Expression of Toll-like receptors in human retinal and choroidal vascular endothelial cells

Toll-like receptors (TLRs) are a family of proteins that initiate the innate immune response in reaction to invading microbes. Studies confirm the expression of TLRs in a variety of ocular tissues and cells, and it has also been suggested that selected TLRs may be associated with geographic atrophy and neovascularisation in age-related macular degeneration, diabetic retinopathy and other vascular and inflammatory diseases of the ocular posterior segment. However, TLR expression and localisation in the retinal and choroidal vasculature has not been defined. A better understanding of differential TLR expression in the choroid and retina, particularly in endothelial cells would improve our knowledge of vascular and inflammatory diseases in the posterior segment of the eye. In this study the gene (mRNA) expression of TLRs 1-10 was investigated using RT-PCR and comparative qPCR and the protein expression and localisation of selected TLRs (3, 4, 6 and 9) were examined using western blotting, flow cytometry and immunofluorescent staining. PCR showed gene expression of TLR1-6 and 9 in human choroidal endothelial cells (hCEC) and TLR2-6, 9 and 10 in human retinal endothelial cells (hREC). Western blotting detected TLR3, 4 and 9 proteins in both hCEC and hREC with higher levels in hCEC, whilst TLR6 protein was not detectable in either endothelial cell type. Flow cytometry detected all four TLRs (3, 4, 6 and 9) on the cell surface and intracellularly, TLR6 expression was detectable but low. The expression and localisation of TLR3, 4 and 9 were confirmed by immunofluorescent staining in endothelial cells and whole tissue sections and their functionality tested by expression of IL-6 (ELISA) in response to stimulation with specific TLR ligands. This study has, for the first time, identified the differential expression and localisation of TLRs in intraocular endothelial cells. This profiling will help inform our understanding of different retinal and choroidal vascular diseases, as well as the development of future treatments for intraocular vascular diseases.