key: cord-0788269-jg8r0jel
authors: Kala, Smriti; Meteleva, Ksenia; Serghides, Lena
title: ACE2, TMPRSS2 and L-SIGN expression in placentae from HIV-positive pregnancies exposed to antiretroviral therapy–implications for SARS-CoV-2 placental infection
date: 2021-04-21
journal: J Infect Dis
DOI: 10.1093/infdis/jiab166
sha: 907614f9d33c2bb4e429a286dff8b1fca4a7d076
doc_id: 788269
cord_uid: jg8r0jel

BACKGROUND: SARS-CoV-2 binding receptor ACE2 and the spike protein priming protease TMPRSS2 are co-expressed in human placentae. It is unknown whether their expression is altered in the context of HIV infection and antiretroviral therapy (ART). METHODS: We compared mRNA levels of SARS-CoV-2 cell-entry mediators ACE2, TMPRSS2 and L-SIGN (an alternative entry receptor) by qPCR in 105 placentae: 45 from pregnant women with HIV (WHIV) exposed to protease inhibitor (PI)-based ART, 17 from WHIV on non-PI-based ART, and 43 from HIV-uninfected women. RESULTS: ACE2 levels were lower, while L-SIGN levels were higher in placentae from WHIV on PI-based ART as compared to those on non-PI-based ART and to HIV-uninfected women. TMPRSS2 levels were similar between groups. Black race was significantly associated with lower expression of ACE2 and higher expression of L-SIGN. ACE2 levels were significantly higher in placentae of female fetuses. DISCUSSION: We have identified pregnant women of Black race and WHIV who are on PI-based ART to have relatively lower expression of placental ACE2 than those of White race and HIV-uninfected women. This effect may potentially contribute to altered susceptibility to COVID-19 in these women, either favorably; by reduced viral entry, or detrimentally; by loss of ACE2 protection against hyperinflammation.

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, which has posed a serious threat globally [1, 2] . Data are emerging on the clinical manifestations of in pregnant women. SARS-CoV-2 infection during pregnancy is associated with increased risk of preterm labor, and babies born to infected mothers have a higher risk of admission to the neonatal unit [3] [4] [5] [6] .

There are sporadic reports of miscarriage, stillbirth, fetal demise, and neonates testing positive for the virus [7] [8] [9] . The risk of SARS-CoV-2 placental infection seems to be low [10] [11] [12] [13] , although reports of electron microscopy observations of virions invading the syncytial layer [14] [15] [16] , and presence of strong staining of SARS-CoV-2 nucleocapsid/spike glycoprotein in the syncytial layer [16] [17] [18] [19] , have contributed to growing evidence that SARS-CoV-2 can infect the placenta.

The placenta also seems to be susceptible to the effects of maternal COVID-19 disease, even in the absence of detectable or very low levels of SARS-CoV-2 mRNA or protein in the placenta [8, [20] [21] [22] [23] . This is evident from histopathological abnormalities such as villous fibrin deposition, maternal vascular malperfusion, fetal vascular malperfusion, and villitis/intervillositis observed in placentae from women with even mild COVID-19 disease [8, [20] [21] [22] [23] [24] . Based on current evidence, the rate of SARS-CoV-2 placental infection is considered low [10] [11] [12] [13] ; although it appears that there is considerable potential for SARS-CoV-2 to affect placental function and fetal development [25] .

SARS-CoV-2 invades host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor [26] [27] [28] , a component of the renin-angiotensin system (RAS), which is a critical regulator of blood pressure, electrolyte balance, and fluid homeostasis [29] [30] [31] . Many components of RAS including ACE2 are upregulated in normal pregnancy [32, 33] . Upregulation of ACE2 mediates conversion of angiotensin IIa vasoconstrictor, to angiotensin-(1-7)a vasodilator, and contributes to relatively low blood pressures in pregnancy [34] . Upon binding ACE2, SARS-CoV-2 causes its downregulation, A c c e p t e d M a n u s c r i p t 5 enhancing RAS imbalance with increased angiotensin II relative to decreased angiotensin-(1-7), which can cause vasoconstriction, inflammation, and coagulopathy [35, 36] .

