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.

Background-Inflammation is implicated in atherogenesis and plaque disruption. Toll-like receptor 2 (TLR-2) and TLR-4, a human homologue of drosophila Toll, play an important role in the innate and inflammatory signaling responses to microbial agents. To investigate a potential role of these receptors in atherosclerosis, we assessed the expression of TLR-2 and TLR-4 in murine and human atherosclerotic plaques. Methods and Results-Aortic root lesions of high-fat diet-fed apoE-deficient mice (nϭ5) and human coronary atherosclerotic plaques (nϭ9) obtained at autopsy were examined for TLR-4 and TLR-2 expression by immunohistochemistry. Aortic atherosclerotic lesions in all apoE-deficient mice expressed TLR-4, whereas aortic tissue obtained from control C57BL/6J mice showed no TLR-4 expression. All 5 lipid-rich human plaques expressed TRL-4, whereas the 4 fibrous plaques and 4 normal human arteries showed no or minimal expression. Serial sections and double immunostaining showed TLR-4 colocalizing with macrophages both in murine atherosclerotic lesions and at the shoulder region of human coronary artery plaques. In contrast to TLR-4, none of the plaques expressed TLR-2. Furthermore, basal TLR-4 mRNA expression by human monocyte-derived macrophages was upregulated by ox-LDL in vitro. A potential role for infection in the development of atherosclerosis has been considered for several decades, but interest in this topic has recently reemerged because of several recent observations. Accumulating evidence has implicated specific infectious agents, including Chlamydia pneumoniae, in the progression and/or destabilization of atherosclerosis. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] Recent studies suggest that chlamydia lipopolysaccharide (LPS) induces foam-cell formation, whereas its heat-shock protein (chlamydia HSP60) induces oxidative modification of LDL. 5,18 Chlamydia HSP60 has been implicated in the induction of deleterious immune responses in human chlamydial infection and has been found to colocalize with infiltrating macrophages in the atheroma lesions. 19 Collectively, these data support a potential role for C pneumoniae in the development and progression of atherosclerosis and suggest that this organism may indeed play an active role in atheroma development. Available data, however, also underscore the current lack of a complete understanding of the molecular mechanisms that link C pneumoniae infection to innate immunity and trigger the signals for enhanced inflammation and atherogenesis. Conclusions-OurLPS, a major component of the outer surface of Gramnegative bacteria, activates the proinflammatory transcription factor nuclear factor (NF)-B in endothelial cells and macrophages. 20,21 Recently, human Toll-like receptor-4 (TLR-4), a human homologue of drosophila Toll, has been identified as Currently, more than 10 human TLRs have been identified, and at least 10 human homologues of drosophila Toll have been sequenced. Whereas TLR-4 is used by enteric Gramnegative bacteria and LPS, TLR-2 is use...

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Inflammatory bowel disease (IBD) arises from a dysregulated mucosal immune response to luminal bacteria. Toll-like receptor (TLR)4 recognizes LPS and transduces a proinflammatory signal through the adapter molecule myeloid differentiation marker 88 (MyD88). We hypothesized that TLR4 participates in the innate immune response to luminal bacteria and the development of colitis. TLR4-/- and MyD88-/- mice and littermate controls were given 2.5% dextran sodium sulfate (DSS) for 5 or 7 days followed by a 7-day recovery. Colitis was assessed by weight loss, rectal bleeding, and histopathology. Immunostaining was performed for macrophage markers, chemokine expression, and cell proliferation markers. DSS treatment of TLR4-/- mice was associated with striking reduction in acute inflammatory cells compared with wild-type mice despite similar degrees of epithelial injury. TLR4-/- mice experienced earlier and more severe bleeding than control mice. Similar results were seen with MyD88-/- mice, suggesting that this is the dominant downstream pathway. Mesenteric lymph nodes from TLR4-/- and MyD88-/- mice more frequently grew gram-negative bacteria. Altered neutrophil recruitment was due to diminished macrophage inflammatory protein-2 expression by lamina propria macrophages in TLR4-/- and MyD88-/- mice. The similarity in crypt epithelial damage between TLR4-/- or MyD88-/- and wild-type mice was seen despite decreased epithelial proliferation in knockout mice. TLR4 through the adapter molecule MyD88 is important in intestinal response to injury and in limiting bacterial translocation. Despite the diversity of luminal bacteria, other TLRs do not substitute for the role of TLR4 in this acute colitis model. A defective innate immune response may result in diminished bacterial clearance and ultimately dysregulated response to normal flora.

