Abstract
Neutrophils are short-lived innate immune cells. These cells respond quickly to stimuli, and die within minutes to hours; the relevance of DNA transcription in dying neutrophils remains an enigma for several decades. Here we show that the transcriptional activity reflects the degree of DNA decondensation occurring in both NADPH oxidase 2 (Nox)-dependent and Nox-independent neutrophil extracellular trap (NET) formation or NETosis. Transcriptomics analyses show that transcription starts at multiple loci in all chromosomes earlier in the rapid Nox-independent NETosis (induced by calcium ionophore A23187) than Nox-dependent NETosis (induced by PMA). NETosis-specific kinase cascades differentially activate transcription of different sets of genes. Inhibitors of transcription, but not translation, suppress both types of NETosis. In particular, promoter melting step is important to drive NETosis (induced by PMA, E. coli LPS, A23187, Streptomyces conglobatus ionomycin). Extensive citrullination of histones in multiple loci occurs only during calcium-mediated NETosis, suggesting that citrullination of histone contributes to the rapid DNA decondensation seen in Nox-independent NETosis. Furthermore, blocking transcription suppresses both types of NETosis, without affecting the reactive oxygen species production that is necessary for antimicrobial functions. Therefore, we assign a new function for transcription in neutrophils: Transcriptional firing, regulated by NETosis-specific kinases, helps to drive NETosis.
Highlights
In this study, we considered that the process of transcription itself is important to drive NETosis, and specific transcriptional steps could contribute to the decondensation of chromatin required for NETosis
Qualitative examination shows that at the 30-min time point more neutrophil nuclei delobulated and decondensed in A23187-mediated NETosis compared to PMA-mediated NETosis
At the 60-min time point, some of the same kinases p38, cSrc, Akt, PyK2 (FAK2) Jnk and Erk continue to activate the transcription of 334, 286, 203, 190, 148 and 138 genes, respectively (Fig. 4D). These analyses show that p38, cSrc, Akt, Jnk and Erk1/2 are some of the major kinases that contribute to the transcription of A23187-mediated NADPH oxidase 2 (Nox)-independent NETosis
Summary
We considered that the process of transcription itself is important to drive NETosis, and specific transcriptional steps could contribute to the decondensation of chromatin required for NETosis. We examined whether specific kinases that regulate different sets of transcription factors could facilitate transcriptional firing in multiple loci of chromosomes to promote chromatin decondensation. To answer these questions, we used the prototypic agonists PMA and A23187 to induce Nox-dependent and Nox-independent NETosis, respectively. We confirmed the importance of transcription for biologically relevant agonist-induced NETosis (LPS and ionomycin to induce Nox-dependent and calcium-mediated Nox-independent NETosis, respectively).
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