A comparison of neutrophil secretion of cytokines and NET formation in response to Lipopolysaccharide and Group B Streptococcus (GBS) in mice and rats in vitro

Abstract

Introduction: Neutrophil granulocytes (PMNs) play a crucial role in initial pathogen defense, in part through the release of neutrophil extracellular traps (NETs). These web-like structures are essential for pathogen clearance and preventing dissemination but can also elicit excessive inflammation resulting in tissue damage. Purpose: Here, we aimed to investigate the link between NETosis and inflammation induced by gram-negative (lipopolysaccharide, LPS) and gram-positive (Group B Streptococcus lysates, GBS) bacterial mimetics. As mice and rats are common models to investigate inflammation and sickness responses, we aimed to compare the inflammatory reaction of neutrophils derived from these two species. Methods: Bone marrow-derived PMNs from adult mice and rats were cultured after isolation by negative magnetic bead selection. NET formation was evaluated through immunofluorescence detecting the NET markers myeloperoxidase (MPO) and DNA/histone-1-complexes. The secretion of pro-inflammatory cytokines (TNF-α and IL-6) in the supernatants were analyzed using bioassays. Results: We revealed a significant increase in NET formation after GBS treatment in both rat and mouse PMN cultures, whereas LPS had minimal effects on NET induction. Gram-negative and gram-positive inflammatory conditions increased TNF-α and IL-6 secretion in rat PMN cultures. For mouse PMNs, TNF-α levels were increased after stimulation with LPS, while IL-6 was only elevated upon treatment with the highest dose of LPS (20μg/mL) applied. GBS did not significantly alter IL-6 and TNF-α secretion in mouse PMN cultures. Conclusion: These findings suggest that PMNs derived from mice and rats exhibit species-specific distinct responses to inflammatory stimuli. GBS induced NET formation in both rat and mouse PMNs. However, the main difference in rat and mouse PMNs inflammatory response was observed regarding GBS- and LPS-induced secretion of TNF-α and IL-6, suggesting a greater cytokine responsiveness in rat PMNs relative to mouse PMNs.