NATURAL KILLER CELLS, HYPOXIA, AND EPIGENETIC REGULATION OF HEMOCHORIAL PLACENTATION

Abstract

During the establishment of pregnancy, uterine stromal cells differentiate into decidual cells and recruit natural killer (NK) cells. These NK cells are characterized by low cytotoxicity and distinct cytokine production. In rodent as well as in human pregnancy, the uterine NK cells peak in number around mid-gestation after which they decline. NK cells associate with uterine spiral arteries and are implicated in pregnancy associated vascular remodeling processes and potentially in modulating trophoblast invasion. Failure of trophoblast invasion and vascular remodeling has been shown to be associated with pathological conditions like preeclampsia syndrome, hypertension in mother and/or fetal growth restriction. We hypothesize that NK cells fundamentally contribute to the organization of the placentation site. In order to study the in vivo role of NK cells during pregnancy, gestation stage-specific NK cell depletion was performed in rats using anti asialo GM1 antibodies. Robust endovascular trophoblast invasion and vascular remodeling were observed in NK cell depleted animals. This depletion affected mesometrial vasculature, lowered relative oxygen concentrations at the placentation site and altered trophoblast lineage commitment at gestation d9.5. There was also a significant change in the organization of the chorioallantoic placenta on d13.5. Delivery of oxygen appears to be a key signal influencing both trophoblast cell differentiation and organization of the placentation site. Hypoxia promotes development of the invasive trophoblast lineage. We next evaluated the impact of hypoxia on the trophoblast stem (TS) cell transcriptome. DNA microarray analysis was performed on rat TS cells exposed to atmospheric and low oxygen (0.5%). Upregulation of genes characteristic of an invasive phenotype and a marked downregulation of stem state-associated genes were observed. Matrix metalloproteinase 12 (Mmp12) and lysine demethylase 3A (Kdm3a) were markedly upregulated in response to low oxygen, while E-cadherin (Cdh1) expression was dramatically decreased. These responses were dependent upon hypoxia inducible factor (HIF) signaling. Several of the HIF targets were identified to be Kdm3a targets. KDM3A acts on dimethyl and monomethyl histone H3K9 substrates, which prompted an examination of global histone H3K9 methylation status of trophoblast cells developing in vitro and in vivo. Hypoxia significantly impacted histone H3K9 methylation. Knockdown of KDM3A in TS cells inhibited hypoxia-induced Mmp12 gene expression and disrupted histone H3K9 methylation status at the Mmp12 locus. Ectopic expression of KDM3A stimulated MMP12 expression. KDM3A knockdown also significantly decreased hypoxia-activated TS cell invasion through Matrigel chambers. Finally, blastocysts transduced with Kdm3a shRNA expressing lentiviral particles showed reduced outgrowth when exposed to low oxygen tensions. In summary, NK cells impact oxygen delivery and uteroplacental adaptive responses, influencing critical lineage decisions affecting trophoblast differentiation and the ultimate structure and function of the chorioallantoic placenta. Hypoxia/HIF-directed epigenetic remodeling via KDM3A contributes to the control of TS cell adaptations during placentation modulating trophoblast lineage commitment and invasion.