Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH.

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" defi ned as hepatic steatosis without substantial infl ammation or fi brosis. Simple steatosis carries a very low risk of progression to cirrhosis and liver dysfunction ( 1 ). However, 10-30% of patients with NAFLD have or develop nonalcoholic steatohepatitis (NASH), characterized by hepatic lobular infl ammation and fi brosis in addition to steatosis. Progression to cirrhosis occurs in a proportion of patients ( 1,2 ).
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 proinfl ammatory or profi brotic 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 signifi cantly 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 (unesterifi ed) cholesterol (FC) were increased in NASH but not in simple steatosis, whereas levels of 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 ( 8 ): a ) vehicle control (n = 8); b ) atorvastatin (n = 10), 20 mg/kg body weight/day (Lipitor®; Pfi zer 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).

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
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 ( 23 ). 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 ( 6 ). Thus, foz/foz mice on the Ath diet mirror the metabolic and histological features of human NASH. Similar fi ndings have been noted in mice with other hypothalamic appetite defects, such as Mc4r-ko mice ( 13 ), refl ecting 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).

Hepatic histological assessment
Formalin-fi xed, paraffi n-embedded liver sections were stained with hematoxylin and eosin and Sirius red (for collagen). Histological steatosis, infl ammation, ballooning degeneration, and fi brosis 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. ( 24 ).

Assessment of hepatic cholesterol crystals and FC
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 fi lter to evaluate for the presence of birefringent crystals that we have previously shown to represent cholesterol crystals ( 18 ). With the polarizing fi lter in place, images of 10 random fi elds were taken (200× original magnifi cation; 0.26 mm 2 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 fi lipin to visualize FC ( 18 ). Filipin interacts with the 3 ␤hydroxy group of cholesterol to fl uoresce blue ( 25 ).
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.
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 ( 18 ). 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 infl amed visceral adipose tissue ( 19,20 ). 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 fi ndings are particularly relevant because cholesterol crystals have recently been shown to activate the NLRP3 infl ammasome in animal models of atherosclerosis ( 21,22 ), thus providing a mechanism by which exposure of KCs to cholesterol crystals could lead to chronic infl ammation and resultant fi brosis 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 fi ndings 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 fi brosis.

Animal procedures
These experiments were performed on mice used previously to analyze the therapeutic effects of cholesterol-lowering drugs on NASH pathology ( 8 ). 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 We used real-time PCR to quantify mRNA gene expression levels as previously described ( 8 ) of the following components of the NLRP3 infl ammasome in liver tissue: Caspase-1 , Nalp3 , and Asc (apoptosis-associated speck-like, caspase recruitment domain containing protein) ( 26 ).

Statistical analyses
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 signifi cant.

Treatment of obese, diabetic mice with ezetimibe and atorvastatin to lower hepatic FC levels resolves hepatocyte cholesterol crystals in association with resolution of NASH
As previously reported ( 6,8 ), Ath-fed foz/foz mice develop fi brotic NASH after 24 weeks in association with obesity, hyperinsulinemia, hyperglycemia, hypoadiponectinemia, and increased hepatic FC content ( Table 1 ). Administration of KCs) and with anti-TNF-␣ antibodies, which identify activated M1 macrophages, as described previously ( 8,18 ). 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 fi elds (200× original magnifi cation) per liver. The percent area that stained with anti-TNF ␣ in 10 random fi elds 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 Scientifi c, 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 infl ammasome 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 infl ammasome: cleaved caspase-1 can mediate the downstream effects of the NLRP3 infl ammasome by cleaving and activating pro-interleukin (IL)-1 and pro-IL-18. All primary antibodies were identifi ed using secondary antibodies labeled with AlexaFluor 488 (Invitrogen, Camarillo, CA) examined with the FITC fi lters on a Nikon fl uorescent microscope.  remaining had specks of birefringence in the periphery of the lipid droplet. The foz/foz mice that were treated with both ezetimibe and atorvastatin for the last 8 weeks of the experiment had almost complete resolution of fi lipin staining and of cholesterol crystals ( Fig. 2 ), which accounted for 0.01% (SD, 0.02) of liver surface area as compared with 3.3% (SD, 2.2) in the vehicle-treated mice ( P < 0.05) ( Table 1 ). Intermediate levels of cholesterol crystallization were observed in mice treated with atorvastatin alone (1%), and even lower levels (0.1%) of cholesterol crystallization were observed with ezetimibe treatment alone.

