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Keywords:

  • infection;
  • inflammation;
  • placenta;
  • pregnancy;
  • TLR;
  • trophoblast

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

During normal pregnancy, the decidua is populated by a variety of leucocytes; however, cells of the innate immune system seem to dominate this tissue. Their presence suggests that the innate immune system is not indifferent to the fetus and has been associated with a response of the maternal immune system to the ‘semi-allograft’ fetus. New evidences, however, indicate that these immune cells are critical for decidual and trophoblast development rather than induction of tolerance. We hypothesized that during implantation, an inflammatory environment is necessary for the attachment and invasion of the blastocyst. The existence of an ‘inflammatory-mediated embryo implantation’ condition is dependent on the proper ‘education’ of the innate immune system which we propose is mediated by the trophoblast. Here we postulate that trophoblast cells successfully orchestrate their inflammatory environment and regulate immune cells differentiation and activation through Toll-like receptors (TLR). We will describe potential functions of TLR in trophoblast cells, their recognition and response to microorganisms, and their involvement in innate immunity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

The definition of pregnancy as a ‘Th-2’ state postulates that pregnancy is an anti-inflammatory condition,1 and a shift to a ‘Th-1’ state would lead to abortion or pregnancy complications. This hypothesis was enthusiastically embraced and numerous studies attempted to prove and support this premise. While many studies confirmed this hypothesis, a similar number of studies argued against this notion.2,3 The reason for these contradictory results may be due to oversimplification of disparate observations made during pregnancy. In the aforementioned studies, pregnancy was evaluated as a single event, when in reality it has three distinct immunological phases that are characterized by distinct biological processes and can be symbolized by how the pregnant woman feels.

We evaluated the expression of cytokines in the blood of pregnant women in three separate periods: early, mid, and late pregnancy, and found the cytokine profile was different for each stage. Early pregnancy was characterized by a dominant pro-inflammatory profile with high levels of cytokines and chemokines, such as IL-8 and MCP-1. The circulating levels of all these cytokines decreased significantly during mid-pregnancy but increased again at the end of the pregnancy (Fig. 1). It is important to take in consideration the fact that these changes may be associated with the local alterations undergoing at the implantation site and may not represent a systemic profile.2–4

image

Figure 1. Cytokine profile during pregnancy. Model of a cytokine profile of normal pregnancies determined according to gestational age.

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Implantation resembles an open wound that requires a strong inflammatory response. During the early stage of implantation, the trophoblast has to break the epithelial lining of the uterus in order to adhere, and invade into the endometrial tissue, as well as replace vascular smooth muscle of the uterine spiral arteries in order to secure an adequate blood supply. All these activities create a veritable ‘battleground’ of invading, dying, and repairing cells. During this period, the mother's wellbeing is affected: she feels terrible because her whole body is struggling to adapt to the presence of the fetus (in addition to hormone changes and other factors, this immune response is responsible for ‘morning sickness’). Thus, the first trimester of pregnancy is a pro-inflammatory phase (Fig. 2).

image

Figure 2. Immunological phases of pregnancy. Early pregnancy is associated with a pro-inflammatory environment; a second immunologic phase is represented by mid-pregnancy when an anti-inflammatory environment dominates. A third immunological phase is characterized by a pro-inflammatory environment and is present at the end of the pregnancy.

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The second immunological phase of pregnancy is, in many ways, the optimal time for the mother. This is a period of rapid fetal growth and development. The mother, placenta and fetus are symbiotic, and the predominant immunological feature is induction of an anti-inflammatory state. The woman no longer suffers from nausea and fever as she did in the first stage, in part because the immune response is no longer the predominant endocrine feature.

During the last immunological phase of pregnancy, the fetus has completed its development; all the organs are functional and ready to deal with the external world. Now the mother needs to deliver the baby and this can only be achieved through renewed inflammation. Parturition is characterized by an influx of immune cells into the myometrium in order to promote initiation of an inflammatory process. This pro-inflammatory environment promotes the contraction of the uterus, expulsion of the baby, and rejection of the placenta. In conclusion, pregnancy is a pro-inflammatory and anti-inflammatory condition, depending upon the stage of gestation4 (Fig. 2).

