NADPH Oxidase-Derived Peroxynitrite Drives Inflammation in Mice and Human Nonalcoholic Steatohepatitis via TLR4-Lipid Raft Recruitment.

The molecular events that link NADPH oxidase activation and the induction of Toll-like receptor (TLR)-4 recruitment into hepatic lipid rafts in nonalcoholic steatohepatitis (NASH) are unclear. We hypothesized that in liver, NADPH oxidase activation is key in TLR4 recruitment into lipid rafts, which in turn up-regulates NF-κB translocation to the nucleus and subsequent DNA binding, leading to NASH progression. Results from confocal microscopy showed that liver from murine and human NASH had NADPH oxidase activation, which led to the formation of highly reactive peroxynitrite, as shown by 3-nitrotyrosine formation in diseased liver. Expression and recruitment of TLR4 into the lipid rafts were significantly greater in rodent and human NASH. The described phenomenon was NADPH oxidase, p47phox, and peroxynitrite dependent, as liver from p47phox-deficient mice and from mice treated with a peroxynitrite decomposition catalyst [iron(III) tetrakis(p-sulfonatophenyl)porphyrin] or a peroxynitrite scavenger (phenylboronic acid) had markedly less Tlr4 recruitment into lipid rafts. Mechanistically, peroxynitrite-induced TLR4 recruitment was linked to increased IL-1β, sinusoidal injury, and Kupffer cell activation while blocking peroxynitrite-attenuated NASH symptoms. The results strongly suggest that NADPH oxidase-mediated peroxynitrite drove TLR4 recruitment into hepatic lipid rafts and inflammation, whereas the in vivo use of the peroxynitrite scavenger phenylboronic acid, a novel synthetic molecule having high reactivity with peroxynitrite, attenuates inflammatory pathogenesis in NASH.