Decidual NK Cells Research Articles

Compromised C3b-VSIG4 axis between decidual NK cells and macrophages contributes to recurrent spontaneous abortion.

NK cells and macrophages constitute the predominant immune cell subsets in the decidua during the first trimester of pregnancy, with macrophages typically adopting an anti-inflammatory phenotype. Conversely, in the third trimester, macrophages undergo a shift towards a pro-inflammatory phenotype concurrent with a reduction in NK cell numbers. The direct regulatory impact of NK cells on macrophage phenotype remains poorly explored. In our investigation, we observed that ICAM1+ macrophages stimulate the expression of intracellular C3 in LFA1+ decidual NK cells. Notably, Cathepsin W within NK cells exhibit the potential to generate active C3b fragments, effectively inhibit the proinflammatory phenotype of macrophages by binding to VSIG4. Our study unveils a direct regulatory mechanism orchestrated by decidual NK cells over macrophages, providing a potential pathogenic explanation for recurrent spontaneous abortion.

The effect of Toxoplasma gondii infection on galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

BackgroundToxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3). However, the effects of T. gondii infection on Gal-9 expression in dMφ, and the impact of altered Gal-9 expression levels on the maternal–fetal tolerance function of decidual natural killer (dNK) cells, are still unknown.MethodsPregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9−/− pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation–polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting.ResultsToxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy.ConclusionsToxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.Graphical

Deficiency of UCHL1 results in insufficient decidualization accompanied by impaired dNK modulation and eventually miscarriage

BackgroundMiscarriage is a frustrating complication of pregnancy that is common among women of reproductive age. Insufficient decidualization which not only impairs embryo implantation but disturbs fetomaternal immune-tolerance, has been widely regarded as a major cause of miscarriage; however, the underlying mechanisms resulting in decidual impairment are largely unknown.MethodsWith informed consent, decidual tissue from patients with spontaneous abortion or normal pregnant women was collected to detect the expression profile of UCHL1. Human endometrial stromal cells (HESCs) were used to explore the roles of UCHL1 in decidualization and dNK modulation, as well as the mechanisms involved. C57/BL6 female mice (7–10 weeks old) were used to construct pregnancy model or artificially induced decidualization model to evaluate the effect of UCHL1 on mice decidualization and pregnancy outcome.ResultsThe Ubiquitin C-terminal hydrolase L1 (UCHL1), as a deubiquitinating enzyme, was significantly downregulated in decidua from patients with miscarriage, along with impaired decidualization and decreased dNKs. Blockage of UCHL1 led to insufficient decidualization and resultant decreased expression of cytokines CXCL12, IL-15, TGF-β which were critical for generation of decidual NK cells (dNKs), whereas UCHL1 overexpression enhanced decidualization accompanied by increase in dNKs. Mechanistically, the promotion of UCHL1 on decidualization was dependent on its deubiquitinating activity, and intervention of UCHL1 inhibited the activation of JAK2/STAT3 signaling pathway, resulting in aberrant decidualization and decreased production of cytokines associated with dNKs modulation. Furthermore, we found that inhibition of UCHL1 also disrupted the decidualization in mice and eventually caused adverse pregnancy outcome.ConclusionsUCHL1 plays significant roles in decidualization and dNKs modulation during pregnancy in both humans and mice. Its deficiency indicates a poor pregnancy outcome due to defective decidualization, making UCHL1 a potential target for the diagnosis and treatment of miscarriage.Graphical

Single-cell transcriptomic analysis of decidual immune cell landscape in the occurrence of adverse pregnancy outcomes induced by Toxoplasma gondii infection

BackgroundToxoplasma gondii is an obligate intracellular parasite that can lead to adverse pregnancy outcomes, particularly in early pregnancy. Previous studies have illustrated the landscape of decidual immune cells. However, the landscape of decidual immune cells in the maternal–fetal microenvironment during T. gondii infection remains unknown.MethodsIn this study, we employed single-cell RNA sequencing to analyze the changes in human decidual immune cells following T. gondii infection. The results of scRNA-seq were further validated with flow cytometry, reverse transcription-polymerase chain reaction, western blot, and immunofluorescence staining.ResultsOur results showed that the proportion of 17 decidual immune cell clusters and the expression levels of 21 genes were changed after T. gondii infection. Differential gene analysis demonstrated that T. gondii infection induced the differential expression of 279, 312, and 380 genes in decidual NK cells (dNK), decidual macrophages (dMφ), and decidual T cells (dT), respectively. Our results revealed for the first time that several previously unknown molecules in decidual immune cells changed following infection. This result revealed that the function of maternal–fetal immune tolerance declined, whereas the killing ability of decidual immune cells enhanced, eventually contributing to the occurrence of adverse pregnancy outcomes.ConclusionsThis study provides valuable resource for uncovering several novel molecules that play an important role in the occurrence of abnormal pregnancy outcomes induced by T. gondii infection.Graphical

