Research ArticleNLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice
Graphical abstract
Introduction
Non-alcoholic fatty liver disease (NAFLD) increases standardised mortality from cardiovascular events, common cancers, cirrhosis and hepatocellular carcinoma [1], [2]. Adverse liver outcomes are confined to the 10–25% of NAFLD patients with liver fibrosis, particularly with the pathology of steatohepatitis (NASH) [3], [4]. NASH occurs when overnutrition is complicated by insulin resistance and metabolic syndrome [2], [5], [6], particularly with a personal or family history of type 2 diabetes. Despite these connections, detailed mechanisms linking metabolic obesity to liver pathology are unclear. The most compelling concept is that hepatocyte injury results from lipotoxicity, a consequence of accumulated toxic lipid species [6], [7]. Lipotoxicity, via release of danger-activated molecular patterns (DAMPs) from injured hepatocytes, and the gut microbiome via release of pathogen-activated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) activate innate immunity to cause liver inflammation [8], [9], [10].
Innate immunity involves signalling via pattern recognition receptors, such as the Toll-like receptors (TLRs). During the last few years another trigger for liver inflammation in NAFLD has been identified as the NOD-like receptor protein 3 (NLRP3) inflammasome [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Inflammasomes are multiprotein scaffolds that respond to noxious signals (PAMPs, DAMPs) to recruit the adapter protein ASC and pro-caspase 1, thereby activating caspase 1 by autocatalysis [18], [21], [22]. Since the outcomes include programed cell death (pyroptosis), inflammation and fibrosis [16], [19], [22], the threshold for inflammasome activation requires a double activation signal. For the NLRP3 inflammasome, which is highly expressed in liver, the first signal is often LPS, but TNF-α and IL-1β are among other signal-1 molecules pertinent to NASH [16], [19], [22]. Factors that potentially provide the second signal include ATP, amyloid, uric acid and cholesterol crystals [23], [24], while oxidative stress and potassium ion transport are related to the effector pathways of NLRP3 activation. In dietary and nutrient deficiency fatty liver diseases, increased hepatic expression of Nlrp3, Asc and Casp1 accompanies liver inflammation [11], [14], [18], [25]. NLRP3 activation occurs in human NASH [11], [20], while experiments in Nlrp3−/−, Casp1−/− and Asc−/− mice indicate NLRP3 activation is mechanistically important for NAFLD [11], [13], [20]. Until now, however, it has not been possible to test whether requirement for NLRP3 activation in the initiation and perpetuation of liver inflammation in NASH is obligatory, using intervention experiments. Further, we note that earlier studies did not use mice with metabolic syndrome, the invariable context of NASH in humans [1], [2], [5], [6]. Development of MCC950 as a first-in-class, highly potent and selective small molecule inhibitor of NLRP3 [26], and characterization of a hyperphagic obesity mouse model of NASH linked to type 2 diabetes and metabolic syndrome [27], [28], [29], [30], allowed us to conduct such studies, as reported here.
Cholesterol crystals activate NLRP3 in LPS-exposed macrophages [23], [24]. Recently, cholesterol crystals have been observed in livers of human NASH and murine NASH models, including the one used for the present experiments [27], [31]. Here, we first showed that cholesterol crystals activate NLRP3 in LPS-exposed Kupffer cells (KCs), bone marrow macrophages (BMMs), but minimally (if at all) in hepatocytes; 10 nM MCC950 inhibited such activation, as well as the resultant neutrophil migration in response to conditioned media from macrophages exposed to cholesterol crystals. We then conducted simultaneous studies in San Diego, CA and Canberra, Australia to test the efficacy of MCC950 for preventing or reversing liver injury, inflammation and fibrosis in two entirely different models of experimental steatohepatitis. Appetite-defective foz/foz mice are an overnutrition model in which NASH accompanies the onset of obesity and its metabolic complications, including diabetes and hypercholesterolemia [27], [28], [29], [30]. The methionine and choline deficient (MCD) dietary model causes severe steatohepatitis with liver fibrosis, whose pathogenesis involves hepatic oxidative stress as observed in human NASH [32], [33]. Taken together, these novel findings provide robust 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.
Section snippets
Animal models
Animal experiments were approved by Animal Experimentation Ethics Committees of Australian National University (Canberra) and UC San Diego.
Atherogenic diet-fed foz/foz model
Groups (n = 11–13) of female Alms1 mutant (foz/foz) mice or wild-type (Wt) littermates were fed atherogenic diet (23% fat, 45% carbohydrate, 20% protein, 0.2% cholesterol; SF03020, Specialty Feeds, Glenn Forrest, Western Australia) for 16 weeks, when foz/foz mice weighed >60 g and exhibited hyperinsulinemia, diabetes, hypertension, hypercholesterolemia,
Cholesterol crystals activate NLRP3 in wild-type but not Myd88−/− macrophages, Kupffer cells and hepatocytes
The high free cholesterol content of human and foz/foz mouse NASH livers [30], [38], [39], [40], [41] results in cholesterol crystallization [27], [31]. Cholesterol crystals activate NLRP3 in LPS-primed macrophages [23], [24]. We first demonstrated such activation (measured as IL-1β release) (Fig. 1A) and showed it is substantially inhibited by 10 nM MCC950 (Fig. 1B). Culture supernatant from crystal-exposed BMMs stimulated neutrophil chemotaxis; prior addition of MCC950 blocked this effect (Fig. 1
Discussion
The most important finding in these studies is that pharmacological inhibition of NLRP3 in vivo reduces liver inflammation and hepatocyte injury in metabolic syndrome-related NAFLD with significant reduction in resultant liver fibrosis. Hepatic NLRP3 expression increases during development of experimental and clinical NAFLD [11], [13], [14], [18], [19], [20], [25], and mice deficient in NLRP3 (Nlrp3−/−) or its essential components (Asc−/− and Casp1−/− mice) are protected against fatty liver
Financial support
Supported by NIH Project grants R2 AA023574 and U01 AA022489 (to AEF), Australian NHMRC project grants 1084136 and 1044288 (to GCF), and 1086786 (to MAC and AABR), and Deutsche Forschungsgemeinschaft WR 173/3-1 (to AW). Matthew Cooper is an NHMRC Principle Research Fellow (1059354).
Conflict of interest
ARM, AW, MMY, CDJ, DVR, FH, NCT, CS, GNI, SLM, AEF and GCF have nothing to disclose. AABR, KS, and MAC are co-inventors on a patent application filed by The University of Queensland describing novel small molecules inhibitors of the NLRP3 inflammasome. However, none of these new molecules are described in the present work, which is limited to the public domain, non-patented compound MCC950. Hence there is no direct conflict of interest in this work describing the role of MCC950 in NASH, but the
Authors’ contributions
Auvro R Mridha: experimental design and acquisition of data; analysis and interpretation of data, statistical analysis, drafting of figures, intellectual input and critical review of manuscript. Alexander Wree: experimental design and acquisition of data; analysis and interpretation of data, statistical analysis, drafting of figures and critical review of manuscript. Avril AB Robertson: experimental design and acquisition of data; interpretation of data and critical review of manuscript.
Acknowledgements
David Povero and Maria Eugenia Inzaugarat kindly assisted with conduct of additional studies in the MCD model in San Diego and assisted with preparation of the figures. The authors also gratefully acknowledge the excellent support of Vanessa Barns and Hans Wang for conduct of animal studies in Canberra.
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These authors contributed equally as joint first authors.