CD40 siRNA Research Articles

PD-L1 Inhibits T Cell-Induced Cytokines and Hyaluronan Expression via the CD40-CD40L Pathway in Orbital Fibroblasts From Patients With Thyroid Associated Ophthalmopathy

Thyroid associated ophthalmopathy (TAO), characterized by T cell infiltration and orbital fibroblast activation, is an organ-specific autoimmune disease which is still short of effective and safety therapeutic drugs. The PD-1/PD-L1 pathway has been reported hindering the progression of Graves’ disease to some extent by inhibiting T cell activity, and tumor therapy with a PD-1 inhibitor caused some adverse effects similar to the symptoms of TAO. These findings suggest that the PD-1/PD-L1 pathway may be associated with the pathogenesis of TAO. However, it remains unknown whether the PD-1/PD-L1 pathway is involved in orbital fibroblast activation. Here, we show that orbital fibroblasts from patients with TAO do not express PD-L1. Based on in vitro OF-T cell co-culture system, exogenous PD-L1 weakens T cell-induced orbital fibroblast activation by inhibiting T cell activity, resulting in reduced production of sICAM-1, IL-6, IL-8, and hyaluronan. Additionally, exogenous PD-L1 treatment also inhibits the expression of CD40 and the phosphorylation levels of MAPK and NF-κB pathways in orbital fibroblasts of the OF-T cell co-culture system. Knocking down CD40 with CD40 siRNA or down-regulating the phosphorylation levels of MAPK and NF-κB pathways with SB203580, PD98059, SP600125, and PDTC can both reduce the expression of these cytokines and hyaluronan. Our study demonstrates that the orbital immune tolerance deficiency caused by the lack of PD-L1 in orbital fibroblasts may be one of the causes for the active orbital inflammation in TAO patients, and the utilization of exogenous PD-L1 to reconstruct the orbital immune tolerance microenvironment may be a potential treatment strategy for TAO.

The Role of CD40 in Allergic Rhinitis and Airway Remodelling.

Background Allergic rhinitis (AR) affects millions of people and is lack of effective treatment. CD40 is an important costimulatory molecule in immunity. However, few studies have focused on the role of CD40 in AR. Methods In this study, we built mouse model of chronic AR. The mice were divided into the AR, control, intravenous CD40 siRNA, and nasal CD40 siRNA groups (n = 6 each). We detected OVA-sIgE, IL-4, IL-5, IL-13, IL-10, IFN-γ, and TGF-β levels in serum and supernatant by ELISA, CD40+ splenic DCs, and Foxp3+ Tregs by flow cytometry and CD40 mRNA by RT2-PCR. We also used PAS and MT stains to assess tissue remodelling. Results (1) The OVA-sIgE, IL-4, IL-5, and IL-13 levels in the serum or supernatant of nasal septal membrane of AR mice were significantly higher than control. After treated with CD40 siRNA, those indicators were significantly decreased. The IFN-γ, IL-10, and TGF-β levels in AR mice were significantly lower than that in control and were increased by administration of CD40 siRNA. (2) AR mice had significantly fewer Foxp3+ Tregs in the spleen than control mice. After treated with CD40 siRNA, AR mice had significantly more Foxp3+ Tregs. (3) AR mice exhibited a significantly higher CD40 mRNA levels than control. Administration of CD40 siRNA significantly reduced the CD40 mRNA level. (4) The AR mice showed significantly greater collagen deposition than the control in MT staining. Applications of CD40 siRNA significantly reduced the collagen deposition in AR mice. Conclusion CD40 siRNA therapy shows promise for chronic AR as it significantly attenuated allergic symptoms and Th2-related inflammation and upregulated Foxp3+ Tregs. CD40 plays a role in tissue remodelling in AR, which can be inhibited by CD40 siRNA application.

In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining.

