In vitro and in vivo lipopolysaccharide-driven activation of human neutrophils in healthy volunteers as a tool for clinical drug development

Abstract

Neutrophils are an emerging target for therapeutical intervention in both autoimmune diseases as well as cancer. Since healthy humans lack constitutive neutrophil activation, induction of neutrophil activation is necessary to evaluate investigational compounds and can be achieved via intravenous administration of lipopolysaccharides (LPS). Furthermore, LPS stimulation can be performed ex vivo during clinical trials, and in vitro for pre-clinical analysis. Therefore, we aimed to provide a time course of neutrophil responses after in vivo LPS administration using samples from human endotoxemia trials and compared this to in vitro LPS stimulated whole blood cultures. We performed shotgun proteomics on in vivo stimulated neutrophils, and measured neutrophil activation by flow cytometry using CD11b and CD62L as activation markers and elastase, MPO, LL37 and nucleosome levels as degranulation and NETosis markers. Neutrophil numbers rapidly increased after LPS administration. In line, we found significant increases in neutrophil activation and degranulation markers both in vitro as well as in vivo, which all returned to baseline within 24 h. Degranulation proteins and NETosis related nucleosomes rapidly increased after LPS administration (1 h after exposure) in vivo, while higher concentrations of LPS were necessary in vitro. Lastly, shotgun proteomics revealed little but significant differences in the neutrophil proteome after in vivo LPS administration, pointing to degranulation after LPS stimulation. Both, the in vitro whole blood LPS stimulation assay and the human endotoxemia model, could be valuable tools for evaluation of the effects of future drugs modulating neutrophil responses during preclinical and clinical development.