Feto-placental Hofbauer cells (HBCs) are macrophages residing in placental stroma. They are generally described as anti-inflammatory M2 polarized cells, promoting tolerance and tissue remodeling. In ...certain pathologies, however, a possible phenotypical switch towards pro-inflammatory M1 macrophages has been proposed. The study aimed to determine if HBCs can acquire an M1 phenotype under pro-inflammatory conditions in vitro. HBCs were isolated from healthy human term placentas. Cells were cultivated upon addition of LPS and INF-γ or IL-4 and IL-13 to induce the M1 and M2 phenotype, respectively. Specific cell polarization markers and cytokines, associated with respective phenotypes, were investigated by flow cytometry and ELISA. THP-1 macrophages served as positive control. Pro-inflammatory stimuli reduced M2 markers CD163 and DC-SIGN, but did not induce M1 markers. TNF-α release was increased, but at the same time TGF-β and IL-10 release was upregulated, resembling in part the M2b sub-phenotype. Anti-inflammatory stimuli had no effect on HBC polarization. HBCs maintain their M2 phenotype in vitro despite inflammatory stimuli, which might represent a state of adaption and tolerance to avoid rejection of the semiallogeneic feto-placental unit.
Hofbauer cells (HBCs) are resident macrophages of the human placenta, regulating immune tolerance and tissue homeostasis. HBCs of a normal placenta (CTR) exhibit mainly an anti-inflammatory M2 ...phenotype. Under exaggerated chronic inflammation during pregnancy, as in preeclampsia (PE), a phenotypic switch towards M1 polarization has been proposed. PE, defined as maternally derived syndrome can be distinguished into two different entities: early-onset (EO) preeclampsia and late-onset (LO) preeclampsia. Although the clinical presenting characteristics overlap, both can be identified by biochemical markers, heritability, and different maternal and fetal outcomes. To date, no study has specifically investigated polarization and phenotype of EO- and LO-PE HBCs and looked at possible changes in HBC functionality. Primary HBCs were isolated from CTR and PE placentae. First,
morphological differences were observed between CTR and PE HBCs, with both PE groups exhibiting features of M1 macrophages alongside M2 forms. Interestingly, a different polarization pattern was observed between EO- and LO-PE HBCs. EO-PE HBCs develop a tissue remodeling M2 phenotype that is strongly shifted toward M1 polarization and showed a significant upregulation of CD86, TLR4, and HLA-DR. Furthermore, this pro-inflammatory signature is corroborated by higher expression of IRF5 and of
(p ≤ 0.05). However, their M2 characteristics is reflected by significant TGF-β secretion and
expression. In contrast, LO-PE HBCs developed a phagocytic CD209-low M2 phenotype in which the M1 pattern was not as pronounced as they downregulated the
gene, but expressed increased levels of pro-inflammatory CD80 and TLR1 (p ≤ 0.05). The enhanced phagocytosis and MMP-9 secretion alongside the increased secretion of anti-inflammatory IL -4, IL -13 and TGF-β in both EO- and LO-PE HBCs suggests their adaptive role and plasticity in resolving inflammation and tissue homeostasis.
Hofbauer cells (HBCs) are macrophages of the feto-placental unit. Despite the general view that these cells have an anti-inflammatory M2 phenotype, recent studies have claimed that pregnancy ...pathologies-e.g., gestational diabetes mellitus (GDM)-cause a switch from an M2 to an M1 pro-inflammatory phenotype in HBCs. The pilot-study presented here challenges this claim, showing that HBCs maintain anti-inflammatory properties in spite of the hyperglycemic, low-grade inflammatory environment of GDM.
HBCs were isolated from placentae of healthy women (
= 5) and women with GDM (
= 6) diagnosed in the second trimester. FACS was used to measure surface markers associated with either M1 or M2 phenotype on the cells. In addition, placental tissue sections were subjected to immune histochemical imaging to assess the phenotype within the tissue context. Supernatant from control and GDM HBCs was collected at defined time points and used in a multiplex ELISA-on-beads approach to assess secretion of cytokines, chemokines, and growth factors. The effect of HBC cell culture supernatant on placental endothelial activation was investigated.
