Immune‑related pathophysiological causes relevant to a subset of patients with preeclampsia (Review)

Preeclampsia is characterized by inadequate extravillous trophoblast (EVT) invasion, leading to feto-placental hypoxia, a release of an excessof anti-angiogenic factors, and finally eliciting maternal endothelial dysfunction and maternal symptoms, such as new-onset hypertension and proteinuria. Despite intensive research over the past decade, the initial pathogenesis of this disorder remains elusive. There are likely different subtypes of preeclampsia and maternal, fetal and placental factors all play an important role in the disease development. In this review, we focus on an immune-related pathway or pathophysiological causes relevant to a subset of patients with preeclampsia. Using the PubMed database, we conducted a literature review of various studies related to the pathogenesis of preeclampsia. The two-stage theory is a notable postulate of the disease. The antecedents of poor placentation are immunological, epigenetic and environmental factors in origin. The causes of inadequate immune mechanisms at the maternal-fetal interface in preeclampsia is considered to be a lack of human leucocyte antigen (HLA) class I molecules, such as HLA-G and HLA-C. A reduction in HLA molecules facilitate EVTs to elicit an immune attack that furthers cell killing. A series of the HLA-G promoter region on EVTs has been found to be more highly methylated in preeclampsia than in normal pregnancy, possibly due to the increased expression of DNA methyltransferase-1 (DNMT-1). Promoter hypermethylation is one of the major epigenetic alterations that may prime for HLA-G gene inactivation. Epigenetic alterations are reversible, and nutrition, lifestyle and environmental factors may be the epigenetic regulators that modify gene expression. However, the timing, cause and underlying mechanisms of HLA gene methylation have not yet been fully established. The adverse intrauterine environment that contributes to immune responses, inflammation, or oxidative stress may be associated with increased susceptibilities for a number of adult diseases, including preeclampsia. We have hypothesized that close interactions between the inherited epigenetic architecture (hypermethylation of HLA molecules) and adverse intrauterine environmental exposures (oxidative stress and inflammation) leading to epigenetic modifications and to the aberrant DNA methylation of HLA class I molecules, may act as an early event of preeclampsia development.

Related papers:

title: Adverse metabolic phenotype of female offspring exposed to preeclampsia in utero: a characterization of the BPH/5 mouse in postnatal life.
abstract: Preeclampsia (PE) is a devastating disorder of pregnancy that classically presents with maternal hypertension and proteinuria after 20 wk of gestation. In addition to being a leading cause of maternal and fetal morbidity/mortality, epidemiological and prospective studies have revealed long-term consequences for both the mother and baby of preeclamptic pregnancies, including chronic hypertension as well as other cardiovascular diseases and metabolic derangements. To better understand the effect of in utero exposure of PE on offspring, we utilized the BPH/5 mouse, a spontaneous model of the maternal and fetal PE syndrome. We hypothesized that young BPH/5 offspring would have altered metabolic and cardiovascular phenotypes. Indeed, BPH/5 growth-restricted offspring showed excess catch-up growth by early adulthood due to hyperphagia and increased white adipose tissue (WAT) accumulation, with inflammation markers isolated to the reproductive WAT depot only. Both excessive WAT accumulation and the inflammatory WAT phenotype were corrected by pair-feeding young BPH/5 female mice. We also found that young BPH/5 female mice showed evidence of leptin resistance. Indeed, chronic hyperleptinemia has been shown to characterize other rodent models of PE; however, the maternal metabolic profile before pregnancy has not been fully understood. Furthermore, we found that these mice show signs of cardiovascular anomalies (hypertension and cardiomegaly) and altered signaling within the reproductive axis in early life. Future studies will involve challenging the physiological metabolic state of BPH/5 mice through pair-feeding to reduce WAT before pregnancy and determining its causal role in adverse pregnancy outcomes.

title: Preeclampsia and Diabetes
abstract: Preeclampsia is diagnosed in women presenting with new onset hypertension accompanied by proteinuria or other signs of severe organ dysfunction in the second half of pregnancy. Preeclampsia risk is increased 2- to 4-fold among women with type 1 or type 2 diabetes. The limited number of pregnant women with preexisting diabetes and the difficulties associated with diagnosing preeclampsia in women with proteinuria prior to pregnancy are significant barriers to research in this high-risk population. Gestational diabetes mellitus (GDM) also increases preeclampsia risk, although it is unclear whether these two conditions share a common pathophysiological pathway. Nondiabetic women who have had preeclampsia are more likely to develop type 2 diabetes later in life. Among women with type 1 diabetes, a history of preeclampsia is associated with an increased risk of retinopathy and nephropathy. More research examining the pathophysiology, treatment, and the long-term health implications of preeclampsia among women with preexisting and gestational diabetes is needed.

title: Preeclampsia and health risks later in life: an immunological link
abstract: Pregnancy represents a period of physiological stress, and although this stress is experienced for a very modest portion of life, it is now recognized as a window to women’s future health, often by unmasking predispositions to conditions that only become symptomatic later in life. In normal pregnancy, the mother experiences mild metabolic syndrome-like condition through week 20 of gestation. A pronounced phenotype of metabolic syndrome may program pregnancy complications such as preeclampsia. Preeclampsia is a serious complication with a myriad of manifestations for mother and offspring. This pregnancy syndrome is a polygenic disease and has been now linked to higher incidence of cardiovascular disease, diabetes, and several other disorders associated with vulnerable organs. Furthermore, the offspring born to preeclamptic mothers also exhibit an elevated risk of cardiovascular disease, stroke, and mental disorders during adulthood. This suggests that preeclampsia not only exposes the mother and the fetus to complications during pregnancy but also programs chronic diseases in later life. The etiology of preeclampsia is thought to be primarily associated with poor placentation and entails excessive maternal inflammation and endothelial dysfunction. It is well established now that the maternal immune system and the placenta are involved in a highly choreographed cross-talk that underlies adequate spiral artery remodeling required for uteroplacental perfusion and free flow of nutrients to the fetus. Since normal pregnancy is associated with a sequence of events represented by temporal events of inflammation (implantation), anti-inflammation (gestation), and inflammation (parturition), it is quite possible that unscheduled alterations in these regulatory responses may lead to pathologic consequences. Although it is not clear whether immunological alterations occur early in pregnancy, it is proposed that dysregulated systemic and placental immunity contribute to impaired angiogenesis and the onset of preeclampsia. This review will focus on important aspects of the immune system that coordinate with placental dysfunction to program preeclampsia and influence health in later life.

title: Pre-eclampsia: is it all in the placenta?
abstract: Hypertensive disorders of pregnancy complicate almost 7 - 10 % of all pregnancies. The dyad of hypertension and proteinuria after 20 weeks of gestation is referred to as pre-eclampsia. It is a major cause of maternal morbidity and mortality and is also associated with increased perinatal problems. Despite intensive research over the years the exact cause of pre-eclampsia remains unknown. Nevertheless, information gleaned from published studies point to the placenta as the probable pathogenetic focus of pre-eclampsia, as the disease usually resolves within 24 - 48 hours after delivery of the placenta. Although the precise involvement of the placenta in pre-eclampsia remains unclear there are indications that the trophoblastic invasion of the uterine spiral arteries is abnormal in women who develop pre-eclampsia. This impaired invasion leads to decreased placental perfusion and ultimately to placental hypoxia. The distressed or ischaemic placenta then secretes a factor(s) into the maternal circulation, which cause/s widespread endothelial cell dysfunction characterized by vasospasm, activation of coagulation system and organ ischaemia. The cause of the defective cytotrophoblastic invasion of the spiral arteries and the link between placental ischaemia and generalized maternal endothelial dysfunction remain unknown. Although the placenta appears to have a major role in the pathogenesis of pre-eclampsia, evidence also suggests that factors like maternal genetic predisposition, dietary, environmental and behaviour, which surface during the stress of pregnancy might also be involved in the development of pre-eclampsia. It is known that not all women with poor cytotrophoblast invasion develop pre-eclampsia and not all women with preeclampsia show poor cytotrophoblast invasion. Over the years, a number of potential risk factors associated with the development of pre-eclampsia are being recognized and it might be appropriate now to develop some preventative strategies based upon the available information.

title: Circulating angiogenic factors and the risk of preeclampsia*
abstract: The cause of preeclampsia remains unclear. Limited data suggest that excess circulating soluble fms-like tyrosine kinase 1 (sFlt-1), which binds placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), may have a pathogenic role.



We performed a nested case-control study within the Calcium for Preeclampsia Prevention trial, which involved healthy nulliparous women. Each woman with preeclampsia was matched to one normotensive control. A total of 120 pairs of women were randomly chosen. Serum concentrations of angiogenic factors (total sFlt-1, free PlGF, and free VEGF) were measured throughout pregnancy; there were a total of 655 serum specimens. The data were analyzed cross-sectionally within intervals of gestational age and according to the time before the onset of preeclampsia.



During the last two months of pregnancy in the normotensive controls, the level of sFlt-1 increased and the level of PlGF decreased. These changes occurred earlier and were more pronounced in the women in whom preeclampsia later developed. The sFlt-1 level increased beginning approximately five weeks before the onset of preeclampsia. At the onset of clinical disease, the mean serum level in the women with preeclampsia was 4382 pg per milliliter, as compared with 1643 pg per milliliter in controls with fetuses of similar gestational age (P<0.001). The PlGF levels were significantly lower in the women who later had preeclampsia than in the controls beginning at 13 to 16 weeks of gestation (mean, 90 pg per milliliter vs. 142 pg per milliliter, P=0.01), with the greatest difference occurring during the weeks before the onset of preeclampsia, coincident with the increase in the sFlt-1 level. Alterations in the levels of sFlt-1 and free PlGF were greater in women with an earlier onset of preeclampsia and in women in whom preeclampsia was associated with a small-for-gestational-age infant.



Increased levels of sFlt-1 and reduced levels of PlGF predict the subsequent development of preeclampsia.

title: The role of inflammation in the pathology of preeclampsia.
abstract: Preeclampsia (PE) affects 5-7% of all pregnancies in the United States and is the leading cause of maternal and prenatal morbidity. PE is associated with hypertension after week 20 of gestation, decreased renal function and small-for-gestational-age babies. Women with PE exhibit chronic inflammation and production of autoantibodies. It is hypothesized that during PE, placental ischaemia occurs as a result of shallow trophoblast invasion which is associated with an immune imbalance where pro-inflammatory CD4(+) T-cells are increased and T regulatory cells (Tregs) are decreased. This imbalance leads to chronic inflammation characterized by oxidative stress, pro-inflammatory cytokines and autoantibodies. Studies conducted in our laboratory have demonstrated the importance of this immune imbalance in causing hypertension in response to placental ischaemia in pregnant rats. These studies confirm that increased CD4(+) T-cells and decreased Tregs during pregnancy leads to elevated inflammatory cytokines, endothelin (ET-1), reactive oxygen species (ROS) and agonistic autoantibodies to the angiotensin II (Ang II), type 1 receptor (AT1-AA). All of these factors taken together play an important role in increasing the blood pressure during pregnancy. Specifically, this review focuses on the decrease in Tregs, and their associated regulatory cytokine interleukin (IL)-10, which is seen in response to placental ischaemia during pregnancy. This study will also examine the effect of regulatory immune cell repopulation on the pathophysiology of PE. These studies show that restoring the balance of the immune system through increasing Tregs, either by adoptive transfer or by infusing IL-10, reduces the blood pressure and pathophysiology associated with placental ischaemia in pregnant rats.

title: Role of Innate Immunity in Preeclampsia: A Systematic Review
abstract: Innate immune system dysfunction has been known to be a key player in preeclampsia (PE). Activation of the maternal innate immunity may be triggered by invading microorganisms or endogenous ligands, which are detected by different pattern recognition receptors (PRRs). Although some studies have linked PRR activation to PE, it is still unclear if dysregulated PRR expression is associated with the development of this complication. Therefore, we conducted a systematic review of the literature, searching articles that evaluated associations of PRRs with PE. Twenty-six studies met the inclusion criteria: 20 of them analyzed PRR expressions and 6 studies investigated the association between PRR polymorphisms and PE. Among the PRRs, only few studies analyzed retinoic acid–inducible gene I-like helicase (RLH) and/or toll-like receptor (TLR)-1, 5, 6, 7, 8, and 9 expressions in immune cells or placentas from women with PE and controls; thus, it is inconclusive if these PRRs are involved in PE. Results from the 10 studies that analyzed TLR-2 expressions in women with PE and controls are also contradictory. The majority of the studies that investigated TLR-3 and -4 expressions indicate that these PRRs are increased in placenta or immune cells from women with PE compared to pregnant control woman. To date, polymorphisms in TLR-2, -3, and -4 and nucleotide-binding oligomerization domain-like receptor 2 genes do not seem to be associated with PE development. No study has evaluated the association between polymorphisms in genes codifying other TLRs or RLHs genes. In conclusion, available data in literature support a role for TLR-3 and TLR-4 in the pathogenesis of PE.

title: Natural killer T cells in Preeclampsia: An updated review.
abstract: Preeclampsia (PE), as a pregnancy-specific syndrome, has become one of the main causes of maternal and fetal mortality worldwide and is known as a major risk factor for preterm birth. PE is typically characterized by hypertension, significant proteinuria, and an excessive maternal systemic inflammatory response. Recent evidences provide support for the notion that Natural killer T (NKT) cells (a small, but significant immunoregulatory T cell subset of human peripheral blood lymphocytes) play pivotal roles in pregnancy. NKT cells with unique transcriptional and cytokine profiles exist in different peripheral tissues acting as mediators between the innate and adaptive immune systems. NKT cells secrete Interleukin-4 (IL-4) and Interferon-γ (IFN-γ) which might regulate the balance between Type 1T helper (Th1) and Type 2T helper (Th2) responses. During pregnancy, maternal immunity is biased towards type II cytokine production to inhibit the function of type I cytokines that could be harmful for the developing fetus. This shift to type II cytokines does not occur in preeclamptic patients. In this review, we discuss the numbers, phenotype, changes, and the functional activity of Natural killer T (NKT) cells during normal pregnancy and preeclampsia.

title: Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma.
abstract: Pre-eclampsia is a hypertensive disorder characterized by the new onset of hypertension >140/90 mmHg and proteinuria after the 20th week of gestation. The disorder is multifactorial and originates with abnormal placentation. Comparison of the placental proteome of normotensive (n = 25) and pre-eclamptic (n = 25) patients by gel-free proteomic techniques identified a total of 2145 proteins in the placenta of which 180 were differentially expressed (>1.3 fold, p < 0.05). Gene ontology enrichment analysis of biological process suggested that the differentially expressed proteins belonged to various physiological processes such as angiogenesis, apoptosis, oxidative stress, hypoxia, and placental development, which are implicated in the pathophysiology of pre-eclampsia. Some of the differentially expressed proteins were monitored in the plasma by multiple reaction monitoring analysis, which showed an increase in apolipoproteins A-I and A-II in gestational weeks 26-30 (2-fold, p < 0.01), while haptoglobin and hemopexin decreased in gestational weeks 26-30 and week 40/at delivery (1.8 fold, p < 0.01) in pre-eclamptic patients. This study provides a proteomic insight into the pathophysiology of pre-eclampsia. Identified candidate proteins can be evaluated further for the development of potential biomarkers associated with pre-eclampsia pathogenesis.

title: Oxidative stress in the placenta
abstract: Pregnancy is a state of oxidative stress arising from increased placental mitochondrial activity and production of reactive oxygen species (ROS), mainly superoxide anion. The placenta also produces other ROS including nitric oxide, carbon monoxide, and peroxynitrite which have pronounced effects on placental function including trophoblast proliferation and differentiation and vascular reactivity. Excessive production of ROS may occur at certain windows in placental development and in pathologic pregnancies, such as those complicated by preeclampsia and/or IUGR, overpowering antioxidant defenses with deleterious outcome. In the first trimester, establishment of blood flow into the intervillous space is associated with a burst of oxidative stress. The inability to mount an effective antioxidant defense against this results in early pregnancy loss. In late gestation increased oxidative stress is seen in pregnancies complicated by diabetes, IUGR, and preeclampsia in association with increased trophoblast apoptosis and deportation and altered placental vascular reactivity. Evidence for this oxidative stress includes increased lipid peroxides and isoprostanes and decreased expression and activity of antioxidants. The interaction of nitric oxide and superoxide produces peroxynitrite, a powerful prooxidant with diverse deleterious effects including nitration of tyrosine residues on proteins thus altering function. Nitrative stress, subsequent to oxidative stress is seen in the placenta in preeclampsia and diabetes in association with altered placental function.

title: A leading role for the immune system in the pathophysiology of preeclampsia
abstract: Preeclampsia syndrome is characterized by inadequate placentation, because of deficient trophoblastic invasion of the uterine spiral arteries, leading to placental hypoxia, secretion of proinflammatory cytokines, the release of angiogenic and antiangiogenic factors and miRNAs. Although immune‐system alterations are associated with the origin of preeclampsia, other factors, including proinflammatory cytokines, neutrophil activation, and endothelial dysfunction, are also related to the pathophysiology of this syndrome. The pathophysiology of preeclampsia may involve several factors, including persistent hypoxia at the placental level and the release of high amounts of STBMs. DAMP molecules released under hypoxic conditions and STBMs, which bind TLRs, may activate monocytes, DCs, NK cells, and neutrophils, promoting persistent inflammatory conditions in this syndrome. The development of hypertension in preeclamptic women is also associated with endothelial dysfunction, which may be mediated by various mechanisms, including neutrophil activation and NET formation. Furthermore, preeclamptic women have higher levels of nonclassic and intermediate monocytes and lower levels of lymphoid BDCA‐2+ DCs. The cytokines secreted by these cells may contribute to the inflammatory process and to changes in adaptive‐immune system cells, which are also modulated in preeclampsia. The changes in T cell subsets that may be seen in preeclampsia include low Treg activity, a shift toward Th1 responses, and the presence of Th17 lymphocytes. B cells can participate in the pathophysiology of preeclampsia by producing autoantibodies against adrenoreceptors and autoantibodies that bind the AT1‐R.

title: Regulatory T cells in embryo implantation and the immune response to pregnancy
abstract: At implantation, the embryo expresses paternally derived alloantigens and evokes inflammation that can threaten reproductive success. To ensure a robust placenta and sustainable pregnancy, an active state of maternal immune tolerance mediated by CD4+ regulatory T cells (Tregs) is essential. Tregs operate to inhibit effector immunity, contain inflammation, and support maternal vascular adaptations, thereby facilitating trophoblast invasion and placental access to the maternal blood supply. Insufficient Treg numbers or inadequate functional competence are implicated in idiopathic infertility and recurrent miscarriage as well as later-onset pregnancy complications stemming from placental insufficiency, including preeclampsia and fetal growth restriction. In this Review, we summarize the mechanisms acting in the conception environment to drive the Treg response and discuss prospects for targeting the T cell compartment to alleviate immune-based reproductive disorders.

