Differential Expression of Rab5 and Rab7 Small GTPase Proteins in Placental Tissues From Pregnancies Affected by Maternal Coronavirus Disease 2019.

PURPOSE: The majority of pregnancies affected by maternal coronavirus disease 2019 (COVID-19) do not result in fetal transmission. However, several studies have identified parenchymal changes in their placental tissues, suggesting a placental response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the maternal-fetal interface. Although many COVID-19 placental studies have focused on the expression of the canonical SARS-CoV-2 entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2, further characterization of subcellular molecules involved in viral trafficking have not yet been investigated in these tissues. Of interest are Rab proteins, a family of small GTPase proteins that direct intracellular transport between different endocytic organelles. Rab5 and Rab7 in particular have previously been implicated in HIV and cytomegalovirus invasion of placental trophoblast cells in vitro; the localization of these molecules has not been fully characterized within the human maternal-fetal interface, however, or within placental tissues from SARS-CoV-2-infected pregnancies.
METHODS: Using fluorescent immunohistochemistry, Rab5 and Rab7 placental localization and comparative fluorescence intensity were explored in a cohort of placental tissues from pregnancies affected by maternal COVID-19 disease (COVID, n = 15) compared with contemporary control subjects (Control, n = 10). Fluorescence intensity was quantified by using corrected total cell fluorescence values.
FINDINGS: Within placental villi, Rab5 was consistently localized in syncytiotrophoblast and cytotrophoblast cells. Rab5 had significantly higher mean (SEM) fluorescence intensity in the COVID cohort (Control, 1.96 [0.16]; COVID, 2.62 [0.09]; P = 0.0014). In contrast, although Rab7 was also localized within placental villous syncytiotrophoblast and cytotrophoblast cells, mean (SEM) Rab7 fluorescence intensity was significantly downregulated in COVID vs Control placentas (Control, 35.9 [4.1]; COVID, 20.1 [0.52]; P = 0.0001). IMPLICATIONS: This differential expression of Rab5 and Rab7 suggests that placental endocytic pathways may be altered at the maternal-fetal interface in pregnancies affected by maternal SARS-CoV-2 infection. As key molecules governing intracellular vesicle transport, including viral trafficking, Rab GTPase proteins may be of interest for ongoing studies examining placental responses to COVID-19 in pregnancy.

Introduction

With >10 million confirmed cases and several hundred thousand deaths reported worldwide, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel infectious agent responsible for coronavirus disease 2019 (COVID-19), remains a significant global health burden. Of particular interest is the study of COVID-19 in pregnancy. With prolonged maternal–fetal exposures being a physiological necessity for healthy gestation, viral transmission of SARS-CoV-2 in pregnancy cannot be combated through normal quarantine measures. However, low rates of fetal transmission (1%–4%) have been consistently reported throughout the pandemic. , As the main exchange interface between mother and fetus in pregnancy, the placenta seems to be creating a physiological barrier against fetal infection of SARS-CoV-2. However, the mechanisms governing the lack of fetal transmission remain unclear. The present article reviews the current literature on COVID-19 in pregnancy and placental responses to SARS-CoV-2 infection. We also share novel data from a pilot study examining the expression of selected proteins involved in endocytic vesicle trafficking in a cohort of placental tissues from pregnancies affected by maternal COVID-19, highlighting a potential new direction for the study of SARS-CoV-2 responses at the maternal–fetal interface.

Although pregnant women have been estimated to comprise only 9% of SARS-CoV-2–positive cases among women of reproductive age in the United States, they seem to be particularly susceptible to complications of COVID-19 disease, with reports of increased disease severity and higher rates of hospitalization compared with other adult populations. , Regarding perinatal clinical signs in newborns of mothers with positive SARS-CoV-2 test results, studies have reported mixed results. Interestingly, infants appear to be asymptomatic with low rates of positive SARS-CoV-2 test results. In those who have tested positive, there have been reports of complications, including preterm birth, respiratory distress syndrome, meconium-stained amniotic fluid, fever, tachycardia, and tachypnea in case series.7, 8, 9, 10 In other clinical literature investigating SARS-CoV-2–negative infants born to SARS-CoV-2–positive mothers, clinical findings such as chronic in utero fetal distress, chorioamnionitis, preterm birth, dyspnea, thrombocytopenia, vomiting, fever, tachycardia, and low birth weight have been reported.11, 12, 13 However, the majority of the literature has reported normal findings (eg, APGAR scores, birth weight) in neonates born to SARS-CoV-2–positive mothers. , , 14, 15, 16, 17, 18

