Reduced severe acute respiratory syndrome coronavirus 2 entry factors and enhanced innate immune gene expression in the nasal epithelium of pregnant rats

Abstract

An enigmatic epidemiological feature of the ongoing COVID-19 pandemic is the high rate of asymptomatic infection in pregnant women.1 This is puzzling because systemic immune changes predispose pregnant women to increased severity of respiratory viral infections, especially influenza A.2 A major roadblock in understanding this atypical clinical presentation is the poor characterization of cellular entry factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - angiotensin-converting enzyme 2 (ACE2) and the androgen-sensitive transmembrane protease serine 2 (TMPRSS2) - in the respiratory tract during pregnancy. Motivated by a recent report showing estradiol-mediated downregulation of ACE2 in the airway epithelium,3 we hypothesized that the hormonal changes of pregnancy will decrease the expression of SARS-CoV-2 cell entry factors. Here, we compare their expression and examine the innate immune system in the nasal epithelium of term pregnant (gestational day 20) vs. non-pregnant 2-month old female rats STUDY DESIGN: All experiments were conducted after appropriate institutional approval (protocol ID:19-1071) and comply with ARRIVE guidelines. Briefly, nasal epithelia from euthanized rats (n=9 each) were dissected according to the protocol described by Dunston et al. with modifications.4 Collected samples were assayed for the expression of SARS-CoV-2 entry factors (Ace2, Tmprss2), innate antiviral immune genes that are highly co-expressed with Ace2 (Tnfsf10, Mx1, Nos2),5 and genes involved in SARS-CoV-2 detection and defense (Rig-1, Tlr7, MyD88, Irf7) with TaqMan qPCR (ThermoFisher Scientific, Inc.). In addition, we determined the expression of ACE2 (LS-c763699, LifeSpan BioSciences, Inc.; 1:1000 dilution) and TMPRSS2 (sc-515727, Santa Cruz Biotechnology, Inc.; 1:250 dilution) protein with immunoblots. Finally, we assayed ACE2 enzyme activity with a fluorometric assay (K897-100, Biovision Inc.). We observed a marked downregulation of the expression of Ace2 and Tmprss2 genes (Fig 1A) along with concomitant changes in protein expression (1B), and a significant decrease in ACE2 enzyme activity in the nasal epithelium during pregnancy (1C). Innate immune genes with antiviral function that are highly co-expressed with Ace2 (Tnfsf10, Mx1, Nos2) were significantly elevated in the nasal epithelium from pregnant rats (1D). Similarly, the expression of cytoplasmic (Rig-1) and endosomal viral sensors (Tlr7, MyD88, and Irf7) involved in the detection of SARS-CoV-2 were substantially upregulated with pregnancy (1E). Collectively, our results show a decrease in cell entry factors for SARS-CoV-2 and a surprisingly robust expression of innate immune response genes in the nasal epithelium of pregnant rats. Based on our preclinical findings, we surmise that the high rate of asymptomatic infection in pregnant women is likely due to decreased SARS-CoV-2 tropism secondary to reduced expression of cell entry factors. Our observation of upregulated innate immune defense in the nasal epithelium, in contrast to the immunological indolence at the placental-fetal interface, was unexpected and novel. Considering the exquisite vulnerability of pregnant women to influenza A virus, another single-stranded RNA virus, but not SARS-CoV-2, our findings set the stage for comprehensive characterization of respiratory mucosal immunology in pregnant women to better understand host-pathogen interaction in this unique demographic subset.