title: Combination of roflumilast with a beta-2 adrenergic receptor agonist inhibits proinflammatory and profibrotic mediator release from human lung fibroblasts.
abstract: BACKGROUND
  Small airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD). The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting β2 adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis.
  
  
  METHODS
  The effects of roflumilast and indacaterol treatment were characterized on transforming growth factor β1 (TGFβ1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed.
  
  
  RESULTS
  Evaluation of roflumilast (1-10 μM) showed no significant inhibition alone on TGFβ1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF.
  
  
  CONCLUSIONS
  These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.
authors.affiliation: Respiratory Research, Gilead Sciences, 199 East Blaine St, Seattle, WA 98102, USA. Tannheimer@gilead.com
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Tannheimer
authors.forename: Clifford D
authors.initials: CD
authors.lastname: Wright
authors.forename: Michael
authors.initials: M
authors.lastname: Salmon
mesh_terms: D000199:Actins / Q000378:metabolism; D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology*; D000631:Aminopyridines / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D003521:Cyclopropanes / Q000494:pharmacology; D019332:Endothelin-1 / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007189:Indans / Q000494:pharmacology*; D007249:Inflammation / Q000378:metabolism / Q000517:prevention & control*; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058988:Phosphodiesterase 4 Inhibitors / Q000494:pharmacology*; D011658:Pulmonary Fibrosis / Q000378:metabolism / Q000517:prevention & control*; D015363:Quinolones / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D058666:Adrenergic beta-2 Receptor Agonists; D000631:Aminopyridines; D001549:Benzamides; C483024:CCL5 protein, human; C526317:CCN2 protein, human; C114753:CXCL10 protein, human; D018946:Chemokine CCL5; D054357:Chemokine CXCL10; D003521:Cyclopropanes; D019332:Endothelin-1; D005353:Fibronectins; D007189:Indans; D058988:Phosphodiesterase 4 Inhibitors; D015363:Quinolones; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; C424423:Roflumilast; D055513:Connective Tissue Growth Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C510790:indacaterol