Human Placental Macrophages Research Articles

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.

Host inflammatory dynamics reveal placental immune modulation by Group B Streptococcus during pregnancy

Group B Streptococcus (GBS) is a pathobiont that can ascend to the placenta and cause adverse pregnancy outcomes, in part through production of the toxin β‐hemolysin/cytolysin (β‐h/c). Innate immune cells have been implicated in the response to GBS infection, but the impact of β‐h/c on their response is poorly defined. We show that GBS modulates innate immune cell states by subversion of host inflammation through β‐h/c, allowing worse outcomes. We used an ascending mouse model of GBS infection to measure placental cell state changes over time following infection with a β‐h/c‐deficient and isogenic wild type GBS strain. Transcriptomic analysis suggests that β‐h/c‐producing GBS elicit a worse phenotype through suppression of host inflammatory signaling in placental macrophages and neutrophils, and comparison of human placental macrophages infected with the same strains recapitulates these results. Our findings have implications for identification of new targets in GBS disease to support host defense against pathogenic challenge.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

Streptococcus agalactiae npx Is Required for Survival in Human Placental Macrophages and Full Virulence in a Model of Ascending Vaginal Infection during Pregnancy.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx, which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δnpx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δnpx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host. IMPORTANCE This study sheds new light on the way that group B Streptococcus (GBS) defends itself against oxidative stress in the infected host. The enzyme encoded by the GBS gene npx is an NADH peroxidase that, our study reveals, provides defense against macrophage-derived reactive oxygen stress and facilitates infections of the uterus during pregnancy. This enzyme could represent a tractable target for future treatment strategies against invasive GBS infections.

SOC-II-10 Human placental macrophages as targets of cadmium toxicity

SOC-II-10 Human placental macrophages as targets of cadmium toxicity

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.

Gestational diabetes is associated with changes in inflammatory gene expression related to human placental macrophages

Gestational diabetes (GDM) is associated with adverse pregnancy outcomes. Metabolic dysfunction with diabetes can alter immune responses. With GDM, changes in the placental immune system are thought to drive pregnancy complications. The objective of this study as to evaluate placental pro and anti-inflammatory gene expression in pregnancies complicated by GDM.

Impact of Gestational Diabetes Mellitus (GDM) on M2-Phenotype and number of human placental macrophages assessed in situ by multiplex labelling and automated image analysis

Impact of Gestational Diabetes Mellitus (GDM) on M2-Phenotype and number of human placental macrophages assessed in situ by multiplex labelling and automated image analysis

Group B Streptococcus cpsE Is Required for Serotype V Capsule Production and Aids in Biofilm Formation and Ascending Infection of the Reproductive Tract during Pregnancy.

Group B Streptococcus (GBS) is an encapsulated Gram-positive pathogen that causes ascending infections of the reproductive tract during pregnancy. The capsule of this organism is a critical virulence factor that has been implicated in a variety of cellular processes to promote pathogenesis. Primarily comprised of carbohydrates, the GBS capsule and its synthesis is driven by the capsule polysaccharide synthesis (cps) operon. The cpsE gene within this operon encodes a putative glycosyltransferase that is responsible for the transfer of a Glc-1-P from UDP-Glc to an undecaprenyl lipid molecule. We hypothesized that the cpsE gene product is important for GBS virulence and ascending infection during pregnancy. Our work demonstrates that a GBS cpsE mutant secretes fewer carbohydrates, has a reduced capsule, and forms less biofilm than the wild-type parental strain. We show that, compared to the parental strain, the ΔcpsE deletion mutant is more readily taken up by human placental macrophages and has a significantly attenuated ability to invade and proliferate in the mouse reproductive tract. Taken together, these results demonstrate that the cpsE gene product is an important virulence factor that aids in GBS colonization and invasion of the gravid reproductive tract.

