Toll-like Receptor 2 Signalling and the Lysosomal Machinery in Barrett’s Esophagus

Abstract

Background & Aims: Inflammation plays an important role in the development of esophageal adenocarcinoma and its metaplastic precursor lesion, Barrett’s esophagus. Toll-like receptor (TLR) 2 signalling and lysosomal function have been linked to inflammation-associated carcinogenesis. We examined the expression of TLR2 in the esophagus and the effect of long-term TLR2 activation on morphological changes and expression of factors involved in lysosomal function in a Barrett’s esophagus epithelium cell line.

Methods: TLR2 expression in normal squamous esophagus, reflux esophagitis, Barrett’s esophagus and esophageal adenocarcinoma biopsies was assessed with Q-RT-PCR, in situ hybridization and immunohistochemistry. Barrett’s esophagus epithelium cells (BAR-T) were incubated with acid and bile salts in the presence or absence of the TLR2 agonist Pam3CSK4 for a period up to 4 weeks. Morphological changes were assessed with electron microscopy, while Q-RT-PCR was used to determine the expression of lysosomal enzymes (Cathepsin B and C) and factors involved in endocytosis (LAMP-1 and M6PR) and autophagy (LC3 and Rab7).

Results: TLR2 was expressed in normal squamous esophagus, reflux esophagitis, Barrett’s esophagus but was most prominent in esophageal adenocarcinoma. Long-term TLR2 activation in acid and bile salts exposed BAR-T cells resulted in more and larger lysosomes, more mitochondria and increased expression of LAMP-1, M6PR, Cathepsin B and C when compared to BAR-T cells incubated with acid and bile salts but no TLR2 agonist. Factors associated with autophagy (LC3 and Rab7) expression remained largely unchanged.

Conclusion: Activation of TLR2 in acid and bile salts exposed Barrett epithelium cells resulted in an increased number of mitochondria and lysosomes and increased expression of lysosomal enzymes and factors involved in endocytosis.

Abbreviations: BE: Barrett’s esophagus; DCA: deoxycholic acid; EAC: esophageal adenocarcinoma; GCA: glycocholic acid; IL8: interleukin 8; IHC: immunohistochemistry; ISH: in situ hybridization; LAMP-1: lysosomal-associated membrane protein-1; LC3: microtubule-associated protein 1A/1B-light chain 3; M6PR: mannose-6-phosphate receptor; NF-κB: Nuclear Factor–κB; PPIs: proton pump inhibitors; Q-RT-PCR: Quantitative reverse transcriptase polymerase chain reaction; RE: reflux esophagitis; SQ: normal squamous esophagus; TCDCA: taurochenodeoxycholic acid; TLR: Toll-like receptor; TNFα: tumor necrosis factor α.