The transcription factor XBP1 is selectively required for eosinophil differentiation.

The transcription factor XBP1 has been linked to the development of highly secretory tissues such as plasma cells and Paneth cells, yet its function in granulocyte maturation has remained unknown. Here we discovered an unexpectedly selective and absolute requirement for XBP1 in eosinophil differentiation without an effect on the survival of basophils or neutrophils. Progenitors of myeloid cells and eosinophils selectively activated the endoribonuclease IRE1α and spliced Xbp1 mRNA without inducing parallel endoplasmic reticulum (ER) stress signaling pathways. Without XBP1, nascent eosinophils exhibited massive defects in the post-translational maturation of key granule proteins required for survival, and these unresolvable structural defects fed back to suppress critical aspects of the transcriptional developmental program. Hence, we present evidence that granulocyte subsets can be distinguished by their differential reliance on secretory-pathway homeostasis.

Advances and ongoing challenges in eosinophilic gastrointestinal disorders presented at the CEGIR/TIGERs Symposium at the 2024 American Academy of Allergy, Asthma & Immunology meeting.

The Consortium of Eosinophilic Gastrointestinal disease Researchers (CEGIR) and The International Gastrointestinal Eosinophil Researchers (TIGERs) organized a daylong symposium at the 2024 annual meeting of the American Academy of Allergy, Asthma & Immunology. The symposium featured new discoveries in basic and translational research as well as debates on the mechanisms and management of eosinophilic gastrointestinal diseases. Updates on recent clinical trials and consensus guidelines were also presented. We summarize the updates on eosinophilic gastrointestinal diseases presented at the symposium.

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.

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.

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.

An algorithm for the classification of mRNA patterns in eosinophilic esophagitis: Integration of machine learning.

BACKGROUND: Diagnostic evaluation of eosinophilic esophagitis (EoE) remains difficult, particularly the assessment of the patient's allergic status. OBJECTIVE: This study sought to establish an automated medical algorithm to assist in the evaluation of EoE. METHODS: Machine learning techniques were used to establish a diagnostic probability score for EoE, p(EoE), based on esophageal mRNA transcript patterns from biopsies of patients with EoE, gastroesophageal reflux disease and controls. Dimensionality reduction in the training set established weighted factors, which were confirmed by immunohistochemistry. Following weighted factor analysis, p(EoE) was determined by random forest classification. Accuracy was tested in an external test set, and predictive power was assessed with equivocal patients. Esophageal IgE production was quantified with epsilon germ line (IGHE) transcripts and correlated with serum IgE and the Th2-type mRNA profile to establish an IGHE score for tissue allergy. RESULTS: In the primary analysis, a 3-class statistical model generated a p(EoE) score based on common characteristics of the inflammatory EoE profile. A p(EoE) ≥ 25 successfully identified EoE with high accuracy (sensitivity: 90.9%, specificity: 93.2%, area under the curve: 0.985) and improved diagnosis of equivocal cases by 84.6%. The p(EoE) changed in response to therapy. A secondary analysis loop in EoE patients defined an IGHE score of ≥37.5 for a patient subpopulation with increased esophageal allergic inflammation. CONCLUSIONS: The development of intelligent data analysis from a machine learning perspective provides exciting opportunities to improve diagnostic precision and improve patient care in EoE. The p(EoE) and the IGHE score are steps toward the development of decision trees to define EoE subpopulations and, consequently, will facilitate individualized therapy.

Resident intestinal eosinophils constitutively express antigen presentation markers and include two phenotypically distinct subsets of eosinophils.

Intestinal eosinophils are implicated in homeostatic and disease-associated processes, yet the phenotype of intestinal tissue-dwelling eosinophils is poorly defined and their roles in intestinal health or disease remain enigmatic. Here we probed the phenotype and localization of eosinophils constitutively homed to the small intestine of naive mice at baseline, and of antigen-sensitized mice following intestinal challenge. Eosinophils homed to the intestinal lamina propria of naive mice were phenotypically distinguished from autologous blood eosinophils, and constitutively expressed antigen-presenting cell markers, suggesting that intestinal eosinophils, unlike blood eosinophils, may be primed for antigen presentation. We further identified a previously unrecognized resident population of CD11chi eosinophils that are recovered with intraepithelial leucocytes, and that are phenotypically distinct from both lamina propria and blood eosinophils. To better visualize intestinal eosinophils in situ, we generated eosinophil reporter mice wherein green fluorescent protein expression is targeted to both granule-delimiting and plasma membranes. Analyses of deconvolved fluorescent z-section image stacks of intestinal tissue sections from eosinophil reporter mice revealed eosinophils within intestinal villi exhibited dendritic morphologies with cellular extensions that often contacted the basement membrane. Using an in vivo model of antigen acquisition in antigen-sensitized mice, we demonstrate that both lamina propria-associated and intraepithelium-associated eosinophils encounter, and are competent to acquire, lumen-derived antigen. Taken together these data provide new foundational insights into the organization and functional potential of intestinal tissue-dwelling eosinophils, including the recognition of different subsets of resident intestinal eosinophils, and constitutive expression of antigen-presenting cell markers.

