title: Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization.
abstract: The potential induction of inflammatory cytokines and interferon responses by small-interfering RNAs (siRNAs) represents a major obstacle for their use as inhibitors of gene expression. Therapeutic applications of siRNAs will require a better understanding of the mechanisms that trigger such unwanted effects, especially in freshly isolated human cells. Surprisingly, the induction of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) in adherent peripheral blood mononuclear cells (PBMC) was not restricted to double-stranded siRNAs, because induction was also obtained with single-stranded siRNAs (sense or antisense strands). The immunostimulatory effects were sequence-dependent, since only certain sequences are prone to induce inflammatory responses while others are not. The induction of TNF-alpha, IL-6 and interferon alpha (IFN-alpha) was chloroquine-sensitive and dependent more likely on endosomal Toll-like receptor signaling in particular TLR8. Indeed, no significant immunostimulatory effects were detected when either double or single-stranded siRNAs were delivered directly to cytoplasm via electroporation. Both RNA types activated a NF-kappaB promoter-driven luciferase gene in transiently transfected human adherent PBMC. Moreover, culture of immature dendritic cells with either double or single-stranded siRNAs stimulated interleukin-12 production and induced the expression of CD83, an activation marker. Interestingly, several double-stranded siRNAs did not induce TNF-alpha, IL-6 and IFN-alpha production, however, their single-stranded sense or antisense did. Taken together, the present data indicate for the first time that the induction of inflammatory cytokines and IFN-alpha responses by either double-stranded or single-stranded siRNAs in adherent PBMC is sequence-dependent and requires endosomal intracellular signaling. The finding that endosomal localization of self-RNAs (sense strands) can trigger Toll-like receptor signaling in adherent human PBMC is intriguing because it indicates that endosomal self-RNAs can display a molecular pattern capable for activating innate immunity.
authors.affiliation: The Norwegian Radium Hospital, Department of Immunology, Molecular Medicine Group, Montebello, N-0310 Oslo, Norway. mosioud@ulrik.uio.no
authors.forename: Mouldy
authors.initials: M
authors.lastname: Sioud
mesh_terms: D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology; D016328:NF-kappa B / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012330:RNA, Double-Stranded / Q000032:analysis / Q000235:genetics / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology; D051200:Toll-Like Receptor 8; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D016207:Cytokines; D016898:Interferon-alpha; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; C495349:TLR8 protein, human; D051200:Toll-Like Receptor 8; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha