April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Vitamin D Activation and TLR2 Activity in Human Corneal Epithelial Cells
Author Affiliations & Notes
  • Rose Y Reins
    College of Optometry, Univ of Houston, Houston, TX
  • Hasna Badouri
    College of Optometry, Univ of Houston, Houston, TX
  • Alison M McDermott
    College of Optometry, Univ of Houston, Houston, TX
  • Footnotes
    Commercial Relationships Rose Reins, None; Hasna Badouri, None; Alison McDermott, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5545. doi:
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      Rose Y Reins, Hasna Badouri, Alison M McDermott; Vitamin D Activation and TLR2 Activity in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5545.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Vitamin D has been shown to play a role in the wound healing response, increasing effectors of the innate immune system important for host protection. Toll-like receptors (TLR), sensors of the innate immune system, are also activated during wounding, and can interact with vitamin D signaling. In particular, TLR2 engagement has been demonstrated to enhance vitamin D activation and antimicrobial peptide production (LL-37) in other tissues. LL-37 itself is able to aid in corneal epithelial wound healing, increasing cell migration. Therefore, we examined the interaction between vitamin D and TLR2 signaling, as well as the involvement of the vitamin D receptor (VDR) in antimicrobial peptide expression.

Methods: Telomerase-immortalized human corneal epithelial cells (hTCEpi) were stimulated with 1,25D3 (10-7M) or TLR2 agonists FSL1, Pam3CSK4 (1μg/ml), and zymosan (10μg/ml) alone or concurrently for 24 hours. RNA was collected from control and treated cells and expression of CYP27B1, TLR2, and LL-37 were analyzed by real time PCR. For VDR silencing, 10nM control or VDR-specific siRNA were transfected into hTCEpi using Lipofectamine. VDR knock-down was confirmed by RT-PCR and Western analysis at 24, 48, and 72 hours post-transfection. Cells were stimulated with 1,25D3 24 hours after transfection.

Results: Stimulation of hTCEpi with TLR2 agonists resulted in a significant increase (~2.5 fold) of CYP27B1, the vitamin D activating hydroxylase. In addition, the coordinated response of both 1,25D3 and TLR2 agonists resulted in increased production of the antimicrobial peptide, LL-37 (8-12 fold increase), above 1,25D3 stimulation alone (2.5 fold). 1,25D3 did not modulate TLR2 expression, however. VDR expression was effectively knocked down with siRNA (77% at 24 hours) and 1,25D3 did not increase LL-37 in these samples, compared to cells with normal VDR expression.

Conclusions: Activation of hTCEpi through TLR2 ligation resulted in an increase in CYP27B1, the enzyme responsible for producing the active form of vitamin D, 1,25D3. Also, vitamin D and TLR2 acted synergistically to produce LL-37. The vitamin D-mediated expression of LL-37 was shown to be dependent on VDR, demonstrating functional activity of the receptor in hTCEpi. These results suggest that upon TLR2 activation, such as during wound healing, vitamin D activity can be augmented and can cooperate with TLR2 to enhance innate immunity and antimicrobial peptide production.

Keywords: 482 cornea: epithelium • 480 cornea: basic science  
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