The interplay of inflammation and placenta in maternal diabetes: insights into Hofbauer cell expression patterns.

Introduction: Inflammation of the placenta is harmful to both the fetus and the mother. Inflammation is strongly associated with diabetes, a common complication of pregnancy. Hofbauer cells (HBCs), unique immune system cells of fetal origin in the placenta, play complex roles, including growth of placental villi and their branching, stromal remodelling, and angiogenesis. Methods: Our study investigated the expression of IL-1ß, IL-10, CYP2C8, CYP2C9, CYP2J2 and sEH in HBCs from patients with type 1 diabetes mellitus (T1DM) and gestational diabetes mellitus (GDM) compared to healthy controls using immunohistochemistry. We also assessed the structure of the villus stroma using Masson´s trichrome. Results: In T1DM, HBCs showed inflammatory activation characterised by increased IL-1ß and decreased CYP epoxygenase expression compared to normal placentas. Conversely, significant inflammation in HBCs appeared less likely in GDM, as levels of IL-1ß and CYP epoxygenases remained stable compared to normal placentas. However, GDM showed a significant increase in sEH expression. Both types of diabetes showed delayed placental villous maturation and hypovascularisation, with GDM showing a more pronounced effect. Conclusion: The expression profiles of IL-1ß, CYP epoxygenases and sEH significantlly differ between controls and diabetic placentas and between T1DM and GDM. These facts suggest an association of the CYP epoxygenase-EETs-sEH axis with IL-1ß expression as well as villous stromal hypovascularisation. Given the stable high expression of IL-10 in both controls and both types of diabetes, it appears that immune tolerance is maintained in HBCs.

Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Altered placental immune cell composition and gene expression with isolated fetal spina bifida.

PROBLEM: Fetal spina bifida (SB) is more common in pregnant people with folate deficiency or anomalies of folate metabolism. It is also known that fetuses with SB have a higher risk of low birthweight, a condition that is typically placental-mediated. We therefore hypothesized that fetal SB would associate with altered expression of key placental folate transporters and an increase in Hofbauer cells (HBCs), which are folate-dependent placental macrophages. METHOD OF STUDY: Folate receptor-α (FRα), proton coupled folate receptor (PCFT), and reduced folate carrier (RFC) protein localization and expression (immunohistochemistry) and HBC phenotypes (HBC abundance and folate receptor-ß [FRß] expression; RNA in situ hybridization) were assessed in placentae from fetuses with SB (cases; n = 12) and in term (n = 10) and gestational age (GA) - and maternal body mass index - matched (n = 12) controls without congenital anomalies. RESULTS: Cases had a higher proportion of placental villous cells that were HBCs (6.9% vs. 2.4%, p = .0001) and higher average HBC FRß expression (3.2 mRNA molecules per HBC vs. 2.3, p = .03) than GA-matched controls. HBCs in cases were largely polarized to a regulatory phenotype (median 92.1% of HBCs). In sex-stratified analyses, only male cases had higher HBC levels and HBC FRß expression than GA-matched controls. There were no differences between groups in the total percent of syncytium and stromal cells that were positive for FRα, PCFT, or RFC protein immunolabeling. CONCLUSIONS: HBC abundance and FRß expression by HBCs are increased in placentae of fetuses with SB, suggesting immune-mediated dysregulation in placental phenotype, and could contribute to SB-associated comorbidities.

CD68 expression in the placenta of gestational diabetic mothers: A case-control study.

