Extravillous Trophoblast and Endothelial Cell Crosstalk Mediates

Extravillous Trophoblast and Endothelial Cell Crosstalk Mediates Leukocyte Infiltration to the Early Remodeling Decidual Spiral Arteriole Wall This information is current as of September 24, 2021. Ruhul H. Choudhury, Caroline E. Dunk, Stephen J. Lye, John D. Aplin, Lynda K. Harris and Rebecca L. Jones J Immunol published online 10 April 2017 http://www.jimmunol.org/content/early/2017/04/07/jimmun ol.1601175 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 10, 2017, doi:10.4049/jimmunol.1601175 The Journal of Immunology

Extravillous Trophoblast and Endothelial Cell Crosstalk Mediates Leukocyte Inﬁltration to the Early Remodeling Decidual Spiral Arteriole Wall

Ruhul H. Choudhury,*,† Caroline E. Dunk,‡ Stephen J. Lye,‡ John D. Aplin,*,† Lynda K. Harris,*,†,x and Rebecca L. Jones*,†

Decidual spiral arteriole (SpA) remodeling is essential to ensure optimal uteroplacental blood flow during human pregnancy, yet very little is known about the regulatory mechanisms. Uterine decidual NK (dNK) cells and macrophages infiltrate the SpAs and are proposed to initiate remodeling before colonization by extravillous trophoblasts (EVTs); however, the trigger for their infiltration is unknown. Using human first trimester placenta, decidua, primary dNK cells, and macrophages, we tested the hypothesis that EVTs activate SpA endo-

thelial cells to secrete chemokines that have the potential to recruit maternal immune cells into SpAs. Gene array, real-time PCR, and Downloaded from ELISA analyses showed that treatment of endothelial cells with EVT conditioned medium signiﬁcantly increased production of two chemokines, CCL14 and CXCL6. CCL14 induced chemotaxis of both dNK cells and decidual macrophages, whereas CXCL6 also induced dNK cell migration. Analysis of the decidua basalis from early pregnancy demonstrated expression of CCL14 and CXCL6 by endothelial cells in remodeling SpAs, and their cognate receptors are present in both dNK cells and macrophages. Neutralization studies identiﬁed IL-6 and CXCL8 as factors secreted by EVTs that induce endothelial cell CCL14 and CXCL6 expression. This study

has identiﬁed intricate crosstalk between EVTs, SpA cells, and decidual immune cells that governs their recruitment to SpAs in the http://www.jimmunol.org/ early stages of remodeling and has identiﬁed potential key candidate factors involved. This provides a new understanding of the interactions between maternal and fetal cells during early placentation and highlights novel avenues for research to understand defective SpA remodeling and consequent pregnancy pathology. The Journal of Immunology, 2017, 198: 000–000.

uring the first half of pregnancy, uterine spiral arterioles of vasoreactivity (2, 3). Failure of remodeling is associated with fetal (SpAs) undergo remodeling to optimize maternal perfusion growth restriction, late miscarriage, and/or preeclampsia (4–6). D of the placenta, ensuring that nutrient and oxygen supply The highly specialized immune environment of the uterus reflects can increase to meet fetal needs in later gestation (1). Disruption and its dual role in protecting the mother from blood-borne and mucosally by guest on September 24, 2021 loss of extracellular matrix, vascular endothelium, and smooth muscle transmitted pathogens, while supporting the development of the cells allow irreversible expansion of the arterial channels with loss hemiallogeneic conceptus. The placenta is not immunologically si- lent; rather its active recognition by the maternal immune system is *Maternal and Fetal Health Research Centre, Division of Developmental Biology and central to successful pregnancy outcome (7). Thus immune cells are Medicine, School of Medical Sciences, Faculty of Biology, Medicine and Health, abundant in the decidua, comprising almost 40% of the total (8). University of Manchester, Manchester M13 9WL, United Kingdom; †Academic Health Science Centre, St. Mary’s Hospital, Manchester M13 9WL, United Kingdom; They include decidual NK (dNK) cells, macrophages, dendritic ‡Research Centre for Women’s and Infants’ Health, Lunenfeld-Tanenbaum Research cells, and T cells, the former two comprising almost 90% of the total Institute, Mount Sinai Hospital, Toronto, Ontario M5T 3H7, Canada; and xManchester Pharmacy School, University of Manchester, Manchester M13 9PT, United Kingdom leukocyte population (9). dNK cells are phenotypically distinct from peripheral blood NK (PBNK) cells, coupling reduced cytotoxicity ORCIDs: 0000-0002-4146-099X (C.E.D.); 0000-0001-8777-9261 (J.D.A.); 0000- 0001-7709-5202 (L.K.H.); 0000-0002-8871-0589 (R.L.J.). with an ability to secrete cytokines and angiogenic factors (10). Received for publication July 5, 2016. Accepted for publication March 14, 2017. SpA remodeling is now understood to result from the cooperative This work was supported by a grant from the Canadian Institutes for Health Re- activity of placental-derived extravillous trophoblasts (EVTs) and de- search. The Maternal and Fetal Health Research Centre is supported by Tommy’s cidual immune cells (11–13). Although EVTs actively contribute to Fund, an Action Medical Research Endowment, and the Greater Manchester Clinical extracellular matrix breakdown and vascular cell loss (3, 10), recent Research Network. L.K.H. was supported by the Biotechnology and Biological Sci- ences Research Council’s David Phillips Research Fellowship (BB/H022627/1). evidence shows SpA remodeling may be initiated by infiltration of The gene expression data presented in this article have been submitted to ArrayExpress decidual leukocytes into arteriolar walls before migrating endovascular (https://www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-5467. (v)EVTs reach the immediate locality (13, 14). Mice deficient in dNK Address correspondence and reprint requests to Ruhul H. Choudhury, Maternal and and T cells exhibit impaired SpA remodeling and subsequently develop Fetal Health Research Centre, Division of Developmental Biology and Medicine, a preeclampsia-like disorder (15), and impaired regulation of macro- School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, St. Mary’s Hospital, 5th Floor Research, Oxford Road, Manchester M13 phages is also associated with poor SpA remodeling (16, 17). However, 9WL, U.K. E-mail address: [email protected] the trigger that initiates immune cell infiltration is unknown (13). The online version of this article contains supplemental material. Using a human placenta-decidua coculture model in which EVTs Abbreviations used in this article: AU, arbitrary unit; dNK, decidual NK; EVT, detach from anchoring villus tips and invade decidual arteries, we extravillous trophoblast; EVT-CM, EVT conditioned medium; HUtMvEC, human previously demonstrated that leukocyte infiltration of SpAs depends uterine microvascular endothelial cell; HUtMvEC-CM, HUtMvEC conditioned medium; MIC-1, macrophage inhibitory cytokine-1; PBNK, peripheral blood NK; on the presence of EVTs in adjacent tissue (18). This is consistent PPLR, probability of positive log ratio; rh, recombinant human; a-SMA, a-smooth with histological evidence showing that SpA remodeling takes place muscle actin; SpA, spiral arteriole; vEVT, endovascular EVT. in the decidua basalis, where EVTs invade the decidual stroma (as Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 interstitial EVTs) and plug (as vEVTs) the SpAs (19), but not in parts