Role of the Innate Immune System in Pregnancy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

During normal pregnancy, several of the cellular components of the innate immune system are found at the site of implantation and this has been taken as conclusive proof that the maternal immune system responds to the allograft fetus. During the first trimester, 70% of decidual leukocytes are natural killer (NK) cells, 20–25% are macrophages and approximately 1.7% are dendritic cells.5–7 These cells infiltrate the decidua and accumulate around the invading trophoblast cells. Furthermore, from the first trimester onwards, circulating monocytes, granulocytes and NK cells increase in number and acquire an activated phenotype. What this evidence suggests is that the maternal innate immune system is not indifferent to the fetus. Indeed, many studies have shown that maternal systemic and local immune response is not against the allograft fetus, but supporting fetus in order to establish proper maternal and fetal immunological relationships. For instance, depletion of NK cells during pregnancy, instead of being protective, has been shown to be detrimental for pregnancy outcome.8 Much effort was focused on the susceptibility of the trophoblast to NK cell mediated cytotoxicity,9,10 until it was found that uterine NK cells are not cytotoxic.11 Moreover, recently it was shown that uterine NK cells are important for mediating angiogenesis and trophoblast invasion; the two critical events in early pregnancy.12 Similar findings have been observed with other immune cells. For example, macrophages within the decidua are important for clearing apoptotic and cellular debris, as well as facilitating trophoblast migration throughout gestation,13,14 while dendritic cells play an important role in the early implantation stage.15 More recent observations suggest that during implantation and early pregnancy DC are not associated with the process of ‘fetal tolerance’ but may play a critical role on the development of the decidua.16

Therefore, we can conclude that the innate immune cells may play a critical role in the fetal-maternal immune adjustment and in successful placentation. These findings, the exact opposite of the classical reproductive immunology dogma, question the whole paradigm of pregnancy that has until now assumed the maternal immune system a threat to the developing fetus.

Redefining Medawar's Hypothesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Medawar's original observation was based on the assumption that the placenta was analogous to a ‘piece of skin’ with paternal antigens, which under normal immunological conditions, should be rejected. However, the placenta is more than just a transplanted organ. Our knowledge of placental biology has significantly increased over the last 50 years. We now know that the placenta is a complex organ, which has evolved from the original ‘egg cover’. Pregnancy and implantation, contrary to ‘graft implants’, has been taking place for more than 30 000 000 years.17 Therefore, from an evolutionary point of view it is difficult to conceive that the placenta and the maternal immune system still maintain an antagonistic status. Thus, while there should be an active mechanism preventing the potential recognition of paternal antigens by the maternal immune system, the trophoblast and the maternal immune system have evolved and established a cooperative status, helping each other against common enemies, that is: infectious microorganisms.

Infection and Pregnancy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Bacterial and viral infections pose a significant threat to a pregnancy, and to the wellbeing of the fetus, by gaining access to the placenta through one of three major routes: through maternal circulation; by ascending into the uterus from the lower reproductive tract; or by descending into the uterus from the peritoneal cavity.18 Clinical studies have established a strong association between pregnancy complications and intrauterine infections.18–20 Indeed, infections have been reported as responsible for up to 40% of preterm labor cases. Furthermore, 80% of preterm deliveries occurring at less than 30 weeks of gestation have evidence of infection.19–21 In addition, other pregnancy complications, such as preeclampsia, may have an underlying infectious trigger.22–24 Thus, we asked ourselves, how a microorganism may initiate a response that would induce preterm labor or abortion, or even preeclampsia? To our surprise, we have found that the same cells that promote fetal acceptance under normal conditions – the trophoblast – may initiate the signals promoting such fetal rejection in the presence of an infection.25

Today, our research is focused on understanding how the trophoblast and the maternal immune system can work together to protect the fetus against infection. The results of our studies suggest that the trophoblast functions as a conductor of a symphony, where the musicians are the cells of the maternal immune system. The success of the pregnancy depends on how well the trophoblast communicates with each immune cell type and then how all of them work together. At the molecular level, we try to understand how the trophoblast recognizes who and what is present, and based on that information what type of signals are sent that would then coordinate the activities of each of the cellular components at the implantation site.

