Polymorphonuclear neutrophils are the most numerous leucocytes present in human blood, and function as crucial players in innate immune responses. Neutrophils are indispensable for the defence towards microbes, as they effectively counter them by releasing toxic enzymes, by synthetizing reactive oxygen species and by producing inflammatory mediators. Interestingly, recent findings have highlighted an important role of neutrophils also as promoters of the resolution of inflammation process, indicating that their biological functions go well beyond simple pathogen killing. Consistently, data from the last decades have highlighted that neutrophils may even contribute to the development of adaptive immunity by performing previously unanticipated functions, including the capacity to extend their survival, directly interact with other leucocytes or cell types, and produce and release a variety of cytokines. In this article, we will summarize the main features of, as well as emphasize some important concepts on, the production of cytokines by human neutrophils.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse. However, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer, and viral infection. We uncover an extreme diversity of human neutrophils in vivo , reflecting the rates of cell mobilization, differentiation, and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of context-dependent functional responses. In this context, we detected an acute interferon (IFN) response in the blood of HSC-T patients that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Neutrophils are known to perform a series of effector functions that are crucial for the innate and adaptive responses, including the synthesis and secretion of a variety of cytokines. In light of the controversial data in the literature, the main objective of this study was to more in-depth reevaluate the capacity of human neutrophils to express and produce cytokines of the IL-17 family in vitro. By reverse transcription quantitative real-time PCR, protein measurement via commercial ELISA, immunohistochemistry (IHC) and immunofluorescence (IF), flow cytometry, immunoblotting, chromatin immunoprecipitation (ChIP), and ChIP-seq experiments, we found that highly pure (>99.7%) populations of human neutrophils do not express/produce IL-17A, IL-17F, IL-17AF, or IL-17B mRNA/protein upon incubation with a variety of agonists. Similar findings were observed by analyzing neutrophils isolated from active psoriatic patients. In contrast with published studies, IL-17A and IL-17F mRNA expression/production was not even found when neutrophils were incubated with extremely high concentrations of IL-6 plus IL-23, regardless of their combination with inactivated hyphae or conidia from Aspergillus fumigatus. Consistently, no deposition of histone marks for active (H3K27Ac) and poised (H3K4me1) genomic regulatory elements was detected at the IL-17A and IL-17F locus of resting and IL-6 plus IL-23-stimulated neutrophils, indicating a closed chromatin conformation. Concurrent experiments revealed that some commercial anti-IL-17A and anti-IL-17B antibodies (Abs), although staining neutrophils either spotted on cytospin slides or present in inflamed tissue samples by IHC/IF, do not recognize intracellular protein having the molecular weight corresponding to IL-17A or IL-17B, respectively, in immunoblotting experiments of whole neutrophil lysates. By contrast, the same Abs were found to more specifically recognize other intracellular proteins of neutrophils, suggesting that their ability to positively stain neutrophils in cytospin preparations and, eventually, tissue samples derives from IL-17A- or IL-17B-independent detections. In sum, our data confirm and extend, also at epigenetic level, previous findings on the inability of highly purified populations of human neutrophils to express/produce IL-17A, IL-17B, and IL-17F mRNAs/proteins in vitro, at least under the experimental conditions herein tested. Data also provide a number of justifications explaining, in part, why it is possible to false positively detect IL-17A+-neutrophils.

Human neutrophils contribute to the regulation of inflammation via the generation of a range of cytokines that affect all elements of the immune system. Here, we investigated their ability to express some of the members of the IL‐12 family after incubation with TLR8 agonists. Highly pure human neutrophils were thus incubated for up to 48 h with or without R848, or other TLR8 agonists, to then measure the expression levels of transcripts and proteins for IL‐12 family member subunits by RNA‐seq, reverse transcription quantitative PCR, and ELISA. We show a TLR8‐mediated inducible expression of IL‐12B and IL‐23A, but not IL‐12A, mRNA, which occurs via chromatin remodeling (as assessed by ChIP‐seq), and subsequent production of IL‐23 and IL‐12B, but no IL‐12, proteins. Induction of IL‐23 requires endogenous TNF‐α, as both mRNA and protein levels were blocked in TLR8‐activated neutrophils via a TNF‐α‐neutralizing Ab. We also show that supernatants from TLR8‐activated neutrophils, but not autologous monocytes, induce the differentiation of Th17 cells from naïve T cells in an IL‐23‐dependent fashion. This study unequivocally demonstrates that highly pure human neutrophils express and produce IL‐23, further supporting the key roles played by these cells in the important IL‐17/IL‐23 network and Th17 responses.

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBPb, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTFmarked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/ POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor kB (NF-kB) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation.