SARS-CoV-2 Coronavirus Spike Protein-Induced Apoptosis, Inflammatory, and Oxidative Stress Responses in THP-1-Like-Macrophages: Potential Role of Angiotensin-Converting Enzyme Inhibitor (Perindopril)

Abstract

A purified spike (S) glycoprotein of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) coronavirus was used to study its effects on THP-1 macrophages, peripheral blood mononuclear cells (PBMCs), and HUVEC cells. The S protein mediates the entry of SARS-CoV-2 into cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptors. We measured the viability, intracellular cytokine release, oxidative stress, proinflammatory markers, and THP-1-like macrophage polarization. We observed an increase in apoptosis, ROS generation, MCP-1, and intracellular calcium expression in the THP-1 macrophages. Stimulation with the S protein polarizes the THP-1 macrophages towards proinflammatory futures with an increase in the TNFα and MHC-II M1-like phenotype markers. Treating the cells with an ACE inhibitor, perindopril, at 100 µM reduced apoptosis, ROS, and MHC-II expression induced by S protein. We analyzed the sensitivity of the HUVEC cells after the exposure to a conditioned media (CM) of THP-1 macrophages stimulated with the S protein. The CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, the direct stimulation of the HUVEC cells with the S protein, slightly increased HIF1α and MCP-1 expression, which was significantly increased by the ACE inhibitor treatment. The S protein stimulation induced ROS generation and changed the mitogenic responses of the PBMCs through the upregulation of TNFα and interleukin (IL)-17 cytokine expression. These effects were reduced by the perindopril (100 µM) treatment. Proteomic analysis of the S protein stimulated THP-1 macrophages with or without perindopril (100 µM) exposed more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that the blood and vascular components could be activated directly through S protein systemically present in the circulation and that the activation of the local renin angiotensin system may be partially involved in this process.

Graphical: Suggested pathways that might be involved at least in part in S protein inducing activation of inflammatory markers (red narrow) and angiotensin-converting enzyme inhibitor (ACEi) modulation of this process (green narrow).

Keywords: HUVEC cells; ROS; SARS-CoV-2; angiotensin-converting enzyme inhibitor; inflammation; monocyte/macrophages; spike protein.

Graphical Abstract

Figure 1

Effects of spike (S) protein on cell viability and inflammatory markers. Thp-1 macrophages were mock treated (Ctrl), stimulated with S protein (SP) (100 nM) or SP with ACE inhibitor (SP Perindopril, 100 µM), perindopril, added 2 h before SP. (A) Apoptosis was measured by Annexin V binding essay. (B) Oxidative stress was evaluated by reactive oxygen species (ROS) expression. (C) Monocyte chemoattractant protein-1 (MCP-1) expression essay. (D) Fluo4 fluorescence as a function of cytosolic free Ca²⁺ intracellular calcium was performed using flow cytometry analysis. The data are presented as mean ± SEM, n = 4–5. Ordinary one-way ANOVA followed by Dunnett’s test: *p < 0.05, **p < 0.01,***p < 0.001.

Figure 2

Effects of spike (S) protein on cell polarization of THP-1-like macrophages. Thp-1 macrophages were mock treated (Ctrl), stimulated with S protein (SP) (100 µM) or SP with ACE inhibitor (SP Perindopril, 100 µM), perindopril, added 2 h before SP. After cell differentiation, the effect of SP stimulation on (A) TNFα, (B) MHCII, (C) HLA-DR, and (D) CD206 expression was measured by flow cytometry. The data are presented as mean ± SEM, n = 4–5. Ordinary one-way ANOVA followed by Dunnett’s test *p < 0.05, **p < 0.01.

Figure 3

Effects of spike (S) protein stimulation on PBMCs. PBMCs were mock treated controls (Ctrl), stimulated with S protein (SP) (100 µM), or SP with ACE inhibitor (SP Perindopril, 100 µM), perindopril, added 2 h before SP. Flow cytometry analysis was performed for measurement of (A) expression of TNFα in PBMCs, (B) expression of TNFα on CD4 positive T cells, (C) expression of intracellular IL-17 on CD4 T cells, and (D) ROS production on PBMCs. The data are presented as mean ± SEM, n = 4. Ordinary one-way ANOVA followed by Dunnett’s test: *p < 0.05, **p < 0.01.

Figure 4

Effects of conditioned media of Thp-1 macrophages cells stimulated with spike (S) protein on HUVEC cells. Thp-1 macrophages were mock treated (Ctrl), stimulated with S protein (SP) (100 µM), or SP with ACE inhibitor (SP Perindopril, 100 µM), perindopril, added 2 h before SP. After 24 h incubation, conditioned media (CM) was collected and used for HUVEC cell culture. (A) Apoptosis was measured by Annexin V binding essay. (B) Necrosis detection was evaluated by internalization of propidium iodide (PI). (C) Monocyte chemoattractant protein-1 (MCP-1) expression. (D) ROS detection was measured by flow cytometry. The data are presented as mean ± SEM, n = 4. Ordinary one-way ANOVA followed by Dunnett’s test: *p < 0.05, **p < 0.01.

Figure 5

Effect of spike (S) protein stimulation on HUVEC cells. HUVEC cells were mock treated (Ctrl), stimulated with S protein (SP) (100 µM) or SP with ACE inhibitor (SP Perindopril, 100 µM), perindopril, added 2 h before SP. (A) Hypoxia-inducible factor 1-alpha (HIF1α) expression. (B) Monocyte chemoattractant protein-1 (MCP-1) expression. (C) ROS detection. (D) Apoptosis evaluated by Annexin V binding essay was measured by flow cytometry. The data are presented as mean ± SEM, n = 4–5. Ordinary one-way ANOVA followed by Dunnett’s test *p < 0.05, ***p < 0.001.

Figure 6

Mass spectrometry analysis of THP-1 macrophages cells stimulated with spike (S) protein. Volcano plot display differentially expressed proteins in cells stimulated with S protein (SP) (100 µM) versus mock treated (Ctrl) or in presence of ACE inhibitor (SP Perindopril, 100 µM): colored circles are significant proteins (p < 0.05), red is upregulated proteins with fold change ≥1.25, and blue is downregulated protein with fold change ≤0.83. Student’s t-test used for statistical analysis. Gray circles are non-regulated proteins, and black are proteins with non-significant p-values.