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

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.

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.
Keywords: neutrophils, il-17 members, il-17a, il-17B, il-17F inTrODUcTiOn The IL-17 family of cytokines consists of six members, namely IL-17A (usually referred to as IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also known as IL- 25), and IL-17F (1). After the discovery of a subtype of CD4 + T helper, expressing IL-17A and IL-17F (currently known as Th17 cells), plenty of studies have been published correlating Th17 cells with a wide range of physiological and pathological processes. IL-17A and IL-17F are not only the most studied but also the most closely related, since they share 50% of amino acid sequence identity, adjacent gene localization (2) and binding to the same IL-17R, in this case composed by the IL-17RA and IL-17RC subunits (1). The IL-17R group comprises, in fact, five receptor subunits, IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE (3). IL-17RA was the first to be described, is ubiquitously expressed (particularly in hematopoietic cells), and functions as a common receptor subunit used by at least four ligands, namely IL-17A, IL-17C, IL-17E, and IL-17F (3). IL-17F is often coproduced with IL-17A, so that together they can also form an IL-17F/IL-17A heterodimer (4) binding to the IL-17RA/ IL17RC complex as either homodimers or heterodimers (3). IL-17A and IL-17F are proinflammatory cytokines that play key regulatory roles in host defense and inflammatory diseases. They mainly mediate immune regulatory functions by promoting the generation of proinflammatory cytokines/growth factors (including G-CSF, GM-CSF, and IL-6) and chemokines (such as CXCL8, CXCL6, and CXCL1) by epithelial and other stromal cells, which ultimately lead to the attraction and activation of neutrophils and macrophages into the inflammatory site (5), as well as to granulopoeisis (6). Although crucial in protecting the host from invasion by many types of pathogens, including bacteria and fungi (7), dysregulated IL-17A and IL-17F production can lead to the development of autoimmune diseases, such as psoriasis, multiple sclerosis, and rheumatoid arthritis (RA), as well as cancer progression (5,8). The latter observations hence make IL-17A/F as a very important target for the development of new therapies (1,8).
As mentioned, Th17 cells are considered the main sources of IL-17A and IL-17F. However, other innate immune cells produce these cytokines, including γδ T cells, natural killer T cells, invariant natural killer cells, Paneth cells, TCRβ + natural Th17 cells, lymphoid-tissue inducer-like cells, IL-17-expressing type 3 innate lymphoid cells, and mast cells (8,9). By contrast, it is still questionable whether human polymorphonuclear neutrophils represent sources of IL-17A or IL-17F. It is currently well established that neutrophils are crucial players in innate immune responses, not only for their capacity to perform defensive functions (10) but also for their ability to produce a large variety of cytokines (11). Concerning IL-17A and/or IL-17F, in 2010, we reported that highly purified populations of human neutrophils (>99.7%), incubated for up to 20 h with IFNγ and/or LPS in vitro, do not produce IL-17A (12). While a few papers substantially confirm our findings (13)(14)(15)(16)(17), the majority of the subsequent studies report that human neutrophils may represent sources of IL-17A . Experimental evidence proving that human neutrophils express IL-17A mostly, but not only, derives by immunohistochemistry (IHC) and/or immunofluorescence (IF) studies documenting IL-17A + -neutrophils in tissue specimens from a variety of pathological conditions (18, 19, 21, 22, 25, 27, 28, 30-32, 34, 36-39, 41-43, 45, 47-49, 51, 53). Interestingly, many of these studies focus on psoriasis (20,25,30,32,35,49), a disease characterized by an early accumulation of neutrophils in skin lesions in which neutrophil-derived mediators (such as reactive oxygen species, granule proteins, and cytokines) may alter the homeostatic state of keratinocytes and endothelial cells (54). At the end of 2014, however, Tamarozzi et al. (13) not only reported the absence of IL-17A mRNA expression and production by highly pure (99.9%) populations of resting or activated neutrophils but also demonstrated that some of the commercial polyclonal anti-IL-17A antibodies (Abs) stain neutrophils for their non-specific recognition of various intracellular proteins different from antigenic IL-17A. Nevertheless, reports describing either IL-17A-positive neutrophils in tissue samples from diseases or in vitro-stimulated neutrophils as sources of IL-17A, continue to be published (20,23,24,26,29,33,35,40,44,46,50,52). Based on these premises, we decided to more accurately analyze the issue of whether human neutrophils produce IL-17A, as well as other IL-17 members in vitro.

