Maternal-Fetal Immunology

Toll-like Receptors

TLRs are one of the best studied systems of PRRs in pregnancy and play a key role in innate immunity. TLRs are a key early sensor of pathogens and can activate both the innate and adaptive immune system. Ten functional toll homologs are found in humans, and they recognize a wide range of pathogen ligands (see Figs. 4.1B, 4.3, and 4.4 ); all 10 TLRs have been described to be expressed in term placenta or choriocarcinoma cell lines. TLR4 is a TLR that recognizes LPS from gram-negative bacteria, which triggers a signaling cascade leading to cytokine gene expression (see Fig. 4.4 ). TLR4 is expressed on macrophages, DC, endothelium, and numerous epithelial tissues. TLR2 recognizes motifs from gram-positive bacteria, including lipoteichoic acid and peptidoglycan. Several bacteria evade TLR recognition by producing proteins or LPS mutants that interfere with TLR signaling. For example, Yersinia pestis (bacteria responsible for “the plague”) expresses a tetraacetylated LPS that is poorly recognized by TLR4 and results in TLR4 antagonism. Brucella abortus , known to induce recurrent abortion in cattle, produces at least two proteins that are potent inhibitors of TLR signaling, which gives the bacteria a survival advantage in evading immune detection.

Both TLR2 expression and TLR4 expression have been demonstrated in the placenta, and first-trimester trophoblast expresses both TLR2 and TLR4. Activation of TLR2 triggers Fas-mediated apoptosis, whereas TLR4 activation induces proinflammatory cytokine production. The immunologic capability of first-trimester trophoblast to recognize pathogens and induce apoptosis suggests that innate immunity is an important placental mechanism for triggering infection-associated spontaneous abortion. TLR4 is also expressed in villous macrophages, villous and extravillous trophoblast, and the chorioamniotic membranes. Expression of TLR4 and TLR2 increases in the chorioamniotic membranes of women with intraamniotic infection and also in term labor. Although intrauterine injection of LPS induces preterm birth in murine and nonhuman primate models, administration of LPS to TLR4 mutant mice or LPS blockade with a TLR4 antagonist does not result in preterm delivery. This finding suggests TLR4 is required for LPS-induced preterm birth in mice and an important driver of the inflammatory cascade resulting from intraamniotic infection.

Maturation of TLR expression in the fetal membranes over time may explain the tendency for infection-associated preterm births to occur no earlier than the late second or early third trimester . Although TLR4 is expressed in the cytoplasm of amniotic epithelium in the first trimester, not until 25 weeks is there TLR4 expression on the apical membrane, which is bathed in amniotic fluid and potential pathogens. A similar ontogeny in TLR4 expression is seen in the fetal lung. When mouse fetal lung is exposed to LPS on fetal day 14 (term is 20 days), TLR4 expression and cytokines are undetectable. By day 17, TLR4 is expressed and an acute cytokine response occurs in fetal lungs. TLR4 likely controls the magnitude of the LPS-induced cytokine response during the perinatal period, and TLR4 placental expression appears to be dependent on gestational age.

Antimicrobial Peptides

Antimicrobial peptides are secreted by neutrophils and epithelial cells to kill bacteria by damaging pathogen membranes. Defensins are a major family of antimicrobial peptides that protect against bacterial, fungal, and viral pathogens. Neutrophils secrete α-defensins and epithelial cells in the gut and lung secrete β-defensins. Both α- and β-defensins are temporally expressed by endometrial epithelial cells during the menstrual cycle. Susceptibility to upper genital tract infection may be related in part to the decreased expression of antimicrobial peptides in response to hormonal changes during the menstrual cycle. Many other tissues of the female reproductive tract and the placenta secrete defensins, including the vagina, cervix, fallopian tubes, decidua, and chorion. Elevated concentrations of vaginal and amniotic fluid defensins have been associated with intraamniotic infection and preterm birth.

Macrophages

Macrophages mature from circulating monocytes that leave the circulation to migrate into tissues throughout the body. Macrophages have critical scavenger functions that help to prevent bacteria from establishing an intrauterine infection during pregnancy. Macrophages are one of the most abundant immune cell types in the placenta and can directly recognize, ingest, and destroy pathogens. Pathogen recognition may occur through PRRs, such as TLRs, scavenger receptors, and mannose receptors. Macrophages also internalize pathogens or pathogen particles through phagocytosis, macropinocytosis, and receptor-mediated endocytosis. Multiple receptors on the macrophage can induce phagocytosis, including the mannose receptor, scavenger receptor, CD14, and complement receptors. Macrophages also release many bactericidal agents after ingesting a pathogen, such as oxygen radicals, nitric oxide, antimicrobial peptides, and lysozyme.

