Activation of vitamin D regulates response of human bronchial epithelial cells to Aspergillus fumigatus in an autocrine fashion

Abstract

Aspergillus fumigatus (A. fumigatus) is one of the most common fungi to cause diseases in humans. Recent evidence has demonstrated that airway epithelial cells play an important role in combating A. fumigatus through inflammatory responses. Human airway epithelial cells have been proven to synthesize the active vitamin D, which plays a key role in regulating inflammation. The present study was conducted to investigate the impact of A. fumigatus infection on the activation of vitamin D and the role of vitamin D activation in A. fumigatus-elicited antifungal immunity in normal human airway epithelial cells. We found that A. fumigatus swollen conidia (SC) induced the expression of 1α-hydroxylase, the enzyme catalyzing the synthesis of active vitamin D, and vitamin D receptor (VDR) in 16HBE cells and led to increased local generation of active vitamin D. Locally activated vitamin D amplified SC-induced expression of antimicrobial peptides in 16HBE cells but attenuated SC-induced production of cytokines in an autocrine fashion. Furthermore, we identified β-glucan, the major A. fumigatus cell wall component, as the causative agent for upregulation of 1α-hydroxylase and VDR in 16HBE cells. Therefore, activation of vitamin D is inducible and provides a bidirectional regulation of the responses to A. fumigatus in 16HBE cells.

Figure 1

A. fumigatus induces the expression of 1α-hydroxylase and VDR and the conversion of 25D₃ to 1,25D₃ in 16HBE cells. (a) 16HBE cells were stimulated with resting conidia (RC) or swollen conidia (SC) [multiplicity of infection (MOI) = 1-2] for 2, 4, 8, 16, and 24 h. Nontreated 16HBE cells (basal) were used as controls. After treatment, cells were 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 illustrated are from one representative experiment of three separate ones, which are converted to densitometry units in graphs shown. 1α-Hydroxylase and VDR protein and mRNA expression was significantly upregulated in a time-dependent manner and peaked at 24 h after treatment with SC, whereas cells stimulated with RC showed no upregulation of 1α-hydroxylase and VDR protein and mRNA expression. All experiments were performed in triplicate on three consecutive days. Data shown are mean ± SE. Student's t-test, ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001, for comparison with baseline. (b) 16HBE cells were stimulated with RC or SC (MOI = 1-2) for 24 h in the presence of increasing doses of inactive vitamin D (25D₃), and active vitamin D (1,25D₃) was measured by ELISA in supernatants 24 h later. 16HBE cells converted the inactive vitamin D to the active form without other stimuli. 16HBE cells stimulated with SC generated greater amounts of active vitamin D in the presence of inactive vitamin D than cells without any treatment. RC stimulation did not influence the synthesis of active vitamin D in 16HBE cells. The graph reflects mean 1,25D₃ levels and SEM of three independent experiments. Student's t-test, ^∗ P < 0.05 and ^∗∗ P < 0.01, for comparison of SC challenged cells with cells without any treatment.

Figure 2

Locally activated vitamin D synergistically increases the expression of LL-37 and HBD2 in 16HBE cells infected with A. fumigatus but attenuates A. fumigatus-induced production of chemokines and cytokines. (a)–(c) 16HBE cells were untreated (basal) or stimulated with resting conidia (RC) or swollen conidia (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). Locally activated vitamin D synergistically induces protein expression of LL-37 and β-defensin-2 (HBD2) to a similar extent as exogenous active vitamin D (a). The protein expression of LL-37 and β-defensin-2 (HBD2) was evaluated by Western blot analysis. The Western blots illustrated are from one representative experiment out of three and converted to densitometry units in respective graphs. (b)-(c) Locally activated vitamin D attenuates A. fumigatus-induced production of chemokines and cytokines in 16HBE cells to a similar extent as exogenous active vitamin D. TNF-α, IL-1β, IL-6, and IL-8 mRNA expression was measured using quantitative real-time PCR (b). The concentrations of TNF-α and IL-8 in culture supernatants of 16HBE cells were measured by ELISA (c). Values reflect mean fold change from control and SEM of three independent experiments. Student's t-test, ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001, for comparison with baseline; ^# P < 0.05 and ^## P < 0.01, for comparison with control.

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 t-test, ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001, for comparison with baseline; ^# P < 0.05 and ^## P < 0.01, 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 t-test, ^∗ P < 0.05, for comparison with control siRNA transfected cells.

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 t-test, ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001, 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 t-test, ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001, for comparison with baseline; ^# P < 0.05 and ^## P < 0.01, for comparison with control.