Decidual Natural Killer Cells Research Articles

The Multiple Faces of the Decidual Natural Killer Cell

The decidual NK (dNK) cell is called on to support placental growth by providing an array of growth factors that directly transform the spiral artery and direct trophoblast invasion. Successful transformation is dependent upon adequate stimulation paradoxically stimulating the cell for placental support rather than cytotoxicity. With the identification of its supportive role, the presence of an intact cytotoxic mechanism has been confusing. Investigators have found that the cell remains fully capable of cytotoxic responses particularly in response to pathogen-specific signals. We postulate a dual threshold model where moderate stimulation results in release of stimulatory factors supporting placentation while intense stimulation, particularly triggered through pathogen-specific receptors, restores the cell to its protective, cytotoxic, role. Individual dNK cells mature attaining the capacity to respond to the delivery of cognate signals. The process, known as 'licensing' tunes responsiveness to the degree to which stochastically selected inhibitory receptors block cytotoxic response to self. A changing licensing milieu within the decidua may result in altered and unsuitable receptor expression. We postulate that a heterogeneous population of dNK cells where cells inappropriately licensed for the milieu contributes to pathology.

Toxoplasma gondii Infection Regulates the Balance of Activating and Inhibitory Receptors on Decidual Natural Killer Cells

Inhibitory receptors and activating receptor expressed on decidual natural killer (dNK) cells are generally believed to be important in abnormal pregnancy outcomes and induced adverse pregnancy. However, if Toxoplasma gondii (T. gondii) infection induced abnormal pregnancy was related to dNK cells changes is not clear. In this study, we used human dNK cells co-cultured with human extravillous cytotrophoblast (EVT) cells following YFP-Toxoplasma gondii (YFP-T. gondii) infection in vitro and established animal pregnant infection model. Levels of inhibitory receptors KIR2DL4 and ILT-2, their ligand HLA-G, and activating receptor NKG2D in human decidua, and NKG2A and its ligand Qa-1 and NKG2D in mice uterine were analyzed by real-time PCR and flow cytometry with levels of NKG2D significantly higher than those of KIR2DL4 and ILT-2 in vitro and in invo. The level of NKG2D was positively correlated with cytotoxic activity of dNK cells in vitro. Numbers of abnormal pregnancies were significantly greater in the infected group than in the control group. This result demonstrated that the increased NKG2D expression and imbalance between inhibitory receptors of dNK cells and HLA-G may contribute to abnormal pregnancy outcomes observed upon maternal infection with T. gondii.

The Proangiogenic Phenotype of Natural Killer Cells in Patients with Non-Small Cell Lung Cancer

The tumor microenvironment can polarize innate immune cells to a proangiogenic phenotype. Decidual natural killer (dNK) cells show an angiogenic phenotype, yet the role for NK innate lymphoid cells in tumor angiogenesis remains to be defined. We investigated NK cells from patients with surgically resected non-small cell lung cancer (NSCLC) and controls using flow cytometric and functional analyses. The CD56+CD16- NK subset in NSCLC patients, which represents the predominant NK subset in tumors and a minor subset in adjacent lung and peripheral blood, was associated with vascular endothelial growth factor (VEGF), placental growth factor (PIGF), and interleukin-8 (IL-8)/CXCL8 production. Peripheral blood CD56+CD16- NK cells from patients with the squamous cell carcinoma (SCC) subtype showed higher VEGF and PlGF production compared to those from patients with adenocarcinoma (AdC) and controls. Higher IL-8 production was found for both SCC and AdC compared to controls. Supernatants derived from NSCLC CD56+CD16- NK cells induced endothelial cell chemotaxis and formation of capillary-like structures in vitro, particularly evident in SCC patients and absent from controls. Finally, exposure to transforming growth factor-β1 (TGFβ1), a cytokine associated with dNK polarization, upregulated VEGF and PlGF in peripheral blood CD56+CD16- NK cells from healthy subjects. Our data suggest that NK cells in NSCLC act as proangiogenic cells, particularly evident for SCC and in part mediated by TGFβ1.

