Reproductive Immunology: Basic Concepts

Reproductive Immunology: Basic Concepts gives a holistic insight into the understanding of the complex interactions between the maternal immune system and the fetal/placental unit necessary for the success of pregnancy. This interaction is critical for the support of the human fetal semiallograft and the protection against infections. The book covers various topics such as B cells, macrophages, T cells, discussion on fetal signals and their impact on maternal reproductive cells such as endometrial cells, mast cells, and the role of fetal Hofbauer cells, the immune regulatory role of glucorticoids, and many other novel topics within the field of reproductive immunology.

Edited and written by experts in the field, this book introduces the up-to-date knowledge of the role of the immune system during pregnancy and provides the necessary background to understand pregnancy complications associated with alterations in the functioning of the immune system. The book provides a complete discussion on the immunological aspects of pregnancy and serves as a great tool for research scientists, students, reproductive immunologists and OBGYNs.

• Shows the detailed evaluation of the knowledge related to each immune cell type in the pregnant and not pregnant uterus
• Evaluates each immune cell type and its function during specific reproductive events
• Provides the biological background for understanding the clinical aspects that will be discussed in subsequent volumes in the series

• Language: English
• Publisher: Academic Press
• Release date: Feb 12, 2021
• ISBN: 9780128189306

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Europe.

Chapter 1: The role of the immune system during pregnancy: General concepts

Anthony J. Maxwella; Yuan Youa; Paulomi Bole Aldob; Yonghong Zhangc; Jiahui Dinga; Gil Mora    a C.S. Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, United States

b Department of Obstetrics, Gynecology and Reproductive Sciences, Division of Reproductive Sciences, Yale School of Medicine, New Haven, CT, United States

c Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University, School of Medicine, Nanjing, China

Abstract

The interaction between the fetus and the maternal immune system is a dynamic process that shows a high degree of plasticity, which is required for proper fetal development. The maternal immune system must continuously adapt and change as it interacts with the fetus. This is especially true during environmental challenges, such as infections. This book goes into detail about the roles of specific immune cells and their contribution to a successful pregnancy. We will first build a base on normal physiology during these processes, so that the pathophysiology of different pregnancy complications can better be understood. Future books in this series will discuss how dysregulation of specific immunological process can impact the success of a pregnancy. In this first chapter, we will discuss how the microenvironment created by maternal immune cells is essential for the different developmental stages of gestation. Furthermore, we discuss how crosstalk between trophoblasts, which are fetal cells, and maternal immune cells are required to maintain a microenvironment that facilitates adequate fetal growth and provides protection against infections.

Keywords

Inflammation; Macrophages; Trophoblast; Implantation; Pregnancy; Maternal immune system; Tolerance

1: Introduction

In the last decade, our understanding of how the immune system is involved in pregnancy, especially during early pregnancy, has undergone a major transformation. From being focused on the process of tolerance to paternal antigens and the breach of tolerance being considered as the main cause of pregnancy loses and other clinical complications, we now know that the immune system plays a critical role supporting many of the physiological changes taking place at the uterus necessary for implantation, placentation, and fetal grow.

Successful implantation and placentation are critical events necessary for establishment of pregnancy. For this process to be successfully accomplished, multiple cellular events take place to facilitate the interaction between the blastocyst and the endometrium. The embryo is capable of sensing and responding to local signals, which will shift the endometrial microenvironment toward one that is better suited for implantation [1]. Similarly, the female reproductive tract is not passive and plays an active role in each of the biological steps that lead to implantation. This reactivity demonstrates a high degree of plasticity that is required to respond to the signals coming from the fetus as well as from the maternal microenvironment [2]. This degree of plasticity is a powerful force that is capable of selectively supporting viable embryos or eliminating abnormal embryos. Indeed, there is a strong data from both experimental and clinical research indicating that uterine receptivity, rather than ovulation and conception, is a main rate-limiting process for the reproductive success.

Throughout this book we review the specific characteristics of the different types of immune cells present at the implantation site and their role in normal pregnancy. This information constitutes the base for understanding pathological conditions in pregnancy that are associated with dysregulation of the immune system. In this first chapter, we will discuss the role of different immune cells and the process of inflammation during the most critical period of reproduction: implantation, and early placentation. Additionally, we will examine why proper immune regulation is critical for successful pregnancy.

2: Immunological stages of pregnancy

The interaction between the fetus and the maternal immune system is a dynamic and evolving process. The interactions between these two systems shows a high degree of plasticity that adapts to the continuous changes that the fetus is subject to during gestation. Therefore, defining the characteristics of the maternal immune system throughout gestation as a monolithic condition is inadequate. The maternal immune system is continuously changing as it interacts with fetal signals and environmental challenges, such as infections. We have defined at least three distinct immunological states that correspond to the stages of fetal development: first, a pro-inflammatory stage associated with implantation and placentation; second, an antiinflammatory stage associated with fetal growth; and third, a second pro-inflammatory stage responsible for the initiation of parturition [3, 4] (Fig. 1). As we will discuss later, shortening or extending a specific period might have a detrimental effect on the normal development of the fetus as well as on the outcome of the pregnancy. Between each stage there is a transitional period, which may play a critical role in the adaptation process. However, the characteristics of these transitional periods are still poorly understood.

