The Effect Of Cyclin-dependent Kinase 9 On Allotransplantation Rejection And Molecular Mechanism

Organ transplantation is still the most effective therapy for end-organ failure.There are three types of organ transplantation,including allogeneic transplantation,xenotransplantation and autotransplantation.Among these,allogeneic transplantation is the most common type.Allotransplantation rejection is a complex pathophysiological process.A large number of genes interactions forming gene regulatory networks are involved in the occurrence and development of allorejection,which is the threat of graft survival and the main cause of treatment failure.Recently,immunosuppressive therapy has made significant progress in short-term graft survival,but long-term survival has not been apparently improved.At present,the common clinical immunosuppressants could not eliminate persistent immune injury caused by complex gene regulation,while the immune inhibition seriously affected patients life.These are the key challenges in current clinical organ transplantation practice.Therefore,it is urgent to find a new strategy that effectively suppresses de novo inflammation by targeting many more primary response genes in allografts and design a higher selectivity inhibitor.CDK9?Cyclin-dependent kinase 9?,a component of the positive transcription elongation factor b?P-TEFb?kinase,phosphorylates the RNA polymerase ?C-terminal domain and negative transcription elongation factors to start transcription,is the critical speed limitation in the process of cell transcription elongation.Primary response genes?PRGs?are a set of genes that are induced in response to both cell-extrinsic and cell-intrinsic signals and do not require de novo protein synthesis for their expression.These 'first responders' in the waves of transcription of signal responsive genes play pivotal roles in a wide range of biological responses,including neuronal survival and plasticity,cardiac stress response,innate and adaptive immune responses and glucose metabolism.Accumulating evidences support a central role for CDK9 in monitoring the activation of primary inflammatory response genes to control inflammatory processes.There are two isoforms of CDK9,CDK9₄₂ and CDK9₅₅,which have different distribution in organs,tissues and cells.Recent studies have shown that in vitro cultured rat hepatocytes,with the increased incubation time,the expression of CDK9₅₅ decreased gradually,and that of CDK9₄₂ gradually increased.In view of the central regulatory role of CDK9 in inflammation,the molecular mechanism of CDK9 on allograft rejection was studied in this paper.The research purposes are:to clarify the effects of CDK9 inhibitors on allograft survival,and to explore the related signaling pathways regulating factors of the undergoing mechanism,and the effect and influence of the alternative expression of CDK9 two isoforms during allorejection,so as to study the induction of immune tolerance by targeting CDK9 specific isofrom to provide theoretical and experimental basis for monitoring and treatment,and provide a new strategy for clinical organ transplant rejection.Part One Study on the relationship between grafts survival and the expression of CDK9 and Cyclin T1 in allorecipientsMethods1.Dynamic expression of CDK9 and Cyclin T1 during allograft rejection1.1 Establishment of murine skin isograft and allograft modelsUnder sterile conditions,preparation of full thickness skin of BALB/c and C57BL/6 mice.Grafts were transplanted into BALB/c mice with bandage to be as isograft group and allograft group respectively.The allotransplantation group was divided into PHA747691?CDK9 inhibitor?treatment group and normal saline control group:the experimental group by administration of PHA767491?3,10,30mg/kg?to allotransplantation mice at day-1,1,3,5,7,9,11 and normal saline group as control group.On day4,8,12 and 16 after transplantation,all the mice were sacrificed and skin grafts and spleens were isolated.1.2 Expression of CDK9 and its chaperone Cyclin T1 in the graftsIsograft and allograft group skin were isolated and total RNA were harvested on day 4,8,12 and 16 post transplantation.The mRNA levels of CDK9 and its chaperone Cyclin T1 were detected by RT-qPCR.2.Impact of CDK9 inhibition on the alternative expression genes of CD4⁺ T