Roflumilast N‐oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro

Abstract

BACKGROUND AND PURPOSEMucociliary malfunction occurs in chronic obstructive pulmonary disease (COPD) and compromised functions of ciliated bronchial epithelial cells may contribute to this. Cigarette smoke, a major risk factor for COPD, impairs ciliary beat frequency (CBF). cAMP augments CBF. This in vitro study addressed, in differentiated, primary human bronchial epithelial cells, whether roflumilast N-oxide, a PDE4 inhibitor, (i) augments CBF; (ii) prevents the reduction in CBF induced by cigarette smoke extract (CSE); and (iii) protects against the loss of the ciliated phenotype following long-term CSE exposure. EXPERIMENTAL APPROACHAir-liquid interface cultured human bronchial epithelial cells were incubated with roflumilast N-oxide and exposed to CSE. CBF was assessed by digital high speed video microscopy (DHSV). Ciliated cells were characterized by b-tubulin IV staining and analyses of Foxj1 and Dnai2 mRNA and protein (real-time quantitative PCR, Western blotting). KEY RESULTSRoflumilast N-oxide concentration-dependently triggered a rapid and persistent increase in CBF and reversed the decrease in CBF following CSE. Long-term incubation of bronchial epithelial cells with CSE resulted in a loss in ciliated cells associated with reduced expression of the ciliated cell markers Foxj1 and Dnai2. The PDE4 inhibitor prevented this loss in the ciliated cell phenotype and the compromised Foxj1 and Dnai2 expression. The enhanced release of IL-13 following CSE, a cytokine that diminishes the proportion of ciliated cells and in parallel, reduces Foxj1 and Dnai2, was reversed by roflumilast N-oxide. CONCLUSION AND IMPLICATIONSRoflumilast N-oxide protected differentiated human bronchial epithelial cells from reduced CBF and loss of ciliated cells following CSE. AbbreviationsCBF, ciliary beat frequency; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; DHSV, digital high speed video imaging technique; Dnai2, axonemal dynein intermediate polypeptide 2; DUOX, dual oxidase; Foxj1, forkhead transcription factor 1; NAC, N-acetyl cysteine; NOX, NADPH oxidase; ROS, reactive oxygen species BJP British Journal of Pharmacology

Cigarette smoke up‐regulates PDE3 and PDE4 to decrease cAMP in airway cells

“…As reported earlier (Milara et al ., 2012), CBF was reduced significantly by the CS extract (Figure 10B) and the PDE4 inhibitor rolipram (10 μM) fully reversed the reduction of CBF (Figure 10B). As illustrated in Figure 10C, methacholine did not significantly alter cAMP levels in both control and CS extract‐exposed airways.…”

“…PDE4 inhibitors are currently used for the treatment of COPD, and additional compounds are under development (Page, 2014). The PDE4 inhibitor roflumilast N‐oxide partly reverses CS‐induced epithelial dysfunction (Milara et al ., 2014, 2012; Schmid et al ., 2015; Tyrrell et al ., 2015), and the PDE4 inhibitors, GPD‐1116 and piclamilast can prevent the development of CS‐induced emphysema and pulmonary hypertension in mice (Mori et al ., 2008; Seimetz et al ., 2015, p. 4). Zl‐n‐91, a selective PDE4 inhibitor can also suppress CS‐induced lung inflammatory in rats (Wang et al ., 2010; Bucher et al ., 2016).…”

“…As part of this effort, our new findings point to a key role of PDE4D as a contributor to CS‐induced lung diseases. We observed an increase in PDE4A and PDE4B in the CS exposure models, and we thus propose that PDE4 subtypes may be effective drug targets, for example, to improve cilia motility of ciliated cells (Milara et al ., 2012), to inhibit the secretion of proinflammatory cytokines (Jin and Conti, 2002; Ariga et al ., 2004; Ma et al ., 2014) and to diminish cell proliferation (Selige et al ., 2011). To correlate cAMP changes with physiological responses, we measured CBF, as a marker for epithelial function.…”

“…To correlate cAMP changes with physiological responses, we measured CBF, as a marker for epithelial function. We found that inhibition of PDE4 fully reversed CBF down‐regulation induced by ex vivo exposure to CS extract (Figure 10B), which was in agreement with previous studies (Milara et al ., 2012). …”

“…COPD is associated with alterations in airway smooth muscle and epithelial cell function, a process paralleled by changes in activity and/or expression of cAMP signalling pathway effectors (Barnes, 2000; Qaseem et al ., 2011). Though CS can reportedly reduce cAMP in association with activation of PDE4 in cultured human bronchial epithelial cells (Milara et al ., 2014, 2012; Schmid et al ., 2015), the full repertoire of mechanisms that modulate cAMP in response to CS in multi‐cellular airway are not known.…”