Purpose:
Toll-like receptor 3 (TLR3) is a highly conserved innate immune sensor for pathogenic double-stranded RNA (dsRNA). Upon TLR3 activation, RPE cells undergo apoptosis via IFN induction resulting in a geographic atrophy (GA) like phenotype in mice. We recently discovered increased expression of endogenous Alu-derived dsRNAs in RPE cells from human eyes with GA and demonstrated direct cytotoxic effects on RPE in vivo and in vitro. In this study, we investigated whether the Alu-derived dsRNAs function via TLR3 to induce RPE apoptosis.
Methods:
Wild-type C57BL/6 and TLR3 deficient mice received subretinal injections of purified Alu-derived dsRNAs, correlate plasmid constructs, appropriate controls, or a TLR3 agonist (poly I:C) followed by fundoscopic analysis at 7 days. Primary isolates of human RPE (hRPE) were treated with purified Alu-derived dsRNAs or poly I:C with appropriate controls followed by RNA (8 h) and protein harvest (24 h) (n=3/group). Relative quantitative PCR was performed for 84 genes related to pro-apoptotic and TLR induced pathways. For protein analyses, multiplex bead arrays were used to measure IL-1β, IL-6, IFN-γ, TNF-α, CXCL10, IL-8 (or mouse homolog KC), and MCP-1 with normalization to total protein. Data were analyzed using hierarchical clustering and Mann-Whitney U tests (p<0.05).
Results:
Treatment with Alu-derived dsRNAs induced RPE degeneration in both wild-type and TLR3 deficient mice whereas poly I:C only exhibited effects in wild-type mice. In hRPE cultures, TLR3 activation resulted in a reproducible expression signature with upregulation of caspase/inflammasome pathways and significant increases in Fas, Fas ligand, FLIP, RIPK2, and caspases 1, 5, 7, 8, and 10. Multiplex protein analyses demonstrated acute immune activation with TLR3 agonists with increased concentrations of IL-6, IFN-γ, IP-10, IL-8 and MCP-1. Conversely, Alu-derived dsRNAs did not significantly upregulate these cytokines nor did they produce an expression signature consistent with TLR3 activation.
Conclusions:
These data suggest that Alu-derived dsRNAs may not function via TLR3 to induce RPE cytotoxicity. The mammalian immune system harbors redundant mechanisms to enable the efficient detection of pathogenic dsRNA, thus alternate receptors for Alu-derived dsRNAs are currently being explored.
Keywords: age-related macular degeneration • immunomodulation/immunoregulation • apoptosis/cell death