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our group has broad experience in silencing CD40 using RNAi technology, and here we summarize protocols for the systemic administration of a specific anti-CD40 siRNA in different rodents models, in addition to the subsequent quantification of CD40 expression in murine kidneys by immunostaining. The use of RNAi technology with specific siRNAs to silence genes is becoming an essential method to investigate gene functions and is rapidly emerging as a therapeutic tool. Graphic abstract: CD40 siRNA mechanism.

A novel allergen-specific therapy with regulatory T cells induced by CD40-silenced dendritic cells.

We previously reported that dendritic cells (DCs) transfected with CD40 siRNA and pulsed by ovalbumin (OVA) (CD40-silenced OVA DCs) inhibited allergic responses through facilitation of regulatory T cells (Tregs). However, to our knowledge, no prior study has examined allergen-specific therapy by administration of siRNA-induced Tregs for the control of allergy. We aimed to investigate the effect of Tregs induced in vitro on allergic responses and symptoms in vivo. Mice were treated with Tregs (OVA DCs-induced Tregs) induced by CD40-silenced OVA DCs or Tregs (nonantigen DCs-induced Tregs) induced by DCs transfected with CD40 siRNA and pulsed with no antigen, and the effects of these Tregs on allergic responses were estimated. Administration of nonantigen DCs-induced Tregs prevented not only OVA-induced allergy but also keyhole limpet hemocyanin-induced allergy. Administration of OVA DCs-induced Tregs significantly reduced the number of sneezes and nasal rubbing movements, eosinophilia in the nasal mucosa, and the level of OVA-specific IgE in mice with OVA-induced allergy, compared with CD40-silenced nonantigen DC-induced Tregs in numbers 20 times greater, even in mice with established allergic rhinitis. Furthermore, Tregs induced by CD40-silneced DCs pulsed with Cry j 1, a major allergen of Japanese cedar pollen, inhibited Japanese cedar-induced allergy. This study shows for the first time that both antigen-independent Tregs and antigen-specific Tregs can be induced by siRNA, and that therapy with siRNA-induced Tregs inhibits allergic responses and symptoms. It also shows that antigen-specific Tregs have more potent effects in inhibiting allergic responses than antigen-nonspecific Tregs.

Regulating the expression of CD80/CD86 on dendritic cells to induce immune tolerance after xeno-islet transplantation

BackgroundAntigen present cells (APCs) have been demonstrated to play dual roles in immune tolerance. Recently, compelling evidence indicates that APCs that express CD80, but not CD86 can protect allograft. We investigated whether modulation of CD80 in dendritic cells (DCs) offer protection for xeno-islets. MethodsIn vitro, isolated mature murine DCs received untransfection, transfection with CD86 siRNA or negative control siRNA. The DCs were used in mixed lymphocyte reaction in which rat islets and murine splenocytes were further added. On day 3 of co-culturing, the proliferation of lymphocytes was measured and interleukin (IL)-2, IL-4, IL-10, transforming growth factor β (TGF-β), interferon γ (INF-γ) and indoleamine 2,3-dioxygenase (IDO) from the supernatants were determined. Islets viability and function were also assessed. In vivo, streptozotocin-induced diabetic mice underwent rat islets transplantation were pre-treated with above DCs. At designated time, xeno-islets were subjected to histopathology, immunohistochemistry, survival time and functional tests. Peripheral blood T lymphocyte profiles were also examined. ResultsCD86-silenced-DCs had unchanged expression of CD80 and significantly suppressed the proliferation of lymphocytes. CD86-silenced-DCs simultaneously reduced IL-2 and INF-γ and increased IL-10, TGF-β and IDO, while had minimal effect on IL-4. The CD86-silenced-DCs also improved cell viability and function of xeno-islets when compared to untransfection and transfection control groups. In xeno-islets transplanted diabetic mice, transfer of CD86-silenced-DCs resulted in improved histopathology and dramatically prolonged survival time of the islets. These effects were also mirrored by the functional tests. Further analysis revealed that CD86-silenced-DCs had up-regulated levels of CD4+CD25+T cells in the peripheral blood compared to the other groups. ConclusionsCD86-silenced-DCs induced immune tolerance of rat xeno-islets in recipient diabetic mice with up-regulated peripheral blood CD4+CD25+T cells.

