Extracellular Sombrero Vesicles are Hallmarks of Eosinophilic Cytolytic Degranulation in Tissue Sites of Human Diseases.

Eosinophil sombrero vesicles (EoSVs) are large tubular carriers resident in the cytoplasm of human eosinophils, identifiable by transmission electron microscopy (TEM), and important for immune mediator transport. Increased EoSV formation occurs in activated eosinophils in vitro and in vivo. In tissue sites of eosinophilic cytolytic inflammation, extracellular EoSVs are noted, but their frequency and significance in eosinophil-associated diseases (EADs) remain unclear. Here, we performed comprehensive quantitative TEM analyses and electron tomography to investigate the numbers, density, integrity, and three-dimensional (3D) structure of EoSVs in different biopsy tissues from five prototypic EADs (eosinophilic chronic rhinosinusitis/nasal sinuses, ulcerative colitis/intestines, hypereosinophilic syndrome/skin, dermatitis/skin, and schistosomiasis/rectum). The morphology of extracellular EoSVs was also compared with that of cytoplasmic EoSVs, isolated by subcellular fractionation from peripheral blood eosinophils. We demonstrated that: i) eosinophil cytolysis, releasing intact EoSVs and membrane-bound granules, is a consistent event in all EADs; ii) EoSVs persist intact even after complete disintegration of all cell organelles, except granules (late cytolysis); iii) the EoSV population, composed of elongated, curved, and typical sombreros, and the EoSV 3D architecture, diameter, and density remain unchanged in the extracellular matrix; iv) free EoSVs closely associate with extracellular granules; and v) free EoSVs also associate with externalized chromatin during eosinophil ETosis. Remarkably, EoSVs appeared on the surface of other cells like plasma cells. Thus, eosinophil cytolysis/ETosis can secrete intact EoSVs, alongside granules, in inflamed tissues of EADs, potentially serving as propagators of eosinophil immune responses post-cell death.

Aggregated eosinophils and neutrophils characterize the properties of mucus in chronic rhinosinusitis.

BACKGROUND: Airway obstruction caused by viscous mucus is an important pathophysiologic characteristic of persistent inflammation, which can result in organ damage. OBJECTIVE: We investigated the hypothesis that the biophysical characteristics of accumulating granulocytes affect the clinical properties of mucus. METHODS: Surgically acquired nasal mucus samples from patients with eosinophilic chronic rhinosinusitis and neutrophil-dominant, noneosinophilic chronic rhinosinusitis were evaluated in terms of computed tomography density, viscosity, water content, wettability, and protein composition. Isolated human eosinophils and neutrophils were stimulated to induce the formation of extracellular traps, followed by the formation of aggregates. The biophysical properties of the aggregated cells were also examined. RESULTS: Mucus from patients with eosinophilic chronic rhinosinusitis had significantly higher computed tomography density, viscosity, dry weight, and hydrophobicity compared to mucus from patients with noneosinophilic chronic rhinosinusitis. The levels of eosinophil-specific proteins in mucus correlated with its physical properties. Eosinophil and neutrophil aggregates showed physical and pathologic characteristics resembling those of mucus. Cotreatment with deoxyribonuclease and heparin, which slenderizes the structure of eosinophil extracellular traps, efficiently induced reductions in the viscosity and hydrophobicity of both eosinophil aggregates and eosinophilic mucus. CONCLUSIONS: The present study elucidated the pathogenesis of mucus stasis in infiltrated granulocyte aggregates from a novel perspective. These findings may contribute to the development of treatment strategies for eosinophilic airway diseases.

Biologics and Hypereosinophilic Syndromes: Knowledge Gaps and Controversies.

Hypereosinophilic syndromes (HES) are a heterogeneous group of disorders defined by blood and/or tissue hypereosinophilia and clinical manifestations attributable to the eosinophilia. Although various clinical subtypes of HES have been described, the general approach to therapy in all subtypes has focused on the reduction of blood and tissue eosinophilia to improve symptoms and halt disease progression. Until recently, this typically involved the use of corticosteroids and/or other immunosuppressive or cytotoxic drugs with significant toxicity. Whereas imatinib, the first targeted therapy approved for treatment of HES, has dramatically changed the prognosis of patients with primary (myeloid) forms of HES, it is ineffective in patients with other HES subtypes. For these nonmyeloid patients with HES, the development of eosinophil-targeting biologics (most notably, mepolizumab, the first biologic approved for the treatment of HES) has been transformative. Nevertheless, important issues remain with respect to the efficacy and safety of these biologics in the treatment of the varied subtypes of HES. Moreover, with the increasing number of commercially available biologics with direct or indirect effects on eosinophils, questions related to the choice of initial biologic, potential reasons for biologic failure, and treatment options in the setting of incomplete response are becoming increasingly common.

