Variable Induction of Pro-inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

Ouyang, Weiming; Xie, Tao; Fang, Hui; Gao, Chunling; Stantchev, Tzanko; Clouse, Kathleen A.; Yuan, Kun; Ju, Tongzhong; Frucht, David M.

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of endotoxin, which correlated directly with their abilities to induce cytokine production. The lipopolysaccharide (LPS) inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.