ESTABLISHMENT OF A SYSTEM FOR STABLE EXPRESSION OF SARS-COV-2 EFFECTOR PROTEINS IN HUMAN MONOCYTIC CELL LINE

The process of efferocytosis, which involves the efficient removal, processing, and degradation of dead cells, is crucial for promoting tissue renewal and protection against complications caused by excessive tissue damage. Macrophages play an important role in the phagocytosis and removal of these cell corpses. Efferocytosis is coupled to other environmental signals to functionally wire macrophages towards an anti-inflammatory and tissue repair program. Of note, the phagocytosis of apoptotic cells infected with viable SARS-CoV-2, compared to the phagocytosis of sterile apoptotic cells, reduces the expression of efferocytic receptors in macrophages. This repression causes a failure in the capacity of macrophages to sequentially remove other cell corpses, potentially interfering with the ability of these phagocytes to promote efficient tissue repair. In this context, our hypothesis is that specific proteins from the SARS-CoV-2 genome negatively modulate the expression of genes coding for these efferocytic receptors, thus impairing the continual efferocytosis. According to this hypothesis, we adapted and standardized a protocol for the stable expression of each SARS-CoV-2 protein in human monocytic THP-1 cell line using a lentiviral expression system. Expression of SARS-CoV-2 constructs was confirmed by immunoblotting. Using RT-qPCR, we are currently evaluating the role of these viral proteins in modulating the transcription of efferocytic receptors induced by different stimuli. As next steps, we will perform functional assays using flow cytometry to assess the rate of apoptotic cell internalization by macrophages differentiated from transformed THP-1 cells to determine if reduced gene expression results in impaired efferocytosis.