Although pre-eclampsia (PE), a hypertensive disorder of pregnancy, has significant maternal and fetal morbidity and mortality worldwide, the mechanisms contributing to this disease have not been fully elucidated. Studies in patients and experimental models have shown that changes in the number or function of immune cells of both the adaptive and innate immune systems contribute to the development and pathogenesis of PE. This commentary summarizes our current understanding of the role of the immune system in the pathogenesis of PE, specifically focussing on dysfunction of natural killer (NK) cells and T lymphocyte populations.
Pre-eclampsia (PE) is a highly variable and heterogeneous syndrome that is defined by new-onset hypertension after 20 weeks of gestation and one or more of the following diagnostic criteria: new-onset proteinuria, thrombocytopenia, impaired liver function, renal insufficiency, pulmonary edema, or visual or cerebral disturbances . PE affects 2–8% of pregnancies worldwide and is a leading cause of maternal and perinatal morbidity and mortality . The exact etiology of PE is unknown, partially due to the heterogeneous nature of the disease; however, it is thought that a defect in trophoblast differentiation and invasion into the maternal myometrium, leading to poor uterine spiral artery remodeling, is an initiating event in the pathogenesis of the disease . The impaired spiral artery remodeling leads to placental ischemia, resulting in the release of factors into the maternal circulation that account for the clinical manifestations of the disease. It is believed by many investigators that an abnormal maternal immune response against the fetus is an initiating factor in the disease process, which is then followed by a systemic inflammatory response involving leukocytes and the endothelium . Cells of both the innate and adaptive immune systems, including neutrophils, monocytes, natural killer (NK) cells, and T lymphocytes, have been implicated in both the initiation of the disease process and in the later pathogenesis of PE. This commentary will focus on recent advances in our understanding of the role of the immune system in the pathogenesis of PE, specifically NK and T lymphocyte dysfunction.
NK cells in pregnancy and PE
NK cells are large granular lymphocytes that develop in the bone marrow from common lymphoid progenitor cells. NK cells are cytotoxic toward tumor cells and infected cells and also have the ability to produce cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ, which can influence adaptive immune responses . Approximately 90% of NK cells in the peripheral blood are highly cytotoxic and possess the CD56dimCD16+ surface phenotype, while the other 10% have a CD56brightCD16− phenotype and limited cytotoxicity [5,6]. In a healthy pregnancy, the percentages of NK cells in the peripheral blood tend to increase during the first trimester, decrease in the second trimester, and decrease again in the third trimester . Studies in patients with recurrent spontaneous abortion have shown that pregnancy loss is associated with increased activity of peripheral blood NK cells . In addition, alterations in circulating NK cells have been suggested by Sargent et al.  to have a causative role in the pathogenesis of PE. Specifically, women with PE have a lower level of CD56bright cells that express the natural cytotoxicity receptor NKp46. This phenotypic change in the circulating NK cells is apparent several months before the onset of symptoms .
Uterine NK (uNK) cells are a unique uterine-specific lymphoid lineage that are present in the human endometrium prior to pregnancy and progressively expand during implantation and throughout the first trimester [5,11]. After the first trimester, the uNK cells become progressively less granular and decrease in number, leaving very few uNK cells at term . The origin of uNK cells has been studied for several decades, and it is currently thought that they are a heterogeneous population of cells derived from both peripheral blood NK cells that home to the uterus as well as in situ progenitors . uNK cells have features of both NK cell subsets present in the peripheral blood and have a CD56brightCD16− surface phenotype. Similar to the minor peripheral blood population of CD56brightCD16− NK cells, uNK cells display limited cytotoxicity, as they do not kill trophoblast cells and are only weakly cytotoxic against cancer cell lines . The cytotoxic activity is preserved, however, because the stimulation of the NKp46 receptor in freshly isolated uNK cells results in effective target cell lysis . Siewiera et al.  found that uNK cells can efficiently lyse autologous target cells infected with human cytomegalovirus (HCMV) in vitro, and that the uNK cells also infiltrate HCMV infected trophoblast cells in trophoblast villi explant cultures. These data suggest that uNK cells may be important in preventing the spread of viral infections.
