Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria

Abstract

The colonic epithelium provides an interface between the host and micro-organisms colonising the gastrointestinal tract. Molecular recognition of bacteria is facilitated through Toll-like receptors (TLR). The colonic epithelium expresses relatively high levels of mRNA for TLR3 and less for TLR2 and -4. Little is known of the expression patterns and mode of induction of expression for these pattern recognition receptors in human colon. The aim of this study was to investigate their localization in the gut and induction of expression in epithelial cell lines by mucosal bacteria. TLR2 and -4 were expressed only in crypt epithelial cells, expression was lost as the cells matured and moved towards the gut lumen. In contrast, TLR3 was only produced in mature epithelial cells. HT29 and CACO-2 had different levels of expression for TLR1-4. Co-culture of HT29 cells with different mucosal isolates showed that they were highly responsive to bacterial challenge, with up-regulation of mRNA for TLR1-4. In contrast, CACO-2 cells were refractive to bacterial challenge, showing little difference in mRNA levels. TLR3 was induced in HT29 only by Gram-positive commensals with up-regulation of both mRNA and protein and an enhancement of the antiviral immune response. This pattern of expression allows induction of responsiveness to bacteria only by the crypt epithelium so that tolerance to commensal organisms can be maintained. In contrast, mature columnar epithelium is able to respond to viral pathogens, which are not part of the normal gut commensal microbiota.

Figure 1

Immunohistochemical analysis of normal healthy human colon stained with a monoclonal mouse anti-human TLR2 IgG2a antibody, visualized using immunoperoxidase. (a) Transverse section (×40) across the crypt region with staining in the epithelium and lamina propria. (b and c) Respectively, a transverse and longitudinal section of more mature epithelial cells that interface with the gut lumen (×40). (d) A longitudinal section (×20) from crypt to lumen.

Figure 2

Immunohistochemical analysis of normal healthy human colon stained with a monoclonal mouse antihuman TLR3 IgG1 antibody, visualized using immunoperoxidase. (a and b) Longitudinal sections (×40) through the epithelium at the luminal surface. (c) A longitudinal section (×20) from crypt to lumen, and (d) a longitudinal section with a transverse region (×40) showing staining seen from the gut lumen.

Figure 3

Immunohistochemical analysis of normal healthy human colon stained with a monoclonal mouse anti-human TLR4 IgG2a antibody, visualized using immunoperoxidase. Expression patterns of human TLR4. (a)A transverse section (×40) across the crypt region with staining in the epithelium and lamina propria. (b) A longitudinal section through the crypt region (×40). (c) (×40) A longitudinal section of more mature epithelial cells. (d) A longitudinal section (×20) from crypt to lumen.

Figure 4

(a–d) Negative controls, both transverse and longitudinal, of healthy human colon stained with IgG1 and IgG2a isotype controls followed by appropriate secondary antibody plus immunoperoxidase visualization.

Figure 5

Levels of gene expression for TLR1 (a), TLR2 (b), TLR3 (c) and TLR4 (d) by HT29 cells in coculture with six different bacterial isolates from the rectal mucosa for three hrs. Results are expressed as a mean plus standard deviation of three different experiments.

Figure 6

Levels of gene expression for TLR1 (a), TLR2 (b), TLR3 (c) and TLR4 (d) by CACO-2 cells in coculture. See legend to Fig. 5.

Figure 7

Levels of gene expression for TLR1 (a), TLR2 (b), TLR3 (c) and TLR4 (d) by HT29 cells in coculture with different TLR ligands for 3 hr. Results are expressed as a mean plus standard deviation of three different experiments.

Figure 8

Confocal microscopy of an 18-hr coculture of HT29 cells with either Bacteroides fragilis (a, b) or Bifidobacterium bifidum (c, d). The cells were stained with goat antihuman TLR3 antibody, followed by a secondary FITC-conjugated rabbit anti-goat immunoglobulin antibody giving green staining where TLR3 is expressed. Cell nuclei have been counterstained in red with propidium iodide.

Figure 9

Levels of mRNA for interferon beta in HT29 cells in coculture with B. bifidum for 18 hr followed by a 3-hr stimulation with poly I:C. The results are expressed as a mean plus standard deviation of three different experiments.