NADPH oxidase limits lipopolysaccharide-induced lung inflammation and injury in mice through reduction-oxidation regulation of NF-κB activity

W Han, H Li, J Cai, LA Gleaves… - The Journal of …, 2013 - journals.aai.org
W Han, H Li, J Cai, LA Gleaves, VV Polosukhin, BH Segal, FE Yull, TS Blackwell
The Journal of Immunology, 2013journals.aai.org
Although reactive oxygen species (ROS) produced by NADPH oxidase are known to
regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is
incompletely understood. In these studies, we treated wild-type (WT) and p47 phox-deficient
mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling
through the NF-κB pathway. After intratracheal instillation of LPS, ROS generation was
impaired in p47 phox−/− mice, whereas these mice had increased neutrophilic alveolitis and …
Abstract
Although reactive oxygen species (ROS) produced by NADPH oxidase are known to regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is incompletely understood. In these studies, we treated wild-type (WT) and p47 phox-deficient mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling through the NF-κB pathway. After intratracheal instillation of LPS, ROS generation was impaired in p47 phox−/− mice, whereas these mice had increased neutrophilic alveolitis and greater lung injury compared with WT controls. In mice interbred with transgenic NF-κB reporters (HIV-long terminal repeat/luciferase [HLL]), we found exaggerated LPS-induced NF-κB activation and increased expression of proinflammatory cytokines in lungs of p47 phox−/−/HLL mice compared with controls. Both lung macrophages and bone marrow–derived macrophages (BMDMs) isolated from p47 phox−/−/HLL mice showed enhanced LPS-stimulated NF-κB activity compared with controls. Although nuclear translocation of NF-κB proteins was similar between genotypes, EMSAs under nonreducing conditions showed increased DNA binding in p47 phox−/−/HLL BMDMs, suggesting that ROS production reduces NF-κB binding to DNA without affecting nuclear translocation. Increased intracellular reduced glutathione/glutathione disulfide ratio and greater nuclear redox factor 1 (Ref-1) levels were present in p47 phox−/−/HLL compared with WT BMDMs, pointing to NADPH oxidase modulating intracellular redox status in macrophages. Treatment with the Ref-1–specific inhibitor E3330 or hydrogen peroxide inhibited LPS-induced NF-κB activation in p47 phox−/−/HLL BMDMs but not in WT/HLL cells. Consistent with these findings, small interfering RNA against Ref-1 selectively reduced NF-κB activity in LPS-treated p47 phox−/−/HLL BMDMs. Together, these results indicate that NADPH oxidase limits LPS-induced NF-κB transcriptional activity through regulation of intracellular redox state.
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