Role Of Toll-like Receptor Signal Transduction In Cardiac Allograft Rejection In Mice

Heart transplantation which is considered as an effective means for treating terminal heart disease has improved greatly rencently , but the acute or chronic immunologic rejection are still a major disturbance which affect operative result. With the development of studying innate immunity, toll-like receptor(TLR) signal pathway is consider as a bridge linked innate immunity and adaptive immunity.The cells which expressed TLR, for example dendritic cell(DC) play important role in allograft rejection. Our project will study the role of TLR signal pathway in cardiac rejection, and try to decrease the morbidity of allograft rejection by inhibiting TLR signal pathway through using MyD88siRNA gene. This study was divided into 4 parts as follows:Role of Toll-like Receptor Signal Transduction In Cardiac Allograft Rejection in MicePart 1.Experiment 1.Study on generation , identification and cytobiological characteristics of dendritic cells from mouse boneObjectives: To obtain and identify dendritic cells(DCs)from mouse bone marrow in vitro and study its cytobiological characteristics. Methods: Mouse bone marrow cells were induced to form dendritic cells by recombinant mouse-granulocyte and macrophage colony-stimulus factor (GM-CSF) and interleukin-4 (IL-4) in vitro. Dendritic cells were divided into control group and LPS group. The morphological changes were observed with light inverted microscope. CD11c, CD80, CD86 and MHC-Ⅱwere identified with flow cytometry. The biological function was studied with mixed lymphocyte reaction (MLR).Results: The cultured DCs from mouse bone marrow displayed the typical morphological characteristics of DCs. The expression of CD1lc is no deference in two groups, while the expression of CD80,CD86and MHC-Ⅱin LPS group are higher than that of control group.The DCs in LPS group could stimulate allogenic mixed lymphocyte proliferation. Conclusions:The DCs generated have cytobiologlcal characteristics of myeloid lineage of DCs,and it will be widely used in the clinical study of and experiment. Experiment 2.The impact of hypoxia /reoxygenation on maturation of dendritic cells cultured from murine bone marrowObjective: To explore the impact and mechanism of hypoxia /reoxygenation stimulation on maturation of dendritic cells( DCs) cultured from murine bone marrow. Methods: Mouse DCs were generated from bone marrow cells and were divided into control group and hypoxia /reoxygenation group. DCs in control group was cultured at normal condition, and in hypoxia /reoxygenation group was cultured at hypoxic condition for 4 hours followed by cultured at normal condition for 24 hours. Flow cytometry and mixed lymphocyte reaction (MLR) was used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γand IL-12 in the supernatant. Immunochemistry and western blot was used to detect the concentration of NF-κB. Results: Hypoxia /reoxygen stimulation increased the CD80, CD86, MHC-Ⅱ, CD14 and TLR4 in the cytomembrane of DCs and TNF-α, IFN-γ, IL-12 concentration in the supernatant. Hypoxia /reoxygen stimulation also promoted the shift of NF-κB to karyon. Conclusions: Hypoxia /reoxygen stimulation perhaps promots the maturation of DCs through toll-like receptors signal pathway. Inhibition maturation of DCs may be a novel way for treatment of ischemia-reperfusion injury and rejection.Experiment 3.Role of Myeloid differentiation factor 88 in HSP60 signal transduction in dendritic cellsObjective: To explore the role and mechanism of myeloid differentiation factor 88(MyD88) in HSP60 signal transduction in dendritic cells. Methods: Mouse DCs were cultured from murine bone marrow cells. The DC marker CD11c was detected by flow cytometry, then DCs were divided into control group, HSP60 groupand RNA interference group. Control group was cultured under normal condition, and HSP60 group was cultured with 10μg/ml of HSP60. RNA interference group was first cultured with MyD88 siRNA for12 hours and then HSP60 was added into the culture mixture. All groups were cultured for 48 hours. Immunochemistry was used to detect the concentration of MyD88 and NF-κB. Western blot was used to detect the concentration of MyD88.Flow cytometry and mixed lymphocyte reaction (MLR) were used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γand IL-12 in the supernatant. Results: The expression of CD11c in murine bone marrow DCs was 88.76%. HSP60 stimulation increased the expression of CD80, CD86, MHC-Ⅱin DCs and TNF-α, IFN-γ, IL-12 secretion in the supernatant. HSP60 stimulation also increased the level of MyD88 in the cytoplasm and promoted the shift of NF-κB to karyon and the proliferation of allogeneic T cells. MyD88 siRNA could decrease MyD88 and inhibit these effects induced by HSP60. Conclusions: HSP60 activates DCs through MyD88-dependent pathway. MyD88 is a critical role in HSP60 signal transduction. Inhibition of MyD88 may be a novel way for treating disease correlated with HSP60.Part 2. Expression and