The trophoblast, just like an innate immune cell, expresses pattern recognition receptors (PRR) that function as ‘sensors’ of the surrounding environment.25–29 Through these sensors, the trophoblast can recognize the presence of bacteria, viruses, dying cells and damaged tissue. Upon recognition, the trophoblast will secrete a specific set of cytokines that in turn, will act upon the immune cells within the decidua (i.e. macrophages, T regulatory cells, NK cells), ‘educating’ them to work together in support of the growing fetus30 (Fig. 3). Indeed, each immune cell type acquires specific properties related to implantation and placentation, as already discussed. However, a viral or bacterial infection may perturb the harmony of these interactions. There are a number of different PRR, including the mannose-binding receptor and the scavenger receptor;31 however, this review will focus on the major family of PRR, the Toll-like receptors (TLR). We will discuss the expression and function of TLR at the maternal–fetal interface and their roles in the interaction between the trophoblast and the maternal immune system.

image

Figure 3. Toll-like receptor (TLR) signaling. Membranal TLR: TLR-1, -2, -4, -5 and -6, can recognize external signals, while cytoplasmic TLR TLR-3, -7, -8, and -9 will recognize intracellular signals. Following ligation, the majority of TLR induce activation of NFκB and cytokine production in a MyD88-dependent manner. TLR-3, as well as TLR-4, signal in a MyD88-independent manner, which induces the expression of type I interferons (IFN) and IFN-inducible proteins. IFN (interferon), TRIF (Toll/IL-1 receptor domain-containing adaptor inducing IFN-β), IRF-3 (IFN regulatory factor).

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Toll-like Receptors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Toll-like receptors are transmembrane proteins with extracellular domains of leucine-rich repeat motifs, that are evolutionarily conserved to recognize pathogen-associated molecular patterns (PAMP) in bacteria, viruses, fungi and parasites. Eleven mammalian TLR have been identified to date (TLR-1 to TLR-11),32,33 however, no functional TLR-11 proteins have been documented in humans.34,35

Each TLR differs in its specificity, so while individually TLR respond to limited ligands, collectively the family of TLR can respond to a wide range of PAMP associated with bacteria, viruses, fungi and parasites (Table 1). TLR-4, the first to be identified,36 is the specific receptor for gram-negative bacterial lipopolysaccharide (LPS).37 TLR-2 has the widest specificity, recognizing bacterial lipoproteins, gram-positive bacterial peptidoglycan (PGN) and lipoteichoic acid (LTA), and fungal zymosan.38–40 The range of ligands to which TLR-2 responds appears to be broadened by its heterodimerization with other TLR, so that TLR-1/-2 heterodimers respond to a panel of lipoproteins different from those recognized by TLR-2/-6.41,42 TLR -3, -7, and -8 appear to play important roles in response to viruses. TLR-3 is known to bind viral double-stranded RNA,43 while TLR -7 and -8 interact with single-stranded RNA.44,45 TLR-5 recognizes bacterial flagellin, and TLR-9 mediates cell responses to bacterial DNA through recognition of cytosine-guanine pairs (‘CpG’ motifs),46 and can also be activated by the Herpes virus.47,48 In addition to detecting PAMP, TLR-4 and TLR-2 can bind endogenous molecules called DAMP (danger-associated molecular patterns), such as reactive oxygen species (ROS)49 high mobility group box protein 1 (HMGB1),50 and Heat shock proteins (Hsp) such as Hsp60, Hsp70 and Hsp90.51

Table 1.  Toll-like receptors (TLR) and ligands
TLRLigands
ExogenousEndogenous
  1. ND, no data.