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice

ObjectiveRecurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models.MethodsExosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR.ResultsIFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3′ UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity.ConclusionOur study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal–fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.Graphical

Pregnancy trained decidual NK cells protect pregnancies from harmful Fusobacterium nucleatum infection.

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.

Deciphering decidual leukocyte traffic with serial intravascular staining.

The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, the degree, timing, and subset-specific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques (Macaca fascicularis) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy.

Autophagy Inhibition in Trophoblasts Induces Aberrant Shift in CXCR4+ Decidual NK Cell Phenotype Leading to Pregnancy Loss

Background: Pregnancy, a complex biological phenomenon, relies on intricate maternal–fetal interactions for success. Decidual natural killer (dNK) cells and trophoblasts are pivotal in establishing immune tolerance at the maternal–fetal interface. The chemokine receptor CXCR4 plays a crucial role in NK cell development and immune tolerance during early placental development. Methods: Primary decidual immune cells from 42 women with normal pregnancies and 20 patients experiencing recurrent spontaneous abortions (RSAs) were studied. Gene transcription in NK cells was assessed using real-time polymerase chain reaction. In a co-culture system, we examined the influence of trophoblasts on CXCR4 expression in dNK cells, with subsequent analysis conducted via flow cytometry. The proportion of CXCR4+ NK cells was assessed using flow cytometry after co-culture with trophoblasts pre-treated with 3-MA or a p53 activator. Results: Our study confirmed a diminished presence of decidual CXCR4+ NK cells in RSA patients during early pregnancy. Co-culturing with a trophoblast-derived supernatant increased CXCR4 expression in dNK cells. In addition, trophoblast autophagy plays an educative role in regulating the dNK landscape via the IGF2-TP53-CXCR4 axis. Conclusion: Autophagy inhibition in trophoblasts induces an aberrant shift in the CXCR4+ dNK phenotype, potentially contributing to pregnancy loss. This sheds light on the nuanced behavior of dNK cells during pregnancy, offering promising therapeutic avenues to mitigate pregnancy complications.

Maternal CXCR4 deletion results in placental defects and pregnancy loss mediated by immune dysregulation.

CXCR4 is a key regulator of the development of NK cells and DCs, both of which play an important role in early placental development and immune tolerance at the maternal-fetal interface. However, the role of CXCR4 in pregnancy is not well understood. Our study demonstrates that adult-induced global genetic CXCR4 deletion, but not uterine-specific CXCR4 deletion, was associated with increased pregnancy resorptions and decreased litter size. CXCR4-deficient mice had decreased NK cells and increased granulocytes in the decidua, along with increased leukocyte numbers in peripheral blood. We found that CXCR4-deficient mice had abnormal decidual NK cell aggregates and NK cell infiltration into trophoblast areas beyond the giant cell layer. This was associated with low NK cell expression of granzyme B, a NK cell granule effector, indicative of NK cell dysfunction. Pregnancy failure in these mice was associated with abnormalities in placental vascular development and increased placental expression of inflammatory genes. Importantly, adoptive BM transfer of WT CXCR4+ BM cells into CXCR4-deficient mice rescued the reproductive deficits by normalizing NK cell function and mediating normal placental vascular development. Collectively, our study found an important role for maternal CXCR4 expression in immune cell function, placental development, and pregnancy maintenance.

Paradoxical expression of NRP1 in decidual stromal and immune cells reveals a novel inflammation balancing mechanism during early pregnancy.

