Chandrakant Tayade

Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones

Summary:  In adult females of many species, a transient population of natural killer (NK) cells appears in cycles within the uterine endometrium (lining). Appearance of these lymphocytes coincides with specific phases of the ovarian hormone cycle and/or early pregnancy. Studies in rodents, women, and pigs dominate the literature and suggest the uterine (u)NK cells are an activated subset sharing many but not all features with circulating or lymphoid organ‐residing NK cells. During successful murine pregnancy, uNK cells appear to regulate initiation of structural changes in the feed arterial systems that support maternal endometrial tissue at sites of implantation and subsequent placental development. These changes, which reverse after pregnancy, create a higher volume arterial bed with flaccid vessels unresponsive to vasoactive compounds. These unique pregnancy‐associated arterial changes elevate the volume of low‐pressure, nutrient‐rich, maternal arterial blood available to conceptuses. Regulation of the differentiation, activation, and functions of uNK cells is only partially known, and there is lively debate regarding whether and how uNK cells participate in infertility or spontaneous abortion. This review highlights the biology of uNK cells during successful pregnancy.

Interferon Gamma in Successful Pregnancies

Abstract Interferon gamma (IFNG) is a proinflammatory cytokine secreted in the uterus during early pregnancy. It is abundantly produced by uterine natural killer cells in maternal endometrium but also by trophoblasts in some species. In normal pregnancies of mice, IFNG plays critical roles that include initiation of endometrial vasculature remodeling, angiogenesis at implantation sites, and maintenance of the decidual (maternal) component of the placenta. In livestock and in humans, deviations in these processes are thought to contribute to serious gestational complications, such as fetal loss or preeclampsia. Interferon gamma has broader roles in activation of innate and adaptive immune responses to viruses and tumors, in part through upregulating transcription of genes involved in cell cycle regulation, apoptosis, and antigen processing/presentation. Despite this, rodent and human trophoblast cells show dampened responses to IFNG that reflect the resistance of these cells to IFNG-mediated activation of major histocompatibility complex (MHC) class II transplantation antigen expression. Lack of MHC class II antigens on trophoblasts is thought to facilitate survival of the semiallogeneic conceptus in the presence of maternal lymphocytes. This review describes the dynamic roles of IFNG in successful pregnancy and briefly summarizes data on IFNG in gestational pathologies.

Differential Gene Expression in Endometrium, Endometrial Lymphocytes, and Trophoblasts during Successful and Abortive Embryo Implantation

Prenatal mortality reaching 30% occurs during the first weeks of gestation in commercial swine. Mechanisms for this are unknown although poor uterine blood supply has been postulated. In other species, vascular endothelial growth factor, hypoxia-inducible factor 1-α, and IFN-γ regulate gestational endometrial angiogenesis. Vascular endothelial growth factor and hypoxia-inducible factor 1-α are also important for placental angiogenesis while trophoblastic expression of Fas ligand is thought to protect conceptuses against immune-mediated pregnancy loss. In this study, we document dynamic, peri-implantation differences in transcription of genes for angiogenesis, cytokine production, and apoptosis regulation in the endometrium, and laser capture microdissected endometrial lymphocytes and trophoblasts associated with healthy or viable but arresting porcine fetuses. In healthy implantation sites, endometrial gene expression levels differed between anatomic subregions and endometrial lymphocytes showed much greater transcription of angiogenic genes than trophoblasts. In arresting fetal sites, uterine lymphocytes had no angiogenic gene transcription and showed rapid elevation in transcription of proinflammatory cytokines Fas and Fas ligand while trophoblasts showed elevated transcription of IFN-γ and Fas. This model of experimentally accessible spontaneous fetal loss, involving blocked maternal angiogenesis, should prove valuable for further investigations of peri-implantation failure of normally conceived and surgically transferred embryos in many species, including the human.

