Motohiko Suzuki

Immune Modulation and Tolerance Induction by RelB-Silenced Dendritic Cells through RNA Interference

Dendritic cells (DC), the most potent APCs, can initiate the immune response or help induce immune tolerance, depending upon their level of maturation. DC maturation is associated with activation of the NF-κB pathway, and the primary NF-κB protein involved in DC maturation is RelB, which coordinates RelA/p50-mediated DC differentiation. In this study, we show that silencing RelB using small interfering RNA results in arrest of DC maturation with reduced expression of the MHC class II, CD80, and CD86. Functionally, RelB-silenced DC inhibited MLR, and inhibitory effects on alloreactive immune responses were in an Ag-specific fashion. RelB-silenced DC also displayed strong in vivo immune regulation. An inhibited Ag-specific response was seen after immunization with keyhole limpet hemocyanin-pulsed and RelB-silenced DC, due to the expansion of T regulatory cells. Administration of donor-derived RelB-silenced DC significantly prevented allograft rejection in murine heart transplantation. This study demonstrates for the first time that transplant tolerance can be induced by means of RNA interference using in vitro-generated tolerogenic DC.

Gene Silencing of Complement C5a Receptor Using siRNA for Preventing Ischemia/Reperfusion Injury

Ischemia/reperfusion (I/R) injury in organ transplantation significantly contributes to graft failure and is untreatable using current approaches. I/R injury is associated with activation of the complement system, leading to the release of anaphylatoxins, such as C5a, and the formation of the membrane attack complex. Here, we report a novel therapy for kidney I/R injury through silencing of the C5a receptor (C5aR) gene using siRNA. Mice were injected with 50 microg of C5aR siRNA 2 days before induction of ischemia. Renal ischemia was then induced through clamping of the renal vein and artery of the left kidney for 25 minutes. The therapeutic effects of siRNA on I/R were evaluated by assessment of renal function, histopathology, and inflammatory cytokines. siRNA targeting C5aR efficiently inhibited C5aR gene expression both in vitro and in vivo. Administering C5aR siRNA to mice preserved renal function from I/R injury, as evidenced by reduced levels of serum creatinine and blood urea nitrogen in the treated groups. Inhibition of C5aR also diminished in vivo production of the pro-inflammatory cytokine tumor necrosis factor-alpha and chemokines MIP-2 and KC, resulting in the reduction of neutrophils influx and cell necrosis in renal tissues. This study demonstrates that siRNA administration represents a novel approach to preventing renal I/R injury and may be used in a variety of clinical settings, including transplantation and acute tubular necrosis.

Protection of renal ischemia injury using combination gene silencing of complement 3 and caspase 3 genes.

Background. Ischemia/reperfusion (I/R) injury occurs in clinical kidney transplantation, which results in graft dysfunction and rejection. It has been documented that I/R injury is associated with complement activation and renal cell apoptosis. The purpose of this study was to develop a strategy to prevent I/R injury using small interfering RNA (siRNA) that target complement 3 (C3) and caspase 3 genes. Methods. siRNA-expression vectors were constructed to target C3 and caspase 3 genes. Gene silencing efficacy was assessed using real-time polymerase chain reaction. In vivo gene silencing was performed by hydrodynamic injection with C3 and caspase 3 siRNA. Renal I/R injury was induced through clamping the renal vein and artery for 25 min. I/R injury was evaluated using kidney histopathology, blood urea nitrogen (BUN), serum levels of creatinine, and survival. Results. Effective gene silencing was first confirmed in vitro. Notably upregulated expression of C3 and caspase 3 genes was observed from 2 to 48 hr after I/R injury, which were effectively and specifically inhibited by C3 and caspase 3 siRNA. In comparison with control mice, serum levels of creatinine and BUN were also significantly decreased in C3 and caspase 3 siRNA-treated mice. Furthermore, the therapeutic effect of siRNA was assessed in a severe, lethal I/R injury experiment, in which siRNA treatment significantly reduced mortality. Tissue histopathology showed an overall reduction in injury area in siRNA-treated mice. Conclusions. This is the first demonstration that renal I/R injury can be prevented through silencing the complement gene and apoptosis gene, highlighting the potential for siRNA-based clinical therapy.

A novel in vivo siRNA delivery system specifically targeting dendritic cells and silencing CD40 genes for immunomodulation

Translation of small interfering RNA (siRNA)-based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.

Preventing autoimmune arthritis using antigen-specific immature dendritic cells: a novel tolerogenic vaccine

Conventional treatments for autoimmune diseases have relied heavily on nonspecific immune suppressants, which possess a variety of adverse effects without inhibiting the autoimmune process in a specific manner. In the present study we demonstrate the effectiveness of antigen-specific, maturation-resistant, tolerogenic dendritic cells (DC) in suppressing collagen-induced arthritis, a murine model of rheumatoid arthritis. Treatment of DC progenitors with the NF-κB inhibiting agent LF 15-0195 (LF) resulted in a population of tolerogenic DC that are characterized by low expression of MHC class II, CD40, and CD86 molecules, as well as by poor allostimulatory capacity in a mixed leukocyte reaction. Administering LF-treated DC pulsed with keyhole limpet hemocyanin antigen to naïve mice resulted hyporesponsiveness specific for this antigen. Furthermore, administration of LF-treated DC to mice with collagen-induced arthritis resulted in an improved clinical score, in an inhibited antigen-specific T-cell response, and in reduced antibody response to the collagen. The efficacy of LF-treated DC in preventing arthritis was substantiated by histological examination, which revealed a significant decrease in inflammatory cell infiltration in the joints. In conclusion, we demonstrate that in vitro-generated antigen-specific immature DC may have important potential as a tolerogenic vaccine for the treatment of autoimmune arthritis.

