Epithelial Toll-Like Receptor 5 Is Constitutively Localized in the Mouse Cecum and Exhibits Distinctive Down-Regulation during Experimental Colitis

Abstract

We recently demonstrated that the pattern recognition receptors (PRRs) toll-like receptor 2 (TLR2), TLR4, and CD14 are expressed in mouse colonic epithelium in a compartmentalized manner. Here we report the localization of TLR5, the receptor for bacterial flagellin, and its distinctive down-regulation during experimental colitis. Guts from normal BALB/c mice and those with dextran sodium sulfate (DSS)-induced colitis were compared. Each gut was divided into seven segments (stomach, small intestine [three parts], and colon [three parts]), and epithelial cells and crypt units were collected by scraping and EDTA treatment, respectively. Northern blotting showed that TLR5 mRNA was preferentially expressed in the epithelium of the proximal colon in normal mice. Laser capture microdissection coupled to reverse transcriptase PCR confirmed this localization. TLR5 protein expression reflected mRNA expression, as evidenced by Western blotting. In mice with acute colitis, inflammation occurred mainly in the distal colon. Interestingly, while TLR2, TLR4, and CD14 were up-regulated in the inflamed colon, TLR5 was down-regulated at both the mRNA and protein levels. Decreased TLR5 expression was more evident during chronic colitis. Additional in vitro studies using a mouse cell line, Colon-26, showed that gamma interferon (IFN-␥) time-and dose-dependently down-regulates TLR5. In conclusion, epithelial cells, mainly in the proximal colon, constitutively express TLR5. TLR5 expression is down-regulated in vivo during acute and chronic DSS-induced colitis, in contrast to the expression of TLR2, TLR4, and CD14. The mechanism governing TLR5 regulation may therefore differ from that controlling other PRRs. Finally, IFN-␥ may be involved in down-regulating TLR5 expression.The concept of a highly efficient innate immune system that recognizes potential pathogens by detecting lipopolysaccharides (LPSs), peptidoglycans, lipopeptides, flagellin, or many other highly preserved and unvarying structural molecules (27) is now widely accepted, and innate immunity acts as the firstline defense against pathogenic microorganisms in mammals. Although the innate immune system is basically activated by pathogen-related molecules, this activation seems to be driven by several kinds of strategically regulated pattern recognition receptors (PRRs). Because of the variety of PRRs and their sophisticated intracellular signal transduction systems, the innate immune system is increasingly regarded as highly complex.Since the discovery of human toll (22), several new receptors and ligands have been recognized, and more are constantly being added to the arena. Toll-like receptors (TLRs) are transmembrane proteins that share a leucine-rich repeat ectodomain, which confers high specificity for particular ligands (4). TLRs also share an intracellular domain which contains a toll-interleukin-1 (IL-1) receptor homology domain common to the IL-1 receptor family and the IL-18 receptor (36). Ligand binding to TLRs triggers activation of the IL-1 receptor-associated k...

The Roles of Bacteria and TLR4 in Rat and Murine Models of Necrotizing Enterocolitis

“…Similar to our findings of increased epithelial TLR4 and unchanged TLR5 expression in experimental NEC, there is increased enterocyte-specific expression of TLR4 and decrease of TLR5 expression in UC and CD (18,19) and in the DSS model of colitis (20). However, in our experimental NEC model, TLR4 deficiency appears to be clearly protective, while in a murine model of colitis, the role of TLR4 is less clear.…”

TLR5 is not required for flagellin-mediated exacerbation of DSS colitis

“…This is supported by the finding that DSS-colitis actually downregulates TLR expression in colonic tissue, 29 and also by a recent report that an intraperitoneal injection of flagellin administered immediately before the start of DSS treatment can actually be protective. 13 The fact that intrarectal flagellin worsens colitis while intraperitoneal flagellin is protective is likely the consequence of different routes of administration; TLR5 distribution in mouse tissues remains somewhat uncertain due to the lack of high-affinity detection antibodies, but there are reports that expression on the colonic mucosa is low and mainly confined to the cecum.…”

Increased expression of midkine in the rat colon during healing of experimental colitis

“…Samples captured by 5,000 shots on each transfer film cap were immersed in RNA extraction solution. To exclude the possibility that contaminating cells within the colonic epithelial preparation may be responsible for the detection of RPTP-␤ expression, we performed RT-PCR to detect the cytokeratin-20 (an epithelial maker) and CD45 (pan-leukocyte maker) mRNA simultaneously in LCM samples, as reported previously (13,18,36).…”