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.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

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 a 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 stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T 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.

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.

Biological Roles of Neutrophil-Derived Granule Proteins and Cytokines.

Neutrophils, the most abundant white blood cells in human circulation, entertain intense interactions with other leukocyte subsets, platelets, and stromal cells. Molecularly, such interactions are typically communicated through proteins generated during granulopoiesis, stored in granules, or produced on demand. Here, we provide an overview of the mammalian regulation of granule protein production in the bone marrow and the de novo synthesis of cytokines by neutrophils recruited to tissues. In addition, we discuss some of the known biological roles of these protein messengers, and how neutrophil-borne granule proteins and cytokines can synergize to modulate inflammation and tumor development. Decoding the neutrophil interactome is important for therapeutically neutralizing individual proteins to putatively dampen inflammation, or for delivering modified neutrophil-borne proteins to boost host defense.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

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.

Epigenetic regulation of neutrophil development and function.

In addition to performing well-defined effector functions, neutrophils are now recognized as versatile and sophisticated cells with critical immunoregulatory roles. These include the release of a variety of proinflammatory or immunosuppressive cytokines, as well as the expression of genes with regulatory functions. Neutrophils share broad transcriptional features with monocytes, in keeping with the close developmental relation between the two cell types. However, neutrophil-specific gene expression patterns conferring cell type-specific responses to bacterial, viral or fungal components have been identified. Accumulating evidence suggest that these differences reflect the peculiar epigenomic and regulatory landscapes of neutrophils and monocytes, in turn controlled by the specific lineage-determining transcription factors shaping their identity. In this review, we will describe current knowledge on how neutrophil identity and function are controlled at the molecular level, focusing on transcriptional and chromatin regulation of neutrophil development and activation in response to inflammatory stimuli.

Mature CD10+ and immature CD10- neutrophils present in G-CSF-treated donors display opposite effects on T cells.

The identification of discrete neutrophil populations, as well as the characterization of their immunoregulatory properties, is an emerging topic under extensive investigation. In such regard, the presence of circulating CD66b+ neutrophil populations, exerting either immunosuppressive or proinflammatory functions, has been described in several acute and chronic inflammatory conditions. However, due to the lack of specific markers, the precise phenotype and maturation status of these neutrophil populations remain unclear. Herein, we report that CD10, also known as common acute lymphoblastic leukemia antigen, neutral endopeptidase, or enkephalinase, can be used as a marker that, within heterogeneous populations of circulating CD66b+ neutrophils present in inflammatory conditions, clearly distinguishes the mature from the immature ones. Accordingly, we observed that the previously described immunosuppressive neutrophil population that appears in the circulation of granulocyte colony-stimulating factor (G-CSF)-treated donors (GDs) consists of mature CD66b+CD10+ neutrophils displaying an activated phenotype. These neutrophils inhibit proliferation and interferon γ (IFNγ) production by T cells via a CD18-mediated contact-dependent arginase 1 release. By contrast, we found that immature CD66b+CD10- neutrophils, also present in GDs, display an immature morphology, promote T-cell survival, and enhance proliferation and IFNγ production by T cells. Altogether, our findings uncover that in GDs, circulating mature and immature neutrophils, distinguished by their differential CD10 expression, exert opposite immunoregulatory properties. Therefore, CD10 might be used as a phenotypic marker discriminating mature neutrophils from immature neutrophil populations present in patients with acute or chronic inflammatory conditions, as well as facilitating their isolation, to better define their specific immunoregulatory properties.

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.

Endothelin-1 receptor blockade as new possible therapeutic approach in multiple myeloma.

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin-1 (EDN1) axis, consisting of EDN1 acting through EDN-receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone-marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down-regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB-expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI-8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1-mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.

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.

Neutrophil-Expressed p21/waf1 Favors Inflammation Resolution in Pseudomonas aeruginosa Infection.

Neutrophil-associated inflammation during Pseudomonas aeruginosa lung infection is a determinant of morbidity in cystic fibrosis (CF). Neutrophil apoptosis is a key factor in inflammation resolution and is controlled by cytosolic proliferating cell nuclear antigen (PCNA). p21/Waf1, a cyclin-dependent kinase inhibitor, is a partner of PCNA, and its mRNA is up-regulated in human neutrophils during LPS challenge. We show here that, after 7 days of persistent infection with P. aeruginosa, neutrophilic inflammation was more prominent in p21(-/-) compared with wild-type (WT) mice. Notably, no intrinsic defect in the phagocytosis of apoptotic cells by macrophages was found in p21(-/-) compared with WT mice. Inflammatory cell analysis in peritoneal lavages after zymosan-induced peritonitis showed a significantly increased number of neutrophils at 48 hours in p21(-/-) compared with WT mice. In vitro analysis was consistent with delayed neutrophil apoptosis in p21(-/-) compared with WT mice. Ectopic expression of p21/waf1 in neutrophil-differentiated PLB985 cells potentiated apoptosis and reversed the prosurvival effect of PCNA. In human neutrophils, p21 messenger RNA was induced by TNF-α, granulocyte colony-stimulating factor, and LPS. Neutrophils isolated from patients with CF showed enhanced survival, which was reduced after treatment with a carboxy-peptide derived from the sequence of p21/waf1. Notably, p21/waf1 was detected by immunohistochemistry in neutrophils within lungs from patients with CF. Our data reveal a novel role for p21/waf1 in the resolution of inflammation via its ability to control neutrophil apoptosis. This mechanism may be relevant in the neutrophil-dominated inflammation observed in CF and other chronic inflammatory lung conditions.

