| Part one The Expression of PD-1, PD-L1, Foxp3 and RORyt in Placenta of Preeclamptic WomenObjective:To explore the role of PD-1/PD-L1 signaling pathway on the pathogenesis of preeclampsia, the expression levels of PD-1 and its ligand PD-L1, as well as Foxp3 and RORyt, the specific nuclear transcription factors of Treg and Th17 cells respectively, in the placental tissues of women with preeclampsia and normal pregnancy were detected.Methods:The placentas from women with normal pregnancy (n=16) and preeclampsia (n=10) were collected. The expression of PD-1, PD-L1, Foxp3 and RORyt in placentas were analyzed by immunohistochemistry, and quantitative real-time PCR and western blot were used to determine the mRNA and protein expression levels of PD-1, PD-L1, Foxp3 and RORyt.Results:1. The PD-1, PD-L1, Foxp3 and RORyt proteins were expressed in the placentas of normal pregnant and preeclamptic women, PD-1 and PD-L1 were mainly located in the cytoplasm and cell membrane of the villous trophoblast cells, while Foxp3 and RORyt were mainly located in the nuclear and cytoplasm of the villous trophoblast cells and villous stromal cells.2. Compared with the normal pregnant group, the mRNA expression levels of PD-1 (P < 0.05) and PD-L1 (P< 0.05) were lower in the preeclamptic group. The mRNA expression levels of Foxp3 decreased (P< 0.01), while those of RORyt increased (P< 0.05) in the preeclamptic group.3. Compared with the normal group, the protein expression levels of PD-1 and PD-L1 in the preeclamptic group were lower (P< 0.01). The protein expression levels of Foxp3 in preeclamptic group were significantly decreased (P< 0.01), while those of RORyt were elevated (P< 0.01).Conclusion:The findings indicated that the proteins of PD-1 and PD-L1 were mainly expressed in the villous trophoblast cells of placentas; both of them decreased in preeclamptic women, which were characterized by the decreased Foxp3 and increased RORyt expression in placentas. Furthermore, the altered expression of PD-1 and PD-L1 might contribute to preeclampsia in the immune-dependent and immune-independent ways.Part two The Therapeutic Effect and Molecular Mechanisms of PD-L1 Fc on Preeclampsia-like Rats via Activation of PD-1/PD-L1 Signaling PathwayObjective:To explore the potential therapeutic value of PD-L1 Fc on preeclampsia, the preeclampsia-like rats were established by L-NG-Nitroarginine Methyl Ester (L-NAME) and treated with PD-L1 Fc. The maternal blood pressure, urine protein, weight gain, liver and kidney function, and the changes of Treg/Th17 cell balance in peripheral blood and the placentas, as well as the offspring outcomes were evaluated. The underlying molecular mechanism of PD-L1 Fc regarding the therapeutic effects on the preeclampsia-like rats was also investigated.Methods:1. SD female rats (n=20) were randomly divided into four groups:normal pregnancy group, preeclampsia-like group, magnesium sulfate (MgSO4) treatment group and PD-L1 Fc treatment groups. Each group included five rats. The latter three groups were subcutaneously injected with L-NAME to establish rat model of preeclampsia. On the 16th day of pregnancy, the first and second groups were given saline as controls, and the latter two groups were given MgSO4 and PD-L1 Fc fusion proteins, respectively. All the goups were given subcutaneous injection.2. Maternal blood pressure was measured using noninvasive rat tail artery manometry before pregnancy, and every other day after pregnancy. Before pregnancy, and on the 10th,17th and 20th day of pregnancy,24-h urinary were collected and urine protein excretion was analyzed. In addition, maternal body weight was determined every other day from the second day of gestation until sacrificed.3. On the 21th day of gestations, all the rats were sacrificed. The pathological examination in livers and kidneys of maternal rats were analyzed by HE staining.4. The profiles of Treg and Th17 cells in peripheral blood were determined by flow cytometry. In the placentas, the expression of Foxp3 and RORyt were evaluated by