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601175 2 EVT-EC CROSSTALK DURING SPIRAL ARTERY REMODELING

Table I. Details of primer sequences for real-time PCR

Primer Set Sequence Reference CCL14 59-GACTGAATCCTCCTCACGGG TGGCATCTTCTCTTTATGTCTCTG-39 (20) CCL20 59-TGCTGTACCAAGAGTTTGCTC CGCACACAGACAACTTTTTCTTT-39 Primer Bank ID: 4759076a1 CXCL2 59-CGCATCGCCCATGGTTAAGA CTGTTCCTGTAAGGCAGGGC-39 NCBI Primer-BLAST CXCL6 59-GCAGTTTACCAATCGTTTTGGGG AGAGCTGCGTTGCACTTGTT-39 (21) CX3CL1 59-GCTTTGCTCATCCACTATCAACA GCTCCAGGCTACTGCTTTCG-39 (21) CXCR7 59-TTCGTCATCGGCATGATTGC AGTGCGTGTCATAGCCTGTG-39 Primer Bank ID: 114155149c2 YWHAZ 59-CCTGCATGAAGTCTGTAACTGAG TTGAGACGACCCTCCAAGATG-39 (22) HMBS 59-ATTCGGGGAAACCTCAACACC ATGCAGCGAAGCAGAGTCTC-39 Primer Bank ID: 66933008c3 ZNF8232 59-AAGGACAAAGGATCATTGCCAC ACCCTCAGAGGTAGCTTCAAAT-39 Primer Bank ID: 37574600c3 ID, identification; NCBI, National Center for Biotechnology Information. of the decidua distant from the placental site. These findings suggest were cultured in a 1:1 mixture of serum-free DMEM/F12 and EBM-2 for a an indirect interaction between EVTs and immune cells that drives further 24 h, then treated with a 1:1 mixture of serum-free EBM-2 and either immune cell infiltration of the SpA media. control medium (DMEM/F12) or EVT-CM for 24 h. After washing in PBS, RNA was extracted using the RNeasy mini kit (Qiagen, Manchester, U.K.), EVTs produce a large repertoire of paracrine factors including followed by treatment with the DNase kit (Ambion, Austin) to remove cytokines, growth factors, and proteases, many of which have the contaminating genomic DNA, both according to the manufacturer’s in- Downloaded from potential to alter endothelial cell function. Chemokines have established structions. The RNA sample was assessed for purity and concentration using roles in decidual function and leukocyte recruitment during pregnancy spectrophotometry and a Nanodrop (2000c; Thermo Scientific). (20, 21, 23, 24). We therefore hypothesized that cytokines released by Ab array. A proteome profiler cytokine array (ARY022; R&D Systems, Minneapolis) was carried out according to the manufacturer’s instructions invading EVTs activate endothelial cells to upregulate the expression on pooled EVT-CM (n = 6) to identify EVT-secreted factors. The presence of dNK cells and macrophage chemoattractants, thus inducing infil- of candidate proteins identified in the array was validated using individual

tration of the SpA wall from the surrounding decidual stroma. samples; protein concentration was measured by ELISA, as described (27). http://www.jimmunol.org/ Macrophage inhibitory cytokine-1 (MIC-1) concentration in EVT-CM was quantiﬁed using ELISA (R&D Systems, Abingdon, U.K.) according to the Materials and Methods manufacturer’s instructions. Primary tissues Endothelial cell culture with IL-6, CXCL8, and MIC-1 First trimester placental tissue (5–9 wk gestation) (n = 16) was collected neutralizing Abs from women undergoing elective medical and surgical termination of pregnancy. Decidual tissue (6–12 wk gestation) (n = 22) was collected HUtMvECs were treated with EVT-CM as described above, with the addition following surgical termination of pregnancy only. Placental and decidual of neutralizing Abs for IL-6 and CXCL8 (6708 and 48311, mouse mono- tissues were dissected as described previously and processed for cell and clonal; R&D Systems), both singularly and in combination, at an excess of

tissue culture (13). Approval was obtained from North West Research 10 3 (1 ng/ml) the original cytokine concentration. The experiment was also by guest on September 24, 2021 Ethics Committee (08/H1010/28). Written informed consent was obtained repeated using MIC-1 neutralizing Abs (20 ng/ml, (147627, mouse mono- from all women undergoing termination of pregnancy. clonal; R&D Systems). HUtMvECs were treated with EVT-CM containing mouse IgG at a matching concentration as a negative control. EVT outgrowths and production of conditioned medium Gene expression analysis EVT conditioned medium (EVT-CM) (n = 9) was generated as described previously (25). Briefly, the terminal portions of villi from ,9 wk pla- RNA extracted from HUtMvECs treated with EVT-CM for 24 h (EVT-CM centae were dissected out and laid over a collagen I (Corning, Bedford, from nine different placentae), and untreated control cells were separately U.K.) surface in a 24-well plate. The explants were cultured in DMEM/ pooled and gene profiling performed using Affymetrix Human Genome U133 F12 (Sigma, Gillingham, Dorset) with 10% FBS in 5% CO2 and 20% O2. Plus 2.0 microarray. Quality control, normalization, and expression analysis After 48 h, outgrowths with 30% or more EVT coverage were washed in in control and EVT-CM–treated groups were as previously described (28– PBS and cultured in serum-free DMEM/F12 for a further 48 h. EVT-CM 30). Using positional update and matching algorithms, the probability of was then collected and centrifuged at 3000 3 g for 5 min to remove any positive log ratio (PPLR) was calculated (30). Thresholds for significant debris and stored at 280˚C for further experiments. changes in expression were predefined as PPLR scores of .0.9 (for upregulated genes) and ,0.1 (for downregulated genes) and were used to Endothelial cell culture select candidate chemokines. Chemokine expression was considered to be above background levels at a signal intensity of .50 arbitrary unit (AU). Human uterine microvascular endothelial cells (HUtMvECs) (Lonza, Co- logne, Germany) were cultured as described previously (26). Upon reaching Reverse transcription, real-time PCR, and ELISA confluency (passages 8–10), 1 3 105 cells were seeded per well in a six- well plate. After culturing in serum-containing EBM-2 (Lonza) for 24 h, To validate the changes in mRNA expression of candidate chemokines the medium was removed and the cells were washed with PBS. The cells detected by gene array, real-time PCR was carried out on individual RNA

