Science.gov

Sample records for receptor tlr genes

  1. Characterization, expression analysis and localization pattern of toll-like receptor 1 (tlr1) and toll-like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella.

    PubMed

    He, L B; Wang, H; Luo, L F; Jiang, S H; Liu, L Y; Li, Y M; Huang, R; Liao, L J; Zhu, Z Y; Wang, Y P

    2016-08-01

    In this study, the toll-like receptor 1 (tlr1) and toll-like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up-regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells. PMID:27221024

  2. Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

    PubMed Central

    Cheng, Yuqiang; Sun, Yingjie; Wang, Hengan; Shi, Shuduan; Yan, Yaxian; Li, Jing

    2015-01-01

    Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-κB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation. PMID:25269719

  3. MAPPING OF TOLL LIKE RECEPTOR (TLR) GENES IN RAINBOW TROUT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize conserved pathogen structures to induce innate immune effector molecules. In vertebrates, TLRs can distinguish among classes of pathogens and serve an important role in orchestrating the appropriate adaptive immune resp...

  4. SNP identification, genetic mapping and tissue expression of the rainbow trout TLR9 gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes have been reported to be associated with disease resistance in human and livestock. A number of TLR genes have been identified in rainbow trout including TLR2, TLR3, TLR5, TLR20, TLR22 and TLR23. The rainbow trout (Oncorhynch...

  5. Cloning and expression analysis of a Toll-like receptor 22 (tlr22) gene from turbot, Scophthalmus maximus.

    PubMed

    Hu, Guo-Bin; Zhang, Shou-Feng; Yang, Xi; Liu, Da-Hai; Liu, Qiu-Ming; Zhang, Shi-Cui

    2015-06-01

    Toll-like receptor 22 (TLR22) exists exclusively in aquatic animals and recognizes double stranded RNA (dsRNA). In the present study, a tlr22 gene and its 5'-flanking sequence were cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (sm)tlr22 gene spans over 5.6 kb with a structure of 4 exon-3 intron and encodes 962 amino acids. The deduced protein shows the highest sequence identity (76.7%) to Japanese flounder Tlr22 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 27 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr22s. The interferon-stimulated response element (ISRE) and signal transducer and activator of transcription (STAT) binding site important for the basal transcriptional activity of TLR3 were predicted in the 5'-flanking sequence of smtlr22 gene. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr22 mRNA in all examined tissues with higher levels in the head kidney, kidney and spleen. Further, smtlr22 expression was significantly up-regulated following challenge with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) or turbot reddish body iridovirus (TRBIV) in the gills, head kidney, spleen and muscle, with maximum increases ranging from 2.56 to 6.24 fold upon different immunostimulants and organs. These findings suggest a possible role of Smtlr22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and Gram-negative bacteria. PMID:25770871

  6. SNP Marker Discovery in Koala TLR Genes

    PubMed Central

    Cui, Jian; Frankham, Greta J.; Johnson, Rebecca N.; Polkinghorne, Adam; Timms, Peter; O’Meally, Denis; Cheng, Yuanyuan; Belov, Katherine

    2015-01-01

    Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases. PMID:25799012

  7. SNP marker discovery in koala TLR genes.

    PubMed

    Cui, Jian; Frankham, Greta J; Johnson, Rebecca N; Polkinghorne, Adam; Timms, Peter; O'Meally, Denis; Cheng, Yuanyuan; Belov, Katherine

    2015-01-01

    Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases. PMID:25799012

  8. TLR2, TLR3, TLR4 and CD14 gene polymorphisms associated with oral lichen planus risk.

    PubMed

    Stanimirovic, Dragan; Zeljic, Katarina; Jankovic, Ljiljana; Magic, Marko; Hadzi-Mihajlovic, Milos; Magic, Zvonko

    2013-10-01

    The aim of this study was to assess whether polymorphisms in toll-like receptor (TLR) and cluster of differentiation 14 (CD14) genes are associated with oral lichen planus (OLP) risk and clinical course of the disease. The study group consisted of 101 patients with confirmed OLP and 104 healthy blood donors without systemic or oral mucosal diseases. Single nucleotide polymorphisms of TLR2 (rs3804099), TLR3 (rs3775291 and rs5743312), TLR4 (rs4986790 and rs4986791), and CD14 (rs2569190) genes were genotyped using real-time PCR or PCR-restriction fragment length polymorphism (PCR-RFLP). The rs5743312 TLR3 gene polymorphism was associated with increased OLP risk in comparison with the wild type genotype (OR = 15.984, P = 0.011). No association with OLP risk was observed for the polymorphisms studied in TLR2, TLR4 and CD14 genes or for the rs3775291 polymorphism of the TLR3 gene. The polymorphisms of the TLR3 gene were in linkage disequilibrium (D' = 1, r(2) = 0.1). Identified haplotypes were not associated with the risk of OLP. The findings of the current study suggest that the TT genotype of the rs5743312 TLR3 gene polymorphism may play a significant role in the aetiology of OLP. PMID:24028589

  9. Hypoxic stress: impact on the modulation of TLR2, TLR4, NOD1 and NOD2 receptor and their down-stream signalling genes expression in catla (Catla catla).

    PubMed

    Basu, Madhubanti; Paichha, Mahismita; Lenka, Saswati S; Chakrabarty, Rina; Samanta, Mrinal

    2016-01-01

    The damage-associated molecular patterns (DAMPs) released from the damaged tissue/cells are recently reported as endogenous ligands to activate toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors signaling pathways. In the aquatic environment, reduction in dissolved oxygen (DO) concentration causes hypoxic stress resulting in tissue damage and patho-biological changes in fish. We envisaged the critical role of TLR and NOD receptors in recognizing DAMPs as endogenous ligands during hypoxic stress in fish. Catla (Catla catla) fingerlings (avg. wt ~56 g) was exposed to hypoxic stress (DO: 1-3 mg/L) for 1 and 24 h. After the designated time course, total RNA was extracted from gill, liver, kidney and blood, and modulation of TLRs (TLR2 and TLR4), NOD (NOD1 and NOD2) receptors, MyD88 (myeloid differentiation primary response gene 88), RICK (receptor interacting serine-threonine protein kinase-2), interleukin (IL)-6, IL-8 and IL-10 gene expression were analyzed by quantitative reverse transcriptase PCR assay. Significant (p < 0.05) up-regulation of some DAMPs {high-mobility group box 1 and heat shock protein-70}, TLRs and NOD receptors genes expressions were observed in the hypoxic fish tissues as compared to the control. Further investigation revealed inductive expression of MyD88, RICK, IL-6, IL-8 and IL-10 genes in the TLRs and NODs activated tissues of the hypoxic fish. These data together may suggest the important role of TLRs and NOD receptors signaling pathway in sterile inflammation and pathobiology of fish in hypoxic stress, and warrant further study to investigate the role of TLR and NOD receptors in abiotic stress management in aquaculture. PMID:26590162

  10. [The role of TLR-2, TLR-3, TLR-4 genes polymorphism of grippe].

    PubMed

    2014-01-01

    Distribution and prognostic value of Arg753Gln TLR-2, Leu412Рhe TLR-3, Asp299Gly TLR-4 genes polymorphism in case of grippe have been studied in the present article. The examination has comprised 112 patients with grippe including 63 cases with uncomplicated course and 49 cases with grippe-associated pneumonia. The population control group has included 90 scarcely healthy persons to study Arg753Gln TLR-2 gene polymorphism and 80 ones to study Leu412Phe TLR-3 gene polymorphism. The conducted researches results have shown reliably higher frequency of heterozygous genotype Asp/Gly TLR-4 in patients with grippe (12,69%) and grippe-associated pneumonia (14,28%) compared with population control (3,33%). The frequency of homozygous genotype Phe/Phe TLR-3 in patients with grippe-associated pneumonia (18,37%) has exceeded the indices of patients with uncomplicated grippe (4,76%, р=0,02) and healthy persons (5,00%, р=0,03). Mutant genotypes combinations TLR-2, TLR-3, TLR-4 have been determined barely in patients with grippe and grippe-associated pneumonia with the frequency 11,11%-14,28%. It has been established that there is the increased risk of grippe development for persons with Asp/Gly TLR-4 genotype and TLR-2, TLR-3, TLR-4 mutant genotypes combinations; there is the increased risk of grippe-associated pneumonia for patients with mutant homozygous genotype Phe/Phe TLR-3. PMID:25214272

  11. Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Palti, Yniv; Gahr, Scott A; Purcell, Maureen K; Hadidi, Sima; Rexroad, Caird E; Wiens, Gregory D

    2010-02-01

    Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated

  12. Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

    USGS Publications Warehouse

    Palti, Yniv; Gahr, Scott A.; Purcell, Maureen K.; Hadidi, Sima; Rexroad, Caird E., III; Wiens, Gregory A.

    2010-01-01

    Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated

  13. Sequence characterization and phylogenetic analysis of Toll-like receptor (TLR) 4 gene in the Tibetan macaque (Macaca thibetana).

    PubMed

    Dai, Q X; Yao, Y F; Qi, Z C; Huang, Y; Ni, Q Y; Zhang, M W; Xu, H L

    2015-01-01

    In this study, the complete coding region sequence of an innate immune-related TLR4 gene was obtained from the Tibetan macaque (Macaca thibetana) genome via PCR and direct sequencing. The sequence had a total length of 2481 bp, contained 3 complete exons, and encoded 826 amino acids (AAs); its isoelectric point was 5.703, and the molecular weight was 94.72 kDa. The high structure prediction showed that the protein was comprised of one extracellular region, one transmembrane region, and one intracellular region. There were 48 potential functional sites in the protein, including glycosylation, phosphorylation, and acetylation sites. A homology analysis among 9 primate species, including the Tibetan macaque, human, chimpanzee, gibbon, rhesus macaque, cynomolgus monkey, pig-tailed monkey, squirrel monkey, and small-eared galago, showed that the homology of the nucleotide and AA sequences ranged from 60.9-99.5% and 51.4- 99.0%, respectively. Higher variability was identified in the extracellular region of the TLR4 protein, and its variable sites accounted for 88.79% (AA) of the total variable sites. Additionally, the number of AAs at the 3' end of the intracellular region was notably different among the primate lineages. The phylogenetic tree based on TLR4 gene exons of 9 primate species showed that the Tibetan macaque clustered with the rhesus macaque, cynomolgus monkey, and pig-tailed monkey; it was most distant from the small-eared galago. This study will provide an important basis for further study on the expression, regulation, and polymorphism of the TLR4 gene and the relationship between polymorphisms and host disease susceptibility. PMID:25867333

  14. Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effector nuclease- (TALEN)-mediated gene inactivation

    PubMed Central

    Ferguson, Carolyn; McKay, Matthew; Harris, R. Adron; Homanics, Gregg E.

    2013-01-01

    Genetically engineered mice are a valuable resource for studies of the behavioral effects of ethanol. However, for some behavioral tests of ethanol action, the rat is a superior model organism. Production of genetically engineered rats has been severely hampered due to technical limitations. Here we utilized a promising new technique for efficient site-specific gene modification to create a novel gene knockout rat line. This approach is based on Transcriptional Activator-Like Effector Nucleases (TALENs). TALENs function in pairs and bind DNA in a sequence-specific manner. Upon binding to the target sequence, a functional nuclease is reconstituted that creates double-stranded breaks in the DNA that are efficiently repaired by non-homologous end joining. This error-prone process often results in deletions of varying lengths at the targeted locus. The toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and neurodegeneration, as well as multiple ethanol-induced behavioral effects. To generate Tlr4 knockout rats, a pair of TALEN constructs was created that specifically target Exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. The mutated allele consisted of a 13 base-pair deletion that was predicted to create a frameshift mutation after amino acid 25. This founder rat successfully transmitted the mutation to F1 offspring. Heterozygous F1 offspring were interbred to produce homozygous F2 animals. Homozygous mutants expressed the 13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. Thus, the mutant Tlr4 allele generated by TALEN-mediated gene

  15. Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9.

    PubMed

    Keestra, A Marijke; de Zoete, Marcel R; Bouwman, Lieneke I; van Putten, Jos P M

    2010-07-01

    TLRs comprise a family of evolutionary conserved sensory receptors that respond to distinct classes of ligands. For one major evolutionary branch of TLRs, the ligands are still largely unknown. Here we report the cloning and function of one member of this group, chicken TLR21 (chTLR21). This TLR is absent in the human species but has homologs in fish and frog and displays similarity with mouse TLR13. Expression of chTLR21 in HEK293 cells resulted in activation of NF-kappaB in response to unmethylated CpG DNA, typically recognized by mammalian TLR9. Silencing of chTLR21 (but not chTLR4) in chicken macrophages inhibited the response to CpG-DNA (but not to LPS), indicating similar functionality of the endogenous receptor. ChTLR21 responded to human- and murine-specific TLR9 ligands, as well as to bacterial genomic DNA isolated from Salmonella enterica serovar Enteritidis. Confocal microscopy located chTLR21 in the same intracellular compartments as human TLR9. Inhibition of the chTLR21 response by the endosomal maturation inhibitor chloroquine suggested that the receptor is functional in endolysosomes, as known for TLR9. The analogous localization and function of the phylogenetically only distantly related chTLR21 and mammalian TLR9 suggest that during evolution different classes of TLRs have emerged that recognize the same type of ligands. PMID:20498358

  16. Deletion of scavenger receptor A gene in mice resulted in protection from septic shock and modulation of TLR4 signaling in isolated peritoneal macrophages

    PubMed Central

    Drummond, Robert; Cauvi, David M; Hawisher, Dennis; Song, Donghuan; Niño, Diego F; Coimbra, Raul; Bickler, Stephen; De Maio, Antonio

    2015-01-01

    Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(−/−) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(−/−) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMϕs) were isolated from Sra(−/−) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMϕs isolated from B6 mice. No differences were found in LPS binding between PMϕs derived from Sra(−/−) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity. PMID:22751446

  17. Plasma cell toll-like receptor (TLR) expression differs from that of B cells, and plasma cell TLR triggering enhances immunoglobulin production.

    PubMed

    Dorner, Marcus; Brandt, Simone; Tinguely, Marianne; Zucol, Franziska; Bourquin, Jean-Pierre; Zauner, Ludwig; Berger, Christoph; Bernasconi, Michele; Speck, Roberto F; Nadal, David

    2009-12-01

    Toll-like receptors (TLRs) are key receptors of the innate immune system and show cell subset-specific expression. We investigated the messenger RNA (mRNA) expression of TLR genes in human haematopoietic stem cells (HSC), in naïve B cells, in memory B cells, in plasma cells from palatine tonsils and in plasma cells from peripheral blood. HSC and plasma cells showed unrestricted expression of TLR1-TLR9, in contrast to B cells which lacked TLR3, TLR4 and TLR8 but expressed mRNA of all other TLRs. We demonstrated, for the first time, that TLR triggering of terminally differentiated plasma cells augments immunoglobulin production. Thus, boosting the immediate antibody response by plasma cells upon pathogen recognition may point to a novel role of TLRs. PMID:19950420

  18. Differential adapter recruitment by TLR2 co-receptors.

    PubMed

    Piao, Wenji; Ru, Lisa W; Toshchakov, Vladimir Y

    2016-07-01

    TLR2 heterodimers with TLR1 or TLR6 recognize distinct pathogen-associated molecules such as tri- and di-acylated lipopeptides. The activated TLR2 heterodimers recruit Toll-IL-1R domain- (TIR-) containing adapter proteins, TIRAP and MyD88, through the receptor TIR domains. Molecular recognition mechanisms responsible for agonist-driven, TIR domain-mediated receptor-adapter interactions as well as the structure of resultant signaling complexes remain unknown. We previously reported that the cell-permeable peptide derived from helix D of TLR2 TIR (2R9) specifically binds TIRAP in vitro and in cells and thereby inhibits TIRAP-dependent TLR signaling. This study demonstrates that cell-permeable peptides from D helix of TLR1 or TLR6, peptides 1R9 and 6R9 respectively, inhibit signaling mediated by cognate TLR2 co-receptors. Interestingly, 1R9 and 6R9 bind different TLR2 adapters, as they selectively bind MyD88 and TIRAP TIR, respectively. Both peptides block the agonist-induced co-immunoprecipitation (co-IP) of TLR2 with TIRAP or MyD88, but not TLR2 co-IP with co-receptors. Our data suggest that D helices of TLR1 and TLR6 TIR domains are adapter recruitment sites in both co-receptors; yet the sites recruit different adapters. The D helix in TLR1 is the MyD88 docking site, whereas in TLR6 this site recruits TIRAP. PMID:27150837

  19. Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behçet disease.

    PubMed

    Kirino, Yohei; Zhou, Qing; Ishigatsubo, Yoshiaki; Mizuki, Nobuhisa; Tugal-Tutkun, Ilknur; Seyahi, Emire; Özyazgan, Yilmaz; Ugurlu, Serdal; Erer, Burak; Abaci, Neslihan; Ustek, Duran; Meguro, Akira; Ueda, Atsuhisa; Takeno, Mitsuhiro; Inoko, Hidetoshi; Ombrello, Michael J; Satorius, Colleen L; Maskeri, Baishali; Mullikin, James C; Sun, Hong-Wei; Gutierrez-Cruz, Gustavo; Kim, Yoonhee; Wilson, Alexander F; Kastner, Daniel L; Gül, Ahmet; Remmers, Elaine F

    2013-05-14

    Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behçet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were evaluated for BD association. A differential distribution of the rare and low-frequency NSVs of a gene in 2,461 BD cases compared with 2,458 controls indicated their collective association with disease. By stringent criteria requiring at least a single burden test with study-wide significance and a corroborating test with at least nominal significance, rare and low-frequency NSVs in one GWAS-identified gene, IL23R (P = 6.9 × 10(-5)), and one gene involved in innate immunity, TLR4 (P = 8.0 × 10(-4)), were associated with BD. In addition, damaging or rare damaging NOD2 variants were nominally significant across all three burden tests applied (P = 0.0063-0.045). Furthermore, carriage of the familial Mediterranean fever gene (MEFV) mutation Met694Val, which is known to cause recessively inherited familial Mediterranean fever, conferred BD risk in the Turkish population (OR, 2.65; P = 1.8 × 10(-12)). The disease-associated NSVs in MEFV and TLR4 implicate innate immune and bacterial sensing mechanisms in BD pathogenesis. PMID:23633568

  20. Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE)

    PubMed Central

    2013-01-01

    Background In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs). Results We sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini

  1. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

    PubMed Central

    Chen, Nai-Yu; Nagarajan, Govindarajulu; Chiou, Pinwen Peter

    2015-01-01

    Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish. PMID:25955250

  2. Association of TLR and TREM-1 gene polymorphisms with atherosclerosis severity in a Russian population

    PubMed Central

    Kutikhin, Anton G.; Ponasenko, Anastasia V.; Khutornaya, Maria V.; Yuzhalin, Arseniy E.; Zhidkova, Irina I.; Salakhov, Ramil R.; Golovkin, Alexey S.; Barbarash, Olga L.; Barbarash, Leonid S.

    2016-01-01

    Local vascular immune response is primarily initiated via Toll-like receptors (TLRs) and triggering receptor expressed on myeloid cells-1 (TREM-1). We previously showed that certain TLR and TREM-1 gene polymorphisms are associated with coronary artery disease (CAD). Therefore, we hypothesized that these gene polymorphisms are associated with atherosclerosis severity. This study included 292 consecutive patients with CAD who were admitted to the Research Institute for Complex Issues of Cardiovascular Diseases (Kemerovo, Russian Federation) during 2011–2012. Sample genotyping was performed in 96-well format using the TaqMan SNP genotyping assay. We found that C/C genotype of the rs3804099 polymorphism within TLR2 gene and T/T genotype of the rs4711668 polymorphism within TREM-1 gene were significantly associated with severe coronary atherosclerosis while C allele of the rs5743551 polymorphism within TLR1 gene, A/G genotype of the rs4986790 polymorphism and C/T genotype of the rs4986791 polymorphism within TLR4 gene, and C allele of the rs3775073 polymorphism within TLR6 gene were significantly associated with severe noncoronary atherosclerosis. However, A/A genotype of the rs5743810 polymorphism within TLR6 gene was significantly associated with mild noncoronary atherosclerosis. We conclude that certain TLR and TREM-1 gene polymorphisms are significantly associated with atherosclerosis severity in a Russian population. PMID:27200266

  3. Association of TLR and TREM-1 gene polymorphisms with atherosclerosis severity in a Russian population.

    PubMed

    Kutikhin, Anton G; Ponasenko, Anastasia V; Khutornaya, Maria V; Yuzhalin, Arseniy E; Zhidkova, Irina I; Salakhov, Ramil R; Golovkin, Alexey S; Barbarash, Olga L; Barbarash, Leonid S

    2016-09-01

    Local vascular immune response is primarily initiated via Toll-like receptors (TLRs) and triggering receptor expressed on myeloid cells-1 (TREM-1). We previously showed that certain TLR and TREM-1 gene polymorphisms are associated with coronary artery disease (CAD). Therefore, we hypothesized that these gene polymorphisms are associated with atherosclerosis severity. This study included 292 consecutive patients with CAD who were admitted to the Research Institute for Complex Issues of Cardiovascular Diseases (Kemerovo, Russian Federation) during 2011-2012. Sample genotyping was performed in 96-well format using the TaqMan SNP genotyping assay. We found that C/C genotype of the rs3804099 polymorphism within TLR2 gene and T/T genotype of the rs4711668 polymorphism within TREM-1 gene were significantly associated with severe coronary atherosclerosis while C allele of the rs5743551 polymorphism within TLR1 gene, A/G genotype of the rs4986790 polymorphism and C/T genotype of the rs4986791 polymorphism within TLR4 gene, and C allele of the rs3775073 polymorphism within TLR6 gene were significantly associated with severe noncoronary atherosclerosis. However, A/A genotype of the rs5743810 polymorphism within TLR6 gene was significantly associated with mild noncoronary atherosclerosis. We conclude that certain TLR and TREM-1 gene polymorphisms are significantly associated with atherosclerosis severity in a Russian population. PMID:27200266

  4. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  5. Identification and functional characterization of three TLR signaling pathway genes in Cyclina sinensis.

    PubMed

    Ren, Yipeng; Pan, Heting; Pan, Baoping; Bu, Wenjun

    2016-03-01

    Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play a critical role in initiating and activating the innate immune system. In this study, we identified two TLR genes (CsTLR4 and CsTLR13) and the MyD88 (CsMyD88) gene using a transcriptome library from Cyclina sinensis. The sequence features and mRNA expression profiles of the genes were characterized, and their functions in the immune response were investigated to validate the TLR signaling pathway and its potential role in immune defense. The expression patterns of CsTLR4, CsTLR13 and CsMyD88 were detected in all the tissues examined from healthy clams and were primarily expressed in the hemocytes (P < 0.05), as shown by real-time PCR. Upon challenge with Vibrio anguillarum and Micrococcus luteus, they were significantly increased in hemocytes (P < 0.01), whereas only CsTLR13 and CsMyD88 were up-regulated (P < 0.01) by poly (I:C) challenge. In addition, the mRNA expression level of CsC-LYZ and CsAMP was down-regulated at 72 h (P < 0.01) after injection with CsMyD88 RNAi. These findings might be valuable for understanding the innate immune signaling pathways of C. sinensis and enabling future studies on host-pathogen interactions. PMID:26804650

  6. Donor TLR9 gene tagSNPs influence susceptibility to aGVHD and CMV reactivation in the allo-HSCT setting without polymorphisms in the TLR4 and NOD2 genes.

    PubMed

    Xiao, H W; Luo, Y; Lai, X Y; Shi, J M; Tan, Y M; He, J S; Xie, W Z; Zheng, W Y; Ye, X J; Yu, X H; Cai, Z; Lin, M F; Huang, H

    2014-02-01

    Owing to ethnicity of the population, those best confirmed polymorphisms in the TLR (toll-like receptor)4 and NOD2 genes with significantly prognostic impact on allogeneic hematopoietic SCT (allo-HSCT) seem to be more applicable to Europeans and are nonpolymorphic in the Asian population. The influence of innate immunity gene polymorphisms on the outcomes of allo-HSCT in those populations has been questioned. We evaluated the influence of 10 candidate single nucleotide polymorphisms (SNPs) in the TLR1, TLR2, TLR3, TLR8 and TLR9 genes on the outcomes of allo-HSCT in a Chinese population including 138 pairs of patients and unrelated donors and a second cohort of 102 pairs of patients and HLA-identical sibling donors. We found that two tagSNPs in the TLR9 gene in the donor side, +1174 A/G (rs352139) and +1635 C/T (rs352140), influenced the risk of acute GVHD (aGVHD) and CMV reactivation. Furthermore, the presence of the susceptible haplotype (A-C) in donor may be an informative predicator of worse OS at 5 years compared with those with the G-C and G-T haplotypes (58% vs 82.9%, P=0.024). Our data suggested an unrecognized association between donor TLR9 tagSNPs and the risk of HSCT-related complications in a population without polymorphisms in the TLR4 and NOD2 genes. PMID:24121213

  7. Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

    PubMed Central

    Fisher, Colleen A.; Bhattarai, Eric K.; Osterstock, Jason B.; Dowd, Scot E.; Seabury, Paul M.; Vikram, Meenu; Whitlock, Robert H.; Schukken, Ynte H.; Schnabel, Robert D.; Taylor, Jeremy F.; Womack, James E.; Seabury, Christopher M.

    2011-01-01

    Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several

  8. Association between TLR4 and TLR9 Gene Polymorphisms with Development of Pulmonary Tuberculosis in Zahedan, Southeastern Iran

    PubMed Central

    Jahantigh, Danial; Salimi, Saeedeh; Alavi-Naini, Roya; Emamdadi, Abolfazl; Owaysee Osquee, Hamid; Farajian Mashhadi, Farzaneh

    2013-01-01

    Some evidence suggests that a variety of genetic factors contribute to development of the tuberculosis (TB). TLR4 and TLR9 have been proposed as susceptibility genes for TB. This study was performed in 124 newly diagnosed TB cases and 149 healthy controls in a TB-endemic region of Iran. The TLR4 genes Asp299Gly, Thr399Ile, and TLR9 gene T-1486C polymorphisms were amplified by polymerase chain reaction (PCR) and then detected by PCR-restriction fragment length polymorphism (RFLP). The frequencies of the mutant alleles of TLR4 Arg299Gly, Thr399Ile, and TLR9 T-1486C polymorphisms were 0.8 versus 0.1, 5.6 versus 3, and 28.6 versus 25.2 in patients and controls, respectively, that were not significant. The synergic effect of TI,II/CC genotypes for TLR4 Thr399Ile and TLR9 T-1486C polymorphisms showed increased risk of PTB susceptibility. In conclusion, no significant relation was found between TLR4 and TLR9 polymorphisms alone and PTB. However, synergic effects of TLR4 Thr399Ile and TLR9-1486T/C polymorphisms might increase risk of PTB. PMID:23766695

  9. Acanthamoeba infection in lungs of mice expressed by toll-like receptors (TLR2 and TLR4).

    PubMed

    Derda, Monika; Wojtkowiak-Giera, Agnieszka; Kolasa-Wołosiuk, Agnieszka; Kosik-Bogacka, Danuta; Hadaś, Edward; Jagodziński, Paweł P; Wandurska-Nowak, Elżbieta

    2016-06-01

    Toll-like receptors (TLRs) play a key role in the innate immune responses to a variety of pathogens including parasites. TLRs are among the most highly conserved in the evolution of the receptor family, localized mainly on cells of the immune system and on other cells such as lung cells. The aim of this study was to determine for the first time the expression of TLR2 and TLR4 in the lung of Acanthamoeba spp. infected mice using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemical (IHC) staining. The Acanthamoeba spp. were isolated from a patient with Acanthamoeba keratitis (AK) (strain Ac 55) and from environmental samples of water from Malta Lake (Poznań, Poland - strain Ac 43). We observed a significantly increased level of expression of TLR2 as well as TLR4 mRNA from 2 to 30 days post Acanthamoeba infection (dpi) in the lungs of mice infected with Ac55 (KP120880) and Ac43 (KP120879) strains. According to our observations, increased TLR2 and TLR4 expression in the pneumocytes, interstitial cells and epithelial cells of the bronchial tree may suggest an important role of these receptors in protective immunity against Acanthamoeba infection in the lung. Moreover, increased levels of TLR2 and TLR4 mRNA expression in infected Acanthamoeba mice may suggest the involvement of these TLRs in the recognition of this amoeba pathogen-associated molecular pattern (PAMP). PMID:26940205

  10. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression.

    PubMed

    Le, Hai Van; Kim, Jae Young

    2016-01-01

    Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C-C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10. PMID:27258267

  11. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

    PubMed Central

    Le, Hai Van; Kim, Jae Young

    2016-01-01

    Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C–C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10. PMID:27258267

  12. Identification and characterisation of TLR18-21 genes in Atlantic salmon (Salmo salar).

    PubMed

    Lee, P T; Zou, J; Holland, J W; Martin, S A M; Collet, B; Kanellos, T; Secombes, C J

    2014-12-01

    Teleost fish possess many types of toll-like receptor (TLR) some of which exist in other vertebrate groups and some that do not (ie so-called "fish-specific" TLRs). In this study, we identified in Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs seven TLRs that are not found in mammals, including six types of fish-specific TLRs (one TLR18, one TLR19, and four TLR20 members (two of which are putative soluble forms (s)) and one TLR21. Phylogenetic analysis revealed that teleost TLR19-21 are closely related with murine TLR11-TLR13, whilst teleost TLR18 groups with mammalian TLR1, 2, 6 and 10. A typical TLR protein domain structure was found in all these TLRs with the exception of TLR20b(s) and TLR20c(s). TLR-GFP expression plasmids transfected into SHK-1 cells showed that salmon TLR19, TLR20a and TLR20d were preferentially localised to the intracellular compartment. Real time PCR analysis suggested that salmon TLR19-TLR21 are mainly expressed in immune related organs, such as spleen, head kidney and gills, while TLR18 transcripts are more abundant in muscle. In vitro stimulation of primary head kidney cells with type I IFN, IFNγ and IL-1β had no impact on TLR expression. Infectious salmon anaemia virus (ISAV) infection, in vivo, down-regulated TLR20a, TLR20b(s), TLR20d and TLR21 in infected salmon kidney tissue. In contrast, up-regulation of TLR19 and TLR20a expression was found in posterior kidney in rainbow trout with clinical proliferative kidney disease (PKD). PMID:25450999

  13. Association between TLR2 and TLR4 Gene Polymorphisms and the Susceptibility to Inflammatory Bowel Disease: A Meta-Analysis

    PubMed Central

    Huang, Xiuping; Zhang, Wei; Han, Zelong; Liu, Side

    2015-01-01

    Background The associations between toll-like receptor 2 (TLR2) and toll-like receptor 4(TLR4) polymorphisms and inflammatory bowel disease (IBD) susceptibility remain controversial. A meta-analysis was performed to assess these associations. Methods A systematic search was performed to identify all relevant studies relating TLR2 and TLR4 polymorphisms and IBD susceptibility. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Subgroup analyses were performed by ethnicity and publication quality. Results Thirty-eight eligible studies, assessing 10970 cases and 7061 controls were included. No TLR2 Arg677Trp polymorphism was found. No significant association was observed between TLR2 Arg753Gln polymorphism and Crohn’s disease (CD) or ulcerative colitis (UC) in all genetic models. Interestingly, TLR4 Asp299Gly polymorphism was significantly associated with increased risk of CD and UC in all genetic models, except for the additive one in CD. In addition, a statistically significant association between TLR4 Asp299Gly polymorphism and IBD was observed among high quality studies evaluating Caucasians, but not Asians. Associations between TLR4 Thr399Ile polymorphisms and CD risk were found only in the allele and dominant models. The TLR4 Thr399Ile polymorphism was associated with UC risk in pooled results as well as subgroup analysis of high quality publications assessing Caucasians, in allele and dominant models. Conclusions The meta-analysis provides evidence that TLR2 Arg753Gln is not associated with CD and UC susceptibility in Asians; TLR4 Asp299Gly is associated with CD and UC susceptibility in Caucasians, but not Asians. TLR4 Thr399Ile may be associated with IBD susceptibility in Caucasians only. Additional well-powered studies of Asp299Gly and other TLR4 variants are warranted. PMID:26023918

  14. No evidence of major effects in several Toll-like receptor gene polymorphisms in rheumatoid arthritis

    PubMed Central

    Jaen, Olivier; Petit-Teixeira, Elisabeth; Kirsten, Holger; Ahnert, Peter; Semerano, Luca; Pierlot, Céline; Cornelis, Francois; Boissier, Marie-Christophe; Falgarone, Geraldine

    2009-01-01

    Introduction The objective was to study the potential genetic contribution of Toll-like receptor (TLR) genes in rheumatoid arthritis (RA). TLRs bind to pathogen-associated molecular patterns, and TLR genes influence both proinflammatory cytokine production and autoimmune responses. Host–pathogen interactions are involved in RA physiopathology. Methods We tested SNPs of five TLR genes (TLR9, TLR2, TLR6, TLR1, and TLR4) in a cohort of 100 French families with RA. Genotypes were analyzed using the transmission disequilibrium test. As TLR2, TLR6, and TLR1 are located on chromosome 4, we determined the haplotype relative risk. Analyses were performed in subgroups defined by status for rheumatoid factor, anti-cyclic citrullinated peptide autoantibodies, and erosions. Results We found no disequilibrium in allele transmission for any of the SNPs of the five TLR genes. In subgroup analyses, no associations were detected linking TLR9, TLR2, or TLR9/TLR2 to rheumatoid factor, anti-cyclic citrullinated peptide autoantibodies, or erosions. Haplotype analysis of the polymorphisms showed no haplotype associations in any of the subgroups. Conclusions We found no evidence of major effects of TLR gene polymorphisms in RA, although we tested different TLR phenotypes. Moreover, no associations were noted with autoantibody production or erosions. PMID:19134200

  15. TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

    PubMed Central

    Xu, Jia; Benabou, Kelly; Cui, Xiangdong; Madia, Marissa; Tzeng, Edith; Billiar, Timothy; Watkins, Simon; Sachdev, Ulka

    2015-01-01

    Toll-like receptors (TLRs) play an important role in regulating muscle regeneration and angiogenesis in response to ischemia. TLR2 knockout mice exhibit pronounced skeletal muscle necrosis and abnormal vessel architecture after femoral artery ligation, suggesting that TLR2 signaling is protective during ischemia. TLR4, an important receptor in inflammatory signaling, has been shown to regulate TLR2 expression in other systems. We hypothesize that a similar relationship between TLR4 and TLR2 may exist in hindlimb ischemia in which TLR4 upregulates TLR2, a mediator of angiogenesis and perfusion recovery. We examined the expression of TLR2 in unstimulated and in TLR-agonist treated endothelial cells (ECs). TLR2 expression (low in control ECs) was upregulated by lipopolysaccharide, the danger signal high mobility group box-1, and hypoxia in a TLR4-dependent manner. Endothelial tube formation on Matrigel as well as EC permeability was assessed as in vitro measures of angiogenesis. Time-lapse imaging demonstrated that ECs lacking TLR4 formed more tubes, whereas TLR2 knockdown ECs exhibited attenuated tube formation. TLR2 also mediated EC permeability, an initial step during angiogenesis, in response to high-mobility group box-1 (HMGB1) that is released by cells during hypoxic injury. In vivo, ischemia-induced upregulation of TLR2 required intact TLR4 signaling that mediated systemic inflammation, as measured by local and systemic IL-6 levels. Similar to our in vitro findings, vascular density and limb perfusion were both enhanced in the absence of TLR4 signaling, but not if TLR2 was deleted. These findings indicate that TLR2, in the absence of TLR4, improves angiogenesis and perfusion recovery in response to ischemia. PMID:26181630

  16. Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance.

    PubMed

    Murphy, Michael; Xiong, Yanbao; Pattabiraman, Goutham; Qiu, Fu; Medvedev, Andrei E

    2015-07-31

    Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and

  17. Heterogeneous functional effects of concomitant B cell receptor and TLR stimulation in chronic lymphocytic leukemia with mutated versus unmutated Ig genes.

    PubMed

    Chatzouli, Maria; Ntoufa, Stavroula; Papakonstantinou, Nikos; Chartomatsidou, Elisavet; Anagnostopoulos, Achilles; Kollia, Panagoula; Ghia, Paolo; Muzio, Marta; Stamatopoulos, Kostas; Belessi, Chrysoula

    2014-05-15

    We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression, function, and/or tolerance. In this study, to explore whether specific types of BCR/TLR collaboration exist in CLL, we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM, imiquimod, and CpG oligodeoxynucleotide for BCR, TLR7, and TLR9, respectively, alone or in combination for different time points. After in vitro culture in the absence of stimulation, differences in p-ERK were identified at any time point, with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL, whereas BCR/TLR synergism was required in M-CLL, in which the effect was overall limited in scale. An opposite pattern was observed regarding induction of apoptosis, as studied by Western blotting for the cleaved fragment of poly(ADP-ribose) polymerase, and the active isoform of caspase-8, with M-CLL responding even to single stimulation, contrasting with U-CLL that showed minimal response. Our findings suggest that concomitant engagement of BCR and TLR leads to differential responses in CLL depending on the mutational status of the BCR. Differential intensity and duration of responses in M-CLL versus U-CLL indicates that the differences in signal transduction between the two subgroups may be primarily quantitative rather than qualitative. PMID:24719462

  18. Metalloproteinase-dependent TLR2 ectodomain shedding is involved in soluble toll-like receptor 2 (sTLR2) production.

    PubMed

    Langjahr, Patricia; Díaz-Jiménez, David; De la Fuente, Marjorie; Rubio, Estefhany; Golenbock, Douglas; Bronfman, Francisca C; Quera, Rodrigo; González, María-Julieta; Hermoso, Marcela A

    2014-01-01

    Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14+ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders. PMID:25531754

  19. Metalloproteinase-Dependent TLR2 Ectodomain Shedding is Involved in Soluble Toll-Like Receptor 2 (sTLR2) Production

    PubMed Central

    Langjahr, Patricia; Díaz-Jiménez, David; De la Fuente, Marjorie; Rubio, Estefhany; Golenbock, Douglas; Bronfman, Francisca C.; Quera, Rodrigo; González, María-Julieta; Hermoso, Marcela A.

    2014-01-01

    Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14+ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders. PMID:25531754

  20. Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution.

    PubMed

    Wu, Baojun; Huan, Tianxiao; Gong, Jing; Zhou, Pin; Bai, Zengliang

    2011-12-01

    Domain shuffling, which is an important mechanism in the evolution of multi-domain proteins, has shaped the evolutionary development of the immune system in animals. Toll and Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate and adaptive immune systems. Draft genome sequences provide the opportunity to compare the Toll/TLR gene repertoire among representative metazoans. In this study, we investigated the combination of Toll/interleukin-1 receptor (TIR) and leucine-rich repeat (LRR) domains of metazoan Toll/TLRs. Before Toll with both domains occurred in Cnidaria (sea anemone, Nematostella vectensis), through domain combinations, TIR-only and LRR-only proteins had already appeared in sponges (Amphimedon queenslandica). Although vertebrate-like TIR (V-TIR) domain already appeared in Cnidaria, the vertebrate-like TLR (V-TLR) with both domains appeared much later. The first combination between V-TIR domain and vertebrate-like LRR (V-LRR) domain for V-TLR may have occurred after the divergence of Cnidaria and bilateria. Then, another combination for V-TLR, a recombination of both domains, possibly occurred before or during the evolution of primitive vertebrates. Taken together, two rounds of domain combinations may thus have co-shaped the vertebrate TLRs. PMID:22227927

  1. Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus

    PubMed Central

    Wong-Baeza, Carlos; Tescucano, Alonso; Astudillo, Horacio; Reséndiz, Albany; Landa, Carla; España, Luis; Serafín-López, Jeanet; Estrada-García, Iris; Estrada-Parra, Sergio; Flores-Romo, Leopoldo; Wong, Carlos; Baeza, Isabel

    2015-01-01

    Systemic lupus erythematosus is characterized by dysregulated activation of T and B cells and autoantibodies to nuclear antigens and, in some cases, lipid antigens. Liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice, including IgM and IgG antibodies against nonbilayer phospholipid arrangements. As the effect of these liposomes on the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation, we evaluated the effect of these liposomes on Toll-like receptor (TLR) signaling, cytokine production, proinflammatory gene expression, and T, NKT, dendritic, and B cells. Liposomes induce TLR-4- and, to a lesser extent, TLR-2/TLR-6-dependent signaling in TLR-expressing human embryonic kidney (HEK) cells and bone marrow-derived macrophages. Mice with the lupus-like disease had increased serum concentrations of proinflammatory cytokines, C3a and C5a; they also had more TLR-4-expressing splenocytes, a higher expression of genes associated with TRIF-dependent TLR-4-signaling and complement activation, and a lower expression of apoptosis-related genes, compared to healthy mice. The percentage of NKT and the percentage and activation of dendritic and B2 cells were also increased. Thus, TLR-4 and TLR-2/TLR-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus-like disease in mice. PMID:26568960

  2. Role of the Toll Like receptor (TLR) radical cycle in chronic inflammation: possible treatments targeting the TLR4 pathway.

    PubMed

    Lucas, Kurt; Maes, Michael

    2013-08-01

    Activation of the Toll-like receptor 4 (TLR4) complex, a receptor of the innate immune system, may underpin the pathophysiology of many human diseases, including asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation. TLRs are pattern recognition receptors that recognize damage-associated molecular patterns and pathogen-associated molecular patterns, including lipopolysaccharide (LPS) from gram-negative bacteria. Here we focus on the environmental factors, which are known to trigger TLR4, e.g., ozone, atmosphere particulate matter, long-lived reactive oxygen intermediate, pentachlorophenol, ionizing radiation, and toluene. Activation of the TLR4 pathways may cause chronic inflammation and increased production of reactive oxygen and nitrogen species (ROS/RNS) and oxidative and nitrosative stress and therefore TLR-related diseases. This implies that drugs or substances that modify these pathways may prevent or improve the abovementioned diseases. Here we review some of the most promising drugs and agents that have the potential to attenuate TLR-mediated inflammation, e.g., anti-LPS strategies that aim to neutralize LPS (synthetic anti-LPS peptides and recombinant factor C) and TLR4/MyD88 antagonists, including eritoran, CyP, EM-163, epigallocatechin-3-gallate, 6-shogaol, cinnamon extract, N-acetylcysteine, melatonin, and molecular hydrogen. The authors posit that activation of the TLR radical (ROS/RNS) cycle is a common pathway underpinning many "civilization" disorders and that targeting the TLR radical cycle may be an effective method to treat many inflammatory disorders. PMID:23436141

  3. Differential expression of Toll-like receptor (TLR) and B cell receptor (BCR) signaling molecules in primary diffuse large B-cell lymphoma of the central nervous system.

