Cytokines from SARS-CoV-2 Spike-Activated Macrophages Hinder Proliferation and Cause Cell Dysfunction in Endothelial Cells

Abstract

Endothelial dysfunction plays a central role in the severity of COVID-19, since the respiratory, thrombotic and myocardial complications of the disease are closely linked to vascular endothelial damage. To address this issue, we evaluate here the effect of conditioned media from spike S1-activated macrophages (CM_S1) on the proliferation of human umbilical endothelial cells (HUVECs), focusing on the specific role of interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Results obtained demonstrate that the incubation with CM_S1 for 72 h hinders endothelial cell proliferation and induces signs of cytotoxicity. Comparable results are obtained upon exposure to IFN-γ + TNF-α, which are thus postulated to play a pivotal role in the effects observed. These events are associated with an increase in p21 protein and a decrease in Rb phosphorylation, as well as with the activation of IRF-1 and NF-kB transcription factors. Overall, these findings further sustain the pivotal role of a hypersecretion of inflammatory cytokines as a trigger for endothelial activation and injury in the immune-mediated effects of COVID-19.

Keywords: COVID-19; cytokines; endothelial dysfunction; macrophages.

Figure 1

HUVECs were maintained in growth medium (control) or treated with conditioned medium obtained by incubating monocyte-derived macrophages in the absence (CM_cont) or in the presence of 5 nM S1 (CM_S1). (A) Cell proliferation was determined as specified in Section 2 at the indicated incubation times. Each point represents the media ± SD of three determinations in a representative experiment that, repeated three times, gave comparable results. Right: phase contrast microscopy images of cells treated for 72 h are shown. Bar = 100 µM. (B) After 4 h, the expression of cell cycle inhibitor p21 and p27 mRNA (left) was measured by means of RT-qPCR and expressed as fold change of control cells (=1). Bars are means ± SEM of four independent experiments, each performed in duplicate. * p < 0.05 vs. control with Kruskal–Wallis test. At the indicated times, the expression of p21^WAF1 and phosphorylated Rb proteins was evaluated with Western blot analysis (right), as described in Section 2. Representative blots are shown, along with the mean ± SD of the densitometry analysis of three different experiments. * p < 0.05 vs. control cells with Kruskal–Wallis test. (C) After 48 h treatment, cell death was assessed with flow cytometry upon staining with Annexin V-FITC/Propidium iodide (see Section 2). Representative scatter plots are shown with the indicated % of viable and apoptotic/necrotic cells of 10,000 events. The experiments were replicated three times with comparable results; right panel shows the mean ± SD of viable (LL) and dead (UL + UR + LR) cells in the three experiments.

Figure 2

HUVECs were maintained in growth medium (control) or incubated in CM_cont or in CM_S1 for the indicated times. Protein expression was assessed by means of Western blot analysis, as detailed in Section 2. Representative blots are shown (left); mean ± SD of the densitometry analysis of three different experiments is also shown (right). * p < 0.05 vs. control cells with Kruskal–Wallis test.

Figure 3

HUVECs were incubated in growth medium, either in the absence (control) or in the presence of the indicated cytokines. (A) After 72 h, the number of adherent cells was measured to calculate cell proliferation, as described in Section 2. Data are expressed as percentage of control, untreated cells; each bar represents the media ± SEM of three experiments, each performed in triplicate. * p < 0.05 vs. control, untreated cells with Kruskal–Wallis test. (B) The expression of IRF-1 and p21 mRNAs was measured after 4 h of incubation with RT-qPCR. Data are expressed as fold change of control (=1). Bars are means ± SEM of three independent experiments, each performed in duplicate. * p < 0.05, ** p < 0.01, vs. control, untreated cells with Kruskal–Wallis test. (C) HUVECs were maintained in the absence (control) or in the presence of IFN-γ + TNF-α. The amount of the indicated proteins was assessed with Western Blot analysis, as detailed in Section 2. Representative blots are shown (left), along with mean ± SD of the densitometry analysis of three different experiments (right). * p < 0.05, ** p < 0.01, vs. control cells with Kruskal–Wallis test. Original Western Blot images can be found in the Supplementary Materials.

Figure 4

Cells were maintained in the absence (control) or in the presence of IFN-γ + TNF-α. (A) After 72 h, phase contrast microscopy images were taken (Bar = 100 µM). (B) After 24 h of incubation, cell cycle was analyzed with flow cytometry, as detailed in Section 2. Plots obtained in a representative experiment are shown (left), while cell distribution among the different phases of the cell cycle is shown (right), with bars corresponding to the mean ± SEM of data obtained in four independent experiments. * p < 0.05 vs. control cells with Mann–Whitney test. (C) After 48 h, apoptotic cell death was evaluated with flow cytometry through Annexin V-FITC/PI staining, as detailed in Section 2. Representative scatter plots are shown with the indicated % of viable and apoptotic/necrotic cells of 10,000 events. The experiments were replicated three times with comparable results. (D) At the indicated times, the expression of Bak, Bid and Bcl-2 mRNAs was measured by means of RT-qPCR. Data are expressed as fold change of untreated control cells (=1). Bars are means ± SEM of three independent experiments, each performed in duplicate. * p < 0.05 vs. control cells with Kruskal–Wallis test.