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PubMed2002

Regulation of Toll-like receptor 4 expression in the lung following hemorrhagic shock and lipopolysaccharide.

The Toll-like receptor 4 (TLR4) has recently been shown to function as the major upstream sensor for LPS. In this study, a rodent model of lung injury following resuscitated hemorrhagic shock was used to examine the regulation of TLR4 gene and protein expression in vivo and in vitro. Intratracheal LPS alone induced a rapid reduction in whole lung TLR4 mRNA, an effect which is also observed in recovered alveolar macrophages. This effect appeared to be due to a lowering of TLR4 mRNA stability by approximately 69%. By contrast, while shock/resuscitation alone had no effect on TLR4 mRNA levels, it markedly altered the response to LPS. Specifically, antecedent shock prevented the LPS-induced reduction in TLR4 mRNA levels. This reversal was explained by the ability of prior resuscitated shock both to prevent the destabilization of TLR4 mRNA by LPS and also to augment LPS-stimulated TLR4 gene transcription compared with LPS alone. Oxidant stress related to shock/resuscitation appeared to contribute to the regulation of TLR4 mRNA, because supplementation of the resuscitation fluid with the antioxidant N-acetylcysteine reversed the ability of shock/resuscitation to preserve TLR4 mRNA levels following LPS. TLR4 protein levels in whole lung mirrored the changes seen for TLR4 mRNA. Considered in aggregate, these data suggest that levels of tlr4 expression are controlled both transcriptionally as well as posttranscriptionally through altered mRNA stability and that antecedent shock/resuscitation, a form of global ischemia/reperfusion, might influence regulation of this gene.

Animals↗

PubMed2005

Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia.

We hypothesized that PU.1 and PU.1 interacting proteins (PIP) binding to the Toll-like receptor 4 (TLR4) promoter is involved in endotoxin-induced upregulation of TLR4 gene expression. Our results employing chromatin immunoprecipitation assays indicate that PU.1 binds to the murine TLR4 promoter both in macrophage cells and, most importantly, in whole lung tissue. Treatment of RAW 264.7 cells with endotoxin induced the association of PU.1 and the TLR4 promoter in a time-dependent manner, and this was closely tied to interactions between the TLR4 promoter and the PIP interferon regulatory factors (IRF)4 and IRF8. PU.1 binding was related to increases in steady-state TLR4 mRNA and total TLR4 protein in RAW cells. Endotoxemia in animals caused the similar inducible interaction between PU.1 and IRF4 and the TLR4 promoter in lung tissue of mice that was treated with a single intraperitoneal injection of endotoxin. PU.1 binding to the TLR4 promoter was not enhanced in the lung tissue of endotoxin-resistant C3H/HeJ mice in response to endotoxemia. Transient transfection studies in RAW cells indicate that inducible binding of PU.1 to the TLR4 promoter is abrogated by a Ser148 to Ala mutation in PU.1. These data suggest that induction of PU.1/PIP binding to the TLR4 promoter is involved in endotoxin response in vivo and may mediate transcriptional changes in TLR4 gene expression.

Amino Acid Sequence↗

PubMed2006

The role of human antigen R, an RNA-binding protein, in mediating the stabilization of toll-like receptor 4 mRNA induced by endotoxin: a novel mechanism involved in vascular inflammation.

OBJECTIVE: Lipopolysaccharide (LPS) interacts with toll-like receptor 4 (TLR4) and induces proliferation of vascular smooth muscle cells (VSMCs) which plays a causal role in atherogenesis. The role of TLR4 expression and regulation in LPS-stimulated VSMCs remains unclear. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3' untranslated regions (3'UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs). METHODS AND RESULTS: Stimulation of HASMCs with LPS significantly increased the cytosolic HuR level in vitro. Immunoprecipitation and RT-PCR demonstrated that LPS markedly increased the interaction of HuR and 3'UTR of TLR4 mRNA. The reporter plasmid, which contains the 3'UTR of TLR4 mRNA, significantly increased luciferase reporter gene expression in LPS-induced HASMCs. These data suggest that the 3'UTR of TLR4 mRNA confers LPS responsiveness and that HuR modulates 3'UTR-mediated gene expression. Knock-down of HuR inhibited LPS-induced TLR4 mRNA stability in HASMCs and luciferase reporter gene expression in CMV-Luciferase-TLR4 3'UTR-transfected HASMCs. In addition, inhibition of NADPH oxidase activity by diphenylene iodonium, knock-down of Rac1 gene expression by siRNA, and decrease of p38 MAPK activity by SB203580 significantly decreased the cytosolic HuR level, which mediates TLR4 mRNA stability. CONCLUSIONS: Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.