    PubMed

    Akhter, Ariz; Masir, Noraidah; Elyamany, Ghaleb; Phang, Kean-Chang; Mahe, Etienne; Al-Zahrani, Ali Matar; Shabani-Rad, Meer-Taher; Stewart, Douglas Allan; Mansoor, Adnan

    2015-01-01

    Primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) is a distinct and aggressive lymphoma that is confined to CNS. Since, central nervous system is barrier-protected and immunologically silent; role of TLR/BCR signaling in pathogenesis and biology of CNS DLBCL is intriguing. Genomic mutations in key regulators of TLR/BCR signaling pathway (MYD88/CD79B/CARD11) have recently been reported in this disease. These observations raised possible implications in novel targeted therapies; however, expression pattern of molecules related to TLR/BCR pathways in this lymphoma remains unknown. We have analyzed the expression of 19 genes encoding TLR/BCR pathways and targets in CNS DLBCLs (n = 20) by Nanostring nCounter™ analysis and compared it with expression patterns in purified reactive B-lymphocytes and systemic diffuse large B cell lymphoma (DLBCL) (n = 20). Relative expression of TLR4, TLR5, TLR9, CD79B and BLNK was higher in CNS DLBCLs than in control B-lymphocytes; where as TLR7, MALT1, BCL10, CD79A and LYN was lower in CNS DLBCLs (P < 0.0001). When compared with systemic DLBCL samples, higher expression of TLR9, CD79B, CARD11, LYN and BLNK was noted in CNS DLBCL (>1.5 fold change; P < 0.01). The B cell receptor molecules like BLNK and CD79B were also associated with higher expression of MYD88 dependent TLRs (TLR4/5/9). In conclusion, we have shown over expression of TLR/BCR related genes or their targets, where genomic mutations have commonly been identified in CNS DLBCL. We have also demonstrated that TLR over expression closely relate with up regulation of genes associated with BCR pathway like CD79B/BLNK and CARD11, which play an important role in NF-kB pathway activation. Our results provide an important insight into the possibility of TLR and/or B-cell receptor signaling molecules as possible therapeutic targets in CNS DLBCL. PMID:25391967

  4. Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

    PubMed Central

    Sarvestani, Soroush T.; Tate, Michelle D.; Moffat, Jessica M.; Jacobi, Ashley M.; Behlke, Mark A.; Miller, Alistair R.; Beckham, Simone A.; McCoy, Claire E.; Chen, Weisan; Mintern, Justine D.; O'Keeffe, Meredith; John, Matthias

    2014-01-01

    RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA. PMID:24227841

  5. Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.

    PubMed

    Chakrapani, Vemulawada; Patra, Swagat Kumar; Panda, Rudra Prasanna; Rasal, Kiran Dashrath; Jayasankar, Pallipuram; Barman, Hirak Kumar

    2016-08-01

    Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes. PMID:27079451

  6. Elevated CD14 (Cluster of Differentiation 14) and Toll-Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice.

    PubMed

    Rider, Daniel; Furusho, Hisako; Xu, Shuang; Trachtenberg, Alexander J; Kuo, Winston Patrick; Hirai, Kimito; Susa, Mako; Bahammam, Laila; Stashenko, Philip; Fujimura, Akira; Sasaki, Hajime

    2016-09-01

    Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc. PMID:27314637

  7. Deviation from major codons in the Toll-like receptor genes is associated with low Toll-like receptor expression

    PubMed Central

    Zhong, Fei; Cao, Weiping; Chan, Edmund; Tay, Puei Nam; Cahya, Florence Feby; Zhang, Haifeng; Lu, Jinhua

    2005-01-01

    Microbial structures activate Toll-like receptors (TLRs) and TLR-mediated cell signalling elicits and regulates host immunity. Most TLRs are poorly expressed but the underlying expression mechanism is not clear. Examination TLR sequences revealed that most human TLR genes deviated from using major human codons. CD14 resembles TLRs in sequence but its gene preferentially uses major codons. Indeed, CD14 expression on monocytes was higher than expression of TLR1 and TLR2. The TLR9 gene is abundant in major codons and it also showed higher expression than TLR1, TLR2 and TLR7 in transfected 293T cells. Change of the 5′-end 302 base pairs of the TLR2 sequence into major human codons markedly increased TLR2 expression, which led to increased TLR2-mediated constitutive nuclear factor-κB activation. Change of the 5′-end 381 base pairs of the CD14 sequence into prevalent TLR codons markedly reduced CD14 expression. These results collectively show that the deviation of TLR sequences from using major codons dictates the low TLR expression and this may protect the host against excessive inflammation and tissue damages. PMID:15606798

  8. [Incidence of odontogenic phlegmon associated with polymorphic variant 896A/G of gene TLR4, but not with 2258G/A of gene TLR2].

    PubMed

    Kuong, Vu V'et; Avetikov, D S; Shlykova, O A; Izmaĭlova, O V; Kaĭdashev, I P

    2014-10-01

    The problem actuality is caused by significant enhancement of the incidence rate for inflammatory diseases of the head and neck tissues, first of all of the oral cavity floor abscesses and phlegmons, which causes severe forms of mediastinitis while inadequate treatment. The authors have had established, that Toll-like receptors (TLR) initiate a cascade of anti-inflammatory reactions of the inborn immunity, followed by synthesis of a certain cytokines, and their genetic polymorphism changes the immune reactivity of the organism. Trustworthy correlation of the gene TLR4 (rs4986790) polymorphism 896A/G was proved with high risk of the odontogenic phlegmon of the oral cavity floor occurrence, what would permit to prognosticate the disease course in early terms, to optimize the schemes of its prophylaxis and treatment. PMID:25675790

  9. Molecular cloning and expression of toll-like receptor 4 (tlr4) in the blunt snout bream (Megalobrama amblycephala).

    PubMed

    Lai, Ruifang; Liu, Han; Jakovlić, Ivan; Zhan, Fanbin; Wei, Jin; Yang, Pinhong; Wang, Weimin

    2016-06-01

    Toll-like receptors (TLRs) play a pivotal role in teleost innate immune system. In this study, Megalobrama amblycephala (ma) tlr4 gene was cloned, its putative polypeptide product characterized, and expression analysed. Matlr4 cDNA is 2862 bp long, with an open reading frame of 2364 bp encoding 787 amino acids. MaTlr4 is a typical TLR protein, including the extracellular part with nine leucine-rich repeat motifs, a transmembrane region and a cytoplasmic Toll/interleukin-1 receptor domain. MaTlr4 has the highest level of identity (94%) and similarity (97%) with the grass carp Tlr4.2 homolog. This was also corroborated by the phylogenetic analysis, which placed MaTlr4 in a cluster with other cyprinid homologs. Matlr4 mRNA was ubiquitously expressed in all examined tissues and during all sampled developmental stages. The observed peak in matlr4 mRNA expression during gastrula and somite stages is in good agreement with its proposed role in the development of the neural system. Temporal expression patterns of matlr4 and maMyD88 mRNAs and proteins were analyzed in liver, spleen, head kidney, trunk kidney and intestine after Aeromonas hydrophila infection. And mRNA expression varied between different time-points. Both MaTlr4 and MaMyD88 protein expressions at 12 hpi were significantly enhanced in head kidney and intestine. These results indicate that matlr4 is involved in the immune response in M. amblycephala, and that it is indeed a functional homologue of tlr4s described in other animal species. PMID:26802439

  10. Molecular characterization and expression profile of partial TLR4 gene in association to mastitis in crossbred cattle.

    PubMed

    Panigrahi, Manjit; Sharma, Arjava; Bhushan, Bharat

    2014-01-01

    Crossbred cattle are more prone to mastitis in comparison to indigenous cattle. Toll-like receptor 4 (TLR4) recognizes pathogen ligands, for example, lipopolysaccharide (LPS) endotoxin from Escherichia coli and mediates signaling to initiate innate and adaptive immune responses. Mutations in TLR4 can compromise the host immune response to certain pathogens, so it may be a potential candidate for marker assisted selection to enhance mastitis resistance in dairy cattle. Hence, in this study role of bovine TLR4 gene in mastitis resistance was investigated by association as well as expression profiling analysis in crossbred cattle. The animals were divided into mastitis affected and unaffected groups on the basis of history of animals and California Mastitis Test (CMT). PCR-SSCP and Sequence analysis revealed three genotypes of coreceptor binding region 1 (CRBR1) fragment of TLR4 gene namely AA, AB, and BB in both groups of cattle. The logistic regression model did not show any significant effect of these genotypes on the occurrence of clinical mastitis. Moreover, in vitro challenge of peripheral blood mononuclear cells (PBMCs) with LPS failed to show any association of the genotypes with TLR4 gene expression. In a nutshell, in the present study enough evidence was not found for association of the SNP variants of CRBR1 fragment of TLR4 gene with mastitis susceptibility in crossbred cattle. PMID:24669869

  11. Toll-Like Receptor (TLR)-7 and -8 Modulatory Activities of Dimeric Imidazoquinolines

    PubMed Central

    Shukla, Nikunj M.; Mutz, Cole A.; Malladi, Subbalakshmi S.; Warshakoon, Hemamali J.; Balakrishna, Rajalakshmi; David, Sunil A.

    2012-01-01

    Toll-like receptors (TLRs) are pattern recognition receptors that recognize specific molecular patterns present in molecules that are broadly shared by pathogens, but are structurally distinct from host molecules. The TLR7-agonistic imidazoquinolines are of interest as vaccine adjuvants given their ability to induce pronounced Th1-skewed humoral responses. Minor modifications on the imidazoquinoline scaffold result in TLR7-antagonistic compounds which may be of value in addressing innate immune activation-driven immune exhaustion observed in HIV. We describe the syntheses and evaluation of TLR7 and TLR8 modulatory activities of dimeric constructs of imidazoquinoline linked at the C2, C4, C8, and N1-aryl positions. Dimers linked at the C4, C8 and N1-aryl positions were agonistic at TLR7; only the N1-aryl dimer with a 12-carbon linker was dual TLR7/8 agonistic. Dimers linked at C2 position showed antagonistic activities at TLR7 and TLR8; the C2 dimer with a propylene spacer was maximally antagonistic at both TLR7 and TLR8. PMID:22239408

  12. Evolution of the chicken Toll-like receptor gene family: A story of gene gain and gene loss

    PubMed Central

    Temperley, Nicholas D; Berlin, Sofia; Paton, Ian R; Griffin, Darren K; Burt, David W

    2008-01-01

    Background Toll-like receptors (TLRs) perform a vital role in disease resistance through their recognition of pathogen associated molecular patterns (PAMPs). Recent advances in genomics allow comparison of TLR genes within and between many species. This study takes advantage of the recently sequenced chicken genome to determine the complete chicken TLR repertoire and place it in context of vertebrate genomic evolution. Results The chicken TLR repertoire consists of ten genes. Phylogenetic analyses show that six of these genes have orthologs in mammals and fish, while one is only shared by fish and three appear to be unique to birds. Furthermore the phylogeny shows that TLR1-like genes arose independently in fish, birds and mammals from an ancestral gene also shared by TLR6 and TLR10. All other TLRs were already present prior to the divergence of major vertebrate lineages 550 Mya (million years ago) and have since been lost in certain lineages. Phylogenetic analysis shows the absence of TLRs 8 and 9 in chicken to be the result of gene loss. The notable exception to the tendency of gene loss in TLR evolution is found in chicken TLRs 1 and 2, each of which underwent gene duplication about 147 and 65 Mya, respectively. Conclusion Comparative phylogenetic analysis of vertebrate TLR genes provides insight into their patterns and processes of gene evolution, with examples of both gene gain and gene loss. In addition, these comparisons clarify the nomenclature of TLR genes in vertebrates. PMID:18241342

  13. Modulation of TLR3/TLR4 inflammatory signaling by the GABAB receptor agonist baclofen in glia and immune cells: relevance to therapeutic effects in multiple sclerosis

    PubMed Central

    Crowley, Tadhg; Fitzpatrick, John-Mark; Kuijper, Teun; Cryan, John F.; O’Toole, Orna; O’Leary, Olivia F.; Downer, Eric J.

    2015-01-01

    The GABAB receptor agonist, baclofen, is used to treat muscle tightness and cramping caused by spasticity in a number of disorders including multiple sclerosis (MS), but its precise mechanism of action is unknown. Neuroinflammation drives the central pathology in MS and is mediated by both immunoreactive glial cells and invading lymphocytes. Furthermore, a body of data indicates that the Toll-like receptor (TLR) family of innate immune receptors is implicated in MS progression. In the present study we investigated whether modulation of GABAB receptors using baclofen can exert anti-inflammatory effects by targeting TLR3 and(or) TLR4-induced inflammatory signaling in murine glial cells and human peripheral blood mononuclear cells (PBMCs) isolated from healthy control individuals and patients with the relapse-remitting (RR) form of MS. TLR3 and TLR4 stimulation promoted the nuclear sequestration of NF-κB and pro-inflammatory cytokine expression in murine glia, while TLR4, but not TLR3, promoted pro-inflammatory cytokine expression in PBMCs isolated from both healthy donors and RR-MS patients. Importantly, this effect was exacerbated in RR-MS patient immune cells. We present further evidence that baclofen dose-dependently attenuated TLR3- and TLR4-induced inflammatory signaling in primary glial cells. Pre-exposure of PBMCs isolated from healthy donors to baclofen attenuated TLR4-induced TNF-α expression, but did not affect TLR4-induced TNF-α expression in RR-MS patient PBMCs. Interestingly, mRNA expression of the GABAB receptor was reduced in PBMCs from RR-MS donors when compared to healthy controls, an effect that might contribute to the differential sensitivity to baclofen seen in healthy and RR-MS patient cells. Overall these findings indicate that baclofen differentially regulates TLR3 and TLR4 signaling in glia and immune cells, and offers insight on the role of baclofen in the treatment of neuroinflammatory disease states including MS. PMID:26283920

  14. Toll-like receptors 2, 3 and 4 (TLR-2, TLR-3 and TLR-4) are expressed in the microenvironment of human acquired cholesteatoma.

    PubMed

    Szczepański, Mirosław; Szyfter, Witold; Jenek, Renata; Wróbel, Maciej; Lisewska, Iwona Mozer; Zeromski, Jan

    2006-07-01

    Human toll-like receptors (TLR 1-10) are crucial in the induction and activation of innate immunity in the course of an infection. They are expressed mainly on the cells of the immune system, and also on some epithelia and endothelia. Their ligands so called pathogen associated molecular patterns are abundant on invading microbes. TLR-ligand binding results in cell signal transduction and subsequent production of various proinflammatory cytokines such as IL-1 and TNF-alpha. Acquired cholesteatoma is formed during chronic otitis media in the proportion of cases. It has adverse effects on ear structures, resulting in osteolysis and bone resorption. Its formation and pathogenesis are not fully understood. The current study attempted to search the possible role of TLRs in this somewhat awkward pathological condition. Surgical specimens of human acquired cholesteatoma (n=15) and normal external auditory canal skin (n=5, control tissues) were tested by immunohistochemistry for the presence of TLRs. Three TLRs were examined: TLR-2, TLR-3 and TLR-4. All TLRs tested were demonstrated in matrix (layer of keratinizing epithelium) and perimatrix (granulation tissue) of this inflammatory tumour. Expression of particular TLRs within the keratinizing epithelium was distinct and uneven. In the perimatrix, numerous T (CD3+) cells were seen and relatively few macrophages (CD11c+, HLA-DR+). There was a weak expression of all TLRs on normal (non-inflammatory) skin. Expression of TLR-3 both on the epithelium and some cells within the perimatrix and the presence of T cells may suggest that apart from innate immune responses, mechanisms of adaptive immunity also operate in cholesteatoma. Weak expression of these receptors on normal skin may also suggest the important role of TLRs in the etiopathogenesis of cholesteatoma. PMID:16538507

  15. Toll-Like Receptor Gene Expression during Trichinella spiralis Infection

    PubMed Central

    Kim, Sin; Park, Mi Kyung; Yu, Hak Sun

    2015-01-01

    In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation. PMID:26323841

  16. Toll-Like Receptor Gene Expression during Trichinella spiralis Infection.

    PubMed

    Kim, Sin; Park, Mi Kyung; Yu, Hak Sun

    2015-08-01

    In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP(-/-) MEF cells, and quite substantially decreased in TRIF(-/-) MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP(-/-) MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation. PMID:26323841

  17. Toll-like receptor (TLR)-2/4 expression in retinal ganglion cells in a high-glucose environment and its implications.

    PubMed

    Zhao, M; Li, C H; Liu, Y L

    2016-01-01

    Diabetic retinopathy (DR), a major complication of diabetes mellitus, is the leading cause of adult blindness. The Toll-like receptor (TLR) family is believed to be involved in the pathogenesis and progression of DR. Here, we investigated the expression profiles of TLR-2 and TLR-4 in retinal ganglion cells (RGCs), in an attempt to elucidate the role of these molecules in the etiology of DR. In vitro cultured RGCs were divided into control and high-glucose groups. The mRNA and protein levels of TLR-2, TLR-4, and nuclear factor (NF)-κB were detected by real-time PCR and western blotting. RGCs were further transfected with specific siRNA targeting TLR2/TLR4; the proliferation of transfected RGCs and their tumor necrosis factor (TNF)-α and interleukin (IL)-8 secretory capacity were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and enzyme-linked immunosorbent assays (ELISA), respectively. In a high-glucose environment, TLR-2/4 expression was significantly upregulated in RGCs (while their viability decreased); additionally, NF-κB expression and secretion of TNF-α and IL-8 were significantly increased. Co-silencing of the TLR-2 and TLR-4 genes inhibited NF- κB expression and TNF-α/IL-8 secretion, while increasing the survival rate of RGCs. Therefore, a high-glucose environment can potentiate the expression of TLR-2 and TLR-4 in RGCs, activate the downstream signaling pathway, and increase the secretion of pro-inflammatory factors, thereby aggravating DR. PMID:27173229

  18. Plasmacytoid dendritic cell precursors/type I interferon-producing cells sense viral infection by Toll-like receptor (TLR) 7 and TLR9.

    PubMed

    Ito, Tomoki; Wang, Yui-Hsi; Liu, Yong-Jun

    2005-01-01

    Plasmacytoid dendritic cell (pDC) precursors, also called type I IFN (alpha/beta/omega)-producing cells (IPCs), are the key effectors in the innate immune system because of their extraordinary capacity to produce type I IFNs against microbial infection, particularly viral infection. In contrast to myeloid DCs, human pDC/IPCs selectively express Toll-like receptor (TLR) 7 and TLR9 within the endosomal compartment. These receptors are specifically designed to recognize the nucleoside-based products derived from RNA viruses and DNA viruses. Therefore, this expression profile potentially enables pDC/IPCs to sense a variety of viruses. Stimulation of TLR7 or TLR9 leads to type I IFN responses through the MyD88 pathway. Thus, pDC/IPCs may play a central role in host defense against viral infection through the TLR7 and TLR9 system. PMID:15592841

  19. Identification, characterization, and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Induction of the innate immune pathways is critical for early antiviral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by s...

  20. Combined effect of TLR2 gene polymorphism and early life stress on the age at onset of bipolar disorders.

    PubMed

    Oliveira, José; Etain, Bruno; Lajnef, Mohamed; Hamdani, Nora; Bennabi, Meriem; Bengoufa, Djaouida; Sundaresh, Aparna; Chaabane, Arij Ben; Bellivier, Frank; Henry, Chantal; Kahn, Jean-Pierre; Charron, Dominique; Krishnamoorthy, Rajagopal; Leboyer, Marion; Tamouza, Ryad

    2015-01-01

    Gene-environment interactions may play an important role in modulating the impact of early-life stressful events on the clinical course of bipolar disorder (BD), particularly associated to early age at onset. Immune dysfunction is thought to be an important mechanism linking childhood trauma with early-onset BD, thus the genetic diversity of immune-related loci may account for an important part of the interindividual susceptibility to this severe subform. Here we investigated the potential interaction between genetic variants of Toll-like receptors 2 (TLR2) and 4 (TLR4), major innate immune response molecules to pathogens, and the childhood trauma questionnaire (CTQ) in age at onset of BD. We recruited 531 BD patients (type I and II or not otherwise specified), genotyped for the TLR2 rs4696480 and rs3804099 and TLR4 rs1927914 and rs11536891 single-nucleotide polymorphisms and recorded for history of childhood trauma using the CTQ. TLR2 and TLR4 risk genotype carrier state and history of childhood emotional, physical and sexual abuses were evaluated in relation to age at onset as defined by the age at first manic or depressive episode. We observed a combined effect of TLR2 rs3804099 TT genotype and reported sexual abuse on determining an earlier age at onset of BD by means of a Kaplan-Meier survival curve (p = 0.002; corrected p = 0.02). Regression analysis, however, was non-significant for the TLR2-CTQ sexual abuse interaction term. The negative effects of childhood sexual abuse on age at onset of BD may be amplified in TLR2 rs3804099 risk genotype carriers through immune-mediated pathways. Clinical characteristics of illness severity, immune phenotypes and history of early life infectious insults should be included in future studies involving large patient cohorts. PMID:25790282

  1. Combined Effect of TLR2 Gene Polymorphism and Early Life Stress on the Age at Onset of Bipolar Disorders

    PubMed Central

    Oliveira, José; Etain, Bruno; Lajnef, Mohamed; Hamdani, Nora; Bennabi, Meriem; Bengoufa, Djaouida; Sundaresh, Aparna; Chaabane, Arij Ben; Bellivier, Frank; Henry, Chantal; Kahn, Jean-Pierre; Charron, Dominique; Krishnamoorthy, Rajagopal; Leboyer, Marion; Tamouza, Ryad

    2015-01-01

    Gene-environment interactions may play an important role in modulating the impact of early-life stressful events on the clinical course of bipolar disorder (BD), particularly associated to early age at onset. Immune dysfunction is thought to be an important mechanism linking childhood trauma with early-onset BD, thus the genetic diversity of immune-related loci may account for an important part of the interindividual susceptibility to this severe subform. Here we investigated the potential interaction between genetic variants of Toll-like receptors 2 (TLR2) and 4 (TLR4), major innate immune response molecules to pathogens, and the childhood trauma questionnaire (CTQ) in age at onset of BD. We recruited 531 BD patients (type I and II or not otherwise specified), genotyped for the TLR2 rs4696480 and rs3804099 and TLR4 rs1927914 and rs11536891 single-nucleotide polymorphisms and recorded for history of childhood trauma using the CTQ. TLR2 and TLR4 risk genotype carrier state and history of childhood emotional, physical and sexual abuses were evaluated in relation to age at onset as defined by the age at first manic or depressive episode. We observed a combined effect of TLR2 rs3804099 TT genotype and reported sexual abuse on determining an earlier age at onset of BD by means of a Kaplan-Meier survival curve (p = 0.002; corrected p = 0.02). Regression analysis, however, was non-significant for the TLR2-CTQ sexual abuse interaction term. The negative effects of childhood sexual abuse on age at onset of BD may be amplified in TLR2 rs3804099 risk genotype carriers through immune-mediated pathways. Clinical characteristics of illness severity, immune phenotypes and history of early life infectious insults should be included in future studies involving large patient cohorts. PMID:25790282

  2. Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3.

    PubMed

    Dang, Jason; Tiwari, Shashi Kant; Lichinchi, Gianluigi; Qin, Yue; Patil, Veena S; Eroshkin, Alexey M; Rana, Tariq M

    2016-08-01

    Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly. PMID:27162029

  3. Toll-Like Receptor (TLR)-Associated Sequence Variants and Prostate Cancer Risk among Men of African Descent

    PubMed Central

    Rogers, Erica N.; Jones, Dominique; Kidd, Nayla C.; Yeyeodu, Susan; Brock, Guy; Ragin, Camille; Jackson, Maria; McFarlane-Anderson, Norma; Tulloch-Reid, Marshall; Kimbro, K. Sean; Kidd, LaCreis R.

    2013-01-01

    BACKGROUND Recent advances demonstrate a relationship between chronic/recurrent inflammation and prostate cancer (PCA). Among inflammatory regulators, toll-like receptors (TLRs) play a critical role in innate immune responses. However, it remains unclear whether variant TLR genes influence PCA risk among men of African descent. Therefore, we evaluated the impact of 32 TLR-associated single nucleotide polymorphisms (SNPs) on PCA risk among African-Americans and Jamaicans. METHODS SNP profiles of 814 subjects were evaluated using Illumina’s Veracode genotyping platform. Single and combined effects of SNPs in relation to PCA risk were assessed using age-adjusted logistic regression and entropy-based multifactor dimensionality reduction (MDR) models. RESULTS Seven sequence variants detected in TLR6, TOLLIP, IRAK4, IRF3 were marginally related to PCA. However, none of these effects remained significant after adjusting for multiple hypothesis testing. Nevertheless, MDR modeling revealed a complex interaction between IRAK4 rs4251545 and TLR2 rs1898830 as a significant predictor of PCA risk among U.S. men (permutation testing p-value = 0.001). CONCLUSIONS MDR identified an interaction between IRAK4 and TLR2 as the best two factor model for predicting PCA risk among men of African descent. However, these findings require further assessment and validation. PMID:23657238

  4. Study of Toll-like receptor gene loci in sarcoidosis

    PubMed Central

    Schürmann, M; Kwiatkowski, R; Albrecht, M; Fischer, A; Hampe, J; Müller-Quernheim, J; Schwinger, E; Schreiber, S

    2008-01-01

    Sarcoidosis is a multi-factorial systemic disease of granulomatous inflammation. Current concepts of the aetiology include interactions of unknown environmental triggers with an inherited susceptibility. Toll-like receptors (TLRs) are main components of innate immunity and therefore TLR genes are candidate susceptibility genes in sarcoidosis. Ten members of the human TLR gene family have been identified and mapped to seven chromosomal segments. The aim of this study was to investigate all known TLR gene loci for genetic linkage with sarcoidosis and to follow positive signals with different methods. We analysed linkage of TLR gene loci to sarcoidosis by use of closely flanking microsatellite markers in 83 families with 180 affected siblings. We found significant linkage between sarcoidosis and markers of the TLR4 gene locus on chromosome 9q (non-parametric linkage score 2·63, P = 0·0043). No linkage was found for the remaining TLR gene loci. We subsequently genotyped 1203 sarcoidosis patients from 997 families, 1084 relatives and 537 control subjects for four single nucleotide polymorphisms of TLR4, including Asp299Gly and Thr399Ile. This genotype data set was studied by case–control comparisons and transmission disequilibrium tests, but showed no significant results. In summary, TLR4 − w ith significant genetic linkage results − appears to be the most promising member of the TLR gene family for further investigation in sarcoidosis. However, our results do not confirm the TLR4 polymorphisms Asp299Gly and Thr399Ile as susceptibility markers. Our results rather point to another as yet unidentified variant within or close to TLR4 that might confer susceptibility to sarcoidosis. PMID:18422738

  5. Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

    PubMed Central

    Turner, Matthew L.; Cronin, James G.; Healey, Gareth D.

    2014-01-01

    Bacteria often infect the endometrium of cattle to cause endometritis, uterine disease, and infertility. Lipopeptides are commonly found among bacteria and are detected by the Toll-like receptor (TLR) cell surface receptor TLR2 on immune cells. Heterodimers of TLR2 with TLR1 or TLR6 activate MAPK and nuclear factor-κB intracellular signaling pathways to stimulate inflammatory responses. In the endometrium, epithelial and stromal cells are the first to encounter invading bacteria, so the present study explored whether endometrial cells can also mount inflammatory responses to bacterial lipopeptides via TLRs. The supernatants of pure populations of primary bovine endometrial epithelial and stromal cells accumulated the cytokine IL-6 and the chemokine IL-8 in response to triacylated or diacylated bacterial lipopeptides. The accumulation of IL-6 and IL-8 in response to triacylated lipopeptides was reduced by small interfering RNA targeting TLR2 or TLR1 but not TLR6, whereas cellular responses to diacylated lipopeptide were reduced by small interfering RNA targeting TLR2, TLR1, or TLR6. Both lipopeptides induced rapid phosphorylation of ERK1/2, p38, and nuclear factor-κB in endometrial cells, and inhibitors of ERK1/2 or p38 limited the accumulation of IL-6. The ovarian steroids estradiol and progesterone had little impact on inflammatory responses to lipopeptides. The endometrial epithelial and stromal cell responses to lipopeptides via TLR2, TLR1, and TLR6 provide a mechanism linking a wide range of bacterial infections to inflammation of the endometrium. PMID:24437488

  6. Molecular cloning of Salmo salar Toll-like receptors (TLR1, TLR22, TLR5M and TLR5S) and expression analysis in SHK-1 cells during Piscirickettsia salmonis infection.

    PubMed

    Salazar, C; Haussmann, D; Kausel, G; Figueroa, J

    2016-02-01

    In fish, the innate immune system is the primary response against infection. Toll-like receptors (TLRs) recognize pathogens through pathogen-associated molecular patterns (PAMPs), and some target molecules of TLRs are homologous between fish and mammals. Piscirickettsia salmonis is one of the main pathogens affecting the salmon industry in Chile. Better knowledge of mechanisms underlying its invasive capacity and recognition of target cells is crucial for vaccine development. Therefore, Salmo salar L. TLR1, TLR22, membrane TLR5M and soluble TLR5S sequences were cloned, and expression kinetics were analysed by RT-qPCR in salmon head kidney cells (SHK-1) infected with three different P. salmonis preparations: alive, formaldehyde treated, extract. Clearly, all analysed TLRs were expressed and transcription level changes were revealed at 2 hpi, 12 or 16 hpi and 24 hpi depending on P. salmonis infection scheme. Increased IL1-beta expression confirmed TLR pathway response. Furthermore, significant expression modulations of several members of the TLR pathway in this in vitro model suggest that P. salmonis extract rather than formaldehyde-inactivated bacteria might strengthen the salmon immune system. PMID:25903926

  7. Functional polymorphisms of the TLR7 and TLR8 genes contribute to Mycobacterium tuberculosis infection.

    PubMed

    Lai, Yung-Fa; Lin, Tsun-Mei; Wang, Chiou-Huey; Su, Pei-Yi; Wu, Jiun-Ting; Lin, Meng-Chih; Eng, Hock-Liew

    2016-05-01

    Tuberculosis (TB) has recently re-emerged as a major global public health threat and Mycobacterium tuberculosis (MTB) is a highly successful pathogen that evolved remarkable strategies to establish persistent infection. There is strong evidence that host genetic factors influence individual susceptibility to TB. In this study, we evaluated the associations between the TLR7 and TLR8 genetic polymorphisms and TB susceptibility in Chinese individuals. The results demonstrated that the frequency of the TLR8-129C allele was higher in male patients with pulmonary TB than in healthy controls (22.9% vs. 6.8%, p < 0.001). Based on haplotype analysis, the frequency of the TLR7 IVS2-151A/TLR8 -129C haplotype increased the risk for TB infection compared to the wild-type allele (TLR7 IVS2-151A/TLR8 -129G), with OR = 3.23 (95% CI = 1.58-6.61; p = 0.001). An ex vivo phagocytosis assay that examined the functional effects of these polymorphisms on the defense against MTB revealed higher phagocytosis in monocytes from males with the TLR7 IVS2-151A/TLR8 -129C genotype than in those with the wild-type allele (73.0 ± 20.3% versus 34.6 ± 8.1%; p = 0.03). In addition, mRNA expression and cytokine production were analyzed in the whole blood of male healthy volunteers stimulated with inactivated MTB ex vivo. TNFα production was lower in TLR7 IVS2-151A/TLR8 -129C subjects than in those with the wild-type allele (578.4 ± 90.3 pg/ml versus 1043 ± 136 pg/ml; p = 0.03), and the expression of TLR7 was significantly impaired (0.8 ± 0.1 folds, p = 0.05) after MTB stimulation. In conclusion, these findings provide evidence that TLR7 and TLR8 genetic polymorphisms are associated with susceptibility to MTB infection, and the link is shaped by less effective MTB phagocytosis and impaired TLR signaling. PMID:27156628

  8. In Vitro Reconstitution of the Toll/Interleukin-1 Receptor (TIR) Domain Complex Between TLR5/6 and Myd88.

    PubMed

    Jang, Tae-Ho; Narayanan, Kannan Badri; Park, Hyun Ho

    2016-01-01

    Toll-like receptors (TLRs) are evolutionarily conserved receptors with trimodular structure to respond to endogenous ligands and exogenous ligands from microbial pathogens. The highly conserved cytoplasmic C-terminal Toll/interleukin-1 receptor (TIR) domain of TLRs plays a crucial role in inflammatory reactions. In myeloid differentiation primary-response protein 88 (MyD88)- dependent signaling pathway, the interaction of TLRsTIR with cytosolic adaptor protein, MyD88TIR recruits IL-1R-associated kinases (IRAK) for subsequent activation of transcription factors nuclear factor kB (NF-kB) and activation protein 1 (AP-1) and other effector molecules. In the present investigation, TLR5TIR, TLR6TIR and MyD88TIR genes were subcloned and overexpressed in bacterium Escherichia coli strain BL- 21 (DE3). The purification and biochemical characterization of TLR5TIR and TLR6TIR&, and MyD88TIR proteins were also performed. The protein-protein interactions between TIR domains of TLR5 and TLR6 with MyD88, respectively, were evaluated in vitro at physiological pH and salt concentration. The in vitro reconstitution results showed that under physiological pH and salt concentration, MyD88TIR interacted with TLR5TIR, and did not interact with TLR6TIR protein. Both TIR domain-containing TLR5 and TLR6 proteins were prone to aggregation in a temperature-dependent manner at room temperature. At normal physiological pH and salt concentration, with the addition of binding partner MyD88TIR to TLR5/6TIR, time-dependent aggregation was not observed in both TLRsTIR at both room temperature and 4 ºC for 2 d, influencing the solubility of TLR5/6TIR. Moreover, TLR5TIR alone exhibited increase in solubility of the protein with increase in the salt concentration of the buffered solution from 0.025 M to 1.25 M at room temperature. PMID:26548862

  9. A Novel Toll-Like Receptor (TLR) Influences Compatibility between the Gastropod Biomphalaria glabrata, and the Digenean Trematode Schistosoma mansoni

    PubMed Central

    Pila, Emmanuel A.; Tarrabain, Mahmoud; Kabore, Alethe L.; Hanington, Patrick C.

    2016-01-01

    Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to β-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which

  10. A Novel Toll-Like Receptor (TLR) Influences Compatibility between the Gastropod Biomphalaria glabrata, and the Digenean Trematode Schistosoma mansoni.

    PubMed

    Pila, Emmanuel A; Tarrabain, Mahmoud; Kabore, Alethe L; Hanington, Patrick C

    2016-03-01

    Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to β-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which

  11. TLR21's agonists in combination with Aeromonas antigens synergistically up-regulate functional TLR21 and cytokine gene expression in yellowtail leucocytes.

    PubMed

    Reyes-Becerril, Martha; Ascencio-Valle, Felipe; Hirono, Ikuo; Kondo, Hidehiro; Jirapongpairoj, Walissara; Esteban, Maria Angeles; Alamillo, Erika; Angulo, Carlos

    2016-08-01

    The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1β and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1β and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail. PMID:26987525

  12. Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine mammary epithelial cells contribute to the innate immune response to intramammary infections by recognizing pathogens through specialized pattern recognition receptors. Toll-like receptor 4 (TLR4) is one such receptor that binds and is activated by lipopolysaccharide (LPS), a component of the...

  13. Escherichia coli lipopolysaccharide upregulates the expression of both toll like receptor 4 and 2 (TLR4 and TLR2) in cultured bovine mammary epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine mammary epithelial cells contribute to the innate immune response to intramammary infection. Their ability to mount such a response is dependent upon mammary epithelial recognition of the invading pathogen by specialized receptors. Toll-like receptor 4 (TLR4) is one such receptor that recog...

  14. A critical role of toll-like receptor 2 (TLR2) and its’ in vivo ligands in radio-resistance

    PubMed Central

    Gao, Fu; Zhang, Chaoxiong; Zhou, Chuanfeng; Sun, Weimin; Liu, Xin; Zhang, Pei; Han, Jiaqi; Xian, Linfeng; Bai, Dongchen; Liu, Hu; Cheng, Ying; Li, Bailong; Cui, Jianguo; Cai, Jianming; Liu, Cong

    2015-01-01

    The role of Toll-like receptor-2 (TLR2) in radio-resistance remained largely unknown. TLR2 knockout (TLR2−/−) mice received radiation of 6.5 Gy, and then were studied. We found that radiation resulted in more severe mortality and morbidity rates in TLR2−/− mice. The cause of death in TLR2−/− mice may be severe and persistent bone marrow cell loss. Injection of the TLR2 agonist Pam3CSK4 into wild type (WT) mice induced radio-resistance. Myd88−/− mice were more susceptible to radiation. In conclusion, our data indicate that, similar to TLR4, TLR2 plays a critical role in radio-resistance. PMID:26268450

  15. A critical role of toll-like receptor 2 (TLR2) and its' in vivo ligands in radio-resistance.

    PubMed

    Gao, Fu; Zhang, Chaoxiong; Zhou, Chuanfeng; Sun, Weimin; Liu, Xin; Zhang, Pei; Han, Jiaqi; Xian, Linfeng; Bai, Dongchen; Liu, Hu; Cheng, Ying; Li, Bailong; Cui, Jianguo; Cai, Jianming; Liu, Cong

    2015-01-01

    The role of Toll-like receptor-2 (TLR2) in radio-resistance remained largely unknown. TLR2 knockout (TLR2(-/-)) mice received radiation of 6.5 Gy, and then were studied. We found that radiation resulted in more severe mortality and morbidity rates in TLR2(-/-) mice. The cause of death in TLR2(-/-) mice may be severe and persistent bone marrow cell loss. Injection of the TLR2 agonist Pam3CSK4 into wild type (WT) mice induced radio-resistance. Myd88(-/-) mice were more susceptible to radiation. In conclusion, our data indicate that, similar to TLR4, TLR2 plays a critical role in radio-resistance. PMID:26268450

  16. Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor

    SciTech Connect

    Ivičak-Kocjan, Karolina; Panter, Gabriela; Benčina, Mojca; Jerala, Roman

    2013-05-24

    Highlights: •The chimeric protein fusing flagellin to the TLR5 ectodomain is constitutively active. •Mutation P736H within the BB-loop of TLR5 TIR domain renders the receptor inactive. •The R90D mutation in flagellin inactivated autoactivation of the chimeric protein. •The 2:2 stoichiometry of the TLR5:flagellin complex is physiologically relevant. -- Abstract: Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation.

  17. Synaptic abnormalities of mice lacking toll-like receptor (TLR)-9.

    PubMed

    Patel, V; Patel, A M; McArdle, J J

    2016-06-01

    Motor, sensory, and autonomic abnormalities are reported for toll-like receptor 9 (TLR9) knock-out (KO) mice. However, a physiological role of TLR9 in the nervous system is largely unknown. Since altered synaptic transmission can contribute to sensory and motor abnormalities, we evaluated neuromuscular junction (NMJ) function and morphology of TLR9 KO mice. Triangularis sterni nerve-muscle preparations were dissected from TLR9 KO and age-matched control mice. Two-electrode voltage clamp of the motor endplate revealed that the amplitude and frequency of miniature end plate currents (mEPCs) for TLR9 KO NMJs were significantly greater than control. In contrast, mean endplate current (EPC, 1Hz) amplitude was equivalent to control. The ratio of mean EPC to mean mEPC amplitude indicated a decline of quantal content (m) for TLR9 KO NMJs. Furthermore, m declined more rapidly than control in response to 50-Hz stimulus trains. A rightward shift of the mEPC amplitude distribution suggested formation of vesicles containing larger amounts of acetylcholine (ACh). Staining with rhodamine α-bungarotoxin revealed a significant decline of endplate size in TLR9 KO mice. This alteration may result from ACh-induced decline of acetylcholine receptor (AChR) expression resulting from increased frequency and amplitude of mEPCs. At the same time, excessive spontaneous vesicular ACh release may initiate retrograde suppression of excitation-secretion coupling. These data suggest a novel role of TLR9 in the development of the NMJ. PMID:26955780

  18. TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes.

    PubMed

    Oosting, Marije; Ter Hofstede, Hadewych; Sturm, Patrick; Adema, Gosse J; Kullberg, Bart-Jan; van der Meer, Jos W M; Netea, Mihai G; Joosten, Leo A B

    2011-01-01

    After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs), such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2. Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production. This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans. PMID:21998742

  19. Interleukin-18 Increases TLR4 and Mannose Receptor Expression and Modulates Cytokine Production in Human Monocytes

    PubMed Central

    Dias-Melicio, Luciane Alarcão; Fernandes, Reginaldo Keller; Rodrigues, Daniela Ramos; Golim, Marjorie Assis; Soares, Angela Maria Victoriano Campos

    2015-01-01

    Interleukin-18 is a proinflammatory cytokine belonging to the interleukin-1 family of cytokines. This cytokine exerts many unique biological and immunological effects. To explore the role of IL-18 in inflammatory innate immune responses, we investigated its impact on expression of two toll-like receptors (TLR2 and TLR4) and mannose receptor (MR) by human peripheral blood monocytes and its effect on TNF-α, IL-12, IL-15, and IL-10 production. Monocytes from healthy donors were stimulated or not with IL-18 for 18 h, and then the TLR2, TLR4, and MR expression and intracellular TNF-α, IL-12, and IL-10 production were assessed by flow cytometry and the levels of TNF-α, IL-12, IL-15, and IL-10 in culture supernatants were measured by ELISA. IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-α and IL-10 was also increased by cytokine treatment. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Taken together, these results show an important role of IL-18 on the early phase of inflammatory response by promoting the expression of some pattern recognition receptors (PRRs) that are important during the microbe recognition phase and by inducing some important cytokines such as TNF-α and IL-10. PMID:25873755

  20. CD14+ cells are required for IL-12 response in bovine blood mononuclear cells activated with Toll-like receptor (TLR) 7 and TLR8 ligands.

