600 Esophageal Epithelial and Mesenchymal Cross-Talk Leads to Features of Epithelial to Mesenchymal Transition (EMT) In Vitro

Abstract

Background and Aims: Subepithelial fibrosis has recently been recognized as a histologic feature of eosinophilic esophagitis (EoE), a chronic inflammatory disease which leads to esophageal stricture, dysphagia, and recurrent food impaction. It is widely believed that activation by pro-fibrogenic cytokines including TGF-β, TNF-α, and IL-1β, stimulates fibroblasts to acquire the activated phenotype of myofibroblasts, morphologic intermediates between fibroblasts and smooth muscle cells which synthesize extracellular matrix (ECM) components. In the context of EoE, it has been suggested that these pro-fibrotic cytokines are released by circulating innate immune effector cells including mast cells and eosinophils, leading to fibroblast differentiation to the myofibroblast phenotype. To date, the potential role of esophageal epithelial cells as effector cells in EoE-associated tissue remodeling has never been investigated. In the current study, we investigated the consequences of crosstalk between esophageal epithelial cells and esophageal fibroblasts, and explored the ability of esophageal epithelial cells to develop features characteristic of epithelial to mesenchymal transition (EMT) In Vitro. Methods and Results: To determine whether factors secreted by esophageal epithelial cells can influence fibroblast expression of pro-fibrotic genes, we stimulated primary human fetal esophageal fibroblasts (FEF3 cells) with conditioned epithelial media (CEM) harvested from confluent non-transformed immortalized esophageal epithelial cells (EPC2-hTERT). Using qRT-PCR, we quantified FEF3 expression of profibrotic cytokines TGF-β, TNF-α, and IL-1β at various time points after CEM stimulation. Surprisingly, we observed a robust induction in mRNA expression of IL-1β and TNF-α, with a modest induction of TGF-β after stimulation. Based upon this unexpected result, we hypothesized that fibroblast derived factors might induce epithelial to mesenchymal transition (EMT) In Vitro. To test this hypothesis, we cultured FEF3 cells in CEM for 2 days, and transferred this fibroblast-conditioned CEM onto EPC2-hTERT cells for 5 days. Surprisingly, this led to enhanced esophageal epithelial expression of α-smooth muscle actin (SMA) and decreased expression of epithelial-specific e-cadherin, features suggestive of EMT. Similar changes in α-SMA and e-cadherin expression were observed after EPC2-hTERT cells were cultured with recombinant TGF-β, TNF-α, and IL-1β. Conclusions: There is a complex interplay between the esophageal epithelium and its underlying matrix. Our results suggest for the first time that paracrine signals from epithelium prime adjacent fibroblasts to express profibrotic cytokines and that these cytokines may, in turn, play a role in epithelial to mesenchymal transition by human esophageal epithelial cells In Vitro.