Background: Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with the onset or first recognition during pregnancy and is the most common metabolic complication of pregnancy. Significant maternal and fetal complications can result from undiagnosed or inadequately treated GDM. Aim: To investigate the difference in the expression of the CD-68 marker in the Hofbauer cells (HCs) and their distribution within the villi in the placentas of diabetic and non-diabetic mothers. Materials and Methods: Sixty placentas were included in the study, 30 as controls and 30 from mothers with diagnosed GDM as cases. Full-thickness cross sections of placentas were obtained. Tissue processing was done, followed by haematoxylin and eosin (H&E). A study of CD68 markers (placental macrophages) was done using standard protocols of immunohistochemistry. Statistical Analysis: Frequencies and percentages of Hofbauer cells (HCs) found in case and control placental tissue were calculated. Student's t-test was used to compare two groups using SPSS 13.0 software. When P is 0.0001, differences were considered statistically significant. Results and Conclusion: We studied the distribution and number of fetal macrophages (CD68+) in diabetic and non-diabetic placentas. The immunostained CD68+ cell count was identified to be significantly higher in the GDM placenta. In relation to fetal blood vessels in the villus stroma of the GDM placenta in comparison to control, CD68+ cells were found more frequently. This study shows a significant increase in the number of Hofbauer cells in the placenta of mothers with GDM in comparison to control (P < 0.0001). An increase in macrophages in these placentae might be related to the protective mechanism against inflammation. Further studies are required to investigate the mechanism in detail.

Sex-specific impact of maternal obesity on fetal placental macrophages and cord blood triglycerides.

INTRODUCTION: Maternal obesity is associated with increased risk of offspring obesity and cardiometabolic disease. Altered fetoplacental immune programming is a potential candidate mechanism. Differences in fetal placental macrophages, or Hofbauer cells (HBCs), have been observed in maternal obesity, and lipid metabolism is a key function of resident macrophages that may be deranged in inflammation/immune activation. We sought to test the following hypotheses: 1) maternal obesity is associated with altered HBC density and phenotype in the term placenta and 2) obesity-associated HBC changes are associated with altered placental lipid transport to the fetus. The impact of fetal sex was evaluated in all experiments. METHODS: We quantified the density and morphology of CD163-and CD68-positive HBCs in placental villi in 34 full-term pregnancies undergoing cesarean delivery (N = 15, maternal BMI ≥30 kg/m2; N = 19, BMI <30 kg/m2). Antibody-positive cells in terminal villi were detected and cell size and circularity analyzed using a semi-automated method for thresholding of bright-field microscopy images (ImageJ). Placental expression of lipid transporter genes was quantified using RTqPCR, and cord plasma triglycerides (TGs) were profiled using modified Wahlefeld method. The impact of maternal obesity and fetal sex on HBC features, lipid transporters, and cord TGs were evaluated by two-way ANOVA. Spearman correlations of cord TGs, HBC metrics and gene expression levels were calculated. RESULTS: Maternal obesity was associated with significantly increased density of HBCs, with male placentas most affected (fetal sex by maternal obesity interaction p = 0.04). CD163+ HBCs were larger and rounder in obesity-exposed male placentas. Sexually dimorphic expression of placental FATP4, FATP6, FABPPM, AMPKB1 and AMPKG and cord TGs was noted in maternal obesity, such that levels were higher in males and lower in females relative to sex-matched controls. Cord TGs were positively correlated with HBC density and FATP1 expression. DISCUSSION: Maternal obesity is associated with sex-specific alterations in HBC density and placental lipid transporter expression, which may impact umbilical cord blood TG levels and offspring cardiometabolic programming.

Examining the Effects of Gestational Physical Activity and Hofbauer Cell Polarization on Angiogenic Factors.

While gestational physical activity (PA) has demonstrated health benefits for both birthing parent and fetus, the mechanisms still need to be fully understood. Placental macrophages, or Hofbauer cells (HBCs), comprise a heterogenous population containing inflammatory (CD206-) and anti-inflammatory (CD206+) phenotypes. Similar to other tissue-resident macrophages (TRMs), HBCs are potential mediators of angiogenesis due to their secretion of both pro- and anti-angiogenic factors, including FGF2, VEGF, and SPRY2. While PA is associated with an increase in the proportion of VEGF- and FGF2-producing CD206+ macrophages in other tissues, the phenotypes producing FGF2, VEGF, and SPRY2 in the placenta and the associated relationships with gestational PA have not been studied. Using accelerometry, pregnant participants were classified as physically active or inactive in mid- and late-gestation. Term placenta tissue was collected at delivery and used for Western blotting and immunofluorescence to examine the protein expression of FGF2 and SPRY2, and to localize FGF2 in histological samples, respectively. Primary cultures of HBCs were used to examine the phenotypic differences in FGF2, SPRY2, and VEGF production. While no differences in the placental expression of SPRY2, total FGF2, or high-molecular-weight FGF2 were observed based on PA status, active individuals had significantly reduced levels of low-molecular-weight FGF2. Additionally, HBCs of all polarizations produce VEGF, FGF2, and SPRY2, and can form intercellular junctions and multinucleated giant cells. These findings suggest a possible relationship between PA and HBC-driven angiogenesis, providing an avenue for future exploration.

Characterization of Hofbauer cell polarization and VEGF localization in human term placenta from active and inactive pregnant individuals.

Physical activity (PA) during pregnancy is associated with parental and fetal health benefits; however, the mechanisms through which these benefits arise are yet to be fully understood. In healthy pregnancies Hofbauer cells (HBCs) comprise a heterogenous population containing CD206+ and CD206- phenotypes. In healthy pregnancies, CD206+ represent the majority, while dysregulations have been associated with pathological conditions. HBCs have also been identified as potential drivers of angiogenesis. As PA induces changes in macrophage polarization in non-pregnant populations, this novel study examined the relationship between PA and HBC polarization and to identify which HBC phenotypes express VEGF. Participants were classified as active or inactive, and immunofluorescence cell-labelling was used to quantify total HBCs, CD206+ HBCs, and the proportion of total HBCs expressing CD206. Immunofluorescent colocalization assessed which phenotypes expressed VEGF. Protein and mRNA expression of CD68 and CD206 were measured in term placenta tissue using Western blot and RT-qPCR, respectively. Both CD206+ and CD206- HBCs expressed VEGF. The proportion of CD206+ HBCs was elevated in active individuals; however, CD206 protein expression was observed to be lower in active participants. Combined with a lack of significant differences in CD206 mRNA levels, these findings suggest potential PA-mediated responses in HBC polarization and CD206 translational regulation.

Placenta analysis of Hofbauer cell profile according to the class of antiretroviral therapy used during pregnancy in people living with HIV.

INTRODUCTION: The use of antiretroviral therapy drastically reduces vertical transmission of Human Immunodeficiency Virus. However, recent studies demonstrate associations between ART use during pregnancy and placental inflammation, particularly within protease inhibitor (PI)-based regimens. We sought to characterize placental macrophages, namely Hofbauer cells, according to the class of ART used during pregnancy. METHODS: Using immunofluorescence and immunohistochemistry, placentas from 79 pregnant people living with HIV (PPLWH) and 29 HIV-uninfected people were analyzed to quantify the numbers and frequencies of leukocytes (CD45+) and Hofbauer cells (CD68+ and/or CD163+). PPLWH were stratified into three groups based on class of ART: non-nucleoside reverse transcriptase inhibitor (NNRTI)-based, integrase strand-transfer inhibitor (INSTI)-based, and PI-based regimens. RESULTS: Placentas of PPLWH contained significantly more leukocytes and Hofbauer cells than controls. Multivariable analyses revealed that this increase in immune cells was associated with a predominantly CD163+ profile in all ART subgroups compared to the HIV-negative group. This was characterized by an increase in total CD163+ cells in the PI and INSTI subgroups, and a higher frequency of CD163+ cells and CD163+/CD68+ ratio in the NNRTI and PI subgroups. DISCUSSION: Placentas of PPLWH treated with any ART regimen during their entire pregnancy displayed a selection for CD163+ cells compared to the HIV-negative group, regardless of class of ART, suggesting that class of ART does not intrinsically affect selection of CD163+ and CD68+ Hofbauer cells. Further investigations into the role of Hofbauer cells in ART-associated placental inflammation are warranted to identify the mechanisms behind their potential involvement in maternal-fetal tolerance maintenance.

Mild/Asymptomatic Maternal SARS-CoV-2 Infection Leads to Immune Paralysis in Fetal Circulation and Immune Dysregulation in Fetal-Placental Tissues.

