Transcript levels of Toll-Like Receptors 5, 8 and 9 correlate with inflammatory activity in Ulcerative Colitis

Abstract

Background: Dysregulation of innate immune response by Toll-Like Receptors (TLRs) is a key feature in Ulcerative Colitis (UC). Most studies have focused on TLR2, TLR3, and TLR4 participation in UC. However, few studies have explored other TLRs. Therefore, the aim of this study was to evaluate the mRNA profiles of TLR1 to 9 in colonic mucosa of UC patients, according to disease activity.

Methods: Colonic biopsies were taken from colon during colonoscopy in 51 patients with Ulcerative Colitis and 36 healthy controls. mRNA levels of TLR1 to 9, Tollip, inflammatory cytokines IL6 and TNF were assessed by RT-qPCR with hydrolysis probes. Characterization of TLR9 protein expression was performed by Immunohistochemistry.

Results: Toll-like receptors TLR8, TLR9, and IL6 mRNA levels were significantly higher in the colonic mucosa from UC patients (both quiescent and active) as compared to healthy individuals (p < 0.04). In the UC patients group the TLR2, TLR4, TLR8 and TLR9 mRNA levels were found to be significantly lower in patients with quiescent disease, as compared to those with active disease (p < 0.05), whereas TLR5 showed a trend (p = 0.06). IL6 and TNF mRNA levels were significantly higher in the presence of active disease and help to discriminate between quiescent and active disease (p < 0.05). Also, IL6 and TNF mRNA positively correlate with TLRs mRNA with the exception for TLR3, with stronger correlations for TLR5, TLR8, and TLR9 (p < 0.0001). TLR9 protein expression was mainly in the lamina propria infiltrate.

Conclusions: This study demonstrates that TLR2, TLR4, TLR8, and TLR9 expression increases in active UC patients, and that the mRNA levels positively correlate with the severity of intestinal inflammation as well as with inflammatory cytokines.

Figure 1

Toll-like receptors TLR5, TLR8, and TLR9 mRNA levels in Ulcerative Colitis correlate with Endoscopic Activity. RT-qPCR was performed to assess mRNA levels in colonic mucosa biopsies from UC patients according to Mayo subscore endoscopic activity, bars show means with standard error of the mean of TLRs and IL6 transcript levels with RPLP0 as housekeeping gene determined by 2^-ΔΔCt, differences among groups were assessed by Mann-Whitney U test, and p values are presented in the figure.

Figure 2

Toll-like receptors TLR5, TLR8, and TLR9 mRNA levels in Ulcerative Colitis correlate with Histological Activity. RT-qPCR was performed to assess mRNA levels in colonic mucosa biopsies from UC patients according to Riley Histological Activity, bars show means with standard error of the mean of TLRs and IL6 transcript levels with RPLP0 as housekeeping gene determined by 2^-ΔΔCt, differences among groups were assessed by Mann-Whitney U test, and p values are presented in the figure.

Figure 3

TLR9 increased detection in positive infiltrating immune cells in active UC. Representative immunoperoxidase analysis of TLR9 expression of inflamed colonic tissue from six patients (panel A-D) uninflamed colonic tissue (panel E-F) and taken from a representative patient with UC. In the negative controls (neg. ctrl), the secondary antibody was omitted. Original magnification was 20 × (panels A, C, D).