Abstract
Purpose:
Vitamin D3 has been shown to have an important immunomodulatory role during infection and inflammation in many tissues. This study evaluated the in vitro anti-inflammatory actions of vitamin D3 on human corneal epithelial cells in response to Toll-like receptor (TLR) 3 activation.
Methods:
Telomerase-immortalized human corneal epithelial cells (hTCepi) were stimulated with 1,25D3 (10-7M) and/or TLR3 agonist PolyI:C (1μg/ml) for 24 hours. RNA was collected from control and treated cells and expression of IL-1β, IL-6, IL-8, IL-23, IFNβ, TNFα, MIP3α, MMP-9, LL-37, and TLR3 were analyzed by real time RT-PCR. Protein expression was examined using ELISA (IL-8, MMP9), Luminex (IL-1β, IL-6, TNFα), western (LL-37), and flow cytometry (TLR3). For evaluation of NFκΒ signaling, cells were incubated for 2 hours with PolyI:C and 1,25D3 and p65 nuclear translocation was visualized through immunofluorescence. All experiments were performed a minimum of three times.
Results:
Stimulation with PolyI:C resulted in a significant increase (range 38 to 1025 fold) in pro-inflammatory cytokine (IL-1β, IL-6, IL-8, IL-23, IFNβ, TNFα, and MIP3α) expression in hTCepi. Addition of 1,25D3 downregulated both the RNA and protein levels of these cytokines following TLR3 activation (41-87%). MMP-9 expression was also attenuated by 1,25D3 during PolyI:C stimulation by 51%. Additionally, 1,25D3 decreased TLR3 expression by 39%. Interestingly, the coordinated response of both 1,25D3 and PolyI:C resulted in increased production of the anti-microbial peptide, LL-37 (9.1±2 fold), above 1,25D3 stimulation alone (5.6±0.4 fold). Finally, NFκΒ p65 nuclear translocation did not appear to be disrupted by 1,25D3 at the time points tested following PolyI:C stimulation.
Conclusions:
Activation of hTCepi through TLR3 resulted in a robust induction of inflammatory mediators. Vitamin D3 attenuated this response by decreasing the production of these cytokines as well as TLR3 expression during PolyI:C stimulation. At the same time, LL-37 expression was augmented. While an effect on NFκΒ nuclear localization was not seen at the times indicated, further studies are ongoing to determine how vitamin D3 affects this signaling pathway. These results suggest an important role for vitamin D3 in protecting the ocular surface during inflammation and demonstrate its ability to dampen the potentially harmful effects of an inflammatory response.
Keywords: 482 cornea: epithelium •
555 immunomodulation/immunoregulation •
557 inflammation