Decidual Natural Killer Cells Research Articles

Gene Regulatory Network Analysis of Decidual Stromal Cells and Natural Killer Cells.

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.

TNFSF14+ natural killer cells prevent spontaneous abortion by restricting leucine-mediated decidual stromal cell senescence.

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.

Decidual natural killer cells drive extravillous trophoblast developmental pathways: evidence from trophoblast organoid co-cultures

Decidual natural killer cells drive extravillous trophoblast developmental pathways: evidence from trophoblast organoid co-cultures

Targeting Decidual CD16+ Immune Cells with Exosome-Based Glucocorticoid Nanoparticles for Miscarriage.

Immune dysfunction in early pregnancy including overactivation of cytotoxic CD16+ NK cells and proinflammatory M1 macrophages at the maternal-fetal interface interferes with trophoblast invasion, spiral artery remodeling, and decidualization, potentially leading to miscarriage. Immunosuppressants like glucocorticoids (GCs) are used to regulate the immune microenvironment in clinical treatment, but the lack of safe and efficient tissue-specific drug delivery systems, especially immune cell-specific vectors, limits their widespread clinical application. Here, a previously uncharacterized delivery system is reported, termed GC-Exo-CD16Ab, in which GCs are loaded into purified exosomes derived from human umbilical cord mesenchymal stem cells, and subsequently decorated with antibody CD16Ab. GC-Exo-CD16Ab is biocompatible and has remarkable delivery efficiency toward CD16+ decidual natural killer (NK) cells and CD16+ macrophages in mice. This innovative approach effectively suppresses the cytotoxicity of decidual NK cells, inhibits M1 macrophage polarization, and regulates the decidual microenvironment, thereby enhancing placental and fetal morphology, and ultimately mitigating miscarriage risk in the abortion-prone mice. The developed GC-Exo-CD16Ab provides a feasible platform for precise and tissue-specific therapeutic strategies for miscarriage and pregnancy-related diseases.

Some etiological features of spontaneous abortions (literature review)

The review article examines some etiological features of spontaneous abortions. There are many mechanisms of this pathology, and the article presents some of them: a shift in the balance of decidual natural killer cells (dNK), natural killer T (NKT) cells, regulatory T cells (Tregs), monocytes, macrophages, lymphocytes, and dendritic cells at the fetal-maternal border. There are several pathologies associated with Tregs: expression of soluble Tim-3, imbalance between Th1/Th2 cells and Th17/Tregs at different stages of pregnancy. Spontaneous abortions are associated with defects in fetal sHLA class I, which affect placental vessels and maternal immune cells through dNK and INF-γ, as well as defects in decidual cells, which are precursors of immune cells at the fetal-maternal border. The B7‑H4 protein, a regulator of T cell activity, also plays an important role. In addition, some researchers have noted the presence of immune reactions against the Y chromosome of the fetus. Poorly studied mechanisms of spontaneous abortions are defective LIF and CD95/CD95 ligand system.

Multi-Layered Mechanisms of Immunological Tolerance at the Maternal-Fetal Interface.

Pregnancy represents an immunological paradox where the maternal immune system must tolerate the semi-allogeneic fetus expressing paternally-derived Ags. Accumulating evidence over decades has revealed that successful pregnancy requires the active development of robust immune tolerance mechanisms. This review outlines the multi-layered processes that establish fetomaternal tolerance, including the physical barrier of the placenta, restricted chemokine-mediated leukocyte trafficking, lack of sufficient alloantigen presentation, the presence of immunosuppressive regulatory T cells and tolerogenic decidual natural killer cells, expression of immune checkpoint molecules, specific glycosylation patterns conferring immune evasion, and unique metabolic/hormonal modulations. Interestingly, many of the strategies that enable fetal tolerance parallel those employed by cancer cells to promote angiogenesis, invasion, and immune escape. As such, further elucidating the mechanistic underpinnings of fetal-maternal tolerance may reciprocally provide insights into developing novel cancer immunotherapies as well as understanding the pathogenesis of gestational complications linked to dysregulated tolerance processes.

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

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

Decidual natural killer cells dysfunction is caused by IDO downregulation in dMDSCs with Toxoplasma gondii infection

Myeloid-derived suppressor cells (MDSCs) play a crucial role in maintaining maternal-fetal tolerance by expressing some immune-suppressive molecules, such as indoleamine 2,3-dioxygenase (IDO). Toxoplasma gondii (T. gondii) infection can break the immune microenvironment of maternal-fetal interface, resulting in adverse pregnancy outcomes. However, whether T. gondii affects IDO expression in dMDSCs and the molecular mechanism of its effect are still unclear. Here we show, the mRNA level of IDO is increased but the protein level decreased in infected dMDSCs. Mechanistically, the upregulation of transcriptional levels of IDO in dMDSCs is regulated through STAT3/p52-RelB pathway and the decrease of IDO expression is due to its degradation caused by increased SOCS3 after T. gondii infection. In vivo, the adverse pregnancy outcomes of IDO−/− infected mice are more severe than those of wide-type infected mice and obviously improved after exogenous kynurenine treatment. Also, the reduction of IDO in dMDSCs induced by T. gondii infection results in the downregulation of TGF-β and IL-10 expression in dNK cells regulated through Kyn/AhR/SP1 signal pathway, eventually leading to the dysfunction of dNK cells and contributing the occurrence of adverse pregnancy outcomes. This study reveals a novel molecular mechanism in adverse pregnancy outcome induced by T. gondii infection.