Clinical heterogeneity of dominant chronic mucocutaneous candidiasis disease: presenting as treatment-resistant candidiasis and chronic lung disease.

In gain-of-function STAT1 mutations, chronic mucocutaneous candidiasis disease (CMCD) represents the phenotypic manifestation of a complex immunodeficiency characterized by clinical and immunological heterogeneity. We aimed to study clinical manifestations, long-term complications, molecular basis, and immune profile of patients with dominant CMCD. We identified nine patients with heterozygous mutations in STAT1, including novel amino acid substitutions (L283M, L351F, L400V). High risk of azole-resistance was observed, particularly when intermittent regimens of antifungal treatment or use of suboptimal dosage occurs. We report a case of Cryptococcosis and various bacterial and viral infections. Risk of developing bronchiectasis in early childhood or gradually evolving to chronic lung disease in adolescent or adult ages emerges. Lymphopenia is variable, likely progressing by adulthood. We conclude that continuous antifungal prophylaxis associated to drug monitoring might prevent resistance to treatment; prompt diagnosis and therapy of lung disease might control long-term progression; careful monitoring of lymphopenia-related infections might improve prognosis.

Synergistic production of TNFα and IFNα by human pDCs incubated with IFNλ3 and IL-3.

In this study, we investigated whether IFNλ3 and IL-3 reciprocally influence their capacity to activate various functions of human plasmacytoid dendritic cells (pDCs). In fact, we preliminarily observed that IFNλ3 upregulates the expression of the IL-3Rα (CD123), while IL-3 augments the expression of IFNλR1 in pDCs. As a result, we found that combination of IFNλ3 and IL-3 induces a strong potentiation in the production of TNFα, IFNα, as well as in the expression of Interferon-Stimulated Gene (ISG) mRNAs by pDCs, as compared to either IFNλ3 or IL-3 alone. In such regard, we found that endogenous IFNα autocrinally promotes the expression of ISG mRNAs in IL-3-, but not in IFNλ3 plus IL-3-, treated pDCs. Moreover, we uncovered that the production of IFNα by IFNλ3 plus IL-3-treated pDCs is mostly dependent on endogenously produced TNFα. Altogether, our data demonstrate that IFNλ3 and IL-3 collaborate to promote, at maximal levels, discrete functional responses of human pDCs.

IL-10 disrupts the Brd4-docking sites to inhibit LPS-induced CXCL8 and TNF-α expression in monocytes: Implications for chronic obstructive pulmonary disease.

BACKGROUND: IL-10 is well known for its ability to block the expression and production of numerous proinflammatory cytokines, in this manner preventing the development of excessive or chronic immune activation. IL-10-induced transcriptional repression of CXCL8 and TNFA genes consists of 2 distinct phases: an early phase, occurring rapidly and in a protein synthesis-independent manner, followed by a second phase that is more delayed and dependent on protein synthesis. OBJECTIVE: We sought to identify the mechanisms through which IL-10 rapidly and directly suppresses LPS-induced CXCL8 and TNF-α transcription, which might be defective under pathologic conditions. METHODS: The molecular events triggered by IL-10 in LPS-activated monocytes at the CXCL8 and TNFA loci were investigated by using the chromatin immunoprecipitation assay. RESULTS: Inhibition of LPS-induced CXCL8 and TNF-α expression by IL-10 proceeds through a common mechanism targeting LPS-induced phosphorylation of the nuclear factor κB p65 serine 276 residue (pS276p65). As a result, all the pS276p65-dependent events occurring at the CXCL8 and TNFA loci are consistently reduced, ultimately leading to a reduction in transcript elongation. Additionally, IL-10 selectively controls CXCL8 transcript elongation through histone deacetylase (HDAC) 2-dependent covalent chromatin modifications, disrupting the assembly of the transcriptional machinery. Remarkably, PBMCs from patients with acute-phase chronic obstructive pulmonary disease, which express negligible HDAC2 levels, are scarcely affected by IL-10 in terms of inhibition of CXCL8 expression. CONCLUSIONS: This study provides mechanistic evidence that IL-10 creates a chromatin environment that decreases the transcriptional rate of CXCL8 and TNF-α to Toll-like receptor 4-activating signals. Data identify novel molecular targets for therapeutic strategies aimed at dampening inflammation in pathologies such as chronic obstructive pulmonary disease, in which reduced intracellular HDAC2 levels have been described.

Cutting edge: An inactive chromatin configuration at the IL-10 locus in human neutrophils.

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10-producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.