Immunomodulatory properties of extracellular vesicles in the dialogue between placental and immune cells

1.1 Intercellular communication mediated by extracellular vesicles

Cells have developed an efficient strategy to transfer sets of molecular information through biological fluids without their disintegration and degradation by messaging via EVs. Moreover, molecules that are cell membrane-impermeable can be incorporated effectively and at adequate amounts. This mechanism of intercellular communication is achieved by packing a rich assortment of ncRNAs, mRNAs, proteins, and lipids into vesicular bodies that are constitutively exported by donor cells and incorporated by recipient cells.^1-4 EVs modulate signaling pathways, mRNA levels, and protein expression influencing in this way cell metabolism, proliferation, apoptosis, and immunological functions.⁵ Besides their biological roles, EVs are promising vehicles for therapeutic delivery of biological materials and synthetic compounds to cells without triggering unwanted immunological responses.^{6, 7}

Based on their size, content, and origin, EVs can be classified into exosomes (40–150 nm), microvesicles (150–1000 nm), and apoptotic bodies (1000–5000 nm).^{4, 8} The placenta also releases a particular population of large vesicles denominated syncytial nuclear aggregates (SNAs) containing fetal DNA, RNA, and organelles, which are associated with maternal-fetal communication.^{9, 10} Other EV types/subtypes have been recognized and updated classifications are likely to arise with further advancements in the field. Due to their reduced dimensions and heterogeneity, the analysis of EVs imposes several methodological challenges. Limitations to isolate specific EV populations have led to the proposal to classify EV fractions in exosome-enriched small vesicles (sEVs) and in microvesicles-enriched large vesicles (lEVs).¹¹ Analytical approaches are being refined to handle these constraints allowing a better resolution, isolation, and characterization of EV populations.^{12, 13}

Exosomes are generated through endosomal and secretory pathways. Membrane invagination of late endosomes leads to the formation of multivesicular bodies (MVBs) filled with intra-luminal vesicles (ILVs). Following the docking and fusion of MVBs with the plasma membrane, ILVs are secreted in the extracellular space as exosomes. Cytoskeleton components, tetraspanins (CD9, CD63, CD81, and CD82), and the endosomal sorting complex required for transport (ESCRT), including ALIX, Tsg101, CHMP4, as well as Flotillin-1, Syntenin-1, and RAB small GTPases are part of molecular complexes that drive EV biogenesis and secretion.^14-16 Some of these molecules are conserved on the surface of secreted EVs and can serve as markers for their identification and isolation. Differently from exosomes, microvesicles are released from outward budding and fission of plasma membrane blebs. Nevertheless, there is a high correspondence in the molecular complexes engaged in both exosome and microvesicle biogenesis. Microvesicle biogenesis and release also involve myosin light chain, RhoA GTPases, and their downstream mediators ROCK (Rho-associated coiled-coil containing kinase) and cofilin.^{17, 18} Different markers have been proposed to distinguish between exosomes (CD9, CD63, and CD81)¹⁹ and microvesicles (annexin A1),⁴ but they may be not universally appropriated for EVs from different sources. Therefore, the applicability of EV markers should be validated within each experimental context.

Viruses exploit the cellular machinery to replicate sharing similar routes with EV biogenesis.²⁰ Considering that around half or more of the human genome originated from retroviruses,²¹ it is tempting to speculate that viral infections may be engaged in an evolutionary symbiosis²² with cellular EV biogenesis. The exportation of the endogenous retroviral envelope protein syncitin, which participates in trophoblast syncytialization, in placental exosomes²³ is an evidence of this process. Viral infections may have contributed to the development of EV biogenesis pathways and, simultaneously, the development of EV biogenesis pathways may have aided in the evolution and transmission of viruses.