Uterine macrophages represent up to one-third of total leukocytes in pregnancy-associated tissue during the later parts of pregnancy and have many critical functions to support the pregnancy. Macrophages are a major source of inducible nitric oxide synthetase, a rate-limiting enzyme for nitric oxide production. During pregnancy, nitric oxide is thought to relax uterine smooth muscle, and uterine nitric oxide synthetase activity and expression decreases before parturition. Uterine macrophages are also a major source of prostaglandins, inflammatory cytokines, and matrix metalloproteinases that are prominent during term and preterm parturition. Throughout pregnancy, macrophages are in close proximity to invading trophoblasts that establish placentation. Placental growth involves trophoblast remodeling and programmed cell death (apoptosis). Macrophages in the placenta phagocytose apoptotic trophoblast cells, which also programs the macrophage to release antiinflammatory cytokines (e.g., IL-10) promoting fetal tolerance.

Natural Killer Cells

The NK cell has important functions during pregnancy and becomes the most abundant leukocyte in the pregnant uterus. NK cells differ from T and B cells in that they do not express clonally distributed receptors for foreign antigens and can lyse target cells without prior sensitization. The phenotype of decidual NK (dNK) cells is different from that of NK cells in peripheral blood, which correlates with different primary functions. Most NK cells in blood (90%) have low CD56 and high CD16 expression (CD56 ^dim /CD16 ^bright ); in the uterine decidua, dNK cells have high CD56 expression (CD56 ^bright ). The level of CD56 expression determines whether an NK cell has a primary cytolytic (CD56 ^dim ) or cytokine-producing function (CD56 ^bright ). During pregnancy, dNK cells are the predominant immune cell in the decidua with peak levels in early pregnancy (∼85%) that gradually decline by mid-gestation, but remain at approximately 50% of total decidual immune cells. In addition, dNK cells are thought to play a major role in remodeling of the spiral arteries to establish normal placentation in a highly regulated process involving decidual macrophages. Mice with genetically defective or low numbers of dNK cells fail to undergo spiral artery remodeling and normal development of the decidua, which are critical processes for normal placentation. This defect is corrected with administration of interferon-γ (IFN-γ), a prominent NK cell cytokine, suggesting that dNK cells play an important role in the angiogenesis necessary for trophoblast invasion. The cytolytic activity of dNK cells is low and further inhibited by interactions with human leukocyte antigen (HLA)-G. Placentation and spiral artery remodeling relies upon the action of dNK cells, which is a critical immune cell subset in pregnancy.

Complement System

An important component of the innate immune system is the complement system, which consists of a large number of plasma proteins that cooperate to destroy and facilitate the removal of pathogens (see Fig. 4.1C ). Complement proteins are detected in the amniotic fluid during intraamniotic infection, and regulation of complement is necessary to protect placental and fetal tissues from inflammation and destruction. The nature of the initial pathogen trigger determines one of three activation pathways: classical, alternate, and lectin-binding pathways. The classical pathway of complement activation is triggered when the complement protein, C1q, binds to antigen-antibody complexes on the surface of pathogens. This binding then results in a series of activation and amplification steps that result in production of the membrane attack complex (MAC), which creates a pore in the pathogen membrane leading to cell lysis. Formation of the MAC is an important mechanism of host defense against Neisseria species. Genetic deficiencies in C5 to C9 complement proteins have been associated with susceptibility to Neisseria gonorrhea and Neisseria meningitidis.

Regulatory proteins exist to protect cells from the deleterious effects of complement and are expressed on the placental membranes. Placental tissues at the maternal-fetal interface strongly express several negative regulators of complement activation, including CD59 (MAC antagonist), membrane cofactor protein, and decay accelerating factor (inhibitor of C3 and C5 convertases). Whether these regulatory proteins might become overwhelmed during an intraamniotic infection, leading to weakening of the membranes by complement proteins, is unknown.

Cytokines

The release of cytokines and chemokines by macrophages and other immune cells represents an important induced innate immune response ( Table 4.1 ; see Fig. 4.1D ). Activated macrophages secrete cytokines (i.e., IL-1β, IL-6, IL-12, and TNF-α) that initiate inflammatory responses to control infections. These cytokines are often referred to as proinflammatory because they mediate fever, lymphocyte activation, tissue destruction, and shock. Higher levels of several cytokines and chemokines have been implicated in the increased morbidity and mortality to influenza during pregnancy. In lung homogenates of pregnant mice infected with the 2009 H1N1 influenza virus strain, there were very high levels of IL-6 and IL-8; regulated on activation, normal T-cell expressed and secreted (RANTES [CCL5]); and monocyte chemotactic protein 1 (MCP-1 [CCL2]). Dramatic elevations in IL-6 have been implicated in deaths due to the 1918 influenza virus with an estimated mortality in pregnancy of 27%. An increase in cytokine levels is likely not the only explanation for increased morbidity and mortality from influenza infection during pregnancy. Enhanced NK and T-cell responses to influenza vaccination in pregnant women were reported, suggesting that robust cellular immune responses also play a role and may exacerbate tissue injury during an active infection.