To investigate the balancing mechanisms between decidualization-associated inflammation and pregnancy-related immunotolerance. Decidual samples from women with normal pregnancy (n = 58) or unexplained spontaneous miscarriage (n = 13), peripheral blood from normal pregnancy and endometria from non-pregnancy (n = 10) were collected. Primary endometrial stromal cells (ESCs), decidual stromal cells (DSCs), decidual immune cells (DICs) and peripheral blood mononuclear cells (PBMCs) were isolated. The plasmid carrying neuropilin-1 (NRP1) gene was transfected into ESC for overexpression. To induce decidualization in vitro, ESCs were treated with a combination of 10nM estradiol, 100nM progesterone and 0.5mM cAMP. Anti-Sema3a and anti-NRP1 neutralizing antibodies were applied to block the ligand-receptor interactions. RNA-seq analysis was performed to identify differentially expressed genes in DSCs and DICs, and NRP1 expression was verified by Western blotting and flow cytometry. The secretion of inflammatory mediators was measured using a multifactor cytometric bead array. The effects of Sema3a-NRP1 pathway on DICs were determined by flow cytometry. Statistical differences between groups were compared using the T test and one way or two-way ANOVA. Combined with five RNA-seq datasets, NRP1 was the only immune checkpoint changing oppositely between DSCs and DICs. The decreased expression of NRP1 in DSCs allowed intrinsic inflammatory responses required for decidualization, while its increased expression in DICs enhanced tolerant phenotypes beneficial to pregnancy maintenance. DSC-secreted Sema3a promoted immunosuppression in DICs via NRP1 binding. In women with miscarriage, NRP1 was abnormally elevated in DSCs but diminished in decidual macrophages and NK cells. NRP1 is a multifunctional controller that balances the inflammatory states of DSCs and DICs in gravid uterus. Abnormal expression of NRP1 is implicated in miscarriage.

Mechanisms of aberrant tissue residency programming of uterine natural killer cells in uterus transplantation

Abstract Uterine natural killer cells (uNK) are the most common human decidual lymphocytes, but the origin and ontogeny of uNKs remain unclear. Studies of human uterus transplant recipients (UTx) suggest that uNKs originate in the blood, but how these cells become tissue resident and differentiate into decidual NK cells is unknown. We studied the molecular mechanisms governing uNK tissue residency in human endometrium using single-cell RNA-seq and flow cytometry analyses. trNK cells with a CD49a+ CD16− phenotype were the most abundant CD56+ cells in the endometrium of 5 healthy control volunteers (HCs) [27±17% trNK vs. 4±2.7% CD49a− CD16+ conventional NK cells (cNK); p<0.02]. trNK cells differentially expressed genes associated with T cell tissue residency, such as ITGAE and ZNF683, compared to cNK. Although peripheral blood NK cells can express CD103 when incubated with TGF-β, trNKs upregulated CD103 expression in response to IL-15 alone in vitro. Given the dependency of CD103 expression on NFAT signals, we studied CD103 expression on trNKs by incubating human endometrium (n=7) with the calcineurin inhibitor FK506, which decreased IL-15-induced upregulation of CD103 (81% CD103+ [IL-15 alone] vs. 70% CD103− [IL-15 + FK506]; p=0.02). We next tested the in vivo relevance of these findings by comparing the frequency of trNKs in endometrial biopsies of five HCs with two biopsies of one UTx on FK506 immunosuppression. The UTx recipient had significantly fewer trNKs among CD45+ cells in two biopsies (HC: 27±17% vs. UTx: 4.3±2%; p=0.04). Altogether these findings suggest that IL-15-induced calcium signals influence uNK tissue residency, with significant implications for transplant recipients. This work was supported from UAB AMC21 and support of the uterus transplant program from the University of Alabama at Birmingham. P. Porrett is additionally supported by NIH R01 AI145905.

Cellular mechanisms for maintenance of feto-maternal tolerance during pregnancy

Pregnancy is an immunological paradox, since a fetus carrying paternal antigens is a semiallogeneic transplant that should be rejected by the mother’s body. However, the fetus is completely protected from immune attack, thus suggesting some complex mechanisms of feto-maternal interaction. Hormonal, autocrine and paracrine immune signals and neuronal pathways play an important role in the development and maintenance of pregnancy. Pregnancy is considered a dynamic and actively modulated immunological process at each stage of pregnancy, including embryo implantation, placentation, fetal development, and delivery, being represented by a unique immune status. Studying the mechanisms of maintenance of pregnancy is vital to address the problems of miscarriage of unknown etiology. Successful pregnancy is closely related to the ability of the maternal immune system to properly adapt for each distinct stage of gestation. This review considers the main cell populations, such as regulatory subtypes of T and B cells, T helper cells, decidual natural killers, myeloid suppressors, erythroid nucleated cells which provide feto-maternal tolerance via various intercellular and humoral mechanisms. Maternal immune cells in the placenta do not attack fetal cells (trophoblasts) due to the tolerogenic microenvironment created by regulatory T cells and other immune cells. During pregnancy, each subpopulation of T helper cells plays a key role in promotion of fetal development through the production of angiogenic factors, providing immune surveillance and suppressing aberrant effector cell responses against a semi-allogeneic fetus. Accumulation of myeloid suppressor cells is especially relevant, when the immune tolerance is required for survival. Decidual NK cells closely interact with trophoblast cells and secrete cytokines that promote growth, mediate differentiation, trophoblast invasion, and remodeling of the spiral arteries. The favorable tolerogenic state in utero predisposes the newborn to severe infections, especially those caused by intracellular pathogens. Hence, the fetal tolerance may differ from other types of tolerance due to the presence of various immunosuppressive cells, such as erythroid suppressor cells in newborns. In the course of pregnancy, the properties of these cells change dynamically in order to meet the demands that arise during pregnancy in a timely manner. Understanding the immunological changes induced by pregnancy may not only reveal new therapeutic strategies to improve pregnancy outcomes, but also highlight new aspects of how the immune tolerance works being applicable in other physiological and pathological contexts.

