Nox1 Oxidase Suppresses Influenza A Virus-Induced Lung Inflammation and Oxidative Stress

et al. (2013) Nox1 Oxidase Suppresses Influenza A Virus-Induced Lung Inflammation and Oxidative Stress. PLoS ONE 8(4): e60792. doi:10.1371/journal.pone.0060792 Nox1 Oxidase Suppresses Influenza A Virus-Induced Lung Inflammation and Oxidative Stress Stavros Selemidis 0 Huei Jiunn Seow 0 Brad R. S. Broughton 0 Antony Vinh 0 Steven Bozinovski 0 Christopher G. Sobey 0 Grant R. Drummond 0 Ross Vlahos 0 Dennis W. Metzger, Albany Medical College, United States of America 0 1 Department of Pharmacology, Monash University , Clayton, Victoria , Australia , 2 Department of Pharmacology, University of Melbourne , Parkville, Victoria , Australia Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox12/y) mice were infected with the moderately pathogenic HkX-31 (H3N2, 16104 PFU) influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox12/y mice resulted in significantly greater: loss of bodyweight (Day 3); BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1b, IL-6, GM-CSF and TNF-a was higher in Nox12/y lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-c and the antiinflammatory cytokine IL-10 were lower in lungs of Nox12/y mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8+ and CD4+ T lymphocytes, and of Tregs were similar between WT and Nox12/y mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear to have opposing roles in the regulation of inflammation caused by influenza A viruses. - Funding: This work was supported by National Health and Medical Research Council of Australia (NHMRC; nhmrc.gov.au) for Fellowship and Project Grant Support (I.D. 606472, 1006017, 545942, 570861, 606488, 1010984 and 509226) and the Australian Research Council (arc.gov.au) for Fellowship support (I.D. FT120100876). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Influenza A virus infections represent important infectious diseases that continue to inflict significant global morbidity and mortality [1]. The effects of influenza infection vary from strain to strain, ranging from transient debilitating respiratory illness to more severe respiratory complications that are sometimes fatal. Seasonal and pandemic influenza infections over the last century have claimed over 50 million lives and impose a huge socioeconomic burden [2]. Accumulated animal and human studies provide compelling evidence for a new paradigm whereby excessive production of reactive oxygen species (ROS), such as superoxide anion, and its derivatives hydrogen peroxide and peroxynitrite, are crucial mediators of the acute lung injury to influenza A virus infection [35]. However, although the evidence supporting a pathogenic role for ROS in lung injury to influenza A virus is strong, little attention has been directed towards identifying the key enzymes that generate ROS. Knowledge of the culprit enzymes could give rise to novel pharmacological strategies for manipulating oxidative stress and the associated lung injury following influenza A virus infection. Recently the Nox2 isoform of the NADPH oxidase family of superoxide-generating enzymes was identified as a major player in the lung pathology caused by influenza A virus infection. Mice genetically deficient in the Nox2 subunit or in a key regulatory subunit of Nox2 activity, p47phox, demonstrated substantially lower: 1) superoxide production by bronchoalveolar lavage fluid (BALF) inflammatory cells and lung oxidative stress; 2) lung oedema and injury; 3) alveolar lung epithelial apoptosis; and 4) peribronchial inflammation compared to WT mice [4,6,7]. Moreover, a lack of Nox2 oxidase activity resulted in improved lung function [8]. Despite the reduction in airways and BALF inflammation, viral clearance was not compromised but was significantly improved in the Nox2 deficient mice [6,8]. Finally, the protective effects of Nox2 deficiency against influenza A virus infection appeared to occur irrespective of the infecting strain, highlighting the exciting therapeutic potential of targeting Nox2 oxidase [6,7]. Notwithstanding the important role of Nox2, a number of additional sources of superoxide, such as the Nox1 isoform of the NADPH oxidase family, are expressed in lungs and may therefore influence the inflammatory response to influenza A virus infection. It is noteworthy that Nox1 mRNA has been identified in lung epithelial and endothelial cells, potentially placing the enzyme at key sites to regulate cytokine production and lung inflammation following an influenza viral infection [9]. However, it is so far unknown if Nox1 influences lung inflammation in response to influenza A virus infection. Thus, in the present study we performed an extensive phenotypic analysis of the clinical features of influenza A virus infection in the novel Nox1-deficient mouse. The present study shows that Nox1 oxidase critically inhibits the early burst in lung pro-inflammatory cytokine expression, inflammation and oxidative stress caused by influenza A virus infection and therefore, as opposed to the Nox2 oxidase, Nox1 is a protective mechanism against such infections. Expression of Nox1 A Nox1 specific antibody revealed strong immunofluorescence in the endothelium of blood vessels and in alveolar epithelial cells of lung sections taken from WT mice that was not evident in equivalent lung sections from Nox12/y mice (Figure 1). The lack of fluorescence in the Nox12/y lung sections verifies the specificity of the antibody for Nox1. Pro-inflammatory Cytokine and Chemokine Expression in Lungs from Nox12/y Mice We assessed mRNA levels of several pro-inflamm (...truncated)