Table II. Chemokine ligands/receptors differentially expressed in EVT-CM–treated HUtMvECs

Control HUtMvECs EVT-Treated HUtMvECs Chemokine Alternative Name (Expression Intensity AU) (Expression Intensity AU) PUMA Ratio PPLR CCL14 HCC-1 281 450 2.13 0.93 CCL20 MIP-3a 496 267 22.73 0.05 CXCL2 GROb 912 663 21.51 0.04 CXCL6 GCP-2 559 681 1.23 0.99 CX3CL1 Fractalkine 302 160 22.59 0.04 CXCR7 ACKR3 442 621 1.46 0.97 Expression intensity is measured in arbitrary units (AU). Positional update and matching algorithm (PUMA) ratio (fold change) represents expression from control to treated cells. The PPLR score represents conﬁdence in the observed changes, where PPLR score .0.9 or ,0.1 indicates a signiﬁcant upregulation or downregulation, respectively. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Alterations in chemokine mRNA and protein expression by HUtMvECs in response to EVT-CM. Real-time PCR verified that EVT-CM treatment significantly increased mRNA expression of (A) CCL14, (B) CXCL6, and (F) CXCR7 and decreased the expression of (C) CXCL2, (D) CCL20, and (E) CX3CL1 (n = 8). ELISA demonstrated increased concentrations of CCL14 and CXCL6 in EVT-CM–treated HUtMvEC lysates (G and H) and conditioned medium (I and J)(n = 6). Data are median 6 interquartile range. *p , 0.05, **p , 0.01, Wilcoxon matched-pairs test. samples from EVT-CM–treated and untreated HUtMvECs (n = 8), as de- and shaken at 37˚C for 30 min before passing through a 200-mmsieve. scribed previously (21). A total of 250 ng of RNA was used for reverse Stromal cells and leukocytes were separated from RBCs using a Lym- transcription using an AffinityScript Multi Temperature cDNA synthesis kit phoprep density gradient (Cambridge, U.K.). CD56+ dNK cells and (Agilent, Berkshire, U.K.). PCR was performed using specific primers CD14+ macrophages were isolated by positive selection using magnetic (Table I) with Brilliant III Ultra-Fast SYBR Green QPCR Master Mix microbeads (MACS Miltenyi Biotec, Surrey, U.K.) from each decidua. (Agilent), and an annealing temperature of 60˚C. The PCR data were nor- The purity of the cell isolation was measured using flow cytometry after malized using the geometric mean of three housekeeping genes: YWHAZ, immunostaining for CD56 and CD14, as previously described (32) HMBS, and ZNF8232 (Table I) (22, 31). An ELISA (RayBiotech, Wembley, (Supplemental Fig. 3). U.K.) was carried out according to the manufacturer’s instructions to quantify CXCL6 and CCL14 protein expressions in cell lysates and con- Chemotaxis assay ditioned medium from EVT-CM–treated and untreated HUtMvECs (n =6). Isolated dNK cells and macrophages were used for in vitro migration Isolation of primary dNK cells and decidual macrophages assays with recombinant human (rh) CCL14 (Canton) and CXCL6 (San Diego). CD56+ dNK cells were labeled with the nuclear fluorophore Primary dNK cells and decidual macrophages were isolated from decidual YOYO1 (Invitrogen) at 1:1000 in serum-free medium for 15 min at 37˚C. samples as described (32). Briefly, decidual samples were washed, minced, The chemotaxis assay was carried out in 24-well plates containing 4 EVT-EC CROSSTALK DURING SPIRAL ARTERY REMODELING Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. CCL14 and CXCL6 are expressed by decidua basalis spiral arteriolar endothelial cells. (A–D) Representative images of remodeling SpAs. (A) Disorganized smooth muscle cell layer and (B) disorganized endothelium. (C and D) Endothelial cells are positive for CCL14 and CXCL6 (black arrow). (E–L) Representative images of an unremodeled SpA in (E–H) decidua basalis and (I–L) decidua parietalis; endothelial cells are positive for CCL14 and CXCL6. (O and P) Representative images of vein in decidua, endothelial cells are positive for CCL14 and CXCL6. (M)MouseIgGand(N) Rabbit IgG acted as negative controls. Original magnification 320. Scale bar, 50 mm. a-SMA stains for smooth muscle cells and CD31 stains for endothelial cells. transwell inserts with 3-mm pores. rhCCL14 and rhCXCL6 were added to ing colorimetric detection as described previously (13). Serial tissue sections the bottom of