TLR1Triacetylated lipoproteins (with TLR2)ND
TLR2Peptidoglycan, lipopeptides, lipoteichoic acids, zymosanND
TLR3Double-stranded RNAHost RNA
TLR4Lipopolysaccharides, paclitaxelHsp60, Hsp70, Hsp90, ROS, HMGB1, surfactant protein A, fibrinogen, fibronectin, hyaluronic acid oligosaccharides, eosinophil derived neurotoxin
TLR5FlagellinND
TLR6Diacylated lipoprotein (with TLR2)ND
TLR7Single-stranded RNAND
TLR8Single-stranded RNAND
TLR9Non-methylated CpG DNA, herpes virusAutoimmune chromatine-IgG complex
TLR10NDND

Toll-like Receptor Signaling

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Binding of TLR commonly results in the production of cytokines and anti-microbial factors via a common intracellular signaling pathway (Fig. 3). Upon ligand recognition, the TLR recruit the intracellular signaling adapter protein, MyD88, and a subsequent kinase cascade triggers activation of the NFκB pathway,which results in the generation of an inflammatory response.52 However, TLR-3 and TLR-4 can also signal in a MyD88-independent manner.53 Such MyD88-independent signaling occurs through another adapter protein, Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), which not only can activate the NFκB pathway, but also results in the phosphorylation of IFN regulatory factor-3 (IRF-3). This alternative pathway generates an anti-viral response associated with the production of type I IFN and IFN-inducible genes.54

Toll-like Receptor Expression in the Placenta

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

The expression of all 10 TLR has been described in the human placenta, and the dominant cell type expressing these TLR are the trophoblast.55–57 However, the expression pattern of TLR by the trophoblast varies by gestational age as well as by the stage of trophoblast differentiation. For example, first trimester trophoblast cells do not express TLR-6, while this receptor is expressed by third trimester trophoblast cells.55,58 Furthermore, in first trimester placentas, villous cytotrophoblast and extravillous trophoblast cells express TLR-2 and TLR-455,59,60 while syncytiotrophoblast cells do not express these receptors. The lack of TLR expression by the outer trophoblast layer suggest that early pregnancy placenta will only respond to a microbe that has broken through this outer layer. Thus, a microorganism will only pose a threat to the fetus if the TLR-negative syncytiotrophoblast layer is breached and the pathogen has entered either the decidual or the placental villous compartments.25,55

Term placenta, on the other hand, has a different pattern of TLR expression, characterized by positive immunoreactivity for TLR-2 and -4 on the cytoplasm of syncytiotrophoblast.56,60 More recently, Ma et al. evaluated the expression of TLR-2 and TLR-4 in third trimester placentas and described the expression of TLR-2 in endothelial cells, macrophages, syncytiotrophoblast and fibroblast, while TLR-4 expression was prominently expressed in syncytiotrophoblast and endothelial cells.61

These findings suggest that not only immune cells, but also trophoblasts within the placenta have a capacity to respond to the invading pathogens and may be involved, as a part of the innate immune system, in the physiological protection of the placenta.

Toll-like Receptor Signaling and Function at the Maternal-fetal Interface

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Given that TLR are widely expressed at the maternal-fetal interface, not only by immune cells but also by trophoblasts and decidual cells, the next question is ‘what is the role of TLR in these cells and their effect in immune responses during pregnancy?’. Here we will discuss possible functions of TLR at the maternal-fetal interface.

Holmlund et al. first demonstrated that stimulation with zymosan and LPS induced IL-6 and IL-8 cytokine production by third trimester placental cultures, without affecting TLR-2 and TLR-4 mRNA and protein expression levels,56 suggesting functional TLR in the placenta. Our group demonstrated that TLR-expressing first trimester trophoblast generate very distinct patterns of response, depending upon the type of stimuli and, therefore, the specific TLR that is activated. For example, following ligation of TLR-4 with LPS, trophoblast cells generate a slow inflammatory response, characterized by a modest up-regulation of chemokines.27,55 In contrast, peptidoglycan, which signals through TLR-2, induces trophoblast cells to undergo apoptosis. Further studies have suggested that the pro-apoptotic effect observed following PDG treatment is mediated by TLR-1/TLR-2 heterodimers whereas the presence of TLR-6 prevented cell death and a cytokine response ensues through NFκB activation (Abrahams et al. Journal of Immunology, in press). Others studies reporting the induction of trophoblast apoptosis through TLR-2 includes ultraviolet-inactivated human cytomegalovirus (HCMV),62 and a recent report using recombinant Chlamydia Hsp60 through TLR-4.63