superoxide anion Measurement
After isolation, neutrophils were suspended at the concentration of 2 × 10 6 cells/ml in HBSS buffer containing 0.5 mM CaCl2 and 1 mg/ml glucose. Neutrophils (100 µl/well) were then distributed in a 96-well plate and incubated for 10 min at 37°C prior to the addition of 80 µM cytochrome c, 2 mM NaN3 (Sigma) and the indicated stimuli, including 20 ng/ml PMA as control. Plates were then incubated at 37°C in an automated ELx808IU microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) to record cytochrome c reduction (via absorbance at 550 and 468 nm, at intervals of 5 min for 90 min). O2 − production was finally calculated using an extinction coefficient of 24.5 mM (60).

immunocitochemistry, ihc and iF
Cytospin preparations of neutrophils (61) previously cultured with the indicated stimuli were stained by ematoxylin and eosin for morphological evaluation. After coverslip removal, specimens were rehydrated through a scale of alcohols, with endogenous peroxidase activity blocked by treatment with 0.3% H2O2 in methanol for 20 min. Anti-human IL-17A (AF-317-NA), IL-17B (AF1248), and CXCL8 (AF-208) goat IgG pAbs from R&D Systems were 1:50 diluted, added to specimens for 60 min and then revealed using the goat-on-Rodent HRP-polymer (Biocare Medical, Pacheco, CA, USA) followed by diaminobenzidine. Omission of the primary antibody, as well as isotype control staining, was also performed as negative controls. For IL-17A and IL-17B tissue immunostaining, 4-µm tissue sections from two FFPE cases of pustular psoriasis were deparaffinized and rehydrated through a scale of alcohols. Endogenous peroxidase activity was then blocked by treatment with 0.3% H2O2 in methanol for 20 min. Epitope retrieval was performed using a microwave oven in 1.0 mM EDTA buffer (pH 8.0), for 3 cycles of 5 min at 750 W. IL-17A and IL-17B were diluted 1:50 and revealed using the goat HRP-polymer (IHC) or the horse anti-goat IgG biotinylated (Vector Laboratories, Peterborough, UK) followed by streptavidin-FITC (Southern Biotech, Birmingham, AL, USA). DAPI was used for counterstaining. For double IHC, anti-IL-17A and IL-17B Abs were diluted 1:500, and after revelation (as detailed above), anti-CD66b Abs (diluted 1:80 from BioLegend) were added to the sections. Mach4 AP polymer was used as secondary antibody followed by Ferangi Blue as chromogen. Ematoxylin was used for counterstaining.
reverse Transcription Quantitative real-Time Pcr (rT-qPcr) Total RNA was extracted from neutrophils by the RNeasy Mini Kit (Qiagen, Venlo, Limburg, Netherlands), as previously detailed (62). To completely remove any possible contaminating DNA, an on-column DNase digestion with the RNase-free DNase set (Qiagen) was performed during total RNA isolation. Total RNA was then reverse-transcribed into cDNA using Superscript III (Life Technologies, Carlsbad, CA, USA) and random hexamer primers (Life Technologies), while qPCR was carried out using Fast SYBR ® Green Master Mix (Life Technologies). Sequences of gene-specific primer pairs (Life Technologies) are listed in Table  S1 in Supplementary Material. Data were calculated by Q-Gene software 1 and expressed as mean normalized expression units after GAPDH normalization (63).

chiP assays
Chromatin immunoprecipitation experiments were performed exactly as previously described (62). Briefly, nuclear extracts from 2 × 10 6 neutrophils or Th17 cell lines were immunoprecipitated using 1 µl anti-H3K4me1 (ab8895) and anti-H3K27Ac (ab4729) pAbs (both from Abcam, Cambridge, United Kingdom). Coimmunoprecipitated material was subjected to qPCR analysis using the specific promoter primers (purchased from Life Technologies) listed in Table S2 in Supplementary Material. Data from qPCR were expressed as percentage over input DNA and are displayed as mean ± SEM.

chiP-seq
Purified DNA from H3K27Ac and H3K4me1 ChIP assays (performed as described in the previous paragraph) was adapterligated and PCR-amplified for sequencing on HiSeq2000 platform (Illumina, Cambridge, UK) using TruSeq DNA Library Prep Kit (Illumina). After sequencing, reads were quality-filtered according 1 http://www.gene-quantification.de/download.html (Accessed: February 10, 2018). to the Illumina pipeline. Single end (51 bp) reads were then mapped to the human genome (Genome Reference Consortium GRCh37, Feb/2009) using BOWTIE v1.0.0 (64). Only reads with no more than two mismatches (when compared to the reference genome) were converted to tag directories using HOMER's module known as "makeTagDirectory, " and then converted to BedGraph format using HOMER's module known as "makeUCSCfile, " to be finally normalized to 10 7 total tag counts. ChIP-seq signals were visualized using Integrative Genomics Viewer. For H3K4me1 and H3K27Ac ChIP-seqs of Th17 cells, 36 bp reads, already filtered and mapped, were downloaded from database of the "roadmap epigenomics project" 2 (NIH Epigenomics Roadmap Initiative). Aligned reads were then converted to BedGraph format and normalized to 10 7 total tag counts.

statistical analysis
Data are expressed as mean ± SEM or mean ± SD. Statistical evaluation was performed by using, depending on the experiment type, Student's t-test or two-way ANOVA followed by Bonferroni's post hoc test. P values <0.05 were considered as statistically significant.

study approval
Human samples were obtained following informed written consent by both HDs and psoriatic patients. This study was carried out in accordance with the recommendations of Ethic Committee of the Azienda Ospedaliera Universitaria Integrata di Verona (Italy). All the experimental protocols were approved by the Ethic Committee and all subjects gave written informed consent in accordance with the Declaration of Helsinki.

resUlTs human neutrophils incubated With a Variety of agonists In Vitro Do not express il-17 Members at Both mrna and Protein levels
We have previously shown that human neutrophils (>99.7% purity), incubated with 100 U/ml IFNγ and/or 100 ng/ml ultrapure LPS for up to 20 h in vitro, do not produce IL-17A protein (12). Additional RT-qPCR experiments not only confirmed our previous data ( Figure 1A) but also revealed that other agonists, including 5 µM R848 and/or 1,000 U/ml IFNα ( Figure 1B), 10 ng/ml GM-CSF, 100 nM fMLF ( Figure 1C),   Figure S1 in Supplementary Material), or IL-17A and IL-17F proteins in supernatants from CD4 + T cells activated with anti-CD3/anti-CD28 mAbs ( Table 1). We could also detect intracellular IL-17B in lysates of human cerebral cortex (data not shown), as expected (68).
In other experiments, neutrophils were incubated for 3 h with 20 µg/ml IL-6 plus 2 µg/ml IL-23, in the presence or the absence of inactivated conidia, or hyphae, from A. fumigatus. These experiments were done with the purpose to mimic, as much as possible, recently described experimental conditions shown to induce not only IL-17A and IL-17F but also IL-17RC, mRNA expression (23,24,29,39,40,44). Neutrophils were also incubated with 100-500 ng/ml IL-17A to reinvestigate (12) whether they respond to IL-17A or not. As shown in Figure 2, neutrophils treated with either IL-17A or IL-6 plus IL-23 (in the presence or the absence of inactivated A. fumigatus conidia/hyphae), showed neither induction of IL-17A (Figure 2A), IL-17F (Figure 2B), and IL-17RC ( Figure 2C) mRNAs nor upregulation of the constitutively expressed IL-17RA transcript levels ( Figure 2D). Similar results were obtained when incubation was prolonged up to 6 h (data not shown), or when neutrophils were stimulated with PMA/ionomycin after pretreatment for 1 h with IL-6 plus IL-23 ( Figure S2 in Supplementary Material). Elevated levels of IL-17RC mRNAs were, however, detected in HBECs (data not shown), used