Article

Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts

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Abstract

Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.

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... In the current study, we analyzed the full spectrum of CD127+ ILCs and CD94+ NK cells in the human intestinal lamina propria from healthy donors and Crohn's disease patients. Using scRNAseq we identified CD127+CD94+ ILC1s that highly expressed granulysin, a cytotoxic molecule that is linked to bacterial lysis and/or chemotaxis of monocytes [12][13][14][15][16] . These cells were isolated using antibodies against cell surface molecules identified with scRNAseq and further characterization confirmed that these cells highly express granulysin protein, indicating they are in an activated state in vivo. ...
... IL2RB, encoding for the beta subunit of the IL-2 and IL-15 receptor, was expressed in both populations A and B, but highest in NK cells (Fig. 5a). Stimulation with IL-15 has been shown to induce granulysin expression and secretion 15 . Indeed, cultures of freshly isolated CD127+CD94+ CD117− ILCs (population B) from LPMCs on OP-9 cells in the presence of low IL-2 and IL-15, released substantial amounts of granulysin protein as compared to NK cells and ILC3s (Fig. 5b) whereas their intracellular protein expression remained high after culture (Fig. 5c). ...
... Although granulysin was secreted, the expression of intracellular granulysin remained very high, suggesting a constant production and refill of granulysin within these cells. Granulysin was shown to induce bacterial lysis, either alone in high concentrations, or synergistically with granzyme B or perforin [12][13][14][15][16] . Defects in the epithelial barrier during active inflammation in Crohn's disease patients can result in infiltration of bacteria into the lamina propria. ...
Article
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Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.
... While the normal physiological role of dNK cells appears to be in tissue remodeling during early pregnancy, including spiral artery remodeling and regulation of EVT invasion, the question remains as to what happens in times of intrauterine infection? A recent study elegantly demonstrated that dNK cells are able to selectively kill EVT infected with Listeria monocytogenes (Crespo et al., 2020). On recognition of an infected EVT cell, the dNK cells can inject cytotoxic granules into the EVT via nanotubes, therefore clearing infection and leaving uninfected EVT to continue to maintain a successful pregnancy (Crespo et al., 2020). ...
... A recent study elegantly demonstrated that dNK cells are able to selectively kill EVT infected with Listeria monocytogenes (Crespo et al., 2020). On recognition of an infected EVT cell, the dNK cells can inject cytotoxic granules into the EVT via nanotubes, therefore clearing infection and leaving uninfected EVT to continue to maintain a successful pregnancy (Crespo et al., 2020). Whether this is a general dNK cell phenomenon or restricted to one of the identified dNK cell subsets is not yet known. ...
Article
Decidual leukocytes make up approximately 30% of all decidual stromal cells in early pregnancy, of which 70% are uterine natural killer (uNK) cells. uNK cells are phenotypically distinct from peripheral blood NK cells, being CD56[Formula: see text]CD16[Formula: see text]. A recent single-cell sequencing project of the decidua identified three subsets of uNK cells, but we are yet to determine how they differ functionally. Several roles for uNK cells in implantation are starting to emerge including biosensing of poor-quality embryos, killing of bacteria infected trophoblast, spiral artery remodeling, and regulation of trophoblast invasion. Altered numbers of uNK cells have been identified in several pathological conditions, but whether this is causative of the condition is currently unclear.
... In other systems, nanotube-mediated transport of small cytosolic molecules depends on the cationic cytoskele-ton, while the transportation of organelles depends on microtubules. 138 Thus, we hypothesize that nanotubes may play a central role in promoting cell cytolysis, maintaining the interaction with target cells, and thereby killing target cells. Another speculative effect may allow effector cells to patrol multiple target cells at the same time, allowing them to interact with one cell while maintaining interaction with previously encountered cells. ...
... 93 (iv) Crespo et al. revealed that human dNK cells with high expression of GNLY can selectively transfer GNLY via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes without killing the trophoblast, thereby protecting against infection while leaving the maternal-fetal barrier intact. 138 (v) Schiller et al. demonstrated that class I MHC molecules can be directly transferred from one cell to another through TNTs, and the transfer rate can be significantly reduced by inhibiting actin polymerization, while overexpression of the nanotube-inducing protein LST1 can promote the transfer rate. This discovery provided a new strategy for enhancing antigen presentation and enhancing immune effects in immunotherapy 64 ( Figure 6). ...
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Direct intercellular communication is an important prerequisite for the development of multicellular organisms, the regeneration of tissue, and the maintenance of various physiological activities. Tunnel nanotubes (TNTs), which have diameters of approximately 50-1500 nm and lengths of up to several cell diameters, can connect cells over long distances and have emerged as one of the most important recently discovered types of efficient communication between cells. Moreover, TNTs can also directly transfer organelles, vehicles, proteins, genetic material, ions, and small molecules from one cell to adjacent and even distant cells. However, the mechanism of intercellular communication between various immune cells within the complex immune system has not been fully elucidated. Studies in the past decades have confirmed the existence of TNTs in many types of cells, especially in various kinds of immune cells. TNTs display different structural and functional characteristics between and within different immunocytes, playing a major role in the transmission of signals across various kinds of immune cells. In this review, we introduce the discovery and structure of TNTs, as well as their different functional properties within different immune cells. We also discuss the roles of TNTs in potentiating the immune response and their potential therapeutic applications.
... 146 However, very recently, dNK cells were shown to kill L. monocytogenes from within EVT, by injecting anti-microbial Granulysin through nanotubes. 148 NK cells may influence the response of myeloid cells to L. monocytogenes by secreting IL-10. Indeed, L. monocytogenes stimulated IL-10 production by mouse splenic, liver, and pbNK cells at 72 and 96 h post infection, but not at 24 h, which coincides with the peak of IFN-ɣ production by NK cells. ...
... However, temporal changes in the frequencies of the two types of cells in the decidua suggest that tissue-resident T cells may be more important in late gestation, while NK cells may specialize in certain defense mechanisms, like the recently reported nanotube-mediated transfer of granulysin into Listeria-infected trophoblast. 148 Decidual CD8 + T cells are able to degranulate and produce pro-inflammatory cytokines in response to stimulation and are therefore competent in their response to pathogen invasion. 319 How decidual NK and T cells operate during infections and which cells they interact with can be inferred by looking at the sets of MHC class I molecules expressed on cells in the decidua. ...
Article
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Many maternal immune cells populate the decidua, which is the mucosal lining of the uterus transformed during pregnancy. Here, abundant natural killer (NK) cells and macrophages help the uterine vasculature adapt to fetal demands for gas and nutrients, thereby supporting fetal growth. Fetal trophoblast cells budding off the forming placenta and invading deep into maternal tissues come into contact with these and other immune cells. Besides their homeostatic functions, decidual NK cells can respond to pathogens during infection, but in doing so, they may become conflicted between destroying the invader and sustaining fetoplacental growth. We review how maternal NK cells balance their double duty both in the local microenvironment of the uterus and systemically, during toxoplasmosis, influenza, cytomegalovirus, malaria and other infections that threat pregnancy. We also discuss recent developments in the understanding of NK-cell responses to SARS-Cov-2 infection and the possible dangers of COVID-19 during pregnancy.
... Activation of dNK cells could increase their abilities to respond to placental HCMV infection and limit the subsequent virusinduced placental pathology [37]. In addition, dNK cells can release IFN-γ to limit HIV replication in decidual macrophages and directly transfer granulysin (GNLY) to fetal cells by nanotubes, killing intracellular Listeria monocytogenes without killing the host cell [38,39]. Taken together, dNK cells may reduce bacterial loads at the maternal-fetal interface as a result of their cytotoxic functions. ...
... This lack may be related to the difficulty in purifying enough primary dNK cells to meet the requirements of deep sequencing. It is also challenging to conduct gene knockout experiments in primary dNK cells [38]. Additionally, many small molecules including circular RNAs and methylation modifiers (such as N6-methyladenosine and 5-hydroxymethylcytosine) have not been examined in dNK cells. ...
Article
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The critical immune effectors, including T, B, and natural killer (NK) cells, dendritic cells, and macrophages participate in regulating immune responses during pregnancy. Among these immune cells, decidual NK (dNK) cells are involved in key placental development processes at the maternal–fetal interface, such as uterine spiral artery remodeling, trophoblast invasion, and decidualization. Mechanistically, dNK cells significantly influence pregnancy outcome by secreting cytokines, chemokines, and angiogenic mediators and by their interactions with trophoblasts and other decidual cells. MicroRNAs (miRNAs) are small non-coding RNA molecules that participate in the initiation and progression of human diseases. Although the functions of circulating miRNAs in pathological mechanism has been extensively studied, the regulatory roles of miRNAs in NK cells, especially in dNK cells, have been rarely reported. In this review, we analyze the effects of miRNA regulations of dNK cell functions on the immune system during gestation. We discuss aberrant expressions of certain miRNAs in dNK cells that may lead to pathological consequences, such as recurrent pregnancy loss (RPL). Interestingly, miRNA expression patterns are also different between dNK cells and peripheral NK (pNK) cells, and pNK cells in the first- and third‐trimester of gestation. The dysregulation of miRNA plays a pivotal regulatory role in driving immune functions of dNK and pNK cells. Further understanding of the molecular mechanisms of miRNAs in dNK cells may provide new insights into the development of therapeutics to prevent pregnancy failure.
... For example, administration of live pathogen, purified microbial ligands and purified cytokines can each trigger preterm birth in rodents, and larger animals including non-human primates (39,40). Recent studies highlight the unique transfer of antimicrobial peptides between decidual natural killer cells and trophoblast cells to sustain immunity at the maternal fetal interface (41). Increased susceptibility of women during pregnancy to specific prenatal pathogens, such as Listeria monocytogenes, is also recapitulated in animals where fetal wastage and congenital fetal invasion occur with experimental infection during pregnancy (31,42,43). ...
