Single-Cell Transcriptome of Preeclamptic and Control Placentas

Single-cell RNA-sequencing data from GSE173193 database including 4 samples (2 non-preeclampsia samples, ncontrol=2, and 2 preeclampsia samples, npreeclampsia=2) were analyzed, and low-quality or dead cells were removed after quality control (Fig. 1A). Cell cycle assessment revealed that the cell cycle (G1, G2/M, S) had no effect on clustering (Fig. 1B), and both the gene expression levels and the number of genes showed high correlation between different group (Fig. 1C). UMAP visualized and clustered all the cells, 35 cell clusters were obtained. By characterizing marker genes, they were annotated to 10 major cell types, namely epithelial cells, erythroid-like cells, chorionic extravillous trophoblast cells (EVT), fibroblasts, macrophages, monocytes, neutrophils, syncytiotrophoblast (SCT), T/NK cells, and villous cytotrophoblast cells (VCT) (Fig. 1D, F). Epithelial cells and neutrophils were higher in all cell types, and epithelial cells were much higher than the control in the preeclampsia group, whereas monocytes were significantly more numerous in the control group, and there was no clear pattern of change in the other cell types (Fig. 1E, G-H).

Fig. 1

Single-cell transcriptome of preeclamptic and control placentas. (A) Quality control of single-celldata; (B) Cell cycle scores; (C) Correlation plots of nCount_RNA with nFeauture_RNA; (D) Cell clustering and cell type annotation results; (E) Distribution of all cell types in control and preeclampsia groups; (F) Bubble plots of marker gene expression corresponding to each cell type. Epithelial cells: DUSP9, SMAGP, HSD17B1; Erythroid-like cells: HBA1/2, HBB/SNCA; EVT: HLA-G, PAPPA2, HTRA4, fibroblasts: COL6A2, HGF, PDGFR8; Macrophages: CD163, CD209, CSF1R, Monocytes: CD300E, CD14, S100A12; Neutrophils: LTF, LCN2, DEFA4; SCT: CYP19A1, CGA, ERVFRP-1; T/NK cells: CD3E, CD3G; VCT: NRP2, EGFR, SCL27A2. (G) The percentage of cell types in each sample; (H) The percentage of cell types in the preeclampsia and control groups

Immunoscoring Results for Single Cells

Since study have been pointed out that the development of preeclampsia may be related to abnormal immune regulation, so we scored the expression of immune-related genes in all cell types. As shown in Fig. 2A and B, we obtained the distribution of the number of immune genes contained in each cell and the immune-related AUC score, in which 250 cells were more relevant to immunity. The scoring results were subsequently presented in a heatmap, which revealed that the highest immune score was macrophages, followed closely by monocytes, and the lowest was epithelial cells (Fig. 2C). Therefore, the role of macrophages and monocytes in preeclampsia will be analyzed further.

Fig. 2

Immunoscoring results for single cells. (A) The number of immune genes contained in each cell; (B) Distribution of immune-related AUC scores in each cell; (C) The immune-related scores of all cell types

Preeclampsia Is Associated with Immune and Inflammatory Response in Placenta

Analysis of differentially expressed genes (DEGs) and functional enrichment of DEGs were performed for macrophages and monocytes between the control and preeclampsia group. The GO enrichment results revealed that compared with the control group, DEGs of macrophages in the preeclampsia group were enriched in “positive regulation of cytokine production”, “regulation of immune effector processes”, “cell activation involved in immune response”. Similarly, DEGs of monocytes in the preeclampsia group were enriched in the “immune response-regulating cell surface receptor signaling pathway” and “immune response-regulating signaling pathway”. “Chemokine signaling pathway” was enriched in both macrophages and monocytes in KEGG enrichment results (Fig. 3A-B). Therefore, macrophages and monocytes may be participated in the over-activation of immune and inflammatory responses in preeclampsia placenta. We then redefined all the macrophages in the placenta and obtained 16 macrophages subclusters, which could be subdivided into macrophages, monocytes, and the placenta-unique Hofbauer cells after cell annotation [23, 24] (Fig. 3C, E). In the control group, there were more monocytes and Hofbauer cells, whereas macrophages were more prevalent in the preeclamptic group, which suggested that macrophages play an important role in preeclampsia (Fig. 3D).

