Retinal pigment epithelium cells alter the pro-inflammatory response of retinal microglia to TLR-3 stimulation.

Abstract

Microglia are the local cells of the innate immunity in the retina. Toll-like receptor (TLR) 3 is a receptor of the innate immune system, recognizing viral double-stranded RNA. Retinal pigment epithelium (RPE) cells express TLR-3 and react to TLR-3 stimulation. In this study, we investigated the effect of TLR-3-activated RPE on microglia. Primary porcine RPE cells were prepared from freshly prepared pigs' eyes. Retinal microglia were prepared from porcine retina. Expression of the microglia marker Iba1 was evaluated using immunocytochemistry. RPE cells were treated with polyinosinic/polycytidylic acid (Poly I:C; 100 ng/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) for 24 hr. Either the supernatant was applied to microglia for 6 or 24 hr or microglia cells were directly treated with Poly I:C for 6 and 24 hr. Expression of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)α, IL-10, Cox2 and iNOS was evaluated in quantitative PCR. Phagocytosis was evaluated with a microscope-based and a fluoroscan-based phagocytosis assay. Retinal pigment epithelium (RPE) cells induce the expression of IL-6, IL-1ß and IL-10 in microglia cells. Microglia cells respond to Poly I:C stimulation in a concentration-dependent manner with the induction of IL-1ß, IL-6, TNFα, Cox2, iNOS and, to a lesser degree, IL-10. Stimulation of microglia cells with supernatant of Poly I:C-treated RPE cells further elevated IL-6, IL-1ß and Cox2 expression, while it reduced the expression of iNOS. No changes in phagocytosis could be detected. TLR-3-activated RPE exacerbates inflammatory response of microglia in a differentiated manner. This indicates that viral infections in the RPE may have a proinflammatory influence on retinal microglia.