FUNCTIONAL PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HUMAIN CYTOMEGALOVIRUS INFECTION

Event Abstract Back to Event FUNCTIONAL PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HUMAIN CYTOMEGALOVIRUS INFECTION Nabila Jabrane-Ferrat1* and Johan Siewiera1 1 Inserm UMR1043 - CNRS UMR5282 - Université Toulouse III, Immunology, France Circulating Natural Killer cells constitute the first line of defense against invading pathogens. These effectors of the innate immune system recognize and kill tumor or viral-infected cells. In addition to peripheral blood and lymphoid organs, NK cells (dNK) are found in the decidua basalis of the human pregnant uterus. Decidual NK cells secrete a large variety of cytokines, chemokines and growth factors but are poorly cytotoxic. We previously showed that dNK cells freshly isolated from the 1st trimester decidua basalis are able to kill target cells upon ligation of NKp46 activating receptor. This cytotoxic capacity is under the control of CD94/NKG2A inhibiting receptor. We challenged the hypothesis that upon viral infection, dNK cells receive activating signals that overcome the inhibitory signal and trigger their cytotoxic effector function. Using an ex vivo autologous system, we describe a mechanism by which dNK cells kill HCMV infected targets. We show that dNK cells engage activating immune synapse with HCMV-infected autologous targets. dNK cells stimulated with HCMV-infected targets are able to reorganize rapidly both their microtubule organizing center and their Golgi apparatus. These changes in cell morphology result in focused delivery of lytic granules and subsequent killing of target cells. Furthermore, dNK cells stimulated with infected autologous fibroblasts acquired a cytotoxic phenotype. Taken together our data provide the first evidence that dNK cells may be involved in the protection of the fetus against maternal viral infections. Immune synapse between dNK cell (perforin in red) and uterin fibroblast that was infected with human cytomegalovirus. Actin in green, alpha tubulin in blue. Images were taken on a Zeiss confocal microscope (x63) at the U1043 imaging core facility. Figure 1 References Siewiera J, El Costa H, Tabiasco J, Berrebi A, Cartron G, Le Bouteiller P, Jabrane-Ferrat N. Human cytomegalovirus infection elicits new decidual natural killer cell effector functions.PLoS Pathog. 2013 Apr;9(4):e1003257. Keywords: Natural Killer cells, Pregnancy, congenital infection, Cytomegalovirus, Cytotoxicity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Jabrane-Ferrat N and Siewiera J (2013). FUNCTIONAL PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HUMAIN CYTOMEGALOVIRUS INFECTION. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01137 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Nabila Jabrane-Ferrat, Inserm UMR1043 - CNRS UMR5282 - Université Toulouse III, Immunology, Toulouse, Midi-Pyrenees, 31000, France, nabila.jabrane-ferrat@inserm.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nabila Jabrane-Ferrat Johan Siewiera Google Nabila Jabrane-Ferrat Johan Siewiera Google Scholar Nabila Jabrane-Ferrat Johan Siewiera PubMed Nabila Jabrane-Ferrat Johan Siewiera Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Profiling Lgals9 Splice Variant Expression at the Fetal-Maternal Interface: Implications in Normal and Pathological Human Pregnancy1

Disruption of fetal-maternal tolerance mechanisms can contribute to pregnancy complications, including spontaneous abortion. Galectin-9 (LGALS9), a tandem repeat lectin associated with immune modulation, is expressed in the endometrium during the mid and late secretory phases and in decidua during human early pregnancy. However, the role of LGALS9 during pregnancy remains poorly understood. We used real-time PCR and immunohistochemical staining to analyze the expression of Lgals9/LGALS9 during mouse gestation as well as in human tissues obtained from normal pregnancy and spontaneous abortions. In mice, three Lgals9 splice variants were detected, the expression of which was differentially regulated during gestation. Furthermore, decidual Lgals9 expression was deregulated in a mouse model of spontaneous abortion, whereas placental levels did not change. We further found that the LGALS9 D5 isoform suppresses interferon gamma production by decidual natural killer cells. In human patients, six Lgals9 splice variants were detected, and a decrease in Lgals9 D5/10 was associated with spontaneous abortion. Altogether, these results show a differential regulation of Lgals9 isoform expression during normal and pathological pregnancies and designate Lgals9 as a potential marker for adverse pregnancy outcomes.

PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HCMV INFECTION

Event Abstract Back to Event PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HCMV INFECTION Nabila Jabrane-Ferrat1* and Johan Siewiera1 1 INSERM 1043 / CNRS 5282, Centre de Physiopathologie de Toulouse-Purpan, France Circulating Natural Killer cells constitute the first line of defense against invading pathogens. These effectors of the innate immune system recognize and kill tumor or viral-infected cells. In addition to peripheral blood and lymphoid organs, NK cells are found in the decidua basalis of the human pregnant uterus. Decidual NK cells secrete a large variety of cytokines, chemokines and growth factors but are poorly cytotoxic. We previously showed that dNK cells freshly isolated from the 1st trimester decidua basalis are able to kill target cells upon ligation of NKp46 activating receptor. This cytotoxic capacity is under the control of CD94/NKG2A inhibiting receptor. We challenged the hypothesis that upon viral infection, dNK cells receive activating signals that overcome the inhibitory signals and trigger their cytotoxic effector function. Using an ex vivo autologous system, we describe a mechanism by which dNK cells kill HCMV infected targets. We show that dNK cells engage activating immune synapse with HCMV-infected autologous targets. dNK cells stimulated with HCMV-infected targets are able to reorganize rapidly both their microtubule organizing center and their Golgi apparatus. These changes in cell morphology result in focused delivery of lytic granules and subsequent killing of target cells. Furthermore, dNK cells stimulated with infected autologous fibroblasts acquired a cytotoxic phenotype. Taken together our data provide the first evidence that dNK cells may be involved in the protection of the fetus against maternal viral infections. Figure 1 References Siewiera J, El Costa H, Tabiasco J, Berrebi A, Cartron G, Bouteiller P, Jabrane-Ferrat N. Human cytomegalovirus infection elicits new decidual natural killer cell effector functions. PLoS Pathog. 2013 Apr;9(4):e1003257. doi: 10.1371/journal.ppat.1003257. Epub 2013 Apr 4. Keywords: NK cells, Cytomegalovirus, preganancy, Nk receptors, decidua basalis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Jabrane-Ferrat N and Siewiera J (2013). PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HCMV INFECTION. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00542 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Nabila Jabrane-Ferrat, INSERM 1043 / CNRS 5282, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, Midi-Pyrenees, 31000, France, nabila.jabrane-ferrat@inserm.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nabila Jabrane-Ferrat Johan Siewiera Google Nabila Jabrane-Ferrat Johan Siewiera Google Scholar Nabila Jabrane-Ferrat Johan Siewiera PubMed Nabila Jabrane-Ferrat Johan Siewiera Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Natural killer cells promote immune tolerance by regulating inflammatory TH17 cells at the human maternal–fetal interface

Natural killer (NK) cells accumulate at the maternal-fetal interface in large numbers, but their exact roles in successful pregnancy remain poorly defined. Here, we provide evidence that T(H)17 cells and local inflammation can occur at the maternal-fetal interface during natural allogenic pregnancies. We found that decidual NK cells promote immune tolerance and successful pregnancy by dampening inflammatory T(H)17 cells via IFN-γ secreted by the CD56(bright)CD27(+) NK subset. This NK-cell-mediated regulatory response is lost in patients who experience recurrent spontaneous abortions, which results in a prominent T(H)17 response and extensive local inflammation. This local inflammatory response further affects the regulatory function of NK cells, leading to the eventual loss of maternal-fetal tolerance. Thus, our data identify NK cells as key regulatory cells at the maternal-fetal interface by suppressing T(H)17-mediated local inflammation.

Cellular senescence induced by CD158d reprograms natural killer cells to promote vascular remodeling

Natural killer (NK) cells, which have an essential role in immune defense, also contribute to reproductive success. NK cells are abundant at the maternal-fetal interface, where soluble HLA-G is produced by fetal trophoblast cells during early pregnancy. Soluble HLA-G induces a proinflammatory response in primary, resting NK cells on endocytosis into early endosomes where its receptor, CD158d, resides. CD158d initiates signaling through DNA-PKcs, Akt, and NF-κB for a proinflammatory and proangiogenic response. The physiological relevance of this endosomal signaling pathway, and how activation of CD158d through soluble ligands regulates NK cell fate and function is unknown. We show here that CD158d agonists trigger a DNA damage response signaling pathway involving cyclin-dependent kinase inhibitor p21 expression and heterochromatin protein HP1-γ phosphorylation. Sustained activation through CD158d induced morphological changes in NK cell shape and size, and survival in the absence of cell-cycle entry, all hallmarks of senescence, and a transcriptional signature of a senescence-associated secretory phenotype (SASP). SASP is a program that can be induced by oncogenes or DNA damage, and promotes growth arrest and tissue repair. The secretome of CD158d-stimulated senescent NK cells promoted vascular remodeling and angiogenesis as assessed by functional readouts of vascular permeability and endothelial cell tube formation. Retrospective analysis of the decidual NK cell transcriptome revealed a strong senescence signature. We propose that a positive function of senescence in healthy tissue is to favor reproduction through the sustained activation of NK cells to remodel maternal vasculature in early pregnancy.