as control cells (12), thus confirming that our primers were correctly designed. Importantly, the capacity of IL-6 plus IL-23 to stimulate neutrophils was evidenced by their ability to time-dependently promote STAT3 phosphorylation (Figure 2E), as well as to upregulate SOCS3 mRNA expression (Figure 2F), such an effect being potentiated by inactivated A. fumigatus conidia/hyphae ( Figure 2F). By contrast, IL-17A-treatment influenced neither SOCS3 ( Figure 2F) nor CXCL8 (data not shown) mRNA levels in neutrophils. Furthermore, no IL-17A (Figure 3A), IL-17F, or IL-17AF (data not shown) proteins were detected by ELISA either intracellularly or in supernatants harvested from neutrophils incubated with IL-6 plus IL-23, in the presence or the absence of inactivated A. fumigatus conidia/ hyphae. Under the same experimental conditions, CXCL8 protein was newly synthesized and released by neutrophils incubated with IL-6 plus IL-23 in the presence of inactivated A. fumigatus conidia/hyphae, but not in their absence ( Figure 3B). Finally, no IL-17A was detected in IL-6 plus IL-23-stimulated neutrophils by intracellular staining experiments (data not shown), using the anti-human IL-17A eBio64DEC17 mouse IgG1 (from eBioscience) previously shown to function under identical experimental conditions by Taylor et al. (39). We have no clues explaining why we did not reproduce the positive effects on IL-17 expression by IL-6 plus IL-23 (23,24,29,39,40), with or without inactivated A. fumigatus conidia/hyphae. One possibility is that the hyphal extracts from A. fumigatus used by Taylor and colleagues (39), but not our inactivated conidia/hyphae, contain some undefined PAMP(s) that effectively promote(s) IL-17A production/IL-17RC expression by human neutrophils.
Taken together, our data extend previous findings on the inability of human neutrophils to express IL-17 members at the mRNA and protein levels under various activating conditions (13)(14)(15)(16). Data also confirm and extend our previous findings (12) on the inability of IL-17A to directly modify IL-17A, IL-17F, IL-17RA, IL-17RC, SOCS3, and CXCL8 gene expression in human neutrophils.
for both IL-17RA and IL-17RC surface expression (data not shown) (12). It should be pointed out that, for the investigation of surface IL-17RC, we have been using the same anti-IL-17RC, directly PE-conjugated, Abs used in Taylor et al.'s study (39), other than the anti-IL-17RC biotin-conjugated Abs that necessitate PE-conjugated streptavidin for detection (12), without noticing any difference between them. By the way, IFNγ plus LPS and R848 ( Figure S3A in Supplementary Material), as well as IL-6 plus IL-23 in the presence of inactivated A. fumigatus conidia/hyphae ( Figure S3B in Supplementary Material),    Figure 2F), do not induce the expression of IL-17RC in our hands, contradicting some studies (39,44).

O2
-Production by neutrophils stimulated With inactivated A. fumigatus hyphae after Preincubation With il-6 plus il-23 is not Modified by either exogenous il-17a or il-17a inhibitors We then measured the capacity to release O2 − by neutrophils preincubated with or without IL-6 plus IL-23 for 1 h, and then treated for one additional hour with inactivated A. fumigatus hyphae, in the presence or the absence of either IL-17A or anti-IL-17A neutralizing Abs ( Figure S4 in Supplementary Material). As control, neutrophils were also stimulated with either inactivated A. fumigatus hyphae alone or 20 ng/ml PMA. As shown in Figure S4 in Supplementary Material, inactivated A. fumigatus hyphae were found to trigger a remarkable O2 − production by neutrophils, even though lower than PMA. However, A. fumigatus hyphae-stimulated O2 − release was not potentiated by the preincubation of neutrophils with IL-6 plus IL-23 (which, by themselves, did not trigger any O2 − production) ( Figure S4  chromatin Organization at the il-17a and il-17F genomic