(The) roles of hepatic macrophages and hepatic stellate cells in the pathogenesis of non-alcoholic fatty liver disease

Abstract

Part Ⅰ: Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4-MD2 complex Reactive oxygen species (ROS) contribute to the development of non-alcoholic fatty liver disease (NAFLD). ROS generation by infiltrating macrophages involves multiple mechanisms including Toll-like receptor 4 (TLR4)-mediated NADPH oxidase (NOX) activation. However, the mechanisms by which palmitate stimulates TLR4-mediated NOX2 activation are not fully understood. Here, we report that palmitate-stimulated $CD11b^+F4/80^{low}$ hepatic infiltrating macrophages, but not $CD11b^+F4/80^{high}$ Kupffer cells, generate ROS via dynamin-mediated endocytosis of TLR4 and NOX2, without the involvement of MYD88 and TRIF. Using size exclusion chromatography, we demonstrate that unlike LPS-mediated dimerization of the TLR4-MD2 complex, palmitate binds a monomeric TLR4-MD2 complex that triggers endocytosis, ROS generation and increased pro-interleukin-1$\beta$ expression in macrophages. Moreover, palmitate-induced ROS generation in human $CD68^{low}CD14^{high}$ macrophages is strongly suppressed by inhibition of dynamin. Furthermore, NOX2-deficient mice are protected against high-fat diet-induced hepatic steatosis and insulin resistance. Therefore, endocytosis of TLR4 and NOX2 into macrophages might be a novel therapeutic target for NAFLD

Part Ⅱ. Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells Non-alcoholic fatty liver disease (NAFLD) is a dominant cause of chronic liver disease, but the exact mechanism of progression from simple steatosis to nonalcoholic steatohepatitis (NASH) remains unknown. Here, we investigated the role of exosomes in NAFLD progression. Exosomes were isolated from a human hepatoma cell line treated with palmitic acid (PA) and their miRNA profiles examined by microarray. The human hepatic stellate cell (HSC) line (LX-2) was then treated with exosome isolated from hepatocytes. Compared with controls, PA-treated hepatocytes displayed significantly increased CD36 and exosome production. The microarray analysis showed there to be distinctive miRNA expression patterns between exosomes from vehicle- and PA-treated hepatocytes. When LX-2 cells were cultured with exosomes from PA-treated hepatocytes, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with exosomes from vehicle-treated hepatocytes. In conclusion, PA treatment enhanced the production of exosomes in these hepatocytes and changed their exosomal miRNA profile. Moreover, exosomes derived from PA-treated hepatocytes caused an increase in the expression levels of fibrotic genes in HSCs. Therefore, exosomes may have important roles in the crosstalk between hepatocytes and HSCs in the progression from simple steatosis to NASH.