**4. Gene expression of placental immune cells**

Beside the investigation of gene expression in whole placenta, several studies investigated transcriptomic profiles of isolated placental cells including immune and nonimmune cells. We focused here only on immune cells. Placental immune cells are necessary for development of the placenta bed and the semi-allogeneic tolerance of the fetal-placental unit. In addition, they are involved in the regulation of trophoblast invasion, angiogenesis and spiral artery remodeling. The distribution of immune placental cells is markedly distinct from that of circulating immune cells: NK cells and macrophages are found at high density, whereas lymphocytes, dendritic cells and mast cells are less represented. Therefore, we studied the transcriptomic response of macrophages, dendritic cells and mast cells.

#### **4.1. Macrophages**

Placental macrophages include decidual and Hofbauer cells from decidua (basalis) or villous stroma, respectively. They are the most abundant cells that persist throughout pregnancy [114, 115]. They play a central role in maintaining a homeostasis during pregnancy by controlling imbalance between anti- and pro-inflammatory features of placenta environment [116, 117]. Indeed, the pro-inflammatory (M1) polarization of macrophages or the prevention of M2 polarization (anti-inflammatory and/or immunoregulatory properties) leads to spontaneous abortion or miscarriage [118], inadequate remodeling of the uterine vessels during placentation and protection against infection [119].

#### *4.1.1. First and second trimesters*

After placentation, placental macrophages exhibit a M2 profile during the first and second trimesters. This M2 profile contributes to the anti-inflammatory environment of the first part of pregnancy and the prevention of the rejection of the fetus by maternal immune system [120]. The first study of gene expression pattern in human first trimester decidual macrophages was conducted in 2008 [121]. The authors identified 14,000 genes modulated in placental macrophages compared to blood monocytes. Genes involved in immunomodulation and remodeling categories are associated with M2 phenotype. Hence, the genes encoding CCL18, CD209, insulin-like growth factor-1 (IGF-1), mannose receptor c type (MRC)-1 and fibronectin-1 are up-modulated. Houser et al. investigated the polarization of macrophage subsets isolated from decidua from first trimester (6–12 weeks) [122]. They identified two distinct macrophage subsets according to CD11c expression, namely CD11clow and CD11chigh subsets. Both subsets expressed two M2 markers, CD209 (DC-SIGN) and CD206 (mannose receptor). Genes involved in invasion, mobility, inflammatory process and lipid metabolism are enriched in the two subsets, but genes involved in antiapoptotic pathways are found only in CD11high macrophages. In contrast, CD11clow macrophages express genes involved in growth regulation and development, and extracellular communication. These findings are in agreement with those of Svensson et al. who found two macrophage subsets, ICAM-3high and ICAM-3low, in macrophages from first trimester (7–12 weeks) [123]. Both macrophage populations express genes and cytokines associated with M2 profile and are related to CD11c expression.

#### *4.1.2. Third trimester (term placentas)*

Altered circulating levels of specific microRNAs have been reported in compromised pregnancies. For example, the low detection of miR-376c level in blood of 16–18 weeks pregnant women is related with the outcome of preeclampsia [112]. In similar conditions, three miRNAs, including miR-132, miR-29a and miR-222, are decreased in blood from women at 16–19 gestational weeks who were diagnosed a GDM at 25–28 weeks of gestation [113]. Thus, despite the promising perspectives to use microRNAs in diagnosis of complicated pregnan-

Beside the investigation of gene expression in whole placenta, several studies investigated transcriptomic profiles of isolated placental cells including immune and nonimmune cells. We focused here only on immune cells. Placental immune cells are necessary for development of the placenta bed and the semi-allogeneic tolerance of the fetal-placental unit. In addition, they are involved in the regulation of trophoblast invasion, angiogenesis and spiral artery remodeling. The distribution of immune placental cells is markedly distinct from that of circulating immune cells: NK cells and macrophages are found at high density, whereas lymphocytes, dendritic cells and mast cells are less represented. Therefore, we studied the transcriptomic

