Estrogen-dependent regulation of human uterine natural killer cells promotes vascular remodelling via secretion of CCL2.

study questionDoes intrauterine biosynthesis of estrogen play an important role in early pregnancy by altering the function of uterine natural killer (uNK) cells? SUMMARY ANSWER: Estrogens directly regulate the function of human uNK cells by increasing uNK cell migration and secretion of uNK cell-derived chemokine (C-C motif) ligand 2 (CCL2) that critically facilitates uNK-mediated angiogenesis. WHAT IS KNOWN ALREADY: uNK cells are a phenotypically distinct population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. Recently we discovered that decidualisation of human endometrial stromal cells results in the generation of an estrogen-rich microenvironment in areas of decidualised endometrium. We hypothesize that intrauterine biosynthesis of estrogens plays an important role in early pregnancy by altering the function of uNK cells. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used primary human uNK cells which were isolated from first trimester human decidua (n = 32). PARTICIPANTS/MATERIALS, SETTING,

methodsPrimary uNK cells were isolated from first trimester human decidua using magnetic cell sorting. The impact of estrogens on uNK cell function was assessed. Isolated uNK cells were treated with estrone (E1, 10(-8) M) or estradiol (E2, 10(-8) M) alone or in combination with the anti-estrogen ICI 182 780 (ICI, 10(-6) M). uNK cell motility was assessed by transwell migration assay and time-lapse microscopy. Expression of chemokine receptors was assessed by quantitative PCR (qPCR) and immunohistochemistry, and angiogenic factors were assessed by qPCR and cytokine array. Concentrations of CCL2 in supernatants were measured by enzyme-linked immunosorbent assay. Angiogenesis was assessed in a human endometrial endothelial cell network formation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with either E1 or E2 increased uNK cell migration (P = 0.0092 and P = 0.0063, respectively) compared with control. Co-administration of the anti-estrogen ICI blocked the effects of E1 and E2 on cell migration. Concentrations of C-X-C chemokine receptor type 4 (CXCR4) mRNA in uNK cells were increased by E2 treatment. The network formation assay revealed that conditioned media from uNK cells treated with E2 significantly increased human endometrial endothelial cell (HEEC) angiogenesis (P = 0.0029 versus control). Analysis of media from uNK cells treated with E2 using an antibody array identified CCL2 as the most abundant cytokine. Validation assays confirmed concentrations of CCL2 mRNA and protein were increased by E2 in uNK cells (P < 0.05 versus controls). Compared with the control, recombinant human CCL2 was found to increase HEEC network formation (P < 0.05) and neutralization of CCL2 in uNK conditioned media significantly decreased E2-dependent uNK-mediated network formation (P = 0.0006). LIMITATIONS, REASONS FOR CAUTION: Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in vivo in humans. Primary human uNK cells were isolated from first trimester decidua at a range of gestations (8-12 weeks), which may be a source of variation. Primary human uNK cells from non-pregnant endometrium were not assessed and therefore the responses of uNK cells to E2 treatment described in this study may be distinct to uNK cells from first trimester decidua. WIDER IMPLICATIONS OF THE

findingsE2 is an essential regulator of reproductive competence. This study demonstrates a critical role for E2 in regulating cellular cross-talk within the endometrium during early pregnancy. We provide the first evidence that E2 directly regulates the function of human uNK cells by altering uNK cell migration and the secretion of uNK-derived angiogenic factors. We describe a novel mechanism of estrogen-dependent secretion of CCL2 which critically mediates uNK-dependent endometrial angiogenesis. Dysregulation of uNK cell function has been implicated in the aetiology of early implantation disorders and disorders of pregnancy. These novel findings provide unique insight into the regulation of uNK cell activity during the establishment of pregnancy in women and highlight key processes which may be targeted in future therapeutic strategies. STUDY FUNDING/COMPETING INTERESTS: Studies undertaken in the authors' laboratory were supported by MRC Programme Grant G1100356/1 to P.T.K.S. The authors have no conflicts of interest to disclose.