cells in allotransplantation recipientsThe control group mice were sacrificed at the peak of rejection,and the spleen cells were prepared.CD4⁺ T cells were isolated and purified by CD4⁺ T Cell Isolation Kit,and then treated with 3 ?M PHA767491 or equal volume PBS.A number of genes were validated from a CD4⁺ T cell-mediated allorejection SAGE?serial analysis of gene expression?library previously published by our lab,the expression of these genes in allorecipients splenic CD4⁺ T cells after inhibiting CDK9 was detected by RT-qPCR.3.Impact of CDK9 inhibition on survival of allograftsThe growth status and the rejection of skin grafts were observed in the mice of the allorejection group on day 7 post transplantation,and the rejection percentage and survival of the grafts were recorded.The 90%necrosis of the skin graft was considered as a complete rejection.The control group of mice skin allograft rejection occurred at day 12.On the same day,all the mice were sacrificed by cervical dislocation,removed the complete donor grafts and recipients bed tissues,fixed in 10%neutral formalin and paraffin-embedded.Sections were prepared to observe skin graft pathology and CDK9 expression with HE staining and immunohistochemistry assay,respectively.4.Effects of CDK9 on the frequency of splenic CD4⁺ T cells in allorecipientsThe spleens were collected on day 14 after transplantation,and the splenocytes were isolated.The level of CD4⁺ T cells was detected by flow cytometry under CDK9 inhibition.5.Effects of CDK9 on CD4⁺ T cells in allograftsAll the mice of allotransplantation were sacrificed on day 12 post transplantation.Removed donor grafts and recipients bed tissues,fixed in 10%neutral formalin and the paraffin sections were prepared to observe CD4 expression in allografts with immunohistochemistry assay.Results1.There was a significant positive correlation between CDK9 expression and allograft rejection:the expression of CDK9 and its chaperone protein Cyclin T1 in allograft group were significantly higher than that of isograft group.The expression level of CDK9 reached the highest at the peak of allorejection,and decreased with the rejection resolution.The expression of Cyclin T1 and CDK9 were consistent in the the occurrence and development of rejection.This suggests that CDK9 expression is positively correlated with allorejection.2.CDK9 regulated allorejection related genes of CD4⁺ T cells:Because CD4⁺ T cell determined whether allograft rejection occurs or not,in this paper,we selected several allorejection-related genes from a CD4⁺ T cell-mediated allorejection SAGE library previously established by our lab,and detected the role of CDK9 on these genes.The results showed that compared with the control group,the mRNA levels of STAT1,JAk2,Socs5,RhoA,Ly6e,Med8 and Medll were significantly decreased in PHA767491-treated group,while the levels of Gna13 and Fbxw4 were obviously increased,which indicated that CDK9 up-regulated CD4⁺ T cells-mediated allorejection related genes and down-regulated transplantation tolerance genes.3.CDK9 inhibitor significantly prolonged allograft survival:Isograft group without rejection.The skin allograf't graft in control group of mice appeared early hardening,crusting,shrinkage,after unpacking,it was completely excluded.Compared with the control group,skin allografts had good growth in the PHA767491 group.The rejection was weak,the rejection process was slow,and the skin graft survival was significantly longer than that of the control group?p<0.05?.The results indicated that CDK9 inhibitor significantly prolonged the survival time of skin allograft and improved the survival condition of the graft.4.Pathological and immunohistochemical staining results:skin graft tissue in control group mice showed obviously necrosis,graft loose connections with recipient tissue,and a large number of mononuclear cell infiltration;while PHA767491 treatment group mice showed no obvious necrosis of skin graft,graft and recipient tissue were closely connected and the infiltration of inflammatory cells was slight.The results showed that the expression of CDK9 was positively correlated with the severity of allorejection,and that CDK9 inhibitor could protect allografts from rejection.5.CDK9 inhibitor significantly inhibited the frequency of CD4⁺ T cells:the