Changes of human decidual natural killer cells cocultured with YFP-Toxoplasma gondii: implications for abnormal pregnancy

To investigate the changes of human decidual natural killer (dNK) cells cocultured with Toxoplasma gondii in vitro and to infer implications on pregnancy. Case-control study. College and hospital. Decidual tissue was obtained from 85 patients undergoing voluntary abortion during the first trimester of gestation (6-12 weeks). None. The dNK cells were isolated and infected with YFP-Toxoplasma gondii. Cells were observed by fluorescence and confocal microscopy. The CD56(bright)CD16(-)/CD56(dim)CD16(+) dNK ratio, expression of KIR2DL4, ILT-2, and NKG2D on dNK cells were analyzed by flow cytometry and the cytotoxic activity of infected dNK cells were evaluated. The CD56(dim)CD16(+)/CD56(bright)CD16(-) dNK ratio was significantly elevated at 12, 24, and 48 hours after YFP-T. gondii infection. Expression of KIR2DL4, ILT-2, and NKG2D were increased after infection, but NKG2D were significantly higher than those of KIR2DL4 and ILT-2. Both the CD56(dim)CD16(+)/CD56(bright)CD16(-) dNK ratio and NKG2D expression were correlated with dNK cytotoxic activity. Enhanced dNK cytotoxicity due to increased CD16 and NKG2D expression may contribute to abnormal pregnancy outcomes observed with maternal infection with T. gondii.

Increased expression of Toll-like receptor 3 in decidual natural killer cells of patients with unexplained recurrent spontaneous miscarriage

To investigate whether the expression of Toll-like receptor 3 (TLR3) in decidual natural killer (dNK) cells plays a role in the pathogenesis of unexplained recurrent spontaneous miscarriage (URSM). NK cells were isolated from human decidual tissue through magnetic cell sorting and analyzed for phenotypes through flow cytometry. TLR3 expression in dNK cells was detected by quantitative reverse-transcriptase polymerase chain reaction and western blotting. The cytotoxicity of dNK was detected using a lactate dehydrogenase release test. The CD56(+) dNK percentages varied between 85% and 96%. The expression of TLR3 mRNA and protein in dNK cells of URSM patients was statistically significantly higher than that in control women (p=0.005 and p=0.01, respectively). Meanwhile, the mean cytotoxicity percentage of freshly isolated dNK cells from the patients was significantly higher than that from normal controls (31.24 ± 7.46% vs. 16.20 ± 6.03%; p=0.03). The expression of TLR3 in dNK cells is abnormally higher in URSM patients, which indicates that TLR3 expression may be critical to the activation of dNK cells infiltrating the maternal-fetal interface and account for the loss of immune tolerance.

Specific decidual CD14+ cells hamper cognate NK cell proliferation and cytolytic mediator expression after mucin 1 treatment in vitro

Mucin 1 (MUC1) forms a glycocalyx on the surface of decidual epithelial cells that needs to be removed for successful embryo attachment. We investigated whether MUC1 affects human early pregnancy decidual CD14+ cells and their interactions with cognate decidual natural killer (NK) cells. FITC-dextran internalisation, surface and intracellular antigen levels, and proliferation of CD14+ and/or CD56+ cells were analysed by flow cytometry. Magnetic separation was used to purify CD56+ and CD14+ cells. Uncultured CD14+ cells expressed a negligible percentage of CD1a and CD83 molecules. They expressed lower levels of CD16, and higher levels of endocytic mannose receptors (MR), dendritic cell-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN), proinflammatory chemokine CC receptor 5 (CCR5), and CD163 receptor, than their peripheral blood counterparts. Lipopolysaccharide stimulation did not affect FITC-dextran internalisation in CD14+ cells. MUC1 bound and internalised, in a dose-dependent manner, the carbohydrate recognition domain of MR, increasing the decoy IL-1 receptor type II and decreasing IL-15 expression in CD14+ cells. In the presence of MUC1-treated macrophages, the expression levels of the proliferation and cytotoxic mediators (perforin, Fas ligand and TNF-related activation-induced ligand or TRAIL) was attenuated, while that of the anti-inflammatory chemokine CCL17 was increased, in NK cells compared with untreated macrophages. In conclusion, MUC1 supports the alternative activation of tissue-specific CD14+ cells, and may restrict proliferation of NK cells and regulate their content of cytotoxic mediators. Based on the experiments with first-trimester decidual cells in vitro, we conclude that removing MUC1 from decidual tissue might help control trophoblast invasion by NK cells.