Fig. 1 Inflammatory stages of pregnancy: Pregnancy is characterized by three stages of inflammation. Early pregnancy is associated with an inflammatory stage. The second trimester involves an antiinflammatory environment, which then changes to an inflammatory environment at the end of the pregnancy in order to prepare for parturition.

3: Implantation: A wound that needs repair

Implantation is the process when an embryo attaches to the luminal epithelium of the endometrium, followed by migration and invasion into the deeper layer of the endometrium to become implanted [5–9]. In order for the process above to successfully take place, the endometrium must be receptive. Uterine receptivity refers to the status of the uterus when the endometrium is available to accept the embryo for implantation. In humans, the uterus becomes receptive during the mid-secretory phase (days 19–23) of the menstrual cycle, which known as the window of implantation (WOI). This period is characterized by morphological and transcriptional changes in the endometrium, such as the presence of apical protrusions called pinopodes on the cells of the luminal epithelium [10], stromal cell proliferation, and differentiation. Apart from the morphological changes, the genomic signature of a receptive endometrium exhibits characteristics that are similar to inflammatory responses. Specific immune cell, such as macrophages (Mac), dendritic cells (DCs), and natural killer (NK) cells, will migrate and accumulate in the endometrium. Additionally, the receptive endometrium will express different cytokines/chemokines, growth factors, and adhesion molecules [9, 11, 12]. All the above changes will facilitate proper embryo-endometrium interaction and, thus, enabling implantation [13, 14].

Although the whole process of embryo implantation is still considered as a single biological process, growing evidences suggest that embryo attachment is one unique process which differs from trophoblast migration invasion and placentation. Each of these two stages are different and each one involves complex sequence of cellular and molecular changes. This complexity is exemplified by two medical conditions associated with abnormal implantation: recurrent implantation failure (RIF) and recurrent miscarriage (RM). RIF refers to failure to achieve a clinical pregnancy after transfer of at least four good-quality embryos in a minimum of three cycles in a woman under the age of 40 years [15]. RM is defined as three or more consecutive clinical miscarriages [16]. The two complications have been defined as similar conditions sharing common pathological changes. Indeed, both uterine natural killer (uNK) cell count and interleukin (IL) 15 expressions have been reported to be increased in the two conditions [17]. However, new more sensitive approaches and more efficient sample collection showed that certain molecules were dysregulated in one of the conditions but not the other [11]. Although RIF is mainly associated to a failure of the epithelium to support the attachment of the fetus, RM represents an abnormal adaptation of the endometrium to the invading trophoblast and establishment of a functional placenta. Therefore, it is important to remember that implantation involves several steps and each one has its unique characteristics and biological processes.

In general, it is accepted that the process of implantation involves four main steps: rolling, apposition, attachment, and trophoblast migration [18]. Each of these steps encompasses unique immunological/inflammatory processes, which requires different cell types and signals [5]. Furthermore, as indicated above, it is a continuously evolving process that reacts to the microenvironment, which highlights the complexity of the implantation process. In the following section, we will discuss the role of inflammation and its cellular players in each individual step.

Since implantation happens through disruption of maternal tissue, it is understandable that implantation of the blastocyst activates, at least partially, the inflammatory pathway in a way that resembles a similar response observed during tissue injury. Because receptivity is achieved just before menstruation and its molecular signature is characteristic of inflamed or injured tissue, it is plausible that the inflammatory process taking place at this stage is essential for the modification of the epithelium and the stroma, which is required for proper interaction with the embryo.

Given that inflammatory pathways are activated at the time of implantation, and inflammation is thought to be associated with a rejection process (brake of tolerance), the questions that arise are: (1) how does tolerance to an invasive embryo evolve within an inflammatory environment without an immediate rejection of the fetus?; (2) What are the target cells of the inflammatory process?; (3) What are the sources of the inflammatory process? Understanding the mechanisms of how inflammation is timely and spatially triggered and controlled in the uterus is fundamental for developing effective therapeutics to improve fertility and decrease poor obstetrical outcomes.

The implantation site is characterized by infiltration of immune cells, which are important for proper regulation of trophoblast invasion and spiral artery remodeling and thus for placentation [19]. The immune cells that are relevant for implantation are present already before pregnancy. Immune cells that are present in the uterus usually display a unique, uterine phenotype that differs from the phenotype of their counterparts located either in the periphery or in other tissues. The unique phenotypes and possible unique functional properties are likely provoked by the environment of the female reproductive tract, which modifies these immune cells to be more suitable for a particular tissue.

Immune cells represent an important cellular component present in the uterus, before, during, and after pregnancy [3]. These immune cells have unique characteristics, which are discussed in detail throughout this book. Innate immune cells and their function are covered in Chapter 3 (monocytes/macrophages in the pregnant and nonpregnant uterus); Chapter 4 (NK cells), and Chapter 6 discuss about neutrophils. Chapters 5, 7, 12, and 16 examine the role of adaptive immune cells.

