Regulation Of Hyaluronan On Maternal-Fetal Immunotolerance In Human Early Pregnancy

The embryo expresses foreign antigens to mother, and thus has been viewed as allograft. Induction of maternal tolerance to persistence of the alloantigen is a main purpose for maintenance of pregnancy. It has been proven that maternal-fetal interface exhibits a Th2bias, including IL-4, IL-5and IL-10cytokines during pregnancy. Failure to establish such an immune milieu or Thl bias, such as interferon (IFN)-y and tumor necrosis factor (TNF)-a, is associated with miscarriage. Regulatory T cells (Treg) play an important role in preventing from the autoimmune rejection to transplantation. It has been found that Treg expands during pregnancy, but the mechanism underlining the expansion still needs to be elucidated.Hyaluronan (HA) is a non-sulfated glycosaminoglycan polymer of repeating disaccharide units of N-acetylglucosamine and P-glucuronic acid. HA is a prominent component of extracellular matrices (ECM), particularly in rapidly growing and remodeling tissues. The transmembrane glycoprotein, CD44, is the predominant receptor for HA on cell surfaces. Binding of HA to CD44has been implicated in lymphocyte homing, tumorigenesis, and monocyte activation, and also is involved in cell proliferation, differentiation, migration and cell adhesion, contributing to tumor angiogenesis and metastatic spread. Evidence shows that HA accumulates at maternal-fetal interface, and CD44expresses in the placenta throughout the pregnancy. However, the regulation of HA-CD44interaction on maternal-fetal immunotolerance remains unknown. In the present study, we propose to investigate the expression and regulation of HA and its receptor at maternal-fetal interface in human early pregnancy.Part I The expression of HA and its receptor CD44at maternal-fetal interface Objective:To determine the expression of HA and its receptor CD44at maternal-fetal interface and to analyze the regulation of pregnancy-associated hormones on hyaluronan synthetase (HAS) expression and HA production.Methods:Immunochemistry, Real Time RT PCR, ELISA, polyacrylamide gel electrophoresis and flow cytometry (FCM) were used to assess the HA expression, HAS transcription level, HA content, HA molecular size and CD44expression in decidua and villi from normal pregnancy and unexplained miscarriage, respectively. We also investigated the effect of pregnancy-associated hormones on HAS expression and HA level in the primary human DSCs.Results:HA and CD44were both positive staining in decidua and villi; HA is expressed in the cytoplasm and extracellular matrix while CD44was expressed on the cytomembrane. The primary DSCs and trophoblasts secreted HA constitutively with the level of HA produced by DSCs and trophoblasts peaked at1032.0±73.83ng/ml and686.4±57.1ng/ml, respectively. Either the mRNA level of HAS2or the level or molecular size of HA was significantly higher in normal pregnancy than in miscarriage. Progesterone,17(β-estrodial and β-hCG all promoted the transcription of HAS2and secretion of HA from the primary DSCs.Conclusion:DSCs were the major source of HA at maternal-fetal interface, and CD44highly expressed on DSCs. There was an significant increase of level and molecular size of HA in normal pregnancy compared with that from miscarriage. Pregnancy-related hormones enhance HA production.Part Ⅱ Autocrine regulation of hyaluronan on the biological behaviors of decidual stromal cells and trophoblasts via binding to CD44Objective:To investigate the effect of HA on the proliferation, apoptosis of DSCs and trophoblasts, as well as the invasion of trophoblasts, and to investigate the involved intracellular signal pathways.Methods:BrdU proliferation assay, Annexin V-FITC apoptosis assay and transwell matrigel invasion assay were used to analyze the effects of different molecular weight HA on proliferation, apoptosis of DSC and trophoblasts, and invasion of trophoblasts in vitro, respectively. In cell western was utilized to analyze the signal pathways activated by HA. Specific signaling antagonists were used to block intracellular signal transduction to confirm the involved intracellular signal pathway.Results:High molecular weight-HA (HMW-HA) promoted proliferation and inhibited apoptosis of DSCs, but both HMW-HA and MMW-HA increased the proliferation and deceased apoptosis of trophoblasts significantly, and MMW-HA showed a more obvious enhancement on the invasion of trophoblasts than HMW-HA. HA antagonist peptide and