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PubMed2005

TLR4 mediates human retinal pigment epithelial endotoxin binding and cytokine expression.

PURPOSE: We previously demonstrated toll-like receptor 4 (TLR4) to be involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classic role of TLR4 in HRPE endotoxin (lipopolysaccharide [LPS]) binding leading to HRPE proinflammatory cytokine secretion. METHODS: Cultured human HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). HRPE cells labeled with fluorescent monoclonal antibodies (mAb) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin-8 (IL-8) was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14. RESULTS: All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < .05) in the presence of blocking mAb to TLR4 or CD14. Up-regulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14. CONCLUSION: HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.

Blotting, Western↗
PubMed2005

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

Animals↗
PubMed2004

Toll-like receptor 4 (TLR4) of retinal pigment epithelial cells participates in transmembrane signaling in response to photoreceptor outer segments.

Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS-RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS-human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS-RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS-human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS-RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS-human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS.

Animals↗
PubMed2005

Hepatic ischemia/reperfusion injury involves functional TLR4 signaling in nonparenchymal cells.

Endogenous ligands from damaged cells, so-called damage-associated molecular pattern molecules, can activate innate immunity via TLR4 signaling. Hepatic warm ischemia and reperfusion (I/R) injury and inflammation is largely TLR4 dependent. We produced TLR4 chimeric mice to assess whether the TLR4-dependent injury required TLR4 expression on liver parenchymal or nonparenchymal cells. Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mouse bone marrow. Wild-type chimeric mice bearing TLR4 mutant hemopoietic cells and TLR4 mutant mice transplanted with their own bone marrow-derived cells were protected from hepatic I/R and exhibited decreased JNK and NF-kappaB activation compared with WT chimeric mice transplanted with their own bone marrow. In contrast, TLR4 mutant mice transplanted with TLR4 WT bone marrow were not protected from liver I/R and demonstrated pronounced increases in JNK and NF-kappaB activation when compared with autochthonous transplanted mutant mice. In addition, depletion of phagocytes taking up gadolinium chloride failed to provide any additional protection to TLR4 mutant mice, but substantially reduced damage in WT mice after hepatic I/R. Together, these results demonstrate that TLR4 engagement on actively phagocytic nonparenchymal cells such as Kupffer cells is required for warm I/R-induced injury and inflammation in the liver.

Animals↗
PubMed2006

TLR2- and TLR4-mediated signals determine attenuation or augmentation of inflammation by acute alcohol in monocytes.

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-alpha protein and mRNA levels and NF-kappaB activation. In contrast, acute alcohol augmented TNF-alpha production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- plus TLR4-stimulated monocytes by alcohol. This was supported by decreased IRAK-1:TRAF6 association in TLR4 induced but sustained presence of IRAK-1:TRAF6 complexes in TLR2- plus TLR4-stimulated monocytes after alcohol treatment. Phosphorylation of MAPKs such as ERK1/2 was selectively inhibited by acute alcohol in TLR4-stimulated cells. In contrast, JNK phosphorylation as well as AP-1 nuclear binding were augmented by acute alcohol in the presence of combined TLR4 and TLR2 stimulation. Consistent with this result, the JNK inhibitor prevented alcohol-induced augmentation of TNF-alpha production. These results suggest that acute alcohol attenuates TLR4-induced inflammation via inhibition of IRAK-1 and ERK1/2 kinases and increases in IRAK-monocyte levels in monocytes. Conversely, in the presence of TLR2 and TLR4 ligands, acute alcohol augments inflammatory responses via IRAK-1 activation and JNK phosphorylation. Thus, the complexity of TLR-mediated signals may determine attenuation or augmentation of inflammatory responses by acute alcohol.

