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.

Mitochondria are multifunctional organelles of which ultrastructure is tightly linked to cell physiology. Accumulating evidence shows that mitochondrial remodeling has an impact on immune responses, but our current understanding of the mitochondrial architecture, interactions, and morphological changes in immune cells, mainly in eosinophils, is still poorly known. Here, we applied transmission electron microscopy (TEM), single-cell imaging analysis, and electron tomography, a technique that provides three-dimensional (3D) views at high resolution, to investigate mitochondrial dynamics in mouse eosinophils developing in cultures as well as in the context of inflammatory diseases characterized by recruitment and activation of these cells (mouse models of asthma, H1N1 influenza A virus (IAV) infection, and schistosomiasis mansoni). First, quantitative analyses showed that the mitochondrial area decrease 70% during eosinophil development (from undifferentiated precursor cells to mature eosinophils). Mitophagy, a consistent process revealed by TEM in immature but not in mature eosinophils, is likely operating in mitochondrial clearance during eosinophilopoiesis. Events of mitochondria interaction (inter-organelle membrane contacts) were also detected and quantitated within developing eosinophils and included mitochondria-endoplasmic reticulum, mitochondria-mitochondria, and mitochondria-secretory granules, all of them significantly higher in numbers in immature compared to mature cells. Moreover, single-mitochondrion analyses revealed that as the eosinophil matures, mitochondria cristae significantly increase in number and reshape to lamellar morphology. Eosinophils did not change (asthma) or reduced (IAV and Schistosoma infections) their mitochondrial mass in response to inflammatory diseases. However, asthma and schistosomiasis, but not IAV infection, induced amplification of both cristae numbers and volume in individual mitochondria. Mitochondrial cristae remodeling occurred in all inflammatory conditions with the proportions of mitochondria containing only lamellar or tubular, or mixed cristae (an ultrastructural aspect seen just in tissue eosinophils) depending on the tissue/disease microenvironment. The ability of mitochondria to interact with granules, mainly mobilized ones, was remarkably captured by TEM in eosinophils participating in all inflammatory diseases. Altogether, we demonstrate that the processes of eosinophilopoiesis and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils leading to cristae remodeling and inter-organelle contacts. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored.

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.

BACKGROUND: Eosinophils play an important role in allergic inflammation. Glucocorticosteroids have been used as an anti-inflammatory medication for inflammatory diseases involving eosinophil infiltration. Some effect of nebulized lidocaine has been reported when treating certain patients with asthma, which is also an inflammatory disease. The goal of this study was to examine the effects of dexamethasone and lidocaine on eosinophil proliferation and differentiation using a model of human umbilical cord blood mononuclear cells (UCMC) cultured with IL-5.
METHODS: UCMC were cultured with IL-5 (5 ng/mL) for 4 weeks. The effects of dexamethasone and lidocaine on the number and morphology of eosinophilic cells were visualized with Wright-Giemsa and cyanide-resistant peroxidase stains. Moreover, the effect on eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPX) contents in cultured cells were evaluated using radioimmunoassay.
RESULTS: The number of eosinophilic cells and EDN and EPX content in cultured cells increased in a time-dependent manner in the presence of IL-5. Dexamethasone treatment slightly decreased the number of eosinophilic cells in one week, but this effect was lost in 2-4 weeks. Macrophages in cultured UCMC treated with dexamethasone contained more eosinophil granule proteins. Both EDN and EPX content in cultured cells were reduced by dexamethasone. Lidocaine decreased the number of eosinophilic cells and reduced both EDN and EPX contents in cultured cells.
CONCLUSIONS: Dexamethasone suppressed the production of eosinophil granule proteins and may also induce apoptosis of eosinophils, while lidocaine suppresses eosinophilopoiesis.

Acute infection is characterized by eosinopenia. However, the underlying mechanism(s) are poorly understood and it is unclear whether decreased mobilization/production of eosinophils in the bone marrow (BM) and/or increased homing to the tissues play a role. The objective of this study was to investigate the differentiation and activation status of eosinophils in the human BM and blood upon experimental human endotoxemia, a standardized, controlled, and reproducible model of acute systemic inflammation. A BM aspirate and venous blood was obtained from seven healthy volunteers before, 4 h after, and 1 week after intravenous challenge with 2 ng/kg endotoxin. Early progenitors (CD34+/IL-5Rα+), eosinophil promyelocytes, myelocytes, metamyelocytes, and mature eosinophils were identified and quantified in the bone marrow and blood samples using flowcytometry based on specific eosinophil markers (CD193 and IL-5Rα). Activation status was assessed using antibodies against known markers on eosinophils: Alpha-4 (CD49d), CCR3 (CD193), CR1 (CD35), CEACAM-8 (CD66b), CBRM 1/5 (activation epitope of MAC-1), and by plasma cytokine analysis. Four hours after endotoxin administration, numbers of mature eosinophils in the blood and in the BM markedly declined compared with baseline, whereas numbers of all eosinophil progenitors did not change. The remaining eosinophils did not show signs of activation or degranulation despite significantly increased circulating levels of eotaxin-1. Furthermore, the expression of CD49d and CD193 on eosinophils was lower compared to baseline, but normalized after 7 days. Together these data imply that circulatory eosinopenia after an innate immune challenge is mediated by CD49d-mediated homing of eosinophils to the tissues.