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In the fifteen minutes it takes to read this short commentary, more than 400 babies will have been born too early, another 300 expecting mothers will develop preeclampsia, and 75 unborn third trimester fetuses will have died in utero (stillbirth). Given the lack of meaningful progress in understanding the physiological changes that occur to allow a healthy, full term pregnancy, it is perhaps not surprising that effective therapies against these great obstetrical syndromes that include prematurity, preeclampsia, and stillbirth remain elusive. Meanwhile, pregnancy complications remain the leading cause of infant and childhood mortality under age five. Does it have to be this way? What more can we collectively, as a biomedical community, or individually, as clinicians who care for women and newborn babies at high risk for pregnancy complications, do to protect individuals in these extremely vulnerable developmental windows? The problem of pregnancy complications and neonatal mortality is extraordinarily complex, with multiple unique, but complementary perspectives from scientific, epidemiological and public health viewpoints. Herein, we discuss the epidemiology of pregnancy complications, focusing on how the outcome of prior pregnancy impacts the risk of complication in the next pregnancy — and how the fundamental immunological principle of memory may promote this adaptive response.
... Therefore, extravillous trophoblasts, which express high E-cadherin levels, are more permissive to initial L. monocytogenes uptake, but then restrict intracellular bacterial proliferation, a property not shared with third trimester SYN and sub-syncytial trophoblasts (our study). In addition, decidual natural killer cells, which are in close proximity to extravillous trophoblasts at the chorionic villi anchoring sites, were recently shown to transfer granulysin into infected extravillous trophoblasts leading to the restriction of L. monocytogenes cytosolic growth (93). ...
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The placenta controls the growth of the fetus and ensures its immune protection. Key to these functions, the syncytiotrophoblast (SYN) is a syncytium formed by fusion of underlying mononuclear trophoblasts. The SYN covers the placental surface and is bathed in maternal blood to mediate nutritional and waste exchanges between the mother and fetus. The bacterial pathogen Listeria monocytogenes breaches the trophoblast barrier and infects the placental/fetal unit resulting in poor pregnancy outcomes. In this work, we analyzed the L. monocytogenes intracellular lifecycle in primary human trophoblasts. In accordance with previous studies, we found that the SYN is 20-fold more resistant to infection compared to mononuclear trophoblasts, forming a protective barrier to infection at the maternal interface. We show for the first time that this is due to a significant reduction in L. monocytogenes uptake by the SYN rather than inhibition of the bacterial intracellular division or motility. We here report the first transcriptomic analysis of L. monocytogenes-infected trophoblasts (RNA sequencing). Pathway analysis showed that infection upregulated TLR2, NOD-like, and cytosolic DNA sensing pathways, as well as downstream pro-inflammatory circuitry (NF-κB, AP-1, IRF4, IRF7) leading to the production of mediators known to elicit the recruitment and activation of maternal leukocytes (IL8, IL6, TNFα, MIP-1). Signature genes associated with poor pregnancy outcomes were also upregulated upon infection. Measuring the release of 54 inflammatory mediators confirmed the transcriptomic data and revealed sustained production of tolerogenic factors (IL-27, IL-10, IL-1RA, TSLP) despite infection. Both the SYN and mononuclear trophoblasts produced cytokines, but surprisingly, some cytokines were predominantly produced by the SYN (IL-8, IL-6) or by non-fused trophoblasts (TNFα). Collectively, our data support that trophoblasts act as placental gatekeepers that limit and detect L. monocytogenes infection resulting in a pro-inflammatory response, which may contribute to the poor pregnancy outcomes if the pathogen persists.
... In addition to their role during viral infection, dNK cells are able to balance the contradictory demands of fetal tolerance and protection against bacterial infection. Recent work by the group of Lieberman demonstrated that dNK cells are able to kill intracellular Listeria monocytogenes in trophoblasts and dMϕ, through the transfer of granulysin cytotoxic molecules via tunneling nanotube (75). ...
Article
The placenta, the first and largest organ to develop after conception, not only nurtures and promotes the development of the conceptus, but, it also functions as a barrier against invading pathogens. Early phases of pregnancy are associated with expansion of specific subsets of Natural Killer cells (dNK) and macrophages (dMφ) at the maternal uterine mucosa, the basal decidua. In concert with cells of fetal origin, dNK cells, and dMφ orchestrate all steps of placenta and fetus development, and provide the first line of defense to limit vertical transmission. However, some pathogens that infect the mother can overcome this protective barrier and jeopardize the fetus health. In this review, we will discuss how members of the classical TORCH family (Toxoplasma, Other, Rubella, Cytomegalovirus, and Herpes simplex virus) and some emerging viruses (Hepatitis E virus, Zika virus, and SARS-CoV2) can afford access to the placental fortress. We will also discuss how changes in the intrauterine environment as a consequence of maternal immune cell activation contribute to placental diseases and devastating pregnancy outcomes.
... Crespo et al. collected placental and decidual samples (6-12 weeks) and revealed that the antimicrobial peptide granulysin was highly expressed in human dNK cells. Antimicrobial peptide granulysin was selectively transferred via nanotubes to extravillous trophoblasts (EVTs) to kill intracellular Listeria monocytogenes without killing the trophoblasts (17,18). Also, when pregnant women were infected with human cytomegalovirus (HCMV), the expression of killer cell immunoglobulin-like receptor 2DS1 (KIR2DS1) by dNK cells increased the ability to prevent placental HCMV infection. ...
Article
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The immune system recognizes and attacks non-self antigens, making up the cornerstone of immunity activity against infection. However, during organ transplantation, the immune system also attacks transplanted organs and leads to immune rejection and transplantation failure. Interestingly, although the embryo and placenta are semi-allografts, like transplanted organs, they can induce maternal tolerance and be free of a vigorous immune response. Also, embryo or placenta-related antibodies might adversely affect subsequent organ transplantation despite the immune tolerance during pregnancy. Therefore, the balance between the immune tolerance in maternal-fetal interface and normal infection defense provides a possible desensitization and tolerance strategy to improve transplantation outcomes. A few studies on mechanisms and clinical applications have been performed to explore the relationship between maternal-fetal immune tolerance and organ transplantation. However, up to now, the mechanisms underlying maternal-fetal immune tolerance remain vague. In this review, we provide an overview on the current understanding of immune tolerance mechanisms underlying the maternal-fetal interface, summarize the interconnection between immune tolerance and organ transplantation, and describe the adverse effect of pregnancy alloimmunization on organ transplantation.
... For example, dNKs display an elegant and unique mechanism to control Listeria monocytogenes through the antimicrobial peptide granulysin. Granulysin, stored in intracellular granules, can transfer to infected EVTs via nanotubes to selectively kill intracellular Listeria, without killing the infected EVTs [155]. Here we take human cytomegalovirus (HCMV) as an example to summarize the interactions between intrauterine infection and uNK cells during pregnancy. ...
Article
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Tissues are the new frontier of discoveries in immunology. Cells of the immune system are an integral part of tissue physiology and immunity. Determining how immune cells inhabit, housekeep, and defend gut, lung, brain, liver, uterus, and other organs helps revealing the intimate details of tissue physiology and may offer new therapeutic targets to treat pathologies. The uterine microenvironment modulates the development and function of innate lymphoid cells [ILC, largely represented by natural killer (NK) cells], macrophages, T cells, and dendritic cells. These immune cells, in turn, contribute to tissue homeostasis. Regulated by ovarian hormones, the human uterine mucosa (endometrium) undergoes ~400 monthly cycles of breakdown and regeneration from menarche to menopause, with its fibroblasts, glands, blood vessels, and immune cells remodeling the tissue into the transient decidua. Even more transformative changes occur upon blastocyst implantation. Before the placenta is formed, the endometrial glands feed the embryo by histiotrophic nutrition while the uterine spiral arteries are stripped of their endothelial layer and smooth muscle actin. This arterial remodeling is carried out by invading fetal trophoblast and maternal immune cells, chiefly uterine NK (uNK) cells, which also assist fetal growth. The transformed arteries no longer respond to maternal stimuli and meet the increasing demands of the growing fetus. This review focuses on how the everchanging uterine microenvironment affects uNK cells and how uNK cells regulate homeostasis of the decidua, placenta development, and fetal growth. Determining these pathways will help understand the causes of major pregnancy complications.
... The placenta may harbor pathogenic microbes and potentially have its own microbiome. To fight placental infection, decidual natural killer cells protect the placenta through cytolytic effectors such as perforin and granzymes to selectively kill pathogens, sparing normal host cells thus protecting the placenta from intracellular infections (Crespo et al., 2020) such as Listeria monocytogenes that have been previously detected in placental trophoblasts (Cao & Mysorekar, 2014). However, this mechanism is said to be protective only when it occurs in earlier stages of pregnancy. ...
Article
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Preeclampsia is a devastating hypertensive pregnancy disorder that currently affects 2%–8% of pregnancies worldwide. It is associated with maternal and fetal mortality and morbidity and adverse health outcomes both in mom and offspring beyond pregnancy. The pathophysiology is not completely understood, and there are no approved therapies to specifically treat for the disease, with only few therapies approved to manage symptoms. Recent advances suggest that aberrations in the composition of the microbiome may play a role in the pathogenesis of various diseases including preeclampsia. The maternal and uteroplacental environments greatly influence the long‐term health outcomes of the offspring through developmental programming mechanisms. The current review summarizes recent developments on the role of the microbiome in adverse pregnancy outcomes with a focus on preeclampsia. It also discusses the potential role of the maternal microbiome in fetal programming; explores gut‐targeted therapeutics advancement and their implications in the treatment of preeclampsia. Preeclampsia is a devastating hypertensive pregnancy disorder with maternal and fetal mortality and morbidity, and the maternal and uteroplacental environments greatly influence the long‐term health outcomes of the mom and offspring. Recent advances suggest that aberrations in the composition of the microbiome may play a role in the pathogenesis of preeclampsia. The current review summarizes recent developments on the role of the microbiome in adverse pregnancy outcomes with a focus on preeclampsia and explores gut‐targeted therapeutics advancement and their implications in the treatment of preeclampsia.