    PubMed

    Buza, Joram; Benjamin, Ponn; Zhu, Jianzhung; Wilson, Heather L; Lipford, Grayson; Krieg, Arthur M; Babiuk, Lorne A; Mutwiri, George K

    2008-12-15

    Single-stranded viral RNA (ssRNA) was recently identified as the natural ligand for TLR7 and TLR8. ssRNA sequences from viruses, as well as their synthetic analogues stimulate innate immune responses in immune cells from humans and mice, but their immunostimulatory activity has not been investigated in ruminants. In the present investigations, we tested whether synthetic RNA oligoribonucleotides (ORN) can activate immune cells from cattle. In vitro incubation of bovine peripheral blood mononuclear cells (PBMCs) with ORN-induced production of IL-12, IFN-gamma and TNF-alpha. No significant induction of IFN-alpha was observed. Depletion of CD14+ cells from PBMC abrogated the IL-12 response and consequently the IFN-gamma response, suggesting that CD14+ cells are required for PBMC immune activation with ORN. Consistent with these findings, the putative receptors for ORN (TLR7 and TLR8) were expressed at higher levels in the CD14+ fraction than the CD14- PBMC fraction. Pre-treatment of PBMC with bafilomycin (an inhibitor of phagosomal acidification) prior to stimulation with ORN abolished the cytokine responses, confirming that the receptor(s) which mediate the ORN-induced responses are intracellular. These results demonstrate for the first time that the TLR7/8 agonist ORN's have strong immune stimulatory effects in cattle, and suggest that further investigation on the potential of TLR7/8 ligands to activate innate and adaptive immune responses in domestic animals are warranted. PMID:18789542

  1. Toll-Like Receptor (TLR) 2 and TLR9 Expressed in Trigeminal Ganglia are Critical to Viral Control During Herpes Simplex Virus 1 Infection

    PubMed Central

    Lima, Graciela Kunrath; Zolini, Guilherme Pimenta; Mansur, Daniel Santos; Freire Lima, Bráulio Henrique; Wischhoff, Uschi; Astigarraga, Ruiz Gerhardt; Dias, Marcela França; Silva, Mariana das Graças Almeida; Béla, Samantha Ribeiro; do Valle Antonelli, Lis Ribeiro; Arantes, Rosa Maria; Gazzinelli, Ricardo Tostes; Báfica, André; Kroon, Erna Geessien; Campos, Marco Antônio

    2010-01-01

    Herpes simplex virus 1 (HSV-1) is a neurotropic DNA virus that is responsible for several clinical manifestations in humans, including encephalitis. HSV-1 triggers toll-like receptors (TLRs), which elicit cytokine production. Viral multiplication and cytokine expression in C57BL/6 wild-type (WT) mice infected with HSV-1 were evaluated. Virus was found in the trigeminal ganglia (TG), but not in the brains of animals without signs of encephalitis, between 2 and 6 days postinfection (d.p.i.). Cytokine expression in the TG peaked at 5 d.p.i. TLR9−/− and TLR2/9−/− mice were more susceptible to the virus, with 60% and 100% mortality, respectively, as opposed to 10% in the WT and TLR2−/− mice. Increased levels of both CXCL10/IP-10 and CCL2/MCP-1, as well as reduced levels of interferon-γ and interleukin 1-β transcripts, measured in both the TG and brains at 5 d.p.i., and the presence of virus in the brain were correlated with total mortality in TLR2/9−/− mice. Cytokine alterations in TLR2/9−/− mice coincided with histopathological changes in their brains, which did not occur in WT and TLR2−/− mice and occurred only slightly in TLR9−/− mouse brain. Increased cellularity, macrophages, CD8 T cells producing interferon-γ, and expression levels of TLR2 and TLR9 were detected in the TG of WT-infected mice. We hypothesize that HSV-1 infection is controlled by TLR-dependent immune responses in the TG, which prevent HSV-1 encephalitis. PMID:20864677

  2. β-adrenergic receptor agonist, Compound 49b, inhibits TLR4 signaling pathway in diabetic retina

    PubMed Central

    Berger, Elizabeth A.; Carion, Thomas W.; Jiang, Youde; Liu, Li; Chahine, Adam; Walker, Robert Jason; Steinle, Jena J.

    2016-01-01

    Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. While it is clear that tumor necrosis factor- alpha (TNF-α) is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase toll-like receptor 4 (TLR4) signaling, which could be inhibited by a β-adrenergic receptor agonist (Compound 49b) previously shown to have anti-inflammatory actions. In order to investigate β-adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin-injected diabetic mice, as well as human primary retinal endothelial cells (REC) and rat retinal Müller cells (rMC-1) exposed to high glucose (25mM), were treated with a novel β-adrenergic receptor agonist, Compound 49b (50nM), or PBS (control). TLR4 and its downstream signaling partners (MyD88, IRAK1, TRAF6, total and phosphorylated NF-κB) were examined. In addition, we assessed high mobility box group 1 (HMGB1) protein levels. Our data showed that diabetes or high glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in REC and Müller cells grown in high glucose, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway providing a potential mechanism for its protective actions. PMID:26888251

  3. Diversity in the Toll-like receptor genes of the Tasmanian devil (Sarcophilus harrisii).

    PubMed

    Cui, Jian; Cheng, Yuanyuan; Belov, Katherine

    2015-03-01

    The Tasmanian devil is an endangered marsupial species that has survived several historical bottlenecks and now has low genetic diversity. Here we characterize the Toll-like receptor (TLR) genes and their diversity in the Tasmanian devil. TLRs are a key innate immune gene family found in all animals. Ten TLR genes were identified in the Tasmanian devil genome. Unusually low levels of diversity were found in 25 devils from across Tasmania. We found two alleles at TLR2, TLR3 and TLR6. The other seven genes were monomorphic. The insurance population, which safeguards the species from extinction, has successfully managed to capture all of these TLR alleles, but concerns remain for the long-term survival of this species. PMID:25563844

  4. Role of Toll-Like Receptor (TLR) 2 in Experimental Bacillus cereus Endophthalmitis

    PubMed Central

    Novosad, Billy D.; Astley, Roger A.; Callegan, Michelle C.

    2011-01-01

    Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known Gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2-/- mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to108 CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2-/- eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2-/- eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2-/- eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial

  5. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

    PubMed

    Baroni, Adone; Orlando, Manuela; Donnarumma, Giovanna; Farro, Pietro; Iovene, Maria Rosaria; Tufano, Maria Antonietta; Buommino, Elisabetta

    2006-01-01

    Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the beta-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human beta-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity. PMID:16283346

  6. Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions

    PubMed Central

    Solbakken, Monica H.; Tørresen, Ole K.; Nederbragt, Alexander J.; Seppola, Marit; Gregers, Tone F.; Jakobsen, Kjetill S.; Jentoft, Sissel

    2016-01-01

    Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity. PMID:27126702

  7. Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

    PubMed

    Solbakken, Monica H; Tørresen, Ole K; Nederbragt, Alexander J; Seppola, Marit; Gregers, Tone F; Jakobsen, Kjetill S; Jentoft, Sissel

    2016-01-01

    Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity. PMID:27126702

  8. Localization of type I interferon receptor limits interferon-induced TLR-3 in epithelial cells

    EPA Science Inventory

    This study aimed to expand on the role of type I IFNs in the influenza-induced upregulation of TLR3 and determine whether and how the localization of the IFN-alpha/beta receptor (IFNAR) in respiratory epithelial cells could modify IFN-induced responses. Using differentiated prima...

  9. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain.

    PubMed

    Mistry, Pragnesh; Laird, Michelle H W; Schwarz, Ryan S; Greene, Shannon; Dyson, Tristan; Snyder, Greg A; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y; MacKerell, Alexander D; Vogel, Stefanie N

    2015-04-28

    Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

  10. Multiple Sclerosis: Modulation of Toll-Like Receptor (TLR) Expression by Interferon-β Includes Upregulation of TLR7 in Plasmacytoid Dendritic Cells

    PubMed Central

    Hecker, Michael; Paap, Brigitte K.; Thamilarasan, Madhan; Koczan, Dirk; Schott, Eckart; Deuschle, Katrin; Bellmann-Strobl, Judith; Paul, Friedemann; Zettl, Uwe K.; Ruprecht, Klemens; Lehnardt, Seija

    2013-01-01

    Interferon-β is an established treatment for patients with multiple sclerosis (MS) but its mechanisms of action are not well understood. Viral infections are a known trigger of MS relapses. Toll-like receptors (TLRs) are key components of the innate immune system, which sense conserved structures of viruses and other pathogens. Effects of interferon-β on mRNA levels of all known human TLRs (TLR1-10) and the TLR adaptor molecule MyD88 were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy donors by quantitative real-time PCR and by transcriptome analysis in PBMCs of 25 interferon-β-treated patients with relapsing-remitting MS. Regulation of TLR protein expression by interferon-β was investigated by flow cytometry of leukocyte subsets of healthy subjects and of untreated, interferon-β-, or glatiramer acetate-treated patients with MS. Interferon-β specifically upregulated mRNA expression of TLR3, TLR7, and MyD88 and downregulated TLR9 mRNA in PBMCs of healthy donors as well as in PBMCs of patients with MS. Plasmacytoid dendritic cells (pDCs) were identified as the major cell type responding to interferon-β with increased expression of TLR7 and MyD88 protein. In line with this, expression of TLR7 protein was increased in pDCs of interferon-β-treated, but not untreated or glatiramer acetate-treated patients with MS. Interferon-β-induced upregulation of TLR7 in pDCs is of functional relevance since pre-treatment of PBMCs with interferon-β resulted in a strongly increased production of interferon-α upon stimulation with the TLR7 agonist loxoribine. Flow cytometry confirmed pDCs as the cellular source of interferon-α production induced by activation of TLR7. Thus, upregulation of TLR7 in pDCs and a consequently increased activation of pDCs by TLR7 ligands represents a novel immunoregulatory mechanism of interferon-β. We hypothesize that this mechanism could contribute to a reduction of virus-triggered relapses in patients with MS. PMID:23950974

  11. Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

    PubMed Central

    Neal, Matthew D.; Jia, Hongpeng; Eyer, Benjamin; Good, Misty; Guerriero, Christopher J.; Sodhi, Chhinder P.; Afrazi, Amin; Prindle, Thomas; Ma, Congrong; Branca, Maria; Ozolek, John; Brodsky, Jeffrey L.; Wipf, Peter; Hackam, David J.

    2013-01-01

    Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases. PMID:23776545

  12. Toll-like receptor (TLR)7 and TLR9 agonists enhance interferon (IFN) beta-1a's immunoregulatory effects on B cells in patients with relapsing-remitting multiple sclerosis (RRMS).

    PubMed

    Tao, Yazhong; Zhang, Xin; Markovic-Plese, Silva

    2016-09-15

    We report that B cells from patients with RRMS have decreased endogenous IFN-β secretion and deficient IFN receptor (IFNAR)1/2 and TLR7 gene expression in comparison to healthy controls (HCs), which may contribute to disregulation of cytokine secretion by B cells. We propose that TLR7 and TLR9 stimulation with loxorubin (LOX) and CpG, in combination with exogenous IFN-β may effectively reconstitute endogenous IFN-β production deficit and induce the secretion of immunoregulatory cytokines by B cells. Both LOX/IFN-β and CpG/IFN-β in-vitro treatments of B cells from RRMS patients induced higher endogenous IFN-β gene expression in comparison to the exogenous IFN-β alone. CpG/IFN-β combination induced higher secretion of IL-10, TGF-β, and IL-27 in comparison to stimulation with IFN-β. Our study provides a basis for future clinical studies employing IFN-β and TLR7/9 agonists, which may enhance the resolution of the inflammatory response in RRMS. PMID:27609294

  13. Common mechanisms activate plant guard receptors and TLR4

    PubMed Central

    Kagan, Jonathan C.

    2014-01-01

    In metazoans, the innate immune system uses Pattern Recognition Receptors to detect conserved microbial products, whereas in plants Guard Receptors detect virulence factors or activities encoded by pathogens. In a recent study, Williams and colleagues report that plant Guard receptors can be activated by a mechanism remarkably similar to that of mammalian Toll-like Receptor 4. PMID:25224694

  14. Mapping toll-like receptor signaling pathway genes of Zhikong scallop ( Chlamys farreri) with FISH

    NASA Astrophysics Data System (ADS)

    Zhao, Bosong; Zhao, Liang; Liao, Huan; Cheng, Jie; Lian, Shanshan; Li, Xuan; Huang, Xiaoting; Bao, Zhenmin

    2015-12-01

    Toll-like receptor (TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop ( Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes ( CfTLR, CfMyd88, CfTRAF6, CfNFκB, and CfIκB) containing bacterial artificial chromosomes (BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species.

  15. TLR4-mediated induction of TLR2 signaling is critical in the pathogenesis and resolution of otitis media.

    PubMed

    Leichtle, Anke; Hernandez, Michelle; Pak, Kwang; Yamasaki, Kenshi; Cheng, Chun-Fang; Webster, Nicholas J; Ryan, Allen F; Wasserman, Stephen I

    2009-08-01

    Otitis media is the most prevalent childhood disease in developed countries. The involvement of Toll-like receptors (TLRs) in otitis media pathophysiology has been implicated by studies in cell lines and association studies of TLR gene polymorphisms. However, precise functions of TLRs in the etiology of otitis media in vivo have not been examined. We investigated the inflammatory response to nontypeable Haemophilus influenzae using a model of otitis media in wild-type, TLR2(- /-) and TLR4(-/ -) mice by gene microarray, qPCR, immunohistochemistry, Western blot analysis and histopathology. Toll-like receptor-2(- /-) and TLR4(- /-) mice exhibited a more profound, persistent inflammation with impaired bacterial clearance compared to controls. While wild-type mice induced tumor necrosis factor-a (TNF) after non-typeable H. influenzae challenge, TLR2(-/-) and TLR4(-/-) mice lack TNF induction in the early phase of otitis media. Moreover, lack of TLR2 resulted in a late increase in IL-10 expression and prolonged failure to clear bacteria. Toll-like receptor-4(-/- ) mice showed impaired early bacterial clearance and loss of TLR2 induction in early otitis media. Our results demonstrate that both TLR2 and TLR4 signalling are critical to the regulation of infection in non-typeable H. influenzae-induced otitis media. Toll-like receptor-4 signalling appears to induce TLR2 expression, and TLR2 activation is critical for bacterial clearance and timely resolution of otitis media. PMID:19586996

  16. Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1

    PubMed Central

    Chen, Guo-Yun; Brown, Nicholas K; Wu, Wei; Khedri, Zahra; Yu, Hai; Chen, Xi; van de Vlekkert, Diantha; D'Azzo, Alessandra; Zheng, Pan; Liu, Yang

    2014-01-01

    Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection. DOI: http://dx.doi.org/10.7554/eLife.04066.001 PMID:25187624

  17. Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

    SciTech Connect

    Ohnishi, Koji; Komohara, Yoshihiro; Fujiwara, Yukio; Takemura, Kenichi; Lei, XiaoFeng; Nakagawa, Takenobu; Sakashita, Naomi; Takeya, Motohiro

    2011-08-05

    Highlights: {yields} We focused on the interaction between SR-A and TLR4 signaling in this study. {yields} SR-A deletion promoted NF{kappa}B activation in macrophages in septic model mouse. {yields} SR-A suppresses both MyD88-dependent and -independent TLR4 signaling in vitro. {yields} SR-A clears LPS binding to TLR4 which resulting in the suppression of TLR4 signals. -- Abstract: The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A{sup -/-}) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6 and interferon (IFN)-{beta} were significantly increased in SR-A{sup -/-} mice compared to wild-type mice, and elevated nuclear factor kappa B (NF{kappa}B) activation was detected in SR-A{sup -/-} macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NF{kappa}B in vitro. SR-A deletion also promoted the nuclear translocation of NF{kappa}B and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A{sup -/-} macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.

  18. The TLR2 and TLR4 gene polymorphisms in Moroccan visceral leishmaniasis patients.

    PubMed

    Ejghal, Rajaâ; Hida, Moustapha; Bennani, Mounya Lahkim; Meziane, Mariame; Aurag, Rabia; Lemrani, Meryem

    2016-06-01

    Visceral leishmaniasis (VL) is endemic in the Mediterranean basin and leads to the most severe form of Leishmania infection, lethal if left untreated. However, most infections are sub-clinical or asymptomatic, reflecting the influence of host genetic background on disease outcome. This study aimed to investigate possible association of TLR4 Asp299Gly, TLR4 Thr399Ile and TLR2 Arg753Gln polymorphisms with VL in Moroccan children. We enrolled 119 children with VL caused by Leishmania infantum as well as 138 unrelated children, 95 asymptomatic subjects and 43 healthy individuals who had no evidence of present or past infection. Polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system assay (ARMS-PCR). Results showed significant differences in genotype Thr399Ile and recessive model frequencies between VL and delayed-type hypersensitivity (DTH+) groups (p=0.018, OR=0.414CI 0.195-0.880; p=0.029, OR=0.448CI 0.214-0.938], respectively) by having the amino-acid threonine polymorphism as a reference in the VL group. Concerning the Asp299Gly there were a significant associations when comparing VL vs DTH+ (Asp299Gly genotype p=0.002, OR=0.326CI 0.158-0.671, allele frequencies p=0.033, OR=0.396CI 0.164-0.959, recessive model p=0.002, OR=0.343CI 0.172-0.681) and DTH+ vs DTH- groups (Asp299Gly genotype p=2.160E-4, OR=3.065CI 1.672-5.618, Gly299Gly genotype p=0.047, OR=0.368CI 0.299-0.452, allele frequencies p=1.406E-7, OR=29.571CI 3.907-223.8, recessive model p=4.370E-14, OR=36.965CI 8.629-158.3), by having the aspartic acid polymorphism as a reference these results suggest that the allele A (savage) confer protection against the clinical manifestations but not against the infection. Furthermore, there was a significant association regarding the Arg753Gln genotype (p=0.002, OR=0.326CI 0.158-0.671), allele frequencies (p=0.033, OR=0.396CI 0.164-0.959) and when applying a recessive

  19. Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

    PubMed Central

    Ménager, Pauline; Roux, Pascal; Mégret, Françoise; Bourgeois, Jean-Pierre; Le Sourd, Anne-Marie; Danckaert, Anne; Lafage, Mireille; Préhaud, Christophe; Lafon, Monique

    2009-01-01

    Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit. PMID:19247444

  20. MAP1S Protein Regulates the Phagocytosis of Bacteria and Toll-like Receptor (TLR) Signaling.

    PubMed

    Shi, Ming; Zhang, Yifan; Liu, Leyuan; Zhang, Tingting; Han, Fang; Cleveland, Joseph; Wang, Fen; McKeehan, Wallace L; Li, Yu; Zhang, Dekai

    2016-01-15

    Phagocytosis is a critical cellular process for innate immune defense against microbial infection. The regulation of phagocytosis process is complex and has not been well defined. An intracellular molecule might regulate cell surface-initiated phagocytosis, but the underlying molecular mechanism is poorly understood (1). In this study, we found that microtubule-associated protein 1S (MAP1S), a protein identified recently that is involved in autophagy (2), is expressed primarily in macrophages. MAP1S-deficient macrophages are impaired in the phagocytosis of bacteria. Furthermore, we demonstrate that MAP1S interacts directly with MyD88, a key adaptor of Toll-like receptors (TLRs), upon TLR activation and affects the TLR signaling pathway. Intriguingly, we also observe that, upon TLR activation, MyD88 participates in autophagy processing in a MAP1S-dependent manner by co-localizing with MAP1 light chain 3 (MAP1-LC3 or LC3). Therefore, we reveal that an intracellular autophagy-related molecule of MAP1S controls bacterial phagocytosis through TLR signaling. PMID:26565030

  1. The common food additive carrageenan is not a ligand for Toll-Like- Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model.

    PubMed

    McKim, James M; Wilga, Paul C; Pregenzer, Jeffrey F; Blakemore, William R

    2015-04-01

    Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter molecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins. PMID:25640528

  2. Expression of CCL20 and Its Corresponding Receptor CCR6 Is Enhanced in Active Inflammatory Bowel Disease, and TLR3 Mediates CCL20 Expression in Colonic Epithelial Cells

    PubMed Central

    Skovdahl, Helene Kolstad; Granlund, Atle van Beelen; Østvik, Ann Elisabet; Bruland, Torunn; Bakke, Ingunn; Torp, Sverre Helge; Damås, Jan Kristian; Sandvik, Arne Kristian

    2015-01-01

    Background The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production. Methods A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using in situ hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay. Results CCL20 and CCR6 mRNA abundances were increased during active inflammation (CCL20 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; CCR6 1.8 and 2.0, respectively). CCL20 and CCR6 mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and TLR3 silencing reduced CCL20 mRNA and protein levels. Conclusions The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3. PMID:26536229

  3. Identification, characterization and genetic mapping of the TLR1 gene in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Induction of innate immune pathways is critical for early antimicrobial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and l...

  4. Expression of Toll-like receptor 9 (TLR9) in the lungs and lymphoid tissue of pigs.

    PubMed

    Kuzemtseva, Liudmila; Pérez, Mónica; Mateu, Enric; Segalés, Joaquim; Darwich, Laila

    2015-02-01

    The pattern of distribution of Toll-like receptor 9 (TLR9) in different tissues varies between species. The aim of the present study was to describe the distribution of TLR9 expression in selected tissues and organs of healthy pigs at 3 weeks and 3 months of age. Representative formalin-fixed samples of lung, thymus and secondary lymphoid tissues were evaluated by immunohistochemistry. TLR9 positive staining was observed in epithelial cells, vascular endothelium and myoepithelial-like cells, as well as in cells of the alveolar septa of the lung. Antigen presenting cells of perifollicular zones (interdigitating, macrophage and dendritic-like cells) of the Peyer's patches, lymph nodes, spleen and thymus were also immunoreactive for TLR9. No differences were seen in TLR9 protein expression in tissues from the two age groups. PMID:25576141

  5. Should a Toll-like receptor 4 (TLR-4) agonist or antagonist be designed to treat cancer? TLR-4: its expression and effects in the ten most common cancers

    PubMed Central

    Mai, Chun Wai; Kang, Yew Beng; Pichika, Mallikarjuna Rao

    2013-01-01

    Toll-like receptor 4 (TLR-4) is well known for its host innate immunity. Despite the fact that TLR-4 activation confers antitumor responses; emerging evidence suggests that TLR-4 is associated with tumor development and progression. It is now clear that overactivation of TLR-4, through various immune mediators, may cause immune response dysfunction, resulting in tumorigenesis. Different cancers could have different extents of TLR-4 involvement during tumorigenesis or tumor progression. In this review, we focus on infection- and inflammation-related TLR-4 activation in noncancer and cancer cells, as well as on the current evidence about the role of TLR-4 in ten of the most common cancers, viz, head and neck cancer, lung cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, skin cancer, breast cancer, ovarian cancer, cervical cancer, and prostate cancer. PMID:24235843

  6. Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5.

    PubMed

    Cabral, José Miguel; Grácio, Daniela; Soares-da-Silva, Patrício; Magro, Fernando

    2015-10-15

    Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 μg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics. PMID:26294670

  7. Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils*

    PubMed Central

    Ueno-Shuto, Keiko; Kato, Kosuke; Tasaki, Yukihiro; Sato, Miki; Sato, Keizo; Uchida, Yuji; Sakai, Hiromichi; Ono, Tomomi; Suico, Mary Ann; Mitsutake, Kazunori; Tokutomi, Naofumi; Kai, Hirofumi; Shuto, Tsuyoshi

    2014-01-01

    Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells. PMID:24821721

  8. Post-bronchiolitis wheezing is associated with toll-like receptor 9 rs187084 gene polymorphism.

    PubMed

    Nuolivirta, Kirsi; Törmänen, Sari; Teräsjärvi, Johanna; Vuononvirta, Juho; Koponen, Petri; Korppi, Matti; Helminen, Merja; Peltola, Ville; He, Qiushui

    2016-01-01

    Innate immunity receptors play a critical role in host defence, as well as in allergy and asthma. The aim of this exploratory study was to evaluate whether there are associations between TLR7 rs179008, TLR8 rs2407992, TLR9 rs187084 or TLR10 rs4129009 polymorphisms and viral findings, clinical characteristics or subsequent wheezing in infants with bronchiolitis. In all, 135 full-term infants were hospitalized for bronchiolitis at age less than 6 months: 129 of them were followed-up until the age of 1.5 years. The outcome measures were repeated wheezing, use of inhaled corticosteroids, atopic dermatitis during the first 1.5 years of life and total serum immunoglobulin E (IgE). There were no significant associations between the genotypes or allele frequencies of TLR7 rs179008, TLR8 rs2407992, TLR9 rs187084 or TLR10 rs4129009 polymorphisms and clinical characteristics or the severity of bronchiolitis during hospitalization. During follow-up, repeated wheezing was more common in children with TLR9 rs187084 variant genotype CC (30.5%) than in children with TLR9 wild-type genotype TT (12.2%) (p = 0.02, aOR 2.73, 95% CI 1.02-7.29). The TLR10 rs4129009 minor allele G was associated with elevated total serum IgE. TLR9 rs187084 gene polymorphism may be associated with post-bronchiolitis wheezing, and TLR10 rs4129009 gene polymorphism may be associated with atopy. PMID:27498757

  9. Post-bronchiolitis wheezing is associated with toll-like receptor 9 rs187084 gene polymorphism

    PubMed Central

    Nuolivirta, Kirsi; Törmänen, Sari; Teräsjärvi, Johanna; Vuononvirta, Juho; Koponen, Petri; Korppi, Matti; Helminen, Merja; Peltola, Ville; He, Qiushui

    2016-01-01

    Innate immunity receptors play a critical role in host defence, as well as in allergy and asthma. The aim of this exploratory study was to evaluate whether there are associations between TLR7 rs179008, TLR8 rs2407992, TLR9 rs187084 or TLR10 rs4129009 polymorphisms and viral findings, clinical characteristics or subsequent wheezing in infants with bronchiolitis. In all, 135 full-term infants were hospitalized for bronchiolitis at age less than 6 months: 129 of them were followed-up until the age of 1.5 years. The outcome measures were repeated wheezing, use of inhaled corticosteroids, atopic dermatitis during the first 1.5 years of life and total serum immunoglobulin E (IgE). There were no significant associations between the genotypes or allele frequencies of TLR7 rs179008, TLR8 rs2407992, TLR9 rs187084 or TLR10 rs4129009 polymorphisms and clinical characteristics or the severity of bronchiolitis during hospitalization. During follow-up, repeated wheezing was more common in children with TLR9 rs187084 variant genotype CC (30.5%) than in children with TLR9 wild-type genotype TT (12.2%) (p = 0.02, aOR 2.73, 95% CI 1.02–7.29). The TLR10 rs4129009 minor allele G was associated with elevated total serum IgE. TLR9 rs187084 gene polymorphism may be associated with post-bronchiolitis wheezing, and TLR10 rs4129009 gene polymorphism may be associated with atopy. PMID:27498757

  10. TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Toll-like receptor 4 (TLR4) is a key factor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria. Previous studies from our group identified differences in the expression profile of TLR4 and genes affected by the TLR4 signaling pathway among pigs that shed varying...

  11. A regulatory role for macrophage class A scavenger receptors in TLR4-mediated LPS responses.

    PubMed

    Chen, Yunying; Wermeling, Fredrik; Sundqvist, Johanna; Jonsson, Ann-Beth; Tryggvason, Karl; Pikkarainen, Timo; Karlsson, Mikael C I

    2010-05-01

    Recognition of microbial components by TLR, key sensors of infection, leads to induction of inflammatory responses. We found that, in vivo, TLR4 engagement by LPS induces up-regulation of the class A scavenger receptors (SR) macrophage receptor with a collagenous structure (MARCO) and SR-A, which occurs, at least in the case of MARCO, via both MyD88-dependent and -independent pathways. When challenging mice with a low dose of LPS followed by a high dose, class A SR-deficient mice showed a higher survival rate than WT mice. This was paired with increased production of IL-10 and anti-LPS Ab, as well as increased activation status of marginal zone B cells. However, the receptors were not crucial for survival when challenging mice i.p. with Neisseria meningitidis or Listeria monocytogenes, but they were found to contribute to microbial capture and clearance. This indicates physiological significance for the up-regulation of class A SR during early stages of bacterial infection. Thus, we believe that we have revealed a mechanism where SR regulate the activation status of the immune system and are involved in balancing a proper immune response to infection. This regulation could also be important in maintaining tolerance since these receptors have been shown to be involved in regulation of self-reactivity. PMID:20162551

  12. Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR2) is involved in vascular function

    PubMed Central

    Bucci, M; Vellecco, V; Harrington, L; Brancaleone, V; Roviezzo, F; Mattace Raso, G; Ianaro, A; Lungarella, G; De Palma, R; Meli, R; Cirino, G

    2013-01-01

    Background and Purpose Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR2 and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Experimental Approach Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR2 activating peptide (AP) was used as a PAR2 agonist. Aortas harvested from TLR4–/– mice were also used to characterize the PAR2 response. Key Results PAR2, but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR2AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR2AP-induced vasorelaxation and PAR2AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4–/– mice, the expression of PAR2 was reduced and the PAR2AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Conclusions and Implications Cross-talk between PAR2 and TLR4 contributes to vascular homeostasis. PMID:22957757

  13. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    PubMed

    Duriez, Marion; Quillay, Héloïse; Madec, Yoann; El Costa, Hicham; Cannou, Claude; Marlin, Romain; de Truchis, Claire; Rahmati, Mona; Barré-Sinoussi, Françoise; Nugeyre, Marie-Thérèse; Menu, Elisabeth

    2014-01-01

    Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface. PMID:25071732

  14. Polymorphism and haplotype structure in River Buffalo (Bubalus bubalis) toll-like receptor 5 (TLR5) coding sequence.

    PubMed

    Jones, B C; Womack, J E

    2012-04-01

    Most of the 160 million river buffalo in the world are in Asia where they are used extensively, both as a food source and for draught power. Only recently have investigations begun exploring the buffalo genome for variation that might influence health and productivity of these economically important animals. This paper describes the sequence variability of the toll-like receptor 5 (TLR5) gene, which recognizes bacterial flagellin and is a key player in the immune system. TLR5 is comprised of a single exon that is 2577 bp and codes 858 amino acids. We examined single-nucleotide polymorphisms (SNPs) located within the coding region. Overall, 17 SNPs were discovered, seven of which are non-synonymous. Our study population yielded four different haplotypes. We examined predicted protein domain structure and found that river buffalo, swamp buffalo, and African Forest buffalo shared the same protein domain structure and are more similar to each other than they are to cattle and American bison, which are similar to each other. PolyPhen 2 analysis revealed one amino acid substitution in the river buffalo population with potential functional significance. PMID:22537062

  15. Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

    PubMed

    Peng, Jian; Tao, Xin; Li, Rui; Hu, Jingru; Ruan, Jie; Wang, Ruihua; Yang, Manyi; Yang, Rirong; Dong, Xiangru; Chen, Shangwu; Xu, Anlong; Yuan, Shaochun

    2015-10-01

    Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate. PMID:26324776

  16. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Breyer, Richard M.; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2012-01-01

    A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor. PMID:22499581

  17. Polymorphisms of Chicken TLR3 and 7 in Different Breeds

    PubMed Central

    Ruan, Wenke; An, Jian; Wu, Yanhua

    2015-01-01

    Toll-like receptors (TLRs) mediate immune responses via the recognition of pathogen-associated molecular patterns (PAMPs), thus playing important roles in host defense. Among the chicken (Ch) TLR family, ChTLR3 and 7 have been shown to recognize viral RNA. In our earlier studies, we have reported polymorphisms of TLR1, 2, 4, 5, 15 and 21. In the present study, we amplified TLR3 and 7 genes from different chicken breeds and analyzed their sequences. We identified 7 amino acid polymorphism sites in ChTLR3 with 6 outer part sites and 1 inner part site, and 4 amino acid polymorphism sites in ChTLR7 with 3 outer part sites and 1 inner part site. These results demonstrate that ChTLR genes are polymorphic among different chicken breeds, suggesting a varied resistance across numerous chicken breeds. This information might help improve chicken health by breeding and vaccination. PMID:25781886

  18. Characterization of Toll-like receptor gene expression in goldfish (Carassius auratus) during Dactylogyrus intermedius infection.

    PubMed

    Tu, Xiao; Liu, Lei; Qi, Xiaozhou; Chen, Weichao; Wang, Gaoxue; Ling, Fei

    2016-10-01

    Toll-like receptors (TLRs), the first and best understood innate immune receptors, play a notable role in the innate immune system by sensing pathogenic agents and initiating appropriate immune responses. However, studies about the roles of fish TLRs in response to the infection of the ectoparasitic monogenean Dactylogyrus intermedius have been surprisingly vacant. In the present study, cDNA fragments of five members of TLRs family in goldfish (Carassius auratus) were cloned and the expression patterns of nine TLRs in five tissues at different time points during D. intermedius infection were subsequently investigated. We found that the expressions of TLR4, TLR5, TLR20 and TLR22 were significantly elevated after infection at some time points, of which the transcription of TLR5 was progressively increased nearly in all tissues, whereas the mRNA levels of other TLRs (TLR2, 3, 7, 9 and 21) were down-regulated or showed no significant change compared with the control at most time points. Additionally, this paper was also conducted to explore the expression of above TLRs after re-infected with D. intermedius. The results showed a significant upregulation of TLR4, TLR5 and TLR22 in all tested tissues at these two time points, especially the levels of TLR4 and TLR22 expression, were even higher comparing with the first infection. Besides, tissue-specific expression analysis revealed that spleen featured the highest expressions of almost all the TLR-encoding genes among detected tissues. The informations obtained here could be helpful towards understanding the functions of TLRs in response to parasitic infection in goldfish and provide new insights for the development of preventive and therapeutic approaches against D. intermedius infection. PMID:27238769

  19. The role of Toll-like receptor 4 (TLR4) in cardiac ischaemic-reperfusion injury, cardioprotection and preconditioning.

    PubMed

    Lee, Sam Man; Hutchinson, Mark; Saint, David A

    2016-09-01

    Cardiac ischaemic-reperfusion injury (IRI) remains the primary cause of mortality throughout the developed world. Molecular mechanisms underlying IRI are complex and are often interlinked with each other driving a synergistic response. Toll-like receptor 4 (TLR4), an immunosurveillance receptor, is known to enhance tissue injury during IRI by enhancing the inflammatory response. The release of endogenous components during IRI bind onto TLR4 leading to the activation of multiple signalling kinases. Once this event occurs these proteins are defined as danger associated molecular patterns molecules (DAMPs) or alarmins. Examples include heat shock proteins, high mobility group box one (HMGB1) and extracellular matrix proteins, all of which are involved in IRI. However, literature in the last two decades suggests that transient stimulation of TLR4 may suppress IRI and thus improve cardiac recovery. Furthermore, it remains to be seen what role TLR4 plays during ischaemic-preconditioning where acute bouts of ischaemia, preceding a harmful bout of ischaemic-reperfusion, is cardioprotective. The other question which also needs to be considered is that if transient TLR4 signalling drives a preconditioning response then what are the ligands which drive this? Hence the second part of this review explores the possible TLR4 ligands which may promote cardioprotection against IRI. PMID:27249055

  20. Preconditioning of Microglia by α-Synuclein Strongly Affects the Response Induced by Toll-like Receptor (TLR) Stimulation

    PubMed Central

    Gonzalez-Rey, Elena; Lachaud, Christian C.; Guilliams, Tim; Fernandez-Montesinos, Rafael; Benitez-Rondan, Alicia; Robledo, Gema; Hmadcha, Abdelkrim; Delgado, Mario; Dobson, Christopher M.; Pozo, David

    2013-01-01

    In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson’s disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer’s disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies. PMID:24236103

  1. Structural characterisation of Toll-like receptor 1 (TLR1) and Toll-like receptor 6 (TLR6) in elephant and harbor seals.

    PubMed

    Woodman, Sally; Gibson, Amanda J; García, Ana Rubio; Contreras, Guillermo Sanchez; Rossen, John W; Werling, Dirk; Offord, Victoria

    2016-01-01

    Pinnipeds are a diverse clade of semi-aquatic mammals, which act as key indicators of ecosystem health. Their transition from land to marine environments provides a complex microbial milieu, making them vulnerable to both aquatic and terrestrial pathogens, thereby contributing to pinniped population decline. Indeed, viral pathogens such as influenza A virus and phocine distemper virus (PDV) have been identified as the cause of several of these mass mortality events. Furthermore, bacterial infection with mammalian Brucella sp. and methicillin-resistant Staphylococcus aureus strains have also been observed in marine mammals, posing further risk to both co-habiting endangered species and public health. During these disease outbreaks, mortality rates have varied amongst different pinniped species. Analyses of innate immune receptors at the host-pathogen interface have previously identified variants which may drive these species-specific responses. Through a combination of both sequence- and structure-based methods, this study characterises members of the Toll-like receptor (TLR) 1 superfamily from both harbour and elephant seals, identifying variations which will help us to understand these species-specific innate immune responses, potentially aiding the development of specific vaccine-adjuvants for these species. PMID:26827833

  2. Association of Toll-like receptors 2, 3, and 4 genes polymorphisms with periapical pathosis risk

    PubMed Central

    Özan, Ülkü; Ocak, Zeynep; Özan, Fatih; Oktay, Elif-Aybala; Şahman, Halil; Yikilgan, İhsan; Oruçoğlu, Hasan; Er, Kürşat

    2016-01-01

    Background The aim of this study was to investigate the role of gene variations of Toll-like receptors (TLR) 2, 3, and 4 on genetic susceptibility to periapical pathosis. Material and Methods One hundred patients were included in the study and divided into two groups as follows; Control Group (n=50) that have root canal treatment and no periapical lesion, Patient Group (n=50) that have root canal treatment and periapical lesion. TLR2 Arg753Gln, TLR3 (c.1377C/T) and TLR4 Asp299Gly and Thr399Ile polymorphisms were genotyped by using PCR-RFLP. Genotypical analysis of control and patient groups were investigated to disclose whether there is any association between periapical lesions and gene variations. Results There are no significant statistical differences between control and patient groups according to TLR 2 and 4 gene sequence. On the contrary, CC allele detected 74% for TLR 3 in patient group, and this difference was found to be statistically significant (p < 0.005). Conclusions According to these results, it can be suggested that patients with Toll-like receptor 3 gene polymorphisms could be susceptible to periapical pathosis. Key words:Toll-like receptors, periapical pathosis, endodontics. PMID:27031066

  3. TLR Stimulation Dynamically Regulates Heme and Iron Export Gene Expression in Macrophages.

    PubMed

    Philip, Mary; Chiu, Edison Y; Hajjar, Adeline M; Abkowitz, Janis L

    2016-01-01

    Pathogenic bacteria have evolved multiple mechanisms to capture iron or iron-containing heme from host tissues or blood. In response, organisms have developed defense mechanisms to keep iron from pathogens. Very little of the body's iron store is available as free heme; rather nearly all body iron is complexed with heme or other proteins. The feline leukemia virus, subgroup C (FeLV-C) receptor, FLVCR, exports heme from cells. It was unknown whether FLVCR regulates heme-iron availability after infection, but given that other heme regulatory proteins are upregulated in macrophages in response to bacterial infection, we hypothesized that macrophages dynamically regulate FLVCR. We stimulated murine primary macrophages or macrophage cell lines with LPS and found that Flvcr is rapidly downregulated in a TLR4/MD2-dependent manner; TLR1/2 and TLR3 stimulation also decreased Flvcr expression. We identified several candidate TLR-activated transcription factors that can bind to the Flvcr promoter. Macrophages must balance the need to sequester iron from systemic circulating or intracellular pathogens with the macrophage requirement for heme and iron to produce reactive oxygen species. Our findings underscore the complexity of this regulation and point to a new role for FLVCR and heme export in macrophages responses to infection and inflammation. PMID:27006955

  4. TLR Stimulation Dynamically Regulates Heme and Iron Export Gene Expression in Macrophages

    PubMed Central

    Philip, Mary; Chiu, Edison Y.; Hajjar, Adeline M.; Abkowitz, Janis L.

    2016-01-01

    Pathogenic bacteria have evolved multiple mechanisms to capture iron or iron-containing heme from host tissues or blood. In response, organisms have developed defense mechanisms to keep iron from pathogens. Very little of the body's iron store is available as free heme; rather nearly all body iron is complexed with heme or other proteins. The feline leukemia virus, subgroup C (FeLV-C) receptor, FLVCR, exports heme from cells. It was unknown whether FLVCR regulates heme-iron availability after infection, but given that other heme regulatory proteins are upregulated in macrophages in response to bacterial infection, we hypothesized that macrophages dynamically regulate FLVCR. We stimulated murine primary macrophages or macrophage cell lines with LPS and found that Flvcr is rapidly downregulated in a TLR4/MD2-dependent manner; TLR1/2 and TLR3 stimulation also decreased Flvcr expression. We identified several candidate TLR-activated transcription factors that can bind to the Flvcr promoter. Macrophages must balance the need to sequester iron from systemic circulating or intracellular pathogens with the macrophage requirement for heme and iron to produce reactive oxygen species. Our findings underscore the complexity of this regulation and point to a new role for FLVCR and heme export in macrophages responses to infection and inflammation. PMID:27006955

  5. Coordinated Activation of Toll-Like Receptor8 (TLR8) and NLRP3 by the TLR8 Agonist, VTX-2337, Ignites Tumoricidal Natural Killer Cell Activity

    PubMed Central

    Dietsch, Gregory N.; Lu, Hailing; Yang, Yi; Morishima, Chihiro; Chow, Laura Q.; Disis, Mary L.; Hershberg, Robert M.

    2016-01-01

    VTX-2337 (USAN: motolimod) is a selective toll-like receptor 8 (TLR8) agonist, which is in clinical development as an immunotherapy for multiple oncology indications, including squamous cell carcinoma of the head and neck (SCCHN). Activation of TLR8 enhances natural killer cell activation, increases antibody-dependent cell-mediated cytotoxicity, and induces Th1 polarizing cytokines. Here, we show that VTX-2337 stimulates the release of mature IL-1β and IL-18 from monocytic cells through coordinated actions on both TLR8 and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome complex. In vitro, VTX-2337 primed monocytic cells to produce pro-IL-1β, pro-IL-18, and caspase-1, and also activated the NLRP3 inflammasome, thereby mediating the release of mature IL-1β family cytokines. Inhibition of caspase-1 blocked VTX-2337-mediated NLRP3 inflammasome activation, but had little impact on production of other TLR8-induced mediators such as TNFα. IL-18 activated natural killer cells and complemented other stimulatory pathways, including FcγRIII and NKG2D, resulting in IFNγ production and expression of CD107a. NLRP3 activation in vivo was confirmed by a dose-related increase in plasma IL-1β and IL-18 levels in cynomolgus monkeys administered VTX-2337. These results are highly relevant to clinical studies of combination VTX-2337/cetuximab treatment. Cetuximab, a clinically approved, epidermal growth factor receptor-specific monoclonal antibody, activates NK cells through interactions with FcγRIII and facilitates ADCC of tumor cells. Our preliminary findings from a Phase I open-label, dose-escalation, trial that enrolled 13 patients with recurrent or metastatic SCCHN show that patient NK cells become more responsive to stimulation by NKG2D or FcγRIII following VTX-2337 treatment. Together, these results indicate that TLR8 stimulation and inflammasome activation by VTX-2337 can complement FcγRIII engagement and may augment clinical responses in SCCHN

  6. Bryostatin-1, a Naturally Occurring Antineoplastic Agent, Acts as a Toll-like Receptor 4 (TLR-4) Ligand and Induces Unique Cytokines and Chemokines in Dendritic Cells*

    PubMed Central

    Ariza, Maria Eugenia; Ramakrishnan, Rupal; Singh, Narendra P.; Chauhan, Ashok; Nagarkatti, Prakash S.; Nagarkatti, Mitzi

    2011-01-01

    Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11chighCD8α− CD11b+CD4+ phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent. PMID:21036898

  7. TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

    PubMed Central

    de Oliveira, Cristiane Beatriz; Vasconcellos, Cídia; Sakai-Valente, Neusa Y.; Sotto, Mirian Nacagami; Luiz, Fernanda Guedes; Belda, Walter; de Sousa, Maria da Gloria Teixeira; Benard, Gil; Criado, Paulo Ricardo

    2015-01-01

    There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections. PMID:25651327

  8. Toll like receptor (TLR)-4 as a regulator of peripheral endogenous opioid-mediated analgesia in inflammation

    PubMed Central

    2014-01-01

    Background Leukocytes containing opioid peptides locally control inflammatory pain. In the early phase of complete Freund’s adjuvant (CFA)-induced hind paw inflammation, formyl peptides (derived e.g. from Mycobacterium butyricum) trigger the release of opioid peptides from neutrophils contributing to tonic basal antinociception. In the later phase we hypothesized that toll-like-receptor-(TLR)-4 activation of monocytes/macrophages triggers opioid peptide release and thereby stimulates peripheral opioid-dependent antinociception. Results In Wistar rats with CFA hind paw inflammation in the later inflammatory phase (48–96 h) systemic leukocyte depletion by cyclophosphamide (CTX) or locally injected naloxone (NLX) further decreased mechanical and thermal nociceptive thresholds. In vitro β-endorphin (β-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14+CD16- or non-classical M2 macrophages. Monocytes expressing TLR4 dose-dependently released β-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. Despite TLR4 expression proinflammatory M1 and anti-inflammatory M2 macrophages only secreted opioid peptides in response to ionomycin, a calcium ionophore. Intraplantar injection of LPS as a TLR4 agonist into the inflamed paw elicited an immediate opioid- and dose-dependent antinociception, which was blocked by TAK-242, a small-molecule inhibitor of TLR4, or by peripheral applied NLX. In the later phase LPS lowered mechanical and thermal nociceptive thresholds. Furthermore, local peripheral TLR4 blockade worsened thermal and mechanical nociceptive pain thresholds in CFA inflammation. Conclusion Endogenous opioids from monocytes/macrophages mediate endogenous antinociception in the late phase of inflammation. Peripheral TLR4 stimulation acts as a transient counter-regulatory mechanism for inflammatory pain in vivo, and increases the release of opioid peptides from monocytes in vitro. TLR4

  9. Species-specific engagement of human nucleotide oligomerization domain 2 (NOD)2 and Toll-like receptor (TLR) signalling upon intracellular bacterial infection: role of Crohn's associated NOD2 gene variants.

    PubMed

    Salem, M; Seidelin, J B; Eickhardt, S; Alhede, M; Rogler, G; Nielsen, O H

    2015-03-01

    Recognition of bacterial peptidoglycan-derived muramyl-dipeptide (MDP) by nucleotide oligomerization domain 2 (NOD2) induces crucial innate immune responses. Most bacteria carry the N-acetylated form of MDP (A-MDP) in their cell membranes, whereas N-glycolyl MDP (G-MDP) is typical for mycobacteria. Experimental murine studies have reported G-MDP to have a greater NOD2-stimulating capacity than A-MDP. As NOD2 polymorphisms are associated with Crohn's disease (CD), a link has been suggested between mycobacterial infections and CD. Thus, the aim was to investigate if NOD2 responses are dependent upon type of MDP and further to determine the role of NOD2 gene variants for the bacterial recognition in CD. The response pattern to A-MDP, G-MDP, Mycobacterium segmatis (expressing mainly G-MDP) and M. segmatisΔnamH (expressing A-MDP), Listeria monocytogenes (LM) (an A-MDP-containing bacteria) and M. avium paratuberculosis (MAP) (a G-MDP-containing bacteria associated with CD) was investigated in human peripheral blood mononuclear cells (PBMCs). A-MDP and M. segmatisΔnamH induced significantly higher tumour necrosis factor (TNF)-α protein levels in healthy wild-type NOD2 PBMCs compared with G-MDP and M. segmatis. NOD2 mutations resulted in a low tumour necrosis factor (TNF)-α protein secretion following stimulation with LM. Contrary to this, TNF-α levels were unchanged upon MAP stimulation regardless of NOD2 genotype and MAP solely activated NOD2- and Toll-like receptor (TLRs)-pathway with an enhanced production of interleukin (IL)-1β and IL-10. In conclusion, the results indicate that CD-associated NOD2 deficiencies might affect the response towards a broader array of commensal and pathogenic bacteria expressing A-MDP, whereas they attenuate the role of mycobacteria in the pathogenesis of CD. PMID:25335775

  10. Species-specific engagement of human nucleotide oligomerization domain 2 (NOD)2 and Toll-like receptor (TLR) signalling upon intracellular bacterial infection: role of Crohn’s associated NOD2 gene variants

    PubMed Central

    Salem, M; Seidelin, J B; Eickhardt, S; Alhede, M; Rogler, G; Nielsen, O H

    2015-01-01

    Recognition of bacterial peptidoglycan-derived muramyl-dipeptide (MDP) by nucleotide oligomerization domain 2 (NOD2) induces crucial innate immune responses. Most bacteria carry the N-acetylated form of MDP (A-MDP) in their cell membranes, whereas N-glycolyl MDP (G-MDP) is typical for mycobacteria. Experimental murine studies have reported G-MDP to have a greater NOD2-stimulating capacity than A-MDP. As NOD2 polymorphisms are associated with Crohn's disease (CD), a link has been suggested between mycobacterial infections and CD. Thus, the aim was to investigate if NOD2 responses are dependent upon type of MDP and further to determine the role of NOD2 gene variants for the bacterial recognition in CD. The response pattern to A-MDP, G-MDP, Mycobacterium segmatis (expressing mainly G-MDP) and M. segmatisΔnamH (expressing A-MDP), Listeria monocytogenes (LM) (an A-MDP-containing bacteria) and M. avium paratuberculosis (MAP) (a G-MDP-containing bacteria associated with CD) was investigated in human peripheral blood mononuclear cells (PBMCs). A-MDP and M. segmatisΔnamH induced significantly higher tumour necrosis factor (TNF)-α protein levels in healthy wild-type NOD2 PBMCs compared with G-MDP and M. segmatis. NOD2 mutations resulted in a low tumour necrosis factor (TNF)-α protein secretion following stimulation with LM. Contrary to this, TNF-α levels were unchanged upon MAP stimulation regardless of NOD2 genotype and MAP solely activated NOD2- and Toll-like receptor (TLRs)-pathway with an enhanced production of interleukin (IL)-1β and IL-10. In conclusion, the results indicate that CD-associated NOD2 deficiencies might affect the response towards a broader array of commensal and pathogenic bacteria expressing A-MDP, whereas they attenuate the role of mycobacteria in the pathogenesis of CD. PMID:25335775

  11. Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC).

    PubMed

    Bonnefont-Rebeix, Catherine; Marchal, Thierry; Bernaud, Janine; Pin, Jean-Jacques; Leroux, Caroline; Lebecque, Serge; Chabanne, Luc; Rigal, Dominique

    2007-07-15

    Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs. PMID:17521746

  12. Toll-like receptor 4 (TLR4) is required for protective immunity to larval Strongyloides stercoralis in mice.

    PubMed

    Kerepesi, Laura A; Hess, Jessica A; Leon, Ofra; Nolan, Thomas J; Schad, Gerhard A; Abraham, David

    2007-01-01

    TLR4 is important for immunity to various unicellular organisms and has been implicated in the immune responses to helminth parasites. The immune response against helminths is generally Th2-mediated and studies have shown that TLR4 is required for the development of a Th2 response against allergens and helminth antigens in mice. C3H/HeJ mice, which have a point mutation in the Tlr4 gene, were used in this study to determine the role of TLR4 in protective immunity to the nematode Strongyloides stercoralis. It was demonstrated that TLR4 was not required for killing larval S. stercoralis during the innate immune response, but was required for killing the parasites during the adaptive immune response. No differences were seen in the IL-5 and IFN-gamma responses, antibody responses or cell recruitment between wild type and C3H/HeJ mice after immunization. Protective immunity was restored in immunized C3H/HeJ mice by the addition of wild type peritoneal exudate cells in the environment of the larvae. It was therefore concluded that the inability of TLR4-mutant mice to kill larval S. stercoralis during the adaptive immune response is due to a defect in the effector cells recruited to the microenvironment of the larvae. PMID:17196865

  13. Single nucleotide polymorphisms in toll-like receptor genes and case-control association studies with bovine tuberculosis

    PubMed Central

    Bhaladhare, Ashish; Sharma, Deepak; Kumar, Amit; Sonwane, Arvind; Chauhan, Anuj; Singh, Ranvir; Kumar, Pushpendra; Yadav, Ramji; Baqir, Mohd; Bhushan, Bharat; Prakash, Om

    2016-01-01

    Aim: Toll-like receptor 2 (TLR2) and TLR4 genes play critical roles in host recognition of Mycobacterium bovis infection and initiation of innate and adaptive immune response. The present study was aimed at exploring the association of seven single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 genes with susceptibility/resistance against bovine tuberculosis (bTB) infection in cattle. Materials and Methods: A case-control resource population of 35 positive and 45 negative animals was developed after screening with single intradermal tuberculin test for bTB. Resource population was screened for SNPs in TLR2 and TLR4 genes using polymerase chain reaction-restriction fragment length polymorphism. The PROC LOGISTIC procedure of SAS 9.3 was used to find an association of allelic and genotypic frequencies with bTB. Results: In TLR2 gene, two of SNPs under study (rs55617172 and rs68268253) revealed polymorphism while in the case of TLR4 gene all four SNPs under investigation (rs8193041, rs207836014, rs8193060, and rs8193069) were found to be polymorphic in case-control population. SNP locus rs55617172 in TLR2 gene was found significantly (p<0.01) associated with susceptibility/resistance to TB in cattle. Conclusion: These findings indicate the presence of SNPs in TLR2 and TLR4 genes in our resource population. Upon validation in independent, large resource population and following biological characterization, SNP rs55617172 can be incorporated in marker panel for selection of animals with greater resistance to bTB. PMID:27284220

  14. Lack of Association between Polymorphisms of the TLR4 Gene and Infection with the Hepatitis B and C Viruses

    PubMed Central

    Pires-Neto, Orlando de Souza; de Sá, Keyla Santos Guedes; Santana, Barbara Brasil; Tatielle Monteiro Gomes, Samara; da Silva Graça Amoras, Ednelza; da Silva Conde, Simone Regina; Demachki, Sâmia; Azevedo, Vânia Nakauth; Martins-Feitosa, Rosimar Neris; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

    2015-01-01

    Toll-like receptor 4 (TLR4) plays a crucial role in the early recognition of pathogenic microorganisms and provides an ideal model to investigate the consequences of genetic variation and susceptibility to diseases. The present study investigated the occurrence of the single nucleotide polymorphisms (SNPs) rs4986790 (A>G) and rs4986791 (C>T) in the TLR4 gene in chronic carriers of the hepatitis B (HBV) and C (HCV) viruses. A total of 420 blood samples were collected (HBV, 49; HCV, 72; and controls, 299) at the liver disease outpatient clinic of Hospital da Fundação Santa Casa de Misericórdia do Pará (FSCMPA). Genomic DNA extracted from leukocytes was subjected to real-time polymerase chain reaction (qPCR) analysis to identify the genetic profile of the participants. No significant differences were found in the allele and genotype frequencies between the infected participants and controls. No significant associations were found between the investigated polymorphisms and inflammatory activity, fibrosis, and the presence of cirrhosis; the same results were obtained in the haplotype analysis. The results showed a lack of association between the rs4986790 and rs4986791 SNPs and susceptibility to infection with HBV and HCV, as well as clinical and laboratory information of the patients. PMID:26347404

  15. The NRAMP1, VDR, TNF-α, ICAM1, TLR2 and TLR4 gene polymorphisms in Iranian patients with pulmonary tuberculosis: A case-control study.

    PubMed

    Jafari, Mohammad; Nasiri, Mohammad Reza; Sanaei, Roozbeh; Anoosheh, Saber; Farnia, Parisa; Sepanjnia, Adel; Tajik, Nader

    2016-04-01

    The innate immune response drives early events in Mycobacterium tuberculosis infection. Since human genetic variation is an important determinant in the outcome of infection with M. tuberculosis, we typed polymorphisms in the innate immune molecules, such as natural-resistance-associated macrophage protein 1 (NRAMP1), Vitamin D receptor (VDR), Tumor necrosis factor alpha (TNF-α), intercellular adhesion molecule1 (ICAM-1), Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in a case-control study of pulmonary tuberculosis in Iranian population. We conducted an association study and included 96 patients and 122 matched healthy individuals. We used single ARMS-PCR technique to simultaneously genotype fourteen polymorphisms in this survey. Among all fourteen polymorphisms that were examined, three polymorphisms were significantly different between case and control groups. The TNF -308A polymorphism showed significant increase in allele and genotype frequencies among patients compared to control individuals [-308A allele: 19.3 vs. 9.4%, GA genotype: 28.1 vs. 17.2%, AA genotype: 5.2 vs. 0.8%; Corrected P (Pc)<0.05], and the TLR4 variant allele and genotypes prevalence (D299G and T399I) were significantly higher among patients compared to controls [DG genotype: 14.6 vs. 5.7%, Pc<0.05 and I399 allele: 4.2 vs. 0.8%, TI genotype: 8.3 vs. 1.6%; Pc<0.05], respectively. In conclusion, our data suggest that TLR4 (D299G and T399I) and TNF (-308G/A) genetic polymorphisms may influence the risk of developing tuberculosis after exposure to Mycobacterium. PMID:26774366

  16. Toll-like receptor 4 gene polymorphism is associated with chronic periodontitis.

    PubMed

    Ding, Yuan-Sheng; Zhao, Yue; Xiao, Yuan-Yuan; Zhao, Gang

    2015-01-01

    Toll-like receptors (TLRs) contribute to the immune response by recognizing patterns presented by bacteria and other pathogens. These receptors have been implicated in the inflammatory response that contributes to gingivitis and periodontitis. Conflicting reports have suggested that variations in the genes encoding TLRs, particularly TLR2 and TLR4, may influence susceptibility to periodontitis. In this study, the contribution of variations in the genes encoding TLR2 and TLR4 in the context of periodontitis was examined in 254 patients with moderate periodontitis, 418 patients with severe periodontitis, and 260 healthy controls free of gum disease. Genomic DNA was extracted from participants' whole blood, and genotyping of TLR2/TLR4 as performed using real-time polymerase chain reaction with TaqMan MGB primer. The genotype, allele, and haplotype frequencies were compared among control, moderate periodontitis, and severe periodontitis groups. Statistical analysis was performed using chi-square and logistic regression analyses. Of the 9 polymorphic loci detected in the two genes, one, rs11536889 (G>C) in TLR4, displayed a statistically significant difference in distribution between individuals with moderate periodontitis and severe periodontitis (P<0.05). The distribution of the GG genotype in moderate periodontitis was higher than in the severe periodontitis group (P<0.05). Further, for the haplotype rs7873784, rs1927907, and rs1153688 of TLR4, the distribution of haplotype GCG was statistically different between moderate periodontitis and severe periodontitis (P<0.05, OR=1.501). These findings indicate that variation in TLR4 may affect chronic periodontitis susceptibility in a Han Chinese population. PMID:26131223

  17. β-Glucan-supplemented diets increase poly(I:C)-induced gene expression of Mx, possibly via Tlr3-mediated recognition mechanism in common carp (Cyprinus carpio).

    PubMed

    Falco, Alberto; Miest, Joanna J; Pionnier, Nicolas; Pietretti, Danilo; Forlenza, Maria; Wiegertjes, Geert F; Hoole, David

    2014-02-01

    We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard®) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection, particularly in the gut. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGard® was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly(I:C)), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response. Subsequently, a set of immune related genes, including mx, were analysed by real-time PCR on liver, spleen, head kidney and mid gut tissues. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of selected cytokines when compared to untreated fish, while mx gene expression remained stable or was slightly up-regulated. Injection with poly(I:C) induced a similar down-regulated gene expression pattern for cytokines in samples from β-glucan fed fish. In contrast, poly(I:C) injection markedly increased mx gene expression in samples from β-glucan fed fish but hardly in samples from fish fed control feed. In an attempt to explain the high induction of mx, we studied Toll-like receptor 3 (TLR3) gene expression in these carp. TLR3 is a prototypical pattern recognition receptor considered important for the binding of viral double-stranded RNA and triggering of a

  18. [Nle4, D-Phe7]-α-MSH Inhibits Toll-Like Receptor (TLR)2- and TLR4-Induced Microglial Activation and Promotes a M2-Like Phenotype

    PubMed Central

    Carniglia, Lila; Ramírez, Delia; Durand, Daniela; Saba, Julieta; Caruso, Carla; Lasaga, Mercedes

    2016-01-01

    α-melanocyte stimulating hormone (α-MSH) is an anti-inflammatory peptide, proved to be beneficial in many neuroinflammatory disorders acting through melanocortin receptor 4 (MC4R). We previously determined that rat microglial cells express MC4R and that NDP-MSH, an analog of α-MSH, induces PPAR-γ expression and IL-10 release in these cells. Given the great importance of modulation of glial activation in neuroinflammatory disorders, we tested the ability of NDP-MSH to shape microglial phenotype and to modulate Toll-like receptor (TLR)-mediated inflammatory responses. Primary rat cultured microglia were stimulated with NDP-MSH followed by the TLR2 agonist Pam3CSK4 or the TLR4 agonist LPS. NDP-MSH alone induced expression of the M2a/M2c marker Ag1 and reduced expression of the M2b marker Il-4rα and of the LPS receptor Tlr4. Nuclear translocation of NF-κB subunits p65 and c-Rel was induced by LPS and these effects were partially prevented by NDP-MSH. NDP-MSH reduced LPS- and Pam3CSK4-induced TNF-α release but did not affect TLR-induced IL-10 release. Also, NDP-MSH inhibited TLR2-induced HMGB1 translocation from nucleus to cytoplasm and TLR2-induced phagocytic activity. Our data show that NDP-MSH inhibits TLR2- and TLR4-mediated proinflammatory mechanisms and promotes microglial M2-like polarization, supporting melanocortins as useful tools for shaping microglial activation towards an alternative immunomodulatory phenotype. PMID:27359332

  19. Impact of TLR5 rs5744174 on stroke risk, gene expression and on inflammatory cytokines, and lipid levels in stroke patients.

    PubMed

    Gu, Lian; Huang, Jingyan; Tan, Jinjing; Wei, Qiugui; Jiang, Haiyun; Shen, Tingting; Liang, Baoyun; Tang, Nong

    2016-09-01

    Many studies reported that toll-like receptors (TLRs) played an important role in the process of ischemic stroke (IS). However, the impact of TLR5 rs5744174 on stroke risk, gene expression and on inflammatory cytokines, and lipid levels in ischemic stroke patients has not yet been reported and was therefore the subject of this study. In this case-control study, a total of 816 ischemic stroke patients and 816 healthy controls were genotyped using Sequenom MassArray technology. The mRNA expression of TLR5 was detected through quantitative real-time PCR among 52 ischemic stroke patients. The levels of IL-1b, IL-6, IL-8, and TNFα were measured by ELISA among 62 IS patients. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were determined among 816 IS patients using a Hitachi 7600 Automatic Biochemistry Analyzer. Our result showed TLR5 rs5744174 polymorphism was not associated with stroke risk, TLR5 mRNA expression and inflammatory cytokines of IS patients (P > 0.050), but was significantly associated with HDL-C (recessive model: β = - 0.14, 95 % CI: -0.24 to -0.03, P = 0.009). TLR5 rs5744174 polymorphism may have no impact on the stroke risk, gene expression and inflammatory cytokines, but may influence the HDL-C serum level of IS patients in Chinese Han population. PMID:27262705

  20. MGN1703, an immunomodulator and toll-like receptor 9 (TLR-9) agonist: from bench to bedside.

    PubMed

    Wittig, Burghardt; Schmidt, Manuel; Scheithauer, Werner; Schmoll, Hans-Joachim

    2015-04-01

    The adaptive immune system has been the main focus of immunological strategies in oncology with only more recent approaches targeting innate immunity. Endosomal toll-like receptors (TLR-7, TLR-9) activate innate immune responses by signaling damage-associated molecular patterns (DAMP) from decaying tumor cells. This has led to the development of DNA-based TLR-9 agonists, which induce antitumor activity through innate and subsequent adaptive immune responses. Early clinical trials with CpG-ODN as TLR-9 agonists were associated with unfavorable tolerability and narrow clinical efficacy, leading to failure in pivotal trials. dSLIM, the active ingredient of MGN1703, is a DNA-based, radically different molecular alternative to CpG-ODN, which results in genuine antitumor immunomodulation. Preclinical and clinical studies of MGN1703 have confirmed that this TLR-9 agonist has therapeutic potential in a variety of solid tumors, while long-term treatment with high doses was very well tolerated. A pivotal trial of first-line maintenance treatment with MGN1703 in patients with metastatic colorectal cancer is underway. PMID:25577571

  1. Differential Effect of Lactobacillus johnsonii BFE 6128 on Expression of Genes Related to TLR Pathways and Innate Immunity in Intestinal Epithelial Cells.

    PubMed

    Seifert, Stephanie; Rodriguez Gómez, Manuel; Watzl, Bernhard; Holzapfel, Wilhelm H; Franz, Charles M A P; Vizoso Pinto, María G

    2010-12-01

    Probiotics have been shown to enhance immune defenses, but their mechanisms of action are only partially understood. We investigated the modulation of signal pathways involved in innate immunity in enterocytes by Lactobacillus johnsonii BFE 6128 isolated from 'Kule naoto', a Maasai traditional fermented milk product. This lactobacillus sensitized HT29 intestinal epithelial cells toward recognition of Salmonella enterica serovar Typhimurium by increasing the IL-8 levels released after challenge with this pathogen and by differentially modulating genes related to toll-like receptor (TLR) pathways and innate immunity. Thus, the modulation of pro-inflammatory mediators and TLR-pathway-related molecules may be an important mechanism contributing to the potential stimulation of innate immunity by lactobacilli at the intestinal epithelial level. PMID:26781315

  2. Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex*

    PubMed Central

    Ranoa, Diana Rose E.; Kelley, Stacy L.; Tapping, Richard I.

    2013-01-01

    Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLRTLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins. PMID:23430250

  3. Combined Tlr2 and Tlr4 Deficiency Increases Radiation-Induced Pulmonary Fibrosis in Mice

    SciTech Connect

    Paun, Alexandra; Fox, Jessica; Balloy, Viviane; Chignard, Michel; Qureshi, Salman T.; Haston, Christina K.

    2010-07-15

    Purpose: To determine whether Toll-like receptor 2 or 4 genotype alters the lung response to irradiation in C57BL/6 mice using a model developing a phenotype that resembles radiotherapy-induced fibrosis in both histological characteristics and onset post-treatment. Methods and Materials: The pulmonary phenotype of C57BL/6 mice deficient in each or both of these genes was assessed after an 18-Gy single dose to the thoracic cavity by survival time postirradiation, bronchoalveolar lavage cell differential, histological evidence of alveolitis and fibrosis, and gene expression levels, and compared with that of wild-type mice. Results: The lung phenotype of Tlr4-deficient and Tlr2-deficient mice did not differ from that of wild-type mice in terms of survival time postirradiation, or by histological evidence of alveolitis or fibrosis. In contrast, mice deficient in both receptors developed respiratory distress at an earlier time than did wild-type mice and presented an enhanced fibrotic response (13.5% vs. 5.8% of the lung by image analysis of histological sections, p < 0.001). No differences in bronchoalveolar cell differential counts, nor in numbers of apoptotic cells in the lung as detected through active caspase-3 staining, were evident among the irradiated mice grouped by Tlr genotype. Gene expression analysis of lung tissue revealed that Tlr2,4-deficient mice have increased levels of hyaluronidase 2 compared with both wild-type mice and mice lacking either Tlr2 or Tlr4. Conclusion: We conclude that a combined deficiency in both Tlr2 and Tlr4, but not Tlr2 or Tlr4 alone, leads to enhanced radiation-induced fibrosis in the C57BL/6 mouse model.

  4. mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo

    PubMed Central

    Will, Clara; Carevic, Melanie; Rottenberger, Jennifer; Nürnberg, Bernd; Hartl, Dominik; Handgretinger, Rupert; Beer-Hammer, Sandra; Kormann, Michael S. D.

    2016-01-01

    Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for in vivo gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient’s individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is–in contrast to conventional viral vectors–non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either Tlr1/2 mRNA or Tlr2/6 mRNA, but not Tlr2 mRNA alone, resulted in better lung function as well as reduced airway inflammation in vivo. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis. PMID:27101288

  5. mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo.

    PubMed

    Zeyer, Franziska; Mothes, Benedikt; Will, Clara; Carevic, Melanie; Rottenberger, Jennifer; Nürnberg, Bernd; Hartl, Dominik; Handgretinger, Rupert; Beer-Hammer, Sandra; Kormann, Michael S D

    2016-01-01

    Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for in vivo gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient's individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is-in contrast to conventional viral vectors-non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either Tlr1/2 mRNA or Tlr2/6 mRNA, but not Tlr2 mRNA alone, resulted in better lung function as well as reduced airway inflammation in vivo. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis. PMID:27101288

  6. Taste Receptor Genes

    PubMed Central

    Bachmanov, Alexander A.; Beauchamp, Gary K.

    2009-01-01

    In the past several years, tremendous progress has been achieved with the discovery and characterization of vertebrate taste receptors from the T1R and T2R families, which are involved in recognition of bitter, sweet, and umami taste stimuli. Individual differences in taste, at least in some cases, can be attributed to allelic variants of the T1R and T2R genes. Progress with understanding how T1R and T2R receptors interact with taste stimuli and with identifying their patterns of expression in taste cells sheds light on coding of taste information by the nervous system. Candidate mechanisms for detection of salts, acids, fat, complex carbohydrates, and water have also been proposed, but further studies are needed to prove their identity. PMID:17444812

  7. Comparative genomic evidence for duplication of TLR1 subfamily and miiuy croaker TLR1 perceives LPS stimulation via MyD88 and TIRAP.

    PubMed

    Xu, Tianjun; Wang, Yanjin; Li, Jinrui; Shu, Chang; Han, Jingjing; Chu, Qing

    2016-09-01

    Being indispensable pattern recognition receptors in innate immune responses in host protection, Toll-like receptors (TLRs) play an important role in pathogen recognition. Fish TLRs exhibit high variety and distinct features, although little is known about their function on ligand recognition and signaling pathway in fish. This paper reports the evolutionary spectrum of the TLR1 subfamily (referred to as TLR1, TLR6, and TLR10) as determined using the comparative genomic approach. We hypothesized that the TLR1 subfamily underwent two rounds of gene duplication events; the first duplication occurred prior to the divergence of amphibians, and the second one occurred prior to the divergence of eutherians. To further study the function of fish TLR1, we identified miiuy croaker (Miichthys miiuy) TLR1 (mmiTLR1) and determined its potential ability to perceive Vibrio anguillarum and lipopolysaccharide stimulation. Data further suggested that mmiTLR1 is dependent on TIRAP and MyD88 for signal transmission. In addition, immunocytochemistry showed the speculative interaction between MyD88 and mmiTLR1 TIR domain. Overall, we systematically and comprehensively analyzed evolution of TLR1 subfamily and the function of mmiTLR1, which will provide the basis for future scientific research on fish TLRs. PMID:27431585

  8. Blockade of Toll-Like Receptors (TLR2, TLR4) Attenuates Pain and Potentiates Buprenorphine Analgesia in a Rat Neuropathic Pain Model

    PubMed Central

    Jurga, Agnieszka M.; Rojewska, Ewelina; Makuch, Wioletta; Pilat, Dominika; Przewlocka, Barbara

    2016-01-01

    Accumulating evidence indicates that microglial TLR2 and TLR4 play a significant role in nociception. Experiments were conducted to evaluate the contribution of TLR2 and TLR4 and their adaptor molecules to neuropathy and their ability to amplify opioid effectiveness. Behavioral tests (von Frey's and cold plate) and biochemical (Western blot and qRT-PCR) analysis of spinal cord and DRG tissue were conducted after chronic constriction injury (CCI) to the sciatic nerve. Repeated intrathecal administration of LPS-RS (TLR2 and TLR4 antagonist) and LPS-RS Ultrapure (TLR4 antagonist) attenuated allodynia and hyperalgesia. Biochemical analysis revealed time-dependent upregulation of mRNA and/or protein levels of TLR2 and TLR4 and MyD88 and TRIF adaptor molecules, which was paralleled by an increase in IBA-1/CD40-positive cells under neuropathy. LPS-RS and LPS-RS Ultrapure similarly influenced opioid analgesia by enhancing the effectiveness of buprenorphine but not morphine. Summing up, in light of their upregulation over the course of pain, both TLR2 and TLR4 may indeed play a significant role in neuropathy, which could be linked to the observed activation of IBA-1/CD40-positive cells. Blockade of TLR2 and TLR4 produced analgesia and enhanced buprenorphine's effectiveness, which suggests that they may be a putative target for future pharmacological pain relief tools, especially for opioid rotation, when the effect of morphine is tolerated. PMID:26962463

  9. Lack of Association between Toll Like Receptor-2 and Toll Like Receptor-4 Gene Polymorphisms and Other Feature in Iranian Asthmatics Patients.

    PubMed

    Bahrami, Hamid; Daneshmandi, Saeed; Heidarnazhad, Hasan; Pourfathollah, Ali Akbar

    2015-02-01

    Asthma as a chronic inflammatory airway disease is considered to be the most common chronic disease that is involving genetic and environmental factors. Toll like receptors (TLRs) and other inflammatory mediators are important in modulation of inflammation. In this study, we evaluated the role of TLR2 Arg753Gln and TLR4 Asp299Gly polymorphisms in the asthma susceptibility, progress, control levels and lung functions in Iranian patients. On 99 asthmatic patients and 120 normal subjects, TLR2 Arg753Gln and TLR4 Asp299Gly polymorphisms were evaluated by PCR-RFLP method recruiting Msp1 and Nco1 restriction enzymes, respectively. IgE serum levels by ELISA technique were determined and asthma diagnosis, treatment and control levels were considered using standard schemes and criteria. Our results indicated that the genotype and allele frequencies of the TLR2 Arg753Gln and TLR4 Asp299Gly polymorphisms were not significantly different between control subjects and asthmatics and were not related to in asthma features such as IgE levels, asthma history and pulmonary factors. Wherease some previous studies indicated TLRs and their polymorphisms might have some role in asthma incidence and features, our data demonstrated that TLR2 Arg753Gln and TLR4 Asp299Gly gene variants were not risk factors for asthma or its features in Iranian patients. Genetic complexity, ethnicity, influence of other genes or polymorphisms may overcome these polymorphisms in our asthmatics. PMID:25530138

  10. Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss)

    USGS Publications Warehouse

    Palti, Yniv; Rodriguez, M. Fernanda; Gahr, Scott A.; Purcell, Maureen K.; Rexroad, Caird E., III; Wiens, Gregory D.

    2010-01-01

    Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam2CSK4) and triacylated lipoprotein (Pam3CSK4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are

  11. Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss)

    USGS Publications Warehouse

    Palti, Y.; Rodriguez, M.F.; Gahr, S.A.; Purcell, M.K.; Rexroad, C. E., III; Wiens, G.D.

    2010-01-01

    Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5??? UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam2CSK4) and triacylated lipoprotein (Pam3CSK4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling

  12. Gene Polymorphism of Toll-Like Receptors and Lung Function at Five to Seven Years of Age after Infant Bronchiolitis

    PubMed Central

    Lauhkonen, Eero; Koponen, Petri; Vuononvirta, Juho; Teräsjärvi, Johanna; Nuolivirta, Kirsi; Toikka, Jyri O.; Helminen, Merja; He, Qiushui; Korppi, Matti

    2016-01-01

    Aim Toll-like receptors (TLR) play a crucial role in innate immunity, protecting the host from pathogens such as viruses. Genetic variations in TLRs have been associated with the severity of viral bronchiolitis in infancy and with the later occurrence of post-bronchiolitis asthma. The aim of the present study was to evaluate if there are any exploratory associations between TLR gene polymorphisms and lung function at 5 to 7 years of age in former bronchiolitis patients. Methods We performed impulse oscillometry (IOS) at the median age of 6.3 years for 103 children who had been hospitalized for bronchiolitis at less than six months of age. The main parameters evaluated were airway resistance and reactance at 5Hz in baseline and post-exercise measurements. Data on single nucleotide polymorphisms (SNP) of TLR1 rs5743618, TLR2 rs5743708, TLR6 rs5743810 and TLR10 rs4129009 (TLR2 subfamily) and TLR3 rs3775291, TLR4 rs4986790, TLR7 rs179008, TLR8 rs2407992 and TLR 9 rs187084 were available for analyses. Results The TLR4 rs4986790 wild genotype A/A was associated with a greater Rrs5 response (0.72 vs. -0.42, p = 0.03) to exercise. In TLR6 rs5743810, the minor allele T was associated with greater Rrs5 response (0.80 vs. -0.03, p = 0.04) to exercise. In TLR7 rs179008, the major allele A was associated with baseline decline in dRrs/df (-1.03 vs 0.61, p = 0.01) and increased Fres (2.28 vs. 0.89, p = 0.01) in girls. Conclusion Among the nine studied TLRs, only TLR7 rs179008 showed some exploratory associations with post-bronchiolitis lung function deficiency, and polymorphisms of TLR4 rs4986790, and TLR6 rs5743810 in particular, with airway reactivity. These findings call for further confirmatory studies. PMID:26741133

  13. TLR4 antagonist attenuates atherogenesis in LDL receptor-deficient mice with diet-induced type 2 diabetes.

    PubMed

    Lu, Zhongyang; Zhang, Xiaoming; Li, Yanchun; Lopes-Virella, Maria F; Huang, Yan

    2015-11-01

    Although a large number of studies have well documented a key role of toll-like receptor (TLR)4 in atherosclerosis, it remains undetermined if TLR4 antagonist attenuates atherogenesis in mouse model for type 2 diabetes. In this study, we induced type 2 diabetes in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice by high-fat diet (HFD). At 8 weeks old, 20 mice were fed HFD and 20 mice fed regular chow (RC) for 24 weeks. In the last 10 weeks, half HFD-fed mice and half RC-fed mice were treated with Rhodobacter sphaeroides lipopolysaccharide (Rs-LPS), an established TLR4 antagonist. After the treatment, atherosclerotic lesions in aortas were analyzed. Results showed that the HFD significantly increased bodyweight, glucose, lipids including total cholesterol, triglycerides and free fatty acids, and insulin resistance, indicating that the HFD induced type 2 diabetes in LDLR(-/-) mice. Results also showed that Rs-LPS had no effect on HFD-increased metabolic parameters in both nondiabetic and diabetic mice. Lipid staining of aortas and histological analysis of cross-sections of aortic roots showed that diabetes increased atherosclerotic lesions, but Rs-LPS attenuated atherogenesis in diabetic mice. Furthermore, immunohistochemical studies showed that Rs-LPS reduced infiltration of monocytes/macrophages and expression of interleukin (IL)-6 and matrix metalloproteinase-9 in atherosclerotic lesions of diabetic mice. Finally, the antagonistic effect of Rs-LPS on TLR4 was demonstrated by our in vitro studies showing that Rs-LPS inhibited IL-6 secretion from macrophages and endothelial cells stimulated by LPS or LPS plus saturated fatty acid palmitate. Taken together, our study demonstrated that TLR4 antagonist was capable of attenuating vascular inflammation and atherogenesis in mice with HFD-induced type 2 diabetes. PMID:26162692

  14. [TOLL-LIKE RECEPTORS IN COSMONAUT'S PERIPHERAL BLOOD CELLS AFTER LONG-DURATION MISSIONS TO THE INTERNATIONAL SPACE STATION].

    PubMed

    Berendeeva, T A; Ponomarev, S A; Antropova, E N; Rykova, M P

    2015-01-01

    Studies of Toll-like receptors (TLR) in 20 cosmonauts-members of long-duration (124-199-day) missions to the International space station evidenced changes in relative and absolute counts of peripheral blood monocytes with TLR2, TLR4 and TLR6 on the surface, expression of TLR2 and TLR6 genes, and genes of molecules involved in the TLR signaling pathway and TLR-related NF-KB-, JNK/p38- and IRF pathways on the day of return to Earth. The observed changes displayed individual variability. PMID:26934790

  15. Characterization of promoter sequence of toll-like receptor genes in Vechur cattle

    PubMed Central

    Lakshmi, R.; Jayavardhanan, K. K.; Aravindakshan, T. V.

    2016-01-01

    Aim: To analyze the promoter sequence of toll-like receptor (TLR) genes in Vechur cattle, an indigenous breed of Kerala with the sequence of Bos taurus and access the differences that could be attributed to innate immune responses against bovine mastitis. Materials and Methods: Blood samples were collected from Jugular vein of Vechur cattle, maintained at Vechur cattle conservation center of Kerala Veterinary and Animal Sciences University, using an acid-citrate-dextrose anticoagulant. The genomic DNA was extracted, and polymerase chain reaction was carried out to amplify the promoter region of TLRs. The amplified product of TLR2, 4, and 9 promoter regions was sequenced by Sanger enzymatic DNA sequencing technique. Results: The sequence of promoter region of TLR2 of Vechur cattle with the B. taurus sequence present in GenBank showed 98% similarity and revealed variants for four sequence motifs. The sequence of the promoter region of TLR4 of Vechur cattle revealed 99% similarity with that of B. taurus sequence but not reveals significant variant in motifregions. However, two heterozygous loci were observed from the chromatogram. Promoter sequence of TLR9 gene also showed 99% similarity to B. taurus sequence and revealed variants for four sequence motifs. Conclusion: The results of this study indicate that significant variation in the promoter of TLR2 and 9 genes in Vechur cattle breed and may potentially link the influence the innate immunity response against mastitis diseases. PMID:27397987

  16. 7-Ketocholesterol-Induced Inflammation Signals Mostly through the TLR4 Receptor Both In Vitro and In Vivo

    PubMed Central

    Huang, Jiahn-Dar; Amaral, Juan; Lee, Jung Wha; Rodriguez, Ignacio R.

    2014-01-01

    The cholesterol oxide 7-ketocholesterol (7KCh) has been implicated in numerous age-related diseases such as atherosclerosis, Alzheimer's disease, Parkinson's disease, cancer and age-related macular degeneration. It is formed by the autooxidation of cholesterol and especially cholesterol-fatty acid esters found in lipoprotein deposits. This molecule causes complex and potent inflammatory responses in vitro and in vivo. It is suspected of causing chronic inflammation in tissues exposed to oxidized lipoprotein deposits. In this study we have examined the inflammatory pathways activated by 7KCh both in cultured ARPE19 cells and in vivo using 7KCh-containing implants inserted into the anterior chamber of the rat eye. Our results indicate that 7KCh-induced inflammation is mediated mostly though the TLR4 receptor with some cross-activation of EGFR-related pathways. The majority of the cytokine inductions seem to signal via the TRIF/TRAM side of the TLR4 receptor. The MyD88/TIRAP side only significantly effects IL-1β inductions. The 7KCh-induced inflammation also seems to involve a robust ER stress response. However, this response does not seem to involve a calcium efflux-mediated UPR. Instead the ER stress response seems to be mediated by yet identified kinases activated through the TLR4 receptor. Some of the kinases identified are the RSKs which seem to mediate the cytokine inductions and the cell death pathway but do not seem to be involved in the ER stress response. PMID:25036103

  17. Synergistic Stimulation with Different TLR7 Ligands Modulates Gene Expression Patterns in the Human Plasmacytoid Dendritic Cell Line CAL-1

    PubMed Central

    Hilbert, Tobias; Steinhagen, Folkert; Weisheit, Christina; Baumgarten, Georg; Hoeft, Andreas; Klaschik, Sven

    2015-01-01

    Objective. TLR7 ligation in plasmacytoid dendritic cells is promising for the treatment of cancer, allergy, and infectious diseases; however, high doses of ligands are required. We hypothesized that the combination of structurally different TLR7 ligands exponentiates the resulting immune response. Methods. CAL-1 (human pDC line) cells were incubated with the TLR7-specific adenine analog CL264 and single-stranded 9.2s RNA. Protein secretion was measured by ELISA. Microarray technique was used to detect modified gene expression patterns upon synergistic stimulation, revealing underlying functional groups and networks. Cell surface binding properties were studied using FACS analysis. Results. CL264 in combination with 9.2s RNA significantly enhanced cytokine and interferon secretion to supra-additive levels. This effect was due to a stronger stimulation of already regulated genes (by monostimulation) as well as to recruitment of thus far unregulated genes. Top scoring canonical pathways referred to immune-related processes. Network analysis revealed IL-1β, IL-6, TNF, and IFN-β as major regulatory nodes, while several minor regulatory nodes were also identified. Binding of CL264 to the cell surface was enhanced by 9.2s RNA. Conclusion. Structurally different TLR7 ligands act synergistically on gene expression patterns and on the resulting inflammatory response. These data could impact future strategies optimizing TLR7-targeted drug design. PMID:26770023

  18. Gangliosides trigger inflammatory responses via TLR4 in brain glia.

    PubMed

    Jou, Ilo; Lee, Jee Hoon; Park, Soo Young; Yoon, Hee Jung; Joe, Eun-Hye; Park, Eun Jung

    2006-05-01

    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. PMID:16651628

  19. Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia.

