Pathogenic Conversion From Treg Cells To Th17 Cells In The Circulation Of Women With Pre-eclampsia

Pre-eclampsia(PE) remains to be the major cause that is responsible for the high morbidity and mortality rates of mother and fetus. Each year, about 5~10% of pregnancy women are affected. However, the pathogenesis of PE has not yet been completely elucidated, and the clinical treatment of PE is still being focused on the symptomatic treatment state. Therefore, improving and enriching the pathogenesis of PE has an important significance.In the past decades, considerable evidences have proved the prominent roles that Treg/Th17 cells balance plays in normal pregnancy as well as PE. The deficiency or insufficiency of Treg cells in the frequency and/or function are closely related to the progress and outcome of PE. Th17 cells, by contrast, have an opposite effect in PE compared with Treg cells. Recently, accumulating data have demonstrated that Treg cells pose high plasticity and can convert to Th17 cells, which involve in autoimmune/inflammatory diseases. Whether the plasticity and conversion exist in PE remains unknown.CD4~+Foxp3~+IL-17~+ cells are newly defined subsets of T cell population. They own the characteristics of Treg and Th17 cells, and are the intermediate phenotypes of conversion from Treg cells to Th17 cells.In the present study, we first detect the quantities of CD4~+Foxp3~+IL-17~+ intermediate cells in the circulation of women with PE; then analyze the correlation between the intermediate cells and Treg cells as well as Th17 cells; finally, by in-vitro study, we determine the presence of pathogenic conversion from Treg cells to Th17 cells and the factors involved in the transformation.PartⅠ Quantitative changes of CD4~+Foxp3~+IL-17~+intermediate cells in the peripheral blood of women with pre-eclampsiaObjective: To determine the proportion of CD4~+Foxp3~+IL-17~+ intermediate cells in the peripheral blood of women with pre-eclampsia in-vivo.Methods: Peripheral blood of PE patients(characterized by blood pressure ≥ 40/90 mm Hg on two or more consecutive occasions, measured 4 hours apart from each other and proteinuria ≥0.3 g L-1 or at least 1+ protein by dipstick)(n = 23) was collected in heparinzed tubes. Women with normal pregnancy(NP)(n = 40) with matched age, gestational age and parity were recruited as controls. The peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation. Then PBMCs were stained with m Abs of CD4-FITC, Foxp3-APC and IL-17-PE-Cy7. Finally, the proportion of CD4~+Foxp3~+IL-17~+ intermediate cells within CD4~+ T cells were detected by multicolour flow cytometry assay.Results: CD4~+Foxp3~+IL-17~+ intermediate cells were detected in the peripheral blood of women with both NP and PE. Compared with NP, the percentage of CD4~+Foxp3~+IL-17~+ intermediate cells in PE was significantly higher(P < 0.01). Similarly, the percentage of CD4~+IL-17A+ Th17 cells was also higher in PE compared with NP(P < 0.01). In contrast, the Treg/Th17 ratio decreased significantly(P < 0.01) while the proportions of CD4~+Foxp3~+IL-17~+ intermediate cells occupying in CD4~+Foxp3+ Treg cells and in Th17 cells all increased significantly in PE compared with NP(P < 0.01 each).Conclusion: CD4~+Foxp3~+IL-17~+ intermediate cells are present in the peripheral blood of women with both NP and PE. Besides, the percentage of CD4~+Foxp3~+IL-17~+ intermediate cells obviously increased in PE compared with NP.Part II Correlation between the quantities of CD4~+Foxp3~+IL-17~+ intermediate cells and the quantities of CD4~+Foxp3+Treg cells and CD4~+IL-17A+Th17 cells in women with pre-eclampsiaObjectives: To evaluate the correlation between the percentage of circulating CD4~+Foxp3~+IL-17~+ intermediate cell and that of CD4~+IL-17A+ Th17 cells in PE.Methods: According to the data of PartⅠ, the Spearman correlation test was used to assess the correlation between the proportion of circulating CD4~+Foxp3~+IL-17~+ intermediate cell and that of CD4~+Foxp3+ Treg and CD4~+IL-17A+ Th17 cells in PE.Results: The percentage of CD4~+Foxp3~+IL-17~+ intermediate cells showed a negative tendency with CD4~+Foxp3+ Treg cells in PE, but not statistically significant(P > 0.05). The percentage of CD4~+Foxp3~+IL-17~+ intermediate cells was positively correlated with that of CD4~+IL-17A+ Th17 cells in normal late pregnant women(r = 0.604, P < 0.05). Moreover, this positive correlation was more obvious in PE compared to normal controls.(r = 0.803, P < 0.01). In addition, percentage of CD4~+Foxp3~+IL-17~+ intermediate cells in CD4~+Foxp3+ Treg cells was more positively correlated with the number of Th17 cells in PE compared with NP(r = 0.741, P < 0.01 vs. r = 0.397, P < 0.01).Conclusion: There was a positive correlation between the quantities of CD4~+Foxp3~+IL-17~+ intermediate cells and Th17 cells in the peripheral blood of PE, indicating the presence of the transformation from Treg into Th17 cells in PE.Part Ⅲ Factors influencing the pathogenic conversion from Treg to Th17 cellsObjectives: To investigate the factors that influence the pathogenic conversion from Treg to Th17 cells.Methods: The purified CD4~+CD25~+ Treg cells in the peripheral blood of normal pregnant women were isolated by MACS kit. Then, the cells were cultured in conditioned medium containing such cytokines as IL-6/IL-2/IL-1β(to mimic the inflammation condition in PE) for 4.5 days. Finally, the key genes associated with the function of Treg and Th17 cells as Foxp3, IL-10 and ROR-γt, IL-17 A, respectively, were evaluated by fluorescence quantitative PCR technique before and after culture.Results: The relative m RNA expression of ROR-γt and IL-17 A in Treg cells was significantly increased after in-vitro culture(P < 0.01), and that of IL-10 m RNA decreased obviously(P < 0.01). However, the relative expression of Foxp3 m RNA did not change significantly(P > 0.05).Conclusion: IL-6/IL-2/IL-1β can induce the pathogenic conversion of Treg cells in the peripheral blood of women with normal pregnancy to Th17 cells.