... Studies suggest that the decidual composition of immune cells and their interaction with trophoblasts may alter their susceptibility to pathogens. For example, decidual NK cells possess the remarkable ability to transfer granulysins to trophoblasts to protect from L. monocytogenes infection without actively kill ing these cells 41 . These and other studies highlight the often unique and complex strategies of antimicrobial protection that exist at the maternal-fetal interface. ...
Article
Infections are a major threat to human reproductive health, and infections in pregnancy can cause prematurity or stillbirth, or can be vertically transmitted to the fetus leading to congenital infection and severe disease. The acronym ‘TORCH’ (Toxoplasma gondii, other, rubella virus, cytomegalovirus, herpes simplex virus) refers to pathogens directly associated with the development of congenital disease and includes diverse bacteria, viruses and parasites. The placenta restricts vertical transmission during pregnancy and has evolved robust mechanisms of microbial defence. However, microorganisms that cause congenital disease have likely evolved diverse mechanisms to bypass these defences. In this Review, we discuss how TORCH pathogens access the intra-amniotic space and overcome the placental defences that protect against microbial vertical transmission.
... Siewiera et al. provided the first evidence that dNK cells can clear HCMV-infected DSCs (94) and that HCMV-infected EVTs cannot be cleared (75). A recent study suggested that dNK cells killed bacteria in trophoblasts by transferring granulysin without killing placental cells (95). Conversely, viruses can induce expression of activating ligands (e.g., major histocompatibility class I polypeptide-related sequence A (MICA) and MICB) on the surface of infected cells that bind directly to activating NK receptors and promote NK-cell cytotoxicity (96). ...
Article
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Pregnancy is a unique type of immunological process. Healthy pregnancy is associated with a series of inflammatory events: implantation (inflammation), gestation (anti-inflammation), and parturition (inflammation). As the most abundant leukocytes during pregnancy, natural killer (NK) cells are recruited and activated by ovarian hormones and have pivotal roles throughout pregnancy. During the first trimester, NK cells represent up to 50–70% of decidua lymphocytes. Differently from peripheral-blood NK cells, decidual natural killer (dNK) cells are poorly cytolytic, and they release cytokines/chemokines that induce trophoblast invasion, tissue remodeling, embryonic development, and placentation. NK cells can also shift to a cytotoxic identity and carry out immune defense if infected in utero by pathogens. At late gestation, premature activation of NK cells can lead to a breakdown of tolerance of the maternal–fetal interface and, subsequently, can result in preterm birth. This review is focused on the role of dNK cells in normal pregnancy and pathological pregnancy, including preeclampsia, recurrent spontaneous abortion, endometriosis, and recurrent implantation failure. dNK cells could be targets for the treatment of pregnancy complications.
... During pregnancy, any dysregulation of immunity might affect placentation and thus fetal development (21,(58)(59)(60). Gestational immune adaptations are highly specialized to enable selective tolerance towards invading fetal cells, immunecompetence to overcome pathogenic invasion, and immunemediated support of establishing vascularization during placenta formation (61)(62)(63)(64). Imbalance of immunity is thought to hamper correct placentation and thus contribute to the etiology of preeclampsia (PE) (65,66). ...
Article
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Background: Pregnancy is a portentous stage in life, during which countless events are precisely orchestrated to ensure a healthy offspring. Maternal microbial communities are thought to have a profound impact on development. Although antibiotic drugs may interfere in these processes, they constitute the most frequently prescribed medication during pregnancy to prohibit detrimental consequences of infections. Gestational antibiotic intervention is linked to preeclampsia and negative effects on neonatal immunity. Even though perturbations in the immune system of the mother can affect reproductive health, the impact of microbial manipulation on maternal immunity is still unknown. Aim: To assess whether antibiotic treatment influences maternal immunity during pregnancy. Methods: Pregnant mice were treated with broad-spectrum antibiotics. The maternal gut microbiome was assessed. Numerous immune parameters throughout the maternal body, including placenta and amniotic fluid were investigated and a novel machine-learning ensemble strategy was used to identify immunological parameters that allow distinction between the control and antibiotic-treated group. Results: Antibiotic treatment reduced diversity of maternal microbiota, but litter sizes remained unaffected. Effects of antibiotic treatment on immunity reached as far as the placenta. Four immunological features were identified by recursive feature selection to contribute to the most robust classification (splenic T helper 17 cells and CD5+ B cells, CD4+ T cells in mesenteric lymph nodes and RORγT mRNA expression in placenta). Conclusion: In the present study, antibiotic treatment was able to affect the carefully coordinated immunity during pregnancy. These findings highlight the importance of inclusion of immunological parameters when studying the effects of medication used during gestation.
... The antibacterial and antiparasitic function of 9 kDa GRNLY has long been known, a property that has been confirmed and expanded in recent studies [33][34][35]. Our group has shown that recombinant GRNLY is capable of killing tumor cells in vitro through the mitochondrial apoptotic pathway [17,18,27]. ...
Article
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Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosylated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.
... GNLY and PRF1 were decreased in decidua of women with PE but, given the number, they represent two main cytolytic molecules. Additionally, we noticed that GNLY is a cytotoxic protein that is, besides in decidual lymphocytes, significantly expressed and visible as diffuse staining in the cytoplasm of EVT cells, which is consistent with other recent studies [56]. ...
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In our study, we aimed to establish expression of cytotoxic CD8+ T cells in the decidua basalis and the maternal peripheral blood (mPBL) of severe and mild preeclampsia (PE) and compare to healthy pregnancies. Decidual tissue and mPBL of 10 women with mild PE, 10 women with severe PE, and 20 age-matched healthy pregnancy controls were analyzed by double immunofluorescence and qPCR, respectively. By double immunofluorescence staining, we found a decreased total number of cells/mm2 in decidua basalis of granulysin (GNLY)+ (p ˂ 0.0001), granzyme B (GzB)+(p ˂ 0.0001), GzB+CD8+(p ˂ 0.0001), perforin (PRF1)+ (p ˂ 0.0001), and PRF1+CD8+ (p ˂ 0.01) in the severe PE compared to control group. Additionally, we noticed the trend of lower mRNA expression for GNLY, granzyme A (GZMA), GzB, and PRF1 in CD8+ T cells of mPBL in mild and severe PE, with the latter marker statistically decreased in severe PE (p ˂ 0.001). Forkhead box P3 (FOXP3) mRNA in CD8+ T cells mPBL was increased in mild PE (p ˂ 0.001) compared to controls. In conclusion, severe PE is characterized by altered expression of cytotoxic CD8+ T cells in decidua and mPBL, suggesting their role in pathophysiology of PE and fetal-maternal immune tolerance.
... The role of dNK cells in the adaptive immune response, particularly the production of proinflammatory and anti-inflammatory cytokines, has been characterized (5,6). It has been reported that dNK cells exhibit lower cytotoxicity, but higher secretion potential, than pNK cells in physiological settings (9)(10)(11). The dNK cells express a variety of surface receptors, such as NKp44 (CD336), NKp30 (CD337) and NKG2D (CD314) (1,2). ...
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Unexplained recurrent spontaneous abortion (URSA) is believed to be associated with impaired immunosuppression at the maternal-fetal interface, but the detailed molecular mechanism remains unclear. The ATP-adenosine metabolic pathway regulated by CD39/CD73 has recently been recognized to be important in immunosuppression. This study aimed to investigate the regulation of decidual natural killer (dNK) cells and fetal extravillous trophoblast (EVT) cells by CD39 and CD73 in URSA, as well as the possible regulatory mechanism of CD39/CD73 via the TGF-β-mTOR-HIF-1α pathway using clinical samples and cell models. Fewer CD39 ⁺ and CD73 ⁺ cells were found in the URSA decidual and villous tissue, respectively. Inhibition of CD39 on dNK cells transformed the cells to an activated state with increased toxicity and decreased apoptosis, and changed their cytokine secretion, leading to impaired invasion and proliferation of the co-cultured HTR8/SVneo cells. Similarly, inhibition of CD73 on HTR8/SVneo cells decreased the adenosine concentration in the cell culture media, increased the proportion of CD107a ⁺ dNK cells, and decreased the invasion and proliferation capabilities of the HTR8/SVneo cells. In addition, transforming growth factor-β (TGF-β) triggered phosphorylation of mammalian target of rapamycin (mTOR) and Smad2/Smad3, which subsequently activated hypoxia-inducible factor-1α (HIF-1α) to induce the CD73 expression on the HTR8/SVneo cells. In summary, reduced numbers of CD39 ⁺ and CD73 ⁺ cells at the maternal-fetal interface, which may be due to downregulated TGF-β-mTOR-HIF-1α pathway, results in reduced ATP-adenosine metabolism and increased dNK cytotoxicity, and potentially contributes to URSA occurrences.
... GNLY is an antimicrobial protein that forms pores in microbial membranes, allowing delivery of granzymes (such as GZMB, which we also found upregulated during CVR use).[36][37][38] A recent study highlighted the importance of GNLY in the female genital tract, showing that decidual NK cells transfer GNLY to bacterially infected placental trophoblasts, leading to clearance of the bacteria without death of the infected cell.39 The increased levels of GNLY we observed during the follicular phase and during CVR use may indicate recruitment of GNLY-expressing cells to the CVT or increased expression of GNLY by resident cells. ...
