Study The Mechanism For Up-regulation Of CD81 In Trophoblasts Triggers The Imbalance Of Treg/Th17 Leading To Preeclampsia

Background:Preeclampsia(PE)is a main cause of maternal and fetal morbidity and mortality.The disturbance of maternal immune tolerance to semi-allogeneic fetus is recognized as one of the key pathologies of preeclampsia(PE),in which an imbalance between the inflammation-limiting regulatory T cells(Treg)and the inflammation-mediating Th17 cells plays an essential role.Previously we reported that the abnormal upregulation of tetraspannin CD81 in trophoblasts cells(fetal component)participated in the pathogenesis of PE.However,as one of the potential immune regulatory molecules,whether CD81 induces PE by interfering the balance of the maternal immune system need to be clarified.Objective:To investigate the molecular mechanism that CD81 triggers maternal Treg-Th17 immune imbalance leading to the pathogenesis of pre-eclampsia.Methods:1.We investigated the relationship between upregulation of CD81 in trophoblast cells and the imbalance of Treg and Th17 both in the decidua and peripheral blood of pregnant women with severe early-onset preeclampsia by q-PCR,immunohistology,ELISA and FACS.2.In vitro culture of'naive T cells with medium from CD81-overexpressing trophoblast cell line HTR-8 cells,then detected the proportion of Treg or Th17 cells differentiated from naive T cells by FACS.Also we detected the expression of FOXP3 and RORC in the differentiated T cells by q-PCR.3.In vitro study further explored the mechanism that overexpression of CD81 in trophoblasts inhibits Treg cells but promotes Th17 cells differentiation.The supernatant from AdCTL and AdCD81 was separated into exosomes and exosome-free culture medium by ultracentrifugation.These were used to treat nai've T cells separately,then detected the proportion of Treg and Th17 cells.Recombinant human CD81 protein was also used.As CD81 functioned through paracrine pathway,we detected the interleukins in HTR-8 cells after AdCD81 transfection by q-PCR and ELISA.Further,we also used neutralization antibody to block the effect of IL-6,then detected the proportion of Treg and Th17 cells by FACS.4.The adenovirus AdCD81 was injected into rats through tail vein in gestational day 5th(GD5),then we detected the blood pressure,urine protein,fetal weights,the proportion of Treg and Th17 cells in decidua and peripheral blood.And then we used the neutralizing antibody of IL-6 through intraperitoneal administration,and detected the phenotype of PE and balance of Treg/Th17.Results:1.We demonstrated that upregulation of CD81 in trophoblast cells was accompanied by decrease of Treg and increase of Th17 in both decidua and peripheral blood of patients with PE.2.In vitro culture of naive T cells with medium from CD81-overexpressing trophoblast cell line HTR-8 cells resulted in enhanced differentiation of T cells into Th17 cells and dampened formation of Treg,which was dependent on the paracrine of IL-6 in trophocytes induced by CD81.3.In CD81-induced PE rat model,we found a significant shift of T cell differentiation towards Th17 cells and administration of IL-6 antibody mitigated PE phenotype and the imbalance of Treg/Th17.Conclusions:These results define a vital regulatory cascade involving trophocytes-derived CD81,IL-6 and maternal Treg/Th17 in pathogenesis of PE and suggest new therapeutic approaches based on CD81 and IL-6 down-regulation to prevent PE.