Edited by: Jerrold Weiss, University of Iowa, United States
Reviewed by: Jaya Talreja, Wayne State University School of Medicine, United States; Chaofeng Han, Second Military Medical University, China
This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Unlike organ transplants where an immunosuppressive environment is required, a successful pregnancy involves an extremely robust, dynamic, and responsive maternal immune system to maintain the development of the fetus. A specific set of hormones and cytokines are associated with a particular stage of pregnancy. Any disturbance that alters this fine balance could compromise the development and function of the placenta. Although there are numerous underlying causes of pregnancy-related complications, untimely activation of Toll-like receptors (TLR), primarily TLR4, by intrauterine microbes poses the greatest risk. TLR4 is an important Pattern Recognition Receptor (PRR), which activates both innate and adaptive immune cells. TLR4 activation by LPS or DAMPs leads to the production of pro-inflammatory cytokines via the MyD88 dependent or independent pathway. Immune cells modulate the materno–fetal interface by TLR4-mediated cytokine production, which changes at different stages of pregnancy. In most pregnancy disorders, such as PTB, PE, or placental malaria, the TLR4 expression is upregulated in immune cells or in maternal derived cells, leading to the aberrant production of pro-inflammatory cytokines at the materno–fetal interface. Lack of functional TLR4 in mice has reduced the pro-inflammatory responses, leading to an improved pregnancy, which further strengthens the fact that abnormal TLR4 activation creates a hostile environment for the developing fetus. A recent study proposed that endothelial and perivascular stromal cells should interact with each other in order to maintain a homeostatic balance during TLR4-mediated inflammation. It has been reported that depleting immune cells or supplying anti-inflammatory cytokines can prevent PTB, PE, or fetal death. Blocking TLR4 signaling or its downstream molecule by inhibitors or antagonists has proven to improve pregnancy-related complications to some extent in clinical and animal models. To date, there has been a lack of knowledge regarding whether TLR4 accessories such as CD14 and MD-2 are important in pregnancy and whether these accessory molecules could be promising drug targets for combinatorial treatment of various pregnancy disorders. This review mainly focuses on the activation of TLR4 during pregnancy, its immunomodulatory functions, and the upcoming advancement in this field regarding the improvement of pregnancy-related issues by various therapeutic approaches.
Pregnancy is an immunologically unique state owing to the fact that it requires the maternal immune system to be highly active so as to fight the upcoming intrauterine microbial challenges, but it is also simultaneously required to be immunosuppressed to maintain the semi-allogenic fetal development (
The
Toll-like receptors (TLRs) comprise of an extracellular domain, including leucine-rich repeats and a Toll/interleukin-1 receptor (TIR) domain at the cytoplasmic end. Following ligand recognition, TLRs relay the signaling either via the intracellular signaling adapter protein, the myeloid differentiation factor 88 (MyD88)-dependent pathway, or the MyD88-independent pathway, which is also known as the TLR-mediated TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathway. The MyD88-dependent pathway leads to the activation of early phase nuclear factor-κB (NF-κB), resulting in the production of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α. The TRIF-dependent pathway generates Type I IFNs (IFNα /β) through interferon regulatory factor (IRF-3) and via activation of late-phase NF-κB (
Proper release of these cytokines by the activated leukocytes or uterine epithelial cells plays a key role in attaining a successful pregnancy by facilitating the fetus implantation. But there is increasing evidence to suggest that uncontrolled activation of TLRs—either on leukocytes or uterine epithelial and stromal cells, specifically TLR4—at the materno-uterine junction is associated with pregnancy-related problems (
TLR4 in itself is unable to recognize LPS, and it therefore requires numerous other proteins for ligand recognition. The LPS-binding protein (LBP) is one such soluble plasma protein that first interacts with LPS and then transfers it to a cluster of differentiation 14 (either sCD14 or membrane bound). CD14 is a GPI-linked protein that is also one of the PRRs that can bind to the LPS-LBP complex; finally, it also chaperones the LPS molecule to MD-2/TLR4 signaling complex. Myeloid differentiation 2 (MD-2) is an adapter protein that directly recognizes and binds to the conserved lipid A moiety of LPS (
TLR4, the first identified human Toll-like receptor, is the only TLR that can signal via an MyD88-dependent as well as an MyD88-independent manner. It acts as a specific receptor for gram-negative bacterial lipopolysaccharide (LPS) and can also bind DAMPs, such as hyaluronic acid and β-defensin 2, fibrinogen, and heat shock proteins hsp60 and hsp70 (
TLR4 Signaling pathway. The LPS Binding Protein (LBP) binds to LPS and transfers it to CD14 or MD-2, which are the accessory proteins involved in the ligand recognition, dimerization, and endocytosis of TLR4. TLR4, upon dimerization, can signal via two separate pathways, the MyD88-dependent and the MyD88-independent pathway. The MyD88-dependent pathway involves the activation of IRAKs and TRAF6, which results in the phosphorylation of transcription factors, such as NF-κB and AP-1. These transcription factors upon phosphorylation translocate to the nucleus and are involved in triggering the transcription of pro-inflammatory cytokine genes. The MyD88-independent pathway, or the TRIF-dependent pathway, however, involves TRAF3 for the activation of transcription factor IRF-3, which favors the production of Type I interferons, such as IFN α, β.