In both humans and mice, uNK cells participate in spiral artery remodeling with trophoblast cells . Hanna et al.  demonstrated that uNK cells have a limited ability to kill semiallogeneic trophoblast cells, and instead they regulate trophoblast invasion by producing the chemokines interleukin (IL)-8 and IFN-inducible protein (IP)-10. In addition, uNK cells induce vascular growth by secreting angiogenic factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) . These results were echoed in studies by Lash et al. , which found that uNK cells are the primary decidual source of the angiogenic factors angiogenin, transforming growth factor-β (TGF-β1), and VEGF-C within the placental bed. Both peripheral and uNK cells express an array of receptors, including killer immunoglobulin-like receptors (KIR) and NK group 2 (NKG2) A/C/E receptors that recognize MHC Class I, stress, and adhesion molecules on the surface of cells. Trophoblast cells express a unique combination of three MHC Class I molecules: human leukocyte antigen (HLA)-G, HLA-E, and HLA-C [19–21], which are recognized by KIR and NKG2 A/C/E receptors. Studies by Hiby et al.  indicated that pregnancies in which the maternal KIR genotype is AA and the fetus possesses HLA-C2 on trophoblast cells have a markedly increased risk of becoming preeclamptic. HLA-G protects trophoblast cells from uNK cell lysis, but studies by Pazmany et al.  showed that patients with severe PE have reduced expression of HLA-G. Based on these studies, it has been hypothesized that appropriate interactions between uNK cells and trophoblasts are crucial to placental development and maternal spiral artery modifications, and these interactions are not optimal in patients with PE . Furthermore, PE is associated with the elevated expression of anti-angiogenic factors such as soluble endoglin (sENG) and soluble fms-like tyrosine kinase-1 (sFLT1) by placental syncytiotrophoblasts, which could also contribute to the impaired vascular remodeling observed in PE .
In mice, two different populations of uNK cells have been identified, which are defined by their reactivity to Dolichos biflorus agglutinin (DBA). DBA+ uNK cells express angiogenic factors, while DBA− uNK cells express IFN-γ . It was established in elegant experiments using alymphoid mice that received bone marrow from either IFN-γ−/− mice or severe combined immunodeficient mice, which lack T and B lymphocytes, that NK cell derived IFN-γ is specifically required for uterine spiral artery remodeling . More recently, studies using BPH/5 mice, which display many of the cardinal features of PE, showed that these mice have lower levels of uNK cells in the decidua. The decrease in uNK cells is associated with up-regulation of Cox2 and IL-15 at the maternal–fetal interface. Administration of a Cox2 inhibitor lowered Cox2 and IL-15 expression and restored uNK cell numbers . Whether PE in women is associated with altered uNK cell number or function still remains to be determined .
T lymphocytes in pregnancy and PE
In addition to the aberrant function of uNK cells, PE is also associated with a dysregulation of cytokines, which can affect the homeostasis of T cell populations, including CD4+ β T cells, CD8+ β T cells, and γδ T cells. The proportion of circulating naïve, effector, central memory, or effector memory T cells does not change over the course of a healthy human pregnancy ; however, there are many studies that indicate changes in the relative frequencies of circulating T cell subsets, especially CD4+ TH cells . On the other hand, there are alterations in T cell populations in the human decidua. T cells make up 10–20% of the total leukocytes in the human decidua in early pregnancy, and of these T cells, 30–45% are CD4+ and 45–75% are CD8+ . As pregnancy progresses, the percentage of T cells increases in the decidua, with levels up to 40–80% of total leukocytes at full term .
The traditional model of the TH1/TH2 paradigm during pregnancy is that the maternal immune system has a shift toward a TH2-dominated response because of the presence of the placenta, which produces progesterone and cytokines such as IL-4 that promote TH2 responses . It has been proposed that women with pre-eclamptic pregnancies do not have the suppression of TH1 cytokines that is present in healthy pregnancy. For example, Arriaga-Pizano et al. showed that the cytokine profiles in the peripheral blood in women with PE are skewed toward a TH1 phenotype, with an increase in IFN-γ and a decrease in IL-4 . On a cellular level, pre-eclamptic women have been shown to have a higher circulating TH1/TH2 lymphocyte ratio as compared with women experiencing healthy pregnancy . NK cells, cytotoxic T lymphocytes (CTLs), and NK T cells can also be distinguished into type 1 and type 2 subsets [37–40]. Type 1 cells are defined by surface expression of the IL-18 receptor, and type 2 cells express an IL-1R-like protein on their surface . A study by Borzychowski et al.  revealed that the total number of type 1 lymphocytes, (defined by expression of the IL-18 receptor) was higher in pre-eclamptic patients as compared with healthy pregnant controls, and was at a level similar to non-pregnant women. The role of TH1 cells in the development of PE was demonstrated in rodent studies in which T cells isolated from normal pregnant animals were cultured in the presence of cytokines to promote differentiation in activated TH1 cells. These cells were adoptively transferred into normal pregnant animals, which then developed symptoms of PE, including increased blood pressure, altered kidney function, and decidual inflammation . Importantly, transfer of the TH1 cells to non-pregnant animals did not induce changes in blood pressure or kidney function, indicating that the pregnancy induces unique changes in reactivity toward T cells. This shift toward a TH1 predominance during pregnancy could contribute to both the impaired placentation and the maternal syndrome of inflammation and endothelial dysfunction observed during PE.
