The pregnancy-specific glycoproteins (PSGs) are a family of proteins secreted by the syncytiotrophoblast of the placenta and are the most abundant trophoblastic proteins in maternal blood during the third trimester. The human PSG family consists of 10 protein-coding genes, some of which have a possible role in maintaining maternal immune tolerance to the fetus. PSG9 was reported as a potential predictive biomarker of pre-eclampsia, a serious complication of pregnancy that has been related to immunological dysfunction at the fetal-maternal interface. Therefore, we hypothesized that PSG9 may have an immunoregulatory role during pregnancy. We found that PSG9 binds to LAP and activates the latent form of TGF-β1. In addition, PSG9 induces the secretion of TGF-β1 from macrophages but not from CD4+ T-cells. TGF-β1 is required for the ex vivo differentiation of regulatory T-cells and, consistent with the ability of PSG9 to activate this cytokine, we observed that PSG9 induces the differentiation of FoxP3+ regulatory T-cells from naïve murine and human T-cells. Cytokines that are associated with inflammatory responses were also reduced in the supernatants of T-cells treated with PSG9, suggesting that PSG9, through its activation of TGFβ-1, could be a potent inducer of immune tolerance.

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin α5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to α5β1 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to α5β1 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy.

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We previously reported that binding to heparan sulfate (HS) is required for the ability of the placentally-secreted pregnancy-specific glycoprotein 1 (PSG1) to induce endothelial tubulogenesis. PSG1 is composed of four immunoglobulin-like domains but which domains of the protein bind to HS remains unknown. To analyze the interaction of PSG1 with HS, we generated several recombinant proteins, including the individual domains, chimeric proteins between two PSG1 domains, and mutants. Using flow cytometric and surface plasmon resonance studies, we determined that the B2 domain of PSG1 binds to HS and that the positively charged amino acids encompassed between amino acids 43-59 are required for this interaction. Furthermore, we showed that the B2 domain of PSG1 is required for the increase in the formation of tubes by endothelial cells (EC) including a human endometrial EC line and two extravillous trophoblast (EVT) cell lines and for the pro-angiogenic activity of PSG1 observed in an aortic ring assay. PSG1 enhanced the migration of ECs while it increased the expression of matrix metalloproteinase-2 in EVTs, indicating that the pro-angiogenic effect of PSG1 on these two cell types may be mediated by different mechanisms. Despite differences in amino acid sequence, we observed that all human PSGs bound to HS proteoglycans and confirmed that at least two other members of the family, PSG6 and PSG9, induce tube formation. These findings contribute to a better understanding of the pro-angiogenic activity of human PSGs and strongly suggest conservation of this function among all PSG family members.

SUMMARYTrophoblast invasion is a complex biological process necessary for establishment of pregnancy; however, much remains unknown regarding what signaling factors coordinate the extent of invasion. Pregnancy-specific glycoproteins (PSGs) are some of the most abundant circulating trophoblastic proteins in maternal blood during human pregnancy, with maternal serum concentrations rising to as high as 200-400 μg/mL at term. Here, we employ three-dimensional (3D) trophoblast motility assays consisting of trophoblast spheroids encapsulated in 3D gelatin hydrogels to quantify trophoblast outgrowth area, viability, and cytotoxicity in the presence of PSG1 and PSG9 as well as epidermal growth factor and Nodal. We show PSG9 reduces trophoblast motility whereas PSG1 increases motility. Further, we assess bulk nascent protein production by encapsulated spheroids to highlight the potential of this approach to assess trophoblast response (motility, remodeling) to soluble factors and extracellular matrix cues. Such models provide an important platform to develop a deeper understanding of early pregnancy.Graphical Abstract

Hematopoietic stem cell transplantation is curative for many disorders; however, it can be associated with significant morbidity and mortality, often as a result of graft versus host disease (GvHD). GvHD is an immune mediated reaction in which donor T-cells recognize the host antigens as foreign, causing donor T-cells to proliferate and attack host tissues. Establishment of a tolerogenic immune environment while preserving immune response to infectious agents and malignancy is required for successful bone marrow transplantationand GvHD presents a significant obstacle to this. Pregnancy specific glycoproteins (PSGs) are synthesized by the placenta at the onset of pregnancy and are believed to play a role in maintaining a tolerogenic immune environment to prevent rejection of the fetus by the maternal immune system. Two specific PSG proteins, PSG1 and PSG9, are of particular note as they have both been shown to be involved in pathways devoted to induction of immune tolerance. Both PSG1 and PSG9 are involved in activation of transforming growth factor-β (TGFβ), a cytokine essential to suppression of inflammatory T-cells and important for differentiation of tolerance inducing CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs), a cell population shown to be important in the prevention of GvHD. We thus hypothesized that PSG1 and PSG9 (PSG1/9) could be used to treat or prevent GvHD by inducing immune tolerance through TGFβ. Using surface plasmon resonance (SPR) analysis, we have shown that PSG1/9 bind directly to the latency associated peptide (LAP), a protein that confers latency to mature TGFβ, effectively blocking binding of TGFβ to its receptors. Further, bioassays and ELISA data show an increase in the levels of bioactive TGFβ after treatment with PSG1/9, indicating that these proteins have a role in TGFβ activation through their interaction with LAP. In vitro data using naïve mouse T-cells showed that upon treatment with PSG1, there was a significant increase of CD4+ CD25+ cells expressing FoxP3 (18%±1.5; n=3) compared to only 2% in untreated controls (2%±0.4; n=6) (p<0.0001). Similarly, PSG9 treatment also increased FoxP3 expression (11%±2.8; n=3) over controls (p<0.0001). The increase in FoxP3 expression was also observed upon treatment of primary naïve human T-cells with both PSG1 and PSG9 (PSG1, p=0.0073; PSG9, p=0.0028). When a TGFβ receptor inhibitor was added to cell culture, the increase in FoxP3 expression was effectively blocked, further supporting the hypothesis that PSG1/9 induce expression of FoxP3+ Tregs through regulation of TGFβ. As IL-2 is important for the stability and differentiation of Tregsin vivo and its transcription is suppressed by TGFβ, we performed bioassays on CD4+ naïve T-cells with no added IL-2. We observed that in the absence of added IL-2, treatment with PSG1/9 resulted in an inhibition of IL-2 secretion by activated CD4+ T-cells, and there was no increase in the number and percentage of FoxP3 expressing cells. SBE-luc mice, which express luciferase in response to activation of the TGFβ-SMAD pathway, exhibited significantly increased luciferase expression in the abdomen when injected intraperitoneally with PSG1. This supports the idea that PSG1 induces expression of FoxP3 through the TGFβ receptor pathway in vivo. Finally, using a murine model of GvHD, we observed that mice receiving PSG1 had reduced numbers of infiltrating inflammatory CD3+ T-cells in the colon and showed a marked improvement physically and histologically over untreated controls. In addition, PSG1 treated mice had significantly higher expression of FoxP3 in CD4+ CD25+ splenic cells (30%±9.6; n=4) when compared to untreated GvHD controls (8.6%±3.2; n=3) (p=0.0287). Currently, we are studying the cytokine profiles of mice treated with PSG to determine the efficacy of PSG1/9 in reducing the release of pro-inflammatory cytokines during GvHD. Overall, our data suggests that PSG1/9 induce immune tolerance in GvHD and may be a future treatment option for these patients. Disclosures No relevant conflicts of interest to declare.