RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak-Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-beta in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use.

Leishmania major is an obligate intracellular eukaryotic pathogen of mononuclear phagocytes. Invasive promastigotes gain entry into target cells by receptor-mediated phagocytosis, transform into non-motile amastigotes and establish in the phagolysosome. Glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG) is a virulence factor and a major parasite molecule involved in this process. We observed that mice lacking the Toll-like receptor (TLR) pathway adaptor protein MyD88 were more susceptible to infection with L. major than wild-type C57BL/6 mice, demonstrating a central role for this innate immune recognition pathway in control of infection, and suggesting that L. major possesses a ligand for TLR. We sought to identify parasite molecules capable of activating the protective Toll pathway, and found that purified Leishmania LPG, but not other surface glycolipids, activate innate immune signaling pathways via TLR2. Activation of cytokine synthesis by LPG required the presence of the lipid anchor and a functional MyD88 adaptor protein. LPG also induced the expression of negative regulatory pathways mediated by members of the suppressors of cytokine signaling family SOCS-1 and SOCS-3. Thus, the Toll pathway is required for resistance to L. major and LPG is a defined TLR agonist from this important human pathogen.

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Background: Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-κB is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-κB during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-κB activation in all the cells; invasion of cells by the bacteria is not required to activate NF-κB.