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Problem Changes in sex hormones during the menstrual cycle and contraceptive vaginal ring (CVR) use influence immunity within the female genital tract, but the magnitude of these effects and their anatomical location are unclear. Method of Study In a prospective study, 29 women were assessed at three time points: follicular phase, luteal phase, and one month after initiation of the ethinyl estradiol/etonogestrel CVR (NuvaRing®, Merck). We performed microarrays on endocervical cytobrushes and measured immune mediators in cervicovaginal fluid, adjusting for bacterial vaginosis and presence of blood. We compared these results to public gene expression data from the fallopian tubes, endometrium, endo‐ and ectocervix, and vagina. Results Immune‐related gene expression in the endocervix and immune mediators in cervicovaginal fluid increased during CVR use versus both menstrual phases, and in the follicular versus luteal phase. The antimicrobial protein granulysin was high during CVR use, intermediate in the follicular phase, and nearly absent from the luteal phase. Re‐analysis of public gene expression data confirmed increased immune‐related gene expression in the endocervix during the follicular phase. However, in the fallopian tube, endometrium and vagina, the follicular phase showed immunosuppression. Conclusions Immune‐related genes in the cervicovaginal tract were highest during CVR use, intermediate in the follicular phase, and lowest in the luteal phase. Granulysin is a potential biomarker of menstrual phase: frequently detected in follicular samples, but rare in luteal. Lastly, immunological differences between the follicular and luteal phases vary throughout the female genital tract.
... This indicates that tunnelling nanotubes may be relevant for this process (Supplementary Fig. S13). It has previously been shown that actin plays an important role in nanotube formation [85][86][87] . Therefore, we hypothesized that actin-driven protrusions from donor cells to nearby acceptor cells can facilitate transfer of Sterolight. ...
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Information about cholesterol subcellular localization and transport pathways inside cells is essential for understanding and treatment of cholesterol-related diseases. However, there is a lack of reliable tools to monitor it. This work follows the fate of Sterolight, a BODIPY-labelled sterol, within the cell and demonstrates it as a suitable probe for visualization of sterol/lipid trafficking. Sterolight enters cells through an energy-independent process and knockdown experiments suggest caveolin-1 as its potential cellular carrier. Intracellular transport of Sterolight is a rapid process, and transfer from ER and mitochondria to lysosomes and later to lipid droplets requires the participation of active microtubules, as it can be inhibited by the microtubule disruptor nocodazole. Excess of the probe is actively exported from cells, in addition to being stored in lipid droplets, to re-establish the sterol balance. Efflux occurs through a mechanism requiring energy and may be selectively poisoned with verapamil or blocked in cells with mutated cholesterol transporter NPC1. Sterolight is efficiently transferred within and between different cell populations, making it suitable for monitoring numerous aspects of sterol biology, including the live tracking and visualization of intracellular and intercellular transport.
... Instead, dNK cells selectively deliver antimicrobial granulysin to infected cells via direct contact, thereby killing the Listeria without harming the trophoblast. 316 However, they are not currently known to have a direct role against preventing in utero infection by respiratory viruses. SARS-CoV-2 shows only rare vertical transmission, suggesting that there is some maternal-fetal barrier immune activity against the virus. ...
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Pregnant women infected with pathogenic respiratory viruses, such as influenza A viruses (IAV) and coronaviruses, are at higher risk for mortality, hospitalization, preterm birth, and stillbirth. Several factors are likely to contribute to the susceptibility of pregnant individuals to severe lung disease including changes in pulmonary physiology, immune defenses, and effector functions of some immune cells. Pregnancy is also a physiologic state characterized by higher levels of multiple hormones that may impact the effector functions of immune cells, such as progesterone, estrogen, human chorionic gonadotropin, prolactin, and relaxin. Each of these hormones acts to support a tolerogenic immune state of pregnancy, which helps prevent fetal rejection, but may also contribute to an impaired antiviral response. In this review, we address the unique role of adaptive and innate immune cells in the control of pathogenic respiratory viruses and how pregnancy and specific hormones can impact their effector actions. We highlight viruses with sex‐specific differences in infection outcomes and why pregnancy hormones may contribute to fetal protection but aid the virus at the expense of the mother's health.
... The antibacterial and antiparasitic function of 9 kDa GRNLY has long been known, a property that has been confirmed and expanded in recent studies [33][34][35]. Our group has shown that recombinant GRNLY is capable of killing tumor cells in vitro through the mitochondrial apoptotic pathway [17,18,27]. ...
Preprint
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Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosilated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.
... 146 Considering the proximity of NK cells to the extravillous trophoblasts and spiral arteries, and with their key role in implantation, trophoblast invasion, spiral artery remodeling, and protection against microbial infections, it is not surprising that these cells were identified as pivotal targets of EVs. 5,147,148 Beginning in the first trimester of pregnancy, the human placenta constitutively releases sEVs that contain Fas-L (CD178) and TNFα-related apoptosis-inducing ligand (TRAIL or CD253) and that were shown to efficiently induce apoptosis of human leukocytes, PBMCs, and T cell lines in vitro 37,149 (and reviewed in Mincheva-Nillson 61 ). Both proteins are anchored to the sEV surface as highly bioactive membrane-bound trimers, which may protect them from degradation by matrix metalloproteinases. ...
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The mechanisms underlying maternal tolerance of the semi‐ or fully‐allogeneic fetus are intensely investigated. Across gestation, feto‐placental antigens interact with the maternal immune system locally within the trophoblast‐decidual interface and distantly through shed cells and soluble molecules that interact with maternal secondary lymphoid tissues. The discovery of extracellular vesicles (EVs) as local or systemic carriers of antigens and immune‐regulatory molecules has added a new dimension to our understanding of immune modulation prior to implantation, during trophoblast invasion, and throughout the course of pregnancy. New data on immune‐regulatory molecules, located on EVs or within their cargo, suggest a role for EVs in negotiating immune tolerance during gestation. Lessons from the field of transplant immunology also shed light on possible interactions between feto‐placentally derived EVs and maternal lymphoid tissues. These insights illuminate a potential role for EVs in major obstetrical disorders. This review provides updated information on intensely studied, pregnancy‐related EVs, their cargo molecules, and patterns of fetal‐placental‐maternal trafficking, highlighting potential immune pathways that might underlie immune suppression or activation in gestational health and disease. Our summary also underscores the likely need to broaden the definition of the maternal‐fetal interface to systemic maternal immune tissues that might interact with circulating EVs.
... Of central importance are intrauterine immune responses, governed by maternal decidual leukocytes adjacent to the villous placenta. Among these, decidual T cells, NK cells, and macrophages are known to respond to viral infection at the maternal-fetal interface, either through cellular or cytokine-mediated responses (Crespo et al., 2016;Yockey and Iwasaki, 2018;Jabrane-Ferrat, 2019;Crespo et al., 2020;Parker et al., 2020;Granja et al., 2021). ...
Article
While COVID-19 infection during pregnancy is common, fetal transmission is rare, suggesting that intrauterine mechanisms form an effective blockade against SARS-CoV-2. Key among these is the decidual immune environment of the placenta. We hypothesize that decidual leukocytes are altered by maternal SARS-CoV-2 infection in pregnancy and that this decidual immune response is shaped by the timing of infection during gestation. To address this hypothesis, we collected decidua basalis tissues at delivery from women with symptomatic COVID-19 during second (2nd Tri COVID, n=8) or third trimester (3rd Tri COVID, n=8) and SARS-CoV-2-negative controls (Control, n=8). Decidual natural killer (NK) cells, macrophages and T cells were evaluated using quantitative microscopy, and pro- and anti-inflammatory cytokine mRNA expression was evaluated using quantitative reverse transcriptase PCR (qRT-PCR). When compared with the Control group, decidual tissues from 3rd Tri COVID exhibited significantly increased macrophages, NK cells and T cells, whereas 2nd Tri COVID only had significantly increased T cells. In evaluating decidual cytokine expression, we noted that IL-6, IL-8, IL-10 and TNF-α were significantly correlated with macrophage cell abundance. However, in 2nd Tri COVID tissues, there was significant downregulation of IL-6, IL-8, IL-10, and TNF-α. Taken together, these results suggest innate and adaptive immune responses are present at the maternal-fetal interface in maternal SARS-CoV-2 infections late in pregnancy, and that infections earlier in pregnancy show evidence of a resolving immune response. Further studies are warranted to characterize the full scope of intrauterine immune responses in pregnancies affected by maternal COVID-19.
... The antibacterial and antiparasitic function of 9 kDa GRNLY has long been known, a property that has been confirmed and expanded in recent studies [33][34][35]. Our group has shown that recombinant GRNLY is capable of killing tumor cells in vitro through the mitochondrial apoptotic pathway [17,18,27]. ...
Preprint
Full-text available
Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosilated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.
... 25 the directional migration of trophoblast cells, 26 and protection of the fetus from infection. 27 Because the fetus is similar to a semiallograft, maintaining normal pregnancy requires DICs to form an immune tolerance microenvironment in the maternal interface to avoid the fetus's maternal immune rejection. For example, decidua natural killer Cells (dNK cells) show low-toxicity CD16 dim CD56 high phenotype. ...
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Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a new type of coronavirus that has caused fatal infectious diseases and global spread. This novel coronavirus attacks target cells through the interaction of spike protein and angiotensin‐converting enzyme II (ACE2), leading to different clinical symptoms. However, for a successful pregnancy, a well‐established in‐uterine environment includes a specific immune environment, and multi‐interactions between specific cell types are prerequisites. The immune‐related changes in patients infected with novel coronavirus could interfere with the immune microenvironment in the uterus, leading to fetal loss. We first reviewed the intrauterine environment in the normal development process and the possible pregnancy outcome in the infection state. Then, we summarized the immune response induced by SARS‐CoV‐2 in patients and analyzed the changes in ACE2 expression in the female reproductive system. Finally, the present observational evidence of infection in pregnant women was also reviewed. This article is protected by copyright. All rights reserved
... Decidual NK cells can produce large amounts of cytokines, chemokines and angiogenic factors, and gain cytotoxic function in the presence of specific pathogens (12,13). Human dNK cells were recently shown to express the antimicrobial peptide granulysin and selectively transfer this peptide via nanotubes to EVTs to kill intracellular Listeria monocytogenes without killing the trophoblast (14). ...
