Immune Modulation by Silencing IL-12 Production in Dendritic Cells Using Small Interfering RNA

Abstract

RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human and mouse. Specific gene silencing is mediated by short strands of duplex RNA of ∼21 nt in length (termed small interfering RNA or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 production were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.

Small interference RNA modulation of IL‐10 in human monocyte‐derived dendritic cells enhances the Th1 response

“…A lingering concern for siRNA transfection is that transfection reagents may alter DC viability and their induction of a type 1 IFN response. We and others also demonstrated that siRNA transfection did not affect cell viability or the ability of DC to induce a type 1 IFN response [17,18]. Furthermore, unlike antisense oligodeoxyribonucleotides (oligo-dNTP), siRNA appears to require doses 10-to 100-fold lower than such nucleotides to achieve a similar effect [18].…”

“…It is well known that immature DC are highly phagocytic and can internalize a variety of molecules including nucleic acids. However, we and others have found that immature DC are unable to internalize naked siRNA [17]. Long double-stranded RNA ( G 38 bp) could induce a nonspecific type 1 IFN response in mammalian cells, leading to arrest in transcription and cell death.…”

“…Inhibition of IL-10 expression could increase CD40 expression, increasing the allogeneic T cell response and antigen presentation to induce a more potent Th1 immune response. Although many studies have shown a limited ability of DC to be transfected with DNA, Hill et al showed that RNA transfection of bone marrow-derived murine DC by using GenePorter reagents (Gene Therapy Systems, San Diego, CA) was extremely efficient [17]. siRNA has been successfully transfected into human monocyte-derived DC by electroporation [18].…”

“…Transfection of 21-nt chemically synthesized siRNA has been shown to knock down the expression of the translated protein in cultured mammalian cells [10,11,16]. siRNA technology has been used as a powerful tool to modulate the immune response in DC [17,18]. In this study, we assessed the induction of RNAi using siRNA specific for IL-10 in human monocytederived DC.…”

Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG

“…Moreover, this technique is not difficult to perform and its effect can be confirmed within days using simple detection systems, such as RT-PCR, ELISA, and Western blot (18). To date, RNAi technology has generated much insight into the development, activation and function of cells comprising the innate and adaptive immune systems (19).…”