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Background: Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA-sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality., Objectives: We sought to comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (ie, eosinophilic esophagitis [EoE])., Methods: We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings., Results: In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n = 6 individuals] and 398 from esophagus [n = 6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably IFN-γ, IL-10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and the hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DNAX-activation protein-12 (also known as TYROBP) interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling., Conclusions: In EoE, esophageal eosinophils exist as 2 populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types., Competing Interests: Disclosure statement Funding for this study was derived from National Institutes of Health grant R01 AI045898 and the Campaign Urging Research for Eosinophilic Diseases (CURED) awarded to M.E.R. This project was supported in part by National Institutes of Health grant P30 DK078392 (Gene Expression Core, Research Flow Cytometry Core) of the Digestive Diseases Research Core Center in Cincinnati. Disclosure of potential conflict of interest: M.E. Rothenberg is a consultant for Pulm One, Spoon Guru, ClostraBio, Serpin Pharm, Allakos, Celldex, Nexstone One, Santa Ana Bio, Enzen Pharmaceuticals, Bristol Myers Squibb, Astra Zeneca, Pfizer, GlaxoSmith Kline, Regeneron/Sanofi, Revolo Biotherapeutics, and Guidepoint and has an equity interest in the first 9 listed and royalties from reslizumab (Teva Pharmaceuticals), PEESSv2 (Mapi Research Trust), and UpToDate. M.E. Rothenberg is an inventor of patents owned by Cincinnati Children’s Hospital. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Eosinophils accumulate adjacent to epithelial cells in the mucosa of patients with eosinophilic esophagitis (EoE), yet the bidirectional communication between these cells is not well understood. Herein, we investigated the crosstalk between human eosinophils and esophageal epithelial cells. We report that blood-derived eosinophils have prolonged survival when cocultured with epithelial cells; 96 ± 1% and 30 ± 6% viability was observed after 7 and 14 days of coculture, respectively, compared with 1 ± 0% and 0 ± 0% of monoculture. In the presence of IL-13 and epithelial cells, eosinophils had greater survival (68 ± 1%) at 14 days compared with cocultures lacking IL-13. Prolonged eosinophil viability did not require cellular contact and was observed when eosinophils were cultured in conditioned media from esophageal epithelial cells; neutralizing GM-CSF attenuated eosinophil survival. The majority of eosinophil transcripts (58%) were dysregulated in cocultured eosinophils compared with freshly isolated cells. Analysis of epithelial cell transcripts indicated that exposure to eosinophils induced differential expression of a subset of genes that were part of the EoE esophageal transcriptome. Collectively, these results uncover a network of crosstalk between eosinophils and esophageal epithelial cells involving epithelial mediated eosinophil survival and reciprocal changes in cellular transcripts, events likely to occur in EoE., (© 2021. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

Eosinophils are traditionally studied in the context of type 2 immune responses. However, recent studies highlight key innate immune functions for eosinophils especially in colonic inflammation. Surprisingly, molecular pathways regulating innate immune activities of eosinophil are largely unknown. We have recently shown that the CD300f is highly expressed by colonic eosinophils. Nonetheless, the role of CD300f in governing innate immune eosinophil activities is ill-defined. RNA sequencing of 162 pediatric Crohn's disease patients revealed upregulation of multiple Cd300 family members, which correlated with the presence of severe ulcerations and inflammation. Increased expression of CD300 family receptors was also observed in active ulcerative colitis (UC) and in mice following induction of experimental colitis. Specifically, the expression of CD300f was dynamically regulated in monocytes and eosinophils. Dextran sodium sulfate (DSS)-treated Cd300f -/- mice exhibit attenuated disease activity and histopathology in comparison with DSS-treated wild type (WT). Decreased disease activity in Cd300f -/- mice was accompanied with reduced inflammatory cell infiltration and nearly abolished production of pro-inflammatory cytokines. Monocyte depletion and chimeric bone marrow transfer experiments revealed a cell-specific requirement for CD300f in innate immune activation of eosinophils. Collectively, we uncover a new pathway regulating innate immune activities of eosinophils, a finding with significant implications in eosinophil-associated gastrointestinal diseases.

Eosinophilic esophagitis (EoE) is a Th2 cytokine-associated disease characterized by eosinophil infiltration, epithelial cell hyperplasia, and tissue remodeling. Recent studies highlighted a major contribution for IL-13 in EoE pathogenesis. Paired Ig-like receptor B is a cell surface immune-inhibitory receptor that is expressed by eosinophils and postulated to regulate eosinophil development and migration. We report that Pirb is upregulated in the esophagus after inducible overexpression of IL-13 (CC10-Il13(Tg) mice) and is overexpressed by esophageal eosinophils. CC10-Il13(Tg)/Pirb(-/-) mice displayed increased esophageal eosinophilia and EoE pathology, including epithelial cell thickening, fibrosis, and angiogenesis, compared with CC10-Il13(Tg)/Pirb(+/+) mice. Transcriptome analysis of primary Pirb(+/+) and Pirb(-/-) esophageal eosinophils revealed increased expression of transcripts associated with promoting tissue remodeling in Pirb(-/-) eosinophils, including profibrotic genes, genes promoting epithelial-to-mesenchymal transition, and genes associated with epithelial growth. These data identify paired Ig-like receptor B as a molecular checkpoint in IL-13-induced eosinophil accumulation and activation, which may serve as a novel target for future therapy in EoE., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell-associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of β₂-microglobulin (β₂M) molecules, PIR-B served as a permissive checkpoint for IL-5-induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B-deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb(-/-) mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5-induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.