The EV content reflects the expression profile, functional status, and pathological conditions of the cells from which they originate. In this context, they are being explored as biomarkers to monitor the development and progress of diseases, including pregnancy complications.^{24, 25} Mechanisms of cargo selection may take place during EV biogenesis leading to the enrichment of certain molecular components. For example, small RNA species are more abundant than the far longer mRNA sequences.²⁶ Loading of RNA molecules is assumed to occur via passive incorporation as well as RNA-binding proteins.^{14, 27} Although the cargo packed into exosomes and microvesicles presents a significant overlap, there are also differences leading to distinct functions.²⁸

The molecular signature carried by EVs to direct them to recipient cells and their intracellular targets are largely unknown, but many molecular components and main cellular routes of EV uptake have been unveiled. Cells employ a combination of several mechanisms to incorporate EVs, namely: i) clathrin-dependent or -independent endocytosis, ii) caveolin-dependent uptake, iii) lipid raft-mediated endocytosis, iv) phagocytosis, and v) micropinocytosis. The specific contribution of these processes to EV uptake by immune cell populations warrants further investigations. Immune cells present distinct EV production and uptake ratios.²⁹ Monocytes and immature dendritic cells (DCs) incorporate fewer EVs than macrophages and mature DCs, indicating that the functional status of immune cells modulates their “appetite” for EVs.³⁰ Similarly, the activation of T cell receptors (TCR) and B cell receptors (BCR) on the respective cells stimulates exosome secretion.^{31, 32}

1.2 Immunomodulatory properties of EVs

Early reports have shown that syncytiotrophoblast EVs inhibit the expression of activation markers, cytokine production, and proliferation of lymphocytes^{33, 34} and endothelial cells.³⁵ In the search for the biological functions and mechanisms of action, further studies have demonstrated the immunomodulatory properties of EVs and their involvement in antigen presentation to immune cells. Exosomes produced by B lymphocytes and DCs are loaded with functional peptide-bound major histocompatibility complex (MHC) II as well as with co-stimulatory factors CD80 and CD86 and promote T cell proliferation.^{36, 37} EVs are exchanged by DCs to synchronize and amplify their response and, when incorporated by T cells, stimulate their activation.^{37, 38} More recently, the anti-viral properties of trophoblast EVs mediated by miRNAs of the C19MC cluster were unveiled.^{39, 40}

Collectively, several immunomodulatory roles have been attributed to EVs, including immune suppression or activation, apoptosis induction, and trafficking of cytokines, their receptors, and proinflammatory miRNAs.^41-43 A growing body of evidence demonstrates the involvement of EVs in maternal-fetal adaptations to pregnancy and immune cell interactions leading to maternal tolerance.

1.3 Extracellular vesicles in maternal adaptation to pregnancy

In species with hemochorial placentation—such as humans, primates, and mice—trophoblast cells come into direct contact with maternal blood and circulating immune cells. Furthermore, in their way through the endometrium, invading trophoblast cells interact with different tissue-resident cells.^{44, 45} The endometrium has a rich and dynamic population of immune cells, including Natural Killer (NK) cells, macrophages, lymphocytes, DCs, and others. Although trophoblast cells are recognized as foreign elements, they trigger a tolerogenic response and establish a cooperative alliance with maternal immune cells during pregnancy. The development of maternal tolerance represents one of the most complex and fascinating immunological phenomena. During pregnancy, the maternal immune system is finely tuned to allow the development of a semi-allogenic organism inside the uterus and, at the same time, to maintain its defense mechanisms against pathogens.^46-48 A Th1 proinflammatory environment promoted during decidualization and embryo implantation is followed by a tolerogenic Th2 profile over the course of pregnancy. Finally, an additional Th1 response develops to promote labor.⁴⁹ The breakdown of the immunological harmony at the maternal-fetal interface has been associated with abortions and pregnancy complications.^{50, 51} Therefore, deciphering the dialogue between mother and fetus contributes to the comprehension of pregnancy establishment and maintenance as well as of fertility disorders and gestational diseases.

Ex-vivo perfusion of human placenta demonstrates that syncytiotrophoblast cells covering the maternal villi launch a substantial amount of EVs into the intervillous space, from where they are dispersed to other organs and cells through the maternal bloodstream.⁵² Proportionally to its growth and the expansion of villous surface area, the human placenta releases rising concentrations of EVs during pregnancy.⁵³ In the third trimester, the concentration of EVs in maternal blood reaches values far higher than in non-pregnant women.⁵⁴ The molecular information delivered by placental EVs contributes to pregnancy-driven adaptations in the maternal organism. EVs released by endometrial resident immune cells and invading trophoblast cells have received less attention, but may also be important for maternal tolerance and placentation.

