IL-18 but Not IL-1 Signaling Is Pivotal for the Initiation of Liver Injury in Murine Non-Alcoholic Fatty Liver Disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r^-/-- but not Il-1r^-/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Keywords: ALiOS; NAFLD; NASH; NLRP3; Western diet; inflammasome; interleukin 1; interleukin 18.

Figure 1

American lifestyle-induced obesity syndrome (ALiOS) diet in mice induces obesity, metabolic syndrome and fatty liver. Wildtype mice were fed standard diet or ALiOS diet for 24 weeks. (A) Body weight (n = 14, each), (B) visceral fat weight (n = 8, each), (C) serum concentrations of insulin, leptin and adiponectin (n = 8, each, 12–24 weeks combined) and (D) liver triglycerides (n = 4, each) at the end of the study are shown as mean ± standard deviation (* p < 0.01, t-test). (E) Representative liver sections following H&E and Sudan red staining showing micro- and macrovesicular steatosis and heterogenicity in cell and nuclear size but a lack of ballooning or an inflammatory infiltrate (200×, bar represents 100 μM).

Figure 2

Comparison of the liver lipidome in murine and human NAFLD. (A) Wildtype mice were fed standard diet or ALiOS diet for 24 weeks. Liver lipidome was characterized by gas chromatography (GC) as described. Composition of the lipidome is depicted in categories of saturated, monounsaturated, diunsaturated and polyunsaturated fatty acids. (B) Fatty acid composition of mouse and human livers without steatosis (white bars) and with steatosis (black bars) was determined. Previously described fatty acid ratios associated with NAFLD are depicted (mean ± standard deviation; n = 5 for mouse, n = 9–10 for human liver tissue; * p < 0.05; t-test).

Figure 3

Engagement of the inflammasome pathway following ALiOS diet Wildtype mice were fed standard diet (white bars) or ALiOS diet (black bars) for 24 weeks. (A) Expression of indicated genes in liver tissue and (B) serum concentrations of IL-1β and IL-18 (n = 8, each) at the end of the study are shown (mean ± standard deviation; n = 6, each; * p < 0.01, t-test).

Figure 4

IL-18R- but not IL-1R-dependent signaling promotes liver damage in ALiOS diet-induced NAFLD. Mice were fed standard diet (white bars) or ALiOS diet (black bars) for 24 weeks. (A) Liver triglycerides (n = 4, each; * p < 0.01; t-test) and (B) serum ALT levels (n = 8–14; * p < 0.05; t-test) are shown for indicated genotypes as mean ± standard deviation. (C) Depiction of gene expression in liver tissue of standard diet- (white bars) and ALiOS diet-fed Il-18r ^−/− mice (n = 6, each; * p < 0.01, t-test). (D) ATP productivity was determined in mitochondria isolated from liver tissue following 24 weeks of standard diet (white bars) and ALiOS diet (black bars) in wildtype and Il-18r ^−/− mice. (E) Ratios of major hepatic lipidome components (saturated, monounsaturated, diunsaturated and polyunsaturated fatty acids) in ALiOS diet-fed wildtype (black bars) and Il-18r ^−/− (chess field) mice (n = 5, each, no statistical difference).