Effects And The Molecular Mechanism Of Decidual Stromal Cells-derived IL-33 On Inducing Maternal-fetal Immune Tolerance In Human Early Pregnancy

From the immunological point of view, pregnancy is similar to the process of allogeneic transplantation. The process that the fetus with paternal antigen is alive until delivery is a state of tolerance of maternal immune system toward allogeneic tissues of implanted embryo, and the maternal immune rejection will lead to pregnancy failure. To elucidate the immune tolerance mechanism is of great significance not only to reproductive immunology, but also to oncological immunology and autoimmune disease.The maternal-fetal interface is composed principally of decidual stromal cells (DSCs), decidual immune cells (DICs) and trophoblasts (Tros), of which DSCs and DICs are derived from mother while Tros are derived from fetus. To understand mechanisms of tolerance to the fetal allograft, many reviews have researched the biological events occurred at maternal-fetal interface for years. Serving as an immunologically privileged tissue, the decidua and its components, especially DICs play essential functions in pregnancy maintenance. During the first trimester of pregnancy, the human decidua is rich in immune cells which comprise 70%-80% decidual natural killer (dNK) cells,15% macrophages and 10% T cells. These immune-competent cells obtain distinct phenotype and functionality in course of interaction with each other during pregnancy process and maintain the Th2 type immune bias at the maternal-fetal interface.Interleukin (IL)-33, a newly described member of the IL-1 family, has been reported to facilitate primary tumor progression and metastatic dissemination through interaction with its receptor ST2L and decoy receptor sST2. In addition, it has been reported that dysregulation of IL-33/ST2L prolongs uterine receptivity in recurrent pregnancy loss (RPL) patients and disordered levels of serum IL-33 and ST2 are associated with miscarriage and preeclampsia. However, the role of IL-33 at the maternal-interface has not been elucidated yet.Part 1. Expression of IL-33/ST2 in decidual stromal cells (DSCs) from normal pregnancy and recurrent spontaneous abortion (RSA) patientsObjective To investigate whether IL-33/ST2 is expressed and the expression level of IL-33/ST2 in DSCs from normal pregnancy and RSA patientsMethods Expression of IL-33/ST2 in decidua from normal early pregnancy and RSA was detected by immunohistochemistry. ELISA, Real-time PCR and Western Blot were performed to further confirm the aforementioned results and compare the expression of IL-33/ST2 in DSCs from normal pregnancy with that from RSA patients.Results Immunohistochemistry showed that decidual tissues were positively stained with anti-IL-33 and anti-ST2 antibody. ELISA, Real-time PCR and Western Blot further confirmed the expression of IL-33/ST2 in DSCs. Moreover, IL-33/ST2 expression was decreased in DSCs from RSA patients at both gene and protein levels.Conclusion Appropriate expression of IL-33/ST2 in decidua, especially in DSCs is necessary for maintenance of normal pregnancy.Part 2. IL-33 involved in the regulation of decidual stromal cells (DSCs) biological behaviorObjective To probe into the effect of IL-33 on the biological behavior of human first-trimester DSCs.Methods We collected the decidua of human normal early pregnancy, and cultured the primary DSCs. After treatment with IL-33 or sST2 for 48h, DSCs were collected and proliferation, invasion, cell cycle of DSCs were examined by BrdU assay, Transwell assay and flow cytometry respectively. To further validate the regulation effect of IL-33 on DSCs biological behavior, Real-time PCR was applied to detect the expression of proliferation relative (PCNA, survivin) and invasion relative molecules (titin, MMP2) in DSCs.Results BrdU and Transwell assay showed that IL-33 promoted DSCs proliferation as well as invasiveness in a dose-dependent manner, which were eliminated by pretreatment with sST2 against IL-33. In addition, flow cytometry results revealed that the proliferation index increased with increasing IL-33 concentration and showed a peak at lng/mL. Furthermore, treatment of DSCs with IL-33 significantly elevated the mRNA expression of PCNA, survivin, titin and MMP2 in a dose-dependent manner.Conclusion IL-33 promotes proliferation of DSCs through ST2L, and IL-33/ST2L interaction is also involved in invasion of DSCs.Part 3. The regulating mechanism of IL-33 on decidual stromal cells biological functionObjective To elucidate the molecular mechanisms of IL-33 involved in regulating decidual stromal cells biological function.Methods ELISA and flow cytometry were conducted to study the expression level of CCL2/CCR2 by DSCs treated with IL-33. Furthermore, to demonstrate the effect of up regulated CCL2/CCR2 on DSCs biological function, BrdU assay, Transwell assay and flow cytometry were applied once again after adding anti-CCL2 neutralizing antibody or CCR2 blocker RS102895. The IL-33-induced activation of NF-κB, ERK1/2 or JNK was examined by western blot. BAY 11-7082, U0126 and SP600125 were used to block the activation of NF-κB, ERK1/2 and JNK, respectively. After pretreated with above mentioned inhibitors, CCL2 secretion was measured by ELISA and CCR2 expression was analyzed by flow cytometry.Results CCL2/CCR2 was significantly increased with IL-33 administration. Moreover, inhibition of CCL2/CCR2 activation using CCL2 neutralizing antibody or CCR2 blocker prevented IL-33-stimulated proliferation and invasiveness capacity of DSCs. Increasing phosphorylation of NF-κB, ERK1/2 and JNK after treatment with IL-33 was confirmed by western blotting. And the IL-33-induced CCL2/CCR2 expression was abrogated by treatment with the NF-κB inhibitor BAY 11-7082 and ERK1/2 inhibitor U0126. However, the JNK inhibitor, SP600125, didn’t affect IL-33-induced CCL2/CCR2 expression.Conclusion IL-33 promotes proliferation and invasiveness of DSCs by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signal pathways.Part 4. IL-33 regulates the function and phenotype of decidual NKObjective To investigate the functional modulation of IL-33 on decidual NK cellsMethods The expression level of ST2L on decidual and peripheral NK cells was detected by flow cytometry. First-trimester human DSCs were isolated and cultured with IL-33, sST2 or DSCs media for 48h, afterward NK cells were collected and analyzed by flow cytometry and cytotoxicity assay to assess the functionality related molecule expression and cytotoxicity of dNKs.Results We discovered that ST2L was expressed on NK cells. Besides, we compared ST2L expression on peripheral NK with that on decidual NK cells, and found that decidual NK cells expressed ST2L higher than peripheral NK. Treatment with IL-33 or DSCs media resulted in an increase in type 2 cytokines (IL-4, IL-13) as well as in regulatory cytokine (IL-10). While IL-33 treated decidual NK showed marked influence on generating Th2 responses, the effect of IL-33 on TNF-a and IFN-y production seemed inconsistent. Although IL-33 and DSCs media markedly decreased TNF-a production in decidual NK, IL-33 did increase IFN-y expression. In addition, IL-33 down-regulated the activation receptors (NKP30, NKG2D) expression, whereas up-regulated the inhibition receptor (KIR2DL1) expression on decidual NK. Furthermore, both cytotoxicity and cytolytic granules expression (granzyme A, perforin) of dNKs went down after cultured with IL-33 or DSCs-conditional media.Conclusion DSCs-derived IL-33 could modulate the phenotype, Thl/Th2 cytokine expression and cytotoxicity of decidual NK cells, contributing to maternal-fetal immune tolerance.Part 5. The regulating mechanism of IL-33 on the function and phenotype of decidual NK cells.Objective To explore the signaling pathway by which IL-33 regulates the function and phenotype of decidual NK cells.Methods Isolated decidual NK cells were treated with IL-33 or sST2 for 48h. Following the treatment, the mRNA expression of differentiation and development related transcription factors were detected by Real-time PCR. The expression and activation of signaling molecules, including NF-κB, ERK1/2, JNK and P38, were examined by western blot. To further prove the exact signal pathway, dNKs were pretreated with BAY 11-7082, U0126 or SP600125 to block the activation of NF-κB, ERK1/2 and JNK respectively, and then incubated with IL-33 for another 48h. The expression of cytokines and receptors producted by decidual NK were detected by Flow cytometry.Results It was discovered that Nfil3, Id2 and Gata3 mRNA production of decidual NK cells treated with IL-33 were increased when compared with control group. Increasing phosphorylation of NF-κB, ERK1/2 and JNK after treatment with IL-33 was confirmed by western blotting. And the regulative effect of IL-33 on the function and phenotype of decidual NK cells was abrogated by pre-treatment with the NF-κB inhibitor BAY 11-7082.Conclusion NF-KB/Nfi13/Id2/Gata3 signal pathway is involved in the regulation of IL-33 on the function and phenotype of decidual NK cells.