Effect of gene silence of CD40 by small interfering RNA on balance between Th1/Th2 and Th17/Treg in rats with experimental autoimmune myocarditis

Objective To investigate how gene silence of CD40 by small interfering RNA(siRNA) influences the balance between Th1/Th2 and Th17/Treg in rats with experimental autoimmune myocarditis(EAM). Methods Lewis rats were divided into a normal-control group, EAM group, CD40siRNA-therapy group and siRNA-control group by using random number table.EAM, CD40siRNA-therapy and siRNA-control groups were immunized on day 0 and day 7 with purified porcine cardiac myosin to establish EAM, and CD40siRNA was administered intravenously on day 7.The basic data of each group were observed, and the rats were executed on day 21 to study the pathology of myocardial tissues and the myocardial histopathology scores were calculated, and than the changes in electrocardiograms (ECG) and echocardiogram were recorded.Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of interferon (IFN) -γ, interleukin(IL) -10, IL-17 and transforming growth factor(TGF)-β in peripheral blood. Results (1) The overall incidence of EAM, CD40siRNA-therapy and siRNA-control group was 100%, and no rats died from day 0 to day 21.(2) There were only 2 premature rats in EAM and siRNA-control groups, and the ECGs of normal-control group and CD40siRNA-therapy group were normal.(3) The echocardiogram of EAM, CD40siRNA-therapy and siRNA-control group, displayed the left ventricular dilatation, hypertrophy of the left ventricle, the weakening of the left ventricular wall motion, and heart failure, and pericardial effusion was discovered.The left ventricular fractional shortening(LVFS) and the left ventricular ejection fraction (LVEF) of rats treated by CD40siRNA were higher than those of rats in the EAM group[(46.70±8.25)% vs (34.28±11.81)%, (80.92±11.76)% vs (64.03±12.60)%, F=0.652, 0.234, all P<0.05]. (4) Pathologic examination of the EAM group, CD40siRNA therapy group and siRNA control groups, showed myocardial fiber fracture, myocardial tissue necrosis and inflammatory cell infiltration.But myocardial tissue pathological scores of the cardiac tissue of CD40siRNA therapy group was lower than those of the EAM group(2.10±1.07 vs 3.40±0.72, F=1.290, P<0.05). (5) ELISA method examination revealed that the levels of IFN-γ, IL-4, IL-17 and TGF-β in EAM group were increased compared with those of normal control group[(1 245.55±244.56) ng/L vs (501.53±18.93) ng/L, (78.03±10.47) ng/L vs (30.77±1.74) ng/L, (80.82±13.33) ng/L vs (27.98±3.01) ng/L, (156.27±12.11) ng/L vs (4.33±0.79) ng/L, t=7.408, 10.764, 9.250, 30.608, all P<0.05]. However, the levels of IFN-γ and IL-17 in CD40 siRNA therapy group were lower than those in the EAM group[(940.62±128.40) ng/L vs (1 245.55±244.56) ng/L, (60.42±12.40) ng/L vs (80.82±13.33) ng/L, t=2.704, 2.745, all P<0.05], while the levels of IL-4 and TGF-β were higher[(97.91±13.62) ng/L vs (78.03±10.47) ng/L, (178.84±12.10) ng/L vs (156.27±12.11) ng/L, t=2.835, 3.229, all P<0.05]. Conclusions The administration of CD40 siRNA markedly reduces myocardial inflammation of EAM rats and improves their cardiac function, which can be explained by Th1-type and Th17-type cytokines inhibited, Th2-type and Treg-type cytokines increased, and so then the imbalance of Th1/Th2 and Th17/Treg corrected. Key words: Autoimmune myocarditis; Rat; CD40 small interfering RNA; Th1/Th2; Th17/Treg

Effect of CD40 silenced dendritic cells by RNA interference on mice skin allograft rejection.

