Amphiregulin-producing T helper 2 cells have critical role in esophageal fibrosis of EoE

Background: Massive eosinophil infiltration into the esophagus causes subepithelial fibrosis and esophageal stricture in patients with eosinophilic esophagitis (EoE). However, the pathogenesis of esophageal fibrosis remains unclear. Objective: The present study aimed to elucidate the cellular and molecular mechanisms underlying the induction of esophageal fibrosis. Methods: We established a murine model of EoE accompanied by fibrotic responses following long-term intranasal administration of house dust mite (HDM) antigen. Using this murine model, we investigated the characteristics of immune cells infiltrating the fibrotic region of the inflamed esophagus using flow cytometry and histological analyses. We also analyzed the local inflammatory sites in the esophagus of patients with EoE using single-cell RNA sequencing, flow cytometry, and immunohistochemistry. Overall design: Murine entire esophagus or human biopsy samples were placed in a C tube containing digestion buffer (Multi Tissue Dissociation Kit , and tissues were dissociated by a gentleMACS™ octo dissociator with heaters. Esophageal mononuclear cells in cell suspension were separated using Percoll solution.Libraries were then prepared using Chromium Single Cell 3' Reagent Kits v3.1 following the manufacturer's protocol. The generated scRNA-seq libraries were sequenced using a total 127 cycle (paired-end reads) with a NovaSeq 6000.Sequence reads from all samples were processed and aggregated using a Cell Ranger. Aggregated data were further analyzed by Seurat. Specifically, we log-normalized the expression matrix, regressed the data against the total number of unique molecular identifier (UMI)'s detected per cell, performed a PCA analysis, and used PCA dimensions 1-50 to find clusters on UMAP. We visualized the single-cell gene expression as UMAP overlays. The single-cell gene expression was also visualized as density plots using the Nebulosa R software package algorithm.