the wells at 10 ng/ml and 100 ng/ml, and 200,000 immune (5 mm) were immunostained for vascular cells (a-smooth muscle actin cells were added to the inserts. Ten percent FBS and serum-free medium [a-SMA] and CD31 [endothelial cells]), EVTs (HLA-G), and leukocyte (Life Technologies RPMI 1640, Leicestershire, U.K.) were used as common Ag (CD45) using mouse monoclonal Abs (Dako, Cambridgeshire, positive and negative controls, respectively. After an overnight incuba- U.K.), and for CCL14 and CXCL6 using rabbit polyclonal Abs (Thermo tion, cells that had migrated through the filter were collected and their Fisher Scientific, Cheshire, U.K.). Serial tissue sections were also fluorescence read using a M200 plate reader (Tecan) with excitation at immunostained for CXCL8 (goat anti-human polyclonal; Santa Cruz, 480 nm and measurement at 525 nm. The cell number was determined Wembley U.K.) and IL-6 (mouse anti–IL-6 monoclonal; Novus Biolog- using a standard curve. The assay was also repeated using culture me- icals, Abingdon, U.K.). Immunocytochemistry was also carried out on dium from EVT-CM–treated HUtMvECs and in the presence of neu- 5 3 104 HUtMvECs seeded on 13-mm cover glass (Thermo Fisher tralizing Abs to CCL14 (1.5 ng/ml) (R&D Systems) and CXCL6 (1 ng/ Scientific) for CD31 and a-SMA (Supplemental Fig. 1) to assess the ml) (R&D Systems) at an excess of 10 3 the original chemokine con- morphology of the cells. centration. Chemotaxis assays with decidual CD14+ macrophages were For dual immunofluorescence, 5-mm formalin-fixed, paraffin-embedded also performed using the same method except for the transwell inserts, decidua basalis samples were dual-stained for dNK cells or macrophages which contained 8-mm pores. using mouse monoclonal Abs against CD56 (Invitrogen) or CD163 (AbD Serotech, Kidlington, U.K.) and the chemokine receptors CCR1, Primary decidual endothelial cell isolation and culture CCR5 (goat polyclonals; Santa Cruz), CXCR1, or CXCR2 (mouse Primary decidual endothelial cells were isolated as previously de- monoclonals; R&D Systems). As Abs for CXCR1, CXCR2, and CD56 m were raised in the same species, tissues sections were incubated with scribed (33). The cells were seeded on collagen (250 g/ml)-coated m six-well plates and cultured with EVT-CM or control DMEM/F12 10 g/ml of unlabeled goat anti-mouse IgG after addition of the first primary Ab for 30 min at 37˚C, to eliminate cross-reactivity. For im- medium as described above (n = 6). RNA from pooled and individual samples was collected for PCR prior to real-time PCR for chemo- munofluorescence detection, donkey anti-mouse Alexa Fluor 488, don- kines, as above. key anti-mouse Alexa Fluor 568, and rabbit anti-goat Alexa Fluor 568 (Life Technologies, Cheshire, U.K.) conjugates were used. Whole mount Immunohistochemistry and dual immunofluorescence immunofluorescence was also carried out using the same method on EVT outgrowth (n = 3) for EVT identity and cell death using monoclonal Immunohistochemistry was carried out on formalin-fixed, paraffin- Abs against HLA-G and M30 (Roche, West Sussex, U.K.) (Supplemental embedded human first trimester decidua basalis (6–12 wk gestation), us- Fig. 2), respectively. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. Quantification of chemokine expression by vascular endothelial cells and decidual cells. (A and B) Mean immunostaining intensity for CCL14 and CXCL6 was significantly higher in endothelial cells of remodeling SpAs than surrounding decidual cells. Mean immunostaining intensity for CCL14 and CXCL6 in arteriole endothelial cells of (C and D) decidua basalis and (E and F) decidua parietalis was not significantly different from the surrounding decidual cells. Immunostaining intensity for (G) CCL14, but not (H) CXCL6 was higher in decidual cells than vein endothelial cells. (I) CCL14, but not (J) CXCL6 immunostaining intensity was significantly higher in endothelial cells in remodeling arteries compared with those in unre- by guest on September 24, 2021 modeled arteries. Data are median 6 interquartile range (n = 7). *p , 0.05, **p , 0.01, Wilcoxon matched-pairs test.