There are numerous reports that emphasizes the role of immune activation in the intestinal and respiratory tract, and a wealth of knowledge is currently focused on uterine epithelial cells of the female reproductive tract (FRT) being an essential immunological site (
After the dimerization of TLR4 on ligand binding, MyD88 is recruited, and it interacts via its TIR domain to the cytoplasmic region of TLR4 through a homophilic interaction. Several other accessory molecules are also employed, including various IL-1 receptor-associated kinases (IRAKs), TRAFs, and mitogen-activated protein kinases (MAPKs). Next, NF-κB is activated and translocated to the nucleus via initiating the degradation of its inhibitory protein Iκ-Bα by inhibitory kappa B kinase (IKK). Activating protein-1 (AP-1) is one of the transcription factors that is activated by MAPKs (
TLR4/TRIF dependent signaling is only initiated after the receptor complex is internalized into the endosomes. Only TLR3 and TLR4 utilizes this pathway, involving the participation of TRIF and IRF-3 and resulting in the production of type I interferons (IFN) along with pro-inflammatory cytokines. They have the capability to stimulate IFN-β and Interferon-inducible genes in
Histological and functional changes of different parts of the female reproductive tract involving the perimetrium, myometrium, endometrium, cervix, and vagina take place throughout normal pregnancy. Several pregnancy-related tissues are also formed, including the amnion, chorion, and placenta, to support the development of the fetus. Any dysregulation in the usual scenario results in adverse pregnancy outcomes. Hence, in the current review, we have focused on the investigations that have been carried out to look into the function and expression profile of TLR4 during the course of pregnancy, exploring specific materno–fetal tissues of the female reproductive tract that have a close relationship with the developing embryo (
Schematic showing distribution of immune cells across materno–fetal interface during early pregnancy. Macrophages, Treg cells, and dendritic cells are present in the myometrium as well as decidua, while uterine natural killer (uNK) cells are restricted to decidua. The effective crosstalk between various immune cells and extravillous trophoblast cells creates an immunosuppressive environment and helps in the formation of various pregnancy-related tissues, both of which are essential for a successful pregnancy. Extravillous cytotrophoblasts enter the decidua to reach out to maternal spiral arteries for obtaining required nutrient to support developing embryo. Other nutrients, gas, and waste exchange happens via placental villus, which interacts with the maternal blood directly. The villus has double layer of cells consisting of syncytiotrophoblasts and cytotrophoblasts. It encloses the fetal blood vessels along with fibroblasts and fetal macrophages (Hofbauer cells). Immune cells as well as placental cells protect the fetus by expressing PRRs, such as Toll-like receptors, on their surface throughout the pregnancy.
