Decidual Macrophages Research Articles

MiR-146a-5p enhances embryo survival in unexplained recurrent spontaneous abortion by promoting M2 polarization of decidual macrophages

Unexplained recurrent spontaneous abortion (URSA) is one of the most challenging conditions in the reproductive field, and macrophage M1/M2 polarization disorder is involved in URSA pathogenesis, although the relevant mechanisms are undefined. miR-146a-5p possesses an immunoregulatory role and is expressed in decidual immune cells, and this study aims to investigate its effect on decidual macrophage polarization and therapeutic prospects in URSA, which has never been reported. The levels of M1/M2 markers in the deciduae and the miR-146a-5p expression in the decidual macrophages of URSA and healthy pregnant women were first detected and analyzed. Then, the in vitro effect of miR-146a-5p on the M1/M2 polarization and the secretion of inflammatory cytokines was investigated in Tamm-Horsfall protein-1 (THP-1)-induced macrophages. Finally, the in vivo immunotherapeutic effect of miR-146a-5p on embryo survival and the potential mechanisms were evaluated in a murine model of immune-based URSA. As a result, the abnormal M1/M2 polarization, which showed a shift towards the M1 phenotype and correlated with the decreased expression of miR-146a-5p, was verified in human URSA decidual macrophages. miR-146a-5p could inhibit M1 polarization, promote M2 polarization, and result in an anti-inflammatory microenvironment in THP-1-induced macrophages. The intravenous injection of exogenous miR-146a-5p in the first trimester of pregnant URSA mice significantly reduced the embryo resorption rate and promoted the M2 polarization of decidual macrophages. In conclusion, miR-146a-5p enhances embryo survival in URSA by promoting decidual macrophage polarization toward an M2 phenotype, giving new ideas and potential targets for subsequent research on the pathogenesis and immunotherapeutic strategies of URSA.

Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection.

While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

MiR-196a-5p-Rich Extracellular Vesicles from Trophoblasts Induce M1 Polarization of Macrophages in Recurrent Miscarriage.

Purpose Numerous studies have described the presence of crosstalk between trophoblasts and macrophages and the critical role it plays in recurrent miscarriage (RM). However, the mechanism of trophoblast-derived extracellular vesicle (EV) miRNAs and their interactions with decidual macrophages in the pathogenesis of RM remains unclear. Materials and Methods miRNA-seq was used to identify the differentially expressed miRNAs between RM patients and healthy controls. qPCR and in situ hybridization assays were performed to analyze the expression levels of miR-196a-5p in RM. THP-1 cells were treated with EVs, and qPCR and flow cytometry were performed to explore the polarization of macrophages. To explore the crosstalk between trophoblasts and macrophages, a coculture model and a series of cell function assays were performed. Results We first demonstrated that miR-196a-5p expression was higher in the cytotrophoblasts of villous tissues and plasma EVs from RM patients. miR-196a-5p derived from trophoblasts could be transferred into macrophages via EVs to induce M1 polarization via IκBα-mediated NF-κB pathway. Moreover, we found that M1 macrophages induced by EV miR-196a-5p derived from trophoblasts conversely regulated the proliferation, migration, and apoptosis of trophoblasts via TNF-α. Conclusions This study indicated that trophoblast-derived EV miR-196a-5p was positively associated with RM and functioned by regulating the crosstalk between trophoblasts and macrophages. These findings may attribute to identify a novel biomarker specific for RM.

Host-Viral Interactions at the Maternal-Fetal Interface. What We Know and What We Need to Know.

In humans, the hemochorial placenta is a unique temporary organ that forms during pregnancy to support fetal development, gaseous exchange, delivery of nutrition, removal of waste products, and provides immune protection, while maintaining tolerance to the HLA-haploidentical fetus. In this review, we characterize decidual and placental immunity during maternal viral (co)-infection with HIV-1, human cytomegalovirus (HCMV), and Zika virus. We discuss placental immunology, clinical presentation, and epidemiology, before characterizing host susceptibility and cellular tropism, and how the three viruses gain access into specific placental target cells. We describe current knowledge on host-viral interactions with decidual and stromal human placental macrophages or Hofbauer cells, trophoblasts including extra villous trophoblasts, T cells, and decidual natural killer (dNK) cells. These clinically significant viral infections elicit both innate and adaptive immune responses to control replication. However, the three viruses either during mono- or co-infection (HIV-1 and HCMV) escape detection to initiate placental inflammation associated with viral transmission to the developing fetus. Aside from congenital or perinatal infection, other adverse pregnancy outcomes include preterm labor and spontaneous abortion. In addition, maternal HIV-1 and HCMV co-infection are associated with impaired fetal and infant immunity in postnatal life and poor clinical outcomes during childhood in exposed infants, even in the absence of vertical transmission of HIV-1. Given the rapidly expanding numbers of HIV-1-exposed uninfected infants and children globally, further research is urgently needed on neonatal immune programming during maternal mono-and co-infection. This review therefore includes sections on current knowledge gaps that may prompt future research directions. These gaps reflect an emerging but poorly characterized field. Their significance and potential investigation is underscored by the fact that although viral infections result in adverse consequences in both mother and developing fetus/newborn, antiviral and immunomodulatory therapies can improve clinical outcomes in the dyad.