Adjuvants, Immunologic↗
PubMed2006

Murine TLR4 is implicated in cigarette smoke-induced pulmonary inflammation.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with an abnormal inflammatory response of the lungs to noxious particles or gases. We investigated whether Toll-like receptor 4 (TLR4) is implicated in cigarette smoke (CS)-induced pulmonary inflammation in a murine model of COPD. METHODS: C3H/HeOuJ (Tlr4(WT)) and C3H/HeJ (Tlr4(defective)) mice were exposed to air or CS for 5 weeks (subacute) and 26 weeks (chronic), and pulmonary inflammation was evaluated. RESULTS: In Tlr4(WT) mice, subacute and chronic CS exposure induced a substantial pulmonary infiltration of macrophages, neutrophils, lymphocytes and dendritic cells (DCs), that was absent in air-exposed mice. CS exposure increased the costimulatory marker expression on DCs, the levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) in bronchoalveolar lavage (BAL) fluid and induced the pulmonary expression of matrix metalloproteinase-12 (MMP-12), TLR4 and TLR2. In contrast, after subacute CS exposure, Tlr4(defective) mice showed a limited (5-fold lower) increase of DCs and lymphocytes in BAL fluid, lower costimulatory marker expression on DCs and lower MCP-1 and TNF-alpha levels in BAL fluid compared to Tlr4(WT) animals. After chronic CS exposure, however, the difference in pulmonary inflammation between Tlr4(WT) and Tlr4(defective) mice was less pronounced and both strains showed similar MCP-1 and TNF-alpha levels in BAL and similar pulmonary MMP-12, TLR4 and TLR2 expression. CONCLUSIONS: We demonstrated that the TLR4 mutation in C3H/HeJ mice is protective against CS-induced pulmonary influx of neutrophils, DCs and lymphocytes upon subacute CS exposure. However, TLR4 is only of minor importance in chronic CS-induced inflammation in mice.

Animals↗
PubMed2005

Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2.

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be approximately 50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.

Cell Line↗
PubMed2005

Dioscorin isolated from Dioscorea alata activates TLR4-signaling pathways and induces cytokine expression in macrophages.

The Toll-like receptor 4 (TLR4)-signaling pathway is crucial for activating both innate and adaptive immunity. TLR4 is a promising molecular target for immune-modulating drugs, and TLR4 agonists are of therapeutic potential for treating immune diseases and cancers. Several medicinal herb-derived components have recently been reported to act via TLR4-dependent pathways, suggesting that medicinal plants are potential resources for identifying TLR4 activators. We have applied a screening procedure to systematically identify herbal constituents that activate TLR4. To exclude possible LPS contamination in these plant-derived components, a LPS inhibitor, polymyxin B, was added during screening. One of the plant components we identified from the screening was dioscorin, the glycoprotein isolated from Dioscorea alata. It induced TLR4-downstream cytokine expression in bone marrow cells isolated from TLR4-functional C3H/HeN mice but not from TLR4-defective C3H/HeJ mice. Dioscorin also stimulated multiple signaling molecules (NF-kappaB, ERK, JNK, and p38) and induced the expression of cytokines (TNF-alpha, IL-1beta, and IL-6) in murine RAW 264.7 macrophages. Furthermore, the ERK, p38, JNK, and NF-kappaB-mediated pathways are all involved in dioscorin-mediated TNF-alpha production. In summary, our results demonstrate that dioscorin is a novel TLR4 activator and induces macrophage activation via typical TLR4-signaling pathways.

Animals↗
PubMed2001

Genes other than TLR4 are involved in the response to inhaled LPS.

For several decades, the mouse strains C3H/HeJ and C57BL/10ScNCr have been known to be hyporesponsive to endotoxin or lipopolysaccharide (LPS). Recently, mutations in Toll-like receptor (TLR) 4 have been shown to underlie this aberrant response to LPS. To further determine the relationship between TLR4 and responsiveness to LPS, we genotyped 18 strains of mice for TLR4 and evaluated the physiological and biological responses of these strains to inhaled LPS. Of the 18 strains tested, 6 were wild type for TLR4 and 12 had mutations in TLR4. Of those strains with TLR4 mutations, nine had mutations in highly conserved residues. Among the strains wild type for TLR4, the inflammatory response in the airway induced by inhalation of LPS showed a phenotype ranging from very sensitive (DBA/2) to hyporesponsive (C57BL/6). A broad spectrum of airway hyperreactivity after inhalation of LPS was also observed among strains wild type for TLR4. Although the TLR4 mutant strains C3H/HeJ and C57BL/10ScNCr were phenotypically distinct from the other strains with mutations in the TLR4 gene, the other strains with mutations for TLR4 demonstrated a broad distribution in their physiological and biological responses to inhaled LPS. The results of our study indicate that although certain TLR4 mutations can be linked to a change in the LPS response phenotype, additional genes are clearly involved in determining the physiological and biological responses to inhaled LPS in mammals.

