NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims

NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis.

Methods

We fed foz/foz and wild-type mice an atherogenic diet for 16 weeks, gavaged MCC950 or vehicle until 24 weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6 weeks and determined the effects on liver fibrosis.

Results

In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24 weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis.

Conclusion

MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH.

Lay summary

Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

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.