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In humans, the hemochorial placenta is a unique temporary organ that forms during pregnancy to support fetal development, gaseous exchange, delivery of nutrition, removal of waste products, and provides immune protection, while maintaining tolerance to the HLA-haploidentical fetus. In this review, we characterize decidual and placental immunity during maternal viral (co)-infection with HIV-1, human cytomegalovirus (HCMV), and Zika virus. We discuss placental immunology, clinical presentation, and epidemiology, before characterizing host susceptibility and cellular tropism, and how the three viruses gain access into specific placental target cells. We describe current knowledge on host-viral interactions with decidual and stromal human placental macrophages or Hofbauer cells, trophoblasts including extra villous trophoblasts, T cells, and decidual natural killer (dNK) cells. These clinically significant viral infections elicit both innate and adaptive immune responses to control replication. However, the three viruses either during mono- or co-infection (HIV-1 and HCMV) escape detection to initiate placental inflammation associated with viral transmission to the developing fetus. Aside from congenital or perinatal infection, other adverse pregnancy outcomes include preterm labor and spontaneous abortion. In addition, maternal HIV-1 and HCMV co-infection are associated with impaired fetal and infant immunity in postnatal life and poor clinical outcomes during childhood in exposed infants, even in the absence of vertical transmission of HIV-1. Given the rapidly expanding numbers of HIV-1-exposed uninfected infants and children globally, further research is urgently needed on neonatal immune programming during maternal mono-and co-infection. This review therefore includes sections on current knowledge gaps that may prompt future research directions. These gaps reflect an emerging but poorly characterized field. Their significance and potential investigation is underscored by the fact that although viral infections result in adverse consequences in both mother and developing fetus/newborn, antiviral and immunomodulatory therapies can improve clinical outcomes in the dyad.
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Decidual natural killer cell (dNK), the predominant decidual lymphocytes in early pregnancy, are primarily identified based on their CD56bright CD16- phenotype and play an important role in maintaining immune tolerance at the maternal-fetal interface. dNK dysfunction reportedly leads to pathological pregnancy. Indeed, various dNK-derived soluble factors are involved in a series of key processes related to pregnancy outcomes. In this review, we summarize the roles of these dNK-derived factors in immune tolerance and embryonic development to improve the current understanding regarding the physiological and pathological mechanisms that occur during pregnancy, while potentially informing the development of effective therapeutics.
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To establish a healthy pregnancy, the maternal immune system must tolerate fetal and placental allo-antigens, yet remain competent to respond to infections. Thus, immune recognition and presentation of placental, fetal, and pathogen-derived antigens in the right context is essential to establish maternal-fetal tolerance and immunity throughout pregnancy. Here, we review new insights into how maternal decidual immune cells at the maternal-fetal interface can recognize and respond to nonself antigens. A major focus is placed on HLA-C as the key molecule that can elicit allogeneic immune responses by maternal T and NK cells and for which maternal-fetal immune tolerance needs to be established. In addition, HLA-C is also the only classical major histocompatibility molecule expressed by extravillous trophoblast that can present a wide variety of pathogen-derived peptides and activate maternal T and NK cells and is crucial in generation of a protective immune responses to intracellular pathogens.
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Tunneling nanotubes (TNTs), open membranous channels between connected cells, represent a novel direct way of communication between distant cells for the diffusion of various cellular material, including survival or death signals, genetic material, organelles, and pathogens. Their discovery prompted us to review our understanding of many physiological and pathological processes involving cellular communication but also allowed us to discover new mechanisms of communication at a distance. While this has enriched the field, it has also generated some confusion, as different TNT-like protrusions have been described, and it is not clear whether they have the same structure–function. Most studies have been based on low-resolution imaging methods, and one of the major problems is the inconsistency in demonstrating the capacity of these various connections to transfer material between cells belonging to different populations. This brief review examines the fundamental properties of TNTs. In adult tissues, TNTs are stimulated by different diseases, stresses, and inflammatory signals. ‘Moreover’, based on the similarity of the processes of development of synaptic spines and TNT formation, we argue that TNTs in the brain predate synaptic transmission, being instrumental in the orchestration of the immature neuronal circuit.
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The non-classical HLA class I molecule HLA-G is expressed in trophoblasts where it contributes to maternal-fetal tolerance. HLA-G has been implicated in the control of trophoblast invasion, uterine vascular remodeling, and maintenance of a local immunosuppressive state. Understanding HLA-G biology at the maternal-fetal interface is therefore a critical issue in reproduction. In this regard, we review here: (i) the effects of HLA-G on decidual leucocytes and stromal cells, (ii) the contribution of trogocytosis in HLA-G expression on decidual cells, (iii) its interaction with the ILT2, ILT4 and KIR2DL4 receptors, (iv) the link between HLA-G polymorphism and pregnancy disorders, and (v) the expression of newly-described HLA-G isoforms at the maternal-fetal interface.
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(N Engl J Med. 2020;182:1125.e18–1139.e18) During pregnancy, the maternal immune system must protect from bacteria, viruses and other pathogens, while not rejecting the fetus. Decidual natural killer (NK) cells, the most abundant immune cells at the maternal-fetal interface in the first trimester of pregnancy invade the maternal side of the placenta and release cytotoxic granules. Recently, Crespo and colleagues have reported on how NK cells are able to kill harmful pathogens without damaging the placenta.
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Decidual natural killer (dNK) cells are the tissue-resident and major subpopulation of NK cells at the maternal-fetal interface. It has been demonstrated that dNK cells play pivotal roles in pregnancy, including keeping maternal-fetal immune tolerance, promoting extravillous trophoblast (EVT) cell invasion, and driving uterine spiral artery remodeling. However, the molecular mechanisms haven’t been elucidated until recent years. In this review, we systemically introduce the generation, subsets, and surface or soluble molecules of dNK cells, which are critical for maintaining the functions of dNK cells. Further, new functions of dNK cells including well-controlled cytotoxicity, immunosurveillance and immunotrophism supporting via the cell-cell interaction between dNK cells and EVT cells are mainly focused. The molecular mechanisms involved in these functions are also illustrated. Moreover, pregnancy-associated diseases caused by the dNK cells abnormalities are discussed. It will be important for future investigations about the mechanism of maintenance of pregnancy and parturition and potential clinical applications of dNK cells.
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Exosomes are a subset of extracellular vesicles with an average diameter of ~100nm. Exosomes are released by all cells through an endosome-dependent pathway and carry nucleic acids, proteins, lipids, cytokines and metabolites, mirroring the state of the originating cells. The function of exosomes has been implicated in various reproduction processes, such as embryo development, implantation, decidualization and placentation. Placenta-derived exosomes (pEXO) can be detected in the maternal blood as early as 6 weeks after conception and their levels increase with gestational age. Importantly, alternations in the molecular signatures of pEXO are observed in pregnancy-related complications. Thus, these differentially expressed molecules could be the potential biomarkers for diagnosis of the pregnancy-associated diseases. Recent studies have demonstrated that pEXO play a key role in the establishment of maternal immune tolerance, which is critical for a successful pregnancy. To gain a better understanding of the underlying mechanism, we highlighted the advanced studies of pEXO on immune cells in pregnancy.
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Uterine natural killer (uNK) cells are an immune subset located in the uterus. uNK cells have distinct tissue-specific characteristics compared to their counterparts in peripheral blood and lymphoid organs. Based on their location and the pregnancy status of the host, uNK cells are classified as endometrial NK (eNK) cells or decidua NK (dNK) cells. uNK cells are important in protecting the host from pathogen invasion and contribute to a series of physiological processes that affect successful pregnancy, including uterine spiral artery remodeling, fetal development, and immunity tolerance. Abnormal alterations in uNK cell numbers and/or impaired function may cause pregnancy complications, such as recurrent miscarriage, preeclampsia, or even infertility. In this review, we introduce recent advances in human uNK cell research under normal physiological or pathological conditions, and summarize their unique influences on the process of pregnancy complications or uterine diseases. Finally, we propose the potential clinical use of uNK cells as a novel cellular immunotherapeutic approach for reproductive disorders.
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Microglia play critical roles in the defense against neurodegenerative diseases. In this issue of Cell, Scheiblich et al. focus on microglia that ingest toxic aggregates of α-synuclein, finding that α-synuclein-replete microglia exchange aggregates for healthy mitochondria via nanotube connections to unaffected microglia. This communication enables a shared approach to aggregates disposal while preserving the health of the microglial population.
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Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.
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Fetal implantation requires carefully orchestrated involvement of the maternal immune system. Aberrant function within implantation has been suggested as a cause of implantation failure. The emergence of immunological theories of miscarriage has led to immunological testing as an adjuvant treatment in assisted reproductive technology; however, it remains controversial, with mixed evidence both for immunological cause and the benefits of immunological testing. Literature on common methods of immunological testing within assisted reproductive technology is reviewed including those of peripheral and uterine natural killer cells, chronic endometritis, and T-helper cells cytokine ratio. There is little consensus in the evidence on immunological testing in the context of recurrent implantation failure. The field is limited by a lack of uniformity in approach to testing and heterogeneity of the pathophysiological cause. Nevertheless, the maternal immune system is heavily involved in implantation and the new era of personalized medicine ensures that a more defined approach to immunological testing will be achieved.
