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Amit K. Patel, Abigail S. Hackam; Analysis of Toll-like Receptor 3 and Cellular Signaling Pathways in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1660.
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A leading cause of visual impairment is age-related macular degeneration (AMD). The overall goal of this project is to examine the activity of toll-like receptor 3 (TLR3), a mediator of innate immunity, in a cellular model of AMD injury. Although genetic polymorphisms in TLR3 are associated with AMD, the precise role of TRL3 in AMD is unknown. Several reports show that TLR3 activation leads to retina pigment epithelium (RPE) cell death, but other studies indicate that TLR3 has cytoprotective activity. Here, we tested the hypothesis that aberrant TLR3 activation regulates Wnt and STAT3 cellular survival pathways, leading to altered RPE cell viability.
Viability was measured in primary RPE cultures prepared from wild-type mice and the ARPE19 cell line, using Cell Titer Blue assays. TLR3 signaling was activated by 2-100 µg/ml Poly I:C and TLR3 expression was downregulated using siRNA. Cell cultures were exposed to oxidative stress using 0.4mM H2O2 and 0.8mM paraquat to simulate AMD-like injury. STAT3 and p65 expression was identified using IHC. Wnt signaling was induced using the Wnt3a ligand and quantified with luciferase reporter assays.
Activation of TLR3 resulted in a modest dose-dependent decrease of RPE viability by up to 20% (n=5, p<0.05). In contrast, TLR3 activation combined with oxidative stress significantly increased RPE cell viability by 50% (n=5, p<0.05) compared with oxidative stress conditions alone. This protective effect was reversed using TLR3 siRNA (n=5). STAT3 signaling was increased by TLR3 activation by about 60% (n=2). Furthermore, although Wnt signaling significantly increased ARPE19 cell viability when oxidative stress was induced by 0.4mM H2O2 (n=4, p<0.05) and 0.4mM paraquat (n=9, p<0.05), TLR3 activation suppressed Wnt signaling by about 50% (n=3, p<0.05).
We have demonstrated that activation of the innate immunity receptor TLR3 in the presence of AMD-like injury increased the viability of RPE cells. Furthermore, TLR3 activation regulates the Wnt and STAT3 cell survival pathways. TLR3 signaling and related downstream pathways could be further investigated as targets for developing novel therapeutic strategies for AMD.
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