Costin Vladau

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.

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.

Reinstalling Antitumor Immunity by Inhibiting Tumor-Derived Immunosuppressive Molecule IDO through RNA Interference

Tumor-derived immune suppression is a major impediment to successful immune/gene cancer therapy. In the present study, we describe a novel strategy to disrupt tumor-derived immune suppression by silencing a tolerogenic molecule of tumor origin, IDO, using small interfering RNA (siRNA). Silencing of IDO in B16F10 cells in vitro using IDO-siRNA prevented catabolism of tryptophan and inhibited apoptosis of T cells. IDO-siRNA treatment of B16F10 cells in vitro inhibited subsequent growth, tumor formation, and the size of tumor formed, by those cells when transplanted into host mice. In vivo treatment of B16F10 tumor-bearing mice successfully postponed tumor formation time and significantly decreased tumor size. Furthermore, in vivo IDO-siRNA treatment resulted in recovery of T cells responses and enhancement of tumor-specific killing. Thus, silencing IDO may break tumor-derived immune suppression. These data indicate that RNA interference has potential to enhance cancer therapy by reinstalling anticancer immunity.

Preventing renal ischemia-reperfusion injury using small interfering RNA by targeting complement 3 gene.

The complement system is one of the important mediators of renal ischemia–reperfusion injury (IRI). We hypothesized that efficient silencing of C3, which is the central component on which all complement activation pathways converge, could be achieved using small interfering RNA (siRNA), and that this would result in overall inhibition of complement activation, thereby preventing IRI in kidneys. A series of experiments was conducted, using a mouse model of IRI and vector‐delivered C3‐specific siRNA. We demonstrated the following: (1) renal expression of C3 increases as a result of IRI; (2) by incorporation into a pRNAT U6.1 vector, siRNA can be delivered to renal cells in vivo; (3) systemically delivered siRNA is effective in reducing the expression of C3 in an experimentally induced mouse kidney model of IRI; (4) similarly, siRNA reduces complement‐mediated IRI‐related effects, both in terms of renal injury (as evidenced by renal function and histopathology examination) and mouse mortality and (5) silencing the production of C3 diminishes in vivo production of TNF‐α. This study implies that siRNA represents a novel approach to preventing IRI in kidneys and might be used in a variety of clinical settings, including transplantation and acute tubular necrosis.

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.

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.

Tolerogenic dendritic cells transferring hyporesponsiveness and synergizing T regulatory cells in transplant tolerance

Dendritic cells are among the most potent antigen-presenting cells and are important in the development of both immunity and tolerance. Tolerogenic dendritic cell (Tol-DC) is a key factor in the induction and maintenance of tolerance during transplantation. However, the precise mechanism and direct evidence of in vivo immune modulation remain unclear. In the present study, we identified critical roles of immune modulation on transplant tolerance through interactions between Tol-DCs and regulatory T (Treg) cells. Tol-DCs remained in an immature state and were insensitive to maturation stimuli. Tol-DCs in tolerant recipients heightened the expression of indoleamine 2,3-dioxygenase (IDO) that induced allogeneic T-cell apoptosis. Adoptive transfer of Tol-DCs isolated from primary tolerant recipients resulted in augmentation of CD4(+)CD25(+)CTLA4(+) Treg cells and prolonged graft survival in secondary allogeneic heart transplantation and synergized with Treg cells to induce tolerance in secondary recipients. This study indicates that Tol-DC offers two functions during the process of tolerogenesis: suppression of anti-donor T-cell responses through production of IDO and interaction with Treg cells, which provides a framework for future research into tolerance induction.

Identification of viruses in patients with postviral olfactory dysfunction.

Objective: Causative viruses of postviral olfactory dysfunction (PVOD) have not yet been identified. The aim of this study was to investigate causative viruses in patients with PVOD.