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

Abstract

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 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks 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 24weeks, 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.

Keywords: Cholesterol crystals; Diet, atherogenic; Fibrosis; Hepatocytes; Inflammasomes; Interleukin-1β; Kupffer cells; Methionine; NAFLD; NLR proteins; NLRP3.

Fig. 1.. Cholesterol crystals provoke IL-1β release and neutrophil recruitment by macrophages and Kupffer cells, which is inhibited by MCC950.

(A) Effects of cholesterol crystals on IL-1β release for LPS-primed bone marrow macrophages (BMM) from wild-type (Wt), Tlr4^−/−, and Myd88^−/− mice. (B) MCC950 (10 nM) inhibits IL-1β production from Wt BMMs. (C) Enriched culture medium from crystal activated BMMs stimulates neutrophil chemotaxis. (D) Cholesterol crystals stimulate IL-1β release from Kupffer cells, but (E) minimally in hepatocytes. *p <0.05 vs. no crystals. ^Δp <0.05 vs. Wt. ^#p <0.05 MCC950 vs. untreated, by Mann-Whitney U test, n = 9–12.

Fig. 2.. Effects of conditions causing NASH on hepatic expression of NLRP3, pro-caspase 1, active caspase 1, pro-IL-1β and IL-1β.

(A) NLRP3 in wild-type (Wt) and foz/foz mice after 24 weeks atherogenic diet, and gavage during the last 8 weeks with vehicle (saline) or MCC950. (B) Hepatic pro-caspase 1 (p45), (C) active caspase 1 (p10), (D) pro-IL-1β (p30), and (E) IL-1β (p17). (F) Representative Western blot analyses from same experiments, with heat shock protein 90 (HSP90) as loading controls. *p <0.05 vs. Wt. ^#p <0.05 MCC950 vs. vehicle, by one-way ANOVA, n = 6–10 animals/group.

Fig. 3.. Effects of MCC950 vs. vehicle on circulating cytokines/chemokines and transaminases in mice with NASH.

(A) Plasma IL-1β, (B) plasma IL-18, (C) ALT, (D) AST, (E) macrophage chemotactic protein 1 (MCP-1), (F) IL-6. Same mice as in Fig. 2. *p <0.05 or less vs. Wt. ^#p <0.05 MCC950 vs. vehicle, by one-way ANOVA, n = 11–13 animals/group, same animals as in Fig. 2.

Fig. 4.. MCC950 reverses NASH pathology.

(A) (i) Steatosis, (ii) hepatocyte ballooning, and (iii) NAFLD activity score (NAS) in Wt and foz/foz mice after 24 weeks atherogenic diet, and gavage during last 8 weeks with vehicle or MCC950. (B) Representative H&E-stained liver sections demonstrating inflammation in atherogenic foz/foz mouse liver (vehicle) is abolished by MCC950, with reduction of lobular inflammation score. (C) NF-κB activation (nuclear p65) is suppressed, as are, (D) myeloperoxidase (MPO) positive neutrophils, and (E) F4/80 positive macrophages and Kupffer cells as crown-like structures (CLS). Same experiments as Figs. 2 and 3. *p <0.05 vs. Wt. ^#p <0.05 MCC950 vs. vehicle, by one-way ANOVA, n = 11–13 animals/group. Scale bar: 100 μm.

Fig. 5.. In MCD-fed mice MCC950 suppresses hepatic NLRP3 inflammasome activation, inflammatory cell infiltration and fibrosis.

(A) Hepatic Asc, Casp1 and Il1b mRNA in mice fed MCD or MCS diets, and gavaged MCC950 or vehicle for 6 weeks. To right, representative Western blot analyses of pro-caspase 1, active caspase 1, pro-IL-1β and IL-1β: GAPDH loading control. (B) Representative H&E-stained liver sections. To right number of infiltrating neutrophils and F4/80-positive cells. (C) Representative Sirius Red-stained liver sections, and percentage fibrotic area. (D) mRNA expression of Col1a1, Ctgf and Timp1. Data: mean ± SEM, *p <0.05 MCS vs. MCD, ^#p <0.05 vehicle vs. MCC950 by one-way ANOVA, n = 8 animals/group. Scale bar: 100 μm.

Fig. 6.. MCC950 suppresses liver fibrosis in obese, diabetic mice with NASH.

(A) Sirius Red-stained liver sections from Wt and foz/foz mice after 24 weeks atherogenic diet intake and gavage during the last 8 weeks with vehicle or MCC950 (same mice as in Figs. 2–4). (B) Collagen area by Sirius Red densitometry. (C) Changes in collagen 1α (Col-1), (D) alpha-smooth muscle actin (α-SMA), (E) connective tissue growth factor (CTGF), and (F) representative Western blot analyses. Heat shock protein 90 (HSP90) as loading control. *p <0.05 or less vs. Wt. ^#p <0.05 MCC950 vs. vehicle, by one-way ANOVA, n = 6–10 animals/group. Scale bar: 100 μm.