Urine eosinophil-derived neurotoxin: A potential marker of activity in select eosinophilic disorders.

BACKGROUND: Biomarkers of eosinophilic disease activity, especially in the context of novel therapies that reduce blood eosinophil counts, are an unmet need. Absolute eosinophil count (AEC) does not accurately reflect tissue eosinophilia or eosinophil activation. Therefore, the aims of this study were to compare the reliability of plasma and urine eosinophil major basic protein 1, eosinophil cationic protein, eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase measurement and to evaluate the usefulness of eosinophil granule protein (EGP) measurement for the assessment of disease activity in patients with eosinophil-associated diseases treated with mepolizumab, benralizumab, or dexpramipexole. METHODS: Eosinophil granule protein concentrations were measured in serum, plasma, and urine from healthy volunteers and patients with hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and eosinophilic asthma using a multiplex assay. RESULTS: Urine EGP concentrations remained stable, whereas serum and plasma EGP concentrations increased significantly with delayed processing. Plasma (p) EDN, but not urine (u) EDN, concentration correlated with AEC and negatively correlated with prednisone dose. Both pEDN and uEDN decreased significantly following treatment of HES patients with benralizumab and EGPA patients with mepolizumab. uEDN appeared to increase with clinical relapse in both patient groups. CONCLUSIONS: Measurement of EGP in urine is noninvasive and unaffected by cellular lysis. Although plasma and urine EDN concentrations showed a similar pattern following benralizumab and mepolizumab treatment, the lack of correlation between AEC or prednisone dose and uEDN concentrations suggests that measurement of uEDN may provide a potential biomarker of disease activity in patients with HES and EGPA.

In Vivo ETosis of Human Eosinophils: The Ultrastructural Signature Captured by TEM in Eosinophilic Diseases.

Eosinophilic diseases, also termed eosinophil-associated diseases (EADs), are characterized by eosinophil-rich inflammatory infiltrates and extensive eosinophil degranulation with clinically relevant organ pathology. Recent evidence shows that eosinophil cytolytic degranulation, that is, the release of intact, membrane-delimited granules that arises from the eosinophil cytolysis, occurs mainly through ETosis, meaning death with a cytolytic profile and extrusion of nucleus-originated DNA extracellular traps (ETs). The ultrastructural features of eosinophil ETosis (EETosis) have been studied mostly in vitro after stimulation, but are still poorly understood in vivo. Here, we investigated in detail, by transmission electron microscopy (TEM), the ultrastructure of EETosis in selected human EADs affecting several tissues and organ systems. Biopsies of patients diagnosed with eosinophilic chronic rhinosinusitis/ECRS (frontal sinus), ulcerative colitis/UC (intestine), and hypereosinophilic syndrome/HES (skin) were processed for conventional TEM. First, we found that a large proportion of tissue-infiltrated eosinophils in all diseases (~45-65% of all eosinophils) were undergoing cytolysis with release of free extracellular granules (FEGs). Second, we compared the morphology of tissue inflammatory eosinophils with that shown by in vitro ETosis-stimulated eosinophils. By applying single-cell imaging analysis, we sought typical early and late EETosis events: chromatin decondensation; nuclear delobulation and rounding; expanded nuclear area; nuclear envelope alterations and disruption; and extracellular decondensed chromatin spread as ETs. We detected that 53% (ECRS), 37% (UC), and 82% (HES) of all tissue cytolytic eosinophils had ultrastructural features of ETosis in different degrees. Eosinophils in early ETosis significantly increased their nuclear area compared to non-cytolytic eosinophils due to excessive chromatin decondensation and expansion observed before nuclear envelope disruption. ETosis led not only to the deposition of intact granules, but also to the release of eosinophil sombrero vesicles (EoSVs) and Charcot-Leyden crystals (CLCs). Free intact EoSVs and CLCs were associated with FEGs and extracellular DNA nets. Interestingly, not all cytolytic eosinophils in the same microenvironment exhibited ultrastructure of ETosis, thus indicating that different populations of eosinophils might be selectively activated into this pathway. Altogether, our findings captured an ultrastructural signature of EETosis in vivo in prototypic EADs highlighting the importance of this event as a form of eosinophil degranulation and release of inflammatory markers (EoSVs and CLCs).