Unique metabolism and protein expression signature in human decidual NK cells.

Human decidual natural killer (dNK) cells are a unique type of tissue-resident NK cells at the maternal-fetal interface. dNK cells are likely to have pivotal roles during pregnancy, including in maternal-fetal immune tolerance, trophoblast invasion, and fetal development. However, detailed insights into these cells are still lacking. In this study, we performed metabolomic and proteomic analyses on human NK cells derived from decidua and peripheral blood. We found that 77 metabolites were significantly changed in dNK cells. Notably, compared to peripheral blood NK (pNK) cells, 29 metabolites involved in glycerophospholipid and glutathione metabolism were significantly decreased in dNK cells. Moreover, we found that 394 proteins were differentially expressed in dNK cells. Pathway analyses and network enrichment analyses identified 110 differentially expressed proteins involved in focal adhesion, cytoskeleton remodeling, oxidoreductase activity, and fatty acid metabolism in dNK cells. The integrated proteomic and metabolomic analyses revealed significant downregulation in glutathione metabolism in dNK cells compared to pNK cells. Our data indicate that human dNK cells have unique metabolism and protein-expression features, likely regulating their function in pregnancy and immunity.

Метаболическая регуляция функций натуральных киллеров

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.

Research progress in the mechanism of action of NK cell-related receptors during recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) refers to multiple spontaneous abortions with the same sexual partner; In 2020, Chinese expert consensus suggested that it should be defined as the loss of the embryo or fetus before 28 weeks of gestation with the same sexual partner for two or more times in a row, including the biochemical pregnancy that occurred several times in a row. The pathogenesis of RSA is complex and diverse. At present, the pathogenesis factors have been widely recognized by the industry, including genetic factors, endocrine factors, anatomical abnormalities, infectious factors and immune factors. Among them, the role of immune factors such as T lymphocytes, dendritic cells and natural killer cells (NK) in the occurrence and development of RSA has attracted wide attention, especially NK cells. NK cells are an important part of human immune system. The specific surface molecules of Decidual natural killer cell (dNK) play an immunomodulatory role in the invasion of trophoblast cells and the remodeling of uterine spiral artery during pregnancy, which is particularly important in the early gestation period. The function of dNK cells is tightly regulated by a balance between activation and inhibition signals transmitted by the various family of receptors (NK cell receptors, NKRS) that DNK cells express. In this paper, the existing research progress of decidual NK cells on the mechanism of its receptors in the process of recurrent spontaneous abortion will be reviewed as follows.

The Update Immune-Regulatory Role of Pro- and Anti-Inflammatory Cytokines in Recurrent Pregnancy Losses.

Recurrent pregnancy losses (RPL) is a common reproductive disorder with various underlying etiologies. In recent years, rapid progress has been made in exploring the immunological mechanisms for RPL. A propensity toward Th2 over Th1 and regulatory T (Treg) over Th17 immune responses may be advantageous for reproductive success. In women with RPL and animals prone to abortion, an inordinate expression of cytokines associated with implantation and early embryo development is present in the endometrium or decidua secreted from immune and non-immune cells. Hence, an adverse cytokine milieu at the maternal-fetal interface assaults immunological tolerance, leading to fetal rejection. Similar to T cells, NK cells can be categorized based on the characteristics of cytokines they secrete. Decidual NK (dNK) cells of RPL patients exhibited an increased NK1/NK2 ratio (IFN-γ/IL-4 producing NK cell ratios), leading to pro-inflammatory cytokine milieu and increased NK cell cytotoxicity. Genetic polymorphism may be the underlying etiologies for Th1 and Th17 propensity since it alters cytokine production. In addition, various hormones participate in cytokine regulations, including progesterone and estrogen, controlling cytokine balance in favor of the Th2 type. Consequently, the intricate regulation of cytokines and hormones may prevent the RPL of immune etiologies. Local or systemic administration of cytokines or their antagonists might help maintain adequate cytokine milieu, favoring Th2 over Th1 response or Treg over Th17 immune response in women with RPL. Herein, we provided an updated comprehensive review regarding the immune-regulatory role of pro- and anti-inflammatory cytokines in RPL. Understanding the roles of cytokines involved in RPL might significantly advance the early diagnosis, monitoring, and treatment of RPL.

