SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders.
Gloria Cinquegrani, Valentina Spigoni, Nicolas Thomas Iannozzi, Vanessa Parello, Riccardo C Bonadonna, Alessandra Dei Cas
Author Information
Gloria Cinquegrani: Endocrinology and Metabolic Diseases, Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126, Parma, Italy.
Valentina Spigoni: Endocrinology and Metabolic Diseases, Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126, Parma, Italy.
Nicolas Thomas Iannozzi: Endocrinology and Metabolic Diseases, Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126, Parma, Italy.
Vanessa Parello: Endocrinology and Metabolic Diseases, Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126, Parma, Italy.
Riccardo C Bonadonna: Endocrinology and Metabolic Diseases, Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126, Parma, Italy. riccardo.bonadonna@unipr.it. ORCID
Alessandra Dei Cas: Endocrinology and Metabolic Diseases, Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126, Parma, Italy.
INTRODUCTION: The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). 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 MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation. METHODS: Human primary MΦ 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 MΦ. 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 MΦ inflammation. CONCLUSIONS: Glycosylated Spike per se is not pro-inflammatory for human MΦ, 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.
