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N. Yi, S. Nagar, J. H. Salmon, B. C. Gilger; Expression of Toll-like Receptor (TLR)-4 in Equine Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5913.
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mRNA expression of TLR-2 and -9, but not TLR-4, was shown to be up-regulated in retinal tissues with equine recurrent uveitis (ERU) (Yi NY, et al., Proceedings of the ACVO, October 2008), a presumed immune-mediated disease similar to human uveitis. The purpose of this study was to further define the role of TLR-4 in the equine retina by localizing TLR-4 in the normal equine retina and to determine if retinal pigment epithelial (RPE) cells can be induced by LPS to express TLR-4 in vitro.
Localization of TLR-4 was done by immunohistochemistry (IHC) using antibody against TLR-4 (1:500) via standard ABC technique. Equine RPE cells were cultured and maintained until confluency. Second passage cells were stimulated with 0.01, 1, and 10 µg/ml of LPS. TLR4 protein expression was measured by western blot and IL-1β, IL-6 and TNF in cell culture supernatants was evaluated by ELISA at 0, 6, 12, and 24 hours after application of LPS.
On IHC, TLR-4 expression was localized in RPE, photoreceptors, Müller cells, ganglion cell layer and nerve fiber layer in normal equine retina. TLR-4 Western blot analysis showed no differences between unstimulated and stimulated RPE second passage cells regardless of the LPS concentration. In contrast, there was a strong signal for TLR-4 in unstimulated primary RPE cells. Furthermore, there was no upregulation of IL-1β, IL-6, or TNF in supernatants of LPS stimulated second passage RPE cultures.
TLR-4 is present in normal equine retina. However because of the weak expression of TLR-4 and the lack of inflammatory response in second passage cells, primary cell cultures of RPE, which had a strong TLR-4 signal, may be required to study the effects of LPS on TLR-4 in RPE. Alternatively, these results may suggest that TLR-4 does not play a strong role in the pathogenesis of immune-mediated retinal disease in the horse.
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