title: Influenza A virus and TLR7 activation potentiate NOX2 oxidase-dependent ROS production in macrophages.
abstract: Influenza A virus infects resident alveolar macrophages in the respiratory tract resulting in Toll like receptor 7 (TLR7) activation that triggers an inflammatory response to resolve the infection. Macrophages are also major sources of reactive oxygen species (ROS) via the NOX2-containing NADPH oxidase. Although ROS are crucial for pathogen clearance, in response to influenza A virus, ROS are touted as being culprit mediators of the lung tissue injury. The aim of the present study was to determine whether influenza A virus infection and TLR7 activation of macrophages, results in alterations in their ROS production. Here we demonstrate using immunofluorescence that influenza A virus (Hong Kong X-31 strain; H3N2) internalizes in RAW264.7 cells and mouse alveolar macrophages within 1 h, resulting in a significant enhancement in the stimulated NOX2 oxidase-dependent oxidative burst, although virus had no effect on basal ROS. The specific TLR7 agonist imiquimod (10 μg/ml) elevated basal superoxide production and, in a similar fashion to influenza A virus, enhanced NOX2 oxidase-dependent oxidative burst. By contrast, the TLR3 agonist, poly I:C (1-100 μg/ml) failed to influence the oxidative burst to NOX2 oxidase. A peptide corresponding to the region 337-348 on p47phox conjugated to a HIV-tat, designed to inhibit the phosphorylation of Ser346 on p47phox suppressed the influenza A virus- and imiquimod-induced enhancement in the oxidative burst. In conclusion, this study demonstrates for the first time that influenza A virus and TLR7 activation enhance the NOX2 oxidase-dependent oxidative burst in macrophages, which might underpin the acute lung injury to influenza A virus infection.
authors.affiliation: Department of Pharmacology, Monash University , Victoria , Australia.
authors.forename: E E
authors.initials: EE
authors.lastname: To
authors.forename: B R S
authors.initials: BR
authors.lastname: Broughton
authors.forename: K S
authors.initials: KS
authors.lastname: Hendricks
authors.forename: R
authors.initials: R
authors.lastname: Vlahos
authors.forename: S
authors.initials: S
authors.lastname: Selemidis
mesh_terms: D000818:Animals; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D051199:Toll-Like Receptor 7 / Q000235:genetics / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; C495348:TLR7 protein, human; C477038:Tlr7 protein, mouse; D051199:Toll-Like Receptor 7; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases
keywords: imiquimod; lung pathology; oxidative stress; poly I:C; superoxide