An Endothelial Hsp70-TLR4 Axis Limits Nox3 Expression and Protects Against Oxidant Injury in Lungs

Aims: Oxidants play a critical role in the pathogenesis of acute lung injury (ALI). Nox3 is a novel member of the NADPH oxidase (Nox) family of oxidant-generating enzymes, which our laboratory had previously identified to be induced in the lungs of TLR4(-/-) mice. However, the physiologic role of Nox3 induction in lungs and its precise relationship to TLR4 are unknown. Furthermore, the cell compartment involved and the signaling mechanisms of Nox3 induction are unknown. Results: We identified that Nox3 is regulated by heat shock protein 70 (Hsp70) signaling via a TLR4-Trif-signal transducer and activator of transcription 3 (Stat3) pathway and that Nox3 induction leads to increased oxidant injury and death in mice and lung endothelial cells. We generated Nox3(-/-)/TLR4(-/-) double knockout mice, endothelial-targeting lentiviral silencing constructs, and endothelial-targeted Stat3(-/-) mice to specifically demonstrate that Nox3 induction is responsible for the pro-oxidant, proapoptotic phenotype of TLR4(-/-) mice. We also show that an endothelial Hsp70-TLR4-Trif-Stat3 axis is required to suppress deleterious Nox3 induction. Innovation: To date, a physiologic role for Nox3 in oxidant-induced ALI has not been identified. In addition, we generated unique double knockout mice and endothelial-targeted lentiviral silencing constructs to specifically demonstrate the role of a TLR4 signaling pathway in regulating pro-oxidant generation. Conclusions: We identified an endothelial TLR4-Trif antioxidant pathway that leads to the inhibition of a novel NADPH oxidase, Nox3, in lungs and lung endothelial cells. We also identified the role of a TLR4 ligand, Hsp70, in suppressing Nox3 in basal and pro-oxidant conditions. These studies identify potentially new therapeutic targets in oxidant-induced ALI.