Fig. 3

Preeclampsia is associated with immune and inflammatory response in placenta. The GO and KEGG enrichment result of DEGs in (A) macrophages and (B) monocytes; (C) Macrophages re-clustering and annotation, with the distribution of cells in the sample on the left, all cell clusters in the middle, and subtype annotation results on the right; (D) Distribution of macrophages subtypes in the control and preeclampsia groups; (E) Bubble plots of marker genes expression corresponding to the three subtypes (Macrophages: HLA-DRA, FCGR3A; Monocytes: S100A12, CD300E; Hofbauer cells: HPGDS, VSIG4)

Differentiation of Macrophages in the Preeclamptic Placenta

With the aim to explore which macrophage subtype plays a crucial role in preeclampsia, pseudotime was used to analyze the differentiation of macrophage subtypes, and found that monocytes could differentiated into macrophages and Hofbauer cells (Fig. 4A). By modelling the expression of genes in each of the three subtypes, it was found that the expression levels of these genes changed as the subtypes diverged (Fig. 4B). HLA-DRA, HLA-DPB1, and HLA-DQB1 were highly expressed in the monocytes but reduced in the differentiated macrophages and Hofbauer cells, they were expressed on the surface or inside antigen-presenting cells and played central roles in the immune system. We also tagged the gene expressions related to immune and inflammatory responses. The immune receptor activation signal transduction, such as CEBPB, FCER1G, and CCL4 decreased in their levels following cell differentiation. Besides, genes such as APLP2 and HIF1A that played a role in the regulation of vascular endothelial growth factor functions decreased expressions. In summary, macrophage subtypes were related to the aberrant immune regulation in preeclampsia, in which monocytes mediated the imbalance of immune and inflammatory responses and placental angiogenesis through differentiation into macrophages and Hofbauer cells.

Fig. 4

Differentiation of macrophages in the preeclamptic placenta. (A) Differentiation trajectories of macrophages, monocytes, and Hofbauer cells. The left: simulated time phase, each color represented a different phase; The middle: distribution of different subtypes in the pseudotime; The right: cell differentiation trajectory, the darker the color the earlier the period; (B) Cluster time simulations were performed according to differentiation scenarios and plotted as heatmaps

Interaction of Macrophages with Other Cell Types in Preeclamptic Placenta

To further discover the function of macrophages in preeclampsia, we analyzed the intercellular communication between cell types in the control and preeclampsia groups. Results suggested that the number of communications between cell types in the control group was more numerous and stronger (Fig. 5A-B). These cell types mainly functioned in SPP1, Annexin, MIF, Visfatin, VEGF, Galectin, PTN, and CCL signalings. Compared with the control group, the preeclampsia group showed a decrease in communication in different signaling pathways, with a higher decrease in CCL and Visfatin signalings (Fig. 5C), indicating that preeclampsia inhibited normal communication between placental cells. Additionally, communication between the three subtypes of macrophages frequently, which implied that they play a very critical role in the overall function (Fig. 5D-E). So, we assessed the receptor-ligand pairs that associated with these three cell subtypes, and the SPP1-CD44 pairs had the strongest roles in communication, and thus may be a potential point of action for the progression preeclampsia.

Fig. 5

Interaction of macrophages with other cell types in preeclamptic placenta. (A) Statistical plots of the total number of communications and strength of communications for all cell types in the control and preeclampsia groups; (B) Two-dimensional projection plots of efferent and afferent signals of different cell types; (C) The strength of the different signaling pathways in each cell type; (D) Other cells influenced the receptor-ligand pairs of three macrophage subpopulations; (E) Three types of macrophage subpopulations influenced the receptor-ligand pairs of other cells

Macrophages as a Major Cell Type to Regulated Preeclampsia

In order to clarify the mechanism of macrophages in regulating preeclampsia, we analyzed the transcription factors target genes of the three subtypes in the preeclampsia group (Fig. 6A). In the previous analysis, we got the DEGs of macrophages and monocytes between the control and the preeclampsia group. Then intersected the DEGs and the differential transcription factor target genes, and screened out 10 differential target genes related to preeclampsia in monocytes and 116 in macrophages (Fig. 6B-C). These genes were further assessed based on the GSE234729 and GSE25906 datasets and identified 7 genes (SLC9A9, SH2B3, SDC3, RCC2, F13A1, CCL2, and CBLB) were differentially expressed with a consistent trend in the transcriptome, and that they were highly expressed in macrophages (Fig. 6D-F). Thus, macrophages participated in the regulation of preeclampsia as an important functional cell type in the placenta.

Fig. 6

Macrophages as a major cell population to regulated preeclampsia. (A) Heatmap display of differential transcription factors regulating macrophage, monocyte, and Hofbauer cells; (B) Transcription factor target genes of monocytes and (C) macrophages were intersected with DEGs; (D) Analysis of key differential genes in the GSE25906 and (E) GSE234729 datasets; (F) Differential expression of screened key differential genes in different grouped cell types