A nox2/cybb zebrafish mutant with defective myeloid cell reactive oxygen species production displays normal initial neutrophil recruitment to sterile tail injuries.

Reactive oxygen species are important effectors and modifiers of the acute inflammatory response, recruiting phagocytes including neutrophils to sites of tissue injury. In turn, phagocytes such as neutrophils are both consumers and producers of reactive oxygen species. Phagocytes including neutrophils generate reactive oxygen species in an oxidative burst through the activity of a multimeric phagocytic nicotinamide adenine dinucleotide phosphate oxidase complex. Mutations in the NOX2/CYBB (previously gp91phox) nicotinamide adenine dinucleotide phosphate oxidase subunit are the commonest cause of chronic granulomatous disease, a disease characterized by infection susceptibility and an inflammatory phenotype. To model chronic granulomatous disease, we made a nox2/cybb zebrafish (Danio rerio) mutant and demonstrated it to have severely impaired myeloid cell reactive oxygen species production. Reduced early survival of nox2 mutant embryos indicated an essential requirement for nox2 during early development. In nox2/cybb zebrafish mutants, the dynamics of initial neutrophil recruitment to both mild and severe surgical tailfin wounds was normal, suggesting that excessive neutrophil recruitment at the initiation of inflammation is not the primary cause of the "sterile" inflammatory phenotype of chronic granulomatous disease patients. This nox2 zebrafish mutant adds to existing in vivo models for studying reactive oxygen species function in myeloid cells including neutrophils in development and disease.

Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification.

Hematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain-containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells.

Biallelic deleterious germline SH2B3 variants cause a novel syndrome of myeloproliferation and multi-organ autoimmunity.

SH2B3 is a negative regulator of multiple cytokine receptor signalling pathways in haematopoietic tissue. To date, a single kindred has been described with germline biallelic loss-of-function SH2B3 variants characterized by early onset developmental delay, hepatosplenomegaly and autoimmune thyroiditis/hepatitis. Herein, we described two further unrelated kindreds with germline biallelic loss-of-function SH2B3 variants that show striking phenotypic similarity to each other as well as to the previous kindred of myeloproliferation and multi-organ autoimmunity. One proband also suffered severe thrombotic complications. CRISPR-Cas9 gene editing of zebrafish sh2b3 created assorted deleterious variants in F0 crispants, which manifest significantly increased number of macrophages and thrombocytes, partially replicating the human phenotype. Treatment of the sh2b3 crispant fish with ruxolitinib intercepted this myeloproliferative phenotype. Skin-derived fibroblasts from one patient demonstrated increased phosphorylation of JAK2 and STAT5 after stimulation with IL-3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these additional probands and functional data in combination with the previous kindred provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association for a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations.

Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

Antibiotic-chemoattractants enhance neutrophil clearance of Staphylococcus aureus.

The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term 'antibiotic-chemoattractants', consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development.

Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions.

Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.

Pioneer neutrophils release chromatin within in vivo swarms.

Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

The Resistance to Host Antimicrobial Peptides in Infections Caused by Daptomycin-Resistant Staphylococcus aureus.

Daptomycin is an important antibiotic for the treatment of infections caused by Staphylococcus aureus. The emergence of daptomycin resistance in S. aureus is associated with treatment failure and persistent infections with poor clinical outcomes. Here, we investigated host innate immune responses against clinically derived, daptomycin-resistant (DAP-R) and -susceptible S. aureus paired isolates using a zebrafish infection model. We showed that the control of DAP-R S. aureus infections was attenuated in vivo due to cross-resistance to host cationic antimicrobial peptides. These data provide mechanistic understanding into persistent infections caused by DAP-R S. aureus and provide crucial insights into the adaptive evolution of this troublesome pathogen.

Utility of clinical comprehensive genomic characterization for diagnostic categorization in patients presenting with hypocellular bone marrow failure syndromes.

Bone marrow failure (BMF) related to hypoplasia of hematopoietic elements in the bone marrow is a heterogeneous clinical entity with a broad differential diagnosis including both inherited and acquired causes. Accurate diagnostic categorization is critical to optimal patient care and detection of genomic variants in these patients may provide this important diagnostic and prognostic information. We performed real-time, accredited (ISO15189) comprehensive genomic characterization including targeted sequencing and whole exome sequencing in 115 patients with BMF syndrome (median age 24 years, range 3 months - 81 years). In patients with clinical diagnoses of inherited BMF syndromes, acquired BMF syndromes or clinically unclassifiable BMF we detected variants in 52% (12/23), 53% (25/47) and 56% (25/45) respectively. Genomic characterization resulted in a change of diagnosis in 30/115 (26%) including the identification of germline causes for 3/47 and 16/45 cases with pre-test diagnoses of acquired and clinically unclassifiable BMF respectively. The observed clinical impact of accurate diagnostic categorization included choice to perform allogeneic stem cell transplantation, disease-specific targeted treatments, identification of at-risk family members and influence of sibling allogeneic stem cell donor choice. Multiple novel pathogenic variants and copy number changes were identified in our cohort including in TERT, FANCA, RPS7 and SAMD9. Whole exome sequence analysis facilitated the identification of variants in two genes not typically associated with a primary clinical manifestation of BMF but also demonstrated reduced sensitivity for detecting low level acquired variants. In conclusion, genomic characterization can improve diagnostic categorization of patients presenting with hypoplastic BMF syndromes and should be routinely performed in this group of patients.

Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy.

Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.

ß-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

The Neutrophil Nucleus: An Important Influence on Neutrophil Migration and Function.

Neutrophil nuclear morphology has historically been used in haematology for neutrophil identification and characterisation, but its exact role in neutrophil function has remained enigmatic. During maturation, segmentation of the neutrophil nucleus into its mature, multi-lobulated shape is accompanied by distinct changes in nuclear envelope composition, resulting in a unique nucleus that is believed to be imbued with extraordinary nuclear flexibility. As a rate-limiting factor for cell migration, nuclear morphology and biomechanics are particularly important in the context of neutrophil migration during immune responses. Being an extremely plastic and fast migrating cell type, it is to be expected that neutrophils have an especially deformable nucleus. However, many questions still surround the dynamic capacities of the neutrophil nucleus, and which nuclear and cytoskeletal elements determine these dynamics. The biomechanics of the neutrophil nucleus should also be considered for their influences on the production of neutrophil extracellular traps (NETs), given this process sees the release of chromatin "nets" from nucleoplasm to extracellular space. Although past studies have investigated neutrophil nuclear composition and shape, in a new era of more sophisticated biomechanical and genetic techniques, 3D migration studies, and higher resolution microscopy we now have the ability to further investigate and understand neutrophil nuclear plasticity at an unprecedented level. This review addresses what is currently understood about neutrophil nuclear structure and its role in migration and the release of NETs, whilst highlighting open questions surrounding neutrophil nuclear dynamics.

Toxicological assessment of additively manufactured methacrylates for medical devices in dentistry.

The paucity of information on the biological risks of photopolymers in additive manufacturing is a major challenge for the uptake of the technology in the construction of medical devices in dentistry. In this paper, the biocompatibility of methacrylates for denture bases, splints, retainers and surgical guides were evaluated using the innovative zebrafish embryo model, which is providing a high potential for toxicity profiling of photopolymers and has high genetic similarity to humans. Toxicological data obtained confirmed gradations of toxicity influenced by ethanol treatment, exposure scenarios and extraction vehicles. In direct exposure tests, juvenile fish exposed to non-treated methacrylates in ultrapure water showed accelerated toxicity endpoints compared to fish in transparent E3 medium. Similarly, toxic extracts induced mostly acute responses (embryonic mortality) in contrast to cumulative chronic (sublethal and teratogenic effects) in direct exposure. Methacrylates composed of >60% Ethoxylated bisphenol A dimethacrylate produced a relatively lower conversion rate in FTIR spectroscopy, but were safe in zebrafish bioassays after ethanol treatment. The study affirms that biocompatibility was influenced primarily by physico-chemical characteristics of the materials, which subsequently influenced their residual monomer content before and after immersion in ethanol. Given the precautionary implications of the study, we propose a 3-tiered approach i.e. using approved materials, apposite manufacturing parameters and post-processing techniques that together guarantee optimal results for medical devices. STATEMENT OF SIGNIFICANCE: This study is timely and relevant since there is limited published literature that precisely describes the toxicological properties of additively manufactured methacrylates despite their increased popularity for medical devices. While it is generally accepted that the zebrafish excels as a model system for developmental toxicity, a further examination of its utility in this study using different protocols provides basis for its consideration and adoption at a crucial time when there is a lack of consensus regarding the most suited biological assessment methods for medical devices.

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation.

Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB-driven production of IL-1ß and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.

Hematopoietic growth factors: the scenario in zebrafish.

Humoral regulation by ligand/receptor interactions is a fundamental feature of vertebrate hematopoiesis. Zebrafish are an established vertebrate animal model of hematopoiesis, sharing with mammals conserved genetic, molecular and cell biological regulatory mechanisms. This comprehensive review considers zebrafish hematopoiesis from the perspective of the hematopoietic growth factors (HGFs), their receptors and their actions. Zebrafish possess multiple HGFs: CSF1 (M-CSF) and CSF3 (G-CSF), kit ligand (KL, SCF), erythropoietin (EPO), thrombopoietin (THPO/TPO), and the interleukins IL6, IL11, and IL34. Some ligands and/or receptor components have been duplicated by various mechanisms including the teleost whole genome duplication, adding complexity to the ligand/receptor interactions possible, but also providing examples of several different outcomes of ligand and receptor subfunctionalization or neofunctionalization. CSF2 (GM-CSF), IL3 and IL5 and their receptors are absent from zebrafish. Overall the humoral regulation of hematopoiesis in zebrafish displays considerable similarity with mammals, which can be applied in biological and disease modelling research.

Splicing dysfunction and disease: The case of granulopoiesis.

Splicing is a ubiquitous process in eukaryotic cells, long recognised as contributing to diversity of the transcriptome. More specifically, splicing fine-tunes the transcriptome output for highly individual outcomes at different stages of cell development, in specific timeframes, which when perturbed result in significant human diseases. Granulopoiesis provides a particularly well studied example of how splicing can be a highly flexible but tightly regulated process. Focusing on the specific case of granulopoiesis, this review surveys the contribution of cis-splicing variations in individual genes and the trans-regulation of global splicing outcomes during the normal development of neutrophils. Further, the contribution of splicing dysfunction to the pathogenesis of diseases of neutrophil number, function and maturation including hereditary neutropenia, myelodysplasia, and acute myeloid leukaemia is explored.