Nathalie Lejal1, Sandrine Truchet1, Edna Bechor2, Edwige Bouguyon1, Vijay Khedkar1, Nicolas Bertho1, Jasmina Vidic1, Pierre Adenot3, Stéphanie Solier4, Edgar Pick2, Anny Slama-Schwok5. 1. Paris Saclay University, U892 INRA, Jouy en Josas, France. 2. Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Sackler Faculty of Medicine, Tel-Aviv University, Israel. 3. Paris-Saclay University, UMR BDR, INRA, ENVA, Jouy en Josas, France; Paris-Saclay University, MIMA2 Plateform, INRA, Jouy en Josas, France. 4. Paris Saclay University, Gustave Roussy Institute, U1170 INSERM, Villejuif, France. 5. Paris Saclay University, U892 INRA, Jouy en Josas, France; Paris Saclay University, Gustave Roussy Institute, UMR 8200 CNRS, Villejuif, France. Electronic address: Anny.Slama-Schwok@gustaveroussy.fr.
Abstract
BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS: IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.
BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS:IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION:NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.
Authors: Eunice E To; John J O'Leary; Luke A J O'Neill; Ross Vlahos; Steven Bozinovski; Christopher J H Porter; Robert D Brooks; Doug A Brooks; Stavros Selemidis Journal: Antioxid Redox Signal Date: 2020-03-06 Impact factor: 8.401