1 Hepatic TLR 4 signaling in Obese NAFLD 1 2

16 Nonalcoholic fatty liver disease (NAFLD) occurs frequently in the setting of metabolic syndrome, but the factors 17 leading to nonalcoholic steatohepatitis (NASH) are not fully understood. This study investigated toll-like 18 receptor 4 (TLR4) signaling in human liver with the goal of delineating whether activation of this pathway 19 segregates those with nonalcoholic fatty liver (NAFL) from those with NASH. Experiments were performed 20 using liver biopsy tissue obtained from Class III obese subjects undergoing bariatric surgery, and extended to 21 an immortalized human hepatocyte HepaRG cell line and primary human hepatocytes. The bacterial 22 endotoxin lipopolysaccharide (LPS) and total free fatty acid levels were significantly increased in plasma of 23 NASH patients. TLR4 mRNA levels were significantly increased in subjects with NASH compared to NAFL as 24 was IRF3 in the myeloid differentiation factor 88(Myd88-) independent signaling pathway. In HepaRG cells, 25 NF-ҡB nuclear translocation and functional activity increased following treatment with the fatty acid, palmitate, 26 and following exposure to LPS, when compared to hepatocytes stimulated with a lipogenic treatment that 27 induced de novo lipogenesis. Palmitateand LPS-induction of NF-ҡB activity was partially attenuated by 28 chemicalor siRNA-mediated inhibition of TLR4. Expression of TLR4 and its downstream mediators was up29 regulated with palmitate and LPS. Similar results were observed using primary human hepatocytes from a 30 lean donor. Interestingly, NF-ҡi activity assays showed obese donor hepatocytes were resistant to chemical 31 TLR4 inhibition. Conclusion: TLR4 expression is up-regulated in a large cohort of NASH patients, when 32 compared to those with NAFL, and this occurs within the setting of increased LPS and fatty acids. 33 34