Influence of Sex and 1,25α Dihydroxyvitamin D3 on SARS-CoV-2 Infection and Viral Entry

Abstract

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the etiologic agent that causes the coronavirus disease (COVID-19) identified in Wuhan, in 2019. Men are more prone to developing severe manifestations than women, suggesting a possible crucial role of sex hormones. 17,β-Estradiol (E2) and 1,25 α dihydroxyvitamin D₃ (calcitriol) act upon gene pathways as immunomodulators in several infectious respiratory diseases. In this study, we aimed to evaluate the influence of E2 and calcitriol on the VSV-based pseudovirus SARS-CoV-2 and SARS-CoV-2 infection in vitro. We infected Vero E6 cells with the recombinant VSV-based pseudovirus SARS-CoV-2 and the SARS-CoV-2 viruses according to the pre-treatment and pre-post-treatment models. The Angiotensin-Converting Enzyme 2 (ACE2) and Vitamin D Receptor (VDR) gene expression did not change under different treatments. The VSV-based pseudovirus SARS-CoV-2 infection showed a significant decrease in the focus-forming unit count in the presence of E2 and calcitriol (either alone or in combination) in the pre-treatment model, while in the pre-post-treatment model, the infection was inhibited only in the presence of E2. Th SARS-CoV-2 infection highlighted a decrease in viral titres in the presence of E2 and calcitriol only in the pre-post-treatment model. 17,β-Estradiol and calcitriol can exert an inhibitory effect on SARS-CoV-2 infections, demonstrating their protective role against viral infections.

Keywords: 1,25α dihydroxyvitamin D3; COVID-19; E2; SARS-CoV-2; calcitriol; estrogen; sex; sex hormones; viral entry.

Figure A1

The two sets of graphs above show the ACE2 (a,b) and VDR (c,d) gene expression on Vero E6 cells also evaluated on non-infected cells at 12 h (mock-infected cells).

Figure A2

The two sets of graphs above show the ACE2 (a,b), and VDR (c,d) gene expression on Vero E6 cells also evaluated on non-infected cells at 24 h (mock-infected cells).

Figure 1

The analysis of the results of the MTT assay: the concentration of the E2 is shown on the x-axis in panel (a), and the calcitriol in panel (b) is expressed on the semi-logarithmic scale, while the y-axis represents the percentage of cell viability normalized to the control.

Figure 2

Vero E6 cell line infected with VSV-based pseudovirus SARS-CoV-2 expressing GFP (MOI 0.1) after 12 h (A) and 24 h (B) of infection visualized through fluorescence microscope. Images were acquired with digital microscope EVOS FLOID (Life Technologies), zoom 100%.

Figure 3

GFP-expressing VSV-based pseudovirus SARS-CoV-2 infection with MOI 0.1 for 12 h in pre-treated setting with different compounds, in particular (A) control (B), DMSO at 1:40,000 dilution (vehicle) (C), E2 at 100 nM (D), calcitriol at 100 nM, and (E) combination of E2 and calcitriol. Images were acquired with digital microscope EVOS FLOID (Life Technologies), zoom 100%.

Figure 4

GFP-expressing VSV-based pseudovirus SARS-CoV-2 viral infection with MOI 0.1 for 12 h in pre-and post-treated setting with different compounds: (A) control (B), DMSO at 1:40,000 dilution (vehicle) (C), E2 at 100 nM (D), calcitriol at 100 nM, and (E) combination of E2 and calcitriol. Images were acquired with digital microscope EVOS FLOID (Life Technologies), zoom 100%.

Figure 5

FFU/mL count after 12 h of infection with VSV-based pseudovirus SARS-CoV-2 at MOI 0.1 in presence of different compounds in pre-treatment model (E2 100 nM, calcitriol 100 nM, and same concentration in combination). p-value cut-off 0.05. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 6

FFU/mL count after 12 h of infection VSV-based pseudovirus SARS-CoV-2 at MOI 0.1 in presence of different compounds in pre- and post-treatment model (E2 100 nM, calcitriol 100 nM, and same concentration in combination). p-value cut-off 0.05. **** p < 0.0001.

Figure 7

SARS-CoV-2 titre after 24 h of infection in presence of different compounds in pre- (a) and pre–post-(b) treatment models (E2 100 nM, calcitriol 100 nM, and same concentration in combination). Although not statistically significant, we observed reduction in viral titre with E2 and calcitriol alone in pre–post-treatment model, while their combination was associated with trend toward increased viral production.

Figure 8

The graph shows the ACE2 gene expression (left y axis) at the baseline (t0) in the presence of the different compounds under study, and the FFU/mL count is shown on the right y axis (red line) after 12 h of infection with the VSV-based pseudovirus SARS-CoV-2 at an MOI of 0.1 in pre-treatment (a) and pre–post-treatment (b) models.

Figure 9

The graph shows the VDR gene expression (left y axis) at the baseline (t0) in the presence of the different compounds under study, and the FFU/mL is shown on the right y axis (red line) after 12 h of infection with the VSV-based pseudovirus SARS-CoV-2 at an MOI of 0.1 in pre-treatment (a) and pre–post-treatment (b) models.

Figure 10

The graph shows the ACE2 gene expression (left y axis) at the baseline (t0) in the presence of the different compounds under study, and the viral titre SARS-CoV-2 (expressed in TCID 50/mL) after 24 h of infection in pre-treatment (a) and pre–post-treatment (b) models is shown on the right y axis (red line).

Figure 11

The graph shows the VDR gene expression (left y axis) at the baseline (t0) in the presence of the different compounds under study, and the viral titre of SARS-CoV-2 (expressed in TCID 50/mL) after 24 h of infection in pre-treatment (a) and pre–post-treatment (b) models is shown on the right y axis (red line).

Figure 12

A heat map showing the increase or decrease in cytokine levels in the supernatant of Vero E6 cells infected by SARS-CoV-2 compared to non-infected ones. Each column of the heat map represents different cytokines, and rows indicate the different treatments in the two models: the pre-treatment and pre–post-treatment. The blue–yellow color code represents low-to-high average cytokine/chemokine/growth factors levels.

Figure 13

The PD-L1 gene expression in pre-treatment (a) and pre–post-treatment (b) models in the presence and absence of a SARS-CoV-2 infection in the Vero E6 cell line. * p < 0.05.