CD66b-CD64dimCD115- cells in the human bone marrow represent neutrophil-committed progenitors.

Here we report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

Clinical and transcriptomic characteristics of a novel SMARCD2 mutation that disrupts neutrophil maturation and function.

We report a novel case of SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) mutation successfully treated with hematopoietic stem cell transplantation. The female patient presented delayed cord separation, chronic diarrhea, skin abscesses, skeletal dysmorphisms, and neutropenia with specific granule deficiency. Analysis of the transcriptomic profile of peripheral blood sorted mature and immature SMARCD2 neutrophils showed defective maturation process that associated with altered expression of genes related to specific, azurophilic, and gelatinase granules, such as LTF, CRISP3, PTX3, and CHI3L1. These abnormalities account for the prevalence of immature neutrophils in the peripheral blood, impaired function, and deregulated inflammatory responses.

CD14+-Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor.

Apolipoprotein CIII (ApoCIII) represents a key regulator of plasma lipid metabolism and a recognized risk factor for atherosclerosis and cardiovascular diseases. Beyond the regulation of lipoprotein trafficking, ApoCIII is also involved in endothelial dysfunction and monocyte recruitment related to atherothrombosis. With tissue factor (TF) being the primary initiator of the blood coagulation cascade, we hypothesized that ApoCIII-treated monocytes could express it. Hence, human CD14+-monocytes and autologous neutrophils were incubated with ApoCIII and sera from human subjects containing previously measured ApoCIII amounts. By RT-qPCR and ELISA, CD14+-monocytes, but not neutrophils, were found to show increased mRNA expression and production of TNFα, IL-1ß and IL-6 as well as TF mRNA once exposed to ultra-purified ApoCIII. By flow cytometry, CD14+-monocytes were found to rapidly express TF on their cell surface membrane when incubated with either ApoCIII or sera with known concentrations of ApoCIII. Finally, preincubation with specific ApoCIII-neutralizing antibodies significantly reduced the ability of most sera with known concentrations of ApoCIII to upregulate TF protein, other than partially inhibiting cytokine release, in CD14+-monocytes. In sum, herein we demonstrate that ApoCIII activates CD14+-monocytes to express TF. The data identify a potential mechanism which links circulating apolipoproteins with inflammation and atherothrombosis-related processes underlying cardiovascular risk.

Deciphering the fate of slan+ -monocytes in human tonsils by gene expression profiling.

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan+ -monocytes. In recent studies, we and other groups have reported that, in tonsils, slan marks dendritic cell (DC)-like cells, as defined by morphological, phenotypical, and functional criteria. However, subsequent investigations in lymphomas have uncovered a significant heterogeneity of tumor-infiltrating slan+ -cells, including a macrophage-like state. Based on their emerging role in tissue inflammation and cancer, herein we investigated slan+ -cell fate in tonsils by using a molecular-based approach. Hence, RNA from tonsil slan+ -cells, conventional CD1c+ DCs (cDC2) and CD11b+ CD14+ -macrophages was subjected to gene expression analysis. For comparison, transcriptomes were also obtained from blood cDC2, classical (CL), intermediate (INT), NC, and slan+ -monocytes. Data demonstrate that the main trajectory of human slan+ -monocytes infiltrating the tonsil tissue is toward a macrophage-like population, displaying molecular features distinct from those of tonsil CD11b+ CD14+ -macrophages and cDC2. These findings provide a novel view on the terminal differentiation path of slan+ -monocytes, which is relevant for inflammatory diseases and lymphomas.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2-· production in cancer cells.

BACKGROUND: The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy. METHODS: A panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection. RESULTS: We demonstrate that oncogenic mutant p53 isoforms are able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1α/UCP2 axis and mitochondrial O2-· production. We also show a correlation between the decrease of reduced thiols with a poorer clinical outcome of CLL patients bearing mutant TP53 gene. The restoration of the mitochondrial uncoupling protein 2 (UCP2) expression, as well as the addition of the radical scavenger N-acetyl-L-cysteine, reversed the oncogenic effects of mutant p53 as cellular hyper-proliferation, antiapoptotic effect, and resistance to drugs. CONCLUSIONS: The inhibition of the SESN1/AMPK/PGC-1α/UCP2 axis contributes to the pro-oxidant and oncogenic effects of mutant p53, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing mutant TP53 gene.