Few studies have addressed the impact of maternal mild/asymptomatic SARS-CoV-2 infection on the developing neonatal immune system. In this study, we analyzed umbilical cord blood and placental chorionic villi from newborns of unvaccinated mothers with mild/asymptomatic SARSCoV-2 infection during pregnancy using flow cytometry, single-cell transcriptomics, and functional assays. Despite the lack of vertical transmission, levels of inflammatory mediators were altered in cord blood. Maternal infection was also associated with increased memory T, B cells, and non-classical monocytes as well as increased activation. However, ex vivo responses to stimulation were attenuated. Finally, within the placental villi, we report an expansion of fetal Hofbauer cells and infiltrating maternal macrophages and rewiring towards a heightened inflammatory state. In contrast to cord blood monocytes, placental myeloid cells were primed for heightened antiviral responses. Taken together, this study highlights dysregulated fetal immune cell responses in response to mild maternal SARS-CoV-2 infection during pregnancy.

Hyperglycemia disturbs trophoblast functions and subsequently leads to failure of uterine spiral artery remodeling.

Uterine spiral artery remodeling is necessary for fetal growth and development as well as pregnancy outcomes. During remodeling, trophoblasts invade the arteries, replace the endothelium and disrupt the vascular smooth muscle, and are strictly regulated by the local microenvironment. Elevated glucose levels at the fetal-maternal interface are associated with disorganized placental villi and poor placental blood flow. Hyperglycemia disturbs trophoblast proliferation and invasion via inhibiting the epithelial-mesenchymal transition, altering the protein expression of related proteases (MMP9, MMP2, and uPA) and angiogenic factors (VEGF, PIGF). Besides, hyperglycemia influences the cellular crosstalk between immune cells, trophoblast, and vascular cells, leading to the failure of spiral artery remodeling. This review provides insight into molecular mechanisms and signaling pathways of hyperglycemia that influence trophoblast functions and uterine spiral artery remodeling.

Immunofluorescence Studies on the Expression of the SARS-CoV-2 Receptors in Human Term Placenta.

Until September 2021, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2; COVID-19) pandemic caused over 217 million infections and over 4.5 million deaths. In pregnant women, the risk factors for the need of intensive care treatment are generally the same as in the overall population. Of note, COVID-19-positive women deliver earlier than COVID-19-negative women, and the risk for severe neonatal and perinatal morbidity and mortality is significantly higher. The probability and pathways of vertical transmission of the virus from the pregnant woman to the fetus are highly controversial. Recent data have shown that 54 (13%) of 416 neonates born to COVID-19-positive women were infected. Here, we investigated term placentas collected before the SARS-CoV-2 pandemic and studied the main COVID-19 receptors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine subtype 2 (TMPRSS2), as well as neuropilin 1 (NRP1). We performed real-time PCR and immunofluorescence on cryosections in combination with markers for syncytiotrophoblast, endothelial cells, macrophages and stromal cells. The PCR studies showed expression of both the truncated delta form of ACE2, which does not bind the COVID-19 spike protein, and the long form. The ACE2 antibody used does not distinguish between the two forms. We did not observe expression of the canonical SARS-CoV-2 entry machinery on syncytio- and cytotrophoblast. ACE2 and TMPRSS2 are co-expressed in a subpopulation of stromal cells, which in part are CD68-positive macrophages. NRP1 is localized to endothelial cells. In sum, the term placenta is not an organ that directly favors vertical transmission of COVID-19; however, microtraumas and placentitis may weaken its barrier function.

Hofbauer cells and fetal brain microglia share transcriptional profiles and responses to maternal diet-induced obesity.

Maternal immune activation is associated with adverse offspring neurodevelopmental outcomes, many mediated by in utero microglial programming. As microglia remain inaccessible throughout development, identification of noninvasive biomarkers reflecting fetal brain microglial programming could permit screening and intervention. We used lineage tracing to demonstrate the shared ontogeny between fetal brain macrophages (microglia) and fetal placental macrophages (Hofbauer cells) in a mouse model of maternal diet-induced obesity, and single-cell RNA-seq to demonstrate shared transcriptional programs. Comparison with human datasets demonstrated conservation of placental resident macrophage signatures between mice and humans. Single-cell RNA-seq identified common alterations in fetal microglial and Hofbauer cell gene expression induced by maternal obesity, as well as sex differences in these alterations. We propose that Hofbauer cells, which are easily accessible at birth, provide novel insights into fetal brain microglial programs, and may facilitate the early identification of offspring vulnerable to neurodevelopmental disorders in the setting of maternal exposures.

