A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It

Abstract

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.

Cytokine production by human neutrophils: Revisiting the “dark side of the moon”

“…We could also fully confirm and extend the observations concerning the AF‐317‐NA antibody . Accordingly, by IHC staining using AF‐317‐NA antibody we detected IL‐17A + ‐neutrophils not only in skin sections of psoriasis patients, but also in cytospin slides of peripheral cells isolated from healthy donors and incubated for 3 hours with or without R848 . However, whole lysates of the same neutrophil populations displayed major signals at levels of proteins having MW not corresponding to that of IL‐17A when immunoblotted with AF‐317‐NA antibody .…”

“…Interestingly, we made similar findings in relation to the expression of IL‐17B by human neutrophils . Again, we tested a commercial anti‐IL‐17B antibody (#AF1248) previously shown to detect IL‐17B + ‐neutrophils in synovial membranes of RA patients and in the stroma of colon carcinoma cancer by IHC/IF, as well as by immunoblotting of freshly isolated cells .…”

“…Again, we tested a commercial anti‐IL‐17B antibody (#AF1248) previously shown to detect IL‐17B + ‐neutrophils in synovial membranes of RA patients and in the stroma of colon carcinoma cancer by IHC/IF, as well as by immunoblotting of freshly isolated cells . As expected, by IHC experiments using #AF1248 antibody we could detect IL‐17B‐positive neutrophils in psoriasis plaques and cytospin slides of freshly isolated neutrophils . By contrast, we could not measure any IL‐17B in lysates of freshly isolated/activated neutrophils, either by immunoblot using #AF1248 antibody or using 2 different commercial ELISA .…”

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23

“…More recently, human neutrophils (whose purity was not stated) were shown to express IL‐23 (presumably IL‐23p19) mRNA, as well as to produce very elevated levels of IL‐23 protein, when infected with Mycobacterium tuberculosis H37Rv or when incubated with either LPS or Pam3CSK4 (a TLR2 agonist) . However, in these experiments the infected neutrophils were also reported to express IL‐17, which, according to our previous work, does not occur. Finally, immunofluorescence and flow cytometry analyses revealed that, in patients with castration‐resistant prostate cancer (CRPC), tumor‐infiltrating myeloid‐derived suppressor cells with the neutrophil phenotype (CD11b + CD33 + CD15 + cells, polymorphonuclear cell‐myeloid‐derived suppressor cells) express IL‐23 …”

Interleukin‐23 pathway at the enthesis: The emerging story of enthesitis in spondyloarthropathy

“…Although IL‐17A production has historically been conceptualized in terms of upstream IL‐23 signaling, it is increasingly evident that IL‐17 family production, especially by innate cells, may be independent of IL‐23, but the in vivo mechanisms are not clear . The concept that neutrophils are also a IL‐17–expressing cell is highly controversial and heavily debated, and at this time, there are no data on the immunobiology of entheseal‐resident neutrophils and the cytokines they produce. In humans, cytokine stimuli such as IL‐1β, IL‐6, IL‐21, and/or IL‐23 can drive IL‐17 production .…”