. 46: Full Text Full Text PDF PubMed Scopus Google Scholar, K.J. H. J. adipose and obesity 2007; PubMed Scopus Google Scholar). Kupffer cells appeared to be lipid which hepatocyte Kupffer cells aggregated around strongly birefringent hepatocytes, forming crown-like similar to the of dead by in inflamed visceral adipose S. G. G. E. E. Wang S. M. and in adipose of obese mice and Lipid Res. 46: Full Text Full Text PDF PubMed Scopus Google Scholar, K.J. H. J. adipose and obesity 2007; PubMed Scopus Google Scholar). those however, was with cholesterol or cholesterol livers with such activated Kupffer cells the inflammation and fibrosis that distinguish NASH from simple steatosis. Our of these is that the Kupffer cells the free cholesterol cholesterol and from the large lipid droplets of dead Kupffer cells the cholesterol and by of the fatty for the absence of in the droplets within the Kupffer cells. whether from cholesterol crystals, of cholesterol or of not be and in the droplets within the Kupffer cells. Kupffer cells cholesterol crystals into activated foam which in activated the and that NAFLD to progress into NASH. of to free cholesterol and cholesterol crystals has been shown to to their activation (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). studies that cholesterol or inflammasome activation be required to of In a of Kupffer cells in the liver of LDL receptor-deficient mice fed a high-fat high-cholesterol K. van M. van A. R. cholesterol inhibition and of non-alcoholic steatohepatitis in 2010; 584: PubMed Scopus Google Scholar, K. van H. A. van R. N. et al.Dietary liver leads to hepatic inflammation in mouse models of nonalcoholic 2008; PubMed Scopus Google Scholar, F. van T. K. M. et of by and leads to hepatic inflammation and cholesterol storage in Kupffer PubMed Scopus Google Scholar, K. van M. K.J. van et of and in diet-induced nonalcoholic steatohepatitis in 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, T. van F. S.M. M. A. J. cholesterol steatohepatitis in Full Text Full Text PDF PubMed Scopus Google Scholar). However, that cholesterol was from of by the Kupffer cells F. van T. K. M. et of by and leads to hepatic inflammation and cholesterol storage in Kupffer PubMed Scopus Google Scholar, K. van M. K.J. van et of and in diet-induced nonalcoholic steatohepatitis in 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). process may also be our results suggest that of lipid droplets from dead hepatocytes an important mechanism by which are and potentially between the hepatic crown-like structures we in NASH and the crown-like structures recently described in inflamed adipose S. G. G. E. E. Wang S. M. and in adipose of obese mice and Lipid Res. 46: Full Text Full Text PDF PubMed Scopus Google Scholar, K.J. H. J. adipose and obesity 2007; PubMed Scopus Google Scholar). In the dead were shown to be by that the lipid and a of We the and of dead hepatocytes by Kupffer cells in NASH. However, we that the Kupffer cells with cholesterol-containing droplets which were not described in the of adipose We that is to the high of cholesterol and in the hepatic lipid droplets. are the and of activation of Recent studies have that activation of the NLRP3 inflammasome in hepatic Kupffer cells is an important for inflammation in NASH and may therefore to the progression from simple steatosis to NASH M. S. of in diet-induced PubMed Scopus Google Scholar, P. M. inflammasome as a novel for the and treatment of non-alcoholic steatohepatitis in Scholar, C. H. NLRP3 in cells leads to liver inflammation and fibrosis in Scholar). However, may be the inflammasome in NASH was unclear. In the of cholesterol crystals have been shown recently to the NLRP3 inflammasome in (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). these and our of cholesterol lipid by Kupffer cells in NASH, it is to that cholesterol crystals the NLRP3 inflammasome in Kupffer cells an and progression from simple steatosis to NASH. We increased of NLRP3 in our mice with NASH compared with the mice with simple but the was not of may not be that NLRP3 are by Kupffer which of liver cells. studies be to Our is by the small of patients and the that patients with NASH were not to those with simple steatosis with to or insulin human studies with a be required to the that we have described between hepatic cholesterol crystals and NASH, and to determine these are of such as insulin and Our results suggest that an important for progression of simple steatosis to steatohepatitis (NASH) be the of concentrations of free cholesterol within hepatocytes to crystallization of We a strong between the presence of cholesterol crystals within hepatocyte lipid aggregation and activation of Kupffer cells in crown-like structures around such and the presence of NASH simple in and hepatocyte lipid droplets were abundant in NASH and simple cholesterol crystals were in the lipid droplets of livers with NASH. These to be in of human liver and in animal role of these in In of our and J. who and to our We also for with with and high-fat high-fat high-cholesterol nonalcoholic fatty liver disease nonalcoholic fatty liver disease activity score nonalcoholic steatohepatitis