1,25(OH)2D3 and VDR Signaling Pathways Regulate the Inhibition of Dectin-1 Caused by Cyclosporine A in Response to Aspergillus Fumigatus in Human Corneal Epithelial Cells

Abstract

Background: The objective of this study is to observe whether cyclosporine A (CsA) inhibits the expression of dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (A. fumigatus) and to investigate the molecular mechanisms of the inhibition.

Methods: Immortalized human corneal epithelial cells (HCECs) were pretreated with 1,25(OH)2D3 and VDR inhibitor for 1 h, and then they were pretreated with CsA for 12h. After these pretreatments, the HCECs were stimulated with A. fumigatus and curdlan respectively, and the expression of dectin-1 and proinflammatory cytokines (IL-1β and TNF-α) were detected by RT-PCR, western blot and ELISA.

Results: Dectin-1 mRNA and dectin-1 protein expression increased when HCECs were stimulated with A. fumigatus or curdlan, and CsA inhibited the dectin-1 expression both in mRNA and protein levels specifically. Dectin-1 and proinflammatory cytokine expression levels were higher when HCECs were pretreated with VDR inhibitor and CsA compared to pretreatment with CsA alone, while dectin-1 and proinflammatory cytokine levels were lower when HCECs were pretreated with 1,25(OH)2D3 and CsA compared to pretreatment with CsA alone.

Conclusions: These data provide evidence that CsA can inhibit the expression of dectin-1 and proinflammatory cytokines through dectin-1 when HCECs are stimulated by A. fumigatus or curdlan. The active form of vitamin D, 1,25(OH)2D3, and VDR signaling pathway regulate the inhibition of CsA. The inhibition is enhanced by 1,25(OH)2D3, and the VDR inhibitor suppresses the inhibition.

Fig 1

(A-F) The mRNA and protein fold change of dectin-1 and proinflammatory cytokines when HCECs were challenged with CsA and A. fumigatus. The expression of dectin-1 and proinflammatory cytocines decreased when stimulated by CsA. N in each graph represents the control group, and F represents the fungal-stimulation group. The mRNA fold change (A) and protein fold change (E) of dectin-1 were significantly inhibited when pretreated with CsA before stimulation of A. fumigatus. B shows dectin-1 expression when stimulated with curdlan. The mRNA fold change (B) and protein fold change (F) of dectin-1 is also inhibited significantly. C and D were the mRNA fold change of proinflammatory cytokines when challenged with curdlan, TNF-α (C) and IL-1β (D) are both inhibited significantly.

Fig 2

(A-B) The change of dectin-1mRNA and protein expression when HCECs were pretreated with VDR inhibitor. We pretreated the HCECs with VDR inhibitor for 1 h before CsA and 13 h before A. fumigatus stimulation. A was the mRNA fold change of dectin-1. B was the relative protein change of dectin-1.

Fig 3. The change of IL-1β and TNF-α mRNA and protein expression when HCECs were pretreated with VDR inhibitor.

We pretreated the HCECs with VDR inhibitor for 1 h before CsA and 13 h before A. fumigatus stimulation. A was the RNA fold change of IL-1β. C was the protein change of IL-1β. The IL-1β inhibition of the group pretreated with VDR inhibitor was significantly weakened compared to the group without VDR inhibitor stimulation. The same change could be seen in TNF-α (B and D).

Fig 4. The change of dectin-1 mRNA and protein expression when HCECs were challenged with 1,25(OH)₂D₃ before A. fumigatus and CsA stimulation.

We pretreated the HCECs with 1,25(OH)₂D₃ for 1h before CsA and 13 h before A. fumigatus stimulation. A depicts the mRNA fold change of dectin-1. Dectin-1 inhibition of the group pretreated with 1,25(OH)₂D₃ was significantly enhanced compared to the group without 1,25(OH)₂D₃stimulation. The same change could be seen in the protein expression of dectin-1 (B).

Fig 5. The change of IL-1β and TNF-α mRNA and protein expression when HCECs were pretreated with vitamin D.

We pretreated the HCECs with1,25(OH)₂D₃ for 1 h before CsA and 13 h before A. fumigatus stimulation. A was the mRNA fold change of IL-1β. C was the protein change of IL-1β. The IL-1β inhibition of the group pretreated with 1,25(OH)₂D₃ was significantly enhanced compared to the group without 1,25(OH)₂D₃ stimulation. The same could be seen in the change of TNF-α (B and D).