uNK Cell Differentiation Research Articles

Continuous human uterine NK cell differentiation in response to endometrial regeneration and pregnancy.

Immune cell differentiation is critical for adequate tissue-specific immune responses to occur. Here, we studied differentiation of human uterine natural killer cells (uNK cells). These cells reside in a tissue undergoing constant regeneration and represent the major leukocyte population at the maternal-fetal interface. However, their physiological response during the menstrual cycle and in pregnancy remains elusive. By surface proteome and transcriptome analysis as well as using humanized mice, we identify a differentiation pathway of uNK cells in vitro and in vivo with sequential acquisition of killer cell immunoglobulin-like receptors and CD39. uNK cell differentiation occurred continuously in response to the endometrial regeneration and was driven by interleukin-15. Differentiated uNK cells displayed reduced proliferative capacity and immunomodulatory function including enhanced angiogenic capacity. By studying human uterus transplantation and monozygotic twins, we found that the uNK cell niche could be replenished from circulation and that it was under genetic control. Together, our study uncovers a continuous differentiation pathway of human NK cells in the uterus that is coupled to profound functional changes in response to local tissue regeneration and pregnancy.

Maternal exposure to CeO2NPs during early pregnancy impairs pregnancy by inducing placental abnormalities

Cerium dioxide nanoparticles (CeO2NPs) has been widely used in many fields, and also recommended as a promising carrier for cancer targeted drugs in human medicine for its excellent properties. However, its biological safety to human health remains controversial. In this study, we propose a mouse model exposed to CeO2NPs during early pregnancy, to clarify the effect of maternal CeO2NPs exposure and related molecular mechanism. Pregnant mice are injected intravenously with CeO2NPs by once a day on D5, D6, and D7. The effects of CeO2NPs exposure on pregnancy outcomes are observed on D8, D9, D10 and D12. The results show that CeO2NPs exposure during early pregnancy would lead to poor pregnancy outcomes. Further study find that low-quality decidualization, including the imbalance of trophoblast invasion regulators secreted by decidual cells and abnormal recruitment and differentiation of uNK cells, leads to subsequent biological negative “ripple effects”, including placental dysfunction, fetal loss or growth restriction. This study broadens the understanding of the biological safety of CeO2NPs, and provide clues for the prevention of its negative biological effects. Improving the function of uNK cells can be used as one of the therapeutic targets to prevent negative effects of CeO2NPs on pregnancy.

Low-Dose Aspirin and Uterine Natural Killer Cells in Mice at Day Seven of Pregnancy

Background: Inhibiting the effect of aspirin on prostaglandin results in corpus luteum maintenance in pregnant mice. Uterine natural killer (uNK) cells activate between 5 - 7 days of pregnancy. uNK cell differentiation is indirectly regulated by ovarian hormones. Immune cells have important roles in the endometrium at early pregnancy stages. Although low-dose aspirin is effective on ovarian hormones, the effect of low-dose aspirin on the uNK cell population at early pregnancy stages is unknown. Objectives: The aim of this study was to assess the effect of low-dose aspirin on uNK cells at day seven of pregnancy. Materials and Methods: Adult female mice were coupled and divided into two groups (control and experimental). Thereafter, mice in the experimental group received 7.5 mg/kg aspirin twice a day for one week. Blood samples were collected for the measurement of the level of ovarian hormones at day seven of pregnancy and finally the animals were sacrificed by cervical dislocation and uterine horns were removed for histological study. Periodic acid-schiff (PAS) staining was performed for uNK cell counting. Data analysis was conducted by SPSS software. Results: The ovarian hormones level showed a significant difference between the experimental and control groups (P < 0.05). Furthermore, the uNK cell population showed a significant difference between the two groups (P < 0.05). Conclusions: Increased ovarian hormones due to low-dose aspirin administration resulted in decline in the uNK cells number, which may be effective in successful pregnancy.