Humoral Immunity Provides Resident Intestinal Eosinophils Access to Luminal Antigen via Eosinophil-Expressed Low-Affinity Fcγ Receptors.

Eosinophils are native to the healthy gastrointestinal tract and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g., food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct Ag engagement elicits eosinophil effector functions, including degranulation and Ag presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food Ags by a columnar epithelium might similarly engage food Ags. Using an intestinal ligated loop model in mice, in this study we determined that resident intestinal eosinophils acquire Ag from the lumen of Ag-sensitized but not naive mice in vivo. Ag acquisition was Ig-dependent; intestinal eosinophils were unable to acquire Ag in sensitized Ig-deficient mice, and passive immunization with immune serum or Ag-specific IgG was sufficient to enable intestinal eosinophils in otherwise naive mice to acquire Ag in vivo. Intestinal eosinophils expressed low-affinity IgG receptors, and the activating receptor FcγRIII was necessary for Ig-mediated acquisition of Ags by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food Ags in sensitized mice via FcγRIII Ag focusing and that they may therefore participate in Ag-driven secondary immune responses to oral Ags.

Eosinophil ETosis and DNA Traps: a New Look at Eosinophilic Inflammation.

The traditional paradigm of eosinophils as end-stage damaging cells has mainly relied on their release of cytotoxic proteins. Cytokine-induced cell survival and secretion of granular contents from tissue-dwelling eosinophil are thought to be important mechanisms for eosinophilic inflammatory disorders, although the occurrence of cytolysis and its products (i.e., free extracellular granules) has been observed in affected lesions. Recent evidence indicates that activated eosinophils can exhibit a non-apoptotic cell death pathway, namely extracellular trap cell death (ETosis) that mediates the eosinophil cytolytic degranulation. Here, we discuss the current concept of eosinophil ETosis which provides a new look at eosinophilic inflammation. Lessons from eosinophilic chronic rhinosinusitis revealed that ETosis-derived DNA traps, composed of stable web-like chromatin, contribute to the properties of highly viscous eosinophilic mucin and impairments in its clearance. Intact granules entrapped in DNA traps are causing long-lasting inflammation but also might have immunoregulatory roles. Eosinophils possess a way to have post-postmortem impacts on innate immunity, local immune response, sterile inflammation, and tissue damage.

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans.

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Pathophysiology of Non-Esophageal Eosinophilic Gastrointestinal Disorders.

Eosinophilic gastrointestinal disorder (EGID) is an umbrella term encompassing a group of chronic, immune-mediated disorders characterized by eosinophil-rich inflammation affecting one or more segments of the gastrointestinal tract. A recent consensus in nomenclature and emerging data made possible through multi-center consortia are beginning to unravel the molecular and cellular underpinnings of EGIDs below the esophagus. These emerging findings are revealing both overarching commonalities related to a food allergen-driven, chronic, Th2-mediated immune response as well as location-specific nuances in the pathophysiology of the collective EGIDs. Altogether, these advances offer promise for improved diagnoses and more efficacious interventional strategies.

Notch 2 signaling contributes to intestinal eosinophil adaptations in steady state and tissue burden following oral allergen challenge.

Eosinophils not only function as inflammatory effectors in allergic diseases, but also contribute to tissue homeostasis in steady state. Emerging data are revealing tissue eosinophils to be adaptive cells, imprinted by their local tissue microenvironment and exhibiting distinct functional phenotypes that may contribute to their homeostatic vs. inflammatory capacities. However, signaling pathways that regulate eosinophil tissue adaptations remain elusive. Notch signaling is an evolutionarily conserved pathway that mediates differential cell fate programming of both pre- and postmitotic immune cells. This study investigated a role for notch receptor 2 signaling in regulating eosinophil functions and tissue phenotype in both humans and mice. Notch 2 receptors were constitutively expressed and active in human blood eosinophils. Pharmacologic neutralization of notch 2 in ex vivo stimulated human eosinophils altered their activated transcriptome and prevented their cytokine-mediated survival. Genetic ablation of eosinophil-expressed notch 2 in mice diminished steady-state intestine-specific eosinophil adaptations and impaired their tissue retention in a food allergic response. In contrast, notch 2 had no effect on eosinophil phenotype or tissue inflammation within the context of allergic airways inflammation, suggesting that notch 2-dependent regulation of eosinophil phenotype and function is specific to the gut. These data reveal notch 2 signaling as a cell-intrinsic mechanism that contributes to eosinophil survival, function, and intestine-specific adaptations. The notch 2 pathway may represent a viable strategy to reprogram eosinophil functional phenotypes in gastrointestinal eosinophil-associated diseases.

Biologic therapy in rare eosinophil-associated disorders: remaining questions and translational research opportunities.

