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Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts
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.
... It is known that NK cells eliminate intracellular bacteria by releasing granzymes into infected cells through the pores punched by perforins (53). We hypothesize that NKMs could kill intracellular S. aureus in a similar pattern. ...
... Together, NKMs are a highly potent immune stimulator that activates the innate and adaptive antibacterial immunity in bacteria-bearing mice. The augmented NK cell activation and cytotoxic T cell responses benefit the eradication of intracellular bacterial infections by releasing perforin and granzymes, which results in largely enhanced therapeutic efficacy of NKMs (53,77). The efficacy achieved by this synergistic strategy through the spatiotemporally controlled release of NKMs is also stronger than that of multiple doses of antibiotics and the combined administration of antibiotics and DP (figs. ...
... Such an intracellular survival of S. aureus could be one of the major mechanisms of its resistances to the intravenous antibiotics as well as the host defenses such as engulfment by white cells (81). NK cell is known to kill intracellular bacteria by selectively transferring antimicrobial granzymes into infected cells via the nanotubes formed by perforin (53). However, the clinical translation of NK cell-based immunotherapy has remained challenging, at least in part due to the difficulty in NK cell isolation, high cost, and systemic toxicities (82). ...
In the competition between the pathogen infection and the host defense, infectious microorganisms may enter the host cells by evading host defense mechanisms and use the intracellular biomolecules as replication nutrient. Among them, intracellular Staphylococcus aureus relies on the host cells to protect itself from the attacks by antibiotics or immune system to achieve long-term colonization in the host, and the consequent clinical therapeutic failures and relapses after antibiotic treatment. Here, we demonstrate that intracellular S. aureus surviving well even in the presence of vancomycin can be effectively eliminated using an emerging cell-mimicking therapeutic strategy. These cell mimics with natural killer cell–like activity (NKMs) are composed of a redox-responsive degradable carrier, and perforin and granzyme B within the carrier. NKMs perform far more effectivly than clinical antibiotics in treating intracellular bacterial infections, providing a direct evidence of the NK cell–mimicking immune mechanism in the treatment of intracellular S. aureus .
... Regarding the question how dNK cells are able to selectively kill intracellular bacteria without harming the fetus, the antimicrobial peptide granulysin (GNLY) seems to be the part of the answer. It was shown that GNLY on its own was able to annihilate intracellular bacteria inside of infected trophoblasts, without lysing the host cells through insertion into the infected cell via nanotubes [31]. All in all, these experiments showed that the presence of GNLY improved the pregnancy outcome in a murine model infected with Listeria monocytogenes [32]. ...
... We also prove that the bacteriolytic abilities of PTdNKs are mediated by the GNLY secretion upon interaction with WT FN. It was demonstrated by the Lieberman group that dNK cells kill intracellular pathogens found inside trophoblasts in a process which is independent of degranulation [31]. Here, we show that the GNLY-mediated direct killing of FN by PTdNKs is also degranulation independent and that PTdNKs can kill intracellular as well as extracellular FN. ...
... Their DNK1 cell population strongly resembles the PTdNK cells [21]. Combining these recent findings with the work from the Lieberman group regarding the involvement of GNLY in the prevention of adverse pregnancy outcomes after infection with Listeria monocytogenes [31], we can position our research in the realm of further investigating and characterizing the mechanisms at play during dNK cell education. So far, the exact mechanism of education, as well as the precise pathway involved in activating the enhanced bacteriolytic properties of the trained dNK cells, have not been unveiled. ...
Natural killer cells (NKs) found during pregnancy at the maternal-fetal interface named decidual (d)NKs, show signs of education following first pregnancy, resulting in better placentation and fetus-growth, hence termed pregnancy trained dNKs (PTdNKs). Here we show that PTdNKs provide increased protection of the fetus from Fusobacterium nucleatum (FN) infection. We demonstrate that PTdNKs secrete elevated amounts of the bacteriocidal protein granulysin (GNLY) upon incubation with FN compared to dNKs derived from first pregnancies, which leads to increased killing of FN. Furthermore, we showed mechanistically that the GNLY secretion is mediated through the interaction of the FN’s Fap2 protein with Gal-GalNAc present on PTdNKs. Finally, we show in vivo, using GNLY-tg mice that enhanced protection of the fetuses from FN infection is observed, as compared to wild type and that this enhance protection is NK cell dependent. Altogether, we show a new function for PTdNKs as protectors of the fetus from bacterial infection.
... This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts. For complete details on the use and execution of this protocol, please refer to Ikumi et al., 1 Tilburgs et al., 2 Salvany-Celades et al., 3 Crespo et al., 4 van der Zwan et al. 5 ...
... Placental tissues from 5 -18 weeks of gestation can also be used, however often using these earlier placental tissues, collection of placental membranes is difficult and only decidua basalis can be used for leukocyte isolation. 2 incubator at 37 C. 3. Set up a shaking water bath at 37 C. 4. Prepare all media and stock solutions. ...
... This step details how to determine degranulation and intracellular cytokines responses of decidual immune cells in response to HLA-G+ EVT. 4,8 53. To detect degranulation and cytokine production of NK and T cells in response to EVT, to each co-culture described in 35-37, add CD107a PerCP/Cy5.5 or IgG2a PerCP/Cy5.5 control antibodies at a final concentration of 250 ng/mL. ...
Decidual leukocytes play key roles in maternal-fetal tolerance and immunity. Here, we present detailed methods to purify, culture, and functionally analyze human placental dNK, dTreg, dTem, and dMɸ from decidua parietalis, the maternal part of the placental membranes; decidua basalis, the maternal part of the placenta; and placental villi. These sites have high clinical relevance in the development of villitis and chorioamnionitis. This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts.
For complete details on the use and execution of this protocol, please refer to Ikumi et al.,¹ Tilburgs et al.,² Salvany-Celades et al.,³ Crespo et al.,⁴ van der Zwan et al.⁵
... To examine the exact form of GNLY in CD8 + T cells, we first selected two monoclonal antibodies of GNLY for flow cytometry staining, which have been reported that clone RB1 recognizes total GNLY protein, while clone DH2 only recognizes the 9 kDa isoform [37]. We gated the cells with substantial GNLY expression, including NK cells and CD8+ T cells, and compared the proportions of different isoforms of GNLY. ...
People living with HIV (PLWH) exhibit accelerated aging, characterized by systemic inflammation, termed “inflammaging.” While T-cell expansion is prevalent in PLWH, its connection to inflammaging remains unclear. In this study, we analyzed the TCRβ repertoire of 257 healthy controls (HC) and 228 PLWH, revealing pronounced T cell clonal expansion in PLWH. The expansion was only partially reversed following antiretroviral therapy (ART) and closely associated with ART duration, CD4+ T and CD8+ T cell counts and the CD4/CD8 ratio. TCR-based age modeling showed a continuous accelerated trajectory of aging in PLWH, especially in younger individuals, in stark contrast to the nonlinear aging acceleration pattern seen in HC. Furthermore, using single-cell RNA combined TCR sequencing and in vitro experiments, we identified GNLY+CD8+ T cells as the primary population driving clonal expansion and maintenance in PLWH. These cells are characterized by high cytotoxicity and low exhaustion and are activated by interleukin-15 (IL-15) in vitro. Notably, GNLY+CD8+ T cells predominantly express the pro-inflammatory 15 kDa form of granulysin(GNLY). The supernatant from IL-15-stimulated CD8+ T cells induces monocytes to secrete inflammatory factors and disrupts the integrity of intestinal epithelial cells, which can be partially restored by the anti-GNLY antibodies. These findings identify GNLY+CD8+ T cells as the central drivers of persistent clonal expansion, highlighting their crucial role for mitigating inflammaging in PLWH.
... Further, TNT-like structures have been observed in co-cultures of primary human decidual immune cells and trophoblast cells in placental organon-chip models 117 . The functional relevance of TNTs in maternal and fetal cell-to-cell interactions during infection has been further demonstrated by the decidual NK cell immune response to Listeria monocytogenes infection of trophoblast cells 118 . Further investigation of other viruses known to infect the placenta and induce TNT formation, such as SARS-CoV-2 and HIV, is warranted 45,49,119,120 . ...