loci of human neutrophils Signatures of histone posttranslational modifications at a specific gene locus provide indicative elements to predict whether such a gene can be transcribed or not (69,70). Therefore, we evaluated the presence of histone marks associated to active (e.g., H3K27Ac) and poised (e.g., H3K4me1) genomic regulatory elements (71) at the IL17A and IL17F loci of human neutrophils. Genome-wide ChIP-seq assays demonstrated that, in freshly isolated neutrophils, the entire genomic region containing IL17A and IL17F loci is completely devoid of H3K27Ac and H3K4me1 (Figure 5). By contrast, based on data available from the NIH Epigenomics Roadmap Initiative (72), multiple H3K4me1 peaks are present in the same genomic regions of PMA/ionomcyin-stimulated Th17 cells, while H3K27Ac peaks localize at the IL17A locus only (Figure 5). To validate the previous data, we performed H3K27Ac and H3K4me1 qPCR ChIPs using neutrophils incubated for 1 h either with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23, as well as Th17 cell lines (in which IL-17A and IL-17F mRNA is constitutively transcribed), used as positive controls (Figure 6). Based on the H3K4me1 peaks from the ChIP-seqs of Th17 cell lines (72) (Figure 5), we designed specific primers amplifying potential regulatory regions at the IL17A and IL17F genomic loci, namely IL-17A#1 and IL-17F#1 for enhancers, and IL-17A#2, IL-17A#3, and IL-17F#2 for promoters (Figures 6A,B). As expected, Th17 cell lines displayed constitutively bound H3K4me1 at their IL-17A and IL-17F promoters and enhancers (Figures 6A,B, left panels). We also detected high levels of H3K27Ac at the IL-17A and IL-17F promoters and enhancers of Th17 cell lines (Figures 6A,B, right panels), in line with their constitutive expression of both IL-17A and IL-17F mRNA (data not shown). By contrast, we did not observe any H3K4me1 or H3K27Ac at the IL17A and IL17F loci of neutrophils, either under resting conditions (thus confirming the ChIP-seq data shown in Figure 5) or after incubation with IL-6 plus IL-23 (Figures 6A,B). In fact, the H3K4me1 and H3K27Ac levels at the IL-17A and IL-17F enhancers in neutrophils were similar to those ones present at the PRL promoter, a genomic region with a closed chromatin conformation in myeloid cells, herein used to determine the signal background (Figures 6A,B). Notably, measurable amounts of H3K4me1 and H3K27Ac were found at the SOCS3 promoter of neutrophils under resting conditions, as well as in Th17 cell lines ( Figure 6C). Interestingly, H3K27Ac levels tended to increase in neutrophils incubated with IL-6 plus IL-23 (Figure 6C), in accordance with a supposed STAT3dependent induction of SOCS3 mRNA (73). Taken together, data indicate that the organization of the IL17A and IL17F loci in human neutrophils is characterized by the absence of poised chromatin marks, unlike that of IL-17A-and IL-17F-producing Th17 cell lines. Data also indicate that human neutrophils do not reorganize the chromatin of the IL17A and IL17F loci in response to IL-6 plus IL-23, consistent with their inability to de novo accumulate IL-17A and IL-17F mRNA.

human neutrophils From Patients With Psoriasis Do not express il-17a and/or il-17F mrna
We subsequently investigated whether neutrophils isolated from patients with active psoriasis could express/produce IL-17A, IL-17F, and/or IL-17RC mRNA, either constitutively or upon incubation for 20 h with IFNγ plus LPS, R848, or IL-17A. As shown in Figure 7A, the latter was not the case, as psoriatic neutrophils behaved similarly to neutrophils from HDs. Psoriatic neutrophils did not also respond to IL-17A (Figure 7A), due to their lack of surface IL-17RC expression ( Figure 7B). Nonetheless, psoriatic neutrophils fully responded to either R848 or IFNγ plus LPS, as they accumulated CXCL8, TNFα, and SOCS3 transcripts at levels comparable to those in HD neutrophils ( Figure 7A).