At the forefront of interactions with immune cell populations, trophoblast cells employ an arsenal of strategies to instruct the maternal immune system toward fetal tolerance. Increasing evidence demonstrates that EV-mediated intercellular communication plays a major role in this process. Trophoblast-derived EVs are incorporated by maternal immune cells and regulate their proliferation, differentiation, apoptosis, cytokine secretion, and immune functions.^55-58 EVs produced by immune cells, in turn, are able to modulate placental responses, thus, establishing a cross-talk to coordinate placental and immune cell behavior.^{59, 60}

Placental alkaline phosphatase (PLAP),⁶¹ syncitin-1/2,⁶² and HLA-G⁶³ are markers used to identify and isolate trophoblast-derived EVs. Fractions of sEVs and lEVs enriched from human placenta perfusion via ultracentrifugation have mean diameters of 117 ± 47 nm and 236 ± 54 nm, respectively.⁵² The exosomal marker CD63 is predominantly detected in sEVs and Annexin A1, a proposed marker for microvesicles (within the lEV population), is present in both fractions.⁵² Annexin V and calcireticulin are also loaded in all populations of placenta-derived EVs, whereas CD31 and, especially, CD47 are barely detected in nanovesicles (term originally used in the cited paper, corresponding to exosomes).⁶⁴ Numerous proteins and RNA species have been reported in placental EVs. It is estimated that around 1600 average-sized proteins (65 kDa) or approximately 29 000 22nt RNAs are loaded into a 100-nm exosome.⁶⁵ Investigation of EV cargo by mass spectrometry-based proteomics shows that defense and inflammatory responses are among the overrepresented functions of placental EV proteins.^{64, 66} RNA cargo is composed by miRNAs including those from the eutherian-associated chromosome 14 miRNA cluster (C14MC) and primate-associated C19MC as well as placenta-specific and associated miRNAs.⁶⁷ EV content changes in pregnancy pathologies, indicating potential pathogenic mediators as well as diagnostic targets.^{24, 25}

1.4 Maternal immune cell populations

Placental EVs can be recognized and internalized by several immune cell populations in the peripheral blood or the local environment. In early pregnancy, leukocytes in the decidua consist mainly of uterine natural killer (uNK) cells (65%–70%), macrophages (15%–20%), and T cells (10%–20%).^{68, 69} At term, uNK cells are reduced and T cells are the predominant immune subtype. Leukocyte count increases above the normal range because of the physiological stress in pregnancy, but the overall change is the result of modifications in specific subpopulations. Lymphocyte count decreases in the first and second trimester and increases again at the end of pregnancy. Eosinopenia, basopenia, and monocytosis are observed with advanced gestational age.⁷⁰

Current studies are focused on describing which immune cell subpopulations are more likely to internalize placental EVs and which cellular functions are modified therein. The uptake of placental EVs by maternal immune cells and other targets presents some degree of selectivity. The injection of placental EVs into the circulation of recipient mice leads to a preferential accumulation in liver, lungs, and kidneys.⁷¹ Some authors described the incorporation of placental microvesicles by T lymphocytes, but not by B and NK cells,⁶³ while others reported the uptake of placental EVs by B lymphocytes and monocytes.⁷²

Following, we review the immunological properties of EVs and their role in the communication between placenta and T cells, DCs, NK cells, and monocytes/macrophages.

