Granulocyte-Monocyte Progenitors and Monocyte-Dendritic Cell Progenitors Independently Produce Functionally Distinct Monocytes.

Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar but distinct monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6Chi) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of "neutrophil-like" monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection.

Multicenter Systems Analysis of Human Blood Reveals Immature Neutrophils in Males and During Pregnancy.

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20-30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women.

The Granulocyte Progenitor Stage Is a Key Target of IRF8-Mediated Regulation of Myeloid-Derived Suppressor Cell Production.

Alterations in myelopoiesis are common across various tumor types, resulting in immature populations termed myeloid-derived suppressor cells (MDSCs). MDSC burden correlates with poorer clinical outcomes, credited to their ability to suppress antitumor immunity. MDSCs consist of two major subsets, monocytic and polymorphonuclear (PMN). Intriguingly, the latter subset predominates in many patients and tumor models, although the mechanisms favoring PMN-MDSC responses remain poorly understood. Ordinarily, lineage-restricted transcription factors regulate myelopoiesis that collectively dictate cell fate. One integral player is IFN regulatory factor (IRF)-8, which promotes monocyte/dendritic cell differentiation while limiting granulocyte development. We recently showed that IRF8 inversely controls MDSC burden in tumor models, particularly the PMN-MDSC subset. However, where IRF8 acts in the pathway of myeloid differentiation to influence PMN-MDSC production has remained unknown. In this study, we showed that: 1) tumor growth was associated with a selective expansion of newly defined IRF8lo granulocyte progenitors (GPs); 2) tumor-derived GPs had an increased ability to form PMN-MDSCs; 3) tumor-derived GPs shared gene expression patterns with IRF8-/- GPs, suggesting that IRF8 loss underlies GP expansion; and 4) enforced IRF8 overexpression in vivo selectively constrained tumor-induced GP expansion. These findings support the hypothesis that PMN-MDSCs result from selective expansion of IRF8lo GPs, and that strategies targeting IRF8 expression may limit their load to improve immunotherapy efficacy.

Regulation of eosinophil development and survival.

PURPOSE OF REVIEW: Eosinophils are a subset of granulocytes generally associated with type 2 immune responses. They can contribute to protection against helminths but also mediate pro-inflammatory functions during allergic immune responses. Only recently, eosinophils were also found to exert many other functions such as regulation of glucose and fat metabolism, thermogenesis, survival of plasma cells, and antitumor activity. The mechanisms that control eosinophil development and survival are only partially understood. RECENT FINDINGS: Here we review new findings regarding the role of cell-extrinsic and cell-intrinsic factors for eosinophilopoiesis and eosinophil homeostasis. Several reports provide new insights in the regulation of eosinophil development by transcription factors, miRNAs and epigenetic modifications. Danger signals like lipopolysaccharide or alarmins can activate eosinophils but also prolong their lifespan. We further reflect on the observations that eosinophil development is tightly controlled by the unfolded protein stress response and formation of cytoplasmic granules. SUMMARY: Eosinophils emerge as important regulators of diverse biological processes. Their differentiation and survival is tightly regulated by factors that are still poorly understood. Newly identified pathways involved in eosinophilopoiesis and eosinophil homeostasis may lead to development of new therapeutic options for treatment of eosinophil-associated diseases.

miRNA-130a regulates C/EBP-ε expression during granulopoiesis.

