Inflammatory responses in the placenta upon SARS-CoV-2 infection late in pregnancy

Abstract

The effect of SARS-CoV-2 infection on placental function is not well understood. Analysis of placentas from women who tested positive at delivery showed SARS-CoV-2 genomic and subgenomic RNA in 22 out of 52 placentas. Placentas from two mothers with symptomatic COVID-19 whose pregnancies resulted in adverse outcomes for the fetuses contained high levels of viral Alpha variant RNA. The RNA was localized to the trophoblasts that cover the fetal chorionic villi in direct contact with maternal blood. The intervillous spaces and villi were infiltrated with maternal macrophages and T cells. Transcriptome analysis showed an increased expression of chemokines and pathways associated with viral infection and inflammation. Infection of placental cultures with live SARS-CoV-2 and spike protein-pseudotyped lentivirus showed infection of syncytiotrophoblast and, in rare cases, endothelial cells mediated by ACE2 and Neuropilin-1. Viruses with Alpha, Beta, and Delta variant spikes infected the placental cultures at significantly greater levels.

Keywords: Health sciences; Immunology; Transcriptomics.

Graphical abstract

Figure 1

SARS-CoV-2 virus is present in placentas from infected mothers and results in inflammatory responses (A) Graph showing ΔΔCT values of RNA samples isolated from FFPE patient placenta slides from the different patient cohorts included in this study. Positive control lung samples (n = 5), high positive samples (n = 2), and positive samples (n = 20) are significantly higher than negative samples (n = 30). Negative (n = 4) and inflammatory controls (n = 4). Statistical analysis was performed using a one-way ANOVA and adjusted for by multiple comparisons test using the Benjamini–Hochberg FDR method ∗∗∗∗p value < 0.0001. Data are presented as mean ± SEM. (B) Brightfield microscopy images of a representative COVID high positive placental sample H_2 and a representative negative control placental sample C_4. Slides were stained by H&E, in situ hybridization for SARS-CoV-2-RNA and counterstained for syncytial trophoblast marker cytokeratin (KRT7, red), and by immunohistochemistry for SARS-CoV-2-N protein (brown) as well as for CD163-positive Hofbauer cells (HBC). Scale bars = 100 μm. See also Figure S1.

Figure 2

Immune cell infiltration of CD3⁺ and CD4⁺ T cells, CD56⁺ NK, and CD163⁺ Hofbauer cells in a high positive placenta from a COVID-infected mother versus a normal healthy control placenta (A–D) Representative brightfield images of IHC stained FFPE placental tissue sections showing infiltration within maternal blood space (MB), fetal intravillous space (FV), chorionic plate (CP), and maternal decidua (MD) from high positive sample H_2. (E–H) Representative images of the same regions within the placenta from a non-infected negative control, C_3. Sections were stained for CD3 (A and E), CD4 (B and F), CD56 (C and G), CD163 (D and F). Scale bars = 100 μm. (I) Quantification of CD3⁺ T cells, CD4⁺ helper T cells, CD56⁺ NK cells, and CD163 + macrophages/HBC detectable by DAB staining across the indicated regions of placental tissue listed along the top X axis. The number of images analyzed for each antibody are listed in Table S1. Values represent the percentage of DAB positive nuclei out of total number of cells based on nuclear detection by a hematoxylin signal. The grey bar in the boxplots represents the median, and the inner, colored boxes represent the interquartile range (25th and 75th percentiles). Statistical analysis was performed using a two-tailed Mann–Whitney U-test and adjusted for multiple testing with the Benjamini–Hochberg FDR method. ∗p < 0.05, ∗∗p < 0.01. See also Table S1 and Figure S2.

