Figure 4

Roflumilast N-oxide inhibited IL-8 release in human bronchial epithelial cells and demonstrated additive anti-inflammatory properties in corticosteroid resistance conditions. Human bronchial epithelial cells (HBECs) from non-smokers (n = 4), smokers (n = 4), and COPD patients (n = 5) were isolated from lung tissues. (A) HBECs from different patients were stimulated with TLR3 agonist poly I:C or the TLR7/8 dual agonist CL097 for 24 hours and IL-8 levels in the supernatants were measured by ELISA. (B, C, D) HBECs from different patients were incubated with roflumilast N-oxide (RNO) or dexamethasone (DEX) at different concentrations for 1 hour followed by the stimulation with poly I:C 10 μg/ml for 24 hours to measure IL-8 release. (D) In COPD patients, fixed concentrations of DEX 10 nM were combined with different RNO concentrations. Results are expressed as means ± SEM of n = 4–5 (4 non-smoker, 4 smokers, and 5 COPD patients) run in triplicate experiments. Two-way repeated measures analysis of variance (ANOVA) were performed. Post hoc Bonferroni test: *P < 0.05 compared with solvent controls. #P < 0.05 compared with monotherapy.