FIG. 2.

Intranasal treatment with MitoTEMPO reduces mitochondrial and NOX2-derived ROS generation at day 3 postinfection. C57Bl/6J mice were treated once daily via intranasal administration of MitoTEMPO (100 μg) over a 4-day period 1 day before virus infections. Mice were intranasally infected with X31 (10⁴ PFUs) or PBS control. At day 3 p.i, BALF was collected for (A) mitochondrial ROS measured by staining cells with a fluorescent probe (MitoSOX) and assessed using flow cytometric analysis. The populations are measured as a percentage of MitoSOX⁺ cells gated from the CD45⁺ population. (B) MitoSOX⁺ cells were further characterized in CD45⁺ F4/80⁺ macrophages and (C) CD45⁺ Ly6G⁺ neutrophils. (D) 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 = 6–8; MitoTEMPO, n = 6–8; X31 n = 10 X31+mito n = 12–14). Statistical analysis was conducted using (A–C) Students' unpaired t-test and in (D) one-way ANOVA test followed by Tukey's post hoc test for multiple comparisons. Statistical significance was taken where p < 0.05 (*p < 0.05 and **p < 0.01). BALF, bronchoalveolar lavage fluid; PDB, phorbol dibutyrate; ROS, reactive oxygen species.