commercial anti-il-17a abs (aF-317-na) Positively stain cytospins of resting and activated neutrophils due to Their nonspecific recognition of neutrophil intracellular Proteins Different From il-17a In additional experiments, cytospin slides of resting and R848stimulated neutrophils were incubated with goat anti-human IL-17A AF-317-NA Abs, previously shown to stain neutrophils in pathological tissues (18-22, 25, 27, 28, 30-33, 35, 36, 41-43, 45, 47-50), as also confirmed by our IHC/IF staining of inflamed psoriatic tissue (Figure 8A). Consistently, neutrophil cytospin slides became strongly positive upon incubation with AF-317-NA, yet with no difference between resting or R848-activated neutrophils ( Figure 8B). By contrast, immunostaining of the same cytospins slides with anti-human CXCL8 Abs showed a strong positivity only in R848-treated neutrophils (Figure 8B), thus excluding methodological artifacts. Not surprisingly, neutrophils from the same experiments were found totally negative for both IL-17A mRNA expression and IL-17A production once processed for RT-qPCR analysis and ELISA. The detection of IL-17A-positive neutrophils by IHC, in the absence of IL-17A mRNA, could  be explained by the fact that the cytokine may be synthesized in bone marrow neutrophil precursors, at stages during which granule proteins, such as myeloperoxidase (MPO), elastase, and azurocidin 1, are formed (74). However, in transcriptomes made by Rapin et al., generated from cells isolated at different stages during granulopoiesis (65), we did not identify any IL-17A mRNA accumulation (Figure 9). In the same database, we not even detected IL-17RC and IL-10 mRNA (Figure 9), consistent with the inability of mature neutrophils to express them (12,70). By contrast, we did find MPO, elastase, and azurocidin 1 mRNA expression only in transcriptomes of neutrophil precursors, as expected (74), thus validating the reliability of the database (65) (Figure 9). In any case, consistent with the absence of intracellular IL-17 ( Figure 3A), immunoblots performed with AF-317-NA revealed that whole neutrophil lysates did not show any positive signal in correspondence of recombinant human IL-17A (rhIL-17A) molecular weight (MW) (Figure 8C). In these experiments, neutrophils were either freshly isolated (D1 and D2 in Figure 8C), or cultured for 3 h with or without R848, 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low IL-6 plus IL-23 in Figure 8C), or 20 µg/ ml IL-6 plus 2 µg/ml IL-23 (high IL-6 plus IL-23 in Figure 8C). By contrast, AF-317-NA strongly reacted in correspondence of neutrophil proteins displaying higher MW than that of rIL-17A, with no difference in signals among freshly isolated, stimulated, or untreated neutrophils ( Figure 8C). While these data confirm the observations reported by Tamarozzi

human neutrophils Do not express/ Produce il-17B
In a separate set of experiments, we also tested goat anti-IL-17B (AF1248) Abs that, in recent publications, have been shown to positively stain, by IHC and IF, neutrophils present in tissue samples from RA (75) and colon carcinoma (CCR) (76) patients.
Consistently, we found that also neutrophils present in inflamed psoriatic tissue were strongly detectable by IHC and IF stainings with AF1248 ( Figure 10A). On cytospin slides, AF1248 stained neutrophils isolated from the blood of HDs and incubated for 3 h with or without 5 µM R848 in an equivalent manner ( Figure 10B). However, by immunoblotting of whole lysates prepared from neutrophils treated with R848 or IL-6 plus IL-23, AF1248 did not recognize any protein corresponding to the rhIL-17B MW ( Figure 10C). These negative observations were also confirmed by measurement of intracellular, as well as, released IL-17B by two commercial ELISA (see Materials and Methods). Accordingly, no antigenic IL-17B could be measured in supernatants and whole lysates from neutrophils incubated with 5 µM R848 with or without 1,000 U/ml IFNα, 100 µg/ml LPS with or without 100 U/ml IFNγ, 2/20 μg/ml IL-6 plus 2 µg/ml IL-23 (data not shown), in agreement with the lack of IL-17B mRNA induction. Detectable IL-17B levels were, however, measured in lysates of human cerebral cortex (68), demonstrating that our two IL-17B ELISA kits were sensitive enough. Altogether, our data indicate that, similarly to the case of AF-317-NA, the positive stainings of neutrophils in cytospin slides and, possibly, tissue samples by AF1248, likely stand for an IL-17B-unrelated, non-specific, recognition occurring in human neutrophils.

DiscUssiOn
In this study, we have reinvestigated in-depth whether human neutrophils produce IL-17A, IL-17B, IL-17F, and IL-17A/F in vitro. According to the literature, in fact, information on such an issue appears discordant, as the majority of papers sustain that human neutrophils do express/produce IL-17A , while a minority fail to detect it (12)(13)(14)(15)(16)(17). This issue is even more critical if one takes into account that also the capacity of murine neutrophils to produce IL-17A, shown in a variety of mouse models of infectious and autoimmune inflammation (24,39,40,(77)(78)(79)(80)(81), has been recently questioned (82,83). Preclinical models evidencing neutrophil-derived IL-17 as pathogenic in diseases might be, in fact, prematurely taken as proof-of-concept for immediate translational applications in humans.
