key: cord-0743281-lgbrlwt9
authors: Taglauer, Elizabeth S.; Wachman, Elisha M.; Juttukonda, Lillian; Klouda, Timothy; Kim, Jiwon; Wang, Qiong; Ishiyama, Asuka; Hackam, David J.; Yuan, Ke; Jia, Hongpeng
title: Acute SARS-CoV-2 infection in pregnancy is associated with placental ACE2 shedding.
date: 2022-01-26
journal: Am J Pathol
DOI: 10.1016/j.ajpath.2021.12.011
sha: fd46f8a3c035c10ad18ac077185799424634777f
doc_id: 743281
cord_uid: lgbrlwt9

Although human placental tissues may be infected by SARS-CoV-2, the rate of fetal transmission is low, suggesting either viral neutralization or a barrier at the maternal-fetal interface. Angiotensin-converting enzyme (ACE)-2, the main receptor for SARS-CoV-2, is expressed as cell surface and soluble forms regulated by a metalloprotease cleavage enzyme, ADAM17. ACE2 is expressed in the human placenta, but the regulation of placental ACE2 expression in relation to timing of maternal SARS-CoV-2 infection in pregnancy is not well understood. In this study, we evaluated ACE2 expression, ADAM17 activity and serum ACE2 abundance in a cohort of matched villous placental and maternal serum samples from Control pregnancies (SARS-CoV-2 negative, n=8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the 2(nd) trimester (“2(nd)Tri COVID”, n=8) and 3rd trimester (“3(rd)Tri COVID”, n=8). In 3(rd)Tri COVID as compared to control and 2(nd)Tri-COVID villous placental tissues ACE2 mRNA expression was remarkably elevated, however, ACE2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE2 was also significantly increased in the maternal serum from 3(rd)Tri COVID infections as compared to control and 2(nd)Tri-COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE2 protein may be the result of ACE2 shedding. Overall, this work highlights the importance of ACE2 for ongoing studies on SARS-CoV-2 responses at the maternal-fetal interface.

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than one hundred million infections and claimed more than 3 million lives worldwide 1 . Despite effective vaccination and relatively rapid and accurate detection of the virus, the pandemic slowdown is hindered by frequent mutations, which led to new outbreaks in certain areas and then spread globally [2] [3] [4] [5] [6] . Therefore, further understanding the pathogenesis of and the host defense mechanisms to the viral infection is key in developing novel therapies to curb the spread of COVID-19.

Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 7, 8 . This receptor's inducibility and variability are proposed to be an essential element for viral tropism, infectivity, and COVID-19 disease progression and outcomes [9] [10] [11] [12] [13] [14] . ACE2, a vital member of the renin-angiotensin system (RAS), is a monocarboxyl peptidase and type I transmembrane protein 15 . As a proteinase, ACE2 cleaves one amino acid from carboxyl terminals of its substrates, such as Angiotensin II (Ang II), to generate the bioactive peptide, Ang 1-7, which counters the action of Ang II in many physiological processes, including host defense and inflammatory responses 16 . Therefore, the ACE2/Ang 1-7 axis is a negative regulatory mechanism of RAS to alleviate detrimental effects of the over-activated angiotensinconverting enzyme (ACE)/Ang II/ angiotensin receptor 1(AT1R) axis.

A majority of ACE2, after post-translational modification, migrates to the cell membrane. As a type I transmembrane protein, the anchored ACE2 contains a large ectodomain, in which the SARS-CoV-1 and -2 binding domain and enzymatic active domain separate distally, so the respective functions are not interfered with by the other 17 . Notably, the ectodomain of ACE2 undergoes shedding constitutively, and the shedding is inducible in response to various stimuli, including bacteria and viruses. The shed or soluble ACE2 is both enzymatically active and capable of binding to existing viral particles, such as SARS-CoV-2 18 . The soluble ACE2 has been proposed as a decoy receptor to trap the SARS-CoV virus as a promising therapy for COVID-19, and several clinical trials are underway to test the possibility 17 .