Tolerogenic dendritic cells (DCs) play a critical role in inducing and maintaining tolerance. CD40 is a member of tumor necrosis factor receptor super family and is a potent T-cell costimulatory molecule. Therefore, in this study we evaluated the effect of CD40 silenced DCs by RNA interference on mice skin allograft rejection. Skin transplantation was performed from C57BL/6 to BALB/c mouse. Skin allograft recipients were assigned to four groups (n = 5). CD40 downregulated DCs were injected to the BALB/c mice intravenously 7 days before transplantation. Then, graft survival time, Treg generation, CD4(+) and CD8(+) T cells infiltration and cytokine levels in serum of this group were compared with those of untreated and cyclosporine groups. In comparison with untreated group, BALB/c mice injected with CD40 siRNA transfected DCs showed an increased graft survival time, Treg cells, IL-4 and IL-10 cytokine levels as well as decreased number of intragraft CD4(+) and CD8(+) T cells. IFN-γ and IL-12 secretion were diminished, too. Taken together, these data demonstrate that downregulation of CD40 in DCs can expand Treg cells and increase skin allograft survival.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma.

BackgroundCD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.MethodsWe investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.ResultsTreatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.ConclusionThese results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection

BackgroundWhile 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.MethodVectors 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.ResultAllogeneic 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.ConclusionThis 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.

Attenuation of experimental autoimmune myocarditis by si-RNA mediated CD40 silencing.

CD40 plays an important role in the pathogenesis of myocarditis, and inhibition of CD40 expression could be a promising treatment for myocarditis. In this study, we used an animal model, experimental autoimmune myocarditis (EAM), to investigate whether CD40 siRNA could be exploited for myocarditis therapy.Lewis rats were immunized with purified porcine cardiac myosin to induce EAM or were injected with phosphate buffered saline (PBS) alone (PBS group), scrambled small interfering RNA (siRNA) (negative control group), or CD40siRNA (CD40 siRNA group).CD40 siRNA treatment suppressed the increase in heart weight/body weight ratio, and attenuated the severity of myocardial lesions. Cytokine production, including Th1-type cytokines, was significantly suppressed in rats with myocarditis after CD40 siRNA treatment; however, production of Th2-type cytokines was higher. Specific knockdown of CD40 in EAM rats resulted in increased FOXP3 gene expression and the CD25+ CD4+ subpopulation of T cells but also a decrease in CD80 and CD86 expression. Lymphocyte (T and B cell) proliferation in response to myosin stimulation was significantly inhibited by CD40 silencing.CD40-siRNA is a useful tool for inhibiting in vivo CD40 expression, and it could have therapeutic potential in the prevention and treatment of myocarditis in humans.

Effect of CD40 siRNA on T helper lymphocyte-17 cells and interleukin-17, interleukin-23 in rats with experimental autoimmune myocarditis

Objective To investigate the effect of CD40siRNA on the pathologic changes and T helper lymphocyte(Th)-17 cells of myocardium and IL-17, IL-23 of serum in rats with experimental autoimmune myocarditis(EAM). Methods Forty 6-8 week old healthy male Lewis rats with body weight of 185-210 g were divided into EAM group, CD40siRNA group, siRNA group, and normal control group randomly, with 10 rats in each group.The rats in EAM group, CD40siRNA group and siRNA group were induced by immunization with cardiac C protein and completed Freund adjuvant in double foot pads.The rats in normal control group were injected with PBS buffer in double foot pads.On the 8th day after immunization, the rats in CD40siRNA group were injected with CD40siRNA expression vector, and the rats in siRNA group were injected with siRNA expression vector.The rats were sacrificed on day 21 after inoculation.The histopathologic changes were observed by light microscope and the myocardial histopathology scores were calculated.The expression of RORC mRNA of myocardium was detected by real-time quantitative polymerase chain reaction(RT-PCR). Enzyme linked immunoabsorption assay was used to determine the serum level of IL-17 and IL-23. Results Compared with EAM group, the myocardial histopathology score(2.34±0.60 vs 3.40±0.35, P<0.05), the expression of RORC mRNA(2.13±0.28 vs 2.93±0.36, P<0.05) and the serum level of IL-17(114.38±8.29 vs 148.70±5.04, P<0.05)and IL-23(107.00±7.69 vs 136.98±23.16, P<0.05)were significantly lower in CD40siRNA group. Conclusions It is suggested that CD40siRNA expression vector might reduce myocardial injury by inhibiting Th-17 activation and down-regulating the expression of IL-17 and IL-23. Key words: Experimental autoimmune myocarditis; CD40siRNA; RORC; Interleukin-17; Interleukin-23

