**2. Pregnancy and Inflammation**

The inflammatory response of the PlGF-Flt-1/VEGFR-1 axis is mediated by the transcription factor NF-κB involved in genes related to inflammation and immune response regulation. NF-κB has regulatory binding sites in the promoter of the PlGF gene and can modulate the transcriptional activity of PlGF via Rel-A in hypoxic human monocytes [107,108]. PlGF, by increasing the degradation of IκB-α, can increase the DNA binding activity of p65, an NF-κB subunit, thus, generating a self-feeding mechanism [107]. NF-κB is involved in several cellular pathways including inflammation, hypoxia, and angiogenesis, all processes implicated in placental development, thus consequently, when dysregulated, it is considered to be one of the main factors responsible for PE [109–111]. In fact, NF-κB is involved in the production of both TNF-α and PlGF resulting in aberrant activation of innate immune cells and imbalanced differentiation of CD4<sup>+</sup> T lymphocyte subsets, which may account for high cytokine levels and the cytotoxic environment in utero [48,112–114].

In normal pregnancy, three different phases can be identified, each characterized by a specific correlation between NF-κB and PlGF. The first trimester is characterized by a proinflammatory profile in which NF-κB is activated by inflammation and hypoxia that occurs during the development of the placenta. The second trimester shows an anti-inflammatory state as the pregnancy progresses. During the third trimester, NF-κB returns to be expressed at high levels in the decidua in preparation for parturition [115]. In addition, NF-κB can also directly upregulate the expression of TNF-α by lymphocytes during hypoxic stress, thus, establishing a vicious circle that feeds inflammation [116,117]. Therefore, the expression of NF-κB is critical in maintaining an adequate level of cytokines/PlGF required during the different periods of pregnancy.

A further link between the immune response and PlGF results from the involvement of another transcription factor, namely nuclear factor of activated T cells (NFAT)-1, which was initially identified in activated T cells [118,119] and has also been shown to be involved in the control of innate immunity [120]. Cytoplasmic NFAT is activated through calcineurin-mediated dephosphorylation, then, NFAT translocates into the nucleus, where it upregulates the expression of IL-2 and stimulates the growth and differentiation of the T cells [118]. Ding et al. reported that TNF-α was upregulated by tumor-derived PlGF in myelomonocytic cells via NFAT-1, which in turn contributed to the recruitment of PlGF-induced myelomonocytic cells [34]. Moreover, a region of the Flt-1/VEGFR-1 promoter contains a binding site for the transcription factor NFAT-1, thus, providing evidence that Flt-1/VEGFR-1 represents a NFAT-1 target gene [121]. The definitive confirmation of the role played by the PlGF-Flt-1/VEGFR-1-NFAT-1 axis in the placenta derives from the evidence that the inhibition of NFAT reduces both Flt-1/VEGFR-1 and sFlt-1/sVEGFR-1 splice variant e15a transcript secretion from primary human cytotrophoblasts [122]. In addition to being involved in cell proliferation, invasive migration, and angiogenesis, NFAT-1 mediates both the induced anergy of CD4<sup>+</sup> T cells through the expression of different inflammatory cytokines and the Treg-mediated suppression of T-helper (Th) cells activation [123–125]. Thus, PlGF, in addition to mediating inflammation, could contribute to induce a state of tolerance via NFAT-1, by binding Flt-1/VEGFR-1. Overall, these results further confirm the effects of PlGF on the immune response.