Background-We have recently demonstrated that mice deficient in TLR4 or its adapter molecule MyD88 have increased signs of colitis compared to wild-type (WT) mice following dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that Cox-2 derived PGE 2 is important in TLR4-related mucosal repair.

Toll-like receptor (TLR) 4 has been identified as the primary receptor for enteric LPS, whereas TLR2 has been implicated as the receptor for Gram-positive and fungal cell wall components and for bacterial, mycobacterial, and spirochetal lipoproteins. Vascular endothelial cell (EC) activation or injury by microbial cell wall components such as LPS is of critical importance in the development of sepsis and septic shock. We have previously shown that EC express predominantly TLR4, and have very little TLR2. These cells respond vigorously to LPS via TLR4, but are unresponsive to lipoproteins and other TLR2 ligands. Here we show that LPS, TNF-α, or IFN-γ induce TLR2 expression in both human dermal microvessel EC and HUVEC. Furthermore, LPS and IFN-γ act synergistically to induce TLR2 expression in EC, and LPS-induced TLR2 expression is NF-κB dependent. LPS and IFN-γ also up-regulate TLR4 mRNA expression in EC. These data indicate that TLR2 and TLR4 expression in ECs is regulated by inflammatory molecules such as LPS, TNF-α, or IFN-γ. TLR2 and TLR4 molecules may render EC responsive to TLR2 ligands and may help to explain the synergy between LPS and lipoproteins, and between LPS and IFN-γ, in inducing shock associated with Gram-negative sepsis.

Intestinal epithelial cells (IEC) interact with a high density of Gram-positive bacteria and are active participants in mucosal immune responses. Recognition of Gram-positive organisms by Toll-like receptor (TLR)2 induces proinflammatory gene expression by diverse cells. We hypothesized that IEC are unresponsive to Gram-positive pathogen-associated molecular patterns and sought to characterize the functional responses of IEC to TLR2-specific ligands. Human colonic epithelial cells isolated by laser capture microscopy and IEC lines (Caco-2, T84, HT-29) were analyzed for expression of TLR2, TLR6, TLR1, and Toll inhibitory protein (Tollip) mRNA by RT-PCR and quantitative real-time PCR. Response to Gram-positive bacterial ligands was measured by NF-κB reporter gene activation and IL-8 secretion. TLR2 protein expression was analyzed by immunofluorescence and flow cytometry. Colonic epithelial cells and lamina propria cells from both uninflamed and inflamed tissue demonstrate low expression of TLR2 mRNA compared with THP-1 monocytes. IECs were unresponsive to TLR2 ligands including the staphylococcal-derived Ags phenol soluble modulin, peptidoglycan, and lipotechoic acid and the mycobacterial-derived Ag soluble tuberculosis factor. Transgenic expression of TLR2 and TLR6 restored responsiveness to phenol soluble modulin and peptidoglycan in IEC. In addition to low levels of TLR2 protein expression, IEC also express high levels of the inhibitory molecule Tollip. We conclude that IEC are broadly unresponsive to TLR2 ligands secondary to deficient expression of TLR2 and TLR6. The relative absence of TLR2 protein expression by IEC and high level of Tollip expression may be important in preventing chronic proinflammatory cytokine secretion in response to commensal Gram-positive bacteria in the gut.

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