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Background Innate lymphoid cells (ILCs) are comprised of five distinct subsets. ILCs are found at mucosal barriers and may fight invading pathogens. Chlamydia is an intracellular bacterium that infects the mucosa of the genital tract and can cause severe tissue damage. Methods We used a mouse infection model with Chlamydia muridarum ( Cmu ) to measure the reaction of genital tract ILCs to the infection. Results Tissue resident natural killer cells were the largest group in the uninfected female genital tract, and their number did not substantially change. Conventional NK cells were present at the greatest numbers during acute infection, while ILC1 cells continuously increased to high numbers. ILC2 and ILC3 cells were found at lower numbers that oscillated by a factor of 2-4. The majority of ILC3 transdifferentiated into ILC1 cells. NK cells and ILC1 cells produced IFN-γ and, rarely, TNF, but only early in the infection. Lack of B and T cells increased, while the loss of myeloid cells decreased ILC numbers. ILCs accumulated to high density in the oviduct, a main site of tissue destruction. Conclusions ILC subsets are part of the inflammatory and immune reaction during infection with Cmu and may contribute to tissue damage during chlamydial infection.
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The human maternal-fetal interface is an immunologically complex environment that must balance the divergent demands of tolerance towards the developing fetus with anti-pathogen defense. The innate immune responses at the maternal-fetal interface that function in anti-microbial defense have been understudied to-date and how ‘TORCH’ pathogens evade maternal innate immunity to infect the fetus remains poorly understood. Herein, we discuss how newly described decidual innate lymphoid cells and maternal placenta-associated macrophage subsets may be involved in anti-pathogen defense. Moreover, we outline recent advances in our understanding of how placental trophoblasts and fetal-derived macrophages (Hofbauer cells) function in anti-microbial defense. In summary, we highlight current gaps in knowledge and describe novel experimental models of the human decidua and placenta that are poised to advance our knowledge of innate immune defenses at the maternal-fetal interface.
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Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal–fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1 −/− pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.
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The microbiome is believed to be established during the birthing process through exposure to the maternal microbiome and immediate external environment. The absence of a microbiome prior to birth is based on the sterile womb hypothesis, which was formulated at the beginning of the 20th century and is supported primarily by the culture-based approach in microbiological studies. Findings of bacterial presence in products of fertilization such as the placenta, amniotic fluid, foetal membranes, and umbilical cord blood in studies using next-generation DNA sequencing technologies began to challenge the sterile nature of the intrauterine environment during gestation. These studies have been mainly criticized by their approach to contamination and inconclusive evidence of viability. The implications of bacterial presence in utero are far reaching in medicine and basic sciences. If commensal bacteria exist in the foetus, antibiotic therapies in pregnancy particularly for asymptomatic cases will need to be re-evaluated. Experimental studies utilizing gnotobiology may also be impacted by a realignment of theory. This review of existing literature aims to provide insight into the existence of bacteria in utero, specifically the foetal microbiome through analysis of experimental evidence and theoretical concepts, and to suggest approaches that may further provide clarity into this inquiry.
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Cellular metabolism plays an important role in regulating both human and murine NK cell functions. However, it remains unclear whether cellular metabolic process impacts on the function of decidual NK cells (dNK), essential tissue-resident immune cells maintaining the homeostasis of maternal-fetal interface. Remarkably, we found that glycolysis blockage enhances dNK VEGF-A production but restrains its proliferation. Furthermore, levels of IFN-g and TNF-a secreted by dNK get decreased when glycolysis or oxidative phosphorylation (OXPHOS) is inhibited. Additionally, glycolysis, OXPHOS, and fatty acid oxidation disruption has little effects on the secretion and the CD107a-dependent degranulation of dNK. Mechanistically, we discovered that the mammalian target of rapamycin complex 1 (mTORC1) signaling inhibition leads to decreased glycolysis and OXPHOS in dNK. These limited metabolic processes are associated with attenuated dNK functions, which include restricted production of cytokines including IFN-g and TNF-a, diminished CD107a-dependent degranulation, and restrained dNK proliferation. Finally, we reported that the protein levels of several glycolysis-associated enzymes are altered and the mTORC1 activity is significantly lower in the decidua of women with recurrent pregnancy loss (RPL) compared with normal pregnancy, which might give new insights about the pathogenesis of RPL. Collectively, our data demonstrate that glucose metabolism and mTORC1 signaling support dNK functions in early pregnancy.
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Tissue-resident immune cells reside in distinct niches across organs, where they contribute to tissue homeostasis and rapidly respond to perturbations in the local microenvironment. Innate lymphoid cells (ILCs) are a family of innate immune cells that regulate immune and tissue homeostasis. Across anatomical locations throughout the body, ILCs adopt tissue-specific fates, differing from circulating ILC populations. Adaptations of ILCs to microenvironmental changes have been documented in several inflammatory contexts, including obesity, asthma, and inflammatory bowel disease. While our understanding of ILC functions within tissues have predominantly been based on mouse studies, development of advanced single cell platforms to study tissue-resident ILCs in humans and emerging patient-based data is providing new insights into this lymphocyte family. Within this review, we discuss current concepts of ILC fate and function, exploring tissue-specific functions of ILCs and their contribution to health and disease across organ systems.
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Overt and subclinical maternal infections in pregnancy can have multiple and significant pathological consequences for the developing fetus, leading to acute perinatal complications and/or chronic disease throughout postnatal life. In this context, the current concept of pregnancy as a state of systemic immunosuppression seems oversimplified and outdated. Undoubtedly, in pregnancy the maternal immune system undergoes complex changes to establish and maintain tolerance to the fetus while still protecting from pathogens. In addition to downregulated maternal immunity, hormonal changes, and mechanical adaptation (e.g., restricted lung expansion) make the pregnant woman more susceptible to respiratory pathogens, such as influenza virus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Depending on the infectious agent and timing of the infection during gestation, fetal pathology can range from mild to severe, and even fatal. Influenza is associated with a higher risk of morbidity and mortality in pregnant women than in the general population, and, especially during the third trimester of pregnancy, mothers are at increased risk of hospitalization for acute cardiopulmonary illness, while their babies show higher risk of complications such as prematurity, respiratory and neurological illness, congenital anomalies, and admission to neonatal intensive care. RSV exposure in utero is associated with selective immune deficit, remodeling of cholinergic innervation in the developing respiratory tract, and abnormal airway smooth muscle contractility, which may predispose to postnatal airway inflammation and hyperreactivity, as well as development of chronic airway dysfunction in childhood. Although there is still limited evidence supporting the occurrence of vertical transmission of SARS-CoV-2, the high prevalence of prematurity among pregnant women infected by SARS-CoV-2 suggests this virus may alter immune responses at the maternal-fetal interface, affecting both the mother and her fetus. This review aims at summarizing the current evidence about the short- and long-term consequences of intrauterine exposure to influenza, RSV, and SARS-CoV-2 in terms of neonatal and pediatric outcomes.
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Knowledge of the MHC class I ligands of rhesus macaque killer-cell Ig-like receptors (KIRs) is fundamental to understanding the role of natural killer (NK) cells in this species as a nonhuman primate model for infectious diseases, transplantation and reproductive biology. We previously identified Mamu-AG as a ligand for KIR3DL05. Mamu-AG is a nonclassical MHC class I molecule that is expressed at the maternal-fetal interface of the placenta in rhesus macaques similar to HLA-G in humans. Although Mamu-AG and HLA-G share similar molecular features, including limited polymorphism and a short cytoplasmic tail, Mamu-AG is considerably more polymorphic. To determine which allotypes of Mamu-AG serve as ligands for KIR3DL05, we tested reporter cell lines expressing five different alleles of KIR3DL05 (KIR3DL05*001, KIR3DL05*004, KIR3DL05*005, KIR3DL05*008 and KIR3DL05*X) for responses to target cells expressing eight different alleles of Mamu-AG. All five allotypes of KIR3DL05 responded to Mamu-AG2*01:01, two exhibited dominant responses to Mamu-AG1*05:01, and three had low but detectable responses to Mamu-AG3*03:01, -AG3*03:02, -AG3*03:03 and -AG3*03:04. Since KIR3DL05*X is the product of recombination between KIR3DL05 and KIR3DS02 , we also tested an allotype of KIR3DS02 (KIR3DS02*004) and found that this activating KIR also recognizes Mamu-AG2*01:01. Additional analysis of Mamu-AG variants with single amino acid substitutions identified residues in the α1-domain essential for recognition by KIR3DL05. These results reveal variation in KIR3DL05 and KIR3DS02 responses to Mamu-AG and define Mamu-AG polymorphisms that differentially affect KIR recognition.
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Decidual NK cells (dNK) are a unique type of NK cells found at the maternal-fetal interface during pregnancy. dNK play a key role in placental development, trophoblast invasion, and immunity to viral and bacterial infection of the placenta. dNK are the predominant leukocyte population in first trimester placental tissues and comprise around 70% of the total CD45+ leukocytes. dNK remain present throughout pregnancy but their proportion decreases to 20-40% of term placenta decidual tissue leukocytes. Investigation of dNK function throughout pregnancy is of high clinical relevance for understanding the development of placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens. In this chapter, we describe in detail the methods we developed to purify dNK from first trimester and term pregnancy placental tissues. These methods are suitable to assess their protein and gene expression profiles as well as their function.
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The immune cells, especially innate immune cells (Natural Killer cells and Macrophages) residing at the maternal-fetal interface are playing critical roles during pregnancy. Here we discuss the immunological characteristic at the maternal-fetal interface during normal pregnancy. These key decidual immune cells are reshaped of their uterus-specific homeostatic functions within this uterus microenvironment. Through emphasizing the similarities and differences between decidua immune microenvironments with tumor or transplantation immune microenvironments, distinctive immune cell niche with activated, tolerant, proangiogenic and nurturing characteristic at the maternal interface is exhibited. Deeper understanding of the immunological microenvironment during pregnancy yield important insight not only into the pathogenesis of various human pregnancy complications, but also suggest ways to better manipulate these immune cells in cancer and transplant organs.
