{"doi":"10.1111/all.70010","title":"Unsupervised Clustering Reveals Intestine‐Adapted Eosinophil Subsets Shaped by Local Inflammation","abstract":"Steady-state resident intestinal eosinophils sustain a healthy epithelial barrier, mediate anti-microbial immunity, and repair and resolve tissue injury. Juxtaposed to these homeostatic functions, activated eosinophils prone to degranulation contribute to tissue pathology in inflamed settings.S1 Extra-physiologic infiltration of eosinophils is a diagnostic criterion of eosinophilic gastrointestinal disorders (EGIDs; eosinophilic esophagitis (EoE), enteritis (EoN), and colitis (EoC)) and a common characteristic of inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis). Although generally considered pathologic in EGIDs and IBD, contributions of eosinophils to the pathophysiology of either condition remain incompletely understood. Despite their post-mitotic state, tissue-immigrated eosinophils exhibit incompletely understood organ-specific functional adaptationsS2,S3 shaped in part by nutrient-, microbiome-, and tissue-derived factors and mediated in part via eosinophil-intrinsic aryl hydrocarbon receptor- and notch 2 receptor-dependent signaling in steady-state [1-3]. Separate prior studies identified CD22 expressing homeostatic [1], CLEC4a4 expressing immunoregulatory [4], and PD-L1+CD80+ expressing anti-microbial [5], murine intestinal eosinophil subsets that likely contribute to their dichotomous homeostatic and pathologic functions; however, a unified reconciliation of these reported subsets is lacking. Therefore, despite recognition of functional heterogeneity, clinical histopathological assessments remain agnostic to eosinophil phenotypes. Here we applied targeted multidimensional flow cytometry and unsupervised clustering to discriminate intestinal eosinophil phenotypes within and across inflammatory states (Figure 1A). Single cell suspensions were recovered from digested whole small intestines of mice under conditions of health, an allergen-induced model of eosinophilic enteritis (EoN), or TNF-α overexpression (TNFΔARE/+) resulting in spontaneous ileitis (Supporting Information). Eosinophils were gated as described (Figure S1A), and t-distributed stochastic neighbor embedding (t-SNE) plots were generated from data concatenated across all experimental conditions. Eosinophils from allergen-challenged mice clustered closely with those from healthy mice, while a notable divergence appeared within the context of ileitis (Figure 1B,Bi). Receptor expression thresholds were defined manually for each surface receptor (Figure S1B) and expression profiles were overlayed. We previously demonstrated intestinal eosinophils upregulate CD11c along a continuum of expression over time in parallel with their villus migration [6]; we therefore utilized CD11c as a primary indicator of intestinal adaptation. A CD11c− population was observed that also resembled blood eosinophils (e.g., β7 integrin+), and we interpreted these cells to be newly recruited (pre-adapted). Across all conditions, expression profile alignments revealed strong overlap between CD11c, programmed death ligand 1 (PD-L1), and CD80 (Figure 1C). This population likely represents the PD-L1+CD80+ intestine-restricted “active” subset previously reported by Gurtner et al. in steady-state, which was further expanded in bacterial infections and IBD [5]. Frequencies of PD-L1+CD80+ cells among CD11c+ eosinophils were highest in CD11c intermediate to high expressing eosinophils (Figure 1D). Approximately 23% of eosinophils from the concatenated pool expressed low levels of CD11c in the absence of PD-L1 or CD80, ostensibly representing an intermediate population. CD11c+PDL1+CD80+ intestine-adapted eosinophils were further partitioned by expression of the C-type lectin CLEC4a4 (Figure 1E), with CLEC4a4+ cells enriched among intestine-adapted eosinophils from healthy mice (90% overlay) but nearly absent within the context of ileitis (12% overlay). 18%–32% of intestinal eosinophils from healthy mice (compared to 3%–9% from TNFΔARE/+) expressed CLEC4a4 in the absence of P","journal":"Allergy","year":2025,"id":532853,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9371,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1414369,"name":"Arman Kian","orcid":"0009-0000-3584-7461","position":1,"is_corresponding":false},{"id":1393842,"name":"Natalie A. Falta","orcid":"0009-0004-7413-4527","position":2,"is_corresponding":false},{"id":1414370,"name":"Barbara J. Fox","orcid":"0000-0003-2795-4245","position":3,"is_corresponding":false},{"id":339482,"name":"Lisa A. Spencer","orcid":"0000-0002-2365-9559","position":4,"is_corresponding":false},{"id":690176,"name":"Leigha D. Larsen","orcid":null,"position":0,"is_corresponding":true}],"reference_count":8,"raw_metadata":null,"created_at":"2026-07-19T02:51:27.893975Z","pmid":"40878357","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}