The placenta may become exposed not only to bacteria but also to virus, which may pose a substantial threat to the fetus. TLR-3, a receptor known to mediate immune responses towards viral dsRNA,43 is expressed by first trimester trophoblasts.26 The trophoblast presents unique characteristics in terms of their responses to viral infections. TLR-3 ligation by small amounts of viral dsRNA induces a rapid and highly potent inflammatory response characterized by a strong up-regulation of chemokines and the production of type I interferons and interferon-inducible genes.64 The production of IFN-β is critical to mount an antiviral response; therefore suggesting that the trophoblast upon recognition of a virus may initiate a classical antiviral reaction. In addition to interferon, the trophoblast has an ability to produce anti-microbial factors, such as secretory leukocyte protease inhibitor (SLPI), 2′, 5′-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G(APOBEC3G). These all have a direct effect on viral activity suggesting that the placenta, more specifically trophoblasts, can actively prevent the transmission of certain viral infections to the fetus.

In summary, all these studies suggest that trophoblasts are able to recognize bacterial or viral products through TLR and induce differential responses. The factor(s) associated with the type of response may determine the final outcome and be associated with pregnancy complications, such as preterm labor, preeclampsia or intrauterine growth retardation.

Toll-like Receptor Signaling Modulates Immune Cell Function

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Recently we proposed that trophoblast cells are potentially able to modulate the immune system at the maternal-fetal interface, by regulating various immune cell functions.28 Our earlier studies demonstrated that first trimester trophoblasts constitutively secrete cytokines/chemokines such as GRO-α, MCP-1, and IL-8, and that these trophoblasts are also able to recruit monocytes/macrophages, NK cells and neutrophils.28,30 This cytokine/chemokine expression in trophoblasts is further enhanced upon ligation by TLR-4 or TLR-3 agonists, followed by a significant increase in the recruitment of immune cells.28 Moreover, the factors produced by trophoblasts have a potent modulatory effect on the maternal immune cells by determining their differentiation and state of activation. For example, monocytes/macrophages incubated in the presence of trophoblasts or their condition media become less sensitive to LPS stimulation.30 However, under the same conditions, the monocyte response to viral products such as poly(I:C) is significantly enhanced (Koga et al. in preparation).

Based on these observations we propose that the trophoblast is able to ‘educate’ immune cells, where signals originated from trophoblast could determine the subsequent immune cell behavior. This proper trophoblast-immune cell cross talk may be essential for a normal pregnancy. However, a placental response to an infection, if intense enough or left unresolved, may subsequently alter the normal cross talk between the trophoblast and decidual immune cells. TLR-mediated trophoblast inflammatory or apoptotic responses to an infection may impact the resident and recruited maternal immune cells by changing them from a ‘supportive’ into an aggressive phenotype.30 This may further promote a strong pro-inflammatory and pro-apoptotic microenvironment, and may ultimately prove detrimental to pregnancy outcome by facilitating fetal rejection.

Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

A number of animal models have established that gram-negative bacteria can trigger preterm labor, and this has been shown to be mediated by TLR-4.56,65 Intrauterine injection of heat-killed E. coli into wild type pregnant mice at gestational day 14 induced preterm delivery in 100%, whereas in C3H/HeJ mice, which have a spontaneous mutation in TLR-4, none of the mice underwent preterm delivery.

