Roflumilast N-oxide reverses corticosteroid resistance in neutrophils from patients with chronic obstructive pulmonary disease

Background: Glucocorticoid functions are markedly impaired in patients with chronic obstructive pulmonary disease (COPD). The phosphodiesterase 4 inhibitor roflumilast N-oxide (RNO) is the active metabolite of roflumilast approved as a treatment to reduce the risk of exacerbations in patients with severe COPD. Objective: We sought to characterize the differential effects of RNO versus corticosteroids and their potential additive/synergistic effect in neutrophils from patients with COPD, thus providing scientific rationale for the combination of roflumilast with corticosteroids in the clinic. Methods: Peripheral blood neutrophils were isolated from patients with COPD (n = 32), smokers (n = 7), and healthy nonsmokers (n = 25). Levels of IL-8, matrix metallopeptidase 9 (MMP-9), and biomarkers of glucocorticoid resistance were determined by using ELISA and RT-PCR. Neutrophils were incubated with dexamethasone (0.1 nmol/L to 1 mu mol/L), RNO (0.1 nmol/L to 1 mu mol/L), or the combination of 1nmol/L RNO plus 10 nmol/L DEX and stimulated with LPS (1 mu g/mL) or cigarette smoke extract 5%; levels of IL-8, MMP-9, and other biomarkers were measured at the end of the incubation period. Results: Peripheral neutrophils from patients with COPD showed a primed phenotype with an increased basal release of IL-8 and MMP-9 and expressed a corticosteroid resistance molecular profile characterized by an increase in phosphoinositide 3-kinase delta, macrophage migration inhibitory factor, and glucocorticoid receptor beta expression and a decrease in HDAC activity and mitogen-activated protein kinase phosphatase 1 expression. RNO demonstrated robust anti-inflammatory effects on neutrophils from patients with COPD, reversing their resistance to corticosteroids. The combination of RNO and dexamethasone showed additive/synergistic effects, which were consistent with the reversal of corticosteroid-resistant molecular markers by RNO. Conclusion: RNO reverses corticosteroid resistance and shows strong anti-inflammatory effects alone or in combination with corticosteroids on neutrophils from patients with COPD.