Regulation Of RANKL/RANK Interaction On The Functional Phenotype Of Decidual Macrophage In Early Pregnancy

Normal pregnancy is similar to the process of allogeneic transplantation. The process that the fetus with paternal antigen is alive until delivery shows actually maternal immunotolerance to the fetus, and the maternal immune rejection will lead to pregnancy failure, which suggests that the maternal-fetal immune tolerance plays an important role in a successful pregnancy. If the immune tolerance mechanism has been elucidated, it is very significant not only to reproductive immunology, but also to research on tumor immunity and autoimmune disease.The maternal-fetal interface is composed of decidual stromal cells (DSCs), trophoblasts (Tros) and decidual immunocompetent cells (DICs), in which decidual stromal cells and decidual immunocompetent cells are derived from mother, and trophoblast is derived from her fetus. They interact with each other and maintain the Th2type immune bias at the maternal-fetal interface. Decidual macrophages (dMcp) account for10%of the decidual immunocompetent cells, and involved in implantation, placentation and the cervical ripening.The receptor activator of NF-κB ligand (RANKL, also known as TNFSF11, OPGL, TRANCE, or ODF) is a member of tumor necrosis factor (TNF) superfamily, its receptor RANK (also known as TNFRSF11A, OFE, PDFR, TRANCE-R, ODAR, CD265) and decoy receptor osteoprotegerin (OPG; also known as TNFRSF11B) represent the essential molecule unit that controls osteoclast differentiation and thereby fundamental aspects of bone metabolism. RANKL/RANK interacton plays an important role in the immune regulation. RANKL or RANK-mutant mice displayed lack of lymph nodes. RANKL promotes the survival of dendritic cells (DC) by activating Bcl-XL and induces T cells proliferation. There are a variety of immune cells at the maternal-fetal interface, and the role of RANKL at the maternal-fetal interface has not been elucidated yet. 1. RANKL involved in the regulation of decidual sromal cells and trophoblast biological behaviorWe collected the villi and decidua of human normal early pregnancy, and cultured the primary trophoblasts (Tros) and decidual stromal cells (DSCs). The expression of RANKL in tissues and cells was detected by semi-quantified RT-PCR, immunohistochemistry and flow cytometry, respectively. We found that the primary decidual stromal cells co-expressed RANKL and its receptor RANK in transcriptional and translational level. The trophoblasts also co-expressed RANKL and its receptor RANK in the both levels. BrdU kit was used to measure the proliferation of DSCs and trophoblasts after stimulation with RANKL for48h. It showed that RANKL could promote the proliferation of DSCs and trophoblasts in a dose-dependent manner. Annexin V-FITC/PI kit was exploited to detect the apoptosis of DSCs. Meanwhile, the transwell chamber was used to test the invasiveness of primary trophoblasts. We observed that RANKL could significantly inhibit the apoptosis of DSCs at the concentration of100ng/ml, and obviously enhanced the invasiveness of trophoblasts.2. RANKL regulates the function of decidual macrophage and its polarization.We analyzed the expression level of RANK on decidual immunocompetent cells by flow cytometry, and discovered that RANK was expressed higher on decidual macrophages. Thereafter we compared RANK expression on peripheral monocytes or decidual macrophages, and found that decidual macrophages expressed RANK higher than peripheral monocytes. Based on RANK expression or not, decidual macrophages were divided into RANK+macrophage and RANK-macrophage. RANK+macrophage expressed higher levels of CD206, CD209and CD11c, which indicates that it polarized to alternatively activated M2macrophages. DSCs and trophoblasts were co-cultured with decidual macrophages, adding or not rhOPG or anti-RANNKL neutralizing antibody for48h, and then the expression of co-stimulatory molecules CD80and CD86on decidual macrophage was detected by flow cytometry. It was found that the DSCs and trophoblast-derived RANKL could restrain the expression of CD80and CD86on the decidual macrophage. On the other hand, the secretion level of IL-10, IL-12and IL-23were measured by ELISA. We observed that the RANKL derived from DSCs and trophoblasts significantly facilitated