TREG cells express the X-chromosome encoded transcription factor forkhead box P3 (FoxP3) and play an important role in the suppression of inflammatory responses and the maintenance of immune homeostasis . TREG are generated in both the thymus and the periphery, and a normal pregnancy is associated with elevated numbers of TREG . Kahn and Baltimore  explored the role of TREG in maintaining fetal tolerance using inbred mice undergoing their first syngenic pregnancy. Pregnant mice produce an antigen-specific TREG response to the minor antigen (H-Y) that is present on male cells, and depletion of TREG during pregnancy leads to the rejection of male fetuses . The specific importance of peripherally induced TREG in pregnancy was highlighted in studies by Samstein et al. . CNS1, an intronic FoxP3 enhancer, facilitates TGF-β dependent Foxp3 expression and peripheral TREG differentiation, but is not needed for the differentiation of thymic TREG. Pregnancy in CNS1-deficient mice results in high abortion rates in allogenic matings. In addition, the CNS1-deficient females have abnormal spiral artery remodeling and signs of inflammation . These data indicate a specific role for peripheral TREG in maintaining tolerance during pregnancy. Alterations in TREG have been reported during pre-eclamptic pregnancy and may contribute to the development and pathogenesis of the disease. While multiple studies have reported a decrease in circulating TREG cells in pre-eclamptic patients as compared with healthy pregnancy, others have reported no differences in TREG cell percentages. These discrepancies are likely to be due to the markers used to define TREG . Santner-Nanan et al.  quantitated TREG cells using three different sets of markers and found that pregnant women have elevated levels of TREG cells as compared to both pre-eclamptic women and non-pregnant women. More recent studies point to an impairment in the expansion of peripheral TREG cells in PE, which is at least partially due to altered antigen presentation in the decidua . At the maternal-fetal interface, TREG cells are a minor population of lymphocytes, and it is currently unclear whether TREG percentages are altered in PE at the maternal-fetal interface.
TH17 cells are a subset of proinflammatory TH cells that secrete IL-17A, IL-17F, IL-21, and IL-22. While these cells play an important physiological role in the clearance of mucosal infections, abnormal TH17 activity has been implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. Healthy pregnant women have lower levels of TH17 cells, which is not found in pre-eclamptic women . However, it should be noted that the percentage of TH17 cells increases during late pregnancy and may possibly play a role, along with other inflammatory cytokines, in the initiation of labor at term . Rodent studies have shown that infusion of IL-17 or adoptive transfer of TH17 cells isolated from rats exposed to reduced uterine perfusion pressure during pregnancy results in increased blood pressure and oxidative stress in pregnant rats [52,53]. The TH1-dominated immune response reported in PE, which is characterized by increased secretion of the inflammatory cytokines IL-6 and IL-1β, may further promote the differentiation and proliferation of TH17 cells . Likewise, changes in the function of uNK cells may contribute to a TH17/TREG imbalance. uNK cells have been shown to interact with CD14+ monocytic cells to promote induction of TREG cells, and IFN-γ secreted by decidual NK cells has been shown to suppress TH17 cells to reduce inflammation and promote maternal–fetal tolerance [29,55]. The cytokine TGF-β has been shown to drive the differentiation of human TREG cells  and inhibit the differentiation of TH17 cells . However, sENG, which is increased in PE, is a potent TGF-β inhibitor. It has been proposed that the increase in sENG might inhibit the differentiation of TREG and promote the differentiation of TH17 cells during PE ; however, this mechanism has not been formally proven.
While much is known about CD4+ TH subsets in PE, less is known about CD8+ CTLs. CTLs are responsible for antigen-specific cytotoxicity toward infected or dysfunctional host cells and kill by release of proteins from cytolytic granules or by Fas/Fas ligand interactions. Although CD8+ T cells are less abundant than CD4+ T cells in the periphery, they are the most prevalent T cell population in the human decidua at term, with most of the cells being of the activated effector memory phenotype [30,58]. Despite their ability to recognize allogeneic MHC molecules in the context of transplantation, they do not attack fetal cells during normal pregnancy . This is at least partially because of limited classical MHC Class I expression by fetal trophoblast cells . T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are negative co-stimulatory molecules that impair stimulation, activation, and function of CD8+ T cells . CD8+ T cells that express Tim-3 and PD-1 accumulate in the decidua of both mice and humans, and in vitro studies have shown that Tim-3+PD-1+CD8+ T cells secrete high levels of anti-inflammatory cytokines. The ability of these cells to produce anti-inflammatory cytokines is impaired in patients with recurrent spontaneous abortion . While there are few studies on decidual CD8+ T cells in PE, one study reported that CD8+ T cells were decreased in third trimester placental biopsies of pre-eclamptic women as compared with healthy pregnant women , but it is currently unknown whether altered CD8+ T-cell activity at the maternal–fetal interface contributes to the pathogenesis of PE.
