Abstract 15524: SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages

Abstract

Coronavirus Disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of this virus has led to millions of deaths and the long-term cardiovascular and respiratory consequences of COVID-19 continue to pose a massive threat to public health. While recent studies have demonstrated that SARS-CoV-2 may enter kidney epithelial cells via angiotensin converting enzyme 2 (ACE2)-independent mechanisms by inducing receptor-independent macropinocytosis, it is currently unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated cardiovascular disease or pneumonia. Here, we investigated the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in alveolar epithelial cells and macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages, but not in human lung epithelial cells. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike protein-induced macropinocytosis in macrophages both in vitro and in vivo . High resolution scanning electron microscopy (SEM) imaging confirmed spike protein-induced plasma membrane activities characteristic of macropinocytosis. Mechanistic studies demonstrated that inhibition of protein kinase C and phosphoinositide 3-kinase in macrophages blocks SARS-CoV-2 spike protein-induced macropinocytosis. Further, pharmacological blockade of ACE2 did not inhibit macropinocytosis in SARS-CoV-2 spike protein-treated cells. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and its cardiovascular and respiratory complications.