NK cells, hypoxia and trophoblast cell differentiation

Abstract

Hemochorial placentation is characterized by extensive remodeling of the maternal vasculature, converting them to flaccid low resistance vessels. This process greatly facilitates exchange of nutrients and gases between the mother and the fetus. Two key modulators that orchestrate these vascular changes have been identified at the maternal fetal interface, natural killer (NK) cells and invasive trophoblast cells. Hypoxia-inducible factor (HIF) transcription factors direct cellular responses to low oxygen, influencing trophoblast lineage commitment and promoting development of the invasive trophoblast lineage. This short review focuses on role of NK cells on uterine spiral artery development and subsequent modulation of oxygen tensions at the maternal fetal interface.

Growth and development of the embryo within the female reproductive tract are possible because of specific contributions from the placenta.^1,2 Among mammals, the placenta has taken on several shapes, sizes and organizational structures. The functional cell type of the placenta is the trophoblast cell. Trophoblast cells are part of a multiple lineage differentiation pathway and can specialize to modify the maternal environment and facilitate nutrient flow to the developing fetus. Humans and laboratory rodents possess a hemochorial placenta.^1,2 Hemochorial placentation is characterized by extensive remodeling of the maternal uterine vasculature, yielding flaccid, low-resistance blood vessels that facilitate the exchange of nutrients and wastes between mother and fetus. These pregnancy-dependent vascular modifications are orchestrated by the cooperative effort of two cell types: maternal natural killer (NK) cells and invasive trophoblast cells (also called extravillous trophoblast).

Maternal NK cells and invasive trophoblast cells exhibit dynamic changes during the course of gestation.^2,3 Pregnancy is associated with a redistribution/recruitment of immune cells within the placentation site.⁴ Among these changes is an expansion of NK cells in the uterus and their association with uterine spiral arteries. This pregnancy-dependent uterine NK cell expansion is conserved in laboratory rodents (mouse and rat) and primates, including the human.⁵ Among the specialized differentiated trophoblast cells of the placentation site are populations of cells with the capacity to migrate, invade the uterine parenchyma and associate with the vasculature.^3,6-8 These cell types are defined by their relationships with the uterine spiral arteries. Those cells located between blood vessels are referred to as interstitial invasive trophoblast cells, and those cells embedded within vessels and contributing to the replacement of the endothelium are termed endovascular invasive trophoblast cells. Invasive trophoblast cells have their origins in stem and precursor cells arising from trophectoderm during the first differentiation events of embryo development and are subsequently situated within a region of the rodent chorioallantoic placenta termed the junctional zone.^9,10 These stem/precursor cells also seed distinct trophoblast cell lineages in the labyrinth zone, which specialize in bidirectional transport of nutrients and wastes between maternal and fetal compartments.¹¹

Compromised uterine spiral artery remodeling is associated with placentation-related diseases such as early pregnancy loss, preeclampsia and intrauterine growth restriction.^7,8 Failures in NK cell and invasive trophoblast cell lineage development are linked to these disorders.^4,7,8,12

NK Cells Modulate the Timing of Critical Events during Hemochorial Placentation

Strategies have been employed to investigate roles for NK cells during the establishment of pregnancy. These have included genetic mouse models possessing NK cell deficiencies¹³ and systemic immunodepletion of NK cells using administration of antibodies to asialo GM1 during rat gestation.¹⁴ The success of the immunodepletion approach is gestation stage-dependent and most effective when administered immediately prior to or concurrent with embryo implantation. From the mouse genetic models, we learned that NK cells promote uterine spiral artery remodeling, including decreasing arterial smooth muscle wall thickness and increasing vessel lumen diameter.¹⁵ NK cell immunodepletion in the rat results in striking changes in the uterine spiral arteries, characterized initially by a delay in uterine spiral artery development and progression toward the placenta and, subsequently, by an acceleration of endovascular invasive trophoblast cell movement deep into the uterine spiral arteries and extensive uterine spiral artery structural remodeling.¹⁴ These physical changes include replacement of the endothelium with endovascular trophoblast cells, their acquisition of a pseudo-endothelial phenotype, the disappearance of smooth muscle cells in the tunica media of the modified uterine spiral artery, distention of the uterine spiral artery lumen and an increased capacity for blood delivery to the placenta. Thus, NK cells possess two apparently contradictory actions in establishing the hemochorial placenta: (1) they promote uterine spiral artery development and remodeling, and (2) they restrain trophoblast-directed uterine spiral artery remodeling. The relative impact of each action on uterine spiral artery remodeling is not equivalent. Invasive trophoblast-directed uterine spiral artery restructuring is far more extensive than NK cell-directed uterine spiral artery restructuring. It is apparent that these opposing efforts are interconnected in an important way. The presence of NK cells delays and reduces the magnitude of trophoblast-directed uterine spiral arterial modifications, which is viewed as a maternally protective adaptation.¹⁴

Oxygen Tension is a Key Mediator of NK Cell Actions at the Placentation Site

NK cells have profound effects on the organization of the placentation site, including the development and function of the invasive trophoblast cell lineage. These cells have been proposed to affect invasive trophoblast cells through their secretion of an assortment of cytokines/chemokines and via direct cell-cell interactions.^5,16,17 Some factors produced by NK cells stimulate invasive properties of trophoblast cells, whereas other NK cell-derived factors inhibit trophoblast invasion. These observations are based exclusively on in vitro experimentation and are difficult to reconcile. A compelling hypothesis has also been proposed for direct NK cell-invasive trophoblast cell engagement.^16,18 NK cells possess surface receptors, termed killer inhibitory receptors (KIRs), which specifically recognize histocompatibility antigens on the surface of trophoblast located at the maternal-fetal interface. This cell-cell interaction modifies NK cell and trophoblast cell function. Two observations support an involvement of NK cell-invasive trophoblast cell adhesion in hemochorial placentation: (1) the existence of specific NK cell KIR receptor and histocompatibility antigen isoform combinations in successful pregnancies vs. pregnancies afflicted with preeclampsia^18-20 and (2) the observation that endovascular trophoblast invasion and uterine spiral artery remodeling is affected by maternal and paternal histocompatibility antigen combinations.²¹ These paracrine and cell-cell modes of NK cell action on trophoblast cells may be operative during the establishment of the hemochorial placenta; however, they are probably secondary to the more fundamental action of NK cells on uterine spiral artery development. The latter activities are likely mediated by a myriad of angiogenic and vasoactive factors secreted by NK cells.⁵

NK cells regulate uterine spiral artery development and, in doing so, control oxygen tension at the early placentation site. The absence of NK cells at the maternal-fetal interface results in a transient hypoxia.¹⁴ The effects of NK cell depletion on invasive trophoblast-directed uterine spiral artery remodeling can be mimicked by appropriately timed in vivo hypoxia exposure.²² A critical developmental window of sensitivity to in vivo hypoxia exposure exists between E8.5 and E9.5 of rat pregnancy. This gestational interval represents a key developmental phase associated with commitment of trophoblast stem (TS)/precursor cells to differentiated trophoblast cell lineages of the junctional zone vs. the labyrinth zone. In addition to the robust extension of endovascular trophoblast invasion deep into the uterine spiral arteries in both NK cell-depleted and maternal hypoxia in vivo models, the junctional zone also undergoes an expansion relative to the labyrinth zone.^14,22

TS cell populations are an excellent in vitro model system for elucidating mechanisms regulating differentiation.^23,24 TS cell exposure to a range of oxygen tensions can differentially influence cell proliferation and differentiation.^14,25-27 The impact of oxygen tension on development of invasive trophoblast lineages from TS cell populations has received little attention. Low oxygen tensions can promote differentiation of TS cells toward junctional zone trophoblast cell lineages.¹⁴ At least some of these hypoxia-stimulated actions are mediated by the transcription factor complex, hypoxia-inducible factor (HIF).^14,25 HIFs are basic helix loop helix Per-Arnt/AhR-Sim (bHLH-PAS) transcription factors.²⁸ Disruption of HIF signaling impairs TS cell responses to low oxygen and acquisition of an invasive phenotype.¹⁴ Whether these HIF actions are at specific gene loci controlling the invasive trophoblast cell lineage or are indirect through more widespread epigenetic modifications and how they differentially affect TS cell proliferation vs differentiation remain to be determined.²⁹ Identifying the biology of specific hypoxia-sensitive and HIF target genes should provide insight into the regulatory machinery controlling the invasive trophoblast cell lineage. (Fig. 1)

Figure 1. Schematic diagram showing the role of NK cells and hypoxia/HIF signaling in the regulation of trophoblast cell differentiation. NK cells regulate uterine vascular development, which impacts oxygen delivery (ΔO₂) and trophoblast lineage decisions. Differentiated trophoblast lineages in the labyrinth zone include syncytial trophoblast. Differentiated trophoblast lineages in the junctional zone include trophoblast giant cells, spongiotrophoblast cells, glycogen cells and precursors for invasive trophoblast cells.

Acknowledgments

Our research efforts were supported by grants from the NIH (HD20676, HD048861). We thank past and present members of our laboratory for their valuable contributions to the ideas presented in this short review.