Bipotential B-neutrophil progenitors are present in human and mouse bone marrow and emerge in the periphery upon stress hematopoiesis.

Hematopoiesis is a tightly regulated process that gets skewed toward myelopoiesis. This restrains lymphopoiesis, but the role of lymphocytes in this process is not well defined. To unravel the intricacies of neutrophil responses in COVID-19, we performed bulk RNAseq on neutrophils from healthy controls and COVID-19 patients. Principal component analysis revealed distinguishing neutrophil gene expression alterations in COVID-19 patients. ICU and ward patients displayed substantial transcriptional changes, with ICU patients exhibiting a more pronounced response. Intriguingly, neutrophils from COVID-19 patients, notably ICU patients, exhibited an enrichment of immunoglobulin (Ig) and B cell lineage-associated genes, suggesting potential lineage plasticity. We validated our RNAseq findings in a larger cohort. Moreover, by reanalyzing single-cell RNA sequencing (scRNAseq) data on human bone marrow (BM) granulocytes, we identified the cluster of granulocyte-monocyte progenitors (GMP) enriched with Ig and B cell lineage-associated genes. These cells with lineage plasticity may serve as a resource depending on the host's needs during severe systemic infection. This distinct B cell subset may play a pivotal role in promoting myelopoiesis in response to infection. The scRNAseq analysis of BM neutrophils in infected mice further supported our observations in humans. Finally, our studies using an animal model of acute infection implicate IL-7/GM-CSF in influencing neutrophil and B cell dynamics. Elevated GM-CSF and reduced IL-7 receptor expression in COVID-19 patients imply altered hematopoiesis favoring myeloid cells over B cells. Our findings provide novel insights into the relationship between the B-neutrophil lineages during severe infection, hinting at potential implications for disease pathogenesis. IMPORTANCE: This study investigates the dynamics of hematopoiesis in COVID-19, focusing on neutrophil responses. Through RNA sequencing of neutrophils from healthy controls and COVID-19 patients, distinct gene expression alterations are identified, particularly in ICU patients. Notably, neutrophils from COVID-19 patients, especially in the ICU, exhibit enrichment of immunoglobulin and B cell lineage-associated genes, suggesting potential lineage plasticity. Validation in a larger patient cohort and single-cell analysis of bone marrow granulocytes support the presence of granulocyte-monocyte progenitors with B cell lineage-associated genes. The findings propose a link between B-neutrophil lineages during severe infection, implicating a potential role for these cells in altered hematopoiesis favoring myeloid cells over B cells. Elevated GM-CSF and reduced IL-7 receptor expression in stress hematopoiesis suggest cytokine involvement in these dynamics, providing novel insights into disease pathogenesis.

Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation.

Nucleoporins, the components of nuclear pore complexes (NPCs), can play cell type- and tissue-specific functions. Yet, the physiological roles and mechanisms of action for most NPC components have not yet been established. We report that Nup358, a nucleoporin linked to several myeloid disorders, is required for the developmental progression of early myeloid progenitors. We found that Nup358 ablation in mice results in the loss of myeloid-committed progenitors and mature myeloid cells and the accumulation of myeloid-primed multipotent progenitors (MPPs) in bone marrow. Accumulated MPPs in Nup358 knockout mice are greatly restricted to megakaryocyte/erythrocyte-biased MPP2, which fail to progress into committed myeloid progenitors. Mechanistically, we found that Nup358 is required for histone deacetylase 3 (HDAC3) nuclear import and function in MPP2 cells and established that this nucleoporin regulates HDAC3 nuclear translocation in a SUMOylation-independent manner. Our study identifies a critical function for Nup358 in myeloid-primed MPP2 differentiation and uncovers an unexpected role for NPCs in the early steps of myelopoiesis.

Single-cell proteomics and transcriptomics capture eosinophil development and identify the role of IL-5 in their lineage transit amplification.

The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.