Impaired decidual natural killer cell regulation of vascular remodelling in early human pregnancies with high uterine artery resistance

During human pregnancy, natural killer (NK) cells accumulate in the maternal decidua, but their specific roles remain to be determined. Decidual NK (dNK) cells are present during trophoblast invasion and uterine spiral artery remodelling. These events are crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Remodelling of spiral arteries is impaired in the dangerous pregnancy complication pre-eclampsia. We studied dNK cells isolated from pregnancies at 9–14 weeks' gestation, screened by uterine artery Doppler ultrasound to determine resistance indices which relate to the extent of spiral artery remodelling. dNK cells were able to promote the invasive behaviour of fetal trophoblast cells, partly through HGF. Cells isolated from pregnancies with higher resistance indices were less able to do this and secreted fewer pro-invasive factors. dNK cells from pregnancies with normal resistance indices could induce apoptotic changes in vascular smooth muscle and endothelial cells in vitro, events of importance in vessel remodelling, partly through Fas signalling. dNK cells isolated from high resistance index pregnancies failed to induce vascular apoptosis and secreted fewer pro-apoptotic factors. We have modelled the cellular interactions at the maternal-fetal interface and provide the first demonstration of a functional role for dNK cells in influencing vascular cells. A potential mechanism contributing to impaired vessel remodelling in pregnancies with a higher uterine artery resistance is presented. These findings may be informative in determining the cellular interactions contributing to the pathology of pregnancy disorders where remodelling is impaired, such as pre-eclampsia.Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chemerin Regulates NK Cell Accumulation and Endothelial Cell Morphogenesis in the Decidua during Early Pregnancy

Although decidual natural killer (NK) cell accumulation and vascular remodeling are critical steps to ensure successful pregnancy, the molecular mechanisms controlling these events are poorly defined. Herein we analyzed whether chemerin, a recently identified chemoattractant involved in many pathophysiological processes, could be expressed in the uterine compartment and could regulate events relevant for the good outcome of pregnancy. Chemerin expression in human primary culture of stromal (ST) cells, extravillous trophoblast cells, and decidual endothelial cells (DEC) was analyzed by RT-PCR, ELISA, and Western blot. Migration through ST or DEC of peripheral blood and decidual (d) NK cells from pregnant women was performed using a transwell assay. A DEC capillary-like tube formation assay was used to evaluate endothelial morphogenesis. Chemerin is differentially expressed by decidual cells during early pregnancy being present at high levels in ST and extravillous trophoblast cells but not in DEC. Notably, ST cells from pregnant women exhibit and release higher levels of chemerin as compared with ST cells from menopausal or fertile nonpregnant women. Chemerin can support peripheral blood NK cell migration through both DEC and ST cells. Although dNK cells exhibit lower chemerin receptor (CMKLR1) expression than their blood counterpart, CMKLR1 engagement on dNK cells resulted in both ERK activation and migration through decidual ST cells. Interestingly, DEC also express CMKLR1 and undergo ERK activation and capillary-like tube structure formation upon exposure to chemerin. Our data indicate that chemerin is up-regulated during decidualization and might contribute to NK cell accumulation and vascular remodeling during early pregnancy.

PP065. dNK and dNK-CM mediated alterations of DNA methylation in extravillous cytotrophoblasts (EVTS)