Despite a significant increase in research on COVID-19 disease in pregnancy in 2020, questions remain about the risk for transmission of SARS-CoV-2 from mother to fetus (ie, vertical transmission). Before the current pandemic, coronavirus was not associated with vertical transmission. This was true for both SARS-CoV-1 and Middle East respiratory syndrome-coronavirus, which were responsible for major outbreaks in 2002 and 2012. , Selected studies to date have suggested vertical transmission of SARS-CoV-2 based on positive immunoglobulin M antibody laboratory test results in 3 neonates; however, the sensitivity and specificity of these serologic tests have been questioned. , , Other case series and reports have further identified positive results for SARS-CoV-2 from infants’ pharyngeal swabs by real time reverse transcription polymerase chain reaction (real time RT-PCR). , , , In 1 patient, placental biopsy specimens from a stillbirth tested positive immediately after expulsion and at 24 h. Another study reported SARS-CoV-2–positive samples from placental or membrane swabs in 3 pregnant patients, but results of neonatal swabs were all negative. Patanè et al reported positive RT-PCR test results for 2 of 22 mother–neonate dyads, as well as both of their placentas. Nevertheless, the majority of COVID-19 studies in neonatal settings have found no evidence of viral RNA in pharyngeal swabs, blood, feces, or urine. , , 10, 11, 12, 13 , 16, 17, 18 , , Furthermore, breast milk and vaginal secretions have yielded negative SARS-CoV-2 results. ,

Pathology evaluations of placentas from pregnancies affected by maternal COVID-19 have reported a range of placental findings. These reports have included tissue cohorts showing minimal changes as well as various placental abnormalities, including features of maternal vascular malperfusion, increased intervillous thrombi, diffuse perivillous fibrin and inflammatory infiltrates, and increased incidence of chorangiosis compared with controls. , , 28, 29, 30 Other COVID-19 placental case series also observed low-grade fetal vascular malperfusion, maternal vascular malperfusion, villitis, and fetal vascular thrombosis. ,

The COVID-19 pregnancy literature to date has largely focused on various clinical manifestations, epidemiology, and placental pathology; few studies have fully evaluated mechanisms governing the low rates of fetal transmission and/or the placental cellular responses to maternal SARS-CoV-2 infection. The symbiotic maternal–fetal physiology required for pregnancy maintenance and normal fetal development necessitates close and prolonged exposures between mother and fetus throughout gestation. However, despite these exposures, vertical transmission between mother and fetus remains a heterogeneous process, with a broad variability in maternal infections that result in true fetal pathology. In viral infections in which vertical transmission is prevented, the placenta can be a key boundary providing physiological viral blockade.

Placenta villi are the main anatomical interface between mother and infant during pregnancy (Figure 1 A) and are composed of placental cell layers covering a central core of fetal blood vessels. These placental cells, termed villous trophoblasts, are subdivided into syncytiotrophoblast cells (sTBs) that are fed by an underlying layer of cytotrophoblast cells (cTBs) (Figure 1B). sTBs form a monolayer on the outer aspect of the placental villous, which lies in direct contact with maternal blood and is juxtaposed with cTBs and fetal blood vessels. Trafficking between the sTB layer, cTBs, and fetal blood vessels thus comprises the main cellular exchange interface between mother and fetus during pregnancy. Thus, it is important to evaluate the more sub-anatomical aspects of placental tissues to evaluate where a potential SARS-CoV-2 blockade could be occurring at the maternal–fetal interface and identify areas for future investigation.