Cytokine production by human neutrophils: Revisiting the "dark side of the moon".

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.

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations.

BACKGROUND: Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. OBJECTIVE: We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. METHODS: Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. RESULTS: Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN-γ in response to IL-15 and reduced proliferation after stimulation with IL-2 or IL-15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN-α is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL-15, whereas STAT5 phosphorylation and DNA binding to IL-2 receptor α (IL2RA) are reduced or not affected in response to the same cytokine. CONCLUSION: These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities.

Reduction of CRKL expression in patients with partial DiGeorge syndrome is associated with impairment of T-cell functions.

BACKGROUND: Partial DiGeorge syndrome (pDGS) is caused by deletion of the 22q11.2 region. Within this region lies CrK-like (CRKL), a gene encoding an adapter protein belonging to the Crk family that is involved in the signaling cascade of IL-2, stromal cell-derived factor 1α, and type I interferon. Although recurrent infections can be observed in patients with deletion of chromosome 22 syndrome, the immune pathogenesis of this condition is yet not fully understood. OBJECTIVE: We aimed to investigate the role of CRKL in T-cell functional responses in patients affected with pDGS. METHODS: Protein expression levels and phosphorylation of CRKL were evaluated in patients with pDGS. T-cell functional assays in vitro and gene-silencing experiments were also performed. RESULTS: CRKL protein expression, as well as its phosphorylation, were reduced in all patients with pDGS, especially on IL-2 stimulation. Moreover, T cells presented impaired proliferation and reduced IL-2 production on anti-CD3/CD28 stimulation and decreased c-Fos expression. Finally, CRKL silencing in Jurkat T cells resulted in impaired T-cell proliferation and reduced c-Fos expression. CONCLUSIONS: The impaired T-cell proliferation and reduction of CRKL, phosphorylated CRKL, and c-Fos levels suggest a possible role of CRKL in functional deficiencies of T cells in patients with pDGS.

IL-10-induced microRNA-187 negatively regulates TNF-α, IL-6, and IL-12p40 production in TLR4-stimulated monocytes.

IL-10 is a potent anti-inflammatory molecule that, in phagocytes, negatively targets cytokine expression at transcriptional and posttranscriptional levels. Posttranscriptional checkpoints also represent the specific target of a recently discovered, evolutionary conserved class of small silencing RNAs known as "microRNAs" (miRNAs), which display the peculiar function of negatively regulating mRNA processing, stability, and translation. In this study, we report that activation of primary human monocytes up-regulates the expression of miR-187 both in vitro and in vivo. Accordingly, we identify miR-187 as an IL-10-dependent miRNA playing a role in IL-10-mediated suppression of TNF-α, IL-6, and the p40 subunit of IL-12 (IL-12p40) produced by primary human monocytes following activation of Toll-like receptor 4 (TLR4). Ectopic expression of miR-187 consistently and selectively reduces TNFα, IL-6, and IL-12p40 produced by LPS-activated monocytes. Conversely, the production of LPS-induced TNF-α, IL-6, and IL-12p40 is increased significantly when miR-187 expression is silenced. Our data demonstrate that miR-187 directly targets TNF-α mRNA stability and translation and indirectly decreases IL-6 and IL-12p40 expression via down-modulation of IκBζ, a master regulator of the transcription of these latter two cytokines. These results uncover an miRNA-mediated pathway controlling cytokine expression and demonstrate a central role of miR-187 in the physiological regulation of IL-10-driven anti-inflammatory responses.

Human CD34+/dim neutrophil-committed progenitors do not differentiate into neutrophil-like CXCR1+CD14+CD16- monocytes in vitro.