Zika virus (ZIKV) is unique among orthoflaviviruses in its vertical transmission capacity in humans, yet the underlying mechanisms remain incompletely understood. Here, we show that ZIKV induces tunneling nanotubes (TNTs) in placental trophoblasts which facilitate transfer of viral particles, proteins, mitochondria, and RNA to neighboring uninfected cells. TNT formation is driven exclusively via ZIKV non-structural protein 1 (NS1). Specifically, the N-terminal 1-50 amino acids of membrane-bound ZIKV NS1 are necessary for triggering TNT formation in host cells. Trophoblasts infected with TNT-deficient ZIKVΔTNT mutant virus elicited a robust antiviral IFN-λ 1/2/3 response relative to WT ZIKV, suggesting TNT-mediated trafficking allows ZIKV cell-to-cell transmission camouflaged from host defenses. Using affinity purification-mass spectrometry of cells expressing wild-type NS1 or non-TNT forming NS1, we found mitochondrial proteins are dominant NS1-interacting partners. We demonstrate that ZIKV infection or NS1 expression induces elevated mitochondria levels in trophoblasts and that mitochondria are siphoned via TNTs from healthy to ZIKV-infected cells. Together our findings identify a stealth mechanism that ZIKV employs for intercellular spread among placental trophoblasts, evasion of antiviral interferon response, and the hijacking of mitochondria to augment its propagation and survival and offers a basis for novel therapeutic developments targeting these interactions to limit ZIKV dissemination.
... Unlike macrophages, NK cells are a unique class of lymphocytes capable of indiscriminately eliminating tumor cells without prior sensitization, and their primary roles include the indiscriminate identification of target cells and the subsequent deployment of cytotoxic mediators [120,121]. In tumor immunity, Fas and Fas ligands are classified as type I and type II transmembrane proteins, respectively. ...
... Although the expansion of CD4 + GNLY + T cells with ART excludes HIV-1 antigen -induced cell proliferation, other mechanisms remain to be elucidated. Moreover, INRs cause severe destruction of the intestinal barrier, and bacterial translocation contributes to the proliferation of GNLY + T cells, which is also powered by antibiotic function [25]. In addition, CD4 + GNLY + T cells express high levels of SERPINB9, which significantly antagonizes GZMB production by CD8 + T cells [10]. ...
Increased CD4⁺GNLY⁺ T cells have been confirmed to be inversely associated with CD4⁺ T cell count in immunological non-responders (INRs), however, the underlying mechanisms are unknown. This study aimed to elucidate the characteristics of CD4⁺GNLY⁺ T cells and their relationship with immune restoration. Single-cell RNA sequencing, single-cell TCR sequencing, and flow cytometry were used to analyze the frequency, phenotypes, and function of CD4⁺GNLY⁺ T cells. Moreover, Enzyme linked immunosorbent assay was performed to detect plasma cytokines production in patients. CD4⁺GNLY⁺ T cells were found to be highly clonally expanded, characterized by higher levels of cytotoxicity, senescence, P24, and HIV-1 DNA than CD4⁺GNLY⁻ T cells. Additionally, the frequency of CD4⁺GNLY⁺ T cells increased after ART, and further increased in INRs, and were positively associated with the antiretroviral therapy duration in INR. Furthermore, increased IL-15 levels in INRs positively correlated with the frequency and senescence of CD4⁺GNLY⁺ T cells, suggesting that CD4⁺GNLY⁺ T cells may provide new insights for understanding the poor immune reconstitution of INRs. In conclusion, increased, highly clonally expanded, and senescent CD4⁺GNLY⁺ T cells may contribute to poor immune reconstitution in HIV-1 infection.
... The detection of several NK cell markers in this cluster, such as GLNY and NKG7 (Fig. 1B, Supplementary Table S3), supported the interpretation that mRNAs in the cells of this stromal cluster could partly originate from NK cells. Furthermore, NK cells are known to transmit protein and mRNA [3,30]. Thus, because of the possibility of mRNA import, and the small number of cells in this cluster, we did not further focus on the GRNs of this cluster but conducted a functional enrichment analysis of the predicted core regulon targets to generally identify the functional differences between dSsen/nk and dS3. ...
Human reproductive success relies on the proper differentiation of the uterine endometrium to facilitate implantation, formation of the placenta, and pregnancy. This process involves two critical types of decidual uterine cells: endometrial/decidual stromal cells (dS) and uterine/decidual natural killer (dNK) cells. To better understand the transcription factors governing the in vivo functions of these cells, we analyzed single-cell transcriptomics data from first-trimester terminations of pregnancy, and for the first time conducted gene regulatory network analysis of dS and dNK cell subpopulations. Our analysis revealed stromal cell populations that corresponded to previously described in vitro decidualized cells and senescent decidual cells. We discovered new decidualization driving transcription factors of stromal cells for early pregnancy, including DDIT3 and BRF2, which regulate oxidative stress protection. For dNK cells, we identified transcription factors involved in the immunotolerant (dNK1) subpopulation, including IRX3 and RELB, which repress the NFKB pathway. In contrast, for the less immunotolerant (dNK3) population we predicted TBX21 (T-bet) and IRF2-mediated upregulation of the interferon pathway. To determine the clinical relevance of our findings, we tested the overrepresentation of the predicted transcription factors target genes among cell type-specific regulated genes from pregnancy disorders, such as recurrent pregnancy loss and preeclampsia. We observed that the predicted decidualized stromal and dNK1-specific transcription factor target genes were enriched with the genes downregulated in pregnancy disorders, whereas the predicted dNK3-specific targets were enriched with genes upregulated in pregnancy disorders. Our findings emphasize the importance of stress tolerance pathways in stromal cell decidualization and immunotolerance promoting regulators in dNK differentiation.
... We identi ed 10 primary cell populations ( Fig. 4A) based on speci c markers ( Fig. 4B), such as EpCAM + epithelial cells, VWF + endothelial cells (23) , and ACTA2 + broblasts (24) (Fig. 4B). We also identi ed various immune cell subsets, including CD3 + T cells, CD79B + B cells, IGHG1 + plasma B cells, CD68 + macrophages, GNLY + natural killer (NK) cells (25), and CPA3 + mast cells (26) (Fig. 4B). Additional clustering of T cells revealed 5 subsets ( Fig. 4C and D): Granzyme A/K + CD8 + effector T cells, CD127 + CCR7 + central memory CD8 + T cells (CD8 + Tcm), Granzyme A/K + IL7R + effector memory CD8 + T cells (CD8 + Tem) (27) , CTLA4 + exhausted CD4 + T cells, and FOXP3 + regulatory T cells (Treg) ( Fig. 4C and D). ...
Hepatocellular carcinoma (HCC) management is challenging due to its heterogeneous tumor microenvironment and poor treatment responses. To develop personalized approaches, we conducted a clinical trial of transarterial chemoembolization (TACE) combined with immune checkpoint blockade (ICB) where we collected tumor specimens for comprehensive multi-omic profiling. Treatment response was associated with increased infiltration of anti-tumor T cells, driven by cGAS-STING activation within immune-suppressive epithelial cells. However, fibrotic processes impaired these responses in some patients. Based on this insight, we conducted an animal trial in an HCC model where we added an anti-fibrotic agent to ICB and TACE mimic. This improved efficacy compared to ICB and TACE mimic alone. To identify patients who would benefit from such treatment, we constructed a predictive model using data from a limited immunohistochemistry panel and a group sparse learning algorithm. Our findings provide a blueprint for crafting personalized therapies for HCC and other cancers.
... Continuing with the functions of uNK cells, in addition to this immunomodulatory activity, shared with Tregs, they are also involved in the defense against pathogens in the uterine environment, being able to recover their cytotoxic capacity in case of infections. This last scenario, together with the promotion of other immune responses by uNKs, can be observed in cases of placental infection by human cytomegalovirus (HCMV), Listeria monocytogenes, or ZIKV [31,[36][37][38] (Figure 1). ...
