Fig. 5. H1N1 virus load and histologic changes of the lung in H1N1-infected and CS-exposed mice after treatment with tiotropium, fluticasone, or roflumilast. (A) Mice were exposed to CS and infected with H1N1 and additionally treated with either tiotropium (0.1 mg/ml or 0.3 mg/ml), fluticasone (0.3 mg/kg or 0.5 mg/kg), or roflumilast (0.2 mg/kg or 1.0 mg/kg). Shown is the mean +/− SEM viral load in lung homogenate of n = 7 or 8 mice per group measured by immunofocus assay. (B) NCI-H292 cells were infected with H1N1 and treated with tiotropium, fluticasone, or roflumilast as described in Materials and Methods. Changes in virus load were measured in cell lysates using qPCR (mean ± S.E.M. of n = 4 separate wells per group). (C) H&E stains of lung sections from untreated negative control (NC) mice, mice exposed to CS and H1N1, or CS/H1N1-exposed/infected mice, which were additionally treated with tiotropium, fluticasone, or roflumilast. Arrows indicate areas of inflammation or structural changes. Lungs of n = 6–8 mice per group were analyzed and representative sections shown. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 represent significant differences compared with the CS-exposed and H1N1-infected group or the H1N1-infected cells.