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THE CD73-ADENOSINE AXIS PROMOTES DIFFERENTIATION OF ESOPHAGEAL EPITHELIUM IN EOSINOPHILIC ESOPHAGITIS
Date
May 20, 2024
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Background: Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus characterized by eosinophilic inflammation as well as impaired epithelial differentiation and barrier integrity. These epithelial defects are due to the inflammatory infiltrate and specifically Type 2 cytokines, including IL-13. We have identified a CD73+ progenitor population within the basal zone of esophageal epithelium that is critical for tissue renewal. CD73+ basal cells are depleted in human subjects and mice with EoE inflammation with the EoE-associated cytokine IL-13 causing a shift from CD73+ to CD73- basal cells in human 3D organoids. CD73 is an enzyme on the cell surface that hydrolyzes extracellular AMP to adenosine. Herein, we sought to determine the functional consequence of CD73 loss in the EoE epithelium and determine the role of adenosine in supporting epithelial homeostasis. Methods and Results: Utilizing CRISPR-Cas9 technique we established CD73 knockout in the well characterized immortalized non-transformed human esophageal epithelial cells (EPC2-hTERT). We assessed growth, differentiation and barrier in organoid and air-liquid interface cultures using organoid formation assays, RT-PCR, and immunofluorescence. We found that in the KO cultures there was decreased involucrin (IVL) expression by PCR (p<0.01) and IF as well as diminished barrier integrity as measured by transepithelial electrical resistance (p<0.01). Addition of the CD73 enzymatic end-product, adenosine, restored IVL expression by PCR (p<0.001) and IF, organoid formation (p<0.001), and barrier integrity (p<0.01). Because there are 4 adenosine receptors, we interrogated single cell RNA sequencing of human esophageal biopsies as well as organoids to assess the expression of the receptors. Adenosine receptor 2b (A2B) mRNA was found to be expressed in the basal epithelium in both biopsies and organoids. We evaluated an in vitro therapeutic model of EoE wherein esophageal organoids are stimulated with IL-13 and then treated with A2B agonist(BAY60-6583) or adenosine. Adenosine and BAY60-6583 treatment restored involucrin expression diminished by IL-13 (Figure 1). In a murine model of EoE (n=3 per group) utilizing dermal sensitization with OVA-albumin, mice were treated with adenosine or NH4OH (vehicle control) in their drinking water for the duration of EoE induction. Mice treated with adenosine were found to have less basal cell hyperplasia as demonstrated by reduced TP63 staining and increased IVL expression (Figure 2). Conclusions: Currently, nothing is known about the role of purinergic signaling in the esophageal epithelium. These results demonstrate that restoration of adenosine signaling through the CD73-A2B axis may present a promising avenue for EoE therapy by restoring epithelial homeostasis.
EPC-2hTERT organoids stimulated with IL-13 and treated with A2B agonist, BAY 60-6583. Staining performed for marker of terminal differentiation, IVL as well as basal marker TP63.
EoE-like inflammation was induced by dermal sensitization and oral challenge with Ova albumin. Mice were treated with Adenosine or vehicle control. Staining performed for Involucrin (IVL) or TP63.
BACKGROUND: Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder in which chronic inflammation leads the development of fibrosis and stricture formation. Unfortunately, the mechanisms underlying fibrosis remain elusive…
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