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. 2024 Oct;154(4):974-987.
doi: 10.1016/j.jaci.2024.05.029. Epub 2024 Jun 11.

Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus

Netali Ben-Baruch Morgenstern  1 , Mark Rochman  1 , Michael Kotliar  1 , Julia L M Dunn  1 , Lydia Mack  1 , John Besse  1 , Mia A Natale  1 , Andrea M Klingler  1 , Jennifer M Felton  1 , Julie M Caldwell  1 , Artem Barski  1 , Marc E Rothenberg  2
Affiliations

Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus

Netali Ben-Baruch Morgenstern et al. J Allergy Clin Immunol. 2024 Oct.

Abstract

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.

Keywords: CCR3; CEBPE; CLC; DNAAF; Eosinophils; HK3; HRH4; IDO1; OLIG1 and OLIG2; PRSS33; SUCNR1; VSTMs; esophagitis; mast cells; single-cell RNA-sequencing; transcriptional signature.

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Conflict of interest statement

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.

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