CD44neutralizing antibody could completely or partially abolish the up-regulation of exogenous HA. PI3K/AKT and MAPK/ERK1/2signal pathway was activated by HMW-HA stimulation, and LY294002and U0126abrogated the regulation of HA-CD44on the biological behaviors of DSCs and trophoblasts.Conclusion:HMW-HA and MMW-HA enhanced growth of DSCs, and growth and invasion trophoblasts via binding to CD44. The PI3K/AKT and MAPK/ERK1/2signal pathways were differently involved in the functional regulation of HA-CD44on DSCs and trophoblasts biological behaviors.Part III Induction of HA on the immunotolerant phenotype of CD14~+monocytes and macrophages at maternal-fetal interface*Objective:To explore the regulation of different molecular weight HA and the supernatants from DSCs on the phenotype of CD14~+peripheral monocytes and decidual macrophages.Methods:The differential expression of CD44on all types of decidual immune cells (DICs) was analyzed by FCM. CD14~+peripheral monocytes and decidual macrophages, isolated by Miltenyi magnetic beads, were stimulated by exogenous different molecular weight HA and supernatants from DSCs for48h. The expression of the MHC-II, co-stimulatory molecules and the intracellular production of cytokines was detected by FCM.Results:CD44was highly expressed on decidual CD14~+macrophages and regulatory T cells. HMW-HA and supernatants from DSCs down-regulated CD80and CD86, up-regulated PD-L1and PD-L2, with no significant influence on the expression of HLA-DR and ICOS-L on CD14~+peripheral monocytes and decidual macrophages. HA antagonist peptide and CD44neutralizing antibody could attenuate the effects. HMW-HA promoted the secretion of IL-10and TGF-β1of CD14~+monocytes, whereas LMW-HA suppressed the production of IL-10and TGF-β1of CD14~+monocytes. CD14~+decidual macrophages from normal early pregnancy exhibited lower expression of CD80,CD86and IL-12p40, whereas higher level of PD-L1, PD-L2, IL-10and TGF-β1compared to that from unexplained miscarriage.Conclusion:HMW-HA and supernatants from DSCs instructed CD14+peripheral monocytes and decidual macrophages to immunotolerant phenotype.Part IV pCD14~+monocytes educated by HMW-HA and DCM induced pNaive CD4~+T cells to differentiate into a Th2bias and Treg expansionObjective:To elucidate the induction of HMW-HA-pCD14~+monocytes or DCM-pCD14~+monocytes to pNaive CD4~+T cells differentiation.Methods:pCD14~+monocytes educated by HMW-HA and supernatants from DSCs for48h, after washing, co-cultured with pNaive CD4~+T cells for5days. The expression of the co-stimulatory molecules receptors and the intracellular cytokines and transcription factor was analyzed by FCM.Results:HMW-HA-pCD14~+and DCM-pCD14~+monocytes induced the pNaive CD4~+T cells to up-regulate the expression of CTLA-4and PD-1, to secret significantly higher level of IL-4and IL-10, to increase the transcription factor GATA-3, meanwhile, to inhibit the production of IFN-γ and TNF-a, to down-regulate transcription factor T-bet, thus attributing to a Th2immune bias. pNaive CD4~+T cells induced by HMW-HA-pCD14~+and DCM-pCD14~+monocytes secreted higher level of TGF-β1and showed higher expression of Foxp3, which is beneficial to boost Treg enrichment and maintain normal pregnancy.Conclusion:pCD14~+monocytes educated by HMW-HA and supernatants from DSCs can induce pNaive CD4+T cells to differentiation into a Th2bias and Treg expansion, which contributes to immunotolerance at maternal-fetal interface.In summary, primary DSCs and trophoblasts from normal pregnancy produce higher level of HA, whose molecular size averages higher than that from miscarriage. CD44, the main receptor of HA, is detected at maternal-fetal interface. HA-CD44interaction enhances proliferation of DSCs and invasion of trophoblasts while inhibits their apoptosis through P13K/AKT and MAPK/ERK1/2signal pathways, implying that HA plays an important role in implantation and placentation. HMW-HA and supernatants from DSCs instruct CD14~+peripheral monocytes and decidual macrophages to up-regulate the negative co-stimulatory molecules, to down-regulate the positive co-stimulatory molecules, and to secret high level of IL-10and TGF-β1, thus leading the allogenous pNaive CD4~+T cells to differentiate into Th2and Treg subtypes, which builds an immune environment favoring successful pregnancy. Our research indicates that high level and mature state of HA is beneficial to the maintenance of normal pregnancy, while low content and degradation of HA may associate with unexplained miscarriage.