    PubMed

    Di Lorenzo, Flaviana; Kubik, Łukasz; Oblak, Alja; Lorè, Nicola Ivan; Cigana, Cristina; Lanzetta, Rosa; Parrilli, Michelangelo; Hamad, Mohamad A; De Soyza, Anthony; Silipo, Alba; Jerala, Roman; Bragonzi, Alessandra; Valvano, Miguel A; Martín-Santamaría, Sonsoles; Molinaro, Antonio

    2015-08-28

    Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium. PMID:26160169

  20. Zinc oxide influences intestinal integrity, the expressions of genes associated with inflammation and TLR4-myeloid differentiation factor 88 signaling pathways in weanling pigs.

    PubMed

    Hu, Cai Hong; Song, Ze He; Xiao, Kan; Song, Juan; Jiao, Le Fei; Ke, Ya Lu

    2014-07-01

    This study explored whether zinc oxide (ZnO) supplementation could alleviate weanling-induced intestinal injury through TLR and NOD-like receptor signaling pathways. Twelve early-weanling piglets were allotted to two dietary treatments (control vs 2200 mg Zn/kg from ZnO) for 1 wk. The results showed that supplemental ZnO improved daily gain and feed intake, decreased post weaning scour scores, increased villus height and villus height:crypt depth ratio at the jejunal mucosa, and decreased diamine oxidase activity and endotoxin concentration in plasma. The intestinal mRNA levels of TLR4 and its downstream signals, including MyD88, IL-1 receptor-associated kinase 1 and TNF-α receptor-associated factor 6, were decreased, and the expressions of intestinal pro-inflammatory cytokines and chemokines were decreased simultaneously in the ZnO-supplemented piglets. Although NF-κB p65 mRNA abundance was not affected by ZnO supplementation, NF-κB p65 protein expression was down-regulated by ZnO. However, ZnO supplementation had no effect on intestinal expressions of NOD1 and NOD2, and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2, as well as protein expressions of caspase-3 and heat shock protein 70. The results indicated that the protective effects of ZnO on intestinal integrity were closely related to decreasing the expressions of genes associated with inflammation through inhibiting the TLR4-MyD88 signaling pathways. PMID:23956359

  1. Inhibition of LPS binding to MD-2 co-receptor for suppressing TLR4-mediated expression of inflammatory cytokine by 1-dehydro-10-gingerdione from dietary ginger

    SciTech Connect

    Park, Sun Hong; Kyeong, Min Sik; Hwang, Yuri; Ryu, Shi Yong; Han, Sang-Bae; Kim, Youngsoo

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer 1-Dehydro-10-gingerdione (1D10G) from ginger inhibits LPS binding to MD-2. Black-Right-Pointing-Pointer 1D10G suppresses MyD88- or TRIF-dependent signaling in LPS-activated macrophages. Black-Right-Pointing-Pointer 1D10G down-regulates the expression of NF-{kappa}B-, AP1- or IRF3-target genes. Black-Right-Pointing-Pointer MD-2 is a molecular target in the anti-inflammatory action of 1D10G. -- Abstract: Myeloid differentiation protein 2 (MD-2) is a co-receptor of toll-like receptor 4 (TLR4) for innate immunity. Here, we delineated a new mechanism of 1-dehydro-10-gingerdione (1D10G), one of pungent isolates from ginger (Zingiber officinale), in the suppression of lipopolysaccharide (LPS)-induced gene expression of inflammatory cytokines. 1D10G inhibited LPS binding to MD-2 with higher affinity than gingerol and shogaol from dietary ginger. Moreover, 1D10G down-regulated TLR4-mediated expression of nuclear factor-{kappa}B (NF-{kappa}B) or activating protein 1 (AP1)-target genes such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin-1{beta}, as well as those of interferon (IFN) regulatory factor 3 (IRF3)-target IFN-{beta} gene and IFN-{gamma} inducible protein 10 (IP-10) in LPS-activated macrophages. Taken together, MD-2 is a molecular target in the anti-inflammatory action of 1D10G.

  2. The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2

    PubMed Central

    Shamsul, Haque M; Hasebe, Akira; Iyori, Mitsuhiro; Ohtani, Makoto; Kiura, Kazuto; Zhang, Diya; Totsuka, Yasunori; Shibata, Ken- ichiro

    2010-01-01

    Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-β-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1. PMID:20113368

  3. Simvastatin Inhibits Toll-like Receptor 8 (TLR8) Signaling in Primary Human Monocytes and Spontaneous Tumor Necrosis Factor Production from Rheumatoid Synovial Membrane Cultures

    PubMed Central

    Mullen, Lisa; Ferdjani, Jason; Sacre, Sandra

    2015-01-01

    Simvastatin has been shown to have antiinflammatory effects that are independent of its serum cholesterol lowering action, but the mechanisms by which these antiinflammatory effects are mediated have not been elucidated. To explore the mechanism involved, the effect of simvastatin on toll-like receptor (TLR) signaling in primary human monocytes was investigated. A short pretreatment with simvastatin dose-dependently inhibited the production of tumor necrosis factor (TNF)-α in response to TLR8 activation (but not TLR2, -4 or -5). Statins are known inhibitors of the cholesterol biosynthetic pathway, but, intriguingly, TLR8 inhibition could not be reversed by addition of mevalonate or geranylgeranyl pyrophosphate, downstream products of cholesterol biosynthesis. TLR8 signaling was examined in HEK 293 cells stably expressing TLR8, where simvastatin inhibited I kappa B kinase (IKK)α/β phosphorylation and subsequent nuclear factor (NF)-κB activation without affecting the pathway to activating protein-1 (AP-1). Because simvastatin has been reported to have antiinflammatory effects in RA patients and TLR8 signaling contributes to TNF production in human RA synovial tissue in culture, simvastatin was tested in these cultures. Simvastatin significantly inhibited the spontaneous release of TNF in this model, which was not reversed by mevalonate. Together, these results demonstrate a hitherto unrecognized mechanism of simvastatin inhibition of TLR8 signaling that may in part explain its beneficial antiinflammatory effects. PMID:26322850

  4. Identification of Adjuvantic Activity of Amphotericin B in a Novel, Multiplexed, Poly-TLR/NLR High-Throughput Screen

    PubMed Central

    Salyer, Alex C. D.; Caruso, Giuseppe; Khetani, Karishma K.; Fox, Lauren M.; Malladi, Subbalakshmi S.; David, Sunil A.

    2016-01-01

    Small-molecule agonists have been identified for TLR7, TLR8, TLR4 and TLR2 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. These results point to its potential applicability as a safe and effective adjuvant for human vaccines. PMID:26919709

  5. Identification of Adjuvantic Activity of Amphotericin B in a Novel, Multiplexed, Poly-TLR/NLR High-Throughput Screen.

    PubMed

    Salyer, Alex C D; Caruso, Giuseppe; Khetani, Karishma K; Fox, Lauren M; Malladi, Subbalakshmi S; David, Sunil A

    2016-01-01

    Small-molecule agonists have been identified for TLR7, TLR8, TLR4 and TLR2 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. These results point to its potential applicability as a safe and effective adjuvant for human vaccines. PMID:26919709

  6. TLR agonist–Stat3 siRNA conjugates: cell-specific gene silencing and enhanced antitumor immune responses

    PubMed Central

    Kortylewski, Marcin; Swiderski, Piotr; Herrmann, Andreas; Wang, Lin; Kowolik, Claudia; Kujawski, Maciej; Lee, Heehyoung; Scuto, Anna; Liu, Yong; Yang, Chunmei; Deng, Jiehui; Soifer, Harris S.; Raubitschek, Andrew; Forman, Stephen; Rossi, John J.; Pardoll, Drew M.; Jove, Richard; Yu, Hua

    2010-01-01

    Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment. PMID:19749770

  7. Involvement of TLR2 and TLR4 in inflammatory immune responses induced by fine and coarse ambient air particulate matter

    PubMed Central

    Shoenfelt, Joanna; Mitkus, Robert J.; Zeisler, Rolf; Spatz, Rabia O.; Powell, Jan; Fenton, Matthew J.; Squibb, Katherine A.; Medvedev, Andrei E.

    2009-01-01

    Induction of proinflammatory mediators by alveolar macrophages exposed to ambient air particulate matter has been suggested to be a key factor in the pathogenesis of inflammatory and allergic diseases in the lungs. However, receptors and mechanisms underlying these responses have not been fully elucidated. In this study, we examined whether TLR2, TLR4, and the key adaptor protein, MyD88, mediate the expression of proinflammatory cytokines and chemokines by mouse peritoneal macrophages exposed to fine and coarse PM. TLR2 deficiency blunted macrophage TNF-α and IL-6 expression in response to fine (PM2.5), while not affecting cytokine-inducing ability of coarse NIST Standard Reference Material (SRM 1648) particles. In contrast, TLR4−/− macrophages showed inhibited cytokine expression upon stimulation with NIST SRM 1648 but exhibited normal responses to PM2.5. Preincubation with polymyxin B markedly suppressed the capacity of NIST SRM 1648 to elicit TNF-α and IL-6, indicating endotoxin as a principal inducer of cytokine responses. Overexpression of TLR2 in TLR2/4-deficient human embryonic kidney 293 cells imparted PM2.5 sensitivity, as judged by IL-8 gene expression, whereas NIST SRM 1648, but not PM2.5 elicited IL-8 expression in 293/TLR4/MD-2 transfectants. Engagement of TLR4 by NIST SRM 1648 induced MyD88-independent expression of the chemokine RANTES, while TLR2-reactive NIST IRM PM2.5 failed to up-regulate this response. Consistent with the shared use of MyD88 by TLR2 and TLR4, cytokine responses of MyD88−/− macrophages to both types of air PM were significantly reduced. These data indicate differential utilization of TLR2 and TLR4 but shared use of MyD88 by fine and coarse air pollution particles. PMID:19406832

  8. Toll-like receptor stimulation in splenic marginal zone lymphoma can modulate cell signaling, activation and proliferation

    PubMed Central

    Fonte, Eleonora; Agathangelidis, Andreas; Reverberi, Daniele; Ntoufa, Stavroula; Scarfò, Lydia; Ranghetti, Pamela; Cutrona, Giovanna; Tedeschi, Alessandra; Xochelli, Aliki; Caligaris-Cappio, Federico; Ponzoni, Maurilio; Belessi, Chrysoula; Davis, Zadie; Piris, Miguel A.; Oscier, David; Ghia, Paolo; Stamatopoulos, Kostas; Muzio, Marta

    2015-01-01

    Recent studies on splenic marginal zone lymphoma identified distinct mutations in genes belonging to the B-cell receptor and Toll-like receptor signaling pathways, thus pointing to their potential implication in the biology of the disease. However, limited data is available regarding the exact role of TLRs. We aimed at characterizing the expression pattern of TLRs in splenic marginal zone lymphoma cells and their functional impact on the activation, proliferation and viability of malignant cells in vitro. Cells expressed significant levels of TLR1, TLR6, TLR7, TLR8, TLR9 and TLR10 mRNA; TLR2 and TLR4 showed a low, variable pattern of expression among patients whereas TLR3 and TLR5 mRNAs were undetectable; mRNA specific for TLR signaling molecules and adapters was also expressed. At the protein level, TLR1, TLR6, TLR7, TLR9 and TLR10 were detected. Stimulation of TLR1/2, TLR2/6 and TLR9 with their respective ligands triggered the activation of IRAK kinases, MAPK and NF-κB signaling pathways, and the induction of CD86 and CD25 activation molecules, although in a heterogeneous manner among different patient samples. TLR-induced activation and cell viability were also inhibited by a specific IRAK1/4 inhibitor, thus strongly supporting the specific role of TLR signaling in these processes. Furthermore, TLR2/6 and TLR9 stimulation also significantly increased cell proliferation. In conclusion, we demonstrate that splenic marginal zone lymphoma cells are equipped with functional TLR and signaling molecules and that the stimulation of TLR1/2, TLR2/6 and TLR9 may play a role in regulating disease pathobiology, likely promoting the expansion of the neoplastic clone. PMID:26294727

  9. Toll-like receptor stimulation in splenic marginal zone lymphoma can modulate cell signaling, activation and proliferation.

    PubMed

    Fonte, Eleonora; Agathangelidis, Andreas; Reverberi, Daniele; Ntoufa, Stavroula; Scarfò, Lydia; Ranghetti, Pamela; Cutrona, Giovanna; Tedeschi, Alessandra; Xochelli, Aliki; Caligaris-Cappio, Federico; Ponzoni, Maurilio; Belessi, Chrysoula; Davis, Zadie; Piris, Miguel A; Oscier, David; Ghia, Paolo; Stamatopoulos, Kostas; Muzio, Marta

    2015-11-01

    Recent studies on splenic marginal zone lymphoma identified distinct mutations in genes belonging to the B-cell receptor and Toll-like receptor signaling pathways, thus pointing to their potential implication in the biology of the disease. However, limited data is available regarding the exact role of TLRs. We aimed at characterizing the expression pattern of TLRs in splenic marginal zone lymphoma cells and their functional impact on the activation, proliferation and viability of malignant cells in vitro. Cells expressed significant levels of TLR1, TLR6, TLR7, TLR8, TLR9 and TLR10 mRNA; TLR2 and TLR4 showed a low, variable pattern of expression among patients whereas TLR3 and TLR5 mRNAs were undetectable; mRNA specific for TLR signaling molecules and adapters was also expressed. At the protein level, TLR1, TLR6, TLR7, TLR9 and TLR10 were detected. Stimulation of TLR1/2, TLR2/6 and TLR9 with their respective ligands triggered the activation of IRAK kinases, MAPK and NF-κB signaling pathways, and the induction of CD86 and CD25 activation molecules, although in a heterogeneous manner among different patient samples. TLR-induced activation and cell viability were also inhibited by a specific IRAK1/4 inhibitor, thus strongly supporting the specific role of TLR signaling in these processes. Furthermore, TLR2/6 and TLR9 stimulation also significantly increased cell proliferation. In conclusion, we demonstrate that splenic marginal zone lymphoma cells are equipped with functional TLR and signaling molecules and that the stimulation of TLR1/2, TLR2/6 and TLR9 may play a role in regulating disease pathobiology, likely promoting the expansion of the neoplastic clone. PMID:26294727

  10. Transduction of Functionally Contrasting Signals by Two Mycobacterial PPE Proteins Downstream of TLR2 Receptors.

    PubMed

    Udgata, Atul; Qureshi, Rahila; Mukhopadhyay, Sangita

    2016-09-01

    As pathogen-associated molecular pattern sensors, the TLRs can detect diverse ligands to elicit either proinflammatory or anti-inflammatory responses, but the mechanism that dictates such contrasting immune responses is not well understood. In this work, we demonstrate that proline-proline-glutamic acid (PPE)17 protein of Mycobacterium tuberculosis induces TLR1/2 heterodimerization to elicit proinflammatory-type response, whereas PPE18-induced homodimerization of TLR2 triggers anti-inflammatory type responses. Ligation of TLR1/2 caused an increased recruitment of IL-1R-associated kinase (IRAK)1, MyD88, and protein kinase C (PKC)ε to the downstream TLR-signaling complex that translocated PKCε into the nucleus in an IRAK1-dependent manner. PKCε-mediated phosphorylation allowed the nuclear IRAK3 to be exported to the cytoplasm, leading to increased activation of ERK1/2, stabilization of MAPK phosphatase 1 (MKP-1), and induction of TNF-α with concomitant downregulation of p38MAPK. Silencing of TLR1 inhibited PPE17-triggered cytoplasmic export of IRAK3 as well as TNF-α induction, suggesting an important role of TLR1/2 heterodimer in regulating proinflammatory responses via the IRAK3-signaling pathway. In contrast, PPE18-mediated homodimerization of TLR2 caused poorer cytoplasmic export of nuclear IRAK3 and MKP-1 stabilization, resulting in increased p38MAPK activation. Our study hints to a novel mechanism that implicates PKCε-IRAK3-MKP-1 signaling in the regulation of MAPK activity and inflammatory cascades downstream of TLR2 in tuberculosis. PMID:27481848

  11. Insights into the European rabbit (Oryctolagus cuniculus) innate immune system: genetic diversity of the toll-like receptor 3 (TLR3) in wild populations and domestic breeds

    PubMed Central

    2013-01-01

    Background Toll-like receptors (TLRs) belong to the innate immune system and are a major class of pattern recognition receptors representing the first line of the innate immune response. The TLR molecule is structurally composed by an ectodomain that contains leucine rich repeats (LRRs) that interact with pathogen associated molecular patterns (PAMPs), a transmembrane domain and a conserved cytoplasmic domain designated TIR (Toll-IL1 receptor) that is responsible for the intracellular signaling. TLR3 has been associated with the direct recognition of double-stranded viral RNA resulting from viral replication, while TLR7 and TLR8 target single-stranded viral RNA. In the European rabbit (Oryctolagus cuniculus), TLR7 and TLR8 were reported to be absent and pseudogenised, respectively, making TLR3 the only available TLR for the recognition of viral RNA. Thus, the levels of diversity of TLR3 were evaluated in the European rabbit by analysing different genetic backgrounds and exposure to pathogen pressures. Results We detected 41 single nucleotide polymorphisms (SNPs) in the coding sequence of TLR3. The highest diversity was observed in the wild populations of Iberian Peninsula, between 22–33 polymorphic positions. In the French population, 18 SNPs were observed and only 4 polymorphic positions were detected in the domestic breeds. 14 non-synonymous substitutions were observed, most of them in the LRR molecules. The remaining were scattered across the transmembrane and TIR domains. Conclusion The study of TLR3 in European rabbit populations might be relevant to understand the interplay between RNA viruses and innate immunity. Wild rabbit populations presented more diversity than domestic breeds and other mammals previously studied. This might be linked to the absence of population bottlenecks during their evolution and to the almost inexistence of man-mediated selection. The observed variability might have also been potentiated by the contact of the wild populations

  12. The Effect of Estradiol and Progesterone on Toll Like Receptor Gene Expression in A Human Fallopian Tube Epithelial Cell Line

    PubMed Central

    Zandieh, Zahra; Amjadi, Fatemehsadat; Ashrafi, Mahnaz; Aflatoonian, Abbas; Fazeli, Alireza; Aflatoonian, Reza

    2016-01-01

    Objective Toll like receptors (TLRs) are one of the main components of the innate im- mune system. It has been reported that expression of these receptors are altered in the female reproductive tract (FRT) during menstrual cycle. Here we used a fallopian tube epithelial cell line (OE-E6/E7) to evaluate the effect of two sex hormones in modulating TLR expression. Materials and Methods In this experimental study, initially TLR gene expression in OE- E6/E7 cells was evaluated and compared with that of fallopian tube tissue using quanti- tative real time-polymerase chain reaction (qRT-PCR) and immunostaining. Thereafter, OE-E6/E7 cells were cultured with different concentrations of estradiol and progesterone, and combination of both. qRT-PCR was performed to reveal any changes in expression of TLR genes as a result of hormonal treatment. Results TLR1-10 genes were expressed in human fallopian tube tissue. TLR1-6 genes and their respective proteins were expressed in the OE-E6/E7 cell line. Although estradiol and progesterone separately had no significant effect on TLR expression, their combined treatment altered the expression of TLRs in this cell line. Also, the pattern of TLR expres- sion in preovulation (P), mensturation (M) and window of implantation (W) were the same for all TLRs with no significant differences between P, M and W groups. Conclusion These data show the significant involvement of the combination of es- tradiol and progesterone in modulation of TLR gene expression in this human fal- lopian tube cell line. Further experiments may reveal the regulatory mechanism and signalling pathway behind the effect of sex hormones in modulating TLRs in the hu- man FRT. PMID:26862527

  13. Characterization of Toll-like receptor gene expression and the pathogen agonist response in the antarctic bullhead notothen Notothenia coriiceps.

    PubMed

    Ahn, Do Hwan; Shin, Seung Chul; Park, Hyun

    2014-10-01

    Notothenia coriiceps, a typical Antarctic notothenioid teleost, has evolved to adapt to the extreme Antarctic marine environment. We previously reported an extensive analysis of the Antarctic notothenioid transcriptome. In this study, we focused on a key component of the innate immune system, the Toll-like receptors (TLRs). We cloned the full-length sequence of 12 TLRs of N. coriiceps. The N. coriiceps transcriptome for TLR homologue (ncTLR) genes encode a typical TLR structure, with multiple extracellular leucine-rich regions and an intracellular Toll/IL-1 receptor (TIR) domain. Using phylogenetic analysis, we established that all of the cloned ncTLR genes could be classified into the same orthologous clade with other teleost TLRs. ncTLRs were widely expressed in various organs, with the highest expression levels observed in immune-related tissues, such as the skin, spleen, and kidney. A subset of the ncTLR genes was expressed at higher levels in fish exposed to pathogen-mimicking agonists, heat-killed Escherichia coli, and polyinosinic-polycytidylic acid (poly(I:C)). However, the mechanism involved in the upregulation of TLR expression following pathogen exposure in fish is currently unknown. Further research is required to elucidate these mechanisms and to thereby increase our understanding of vertebrate immune system evolution. PMID:25073429

  14. TLR9 is important for protection against intestinal damage and for intestinal repair.

    PubMed

    Rose, William Alfred; Sakamoto, Kaori; Leifer, Cynthia Anne

    2012-01-01

    Toll-like receptors (TLRs) are innate receptors critical for host defense, and play a role in normal biological processes. For example, host DNA, a TLR9 ligand, stimulates epithelial repair following skin wounding. TLR signaling also plays a crucial role in regulating intestinal homeostasis. We therefore asked whether TLR9 is important for intestinal wound repair using a dextran sulfate sodium (DSS)-induced intestinal damage and repair model. We showed that TLR9-deficient mice are more susceptible to DSS, and exhibited delayed wound repair at both the clinical and histologic levels. TLR9-deficient mice showed reduced gene expression of hairy enhancer of split 1, an intestinal progenitor cell differentiation factor, and vascular endothelial growth factor, a growth factor important for epithelial cell restitution. Therefore, we conclude that TLR stimulation may play a normal role in regulating intestinal homeostasis and could potentially be a novel therapeutic target to enhance intestinal wound repair in inflammatory bowel diseases. PMID:22893852

  15. Predisposition to Childhood Otitis Media and Genetic Polymorphisms within the Toll-Like Receptor 4 (TLR4) Locus

    PubMed Central

    Kentala, Erna; Hammarén-Malmi, Sari; Bhutta, Mahmood F.; MacArthur, Carol J.; Wilmot, Beth; Casselbrant, Margaretha; Conley, Yvette P.; Weeks, Daniel E.; Mandel, Ellen M.; Vaarala, Outi; Kallio, Anna; Melin, Merit; Nieminen, Janne K.; Leinonen, Eira; Kere, Juha; Mattila, Petri S.

    2015-01-01

    Background Predisposition to childhood otitis media (OM) has a strong genetic component, with polymorphisms in innate immunity genes suspected to contribute to risk. Studies on several genes have been conducted, but most associations have failed to replicate in independent cohorts. Methods We investigated 53 gene polymorphisms in a Finnish cohort of 624 cases and 778 controls. A positive association signal was followed up in a tagging approach and tested in an independent Finnish cohort of 205 cases, in a British cohort of 1269 trios, as well as in two cohorts from the United States (US); one with 403 families and the other with 100 cases and 104 controls. Results In the initial Finnish cohort, the SNP rs5030717 in the TLR4 gene region showed significant association (OR 1.33, P = .003) to OM. Tagging SNP analysis of the gene found rs1329060 (OR 1.33, P = .002) and rs1329057 (OR 1.29, P = .003) also to be associated. In the more severe phenotype the association was stronger. This finding was supported by an independent Finnish case cohort, but the associations failed to replicate in the British and US cohorts. In studies on TLR4 signaling in 20 study subjects, the three-marker risk haplotype correlated with a decreased TNFα secretion in myeloid dendritic cells. Conclusions The TLR4 gene locus, regulating the innate immune response, influences the genetic predisposition to childhood OM in a subpopulation of patients. Environmental factors likely modulate the genetic components contributing to the risk of OM. PMID:26177520

  16. [Effect of baicalin on pattern recognition receptor TLR2/4-NOD2 and its significance of druggability].

    PubMed

    Chai, Yu-Shuang; Lei, Fan; Xing, Dong-Ming; Ding, Yi; Du, Li-Jun

    2013-08-01

    Activation pattern recognition receptors can cause the startup of downstream signaling pathways, the expression of inflammatory factors, and finally immunological inflammatory reaction. Either exogenous pathogenic microorganisms or endogenous tissue components can activate these pattern recognition receptors as ligands at varying degrees, and then cause the immunological inflammatory reaction. Therefore, it is of great significance to inhibit relevant receptors, as well as the immunological inflammatory reaction, in order to avoid tissue injury during the course of disease. Baicalin is able to specifically inhibit the expression of TLR2/4-NOD2, inhibit the expression of inflammatory factors IL-1beta, IL-6 and TNF-alpha, and thereby reducing the injury of the tissue cells during the course of disease. This effect is non-specific with tissues, which is of great theoretical and practical significance in druggability. In addition, the drug metabolism and toxicity of baicalin are also discussed for its druggability in this article. PMID:24228579

  17. TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression

    PubMed Central

    Souza, Ana C P; Tsuji, Takayuki; Baranova, Irina N; Bocharov, Alexander V; Wilkins, Kenneth J; Street, Jonathan M; Alvarez-Prats, Alejandro; Hu, Xuzhen; Eggerman, Thomas; Yuen, Peter S T; Star, Robert A

    2015-01-01

    Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD-1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD. PMID:26416975

  18. Activation of adult rat CNS endothelial cells by opioid-induced toll-like receptor 4 (TLR4) signaling induces proinflammatory, biochemical, morphological, and behavioral sequelae

    PubMed Central

    Grace, Peter M.; Ramos, Khara M.; Rodgers, Krista M.; Wang, Xiaohui; Hutchinson, Mark R.; Lewis, Makenzie T.; Morgan, Kelly N.; Kroll, Juliet L.; Taylor, Frederick R.; Strand, Keith A.; Zhang, Yingning; Berkelhammer, Debra; Huey, Madeline G.; Greene, Lisa I.; Cochran, Thomas A.; Yin, Hang; Barth, Daniel S.; Johnson, Kirk W.; Rice, Kenner; Maier, Steven F.; Watkins, Linda R.

    2014-01-01

    CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at TLR4, presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (-)-morphine or its mu-opioid receptor (MOR) inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated NFκB, increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 from these cells. (-)-Morphine-induced upregulation of TNFα mRNA and prostaglandin E2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequalae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by

  19. Improved outcome of hematopoietic SCT in patients with homozygous gene variant of Toll-like receptor 9.

    PubMed

    Elmaagacli, A H; Koldehoff, M; Beelen, D W

    2009-09-01

    Toll-like receptor 9 (TLR9) is part of the innate immune system, which is activated by CpG oligonucleotides (ODNs) and produces potent Th1-type innate and adaptive immune responses. It is reported that TLR9 gene variants, T1486C and T1237C, are associated with a reduced TLR9 expression compared with the wild-type gene. In two cohort analyses, we evaluated the influence of these gene variants on the outcome of transplant in 413 patients and donors. A retrospective analysis of the first cohort (n=293) showed that the homozygous CC gene variant of TLR9 (1486) compared with TC/TT gene variants was significantly associated with a markedly improved 5-year TRM (11.7 versus 36.4%, P<0.003), 5-year OS (86.1 vs 48.3%, P<0.001) and a lower relapse rate (13.2 vs 33.3%, P<0.007), whereas the occurrence of acute GVHD was not different. A prospectively performed analysis of the second cohort (n=120) and multivariate analyses confirmed the influence of the CC gene variant on these end points. Compared with patients with TC/TT gene at position 1486 of TLR9, patients with the homozygous CC gene variant had a lower TLR9 mRNA expression and a delayed T-cell immune reconstitution after transplant, which might prevent them from overwhelming immune responses as sepsis or systemic inflammatory response syndrome (SIRS) associated with an increased TRM. In vitro studies using CpG-rich ODNs showed an upregulation of TLR9 expression in cell lines with CC gene variant, but not in cell lines with wild-type gene. PMID:19252531

  20. Negative regulation of the type I interferon signaling pathway by synthetic Toll-like receptor 7 ligands.

    PubMed

    Forsbach, Alexandra; Müller, Christian; Montino, Carmen; Kritzler, Andrea; Nguyen, Toan; Weeratna, Risini; Jurk, Marion; Vollmer, Jörg

    2012-06-01

    Ten Toll-like receptor (TLR) family members have been reported in humans. Here, the endoplasmatic receptors TLR9, TLR8, TLR7, and TLR3 respond to nucleic acids and derivatives or to small molecules (TLR7 and 8). Another cytoplasmic RNA receptor, retinoic acid inducible gene I (RIG-I), is stimulated by 5' triphosphate double-stranded RNA. We discovered that TLR7 small-molecule agonists inhibit nucleic acid-mediated TLR3, TLR7, TLR9, or RIG-I-dependent interferon-α (IFN-α) immune response. Other cytokines and chemokines stimulated by nucleic acid agonists remained unaffected. The observed blockage of TLR3, TLR7, TLR9, and RIG-I-mediated IFN-α response appears to be driven by a competitive mechanism at the type I IFN pathway. Besides type I IFN, IFN response genes such as IFIT-1, Mx1, OAS1, or IRF7 were affected, which indicates that the key element driving the inhibition is located in the type I IFN pathway. Indeed, the heterotrimeric complex formation of phosphor-signal transducer and activator of transcription factor 1 (STAT1), phosphor-STAT2, and IRF9 (called ISGF3, IFN-stimulated gene factor 3) is inhibited through the TLR7 small-molecule agonists by phosphor-STAT2 blockage. These findings provide novel insights into the use of synthetic TLR7 or TLR7/8 small molecules as ligands for immune activation and suppression. PMID:22540943

  1. Identification and expression analysis of two Toll-like receptor genes from sea cucumber (Apostichopus japonicus).

    PubMed

    Sun, Hongjuan; Zhou, Zunchun; Dong, Ying; Yang, Aifu; Jiang, Bei; Gao, Shan; Chen, Zhong; Guan, Xiaoyan; Wang, Bai; Wang, Xiuli

    2013-01-01

    Toll-like receptors (TLRs) are a family of type I integral membrane glycoproteins which play pivotal roles in innate immunity. In this study, two TLRs named AjTLR3 and AjToll were cloned from sea cucumber (Apostichopus japonicus). The full-length cDNA sequences of AjTLR3 and AjToll are 3484 bp and 4211 bp, with an open reading frame (ORF) of 2679 bp and 2853 bp, encoding 892 and 950 amino acids, respectively. Both AjTLR3 and AjToll are composed of a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain and an intracellular Toll/interleukin-1 receptor (TIR) domain. Evolution analysis revealed that AjTLR3 and AjToll were clustered with the vertebrate-like TLRs (V-TLRs) and the protostome-like TLRs (P-TLRs), respectively. These two genes were widely expressed in all five tested tissues (body wall, coelomocytes, tube feet, intestine and respiratory tree), but showed different expression patterns. The significantly up-regulated expressions of AjTLR3 and AjToll after peptidoglycan (PGN), lipopolysaccharides (LPS), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C) challenges suggested that they were functionally involved in the immune responses to the Cram-positive bacteria, Gram-negative bacteria, fungi and double-stranded RNA (dsRNA) viruses, respectively. PMID:23103635

  2. Mutations in TLR/MYD88 pathway identify a subset of young chronic lymphocytic leukemia patients with favorable outcome.

    PubMed

    Martínez-Trillos, Alejandra; Pinyol, Magda; Navarro, Alba; Aymerich, Marta; Jares, Pedro; Juan, Manel; Rozman, María; Colomer, Dolors; Delgado, Julio; Giné, Eva; González-Díaz, Marcos; Hernández-Rivas, Jesús M; Colado, Enrique; Rayón, Consolación; Payer, Angel R; Terol, Maria José; Navarro, Blanca; Quesada, Victor; Puente, Xosé S; Rozman, Ciril; López-Otín, Carlos; Campo, Elías; López-Guillermo, Armando; Villamor, Neus

    2014-06-12

    Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor κB pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ≤50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ≤50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome. PMID:24782504

  3. Differences in codon bias and GC content contribute to the balanced expression of TLR7 and TLR9.

    PubMed

    Newman, Zachary R; Young, Janet M; Ingolia, Nicholas T; Barton, Gregory M

    2016-03-01

    The innate immune system detects diverse microbial species with a limited repertoire of immune receptors that recognize nucleic acids. The cost of this immune surveillance strategy is the potential for inappropriate recognition of self-derived nucleic acids and subsequent autoimmune disease. The relative expression of two closely related receptors, Toll-like receptor (TLR) 7 and TLR9, is balanced to allow recognition of microbial nucleic acids while limiting recognition of self-derived nucleic acids. Situations that tilt this balance toward TLR7 promote inappropriate responses, including autoimmunity; therefore, tight control of expression is critical for proper homeostasis. Here we report that differences in codon bias limit TLR7 expression relative to TLR9. Codon optimization of Tlr7 increases protein levels as well as responses to ligands, but, unexpectedly, these changes only modestly affect translation. Instead, we find that much of the benefit attributed to codon optimization is actually the result of enhanced transcription. Our findings, together with other recent examples, challenge the dogma that codon optimization primarily increases translation. We propose that suboptimal codon bias, which correlates with low guanine-cytosine (GC) content, limits transcription of certain genes. This mechanism may establish low levels of proteins whose overexpression leads to particularly deleterious effects, such as TLR7. PMID:26903634

  4. An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept.

    PubMed

    Smith, Nikaïa; Vidalain, Pierre-Olivier; Nisole, Sébastien; Herbeuval, Jean-Philippe

    2016-01-01

    Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce massive levels of type I interferon in response to pathogens. Unfortunately, pDC are fragile and extremely rare, rendering their functional study a tough challenge. However, because of their central role in numerous pathologies, there is a considerable need for an efficient and reproducible protocol for gene silencing in these cells. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation, and poly-ethylenimine (PEI) technologies. We show that lipid-based reagent DOTAP was extremely efficient for siRNA delivery into pDC, and did not induce cell death or pDC activation. We successfully silenced Toll-Like Receptor 7 (TLR7), CXCR4 and IFN regulatory factor 7 (IRF-7) gene expression in pDC as assessed by RT-qPCR or cytometry. Finally, we showed that TLR7 or IRF-7 silencing in pDC specifically suppressed IFN-α production upon stimulation, providing a functional validation of our transfection protocol. PMID:27412723

  5. An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept

    PubMed Central

    Smith, Nikaïa; Vidalain, Pierre-Olivier; Nisole, Sébastien; Herbeuval, Jean-Philippe

    2016-01-01

    Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce massive levels of type I interferon in response to pathogens. Unfortunately, pDC are fragile and extremely rare, rendering their functional study a tough challenge. However, because of their central role in numerous pathologies, there is a considerable need for an efficient and reproducible protocol for gene silencing in these cells. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation, and poly-ethylenimine (PEI) technologies. We show that lipid-based reagent DOTAP was extremely efficient for siRNA delivery into pDC, and did not induce cell death or pDC activation. We successfully silenced Toll-Like Receptor 7 (TLR7), CXCR4 and IFN regulatory factor 7 (IRF-7) gene expression in pDC as assessed by RT-qPCR or cytometry. Finally, we showed that TLR7 or IRF-7 silencing in pDC specifically suppressed IFN-α production upon stimulation, providing a functional validation of our transfection protocol. PMID:27412723

  6. Discovery and Structure-Activity Relationships of the Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists.

    PubMed

    Morin, Matthew D; Wang, Ying; Jones, Brian T; Su, Lijing; Surakattula, Murali M R P; Berger, Michael; Huang, Hua; Beutler, Elliot K; Zhang, Hong; Beutler, Bruce; Boger, Dale L

    2016-05-26

    Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor. PMID:27050713

  7. TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice

    PubMed Central

    Liu, Tong; Berta, Temugin; Xu, Zhen-Zhong; Park, Chul-Kyu; Zhang, Ling; Lü, Ning; Liu, Qin; Liu, Yang; Gao, Yong-Jing; Liu, Yen-Chin; Ma, Qiufu; Dong, Xinzhong; Ji, Ru-Rong

    2012-01-01

    Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. TLRs mediate innate immunity and regulate neuropathic pain, but their roles in pruritus are elusive. Here, we report that scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Notably, we found that treatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3–/– mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3–/– mice but not Tlr7–/– mice. Consequently, central sensitization–driven pain hypersensitivity, but not acute pain, was impaired in Tlr3–/– mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3–/– mice. Our findings demonstrate a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization. TLR3 may serve as a new target for developing anti-itch treatment. PMID:22565312

  8. Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer.

    PubMed

    Kim, Tae Woo; Lee, Seon-Jin; Oh, Byung Moo; Lee, Heesoo; Uhm, Tae Gi; Min, Jeong-Ki; Park, Young-Jun; Yoon, Suk Ran; Kim, Bo-Yeon; Kim, Jong Wan; Choe, Yong-Kyung; Lee, Hee Gu

    2016-01-26

    Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells. PMID:26675260

  9. Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer

    PubMed Central

    Oh, Byung Moo; Lee, Heesoo; Uhm, Tae Gi; Min, Jeong-Ki; Park, Young-Jun; Yoon, Suk Ran; Kim, Bo-Yeon; Kim, Jong Wan; Choe, Yong-Kyung; Lee, Hee Gu

    2016-01-01

    Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells. PMID:26675260

  10. Humoral immune response and TLR9 gene expression in Pacific red snapper (Lutjanus peru) experimentally exposed to Aeromonas veronii.

    PubMed

    Reyes-Becerril, Martha; Angulo, Carlos; Ascencio, Felipe

    2015-02-01

    Aquaculture production of Pacific red snapper Lutjanus peru is growing rapidly in Mexico, especially in Gulf of California. As it is a relatively new aquaculture species there are few reports evaluating its immune response to pathogens. The Gram-negative bacteria Aeromonas veronii is a heterogeneous organism that causes the disease known as motile aeromonad septicemia, which is responsible for serious economic loss in seabream culture due to bacterial infections. For the purpose of this study, juvenile Pacific red snapper specimens were intraperitoneally injected with low doses of A. veronii (1 × 10(6) CFU ml(-1)). Changes in humoral immune parameters (total protein, myeloperoxidase, lisozyme and antiprotease activities and IgM levels), as well as superoxide dismutase and catalase activities, and TLR9 gene expression were evaluated 24 and 48 h after injection. Overall, the results showed an enhanced in humoral immune parameters and SOD and CAT activities in fish infected with A. veronii compared with control group at 24 or 48 h. By real time PCR assays, the basal mRNA transcripts of TLR9 showed that were highly expressed in intestine and leucocytes compared to skin, head kidney, liver and gill. Then, the mRNA expression levels of TLR9 in head kidney, skin, liver and intestine were analyzed in non-infected and experimentally infected fish 24 and 48 h after injection. A. veronii up-regulated the expression of TLR9 at 24 or 48 h of exposure in all samples analyzed except in liver. Interestingly, intestine produced the greatest increase in transcript levels upon exposure (48 h) to A. veronii. Taken together, our results suggest that low doses of A. veronii infection inducing humoral immune system and TLR9 immune gene in Pacific red snapper that can be useful in the health control of this species. PMID:25462554

  11. Copy Number Variation of TLR-7 Gene and its Association with the Development of Systemic Lupus Erythematosus in Female Patients from Yucatan Mexico

    PubMed Central

    Pacheco, Guillermo Valencia; Cruz, Darig Cámara; González Herrera, Lizbeth J; Pérez Mendoza, Gerardo J; Adrián Amaro, Guadalupe I; Nakazawa Ueji, Yumi E; Angulo Ramírez, Angélica V

    2014-01-01

    Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies against self-antigens, which occurs most often in women between 15 and 40 years of age. The innate immunity is involved in the pathogenesis of SLE through TLR- 7. Genetic factors such as copy number variation (CNV) of target genes may contribute to disease development, but this possible risk has not yet been studied in SLE patients from Yucatan, Mexico. The CNV of TLR-7 gene was determined by quantitative polymerase chain reaction assay using TaqMan probes in 80 SLE women and 150 control subjects. The results showed that 10% of SLE patients exhibited more than two copies of TLR-7 gene, whereas no mRNA overexpression was detected. These data suggested that increased CNV of the TLR-7 gene in Yucatan SLE women can be a risk factor for this disease. PMID:25512712

  12. Oligonucleotides Designed to Inhibit TLR9 Block Herpes Simplex Virus type 1 Infection at Multiple Steps

    PubMed Central

    Sauter, Monica M.; Gauger, Joshua J. L.; Brandt, Curtis R.