The developing embryo is protected from the surrounding environment effectively by the placenta. Numerous PRRs are contemplated to take part in this interface, including Nod-like receptors (NLRs) and TLRs (
The differential expression of TLRs persists till the end of the second trimester. There are various reports that suggest that TLR4 is expressed during the second and third trimester in human placentas obtained from normal and preterm pregnancies. The expression of TLR4 has also been found in the syncytiotrophoblast layer by the third trimester. These studies have signified that placental cells can effectively counter the intrauterine infections (
Chorioamnionitis is characterized by the inflammatory response generated in the amnion and chorion membranes by the invading pathogenic microbes, resulting in preterm labor (
A recent report demonstrated that human fetal membranes and neutrophils that interact directly, and LPS-stimulated factors originating from the fetal membrane, can effectively recruit, and trigger neutrophils to induce inflammatory cytokines and helps them build neutrophil extracellular traps. The effect of TLR activation in preterm infants has also been studied by checking the level of the immunomodulatory factor, such as cAMP concentration in cord blood samples along with peripheral blood samples of preterm babies for the first month after delivery (
Decidua harbors most of the immune cells, which have the capability to generate an instant immune response against invading pathogens. Immune cells, such as macrophages, dendritic cells, uterine Natural Killer (uNK) cells, and Regulatory T cells, present in decidua differentially express TLR2 and TLR4 on their surface during pregnancy (
The host graft model of pregnancy is an old paradigm that suggest that immune cells recognize the fetus as semi-allogenic and hence try to eliminate it. In the current school of thought, however, the immune cells facilitate the implantation, formation, and development of the blastocyst for the sustenance of the pregnancy. In the normal condition, there are three immunological stages: (i) the pro-inflammatory condition in the decidua that aids in implantation and placentation; (ii) the growth of the fetus occurs in an anti-inflammatory environment; and (iii) there is finally a change back to the pro-inflammatory state for parturition (
Immunological stages of pregnancy: during first trimester of pregnancy, the inflammatory response is required for blastocyst implantation.
Fetus implantation in the early stages of pregnancy triggers the immune response at the junction of the decidual endometrium and extravillous trophoblast (EVT). Early contact of EVT with the maternal cells activates the immune system, primarily the innate immunity (
Natural Killer (NK) cells were initially derived by their cytotoxic activity against transformed cells. These cells have a unique ability to produce cytokines and perform cytotoxic functions other than T and B cells of lymphocyte origin.
Uterine NK cells are similar to systemic NK cells, but they do not express CD16 on their surfaces. They are translocated to the endometrium lining and placenta by the chemokine secreted from trophoblast cells. Uterine NK (uNK) cells are different in that they are highly granulated and are considered to play an essential role in maintaining a successful pregnancy by cytokine production in a temporal manner (
In contrast to inflammatory cells, there is an abundant population of decidual macrophages, and these are critical to maintaining pregnancy after successful implantation. Decidual macrophages express CD206 and CD209 molecules on the surface along with CD11c hi/lo antigen. These cells act as antigen-presenting cells to innate (NK cells) and adaptive immune (T cells) cells at the materno–fetal interface during early pregnancy. Unlike circulating macrophages, decidual macrophages have a more M2-like phenotype and perform a “cleanup” function of apoptotic cells to prevent pro-inflammatory condition in the decidua (
Immunology during pregnancy is similar to tumor immunology. In cancer, the adaptive immunity plays a critical role in graft rejection, but cancer cells modulate the immune cells for its establishment. As opposed to rejection, maintaining pregnancy is also a kind of allograft tolerance (
The temporal existence of Treg cells is regulated by TLR4 expression, which is upregulated during early pregnancy in decidual stromal cells and thus decreases the Treg population. This increased TLR4 signaling inhibits the transcription factor Foxp3, which in turn reduces regulatory T-cell polarization (
During normal pregnancy, a large number of cytokines and chemokines are secreted by trophoblasts, which helps in the proper implantation of the embryo on the uterine wall. These cytokines also help in the training of immune cells that are essential for the establishment of different stages of pregnancy (
TLR2 and TLR4 are widely expressed on various innate immune cells, including decidual macrophages and dendritic cells. Along with these immune cells, TLR4 is reported to express in decidual cells during the first trimester, EVTs, Villous cytotrophoblasts, and hofbauer cells, though not in syncytiotrophoblasts (
TLR4 expression is found in various types of cells and at different time points. Any changes in this expression or perturbation in signaling causes pregnancy disorders like preterm birth, preeclampsia, and abortion. Recently, TLR4-mediated IFN-β production and its role in pregnancy has been widely elucidated. There is an increase in the production of IFN-β by trophoblast cells upon LPS-mediated TLR4 activation via the MyD88-independent (TRIF-TBK1-IRF-3 axis) pathway. Increased IFN-β induces downstream interferon stimulating genes and also triggers negative regulators of the TAM receptor, such as Mer and Axl. Absence of these negative regulators were found to be detrimental, as fetal rejection occur in the presence of increased IFN-β in the placenta (
A major problem of neonatal mortality is due to preterm labor (gestation at <37 weeks). PTB is marked by increased pro-inflammatory factors due to local or systemic infection or inflammation, such as infection in intra-amniotic (chorioamnionitis) or periodontitis, which interacts via maternal sera (
Alteration of TLR4 signaling in pregnancy disorders.