FACS and immune staining showed that, indeed, M2 markers, such as CD206 and CD209, are increased in HBCs isolated from GDM placentae. Also, the M1 marker CD86 was increased, but only by trend. Secretion of numerous cytokines, chemokines and growth factors was not changed; pro-inflammatory interleukin (IL)-1β and IL-6 release form GDM HBC was increased but not significant. Exposure to GDM HBC supernatant did not induce cell adhesion molecules (VCAM-1, selectins, vascular endothelial-cadherin) in placental endothelial cells compared to supernatant from control HBCs, an induction of intracellular adhesion molecule 1 was observed however.
Our study-although performed in a small set of patients-shows that placental macrophages maintain their anti-inflammatory, tissue remodeling M2 phenotype even in pregnancies affected by gestational diabetes. This consistent phenotype might be important for propagation of maternal tolerance toward the fetus and for protection of the fetus from a low-grade inflammatory environment.
As opposed to adults, high-density lipoprotein (HDL) is the main cholesterol carrying lipoprotein in fetal circulation. The major HDL receptor, scavenger receptor class B type I (SR-BI), contributes ...to local cholesterol homeostasis. Arterial endothelial cells (ECA) from human placenta are enriched with cholesterol compared to venous endothelial cells (ECV). Moreover, umbilical venous and arterial plasma cholesterol levels differ markedly. We tested the hypothesis that the uptake of HDL-cholesteryl esters differs between ECA and ECV because of the differential expression of SR-BI. We aimed to identify the key regulators underlying these differences and the functional consequences. Immunohistochemistry was used for visualization of SR-BI in situ. ECA and ECV were isolated from the chorionic plate of human placenta and used for RT-qPCR, Western Blot, and HDL uptake assays with
H- and
I-labeled HDL. DNA was extracted for the methylation profiling of the SR-BI promoter. SR-BI regulation was studied by exposing ECA and ECV to differential oxygen concentrations or shear stress. Our results show elevated SR-BI expression and protein abundance in ECA compared to ECV in situ and in vitro. Immunohistochemistry demonstrated that SR-BI is mainly expressed on the apical side of placental endothelial cells in situ, allowing interaction with mature HDL circulating in the fetal blood. This was functionally linked to a higher increase of selective cholesterol ester uptake from fetal HDL in ECA than in ECV, and resulted in increased cholesterol availability in ECA. SR-BI expression on ECV tended to decrease with shear stress, which, together with heterogeneous immunostaining, suggests that SR-BI expression is locally regulated in the placental vasculature. In addition, hypomethylation of several CpG sites within the SR-BI promoter region might contribute to differential expression of SR-BI between chorionic arteries and veins. Therefore, SR-BI contributes to a local cholesterol homeostasis in ECA and ECV of the human feto-placental vasculature.
Hofbauer cells (HBCs) are macrophages of fetal origin that reside in the villous tissue. They are the only immune cells within healthy villi. While HBCs perform innate immune functions such as ...phagocytosis and antigen presentation, they are increasingly recognized for their diverse roles in placental physiology e.g. vascular functionality, tissue homeostasis, tolerance. Consequently, HBCs are of utmost interest in a variety of non-physiological placental conditions.
Villous tissue is collected freshly after delivery and finely minced. The resulting tissue is digested in a two-step process, using Trypsin/DNase to separate cytotrophoblasts and collagenase/DNase to penetrate deeper into the villous stroma, containing HBCs, and obtain a single cell suspension. After a density gradient centrifugation, the corresponding cell layer is collected and subjected to negative immune selection of HBCs, yielding unaffected cells that have not been activated during the isolation process.
In addition to a classical immunocytochemistry (ICC) approach including macrophage markers, and markers for potentially contaminating cell types (e.g. fibroblasts, muscle, mesenchymal cells), we have developed a multi-color flow cytometry (FC) panel. This panel assesses Hofbauer cell purity and polarization states more accurately and comprehensively than qualitative ICC, using percentage analysis of parent cells to estimate the expression levels of specific markers.
The presented protocol allows us to isolate HBCs in significant numbers and high purity, even from placentae compromised by preeclampsia (PE) with limited placental volume. We have successfully developed and implemented this protocol to study healthy, diabetic and PE macrophages, aiding a better understanding of the underlying placental pathophysiology at the cellular level.
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•Isolation of primary Hofbauer cells (HBCs) from human term placenta.•HBCs are not activated immunologically and highly pure.•Purity and polarization are quickly assessed using the presented FACS protocol.•Possibility to cultivate HBCs from different pathologies.