    2014-01-01

    Herpes simplex virus type 1 (HSV-1) is an important human pathogen which requires activation of nuclear factor–kappa B (NFκB) during its replication cycle. The persistent nature of HSV-1 infection, and the emergence of drug-resistant strains, highlights the importance of research to develop new antiviral agents. Toll-like receptors (TLR) play a prominent role during the early antiviral response by recognizing viral nucleic acid and gene products, activating NFκB, and stimulating the production of inflammatory cytokines. We demonstrate a significant effect on HSV-1 replication in ARPE-19 and Vero cells when oligonucleotides designed to inhibit TLR9 are added 2 hours prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10 to 20 micromolar. TLR9 inhibitory oligonucleotides prevented expression of essential immediate early herpes gene products as determined by immunofluorescence microscopy and Western blotting. TLR9 oligonucleotides also interfered with viral attachment and entry. A TLR9 inhibitory oligonucleotide containing five adjacent guanosine residues (G-ODN) exhibited virucidal activity and inhibited HSV-1 replication when added post-infection. The antiviral effect of the TLR9 inhibitory oligonucleotides did not depend on the presence of TLR9 protein, suggesting a mechanism of inhibition that is not TLR9 specific. TLR9 inhibitory oligonucleotides also reduced NFκB activity in nuclear extracts. Studies using these TLR inhibitors in the context of viral infection should be interpreted with caution. PMID:24995383

  13. Esophageal Cancer Related Gene-4 (ECRG4) Interactions with the Innate Immunity Receptor Complex

    PubMed Central

    Podvin, Sonia; Dang, Xitong; Meads, Morgan; Kurabi, Arwa; Costantini, Todd; Eliceiri, Brian P.; Baird, Andrew; Coimbra, Raul

    2014-01-01

    Objective and design The human c2orf40 gene encodes a tumor suppressor gene called esophageal cancer-related gene-4 (ECRG4) with pro- and anti-inflammatory activities that depend on cell surface processing. Here, we investigated its physical and functional association with the innate immunity receptor complex. Methods Interactions between ECRG4 and the innate immunity receptor complex were assessed by flow cytometry, immunohistochemistry, confocal microscopy, co-immunoprecipitation. Phage display was used for ligand-targeting to cells that over express the TLR4-MD2-CD14. Results Immunoprecipitation and immunohistochemical studies demonstrate a physical interaction between ECRG4 and TLR4-MD2-CD14 on human granulocytes. Flow cytometry shows ECRG4 on the cell surface of a subset of CD14+ and CD16+ leukocytes. In a cohort of trauma patients, the C-terminal 16 amino acid domain of ECRG4 (ECRG4133–148), appears processed and shed, presumably at a thrombin-like consensus sequence. Phage targeting this putative ligand shows that this peptide sequence can internalizes into cells through the TLR4/CD14/MD2 complex but modulates inflammation through non-canonical, NFκB signal transduction. Conclusions ECRG4 is present on the surface of human monocytes and granulocytes. Its interaction with the human innate immunity receptor complex supports a role for cell surface activation of ECRG4 during inflammation and implicates this receptor in its mechanism of action. PMID:25511108

  14. Association between Toll-like receptor 9 gene polymorphisms and risk of bacterial meningitis in a Chinese population.

    PubMed

    Wang, X H; Shi, H P; Li, F J

    2016-01-01

    We determined whether two common single nucleotide polymorphisms (SNPs) in the Toll-like receptor 9 gene (TLR9) (TLR9+2848 rs352140 and TLR9-1237 rs5743836) influenced susceptibility to bacterial meningitis in a Chinese population. The study comprised 126 patients with bacterial meningitis and 252 control subjects, all of whom were recruited from the Tuberculosis Hospital of Shanxi Province. Genotyping of TLR9+2848 rs352140 and TLR9-1237 rs5743836 was performed by polymerase chain reaction coupled with restriction fragment length polymorphism. Using logistic regression analysis, we found that individuals with the AA genotype were associated with an increased risk of bacterial meningitis compared with those with the GG genotype (OR = 0.43, 95%CI = 0.19-0.95; P = 0.03). In a recessive model, the AA genotype was correlated with an elevated risk of bacterial meningitis compared with the GG+GA genotype (OR = 0.49, 95%CI = 0.22-0.99; P = 0.04). However, no significant differences were observed in the association between the TLR9-1237 rs5743836 polymorphism and the risk of bacterial meningitis in the codominant, dominant, or recessive models. In conclusion, the results of our study suggest an association between the TLR9+2848 polymorphism and a reduced risk of bacterial meningitis in the codominant and recessive models. PMID:27525854

  15. CD14 Is a Co-Receptor for TLR4 in the S100A9-Induced Pro-Inflammatory Response in Monocytes

    PubMed Central

    He, Zhifei; Riva, Matteo; Björk, Per; Swärd, Karl; Mörgelin, Matthias; Leanderson, Tomas; Ivars, Fredrik

    2016-01-01

    The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response. PMID:27228163

  16. Evidence for TLR4 and FcRγ-CARD9 activation by cholera toxin B subunit and its direct bindings to TREM2 and LMIR5 receptors.

    PubMed

    Phongsisay, Vongsavanh; Iizasa, Ei'ichi; Hara, Hiromitsu; Yoshida, Hiroki

    2015-08-01

    Cholera toxin (CTX) is a virulent factor of Vibrio cholerae that causes life-threatening diarrheal disease. Its non-toxic subunit CTB has been extensively studied for vaccine delivery. In immune cells, CTB induces a number of signaling molecules related to cellular activation and cytokine production. The mechanisms by which CTB exerts its immunological effects are not understood. We report here the immunological targets of CTB. The unexpected finding that GM1 ganglioside inhibited NF-κB activation in human monocytes stimulated with CTX and agonists of Toll-like receptors (TLR) suggests the possibility of CTX-TLR interaction. Indeed, CTX-induced IL-6 production was substantially reduced in MyD88(-/-) or TLR4(-/-) macrophages. Ectopic expression of TLR4 was required for CTX-induced NF-κB activation in HEK 293 cells. Furthermore, the inflammatory capacity of CTB was lost in the absence of TLR4, adaptor protein FcRγ, or its downstream signaling molecule CARD9. Attempts have been made to identify CTB-binding targets from various C-type lectin and immunoglobulin-like receptors. CTB targeted not only GM1 and TLR4 but also TREM2 and LMIR5/CD300b. CTB-TREM2 interaction initiated signal transduction through adaptor protein DAP12. The binding of CTB inhibited LMIR5 activation induced by its endogenous ligand 3-O-sulfo-β-d-galactosylceramide C24:1. In summary, CTB targets TLR4, FcRγ-CARD9, TREM2, and LMIR5. These findings provide new insights into the immunobiology of cholera toxin. PMID:26021803

  17. PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2.

    PubMed

    Darehgazani, Reyhaneh; Peymani, Maryam; Hashemi, Motahare-Sadat; Omrani, Mir Davood; Movafagh, Abolfazl; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

    2016-08-01

    TLR4 is transmembrane pattern-recognition receptor that initiates signals in response to diverse pathogen-associated molecular patterns especially LPS. Recently, there have been an increasing number of studies about the role of TLRs in the pathogenesis of several disorders as well as the therapeutic potential of TLR intervention in such diseases. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a ligand-activated transcription factor with numerous biological effects. PPARγ has been shown to exert a potential anti-inflammatory effect through suppression of TLR4-mediated inflammation. Therefore, PPARγ agonists may have a potential to combat inflammatory conditions in pathologic states. The current study aims to show the decrease of inflammation by overexpression of PPARγ in a cell reporter model. To reach this goal, recombinant pBudCE4.1 (+) containing encoding sequences of human TLR4 and MD2 was constructed and used to transfect HEK cells. Subsequently, inflammation was induced by LPS treatment as control group. In the treatment group, overexpression of PPARγ prior to inflammation was performed and the expression of inflammatory markers was assessed in this condition. The expression of inflammatory markers (TNFα and iNOS) was defined by quantitative real time PCR and the amount of phosphorylated NF-κB was measured by western blot. Data indicated expression of TNFα and iNOS increased in LPS induced inflammation of stably transformed HEK cells with MD2 and TLR4. In this cell reporter model overexpression of PPARγ dramatically prevented LPS-induced inflammation through the blocking of TLR4/NF-κB signaling. PPARγ was shown to negatively regulate TLR4 activity and therefore exerts its anti-inflammatory action against LPS induced inflammation. PMID:26224481

  18. Lectin-like ox-LDL receptor-1 (LOX-1)-Toll-like receptor 4 (TLR4) interaction and autophagy in CATH.a differentiated cells exposed to angiotensin II.

    PubMed

    Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Deng, Xiaoyan; Fan, Yubo; Xiang, David; Mehta, Jawahar L

    2015-04-01

    Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs has also been linked to autophagy. As the main receptor for oxidized low-density lipoprotein (ox-LDL) on the cell surface, lectin-like ox-LDL receptor-1 (LOX-1) is upregulated by proinflammatory cytokines and has been linked to the development of autophagy. However, the relationship between LOX-1, autophagy, and TLR4 in neurons has not been defined. Here, we show that Angiotensin II (Ang II) treatment of CATH.a differentiated neuronal cells resulted in the expression of TLR4 (and associated signals MyD88 and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon (TRIF)), LOX-1 autophagy. LOX-1 knockdown (transfection with specific small interfering RNA (siRNA)) resulted in reduced expression of TLR4 (and associated signals MyD88 and TRIF) and P-P38 mitogen-activated protein kinase (MAPK) and autophagy. TLR4 knockdown with siRNA resulted in reduced LOX-1 expression and autophagy, indicating a positive feedback between LOX-1 and TLR4. Knockdown of TRIF as well as MyD88 or inhibition of P38 MAPK also inhibited the expression of LOX-1 and TLR4 and autophagy. Importantly, pretreatment with 3-methyladenine (autophagy inhibitor) enhanced while rapamycin (autophagy inducer) decreased the expression of LOX-1, TLR4, and P-P38 MAPK. These studies suggest the presence of a bidirectional link between LOX-1and TLR4 in cultured CATH.a differentiated cells exposed to Ang II with an important role for autophagy in this link. PMID:24902807

  19. Toll-like receptor 5 (TLR5), IL-1β secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing.

    PubMed

    Descamps, Delphyne; Le Gars, Mathieu; Balloy, Viviane; Barbier, Diane; Maschalidi, Sophia; Tohme, Mira; Chignard, Michel; Ramphal, Reuben; Manoury, Bénédicte; Sallenave, Jean-Michel

    2012-01-31

    A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1(-/-) and AEP(-/-) mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1β formation. PMID:22307620

  20. Toll-like receptor 5 (TLR5), IL-1β secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing

    PubMed Central

    Descamps, Delphyne; Le Gars, Mathieu; Balloy, Viviane; Barbier, Diane; Maschalidi, Sophia; Tohme, Mira; Chignard, Michel; Ramphal, Reuben; Manoury, Bénédicte; Sallenave, Jean-Michel

    2012-01-01

    A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5−/− AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1−/− and AEP−/− mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1β formation. PMID:22307620

  1. DAMPening Inflammation by Modulating TLR Signalling

    PubMed Central

    Piccinini, A. M.; Midwood, K. S.

    2010-01-01

    Damage-associated molecular patterns (DAMPs) include endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix (ECM) molecules that are upregulated upon injury or degraded following tissue damage. DAMPs are vital danger signals that alert our immune system to tissue damage upon both infectious and sterile insult. DAMP activation of Toll-like receptors (TLRs) induces inflammatory gene expression to mediate tissue repair. However, DAMPs have also been implicated in diseases where excessive inflammation plays a key role in pathogenesis, including rheumatoid arthritis (RA), cancer, and atherosclerosis. TLR activation by DAMPs may initiate positive feedback loops where increasing tissue damage perpetuates pro-inflammatory responses leading to chronic inflammation. Here we explore the current knowledge about distinct signalling cascades resulting from self TLR activation. We also discuss the involvement of endogenous TLR activators in disease and highlight how specifically targeting DAMPs may yield therapies that do not globally suppress the immune system. PMID:20706656

  2. Nucleotide Sequencing and SNP Detection of Toll-Like Receptor-4 Gene in Murrah Buffalo (Bubalus bubalis)

    PubMed Central

    Mitra, M.; Taraphder, S.; Sonawane, G. S.; Verma, A.

    2012-01-01

    Toll-like receptor-4 (TLR-4) has an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections. The present investigation was carried out to study nucleotide sequencing and SNP detection by PCR-RFLP analysis of the TLR-4 gene in Murrah buffalo. Genomic DNA was isolated from 102 lactating Murrah buffalo from NDRI herd. The amplified PCR fragments of TLR-4 comprised of exon 1, exon 2, exon 3.1, and exon 3.2 were examined to RFLP. PCR products were obtained with sizes of 165, 300, 478, and 409 bp. TLR-4 gene of investigated Murrah buffaloes was highly polymorphic with AA, AB, and BB genotypes as revealed by PCR-RFLP analysis using Dra I, Hae III, and Hinf I REs. Nucleotide sequencing of the amplified fragment of TLR-4 gene of Murrah buffalo was done. Twelve SNPs were identified. Six SNPs were nonsynonymous resulting in change in amino acids. Murrah is an indigenous Buffalo breed and the presence of the nonsynonymous SNP is indicative of its unique genomic architecture. Sequence alignment and homology across species using BLAST analysis revealed 97%, 97%, 99%, 98%, and 80% sequence homology with Bos taurus, Bos indicus, Ovis aries, Capra hircus, and Homo sapiens, respectively.

  3. The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6.

    PubMed

    Irvine, Katherine Lucy; Hopkins, Lee Jason; Gangloff, Monique; Bryant, Clare Elizabeth

    2013-01-01

    TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and humans dose-dependently responded to lipoteichoic acid from Staphylococcus aureus, with no significant species difference in EC50 at either receptor pair. The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species' TLRs do not significantly affect ligand recognition. Species differences were seen between the responses to Pam2CSK4 and Pam3CSK4 at TLR2/1. Human TLR2/1, as expected, responded to Pam3CSK4 with greater potency and efficacy than Pam2CSK4. At equine TLR2/1, however, Pam3CSK4 was less potent than Pam2CSK4, with both ligands having similar efficacies. Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning. These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections. PMID:23826682

  4. The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6

    PubMed Central

    2013-01-01

    TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and humans dose-dependently responded to lipoteichoic acid from Staphylococcus aureus, with no significant species difference in EC50 at either receptor pair. The EC50 of Pam2CSK4 was the same for equine and human TLR2/6, indicating amino acid differences between the two species’ TLRs do not significantly affect ligand recognition. Species differences were seen between the responses to Pam2CSK4 and Pam3CSK4 at TLR2/1. Human TLR2/1, as expected, responded to Pam3CSK4 with greater potency and efficacy than Pam2CSK4. At equine TLR2/1, however, Pam3CSK4 was less potent than Pam2CSK4, with both ligands having similar efficacies. Molecular modelling indicates that the majority of non-conserved ligand-interacting residues are at the periphery of the TLR2 binding pocket and in the ligand peptide-interacting regions, which may cause subtle effects on ligand positioning. These results suggest that there are potentially important species differences in recognition of lipopeptides by TLR2/1, which may affect how the horse deals with bacterial infections. PMID:23826682

  5. Toll-Like Receptor-4 Dependent Intestinal Gene Expression During Arcobacter Butzleri Infection of Gnotobiotic Il-10 Deficient Mice

    PubMed Central

    Gölz1, Greta; Alter, Thomas; Bereswill, Stefan; Heimesaat, Markus M.

    2016-01-01

    We have previously shown that Arcobacter butzleri infection induces Toll-like receptor (TLR) -4 dependent immune responses in perorally infected gnotobiotic IL-10–/– mice. Here, we analyzed TLR-4-dependent expression of genes encoding inflammatory mediators and matrix-degrading gelatinases MMP-2 and -9 in the small and large intestines of gnotobiotic TLR-4-deficient IL-10–/– mice that were perorally infected with A. butzleri strains CCUG 30485 or C1, of human and chicken origin, respectively. At day 6 following A. butzleri infection, colonic mucin-2 mRNA, as integral part of the intestinal mucus layer, was downregulated in the colon, but not ileum, of IL-10–/– but not TLR-4–/– IL-10–/– mice. CCUG 30485 strain-infected TLR-4-deficient IL-10–/– mice displayed less distinctly upregulated IFN-γ, IL-17A, and IL-1β mRNA levels in ileum and colon, which was also true for colonic IL-22. These changes were accompanied by upregulated colonic MMP-2 and ileal MMP-9 mRNA exclusively in IL-10–/– mice. In conclusion, TLR-4 is essentially involved in A. butzleri mediated modulation of gene expression in the intestines of gnotobiotic IL-10–/– mice. PMID:27141316

  6. Establishment of the model of white blood cell membrane chromatography and screening of antagonizing TLR4 receptor component from Atractylodes macrocephala Koidz.

    PubMed

    Li, Cuiqin; He, Langchong

    2006-04-01

    A model of white blood cell membrane chromatography (WB-CMC) was established to screen active component from Atractylodes macrocephala Koidz. The component can antagonize Toll-like receptor 4 (TLR4) and inhibit inflammatory reaction. In the model of WB-CMC, cell membrane stationary phase (CMSP) was prepared by immobilizing the rabbit white blood cell membrane (WBCM) onto the surface of silica carrier and taxinol was used as a model molecule. The active component which can act on WBCM and its receptor (such as TLR4) as an effective target in A. macrocephala was determined by using a replacement experiment. The anti-inflammatory effects of the active component were tested by using pharmacological methods in vivo. The results indicated that the retention characteristics of atractylenolide I as active component was similar to that of taxinol in the model of WB-CMC. And so, atractylenolide I acted on the WBCM and TLR4 and its anti-inflammatory activity was related with antagonizing TLR4. Therefore, the interaction between the active component and WBCM and its receptor can be simulated by the model of WB-CMC in vitro. This model can be used to screen active components and to study effective characteristics for acting on definite targets. PMID:16704122

  7. Cleavage and activation of a Toll-like receptor by microbial proteases

    PubMed Central

    de Zoete, Marcel R.; Bouwman, Lieneke I.; Keestra, A. Marijke; van Putten, Jos P. M.

    2011-01-01

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB–dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  8. Cleavage and activation of a Toll-like receptor by microbial proteases.

    PubMed

    de Zoete, Marcel R; Bouwman, Lieneke I; Keestra, A Marijke; van Putten, Jos P M

    2011-03-22

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB-dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  9. Toll-Like Receptor (TLR)-1/2 Triggering of Multiple Myeloma Cells Modulates Their Adhesion to Bone Marrow Stromal Cells and Enhances Bortezomib-Induced Apoptosis

    PubMed Central

    Abdi, Jahangir; Mutis, Tuna; Garssen, Johan; Redegeld, Frank A.

    2014-01-01

    In multiple myeloma (MM), the malignant plasma cells usually localize to the bone marrow where they develop drug resistance due to adhesion to stromal cells and various environmental signals. Hence, modulation of this interaction is expected to influence drug sensitivity of MM cells. Toll-like receptor (TLR) ligands have displayed heterogeneous effects on B-cell malignancies and also on MM cells in a few recent studies, but effects on adhesion and drug sensitivity of myeloma cells in the context of bone marrow stromal cells (BMSCs) have never been investigated. In the present study, we explored the modulatory effects of TLR1/2 ligand (Pam3CSK4) on adhesion of human myeloma cells to BMSCs. It is shown that TLR1/2 triggering has opposite effects in different HMCLs on their adhesion to BMSCs. Fravel, L363, UM-6, UM-9 and U266 showed increased adhesion to BMSC in parallel with an increased surface expression of integrin molecules α4 and αVβ3. OPM-1, OPM-2 and NCI-H929 showed a dose-dependent decrease in adhesion upon TLR activation following a downregulation of β7 integrin expression. Importantly, TLR1/2 triggering increased cytotoxic and apoptotic effects of bortezomib in myeloma cells independent of the effect on stromal cell adhesion. Moreover, the apoptosis-enhancing effect of Pam3CSK4 paralleled induction of cleaved caspase-3 protein in FACS analysis suggesting a caspase-dependent mechanism. Our findings uncover a novel role of TLR activation in MM cells in the context of bone marrow microenvironment. Stimulation of TLR1/2 bypasses the protective shield of BMSCs and may be an interesting strategy to enhance drug sensitivity of multiple myeloma cells. PMID:24794258

  10. Identification and immune functional characterization of pigeon TLR7.

    PubMed

    Xiong, Dan; Song, Li; Pan, Zhiming; Chen, Xiang; Geng, Shizhong; Jiao, Xinan

    2015-01-01

    Toll-like receptor 7 (TLR7) is activated by single-stranded RNA and synthetic imidazoquinoline components, and induces interferon production. In this study, we cloned the TLR7 gene from King pigeon (Columba livia). The TLR7 open reading frame is 3144 bp and encodes a 1047-amino acid protein, consisting of a canonical TLR composition with 15 leucine-rich repeats (LRRs). Amino acid-inserting modifications were found at position 15 of LRR2, LRR11, LRR13, and LRR14 and position 10 of LRR10. The tissue distribution of pigeon TLR7 suggests that immune-associated tissues, especially the spleen and liver, have high TLR7 expression. HEK293T cells transfected with pigeon TLR7 plasmid responded to the agonist R848, indicating a functional TLR7 homolog. Following R848 stimulation of pigeon peripheral blood mononuclear cells, the levels of IFN-γ, IL-6, IL-8, CCL5, and IL-10 mRNA, assessed using quantitative real-time PCR, were significantly up-regulated. After Newcastle disease virus vaccine strain LaSota inoculation and agonist R848 injection, the level of TLR7 mRNA in the spleen of pigeons increased significantly in the R848-injected group, but decreased in the LaSota-inoculated group at three day post-infection (d.p.i.). The mRNA levels of inflammatory cytokines and chemokines were significantly upregulated in both LaSota-inoculated and R848-injected groups. Triggering pigeon TLR7 leads to robust up-regulation of inflammatory cytokines and chemokines, suggesting an important role in the innate immune response. PMID:25874762

  11. Modulation of Innate Immune Responses via Covalently Linked TLR Agonists

    PubMed Central

    2015-01-01

    We present the synthesis of novel adjuvants for vaccine development using multivalent scaffolds and bioconjugation chemistry to spatially manipulate Toll-like receptor (TLR) agonists. TLRs are primary receptors for activation of the innate immune system during vaccination. Vaccines that contain a combination of small and macromolecule TLR agonists elicit more directed immune responses and prolong responses against foreign pathogens. In addition, immune activation is enhanced upon stimulation of two distinct TLRs. Here, we synthesized combinations of TLR agonists as spatially defined tri- and di-agonists to understand how specific TLR agonist combinations contribute to the overall immune response. We covalently conjugated three TLR agonists (TLR4, 7, and 9) to a small molecule core to probe the spatial arrangement of the agonists. Treating immune cells with the linked agonists increased activation of the transcription factor NF-κB and enhanced and directed immune related cytokine production and gene expression beyond cells treated with an unconjugated mixture of the same three agonists. The use of TLR signaling inhibitors and knockout studies confirmed that the tri-agonist molecule activated multiple signaling pathways leading to the observed higher activity. To validate that the TLR4, 7, and 9 agonist combination would activate the immune response to a greater extent, we performed in vivo studies using a vaccinia vaccination model. Mice vaccinated with the linked TLR agonists showed an increase in antibody depth and breadth compared to mice vaccinated with the unconjugated mixture. These studies demonstrate how activation of multiple TLRs through chemically and spatially defined organization assists in guiding immune responses, providing the potential to use chemical tools to design and develop more effective vaccines. PMID:26640818

  12. [THE EXPRESSION OF TLR-4 GENE MONONUCLEAR CELLS PERIPHERAL BLOOO IN PATIENTS BY HIGH RISK OF PURULENT-INFLAMMATORY COMPLICATIONS AFTER SURGERY FOR ACUTE DISEASES OF THE ABDOMINAL ORGANS].

    PubMed

    Sheyko, V D; Sytnik, D A; Pryhidko, R A; Shkurupiy, O A; Shlykova, O A; Izmailova, O V

    2015-06-01

    The specified level of gene expression TLR-4 in peripheral blood mononuclear cells in 77 patients operated on acute diseases of the abdominal organs in the 1st and the 4th day after surgery was determined. Established dynamic changes of gene expression TLR-4. Adverse course early postoperative period in patients initially high and medium risk of purulent-septic complications was accompanied by activation of gene expression TLR-4 in peripheral blood mononuclear cells. PMID:26521462

  13. Prevention and Mitigation of Acute Radiation Syndrome in Mice by Synthetic Lipopeptide Agonists of Toll-Like Receptor 2 (TLR2)

    PubMed Central

    Shakhov, Alexander N.; Singh, Vijay K.; Bone, Frederick; Cheney, Alec; Kononov, Yevgeniy; Krasnov, Peter; Bratanova-Toshkova, Troitza K.; Shakhova, Vera V.; Young, Jason; Weil, Michael M.; Panoskaltsis-Mortari, Angela; Orschell, Christie M.; Baker, Patricia S.; Gudkov, Andrei; Feinstein, Elena

    2012-01-01

    Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios. PMID:22479357

  14. Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling.

    PubMed

    Mcdonald, Kerry-Ann; Huang, Hai; Tohme, Samer; Loughran, Patricia; Ferrero, Kimberly; Billiar, Timothy; Tsung, Allan

    2014-01-01

    Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1-TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1's release from hypoxic hepatocytes in vitro and thereby weakened HMGB1's activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury. PMID:25375408

  15. Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells

    PubMed Central

    Rashidi, Nesa; Mirahmadian, Mahroo; Jeddi-Tehrani, Mahmood; Rezania, Simin; Ghasemi, Jamileh; Kazemnejad, Somaieh; Mirzadegan, Ebrahim; Vafaei, Sedigheh; Kashanian, Maryam; Rasoulzadeh, Zahra; Zarnani, Amir-Hassan

    2015-01-01

    Background Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. Results WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p < 0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p < 0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner (p < 0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p < 0.05). Conclusion Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus. PMID:25927023

  16. Identification and functional characterization of nonmammalian Toll-like receptor 20.

    PubMed

    Pietretti, Danilo; Scheer, Marleen; Fink, Inge R; Taverne, Nico; Savelkoul, Huub F J; Spaink, Herman P; Forlenza, Maria; Wiegertjes, Geert F

    2014-02-01

    Like other vertebrate Toll-like receptors (TLRs), the TLRs of teleost fish can be subdivided into six major families, each of which recognize a general class of molecular patterns. However, there also are a number of Tlrs with unknown function, the presence of which seems unique to the bony fish, among which is Tlr20. We identified full-length complementary DNA (cDNA) sequences for tlr20 of zebrafish and common carp, two closely related fish species. Zebrafish have six copies of tlr20, whereas carp express only a single copy. Both zebrafish Tlr20 (at least Tlr20a-d) and carp Tlr20 have 26 leucine-rich repeats (LRRs). Three-dimensional modeling indicates a best fit to the crystal structure of TLR8. Phylogenetic analyses place Tlr20 in the TLR11 family closest to Tlr11 and Tlr12, which sense ligands from protozoan parasites in the mouse. Conservation of genes on zebrafish chromosome 9, which carries tlr20, with genes on mouse chromosome 14, which carries tlr11, indicates Tlr11 could be a possible ortholog of Tlr20. Confocal microscopy suggests a subcellular localization of Tlr20 at the endoplasmatic reticulum. Although in vitro reporter assays could not identify a ligand unique to Tlr20, in vivo infection experiments indicate a role for Tlr20 in the immune response of carp to protozoan parasites (Trypanoplasma borreli). Carp tlr20 is mainly expressed in peripheral blood leukocytes (PBL) with B lymphocytes, in particular, expressing relatively high levels of Tlr20. In vitro stimulation of PBL with T. borreli induces an upregulation of tlr20, supportive of a role for Tlr20 in the immune response to protozoan parasites. PMID:24327191

  17. Identification and characterization of a novel Toll-like receptor 2 homologue in the large yellow croaker Larimichthys crocea.

    PubMed

    Ao, Jingqun; Mu, Yinnan; Wang, Kunru; Sun, Min; Wang, Xianhui; Chen, Xinhua

    2016-01-01

    Toll-like receptors (TLRs) are key components of innate immunity that play significant roles in immune defence against pathogen invasion. In the present study, we identified a novel TLR2 homologue (LycTLR2b) in large yellow croaker (Larimichthys crocea) that shared low sequence identity with the previously reported large yellow croaker TLR2 (tentatively named LycTLR2a). The full-length cDNA of LycTLR2b was 2926 nucleotides (nt) long and encoded a protein consisting of 797 amino acids (aa). The deduced LycTLR2b protein exhibited a typical TLR domain architecture including a signal peptide, seven leucine-rich repeats (LRRs) in the extracellular region, a transmembrane domain, and a Toll-Interleukin 1 receptor (TIR) domain in the cytoplasmic region. Phylogenetic analysis showed that both LycTLR2a and LycTLR2b fall into a major clade formed by all TLR2 sequences, and are divided into two distinct branches. Genomic organization revealed that the LycTLR2b gene lacks intron, which is similar to zebrafish and human TLR2 genes, whereas the LycTLR2a gene contains multiple introns, as found in damselfish TLR2a and Fugu TLR2 genes. Syntenic analysis suggested that the occurrence of LycTLR2a and LycTLR2b may result from a relatively recent genome duplication event. LycTLR2b mRNA was constitutively expressed in all tissues examined although at different levels. Following bacterial vaccine challenge, LycTLR2b expression levels were significantly up-regulated in both spleen and head kidney tissues. Taken together, these results indicated that two different TLR2 homologues, which may play roles in antibacterial immunity, exist in large yellow croaker. PMID:26551050

  18. Functional TLR5 genetic variants affect human colorectal cancer survival.

    PubMed

    Klimosch, Sascha N; Försti, Asta; Eckert, Jana; Knezevic, Jelena; Bevier, Melanie; von Schönfels, Witigo; Heits, Nils; Walter, Jessica; Hinz, Sebastian; Lascorz, Jesus; Hampe, Jochen; Hartl, Dominik; Frick, Julia-Stefanie; Hemminki, Kari; Schafmayer, Clemens; Weber, Alexander N R

    2013-12-15

    Toll-like receptors (TLR) are overexpressed on many types of cancer cells, including colorectal cancer cells, but little is known about the functional relevance of these immune regulatory molecules in malignant settings. Here, we report frequent single-nucleotide polymorphisms (SNP) in the flagellin receptor TLR5 and the TLR downstream effector molecules MyD88 and TIRAP that are associated with altered survival in a large cohort of Caucasian patients with colorectal cancer (n = 613). MYD88 rs4988453, a SNP that maps to a promoter region shared with the acetyl coenzyme-A acyl-transferase-1 (ACAA1), was associated with decreased survival of patients with colorectal cancer and altered transcriptional activity of the proximal genes. In the TLR5 gene, rs5744174/F616L was associated with increased survival, whereas rs2072493/N592S was associated with decreased survival. Both rs2072493/N592S and rs5744174/F616L modulated TLR5 signaling in response to flagellin or to different commensal and pathogenic intestinal bacteria. Notably, we observed a reduction in flagellin-induced p38 phosphorylation, CD62L shedding, and elevated expression of interleukin (IL)-6 and IL-1β mRNA in human primary immune cells from TLR5 616LL homozygote carriers, as compared with 616FF carriers. This finding suggested that the well-documented effect of cytokines like IL-6 on colorectal cancer progression might be mediated by TLR5 genotype-dependent flagellin sensing. Our results establish an important link between TLR signaling and human colorectal cancer with relevance for biomarker and therapy development. PMID:24154872

  19. TLR-4 cooperates with Dectin-1 and mannose receptor to expand Th17 and Tc17 cells induced by Paracoccidioides brasiliensis stimulated dendritic cells.

    PubMed

    Loures, Flávio V; Araújo, Eliseu F; Feriotti, Claudia; Bazan, Silvia B; Calich, Vera L G

    2015-01-01

    The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the mannose receptor (MR) are C-type lectin receptors (CLRs) previously reported to cooperate with toll-like receptors (TLRs) signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4(+) and CD8(+) T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1, and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs) from C57BL/6 mice. Then, wild type (WT), Dectin-1(-/-), TLR-2(-/-), and TLR-4(-/-) DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4(+) and CD8(+) T cells. In addition, treatment of WT, TLR-2(-/-), and TLR-4(-/-) DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1(-/-) DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was

  20. Toll-like Receptor 2 (TLR2), Transforming Growth Factor-β, Hyaluronan (HA), and Receptor for HA-mediated Motility (RHAMM) Are Required for Surfactant Protein A-stimulated Macrophage Chemotaxis*

    PubMed Central

    Foley, Joseph P.; Lam, David; Jiang, Hongmei; Liao, Jie; Cheong, Naeun; McDevitt, Theresa M.; Zaman, Aisha; Wright, Jo Rae; Savani, Rashmin C.

    2012-01-01

    The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA- mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFβ. In turn, TGFβ1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2−/− mice failed to migrate in response to SPA but responded normally to TGFβ1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44−/− mice had similar responses to SPA, whereas those from RHAMM−/− mice had decreased chemotaxis to SPA, TGFβ1, and HA. In primary macrophages, SPA-stimulated TGFβ production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFβ production. TGFβ1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix. PMID:22948158

  1. TLR2 Activation Limits Rhinovirus-Stimulated CXCL-10 by Attenuating IRAK-1-Dependent IL-33 Receptor Signaling in Human Bronchial Epithelial Cells.

    PubMed

    Ganesan, Shyamala; Pham, Duc; Jing, Yaxun; Farazuddin, Mohammad; Hudy, Magdalena H; Unger, Benjamin; Comstock, Adam T; Proud, David; Lauring, Adam S; Sajjan, Uma S

    2016-09-15

    Airway epithelial cells are the major target for rhinovirus (RV) infection and express proinflammatory chemokines and antiviral cytokines that play a role in innate immunity. Previously, we demonstrated that RV interaction with TLR2 causes ILR-associated kinase-1 (IRAK-1) depletion in both airway epithelial cells and macrophages. Further, IRAK-1 degradation caused by TLR2 activation was shown to inhibit ssRNA-induced IFN expression in dendritic cells. Therefore, in this study, we examined the role of TLR2 and IRAK-1 in RV-induced IFN-β, IFN-λ1, and CXCL-10, which require signaling by viral RNA. In airway epithelial cells, blocking TLR2 enhanced RV-induced expression of IFNs and CXCL-10. By contrast, IRAK-1 inhibition abrogated RV-induced expression of CXCL-10, but not IFNs in these cells. Neutralization of IL-33 or its receptor, ST2, which requires IRAK-1 for signaling, inhibited RV-stimulated CXCL-10 expression. In addition, RV induced expression of both ST2 and IL-33 in airway epithelial cells. In macrophages, however, RV-stimulated CXCL-10 expression was primarily dependent on TLR2/IL-1R. Interestingly, in a mouse model of RV infection, blocking ST2 not only attenuated RV-induced CXCL-10, but also lung inflammation. Finally, influenza- and respiratory syncytial virus-induced CXCL-10 was also found to be partially dependent on IL-33/ST2/IRAK-1 signaling in airway epithelial cells. Together, our results indicate that RV stimulates CXCL-10 expression via the IL-33/ST2 signaling axis, and that TLR2 signaling limits RV-induced CXCL-10 via IRAK-1 depletion at least in airway epithelial cells. To our knowledge, this is the first report to demonstrate the role of respiratory virus-induced IL-33 in the induction of CXCL-10 in airway epithelial cells. PMID:27503209

  2. TLR3-/4-Priming Differentially Promotes Ca2+ Signaling and Cytokine Expression and Ca2+-Dependently Augments Cytokine Release in hMSCs

    PubMed Central

    Park, Kyoung Sun; Kim, Sun Hwa; Das, Amitabh; Yang, Shao-Nian; Jung, Kyoung Hwa; Kim, Mi Kyung; Berggren, Per-Olof; Lee, YoungSeek; Chai, Jin Choul; Kim, Hyun Jin; Chai, Young Gyu

    2016-01-01

    In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs. Poly(I:C) exposure rather than LPS incubation not only elevated inositol 1,4,5-triphosphate receptor (IP3R) expression and IP3R-mediated Ca2+ release, but also promoted Orai and STIM expression as well as store-operated Ca2+ entry into hMSCs. In addition, we also observed that 21 Ca2+ signaling genes were significantly up-regulated in response to TLR3 priming of hMSCs by RNA sequencing analysis. Both poly(I:C) and LPS exposure enhanced cytokine release from hMSCs. The enhanced cytokine release vanished upon siRNA knockdown and chelation of intracellular Ca2+. These data demonstrate that TLR3- and TLR4-priming differentially enhance Ca2+ signaling and cytokine expression, and Ca2+ -dependently potentiates cytokine release in hMSCs. PMID:26980664

  3. Evaluation of Toll-Like Receptors 2/3/4/9 Gene Polymorphisms in Cervical Cancer Evolution.

    PubMed

    Zidi, Sabrina; Sghaier, Ikram; Gazouani, Ezzedine; Mezlini, Amel; Yacoubi-Loueslati, Besma

    2016-04-01

    Accumulative epidemiological evidence suggests that polymorphisms of Toll-like receptors signaling pathway elucidated the cellular and molecular mechanisms of human diseases whose gaining a primordial importance. The aim of our study is to identify the role of TLR 2 (-196 to -174 del), TLR 3 (1377 C>T), TLR 4 (Asp299Gly) and TLR 9 (G2848A) gene polymorphisms with the evolution of cervical cancer in Tunisian women. Blood samples were collected from histopathologically confirmed patients with cervical cancer and unrelated healthy female controls of similar ethnicity. Genotyping of the analyzed polymorphisms were done using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism. For the TLR 2, Ins/Ins genotype is a protector factor [p = 0.006; OR: 0.35(0.16-0.73)] and the dominant genotype of TLR 3 increased the risk of CC in stage (III+IV); C/C versuss C/T [p = 0.033; OR: 2.03(1.00-4.13)] and C/C versus C/T+T/T [p = 0.036; OR: 1.93(1.00-3.74)]. For TLR 4, the dominant genotype Asp/Asp is implicated in the occurrence of CC in stage (I+II) [p = 0.000; OR: 4.55(1.58-13.06)], [p = 0.001; OR: 3.49(1.44-8.45)] and in stage (III+IV) [p = 0.038; OR: 3.77(0.87-16.29)], [p = 0.007; OR: 5.21(1.65-16.46)] and the major allele Asp is a risk factor for the development of tumor in stage (I+II). The TLR2 Ins/Del genotype is associated with tumor evolution to stage (III+IV) [p = 0.003; OR: 3.00 (1.22-7.35)] and the genotypes Gly/Gly and Asp/Gly+Gly/Gly and Gly allele of TLR 4 are implicated in tumor evolution to the advanced stages. Further, TLR 2, TLR 3, TLR 4 and TLR 9 gene polymorphisms are implicated in the modulation of CC risk due to tobacco usage and statue of menopause among cases. Our study suggests a relationship between the incidence of the TLR2, TLR 3, TLR 4 and TLR9 mutations and the clinical progression of CC according to the FIGO classification. However, future studies with different demographic and clinical characteristics in ethnically

  4. The emerging role of Toll-like receptor 4 in myocardial inflammation

    PubMed Central

    Yang, Y; Lv, J; Jiang, S; Ma, Z; Wang, D; Hu, W; Deng, C; Fan, C; Di, S; Sun, Y; Yi, W

    2016-01-01

    Toll-like receptors (TLRs) are a family of pattern recognition receptors involved in cardiovascular diseases. Notably, numerous studies have demonstrated that TLR4 activates the expression of several of pro-inflammatory cytokine genes that play pivotal roles in myocardial inflammation, particularly myocarditis, myocardial infarction, ischemia-reperfusion injury, and heart failure. In addition, TLR4 is an emerging target for anti-inflammatory therapies. Given the significance of TLR4, it would be useful to summarize the current literature on the molecular mechanisms and roles of TLR4 in myocardial inflammation. Thus, in this review, we first introduce the basic knowledge of the TLR4 gene and describe the activation and signaling pathways of TLR4 in myocardial inflammation. Moreover, we highlight the recent progress of research on the involvement of TLR4 in myocardial inflammation. The information reviewed here may be useful to further experimental research and to increase the potential of TLR4 as a therapeutic target. PMID:27228349

  5. Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing β-1,3 Glucan.

    PubMed

    Khan, Nida S; Kasperkovitz, Pia V; Timmons, Allison K; Mansour, Michael K; Tam, Jenny M; Seward, Michael W; Reedy, Jennifer L; Puranam, Sravanthi; Feliu, Marianela; Vyas, Jatin M

    2016-03-01

    Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate β-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that β-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing β-1,3 glucan beads. Dectin-1-mediated spleen tyrosine kinase activation is required for TLR9 trafficking to β-1,3 glucan-, A. fumigatus-, and C. albicans-containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of β-1,3 glucan by Dectin-1 triggers TLR9 trafficking to β-1,3 glucan-containing phagosomes, which may be critical in coordinating innate antifungal defense. PMID:26829985

  6. The TIR-Domain Containing Adaptor TRAM Is Required for TLR7 Mediated RANTES Production

    PubMed Central

    Shevlin, Enda; Miggin, Sinéad M.

    2014-01-01

    Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM−/− murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM−/− cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity. PMID:25211222

  7. Comparison of Gene Expression by Sheep and Human Blood Stimulated with the TLR4 Agonists Lipopolysaccharide and Monophosphoryl Lipid A

    PubMed Central

    Enkhbaatar, Perenlei; Nelson, Christina; Salsbury, John R.; Carmical, Joseph R.; Torres, Karen E. O.; Herndon, David; Prough, Donald S.; Luan, Liming; Sherwood, Edward R.

