BACKGROUND

Airway fibroblasts are major contributors to the histopathological feature of airway remodeling in asthma by their implication in the cell invasiveness and profibrogenic secretory phenotype observed in subepithelial fibrosis. 1,25 Dihydroxy vitamin D (1,25(OH)D) is an important therapeutic agent that blocks many features of airway remodeling induced by profibrogenic mediators, such as transforming growth factor beta 1 (TGF-β1) or T helper type 1 inflammatory cytokines.

OBJECTIVE

We hypothesized that 1,25(OH)D opposes the TGF-β1 or tumor necrosis factor alpha (TNF-α)-Interleukin 1 beta (IL-1β) stimulation on airway fibroblast profibrogenic secretory phenotype observed in severe asthmatic patients. Our aim was to investigate the anti-fibrogenic effect of 1,25(OH)D in TGF-β1 or TNF-α-IL-1β-stimulated human bronchial fibroblast cells (HBFCs) from severe asthmatic compared with non-asthmatic subjects.

PATIENTS AND METHODS

All experiments were performed on primary HBFCs from asthmatic (DHBFCs, n=4) and non-asthmatic subjects (NHBFCs, n=4). mRNA expression and protein quantification of key fibrogenic markers were analyzed by RT-qPCR and ELISA, comparing HBFCs from asthmatic and non-asthmatic subjects. Vitamin D receptor () mRNA expression and its functionality in HBFCs were assessed by RT-qPCR. HBFCs proliferation was assessed by flow cytometry using BrdU-FITC/7AAD bivariate staining, while HBFCs apoptosis by Annexin V-FITC/7AAD.

RESULTS

VDR is constitutively expressed in HBFCs and the addition of 1,25(OH)D significantly increased mRNA expression of (a direct VDRs' target gene) in both HBFCs groups. DHBFCs cultured in the presence of TGF-β1 or TNF-α-IL-1β showed increased mRNA expression and protein secretion of fibrogenic markers when compared to NHBFCs. Additionally, we observed decreased mRNA expression of and CC-chemokines (, CCL11) in response to 1,25(OH)D addition to the TGF-β1 or TNF-α-IL-1β-stimulated HBFCs. Cell culture media obtained from TGF-β1 or TNF-α-IL-1β-stimulated DHBFCs showed decreased protein secretion (fibronectin 1, lumican, MCP1, RANTES and eotaxin-1) in response to 1,25(OH)D when compared to NHBFCs. 1,25(OH)D inhibited proliferation in TGF-β1-stimulated HBFCs through G0/G1 cell cycle arrest and these effects were not correlated with the induction of apoptosis.

CONCLUSION

DHBFCs under TGF-β1 or TNF-α-IL-1β stimulation showed higher fibrogenic capacity when compared to NHBFCs. 1,25(OH)D significantly blocked these effects and highlight 1,25(OH)D as a possible therapeutic target for severe asthma.