Eosinophils are currently regarded as versatile mobile cells controlling and regulating multiple biological pathways and responses in health and disease. These cells store in their specific granules numerous biologically active substances (cytotoxic cationic proteins, cytokines, growth factors, chemokines, enzymes) ready for rapid release. The human gut is the main destination of eosinophils that are produced and matured in the bone marrow and then transferred to target tissues through the circulation. In health the most important functions of gut-residing eosinophils comprise their participation in the maintenance of the protective mucosal barrier and interactions with other immune cells in providing immunity to microbiota of the gut lumen. Eosinophils are closely involved in the development of inflammatory bowel disease (IBD), when their cytotoxic granule proteins cause damage to host tissues. However, their roles in Crohn\'s disease and ulcerative colitis appear to follow different immune response patterns. Eosinophils in IBD are especially important in altering the structure and protective functions of the mucosal barrier and modulating massive neutrophil influx to the lamina propria followed by transepithelial migration to colorectal mucus. IBD-associated inflammatory process involving eosinophils then appears to expand to the mucus overlaying the internal gut surface. The author hypothesises that immune responses within colorectal mucus as well as ETosis exerted by both neutrophils and eosinophils on the both sides of the colonic epithelial barrier act as additional pathogenetic factors in IBD. Literature analysis also shows an association between elevated eosinophil levels and better colorectal cancer (CRC) prognosis, but mechanisms behind this effect remain to be elucidated. In conclusion, the author emphasises the importance of investigating colorectal mucus in IBD and CRC patients as a previously unexplored milieu of disease-related inflammatory responses.

Eosinophilic asthma has conventionally been proposed to be a T helper 2 driven disease but emerging evidence supports a central role of type 2 innate lymphoid cells (ILC2s). These are non-T, non-B cells that lack antigen specificity and produce more IL-5 and IL-13 than CD4+ T lymphocytes, on a cell per cell basis, in vitro. Although it is clear that ILC2s and CD4+ T cells work in concert with each other to drive type 2 immune responses, kinetic studies in allergic asthma suggest that ILC2s may act locally within the airways to \"initiate\" eosinophilic responses, whereas CD4+ T cells act locally and systemically to \"perpetuate\" eosinophilic inflammatory responses. Importantly, ILC2s are increased within the airways of severe asthmatics, with the greatest number of IL-5+ IL-13+ ILC2s being detected in sputum from severe asthmatics with uncontrolled eosinophilia despite high-dose steroid therapy. Although the precise relationship between ILC2s and steroid sensitivity in asthma remains unclear, controlling the activation of ILC2s within the airways may provide an effective therapeutic target for eosinophilic inflammation in airways diseases.

Human eosinophils have characteristic morphologic features, including a bilobed nucleus and cytoplasmic granules filled with cytotoxic and immunoregulatory proteins that are packaged in a specific manner. Eosinophil production in the bone marrow is exquisitely regulated by timely expression of a repertoire of transcription factors that work together via collaborative and hierarchical interactions to direct eosinophil development. In addition, proper granule formation, which occurs in a spatially organized manner, is an intrinsic checkpoint that must be passed for proper eosinophil production to occur. In eosinophil-associated disorders, eosinophils and their progenitors can be recruited in large numbers into tissues where they can induce proinflammatory organ damage in response to local signals. Eosinophils are terminally differentiated and do not proliferate once they leave the bone marrow. The cytokine IL-5 specifically enhances eosinophil production and, along with other mediators, promotes eosinophil activation. Indeed, eosinophil depletion with anti-IL-5 or anti-IL-5Rα is now proven to be clinically beneficial for several eosinophilic disorders, most notably severe asthma, and several therapeutics targeting eosinophil viability and production are now in development. Significant progress has been made in our understanding of eosinophil development and the consequences of tissue eosinophilia. Future research efforts focused on basic eosinophil immunobiology and translational efforts to assist in the diagnosis, treatment selection, and resolution of eosinophil-associated disorders will likely be informative and clinically helpful.

The goal of this series is to offer a survey of the latest literature for clinicians and scientists alike, providing a list of important recent advances relevant to the broad field of allergy and immunology. This particular assignment was to cover the topic of eosinophils. In an attempt to highlight major ideas, themes, trends, and advances relevant to basic and clinical aspects of eosinophil biology, a search of articles published since 2015 in the Journal of Allergy and Clinical Immunology and other high-impact journals was performed. Articles were then reviewed and organized, and then key findings were summarized. Given space limitations, many outstanding articles could not be included, but the hope is that what follows provides a succinct overview of recently published work that has significantly added to our knowledge of eosinophils and eosinophil-associated diseases.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.