Advertisement

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

Published:September 14, 2021DOI:https://doi.org/10.1053/j.semtcvs.2021.07.033
      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

      Keywords

      Abbreviations:

      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)
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Seminars in Thoracic and Cardiovascular Surgery
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Souza R.F.
        From reflux esophagitis to esophageal adenocarcinoma.
        Dig Dis. 2016; 34: 483-490
        • Abdel-Latif M.M.M.
        • Duggan S.
        • Reynolds J.V.
        • et al.
        Inflammation and esophageal carcinogenesis.
        Curr Opin Pharmacol. 2009; 9: 396-404
        • Quante M.
        • Bhagat G.
        • Abrams J.A.
        • et al.
        Bile acid and inflammation activate gastric cardia stem cells in a mouse model of Barrett-like metaplasia.
        Cancer Cell. 2012; 21: 36-51
        • Kauer W.K.
        • Peters J.H.
        • DeMeester T.R.
        • et al.
        Mixed reflux of gastric and duodenal juices is more harmful to the esophagus than gastric juice alone. The need for surgical therapy re-emphasized.
        Ann Surg. 1995; 222 (discussion 531-523): 525-531
        • Bajpai M.
        • Aviv H.
        • Das K.M.
        Prolonged exposure to acid and bile induces chromosome abnormalities that precede malignant transformation of benign Barrett's epithelium.
        Mol Cytogenet. 2012; 5: 43
        • Yen C.J.
        • Izzo J.G.
        • Lee D.F.
        • et al.
        Bile acid exposure up-regulates tuberous sclerosis complex 1/mammalian target of rapamycin pathway in Barrett's-associated esophageal adenocarcinoma.
        Cancer Res. 2008; 68: 2632-2640
        • Hu Q.
        • Sun T.-T.
        • Hong J.
        • et al.
        Proton pump inhibitors do not reduce the risk of esophageal adenocarcinoma in patients with Barrett's esophagus: A systematic review and meta-analysis.
        PloS one. 2017; 12e0169691
        • Stamp D.H.
        Bile acids aided by acid suppression therapy may be associated with the development of esophageal cancers in westernized societies.
        Med Hypotheses. 2006; 66: 154-157
        • Matei D.
        • Dadu R.
        • Prundus R.
        • et al.
        Alkaline reflux esophagitis in patients with total gastrectomy and Roux en Y esojejunostomy.
        J Gastrointestin Liver Dis. 2010; 19: 247-252
        • Fukata M.
        • Abreu M.T.
        Pathogen recognition receptors, cancer and inflammation in the gut.
        Curr Opin Pharmacol. 2009; 9: 680-687
        • Kamdar K.
        • Nguyen V.
        • DePaolo R.W.
        Toll-like receptor signaling and regulation of intestinal immunity.
        Virulence. 2013; 4: 207-212
        • Kohtz P.D.
        • Halpern A.L.
        • Eldeiry M.A.
        • et al.
        Toll-like receptor-4 is a mediator of proliferation in esophageal adenocarcinoma.
        Ann Thorac Surg. 2019; 107: 233-241
        • Yu J.A.
        • Li H.
        • Meng X.
        • et al.
        Group IIa secretory phospholipase expression correlates with group IIa secretory phospholipase inhibition–mediated cell death in K-ras mutant lung cancer cells.
        J Thorac Cardiovasc Surg. 2012; 144: 1479-1485
        • Bennett D.T.
        • Reece T.B.
        • Foley L.S.
        • et al.
        C-terminal tensin-like protein mediates invasion of human lung cancer cells and is regulated by signal transducer and activator of transcription 3.
        J Thorac Cardiovasc Surg. 2015; 149: 369-375
        • Babu A.
        • Meng X.
        • Banerjee A.M.
        • et al.
        Secretory phospholipase A2 is required to produce histologic changes associated with gastroduodenal reflux in a murine model.
        J Thorac Cardiovasc Surg. 2008; 135: 1220-1227
        • Babu A.
        • Mauchley D.
        • Meng X.
        • et al.
        The secretory phospholipase A2 gene is required for gastroesophageal reflux-related changes in murine esophagus.
        J Gastrointest Surg. 2009; 13: 2212-2218
        • Mauchley D.
        • Meng X.
        • Babu A.
        • et al.
        Gastroduodenal reflux induces group IIa secretory phospholipase A(2) expression and activity in murine esophagus.
        Dis Esophagus. 2010; 23: 430-436
        • Alasehirli B.
        • Oğuz E.
        • Oksuzler E.
        • et al.
        Investigation of intercellular adhesion molecules (ICAMs) gene expressions in patients with Barrett's esophagus.
        Tumor Biol. 2014; 35: 4907-4912
        • Hosch S.B.
        • Meyer A.J.
        • Schneider C.
        • et al.
        Expression and prognostic significance of HLA class I, ICAM-1, and tumor-infiltrating lymphocytes in esophageal cancer.
        J Gastrointest Surg. 1997; 1: 316-323
        • Rockett J.C.