Intrinsic antibody mediated enhancement of ZIKV infection in placental macrophages

Abstract Zika virus (ZIKV) vertical transmission from mother to child in utero is associated with a number of neonatal abnormalities, including congenital microcephaly. Yet, the mechanism by which ZIKV infects the placenta and subsequently the fetus remains poorly understood. ZIKV and Dengue virus (DENV) endemic regions are commonly overlapping, and DENV specific antibodies can cross-react with ZIKV. We and others have previously demonstrated that cross-reactive DENV IgG can mediate ZIKV infection of placental cells though increased viral entry via Fc receptors. Specifically, we found that infection with ZIKV immune complexes (ZIKV-IC) generated from cross-reactive anti-DENV monoclonal IgG was enhanced in human placental macrophages called Hofbauer cells, which are found within the chorionic villus stroma near the fetal vasculature. In this study, we aimed to characterize the intrinsic cellular changes that occur in Hofbauer cells as a result of ZIKV-IC infection. We performed RNA sequencing on Hofbauer cells infected with ZIKV complexed with different concentrations of anti-DENV monoclonal IgG. Principal component analysis revealed that while ZIKV incubated with anti-influenza IgG had a similar gene expression profile to ZIKV alone, ZIKV-IC infection with increasing anti-DENV IgG skewed gene expression towards a unique phenotype. We identified a number of transcriptional profiles related to anti-DENV IgG dose. Notably, we observed a pattern of decreased interferon stimulated genes with increasing anti-DENV IgG dose. Yet, increasing anti-DENV IgG also increased ZIKV viral RNA. These results may indicate that ZIKV-IC infection makes Hofbauer cells more permissive to ZIKV replication by modulating the innate antiviral response.

Cross-reactive dengue immune complexes modulate innate antiviral responses to Zika virus infection in human placental macrophages

Abstract Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged in the Americas and is a pathogen of significant public health concern. Unique amongst flaviviruses, ZIKV can be vertically transmitted from an infected mother to the developing fetus in utero, in some cases resulting in adverse pregnancy outcomes, including spontaneous abortion and fetal brain abnormalities. We recently found that Hofbauer cells (HCs), a fetal-derived placental macrophage, are primary targets for ZIKV infection. Further, cross-reactive dengue antibodies facilitate ZIKV transcytosis across the placental barrier to seed infection within HCs. However, little is known about the innate immune mechanisms of HCs to limit virus replication and spread within the placenta. Here, we report that HCs express the RIG-I-like receptors, RIG-I, MDA5, and LGP2, and the cytosolic DNA sensor, cGAS, and can induce a potent innate immune response characterized by production of monocyte and macrophage chemoattractants, MCP-1, MIP-1α, and MIP-1β. In contrast, HCs infected in the presence or absence of ZIKV immune complexes induced little to no type I interferon (IFN), pro-inflammatory cytokines, or chemokines. Notably, HCs infected with ZIKV immune complexes displayed reduced antiviral gene induction as compared to HCs infected in the absence of immune complexes despite similar kinetics of virus replication. We are currently performing RNA sequencing analyses to determine how binding of viral immune complexes can alter the antiviral gene signature within HCs to enhance productive ZIKV infection. These studies will further our understanding of innate immunologic mechanisms and modulation during vertical transmission of flaviviruses in the placenta.

Palmitate induces apoptotic cell death and inflammasome activation in human placental macrophages

IntroductionThere is an increasing prevalence of non-communicable diseases worldwide. Metabolic diseases such as obesity and gestational diabetes mellitus (GDM) increasingly affect women during pregnancy, which can harm pregnancy outcomes and the long-term health and wellbeing of exposed offspring. Both obesity and GDM have been associated with proinflammatory effects within the placenta, the critical organ governing fetal development. MethodsThe purpose of these studies was to model, in vitro, the effects of metabolic stress (high levels of glucose, insulin and saturated lipids) on placental macrophage biology, since these cells are the primary innate immune phagocyte within the placenta with roles in governing maternofetal immune tolerance and antimicrobial host defense. Macrophages were isolated from the villous core of term, human placentae delivered through nonlaboring, elective Cesarean sections and exposed to combinations of elevated glucose (30 mM), insulin (10 nM) and the saturated lipid palmitic acid (palmitate, 0.4 mM). ResultsWe found that palmitate alone induced the activation of the nucleotide-binding oligomerization domain-like receptor (NLR) Family Pyrin Domain Containing 3 (NLRP3) inflammasome in placental macrophages, which was associated with increased interleukin 1 beta release and an increase in apoptotic cell death. Glucose and insulin neither provoked these effects nor augmented the impact of palmitate itself. DiscussionOur findings confirm an impact of saturated fat on placental macrophage immune activation and could be relevant to the impact of metabolic stress in vivo.

A Fast and Reliable Method to Isolate Human Placental Macrophages.

Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are characterized by flow cytometry. Ninety eight percent of isolated CD14+ placental macrophages also express the macrophage marker CD68. Thus, this efficient and reliable method yields placental macrophages at high purity and sufficient quantity for functional studies. © 2019 by John Wiley & Sons, Inc.