However, controversy regarding such a strategy remains because report indicates that soluble ACE2 can facilitate viral entry via alternative routes 19 . J o u r n a l P r e -p r o o f ACE2 shedding predominantly depends upon the activity of a disintegrin and metalloprotease domain 17 (ADAM17), or tumor necrosis factor-alpha (TNF-) converting enzyme (TACE) 18, 20 . ADAM17 is one of 13 genes in the family that encode functional proteases and are involved in ectodomain shedding of an array of growth factors, cytokines, receptors, and adhesion molecules, including TNF-and ACE2 20, 21 . ADAM17 enzymatic activity is controlled by posttranscriptional tissue inhibitors of metalloprotease (TIMPs). However, its gene expression is affected by many stimuli, and the sex hormonal regulation of ADAM17 is not well understood 22, 23 .

In the placenta, ACE2 is found primarily in the outer trophoblast epithelial cell layers of the villous placenta, directly juxtaposed with maternal blood at the main functional interface between mother and fetus 24 . Placental expression levels of ACE2 (and its partner components of the RAS) are prominent in early pregnancy and gradually decrease throughout gestation, implicating role for ACE2 in key events of placentation 24 . Several other groups have also identified ACE2 expression in the villous placenta primarily in trophoblast epithelial cells with a lack of expression among other stromal cells and fetal endothelium [25] [26] [27] . ADAM17 is also found within the villous placental trophoblast cells and its expression and its activity in the placenta has primarily been linked to TNF-a production in inflammatory pregnancy states [28] [29] [30] .

To date, the majority of studies evaluating ACE2 in pregnancies affected by COVID-19 have focused on maternal SARS-CoV-2 infections in the third trimester 26, 27, 31 . As the pandemic has ensued, women have gradually experienced COVID-19 disease throughout their pregnancies with consistent reports of low SARS-CoV-2 fetal transmission. Yet ACE2 expression relative to gestational stage of maternal SARS-CoV-2 infection remains unknown. Further, while the phenomenon of ACE2 shedding, ADAM17 activity and serum ACE2 levels have been characterized in multiple tissue types and COVID-19 patient populations 32 , they are not well defined in the human placenta or in relation to the timing of maternal COVID-19 infections in pregnancy.

Given the dynamic nature of ACE2 and ADAM17 activity among multiple tissues impacted by SARS-CoV-2 infection and COVID-19 disease evolution 17, 32 we hypothesized that ACE2 expression and J o u r n a l P r e -p r o o f ADAM17 activity in the placenta are affected by the timing of maternal SARS-CoV-2 infection in pregnancy relative to delivery. In the present study, ACE2 expression/ADAM17 activity were evaluated in placental villous tissues and maternal serum ACE2 levels in a cohort of maternal-fetal dyads with 2nd and 3rd trimester maternal SARS-CoV-2 infections in comparison with control pregnancies ( Figure 1A ).

This study design allowed for analysis of ACE2 placental expression dynamics in states of acute vs remote SARS-CoV-2 infections in pregnancy to further understand the trajectory of responses to COVID-19 at the maternal-fetal interface.

The current study was approved by the Boston University Medical School Institutional Review Board and written informed consent was obtained from all subjects. Prospective mother-infant dyads were enrolled at Boston Medical Center (BMC) between July 2020 and April 2021. Eligibility criteria were: ≥ 18 years of age, documented symptomatic SARS-CoV-2 infection during the second or third trimester of pregnancy (testing via nasal swab PCR, COVID groups) or no documented SARS-CoV-2 infection during pregnancy (testing via nasal swab PCR, Control group) singleton gestation pregnancy, English or Spanish speaking. Exclusion criteria included inability to provide informed consent.

Maternal blood samples were collected in EDTA collection tubes by trained staff within 24 hours pre or post-delivery. Following collection, blood samples were then stored at 4 ºC and centrifuged within 6 hours of collection. Plasma was then extracted and frozen at -80ºC until analysis.

For placental collection, all tissues were collected within an average time of 3.1 hours (SEM +/-0.14) following delivery and all samples were taken midway between umbilical cord insertion site and edge of placental disk. For histological analysis, full thickness placental biopsies (1cm x 3cm) were collected and fixed in 10% neutral buffered formalin (ThermoFisher) for 72 hours, soaked in 18% sucrose for 24 hours, embedded in Tissue-Plus optimal cutting temperature (OCT) compound (ThermoFisher) and Immunoresearch) were applied for 6h at room temperature. Slides were then washed five times with PBST again for 10 minutes each and mounted with DAPI (AF806, Vector Laboratories). Sections were imaged on a Nikon deconvolution wide-field epifluorescence microscope and processed using NIS-Elements Software (Nikon).

