Genetic inactivation of toll-like receptor 4 alleviates diet-induced nonalcoholic steatohepatitis by inhibiting reactive oxygen species-evoked activation of X-box binding protein-1 in ApoE-deficient mice

Abstract

RESULTS: Nonalcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome characterized by steatosis and necroinflammation. However, the precise mechanism underlying the progression from steatosis to inflammation remains poorly understood. Here we investigated the role of Toll like receptor 4 (TLR4), a key player in innate immunity and inflammation, in the pathogenesis of NASH associated with obesity. We generated ApoE-/-/TLR4-/- mice and ApoE-/-/TLR wild type mice (ApoE-/-/TLR-WT) by cross-breeding the ApoE deficient (ApoE-/-) strain with TLR4-deficient mice, which were then fed a high fat high cholesterol (HFHC) diet for 12 weeks to induce obesity. The results showed that HFHC diet feeding induced typical symptoms of NASH in ApoE-/- mice, including elevated serum level of Alanine aminotransferase and increased NASH score, and this change was accompanied by augmented expression of TLR4 in Kupffer cells. TLR4 inactivation protected against HFHC diet-induced liver injury and reduced hepatic lipid contents in ApoE-/- mice. Furthermore, ApoE-/-/TLR4-/- displayed a significant reduction in hepatic resident of macrophages and were less susceptible to diet-induced production of reactive oxygen species (ROS) and proinflammatory cytokines. In ApoE-/-/TLR4-/- mice on HFHC diet, the splicing of X-box binding protein-1 (XBP-1), a transcription factor involved in ER stress and inflammation, was repressed in the liver tissue compared to those in ApoE-/-/TLR4-WT mice. Ex vivo experiments in primary rat Kupffer cells showed that treatment with antioxidants significantly abrogated endotoxin-induced mRNA splicing of XBP-1 and attenuated production of cytokines. Furthermore, endotoxin-induced production of cytokines and activation of NF-κB were significantly blunted by siRNA-mediated knocking down XBP-1 expression. These findings suggest that TLR4 in Kupffer cells mediates the progression from simple steatosis to inflammation, partly by inducing the production of ROS, thereby leading to the activation of XBP-1.