number of CD4⁺ T cells in allograft control group was significantly higher than that in isograft group,PHA767491 can significantly inhibit high expression of CD4 caused by alloresponse.Immunohistochemical staining result showed that PHA767491 significantly inhibited CD4⁺ T cells infiltration in allografts.This result further confirmed that CDK9 promotes allorejection.Part Two Effects of CDK9 inhibitors on CD4⁺ T cells-mediated allorejectionMethods1.Establishment of CD4⁺ T cells adoptive transferred-C57BL/6-SCID skin allograft mice modelUnder sterile conditions,full thickness skin grafts of C57BL/6 mice were transplanted to SCID mice and bandages were removed on day 7.Grafts healed well 3 weeks post transplantation,indicating SCID murine skin allograft model was successfully established.CD4⁺ T cells were isolated from BALB/c mouse spleen by CD4⁺ T Cell Isolation Kit,and treated with PHA767491?3?M?and PBS for 6 hours respectively.And then,these cells were intraperitoneal injected to SCID allograft recipients respectively,.These mice were devided into PHA767491 treatment group and control group.5.Impact of CDK9 inhibition on the survival of allograft mediated by CD4⁺ T cellsAfter adoptive transfer,growth and rejection of the two groups of C57BL/6-SCID mice skin grafts were daily observed,and the percentage of survival was recorded.At the day that allorejection occured in control group?adoptive transfer of cells at day 12?,all the mice were sacrificed by cervical dislocation,complete donor grafts and recipients bed tissues were harvested.And then fixed grafts in 10%neutral formalin and paraffin sections were prepared to observe skin graft pathology by HE staining.6.Effect of CDK9 inhibition on CD4⁺ T cells mediated-allorejection3.1 Effect of CDK9 inhibition on cytokines profile of CD4⁺ T cellsAt day 12 after adoptive transfer,two groups of mice were sacrificed to obtain C57BL/6-SCID mice skin graft,axillary draining lymph nodes and spleen.Splenocytes suspension was prepared to isolate CD4⁺ T cells using CD4⁺ T Cell Isolation Kit,and then the levels of IFN-?,IL-4,IL-10,IL-17 and IL-22 in skin graft,axillary lymph node and spleic CD4⁺ T cells were detected by cytokine protein chip.3.2 CDK9 inhibition on the activation of alloreactive Teff cellsAt day 7 after adoptive transfer,alloreactive spleic CD4⁺CD25-Teff cells were isolated,and then the levels of CD69,CD25 and intra-cellular IL-2 were detected by flow cytometry.3.3 CDK9 inhibition on the proliferation of Teff cellsTeff cells were isolated from wild type BALB/c mice splenocytes,and then cells were treated with PHA767491?3?M?or equal amount of PBS respectively for 2 hours.After that the anti-CD3 antibody?2 ?g/ml?and anti-CD28 antibody?1?g/ml?were added to stimulate the cells,and the proliferation of Teff cells was detected by CCK-8 assay.7.CDK9 inhibition on CD4⁺CD25+Foxp3+ T cells4.1 Impact of CDK9 inhibition on alloreactive CD4⁺CD254+Foxp3+ Tregs frequencyAt day 12?allorejection occured in control group?,all the mice were sacrificed,splenocytes suspension was prepared.And then cells were stained with anti-CD4,CD25.Foxp3 monoclonal antibody and the frequency of CD4⁺CD25+Foxp3+ Treg cells in CD4⁺ cells proportion was detected by flow cytometry.4.2 CDK9 inhibition on the proliferation of TregsCD4⁺CD25+ Treg cells were isolated from wild-type BALB/c mice splenocytes.Treg cells were treated with PHA767491?3?M?or equal volume of PBS for about 2 hours,and then the anti-CD3 antibody?2 ?g/ml?and anti-CD28 antibody?1 ?g/ml?were added to stimulate cells,and the proliferation of Tregs was detected by CCK-8 assay.4.3 CDK9 inhibition on the suppressive capacity of TregsCD4?CD25+ Tregs and CD4⁺CD25-Teffs were isolated from wild-type BALB/c mice splenocytes.Treg cells were treated with PHA767491?3 ?M or 5?M?or equal volume of PBS for about 2 hours,and then the anti-CD3 antibody?2?g/ml?and anti-CD28 antibody?1 ?g/ml?were added to stimulate the co-cultured cells,and the suppressive capacity of Tregs on Teffs was detected by CCK-8 assay.Results1.Successful establishment of allogeneic transplantation model:3 weeks after transplantation,skin grafts of C57BL/6-SCID mice were in good condition,which indicated the successful establishment of SCID mouse skin allograft model.The control group of C57BL/6-SCID mice skin