Activation of cell membrane-localized Toll-like receptor 3 by siRNA

Small interfering RNA molecules (siRNA) are short dsRNAs that are used for different therapeutic applications. On the other hand, dsRNAs can bind to and activate cell RNA sensors and consequently trigger inflammatory response. Here we show that siRNA activates primary human endothelial cells and human lymphatic endothelial cells and that this response is inhibited by antibodies against TLR3. In contrast, the activation of human lymphatic endothelial cells by poly(I:C) was inhibited by bafilomycin but not by anti-TLR3 antibodies. Bafilomycin also inhibited poly(I:C) but not siRNA cell stimulation in TLR3-transfected HEK293. The response to siRNA required the expression of UNC93B1, which directs TLR3 to the surface of HEK293 cells. We propose that the engaged signaling pathway of TLR3 depends on the receptor localization and on the length of the dsRNA, where the activation of cell membrane TLR3 by short dsRNA leads to a predominantly proinflammatory response, whereas TLR3 activation in endosomal compartments by long dsRNA is characterized by the production of type I IFN. A molecular model suggests that the siRNA can bind to the binding sites of the TLR3 ectodomain and trigger receptor dimerization. These results contribute to understanding of the mechanism of side effects seen in the therapeutic application of naked, unmodified siRNA as a result of the activation of TLR3 localized at the plasma membrane.