CCAAT/enhancer binding protein-ε (C/EBP-ε) is considered a master transcription factor regulating terminal neutrophil maturation. It is essential for expression of secondary granule proteins, but it also regulates proliferation, cell cycle, and maturation during granulopoiesis. Cebpe(-/-) mice have incomplete granulocytic differentiation and increased sensitivity toward bacterial infections. The amount of C/EBP-ε messenger RNA (mRNA) increases with maturation from myeloblasts with peak level in myelocytes (MC)/metamyelocytes (MM), when the cells stop proliferating followed by a decline in more mature cells. In contrast, C/EBP-ε protein is virtually detectable only in the MC/MM population, indicating that expression in more immature cells could be inhibited by microRNAs (miRNAs). We found that miRNA-130a (miR-130a) regulates C/EBP-ε protein expression in both murine and human granulocytic precursors. Overexpression of miR-130a in a murine cell line downregulated C/EBP-ε protein and lactoferrin (Ltf), cathelicidin antimicrobial protein (Camp), and lipocalin-2 (Lcn2) mRNA expression giving rise to cells with a more immature phenotype, as seen in the Cebpe(-/-) mouse. Introduction of a C/EBP-ε mRNA without target site for miR-130a restored both C/EBP-ε production, expression of Camp and Lcn2, and resulted in the cells having a more mature phenotype. We conclude that miR-130a is important for the regulation of the timed expression of C/EBP-ε during granulopoiesis.

Analysis of human samples reveals impaired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome.

Joubert syndrome (JS) and Meckel syndrome (MKS) are pleiotropic ciliopathies characterized by severe defects of the cerebellar vermis, ranging from hypoplasia to aplasia. Interestingly, ciliary conditional mutant mice have a hypoplastic cerebellum in which the proliferation of cerebellar granule cell progenitors (GCPs) in response to Sonic hedgehog (SHH) is severely reduced. This suggests that Shh signaling defects could contribute to the vermis hypoplasia observed in the human syndromes. As existing JS/MKS mutant mouse models suggest apparently contradictory hypotheses on JS/MKS etiology, we investigated Shh signaling directly on human fetal samples. First, in an examination of human cerebellar development, we linked the rates of GCP proliferation to the different levels and localizations of active Shh signaling and showed that the GCP possessed a primary cilium with CEP290 at its base. Second, we found that the proliferation of GCPs and their response to SHH were severely impaired in the cerebellum of subjects with JS/MKS and Jeune syndrome. Finally, we showed that the defect in GCP proliferation was similar in the cerebellar vermis and hemispheres in all patients with ciliopathy analyzed, suggesting that the specific cause of vermal hypo-/aplasia precedes this defect. Our results, obtained from the analysis of human samples, show that the hemispheres and the vermis are affected in JS/MKS and provide evidence of a defective cellular mechanism in these pathologic processes.

Neutrophil survival and c-kit(+)-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.

Polymorphonuclear neutrophils (PMNs) are critical for the formation, maintenance, and resolution of bacterial abscesses. However, the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound, combined with real-time imaging of genetically tagged PMNs, we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound, where their lifespan was markedly extended. A continuous rise in wound PMN number, which was not accounted for by trafficking from the bone marrow or by prolonged survival, was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound, where they proliferated and formed mature PMNs. Furthermore, by blocking their recruitment with an antibody to c-kit, which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival, we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound, but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells.

Increased promyelocytic-derived microparticles: a novel potential factor for coagulopathy in acute promyelocytic leukemia.

The frequent serious bleeding and thrombotic complications in acute promyelocytic leukemia (APL) are major causes of early mortality, but the complex mechanisms causing the bleeding have not been completely elucidated. Because microparticles (MPs) are known to be elevated in thromboembolic disorders, we hypothesized a role for MPs in the pathogenesis of coagulopathy in APL. MPs were isolated from 30 APL patients and 20 healthy subjects and from cultured NB4/APL cells. The morphology of the MPs was examined, and they were quantified and analyzed for their thrombin-generating potential. We confirmed the existence of promyelocytic-derived MPs by morphology using transmission electron microscopy and laser scanning confocal microscopy. Counts of MPs in APL were elevated and were typically from promyelocytic cells (CD33(+) TF(+) MPs). Importantly, the CD33(+) MPs strongly correlated with patient leukocyte count (R = 0.64, p = 0.002) and D-dimer (R = 0.51, p = 0.0038). Moreover, the MPs from patients with APL decreased the coagulation times and induced thrombin generation. APL MP-associated thrombin generation was reduced by 54 % when the extrinsic pathway was blocked using an anti-human tissue factor (TF) antibody. However, neither anti-factor XI nor anti-tissue factor pathway inhibitor had any significant inhibitory effect. Our results show that the procoagulant state in APL is partially due to the TF-dependent procoagulant properties of circulating promyelocytic-derived MPs. TF(+) MPs may be a novel potential risk factor for coagulopathy in APL.

