Abstract
Asthma is a chronic inflammatory lung disease of airways associated with reversible airway obstruction, functional and structural changes in the airways, and airway remodeling. Increased infiltration of CD4+ lymphocytes, especially T helper type 17 (Th17) subsets, in asthmatic lungs suggests their critical role in the pathophysiology of allergic asthma. Vitamin D is a potent immunoregulator modulating the functional responses of immune cells. Vitamin D manifests inhibitory effects on Th17 cells, whereas regulatory T helper subsets (Tregs) are stimulated by vitamin D. However, how vitamin D supplementation affects Th17/Tregs cell densities in asthma is ill-defined. From our laboratory an increase in blood T regulatory cells was reported in vitamin D supplemented OVA (Ovalbumin)-sensitized and challenged mice compared to vitamin D deficient OVA-sensitized and challenged mice. But the exact role of vitamin D deficiency, sufficiency and supplementation on Th17 and Tregs cell densities in a clinically relevant model and at the site of allergic airway inflammation is not well understood.|In this study, the female Balb/c mice were fed either vitamin D supplemented (10,000 IU/kg) diet or vitamin D sufficient (2000 IU/kg) or deficient diets (0 IU/kg). Allergic airway inflammation in mice was induced by clinically relevant cockroach allergen (CRA)-sensitization and challenge till acute and chronic stages of asthma. The serum vitamin D levels (25(OH)D), lung histology, mucus hypersecretion, collagen deposition, total number of broncho-alveolar lavage fluid (BALF) cells, BALF and serum cytokines, suppressor of cytokine signaling (SOCS)-1,3 and 5 mRNA and protein expression were examined in all different groups of mice. Also, we addressed the role of vitamin D supplementation on Th17 and Tregs cell densities in the mice sensitized and challenged with CRA.|Vitamin D-deficient, CRA-sensitized and challenged mice exhibited increased AHR to methacholine and exaggerated features of allergic airway inflammation such as airway obstruction, due to mucus and collagen deposition. On the other hand, AHR and the allergic airway inflammatory features were reversed by vitamin D supplementation in CRA-sensitized and challenged mice. Total number of BALF cells in the vitamin supplemented CRA-sensitized and challenged individuals were significantly increased compared to the number of BALF cells in the vitamin D deficient animals.|SOCS-1,3 and 5 mRNA and protein expression were also analyzed in the lungs of all vitamin D groups. SOCS-1 exhibits a pivotal role for IL-6 signaling pathway to further activate Th17 cell development. SOCS-3 expression is crucial for the development of pathogenic Th2 cells. High SOCS-5 expression is involved in the eosinophilic airway inflammation. Vitamin D deficiency increased the mRNA and protein expression of SOCS-1, 3 and 5 in the lungs of mice with acute or chronic asthma, whereas both mRNA and protein expression of SOCS-1, 3 and 5 were attenuated in vitamin D-supplemented mice sensitized and challenged with CRA.|The mRNA transcripts of IL-21R, IL-23R and RORγT from purified CD4+CD25- lymphocytes and mRNA transcripts of Foxp3 from purified CD4+CD25+ lymphocytes isolated from lungs and spleens of the mice from all vitamin D CRA-sensitized and challenged, and PBS groups were compared using qPCR. CD4+CD25- cells from vitamin D-supplemented, CRA-sensitized and challenged mice exhibited a decrease in IL-21R, IL-23R and RORγT mRNA transcripts compared to the levels of these transcripts from vitamin D-deficient, CRA-sensitized and challenged mice. On the other hand, the CD4+CD25+ cells from vitamin D-supplemented, CRA-sensitized and challenged mice had a significant upregulation of Foxp3 mRNA levels compared to the cells from vitamin D-deficient, CRA-sensitized and challenged mice. The markers for both Th17 and Tregs cells were also compared in all vitamin D groups by flow cytometry. The Th17 cell markers IL-21R, IL-23R and RORγT were increased on CD4+CD25- cells from vitamin D-supplemented, CRA-sensitized and challenged mice compared to the cells from vitamin D-deficient, CRA-sensitized and challenged mice. However the higher expression of Tregs cell markers such as Foxp3, ICOS, PD-1, GITR, Nrp-1 and CTLA4 was observed on CD4+CD25+ cells from vitamin Dvi supplemented CRA-sensitized and challenged mice compared to the cells from vitamin D-deficient, CRA-sensitized and challenged mice.|Finally, the expression of the vitamin D receptor (VDR) and Smad3 in the lungs and spleens of all experimental mice were analyzed. Higher expression of both VDR+ and CD4+CD25+Smad3+ cells were observed in vitamin D-supplemented, CRA-sensitized and challenged mice compared to the vitamin D-deficient, CRAsensitized and challenged group of mice. Also, splenocytes were isolated from CRA-sensitized, vitamin D deficient and sufficient mice, and treated with CRA and TGF-β and/or calcitriol in vitro to examine the number of CD4+CD25-RORγT, CD4+CD25+Foxp3 and CD4+CD25+Smad3. The TGF-β and/or calcitriol treatment reversed the effect of CRA by increasing the Smad3 and Foxp3 expression on CD4+CD25+ cells and downregulated RORγT on CD4+CD25- cells.|In conclusion, findings from this study demonstrated the potential therapeutic properties of vitamin D and the underlying cellular and molecular mechanism involved in reversing the deleterious effects of CRA-induced allergic airway inflammation and AHR in a murine model of asthma. Furthermore, the finding of the immunomodulatory effect of vitamin D supplementation opens a new direction for researching the Th17/Tregs cell imbalance in asthma. The outcomes of this study may be useful in developing better therapeutic modalities applicable to allergic asthma immunomodulation.