The Distributional Characteristics of M2 Macrophages in the Placental Chorionic Villi are Altered Among the Term Pregnant Women With Uncontrolled Type 2 Diabetes Mellitus.

AimNo definite conclusions have been drawn regarding how prolonged exposure to hyperglycemia affects the distribution of macrophages in the placenta, especially in pregnant women with uncontrolled type 2 diabetes mellitus (T2DM). Herein, we explored the distributional characteristics of placental M2 macrophages, including hofbauer cells (HBCs) in the chorionic villi and decidual macrophages, in pregnant women with uncontrolled T2DM.MethodsSix healthy singleton pregnancies and five uncontrolled T2DM singleton pregnancies were collected. Multicolor immunofluorescence and immunohistochemistry were performed to record M1 macrophages by CD80 and CD86, the general M2 macrophages by CD163, M2a macrophages by CD163 and DG-SIGN, M2b macrophages by CD163 and CD86, and M2c macrophages by CD163 and CD206. Meanwhile, the monocyte marker of CD14 and the general macrophage marker of CD68 were also documented on placenta.ResultsIn the chorionic villi and decidua, the most common infiltrated macrophages was the general M2. There were only few M1 and M2b macrophages distributed in the placenta of both the healthy and uncontrolled T2DM groups. The infiltrated degree of M2c macrophages was moderate in chorionic villi and decidua. The uncontrolled T2DM and healthy pregnant women had a comparable amount of M2c macrophages infiltration in the chorionic villi (p = 0.158). Notedly, in both of the healthy and uncontrolled T2DM pregnant women, the predominant subtype of M2 macrophages in the chorionic villi was M2a, where it mainly infiltrated around vessels and syncytiotrophoblasts. The uncontrolled T2DM pregnant women had more M2a macrophage infiltration than the healthy pregnant women (p = 0.016). The M2a macrophages in the decidua of the uncontrolled T2DM group were similar to those of the normal group (p = 0.800). Meanwhile, it was in the chorionic villi but not the decidua, that the CD68+ macrophages and CD14+ M2a macrophages were also elevated in the uncontrolled T2DM group (p = 0.035 and 0.044, respectively).ConclusionThese results confirmed that the M2 macrophages exhibited increased in the chorionic villi of pregnant women with uncontrolled T2DM. The subsets of M2 macrophages in the placental decidua were similar between uncontrolled T2DM pregnant women and normal groups. It may provide a basis for exploring the functions of different subsets of macrophages in the placental chorionic villi.

UHRF1 shapes both the trophoblast invasion and decidual macrophage differentiation in early pregnancy.

Trophoblasts play critical roles in establishment and maintenance of a normal pregnancy. Their dysfunction in early pregnancy is closely related to pregnancy-related diseases, including recurrent pregnancy loss (RPL). Epigenetic modifications dynamically change during pregnancy; however, the role of the epigenetic modifier UHRF1 in trophoblast regulation remains unknown. This is the first study to show that UHRF1 expression was localized in the cytoplasm of cytotrophoblasts, syncytiotrophoblasts, and villi columns, and decreased in the villi of patients with RPL. The invasion and cell viability in a UHRF1knockdown trophoblast cell line were significantly decreased. In addition, the mRNA expression profiles of Swan71 cells were partially altered by UHRF1knockdown. The altered immune-related genes were screened out and the pro-inflammatory TH1-type chemokine/cytokines CXCL2 and IL-1β were identified as the most promising targets of UHRF1 in the trophoblasts, which were significantly increased in the UHRF1knockdown Swan71 cells, villi, and serum from patients with RPL. The macrophages treated with the supernatants of UHRF1knockdown Swan71 cells were polarized to the M1 phenotype and secreted high levels of pro-inflammatory cytokines, which might be driven by the activated MyD88/NF-κB signaling pathway and mediated by the increased expression of CXCR2 and IL-1R1 (CXCL2 and IL-1β receptors, respectively). In addition, the supernatants of UHRF1knockdown Swan71 cells showed stronger chemotaxis to macrophages than those from the controls. Our findings highlight the previously unknown roles of UHRF1 as one of the key regulators on the trophoblasts and their cross-talk with local immune cells, and demonstrate a potential approach for RPL intervention.

