Eosinophil Lineage-committed Progenitors Research Articles

Eosinophil Lineage-Committed Progenitors as a Therapeutic Target for Asthma.

Eosinophilic asthma is the most prevalent phenotype of asthma. Although most asthmatics are adequately controlled by corticosteroid therapy, a subset (5–10%) remain uncontrolled with significant therapy-related side effects. This indicates the need for a consideration of alternative treatment strategies that target airway eosinophilia with corticosteroid-sparing benefits. A growing body of evidence shows that a balance between systemic differentiation and local tissue eosinophilopoietic processes driven by traffic and lung homing of bone marrow-derived hemopoietic progenitor cells (HPCs) are important components for the development of airway eosinophilia in asthma. Interleukin (IL)-5 is considered a critical and selective driver of terminal differentiation of eosinophils. Studies targeting IL-5 or IL-5R show that although mature and immature eosinophils are decreased within the airways, there is incomplete ablation, particularly within the bronchial tissue. Eotaxin is a chemoattractant for mature eosinophils and eosinophil-lineage committed progenitor cells (EoP), yet anti-CCR3 studies did not yield meaningful clinical outcomes. Recent studies highlight the role of epithelial cell-derived alarmin cytokines, IL-33 and TSLP, (Thymic stromal lymphopoietin) in progenitor cell traffic and local differentiative processes. This review provides an overview of the role of EoP in asthma and discusses findings from clinical trials with various therapeutic targets. We will show that targeting single mediators downstream of the inflammatory cascade may not fully attenuate tissue eosinophilia due to the multiplicity of factors that can promote tissue eosinophilia. Blocking lung homing and local eosinophilopoiesis through mediators upstream of this cascade may yield greater improvement in clinical outcomes.

MTOR complexes differentially orchestrates eosinophil development in allergy

Eosinophil infiltration is considered a hallmark in allergic airway inflammation, and the blockade of eosinophil differentiation may be an effective approach for treating eosinophil-related disorders. Mammalian target of rapamycin (mTOR) is a vital modulator in cell growth control and related diseases, and we have recently demonstrated that rapamycin can suppress eosinophil differentiation in allergic airway inflammation. Considering its critical role in haematopoiesis, we further investigated the role of mTOR in eosinophil differentiation in the context of asthmatic pathogenesis. Intriguingly, the inhibition of mTOR, either by genetic deletion or by another pharmacological inhibitor torin-1, accelerated the eosinophil development in the presence of IL-5. However, this was not observed to have any considerable effect on eosinophil apoptosis. The effect of mTOR in eosinophil differentiation was mediated by Erk signalling. Moreover, myeloid specific knockout of mTOR or Rheb further augmented allergic airway inflammation in mice after allergen exposure. Ablation of mTOR in myeloid cells also resulted in an increased number of eosinophil lineage-committed progenitors (Eops) in allergic mice. Collectively, our data uncovered the differential effects of mTOR in the regulation of eosinophil development, likely due to the distinct functions of mTOR complex 1 or 2, which thus exerts a pivotal implication in eosinophil-associated diseases.

Elevated Eosinophil Lineage-Committed Progenitors In Skin Lesions Of Patients With Moderate-To-Severe Atopic Dermatitis

Elevated Eosinophil Lineage-Committed Progenitors In Skin Lesions Of Patients With Moderate-To-Severe Atopic Dermatitis

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

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. 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 (100mg, n=9) or placebo (n=8), as six monthly subcutaneous injections. 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. Patients with severe eosinophilic asthma have an exaggerated eosinophilopoeitic process in their airways. Treatment with 100mg 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.

Eosinophil differentiation in the bone marrow is promoted by protein tyrosine phosphatase SHP2.