title: Why is placentation abnormal in preeclampsia?
abstract: The causes of preeclampsia remain one of the great medical mysteries of our time. This syndrome is thought to occur in 2 stages with abnormal placentation leading to a maternal inflammatory response. Specific regions of the placenta have distinct pathologic features. During normal pregnancy, cytotrophoblasts emigrate from the chorionic villi and invade the uterus, reaching the inner third of the myometrium. This unusual process is made even more exceptional by the fact that the placental cells are hemiallogeneic, coexpressing maternal and paternal genomes. Within the uterine wall, cytotrophoblasts deeply invade the spiral arteries. Cytotrophoblasts migrate up these vessels and replace, in a retrograde fashion, the maternal endothelial lining. They also insert themselves among the smooth muscle cells that form the tunica media. As a result, the spiral arteries attain the physiologic properties that are required to perfuse the placenta adequately. In comparison, invasion of the venous side of the uterine circulation is minimal, sufficient to enable venous return. In preeclampsia, cytotrophoblast invasion of the interstitial uterine compartment is frequently shallow, although not consistently so. In many locations, spiral artery invasion is incomplete. There are many fewer endovascular cytotrophoblasts, and some vessels retain portions of their endothelial lining with relatively intact muscular coats, although others are not modified. Work from our group showed that these defects mirror deficits in the differentiation program that enables cytotrophoblast invasion of the uterine wall. During normal pregnancy, invasion is accompanied by the down-regulation of epithelial-like molecules that are indicative of their ectodermal origin and up-regulation of numerous receptors and ligands that typically are expressed by endothelial or vascular smooth muscle cells. For example, the expression of epithelial-cadherin (the cell-cell adhesion molecule that many ectodermal derivatives use to adhere to one another) becomes nearly undetectable, replaced by vascular-endothelial cadherin, which serves the same purpose in blood vessels. Invading cytotrophoblasts also modulate vascular endothelial growth factor ligands and receptors, at some point in the differentiation process expressing every (mammalian) family member. Molecules in this family play crucial roles in vascular and trophoblast biology, including the prevention of apoptosis. In preeclampsia, this process of vascular mimicry is incomplete, which we theorize hinders the cells interactions with spiral arterioles. What causes these aberrations? Given what is known from animal models and human risk factors, reduced placental perfusion and/or certain disease states (metabolic, immune and cardiovascular) lie upstream. Recent evidence suggests the surprising conclusion that isolation and culture of cytotrophoblasts from the placentas of pregnancies complicated by preeclampsia enables normalization of their gene expression. The affected molecules include SEMA3B, which down-regulates vascular endothelial growth factor signaling through the PI3K/AKT and GSK3 pathways. Thus, some aspects of the aberrant differentiation of cytotrophoblasts within the uterine wall that is observed in situ may be reversible. The next challenge is asking what the instigating causes are. There is added urgency to finding the answers, because these pathways could be valuable therapeutic targets for reversing abnormal placental function in patients.

title: Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy
abstract: Tissue-specific NK cells are abundant in the pregnant uterus and interact with invading placental trophoblast cells that transform the maternal arteries to increase the fetoplacental blood supply. Genetic case-control studies have implicated killer cell Ig-like receptor (KIR) genes and their HLA ligands in pregnancy disorders characterized by failure of trophoblast arterial transformation. Activating KIR2DS1 or KIR2DS5 (when located in the centromeric region as in Africans) lower the risk of disorders when there is a fetal HLA-C allele carrying a C2 epitope. In this study, we investigated another activating KIR, KIR2DS4, and provide genetic evidence for a similar effect when carried with KIR2DS1. KIR2DS4 is expressed by ∼45% of uterine NK (uNK) cells. Similarly to KIR2DS1, triggering of KIR2DS4 on uNK cells led to secretion of GM-CSF and other chemokines, known to promote placental trophoblast invasion. Additionally, XCL1 and CCL1, identified in a screen of 120 different cytokines, were consistently secreted upon activation of KIR2DS4 on uNK cells. Inhibitory KIR2DL5A, carried in linkage disequilibrium with KIR2DS1, is expressed by peripheral blood NK cells but not by uNK cells, highlighting the unique phenotype of uNK cells compared with peripheral blood NK cells. That KIR2DS4, KIR2DS1, and some alleles of KIR2DS5 contribute to successful pregnancy suggests that activation of uNK cells by KIR binding to HLA-C is a generic mechanism promoting trophoblast invasion into the decidua.

title: Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation.
abstract: Reduced trophoblast invasion and vascular conversion in decidua are thought to be the primary defect of common pregnancy disorders including preeclampsia and fetal growth restriction. Genetic studies suggest these conditions are linked to combinations of polymorphic killer cell Ig-like receptor (KIR) genes expressed by maternal decidual NK cells (dNK) and HLA-C genes expressed by fetal trophoblast. Inhibitory KIR2DL1 and activating KIR2DS1 both bind HLA-C2, but confer increased risk or protection from pregnancy disorders, respectively. The mechanisms underlying these genetic associations with opposing outcomes are unknown. We show that KIR2DS1 is highly expressed in dNK, stimulating strong activation of KIR2DS1+ dNK. We used microarrays to identify additional responses triggered by binding of KIR2DS1 or KIR2DL1 to HLA-C2 and found different responses in dNK coexpressing KIR2DS1 with KIR2DL1 compared with dNK only expressing KIR2DL1. Activation of KIR2DS1+ dNK by HLA-C2 stimulated production of soluble products including GM-CSF, detected by intracellular FACS and ELISA. We demonstrated that GM-CSF enhanced migration of primary trophoblast and JEG-3 trophoblast cells in vitro. These findings provide a molecular mechanism explaining how recognition of HLA class I molecules on fetal trophoblast by an activating KIR on maternal dNK may be beneficial for placentation.

title: HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells.
abstract: Natural killer (NK) cells recognize alloantigens on normal cells. One of these alloantigens correlates with homozygosity for a dimorphism of HLA-C at positions 77-80, which is shared by a number of HLA-C alleles. A second allelic alloantigen correlates with homozygosity for the alternative HLA-C dimorphism, which is shared by the remaining HLA-C alleles. Moreover, NK1- and NK2-specific NK cell lines can be generated by mixed leukocyte cultures in which donor and stimulator are homozygous for the alternative dimorphisms at positions 77-80 of HLA-C. In the present work, the role of HLA-C in NK cell-mediated allorecognition was directly investigated by analyzing the effects produced by transfection of several HLA-C alleles on NK sensitivity of class I-deleted mutant cell lines. Transfection of cells with HLA-C alleles encoding Asn-77-Lys-80 (including HLA-Cw4, -Cw5, and -Cw6) inhibited the lysis of the targets by NK1-specific NK cells, whereas HLA-C alleles encoding Ser-77-Asn-80 (including HLA-Cw1, -Cw7, and -Cw13) protected the targets from NK2-specific NK cells. Thus, HLA-C alleles are the dominant inhibitory ligands that protect targets from lysis by these allospecific NK cells.

title: Crosstalk between human endometrial stromal cells and decidual NK cells promotes decidualization in vitro by upregulating IL-25
abstract: Embryo implantation is essential for a successful pregnancy, and leads to the decidualization of endometrial stromal cells (ESCs) in the secretory phase of the menstrual cycle. It has previously been demonstrated that decidual stromal cells (DSCs) co-express interleukin (IL)-25/IL-17RB and that IL-25 further promotes the proliferation of DSCs via activating c-Jun n-terminal kinase and protein kinase B signals, therefore the present study primarily focused on the role of IL-25 in the process of decidualization in vitro. It was demonstrated that the expression of IL-25/IL-17RB in ESCs was decreased compared with DSCs. In addition, following decidualization, the expression levels of IL-25/IL-17RB in ESCs were significantly elevated. Recombinant human (rh) IL-25 promoted the decidualization of ESCs in the presence of 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt and 6α-methyl17α-acetoxyprogesterone, which was partially inhibited by anti-human IL-25 neutralizing antibody (anti-IL-25) or anti-IL-17RB. In addition, decidual natural killer (dNK) cells not only secreted IL-25, however also further accelerated the decidualization in vitro. Therefore, these findings indicated that ESCs differentiate into DSCs in the presence of ovarian hormones, resulting in the upregulation of IL-25/IL-17RB expression in ESCs. Furthermore, IL-25 secreted by ESCs and dNK cells further facilitates the decidualization of ESCs, which may form a positive feedback mechanism at the maternal-fetal interface and thus contribute to the establishment and maintenance of normal pregnancy.

title: Uterine NK cells and macrophages in pregnancy.
abstract: The presence of immune cells in the placental bed is important for both mother and child. Although various immune cells can be found in the placental bed, such as regulatory T cells and dendritic cells, uterine NK cells and macrophages are the most prominent immune cells in the placental bed in early pregnancy. uNK cell and macrophage numbers in the placental bed decrease in the third trimester. These cells seem to be specifically adapted for their function and environment. uNK cells do not show cytotoxic activity, but are producers of cytokines, growth factors and many other factors. uNK cell function is regulated by inhibitory and activating receptors binding to HLA class I on trophoblast cells. uNK cells are also involved in regulating trophoblast invasion. Macrophages mainly show an M2-like phenotype and also produce cytokines and various other factors. They are important in phagocytosis of various cells and cell debris in the placental bed. Both cell types are also involved in angiogenesis and spiral artery remodeling in the placental bed. In this review we will elaborate on the most important functions of uNK cells and macrophages in the placental bed in humans. We will also discuss animal models, since they may provide clues for function of uNK cells and macrophages in humans.

title: Uterine NK cells: active regulators at the maternal-fetal interface.
abstract: Pregnancy presents an immunological conundrum because two genetically different individuals coexist. The maternal lymphocytes at the uterine maternal-fetal interface that can recognize mismatched placental cells are T cells and abundant distinctive uterine NK (uNK) cells. Multiple mechanisms exist that avoid damaging T cell responses to the fetus, whereas activation of uNK cells is probably physiological. Indeed, genetic epidemiological data suggest that the variability of NK cell receptors and their MHC ligands define pregnancy success; however, exactly how uNK cells function in normal and pathological pregnancy is still unclear, and any therapies aimed at suppressing NK cells must be viewed with caution. Allorecognition of fetal placental cells by uNK cells is emerging as the key maternal-fetal immune mechanism that regulates placentation.

title: Microarray Screening for Novel Preeclampsia Biomarker Candidates
abstract: Our aim was to identify novel biomarker candidates for the near-term prediction of preeclampsia in a homogenous collective. In this study, we screened at the genome-wide level for gene expression in placental villous tissue from patients with severe preeclampsia in comparison to normal healthy pregnancies.  Total RNA was extracted from placental villous tissue from 9 preeclamptic patients and 7 normotensive controls after scheduled cesarean sections. After sample pooling, gene expression analysis was performed using six Affymetrix Human Gene 1.0 ST arrays, followed by quantitative RT-PCR and validation of selected markers in the serum of patients at the protein level.  In total, 896 significantly differentially expressed genes were identified (p ≤ 0.05). After restricting these to molecules present in the circulation, 9 upregulated and 5 downregulated genes were selected. Four of them (β-hCG, HTRA4, LHB1, all upregulated; and NOX4, downregulated) were validated by quantitative real-time RT-PCR. Finally, the maternal plasma protein levels of 2 of these genes (LHB and β-hCG) were confirmed to be significantly different between preeclampsia cases and controls.  We identified 14 potential new biomarker candidates for preeclampsia and validated 4 of them by quantitative RT-PCR and 2 of them with subsequent serum protein analyses. Further studies will assess the optimal marker combination for the imminent prediction of impending preeclampsia.

