Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Signalling pathways modulated by the family of IL-1/TLR receptors are central to innate immune responses. Novel components playing key regulatory roles in these pathways continue to be isolated. Here we describe the use of autocatalytic vectors for identifying critical components of these signalling pathways. The method was tested with a vector system where cDNA clones are expressed as EGFP fusion proteins, or in an IRES containing mRNA, combined with a transcription reporter. These constructs are placed under the control of an inducible promoter, responsive to activation of TIR receptors such as IL-1RI or TLR-4. cDNAs which activate the promoter will, when transcribed, form a positive feedback loop. We introduced TIR signalling pathway components into both types of vectors. The components tested regulated reporter (EGFP/luciferase) expression in both cases. Our data suggest that this type of system is capable of selective identification of components from signal transduction pathways once the promoter of a relevant inducible gene is identified from, for example, micro-array experiments.
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PMID:Analysis of innate immune signal transduction with autocatalytic expression vectors. 1815 20

Intracellular heat shock protein 72 (Hsp72) is known to serve a broad cytoprotective role. Recent data indicate that stressed cells can release Hsp72 into the extracellular compartment, although the biological function of extracellular Hsp72 remains to be fully elucidated. Because extracellular Hsp72 has been demonstrated to interact with Toll-like receptor 4, we hypothesized that endogenously produced and released Hsp72 would reprogram the mononuclear cell responses to LPS. THP-1 cells treated with LPS were used as a model for nuclear factor (NF)-kappaB activation. Heat shock conditions consisted of incubation at 43 degrees C for 1 h. Control cells were incubated at 37 degrees C. Twenty four hours after incubation, heat shock conditioned media (HSCM) and control media (CM) were centrifuged, and the respective cells were discarded. A separate group of naive THP-1 cells were then incubated with either HSCM or CM for 18 h and then stimulated with LPS (1 mug/mL). Heat shock significantly increased Hsp72 in HSCM compared with CM. In THP-1 cells transfected with an NF-kappaB luciferase reporter plasmid, the addition of HSCM attenuated subsequent LPS-mediated luciferase activity compared with cells incubated in CM. The addition of HSCM also attenuated LPS-mediated NF-kappaB-DNA binding and IkappaBalpha degradation. Heat shock protein 72-mediated inhibition of NF-kappaB activation was further corroborated by a significant decrease in TNF-alpha production. When HSCM and CM were subjected to Hsp72 depletion via adenosine triphosphate-agarose binding, LPS-mediated activation of NF-kappaB was partially restored, suggesting that Hsp72 is partially responsible for cellular reprogramming in response to HSCM. These data demonstrate that endogenously produced and released extracellular Hsp72 has the ability to reprogram the in vitro response to endotoxin in cultured human mononuclear cells.
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PMID:The role of endogenously produced extracellular hsp72 in mononuclear cell reprogramming. 1832 37

Phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) is essential for the induction of promoters which contain the interferon-stimulated response element (ISRE). IRF3 can be activated by Toll-like receptor 3 (TLR3) in response to the double-stranded RNA mimic poly(I-C) and by TLR4 in response to lipopolysaccharide (LPS). Here we have analyzed the effect of the glucocorticoid dexamethasone on this response. Dexamethasone inhibited the induction of the ISRE-dependent gene RANTES (regulated on activation normal T cell expressed and secreted) in both U373-CD14 cells and human peripheral blood mononuclear cells and also an ISRE luciferase construct, activated by either TLR3 or TLR4. It also inhibited increased phosphorylation of IRF3 in its N terminus in response to LPS and in its C terminus on Ser-396 in response to either poly(I-C) or LPS. Several dexamethasone-induced phosphatases were tested for possible involvement in these effects; MKP1 did not appear to be involved, although MKP2 and MKP5 both partially inhibited induction of the ISRE, pointing to their possible involvement in the effect of dexamethasone. Importantly, we found that dexamethasone could inhibit TBK1 kinase activity and TBK1 phosphorylation on Ser-172, both of which are required for IRF3 phosphorylation downstream of TLR3 and TLR4 stimulation. Our study, therefore, demonstrates that TBK1 is a target for dexamethasone, common to both TLR3 and TLR4 signaling.
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PMID:Glucocorticoids inhibit IRF3 phosphorylation in response to Toll-like receptor-3 and -4 by targeting TBK1 activation. 1835 63

