Novel endosomal NOX2 oxidase inhibitor ameliorates pandemic influenza A virus-induced lung inflammation in mice

Abstract

Background and objective: Influenza A viruses (IAV) cause respiratory tract infections that can be fatal when the virus spreads to the alveolar space (i.e. alveolitis), and this is mainly observed with highly pathogenic strains. Reactive oxygen species (ROS) production by the NOX2 NADPH oxidase in endosomes has been directly implicated in IAV pathology. Recently, we demonstrated that treatment with a novel endosome-targeted NOX2 oxidase inhibitor, cholestanol-conjugated gp91dsTAT (Cgp91ds-TAT), attenuated airway inflammation and viral replication to infection with a low pathogenic influenza A viral strain. Here, we determined whether suppression of endosome NOX2 oxidase prevents the lung inflammation following infection with a highly pathogenic IAV strain.

Methods: C57Bl/6 mice were intranasally treated with either DMSO vehicle (2%) or Cgp91ds-TAT (0.2 mg/kg/day) 1 day prior to infection with the high pathogenicity PR8 IAV strain (500 PFU/mouse). At Day 3 post-infection, mice were culled for the evaluation of airway and lung inflammation, viral titres and ROS generation.

Results: PR8 infection resulted in a marked degree of airway inflammation, epithelial denudation, alveolitis and inflammatory cell ROS production. Cgp91ds-TAT treatment significantly attenuated airway inflammation, including neutrophil influx, the degree of alveolitis and inflammatory cell ROS generation. Importantly, the anti-inflammatory phenotype affected by Cgp91ds-TAT significantly enhanced the clearance of lung viral mRNA following PR8 infection.

Conclusion: Endosomal NOX2 oxidase promotes pathogenic lung inflammation to IAV infection. The localized delivery of endosomal NOX2 oxidase inhibitors is a novel therapeutic strategy against IAV, which has the potential to limit the pathogenesis caused during epidemics and pandemics.

Keywords: NOX2 oxidase; endosome; inflammation; influenza; respiratory infections.

Figure 1

Cgp91ds‐TAT treatment suppressed BALF inflammation in mice infected with PR8 virus. Mice were treated daily with intranasal administration of Cgp91ds‐TAT (0.2 mg/kg) or DMSO (2%; control) over a 4‐day period. Mice were intranasally infected with PR8 (500 PFU) or PBS control 1 day post initial drug treatment. BALF inflammation was assessed via counting the total number of (A) live cells and differential cell counts of (B) macrophages, (C) neutrophils, (D) lymphocytes and (E) eosinophils. A total of 500 cells were counted from random fields by standard morphological criteria. Data are expressed as mean ± SEM (control, n = 7; Cgp91ds‐TAT, n = 5; PR8, n = 9; PR8 + Cgp91ds‐TAT, n = 10). Statistical analysis was conducted using one‐way ANOVA followed by Tukey's post hoc test for multiple comparison tests. Statistical significance was taken where P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ANOVA, analysis of variance; BALF, bronchoalveloar lavage fluid; Cgp91ds‐TAT, cholestanol‐conjugated gp91ds‐TAT; DMSO, dimethyl sulphoxide; PFU, plaque forming units.

Figure 2

Lung histopathological stains reveal Cgp91ds‐TAT reduces airway inflammation in PR8‐infected mice. Histopathological analysis of lungs from WT C57Bl/6J mice treated daily via intranasal administration Cgp91ds‐TAT (0.2 mg/kg) or DMSO (2%; control) over a 4‐day period. Mice were infected with PR8 (500 PFU) or PBS control 1 day post initial drug treatment and analysed at Day 3 post‐infection. Representative H&E images displaying the inflammation in lung sections following H&E staining. Each sample was assigned a score of 0–5 for each individual mouse (higher numbers indicate increased disease severity), as assessed by two independent assessors. Sections were scored for alveolitis, inflammatory cell infiltrate and peribronchiolar inflammation. Magnifications of images are at ×1, ×3, ×6, ×10. The black arrows show peribronchial inflammation, red arrow perivascular inflammation and blue alveolitis. Data were expressed as mean ± SEM (control, n = 7; Cgp91ds‐TAT, n = 5; PR8, n = 9; PR8 + Cgp91ds‐TAT, n = 10). Statistical analysis was conducted using one‐way ANOVA followed by Tukey's post hoc test for multiple comparison tests. Statistical significance was taken where P < 0.05. *P < 0.05; ***P < 0.001. ANOVA, analysis of variance; Cgp91ds‐TAT, cholestanol‐conjugated gp91ds‐TAT; DMSO, dimethyl sulphoxide; HE, haematoxylin and eosin; PFU, plaque forming units; WT, wild type.

Figure 3

Cgp91ds‐TAT markedly reduces viral mRNA expression and ROS generation. WT C57Bl/6J mice (8–12 weeks) were treated daily via intranasal administration of Cgp91ds‐TAT (0.2 mg/kg) or DMSO (2%) control. Mice were intranasally infected with PR8 (500 PFU) or PBS control 1 day post initial drug treatment. (A) Quantitative PCR analysis in lung tissue of mRNA from the gene encoding polymerase of influenza virus strain PR8; results were presented relative to those of GAPDH mRNA. (B) BALF was collected for PDB (10⁻⁶ M)‐stimulated ROS production that was quantified by L‐O12 enhanced chemiluminescence. Data were expressed as mean ± SEM (control, n = 7; Cgp91ds‐TAT, n = 5; PR8, n = 9; PR8 + Cgp91ds‐TAT, n = 10). Statistical analysis was conducted using one‐way ANOVA test followed by Tukey's post hoc test for multiple comparison. Statistical significance was taken where P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001. ANOVA, analysis of variance; BALF, bronchoalveloar lavage fluid; Cgp91ds‐TAT, cholestanol‐conjugated gp91ds‐TAT; DMSO, dimethyl sulphoxide; PCR, polymerase chain reaction; PDB, phorbol dibutyrate; PFU, plaque forming units; RLU, relative light units; ROS, reactive oxygen species; WT, wild type.