Animals↗

PubMed2004

Immune cell Toll-like receptor 4 is required for cardiac myocyte impairment during endotoxemia.

The aim of this study was to investigate the importance of Toll-like receptor 4 (TLR4) signaling on cardiac myocytes versus immune cells in lipopolysaccharide (LPS)-induced cardiac dysfunction. Cardiac myocytes isolated from LPS-treated C57Bl/6 mice showed reduced shortening and calcium transients as compared with myocytes from untreated mice. In addition, LPS-treated C57Bl/6 mice showed impaired cardiac mitochondrial function, including reduced respiration and reduced time of induction of permeability transition. All of the aforementioned cardiac dysfunction was dependent on TLR4, because LPS-treated TLR4-deficient mice did not have reduced myocyte shortening or mitochondrial dysfunction. To evaluate the role of cardiac myocyte versus leukocyte TLR4, LPS was injected into chimeric mice with TLR4-positive leukocytes and TLR4-deficient myocytes. These mice showed reduced myocyte shortening in response to LPS. Myocytes from chimeric mice with TLR4-deficient leukocytes and TLR4-positive myocytes had no response to LPS. In addition, isolated myocytes from C57Bl/6 mice subsequently treated with LPS and serum for various times did not have reduced shortening, despite the presence of TLR4 mRNA and protein, as determined by reverse-transcription polymerase chain reaction and fluorescent-activated cell sorting. In fact, cardiac myocytes had equivalent amounts of TLR4 as endothelium; however, only the latter is responsive to LPS. Furthermore, signaling pathways downstream of TLR4 were not activated during direct LPS treatment of myocytes. In conclusion, TLR4 on leukocytes, and not on cardiac myocytes, is important for cardiac myocyte impairment during endotoxemia.

Animals↗

PubMed2005

Chlamydophila pneumoniae induces expression of toll-like receptor 4 and release of TNF-alpha and MIP-2 via an NF-kappaB pathway in rat type II pneumocytes.

BACKGROUND: The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-kappaB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-alpha and MIP-2. METHODS: Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-kappaB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-kappaB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-alpha and MIP-2 release by ELISA. RESULTS: Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1-2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56 degrees C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 mug/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-kappaB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-alpha and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-alpha and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. CONCLUSION: The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca2+-mediated NF-kappaB activity, necessary for maintenance of TLR4 expression and TNF-alpha and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.

Animals↗

PubMed2004

Evidence for the presence of toll-like receptor 4 system in the human endometrium.

We investigated whether Toll-like receptor (TLR) 4 is at work in the human endometrium. The expression of TLR4 mRNA in endometrial epithelial cells (EECs) and stromal cells (ESCs) was detected by RT-PCR and in situ hybridization. Western blotting analysis revealed the TLR4 protein expression in both cell populations. Treatment of lipopolysaccharide (LPS), the actions of which are mediated through TLR4, significantly increased IL-8 secretion from cultured ESCs in a dose-dependent fashion. The stimulatory effect of LPS was inhibited by the addition of neutralizing antibodies for TLR4 and CD14. LPS also stimulated nuclear translocation of nuclear factor-kappaB in ESCs, which was also inhibited by these antibodies. On the other hand, LPS did not stimulate IL-8 secretion in cultured EECs. However, LPS stimulated IL-8 secretion from EECs in the presence of soluble CD14. Flow cytometric analysis revealed that CD14 was expressed on the cell surface of ESCs but not EECs. In addition, immunohistochemical analysis showed that CD14 was stained in ESCs but not EECs. Pretreatment of interferon-gamma (IFN-gamma) enhanced LPS-induced IL-8 secretion from ESCs. IFN-gamma increased the expression of TLR4 mRNA. It also increased the amounts of mRNA for CD14, MD2, and MyD88, which are needed for LPS recognition in concert with TLR4. In summary, we demonstrated that both ESCs and EECs express TLR4 and respond to LPS through TLR4. We further showed that EECs, not ESCs, required soluble CD14 for TLR4 activation. Interestingly, IFN-gamma, an antiinfectious cytokine, was found to activate the TLR4 system in ESCs. Altogether, the results imply that the TLR4 system might represent local immunity in the human endometrium with differential modes of TLR4 actions between ESCs and EECs.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Toll-like receptor 4 expression levels determine the degree of LPS-susceptibility in mice.

