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SARS-Cov2 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 for SARS-COV-2 coronavirus was used to studying its effects on THP-1 macrophages, PBMCs and HUVEC cells. The S protein mediates SARS-CoV-2 entry into cells through binding to angiotensin converting enzyme 2 (ACE2) receptors. We measured viability, intracellular cytokines release, oxidative stress, pro-inflammatory markers and THP-1-like macrophage polarization. We identified an increase in apoptosis, ROS generation, MCP-1 and intracellular calcium expression in THP-1 macrophages. Furthermore, stimulation with the S protein polarizes THP-1 macrophages towards pro-inflammatory futures with an increase in TNFα and MHC-II M1-like phenotype markers. Treatment of cells with an ACE inhibitor, perindopril at 100nM reduced apoptosis, ROS and MHC-II expression. We further analyzed the sensitivity of HUVEC cells after exposure to a conditioned media (CM) of THP-1 macrophages stimulated with S protein. CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, direct stimulation of HUVEC cells with S protein slightly increased HIF1α and MCP-1 expression which was significantly exaggerated by ACE inhibitor treatment. S protein stimulation induced ROS generation and changed mitogenic responses of PBMCs through upregulation of TNFα and IL-17 cytokine expressions. These effects were blunted by perindopril (100nM) treatment. Proteomic analysis of S protein stimulated THP-1 macrophages with or without perindopril (100nM) uncovered more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that blood and vascular component could be activated directly through S protein systemically present in circulation and that activation of local renin angiotensin system might be partially involved in this process.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human) Severe Acute Respiratory Syndrome Coronavirus 2

TISSUE(S): Monocytic Leukemia Cell Line, Macrophage

SUBMITTER: Bandar Alghanem

LAB HEAD: MOHAMED BOUDJELAL

PROVIDER: PXD025516 | Pride | 2021-11-16

REPOSITORIES: Pride

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Project description:Osteopontin depletion in macrophages perturbs proteostasis via regulation of UCHL1-UPS pathway and activation of mitochondria-mediated apoptosis Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow derived macrophages (BMM), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMM upregulates OPN expression, promoting an anti inflammatory, phagocytic, pro-healing phenotype, whereas OPN inhibition triggered a pro-inflammatory phenotype. Here, we applied a global proteome profiling via mass spectrometry analysis to gain a mechanistic understanding of OPN suppression versus induction in macrophages. We identified over 630 differentially expressed proteins (DEPs) in OPN knockout (OPNKO) or GA stimulated versus wild type (WT) macrophages. Two topmost downregulated DEPs in OPN deficient macrophages were ubiquitin C terminal hydrolase L1 (UCHL1), a crucial component of ubiquitin proteasome system (UPS), and the anti inflammatory Heme oxygenase 1 (HMOX 1), whereas GA stimulation upregulated their expression. We further confirmed UCHL1 expression in BMM, and its direct interaction with OPN in protein complex. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome mitochondria-mediated apoptosis (e.g. ROS, Lamp1/2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g. 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome bioinformatics data, Western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages, inducing apoptosis and inhibiting translation, whereas GA stimulated OPN induction restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via regulation of cell viability, UCHL1 UPS pathway, and protein synthesis, indicating its potential application in immune-based therapy.

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SARS-Cov2 coronavirus spike protein-induced apoptosis, inflammatory and oxidative stress responses in THP-1-Like-Macrophages: potential role of angiotensin converting enzyme inhibitor (perindopril)

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