Genetic and epigenetic orchestration of Gfi1aa-Lsd1-cebpa in zebrafish neutrophil development.

Neutrophils are the most abundant vertebrate leukocytes and they are essential to host defense. Despite extensive investigation, the molecular network controlling neutrophil differentiation remains incompletely understood. GFI1 is associated with several myeloid disorders, but its role and the role of its co-regulators in granulopoiesis and pathogenesis are far from clear. Here, we demonstrate that zebrafish gfi1aa deficiency induces excessive neutrophil progenitor proliferation, accumulation of immature neutrophils from the embryonic stage, and some phenotypes similar to myelodysplasia syndrome in adulthood. Both genetic and epigenetic analyses demonstrate that immature neutrophil accumulation in gfi1aa-deficient mutants is due to upregulation of cebpa transcription. Increased transcription was associated with Lsd1-altered H3K4 methylation of the cebpa regulatory region. Taken together, our results demonstrate that Gfi1aa, Lsd1 and cebpa form a regulatory network that controls neutrophil development, providing a disease progression-traceable model for myelodysplasia syndrome. Use of this model could provide new insights into the molecular mechanisms underlying GFI1-related myeloid disorders as well as a means by which to develop targeted therapeutic approaches for treatment.

Extracellular vesicles from dHL-60 cells as delivery vehicles for diverse therapeutics.

Extracellular vesicles (EVs) are membrane-derived heterogeneous vesicles that mediate intercellular communications. They have recently been considered as ideal vehicles for drug-delivery systems, and immune cells are suggested as a potential source for drug-loaded EVs. In this study, we investigated the possibility of neutrophils as a source for drug-loaded EVs. Neutrophil-like differentiated human promyelocytic leukemia cells (dHL-60) produced massive amounts of EVs within 1 h. The dHL-60 cells are also easily loaded with various cargoes such as antibiotics (penicillin), anticancer drug (paclitaxel), chemoattractant (MCP-1), miRNA, and Cas9. The EVs derived from the dHL-60 cells showed efficient incorporation of these cargoes and significant effector functions, such as bactericidal activity, monocyte chemotaxis, and macrophage polarization. Our results suggest that neutrophils or neutrophil-like promyelocytic cells could be an attractive source for drug-delivery EVs.

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Prdm3 and Prdm16 cooperatively maintain hematopoiesis and clonogenic potential.

Mds1-Evi1 (also known as Prdm3) and Prdm16 are two highly related zinc finger transcription factors that, within the hematopoietic system, are both expressed primarily in hematopoietic stem cells (HSCs). Our laboratory previously found that constitutive Mds1-Evi1 knockout mice are viable, but their HSCs are unable to withstand myeloablative chemotherapy or effectively transplant irradiated recipient mice. A similar phenotype has been observed for Prdm16, except that the Prdm16 constitutive knockout is lethal. Here, we created a novel double-knockout model of Mds1-Evi1 and Prdm16 in the bone marrow, in which double knockout occurs only in cells that endogenously express Mds1-Evi1 and only upon induction with tamoxifen. We show that combined Mds1-Evi1/Prdm16 deficiency causes bone marrow failure within 15 days, with rapid loss in all progenitor compartments, while the peripheral blood exhibits progressive reductions in peripheral monocytes and granulocytes. We found that surviving hematopoietic stem cells and granulocytic progenitors had elevated apoptosis and cell division, and were unable to form colonies in vitro; adding back wild-type Mds1-Evi1 or Prdm16 to double-knockout bone marrow restores colony formation, and for MDS1-EVI1, this activity depends on a functional PR domain. All of these phenotypic effects were exhibited at milder levels in Mds1-Evi1 and Prdm16 single-knockout controls. Overall, these results illustrate that Mds1-Evi1 and Prdm16 play additive roles in maintaining normal hematopoietic stem cell survival.

TNFAIP3 Deficiency Affects Monocytes, Monocytes-Derived Cells and Microglia in Mice.

The intracellular-ubiquitin-ending-enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a potent inhibitor of the pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway. Single nucleotide polymorphisms in TNFAIP3 locus have been associated to autoimmune inflammatory disorders, including Multiple Sclerosis (MS). Previously, we reported a TNFAIP3 down-regulated gene expression level in blood and specifically in monocytes obtained from treatment-naïve MS patients compared to healthy controls (HC). Myeloid cells exert a key role in the pathogenesis of MS. Here we evaluated the effect of specific TNFAIP3 deficiency in myeloid cells including monocytes, monocyte-derived cells (M-MDC) and microglia analyzing lymphoid organs and microglia of mice. TNFAIP3 deletion is induced using conditional knock-out mice for myeloid lineage. Flow-cytometry and histological procedures were applied to assess the immune cell populations of spleen, lymph nodes and bone marrow and microglial cell density in the central nervous system (CNS), respectively. We found that TNFAIP3 deletion in myeloid cells induces a reduction in body weight, a decrease in the number of M-MDC and of common monocyte and granulocyte precursor cells (CMGPs). We also reported that the lack of TNFAIP3 in myeloid cells induces an increase in microglial cell density. The results suggest that TNFAIP3 in myeloid cells critically controls the development of M-MDC in lymphoid organ and of microglia in the CNS.

