2023 Volume 46 Issue 12 Pages 1842-1845
Women are less susceptible than men to coronavirus disease 2019 (COVID-19), which might be due to the female steroid hormone 17β-estradiol. We hypothesized that 17β-estradiol removes the soluble portion of angiotensin-converting enzyme 2 (sACE2) to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds in host cells and that sACE2 then binds to the virus, thereby reducing the infectivity. In the present study, VeroE6/serine protease 2 transmembrane protein (TMPRSS2) cells were infected with pseudo SARS-CoV-2 viruses and used as our model system. This infectivity was reduced by the application of 17β-estradiol. After applying 17β-estradiol to the VeroE6/TMPRSS2 cells, we used a sandwich enzyme-linked immunosorbent assay (ELISA) to measure the sACE2 concentration in the culture medium. Our findings revealed that 17β-estradiol removes sACE2 from VeroE6/TMPRSS2 cells. Furthermore, the pseudo SARS-CoV-2 viruses were incubated in culture medium with ACE2 collected from 17β-estradiol-treated VeroE6/TMPRSS2 cells, and the viruses were measured with an ultrasensitive ELISA using anti-spike protein antibodies. The amount of spike proteins decreased according to the concentration of 17β-estradiol applied. These results clearly demonstrated that the soluble portion of ACE2, which was removed from 17β-estradiol-treated VeroE6/TMPRSS2 cells, bound to the spike proteins of SARS-CoV-2, thereby reducing COVID-19 infectivity.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the single-stranded RNA virus causing coronavirus disease 2019 (COVID-19).1) The spike protein on the viral envelope binds to the angiotensin-converting enzyme receptor 2 (ACE2) on the host cell.2) ACE2 is expressed in most organs but abundantly in lung respiratory epithelium, and functions systemically to lower blood pressure. The spike protein is activated by the serine protease 2 transmembrane protein (TMPRSS2) on the host cell, which allows for internalization of the viral particles.3) The viral RNA is subsequently released into the host cytoplasm, from where it moves to the host ribosome for the production of new virions to infect other cells.
The infectivity and severity of COVID-19 varies between men and women, with women being less susceptible and less severely infected than men.4) A detailed survey in the United States showed that, within race groups, men have a higher COVID-19 mortality rate than women and that black men have the highest COVID-19 mortality rate of all race-sex groups.5) Several possible factors may contribute to this difference, including diet, obesity, and smoking habit.6) Another important factor is the sex difference in the strength of the immune response. The immune response to infection tends to be stronger in women than in men.7) Some studies have looked at the X chromosome to explain the differences in the immune response.8,9) Due to higher expression of a key innate immunity gene (e.g., Toll-like receptor 7 (TLR7) gene) located on the X chromosome, women are able to synthesize more antiviral proteins and interferons in dendritic cells, resulting in a more robust immune system capable of preventing severe SARS-CoV-2 viral disease.
The difference in sex hormones has also been considered. For example, 17β-estradiol, which is more abundant in women, is thought to play an important role in the prevention of SARS-CoV-2 infection, although its mechanism of action is not well understood. Lemes and colleagues showed that 17β-estradiol treatment of VeroE6 cells derived from African green monkey kidney epithelial cells reduced the viral load of SARS-CoV-2 infection.10) SARS-CoV-2 infection per se leads to increased gene expression of ACE2 and TMPRSS2 in VeroE6 cells, which could generate a cycle of viral infection in the host cells.
In the present study, to clarify the infection reduction mechanisms by 17β-estradiol application to VeroE6/TMPRSS2 cells, we hypothesized that 17β-estradiol binds to ACE2 and cleaves ACE2 into soluble ACE2 and a remaining membrane portion, thereby reducing SARS-CoV-2 infection. That is, we provide evidence that soluble ACE2 generated by 17β-estradiol can serve as a defense mechanism against SARS-CoV-2 infection.
The present study was approved by the Office of Research Ethics of Waseda University (Genetic Recombination Experimentation, WD22-006).
Reagents and ChemicalsDulbecco’s Modified Eagle’s Medium (low glucose) and 17β-estradiol (14541-74) were purchased from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (S00HL 10003) was purchased from BioWest (Nuaillé, France). Antibiotic G 418 sulfate solution (108321-42-2) was purchased from InvivoGen (Hong Kong, China). Alkaline phosphatase (ALP) and nicotinamide adenine dinucleotide (reduced form, NADH) were purchased from Roche (Basel, Switzerland). Thio-NAD was obtained from Oriental East (Tokyo, Japan). 3α-Hydroxysteroid dehydrogenase (HSD) was purchased from Asahi Kasei Pharma (Tokyo, Japan). 17β-Methoxy-5β-androstan-3α-ol 3-phosphate was synthesized by one of the authors (T.Y.). The first antibody (i.e., capture side, Clone #A1-1C7) and the second antibody (i.e., detection side, Clone #A2-7G6) for SARS-CoV-2 spike proteins were obtained from Hakarel (Osaka, Japan). Pseudo SARS-CoV-2 and pseudo SARS-CoV-2 green reporter (#C1110G) were purchased from Montana Molecular (Bozeman, MT, U.S.A.). Cellstain-Hochest 33258 (23491-45-4) for nuclear staining was purchased from Dojindo (Kumamoto, Japan). VeroE6 cells expressing human TMPRSS2 (JCRB1819) were purchased from JCRB Cell Bank (Osaka, Japan).11) Human soluble ACE2 enzyme-linked immunosorbent assay kit (SK00707-01) was purchased from Aviscera Bioscience (Santa Clara, CA, U.S.A.).
