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PubMed2006

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways.

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.

Animals↗
PubMed2005

Biological importance of the two Toll-like receptors, TLR2 and TLR4, in macrophage response to infection with Candida albicans.

The aim of this study was to assess the role of TLR2, TLR4 and MyD88 accessory molecule in the effector and secretory response of macrophages to viable microbial agents. Using TLR-deleted macrophage cell lines generated from the bone marrow of genetically engineered mice (TLR4 gene-deficient, MyD88- and TLR2-knockout mice) and wild-type control mice, we found that TLR2-deleted macrophages exhibit increased ability to contain Candida albicans infection compared to TLR2+/+ counterpart. In contrast, both MyD88-/- and TLR4-/- macrophages retain levels of functional activity comparable to that of the respective wild-type MyD88+/+ and TLR4+/+ controls. The difference in anticandidal effector functions observed between TLR2-/- and TLR2+/+ macrophages is abrogated upon opsonization of the fungal target and interestingly is not observed when using other microbial targets, such as Streptococcus pneumoniae and Helicobacter pylori. When tested for secretory response to C. albicans, TLR2-deleted macrophages show a pattern of cytokine production similar to that of TLR2+/+ controls. Finally, flow cytometry analysis reveals that TLR2-deleted macrophages express only TLR4, while, as expected, TLR2+/+ macrophages are both TLR2 and TLR4 positive; in no cases, modulation of such markers occurs in macrophages exposed to C. albicans infection. In conclusion, these data indicate that TLR2 and TLR4 have different biological relevance, in which TLR2 but not TLR4, is involved in the accomplishment of macrophage-mediated anticandidal activity, while the secretory response to C. albicans appears to be TLR4 but not TLR2-dependent.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Control of Mycobacterium bovis BCG infection with increased inflammation in TLR4-deficient mice.

Live mycobacteria have been reported to signal through several pattern recognition receptors (PRR), among them toll-like receptor 4 (TLR4) and TLR2 in vitro. Here, we investigated the role of TLR4 in host resistance to Mycobacterium bovis (BCG) infection in vivo. In vitro, macrophages of TLR4 mutant C3H/HeJ mice infected with BCG expressed lower levels of TNF than controls, and TNF release was further decreased, although not completely absent, in the absence of TLR2. In vivo, TLR4 mutant C3H/HeJ and control C3H/HeOUJ mice were infected with BCG (2 x 10(6) CFU i.v.). Both TLR4 mutant and wild-type mice were able to control the infection and survived 8 months post-BCG infection. Macrophage activation with abundant acid-fast bacilli and expression of inducible nitric oxide synthase (iNOS) and MHC class II antigens was seen in both groups of mice. However, TLR4 mutant mice experienced an arrest of body weight gain and showed signs of increased inflammation, with persistent splenomegaly, increase in granuloma number and augmented neutrophil infiltration. Infection of TLR4-deficient mice with higher doses of BCG (1 and 3 x 10(7) CFU, i.v.) increased the inflammation in spleen and liver, associated with a transient, higher bacterial load in the liver. In summary, TLR4 mutant mice show normal macrophage recruitment and activation, granuloma formation and control of the BCG infection, but this is associated with persistent inflammation. Therefore, TLR4 signaling is not essential for early control of BCG infection, but it may have a critical function in fine tuning of inflammation during chronic mycobacterial infection.

Animals↗
PubMed2006

LPS induces translocation of TLR4 in amniotic epithelium.

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) induced immune responses, which may contribute to preterm labor associated with intraamniotic gram-negative bacterial infections. The study objective was to investigate gestational age and LPS-induced changes in TLR4 subcellular localization within amniotic epithelium, the first line of host defense against intraamniotic bacteria. TLR4 localization in amniotic epithelium was assessed using immunohistochemistry on 24 placentas of different gestational ages: first trimester (n=6), second trimester (n=6), and third trimester (n=12). Immunofluorescence was used to determine TLR4 localization following ex vivo LPS stimulation of amnion from women undergoing cesarean section without labor at term. TLR4 was expressed in the cytoplasm of amniotic epithelium starting at 9weeks with apical polarization by 25weeks gestation. TLR4 localization to the basal membrane was significantly associated with chorioamnionitis (p=0.01). After LPS stimulation, TLR4 was expressed sequentially within the apical membrane, cytoplasm, and finally in the basal cellular compartment. This suggests that TLR4 expression in amniotic epithelium is poised to monitor amniotic fluid for pathogens. TLR4 translocation to the basal membrane may decrease LPS signaling early in an infection, but allow the amniotic epithelium to remain competent to invasive or intracellular bacteria.

Amnion↗
PubMed2001

MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.

Antigens, Surface↗
PubMed2006

Role of TLR4 in allergic sensitization to food proteins in mice.

Allergic sensitization to food proteins and other allergens is increasing in prevalence. One hypothesis for this increase is that the decreased rate of infections or exposure to microbial products leaves the immune system susceptible to inappropriate reactivity to innocuous antigens through the lack of development of regulatory cells. We hypothesized that constitutive Toll-like receptor (TLR)4 signaling (presumably via the commensal flora) could inhibit the development of allergic sensitization to food proteins. We tested this hypothesis by sensitizing TLR4+ and TLR4- mice on two genetic backgrounds, C3H and BALB/c, to two common food allergens [beta-lactoglobulin (betaLG) and peanut (PN)]. B-cell responses were not significantly influenced by TLR4 status. T-cell responses were Th2 skewed in TLR4-deficient C3H mice compared with TLR4 sufficient C3H mice, but this pattern of Th2 skewing was not observed in TLR4-deficient mice on a BALB/c background. In anaphylaxis-susceptible C3H mice, TLR4 deficiency was associated with increased severity of anaphylaxis to PN, and decreased severity of anaphylaxis to betaLG. In anaphylaxis-resistant BALB/c mice, TLR4 deficiency was not sufficient to render mice susceptible to PN-induced anaphylaxis. We conclude that although TLR4 status can influence T-cell responses and anaphylaxis severity, the nature of the influence is highly antigen- and strain-dependent.

Allergens↗
PubMed2005

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex.

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.

Acylation↗
PubMed2006

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.

Amino Acid Substitution↗
PubMed2002

Relevance of mutations in the TLR4 receptor in patients with gram-negative septic shock.

BACKGROUND: Septic shock remains a significant health concern worldwide, and despite progress in understanding the physiological and molecular basis of septic shock, the high mortality rate of patients with septic shock remains unchanged. We recently identified a common polymorphism in toll-like receptor 4 (TLR4) that is associated with hyporesponsiveness to inhaled endotoxin or lipopolysaccharide in humans. METHODS: Since TLR4 is a major receptor for lipopolysaccharide in mammals and gram-negative bacteria are the prevalent pathogen associated with septic shock, we investigated whether these specific TLR4 alleles are associated with a predisposition to a more severe disease outcome for patients with septic shock. We genotyped 91 patients with septic shock as well as 73 healthy blood donor controls for the presence of the TLR4 Asp299Gly and TLR4 Thr399Ile mutations. RESULTS: We found the TLR4 Asp299Gly allele exclusively in patients with septic shock (P =.05). Furthermore, patients with septic shock with the TLR4 Asp299Gly/Thr399Ile alleles had a higher prevalence of gram-negative infections. CONCLUSION: Mutations in the TLR4 receptor may predispose people to develop septic shock with gram-negative microorganisms.

Adult↗
PubMed2007

Downregulation of TLR4-dependent ATP production is critical for estrogen-mediated immunoprotection in Kupffer cells following trauma-hemorrhage.

