A robust intracellular metabolite extraction protocol for human neutrophil metabolic profiling

Neutrophils are phagocytic innate immune cells that play essential roles in host defence, but are also implicated in inflammatory diseases such as rheumatoid arthritis (RA) where they contribute to systemic inflammation and joint damage. Transcriptomic analysis of neutrophils has revealed significant changes in gene expression in neutrophils activated in vitro by cytokines and in vivo during inflammation in RA. However, there are no reports on the global metabolomic changes that occur as a consequence of this activation. The aim of this study was to establish protocols for the study of changes in the metabolome of human neutrophils using 1H NMR spectroscopy. Sample preparation and spectral analysis protocols were optimised using neutrophils isolated by Ficoll-Paque, with decreased washing steps and inclusion of a heat-shock step to quench metabolite turnover. Cells were incubated ± PMA for 15 min in HEPES-free media and samples were analysed by NMR using a 700 MHz NMR Avance IIIHD Bruker NMR spectrometer equipped with a TCI cryoprobe. Chenomx, Bruker TopSpin and AMIX software were used to process spectra and identify metabolites. Principal Component Analysis (PCA) and signalling pathway analysis was carried out using Metaboanalyst. Cell number and number of scans (NS) were optimised as >3.6 million cells and 512 NS. 327 spectral bins were defined in the neutrophil spectra, of which 287 (87.7%) were assigned to 110 metabolites that included: amino acids, peptides and analogues; carbohydrates, carbonyls and alcohols; nucleotides, nucleosides and analogues; lipids and lipid-like molecules; benzenoids; and other organic compounds. 43 metabolites changed at least 1.5 fold (increase or decrease) after the addition of PMA for 5 or 15 min. Pathway analysis revealed that PMA affected nicotinate and nicotinamide metabolism, aminoacyl-tRNA biosynthesis and glycolysis, suggesting a redirection of glucose metabolism from glycolysis to the pentose phosphate pathway and production of NADPH for activation of the NADPH oxidase and subsequent respiratory burst. We have developed protocols for the study of human neutrophils by 1H NMR spectroscopy. Importantly, this methodology has sufficient sensitivity and reproducibility to detect changes in metabolite abundance from cell numbers typically collected from clinical samples or experiments with multiple assay conditions.