Figure 1

Viral entry viewpoint of the maternal–fetal interface. (A) Schematic of human placental villi. (B) Enlarged view of placental villous anatomy. (C) Rab 11 expression in human placental villous. Red, Rab11; Green, E-cadherin; Blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. cTB = cytotrophoblast cell; EC = endothelial cell; FBV = fetal blood vessel; MBS = maternal blood space; sTB = syncytiotrophoblast cell. Modified from Taglauer et al

In ongoing evaluations of COVID-19 in pregnancy, there has been varied evidence of SARS-CoV-2 entry within the placental compartment.23, 24, 25 , , , 35, 36, 37, 38, 39, 40 When SARS-CoV-2 has been identified within placental tissues, multiple studies have found localization within both sTB and cTB placental villous subtypes. , Although the reported variation of SARS-CoV-2 in placental tissues could be due to differences in patient populations and/or experimental techniques, recent meta-analyses have estimated an overall range of 7%–21% of placental tissues showing evidence of SARS-CoV-2 placental invasion in the current literature. , Interestingly, these estimates are still fourfold to fivefold higher than the reported percentages of fetal transmission (1%–4%), , suggesting that even if SARS-CoV-2 gains access to the placental compartment, other mechanisms may still be in place preventing its subsequent transmission to the developing fetus.

SARS-CoV-2 cellular entry proteins have also been investigated in placental tissues. It has been well established that the SARS-CoV-2 spike protein and the host cell proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 are required for cell entry and processing. , Studies examining historical single-cell molecular datasets have reported varying mRNA expression patterns of ACE2 and transmembrane serine protease 2 in placental cell subtypes throughout gestation. , , Subsequent histologic evaluations have identified the presence of both receptors in villous tissues from term control and COVID-19 placental samples, with a comparative predominance of ACE2. ,

Although the majority of studies examining SARS-CoV-2 infection in the placenta have focused on the presence of viral contents and cell surface entry molecules, there has been an overall lack of more detailed exploration on other intracellular molecules, which could determine viral transmission in trophoblast cells and subsequent entry to the fetal circulation (Figure 1B). Of particular interest are pathways involved in endocytosis, which is a common host cell entry mechanism for many viruses. Indeed, a recent review emphasized the importance of examining endocytic pathways in studies on COVID-19, highlighting endocytosis as a high-yield area to identify cell biological mechanism(s) governing SARS-CoV-2 infection.

Central to the process of endocytosis are Rab proteins, a family of small GTPase molecules that are master regulators of endocytic vesicle trafficking and organelle dynamics in eukaryotic cells. Rab5, Rab7, and Rab11 in particular are involved in viral cell entry/egress in a variety of cell types, and their cellular expression can be altered by cytokine-dependent regulation. , Rab5 and Rab7 regulate the biogenesis of early endosomes and endo-lysosomal trafficking, respectively, whereas Rab11 is critical for endocytic recycling to the plasma membrane. , Our previous study found that Rab11 is prominently expressed within trophoblast cells and fetal blood vessels in normal human placental tissues (Figure 1C). Rab 5 and Rab7 have been implicated in HIV and cytomegalovirus entry into trophoblast cells in vitro, but the localization of these molecules has not been fully characterized within primary human placental tissues. , Given their previously described role in viral entry into trophoblast cells, we hypothesized that evaluation of Rab5 and Rab7 in placental tissues affected by maternal COVID-19 would yield important information on the intracellular trophoblast responses to SARS-CoV-2 in pregnancy. To address this hypothesis, we conducted a comparative evaluation of Rab5 and Rab7 expression in a selected cohort of placental tissues from pregnancies affected by maternal COVID-19 disease compared with contemporary controls.