Herein, by using multiple methodological approaches (RT-qPCR, ChIP, ChIP-seq, ELISA, intracellular staining, and immunoblotting), we confirm and greatly extend our previous findings (12) showing that highly purified (>99.7%) populations of human neutrophils, either resting or activated by a variety of stimulatory conditions, including TLR and dectin ligands, fungal PAMPs and cytokines, used singly or in combinations, neither express IL-17A, IL-17F, IL-17B, IL-17C, IL-17D, and IL-17E mRNA nor produce IL-17A, IL-17F, IL-17A/F, and IL-17B in vitro. Similarly, we show that also neutrophils isolated from  patients with active psoriasis do not express IL-17F, IL-17B,  IL-17C, IL-17D, and IL-17E as well as IL-17RC mRNA when  activated by R848, IFNγ plus LPS, and IL-17A in vitro. In such regard, RNA-Seq experiments made by Tamarozzi et al. (13), using neutrophils isolated by negative-selection (>99.9% pure) from HDs or RA patients (as we do), then treated for 1 h with a range of inflammatory cytokines (TNFα, GM-CSF, G-CSF, IL-6, IL-1β, CXCL8, IFNα, and IFNγ), also failed to detect any of the mRNA for IL-17 cytokine family. By contrast, Yamanaka et al. (15) have been recently reported the presence of constitutive IL-17A transcripts in neutrophils from HDs and psoriasis patients isolated by density gradient cell separation (92% purity). However, when the same cell populations were further purified by magnetic sorting (reaching a 99% purity), they were found totally devoid of IL-17A mRNA (15), indicating that contaminating monocytes/lymphocytes were actually responsible for the IL-17A mRNA expression in unsorted "neutrophils. " Needless to say that Yamanaka et al. 's observations (15) are example of a notion that we have been always recommending in our studies (56,59,84), namely the requirement of using highly purified cell populations if one wants to obtain correct results when examining neutrophil gene expression or neutrophil-derived cytokines.
Interestingly, other studies confirm that human neutrophils do not constitutively contain IL-17A transcripts (13,24,29,30,35,39,40,44), including those ultimately showing a concurrent positivity for IL-17A protein, as revealed by intracellular flow cytometry (24,39,40), ELISA (24,39), confocal microscopy (39), or IHC (29). Some authors (30,35) speculated that the absence of IL-17A mRNA in mature neutrophils indicates that the cytokine is synthesized in bone marrow neutrophil precursors, at the stages when granule proteins are formed (74). However, we would exclude such a hypothesis, as our analysis of transcriptomes generated from all types of bone marrow cell populations (65) failed to identify an IL-17A mRNA accumulation at any stage of neutrophil maturation.
We were unable to detect IL-17A and IL-17F mRNA/production/release even by human neutrophils incubated with IL-6 plus IL-23, in contrast to what repeatedly found (23,24,29,39,40). In our experiments, neutrophils did, however, respond to IL-6 plus IL-23 in terms of STAT3 phosphorylation and SOCS3 mRNA induction, indicating that the two cytokines are effectively stimulatory for neutrophils. It is intriguing that, apart from Halwani et al. (23), who found that either 20 ng/ml IL-6 or 20 ng/ml IL-23, singly used, directly induced IL-17A mRNA and protein in a fraction of neutrophils from asthmatic patients, other groups highlighted the necessity to use at least 20 µg/ml IL-6 plus 2 µg/ml IL-23 (29,39,40) (as we did). In this context, the paper by Hu et al. (24), based on the use of neutralizing Abs and pharmacological inhibitors, identified endogenous IL-6 and IL-23 as indirect inducers of IL-17A expression in a fraction of neutrophils either infected with Mycobacterium tuberculosis (MTB), or stimulated with LPS or Pam3CSK4. In this latter study, however, IL-6 and IL-23 levels corresponded to 1 ng/ml at the best. Herein, we failed to detect IL-17A mRNA expression and production in neutrophils incubated with either LPS or Pam3CSK4, even if it is true that they produce IL-6 (62) and IL-23 (our unpublished observations). Whether stimulation of neutrophils with MTB effectively promotes IL-17A expression via endogenous IL-6 and IL-23 remains to be verified. However, no IL-17A, IL-17B, IL-17C, or IFNγ secretion from Mycobacterium bovis Bacille-Calmette Guerin (BCG)-stimulated neutrophils was recently reported (14). It should be also remarked that the purity of neutrophils in studies showing an IL-17 production in response to IL-6 plus IL-23 (23,29,39,40), reported to be >96% at the best (29), does not sufficiently secure fully genuine results at least in our opinion.