Treatment with ezetimibe and atorvastatin caused resolution of hepatic CLSs
Vehicle-treated foz/foz mice fed a high-fat diet developed CLSs of activated macrophages identifi ed by TNF ␣ staining surrounding hepatocytes that exhibited intense cholesterol crystallization within large lipid droplets ( Fig. 3A , B ). CLSs were completely absent in mice treated with ezetimibe and atorvastatin ( Fig. 3G-, H ). In mice 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 fi brosis (fi brosis stage у 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 signifi cant reduction in hepatic histological infl ammation scores, hepatic levels of the proinfl ammatory transcription factor NF-B, and its downstream proinfl ammatory markers vascular cell adhesion molecule 1 and ICAM-1, whereas hepatic expression of the NF-B inhibitor B-␣ (I B ␣ ) increased ( Table 1 ).
Frozen liver sections of the vehicle-treated foz/foz mice exhibited striking birefringence under polarized light within hepatocyte lipid droplets ( Fig. 1 ). Filipin staining confi rmed that the birefringent material consisted of cholesterol crystals ( Fig. 2 ). Some lipid droplets were entirely birefringent, appearing as "Maltese crosses," and the Fig. 1. Treatment with ezetimibe and atorvastatin eliminates hepatic cholesterol crystals and ameliorates infl ammation and fi brosis. Liver sections of foz/foz mice on an atherogenic diet treated with "vehicle" (control), ezetimibe, or atorvastatin or with a combination of both ezetimibe and atorvastatin ("combination") and stained with hematoxylin and eosin (H&E), Sirius red (for collagen), anti-F4/80 antibodies (for macrophages), or unstained frozen sections viewed with polarized light for birefringent cholesterol crystals. H&E or Sirius red stained liver sections demonstrate that foz/foz mice on an atherogenic diet treated with "vehicle" (control) developed fi brosing NASH. In contrast, foz/foz mice treated with combination of ezetimibe and atorvastatin show resolution of fi brotic NASH. Anti-F4/80 staining, which identifi es macrophages, shows multiple rings of macrophages forming CLSs as they surround hepatocyte lipid droplets in the vehicletreated mice. In the ezetimibe-and/or atorvastatin-treated mice, mostly scattered isolated macrophages were seen, with rare CLSs in the ezetimibe-or atorvastatin-treated mice and none in the mice treated with both drugs. Frozen liver sections viewed with polarized light demonstrate birefringence within a large proportion of lipid droplets in the "vehicle"-treated group (control), including droplets that are entirely birefringent appearing as a "Maltese cross." There was progressively less birefringence in the atorvastatin-treated and ezetimibe-treated groups and almost no birefringence in the group that was treated with both drugs. This birefringence is due to cholesterol crystallization as demonstrated by fi lipin staining in Figure 3 . Black scale bars are 50 µm; white scale bars are 100 µm. IHC, immunohistochemistry.

Macrophages in CLSs surrounding steatotic hepatocytes with cholesterol crystals demonstrate NLRP3 activation
The KCs forming CLSs stained intensely positive for NLRP3 in vehicle-treated mice ( Fig. 4A , B ), whereas practically no NLRP3 staining was observed in mice treated with both ezetimibe and atorvastatin ( Fig. 4C, D ). Furthermore, the KCs in CLSs expressed activated (cleaved) caspase 1 in the vehicle-treated mice ( Fig. 4E, F ) but not in the ezetimibe-and atorvastatin-treated mice ( Fig. 4G, H ). The presence of cleaved caspase 1 confi rms activation of the infl ammasome pathway in KCs within CLS as a possible response to cholesterol crystals in NASH. mRNA gene expression levels of critical components of the NLRP3 infl ammasome ( Nalp3 , Asc , and Caspase1 ) in whole liver treated with either ezetimibe or atorvastatin alone, partial (incomplete) resolution of CLSs was observed, with ezetimibe having a more profound effect than atorvastatin. There was striking correlation between the presence and density of cholesterol crystals and the presence/absence of CLSs. Cholesterol crystal presence and density also correlated with the presence/absence of histological NASH after treatment with vehicle versus atorvastatin and/or ezetimibe. These consistent relationships support a causal association between the formation of cholesterol crystals and CLSs in the pathogenesis of NASH.
Staining for neutrophils (antimyeloperoxidase) or T cells (anti-CD3) demonstrated that these cells were not associated with CLSs and were present in very low numbers relative to macrophages (data not shown). Mice supplemented with vehicle, which developed NASH, had strongly birefringent crystals within a large proportion of lipid droplets (A) that also stain with fi lipin (B), demonstrating that they represent cholesterol crystals. Mice supplemented with both ezetimibe and atorvastatin, in which NASH was almost completely ameliorated, did not have any hepatic cholesterol crystals (G, H). Mice supplemented with only atorvastatin or only ezetimibe, in which NASH was only partly ameliorated, had some evidence of hepatic cholesterol crystals, especially in the atorvastatin group, but reduced as compared with the group that received both drugs. Mice supplemented with vehicle, which developed NASH, had strongly birefringent crystals within a large proportion of lipid droplets (A) that were surrounded by crown-like structures of activated macrophages (B). Mice supplemented with both ezetimibe and atorvastatin, in which NASH was almost completely ameliorated, did not have any hepatic cholesterol crystals (G) or macrophage crown-like structures (H). Mice supplemented with only atorvastatin or only ezetimibe, in which NASH was only partly ameliorated, had some evidence of hepatic cholesterol crystals and CLSs, especially in the atorvastatin group, but this amount was reduced as compared with the group that received both drugs.
hepatocytes. Here we demonstrate that hepatic cholesterol crystals and CLSs are also present in a different mouse model of NAFLD/NASH, the foz/foz mouse on an atherogenic diet. Furthermore, treatment with ezetimibe and atorvastatin, which causes resolution of fi brotic NASH in these mice, also resulted in resolution of hepatic cholesterol crystals and CLSs. This suggests a causative association between hepatic cholesterol crystals and CLSs and the development of NASH. Because ezetimibe and atorvastatin are widely clinically available, our results could also inform future human clinical trials if hepatic cholesterol crystallization and formation of CLSs is further confi rmed tissue was lower in ezetimibe-and atorvastatin-treated mice than in vehicle-treated mice, but this difference did not reach statistical signifi cance ( Fig. 4G-1 ).

