**6. Impact of cytotrophoblast cells and placenta-derived exosomes on regulatory B cells differentiation and function**

The pathophysiology of PE is poorly understood despite the evidences supporting a role of immune system in the development of PE [56]. B cells represent a dominant component in the pathogenesis of PE and studies focused on the number and functions of Bregs during PE are of great interest in understanding the pathophysiology

of PE [139]. Harnessing Bregs functions may lead to the capacity of using Bregs as an immunotherapeutic agent for averting and treating pregnancy pathologies such as PE. Perinatal cells including cells from term placenta and fetal annexes (amniotic membrane, chorionic membrane, umbilical cord, etc) are able to inhibit B cell proliferation, impair B cell differentiation and promote Bregs formation, frequently due to bioactive factors secreted by perinatal cells [11, 146]. These cells are considered as a promising tool for therapeutic approaches in PE [146]. Interactions between maternal immune cells and fetal annexes may result in hijacking naïve B cells and educating them to become Bregs. However, how cytotrophoblast (CT) and/or syncytiotrophoblast (ST) cells regulate Bregs differentiation and function during pregnancy is still unknown. Maybe in case of PE, CT and ST and their derived-vesicles (e.g. exosomes) will prevent adequate Bregs development and function, resulting in reduced and dysfunctional Bregs. This default of Bregs might result in an inflammatory environment, which will increase the susceptibility to PE.

Recent *in vitro* and *in vivo studies* have shown that perinatal cells and perinatal cells derived-vesicles interfere with the activation and differentiation of innate and adaptive immune system cells [11]. Poor knowledge is available about the impact of perinatal cells on B lymphocytes, even if some of the complex cross-talks between perinatal cells and B cells have been described. These studies demonstrated that perinatal cells have a strong antiproliferative capacity on B cells, but were not based on cell–cell contact. The demonstration is based on bioactive factors secreted by perinatal cells. For instance, co-cultured human mesenchymal stromal cells (MSC) isolated from umbilical cord (hUC-MSC) in a contact independent with mouse splenic B cells result in abrogation of the proliferation of activated B cells [147]. Likewise, human umbilical cord matrix cells co-cultured with a B cell cancer line (i.e. Burkitt's lymphoma cell line) [148], or with auto-reactive B cells from PBMC of immune thrombocytopenic patients results in inhibition of these B cells proliferation [149]. These observations were confirmed by using other perinatal cells (e.g., mesenchymal stromal cells (MSC)) purified from the amniotic membrane (hAMSC). This MSC supernatant is able to suppress CD19<sup>+</sup> B cell proliferation in PBMC or purified B cells from PBMC, confirming that cell-to-cell contact was not required and suggesting the role of soluble molecules and vesicles such as exosomes [11]. Similarly, human amniotic fluid stromal cells and their conditioned medium (CM) strongly suppress B cell activation and proliferation, and significantly inhibited the expression of CD80/CD86 costimulatory molecules on activated B lymphocytes [150].

However, some data contradict these observations and showed that human amniotic fluid stromal cells are able to suppress the apoptosis of B lymphocytes, favoring an increase in activated B cell survival. The mechanism underlying this inhibition is based on the decrease of the expression of the negative co-inhibitory molecules B7 homolog 4 (B7H4) and programmed death-ligand 1 (PD-L1) on activated B lymphocytes [150]. Moreover, an increase in B cell proliferation and a reduction in spontaneous apoptosis in the presence of human amniotic epithelial cells (hAEC) were also described [143]. Umbilical cord derived-MSC were not able to affect [144] or in other studies able to highly induce the *in vitro* growth of PBMC derived-B cells [145].

It is also demonstrated that human amniotic fluid stromal cells induce down-regulation of the proportion of B1 cells [150], resulting in the reduction of the B cell subset mainly involved in the production of autoantibodies in PE [151–153]. Many studies have shown that perinatal cell and their CM are able to block antibody-secreting cells CD19+ CD27+ CD38+ and the differentiation of B cells into CD138+ plasma cells, resulting

#### *Perspective Chapter: Role of Cytotrophoblast Cells and Placenta-Derived Exosomes… DOI: http://dx.doi.org/10.5772/intechopen.108335*

in the reduction of secreted immunoglobulin [11, 147, 150]. However, co-culture of purified B cells with human amniotic fluid stromal cells results in reduction of the proportion of CD19+ CD20+ CD27+ memory B cells [150], whereas PBMC cultured in the presence of CM-hAMSC increases CD19+ CD27+ CD38− memory B cells [11]. These different results may be explained by the presence of other immune cells among PBMC instead purified B cells. Moreover, different conditions of stimulation were used to activate B cells, and the lack of consensus in the markers used to characterize the B cell population could also support the distinct results observed by different groups.

