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M. E. Kleinman, W. Cho, B. Fowler, H. Kaneko, S. Dridi, J. Z. Baffi, J. Ambati; Molecular Mapping of Toll-Like Receptor Mediated Retinal Pigment Epithelial Cell Responses With Functional Genomics. Invest. Ophthalmol. Vis. Sci. 2010;51(13):491.
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Toll-like receptors (TLRs) are ubiquitously expressed components of the innate immune system that mediate host cell responses to pathogenic invasion. TLR3 is a membrane receptor found both on the cell surface and in endosomes that recognizes viral double-stranded RNA (dsRNA) and small-interfering RNA (siRNA). We recently showed that TLR3 activation induces death of human choroidal endothelial and retinal pigmented epithelial (RPE) cells (Kleinman et al. Nature 2008; Yang et al. NEJM 2008). In this study, we utilized a functional genomics approach to map TLR3 induced immune and pro-apoptotic pathways in RPE.
Primary isolates of human RPE (hRPE) were cultured and genotyped for TLR3. Protein analyses confirmed functional membrane surface receptor. Cultures were treated with TLR3 agonists with appropriate controls followed by RNA harvest at 8 hours. Wild-type C57BL/6 mice received intravitreous injections of the same TLR3 agonists and controls followed by RNA harvest from RPE/choroid at 16 hours. Relative quantitative PCR was performed for 84 genes related to pro-apoptotic and TLR induced pathways (n=3 for each group). Data were analyzed using hierarchical clustering and principle component analyses (PCA) to determine significant changes (p<0.05) in gene regulation within specific molecular pathways.
In treated primary hRPE isolates, critical molecular components involved in tumor necrosis factor/stress, caspase, p53, and FADD pathways were upregulated. In vivo in mice, there were significant changes in interleukin and interferon profiles coupled with increases in TLR3 expression and various downstream signaling cascades. PCA and heat-map visualization demonstrated a strong pattern of gene expression related to apoptosis and TLR signaling.
The expression and activation of TLR3 on RPE is a potent immune signal that shifts gene expression to pro-apoptotic and TLR mediated molecular pathways. Through further investigations, critical signaling events will be identified in order to target and prevent TLR3 mediated RPE cell death in the presence of specific agonists.
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