C57BL/10ScCr (Cr) mice carry a deletion of the Toll-like receptor 4 (tlr4) gene (i.e. they are tlr4(0/0)) and are thus refractory to LPS effects. Insertion of wild-type tlr4 transgene into the tlr4(0/0) Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4(Tr/0)), wild-type C57BL/10ScSn (Sn) (tlr4(+/+)) and heterozygous F1 (Sn x Cr) (tlr4(+/0)) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS-induced cytokine (IL-6) response revealed a linear relationship between the response and the logarithm of TLR4-MD-2 levels.

Animals↗

PubMed2005

Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation.

Microglia are activated by lipopolysaccharide (LPS) to produce neurotoxic pro-inflammatory factors and reactive oxygen species (ROS). While a multitude of LPS receptors and corresponding pathways have been identified, the detailed mechanisms mediating the microglial response to LPS are unclear. Using mice lacking a functional toll-like receptor 4 (TLR4), we demonstrate that TLR4 and ROS work in concert to mediate microglia activation, where the contribution from each pathway is dependent on the concentration of LPS. Immunocytochemical staining of microglia in neuron-glia cultures with antibodies against F4/80 revealed that while TLR4(+/+) microglia were activated the low concentration of 1 ng/ml of LPS, TLR4(-/-) microglia exhibit activated morphology in response to LPS only at higher concentrations (100-1,000 ng/ml). Additionally, tumor necrosis factor-alpha (TNF-alpha) was only produced from higher concentrations (100-1,000 ng/ml) of LPS in TLR4(-/-) enriched microglia cultures. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, reduced TNF-alpha production from TLR4(-/-) microglia. The influence of TLR4 on LPS-induced superoxide production was tested in rat enriched microglia cultures, where the presence or absence of serum failed to show any effect on the superoxide production. Further, both TLR4(-/-) and TLR4(+/+) microglia showed a similar increase in extracellular superoxide production when exposed to LPS (1-1,000 ng/ml). These data indicate that LPS-induced superoxide production in microglia is independent of TLR4 and that ROS derived from the production of extracellular superoxide in microglia mediates the LPS-induced TNF-alpha response of both the TLR4-dependent and independent pathway.

Animals↗

PubMed2005

The Toll-like receptor 4 region Glu24-Pro34 is critical for interaction with MD-2.

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS) but requires MD-2, a molecule associated with the extracellular TLR4 domain, to respond efficiently to LPS. The purpose of this study was to determine the critical stretch of primary sequence in the TLR4 region involved in MD-2 recognition. TLR4 and TLR4/2a chimera consisting of the TLR4 region Met(1)-Phe(54) and the TLR2 region Ala(53)-Ser(784) were coprecipitated with MD-2, but the deletion mutant TLR4(Delta E24-P34) in which the TLR4 region Glu(24)-Pro(34) was deleted failed to coprecipitate. In agreement with the MD-2 binding, LPS-conjugated beads sedimented TLR4 and TLR4/2a chimera but not TLR2 with MD-2. TLR4(Delta E24-P34) barely coprecipitated with LPS-beads. The cells that had been cotransfected with TLR4(Delta E24-P34) and MD-2 did not induce NF-kappa B activation in response to LPS. These results clearly demonstrate that the amino-terminal TLR4 region of Glu(24)-Pro(34) is critical for MD-2 binding and LPS signaling.

Amino Acid Sequence↗

PubMed2000

Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol.

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4.MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFkappaB activation in these transfectants. Noticeable NFkappaB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4. MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFkappaB activation via TLR4.MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFkappaB activation via TLR4.MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4.MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.

Animals↗

PubMed2006

Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility to Candida albicans in a model of hematogenously disseminated infection.

We have studied the role of TLR4 in murine defenses against Candida albicans in two TLR4-defective mouse strains: C3H/HeJ mice which have defective TLR4 signaling, and TLR4-/- knockout mice. Both TLR4-defective mice strains experimentally infected with virulent C. albicans cells showed no significant difference in survival as compared with their respective controls. Recruitment of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR4-/-animals, but significantly enhanced in C3H/HeJ mice, compared with their control mice. In vitro production of TNF-alpha by macrophages from both types of TLR4-defective mice, in response to yeasts and hyphae of C. albicans, was not diminished as compared with production by macrophages from wild-type mice. In vitro production of TNF-alpha by yeast-stimulated splenocytes from mice intravenously infected with the low-virulence C. albicans PCA2 strain was not affected in TLR4-defective mice, but the TNF-alpha production in response to hyphae was higher in TLR4-defective than in control animals; the production of IFN-gamma by these splenocytes was similar to controls, as well as the frequency of IFN-gamma-producing CD4+T lymphocytes, indicating that TLR4-defective mice are capable of mounting a Th1 adaptive immune response. Our data indicate that TLR4 is dispensable for murine immune resistance to C. albicans.