Protein kinase D mediates inflammatory responses of human placental macrophages to Group B Streptococcus.

During pregnancy, Group B Streptococcus (GBS) can infect fetal membranes to cause chorioamnionitis, resulting in adverse pregnancy outcomes. Macrophages are the primary resident phagocyte in extraplacental membranes. Protein kinase D (PKD) was recently implicated in mediating pro-inflammatory macrophage responses to GBS outside of the reproductive system. This work aimed to characterize the human placental macrophage inflammatory response to GBS and address the extent to which PKD mediates such effects. Primary human placental macrophages were infected with GBS in the presence or absence of a specific, small molecule PKD inhibitor, CRT 0066101. Macrophage phenotypes were characterized by evaluating gene expression, cytokine release, assembly of the NLRP3 inflammasome, and NFκB activation. GBS evoked a strong inflammatory phenotype characterized by the release of inflammatory cytokines (TNFα, IL-1β, IL-6 (P≤0.05), NLRP3 inflammasome assembly (P≤0.0005), and NFκB activation (P≤0.05). Pharmacological inhibition of PKD suppressed these responses, newly implicating a role for PKD in mediating immune responses of primary human placental macrophages to GBS. PKD plays a critical role in mediating placental macrophage inflammatory activation in response to GBS infection.

Decidual stromal cell-derived PGE2 regulates macrophage responses to microbial threat.

Bacterial chorioamnionitis causes adverse pregnancy outcomes, yet host-microbial interactions are not well characterized within gestational membranes. The decidua, the outermost region of the membranes, is a potential point of entry for bacteria ascending from the vagina to cause chorioamnionitis. We sought to determine whether paracrine communication between decidual stromal cells and macrophages shaped immune responses to microbial sensing. Decidual cell-macrophage interactions were modeled in vitro utilizing decidualized, telomerase-immortalized human endometrial stromal cells (dTHESCs) and phorbol ester-differentiated THP-1 macrophage-like cells. The production of inflammatory mediators in response to LPS was monitored by ELISA for both cell types, while phagocytosis of bacterial pathogens (Escherichia coli and Group B Streptococcus (GBS)) was measured in THP-1 cells or primary human placental macrophages. Diclofenac, a non-selective cyclooxygenase inhibitor, and prostaglandin E2 (PGE2 ) were utilized to interrogate prostaglandins as decidual cell-derived paracrine immunomodulators. A mouse model of ascending chorioamnionitis caused by GBS was utilized to assess the colocalization of bacteria and macrophages in vivo and assess PGE2 production. In response to LPS, dTHESC and THP-1 coculture demonstrated enhancement of most inflammatory mediators, but a potent suppression of macrophage TNF-α generation was observed. This appeared to reflect a paracrine-mediated effect of decidual cell-derived PGE2 . In mice with GBS chorioamnionitis, macrophages accumulated at sites of bacterial invasion with increased PGE2 in amniotic fluid, suggesting such paracrine effects might hold relevance in vivo. These data suggest key roles for decidual stromal cells in modulating tissue responses to microbial threat through release of PGE2 .

Zika virus productively infects primary human placenta-specific macrophages.

The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus-associated pregnancy complications.

Zika Virus Infects Human Placental Macrophages.

The recent Zika virus (ZIKV) outbreak in Brazil has been directly linked to increased cases of microcephaly in newborns. Current evidence indicates that ZIKV is transmitted vertically from mother to fetus. However, the mechanism of intrauterine transmission and the cell types involved remain unknown. We demonstrate that the contemporary ZIKV strain PRVABC59 (PR 2015) infects and replicates in primary human placental macrophages, called Hofbauer cells, and to a lesser extent in cytotrophoblasts, isolated from villous tissue of full-term placentae. Viral replication coincides with induction of type I interferon (IFN), pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. Our results suggest a mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting placental cells and disrupting the placental barrier.

Mono-ethylhexyl phthalate stimulates prostaglandin secretion in human placental macrophages and THP-1 cells.