title: Microarray analysis of differentially expressed fetal genes in placental tissue derived from early and late onset severe pre-eclampsia.
abstract: Although it has been well documented that pre-eclampsia is caused by a combination of maternal and fetal susceptibility genes, little is known about the precise etiology of this complicated disorder. To investigate how the expression of fetal genes contributes to the mechanisms underlying the progression of this disease, we have analyzed differentially expressed genes using placentas from 13 normal pregnancies and 14 pregnancies with severe pre-eclampsia. We performed genome-wide expression profiling using high-density oligonucleotide microarrays, followed by validation using real-time PCR. Among the 47,000 genes that were screened in the microarray, 137 genes were found to be differentially expressed between normal and pre-eclamptic tissues. Among these candidates, 70 were up-regulated and 67 were down-regulated. The up-regulated genes included leptin and inhibin A, which are well-known biological markers for pre-eclampsia, as well as FLT1, which was recently proved to be tightly linked with the etiology of this disease. Gene ontology analysis further revealed several biological processes that could be associated with the development of pre-eclampsia, including response to stress, host-pathogen interactions, lipid metabolism, and carbohydrate metabolism. Analyses of biological mechanisms highlighted some important pathways that may be involved in this disorder, such as the TGF-beta and CEBPA-related pathways. Furthermore, when our present subjects were classified as either severe cases of early onset or late onset pre-eclampsia, the expression of 11 genes could be correlated with the severity of this disorder. These genes may therefore prove to be novel biological markers by which the severity of this condition could be predicted. Our data are likely to be a useful future resource in the elucidation of the disease-process and in the identification of novel markers for pre-eclampsia.

title: Large Scale Aggregate Microarray Analysis Reveals Three Distinct Molecular Subclasses of Human Preeclampsia
abstract: (PE) is a life-threatening hypertensive pathology of pregnancy affecting 3–5% of all pregnancies. To date, PE has no cure, early detection markers, or effective treatments short of the removal of what is thought to be the causative organ, the placenta, which may necessitate a preterm delivery. Additionally, numerous small placental microarray studies attempting to identify “PE-specific” genes have yielded inconsistent results. We therefore hypothesize that preeclampsia is a multifactorial disease encompassing several pathology subclasses, and that large cohort placental gene expression analysis will reveal these groups.  To address our hypothesis, we utilized known bioinformatic methods to aggregate 7 microarray data sets across multiple platforms in order to generate a large data set of 173 patient samples, including 77 with preeclampsia. Unsupervised clustering of these patient samples revealed three distinct molecular subclasses of PE. This included a “canonical” PE subclass demonstrating elevated expression of known PE markers and genes associated with poor oxygenation and increased secretion, as well as two other subclasses potentially representing a poor maternal response to pregnancy and an immunological presentation of preeclampsia.  Our analysis sheds new light on the heterogeneity of PE patients, and offers up additional avenues for future investigation. Hopefully, our subclassification of preeclampsia based on molecular diversity will finally lead to the development of robust diagnostics and patient-based treatments for this disorder.

title: A lesson for cancer research: placental microarray gene analysis in preeclampsia
abstract: Tumor progression and pregnancy share many common features, such as immune tolerance and invasion. The invasion of trophoblasts in the placenta into the uterine wall is essential for fetal development, and is thus precisely regulated. Its deregulation has been implicated in preeclampsia, a leading cause for maternal and perinatal mortality and morbidity. Pathogenesis of preeclampsia remains to be defined. Microarray-based gene profiling has been widely used for identifying genes responsible for preeclampsia. In this review, we have summarized the recent data from the microarray studies with preeclamptic placentas. Despite the complex of gene signatures, suggestive of the heterogeneity of preeclampsia, these studies identified a number of differentially expressed genes associated with preeclampsia. Interestingly, most of them have been reported to be tightly involved in tumor progression. We have discussed these interesting genes and analyzed their potential molecular functions in preeclampsia, compared with their roles in malignancy development. Further investigations are warranted to explore the involvement in molecular network of each identified gene, which may provide not only novel strategies for prevention and therapy for preeclampsia but also a better understanding of cancer cells. The trophoblastic cells, with their capacity for proliferation and differentiation, apoptosis and survival, migration, angiogenesis and immune modulation by exploiting similar molecular pathways, make them a compelling model for cancer research.

title: Transcriptomic Profiling in Human Decidua of Severe Preeclampsia Detected by RNA Sequencing
abstract: Maternal decidua plays a critical role in implantation and placentation. Impaired decidualization causes failed intravascular trophoblast invasion and inadequate placentation and then increases the risk of preeclampsia (PE). RNA sequencing (RNA‐Seq) has achieved great advances in the characterization and quantification of transcriptomes; is a powerful tool for new transcript discovery, genome annotation, and expression profiling. In the present study, we conducted a RNA‐Seq analysis to compare gene expression between decidua of PE (n = 3, early‐onset severe PE, EOSPE; n = 3, late‐onset severe PE, LOSPE) and normal pregnancies (n = 3). We revealed that decidual gene transcription profile was altered in severe PE and identified 293 key PE‐related genes involved in 19 differentially regulated pathways relevant for the pathogenesis of PE, among which ENO2, PGK1, and HK2 involved in glycolysis/gluconeogenesis and HIF‐1 signaling pathway which are all highly related with tumorigenesis and are significantly upregulated in cancer cells were severely inhibited in the decidua of PE. Moreover, we identified 22 core regulatory genes, including the newly identified pseudogenes BNIP3P1, HK2P1, and PGK1P1 that encode long non‐coding RNA (lncRNA); interestingly, BNIP3/BNIP3P1, HK2/HK2P1, and PGK1/PGK1P1 appear in pairs in core genes. Subsequent analyses using quantitative PCR validated a portion of these results. This study may provide further insight into the mechanisms of PE and function as preventive, predictive, and therapeutic measures. Future functional studies are needed in order to accomplish a greater understanding of the mechanisms involved.  2018.

title: RNA Sequencing and Analysis.
abstract: RNA sequencing (RNA-Seq) uses the capabilities of high-throughput sequencing methods to provide insight into the transcriptome of a cell. Compared to previous Sanger sequencing- and microarray-based methods, RNA-Seq provides far higher coverage and greater resolution of the dynamic nature of the transcriptome. Beyond quantifying gene expression, the data generated by RNA-Seq facilitate the discovery of novel transcripts, identification of alternatively spliced genes, and detection of allele-specific expression. Recent advances in the RNA-Seq workflow, from sample preparation to library construction to data analysis, have enabled researchers to further elucidate the functional complexity of the transcription. In addition to polyadenylated messenger RNA (mRNA) transcripts, RNA-Seq can be applied to investigate different populations of RNA, including total RNA, pre-mRNA, and noncoding RNA, such as microRNA and long ncRNA. This article provides an introduction to RNA-Seq methods, including applications, experimental design, and technical challenges.

title: Differentially Expressed Genes in the Pre-Eclamptic Placenta: A Systematic Review and Meta-Analysis
abstract: To systematically review the literature on human gene expression data of placental tissue in pre-eclampsia and to characterize a meta-signature of differentially expressed genes in order to identify novel putative diagnostic markers.  Medline through 11 February 2011 using MeSH terms and keywords related to placenta, gene expression and gene expression arrays; GEO database using the term “placent*”; and reference lists of eligible primary studies, without constraints.  From 1068 studies retrieved from the search, we included original publications that had performed gene expression array analyses of placental tissue in the third trimester and that reported on differentially expressed genes in pre-eclampsia versus normotensive controls. Two reviewers independently identified eligible studies, extracted descriptive and gene expression data and assessed study quality. Using a vote-counting method based on a comparative meta-profiling algorithm, we determined a meta-signature that characterizes the significant intersection of differentially expressed genes from the collection of independent gene signatures.  We identified 33 eligible gene expression array studies of placental tissue in the 3rd trimester comprising 30 datasets on mRNA expression and 4 datasets on microRNA expression. The pre-eclamptic placental meta-signature consisted of 40 annotated gene transcripts and 17 microRNAs. At least half of the mRNA transcripts encode a protein that is secreted from the cell and could potentially serve as a biomarker.  In addition to well-known and validated genes, we identified 14 transcripts not reported previously in relation to pre-eclampsia of which the majority is also expressed in the 1st trimester placenta, and three encode a secreted protein.

title: Landscape of Transcriptional Deregulations in the Preeclamptic Placenta
abstract: Preeclampsia is a pregnancy disease affecting 5 to 8% of pregnant women and a leading cause of both maternal and fetal mortality and morbidity. Because of a default in the process of implantation, the placenta of preeclamptic women undergoes insufficient vascularization. This results in placental ischemia, inflammation and subsequent release of placental debris and vasoactive factors in the maternal circulation causing a systemic endothelial activation. Several microarray studies have analyzed the transcriptome of the preeclamptic placentas to identify genes which could be involved in placental dysfunction. In this study, we compared the data from publicly available microarray analyses to obtain a consensus list of modified genes. This allowed to identify consistently modified genes in the preeclamptic placenta. Of these, 67 were up-regulated and 31 down-regulated. Assuming that changes in the transcription level of co-expressed genes may result from the coordinated action of a limited number of transcription factors, we looked for over-represented putative transcription factor binding sites in the promoters of these genes. Indeed, we found that the promoters of up-regulated genes are enriched in putative binding sites for NFkB, CREB, ANRT, REEB1, SP1, and AP-2. In the promoters of down-regulated genes, the most prevalent putative binding sites are those of MZF-1, NFYA, E2F1 and MEF2A. These transcriptions factors are known to regulate specific biological pathways such as cell responses to inflammation, hypoxia, DNA damage and proliferation. We discuss here the molecular mechanisms of action of these transcription factors and how they can be related to the placental dysfunction in the context of preeclampsia.