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG-ODN protect mice from infection with this fungal pathogen by inducing IFN-gamma. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH-S, an alveolar macrophage cell line, was stimulated with CpG-ODN in the presence of IFN-gamma. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH-S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-kappaB in a luciferase reporter assay with HEK293T cells, although the TLR4-mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG-ODN and IFN-gamma-stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.
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PMID:Cryptococcus neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucleotide-stimulated macrophages in a fashion independent of capsular polysaccharides. 1840 99

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-kappaB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.
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PMID:Recruitment and endo-lysosomal activation of TLR9 in dendritic cells infected with Trypanosoma cruzi. 1860 88

The present study was designed to elucidate the role of TLR2, TLR4 and dectin-1 in the production of IL-12p40 by bone marrow-derived dendritic cells (BM-DCs) infected with Penicillium marneffei. IL-12p40 production was almost completely abrogated in BM-DCs from TLR2 gene-knockout (KO) and MyD88KO mice, but not from TLR4-defective C3H/HeJ mice compared to those from control mice. Furthermore, BM-DCs from dectin-1KO mice faintly produced IL-12p40 upon stimulation with this fungus. Using a luciferase reporter assay, P. marneffei activated NF-kappaB in HEK293 cells transfected with the TLR2 gene, but not with the dectin-1 gene, and their co-transfection did not lead to further increase in this response. These results indicate that TLR2 and dectin-1 are essential in sensing P. marneffei for the activation of BM-DCs.
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PMID:Toll-like receptor 2 (TLR2) and dectin-1 contribute to the production of IL-12p40 by bone marrow-derived dendritic cells infected with Penicillium marneffei. 1865 8

High mobility group box protein 1 (HMGB1) modulates the innate immune response when present in the extracellular compartment. Receptors for HMGB1 include TLR4, TLR2, and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce LPS tolerance. HMGB1 dose-response experiments were performed on IFN-gamma-differentiated human monocyte-like THP-1 cells. Treatment with 1 microg/ml HMGB1 18 h before exposure to LPS (1 microg/ml) decreased TNF release, NF-kappaB nuclear DNA-binding activity, phosphorylation, and degradation of IkappaBalpha. Preconditioning with HMGB1 alone and HMGB1 in the presence of polymyxin B decreased LPS-mediated, NF-kappaB-dependent luciferase reporter gene expression. The specificity of HMGB1 in tolerance induction was supported further by showing that boiled HMGB1 failed to induce tolerance, and antibodies against HMGB1 blocked the induction of LPS tolerance. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type mice became LPS-tolerant following HMGB1 exposure ex vivo, but macrophages derived from RAGE-deficient mice failed to develop tolerance and responded normally to LPS. Mice preconditioned with HMGB1 (20 microg) 1 h before LPS injection (10 mg/kg) had lower circulating TNF compared with control mice preconditioned with saline vehicle. Similarly, decreased nuclear DNA binding of hepatic NF-kappaB was observed in mice preconditioned with HMGB1. Taken together, these results suggest that extracellular HMGB1 induces LPS tolerance, and the RAGE receptor is required for this induction.
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PMID:Preconditioning with high mobility group box 1 (HMGB1) induces lipopolysaccharide (LPS) tolerance. 1868 5

