Markers of inflammation and infection : identifying differences in neutrophil RNA profile caused by LPS stimulation

Abstract

The neutrophil plays an important role in immunity, as it is specialised in the destruction of microorganisms by processes such as phagocytosis and neutrophil extracellular traps. Neutrophils communicate with other cells and regulate their effector functions. In this study, whole blood from healthy donors was stimulated with lipopolysaccharide (LPS), the immunogenic component of the bacterial cell wall, leading to an inflammatory response. A negative selection protocol that purified neutrophils whilst keeping them untouched was set up. Neutrophil purity was ascertained using cytospin and Sysmex haematology analyser XN-10. RNA was extracted from the isolated neutrophils, quantified and the quality was checked using Agilent bioanalyser and reverse transcription PCR. RNA sequencing was carried out at EMBL on three pairs of samples chosen after performing quality control checks. A principal component analysis was done on the entire data set that confirmed the differences in RNA profile between control and LPS stimulated samples. The list of differentially expressed genes consisted of 1206 upregulated genes and 458 downregulated genes. The enrichment analysis showed that upregulated genes belonged to categories such as response to cytokine (GO:0034097), cytokine receptor binding (GO:0005126), response to virus (GO:0009615), and antigen processing and presentation of peptide antigen via MHC class I (GO:0005126) while downregulated genes belonged to ubiquitin-protein transferase activity (GO:0004842). Pathway analysis showed that upregulated genes were involved in pathways for TLR signalling and interleukin signalling. Gene families related to inflammatory response such as cytokines and chemokines, transcription factors and TLR signalling pathway were found to be differentially expressed. Since RNA-sequencing was performed, novel markers of inflammation and infection such as antisense type of genes and lincRNA were identified.