During normal pregnancy, many cytokines decrease with advancing gestation including interferon-γ (IFN-γ), vascular endothelium growth factor (VEGF), MCP-1(CCL2), and eotaxin. In contrast, TNF-α and granulocyte-colony stimulating factor levels increase slightly with advancing gestation, which is surprising as they are both linked to proinflammatory responses, and maintaining uterine quiescence during pregnancy is thought to require repression of inflammation. Proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 have also been identified in the amniotic fluid, maternal and fetal blood, and vaginal fluid of women with intraamniotic infection at much higher levels than that observed during normal pregnancy. These cytokines not only serve as a marker of intraamniotic infection, but may trigger preterm labor and lead to neonatal complications. The fetal inflammatory response syndrome describes the connection between elevated proinflammatory cytokines in fetal blood, preterm labor, and increased adverse fetal outcomes (see Chapter 36 ).

The relative contribution of individual cytokines and chemokines to preterm labor was studied in a unique nonhuman primate model. Preterm labor was induced by intraamniotic infusions of IL-1β and TNF-α, but not by IL-6 or IL-8. IL-1β stimulated preterm labor in all cases with an intense contraction pattern. TNF-α induced a variable degree of uterine activity characterized as preterm labor in some animals or as a uterine contraction pattern of moderate intensity. Despite prolonged elevations in amniotic fluid levels, neither IL-6 nor IL-8 induced an increase in uterine contractions until near term. These results suggested a primary role for IL-1β and TNF-α in the induction of infection-associated preterm birth. Other data suggest that parturition and prostaglandin mRNA expression was delayed in IL-6 null mutant mice by 1 day compared to wild-type mice and that LPS failed to induce preterm birth in IL-6 mutant mice. Together, these data indicate that IL-6 plays a role in triggering normal parturition, perhaps in activation of labor pathways.

Investigation of the individual effect of a single cytokine on pregnancy or complications of pregnancy in humans has proved challenging for several reasons. Many cytokines tend to be functionally redundant with one cytokine compensating for the absence of another. Second, there are multiple cytokine receptors (i.e., IL-1 receptor antagonist, IL-18 binding protein) that modulate similar cytokine effects. New families of decoy or silent cytokine receptors and suppressors of cytokine signaling have also been discovered in the placenta and amniotic fluid. Finally, molecular variants of cytokines may act as receptor antagonists. Therefore individual cytokine effects during pregnancy must be interpreted in the context of cytokine receptors, receptor antagonists, silent cytokine receptors, and suppressors of cytokine signaling.

Chemokines

Chemokines are a class of cytokines that act primarily as chemoattractants and direct leukocytes to sites of infection. These chemotactic agents constitute a superfamily of small (8 to 10 kDa) molecules that can be divided into three groups—C, CC, and CXC—based on the position of either one or two cysteine residues located near the amino terminus of the protein. IL-8, CCL2 (also known as macrophage chemoattractant protein-1; MCP-1), and RANTES (CCL5) are a few examples of chemokines. CXC chemokines, such as IL-8, bind to CXC receptors (CXCRs) and are important for neutrophil activation and mobilization. Increases in IL-8 levels have been described in the amniotic fluid, maternal blood, and vaginal fluid with infection-associated preterm birth. IL-8 and CCL2 have also been implicated in uterine stretch-induced preterm labor occurring with multiple gestation. Broad inhibition of chemokines has been suggested as a possible therapeutic to prevent preterm labor. A broad-spectrum chemokine inhibitor has been shown to block preterm labor in a mouse model. Chemokines within gestational tissues are thought to promote preterm labor and broad-spectrum inhibition of chemokines represents an interesting strategy for prevention of preterm birth.

Some chemokine receptors are used as a coreceptor for the viral entry of the human immunodeficiency virus (HIV). The two major chemokine coreceptors for HIV are CXCR4 and CCR5, both of which are expressed on activated T cells. CCR5 is also expressed on DCs and macrophages, which allows HIV to infect these cell types. Rare resistance to HIV infection was discovered to correlate with homozygosity for a nonfunctional variant of CCR5 caused by a gene deletion in the coding region. The gene frequency for this CCR5 variant is highest in Northern Europeans. CCR3 is another chemokine coreceptor for HIV that is expressed by microglia and can be used by some HIV strains to infect the brain.