Effect of CD40 siRNA on Th17 cells and IL-17 and IL-23 in rats with experimental autoimmune myocarditis

Objective: To investigate the effect of CD40 siRNA on the myocardial pathologic changes, expression of Th17 cells specific transfer factor gene RORC mRNA of myocardium and concentration of Th17 cells related cytokines interleukin -17 (IL-17) and interleukin -23 (IL-23) in peripheral blood of rats with EAM. Methods: Forty 6-8 week old healthy male Lewis rats with body weight ranging from 185 to 210 gram were divided into EAM group (n=10), CD40 siRNA group (n=10), siRNA group (n=10), and control group (n=10) randomly. The rats in EAM group, CD40 siRNA group, and siRNA group were immunized with cardiac C protein and completed Freund adjuvant in double foot pads. The rats in control group were injected with PBS buffer in double foot pads. On the eighth day after immunization, rats in CD40 siRNA group and siRNA group were injected with CD40 siRNA expression vector and siRNA expression vector three times a week. These rats were sacrificed on day 21 after inoculation. The myocardial histopathologic changes were observed by light microscope and the myocardial histopathology scores were also calculated. The expression of RORC mRNA of myocardium was detected by real-time quantitative polymerase chain reaction (RQ-PCR). Enzyme linked immunoabsorption assay (ELISA) was used to determine the serum level of IL-17 and IL-23. Results: 1. Compared to EAM group, the myocardial histopathology scores in CD40 siRNA group significantly lower (13.33±5.56 vs. 17.00±2.16, P<0.05. 2. The expression of RORC mRNA in CD40 siRNA group decreased significantly compared to the EAM group (2.13±0.28 vs. 2.93±0.36,P<0.05). 3. The serum level of both IL-17 and IL-23 in CD40 siRNA group also decreased significantly compared to EAM group (both P<0.05). Conclusions: The results indicated that CD40 siRNA expression vector might reduce myocardial injury by inhibiting T helper lymphocyte IL-17 activation and down-regulating the expression of IL-17 and IL-23.

Signal pathways in astrocytes activated by cross-talk between of astrocytes and mast cells through CD40-CD40L

BackgroundAstrocytes, which play an active role in chronic inflammatory diseases like multiple sclerosis, exist close to mast cells with which they share perivascular localization. We previously demonstrated the possibility that astrocytes and mast cells interact in vitro and in vivo. This study aimed to investigate the signaling pathways and the role for astrocytes in the interaction of astrocytes and mast cells.MethodsWe co-cultured human U87 glioblastoma (U87) and human mast cell-1 (HMC-1) cell lines, and mouse cerebral cortices-derived astrocytes and mouse bone marrow-derived mast cells (BMMCs). Intracellular Ca2+ ([Ca2+]i) was measured by confocal microscopy; CD40 siRNA by Silencer Express Kit; small GTPases by GTP-pull down assay; PKCs, MAPKs, CD40, CD40L, Jak1/2, STAT1, TNF receptor 1 (TNFR1) by Western blot; NF-κB and AP-1 by EMSA; cytokines by RT-PCR. An experimental allergic encephalomyelitis (EAE) model was induced using myelin oligodendrocyte glycoprotein (MOG) peptide and pertussis toxin in mice. Co-localization of TNFR1 and astrocytes in EAE brain tissues was determined by immunohistochemistry.ResultsEach astrocyte co-culture had increases in [Ca2+]i levels, release of cytokines and chemokines; activities of Rho-family GTPases, NF-κB/AP-1/STAT1727, and Jack1/2, STAT1701. These effects were inhibited by anti-CD40 antibody or CD40 siRNA, and signaling pathways for Jak1/2 were inhibited by anti-TNFR1 antibody. EAE score, expression of TNFR1, and co-localization of TNFR1 and astrocytes were enhanced in brain of the EAE model. Anti-CD40 antibody or 8-oxo-dG pretreatment reduced these effects in EAE model.ConclusionsThese data suggest that astrocytes activated by the CD40-CD40L interaction in co-culture induce inflammatory cytokine production via small GTPases, and the secreted cytokines re-activate astrocytes via Jak/STAT1701 pathways, and then release more cytokines that contribute to exacerbating the development of EAE. These findings imply that the pro-inflammatory mediators produced by cell-to-cell cross-talk via interaction of CD40-CD40L may be as a promising therapeutic target for neurodegenerative diseases like MS.

