Activation of Vitamin D Regulates Response of Human Bronchial Epithelial Cells to Aspergillus fumigatus in an Autocrine Fashion

Figure 4

β-Glucan increases the expression of 1α-hydroxylase and VDR, and vitamin D synergizes with β-glucan to induce the expression of antimicrobial peptides but attenuates β-glucan-induced expression of chemokines and cytokines in 16HBE cells. (a) β-Glucan increases the expression of 1α-hydroxylase and VDR in 16HBE cells in a dose-dependent manner. 16HBE cells were cultured in the absence or presence of increasing doses of β-glucan as indicated for 24 h. Cells were then harvested and the protein and mRNA expression of 1α-hydroxylase and VDR was evaluated by Western blot analysis and quantitative real-time PCR, respectively. The Western blots shown are from one representative experiment out of three, which are converted to densitometry units in graphs. Values reflect mean fold change from control and SEM of three independent experiments. Student’s -test, ^∗, ^∗∗, and ^∗∗∗, for comparison with baseline. (b) β-Glucan increases the conversion of inactive vitamin D (25D₃) to active vitamin D (1,25D₃) in a dose-dependent manner. 16HBE cells were cultured in the presence of increasing doses of β-glucan as indicated and inactive vitamin D (10⁻⁷ M) for 24 h, and active vitamin D (1,25D₃) in supernatants was measured by ELISA. Graph reflects mean 1,25D₃ levels and SEM of three independent experiments. (c), (d) 16HBE cells were cultured in the absence or presence of β-glucan (8 μg/mL) with or without either form of vitamin D (10⁻⁷M, 25D, and 1,25D) for 24 h. Cells were harvested and the protein expression of LL-37 and HBD2 was evaluated by Western blot analysis. The Western blots illustrated are from one representative experiment out of three, which are converted to densitometry units in graphs. The mRNA expression of TNF-α, IL-1β, IL-6, and IL-8 was evaluated by quantitative real-time PCR. Vitamin D synergizes with β-glucan to induce the expression of LL-37 and HBD2 (c) but attenuates β-glucan-induced expression of TNF-α, IL-1β, IL-6, and IL-8 in 16HBE cells (d). Values reflect mean fold change from control and SEM of three independent experiments. Student’s -test, ^∗, ^∗∗, and ^∗∗∗, for comparison with baseline; ^# and ^##, for comparison with control.

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