2.1 Functions during pregnancy

Members of the adaptive immune system, CD4⁺ T cells, CD8⁺ T cells, B cells, and their associated subsets are involved in adaptation to the reproductive process. These cells regulate maternal tolerance and contribute to placental development. T lymphocytes are influenced by numerous factors from trophoblast cells, such as different HLA molecules, hormones, indoleamine 2,3-dioxygenase (IDO), Fas ligand (FasL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), cytokines, chemokines, galectines, proteinases, and others. They form numerous subpopulations with different specific functions and capacities including regulatory T cells (Tregs) T helper 1 (Th1), Th2, Th17, γδ T cells, cytotoxic T cells, their subtypes, and many others.^73-76 Tregs defined as CD4⁺CD25brightFoxp3⁺ increase in the decidua during a normal gestation. Treg cells interact with DCs and uNK cells playing a major role in immune and trophoblast cell regulation in physiological situations. Excessively abundant Treg cells are found in women with placenta accreta, while insufficiency of these cells are related to complications such as preterm birth and preeclampsia.⁷⁷ For more detailed information see.⁷⁶

2.2 Extracellular vesicle-mediated communication

Several studies on T cell biology have investigated the uptake of primary trophoblast- and trophoblastic cell line-derived EVs by Jurkat T cells, demonstrating transfer of EV content and subsequent cellular responses (Figure 1). In our set of studies, trophoblastic EVs with modified miRNA cargo were used to evaluate their effect on T cell proliferation. EVs containing elevated levels of miR-519d-3p increase Jurkat proliferation,⁷⁸ whereas EVs containing elevated miR-141 decrease it.⁷⁹ Transfer of placenta-specific miR-517a-3p from trophoblastic cells into Jurkat T cells reduces the expression of PRKG1, the gene coding for a serine/threonine kinase that acts as a key mediator of the nitric oxide (NO)/cGMP signaling system, which in turn, negatively regulates T cell activation and proliferation.⁸⁰ Furthermore, a soluble form of FasL was found encapsulated in EVs secreted by primary trophoblast cells. FasL is a member of the tumor necrosis factor (TNF) receptor family and is involved in the regulation of apoptosis and cell death. Release of vesicular FasL by disruption of trophoblast-derived EVs resulted in induced Jurkat apoptosis through activation of the Fas pathway.⁸¹ Likewise, induction of TNF-α secretion after stimulation with PMA/ionomycin was also impaired in Jurkat T cells treated with increasing quantities of exosomes isolated from primary cytotrophoblast cells suggesting a negative effect of trophoblast EVs on the activation of T cells.⁸²

The immunomodulatory effects of trophoblast-derived EVs on T cells were further investigated in peripheral blood mononuclear cells (PBMCs). Remarkably, T cells seem to be not the major placental EV cell target as demonstrated in vitro⁷⁸ and in vivo.⁸⁰ Their uptake and transfer of placental miRNAs are lower compared to other immune cell types such as NK cells. However, evidence is cumulating in favor of the pivotal effect of placental EVs on maternal tolerance. Activation of PBMCs by treatment with anti-CD3/anti-CD28 assessed as secretion of Th1 cytokines (TNF-α, IFN-γ, and IL-2) was hampered by the presence of trophoblast-derived EVs. Depletion of syncytin-2 on EVs decreases this inhibitory potential.⁸² Other immunomodulatory molecules expressed on the surface of placental- and trophoblast-derived EVs are FasL, TRAIL, and HLA-G5.^{63, 83, 84} Similar to FasL, TRAIL is an apoptosis-inducing molecule whose expression is strictly confined to the endosomal compartment of the syncytiotrophoblast and which have been found in its secreted EVs. Treatment of Jurkat cells and PMA/ionomycin-activated PBMC with EVs harboring FasL and TRAIL trigger apoptosis.⁸³ Further, placental EVs expressing FasL suppress activated lymphocytes by modulation of CD3-zeta. This effect is T cell subset specific, targeting CD8⁺ more than CD4⁺ and far more than CD4⁺CD25⁺cells which may be part of a mechanism that leads to preferential expansion of lymphocytes with suppressive phenotypes.⁸⁴ In fact, increase of Treg differentiation from CD4⁺ cells may also be triggered by trophoblastic-derived EVs via a mechanism that involves the HSPE1 protein in their membrane and interior.⁸⁵ Recently, a similar effect was reported using EVs obtained from human amnion epithelial cells (hAECs) which have comparable properties to mesenchymal stem/stromal cells. hAEC-derived exosome uptake coincides with the suppression of CD3/CD28 activated T cell proliferation as well as with the maturation of recipient CD4⁺ T cells into FoxP3-expressing Tregs. Intranasal instillation of hAEC exosomes significantly decreased the percentage of splenic CD4⁺ CD3⁺ CD8⁺ T cells in the lungs, reduced lung inflammation and fibrosis in an in vivo murine model suggesting it as a potential therapy for lung fibrosis.⁸⁶ The therapeutic features of placental EVs remain to be determined but cumulating evidence strongly suggests that their transfer to maternal T cells is part of the mechanisms required for the immune privilege in the gravid uterus as well as for the tolerance needed for physiological pregnancy.

