Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

Introduction: IL-6 is a pleiotropic cytokine displaying 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 is controversial. Materials and Methods: Highly pure human neutrophils and CD14+ monocytes, isolated from buffy coats by immunomagnetic selection, were stimulated with Toll-like receptor (TLR) 4 and TLR8 agonists 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 demonstrate that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively mantained in an inactive configuration. However, upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous tumor necrosis factor α (TNFα), which prolongs the synthesis of the IκBζ co-activator and sustains CCAAT/enhancer binding protein-β (C/ EBPβ) recruitment and histone acetylation at the IL-6 regulatory regions. Conclusions: Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.