Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

Abstract

Despite similar infection rates, COVID-19 has resulted in more deaths in men than women. To understand the underlying mechanisms behind this sex-biased difference in disease severity, we infected K18-human angiotensin converting enzyme 2 (ACE2) mice of both sexes with SARS-CoV-2. Our study revealed a unique protein expression profile in the lung microenvironment of female mice. As a result, they were less vulnerable to severe infection, with higher ACE2 expression and a higher estrogen receptor α (ERα)/androgen receptor (AR) ratio that led to increased antiviral factor levels. In male mice, inhaling recombinant ACE2 neutralized the virus and maintained the ERα/AR ratio, thereby protecting the lungs. Our findings suggest that inhaling recombinant ACE2 could serve as a decoy receptor against SARS-CoV-2 and protect male mice by offsetting ERα-associated protective mechanisms. Additionally, our study supports the potential effectiveness of recombinant ACE2 therapy in human lung organoids infected with the Delta variant.

Keywords: Biological sciences; Biology of gender; Immune response; Virology.

Graphical abstract

Figure 1

SARS-CoV-2 infection in male and female k18-hace2 mice over 7 days post-infection (dpi) (A) Body weight loss in response to intranasal inoculation of SARS-CoV-2 (4 × 10⁵ TCID50/50μL/mouse). ∗p < 0.05 vs. male. Data represented in median ± IQR. (B) Lung injury scoring assessed on a scale of 0–4 for each of the following criteria: 1) neutrophil numbers in the alveolar space, 2) alveolar septal thickening, 3) number of hyaline membranes, 4) alveolar hemorrhage, and 5) cellular hyperplasia. ∗p < 0.05 vs. Ctrl and male. Data represented in mean ± SEM. (C) Viral RNA levels in oropharyngeal swabs at 2, 4, and 6 dpi. ∗p < 0.05 vs. male. Data represented in median ± IQR. (D–G) Copy numbers of E-gene and RdRp-gene in multiple organ tissues. ∗p < 0.05 vs. Ctrl. Data represented in mean ± SEM. (H) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (I) Protein expression in lung tissue detected by western blots in vehicle control (C) and at 7 dpi. (J) Concentration of soluble RAGE protein in plasma in vehicle control and at 7 dpi. ∗p < 0.05 vs. other groups. Data represented in mean ± SEM. (K) Correlation of protein levels of ACE2 and ERα in lung tissue of male mice before and after infection at 7 dpi. (L) Estrogen receptor alpha (ERα)/androgen receptor (AR) ratio assessed from western blot analysis in relation to β-actin loading control. ∗p < 0.05 vs. male. Data represented in mean ± SEM. (M) Concentration of 17β Estradiol in lung tissue homogenates in vehicle control and at 7 dpi. ∗p < 0.05 vs. male. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S1).

Figure 2

Proteomic profiles in male and female mice at 7 dpi (A) A volcano plot illustrates significantly different abundant proteins. The -log10 (Benjamini-Hochberg corrected p value) is plotted against the log2 (fold change: female/male) at baseline. (see also Figure S2). (B and C) Volcano plots illustrate significantly different abundant proteins. The -log10 is plotted against the log2 (fold change: 7 dpi/Ctrl) in male and female mice, respectively. The six unique proteins present in female but not in male mice before and at 7 dpi of SARS-CoV-2 are also shown. (D) Changes of ERα-associated proteins by at least 10% at 7 dpi compared to control.

