Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus - 06/07/24

Doi : 10.1016/j.jaci.2024.05.029

Netali Ben-Baruch Morgenstern, PhD ^a,^{^{∗
These authors contributed equally.}}, Mark Rochman, PhD ^a,^{^{∗
These authors contributed equally.}}, Michael Kotliar, ME ^a,^{^{∗
These authors contributed equally.}}, Julia L.M. Dunn, PhD ^a, Lydia Mack, MS ^a, John Besse, MS ^a, Mia A. Natale, BSc ^a, Andrea M. Klingler, MS ^a, Jennifer M. Felton, PhD ^a, Julie M. Caldwell, PhD ^a, Artem Barski, PhD ^a, Marc E. Rothenberg, MD, PhD ^a,^b,^⁎^{^{∗
These authors contributed equally.}}
^a Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio
^b Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio

^∗Corresponding author: Marc E. Rothenberg, MD, PhD, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 7028, Cincinnati, OH 45229-3039.Division of Allergy and ImmunologyCincinnati Children’s Hospital Medical Center3333 Burnet AvenueMLC 7028CincinnatiOH45229-3039

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Graphical abstract

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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.

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

Abbreviations used : CCHMC, EoE, ERK, GEO, NF-κB, PDBU, RNA-seq, scRNA-seq, UMAP