Lymphocyte contributions to altered endometrial angiogenesis during early and midgestation fetal loss

Peri‐implantation and midgestational fetal losses reduce potential litter sizes up to 40% in commercial swine. Peri‐implantation studies [gestation days (gd)15–23] of porcine RNA from laser capture microdissected uterine lymphocytes and biopsies of mesometrial endometrium and trophoblast previously linked gd21–23 fetal arrest with transcriptional deficits in vascular endothelial growth factor (VEGF) and its regulatory factor, hypoxia inducible factor (HIF)‐1α, and with elevations in IFN‐γ and TNF‐α and suggested endometrial lymphocytes played a pivotal, proangiogenic role in fetal survival. Here, we address more comprehensively porcine endometrial angiogenesis by comparing transcription between endometrial endothelium and lymphocytes during early (gd20) and midgestation (gd50) losses and by incorporation of histopathology and protein immunolocalization of VEGF, placenta growth factor (PlGF), VEGF receptor I (VEGFRI), and VEGFRII. In healthy sites, endometrial lymphocytes transcribed more VEGF at gd50 than gd20, and transcripts were more abundant in lymphocytes than in endothelium or trophoblast. Arterial endothelial cells showed the most abundant transcription of PlGF. With fetal arrest, maternal transcripts for VEGF but not PlGF dropped, and fetal transcripts remained relatively stable. Maternal and fetal HIF‐1α transcription declined. Lymphocytes preferentially transcribed VEGFRI over VEGFRII, and endometrial arterial endothelium and trophoblast preferentially transcribed VEGFRII. IFN‐γ and TNF‐α transcripts were present in gd20 and gd50 healthy‐ and arresting‐implantation sites. gd20 arrest was associated with greater transcription of IFN‐γ than TNF‐α in maternal and fetal tissues. At gd50, this was reversed. Endometrial, vascular pathology was evident only at gd50. These data suggest the critical importance for lymphocyte‐driven endometrial angiogenesis, which extends to midgestation.

A Review of Trafficking and Activation of Uterine Natural Killer Cells

Enrichment of uterine natural killer (uNK) cells occurs during pregnancy in many species. However, functions of uNK cells and regulation of their uterine homing are not fully defined. In mice and women, uNK cells contribute to angiogenesis, a role reviewed here and now addressed in a mammal with an alternative placental type.

Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells

During human and rodent uterine decidualization, transient but abundant numbers of uterine natural killer (uNK) cells appear, proliferate, and differentiate. uNK cells share features with peripheral NK cells but are specialized to promote interferon‐γ (IFN‐γ)‐mediated, pregnancy‐associated, structural changes in maternal placental arteries. In CD8+ T cells and NK cells, the transcription factors T‐bet and eomesodermin (Eomes) regulate maturation and effector functions, including IFN‐γ production. No studies are reported for uNK cells. Implantation sites in T‐bet null mice, which have a defect in NK cell maturation, had uNK cells normal in morphology and number and normally modified spiral arteries. As Eomes null mice are not viable, real‐time polymerase chain reaction comparisons between C57Bl/6J (B6) and alymphoid (Rag20/0γc0/0) mice were used to assess uNK cell expression of T‐bet, Eomes, and the target genes IFN‐γ, granzyme A, and perforin. Gestation dated (gd) uterine tissues (mixed cell composition) and 200 morphologically homogeneous, laser‐capture, microdissected uNK cells of different maturation stages were used. In uterus, Eomes transcripts greatly outnumbered those of T‐bet, whether donors were nonpregnant or pregnant, and increased to gd10. In uNK cells, transcripts for T‐bet, Eomes, and IFN‐γ were most abundant in mature stage cells, and transcripts for granzyme A and perforin were lower at this stage than in immature or senescent cells. Thus, Eomes dominance to T‐bet discriminates regulation of the uNK cell subset from that observed for peripheral NK 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.

