A leading role for the immune system in the pathophysiology of preeclampsia

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1 Review A leading role for the immune system in the pathophysiology of preeclampsia Estibalitz Laresgoiti-Servitje 1 American British Cowdray Medical Center, Mexico City, Mexico; and Department of Immunology, School of Medicine, Universidad Panamericana, Mexico City, Mexico RECEIVED NOVEMBER 28, 2012; REVISED APRIL 8, 2013; ACCEPTED APRIL 9, DOI: /jlb Abbreviations: AT2 angiotensin-2, AT1-AA, angiotensin-1 receptor autoantibody, AT1/2-R angiotensin-1/2 receptor, BDCA-2 blood DC antigen 2, DAMP damage-associated molecular pattern, DC-SIGN DC-specific ICAM-3-grabbing nonintegrin, ENG endoglin, Flt1 Fms-like tyrosine kinase 1, FoxP3 forkhead box P3, HIF-1 hypoxia-inducible factor 1-, HMGB1 high-mobility group box-1, IP-10 IFN- -inducible protein-10, itreg induced regulatory T cell, mirna microrna, NET neutrophil extracellular trap, PlGF placental growth factor, RAGE receptor for advance glycation end products, RAS renin angiotensin system, RORc retinoidrelated orphan receptor C, s soluble, SGA small for gestational age, STBM syncytiotrophoblast microparticle, Treg regulatory T cell, unk uterine NK 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. J. Leukoc. Biol. 94: ; Introduction Preeclampsia, a pregnancy disorder, in which hypertension and proteinuria are present after the 20th week of gestation [1], affects 4 6% of all pregnancies [2]. In the first weeks of pregnancy, normal spiral artery remodeling is achieved by the migration of differentiated cytotrophoblasts into the uterine spiral arteries [3]. However, in preeclampsia, vascular changes may not occur in the spiral arteries that provide blood to the intervillous space, leading to decreased placental perfusion [4] and the maintenance of high uteroplacental intravascular resistance [5]. As preeclampsia has multiple origins, the precise etiology has not been defined clearly [6]. Researchers have suggested that an association exists among impaired angiogenesis, changes in local oxygen tension [4], or oxygen-sensing mechanisms [7] and immunological alterations in the early placental microenvironment, which may all participate in the origins of preeclampsia [8]. Later in the pregnancy, the considerably reduced uteroplacental flow that may be associated with long-term uteroplacental hypoxia promotes the release of chemokines and induces inflammation by activating monocytes and neutrophils [9]. Although it is believed that placental hypoxia plays a relevant role in preeclampsia, recent studies suggest that the presence of hypoxic conditions may not be the key feature in all preeclamptic patients, as a disruption of oxygen-sensing mechanisms that promote overexpression of HIF-1 can be present in some women with this syndrome. As high levels of HIF-1 and alterations in oxygen-sensing pathways are more common in early-onset preeclampsia, differences in these mechanisms may help differentiate early- and late-onset preeclampsia [7]. Two stages of the preeclampsia syndrome have been proposed: (1) poor trophoblastic invasion, as a result of altered production of immunoregulatory cytokines and angiogenic factors and (2) a systemic, maternal-inflammatory response, primarily involving the endothelium, which is apparently stim- 1. Correspondence: ABC Medical Center, Sur 136 No A, Mexico City, 01120, Mexico. elaresgoiti@up.edu.mx; /13/ Society for Leukocyte Biology Volume 94, August 2013 Journal of Leukocyte Biology 247

2 ulated by the release of necrotic and/or apoptotic syncytiotrophoblast cells into the maternal circulation [10]. As the first stage of preeclampsia mostly accounts for its origins, this review will discuss the second stage, as it is when the maternalinflammatory response takes place, and most pathophysiological processes occur. The second stage of preeclampsia may also involve poor fetal growth and can be associated independently with the development of intrauterine growth restriction [11]. However, not all babies from preeclamptic mothers are born SGA, and intrauterine growth restriction may be more frequent in early-onset preeclampsia than in late-onset preeclampsia [12]. Some components of the innate and adaptive immune system that may participate in the physiopathology of preeclampsia will be described here, as they produce certain cytokines, they modulate immune responses, or they have shown a modified function that may lead to the symptoms of this disease. The pathophysiology of preeclampsia also involves altered levels of angiogenic factors, AT2-R autoantibodies [13 15], and the presence of different mirnas [16, 17]. THE ROLE OF ANGIOGENIC/ ANTIANGIOGENIC FACTORS AND THEIR INTERACTION WITH THE IMMUNE SYSTEM IN PREECLAMPSIA The persistence of low-oxygen tensions or altered oxygen-sensing mechanisms in preeclampsia promotes placental overexpression of HIF-1 [7, 18]. Later, an imbalance between proand antiangiogenic factors can be observed in this syndrome. Proangiogenic factors include the VEGF and PlGF, whereas antiangiogenic factors that can be present in preeclamptic patients are the seng [19] and the svegf-r1 [20, 21] and its generic splice variant, known as sflt1 [21]. sflt1 inhibits VEGF and PlGF by binding to these factors in the maternal circulation and blocking their angiogenic effects [22]. Whereas sflt1 levels are relatively low early in normal pregnancy [23], in the presence of hypoxic conditions, seng and sflt1 are released by the placenta [18, 24]. HIF-1 increases VEGF, ENG [25], and sflt1 expression [18]. sflt1, which may be produced by different cells, including endothelial cells [26] and the hypoxic villous trophoblast, participates in the clearing of freematernal VEGF [27]. Two isoforms of svegf-r1 have been described: sflt1, which is generic, and sflt1-14, which is human specific [21]. sflt1-14, the most common VEGF inhibitor produced by the human placenta in preeclampsia, is a C-terminal variant isoform of sflt1, and it is also known as sflt1-e15a [28]. Under hypoxic conditions, sflt1-14 can accumulate in the maternal circulation, neutralizing VEGF in distant organs and extending the consequences of preeclampsia [15]. High levels of sflt1 and low levels of PlGF may predict the subsequent development of preeclampsia, as it may be detected 5 weeks before its onset [29]. sflt1 levels differ in early- and late-onset preeclampsia. sflt1 levels are higher in early-onset preeclampsia, making sflt1 a possible biomarker for the early-onset form of this syndrome [30]. As sflt1 is secreted under hypoxic conditions, the differences in sflt1 levels in women that develop the disease earlier or later in pregnancy may be reflecting different oxygen concentrations in preeclamptic patients. Components of the immune system, especially cytokines, may be interacting with angiogenic and antiangiogenic factors in preeclampsia. As seng is increased in preeclampsia, and it binds TGF-, compromising its function and/or bioavailability [23], it may be possible that seng could affect the levels of TGF- required for itreg. On the other hand, the levels of TNF- present in preeclamptic patients can also promote the release of sflt1 [13], especially under chronic hypoxic conditions [31]. Likewise, the binding of AT1-AA, which will be described later in the manuscript, can promote the secretion of seng and sflt1 through TNF- -mediated mechanisms [32, 33]. THE ROLE OF mirnas IN PREECLAMPSIA mirnas are nonprotein-coding RNAs that regulate gene expression and may play a role in the pathogenesis of preeclampsia, also serving as possible biomarkers for this disease [34]. Several mirnas have been found elevated in placentas with preeclampsia. For example, in severe preeclampsia, mir- 16, mir-29b, mir-195, mir-26b, mir-181a, mir-335, and mir- 222 are increased significantly in the placenta [35]. Placentas with preeclampsia and preeclampsia complicated with SGA newborns express mir-182 and mir-210, which are not present in placentas related to SGA alone, SGA hypertension, or in normal placentas [36]. mir-182 has been related T cell clonal expansion [37], to cell cycle, and to apoptosis pathways [38]. mir-182 is present in placentas with severe preeclampsia, it may regulate angiogenesis via VEGF, and it is also acknowledged as a regulator of the transcript variants 1 and 2 of the B cell lymphoma 2-like gene [16]. On the other hand, mir-210 is a potent mirna up-regulated by hypoxia [17] that can inhibit the migration and invasion ability of trophoblast cells [39]. Another mirna overexpressed in preeclampsia that may contribute to its development by down-regulating the angiogenic-regulating factor CYR61 is mir-155 [40]. mirnas may not only be key players in the pathophysiology of preeclampsia, but they may also help differentiate pathologic aspects in placentas affected by preeclampsia, preeclampsia complicated with SGA, and SGA alone. IMMUNE SYSTEM CELLS AND STBMs In normal pregnancies, STBMs present in the maternal circulation may stimulate the production of several cytokines by peripheral monocytes [41]. The recognition of STBMs by peripheral mononuclear leukocytes has been related to inhibition of IFN- production and to decreased levels of IP-10 in the first trimester of normal pregnancies [42]. These changes promote a shift toward type 2 T cell responses that is essential for gestation [43]. In preeclampsia, however, significantly higher levels of sstbms are present compared with normal pregnancies [44]; suppression of IFN- production by NK cells and other lymphocytes that were stimulated with STBMs does 248 Journal of Leukocyte Biology Volume 94, August

3 Laresgoiti-Servitje The immune system in the pathophysiology of preeclampsia not occur; and these cells continue to secrete IFN-, IL-18, TNF-, and IL-12 [42]. STBMs bind predominantly to receptors on monocytes and some B cells, inducing phagocytosis [43]. The receptors that participate in STBM recognition have not been identified clearly but may include RAGE and TLRs [41]. Higher amounts of STBMs released by the placenta may play a role in promoting a more robust inflammatory response [41] in pregnant women. However, the conditions under which the trophoblast microparticles are released may also be relevant, as microparticles derived from a hypoxic trophoblast induce higher concentrations of IL-6 and TNF- from PBMCs that recognize STBMs than do particles derived from a normal trophoblast [45]. Furthermore, STBMs from preeclamptic placentas exacerbate LPS responses in PBMCs [46]. This may help to explain the increased production of cytokines in preeclampsia, in which the placenta can be hypoxic, and may also be related to the generation of DAMPs under these hypoxic environments. In addition to cytokines derived from activated peripheral granulocytes and monocytes, lymphocyte-derived cytokines are probably secondary to the activation of endothelial cells by STBMs and may be involved in the pathophysiology of preeclampsia [47]. THE INNATE IMMUNE SYSTEM IN THE PATHOPHYSIOLOGY OF PREECLAMPSIA TLRs in preeclampsia According to the danger model [48], hypoxia can lead to a persistent inflammatory response, and this may occur in preeclamptic patients. A key inflammatory factor in preeclampsia is the recognition of DAMPs that can result from endothelial cell dysfunction, changes in glucose metabolism, hypoxia, or oxidative stress [49]. In hypoxic microenvironments, DAMPs may not be oxidized and denatured and thus, may promote inflammation [50] through ligation of receptors, such as RAGE, TLR2, and TLR4, which are expressed in immune system cells [51]. S100 and HMGB1 are proteins that behave as DAMPs [51, 52]. High cytoplasmic expression of HMGB1 occurs in decidual cells of preeclamptic patients [53], and S100B is increased in amniotic fluid during preeclamptic pregnancies [54] in direct relation to oxidative stress [55]. Furthermore, expression of TLR4 [56], TLR2, TLR3, and TLR9 is increased in trophoblasts of preeclamptic patients [57]. The expression of TLRs and RAGE receptors by the placenta shows its potential ability to respond to DAMPs, although the role of the placenta in this matter remains unknown [53]. In mice, TLR3 activation can increase systolic blood pressure and endothelial dysfunction, especially in the absence of IL-10 [58]. Although this alteration has not been proven in humans, the findings may be relevant, as preeclamptic women have decreased levels of IL-10 [59]. Moreover, placentas of preeclamptic women have increased expression of TLR3, TLR7, and TLR8 compared with those of normal human pregnancies, and the activation of TLR3, -7, and -8 by dsrnas and ssrnas promotes pregnancy-dependent, proteinuric hypertension and endothelial dysfunction in mice [60]. Likewise, the binding of circulating fetal DNA to TLR9 in mice can activate an inflammatory response, leading to IL-6 secretion [61]. This may be particularly crucial in human preeclampsia, in which high levels of circulating fetal DNA may be present [62]. Fetal DNA can bind TLR9 promoting inflammation, and TLR9 signaling may represent a potential therapeutic pathway, as it may be blocked by pharmacological agents, such as chloroquine [61]. Maternal infections, especially urinary infections and periodontal disease, have been associated with an increased risk of preeclampsia [63]. As many pathogens are recognized by TLRs [64], pathogens may also be increasing TLR activation in this syndrome. Thus, in preeclampsia, STBMs, pathogens, and DAMPs may participate as important activators of inflammatory processes. These mechanisms involve binding to TLRs, making these receptors possible therapeutic targets for preeclampsia. Transcription factors, NF- B and TLRs, in preeclampsia It has been proposed that transcription factors may help us gain insight into the pathophysiology of preeclampsia. On this matter, microarray studies have shown a higher prevalence of E-47, sterol regulatory element-binding protein, and NF- Bp50 transcription factor-binding sites in placentas complicated with preeclampsia [65]. NF- B is a regulator of inflammatory gene expression, it promotes the production of proinflammatory cytokines, and is highly activated in some inflammatory diseases [66]. In patients with preeclampsia, increased translocation of nuclear NF- B has been found in peripheral bloodactivated leukocytes [67]. Whereas the activation of NF- B may be associated to the presence of increased oxidative stress in preeclampsia [68], it is possible that TLRs may also be promoting an increase of NF- B in this syndrome, as all TLR signaling pathways culminate in the activation of this transcription factor [69]. Regarding possible therapeutic strategies affecting NF- B pathways, 5-deoxy- (12,14)-PGJ(2) has been proposed as a therapeutic alternative to modulate NF- B signaling in pregnancy, as it may decrease IFN- and TNF- production through inhibition of NF- B in PBMCs of pregnant women [70]. Monocytes promoting inflammatory conditions in preeclampsia Leukocytes from the nonspecific or innate immune system are important in normal pregnancy, as they promote successful implantation and participate in several events at the feto-maternal interface [71]. These cells may, however, also be involved in the pathophysiology of pregnancy disorders [72]. Activated monocytes and neutrophils are present in the fetal and placental circulation under hypoxic conditions and may contribute to the increased vascular resistance and morbidity of the fetus observed in preeclampsia [9]. Trophoblast cells under hypoxic conditions, such as those in preeclampsia, produce high concentrations of IL-6 and IL-8 and low IL-10 levels [73]. Nevertheless, the placenta is not the only contributor to the production of inflammatory cytokines Volume 94, August 2013 Journal of Leukocyte Biology 249

4 in preeclampsia [74]. Monocytes may represent an important source of proinflammatory cytokines in preeclampsia, as monocytes from preeclamptic patients secrete high levels of IL-1, IL-6, and IL-8 [75]. These cells have been classified into three subsets, according to their expression of CD14 (LPS receptor) and CD16 (FC RIII). Classical monocytes are CD14 CD16, intermediate monocytes are CD14 CD16, and nonclassical monocytes are CD14 CD16 [76]. Women with normal pregnancies have low percentages of classical monocytes and higher percentages of nonclassical/intermediate monocytes compared with nonpregnant women [77]. Nonclassical and intermediate monocytes are even higher in preeclampsia than in normal pregnancies [77, 78], and they show up-regulated expression of TLR4 [78], reflecting the importance of TLRs and TLR ligands in this syndrome. The exact function of the different subsets of monocytes in pregnancy and preeclampsia is unknown. In nonpregnant humans, intermediate monocytes are predisposed toward antigen presentation. They secrete inflammatory cytokines and ROS and may participate in angiogenesis [79]. In contrast, nonclassical monocytes may exhibit DC characteristics and produce IL-12 and IL-8 [80]. During pregnancy, TNF-, IL-6, and other proinflammatory cytokines derived from monocytes can activate the RAS, promote oxidative stress, and may lead to an increase in endothelium-derived vascular contracting molecules to diminish the bioavailability of vascular-relaxing factors derived from the endothelium [81, 82]. Furthermore, elevated TNF- levels correlate with the activity of AT1-R autoantibodies and an increase in sflt-1 and seng levels through AT1-R-mediated TNF- induction [33]. Neutrophils and oxidative stress in preeclampsia Neutrophils are activated in the peripheral blood [83, 84] and in the decidua of preeclamptic patients, and elastase produced by these cells may contribute to vascular damage [85]. In fact, neutrophils are strongly associated with vascular dysfunction in preeclamptic women, as they adhere to the endothelium in high densities [86]. Increased expression of IL-8 and ICAM-1 in vessels of preeclamptic women contributes to the infiltration of neutrophils into the maternal systemic vasculature [86]. Later, neutrophil adhesion to endothelial cells is linked to increased expression of CD11b, and neutrophil adhesion may be promoted by overproduction of superoxides and hydrogen peroxide [87]. Moreover, oxidants generated by activated neutrophil NADPH oxidase may react with different targets to form toxic metabolites that are products of lipid peroxidation, such as 4-hydroxynonenal, which contributes to microbial death and the damage induced by neutrophils [88]. Lipid peroxidation is elevated before and after childbirth (and delivery of the placenta) in women with preeclampsia, suggesting that these patients are under persistent oxidative stress that contributes to an inflammatory response [89]. Neutrophils may be carriers of cellular oxidative stress from the placenta to the vascular environment of the mother [90]. Neutrophil activation results from exposure to hypoxic or inflammatory conditions [89]. Placental microparticles, such as STBMs, may act as inflammatory agents, as in preeclampsia, the release of STBMs can activate neutrophils and promote formation of NETs [91]. NETs are extracellular structures composed of chromatin and granular proteins released during the death process, which occurs upon neutrophil stimulation. In this process, euchromatin and heterochromatin are homogenized, the nuclear and granular membranes disintegrate, and these components combine to create the NET. The NET is liberated when the cellular membrane breaks; it then binds to and kills microorganisms [92]. In the preeclamptic placenta, many NETs are induced in the intervillous space as a result of stimulation of neutrophils by STBMs and IL-8 [91]. As NETs participate in the pathogenesis of inflammatory disorders and autoimmunity [93], they may also contribute to the pathogenesis of preeclampsia, playing a role in the deficient placental perfusion associated with this disease [94]. In epithelial and endothelial cells, NETs can induce cytotoxicity, which is mostly mediated by histones and MPO [95]. The death process that initiates NET formation, called NETosis, is different from necrosis and apoptosis and depends on autophagy [96], generation of ROS, and NADPH oxidase [92], which is required to increase neutrophil adhesion to the endothelium. Besides oxidative stress-induced inflammation and endothelial dysfunction, preeclamptic patients also have increased levels of MPO. This enzyme is produced by activated monocytes and neutrophils and may contribute to placental and endothelial oxidative damage and the dysfunction of endothelial cells reported in these patients [97]. NK cells Placental NK cells, designated as unk cells, play an important role in the acceptance and rejection of the fetus, as they are in direct contact with the trophoblasts [98]. unk cells produce decidual IFN- in early human pregnancy, during which they may inhibit the invasion of the extravillous trophoblast [99] and probably promote a CD4 Th1 cytokine profile in preeclamptic women. Their participation may be more relevant during the origins of preeclampsia. Peripheral NK cells from preeclamptic women express lower intracellular VEGF levels than those from normal pregnant women [100], a finding that may link these cells with the endothelial dysfunction seen in this syndrome. Moreover, as NK cells express functional TLR3 and TLR9, they can recognize RNA and CpG DNA, which promotes their activation, especially in the presence of IL-8 [101]. Components of STBMs are also ligands for the NK cell receptor NKG2D [102]. Thus, STBMs or fetal DNA may interact with NK cells in patients with preeclampsia. NK cells also express several components of the RAS, such as renin, angiotensinogen, angiotensin-converting enzyme, and AT1-R and AT2-R, making NK cells responsive to AT2 levels [103]. Considering that preeclampsia may be related to dysregulation of the RAS [104], the presence of AT1-R and AT2-R in NK cells could be relevant in this syndrome. DCs Besides B cells and macrophages, DCs function as APCs during pregnancy and can modulate immune responses [105]. DCs are the link between the innate and adaptive immune 250 Journal of Leukocyte Biology Volume 94, August

5 Laresgoiti-Servitje The immune system in the pathophysiology of preeclampsia system, as they can respond to a variety of stimuli, such as TLR ligands, cytokines, and immune complexes [106]. These cells are particularly relevant during pregnancy, as they may modulate immune responses, depending on their activation through different TLRs or depending on the cytokine microenvironments in which their activation occurs [107]. Decidual CD14 DC-SIGN DCs may play an important role in itreg induction, and in preeclampsia, CD14 DC-SIGN and CD14 DC-SIGN decidual DCs induce itreg cells poorly [108]. Regarding peripheral blood DCs, human myeloid DCs (DC-1) are CD4 CD11c high CD123 low CD45RO BDCA-1, and plasmacytoid (lymphoid) DCs (DC-2) in peripheral blood are CD4 CD11c CD123 high CD45RA BDCA-2 [109]. The percentage of BDCA-2 lymphoid DCs is significantly lower in the blood of preeclamptic patients compared with women in the third trimester with normal pregnancies [110]. Considering that CD11c (BDCA-1 ) myeloid DCs produce IL-12 and may modulate toward Th1 responses [111] and that lymphoid CD303 (BDCA-2 ) DCs can promote a shift toward Th2 responses [112], the decreased number of lymphoid DCs in blood of preeclamptic women is noteworthy. Furthermore, suppression of Th1 responses by DCs may be associated with expression of serpin in myeloid APCs, which is a plasminogen activator inhibitor [113] that is decreased in preeclamptic women [114]. Myeloid and plasmacytoid DCs respond to TLR ligands, depending on their TLR expression. Myeloid DCs express TLR1 6 and TLR8, whereas plasmacytoid DCs strongly express TLR7 and TLR9 and have a low expression of TLR4 and TLR2 [115]. TLRs may represent a possible therapeutic target in this syndrome, as DCs in women with preeclampsia also show increased expression of basal TLR3, TLR4, and TLR9 and secrete higher levels of IFN-, TNF-, IL-1, and IL-12 [116]. Because of changes in expression of TLRs in DCs in preeclampsia, it may be relevant to evaluate to what extent TLR9 ligands, such as oligodeoxynucleotides containing unmethylated CpG motifs, or TLR7 ligands, such as RNA [117], may participate in the overproduction of IFNs and the modulation of the immune response in preeclamptic patients. Until recently, the stage of DC maturation was considered to be essential for their ability to induce Tregs or activate inflammatory T cell responses, but the developmental stage is no longer considered a key factor that differentiates between tolerogenic versus immunogenic DCs [118]. It is not the maturation state but the inflammatory factors or cytokines present during DC maturation that may influence the ability of DCs to induce different T cell responses [119]. The role of DCs in preeclampsia requires further investigation. Factors promoting the polarization of lymphoid versus myeloid DCs and the factors present during their maturation in preeclampsia remain unclear. The participation of TLRs and their ligands in DCs may help broaden our perspective regarding the role of these cells in the pathophysiology of preeclampsia. Figure 1 shows possible ligands for TLRs and their roles in preeclampsia. The activation of innate immune system cells that participate in the pathophysiology of preeclampsia is also described. THE ADAPTIVE IMMUNE SYSTEM In preeclampsia, the Th1/Th2 paradigm has been used to explain T cell behavior, as a shift from a Th1 to a Th2 phenotype at the fetal-maternal interface may not occur in this syndrome. Whereas Th1 cytokines, including IL-1, IL-2, and IFN-, are predominant in preeclampsia, the production of Th2 cytokines, including IL-10 and IL-5, can be decreased [120]. The changes in cytokine microenvironments, including elevated IFN- levels [ ], occur during the first weeks of pregnancy and promote a CD4 Th1 lymphocyte cytokine profile that can persist further into the preeclamptic pregnancy. The presence of low IL-10 levels in preeclampsia is also relevant [59], as IL-10 protects the fetus from rejection during normal pregnancy via activation of HLA-G expression in trophoblasts and monocytes at the fetal-maternal interface [124]. Although many women with preeclampsia present a shift toward Th1 cytokines, the Th1/Th2 paradigm does not respond to all of the questions regarding immune regulation in preeclampsia. The sole use of this paradigm to explain immunological aspects participating in the pathophysiology of this syndrome could be oversimplifying the mechanisms involved, as patients with preeclampsia can have changes in other T lymphocyte subsets. The alterations in numbers and function of Th17 cells and Tregs may help us understand more clearly the role of lymphocytes in the pathophysiology of preeclampsia. T lymphocyte subsets One CD4 lymphocyte subset that may be involved in the pathophysiology of preeclampsia is the CD4 CD25 FoxP3 Treg (FoxP3 is a Treg transcription factor) [125]. Some researchers have found no differences in Treg numbers between healthy and preeclamptic pregnancies [126]. Others have reported reduced numbers of Tregs in preeclampsia compared with normal pregnancies [127, 128]. CD4 CD25 FoxP3 Treg function is reduced in preeclampsia, which may be related to the presence of inflammatory conditions [129]. Moreover, the Treg pool in preeclamptic patients consists mostly of CD4 CD25 FoxP3 HLA-DR CD45RA cells. Although these cells express HLA-DR, which is related to suppression activity, they exhibit reduced regulatory capacity [125]. Thus, Tregs and Treg subsets seem to play a role in the pathophysiology of preeclampsia, but their role remains unclear. The presence of increased levels of seng, a protein thought to impair TGF- binding to receptors, could be blocking TGF- signals required for Treg functions and may participate in changes in this cell population in preeclampsia [130]. In addition to Tregs, CD4 IL-17-producing T cells (Th17) may participate in preeclampsia. Preeclamptic patients have a lower ratio of Tregs:Th17 cells [131]. T cell polarization is related to an imbalance of T cell transcription factors in PBMCs and in the decidua of preeclamptic patients. Decreased mrna levels of FoxP3 and increased levels of the Th17 transcription factor RORc and the Th1 transcription factor T-bet are present in preeclamptic women compared with healthy pregnant women [132]. The predominance of Th17 cells in pre- Volume 94, August 2013 Journal of Leukocyte Biology 251

6 Figure 1. In normal pregnancy, low levels of STBMs contribute to the inhibition of IFN- production and a shift toward Th2 responses. In preeclampsia, however, persistent hypoxia, the presence of free fetal DNA, and the shedding of high amounts of STBMs into the maternal circulation promote inflammatory conditions, in which neutrophils (NTs), monocytes (MNs), NK cells, endothelial cells (ECs), and DCs are stimulated. Neutrophil stimulation results in the activation of elastases and the production of superoxides and hydrogen peroxide via NADPH and MPO activation, respectively. Direct stimulation of neutrophils by STBMs may also result in damage through NET formation. Superoxides also promote neutrophil adhesion to the endothelium and NET formation at this level. Consequently, neutrophil activation results in vascular damage and dysfunction. In contrast, the nonclassical and intermediate monocytes in the presence of up-regulated TLR4 secrete cytokines and may contribute to the persistent inflammatory conditions. Plasmacytoid and myeloid DCs (pdcs and mdcs, respectively) also respond to TLR ligands and can modulate T cell responses. NK cells may play a role in the production of IFN- and the shift toward Th1 responses and may also respond to TLR ligands. eclampsia, accompanied by decreased Treg function and an altered balance in the Th17:Treg ratio [129], may be a result of altered levels of cytokines, including IL-6 and IL-1, that promote differentiation of these cells from progenitor cells [133]. However, CD8 lymphocytes and NK cells also secrete IL-17 and may contribute to inflammation in this syndrome [134]. T lymphocytes also possess functional RAS elements, able to produce AT2 at inflammatory sites. As AT2 can promote chemotaxis of NK cells and T cells by binding to AT1-R, a RASmediated inflammatory pathway may also be involved in preeclampsia [103]. The role of B lymphocytes and antibodies in preeclampsia A CD19 CD5 B cell population, which is able to produce AT1-AAs, has been identified in the placenta of preeclamptic patients [135]. These human CD19 CD5 B cells share phenotypic properties with murine B-1a lymphocytes [136]. In mice, B-1a cells have been involved in the generation of autoantibodies [137]. Likewise, human CD19 CD5 B cells may become autoreactive [138], as they are able to activate somatic hypermutation mechanisms that can promote mutations in the variable regions of the BCR [139]. This is a reason why human peripheral blood and spleen CD5 B cells may produce polyspecific, autoreactive antibodies [140]. The identification of CD19 CD5 B cells that can produce AT1-AAs [135] represents an important step in the understanding of the pathophysiology of preeclampsia. However, the mechanisms promoting the development of AT1-R autoantibodies have not been described yet. AT1-AAs of the IgG isotype [141] are present in 70 95% of preeclamptic patients [30, 142], they bind to receptors in human trophoblast and vascular cells [143], and their binding induces sflt1 and seng production by human villous explants through TNF- pathways [32, 33]. Binding of AT1-AA increases TNF- signaling in human placental villous explants, which then promotes IL-6 production that induces endothelin-1 production [144]. AT1-AAs, by ligating to the AT1-R on vascular smooth muscle cells, may also promote vasoconstriction [145] and can mediate hypertension by promoting placental oxidative stress [14, 146]. High levels of AT1-AAs are associated with the presence of hypertension, proteinuria, and sflt1 and may correlate with the severity of the disease [142]. Moreover, AT1-AAs may be a possible biomarker for late-onset preeclampsia [30]. As AT1-AAs can cross the pla- 252 Journal of Leukocyte Biology Volume 94, August

7 Laresgoiti-Servitje The immune system in the pathophysiology of preeclampsia cental barrier [104], these antibodies may also contribute to intrauterine growth restriction in some patients with preeclampsia, directly by activating AT1-R on the surface of fetal organs and indirectly by induction of apoptosis in the placenta [147]. AT1-AAs do not disappear completely after childbirth [148]. AT1-AAs are not the only autoantibodies that have been described in preeclamptic patients. The presence of autoantibodies against 1, 2, and 1 adrenoreceptors has been demonstrated in patients with severe preeclampsia and may increase the risk of neonatal morbidity and mortality [149]. Further studies are needed to identify the factors triggering their production and plausible mechanisms by which these antibodies may promote severe preeclampsia. The participation of B cells and different T cell subsets and the possible mechanisms that modulate their activity are described in Fig. 2. CONCLUSIONS Persistent hypoxia, alterations in oxygen-sensing mechanisms at the placental level, and increased levels of sstbms from the placenta are important factors that can contribute to the pathophysiology of preeclampsia. The increased shedding of STBMs from the placenta during preeclampsia may promote endothelial cell dysfunction and activation of maternal leukocytes, such as monocytes, neutrophils, NK cells, and DCs. Whereas monocytes are involved in the secretion of proinflammatory cytokines and may be promoting persistent inflammatory conditions in the preeclamptic patient, neutrophils play an important role in the vascular damage seen in preeclampsia. Neutrophils can be activated by inflammatory conditions caused by STBMs and persistent hypoxia. They may harm the endothelium through NET formation, elastase activation, or superoxide-related damage, promoting vascular dysfunction that results in increased vascular resistance. On the other hand, changes in DC subtypes may also participate in preeclampsia. Lower levels of lymphoid BDCA-2 DCs in preeclampsia could promote Th1-type responses in this syndrome, and they may be regulated by TLR3 and TLR9 ligands. As TLRs can act as receptors for STBMs, fetal DNA, and DAMPs, TLRs may play a key role in maintaining inflammatory conditions in preeclampsia. Moreover, TLRs may represent important therapeutic targets in this syndrome. Further research is needed regarding the role of TLRs in the recognition of STBMs and DAMPs in preeclampsia and their possible relationship with the modulation of DCs and T cell subsets. CD19 CD5 B cells, by producing AT1-AAs, are important contributors to the pathophysiology of preeclampsia and have rendered preeclampsia as a syndrome with autoimmune characteristics. This concept is also supported by the presence of autoantibodies against adrenoreceptors in patients with severe preeclampsia. However, the factors promoting the production of adrenoreceptor autoantibodies and the mechanisms by which these antibodies participate in preeclampsia still need to be explored. Many questions remain regarding the interaction between angiogenic/antiangiogenic factors with immune system cells and the possible participation of mirnas in immune system regulation in preeclampsia. The immune system plays an important role in many pathophysiological processes occurring in preeclamptic patients. This review aimed to examine the participation of several components of the immune system in the pathophysiology of preeclampsia. However, it has its limitations, as because of the great amount of cells and molecules that may be implicated, it is not possible to give a comprehensive overview of all of the interactions involved in this syndrome. Figure 2. Although changes in T cell subsets may be present at the origin of preeclampsia, the persistence of inflammatory conditions promoted by hypoxia, increased STBMs, or free fetal DNA via activation of the innate immune system cells may affect the generation of different T cell responses. Preeclamptic women have lower mrna levels of FoxP3, increased numbers of Th17, increased RORc mrna, and increased Th1 T-bet mrna. High IFN- concentrations may promote the development of Th1 lymphocytes, whereas IL-1, IL-6, and IL-7 may promote the generation of Th17 lymphocytes in preeclamptic women. These changes can result in a reduced regulatory capacity of Tregs in preeclampsia and a low Treg:Th17 ratio. High levels of seng could be affecting TGF- signaling required for itreg. CD19 CD5 B cells participate in the pathophysiology of preeclampsia by producing AT1-AAs. Factors promoting the development of these antibodies have not been described yet. Volume 94, August 2013 Journal of Leukocyte Biology 253

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