RNA was extracted from fresh frozen villous placental tissue biopsies using an RNAqueous kit (Invitrogen) per manufacturer's protocol. RNA transcripts were subsequently evaluated with Taqman® probes and primers (Forward: 5'-TAATGCTGGGGACAAATGGT-3', Reverse: 5'-CAGCTGAAGCTTGACTGTGAG-3', probe: 5'-TCCACACTTGCCCAAATGTA-3') for ACE2. Target expression was normalized to the transcript for GAPDH and relative expression was quantified via fold change in COVID samples relative to Control using 2 -ΔΔCT calculations.

Villous Placental Tissue ACE2 ELISA: Fresh frozen dissected placental tissue (300-500mg) was homogenized with protein lysis buffer (50mM Tris-HCL pH 8.0, 150mM NaCl, 1% NP-40, 0.5mM PMSF(Sigma) and cOmplete protease inhibitor (Roche). Tissue lysates were then centrifuged at 20,000 x J o u r n a l P r e -p r o o f g x 30min at 4ºC followed by collection of sample supernatants. Protein concentration for each sample was determined using a Micro BCA™ Protein Assay Reagent Kit (ThermoFisher) per manufacturer's instructions. Samples were assayed into a Human ACE2 DuoSet ELISA assay (R&D systems), loading equal amounts of protein in duplicate for all samples.

Serum ACE2 and Estradiol ELISA: Frozen maternal serum aliquots were quick thawed and immediately assayed using either a Human ACE2 DuoSet ELISA assay (R&D systems) or a Parameter Estradiol assay per manufacturer's instructions. All samples were assayed in duplicate. Absorbance for all ELISA assays was evaluated using a Molecular Devices SpectraMax i3x Multi-Mode Microplate Reader. 

Patient demographic characteristics, and maternal and neonatal outcomes were reported as follows:

Continuous variables were reported as either median with interquartile ranges or means with standard deviation and categorical variables were reported as percentages as indicated for each parameter (Table   1) . P-values for continuous variables were generated using a one-way analysis of variance test with Tukey's post hoc analysis. P-values for categorical variables were generated using the Fisher exact tests. 

To evaluate ACE2 expression dynamics at the maternal fetal interface in varied types of SARS-CoV-2 infections during pregnancy, a cohort of matched villous placental tissues and maternal serum samples were collected at time of delivery. SARS-CoV-2 infections in pregnancy were classified according to timing relative to delivery: "remote" infections in the 2nd trimester (2 nd Tri COVID) or "acute" infections in the 3rd trimester (3 rd Tri COVID) ( Figure 1A) .

All SARS-CoV-2 positive patients (COVID group) were symptomatic at time of testing (specified as with fever or reports of respiratory or gastrointestinal symptoms and/or loss of smell/taste documented in the medical chart). All cases were classified as "mild-moderate" infections with only one case requiring hospital admission due to COVID-19 and no patients requiring respiratory support or ICU care during the symptomatic phase of their illness ( Table 1) . The SARS-CoV-2 negative cohort (Control) were tested via universal screening procedures at time of admission to BMC labor and delivery. There was no significant difference in patient race, ethnicity, mode of delivery or gestational age at delivery between groups. Additionally, infant birth outcomes (i.e. APGAR scores and birthweight) and infant sex were also not statistically different between groups. Finally, within the COVID pregnancy group, all infants who met criteria for testing were negative for SARS-CoV-2.

To evaluate the abundance of ACE2 in placental tissues from our patient cohorts, we first examined ACE2 expression in full thickness placental biopsies using immunohistochemistry. Placental tissues were first screened for the presence of SARS-N protein. Only 3 of the 16 placental tissues within our COVID showed the presence of SARS-N expression (n=1 in 2nd trimester cohort; n=2 in 3rd trimester cohort; data not shown), a frequency similar to previously published placental tissue cohorts 31, 33 . We first identified ACE2 expression in villous trophoblast epithelial cells (but not fetal blood vessel J o u r n a l P r e -p r o o f endothelium), consistent with previous publications [25] [26] [27] (Figure 1B) . We also noted that villous placental tissues from remote, 2 nd Tri COVID had similar ACE2 expression intensity to those of Control placental tissues. However, ACE2 expression appeared to be decreased in placentas from acute, 3 rd Tri COVID ( Figure 1B ) as compared to both Control and the 2 nd Tri COVID group.

