Hofbauer Cells Research Articles

4 Placental Hofbauer Cells As a Proxy Cell Type for Fetal Brain Microglia

Both placental and brain immune activation have been reported in maternal obesity-exposed (MATOB) offspring, and MATOB offspring have an increased risk for autism spectrum disorder, ADHD, and cognitive deficits. Microglia, the resident brain immune cells, have been implicated in these morbidities. Hofbauer cells (HBCs), resident fetal placental macrophages, and microglia, resident brain macrophages, share a common embryonic origin in the fetal yolk sac in mice and humans. Because direct evaluation of microglial function in a living human fetus or neonate is impossible, we sought to determine whether HBCs could serve as a more accessible biologic surrogate for fetal brain microglia in MATOB and control murine pregnancy. C57BL6/J dams were fed either a 60% high-fat diet or 10% fat control diet for 14 weeks pre-breeding and during pregnancy. At e17.5, fetal brain microglia and corresponding placental macrophages were isolated from fresh tissue. We performed single-cell RNA-sequencing on microglia and matched placental macrophages (10x Genomics, N=16, 4 replicates/group). 77,999 cells were sequenced. Canonical microglial and HBC markers were expressed in both cell types. Unsupervised analyses identified clusters within and across samples that share a significant fraction of “marker genes” (expressed more highly in cells from that cluster than in all other cells). Subsets of HBCs closely resembled subsets of microglia, with overlap analysis of marker genes demonstrating numerous clusters with 80-100% overlap between microglia and HBCs. This was true for both MATOB and CD. ScRNA-Seq elucidated novel shared gene programs and cell states that define HBCs and microglia. Shared gene programs in HBCs and microglia suggest that HBCs may be used as a proxy cell type to assay fetal brain microglial programming, in both MATOB and control mouse pregnancy. This finding may have broader implications for assaying the impact of maternal exposures beyond obesity on fetal brain development.

886 Placental hofbauer cell density is associated with increased neonatal birthweight and cord blood lipids

Maternal obesity is associated with adverse offspring cardiometabolic programming. Hofbauer cells (HC) are resident fetal tissue macrophages and are altered in pathological pregnancy conditions associated with fetoplacental immune activation. We sought to determine whether the number and activation state of placental Hofbauer cells (HC) as determined by CD163 and CD68 expression are associated with adverse maternal and/or cord blood lipid profiles. This is a cross-sectional study of 27 women undergoing planned cesarean delivery at term; women with hypertension or diabetes were excluded. Fasting maternal plasma, placental samples, and cord blood were collected at delivery. Maternal body mass index (BMI) at delivery and neonatal birthweight were obtained by chart review. Fasting lipid profiles (Total cholesterol, HDL, LDL, VLDL, triglycerides) were measured in maternal and cord blood at delivery. Immunohistochemistry was performed on serial placental sections for CD163 and CD68, markers of HC activation. Numbers of CD163+ and CD68+ cells per mm2 of villous area were quantified in terminal villi using ImageJ software and compared to maternal and fetal parameters using Spearman correlations. 41% of women were obese (N=11, pre-pregnancy BMI ≥ 30 kg/m2), and mean BMI at delivery was 33.2±7. CD163+ cells/mm2 was positively correlated with BMI at delivery (Spearman ρ=0.54, P=0.005), neonatal birthweight (ρ=0.42, P=0.03), and cord blood triglycerides (ρ=0.43, P=0.02), but not cord VLDL (ρ=0.37, P=0.06); Fig 1. CD68+ cells/mm2 was correlated with BMI at delivery (ρ=0.43, P=0.03), cord blood triglycerides (ρ=0.43, P=0.03) and cord blood VLDL (ρ=0.40, P=0.04) but not birthweight (ρ=0.24, P=0.24); Fig 2. Maternal lipids were not correlated with HC density. Fetoplacental immune activation was associated with neonatal birthweight, and altered cord blood lipid profiles in the setting of maternal obesity. Future studies will focus on HC activation as an indicator versus. mediator of offspring cardiometabolic programming.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Placental pathology of the third trimester pregnant women from COVID-19