Much of the literature on how ACE2 levels regulate the pathogenesis of COVID-19 is conflicting. While many authors argue that ACE2 is the underlying reason behind many of the risk factors for severe COVID-19 [37] [38] [39] [40] , there is a growing body of literature which argues that ACE2 upregulation is a protective factor for SARS-CoV-2 outcomes due to its role in limiting the potent vasoconstrictive effect of angiotensin II [36, [41] [42] [43] [44] [45] [46] . Therefore, ACE2 expression may have paradoxical effects, aiding SARS-CoV-2 infection, yet conversely limiting viral pathogenicity. Further studies are needed to elucidate the precise effects that altered ACE2 expression has on the acquisition of SARS-CoV-2 infection and associated severity in COVID-19.

Upon ACE2 binding, SARS-CoV-2 employs the host serine protease TMPRSS2 for spike protein priming facilitating viral fusion and cellular infection [26] . In humans, ACE2 and TMPRSS2 genes are expressed in the placenta throughout the three trimesters of pregnancy [47] [48] [49] , with the highest mRNA expression in the first trimester and decreasing expression with advancing gestation [50, 51] . In two recently published reports investigating localization of ACE2 and TMPRSS2 in COVID-19-exposed term placentae, the ACE2 receptor was consistently localized within the outer syncytiotrophoblast layer of chorionic villi, whereas TMPRSS2 was reported to be absent or only present weakly in the villous endothelium and rarely in the syncytiotrophoblast layer [19, 52] . In spite of the relative absence of TMPRSS2, all 15 placentae in a study tested positive for SARS-CoV-2 infection, and there were five cases of fetal transmission [19] . This suggests that the SARS-CoV-2 virus may be using alternative cellular entry pathway molecules to enter the placenta. It has been identified previously that SARS-CoV, the closely related coronavirus responsible for the SARS outbreak, uses C-type lectins DC-SIGN (also known as CD209) and/or L-SIGN (also known as CLEC4M) as independent receptors, or as enhancer factors that facilitate ACE2 mediated virus infection [53] [54] [55] [56] . A study demonstrated that L-SIGN is endogenously expressed in human endothelial cells and mediates SARS-CoV-2 entry and infection [57, A c c e p t e d M a n u s c r i p t 6 58 ]. Recently, a preprint article has established that the N-terminal domain (NTD) of the spike protein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN. Serum samples from SARS-CoV-2 -infected patients were found to contain antibodies against NTD and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells [59] . L-SIGN also serves as an attachment receptor for other viruses such as HIV [60] .

Emerging data indicate that HIV infection may be associated with increased risk of COVID-19 diagnosis [61] and people with HIV may be at a slightly higher risk of death from COVID-19 [62] [63] [64] [65] [66] . The presence of comorbidities, a low CD4 cell count, and lack of an effective antiretroviral therapy contribute to the risk of severe COVID-19 outcomes among people living with HIV [67] . Currently there are no data on the pathogenesis of SARS-CoV-2 in pregnant women with HIV (WHIV), as well as the risk of vertical transmission in this population. Furthermore, it is not known whether the expression of SARS-CoV-2 cellentry mediators is altered in placentae of WHIV exposed to antiretroviral therapy (ART). We previously reported that pregnant WHIV who received protease inhibitor (PI)-based ART had higher levels of estradiol in the maternal and umbilical cord plasma [68] . As estradiol is known to downregulate the expression of ACE2 [69, 70] , we hypothesized that WHIV exposed to PI-based ART have lower placental expression of ACE2. Here, we compared the gene expression pattern of SARS-CoV-2 cell-entry mediators:

ACE2, TMPRSS2 and L-SIGN, in term placentae of WHIV exposed to PI-ART, non-PI-ART, and HIVuninfected women. Since COVID-19 has disproportionally affected racial and ethnic communities [71] , we further explored associations between placental expression of SARS-CoV-2 cell-entry mediators and race.