To further quantify ACE2 expression in these placenta tissues, we isolated protein from dissected villous placental tissue lysates and quantified ACE2 expression through ELISA assay analysis. Similar to the trend noted in our immunohistochemical analysis, we identified that ACE2 in acute third trimester infections was significantly decreased in comparison with Control and 2 nd Tri COVID groups ( Figure   1C ). Thus, through immunohistochemical and quantitative protein analysis, in our tissue cohort, ACE2 expression in villous epithelial tissues appeared to be decreased in acute, but not remote, SARS-CoV-2 infections in pregnancy as compared with controls. Interestingly, qRT-PCR analysis of ACE2 from villous samples of the same placental tissues showed a significant upregulation of ACE2 mRNA expression in acute 3 rd Tri COVID (Figure 1D) , suggesting the noted decrease placental ACE2 protein was due to post-translational influences rather than alteration of mRNA transcripts.

To next investigate the potential post-translational mechanism behind decreased placental ACE2 expression associated with acute maternal SARS-CoV-2 infections, ADAM17 activity was evaluated in dissected villous placental tissues. Because ADAM17 is a cell-surface enzyme known to cleave surface ACE2 20 , we examined whether the activity of ADAM17 in the placenta varied in relation to the timing of maternal SARS-CoV-2 infections in pregnancy. For this analysis, we used a fluorescence-based assay evaluating kinetic ADAM17 cleavage activity over time 34, 35 . We first noted that in comparison with our control group, placental tissues from remote SARS-CoV-2 infections in the 2nd trimester of pregnancy showed a modest elevation that did not reach statistical significance in ADAM17 activity (Figure 2A) .

However, placental tissues from pregnancies with acute third trimester maternal SARS-CoV-2 infections J o u r n a l P r e -p r o o f had a significant increase in ADAM17 activity over time when compared with control villous placental tissues (Mean fold increase: 1.5, **p < 0.01 Control vs 3 rd Tri COVID) (Figure 2B) .

The combined results of decreased villous placental ACE2 protein expression, increased ACE2 gene expression, and increased ADAM17 activity suggested a cleavage of ACE2 from the villous placental tissue. As villous placental tissue is directly juxtaposed with the maternal blood, we next examined whether the changes we observed in ACE2 expression and ADAM17 activity also correlated with any alterations in circulating ACE2 levels in maternal serum. To conduct this analysis, we evaluated ACE2 abundance in maternal serum samples which matched with placental samples analyzed for ACE2 expression and ADAM17 activity. In line with the trends noted with ADAM17 activity, serum ACE2 was significantly increased in acute 3rd trimester SARS-CoV-2 infections as compared to control and 2nd trimester maternal SARS-CoV-2 infections ( Figure 3A) .

Finally, we evaluated whether changes in placental and serum ACE2 abundance correlated with alterations in circulating estrogen levels in our cohort. Estrogen regulates ACE2 expression within airway epithelial cells 36 and has been implicated in the sex differences noted between COVID-19 severity among adults 37 . However, the role of estrogen on ACE2 regulation in pregnancy has been less well defined. Among our cohort there was no significant difference in maternal serum estrogen (assayed as estradiol) levels between our control and COVID groups (Figure 3B ).

The current study identifies new evidence on the dynamics of placental ACE2 expression in SARS-CoV-2 infections in pregnancy and the influence of the gestational stage of maternal infection relative to delivery. Our findings suggest that the ACE2 protein expression changes are the result of placental ACE2 shedding mediated by ADAM17 in response to maternal SARS-CoV-2.

The dynamic nature of placental ACE2 expression that we have noted in our study could account for the variation of reports identifying the presence or absence of ACE2 within trophoblast epithelial cells 38 . Subsequent studies [25] [26] [27] have strongly substantiated pre-pandemic publications 24 for the presence of ACE2 the maternal fetal interface. The 3 rd -Tri COVID-specific increase in ACE2 mRNA could be due to J o u r n a l P r e -p r o o f compensatory upregulation of ACE2 transcripts in response to the active shedding process in this disease state. Recently, a truncated form of human ACE2, namely delta ACE2 has been reported to be upregulated by interferons 39 . Our primer and probe set flanks only the region in the prototype ACE2, therefore the expression we detected is exclusive of delta ACE2. Additionally, there were no significant differences in the maternal serum estradiol between our patient groups, further suggesting influences other than hormonal signaling affecting the dynamic expression of placental ACE2 in these pregnancies.

