Research In Suppressed T Helper Type 17 Inflammatory Function In Unexplained Recurrent Spontaneous Abortion Model By Regulated IL-7/IL-7R Signal Pathway

Objective1. Evaluate the change about IL-7/IL-7Rα signal pathway of human maternal-fetal interface during the implantation phase, understand that the pathway is one of factors of embryo implantation and maintain pregnancy.2. Research in suppressed T helper type 17 inflammatory function in unexplained recurrent spontaneous abortion model by regulated IL-7/IL-7R signal pathway, and as a result suppressed abortion.Methods1. By immunohischemistry we try to know that IL-7/IL-7Rα signal pathway is one of the important factors in embryo implantation and maintain pregnancy in early pregnancy stage.2. Experimental mice were divided into six groups, the normal pregnant mice group, normal pregnant+IL-7 group, normal pregnant mice+IL-7R group, the group of mice abortion, abortion mice+IL-7 group and abortion mice+IL-7R group, by understanding embryo absorption rate was observed in each group of mice; by flow cytometric analysis of peripheral blood of mice in each group Th17/Treg cells, understanding IL-7 /IL-7Ra effect, the two cells changes; and analyzed by immunohistochemical methods Foxp3 expression in maternal-fetal interface of mice in each group and IL-17A; analyzing changes in Foxp3 and RORyt mRNA in the spleen of mice in each group by quantitative PCR methods, to understand whether regulate IL-7/IL-7R signaling pathway could inhibit inflammatory role of Th17 cells in unexplained recurrent spontaneous abortion model, and then induce immune tolerance, inhibit the occurrence of abortion.Rsults:1 The expression of IL-7/IL-7R in maternal-embryo interface.By immunohistochemical methods found that that recurrent spontaneous abortion patients’endometrial stromal cells was strong positive or moderately positive of IL-7 expression more than normal pregnant women weakly positive or negative expression, that is significantly increase. IL-7 expression was strongly positive or moderately positive in decidual glands both in URSA patients and normal pregnant women. While the expression of IL-7R is different. IL-7R expression of recurrent pregnancy loss patient decidual stromal cells was negative or weakly positive, was significantly lower than normal pregnant women. But in decidua gland, IL-7R expression in two groups is similar, no statistically significant.2 Results of animal experiment2.1 Embryonic absorption rate in each mice groupEmbryonic absorption rate in the normal pregnant mice was 11.1%, after the tail vein injection of IL-7 that the absorption rate was 20.63%, significantly higher compared with the normal pregnant mice group. When injected IL-7Ra blocking IL-7 signaling pathway, embryonic absorption rate was 13.04%, a slight increase compared with the normal pregnant mice group, but without statistically different; the absorption rate of mice abortion group was 26.15%. after IL-7 intervention the absorption rate was 31.67%, a little bit increase but no statistically significant. But after IL-7Ra intervention that the embryo absorption was 13.43%, embryo resorption rate decreased statistical significantly. As a result we found that IL-7-induced inflammation may promote local embryo resorption, leading to miscarriage, and blocking this signaling pathway reduce embryo absorption rate, suppress the occurrence of abortion.2.2 Results of Flow cytometry analysisIn the peripheral blood of normal pregnant mice group that Th17 (CD4+IL-17A+/CD4+) /Treg cells (CD4+CD25highFoxp3+/CD4+) was (1.65±0.15%)/(4.90±0.25%), normal pregnant mice+IL-7 intervention group Thl7/Treg cell was (4.35± 0.22%)/(3.26± 0.13%) in peripheral blood, normal pregnant mice+IL-7Ra group Th17/Treg cell was (1.95±0.09%)/(4.71±0.22%) in peripheral blood, we found that the addition of IL-7 intervention in normal pregnant mice significantly increased inflammatory Th17 cells, while Treg cells was significantly reduced; normal pregnant mice added IL-7Ra found that the Th17/Treg cell was a little bit change,but without significant compare with normal pregnant mice. IL-7 may induce Th17 cells by increasing inflammation and promoting abortion.Abortion mice group of peripheral blood Th17/Treg cell was (4.84±0.18%)/(2.57± 0.13%), after IL-7 intervention that the peripheral blood Th17/Treg cells of abortion mice group was (5.38±0.17%)/(1.93±0.09%), after the intervention of IL-7Ra the peripheral blood Th17/Treg cell of abortion mice group was (2.23±0.08%)/(4.93± 0.16%), the above results after the statistical analysis found that when IL-7Ra blocking IL-7 signaling pathway, Th17 cells significantly reduced meanwhile Treg cells increased significantly, for inhibit Thl7 cells inflammatory and surpress the occurrence of abortion.2.3 Quantitative PCR AnalysisExpression rate of RORγt mRNA and Foxp3 mRNA in mice spleend. The expression rate calculation method based on 2-△△CT respect to change in multiples of normal pregnant mice group.The 2-△△CT value of Foxp3 mRNA in normal pregnant mice+ IL-7 group was 0.75±0.22, significantly lower than normal pregnant mice group, meanwhile 2-△△CT value of RORyt mRNA was 1.79±0.55, and compared with normal pregnant group was significantly higher, and in the result the abortion rate was increased significantly (P<0.05); normal pregnant mice+IL-7Ra group Foxp3 mRNA 2-△△CT value was 0.91±0.17 and RORyt mRNA was 1.11±0.12, gene expression compared with normal pregnant mice group was with no significant difference, no effect on pregnancy outcome;The 2-△△CT value of Foxp3 mRNA in abortion mice was 0.53±0.15, RORyt mRNA of 2-AACT value was 1.87±0.49, compared with the normal pregnancy control group, the difference was significant; the Foxp3 mRNA 2-△△CT value of abortion mice+IL-7 group was 0.39±0.12,2-AACT value of RORγt mRNA was 4.87±0.69 (P<0.05), compared with the abortion mice group was significant difference; The 2-△△CT value of Foxp3 mRNA in abortion mice+IL-7Ra group was 0.92±0.12, RORyt mRNA of 2-AACT value was 1.22±0.61, compared with the abortion mice group was significant difference, blocking signaling pathways of IL-7, Thl7-related cells factor secretion decreased, reduce the local inflammation, inhibit the occurrence of abortion (P<0.05).2.4 Immunohistochemical AnalysisIn the maternal-fetal interface of normal pregnant mice group that IL-17A expression was negative or weakly positive, Foxp3 was moderate positive or strong positive expression of; in normal pregnant mice+IL-7 group that IL-17A weakly or moderately positive expression, Foxp3 negative or moderate positive expression, compared with the normal pregnant mice group were significantly different; normal pregnant mice+IL-7Ra group that IL-17A expression was negative or weakly positive, Foxp3 expression was moderate positive or strong positive expression,and that was no significant difference between the control group. In the maternal-fetal interface of abortion mice group IL-17A was moderately positive or strongly positive, Foxp3 was negative or weakly positive expression; abortion mouse+IL-7 group was moderate positive or strong positive expression of IL-17A, Foxp3 was negative or moderate expression, no significant difference between with the abortion mice group; the expression of IL-17A of abortion mouse+IL-7Ra group was negative or weakly positive, Foxp3 was moderate positive or strong positive expression, compared with abortion group was significantly different.Coculsion:The early pregnancy in humans, IL-7/IL-7R signaling pathway may be one of the factors involved in embryo implantation and maintenance of pregnancy, and patients with recurrent miscarrage expression of local IL-7 enhancements and IL-7R reduce may result pregnancy failure. In animal experiments, under the same experimental conditions regulated IL-7/IL-7Ra signaling pathway, in normal pregnant and abortion group,if we use IL-7 intervene and then found that in peripheral blood Th17 cells was increased, Thl7 specific transcription RORyt factor expression levels also increased, expression of IL-17A in maternal-fetal interface was increased too, IL-7 can induce Th17 inflammatory effect, increased miscarriage; and when IL-7Rα blocking this signaling pathway, found that Th17 cells was decreased, Th17 specific transcription RORyt factor expression levels also decreased, expression of IL-17A in maternal-fetal interface was decreased too, embryo resorption rate decreased, all of these change implay that blocking IL-7/IL-7R signaling pathway, affecting Th17 cell survival and proliferation, apoptosis Th17 cells, leading to reduced Th17 cells, while IL-7/IL-7R signaling pathways also affect the number and function of Treg cells, induced Treg secreting cells and related cytokines induce immune tolerance of pregnancy. Speculate regulate IL-7/IL-7R signaling pathway inhibition effect of unexplained recurrent miscarriage inflammatory Th17 cells, increased expression of Treg cells induce immune tolerance of pregnancy, help maintain pregnancy.