Rare eosinophil-associated disorders (EADs), including hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, and eosinophilic gastrointestinal disorders, are a heterogeneous group of conditions characterized by blood and/or tissue hypereosinophilia and eosinophil-related clinical manifestations. Although the recent availability of biologic therapies that directly and indirectly target eosinophils has the potential to dramatically improve treatment options for all EADs, clinical trials addressing their safety and efficacy in rare EADs have been relatively few. Consequently, patient access to therapy is limited for many biologics, and the establishment of evidence-based treatment guidelines has been extremely difficult. In this regard, multicenter retrospective collaborative studies focusing on disease manifestations and treatment responses in rare EADs have provided invaluable data for physicians managing patients with these conditions and helped identify important questions for future translational research. During the Clinical Pre-Meeting Workshop held in association with the July 2023 biennial meeting of the International Eosinophil Society in Hamilton, Ontario, Canada, the successes and limitations of pivotal multicenter retrospective studies in EADs were summarized and unmet needs regarding the establishment of guidelines for use of biologics in rare EADs were discussed. Key topics of interest included (1) clinical outcome measures, (2) minimally invasive biomarkers of disease activity, (3) predictors of response to biologic agents, and (4) long-term safety of eosinophil depletion. Herein, we report a summary of these discussions, presenting a state-of-the-art overview of data currently available for each of these topics, the limitations of the data, and avenues for future data generation through implementation of multidisciplinary and multicenter studies.

CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules.

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-ß-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

Modulation of surface CD11c expression tracks plasticity in murine intestinal tissue eosinophils.

Intestinal eosinophils are implicated in the inflammatory pathology of eosinophilic gastrointestinal diseases and inflammatory bowel diseases. Eosinophils also contribute to intestinal immunologic and tissue homeostasis and host defense. Recent studies in allergic airway disease suggest functional subphenotypes of eosinophils may underly their pathogenic versus protective roles. However, subphenotypes of intestinal eosinophils have not been defined and are complicated by their constitutive expression of the putative eosinophil inflammatory marker CD11c. Here, we propose a framework for subphenotype characterization of intestinal eosinophils based on relative intensity of surface CD11c expression. Using this flow cytometry framework in parallel with histology and BrdU tracing, we characterize intestinal eosinophil subphenotypes and monitor their plasticity at baseline and within the context of acute allergic and chronic systemic inflammation. Data reveal a conserved continuum of CD11c expression amongst intestinal eosinophils in health and acute disease states that overall tracked with other markers of activation. Oral allergen challenge induced recruitment of eosinophils into small intestinal lamina propria surrounding crypts, followed by in situ induction of CD11c expression in parallel with eosinophil redistribution into intestinal villi. Allergen challenge also elicited eosinophil transepithelial migration and the appearance of CD11clo CD11bhi eosinophils in the intestinal lumen. Chronic inflammation driven by overexpression of TNFα led to a qualitative shift in the relative abundance of CD11c-defined eosinophil subphenotypes favoring CD11chi -expressing eosinophils. These findings provide new insights into heterogeneity of intestinal tissue eosinophils and offer a framework for measuring and tracking eosinophil subphenotype versatility in situ in health and disease.

Eosinophils in innate immunity: an evolving story.

Eosinophils are innate immune leukocytes found in relatively low numbers within the blood. Terminal effector functions of eosinophils, deriving from their capacity to release their content of tissue-destructive cationic proteins, have historically been considered primary effector mechanisms against specific parasites, and are likewise implicated in tissue damage accompanying allergic responses such as asthma. However, the past decade has seen dramatic advancements in the field of eosinophil immunobiology, revealing eosinophils to also be key participants in many other facets of innate immunity, from bridging innate and adaptive immune responses to orchestrating tissue remodeling events. Here, we review the multifaceted functions of eosinophils in innate immunity that are currently known, and discuss new avenues in this evolving story.

Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation.

Eosinophil chemotaxis and survival within tissues are key components in the development of tissue eosinophilia and subsequent effector responses. In this study, we demonstrate a novel mechanism of eosinophil autoregulation affecting migration and survival mediated through Notch signaling. We show for the first time that human blood eosinophils express Notch receptors and Notch ligands, expressions of which are influenced by the presence of eosinophil-activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence of Notch receptor activation and subsequent transcription of the Notch-responsive gene HES1 were observed in GM-CSF-stimulated eosinophils, confirming functionality of eosinophil-expressed Notch-signaling components. Moreover, by inhibiting Notch signaling with gamma-secretase inhibitors or Notch receptor-specific neutralizing antibodies, we demonstrate that autocrine Notch signaling enhances stimulus-mediated actin rearrangement and eosinophil chemokinesis, and impairs eosinophil viability. Taken together, these data suggest autocrine Notch signaling, enhanced in response to tissue- or inflammatory-derived signals, influences eosinophil activity and longevity, which may ultimately contribute to the development of tissue eosinophilia and exacerbation or remediation of eosinophil effector functions.