grafts rejected in subsequent 12-15 days,and PHA767491 treatment group rejected on 23-27 days.These results suggest that CDK9 inhibitor weakens CD4⁺ T cells-induced-rejection and prolongs the survival of allograft.2.CDK9 promoted Thl polarization:compared with the control group,the levels of IFN-y were significantly decreased,while the levels of IL-4 and IL-10 were obviously increased in the skin graft and splenic CD4⁺ T cells of PHA767491 group.The changes of cytokine level were not detected in draining lymph nodes.There was no significant difference of IL-17 and IL-22 at three sites between two groups.These results suggest that CDK9 enhances the rejection of by enhancing the Thl polarization.3.CDK9 promoted the activation and proliferation of alloreactive Teff cells:in order to study whether the CDK9 promote allograft rejection from cell activation stage,we isolated recipients spleen Teff cells at day 7 post transplantation?Teff started to recognize alloantigens?,flow cytometry results showed that the levels of CD69,CD25 and intracellular IL-2 in PHA767491 treatment group were significantly lower than control group;the proliferation of Teff was also significantly lower than that of the control.These results suggest that CDK9 promotes activation and proliferation of Teff.5.CDK9 elevated the frequency of Tregs in allorecipients:the frequency of CD4⁺CD25+Foxp3+ Treg in PHA767491 treatment group was significantly higher than that in control group.The two groups had no significant difference in the proliferation of Treg,and PHA767491 did not affect the suppressive capacity of Tregs.These results suggest that PHA767491 can significantly increase the frequency of Tregs in allograft recipients by inhibiting the activation and proliferation of Teffs.Part Three Regulation mechanism of CDK9₄₂ and CDK9₅₅ isoforms on allotransplantation rejectionMethods1.Expression of two isoforms of CDK9 with alloantigen stimulationThe spleen cells of wild type C57BL/6 mice were collected,and then soluble antigens were prepared by centrifugation after ultrasonic disruption.Wild-type BALB/c mice CD4⁺ T cells were gradient time stimulated with soluble alloantigens after treated with PHA767491?3?M?or equal volume PBS for 2h,and then detected the expression of CDK9₄₂,CDK9₅₅ and RNA polymerase ? at each time point by Western blot assay.2.The translocation of two CDK9 isoforms with alloantigen stimulationWild-type BALB/c mice CD4⁺ T cells were gradient time stimulated with soluble antigens after treated with PHA767491?3? M?or equal volume PBS for 2h.obtained supernatant,and separated nuclear and cytoplasmic protein.detected expressions of CDK9₄₂?CDK9₅₅ and its partner protein Cyclin T1 at each time point by Western blot and ELISA.3.The translocation of CDK9₄₂ promotes inflammatory micro-enviroment formationIn order to investigate the role of CDK9₄₂ translocation in the extracellular micro-environment,ELISA was used to detect the levels of IFN-?,IL-1? and IL-6 in the supernatant.4.CDK9₄₂ activates STAT1/IFN-? signaling pathway4.1 CDK9 activated allorejection related genes were associated with STAT1CDK9 siRNA was transfected into RAW264.7 cell line.To explore the effects of CDK9 on allograft rejection related signaling pathway,we selected several genes from CD4⁺ T cell mediated rejection SAGE library,and detected mRNA levels of these genes in CDK9 siRNA transfected RAW264.7 cell line by RT-qPCR.4.2 PHA767491 significantly inhibited the activation of STAT1Wild-type BALB/c mice splenocytes were stimulated with soluble alloantigens after pretreated by anti-IL-6 antibody,anti-IFN-? antibody,PHA767491?3 ?M?or equal volume PBS for 2h,and then stained by phosphorylated STAT1 and STAT3 flow cytometry antibody,the activation of STAT1 and STAT3 was analyzed by flow cytometry.4.3 CDK9 promotes allorejection mainly by activating STAT112 days after transplantation,all the mice were sacrificed,skin grafts from recipient BALB/c mice were removed,fixed in 10%neutral formalin,paraffin embedding and these sections were prepared to observe the expression of CDK9,STAT1,STAT3 and STAT5 in skin allografts by immunohistochemical staining.Results1.CDK9₄₂ was the major molecular isoform in response to allorejection:the expression level of CDK9₄₂ was gradually increased,while CDK9₅₅ was significantly