Lipid alterations in human blood-derived neutrophils lead to formation of neutrophil extracellular traps

The formation of neutrophil extracellular traps (NETs) as a host innate immune defence mechanism has been shown to be the result of a novel cell death process called NETosis. The objective of this study was to investigate the role of cholesterol in the formation of NETs. To this end, primary human neutrophils were treated with different concentrations of methy-beta-cyclodetxrin (M beta CD) to reduce cholesterol level in the cell. The formation of NETs was studied using immunofluorescence microscopy and Picogreen-quantification of released dsDNA. Neutrophils treated with M beta CD showed a significant release of NETs in a process that is independent of NADPH-oxidase. The effect of M beta CD on the lipid composition of the cells was determined using high performance thin layer chromatography (HPTLC). The identities of lipids separated by HPTLC were confirmed by mass spectrometry. Treatment of neutrophils with M beta CD revealed distinct changes in the lipid composition: The percentage of cholesterol in the cell was significantly reduced; other lipids as sphingomyelin were only slightly affected. Interestingly, neutrophils treated with sphingomyelin-degrading sphingomyelinase also showed significant release of NETs. In conclusion, this study shows that lipid alterations facilitate formation of NETs. (C) 2014 Elsevier GmbH. All rights reserved.