        • Darnton S.J.
        • Crocker J.
        • et al.
        Expression of HLA-ABC, HLA-DR and intercellular adhesion molecule-1 in oesophageal carcinoma.
        J Clin Pathol. 1995; 48: 539-544
        • Heidemann J.
        • Maaser C.
        • Lugering A.
        • et al.
        Expression of vascular cell adhesion molecule-1 (CD 106) in normal and neoplastic human esophageal squamous epithelium.
        Int J Oncol. 2006; 28: 77-85
        • Gergen A.K.
        • Jarrett M.J.
        • Li A.
        • et al.
        Expression of adhesion molecules in a gastroduodenal reflux murine model.
        Ann Thorac Surg. 2021; (Online ahead of print)
        • Yoshida N.
        • Uchiyama K.
        • Kuroda M.
        • et al.
        Interleukin-8 expression in the esophageal mucosa of patients with gastroesophageal reflux disease.
        Scand J Gastroenterol. 2004; 39: 816-822
        • Fitzgerald R.C.
        • Abdalla S.
        • Onwuegbusi B.A.
        • et al.
        Inflammatory gradient in Barrett's oesophagus: Implications for disease complications.
        Gut. 2002; 51: 316-322
        • Verbeek R.E.
        • Siersema P.D.
        • Ten Kate F.J.
        • et al.
        Toll-like receptor 4 activation in Barrett's esophagus results in a strong increase in COX-2 expression.
        J Gastroenterol. 2014; 49: 1121-1134
        • Diakowska D.
        • Nienartowicz M.
        • Grabowski K.
        • et al.
        Toll-like receptors TLR-2, TLR-4, TLR-7, and TLR-9 in tumor tissue and serum of the patients with esophageal squamous cell carcinoma and gastro-esophageal junction cancer.
        Adv Clin Exp Med. 2019; 28: 515-522
        • Arias Á.
        • Vicario M.
        • Bernardo D.
        • et al.
        Toll-like receptors-mediated pathways activate inflammatory responses in the esophageal mucosa of adult eosinophilic esophagitis.
        Clin Transl Gastroenterol. 2018; 9: 147
        • Zu Y.
        • Ping W.
        • Deng T.
        • et al.
        Lipopolysaccharide-induced toll-like receptor 4 signaling in esophageal squamous cell carcinoma promotes tumor proliferation and regulates inflammatory cytokines expression.
        Dis Esophagus. 2017; 30: 1-8
        • Rousseau M.C.
        • Hsu R.Y.
        • Spicer J.D.
        • et al.
        Lipopolysaccharide-induced toll-like receptor 4 signaling enhances the migratory ability of human esophageal cancer cells in a selectin-dependent manner.
        Surgery. 2013; 154: 69-77
        • Wu K.
        • Yang Y.
        • Liu D.
        • et al.
        Activation of PPARγ suppresses proliferation and induces apoptosis of esophageal cancer cells by inhibiting TLR4-dependent MAPK pathway.
        Oncotarget. 2016; 7: 44572-44582
        • Yu H.X.
        • Wang X.L.
        • Zhang L.N.
        • et al.
        Involvement of the TLR4/NF-κB signaling pathway in the repair of esophageal mucosa injury in rats with gastroesophageal reflux disease.
        Cell Physiol Biochem. 2018; 51: 1645-1657
        • Lv J.
        • Guo L.
        • Liu J.-J.
        • et al.
        Alteration of the esophageal microbiota in Barrett's esophagus and esophageal adenocarcinoma.
        World J Gastroenterol. 2019; 25: 2149-2161
        • Anwar M.A.
        • Shah M.
        • Kim J.
        • et al.
        Recent clinical trends in Toll-like receptor targeting therapeutics.
        Med Res Rev. 2019; 39: 1053-1090

      Linked Article

      • Commentary: Does Toll Play the Sole Role?
        Seminars in Thoracic and Cardiovascular SurgeryVol. 34Issue 4
        • Preview
          Gastro esophageal reflux has been associated with esophageal cancer as well as benign complications such as peptic strictures. With new concerns being raised about the safety of long term use high dose PPI as well as limited longevity of anti-reflux procedures, investigation into alternative pathways to mitigate the deleterious effect of gastro-esophageal reflux is most welcome.
        • Full-Text
        • PDF
      • Commentary: Choosing the Right Model for Bile Reflux Induced Esophageal Disease Research
        Seminars in Thoracic and Cardiovascular SurgeryVol. 34Issue 4
        • Preview
          Esophageal cancer has a high mortality and poor prognosis. The incidence of esophageal adenocarcinoma (EAC), the most common form of this cancer in the United States, has dramatically risen in recent years.1 Chronic exposure of the esophagus to gastroduodenal intestinal fluid is an important determinant factor in the development of Barrett's esophagus (BE), which is closely associated with the development of EAC.2 Although the close relationship between bile reflux and EAC has been well established, little is known about the mechanisms that link bile acids to esophageal carcinogenesis due to a lack of reliable models.
        • Full-Text
        • PDF