Lipopolysaccharide administration has also been shown to change the cytokine profile by increasing maternal serum concentration of TNF-α and IL-6, as well as placental expression of TNF-α, IL-6 and IL-1α,66 which imply that systemic and local inflammatory responses followed by LPS administration cause preterm labor. However, this effect seems to be dose-dependent. Interestingly, TLR-4 ligation by intrauterine LPS injections is known to up-regulate TLR-2 expression, which suggests that one cascade operates through multiple branching and redundant pathways to bring about the exaggerated outcome, especially in the setting of combined infection.67

Gram-positive bacterial components have been associated with preterm labor as well. For example, in rodents, lipoteichoic acid (LTA) was shown to induce preterm delivery following cervical ripening and placental abruption.68 These effects on pregnancy seems to be TLR mediated as shown by a recent study where either peptidoglycan (PDG) or LTA, both TLR-2 ligands, induced preterm delivery in mice when injected intrauterus.69 In terms of the mechanism, contrary to the effects of TLR-4 ligation, TLR-2 ligation does not seem to induce inflammatory responses. The expression of TNF-α and IL1-β were examined in uterine tissues but no up-regulation was found in PDG-treated mice.69 We also recently established a novel mouse model where PDG was injected intraperitoneally on gestational day 6 and uterine cytokine production, NK cell activation, and apoptosis was evaluated. In this model, no change in cytokine production or NK cell activation was found in PDG-treated uterus, (Abrahams et al. Journal of Immunology, in press)96, in contrast to the findings in LPS-treated mice where cytokine up-regulation and NK cell activation were observed.65 On the other hand, a significant increase of apoptotic trophoblasts were observed in PDG-treated mice (Abrahams et al. Journal of Immunology, in press)96, which is consistent with in vitro studies showing that PDG treatment to trophoblasts induced TLR-2-mediated apoptosis.55 These results suggest that the mechanism underlying preterm labor triggered by PDG is not the result of an inflammatory reaction but apoptosis of the trophoblast.

Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Early studies demonstrated that poly(I:C) induces fetal loss when injected during early pregnancy in various mating pairs such as ‘resorption-prone’,70 syngeneic mating, and allogeneic mating.71 Recently, Lin et al. demonstrated that poly(I:C) induces resorption in pregnant mice through TLR-3, because injection of a neutralizing antibody for TLR-3 abrogated the effects of poly(I:C).72 In addition, they demonstrated that ligation of TLR-3 with poly(I:C) on gestational day 7 induced IL2 and inhibited IL10 expression in CD45+ cells isolated from the placenta.72 The same authors further demonstrated that poly(I:C) injection in early pregnancy induced uNK cells activation and speculated that this is the cause of poly(I:C) induced embryo resorption.73 Zhang et al. showed that poly(I:C) treatment impaired uterine vascular remodeling through endometrial TNF-α up-regulation, and suggested that this induced fetal loss.74

Administration of poly(I:C) during late pregnancy has also detrimental effects on pregnancy as shown by a recent study using intrauterine injection model. When administrated on gestational day 15.5, poly(I:C) induced preterm labor in 31% of the animals along with induction of interferon-β and RANTES expression in uterine tissues.69

The majority of these studies have focused mainly on the effects of TLR-3 ligation on immune cells at the maternal-fetal interface without taking into consideration the potential role of trophoblast. Our in vitro studies suggest that the placenta, and more specifically the trophoblast, plays an active role on the response to poly(I:C).64 Preliminary studies in our lab with animal models confirmed the existence of a local immune response to poly(I:C) mediated by the trophoblast as well as the amnion. Furthermore, we observed that these responses are mediated by TLR-3 in trophoblasts, since poly(I:C) effects are not observed in TLR-3 KO mice (Koga et al. Journal of Immunology, submitted).

These studies combined with clinical data, make a strong case for TLR as mediators of infection-associated prematurity. Possible therapeutic strategies are now being explored in order to determine whether the inhibition of TLR signaling might help prevent such pregnancy complications.

Outcome of Infection and Toll-like Receptor Ligation on Fetal Development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

The fetus is not indifferent to a viral or bacterial infection and the immunological responses by the maternal immune system or the placenta or fetal immune system may have important consequences on the normal development and survival of the fetus. Several studies have evaluated the long-term effects of TLR ligation in the offspring.