Finally, as the placenta is an organ shared by mother and fetus, we also explored the influence of fetal sex on placental SARS-CoV-2 cell-entry mediator expression.

A c c e p t e d M a n u s c r i p t 7

Placentae included in this study were collected from women recruited to the Angiogenesis and Adverse Pregnancy Outcomes in Women with HIV (AAPH) cohort (recruited in Toronto, Canada). Details on the AAPH cohort have been published previously [72] . Briefly, participants were >18 years, with singleton pregnancy. Exclusion criteria included pre-existing hypertension, diabetes, renal, autoimmune, or collagen vascular disease, active opportunistic infection for the WHIV, BMI>40, current illicit or recreational drug use. None of the women were current tobacco smokers or had alcohol use disorder. All 

This study was approved by the Institutional Research Ethics Board at University Health Network (REB #20-5526) and was performed in accordance with the Tri-Council Policy Statement on Ethical Conduct for Research Involving Humans. All participants gave written informed consent for the AAPH study and for inclusion of their samples and data into a biobank program to support studies in HIV and pregnancy.

Placenta samples were collected immediately after delivery. Placental core sections were sampled from three sites on the maternal surface, rinsed in phosphate buffered saline, further dissected into smaller pieces, and immersed in Allprotect tissue reagent (Qiagen). Samples were stored at -80ºC until processing.

A c c e p t e d M a n u s c r i p t 8

Total RNA was isolated from the placental tissue using the mirVana miRNA Isolation Kit (AM1560, Thermo Fisher Scientific) per the manufacturer's protocol. RNA quality and concentration were determined using the Nano-Drop1000 Spectrophotometer (Thermo Fisher Scientific). 10 µg of total RNA was treated with DNase I, RNase-free (EN0521, Thermo Fisher Scientific), followed by addition of 5mM EDTA and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Table 1 . Relative expression of target genes was obtained using the 2CT method [73] .

For demographic and clinical data medians with interquartile ranges (continuous variables) or frequencies (categorical variables) were calculated and compared using Mann-Whitney U-test or Fisher's exact test, respectively. ACE2, TMPRSS2, and L-SIGN levels were log-transformed and differences between groups were assessed using Kruskal-Wallis test with Dunn's multiple comparison post-test, or Mann-Whitney Utest, as appropriate. Correlations were assessed using Pearson r test. Regression analysis was used to examine relationships between log e -transformed ACE2, TMPRSS2, or L-SIGN and ART-exposure status (categorized as none, PI-ART, non-PI-ART), race (categorized as Black, White, or other), and fetal sex (female or male). Statistical analyses were performed using GraphPad Prism v5.0 and Stata v13.0.

A c c e p t e d M a n u s c r i p t 9

We included 105 placentae from the AAPH cohort (recruited in Toronto, Canada), 43 (41%) from women without HIV (control group), and 62 (59%) from WHIV on ART. Of WHIV, 45 (72%) taking a PI-based regimen, nine (15%) a NNRTI-based regimen, and eight (13%) an INSTI-based regimen. For all analyses, placentae exposed to NNRTI or INSTI regimens were grouped together in the non-PI-based ART group.

Demographic information is shown in Table 1 . Maternal age, maternal pre-pregnancy BMI, race, mode of delivery, and fetal sex were similar between groups. All placentae from the HIV-uninfected group were delivered at term, while 56 (90%) of the HIV-positive group were delivered at term and 6 (10%) were delivered pre-term. The median gestational week at birth was 40 for the HIV-uninfected group and 39 for the HIV-positive group. HIV plasma viral load was below detectable limits for 52 (84%) of WHIV, and unavailable for two (3%) women. Median CD4+ T-cell count at time of recruitment for the WHIV was 565 cells/mm 3 . CD4+ T-cell count was below 250 cells/mm 3 for four (6.4%) women and unavailable for one (2%) woman.

We have previously shown that estradiol levels are elevated in pregnancies exposed to PI-based ART, but not in those exposed to NNRTIs or INSTIs [68, 74, 75] ]. Given that ACE2 expression levels have been shown to be influenced by estradiol levels [69, 70] , we hypothesized that placentae from women exposed to PI-based ART will have lower levels of ACE2. Compared to the control group, ACE2 levels were significantly lower in placentae exposed to PI-based ART (median (interquartile range (IQR)) of log etransformed values in arbitrary units: -0.56 (-1.03 -0.11) for PI-based ART vs. 0.12 (-0.28 -0.50) for control, p<0.01) (Figure 1 ). ACE2 levels were similar between the control group and the group exposed to non-PI-based ART. We next explored if maternal estradiol levels measured between gestational week 33-37 correlated with placental ACE2 expression levels. Estradiol levels were only available for 30 In contrast to ACE2, expression levels of L-SIGN were significantly higher in placentae exposed to PIbased ART compared to those exposed to non-PI-based ART and compared to controls (median (IQR) of Table S2 ) with the exception of pre-pregnancy BMI, which was significantly lower in White women compared to Black women. Differences in ACE2 and L-SIGN levels between Black and White women remained significant when we adjusted for maternal BMI.

We also examined if fetal sex was associated with receptor expression levels ( Figure 4 ). L-SIGN and TMPRSS2 levels did not differ by sex. However, ACE2 levels were significantly higher in placentae associated with a female fetus compared to those associated with a male fetus (median (IQR) of log e - A c c e p t e d M a n u s c r i p t 11 We did not observe significant associations between ACE2, L-SIGN, or TMPRSS2 levels and maternal age, maternal BMI. In WHIV we did not observe significant associations between ACE2, L-SIGN, or TMPRSS2 levels and viral load, CD4 count, or preterm birth. In multivariable regression analysis, exposure to PI-based ART, race, and fetal sex, all remained significantly associated with ACE2 expression levels (Table S2) . Similarly, both exposure to PIs and race remained significantly associated with L-SIGN expression levels (Table S3) .

Emerging evidence points to HIV as a potential risk-factor for death from COVID-19 [62] [63] [64] [65] [66] , yet the impact of HIV infection and ART on the clinical presentation, birth outcomes and placental pathology of pregnancies complicated by COVID-19 remains to be investigated [76, 77] . To better understand the risk of SARS-CoV-2 placental infection in WHIV treated with ART, we performed an integrated analysis of the gene expression of SARS-CoV-2 cell-entry mediators in term placentae of WHIV who received ART compared to the expression in placentae from HIV-uninfected women. We found lower expression of ACE2 and higher expression of L-SIGN in placentae from WHIV on PI-based ART compared to those from HIV-uninfected women, while ACE2 and L-SIGN levels were similar in placentae from WHIV on non-PI-based ART compared to HIV-uninfected women. TMPRSS2 levels were similar across all groups.

In agreement with previous reports that ACE2 expression levels are influenced by estradiol levels [69, 70] , we observed a negative correlation between late third trimester maternal estradiol levels and placental ACE2 levels in WHIV on PI-based ART. This correlation was not observed in the HIVuninfected group. We also observed differential expression profiles for ACE2 and L-SIGN based on race, with Black race significantly associated with lower placental expression of ACE2 and higher expression of L-SIGN compared to White race. TMPRSS2 levels did not vary by race.

Our data may help stratify the risk of SARS-CoV-2 placental infection in pregnant WHIV taking ART. In the event of placental infection due to severe maternal COVID-19, we would speculate that the baseline lower ACE2 levels may emerge as unfavorable for the pregnancy due to further downregulation of ACE2 by SARS-CoV-2 binding and loss of the ACE2 vasodilatory function in the local placental RAS system. This RAS imbalance may result in placental inflammation and vasoconstriction, which, over the course of pregnancy may adversely affect the developing fetus, as reported previously, outside the context of COVID-19 [79] [80] [81] [82] [83] . However, these mechanistic pathways, and their relationship to outcomes in maternal SARS-CoV-2 infection, remain to be examined.

In healthy term placentae, ACE2 has been detected by immunohistochemistry in syncytiotrophoblasts, cytotrophoblasts, and fetal capillary endothelium [84, 85] . Placental expression of L-SIGN is also localized to the fetal capillary endothelium [60] . Therefore, co-localization of these two SARS-CoV-2 receptors in the fetal endothelium may potentiate vertical transmission. It is possible that the significantly higher expression of L-SIGN in pregnant women of Black race and WHIV who are on PI-based ART may increase their susceptibility to vertical transmission of SARS-CoV-2. However, it may manifest only in sporadic cases of placental infection in which the syncytial barrier might be breached perhaps due to placental inflammation. These hypotheses need to be evaluated in research studies.

A c c e p t e d M a n u s c r i p t 13 Mortality from COVID-19 has been particularly high in African-American communities [71, [86] [87] [88] [89] .

Higher mortality among Black people could be due to higher prevalence of the known risk factors for COVID-19 complications, such as hypertension, diabetes, obesity, and cardiovascular disease among the Black ethnic group, as well as socioeconomic factors [90] [91] [92] [93] . Furthermore, a study found that Black people with HIV were more likely to die from COVID-19 than other people with HIV [63] . A recent report states that there is a genetic predisposition for lower expression levels of ACE2 in African populations [94] , which is consistent with our data reporting lower placental expression of ACE2 in Black pregnant women. An ACE2 polymorphism found to be associated with cardiovascular and pulmonary conditions was reported in the African/African-American population [95] . Hence, ACE2 and its genomic variants might influence inter-individual variability in disease susceptibility and severity of COVID-19.

We also detected significantly higher ACE2 levels in placentae of female fetuses compared to those of male fetuses. This finding is consistent with a study reporting higher ACE2 expression in different tissues in Asian females as compared to males [96] , although the association between sex and ACE2 expression is still debatable.

One strength of our study is the simultaneous measurement of the expression levels of all major SARS-CoV-2 cell-entry mediators -ACE2, TMPRSS2 and L-SIGNin a large number of term placentae from WHIV compare to placentae from HIV-uninfected women with similar demographics. Further, the original cohort excluded women with hypertension, diabetes, or obesity and did not include recreational or illicit drug users or current tobacco smokers. While these characteristics limit the influence of potential confounding factors in our findings, they also limit the external validity of our data. A limitation of our study is that we could not assess the placental expression of ACE2, TMPRSS2 and L-SIGN in the first and second trimesters of pregnancy in WHIV treated with ART. Another limitation is that we were not Overall, our data show that pregnant women of Black race and WHIV who are on PI-based ART have lower expression levels of ACE2 but higher levels of L-SIGN, which can alter their susceptibility to SARS-CoV-2 placental infection. Once infection is acquired, clinical manifestations might be worse in these women as they may be at a higher risk of placental abnormalities due to RAS dysregulation, leading to pregnancy complications and possibly, trans-placental transmission. These data may better inform clinical considerations surrounding risk stratification and prevention approaches for SARS-CoV-2affected pregnancies exposed to HIV and ART. However, our data should be interpreted cautiously, because there may be other undefined pathways regulating SARS-CoV-2 placental infection. Caroline Dunk for helpful input into the study, and Dr. Monica Guzman Lenis for technical assistance and critical review of the manuscript. We also thank Dr. Lisa Bebell for critical review of the manuscript. We thank all the women who participated in the AAPH cohort, and the AAPH team for their contributions to the study. 

This study was funded by the Canadian Institutes of Health Research (PJT148684). M a n u s c r i p t M a n u s c r i p t 22 Hispanic).

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A c c e p t e d M a n u s c r i p t 16 A c c e p t e d M a n u s c r i p t 18 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t