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

Figure 3

Silencing VDR or 1α-hydroxylase attenuates the effects of locally activated vitamin D on the responses of 16HBE cells to A. fumigatus. (a), (b) 16HBE cells were transfected with a control siRNA or an siRNA against VDR or 1α-hydroxylase for 24 h and then challenged with SC (MOI = 1-2) for another 24 h. The protein and mRNA levels of VDR (a) and 1α-hydroxylase (b) were measured by Western blot analysis and quantitative real-time PCR, respectively. (c)–(f) 16HBE cells were transfected with a control siRNA or an siRNA against VDR or 1α-hydroxylase for 24 h and then stimulated with SC (MOI = 1-2) for 24 h in the presence of either inactive (25D₃; 10⁻⁷ M) or active vitamin D (1,25D₃; 10⁻⁷ M). 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 respective graphs. The mRNA expression of TNF-α, IL-1β, IL-6, and IL-8 was evaluated by quantitative real-time PCR. When VDR expression was silenced, the modulatory effects of both forms of vitamin D on LL-37, HBD2, TNF-α, IL-1β, IL-6, and IL-8 expression in 16HBE cells induced by SC were reduced ((c), (d)); when 1α-hydroxylase expression was silenced, only the effect of 25D₃ was reduced ((e), (f)). 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 SC. (g) CYP27B1 silencing reduces the synthesis of active vitamin D induced by SC in 16HBE cells. 16HBE cells were transfected with a control siRNA or an siRNA against 1α-hydroxylase for 24 h and then stimulated with SC (MOI = 1-2) for 24 h in the presence of inactive vitamin D (25D₃; 10⁻⁷ M); active vitamin D (1,25D₃) in supernatants was measured by ELISA. Graph reflects mean 1,25D₃ levels and SEM of three independent experiments. Student’s -test, ^∗, for comparison with control siRNA transfected cells.

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