Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization.

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.