Pharmacological activities of roflumilast N-oxide, dexamethasone and formoterol in human neutrophils from healthy non-smokers and COPD patients

Introduction: Roflumilast N-oxide (RNO) has anti-inflammatory activities and reverses corticosteroid resistance in COPD neutrophils. This study further explored the anti-inflammatory effects of RNO, dexamethasone (DEX) and formoterol and their combinations in human neutrophils from healthy non-smokers and COPD patients. Methods: Neutrophils were incubated with DEX (0.1nM-1μM), RNO (0.1nM-1μM), formoterol (0.1nM-1μM) or the combinations of RNO 1nM + DEX 10nM and formoterol 1nM + DEX 10nM for 1h, followed by stimulation with 5% cigarette smoke extract (CSE) for 6h. IL-8 release, MIF and MKP-1 expression and PI3Kδ and HDAC2 activities were measured. Results: RNO inhibited CSE-induced IL-8 secretion, reaching a maximal inhibition of 90.4% in healthy and 88.9% in COPD neutrophils. DEX inhibited CSE-induced IL-8 secretion in healthy neutrophils (94.3%) but showed a weak inhibition of 20.6% in COPD neutrophils. Formoterol did not inhibit CSE-induced IL-8 release. The combination of RNO + DEX showed the most additive effects, nearly abolishing CSE-induced IL-8 release in COPD neutrophils, while the formoterol + DEX combo was less effective at decreasing IL-8 release. In COPD neutrophils, RNO restored DEX sensitivity by reversing CSE-induced MIF over-expression and MKP-1 down-regulation as well as by inhibiting CSE-induced PI3Kδ activation and HDAC2 down-regulation; in contrast, formoterol was less effective at restoring DEX sensitivity. Conclusions: The combination of RNO + DEX showed additive anti-inflammatory properties and RNO was more effective than formoterol at restoring DEX sensitivity in human neutrophils from COPD patients.