Toll-like receptor 4 (TLR4) is the key receptor for the LPS component of Gram-negative bacteria. We have identified a SNP within the 5' UTR region of the bovine TLR4 gene at position 226 (c.-226C>G) relative to start codon, which was found to be associated with health related traits in the Canadian Holstein population. TLR4 gene expression was measured over time intervals in samples collected from 18 cows (6 cows in each genotype) challenged in vitro with LPS (20 ng/ml) to determine if this SNP alters the gene expression. TLR4 gene expression was highest 6 h post LPS challenge, and cows with the GG genotype exhibited significantly higher expression compared to other genotypes. This was further validated by a dual luciferase reporter gene assay in which sequential progressive constructs were cloned into the pGL3 basic vector and transfected into bovine Mac-T cells. The reporter construct containing most of the 5' UTR (-456G) exhibited significantly higher luciferase activity compared to-456C, however, further inclusion of 5' upstream sequence showed a reversal phenomenon where the -913C construct exhibited significantly higher activity compared to -913G. These results indicate that the c.-226C>G SNP affects the expression of TLR4, suggesting that this SNP might be influencing the binding and interaction of transcription factors regulating gene expression.
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PMID:Functional characterization of a single nucleotide polymorphism in the 5' UTR region of the bovine toll-like receptor 4 gene. 1881 22

Conjunctival epithelial cells serve as a first line of defense against pathogens presented to the innate immune system. The inflammatory response to Gram-negative bacteria is initiated by toll-like receptor 4 (TLR4). This study investigated whether a TLR4 ligand induces production of inflammatory cytokines in human conjunctival epithelial cells (HCECs) through nuclear factor kappa-B (NF-kappaB). HCECs were evaluated for TLR4 expression by reverse transcriptase-polymerase chain reaction, Western blot analysis, and flow cytometric analysis. HCECs were stimulated with various concentrations of lipopolysaccharide (LPS) and the innate immune response was quantified by measuring expression of the inflammatory cytokines IL-6 and IL-8. Functional NF-kappaB activation was examined using a luciferase reporter assay. Expression of TLR4-specific mRNA as well as its corresponding protein was observed in HCECs. Surface and intracellular expression of TLR4 was observed in flow cytometric analysis. Incubation of HCECs with LPS led to secretion of IL-6 and IL-8. Blockade of TLR4- and TNFR-associated factor (TRAF) 6 activity abolished LPS-induced inflammatory response in HCECs and incubation of HCECs with LPS led to activation of the NF-kappaB transcription factor. LPS did not enhance the TLR4 expression at both mRNA and protein levels in HCECs. Our results demonstrate that surface expression of TLR4 in HCECs can elicit a TLR4-mediated innate immune response through TRAF6-NF-kappaB and contribute to an inflammatory environment on the ocular surface.
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PMID:Toll-like receptor 4 initiates an innate immune response to lipopolysaccharide in human conjunctival epithelial cells. 1895 93

Innate immune recognition of intracellular pathogens involves both extracellular and cytosolic surveillance mechanisms. The intracellular protozoan parasite Trypanosoma cruzi triggers a robust type I IFN response in both immune and nonimmune cell types. In this study, we report that signaling through TBK1 and IFN regulatory factor 3 is required for T. cruzi-mediated expression of IFN-beta. The TLR adaptors MyD88 and TRIF, as well as TLR4 and TLR3, were found to be dispensable, demonstrating that T. cruzi induces IFN-beta expression in a TLR-independent manner. The potential role for cytosolic dsRNA sensing pathways acting through RIG-I and MDA5 was ruled out because T. cruzi was shown to trigger robust expression of IFN-beta in macrophages lacking the MAVS/IPS1/VISA/CARDif adaptor protein. The failure of T. cruzi to activate HEK293-IFN-beta-luciferase cells, which are highly sensitive to cytosolic triggers of IFN-beta expression including Listeria, Sendai virus, and transfected dsRNA and dsDNA, further indicates that the parasite does not engage currently recognized cytosolic surveillance pathways. Together, these findings identify the existence of a novel TLR-independent pathogen-sensing mechanism in immune and nonimmune cells that converges on TBK1 and IFN regulatory factor 3 for activation of IFN-beta gene expression.
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PMID:A novel IFN regulatory factor 3-dependent pathway activated by trypanosomes triggers IFN-beta in macrophages and fibroblasts. 1901 82


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