immunofluorescence, quantitative real-time PCR and western blot.5. The expression of PD-1 and PD-L1, as well as their related signal moleculars in the placenas were determined by quantitative real-time PCR and Western blot.6. The numbers of fetal and embryo resorption rates were counted, and the weight of fetuses and placentas, as well as fetal body length were measured.Results:1. In normal pregnancy group, the systolic blood pressure (SBP) throughout the entire gestation period was consistent, and the SBP was 116.0 ± 1.581 mmHg on GD 16. The rats treated with L-NAME showed increased SBP, being 138.8 ± 1.643 mmHg on GD16, which were significantly higher than the normal pregnancy group (P< 0.001). L-NAME-induced preeclamptic rats treated with saline showed continuously increase on SBP from GD14 until delivery. In contrast, the injection of PD-L1 Fc or magnesium sulfate ameliorated SBP increase, although the antihypertensive effect of magnesium sulfate was speedy and transient, while the effect of PD-L1 Fc was steady and sustained to GD18.2. L-NAME significantly increased the proteinuria levels in preeclamptic rats (P< 0.001). Treatment with magnesium failed to reverse this condition; however, after PD-L1 Fc treatment, urinary protein significantly decreased on GD 20 (P< 0.05). We also observed less net weight gain in the preeclamptic rats (P< 0.05). Treatment with PD-L1 Fc had a trend on reversing the loss of weight gain although it was not statistically significant (P> 0.05). The analysis of histological scoring revealed that the renal and liver histopathologic injury scores in the L-NAME-administered groups were both significantly elevated (P< 0.001, P< 0.001, respectively). However, PD-L1 Fc treatment had lower scores compared with the other two L-NAME treated groups.3. Compared to normal pregnancy group, preeclampsia-like rats had significantly lower proportion of Treg cells (P< 0.01), and the proportion of Th17 cells significantly increased in periphery blood (P< 0.01). Administration of PD-L1 Fc to preeclamptic rats on GD 16 markedly decreased the percentage of Th 17 cells (P< 0.01), but increased Treg/Th17 cell ratios (P< 0.01).4. The results of immunofluorescence staining show that Foxp3 and RORyt were expressed in representative placental samples. These findings were confirmed at the mRNA levels as shown by higher expression of Foxp3 (P< 0.05) and lower expression of ROR?t (P< 0.01) in placentas after PD-L1 Fc treatment. Interestingly, the mRNA levels of PD-1 and PD-L1 increased in the PD-L1 Fc treated groups compared with untreated preeclamptic rats (P< 0.05, P< 0.05, respectively). We proved that the mRNA expression of PI3K/AKT/mTOR declined and that of PTEN increased in the placentas after PD-L1 Fc administration. Furthermore, the protein expression levels of these molecules were consistent with the corresponding mRNA expression.5. Pups delivered from preeclampsia -like rats are characterized by malformation, low fetus weight and short fetus length. In addition, preeclampsia -like rats had the higher percentage of fetal resorption than that in normal pregnant rats (P< 0.01). Treatment with PD-L1 Fc had major effects on fetus outcomes as shown by the increased trend of fetal weight and length, and decrease in the percentage of fetal resorption, which was not significant different compared with the normal pregnant groups. However, the placental weight was significantly increased (P< 0.05).Conclusion:The use of PD-L1 Fc in the L-NAME-induced preeclampsia -like rat models proves to ameliorate the clinical symptoms, and to be more effective than that of magnesium sulfate in protecting the pregnancy and reversing the Treg/Th17 imbalance. This modulatory effect is mediated via inhibiting the expression of PI3K/AKT/mTOR and promoting PTEN signals. In addition, PD-L1 Fc fusion protein exhibits a protective effect on fetal development, suggesting that it has potential therapeutic value in the treatment of preeclampsia.Part three The PD-1/PD-L1 Pathway Regulates the Differentiation of