The Pathological Events of Preeclampsia in a Two-Stage Model

The pathophysiology of preeclampsia has been formalized in a two-stage model that delineates the evolution from poor placentation to the maternal clinical syndrome (22). As described above, poor placentation is due to the incomplete remodeling of the spiral arteries, resulting in high BP flow and irregular delivery of fully oxygenated arterial blood to the placenta (21). The latter is termed placental ischemia/hypoxia and is associated with distortion of the placental villous architecture and increased oxidative stress (21). The subsequent uteroplacental insufficiency results in compromised blood flow to the uterus, resulting in pregnancies with increased perinatal morbidity and mortality (1). However, incomplete remodeling of the spiral arteries is not only associated with preeclampsia but is also found in IUGR and gestational hypertension (22, 28), suggesting that it is not the only cause of preeclampsia. During the initial stage of preeclampsia, poor placentation occurs with no clinical features, followed by a second stage that involves a maternal clinical syndrome with cardiovascular manifestations and renal features (17, 21, 29).

Inadequate invasion of cytotrophoblasts following the incomplete remodeling of spiral arteries exposes the placenta to oxidative stress (22). The second stage of the disorder ensues because the ischemic placenta and increased placental oxidative stress cause excessive systemic inflammation and endothelial dysfunction that manifest as new-onset hypertension and proteinuria (21, 22, 29, 30). The end result of oxidative stress in preeclampsia is exaggerated placental necrosis or apoptosis, which are common histologic features of the preeclamptic placenta (21, 31). Excessive shedding of syncytiotrophoblast microparticles, reflecting placental ischemia and apoptosis of placental cells, into the maternal circulation, creates an inflammatory burden and indirectly affects endothelial function (32–35). This role of a diseased placenta in preeclampsia is part of the emerging theory classifying preeclampsia as two different diseases, either placentogenic or maternogenic (36). The different phenotypes of preeclampsia can thus either be placentogenic, which usually occurs in early pregnancy with association of poor placentation and different severities of IUGR, or maternogenic, which occurs late in pregnancy with no relation to placental insufficiency or IUGR (37). The metabolic syndrome (defined as the occurrence of hypertension, ischemic heart disease, type 2 diabetes mellitus, obesity, and insulin resistance) has also been associated with increased circulating microparticles and subsequent systemic inflammation (38).

The exact stimuli inducing inflammation in preeclamptic patients are, however, incompletely understood and various other possibilities exist (32, 39). For instance, an injured endothelium may release several factors thought to play a role in this systemic inflammatory response (40). These factors include pro-inflammatory cytokines, markers of oxidative stress, thrombomodulin, fibronectin, endothelin-1, and Von Willebrand factor (40, 41). Other placental factors, such as anti-angiogenic [soluble fms-like tyrosine kinase (sFlt-1)] factors secreted by placental cells can also be considered as possible stimuli, since these factors affect the endothelium, a component of the inflammatory system (22, 29, 34). Protein toxic aggregates such as aggregated transthyretin which, is a placental toxin elevated in preeclampsia, have been suggested to contribute to the pathogenesis of this disorder. Elevated levels of aggregated transthyretin are formed in preeclampsia and transported through the secretion of placental extracellular vesicles by syncytiotrophoblasts (42). Aggregated transthyretin in these vesicles may also allow targeted delivery of these toxic proteins to other maternal organs, conducting a signal of cellular stress from the diseased placenta and contributing to the pathogenesis of preeclampsia (42). Protein toxic aggregates is a novel approach, they have been considered to be pathogenic in other diseases, thus the possibility of contributing to excessive inflammation in preeclampsia through creating cellular stress should also be considered (42–44).

The role of inflammatory markers with elevated levels in preeclampsia has also been investigated, including the role of procalcitonin (PCT) and C-reactive protein (CRP) (45). PCT is a precursor of calcitonin and is used as a marker of bacterial infection and resultant systemic inflammation (45, 46). The exact role of PCT and CRP in preeclampsia remains controversial, with the literature revealing conflicting accounts of their usefulness in predicting preeclampsia and its severity (45).