Toll-like receptor 4 (TLR4) mediates mitochondrial DNA (mtDNA) damage and biogenic responses. Mitochondrial transcription factor A (Tfam) is an essential regulator for mtDNA transcription and ATP production. Increased ATP levels were associated with normalization of immune function following trauma-hemorrhage. Moreover, administration of 17beta-estradiol following trauma-hemorrhage upregulates cardiac Tfam and ATP levels. We therefore hypothesized that the salutary effect of 17beta-estradiol on Kupffer cell function following trauma-hemorrhage is mediated via negative regulation of TLR4, which downregulates iNOS, upregulates Tfam and mtDNA-encoded gene cytochrome c oxidase I (mtCOI), and consequently increases cellular ATP levels. Male C3H/HeN, C3H/HeOuJ (intact TLR4), and C3H/HeJ (TLR4 mutant) mice were subjected to trauma-hemorrhage (mean BP 35 +/- 5 mmHg approximately 90 min, then resuscitation) or sham operation. At the beginning of resuscitation, mice received 17beta-estradiol (25 microg/25 g) or vehicle intravenously and were sacrificed 2 h thereafter. Kupffer cell TLR4, iNOS, IL-6 and TNF-alpha production capacities were increased, and ATP, Tfam, and mtCOI levels were decreased following trauma-hemorrhage. Administration of 17beta-estradiol following trauma-hemorrhage prevented the increase in Kupffer cell TLR4, iNOS, and cytokine production. This was accompanied by normalized ATP, Tfam, and mtCOI levels. Furthermore, the decreased Kupffer cell ATP and mtCOI levels were not observed in TLR4 mutant mice following trauma-hemorrhage. Taken together, these findings suggest that downregulation of TLR4-dependent ATP production is critical to 17beta-estradiol-mediated immunoprotection in Kupffer cells following trauma-hemorrhage.

Adenosine Triphosphate↗
PubMedDate not supplied

Analysis of Tlr4-mediated LPS signal transduction in macrophages by mutational modification of the receptor.

In mouse macrophages (RAW 264.7 cells), toll-like receptor 4 (Tlr4) is a limiting factor in lipopolysaccharide (LPS) signal transduction. The expression of only 1-2 x 10(4) copies of recombinant Tlr4 per cell enhances sensitivity to LPS, shifting the EC50 by 30-fold to the left. Expression of the Tlr4(Lps-d) isoform of Tlr4 (found in C3H/HeJ mice) shifts the EC50 2600-fold to the right, essentially abolishing LPS responses. A truncated form of Tlr4, lacking a cytoplasmic domain, exerts only a weak inhibitory effect on signal transduction. Similarly, the normal or Tlr4(Lps-d) forms of protein lacking an ectodomain [corrected], cause modest inhibition of LPS signaling. Manipulations of Tlr4 structure and expression cause changes in LPS sensitivity that range over 3 to 4 orders of magnitude. These findings support the view that Tlr4 is an integral component of a solitary pathway for LPS signal transduction in macrophages and permit inferences related to the mechanism of signaling and its blockade.

Animals↗
PubMed2005

Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression.

Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.

Alkaline Phosphatase↗
PubMed2001

Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation.

Lipopolysaccharide (LPS) has been implicated in the pathogenesis of graft-versus-host disease (GVHD). The toll-like receptor (TLR)-4 has been recently identified as a major receptor for LPS. Mutations of TLR4 have been associated with LPS hyporesponsiveness. We hypothesized that TLR4 mutations reduce the risk of acute GVHD in allogeneic marrow transplant recipients. In a preliminary study to determine the frequency of TLR4 mutations and their possible association with GVHD, we tested 237 patients and their HLA-identical sibling donors for 2 TLR4 polymorphisms. All patients received methotrexate and cyclosporine for GVHD prophylaxis. One or more mutants were detected in 10.8% of patients and 10.6% of donors. Multivariable logistic regression models were used to analyze the association between TLR4 mutations and probability (1-sided) of GVHD. The odds ratio (adjusted for advanced disease, total body irradiation dose, and patient age) for development of grades II to IV GVHD when a mutation was present in the recipient was 0.63 (95% confidence interval [CI], 0.25-1.60; P = .16). When a mutation was present in the donor, the adjusted odds ratio was 0.88 (95% CI, 0.36-2.17; P = .40). When a mutation was present in both recipient and donor, the odds ratio was 0.72 (95% CI, 0.22-2.32; P = .29). Among 24 patients with TLR4 mutations in either donor or recipient, 4 (16.7%) developed gram-negative bacteremia. Among 213 patients without mutations, 14 (6.6%) developed gram-negative bacteremia (P = .09). The data indicate that a reduced risk of acute GVHD is associated with TLR4 mutations and that TLR4 mutations may increase the risk for gram-negative bacteremia. However, these associations are not statistically significant in recipients of HLA-matched sibling marrow transplants who are prophylactically treated for infections and GVHD. A much larger study population would be needed to confirm the role of LPS in the pathogenesis of GVHD in humans.

Bacteremia↗
PubMed2002

TLR4 and MD-2 expression is regulated by immune-mediated signals in human intestinal epithelial cells.