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During pregnancy, maternal decidual tissue interacts with fetal trophoblasts. They constitute the maternal-fetal interface responsible for supplying nutrition to the fetus. Uterine natural killer (uNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy and play critical roles throughout pregnancy. This review provides current knowledge about the functions of uNK cells. uNK cells have been shown to facilitate remodeling of the spiral artery, control the invasion of extravillous trophoblast (EVT) cells, contribute to the induction and maintenance of immune tolerance, protect against pathogen infection, and promote fetal development. Pregnancy-trained memory of uNK cells improves subsequent pregnancy outcomes. In addition, this review describes the distinct functions of three uNK cell subsets: CD27−CD11b−, CD27+ and CD27−CD11b+ uNK cells.
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Problem Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. Design Retrospective cohort study Method of Study Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA®) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. Results In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16⁺ uNK cells in women with a future ongoing clinical pregnancy compared to non‐pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non‐receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum β‐hCG time point when compared to the non‐pregnant group. Conclusion Higher levels of endometrial Tregs and lower levels of CD16⁺ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.
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The combination of the activating killer cell Ig-like receptor 2DS1 (KIR2DS1) expressed by maternal decidual natural killer cells (dNK) and the presence of its ligand, the HLA-C allotype HLA-C2, expressed by fetal trophoblasts, reduces the risk of developing pregnancy complications. However, no molecular or cellular mechanism explains this genetic correlation. Here we demonstrate that KIR2DS1+ dNK acquired higher cytotoxic function than KIR2DS1- dNK when exposed to human cytomegalovirus (HCMV)-infected decidual stromal cells (DSC), particularly when DSCs express HLA-C2. Furthermore, dNK were unable to degranulate or secrete cytokines in response to HCMV-infected primary fetal extravillous trophoblasts. This emphasizes the immunological challenge to clear placental viral infections within the immune-privileged placenta. Activation of dNK through KIR2DS1/HLA-C2 interaction increases their ability to respond to placental HCMV infection and may limit subsequent virus-induced placental pathology. This mechanism is directly related to how KIR2DS1 expressed by dNK reduces development of severe pregnancy complications such as miscarriages and preterm delivery.
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The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of researchconsented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm³ pieces and placed separately on matrixlined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.
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Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents-granzyme (Gzm) proteases and the pore-forming perforin (PFN)-into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8(+) CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.
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The interaction of noncytotoxic decidual natural killer cells (dNK) and extravillous trophoblasts (EVT) at the maternal-fetal interface was studied. Confocal microscopy revealed that many dNK interact with a single large EVT. Filamentous projections from EVT enriched in HLA-G were shown to contact dNK, and may represent the initial stage of synapse formation. As isolated, 2.5% of dNK contained surface HLA-G. However, surface HLA-G-negative dNK contained internalized HLA-G. Activation of dNK resulted in the disappearance of internalized HLA-G in parallel with restoration of cytotoxicity. Surface HLA-G was reacquired by incubation with EVT. This HLA-G cycle of trogocytosis, endocytosis, degradation, and finally reacquisition provides a transient and localized acquisition of new functional properties by dNK upon interaction with EVT. Interruption of the cycle by activation of dNK by cytokines and/or viral products serves to ensure the NK control of virus infection at the interface, and is illustrated here by the response of dNK to human cytomegalo virus (HCMV)-infected decidual stromal cells. Thus, the HLA-G cycle in dNK can provide both for NK tolerance and antiviral immunity.
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Invading human leukocyte antigen-G+ (HLA-G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA-G+ EVT and HLA-G- villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA-G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA-G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25(HI)FOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal-fetal tolerance, the properties of which are not imitated by HLA-G-expressing surrogate cell lines.
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When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.
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During the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus (HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we demonstrate that CD56(pos) dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through which these cells may operate to limit the spreading of viral infection to fetal tissues.
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Natural killer cells constitute 50-90% of lymphocytes in human uterine decidua in early pregnancy. Here, CD56(bright) uterine decidual NK (dNK) cells were compared with the CD56(bright) and CD56(dim) peripheral NK cell subsets by microarray analysis, with verification of results by flow cytometry and RT-PCR. Among the approximately 10,000 genes studied, 278 genes showed at least a threefold change with P < or = 0.001 when comparing the dNK and peripheral NK cell subsets, most displaying increased expression in dNK cells. The largest number of these encoded surface proteins, including the unusual lectinlike receptors NKG2E and Ly-49L, several killer cell Ig-like receptors, the integrin subunits alpha(D), alpha(X), beta1, and beta5, and multiple tetraspanins (CD9, CD151, CD53, CD63, and TSPAN-5). Additionally, two secreted proteins, galectin-1 and progestagen-associated protein 14, known to have immunomodulatory functions, were selectively expressed in dNK cells.
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Listeria monocytogenes is an important cause of maternal-fetal infections and serves as a model organism to study these important but poorly understood events. L. monocytogenes can infect non-phagocytic cells by two means: direct invasion and cell-to-cell spread. The relative contribution of each method to placental infection is controversial, as is the anatomical site of invasion. Here, we report for the first time the use of first trimester placental organ cultures to quantitatively analyze L. monocytogenes infection of the human placenta. Contrary to previous reports, we found that the syncytiotrophoblast, which constitutes most of the placental surface and is bathed in maternal blood, was highly resistant to L. monocytogenes infection by either internalin-mediated invasion or cell-to-cell spread. Instead, extravillous cytotrophoblasts-which anchor the placenta in the decidua (uterine lining) and abundantly express E-cadherin-served as the primary portal of entry for L. monocytogenes from both extracellular and intracellular compartments. Subsequent bacterial dissemination to the villous stroma, where fetal capillaries are found, was hampered by further cellular and histological barriers. Our study suggests the placenta has evolved multiple mechanisms to resist pathogen infection, especially from maternal blood. These findings provide a novel explanation why almost all placental pathogens have intracellular life cycles: they may need maternal cells to reach the decidua and infect the placenta.
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Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellularMycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.
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The fetus is considered to be an allograft that, paradoxically, survives pregnancy despite the laws of classical transplantation immunology. There is no direct contact of the mother with the embryo, only with the extraembryonic placenta as it implants in the uterus. No convincing evidence of uterine maternal T-cell recognition of placental trophoblast cells has been found, but instead, there might be maternal allorecognition mediated by uterine natural killer cells that recognize unusual fetal trophoblast MHC ligands.
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The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subsequent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phagosomes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells.
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We have identified human monocytic (THP-1) and myelogenous CD34+ (KG-1) leukemia cell lines that can be differentiated rapidly into mature dendritic cells (DCs) when cultured in serum-free medium containing GM-CSF, TNF-alpha, and ionomycin. These hematopoietic cell line-derived DCs are highly pure and monotypic, and display the morphologic, phenotypic, molecular, and functional properties of DCs generated from human donor-derived monocytes or CD34+ hematopoietic progenitor cells. During differentiation into mature DCs, the cells exhibit de novo cell-surface expression of CD83, CD80, CD86, CD40, CD206, CD209, CD120a, CD120b, and intracellular synthesis of IL-10, increase their endocytotic capacity, and acquire characteristic stellate morphology. To further define the cells as DCs, cytosolic induction and upregulation of RelB and RelA (p65), transcription factors of the NF-kappaB/Rel family essential for differentiation and maturation of DCs, as well as upregulation of the immunoproteasome subunits LMP2, LMP7, and MECL-1, and the proteasome activator PA28alpha, components essential for efficient MHC class I peptide antigen processing, were demonstrated during differentiation of the cells. In contrast to the cell lines, the cell line-derived mature DCs are capable of stimulating allogeneic CD4+ and CD8+ T cells, ultimately defining them as potent antigen-presenting cells. The approach to differentiate THP-1 and KG-1 cells into immature and mature DCs may serve as an experimental model to study molecular events and pathways that govern the differentiation of human malignant myeloid precursors, monocytes, and CD34+ hematopoietic progenitor cells into DCs.
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In early pregnancy invading fetal trophoblasts encounter abundant maternal decidual natural killer cells (dNK). dNK express perforin, granzymes A and B and the activating receptors NKp30, NKp44, NKp46, NKG2D, and 2B4 as well as LFA-1. Even though they are granular and express the essential molecules required for lysis, fresh dNK displayed very reduced lytic activity on classical MHC I negative targets K562 and 721.221, approximately 15% of that of peripheral NK cells. dNK formed conjugates and activating immune synapses with 721.221 and K562 cells in which CD2, LFA-1 and actin were polarized toward the contact site. However, in contrast to peripheral NK cells, they failed to polarize their microtubule organizing centers and perforin-containing granules to the synapse, accounting for their lack of cytotoxicity.
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Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.
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Granulysin (GNLY) is a cytolytic molecule expressed by human CTL and NK cells with activity against a variety of tumors and microbes, including Mycobacterium tuberculosis. Although the molecular mechanism of GNLY-induced apoptosis of Jurkat T cells is well defined in vitro, no direct evidence for its in vivo effects has been demonstrated. Because there is no murine homologue of GNLY, we generated mice expressing GNLY using a bacterial artificial chromosome containing the human GNLY gene and its 5' and 3' flanking regions. GNLY is expressed in leukocytes from transgenic mice with similar kinetics as in PBMC from humans: GNLY is constitutively expressed in NK cells and, following stimulation through the TCR, appears in T lymphocytes 8-10 days after activation. Both forms of GNLY (9 and 15 kDa) are produced by activated T cells, whereas the 15-kDa form predominates in freshly isolated NK cells from transgenic animals. GNLY mRNA is highest in spleen, with detectable expression in thymus and lungs, and minimal expression in heart, kidney, liver, muscle, intestine, and brain. Allospecific cell lines generated from GNLY transgenic animals showed enhanced killing of target cells. In vivo effects of GNLY were evaluated using the syngeneic T lymphoma tumor C6VL. GNLY transgenic mice survived significantly longer than nontransgenic littermates in response to a lethal tumor challenge. These findings demonstrate for the first time an in vivo effect of GNLY and suggest that GNLY may prove a useful therapeutic modality for the treatment of cancer.
