Mo1923 Upregulation and the Regulation of Esophageal Epithelial-Derived IL-33 in Non-Erosive Reflux Disease

Abstract

Background and Aims: Interleukin-33 (IL-33) is a tissue-derived cytokine constitutively expressed in epithelial cells of tissues exposed to the environment and plays a role in sensing damage caused by inflammatory diseases. IL-33 acts as both a traditional cytokine and as a chromatin-associated nuclear factor in both innate and adaptive immunity. We recently showed that IL-33 in the esophageal mucosa is up-regulated in reflux esophagitis (J Gastroenterol, 2014). However, IL-33 expression in non-erosive reflux disease (NERD), and the regulation of cytokines by IL-33 has not been examined. We therefore examined the expression of IL-33 in NERD patients and the function of IL-33 in the production of inflammatory cytokines in esophageal epithelial cells using normal esophageal squamous epithelial cells (HEECs). Methods: The expression of IL-33 in the lower esophageal mucosa of NERD patients and controls was examined using real-time RT-PCR and immunofluorescence. Intercellular space diameter (ISD) was measured by blinded light microscopy of esophageal biopsies. Cytokines produced from an esophageal biopsy specimen were determined using the Bio-Plex suspension array system. We also developed and used an air-liquid interface (ALI) in vitro model for culture of stratified squamous epithelium using primary HEECs (Am J Physiol Gastrointest Liver Physiol. 2012). IL-33 production by the cultures was assessed by western blotting and immunofluorescence staining. Cytokines produced by the cell layers after stimulation were measured using ELISA and the Bio-Plex suspension array system. Pharmacological inhibitors were used to examine the regulation of cytokine production by IFNg, and IL-33 siRNA was used to examine its function in HEECs. Results: IL33 expression and mean ISD were significantly increased in the mucosa of the NERD group compared to that of the control group. The upregulated IL-33 expression was mainly located in the nuclei of the basal cell layer in the NERD group. The esophageal biopsy specimen produced IL-8 and IL-6. In vitro, IFNg stimulation from the basal side of the inserts induced nuclear IL-33 expression and IL-33 mRNA in a doseand time-dependent manner in ALIcultured HEECs. Furthermore, IFNg induced IL-8 and IL-6 release from the ALI-cultured HEECs. IFNg-induced IL-33 protein up-regulation in the ALI-cultured HEECs was mediated through JAK and p38MAPK activation, but not through PKA activity. IFNg-induced IL-8 and IL-6 release was JAK, p38MAPK, and STAT1 dependent. IFNg-induced IL-8 and IL-6