The advent of recent cutting-edge technologies has allowed the discovery and characterization of novel progenitors of human neutrophils, including SSCloCD66b+CD15+CD11b-CD49dhiproNeu1s, SSChiCD66b+CD15+CD11b-CD49dintproNeus2s, CD66b+CD15+CD11b+CD49d+CD101-preNeus, and Lin-CD66b+CD117+CD71+eNePs. In this research field, we recently identified CD66b-CD38+CD64dimCD115-, CD34+, and CD34dim/- cells exclusively committed to the neutrophil lineage (which we renamed as CD34+ and CD34dim/- neutrophil-committed progenitors), representing the earliest neutrophil precursors identifiable and sorted by flow cytometry. Moreover, based on their differential CD34 and CD45RA expression, we could identify 4 populations of neutrophil-committed progenitors: CD34+CD45RA-/NCP1s, CD34+CD45RA+/NCP2s, CD34dim/-CD45RA+/NCP3s, and CD34dim/-CD45RA-/NCP4s. This said, a very recent study by Ikeda and coworkers (PMID: 36862552) reported that neutrophil precursors, termed either neutrophil progenitors or "early neutrophil-committed progenitors," would generate immunosuppressive neutrophil-like CXCR1+CD14+CD16- monocytes. Hence, presuming that neutrophil progenitors/"early neutrophil-committed progenitors" correspond to neutrophil-committed progenitors, the selective neutrophil commitment that we attributed to neutrophil-committed progenitors is contradicted by Ikeda and coworkers' article. In this study, by performing a more analytical reevaluation at the phenotypic and molecular levels of the cells generated by neutrophil-committed progenitors 2 and 4 (selected as representatives of neutrophil-committed progenitors), we categorically exclude that neutrophil-committed progenitors generate neutrophil-like CXCR1+CD14+CD16- monocytes. Rather, we provide substantial evidence indicating that the cells generated by neutrophil progenitors/"early neutrophil-committed progenitors" are neutrophilic cells at a different stage of maturation, displaying moderate levels of CD14, instead of neutrophil-like CXCR1+CD14+CD16- monocytes, as pointed by Ikeda and coworkers. Hence, the conclusion that neutrophil progenitors/"early neutrophil-committed progenitors" aberrantly differentiate into neutrophil-like monocytes derives, in our opinion, from data misinterpretation.

Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors.

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs.

Severe impairment of IFN-γ and IFN-α responses in cells of a patient with a novel STAT1 splicing mutation.

Subjects affected by Signal Transducer and Activator of Transcription 1 (STAT1) deficiency have lethal bacterial and viral infections. Complete STAT1 deficiency is inherited as an autosomal recessive disease; partial STAT1 deficiency is inherited as an autosomal recessive or autosomal dominant trait. Here, we report a patient who developed disseminated mycobacteriosis early in life and had several viral infections, including herpetic skin infection and interstitial pneumonia by cytomegalovirus with severe respiratory distress. Molecular analysis of STAT1 showed a novel homozygous mutation affecting a splice site, leading to exon 3 skipping and to synthesis of a lower molecular weight STAT1 protein. This mutation leads to marked reduction of STAT1 phosphorylation; the electromobility shift assay showed a complete defect of DNA-binding activity, which accounts for the complete impairment of peripheral blood mononuclear cell functional response to both IFN-γ and IFN-α. Moreover, analysis of natural killer cells showed a defective STAT1 phosphorylation in response to IFN-α and impaired basal cytolytic activity, suggesting that the STAT1-dependent pathway might be important for natural killer cell function. These results suggested that exon 3 skipping of STAT1 leads to abnormal signaling in response to IFN-γ and IFN-α, which is associated with susceptibility to intracellular pathogens and viruses.

The slan antigen identifies the prototypical non-classical CD16+-monocytes in human blood.

Introduction: Peripheral monocytes in humans are conventionally divided into classical (CL, CD14++CD16-), intermediate (INT, CD14++CD16+) and non-classical (NC, CD14dim/-CD16++) cells, based on their expression levels of CD14 and CD16. A major fraction of the NC-monocytes has been shown to express the 6-sulfo LacNAc (slan) antigen, but whether these slan+/NC-monocytes represent the prototypical non-classical monocytes or whether they are simply a sub-fraction with identical features as the remainder of NC monocytes is still unclear. Methods: We analyzed transcriptome (by bulk and single cell RNA-seq), proteome, cell surface markers and production of discrete cytokines by peripheral slan+/NC- and slan-/NC-monocytes, in comparison to total NC-, CL- and INT- monocytes. Results: By bulk RNA-seq and proteomic analysis, we found that slan+/NC-monocytes express higher levels of genes and proteins specific of NC-monocytes than slan-/NC-monocytes do. Unsupervised clustering of scRNA-seq data generated one cluster of NC- and one of INT-monocytes, where all slan+/NC-monocytes were allocated to the NC-monocyte cluster, while slan-/NC-monocytes were found, in part (13.4%), within the INT-monocyte cluster. In addition, total NC- and slan-/NC-monocytes, but not slan+/NC-monocytes, were found by both bulk RNA-seq and scRNA-seq to contain a small percentage of natural killer cells. Conclusion: In addition to comparatively characterize total NC-, slan-/NC- and slan+/NC-monocyte transcriptomes and proteomes, our data prove that slan+/NC-, but not slan-/NC-, monocytes are more representative of prototypical NC-monocytes.

