Enhanced TLR4 expression in steatotic hepatocytes

Nonalcoholic fatty liver disease (NAFLD) starts with hepatic steatosis, which can progress with inflammation and fibrosis. NAFLD Patients have a higher prevalence of small intestinal bacterial overgrowth and frequently develop a leaky intestinal barrier. Toll-like receptor 4 (TLR4) is crucial for the recognition of lipopolysaccharides (LPS), and it has been shown that TLR4 signaling is a key factor for hepatic inflammation and fibrosis.

The aim of this study was to analyze TLR4 expression in NAFLD.

Methods and Results: In 3 dietary murine NASH-models, namely feeding a Western type high-fat diet, a methionine- and choline-deficient diet or the atherogenic Paigen diet, hepatic TLR4 expression was significantly increased compared to normal livers. Further, we assessed TLR4 expression in a dietary murine model of steatosis without hepatocellular damage and inflammation. Here, TLR4 expression was lower than in the NASH models but still significantly higher than in normal, non-steatotic hepatic tissue. Accordingly, TLR4 expression was significantly up-regulated in human NASH compared to normal hepatic tissue, and also in human fatty livers without inflammation TLR4 expression was significantly enhanced. Next, we assessed an in vitro model in which we induced cellular steatosis in primary human hepatocytes by incubation with free fatty acids, and also in vitro hepatocellular steatosis led to enhanced TLR4 expression levels as evidenced by qPCR and Western blotting. Of note, LPS induced pro-inflammatory gene expression was significantly higher in steatotic hepatocytes compared to control cells.

Conclusions: Our data indicate steatotic hepatocytes as cellular source of enhanced hepatic TLR4 expression during the early stage of NAFLD. Based on the known biological effects of LPS mediated TLR4 activation it appears likely that increased hepatocellular TLR4 expression plays a critical role in NAFLD progression.