Nowadays, recurrent pregnancy loss (RPL) is an undesirable condition suffered by many patients of reproductive age. In this scenario, certain immune cell populations and molecules, involved in maternal–fetal compatibility, have emerged as factors related with the pathogenesis of RPL. Among them, uterine Natural Killer cells (uNKs) appear to be of great relevance. These cells are involved in numerous processes during pregnancy, such as the remodeling of uterine spiral arteries or the control of trophoblast invasion. These functions are regulated by the interactions that these cells establish with the extravillous trophoblast, mainly through their Killer Immunoglobulin-like Receptors (KIRs) and the Human Leukocyte Antigen-C (HLA-C) molecules expressed by the embryo. A high level of polymorphism has been reported for both molecules involved in this interaction, with some of the possible KIR–HLA-C combinations being associated with an increased risk of RPL. However, the complexity of the maternal–fetal interface goes beyond this, as other HLA molecules also appear to be related to this reproductive pathology. In this review, we will discuss the role of uNKs in pregnancy, as well as the polymorphisms and clinical implications of KIR–HLA-C binding. We will also address the involvement of other, different HLA molecules in RPL, and the current advice on the appropriate management of patients with ‘immunological mismatch’, thus covering the main aspects regarding the involvement of maternal–fetal compatibility in RPL.
... Research of the last 6 years succeeded in finding out that there are 3 major subpopulations of dNK while discovering memory dNK in the decidua of multiple pregnancies [18,19]. It was also shown that dNK cells can kill bacteria without killing trophoblast cells through granulysin, therefore limiting the expansion of inflammation in the placenta [20]. Moreover, a certain cluster of differentiation (CD49a + ) present on NK cells has been shown to promote fetal growth [21]. ...
The juxtaposition of two seemingly disparate physiological phenomena within the human body—namely, cancer and pregnancy—may offer profound insights into the intricate interplay between malignancies and the immune system. Recent investigations have unveiled striking similarities between the pivotal processes underpinning fetal implantation and successful gestation and those governing tumor initiation and progression. Notably, a confluence of features has emerged, underscoring parallels between the microenvironment of tumors and the maternal–fetal interface. These shared attributes encompass establishing vascular networks, cellular mobilization, recruitment of auxiliary tissue components to facilitate continued growth, and, most significantly, the orchestration of immune-suppressive mechanisms.
Our particular focus herein centers on the phenomenon of immune suppression and its protective utility in both of these contexts. In the context of pregnancy, immune suppression assumes a paramount role in shielding the semi-allogeneic fetus from the potentially hostile immune responses of the maternal host. In stark contrast, in the milieu of cancer, this very same immunological suppression fosters the transformation of the tumor microenvironment into a sanctuary personalized for the neoplastic cells.
Thus, the striking parallels between the immunosuppressive strategies deployed during pregnancy and those co-opted by malignancies offer a tantalizing reservoir of insights. These insights promise to inform novel avenues in the realm of cancer immunotherapy. By harnessing our understanding of the immunological events that detrimentally impact fetal development, a knowledge grounded in the context of conditions such as preeclampsia or miscarriage, we may uncover innovative immunotherapeutic strategies to combat cancer.
... It is tempting to speculate that conferring a hormoneindependent enhancement in NK cell-intrinsic function in females would increase protection against infection of the mother and trophoblast during pregnancy-induced systemic immunosuppression associated with pregnancy hormones and increased numbers of regulatory T cells. 162,163 While several studies have implicated decidual NK cells in the control of microbial and viral infections in trophoblasts, 141,164,165 whether increased UTX expression is required for protective NK cell responses in females during pregnancy is unknown. On the contrary, decidual-resident NK cells are critical for fetal growth and development by promoting vascularization and remodeling of uterine spiral arteries through IFNγ and VEGFα production, and trophoblast invasion through GM-CSF secretion. ...
NK cells are short‐lived innate lymphocytes that can mediate antigen‐independent responses to infection and cancer. However, studies from the past two decades have shown that NK cells can acquire transcriptional and epigenetic modifications during inflammation that result in increased survival and lifespan. These findings blur the lines between the innate and adaptive arms of the immune system, and suggest that the homeostatic mechanisms that govern the persistence of innate immune cells are malleable. Indeed, recent studies have shown that NK cells undergo continuous and strictly regulated adaptations controlling their survival during development, tissue residency, and following inflammation. In this review, we summarize our current understanding of the critical factors regulating NK cell survival throughout their lifespan, with a specific emphasis on the epigenetic modifications that regulate the survival of NK cells in various contexts. A precise understanding of the molecular mechanisms that govern NK cell survival will be important to enhance therapies for cancer and infectious diseases.
... proangiogenic and less cytotoxic compared to pNKs 81,82 , constitutively express CD69 in mice 53 , and display receptors for MHC I and MHC ligand recognition, enabling cooperation with antigen presenting cells 81,83 . Studies on uNKs in local bacterial infections in pregnancy are scarce; [83][84][85] one study on decidual NKs (dNKs) showed that human dNKs selectively transfer granulysin to kill intracellular Listeria monocytogenes in placental cells 86 . NK depletion in bacteremic non-pregnant mice improves survival against some bacterial species 87,88 , worsens survival in some instances 89 , and does not appear to impact GBS systemic infection 84,87 . ...
Group B Streptococcus (GBS) is a pervasive perinatal pathogen, yet factors driving GBS dissemination in utero are poorly defined. Gestational diabetes mellitus (GDM), a complication marked by dysregulated immunity and maternal microbial dysbiosis, increases risk for GBS perinatal disease. Using a murine GDM model of GBS colonization and perinatal transmission, we find that GDM mice display greater GBS in utero dissemination and subsequently worse neonatal outcomes. Dual-RNA sequencing reveals differential GBS adaptation to the GDM reproductive tract, including a putative glycosyltransferase (yfhO), and altered host responses. GDM immune disruptions include reduced uterine natural killer cell activation, impaired recruitment to placentae, and altered maternofetal cytokines. Lastly, we observe distinct vaginal microbial taxa associated with GDM status and GBS invasive disease status. Here, we show a model of GBS dissemination in GDM hosts that recapitulates several clinical aspects and identifies multiple host and bacterial drivers of GBS perinatal disease.
... Bacterial entry into the placenta also activates innate defense mechanisms within the placenta. A recent study identified decidual NK (dNK) cells as part of the immunity against L. monocytogenes infection [84]. The cells transfer the antimicrobial peptide granulysin to infected trophoblasts without inducing the standard granzyme-B-mediated cytotoxicity. ...
Vertically transmitted infections are a significant cause of fetal morbidity and mortality during pregnancy and pose substantial risks to fetal development. These infections are primarily transmitted to the fetus through two routes: (1) direct invasion and crossing the placenta which separates maternal and fetal circulation, or (2) ascending the maternal genitourinary tact and entering the uterus. Only two bacterial species are commonly found to cross the placenta and infect the fetus: Listeria monocytogenes and Treponema pallidum subsp. pallidum. L. monocytogenes is a Gram-positive, foodborne pathogen found in soil that acutely infects a wide variety of mammalian species. T. pallidum is a sexually transmitted spirochete that causes a chronic infection exclusively in humans. We briefly review the pathogenesis of these two very distinct bacteria that have managed to overcome the placental barrier and the role placental immunity plays in resisting infection. Both organisms share characteristics which contribute to their transplacental transmission. These include the ability to disseminate broadly within the host, evade immune phagocytosis, and the need for a strong T cell response for their elimination.
... 细胞和EVTS细胞. 其机制是dNK细胞形成纳米管, 将 GNLY, 而不是其他细胞死亡诱导细胞毒性颗粒蛋白 转移到受感染的JEG3和EVTSs细胞中 [88] . 最近, Fang . ...
... They also display distinct cytotoxic responses as dNK cells seem to produce high levels of cytokines and be less cytotoxic during trophoblast infection [166,167]. Therefore, dNK cells tend to preserve the placental trophoblasts during the development of an immune response against some pathogens, evidencing the fact that the placenta is considered a highly privileged organ [168][169][170][171]. Despite the fact that dNK cells are an immunotolerant subpopulation of cells, the gaining of a cytotoxic phenotype can occur regarding some specific infections [172,173]. ...
Dengue virus (DENV) and chikungunya (CHIKV) are arthropod-borne viruses belonging to the Flaviviridae and Togaviridae families, respectively. Infection by both viruses can lead to a mild indistinct fever or even lead to more severe forms of the diseases, which are characterized by a generalized inflammatory state and multiorgan involvement. Infected mothers are considered a high-risk group due to their immunosuppressed state and the possibility of vertical transmission. Thereby, infection by arboviruses during pregnancy portrays a major public health concern, especially in countries where epidemics of both diseases are regular and public health policies are left aside. Placental involvement during both infections has been already described and the presence of either DENV or CHIKV has been observed in constituent cells of the placenta. In spite of that, there is little knowledge regarding the intrinsic earlier immunological mechanisms that are developed by placental cells in response to infection by both arboviruses. Here, we approach some of the current information available in the literature about the exacerbated presence of cells involved in the innate immune defense of the placenta during DENV and CHIKV infections.