AimsTo explore the clinical characteristics and placental pathological changes of pregnant women with 2019 novel coronavirus (CoV) disease (COVID-19) in the third trimester, and to assess the possibility of vertical transmission.Methods and resultsThe placenta tissues were evaluated by using immunohistochemistry for inflammatory cells and Hofbauer cells, and using severe acute respiratory syndrome (SARS) CoV-2 RNA Fluorescence In-Situ Hybridization (FISH) and SARS-CoV-2 spike protein immunofluorescence (IF) double staining. All eight placentas from the third trimester pregnancy women were studied. All patients were cured, no clinical or serological evidence pointed to vertical transmission of SARS-CoV-2. Features of maternal vascular malperfusion (MVM) such as increased syncytial knots were present in all 8 cases (8/8), and increased focal perivillous fibrin depositions were presented in 7 cases (7/8). No significate chronic histiocytic intervillositis was noted in the placenta. The number of macrophages and inflammatory cells such as T cells, B cells and plasma cells in the placental villous was not significantly increased in all cases. Moreover, all of eight cases demonstrated negative results by FISH using a SARS-CoV-2 virus RNA probe and by IF using a monoclonal antibody against SARS-CoV-2 spike protein.ConclusionsWe found no evidence of vertical transmission and adverse maternal-fetal outcomes in the placentas of third trimester COVID-19 pregnancy women, which provided further information for the clinical management of those women in the third trimester. However, further studies are still needed for patients with infections in different stage of gestation, especially in first and second trimester.

The expression patterns of IL-1β and IL-10 and their relation to CYP epoxygenases in normal human placenta

IntroductionThe success of pregnancy depends on the regulation of immunological processes in the placenta. Important mediators of an immune response include pro- and anti-inflammatory interleukins which may be regulated by CYP epoxygenases and their metabolites. The relation between interleukins and CYP epoxygenases expression in human placenta has not yet been studied vastly. Material and methodsWe investigated the expression patterns of IL-1β and IL-10 in embryonic (n=8), early foetal (n=16) and term (n=7) human placenta tissue by an immunohistochemical method and evaluated the results by Kruskal-Wallis test. The obtained data was correlated using Spearman’s correlation coefficient to our previously published data of CYP epoxygenases expression in the same samples. To confirm that Hofbauer cells express IL-10 and IL-1β as well as CYP2C8 and IL-10 together, and thus there is a relation between proteins of interests, we used multiplex immunofluorescent staining. ResultsThe expression of IL-1β decreased with gestational age in cytotrophoblast, syncytiotrophoblast, as well as in Hofbauer cells whilst IL-10 decreased in cytotrophoblast, remained at the same levels in syncytiotrophoblast and increased in Hofbauer cells. In trophoblast cells, we found a statistically significant positive correlation between the expression of CYP2J2 and CYP2C9 with IL-1β, whereas there was no relation between IL-10 and any of the tested CYP epoxygenases. In Hofbauer cells, we found a significant positive correlation between CYP2C8 and IL-10 and a significant negative correlation between CYP2C8 and IL-1β. ConclusionOur results showed that the exact role and relation of interleukins and CYP epoxygenases and their metabolites is dependent on their respective cellular context. Because of IL-10, IL-1β, as well as HBCs play a role in various pathological conditions, further investigation of the exact role of CYP epoxygenase, interleukins and their relations is needed.

