203 A STAT6 Inhibitor (AS1517499) and a Tyrosine Kinase Inhibitor (Leflunomide) Block IL-13/STAT6 Signaling and Eotaxin-3 Expression in Esophageal Fibroblasts and Epithelial Cells: Promising Agents to Improve Inflammation and Prevent Fibrostenosis in EoE

Abstract

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.