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W. Cho, M. E. Kleinman, D. Gibbs, D. R. Hinton, K. Zhang, J. Ambati; A Genetic Polymorphism in the Human Toll-Like Receptor 3 Gene Protects Against dsRNA Induced Cell Death. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5156.
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Toll-like receptor 3 (TLR3) is a double-stranded RNA (dsRNA) sensor that mediates the mammalian innate immune response against numerous viruses. Recently, a single nucleotide polymorphism (SNP) within the coding region of TLR3, termed L412F, was associated with a decreased risk of age-related macular degeneration, a disease hallmarked by retinal pigment epithelium (RPE) cell degeneration. There is preliminary evidence that this SNP suppresses dsRNA induced immune system activation and downstream host-cell death pathways; therefore, we sought to determine whether the L412F SNP affects RPE cell survival after exposure to dsRNA in vitro and in vivo.
Primary RPE cells were genotyped for the TLR3 L412F SNP, and homozygous normal and heterozygous isolates were treated with various concentrations of the synthetic dsRNA, poly I:C, after sensitization with interferon α/β (1000U/mL). Annexin-V staining was analyzed by flow cytometry (FACS) at 24 hrs, and cell viability was assayed by BrdU ELISA at 48 hrs after poly I:C treatment. To evaluate the importance of TLR3 signaling in RPE cell death in vivo, we intravitreously injected poly I:C (2µg) or control poly dI:dC (2µg) into wild-type and TLR3 deficient mice. RPE cell survival and caspase-3 activation were quantified using FACS.
Human RPE cell isolates derived from carriers of the L412F SNP demonstrated increased cell viability and decreased apoptosis compared to non-carrier controls after exposure to the synthetic dsRNA, poly I:C. In vivo, we found that intravitreous administration of poly I:C in wild-type mice significantly decreased RPE cell numbers over a 72 hr time-course and increased RPE cell caspase-3 activation compared to poly dI:dC treated wild-type or TLR3 deficient mice.
These data suggest that TLR3 signaling is critical in dsRNA mediated RPE cell death, and that patients with the L412F SNP may exhibit a decreased risk of AMD due to suppression of RPE cell death after exposure to viral dsRNA.
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