Animals↗

PubMed2006

Hypertonic saline enhances host defense to bacterial challenge by augmenting Toll-like receptors.

OBJECTIVE: To determine whether hypertonic saline infusion modulates thermal injury-induced bacterial translocation and host response to bacterial challenge through the augmentation of Toll-like receptors (TLRs). DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the mice. INTERVENTIONS: In experiment 1, mice underwent burn were given with 10 mL/kg hypertonic saline (7.5% NaCl), 10 mg/kg saline (N/S1), or 80 mL/kg saline (N/S2) at 4 or 8 hrs after burn. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay. In experiment 2, mice receiving hypertonic saline or saline after thermal injury received peritoneal challenge with Escherichia coli, and bacterial clearance was measured. In experiment 3, peritoneal cells from mice receiving hypertonic saline or saline after thermal injury were incubated with E. coli, and bacterial count, TLR2, TLR4, MIP2, CXCR2, pp38, and ERK expression were evaluated. In experiment 4, reactive oxygen species production, CXCR2, MIP2, TLR2, and TLR4 expression of bone marrow neutrophil from mice receiving hypertonic saline or saline treatment after thermal injury were evaluated. In experiment 5, neutrophil were cultured with hypertonic saline or N/S and incubated with E. coli. TLR2 and TLR4 expression and bacterial count were evaluated. In experiment 6, mice were fed with oral antibiotics with or without lipopolysaccharide, a TLR ligand, supplements. At 24 hrs after burn, mesenteric lymph nodes were harvested for bacterial translocation assay, and neutrophils were harvested for TLR2 and TLR4 protein assay. MEASUREMENTS AND MAIN RESULTS: Hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased bacterial clearance, phagocytic activity, and TLR2, TLR4, CXCR2, pp38, and p44/42 expression of peritoneal cells. Hypertonic saline treatment at 4 or 8 hrs after thermal injury decreased reactive oxygen species production of neutrophil. Hypertonic saline injection increased TLR2, TLR4, and pp38 expression of neutrophil. In vitro treatment of neutrophil with hypertonic saline increased phagocytic activity and TLR2 and TLR4 expression. Commensal depletion with oral antibiotics decreased TLR2 and TLR4 expression of neutrophil; lipopolysaccharide increased TLR4 expression of neutrophil and decreased thermal injury-induced bacterial translocation. CONCLUSIONS: Restoration of extracellular fluid in burn shock with hypertonic saline decreased thermal injury-induced bacterial translocation. Hypertonic saline increased the phagocytic activity and TLR2, TLR4, CXCR2, pp38, and P44/42 expression of peritoneal cells. Hypertonic saline decreased reactive oxygen species but increased TLR2, TLR4, and pp38 expression and phagocytic activity of bone marrow neutrophil. Stimulation of the TLRs with lipopolysaccharide in commensal depleted mice increased TLRs expression of neutrophil and decreased thermal injury-induced bacterial translocation. Taken together with the fact that stimulation of TLRs with hypertonic saline increases phagocytic activity of systemic inflammatory cells, we conclude that TLRs play a critical role in the innate immunity by recognizing bacteria and that hypertonic saline enhances host response to bacterial challenge by increasing TLRs of inflammatory cells.

Animals↗

PubMed2005

Toll-like receptor 4 detected in exocrine pancreas and the change of expression in cerulein-induced pancreatitis.

OBJECTIVES: To detect Toll-like receptor 4 (TLR4) expression and distribution in rat pancreas and the change of TLR4 expression in cerulein-induced pancreatitis (CIP). METHODS: Acute pancreatitis was induced by subcutaneous injections of cerulein at a total dose of 20 microg/kg. Immunohistochemistry (IHC) was used to detect and localize TLR4 in rat pancreas, and real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitatively determine the expression of TLR4 mRNA in CIP. RESULTS: IHC showed the presence of TLR4 in rat pancreas, and its distribution was specifically localized to pancreatic ductal epithelium, vascular endothelium, and islet. No TLR4 staining was detected in exocrine acinar cells. Real-time RT-PCR results revealed low-level TLR4 mRNA expression in the rat pancreas, and the change of TLR4 in CIP only developed within the first 4 hours, which is a rapid up-regulation process that peaks at the first hour. TLR4 mRNA was sustained at baseline level from 4 to 24 hours. CONCLUSIONS: TLR4 protein was expressed in pancreas and localized to epithelial (pancreatic duct) or endothelial (vessels) tissues; TLR4 responded favorably to the inflammatory process, and the change of expression was characterized as a rapid up-regulation in the early stage of CIP.