Morphology and quantitative composition of hematopoietic cells in murine bone marrow and spleen of healthy subjects.

Laboratory mice play an outstanding role in modeling human development and disease. In contrast to human leukemia, the spleen is involved in almost all cases, and the bone marrow is only variably involved in murine models. Although mice have been used for medical research for over 100 years, there are only few reports with a small number of cases looking at morphology and quantitative composition of murine hematopoietic cells in the bone marrow of non-transplanted animals of most strains. To our knowledge, there is not even a single report describing the splenogram in C57BL/6J mice, one of the most commonly used strains for medical research. The present study illustrates the morphology of the hematopoietic cells in the bone marrow and spleen of non-treated C57BL/6J mice and establishes the murine myelogram from the largest healthy C57BL/6J cohort reported to date. Furthermore, we present the first murine splenogram described for C57BL/6J mice. Our study supports the acceptance of the presence of >5 % blast cells as providing clear evidence of abnormality in bone marrow like in humans. In addition, we are the first to show <1 % blast cells in the normal spleen. Interestingly, classical dysplastic changes were rare in normal healthy mice. Our study of the bone marrow and spleen of healthy non-transplanted animals provides reference ranges of each cell type and for the myeloid/erythroid ratio, which can be used to interpret preclinical gene therapy data, leukemogenesis, and hematopoiesis studies, and may improve the quality of such analyses.

A recombined protein (rSj16) derived from Schistosoma japonicum induces cell cycle arrest and apoptosis of murine myeloid leukemia cells.

rSj16, a recombined protein from Schistosoma japonicum, has been identified as an anti-inflammatory molecule. In this study, we demonstrated that rSj16 strongly suppressed the growth of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. rSj16 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells as well as causing cell cycle arrest at the G0/G1 phase. The expressions of cyclin D1, D2, D3, and E, and Cdk 2, 4, and 6 genes in WEHI-3B JCS cells were significantly down-regulated at 24 h as measured by real-time PCR. Furthermore, apoptosis induced by rSj16 was confirmed by 4,6-diamidino-2-phenylindole nuclear staining assay and annexin V/propidium iodide double staining. A reduction of the mitochondrial membrane potential indicated an active involvement of mitochondria in the apoptosis process. rSj16 treatment induced an increase in the activity of caspase 3, 6, and 9, and expression of pro-apoptotic Bax. Meanwhile, the decreased expression of anti-apoptotic Bcl-2 was observed after rSj16 treatment. Taken together, our results implied that rSj16 can inhibit proliferation by inducing G0/G1 cell cycle arrest and apoptosis of murine myeloid leukemia cells via activation of the caspase-mediated mechanism by regulating the expression of Bcl-2 family.

ImageStream promyelocytic leukemia protein immunolocalization: in search of promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all-trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per-cell quantitative image analysis for statistically large sample sizes, enabling precise and operator-independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment.

Immunomodulatory derivatives induce PU.1 down-regulation, myeloid maturation arrest, and neutropenia.

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.

Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis.

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor, retinoid-inducible nuclear factor (RINF), that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore, short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly, RINF localizes to 5q31.3, a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene.

Gfi1 integrates progenitor versus granulocytic transcriptional programming.