A Review of Molecular Contrasts Between Arresting and Viable Porcine Attachment Sites

Significant spontaneous fetal loss of unknown cause occurs in North American commercial swine. About 30% of conceptuses, thought to be genetically normal, are lost during the peri‐attachment period. An additional 20% are lost at mid‐pregnancy. Littermate endometrial and trophoblast biopsies were studied by quantitative real‐time PCR for gene expression, and immunohistochemistry for protein expression at gestation day (gd)15–23 and 50. RNA analyses were also conducted on endometrial lymphocytes and arterial endothelial cells removed from biopsies by laser capture microdissection. Genes were selected for study from human literature and cloned as required. As in humans, angiogenic, cytokine, chemokine and chemokine decoy receptor gene expression occurs at the porcine maternal–fetal interface. In each tissue studied, distinct patterns of expression are found between early and mid‐pregnancy, as well as between viable and arresting conceptus attachment sites. These changes involve both endometrial lymphocytes and dendritic cells. Restriction in endometrial angiogenesis, reduction in expression of the chemokine decoy receptor D6, and reduction in dendritic cell numbers contribute to fetal arrest. In peri‐attachment loss, interferon‐γ is more abundantly transcribed than tumor necrosis factor‐α, but this ratio is reversed during midgestation failure. Further characterization of spontaneous fetal loss in pigs will identify targets for modification by hog producers and may provide a model for identification of antecedents to fetal loss in humans.

Analysis of uterine natural killer cells in mice.

The term uterine natural killer (uNK) cell is applied in mice to an abundant but transient NK cell population that undergoes unique, terminal differentiation within embryo implantation sites during endometrial decidualization and pregnancy. In mice, decidualization is induced by attachment and implantation of hatched, blastocyst-stage embryos. Within each implantation site, uNK cells proliferate and rapidly differentiate into highly restricted regions called decidua basalis and the mesometrial lymphoid aggregate of pregnancy (MLAp). uNK cells begin to die within healthy decidua basalis by day 8 of the 19-20 day pregnancy of mice. By gestation day 12, uNK cell numbers have peaked and most uNK cells show in situ nuclear fragmentation indicative of disintegration. Morphological studies (standard histology, ultrastructure, immunohistochemistry, in situ hybridization, and RNA analyses from laser capture microdissected uNK cells) have provided most of the current understanding regarding this cell lineage. These approaches identified the special angiogenic properties of uNK cells and their regulatory relationships with normal physiological changes to the uterine (endometrial) arterial tree that accompany successful pregnancy. This chapter highlights key information needed for successful dissection of the dynamically changing decidua basalis that is enriched in uNK cells and special morphological procedures used for uNK cell study. Preparation of viable mouse uNK cell suspensions is difficult but can be achieved. This chapter includes techniques for isolation of uterine leukocyte suspensions and their enrichment for uNK cells that permit immediate downstream applications such as culture, isolation of high quality RNA, or flow cytometry.

Functions of alpha 2 macroglobulins in pregnancy

The alpha 2 macroglobulins (A2M) are a family of abundant plasma proteins produced predominantly by the mammalian liver. Pregnancy zone proteins (PZP) of humans and rats are A2M family members that bind a wide variety of macromolecules including the important pregnancy-associated molecules such as vascular endothelial growth factor, placenta growth factor and glycodelin (also called PP14). Recently, a mouse gene analogous to PZP (A2M of pregnancy or A2Mp) was cloned. A2Mp has a unique pattern of expression in reproductive and cardiovascular tissues and, unexpectedly, is not expressed by liver. Since changes in heart function and remodeling of renal and uterine vasculature are amongst the earliest maternal responses to pregnancy, the product of the A2Mp gene has been postulated to systemically regulate these changes. A2Ms with and without non-covalently bound ligands also down regulate immune cell activation but promote immune cell migration, additional features associated with gestational success. Here, we review the A2M gene families of mice and humans, the predicted structural relationships between A2M and its pregnancy induced forms and the postulated roles for this gene family in normal pregnancy.