    2015-01-01

    Background Animal models that mimic human biology are important for successful translation of basic science discoveries into the clinical practice. Recent studies in rodents have demonstrated the efficacy of TLR4 agonists as immunomodulators in models of infection. However, rodent models have been criticized for not mimicking important characteristics of the human immune response to microbial products. The goal of this study was to compare genomic responses of human and sheep blood to the TLR4 agonists lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA). Methods Venous blood, withdrawn from six healthy human adult volunteers (~ 28 years old) and six healthy adult female sheep (~3 years old), was mixed with 30 μL of PBS, LPS (1μg/mL) or MPLA (10μg/mL) and incubated at room temperature for 90 minutes on a rolling rocker. After incubation, 2.5 mL of blood was transferred to Paxgene Blood RNA tubes. Gene expression analysis was performed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip. Agilent gene expression microarrays were scanned with a G2565 Microarray Scanner. Differentially expressed genes were identified. Results 11,431 human and 4,992 sheep probes were detected above background. Among them 1,029 human and 175 sheep genes were differentially expressed at a stringency of 1.5-fold change (p<0.05). Of the 175 sheep genes, 54 had a known human orthologue. Among those genes, 22 had > 1.5-fold changes in human samples. Genes of major inflammatory mediators, such as IL-1, IL-6 and IL-8, TNF alpha, NF-kappaB, ETS2, PTGS2, PTX3, CXCL16, KYNU, and CLEC4E were similarly (>2-fold) upregulated by LPS and MPLA in both species. Conclusion The genomic responses of peripheral blood to LPS and MPLA in sheep are quite similar to those observed in humans, supporting the use of the ovine model for translational studies that mimic human inflammatory diseases and the study of TLR-based immunomodulators. PMID:26640957

  8. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells.

    PubMed

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-10-01

    Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases. PMID:22960171

  9. Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll-like receptor-2-dependent mechanism

    PubMed Central

    Weiss, Gudrun; Rasmussen, Simon; Zeuthen, Louise Hjerrild; Nielsen, Birgit Nøhr; Jarmer, Hanne; Jespersen, Lene; Frøkiær, Hanne

    2010-01-01

    Lactobacilli are probiotics that, among other health-promoting effects, have been ascribed immunostimulating and virus-preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin-12 (IL-12) -inducing properties. As IL-12 production depends on the up-regulation of type I interferons (IFNs), we hypothesized that the strong IL-12-inducing capacity of Lactobacillus acidophilus NCFM in murine bone-marrow-derived dendritic cells (DCs) is caused by an up-regulation of IFN-β, which subsequently induces IL-12 and the double-stranded RNA binding Toll-like receptor-3 (TLR-3). The expression of the genes encoding IFN-β, TLR-3, IL-12 and IL-10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn-β, Il-12 and Il-10 compared with the synthetic double-stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr-3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up-regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN-β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN-β expression was markedly reduced in TLR-2−/− DCs, dependent on endocytosis, and the major cause of the induction of Il-12 and Tlr-3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR-2 manner dependent on IFN-β. PMID:20545783

  10. Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll-like receptor-2-dependent mechanism.

    PubMed

    Weiss, Gudrun; Rasmussen, Simon; Zeuthen, Louise Hjerrild; Nielsen, Birgit Nøhr; Jarmer, Hanne; Jespersen, Lene; Frøkiaer, Hanne

    2010-10-01

    Lactobacilli are probiotics that, among other health-promoting effects, have been ascribed immunostimulating and virus-preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin-12 (IL-12) -inducing properties. As IL-12 production depends on the up-regulation of type I interferons (IFNs), we hypothesized that the strong IL-12-inducing capacity of Lactobacillus acidophilus NCFM in murine bone-marrow-derived dendritic cells (DCs) is caused by an up-regulation of IFN-β, which subsequently induces IL-12 and the double-stranded RNA binding Toll-like receptor-3 (TLR-3). The expression of the genes encoding IFN-β, TLR-3, IL-12 and IL-10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn-β, Il-12 and Il-10 compared with the synthetic double-stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr-3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up-regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN-β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN-β expression was markedly reduced in TLR-2(-/-) DCs, dependent on endocytosis, and the major cause of the induction of Il-12 and Tlr-3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR-2 manner dependent on IFN-β. PMID:20545783

  11. Polymorphism in the promoter region of the Toll-like receptor 9 gene and cervical human papillomavirus infection

    PubMed Central

    Oliveira, Lucas Boeno; Louvanto, Karolina; Ramanakumar, Agnihotram V.; Franco, Eduardo L.

    2013-01-01

    Polymorphism in the Toll-like receptor (TLR) 9 gene has been shown to have a significant role in some diseases; however, little is known about its possible role in the natural history of human papillomavirus (HPV) infections. We investigated the association between a single-nucleotide polymorphism (SNP) (rs5743836) in the promoter region of TLR9 (T1237C) and type-specific HPV infections. Specimens were derived from a cohort of 2462 women enrolled in the Ludwig–McGill Cohort Study. We randomly selected 500 women who had a cervical HPV infection detected at least once during the study as cases. We defined two control groups: (i) a random sample of 300 women who always tested HPV negative, and (ii) a sample of 234 women who were always HPV negative but had a minimum of ten visits during the study. TLR9 genotyping was performed using bidirectional PCR amplification of specific alleles. Irrespective of group, the WT homozygous TLR9 genotype (TT) was the most common form, followed by the heterozygous (TC) and the mutant homozygous (CC) forms. There were no consistent associations between polymorphism and infection risk, either overall or by type or species. Likewise, there were no consistently significant associations between polymorphism and HPV clearance or persistence. We concluded that this polymorphism in the promoter region of TLR9 gene does not seem to have a mediating role in the natural history of the HPV infection. PMID:23677790

  12. Identification of single nucleotide polymorphisms in the bovine Toll-like receptor 1 gene and association with health traits in cattle

    PubMed Central

    2012-01-01

    Bovine mastitis remains the most common and costly disease of dairy cattle worldwide. A complementary control measure to herd hygiene and vaccine development would be to selectively breed cattle with greater resistance to mammary infection. Toll-like receptor 1 (TLR1) has an integral role for the initiation and regulation of the immune response to microbial pathogens, and has been linked to numerous inflammatory diseases. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) within the bovine TLR1 gene (boTLR1) are associated with clinical mastitis (CM). Selected boTLR1 SNPs were analysed within a Holstein Friesian herd. Significant associations were found for the tagging SNP -79 T > G and the 3'UTR SNP +2463 C > T. We observed favourable linkage of reduced CM with increased milk fat and protein, indicating selection for these markers would not be detrimental to milk quality. Furthermore, we present evidence that some of these boTLR1 SNPs underpin functional variation in bovine TLR1. Animals with the GG genotype (from the tag SNP -79 T > G) had significantly lower boTLR1 expression in milk somatic cells when compared with TT or TG animals. In addition, stimulation of leucocytes from GG animals with the TLR1-ligand Pam3csk4 resulted in significantly lower levels of CXCL8 mRNA and protein. SNPs in boTLR1 were significantly associated with CM. In addition we have identified a bovine population with impaired boTLR1 expression and function. This may have additional implications for animal health and warrants further investigation to determine the suitability of identified SNPs as markers for disease susceptibility. PMID:22417166

  13. TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Toll-like receptor 4 (TLR4) is a key receptor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria; genetic variation in this specific gene has been linked with the host’s response to bacterial infections and disease resistance. Since colonization and ...

  14. FAAH-mediated modulation of TLR3-induced neuroinflammation in the rat hippocampus.

    PubMed

    Henry, Rebecca J; Kerr, Daniel M; Finn, David P; Roche, Michelle

    2014-11-15

    The present study examined the effect of enhancing fatty acid amide hydrolase (FAAH) substrate levels in vivo on Toll-like receptor (TLR)3-induced neuroinflammation. Systemic and central (i.c.v.) administration of the FAAH inhibitor URB597 increased hippocampal levels of the N-acylethanolamines palmitoylethanolamide and oleoylethanolamide, but not anandamide. Systemic URB597 increased IFNα, IFNγ and IL-6 expression following TLR3 activation and attenuated TLR3-induced IL-1β and TNFα expression. In comparison, central URB597 administration attenuated the TLR3-induced increase in TNFα and IFNγ expression (and associated downstream genes IP-10 and SOCS1), while concurrently increasing IL-10 expression. These data support an important role for FAAH-mediated regulation of TLR3-induced neuroinflammatory responses. PMID:25245162

  15. Lactobacillus rhamnosus GG increases Toll-like receptor 3 gene expression in murine small intestine ex vivo and in vivo.

    PubMed

    Aoki-Yoshida, A; Saito, S; Fukiya, S; Aoki, R; Takayama, Y; Suzuki, C; Sonoyama, K

    2016-06-01

    Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-α (IFN-α), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-α and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I:C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium. PMID:27013459

  16. TLR2, TLR4 AND MyD88 Mediate Allergic Airway Disease (AAD) and Streptococcus pneumoniae-Induced Suppression of AAD

    PubMed Central

    Thorburn, Alison N.; Tseng, Hsin-Yi; Donovan, Chantal; Hansbro, Nicole G.; Jarnicki, Andrew G.; Foster, Paul S.; Gibson, Peter G.; Hansbro, Philip M.

    2016-01-01

    Background Exposure to non-pathogenic Streptococcus pneumoniae and vaccination are inversely associated with asthma. Studies in animal models demonstrate that airway administration of S. pneumoniae (live or killed), or its vaccines or components, suppresses the characteristic features of asthma in mouse models of allergic airway disease (AAD). These components could be developed into immunoregulatory therapies. S. pneumoniae components are recognized by Toll-like receptors (TLR) 2 and TLR4, and both induce inflammatory cell responses through the adaptor protein myeloid differentiation primary response gene 88 (MyD88). The involvement of TLR2, TLR4 and MyD88 in the pathogenesis of AAD and asthma is incompletely understood, and has not been studied in S. pneumoniae-mediated suppression of AAD. We investigated the role of TLR2, TLR4 and MyD88 in the development of AAD and S. pneumoniae-mediated suppression of AAD. Methods and Findings OVA-induced AAD and killed S. pneumoniae-mediated suppression of AAD were assessed in wild-type, TLR2-/-, TLR4-/-, TLR2/4-/- and MyD88-/- BALB/c mice. During OVA-induced AAD, TLR2, TLR4 and MyD88 were variously involved in promoting eosinophil accumulation in bronchoalveolar lavage fluid and blood, and T-helper type (Th)2 cytokine release from mediastinal lymph node T cells and splenocytes. However, all were required for the induction of airways hyperresponsiveness (AHR). In S. pneumoniae-mediated suppression of AAD, TLR2, TLR4 and MyD88 were variously involved in the suppression of eosinophilic and splenocyte Th2 responses but all were required for the reduction in AHR. Conclusions These results highlight important but complex roles for TLR2, TLR4 and MyD88 in promoting the development of OVA-induced AAD, but conversely in the S. pneumoniae-mediated suppression of AAD, with consistent and major contributions in both the induction and suppression of AHR. Thus, TLR signaling is likely required for both the development of asthma and the

  17. Corticotropin-Releasing Factor and Toll-Like Receptor Gene Expression Is Associated with Low-Grade Inflammation in Irritable Bowel Syndrome Patients with Depression

    PubMed Central

    Jizhong, Song; Qiaomin, Wang; Chao, Wang

    2016-01-01

    The mechanism of low-grade inflammation in irritable bowel syndrome (IBS) is unclear; our research concentrates on the involvement of the corticotropin-releasing factor (CRF) and Toll-like receptor (TLR) gene expression in the process of low-grade inflammation in IBS patients with depression. This study suggests more IBS patients are presenting with the states of depression and anxiety. IBS patients with depression have shown a lower grade inflammatory response and an imbalance of the inflammatory response. CRF1, CRF2, TLR2, and TLR4 in IBS patients with depression are significantly higher than those without depression and controls. Thus, activation of the CRF-TLR associated pathways produces an inflammatory reaction, which can concurrently affect the digestive tract and the CNS and induce the corresponding digestive and psychiatric symptoms. PMID:27478433

  18. TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells.

    PubMed

    Cruz-Aguilar, Marisa; Castillo-Rodal, Antonia I; Schcolnik-Cabrera, Alejandro; Bonifaz, Laura C; Molina, Gabriela; López-Vidal, Yolanda

    2016-07-01

    Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M. scrofulaceum and we analyzed the effect on the activation of DCs. We found that M. scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M. scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M. scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M. scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M. scrofulaceum. PMID:27450002

  19. The biology of Toll-like receptors.

    PubMed

    Means, T K; Golenbock, D T; Fenton, M J

    2000-09-01

    In 1997, a human homologue of the Drosophila Toll protein was described, a protein later to be designated Toll-like receptor 4 (TLR4). Since that time, additional human and murine TLR proteins have been identified. Mammalian TLR proteins appear to represent a conserved family of innate immune recognition receptors. These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in the activation of genes that mediate innate immune defenses. Numerous studies have now identified a wide variety of chemically-diverse bacterial products that serve as putative ligands for TLR proteins. More recent studies have identified the first endogenous protein ligands for TLR proteins. TLR signaling represents a key feature of innate immune response to pathogen invasion. PMID:10817965

  20. A functional variant at miR-34a binding site in toll-like receptor 4 gene alters susceptibility to hepatocellular carcinoma in a Chinese Han population.

    PubMed

    Jiang, Zi-Cheng; Tang, Xian-Mei; Zhao, Ying-Ren; Zheng, Lei

    2014-12-01

    Toll-like receptor 4 (TLR4) plays a key role in prompting the innate or immediate response. A growing body of evidence suggests that genetic variants of TLR4 gene were associated with the development of cancers. This study aimed to investigate the relationship of a functional variant (rs1057317) at microRNA-34a (miR-34a) binding site in toll-like receptor 4 gene and the risk of hepatocellular carcinoma. A single center-based case-control study was conducted. In this study, the polymerase chain reaction (PCR) and direct sequencing were used to genotype sequence variants of TLR4 in 426 hepatocellular carcinoma cases and 438 controls. The modification of rs1057317 on the binding of hsa-miR-34a to TLR4 messenger RNA (mRNA) was measured by luciferase activity assay. Individuals carrying the AA genotypes for the rs1057317 were associated significantly with increased risk of hepatocellular carcinoma comparing with those carrying wild-type homozygous CC genotypes (adjusted odds ratio [OR] by sex and age, from 1.116 to 2.452, P = 0.013). The activity of the reporter vector was lower in the reporter vector carrying C allele than the reporter vector carrying A allele. Furthermore, the expression of TLR4 was detected in the peripheral blood mononucleated cell of hepatocellular carcinoma (HCC) patients, suggesting that mRNA and protein levels of TLR4 might be associated with SNP rs1057317. Collectively, these results suggested that the risk of hepatocellular carcinoma was associated with a functional variant at miR-34a binding site in toll-like receptor 4 gene. miR-34a/TLR4 axis may play an important role in the development of hepatocellular carcinoma. PMID:25179842

  1. Cloning and differential expression of a novel toll-like receptor gene in noble scallop Chlamys nobilis with different total carotenoid content.

    PubMed

    Lu, Yeqing; Zheng, Huaiping; Zhang, Hongkuan; Yang, Jianqin; Wang, Qiang

    2016-09-01

    To investigate whether toll like receptors (TLRs) genes do have an immune influence on noble scallop Chlamys nobilis under pathogen stress, acute challenges lasting 48 h to Vibrio parahaemolyticus, lipopolysaccharide (LPS), polyinosinic polycytidylic acid (Poly I:C), and PBS were conducted in two scallop stains of orange and brown with different carotenoids content. A novel toll-like receptor gene called CnTLR-1 was cloned and its transcripts under different challenges were determined. Meantime, total carotenoids content (TCC) of different immune responses were determined to investigate whether there was a relationship between gene expression and carotenoids content. The full length cDNA of CnTLR-1 is 2982 bp with an open reading frame (ORF) of 1920 bp encoding 639-deduced amino acids, which contains five leucine-rich repeats (LRR), two LRR-C-terminal (LRRCT) motifs and a LRR-N-terminal (LRRNT) motif in the extracellular domain, a transmembrane domain and a Toll/Interleukin-1 Receptor (TIR) of 138-amino acids in the cytoplasmic region. Phylogenetic tree analysis showed that CnTLR-1 could be clustered with mollusk TLRs into one group and especially was related closely to Crassostrea gigas and Mytilus galloprovincialis TLRs. CnTLR-1 transcripts were detected in decreasing levels in the mantle, hemocytes, gill, kidney, gonad, hepatopancreas, intestines and adductor. Compared with PBS control group, CnTLR-1 transcripts were up-regulated in V. parahaemolyticus, LPS and Poly I:C groups. Further, CnTLR-1 transcripts were significantly higher in orange scallops than that of brown ones with and without pathogenic challenges. TCC, which is higher in orange scallops, was initially increased and then decreased during a 48 h immune challenge in the hemocytes. The present results indicate that CnTLR-1 is an important factor involved in the immune defense against pathogens in the noble scallop. PMID:27403592

  2. The effect of Tlr4 and/or C3 Deficiency and of Neonatal Gene Therapy on Skeletal Disease in Mucopolysaccharidosis VII mice

    PubMed Central

    Xing, Elizabeth M.; Wu, Susan; Ponder, Katherine P.

    2015-01-01

    Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder caused by the deficiency of the enzyme β-glucuronidase (Gusb-/-) and results in glycosaminoglycan (GAG) accumulation. Skeletal abnormalities include stunted long bones and bone degeneration. GAGs have been hypothesized to activate toll-like receptor 4 (Tlr4) signaling and the complement pathway, resulting in upregulation of inflammatory cytokines that suppress growth and cause degeneration of bone. Gusb-/- mice were bred with Tlr4- and complement component 3 (C3)-deficient mice, and the skeletal manifestations of the doubly- and triply-deficient mice were compared to those of purebred Gusb-/- mice. Radiographs showed that purebred Gusb-/- mice had shorter tibias and femurs, and wider femurs, compared to normal mice. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb-/- mice. The glenoid cavity and humerus were scored on a scale from 0 (normal) to +3 (severely abnormal) for dysplasia and bone irregularities, and the joint space was measured. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb-/- mice, and their joint space remained abnormally wide. Gusb-/- mice treated neonatally with an intravenous retroviral vector (RV) had thinner femurs, longer legs, and a narrowed joint space compared with untreated purebred Gusb-/- mice, but no improvement in glenohumeral degeneration. We conclude that Tlr4- and/or C3- deficiency fail to ameliorate skeletal abnormalities, and other pathways may be involved. RV treatment improves some but not all aspects of bone disease. Radiographs may be an efficient method for future evaluation, as they readily show glenohumeral joint abnormalities. PMID:25559179

  3. TLR2 and TLR4 mediate the TNFα response to Vibrio vulnificus biotype 1.

    PubMed

    Stamm, Lola V; Drapp, Rebecca L

    2014-08-01

    Vibrio vulnificus (Vv) is a pathogenic bacterium that can cause life-threatening infections in humans. Most fatal cases are due to septic shock that results from dysregulation of cytokines, particularly TNFα, which plays a critical role in the outcome of Vv infection. The goal of this study was to investigate the Toll-like receptor (TLR)-mediated TNFα response to four Vv biotype 1 strains using mice deficient for TLR2, TLR4, and TLR2/TLR4. Ex vivo assays were performed with blood, splenocytes, and Kupffer cells (KC) from wild-type (WT) and TLR-knockout (KO) mice using formalin-inactivated Vv (f-Vv) as stimulant. All f-Vv biotype 1 strains elicited strong TNFα production by WT mouse blood and cells, which was TLR2 and TLR4 dependent. OxPAPC, an inhibitor of TLR2 and TLR4 signaling, effectively blunted the TLR-mediated TNFα response to f-Vv. Furthermore, TLR2 KO and TLR2/TLR4 KO mice were more resistant to lethal infection with Vv ATCC 27562 than WT mice, perhaps due to attenuation of the TNFα response. These data suggest that it may be possible to devise strategies to specifically target the harmful TLR-mediated TNFα response as an adjunct to antibiotic treatment of severe Vv infection. PMID:24532589

  4. Direct Regulation of TLR5 Expression by Caveolin-1

    PubMed Central

    Lim, Jae Sung; Nguyen, Kim Cuc Thi; Han, Jung Min; Jang, Ik-Soon; Fabian, Claire; Cho, Kyung A

    2015-01-01

    Toll-like receptor 5 (TLR5) is a specific receptor for microbial flagellin and is one of the most well-known receptors in the TLR family. We reported previously that TLR5 signaling is well maintained during aging and that caveolin-1 may be involved in TLR5 signaling in aged macrophages through direct interactions. Therefore, it is important to clarify whether caveolin-1/TLR5 interactions affect TLR5 expression during aging. To assess the effect of caveolin-1 on TLR5, we analyzed TLR5 expression in senescent fibroblasts and aged tissues expressing high levels of caveolin-1. As expected, TLR5 mRNA and protein expression was well maintained in senescent fibroblasts and aged tissues, whereas TLR4 mRNA and protein were diminished in those cells and tissues. To determine the mechanism of caveolin-1-dependent TLR5 expression, we examined TLR5 expression in caveolin-1 deficient mice. Interestingly, TLR5 mRNA and protein levels were decreased dramatically in tissues from caveolin-1 knockout mice. Moreover, overexpressed caveolin-1 in vitro enhanced TLR5 mRNA through the MAPK pathway and prolonged TLR5 protein half-life through direct interaction. These results suggest that caveolin-1 may play a crucial role in maintaining of TLR5 by regulating transcription systems and increasing protein half-life. PMID:26615831

  5. Direct Regulation of TLR5 Expression by Caveolin-1.

    PubMed

    Lim, Jae Sung; Nguyen, Kim Cuc Thi; Han, Jung Min; Jang, Ik-Soon; Fabian, Claire; Cho, Kyung A

    2015-12-01

    Toll-like receptor 5 (TLR5) is a specific receptor for microbial flagellin and is one of the most well-known receptors in the TLR family. We reported previously that TLR5 signaling is well maintained during aging and that caveolin-1 may be involved in TLR5 signaling in aged macrophages through direct interactions. Therefore, it is important to clarify whether caveolin-1/TLR5 interactions affect TLR5 expression during aging. To assess the effect of caveolin-1 on TLR5, we analyzed TLR5 expression in senescent fibroblasts and aged tissues expressing high levels of caveolin-1. As expected, TLR5 mRNA and protein expression was well maintained in senescent fibroblasts and aged tissues, whereas TLR4 mRNA and protein were diminished in those cells and tissues. To determine the mechanism of caveolin-1-dependent TLR5 expression, we examined TLR5 expression in caveolin-1 deficient mice. Interestingly, TLR5 mRNA and protein levels were decreased dramatically in tissues from caveolin-1 knockout mice. Moreover, overexpressed caveolin-1 in vitro enhanced TLR5 mRNA through the MAPK pathway and prolonged TLR5 protein half-life through direct interaction. These results suggest that caveolin-1 may play a crucial role in maintaining of TLR5 by regulating transcription systems and increasing protein half-life. PMID:26615831

  6. Mechanical-Stretch of C2C12 Myoblasts Inhibits Expression of Toll-Like Receptor 3 (TLR3) and of Autoantigens Associated with Inflammatory Myopathies

    PubMed Central

    Liu, Xinghui; Adriouch, Sahil; Liao, Hua

    2013-01-01

    Recent studies in patients suffering from inflammatory autoimmune myopathies suggested that moderate exercise training improves or at least stabilizes muscle strength and function without inducing disease flares. However, the precise mechanisms involved in this beneficial effect have not been extensively studied. Here we used a model of in vitro stretched C2C12 myoblasts to investigate whether mechanical stretch could influence myoblast proliferation or the expression of proinflammatory genes. Our results demonstrated that cyclic mechanical stretch stimulated C2C12 cell cycling and early up-regulation of the molecules related to mechanical-stretch pathway in muscle (calmodulin, nNOS, MMP-2, HGF and c-Met). Unexpectedly, mechanical stretch also reduced the expression of TLR3 and of proteins known to represent autoantigens in inflammatory autoimmune myopathies (Mi-2, HRS, DNA-PKcs, U1-70). Interestingly, stimulation or inhibition of calmodulin, NOS, HGF or c-Met molecules in vitro affected the expression of autoantigens and TLR3 proteins confirming their role in the inhibition of autoantigens and TLR3 during mechanical stretch. Overall, this study demonstrates for the first time that mechanical stretch could be beneficial by reducing expression of muscle autoantigens and of pro-inflammatory TLR3 and may provide new insight to understand how resistance training can reduce the symptoms associated with myositis. PMID:24224022

  7. Toll-like receptor 3 gene polymorphisms in South African Blacks with type 1 diabetes.

    PubMed

    Pirie, F J; Pegoraro, R; Motala, A A; Rauff, S; Rom, L; Govender, T; Esterhuizen, T M

    2005-08-01

    Type 1 diabetes is the consequence of exposure of genetically susceptible individuals to specific environmental precipitants. The innate immune system provides the initial response to exogenous antigen and links with the adaptive immune system. The aim of this study was to assess the role of polymorphisms occurring in the cytoplasmic region of toll-like receptor (TLR) 3 gene and immediate 5' sequence, in subjects of Zulu descent with type 1 diabetes in KwaZulu-Natal, South Africa. Seventy-nine subjects with type 1 diabetes and 74 healthy normal glucose tolerant gender-matched control subjects were studied. Parts of exon 4 and exon 3/intron 3 of the TLR3 gene were studied by polymerase chain reaction, direct sequencing and restriction enzyme digestion with Bts 1. Of the nine polymorphisms studied, a significant association with type 1 diabetes was found for the major allele in the 2593 C/T polymorphism and for the minor alleles in the 2642 C/A and 2690 A/G polymorphisms, which were found to be in complete linkage disequilibrium. Correction of the P-values for the number of alleles studied, however, rendered the results no longer significant. These results suggest that polymorphisms in the TLR3 gene, which is part of the innate immune system, may be associated with type 1 diabetes in this population. PMID:16029432

  8. Variation at Innate Immunity Toll-Like Receptor Genes in a Bottlenecked Population of a New Zealand Robin

    PubMed Central

    Grueber, Catherine E.; Wallis, Graham P.; King, Tania M.; Jamieson, Ian G.

    2012-01-01

    Toll-like receptors (TLRs) are an ancient family of genes encoding transmembrane proteins that bind pathogen-specific molecules and initiate both innate and adaptive aspects of the immune response. Our goal was to determine whether these genes show sufficient genetic diversity in a bottlenecked population to be a useful addition or alternative to the more commonly employed major histocompatibility complex (MHC) genotyping in a conservation genetics context. We amplified all known avian TLR genes in a severely bottlenecked population of New Zealand's Stewart Island robin (Petroica australis rakiura), for which reduced microsatellite diversity was previously observed. We genotyped 17–24 birds from a reintroduced island population (including the 12 founders) for nine genes, seven of which were polymorphic. We observed a total of 24 single-nucleotide polymorphisms overall, 15 of which were non-synonymous, representing up to five amino-acid variants at a locus. One locus (TLR1LB) showed evidence of past directional selection. Results also confirmed a passerine duplication of TLR7. The levels of TLR diversity that we observe are sufficient to justify their further use in addressing conservation genetic questions, even in bottlenecked populations. PMID:23024782

  9. Synthesis of anti-inflammatory α-and β-linked acetamidopyranosides as inhibitors of toll-like receptor 4 (TLR4)§

    PubMed Central

    Wipf, Peter; Eyer, Benjamin R.; Yamaguchi, Yukihiro; Zhang, Feng; Neal, Matthew D.; Sodhi, Chhinder P.; Good, Misty; Branca, Maria; Prindle, Thomas; Lu, Peng; Brodsky, Jeffrey L.; Hackam, David J.

    2014-01-01

    The low-molecular weight isopropyl 2-acetamido-α-glucoside 16 (C34) inhibits toll-like receptor 4 (TLR4) in enterocytes and macrophages in vitro, and reduces systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. We used a copper(II)-mediated solvolysis of anomeric oxazolines and an acid-mediated conversion of β-glucosamine and β-galactosamine pentaacetates to generate analogs of 16 at the anomeric carbon and at C-4 of the pyranose ring. These compounds were evaluated for their influence on TLR4-mediated inflammatory signaling in cultured enterocytes and monocytes. Their efficacy was confirmed using a NF-kB-luciferase reporter mouse, thus establishing the first structure-activity relationship (SAR) study in this series and identifying the more efficacious isopropyl 2-acetamido-α-galactoside 17. PMID:26236050

  10. TLR1, 2, 4, 6 and 9 Variants Associated with Tuberculosis Susceptibility: A Systematic Review and Meta-Analysis

    PubMed Central

    Schurz, Haiko; Daya, Michelle; Möller, Marlo; Hoal, Eileen G.; Salie, Muneeb

    2015-01-01

    Background Studies investigating the influence of toll-like receptor (TLR) polymorphisms and tuberculosis susceptibility have yielded varying and often contradictory results in different ethnic groups. A meta-analysis was conducted to investigate the relationship between TLR variants and susceptibility to tuberculosis, both across and within specific ethnic groups. Methods An extensive database search was performed for studies investigating the relationship between TLR and tuberculosis (TB) susceptibility. Data was subsequently extracted from included studies and statistically analysed. Results 32 articles involving 18907 individuals were included in this meta-analysis, and data was extracted for 14 TLR polymorphisms. Various genetic models were employed. An increased risk of TB was found for individuals with the TLR2 rs3804100 CC and the TLR9 rs352139 GA and GG genotypes, while decreased risk was identified for those with the AG genotype of TLR1 rs4833095. The T allele of TLR6 rs5743810 conferred protection across all ethnic groups. TLR2 rs5743708 subgroup analysis identified the A allele to increase susceptibility to TB in the Asian ethnic group, while conferring protection in the Hispanic group. The T allele of TLR4 rs4986791 was also found to increase the risk of TB in the Asian subgroup. All other TLR gene variants investigated were not found to be associated with TB in this meta-analysis. Discussion Although general associations were identified, most TLR variants showed no significant association with TB, indicating that additional studies investigating a wider range of pattern recognition receptors is required to gain a better understanding of this complex disease. PMID:26430737

  11. Toll-like receptor cascade and gene polymorphism in host–pathogen interaction in Lyme disease

    PubMed Central

    Rahman, Shusmita; Shering, Maria; Ogden, Nicholas H; Lindsay, Robbin; Badawi, Alaa

    2016-01-01

    Lyme disease (LD) risk occurs in North America and Europe where the tick vectors of the causal agent Borrelia burgdorferi sensu lato are found. It is associated with local and systemic manifestations, and has persistent posttreatment health complications in some individuals. The innate immune system likely plays a critical role in both host defense against B. burgdorferi and disease severity. Recognition of B. burgdorferi, activation of the innate immune system, production of proinflammatory cytokines, and modulation of the host adaptive responses are all initiated by Toll-like receptors (TLRs). A number of Borrelia outer-surface proteins (eg, OspA and OspB) are recognized by TLRs. Specifically, TLR1 and TLR2 were identified as the receptors most relevant to LD. Several functional single-nucleotide polymorphisms have been identified in TLR genes, and are associated with varying cytokines types and synthesis levels, altered pathogen recognition, and disruption of the downstream signaling cascade. These single-nucleotide polymorphism-related functional alterations are postulated to be linked to disease development and posttreatment persistent illness. Elucidating the role of TLRs in LD may facilitate a better understanding of disease pathogenesis and can provide an insight into novel therapeutic targets during active disease or postinfection and posttreatment stages. PMID:27330321

  12. Toll-like receptor cascade and gene polymorphism in host-pathogen interaction in Lyme disease.

    PubMed

    Rahman, Shusmita; Shering, Maria; Ogden, Nicholas H; Lindsay, Robbin; Badawi, Alaa

    2016-01-01

    Lyme disease (LD) risk occurs in North America and Europe where the tick vectors of the causal agent Borrelia burgdorferi sensu lato are found. It is associated with local and systemic manifestations, and has persistent posttreatment health complications in some individuals. The innate immune system likely plays a critical role in both host defense against B. burgdorferi and disease severity. Recognition of B. burgdorferi, activation of the innate immune system, production of proinflammatory cytokines, and modulation of the host adaptive responses are all initiated by Toll-like receptors (TLRs). A number of Borrelia outer-surface proteins (eg, OspA and OspB) are recognized by TLRs. Specifically, TLR1 and TLR2 were identified as the receptors most relevant to LD. Several functional single-nucleotide polymorphisms have been identified in TLR genes, and are associated with varying cytokines types and synthesis levels, altered pathogen recognition, and disruption of the downstream signaling cascade. These single-nucleotide polymorphism-related functional alterations are postulated to be linked to disease development and posttreatment persistent illness. Elucidating the role of TLRs in LD may facilitate a better understanding of disease pathogenesis and can provide an insight into novel therapeutic targets during active disease or postinfection and posttreatment stages. PMID:27330321

  13. Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling

    PubMed Central

    McDonald, Kerry-Ann; Huang, Hai; Tohme, Samer; Loughran, Patricia; Ferrero, Kimberly; Billiar, Timothy; Tsung, Allan

    2014-01-01

    Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1–TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury. PMID:25375408

  14. Effects of the TLR2 Agonists MALP-2 and Pam3Cys in Isolated Mouse Lungs

    PubMed Central

    Barrenschee, Martina; Lex, Dennis; Uhlig, Stefan

    2010-01-01

    Background Gram-positive and Gram-negative bacteria are main causes of pneumonia or acute lung injury. They are recognized by the innate immune system via toll-like receptor-2 (TLR2) or TLR4, respectively. Among all organs, the lungs have the highest expression of TLR2 receptors, but little is known about the pulmonary consequences of their activation. Here we studied the effects of the TLR2/6 agonist MALP-2, the TLR2/1 agonist Pam3Cys and the TLR4 agonist lipopolysaccharide (LPS) on pro-inflammatory responses in isolated lungs. Methodology/Principal Findings Isolated perfused mouse lungs were perfused for 60 min or 180 min with MALP-2 (25 ng/mL), Pam3Cys (160 ng/mL) or LPS (1 µg/mL). We studied mediator release by enzyme linked immunosorbent assay (ELISA), the activation of mitogen activated protein kinase (MAPK) and AKT/protein kinase B by immunoblotting, and gene induction by quantitative polymerase chain reaction. All agonists activated the MAPK ERK1/2 and p38, but neither JNK or AKT kinase. The TLR ligands upregulated the inflammation related genes Tnf, Il1β, Il6, Il10, Il12, Ifng, Cxcl2 (MIP-2α) and Ptgs2. MALP-2 was more potent than Pam3Cys in inducing Slpi, Cxcl10 (IP10) and Parg. Remarkable was the strong induction of Tnc by MALP2, which was not seen with Pam3Cys or LPS. The growth factor related genes Areg and Hbegf were not affected. In addition, all three TLR agonists stimulated the release of IL-6, TNF, CXCL2 and CXCL10 protein from the lungs. Conclusions/Significance TLR2 and TLR4 activation leads to similar reactions in the lungs regarding MAPK activation, gene induction and mediator release. Several genes studied here have not yet been appreciated as targets of TLR2-activation in the lungs before, i.e., Slpi, tenascin C, Parg and Traf1. In addition, the MALP-2 dependent induction of Tnc may indicate the existence of TLR2/6-specific pathways. PMID:21124967

  15. Inhibition of TLR4 Signalling-Induced Inflammation Attenuates Secondary Injury after Diffuse Axonal Injury in Rats

    PubMed Central

    Zhao, Yonglin; Zhang, Ming; Zhao, Junjie; Ma, Xudong; Huang, Tingqin; Pang, Honggang

    2016-01-01

    Increasing evidence suggests that secondary injury after diffuse axonal injury (DAI) damages more axons than the initial insult, but the underlying mechanisms of this phenomenon are not fully understood. Recent studies show that toll-like receptor 4 (TLR4) plays a critical role in promoting adaptive immune responses and have been shown to be associated with brain damage. The purpose of this study was to investigate the role of the TLR4 signalling pathway in secondary axonal injury in the cortices of DAI rats. TLR4 was mainly localized in microglial cells and neurons, and the levels of TLR4 downstream signalling molecules, including TLR4, myeloid differentiation primary response gene 88, toll/IR-1-(TIR-) domain-containing adaptor protein inducing interferon-beta, interferon regulatory factor 3, interferon β, nuclear factor κB (NF-κB) p65, and phospho-NF-κB p65, significantly increased and peaked at 1 d after DAI. Inhibition of TLR4 by TAK-242 attenuated apoptosis, neuronal and axonal injury, and glial responses. The neuroprotective effects of TLR4 inhibition were associated with decreases in the levels of TLR4 downstream signalling molecules and inflammatory factors, including interleukin-1β, interleukin-6, and tumour necrosis factor-α. These results suggest that the TLR4 signalling pathway plays an important role in secondary injury and may be an important therapeutic target following DAI. PMID:27478307

  16. Gene/environment interactions in the pathogenesis of autoimmunity: new insights on the role of Toll-like receptors.

    PubMed

    Gianchecchi, Elena; Fierabracci, Alessandra

    2015-11-01

    Autoimmune disorders are increasing worldwide. Although their pathogenesis has not been elucidated yet, a complex interaction of genetic and environmental factors is involved in their onset. Toll-like receptors (TLRs) represent a family of pattern recognition receptors involved in the recognition and in the defense of the host from invading microorganisms. They sense a wide range of pathogen associated molecular patterns (PAMPs) deriving from metabolic pathways selective of bacterial, viral, fungal and protozoan microorganisms. TLR activation plays a critical role in the activation of the downstream signaling pathway by interacting and recruiting several adaptor molecules. Although TLRs are involved in the protection of the host, several studies suggest that, in certain conditions, they play a critical role in the pathogenesis of autoimmune diseases. We review the most recent advances showing a correlation between some single nucleotide polymorphisms or copy number variations in TLR genes or in adaptor molecules involved in TLR signaling and the onset of several autoimmune conditions, such as Type I diabetes, autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy, rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. In light of the foregoing we finally propose that molecules involved in TLR pathway may represent the targets for novel therapeutic treatments in order to stop autoimmune processes. PMID:26184547

  17. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    SciTech Connect

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  18. TLR7 and TLR9 ligands regulate antigen presentation by macrophages.

    PubMed

    Celhar, Teja; Pereira-Lopes, Selma; Thornhill, Susannah I; Lee, Hui Yin; Dhillon, Manprit K; Poidinger, Michael; Connolly, John E; Lim, Lina H K; Biswas, Subhra K; Fairhurst, Anna-Marie

    2016-05-01

    The toll-like receptors (TLRs) are important innate receptors recognizing potentially pathogenic material. However, they also play a significant role in the development of Alzheimer's disease, cancer, autoimmunity and the susceptibility to viral infections. Macrophages are essential for an effective immune response to foreign material and the resolution of inflammation. In these studies, we examined the impact of different TLR ligands on macrophage cell function. We demonstrate that stimulation of all TLRs tested increases the phagocytosis of apoptotic cells by macrophages. TLR7 and TLR9 ligation decreased the levels of the surface co-expression molecules CD86 and MHCII, which was associated with a concomitant reduction in antigen presentation and proliferation of T cells. This down-regulation in macrophage function was not due to an increase in cell death. In fact, exposure to TLR7 or TLR9 ligands promoted cell viability for up to 9 days, in contrast to TLR3 or TLR4. Additionally, macrophages exposed to TLR7/TLR9 ligands had a significantly lower ratio of Il-12/Il-10 mRNA expression compared with those treated with the TLR4 ligand, LPS. Taken together, these data demonstrate that TLR7/TLR9 ligands push the macrophage into a phagocytic long-lived cell, with a decreased capacity of antigen presentation and reminiscent of the M2 polarized state. PMID:26567289

  19. The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2.

    PubMed

    Keestra, A Marijke; de Zoete, Marcel R; van Aubel, Rémon A M H; van Putten, Jos P M

    2007-06-01

    The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-kappaB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)(3)-Lys (Pam(3)CSK(4)), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6-16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2. PMID:17513760

  20. Toll-Like Receptors of Deuterostome Invertebrates

    PubMed Central

    Satake, Honoo; Sekiguchi, Toshio

    2012-01-01

    Defensive systems against pathogens are responsible not only for survival or lifetime of an individual but also for the evolution of a species. Innate immunity is expected to be more important for invertebrates than mammals, given that adaptive immunity has not been acquired in the former. Toll-like receptors (TLRs) have been shown to play a crucial role in host defense of pathogenic microbes in innate immunity of mammals. Recent genome-wide analyses have suggested that TLR or their related genes are conserved in invertebrates. In particular, numerous TLR-related gene candidates were detected in deuterostome invertebrates, including a sea urchin (222 TLR-related gene candidates) and amphioxus (72 TLR-related gene candidates). Molecular phylogenetic analysis verified that most of sea urchin or amphioxus TLR candidates are paralogous, suggesting that these organisms expanded TLR-related genes in a species-specific manner. In contrast, another deuterostome invertebrate, the ascidian Ciona intestinalis, was found to possess only two TLR genes. Moreover, Ciona TLRs, Ci-TLR1 and Ci-TLR2, were shown to possess “hybrid” functionality of mammalian TLRs. Such functionality of Ci-TLRs could not be predicted by sequence comparison with vertebrate TLRs, indicating confounding evolutionary lineages of deuterostome invertebrate TLRs or their candidates. In this review article, we present recent advances in studies of TLRs or their candidates among deuterostome invertebrates, and provide insight into an evolutionary process of TLRs. PMID:22566918

  1. Role of constitutive androstane receptor in Toll-like receptor-mediated regulation of gene expression of hepatic drug-metabolizing enzymes and transporters.

    PubMed

    Shah, Pranav; Guo, Tao; Moore, David D; Ghose, Romi

    2014-01-01

    Impairment of drug disposition in the liver during inflammation has been attributed to downregulation of gene expression of drug-metabolizing enzymes (DMEs) and drug transporters. Inflammatory responses in the liver are primarily mediated by Toll-like receptors (TLRs). We have recently shown that activation of TLR2 or TLR4 by lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, leads to the downregulation of gene expression of DMEs/transporters. However, the molecular mechanism underlying this downregulation is not fully understood. The xenobiotic nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the expression of DMEs/transporter genes. Downregulation of DMEs/transporters by LTA or LPS was associated with reduced expression of PXR and CAR genes. To determine the role of CAR, we injected CAR(+/+) and CAR(-/-) mice with LTA or LPS, which significantly downregulated (~40%-60%) RNA levels of the DMEs, cytochrome P450 (Cyp)3a11, Cyp2a4, Cyp2b10, uridine diphosphate glucuronosyltransferase 1a1, amine N-sulfotransferase, and the transporter, multidrug resistance-associated protein 2, in CAR(+/+) mice. Suppression of most of these genes was attenuated in LTA-treated CAR(-/-) mice. In contrast, LPS-mediated downregulation of these genes was not attenuated in CAR(-/-) mice. Induction of these genes by mouse CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene was sustained in LTA- but not in LPS-treated mice. Similar observations were obtained in humanized CAR mice. We have replicated these results in primary hepatocytes as well. Thus, LPS can downregulate DME/transporter genes in the absence of CAR, whereas the effect of LTA on these genes is attenuated in the absence of CAR, indicating the potential involvement of CAR in LTA-mediated downregulation of DME/transporter genes. PMID:24194512

  2. Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFβ, and IDO1.