... The inhibition of IFN-γ production by progesterone is mediated by the binding of this hormone on the nuclear glucocorticoid receptors (nGR) as uNK cells do not express receptors for progesterone. uNk cells have high number of cytotoxic granules as compared to pNK cells, which can be helpful in controlling infections in a pregnant female [23,24]. IFN-γ activates differentiation of Th1 cells which are crucial in providing immunity against microbes in pregnant females. ...
... 42,952 single cells were captured with high sequencing quality, and we found 9 major cell populations ( Fig. 4b), which was annotated based on their specific markers (Fig. 4c), such as EpCAM + epithelial cells, DCN + smooth muscle cells 38 , CD31 + endothelial cells and SPARC + fibroblasts 39,40 (Fig. 4c). In addition, various immune cell subsets were identified, including CD3 + T cells, CD79B + B cells, CD68 + macrophages, GNLY + natural killer (NK) cells 41 , CPA3 + mast cells 42 (Fig. 4c). ...
Ulcerative colitis is a chronic inflammatory bowel disorder with cellular heterogeneity. To understand the composition and spatial changes of the ulcerative colitis ecosystem, here we use imaging mass cytometry and single-cell RNA sequencing to depict the single-cell landscape of the human colon ecosystem. We find tissue topological changes featured with macrophage disappearance reaction in the ulcerative colitis region, occurring only for tissue-resident macrophages. Reactive oxygen species levels are higher in the ulcerative colitis region, but reactive oxygen species scavenging enzyme SOD2 is barely detected in resident macrophages, resulting in distinct reactive oxygen species vulnerability for inflammatory macrophages and resident macrophages. Inflammatory macrophages replace resident macrophages and cause a spatial shift of TNF production during ulcerative colitis via a cytokine production network formed with T and B cells. Our study suggests components of a mechanism for the observed macrophage disappearance reaction of resident macrophages, providing mechanistic hints for macrophage disappearance reaction in other inflammation or infection situations.
... 21 Decidual NK cells directly cytotoxic to LM through secreting granulysin, have also been reported. 22 The mechanisms NK cells specifically utilise to recognize LM-infected cells are largely unknown. A line of evidence points to indirect activation of NK cells through accessory cells. ...
The study of cellular immune responses in animal disease models demands detailed knowledge of development, function, and regulation of immune cells, including natural killer (NK) cells. Listeria monocytogenes (LM) bacterium has been explored in a large area of research fields, including the host pathogen interaction. Although the importance role of NK cells in controlling the first phase of LM burden has been investigated, the interaction between NK cells and infected cells in details are far from being comprehended. From in vivo and in vitro experiments, we can drive several important pieces of knowledge that hopefully contribute to illuminating the intercommunication between LM-infected cells and NK cells. Experimental studies performed in rats revealed that certain NK cell ligands are influenced in LM-infected cells. These ligands include both classical- and non-classical MHC class I molecules and C-type lectin related (Clr) molecules that are ligands for Ly49- and NKR-P1 receptors respectively. Interaction between these receptors:ligands during LM infection, demonstrated stimulation of rat NK cells. Hence, these studies provided additional knowledge to the mechanisms NK cells utilise to recognise and respond to LM infection outlined in the current review.
... In the endometrium, decidualising stromal cells regulate the proliferative expansion and differentiation of NK cells into functionally and phenotypically distinct uterine NK (uNK) subsets that are intrinsically tolerant to invading trophoblast cells and secrete an abundance of cytokines and angiogenic factors implicated in vascular remodelling (Strunz et al., 2021;Huhn et al., 2020;Moffett and Shreeve, 2022). Remarkably, uNK cells can kill intracellular bacteria in placental trophoblast without compromising the viability of host cells (Crespo et al., 2020). Thus, the emergence of decidual cells in ancestral eutherians not only enabled invasive implantation but converted allorecognition of trophoblast cells by uNK cells into a process that facilitates haemochorial placentation and safeguards the maternalfetal interface against infection. ...
Embryo implantation in humans is interstitial, meaning the entire conceptus embeds in the endometrium before the placental trophoblast invades beyond the uterine mucosa into the underlying inner myometrium. Once implanted, embryo survival pivots on the transformation of the endometrium into an anti-inflammatory placental bed, termed decidua, under homeostatic control of uterine natural killer cells. Here, we examine the evolutionary context of embryo implantation and elaborate on uterine remodelling before and after conception in humans. We also discuss the interactions between the embryo and the decidualising endometrium that regulate interstitial implantation and determine embryo fitness. Together, this Review highlights the precarious but adaptable nature of the implantation process.
... Studies have shown that between NK cells and target cells, membrane nanotubes can form where proteins accumulate, allowing distant interactions between NK cells and target cells and leading to the lysis of distant target cells [24] (Fig. 2). Other studies have shown that maternal decidual NK (dNK) cells can control bacterial infection by forming nanotubes that attach to trophoblast cells, deliver the antimicrobial peptide granulysin, and kill bacteria that infect the cells [25]. The frequency of nanotube formation depends on the number of receptor-ligand interactions. ...
Immune escape is a hallmark of cancer. The dynamic and heterogeneous tumour microenvironment (TME) causes insufficient infiltration and poor efficacy of natural killer (NK) cell-based immunotherapy, which becomes a key factor triggering tumour progression. Understanding the crosstalk between NK cells and the TME provides new insights for optimising NK cell-based immunotherapy. Here, we present new advances in direct or indirect crosstalk between NK cells and 9 specialised TMEs, including immune, metabolic, innervated niche, mechanical, and microbial microenvironments, summarise TME-mediated mechanisms of NK cell function inhibition, and highlight potential targeted therapies for NK-TME crosstalk. Importantly, we discuss novel strategies to overcome the inhibitory TME and provide an attractive outlook for the future.
Natural killer (NK) cells are critical regulators of immune processes during early pregnancy, playing a key role in maintaining maternal‐foetal immune tolerance and supporting successful implantation. In particular, uterine NK cells, a specialised subset of NK cells, facilitate trophoblast invasion, spiral artery remodelling and placental establishment. Dysregulation of NK cell activity, however, has been implicated in pregnancy complications, notably recurrent spontaneous abortion (RSA) and recurrent implantation failure (RIF). Aberrant NK cell functions, such as heightened cytotoxicity or defective immune signalling, can disrupt the balance between immune tolerance and response, leading to impaired placental development, reduced trophoblast activity and compromised uteroplacental blood flow. This review examines the role of NK cells in early pregnancy, emphasising their contributions to immune modulation and placentation. It also investigates the mechanisms by which NK cell dysfunction contributes to RSA and RIF, and explores therapeutic strategies aimed at restoring NK cell balance to improve pregnancy outcomes. A deeper understanding of NK cell interactions during early pregnancy may provide critical insights into the pathogenesis of pregnancy failure and facilitate targeted immunotherapeutic approaches.
Regulatory T (Treg) cells are essential for maternal immune tolerance of the fetus and placenta. In preeclampsia, aberrant Treg cell tolerance is implicated, but whether and how Treg cells affect the uterine vascular dysfunction thought to precede placental impairment and maternal vasculopathy is unclear. We utilized Foxp3DTR mice to test the hypothesis that Treg cells are essential regulators of decidual spiral artery adaptation to pregnancy. Transient Treg cell depletion during early placental morphogenesis caused impaired remodeling of decidual spiral arteries, altered uterine artery function and led to fewer DBA+ uterine natural killer (uNK) cells, resulting in late gestation fetal loss and fetal growth restriction. Replacing the Treg cells by transfer from wild-type donors mitigated the impact on uNK cells, vascular remodeling, and fetal loss. RNA sequencing of decidua revealed genes associated with NK cell function and placental extravillous trophoblasts were dysregulated after Treg cell depletion, and normalized by Treg cell replacement. These data implicate Treg cells as essential upstream drivers of uterine vascular adaptation to pregnancy, through a mechanism likely involving phenotypic regulation of uNK cells and trophoblast invasion. The findings provide insight into mechanisms linking impaired adaptive immune tolerance and altered spiral artery remodeling, two hallmark features of preeclampsia.