The placenta is a unique and pivotal organ in reproduction, controlling crucial growth and cell differentiation processes that ensure a successful pregnancy. Placental development is a tightly ...regulated and dynamic process, in which the transforming growth factor beta (TGFβ) superfamily plays a central role. This family of pleiotropic growth factors is heavily involved in regulating various aspects of reproductive biology, particularly in trophoblast differentiation during the first trimester of pregnancy. TGFβ signalling precisely regulates trophoblast invasion and the cell transition from cytotrophoblasts to extravillous trophoblasts, which is an epithelial-to-mesenchymal transition-like process. Later in pregnancy, TGFβ signalling ensures proper vascularization and angiogenesis in placental endothelial cells. Beyond its role in trophoblasts and endothelial cells, TGFβ signalling contributes to the polarization and function of placental and decidual macrophages by promoting maternal tolerance of the semi-allogeneic foetus. Disturbances in early placental development have been associated with several pregnancy complications, including preeclampsia (PE) which is one of the severe complications. Emerging evidence suggests that TGFβ is involved in the pathogenesis of PE, thereby offering a potential target for intervention in the human placenta.
This comprehensive review aims to explore and elucidate the roles of the major members of the TGFβ superfamily, including TGFβs, bone morphogenetic proteins (BMPs), activins, inhibins, nodals, and growth differentiation factors (GDFs), in the context of placental development and function. The review focusses on their interactions within the major cell types of the placenta, namely trophoblasts, endothelial cells, and immune cells, in both normal pregnancies and pregnancies complicated by PE throughout pregnancy.
A literature search was carried out using PubMed and Google Scholar, searching terms: 'TGF signalling preeclampsia', 'pregnancy TGF signalling', 'preeclampsia tgfβ', 'preeclampsia bmp', 'preeclampsia gdf', 'preeclampsia activin', 'endoglin preeclampsia', 'endoglin pregnancy', 'tgfβ signalling pregnancy', 'bmp signalling pregnancy', 'gdf signalling pregnancy', 'activin signalling pregnancy', 'Hofbauer cell tgfβ signalling', 'placental macrophages tgfβ', 'endothelial cells tgfβ', 'endothelium tgfβ signalling', 'trophoblast invasion tgfβ signalling', 'trophoblast invasion Smad', 'trophoblast invasion bmp', 'trophoblast invasion tgfβ', 'tgfβ preeclampsia', 'tgfβ placental development', 'TGFβ placental function', 'endothelial dysfunction preeclampsia tgfβ signalling', 'vascular remodelling placenta TGFβ', 'inflammation pregnancy tgfβ', 'immune response pregnancy tgfβ', 'immune tolerance pregnancy tgfβ', 'TGFβ pregnancy NK cells', 'bmp pregnancy NK cells', 'bmp pregnancy tregs', 'tgfβ pregnancy tregs', 'TGFβ placenta NK cells', 'TGFβ placenta tregs', 'NK cells preeclampsia', 'Tregs preeclampsia'. Only articles published in English until 2023 were used.
A comprehensive understanding of TGFβ signalling and its role in regulating interconnected cell functions of the main placental cell types provides valuable insights into the processes essential for successful placental development and growth of the foetus during pregnancy. By orchestrating trophoblast invasion, vascularization, immune tolerance, and tissue remodelling, TGFβ ligands contribute to the proper functioning of a healthy maternal-foetal interface. However, dysregulation of TGFβ signalling has been implicated in the pathogenesis of PE, where the shallow trophoblast invasion, defective vascular remodelling, decreased uteroplacental perfusion, and endothelial cell and immune dysfunction observed in PE, are all affected by an altered TGFβ signalling.
The dysregulation of TGFβ signalling in PE has important implications for research and clinical practice. Further investigation is required to understand the underlying mechanisms, including the role of different ligands and their regulation under pathophysiological conditions, in order to discover new therapeutic targets. Distinguishing between clinically manifested subtypes of PE and studying TGFβ signalling in different placental cell types holistically is an important first step. To put this knowledge into practice, pre-clinical animal models combined with new technologies are needed. This may also lead to improved human research models and identify potential therapeutic targets, ultimately improving outcomes for affected pregnancies and reducing the burden of PE.
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