Perinatal cells not only modulate B cell function by favoring their differentiation toward plasma cells, but they also promote the formation of Bregs. Indeed, it was reported that hAEC induced the expansion of CD19+ CD24hiCD38hi Bregs [143]. However, recent data suggested that IL-10+ Bregs were inhibited by human amniotic fluid stromal cells [150]. These observations clearly showed that more knowledge is needed to understand the impact of perinatal cells and other related vesicles on Bregs differentiation and functions. Thereby, it's important to identify the signaling pathways involved in underlying how perinatal cells and derivatives affect B cell proliferation and differentiation. Two signaling pathways were identified to be suppressed through CpG oligodeoxynucleotides (CpG ODN) by hAMSC: 1-the Toll-like receptor 9 (TLR9)-myeloid differentiation primary response 88 (MyD88)-interleukin-1 receptor-associated kinase (IRAK)1/4 and 2- the TLR9-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) pathways [11]. This suppression results in a reduction of uptake of the CpG ODN by CD205, TLR9, and CD14. Consequently, IRAK-4, mitogen-activated protein kinases (MAPK) (c-Jun N-terminal Kinase (JNK), p38 MAPK, extracellular signal-regulated kinase (ERK)) and nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB) pathways were inhibited. This induces an important reduction in the expression of phosphorylated AKT [11, 147]. The exact mechanism by which perinatal cells and derivatives induce Bregs differentiation is still unknown, and needs to be investigated [154]. Few data demonstrate that perinatal cells produce soluble factors including prostanoids (i.e., prostaglandin E2 (PGE2)), and maybe exosomes to immune regulate cells [155–157]. Therefore, we can speculate that Bregs differentiation is also induced by bioactive vesicles.

Based on *in vitro* results showing that perinatal cells have immunomodulatory properties, they were successfully tested in several inflammatory and immunemediated diseases, including lung [158, 159] and liver [160] fibrosis, inflammatory bowel disease, collagen-induced arthritis, experimental autoimmune encephalomyelitis [161], multiple sclerosis, wound healing [157, 162], traumatic brain injury [163], cerebral ischemia [164], Huntington's disease [165], and diabetes [166].

The therapeutic using hAMSC in pathological conditions driven by B cells has demonstrated a reduced idiopathic pulmonary fibrosis progression [158]. This treatment allows low levels of B cells in alveolar spaces and reduced the amount of CD138+ antibody-secreting cells in lung tissues, suggesting a decrease in B cell recruitment and an impairment of the maturation of B cells. Therapy using hAEC has also shown remarkable results in animal models of Hashimoto's thyroiditis and systemic lupus erythematosus (SLE) [167].

hAEC induced significant up-regulation of Bregs in experimental autoimmune thyroiditis mice. In this experiment, authors have shown that B10 cells are the major target of hAEC. In SLE mice, hAEC has shown the reduction in autoantibody production but without effect on B10 cells, suggesting that the mechanism of hAEC immunomodulation depends on the disease [167].

#### **Figure 1.**

*Differentiation and functional properties of Bregs. Through the production of exosomes by fetal annexes including cytotrophoblast cells, naive B cells can differentiate into Bregs. By producing IL-10, TGF-b, and IL-35, Bregs can suppress tumor necrosis factor-a (TNF-a)-producing monocytes, IL-12-producing dendritic cells, Th17 cells, Th1 cells, and cytotoxic CD8+ T cells. Bregs can also induce the differentiation of immunosuppressive Tregs, T regulatory 1 (Tr1), and dNK cells. This figure was created using Biorender.com.*

In the context of chronic graft-versus-host disease (cGVHD) prophylaxis repeated infusion of hUC-MSC seems to minimize the severity and the symptoms of cGVHD by increasing CD27+ memory B cells [168].

The immune modulation properties of perinatal cells depend on the origin of these cells. Indeed, fetal-derived cells induce strong inhibition of T-cell proliferation, cytotoxicity, and switch to M2 macrophages, while maternal-derived cells were more strongly able to induce Tregs [169]. **Figure 1** describes the probable implication of exosomes in Bregs differentiation and function (**Figure 1**).

As PE is pro-inflammatory disease and syncytiotrophoblast-derived exosomes (SDE) contribute to materno-fetal immuno-tolerance, it will be useful to understand how STB cells and SDE contribute to PE by altering Bregs differentiation and function during human pregnancy. These mechanisms may be close to those that inhibit immune flares or chronic inflammation in autoimmune diseases and transplantation.

### **7. Conclusion**

Gestation is a remarkable biological process in which the mother carries a fetus harboring half of a foreign genome belonging to the father. To allow the growth of the fetus, the maternal immune system needs to accommodate the semi-allogeneic fetus by dampening its immune responses. This results in a state of immunological tolerance throughout gravidity while maintaining the capacity to respond to pathogens properly. This paradoxical situation requires a perfect regulation of the balance between immune tolerance and immune activation.

To enable more accurate prediction and prevention of PE, its pathogenesis needs to be more understood. Increasing evidence suggests a consequence of the altered immune system in the development of PE. Today, it is clear that perinatal cells have capacity to regulate B cell response at different levels: by inhibiting B cell multiplication, impairing B cell differentiation, and inducing B regulatory cell formation.

*Perspective Chapter: Role of Cytotrophoblast Cells and Placenta-Derived Exosomes… DOI: http://dx.doi.org/10.5772/intechopen.108335*

Future research should focus on understanding how cytotrophoblast cells and placenta-derived exosomes act on B cells.

Overall, it is clear that cytotrophoblast cells and placenta-derived exosomes harbor the capacity of being a novel therapeutic approach for PE. However, the opposite results and the mainly small number of studies exploring the effect of cytotrophoblast cells and placenta-derived exosomes on the Bregs subset cannot allow deciding on a position. Further *in vitro* and *in vivo* studies are necessary to better decide the immunomodulatory potential of perinatal cells, leading to an important strategy for the treatment of PE.
