Cell-type specific distribution and activation of type I IFN pathway molecules at the placental maternal-fetal interface in response to COVID-19 infection

Abstract

Background and objective: COVID-19 infection in pregnancy significantly increases risks of adverse pregnancy outcomes. However, little is known how the innate immunity at the placental maternal-fetal interface responds to COVID-19 infection. Type I IFN cytokines are recognized as a key component of the innate immune response against viral infection. In this study, we specifically evaluated expression of IFN antiviral signaling molecules in placentas from women infected with COVID-19 during pregnancy.

Methods: Expression of IFN activation signaling pathway molecules, including cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), interferon regulatory factor 3 (IRF3), Toll-like receptor 7 (TLR7), mitochondrial antiviral-signaling protein (MAVS), and IFNβ were determined in formalin-fixed paraffin embedded (FFPE) placental tissue sections (villous and fetal membrane) by immunostaining. A total of 20 placentas were examined, 12 from COVID-19 patients and 8 from non-COVID-19 controls. Patient demographics, clinical data, and placental pathology report were acquired via EPIC medical record review.

Results: Except BMI and placental weight, there was no statistical difference between COVID and non-COVID groups in maternal age, gestational age at delivery, gravity/parity, delivery mode, and newborn gender and weight. In COVID-exposed group, the main pathological characteristics in the placental disc are maternal and fetal vascular malperfusion and chronic inflammation. Compared to non-COVID controls, expression of IFN activation pathway molecules were all upregulated with distinct cell-type specific distribution in COVID-exposed placentas: STING in villous and decidual stromal cells; IRF3 in cytotrophoblasts (CTs) and extra-villous trophoblasts (EVTs); and TLR7 and MAVS in syncytiotrophoblasts (STs), CTs, and EVTs. Upregulation of STING, MAVS and TLR7 was also seen in fetal endothelial cells.

Conclusions: STING, IRF3, TLR7, and MAVS are key viral sensing molecules that regulate type I IFN production. Type I IFNs are potent antiviral cytokines to impair and eradicate viral replication in infected cells. The finding of cell-type specific distribution and activation of these innate antiviral molecules at the placental maternal-fetal interface provide plausible evidence that type I IFN pathway molecules may play critical roles against SARS-CoV-2 infection in the placenta. Our findings also suggest that placental maternal-fetal interface has a well-defined antiviral defense system to protect the developing fetus from SARS-CoV-2 infection.

Keywords: COVID-19; IRF3; MAVS; STING; TLR7; placental maternal-fetal interface; pregnancy; type I IFNs.

Figure 1

Representative villous tissue H&E staining and expression of STING and IFNβ in villous tissue and fetal membrane in placentas exposed to maternal COVID-19 infection. (A) Representative villous tissue H&E staining in COVID-exposed (b and c) placentas compared to non-COVID control (a) showing features of fetal vascular malperfusion including avascular villi, vascular thrombi, and perivascular fibrin deposition in villous tissue from COVID-19 exposed placentas. IVS: intervillous space; double arrow: STs; *: villous stroma; bold arrows: avascular villi and perivascular fibrin deposition; green arrow: fetal vessel thrombi. Bar = 50µm. (B) STING and IFNβ expression in villous tissue and fetal membrane in placentas exposed to maternal COVID-19 infection. a and b: villous tissue; a1 and b1: fetal membrane; a and a1: STING; and b and b1: IFNβ. In villous tissue: double arrow: STs; *: villous stroma. In fetal membrane: ‡: EVTs; #: decidua. Bar = 50µm in a and b; Bar = 100µm in a1 and b1.

Figure 2

STING, CD68, CD16, and vimentin expression in villous tissue of placentas with or without exposure to maternal COVID-19 infection. Active infection: from placentas delivered by women with positive detection of SARS-COV-2 RNA when admitted to Labor and Delivery. Recovered case: from placentas delivered by women with positive detection of SARS-COV-2 RNA at second trimester and negative detection of SARS-COV-2 RNA when the patient was admitted to hospital for delivery. Non-infected control: from placenta delivered by women who was never infected with COVID-19 before and during pregnancy. STING is strongly expressed in villous stromal cells in both active infection (a1) and recovered (b1) cases, but only a few positive cells were seen in villous stromal in non-infected control (c1) placentas. Both CD68 and CD16 are markers for macrophages. Only a few positive CD68 and CD16 cells were detected in villous stromal in active (a2, a3), recovered (b2, b3), and control (c2, c3) placentas. Vimentin is a marker of mesenchymal cells. Vimentin positive cells were detected in villous stroma in all villous tissue examined: active infection (a4), recovered (b4), and non-COVID control (c4) placentas. These results indicate that STING positive cells are villous mesenchymal stromal cells (MSCs), not Hofbauer cells (macrophages), in placentas exposed to COVID infection. Bar = 50µm.

Figure 3

STING, IRF3, TLR7, MAVS, and IFNβ expression in villous tissue of placentas with or without exposure to maternal COVID-19 infection. (A) STING, IRF3, TLR7, MAVS, and IFNβ expression in villous tissues from placentas with or without exposure to maternal COVID-19 infection. Images a to e are representative images of STING, IRF3, TLR7, MAVS, and IFNβ expression in villous tissue sections in non-COVID control placentas, and images a1 to e1 show zoom of enlarged rectangle area in a to e of each, respectively. Images f to j are representative images of STING, IRF3, TLR7, MAVS, and IFNβ expression in villous tissue sections in COVID-exposed placentas, and images f1 to j1 show zoom of enlarged rectangle area in f to j of each, respectively. Bar = 50µm. Activation or upregulation of STING, IRF3, TLR7, MAVS, and IFNβ expression were found in different cell-types in COVID-exposed placental villous tissue compared to non-COVID controls: 1) strong STING expression signal in stromal cells and fetal endothelial cells; 2) activation of IRF3 in CTs; and 3) increased TLR7 expression in STs, CTs, and fetal endothelial cells; 4) upregulation of MAVS expression in STs, CTs, stromal cells, and fetal endothelial cells; and 5) increased IFNβ expression in STCs and fetal endothelial cells, respectively. Red arrow: CTs; Green arrow: fetal endothelial cells. (B) Violin graphs show that relative expression for STING, IRF3, TLR7, MAVS, and IFNβ in villous tissue sections are significantly increased in COVID-exposed placental villous tissue (COVID) vs. non-COVID controls (Cont), * p<0.05 and ** p<0.01, respectively.

Figure 4

STING, IRF3, TLR7, MAVS, and IFNβ expression in fetal membrane of placentas with or without exposure to maternal COVID-19 infection. Images a–e show STING, IRF3, TLR7, MAVS, and IFNβ expression in fetal membrane from non-COVID control placentas, and images f–j show STING, IRF3, TLR7, MAVS, and IFNβ expression in fetal membrane from COVID-exposed placentas. Upregulation of IRF3 in EVTs, and increased STING, TLR7, and IFNβ expression in chorionic mesoderm and decidual stromal cells were detected in fetal membrane of COVID-exposed vs. non-COVID control placentas. Increased IFNβ expression was also noticed in EVTs in fetal membrane of COVID-exposed placentas. ‡: EVTs and # decidual cells. Bar = 100µm.

Figure 5

A summary of type I IFN pathway molecule activation in different cell types at the maternal-fetal interface in placentas from women infected with COVID-19 in pregnancy. ST, Syncytiotrophoblasts; CT, cytotrophoblasts; HC, Hofbauer cells; SC, stromal cells; FEC, fetal endothelial cells; DC, decidual cells; AE, amnion epithelial cells; EVT, extra-villous trophoblasts, respectively.