The Experimental Study On Chemokine Receptor CCR5 Inhibition To Protect Cardiac Allografts In Mice And Primates

PartⅠThe experimental study on CCR5 antibody to prolong cardiac allograft survival in miceObjective: To study the effect of CCR5 monoclonal antibody in protecting cardiac allograft in mice.Methods:Hearts from BALB/c mice were transplanted into C57BL recipients. They were administrated with CCR5 mAb or control antibody and Cyclosporine or PBS, respectively. 8 animals in CCR5 mAb plus CsA treated group were sacrificed on day 18 post-transplantation. The Treg cells in allografts were analyzed by flow cytometry and the levels of foxp3,IL-2,IL-10,TGF-βand INF-y mRNA were assessed by Q-PCR method. Histopathologic and immunohistochemical study were used to study the allograt rejction and the infiltration of CCR5+,CD4+,CD8+ and CD4+CD25+Foxp3+ T cells, respectively.Results:As compared with recipients treated with control Ab plus PBS, allografts treated with CCR5 mAb and Cyclosporine showed significantly prolonged survival (> 90d, P<0.001), markedly decreased CD4+ and CD8+ T cells (P<0.005) and increased frequency of CD4+CD25+Foxp3+ regulatory cells (23.98±1.55% vs 6.30±0.57%, P<0.005). The expression of IL-2, INF-y mRNA in CCR5 mAb plus CsA-treated allograft is much fewer than other three control groups. On the contrary, the expression of IL-10 and foxp3 mRNA was significantly increased in CCR5 mAb plus CsA-treated group. There was also an increased expression of TGF-βmRNA in CCR5 mAb plus CsA-treated group on day 18, but on day 90 it decreased to the level of control groups.Conclusions:CCR5 blockade in combination with Cyclosporine is effective in protecting cardiac allograft in a robust murine model. PartⅡThe experimental study on CCR5 antagonist Maraviroc to protect cardiac allograft in primatesObjective:To study the effect and mechanism of CCR5 antagonist Maraviroc (MVC) alone or in combination with Cyclosporine (CsA) in prolonging cardiac allograft survivals.Methods:Rhesus monkey cardiac allograft models were established and randomized into four groups. MVC + CsA group (MVC/CsA), MVC group, CsA group and Control group. The cardiac allografts in MVC + CsA group were biopsied on days 9,30 and 45 and harvested on day 240 post-transplantation. Both the cardiac allografts treated with MVC alone and with CsA alone were biopsied on day 9. The immunohistochemical method was used to study the expression of C3d, CCR5, CD4, CD8, CD68, Arg1, Mrc1 and PPARy in allograft. And flow cytometry method was used to assess the level of alloantibody and the ratio of T subsets including Th1, Th2, Th17 and Treg in peripheral.Results:In an established rhesus monkey cardiac allograft model, recipients in MVC/CsA group showed significantly prolonged survival of heart allografts (>240 days, P<0.001). These in vivo results in the MVC/CsA group correlated with delayed alloantibody response,markedly decreased graft infiltration by CCR5+, CD4+, CD8+ and CD68+ cells (P<0.05),low numbers of pro-inflammatory Thl and Thl7 cells while increased numbers of Th2 and Treg cells, as compared with other groups. Furthermore, grafts from MVC/CsA group had elevated numbers of alternatively activated macrophages (AAM) and the expression of peroxisome proliferator-activated receptorγ(PPARy).Conclusion:MVC/CsA is effective in protecting cardiac allograft in primates. PartⅢMVC/CsA generated AAM dependent on activation of PPARyObjective:To investigate the ralationship among MVC/CsA, AAM and PPARyMethods:in vitro MLR, MVC/CsA was added to investigate their influence on expression of AAM markers including Argl and Mrcl, in the control group, additional PPARy antagonist BADGE was added to investigate its role in MVC/CsA generating AAM. We also used in vivo experiment to assess the expression of AAM in cardiac allograft with add of BADGE. Next, we investigated the effect of MVC/CsA on generating Th2 cytokines IL-4 and IL-13, and then we assessed the expression of PPARy, Argl and Mrcl with add of IL-4 and IL-13.Results:Both in vitro and in vivo experiments domenstrated that MVC/CsA could generating AAM, and the generation of AAM by MVC/CsA was abrogated with use of PPARy antagonist BADGE. In addition, MVC/CsA could promote the expression of Th2 cytokine IL-4 and IL-13, and then IL-4 and IL-13 could activate PPARy and generating AAM.Conclusion:MVC/CsA promoted the expression of IL-4 and IL-13 which could activate PPARy and generate AAM.