Animals↗
PubMed2005

TLR4, but not TLR2, signals autoregulatory apoptosis of cultured microglia: a critical role of IFN-beta as a decision maker.

TLRs mediate diverse signaling after recognition of evolutionary conserved pathogen-associated molecular patterns such as LPS and lipopeptides. Both TLR2 and TLR4 are known to trigger a protective immune response as well as cellular apoptosis. In this study, we present evidence that TLR4, but not TLR2, mediates an autoregulatory apoptosis of activated microglia. Brain microglia underwent apoptosis upon stimulation with TLR4 ligand (LPS), but not TLR2 ligands (Pam(3)Cys-Ser-Lys(4), peptidoglycan, and lipoteichoic acid). Based on studies using TLR2-deficient or TLR4 mutant mice and TLR dominant-negative mutants, we also demonstrated that TLR4, but not TLR2, is necessary for microglial apoptosis. The critical difference between TLR2 and TLR4 signalings in microglia was IFN regulatory factor-3 (IRF-3) activation, followed by IFN-beta expression: while TLR4 agonist induced the activation of IRF-3/IFN-beta pathway, TLR2 did not. Nevertheless, both TLR2 and TLR4 agonists strongly induced NF-kappaB activation and NO production in microglia. Neutralizing Ab against IFN-beta attenuated TLR4-mediated microglial apoptosis. IFN-beta alone, however, did not induce a significant cell death. Meanwhile, TLR2 activation induced microglial apoptosis with help of IFN-beta, indicating that IFN-beta production following IRF-3 activation determines the apoptogenic action of TLR signaling. TLR4-mediated microglial apoptosis was mediated by MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-beta, and was associated with caspase-11 and -3 activation rather than Fas-associated death domain protein/caspase-8 pathway. Taken together, TLR4 appears to signal a microglial apoptosis via autocrine/paracrine IFN-beta production, which may act as an apoptotic sensitizer.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Selective priming to Toll-like receptor 4 (TLR4), not TLR2, ligands by P. acnes involves up-regulation of MD-2 in mice.

Lipopolysaccharide (LPS) triggers cytokine production through Toll-like receptor 4 (TLR4), which shares downstream signaling pathways with TLR2. We investigated the roles of TLR2 and TLR4 in Propionibacterium acnes (P. acnes)-primed, LPS-induced liver damage using selective TLR ligands. Stock LPS induced interleukin 8 in both TLR4- and TLR2-expressing human embryonic kidney (HEK) 293 cells. Purified LPS (TLR4 ligand) activated HEK/TLR4 cells, while peptidoglycan and lipoteichoic acid (TLR2 ligands) activated HEK/TLR2 cells, respectively. In mice, P. acnes priming resulted in increased liver messenger RNA (mRNA) and serum levels of tumor necrosis factor alpha, interleukin 12, and interferon gamma (IFN-gamma) by both stock LPS and purified LPS challenges compared with nonprimed controls. In contrast, P. acnes failed to sensitize to TLR2 ligands (peptidoglycan + lipoteichoic acid). In the liver, P. acnes-priming was associated with up-regulation of TLR4 and MD-2 proteins, and subsequent LPS challenge further increased MD-2 and CD14 mRNA levels. The lack of sensitization to TLR2 ligands by P. acnes correlated with no increase in hepatic TLR1 or TLR6 mRNA. In vitro, P. acnes pretreatment desensitized RAW macrophages to a secondary stimulation via both TLR2 and TLR4. However, IFN-gamma could selectively prevent desensitization to TLR4 but not to TLR2 ligands. Furthermore, P. acnes induced production of IFN-gamma in vivo as well as in isolated splenocytes. In vitro, P. acnes-primed Hepa 1-6 hepatocytes but not RAW macrophages produced increased MD-2 and CD14 mRNA levels after an LPS challenge. In conclusion, P. acnes priming to selective TLR4-mediated liver injury is associated with up-regulation of TLR4 and MD-2 and is likely to involve IFN-gamma and prevent TLR4 desensitization by P. acnes.

Animals↗
PubMed2006

Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: Role in proliferation and apoptosis in the intestine.

BACKGROUND & AIMS: We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. METHODS: Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. RESULTS: Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. CONCLUSIONS: We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Allelic variation in TLR4 is linked to susceptibility to Salmonella enterica serovar Typhimurium infection in chickens.

Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. We describe here the cloning and characterization of the avian orthologue of mammalian TLR4. Chicken TLR4 encodes a 843-amino-acid protein that contains a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll-interleukin-1R signaling domain characteristic of all TLR proteins. The chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Northern blot analysis reveals that TLR4 is expressed at approximately the same level in all tissues tested, including brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart, and spleen. The probe detected only one transcript of ca. 4.4 kb in length for all tissues except muscle where the size of TLR4 mRNA was ca. 9.6 kb. We have mapped TLR4 to microchromosome E41W17 in a region harboring the gene for tenascin C and known to be well conserved between the chicken and mammalian genomes. This region of the chicken genome was shown previously to harbor a Salmonella susceptibility locus. By using linkage analysis, TLR4 was shown to be linked to resistance to infection with Salmonella enterica serovar Typhimurium in chickens (likelihood ratio test of 10.2, P = 0.00138), suggesting a role of TLR4 in the host response of chickens to Salmonella infection.

Alleles↗
PubMed2006

TLR4 is required for host resistance in Pseudomonas aeruginosa keratitis.

PURPOSE: To determine the role of Toll-like receptor 4 (TLR4) in Pseudomonas aeruginosa (P. aeruginosa) keratitis in resistant (cornea-healing) BALB/c mice. METHODS: Corneal TLR4 mRNA levels were tested by real-time PCR in BALB/c mice before and after infection. Clinical score, slit lamp, histopathology, bacterial counts, and polymorphonuclear neutrophil (PMN) quantitation were performed in the infected cornea of TLR4-deficient (TLR4(lps-d)) and wild-type BALB/c mice. mRNA for IL-1beta, MIP-2, IFN-gamma, IL-18, inducible nitric oxide synthase (iNOS), and beta-defensin-2 levels were measured by real-time PCR. Protein levels for IL-1beta, MIP-2, and IFN-gamma were tested by ELISA. RESULTS: In resistant BALB/c mice, TLR4 mRNA expression was significantly upregulated in the cornea after P. aeruginosa infection. In contrast, TLR4-deficient mice were susceptible to infection with P. aeruginosa and showed increased corneal opacity, PMN infiltration, bacterial counts, and perforated infected corneas. After infection, TLR4-deficient mice also showed increased mRNA expression of proinflammatory cytokines (IL-1beta and MIP-2) and type-1-associated cytokines (IFN-gamma and IL-18) when compared with wild-type BALB/c mice. ELISA analyses showed that IL-1beta, MIP-2, and IFN-gamma protein levels also were significantly upregulated in the cornea of TLR4-deficient versus wild-type mice. In contrast, levels of iNOs and beta-defensin-2 were significantly decreased in TLR4-deficient compared with wild-type mice. CONCLUSIONS: TLR4 is critical in host resistance to P. aeruginosa, as its deficiency results in increased PMN infiltration and proinflammatory cytokine production, decreased iNOs and beta-defensin-2 production, impaired bacterial killing, and a susceptible phenotype.

Animals↗
PubMed2006

Gangliosides trigger inflammatory responses via TLR4 in brain glia.

Gangliosides participate in various cellular events of the central nervous system and have been closely implicated in many neuronal diseases. However, the precise molecular mechanisms underlying the pathological activity of gangliosides are poorly understood. Here we report that toll-like receptor 4 (TLR4) may mediate the ganglioside-triggered inflammation in glia, brain resident immune cells. Gangliosides rapidly altered the cell surface expression of TLR4 in microglia and astrocytes within 3 hours. Using TLR4-specific siRNA and a dominant-negative TLR4 gene, we clearly demonstrate the functional importance of TLR4 in ganglioside-triggered activation of glia. Inhibition of TLR4 expression by TLR4-siRNA suppressed nuclear factor (NF)-kappaB-binding activity, NF-kappaB-dependent luciferase activity, and transcription of inflammatory cytokines after exposure to gangliosides. Transient transfection of dominant-negative TLR4 also attenuated NF-kappaB-binding activity and interleukin-6 promoter activity. In contrast, these activities were slightly elevated in cells with wild-type TLR4. In addition, CD14 was required for ganglioside-triggered activation of glia, and lipid raft formation may be associated with ganglioside-stimulated signal propagation. Taken together, these results suggest that TLR4 may provide an explanation for the pathological ability of gangliosides to cause inflammatory conditions in the brain.