SHP2 participates in multiple signaling events by mediating T-cell development and function, and regulates cytokine-dependent granulopoiesis. To explore whether and how SHP2 can regulate bone-marrow eosinophil differentiation, we investigate the contribution of SHP2 in the bone-marrow eosinophil development in allergic mice. Blockade of SHP2 function by SHP2 inhibitor PHPS-1 or conditional shp2 knockdown by adenovirus-inhibited bone-marrow-derived eosinophil differentiation in vitro, with no detectable effects on the apoptosis of eosinophils. Furthermore, SHP2 induced eosinophil differentiation via regulation of the extracellular signal-regulated kinase pathway. Myeloid shp2 conditional knockout mice (LysMcreshp2flox/flox) failed to induce eosinophilia as well as airway hyper-responsiveness. The SHP2 inhibitor PHPS-1 also alleviated eosinophilic airway inflammation and airway hyper-responsiveness, accompanied by significantly reduced levels of systemic eosinophils and eosinophil lineage-committed progenitors in allergic mice. We demonstrate that inhibition of eosinophil development is SHP2-dependent and SHP2 is sufficient to promote eosinophil formation in vivo. Our data reveal SHP2 as a critical regulator of eosinophil differentiation, and inhibition of SHP2 specifically in myeloid cells alleviates allergic airway inflammation.

Increased numbers of activated group 2 innate lymphoid cells in the airways of patients with severe asthma and persistent airway eosinophilia

In patients with severe eosinophilic asthma, local maturation rather than systemic recruitment of mature cells might contribute to persistent airway eosinophilia. Group 2 innate lymphoid cells (ILC2s) are a major source of type 2 cytokines (IL-5 and IL-13) and can facilitate eosinophilic inflammatory responses in mouse models of asthma in the absence of CD4+ lymphocytes. This study investigated the potential role of ILC2s in driving chronic airway eosinophilia in patients with severe asthma, despite regular high-dose oral corticosteroid therapy. In a cross-sectional study we enumerated blood and sputum ILC2s (lin(-)CD45(+)127(+)ST2(+)) and levels of intracellular IL-5 and IL-13 in patients with severe asthma (n = 25), patients with steroid-naive mild atopic asthma (n = 19), and nonatopic control subjects (n = 5). Results were compared with numbers of CD4+ lymphocytes, eosinophil lineage-committed progenitors (eosinophilopoietic progenitor cells [EoPs]), and mature eosinophils. Significantly greater numbers of total and type 2 cytokine-producing ILC2s were detected in blood and sputum of patients with severe asthma compared to mild asthmatics. In contrast, intracellular cytokine expression by CD4 cells and EoPs within the airways did not differ between the asthmatic groups. In patients with severe asthma, although sputum CD4+ cells were more abundant than ILC2s and EoPs, proportionally, ILC2s were the predominant source of type 2 cytokines. In addition, there were significantly greater numbers of sputum IL-5(+)IL-13(+) ILC2s in patients with severe asthma whose airway eosinophilia was greater than 3%, despite normal blood eosinophil numbers (<300/μL). Our findings suggest that ILC2s can promote the persistence of airway eosinophilia in patients with severe asthma through uncontrolled localized production of the type 2 cytokines IL-5 and IL-13, despite high-dose oral corticosteroid therapy.

Rapamycin inhibition of eosinophil differentiation attenuates allergic airway inflammation in mice

The mammalian target of rapamycin (mTOR) signalling pathway regulates immune responses, and promotes cell growth and differentiation. Inhibition of mTOR with rapamycin modulates allergic asthma, while the underlying molecular mechanisms remain elusive. Here, we demonstrate that rapamycin, effectively inhibits eosinophil differentiation, contributing to its overall protective role in allergic airway inflammation. Rapamycin was administered in a mouse model of ovalbumin-induced allergic airway inflammation, and the eosinophil differentiation was analysed in vivo and in vitro. Rapamycin significantly attenuated allergic airway inflammation and markedly decreased the amount of eosinophils in local airways, peripheral blood and bone marrow, independently of levels of interleukin-5 (IL-5). In vitro colony forming unit assay and liquid culture demonstrated that rapamycin directly inhibited IL-5-induced eosinophil differentiation. In addition, rapamycin reduced the production of IL-6 and IL-13 by eosinophils. Rapamycin was also capable of reducing the eosinophil levels in IL-5 transgenic NJ.1638 mice, again regardless of the constitutive high levels of IL-5. Interestingly, rapamycin inhibition of eosinophil differentiation in turn resulted in an accumulation of eosinophil lineage-committed progenitors in bone marrow. Altogether these results clearly demonstrate a direct inhibitory role of rapamycin in eosinophil differentiation and function, and reemphasize the importance of rapamycin and possibly, mTOR, in allergic airway disease.

Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice

AbstractEosinophil activities are often linked with allergic diseases such as asthma and the pathologies accompanying helminth infection. These activities have been hypothesized to be mediated, in part, by the release of cationic proteins stored in the secondary granules of these granulocytes. The majority of the proteins stored in these secondary granules (by mass) are major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX). Unpredictably, a knockout approach targeting the genes encoding these proteins demonstrated that, unlike in mice containing a single deficiency of only MBP-1 or EPX, the absence of both granule proteins resulted in the near complete loss of peripheral blood eosinophils with no apparent impact on any other hematopoietic lineage. Moreover, the absence of MBP-1 and EPX promoted a concomitant loss of eosinophil lineage-committed progenitors in the marrow, identifying a specific blockade in eosinophilopoiesis as the causative event. Significantly, this blockade of eosinophilopoiesis is also observed in ex vivo cultures of marrow progenitors and is not rescued in vivo by adoptive bone marrow engraftment, suggesting a cell-autonomous defect in marrow progenitors. These observations implicate a role for granule protein gene expression as a regulator of eosinophilopoiesis and provide another strain of mice congenitally deficient of eosinophils.

Negative Regulation of Eosinophil Production by Toll-Like Receptors

Abstract 1237Eosinophilia is a common clinical problem associated with numerous disorders such as atopic diseases, parasitic infections, hypereosinophilic syndrome (HES), and cancer. A marked decrease in circulating eosinophils, or eosinopenia, has long been associated with acute bacterial infections. Eosinopenia has been shown to be a sensitive and reliable marker for distinguishing between non-infectious and infection-associated sepsis in the intensive care unit setting. Eosinopenia with acute infection had been assumed to be secondary to the release of adrenal glucocorticoids in response to the stress of infection. However, animal studies performed in the 1970s demonstrated that inhibition of parasite-associated eosinophilia with acute bacterial infection was independent of adrenal stimulation; the investigator speculated that it was due to a direct effect on the eosinophil precursor. To date, the mechanism of infection-associated eosinopenia, and its potential for suppressing IL-5-mediated eosinophilia, has not been investigated.To study eosinophil development, we established an ex vivo liquid culture system in which we can follow the differentiation of murine eosinophil lineage-committed progenitors (EoPs) to mature functional effector eosinophils that express known eosinophil surface markers and granule proteins and respond to eosinophil-active chemoattractants. We demonstrate that murine eosinophil precursors express mRNA for six TLRs. Ligands for TLR2 heterodimers, TLR4 and TLR7, but not TLR3, inhibited eosinophil ex vivo growth by attenuating EoP proliferation in response to IL-5 stimulation. Exposure to heat-killed E. coli also resulted in diminished EoP proliferation. The developing eosinophils further responded to TLR activation with production of a subset of inflammatory cytokines, including IL-6, IL-10, TNF-alpha and CXCL10. Co-culturing of untreated EoPs with LPS-exposed EoPs resulted in reduced IL-5-mediated proliferation of the untreated EoPs, suggesting that EoPs secrete mediators in response to TLR activation that regulate their proliferation in an autocrine/paracrine manner. Neutralization of IFN-beta, but not IFN-alpha or IFN-gamma, partially protected developing eosinophils from the inhibitory effects of LPS. In vivo studies revealed that endotoxemia and bacteremia reduced numbers of EoPs in the bone marrow of wild-type mice. Further, LPS dose-dependently reduced hypereosinophilia in IL-5 transgenic mice, highlighting the potential therapeutic value of this approach even in an extreme IL-5-driven clinical state. Suppression of eosinophil production by TLR4 ligands is conserved between mice and humans as yield of eosinophils from human EoPs was reduced following LPS exposure. Taken together, these findings identify a mechanistic explanation for eosinopenia following bacterial infections and identify a novel therapeutic strategy for inhibiting eosinophil production and peripheral eosinophilia in eosinophil-associated diseases. Disclosures:No relevant conflicts of interest to declare.

