SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders

Introduction The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (M Phi). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination. Objectives To assess (1) the effects of different Spikes on human primary M Phi inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased M Phi inflammation. Methods Human primary M Phi were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts. Results LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary M Phi. Two (out of five) Spikes were contaminated with endotoxins >= 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced M Phi inflammation. Conclusions Glycosylated Spike per se is not proinflammatory for human M Phi, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination.

Automated summary

The glycosylated Spike protein itself does not cause inflammation in human primary M Phi cells, which may be crucial for evading host innate immunity. However, some Spike proteins contaminated with endotoxins can trigger inflammatory responses. In vitro studies with commercially available Spike proteins should be carefully conducted to avoid potential LPS contamination.