Materials and Methods

Sample Collection

This study was conducted at Boston Medical Center, Boston, Massachusetts, with samples obtained between April and May 2020 during a period of peak COVID-19 admissions. At that time, universal testing for SARS- CoV-2 by PCR of nasopharyngeal swabs was instituted at our hospital for all women admitted in labor. Placental samples were collected from women who tested positive at the time of delivery along with contemporary control subjects who tested negative at the time of delivery who delivered in the same time period. Control subjects were also matched for gestational age at delivery. Within the COVID-19 placental cohort, a subset of five COVID-19 placental tissues were specifically selected from pregnancies with evidence of fetal transmission (ie, positive infant SARS-CoV-2 PCR testing from nasopharyngeal swabs within 24 h of delivery). Tissues were collected with approval from the Boston University Medical Campus Institutional Review Board, with an informed consent waiver specific to this study.

Tissue Processing

Shortly after delivery, placental tissues were placed in 4% buffered formalin followed by grossing, staging, and histologic evaluation by board-certified pathologists at Boston Medical Center. The remaining formalin-fixed placental tissues were then dissected into full thickness placental biopsy specimens (including decidua, villous tissue, and chorionic plate) and soaked in 18% sucrose for 1 week, which was then followed by embedding in Tissue-Tek O.C.T. solution (ThermoFisher, Waltham, MA) and freezing at −80 °C. Tissue blocks were then cryo-sectioned at 10 μm thickness for subsequent immunostaining.

Immunohistochemistry

Placental tissue sections were first subjected to antigen retrieval with 10 mM sodium citrate, 0.05% Tween 20, pH 6, boiling for 20 min, followed by a series of washes with phosphate-buffered saline 0.05% with Tween 20 (PBS-T). Slides were then incubated for 1 h at room temperature in PBS with permeabilization solution 0.2% Triton X-100 along with 10% blocking serum of host species for all correlate secondary antibodies (MilliporeSigma, Burlington, Massachusetts).

Rab5/Rab7 Co-labeling

Slides were then incubated at 4 °C overnight with the following primary antibodies at 1:100 diluted in PBS-T: Rab5A (mouse anti-human, 66,339; Proteintech, Rosemont, Illinois) or Rab7 (rabbit anti-human Ab137029; Abcam, Cambridge, United Kingdom). After a series of washes, slides were incubated with the following corresponding secondary antibodies (Abcam) at a dilution of 1:500: Rab5A, donkey anti-mouse Alexa Fluor 594; and Rab7, donkey anti-rabbit Alexa Fluor 647.

Cytokeratin 7 and Rab Co-labeling

Rab5 and Rab7 primary and secondary staining was performed as described earlier. Following a series of washes after secondary antibody labeling, an Alexa Fluor 488–conjugated anti–cytokeratin-7 primary antibody (mouse anti-human, ab185048) was added to slides at 1:100 diluted in PBS-T. For control staining of placental tissues, slides were incubated with only secondary antibodies (without primary antibodies). After a final series of washes, slides were cover-slipped with Prolong Gold with 4′,6-diamidino-2-phenylindole (ThermoFisher). To ensure consistency for comparative analysis, all cohort slides (COVID-19 and control) were stained together in bulk and imaged within 24–48 h of staining.

Microscopy

Immunofluorescent images were captured by using a Nikon deconvolution wide-field epifluorescence microscope using NIS-Elements Software (Nikon, Tokyo, Japan) with slide cohorts blinded at time of image acquisition. Within the villous compartment, 8 randomized images were captured at 200× magnification, using standardized exposure times. Images were further processed post-acquisition via Fiji software package for ImageJ (US National Institutes of Health, Bethesda, Maryland; https://imagej.nih.gov.ij/).

Quantitative Image Analysis

Using ImageJ software, image area and integrated density were measured for each immunofluorescent 200× image (n = 8 randomized areas per slide). Mean fluorescence values of 5 random background readings per image cohort were also measured. Measurements were obtained of 2 blinded reviewers (Y.B. and E.T.). From these values, a raw corrected total cell fluorescence (CTCF) was calculated per published protocols: CTCF = integrated density – (area of selected image × average mean background fluorescence).52, 53, 54 Average CTCF was also calculated from 3 secondary-only, negative control slides (background CTCF). Final CTCF values for each target were then used to generate a final CTCF value as a ratio of target CTCF/secondary-only control CTCF.