Influence of Atopic Dermatitis on Reproduction and Uterine Natural Killer Cells

ABSTRACTThe causal relationship between severe allergic conditions and successful pregnancy remains unclear. We aimed to evaluate reproductive performance in an experimental mouse model of atopic disease (AD), and the appearance of uterine natural killer (uNK) cells that have crucial roles in placental formation was examined. In the NC/Nga pregnant mice with moderate skin allergic lesions and an 8.6-fold elevation of plasma IgE, significant differences were not detected in the reproductive indices of the number of normal fetuses, abortion rate and placental size. There were few uNK cells in the placenta of AD mice, and they showed a significant decrease regarding the immature subtype as compared with controls. These findings revealed that AD disturbs uNK cell differentiation and provides disadvantageous effects on placental formation, although it does not arrest the pregnancy process. It may be possible that specific immunological conditions behind AD operate favorably to recover the reproductive performance.

Splenectomy Delays Uterine Natural Killer Cell Recruitment to Implantation Sites and Prolongs Pregnancy in Mice

Uterine natural killer (uNK) cells are the dominant lymphocytes found in pregnant mammals that develop uterine decidualization. Four stages of mouse uNK cell differentiation are recognized using Dolichos biflorus agglutinin (DBA) lectin histochemistry. Each uNK cell subtype has a preferential domain. In human and mouse normal pregnancies, uNK cells are activated interferon gamma-producing cells that promote angiogenesis and development of the decidua and placenta. Murine transplant models suggest that uNK cells differentiate from self-renewing progenitors found in peripheral secondary lymphoid tissues, particularly spleen and lymph nodes. In this work, the spleen was removed 7 days before mice were mated to address whether absence of the spleen reduced uNK cell numbers or altered the distributions of maturing uNK cell subsets using quantitative lectin histochemistry. Splenectomy delayed uNK cell maturation within implantation sites. This coincided with delayed decidual and placental development and a significant (48 hr) lengthening of gestation without loss of viability. These studies characterize spleen as a biologically important progenitor tissue for uNK precursor cells.

Maternal Heme Oxygenase 1 Regulates Placental Vasculature Development via Angiogenic Factors in Mice1

The placental vasculature is critical for nutrient, gas, and waste exchange between the maternal and fetal systems. Its development depends on the proper expression and interaction of angiogenesis and associated growth factors. Heme oxygenase (HMOX), the enzyme for heme degradation, plays a role in angiogenesis and is highly expressed in the placenta. To evaluate the role of maternal HMOX1, the inducible HMOX isozyme, on placental vasculature formation, mice with a partial deficiency in Hmox1 (Hmox1(+/-)) were used. Three-dimensional images of placental vasculatures as well as spiral arteries from Hmox1(+/+) or Hmox1(+/-) placentas were created by vascular corrosion casting technique and imaged by micro-computerized tomography (microCT). The structures and morphologies of fetomaternal interfaces were observed by histological staining and the ultrastructure of uterine natural killer (uNK) cells, a major regulator in spiral artery remodeling, was analyzed by transmission electron microscopy. A group of growth factors and angiogenic factors from the decidua/mesometrial lymphoid aggregate of pregnancy (MLAp) as well as labyrinth regions were quantified using an angiogenesis PCR array kit and compared between Hmox1(+/+) or Hmox1(+/-) placentas. In conclusion, a partial deficiency of maternal Hmox1 resulted in the malformation of fetomaternal interface, insufficiency of spiral artery remodeling, and alteration of uNK cell differentiation and maturation. These changes were independent of the fetal genotype, but relied on the maternal HMOX1 level, which determined the balance of expression levels of pro- and antiangiogenic factors in the decidua/MLAp region. These results implied that Hmox1 polymorphisms among the human population might contribute to some unexplained cases of pregnancy disorders, such as fetal growth retardation and preeclampsia.

Differentiation of Uterine Natural Killer Cells in Pregnant SCID (scid/scid) Mice

To determine whether functional T- and B-cells can affect differentiation and/or proliferation of uterine natural killer (uNK) cells, their numbers in SCID mice (genotype, C.B.-17/Icr-scid/scid) were compared with those of control mice (genotype, C.B.-17/Icr-+/+) on days 8, 12 and 16 of pregnancy. Using biotinylated-Dolichos biflorus agglutinin (DBA) lectin staining, uNK cells can be readily classified into 4 subtypes, I to IV, from immature to mature types. The number of uNK cells was significantly lower in the decidua basalis of SCID mice than in that of control mice on day 8 of pregnancy. Particularly, the number of uNK cells of immature subtype II was significantly lower in SCID mice than in the control mice. By day 12, however, the uNK cell number in the SCID mice reached the same level as that of the control mice. It is likely that uNK cell differentiation in SCID mice was delayed during the early placentation period due to a lack of functional T and B cells.