In the remainder of this chapter, we examine our current understanding of how immune cells, stromal cells, and trophoblast cells actively interact to support implantation. We also discuss how the uterine immune cell infiltrates play a central role in the process of tissue renewal and differentiation, in addition to, participating in the development of a receptive endometrium.

4: The inflammatory characteristics of embryo implantation

Distinct immunological and molecular changes are observed in the receptive endometrium before implantation occurs [20]. During apposition, chemokines and cytokines, produced by the endometrial cells, guide the blastocyst to the site of implantation and the embryonic L-selectin binds to its ligands on the luminal epithelium. This enables the initial contact of the blastocyst with the uterus. In mice, leukemia inhibitory factor (LIF) is transiently increased in mouse uterus before implantation [21]. LIF is an IL-6 class cytokine with pro-inflammatory potential [22]. These data indicated that endometrium before implantation is already in an inflammatory state, potentially, under the control of ovarian steroids. In addition to LIF, the immune status at the implantation site is characterized by expression of pro-inflammatory cytokines, such as IL-6, IL-1β, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α [23–25]. These cytokines could be produced by endometrial epithelial cells, endometrial cells, as well as the immune cells that are recruited to the site of implantation.

If this inflammatory process is not present, the endometrium is not receptive and not prepared for implantation. However, by reactivating the inflammatory process we could promote the receptivity of the endometrium [26]. This concept is supported by the fact that endometrial biopsies, a process that causes local injury and inflammation, increase uterine receptivity [27]. Indeed, the mechanism by which the biopsy treatment increases endometrial receptivity demonstrates that the local injury induces an inflammatory response that is characterized by elevated levels of pro-inflammatory cytokines/chemokines, such as macrophage inflammatory protein (MIP)1β, TNFα, growth regulated oncogene α (GROα), osteopontin (OPN), and IL-15 [28]. In addition, there is an increase in the number of innate immune cells, particularly macrophages, dendritic cells (DCs), and a unique population of natural killer (NK) cells with a CD56hiCD57lo phenotype. In many regards, the cytokine and immune cell response to embryo implantation reflects a controlled inflammatory response that is akin to that seen in tissue injury and wound healing [19].

These innate immune cells are abundant in the decidua during the luteal phase of menstrual cycle when implantation commences (reviewed in Chapters 3 and 4). Throughout the menstrual cycle, mature CD83+ DCs and CD68+ macrophages increase, peaking in the late secretory phase [29]. Immediately after coitus, the presence of seminal fluid further enhance the recruitment of macrophages, DCs, and T cells to the endometrium [30].

Macrophages and NK cells are the main cytokine producers in the human endometrium [31]. Through secretion of LIF and IL1β, macrophages enhance endometrial receptivity by increasing cell surface fucosylated structures, which allows for trophectoderm attachment [1]. Specific depletion of CD11b macrophages resulted in implantation failure, owing to the fact that macrophage also play a role in the normal physiology of the ovaries and its depletion altered the luteal microvascular network that is necessary for the integrity of the corpus luteum and progesterone production [32].

The role of endometrial NK cells in implantation remains unclear, because successful implantation is still possible in NK cell deficient mice [33]. Current evidence points toward the involvement of decidual NK cells in the placenta-formation processes after initial implantation [34] (Chapter 4). However, an indirect role for human endometrial NK cells in implantation cannot be overlooked because of their high abundance and ability to secrete large amounts of cytokines, such as IFN-γ, TNF-α, GM-CSF, and IL-10 [35].

T cells represent the third largest fraction of immune cells found in the human endometrium [36, 37] (reviewed in Chapters 2, 5, 7, and 19). Since T cells primarily reside in the deeper layers of the endometrium in the absence of pregnancy, their relation to fertility disorders more likely suggests a role in early placenta formation after implantation, rather than a contribution to the preimplantation period [34]. However, CD4+   CD25+ T regulatory (Treg) cells, a tiny proportion of T lymphocyte population which synthesize copious amounts of transforming growth factor (TGF)-β and IL-10, are essential for embryo implantation by limiting inflammatory activation in macrophages and NK cells [38] and promoting the transition to an antiinflammatory stage of pregnancy (Figs. 1 and 2).

Fig. 2 Switching inflammation: The success of the pregnancy depends on the timely and efficient change from an inflammatory into antiinflammatory stage. Implantation depends on inflammation. Placentation is an antiinflammatory condition, while parturition depends on an adequate inflammatory process. Preterm birth can result from early inflammation. Miscarriage or early pregnancy lose is associated with continued inflammation, while implantation failure can be due to either a lack of inflammation or excessive inflammation.