GAS6-mediated dialogue between decidual stromal cells and macrophages is essential for early pregnancy maintenance by inducing M2-like polarization and cell proliferation of decidual macrophages.

Maternal immunotolerance towards the semi-allogeneic foetus is critical for normal pregnancy (NP). As a secretory protein, growth arrest-specific factor 6 (GAS6) promotes cancer progression by inducing the conversion of tumour-associated macrophages to an immunosuppressive M2-like phenotype. However, little is known about whether GAS6 regulates decidual macrophages (dMφs) in the early maternal-foetal interface. In this study, first-trimester decidual tissues were obtained from normal pregnant women undergoing elective terminations and patients with miscarriages. The expression of GAS6 and its receptors (AXL, TYRO3 and MERTK) in decidua and GAS6 secretion by decidual stromal cells (DSCs) was measured. Then, we investigated the effect of recombinant human GAS6 (rhGAS6) on dMφs isolated from NP and THP-1 cells, and revealed the underlying mechanism. Both the expression of GAS6 in DSCs and MERTK in dMφs, in addition to GAS6 secretion by DSCs, was found to be significantly decreased in miscarriage patients compared to that in NPs. Additionally, we observed that rhGAS6 polarized dMφs and THP-1 cells towards an M2-like phenotype, as evidenced by the up-regulated CD163 expression. Moreover, rhGAS6 enhanced the clearance of toxic cell-free haemoglobin by dMφs by up-regulating CD163 expression, and rhGAS6 also boosted cell proliferation of dMφs and THP-1 cells. Finally, we demonstrated that rhGAS6 stimulated CD163 expression and cell proliferation by activating the PI3K/Akt signalling pathway. Collectively, these findings suggest that GAS6-mediated dialogue between DSCs and dMφs is crucial for the establishment and maintenance of maternal-foetal immunotolerance, and decreased GAS6 secretion by DSCs may lead to the occurrence of miscarriage in the first trimester.

An imbalance of the IL-33/ST2-AXL-efferocytosis axis induces pregnancy loss through metabolic reprogramming of decidual macrophages.

During embryo implantation, apoptosis is inevitable. These apoptotic cells (ACs) are removed by efferocytosis, in which macrophages are filled with a metabolite load nearly equal to the phagocyte itself. A timely question pertains to the relationship between efferocytosis-related metabolism and the immune behavior of decidual macrophages (dMΦs) and its effect on pregnancy outcome. Here, we report positive feedback of IL-33/ST2-AXL-efferocytosis leading to pregnancy failure through metabolic reprogramming of dMΦs. We compared the serum levels of IL-33 and sST2, along with IL-33 and ST2, efferocytosis and metabolism of dMΦs, from patients with normal pregnancies and unexplained recurrent pregnancy loss (RPL). We revealed disruption of the IL-33/ST2 axis, increased apoptotic cells and elevated efferocytosis of dMΦs from patients with RPL. The dMΦs that engulfed many apoptotic cells secreted more sST2 and less TGF-β, which polarized dMΦs toward the M1 phenotype. Moreover, the elevated sST2 biased the efferocytosis-related metabolism of RPL dMΦs toward oxidative phosphorylation and exacerbated the disruption of the IL-33/ST2 signaling pathway. Metabolic disorders also lead to dysfunction of efferocytosis, resulting in more uncleared apoptotic cells and secondary necrosis. We also screened the efferocytotic molecule AXL regulated by IL-33/ST2. This positive feedback axis of IL-33/ST2-AXL-efferocytosis led to pregnancy failure. IL-33 knockout mice demonstrated poor pregnancy outcomes, and exogenous supplementation with mouse IL-33 reduced the embryo losses. These findings highlight a new etiological mechanism whereby dMΦs leverage immunometabolism for homeostasis of the microenvironment at the maternal-fetal interface.

A defective lysophosphatidic acid-autophagy axis increases miscarriage risk by restricting decidual macrophage residence

ABSTRACT Massive infiltrated and enriched decidual macrophages (dMφ) have been widely regarded as important regulators of maternal-fetal immune tolerance and trophoblast invasion, contributing to normal pregnancy. However, the characteristics of metabolic profile and the underlying mechanism of dMφ residence remain largely unknown. Here, we observe that dMφ display an active glycerophospholipid metabolism. The activation of ENPP2-lysophosphatidic acid (LPA) facilitates the adhesion and retention, and M2 differentiation of dMφ during normal pregnancy. Mechanistically, this process is mediated through activation of the LPA receptors (LPAR1 and PPARG/PPARγ)-DDIT4-macroautophagy/autophagy axis, and further upregulation of multiple adhesion factors (e.g., cadherins and selectins) in a CLDN7 (claudin 7)-dependent manner. Additionally, poor trophoblast invasion and placenta development, and a high ratio of embryo loss are observed in Enpp2 ±, lpar1 −/− or PPARG-blocked pregnant mice. Patients with unexplained spontaneous abortion display insufficient autophagy and cell residence of dMφ. In therapeutic studies, supplementation with LPA or the autophagy inducer rapamycin significantly promotes dMφ autophagy and cell residence, and improves embryo resorption in Enpp2 ± and spontaneous abortion mouse models, which should be dependent on the activation of DDIT4-autophagy-CLDN7-adhesion molecules axis. This observation reveals that inactivation of ENPP2-LPA metabolism and insufficient autophagy of dMφ result in resident obstacle of dMφ and further increase the risk of spontaneous abortion, and provides potential therapeutic strategies to prevent spontaneous abortion. Abbreviations: ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; Atg5: autophagy related 5; ATG13: autophagy related 13; BECN1: beclin 1; CDH1/E-cadherin: cadherin 1; CDH5/VE-cadherin: cadherin 5; CFSE: carboxyfluorescein succinimidyl ester; CLDN7: claudin 7; CSF1/M-CSF: colony stimulating factor 1; CSF2/GM-CSF: colony stimulating factor 2; Ctrl: control; CXCL10/IP-10: chemokine (C-X-C) ligand 10; DDIT4: DNA damage inducible transcript 4; dMφ: decidual macrophage; DSC: decidual stromal cells; ENPP2/ATX: ectonucleotide pyrophosphatase/phosphodiesterase 2; Enpp2 ±: Enpp2 heterozygous knockout mouse; ENPP2i/PF-8380: ENPP2 inhibitor; EPCAM: epithelial cell adhesion molecule; ESC: endometrial stromal cells; FGF2/b-FGF: fibroblast growth factor 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPCPD1: glycerophosphocholine phosphodiesterase 1; HE: heterozygote; HIF1A: hypoxia inducible factor 1 subunit alpha; HNF4A: hepatocyte nuclear factor 4 alpha; HO: homozygote; ICAM2: intercellular adhesion molecule 2; IL: interleukin; ITGAV/CD51: integrin subunit alpha V; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11b: integrin subunit alpha X; ITGB3/CD61: integrin subunit beta 3; KLRB1/NK1.1: killer cell lectin like receptor B1; KRT7/cytokeratin 7: keratin 7; LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; lpar1 −/−: lpar1 homozygous knockout mouse; LPAR1i/AM966: LPAR1 inhibitor; LY6C: lymphocyte antigen 6 complex, locus C1; LYPLA1: lysophospholipase 1; LYPLA2: lysophospholipase 2; Lyz2: lysozyme 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MARVELD2: MARVEL domain containing 2; 3-MA: 3-methyladenine; MBOAT2: membrane bound O-acyltransferase domain containing 2; MGLL: monoglyceride lipase; MRC1/CD206: mannose receptor C-type 1; MTOR: mechanistic target of rapamycin kinase; NP: normal pregnancy; PDGF: platelet derived growth factor; PLA1A: phospholipase A1 member A; PLA2G4A: phospholipase A2 group IVA; PLPP1: phospholipid phosphatase 1; pMo: peripheral blood monocytes; p-MTOR: phosphorylated MTOR; PPAR: peroxisome proliferator activated receptor; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGi/GW9662: PPARG inhibitor; PTPRC/CD45: protein tyrosine phosphatase receptor type, C; Rapa: rapamycin; RHEB: Ras homolog, mTORC1 binding; SA: spontaneous abortion; SELE: selectin E; SELL: selectin L; siCLDN7: CLDN7-silenced; STAT: signal transducer and activator of transcription; SQSTM1: sequestosome 1; TJP1: tight junction protein 1; VCAM1: vascular cell adhesion molecule 1; WT: wild type.