BackgroundDiethylhexyl phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure, which is widespread in the US, increases preterm birth risk; however, the mechanisms driving this relationship are unclear. Because cyclooxygenase-2 (COX-2) dependent prostaglandin synthesis is implicated in preterm birth, we evaluated effects of mono-2-ethylhexyl phthalate (MEHP), the active metabolite of DEHP, on prostaglandin E2 (PGE2) synthesis and COX expression in human placental macrophages (PM). In addition, responses in PM were compared to those in a human macrophage-like cell line, THP-1.MethodsPM and THP-1 cells were treated for 2, 4, 8, or 24 h with MEHP concentrations ranging from 10 to 180 micromolar. PGE2 concentrations were assessed in culture medium using ELISA, and COX expression was determined by western blot.ResultsTreatment of PM and THP-1 cells with 180 micromolar MEHP for 24 h significantly increased PGE2 release. Co-treatment of PMs or THP-1 cells with 180 micromolar MEHP and the non-selective COX inhibitor indomethacin reduced MEHP-stimulated PGE2 production. Similarly, co-treatment of PM and THP-1 cells with the COX-2 selective inhibitor NS-398 resulted in a significant decrease in PGE2, suggesting that MEHP-stimulated PGE2 is dependent specifically on increased COX-2 expression. Western blot analysis revealed a significant increase in COX-2 expression in PM and THP-1 cells treated with 180 micromolar MEHP, and no changes in COX-1 expression, supporting the role of COX-2 in MEHP-stimulated PGE2 synthesis.ConclusionsThe findings from this study are the first to demonstrate phthalate-stimulated PGE2 synthesis in PM and warrant future studies into COX-2-dependent prostaglandin synthesis as a mechanism of toxicant-associated preterm birth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0046-8) contains supplementary material, which is available to authorized users.

Placental Hofbauer cells assemble and sequester HIV‐1 in tetraspanin‐positive compartments that are accessible to broadly neutralizing antibodies

IntroductionWithin monocyte-derived macrophages, HIV-1 accumulates in intracellular virus-containing compartments (VCCs) that are inaccessible to the external environment, which implicate these cells as latently infected HIV-1 reservoirs. During mother-to-child transmission of HIV-1, human placental macrophages (Hofbauer cells (HCs)) are viral targets, and have been shown to be infected in vivo and sustain low levels of viral replication in vitro; however, the risk of in utero transmission is less than 7%. The role of these primary macrophages as viral reservoirs is largely undefined. The objective of this study is to define potential sites of viral assembly, accumulation and neutralization in HCs given the pivotal role of the placenta in preventing HIV-1 infection in the mother-infant dyad.MethodsTerm placentae from 20 HIV-1 seronegative women were obtained following caesarian section. VCCs were evaluated by 3D confocal and electron microscopy. Colocalization R values (Pearson's correlation) were quantified with colocalization module of Volocity 5.2.1. Replication kinetics and neutralization studies were evaluated using p24 ELISA.ResultsWe demonstrate that primary HCs assemble and sequester HIV-1BaL in intracellular VCCs, which are enriched in endosomal/lysosomal markers, including CD9, CD81, CD63 and LAMP-1. Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red. Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies. In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI.ConclusionsThese findings suggest that placental HCs possess intrinsic adaptations facilitating unique sequestration of HIV-1, and may serve as a protective viral reservoir to permit viral neutralization and/or antiretroviral drug entry in utero.

Macrophage-Derived IL-33 Is a Critical Factor for Placental Growth

IL-33, the most recently discovered member of the IL-1 superfamily and ligand for the transmembrane form of ST2 (ST2L), has been linked to several human pathologies including rheumatoid arthritis, asthma, and cardiovascular disease. Deregulated levels of soluble ST2, the natural IL-33 inhibitor, have been reported in sera of preeclamptic patients. However, the role of IL-33 during healthy pregnancy remains elusive. In the current study, IL-33 was detected in the culture supernatants of human placental and decidual macrophages, identifying them as a major source of secreted IL-33 in the uteroplacental unit. Because flow cytometry and immunofluorescence stainings revealed membranous ST2L expression on specific trophoblast populations, we hypothesized that IL-33 stimulates trophoblasts in a paracrine manner. Indeed, BrdU incorporation assays revealed that recombinant human IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts and cell column trophoblasts in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. Interestingly, Western blot and immunofluorescence analyses demonstrated that IL-33 activates AKT and ERK1/2 in primary trophoblasts and placental explants. Inhibitors against PI3K (LY294002) and MEK1/2 (UO126) efficiently blocked IL-33-induced proliferation in all model systems used. In summary, with IL-33, we define for the first time, to our knowledge, a macrophage-derived regulator of placental growth during early pregnancy.