Patients with PE have higher circulating levels of the cytolytic molecule granulysin, which is present in the lytic granules of NK cells and CTLs. In addition, the levels of granulysin correlated with blood pressure in PE patients . A later study analyzed the prevalence of granulysin positive NK cell and CTLs in pregnant and pre-eclamptic women and found that there were no differences in granulysin+ NK cells between the two groups, but that women with PE have significantly higher levels of granulysin+ CTLs. The authors speculated that the increased production of granulysin might play a role in the development of a TH1-dominated cytokine response, as granulysin can activate monocytes to produce proinflammatory cytokines such as TNF-α, monocyte chemotactic protein (MCP)-1, and regulated upon activation normal T cell expressed and secreted (RANTES) . Finally, an analysis of CTL specific for paternal antigens determined that found that pre-eclamptic women had more CTLs that were partner specific .
Mucosal tissues are populated with a distinct subset of T cells that possess a γδ T-cell receptor (TCR). Unlike conventional αβ T cells, γδ T cells do not recognize antigens in the context of MHC molecules, but instead bind lipids and phosphoantigens . In addition to being resident lymphocytes at mucosal surfaces, γδ T cells also represent 1–10% of the total circulating peripheral blood mononuclear cells. Different subsets of γδ T cells, defined by different variable (V) gene usage in the TCR, populate distinct compartments throughout the body. For example, in humans, Vγ9/Vδ2 T cells predominate in the peripheral blood, and cells that possess a Vδ1 gene segment predominate in epithelial tissues. γδ T cells are present in the endometrium in non-pregnant mammals and show a dramatic increase during pregnancy . The majority of the γδ T cells in female reproductive tract, placenta, and decidua are resident cells that express the germline encoded Vγ6/Vδ1 receptor with some minor populations expressing Vγ4 or Vγ1 [68,69]. A recent study by Pinget et al.  determined that there is an increase in Vγ4+ γδ T cells in placental tissue during murine pregnancy. This cell population is either expanded during pregnancy or recruited to the site. Surprisingly, the majority of (approximately 70%) of the γδ T cells in the maternal tissues were found to express the transcription factor RAR-related orphan receptor gamma t (RORγt) and produce IL-17. The function of these cells is unknown; however, the authors suggested that these cells likely play a role in host defense against pathogens . A pathogenic role for γδ T cells in PE was recently identified by Chatterjee et al.  in a murine model of PE in which administration of viral DNA/RNA mimetics activate toll-like receptors (TLR) and cause symptoms of PE in a pregnancy-specific manner, including hypertension and proteinuria. Placentas from pre-eclamptic women express significantly higher levels of TLRs [72,73], and some researchers have associated viral infections with placental disorders, including PE [74,75]. In the study by Chatterjee et al. , the authors determined that the primary immune cell type activated by TLR ligation during pregnancy is the γδ T cell. First, they found that pregnant mice administered TLR agonists have an increase in inflammatory γδ T cells that express the MHC-associated invariant chain (CLIP) on their surface; however, the administration of TLR agonists to either CLIPdef or γδ T cell knockout mice does not result in a pre-eclamptic phenotype. Finally, in preliminary data using human placentas, the authors showed that women with PE have significantly higher levels of γδ TCR protein . This study highlights the need for further investigation into the role of this subset of T cells in the pathogenesis of PE.
Many studies have implicated alterations in the innate and adaptive immune systems as major contributors to the inflammation and endothelial dysfunction of PE. While experimental studies in animal models have shown that immune modulation can attenuate the hypertension and end organ damage associated with PE, greater investigation is needed to further define the roles of distinct cell populations in PE and how these cell types interact with each other to regulate vascular remodeling and placentation, systemic inflammation, and endothelial function to impact the overall health of the mother and fetus. By understanding the immune adaptations that occur during early pregnancy and defining the aberrations that occur during PE, we can begin to develop therapies to target these pathways and improve outcomes for women and their offspring.
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the National Institutes of Health [grant numbers F32HL137393 (to E.B.T.), R01HL134711 to (J.M.S.)].
E.B.T. and J.M.S. wrote the manuscript.
MHC-associated invariant chain
cytotoxic T lymphocyte
Dolichos biflorus agglutinin
Forkhead box P3
human leukocyte antigen
killer immunoglobulin-like receptor
NK group 2
programmed cell death-1
RAR-related orphan receptor gamma t
regulatory T cell
T-cell immunoglobulin mucin-3
transforming growth factor-β
tumor necrosis factor
vascular endothelial growth factor