Preeclampsia is a heterogeneous disorder caused after the 20th week of pregnancy due to local or systemic abnormalities. There is much evidence to suggest that TLR signaling activation could cause PE in many ways (
Other than different maternal syndromes, which are associated with PE, serum TLR4 and NF-κB p65 could be used as a biomarker for predicting cytokine environment and its influence on the immune cells (
Parasitic infection caused by
Under the same scenario, it was observed that the TLR4 receptor behaves differently on the maternal and fetal interface. Maternal TLR4 is involved in the pathogenesis of malaria severity, while fetal TLR4 has a protective response against placental parasite burden, which could be due to the paternal allele for
Various TLR4 antagonist and inhibitors have been developed that are currently in different phases of clinical trial for diseases other than pregnancy. There are few options that are currently being studied for immune modulation and inhibition of TLR expression for pregnancy-related disorders. The association of TLR4 was studied in women with aPL (antiphospholipid antibodies), which activate the TLR4 pathway and the inflammatory response in trophoblasts leading to miscarriages, PE, and PTB (
Cytokine-suppressive anti-inflammatory drugs (CSAID's) are a novel group that target the NF-κB and MAP Kinase pathways, making them more effective than Non-Steroidal Anti-inflammatory Drugs (NSAID). CSAIDs that can selectively inhibit TAK1 and the IKK complex are well-studied in animal models, which has resulted in the reduction of cytokines and prostaglandin levels (
List of drugs targeting TLR4, and its downstream signaling molecules during pregnancy disorder.
Hydroxyquinone | Reduces production of TNF and endothelin-1 | PE | ( |
Asprin | Prevents endothelial dysfunction due to TNF | PE | ( |
Curcumin | Downregulates TLR4 expression and NF-κB mediated inflammatory response | PE | ( |
Vitamin D | Calcitriol can modulate innate as well as adaptive response (pro to anti- inflammatory) Decreases TLR4 expression | PTB, PE & spontaneous miscarriages PE | ( |
Rosiglitazone | Reduces TLR4 mediated inflammation Increases antioxidant response by NRF-2 and HO-1 | PTB | ( |
Progesterone | Inhibit TLR4 expression in macrophages Promotes Th2 differentiation Induces tolerance at materno–fetal junction | PE | ( |
Maintain Th17/Treg cell balance | Infection of |
( |
|
IL-10 | Maintains anti-inflammatory condition in decidua | PTB | ( |
NEMO-binding Domain Inhibitor | Reduces Prostaglandin E2 (PGE2) in LPS and |
PTB | ( |
Parthenolide | Reduces inflammatory gene expression in patient derived choriodecidual cells. Decreases TNF-α and COX-2 expression in human urothelial cell stimulated with TNF-α. | PTB | ( |
TPCA-1 | Similar effect as of parthenolide. Reduction in PGE2 level in LPS stimulated ovine pregnancy model | PTB | ( |
Various Drugs that target TLR4 pathway in pregnancy disorders: drugs and anti-inflammatory agents that target TLR4 pathway and its downstream molecules during infection induced preterm birth. Hormones and drugs targeting TLR4 expression help in switching the pro-inflammatory environment to anti-inflammatory in various pregnancy disorders. TNF inhibitors reduce the increased TNF production during altered TLR4 activation in preeclampsia.
Detailed study of spatiotemporal expression of TLRs during normal pregnancy and related disorders using various model systems has increased our understanding of placental infections and furthered our development of strategies to overcome the adverse pregnancy outcomes. Activation of innate immune PRR through TLR4 at the materno–fetal interface ensures that the developing fetus is protected from invading pathogens at early stage of pregnancy. But uncontrolled activation of TLR4 has been proven to trigger chronic inflammation and to result in loss of pregnancy. Hence, increased levels of TLR4 on leucocytes or cells of maternal and fetal origin could be used as a biomarker for pregnancy disorders. Many studies have shown the involvement of innate immune cells for sustaining a successful pregnancy.
It is not yet clear how the TLR4 expression pattern alters during various stages of pregnancy and in what way its uncontrolled activation on immune or other decidual cells at the maternal–fetal interface leads to various pregnancy failures. Addressing this issue may help in developing certain clinical diagnostic markers as well as specific antagonists targeting either TLR4 specifically or its downstream effector molecules for improving pregnancy outcomes.
PF and VS contributed equally in writing the review. Conception of Idea was done by SC, PF, and VS. Manuscript writing and editing was done by all the authors.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are thankful to CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata and National Centre for Cell Science (NCCS), Pune for providing infrastructure facilities.