It has been well established that the human microbiota plays a fundamental role in the functioning of the immune system and the regulation of immune responses (47). Microbiota–immune interaction is mediated through microbial-associated molecular patterns; for instance, the bacterial endotoxin lipopolysaccharide (LPS) is recognized by cellular toll-like receptors (TLRs) (47). Researchers have established LPS to be an external stimulus, which is able to induce hypertension and proteinuria identical to preeclampsia in pregnant rats (48). In this particular animal model of human preeclampsia, lipid A/LPS, a known pro-inflammatory stimulus, provoked systemic inflammation and was associated with the clinical manifestations of preeclampsia (48). In a study by Cotechini et al. (49) performed on pregnant rats, the administration of LPS induced a systemic and local inflammatory response, fetal growth restriction (FGR), and an increase in mean arterial pressure, through a mechanism mediated by tumor necrosis factor (TNF). In addition, the authors observed deficient trophoblast invasion and impaired spiral artery remodeling characteristic of preeclampsia, to be linked to LPS-induced FGR (49). Similar results were found by Xue et al. (50) who demonstrated that a single administration of LPS to pregnant rats induced inflammation specifically by binding to its receptor, the TLR-4 in the placenta. TLR-4 signaling in the placenta was associated with deficient trophoblast invasion and spiral artery remodeling contributing to poor placentation that may result in a preeclampsia-like syndrome (50). Ultimately, all of these secreted placental factors and stimuli, including bacterial endotoxin, are capable of disrupting the fine-tuned balance of the immune system and inducing a systemic inflammatory response.

Angiogenesis as Part of Placentation and Biomarkers Present in Preeclampsia

The extensive angiogenesis that is characteristic of successful placentation is indirectly disrupted by placental ischemia/hypoxia in preeclampsia (1, 27, 51). Angiogenesis is critical for the improvement of placental circulation and blood flow by the formation of new vascular beds to enable vascular growth and development of the placenta (27). Placental factors, specifically pro-angiogenic [vascular endothelial growth factor (VEGF) and placental growth factor (PIGF)] and anti-angiogenic proteins, are responsible for placental growth, vascularization, and maintenance of vessel health (8, 27). These factors act as biomarkers that reflect unique patterns in preeclampsia according to the severity as well as the gestational age at the onset of the disorder (52). Preeclampsia is associated with decreased levels of vasodilators, such as nitric oxide (NO) and prostacyclin, as a result of the disproportionate increase in sFlt-1 and decrease in VEGF (Figure ​(Figure1)1) and PIGF (53–55).

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Figure 1

Molecular mechanisms involved in the upregulation and downregulation of antiangiogenic and angiogenic factors ultimately leading to endothelial dysfunction and reduced vasodilation in the pathogenesis of preeclampsia. Although there is a lack of proof that soluble fms-like tyrosine kinase (sFlt-1) is associated with excessive systemic inflammation, this illustration emphasizes the effect of the immune system on angiogenesis and that dysregulation thereof could contribute to the pathogenesis of preeclampsia.

The growth factor VEGF and its homolog, PIGF, are essential for angiogenesis because of their pro-angiogenic activity that induces vascular permeability and promotes the proliferation and survival of epithelial cells (40). VEGF may cause vasodilation by stimulating the NO-cyclic guanosine monophosphate vascular relaxation pathway as well as by increasing the production of the calcium ion (Ca²⁺)i (53). An increase in (Ca²⁺)i promotes the binding to endothelial NO (eNOS) and subsequently increases eNOS activity and stimulates NO production (Figure ​(Figure1)1) (53). Transforming growth factor (TGF)-β1 is involved in angiogenesis by regulating the expression of VEGF by means of intracellular signaling (40, 56). The inhibition of TGF-β1 signaling will lead to reduced endothelium-dependent vasodilation and increased endothelial cell apoptosis, suggesting that dysregulated TGF-β1 signaling may be involved in the pathogenesis of preeclampsia (40). A decline of VEGF results in glomerular endotheliosis, a specific endothelial renal lesion presenting with proteinuria (21). In addition to low VEGF concentrations, a decline in NO, PIGF, and TGF-β1 levels, in concert with an increase of anti-angiogenic factors, deprives endothelial cells of support leading to endothelial dysfunction (8, 17, 22). Thus, endothelial dysfunction is a result of an antiangiogenic state in the placenta and contributes to the pathology of preeclampsia (Figure ​(Figure1)1) (9, 52, 57).

