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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)
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.
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.
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.
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.
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