The normal intestinal epithelium is not inflamed despite contact with a high density of commensal bacteria. Intestinal epithelial cells (IEC) express low levels of TLR4 and MD-2 and are lipopolysaccharide (LPS)-unresponsive. We hypothesized that immune-mediated signals regulate the expression of TLR4 and MD-2 in IEC. Expression of TLR4 and MD-2 was examined in normal colonic epithelial cells or intestinal epithelial cell lines. The effect of the cytokines interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor-alpha (TNF-alpha) on TLR4 and MD-2 expression was examined by reverse transcription-PCR and Western blot. NF-kappaB transcriptional activation and interleukin-8 secretion were used as measures of LPS responsiveness. Native colonic epithelial cells and IEC lines express a low level of TLR4 and MD-2 mRNA. IFN-gamma regulates MD-2 expression in both IEC lines, whereas IFN-gamma and TNF-alpha regulate TLR4 mRNA expression in IEC lines. Pre-incubation with IFN-gamma and/or TNF-alpha sensitizes IEC to LPS-dependent interleukin-8 secretion. To examine MD-2 transcriptional regulation, we cloned a 1-kb sequence proximal to the MD-2 gene translational start site. This promoter directed expression of a reporter gene in endothelial cells and IEC. IFN-gamma positively regulated MD-2 promoter activity in IEC. Co-expression of a STAT inhibitor, SOCS3, blocked IFN-gamma-mediated MD-2 promoter activation. T cell-derived cytokines lead to increased expression of TLR4 and MD-2 and LPS-dependent pro-inflammatory cytokine secretion in IEC. IFN-gamma regulates expression of the critical TLR4 co-receptor MD-2 through the Janus tyrosine kinase-STAT pathway. Th1 cytokines may initiate or perpetuate intestinal inflammation by altering toll-like receptor expression and bacterial reactivity.

Antigens, Surface↗
PubMed2004

Hypertensive rats are susceptible to TLR4-mediated signaling following exposure to combustion source particulate matter.

Toll-like receptor 4 (TLR4) has been shown to play a role in cell signaling that results in neutrophilic inflammation in response to lipopolysaccharide and respiratory syncytial virus infection. TLR4 also interacts with CD14, which upon complex formation triggers TLR4-associated signaling pathways to produce a proinflammatory response. This mechanism results in the activation of NF-kappa B and subsequent inflammatory gene induction. In order to determine the effect of combustion source particle matter (PM), rich in zinc and nickel but with negligible endotoxin, on a possible activation of TLR4-mediated cell signaling and inflammation, we intratracheally (IT) instilled 3.3 mg/kg of PM into 12-w-old healthy male Wistar Kyoto (WKY) and susceptible spontaneously hypertensive (SH) rats. Inflammation, inflammatory-mediator gene expression, bronchoalveolar lavage fluid (BALF) protein and LDH, TLR4 and CD14 protein, and NF-kappa B activation in the lung were determined after 24 h. Dose-response data (0.0, 0.83, 3.33, and 8.3 mg/kg PM) for BALF LDH were obtained as a marker of lung cell injury in SH rats. BALF neutrophils, but not macrophages, were significantly increased in the PM-exposed WKY and SH rats. SH rats showed a greater PMN increase than WKY rats. Similarly, BALF protein and LDH levels were also increased following PM exposure but to a significantly greater extent in SH rats. Plasma fibrinogen was increased only in SH rats exposed to PM. The increased inflammation seen in PM-exposed SH rats was accompanied by a significant increase in TLR4 protein in the lung tissue, which was primarily localized in alveolar macrophages and epithelial cells. CD14 was also increased by PM exposure in both SH and WKY rats but was significantly greater in the SH rats. These increases were associated with greater translocation of NF-kappa B in the lungs of SH rather than WKY rats. This was accompanied by increased macrophage inhibitory protein (MIP)-2 mRNA expression at 24 h of exposure. These data suggest that the increased inflammation in the lungs of PM-exposed SH rats compared to WKY rats is accompanied by an increase in TLR4-mediated cell signaling. Thus, one of the mechanisms for greater susceptibility of SH rats to PM exposure may involve an increased activation of the TLR4 signaling pathway.