Mitochondrial Population in Mouse Eosinophils: Ultrastructural Dynamics in Cell Differentiation and Inflammatory Diseases.

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.

Eosinophils in Health and Disease: A State-of-the-Art Review.

Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

Eosinophil ETosis-Mediated Release of Galectin-10 in Eosinophilic Granulomatosis With Polyangiitis.

OBJECTIVE: Eosinophils are tissue-dwelling immune cells. Accumulating evidence indicates that a type of cell death termed ETosis is an important cell fate involved in the pathophysiology of inflammatory diseases. Although the critical role of eosinophils in eosinophilic granulomatosis with polyangiitis (EGPA; formerly Churg-Strauss syndrome) is well established, the presence of eosinophil ETosis (EETosis) is poorly understood. We undertook this study to better understand the characteristics of EETosis. METHODS: In vitro studies using blood-derived eosinophils were conducted to characterize EETosis. The occurrence of EETosis in tissues from patients with EGPA was studied by immunostaining and electron microscopy. Serum concentrations of eosinophil-derived proteins in healthy controls, patients with asthma, and EGPA patients with active disease or with disease in remission (n = 15 per group) were examined. RESULTS: EETosis was reliant on reactive oxygen species and peptidylarginine deiminase type 4-dependent histone citrullination, resulting in the cytolytic release of net-like eosinophil extracellular traps, free galectin-10, and membrane-bound intact granules. The signature of EETosis, including loss of cytoplasmic galectin-10 and deposition of granules, was observed in eosinophils infiltrating various tissues from EGPA patients. Serum eosinophil granule proteins and galectin-10 levels were increased in EGPA and positively correlated with disease activity as assessed by the Birmingham Vasculitis Activity Score (r = 0.8531, P < 0.0001 for galectin-10). When normalized to blood eosinophil counts, this correlation remained for galectin-10 (r = 0.7168, P < 0.0001) but not for granule proteins. Galectin-10 levels in active EGPA positively correlated with serum interleukin-5 levels. CONCLUSION: Eosinophils infiltrating diseased tissues in EGPA undergo EETosis. Considering the exclusive expression and large pool of cytoplasmic galectin-10 in eosinophils, elevated serum galectin-10 levels in patients with EGPA might reflect the systemic occurrence of cytolytic EETosis.

How to detect eosinophil ETosis (EETosis) and extracellular traps.

Eosinophils are short-lived and comprise only a small population of circulating leukocytes; however, they play surprisingly multifunctional roles in homeostasis and various diseases including allergy and infection. Recent research has shed light on active cytolytic eosinophil cell death that releases eosinophil extracellular traps (EETs) and total cellular contents, namely eosinophil extracellular trap cell death (EETosis). The pathological contribution of EETosis was made more cogent by recent findings that a classical pathological finding of eosinophilic inflammation, that of Charcot-Leyden crystals, is closely associated with EETosis. Currently no gold standard methods to identify EETosis exist, but "an active eosinophil lysis that releases cell-free granules and net-like chromatin structure" appears to be a common feature of EETosis. In this review, we describe several approaches that visualize EETs/EETosis in clinical samples and in vitro studies using isolated human eosinophils. EETs/EETosis can be observed using simple chemical or fluorescence staining, immunostaining, and electron microscopy, although it is noteworthy that visualization of EETs is greatly changed by sample preparation including the extracellular space of EETotic cells and shear flow. Considering the multiple aspects of biological significance, further study into EETs/EETosis is warranted to give a detailed understanding of the roles played in homeostasis and disease pathogenesis.

The Charcot-Leyden crystal protein revisited-A lysopalmitoylphospholipase and more.