Prevention of Renal Ischemic Injury by Silencing the Expression of Renal Caspase 3 and Caspase 8

Background. Apoptotic pathways mediated by caspases play a critical role in renal ischemia-reperfusion injury (IRI). Downregulation of the caspase cascade, using small interfering RNA (siRNA) to silence the expression of caspase 3 and caspase 8, may have substantial therapeutic potential for limiting renal injury. Methods. IRI was induced in mice by clamping of the renal vein and artery for 25 or 35 min at 37°C. Caspase 3 and caspase 8 (caspase 3/8) siRNA was administrated by hydrodynamic injection. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to analyze the gene silencing efficacy, and the therapeutic effects of siRNA were evaluated by renal function analysis, histological examination, and overall survival of mice suffering from IRI. Results. In this study, we have shown, using quantitative PCR, that IRI is associated with increased levels of renal caspase 3/8 mRNA. Mice treated with caspase 3/8 siRNA showed a significant down-regulation in kidney expression of caspase 3/8 at both, transcriptional and protein levels. Kidney function in IRI was protected by siRNA therapy, as levels of blood urea nitrogen and creatinine were significantly reduced in mice treated with siRNA. Histological examination demonstrated that tissue injury caused by IRI was significantly reduced as a result of caspase 3/8 siRNA treatment. Furthermore, survival data showed that more than 70% of mice in siRNA-treated groups survived until the end of the eight-day observation period. Conclusion. Herein, we have demonstrated the therapeutic potential of using siRNA to knock down the expression of caspases and prevent acute renal injury.

Treatment of Autoimmune Arthritis Using RNA Interference-Modulated Dendritic Cells

Dendritic cells (DCs) have a dual ability to either stimulate or suppress immunity, which is primarily associated with the expression of costimulatory molecules. Ag-loaded DCs have shown encouraging clinical results for treating cancer and infectious diseases; however, the use of these cells as a means of suppressing immune responses is only recently being explored. Here, we describe the induction of RNA interference through administering short interfering RNA (siRNA) as a means of specifically generating tolerogenic DCs. Knockdown of CD40, CD80, and CD86, prior to loading DCs with the arthritogenic Ag collagen II, led to a population of cells that could effectively suppress onset of collagen-induced arthritis. Maximum benefits were observed when all three genes were concurrently silenced. Disease suppression was associated with inhibition of collagen II-specific Ab production and suppression of T cell recall responses. Downregulation of IL-2, IFN-γ, TNF-α, and IL-17 and increased FoxP3+ cells with regulatory activity were observed in collagen-induced arthritis mice treated with siRNA-transfected DCs. Collectively, these data support the use of ex vivo gene manipulation in DCs using siRNA to generate tailor-made tolerogenic vaccines for treating autoimmunity.

Generation of therapeutic dendritic cells and regulatory T cells for preventing allogeneic cardiac graft rejection.

Tolerogenic dendritic cells (Tol-DCs) and regulatory T cells (Treg) are key factors in the induction and maintenance of transplantation tolerance. We previously demonstrated that ex vivo-isolated Tol-DCs promote Treg generation, and vice versa, in an in vitro co-culture system. Here we demonstrate the occurrence of such an immune regulatory feedback loop in vivo. Tol-DC generated in vitro by treatment with LF 15-0195 exhibited features of immature DC and express low levels of MHC class II, CD86 and CD40. These Tol-DCs were capable of augmenting CD4(+)CD25(+)CTLA4(+) and FoxP3(+) Treg cell numbers and activity in cardiac allograft recipients. On the other hand, Tol-DCs possessed an ability to generate Treg cells in vitro. The adoptive transfer of these in vitro-generated Treg cells resulted in an increase of Tol-DC in vivo, suggesting that an immune regulatory feedback loop, between Tol-DC and Treg, exists in vivo. Furthermore, the administration of in vitro-generated Tol-DCs or Treg cells prevented rejection of allografts. Co-administration of Tol-DC and Treg synergized efficacy of promoting allograft survival heart transplantation. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs and Treg.

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis

IntroductionWe have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.MethodsMice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.ResultsSystemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.ConclusionsThese studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.

Inhibition of allergic responses by CD40 gene silencing

Background:u2002 Gene silencing using small interfering RNA (siRNA) is a potent method of specifically knocking down molecular targets. Small interfering RNA is therapeutically promising, however, treatment of allergic diseases with siRNA has not been explored in vivo. The aim of this study was to evaluate therapeutic effects of CD40 siRNA on inhibition of allergic responses.