Effect of roflumilast N-oxide on non-typable haemophilus influenzae (NTHi) interaction with human airway epithelial A549 cells

Background/Aim: NTHi is associated with lung colonisation and exacerbations in COPD. Previously we found that NTHi invasion of airway epithelial cells is PI3-kinase (PI3K)-dependent. Furthermore NTHi survives intracellularly in vacuoles with late endosome features, explaining airway colonisation despite NTHi-negative sputum. Here we explored whether the PDE4 inhibitor roflumilast N-oxide (RNO), the active metabolite of roflumilast (approved in EU for severe COPD), dexamethasone (DEX) and salmeterol (SAL) affect NTHi invasion of A549 cells and NTHi-induced IL-8 release. Methods: For invasion, cells were infected with NTHi (100 bacteria/cell, 2h), incubated with gentamycin (1h) to kill extracellular bacteria, and lysed to determine intracellular bacteria by plating. Intracellular lifestyle was monitored by immunofluorescence with antibodies to late endosome markers (lamp1, CD63). PI3K-dependent Akt phosphorylation was assessed by Western. IL-8 was measured by ELISA. Results: RNO (1μM) reduced NTHi invasion by 50% (c.f.u./well; Control: 5000±100, RNO: 2439±122; n=3, p<0.05) while DEX (1μM) and SAL (1μM) failed. Forskolin (10μM) reduced NTHi invasion by 70%, supporting a role of cAMP. RNO (1μM) curbed NTHi-induced Akt phosphorylation by 40%. Neither drug affected NTHi intracellular lifestyle. RNO (1μM) or DEX (1μM) reversed a 6.5-fold increase in NTHi-induced IL-8 release by 48±3% and 75±3% respectively and NF-κB activation. Conclusions: PDE4 inhibition (1μM RNO) reduced NTHi internalisation in A549 cells by interfering with PI3K signalling. RNO inhibited IL-8 release. DEX, while reducing NTHI-induced IL-8, did not affect internalisation.