Accumulating evidence demonstrates that Treg-derived EVs have immunomodulatory properties and the ability to generate tolerance. Their functions include the modulation of T cell proliferation, differentiation, and cytokine production,^{87, 88} as well as the induction of a tolerogenic phenotype in dendritic cells.⁸⁹ A potential influence of EVs produced by T cells on trophoblast functions has not been investigated yet and warrants future research.

3.1 Functions during pregnancy

DCs, a class of antigen-presenting cells (APC), can induce both antigen-specific activation and suppression in the course of immune responses. During pregnancy, DCs possess the ability to process and present fetal antigens to decidual T cells. DCs constitute nearly 1% of the total decidual immune cells, a percentage that seems to be widely constant throughout gestation, but markedly higher than in peripheral blood.⁹⁰ Two main subpopulations of DCs have been described at the maternal-fetal interface, namely myeloid type 1 and type 2. DCs acquire a tolerogenic phenotype in the absence of stimulatory signals and when exposed to molecules with anti-inflammatory properties, such as IL-10, human chorionic gonadotropin, estradiol, and progesterone.^{91, 92} Myeloid type 2 DCs recognize fetal antigens from trophoblast and exert an immune tolerance effect through the production of anti-inflammatory cytokines that prevent T cell activation.⁹³ Moreover, human decidual DCs appear to have predominantly an immature phenotype characterized by low expression of CD80, CD86, CD40, and CD205.⁹⁴

3.2 Extracellular vesicle-mediated communication

Neutrophils, macrophages, mast cells, eosinophils, basophils, and NK cells, communicate with DCs as well as T and B lymphocytes, linking the innate and adaptive immune responses. In a normal inflammatory context, major effects induced by EVs derived from immune cells are mediated by targeting DCs and influencing their maturation, migration, and antigen delivery.⁹⁵ In addition, EVs released by mature DCs contain MHC class I and II and co-stimulatory molecules, such as CD86, indicating their immune-activating role.^{96, 97} EVs derived from immature DCs induce either no proliferation³⁴ or a low level of proliferation³⁵ in naïve CD4+ T cells.^{37, 98} While the fraction of lEVs from immature DCs promotes the secretion of Th2 cytokines, that of sEVs stimulates Th1-associated IFN-γ secretion. Upon DC activation, both EV populations have a similar effect leading to IFN-γ production.²⁸

To our current knowledge, the potential contribution of trophoblast-derived EVs in the modulation of DC maturation, activity, and regulation of immune responses at the maternal-fetal interface has not been addressed. Proteomic analyses of different EV fractions from first-trimester placenta showed the presence of proteins involved in vesicle internalization, complement pathway, minor histocompatibility antigens, and several others.^{40, 64, 99} HLA-G-bearing EVs released by trophoblast cells represent an additional instrument to modulate immune responses at the materno-fetal interphase. Both, first trimester and term placentas secrete HLA-G5 isoforms via EVs.¹⁰⁰ Cytotrophoblast cells, but not differentiated syncytiotrophoblast, release HLA-G5-positive exosomes.¹⁰¹ HLA-G is thought to orchestrate the cross-talk between embryo trophoblasts, decidual leukocytes, and stromal cells allowing the trophoblast invasion, decidual cell differentiation, vascular remodeling, and the reprograming of local maternal immune responses.¹⁰² The DC10 subset of tolerogenic DCs that secretes high amounts of IL-10 and expresses high levels of HLA-G and its ligand ILT4 has been found at the maternal-fetal interface.¹⁰³ This enforces the idea of a direct interaction of HLA-G⁺ trophoblast-derived EVs with ILT4⁺ DCs and vice versa.