Immunological factors also play a role in the secretion of antiangiogenic factors. For instance, complement activation has been shown to stimulate monocytes to release antiangiogenic factors (20). Studies in murine models of experimental pregnancy loss demonstrated that conditions of hypoxia and inflammation lead to the release of increased amounts of antiangiogenic factors and are therefore strong triggers of angiogenic dysregulation (20, 58). These studies and those in humans demonstrate that pregnancy complications, such as recurrent pregnancy loss (RPL), preterm birth, and preeclampsia, are associated with excessive complement activation, especially enhanced C5a synthesis, that promotes the secretion of the antiangiogenic factor sFlt-1 (20, 59). Activation of C5 contributes to fetal loss and adverse pregnancy outcomes by inducing a dysregulation of angiogenic factors and initiating a complex series of events, as demonstrated in the abortion-prone mouse mating combination CBA/J and DBA/2 (20). The importance of activation of complement through the lectin pathway was further illustrated with the administration of inhibitory factors, such as Polyman2, which neutralizes mannose-binding lectins and anti-C5, a neutralizing recombinant antibody that prevented fetal loss in this mating combination (20, 60). The important role of disruption in the regulation of complement activation in preeclampsia is also demonstrated by the findings of C4A deficiencies and C4b deposits in preeclamptic placentas (61, 62).

Soluble fms-like tyrosine kinase (also referred to as sVEGFR-1) is a soluble form of the VEGF/PIGF receptor Flt-1 and is a potent inhibitor of VEGF and PIGF angiogenic activity (51, 53–55). Elevated levels of sFlt-1 are associated with preeclampsia and have been found to induce hypertension, proteinuria, and glomerular capillary endotheliosis when administered to pregnant animals (63). Increased levels of sFlt-1 are more pronounced at an early stage before the onset of preeclampsia and are associated with several associated risk factors, including diabetes mellitus and gestational hypertension (52, 64, 65). Excess levels of sFlt-1 inhibit cytotrophoblast differentiation and invasion and subsequently contribute to poor placentation (20). A chronic increase in the production of pro-inflammatory cytokine, TNF-α, was found to induce hypertension in rats and stimulate the excessive secretion of sFlt-1, further supporting the effect of the immune system on inadequate angiogenesis in preeclampsia (17). Excessive secretion of sFlt-1 is also suggested to be induced by hypoxic conditions and reflect the response of oxidatively stressed syncytiotrophoblasts during poor placentation. Therefore, possible sources contributing to the higher concentrations of sFlt-1 observed during preeclampsia are the syncytiotrophoblasts in the placenta in the presence of oxidative stress, as well as peripheral blood mononuclear cells, macrophages, and endothelial- and vascular smooth muscle cells (34, 66).

Increased concentrations of another antiangiogenic factor, soluble endoglin (sEng), in preeclampsia contributes to endothelial dysfunction through its inhibition of TGF-β1 signaling (35, 67). Elevated levels of sEng could be attributed to oxidative stress and hypoxic conditions in the placenta as well as to stimulation by inflammatory cytokines, such as TNF-α and interferon (IFN)-γ, from endothelial or placental cells during non-hypoxic conditions (29, 68). The combined action of increased sFlt-1 and sEng concentrations is suggested to characterize preeclampsia better than a single analyte, emphasizing the role of several antiangiogenic factors in clinical preeclampsia (40, 69). Considering that the endothelium is a component of the systemic inflammatory system, elevated levels of antiangiogenic factors (sFlt-1 and sEng) secreted by a diseased placenta may contribute to systemic inflammation (Figure ​(Figure1)1) (29).

In contrast to the pattern of the antiangiogenic factors, pro-angiogenic PIGF and VEGF are present in decreased concentrations in preeclampsia even before clinical manifestation occurs (52). The patterns of these biomarkers are considered relevant and an altered ratio of pro-angiogenic to anti-angiogenic factors is correlated with the severity of the disorder (70). Increased concentrations of sFlt-1 lead to PIGF and VEGF being sequestered (35, 52); hence, an imbalance in the expression of angiogenic and antiangiogenic factors leads to inadequate angiogenesis and disruption of the maternal endothelium and immunological balance advancing to the clinical signs of preeclampsia (Figure ​(Figure1)1) (9, 17, 52).

Imbalances Between Th Cell Subsets

The regulation of pro- and anti-inflammatory immune responses lies in the interplay between different Th cell subsets, especially between Th1 and Th2 cells, characterized by the release of pro-inflammatory (e.g., IL-2, IL-6, IL-8, IFN-γ, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13), respectively (112, 113). Th2 cytokines are involved in antibody production and take part in the regulation of inflammation in cooperation with IL-10-producing Tregs (88). During pregnancy, modulation of immune responses occurs, with distinct immunological features, observed at different stages (114). Implantation and placentation in the first and early second trimester of pregnancy are characterized by a pro-inflammatory environment that ensures adequate repair of the uterine epithelium and removal of cellular debris that accumulates secondary to embryo implantation (114). The second and third trimester are times of rapid fetal growth and development and the predominant immunological profile in the feto-maternal interface is anti-inflammatory (114). Progesterone, estradiol, prostaglandin D2, and leukemic inhibitory factor all promote the development of this Th2 profile (104, 105). The immunological effect of progesterone is mediated through a protein called P-induced blocking factor, which induces dominant Th2 cytokine production and immune modulation in aid of maternal tolerance toward the fetus (115). A Th2 bias in pregnancy modulates cell-mediated immunity, which is responsible for the increased susceptibility of pregnant women to bacterial LPS and intracellular pathogenesis of, for instance, Listeria monocytogenes (75, 104). Finally, during parturition, the immune profile reverts back to a pro-inflammatory state, which is necessary for the processes of delivery, such as contraction of the uterus and expulsion of the fetus and the placenta (104).

It has been proposed that a shift toward a Th2 profile in the second trimester does not occur in preeclampsia or, alternatively, that it is reversed in the early stages of the disease (71). This has the consequences that Th1 responses are not downregulated and cytokines exhibit mostly a pro-inflammatory profile, with elevated levels of IFN-γ and reduced levels of IL-4 and IL-10 reported (71, 116). Increased levels of Th1 type pro-inflammatory cytokines have also been associated with other pregnancy complications, such as preterm birth and IUGR (117). This pro-inflammatory systemic environment contributes to the failure of immune tolerance and results in immune dysregulation (Figure ​(Figure2)2) (59, 118). The mediators that regulate this Th1/Th2 shift are not completely understood and are speculated to involve NK cells and IFN-γ, but also other cytokines, such as IL-12, IL-8, and the TGF-β family (117, 118). Interleukin-12 induces Th1 responses and stimulates the release of IFN-γ by NK and naïve T cells (7). In turn, IFN-γ primes monocytes to release more IL-12, increasing the production of IFN-γ. It has been suggested that this positive feedback cycle contributes to rapid deterioration in severe preeclampsia (7). Another key aspect in this paradigm is the negative feedback of IFN-γ on Th2 cells, in so doing suppressing immune regulation (71).

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Figure 2

An illustrative model of the various components of a dysregulated immune system in preeclampsia. The recognition of antigens is considered a starting point where immune cells are stimulated based on the interaction between specific killer immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) ligand genotypes. Subsequently, a dysregulated immune system in preeclampsia is attributed to an imbalance of pro- and anti-inflammatory cytokines as result of the overactivation of immune cells and pro-inflammatory stimuli that promote inflammation. Therefore, a hyperinflammatory environment in preeclampsia is established by the failure of regulatory mechanisms.

Possible mechanisms by which dominant Th1 immune responses induce the clinical aspects of preeclampsia are not completely known, but many of the respective Th1 cytokines (TNF-α, IL-2, IL-12, IL-18, and IFN-γ) have been reported to induce apoptosis of trophoblasts (25, 118). In addition, IL-12 and IFN-γ inhibit angiogenesis, whereas TNF-α activates endothelial cells and induces glomerular endothelial damage (7, 117). Tumor necrosis factor-α also inhibits the production of NO and thus influences the circulating levels of angiogenic factors in the second stage of preeclampsia (Figure ​(Figure1)1) (118). Studies have, however, reported highly variable results of the circulating levels of TNF-α in preeclampsia, with some showing increased, unchanged, or even insignificantly decreased levels, possibly due to differences in timing and study method used (119–126). Nonetheless, serum TNF-α concentrations have been positively and significantly correlated with diastolic BP and uric acid levels, and it has hence been proposed as a potential marker of the severity of preeclampsia (125). TNF-α plays a critical role in maternal inflammation. Cotechini et al. (49) provided evidence to highlight the importance of this cytokine by using a mouse model where it was observed to induce abnormal inflammation in pregnant rats leading to FGR and features characteristic of preeclampsia (49). Elevated levels of IFN-γ are thought to play a central role in endothelial dysfunction and the exaggerated systemic inflammatory response in preeclampsia (Figure ​(Figure2)2) (71). Overproduction of TNF-α and IFN-γ has also been reported in other adverse pregnancy outcomes, such as preterm birth (127) and IUGR, and could be suggested viewed as a primary target in preeclampsia pathology also because of their ability to inhibit sufficient angiogenesis, as previously mentioned (Figure ​(Figure2)2) (117).

This one-dimensional Th1/Th2 paradigm can, however, not fully explain the immunological changes observed during preeclampsia and recent work has expanded the dichotomy to include the Th17 phenotype and a subset of CD4⁺ T cells known as CD25⁺ Forkhead box P3 gene (Foxp3⁺) Tregs (128). The latter are responsible for the development and regulation of immune responses and hence, the maintenance of immunological tolerance to the fetus (25, 112). Extrathymic and peripherally generated Tregs (pTregs) induce tolerance by suppressing the immune response generated by T-effector cells to foreign antigens, whereas thymic Tregs (tTregs) act to prevent autoimmunity by maintaining MHC class II antigen-specific tolerance (59).

Peripherally generated Tregs are produced within the decidualized endometrium during early pregnancy and are believed to play an essential role in pregnancy (129–131). Others have proposed that both tTregs and pTregs are indispensable, with tTregs potentially initiating a tolerant state and pTregs maintaining tolerance (132). Tregs have to be exposed to antigens presented by “tolerogenic” DCs in an appropriate cytokine environment for them to proliferate, mature, and eventually exert suppressive effects (128). Although HLA II is not expressed in either villous or extravillous cytotrophoblasts, antigenic stimulation is believed to come from the trophoblastic cell debris that contains intracellular fetal HLA-DR (59). It has also been suggested that Tregs recognize paternal HLA-C and can therefore downregulate anti-paternal responses, although the stability of this memory is still unknown and needs further investigation (79).

Regulatory T cells are differentiated according to the expression of surface markers, most notably the IL-2 receptor, CD25, and the transcription factor, forkhead box P3 (Foxp3⁺) (25, 59, 133). It has been suggested that the decidual CD4⁺ CD25⁺ Foxp3⁺ Tregs subsets specifically play a role in pregnancy by mediating immune tolerance to fetal antigens and suppressing inflammatory responses (134).

Regulatory T cells have many suppressive functions and targets, including suppression of activation, proliferation and cytokine release of CD4⁺ and CD8⁺ T cells, suppressing B-cell proliferation and immunoglobulin production, inhibiting the cytotoxic function of NK cells, and inhibiting maturation and function of antigen-presenting cells, such as DCs and macrophages (129). The molecular mechanisms by which Tregs exert their control are, however, incompletely understood. Some propose that their main action is through suppression of specific NK phenotypes and pro-inflammatory cytokines (71) or through the production of immunoregulatory cytokines, such as IL-10 and TGF-β that in turn promote and inhibit the differentiation of Tregs and Th17cells, respectively (135, 136). Others believe the major mechanism to be through the expression of the membrane glycoprotein, cytotoxic T lymphocyte antigen (CTLA)-4 (137). The co-ligation of CTLA-4 with the T cell receptor results in increased expression of IDO, which effectively starves T cells by restricting the accessibility of the essential amino acid, tryptophan (137, 138). The interaction between Tregs and IDO is bidirectional, and the presence of IDO promotes the differentiation of T cells into Tregs, while in its absence, Tregs are reprogrammed to acquire a Th17 pro-inflammatory phenotype (79).

T-helper 17 cells are a CD4⁺ lymphocyte subpopulation, characterized by the production of the pro-inflammatory cytokine, IL-17 (112, 128). The Th17cells induce a protective immune response against bacterial or fungal infections and the excessive upregulation of this subpopulation has been associated with the development and progression of autoimmune and chronic inflammatory diseases, allergies, and graft rejection (128, 139). A portion of IL-17A-producing cells have been found to also produce the Th1 cytokines, such as IFN-γ, and are hence termed Th17/Th1. Conversely, a small proportion of cells that have the ability to produce both IL-17A and IL-4 are termed Th17/Th2 cells. The latter clones originate in the presence of IL-4, suggesting that a microenvironment rich in IL-4 may induce a shift from Th17 to Th17/Th2 cells (140). IL-4 and HLA-G5 in the uterine environment is considered at least partly responsible for the development of specifically Th17/Th2 cells, which is crucial for successful implantation of the embryo in pregnancy (140, 141). Lombardelli et al. (141) reported that in successful pregnancy, a large number of CD4⁺ T cells produce both IL-17 and IL-4, whereas pathogenic decidual Th17/Th1cells are commonly found in patients with unexplained recurrent abortion (141). The same study conducted by Lombardelli et al. (141) propose that the potentially detrimental effects of IL-17 may be counteracted when Th17cells produce IL-17 together with IL-4 in the first trimester of pregnancy (141). IL-17/IL-4-producing CD4⁺ T cells promote an adequate response to extracellular pathogens, whereas IL-4 may induce tolerance toward the paternal HLA-C antigens and IL-17 could promote the proliferation and invasion of extravillous cytotrophoblasts (141).

A similar developmental lineage exists between Th17/Treg subsets, and the linked evolution of these subsets enables a balanced regulation of inflammation and autoimmunity (Figure ​(Figure2)2) (84, 113, 142). Tregs and IL-17⁺ T cells are both involved in the establishment and maintenance of pregnancy, where these cells function as regulatory and effector cells, respectively (84, 143). Tregs act as regulators of Th17cells and other immune cells involved in tolerance at the feto-maternal interfaces (25, 128).

Normal pregnancy is proposed to have a bias away from a Th17 response, while an increased ratio of IL-17⁺/Tregs has been demonstrated in cases of RPL, unexplained miscarriage, preterm birth, and preeclampsia (Figure ​(Figure2)2) (59, 79, 113, 128, 139, 144). It has been shown that IL-17-producing lymphocytes are increased in the peripheral blood of preeclamptic patients in the third trimester of pregnancy and a significant correlation has been found between levels of Th17, IL-2-, and IFN-γ-producing T cells and the development of preeclampsia (145).

In preeclampsia, the uterine microenvironment acts as a contributing factor by influencing the differentiation of T cell subsets, especially Th17cells (146). Therefore, further differentiation of Th17cells could be related to the continuous release of pro-inflammatory cytokines and a dominant Th1 environment in preeclampsia (118, 146). The increased production of IL-1β and IL-6 by activated monocytes that induce an inflammatory microenvironment, as seen in recurrent abortions, can also participate in the expansion of Th17 and their conversion from Tregs (74, 147).

In addition to the regulatory action of Tregs, dNK cells are present in elevated levels in normal pregnancy and inhibit local inflammation at the feto-maternal interface and maintain tolerance by suppressing the development of pro-inflammatory Th17cells (74, 136). The ratio of dNK/Th17cells is decreased in cases of RPL abortion, indicating impaired regulatory activity and increased decidual inflammation (74). Deficient levels of Tregs and the absence of dNK cells lead to a prominent Th17 response, extensive local inflammation, and the failure of maternal–fetal tolerance, which could be possible mechanisms that play a role in the development of preeclampsia (74, 136).

Tolerance at the maternal–fetal interface is achieved through the production of the immunoregulatory cytokines, such as IL-10 and TGF-β1, that promote and inhibit the differentiation of Tregs and Th17cells, respectively (136). Tregs also suppress innate and adaptive immune responses and play a major role in modulating the activity of self-reactive cells (112). The importance of Tregs in pregnancy was confirmed when the administration of these cells was found to prevent spontaneous abortion in pregnant mice (25). It is suggested that Tregs do not directly suppress Th1 responses in normal pregnancy, but regulate immune responses through the suppression of specific NK phenotypes and pro-inflammatory cytokine production (71). Although Tregs cannot reverse the shift toward a Th1 response by promoting Th2, these cells promote a tolerant microenvironment at the feto-maternal interface (148).

Preeclampsia has been associated with an imbalance in the two subpopulations of Tregs (25, 149). In comparison to normal pregnancy, preeclampsia is associated with increased numbers of CD4⁺CD25^highFoxp3⁺ and decreased numbers and functional activity of CD4⁺CD25⁺Foxp3^high+ cells (149). The stability of Tregs is suggested to be influenced by inflammatory environments, as has been demonstrated in inflammatory bowel disease (150). Pro-inflammatory cytokines, such as TNF-α, have been shown to have a direct negative effect on the function of Tregs, especially in cases of rheumatoid arthritis, and this may be an explanation for the altered levels of Tregs observed in preeclampsia (59, 150, 151). Furthermore, it has been suggested that a pro-inflammatory environment tends to suppress the generation of Tregs (79). In the pro-inflammatory environment of preeclampsia, IDO has been shown to be less active in late stages, possibly due to underactive Treg responses (79, 152). A decline in the level of decidual Tregs consequently leads to failed regulatory mechanisms, resulting in the immunological rejection of the fetus (25, 153).

Aberrant Activation of Decidual NK Cells

In the first trimester of pregnancy, a subset of peripheral blood NK cells (decidual CD16⁻CD56^bright NK cells—dNK cells) differentiates to constitute the major immune cell population in the decidua (59, 75). Peripheral NK cells (CD16⁺CD56^dim) are cytotoxic, express high levels of KIRs and CD57 (a marker of cell maturation and potent cytotoxic potential). and do not secrete cytokines (154, 155). By contrast, CD16⁻CD56^bright cells do not induce antibody-mediated cell toxicity and express only low levels of perforin, but high levels of the inhibitory receptor CD94/NKG2 (154). Although CD16⁻CD56^bright NK cells in the decidua possess cytotoxic properties, these cells are an important source of immunoregulatory cytokines, such as IL-10 and TGF-β1, which play an important role in tolerance and successful pregnancy by directly inhibiting the proliferation of Th17cells and suppressing inflammatory responses, as mentioned earlier (7, 25, 74, 81, 87, 136). While dNK cells also secrete their classic, pro-inflammatory cytokine, IFN-γ, its effect is mainly exerted through inducing spiral artery remodeling by initiating vessel instability (74, 156). The secretion of chemokines, IL-8, and the IFN-inducible-protein (IP)-10 by dNK cells facilitates the migration of the extravillous cytotrophoblast into the decidua basalis and results in the invasion of spiral arteries, further contributing to uterine vascular remodeling crucial for placental development (87).

The phenotype of dNK cells can be altered by severe inflammation in the uterine microenvironment caused by severe stress, viral infection, and autoimmune diseases (74). The expression of the marker CD27 on NK cells, which correlates with an increased ability to proliferate and produce IFN-γ and with lower cytotoxic potential (157), has been suggested to be an important marker in distinguishing different subsets of NK cells (74). A large number of CD27⁺ NK cells have been found in the decidua, where this subset of NK cells is an important source of cytokines and shows limited cytotoxicity (74). Studies of RPL as a model pathogenic state demonstrated an altered ratio of CD56^bright CD27⁺ to Th17cells, impaired release of the immunoregulatory cytokines, such as IL-10 and IFN-γ, as well as the failed inhibition of inflammation (74, 158). A possible explanation for these findings is that dNK cells may have increased cytotoxicity toward inflammatory cells together with impaired regulatory capacity, and that prolonged overactivation of dNK cells may result in them becoming immunodepleted and adopting an abnormal phenotype (74).

Preeclampsia is associated with a shift in NK cells toward a cytotoxic phenotype with increased production of IFN-γ, which causes apoptosis of cytotrophoblasts, thus inhibiting trophoblast invasion (25, 159). In addition, the lower expression of the natural cytotoxicity receptor, NKp46, on NK cells in women with preeclampsia has been suggested to increase the production of the pro-inflammatory cytokine, TNF-α, which further drives a shift in NK cells (160). Accordingly, inappropriate recruitment of peripheral blood NK cells to specialized decidual cells may lead to the development of a cytotoxic environment in utero (113). Hence, successful implantation and placentation depend on a fine balance between decidual NK cell infiltration and activation of regulatory phenotypes (113).