Single-cell immune profiling reveals markers of emergency myelopoiesis that distinguish severe from mild respiratory syncytial virus disease in infants.

Whereas most infants infected with respiratory syncytial virus (RSV) show no or only mild symptoms, an estimated 3 million children under five are hospitalized annually due to RSV disease. This study aimed to investigate biological mechanisms and associated biomarkers underlying RSV disease heterogeneity in young infants, enabling the potential to objectively categorize RSV-infected infants according to their medical needs. Immunophenotypic and functional profiling demonstrated the emergence of immature and progenitor-like neutrophils, proliferative monocytes (HLA-DRLow , Ki67+), impaired antigen-presenting function, downregulation of T cell response and low abundance of HLA-DRLow B cells in severe RSV disease. HLA-DRLow monocytes were found as a hallmark of RSV-infected infants requiring hospitalization. Complementary transcriptomics identified genes associated with disease severity and pointed to the emergency myelopoiesis response. These results shed new light on mechanisms underlying the pathogenesis and development of severe RSV disease and identified potential new candidate biomarkers for patient stratification.

Telomerase RNA-based aptamers restore defective myelopoiesis in congenital neutropenic syndromes.

Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus facilitating myeloid gene expression. The CR4/CR5 domain of TERC is known to play this role, since a mutation of this domain found in dyskeratosis congenita (DC) patients decreases its affinity for RNA Pol II, impairing its myelopoietic activity as a result. In this study, we report that two aptamers, short single-stranded oligonucleotides, based on the CR4/CR5 domain were able to increase myelopoiesis without affecting erythropoiesis in zebrafish. Mechanistically, the aptamers functioned as full terc; that is, they increased the expression of master myeloid genes, independently of endogenous terc, by interacting with RNA Pol II and with the terc-binding sequences of the regulatory regions of such genes, enforcing their transcription. Importantly, aptamers harboring the CR4/CR5 mutation that was found in DC patients failed to perform all these functions. The therapeutic potential of the aptamers for treating neutropenia was demonstrated in several preclinical models. The findings of this study have identified two potential therapeutic agents for DC and other neutropenic patients.

MicroRNA-223 limits murine hemogenic endothelial cell specification and myelopoiesis.

Embryonic definitive hematopoiesis generates hematopoietic stem and progenitor cells (HSPCs) that are essential for the establishment and maintenance of the adult blood system. This process requires the specification of a subset of vascular endothelial cells (ECs) to become hemogenic ECs and to have subsequent endothelial-to-hematopoietic transition (EHT), and the underlying mechanisms are largely undefined. We identified microRNA (miR)-223 as a negative regulator of murine hemogenic EC specification and EHT. Loss of miR-223 leads to increased formation of hemogenic ECs and HSPCs, which is associated with increased retinoic acid signaling, which we previously showed as promoting hemogenic EC specification. Additionally, loss of miR-223 leads to the generation of myeloid-biased hemogenic ECs and HSPCs, which results in an increased proportion of myeloid cells throughout embryonic and postnatal life. Our findings identify a negative regulator of hemogenic EC specification and highlight the importance of this process for the establishment of the adult blood system.

Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis.

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and ß-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age.

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the blood system. Despite their multipotency, MPPs display poorly understood lineage bias. Here, we examine whether lineage-specifying transcription factors, such as the B-lineage determinant EBF1, regulate lineage preference in early progenitors. We detect low-level EBF1 expression in myeloid-biased MPP3 and lymphoid-biased MPP4 cells, coinciding with expression of the myeloid determinant C/EBPα. Hematopoietic deletion of Ebf1 results in enhanced myelopoiesis and reduced HSC repopulation capacity. Ebf1-deficient MPP3 and MPP4 cells exhibit an augmented myeloid differentiation potential and a transcriptome with an enriched C/EBPα signature. Correspondingly, EBF1 binds the Cebpa enhancer, and the deficiency and overexpression of Ebf1 in MPP3 and MPP4 cells lead to an up- and downregulation of Cebpa expression, respectively. In addition, EBF1 primes the chromatin of B-lymphoid enhancers specifically in MPP3 cells. Thus, our study implicates EBF1 in regulating myeloid/lymphoid fate bias in MPPs by constraining C/EBPα-driven myelopoiesis and priming the B-lymphoid fate.