Cell-specific characterization of the placental methylome

BackgroundDNA methylation (DNAm) profiling has emerged as a powerful tool for characterizing the placental methylome. However, previous studies have focused primarily on whole placental tissue, which is a mixture of epigenetically distinct cell populations. Here, we present the first methylome-wide analysis of first trimester (n = 9) and term (n = 19) human placental samples of four cell populations: trophoblasts, Hofbauer cells, endothelial cells, and stromal cells, using the Illumina EPIC methylation array, which quantifies DNAm at > 850,000 CpGs.ResultsThe most distinct DNAm profiles were those of placental trophoblasts, which are central to many pregnancy-essential functions, and Hofbauer cells, which are a rare fetal-derived macrophage population. Cell-specific DNAm occurs at functionally-relevant genes, including genes associated with placental development and preeclampsia. Known placental-specific methylation marks, such as those associated with genomic imprinting, repetitive element hypomethylation, and placental partially methylated domains, were found to be more pronounced in trophoblasts and often absent in Hofbauer cells. Lastly, we characterize the cell composition and cell-specific DNAm dynamics across gestation.ConclusionsOur results provide a comprehensive analysis of DNAm in human placental cell types from first trimester and term pregnancies. This data will serve as a useful DNAm reference for future placental studies, and we provide access to this data via download from GEO (GSE159526), through interactive exploration from the web browser (https://robinsonlab.shinyapps.io/Placental_Methylome_Browser/), and through the R package planet, which allows estimation of cell composition directly from placental DNAm data.

Hofbauer Cells and Nuclear Factor of Kappa Beta on Rat's Placenta that Black Carbon Exposure

Hofbauer Cells and Nuclear Factor of Kappa Beta on Rat's Placenta that Black Carbon Exposure

Isolation of First-Trimester and Full-term Human Placental Hofbauer Cells.

The placenta is the crucial organ that regulates the health of both mother and fetus during pregnancy. The human placenta is composed of villous tree-like structures that embed into the maternal decidua. Within the stroma of the villi resides a population of fetally-derived macrophages, the Hofbauer cells (HBC). HBC are the only fetal immune cells found within the placenta in the steady-state and are thought to play a crucial role in placental function. From the 10th week of gestation, maternal blood flow into the intervillous space begins, resulting in the placental villi becoming bathed in maternal blood. To study HBC it is necessary to develop techniques that allow for their specific isolation and distinction from maternal blood monocytes and decidual macrophages. Here, we describe a protocol that explains step-by-step the strategy we have developed that allows the specific isolation of HBC.

Single Cell Profiling of Hofbauer Cells and Fetal Brain Microglia Reveals Shared Programs and Functions

Single Cell Profiling of Hofbauer Cells and Fetal Brain Microglia Reveals Shared Programs and Functions

Gatekeepers of the fetus: Characterization of placental macrophages.

In this issue of JEM, Thomas et al. (https://doi.org/10.1084/jem.20200891) provide elegant technological and conceptual advances that further our understanding of the immune cells enriched at the maternal-fetal interface. Using new isolation strategies to better separate maternal- and fetal-derived cells, the authors identify previously undefined maternal-derived immune cells associated with the fetal-derived placenta and provide an in-depth analysis of the markers and characteristics of placental Hofbauer cells.

Morphometric analysis of Hofbauer cells in normal placenta and chorioamnionitis in humans.

Hofbauer cells are macrophages residing in the stroma of placental villi and play a number of roles during normal pregnancy, as well as pathological conditions. A morphometric analysis of Hofbauer cells, in particular to investigate the number of cells, their size and shape in samples of normal human placenta from 1st trimester, term and with chorioamnionitis was performed. Tissue samples were immunostained for CD206 antigen and evaluated using ImageJ software. We detected significant changes in number and morphology of HBCs between normal placenta and placenta with chorioamnionitis samples. In chorioamnionitis, the cells were unevenly distributed within the villi, generally present in higher numbers, larger and more elongated than those in normal 1st trimester and term placenta.

Stereological and Histological Assessment of the Umbilical Cord in New-Born Rat.

Background:Umbilical cord plays a crucial role in the continuation of pregnancy by transferring nutrition and oxygen across the placenta to the fetus. We aimed to investigate the morphometrical and histological features of the umbilical cords in new-born rats.Materials and Methods:The adult male and female rats were chosen for matting purpose in the present study. Briefly, ten adult Wistar albino rats (female, n = 5; male, n = 5) were randomly assigned into five groups of two animals (female, n = 1; male, n = 1). Immediately after parturition, two umbilical cords of new-born rats (0-day-old) from each group were randomly selected. Finally, ten umbilical cord samples were examined using the histological and stereological methods in the light and electron microscopes.Results:The total numbers of Hofbauer cells and mesenchymal stromal cells was estimated statistically. We also calculated the mean volume of umbilical cords, arteries and veins, as well as arterial and venous lumens. Our histological findings also exhibited the histological features of Hofbauer cells, mesenchymal stromal cell cells, and blood vessels.Conclusion:Our findings showed more detailed information about umbilical cord tissues and their components, and that may contribute to the diagnose of umbilical cord complications in the developing fetus.