Placental macrophages include decidual and Hofbauer cells from decidua (basalis) or villous stroma, respectively. They are the most abundant cells that persist throughout pregnancy [114, 115]. They play a central role in maintaining a homeostasis during pregnancy by controlling imbalance between anti- and pro-inflammatory features of placenta environment [116, 117]. Indeed, the pro-inflammatory (M1) polarization of macrophages or the prevention of M2 polarization (anti-inflammatory and/or immunoregulatory properties) leads to spontaneous abortion or miscarriage [118], inadequate remodeling of the uterine vessels during

After placentation, placental macrophages exhibit a M2 profile during the first and second trimesters. This M2 profile contributes to the anti-inflammatory environment of the first part of pregnancy and the prevention of the rejection of the fetus by maternal immune system [120]. The first study of gene expression pattern in human first trimester decidual macrophages was conducted in 2008 [121]. The authors identified 14,000 genes modulated in placental macrophages compared to blood monocytes. Genes involved in immunomodulation and remodeling categories are associated with M2 phenotype. Hence, the genes encoding CCL18, CD209, insulin-like growth factor-1 (IGF-1), mannose receptor c type (MRC)-1 and fibronectin-1 are up-modulated. Houser et al. investigated the polarization of macrophage subsets isolated from decidua from first trimester (6–12 weeks) [122]. They identified two

cies, future studies are needed to provide a proof of concept.

**4. Gene expression of placental immune cells**

response of macrophages, dendritic cells and mast cells.

placentation and protection against infection [119].

*4.1.1. First and second trimesters*

**4.1. Macrophages**

40 Placenta

The third trimester is associated to a pro-inflammatory environment. This inflammatory state is due to the synchronization of immune-endocrine cross-talk, based on especially estrogens [124], which favors M1 polarization of macrophages [125]. Indeed, a low level of estradiol is sufficient to promote macrophage M1 polarization, as measured by the secretion of inflammatory cytokines such as IL-1β, IL-6 and TNF. The investigation of DNA of at term placenta macrophages reveals the methylation of inflammatory genes including TLR9, IL-1β, IL-12RB2, CD48 and FGR and the hypomethylation of M2 genes including CCL2, CCL13, CCL14 and CD209 [126]. Using microarray analysis, we previously investigated the polarization of macrophages from at term placentas in comparison to macrophage-derived monocytes [127]. We did not find a polarization of at term placenta macrophages. Indeed, both M1 (CXCL9, EDN1, IL-15, IL-15RA and IL-2RA) and M2 (FN1, CTSC and CCL23) genes were up-modulated. Interestingly, we reported for the first time the ability of placental macrophages to formed multinuclear giant cells (MGCs). These MGCs present functional enrichment in genes associated with cytoskeleton reorganization and immune response. In addition, as observed in placental macrophages, MGCs are not polarized. Taking together, although the third trimester is associated with an inflammatory environment, the activation of placental macrophages does not reproduce the classical model of M1/M2 polarization.

#### *4.1.3. Placenta macrophages in pathological pregnancy*

Placental macrophages are likely associated with number of pregnancy complications. Here, we focused on three pathological conditions, preeclampsia, GDM and chorioamnionitis, in which the involvement of macrophages is documented [128] .

#### *4.1.3.1. Preeclampsia*

Although alterations of macrophages are suspected in preeclampsia, the studies of placenta macrophages during preeclampsia are controversial [116, 129]. Some studies reported a decreased [115, 130, 131] or an increased [132–135] number of placental macrophages. In addition, they produce inflammatory cytokines and anti-inflammatory cytokines [136, 137]. It has been also reported that the count of M2 macrophages is decreased in the decidua of preeclamptic placentas [132]. The transcriptomic and protein expression of CD74, a HLA class II molecule, are decreased in placental macrophages of preeclampsia women. This down-regulation of CD74 interferes with trophoblast-macrophage cross-talk [138]. Prins et al. investigated macrophages in early decidua from women who later developed preeclampsia [139]. They observed an increased expression of CD68 mRNA, but decreased CD206/CD68 mRNA ratio, suggesting that the number of M2 macrophages is affected before the onset of preeclampsia. This finding may be related to the increased number of M1 macrophages in preeclamptic placenta found by other authors [140]. A better comprehension of the rupture of the M1/M2 balance may provide insight into understanding of preeclampsia pathogenesis.