Acute Disease↗

PubMed2005

Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction.

Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.

Animals↗

PubMed2004

Toll-like receptor 4 mediates the antitumor host response induced by a 55-kilodalton protein isolated from Aeginetia indica L., a parasitic plant.

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-kappaB-dependent reporter plasmid, AILb-A induced NF-kappaB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-kappaB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Animals↗

PubMed2004

Toll-like receptor 4-dependent early elicited tumor necrosis factor alpha expression is critical for innate host defense against Bordetella bronchiseptica.

Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns. Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined. We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation with Bordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model. Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4. The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response to B. bronchiseptica. However, early tumor necrosis factor alpha (TNF-alpha) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding. TLR4-deficient mice and TNF-alpha-/- mice are similarly susceptible to infection with relatively low doses of B. bronchiseptica and in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-alpha response that is critical for preventing severe pneumonia and limiting bacterial growth. These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-alpha response that is critical to innate host defense during acute gram-negative respiratory infection.

Animals↗

PubMed2004

Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis.

OBJECTIVES: To determine if polymorphisms within the Toll-like receptor 4 (TLR4) gene are associated and linked with juvenile idiopathic arthritis (JIA). To investigate any possible gene-gene (epistatic) interaction between TLR4 and macrophage migration inhibitory factor (MIF) gene polymorphisms. METHODS: 313 simplex families (each containing one affected JIA proband) were genotyped. Two known functionally important single nucleotide polymorphisms (SNPs) within the TLR4 gene (Asp299Gly and Thr399Ile) were typed by SNaPshot ddNTP primer extension and capillary electrophoresis. Single point and multipoint transmission disequilibrium tests (TDT) were carried out through the extended TDT and TDT phase packages for the two TLR4 SNPs. Epistatic interaction between TLR4 haplotypes and the previously JIA associated MIF CATT(7)-MIF-173*C promoter haplotype was investigated by chi(2) test and unconditional logistic regression in Stata version 7. RESULTS: No distortion from random inheritance was observed by single point analysis for TLR4 Asp299Gly (p = 0.89) or TLR4 Thr399Ile (p = 0.40). Similarly, no distortion in transmission was seen when the TLR4 haplotypes were studied (p = 0.54). Additionally, no evidence for gene-gene interaction between TLR4 polymorphisms and the previously associated MIF gene polymorphisms was found (p = 0.40). CONCLUSIONS: No linkage or association was seen for Asp299Gly or Thr399Ile SNPs of TLR4 with JIA susceptibility. No evidence of an epistatic interaction between these TLR4 polymorphisms and MIF polymorphisms was found.

Adolescent↗

PubMed2006

MyD88 and NOS2 are essential for toll-like receptor 4-mediated survival effect in cardiomyocytes.

Innate immune system such as Toll-like receptor 4 (TLR4) represents the first line of defense against infection. In addition to its pivotal role in host immunity, recent studies have suggested that TLR4 may play a broader role in mediating tissue inflammation and cell survival in response to noninfectious injury. We and other investigators have reported that cardiac TLR4 signaling is dynamically modulated in ischemic myocardium and that activation of TLR4 confers a survival benefit in the heart and in isolated cardiomyocytes. However, the signaling pathways leading to these effects are not completely understood. Here, we investigate the role of MyD88, an adaptor protein of TLR4 signaling, and inducible nitric oxide synthase (NOS2) in mediating TLR4-induced cardiomyocyte survival in an in vitro model of apoptosis. Serum deprivation induced a significant increase in the number of apoptotic cardiomyocytes as demonstrated by transferase-mediated dUTP nick-end labeling (TUNEL) assay, nuclear morphology, DNA laddering, and DNA-histone ELISA. Lipopolysaccharide (LPS), a TLR4 agonist, activated TLR4 signaling and led to significant reduction in apoptotic cardiomyocytes and improved cellular function of surviving cardiomyocytes with enhanced Ca(2+) transients and cell shortening. We found that both TLR4 and MyD88 are required for the LPS-induced beneficial effects as demonstrated by improved survival and function in wild-type but not in TLR4(-/-) or MyD88(-/-) cardiomyocytes. Moreover, genetic deletion or pharmacological inhibition of NOS2 abolished survival and functional rescue of cardiomyocytes treated with LPS. Taken together, these data suggest that TLR4 protects cardiomyocytes from stress-induced injury through MyD88- and NOS2-dependent mechanisms.

Adaptor Proteins, Signal Transducing↗