Administration of LPS to pregnant mice was shown to cause acute fetal cardiovascular depression,75 and inhibit structural development of the distal fetal mouse lung in a TLR-4 dependent manner.76 Similarly, cerebral white matter damage, which is one of the biggest problems seen in preterm neonates because of its strong association with their lifetime adverse outcome, is also believed to be caused by TLR-4 activation in the fetus.77 It is worthy to mention that low doses of LPS, which has no adverse effects on pregnancy outcome, dramatically increase brain injury to subsequent hypoxic-ischemic challenge in a newborn rat animal model.77 These findings are compatible with clinical findings showing that maternal exposure to bacteria, not only causes preterm labor, but also contributes to long-term adverse outcome in the offspring such as cerebral white matter damage.

Adverse effects of maternal TLR-3 activation were also found in fetuses in various animal models. Maternal poly(I:C) or virus exposure cause marked behavioral changes in the offspring mouse,78 which is relevant to many epidemiological studies showing that maternal exposure to virus cause not only abortion or preterm birth but also fetal schizophrenia and autism.79,80 Offspring of poly(I:C)-treated pregnant mice were shown to have less expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and TNF-α in their placenta, liver/spleen and brain, which may represent a potential mechanism through which maternal viral infection increases a risk for such neurodevelopmental disorders.81

Given that maternal infections are detrimental to the long-term health of offspring, the next question is whether these adverse effects of maternal infections on the fetus are due to a direct effect of the pathogen (virus, bacteria) or secondary effect through maternal response (inflammation, cytokines). In this context, Kim et al. demonstrated the expression of TLR-2 and TLR-4 in skin samples obtained from preterm delivered babies by immunohystochemistry.82 As for function of TLR in fetus, studies of mouse and human fetal cells show that stimulation of fetal intestinal cells or fetal monocytes with LPS results in production of chemokines and cytokines.83,84 These findings indicate that fetal cells are also capable of recognizing microbial products and participate in innate immune defense in the case of microbial invasion of the amniotic cavity; although the expressions of other pattern recognition receptors in various fetal tissues/organs still need to be elucidated.

Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Intrauterine infection and subsequent chorioaminionitis (CAM) are known to be among the most important causes of preterm delivery.85 We evaluated the expression of TLR-2 and TLR-4 in chorioamniotic membranes in spontaneous labor at term and in preterm parturition that are associated with CAM. TLR-2 and TLR-4 mRNA expression were significantly higher in membranes from women at term with spontaneous labor than women not in labor. TLR-2 expression in chorioamniotic membranes was significantly higher in patients with CAM than those without CAM. The expression of TLR-2 was also restricted to the basal surface of amniotic epithelial cells in non CAM preterm labor whereas in CAM cases, diffuse and strong positive staining for the entire cytoplasm of epithelium was observed.86 On the other hand, Rindsjo et al. demonstrated that TLR-2 expression in trophoblast was decreased in patients with CAM compared to those without CAM.59 These findings suggest that the response to infection varies in the different parts of the maternal-fetal interface. However, we have to take in consideration the possibility that these variations might be the result of technical variations among study groups.

As for TLR-4, Kumasaki et al. showed that TLR-4 expression in the villous Hofbauer cells was higher in preterm placenta with CAM than preterm placenta without CAM, or term placenta with or without CAM.57 Recently TLR-4 expression was shown at the amniotic epithelium and the strongest immunoreactivity for TLR-4 was observed at basal membrane in CAM patients. The authors suggested that an infection may induce the translocation of TLR-4 from apical to basal membrane in order to decrease TLR signaling during early infection but allow the amniotic epithelium to remain competent to invasive bacteria.87

In addition to CAM, we also evaluated the involvement of TLR in the etiology of preeclampsia. Thus, TLR-4 expression in trophoblast was significantly higher in women with preterm delivery associated with preeclampsia than in women with or without CAM preterm delivery. Furthermore, TLR-4 expression was co-localized with activated NFκB, TNF-α and M30 (an apoptosis marker specific for epithelial cells) suggesting that inflammatory cytokines can induce TLR-4 expression and thereby enhance further trophoblast response to TLR ligands.88 Similarly, Wang et al. described a correlation between high levels of TLR-4 expression in microvessel endothelial cells isolated from placental villi, and placental vascular disease, defined by an abnormal umbilical artery Doppler study.89 These findings imply that the level of TLR expression in the placenta is controlled by certain pathogen per se and/or endogenous molecule produced upon inflammation, as a feedback mechanism to enhance or inhibit further immune responses, although precise mechanisms are not clarified yet.

