Therapeutic Targeting of Endosome and Mitochondrial Reactive Oxygen Species Protects Mice From Influenza Virus Morbidity

Abstract

There is an urgent need to develop effective therapeutic strategies including immunomodulators to combat influenza A virus (IAV) infection. Influenza A viruses increase ROS production, which suppress anti-viral responses and contribute to pathological inflammation and morbidity. Two major cellular sites of ROS production are endosomes via the NOX2-oxidase enzyme and the electron transport chain in mitochondria. Here we examined the effect of administration of Cgp91ds-TAT, an endosome-targeted NOX2 oxidase inhibitor, in combination with mitoTEMPO, a mitochondrial ROS scavenger and compared it to monotherapy treatment during an established IAV infection. Mice were infected with IAV (Hkx31 strain; 104PFU/mouse) and 24 h post infection were treated with Cgp91ds-TAT (0.2 mg/kg), mitoTEMPO (100 μg) or with a combination of these inhibitors [Cgp91ds-TAT (0.2 mg/kg)/mitoTEMPO (100 μg)] intranasally every day for up to 2 days post infection (pi). Mice were euthanized on Days 3 or 6 post infection for analyses of disease severity. A combination of Cgp91ds-TAT and mitoTEMPO treatment was more effective than the ROS inhibitors alone at reducing airway and neutrophilic inflammation, bodyweight loss, lung oedema and improved the lung pathology with a reduction in alveolitis following IAV infection. Dual ROS inhibition also caused a significant elevation in Type I IFN expression at the early phase of infection (day 3 pi), however, this response was suppressed at the later phase of infection (day 6 pi). Furthermore, combined treatment with Cgp91ds-TAT and mitoTEMPO resulted in an increase in IAV-specific CD8+ T cells in the lungs. In conclusion, this study demonstrates that the reduction of ROS production in two major subcellular sites, i.e. endosomes and mitochondria, by intranasal delivery of a combination of Cgp91ds-TAT and mitoTEMPO, suppresses the severity of influenza infection and highlights a novel immunomodulatory approach for IAV disease management.

Influenza virus replication is affected by glutaredoxin1‐mediated protein deglutathionylation

“…It is now appreciated that oxidative stress, historically defined as an imbalance between the production of oxidant species, mainly reactive oxygen and nitrogen species (ROS, RNS), and antioxidant systems of the cell, leads to disruption of redox signaling 8 . Several groups, including ours, have reported overproduction of ROS and impairment of antioxidant defenses, such as reduced glutathione (GSH) levels, during influenza virus infection; of note, perturbation of redox‐sensitive pathways, including the immune ones, favors influenza virus replication 9–13 . In particular, depletion of GSH affects the folding and maturation of the viral glycoprotein hemagglutinin (HA) and the progression of the virus life‐cycle 9 …”

“…8 Several groups, including ours, have reported overproduction of ROS and impairment of antioxidant defenses, such as reduced glutathione (GSH) levels, during influenza virus infection; of note, perturbation of redox-sensitive pathways, including the immune ones, favors influenza virus replication. [9][10][11][12][13] In particular, depletion of GSH affects the folding and maturation of the viral glycoprotein hemagglutinin (HA) and the progression of the virus life-cycle. 9 Importantly, GSH is not only an antioxidant molecule but is fundamental in the redox regulation of several cellular functions.…”