A Genetic Polymorphism in the Human Toll-Like Receptor 3 Gene Protects Against dsRNA Induced Cell Death

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: age-related macular degeneration • apoptosis/cell death • mutations