Quantification of chemokine and cytokine staining genes are related to cell cycle regulation, dTMP de novo biosyn- Intensity of CCL14, CXCL6, CXCL8, and IL-6 immunostaining in decidual thesis pathway and cancer signaling. For the purpose of this study, sections was quantified using unbiased HistoQuest image analysis software alterations in chemokine expression were prioritized. Six candidates version 3.5 (TissueGnostics, Vienna, Austria) as previously described (34). with signal intensity .50 AU were selected for validation: CCL14, Briefly, seven images of remodeling SpAs, unremodeled SpAs from the CCL20, CXCL2, CXCL6, CX3CL1 and CXCR7 (Tables I, II). decidua basalis and decidua parietalis, and veins from five different tissues were analyzed for mean DAB staining intensities in endothelial cells and CCL14 and CXCL6 are upregulated by HUtMvECs following the rest of the decidual cells. Mean DAB staining intensities for CXCL8 stimulation with EVT-CM (n = 7, five individual tissues) and IL-6 (n = 7, five individual tissues) in vEVTs and surrounding total decidual cells were also analyzed using the As predicted by the microarray, all six chemokine/receptor can- same methods. didate genes were confirmed by real-time PCR to be altered at the , , Statistical analysis mRNA level by EVT-derived factors (p 0.05 and p 0.01; Fig. 1A–F). CCL14 and CXCL6 were selected for further study, in The Wilcoxon matched-pairs signed rank test was carried out to assess the line with our hypothesis that EVT-CM stimulates production of difference between EVT-CM–treated and control HUtMvEC groups. The Wilcoxon matched-pairs signed rank test was also used for DAB intensity leukocyte chemoattractants by uterine vascular endothelial cells. analysis in chemokine and cytokine staining. The Friedman test and Both chemokines were also upregulated at the protein level in Dunn multiple comparisons post hoc test were used to analyze the dif- HUtMvEC lysates (Fig. 1G, 1H; p , 0.05) and in HUtMvEC ference between groups following chemokine or cytokine neutralization conditioned medium (HUtMvEC-CM) (p , 0.05; Fig. 1I, 1J), experiments. following treatment with EVT-CM. The chemokine levels in the EVT-CM were also measured; EVTs secreted a low concentration Results of CCL14 (4.0 pg/ml) and CXCL6 (8.0 pg/ml) compared with the Treatment of HUtMvECs with EVT-CM alters chemokine higher mean concentration in the EVT-CM–treated HUtMvEC-CM mRNA expression profile (129 and 85 pg/ml respectively). Gene profiling was performed on HUtMvECs following stimulation with EVT-secreted factors. A total of 1179 genes were up- CCL14 and CXCL6 are observed in endothelial cells of regulated and 1140 genes were downregulated by EVT-CM using decidual SpAs and are upregulated following stimulation with PPLR thresholds of .0.9 and ,0.1 to define significant differ- EVT-CM ential expression (Accession number E-MTAB-5467, ArrayEx- HUtMvECs were used as a model of decidual SpA endothelial press, https://www.ebi.ac.uk/arrayexpress/). Most of the altered cells. To assess whether SpA endothelial cells in the decidua 6 EVT-EC CROSSTALK DURING SPIRAL ARTERY REMODELING Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Alterations in chemokine mRNA expression by primary decidual endothelial cells in response to EVT-CM. real-time PCR verified that EVT- CM treatment increased mRNA expression of (A) CCL14, (B) CXCL6, and (F) CXCR7 and decreased mRNA expression of (C) CXCL2, (D) CCL20, and (E) CX3CL1 in all four individual samples. (n = 4). Wilcoxon matched-pairs test. The changes are not significant with p . 0.05. basalis express CCL14 and CXCL6 in vivo, immunohisto- mean immunostaining intensity of either chemokine between chemistry was performed. Unremodeled and early remodeling endothelial cells of unremodeled SpAs and decidual cells SpAs were identified by serial section immunostaining for (Fig. 3C–F). However, immunostaining intensity for CCL14, but vascular, immune cell, and trophoblast markers. Endothelial not CXCL6, (Fig. 3G, 3H) was lower in venous endothelial cells cells in both unremodeled (Fig. 2G, 2H) and early remodeling than decidual cells (p , 0.01). The immunostaining intensity for (Fig. 2C, 2D) SpAs expressed CCL14 and CXCL6. Both che- CCL14 was significantly higher in endothelial cells in remodeling mokines were additionally localized to endothelial cells in arteries compared with those in unremodeled arteries (Fig. 3I, p , unremodeled SpAs in the decidua parietalis (Fig. 2K, 2L), ve- 0.05). A similar pattern was detected for CXCL6 but this did not nous endothelial cells (Fig. 2O, 2P), and decidual stromal cells, reach significance (Fig. 3J). but were markedly absent from SpA smooth muscle cells (20). To verify the relevance of the EVT-induced changes in Quantitative analysis of immunostaining intensity using Histo- chemokine expression identified in HUtMvECs, real-time PCR Quest software demonstrated that CCL14 and CXCL6 protein analysis was performed on EVT-CM–treated primary endothe- expressions were significantly higher in endothelial cells of lial cells isolated from decidual tissues. EVT-CM treatment in- remodeling arteries (p , 0.05, Fig. 3A, 3B) compared with duced increased expression of CCL14 (3.9-fold change) and surrounding decidual cells (34). There was no difference in CXCL6 (2.2-fold change) in pooled samples (n = 6). Analysis of The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. Chemotaxis of dNK cells and decidual macrophages toward recombinant chemokines and HUtMvEC-CM. (A) CCL14 and (C) CXCL6 were chemoattractants for dNK cells at 100 ng/ml; (B) CCL14 but not (D) CXCL6 was a chemoattractant for decidual macrophages; (E) dNK cells and (F) decidual macrophages migrate toward conditioned medium from HUtMvECs stimulated by EVT-secreted factors. dNK cell and macrophage migration can be reduced significantly by cotreatment with neutralizing Abs against (E) CCL14 and CXCL6, and (F) CCL14, respectively. Data are median 6 interquartile range (n = 8). * and ++ represent significant differences compared to serum-free and HUtMvEC CM groups, respectively. *p , 0.05 Friedman test with Dunn’s posthoc test. individual samples (n = 4) demonstrated the consistency of the 5D). Decidual macrophages also migrated toward HUtMvEC- response, although the sample size was too small for robust statis- CM (p , 0.05, Fig. 5F) and cotreatment with CCL14 neutral- tical analysis (Fig. 4). izing Ab significantly reduced their migration (p , 0.05, Fig. 5F). CCL14 and CXCL6 chemoattract dNK cells To investigate whether CCL14 and CXCL6 act as chemoattractants Decidual NK cells express receptors for CXCL6 and CCL14 for dNK cells, in vitro migration assays were carried out. Primary Dual immunofluorescence of the decidua basalis revealed ex- human dNK cells migrated toward rhCCL14 and rhCXCL6 pression of CCR1 and CCR5 (CCL14 receptors), and CXCR1 (Fig. 5A, 5C) at 100 ng/ml (p , 0.05). dNK cells also migrated and CXCR2 (CXCL6 receptors) by dNK cells (Fig. 6A–H). toward HUtMvEC-CM when compared with serum-free medium CXCR1 and a low level of CXCR2 were detectable on the (p , 0.05, Fig. 5E), and this migration could be effectively majority of CD56+ NK cells, whereas only a subset expressed inhibited by cotreatment with CCL14- and CXCL6-neutralizing CCR1 and CCR5. Decidual stromal cells also express these Abs in combination (p , 0.05, Fig. 5E). receptors. CCL14, but not CXCL6, is chemoattractant to decidual macrophages Decidual macrophages express CCR1 and CCR5 Primary decidual macrophages migrated in response to rhCCL14 Almost all decidual macrophages strongly expressed CCR1 and (p , 0.05, Fig. 5B), but were unresponsive to rhCXCL6 (Fig. CCR5 (CXCL6 receptors) (Fig. 6I–L). CCL14 and CXCL6 8 EVT-EC CROSSTALK DURING SPIRAL ARTERY REMODELING

A B M

C D N

E F O FIGURE 6. dNK cells and mac- Downloaded from rophages in decidua basalis express receptors for chemokines. Dual im- munoﬂuorescence of dNK cells (green) with (A and B) CXCR1, (C and D) CXCR2, (E and F) CCR1, and (G and H) CCR5 receptors (all red). Representative immune cells http://www.jimmunol.org/ expressing receptors are indicated by white arrows. Dual immunoﬂuo- G H rescence of decidual macrophages (green) with (I and J) CCR1 and (K and L) CCR5 receptors (all red). Negative controls: (N) mouse or (O) goat IgG. (M) Liver was used as a positive control. by guest on September 24, 2021 I J

K L

receptor expressions in dNK cells and macrophages were con- further study due to their ability to act on endothelial cells (35, 36) sistent at 6–12 wk gestation. and induce chemokine expression in other cell types (37, 38). To assess whether EVTs in the decidua basalis express IL-6 EVTs secrete multiple cytokines and growth factors and CXCL8, immunohistochemistry was performed; both were An Ab array was employed to identify cytokines and growth factors expressed by EVTs in anchoring columns and vEVTs in remodeling present in EVT-CM. These included IL-6 and CXCL8, macrophage decidual SpAs, although IL-6 immunoexpression was weaker than migration inhibitory factor (MIF), MIC-1 and CCL2 (Table III). The IL-8, consistent with the ELISA data (Fig. 7A–H, Table IV). The concentrations of MIC-1, CXCL8, and IL-6 were measured by mean immunostaining intensity for CXCL8 was signiﬁcantly (p , ELISA in individual samples. High and consistent secretion of MIC- 0.05) higher in vEVTs (Fig. 7I) compared with surrounding decidual 1 was observed in all samples (Table IV). CXCL8 and IL-6 secre- cells. However, there was no difference in mean IL-6 expres- tions varied between samples but no gestation-dependent trend was sion between vEVTs and decidual cells (Fig. 7J). Furthermore, observed (Table IV). MIC-1, IL-6, and CXCL8 were selected for the original microarray performed on unstimulated HUtMvECs The Journal of Immunology 9

Table III. Analysis of EVT-CM by cytokine Ab array

Cytokine Abbreviation or Alternative Name Control Medium EVT-CM Fold Change from Control Medium Chitinase 3-like 1 CHI3L1 353.38 6884.59 19.48 Cystatin C CST3 133.60 1006.18 7.53 Dipeptidyl peptidase-4 DPP4 493.57 5794.93 11.74 Epidermal growth factor EGF 2720.51 441.00 0.16 Extracellular matrix protein inducer EMMPRIN 185.43 4006.02 21.60 Fibroblast growth factor 2 FGF-2 9947.07 536.51 0.05 Fibroblast growth factor 19 FGF-19 5685.20 1440.13 0.25 Macrophage inhibitory cytokine-1 MIC-1 42.19 7436.28 176.25 Colony stimulating factor 2 CSF-2 287.43 2484.56 8.64 Growth hormone HGH 71.14 4674.56 65.70 Insulin-like growth factor binding protein 3 IGFBP-3 1493.80 22794.85 15.26 IL 1a IL-1a 1709.38 5793.90 3.39 IL-6 IL-6 274.34 1051.23 3.83 Chemokine (C-X-C motif) ligand 8 CXCL8 171.26 5912.20 34.52 Leptin Leptin 1015.82 5962.88 5.87 Monocyte chemotactic protein 1 MCP-1 770.28 2804.00 3.64 Macrophage migration inhibitory factor MIF 808.06 5935.62 7.35 Osteopontin OPN 1086.06 21018.27 19.35 Platelet-derived growth factor a polypeptide PGDF 53807.81 14723.79 0.27 Downloaded from Serpin E1 PAI-1 502.34 22832.64 45.45 IL-1 receptor-like 1 IL-1R1 309.92 4499.20 14.51 Densitometric analysis of Ab array comparing cytokine concentrations in control culture medium (serum-free) with EVT-CM. showed that these cells expressed receptors for IL-6: IL-6R (311 cruitment of decidual immune cells into the SpA wall during the AU), IL-6-ST (9586 AU) and CXCL8 (CXCR1 (102 AU). early stages of remodeling. We identiﬁed two candidate endothelial http://www.jimmunol.org/ Neutralizing IL-6 and CXCL8 in EVT-CM reduces the chemokines, CCL14 and CXCL6, which are upregulated in response stimulation of CCL14 and CXCL6 expressions in HUtMvECs, to EVT-secreted factors and induce the migration of dNK cells and but MIC-1 neutralization has no effect on chemokine expression decidual macrophages. Inhibition of IL-6 and CXCL8 reduced the stimulatory effect of EVT-CM on the expression of these chemo- Addition of IL-6 and CXCL8 neutralizing Abs in combination to kines indicating that they are two key EVT-derived activating factors. EVT-CM signiﬁcantly reduced its ability to upregulate expressions of Thus we propose a model whereby factors including IL-6 and , CCL14 and CXCL6 mRNA in HUtMvECs (p 0.001; Fig. 8A, 8B). CXCL8, derived from EVTs in the upper segments of decidual Treatment with IL-6 neutralizing Ab alone had a similar effect (p ,

SpAs, activate endothelial cells to release CCL14 and CXCL6, by guest on September 24, 2021 0.01), although this effect did not reach statistical significance when which then attract dNK cells and macrophages from the surrounding CXCL8 neutralizing Ab was used alone (p = 0.067). Neutralization of tissue into the SpAs to initiate remodeling (Fig. 10). To our knowl- either cytokine alone in EVT-CM significantly reduced HUtMvEC edge, the in vitro findings suggest, for the first time, a mechanism , expression of CXCL6 (p 0.01, Fig. 8B). Addition of CXCL8 by which decidual immune cells are stimulated to infiltrate the neutralizing Ab alone or in combination with IL-6 neutralizing Ab had SpAs and provide evidence of complex interactions between fetal no effect on expression of CXCL2, CCL20, CX3CL1, and CXCR7 EVTs and maternal vascular and immune cells in coordinating the mRNA (Fig. 8C–F). Neutralization of MIC-1 did not affect EVT-CM process. It may also explain why arteriolar walls in the decidua induced up- or downregulation of CCL14, CXCL6, CXCL2, CCL20, parietalis, despite being surrounded by tissue rich in dNK cells CX3CL1, and CXCR7 (Fig. 9A–F). The addition of a nonspecific IgG and macrophages, remain free of infiltrating immune cells. had no inhibitory effect. Findings were assembled into a hypothetical Decidual stromal cells and endothelial cells in unremodeled model of the initiation of SpA remodeling (Fig. 10). vessels and veins also expressed both of the chemokines of interest. This is not surprising, as previous studies have demonstrated that Discussion decidual cells express a wide range of chemokines, presumed to be The findings from this study support the hypothesis that EVTs responsible for inducing immune cell recruitment into the decidua interact indirectly with SpA endothelial cells to facilitate the re- and creating the unique decidual immunological environment (20,