5: Dendritic cells and their effect on the epithelium and stroma

Among antigen presenting cells (APC), the CD11chigh dendritic cells (DCs) represent around 5%–10% out of all hematopoietic uterine cells and are the most potent inducers of primary immune responses [39]. DCs are a heterogeneous population of bone marrow-derived cells that are uniquely designed to initiate and coordinate innate and adaptive immune response. DCs are not only essential for the induction of primary immune responses but are also important for the induction of immunological tolerance [40]. DCs are present in the nonpregnant endometrium both in humans and rodents [39–41]. They also accumulate in the pregnant uterus prior to implantation and remain in the decidua throughout most of the pregnancy [31, 40]. Their functions and stages of differentiation are regulated by the cytokine and chemokine profiles in the local microenvironment [42–44]. Moreover, recent evidence points to a pivotal role of DC in shaping the cytokine profile toward the establishment of a tolerogenic microenvironment at the maternal-fetal interface [45]. Two types of tolerogenic DCs are present in the murine uterus: those positive for CD103 (CD11c   + CD103   +) and those double positive for CD11c and CD11b (CD11b   + CD11c   +) [46]. Tolerogenic DCs are potent secretors of antiinflammatory mediators, such as IL-10, and weak producers of pro-inflammatory cytokines, including IL-12 and TNFα [5, 47].

Evaluation of their function during the period of implantation revealed a critical role for DCs in the preparation of the epithelium and stroma of the uterus for embryo implantation [48]. Depletion of uterine DCs (uDC) was associated with impaired decidual proliferation and differentiation, as well as perturbed angiogenesis that was characterized by reduced vascular expansion and maturation. It was suggested that uDC governs uterine receptivity through a mechanism that is independent of their anticipated role as APC by regulating tissue remodeling and angiogenesis through the provision of critical factors, such as sFlt1 and TGFβ1, that synergistically promote coordinated blood vessel maturation [48].

As the blastocyst passages from the fallopian tube to the uterine cavity, the surface epithelium of the uterus represents the first contact responsible for adequate attachment of the trophectoderm to the endometrium. The underlying stroma will then have a critical role on the subsequent trophoblast invasion and then establishment of the placenta or placentation.

Intriguingly, when a mammalian blastocyst enters the uterine cavity, the surface epithelium of the uterus is covered by molecules, such as Mucin 1 (MUC1) carbohydrates, that prevent the attachment of blastocyst to the uterus. Actually, in the human endometrium MUC1 is upregulated during the implantation period [49, 50]. This suggests that the human endometrial surface epithelium prevents blastocyst adhesion, except for the precise spot were the embryo is meant to attach. We observed that cytokines/chemokines produced by DCs/Mo stimulated endometrial epithelial cells to express the adhesive molecule Osteopontin (OPN) and its receptors ITGB3 and CD44. However, MUC16, which interferes with adhesion, was downregulated. Other implantation-associated genes, such as CHST2, MIP1B, and GROα, were also upregulated by DCs [26–28]. As a result of this specific inflammatory process, the blastocyst is able to attach to a specific area of the epithelium that has all the elements necessary to support the attachment of the rolling embryo.

6: Macrophages and their role during implantation

Tissue-resident macrophages are sentinel innate immune cells that display a spectrum of functions and produce a panel of cytokines that orchestrate innate and adaptive immune responses [51]. Macrophage activation and function are influenced by signals received from the local environment [52, 53]. The functional plasticity of macrophages has given rise to the notion of macrophage polarization, which ranges from classically activated pro-inflammatory M1 macrophages to alternatively activated pro-resolving/antiinflammatory M2 macrophage [53]. M1 macrophages have microbicidal activity and secrete a dominant profile of pro-inflammatory cytokines, while M2 macrophages have immunomodulatory functions, including induction of tolerance and resolution of inflammation [54]. Most tissue resident macrophages, brain microglia excluded, are derived from hematopoietic stem cells and not from yolk sac precursors as suggested previously [55]. In homeostatic conditions, macrophages are maintained by self-renewal. Under inflammatory condition, the embryonically derived macrophages could be partially replaced by bone marrow-derived monocytes [56].

A specific developmental function of macrophages is particularly evident in the tissues of the female reproductive tract. The human endometrium matures after birth and then undergoes repeated cycles of breakdown, repair, and regeneration. This continued cycle requires a well-orchestrated process of efficient removal of cellular debris in order to prevent antigen leaking that could induce an auto-immune response and the induction of signals for tissue repair [57, 58]. Macrophages are essential in this process. The number of macrophages fluctuate during the menstrual cycle and are driven by estrogen and progesterone [59]. After fertilization, additional macrophages are recruited into the endometrium in an inflammation-like response to male antigens [60], so that 20–30% of all decidual leukocytes are macrophages during the preimplantation period [61]. Although macrophages are present in the placental bed at all times during pregnancy, the number of decidual macrophages fluctuates with gestational age with the highest numbers found in the first and second trimester. Both M1 and M2 macrophages are present in the uterine decidua during pregnancy, but their relative numbers vary. After an initial inflammatory phase, when M1 macrophages predominate, decidual macrophages have a predominantly M2 phenotype until the onset of parturition [62, 63]. Emerging evidence suggests that macrophage homing and phenotype switching is of paramount importance for successful pregnancy. Furthermore, dysregulation of macrophage polarity is linked to several disorders of pregnancy, including recurrent pregnancy loss and preeclampsia [62, 64–67].

