Transcript expression analysis of the NETotic neutrphils

Neutrophils are short-lived innate immune cells. Upon encountering appropriate stimuli, neutrophils generate and release neutrophil extracellular traps (NETs), primarily via NADPH oxidase (Nox)-dependent (~2 hours) or Nox-independent NETosis (~15-60 minutes). Ironically, DNA transcription in dying neutrophils remains an enigma. We hypothesized that transcriptional activation, regulated by NETosis-specific kinases, is important to drive the chromatin decondensation necessary for NETosis. For the first time, we show here that (i) the degree of NETosis corresponds to the degree of genome-wide transcription; (ii) kinase-specific transcriptional activation reflects transcriptional firing during different types of NETosis; and (iii) Transcriptomics suggests that NETosis could differentially regulate inflammation. Therefore, we propose that the initial steps of transcriptional firing, but neither transcription per se help to drive NETosis. To determine the transcriptional activity during NETosis in the same time period, we extracted total RNA from unstimulated and NETotic neutrophils at 30- and 60-min time points (3 conditions: unstimulated controls, PMA-activated cells, A23187-activated cells; 3 independent donors for each condition; each time point had its own control; a total of 18 samples). Using these samples we conducted transcriptome analysis by using Affymetrix platform (HTA2) gene chips.