Abstract
Chronic inflammation is an important component that contributes to many age-related neurodegenerative diseases, including macular degeneration. Here, we report a role for toll-like receptor 3 (TLR3) in cone-rod dystrophy (CORD) of mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), proteins critical for all-trans-retinal clearance in the retina. Increased expression of toll-like receptor-signaling elements and inflammatory changes were observed in Rdh8(-/-)Abca4(-/-) eyes by RNA expression analysis. Unlike 3-month-old Rdh8(-/-)Abca4(-/-) mice that developed CORD, 6-month-old Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice did not evidence an abnormal retinal phenotype. Light-induced retinal degeneration in Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice was milder than that in Rdh8(-/-)Abca4(-/-) mice, and a 2-fold increased TLR3 expression was detected in light-illuminated retinas of Rdh8(-/-)Abca4(-/-) mice compared with nonilluminated retinas. Poly(I-C), a TLR3 ligand, caused caspase-8-independent cellular apoptosis. Whereas poly(I-C) induced retinal cell death in Rdh8(-/-)Abca4(-/-) and WT mice both in vivo and ex vivo, this was not seen in mice lacking Tlr3. Far fewer invasive macrophage/microglial cells in the subretinal space and weaker activation of Muller glial cells were exhibited by Tlr3(-/-)Rdh8(-/-) Abca4(-/-) mice compared with Rdh8(-/-)Abca4(-/-) mice at 3 and 6 months of age, indicating that loss of TLR3 inhibits local inflammation in the retina. Both poly(I-C) and endogenous products emanating from dying/dead retinal cells induced NF-κB and IRF3 activation. These findings demonstrate that endogenous products from degenerating retina stimulate TLR3 that causes cellular apoptosis and retinal inflammation and that loss of TLR3 protects mice from CORD.
Highlights
Animal models featuring retinal damage to rod/cone photoreceptors have commonly been used to identify and investigate pathogenic mechanisms shared with human blinding diseases
Rdh8Ϫ/ϪAbca4Ϫ/Ϫ mice initially manifest lipofuscin accumulation, drusen, and photoreceptor/retinal pigment epithelial (RPE) atrophy followed by choroidal neovascularization (CNV) that are hallmark features of human macular degeneration exemplified by Stargardt disease and age-related macular degeneration (AMD)
toll-like receptor (TLR) Signaling Is Enhanced in Retinal Degeneration Exhibited by Rdh8Ϫ/ϪAbca4Ϫ/Ϫ Mice—TLRs constitute distinct families of pattern-recognition receptors that recognize components derived from viruses or endogenous cellular degradation and trigger innate immune responses [18]
Summary
Animals—Rdh8Ϫ/ϪAbca4Ϫ/Ϫ double knock-out mice were generated as described previously, and all knock-out mice were genotyped by established methods [4, 23]. After co-incubation, RPE cells were stained with Hoechst 33342 (AnaSpec Inc., Fremont, CA) for 30 min and washed twice with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4). The resulting cells (5 ϫ 104) were cultured on sterile coverslips in 24-well plates and stimulated for 0, 15, 30, 60, and 120 min with either LPS (100 ng/ml), poly(I-C) (10 g/ml), or supernatants containing degraded Y76 cells obtained after a 16-h co-incubation with 30 M all-trans-retinal. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 1 min at room temperature and incubated with rabbit anti-mouse p65 (1:100; Ebioscience) in PBS containing 10% goat serum for 1 h at room temperature. Statistical Analyses—Data representing the means Ϯ S.D. or the means Ϯ S.E. for the results of at least three independent experiments were compared by the one-way analysis of variance test
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