The Roles Of Activated CD4~+Th17/Treg Cells In Spleen And Their Secreted Cytokines In A URSA Mouse Model

Objective:(1) To reveal the changes of spleen CD4+ Th17/Treg cells activation and the secretion of IL-17 A, IL-10, IL-21, IL-23 cytokine in non-pregnant mice, mice with normal pregnancy and URSA model mice;(2) To discuss the roles of activated CD4+ Th17/Treg cells and their secreted cytokines in a URSA mouse model. This research will eventually provide significant scientific basis for the novel strategies of immune prevention and treatments in URSA patients.Methods: Three experimental models were established, including URSA mouse(♀CBA/J×♂DBA/2J), non-pregnant mouse(♀CBA/J) and normal pregnancy mouse(♀CBA/J×♂Balb/c). Spleen CD4+ T cells were isolated from three female mouse groups using immunomagnetic beads sorting technology(MACS). Isolated CD4+ T cells were induced to Th17 and Treg cells in vitro. Flow cytometry technology was used to detect the activation of induced Th17 and Treg cells. Secreted IL-17 A, IL-10, IL-21 and IL-23 in cells culture supernatant and in the supernatant of grinding decidual and placental cells were measured by Luminex x MAP. The protein levels of IDO and Fox P3 in Th17/Treg cells and in decidua and placenta cells in the interface of maternal fetal were detected by western-blot.Results:(1) FCM was used to detect spleen Th17 cells, which were induced from CD4+T cells isolated from non-pregnant mice, normal pregnant mice and URSA mice. Th17 cells from non-pregnant mice, normal pregnant mice and URSA mice were(0.32+0.19) %and(0.13+0.06) % and(0.72+0.38) %, respectively. The number of Th17 cells from URSA group was significantly higher than those in non-pregnant group and normal pregnancy group. There was statistical significant difference between every two groups(P<0.05). The numbers of CD4+CD25+Fox P3+Treg cells in three groups were(0.38+0.15) %,(0.85+0.48) % and(0.17+0.12) %, respectively. The number of CD4+CD25+Fox P3+Treg cells in URSA group was significant lower than those in non-pregnancy group and normal pregnancy group. Statistical analysis showed significant difference between every two groups(P<0.05).(2) Lumina x Map was used to detect secreted cytokines in the supernatant of grinding decidua cells and placental cells that came from mouse maternal-fetal interface. Lumina x Map showed in normal pregnancy group, IL-17A(9.90±5.04)pg / ml, IL-10(6.50±7.03)pg / ml, IL-23(14.84±11.41) pg / ml and in URSA group, IL-17A(14.00±16.73) pg / ml,IL-10(4.10±6.25) pg / ml, IL-23(17.52±16.15) pg / ml. IL-21 concentration was below the detection limit(P>0.05). IL-17 A, IL-10, IL-21, IL-23 cytokine concentrations of the induced Th17/Treg cells supernatants were below the detection limit.(3) The protein levels of IDO and Foxp3 in induced Th17/Treg cells in non-pregnant mouse, normal pregnant mouse and URSA mouse were detectd by western blot. The expression of IDO and Fox P3 in URSA group was significantly lower than those in non-pregnant group and normal pregnancy group. Statistical analysis showed significant difference between every two groups(P<0.05). Also, IDO and Fox P3 protein expression of grinding decidua cells and placental cells in URSA group was significantly lower than that in normal pregnancy group. Statistical analysis showed significant difference(P<0.05)among these groups.Conclusion:(1) The differentiation, proliferation and activation of Th17 cells in URSA mice were associated with the onset of URSA. The increased number of Th17 cells had negative effects on pregnancy. Normal pregnancy clearly showed CD4+CD25+Fox P3+Treg cells tendency, however, in URSA, the number of CD4+CD25+Fox P3+Treg cells was decreased, which was not sufficience to maintain pregnancy.(2) The activated CD4+ T cells in URSA mouse spleen secreted IL-17 A and IL-23,which were closely related to the pathogenesis of URSA. The inceased amount of these two cytokines may induce the occurrence of URSA, while the increased amount of IL-10 may maintain normal pregnancy.(3) The protein levels of IDO and FoxP3 in the grinding decidua cells and placental cells in URSA group were significantly lower than those in normal pregnancy group.Monitoring IDO changes in early pregnancy in URSA patients may predict the outcome of pregnancy.