Expression and Putative Role of 1,25(OH)2 D3 and TLR Signaling in Human Ocular Cells

Abstract

Purpose: : Corneal cells respond to infections by eliciting an innate immune response through Toll-like receptors (TLRs). Vitamin D (1,25(OH)₂D₃) is known to exert an immunomodulatory effect mediated by TLRs. In this study we aim to investigate the role of 1,25(OH)₂D₃ in corneal infections and existence of a cross-talk between vitamin D and TLR signaling.

Methods: : Immunohistochemical analyses for the expression for VDR, active vitamin D anabolizing enzyme (1α-OHase) and its catabolizing enzyme (24-OHase) were performed on paraffin embedded tissue sections of normal human anterior segments. Primary cultures of human corneal epithelial cells (PHCEC), corneal fibroblasts (HKF) and scleral fibroblasts (HSF) derived from donor corneal-scleral tissue, were analyzed for mRNA and protein expression for VDR, 1α-OHase and 24-OHase, using conventional RT-PCR, real-time PCR and Western blotting. The production of various cytokines (IL-8, IL-10, IL-12, IL-13, IL-4, IL-1β, IL-6, IFNγ and TNF-α) were examined by a combination of ELISA and multiplex bead immunoassays of culture supernatants. Pathway-focused gene expression real-time PCR superarray technology enabled study of the cross-talk between TLR and VDR signaling.

Results: : Immunolocalization studies confirmed expression of the VDR, 1α-OHase and 24-OHase in the corneal and limbal epithelium, and corneal endothelium. The PHCEC, HKF and HSF expressed mRNA and protein for VDR, 1α-OHase and 24-OHase. Following TLR activation, mRNA and protein expression of cytokines CSF-2,-3, IL-1α and β, IL-2, IL-6, IL-8, IL-12; chemokine-CXCL10; anti-inflammatory cytokine-IL-10 and anti-microbial peptide-cathelicidin were significantly up-regulated in all cell types. Under these conditions, the mRNAs for 1α-OHase and 24-OHase were also significantly up-regulated. 1,25(OH)₂D₃ (10nM) partially negated the stimulatory effects on cytokine/chemokine production upon TLR stimulation.

Conclusions: : Following TLR activation, naive corneal cells respond with production of pro-inflammatory cytokines capable of eliciting an inflammatory response, which is partially blocked by 1,25(OH)₂D_3. These data suggest a potential therapeutic role for vitamin D in combating corneal infections.

Keywords: cornea: basic science • receptors • cytokines/chemokines