Current status of fertility preservation for women with collagen disease undergoing gonadotoxic therapy in Nagasaki University Hospital

Alterations in the number and protein/gene expression of Hofbauer cells (HBCs) may play a role in microbial-driven/cytokine-mediated placental inflammation, and in subsequent pregnancy complications such as villitis, histologic chorioamnionitis, and the fetal inflammatory response syndrome. Pyroptosis is an inflammatory form of cell death mediated by the inflammasome, a multi-protein complex which drives the processing and secretion of interleukin 1 beta (IL-1β). Pyroptosis can be triggered by bacterial lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in non-placental macrophages through activation of the NLRP3 inflammasome. However, the role of inflammasome activation and pyroptosis in HBC pathophysiology remains unclear. HBCs isolated from human term placentas were treated with or without LPS or ATP, alone or in combination. Treatment of HBCs with both LPS and ATP induced the rapid secretion of high levels of IL-1β and at the same time, cell death associated with nuclear condensation and cellular swelling. HBC treatment with both LPS and ATP induced caspase-1 activation, gasdermin D (GSDMD) cleavage, which mediates pyroptosis, and IL-1β processing. Caspase-1 activation, GSDMD cleavage, IL-1β processing, and IL-1β secretion were all significantly reduced following NLRP3 knockdown; inhibition of caspase-1; and inhibition of P2X7, the receptor that mediates K+ efflux. Together, our data indicate that LPS and ATP treatment stimulated NLRP3 inflammasome activation and pyroptosis in HBCs leading to the rapid release of IL-1β. Since the localization of HBCs confers a unique ability to influence microbial-associated placental and fetal inflammation, these studies suggest a key role for the inflammasome and pyroptosis in mediating HBC driven inflammation.

SARS-CoV-2 can infect the placenta and is not associated with specific placental histopathology: a series of 19 placentas from COVID-19-positive mothers

Congenital infection of SARS-CoV-2 appears to be exceptionally rare despite many cases of COVID-19 during pregnancy. Robust proof of placental infection requires demonstration of viral localization within placental tissue. Only two of the few cases of possible vertical transmission have demonstrated placental infection. None have shown placental expression of the ACE2 or TMPRSS2 protein, both required for viral infection. We examined 19 COVID-19 exposed placentas for histopathologic findings, and for expression of ACE2, and TMPRSS2 by immunohistochemistry. Direct placental SARS-CoV-2 expression was studied by two methods—nucleocapsid protein expression by immunohistochemistry, and RNA expression by in situ hybridization. ACE2 membranous expression in the syncytiotrophoblast (ST) of the chorionic villi is predominantly in a polarized pattern with expression highest on the stromal side of the ST. In addition, cytotrophoblast and extravillous trophoblast express ACE2. No ACE2 expression was detected in villous stroma, Hofbauer cells, or endothelial cells. TMPRSS2 expression was only present weakly in the villous endothelium and rarely in the ST. In 2 of 19 cases, SARS-CoV-2 RNA was present in the placenta focally in the ST and cytotrophoblast. There was no characteristic histopathology present in our cases including the two placental infections. We found that the placenta is capable of being infected but that this event is rare. We propose one explanation could be the polarized expression of ACE2 away from the maternal blood and pronounced paucity of TMPRSS2 expression in trophoblast.

