Protective role of Nox1 oxidase against influenza A virus-induced lung inflammation

S. Selemidis,¹ H.J. Seow,² B.R.S. Broughton,¹ S. Bozinovski,² K.H. Krause,³ J. Stambas,⁴ A. Vinh,¹ C.G. Sobey,¹ G.R. Drummond¹ and R. Vlahos,² ¹Department of Pharmacology, Monash University, Clayton, VIC 3800, Australia, ²Department of Pharmacology, Melbourne University, Parkville, VIC 3010, Australia, ³Department of Pathology and Immunology, Université de Genève, Genève, Switzerland and ⁴School of Medicine, Deakin University, Waurn Ponds, VIC 3216, Australia.

Introduction: The Nox2 isoform of the NADPH oxidase family of superoxide-generating enzymes promotes the acute lung injury and airways inflammation caused by influenza A virus infection (Snelgrove et al., 2006; Vlahos et al., 2011). However, airway epithelial cells and lung endothelial cells also express the Nox1 isoform of NADPH oxidase (Carnesecchi et al., 2009) placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation.

Aim: To investigate the potential regulation of the lung inflammation caused by influenza A viral infection by the Nox1 oxidase in vivo in mice.

Methods: Age-matched male WT (C57BL/6) and Nox1-deficient mice (i.e. Nox1^−/y) were infected intranasally with the moderately pathogenic HkX-31 (H3N2, 1×10⁴ PFU) influenza A virus for analysis of bodyweight, airways inflammation (bronchoalveolar lavage fluid (BALF) cell counting), oxidative stress (L-O12 chemiluminescence and 3-nitrotyrosine staining for superoxide and peroxynitrite production, respectively), viral titers (plaque assay), lung histopathology (H & E staining), cytokine/chemokine expression (Q-PCR) and T lymphocyte subsets including CD8⁺, CD4⁺ and CD25⁺CD4⁺FoxP3⁺ (i.e. T regulatory cells - Tregs; flow cytometry).

Results: HkX-31 virus infection of Nox1^−/y mice resulted in a significantly greater: loss of bodyweight; BALF neutrophilia, peri-bronchial and alveolar inflammation; BALF inflammatory cell superoxide production and peri- bronchial, epithelial and endothelial oxidative stress; and expression of pro-inflammatory cytokines including CCl2, CCl3, CXCl2, IL-1, IL-6 and TNF- when compared to WT control mice. Also, expression of the anti- inflammatory cytokine IL-10 was lower in Nox1^−/y mice. Lung viral titers, and the degree of airways infiltration of active (i.e. CD69⁺ and CD44⁺) CD8⁺ and CD4⁺ T lymphocytes, and of Tregs were similar between influenza infected WT and Nox1^−/y mice.

Discussion: In summary, Nox1 oxidase has anti-inflammatory properties in the lungs and protects against influenza A virus infection in mice. Our findings imply that there are differential roles of Nox1 versus Nox2 oxidases in the regulation of inflammation caused by influenza A viruses.

Carnesecchi S, Deffert C, Pagano A, Garrido-Urbani S, Métrailler-Ruchonnet I, Schäppi M, Donati Y, Matthay MA, Krause KH, Barazzone Argiroffo C. (2009). American Journal of Respiratory and Critical Care Medicine 180: 972-81.

Snelgrove RJ, Edwards L, Rae AJ, Hussell T. (2006). European Journal of Immunology 36: 1364-1373

Vlahos R, Stambas J, Bozinovski S, Broughton BRS, Drummond GR, Selemidis S. (2011). PLoS Pathogens 7: e1001271.