Natural killer (NK) cells, initially identified for their rapid virus-infected and leukemia cell killing and tumor destruction, are pivotal in immunity. They exhibit multifaceted roles in cancer, viral infections, autoimmunity, pregnancy, wound healing, and more. Derived from a common lymphoid progenitor, they lack CD3, B-cell, or T-cell receptors but wield high cytotoxicity via perforin and granzymes. NK cells orchestrate immune responses, secreting inflammatory IFNγ or immunosuppressive TGFβ and IL-10. CD56 dim and CD56 bright NK cells execute cytotoxicity, while CD56 bright cells also regulate immunity. However, beyond the CD56 dichotomy, detailed phenotypic diversity reveals many functional subsets that may not be optimal for cancer immunotherapy. In this review, we provide comprehensive and detailed snapshots of NK cells’ functions and states of activation and inhibitions in cancer, autoimmunity, angiogenesis, wound healing, pregnancy and fertility, aging, and senescence mediated by complex signaling and ligand-receptor interactions, including the impact of the environment. As the use of engineered NK cells for cancer immunotherapy accelerates, often in the footsteps of T-cell-derived engineering, we examine the interactions of NK cells with other immune effectors and relevant signaling and the limitations in the tumor microenvironment, intending to understand how to enhance their cytolytic activities specifically for cancer immunotherapy.
This work was supported by grants from the National Natural Science Foundation of China (No. 81800150 to Bo Cai, No. 81670110 to Kaixun Hu, and No. 31500732 to Yi Wang), Translational Research Grant of NCRCH (No. 2020ZKZB02 to Mei Guo), the Foundation for Young Scientists of Chinese PLA General Hospital (No. QNF19043 to Bo Cai, No. QNF19041 to Yi Wang, No. QNC19034 to Kaixun Hu, and No. 22QNFC022 to Linjun Sheng).
Natural killer (NK) cells have emerged as a promising cellular immunotherapy in the treatment of cancer patients. Recent research has focused on umbilical cord blood (UCB) as a source of NK cells, yielding encouraging results. However, the challenge lies in the acquisition for sufficient functional NK cells. We demonstrated that in vitro activation by a combination of cytokines, followed by prolonged expansion (14–20 days) with high doses of IL-2, can induce and expand memory-like NK cells from UCB. These memory-like NK cells displayed augmented proliferation and sustained cytotoxic efficacy.Via single-cell analysis, we detected considerable heterogeneity among UCB-derived NK cells. Notably, memory-like NK cells exhibited a markedly higher proportion displaying a proliferative phenotype. Additionally, we observed elevated levels of the transcription factor HOPX in memory-like NK cells compared to their non-memory-like counterparts. Moreover, we found distinct biological characteristics among memory-like NK cells correlating with varying levels of HOPX expression. In conclusion, we developed a cytokine-based culture system for obtaining functional memory-like NK cells from UCB which displayed specific characteristics.
In preparation for a potential pregnancy, the endometrium of the uterus changes into a temporary structure called the decidua. Senescent decidual stromal cells (DSCs) are enriched in the decidua during decidualization, but the underlying mechanisms of this process remain unclear. Here, we performed single-cell RNA transcriptomics on ESCs and DSCs and found that cell senescence during decidualization is accompanied by increased levels of the branched-chain amino acid (BCAA) transporter SLC3A2. Depletion of leucine, one of the branched-chain amino acids, from cultured media decreased senescence, while high leucine diet resulted in increased senescence and high rates of embryo loss in mice. BCAAs induced senescence in DSCs via the p38 MAPK pathway. In contrast, TNFSF14+ decidual natural killer (dNK) cells were found to inhibit DSC senescence by interacting with its ligand TNFRSF14. As in mice fed high-leucine diets, both mice with NK cell depletion and Tnfrsf14-deficient mice with excessive uterine senescence experienced adverse pregnancy outcomes. Further, we found excessive uterine senescence, SLC3A2-mediated BCAA intake, and insufficient TNFRSF14 expression in the decidua of patients with recurrent spontaneous abortion. In summary, this study suggests that dNK cells maintain senescence homeostasis of DSCs via TNFSF14/TNFRSF14, providing a potential therapeutic strategy to prevent DSC senescence-associated spontaneous abortion.
Problem
Endometrial immune cells are essential for maintaining homeostasis and the endometrial receptivity to embryo implantation. Understanding regional variations in endometrial immune cell populations is crucial for comprehending normal endometrial function and the pathophysiology of endometrial disorders. Despite previous studies focusing on the overall immune cell composition and function in the endometrium, regional variations in premenopausal women remain unclear.
Method of Study
Endometrial biopsies were obtained from four regions (anterior, posterior, left lateral, and right lateral) of premenopausal women undergoing hysteroscopy with no abnormalities. A 15‐color human endometrial immune cell‐focused flow cytometry panel was used for analysis. High‐dimensional flow cytometry combined with a clustering algorithm was employed to unravel the complexity of endometrial immune cells. Additionally, multiplex immunofluorescent was performed for further validation.
Results
Our findings revealed no significant variation in the distribution and abundance of immune cells across different regions under normal conditions during the proliferative phase. Each region harbored similar immune cell subtypes, indicating a consistent immune microenvironment. However, when comparing normal regions to areas with focal hemorrhage, significant differences were observed. An increase in CD8 ⁺ T cells highlights the impact of localized abnormalities on the immune microenvironment.
Conclusions
Our study demonstrates that the endometrial immune cell landscape is consistent across different anatomical regions during the proliferative phase in premenopausal women. This finding has important implications for understanding normal endometrial function and the pathophysiology of endometrial disorders.
Pregnancy is a fascinating immunological phenomenon because it allows allogeneic fetal and placental tissues to survive inside the mother. As a component of innate immunity with high inflammatory potential, the complement system must be tightly regulated during pregnancy. Dysregulation of the complement system plays a role in pregnancy complications including pre-eclampsia and intrauterine growth restriction. Complement components are also used as biomarkers for pregnancy complications. However, the mechanisms of detrimental role of complement in pregnancy is poorly understood. C5a is the most potent anaphylatoxin and generates multiple immune reactions via two transmembrane receptors, C5aR1 and C5aR2. C5aR1 is pro-inflammatory, but the role of C5aR2 remains largely elusive. Interestingly, murine NK cells have been shown to express C5aR2 without the usual co-expression of C5aR1. Furthermore, C5aR2 appears to regulate IFN-γ production by NK cells in vitro. As IFN-γ produced by uterine NK cells is one of the major factors for the successful development of a vital pregnancy, we investigated the role anaphylatoxin C5a and its receptors in the establishment of pregnancy and the regulation of uterine NK cells by examinations of murine C5ar2–/– pregnancies and human placental samples. C5ar2–/– mice have significantly reduced numbers of implantation sites and a maternal C5aR2 deficiency results in increased IL-12, IL-18 and IFN-γ mRNA expression as well as reduced uNK cell infiltration at the maternal-fetal interface. Human decidual leukocytes have similar C5a receptor expression patterns showing clinical relevance. In conclusion, this study identifies C5aR2 as a key contributor to dNK infiltration and pregnancy success.
Due to the physiological alteration during pregnancy, maternal gut microbiota changes following the metabolic processes. Recent studies have revealed that maternal gut microbiota is closely associated with the immune microenvironment in utero during pregnancy and plays a vital role in specific pregnancy complications, including preeclampsia, gestational diabetes, preterm birth and recurrent miscarriages. Some other evidence has also shown that aberrant maternal gut microbiota increases the risk of various diseases in the offspring, such as allergic and neurodevelopmental disorders, through the immune alignment between mother and fetus and the possible intrauterine microbiota. Probiotics and the high-fiber diet are effective inventions to prevent mothers and fetuses from diseases. In this review, we summarize the role of maternal gut microbiota in the development of pregnancy complications and the health condition of future generations from the perspective of immunology, which may provide new therapeutic strategies for the health management of mothers and offspring.
During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother’s body. The trophoblast’s unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.
Objectives
Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies in circulation and pathological pregnancy. However, the pathogenesis of OAPS remains unknown. We aimed to reveal cellular compositions and molecular features of decidual cells involved in the development of OAPS using single-cell RNA sequencing (scRNA-seq).
Methods
We performed unbiased scRNA-seq analysis on the first-trimester decidua from five OAPS patients and five healthy controls (HCs), followed by validations with flow cytometry, immunohistochemical staining and immunofluorescence in a larger cohort. Serum chemokines and cytokines were measured by using ELISA.