The Charcot-Leyden crystal protein (CLC-P), a constituent of human and not mouse eosinophils, is one of the most abundant proteins within human eosinophils. It has a propensity to form crystalline structures, Charcot-Leyden crystals, which are hallmarks in their distinctive extracellular crystalline forms as markers of eosinophilic inflammation. The functions of CLC-P within eosinophils have been uncertain. Although the action of CLC-P as a lysophospholipase has been questioned, assays of chromatographically purified CLC-P and crystal-derived CLC-P as well as studies of transfected recombinant CLC-P have consistently documented that CLC-P endogenously expresses lysophospholipase activity, releasing free palmitate from substrate lysopalmitoylphosphatidylcholine. Rather than acting solely as a hydrolytic enzyme to release palmitate from a lysolipid substrate, some other lysophospholipases function more dominantly as acyl-protein thioesterases (APTs), enzymes that catalyze the removal of thioester-linked, long chain fatty acids, such as palmitate, from cysteine residues of proteins. As such APTs participate in palmitoylation, a post-translational modification that can affect membrane localization, vesicular transport, and secretion. CLC-P has attributes of an APT. Thus, whereas CLC-P expresses inherent lysophospholipase activity, like some other lysophospholipase enzymes, it likely also functions in regulating the dynamic palmitoylation cycle, including, given its dominant subplasmalemmal location, at the human eosinophil's plasma membrane.

Pulmonary Eosinophilic Granulomatosis with Polyangiitis Has IgG4 Plasma Cells and Immunoregulatory Features.

The immunologic mechanisms promoting eosinophilic granulomatosis with polyangiitis (EGPA) are unclear. To characterize the mechanisms underlying pulmonary EGPA, we examined and compared EGPA paraffin-embedded lung biopsies with normal lung biopsies, using immunostaining, RNA sequencing, and RT-PCR. The results revealed novel type 2 as well as immuneregulatory features. These features included basophils and increased mast cell contents; increased immunostaining for tumor necrosis factor ligand superfamily member 14; sparse mast cell degranulation; numerous forkhead box protein P3 (FoxP3)+ regulatory T cells and IgG4 plasma cells; and abundant arachidonate 15-lipoxygenase and 25-hydroxyvitamin D-1 α hydroxylase, mitochondrial. Significantly decreased 15-hydroxyprostaglandin dehydrogenase [NAD(+)], which degrades eicosanoids, was observed in EGPA samples. In addition, there was significantly increased mRNA for chemokine (C-C motif) ligands 18 and 13 and major collagen genes, IgG4-rich immune complexes coating alveolar macrophages, and increased immunostaining for phosphorylated mothers against decapentaplegic homolog 2/SMAD2, suggesting transforming growth factor-ß activation. These findings suggest a novel self-promoting mechanism of activation of alveolar macrophages by arachidonate 15-lipoxygenase-derived eicosanoids to express chemokines that recruit a combined type 2/immunoregulatory immune response, which produces these eicosanoids. These results suggest that the pulmonary EGPA immune response resembles the immune response to a tissue-invasive parasite infection.

Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils.

A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).

Correction to: Charcot-Leyden Crystals in Eosinophilic Inflammation: Active Cytolysis Leads to Crystal Formation.

The original version of this article incorrectly listed the third author's name. It should be Yohei Yamamoto, not Yamamoto Yohei.

Charcot-Leyden Crystals in Eosinophilic Inflammation: Active Cytolysis Leads to Crystal Formation.

PURPOSE OF REVIEW: Charcot-Leyden crystals (CLCs), slender bipyramidal hexagonal crystals, were first described by Jean-Martin Charcot in 1853, predating Paul Ehrlich's "discovery" of eosinophils by 26 years. To date, CLCs are known as a classical hallmark of eosinophilic inflammation. CLC protein expresses palmitate cleaving lysophospholipase activity and is a member of the family of S-type lectins, galectin-10. We summarize current knowledge regarding the pathological observations of CLCs and their mechanism of generation focusing on eosinophil cell death. RECENT FINDINGS: The presence of CLCs in vivo has been consistently associated with lytic eosinophils. Recent evidence revealed that cytolysis represents the occurrence of extracellular trap cell death (ETosis), an active non-apoptotic cell death process releasing filamentous chromatin structure. Galectin-10 is a predominant protein present within the cytoplasm of eosinophils but not stored in secretory granules. Activated eosinophils undergo ETosis and loss of galectin-10 cytoplasmic localization results in intracellular CLC formation. Free galectin-10 released following plasma membrane disintegration forms extracellular CLCs. Of interest, galectin-10-containing extracellular vesicles are also released during ETosis. Mice models indicated that CLCs could be a novel therapeutic target for Th2-type airway inflammation. The concept of ETosis, which represents a major fate of activated eosinophils, expands our current understanding by which cytoplasmic galectin-10 is crystalized/externalized. Besides CLCs and free galectin-10, cell-free granules, extracellular chromatin traps, extracellular vesicles, and other alarmins, all released through the process of ETosis, have novel implications in various eosinophilic disorders.