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

Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate: immune mechanisms of recurrent early pregnancy loss.

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss (RPL) at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG (NOD/Shi-scid/IL-2Rγnull) mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients (dNK-RPL) remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+ dNK cells. Mechanistic studies revealed that CD56+CD39+ dNK subset facilitates early differentiation of mouse trophoblast stem cells (mTSCs) towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor (M-CSF). Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+ dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium areprecursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

Single-cell transcriptome analyses reveal disturbed decidual homoeostasis in obstetric antiphospholipid syndrome

ObjectivesObstetric 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...

Alterations in the pro-resolving lipid mediator machinery within first trimester maternal tissue: Implications in decidualization and miscarriage risk

A pivotal event in uterine receptivity and human reproduction is the differentiation of endometrial stromal cells into decidual cells, known as decidualization. Decidualization is interlinked with its inflammatory environment. Our study aimed to investigate the presence and role of pro-resolving lipid mediators in first trimester maternal tissue. We assessed the levels of LXA4 and RvD1, along with their metabolic LOX enzymes, in elective (control) and sporadic miscarriage samples. We investigated the effects of LXA4 and RvD1 on decidualization using primary endometrial stromal cells and the immortalized endometrial stromal St-T1b cell line. The upregulation of 12- and 15-LOX expression was observed in pregnancy tissue after sporadic miscarriage, suggesting an inflammatory imbalance. Furthermore, incubation with these lipid mediators led to a decrease in decidualization biomarkers PRL and IGFBP-1, accompanied by morphological changes indicative of aberrant differentiation. The expression of LOX enzymes in decidual natural killer cells suggests their involvement in regulating the inflammatory surroundings and the extent of decidualization.

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.

The novel role of activating receptor KIR2DS5 in preeclampsia

Preeclampsia (PE) is a serious complication of pregnancy. Decidual natural killer (dNK) cells were reported to participate in the remodeling of spiral arteries through producing a group of cytokines, including granulocyte–macrophage colony stimulating factor (GM-CSF). KIR2DS5 is an activating receptor of NK cells that specifically recognizes HLA-C2 on trophoblasts. Currently, there are no reports regarding the precise mechanism of KIR2DS5 in PE. This study included 30 PE patients and 30 healthy pregnant women. We found that the expressions of KIR2DS5 were significantly lower in PE deciduae compared to those of healthy pregnancies. By transfecting knockdown and overexpression lentivirus vectors of KIR2DS5 into dNK cells isolated from deciduae of early pregnancy, we altered the KIR2DS5 expression level in dNK cells. Then, these dNK cells and trophoblast cell lines were co-cultured as trophoblast-dNK cells. In the trophoblast-dNK cells, we examined the influence of KIR2DS5 on the biological manifestations of trophoblasts. As anticipated, overexpression of KIR2DS5 could facilitate cell proliferation, migration, and invasion. Furthermore, increased expression of KIR2DS5 inhibited cell apoptosis and enhanced the progression of cells from theG1 to theS stage. Further mechanistic study demonstrated a positive relationship between KIR2DS5 and GM-CSF in trophoblast-dNK cells. Accordingly, our observations indicated that a decrease in KIR2DS5 could reduce the expression of GM-CSF via the JAK2/STAT5 pathway, resulting in the failure of the activated signal to be transmitted to dNK cells and ultimately leading to the occurrence of PE. KIR2DS5 may be a new contributor for the prediction and diagnosis of PE.

Single-cell characterization of self-renewing primary trophoblast organoids as modeling of EVT differentiation and interactions with decidual natural killer cells