title: Biomarkers in pre-eclampsia: A novel approach to early detection of the disease
abstract: Pre-eclampsia is a unique disorder of human pregnancy with a great impact on maternal and perinatal morbidity and mortality worldwide and especially in developing countries. The aetiology is still unknown and the pathophysiology of the disease is the subject of extensive investigation. Recently, much of the interest of the investigators for the prediction of pre-eclampsia has been aimed at measurable manifestations of abnormal placentation, endothelial dysfunction and feto-maternal unit perfusion. Biomarkers constitute a novel approach to an early detection of the disease. Low maternal serum levels of PAPP-A and PP13 early in pregnancy are predictive for emerging pre-eclampsia. On the other hand, increased levels of homocysteine, ADMA, sEng, leptin and sFlt-1 in the 1st trimester, signal the onset of the disease later in pregnancy. After the onset of pre-eclampsia, increased serum levels of PAPP-A, ADMA, homocysteine and sFlt-1 are associated with the severity of the disease. The identification of biomarkers which can contribute to the early detection of pre-eclampsia is essential. It could then be possible to apply better surveillance and treatment protocols in such patients.

title: Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology
abstract: We provide evidence of a decidualization defect in the endometrium of women with severe preeclampsia (PE) that was detected at the time of delivery and persisted years after the affected pregnancy. We went on to link this defect to impaired cytotrophoblast invasion. The transcriptional signature of the defect could enable its detection before (or after) conception, which would aid the development of therapies focused on improving decidualization and perhaps preventing severe PE. In preeclampsia (PE), cytotrophoblast (CTB) invasion of the uterus and spiral arteries is often shallow. Thus, the placenta’s role has been a focus. In this study, we tested the hypothesis that decidual defects are an important determinant of the placental phenotype. We isolated human endometrial stromal cells from nonpregnant donors with a previous pregnancy that was complicated by severe PE (sPE). Compared with control cells, they failed to decidualize in vitro as demonstrated by morphological criteria and the analysis of stage-specific antigens (i.e., IGFBP1, PRL). These results were bolstered by global transcriptional profiling data that showed they were transcriptionally inert. Additionally, we used laser microdissection to isolate the decidua from tissue sections of the maternal–fetal interface in sPE. Global transcriptional profiling revealed defects in gene expression. Also, decidual cells from patients with sPE, which dedifferentiated in vitro, failed to redecidualize in culture. Conditioned medium from these cells failed to support CTB invasion. To mimic aspects of the uterine environment in normal pregnancy, we added PRL and IGFBP1, which enhanced invasion. These data suggested that failed decidualization is an important contributor to down-regulated CTB invasion in sPE. Future studies will be aimed at determining whether this discovery has translational potential with regard to assessing a woman’s risk of developing this pregnancy complication.

title: Emerging role for dysregulated decidualization in the genesis of preeclampsia.
abstract: In normal human placentation, uterine invasion by trophoblast cells and subsequent spiral artery remodeling depend on cooperation among fetal trophoblasts and maternal decidual, myometrial, immune and vascular cells in the uterine wall. Therefore, aberrant function of anyone or several of these cell-types could theoretically impair placentation leading to the development of preeclampsia. Because trophoblast invasion and spiral artery remodeling occur during the first half of pregnancy, the molecular pathology of fetal placental and maternal decidual tissues following delivery may not be informative about the genesis of impaired placentation, which transpired months earlier. Therefore, in this review, we focus on the emerging prospective evidence supporting the concept that deficient or defective endometrial maturation in the late secretory phase and during early pregnancy, i.e., pre-decidualization and decidualization, respectively, may contribute to the genesis of preeclampsia. The first prospectively-acquired data directly supporting this concept were unexpectedly revealed in transcriptomic analyses of chorionic villous samples (CVS) obtained during the first trimester of women who developed preeclampsia 5 months later. Additional supportive evidence arose from investigations of Natural Killer cells in first trimester decidua from elective terminations of women with high resistance uterine artery indices, a surrogate for deficient trophoblast invasion. Last, circulating insulin growth factor binding protein-1, which is secreted by decidual stromal cells was decreased during early pregnancy in women who developed preeclampsia. We conclude this review by making recommendations for further prospectively-designed studies to corroborate the concept of endometrial antecedents of preeclampsia. These studies could also enable identification of women at increased risk for developing preeclampsia, unveil the molecular mechanisms of deficient or defective (pre)decidualization, and lead to preventative strategies designed to improve (pre)decidualization, thereby reducing risk for preeclampsia development.

title: Identification of key microRNAs and genes in preeclampsia by bioinformatics analysis
abstract: Preeclampsia is a leading cause of perinatal maternal–foetal mortality and morbidity. The aim of this study is to identify the key microRNAs and genes in preeclampsia and uncover their potential functions. We downloaded the miRNA expression profile of GSE84260 and the gene expression profile of GSE73374 from the Gene Expression Omnibus database. Differentially expressed miRNAs and genes were identified and compared to miRNA-target information from MiRWalk 2.0, and a total of 65 differentially expressed miRNAs (DEMIs), including 32 up-regulated miRNAs and 33 down-regulated miRNAs, and 91 differentially expressed genes (DEGs), including 83 up-regulated genes and 8 down-regulated genes, were identified. The pathway enrichment analyses of the DEMIs showed that the up-regulated DEMIs were enriched in the Hippo signalling pathway and MAPK signalling pathway, and the down-regulated DEMIs were enriched in HTLV-I infection and miRNAs in cancers. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses of the DEGs were performed using Multifaceted Analysis Tool for Human Transcriptome. The up-regulated DEGs were enriched in biological processes (BPs), including the response to cAMP, response to hydrogen peroxide and cell-cell adhesion mediated by integrin; no enrichment of down-regulated DEGs was identified. KEGG analysis showed that the up-regulated DEGs were enriched in the Hippo signalling pathway and pathways in cancer. A PPI network of the DEGs was constructed by using Cytoscape software, and FOS, STAT1, MMP14, ITGB1, VCAN, DUSP1, LDHA, MCL1, MET, and ZFP36 were identified as the hub genes. The current study illustrates a characteristic microRNA profile and gene profile in preeclampsia, which may contribute to the interpretation of the progression of preeclampsia and provide novel biomarkers and therapeutic targets for preeclampsia.

title: Consensus strategy in genes prioritization and combined bioinformatics analysis for preeclampsia pathogenesis
abstract: BackgroundPreeclampsia is a multifactorial disease with unknown pathogenesis. Even when recent studies explored this disease using several bioinformatics tools, the main objective was not directed to pathogenesis. Additionally, consensus prioritization was proved to be highly efficient in the recognition of genes-disease association. However, not information is available about the consensus ability to early recognize genes directly involved in pathogenesis. Therefore our aim in this study is to apply several theoretical approaches to explore preeclampsia; specifically those genes directly involved in the pathogenesis.MethodsWe firstly evaluated the consensus between 12 prioritization strategies to early recognize pathogenic genes related to preeclampsia. A communality analysis in the protein-protein interaction network of previously selected genes was done including further enrichment analysis. The enrichment analysis includes metabolic pathways as well as gene ontology. Microarray data was also collected and used in order to confirm our results or as a strategy to weight the previously enriched pathways.ResultsThe consensus prioritized gene list was rationally filtered to 476 genes using several criteria. The communality analysis showed an enrichment of communities connected with VEGF-signaling pathway. This pathway is also enriched considering the microarray data. Our result point to VEGF, FLT1 and KDR as relevant pathogenic genes, as well as those connected with NO metabolism.ConclusionOur results revealed that consensus strategy improve the detection and initial enrichment of pathogenic genes, at least in preeclampsia condition. Moreover the combination of the first percent of the prioritized genes with protein-protein interaction network followed by communality analysis reduces the gene space. This approach actually identifies well known genes related with pathogenesis. However, genes like HSP90, PAK2, CD247 and others included in the first 1% of the prioritized list need to be further explored in preeclampsia pathogenesis through experimental approaches.

title: DNA methylation profiles in preeclampsia and healthy control placentas.
abstract: Preeclampsia is a hypertensive disorder of pregnancy that affects 3-5% of all pregnancies. There is evidence to suggest that epigenetic mechanisms, such as DNA methylation, play a role in placental development and function. This study compared DNA methylation profiles of placentas from preeclampsia-affected pregnancies with placentas from healthy pregnancies to identify gene-specific changes in DNA methylation that may contribute to the development of preeclampsia. The methylation status of eight placental biopsies taken from preeclampsia-affected and 16 healthy pregnancies was analyzed using the Illumina Infinium Methylation 450 BeadChip array. Bisulfite pyrosequencing was used to confirm regions found to be differentially methylated between preeclampsia and healthy placentas. A total of 303 differentially methylated regions, 214 hypermethylated and 89 hypomethylated, between preeclampsia cases and controls were identified, after adjusting for gestational age (adjusted P < 0.05). Functional annotation found cell adhesion, wingless type MMTV Integration Site family member 2 (Wnt) signaling pathway, and regulation of transcription were significantly enriched in these gene regions. Hypermethylation of WNT2, sperm equatorial segment protein (SPESP1), NADPH oxidase 5 (NOX5), and activated leukocyte cell adhesion molecule (ALCAM) in preeclampsia placentas was confirmed with pyrosequencing. This study found differences in methylation in gene regions involved in cell signaling (WNT2), fertilization and implantation (SPESP1), reactive oxygen species signaling (NOX5), and cell adhesion (ALCAM). These results build on recently published studies that have reported significant differences in DNA methylation in preeclampsia placentas.

