Decreased Expression of Toll-Like Receptor-4 and MD-2 Correlates with Intestinal Epithelial Cell Protection Against Dysregulated Proinflammatory Gene Expression in Response to Bacterial Lipopolysaccharide

Abstract

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-κB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1β in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-κB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.

Airway epithelial cells modify immune responses by inducing an anti‐inflammatory microenvironment

“…It has been shown convincingly that epithelial cells at surfaces that regularly or permanently encounter microbes express various TLR and are able to actively sense danger due to infectious agents [4][5][6][7][8]. This has two important implications: First, epithelial cells at non-sterile surfaces have to regulate their TLR sensitivity to avoid continuous threat of inflammation and, second, organ-specific mechanisms of regulating immunity have to exist.…”

“…2A). Furthermore, the observed inhibitory effect was not specific for TLR4 signaling because stimulation of DC by other TLR ligands, including lipoteichoic acid, Pam 3 CSK 4 and poly(dI:dC) was also inhibited ( Fig. 2A).…”

“…Nowadays it is known that prototypical pattern recognition receptors including Toll-like receptors (TLR) are not only expressed on professional innate immune cells but also cover epithelial cells within various organs including the gut [4], the airways [2,5,6], the oral cavity [7] and the skin [8]. In addition, it is now clear that TLR not only sense microbial components derived from pathogenic microbes but also recognize microbial compounds in general [9].…”

“…These hypotheses have now been tested to some extent for the gut where microbial encounter is permanent and strong [4,[10][11][12][13]. However, only limited information is available for the situation in the upper airways.…”

Immunity in the female sheep reproductive tract

“…This has implications for mucosal sites that harbour commensal Gram-negative microorganisms. Tolerance of commensal microflora by the gut mucosal epithelium can be achieved by a down-regulation of TLR4 and MD-2 expression [2]. It has also been proposed that the failure of epithelial cells to express CD14 is to prevent the host mounting unnecessary responses to commensal Gram-negative bacteria [22].…”