We previously isolated decidual dendritic cells from at term placentas of healthy women by combining negative selection with anti-CD14 antibodies and positive selection with anti-CD11c antibodies [149]. We found that 1525 genes are differentially modulated with a specific transcriptional profile as compared to monocyte-derived dendritic cells. It mainly consists of the up-modulation of genes involved in immunomodulatory cytokines, estrogen and progesterone gene pathway. The investigation of gene expression programs in placenta dendritic cells is emerging and will require additional investigation to associate gene expression and

Gene Expression Profiling of Placenta from Normal to Pathological Pregnancies

http://dx.doi.org/10.5772/intechopen.80551

43

Mast cells are found in human placentas [150] and it has been reported that mast cells and their products, especially histamine, could participate in the placenta development. Indeed, histamine is involved in trophoblast invasion and growth [151] and the cross-talk with trophoblasts *via* the expression of adhesion molecules by trophoblasts [152]. The alteration of these specific adhesion molecules is associated with the impairment of placenta invasion and the outcome of preeclampsia. A decreased number of mast cells have been reported in preeclampsia, GDM and intrauterine growth retardation [151, 153]. To our knowledge, no data about gene expression in placenta mast cells has been published. We isolated placenta mast cells from at term women using positive selection with CD117 and IgE antibodies. In a comparative study with a mast cell line (human mast cells, HMC-1.2), we found that a large number of genes are up-modulated in placenta mast cells. The functional analysis reveals an enrichment with three categories, FcεRI signaling, immune response and reproduction processes. In this latter category, we identified specific genes of Wnt pathway and a set of genes involved in the response to estrogen and progesterone (manuscript in preparation). These preliminary results highlight the originality of placenta mast cells among placenta

**4.4. Common transcriptomic signature of macrophages, dendritic cells and** 

To identify a common signature of these three placenta immune cells, we performed a retroanalysis of microarray data deposited in Gene Expression Omnibus at the National Center for Biotechnology Information. We evaluated the transcriptomic signatures of macrophages, dendritic cells and mast cells from at term placentas of healthy women and compared them to those of monocytes, monocyte-derived dendritic cells and the cell line HMC-1, respectively. As depicted in **Figure 2A**, the hierarchical clustering reveals two major branches that distinguish cells of placenta origin from the others (ANOVA, p < 0.001). Focusing on common modulated genes, we observed that 479 (**Figure 2B**) and 5671 (**Figure 2C**) genes are up- and down-regulated, respectively. Interestingly, 45% of down-modulated genes are associated with pregnancy versus 10% of up-modulated genes. Thus, these data suggest that innate immune cells express a core of genes that reflect the influence of placenta

dendritic cells subsets in normal and pathological pregnancy.

**4.3. Mast cells**

innate immune cells.

microenvironment.

**mast cells**

## *4.1.3.2. Gestational diabetes mellitus*

The inflammation associated with GDM leads to macrophage infiltration into placenta, suggesting a key role of these cells during this metabolic complication of pregnancy. The number of macrophages is increased in placentas from women with GDM as compared to normal pregnancy [141]. Placental macrophages, isolated from an experimental model of diabetes (rats receiving an injection of streptozotocin), change from M2 to M1 inflammatory profile under high glucose stimulation [142]. Similarly, isolated placental macrophages from diabetic women present a M1 or an atypical M2 profile [142]. Immunohistochemistry and PCR approaches show that the mRNA expression levels of TNF and IL-6 are higher in placentas from women with GDM than in controls, suggesting an inflammatory profile of placenta macrophages. Further studies are needed to define the role of inflammatory macrophages in GDM.
