title: Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3.
abstract: Small interfering (si) and short hairpin (sh) RNAs induce robust degradation of homologous mRNAs, making them a potent tool to achieve gene silencing in mammalian cells. Silencing by siRNAs is used widely because it is considered highly specific for the targeted gene, although a recent report suggests that siRNA also induce signaling through the type I IFN system. When human embryonic kidney 293 (HEK293) or keratinocyte (HaCaT) cell lines or human primary dendritic cells or macrophages were transfected with siRNA or shRNAs, suppression of nontargeted mRNA expression was detected. Additionally, siRNA and shRNA, independent of their sequences, initiated immune activation, including IFN-alpha and TNF-alpha production and increased HLA-DR expression, in transfected macrophages and dendritic cells. The siRNAs induced low, but significant, levels of IFN-beta in HEK293 and HaCaT cells. Secretion of these cytokines increased tremendously when HEK293 cells overexpressed Toll-like receptor 3 (TLR3), and the increased secretion of IFN-beta was inhibited by coexpression of an inhibitor of TIR domain-containing adapter-inducing IFN-beta, the TLR3 adaptor protein linked to IFN regulatory factor 3 signaling. Although siRNA and shRNA knockdown of genes represents a new and powerful tool, it is not without nonspecific effects, which we demonstrate are mediated in part by signaling through TLR3.
authors.affiliation: Division of Neurosurgery and Infectious Diseases, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Katalin
authors.initials: K
authors.lastname: Karikó
authors.forename: Prakash
authors.initials: P
authors.lastname: Bhuyan
authors.forename: John
authors.initials: J
authors.lastname: Capodici
authors.forename: Drew
authors.initials: D
authors.lastname: Weissman
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000502:physiology; D002478:Cells, Cultured; D020868:Gene Silencing; D006801:Humans; D007372:Interferons / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000502:physiology*; D015398:Signal Transduction; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D019892:eIF-2 Kinase / Q000502:physiology
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; C470409:TICAM1 protein, human; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D007372:Interferons; D019892:eIF-2 Kinase