DCs express IDO and can inhibit T cells by depleting essential tryptophan, by producing toxic metabolites, or by generating Treg cells.¹⁰⁴ Exosomes from DCs containing IDO have immunosuppressive activity when administrated in murine models of arthritis.¹⁰⁵ Moreover, exosomes containing IDO can induce immunosuppressive activity in recipient DCs. Trophoblast cells express IDO during the first and third trimester of pregnancy¹⁰⁶ to mediate fetal tolerance.¹⁰⁷ The production of IDO⁺-transporting EVs by trophoblast cells remains to be examined as a potential mechanism to control DC maturation and immunosuppressive activity in pregnancy. Similarly, the contribution of DC-derived EVs to maternal tolerance during normal and complicated pregnancies deserves further investigation.

4.1 Functions during pregnancy

As part of the innate immune system, NK cells are specialized in the combat of virus-infected and cancer cells and display cytotoxic competence, immunomodulatory functions, and tissue remodeling properties. Two major populations of NK cells have been described: cytotoxic (CD16⁺ CD56^dim) and immunomodulatory (CD16⁻ CD56^bright) subtypes, both with particular specializations in the compartments they settle in.^{108, 109} Their effector functions are regulated by elaborated mechanisms related to the repertoire of activating and inhibitory receptors on their membrane including NKG2D, killer-cell immunoglobulin-like receptors (KIRs), and natural cytotoxicity receptors (NCR).¹¹⁰

Uterine NK (uNK) cells are the most abundant immune population in the endometrium¹¹¹ playing pivotal roles in the reproductive process. uNK cells promote endometrial regeneration through the clearance of senescent decidual cells¹¹² and support placenta development during pregnancy. In addition, to regulate maternal tolerance, NK cells influence trophoblast invasion into the endometrium and regulate the remodeling of spiral arteries to provide an adequate blood supply to the growing fetus.^{113, 114}

4.2 Extracellular vesicle-mediated communication

Trophoblast-derived EVs contain HLA-G molecules. When interacting with inhibitory KIR2DR4 receptor and CD158d, HLA-G prevents uNK cell activation and cytotoxicity, stimulating a senescence-associated secretory phenotype (SASP) response associated with maternal tolerance and placenta development.^{115, 116} Syncitiotrophoblast-derived EVs contain MHC class I chain-related proteins A (MIC A) and MIC B are able downregulate the activating NKG2D receptor in NK, γδ T, and CD8+ αβ T cells, and to reduce NKG2D-dependent cytotoxicity. This mechanism has been associated with the acquisition of a tolerogenic state in NK cells during pregnancy.¹¹⁷ We observed that NK-92 cells have a higher uptake of trophoblastic EVs than Jurkat T cells. While trophoblastic EVs with elevated miR-519d-3p levels, due to the transfection of this miRNA to the donor cells, decrease NK-92 proliferation, they increase that of Jurkat T.⁷⁸ These data indicate the role of placental miRNA-containing EVs in the modulation of maternal immune responses. Despite initial evidence, the role of trophoblast-derived EVs on NK cell responses remains to a great extent unknown.

Thus far, most studies have investigated EVs from peripheral blood NK (pbNK) cells. A comprehensive characterization of EV features from uterine and pbNK cells during pregnancy as well as a comparative evaluation of EVs produced by cytotoxic and immunomodulatory NK cells are still lacking. Although uNK and pbNK cells encompass phenotypically distinct populations,^{118, 119} results from one subtype may offer insightful hints about the functions from the other.

NK cells produce and release EVs in a constitutive manner which can be further enhanced by stimulation with IL-2 and IL-15.^{120, 121} Populations of small EVs, ranging from 40 to 200 nm, predominate in the secretion from pbNK cells in comparison to microvesicles with a mean diameter of 315 nm.^{120, 122} The adhesion molecule CD56 is present on NK cell-derived EVs and can be used for their isolation.¹²³ Both, exosomes and microvesicles, derived from NK cells present the conventional EV surface markers CD9, CD63, CD81,¹²⁰ which distinguish these EV populations in other cell models.