Cell Culture and 17β-Estradiol ApplicationVeroE6/TMPRSS2 were cultured at 37 °C in a 5% CO2 atmosphere using a culture medium comprising Dulbecco’s Modified Eagle’s Medium (low glucose), 10% fetal bovine serum, and 1 mg/mL antibiotic G 418 sulfate solution. During culture, 17β-estradiol was adjusted to a final concentration of 0.1, 1, or 10 µM. The stock solution of 17β-estradiol was prepared using EtOH as a vehicle, and the maximum concentration of EtOH in the 17β-estradiol solutions was 0.5%. Thus, a 0% 17β-estradiol solution means 0.5% EtOH. VeroE6/TMPRSS2 is the best dedicated cell line to perform our experiments. Previous studies showed that VeroE6/TMPRSS2 cells were infected with 10-fold serially diluted SARS-CoV-2 samples, and the infected cells were visualized by indirect immunofluorescence assays. The results showed that VeroE6/TMPRSS2 had about 10-fold more SARS-CoV-2 infected cells than the parental VeroE6 cells. These data suggest that TMPRSS2 may play an important role in SARS-CoV-2 cell entry.11)
Pseudo SARS-CoV-2 Infection AssayVeroE6/TMPRSS2 cells were cultured at an initial concentration of 1.0 × 105 cells/mL. Twenty-four hours later, cells were washed with phosphate buffered saline (PBS), and pseudo SARS-CoV-2 green reporter was added at 1.0 × 104 vg/mL. The cells were placed in an incubator for 1 h, washed with PBS, and observed with FV1000 confocal laser scanning biologic microscope (Olympus, Tokyo, Japan). Acquired images were analyzed using ImageJ (https://imagej.nih.gov/ij/). The cells were also stained by Hoechst 33258 according to the manufacture’s instruction.
Enzyme-Linked Immunosorbent Assay (ELISA) for Soluble ACE2Soluble ACE2 was assayed using a human soluble ACE2 ELISA kit. The standard, samples, and positive control adjusted to 100 µL were added to each well of a 96-well plate in the kit and incubated at room temperature for 2 h. A 100-µL solution of secondary antibody conjugated with biotin in dilution buffer was added to each well and incubated at room temperature for 1.5 h with shaking. Streptavidin (100 µL) conjugated with horseradish peroxidase (HRP) in HRP dilution buffer was added to each well and the plate was incubated at room temperature for 45 min with shaking. To amplify the ELISA signal, 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added to each well and the plate was incubated at room temperature for 20 min with shaking. After reaction of the TMB solution, 100 µL of the stop solution was added to each well. The solution was measured with a microplate reader (SH-1000, Corona Electric, Ibaraki, Japan) at 450 nm. The 450-nm signals were normalized to those at 570 nm.
ELISA with Thio-NAD CyclingAn ELISA with thio-NAD cycling was performed according to the procedures reported previously with slight modification.12,13) Briefly, the capture-side (primary) antibody used for the SARS-CoV-2 spike protein was 1 µg/mL, and the detection-side (secondary) antibody was 100 ng/mL. The target antigen was pseudo SARS-CoV-2 (final concentration of 106 vg/mL) incubated with culture medium of VeroE6/TMPRSS2 cells applied with 17β-estradiol, i.e., culture medium containing sACE2. Pseudo SARS-CoV-2 and the culture medium were mixed for 1 to 2 h prior to the experiments and maintained at room temperature. The primary antibody was added to the microplate wells and incubated at room temperature for 1 h. The microplates were then incubated with 1% bovine serum albumin in Tris-buffered saline at room temperature for 1 h. The antigen was added to each well and the microplates were incubated at room temperature for 1 h with shaking. The secondary antibody conjugated with alkaline phosphatase was then added to the wells and the microplates were incubated at room temperature for 1 h with shaking. To amplify the ELISA signal, thio-NAD cycling solution was added to each well. This thio-NAD cycling solution contained 1.0 mM NADH, 2.0 mM thio-NAD, 0.4 mM 17β-methoxy-5β-androstan-3α-ol 3-phosphate, and 10 U/mL 3α-HSD in 100 mM Tris–HCl (pH 9.0).14,15) In thio-NAD cycling, thio-NADH was measured with a microplate reader at 405 nm. The 405 nm signals were normalized to those at 660 nm.