Animals↗
PubMed2003

Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life span.

Neutrophil responses to commercial LPS, a dual Toll-like receptor (TLR)2 and TLR4 activator, are regulated by TLR expression, but are amplified by contaminating monocytes in routine cell preparations. Therefore, we investigated the individual roles of TLR2 and TLR4 in highly purified, monocyte-depleted neutrophil preparations, using selective ligands (TLR2, Pam(3)CysSerLys(4) and Staphylococcus aureus peptidoglycan; TLR4, purified LPS). Activation of either TLR2 or TLR4 caused changes in adhesion molecule expression, respiratory burst (alone, and synergistically with fMLP), and IL-8 generation, which was, in part, dependent upon p38 mitogen-activated protein kinase signaling. Neutrophils also responded to Pam(3)CysSerLys(4) and purified LPS with down-regulation of the chemokine receptor CXCR2 and, to a lesser extent, down-regulation of CXCR1. TLR4 was the principal regulator of neutrophil survival, and TLR2 signals showed relatively less efficacy in preventing constitutive apoptosis over short time courses. TLR4-mediated neutrophil survival depended upon signaling via NF-kappa B and mitogen-activated protein kinase cascades. Prolonged neutrophil survival required both TLR4 activation and the presence of monocytes. TLR4 activation of monocytes was associated with the release of neutrophil survival factors, which was not evident with TLR2 activation, and TLR2 activation in monocyte/neutrophil cocultures did not prevent late neutrophil apoptosis. Thus, TLRs are important regulators of neutrophil activation and survival, with distinct and separate roles for TLR2 and TLR4 in neutrophil responses. TLR4 signaling presents itself as a pharmacological target that may allow therapeutic modulation of neutrophil survival by direct and indirect mechanisms at sites of inflammation.

Cell Adhesion Molecules↗
PubMed2006

Differential involvement of TLR2 and TLR4 in host survival during pulmonary infection with Chlamydia pneumoniae.

The relevance of TLR2 and TLR4 for recognizing Chlamydia pneumoniae in vivo during pulmonary infection and to survive the infection was explored. We found that early immune responses triggered by C. pneumoniae partially depended on TLR2, but not on TLR4. The chemokines MIP-2 and MIP-1alpha were not induced, while IL-12p40 levels were higher in TLR2(-/-) mice compared to wild-type mice. Secretion of TNF, keratinocyte-derived chemokine and monocyte chemoattractant protein-1 was attenuated in TLR2(-/-) mice, while IFN-gamma was increased as in wild-type mice. The pulmonary cyto- and chemokine response of TLR2(-/-) x TLR4(d/d) was similar to TLR2(-/-) mice. TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice also attracted fewer polymorphonuclear neutrophils into the lung, while TLR4(d/d) mice recruited them. Attenuated recruitment of polymorphonuclear neutrophils correlated with reduced weight loss in TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice and a lower chlamydial burden 3 days post infection. At 9 days post infection, TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice produced cyto- and chemokines as efficiently as wild-type mice, indicating that the involvement of TLR in inflammation varies over time. All TLR2(-/-) x TLR4(d/d) mice succumbed to the infection, while about 50% of TLR2(-/-) mice died. Taken together, the function of TLR2 and TLR4 is required to survive pulmonary infection with C. pneumoniae.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Ligand-independent oligomerization of TLR4 regulated by a short hydrophobic region adjacent to the transmembrane domain.

Toll-like receptors (TLRs) are key receptors for the activation of immune responses directed against pathogens. Among the more than 10 identified TLRs, TLR4 is the most unique because it associates with a variety of adaptor molecules for ligand recognition and signal transduction. However, the relationship between the unique characteristics and structural features of TLR4 is poorly defined. In this study, we demonstrate a novel biochemical characteristic of TLR4. TLR4, but not other TLRs, was observed as highly aggregated forms in immunoblotting. Interestingly, substitution of the transmembrane and cytoplasmic domain of TLR4 with those of other TLRs completely abolished the aggregation of TLR4. Furthermore, we found a short hydrophobic region (HR) adjacent to the transmembrane domain of TLR4; the TLR4 mutant lacking the HR was not aggregated and was nonfunctional in response to lipopolysaccharide. These results suggest that the HR may play a critical role in the functional oligomerization of TLR4.

Animals↗