significance of HSP60 and toll-like receptor 4 transducting system in mouse cardiac transplantationObjective: To detect the expression of HSP60 and toll-like receptor 4 transducting system in mouse cardiac transplantation , and to investigate their role in mouse cardiac transplantation. Methods: Mouse cervical heart transplantation model was established The animals were divided into control group and experimental group . The heart and blood were chosen for study at day 3 and day 7. Pathological analysis were performed.The levels of cytokines in the blood serum were determined using ELISA. The expression of HSP60, TLR4,MyD88 and NF-κB in cardiac transplantation were determined by immunohistochemistry and western blot. Results: Severe rejection could observed in experimental group whereas no distinct rejection in control group. Thl cytokines (TNF-α,IFN-γ,IL-12)increased significantly in experimental group as compared with that in control group. The expression of HSP60, TLR4,MyD88 , NF-κB was higher in experimental group than that in control group. Conclusions: HSP60 increased significantly after heart transplantation which can activate toll-like receptor 4 transducting system in MyD88-dependent pathway and promote allograft rejection. Regulation of HSP60 signal transduction may be a novel way for treating allograft rejection. Part 3.Inhibitory effects of RNA interference on MyD88 expression and biological activity in murine myeloid dendritic cellsObjective: To synthesize small interference RNA aimed directly at myeloid differentiation factor 88 in murine myeloid dendritic cells(DCs) and observe the inhibitory effects on MyD88 expression and biological activity of DCs by RNA interference, and provide basis for clinical applications of DCs. Methods: Three pairs of MyD88 siRNA were synthesized and transfected into DCs with RNAi-mate. The mRNA and protein expressions of MyD88 were analyzed by semi-quantified RT-PCR and Western blot. Mouse DCs were divided into control group and RNA interference group. One of the highest effective siRNA was transfected into RNA interference group. 12 hours later, LPS of the final concentrations of 10μg/ml was added in two groups and continued to culture for 3 days. Flow cytometry and mixed lymphocyte reaction (MLR) were used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γand IL-12 in the supernatant. Immunochemistry was used to detect the concentration of NF-κB. Results: MyD88 mRNA and protein were reduced 90% and 85% by sequence2 siRNA, 92% and 88% by sequence3 siRNA respectively, while no change was found in other groups. LPS stimulation increased the CD80, CD86, MHC-Ⅱin the cytomembrane of DCs and TNF-α, IFN-γ, IL-12 concentration in the supernatant in control group. LPS stimulation also promoted the shift of NF-κB to karyon and the proliferation of allogeneic T cells in control group. MyD88 RNA interference can inhibit these effects. Conclusions: RNA interference can knockdown MyD88 expression in murine myeloid dendritic cells and inhibit maturation of DCs. This may provide a new strategy of gene therapy for relative diseases.Part 4.Pretreatment of donor dendritic cells with MyD88siRNA to induce tolerance in mouse allograft recipientsObjective: To explore the effects of donor dendritic cells treated with MyD88siRNA in tolerance induction in mouse allograft recipients. Methods: MyD88siRNA were synthesized chemically and transfected into DCs derived from BALB/c bone marrow by RNAi-mate. The DCs modified with MyD88siRNA,named MyD88siRNA-DC,were injected into the recipient C57BL/6 mice 7 days before transplantation.The existence of the donor MyD88siRNA-DC in the recipient animal spleens was studied by double- immunofluorescence staining.The responsiveness of the recipient spleen T cell to the donor alloantigen was determined by mixed lymphocyte reaction (MLR). The cervical heterotopic heart transplantation model was established with"cuff"technique and the cardiac allograft survival time was observed. Pathological analysis were performed and the levels of cytokines in the serum were determined using ELISA. Results: The survival rate of the donor-derived MyD88siRNA-DC in the recipient spleens was higher than that of the donor derived Day8-DC. The donor-derived MyD88siRNA-DC induced alloantigen-specific T-cell hypo-responsiveness. The cardiac allograft survival time of the MyD88siRNA-DC treated group was longer than that of the Day8- DC group and PBS treated group (24.50±4.42)days vs(13.67±2.25) days and (6.67±1.3) days (P<0.01). Pathological grade of rejection was significantly lower (P< 0.01).In the MyD88siRNA-DC treated group,the levels of IL-12 and IFN-γin the serum decreased significantly(P< 0.01),but the levels of IL- 4 and IL-10 in the serum increased significantly (P<0.01). Conclusions: The injection of the donor-derived MyD88 siRNA-DC can leads to donor-specific tolerance in transplant recipients.The Polarization of Th2 response and chimerism of recipient may play important roles on immune tolerance to cardiac allografts.