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.

Keywords::

• superoxide
• imiquimod
• poly I:C
• lung pathology
• oxidative stress

Acknowledgments

The authors wish to thank the Australian Research Council for fellowship and funding support (FT120100876) for Dr Stavros Selemidis and the National Health and Medical Research Council of Australia for funding support (APP1027112). Also the authors wish to thank Dr John Stambas for providing stocks of X-31 influenza A virus.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.