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Decreased CSTB, RAGE, and Axl Receptor Are Associated with Zika Infection in the Human Placenta.

Zika virus (ZIKV) compromises placental integrity, infecting the fetus. However, the mechanisms associated with ZIKV penetration into the placenta leading to fetal infection are unknown. Cystatin B (CSTB), the receptor for advanced glycation end products (RAGE), and tyrosine-protein kinase receptor UFO (AXL) have been implicated in ZIKV infection and inflammation. This work investigates CSTB, RAGE, and AXL receptor expression and activation pathways in ZIKV-infected placental tissues at term. The hypothesis is that there is overexpression of CSTB and increased inflammation affecting RAGE and AXL receptor expression in ZIKV-infected placentas. Pathological analyses of 22 placentas were performed to determine changes caused by ZIKV infection. Quantitative proteomics, immunofluorescence, and western blot were performed to analyze proteins and pathways affected by ZIKV infection in frozen placentas. The pathological analysis confirmed decreased size of capillaries, hyperplasia of Hofbauer cells, disruption in the trophoblast layer, cell agglutination, and ZIKV localization to the trophoblast layer. In addition, there was a significant decrease in CSTB, RAGE, and AXL expression and upregulation of caspase 1, tubulin beta, and heat shock protein 27. Modulation of these proteins and activation of inflammasome and pyroptosis pathways suggest targets for modulation of ZIKV infection in the placenta.

The pathologic changes of human placental macrophages in women with hyperglycemia in pregnancy.

As the most common complication of pregnancy, hyperglycemia has a profound impact on maternal and fetal health and the long-term development of the offspring. Placental macrophages, including fetal-derived macrophages also known as Hofbauer cells (HBCs) and placenta-associated maternal monocyte/macrophages (PAMMs), are thought to play a crucial role in regulating pregnancy and maintaining the internal environment, and are critical for fetal development. This review aims to profile existing knowledge on HBCs and PAMMs across gestation, and consider how hyperglycemia may impact their phenotype and function in pregnancy.

Isolation of Decidual Macrophages and Hofbauer Cells from Term Placenta-Comparison of the Expression of CD163 and CD80.

(1) Background: Placental immune cells are playing a very important role in a successful placentation and the prevention of pregnancy complications. Macrophages dominate in number and relevance in the maternal and the fetal part of the placenta. The evidence on the polarization state of fetal and maternal macrophages involved in both, healthy and pregnancy-associated diseases, is limited. There is no representative isolation method for the direct comparison of maternal and fetal macrophages so far. (2) Material and Methods: For the isolation of decidual macrophages and Hofbauer cells from term placenta, fresh tissue was mechanically dissected and digested with trypsin and collagenase A. Afterwards cell enrichment was increased by a Percoll gradient. CD68 is represented as pan-macrophage marker, the surface markers CD80 and CD163 were further investigated. (3) Results: The established method revealed a high cell yield and purity of the isolated macrophages and enabled the comparison between decidual macrophages and Hofbauer cells. No significant difference was observed in the percentage of single CD163+ cells in the distinct macrophage populations, by using FACS and immunofluorescence staining. A slight increase of CD80+ cells could be found in the decidual macrophages. Considering the percentage of CD80+CD163- and CD80-CD163+ cells we could not find differences. Interestingly we found an increased number of double positive cells (CD80+CD163+) in the decidual macrophage population in comparison to Hofbauer cells. (4) Conclusion: In this study we demonstrate that our established isolation method enables the investigation of decidual macrophages and Hofbauer cells in the placenta. It represents a promising method for direct cell comparison, enzyme independently, and unaffected by magnetic beads, to understand the functional subsets of placental macrophages and to identify therapeutic targets of pregnancy associated diseases.

Erythro-myeloid progenitor origin of Hofbauer cells in the early mouse placenta.

