There appeared to be substantial Sirius Red-positive material in liver sections of vehicle-treated controls and very little in MCC950-treated mice, even though apparent difference in collagen density fell short of significance (= 0.056) (Supplementary Fig. including designation as NASH pathology, and liver fibrosis. and accompanies liver inflammation [11,14,18,25]. NLRP3 activation occurs in human NASH [11,20], while experiments in mice are an overnutrition model in which NASH accompanies the onset of obesity and its metabolic complications, including diabetes and hypercholesterolemia [27C30]. The methionine and choline deficient (MCD) dietary model causes severe steatohepatitis with liver fibrosis, whose pathogenesis entails hepatic oxidative stress as observed in human NASH [32,33]. Taken together, these novel findings provide strong support for the proposal that pharmacological blockade of NLRP3 activation in the liver can improve NASH pathology and modulate other forms of steatohepatitis, including its most critical outcome of liver fibrosis. Materials and methods Animal models Animal experiments were approved by Animal Experimentation Ethics Committees of Australian National University or college (Canberra) and UC San Diego. Atherogenic diet-fed foz/foz model Groups (n = 11C13) of female mutant (mice weighed 60 g and exhibited hyperinsulinemia, diabetes, hypertension, hypercholesterolemia, hypoadiponectinemia, and NASH with fibrosis [29,30]. From 16 weeks, mice were gavaged MCC950 (20 mg/kg body weight) or vehicle (0.9% NaCl) every day, 5 days/week until 24 weeks (Supplementary Fig. 1A). Venous blood samples (~0.2 ml) were obtained by cheek bleeding at 15 and 20 weeks. At 24 weeks, mice were fasted 4 h to determine blood glucose (Accu-Check glucometer, Roche Diagnostics), anesthetized (ketamine and xylazine), plasma and liver collected and processed for histological and molecular readouts [29,30]. Methionine and choline deficient diet model Since 2000 [32], this mouse model has been used extensively to cause severe steatohepatitis and fibrosis, resembling human NASH pathology [28]. Groups (n = 8) of 8-week-old male C57BL/6 (B6) mice were placed on MCD diet (TD90262, Teklad Mills, Madison, WI), controls were fed the identical diet supplemented with methionine and choline (MCS) (TD94149, Teklad Mills). MCD-fed mice and MCS controls were divided into groups receiving MCC950 (10 mg/kg body weight in 0.9% NaCl every day for 5 days, followed by 20 mg/kg every second day up to 6 weeks), or vehicle by gavage. At the end of experiments, mice were anesthetized, liver and plasma collected [19,20]. In a second experiment, mice (n = 5C6/group) were fed MCD diet WYE-354 for 6 weeks, during the last 2 weeks they were gavaged every day with MCC950 (20 mg/kg) or vehicle before experiments were completed. Cholesterol crystal preparation Hydrated cholesterol crystals, prepared as explained [24], were homogenized to obtain crystals 10 m in diameter, and suspended in culture media before addition to cells. Isolation and culture of bone marrow-derived macrophages, main hepatocytes, Kupffer cells, and neutrophil chemotaxis studies Using 8C10-week-old female B6 mice, bone marrow cells were extracted and differentiated (7 days) into BMMs in murine L929 media (diluted 1:10 in RPMI with 10% FBS). Morphology was checked by Wright-Giemsa staining, phenotype by Mac1 and F4/80 double-positive cells (circulation cytometry). In selected experiments, we isolated/cultured BMMs from assessments were performed with GraphPad Prism Software Inc, CA, Version 5.03. For experiments involving 3 or more groups, data were evaluated using one-way ANOVA with multiple comparison analysis. Data are expressed as mean SEM, or as complete number or percentage for categorical variables. The number of animals or measurements in each group is usually indicated in the physique legends. The significance level was set at = 5% for all those comparisons. Results Cholesterol crystals activate NLRP3 WYE-354 in wild-type but not Myd88?/? WYE-354 macrophages, Kupffer cells EPAS1 and hepatocytes The high free cholesterol content of human and mouse NASH livers [30,38C41] results in cholesterol crystallization [27,31]. Cholesterol crystals activate NLRP3 in LPS-primed macrophages [23,24]..