Figure 3

Inhalation of rACE2 attenuates SARS-CoV-2 infection, replication, and lung injury and recovers expression of endogenous ACE2 and ERα in K18-hACE2 male mice at 7 dpi (A) Study scheme. Mice were intranasally inoculated with SARS-CoV-2 (4 × 10⁵ TCID50/50μL/mouse) and 48 h later received daily nebulization of rACE2 (12 mg/kg) or vehicle control solution for 5 days. (B and C) Copy numbers of viral envelope (E) gene by RT-qPCR and titers (TCID50) in lung tissue in the SARS-CoV-2 infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (D) Gene expression of IL-6 in lung tissue in the infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (E) Concentration of soluble RAGE protein in plasma in infection alone and rACE2-treated mice. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (F) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (G) Representative lung histology by H&E staining. Arrows indicate neutrophil infiltration in the alveolar space. Lung injury scores from 6 animals per group are also shown. ∗p < 0.05 vs. naive control (C), ♰p < 0.05 vs. SARS-CoV-2 alone & (C). Data represented in mean ± SEM. (H and I) Protein expression in lung tissue detected by western blots in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C & rACE2-treated groups. Data represented in mean ± SEM. (J and K). Fold changes in the concentrations of 17β Estradiol and ERα/AR ratio assessed from western blot analysis in relation to β-actin loading control in lung tissue in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C and SARS-CoV-2, respectively. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S3).

Figure 4

Nebulization of rACE2 partially restores the ERα-associated signaling proteomic profiles in lung tissue at 7 dpi Proteomic analysis revealed that treatment with rACE2 resulted in an increase in 31 ERα-associated proteins and a decrease in 10 proteins. Of note, 16 of these proteins (highlighted in purple) were also found in female mice without rACE2 treatment at 7 dpi.

Figure 5

Treatment with rACE2 attenuates Delta variant replication and viral load and decreases cytopathic effects in human lung organoids (A) Human lung organoids expressing pro-surfactant protein C (SPC) and epithelial cell adhesion molecule (EpCAM). Scale bars, 20 μm (main images). (B) Representative images of human lung organoids in naive control (Ctrl, upper panel), Delta variant-challenged (middle panel), and treated with a pre-mixture of rACE2 (400 μg/mL) and Delta variant (1.8 × 10⁷ TCID50/mL) for 30 min (lower panel). The expression of ACE2 (red) and viral nucleocapsid protein (green) were detected at 4 h post-infection. Scale bars, 5 μm. (see also Figure S4). (C) Levels of viral envelope (E) gene RNA were assessed by qRT-PCR after treatment with various concentrations of rACE2 in human lung organoids at 3 dpi. Supernatants of the Delta variant-infected lung organoids were collected and used to infect Vero E6 cells for TCID50 assay. ∗p < 0.05 vs. Mock; ♰ p < 0.05 vs. Delta variant alone. Data represented in mean ± SEM. (D) Representative images of cytopathic effects of human lung organoids in Delta variant infection and rACE2-treated groups at 3 dpi. Scale bars, 1000 μm (main images) and 400 μm (magnified). Data presented are representatives of three independent experiments.

Figure 6

Proposed mechanisms underlying biological sex differences in outcomes of SARS-CoV-2 and Delta variant infections and the therapeutic potential of inhaled ACE2 (A) Under physiological conditions, ACE2 catalyzes the conversion of Ang II to Ang1-7 for organ protection. The ACE2 gene is located at X chromosome p22.2, where genes are known to escape X-inactivation, contributing to phenotypic differences of ACE2 between biological sexes. Membrane expression of ACE2 can be upregulated by estrogens through estrogen receptor α (ERα), which exerts anti-inflammatory properties and further enhances organ protection. In contrast, androgen receptor (AR) can cleave ACE2 and mediate pro-inflammation and tissue injury.. (B) During SARS-CoV-2 infection, the virus binds to and decreases host cell surface ACE2. For a given infection, the reduction of endogenous ACE2 expression is associated with a dramatic decrease in ERα expression, resulting in loss of organ protection in males. In contrast, expression of endogenous ACE2 was sustained in females, potentially due to the extra X-linked ACE2 and well-maintained ERα expression, leading to compensatory mechanisms and persistent organ protection. (C) Inhalation of rACE2 can restore the balance of endogenous ACE2 and ERα by blocking or attenuating SARS-CoV-2 from binding to membrane ACE2 in males.