Rho and Rac, but not ROCK, are required for secretion of human and mouse eosinophil-associated RNases.

BACKGROUND: Eosinophil-associated RNases (EARs) are stored preformed in eosinophil cytoplasmic secretory granules and have a key role in eosinophil effector functions in host defence and inflammatory disorders. However, the secretion mechanisms of EARs are poorly understood. OBJECTIVE: Our study aimed to understand the involvement of cytoskeleton machinery in EAR secretion. METHODS: Fresh human and mouse eosinophils were stimulated with CCL11, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of cytoskeletal elements or microtubules was probed using specific inhibitors. RESULTS: We found that dynamic polymerization of microtubules and cytoskeletal elements, such as Rho and Rac, is required for chemokine-mediated EAR secretion from human and mouse eosinophils. However, inhibition of ROCK (Rho-associated protein kinase) increased EAR secretion in human and mouse eosinophils even in the absence of chemokine stimulation, suggesting ROCK negatively regulates EAR secretion. CONCLUSIONS: Collectively, these data suggest a cytoskeleton-dependent mechanism of EAR secretion from eosinophils, findings that are pertinent to host defence, allergy and other eosinophil-associated diseases.

Evaluation of clinical benefit from treatment with mepolizumab for patients with eosinophilic granulomatosis with polyangiitis.

BACKGROUND: In a recent phase III trial (NCT02020889) 53% of mepolizumab-treated versus 19% of placebo-treated patients with eosinophilic granulomatosis with polyangiitis (EGPA) achieved protocol-defined remission. OBJECTIVE: We sought to investigate post hoc the clinical benefit of mepolizumab in patients with EGPA using a comprehensive definition of benefit encompassing remission, oral glucocorticoid (OGC) dose reduction, and EGPA relapses. METHODS: The randomized, placebo-controlled, double-blind, parallel-group trial recruited patients with relapsing/refractory EGPA receiving stable OGCs (prednisolone/prednisone, ≥7.5-50 mg/d) for 4 or more weeks. Patients received 300 mg of subcutaneous mepolizumab or placebo every 4 weeks for 52 weeks. Clinical benefit was defined post hoc as follows: remission at any time (2 definitions used), 50% or greater OGC dose reduction during weeks 48 to 52, or no EGPA relapses. The 2 remission definitions were Birmingham Vasculitis Activity Score of 0 plus OGC dose of 4 mg/d or less (remission 1/clinical benefit 1) or 7.5 mg/d or less (remission 2/clinical benefit 2). Clinical benefit was assessed in all patients and among subgroups with a baseline blood eosinophil count of less than 150 cells/µL, baseline OGC dosage of greater than 20 mg/d, or weight of greater than 85 kg. RESULTS: With mepolizumab versus placebo, 78% versus 32% of patients experienced clinical benefit 1, and 87% versus 53% of patients experienced clinical benefit 2 (both P < .001). Significantly more patients experienced clinical benefit 1 with mepolizumab versus placebo in the blood eosinophil count less than 150 cells/µL subgroup (72% vs 43%, P = .033) and weight greater than 85 kg subgroup (68% vs 23%, P = .005); in the OGC greater than 20 mg/d subgroup, results were not significant but favored mepolizumab (60% vs 36%, P = .395). CONCLUSION: When a comprehensive definition of clinical benefit was applied to data from a randomized controlled trial, 78% to 87% of patients with EGPA experienced benefit with mepolizumab.

Leptin Elicits LTC4 Synthesis by Eosinophils Mediated by Sequential Two-Step Autocrine Activation of CCR3 and PGD2 Receptors.

Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.

Charcot-Leyden crystal formation is closely associated with eosinophil extracellular trap cell death.

Protein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.