NLRP3 inflammasome function and pyroptotic cell death in human placental Hofbauer cells

Alterations in the number and protein/gene expression of Hofbauer cells (HBCs) may play a role in microbial-driven/cytokine-mediated placental inflammation, and in subsequent pregnancy complications such as villitis, histologic chorioamnionitis, and the fetal inflammatory response syndrome. Pyroptosis is an inflammatory form of cell death mediated by the inflammasome, a multi-protein complex which drives the processing and secretion of interleukin 1 beta (IL-1β). Pyroptosis can be triggered by bacterial lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in non-placental macrophages through activation of the NLRP3 inflammasome. However, the role of inflammasome activation and pyroptosis in HBC pathophysiology remains unclear. HBCs isolated from human term placentas were treated with or without LPS or ATP, alone or in combination. Treatment of HBCs with both LPS and ATP induced the rapid secretion of high levels of IL-1β and at the same time, cell death associated with nuclear condensation and cellular swelling. HBC treatment with both LPS and ATP induced caspase-1 activation, gasdermin D (GSDMD) cleavage, which mediates pyroptosis, and IL-1β processing. Caspase-1 activation, GSDMD cleavage, IL-1β processing, and IL-1β secretion were all significantly reduced following NLRP3 knockdown; inhibition of caspase-1; and inhibition of P2X7, the receptor that mediates K+ efflux. Together, our data indicate that LPS and ATP treatment stimulated NLRP3 inflammasome activation and pyroptosis in HBCs leading to the rapid release of IL-1β. Since the localization of HBCs confers a unique ability to influence microbial-associated placental and fetal inflammation, these studies suggest a key role for the inflammasome and pyroptosis in mediating HBC driven inflammation.

SARS-CoV2 vertical transmission with adverse effects on the newborn revealed through integrated immunohistochemical, electron microscopy and molecular analyses of Placenta.

Background. The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated.MethodsWe screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations.FindingsSARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence.InterpretationWe provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.

Sexually Dimorphic Crosstalk at the Maternal-Fetal Interface.

Crosstalk through receptor ligand interactions at the maternal-fetal interface is impacted by fetal sex. This affects placentation in the first trimester and differences in outcomes. Sexually dimorphic signaling at early stages of placentation are not defined. Investigate the impact of fetal sex on maternal-fetal crosstalk. Receptors/ligands at the maternal-fetal surface were identified from sexually dimorphic genes between fetal sexes in the first trimester placenta and defined in each cell type using single-cell RNA-Sequencing (scRNA-Seq). Academic institution. Late first trimester (~10-13 weeks) placenta (fetal) and decidua (maternal) from uncomplicated ongoing pregnancies. Transcriptomic profiling at tissue and single-cell level; immunohistochemistry of select proteins. We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. We examined fetal sex differences in 5 major cell types (trophoblasts, stromal cells, Hofbauer cells, antigen-presenting cells, and endothelial cells). Ligands from the CC family chemokine ligand (CCL) family were most highly representative in females, with their receptors present on the maternal surface. Sexually dimorphic trophoblast transcripts, Mucin-15 (MUC15) and notum, palmitoleoyl-protein carboxylesterase (NOTUM) were also most highly expressed in syncytiotrophoblasts and extra-villous trophoblasts respectively. Gene Ontology (GO) analysis using sexually dimorphic genes in individual cell types identified cytokine mediated signaling pathways to be most representative in female trophoblasts. Upstream analysis demonstrated TGFB1 and estradiol to affect all cell types, but dihydrotestosterone, produced by the male fetus, was an upstream regulator most significant for the trophoblast population. Maternal-fetal crosstalk exhibits sexual dimorphism during placentation early in gestation.

Characterization of Placental Infection by Zika Virus in Humans: A Review of the Literature.

The aim of the current review is to present a systematic evaluation of reported human placental findings in cases of zika virus (ZIKV) infection. We reviewed the EMBASE, PUBMED, and SCIELO databases until June 2019, without language restrictions. The search terms placenta AND zika virus were used. The inclusion criteria of the studies were studies that reported placental findings in humans. Experimental studies, reviews, notes or editorials were excluded. A total of 436 studies were retrieved; after duplicate exclusion, 243 articles had their titles screened, and 128 had their abstract read; of those, 32 were included in the final analysis (18 case reports, 10 case series, and 4 cohorts) DATA COLLECTION: We collected data concerning the author, year of publication, study design, number of participants, number of placental samples, onset of symptoms, perinatal outcomes, and main findings on histological analysis. The placental pathologic findings were described as mild and nonspecific, similar to those of other placental infections, including chronic placentitis, chronic villitis, increased Hofbauer cells, irregular fibrin deposits, increased mononuclear cells in the villus stroma, villous immaturity, edema, hypervascularization, stromal fibrosis, calcification, and focal necrosis of syncytiotrophoblasts. Zika infection presents unspecific placental findings, similar to other infections in the toxoplasmosis, other agents, rubella, cytomegalovirus, and herpes (TORCH)group. Characterizing and standardizing placental findings after zika virus infection is key to understanding the mechanisms of congenital diseases.