DISCUSSION
We recently reported that cholesterol crystals were present in hepatocyte lipid droplets in the setting of experimental and human NASH but not in simple steatosis ( 18 ). CLSs of activated KCs surrounded and processed cholesterol crystal-containing remnant lipid droplets of dead Frozen liver sections are shown from foz/foz mice fed a high-fat, high-cholesterol diet for 24 weeks. Each section is viewed with polarized light (to demonstrate cholesterol crystals) or fl uorescent light. A and B: KCs from vehicle-treated mice stained with anti-NLRP3 (identifi es the NLRP3 infl ammasome, colored green) showing that the macrophages that aggregate around heavily crystallized droplets forming CLSs exhibit critical components of the NLRP3 infl ammasome. C and D: KCs from mice treated with both ezetimibe and atorvastatin stained with fl uorescently labeled anti-NLRP3 and showing no staining. E and F: KCs from vehicle-treated mice stained with FLICA caspase 1, which identifi es activated (cleaved) caspase 1 (red), showing that the macrophages in CLSs expressed activated caspase 1 and can potentially cleave pro-IL1 into IL1. G and H: KCs from mice treated with both ezetimibe and atorvastatin showing no staining. I, J, and K: mRNA expression levels (mean ± SEM) for components of the NLRP3 infl ammasome ( Caspase 1 , Nalp3 , and Asc , respectively) demonstrating reduced expression in mice treated with ezetimibe and atorvastatin relative to vehicle-treated mice (this did not reach statistical signifi cance defi ned as P < 0.05).
to be an important pathogenetic mechanism in human NASH. Finally, our results suggest that hepatic cholesterol crystals mediate progression to NASH via activation of the NLRP3 infl ammasome within the KCs that are exposed to cholesterol crystals in CLSs.
High levels of intracellular-free cholesterol can lead to cholesterol crystallization, which has been shown to result in cell toxicity in macrophages in the setting of atherosclerosis, potentially by physically disrupting the integrity of intracellular structures ( 27 ). Crystallized cholesterol originating in foamy macrophages is in fact an important contributor to the atheromatous plaque. By analogy with these mechanisms of cholesterol crystal formation and cellular toxicity in the setting of atherosclerosis, it is tempting to postulate that cholesterol crystallization within hepatocyte lipid droplets may also lead to cell toxicity. Dead hepatocytes containing cholesterol crystals within their remnant lipid droplets stimulate the aggregation of KCs around them, causing formation of the characteristic CLSs that we describe. Processing of the remnant lipid droplets by KCs creates even more free cholesterol as the cholesterol esters within the lipid droplets are hydrolyzed. This additional free cholesterol also crystallizes, leading to the intensely birefringent lipid droplets in the center of CLSs, some appearing as a "Maltese cross," which represents supersaturated, metastable cholesterol in liquid crystalline form that is on the verge of undergoing a phase transition to cholesterol microcrystals. Further experiments are needed to specifi cally determine the factors that infl uence cholesterol crystallization within hepatocyte lipid droplets and how such cholesterol crystallization might affect the structure and function of the lipid droplet and induce cell toxicity or cell death.
Infl ammasomes are molecular platforms activated upon cellular infection or stress that trigger the maturation of proinfl ammatory cytokines such as IL-1 ␤ to engage innate immune defenses ( 28 ). The infl ammasomes sense pathogen-associated molecular patterns in the cytosol as well as host-derived signals, known as "damage-associated molecular patterns," which indicate cellular damage or stress. The NLRP3 infl ammasome also drives infl ammation in response to exposure to crystals that are known to cause human chronic infl ammatory conditions (29)(30)(31)(32). Cholesterol crystals are the latest addition to the types of crystals shown to activate the NLRP3 infl ammasome in the setting of an important, human chronic infl ammatory condition, in this case atherosclerosis ( 21,22 ). We demonstrated that KCs become exposed to crystallized cholesterol as they aggregate into CLSs around remnant lipid droplets containing cholesterol crystals ( Fig. 3 ). We also showed that KCs in CLSs stain strongly positive with NLRP3. Taken together with the recent literature on NLRP3 activation in macrophages by cholesterol crystals in atherosclerosis, our fi ndings suggest that exposure of KCs to cholesterol crystals may activate the NLRP3 infl ammasome, leading to chronic infl ammation and contributing to the development of NASH. Specifi cally designed experiments are needed to prove whether the NLRP3 infl ammasome is an important mediator of chronic infl ammation induced by exposure of hepatocytes and KCs to cholesterol crystals.
