title: Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro.
abstract: OBJECTIVE
  Mucociliary 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.
  
  
  METHODS
  Air-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 β-tubulin IV staining and analyses of Foxj1 and Dnai2 mRNA and protein (real-time quantitative PCR, Western blotting).
  
  
  RESULTS
  Roflumilast 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.
  
  
  CONCLUSIONS
  Roflumilast N-oxide protected differentiated human bronchial epithelial cells from reduced CBF and loss of ciliated cells following CSE.
authors.affiliation: Research Unit, University General Hospital Consortium, Valencia, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Milara
authors.forename: M
authors.initials: M
authors.lastname: Armengot
authors.forename: P
authors.initials: P
authors.lastname: Bañuls
authors.forename: H
authors.initials: H
authors.lastname: Tenor
authors.forename: Rolf
authors.initials: R
authors.lastname: Beume
authors.forename: E
authors.initials: E
authors.lastname: Artigues
authors.forename: J
authors.initials: J
authors.lastname: Cortijo
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D001980:Bronchi / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D002923:Cilia / Q000187:drug effects / Q000502:physiology*; D003521:Cyclopropanes / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D058988:Phosphodiesterase 4 Inhibitors / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012906:Smoke / Q000009:adverse effects*; D013997:Time Factors; D062789:Tobacco Products / Q000009:adverse effects
chemical_list: D000631:Aminopyridines; D001549:Benzamides; D003521:Cyclopropanes; D058988:Phosphodiesterase 4 Inhibitors; D012906:Smoke; C517734:roflumilast N-oxide