OR24-07 Fetal Sex Impacts First Trimester Maternal-Fetal Communication in Humans

The placenta serves as a regulator of fetal growth throughout pregnancy. Signaling at the maternal-fetal interface is critical during placentation and lays the groundwork for placenta function, affecting pregnancy outcomes. Fetal growth is impacted by fetal sex, with males larger than females, and maternal gestational diabetes and obesity independently increase the risk of macrosomia in male fetuses only. We previously demonstrated differentially expressed genes (DEGs) among sexes involves ancient canonical pathways and metabolic functions in placenta tissue. As these are likely impacted by signaling at the maternal-fetal interface, our aim here was to identify sex differences in signaling at the maternal-fetal interface and among individual cell types within the placenta to explain these differences. RNA-sequencing of first trimester placenta and maternal decidua as well as single cell RNA-sequencing in first trimester placenta was performed in ongoing pregnancies. We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. From these, 35 of 115 receptors and/or ligand genes were also found to be upstream regulators of pathways critical in sexually dimorphic placentation which may define regulation. Single cell analysis identified five major cell types (trophoblasts, stromal cells, hofbauer cells, antigen presenting cells, and endothelial cells), and all had sexually dimorphic genes. Among individual cell types, ligands from the CC-family of cytokines were most highly representative in females, with their corresponding receptors present on the maternal surface. Furthermore, upstream regulator analysis of sexually dimorphic genes demonstrated TGFβ1 and estradiol to significantly affect all cell types. Dihydrotestosterone, which is produced by the male fetus, was an upstream regulator that was most significant for the trophoblast population. In addition, gene ontology enrichment analysis identified distinctive enriched functions between male and female trophoblasts, with cytokine mediated signaling pathways most representative. MUC15 and NOTUM were the most highly expressed sexually dimorphic autosomal genes found in distinct cell types of the trophoblast population, cell types critical for placentation and nutrient exchange. Thus, differences in hormone and immune signaling pathways may account for differential gene expression and differences in trophoblast function during placentation, which may in turn explain developmental differences, including fetal size, well-being, and overall outcomes.

Baby’s First Macrophage: characterizing Hofbauer Cell phenotype through gestation using unsupervised dimensionality reduction

Abstract Fetal placental macrophages (Hofbauer cells (HCs)) appear at 18 days post-conception and are maintained throughout pregnancy. Despite their identification more than a century ago, there are few studies characterizing the evolution of human HC phenotype through gestation. Using high-dimensional flow cytometry, we measured expression of CD68, CD80, CD86, HLA-DR, CD163, CD206, and CD209 on human HCs ex vivo. This panel includes markers previously validated on term human HCs for immune activation (CD80, CD86) and modulation (DC-SIGN, HLA-DR). To best capture the spectrum of HC diversity across gestation, we employed an unbiased approach to analyze processed datasets from HCs isolated from placentae 12–17 weeks gestational age (n=5), 17–24 weeks gestational age (n=7) and at term (n=5) using t-distributed stochastic neighbor embedding analysis (tSNE). HC populations were more diverse early in pregnancy and at term, compared to the second trimester; this is anticipated given the changing microenvironment following initial placental anchoring to eventual parturition. Marker expression heatmaps were generated to assess gestation-dependent changes in HC phenotype; HCs expressing activation markers were most frequent in early gestation, and reduced by term. HCs bearing markers of immune modulation were most frequent at mid-gestation, and least in early gestation. These results demonstrate the power of utilizing computational methods to analyze high-dimensional flow cytometry data collected from an understudied cell type and demonstrate how HCs phenotypically evolve during human pregnancy.

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.