Decidual macrophage derived MMP3 contributes to extracellular matrix breakdown in spiral artery remodeling in early human pregnancy

Remodeling of the uterine spiral arteries is required for a successful pregnancy. This process requires the co-ordinated activity of a number of different cell types including uterine natural killer cells, decidual macrophages, extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells. We have previously demonstrated that decidual macrophages facilitate breakdown of fibronectin and laminin in a model of spiral artery remodeling. The aim of the current study was to determine which matrix metalloproteinases (MMPs) decidual macrophages express and play roles in extracellular matrix (ECM) breakdown in vascular remodeling. Decidual macrophages were isolated from first trimester decidua and cultured for 24 h to obtain conditioned medium. MMP secretion was assessed by a membrane based array and immunohistochemistry of decidual sections. In addition, the chorionic plate artery (CPA) model was used with decidual macrophage conditioned medium, with and without a MMP3 inhibitor and ECM protein expression assessed using quickscore. The decidual macrophages secreted a wide range of MMPs, with MMP3 being the most predominant. Co-localization of MMP3 to decidual macrophages was confirmed by immunohistochemistry. Decidual macrophage conditioned medium facilitated breakdown of laminin and fibronectin in the CPA model, an effect that was abrogated by the MMP3 inhibitor. These data further support the role of decidual macrophages in tissue remodeling in the first trimester of pregnancy. An alteration in their numbers or phenotype would impact spiral artery remodeling and contribute to the etiology of a number of complications of pregnancy.

Prostaglandin E2 receptor 3 promotes M1 macrophages polarization in unexplained recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is associated with macrophage polarization, which can be modulated by prostaglandin E2 (PGE2). Our previous study demonstrated that PGE2 receptor 3 (EP3) signaling is induced in the first-trimester placentas of uRPL patients compared with its expression in healthy controls. However, whether EP3 plays a role in macrophage polarization at the maternal-fetal interface of uRPL women remains unknown. The positive expression of EP3 in decidual macrophages was confirmed by double immunofluorescence staining in the first-trimester placentas collected from uRPL patients and healthy controls. Antibodies CD68, iNOS, and CD163 were used as immunofluorescence marker for decidual macrophages, M1, and M2 macrophages. To clarify the effects of EP3 on macrophage polarization, THP-1 monocyte cells were applied as M0 macrophages after phorbol 12-myristate 13-acetate (PMA) treatment for in vitro study. The mRNA levels of representative M1 markers (interleukin-1β and interleukin-6) and M2 markers (interleukin-10 and arginase-1) were quantified with qPCR in M0 macrophages being stimulated with sulprostone (an EP3 agonist) or L-798,106 (an EP3 antagonist). We found that EP3 expression was upregulated in the decidual macrophages of first-trimester placentas from uRPL patients compared with healthy controls. Furthermore, EP3 expression was increased in M1 macrophages compared with that in M2 macrophages in first-trimester placentas of uRPL patients. Sulprostone intensified the mRNA levels of IL-6 together with interferon-γ, whereas L-798,106 stimulated the mRNA expression of IL-10 and Arg-1 in a dose-dependent manner.

Decorin promotes decidual M1-like macrophage polarization via mitochondrial dysfunction resulting in recurrent pregnancy loss.

