Cell-specific epigenetic landscapes drive differential cytokine expression in human neutrophils and monocytes

Polymorphonuclear neutrophils typically function at the frontline of innate defense in host responses to foreign microorganisms. In addition, extensive research performed in the last 20 years has contributed to recognize neutrophils as versatile cells, also displaying an important role in linking the innate and adaptive arms of the immune response. The latter function occurs, in part, by virtue of the capacity of neutrophils to de novo synthesize and release a vast repertoire of cytokines. There is not a general consensus in the literature as to whether human neutrophils produce pro- or anti-inflammatory IL-6 or anti-inflammatory IL-10, both cytokines being highly important for the balanced functioning of the immune system. Consequently, the objective of the study was to clarify, by studies at the level of chromatin structure, whether highly pure human neutrophils express IL-6 in response to various stimuli, including TLR agonists, and whether we could confirm, at chromatin level, our previous observation about neutrophils’ incapacity to produce IL-10. A complete understanding of how IL-6 and IL-10 expression is regulated at molecular level in specific cell types has obvious implications for a better comprehension of inflammatory disease pathogenesis, as well as for immunotherapeutic strategies. As a result of the doctoral study, it was observed that highly pure human neutrophils genuinely produce IL-6 in response to agonists for TLR8 and (less efficiently) TLR4. Interestingly, the kinetics of IL-6 expression and release differed between neutrophils and autologous monocytes, being both significantly delayed in neutrophils. The delayed expression of IL-6 was explained by closed/inactive conformation of chromatin in the entire IL-6 locus in resting neutrophils, while it appeared already poised for transcription and more prone for rapid chromatin remodeling in autologous monocytes, indicated by the lack of H3K4me1, H3K27Ac and H4Ac marks at the IL-6 locus in neutrophils while being readily present in monocytes, as discovered by chromatin immunoprecipitation (ChIP). Remarkably, IL-6 locus in neutrophils underwent chromatin reorganization upon R848-treatment, indicated by recruitment of PU.1, C/EBPβ, NF-κB and acquisition of significant levels of histone marks (H3K27Ac, H4Ac, H3K4me and H3K4me3) at de novo induced enhancers and at the promoter. Furthermore, in the current doctoral study it was discovered that IL-6 expression in neutrophils and monocytes was regulated by cell type-specific enhancer patterns. In fact, amongst others, de novo enhancer at 14kb upstream of transcription start site (TSS) was induced in neutrophils, but not in monocytes. Instead, an enhancer at 64kb upstream of TSS likely represented more prominent regulatory site in monocytes than in neutrophils. Furthermore, endogenous TNFα was found to amplify IL-6 expression at later time points in R848-treated neutrophils by modulating the chromatin landscape at the IL-6 locus and prolonging the induction of IκBζ antigenic expression, leading to enhanced transcription factor binding. The locus of IL-10, similarly to the locus of IL-6, was confirmed to be closed/inactive in resting neutrophils, indicated by the lack of H3K4me3, H3K27Ac, H4Ac marks at IL-10 locus, by ChIP, in neutrophils, contrary to monocytes. Interestingly, IL-10 locus, differently from IL-6 locus, retained inert chromatin organization even after upon LPS, SAA, or Pam3CSK4 stimulation, both in neutrophils from healthy donors and melanoma patients in contrary to autologous monocytes and murine neutrophils, indicated by the absence of induction of histone modifications and TF recruitment by ChIP. Altogether, the results of this doctoral study clarify controversial literature on the ability of human neutrophils to generate IL-6 and IL-10 and uncover that cytokine expression in these cells can be prominently regulated at epigenetic level.