THORACIC – Original Submission| Volume 34, ISSUE 4, P1324-1335, December 2022

Toll-like Receptor 4 Mediates Reflux-Induced Inflammation in a Murine Reflux Model

Published:September 14, 2021DOI:
      Dysregulation of toll-like receptor (TLR) signaling within the gastrointestinal epithelium has been associated with uncontrolled inflammation and tumorigenesis. We sought to evaluate the role of TLR4 in the development of gastroesophageal reflux-mediated inflammation and mucosal changes of the distal esophagus. Verified human esophageal Barrett's cells with high grade dysplasia (CPB) and esophageal adenocarcinoma cells (OE33) were treated with deoxycholic acid for 24 hours. Cells were pretreated with a TLR4-specific inhibitor peptide 2 hours prior to deoxycholic acid treatment. Inflammatory markers were evaluated using immunoblotting and enzyme-linked immunosorbent assay. A surgical reflux mouse model was generated by performing a side-to-side anastomosis between the second portion of the duodenum and the gastroesophageal junction. Control animals underwent laparotomy with incision and closure of the esophagus superior to the gastroesophageal junction (sham procedure). Esophageal sections were evaluated using hematoxylin and eosin staining and immunohistochemistry. Deoxycholic acid increased expression of inflammatory markers including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin 8. Pretreatment with a TLR4 inhibitor significantly decreased deoxycholic acid-induced inflammatory marker expression. C3H/HeNCrl mice demonstrated a significant increase in mucosal hyperplasia and proliferation following DGEA compared to sham procedure. TLR4 mutant mice (C3H/HeJ) undergoing DGEA demonstrated an attenuated hyperplastic and proliferative response compared to C3H/HeNCrl mice. Inhibition of TLR4 signaling attenuates reflux-induced inflammation in vivo. These findings identify TLR4 inhibition as a potential therapeutic target to halt the progression of pathologic esophageal changes developing in the setting of chronic gastroesophageal reflux disease.

      Graphical abstract



      DCA (Deoxycholic acid), DGEA (Duodeno-gastroesophageal anastomosis), EAC (Esophageal adenocarcinoma), ELISA (Enzyme-linked immunosorbent assay), FBS (Fetal bovine serum), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), H&E (Hematoxylin and eosin), ICAM-1 (Intercellular adhesion molecule-1), IL-8 (Interleukin 8), LPS (Lipopolysaccharide), PBS (Phosphate-buffered saline), TLR (Toll-like receptor), TLR4 (Toll-like receptor 4), VCAM-1 (Vascular cell adhesion molecule-1)
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