Decreased B7-H3 promotes unexplained recurrent miscarriage via RhoA/ROCK2 signaling pathway and regulates the secretion of decidual NK cells†.

The cause for at least 50% of recurrent miscarriages is unclear, which is defined as unexplained recurrent miscarriages. The B7-H1 (PD-L1), a molecule of the B7 family, promotes tumor development by modulating immune evasion, and recent researchers have also attached importance to the role of B7-H3, another molecule of B7 family, in tumor. Based on the similarity between growth and immune response in tumors and pregnancy, we first explored the role of B7-H3 in unexplained recurrent miscarriages. We found reduced levels of B7-H3 in the villus tissue of unexplained recurrent miscarriage patients, and it was mainly expressed on the cell membrane of extravillous trophoblasts. Further, the HTR-8/SVneo and JEG-3 cells were selected to explore the role of B7-H3 in proliferation, apoptosis, tube formation, migration, and invasion. We found that B7-H3 regulated trophoblast migration and invasion via RhoA/ROCK2 signaling pathway. Inflammatory cytokines were detected through enzyme-linked immunosorbent assay after co-culturing with decidual natural killer cells and B7-H3-knockout JEG-3. Results showed that B7-H3 inhibited IL-8 and IP-10 secretion from the decidual natural killer cells. In a CBA/J × DBA/2 abortion-prone mice model, treatment with B7-H3-Fc protein successfully reduced the rate of embryo resorption. In conclusion, our results revealed a possible mechanism by which decreased B7-H3 on trophoblasts of unexplained recurrent miscarriages inhibited trophoblast migration and invasion and increased IL-8 and IP-10 secretion from the decidual natural killer cells. Furthermore, B7-H3 may be a promising new therapeutic target in unexplained recurrent miscarriage patients.

Decidual natural killer cells and trophoblast cells: cellular, humoral and molecular mechanisms of interaction

Natural killer cells (NK cells) represent a group of lymphocytes of innate immunity. In addition to NK cells of peripheral blood, tissue-resident populations are described. NK cells of the decidual envelope (decidual NK cells) represent one of the local NK cell populations. Decidual NK cells differ in phenotype and function from peripheral blood NK cells. These cells have, mainly, regulatory functions. At the same time they retain the ability to perform cytotoxic effects. In the uterus, NK cells are located closely to the cells of fetal origin, i.e., trophoblast cells, which differentiate from the outer layer of the invading blastocyst. The purpose of the review article was to analyze the literature data on the studies of the molecular interactions between NK cells and trophoblast cells, as well as potential means of regulating these interactions. The review presents currently available data on receptor-mediated effects (due to adhesion molecules and cytotoxic receptors) and distant interactions (involving cytokines, chemokines and growth factors secreted by the both cell types) between NK population and trophoblast cells. The receptors regulating contacts of NK cells and trophoblast cells with extracellular matrix are also considered. The review provides information on activation of signaling pathways in NK cells and trophoblast cells resulting from their interaction with each other and components of the extracellular matrix. Currently, the molecular mechanisms regulating the NK cell functions and their interaction with trophoblast cells have not been studied sufficiently. The authors attempted to consider molecular regulation of the functional activity of NK cells mediated by the molecular complex of RNA polymerase II. We also describe participation of cyclin-dependent CDK8/19 kinases which comprise a part of the mediator complex which provides functioning of immune cells. The data on the participation of CDK8/19 in regulation of intracellular signaling pathways, as well as influence of CDK8/19 on the NK cell functions, are considered. Summarizing the data presented in the literature, one may emphasize that there is an extensive mutual influence of NK cells and trophoblast cells in decidual lining of uterus during pregnancy, thus leading to a changes in phenotype and functions of these cells. Experimental studies are required on the contribution of molecular mechanisms involved in transcription and translation processes to the biology of NK cells, and their role in maintaining interactions between NK cells and trophoblast cells, including the pathways involving CDK8/19.

An active glutamine/α-ketoglutarate/HIF-1α axis prevents pregnancy loss by triggering decidual IGF1+GDF15+NK cell differentiation.

Deficiency of decidual NK (dNK) cell number and function has been widely regarded as an important cause of spontaneous abortion. However, the metabolic mechanism underlying the crosstalk between dNK cells and embryonic trophoblasts during early pregnancy remains largely unknown. Here, we observed that enriched glutamine and activated glutaminolysis in dNK cells contribute to trophoblast invasion and embryo growth by insulin-like growth factor-1 (IGF-1) and growth differentiation factor-15 (GDF-15) secretion. Mechanistically, these processes are dependent on the downregulation of EGLN1-HIF-1α mediated by α-ketoglutarate (α-KG). Blocking glutaminolysis with the GLS inhibitor BPTES or the glutamate dehydrogenase inhibitor EGCG leads to early embryo implantation failure, spontaneous abortion and/or fetal growth restriction in pregnant mice with impaired trophoblast invasion. Additionally, α-KG supplementation significantly alleviated pregnancy loss mediated by defective glutaminolysis in vivo, suggesting that inactivated glutamine/α-ketoglutarate metabolism in dNK cells impaired trophoblast invasion and induced pregnancy loss.

Single-cell profiling reveals mechanisms of uncontrolled inflammation and glycolysis in decidual stromal cell subtypes in recurrent miscarriage.

Do distinct subpopulations of decidual stromal cells (DSCs) exist and if so, are given subpopulations enriched in recurrent miscarriage (RM)? Three subpopulations of DSCs were identified from which inflammatory DSCs (iDSCs) and glycolytic DSCs (glyDSCs) are significantly enriched in RM, with implicated roles in driving decidual inflammation and immune dysregulation. DSCs play crucial roles in establishing and maintaining a successful pregnancy; dysfunction of DSCs has been considered as one of the key reasons for the development of RM. We collected 15 early decidual samples from five healthy donors (HDs) and ten RM patients to perform single-cell RNA sequencing (scRNA-seq). A total of 43 RM patients and 37 HDs were enrolled in the validation cohort. Non-immune cells and immune cells of decidual tissues were sorted by flow cytometry to perform scRNA-seq. We used tissue microarrays (TMA) to validate three distinct subpopulations of DSCs. The expression of inflammatory and glycolytic proteins by DSCs was validated by immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC). Different subsets of decidual NK (dNK) cells and macrophages were also validated by multicolor flow cytometry and mIHC. Cell ligand-receptor and spatial analyses between DSCs and immune cells were analyzed by mIHC. We classify the DSCs into three subtypes based on scRNA-seq data: myofibroblastic (myDSCs), inflammatory (iDSCs) and glycolytic (glyDSCs), with the latter two being significantly enriched in RM patients. The distribution patterns of DSC subtypes in the RM and HD groups were validated by mIHC. Single-cell analyses indicate that the differentiation of iDSCs and glyDSCs may be coupled with the degrees of hypoxia. Consequently, we propose a pathological model in which a vicious circle is formed and fueled by hypoxic stress, uncontrolled inflammation and aberrant glycolysis. Furthermore, our results show that the inflammatory SPP1+ macrophages and CD18+ dNK cells are preferentially increased in the decidua of RM patients. Cell ligand-receptor and mIHC spatial analyses uncovered close interactions between pathogenic DSCs and inflammatory SPP1+ macrophages and CD18+ NK cells in RM patients. The raw single-cell sequence data reported in this paper were deposited at the National Omics Data Encyclopedia (www.biosino.org), under the accession number OEP002901. The number of decidual samples for scRNA-seq was limited and in-depth functional studies on DSCs are warranted in future studies. Identification of three DSC subpopulations opens new avenues for further investigation of their roles in RM patients. This study was supported by the Strategic Priority Research Program (No. XDB29030302), Frontier Science Key Research Project (QYZDB-SSW-SMC036), Chinese Academy of Sciences; National Key Research and Development Program of China (2021YFE0200600), National Natural Science Foundation of China (No. 31770960), Shanghai Municipal Science and Technology Major Project (No. 2019SHZDZX02, HS2021SHZX001), and Shanghai Committee of Science and Technology (17411967800). All authors report no conflict of interest.