Antioxidants (Jan 2024)

SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages via PKC-NADPH Oxidase Signaling

  • WonMo Ahn,
  • Faith N. Burnett,
  • Ajay Pandey,
  • Pushpankur Ghoshal,
  • Bhupesh Singla,
  • Abigayle B. Simon,
  • Cassandra C. Derella,
  • Stephen A. Addo,
  • Ryan A. Harris,
  • Rudolf Lucas,
  • Gábor Csányi

DOI
https://doi.org/10.3390/antiox13020175
Journal volume & issue
Vol. 13, no. 2
p. 175

Abstract

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Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine 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 in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. 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 COVID-19 pathogenesis.

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