Nuclear Receptor SHP Reduces Proinflammatory Macrophage Polarization in High Fat‐cholesterol‐fructose Diet Induced Mouse Model of Non‐alcoholic Steatohepatitis

Objective The incidence of nonalcoholic fatty liver disease (NAFLD) is rapidly becoming a major health concern in the United States. The mechanisms for progression from simple steatosis to more aggressive non‐alcoholic steatohepatitis (NASH) remain poorly understood. The aim for this study was to develop a mouse model that closely mimics progressive NAFLD in human and to investigate a novel role of nuclear receptor small heterodimer partner (Nr0b2, SHP) in regulating macrophage polarization in NASH. Methods 2 month old male C57Bl/6J mice were fed a high fat diet with high cholesterol and fructose (HF–HC–HF) for 4, 8, 12, and 20 weeks and the extent of steatosis, liver inflammation, and fibrosis were evaluated at each time point. For GFP and SHP groups, mice received 8 weeks HF–HC–HF feeding before tail vein injection with adeno‐associated virus 8 (AAV8)‐alpha‐1‐antitrypsin (AAT)‐SHP or AAV8‐AAT‐GFP control, and followed by additional 4 weeks HF– HC–HF diet. Serum aspartate aminotransferase (AST) and alanine aminotranferease (ALT) levels, liver morphology, fibrosis, and inflammation were evaluated. Mouse macrophage cell line RAW 264.7 cells overexpressed with SHP or control vector was included to evaluate SHP in mediating inflammation response to lipopolysaccharide (LPS) treatment. Results The HF‐HC‐HF feeding resulted in insulin resistance and liver steatosis at 4 weeks, progressing to steatohepatitis at 8 weeks, and developed early fibrosis at 12 weeks. HF‐HC‐HF animal exhibited increase levels of serum ALT and AST as early as 8 weeks post feeding. Steatohepatitis was present in HF‐HC‐HF liver by the increase of hepatocyte ballooning, infiltration of macrophages, and up‐regulation of proinflammatory cytokines (TNFα, CCL2, IL‐1β, IL‐6). At 12, 20, and 20 weeks, increased proinflammatory M1 macrophages and decreased anti‐inflammatory M2 macrophages in HF‐HC‐HF liver were revealed by measures of M1 and M2 genes and proteins, which paralleled the increase of liver fibrosis. Overexpressing SHP in HF‐HC‐HF liver significantly reduced insulin resistance and decreased infiltration of macrophages into the liver as found by immunohistochemistry. In line with lower levels of intrahepatic macrophages, proinflammatory cytokines (TNFα, CCL2) were significantly reduced in AAV8‐AAT‐SHP liver compared to AAV8‐AAT‐GFP liver. Importantly, the mechanism by which SHP inhibited inflammation response to HF‐HC‐HF was through the regulation of macrophage polarization as evidenced by decreased proinflammatory M1 macrophages and increased anti‐inflammatory M2 macrophages in AAV8‐AAT‐SHP liver. The critical role of SHP in regulating macrophage polarization was further confirmed in LPS treated SHP overexpression RAW 264.7 cells. Finally, fibrosis progression in HF‐HC‐HF liver was significantly altered by SHP overexpression without affecting steatosis. Conclusions Our study identified a previous unknown regulatory role of SHP in macrophage polarization that downregulates inflammation in NAFLD. The associated ameliorated NASH development suggests that pharmacological activation of SHP could be an interesting novel approach for NASH treatment. Support or Funding Information NCI K22CA184146 and NIH P20 GM103549