Robust temporal map of human in vitro myelopoiesis using single-cell genomics.

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

TIRAP drives myelosuppression through an Ifnγ-Hmgb1 axis that disrupts the endothelial niche in mice.

Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.

Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis.

In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 T cells toward an activation state that includes IFN-γ production by these T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies.

Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA (terc) binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish terc harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, TERC regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a TERC mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that TERC acts as a transcription factor, revealing a target for therapeutic intervention in DC patients.

Optimized Intracellular Staining Reveals Heterogeneous Cytokine Production Ability of Murine and Human Hematopoietic Stem and Progenitor Cells.

Under stress conditions, hematopoietic stem and progenitor cells (HSPCs) can translate danger signals into a plethora of cytokine signals. These cytokines, or more precisely their combination, instruct HSPCs to modify the magnitude and composition of hematopoietic output in response to the threat, but investigations into the heterogeneous cytokine expression and regulatory mechanisms are hampered by the technical difficulty of measuring cytokine levels in HSPCs at the single-cell level. Here, we optimized a flow cytometry-based method for the simultaneous assessment of multiple intracellular cytokines in HSPCs. By selecting an optimal combination of cytokine restimulation reagents, protein transport inhibitors, and culture supplements, an optimized restimulation protocol for intracellular staining was developed. Using this method, we successfully examined expression levels of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in murine and human HSPC subsets under steady-state or different stress conditions. Different cytokine expression patterns were observed, suggesting distinct regulatory modes of cytokine production dependent on the HSPC subset, cytokine, disease, organ, and species. Collectively, this technical advance may help to obtain a better understanding of the nature of HSPC heterogeneity on the basis of differential cytokine production.

HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.

Runx1 regulates zebrafish neutrophil maturation via synergistic interaction with c-Myb.

Neutrophils play an essential role in the innate immune defense system in vertebrates. During hematopoiesis, the full function of neutrophils involves maturation of granules and related enzymes. Yet, transcription regulators that promote neutrophil maturation remain largely undefined. Here, two hematopoiesis-defective zebrafish mutants, runx1w84x and c-mybhkz3, were used to investigate the in vivo roles of Runx1 in cooperation with c-Myb in regulating neutrophil maturation. Loss of runx1 impairs primitive neutrophil development. Additional regulation of c-myb+/- and c-myb-/- induces a more severe phenotypes suggesting a synergistic genetic interaction between c-myb and runx1 in neutrophil maturation. Further studies revealed that the two transcription factors act cooperatively to control neutrophil maturation processes via transactivating a series of neutrophil maturation-related genes. These data reveal the in vivo roles of Runx1 in regulating primitive neutrophil maturation while also indicating a novel genetic and molecular orchestration of Runx1 and c-Myb in myeloid cell development. The study will provide new evidence on the regulation of neutrophil maturation during hematopoiesis.

Two patients of trisomy 21 with transient abnormal myelopoiesis with hypereosinophilia without blasts in peripheral blood smears.

Clinical diagnosis of transient abnormal myelopoiesis (TAM) relies on the detection of characteristic blasts and leukocytosis in peripheral blood. We report two patients of trisomy 21 with TAM with hypereosinophilia, who had neither circulating blasts nor leukocytosis. Genetic testing of polymorphonuclear leukocytes isolated from whole blood revealed heterozygous mutations in GATA1, suggesting that the mutations were harbored in increased eosinophils. Both patients had direct hyperbilirubinemia and one died of liver fibrosis. Our findings emphasize the importance of screening for GATA1 mutations in neonatal infants with Down syndrome and hypereosinophilia even if blasts are not detected in peripheral blood smears.