Rationale: Recurrent pregnancy loss (RPL) is a distressing disorder that seriously affects the physical and psychological health of women. RPL is also a sentinel risk marker for future obstetric complications and warrants in-depth investigation. Abnormal polarization and functions of decidual macrophages are associated with RPL; however, the underlying mechanisms remain poorly understood. Methods: Decorin expression, localization, and content in the decidua of women with normal pregnancy (NP) and those with RPL were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, immunofluorescence, and enzyme-linked immunosorbent assay. The profiles of decidual macrophage subsets were determined using flow cytometry and immunofluorescence in both groups. The correlation between decorin content and the proportion of decidual macrophage subsets in the decidua of early NP women was determined using Pearson analysis. The effects of decorin on the polarization and functions of macrophages were assessed in an in-vitro model of Raw264.7 cells via flow cytometry, western blotting, and RT-qPCR. Moreover, the mitochondrial metabolism in Raw264.7 cells under decorin administration was measured via flow cytometry, western blotting, and immunofluorescence. Thirty-three pregnant mice were included in the in vivo model and underwent different treatments. The embryo abortion rate, macrophage phenotype in the spleen and uteri, and placental development were evaluated using flow cytometry and hematoxylin-eosin staining. Results: Decorin, derived from decidual stromal cells, was highly expressed in the decidua of women with RPL. A positive correlation between decorin content and the proportion of M1-like macrophages was also observed in the decidua of early NP women. In vitro studies showed that decorin treatment inhibited macrophage polarization to M2-like subsets and boosted the inflammatory response, which was related to enhanced anaerobic glycolysis, increased mitochondrial membrane potential and intracellular reactive oxygen species levels, reduced mitochondrial mass, and activation of the myeloid differentiation primary response 88-nuclear factor-κB signaling pathway. Adoptive transfer of decorin-treated bone marrow-derived macrophages in pregnant C57BL/6 mice increased the embryo absorption, accompanied by impaired fetal vascularization. Conclusions: Decidual stromal cell-derived decorin can polarize decidual macrophages toward the M1 phenotype by regulating mitochondrial metabolism, resulting in the occurrence of RPL.

Trophoblast-derived Lactic Acid Orchestrates Decidual Macrophage Differentiation via SRC/LDHA Signaling in Early Pregnancy.

Lactic acid (LA) metabolism in the tumor microenvironment contributes to the establishment and maintenance of immune tolerance. This pathway is characterized in tumor associated macrophages. However, the role and pathway of LA metabolism at maternal-fetal interface during early pregnancy, especially in decidual macrophage differentiation, are still unclear. Herein, for the first time, we discovered that LA can trigger either M2 or M1 macrophage polarization via oxidative phosphorylation and glycolysis regulation under normoxia or hypoxia, respectively. Also, LA metabolism played a vital role in decidual macrophages-mediated recurrent pregnancy loss (RPL), through HIF-1α/SRC/LDHA pathway. Moreover, blockade of LA intake with AZD3965 (MCT-1 inhibitor) could rescue pregnancy in an abortion-prone mouse model, suggesting a potential therapeutic target in RPL. Collectively, the present study identifies the previously unknown functions of LA metabolism in the differentiation of decidual macrophages in early normal pregnancy and RPL, and provides a potential therapeutic strategy in RPL by manipulating decidual macrophages' functions through LA metabolic pathway.

Decidua Basalis: An Ex Vivo Model to Study HIV-1 Infection During Pregnancy and Beyond.

The human decidua basalis, main uterine mucosa during pregnancy, provides an ex vivo model for studying natural protection of macrophages against HIV-1 infection at the mucosal level. Beyond pregnancy, the decidua constitutes also a valuable tool to assess tissue-resident macrophage infection. Here, we provide a detailed protocol for decidual macrophage purification and tissue infection.

Uncomplicated oocyte donation pregnancies display an elevated CD163-positive type 2 macrophage load in the decidua, which is associated with fetal-maternal HLA mismatches.

ProblemThe embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which may challenge the maternal immune system to tolerize the fetus. Decidual macrophages are essential in maintaining a healthy pregnancy, and type 2 macrophages may exhibit immune suppressive activity. We hypothesized that the composition of decidual macrophages is different between uncomplicated OD pregnancies and non‐OD in vitro fertilization (IVF) pregnancies, and is related to fetal‐maternal incompatibility.Method of studyWomen with uncomplicated pregnancy were enrolled: 25 singleton OD pregnancies and 17 non‐OD IVF pregnancies. The extent of immunohistochemical staining of CD14 (pan‐macrophage marker) and CD163 (type 2 macrophage marker) in both decidua basalis and parietalis was quantitated by digital image analysis. Maternal and fetal DNA was typed for human leukocyte antigen (HLA)‐A, ‐B, C, ‐DRB1, and ‐DQB1, and fetal‐maternal HLA mismatches were calculated.ResultsOD pregnancies showed a higher percentage of CD163+ staining (P = .040) and higher CD163/CD14 ratio (P = .032) in the parietalis than non‐OD IVF. The OD group was separated into a semi‐allogeneic group (≤5 fetal maternal HLA mismatches) and a fully allogeneic group (> 5 mismatches). The HLA‐fully‐allogeneic OD group, but not the HLA‐semi‐allogeneic OD group, showed significantly elevated CD163/CD14 ratio in the parietalis compared with the non‐OD IVF group (P < .05).ConclusionsUncomplicated OD pregnancies display a higher CD163‐positive cell fraction in the total decidual macrophage population compared to autologous pregnancies, which may suggest that a local type 2 macrophage‐related mechanism is needed to compensate for the higher fetal‐maternal HLA mismatch load.