Figure 3

Placental explants and cell clusters infected by SARS-CoV-2 S protein pseudotyped lentiviruses (A) Graph showing relative luminescence units (RLU) from placental explant cultures 72 hpi after infection with lentivirus pseudotyped with SARS-CoV-2 spike or VSV-G protein with or without the addition of reverse transcriptase inhibitor Nevirapine (NVP). Statistical significance was determined by the two-tailed unpaired t-test (∗p ≤ 0.05, ∗∗p ≤ 0.005). Data are presented as mean ± SEM. (B) Graphs showing RLU from infected isolated primary placental cell clusters (left) and infected A549-ACE2 cells (right) 72 hpi with the addition of blocking antibodies against ACE2 and NRP1. Statistical significance was determined by the two-tailed unpaired t-test (∗∗p ≤ 0.005, ∗∗∗p ≤ 0.001). Data are presented as mean ± SEM. (C) Brightfield and live fluorescence microscopy images of cultured placental explants, mock-infected (Mock, left column), 72 hpi with either SARS-CoV-2-S pseudotyped lentivirus (SARS-CoV-2-S, center column) or VSV-G pseudotyped lentivirus (VSV-G, right column). (D) Fluorescence microscopy images on sections from mock-infected (Mock, top row), SARS-CoV-2-S pseudotyped lentivirus-infected (SARS-CoV-2-S, center row), or VSV-G pseudotyped lentivirus-infected (VSV-G, bottom row) explants, stained for the GFP reporter (green) syncytial trophoblast marker, cytokeratin (KRT7, grey), endothelial marker CD31 (red), and DAPI nuclear stain (blue). Scale bars = 500 μm. (E) Graphs showing RLU from placental cell clusters (left) and A549-ACE2 cells (right) infected with lentivirus pseudotyped by VSV-G (control), wild-type SARS-CoV-2 spike (D614G), Alpha variant (B.1.1.7), Beta variant (B.1.351), and Delta variant (B.1.617.2) spike. Lentivirus without a pseudotyped enveloped protein was included as a control (No Env). Statistical analysis was performed using one-way ANOVA (∗p value < 0.05, ∗∗p value < 0.005, ∗∗∗p value < 0.001). Statistical significance was determined by the two-tailed unpaired t-test (∗∗p ≤ 0.005, ∗∗∗p ≤ 0.001). Data are presented as mean ± SEM. See also Figure S3.

Figure 4

Primary human placenta cells can be infected with SARS-CoV-2 ex vivo (A) qRT-PCR analysis of relative viral N subgenomic RNA expression in primary placental cell clusters infected with SARS-CoV-2 ex vivo (MOI = 1) at 24 hpi and normalized to ACTB levels (mean ± SD; n = 12 from four independent experiments; Student’s t test, ∗∗∗∗p < 0.0001). (B) Three-dimensional reconstruction of confocal imaging of primary placental cell clusters infected with SARS-CoV-2 ex vivo (MOI = 1) at 24 hpi, stained for trophoblast marker KRT7 (green), SARS-N (red), endothelial marker CD31 (grey), and DAPI (blue). Scale bar = 30 μm. (C) Confocal imaging of primary placental cell clusters infected with MOCK (top rows) or SARS-CoV-2 (MOI = 1, bottom rows) ex vivo at 24 hpi, stained for trophoblast marker KRT7 (green), SARS-N (red), endothelial marker CD31 (grey), and DAPI (blue). Arrows indicate the presence of SARS-N nucleocapsid protein in trophoblast and endothelial cells. Scale bar = 20 μm. See also Video S1.

Figure 5

SARS-CoV-2 infection of ex vivo placental explants demonstrates a robust inflammatory response (A) SARS-CoV-2 viral RNA FPKM levels in mock and SARS-CoV-2-infected placental cell clusters. Data are presented as mean ± SD. n = 3 biological replicates, Student’s t test (∗∗p = 0.0012). (B) PCA analysis of gene expression profiles in mock infected samples (n = 3) and SARS-CoV-2-infected samples (n = 3 biological replicates). (C) Expression heatmap of sample-to-sample distances for the overall gene expression. (D–G) Heatmap from RNA-seq data of showing placental cell marker gene expression (D), chemokine and cytokine gene expression (E), inflammatory associated gene expression (F), and cell death marker gene expression (G). (H) IPA depicting the top canonical biological pathways affected when comparing SARS-CoV-2-infected placenta cell clusters with mock infected controls. TC, trophoblast cells; EC, endothelial cells; HB, Hofbauer cells.

Figure 6

Transcriptional analysis of term placentas of COVID-19 patients (A) SARS-CoV-2 viral RNA FPKM levels in placentas from healthy control pregnancies (n = 4), COVID⁺ mothers (n = 17), and placentas with non-COVID related inflammatory pathologies (n = 4). B.1.1.07 Alpha variant (red) was detected in the two high positive placentas (H_1, H_2). (B) PCA analysis of gene expression profiles in control samples (C, n = 4), high positive samples (H, n = 2), positive samples (P, n = 15), and inflammatory samples (I, n = 4). (C) Expression heatmap of sample-to-sample distances for the overall gene expression. (D–F) Heatmap from RNA-seq data of placentas from control samples (C, n = 4), high positive samples (H, n = 2), positive samples (P, n = 15), and inflammatory samples (I, n = 4) showing placental cell marker gene expression (D), chemokine and cytokine gene expression (E), and inflammatory associated gene expression (F). (G) Ingenuity pathway analysis (IPA) depicting the top canonical biological pathways affected when comparing high positive SARS-CoV-2 placentas with control placentas. TC, trophoblast cells; EC, endothelial cells; HB, Hofbauer cells. (H) Heatmap of cellular deconvolution estimating the relative abundance of cell types at the maternal–fetal interface. Mean values per group were the Z-score transformed per row. The original mean relative proportion of each cell type is displayed on the right. See also Table S2; Figures S4 and S5.