Nevertheless, we investigated potential mechanisms helping to clarify whether human neutrophils respond to IL-6 plus IL-23 in terms of IL-17A expression or not. ChIP assays revealed that, in resting, as well as in IL-6 plus IL-23-stimulated, neutrophils, but not in Th17 cell lines, the IL17A locus does not contain any H3K4me1 and H3K27Ac, which are two histone marks that are usually present in those genomic regions that act as active enhancers (85). On the other hand, the levels of H3K27Ac were found increased at the SOCS3 promoter of neutrophils incubated with IL-6 plus IL-23, consistent with the potentially inducible SOCS3 mRNA transcription. Notably, the complete absence of H3K4me1 at the IL17A locus of neutrophils is particularly informative, since such a histone modification is known to precede very early, but time-consuming (86), events necessary for the assembly of the transcriptional machinery, including nucleosomal depletion, H3K27Ac deposition, and enhancer activation (85). Based on our data, it appears that the chromatin at the IL17A locus of human neutrophils likely displays a closed conformation, inaccessible to transcription factors and, consequently, RNA polymerase, ultimately preventing IL-17A mRNA transcription in resting as well as stimulated neutrophils. It is thus very unlikely that H3K4me1 modification could be induced within 1 h, e.g., the time-point at which IL-17 mRNA expression in IL-6 plus IL23-stimulated neutrophils has been observed (29,39,40). Obviously, this does not exclude that there could exist some stimulatory conditions able to modify the chromatin at the IL17A or IL17F loci of human neutrophils.
Similar concerns can be made for the, to date, reported IL-17B expression by human neutrophils. Accordingly, IL-17B has been detected in neutrophils infiltrating the synovial membrane of RA patients (75) and the stroma of CCR cancer (76) by IHC/IF, as well as in freshly isolated neutrophils by immunoblotting (75), in all cases using #AF1248 Abs. We also detected IL-17B-positive neutrophils in psoriasis plaques and cytospin slides of freshly isolated neutrophils by IHC using #AF1248. However, we could not measure any IL-17B in lysates of freshly isolated/activated neutrophils either by using two different commercial ELISA or by #AF1248 immunoblotting. In the latter experiments, many proteins with MW different from that of rIL-17B were recognized by #AF1248, thus invalidating at least the cytospin results. Intriguingly, Kouri et al. (75) did detect IL-17B protein in lysates of neutrophils (95% pure), by both ELISA and immunoblotting using #AF1248. However, these authors showed only a portion of the western blot (75), thus rendering impossible to know whether additional major proteins were recognized by #AF1248. Curiously, we, Tamarozzi et al. (13) and Kouri et al. (75), all found that human neutrophils do not transcribe IL-17B mRNA under resting or activating condition. Furthermore, no IL-17B secretion from BCG-stimulated neutrophils was recently shown (14). In such regard, Koury et al. (75) suggested that IL-17B is synthesized only at the promyelocyte and myelocyte stage in the bone marrow, disappearing in mature neutrophils. However, our analysis of transcriptomes generated from all types of bone marrow cell populations (65) revealed that, similarly to IL-17A, also IL-17B is never transcribed during the different stages of neutrophil maturation. Altogether, data suggest that human neutrophils do not express IL-17B in vitro. They also suggest that the positive staining of neutrophils by IHC using AF1248 is likely due to a non-specific, IL-17B independent, binding of these Abs.
In conclusion, data shown in this study are consistent with the notion that human neutrophils are unable to express and produce IL-17A, IL-17B, or IL-17F in vitro.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of Ethic Committee of the Azienda Ospedaliera Universitaria Integrata di Verona (Italy). All the experimental protocols were approved by the Ethic Committee and all subjects gave written informed consent in accordance with the Declaration of Helsinki.

aUThOr cOnTriBUTiOns
All authors were involved in discussing and drafting the article, approved the final version to be published, and had full access to all data, taking responsibility for their integrity and analysis accuracy. In particular, NT, FA-S, SG, EG, SL, LG, and FC performed the experiments; FA-S, FB-A, NT, SL, WV, and MC analyzed the results; GG provided patients; and FS, NT, AM, WV, and MC conceived the experiments and wrote the paper.