A newly intervention strategy in preeclampsia: Targeting PD-1/Tim-3 signaling pathways to modulate the polarization of decidual macrophages.

Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) are important immune checkpoint receptors that prevent an overreacted maternal immune response to fetal antigens during pregnancy. Disruption of complex immune regulation mechanisms is associated with adverse pregnancy outcomes, including preeclampsia (PE). Our recent study showed that the Tim-3 pathway was involved in the regulation of decidual macrophage polarization. Decidual macrophages polarized to the M1 phenotype may impair uterine vessel remodeling during placentation, accounting for the occurrence of PE. Co-blockade of the PD-1/Tim-3 pathway was shown to successfully control tumor growth in preclinical cancer models. However, the effects of activating both PD-1 and Tim-3 pathways as a combined intervention strategy in PE are never reported. Herein, we observed the skew of decidual macrophage polarization toward the M1 phenotype in patients with PE and lipopolysaccharide (LPS)-induced PE-like rat model. Moreover, we found that the activation of PD-1/Tim-3 pathway by using PD-L1 and Gal-9 fusion proteins could alleviate the manifestation of the LPS-induced PE-like rats and protect their offspring. Compared with the single intervention, the combination of PD-L1and Gal-9 fusion proteins exhibited obvious advantages in the relief of PE-like symptoms, trophoblast invasion, and fetal vascular development, indicating a synergistic effect of the activated PD-1/Tim-3 pathway. The in vitro study also revealed that the combined intervention using PD-L1 and Gal-9 fusion proteins inhibited the LPS-induced M1macrophage polarization via the synergic activation of the ERK/GSK3β/β-catenin signaling pathway. Together, our findings provide the first evidence that simultaneous activation of PD-1/Tim-3signaling pathways may have an optimal protective effect and serve as a new potential target for PE intervention.

Downregulation of EZH2 in Trophoblasts Induces Decidual M1 Macrophage Polarization: a Potential Cause of Recurrent Spontaneous Abortion

Macrophages are known to be pivotal for ensuring the establishment of the immune tolerance microenvironment at the maternal–fetal interface. In particular, trophoblasts stay in close contact with decidual macrophages (DMs), which have been reported to play an active role in the modulation of the polarization of DMs. Thus, any dysfunction of trophoblasts might be associated with certain pregnancy‐related complications, such as recurrent spontaneous abortion (RSA). Enhancer of zeste homolog 2 (EZH2) is an important epigenetic regulatory gene that has been previously shown to be related to immune regulation. The present study assessed the expression of EZH2 in villi tissue obtained from healthy controls and RSA patients. Trophoblasts conditioned medium was collected to incubate macrophages differentiated from the THP‐1 cell line. The expression and function of EZH2 in trophoblasts were knocked down either by the use of siRNA or GSK126 as an inhibitor. Our results show a significant decrease in the expression of EZH2 in villi tissue from RSA patients as compared to healthy controls. Further, the inhibition of expression or function of EZH2 in trophoblasts promoted M1 macrophage polarization, which might be involved in the pathogenesis of RSA. Moreover, the suppression of EZH2 was found to affect the secretion of immune and inflammatory cytokines in trophoblasts. Altogether, these results indicated the importance of EZH2 in the regulation of immune functions of trophoblasts and thus highlighted its potential to be explored as a therapeutic target to prevent and treat pregnancy loss.

Decreased nitric oxide content mediated by asymmetrical dimethylarginine and protein l-arginine methyltransferase 3 in macrophages induces trophoblast apoptosis: a potential cause of recurrent miscarriage.

