Phosphorylated STAT3 Regulate The Balance Of Treg/Th17 At Maternal-fetal Interface In Unexplained Recurrent Spontaneous Abortion

Part One The characteristics of phosphorylation of STAT3,the profiles of T cells and related cytokines in the maternal-fetal interface of the unexplained recurrent spontaneous abortion patientsObjective:Comparison of the characteristics of phosphorylation of STAT3,the proportion of Th17 cells and Treg cells,the concentrations of IL-17A and IL-10 between unexplained recurrent spontaneous abortion(URSA)patients and normal pregnancy women.Methods:Collected the decidual tissue of URSA patients and normal pregnancy women.The populations of pSTAT3~+T cells,Th17 cells and Treg cells were detected by flow cytometry.The concentrations of IL-17A and IL-10in decidual tissue were detected by enzyme linked immunosorbent assay(ELISA).Results:The percentage of Th17 cells was 11.57±1.17%in URSA patients which was elevated(P<0.001),compared the percentage of Th17 cells in normal pregnancy women was 4.37±0.73%.The proportion of Treg cells in URSA patients was 8.45±0.94%which was decreased(P=0.004),compared the percentage of Th17 cells in normal pregnancy women was 13.75±1.49%.The result of Pearson correlation analysis between Th17 and Treg cells showed that there was a significant negative correlation(r=-0.876,P<0.001).The percentage of pSTAT3~+T cells in normal pregnancy women was 36.62±3.03%,and the percentage in URSA patients was 56.37±4.42%.There was a significant differential as P value was 0.0001.There was a negative correlation between Treg cells and pSTAT3~+T cells(r=-0.856,P<0.001),meanwhile the percentages of Th17 cells was positive correlated with the pSTAT3+T cells(r=0.883,P<0.001).Compared to the normal pregnancy women,the concentrations of IL-17A in the decidual tissue of URSA patients were much higher(P<0.001),and the concentrations of IL-10 were much lower(P<0.001).There was a negative correlation between IL-10 concentration and pSTAT3~+T cells(r=-0.717,P<0.001),meanwhile the concentration of IL-17A cells was positive correlated with the pSTAT3~+T cells(r=0.754,P<0.001).Conclusions:The pSTAT3 plays a role in the pathogenesis of URSA,probably through the regulation of the balance between Th17 cells and Treg cells.Over-activation of STAT3 leads to increased Th17 proliferation and secretion of IL-17A,and inhibition of Treg cells proliferation and secretion of IL-10.The level of pSTAT3 of T cells was positively correlated with the number of Th17 cells and the concentration of IL-17A,which was negatively correlated with the number of Treg cells and the concentration of IL-10.Part Two Impaired function of regulatory T cells in patients with URSA is mediated by phosphorylation of STAT3Objective:To Investigate the possible role of STAT3 pathway in the pathogenesis of dysfunctional Tregs in URSA.Methods:The suppressive function and the proliferative activity of Tregs were detected from URSA patients and normal pregnancy women.Expression of phospho-STAT3 in URSA Tregs was evaluated by flow cytometry.Furthermore,Tregs were treated with Stattic(STAT3 inhibitor V)to investigate the role of STAT3 pathway in the function of Tregs.Quantitative Real-Time PCR(qRT-PCR)was performed to detect the expression of STAT3,STAT5 and Foxp3 mRNA in Treg cells.The supernatant concentrations of IL-10 and TGF-?1 were detected by ELISA.In addition,IL-6,IL-17 and IL-23 treatments were performed to identify the upstream molecules of STAT3 pathway in Tregs.Results:Tregs from decidual tissue of URSA patients showed decreased suppressive function and poor proliferation activity.There was 86.20±2.09%Tregs proliferated in normal pregnancy women,while the percentage was76.36±3.99%in URSA patients(P<0.01).And the suppression ratio was10.15±1.37%in normal pregnancy women and 3.35±1.43%in URSA patients.There was a significant difference between two groups(P<0.001).The percentage of pSTAT3~+Treg cells was elevated in URSA group which could be decreased by Stattic.Compared to URSA group,the proportion of pSTAT3~+Treg cells in normal pregnancy group was much lower(65.76±5.04%VS 42.4±4.59%,P<0.001).And this proportion of URSA+Stattic group(38.9±4.76%)was much lower than that in URSA group(P<0.001).Additionally,Stattic could repair the suppressive function and proliferation activity of Treg cells.After 72hours cultivated,82.24±3.12%Treg proliferated in URSA group,the proportions in URSA+Stattic group was 88.98±3.27%.There is significant difference between two groups(P<0.01).And the suppression ratio of Tresp+Treg+Stattic group was 72.42±2.89%,which was much higher than Tresp+Stattic group(22.94±3.47%)and Tresp+Treg group(59.77±3.13%).There were significant differences in three groups and pairwise comparison(all P<0.001).Phosphor-STAT3 inhibited the proliferation of Treg cells through downregulated the expression of STAT5 mRNA and Foxp3 mRNA.After Stattic treatment,the expression levels of STAT5 mRNA and Foxp3 mRNA were increased.Furthermore,phosphor-STAT3 inhibited the secretion of TGF-?1 and IL-10.Proinflammatory cytokines,IL-6 and IL-23,could induce the phosphorylation of STAT3.however,IL-17A did not show the effect of regulation the phosphorylation of STAT3.Conclusion:Our finding suggests that the over-activation of STAT3 impaired the functions of regulatory T cells which plays a vital role in the pathogenies of URSA.The Treg cells insolated from the decidual tissue of URSA patients experience a predominant STAT3 phosphorylation by exposure to pro-inflammatory cytokines,leading to their impaired functions in suppressing Tresp and proliferation activation.Part Three Prednisone may induce immunologic tolerance by activating the functions of decidual immune cells in early pregnancyObjective:The objective of this study was to investigate alterations of human first trimester decidual immune cells and relative cytokines by treated with prednisone.Methods:The decidual lymphocytes were divided into four groups, Control,Prednisone,Cytokines and Prednisone+Cytokines groups.After 72hours'cultivation,the mRNA expression level of STAT3,STAT5,RORC and Foxp3 were examined by RT-PCR.The quantities of Th17,Treg and pSTAT3were tested by flow cytometry,and the concentration of IL-17A and IL-10 were detected by ELISA.Results:After prednisone treatment,the expressions of STAT5 and Foxp3 were increased,and the transcription of RORC was inhibited.The STAT3mRNA expression did not show significantly differences between the four groups.Compared to Control group,the Treg cells proportion of the Prednisone group were increased accompanied by a decreasing of Th17 cells.After prednisone treatment,the percentage of CD4~+pSTAT3~+cells are significantly decreased than that in control group and prednisone+cytokines group.There is a negative correlation between CD4~+pSTAT3~+cells and Treg cells,meanwhile a positive correlation between CD4~+pSTAT3~+cells and Th17 cells.Levels of IL-10 correlated positively with prednisone treatment,and the drug decreased the secretion of IL-17A.There is a negative correlation between CD4~+pSTAT3~+cells and the concentration of IL-10,meanwhile a positive correlation between CD4~+pSTAT3~+cells and the concentration of IL-17A.Conclusions:Prednisone could induce the expression of STAT5 and Foxp3 through inhibiting the phosphorylation of STAT3 to facilitate the differentiation of Treg cells and inhibit the differentiation of Th17 cells,with reduction of IL-17A secretion and increasing the secretion of IL-10,thus to rebuild the immunologic homeostasis.