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S. Shiose, K. Okano, T. Maeda, J. Tang, K. Palczewski, A. Maeda; Role of Toll-Like Receptor 3 in Retinal Pigment Epithelial Cell Death in Rdh8-/-Abca4-/- Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6179.
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Toll-like receptor 3 (TLR3) encodes a viral sensor and plays an important role in the immune response cascade. Moreover, a TLR3 polymorphism reportedly is associated with dry type age-related macular degeneration (AMD). We have observed that mice lacking both ATP-binding cassette transporter 4 (Abca4) and retinol dehydrogenase 8 (Rdh8) developed AMD-like retinal degeneration. Complement deposition at Bruch’s membrane in Rdh8-/-Abca4-/- mice suggests a connection to innate immunity as well as to human AMD. The aim of this study is to examine possible TLR3 involvement in the retinal degeneration of Rdh8-/-Abca4-/- mice.
Expression analysis of genes involved in TLR-signaling was performed with RNA extracted from Rdh8-/-Abca4-/- mouse eyes. TLR3 expression in human retinal pigment epithelium (RPE) cells (ARPE19), mouse RPE cells and whole mouse eyes was determined by immunoblot and RT-PCR. RPE cell death, after stimulation with TLR3 ligand (poly I:C), was examined in primary mouse RPE cells by counting the number of cells with nuclei condensed stained by Hoechst33342. The severity of retinal degeneration was evaluated by optical coherence tomography (OCT) and histology in Rdh8-/-Abca4-/-, Tlr3-/-Rdh8-/-Abca4-/- and Tlr3-/- mice.
RNA analysis of retina from light-exposed (degenerated) and dark-adapted (non degenerated) Rdh8-/-Abca4-/-mice, and retina from 2-3-month-old (mimic dry-AMD) and 6-8-month-old (mimic wet-AMD) Rdh8-/-Abca4-/- mice uncovered differential expression levels for many genes involved in TLR-signaling, including TLR3 itself. RT-PCR demonstrated that retina and whole eyes of Rdh8-/-Abca4-/- mice expressed Tlr3, Tlr4 and Tlr8. TLR3 expression was also determined by RT-PCR and immunoblot in ARPE19 and primary cultured RPE cells isolated from Rdh8-/-Abca4-/- mice. Culturing ARPE19 and RPE cells from Rdh8-/-Abca4-/- mice with poly I:C for 24 h caused cell death in a dose dependent manner, whereas poly I:C-induced cell death was not observed in RPE cells from Tlr3-/- and Tlr3-/-Rdh8-/-Abca4-/- mice. Subretinal poly I:C injection caused retinal degeneration in 4-week-old Rdh8-/-Abca4-/- mice but not in 4-week-old Tlr3-/- mice. No retinal degeneration was observed in Tlr3-/-Rdh8-/-Abca4-/- or Tlr3-/- mice at 3 weeks, 6 weeks or 3 months of age, whereas 3-month-old Rdh8-/-Abca4-/- mice showed significant retinal degeneration.
TLR3 plays a role in retinal degeneration of Rdh8-/-Abca4-/- mice.
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