Plasmacytoid Dendritic Cell, Slan+-Monocyte and Natural Killer Cell Counts Function as Blood Cell-Based Biomarkers for Predicting Responses to Immune Checkpoint Inhibitor Monotherapy in Non-Small Cell Lung Cancer Patients.

The advent of immune checkpoint inhibitors (ICIs), for instance, programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockers, has greatly improved the outcome of patients affected by non-small cell lung cancer (NSCLC). However, most NSCLC patients either do not respond to ICI monotherapy or develop resistance to it after an initial response. Therefore, the identification of biomarkers for predicting the response of patients to ICI monotherapy represents an urgent issue. Great efforts are currently dedicated toward identifying blood-based biomarkers to predict responses to ICI monotherapy. In this study, more commonly utilized blood-based biomarkers, such as the neutrophil-to-lymphocyte ratio (NLR) and the lung immune prognostic index (LIPI) score, as well as the frequency/number and activation status of various types of circulating innate immune cell populations, were evaluated in NSCLC patients at baseline before therapy initiation. The data indicated that, among all the parameters tested, low plasmacytoid dendritic cell (pDC), slan+-monocyte and natural killer cell counts, as well as a high LIPI score and elevated PD-L1 expression levels on type 1 conventional DCs (cDC1s), were independently correlated with a negative response to ICI therapy in NSCLC patients. The results from this study suggest that the evaluation of innate immune cell numbers and phenotypes may provide novel and promising predictive biomarkers for ICI monotherapy in NSCLC patients.

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion.

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease.

Neutrophils inhibit γδ T cell functions in the imiquimod-induced mouse model of psoriasis.

Background: Psoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood. Methods: In this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development. Results: By analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice. Conclusions: Overall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells.

Uncovering an IL-10-dependent NF-kappaB recruitment to the IL-1ra promoter that is impaired in STAT3 functionally defective patients.

The interleukin 1 receptor antagonist (IL-1ra) is an important negative regulator of the inflammatory response, whose genetic deficiency has been recently shown to cause a severe autoinflammatory syndrome in humans. In this study we characterized the molecular mechanisms whereby interleukin 10 (IL-10) potentiates IL-1ra transcription in LPS-stimulated monocytes and neutrophils. Using chromatin immunoprecipitation, we show that although NF-kappaBp65 and NF-kappaBp50 proteins accumulate into the nuclei and bind to the IkappaB alpha promoter during LPS stimulation, they are not recruited to the kappaB sites of the IL-1ra promoter. However, in response to LPS plus IL-10, which were found to induce chromatin acetylation, recruitment of both NF-kappaBp65 and NF-kappaBp50 to the IL-1ra promoter efficiently occurs in a STAT3-dependent manner. Accordingly, in neutrophils from hyper-IgE syndrome patients, who carry a nonfunctional STAT3, IL-10 failed to promote NF-kappaBp65 recruitment to the IL-1ra promoter and consequently to potentiate LPS-induced IL-1ra transcription. Altogether our findings uncover a novel mechanism whereby IL-10-activated STAT3 modulates IL-1ra transcription in LPS-treated phagocytes by making IL-1ra promoter accessible to readily available nuclear NF-kappaB.

Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8.

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/EBPß, 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 LDTF-marked 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-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor κB (NF-κB) 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.

Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39.

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL-12A, IL-23A, and IL-27A) with two ß (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFNγ in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFNγ plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo.