Research On The Suppressive Effect On Rejection In Mice Cardiac Transplantation By Indoleamine2,3-Dioxygenase

Suppressive effect of indoleamine2,3-dioxygenase (IDO) on rejection of mice cardiac transplantation was studied in this experiment. Suppressive effect of transfected IDO positive dendritic cells (IDO+DC) and tryptophan catabolites (TC) were used to induce immune tolerance by suppressing T lymphocyte proliferation during the antigen presenting state of DC. Suppression of T lymphocytes proliferation or clone deletion was achieved when T lymphocytes underwent tryptophan starvation state with accumulation of TC. The suppressive effect on rejection of mice cardiac transplantation was evaluated, which provided experimental evidence for further research in the field of immune tolerance.Part I Suppressive Effect of IDO+DC in Combination with TC on T LymphocyteObjective To evaluate the in vitro and in vivo suppressive effect of IDO+DC and TC on T lymphocyte. Method Adenovirus vector containing IDO gene was used to infect donor (C57BL/6) DC to obtain IDO+DC. In vitro, CD4+T lymphocytes were isolated from the spleen of untreated recipient (BALB/c) mouse, and then treated with either donor DC, TC with donor DC, IDO+DC or Co-administration of IDO+DC and TC. In vivo effect of treatments were examined by injection of either donor DC, TC, IDO+DC or Co-administration of IDO+DC and TC into recipient mouse, and CD4+T lymphocytes were isolated and harvested from the spleen five days after injection. Harvested CD4+T lymphocytes of the treated recipient mouse were mixed cultured with donor DC. Meanwhile, CD4+T lymphocytes harvested from recipient mouse treated with Co-administration of IDO+DC and TC were mixed cultured with tertiary origin DC from C3H/He mouse. T lymphocyte apoptosis rate (AR) and proliferation stimulation index (SI) were evaluated for both in vitro and in vivo effect of treatments. Result Both in vitro and in vivo effect of treatments with Co-administration of IDO+DC and TC showed significant decrease in T lymphocytes proliferation SI, and significant increase in T lymphocyte AR (P＜0.01). By using CD4+T lymphocytes from recipient mouse injected with Co-administration of IDO+DC and TC, mixed cultured with donor origin DC showed significant decrease in T lymphocyte SI, and significant increase in T lymphocyte AR (P<0.01) compare with that when mixed cultured with tertiary origin DC. Conclusion IDO+DC or TC alone is less effective comparing with combination administration of IDO+DC and TC, which showed stronger suppressing effect on T lymphocytes and might be able to induce antigen-specific immune suppression.Part II Establishment of Heterotropic Cardiac Transplantation Model in MiceObjective To establish a stable and reliable mouse heterotopic cardiac transplantation model for accurate in vivo results. Method Mouse heterotopic cardiac transplantation (HCT) model was established using C57BL/6mouse as the donor and BALB/c mouse as the recipient. Establishment of model was divided into two phases.100and80pairs of HCT were performed respectively in the training phase and stable phase. Anesthesia technique and method of harvesting the graft heart were also improved accordingly during both phases. Result Surgical success rate of HCT in stable phase (91.25%) was significantly increased when compared with that in training phase (29%)(P＜0.01), and no recipients were dead due to anesthesia in both phases. Vascular anastomosis failure rate, donor heart ischemia time and surgical time all showed significant improvement in stable phase when compare to those in traning phase (P<0.01). Conclusion Long-term training of microsurgical technique avoids wasting of experimental animals and at the same time ensures the quality of the model to be used for further experiments. Prerequisite for successful establishment of HCT model is successful anesthesia for the mouse, and ensuring the quality of vascular anastomosis is the key to successful establishment of HCT model. Mice HCT model is a stable and reliable model for further research in organ transplant rejection.Part III Suppressive Effect of IDO+DC in Combination with TC on Rejection in Mice Heterotopic Cardiac Transplantation Objective To examine the combination effect of IDO+DC and TC on transplantation rejection and possible anti-rejection mechanism. Method Donor mice were divided into two major groups, the control group and treatment group. Control group was further divided into four subgroups, including injection of PBS, untransfected DC, vector-virus DC, or Cyclosporine (10mg/kg/d). Treatment group was divided into three subgroups, including injection of TC, IDO+DC or Co-administration of IDO+DC and TC. Survival time of the donor heart in every groups was observed. Meanwhile, donor hearts were harvested7days post transplantation for different examinations, including pathological examination through H&E, mRNA expression of IDO and different cytokines through qRT-PCR, IDO protein expression through western blot and immunohistochemistry. Peripheral blood of recipient was also harvested for T lymphocyte apoptosis rate examination through FACS. Result Compared with PBS, untransfected DC, vector-virus DC control groups, IDO+DC and TC treatment significantly prolonged the survival time of donor hearts (P＜0.01). Pathological grading was significantly decreased (P<0.05). Boeh IDO mRNA and protein expression showed significant increase (P<0.01). mRNA expression of TNF-a was decreased (P＜0.05), while IL-10was significantly increased (P<0.01). Furthermore, the T lymphocyte apoptosis rate was also significantly increased (P＜0.01). Compared with treatment group with IDO+DC or TC alone, co-administration of IDO+DC and TC treatment significantly prolonged the survival time of the donor heart (P＜0.05). Both IDO mRNA and protein expression showed significant increase (P<0.05). mRNA expression of IL-2, IFN-γ, and TNF-a were significantly decreased (P＜0.05), with the exception for the IDO+DC treatment group of IL-2(P>0.05), and expression of IL-10was also significantly increased (P<0.05). Furthermore, the T lymphocyte apoptosis rate was significantly increased as well (P＜0.01). Pathological grading was decreased, but without significant difference (P>0.05). Conclusion Suppressive effect of co-administration of IDO+DC with TC was much more effective than administration of IDO+DC or TC alone, which suggested that IDO achieved immune suppressive effect through the pathway of tryptophan depletion and accumulation of TC. Part IV Antigen-specific Suppressive Effect of IDO+DC in Combination with TC on Transplant RejectionObjective To observe the suppressive effect of combination treatment with IDO+DC and TC on skin graft rejection of post cardiac transplantation mice. Method C57BL/6or C3H/He mouse was used as the donor, and BALB/c mouse treated with combination of IDO+DC (C57BL/6origin) and TC underwent HCT with donor heart survival>30days was used as the recipient to establish skin transplantation model. Then the condition of skin graft was monitored. Result Skin graft of C57BL/6origin survived>2weeks post transplantation, with mild skin curl and hair loss. Skin graft of C3H/He origin totally scab and fall off10days post transplantation. Conclusion Transplant rejection of skin graft decreased in mouse underwent treatment with combination of IDO+DC and TC, which indicated that combination treatment with IDO+DC and TC might induce antigen-specific immune tolerance.