Regulation And Mechanism Of Autophagy In Allotransplantation Rejection

RESEARCH BACKGROUND AND PURPOSEOrgan transplantation rescued millions of lives who suffered from vital organ failure.However,the graft rejection after surgery was still the most essential factor in transplantation graft survival.Recently,study showed that T cells acted as the main effect cells and played an important role in graft rejection.Focusing on mechanism of T cell mediated transplant rejection will make important progress in inducing long-term graft tolerance.Autophagy is ubiquitous existence and homeostatic mechanism that cells could resist to pressure in eukaryotic cells.Considering that allograft rejection acts as a strong stimulus to host,autophagy would inevitably mediate allotranplantation response in order to maintain the harmonious and stable environment of host.It was reported that autophagy was involved in various regulation of T cells by affecting their activation,proliferation,survival and the secretion of cytokines.Recently study showed that autophagy could participate regulation of T cells function from many fields,and affect T cells activation and survival,and influence cytokines expression.The effect of autophagy on allograft rejection is mainly through regulating the number and activity of Treg cells.Rapamycin was used as an immunosuppressive drug to regulate the immune response and induce allograft tolerance by inhibiting T cells proliferation,altering cytokine secretion and modulate immune response.Rapamycin could stimulate various cytokines production and exhibit a variety of effects in different disease.Rapamycin was found to act as an inducer of autophagy activation.Cytokines are important immune molecules that regulate cell activation,differentiation and function,and play a crucial role in graft tolerance induction.Nevertheless,whether autophagy activation result in the changes of allorejection,the varieties of cytokine expression and the impact of allograft survival,have not been reported yet.3MA was widely used in fundamental research to depress the formation of autopagysome by selectively inhibiting PI3K.It is urgent to clarify that whether immunosuppress effect induced by rapamycin is influenced after autophagy inhibition and how its molecular mechanism was.In recent years,studies have shown that Toll like receptor 5(TLR5)specific activator flagellin can prolong the survival of allograft and increase the number of Treg cells.TLR5 was closely related to transplantation tolerance on account of its higher expression under rapamycin induced allotolerance.As rapamycin induces tolerance meanwhile increases autophagy,it is greatly significant to research whether autophagy has impact on the effect of targeting imaging of TLR5 on transplantation rejection.In a word,our study investigated the effects of rapamycin on the survival condition of allograft and the effective mechanism of autophagy on allograft tolerance in two ways.Firstly,we investigated the survival environment of allograft and the effects of immune cells under the application of rapamycin and 3MA,meanwhile investigating the autophagy molecular expression and modification.Secondly,we studied whether autophagy influence the effect of targeting imaging of TLR5 on acute transplantation rejection with allotransplanted mice models.Part One Impact and molecular mechanism of autophagy modulation on allograft toleranceMETHOD1.Establishment of skin transplantation model:Skin transplantation model was established and divided into Control(Ctrl)group,rapamycin(Rapa)group,3MA group,rapamycin plus 3MA(Rapa+3MA)group and treated with rapamycin and 3MA respectively.2.Cytokine secretion of allograft under rapamycin treatment:Alloskin was separated on day 4 and day 12 after transplantation and cytokines level were determined by RT-PCR.3.Autophagy molecular expression:Splenic CD4+T and CD8+T cells from Control group and Rapa group were extracted on day 4 and day 12 post transplantation and detected autophagy molecule expression with Western Blot.Splenic CD4+T cells were isolated from Control group and Rapa group on day 12 and treated with 3MA ex vivo for 8h in incubator.Western Blot was used to detect the expression of autophagy molecule.Splenic CD4+T and CD8+T cells were extracted from Control group,Rapa group,3MA group and Rapa+3MA group after transplantation on day 12,and were determined autophagy molecule expression by RT-PCR.4.Cytokines detection in rejection period:CD4+T cells were extracted from mice spleen in the four groups on day 12,cytokine was detected by RT-PCR.Mice serum were collected and IL-10 and IFN-? level were detected by ELISA.5.Treg cell assay:CD4+T cells were extracted from spleen of mice in the four groups on day 12,and Foxp3 and TGF-? were detected by RT-PCR.The spleen cells of model mice were separated on day 12 and the ratio of Treg cells were analyzed by flow cytometry.6.Cell proliferation assay:Mixed lymphocyte culture was performed and spleen cells of BALB/c mice were used as effector cells,C57BL/6 mouse spleen cells acted as stimulated cells.Cell suspensions were treated with rapamycin and 3MA and then tested proliferation of effector cells.7.Graft survival:Graft survival were recorded after transplantation and survival condition was observed.RSULTS1.The cytokine expression of allograft:Higher Thl type(IL-2,IFN-?),Th2 type(IL-4,IL-10),Th17 type(IL-17a)cytokine were found on day 12 than day 4 post transplantation.Rapamycin treatment led to lower Thl type(IL-2,IFN-?)and higher Th2 type cytokine(IL-4,IL-10),Th17 type cytokine(IL-17a),TGF-? on allograft on day 12.2.The expression quantity and feature of autophagy in allotolerance:Beclinl and LC3?/1 expression of CD4+T cells in Rapa group were higher than Control group both on day 4 and day 12(P<0.05,vs.Control).Beclinl and LC3?