Is the protein l-arginine methyltransferase 3 (PRMT3)/asymmetrical dimethylarginine (ADMA)/nitric oxide (NO) pathway involved in the development of recurrent miscarriage (RM), and what is the potential mechanism? Elevated levels of PRMT3 and ADMA inhibit NO formation in the decidua, thereby impairing the functions of trophoblast cells at the maternal-foetal interface. Decreased NO bioavailability is associated with RM. ADMA, an endogenous inhibitor of nitric oxide synthase (NOS), is derived from the methylation of protein arginine residues by PRMTs and serves as a predictor of mortality in critical illness. A total of 145 women with RM and 149 healthy women undergoing elective termination of an early normal pregnancy were enrolled. Ninety-six female CBA/J, 24 male DBA/2 and 24 male BALB/c mice were included. CBA/J × DBA/2 matings represent the abortion group, while CBA/J × BALB/c matings represent the normal control group. The CBA/J pregnant mice were then categorised into four groups: (i) normal + vehicle group (n = 28), (ii) abortion + vehicle group (n = 28), (iii) normal + SGC707 (a PRMT3 inhibitor) group (n = 20) and (iv) abortion + SGC707 group (n = 20). All injections were made intraperitoneally on Days 0.5, 3.5 and 6.5 of pregnancy. Decidual tissues were collected on Days 8.5, 9.5 and 10.5 of gestation. The embryo resorption rates were calculated on Day 9.5 and Day 10.5 of gestation. NO concentration, ADMA content, NOS activity, expression levels of NOS and PRMTs in decidual tissues were determined using conventional assay kits or western blotting. PRMT3 expression was further analysed in decidual stromal cells, macrophages and natural killer cells. A co-culture system between decidual macrophages (DMs) and HTR-8/SVneo trophoblasts was constructed to study the roles of the PRMT3/ADMA/NO signalling pathway. Trophoblast apoptosis was analysed via Annexin V-fluorescein isothiocyanate/propidium iodide staining. CBA/J × DBA/2 mouse models were used to investigate the effects of SGC707 on embryo resorption rates. Our results show that NO concentration and NOS activity were decreased, but ADMA content and PRMT3 expression were increased in the decidua of RM patients. Moreover, compared with the normal control subjects, PRMT3 expression was significantly up-regulated in the macrophages but not in the natural killer cells or stromal cells of the decidua from RM patients. The inhibition of PRMT3 results in a significant decrease in ADMA accumulation and an increase in NO concentration in macrophages. When co-cultured with DMs, which were treated with SGC707 and ADMA, trophoblast apoptosis was suppressed and induced, respectively. In vivo experiments revealed that the administration of SGC707 reduced the embryo resorption rate of CBA/J × DBA/2 mice. All sets of experiments were not performed with the same samples. The main reason is that each tissue needs to be reserved for clinical diagnosis and only a small piece of each tissue can be cut and collected for this study. Our results indicate that the PRMT3/ADMA/NO pathway is a potential marker and target for the clinical diagnosis and therapy of RM. This study was supported by the National Key Research and Development Program of China (2017YFC1001401), National Natural Science Foundation of China (81730039, 82071653, 81671460, 81971384 and 82171657) and Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015). The authors have declared no conflict of interest. N/A.

MNSFβ Regulates TNFα Production by Interacting with RC3H1 in Human Macrophages, and Dysfunction of MNSFβ in Decidual Macrophages Is Associated With Recurrent Pregnancy Loss.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal–fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-β (MNSFβ) in embryonic implantation and pregnancy success. MNSFβ is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh (CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFβ and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFβ and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFβ to RC3H1. Specific knockdown of MNSFβ in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFβ expression in dMϕ may promote TNFα production via its interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal–fetal interface and thus pregnancy loss.

The Impact of Hypoxia in Early Pregnancy on Placental Cells.

Oxygen levels in the placental microenvironment throughout gestation are not constant, with severe hypoxic conditions present during the first trimester. This hypoxic phase overlaps with the most critical stages of placental development, i.e., blastocyst implantation, cytotrophoblast invasion, and spiral artery remodeling initiation. Dysregulation of any of these steps in early gestation can result in pregnancy loss and/or adverse pregnancy outcomes. Hypoxia has been shown to regulate not only the self-renewal, proliferation, and differentiation of trophoblast stem cells and progenitor cells, but also the recruitment, phenotype, and function of maternal immune cells. In this review, we will summarize how oxygen levels in early placental development determine the survival, fate, and function of several important cell types, e.g., trophoblast stem cells, extravillous trophoblasts, syncytiotrophoblasts, uterine natural killer cells, Hofbauer cells, and decidual macrophages. We will also discuss the cellular mechanisms used to cope with low oxygen tensions, such as the induction of hypoxia-inducible factor (HIF) or mammalian target of rapamycin (mTOR) signals, regulation of the metabolic pathway, and adaptation to autophagy. Understanding the beneficial roles of hypoxia in early placental development will provide insights into the root cause(s) of some pregnancy disorders, such as spontaneous abortion, preeclampsia, and intrauterine growth restriction.