Decidual macrophages are a heterogeneous population with diverse phenotypes that facilitate adaptive responses to the ever-changing environment. Decidual macrophages do not belong to either of the M1 and M2 subsets, since they are not typically induced by Th2 cytokines, but by macrophage colony-stimulating factor (M-CSF) and IL-10 [68].

Decidual macrophages are implicated in remodeling processes and in inducing expression of epithelial glycoproteins associated with embryo attachment. Additionally, they potentially contribute to subsequent events of the uterine decidual response and placental trophoblast invasion (see Chapter 3 for detailed review of macrophage function during pregnancy).

Macrophages can be found in the vicinity of spiral arteries that are in the process of early remodeling and in close proximity to the extravillous trophoblast [64, 69]. Therefore, they are proposed to be involved in several processes required for a successful pregnancy, including trophoblast invasion, tissue and vascular remodeling, immune tolerance, embryo growth, and initiation of parturition [70–72].

The origin of decidual macrophages is a subject of controversy and it is discussed in Chapter 3. As discussed above, in the nonpregnant uterus, macrophage number increases during the proliferative phase, potentially under the control of sex hormones such as estrogen [73], while in the pregnant uterus, trophoblast derived factors have a significant role in their recruitment and, as discussed below, their differentiation (Fig. 3) [74].

Fig. 3 Trophoblast cells master of Immune modulation: Trophoblast cells function as immune modulators by promoting the recruitment of monocytes into the implantation site. Similarly, trophoblast secreted factors promote M1 macrophage polarization into M2 macrophages, which is a critical stage to ensure the process of placentation.

7: Mechanisms of macrophages mediate implantation success

Trophoblast cell migration is the process of normal cell movement following a chemotactic gradient, whereas trophoblast cell invasion is the capability to navigate through the endometrium’s extracellular matrix and infiltrate into neighboring tissues, such as maternal blood vessels. As we have mentioned, the endometrium before implantation is already in an inflammatory state that may be under the control of ovarian steroids. Hence, by acting like a leukocyte, which attach to the endothelium and migrate into tissues following an inflammatory gradient, the embryo sticks and migrates into the inflamed endometrium. The first step of inflammation is local secretion of pro-inflammatory signals, which is why macrophages may play a role here. Macrophages are capable of secreting pro-inflammatory signals such as TNFα, IL1β, Il-8, IL-6, and others [75]. TNFα is detectable in oviduct and uterine tissues throughout the preimplantation period in mice and in women. Uterine macrophages are a primary source of TNFα in the reproductive tract; and at appropriate levels, local TNFα may contribute to implantation success [64]. However, addition of TNFα to embryo in vitro culture medium increases the percentage of apoptotic blastomeres in mouse and rat blastocysts, particularly in the inner cell mass [76], suggesting a different role of TNFα during the implantation process.

We then asked the question, how could macrophage-derived TNFα promote embryo implantation? Since TNFα was found before and during the process of implantation [77], we postulate that TNFα might contribute to trophoblast migration and invasion through an indirect pathway. The ligation of the TNFα receptors, TNFR1 or TNFR2, in human endometrium stromal cells (HESC)s stimulates transcription and the release of various chemokines and cytokines, such as chemokine C-X-C motif ligand 10 (CXCL10), growth-regulated protein alpha (GRO-α), RANTES, granulocyte-colony stimulating factor (G-CSF), and IL-8. These high levels of pro-inflammatory cytokines and chemokines may contribute to the inflammatory condition of implantation and promote migration and invasion of the embryo. Using an in vitro 3-D blastocyst implantation model, we showed that the supernatant from HESCs treated with TNFα significantly promoted blastocyst-like spheroid migration of trophoblasts and invasion into extracellular matrix [78]. Therefore, we postulated that M1 uterine macrophages may contribute to embryo implantation by stimulating HESCs via TNFα (Fig. 3).

Compared with the luminal epithelium, where epithelial cells tightly adhere to each other and form an effective physical and immunological barrier to microbial invasion [79–81], HESCs display a higher degree of plasticity. At the time of implantation, the endometrium undergoes further decidualization, which is essential for a successful pregnancy. The decidual secretion contain high amounts of pro-inflammatory factors that can enhance invasion, such as IL-1β, IL-5, IL-7, IL-8, IL-15, CXCL10, IL-12, and RANTES, as well as immune modulatory factors like IL-10 and vascular endothelial growth factor (VEGF) [82]. Therefore, the decidua must balance the production of these pro- and antiinvasive molecules in harmonizing fashion to facilitate a timely and regulated invasion [82].

With the progressing of invasion, the embryo carrying paternal antigens will continue to communicate with maternal decidual immune cells, including macrophages. For survival of the semiallogeneic blastocyst, the decidua transforms its innate and adaptive immune system to prevent any immunological reaction to the conceptus. With modulation by various signals from trophoblasts and decidualized stromal cells, the quantity of decidual macrophages increased, which shows a unique immunosuppressed phenotype. This effect was partly mediated by TGF-β [83] and soluble programmed cell death ligand 1 (PD-L1) produced by trophoblasts (Fig. 3).