/?expression of CD8+T cells in Rapa group were higher(P<0.05,vs.Control)on day 12.Higher Beclin 1 expression of CD4+T cells were detected in Rapa group than Control group(P<0.001),while 3MA treatment decreased this effect(P<0.01,vs.untreated group).ATG5 expression of CD4+and CD8+T cells were apparently up-regulated in group of Rapa(P<0.001,P<0.05 vs.Control)and obviously decreased in the group of Rapa plus 3MA(P<0.01,P<0.05 vs.Rapa alone).3.The expression of cytokines:The results showed higher IL-4,IL-10(Th2 cytokine)and lower IFN-y(Thl cytokine)in splenic CD4+ T cells(P<0.05,P<0.001,vs.Control group),and higher IL-10 and lower IFN-y in recipient mice serum in Rapa group(P<0.01,P<0.01,vs.Control group).IL-4,IL-10 of CD4+T cells and IL-10 in serum decreased significantly in group of Rapa plus 3MA(P<0.01,vs.Rapa alone).4.The expression of Treg and Treg-related cytokine:Foxp3 expression,TGF-P expression and CD25+Foxp3sTreg(Regulatory T cells)proportion were remarkably increased in Rapa group than Control group,while 3MA application inhibited Foxp3 expression and the Treg proportion than Rapa group.5.The proliferation of spleen cells:3MA and Rapa alone or combined together could inhibit proliferation of splenic cells(P<0.01 vs.Control).However,proliferation was increased in group of Rapa combined with 3MA(P<0.05 vs Rapa group).6.Allograft condition:Rapamycin administrated in vivo apparently prolonged allograft survival(P<0.01,vs.Control),and 3MA administered alone did not change the survival.3MA combined with Rapa administration kept the Rapa prolonged effect along with soft,well appearance of alloskin grafts.Part two Impact of autophagy on targeting imaging of allotransplantation with 125I-anti-TLR5METHODS1.The preparation and identify of radionuclide probe:Prepare 125I-anti-TLR5 by Iodogen method and analyze the purity and stability of 125I-anti-TLR5.2.Specific combination in vitro assay:Spleen cells were separate from BALB/c mice and treated with rapamycin and 3MA in vitro.Cell uptake and dissociation assays of 125I-anti-TLR5 were analyzed.3.Establishment of skin transplantation model:Skin transplantation model was established and divided into Control(Ctrl)group,rapamycin(Rapa)group,3MA group,rapamycin plus 3MA(Rapa+3MA)group and treated with rapamycin and 3MA.4.Biodistribution assay:125I-anti-TLR5 Ab was injected into the tail vein of the model mice on day 9 post transplantation.At 72h after injection,the biodistribution was measured to calculate the target and non-target(T/NT)ratio.5.Whole-body phosphor-autoradiography assay:At 24h,48h,72h after injection of 125I-anti-TLR5,the whole-body phosphor-autoradiography of was performed to analyze radioactive activity ratio.6.HE and immunohistochemical staining assay:HE and immunohistochemical staining examination of Beclin 1 and TLR5 in allograft were executed on day 12 post transplantation.RESULTS1.Purity and Stability of 125I-anti-TLR5:125I-anti-TLR5 was successfully obtained with good radiochemical purity(95.88%)and stability(over 90%before 72h).2.Combination and dissociation of cells and TLR5:The uptake and dissociation of tracer was not apparently changed with the treatment of Rapa plus 3MA compared with the Control group.3.Biodistribution in mouse model:Biodistribution studies showed mouse model metabolize 125I-anti-TLR5 mainly through liver and kidney,and skin graft have highest radioactivity count.The T/NT ratio of Rapa group(13.25)was higher than Control group(4.32)and Rapa+3MA group(6.46).4.The whole-body phosphor-autoradiography:The whole-body phosphor-autoradiography showed that clear skin grafts imaging in Rapa group and Rapa+3MA group were observed after 48h.Radioactivity ratio showed that at 48h and 72h,Rapa group(2.52,5.33)and Rapa+3MA group(2.22,2.46)significantly increased than Control group(1.30,1.46).At 72h,Rapa group(5.33)apparently increased than Rapa+3MA group(2.46).5.HE and Immunohistochemical staining:Allograft skin HE staining showed that severe inflammatory infiltration was observed in Control and 3MA group than Rapa group and Rapa+3MA group.Inflammatory was not significantly change in Rapa +3MA group than Rapa alone.Immunohistochemical staining of Beclinl and TLR5 showed same expression tendency.Rapa group showed more Beclinl and TLR5 expression,but Rapa+3MA group showed less variation.CONCLUSIONS1.Thl type cytokine expression of allograft in allorejection period was significantly increased than nonspecific inflammatory term in early transplantation.Rapamycin treatment as an autophagy inducer in vivo could suppress the expression of Thl type cytokine and lead higher Th2 and Th17 type cytokine expression.2.Autophagy participated in rapamycin induced allotolerance regulation.Under rapamycin application,autophagy inhibition could not change the capacity of rapamycin to induce tolerance.And autophagy could promote Th2 predominant along with higher Treg and Foxp3 expression to favor allotolerance underrapamycin application.3.Autophagy has no significant effect on 125I-anti-TLR5 targeted allograft rejection under rapamycin application.125I-anti-TLR5 could be applied to targeted imaging of allotransplantation under tolerance.Our studies have great significance about supplying a foundational theory to clarify the impact of autophagy on allorejection and experimental evidences for allograft survival targeted detection by TLR5.INNOVATIVE POINTS1.In vivo treatment of autophagy inducer rapamycin could lead to cytokine variation of allograft and result in lower Thl type cytokine and higher Th2 and Th17 cytokine expression.2.Autophagy participates modulation of rapamycin induced tolerance and autophagy inhibition showed no significantly impact on rapamycin induced tolerance.Autophagy favor allotolerance through promoting Th2 predominant along with higher Treg and Foxp3 expression under Rapa application.3.Autophagy inhibition has no remarkable effect on TLR5 targeted imaging of graft under rapamycin appliance.125I-anti-TLR5 could be used to targeted image of allograft.