Integrated analysis of an in vivo model of intra-nasal exposure to instilled air pollutants reveals cell-type specific responses in the placenta

Abstract

The placenta is a heterogeneous organ whose development involves complex interactions of trophoblasts with decidual, vascular, and immune cells at the fetal-maternal interface. It maintains a critical balance between maternal and fetal homeostasis. Placental dysfunction can lead to adverse pregnancy outcomes including intra-uterine growth restriction, pre-eclampsia, or pre-term birth. Exposure to environmental pollutants contributes to the development of placental abnormalities, with poorly understood molecular underpinning. Here we used a mouse (C57BL/6) model of environmental pollutant exposure by administration of a particulate matter (SRM1649b at 300 μg/day/mouse) suspension intra-nasally beginning 2 months before conception and during gestation, in comparison to saline-exposed controls. Placental transcriptomes, at day 19 of gestation, were determined using bulk RNA-seq from whole placentas of exposed (n = 4) and control (n = 4) animals and scRNAseq of three distinct placental layers, followed by flow cytometry analysis of the placental immune cell landscape. Our results indicate a reduction in vascular placental cells, especially cells responsible for structural integrity, and increase in trophoblast proliferation in animals exposed to particulate matter. Pollution-induced inflammation was also evident, especially in the decidual layer. These data indicate that environmental exposure to air pollutants triggers changes in the placental cellular composition, mediating adverse pregnancy outcomes.

Figure 1

(a) Schematic representation of mouse placenta and location of various cell types in different placental compartments. (b) Uniform Manifold Approximation and Projection (UMAP) plot of cell and tissue clusters detected in scRNAseq in all 6 samples. Sub-clusters of related cell types could be detected for immune cells, decidual cells, endo/epithelial-like cells, erythroid cells and trophoblasts. (c) UMAP projection of cells originating from air pollution (AP) samples (n = 3) and control (CON) samples (n = 3). (d, e) Quantification of each cell type by placental layer, decidual, junctional and labyrinthine (d) or treatment groups, namely AP and CON (e). Cell counts were normalized by the total cell count per sample and depicted as fractions of the total cell count for each cell/tissue type.

Figure 2

Deconvolution of Bulk RNA-Seq data. (a) Cell-type deconvolution of bulk RNAseq data based on pseudobulk scRNAseq as a reference. Numbers present a fraction of the total count. Stars represent an FDR-corrected significant difference (t-test) between the detected cell type fractions in AP (air pollution; n = 4 sequenced samples obtained from 6 pregnant mice) and CON (control; n = 4 sequenced samples obtained from 6 pregnant mice) at a level below 0.05. (b) Overlap between the 50 top marker genes for each tissue (Supplementary Table 2) type in the scRNAseq dataset and genes up- or downregulated in AP versus CON detected by bulk RNAseq (Supplementary Table 3). A higher value (depicted with darker color) represents a higher level of overlap. (c) Differential abundance of cell types based on z-scores calculated using cell specific marker genes from scRNAseq analysis which were significantly expressed among all samples and filtered by p value < 0.05. Students t-test was performed to calculate the significance between two groups InvSpT1 = Invasive Spongiotrophoblasts Type 1. InvSpT2 = Invasive Spongiotrophoblasts Type 2.

Figure 3

Abundance of various immune cells in different placental compartments: (a, b) showing the abundance of NK cells (a) and T cells (b) in placentas of CON or AP group based on Bulk RNA-seq (left panel; n = 4 sequenced from each group obtained from n = 6 pregnant mice) or Flow cytometry data (right panel; n = 6 from each group) from three different compartments of placentas. (c) abundance of total macrophages between CON and AP groups (left panel) as obtained from Bulk RNA-Seq and abundance of M1 and M2 macrophages from three different compartments of mouse placentas by Flow cytometry (right panel). Statistical analysis was undertaken using the Student’s t-test to compare AP versus CON groups with significance achieved at a p value < 0.05.

Figure 4

(a) Differentially expressed genes (DEGs) obtained from placental samples of control (CON) and air pollution (AP) exposed mice. DEGs were selected based on FDR < 0.05 and Log2Foldchange > 2. Genes which are part of any of the pathways in the right panel have been labeled. A full list of differentially expressed genes can be found in Supplementary Table 3. (b) List of Hallmark pathways positively or negatively enriched between AP and CON groups obtained using GSEA are shown.