Physiological expression of miR-130a during differentiation of CD34+ human hematopoietic stem cells results in the inhibition of monocyte differentiation.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

Affinity separation and subsequent terminal differentiation of acute myeloid leukemia cells using the human transferrin receptor (CD71) as a capture target.

The microfluidic detection of myeloblasts in blood via the human transferrin receptor (CD71) can serve as a diagnostic marker for acute myeloid leukemia (AML). Furthermore, CD71 expression is present in all proliferating cells and can capture target cells without prior knowledge of AML subtype. The use of anti-CD71 as the affinity ligand for AML detection in this work yields a capture efficiency and purity during peak CD71 expression of 92% and 62%, respectively. Additionally, target cells were isolated from lysed, preserved blood samples with a capture purity of 32% at a concentration of 10% myeloblasts in blood, half of the current diagnosis threshold determined by the World Health Organization. Cells isolated using this capture ligand were then subjected to post-separation differentiation therapy. HL60 cells were differentiated into non-proliferating, neutrophil-like cells. After 48 hours of incubation with 1.5% DMSO, there was a decrease in the CD71 antigen expression in differentiated cells. This separation approach can be used to screen blood samples for AML cells, and the effluent of the separation is available for post-separation analyses.

TRAF6 Distinctly Regulates Hematopoietic Stem and Progenitors at Different Periods of Development in Mice.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is identified as a signaling adaptor protein that regulates bone metabolism, immunity, and the development of several tissues. Therefore, its functions are closely associated with multiple diseases. TRAF6 is also involved in the regulation of hematopoiesis under steady-state conditions, but the role of TRAF6 in modulating hematopoietic stem and progenitor cells (HSPCs) during the developmental stages remains unknown. Here, we report that the deletion of TRAF6 in hematopoietic lineage cells resulted in the upregulation of HSPCs in the fetal liver at the prenatal period. However, in the early postnatal period, deletion of TRAF6 drastically diminished HSPCs in the bone marrow (BM), with severe defects in BM development and extramedullary hematopoiesis in the spleen being identified. In the analysis of adult HSPCs in a BM reconstitution setting, TRAF6 played no significant role in HSPC homeostasis, albeit it affected the development of T cells. Taken together, our results suggest that the role of TRAF6 in regulating HSPCs is altered in a spatial and temporal manner during the developmental course of mice.

[Identification of a human progenitor strictly committed to monocytic differentiation: a counterpart of mouse cMoPs].

Monocytes give rise to macrophages and dendritic cells (DCs) under steady-state and inflammatory conditions, thereby contributing to host defense and tissue pathology. Inflammation triggers the differentiation of tissue-infiltrating monocytes into monocyte-derived macrophages and DCs, which are associated with homeostatic host defense reactions and inflammatory diseases. In mice, monocytes are divided into classical Ly6chi- and non-classical Ly6clo-expressing subsets. Ly6clo monocytes are present only in the blood; however, Ly6chi monocytes are found in blood and other tissues (wherein they differentiate into macrophages and DCs). In this context, most Ly6clo monocytes are derived from Ly6chi monocytes. In humans, monocytes comprise major CD14+CD16- and other CD14+CD16+ and CD14loCD16+ monocytes. A monocyte lineage-restricted common monocyte progenitor (cMoP) was previously identified in mice; herein, we introduce human cMoP, which was identified as a CLEC12AhiCD64hi subpopulation of conventional granulocyte-monocyte progenitors (cGMPs) in umbilical cord blood and bone marrow. The human cMoP produced monocyte subsets without showing any potential of differentiating into myeloid or lymphoid cells ex vivo. Within the cGMP population, we also identified revised GMPs that completely lacked DC and lymphoid potential, which sequentially produced cMoPs, pre-monocytes, and monocytes. Collectively, our findings enhance the current understanding of human myeloid cell differentiation pathways.

Hhex induces promyelocyte self-renewal and cooperates with growth factor independence to cause myeloid leukemia in mice.

