Toll-like receptors 1, 2, 4 and 6 in esophageal epithelium, Barrett's esophagus, dysplasia and adenocarcinoma

Abstract

Background: Toll-like receptors (TLRs) recognize microbial and endogenous ligands and have already shown to play a role in esophageal cancer. In this study, we evaluated especially TLRs that sense bacterial cell wall components in Barrett's esophagus, dysplasia and esophageal adenocarcinoma.

Methods: TLRs 1, 2, 4 and 6 were stained immunohistochemically and assessed in esophageal specimens from patients with esophageal dysplasia (n = 30) or adenocarcinoma (n = 99). Structures and lesions were evaluated including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37), and esophageal adenocarcinoma (n = 99).

Results: We found TLR1, TLR2, TLR4 and TLR6 expression in all lesions. TLR expression increased in Barrett's mucosa and dysplasia. There was profound increase of TLR expression from gastric- to intestinal-type columnar epithelium. In cancers, high nuclear and cytoplasmic staining of TLR4 associated with metastatic disease and poor prognosis.

Conclusions: TLR1, TLR2, TLR4 and TLR6 are upregulated during malignant changes of esophageal columnar epithelium. Increased TLR4 expression associates with advanced stage and poor prognosis in esophageal adenocarcinoma.

Keywords: Toll-like receptor 1; Toll-like receptor 2; Toll-like receptor 4; Toll-like receptor 6; esophageal adenocarcinoma.

Figure 1. Examples of nuclear TLR4 expression

Immunohistochemical staining showing negative (A) and positive (B) nuclear TLR4 staining. Immunofluorescence confirming variable nuclear expression with examples of both TLR4 (red label) negative (cell 1) and positive (cell 2) nuclei in the same carcinoma sample (C). Nuclei are marked with DNA specific DAPI staining (blue). Corresponding intensity profiles of TLR (D, E) of carcinoma cells with TLR4 positive cytoplasm but negative nucleus (see figure C, Cell 1) and both positive cytoplasm and nucleus (see figure C, Cell 2). Solid line shows the intensity of DAPI and dotted line the intensity of TLR4. Magnification 40x (IHC) and 60x (IF) were used.

Figure 2. Examples of typical expression patterns of TLR1 (A, E), TLR2 (B, F), TLR4 (C, G) and TLR6 (D, H)

(A–D) represent the same sample with normal epithelium (NE), low-grade dysplasia (LGD), high grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) marked in the figure A. Gradual increase is found through normal epithelium – metaplasia – dysplasia sequence. E-H show intestinal type metaplasia (left) and gastric type metaplasia (right). Gastric metaplasia presented a strong polarized staining to the basal cytoplasm in TLR 1, 4 and 6 stainings. TLR1 and TLR2 show basal polarization in intestinal metaplasia, whereas TLR4 and TLR6 are expressed more diffusely. Expression pattern of all studied TLRs in adenocarcinoma is diffuse extending homogenously throughout the cell cytoplasm with no apparent basal polarization. Magnifications 6× and 20× were used.

Figure 3. Kaplan-Meier curve showing esophageal adenocarcinoma survival stratified by nuclear TLR1 (A) or TLR4 (B) expression and TLR4 histoscore (C)