There was a striking correlation between the presence/ absence of hepatic cholesterol crystals and CLSs and the presence/absence of NASH after treatment with ezetimibe and atorvastatin. The foz/foz mice that were treated with ezetimibe and atorvastatin for the last 8 weeks of the experiment had almost complete resolution of cholesterol crystals (0.01% of liver surface area as compared with 3.3% in the vehicle-treated mice) and CLSss, as shown in Figs. 2 and 3 , together with a profound amelioration of fi brotic NASH. Ezetimibe alone and atorvastatin alone had intermediate effects on cholesterol crystallization and formation of CLSs, with the effect of ezetimibe being greater than that of atorvastatin, again correlating with the degree of resolution of fi brotic NASH. These tight correlations point to a causative association between hepatic cholesterol crystals, CLSs, and NASH.
The additive effects of ezetimibe and atorvastatin on ameliorating hepatic cholesterol crystals can be explained by the fact that they reduce the exposure of the liver to cholesterol via different and independent pathways. Atorvastatin inhibits hepatic cholesterol synthesis by inhibiting HMG-CoA reductase, whereas ezetimibe inhibits the intestinal absorption of cholesterol by binding Niemann-Pick C1 like-1 ( 33 ). In addition, because Niemann-Pick C1 like-1 is expressed in hepatocytes and biliary epithelial cells as well as in enterocytes ( 34 ), ezetimibe may interrupt the hepatic recycling of cholesterol secreted into bile and taken up by biliary duct epithelium. Our results do not suggest that the mechanism by which hepatic cholesterol exposure is reduced is important; rather, the degree of reduction of hepatic cholesterol exposure seems to be more important than the mechanism by which it is achieved.
An emerging literature suggests that cholesterol-lowering drugs may be benefi cial for hepatic outcomes in human NASH. Four randomized control trials ( 35-38 ) (a total of 719 participants) assessed statins in patients with NAFLD. Statins improved alanine aminotransferase and radiological steatosis in hyperlipidemic patients with NAFLD and caused dramatic reductions in cardiovascular events ( 35 ). Only one study evaluated histological outcomes and found no improvement in hepatic inflammation or fibrosis after 12 months of simvastatin; however, only 10 patients underwent repeat liver biopsies, and the study was clearly underpowered ( 38 ). In small pilot clinical trials, treatment of patients with biopsy-proven NASH with ezetimibe for 6 ( 16 ) or 24 ( 17 ) months was associated with improvements in hepatic steatosis, infl ammation, and hepatocyte ballooning but not fi brosis ( 17 ). However, these studies were not randomized or controlled. In a small, randomized controlled trial, addition of ezetimibe to a moderate weight loss diet for 16 weeks in obese subjects significantly improved hepatic steatosis, infl ammation, and LDL-apoB-100 metabolism ( 39 ). Finally, in a recent small, open-label randomized control trial, treatment with ezetimibe for 6 months improved hepatic fi brosis but increased hemoglobin A1c levels ( 40 ). In summary, although existing data showing that cholesterol-lowering drugs benefi t NAFLD pathology and disease progression are fragmentary, this approach is worthy of controlled study, particularly using aggressive cholesterol-lowering approaches now recommended for those at high risk of cardiovascular events ( 41 ).
Further work is required in humans to establish more fi rmly that hepatic cholesterol crystals and CLSs are associated with NASH but not with simple steatosis. Our current work in mice might be relevant because ezetimibe and atorvastatin are clinically available and could be used in preliminary clinical trials to determine if they lead to resolution of hepatic cholesterol crystals and CLSs. Finally, the mechanistic importance of cholesterol crystal-mediated activation of NLRP3 infl ammasomes in KCs in NASH requires further elucidation because this could provide a novel, much-needed therapeutic pathway for this common liver disease.