Results
A higher ratio of macrophages but a lower ratio of decidual natural killer (dNK) cells was found in decidua from OAPS compared with HCs. Vascular endothelial cells shrinked in OAPS decidua while having upregulated chemokine expression and conspicuous responses to IFN-γ and TNF-α. Macrophages in OAPS had stronger phagocytosis function, complement activation signals and relied more on glycolysis. dNK cells were more activated in OAPS and had enhanced cytotoxicity and IFN-γ production. Downregulation of granules in OAPS dNK cells could be associated with suppressed glycolysis. Moreover, stromal cells had a prosenescent state with weakened immune surveillance for senescent cells in OAPS. In addition, the cellular interactions between decidual immune cells and those of immune cells with non-immune cells under disease state were altered, especially through chemokines, IFN-γ and TNF-α.
Conclusion
This study provided a comprehensive decidual cell landscape and identified aberrant decidual microenvironment in OAPS, providing some potential therapeutic targets for this disease.
A thoroughly revised third edition of the acclaimed textbook for caregivers involved in the management of pregnant women with uncommon diseases or an unusual or rare condition. The book offers valuable case reports and experience collated by an international team of editors and contributors who are leading experts in the field. This edition contains five additional chapters covering topics like cardiac and neuraxial point of care ultrasound, substance abuse, rare inherited conditions, and anesthesia for rare fetal and placental conditions. Clear, concise management guidelines and algorithms are provided, and each chapter is written from the viewpoint of the obstetric anesthesiologist. Numerous tables, figures and photographs provide visual aids and each chapter contains valuable clinical insights highlighting the essential facts. Featuring updated figures and references, links to useful websites for further reading and a list of commonly used abbreviations. A valuable resource for obstetric anesthetists, perinatologists and other obstetric care providers.
Аннотация.Галектинами называют семейство β-галактозид-связывающих низкомолекулярных белков (лектинов), гомологичных по аминокислотной последовательности углевод-связывающего сайта. Галектины взаимодействуют с гликанами на поверхности клеток и участвуют в регуляции функциональной активностибольшинства клеток организма человека. Галектин-9 играет важнейшую роль в контроле функций клеток иммунной системы. Особое значение приобретают иммунорегуляторные эффекты галектина-9 при беременности, когда его продукция клетками трофобласта значительно увеличивается, что ассоциировано с формированием периферической иммунной толерантности. Основным лигандом для галектина-9 является молекула Tim-3(Т-клеточный иммуноглобулиновый и муциновый домен, содержащий белок 3), высокая экспрессия которой выявляется на натуральных киллерах (NK-клетки). При беременности фенотип и функции NK-клеток существенно меняются, что необходимо для предотвращения цитотоксических реакций на клетки плода. В обзоре систематизированы данные о роли галектина-9 вTim-3-опосредованной регуляции функций NK-клеток в аспекте беременности.Изучение регуляции функциональной активности NK-клеток необходимо для повышения эффективности NK-клеточной терапии.
Ключевые слова: галектин-9,NK-клетки, Tim-3, иммунная толерантность, беременность
Zika virus (ZIKV) infection continues to pose a significant public health concern due to limited available preventive measures and treatments. ZIKV is unique among flaviviruses in its vertical transmission capacity (i.e., transmission from mother to fetus) yet the underlying mechanisms remain incompletely understood. Here, we show that both African and Asian lineages of ZIKV induce tunneling nanotubes (TNTs) in placental trophoblasts and multiple other mammalian cell types. Amongst investigated flaviviruses, only ZIKV strains trigger TNTs. We show that ZIKV-induced TNTs facilitate transfer of viral particles, proteins, and RNA to neighboring uninfected cells. ZIKV TNT formation is driven exclusively via its non-structural protein 1 (NS1); specifically, the N-terminal region (50 aa) of membrane-bound NS1 is necessary and sufficient for triggering TNT formation in host cells. Using affinity purification-mass spectrometry of cells infected with wild-type NS1 or non-TNT forming NS1 (pNS1ΔTNT) proteins, we found mitochondrial proteins are dominant NS1-interacting partners, consistent with the elevated mitochondrial mass we observed in infected trophoblasts. We demonstrate that mitochondria are siphoned via TNTs from healthy to ZIKV-infected cells, both homotypically and heterotypically, and inhibition of mitochondrial respiration reduced viral replication in trophoblast cells. Finally, ZIKV strains lacking TNT capabilities due to mutant NS1 elicited a robust antiviral IFN-λ 1/2/3 response, indicating ZIKV's TNT-mediated trafficking also allows ZIKV cell-cell transmission that is camouflaged from host defenses. Together, our findings identify a new stealth mechanism that ZIKV employs for intercellular spread among placental trophoblasts, evasion of antiviral interferon response, and the hijacking of mitochondria to augment its propagation and survival. Discerning the mechanisms of ZIKV intercellular strategies offers a basis for novel therapeutic developments targeting these interactions to limit its dissemination.
The blastocyst nidation is the most crucial stage to a successful pregnancy, as the white cells work to promote a favorable endometrial microenvironment for this process. Intriguingly, this implantation window lasts, on average, 6 days in most regular women, and its quality is affected by many pathological conditions. Since the grounds of reproductive failure in healthy couples are still uncharted, studies have widely suggested a potential hostile role of the immune system in the equilibrium of the maternal–fetal interface. In recent years, natural killer cells have been the highlight as they represent the greatest lymphocyte in the uterus and have immune surveillance through cytotoxicity during the implantation window. This review explored the main techniques used for natural killer (NK) cell testing in the implantation window over the last 13 years on the PubMed® database. Of 2167 published articles potentially relevant for the review, only thirty-three were about cell evaluation in healthy women, met the inclusion criteria, and had their methodology critically analyzed. Here, we bring a summary from the study group and sample collection to evidence comments about their findings and correlations. Meanwhile, we also summarize the current relationship between NK cells and endometrial receptivity with reproductive failure to help enhance the possibilities for future research. In conclusion, our overview points out that restricted and unstandardized methods support the controversy between the NK population and unsuccessful embryo implantation, which is an obstacle to studying why healthy eggs do not thrive and finding a solution for one of the most controversial topics in human reproduction.
Objective
Autoimmune thyroid disease (AITD) is known to be associated with unexplained infertility in women. Although the presence of antithyroid antibodies have been speculated to be a marker of an immune imbalance that might lead to implantation failure, its underlying mechanism influencing the endometrial receptivity remains to be elucidated. In this study, we used single-cell RNA sequencing (scRNA-seq) to dissect immune microenvironment in endometrium of AITD patients during window of implantation (WOI).
Methods
We collected CD45⁺ immune cell populations of endometrium samples of unexplained infertile women with AITD (n=3), as well as samples of AITD⁻ controls (n=3). The cells were then processed with 10X Genomics Chromium for further analysis.
Results
We characterized 28 distinct immune cell subtypes totally, and uncovered differences in the composition and gene expression patterns between AITD patients and controls. The proportions of T CD4⁺, cNK, ILC3, T CD8⁺ GZMK⁺ , T CD8⁺ Cytotoxic and ILC3 CD3E ⁻ cells were increased, and CD366⁺ uNK1 was decreased in AITD⁺ patients. And the abnormal expression of GNLY and chemokines was observed in AITD patients. In addition, uNK and T CD8⁺ Cytotoxic cells showed lower cytotoxicity but activation of immune response. Genes enriched in cell adhesion of ILC3 and Tregs were downregulated, while the number of ILC3 and Tregs were increased.
Conclusion
Immune imbalance exists in endometrium during WOI, which may impact embryo implantation.
Successful embryo implantation requires both a receptive endometrium and competent blastocysts. After implantation, the maternal decidua undergoes a series of changes, including uterine spiral artery (SA) remodeling to accommodate the fetus and provide nutrients and oxygen for the fetus to survive. Uterine spiral arteries transform from small-diameter, high-resistance arteries to large-diameter and low-resistance arteries during pregnancy. This transformation includes many changes, such as increased permeability and dilation of vessels, phenotypic switching and migration of vascular smooth muscle cells (VSMCs), transient loss of endothelial cells (ECs), endovascular invasion of extravillous trophoblasts (EVTs), and presence of intramural EVT, which are regulated by uterine NK (uNK) cells and EVTs. In this review, we mainly focus on the separate and combined roles of uNK cells and EVTs in uterine SA remodeling in establishing and maintaining pregnancy. New insight into related mechanisms will help us better understand the pathogenesis of pregnancy complications such as recurrent pregnancy loss (RPL) and preeclampsia (PE).
A successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus, allowing for appropriate trophoblasts invasion and protecting the fetus from invading pathogens. Therefore, maternal immunity is critical for the establishment and maintenance of pregnancy, especially at the maternal-fetal interface. Anatomically, the maternal-fetal interface has both maternally- and fetally- derived cells, including fetal originated trophoblasts and maternal derived immune cells and stromal cells. Besides, a commensal microbiota in the uterus was supposed to aid the unique immunity in pregnancy. The appropriate crosstalk between fetal derived and maternal originated cells and uterine microbiota are critical for normal pregnancy. Dysfunctional maternal-fetal interactions might be associated with the development of pregnancy complications. This review elaborates the latest knowledge on the interactions between trophoblasts and decidual immune cells, highlighting their critical roles in maternal-fetal tolerance and pregnancy development. We also characterize the role of commensal bacteria in promoting pregnancy progression. Furthermore, this review may provide new thought on future basic research and the development of clinical applications for pregnancy complications.
Согласно современным представлениям, трансформация фенотипа и функций натуральных киллеров (NK-клеток) ассоциирована с метаболическим репрограммированием, а именно преимущественным использованием клеткой специфических метаболических путей для получения энергии. Ключевыми молекулами, участвующими в контроле метаболических программ NK-клеток, являются мишень для рапамицина в клетках млекопитающих (mTOR) и аденозинмонофосфат-активируемая протеинкиназа (AMPK). Модуляция активности mTOR и AMPK различными агентами определяет метаболическое репрограммирование, изменение фенотипа NK-клеток и функциональной активности. Особенности метаболической регуляции эффекторных функций NK-клеток зависят от их локализации, степени зрелости, продолжительности и специфичности активационных сигналов. Особое внимание уделяется изменению фенотипа, функциональной и метаболической активности NK-клеток при беременности, что ассоциировано с формированием периферической иммунной толерантности. Изучение метаболической регуляции функциональной активности NK-клеток необходимо для повышения эффективности NK-клеточной терапии. Ключевые слова: NK-клетки, метаболизм, децидуальный фенотип, иммунная толерантность, беременность Для цитирования: Орлова Е. Г. Метаболическая регуляция функций натуральных киллеров // Вест-ник Пермского университета. Сер. Биология. 2023. Вып. 1. С. 83-94. http://dx.doi.org/10.17072/1994-9952-2023-1-83-94.
Благодарности: работа выполнена при поддержке Российского научного фонда, проект № 22-25-00694.
Abstract. Changes in the body metabolism affect the metabolic and functional activity of cells of the immune system. Pregnancy is characterized by changes in hormone production, basal metabolism, immunoreactivity, increase of fat mass. During pregnancy, NK cells acquire a regulatory phenotype, migrate to the uterus and become the dominant subpopulation of lymphocytes in uterus (decidual NK), which is necessary to maintain the invasive syncytiotrophoblast growth. This transformation into decidual NK cells is accompanied by increased glucose consumption and a predominant transition from oxidative phosphorylation to glycolysis. The key molecules involved in controlling the implementation of metabolic programs of NK cells are the target for rapamycin in mammalian cells (mTOR) and AMP-activated protein kinase (AMPK). The modularization of mTOR and AMPK activity by various agents (hormones, cytokines) during pregnancy determines the metabolic reprogramming of the NK cell phenotype and functions. The study of the metabolic regulation of the NK functional activity is necessary to increase the effectiveness of NK cell therapy.
Although medical science has been fully developed, due to the high heterogeneity of triple-negative breast cancer (TNBC), it is still difficult to use reasonable and precise treatment. In this study, based on local optimization-feature screening and genomics screening strategy, we screened 25 feature genes. In multiple machine learning algorithms, feature genes have excellent discriminative diagnostic performance among samples composed of multiple large datasets. After screening at the single-cell level, we identified genes expressed substantially in myeloid cells (MCGs) that have a potential association with TNBC. Based on MCGs, we distinguished two types of TNBC patients who showed considerable differences in survival status and immune-related characteristics. Immune-related gene risk scores (IRGRS) were established, and their validity was verified using validation cohorts. A total of 25 feature genes were obtained, among which CXCL9, CXCL10, CCL7, SPHK1, and TREM1 were identified as the result after single-cell level analysis and screening. According to these entries, the cohort was divided into MCA and MCB subtypes, and the two subtypes had significant differences in survival status and tumor-immune microenvironment. After Lasso-Cox screening, IDO1, GNLY, IRF1, CTLA4, and CXCR6 were selected for constructing IRGRS. There were significant differences in drug sensitivity and immunotherapy sensitivity among high-IRGRS and low-IRGRS groups. We revealed the dynamic relationship between TNBC and TIME, identified a potential biomarker called Granulysin (GNLY) related to immunity, and developed a multi-process machine learning package called “MPMLearning 1.0” in Python.
Graphical Abstract
In this review, an attempt was made to substantiate the possibility for neurotrophins to be involved in the development of immune tolerance based on data accumulated on neurotrophin content and receptor expression in the trophoblast and immune cells, in particular, in natural killer cells. Numerous research results are reviewed to show that the expression and localization of neurotrophins along with their high‐affinity tyrosine kinase receptors and low‐affinity p75NTR receptor in the mother‐placenta‐fetus system indicate the important role of neurotrophins as binding molecules in regulating the crosstalk between the nervous, endocrine and immune systems in pregnancy. An imbalance between these systems can occur with tumor growth and pathological processes observed in pregnancy complications and fetal development anomalies. This article is protected by copyright. All rights reserved
Pig industry is facing new challenges that make necessary to reorient breeding programs to produce more robust and resilient pig populations. The aim of the present work was to study the genetic determinism of lymphocyte subpopulations in the peripheral blood of pigs and identify genomic regions and biomarkers associated to them. For this purpose, we stained peripheral blood mononuclear cells to measure ten immune-cell-related traits including the relative abundance of different populations of lymphocytes, the proportions of CD4⁺ T cells and CD8⁺ T cells, and the ratio of CD4⁺/CD8⁺ T cells from 391 healthy Duroc piglets aged 8 weeks. Medium to high heritabilities were observed for the ten immune-cell-related traits and significant genetic correlations were obtained between the proportion of some lymphocytes populations. A genome-wide association study pointed out 32 SNPs located at four chromosomal regions on pig chromosomes SSC3, SSC5, SSC8, and SSCX as significantly associated to T-helper cells, memory T-helper cells and γδ T cells. Several genes previously identified in human association studies for the same or related traits were located in the associated regions, and were proposed as candidate genes to explain the variation of T cell populations such as CD4, CD8A, CD8B, KLRC2, RMND5A and VPS24. The transcriptome analysis of whole blood samples from animals with extreme proportions of γδ T, T-helper and memory T-helper cells identified differentially expressed genes (CAPG, TCF7L1, KLRD1 and CD4) located into the associated regions. In addition, differentially expressed genes specific of different T cells subpopulations were identified such as SOX13 and WC1 genes for γδ T cells. Our results enhance the knowledge about the genetic control of lymphocyte traits that could be considered to optimize the induction of immune responses to vaccines against pathogens. Furthermore, they open the possibility of applying effective selection programs for improving immunocompetence in pigs and support the use of the pig as a very reliable human biomedical model.
Objective:
By analyze the expression levels of immune cells and cytokines secreted by preeclampsia patients before and after delivery. Furthermore, to determine the inflammatory and immunological mechanism responsible for preeclampsia, to provide better future prevention and treatment.
Methods:
Twenty-one preeclampsia-diagnosed pregnant women were involved in this study as an observation group. Twenty pregnant women with no history of PE, age-matched, were added to the control group. Thirty NP women, age-matched were included as the reference. The levels of cytokines secreted by T, B, NK and Treg immune cells and Th1, Th2 and Th17 cells were detected before and after delivery.
Results:
The number of CD3 + cells in women without preeclampsia before delivery was not statistically different from that in PE patients, but the number of CD4 + and CD8 + cells was lower than that in women without preeclampsia. After delivery, CD4 + cells were larger than those in pregnant women without preeclampsia, while CD8 + cells were smaller than those in pregnant women without preeclampsia. The level of cytokines secreted by Th1 in pregnant women without preeclampsia before delivery was higher than that in PE patients. The levels of cytokines produced by Th1, Th2 and Th17 cells before and after delivery were higher in pregnant women without preeclampsia than in NP women, while Th2 and Th17 were in PE group.