Statistical AnalysisThe experimental data were obtained by subtracting the mean value of the blank signals from each of the corresponding measured data points. The data are expressed as mean + standard error of the mean (S.E.M.). Significant differences were determined using R (version 4.2.1; https://www.r-project.org/) with p < 0.05 considered significant. Comparisons were made using a one-way ANOVA with a post-hoc Holm test. The ggplot2 package in R calculations adopts outlying points. That is, the upper whisker extends from the hinge to the largest value no further than 1.5× IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5× IQR of the hinge. The data beyond the end of the whiskers are called outlying points and are plotted individually.
Fluorescence images of VeroE6/TMPRSS2 cells infected with pseudo SARS-CoV-2 green reporter were analyzed by ImageJ to evaluate differences in the infectivity of the simulated virus in vitro with and without estradiol treatment (Fig. 1A). The fluorescence intensity of infected cells decreases with an increase in the 17β-estradiol concentration, even though there was no significant stepwise difference in the fluorescence intensity between applications of 0.1, 1, and 10 µM 17β-estradiol. The fluorescence intensity measured by ImageJ decreased with an increase in the concentration of 17β-estradiol (p < 0.01, N = 3 wells each, Fig. 1B).
(A) Fluorescence images after incubation with different concentration of 17β-estradiol. The upper number shows the concentration of 17β-estradiol. In the case of 0 µM, 0.5% EtOH was added as a vehicle. Scale bars: 100 µm. The bottom two blue images show the Hoechst 33258 staining. (B) Change in SARS-CoV-2 infectivity of VeroE6/TMPRSS2 cells. Fluorescence intensity was measured in 256 steps and normalized by the cell numbers. N = at least 114 cells from 3 different photos. For the fluorescence intensity at 255 of 0 µM 17β-estradiol, there are too much data, so many data are lined up horizontally making a straight line.
Changes in sACE2 were assayed with a sandwich ELISA using the culture medium of the VeroE6/TMPRSS2 cells after applying various concentrations of 17β-estradiol (Fig. 2). Increasing the amount of 17β-estradiol (0.1, 1 and 10 µM) increased the amount of sACE (0 vs. 1 µM: p < 0.01 and 0 vs. 10 µM: p < 0.01, N = 4 wells each).
N = 4 wells each.
To confirm that sACE2 prevents the invasion of coronaviruses into cells, pseudo SARS-CoV-2 were incubated with culture medium of VeroE6/TMPRSS2 cells treated with 17β-estradiol, and the viruses were measured by the ultrasensitive ELISA using the anti-spike protein antibodies. The ELISA absorbance is expected to decrease if ACE2 is bound to the spike proteins. The results showed that the ELISA absorbance was significantly reduced with the addition of 17β-estradiol compared to that without (Fig. 3; p = 0.016 for 0 vs. 0.1 µM, p = 0.014 for 0 vs. 1 µM, and p = 0.045 for 0 vs. 10 µM; N = 3 wells each).
After incubating pseudo SARS-CoV-2 with culture medium containing sACE2 that was removed from VeroE6/TMPRSS2 cells treated with 17β-estradiol, the spike proteins on pseudo SARS-CoV-2 were measured with an ultrasensitive ELISA. N = 3 wells each.
The present study showed that the female hormone 17β-estradiol first removes the soluble portion of ACE2 and then this portion binds to SARS-CoV-2, decreasing its infectivity. Sex hormones have long been thought to play a role in infection and severity, but our results clearly demonstrated that 17β-estradiol is a key factor for infection. Thus, our results clarified a mechanism of the sex difference in COVID-19 infectivity. Further investigation into how 17β-estradiol removes sACE2 is necessary. In addition, it is noteworthy that ACE2 was not detected by the amount of its mRNA but by its protein in the present study. We cannot exclude the possibility that 17β-estradiol enhances the cells’ own antiviral activity. However, in Figs. 2 and 3, we believe that sACE2 sufficiently blocks SARS-CoV-2.
Conversely, a male hormone, androgen, may explain the greater susceptibility to COVID-19 in men. Stopsack et al. developed the following scenario.16) The TMPRSS2 gene is a partner in a gene fusion event in solid tumors, i.e., somatic gene rearrangements involving TMPRSS2 with ERG, a member of the ETS family of oncogenic transcription factors. Although ERG is not normally regulated by androgen, the gene fusion juxtaposes the androgen receptor regulatory elements of TMPRSS2 with the ERG gene. As a result, the ERG gene is controlled by androgen receptor signaling and is highly expressed in prostate cancers harboring TMPRSS2-ERG fusion. The presence of TMPRSS2-ERG in prostate cancer and the strong regulation of TMPRSS2 by androgens led us to hypothesize that TMPRSS2 could partially account for the predominance of COVID-19 in men.
This study was supported by a Grant-in-Aid for JSPS Fellows (JP22J12904) to Y.K., JST SPRING (JPMJSP2128) to Y.K., a Grant-in-Aid for Young Scientists (Early Bird) from the Waseda Research Institute for Science and Engineering, Waseda University to Y.K., the JSPS KAKENHI (20H04556) to E.I., and the Waseda University Grant for Special Research Projects (2023Q-004) to E.I. The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.
The authors declare no conflict of interest.
