Toll-like receptor 3 and geographic atrophy in age-related macular degeneration

Abstract

Background: Age-related macular degeneration is the most common cause of irreversible visual impairment in the developed world. Advanced age-related macular degeneration consists of geographic atrophy and choroidal neovascularization. The specific genetic variants that predispose patients to geographic atrophy are largely unknown.

Methods: We tested for an association between the functional toll-like receptor 3 gene (TLR3) variant rs3775291 (involving the substitution of phenylalanine for leucine at amino acid 412) and age-related macular degeneration in Americans of European descent. We also tested for the effect of TLR3 Leu and Phe variants on the viability of human retinal pigment epithelial cells in vitro and on apoptosis of retinal pigment epithelial cells from wild-type mice and Tlr3-knockout (Tlr3(-/-)) mice.

Results: The Phe variant (encoded by the T allele at rs3775291) was associated with protection against geographic atrophy (P=0.005). This association was replicated in two independent case-control series of geographic atrophy (P=5.43x10(-4) and P=0.002). No association was found between TLR3 variants and choroidal neovascularization. A prototypic TLR3 ligand induced apoptosis in a greater fraction of human retinal pigment epithelial cells with the Leu-Leu genotype than those with the Leu-Phe genotype and in a greater fraction of wild-type mice than Tlr3(-/-) mice.

Conclusions: The TLR3 412Phe variant confers protection against geographic atrophy, probably by suppressing the death of retinal pigment epithelial cells. Since double-stranded RNA (dsRNA) can activate TLR3-mediated apoptosis, our results suggest a role of viral dsRNA in the development of geographic atrophy and point to the potential toxic effects of short-interfering-RNA therapies in the eye.

Figure 1

Negative log p-values from TLR3 rs3775291 for association with GA. A. Negative log p-values (Y-axis) from association analyses for eight SNPs in the TLR3 region on 4q35 (also see table S3). Blue squares represent–log p values of all SNPs around rs3775291 of the Utah case-control series. The red circle represents rs3775291 for the first replication case-control series, while the green triangle represents rs3775291 for the AREDS replication case-control series. The black diamonds represent association for case and control in the three case-control series combined. B. Genomic structure and locations of genes between 187222847 base pairs and 187243847 base pairs (NCBI build 36). C. Pairwise D'Haploview plots for SNPs around TLR3 using combined GA case and normal control data.

Figure 2

The T allele of rs3775291 (L412F) confers protection from poly (I:C)-induced cytotoxicity. A. The TLR3 ligand poly (I:C) dose-dependently (0.5-5 μg/ml) reduced the survival of primary human retinal pigmented epithelial cells (hRPECs) expressing the homozygous major allele, leading to a protein with an L at amino acid 412 of TLR3 (denoted as LL to indicate the product from each allele) compared to vehicle (phosphate buffered saline) or to poly (dI:dC), which does not activate TLR3. * P < 0.01; n=4. B. poly (I:C) (5 μg/ml) reduced viability of hRPECs expressing LL at amino acid 412 of TLR3 to a significantly greater degree than of hRPECs expressing the FF variant (15±1% vs 58±10%; * P = 0.01; n=4). C. The fraction of poly (I:C) (5 μg/ml) stimulated hRPECs undergoing apoptosis (Annexin+ PI−) was significantly greater poly (I:C) (5 μg/ml) in 412LL cells than in 412 LF cells (22±2% vs 11±2%; * P = 0.03; n=4). D. Intravitreous administration of poly (I:C) (2 μg) induced significantly less death of RPE cells (viability defined as fraction of CD147+CD31–cells in the RPE/choroid layers) in Tlr3−/− mice than in wild-type mice (48.6±0.4% vs 78.1±2.0%; * P = 0.03; n=4). E. Intravitreous administration of poly (I:C) (2 μg) induced activated caspase-3 expression in a greater fraction of RPE cells in wild-type mice than in Tlr3−/− mice (5.2±0.6% vs 3.3±0.1%; * P = 0.03; n=4). There was no significant difference in the fraction of activated caspase-3 expressing RPE cells between mice of different genotypes under control (baseline) conditions (*P = 0.76; n=4-6). F. Intravitreous administration of poly (I:C) (2 μg) induced TUNEL expression in a greater fraction of RPE and outer retinal cells in wild-type mice than in Tlr3−/− mice (35.2±1.4% vs 8.6±0.5%; * P = 0.05; n=6). Values are shown as mean ±S.E.M. All P values were calculated by Mann Whitney U test.