Experience with lentivirus-mediated CD40 gene silencing in a mouse model of Graves' disease

CD40 plays an important role in the pathogenesis of Graves' disease (GD). Inhibition of CD40 expression may be a promising treatment for GD. In this study, we used an animal model to investigate whether lentivirus expressing siRNA for CD40 (LV-CD40-siRNA) could be useful for the therapy of GD. BALB/c mice were injected with PBS alone (PBS group), negative lentivirus (control siRNA group), or LV-CD40-siRNA (CD40 siRNA group), 3 days before being treated with adenovirus expressing human TSHR A subunit (Ad-TSHR289) three times at 3-week intervals to induce GD model. Sera thyroxine (T(4)) levels were assayed by RIA. The expression of CD40 was detected at the mRNA level by real-time PCR and protein level by flow cytometry. The expression of CD40, CD80, and CD86 was significantly decreased in the CD40 siRNA group (P<0.05), while FOXP3 expression was increased compared to the control siRNA group (P=0.05). Mean T(4) levels were decreased 14% in the CD40 siRNA group compared to the control siRNA group. The rate of disease induction was similar among the three groups injected with Ad-TSHR289. LV-CD40-siRNA is a useful tool to inhibit the expression of CD40 in vivo, but it cannot decrease the incidence of hyperthyroidism in a limited period of time.

Inducing tolerance with CD40 siRNA

Inducing tolerance with CD40 siRNA

A novel allergen-specific therapy for allergy using CD40-silenced dendritic cells

Induction of RNA interference with small interfering RNA (siRNA) has demonstrated therapeutic potential through the knockdown of target genes. We have previously reported that systemic administration of CD40 siRNA is capable of attenuating allergic symptoms but in an allergen-nonspecific fashion. However, siRNA-based allergen-specific therapy for allergy has not been developed. We attempted to develop a new allergen-specific therapy for allergy using CD40-silenced and allergen-pulsed dendritic cells (DCs). Bone marrow-derived DCs were silenced with CD40 siRNA and pulsed with ovalbumin (OVA). Mice had allergy after intraperitoneal sensitization with OVA and keyhole limpet hemocyanin, followed by intranasal challenge with the same allergens. The mice were treated with CD40-silenced and OVA-pulsed DCs (CD40-silenced OVA DCs) either before allergic sensitization or after establishing allergic rhinitis. Mice receiving CD40-silenced OVA DCs either before or after the establishment of allergic rhinitis showed remarkable reductions in allergic symptoms caused by OVA challenge, as well as anti-OVA IgE levels in sera. Additionally, CD40-silenced OVA DCs suppressed eosinophil infiltration at the nasal septum, OVA-specific T-cell responses, T-cell production of IL-4 and IL-5 after stimulation with OVA, and CD4(+)CD25(-) effector T-cell responses. Furthermore, CD40-silenced OVA DCs facilitated the generation of CD4(+)CD25(+) forkhead box protein 3-positive OVA-specific regulatory T cells, which inhibit allergic responses in vivo. However, CD40-silenced OVA DCs suppressed only OVA-specific allergy but did not inhibit keyhole limpet hemocyanin-induced allergy, suggesting that CD40-silenced OVA DCs induce allergen-specific tolerance. This study is the first to demonstrate a novel allergen-specific therapy for allergy through DC-mediated immune modulation after gene silencing of CD40.

