Cell-specific epigenetic landscapes drive differential IL-6 gene expression in human neutrophils and monocytes.

Purpose: IL-6 is a pleiotropic cytokine with a broad range of pro- and anti-inflammatory functions, currently representing a target to modulate in various inflammatory diseases. While monocytes are major source of IL-6, whether human neutrophils produce this cytokine remains still controversial. Herein, we performed new studies, also at epigenetic level, to clarify whether TLR-activated neutrophils express and produce IL-6. Methods: Human neutrophils and CD14+-monocytes, isolated from buffy coat by immunomagnetic beads at a purity of 99.7 ± 0.2 % and 98 ± 1 % respectively, were stimulated with TLR4 and TLR8 agonists, as well as with other ligands for up to 20 h. IL-6 mRNA expression and production were then measured by RT-qPCR and ELISA, respectively, while analysis of the chromatin status at the IL-6 genomic locus was investigated by chromatin immunoprecipitation assays for histone modifications or transcription factor binding. Results: Our data prove that highly purified neutrophils display the capacity to produce biologically active quantities of IL-6, but only after a prolonged incubation with R848 or, less efficiently, LPS. Data also uncover that IL-6-induction by R848 requires PU.1 recruitment, H3 methylation (e.g., H3K4me1 and H3K4me3) and H3 and H4 acetylation (e.g., H3K27Ac and H4Ac) at the IL-6 locus. Analysis of the distribution of histone modifications within 64 kilobases upstream of the IL-6 transcription start site also revealed that neutrophils and monocytes utilize both common and cell-specific enhancer sites. Discussion: In consideration that TLR8 recognizes single strand RNA from viruses, our observation that neutrophils treated with TLR8 agonists produce IL-6 is consistent with a role of these cells in the context of antiviral responses, as recently highlighted by our previous findings. Conclusions: Data clarify that, at least under TLR-stimulation, neutrophils may generate IL-6 by activating a cascade of events controlled at the epigenetic level.