Vitamin D Activation and Function in Human Corneal Epithelial Cells During TLR-Induced Inflammation

Abstract

Purpose: Vitamin D is recognized to be an important modulator of the immune system. In the eye, studies have shown that deficiencies and genetic differences in vitamin D-related genes have a significant impact on the development of various ocular diseases. Our current study examines the ability of human corneal epithelial cells (HCEC) to activate vitamin D and the effect of vitamin D treatment on antimicrobial peptide production and cytokine modulation during inflammation, with the ultimate goal of using vitamin D therapeutically for corneal inflammation.

Methods: Human corneal epithelial cells were treated with 10-7M vitamin D3 (D3) or 25-hydroxyvitamin D3 (25D3) for 24 hours and 1,25-dihydroxyvitamin D3 (1,25D3) detected by immunoassay. Human cathelicidin (LL-37) expression was examined by RT-PCR, immunoblot, and immunostaining following 1,25D3 treatment and antimicrobial activity of 1,25D3-treated cells was determined. Cells were stimulated with TLR3 agonist polyinosinic-polycytidylic acid (Poly[I:C]) for 24 hours and cytokine levels measured by RT-PCR, ELISA, and Luminex. Immunostaining determined expression of vitamin D receptor (VDR) and retinoic acid inducible gene-1 receptor (RIG-1) as well as NF-κB nuclear translocation.

Results: When treated with inactive vitamin D metabolites, HCEC produced active 1,25D3, leading to enhanced expression of the antimicrobial peptide, LL-37, dependent on VDR. 1,25-D3 decreased the expression of proinflammatory cytokines (IL-1β, IL-6, TNFα, and CCL20) and MMP-9 induced by Poly(I:C) as well as pattern recognition receptor expression (TLR3, RIG-1, MDA5). However, early activation of NF-κB was not affected.

Conclusions: These studies demonstrate the protective ability of vitamin D to attenuate proinflammatory mediators while increasing antimicrobial peptides and antipseudomonas activity in corneal cells, and further our knowledge on the immunomodulatory functions of the hormone.

Figure 1

Human corneal epithelial cells express the enzymes for vitamin D metabolism, a functional VDR, and are able to convert inactive vitamin D compounds to the biologically active metabolite, 1,25D₃. (A) Reverse transcription-PCR analysis of hydroxylase and VDR expression in primary HCEC and hTCEpi. Cytochrome p450 (CYP) hydroxylases: 27A1 and 2R1 (25-hydroxylases—catalyze D₃ to 25D₃): 27B1 (1α-hydroxlyase—catalyzes 25D₃ to 1,25D₃). (B) Human telomerase–immortalized corneal epithelial cells were stimulated with 1,25D₃ (10⁻⁷M) or vehicle for 24 hours, then fixed and stained for VDR (FITC/green) and DAPI nuclear stain (blue). Rat IgG served as a negative control. Images are representative of three independent experiments. Vitamin D receptor nuclear localization is significantly increased with 1,25D₃ treatment compared to control, as determined by nuclear staining intensity of the FITC signal; P < 0.0001, Student's t-test. Scale bars: 40 μm. Human telomerase–immortalized corneal epithelial cells were treated with (C) 25D₃ (10⁻⁶M–10⁻⁹M) or (D) D₃ (10⁻⁷M) for 24 hours and 1,25D₃ (pmol/L) was quantitated in cell supernatants by immunoassay. Data represent mean ± SEM of four independent experiments. Statistical analysis was by ANOVA with Bonferroni's test for multiple comparisons (C) and Student's t-test (D); *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2

Human corneal epithelial cells respond to vitamin D by inducing the expression of CYP24A1, regulated by the VDR. (A) Human telomerase–immortalized corneal epithelial cells were treated with D₃, 25D₃, or 1,25D₃ (10⁻⁷M) for 24 hours and CYP24A1 expression was determined by real-time PCR (left) and Western blotting (right). (B) Human telomerase–immortalized corneal epithelial cells were left untreated (control), transfected with a nonspecific control siRNA (Neg. siRNA), or VDR siRNA for 24, 48, or 72 hours and analyzed for VDR expression by real-time PCR (left) and Western blot (right). (C) Human telomerase–immortalized corneal epithelial cells were left untreated (control and 1,25D₃) or transfected with VDR siRNA for 24 hours and then treated with 1,25D₃ for 24 hours to determine if VDR expression is required for 1,25D₃-mediated gene induction. Expression of CYP24A1 was analyzed by real-time PCR. Data represent mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test (A, B) and ANOVA with Bonferroni's test for multiple comparisons (C), *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3