Conclusion:
PE patients may reduce CD4 + cells and CD8 + cells, and down-regulate the level of cytokines (especially TNF-β) secreted by Th1 cells to reduce the maternal rejection of embryos, thereby reducing the risk of premature delivery, which is a protective adaptive compensation mechanism in favor of embryos.
Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack. Granzyme B, released by cytotoxic immune cells, causes cell death by targeting key proteostatic proteins and pathways in a range of pathogenic bacteria.
Significance
The etiology of pregnancy complications such as miscarriage, preterm birth, and preeclampsia are largely unknown. The combination of the maternal Killer Immunoglobulin-like Receptor-2DS1 (KIR2DS1) with fetal HLA-C was identified as a factor determining risk of pregnancy complications. Thus decidual natural killer cells (dNK) that express high levels of KIR may have a predominant role in pregnancy outcome. Although the dNK effect has been associated with cytokine and growth factor production to promote placentation, here we show that the combination of maternal KIR and HLA-C is also a factor that determines protection against placental human cytomegalovirus (HCMV) infections. Expression of KIR2DS1 by dNK increases the cytotoxic response of dNK to HLA-C2+ HCMV-infected maternal decidual stromal cells, preventing viral spread and placental pathology.
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.
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.
Significance
Decidual natural killer cells (dNK), the largest population of leukocytes at the maternal–fetal interface, have low cytotoxicity. They are believed to play a role in facilitating invasion of fetal HLA-G+ extravillous trophoblasts (EVT) into maternal tissues, a process essential for establishment of healthy pregnancies. dNK are low-cytotoxic while nonetheless containing cytotoxic granules and functioning in immunity to viral infections, illustrated here with human cytomegalo virus (HCMV). Interaction of dNK with EVT leads to acquisition of HLA-G by dNK (trogocytosis), followed by a cycle of internalization, degradation, and reacquisition of HLA-G. Cytokine activation facilitates HLA-G degradation and coincides with increased cytotoxicity by dNK. Thus, the cycle provides both for NK tolerance and antiviral immune function of dNK.
Significance
Fetal extravillous trophoblasts (EVT) invade uterine tissue and interact with maternal immune cells during pregnancy. EVT express human leukocyte antigen-C (HLA-C) and -G (HLA-G). Although polymorphic HLA-C can elicit a maternal immune response, HLA-G has been associated with induction of immune tolerance. We have succeeded in isolating all maternal immune cell types as well as EVT from human placental tissue. These methods were used to elucidate the unique charateristics of EVT as well as their interaction with maternal immune cells. We demonstrate that EVT are specialized cells whose properties are not imitated by HLA‐G–expressing surrogate cell lines. Studies using primary EVT are crucial for understanding maternal–fetal tolerance and development of pregnancy complications such as preeclampsia and miscarriages.
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.
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.
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.
For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the
analysis of scientific images. We discuss the origins, challenges and solutions of these two programs,
and how their history can serve to advise and inform other software projects.
Killer lymphocytes induce apoptosis by the release of cytotoxic mediators from specialized secretory lysosomes, called cytotoxic granules, into the immunological synapse formed with a cell targeted for elimination. Methods are presented here for isolating CTL and NK cell cytotoxic granules using cell disruption by nitrogen cavitation followed by continuous Percoll density gradient fractionation. Protocols are also given for purifying the key cytolytic molecules (perforin, granzyme A, granzyme B, and granulysin) from isolated cytotoxic granules by fast protein liquid chromatography.
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.
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.
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.
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.
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.
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.
In steady-state conditions, the number and distribution of lymphocyte populations are under homeostatic control. New lymphocytes are continuously produced in primary and secondary lymphoid organs and then achieve immune-competence within different tissues, and they must challenge with resident cells for survival. The first step in the study of tissue lymphoid cells is their isolation in intact and viable form appropriate for establishment of in vitro culture systems. For reasons of simplicity, cell purity, cell yields and various purposes, lymphocytes obtained from different tissues in different labs were subjected to diverse protocols. To fully elucidate the nature of the local immune system as well as to adequately study the innate role of lymphocytes in liver, intestine, lung and uterus, we briefly reviewed the characterization of resident lymphocytes, and additional information on those cells from non-lymphoid tissues by using the recommended operation procedure was also presented.
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.
An important mechanism of host defense to Cryptococcus neoformans involves the direct microbicidal activity of lymphocytes. The importance of CD4+ T cells is illustrated by the incidence of this infection in the acquired immunodeficiency syndrome (AIDS) patients; however, the relative activity of microbicidal CD4+ T cells compared with CD8+ T cells and natural killer (NK) cells has not been established. Further, although NK cells and CD8+ T cells use perforin or granulysin, respectively, to kill C neoformans, the effector molecule used by CD4+ T cells is not known. Experiments demonstrated that IL-2-activated peripheral blood lymphocytes from healthy adults acquire anticryptococcal activity, and surprisingly, that CD4+ T cells had the most profound effect on this activity. Using SrCl(2)induced degranulation and siRNA knockdown, granulysin was shown to be the effector molecule. Although activation by anti-CD3 + IL-2 resulted in the additional expression of perforin, this did not improve the anticryptococcal activity. Cryptococcal killing by CD4+ T cells was defective in human immunodeficiency virus (HIV)-infected patients due to dysregulated granulysin and perforin production in response to IL-2 or anti-CD3 + IL-2. In conclusion, CD4+ T cells are the major subset of cells responsible for killing C neoformans in peripheral blood. These cells use granulysin as the effector molecule, and priming is dysregulated in HIV-infected patients, which results in defective microbicidal activity.
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.
Transmission of HIV-1 via intercellular connections has been estimated as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Uterine Natural Killer (uNK) cells are a transient lymphocyte population found in the pregnant uteri of human and rodents. The pregnant uterine environment appears to influence migration, differentiation and suppression of the cytolytic activation of uNK cells but the mechanisms involved in these processes are not well understood. Similarities to circulating NK (cNK) cells are limited. The present study sought to discrimate uNK cells from cNK cells in mice by identification of a unique uNK cell marker. Dolichos biflorus (DBA) lectin, which has high selectivity for glycoconjugates containing N-acetyl D-galactosomine in the terminal position, reacted with the plasma membranes of mouse uNK cells. DBA lectin did not react with other uterine lymphocytes or with cNK cell surfaces in Swiss, CBA-J, C57BL/6, SJL, BALB/c, DBA-2 mice strains. DBA lectin staining was useful for both light and electron microscopy and distinguished 4 uNK cell subtypes that appear to be stages of differentiation. Quantitative evaluation of these 4 uNK cell subtypes over early to late gestational times showed dynamic changes between immature and mature forms in different compartments of the implantation sites and indicated the occurrence of microdomains in the uterus capable of controlling uNK cell proliferation and differentiation. This is the first report showing mouse uNK cells expressing specific molecules not found in other NK cells. Use of this reagent should enhance studies of earlier, non-granulated forms of uNK cells and provide new strategies for purification of mouse uNK cells for functional and molecular studies.
We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.
Cell surfaces in the immune system are richly equipped with a complex mixture of glycans, which can be recognized by diverse glycan-binding proteins. The Siglecs are a family of sialic-acid-binding immunoglobulin-like lectins that are thought to promote cell-cell interactions and regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. In this Review, we describe recent studies on signalling mechanisms and discuss the potential role of Siglecs in triggering endocytosis and in pathogen recognition. Finally, we discuss the postulated functions of the recently discovered CD33-related Siglecs and consider the factors that seem to be driving their rapid evolution.
Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication (for example, by trafficking vesicles or transmitting calcium-mediated signals), but they can also contribute to pathologies (for example, by directing the spread of viruses). Recent data have revealed considerable heterogeneity in their structures, processes of formation and functional properties, in part dependent on the cell types involved. Despite recent progress in this young research field, further research is sorely needed.
Membrane nanotubes: dynamic longdistance connections between animal cells
- D M Davis
- S Sowinski
Davis, D.M., and Sowinski, S. (2008). Membrane nanotubes: dynamic longdistance connections between animal cells. Nat. Rev. Mol. Cell Biol. 9,
431-436.