RESUMO
Objective: Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disorder characterized by eosinophil-rich mucosal inflammation and tissue remodeling. Transcriptional profiling of esophageal biopsies has previously revealed upregulation of type I and II interferon (IFN) response genes. We aim to unravel interactions between immune and epithelial cells and examine functional significance in esophageal epithelial cells. Design: We investigated epithelial gene expression from EoE patients using single-cell RNA sequencing and a confirmatory bulk RNA-sequencing experiment of isolated epithelial cells. The functional impact of interferon signaling on epithelial cells was investigated using in vitro organoid models. Results: We observe upregulation of interferon response signature genes (ISGs) in the esophageal epithelium during active EoE compared to other cell types, single-cell data, and pathway analyses, identified upregulation in ISGs in epithelial cells isolated from EoE patients. Using an esophageal organoid and air-liquid interface models, we demonstrate that IFN-γ stimulation triggered disruption of esophageal epithelial differentiation, barrier integrity, and induced apoptosis via caspase upregulation. We show that an increase in cleaved caspase-3 is seen in EoE tissue and identify interferon gamma (IFNG) expression predominantly in a cluster of majority-CD8+ T cells with high expression of CD69 and FOS. Conclusion: These findings offer insight into the interplay between immune and epithelial cells in EoE. Our data illustrate the relevance of several IFN-γ-mediated mechanisms on epithelial function in the esophagus, which have the potential to impact epithelial function during inflammatory conditions.
RESUMO
BACKGROUND: Germline-encoded innate immune pattern recognition receptors (PRR) are expressed at epithelial surfaces and modulate epithelial defenses. Evidence suggests that stimulation of the Toll-like receptor (TLR) family of PRR may regulate epithelial barrier integrity by upregulating tight junction (TJ) complex protein expression, but it is not known whether this mechanism is utilized in esophageal epithelial cells. TJ complex proteins maintain intact barrier function and are dysregulated in atopic disorders including eosinophilic esophagitis. METHODS: Pattern recognition receptors expression was assessed in EoE and control primary esophageal epithelial cells, demonstrating robust expression of TLR2 and TLR3. The three-dimensional air-liquid interface culture (ALI) model was used to test whether TLR2 or TLR3 stimulation alters epithelial barrier function using an in vitro model of human epithelium. Transepithelial electrical resistance (TEER) and FITC-Dextran permeability were evaluated to assess membrane permeability. ALI cultures were evaluated by histology, immunohistochemistry, Western blotting, and chromatin immunoprecipitation (ChIP). RESULTS: TLR3 stimulation did not change TEER in the ALI model. TLR2 stimulation increased TEER (1.28- to 1.31-fold) and decreased paracellular permeability to FITC-Dextran, and this effect was abolished by treatment with anti-TLR2 blocking antibody. TJ complex proteins claudin-1 and zonula occludens-1 were upregulated following TLR2 stimulation, and ChIP assay demonstrated altered histone 4 acetyl binding at the TJP1 enhancer and CLDN1 enhancer and promoter following zymosan treatment, implying the occurrence of durable chromatin changes. CONCLUSIONS: Our findings implicate the TLR2 pathway as a potential regulator of esophageal epithelial barrier function and suggest that downstream chromatin modifications are associated with this effect.