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

Department of Medicine, Division of Rheumatology, University of Western Ontario, London, Ontario, Canada.
The Journal of Immunology (Impact Factor: 4.92). 08/2003; 171(2):691-6. DOI: 10.4049/jimmunol.171.2.691
Source: PubMed


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 approximately 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.

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Available from: Thomas E Ichim, Oct 01, 2014
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    • "Gene silencing by using small interfering RNA (siRNA) is capable of specifically blocking gene expression in mammalian cells without triggering the nonspecific panic response [16,17]. The strategies of using siRNA have been successful in inducing therapeutic benefits in animal models of various diseases and are currently in clinical trials [18-23]. "
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    ABSTRACT: Background While substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival. Method Vectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed. Result Allogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10–16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4 + CD25 + FoxP3+ regulatory T cell differentiation in vitro. Conclusion This study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.
    Full-text · Article · May 2014 · Journal of Translational Medicine
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    • "BMDMs and BMDCs were obtained from the WT mice as described by Hill et al. (2003). In brief, BM cells were extracted from the femurs of adult mice and washed with the same type of culture media used for experiment 1. "
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    ABSTRACT: The aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Fluorescein isothiocyanate (FITC)-labeled EC-12 was cocultured with peritoneal macrophage and the amount of EC-12 phagocytosed by peritoneal macrophages was measured using a microplate fluorometer. Peritoneal macrophages from toll-like receptor (TLR)2-, TLR7-, and MyD88-deficient knockout (KO) mice exhibited similar levels of EC-12 phagocytosis to those from wild-type mice. Similarly, dectin-1 neutralization of peritoneal macrophages had no effect on EC-12 phagocytosis. However, blockade of the mannose receptor (MR) significantly decreased the amount of EC-12 phagocytosed by peritoneal macrophages; the same effect was observed in bone marrow-derived macrophages and dendritic cells. Our findings suggest that MR plays a major role in EC-12 phagocytosis by the APCs. This aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Our findings suggest that mannose receptor (MR) plays a major role in EC-12 phagocytosis by the APCs.
    Full-text · Article · Aug 2013 · MicrobiologyOpen
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    • "Silencing of IL-12p35 on bone marrow derived DC was previously performed by our group using presynthesized siRNA [35]. Although this approach is widely used, one disadvantage is the relatively low efficacy due to consumption of the exogenously administered siRNA. "
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    ABSTRACT: We have previously demonstrated that immune modulation can be accomplished by administration of gene silenced dendritic cells (DC) using siRNA. In this study, we demonstrate the therapeutic utilization of shRNA-modified DC as an antigen-specific tolerogenic vaccine strategy for autoimmune arthritis. A shRNA that specifically targets IL-12 p35 was designed and cloned into a plasmid vectors (IL-12 shRNA). Bone marrow-derived DC from DBA/1 mice were transfected with the IL-12 shRNA construct in vitro. Mice with collagen II (CII)-induced arthritis (CIA) were treated with the modified DCs expressing the shRNA. Recall response and disease progression were assessed. After gene silencing of IL-12 in DC, DC were shown to selectively inhibit T cell proliferation on recall responses and in an MLR. In murine CIA, we demonstrated that administration of IL-12 shRNA-expressing DC that were pulsed with CII inhibited progression of arthritis. The therapeutic effects were evidenced by decreased clinical scores, inhibition of inflammatory cell infiltration in the joint, and suppression of T cell and B cell responses to CII. We demonstrate a novel tolerance-inducing protocol for the treatment of autoimmune inflammatory joint disease in which the target antigen is known, utilizing DNA-directed RNA interference.
    Full-text · Article · Jan 2012 · Journal of Translational Medicine
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