Placental DNA methylation is thought to be influenced by environmental exposure. Decidual natural killer cells (dNKs) directly contact with cytotrophoblasts in the early stage of pregnancy. dNKs may affect DNA methylation of extravillous cytotrophoblasts (EVTs) directly or indirectly through their secreted soluble factors. Previously, we showed that EVT outgrowth and migration on collagen gel were restricted by exposure to dNK or dNK-derived conditioned medium (dNK-CM) (ref [1]). The aim of this study was to determine if EVT DNA methylation was altered by treating with dNK or dNK-CM. Placental explants collected from 6 first-trimester healthy pregnancy terminations were cultured on a rat collagen gel model. Outgrowth EVTs from each subject were treated with medium or concordant dNK (trapped inside of hollow fibers) or dNK-CM. EVTs were harvested after 96-hour co-culturing and underwent DNA extraction. DNA methylation was quantified using the Infinium Human Methylation 450 BeadChip, which targets over 450,000 CpG sites in the human genome. Differential methylation was defined by having p<0.05 (Student's t-test) and average DNA methylation change >10% before and after treatments. Functional enrichment was assessed by gene ontology analysis with False Discovery Rate <10% defined as significantly enriched. Increased DNA methylation was observed for 360 loci and 572 loci by dNK or dNK-CM respectively, and decreased DNA methylation was shown for 62 loci and 188 loci by dNK or dNK-CM respectively. DNA methylation at 44 loci was altered by both dNK and dNK-CM. The common loci were overrepresented for associations with EVT differentiation, adhesion and migration. Examples of the relevant overlapped loci with increased DNA methylations were MYO15A and PRDM16 (PR domain zinc finger protein 16); and the overlapped loci with reduced DNA methylation were CDH9 and USP29 (ubiquitin specific protein 29). dNK but not dNK-CM reduced IL18 methylation and increased methylation on ITGAL (integrin, alpha L) and ITGB7. dNK-CM but not dNK reduced methylation of ITGAD and PCDH8 (protocadherin 8) and increased methylation of CDH4 and CDH6. DNA methylation of EVT was altered by exposure to surrounding dNK and their secreted soluble molecules. These results serve as a basis for further investigations on whether DNA methylation can mediate the changes in protein expression that influence EVT differentiation, adhesion and migration.

Extravillous trophoblast and decidual natural killer cells: a remodelling partnership

During pregnancy, maternal uterine spiral arteries (SAs) are remodelled from minimal-flow, high-resistance vessels into larger diameter vessels with low resistance and high flow. Fetal extravillous trophoblasts (EVT) have important roles in this process. Decidual natural killer cells (dNK cells) are the major maternal immune component of the decidua and accumulate around SAs before trophoblast invasion. A role for dNK cells in vessel remodelling is beginning to be elucidated. This review examines the overlapping and dissimilar mechanisms used by EVT and dNK cells in this process and how this may mirror another example of tissue remodelling, namely cancer development. The published literature was searched using Pubmed focusing on EVT, dNK cells and SA remodelling. Additional papers discussing cancer development are also included. Similarities exist between actions carried out by dNK cells and EVT. Both interact with vascular cells lining the SA, as well as with each other, to promote transformation of the SA. EVT differentiation has previously been likened to the epithelial-mesenchymal transition in cancer cells, and we discuss how dNK-EVT interactions at the maternal-fetal interface can also be compared with the roles of immune cells in cancer. The combined role that dNK cells and EVT play in SA remodelling suggests that these interactions could be described as a partnership. The investigation of pregnancy as a multicellular system involving both fetal and maternal components, as well as comparisons to similar examples of tissue remodelling, will further identify the key mechanisms in SA remodelling that are required for a successful pregnancy.

Dynamic Shift from CD85j/ILT-2 to NKG2D NK Receptor Expression Pattern on Human Decidual NK during the First Trimester of Pregnancy

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1+/KIR2DL2/L3/S2+ subset towards the double negative subset, coupled with a decrease of the CD85j+/NKG2D− subset in favour of the CD85j−/NKG2D+ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14+ and CD3+ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14+ and CD3+ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14+ cells whereas it was not detected on CD3+ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.

Immunophenotype and Cytokine Profiles of Rhesus Monkey CD56bright and CD56dim Decidual Natural Killer Cells1

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56(bright) natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56(bright) CD16(+)NKp30(-) decidual NK cells, with minor CD56(dim) and CD56(neg) dNK cells. Intracellular cytokine staining demonstrated that CD56(dim) and not CD56(bright) dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.

Decidual spiral artery remodeling during early post-implantation period in mice: Investigation of associations with decidual uNK cells and invasive trophoblast

Circumferential remodeling of spiral arteries (SAs) during pregnancy is crucial for regulating maternal blood flow into the placenta and clinically important. However its mechanism is still ill defined in humans and mice. In mice, several important aspects of decidual SA remodeling (SAR) remain unexplored and were addressed here using morphometrics by examining SAs within the mesometrial half of the decidua basalis between embryonic day 6.5 (E6.5) and midgestation (E10.5). The data presented here provide evidence that supports the following novel conclusions about SAR: (a) SAs (defined by their muscular walls) appear between E6.5 and E7.5, undergo ‘outward hypertrophic’ SAR (SA lumen widening with muscular wall thickening) during the E7.5–E8.5 and E9.5–E10.5 periods, and ‘outward hypotrophic’ SAR (SA lumen widening with muscular wall thinning) during the E8.5–E9.5 interval. (b) ‘Outward hypotrophic’ SAR is associated with decreases in the overall number, but not density, of SA media nuclei, suggesting loss of SA muscular wall cells. Proximity of placenta-derived invasive trophoblast to SAs appears not be involved in SAR, as these cells were undetectable in the mesometrial region of decidua basalis throughout the E6.5–E10.5 period. Although the maternally derived lymphocytes of the decidual uterine natural killer (uNK) cell type are required for decidual SAR, the timing of this, the uNK cell parameter involved and the type of SAR they influence have not been adequately explored. Evidence is presented here that not all decidual SAR during this period is uNK cell-dependent. Rather, the data suggest that uNKs only influence ‘outward hypotrophic’ SAR during the E8.5–E9.5 period. Evidence is presented that the uNK cell parameter involved is the attainment of a certain maturation state (based on uNK cell size) by SA wall uNKs of the Dolichos biflorus agglutinin (DBA) lectin-positive uNK cell subset. This work also suggests that the previously shown loss of contractile mural cell character from the SA wall does not depend on either uNKs or trophoblast proximity. The novel implications of the present data for early mouse pregnancy are discussed.

CYR61 Modulates the Vascular Endothelial Growth Factor C Expression of Decidual NK Cells Via PI3K/AKT Pathway

Either vascular endothelial growth factor C (VEGFC) or CYR61 plays an important role in placental development and may be involved in pre-eclampsia. Decidual natural killer (dNK) cells are the main source of VEGFC in the maternal-fetal interface. However, it is unclear about CYR61 on the regulation of VEGFC secretion in dNK cells. Decidual natural killer cells were isolated from decidual tissues of first trimester of pregnancy with anti-human CD56-conjugated microbeads. Integrin αvβ3 was detected using immunofluorescent staining. dNK cells were cultured in the presence of CYR61, anti-human αvβ3 integrin antibody (LM609), PI3K inhibitor (LY294002), or MEK inhibitor (U0126). VEGFC mRNA and protein were evaluated by real-time PCR and ELISA, respectively. Exogenous CYR61 induced the expression of VEGFC in dNK cells in both mRNA and protein levels. Integrin αvβ3 was strongly expressed on dNK cell surface. Anti-αvβ3 integrin antibody inhibited the effect of CYR61 on VEGFC expression. LY294002, but not U0126, significantly reduced this promotion effect of CYR61 on dNK cells. The upregulation of VEGFC secretion mainly depends on CYR61 binding with integrin αvβ3 on the surface of dNK cells. PI3K/AKT, rather than the ERK/MAPK signal, is involved in the regulation.

Decidual NK Cell‐Derived Conditioned Medium (dNK‐CM) Mediates VEGF‐C Secretion in Extravillous Cytotrophoblasts

The regulatory mechanisms involved in VEGF-C secretion by trophoblasts during placentation are poorly understood. We investigated whether or not decidual natural killer cell conditioned medium (dNK-CM) stimulated VEGF-C secretion in the extravillous cytotrophoblast (EVT) cell line HTR8/SVneo. The effects of dNK-CM and recombinant IFN-γ on VEGF-C induction by HTR8/SVneo were studied in the absence or presence of IFN-γ or its receptor blocking antibodies, p38 inhibitor (SB202190), JAK inhibitor (JAK inhibitor-1, JI-1), and on STAT1 knockdown HTR8/SVneo. VEGF-C was quantified by ELISA. FACS was used to investigate the phosphorylations of Tyr701 or Ser727 of STAT1 on stimulated HTR8/SVneo. dNK-CM facilitated VEGF-C secretion by HTR8/SVneo. IFN-γ and IFN-γR1 or IFN-γR2 blocking antibodies reduced both dNK-CM- and IFN-γ-induced VEGF-C secretion. Phosphorylations on Tyr701 or Ser727 of STAT1 were elevated upon stimulation. Secretion of VEGF-C was reduced by treatment with SB202190, JI-1, or STAT1 knockdown by siRNA. VEGF-C production by trophoblasts is regulated by soluble factors secreted by dNK through p38 and JAK-STAT1 pathways.