Naive CD4+T cells in Preeclampsia WomenObjective:Intervened the naive CD4+T cells under different culture conditions by activating or blocking the PD-1/PD-L1 signal pathway, so as to clarify the effect and mechanism of PD-1/PD-L1 pathway on the differentiation of naive CD4+T cells to Treg and Th17 cells.Methods:Peripheral blood of normal pregnant women (n= 15) and patients with preeclampsia (n=15) were collected. Naive CD4+T cells were isolated by MACS and cultured alone or co-cultured with PD-L1 Fc protein or anti-PD-L1 mAb under the ThO, Treg and Th17 immune environment in vitro, respectively. The mRNA expressions of transcription factors and signaling molecules -Foxp3, ROR?t, PI3K, AKT, m-TOR and PTEN were determined by quantitative real-time -PCR.Results:1. In normal pregnancy group, the administration of PD-L1 Fc significantly enhanced the expression levels of Foxp3 mRNA than the control group under Th0-prone conditions (P< 0.05), while the administration of anti-PD-L1 mAb inhibited the expression levels of Foxp3 mRNA (P< 0.05). Under Th17-prone conditions, the expression levels of Foxp3 mRNA were significantly increased under PD-L1 Fc administration (P< 0.001), although it had no significant change with anti-PD-L1 mAb treatment. Under Treg-prone conditions, the expression levels of Foxp3 mRNA showed no significant difference among the three groups. In pre-eclamptic group, the administration of PD-L1 Fc enhanced the expression levels of Foxp3 mRNA than the corresponding groups under Th0-(P< 0.05) and Th17-prone (P< 0.05) conditions. No difference was found in the expression levels of Foxp3 mRNA among the three groups under Treg-prone conditions.2. In normal pregnancy group, the administration of PD-L1 Fc inhibited the expression levels of RORyt mRNA (P< 0.05), which was increased with anti-PD-L1 mAb treatment (P< 0.05). Under Th17-prone conditions, the expression levels of RORyt mRNA were inhibited (P< 0.05) with PD-L1 Fc treatment, while the administration of anti-PD-L1 mAb promoted the expression levels of RORyt mRNA (P< 0.05) under Treg-prone conditions. In pre-eclamptic group, the administration of anti-PD-L1 mAb enhanced the expression levels of RORyt mRNA under Th0-prone conditions (P< 0.001). The mRNA expression of RORyt was prone to be down-regulated with the administration of PD-L1 Fc under Th17- and Treg-prone conditions, although no significant difference was found (P> 0.05).3. In normal pregnant group, under Th0-prone conditions, the expression levels of PI3K, AKT, m-TOR mRNA were down-regulated with the administration of PD-L1 Fc (P< 0.05, P< 0.05, P< 0.05), and the expression levels of PTEN mRNA were up-regulated (P< 0.05), which was down-regulated with the treatment of anti-PD-L1 mAb (P< 0.01). Under Th17-prone conditions, the administration of PD-L1 Fc significantly inhibited the expression of AKT mRNA (P< 0.05). The expression of m-TOR mRNA was promoted with anti-PD-L1 mAb treatment under the Treg-prone conditions (P< 0.05). In pre-eclamptic group, under Th0-prone conditions, the expression levels of AKT mRNA were inhibited with PD-L1 Fc treatment (P< 0.05), while the treatment of anti-PD-L1 mAb significantly enhanced the expression levels of PI3K, AKT, m-TOR mRNA (P< 0.01, P< 0.05, P< 0.01, respectively). Under Th17-prone conditions, the expression levels of PI3K mRNA were significantly up-regulated with the administration of anti-PD-L1 mAb (P< 0.01). The expression levels of PTEN mRNA were up-regulated with the administration of PD-L1 Fc under Treg-prone conditions (P <0.01).Conclusion:Under Th0-, Th17- and Treg-prone conditions, the expression levels of Foxp3 mRNA were up-regulated and those of RORyt mRNA were down-regulated with the administration of PD-L1 Fc, while the treatment of anti-PD-L1 mAb revised the phenomena, indicating that PD-L1 Fc promoted naive CD4+T cells to differentiate into Treg cells. In addition, PI3K, AKT, m-TOR and PTEN signals underlies the molecular mechanism of PD-1/PD-L1 pathway. |
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