Table IV. EVTs differentially secrete MIC-1, CXCL8, and IL-6

Tissue Number Termination Type Gestational Age (wk) MIC-1 Concentration (pg/ml) CXCL8 Concentration (pg/ml) IL-6 Concentration (pg/ml) EP415 MTOP 8 + 6 1788 168 59 EP420 MTOP 8 + 0 1849 NM NM EP421 MTOP 7 + 0 1855 32 85 EP425 MTOP 6 + 0 1820 56 33 EP433 STOP 9 + 1 NM 56 13 EP454 MTOP 8 + 1 1846 NM NM EP461 MTOP 8 + 1 1790 NM NM EP463 MTOP 8 + 0 NM 76 45 EP469 MTOP 6 + 1 NM 16 12 EP471 MTOP 6 + 2 1877 NM NM EP519 STOP 7 + 6 1861 NM NM MTOP, medical termination of pregnancy; NM, not measured; STOP, surgical termination of pregnancy. 10 EVT-EC CROSSTALK DURING SPIRAL ARTERY REMODELING Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. CXCL8 and IL-6 are expressed by vEVTs. Serial sections of first trimester decidua basalis immunostained for (A and E) HLA-G (EVT marker), (D) a-SMA (smooth muscle cell marker), (B and F) CXCL8, and (C and G) IL-6. (A–C) Invading EVTs from anchoring villus are positive for CXCL8 and IL-6 (black arrow). (D) Extensive disorganization of smooth muscle cell layer (black arrow) in a remodeling arteriole. (F and G) Endovascular EVTs are positive for CXCL8 and IL-6. (H) Mouse IgG was used as a negative control. (A–C) Original magnification 310 and (D–H) original magnification 320. Scale bars, 50 mm. (I) CXCL8 immunostaining intensity is significantly higher in vEVTs than surrounding decidual cells. (J) IL-6 immunostaining intensity is similar in vEVTs and decidual cells. Data are median 6 interquartile range (n = 7). *p , 0.05, Wilcoxon matched-pairs test.

39–41). Moreover, our in vitro studies demonstrated that untreated chemoattractants for dNK cells in vitro. CCL14 is a known HUtMvECs and primary decidual endothelial cells express CCL14 monocyte chemoattractant, whereas CXCL6 classically acts on and CXCL6 mRNA, with increased expression upon EVT-CM neutrophils, acting through the receptors CXCR1 and CXCR2 (42). treatment. These findings were reproduced when immunostaining We identified expression of the cognate receptors (CXCR1, intensity of the chemokines was examined in the decidua basalis, CXCR2, CCR1, and CCR5) for both chemokines by dNK cells. with greater staining intensity detected in the endothelial cells of Previous studies indicated low mRNA expression of CCR1, remodeling SpAs than in surrounding decidual stromal cells and CCR5, and CXCR1 by dNK cells (43); however, at the protein endothelial cells in unremodeled SpAs. This reinforces the concept level CXCR1 was relatively abundant (41). Furthermore, cocul- of a localized chemokine gradient in remodeling arteries that reg- ture of PBNK cells with primary human decidual cells induces a ulates focal SpA leukocyte infiltration. dNK cell–like phenotype and this is accompanied by a significant Our findings using recombinant chemokine ligands and neu- increase in CCR1, CCR5, and CXCR1 protein expression (44). tralizing Abs demonstrate that CCL14 and CXCL6 are endothelial These results support the receptor expression patterns observed in The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ FIGURE 8. Chemokine expression by HUtMvECs in response to EVT-CM in the presence and absence of IL-6 and IL-8 neutralizing Abs. (A) EVT-CM stimulated CCL14 mRNA expression compared with control medium. This effect was blocked by cotreatment with neutralizing Ab against IL-6, alone or in combination with anti–IL-8. (B) EVT-CM stimulated CXCL6 mRNA expression compared with control medium. This effect was blocked by cotreatment with anti–IL-8 or anti–IL-6, or both in combination. EVT-CM also reduced (C) CXCL2, (D) CCL20, and (E) CX3CL1, and stimulated (F) CXCR7 mRNA expression compared with control medium. Cotreatment with neutralizing Ab against IL-6 and/or IL-8 had no effect in the mRNA expression of these chemokines/receptors. Mouse IgG served as a negative control. Data are median 6 interquartile range (n = 8). Friedman test with Dunn post hoc test. +p , 0.05, ++p , 0.01 compared with control. *p , 0.05, **p , 0.01, ***p , 0.001 compared with EVT treatment alone.

our study and also suggest dNK cells express these receptors at their receptors CXCR1, IL-6R, and gp130 by HUtMvECs. Al- by guest on September 24, 2021 higher levels than PBNK cells due to the local decidual micro- though decidual cells express both these cytokines, EVTs from environment. CCL14 induced chemotaxis of decidual macro- anchoring villi and vEVTs express higher levels of CXCL8. phages, consistent with its established actions in peripheral Moreover, their anatomical location within the SpA lumen places monocytes via CCR1 and CCR5 receptors (45). To our knowl- them in closer proximity to the endothelium, suggesting local edge, expression of these receptors by decidual macrophages has interactions between vEVT-secreted cytokines and endothelial not been reported previously. cells. Both cytokines have established actions on the vascular The chemokine receptors studied (CCR1, CCR5, CXCR1, and endothelium, signaling via Jak/stat pathways to activate inflam- CXCR2) have multiple ligands, including chemokines such as matory mediators, including NF-kB, adhesion molecules such as CCL3 and CXCL8 that have been previously reported in decidual ICAM-1 and VCAM-1, angiogenic factors, and chemokines (in- stromal cells (23, 44) or vascular cells (CCL16) (18, 20, 46, 47). cluding CXCL6) (52–56). These chemokines, and others, may contribute to recruitment of Studies of spontaneous miscarriage have identified reduced immune cells to the decidual stroma; however, in the current study IL-6 and CXCL8 in EVTs and lesser expression of their recep- we found no support for their involvement in EVT-stimulated tors by decidual endothelial cells (57); this may impair EVT- homing to remodeling SpAs because they were not identified in endothelial cell crosstalk, leading to dysregulation of endothelial our unbiased gene array. As well as decidual leukocytes, EVTs cell-derived chemokine production and reduced leukocyte re- also express the receptors for CCL14 and CXCL6 (46, 48), hence cruitment. There is evidence for altered levels of cytokines, these chemokines may have further actions in promoting coloni- including CXCL8 and IL-6, and chemokines (CCL2, CCL4, zation and remodeling of SpAs by EVTs. It is important to note CCL7, CXCL10, and CXCL12), in patients with preeclampsia that the corresponding receptors are not specific to dNK cells or (58–63). However, these measurements are restricted to maternal decidual macrophages; T cells express receptors for both che- serum or third trimester decidua and were made after diagnosis mokines. However, very few T cells have been observed in the of preeclampsia, and thus may reflect systemic endothelial dys- remodeling artery wall, presumably because of their relative rarity function and inflammation associated with the disease state. in the decidua (9, 14). Analyzing cytokine and chemokine expressions in the decidua of EVTs plug the openings of SpAs into the intervillous space very patients with a high risk of developing preeclampsia, such as early in pregnancy and also invade interstitially into the decidua. those presenting with high resistance Doppler indices of uterine EVTs are a rich source of cytokines and growth factors, including arteries in the first trimester (62), may help to identify whether IL-6, CXCL8, M-CSF, TGF-b, TNF and IL-13 (49–51). CXCL8 defects in EVT-endothelial signaling contribute to impaired and IL-6 in EVT-CM can activate HUtMvECs to secrete CCL14 remodeling in vivo. and CXCL6. This is consistent with our observation that IL-6 and Other factors secreted by EVTs that could affect endothelial cell CXCL8 are expressed by vEVTs in first trimester decidua and phenotype and behavior (64–67) were identified in our analysis, 12 EVT-EC CROSSTALK DURING SPIRAL ARTERY REMODELING Downloaded from http://www.jimmunol.org/

