The Expression And Functions Of CXCL12/CXCR4/CXCR7 Axis At The Maternal-Fetal Interface Of Human First-Trimester

The developmental potential of the embryo, the receptivity of endometrium and the cross-talk of maternal-fetal immune microenvironment are considered to be essential factors for the goal of building a successful pregnancy. According to immunological view, pregnancy is similar to a successful transplant of semi-allogeneic. Maternal not only accept embryos carrying paternal antigens, but also establish a unique mother-fetal interface immune microenvironment to protect them being attacked by own immune system, providing nutrition continuously through a well maternal-fetal crosstalk. Therefore, the mother-fetal immune regulation thought to be the core of the maternal-fetal crosstalk. At the cellular and molecular level, maternal-fetal immune microenvironment are constituted of father derived trophoblast cells and mother derived immune cells, producing various cytokines, growth factors and hormones work as a network at the immune interface. Maternal may reject fetal possibly lead to infertility, pre-eclampsia, spontaneous abortion, intrauterine growth retardation and other pathological pregnancy-related diseases, while the maternal-fetal interface immune microenvironment disorders.Chemokines are a kind of small molecule cytokines, inducing specific directional movement of immune cells, binding with chemokine receptor, involving in the regulation of biological development, inflammation, immune responses, angiogenesis and tumor cell migration and invasion and other physiological and pathological processes. The chemokine CXCL12 regulates cell migration and homing processes in the immune system, hematopoietic system, brain, and other tissues, playing important role in human being. Our previous research work focus on the chemokine CXCL12/CXCR4 signaling pathway in the mother-fetal interface. CXCR7, a novel receptor for SDF-1, has been identified recently. Research has demonstrated that SDF-1/CXCR7 interaction could play an important role in cancer progression. The latter is also referred to as atypical chemokine receptor 3 (ACKR3) because it does not signal through G proteins and elicits no chemotaxis and its’scavenger’ dual. Their function is unclear in reproductive immune field still, especially at the maternal-fetal interface. In preliminary experiments, we found that and decidua were expressed CXCR7 during human first-trimester pregnancy. Therefore, our study intends to establish isolation and culture system of functional cells in mother-fetal immune microenvironment and explore CXCR7 expression and biological function during human first-trimester pregnancy. We speculate that CXCR7 involved CXCL12 /CXCR4 signaling pathway, and as an important bridge media to induce communication of various function cells on mother-fetal interface. The result will provide theoretical basis for improve immune tolerance adjustment mechanism of chemokine factors and receptors in the maternal-fetal crosstalk, and prevention strategies of immune-related clinical pathological pregnancy.Part I Isolation and culture system of mainly function cells at the maternal-fetal immune microenvironment.Objective To establish a stable system for isolation, purification and culture of trophoblast cells(TCs), decidual stromal cells(DSCs) and decidual epithelial cells(DECs) of human first-trimester pregnancy for further research.Methods Human first-trimester pregnancy villous and decidual tissues were digested with 0.1% Collagenase IV or 0.25% trypsin or 2.5Kunitz/ml DNAase I. Mesh screen filtration, density gradient centrifugation, differential adhesion and Extra Cellular Matrix gel pretreated methods were employed to isolate and purify the aim cells. Three kinds of cells were identified by immunocytochemistry with Anti-vimentin and Anti-cytokeratin 7. Cell Counting-8 Kit was used to analyze the viability of these cells in vitro.Results TCs, DSCs and DECs could be isolated and cultured successfully in vitro through methods above. The cell purity was over 90%. CCK-8 proliferative assay was confirmed the cells growth activity well.Conclusion TCs, DSCs and DECs of human first-trimester pregnancy for further research could be collected at the same time with this economical and efficient method.Part Ⅱ The expression pattern of Chemokine receptor CXCR7 in TCs, DSCs and DECs of the maternal-fetal immune microenvironment during human first-trimester pregnancy.Objective To explore the expression of protein CXCR7 at the maternal-fetal interface during human first-trimester pregnancy.Methods Human first-trimester pregnancy villous and decidual tissues were collected under the management of Ethics Committee department of Zhongnan Hospital of Wuhan University, And TC, DSC and DEC were gained in the isolation and culture system in Part I. The expression of CXCR7 and CXCR4 were examined by Immunohistochemistry(IHC), immunocytochemistry(ICC), Flow cytometry(FCM) and Indirect immunofluorescence, respectively.Results IHC, ICC and FCM disclosed specific staining for both CXCR7 and CXCR4 protein in TCs,DSCs and DECs, respectively. The mean optical density of CXCR7 in TC and DSC was significantly lower than that of CXCR4(P<0.05). However, The mean optical density of CXCR4 in DEC was significantly lower than that of CXCR7(P<0.05).Conclusion Chemokine CXCL12 and its receptor CXCR7 and CXCR4 are co-expressed in human first-trimester pregnancy TCs, DSCs and DECs, Unequal expression model of CXCR7 may contributes to the establishment of maternal-fetal immune microenvironment.Part III The role of CXCL12/CXCR7/CXCR4 axis at the maternal-fetal immune microenvironment.Objective To investigate the role of the chemokine CXCL12/CXCR4/CXCR7 axis on the crosstalk among human first-trimester TCs, DSCs and DECs, to contribute to a better understanding of the molecular mechanisms on the interaction between the mother and embryo during pregnancy.Methods The expression of CXCR4 and CXCR7 on DECs and DSCs membrane were observed with the human recombination CXCL12(rhCXCL12) and TC conditioned medium (TCM), respectively. CXCL12, CXCR4 and CXCR7 neutralizing antibodies were used to block the CXCL12/CXCR4/CXCR7 axis. The effects of rhCXCL12 or TCM on proliferation and invasion of DSCs were examined by measuring CCK-8 assay and an invasion assay, respectively. Finally, a co-culture model was established to investigate the effect of CXCL 12 secreted from TCs on motility of DSCs.Results RhCXCL12 induced an increase in CXCR4 levels on DSCs via binding to CXCR4 (P<0.05) but had no effect on the proliferation of DSCs. On the contrary, rhCXCL12 has no effect of change the expression level of CXCR7(P>0.05). CCK8 assay showed that both rhCXCL12 and TCM nonparticipation in the proliferation process of DSCs and DECs, respectively. Moreover, both rhCXCL12 and TCM reinforced the invasive ability of DSCs via CXCR4 ligation. A co-culture model further confirmed that the enhanced invasiveness of DSCs in co-culture with TCs and inhibited by anti-CXCR4 or anti-CXCL12 neutralizing antibody, respectively (P< 0.05).Conclusion CXCL12/CXCR4/CXCR7 do not participate in the modulation of DSCs and DECs proliferation in various experimental groups. TC-derived CXCL12 promotes CXCR4 expression and invasion of DSCs via ligation with CXCR4. Our data highlight the role of CXCL12/CXCR4 axis on the co-operation between TCs and DSCs during human first-trimester pregnancy. Based on these observations, it is reasonable to propose that on the long journey of TC penetration into maternal deciduals, DSCs are not merely passively invaded by the embryo, instead, they could actively pave the way for TCs through ECM degradation and movement.