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.

Keywords: NADPH oxidase; airway inflammation; endosome; inflammation; influenza A virus; lung inflammation; mitochondria; reactive oxygen.

FIGURE 1

Combination therapy of Cgp91ds-TAT and mitoTEMPO reduces IAV-induced bodyweight loss, airway inflammation and pulmonary edema. (A) C57Bl/6J mice were treated once daily via intranasal administration of Cgp91ds-TAT (0.2 mg/kg) and mitoTEMPO (100 µg) in combination or individually over a 2 days period 1 day post-infection with HKx31 (10⁴ PFUs) or PBS. (B) Daily bodyweight measurements were taken over the experimental period. (C) Pulmonary edema was assessed by measuring the wet lung weight to bodyweight ratio at day 3 pi and day 6 pi. Airway inflammation was assessed via counting the total number of live cells isolated from the bronchoalveolar lavage fluid that were differentiated into macrophages, neutrophils and lymphocytes at (D) day 3 pi and (E) day 6 pi. 500 cells were counted from random fields by standard morphological criteria. Data are expressed as mean ± SEM (Control, n = 8; Cgp91, n = 4; mitoTEMPO n = 4; Cgp91/Mito, n = 8; Hkx31, n = 13; Hkx31 + Cgp91, n = 5; Hkx31 + Mito, n = 5; Hkx31 + Cgp91/Mito, n = 10). Statistical analysis was conducted using a two-way analysis of variance (ANOVA) followed by Holm’s Sidak post-hoc multiple comparison test in ‘B and C’ and one-way ANOVA test followed by Tukey’s post hoc test for multiple comparison test for (D, E). Statistical significance was taken where p < 0.05 (* denotes p < 0.05 and **p < 0.01). ## indicates p < 0.01 against Hk x-31.

FIGURE 2

Dual inhibition of endosomal and mitochondrial ROS reduces lung pathology in IAV infected mice. Histopathological analysis of lungs from C57Bl/6J mice that were infected with Hkx31 (10⁴ PFUs) or PBS via intranasal administration. Animals were treated once daily 1-day following infection via intranasal administration with Cgp91ds-TAT (0.2 mg/kg) and mitoTEMPO (100 µg) in combination or individually over a 2 days period at (A) day 3 pi and (B) day 6 pi. Representative images displaying lung inflammation from paraffin embedded lungs were sectioned (10 μm) longitudinally and stained with H&E. Each sample was scored blindly from 0–5 for each individual mouse (higher numbers indicate increased severity) from two independent assessors. Sections were scored for alveolitis (red arrows), inflammatory cell infiltrate and peribronchiolar inflammation (black arrows). Representative images are presented at three different magnifications [×1 (clear), ×3 (blue), ×6 (green)]. Data are expressed as mean ± SEM. Statistical analysis was conducted using non-parametric Mann-Whitney test. Statistical significance was taken where p < 0.05 (* denotes p < 0.05 and **p < 0.01).

FIGURE 3

Effects of ROS inhibition on mitochondrial and NOX2-derived ROS generation. C57Bl/6J mice were treated once daily via intranasal administration of Cgp91ds-TAT (0.2 mg/kg) and mitoTEMPO (100 µg) in combination or individually over a 2 days period 1 day pi with Hkx31 (10⁴ PFUs) or PBS for assessment at (A) day 3 pi and (B) day 6 pi. Cells isolated from lung tissue were collected for mitochondrial ROS measurements by staining cells with a fluorescent probe (Mitosox) and assesed using flow cytometric analysis. The populations are measured as a % of mitosox positive cells gated from the CD45⁺ population. The percentage of Mitosox + cells were further characterised in Ly6G + neutrophils by gating from the CD45⁺ population. PDB (10⁻⁶ M) stimulated ROS production that was quantified by L-O12 enhanced chemiluminescence from BALF inflammatory cells. Data are expressed as mean ± SEM (Control, n = 6; Cgp91, n = 4; mitoTEMPO n = 4; Cgp91/Mito, n = 6; Hkx31, n = 8 Hkx31 + Cgp91, n = 5; Hkx31 + Mito, n = 5; Hkx31 + Cgp91/Mito, n = 8). Statistical analysis were conducted using one-way ANOVA test followed by Tukey’s post hoc test for multiple comparison test. Statistical significance was taken where p < 0.05 (* denotes p < 0.05 and **p < 0.01).

FIGURE 4

Lung IFN-β and viral mRNA expression at an early and late stage of influenza A virus infection. Mice were infected with Hx x-31 at (10⁴ PFUs) or PBS via intranasal administration. Animals were treated once daily 1 day following infection via intranasal administration with a combination of Cgp91ds-TAT (0.2 mg/kg) and mitoTEMPO (100 µg) or ROS inhibitors alone over a 2 days period for IFN-β analysis at (A) day 3 pi and (B) day 6 pi. and mRNA analysis of the gene encoding polymerase of influenza virus strain by QPCR in (C) at day 3 pi and (D) day 6 pi. IFN-β and viral PA were quantified in lung tissue. Responses are relative to GAPDH and expressed as a fold-change above naïve controls. Data are expressed as mean ± SEM (Control, n = 6; Cgp91, n = 4; mitoTEMPO n = 4; Cgp91/Mito, n = 8; Hkx31, n = 14 Hkx31 + Cgp91, n = 5; Hkx31 + Mito, n = 5; Hkx31 + Cgp91/Mito, n = 12). 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 (* denotes p < 0.05 and **p < 0.01).

FIGURE 5

Combination therapy elevates virus-specific T cells in the lung. C57Bl/6J mice were treated once daily via intranasal administration of Cgp91ds-TAT (0.2 mg/kg) and mitoTEMPO (100 µg) in combination or individually over a 2 days period 1 day pi with Hkx31 (10⁴ PFUs) or PBS for assessment at day 6 pi. Cells isolated from the lung tissue were collected for immune cell differentiation that was assesed using flow cytometric analysis. Virus-specific CD8⁺ T cells were gated as CD8+NP366+. Data are expressed as mean ± SEM (Control, n = 4; Cgp91, n = 4; mitoTEMPO n = 4; Cgp91/Mito, n = 4; Hkx31, n = 5 Hkx31 + Cgp91, n = 6; Hkx31 + Mito, n = 6; Hkx31 + Cgp91/Mito, n = 6). Statistical analysis were conducted using one-way ANOVA test followed by Tukey’s post hoc test for multiple comparison test. Statistical significance was taken where p < 0.05 (* denotes p < 0.05 and **p < 0.01).