Statistical Analysis

All statistical analyses were performed by using Prism 7 software (GraphPad, San Diego, California). CTCF values were compared by using independent sample t tests for normally distributed, continuous data. Differences were considered significant at P < 0.05.

Results

Demographic characteristics of our placental cohort were as previously published by Taglauer et al for the 15 COVID-19 and 10 control mother–infant dyads. Because Rab5 and Rab7 have only been previously described in trophoblast cells in vitro, we first evaluated their in situ localization in our cohort of control placental tissues. Both Rab5 and Rab7 were identified in placental villi, and co-staining with cytokeratin 7, a pan trophoblast marker, revealed localization in villous sTB and cTB cell subpopulations (Figures 2 A and 2B). Placental tissues were then evaluated for comparative Rab5 and Rab7 expression in COVID-19 versus control tissues by using dual immunofluorescence followed by analysis of CTCF values. In both tissue cohorts, Rab5 expression was found throughout sTBs as well as the underlying cTB cell layers, with a small but significant increase in overall fluorescence intensity among COVID-19 placentas (Figures 3 A and 3E; Figure 4 A). In contrast, there was a striking decrease in Rab7 intensity in villous tissues among all COVID-19 placentas evaluated (Figures 3B and 3F). This resulted in a statistically significant decrease in the Rab7 fluorescence intensity (CTCF) values in COVID-19 placentas (Figure 4B).

Figure 2

Rab5 and Rab7 trophoblast localization in placental villi. Evaluation of pan trophoblast marker cytokeratin 7 along with Rab5 and Rab7 expression in healthy term placental tissues (n = 10). (A) Representative images of co-staining with cytokeratin 7 (green) and Rab5 (red). (B) Representative images of co-staining with cytokeratin 7 (green) and Rab7 (red). Blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. Scale bars, 12 μm. Inset images: secondary-only negative control. cTB = cytotrophoblast cell; sTB = syncytiotrophoblast cell.

Figure 3

Rab5 and Rab7 expression in control versus coronavirus disease 2019 (COVID-19) placental villous tissues. (A–D) Representative images of Rab5 and Rab7 co-labeling in control tissues (ie, placental tissues from pregnant women who were severe acute respiratory syndrome coronavirus 2 negative upon admission screening (n = 10). (E–H) Representative images of Rab5 and Rab7 co-labeling in COVID-19 tissues (ie, placental tissues from pregnant women who were severe acute respiratory syndrome coronavirus 2 positive upon admission screening (n = 15). (C and G) Inset images, secondary-only negative control. (D and H) Enlarged placental image of dashed white boxes in panels C and G, respectively. Green, Rab5; Red, Rab7; Blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. Scale bars: panels A through C and panels E through G, 50 μm; panels D and H, 10 μm cTB = cytotrophoblast cell; sTB = syncytiotrophoblast cell.

Figure 4

Rab5 and Rab7 differential expression in coronavirus disease 2019 (COVID-19) placental tissues. Graphical analysis of corrected total cell fluorescence values from Rab5 (A) and Rab7 (B) image analysis. Placental tissue cohorts are as described in Figure 3. ∗P < 0.0014. †P = 0.0001.

Overlay of Rab5 and Rab7 imaging further illustrated the co-localization of Rab5 and Rab7 in the sTB layer of control tissues and the predominance of Rab5 expression in the sTB layer of COVID-19 placental villi (Figures 3C, 3G, 3D, and 3H). Subanalysis of COVI-19 placental tissues (n = 5) from selected pregnancies with evidence of fetal transmission showed a pattern of Rab5 and Rab7 consistent with the rest of the COVID-19 placental cohort (data not shown). Overall, these results identify Rab5 and Rab7 localization within placental villi, a key anatomical area of the maternal–fetal interface. Furthermore, they illustrate altered expression of both proteins in placental tissues affected by maternal COVID-19 disease.

Discussion

This study highlights Rab GTPases as placental targets of interest for ongoing analysis of COVID-19 in pregnancy. The differential expression of Rab5 and Rab7 in COVID-19 placental tissues suggests trophoblast-specific molecular alterations in response to SARS-CoV-2 at the maternal–fetal interface. Although these results require ongoing validation, they are consistent with prior research examining Rab5 and Rab7 expression in host–cell pathogen interactions. Bacterial component muramyl dipeptide (found in both gram-positive and gram-negative bacteria) has been shown to have opposing effects on Rab5 and Rab7 expression in macrophages, corresponding with pathogen lysosomal degradation. Furthermore, although mechanisms dictating Rab protein expression are multifactorial, some key themes emerge from their regulation in response to infection. Rab5 and Rab7 seem to be governed by distinct signaling pathways in response to cytokine production. Rab5 can be upregulated in response to proinflammatory cytokines such as interleukin-6 (IL-6) via activation of the extracellular signal–regulated kinase pathway. In contrast, Rab7 can be altered by an IL-12–dependent, p38 mitogen-activated protein kinase–directed pathway. Because altered levels of both IL-6 and IL-12 are among the systemic cytokine responses associated with COVID-19 disease, intrauterine cytokine changes could be one of the determinants driving altered expression of Rab5 and Rab7 in these placental tissues. However, as the pregnancy-specific cytokine profiles of maternal COVID-19 infection have not been well defined, ongoing studies will be needed to identify factors regulating Rab5 and Rab7 differential expression in placental tissues and the potential role of these proteins in the SARS-CoV-2 response at the maternal–fetal interface.

The current study has several limitations. First, it was a pilot analysis of histologic findings in a selected cohort of fixed placental tissues. Although differences were observed between groups, these initial data require ongoing validation with comparative evaluation of mRNA and protein expression in greater numbers of freshly preserved samples. In addition, these tissues were collected from pregnancies during a specific time frame during a peak of admissions and illness severity in the early stages of the COVID-19 pandemic. Ongoing evaluation of placental tissues from COVID-19–positive pregnancies over a broader time frame and clinical heterogeneity will be informative to capture a more complete picture of Rab5 and Rab7 expression changes. Finally, Rab protein expression was only evaluated in pregnancies with maternal COVID-19 in the third trimester. Analysis across first-, second-, and third-trimester maternal SARS-CoV-2 infections will be informative to evaluate how the placental expression of these proteins changes relative to the gestational timing of maternal COVID-19.

Overall, this work highlights the importance of investigating additional subcellular pathways to more fully understand the placental response to COVID-19 in pregnancy. Because Rab5 regulates early endosome processes and Rab7 directs early to late endosomal transport, future evaluation of endosomal subtype markers will also be a key area of investigation. Comparative analysis of early endosome antigen 1, a marker of early endosomes, and CD63, a late endosome and multivesicular body marker, could be informative to identify whether downregulation of Rab7 in COVID-19 placental tissues results in retention of the virus in early endosomes or trapping within late endosomes and multivesicular bodies. It is important to note that within our COVID-19 placental cohort, Rab5 upregulation and Rab7 downregulation was noted among all tissues, including those selected from pregnancies with evidence of SARS-CoV-2 fetal transmission.

Conclusions

Although the current study was not powered to identify true correlations with these expression changes and fetal transmission, our findings do suggest that mechanisms in addition to altered Rab GTPase expression could be mediating the physiological placental blockade of SARS-CoV-2 in pregnancy. This would be entirely expected as maternal–fetal trafficking is a highly regulated and multifactorial process throughout pregnancy. Continued evaluation of larger COVID-19 placental cohorts using multivariate analyses such as RNA-sequencing and spatial transcriptomic approaches will be highly informative to more fully characterize the physiological placental response to COVID-19 in pregnancy and potentially identify therapeutic targets for other organ systems to help combat systemic SARS-CoV-2–mediated disease.

Disclosures

The authors have indicated that they have no conflicts of interest regarding the content of this article. The funding agencies sponsoring this work had no role in the study design, the collection, analysis or interpretation of data, in the writing of the manuscript or the decision to submit the manuscript for publication.