decreased,and the expression pattern of RNA polymerase II and CDK9₄₂ was basically accordance.PHA767491 can significantly inhibit the expression of these three proteins.These results suggest that CDK9₄₂ is the major isoform that activates the downstream immune response with alloantigen stimulation.2.CDK9₄₂ translocation promoted the formation of an inflammatory microenvironment:with the stimulation of antigen,CDK9₄₂ was translocated from the nucleus to the cytoplasm and released into the extracellular environment,while CDK9₅₅ was expressed stably in the nucleus.Cyclin T1 also has the corresponding translocation,however,its expression pattern is not consistent with the two CDK9 isoforms.The levels of IFN-y,IL-1? and IL-6 in the control group were significantly higher than those in PHA767491-treated group,and their expression trend was consistent with CDK9₄₂.PHA767491 could significantly inhibit the translocation of CDK9₄₂ and the level of pro-inflammatory cytokines in the supernatant.These results suggest that the translocation of CDK9₄₂ promotes the formation of inflammatory microenvironment.3.CDK9 inhibition reduced STAT1 related gene expression:the mRNA levels of CXCL9,CXCL10,CXCL11,GBP4,STAT1,JAk2 and Socs5 were significantly lower in CDK9-block group than the control group,these genes involved in cell proliferation,apoptosis,cell activation,transcription regulation,signal transduction,cell growth and maintenance,and all associated with STAT1.While STAT3-related genes,including C4a,CXCL12 and Socs3,did not alter significantly as like the genes above.4.CDK9 mainly affected allorejection by regulating the STAT1/IFN-? signaling pathway:with alloantigen stimulation,the activation of STAT1 was significantly higher than that of STAT3,the activation of STAT1 and STAT3 were both significantly decreased in IFN-? block group,while only STAT3 activation was inhibited in IL-6 blocked group,and PHA767491 significantly inhibited STAT1 phosphorylation.These results suggest that STAT1 plays a more important role in promoting inflammation and CDK9 in this process may be mainly through the regulation of STAT1/IFN-? signaling pathway to promote allorejection.5.Immunohistochemical staining results:CDK9 and STAT1 were significantly decreased in the PHA767491 treated mice,and the expression of STAT3 was slightly decreased.There was no significant difference in STAT5 expression between the allorejection group and PHA767491 treatment group.These results suggested that CDK9 promotes allorejection predominantly by activating STAT1.Conclusions1.CDK9 inhibition administration in vivo significantly prolonged survival and improved living condition of murine skin allograft.The expression level of CDK9 in graft was positively correlated with the severity of graft damage.2.CDK9 inhibition administration in vivo significantly increased the frequency of CD4⁺CD25+Foxp3+ Treg cells,significantly inhibited the activation and proliferation of Teff cells,but showed no significant effect on the proliferation and inhibitory capacity of Treg.3.CDK9₄₂ was the major isoform that could respond to the alloantigen stimulation and promote the downstream immune response.It regulated many allorejection related genes and its translocation promoted the formation of an inflammatory environment.CDK9 promoted allorejection predominatly by activating the STAT1/IFN-? signal pathway.Points of Innovation1.For the first time to study the effect of CDK9 on allorejection,found that CDK9 Inhibitor,PHA767491,could prolong skin allograft survival and induce allotransplantation immune tolerance.Our study provides a new therapeutic target and method for clinical organ transplantation.2.It is the first time to prove that CDK9 regulates many allorejection related genes,promotes the occurrence and development of allorejection,and plays a central role in the process.Our study provides new research ideas to further investigate the molecular mechanism of allotransplantation rejection.3.It is the first time to prove that CDK9₄₂ is the major isoform responding to alloantigen stimulation and promotes downstream immune response;its translocation promotes the formation of an inflammatory microenvironment,which are important for the research of higher selective CDK9 inhibitors used in anti-inflammation and anti-rejection.