The hematopoietically expressed homeobox (Hhex) transcription factor is overexpressed in human myeloid leukemias. Conditional knockout models of murine acute myeloid leukemia indicate that Hhex maintains leukemia stem cell self-renewal by enabling Polycomb-mediated epigenetic repression of the Cdkn2a tumor suppressor locus, encoding p16Ink4a and p19Arf However, whether Hhex overexpression also affects hematopoietic differentiation is unknown. To study this, we retrovirally overexpressed Hhex in hematopoietic progenitors. This enabled serial replating of myeloid progenitors, leading to the rapid establishment of interleukin-3 (IL-3)-dependent promyelocytic cell lines. Use of a Hhex-ERT2 fusion protein demonstrated that continuous nuclear Hhex is required for transformation, and structure function analysis demonstrated a requirement of the DNA-binding and N-terminal-repressive domains of Hhex for promyelocytic transformation. This included the N-terminal promyelocytic leukemia protein (Pml) interaction domain, although deletion of Pml failed to prevent Hhex-induced promyelocyte transformation, implying other critical partners. Furthermore, deletion of p16Ink4a or p19Arf did not promote promyelocyte transformation, indicating that repression of distinct Hhex target genes is required for this process. Indeed, transcriptome analysis showed that Hhex overexpression resulted in repression of several myeloid developmental genes. To test the potential for Hhex overexpression to contribute to leukemic transformation, Hhex-transformed promyelocyte lines were rendered growth factor-independent using a constitutively active IL-3 receptor common ß subunit (ßcV449E). The resultant cell lines resulted in a rapid promyelocytic leukemia in vivo. Thus, Hhex overexpression can contribute to myeloid leukemia via multiple mechanisms including differentiation blockade and enabling epigenetic repression of the Cdkn2a locus.

CSF-1-induced Src signaling can instruct monocytic lineage choice.

Controlled regulation of lineage decisions is imperative for hematopoiesis. Yet, the molecular mechanisms underlying hematopoietic lineage choices are poorly defined. Colony-stimulating factor 1 (CSF-1), the cytokine acting as the principal regulator of monocyte/macrophage (M) development, has been shown to be able to instruct the lineage choice of uncommitted granulocyte M (GM) progenitors toward an M fate. However, the intracellular signaling pathways involved are unknown. CSF-1 activates a multitude of signaling pathways resulting in a pleiotropic cellular response. The precise role of individual pathways within this complex and redundant signaling network is dependent on cellular context, and is not well understood. Here, we address which CSF-1-activated pathways are involved in transmitting the lineage-instructive signal in primary bone marrow-derived GM progenitors. Although its loss is compensated for by alternative signaling activation mechanisms, Src family kinase (SFK) signaling is sufficient to transmit the CSF-1 lineage instructive signal. Moreover, c-Src activity is sufficient to drive M fate, even in nonmyeloid cells.

A late-lineage murine neutrophil precursor population exhibits dynamic changes during demand-adapted granulopoiesis.

Homeostasis of neutrophils-the blood cells that respond first to infection and tissue injury-is critical for the regulation of immune responses and regulated through granulopoiesis, a multi-stage process by which neutrophils differentiate from hematopoietic stem cells. Granulopoiesis is a highly dynamic process and altered in certain clinical conditions, such as pathologic and iatrogenic neutropenia, described as demand-adapted granulopoiesis. The regulation of granulopoiesis under stress is not completely understood because studies of granulopoiesis dynamics have been hampered by technical limitations in defining neutrophil precursors. Here, we define a population of neutrophil precursor cells in the bone marrow with unprecedented purity, characterized by the lineage-CD11b+Ly6GloLy6BintCD115-, which we call NeuPs (Neutrophil Precursors). We demonstrated that NeuPs differentiate into mature and functional neutrophils both in vitro and in vivo. By analyzing the gene expression profiles of NeuPs, we also identified NeuP stage-specific genes and characterized patterns of gene regulation throughout granulopoiesis. Importantly, we found that NeuPs have the potential to proliferate, but the proliferation decreased in multiple different hematopoietic stress settings, indicating that proliferating NeuPs are poised at a critical step to regulate granulopoiesis. Our findings will facilitate understanding how the hematopoietic system maintains homeostasis and copes with the demands of granulopoiesis.

Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation.

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe2+. We showed that HO-1 knock-out mice (HO-1-/-) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1. The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1-/- mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1-/- mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPß (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPß expression and activity in HO-1-/- myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1-/- granulocytes were functionally competent as determined by oxidative burst capacity. In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPß protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia.

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.

To the Large Nucleolar Bodies in Apoptotic Leukaemic Granulocytic Progenitors without Further Differentiation. Are Large Nucleoli Always Present in Proliferating Cells?

