Abstract
Purpose: :
A leading cause of visual impairment is age-related macular degeneration (AMD). Innate immunity is proposed to play a role in the pathogenesis of AMD, although its precise contribution is unknown. The overall goal of this project is to examine the activity of toll-like receptor 4 (TLR4), a mediator of innate immunity, in a cellular model of AMD injury. TLR4 activation has damaging effects elsewhere in the CNS, leading to our hypothesis that aberrant TLR4 activation in the retina reduces viability of retina cells. Additionally, we propose TLR4 may decrease survival through suppression of survival pathways such as Wnt signaling.
Methods: :
Expression of TLR4 in both wild-type and rd1 retinal degenerating mice was identified using IHC. Viability assays were conducted on photoreceptor-Muller glia cocultures prepared from wild-type mice, and a retinal pigmented epithelium (RPE) cell line using Cell Titre Blue and WST-1 assays. TLR4 signaling was activated by 0.05-50 ug/ml lipopolysaccharide (LPS). Cell cultures were exposed to oxidative stress using 0.4mM H2O2 and 0.8mM paraquat to simulate AMD-like injury.
Results: :
IHC on retina sections and primary cultures showed that TLR4 is expressed in photoreceptors and Muller glia in wild-type and rd1 mice, with strong localization in photoreceptor outer segments. Activation of TLR4 combined with oxidative stress resulted in a significant decrease in viability by 50% (p<0.05, n=4) compared to oxidative stress alone in photoreceptor-Muller glia cocultures. In contrast, Wnt3a protected against H2O2 -mediated toxicity (p<0.05, n=4). Similarly, we demonstrated decreased RPE viability by 20% (p<0.05, n=3) when exposed to LPS and 0.8mM paraquat. Furthermore, activating TLR4 suppressed Wnt signaling in Muller glia by 42% (p<0.05, n=5).
Conclusions: :
We have demonstrated that activation of the innate immunity receptor TLR4 in the presence of AMD-like injury reduces the viability of retinal cell types. Furthermore, we showed that regulation of the neuroprotective Wnt pathway by TLR4 is a potential mechanism. Therefore, TLR4 signaling could be further investigated as a target for developing novel therapeutic strategies for AMD.
Keywords: age-related macular degeneration • cell survival • oxidation/oxidative or free radical damage