Ongoing evaluation of other influences on ACE2 post-translational regulation and ADAM17 activity will be required in future studies to characterize the mechanisms governing this process more clearly. Multiple studies have identified signs of placental inflammation in pregnancies affected by acute maternal SARS-CoV-2 including significant fibrosis 25, 33 , increased mononuclear cell infiltrates 40 and upregulation of interferon response genes 31, 41, 42 . Thus, pro-inflammatory cytokine regulation and immune response signaling pathways will be a key area of focus for future analysis of ACE2 and ADAM17 regulation in these tissues.

Consistent with previous studies [25] [26] [27] we also found that ACE2 was not expressed in the endothelium of fetal blood vessels in Control or COVID placental tissues. These findings highlight an important contrast in the pulmonary and placental responses to SARS-CoV-2. In healthy lung tissue, the pulmonary endothelium has minimal expression of ACE2 but in patients with fatal COVID-19, ACE2 expression is increased on the pulmonary endothelium 43 . Type I interferons alpha and beta induce endothelial ACE2 expression, subsequently facilitating SARS-CoV-2 endothelial invasion in vitro 43 . In stark contrast with the lung, the lack of ACE2 on the fetal endothelium in COVID placental tissues further supports the likely central role of ACE2 regulation as a protective countermeasure against perinatal

This work is also the first study to evaluate serum ACE2 levels in pregnant women affected by COVID-19. Serum ACE2 levels are increased in pregnancy relative to non-pregnant women and further relative increases have been associated with small for gestational age (SGA) fetal growth 44 . While serum ACE2 in non-pregnant adult COVID-19 patients has been widely evaluated [45] [46] [47] , no studies to date have J o u r n a l P r e -p r o o f characterized maternal serum ACE2 content in pregnancies with maternal SARS-CoV-2 infection.

While our combined data suggest that the increase in maternal serum ACE2 in acute 3 rd Tri COVID pregnancies could be the result of increased ACE2 placental shedding, the source of circulating maternal serum ACE2 levels in these pregnancies (lung, placenta or both) requires further investigation.

This study has several limitations. First, the cohort is a small sample size and the gestational evaluation only spanned infections in the 2nd and 3 rd (but not 1 st ) trimesters of pregnancy. Additionally, as stated above, soluble ACE2 in maternal serum cannot be directly identified as placental in origin and could be derived from multiple sources. Ongoing mechanistic studies, including relevant animal models, are needed to evaluate ACE2 expression relative to maternal COVID-19 infection in all trimesters of pregnancy, to clarify the placental origin of maternal serum ACE2, and to functionally correlate ADAM17 activity with ACE2 placental regulation. Further, the impact of these alterations in placental ACE2 on maternal and infant physiology will require close long-term follow up.

In conclusion, our work highlights a previously unrecognized dynamic expression of ACE2 in the human placenta which can be modulated by the timing of maternal SARS-CoV-2 infection in pregnancy relative to delivery. Taken together our data provide support for the growing body of evidence on the importance of ACE2 regulation in the placental response against SARS-CoV-2. The human placenta has many functional and structural parallels with the human lung 48 , and thus continues to be an important primary human tissue for identification of key targets to combat COVID-19 pathogenesis. Error bars: +/-standard error of the mean. * p < 0.05, ** p < 0.01 *** p < 0.001 P-values for continuous variables were generated using ANOVA test (indicated by ‡), or Kruskal Wallis rank test (indicated by §). P-values for categorical variables were generated using the Fisher exact tests. 

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We would like to thank the Department of Pathology at Boston Medical Center (BMC); the Boston University (BU) School of Medicine Cellular Imaging Core, the BU Analytical Instrumentation Core and the Maxwell Finland Laboratory for Pediatric Infectious Disease at BMC, particularly lab manager Yazdan Dasthagrasaheb, and laboratory technician Loc Truong. We would also like to thank the staff on the BMC Labor and Delivery and Postpartum Units, as well as the nursing and physician staff of the nursery and NICU at BMC who assisted with maternal blood sampling and subject recruitment.