A new aspect on TLR function is related to its ability to recognize not only microbial ligands but also host products, also know as ‘danger signals’ released by injured cells,90 suggesting that TLR might be involved not only in infection but also non-infection-related conditions associated with pregnancy. Indeed, recently, Holmlund et al. demonstrated that HMGB1, a ligand for TLR-4, is highly expressed in decidua from preeclampic patients.91

Toll-like Receptor Polymorphisms and Pregnancy Disorders

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

Since polymorphisms in TLR genes were shown to be associated with impaired offspring receptor function and an increased susceptibility to infections, a number of studies evaluated whether polymorphisms in TLR genes are associated with pregnancy disorder. As for preterm labor, most of the studies are focusing on polymorphism in TLR-2 and TLR-4. Interestingly, not only polymorphism in the mother, but also that in the infant was analyzed and proved associations between fetal polymorphism and susceptibility to preterm labor. These findings imply that not only the immune system in the mother, but also that in the fetus or placenta contribute the innate immune response in preventing adverse outcomes in pregnancy. One study evaluated infants' genomic DNA and showed that infants who carried two polymorphic TLR-2 alleles (-16934TA/AA and 2258GA/AA) had significantly shorter gestational ages.92 Another study conducted within the Finish population found that G allele in TLR-4 299, both in infants and mothers, was associated with preterm labor.93

Bacterial vaginosis (BV), known to induce preterm birth, is also reported to be associated with TLR-4 polymorphisms. One study found T allele for TLR-4 399 was significantly less common in women with BV compared with women without BV.94 Another study showed G allele for TLR-4 896, which is known to impair responses to LPS, was associated with an increase in vaginal pH, Gardnerella vaginalis levels and concentration of anaerobic gram-negative rods.95

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References

There is growing evidence that TLR recognition and response occurs not only in immune cells but also in non-immune cells such as the trophoblast. While we have recognized the importance of TLR in the placenta, more specifically the trophoblast, to the defense against pathogens, the role of these receptors in establishing tolerance to the growing fetus is still unknown. It is intriguing to speculate that TLR at the maternal-fetal interface may play a role in establishing normal pregnancy, given the fact that commensal bacteria, which may potentially be bound to the TLR, are present in the reproductive tract, although further studies are required to elucidate this hypothesis. It is also still unclear what regulates the expression pattern and functional activity of TLR during pregnancy, either in physiological or pathological conditions.

Our studies provide an alternative perspective on the role of the maternal innate immune system and its interactions with the trophoblast during pregnancy. The trophoblast, upon identification of potentially dangerous molecular signatures, signals the maternal immune system, which responds with coordinate actions. These cells cross talk to jointly protect against infectious microorganisms. What was originally proposed to be only a graft-host interaction should now include a supportive regulatory interaction between the trophoblast and the maternal immune system. Further studies on TLR at the maternal-fetal interface will shed light on how the balance between tolerance to the allergenic fetus and host defense against possible pathogens is maintained.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of the Innate Immune System in Pregnancy
  5. Redefining Medawar's Hypothesis
  6. Infection and Pregnancy
  7. Toll-like Receptors
  8. Toll-like Receptor Signaling
  9. Toll-like Receptor Expression in the Placenta
  10. Toll-like Receptor Signaling and Function at the Maternal-fetal Interface
  11. Toll-like Receptor Signaling Modulates Immune Cell Function
  12. Bacterial Effects on Pregnancy: Toll-like Receptor-2 and -4 In Vivo Response
  13. Viral Effects on Pregnancy: Toll-like Receptor 3 In Vivo Response
  14. Outcome of Infection and Toll-like Receptor Ligation on Fetal Development
  15. Toll-like Receptor Expression at the Maternal-fetal Interface in Pregnancy Complications
  16. Toll-like Receptor Polymorphisms and Pregnancy Disorders
  17. Summary
  18. References