Large nucleoli have generally been believed to be present in less differentiated and proliferating cells including the malignant ones. Such nucleoli have also been considered to be active in the biosynthetic process and major cell developmental activities. In contrast, after cytostatic treatment, apoptotic leukaemic progenitors still containing nuclei did not exhibit substantial reduction of the nucleolar size but displayed decreased nucleolar biosynthetic activity. The present study was undertaken to provide more information on the large nucleoli in spontaneously occurring apoptotic leukaemic progenitors without further differentiation. Leukaemic progenitors of established cell lineages originating from leukaemic patients represented a very convenient model for such study. Some of them exhibit morphological signs of the spontaneously occurring apoptotic process. Since such signs are expressed by nuclear and cytoplasmic morphological variability, the present study dealt with spontaneously occurring apoptotic progenitors with preserved nuclei characterized by heavy chromatin condensation and occasional fragmentation. Based of nucleolar body and nuclear maximal diameter measurements it seems to be clear that the nucleolar size in these cells was not substantially reduced, contrary to that of the nucleus. However, large nucleolar bodies in spontaneously occurring apoptotic cells were characterized by markedly reduced biosynthetic activity, as expressed by the decreased number of nucleolar transcription markers such as nucleolar fibrillar centres. In conclusion, large nucleoli may be present not only in proliferating, but also in spontaneously occurring apoptotic cells.

Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day.

Response of Hematopoietic Stem and Progenitor Cells to Reserpine in C57Bl/6 Mice.

We studied the response of hematopoietic stem cells and progenitor cells to sympatholytic reserpine in intact C57Bl/6 mice and in animals with cyclophosphamide-induced leukopenia. The count of long-term hematopoietic stem cells (Lin(-)Sca-1(+)c-kit(+)CD34(-)) in the bone marrow of healthy mice increased in 7 min after reserpine injection and remained elevated in 2 h in parallel with elevated content of short-term stem (Lin(-)Sca-1(+)c-kit(+)CD34(+)) and progenitor (Lin(-)Sca-1(+)c-kit(+)) cells. Reserpine produced no effect on recruitment of hematopoietic stem and progenitor cells into the peripheral blood, but increased the serum level of granulocyte CSF and increased the count of metamyelocytes and neutrophilic granulocytes in the blood (in 2 h postinjection). Transplantation of bone marrow hematopoietic stem and progenitor cells from reserpine-treated donor C57Bl/6 mice to recipient CBA mice with 5-fluorouracil-induced leukopenia accelerated regeneration of the granulocytic lineage cells in leukemic mice. In cyclophosphamide-treated C57Bl/6 mice, reserpine reduced the level of short-term hematopoietic stem cells and increased the count of progenitor hematopoietic cells in the bone marrow in parallel with recruitment of the progenitors into the peripheral blood.

Role of local eosinophilopoietic processes in the development of airway eosinophilia in prednisone-dependent severe asthma.

BACKGROUND: In severe asthmatics with persistent airway eosinophilia, blockade of interleukin-5 has significant steroid-sparing effects and attenuates blood and sputum eosinophilia. The contribution of local maturational processes of progenitors within the airways relative to the recruitment of mature cells from the peripheral circulation to the development of airway eosinophilia is not known. We hypothesize that local eosinophilopoiesis may be the predominant process that drives persistent airway eosinophilia and corticosteroid requirement in severe asthmatics. OBJECTIVES: In a cross-sectional study, the number and growth potential of eosinophil-lineage-committed progenitors (EoP) were assayed in 21 severe eosinophilic asthmatics, 19 mild asthmatics, eight COPD patients and eight normal subjects. The effect of anti-IL-5 treatment on mature eosinophils and EoP numbers was made in severe eosinophilic asthmatics who participated in a randomized clinical trial of mepolizumab (substudy of a larger GSK sponsored global phase III trial, MEA115575) where subjects received mepolizumab (100 mg, n = 9) or placebo (n = 8), as six monthly subcutaneous injections. RESULTS: Mature eosinophil and EoP numbers were significantly greater in the sputum of severe asthmatics compared with all other subject groups. In colony-forming assays, EoP from blood of severe asthmatics demonstrated a greater response to IL-5 than mild asthmatics. Treatment of severe asthmatics with mepolizumab significantly attenuated blood eosinophils and increased EoP numbers consistent with blockade of systemic eosinophilopoiesis. There was however no significant treatment effect on mature eosinophils, sputum EoP numbers or the prednisone maintenance dose. CONCLUSIONS AND CLINICAL RELEVANCE: Patients with severe eosinophilic asthma have an exaggerated eosinophilopoeitic process in their airways. Treatment with 100 mg subcutaneous mepolizumab significantly attenuated systemic differentiation of eosinophils, but did not suppress local airway eosinophil differentiation to mature cells. Targeting IL-5-driven eosinophil differentiation locally within the lung maybe of relevance for optimal control of airway eosinophilia and asthma.