Major Histocompatibility Complex

Discriminating cells that are “self” from “nonself” is a critical function of the immune system to determine which cells to destroy and which to leave alone. In pregnancy, this process must be carefully regulated to prevent the killing of fetal cells, which express paternal genes that appear foreign to the maternal immune system, in effect expanding the definition of self to include the fetus. The ability of a lymphocyte to recognize self from nonself is based on the expression of unique major histocompatibility complex (MHC) molecules on a cell's surface, which present small peptides from within the cell. MHC molecules are highly polymorphic proteins produced by a cluster of genes on the short arm of chromosome 6. This gene complex is classically divided into two distinct regions referred to as class I and class II . Class I contains classical HLA genes (e.g., HLA-A, -B, and -C) and nonclassical HLA genes that are distinguished by more limited polymorphism (e.g., HLA-G, -E, and -F). Class II contains polymorphic genes that are often matched for transplantation, including those of the HLA-DR, -DQ, and –DP families of genes. Reduced HLA matching is associated with graft rejection after transplantation through activation of T cells. This system is significantly different from the innate immune system, in which recognition of HLA genes is not necessary for pathogen destruction.

Humoral Immune Responses: B Cells and Antibodies

The function of B cells is to protect the extracellular spaces (e.g., plasma, vagina) in the body through which infectious pathogens usually spread (see Fig. 4.2A ). B cells mainly fight infection by secreting antibodies, also called immunoglobulins . There are many similarities between B and T lymphocytes. Like T cells, B cells also undergo clonal expansion after antigen stimulation and can be identified by a variety of specific cell surface markers (e.g., CD19, CD20, and BCR antigens). Activated B cells may proliferate and differentiate into antibody-secreting plasma cells. Antibodies control infection by several mechanisms, including neutralization, opsonization, and complement activation . Neutralization of a pathogen refers to the process of antibody binding, which prevents the pathogen from binding to a cell surface and internalizing. Alternatively, antibodies coating the pathogen may enhance phagocytosis, also referred to as opsonization . Antibodies may also directly activate the classical complement pathway. Activation of the B cell drives the B cell to proliferate and differentiate into an antibody-secreting plasma cell.

The B cell immune repertoire changes dramatically during pregnancy. Immature B cells, which are the precursors to antigen-specific mature B cells, are significantly reduced with advancing gestation in the maternal bone marrow, blood, and spleens of pregnant mice. Lymphopoiesis of B cells is reduced during pregnancy, which may be mediated by the normal pregnancy rise in estradiol. Estradiol reduces levels of IL-7, a critical factor necessary for B-cell production in the bone marrow. This reduction in immature B cells is further potentiated during the second half of pregnancy by the antigen-induced deletion of immature B cells. In contrast to the reduction in immature B cells, the number of mature B cells increases during pregnancy, particularly in the lymph nodes draining the uterus. Overall, pregnancy is associated with profound changes in the numbers and diversity of B-cell subtypes.

Autoantibodies produced by B cells against angiotensin receptor I (AT1-AA) are thought to play a role in inducing hypertension and proteinuria in women with preeclampsia and fetal intrauterine growth restriction. AT1-AA are present in 70% to 95% of women with preeclampsia and antibody titer correlated with disease severity. AT1-AA can bind to endothelial and placental cells in vitro to induce oxidative stress, cytokine and endothelin production. Transfer of these autoantibodies from women with preeclampsia can also induce hypertension and proteinuria in pregnant mice. Although there is a wide spectrum of immunologic abnormalities in preeclampsia, the concept that an autoantibody can cause disease in pregnancy is well established. For example, Graves disease is the most common causes of thyrotoxicosis in pregnancy. More than 80% of individuals with Graves have antithyroid-stimulating hormone receptor autoantibodies. B cells are likely beneficial in establishing fetal tolerance but may also contribute to the pathogenesis of certain obstetrical complications like preeclampsia.

Regulatory B Cells

A unique population of B cells, called regulatory B cells (B _REG ), act to inhibit inflammatory responses that contribute to autoimmunity and may play a key role in maintenance of fetal tolerance. A key function of B _REG cells is production of IL-10, which is a powerful antiinflammatory cytokine. In addition, B _REG cells control inflammatory responses by inhibiting production of the proinflammatory cytokine TNF-α, maintaining DCs in an immature state, and suppressing antigen-specific CD4 ⁺ T-cell proliferation. The role of B _REG cells in producing IL-10 has particular importance in pregnancy. Although not required for successful pregnancy, IL-10 is protective against adverse pregnancy outcomes if inflammation occurs. In pregnant mice, a low-dose inflammatory stimulus (lipopolysaccharide inoculation) was associated with a significantly greater rate of preterm fetal loss when the mouse was deficient in IL-10 compared to control mice; administration of exogenous IL-10 attenuated proinflammatory cytokine production in the serum and significantly reduced the rate of preterm fetal loss in both IL-10 deficient and control mice. IL-10 production by B _REG cells likely enhances other mechanisms of fetal tolerance and helps to control excessive inflammatory responses that jeopardize successful completion of the pregnancy.