TLR4 and MD-2 Expression Is Regulated by Immune-mediated Signals in Human Intestinal Epithelial Cells

Abstract

The intestinal epithelium is continually exposed to a high intraluminal concentration of diverse bacteria and bacterial products (1, 2). Despite the density of commensal bacteria and their products, the intestinal mucosa maintains a controlled state of inflammation. By contrast, invasive or toxin-producing pathogenic bacteria elicit acute inflammation and secretion of pro-inflammatory cytokines by intestinal epithelial cells and lamina propria mononuclear cells (3, 4). Idiopathic inflammatory bowel disease in humans and animals is characterized by acute and chronic inflammation in the absence of a specific pathogen. Compelling evidence in genetically susceptible animal models of inflammatory bowel disease demonstrates that Th1 cytokines and commensal bacteria are required for the induction of chronic inflammation (5-9). The recent discovery of a genetic association in inflammatory bowel disease patients with a mutation in a gene involved in LPS 1 signaling, NOD2, supports the idea that innate immunity may be defective in patients with idiopathic inflammatory bowel disease (10, 11).We wished to understand the mechanism by which the normal intestinal epithelium guards against chronic activation in the presence of commensal flora. Commensal gut bacteria include both Gram-positive and Gram-negative organisms (2). The cell wall of Gram-negative bacteria contains LPS, a potent pro-inflammatory pathogen-associated molecular pattern responsible for the systemic manifestations of septic shock (12). The response to LPS is mediated by its interaction with tolllike receptor 4 (TLR4) in conjunction with secreted MD-2 and soluble or membrane-bound CD14 and transduced via the IL-1 receptor signaling complex to activate NF-B and pro-inflammatory cytokine secretion (13-16). We and others have previously described that intestinal epithelial cells are unresponsive to purified, protein-free LPS as measured by 18). To determine the reason for LPS unresponsiveness, we assayed for the presence of TLR4 and its co-receptor MD-2 and found that intestinal epithelial cells express low levels of TLR4 and MD-2 (17). Expression of both TLR4 and MD-2 restores the ability of intestinal epithelial cells to respond to LPS, suggesting that the intracellular signaling pathway leading to NF-B is intact in these cells. These in vitro model systems are consistent with findings in normal adult human colonic biopsies, small intestinal resections, and fetal intestinal epithelial cells, which have demonstrated low TLR4 expression by immunohistochemistry and 19). These studies did not examine the expression of the MD-2 co-receptor, which is required for LPS responsiveness, nor did they measure TLR4 function.Little is known about the regulation of TLR4 or MD-2 expression. Whereas normal intestinal epithelial cells express

TLR Signaling in the Gut in Health and Disease

“…Tollip is a Toll/ IL-1R (TIR) domain-containing inhibitory protein that is bound to IRAK (21). We have shown that Tollip expression is high in intestinal epithelial cells that are poorly responsive to LPS (22). Furthermore, Tollip expression increases in LPS-or LTA-treated intestinal epithelial cells and is associated with hyporesponsiveness to PAMPs (9).…”

Synergistic Proinflammatory Effects of the Antiviral Cytokine Interferon-α and Toll-Like Receptor 4 Ligands in the Atherosclerotic Plaque

“…27 The effect of IFN-␣ on TLR4 is unique; other cytokines like TNF-␣ or IFN-␥ lead to little or no TLR4 increase. 28 Although binding of lipopolysaccharide to TLR4 usually results in receptor downregulation, causing endotoxin tolerance, 25 IFN-␣ was capable of counteracting this inhibitory effect. Thus, simultaneous signaling of TLR9, triggering IFN-␣ production, and TLR4, inducing cytokine production, is required for a sustained immune response to TLR4 ligands.…”

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

“…TLRs are expressed by most immune cells including PMN as well as epithelial cells. While recent studies have uncovered a number of different TLRs, a majority of the research has focused on TLR4 which was the first of these receptors to be discovered, and to a lesser extent TLR2 , Abreu et al 2002, Hausmann et al 2002, Lorenz et al 2002, Backhed & Hornef 2003, Fan & Malik 2003. Discovery of a naturally occurring mutation in mice for the TLR4 gene (Takeda et al 2003) was central to our understanding of the importance of TLRs.…”

“…In humans, a number of alleles of TLR4 have been identified and are associated with an increased risk of septic shock (Fenton & Golenbock 1998, Abreu et al 2002, Hausmann et al 2002, Lorenz et al 2002, Backhed & Hornef 2003, Fan & Malik 2003, Sabroe et al 2003. Although TLR4 has been shown to interact with heat shock proteins and taxol, arguably the most important role for TLR4 is as a cellular receptor for bacterial LPS, a central bacterial cell wall component comprising ~13% of the gram negative envelope (Fenton & Golenbock 1998, Viriyakosol et al 2000, Abreu et al 2002, Hausmann et al 2002, Lorenz et al 2002, Backhed & Hornef 2003, Fan & Malik 2003.…”