Air Pollutants↗
PubMed2004

Role for CD14, TLR2, and TLR4 in bacterial product-induced anorexia.

The cell surface component CD14 and the toll-like receptors 2 and 4 (TLR2 and TLR4) are important in mediating the immune responses to bacterial products in mammals. Using mice genetically deficient in CD14, TLR2, or TLR4, we studied the role of these molecules in the anorectic effects of LPS and muramyl dipeptide (MDP). CD14 or TLR2 knockout (KO) and TLR4-deficient (TLR4-DEF) mice as well as corresponding wild-type (WT) colittermates were injected intraperitoneally at dark onset with LPS (2 microg/mouse), MDP (10 mg/kg), interleukin-1 beta (IL-1 beta, 150 ng/mouse), or vehicle, and food intake was recorded. LPS and MDP reduced food intake in WT mice of all genotypes tested. The anorectic effect of LPS was attenuated (P < 0.04) in CD14-KO and TLR4-DEF mice but not in TLR2-KO (P > 0.05). The anorectic effect of MDP was blunted in CD14-KO and TLR2-KO (P < 0.02) mice but not in TLR4-DEF mice. IL-1 beta reduced food intake similarly in all genotypes tested. These results indicate that CD14 is involved in mediating the anorectic effects of both LPS and MDP. Furthermore, TLR4 and TLR2 are specifically involved in mediating the anorectic effects of LPS and MDP, respectively. The results are consistent with the hypothesis that TLR4 functions as the true LPS receptor and that TLR2 is involved in recognition of gram-positive bacterial products.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2006

Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.

This study presents evidence that human platelets bind lipopolysaccharide (LPS) from enterohemorrhagic Escherichia coli (EHEC) through a complex of toll-like receptor 4 (TLR4) and CD62, leading to their activation. TLR4 colocalized with CD62 on the platelet membrane, and the TLR4 specificity of LPS binding to platelets was confirmed using C57BL/10ScN mice lacking Tlr4. Only platelets from TLR4 wild-type mice bound O157LPS in vitro. After in vivo injection, O157LPS bound to platelets from wild-type mice, which had lower platelet counts than did mice lacking TLR4. Mouse experiments confirmed that O157LPS binding to TLR4 is the primary event leading to platelet activation, as shown by CD40L expression, and that CD62 further contributes to this process. Activation of human platelets by EHEC-LPS was demonstrated by expression of the activated GPIIb/IIIa receptor, CD40L, and fibrinogen binding. In perfusion experiments, platelet activation on endothelial cells was TLR4 and CD62 dependent. O157LPS was detected on platelets from 12 of 14 children with EHEC-associated hemolytic uremic syndrome (HUS) and on platelets from 2 children before the development of HUS but not on platelets of EHEC-infected children in whom HUS did not develop (n = 3). These data suggest that O157LPS on platelets might contribute to platelet consumption in HUS.

Adolescent↗
PubMed2000

Phylogenetic variation and polymorphism at the toll-like receptor 4 locus (TLR4).

BACKGROUND: Differences in responses to bacterial surface lipopolysaccharides (LPSs) are apparent between and within mammalian species. It has been shown in mice that resistance to LPS is caused by defects in the Toll-like receptor 4 gene (Tlr4), the product of which is thought to bind LPS and mediate LPS signal transduction in immune system cells. RESULTS: We have sequenced the Toll-like receptor 4 gene of humans (TLR4; 19.0 kilobases, kb) and mice (Tlr4; 91.7 kb), as well as the coding region and splice junctions of Tlr4 from 35 mouse (Mus musculus) strains, from the chimpanzee and from the baboon. No other discernible genes or regions of interspecies conservation lies close to Tlr4 and, in both humans and mice, flanking sequences and introns are rich in repeats of retroviral origin. Interstrain analyses reveal that Tlr4 is a polymorphic protein and that the extracellular domain is far more variable than the cytoplasmic domain, both among strains and among species. The cytoplasmic domain of the Tlr4 protein is highly variable at the carboxy-terminal end. CONCLUSIONS: We suggest that selective evolutionary pressure exerted by microbes expressing structurally distinguishable LPS molecules has produced the high level of variability in the Tlr4 extracellular domain. The highly variable carboxy-terminal region of the cytoplasmic domain is likely to determine the magnitude of the response to LPS within a species.

Animals↗