Inhibition of allergic responses by CD40 gene silencing

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. Mice sensitized with ovalbumin (OVA) and alum were treated with CD40 siRNA, scrambled siRNA, or phosphate buffer saline (PBS) alone, and then challenged intranasally with OVA. A significant reduction in nasal allergic symptoms was observed in the CD40 siRNA treated OVA-allergic mice compared to the controls of scrambled siRNA and PBS alone, which is correlated with the decrease of local eosinophil accumulation. CD40 siRNA treatment knocked down CD40 expression on dendritic cells (DCs) in vivo and impaired their antigen presenting function. Treatment with CD40 siRNA resulted in inhibition of OVA-specific T cell response and decrease of interleukin-4 (IL-4), IL-5, and interferon-gamma production from T cells stimulated with OVA. Administration of CD40 siRNA also suppressed CD40 expression on B cells, resulting in down-regulation of OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a levels. Additionally, increased regulatory T cells were observed in the CD40 siRNA treated mice. The present study demonstrates a novel therapeutic use for siRNA in allergy. CD40 siRNA attenuated allergy through inhibition of DC and B cell functions and generation of regulatory T (Treg) cells.

Topical Application of Cream-emulsified CD86 siRNA Ameliorates Allergic Skin Disease by Targeting Cutaneous Dendritic Cells

Induction of the co-stimulatory molecule CD86 on dendritic cells (DCs) in the peripheral tissues is a critical event in triggering antigen-specific immune responses. In this study, we propose a new small interfering RNA (siRNA)-based therapy using cream-emulsified CD86 siRNA, targeting DCs for murine contact hypersensitivity (CH) and atopic dermatitis (AD)-like disease. Topical application of CD86 siRNA efficiently inhibited CH and markedly decreased the numbers of infiltrating CD86(+) or major histocompatibility complex (MHC) class II(+) cells in murine ear skin. The total number of cells, the percentage of hapten-carrying DCs, and their CD86 expression in the regional lymph nodes (RLNs) also significantly decreased. These results suggest that the silencing of CD86 in local DCs inhibits the recruitment and migration of DCs into the skin and RLNs, respectively, resulting in reduced antigen-specific local inflammation. The therapeutic efficacy of the CD86 siRNA was confirmed in AD-prone NC/Nga mice. Treatment produced marked amelioration in the clinical manifestations of AD and reduced the antigen-specific production of interleukin-4 (IL-4) and serum immunoglobulin E (IgE) and IgG1. Our results suggest that the targeting of cutaneous DCs by CD86 siRNA may be a promising strategy in the treatment of allergic skin disease.

Novel Vaccination for Allergy through Gene Silencing of CD40 Using Small Interfering RNA

Small interfering RNA (siRNA) is a potent means of inducing gene-specific silencing. Gene silencing strategies using siRNA have demonstrated therapeutic benefits in animal models of various diseases, and are currently in clinical trials. However, the utility of gene silencing as a treatment for allergic diseases has not yet been reported. In this study, we report a novel therapy for allergy through gene silencing of CD40, a critical costimulatory molecule and a key factor in allergic immune responses. Silencing CD40 resulted in generation of immunoregulatory dendritic cells (DCs). Administration of CD40 siRNA remarkably reduced nasal allergic symptoms and local eosinophil accumulation in the OVA-induced allergic mice. The OVA-specific T cell response was inhibited after the CD40 siRNA treatment. Additionally, anti-OVA specific IgE and production of IL-4 and IL-5 of T cells stimulated by OVA were significantly decreased in CD40 siRNA-treated mice. Furthermore, we demonstrated that the therapeutic effects by CD40 siRNA were associated with impaired Ag-presenting functions of DCs and B cells, and generation of regulatory T cells. The present study highlights a therapeutic potential of siRNA-based treatment for allergic diseases.