PT  - JOURNAL ARTICLE
AU  - Keshia Hendricks
AU  - Eunice To
AU  - Ross Vlahos
AU  - Brad Broughton
AU  - Hitesh Peshavariya
AU  - Stavros Selemidis
TI  - Influenza A virus causes vascular endothelial cell oxidative stress via NOX2 oxidase
AID  - 10.1183/13993003.congress-2016.PA3967
DP  - 2016 Sep 01
TA  - European Respiratory Journal
PG  - PA3967
VI  - 48
IP  - suppl 60
4099  - http://erj.ersjournals.com/content/48/suppl_60/PA3967.short
4100  - http://erj.ersjournals.com/content/48/suppl_60/PA3967.full
SO  - Eur Respir J2016 Sep 01; 48
AB  - The vascular endothelium has been shown to contribute to influenza A virus (IAV) induced lung inflammation, but the mechanisms by which this occurs remain largely unknown. IAV enters cells via endocytosis and activates toll-like receptors (TLRs), resulting in an inflammatory response. Interactions between TLRs and the ROS-generating NADPH oxidases have a critical role in inflammation. Recently, we have shown that NOX2-deficiency is protective against IAV-induced inflammation. The aim of this study was to determine if IAV causes an increase in ROS production in endothelial cells, and to establish the subcellular localization and enzymatic source.Human microvascular endothelial cells (HMEC), and WT and NOX2-/- mouse lung endothelial cells (MLEC) were infected with IAV (Hong Kong X-31; multiplicity of infection 10) or treated with TLR3 agonist, polyinosinic:polycytidylic acid (poly I:C) (100µg/ml), at various time points. Confocal microscopy was used to localize early endosomes, IAV nucleoprotein and NOX2. OxyBURST fluorescence was used to detect endosomal ROS.IAV nucleoprotein and NOX2 were co-localised with early endosomes in HMEC. IAV infection of WT MLEC caused a significant ∼60% (p=0.02, n=6) increase in endosomal ROS production compared to uninfected cells that was almost abolished in NOX2-/- MLEC (p<0.0001, n=6). There was ∼2 fold increase in endosomal ROS in poly I:C-treated WT MLEC compared to the control (p=0.02, n=8) that was substantially reduced in NOX2-/- MLEC (p=0.001, n=8).This study shows that IAV internalises into endothelial cells via endocytosis, and causes a NOX2-dependent increase in endosomal ROS, potentially by a TLR3-mediated mechanism.