title: Low Placental Growth Factor Across Pregnancy Identifies a Subset of Women With Preterm Preeclampsia: Type 1 Versus Type 2 Preeclampsia?
abstract: Preeclampsia is a heterogeneous syndrome affecting 3% to 5% of all pregnancies. An imbalance of the antiangiogenic and proangiogenic factors, soluble receptor fms-like tyrosine kinase 1 and placental growth factor (PGF), is thought to contribute to the pathophysiology of preeclampsia. Maternal plasma PGF and soluble receptor fms-like tyrosine kinase 1 were quantified by specific immunoassays in cross-sectional samples from 130 preeclamptic subjects and 342 normotensive controls at delivery and longitudinally in samples from 50 women who developed preeclampsia and 250 normotensive controls. Among women who developed preeclampsia, 46% (n=23) evidenced a pattern of consistently low maternal PGF across pregnancy below the lower 95% CI of controls from 15 weeks' gestation to term. In contrast, the remaining 54% (n=27) of women who developed preeclampsia had maternal PGF concentrations similar to or above (n=7) those of normotensive controls. Subjects with low PGF across pregnancy who developed preeclampsia evidenced significantly higher blood pressure in early pregnancy (P<0.05) and, after diagnosis, earlier gestational age at delivery (P<0.05) and more preterm birth (P<0.05) compared with preeclamptic patients with high PGF. A significant subset of women who develop preeclampsia show evidence of consistently low PGF across pregnancy. Low PGF with preeclampsia was associated with preterm delivery compared with preeclamptic patients with high PGF. Identifying women with consistently low plasma PGF during pregnancy may provide a greater understanding of preeclampsia pathophysiology and may provide more focused research and clinical activities.

title: Variation of maternal KIR and fetal HLA-C genes in reproductive failure: too early for clinical intervention.
abstract: A distinctive type of (uterine) natural killer (NK) cell is present in the uterine decidua during the period of placental formation. Uterine NK cells express members of the killer immunoglobulin-like receptor (KIR) family that bind to parental HLA-C molecules on the invading placental trophoblast cells. The maternal KIR genes and their fetal ligands are highly variable, so different KIR/HLA-C genetic combinations occur in each pregnancy. Some women only possess inhibitory KIR genes, whereas other women also express activating KIR genes. The overall signal that NK cells receive from paternal HLA-C on trophoblast depends on the ratio of activating and inhibitory KIR genes expressed by them. Therefore, NK cells provide a balance during placentation to ensure maternal survival and an adequately nourished fetus. Because inhibitory KIRs are found more frequently in women with defective placentation, e.g. pre-eclampsia, fetal growth restriction or recurrent spontaneous abortion, some fertility clinics suggest that women should be 'tissue typed' for their KIR genotypes. We explain why, presently, it is premature to introduce KIR and HLA-C typing to predict pregnancy outcome. In future, however, selecting for certain combinations of KIR and HLA-C variants in surrogacy, egg or sperm donation may prove useful to reduce disorders of pregnancy.

title: Subsets of human natural killer cells and their regulatory effects
abstract: Human natural killer (NK) cells have distinct functions as NKtolerant, NKcytotoxic and NKregulatory cells and can be divided into different subsets based on the relative expression of the surface markers CD27 and CD11b. CD27+ NK cells, which are abundant cytokine producers, are numerically in the minority in human peripheral blood but constitute the large population of NK cells in cord blood, spleen, tonsil and decidua tissues. Recent data suggest that these NK cells may have immunoregulatory properties under certain conditions. In this review, we will focus on these new NK cell subsets and discuss how regulatory NK cells may serve as rheostats or sentinels in controlling inflammation and maintaining immune homeostasis in various organs.

title: The immunosuppressive molecule HLA-G and its clinical implications
abstract: Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) class I molecule that, through interaction with its receptors, exerts important tolerogenic functions. Its main physiological expression occurs in placenta where it seems to participate in the maternal tolerance toward the fetus. HLA-G has been studied as a marker of pregnancy complications such as abortion or pre-eclapmsia. Although HLA-G is not expressed in most adult tissues, its ectopic expression has been observed in some diseases such as viral infections, autoimmune disorders, and especially cancer. HLA-G neo-expression in cancer is associated with the capability of tumor cells to evade the immune control. In this review, we will summarize HLA-G biology and how it participates in these physiopathological processes. Special attention will be paid to its role as a diagnostic tool and also as a therapeutic target.

title: Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2.
abstract: Many common disorders of pregnancy are attributed to insufficient invasion of the uterine lining by trophoblast, fetal cells that are the major cell type of the placenta. Interactions between fetal trophoblast and maternal uterine NK (uNK) cells--specifically interactions between HLA-C molecules expressed by the fetal trophoblast cells and killer Ig-like receptors (KIRs) on the maternal uNK cells--influence placentation in human pregnancy. Consistent with this, pregnancies are at increased risk of preeclampsia in mothers homozygous for KIR haplotype A (KIR AA). In this study, we have demonstrated that trophoblast expresses both paternally and maternally inherited HLA-C surface proteins and that maternal KIR AA frequencies are increased in affected pregnancies only when the fetus has more group 2 HLA-C genes (C2) than the mother. These data raise the possibility that there is a deleterious allogeneic effect stemming from paternal C2. We found that this effect also occurred in other pregnancy disorders (fetal growth restriction and recurrent miscarriage), indicating a role early in gestation for these receptor/ligand pairs in the pathogenesis of reproductive failure. Notably, pregnancy disorders were less frequent in mothers that possessed the telomeric end of the KIR B haplotype, which contains activating KIR2DS1. In addition, uNK cells expressed KIR2DS1, which bound specifically to C2+ trophoblast cells. These findings highlight the complexity and central importance of specific combinations of activating KIR and HLA-C in maternal-fetal immune interactions that determine reproductive success.

title: A deficiency of placental IL-10 in preeclampsia.
abstract: Accommodation of the fetoplacental unit in human pregnancy requires maternal immune tolerance to this "semiallograft". Local antiplacental immunity is modified by synthesis of uncommon histocompatibility Ags (e.g., HLA-G), growth factors, and cytokines by the placenta. Placental interleukins have been identified in reproductive tissues, but their roles in adaptive maternal immunity and determining term pregnancy outcomes have not been fully clarified. This study examined the distribution of IL-10 and TNF-alpha staining in term placentas. Women with proteinuric hypertension (PE, n = 10) were compared with an age-matched group with normal pregnancy (NP, n = 14) and gestational hypertension (GH, n = 6). Using immunohistochemistry of parrafin-fixed tissues, trophoblast cells were identified by cytokeratin 7 and cytokeratin 18 staining. The cytokine binding of villous trophoblast cells was scored depending on the extent of circumferential cytoplasm staining (<25%; intermediate or >75%). The cytokine positive decidual cells were scored as a percentage of total extravillous trophoblast cells. There was a reduction in villous IL-10 immunostaining compared with normal term placenta (PE, 10.2 +/- 1.1, mean +/- SEM; NP, 14.07 +/- 1.16 Mann-Whitney U test; p = 0.02). In these patients, there was an increase in TNF-alpha immunostaining. Sparse endovascular extravillous trophoblast cells demonstrated nuclear IL-10 staining in 30% of patients with preeclampsia. Serum IL-10 was diminished in women with preeclampsia compared with normal pregnancy. In conclusion, villous trophoblast demonstrated diminished immunostaining of IL-10 in preeclampsia. This abnormality may be associated with heightened maternal antifetal immunity and therefore inadequate placental development in preeclampsia.

title: HLA Class Ib Molecules and Immune Cells in Pregnancy and Preeclampsia
abstract: Despite decades of research, the highly prevalent pregnancy complication preeclampsia, “the disease of theories,” has remained an enigma. Indeed, the etiology of preeclampsia is largely unknown. A compiling amount of studies indicates that the pathological basis involves a complex array of genetic predisposition and immunological maladaptation, and that a contribution from the mother, the father, and the fetus is likely to be important. The Human Leukocyte Antigen (HLA)-G is an increasing focus of research in relation to preeclampsia. The HLA-G molecule is primarily expressed by the extravillous trophoblast cells lining the placenta together with the two other HLA class Ib molecules, HLA-E and HLA-F. Soluble isoforms of HLA-G have been detected in the early endometrium, the matured cumulus–oocyte complex, maternal blood of pregnant women, in umbilical cord blood, and lately, in seminal plasma. HLA-G is believed to be involved in modulating immune responses in the context of vascular remodeling during pregnancy as well as in dampening potential harmful immune attacks raised against the semi-allogeneic fetus. In addition, HLA-G genetic variants are associated with both membrane-bound and soluble forms of HLA-G, and, in some studies, with preeclampsia. In this review, a genetic contribution from the mother, the father, and the fetus, together with the presence and function of various immune cells of relevance in pregnancy are reviewed in relation to HLA-G and preeclampsia.

title: HLA non-A,B,C class I genes: Their structure and expression
abstract: Clearly, the human genome includes a group of genes closely related to but distinct from the HLA class I genes encoding the HLA-A, -B, and -C major transplantation antigens. These non-A,B,C class I genes, designated as HLA-E, HLA-F, and HLA-G, are on the short arm of chromosome 6 and part of the HLA class I gene family. Although the human HLA-E, -F, and -G genes have features in common with the murine Qa- and Tla-genes, e.g. little allelic polymorphism, their relationship to the murine Qa- and Tla-region genes remains unclear. It has been suggested that the nonclassical MHC class I molecules function as ligands for gamma-delta T lymphocytes. The speculation is supported by the recent reports of a murine Qa-1 restricted gamma-delta T cell hybridoma and recognition of a TL antigen by gamma delta T cell receptors. The amino acid sequences of the HLA-E, -F, and -G encoded proteins suggest that each protein is likely to fold three-dimensionally into a structure very similar to HLA-A2 and has a capability of presenting a bound peptide at the cell surface. In light of the possible role of bound peptide in the expression of a class I molecule at the cell surface, it is interesting to note that the HLA-E and HLA-F molecules, even in association with beta 2-microglobulin, could not be detected at the cell surface of a transfected B-LCL. In contrast, the HLA-G molecule was found at the surface of transfected B-LCLs. Both HLA-E and HLA-F are less similar in sequence to HLA-A,B,C than is HLA-G. One explanation would be that the HLA-E and -F molecules have a mutation such that they are no longer able to bind peptide. If the HLA-G molecule does function to present peptide to T lymphocytes, there are features unique to HLA-G that should impact on its ability to perform this function. Both the analysis of HLA-G RNA and protein in trophoblasts indicate that HLA-G, unlike HLA-A, -B, -C, is relatively nonpolymorphic. Since HLA-A,B,C polymorphism is thought to increase the number of different peptides that these molecules can bind, HLA-G is likely to be able to bind a relatively limited variety of peptides. HLA-G also differs from HLA-A, -B, and -C in that it seems to only be expressed by placental amniochorionic trophoblasts.(ABSTRACT TRUNCATED AT 400

title: [Expression of NKG2A and NKG2C receptors and their ligand HLA-E in decidua of preeclampsia patients].
abstract: To investigatethe expressions of NKG2A, NKG2C receptors and their ligand HLA-E in decidua of preeclampsia patients.



