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

Eosinophils 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 versus 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 post-mitotic 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 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.

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.

Assessing Phenotypic Heterogeneity in Intestinal Tissue Eosinophils.

Eosinophils are primarily tissue-dwelling leukocytes. Utilization of flow cytometry techniques applied to digested tissues is expanding the scope of organs within which eosinophils are identified at baseline and is providing deeper insights into categorizing phenotypically and functionally distinct tissue-resident eosinophil subpopulations in health and disease. Here we describe a tissue digestion protocol and flow cytometry gating strategy for identification and isolation of tissue eosinophils from the small intestine of mice. This protocol is also amenable to the isolation and characterization of colonic eosinophils, and of intestinal eosinophils from human resected tissues.

Heterogeneity of Intestinal Tissue Eosinophils: Potential Considerations for Next-Generation Eosinophil-Targeting Strategies.

Eosinophils are implicated in the pathophysiology of a spectrum of eosinophil-associated diseases, including gastrointestinal eosinophilic diseases (EGIDs). Biologics that target the IL-5 pathway and are intended to ablate eosinophils have proved beneficial in severe eosinophilic asthma and may offer promise in treating some endotypes of EGIDs. However, destructive effector functions of eosinophils are only one side of the coin; eosinophils also play important roles in immune and tissue homeostasis. A growing body of data suggest tissue eosinophils represent a plastic and heterogeneous population of functional sub-phenotypes, shaped by environmental (systemic and local) pressures, which may differentially impact disease outcomes. This may be particularly relevant to the GI tract, wherein the highest density of eosinophils reside in the steady state, resident immune cells are exposed to an especially broad range of external and internal environmental pressures, and greater eosinophil longevity may uniquely enrich for co-expression of eosinophil sub-phenotypes. Here we review the growing evidence for functional sub-phenotypes of intestinal tissue eosinophils, with emphasis on the multifactorial pressures that shape and diversify eosinophil identity and potential targets to inform next-generation eosinophil-targeting strategies designed to restrain inflammatory eosinophil functions while sustaining homeostatic roles.