the IL-10secretion and suppressed the secretion of IL-12and IL-23of macrophages, which indicates RANKL induces the decidual macrophage polarized to M2phenotype. The DSCs and trophoblasts-primed decidual macrophages was co-cultured with decidual naive T cells for5days, and then IL-4, IL-10, TNF-a and IFN-y were measured by Bioplex system, and it was found that IL-4and IL-10secretion were at high level but TNF-a and IFN-y were at low level. It may be proposed that DSCs and trophoblasts-primed decidual macrophages induces the decidual naive T cells biased to Th2phenotype, which is beneficial to the immune tolerance at maternal-fetal interface.3. The regulating mechanism of RANKL on decidual macrophage polarizationDSCs and trophoblasts were co-cultured with decidual macrophages for24h, by adding or not rhOPG or anti-RANKL neutralizing antibody, and then phosflow technique was exploited to analyze the phosphorylation level of signal molecules. We discovered that RANKL activated Akt (pS473) molecule and its downstream STAT6, a master regulator of M2genes, which induced the expression of transcription factors IRF4and Jmjd3(histone demethylase). We detected the transcription factors IRF4and Jmjd3by quantitative real-time PCR and observed RANKL induced IRF4and Jmjd3transcription that resulted in decidual macrophages polarized to M2phenotype, which was involved in immune tolerance at maternal-fetal interface. The plasmids pCDNA3.1(+)-RANKL was transfected into DSCs or JEG-3cells, a choriocarcinoma cell line, and RANKL was over-expressed successfully. The RANKL over-expressed DSCs and trophoblasts were co-cultured with decidual macrophage, some with adding the specific PI3K/Akt inhibitor Ly294002for12h, and then measure the secretion level of IL-10, IL-12and IL-23by ELISA as well as transcription factors IRF4and Jmjd3by quantitative real-time PCR. It was found that RANKL over-expression could enhance the secretion of IL-10and the transcription factors IRF4and Jmjd3partly through PI3K/Akt signal pathway.4. The decreased expression of RANKL at maternal-fetal interface leads to the dysfunction of macrophages and spontaneous abortion.After normal pregnancy CBA/J♀×BALB/c♂matings and abortion-prone CBA/J♀×DBA/2♂matings were established, we cultured the primary decidual immunocompetent cells at day5and day9of gestation, and analyzed the expression of RANK on decidual macrophages. It was found that decidual macrophage of normal pregnant mice expressed RANK higher than abortion-prone mice at the day5and day9of gestation. The abortion-prone mice expressed MHC-II molecule higher than the normal pregnant mice on the day9, but there was no significant difference of CD80and CD86between these two groups. The one side uterine in normal murine pregnancy at the day3of gestation were given anti-RANKL neutralizing antibody or Ly294002treatment, and we evaluated the embryo absorption rate and the expression of CD80, CD86and MHC-II molecule on decidual macrophage on the day7of gestation. It was showed that the decidual macrophage of uterine treated with anti-RANKL neutralizing antibody expressed higher CD80and CD86higher than that of the control, and the decidual macrophage of uterine treated with Ly294002expressed CD206lower than that of the control. The embryo absorption rate in the anti-RANKL neutralizing antibody or Ly294002treatment were higher than that of the control.In conclusion, RANKL has multiple modulating effects at the maternal-fetal interface:(1) RANKL promotes proliferation of decidual stromal cells and inhibits its apoptosis in an autocrine manner.(2) RANKL enhances the proliferation and invasiveness of trophoblasts.(3) RANKL derived from DSCs and trophoblasts inhibits the expression of CD80and CD86on decidual macrophage and facilitates the secretion of IL-10, a M2phenotype.(4) Decidual macrophage primed by DSCs and trophoblasts can induce decidual naive T cells to Th2bias, which is beneficial to immune tolerance at maternal-fetal interface.(5) RANKL derived from DSCs and trophoblasts activates PI3K/Akt and downstream STAT6signal pathway in the decidual macrophage and enhances the transcription of IRF4and Jmjd3, which drives macrophage polarized to M2phenotype.(6) The decreased expression of RANKL at maternal-fetal interface leads to the dysfunction of macrophages and spontaneous abortion.