The Hepatic Inflammsome Has an Increased NK-T and CD8+ T- Cell Signature in a Murine Model of Fibrotic Nonalcoholic Steatohepatitis (NASH)

Abstract

High fat diet induced models of simple hepatic steatosis have been shown to result in a decreased hepatic natural killerT (NKT) cell population. Interestingly, NKT cells are known to correlate positively with the development of adipose tissue inflammation and glucose intolerance in diet-induced obesity. Recent data implicates refined carbohydrates (Fructose) in the progression of hepatic steatosis to advanced fibrotic NASH in humans. This has also been replicated in a novel model of obesity and NASH with fibrosis in mice. We therefore hypothesized that NKT cells populate the livers in a murine model of advanced NASH with fibrosis. Methods: Adult male C57BL6 mice were randomly assigned to control (C) or high fat high carbohydrate diet (HFHC). The C group received regular chow and water while the HFHC group received diet with Medium Chain Triglycerides (MCT; 58% kcal) and water supplemented with 55% fructose & 45% sucrose. Mice were sacrificed at 16 wks of age and underwent hepatic mononuclear cell phenotyping by flow cytometry. Results: Mice receiving HFHC diet gained significantly more weight than C fed mice starting at 4 wk (p<0.01), with a Mean ± SE weight of 45.84±1.4g at 16 wk compared to 31.33±1.0g for C fed mice (p<0.0001). Fasting glucose was higher in HFHC mice at 16 wks (176.3±9.2 mg/dl; p=0.001). Liver weight at 16 weeks was higher in the HFHC group (2.44±0.2g) compared to the C fed mice (1.47±0.03g; p<0.01). Steatosis as measured by liver triacylglycerol (TG) content was also higher in HFHC mice at 16 wks (2354±487.8 mg/dl/100 mg wet liver) compared to the C fed mice (p= 0.001). Inflammation as measured by serum alanine aminotransferase (ALT) levels was higher in HFHC mice (135.8±24.28 IU/L) at 16 wks compared to the C fed mice (p=0.019). Flow cytometric analysis identified increased NK-T cell (CD3+NK1.1+) signature in mice receiving HFHC (3.7±2.3x104 cells) compared to C fed mice (1.1±0.6x104 cells; p=0.02). While hepatic CD1d-restricted invariant NK-T CD4+ and CD8+ cell populations remained unchanged, total CD3+CD8+ T-cells significantly increased in the HFHC group (1.93±0.79x105) vs. C fed (1.0±0.7x105; p=0.04). A corresponding increase in CD8+ T-cells expressing the activation marker CD69 was also seen [(2.02±1.01x104 (C) vs 6.85±2.6x104 (HFHC); p<0.01]. Liver fibrosis was visible at sacrifice in majority of the HFHC fed mice (Stage 1c and 2) while none was seen in C diet fed mice. Conclusion: HFHC diet produces a relatively rapid and progressive liver injury resulting in NASH with fibrosis in a background of diet-induced obesity. In this system, which closely resembles the human phenotype of advanced NASH with fibrosis, we observed a significant increase in hepatic NK-T and CD8+ T-cells. These cell types may potentially typify the hepatic inflammasome in the progression of human disease to NASH with fibrosis.