Vitamin D increases the expression of vitamin D–regulated genes involved in innate immune defense. (A) Human telomerase–immortalized corneal epithelial cells were treated with 10⁻⁷M D₃, 25D₃, or 1,25D₃ for 24 hours and expression of LL-37 and CD14 was determined by real-time PCR (left) and LL-37 production was determined by dot blot (right). (B) Human telomerase–immortalized corneal epithelial cells were left untreated (control and 1,25D₃) or transfected with VDR siRNA for 24 hours and then treated with 1,25D₃ for 24 hours to determine if VDR expression is required for 1,25D₃-mediated gene induction. LL-37 (left) and CD14 (right) expression was analyzed by real-time PCR. Graphs represent mean ± SEM of three independent experiments. Statistical analysis was by ANOVA with Bonferroni's test for multiple comparisons, *P < 0.05, **P < 0.01 compared to control; ****P < 0.0001 compared to 1,25D₃. (C) Human corneas were treated with 1,25D₃ for 24 hours, fixed in paraformaldehyde, and frozen sections stained for LL-37 or a control rabbit IgG (green) and DAPI nuclear stain (blue).

Figure 4

Vitamin D increases HCEC antimicrobial activity against P. aeruginosa and enhances TLR-mediated LL-37 production. (A) SV40-HCEC were left untreated (Cells) or treated with 1,25D₃ (10⁻⁷M) for 24 hours (Cells+1,25D₃) and supernatants used in an antimicrobial assay. For controls, media only (Media) or 1,25D₃ in media (Media+1,25D₃) were used without cells. Data represent mean ± SEM of three independent experiments. Statistical analysis was by Student's t-test, **P < 0.01. (B) Human telomerase–immortalized corneal epithelial cells were treated with 1 μg/mL TLR agonists for 24 hours (Pam3CSK4 [TLR2/1] [Pam], FSL1 [TLR2/6], Poly[I:C] [TLR3], Flagellin [TLR5] [Flag]) or 50 μg/mL zymosan (TLR2) (Zym) for 6 hours. Expression of CYP27B1 and CYP24A1 was determined by real-time PCR. nd, not determined. (C) Human telomerase–immortalized corneal epithelial cells were treated with TLR agonists (Flag, FSL1, HKLM, Pam, or Poly[I:C]), in combination with 1,25D₃ (10⁻⁷M) for 24 hours and LL-37 expression analyzed by real-time PCR. Data represent mean ± SEM of four to six independent experiments. Statistical analysis was by ANOVA with Bonferroni's test for multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Vitamin D modulates HCEC response to Poly(I:C) through decreased production of proinflammatory cytokines and MMP-9. Human telomerase–immortalized corneal epithelial cells were treated with 1,25D₃ (10⁻⁷M) and/or TLR agonists (1 μg/mL) for 24 hours. (A) Cell lysates were collected for RNA isolation followed by real-time PCR analysis (top row) and supernatants were used to quantify protein levels (bottom row) of IL-8 (ELISA), and IL-1β, IL-6, TNFα, or CCL20 (Luminex assay). (B) For MMP-9 expression, cell lysates were collected for RNA isolation followed by real-time PCR analysis (top) and supernatants were used to quantify protein levels by ELISA (bottom). (C) Human telomerase–immortalized corneal epithelial cells were treated with 1,25D₃ (10⁻⁷M) for 24 hours (top) or 1,25D₃ in combination with FSL1 or Flagellin (Flag, bottom). Relative quantity of MMP-9 was assessed by real-time PCR. Graphs represent mean ± SEM of four to five independent experiments. Statistical analysis was by ANOVA with Bonferroni's test for multiple comparisons or Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Vitamin D lowers the expression of PRRs TLR3 and RIG-1/MDA-5 but does not block NF-κB p65 nuclear translocation following Poly(I:C) stimulation. (A) Human telomerase–immortalized corneal epithelial cells were treated with 1,25D₃ (10⁻⁷M) and/or Poly(I:C) (1 μg/mL) for 24 hours. TLR3 expression was determined by real-time PCR (left) and flow cytometry analyses (middle and right). (B) Cytoplasmic pattern recognition receptors RIG-1 (left) and MDA-5 (right) expression was determined by real-time PCR. Data represent mean ± SEM of five independent experiments. Statistical analysis was by ANOVA with Bonferroni's test for multiple comparisons (A) or Student's t-test (B), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) Human telomerase–immortalized corneal epithelial cells were stimulated with 1,25D₃ (10⁻⁷M) or vehicle for 24 hours, then fixed and stained for RIG-1 (red) and DAPI nuclear stain (blue). (D) Human telomerase–immortalized corneal epithelial cells were stimulated with 1,25D₃ and/or Poly(I:C) for 2 hours and stained with NF-κB subunit p65 (green) and DAPI nuclear stain (blue). Images are representative of two independent experiments. Scale bars: 40 μm.