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.

Current knowledge on the early stages of human neutropoiesis.

Polymorphonuclear neutrophils are no longer considered as a homogeneous population of terminally differentiated and short-lived cells that belong to the innate immune system only. In fact, data from the past decades have uncovered that neutrophils exhibit large phenotypic heterogeneity and functional versatility that render them more plastic than previously thought. Hence, their precise role as effector cells in inflammation, in immune response and in other pathophysiological processes, including tumors, needs to be better evaluated. In such a complex scenario, common knowledge of the differentiation of neutrophils in bone marrow refers to lineage precursors, starting from the still poorly defined myeloblasts, and proceeding sequentially to promyelocytes, myelocytes, metamyelocytes, band cells, segmented neutrophils, and mature neutrophils, with each progenitor stage being more mature and better characterized. Thanks to the development and utilization of cutting-edge technologies, novel information about neutrophil precursors at stages earlier than the promyelocytes, hence closer to the hematopoietic stem cells, is emerging. Accordingly, this review discusses the main findings related to the very early precursors of human neutrophils and provides our perspectives on human neutropoiesis.

On the Improper Use of the Term High-Density Neutrophils.

Recent studies have revealed that neutrophils exhibit an unsuspected heterogeneity. In this context, the term high-density neutrophils (HDNs) has recently gained ground to define nothing more than neutrophils displaying an unaltered normal density. Therefore, as discussed here, we argue that the HDNs term must be avoided, as it is confounding and scientifically inappropriate.

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.

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.

IL-10-producing B cells are characterized by a specific methylation signature.

Among the family of regulatory B cells, the subset able to produce interleukin-10 (IL-10) is the most studied, yet its biology is still a matter of investigation. The DNA methylation profiling of the il-10 gene locus revealed a novel epigenetic signature characterizing murine B cells ready to respond through IL-10 synthesis: a demethylated region located 4.5 kb from the transcription starting site (TSS), that we named early IL10 regulatory region (eIL10rr). This feature allows to distinguish B cells that are immediately prone and developmentally committed to IL-10 production from those that require a persistent stimulation to exert an IL-10-mediated regulatory function. These late IL-10 producers are instead characterized by a delayed IL10 regulatory region (dIL10rr), a partially demethylated DNA portion located 9 kb upstream from the TSS. A demethylated region was also found in human IL-10-producing B cells and, very interestingly, in some B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, characterized by an immunosuppressive microenvironment. Our findings define murine and human regulatory B cells as an epigenetically controlled functional state of mature B cell subsets and open a new perspective on IL-10 regulation in B cells in homeostasis and disease.

Neutrophil-derived chemokines on the road to immunity.

During recent years, it has become clear that polymorphonuclear neutrophils are remarkably versatile cells, whose functions go far beyond phagocytosis and killing. In fact, besides being involved in primary defense against infections-mainly through phagocytosis, generation of toxic molecules, release of toxic enzymes and formation of extracellular traps-neutrophils have been shown to play a role in finely regulating the development and the evolution of inflammatory and immune responses. These latter neutrophil-mediated functions occur by a variety of mechanisms, including the production of newly manufactured cytokines. Herein, we provide a general overview of the chemotactic cytokines/chemokines that neutrophils can potentially produce, either under inflammatory/immune reactions or during their activation in more prolonged processes, such as in tumors. We highlight recent observations generated from studying human or rodent neutrophils in vitro and in vivo models. We also discuss the biological significance of neutrophil-derived chemokines in the context of infectious, neoplastic and immune-mediated diseases. The picture that is emerging is that, given their capacity to produce and release chemokines, neutrophils exert essential functions in recruiting, activating and modulating the activities of different leukocyte populations.

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.

Human neutrophils in the saga of cellular heterogeneity: insights and open questions.

Recent findings have uncovered novel fascinating aspects of the biology of neutrophils, which ultimately attribute to these cells a broader role in inflammation and immunity. One aspect that is currently under intensive investigation is the notion of neutrophil 'heterogeneity'. Studies examining neutrophils in a variety of acute and chronic inflammatory conditions report, in fact, the recovery of CD66b(+) cells displaying neutrophil-like morphology at different degrees of maturation/activation, able to exert either immunosuppressive or proinflammatory properties. These heterogeneous populations of mature and immature neutrophils are indicated with a variety of names, including 'low density neutrophils (LDNs)', 'low density granulocytes (LDGs)', 'granulocytic-myeloid derived suppressor cells (G-MDSCs)', and immunosuppressive neutrophils. However, due to the lack of discrete markers that can unequivocally allow their specific identification and isolation, the precise phenotype and function of all these presumably novel, neutrophil-like, populations have not been correctly defined yet. Aim of this article is to summarize current knowledge on the mature and immature neutrophil populations described to date, featuring immunosuppressive or proinflammatory properties, often defined as 'subsets', as well as to critically discuss unresolved issues in the field.

Recent advances on the crosstalk between neutrophils and B or T lymphocytes.

An increasing body of literature supports a role for neutrophils as players in the orchestration of adaptive immunity. During acute and chronic inflammatory conditions, neutrophils rapidly migrate not only to sites of inflammation, but also to draining lymph nodes and spleen, where they engage bidirectional interactions with B- and T-lymphocyte subsets. Accordingly, a relevant role of neutrophils in modulating B-cell responses under homeostatic conditions has recently emerged. Moreover, specialized immunoregulatory properties towards B or T cells acquired by distinct neutrophil populations, originating under pathological conditions, have been consistently described. In this article, we summarize the most recent data from human studies and murine models on the ability of neutrophils to modulate adaptive immune responses under physiological and pathological conditions and the mechanisms behind these processes.

Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates.

In cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease.

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.

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.

Never underestimate the power of a neutrophil.

Polymorphonuclear neutrophil leukocytes (PMNs) have long been viewed as short-lived effector cells capable of phagocytosing pathogens and mediating tissue damage. In this issue of Immunity, Zhang et al. (2009) shed fresh light on the plasticity and immunoregulatory functions of PMNs.

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.

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.

Social networking of human neutrophils within the immune system.

It is now widely recognized that neutrophils are highly versatile and sophisticated cells that display de novo synthetic capacity and may greatly extend their lifespan. In addition, concepts such as "neutrophil heterogeneity" and "neutrophil plasticity" have started to emerge, implying that, under pathological conditions, neutrophils may differentiate into discrete subsets defined by distinct phenotypic and functional profiles. A number of studies have shown that neutrophils act as effectors in both innate and adaptive immunoregulatory networks. In fact, once recruited into inflamed tissues, neutrophils engage into complex bidirectional interactions with macrophages, natural killer, dendritic and mesenchymal stem cells, B and T lymphocytes, or platelets. As a result of this cross-talk, mediated either by contact-dependent mechanisms or cell-derived soluble factors, neutrophils and target cells reciprocally modulate their survival and activation status. Altogether, these novel aspects of neutrophil biology have shed new light not only on the potential complex roles that neutrophils play during inflammation and immune responses, but also in the pathogenesis of several inflammatory disorders including infection, autoimmunity, and cancer.