A, Scatter plots showing markedly decreased expression of ACE2 and TMPRSS2 in the nasal epithelium during pregnancy. B, Representative immunoblots showing markedly reduced ACE2 and TMPRSS2 protein expression along with quantification as scatter plots. Rat small intestinal lysate was used as positive control and β-actin as the loading control. C, Scatter plot demonstrating a marked reduction in enzymatic ACE2 activity in the nasal epithelium of pregnant rats. D, Scatter plots showing substantial up-regulation of innate immune genes highly coexpressed with ACE2 (TNFSF10, MX1, NOS2). E, Scatter plots showing up-regulation of genes involved with SARS-CoV-2 detection (RIG-1, TLR7, MYD88, IRF7) in pregnant nasal epithelial samples suggesting the possibility of heightened innate immune surveillance at baseline. Expression levels of genes of interest were assayed in duplicate along with 2 endogenous housekeeping control genes (EEF2 and ACTB). All TaqMan primers were acquired from Thermo Fisher Scientific, and thermal cycling was performed in 7500 Fast Real-Time PCR System (Applied Biosystems: Makrogiannis Phil, Foster City, CA). Relative mRNA expression, normalized to the geometric mean of EEF2 and ACTB, was calculated using the 2-ΔΔCT method. Data outliers were eliminated using robust regression and outlier analysis with Q set to 10% and normality of residuals was assessed with D’Agostino-Pearson omnibus test. Normally and nonnormally distributed data were analyzed with Welch’s t test and Mann-Whitney U test, respectively, with P≤.05 accorded statistical significance. Data were analyzed with Prism 8 for macOS (version 8.2.1; GraphPad Software Inc, San Diego, CA) and presented as mean±SEM; aP≤.05; bP≤.01; cP≤.001; and dP≤.0001 (n=9 each for all experiments except western blot where n=6 per group).
ACE2, angiotensin-converting enzyme 2; mRNA, messenger RNA; MYD88, myeloid differentiation primary response 88; NOS2, nitric oxide synthase 2; RIG-1, retinoic acid-inducible gene I; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SEM, standard error of mean; TLR7, toll-like receptor 7; TMPRSS2, transmembrane protease serine 2; TNFSF10, tumor necrosis factor ligand superfamily member 10.
Palanisamy. Reduced SARS-CoV-2 entry factors and innate immune gene expression in the epithelium of pregnant rats. Am J Obstet Gynecol 2021.