Fig. 9

Endosome targeted delivery of a NOX2 oxidase inhibitor protects mice following influenza A virus infection in vivo. a–e Alveolar macrophages from WT mice were treated with the Cy5 cholestanol-PEG linker fluorophore (Cy5-chol; 100 nM) for 30 min and infected with HKx31 influenza A virus (MOI of 10). Cells were then counter labeled with antibodies to either: a and b EEA1, c NOX2 or d influenza A nucleoprotein (NP). All cells were then stained with 4′,6′-diamidino-2-phenylindole (DAPI) and imaged with confocal microscopy. b Cells were pretreated with dynasore (100 μM) for 30 min prior to exposure to Cy5-cholestanol. e Quantification of data from (a–d, n = 5). f RAW 264.7 macrophages were either untreated or treated with various concentrations of cholestanol-conjugated gp91ds-TAT (Cgp91), ethyl conjugated gp91ds-TAT (Egp91) or unconjugated gp91ds-TAT (Ugp91) for 30 min prior to quantifying ROS production by L-O12 (100 μM)-enhanced chemiluminescence (n = 7). g Superoxide production via the xanthine/xanthine oxidase cell-free assay in the absence or presence of Ugp91ds-TAT, (1 μM) or Cgp91ds-TAT (1 μM) (n = 6). h, i Ugp91ds-TAT (0.02 mg/kg/day) or Cgp91ds-TAT (0.02 mg/kg/day) were delivered intranasally to WT mice once daily for 4 days. At 24 h after the first dose of inhibitor, mice were either treated with saline or infected with HKx31 influenza A virus (1 × 10⁵ PFU per mouse). Mice were culled at day 3 post-infection and h airway inflammation was assessed by BALF cell counts and i lung IFN-β mRNA was determined by QPCR (n = 7). (NS) denotes not significant. j–m Mice were subjected to the NOX2 inhibitor treatment regime and virus infection protocol as in h except NOX2 inhibitors were delivered at a dose of 0.2 mg/kg/day (n = 7). Analysis of j airway inflammation by BALF counts, k body weight (% weight change from the value measured at Day -1), l ROS production by BALF inflammatory cells with L-O12 enhanced chemiluminescence and m viral mRNA by QPCR. Data are represented as mean ± S.E.M. e Unpaired t-test; statistical significance taken when the P < 0.05. f, g, h, j, k, l One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. i and m Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons. Statistical significance was accepted when P < 0.05. *P < 0.05; **P < 0.01. Scale bars: 10 µm