Decidua tissues were collected from 30 patients with mild preeclampsia, 42 patients with severe preeclampsia and 46 normal pregnancy as contrast. The expressions of NKG2A, NKG2C protein were detected by immunohistochemitry and mRNAs of NKG2A, NKG2C and HLA-E genes were detected by RT-PCR.



The expression of HLA-E mRNA was significantly decreased in preeclampsia, especially in severe preeclampsia patients (P < 0.05); The mRNA and protein expression of NKG2A, NKG2C in severe preeclampsia group were significantly higher than that in normal pregnancy group (P < 0.05); In the severe preeclampsia group, the ratio of expression level of NKG2A and NKG2C was significantly lower than that of normal group and the mRNA and protein expression of NKG2C receptor were both significantly higher than that of NKG2A (P < 0.05); The mRNA expression level of HLA-E, NKG2A and NKG2C were closely related with clinical and biochemical indexes,such as blood pressure of late pregnancy and 24-hour proteinuria.



The decreased expression of HLA-E and the unevenly increase of NKG2A and NKG2C may involve in the pathogenesis of preeclampsia.

title: Preimplantation Factor (PIF) Promotes HLA-G, -E, -F, -C Expression in JEG-3 Choriocarcinoma Cells and Endogenous Progesterone Activity
abstract: Pregnancy success requires mandatory maternal tolerance of the semi/ allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and –C) and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells.  PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis.  In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling.  PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders.

title: Hypermethylation of the HLA-G promoter is associated with preeclampsia.
abstract: Preeclampsia (PE) is a severe pregnancy-induced disorder characterized by hypertension and proteinuria and a leading cause of perinatal maternal-fetal mortality and morbidity in developing countries. Dysregulated human leukocyte antigen (HLA)-G was found in placentas as well as in maternal sera from PE patients; however, the reason for this difference is unknown. As accumulating evidence has confirmed that DNA methylation is an important mechanism for regulating gene expression, we sought to test the hypothesis that alteration in the DNA methylation of the HLA-G promoter region is responsible for decreased expression of HLA-G in PE. Bisulfite pyrosequencing showed that a series of CpG sites in the HLA-G promoter region were significantly more highly methylated in PE than in normal pregnancy (NP). Interestingly, the hypermethylated CpG sites were mostly reported to be binding sites of active transcription factors. To further investigate the regulation of HLA-G methylation, we also defined the expression patterns of DNA methyltransferases (DNMTs) in placental tissue using immunohistochemistry and quantitative polymerase chain reaction analyses. Here, we demonstrate that DNMT-1 is overexpressed and HLA-G expression is reduced in PE women when compared with NP. Furthermore, both treatment with the DNMT inhibitor 5-aza-2'-deoxycytidine and specific knockdown of DNMT-1 using siRNAs can significantly increase the expression level of HLA-G in a trophoblastic cell line, indicating the potential mechanism of DNMT-1-mediated DNA methylation in HLA-G regulation. Taken together, our research confirms that DNMT-1-mediated promoter hypermethylation of HLA-G is associated with PE. These findings provide new insights into the diagnosis and treatment of PE.

title: Altered Expression of Human Leukocyte Antigen G (HLA‐G) on Extravillous Trophoblasts in Preeclampsia: Immunohistological Demonstration With Anti‐HLA‐G Specific Antibody “87G” and Anti‐cytokeratin Antibody “CAM5.2”
abstract: Human leukocyte antigen‐G (HLA‐G) is suggested to be at play in the materno‐fetal immune relationship during pregnancy. In the light of current concept that disruption of the materno‐fetal immune relationship could account for several complications of pregnancy, including preeclampsia, we asked whether the expression of HLA‐G protein on the trophoblasts is altered in preeclampsia.

title: The maternal HLA-G 1597ΔC null mutation is associated with increased risk of pre-eclampsia and reduced HLA-G expression during pregnancy in African-American women.
abstract: The non-classical major histocompatibility complex molecule, human leukocyte antigen (HLA)-G, is thought to contribute to maternal immune tolerance and successful placentation during pregnancy. Genetic polymorphisms in HLA-G are known to influence expression levels as well as the relative expression of individual protein isoforms. As diminished or aberrant HLA-G expression patterns may contribute to the development of certain pregnancy complications, we sought to investigate the association between functional HLA-G polymorphisms and the risk of pre-eclampsia (PE) in African-American women. The association between maternal and fetal genotype at six HLA-G polymorphisms and risk of PE was assessed in 372 pregnancies (314 normotensive; 58 pre-eclamptic). We observed an elevated risk of PE (P = 0.00027) in pregnancies where the mother carried the 1597ΔC allele, a null allele that abolishes expression of full-length HLA-G isoforms. Furthermore, the frequency of the maternal 1597ΔC allele was highest in the subset of pre-eclamptic pregnancies that were delivered preterm, suggesting an association between the null allele and the severity of PE. We then replicated the association between higher maternal 1597ΔC allele frequency and increased severity of PE (P = 0.038) in an independent sample of 533 African-American women. Finally, to investigate the mechanistic basis of this association, we measured circulating soluble HLA-G (sHLA-G) concentrations in maternal serum collected during pregnancy in 51 healthy, normotensive African-American control women and found significantly lower levels in women carrying the 1597ΔC allele (P = 0.012). These results demonstrate that maternal HLA-G genotype is significantly associated with risk of PE in African-American women and is predictive of circulating sHLA-G levels during pregnancy.

title: Regulatory role of tryptophan degradation pathway in HLA-G expression by human monocyte-derived dendritic cells.
abstract: Dendritic cells (DC) are strong inducers of immunity but they can also be tolerogenic. During monocyte differentiation to DC the immunosuppressive indoleamine-2,3-dioxygenase (IDO) is induced. IDO degrades Trp to kynurenine, which is further metabolized to 3-hydroxyanthranilic acid. DC can also express mRNA and protein of the tolerogenic molecule HLA-G, but there is no surface expression. We studied the effect of the Trp degrading pathway on HLA-G expression by DC. When monocytes were differentiated to immature DC in presence of either Trp or its metabolites kynurenine or 3-hydroxyanthranilic acid they expressed cell surface HLA-G, and Trp also increased shedding of HLA-G1. Trp induced HLA-G cell surface expression when present during maturation with IFN-gamma+LPS, but not with TNF-alpha. Kynurenine increased HLA-G expression in both TNF-alpha and IFN-gamma+LPS matured DC, and 3-hydroxyanthranilic acid had a very weak effect on HLA-G cell surface expression when present during maturation. Shedding of HLA-G1 was more pronounced in IFN-gamma+LPS-matured DC than in immatured DC. Maturation with IFN-gamma+LPS in presence of kynurenine also increased HLA-G5 secretion. The mechanism involved seems to be post-translational as mRNA and cellular HLA-G protein content was not increased with Trp, kynurenine or 3-hydroxyanthranilic acid treatments. Finally, immature DC preincubated with Trp, kynurenine and 3-hydroxyanthranilic acid have after a decreased capacity to stimulate T cells in mixed lymphocyte reaction. In IFN-gamma+LPS-matured DC this decreased capacity was obtained with kynurenine and 3-hydroxyanthranilic acid. These results suggest that IDO can induce HLA-G cell surface expression in DC, and that these two molecules can cooperate in the immune suppression.

title: A Specific Interferon (IFN)-stimulated Response Element of the Distal HLA-G Promoter Binds IFN-regulatory Factor 1 and Mediates Enhancement of This Nonclassical Class I Gene by IFN-β*
abstract: Type I interferons display a broad range of immunomodulatory functions. Interferon β increases gene expression at the transcriptional level through binding of factors to the interferon-stimulated response element (ISRE) within the promoters of interferon-inducible genes, such as HLA class I. Despite mutation of the class I ISRE sequence within the nonclassical HLA-G class I gene promoter, we show that interferon β enhances both transcription and cell surface expression of HLA-G in trophoblasts and amniotic and thymic epithelial cells that selectively express it in vivo. Deletion and mutagenesis analysis of a putative interferon-regulatory factor (IRF)-1 binding site within the HLA-G promoter show that HLA-G transactivation is mediated through an ISRE sequence 746 base pairs upstream from ATG, which is distinct from the interferon-responsive element described within proximal classical class I gene promoters. Electrophoretic mobility shift analysis and supershift analysis further demonstrate that interferon-responsive transcription factors, including IRF-1, specifically bind to the HLA-G ISRE. Our results provide evidence that IRF-1 binding to a functional ISRE within the HLA-G promoter mediates interferon β-induced expression of the HLA-G gene. These observations are of general interest considering the implication of HLA-G in mechanisms of immune escape involved in fetal-maternal tolerance and other immune privilege situations.

title: Quantified Colocalization Reveals Heterotypic Histocompatibility Class I Antigen Associations on Trophoblast Cell Membranes: Relevance for Human Pregnancy1
abstract: Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic associations.

title: NLRP2 is a suppressor of NF-B signaling and HLA-C expression in human trophoblasts
abstract: During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.  By modulating the NF-KB pathway and HLA-C expression on human trophoblasts, NLRP2, may contribute to preventing detrimental inflammatory responses at the maternal-fetal interface.

