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W. G. Cho, M. E. Kleinman, R. J. Albuquergue, J. Z. Baffi, H. Kaneko, K. Saito, M. G. Rich, J. Ambati; Activation of TLR3 by 21nt-siRNA and Intracellular Trafficking of the Human Toll-like Receptor 3 Upon 21nt-siRNA Stimulation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1189.
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Toll-like receptor 3 (TLR3) is a double-stranded RNA (dsRNA) sensor that mediates an anti-viral innate immune response. Recently, 21nt-siRNA has been reported as a TLR3 agonist (Kleinman et al. Nature 2008), but details of how 21nt-siRNA binds or activates TLR3 remains to be elucidated. How 21nt-siRNA differentially engages extracellular and intracellular TLR3, what mechanisms drive internalization of 21nt-siRNA conjugated to cholesterol (a cell-permeating moiety), and how TLR3 traffics in the cell after siRNA exposure are equally unclear. We sought to mechanistically dissect these questions.
Primary human RPE cells were treated with naked or cholesterol-conjugated 21nt-siRNA.TLR3 activation was monitored by western blotting using phospho-specific antibody. Cellular uptake of fluorescein-conjugated siRNA (Fl-siRNA) with or without cholesterol conjugation was visualized by time lapse confocal live imaging. Intracellular trafficking and subcellular localization of Fl-21nt-siRNA and GFP-tagged TLR3 were visualized by immunofluorescent confocal microscopy.
Fl-21nt-siRNA was internalization within 30 minutes if it was conjugated to cholesterol but not if it was unmodified. Yet naked 21-nt siRNA induced cell surface TLR3 phosphorylation. Fl-siRNA-chol internalization requires scavenger receptors and involves vesicular transport. GFP-tagged TLR3 trafficked to different subcellular locations depending on whether it ligated extracellular or internalized 21-nt siRNA.
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