Differential expression of key regulators of Toll-like receptors in ulcerative colitis and Crohn's disease: a role for Tollip and peroxisome proliferator-activated receptor gamma?

Abstract

The innate immune system is currently seen as the probable initiator of events which culminate in the development of inflammatory bowel disease (IBD) with Toll-like receptors (TLRs) known to be involved in this disease process. Many regulators of TLRs have been described, and dysregulation of these may also be important in the pathogenesis of IBD. The aim of this study was to perform a co-ordinated analysis of the expression levels of both key intestinal TLRs and their inhibitory proteins in the same IBD cohorts, both ulcerative colitis (UC) and Crohn's disease (CD), in order to evaluate the potential roles of these proteins in the pathogenesis of IBD. Of the six TLRs (TLRs 1, 2, 4, 5, 6 and 9) examined, only TLR-4 was increased significantly in IBD, specifically in active UC. In contrast, differential alterations in expression of TLR inhibitory proteins were observed. A20 and suppressor of cytokine signalling 1 (SOCS1) were increased only in active UC while interleukin-1 receptor-associated kinase 1 (IRAK-m) and B cell lymphoma 3 protein (Bcl-3) were increased in both active UC and CD. In contrast, expression of both peroxisome proliferator-activated receptor gamma (PPARγ) and Toll interacting protein (Tollip) was decreased in both active and inactive UC and CD and at both mRNA and protein levels. In addition, expression of both PPARγ and A20 expression was increased by stimulation of a colonic epithelial cell line Caco-2 with both TLR ligands and commensal bacterial strains. These data suggest that IBD may be associated with distinctive changes in TLR-4 and TLR inhibitory proteins, implying that alterations in these may contribute to the pathogenesis of IBD.

Keywords: PPARγ; Toll-like receptor; Tollip; inflammatory bowel disease; regulation.

Figure 1

Toll‐like receptor (TLR‐4) expression is increased in active ulcerative colitis (UC) compared to healthy controls. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis of TLR genes was assessed in UC, Crohn's disease (CD) and healthy controls (HC). Data were normalized to a β‐actin and expressed using the 2^−ΔΔCT method. Statistical analysis was performed using one‐way analysis of variance (anova) with Bonferroni post‐test. **P < 0·01.

Figure 2

Expression of Toll‐like receptor (TLR‐4) inhibitory proteins is altered differentially in inflammatory bowel disease (IBD) populations. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis of known TLR inhibitor genes was assessed in ulcerative colitis (UC), Crohn's disease (CD) and healthy controls (HC). A20, suppressor of cytokine signalling 1 (SOCS1), interleukin‐1 receptor‐associated kinase 1 (IRAK‐m) and B cell lymphoma 3 protein (Bcl‐3) mRNA was up‐regulated in IBD (a). Tollip and peroxisome proliferator‐activated receptor gamma (PPARγ) mRNA was downregulated in IBD (b) and RP105 and single immunoglobulin receptor‐related (SIGIRR) mRNA displayed no alteration in expression (c). Data were normalized to a β‐actin and expressed using the 2^−ΔΔCT method. Statistical analysis was performed using one‐way analysis of variance (anova) with Bonferroni post‐test *P < 0·05, **P < 0·01 and ***P < 0·001.

Figure 3

Protein expression of Toll interacting protein (Tollip), peroxisome proliferator‐activated receptor gamma (PPARγ), single immunoglobulin receptor‐related (SIGIRR) and A20. Tollip, PPARγ and SIGIRR protein expression were detected in colonic sections of both inflammatory bowel disease (IBD) and healthy controls (HC). Tollip and SIGIRR expression was assessed by immunofluorescence, magnification ×20 exposure for 3 s (a,c) and PPARγ and A20 expression assessed by immunohistochemistry, magnification ×40 (b,d). Haematoxylin and eosin (H&E) staining was performed (e). Data shown are representative of 10 patients per group.

Figure 4

Peroxisome proliferator‐activated receptor gamma (PPARγ) and A20 expression is increased by Toll‐like receptor (TLR) ligands. Caco‐2 cells were stimulated with lipopolysaccharide (LPS) (100 ng/ml), flagellin (100 ng/ml) and Pam3‐Cys‐Ser‐Lys4 (Pam3Csk4) (1 μg/ml) for 8 h. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis was performed on the stimulated cells and expression of Toll interacting protein (Tollip) (a), single immunoglobulin receptor‐related (SIGIRR) (b), PPARγ (c) and A20 (d) assessed. Results shown are representative of three separate experiments. Values are shown as mean ± standard error of the mean (n = 3). Statistical analysis was performed using one‐way analysis of variance (anova) with Bonferroni post‐test. **P < 0·01.

Figure 5

Single immunoglobulin receptor‐related (SIGIRR) expression is decreased, while peroxisome proliferator‐activated receptor gamma (PPARγ) and A20 expression is increased, by stimulation with commensal bacterial strains. Caco‐2 cells were treated with three different bacterial strains (Lactobacillus salivarius, Escherichia coli K12 and Bacteroides fragilis) at a ratio of bacteria : cells of 10 : 1 for 8 h. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis was performed on the stimulated cells and expression of Toll interacting protein (Tollip) (a), SIGIRR (b), PPARγ (c) and A20 (d) assessed. Results shown are representative of three separate experiments. Values are shown as mean ± standard error of the mean (n = 3). Statistical analysis was performed using one‐way analysis of variance (anova) with Bonferroni post‐test. **P < 0·01 and ***P < 0·001.

Figure 6

The peroxisome proliferator‐activated receptor gamma (PPARγ) agonist, troglitazone, reduces lipopolysaccharide (LPS) and flagellin‐induced interleukin (IL)‐6 production. Caco‐2 cells were pretreated with Troglitazone (10 µM) for 1 h prior to stimulation with lipopolysaccharide (LPS) (100 ng/ml) or flagellin (100 ng/ml). Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis was performed on the stimulated cells and expression of interleukin (IL)‐6 assessed. Results shown are representative of three separate experiments. Values are shown as Mean ± standard error of the mea (n = 3). Statistical analysis was performed using one‐way analysis of variance (anova) with Bonferroni post‐test. ***P < 0·001.