In addition, the blastocyst can also modulate the expression of chemokines from decidual cells through hCG. We demonstrated the existence of crosstalk between the placenta (hCG) and the decidua (CXCL10) that is important for the control of immune cell recruitment. Human and animal studies have demonstrated that the cytokine milieu in the uterus is not only different between the pregnant and nonpregnant uterus but also differs between early and late pregnancy [4, 84, 85]. As discussed above, implantation and early trophoblast invasion has been shown to depend on the presence of inflammatory signals [48]. However, once implantation is achieved, the inflammatory environment needs to be reversed into an antiinflammatory state in order to prevent maternal rejection of the embryo [3, 19]. Human chorionic gonadotropin (hCG) is one of the earliest hormones produced by the blastocyst and has potent immune modulatory effects, especially in relation to T cells [86–88], B cells, and dendritic cells [89, 90]. hCG has been suggested to induce the immune modulatory changes seen in the phenotype of B-cells and also modulate the function of immune cells either through a direct pathway that involves the direct binding of hCG to its receptor on T and B cells [88, 91] or indirect pathways by inducing changes in regulatory cell populations, such as dendritic cells [92–94] or decidual/stromal cells (Chapter 5). Nancy et al. [95] found in mice that the chemokine genes CXCL9 and CXCL10 are transcriptionally silenced in mouse decidual stromal cells (reviewed in Chapter 9). This occurred in association with promoter accrual of tri-methyl histone H3 lysine 27 (H3K27me3), a repressive histone mark generated by polycomb repressive complex 2 (PRC2). H3K27me3 is an epigenetic modification to the DNA packaging protein histone H3 and constitutes a mark that indicates the tri-methylation to the 27th lysine residue of the histone H3 protein. This tri-methylation is associated with the downregulation of nearby genes via the formation of heterochromatic regions [96] (Chapter 8). We recently reported the identification of a specific site on the promoter region of CXCL10 that was modified by H3K27me3 and shows that hCG-induced H3K27me3 modification requires the recruitment of the PRC2 member EZH2. Interestingly, we observed a correlation between circulating hCG and CXCL10 levels during early gestation in normal pregnancies [97]. These findings demonstrate how the fetal-trophoblast unit actively participates in regulating inflammation at the maternal-fetal interface in order to promote pregnancy. The epigenetic inhibition of CXCL10-mediated inflammation is promoted by hCG from stromal cells. However, in cases of infection, LPS can modulate the suppression of CXCL10 and induce inflammatory condition that will recruit cytotoxic T cells to the maternal-fetal interface. Based on this and other findings, we postulated a possible mechanism by which infection can induce pregnancy complications, such as preterm birth [97]. hCG, as an antiinflammatory agent, may have therapeutic potential for prevention of miscarriage and preterm birth in pregnancies that are complicated by infection (discussed in Chapter 5).

With the progression of trophoblast differentiation and invasion into the deeper layers of the endometrium, other types of maternal immune cells are exposed to the trophoblast secreted factors. Direct interactions between trophoblast cells and uNK cells are suggested to reduce NK cytotoxicity and render uNK cells more tolerogenic toward fetal antigens [98]. Furthermore, we demonstrated, using in vitro models, that trophoblasts can recruit and induce differentiation of Treg cells [99]. In addition, regulatory B (Breg) cells were also induced in the presence of factors secreted by trophoblast cells [100]. The trophoblast-derived hCG may impact B cells and further force acquisition of a pregnancy-protective phenotype [101] (Chapter 5). hCG not only induces the secretion of asymmetric Abs but also provokes the conversion of human conventional B cells into Breg cells [90, 102].

Successful remodeling of the uterine spiral arteries is essential for a complication-free pregnancy and is best described in terms of its morphologic features. Decidual macrophages are the key regulators of vascular remodeling in human pregnancy. Instead of altering extravillous trophoblast cell invasion or vascular smooth muscle cell differentiation, they are able to induce extracellular matrix breakdown and phagocytose apoptotic vascular smooth muscle cells. With producing a wide range of cytokines (IL-1β, -2, -4, -6, -8, -10, and TNF-α), proteases (matrix metalloproteinase-1, -2, -7, -9, and -10), and angiogenic growth factors, the spiral artery remodeling process would be further enhanced [103] (Chapter 3).

8: The evolutionary role of inflammation during embryo implantation

Although inflammation is critical for implantation, it must be properly contained and controlled in order for implantation to properly progress [19]. The capacity to resolve decidual inflammation has evolved as a key feature underpinning placentation in viviparous mammals. Interestingly, the inflammatory process necessary for embryo implantation is a well conserved evolutionary process. Work by the Wagner lab using the opossum model have demonstrated that inflammation is observed during the early attachment of the embryo and is characterized by the expression of immune related gene, including IL-1A, IL-6, TNF, PTGS2 (aka COX2), PTGES), IL-17A, and neutrophil elastase [104]. However, implantation in the opossum is short lived. After the conceptus attaches to the endometrium, it is born 2–3 days later. Detachment from the uterine lining is associated with increased inflammatory environment, similar to the parturition process observed in mammals [105, 106]. These observations suggest that inflammation is a necessary process, throughout evolution, for implantation. However, the shift to an antiinflammatory stage is required for the maintenance of the pregnancy, since a continuation of the inflammatory environment will mimic parturition [106]. Therefore, the switch-on and -off for inflammation during implantation is a necessary step in order to maintain the pregnancy. It is also a critical step for the process of placentation (Fig. 2).

