Debra Goldman-Wohl

Regulation of trophoblast invasion: from normal implantation to pre-eclampsia

Conversion of the maternal spiral arteries into larger competent vessels is one of the essential steps in the development of the normal placenta. This process is apparently dependent on the invasion by trophoblasts of the sub-endometrial area and the spiral arteries. Preeclampsia is characterized by shallow trophoblast invasion and unconverted narrow spiral arteries. This leads to fetal hypoxia that causes endothelial injury that eventually manifest as maternal hypertension, edema, and proteinuria. The following steps have been shown to be involved in the breakthrough of the trophoblasts from the uterine cavity into the decidua and the spiral arteries: trophoblast targeting, adhesion, and detachment from the extracellular matrix (ECM), invasion of the ECM and maternal vessels by proteolysis. Abnormal expression and activity of these molecules may explain in part some of the molecular mechanisms leading to abnormal placentation and the development of preeclampsia.

Novel APC-like properties of human NK cells directly regulate T cell activation

Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane-enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell-mediated cytotoxicity and specific ligand recognition by cell surface-activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell-activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2-deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell-activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells.

A Novel Human-Specific Soluble Vascular Endothelial Growth Factor Receptor 1: Cell Type-Specific Splicing and Implications to Vascular Endothelial Growth Factor Homeostasis and Preeclampsia

A human-specific splicing variant of vascular endothelial growth factor (VEGF) receptor 1 (Flt1) was discovered, producing a soluble receptor (designated sFlt1-14) that is qualitatively different from the previously described soluble receptor (sFlt1) and functioning as a potent VEGF inhibitor. sFlt1-14 is generated in a cell type-specific fashion, primarily in nonendothelial cells. Notably, in vascular smooth muscle cells, all Flt1 messenger RNA is converted to sFlt1-14, whereas endothelial cells of the same human vessel express sFlt1. sFlt1-14 expression by vascular smooth muscle cells is dynamically regulated as evidenced by its upregulation on coculture with endothelial cells or by direct exposure to VEGF. Increased production of soluble VEGF receptors during pregnancy is entirely attributable to induced expression of placental sFlt1-14 starting by the end of the first trimester. Expression is dramatically elevated in the placenta of women with preeclampsia, specifically induced in abnormal clusters of degenerative syncytiotrophoblasts known as syncytial knots, where it may undergo further messenger RNA editing. sFlt1-14 is the predominant VEGF-inhibiting protein produced by the preeclamptic placenta, accumulates in the circulation, and hence is capable of neutralizing VEGF in distant organs affected in preeclampsia. Together, these findings revealed a new natural VEGF inhibitor that has evolved in humans, possibly to protect nonendothelial cells from adverse VEGF signaling. Furthermore, the study uncovered the identity of a VEGF-blocking protein implicated in preeclampsia.

Endometrial NK Cells Are Special Immature Cells That Await Pregnancy

NK cells populate the human endometrium before pregnancy. Unlike decidual NK cells that populate the decidua during pregnancy, the NK cells present in the human endometrium, before pregnancy, have not been fully characterized. In this study, we provide a detailed analysis of the origin, phenotype, and function of endometrial NK cells (eNK). We show that eNK cells have a unique receptor repertoire. In particular, they are negative for NKp30 and chemokine receptor expression, which distinguishes them from any other NK subset described so far. We further show that eNK cells lack NK-specific functional phenotype and activity such as cytokine secretion and cytotoxicity, before IL-15 stimulation. Following such stimulation, endometrial NK cells acquire phenotype and function that are similar to those of decidual NK cells. We therefore suggest that eNK cells are inactive cells (before IL-15 activation and in relation to the known NK activity) that are present in the endometrium before conception, waiting for pregnancy.

Complexes of HLA-G Protein on the Cell Surface Are Important for Leukocyte Ig-Like Receptor-1 Function

The nonclassical class I MHC molecule HLA-G is selectively expressed on extravillous cytotrophoblast cells at the maternal-fetal interface during pregnancy. HLA-G can inhibit the killing mediated by NK cells via interaction with the inhibitory NK cell receptor, leukocyte Ig-like receptor-1 (LIR-1). Comparison of the sequence of the HLA-G molecule to other class I MHC proteins revealed two unique cysteine residues located in positions 42 and 147. Mutating these cysteine residues resulted in a dramatic decrease in LIR-1 Ig binding. Accordingly, the mutated HLA-G transfectants were less effective in the inhibition of NK killing and RBL/LIR-1 induced serotonin release. Immunoprecipitation experiments demonstrated the involvement of the cysteine residues in the formation of HLA-G protein oligomers on the cell surface. The cysteine residue located at position 42 is shown to be critical for the expression of such complexes. These oligomers, unique among the class I MHC proteins, probably bind to LIR-1 with increased avidity, resulting in an enhanced inhibitory function of LIR-1 and an impaired killing function of NK cells.

Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions

Lymphocytes in direct contact with embryonic extravillous trophoblasts constitute more than 40% of decidual cells and appear to play major roles in implantation and early gestation. A unique subset of NK cells, making up 70-80% of decidual lymphocytes, express high levels of CD56 but lack CD16. We have recently demonstrated a novel class I MHC-independent inhibitory mechanism of NK cell cytotoxicity that is mediated by CEACAM1 homotypic interactions. This mechanism is used by some melanoma cells to avoid attack, mainly by CD16(-) NK cells. We now demonstrate that CEACAM1 is expressed on primary extravillous trophoblasts and is upregulated on the vast majority of IL-2-activated decidual lymphocytes, including NK, T, and NKT cells. Importantly, we present evidence that CEACAM1 interactions inhibit the lysis, proliferation, and cytokine secretion of activated decidual NK, T, and NKT cells, respectively. In vivo analysis of decidual lymphocytes isolated from cytomegalovirus-infected (CMV-infected) pregnant women revealed a dramatic increase in the expression of CEACAM1. Finally, we suggest that a novel ligand for this adhesion molecule is present on the surface of CMV-infected fibroblasts. These combined results demonstrate a major role for the CEACAM1 protein in controlling local decidual immune responses.

The CD85J/leukocyte inhibitory receptor-1 distinguishes between conformed and beta 2-microglobulin-free HLA-G molecules.

For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with β2m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.

MiRNA-Mediated Control of HLA-G Expression and Function

HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT) of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3′UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3′UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G.

Inhibitory NK receptor recognition of HLA-G: regulation by contact residues and by cell specific expression at the fetal-maternal interface.

The non-classical HLA-G protein is distinguished from the classical MHC class I molecules by its expression pattern, low polymorphism and its ability to form complexes on the cell surface. The special role of HLA-G in the maternal-fetal interface has been attributed to its ability to interact with specific receptors found on maternal immune cells. However this interaction is restricted to a limited number of receptors. In this study we elucidate the reason for this phenomenon by comparing the specific contact residues responsible for MHC-KIR interactions. This alignment revealed a marked difference between the HLA-G molecule and other MHC class I molecules. By mutating these residues to the equivalent classical MHC residues, the HLA-G molecule regained an ability of interacting with KIR inhibitory receptors found on NK cells derived either from peripheral blood or from the decidua. Functional NK killing assays further substantiated the binding results. Furthermore, double immunofluorescent staining of placental sections revealed that while the conformed form of HLA-G was expressed in all extravillous trophoblasts, the free heavy chain form of HLA-G was expressed in more distal cells of the column, the invasion front. Overall we suggest that HLA-G protein evolved to interact with only some of the NK inhibitory receptors thus allowing a control of inhibition, while permitting appropriate NK cell cytokine and growth factor production necessary for a viable maternal fetal interface.

Modeling of Human Cytomegalovirus Maternal-Fetal Transmission in a Novel Decidual Organ Culture

ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital infection, associated with severe birth defects and intrauterine growth retardation. The mechanism of HCMV transmission via the maternal-fetal interface is largely unknown, and there are no animal models for HCMV. The initial stages of infection are believed to occur in the maternal decidua. Here we employed a novel decidual organ culture, using both clinically derived and laboratory-derived viral strains, for the ex vivo modeling of HCMV transmission in the maternal-fetal interface. Viral spread in the tissue was demonstrated by the progression of infected-cell foci, with a 1.3- to 2-log increase in HCMV DNA and RNA levels between days 2 and 9 postinfection, the expression of immediate-early and late proteins, the appearance of typical histopathological features of natural infection, and dose-dependent inhibition of infection by ganciclovir and acyclovir. HCMV infected a wide range of cells in the decidua, including invasive cytotrophoblasts, macrophages, and endothelial, decidual, and dendritic cells. Cell-to-cell viral spread was revealed by focal extension of infected-cell clusters, inability to recover infectious extracellular virus, and high relative proportions (88 to 93%) of cell-associated viral DNA. Intriguingly, neutralizing HCMV hyperimmune globulins exhibited inhibitory activity against viral spread in the decidua even when added at 24 h postinfection—providing a mechanistic basis for their clinical use in prenatal prevention. The ex vivo-infected decidual cultures offer unique insight into patterns of viral tropism and spread, defining initial stages of congenital HCMV transmission, and can facilitate evaluation of the effects of new antiviral interventions within the maternal-fetal interface milieu.