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule-1, P-selectin and ICAM-1 permit alpha(4)beta(7)-integrin-expressing DC, especially those of the myeloid lineage (CD11c(+)CD11b(+) DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule-1 in P-selectin(-/-) mice or experimental approaches with beta7-integrin(-/-) or ICAM-1(-/-) mice revealed that limited access or absence of CD11c(+)CD11b(+) DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into beta7-integrin(-/-) mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c(+)CD11b(+) DC are characterized by decreased IL-15 and IL-12 mRNA and/or protein levels. Chronic administration of IL-15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL-12 positively affects the IFN-gamma expression of uNK cells. Real-time RT-PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL-15 and IL-12 in the pregnant mouse uterus.

Uterine Natural Killer Cells in Perforin and .BETA.2-Microglobulin Deficient Mice

Uterine natural killer (uNK) cells have roles for immune responses at the feto-maternal interface in mice. We studied the effects of beta(2)-microglobulin (beta(2)m) and perforin on proliferation and differentiation of uNK cells in pregnancy, using beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice and perforin-deficient (P(-/-)) mice. The cell population of uNK cells in the metrial gland of P(-/-) mice was tended to be higher than the control B6 mice. The cell population of uNK cells in the metrial gland of beta(2)m(-/-) mice was significantly increased at Day 12 of pregnancy comparing to B6 and P(-/-) mice. On the other hand, the cell population of uNK cells in the decidua basalis of beta(2)m(-/-) mice was tended to be lower than B6 and P(-/-) mice. These results indicate that beta(2)m may be involved in proliferation of uNK cells in the metrial gland, and that beta(2)m may affect the maturation of uNK cells in the decidua basalis.

A Map of Relationships Between Uterine Natural Killer Cells and Progesterone Receptor Expressing Cells During Mouse Pregnancy

Uterine natural killer (uNK) cells appear in implantation sites with decidualization. Initially, they are rare, highly proliferative cells that become abundant by midgestation and then die abruptly. Steps regulating the cyclic appearance of uNK cells are incompletely defined. Although progesterone (P4) has an essential role, mature uNK cells of women and mice lack progesterone receptors (PR). Immunohistochemical studies suggest that mouse PR − uNK cells may co-localize with PR + stromal cells while human PR − uNK cells co-localize with immature, DC-SIGN + dendritic cells (DCs). DCs have the potential to produce progesterone-regulated interleukin (IL)-15, a growth factor essential for uNK cells. Since the high affinity IL-15Rα is presented to differentiating NK cells in trans, requiring cell contact, histologically-detected interactions may be of central importance for uNK cell differentiation. Thus, a pregnancy time course, histological study of uNK cell differentiation and localization was undertaken in PR lacZ transgenic mice. PR + cells and uNK cells were co-localized using LacZ histochemistry and Dolichos biflorus (DBA) lectin staining, respectively. uNK cells appeared mesometrially, where PR + cells were rare, at gestation day 5.5. uNK cells had limited, apparently random contact with PR + cells throughout pregnancy and never themselves expressed PR. Thus, uNK cell differentiation does not appear to require contact with PR + cells.

Uterine NK cells in murine pregnancy

Murine uterine natural killer (uNK) cells are transient, short-lived, terminally differentiated lymphocytes found in decidualized endometrium. Cells expressing natural killer cell surface markers are present in uteri of infant mice. Terminal uNK cell differentiation coincides with mesometrial decidual development subsequent to blastocyst implantation and begins about gestation day 5. uNK cells proliferate rapidly and, within 3 days, senescent uNK cells appear in normal implantation sites. Mid-gestation, senescent cells become dominant and uNK cell numbers decline until term when remaining cells are shed with the placenta. Transplantable uNK cell progenitors occur outside the uterus, suggesting that blood cell homing augments any in-utero progenitors. Early in healthy pregnancies, uNK cells produce cytokines and angiogenic molecules. Their lytic capacity in normal gestation and in pregnancy failure is incompletely defined. A significant shift recently occurred in thinking about major uNK cell functions. Activated uNK cells are now considered critical for appropriate endometrial angiogenesis in early implantation site development and in non-gestational endometrium. Because analogous cells appear in the endometria of women during each menstrual cycle and become abundant in early pregnancy, studies involving experimental pregnancy termination in genetically manipulated mice continue to have great importance for understanding regulation at the human maternal–fetal interface.

Differentiation and Elimination of Uterine Natural Killer Cells in Delayed Implantation and Parturition Mice

To clarify the roles of uterine natural killer (uNK) cells in implantation and parturition, differentiation and elimination of uNK cells in the pregnant uterus was examined using artificial delayed implantation (DI) and delayed parturition (DP) mice. To prepare DI mice, pregnant mice were ovariectomized on the third day of pregnancy (D3) and treated with 2 mg progesterone daily. The same amount of progesterone was administered on D15 or D17 of normal pregnant mice at 24 h intervals until sampling to prepare DP mice. The uNK cells contained PAS-positive granules on D8 in DI mice. The uNK cells in DI mice were smaller in size, and differentiation of these cells was delayed compared to those of the control mice. From D19 to D21 in DP mice, the metrial gland was well developed and uNK cells were present. The number of uNK cell granules decreased on D21, and there were no uNK cells in the normal pregnant mice. This result suggests that differentiation of uNK cells is not directly related to implantation, but elimination of these cells is closely involved in parturition.

Ultrastructural Study of Uterine Natural Killer Cells Found in Pregnant, Interleukin-2 Receptor .BETA.-chain Overexpressed Transgenic Mice

We previously reported that all fetuses died or were resorbed on day 12 of pregnancy (Day 1= the day of plug) in interleukin-2 (IL-2) receptor beta-chain overexpressed transgenic (Tg2Rbeta) mice. In this study, to clarify the role of uterine natural killer (uNK) cells in pregnancy, the ultrastructure of Tg2Rbeta mouse uNK cells was analyzed using a transmission electron microscope. uNK cells and their granules on day 10 of pregnancy were larger in Tg2Rbeta mice than control mice, indicating that differentiation of uNK cells in Tg2Rbeta mice progressed rapidly. Additionally, the granules of uNK cells in Tg2Rbeta mice on day 10 of pregnancy had an irregular morphology. The multivesicular regions were present in the cap structure of these granules, suggesting that the uNK cells of the Tg2Rbeta mice had cytotoxic activity.

Assessment of requirements for IL-15 and IFN regulatory factors in uterine NK cell differentiation and function during pregnancy.

In mouse and human, precursors of NK cell lineage home to decidualizing uteri. To assess the requirement for IL-15, an essential cytokine for NK differentiation in lymphoid tissue, on uterine NK (uNK) cell differentiation, implantation sites from IL-15(-/-) mice were analyzed histologically. IL-15(-/-) implantation sites had no uNK cells, no spiral-artery modification, and lacked the decidual integrity found in normal mice. IL-15(-/-) recipients of C57BL/6 marrow displayed similar pathology. However, implantation sites from recombination-activating gene-2(-/-)gamma(c)(-/-) (alymphoid) recipients of IL-15(-/-) marrow showed normal uNK cells, modified spiral arteries, and well-developed decidua basalis. Deletion of the IFN-regulatory factor (IRF)-1, but not IRF-2 (factors important in peripheral NK cell differentiation) limited but did not prevent uNK cell development. In situ hybridization localized IRF-1 largely to placental trophoblast cells. IRF-1(-/-) marrow transplanted into recombination-activating gene-2(-/-)gamma(c)(-/-) displayed competence for full uNK cell differentiation. IL-15 mRNA expression at implantation sites of IRF-1(-/-) and C57BL/6 was similar, suggesting that, unlike in bone marrow and spleen, IRF-1 does not regulate IL-15 in the pregnant uterus. Terminal differentiation of uNK cells was not promoted in pregnant IRF-1(-/-) mice by 5-day infusion of murine rIL-15, suggesting that IRF-1 deficiency rather than IL-15 deficiency limits uNK cell differentiation in these mice. Further, IRF-1 regulates placental growth, birth weight, and postnatal growth of offspring. These studies indicate that uNK cell development and maturation share some aspects with NK cell development in other tissues, but also display distinctive tissue-specific regulation.