The next question that we wanted to ask was what are the cellular components responsible for the inflammatory switch? Through their potent immune capacity, uterine/decidual macrophages appear to be critical for the switch-on and -off of inflammation during implantation. As mentioned above, the unique macrophage phenotype and plasticity makes these cells a major player for the establishment and maintenance of a successful pregnancy. Being near trophoblasts and the decidua, the environment in which macrophages mature and differentiate will have an impact on the process of trophoblast invasion and placentation.

9: Trophoblast-macrophage interaction: Switching inflammation

Trophoblast cells are a well-established source of regulation at the maternal-fetal interface [3, 107, 108] (Chapter 20). Trophoblasts have immune like functions and are able to respond to bacterial and viral ligands [7, 19, 109–111]. More importantly, they are able to constitutively produce a host of growth factors, chemokines, and cytokines, such as IL-8, IL-6, and TGF-β [70, 108]. Trophoblast-derived factors are considered the main modulators responsible for macrophage differentiation and function [83]. Using in vitro models, we showed that human trophoblasts promote the differentiation of CD14+ monocytes into CD14+   CD206highCD86low macrophages and present an unusual transcriptional profile in response to TLR4/LPS activation that is characterized by the expression of type 1 Interferon beta (IFNβ) expression. IFN-β further enhances the constitutive production of soluble PD-L1 from trophoblast cells. PD-1 blockage inhibits trophoblast induced macrophage differentiation; a phenotype similar to decidual macrophages [83]. Some of the factors responsible for trophoblast-induced differentiation includes TGF-β [83], HLA-G5 [112], PD-L1 [113], hyaluronan [114], IL-34 [115], and CXCL16 [116].

There is emerging evidence suggesting that the PD-1/PD-L1 signaling is a major regulator of macrophage differentiation and function [113]. By ligation with PD-1 receptor on immune cells, PD-1/PD-L1 signaling causes immunosuppression through inhibition of immune cell activation, proliferation, survival, and pro-inflammatory function, as well as contribute to peripheral tolerance. Both membrane and secreted PD-L1 (sPD-L1) are biologically active and capable of triggering apoptotic signals in target T-cells due to retention of PD-1-binding domain [117]. We, among others, have reported that trophoblasts are capable of constitutively expressing both membrane PD-L1, and sPD-L1 [118–120]. However, compared with membrane PD-L1 protein, we observed high levels of sPD-L1 in the supernatant of trophoblast cells, which is further enhanced by exposure to IFNβ. Recent reports have described PD-L1 protein expression to be present in syncytiotrophoblast and extravillous cytotrophoblasts [120], as well as in the serum of pregnant women [119]. Indeed, PD-L1 protein expression increases from the first-trimester placenta through the second- and third-trimester tissues [120, 121]. Furthermore, soluble PD-L1 can be detected in the blood of pregnant women and increases throughout to gestation [118]. Therefore, based on these observations we postulated that the increase in sPD-L1 in the maternal blood likely indicates the status of placental growth and immune modulatory functions that aim to promote/maintain immune tolerance during gestation [119]. An important component of this immune modulatory function is associated with macrophage polarization.

In summary, we postulate that early in pregnancy, macrophages existing at the implantation site presents characteristics associated with M1 phenotype and contributes to the pro-inflammatory environment that is necessary for implantation. However, as pregnancy progresses to a point where the developing placenta invades the endometrium and makes contact with the decidua, the trophoblasts are capable of changing the microenvironment to one where inflammation has been turned off through the induction of M2 polarization of decidual macrophages. This process will eventually lead to the establishment of placentation: the critical stage in mammalian evolution (Figs. 3 and 4).

Fig. 4 The role of macrophage polarization: M1 type macrophage secrete TNFα, which promotes stromal cells to secrete a range of chemokines and cytokines that regulate trophoblast migration and invasion. These processes are important the early stages of placentation.

10: The role of the microbiome during pregnancy: Maintaining the immunological balance

There is accumulating evidence that demonstrates the existence and importance of a normal microbiota in both the pregnant and the nonpregnant uterus (Chapter 8). Recent animal studies suggest that the maternal microbiota may aid the induction of a tolerogenic immune system, thereby allowing receptivity and preventing the rejection of the fetal-placental unit [34, 122].

To detect different types of pathogens, the innate immune system senses pathogen-associated molecular patterns through pattern recognition receptors, such as toll-like receptors (TLRs), NOD-like receptors, and C-type lectin receptors [123–125] (Chapter 17). Activation of TLRs usually initiates a classical response, which involves the MyD88/nuclear factor-kappa-B pathway. This pathway leads to an inflammatory cascade that includes the upregulation of chemokines (e.g., IL-8 and CCL2) and cytokines (e.g., IL-6 and TNF-α) [126, 127]. However, a second major group of cytokines produced by LPS through TLR4 are the type IFNα and IFNβ [128], which can be detected at the maternal-fetal interface in the trophoblast and decidual macrophages [19, 83, 109].

IFN-β can further enhance the immune-regulatory activities of macrophages by promoting the expression of immune checkpoint molecules (e.g., PD-L1 and Gal-9) and scavenger receptors (e.g., CD206), as well as the tissue repair activity [83]. The role of LPS-induced IFN-β as a crucial immune modulator during pregnancy was demonstrated in studies using IFN receptor-deficient mice (IFNAR−/−). The IFNAR−/− mice are characterized by increased sensitivity to LPS challenge and undergo fetal death within 24 h following a challenge with low doses of LPS [109]. The absence of IFN receptor was also associated with the expression of pro-inflammatory cytokines and chemokines (including TNF, IL-6 and IL-8) that are responsible for the induction of preterm birth [109], suggesting an immune regulatory role for IFNβ in the prevention of excessive inflammation at the maternal/fetal interface.

11: Inadequate macrophage polarization and implantation failure

Implantation failure is an intriguing clinical quandary in reproductive medicine that remains poorly characterized in otherwise healthy women. The Human Fertilization and Embryology Authority for the United Kingdom indicated that around one in four attempted in vitro fertilization (IVF) cycles results in live birth and only 50% of women under the age of 35 years who receive a blastocyst transfer achieve a pregnancy. Inadequate endometrial receptivity is considered one of the main causes of implantation failure. Recurrent pregnancy loss (RPL), also referred to as recurrent miscarriage or habitual abortion, is defined as three consecutive pregnancy losses prior to 20 weeks from the last menstrual period. Both, implantation failure and recurrent miscarriages are two major medical challenges in reproductive medicine. From an immunologic/inflammatory point of view, although the two complications share the same outcome, they have an opposite inflammatory process.

As discussed above, inflammation is necessary during implantation, because it is supposed to induce the transition of the endometrium from its nonreceptive to its receptive state. But this raises the question, what is the immune status for implantation failure? Either compromised or excessive immune response may underlie the occurrence of implantation failure [6]. This could be further demonstrated by the clinical data: (1) performing endometrial biopsies increases inflammation and uterine receptivity [27]; (2) immunotherapy (e.g., glucocorticoids, intravenous immunoglobulin, intralipids, TNF-α inhibitor) inhibiting immune response lead to improvements in live birth rate in some women undergoing IVF treatment or in the prevention of idiopathic recurrent pregnancy loss [129–133]. Therefore, both compromised and excessive immune responses induce the occurrence of implantation failure. Considering the extant evidence, it is reasonable to infer that dysfunctional macrophages in the periconception phase is a key upstream driver of the altered uterine/decidual environment. Thus, failure to achieve an appropriate inflammatory condition that precedes limited attachment of the blastocyst will subsequently cause implantation failure.

RPL, on the other hand, is the result of continued and enhanced inflammation, which leads to an early rejection/parturition, similar to the process observed with the opossum [105]. Indeed, excessive activation of M1 macrophages were found in women with recurrent miscarriages [113, 134], demonstrating the essential role of switching inflammation off after completion of implantation.

In a recent study [77], we described a normative longitudinal serum concentrations that examined IL-10 and TNFα in early pregnancy. We illustrated how the IL-10:TNFα ratio develops throughout the first trimester and how this profile differs in pregnancies that are destined for loss, before a diagnosis or any symptoms. Normal pregnancy showed an early pro-inflammatory profile followed by a clear shift toward an antiinflammatory profile immediately after implantation. This was characterized by an increase in IL-10, decrease in TNFα, and an increasing IL-10:TNFα ratio. Interestingly, pregnancy loss was associated with a failure in this shift and with no increase in neither IL-10 alone nor in the IL-10:TNFα ratio. These findings provide evidence that support the idea that the shift from inflammation to antiinflammation is necessary for the maintenance of the pregnancy [19] and support earlier findings defining normal pregnancy as an antiinflammatory condition [135, 136]. Furthermore, these findings illustrate that the shift happens very early, right after implantation.

12: Conclusion

The maternal immune system must continuously adapt and change for the success of the pregnancy. This book goes into detail about the roles of specific immune cells and their contribution to a successful pregnancy. We intent to build a base on normal physiology during pregnancy, so that the pathophysiology of different pregnancy complications can better be understood. Future books in this series will discuss how dysregulation of specific immunological process can impact the success of a pregnancy. In this first chapter, we have discussed new concepts related to how the microenvironment created by maternal immune cells is essential for the different developmental stages of gestation.

Clinical relevance

•Understanding the process of implantation failure

•Mechanisms to prevent infection while maintaining tolerance

•How to improve the process of implantation and prevent miscarriages.

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