title: Epigenetic mechanisms for nutrition determinants of later health outcomes.
abstract: Epigenetic marking on genes can determine whether or not genes are expressed. Epigenetic regulation is mediated by the addition of methyl groups to DNA cytosine bases, of methyl and acetyl groups to proteins (histones) around which DNA is wrapped, and by small interfering RNA molecules. Some components of epigenetic regulation have evolved to permit control of whether maternal or paternal genes are expressed. The epigenetic imprinting of IGF2 expression is an example of maternal and paternal epigenetic marking that modulates fetal growth and fetal size. However, epigenetic regulation also permits the fetus and the infant to adapt gene expression to the environment in which it is growing; sometimes when this adjustment goes awry, the risk of chronic disease is increased. Recent progress in the understanding of nutritional influences on epigenetics suggests that nutrients that are part of methyl-group metabolism can significantly influence epigenetics. During critical periods in development, dietary methyl-group intake (choline, methionine, and folate) can alter DNA and histone methylation, which results in lifelong changes in gene expression. In rodent models, pregnant dams that were fed diets high in methionine, folic acid, and choline produced offspring with different coat colors or with kinked tails. A number of syndromes in humans can be caused by defective epigenetic regulation, including Rett syndrome. There are interesting examples of the effects of nutrition in early life that result in altered health in adults, and some of these could be the result of altered epigenetic regulation of gene expression.

title: 5-Hydroxymethylcytosine-mediated alteration of transposon activity associated with the exposure to adverse in utero environments in human.
abstract: Preeclampsia and gestational diabetes mellitus (GDM) are the most common clinical conditions in pregnancy that could result in adverse in utero environments. Fetal exposure to poor environments may raise the long-term risk of postnatal disorders, while epigenetic modifications could be involved. Recent research has implicated involvement of 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine, via oxidation by ten-eleven translocation (TET) enzymes, in DNA methylation-related plasticity. Here, we show that the TET2 expression and 5hmC abundance are significantly altered in the umbilical veins of GDM and preeclampsia. Genome-wide profiling of 5hmC revealed its specific reduction on intragenic regions from both GDM and preeclampsia compared to healthy controls. Gene Ontology analysis using loci bearing unique GDM- and preeclampsia-specific loss-of-5hmC indicated its impact on several critical biological pathways. Interestingly, the substantial alteration of 5hmC on several transposons and repetitive elements led to their differential expression. The alteration of TET expression, 5hmC levels and 5hmC-mediated transposon activity was further confirmed using established hypoxia cell culture model, which could be rescued by vitamin C, a known activator of TET proteins. Together, these results suggest that adverse pregnancy environments could influence 5hmC-mediated epigenetic profile and contribute to abnormal development of fetal vascular systems that may lead to postnatal diseases.

title: Ten-eleven translocation 2 demethylates the MMP9 promoter, and its down-regulation in preeclampsia impairs trophoblast migration and invasion
abstract: Preeclampsia is the most common clinical disorder in pregnancy and might result from disordered uterine environments caused by epigenetic modifications, including deregulation of DNA methylation/demethylation. Recent research has indicated that 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) via oxidation by ten-eleven translocation (TET) enzymes, is involved in DNA methylation–related plasticity. Here, we report that TET2 expression and 5hmC abundance are significantly altered in the placentas from preeclampsia patients. shRNA-mediated TET2 knockdown (shTET2) reduced trophoblast migration and invasion when cultured in Matrigel. Both real-time PCR of matrix metalloproteinase (MMP)–related transcripts and a human angiogenesis antibody array indicated that TET2 knockdown in trophoblasts inhibits the expression of MMP transcript, of which MMP9 represented one of the most significant TET2 downstream targets. Using an established shTET2 HTR-8/SVneo cell model, we further confirmed alterations of 5hmC levels and MMP9 expression at both mRNA and protein levels. In particular, we found that TET2 bound to and removed 5mC modifications at the MMP9 promoter region. Interestingly, in TET2 knockdown cells, both MMP9 expression and the compromised trophoblast phenotype could be rescued by vitamin C, an activator of TET enzyme activity. Finally, TET2 expression correlated with MMP9 levels in placenta samples from the preeclampsia patients, indicating that TET2 deregulation is critically involved in the pathogenesis of preeclampsia through down-regulation of MMP9 expression. Our findings highlight a critical role of TET2 in regulating trophoblast cell migration through demethylation at the MMP9 promoter, and suggest that down-regulation of the TET2–MMP9–mediated pathway contributes to preeclampsia pathogenesis.

title: The role of methylation, DNA polymorphisms and microRNAs on HLA-G expression in human embryonic stem cells.
abstract: The human leukocyte antigen (HLA)-G gene seems to play a pivotal role in maternal tolerance to the fetus. Little is known about HLA-G expression and its molecular control during in vivo human embryogenesis. Human embryonic stem cells (hESC) provide an interesting in vitro model to study early human development. Different studies reported discrepant findings on whether HLA-G mRNA and protein are present or absent in hESC. Several lines of evidence indicate that promoter CpG methylation and 3' untranslated region (3'UTR) polymorphisms may influence HLA-G expression. We investigated how HLA-G expression is linked to the patterns of promoter methylation and explored the role of the 3'UTR polymorphic sites and their binding microRNAs on the post-transcriptional regulation of HLA-G in eight hESC lines. We showed that, while the gross expression levels of HLA-G are controlled by promoter methylation, the genetic constitution of the HLA-G 3'UTR, more specifically the 14bp insertion in combination with the +3187A/A and +3142G/G SNP, plays a major role in HLA-G mRNA regulation in hESC. Our findings provide a solid first step towards future work using hESC as tools for the study of early human developmental processes in normal and pregnancy-related disorders such as preeclampsia.

title: Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-gamma inducible promoter (PIV) of CIITA.
abstract: Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

title: Hypermethylation of HLA class I gene is associated with HLA class I down-regulation in human gastric cancer.
abstract: Down-regulated expression of human leukocyte antigen (HLA) class I molecules in many human cancers facilitate tumor cells to escape from immune attack. Promoter hypermethylation, one of the major epigenetic changes responsible for gene inactivation, plays an important role in gastric carcinogenesis. This study evaluated the expression and alteration of HLA class I molecules in a panel of 47 pairs of gastric cancer specimens with their noncancerous parts from Chinese patients by using immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP) analysis. The expression of HLA-A, HLA-B/C and HLA class I complex was lost or down-regulated in human gastric cancer. The percentage of promoter methylation was 59.57% for HLA-A gene, 55.32% for HLA-B gene and 48.94% for HLA-C gene in gastric cancer, while it was decreased to 19.15%, 12.77% and 6.38% in the adjacent nontumor tissues, respectively. Seven of 10 (70%), 4 of 6 (66.7%) and 3 of 4 (75%) gastric cancer specimens with promoter hypermethylation at HLA-A, -B and -C loci showed transcriptional inactivation of HLA-A,-B and -C genes, suggesting an association between promoter hypermethylation and down-regulated expression of HLA class I molecules. Human gastric cancer cell line BGC-823 showed HLA-A down-regulation with promoter methylation of HLA-A locus. Treatment with DNA methyltransferase inhibitor restored the expression of HLA-A mRNA and surface HLA-A complex. Thus, our results showed that promoter hypermethylation might be one of the mechanisms that lead to HLA class I antigen down-regulation in gastric cancer.

title: Mechanisms of Endothelial Dysfunction in Hypertensive Pregnancy and Preeclampsia.
abstract: Preeclampsia is a pregnancy-related disorder characterized by hypertension and could lead to maternal and fetal morbidity and mortality. Although the causative factors and pathophysiological mechanisms are unclear, endothelial dysfunction is a major hallmark of preeclampsia. Clinical tests and experimental research have suggested that generalized endotheliosis in the systemic, renal, cerebral, and hepatic circulation could decrease endothelium-derived vasodilators such as nitric oxide, prostacyclin, and hyperpolarization factor and increase vasoconstrictors such as endothelin-1 and thromboxane A2, leading to increased vasoconstriction, hypertension, and other manifestation of preeclampsia. In search for the upstream mechanisms that could cause endothelial dysfunction, certain genetic, demographic, and environmental risk factors have been suggested to cause abnormal expression of uteroplacental integrins, cytokines, and matrix metalloproteinases, leading to decreased maternal tolerance, apoptosis of invasive trophoblast cells, inadequate spiral arteries remodeling, reduced uterine perfusion pressure (RUPP), and placental ischemia/hypoxia. RUPP may cause imbalance between the antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin and the proangiogenic factors vascular endothelial growth factor and placental growth factor, or stimulate the release of other circulating bioactive factors such as inflammatory cytokines, hypoxia-inducible factor-1, reactive oxygen species, and angiotensin AT1 receptor agonistic autoantibodies. These circulating factors could then target endothelial cells and cause generalized endothelial dysfunction. Therapeutic options are currently limited, but understanding the factors involved in endothelial dysfunction could help design new approaches for prediction and management of preeclampsia.

title: Inflammatory and Immune System Markers.
abstract: Since preeclampsia was first described by Hippocrates in 400 BC, the theory of its causation has shifted from toxins to a current theory that incorporates both vascular and immunological causation. Poor placentation whether it is genetically predisposed or due to low expression of defective HLA-G on fetal trophoblasts is believed to be the initial insult. Oxidative stress from placental ischemia/hypoxia leads to an overload of trophoblast debris by stimulating apoptosis or necrosis. Partial failure of the maternal immune system to tolerate the paternal alloantigens activates maternal immune cells to secrete cytokines whose pleiotropic functions lead to dysfunction of the maternal vascular and placental endothelium, blood coagulation, and fibrinolytic system. This chapter describes some of the key methodologies (flow cytometry, ELISAs, and multiplex immunoassays) for the identification and quantification of inflammation and immune system markers in the study of preeclampsia pathogenesis, as well as diagnostic and therapeutic development. The methodologies may be utilized for a variety of tissue sources in the study of preeclampsia: maternal peripheral blood, umbilical cord blood, intervillous blood, decidua, chorionic villous, amnion and chorion membranes, and cell culture supernatant.