Regulation of leukocyte recruitment to the murine maternal/fetal interface by specialized patterns of vascular adhesion receptors

Our findings reveal a remarkable regulation of leukocyte recruitment to specialized microdomains at the maternal/fetal interface during initial placenta development. Each of these microdomains is associated with unique patterns of vascular differentiation, including distinctive populations of leukocytes. A striking observation is the coexpression of the a4b7 ligand MAdCAM‐1 and P‐selectin on vessels of the vascular zone within the decidua basalis. This combination may restrict interactions to leukocytes combining the unusual ability to bind P‐selectin and MAdCAM‐1 (e.g. a4b7+ monocytes observed in these vessels). The maternal vessels of the central decidua basalis surrounded by a4b1+ uNK cells express the a4b1 ligand VCAM‐1. To assess directly the importance of identified vascular adhesion receptors TRITC‐labeled a4b7hi TK1 or a4b1+ L1/2 cells were injected i.v. and allowed to circulate for 1 hr. Then the uterus was sectioned to identify vascular sites of accumulation. TK1 cells were localized in the MAdCAM‐1 + P‐selectin+ vessels of the vascular zone whereas L1/2 cells interacted selectively with vessels in the central decidua basalis. Prior injection of blocking mABs against MAdCAM‐1 or VCAM‐1 inhibited accumulation of TK1 cells or L1/2 cells, respectively. Long‐term inhibition studies using blocking mABs against MAdCAM‐1, P‐selectin or a4b7 integrin as well as experiments with b7ko mice suggest a critical role of a4b7+ monocytes for the differentiation of uNK cells and placentation.

Differentiation of Murine Uterine NK Cells in Ectopically Grafted Uterine Tissues.

To investigate whether progesterone plays an essential role in differentiation of uNK cells, we ectopically transplanted tissue grafts from different developmental stages of uteri under various conditions. When female mice were used as recipients, decidualization and differentiation of uNK cells were observed in grafts from mature (8 weeks old) and immature (4 weeks old) donors, but there was no change in grafts from postnatal (10 days after birth) donors. When immature female mice were used as recipients, uNK cell differentiation was observed in grafts from both mature and immature donors, but decidualization and differentiation of uNK cells were not observed in the recipient uterus even in the presence of progesterone. These results suggest that uNK cell differentiation is not associated with the sexual maturation of recipients, and that the effect of progesterone on uNK cell differentiation is not direct, even though progesterone is an essential factor for uNK cell differentiation. When male mice were used as recipients, decidualization and differentiation of uNK cells were not observed in uterine grafts from normal mature mice. On the other hand, in castrated recipients uNK differentiation was induced in the graft, suggesting that uNK cell cannot fully differentiate in the presence of testosterone.

Uterine natural killer cells do not require interleukin-2 for their differentiation or maturation.

Natural Killer lymphocytes (NK cells) from the pregnant uterus and from other tissues in pregnant and nonpregnant mammals can be stimulated by interleukin-2 (IL-2) during culture to become Lymphokine Activated Killer (LAK) cells. The susceptibility of cultured trophoblast cells to lysis by LAK cells raises the enigma of why uterine (u) NK cells that are characterized by morphology and by surface phenotyping as "activated," and thus potentially damaging to the placenta, become localized to implantation sites during normal rodent gestation. uNK cells migrating from explant cultures of the metrial gland were assessed for expression (mRNA and protein) of each chain of the IL-2 receptor (IL-2R). Implantation sites from transgenic mice lacking a functional IL-2 gene were examined histologically for the differentiation of mature, granulated uNK cells. Early post-implantation, mRNA from migrating uNK cells contains transcripts for all three chains of the IL-2R. Only IL-2Rgamma was expressed at day 12 of gestation; expression of this gene was also lost by day 16. Loss of IL-2R transcription did not result in loss of protein expression; however, it did coincide with loss of uNK cell viability in vivo. Apparently normal differentiation of uNK cells occurred in IL-2(-/-) mice and in doubly mutant IL-2(-/-).beta2m(-/-) mice. Thus, despite uNK cell expression of the full IL-2R at day 8 of gestation, IL-2 is not required for the maturation of uNK cells to their fully granulated form or for normal placental development.