In patients with severe congenital neutropenia (SCN) and mice with growth factor independent-1 (Gfi1) loss of function, arrested myeloid progenitors accumulate, whereas terminal granulopoiesis is blocked. One might assume that Gfi-null progenitors accumulate because they lack the ability to differentiate. Instead, our data indicate that Gfi1 loss of function deregulates 2 separable transcriptional programs, one of which controls the accumulation and lineage specification of myeloid progenitors, but not terminal granulopoiesis. We demonstrate that Gfi1 directly represses HoxA9, Pbx1, and Meis1 during normal myelopoiesis. Gfi1-/- progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and progenitor transformation in collaboration with oncogenic K-Ras. Limiting HoxA9 alleles corrects, in a dose-dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1 in myeloid progenitor cells but did not rescue Gfi1-/- blocked granulopoiesis. Thus, Gfi1 integrates 2 events during normal myeloid differentiation; the suppression of a HoxA9-Pbx1-Meis1 progenitor program and the induction of a granulopoietic transcription program.

Coordinated regulation of transcription factors through Notch2 is an important mediator of mast cell fate.

Mast cells are thought to participate in a wide variety of pathophysiological conditions. Mechanisms of regulation, however, of mast cell production and maturation are still to be elucidated. Mast cell developmental process is likely to be profoundly affected by cell-autonomous transcriptional regulators such as the GATA family and CCAAT/enhancer binding protein (C/EBP) family members. Extracellular regulators such as stem cell factor and IL-3 have essential roles in basal and inducible mast cell generation, respectively. The relationship, however, between the extracellular signaling and cellular transcriptional control is unclear, and the trigger of the mast cell development remains elusive. Notch signaling plays a fundamental role in the lymphopoietic compartment, but its role in myeloid differentiation is less clear. Here, we demonstrate that Notch signaling connects environmental cues and transcriptional control for mast cell fate decision. Delta1, an established Notch ligand, instructs bone marrow common myeloid progenitors and granulocyte-macrophage progenitors toward mast cell lineage at the expense of other granulocyte-macrophage lineages, depending on the function of the Notch2 gene. Notch2 signaling results in the up-regulation of Hes-1 and GATA3, whereas simultaneous overexpression of these transcription factors remarkably biases the progenitor fate toward the mast cell-containing colony-forming cells. C/EBPalpha mRNA was down-regulated in myeloid progenitors as a consequence of Hes-1 overexpression, in agreement with the recent proposal that the down-regulation of C/EBPalpha is necessary for mast cell fate determination. Taken together, signaling through Notch2 determines the fate of myeloid progenitors toward mast cell-producing progenitors, via coordinately up-regulating Hes-1 and GATA3.

A comparative study of bone marrow and peripheral blood CD34+ myeloblasts in acute myeloid leukaemia.

To examine the differences between primitive bone marrow (BM) and peripheral blood (PB) myeloblasts in acute myeloid leukaemia (AML), we compared CD34(+) myeloblasts of paired BM and PB samples from 14 AML patients in terms of surface phenotype, homing and engraftment in a xenogeneic transplantation model, and gene expression, based on microarray studies and quantitative polymerase chain reaction. While there was no significant difference in surface phenotypes between these two populations, in vivo assay showed significantly better homing potential of PB CD34(+) cells than BM CD34(+) cells. Significant correlation between homing and engraftment in AML samples was also noted. In addition, gene expression profiling of CD34(+) cells from five paired BM and PB leukaemic samples showed that genes involved in G-protein and prostaglandin signalling, chemotaxis and stress response, cell proliferation and apoptosis were down-regulated in PB CD34(+) myeloblasts. These data suggested that circulating primitive myeloblasts in AML are functionally different from those residing in the marrow compartment, and such differences may be partly regulated by the BM microenvironment.

MicroRNA-27 enhances differentiation of myeloblasts into granulocytes by post-transcriptionally downregulating Runx1.