BackgroundExtravillous trophoblast cell (EVT) differentiation and its communication with maternal decidua especially the leading immune cell type natural killer (NK) cell are critical events for placentation. However, appropriate in vitro modelling system and regulatory programs of these two events are still lacking. Recent trophoblast organoid (TO) has advanced the molecular and mechanistic research in placentation. Here, we firstly generated the self-renewing TO from human placental villous and differentiated it into EVTs (EVT-TO) for investigating the differentiation events. We then co-cultured EVT-TO with freshly isolated decidual NKs for further study of cell communication. TO modelling of EVT differentiation as well as EVT interaction with dNK might cast new aspect for placentation research.ResultsSingle-cell RNA sequencing (scRNA-seq) was applied for comprehensive characterization and molecular exploration of TOs modelling of EVT differentiation and interaction with dNKs. Multiple distinct trophoblast states and dNK subpopulations were identified, representing CTB, STB, EVT, dNK1/2/3 and dNKp. Lineage trajectory and Seurat mapping analysis identified the close resemblance of TO and EVT-TO with the human placenta characteristic. Transcription factors regulatory network analysis revealed the cell-type specific essential TFs for controlling EVT differentiation. CellphoneDB analysis predicted the ligand-receptor complexes in dNK-EVT-TO co-cultures, which relate to cytokines, immunomodulation and angiogenesis. EVT was known to affect the immune properties of dNK. Our study found out that on the other way around, dNKs could exert effects on EVT causing expression changes which are functionally important.ConclusionOur study documented a single-cell atlas for TO and its applications on EVT differentiation and communications with dNKs, and thus provide methodology and novel research cues for future study of human placentation.

Bioinformatics analysis of competing endogenous RNA network in decidual natural killer cell from unexplained recurrent spontaneous abortion.

Decidual natural killer (dNK) cell plays a pivotal role in maintaining pregnancy, especially in the first trimester. Noncoding-RNAs (ncRNAs) are critical regulators of transcription and protein expression. Dysregulation of ncRNAs may be involved in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). However, the role of competing endogenous RNA (ceRNA) based on mRNA-miRNA-lncRNA network in regulating the incidence and progression of URSA remains elusive. The aim of the study is to identify the regulatory network of mRNA-miRNA-LncRNA ceRNA based on bioinformatics analysis in dNK from patients with URSA. Eligible studies were retrieved from PubMed, Embase, and the Gene Expression Omnibus (GEO) databases to identify differentially expressed genes (DEGs), miRNAs and LncRNAs in dNK cells of patients with URSA. Protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software. Potential regulatory miRNAs and lncRNAs of mRNAs were predicted by miRTarBase and RNA22 and subject to bioinformatics analysis. A total of 634 DEGs were screened, including 290 upregulated and 344 downregulated DEGs. Among 207 differentially expressed lncRNAs, 110 lncRNAs were upregulated and 97 were downregulated. According to node degree, 30 hub genes were identified for subsequent research. After drawing the Venn diagram and matching to Cytoscape, an mRNA-miRNA-lncRNA network linked to the pathogenesis of URSA in dNK cells was constructed. A novel regulatory network of mRNA-miRNA-lncRNA ceRNA is established in dNK cells from patients with URSA. All RNAs might be used as the biomarkers of the pathogenesis of URSA.

Toxoplasma gondii Causes Adverse Pregnancy Outcomes by Damaging Uterine Tissue-Resident NK Cells That Secrete Growth-Promoting Factors.

Vertical transmission of the intracellular parasite, Toxoplasma gondii can lead to adverse pregnancy outcomes especially when infection occurs in early pregnancy. Decidual natural killer (dNK) cells accumulate at the maternal-fetal interface in large numbers during early pregnancy. Their nutritional roles during infection with T. gondii remain poorly defined. In the present study, we demonstrated that a functional deficiency of the uterine tissue-resident NK (trNK) cells, a subset of dNK cells, contributes to the adverse pregnancy outcomes induced by T. gondii in early pregnancy. Adverse pregnancy outcomes could be ameliorated by adoptive transfer of trNK cells. Moreover, fetal growth restriction could be improved after supplementation of growth-promoting factors. In addition to the widely recognized disturbance of the immune balance at the interface between the mother and the fetus, our study reveals a novel mechanism in T. gondii that contributes to the adverse pregnancy outcomes.

Pyruvate dehydrogenase kinase 1 regulates the function of human decidual natural killer cells.

Pyruvate dehydrogenase kinase 1 (PDK1) is an important enzyme for immune cell development. However, PDK1's role in human decidual natural killer (dNK) cells remains largely unknown. PDK1 expression in dNK cells from patients with recurrent spontaneous abortions (RSA) and age-matched healthy controls was analyzed by qRT-PCR, western bolt and flow cytometry. Moreover, dNK cells were treated with PDK1 inhibitoror the PDK1 siRNA followed by functional assays. The dNK cells from patients who underwent RSAs had higher mRNA expression and increased proteinof PDK1, perforin (PRF1), Granzyme B (GZMB), IFN-γ (IFNG), and CD107a expression compared to dNK cells from age-matched healthy controls. Perforin, Granzyme B, IFN-γ and CD107a expression levels in dNK cells were down-regulated when dNK cells were treated with a PDK1 inhibitor. As measured by the 51 Cr release assay, the killing activity of dNK cells was found to be decreased. We also demonstrated that PDK1 blockade could up-regulate the migration and adhesion of dNK cells. Furthermore, PDK1 inhibition reduced the glycolysis of dNK cells. This study suggested that PDK1 plays an important role in regulating dNK cell functions and human RSA.