Eosinophil differentiation in the bone marrow is inhibited by T cell–derived IFN-γ

AbstractTo explore whether and how T cells can affect myelopoiesis, we investigated myeloid differentiation in a model for T cell-mediated immune activation. We found that CD70-transgenic (CD70TG) mice, which have elevated numbers of interferon-γ (IFN-γ)–producing effector T cells in the periphery and bone marrow, are almost devoid of eosinophilic granulocytes. Induction of allergic airway inflammation in these mice failed to induce eosinophilia as well as airway hyperresponsiveness. CD70TG mice also have strongly reduced numbers of eosinophil lineage-committed progenitors, whereas granulocyte/macrophage progenitors from these mice are unable to generate eosinophils in vitro. We found that granulocyte/macrophage progenitors express IFN-γR1 and that IFN-γ is sufficient to inhibit eosinophil differentiation of both murine and human progenitor cells in vitro. We demonstrate that inhibition of eosinophil development in CD70TG mice is IFN-γ–dependent and that T cell–derived IFN-γ is sufficient to inhibit eosinophil formation in vivo. Finally, we found that IFN-γ produced on anti-CD40 treatment and during viral infection can also suppress eosinophil formation in wild-type mice. These data demonstrate that IFN-γ inhibits the differentiation of myeloid progenitors to eosinophils, indicating that the adaptive immune system plays an important role in orchestrating the formation of the appropriate type of myeloid cells during immune activation.

Eosinophilopoiesis at the Cross-Roads of Research on Development, Immunity and Drug Discovery

Eosinophils are a minority subpopulation of leukocytes whose roles in host defense against infection remain controversial, but which have been implicated in the pathogenesis of both acute allergic inflammation and the chronic bronchopulmonary remodelling in asthma. Eosinophilia, a hallmark of both helminth infections and atopic diseases, is maintained through upregulation of eosinophilopoiesis by means of increased production and effectiveness of Interleukin-5 (IL-5), a major Th2 cytokine. These mechanisms are further modulated by a wide variety of agents, including glucocorticoids, nonsteroidal anti-inflammatory drugs and mediators of inflammation. We review recent progress made by different groups in the study of eosinophilopoiesis that led to the identification of the heterogeneous targets for developmental regulation by IL-5 and other agents, and to the ongoing characterization of the molecular mechanisms that ensure their commitment to the eosinophil lineage. We argue that the study of eosinophilopoiesis provides insight into basic developmental processes, and especially into how modulators influence the constitutive rate of eosinophil production by controlling the rates of apoptosis and terminal differentiation. The mechanisms underlying the apparently paradoxical effects of dexamethasone, a drug widely employed to control inflammation, as well as the role of specific molecular targets (including inducible NO synthase and CD95/Fas) in developmental regulation, are discussed in detail. We further argue that eosinophilopoiesis offers unique insights of how immune and endocrine effector loops interact to control both the steady-state responses to IL-5 and the susceptibility to modulation of these responses by drugs and cytokines. We also review the existing evidence on the recruitment of circulating stem cells and progenitors into inflammatory sites, and on a critical role for IL-5 in the accumulation of eosinophil lineage-committed progenitors in lungs of allergic mice. Finally, we review recent progress in the study of the regulatory T cell populations present in bone-marrow, and discuss alternative mechanisms through which cellular immunity may influence eosinophilopoiesis.

Sputum CD34+IL-5Ralpha+ cells increase after allergen: evidence for in situ eosinophilopoiesis.

Eosinophil lineage-committed progenitors increase in the bone marrow of subjects with asthma developing allergen-induced airway hyperresponsiveness and eosinophilia. Also, higher numbers of circulating eosinophil/basophil cfu have been demonstrated 24 hours after allergen inhalation and in bronchial and nasal biopsies of allergic individuals. These cells may undergo in situ eosinophilopoiesis, suggesting that after allergen inhalation, progenitor cells traffic from the bone marrow to the airways, providing an ongoing source of effector cells. To examine this possibility, CD34(+) and CD34(+)IL-5Ralpha(+) cells were measured in induced sputum from allergic subjects with asthma at baseline and at 7 and 24 hours after allergen and diluent inhalation, using flow cytometry. Isolated early responders (n = 9) were contrasted to dual responders (n = 9), who develop allergen-induced sputum and blood eosinophilia and airway hyperresponsiveness, and to normal control subjects. At baseline, there were significantly fewer sputum eosinophils and CD34(+) cells in normal control subjects compared with subjects with asthma. Sputum CD34(+) cells increased at 7 hours after allergen inhalation in both groups of subjects with asthma, which was sustained at 24 hours in the dual responder group only, associated with sustained increases in sputum CD34(+)IL-5Ralpha(+) cells, eosinophils, and interleukin-5. These results indicate that eosinophil progenitors can migrate to the airways and may differentiate toward an eosinophilic phenotype.