NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice

Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared with wild-type (Nrf2^+/+) macrophages, we observed greater protein kinase C-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2^-/-) macrophages that was modulated by glutathione levels. To address the NADPH oxidase-mediated hyperinflammatory response and sepsis-induced lung injury and mortality in Nrf2^-/- mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91^phox (Nrf2^-/-//gp91 ^phox-/-). Compared with Nrf2^+/+ macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MyD88 and Toll/IL-1R domain-containing adaptor in Nrf2 ^-/- macrophages that was diminished by ablation of gp91 ^phox. Similarly, phosphorylation of IκB and IFN regulatory factor 3 as well as cytokine expression was markedly higher in Nrf2 ^-/- macrophages; whereas, it was similar in Nrf2^+/+ and Nrf2^-/-//gp91^phox-/-. In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2^-/- mice that was significantly reduced by ablation of gp91^phox. Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2 ^-/- mice; whereas, Nrf2^-/-//gp91^phox-/- showed prolong survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.