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Primary T cell immunodeficiency and HIV-infected patients are plagued by non-viral infections caused by bacteria, fungi, and parasites, suggesting an important and underappreciated role for T lymphocytes in controlling microbes. Here, we review recent studies showing that killer lymphocytes use the antimicrobial cytotoxic granule pore-forming peptide granulysin, induced by microbial exposure, to permeabilize cholesterol-poor microbial membranes and deliver death-inducing granzymes into these pathogens. Granulysin and granzymes cause microptosis, programmed cell death in microbes, by inducing reactive oxygen species and destroying microbial antioxidant defenses and disrupting biosynthetic and central metabolism pathways required for their survival, including protein synthesis, glycolysis, and the Krebs cycle.
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ABSTRACT. Recent literature data reveals the most common etiological agents of congenital parasitoses to be Toxoplasma gondii, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. An analysis of clinical data indicates that parasitic congenital infections are often asymptomatic, whereas symptomatic newborns usually display nonspecific symptoms, which greatly hinders correct diagnosis. The long-term consequences of prenatal infections are serious clinical problems. This article presents the possible routes of vertical transmissions (mother-to-child) of pathogens including prenatal, perinatal, as well as postnatal routes. It highlights the role of factors involved in protozoa transmission and development of congenital parasitic diseases, such as parasite genotypes, the relationship between the timing of maternal infection and the probability of passage of the parasite through the placental barrier, and the immunological features of pregnant women. Acquired and congenital babesioses in human and experimental animals are presented. It emphasises that the mechanisms by which parasites infect the placenta and cross from mother to fetus are still poorly understood. It also describes the cellular mechanisms of infection by T. gondii, such as tachyzoites crossing biological barriers, the expression of Toll-Like Receptors (TLR) family on trophoblasts and syncytiotrophoblasts as an immune response to intrauterine infection and cases of congenital and acquired toxoplasmosis, as well as the long-term consequences of congenital invasion with T. gondii, episodes of reactivation of latent toxoplasmosis and T. gondii reinvasions. Mycological topics include a rare case of in utero fungal infection of offspring by a mother with vaginal candidosis, and the fungal contamination of ward facilities and medical equipment as potential sources of exogenous infections of newborn children.
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We previously showed that intracellular bacteria are present in the human placental maternal-fetal interface (basal plate). To determine the bacterial niche, basal plate biopsies were 1) examined histologically, and 2) cultured ex vivo, infected with either gram negative (E. coli) or positive (L. monocytogenes) bacteria, and examined by histological staining, immunofluorescence, and transmission electron microscopy. We found bacteria in fetal extravillous trophoblasts (EVTs) in basal plate biopsies. Both E. coli and L. monocytogenes also predominantly invaded EVTs in basal plate explants where they replicated and formed clusters or existed as single organisms. EVTs are the cell type most susceptible to bacterial colonization, likely due to their expression of major histocompatibility antigen and immune-privileged status. Pathogens persisting and replicating in the EVTs may constitute a source of intrauterine colonization that leads to adverse outcomes such as preterm birth.
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The immune cells that reside at the interface between the placenta and uterus are thought to play many important roles in pregnancy. Recent work has revealed that the composition and function of these cells is locally controlled by the specialized uterine stroma (the decidua) that surrounds the implanted conceptus. Here, I discuss how key immune cell types (natural killer cells, macrophages, dendritic cells, and T cells) are either enriched or excluded from the decidua, how their function is regulated within the decidua, and how they variously contribute to pregnancy success or failure. The discussion emphasizes the relationship between human and mouse studies. Deeper understanding of the immunology of the maternal-fetal interface promises to yield significant insight into the pathogenesis of many human pregnancy complications, including preeclampsia, intrauterine growth restriction, spontaneous abortion, preterm birth, and congenital infection. Expected final online publication date for the Annual Review of Immunology Volume 31 is March 19, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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Granulysin (GNLY) is a novel cytolytic protein lytic against a variety of tumor cells and microbes. The role of GNLY during pregnancy has not been extensively explored. The aim of this study is to examine GNLY expression and distribution in the first trimester pregnancy peripheral blood (PB) and decidua, the ability of decidual and PB natural killer (NK) cells to secrete GNLY spontaneously, and the role of antigen-presenting cells (APC) in the regulation of GNLY expression in decidual NK cells. GNLY expression was analyzed using cell permeabilization method, flow cytometry, and immunohistochemistry. GNLY secretion by purified NK cells was detected by ELISA method. GNLY is abundantly expressed at the maternal-fetal interface in the first trimester pregnancy. Decidual T lymphocytes express significantly higher levels of GNLY (58%) then PB T lymphocytes (11%). Over 85% of decidual CD56(+) cells express GNLY and when cultured spontaneously release high quantities of GNLY. Decidual APC participate in the control of GNLY expression in CD56(+) cells. Abundant expression of GNLY in the decidual immunocompetent cells and the capacity of decidual CD56(+) cells to spontaneously secrete high quantities of GNLY point to important protective and immunomodulatory role that this molecule could play at the maternal-fetal interface.
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Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells.
Article
Citation Manaster I, Mandelboim O. The unique properties of uterine NK cells. Am J Reprod Immunol 2009 Natural killer (NK) cells are lymphocytes of the innate immunity system that are able to kill various hazardous pathogens and tumors. However, it is now widely accepted that NK cells also possess non-destructive functions, as has been demonstrated for uterine NK cells. Here, we review the unique properties of the NK cells in the uterine mucosa, prior to and during pregnancy. We discuss the phenotype and function of mouse and human endometrial and decidual NK cells and suggest that the major function of decidual NK cells is to assist in fetal development. We further discuss the origin of decidual NK cells and suggest several possibilities that might explain their accumulation in the decidua during pregnancy.
Article
Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class-I (HLA-I) loci has made it difficult to generate locus-specific monoclonal antibodies (mAbs). The problem of defining an antibody's reactivity against the thousands of existing HLA-I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA-I to characterize experimentally the reactivity of nine mAb against 96 common HLA-I allotypes. In conjunction with donor HLA-I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG-3 and JAR; and the placental cell lines HTR-8/SVneo, Swan-71 and TEV-1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA-C, HLA-G and HLA-E, but not HLA-A, HLA-B or HLA-DR molecules in normal pregnancy. Tumour-derived JEG-3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.
Article
Considerable evidence indicates that cloned CTL cell lines kill target cells by releasing toxic granules that contain a cytolytic protein, called perforin, and several serine esterases (granzymes A to F). However, primary CTL, such as the highly cytolytic peritoneal exudate lymphocyte (PEL) cell population, have been found by a hemolytic assay to have no perforin, or perhaps only borderline levels of that protein, suggesting that these cells use a different lytic mechanism. To determine whether or not primary CTL express the perforin gene, we have here compared mRNA from PEL CTL and from a cloned CTL cell line, 2C, by Northern blot analysis using a perforin cDNA probe. CD8+ PEL CTL contain approximately 30% of the amount of perforin message present in 2C. Moreover, depletion of CD8+ T cells from the total peritoneal exudate cell population removes both cytolytic activity and perforin message. We have previously shown that PEL CTL elicit the same changes in target cells as cloned CTL cell lines and are resistant to lysis by the toxic granules purified from these cells lines. Taken together these results are consistent with the view that primary CTL, as well as long term cloned CTL cell lines, exercise their cytolytic activity by means of perforin.
Article
At the time the human placenta is established, the uterine mucosal lining (decidua) is infiltrated by abundant CD3- CD56bright natural killer (NK) cells. NK cells circulating in blood are known to contain perforin and granzyme A in their cytoplasmic granules. TIA-1, an RNA-binding protein capable of inducing DNA fragmentation, has also been found in the granules of cytolytic cells. In this paper, we demonstrate the presence of perforin, granzyme A and TIA-1 in the granules of uterine NK cells. Sixteen sections of non-pregnant endometrium throughout the menstrual cycle and six sections of early decidua, together with cytospins of four preparations of isolated decidual leukocytes were stained by both immunohistology and immuno-electron microscopy to localize perforin, granzyme A and TIA-1 to the cytoplasmic granules of CD56+ cells. The presence in vivo of these cytolytic molecules in a normal physiological situation implies that these uterine NK cells may have effector functions in the control of normal placentation.
Article
In mice and humans, expression of the tumour necrosis factor receptor-1 (TNF-R1) gene in placental trophoblast cells is constitutive whereas expression of the TNF-R2 gene is developmentally programmed. In order to study the individual functions of TNF-R1 and -R2 in this lineage, cell lines were generated from placental explants of homozygous matings of gestation day 10 outbred mice (Swiss-Webster), TNF-R1-deficient (TNF-R1-/-) and TNF-R2-/- transgenic mice as well as the background strain for the TNF-R2-/- mice (WT, C57BL/6x129). All of the cells exhibited trophoblast markers; they contained cytokeratin intermediate filaments, expressed alkaline phosphatase activity and displayed transferrin receptors, but were negative for vimentin filaments and the macrophage marker, F4/80. Analysis of DNA by polymerase chain reaction demonstrated the expected TNF-R genotype in each line. In experiments testing the effects of recombinant mouse TNF-alpha (rmTNF-alpha) on viability and proliferation of the cell lines, rmTNF-alpha modestly but dose-dependently inhibited the growth of WT and TNF-R2-/- cells while having no effect on TNF-R1-/- cells. Actinomycin D-treated WT and, to a lesser extent, TNF-R2-/- cells, were more sensitive to growth inhibition than untreated cells whereas TNF-R1-/- cell responses remained unchanged. These data indicated that rmTNF-alpha inhibits growth of trophoblastic cells through TNF-R1 and that newly synthesized protein(s) provide partial protection against toxicity. In contrast to the receptor species-specific effects on cell growth exerted by rmTNF-alpha, both TNF-R mediated inhibition of alkaline phosphatase activity. Collectively, the observations support the postulate that receptor expression is the key factor which determines the nature and extent of TNF-alpha effects on trophoblast cell growth and function.