We investigated the regulation of the transcription factor Runx1 by microRNA (miR)-27 and the resulting effects upon the differentiation of myeloblasts into granulocytes. When 32D.cl3 cell differentiation was induced using granulocyte colony-stimulating factor (CSF3), Runx1 transcription was moderately downregulated, while Runx1 protein levels were completely inhibited, suggesting an involvement of post-transcriptional regulation. Simultaneously, levels of miR-27 and its precursor increased substantially. Reporter assays revealed that miR-27 targets the 3'UTR of the Runx1 transcript. Furthermore, introduction of pre-miR-27 alone into 32D.cl3 cells resulted in downregulation of Runx1 protein, thereby allowing the cell differentiation even in the absence of CSF3. Conversely, transduction of anti-miR-27 caused upregulation of Runx1 protein, thereby antagonizing the CSF3-mediated granulocyte differentiation. Finally, the CSF3-induced transcription factor C/EBPalpha enhanced transcription of a host gene of miR-27, C9orf3, via activation of its promoter. Thus, miR-27 enhances differentiation of myeloblasts into granulocytes via post-transcriptional downregulation of Runx1.

Effects of Obesity and Exercise on Bone Marrow Progenitor Cells after Radiation.

INTRODUCTION: The late effects of radiation therapy can have significant consequences for the health and quality of life of long-term cancer survivors. Radiation induces persistent alterations in hematopoietic stem and progenitor cells (HSPC) and the bone marrow environment; however, how relevant host factors such as obesity and exercise differentially regulate HSPC content and the bone marrow environment after radiation exposure remains unknown. The purpose of this investigation was to evaluate how the combination of obesity and exercise training modulates HSPC and their niche after sublethal radiation exposure in mice. METHODS: Mice fed either a control or a high-fat diet to induce obesity remained sedentary or underwent a progressive treadmill exercise program. At 13 wk of age, mice were irradiated (3 Gy) and continued their specific diets and exercise program for four more weeks. RESULTS: Exercise-trained mice had significantly higher quantities of several HSPC subpopulations and bone marrow stromal cell populations, whereas HSPC subpopulations were significantly lower in obese mice after radiation. Reactive oxygen species content was significantly decreased in HSPC with exercise training. Proteomics analysis of bone marrow supernatant revealed clustering of biologically relevant changes in exercise-trained mice. Functional evaluation of bone marrow supernatant revealed a significant increase in leukemia blast viability in obese mice but not in the exercise-trained mice (P < 0.05). CONCLUSION: Together, these data suggest that exercise training partially restores the negative effects of obesity on HSPC and their niche after radiation exposure. As such, exercise training should be considered to mitigate the late effects of radiation therapy on the hematopoietic system for cancer survivors with or without obesity who have undergone radiation therapy.

PLC-beta2 activity on actin-associated polyphosphoinositides promotes migration of differentiating tumoral myeloid precursors.

During both maturation and function, neutrophils are subjected to reorganization of the actin cytoskeleton. Among the molecules that influence cytoskeletal architecture, the amount and subcellular localization of phosphoinositides, regulated by specific kinases and phosphatases, may play a crucial role. In neutrophils, PLC-beta2 is a major phosphoinositide-dependent phospholipase C isoform activated in response to chemoattractants, even though its role in the modifications of cell morphology and motility that occur during the inflammatory process has not been fully elucidated. In APL-derived promyelocytes induced to complete their maturation program, we have found that the expression levels of PLC-beta2 positively correlate with the degree of the reached neutrophil differentiation. Here, we demonstrate that PLC-beta2 modulates the migration capability of promyelocytes induced to differentiate with ATRA. In differentiating cells, the association of PLC-beta2 with actin, mediated by the PH domain, seems crucial for catalytic activity. We conclude that phosphodiesterase activity of PLC-beta2 on the actin-associated PIP2 may be responsible, by modifying the phosphoinositide pools, for the modifications of cytoskeleton architecture that take place during motility of differentiating promyelocytes.