FIGURE 9. Chemokine expression by HUtMvECs in response to EVT-CM in the presence and absence of MIC-1 neutralizing Abs. EVT-CM treatment significantly increased expression of (A) CCL14, (B) CXCL6, and (F) CXCR7 and decreased expression for (C) CXCL2, (D)CCL20,and(E) CX3CL1. Cotreatment with neutralizing Ab against MIC-1 had no effect on the expression of the chemokines/receptors. Mouse IgG served as a negative control for the neutralizing Abs. Data are median 6 interquartile range (n = 8). Friedman test with Dunn post hoc test. +p , 0.05, ++p , 0.01, compared with control. by guest on September 24, 2021 including MIF, MIC-1, CD147, IL-1a and CCL2, all potential the chemotaxis assay to minimize phenotypic drift. Migrated candidates for contributing to EVT-SpA crosstalk. A low con- cells were in HUtMvEC-CM for a maximum of 16 h before centration of MIC-1 in maternal circulation is a strong predictor collection and counting. Cytokine production reflecting NK of miscarriage (68, 69). However, despite being present in high cell/macrophage activation due to a possible allogeneic re- concentrations in EVT-CM, we did not observe any effect of sponse was not measured as the study focused on migration MIC-1 on endothelial expression of the chemokines of interest. only. The purity of the dNK cells and macrophage populations A strength of this study is the use of primary EVT cultures, used for the chemotaxis assay was 80%, suggesting a strong generated using the established EVT outgrowth model (25, 70), enrichment for the specific immune cells of interest, but the thus closely resembling physiological conditions in pregnancy. presence of small numbers of other decidual cells. One further Similar studies of EVT-endothelial cell interactions have used caution for data interpretation is the use of a standard 20% EVT cell lines and have identified upregulation of CXCL10 in oxygen tension for EVT outgrowths. The maternofetal interface response to EVT-CM exposure (71). Although CXCL10 ex- is physiologically hypoxic in the first trimester (73), but oxygen pression was identified in EVT-primed endothelial cells, the tension in the decidua basalis is unknown. As previously de- change in expression (PPLR = 0.55) did not meet our threshold scribed (74), we found high rates of EVT outgrowth at 20% for further analysis. This may be due to differences in endo- oxygen, allowing us to obtain conditioned medium in a 48-h thelial cell lines used, or in the cytokine repertoire of primary time frame, prior to EVT-induced matrix digestion and loss of EVTs compared with SGHPL-4 cells. HUtMvECs are derived cell viability. Furthermore, we validated observations in first from the uterus and have been widely used in similar studies trimester decidua basalis tissue sections. (72). In addition, we were able to replicate the upregulation of In summary, this study demonstrates a complex localized CXCL6andCCL14expressionbyEVT-CMinprimarydecidual communication between EVTs, SpA endothelial cells, and de- endothelial cells. Furthermore, immunostaining of the decidua cidual leukocytes that potentially regulates leukocyte recruit- basalis revealed CCL14 and CXCL6 on SpA endothelial cells, ment to SpAs in early pregnancy and identifies key candidate supporting our in vitro observations. We were unable to pair chemokines and cytokines involved. Most studies have focused leukocytes and EVT outgrowths from the same samples because on EVT migration to the SpA wall and the consequences of poor EVT outgrowth takes a total of 96 h, and it is not possible to EVT invasion. We suggest initiation of SpA remodeling depends culture decidual leukocytes for that long without affecting their on activation of innate immune cells by paracrine factors re- phenotype and viability. EVT-CM and HUtMvEC-CM were leased from trophoblasts. The findings identify novel avenues for cleared of cellular debris so the medium contained only soluble research and potential therapeutic interventions to treat inade- proteins. NK cells and macrophages were freshly isolated for quate SpA remodeling in pathological pregnancies. The Journal of Immunology 13

Acknowledgments We thank all the research nurses in St. Mary’s Hospital and Mount Sinai Hospital for efforts in obtaining consents and with collecting tissues. We also thank all the donors for consenting to participate and making this study possible. We would like to acknowledge Dr. Samantha D Smith, Dr. Leo Zeef and Bona Kim for assistance with experiments.

Disclosures The authors have no ﬁnancial conﬂicts of interest.

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CD31 αSMA

Supplementary figure 1: CD31 is expressed by HUtMvEC. (A) All HUtMvEC are positive for CD31 (black arrow) and negative for (B) αSMA. Mouse IgG was used as a negative control. 20 x original magnification. Scale bar represents 50μm. IgG

HLA-G+DAPI M30+DAPI

Supplementary figure 2: HLA-G is expressed by Extravillous trophoblasts (EVTs) in EVT outgrowth. (A) EVTs are positive for HLA-G (white arrow) and negative for (B) M30. Mouse IgG was used as a negative control. 40 x original magnification. Scale bar represents 50μm. A) B) C)

D) E) F)

Supplementary figure 3: Flow cytometry analysis for dNK cells and macrophage purity. (A,B,C) 81.7% of the isolated dNK cells are CD56 positive. (D,E,F) 83.5% of the isolated macrophages are CD14 positive. Mouse IgG was used as negative control , black peak (C & F).