title: NADPH oxidase limits lipopolysaccharide-induced lung inflammation and injury in mice through reduction-oxidation regulation of NF-κB activity.
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.
authors.affiliation: Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. wei.han@vanderbilt.edu
authors.forename: Wei
authors.initials: W
authors.lastname: Han
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Jiyang
authors.initials: J
authors.lastname: Cai
authors.forename: Linda A
authors.initials: LA
authors.lastname: Gleaves
authors.forename: Vasiliy V
authors.initials: VV
authors.lastname: Polosukhin
authors.forename: Brahm H
authors.initials: BH
authors.lastname: Segal
authors.forename: Fiona E
authors.initials: FE
authors.lastname: Yull
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Blackwell
mesh_terms: D000818:Animals; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology* / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D055370:Lung Injury / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D019255:NADPH Oxidases / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism*; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D010084:Oxidation-Reduction; D011014:Pneumonia / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase