*2.2. Natural Killer (NK) Cells*

During pregnancy, NK cells are the predominant population of lymphoid immune cells in the uterus at the maternal-fetal interface and are involved both in placental vascular remodeling and in a mother's immune tolerance [128–134].

NK cells are members of a rapidly expanding family of innate lymphoid cells (ILCs), derived from early common lymphoid progenitors, the CD34<sup>+</sup> hematopoietic stem cell [135,136]. Given their heterogeneous characteristics, a new classification that categorizes ILCs into five subsets based on transcription factors and cytokines production has been proposed [137]. These subsets include ILCs1, ILCs2, ILCs3, lymphoid tissue-inducer (LTi) cells, and conventional NK (cNK) cells [138,139]. ILCs interact with the tissue cells to ensure, in addition to the immune response, homeostasis and tissue repair [140,141]. Among these subsets, cNKs are very similar to Tbet<sup>+</sup> ILCs1 producing IFN-γ but differ in their higher cytotoxic potential. A common feature of cNK cells and tissue resident NK (trNK) cells is their IL-15-dependent signaling during early development, however, they differ in their ability to recirculate [142–144]. In fact, cNK cells circulate freely, while trNK cells are resident in the liver, skin, kidney, and virgin uterus [135,136,140,143–145].

On the basis of different receptors and transcription factors expression, it is possible to further distinguish cNK cells from trNK cells. In contrast to cNK cells, where CD56 and CD16 expression allows for discrimination of cell cytotoxic and regulatory subsets, uterine trNK cells are almost exclusively CD16−CD56bright [146]. In addition, whereas Tbet is required for the development of trNK cells in the liver and skin, uterine trNK develop independently of the Tbet transcription factor [147]. All these differences indicate that cNK and trNK cells in the uterus represent different lineages of NK cells rather than different differentiation states [130,140,145].

In mice, the onset of decidualization is characterized by a series of events such as the extracellular matrix (ECM) remodeling of the endometrial stroma, the induction of angiogenesis, and a significant increase in uNK cells, which originates mainly from local proliferating trNK cells, [73,143,144,148–150] while the recruitment of cNK cells takes place later [144,151].

Unlike cytotoxic T lymphocytes, NK cells can eliminate tumor cells, infected cells or nonself cells, by direct contact without a previous activation, due to their natural cytotoxic activity. Direct contact between NK and the target cells may engage activating or inhibitory receptors expressed on NK cells. Each NK cell can express simultaneously several different activating or inhibitory receptors, resulting in the potential for many specificities. The uNK cells cytotoxic ability is regulated by an education process where only those cells that recognize the "self" are promoted to have cytotoxic ability, become tolerant, and act when there is a "dangerous self" or "missing self" signals [152,153]. This ability of NK cells is tightly regulated by a complex of interactions among the target cell and activating or inhibitory receptors expressed on the NK cell surface. If the strength of the activating signals outweighs the inhibitory signals, the cell releases cytolytic granules directed against the target cell and produces cytokines [154,155]. Among these receptors, the expression of killer immunoglobulin-like receptors (KIRs) confers to uNK cells an important function, by inhibiting the production of cytotoxic cytokines and stimulating the production of angiogenic factors [75,131,156]. Thus, resting uNK cells have a low cytotoxic ability as compared with "primed" uNK cells. Additionally, uNK cells priming is regulated by the microenvironment in which the NK cell is present and can also be regulated by the proximity of other immune cell types such as monocytes, DCs, and T cells [157–161].

Genetic association studies have indicated that both uNK cell-activating receptors, KIR2DS4 and KIR2DS1, recognize fetal HLA-A ligands and protect from PE [157,162]. Conversely, pregnant women with a specific KIR haplotype and fetal HLA-C2 genotype combination have a significantly higher risk of PE [163]. Thus, uNK cells can respond to fetal MHC class I via their inhibitory and activating receptors to control appropriate placental vascularization and development.

During the first trimester of pregnancy, uNK cells represent as much as 50–70% of decidual infiltrating lymphocytes and are characterized by CD56brightCD16−KIR+CD9<sup>+</sup> phenotype [146,164,165]. Unlike cNK cells, uNK cells are poorly cytolytic; they release cytokines/chemokines that regulate the immune environment and angiogenic growth factors, such as PlGF, for placentation [73,129,148–150,156,166,167]. One of the plausible mechanisms by which uNK cells are inhibited in their cytotoxic activity is through hypoxia, a condition that is able to stimulate angiogenesis and which is normally present during decidualization. Indeed, it was shown that the cytolytic capacities of uNK cells were markedly and significantly impaired under hypoxic conditions and this inhibition was associated with the activation of transcriptional factor STAT3 [168]. Of interest, as mentioned above, hypoxia upregulates the expression of PlGF, its receptor Flt-1/VEGFR-1 and co-receptor NRP1 [68,69,82]. A further mechanism by which uNK

cells induce maternal tolerance is through the crosstalk between uNK and CD14<sup>+</sup> myelomonocytic cells. These cells are in close contact in the decidua and their interaction is mediated by IFN-γ. Following interaction with uNK cells, decidual CD14<sup>+</sup> cells express indoleamine 2,3-dioxygenase (IDO) resulting in the induction of Treg cells and immunosuppression [169]. In addition, decidual CD14<sup>+</sup> cells may also induce Treg cells through transforming growth factor-β (TGF-β) production or Cytotoxic T-Lymphocyte Antigen (CTLA)-4-mediated interactions. Notably, only the interaction between uNK and decidual CD14<sup>+</sup> cells results in Treg cell induction, whereas cNK or CD14<sup>+</sup> cells isolated from peripheral blood are ineffective [169]. Therefore, uNK cells, in addition to being involved in the regulation of invading trophoblastic cells and in providing immunity during pregnancy, play an essential role in modulating maternal tolerance [7,128,132,152].

Although the details of signaling pathway triggering cytokines production are still elusive, IFN-γ and angiogenic growth factors such as PlGF produced by uNK cells contribute to spiral arteriole remodeling by acting on endothelial cells and decidual stromal cells [73,129,148,159,170]. By contrast, genetic evidence in humans and mice suggests that excessive inhibition of uNK cells function impedes both decidual arterial remodeling and fetal growth [75,148,167,170–172] (Figure 2). However, it has been reported that uNK cells depletion did not reproduce a deficiency in uterine spiral artery remodeling but was associated with a marked maternal uterine vasculopathy at a later time point in gestation in a rat model of PE induced by antibody-based NK cells depletion [171]. This evidence could account for the onset of endothelial dysfunction, and subsequently, PE in late pregnancy. *Int. J. Mol. Sci.* **2020**, *21*, 8714 8 of 25

**Figure 2.** Reduced invasiveness and remodeling of maternal spiral arteries by trophoblastic cells is associated with a risk of pathological pregnancy. **Figure 2.** Reduced invasiveness and remodeling of maternal spiral arteries by trophoblastic cells is associated with a risk of pathological pregnancy.

During a normal pregnancy (Figure 2, left panel) with adequate PlGF levels, EVT cells migrate to the myometrium and infiltrate the endothelium of the maternal spiral arteries. This results in dilatation and increased flow of maternal blood at low pressure into the intervillous space. Uterine NK cells (CD56+CD16−) and M2 macrophages facilitate deep invasion of trophoblast cells into the myometrium. Moreover, tolerogenic immature dendritic cells (iDCs) promote Treg lymphocytes. Conversely, in pathological pregnancies (Figure 2, right panel) with unbalanced levels of PlGF, the immune cells in the uterine microenvironment (uNK CD56lowCD16+ cells, M1 macrophages, mature DCs, Th1 and Th17 lymphocytes, and CD8+ cells) fuel an excessive inflammatory response and reduce During a normal pregnancy (Figure 2, left panel) with adequate PlGF levels, EVT cells migrate to the myometrium and infiltrate the endothelium of the maternal spiral arteries. This results in dilatationand increased flow of maternal blood at low pressure into the intervillous space. Uterine NK cells (CD56+CD16−) and M2 macrophages facilitate deep invasion of trophoblast cells into the myometrium. Moreover, tolerogenic immature dendritic cells (iDCs) promote Treg lymphocytes. Conversely, in pathological pregnancies (Figure 2, right panel) with unbalanced levels of PlGF, the immune cells in the uterine microenvironment (uNK CD56lowCD16<sup>+</sup> cells, M1 macrophages, mature DCs, Th1 and Th17 lymphocytes, and CD8<sup>+</sup> cells) fuel an excessive inflammatory response and reduce the invasion

[173]. They also produce several cytokines that recruit and regulate cells of the adaptive immune system as well [161,174]. In addition to the production of PlGF and cytokines, an additional mechanism by which uNK could induce maternal-fetal tolerance appears to promote the induction of Treg cells and suppress Th17 cells [175,176]. Indeed, PE is associated with a reduction in Treg cells in the circulation and decidua and the severity of the disease is related to their reduced number [177]. It is plausible to assume that one of the mechanisms by which uNK cells induce maternal tolerance is through the production of an adequate level of PlGF. In fact, as previously mentioned, the involvement of Flt-1/VEGFR-1 and NRP1 receptors induces the secretion of IL-10 by T lymphocytes [88] and the delayed maturation of DCs [93], respectively, thus, favoring the development of Treg

Of note, serum IL-17 levels have been shown to be significantly higher in PE patients than in healthy non-pregnant and pregnant women, and elevated IL-17 and sFlt-1/PlGF ratio serum level had an additive effect on the risk of PE [178]. Predictably, Th17 cells are higher in PE than in non-PE pregnancy, resulting in an imbalance between Treg/Th17 with a proinflammatory phenotype and an increased secretion of inflammatory cytokines [176]. This evidence has been recently confirmed by Yoo et al. Indeed, Th17 cells selectively secrete PlGF, and PlGF in turn, specifically induces the differentiation of inflammatory Th17 cells by activating the transcription factor STAT3 via binding to Flt-1/VEGFR-1. Moreover, PlGF replaces the activity of IL-6 in the production of IL-17, suppressing

pressure resulting in placental stress and reduced placental development.

cells.

the invasion of the trophoblast with less remodeling of the spiral artery. Blood flows at higher

of the trophoblast with less remodeling of the spiral artery. Blood flows at higher pressure resulting in placental stress and reduced placental development.

Uterine NK cells have also been reported to contribute directly to fetal growth by producing growth-promoting factors essential for embryo development prior to the development of the placenta [173]. They also produce several cytokines that recruit and regulate cells of the adaptive immune system as well [161,174]. In addition to the production of PlGF and cytokines, an additional mechanism by which uNK could induce maternal-fetal tolerance appears to promote the induction of Treg cells and suppress Th17 cells [175,176]. Indeed, PE is associated with a reduction in Treg cells in the circulation and decidua and the severity of the disease is related to their reduced number [177]. It is plausible to assume that one of the mechanisms by which uNK cells induce maternal tolerance is through the production of an adequate level of PlGF. In fact, as previously mentioned, the involvement of Flt-1/VEGFR-1 and NRP1 receptors induces the secretion of IL-10 by T lymphocytes [88] and the delayed maturation of DCs [93], respectively, thus, favoring the development of Treg cells.

Of note, serum IL-17 levels have been shown to be significantly higher in PE patients than in healthy non-pregnant and pregnant women, and elevated IL-17 and sFlt-1/PlGF ratio serum level had an additive effect on the risk of PE [178]. Predictably, Th17 cells are higher in PE than in non-PE pregnancy, resulting in an imbalance between Treg/Th17 with a proinflammatory phenotype and an increased secretion of inflammatory cytokines [176]. This evidence has been recently confirmed by Yoo et al. Indeed, Th17 cells selectively secrete PlGF, and PlGF in turn, specifically induces the differentiation of inflammatory Th17 cells by activating the transcription factor STAT3 via binding to Flt-1/VEGFR-1. Moreover, PlGF replaces the activity of IL-6 in the production of IL-17, suppressing the generation of Treg cells [37]. Remarkably, as mentioned above, the hypoxic microenvironment reduces the killing capacity of uNK cells via STAT3 and, at the same time, upregulates PlGF production by uNK cells. By contrast, PlGF secreted by Th17 cells suppresses the generation of Treg cells via STAT3 and induces proinflammatory IL-17 secretion. Overall, these results suggest a refined regulation of the inflammatory response in the early stages of pregnancy, confirming the important role of PlGF in modulating the vascular development of the placenta, and also the behavior of the immune cells.