A comprehensive proteomic analysis revealed the presence of 3830 proteins in pbNK-derived EVs, where 2645 were shared between exosomes and microvesicles, 279 were expressed exclusively by exosomes, and 906 by microvesicles. The enrichment of hundreds of proteins was also observed in EV content when compared with cellular extracts from EV donor NK cells,¹²⁰ indicating the existence of mechanisms of protein cargo selection. NK-derived EV proteins were associated with several biological processes, including signal transduction and immunological processes.¹²⁰ MIC A and MIC B, lymphocyte function-associated antigen (LFA-1), DNAX accessory molecule-1 (DNAM1) and programmed cell death protein (PD-1), FasL, CD16, NKG2D, NKp30, and NKp40 as well as immune checkpoint proteins CD47 and CD276 are among the immunomodulatory molecules present in NK cell-derived EVs.^{122, 124} They also contain the cytolytic enzymes perforin, granzyme A and B, and granulysin.¹²³

EVs secreted by pbNK cells modulate immune responses and induce caspase-dependent and -independent apoptosis of cancer and activated PBMCs. Non-malignant cells and resting immune cells are resistant to NK-derived EV-mediated apoptosis,^{121, 125} indicating that NK cells have adjusted their EV content to target cells, for minimizing widespread and unspecific effects. Similarly to EVs derived from other immune cell types, the effects of NK cell-derived EVs on trophoblast cell responses remain to be determined under physiological and pathological conditions.

5.1 Functions during pregnancy

Macrophages are mononuclear phagocytic cells deriving from circulating monocytes. They are important for detection, ingestion and processing of extracellular material, cell debris, foreign bodies, and pathogens.¹²⁶ Upon entering into a tissue, specific factors control the differentiation and functions of the final mature tissue-macrophage, regulating their proliferation, motility, and phenotype.^{127, 128} Macrophages are present in the endometrium and placental bed during the entire pregnancy, being the second most abundant leukocyte population with approximately 20% of all decidual immune cells.¹²⁹ Among the main functions of decidual macrophages are the clearance of apoptotic cells from the growing placenta,¹³⁰ and the production of cytokines, angiogenic factors, and matrix metalloproteinases for supporting remodeling of the spiral arteries and maternal tolerance.^{126, 128}

Decidual macrophages are mostly of the anti-inflammatory M2 subtype and maintain a phenotypic balance between M1 and M2 phenotype during pregnancy.¹³⁰ Placental macrophages of fetal origin, the Hofbauer cells,¹³¹ also display a M2 phenotype, which supports the maternal tolerance toward the fetus.¹³² Macrophages use phagocytosis to process cell debris by an actin-mediated mechanism which has also been described for exosome internalization.¹³³

5.2 Extracellular vesicle-mediated communication

Trophoblast cells produce diverse substances and extracellular vesicles, which can alter the polarization of decidual macrophages, favoring the M2 phenotype which supports the gestational process.¹³⁴ Trophoblast-derived exosomes interact with monocytes and macrophages, promoting their migration and the expression of CD54, IL-1beta, IL-6, IL-8.¹³⁵ Fibronectin can be transferred to macrophages via trophoblast-derived exosomes, modulating the production of interleukin-1β.¹³⁶ EVs from the trophoblastic cell line BeWo expressing syncytin-1 are taken up by PBMCs and modulate their activation and response to lipopolysaccharide (LPS).¹³⁷

Macrophages do not only receive, but also release EVs. Macrophage-derived exosomes stimulate placental cytokine release and activation of naive recipient immune cells. The placenta responds to EV-mediated messages from activated macrophages which may contribute to protection of the fetus in maternal infection or inflammation.⁵⁹ EVs isolated from Swan71 trophoblastic cells stimulate monocyte migration and the secretion of the proinflammatory cytokines IL-1, IL-6 IL-18, and IL-12 as well as TNF-α, CSF2, and SERPINE1.¹³⁸ In the opposite direction, clathrin-dependent endocytosis of macrophage-derived exosomes by trophoblast cells promotes the production of IL-6, IL-8, and IL-10.⁵⁹ Monocyte-derived EVs do not induce such effects,⁶⁰ demonstrating that the differentiation status of these cells influences their EV functions.