Hofbauer cells (HBCs) are tissue macrophages of the placenta thought to be important for fetoplacental vascular development and innate immune protection. The developmental origins of HBCs remain unresolved and could implicate functional diversity of HBCs in placenta development and disease. In this study, we used flow cytometry and paternally inherited reporters to phenotype placenta macrophages and to identify fetal-derived HBCs and placenta-associated maternal macrophages in the mouse. In vivo pulse-labeling traced the ontogeny of HBCs from yolk sac-derived erythro-myeloid progenitors, with a minor contribution from fetal hematopoietic stem cells later on. Single-cell RNA-sequencing revealed transcriptional similarities between placenta macrophages and erythro-myeloid progenitor-derived fetal liver macrophages and microglia. As with other fetal tissue macrophages, HBCs were dependent on the transcription factor Pu.1, the loss-of-function of which in embryos disrupted fetoplacental labyrinth morphology, supporting a role for HBC in labyrinth angiogenesis and/or remodeling. HBC were also sensitive to Pu.1 (Spi1) haploinsufficiency, which caused an initial deficiency in the numbers of macrophages in the early mouse placenta. These results provide groundwork for future investigation into the relationship between HBC ontogeny and function in placenta pathophysiology.

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.

Hofbauer Cells Spread Listeria monocytogenes among Placental Cells and Undergo Pro-Inflammatory Reprogramming while Retaining Production of Tolerogenic Factors.

Pregnant women are highly susceptible to infection by the bacterial pathogen Listeria monocytogenes, leading to miscarriage, premature birth, and neonatal infection. L. monocytogenes is thought to breach the placental barrier by infecting trophoblasts at the maternal/fetal interface. However, the fate of L. monocytogenes within chorionic villi and how infection reaches the fetus are unsettled. Hofbauer cells (HBCs) are fetal placental macrophages and the only leukocytes residing in healthy chorionic villi, forming a last immune barrier protecting fetal blood from infection. Little is known about the HBCs' antimicrobial responses to pathogens. Here, we studied L. monocytogenes interaction with human primary HBCs. Remarkably, despite their M2 anti-inflammatory phenotype at basal state, HBCs phagocytose and kill non-pathogenic bacteria like Listeria innocua and display low susceptibility to infection by L. monocytogenes. However, L. monocytogenes can exploit HBCs to spread to surrounding placental cells. Transcriptomic analyses by RNA sequencing revealed that HBCs undergo pro-inflammatory reprogramming upon L. monocytogenes infection, similarly to macrophages stimulated by the potent M1-polarizing agents lipopolysaccharide (LPS)/interferon gamma (IFN-γ). Infected HBCs also express pro-inflammatory chemokines known to promote placental infiltration by maternal leukocytes. However, HBCs maintain the expression of a collection of tolerogenic genes and secretion of tolerogenic cytokines, consistent with their tissue homeostatic role in prevention of fetal rejection. In conclusion, we propose a previously unrecognized model in which HBCs promote the spreading of L. monocytogenes among placental cells and transition to a pro-inflammatory state likely to favor innate immune responses, while maintaining the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity. IMPORTANCE Infection of the placental/fetal unit by the facultative intracellular pathogen Listeria monocytogenes results in severe pregnancy complications. Hofbauer cells (HBCs) are fetal macrophages that play homeostatic anti-inflammatory functions in healthy placentas. HBCs are located in chorionic villi between the two cell barriers that protect fetal blood from infection: trophoblast cells at the maternal interface (in contact with maternal blood), and fetal endothelial cells at the fetal interface (in contact with fetal blood). As the only leukocytes residing in chorionic villi, HBCs form a critical immune barrier protecting the fetus from infection. Here, we show that although HBCs display low susceptibility to L. monocytogenes, the bacterium still replicates intracellularly and can spread to other placental and fetal cells. We propose that HBCs are permissive to L. monocytogenes transplacental propagation and can repolarize toward a pro-inflammatory phenotype upon infection. However, consistent with their placental homeostatic functions, repolarized HBCs maintain the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity, at least at early stages of infection.

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.