    PubMed

    Moyer, Benjamin J; Rojas, Itzel Y; Kerley-Hamilton, Joanna S; Hazlett, Haley F; Nemani, Krishnamurthy V; Trask, Heidi W; West, Rachel J; Lupien, Leslie E; Collins, Alan J; Ringelberg, Carol S; Gimi, Barjor; Kinlaw, William B; Tomlinson, Craig R

    2016-06-01

    Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity

  3. Synergistic TLR2/6 and TLR9 Activation Protects Mice against Lethal Influenza Pneumonia

    PubMed Central

    Tuvim, Michael J.; Gilbert, Brian E.; Dickey, Burton F.; Evans, Scott E.

    2012-01-01

    Lower respiratory tract infections caused by influenza A continue to exact unacceptable worldwide mortality, and recent epidemics have emphasized the importance of preventative and containment strategies. We have previously reported that induction of the lungs' intrinsic defenses by aerosolized treatments can protect mice against otherwise lethal challenges with influenza A virus. More recently, we identified a combination of Toll like receptor (TLR) agonists that can be aerosolized to protect mice against bacterial pneumonia. Here, we tested whether this combination of synthetic TLR agonists could enhance the survival of mice infected with influenza A/HK/8/68 (H3N2) or A/California/04/2009 (H1N1) influenza A viruses. We report that the TLR treatment enhanced survival whether given before or after the infectious challenge, and that protection tended to correlate with reductions in viral titer 4 d after infection. Surprisingly, protection was not associated with induction of interferon gene expression. Together, these studies suggest that synergistic TLR interactions can protect against influenza virus infections by mechanisms that may provide the basis for novel therapeutics. PMID:22299046

  4. TLR3 deficiency increases voluntary alcohol consumption.

    PubMed

    Jang, Yujin; Lee, Min Hee; Park, Jong-Hwan; Han, Seung-Yun; Kim, Dong Kwan

    2016-03-23

    Toll-like receptors (TLRs) are innate immunity-related receptors. Many studies have indicated the involvement of TLRs in neurophysiology and neuropathology. One study showed that TLR3 regulates hippocampal memory and is highly expressed in the mesolimbic dopamine system, suggesting that TLR3 signaling may regulate alcohol consumption. The present study assessed the potential role of TLR3 in alcohol intake pattern. We used adult BalbC wild-type mice and TLR3 knockout mice and tested two-bottle alcohol preference over 15 days and one-bottle 2 or 4 h drinking in the dark over 4 consecutive days. The 10% alcohol consumption rate of TLR3 knockout mice increased on the 24 h free-choice test. Our findings support a potential regulatory role of TLR3 in alcohol consumption. PMID:26885867

  5. Evaluating Effect of Mesenchymal Stem Cells on Expression of TLR2 and TLR4 in Mouse DCs

    PubMed Central

    Karimi, Mohammad Hossein; Barzkar, Zahra; Babaee, Maryam; Naghdi, Majid

    2016-01-01

    Purpose: Mesenchymal stem cells (MSCs) are multipotent cells and recent findings suggest immunomodulatory effect of them on immune cells including T cells and dendritic cells (DCs). DCs are the most potent antigen presenting cells. It seems because of immunoregulatory properties of MSCs, they can affect the maturation and differentiation of DCs. DCs express a kind of surface receptors called toll-like receptors (TLRs) and play a key role in maturation process and activation of DCs. The aim of this study was to evaluate expression of TLR2 and TLR4 on DCs after exposure to mesenchymal stem cell’s supernatant in culture media containing LPS and devoid of it. Methods: In this experimental study, MSCs and DCs were extracted from adult Balb/c mouse bone marrow and spleen, respectively. MSCs supernatant were collected 24 and 48 h after 5th passage, and in adjusted with DCs culture. Isolated DCs were co-cultured with MSCs supernatant, incubation time were 24 and 48 hours. mRNA levels of TLR2 and TLR4 were evaluated using real time PCR technique. Results: The results demonstrated that although, expressions of these two receptors were up-regulated in culture media lacking LPS in comparison with the control group but the increase was not significant. There were no significant associations between LPS stimulated DCs with and without MSCs supernatants. Conclusion: According to the results presented here, it appears that TLR2 and TLR4 gene expressions on the DCs are not affected by MSCs supernatant. PMID:27478779

  6. Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

    PubMed

    Mellett, Mark; Atzei, Paola; Bergin, Ronan; Horgan, Alan; Floss, Thomas; Wurst, Wolfgang; Callanan, John J; Moynagh, Paul N

    2015-01-01

    Receptor families of the innate immune response engage in 'cross-talk' to tailor optimal immune responses against invading pathogens. However, these responses are subject to multiple levels of regulation to keep in check aberrant inflammatory signals. Here, we describe a role for the orphan receptor interleukin-17 receptor D (IL-17RD) in negatively regulating Toll-like receptor (TLR)-induced responses. Deficiency of IL-17RD expression in cells leads to enhanced pro-inflammatory signalling and gene expression in response to TLR stimulation, and Il17rd(-/-) mice are more susceptible to TLR-induced septic shock. We demonstrate that the intracellular Sef/IL-17R (SEFIR) domain of IL-17RD targets TIR adaptor proteins to inhibit TLR downstream signalling thus revealing a paradigm involving cross-regulation of members of the IL-17R and TLR families. PMID:25808990

  7. Flavonoids Affect Host-Microbiota Crosstalk through TLR Modulation

    PubMed Central

    Pérez-Cano, Francisco J.; Massot-Cladera, Malen; Rodríguez-Lagunas, Maria J.; Castell, Margarida

    2014-01-01

    Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs) in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases. PMID:26785232

  8. Local Innate Responses to TLR Ligands in the Chicken Trachea

    PubMed Central

    Barjesteh, Neda; Alkie, Tamiru Negash; Hodgins, Douglas C.; Nagy, Éva; Sharif, Shayan

    2016-01-01

    The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV. PMID:27455308

  9. Local Innate Responses to TLR Ligands in the Chicken Trachea.

    PubMed

    Barjesteh, Neda; Alkie, Tamiru Negash; Hodgins, Douglas C; Nagy, Éva; Sharif, Shayan

    2016-01-01

    The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV. PMID:27455308

  10. Folate Receptor Targeted Delivery of siRNA and Paclitaxel to Ovarian Cancer Cells via Folate Conjugated Triblock Copolymer to Overcome TLR4 Driven Chemotherapy Resistance.

    PubMed

    Jones, Steven K; Lizzio, Vincent; Merkel, Olivia M

    2016-01-11

    This paper focuses on the ability of a folate-decorated triblock copolymer to deliver a targeted dose of siRNA in order to overcome chemotherapy resistance which can commonly cause complications in ovarian cancer patients. The micelleplexes that are formed upon electrostatic interaction with siRNA are used to deliver siRNA in a targeted manner to SKOV-3 ovarian cancer cells that overexpress folate receptor-α (FRα). The triblock copolymer consists of polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (PEI-g-PCL-b-PEG-Fol). In this work, polymers of different molecular weights of PEG, as well as different grafting degrees of the (g-PCL-b-PEG-Fol) chains to PEI, were analyzed to optimize targeted siRNA delivery. The polymers, their micelleplexes, and the in vitro performance of the latter were characterized by nuclear magnetic resonance, dynamic light scattering, transmission electron microscopy, flow cytrometry, western blot, confocal microscopy, and in luciferase assays. The different PEI-g-PCL-b-PEG-Fol conjugates showed suitable sizes below 260 nm, especially at N/P 5, which also allowed for full siRNA condensation. Furthermore, flow cytometry and Western blot analysis demonstrated that our best polymer was able to effectively deliver siRNA and that siRNA delivery resulted in efficient protein knockdown of toll-like receptor 4 (TLR4). Consequently, TLR4 knock down within SKOV-3 cells resensitized them toward paclitaxel (PTX) treatment, and apoptotic events increased. This study demonstrates that PEI-g-PCL-b-PEG-Fol conjugates are a reliable delivery system for siRNA and are able to mediate therapeutic protein knockdown within ovarian cancer cells. Additionally, this study provides further evidence to link TLR4 levels to chemotherapy resistance. PMID:26636884

  11. A member of the Tlr family is involved in dsRNA innate immune response in Paracentrotus lividus sea urchin.

    PubMed

    Russo, Roberta; Chiaramonte, Marco; Matranga, Valeria; Arizza, Vincenzo

    2015-08-01

    The innate immune response involves proteins such as the membrane receptors of the Toll-like family (TLRs), which trigger different intracellular signalling pathways that are dependent on specific stimulating molecules. In sea urchins, TLR proteins are encoded by members of a large multigenic family composed of 60-250 genes in different species. Here, we report a newly identified mRNA sequence encoding a TLR protein (referred to as Pl-Tlr) isolated from Paracentrotus lividus immune cells. The partial protein sequence contained the conserved Toll/IL-1 receptor (TIR) domain, the transmembrane domain and part of the leucine repeats. Phylogenetic analysis of the Pl-Tlr protein was accomplished by comparing its sequence with those of TLRs from different classes of vertebrates and invertebrates. This analysis was suggestive of an evolutionary path that most likely represented the course of millions of years, starting from simple organisms and extending to humans. Challenge of the sea urchin immune system with poly-I:C, a chemical compound that mimics dsRNA, caused time-dependent Pl-Tlr mRNA up-regulation that was detected by QPCR. In contrast, bacterial LPS injury did not affect Pl-Tlr transcription. The study of the Tlr genes in the sea urchin model system may provide new perspectives on the role of Tlrs in the invertebrate immune response and clues concerning their evolution in a changing world. PMID:25907136

  12. Association of TLR4 (896A/G and 1196C/T) Gene Polymorphisms with Asthma Risk: A Meta-Analysis

    PubMed Central

    Li, Shaojun; Xie, Xinming; Song, Yang; Jiang, Haoxiang; Wu, Xiaojuan; Su, Xiaofan; Yang, Lan; Li, Manxiang

    2015-01-01

    Background Conflicting data have been reported on the association between Toll-like receptor 4 (TLR4) +896A/G and +1196C/T polymorphisms and the risk of asthma. Therefore, we conducted this meta-analysis to clarify the effect of TLR4 +896A/G and +1196C/T polymorphisms on the risk of asthma. Material/Methods An electronic literature search was performed using PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure (CNKI), and Wanfang Data to find relevant studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. All statistical analyses were conducted using STATA software version 12.0. Results A total of 14 studies with 2873 asthma cases and 3110 controls were included. The pooled results indicated a significant association between TLR4 +1196C/T polymorphism and the risk of asthma (T vs. C: OR=0.79, 95%CI=0.63–0.99, P=0.04; TT+CT vs. CC: OR=0.76, 95%CI=0.59–0.96, P=0.03; CT vs. CC: OR=0.74, 95%CI=0.58–0.95, P=0.02). In subgroup analysis by ethnicity, TLR4 +1196C/T polymorphism was significantly associated with asthma risk in Asians (T vs. C: OR=0.73, 95%CI=0.54–0.98, P=0.04; TT+CT vs. CC: OR=0.70, 95%CI=0.51–0.96, P=0.03; CT vs. CC: OR=0.69, 95%CI=0.50–0.96, P=0.03), but not in whites. For TLR4 +896A/G polymorphism, no significant association was found between TLR4 +896A/G polymorphism and asthma risk under any genetic models. Conclusions The results of this meta-analysis suggest that T allele of the TLR4 +1196C/T might act as a protective factor against the development of asthma. PMID:26588249

  13. TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

    PubMed

    Gomes, Marco Túlio; Campos, Priscila Carneiro; Pereira, Guilherme de Sousa; Bartholomeu, Daniella Castanheira; Splitter, Gary; Oliveira, Sergio Costa

    2016-05-01

    Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9. PMID:26578650

  14. The Protective Role of TLR3 and TLR9 Ligands in Human Pharyngeal Epithelial Cells Infected with Influenza A Virus

    PubMed Central

    Bo, Zhi-jian; Xu, Ming-yu; Sun, Nan; Liu, Dan-hong

    2014-01-01

    In this study we aim to extensively investigate the anti-influenza virus immune responses in human pharyngeal epithelial cell line (Hep-2) and evaluate the protective role of Toll-like receptor (TLR) ligands in seasonal influenza A H1N1 (sH1N1) infections in vitro. We first investigated the expression of the TLRs and cytokines genes in resting and sH1N1 infected Hep-2 cells. Clear expressions of TLR3, TLR9, interleukin (IL)-6, tumour necrosis factor (TNF)-α and interferon (IFN)-β were detected in resting Hep-2 cells. After sH1N1 infection, a ten-fold of TLR3 and TLR9 were elicited. Concomitant with the TLRs activation, transcriptional expression of IL-6, TNF-α and IFN-β were significantly induced in sH1N1-infected cells. Pre-treatment of cells with poly I:C (an analog of viral double-stranded RNA) and CpG-ODN (a CpG-motif containing oligodeoxydinucleotide) resulted in a strong reduction of viral and cytokines mRNA expression. The results presented indicated the innate immune response activation in Hep-2 cells and affirm the antiviral role of Poly I:C and CpG-ODN in the protection against seasonal influenza A viruses. PMID:24976762

  15. Inflammatory Signalling in Fetal Membranes: Increased Expression Levels of TLR 1 in the Presence of Preterm Histological Chorioamnionitis

    PubMed Central

    Waring, Gareth J.; Robson, Stephen C.; Bulmer, Judith N.; Tyson-Capper, Alison J.

    2015-01-01

    Histological chorioamnionitis (HCA) is an established marker of ascending infection, a major cause of preterm birth. No studies have characterised the global change in expression of genes involved in the toll-like receptor (TLR) signalling pathways in the presence of HCA in the setting of preterm birth (pHCA). Fetal membranes were collected immediately after delivery and underwent histological staging for inflammation to derive 3 groups; term spontaneous labour without HCA (n = 9), preterm birth <34 weeks gestation without HCA (n = 8) and pHCA <34 weeks (n = 12). Profiling arrays ran in triplicate for each group were used to determine the expression of 84 genes associated with TLR signalling and screen for genes of interest (fold change >2; p<0.1). Expression of identified genes was validated individually for all samples, relative to GAPDH, using RT-PCR. Expression of TLR 1, TLR 2, lymphocyte antigen 96, interleukin 8 and Interleukin-1 receptor-associated kinase-like 2 was increased in pHCA (p<0.05). Degree of expression was positively associated with histological staging of both maternal and fetal inflammation (p<0.05). The inflammatory expression profile at the maternal/fetal interface associated with pHCA, a reflection of ascending infection, is extremely heterogeneous suggesting polymicrobial involvement with activation of a common pathway. Antagonism of TLR 1 and TLR 2 signalling in this setting warrants further assessment. PMID:25965269

  16. TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16INK4a protein expression

    PubMed Central

    Parroche, P; Roblot, G; Le Calvez-Kelm, F; Tout, I; Marotel, M; Malfroy, M; Durand, G; McKay, J; Ainouze, M; Carreira, C; Allatif, O; Traverse-Glehen, A; Mendiola, M; Pozo-Kreilinger, J J; Caux, C; Tommasino, M; Goutagny, N; Hasan, U A

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16INK4a was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16INK4a post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis. PMID:27454079

  17. TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16(INK4a) protein expression.

    PubMed

    Parroche, P; Roblot, G; Le Calvez-Kelm, F; Tout, I; Marotel, M; Malfroy, M; Durand, G; McKay, J; Ainouze, M; Carreira, C; Allatif, O; Traverse-Glehen, A; Mendiola, M; Pozo-Kreilinger, J J; Caux, C; Tommasino, M; Goutagny, N; Hasan, U A

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16(INK4a) was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16(INK4a) post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis. PMID:27454079

  18. Structure of the human progesterone receptor gene.

    PubMed

    Misrahi, M; Venencie, P Y; Saugier-Veber, P; Sar, S; Dessen, P; Milgrom, E

    1993-11-16

    The complete organization of the human progesterone receptor (hPR) gene has been determined. It spans over 90 kbp and contains eight exons. The first exon encodes the N-terminal part of the receptor. The DNA binding domain is encoded by two exons, each exon corresponding to one zinc finger. The steroid binding domain is encoded by five exons. The nucleotide sequence of 1144 bp of the 5' flanking region has been determined. PMID:8241270

  19. Interferon-stimulated gene (ISG) 60, as well as ISG56 and ISG54, positively regulates TLR3/IFN-β/STAT1 axis in U373MG human astrocytoma cells.

    PubMed

    Imaizumi, Tadaatsu; Yoshida, Hidemi; Hayakari, Ryo; Xing, Fei; Wang, Lian; Matsumiya, Tomoh; Tanji, Kunikazu; Kawaguchi, Shogo; Murakami, Manabu; Tanaka, Hiroshi

    2016-04-01

    Treatment of cells with interferons (IFNs) induces the phosphorylation of signal transducer and activator of transcription 1 (STAT1), leading to the expression of hundreds of IFN-stimulated genes (ISGs). ISGs exert various antiviral and pro-inflammatory reactions. We have previously reported that ISG56 and ISG54 are induced by polyinosinic-polycytidylic acid (poly IC), an authentic agonist for Toll-like receptor 3 (TLR3), in U373MG human astrocytoma cells. ISG56 and ISG54 are also named as IFN-induced proteins with tetratricopeptide repeats (IFIT) 1 and IFIT2, respectively. In the present study, we demonstrated that poly IC induces the expression of ISG60, also named as IFIT3, in U373MG cells. RNA interference experiments showed that the induction of ISG60 by poly IC was mediated by TLR3, IFN-β, ISG56 and ISG54, whereas ISG60 is involved in poly IC-induced expression of ISG56, ISG54 and a chemokine CXCL10. The level of phosphorylated STAT1 was enhanced by poly IC, and it was inhibited by knockdown of ISG56, ISG54 or ISG60. These results suggest that there is a positive feedback loop between phosphorylated STAT1 and these ISGs. PMID:26423178

  20. Assessment of the effect of TLR7/8, TLR9 agonists and CD40 ligand on the transformation efficiency of Epstein-Barr virus in human B lymphocytes by limiting dilution assay.

    PubMed

    Younesi, Vahid; Shirazi, Forough Golsaz; Memarian, Ali; Amanzadeh, Amir; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2014-01-01

    Infection of human B cells with Epstein-Barr virus (EBV) induces polyclonal activation in almost all infected cells, but a small proportion of infected cells are transformed to immortalized lymphoblastoid cell lines. Since B cells are activated also by CD40 ligand (CD40L) and Toll-like receptor (TLR) agonists via a similar signaling pathway, it is likely that costimulation through these molecules could result in synergistic enhancement of the transformation efficiency of EBV. In this study, the stimulatory effect of TLR7/8 (R848), TLR9 (CpG) agonists and/or CD40L on transformation efficiency of EBV in normal human B cells was assessed using the limiting dilution assay. Costimulation of peripheral blood mononuclear cells (PBMCs) with CpG and R848, but not CD40L, increased significantly the frequency of EBV transformed B cells (p < 0.001). Neither synergistic nor additive effects were observed between TLR agonists and CD40L and also TLR7/8 and TLR9 agonists. Costimulation with R848, CpG and CD40L enhanced the proliferative response of B cells infected with EBV. This effect was more evident when enriched B cells were employed, compared to PBMCs. The promoting effect of TLR agonists stimulation, implies that EBV may take advantage of the genes induced by the TLR stimulation pathway for viral latency and oncogenesis. PMID:23404520

  1. Sequence characterization of river buffalo Toll-like receptor genes 1-10 reveals distinct relationship with cattle and sheep.

    PubMed

    Dubey, P K; Goyal, S; Kathiravan, P; Mishra, B P; Gahlawat, S K; Kataria, R S

    2013-04-01

    The present study was undertaken to characterize the full-length transcripts of Toll-like receptor (TLR) genes 1-10 of river buffalo. The conceptualized amino acid identity of bubaline TLRs ranged between 86% to 100% with ruminants, while it ranged between 45% to 91% with other vertebrate species. Simple modular architecture tool (SMART) analysis revealed the presence of TIR domains and varying numbers of leucine-rich repeat motifs in all the buffalo TLRs. With respect to TIR domains, TLRs 1, 2 and 3 of river buffalo were found to have 99.3% identity with cattle and 100% identity of TLRs 4, 6 and 10 with sheep. Phylogenetic analysis of TLRs of buffalo and different vertebrate species revealed the clustering of major TLR gene subfamilies with high bootstrap values. The evolutionary relationship between buffalo and other ruminant species was found to vary among different TLRs. In order to understand the relationship between TLRs of different ruminant species, multidimensional scaling (MDS) analysis of pairwise amino acid differences between different species within each TLR was performed. Buffalo and cattle were found to be closely related only with respect to TLRs 1, 2 and 7, while buffalo and sheep were found to be clustering together with respect to TLRs 3, 6, 8 and 10. The distinct relationship of bubaline TLRs with cattle and sheep revealed the possible differences in the pathogen recognition receptor systems in these animals and consequently the differences in their susceptibility/resistance to various invading organisms. PMID:22694123

  2. Association of Single Nucleotide Polymorphisms in Toll-like Receptors with Acinetobacter baumanii Infectionin a Chinese Population

    PubMed Central

    HE, Lei; LIN, Maohu; FAN, Wensheng; LIU, Yunxi; SUO, Jijiang; XING, Yubin; JIA, Ning

    2016-01-01

    Background: During recent years, infection of Acinetobacter baumanii showed a rapid growth in hospitals and community. Toll-like receptors (TLRs) are the most important pattern recognition receptors, which play a critical role during recognizing invading pathogens by the natural immune system. Our objective was to determine the associations of TLRs polymorphisms with the susceptibility to A. baumanii infection in a Chinese population. Methods: We carried out a case-control study, genotyping 13 polymorphisms of TLR-2, TLR-4, TLR-5 and TLR-9 genes on 423 A. baumanii-infected patients and 385 exposed controls. Thirteen SNPs at the TLR-2 (rs3804099, rs7656411 and rs76112010), TLR-4 (rs1927914, rs10759932 and rs11536889), TLR-5 (rs1341987, rs1640827, rs1861172, rs2241097, rs5744174 and rs17163737) and TLR9 (rs187084) genes were analyzed. SNP genotyping was performed using an improved multiplex ligation detection reaction (iMLDR) technique. Results: The SNP of TLR-9, rs187084, was related to A. baumanii-infection significantly under recessive model (G/G, to A/A + G/A, P = 0.0064, OR = 0.59, 95% CI = 0.40–0.86) after adjustment with age. Besides, the haplotype GCG of TLR-4 was significantly associated with A. baumanii infection (P = 0.027). Conclusion: TLR-4 and TLR-9 may be related to the susceptibility to A. baumanii infection in a Chinese population. PMID:27057517

  3. Polo-Like Kinase 1 (PLK1) Is Involved in Toll-like Receptor (TLR)-Mediated TNF-α Production in Monocytic THP-1 Cells

    PubMed Central

    Hu, Jinyue; Wang, Guihua; Liu, Xueting; Zhou, Lina; Jiang, Manli; Yang, Li

    2013-01-01

    Polo-like kinases (PLKs) have been reported to be essential components of anti-viral pathways. However, the role of PLKs in the production of pro-inflammatory cytokines induced by TLR activation is uncertain. We report here that monocytic THP-1 cells expressed PLK1, PLK2, PLK3 and PLK4. When THP-1 cells were treated with GW843682X, an inhibitor of PLK1 and PLK3, the results showed that GW843682X down-regulated Pam3CSK4- and LPS-induced TNF-α at both the gene and protein levels. GW843682X did not impact Pam3CSK4-induced IL-1β and IL-8 or LPS-induced IL-1β, but it down-regulated LPS-induced IL-8 significantly. Moreover, western blot results showed that TLRs activated PLK1, and PLK1 inhibition by RNA interference down-regulated Pam3CSK4-induced TNF-α production, suggesting the involvement of PLK1 in TNF-α up-regulation. In addition, GW843682X treatment for 12 to 24 h induced cell death and down-regulated MyD88, but neither of these roles contributed to the down-regulation of TNF-α, as TNF-α gene expression was up-regulated at 1 h. Furthermore, GW843682X inhibited Pam3CSK4-induced activation of ERK and NF-κB, which contributed to Pam3CSK4-induced up-regulation of TNF-α. GW843682X also inhibited LPS-induced activation of ERK, p38 and NF-κB, which contributed to LPS-induced up-regulation of TNF-α. Taken together, these results suggested that PLK1 is involved in TLR2- and TLR4-induced inflammation, and GW843682X may be valuable for the regulation of the inflammatory response. PMID:24205328

  4. Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4 signalling pathway to alleviate microglial activation

    PubMed Central

    Sun, Meiyan; Deng, Bin; Zhao, Xiaoyong; Gao, Changjun; Yang, Lu; Zhao, Hui; Yu, Daihua; Zhang, Feng; Xu, Lixian; Chen, Lei; Sun, Xude

    2015-01-01

    Excessive microglial activation often contributes to inflammation-mediated neurotoxicity in the ischemic penumbra during the acute stage of ischemic stroke. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation via the NF-κB pathway. Isoflurane preconditioning (IP) can provide neuroprotection and inhibit microglial activation. In this study, we investigated the roles of the TLR4 signalling pathway in IP to exert neuroprotection following ischemic stroke in vivo and in vitro. The results showed that 2% IP alleviated neurological deficits, reduced the infarct volume, attenuated apoptosis and weakened microglial activation in the ischemic penumbra. Furthermore, IP down-regulated the expression of HSP 60, TLR4 and MyD88 and up-regulated inhibitor of IκB-α expression compared with I/R group in vivo. In vitro, 2% IP and a specific inhibitor of TLR4, CLI-095, down-regulated the expression of TLR4, MyD88, IL-1β, TNF-α and Bax, and up-regulated IκB-α and Bcl-2 expression compared with OGD group. Moreover, IP and CLI-095 attenuated microglial activation-induced neuronal apoptosis, and overexpression of the TLR4 gene reversed the neuroprotective effects of IP. In conclusion, IP provided neuroprotection by regulating TLR4 expression directly, alleviating microglial activation and neuroinflammation. Thus, inhibiting the activation of microglial activation via TLR4 may be a new avenue for stroke treatment. PMID:26086415

  5. MDA5 complements TLR3 in suppression of neuroblastoma

    PubMed Central

    Hsu, Wen-Ming; Huang, Chao-Cheng; Lee, Hsin-Yu; Wu, Pei-Yi; Wu, Min-Tsui; Chuang, Hui-Ching; Lin, Li-Ling; Chuang, Jiin-Haur

    2015-01-01

    Toll-like receptor3 (TLR3) has been confirmed to be differentially expressed in neuroblastoma (NB), and predicts a favorable prognosis with a high expression in tumor tissues. Treatment with TLR3 agonist - polyinosinic-polycytidylic acid [poly(I:C)] could induce significant but limited apoptosis in TLR3-expressing NB cells, suggesting that other viral RNA sensors, including melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) in the cytosolic compartment might also be implicated in poly(I:C)-induced NB cell death. MDA5 and RIG-I were induced by poly(I:C) to express in two of six NB cell lines, SK-N-AS (AS) and SK-N-FI, which were associated with up-regulation of caspase9 and active caspase3. While knockdown of either MDA5 or RIG-I alone is ineffective to decrease caspase9 and active caspase3, simultaneously targeting MDA5 and TLR3 showed the best effect to rescue poly(I:C) induced up-regulation of mitochondrial antiviral signaling protein (MAVS), caspase9, active caspase3, and apoptosis in AS cells. Over-expression of MDA5 in FaDu cells resulted in significantly less colony formation and more poly(I:C)-induced cell death. Further studies in human NB tissue samples revealed that MDA5 expression in NB tissues predicted a favorable prognosis synergistically with TLR3. Our findings indicate that MDA5 may serve as a complementary role in the TLR3 activated suppression of NB. PMID:26208481

  6. Discovery of toll-like receptor 13 exists in the teleost fish: Miiuy croaker (Perciformes, Sciaenidae).

    PubMed

    Wang, Yanjin; Bi, Xueyi; Chu, Qing; Xu, Tianjun

    2016-08-01

    Toll-like receptors (TLRs) play an indispensable role in the immune response for pathogen recognition and triggering not only innate immunity but also adaptive immunity. Here we report the TLR13 homologue, one member of TLRs, in Perciformes (especially Sciaenidae). And we used the miiuy croaker as represented species for further functional experiments. Former study reported the TLR13 only expressed in murine, and we are the first to report the teleost TLR13 (mmiTLR13). MmiTLR13 expressed highly in immune defense related tissues, such as the liver, spleen, and kidney, and Vibrio anguillarum or poly(I:C) infection showed the immune response of mmiTLR13. Further luciferase reporter assays showed the ability for activation of ISRE luciferase reporter, but it failed to active NF-κB. And further gene silence by short hairpin RNA (shRNA) confirmed the results. Immunofluorescence of mmiTLR13 presents the cytoplasmic distribution in Hela cell. In addition, the Toll/interleukin 1 receptor (TIR) domain of mammal TLR5 exhibits high identity with TLR13, which indicated the high homology between TLR5 and TLR13. These findings will lay the fundamental cornerstone for further research of teleost TLR13 and expand the horizon for better understand the teleost TLRs system. PMID:26952767

  7. Human and chicken TLR pathways: manual curation and computer-based orthology analysis

    PubMed Central

    Gillespie, Marc; Shamovsky, Veronica; D’Eustachio, Peter

    2011-01-01

    The innate immune responses mediated by Toll-like receptors (TLR) provide an evolutionarily well-conserved first line of defense against microbial pathogens. In the Reactome Knowledgebase we previously integrated annotations of human TLR molecular functions with those of over 4000 other human proteins involved in processes such as adaptive immunity, DNA replication, signaling, and intermediary metabolism, and have linked these annotations to external resources, including PubMed, UniProt, EntrezGene, Ensembl, and the Gene Ontology to generate a resource suitable for data mining, pathway analysis, and other systems biology approaches. We have now used a combination of manual expert curation and computer-based orthology analysis to generate a set of annotations for TLR molecular function in the chicken (Gallus gallus). Mammalian and avian lineages diverged approximately 300 million years ago, and the avian TLR repertoire consists of both orthologs and distinct new genes. The work described here centers on the molecular biology of TLR3, the host receptor that mediates responses to viral and other doubled-stranded polynucleotides, as a paradigm for our approach to integrated manual and computationally based annotation and data analysis. It tests the quality of computationally generated annotations projected from human onto other species and supports a systems biology approach to analysis of virus-activated signaling pathways and identification of clinically useful antiviral measures. PMID:21052677

  8. Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor

    PubMed Central

    2014-01-01

    Background Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. Methods The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. Results Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNβ in the RAW264.7 challenged with LPS. Conclusion Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action. PMID:24602493

  9. Molecular cloning and expression of TLR in the Eisenia andrei earthworm.

    PubMed

    Škanta, František; Roubalová, Radka; Dvořák, Jiří; Procházková, Petra; Bilej, Martin

    2013-12-01

    Toll-like receptors (TLRs) play an important role in defense responses to pathogens in invertebrates. Here we characterize the first TLR isolated from an oligochaete annelid, namely, Eisenia andrei (EaTLR) and show its expression pattern. The full-length EaTLR cDNA consists of 2615 bp encoding a putative protein of 675 amino acids. The predicted amino acid sequence comprises of an extracellular domain containing 31 amino acid signal peptide and seven leucine-rich repeats (LRR), capped with cysteine-rich N- and C-terminal LRRs followed by a transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). TIR domains of twenty individual earthworms were sequenced and the variability suggesting the presence of a high number of TLR genes in the genome of E. andrei was observed. Phylogenetic analysis revealed the highest similarity of EaTLR with polychaete annelid, Capitella teleta and TLRs of mollusks and echinoderms. Finally, the highest constitutive expression of EaTLR was observed in the digestive tract. Gene expression was significantly increased in coelomocytes of E. andrei after the challenge with Gram-positive bacteria. PMID:23969138

  10. High basal levels of functional toll-like receptor 3 (TLR3) and noncanonical Wnt5a are expressed in papillary thyroid cancer and are coordinately decreased by phenylmethimazole together with cell proliferation and migration.

    PubMed

    McCall, Kelly D; Harii, Norikazu; Lewis, Christopher J; Malgor, Ramiro; Kim, Won Bae; Saji, Motoyasu; Kohn, Aimee D; Moon, Randall T; Kohn, Leonard D

    2007-09-01

    High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation. PMID:17525119

  11. The rationale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes in advanced ovarian cancer.

    PubMed

    Adams, M; Navabi, H; Croston, D; Coleman, S; Tabi, Z; Clayton, A; Jasani, B; Mason, M D

    2005-03-18

    A clinical trial employing an immunotherapeutic approach based on the use of a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes carrying tumour-associated antigens is planned in advanced ovarian cancer in conjunction with conventional first line chemotherapy. Most patients with ovarian cancer present with advanced disease and despite high initial response rate to chemotherapy the majority will relapse within 2 years with poor overall survival. Tumour antigen-specific T cells are naturally occurring in ovarian cancer patients and T cell infiltration of the tumour is highly prognostic. Novel immunotherapy to expand and activate tumour antigen-specific T cells combined with adjuvant treatment to overcome tumour-induced immunosuppression is considered to be therapeutically beneficial. The rationale for adopting such a combined approach is discussed here. PMID:15755631

  12. Roles of Toll-like receptors 2 and 6 in the inflammatory response to Mycoplasma gallisepticum infection in DF-1 cells and in chicken embryos.

    PubMed

    Tian, Wei; Zhao, Chengcheng; Hu, Qingchuang; Sun, Jianjun; Peng, Xiuli

    2016-06-01

    While Mycoplasma gallisepticum (MG) is a major pathogen that causes chronic respiratory diseases in chicken, the molecular mechanism of MG infection is not clear. In this study, we investigated the roles of Toll-like receptor 2 (TLR2) and 6 (TLR6) in MG infection. We found that TLR2 type 2 (TLR2-2) and TLR6 had differential expressions in chicken embryo fibroblasts (DF-1 cells), where TLR6 was highly expressed, but TLR2-2 was barely expressed. Upon MG infection, TLR6 expression was upregulated, followed by upregulation of downstream factors, MyD88, NF-κB, IL2, IL6, and TNF-α. Knockdown of TLR6 expression by shRNA abolished the MG-induced inflammatory responses. More interestingly, in the presence of TLR6, TLR2-2 didn't respond to MG infection in DF-1 cells. When TLR6 was knocked down by shRNA, however, TLR2 was upregulated upon MG infection, which was followed by upregulation of proinflammatory genes. Finally, we tested effects of the MG infection on expression of TLR2-2 and TLR6 in the lungs and trachea tissues of chicken embryos. We found both TLR2-2 and TLR6 were upregulated upon MG infection, followed by upregulation of the downstream NF-κB-mediated inflammatory responses. This study was the first to report the differential roles of TLR2-2 and TLR6 in MG-infected DF-1 cells and chicken embryos. PMID:26797426

  13. Gene polymorphisms of Toll-like and related recognition receptors in relation to the vaginal carriage of Gardnerella vaginalis and Atopobium vaginae.

    PubMed

    Verstraelen, Hans; Verhelst, Rita; Nuytinck, Lieve; Roelens, Kristien; De Meester, Els; De Vos, Daniel; Van Thielen, Martine; Rossau, Rudi; Delva, Wim; De Backer, Ellen; Vaneechoutte, Mario; Temmerman, Marleen

    2009-01-01

    Host genetic factors have previously been found to act as determinants of differential susceptibility to major infectious diseases. It is less clear whether such polymorphisms may also impose on pathogen recognition in mucosal overgrowth conditions such as bacterial vaginosis, an anaerobic overgrowth condition characterised by the presence of a vaginal biofilm consisting of the Gram-positive anaerobes Gardnerella vaginalis and Atopobium vaginae. We selected 34 single nucleotide polymorphisms pertaining to 9 genes involved with Toll-like receptor-mediated pathogen recognition and/or regulation (LBP, CD14, TLR1, TLR2, TLR4, TLR6, MD2, CARD15 and SIGIRR) and assessed in a nested case-control study their putative association with bacterial vaginosis, as diagnosed by Gram staining, and with the vaginal carriage of A. vaginae and G. vaginalis, as determined by species-specific PCR, among 144 pregnant women. Carriage of G. vaginalis during early pregnancy was associated with the -1155A>G substitution in the promoter region of the MD2 gene (p=0.041). The presence of A. vaginae during the first half of the pregnancy was significantly associated with the CD14 intron 2 1342G>T (p=0.039), the TLR1 exon 4 743A>G (p=0.038), and the CARD15 exon 4 14772A>T (p=0.012) polymorphisms, and marginally significantly associated with the LBP exon13 26842C>T (p=0.056), the CD14 promoter -260C>T (p=0.052), and the TLR1 promoter -7202A>G (p=0.062) polymorphisms. However, no association between gene polymorphisms and bacterial vaginosis as such could be documented. Our data suggest that some degree of genetic susceptibility involving pathogen recognition may occur with the key bacterial vaginosis organism, A. vaginae. PMID:19200604

  14. Pterostilbene, a novel natural plant conduct, inhibits high fat-induced atherosclerosis inflammation via NF-κB signaling pathway in Toll-like receptor 5 (TLR5) deficient mice.

    PubMed

    Zhang, Yuan; Zhang, Yi

    2016-07-01

    Atherosclerosis is a specific form of an artery wall thickens, a syndrome affecting arterial blood vessels due to a chronic inflammatory response in the walls of arteries, which is promoted by fat accumulation. Toll-like receptors (TLRs) play prominent roles in inflammatory responses. And TLR5 is overexpressed in several diseases. Here in our study, we investigated the effect of TLR5 in high fat-induced atherosclerosis via NF-κB signaling pathway modulating pro-inflammatory cytokines releasing. Our results found that high fat induced atherosclerosis in wild type mice with fat accumulation and inflammatory response through NF-κB activation. Contrastly, TLR5 knockout mice displayed lower fat accumulation and ameliorated inflammation after high fat feeding with NF-κB inactivation. In addition, pterostilbene, as a natural dimethyl ether derivative of resveratrol mainly from blueberries, has diverse pharmacological activities, especially anti-inflammation. Our study also found that pterostilbene displayed inhibited role in suppressing inflammatory response through inactivating NF-κB signaling pathway regulated by TLR5 down-regulation in high fat-induced mice. Moreover, in vitro experiments of vascular smooth muscle cells (VSMCs) challenged with LPS or TNF-α, further indicated that NF-κB was involved in atherosclerosis progression, leading to high secretion of pro-inflammatory cytokines. However, VSMCs from TLR5 deficient mice inhibited phosphorylated levels of NF-κB signalilng pathway, finally resulting in down-regulation of inflammatory cytokines. Notably, pterostilbene also displayed suppressed role in inflammatory response via NF-κB inactivity in LPS or TNF-α-induced VSMCs by decreasing TLR5 expression. The results above indicated a novel therapeutic strategy of pterostilbene to protect against atherosclerosis via TLR5 regulation for clinic treatment in the future. PMID:27261612

  15. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    PubMed Central

    Gupta, Pooja H.; Patel, Nirmal A.; Rank, D. N.; Joshi, C. G.

    2015-01-01

    Aim: An attempt has been made to study the toll-like receptors 4 (TLR4) gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR), respectively from Kankrej (22) and Triple cross (24) cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B) of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p<0.05). Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS) indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05). Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance. PMID:27047144

  16. Transcriptional profiling of TLR-4/7/8-stimulated guinea pig splenocytes and whole blood by bDNA assay

    PubMed Central

    Ching, Lance K.; Mompoint, Farah; Guderian, Jeffrey A.; Picone, Alex; Orme, Ian M.; Coler, Rhea N.; Reed, Steven G.; Baldwin, Susan L.

    2011-01-01

    Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens. PMID:21839740

  17. Characterisation of Toll-like receptors 4, 5 and 7 and their genetic variation in the grey partridge.

    PubMed

    Vinkler, Michal; Bainová, Hana; Bryjová, Anna; Tomášek, Oldřich; Albrecht, Tomáš; Bryja, Josef

    2015-02-01

    Toll-like receptors (TLRs) are a cornerstone of vertebrate innate immunity. In this study, we identified orthologues of TLR4, TLR5 and TLR7 (representing both bacterial- and viral-sensing TLRs) in the grey partridge (Perdix perdix), a European Galliform game bird species. The phylogeny of all three TLR genes follows the known phylogeny of Galloanserae birds, placing grey partridge TLRs (PePeTLRs) in close proximity to their turkey and pheasant orthologues. The predicted proteins encoded by the PePeTLR genes were 843, 862-863 and 1,047 amino acids long, respectively, and clearly showed all TLR structural features. To verify functionality in these genes we mapped their tissue-expression profiles, revealing generally high PePeTLR4 and PePeTLR5 expression in the thymus and absence of PePeTLR4 and PePeTLR7 expression in the brain. Using 454 next-generation sequencing, we then assessed genetic variation within these genes for a wild grey partridge population in the Czech Republic, EU. We identified 11 nucleotide substitutions in PePeTLR4, eight in PePeTLR5 and six in PePeTLR7, resulting in four, four and three amino acid replacements, respectively. Given their locations and chemical features, most of these non-synonymous substitutions probably have a minor functional impact. As the intraspecific genetic variation of the three TLR genes was low, we assume that either negative selection or a bottleneck may have reduced TLR population variability in this species. PMID:25626717

  18. Postnatal TLR2 activation impairs learning and memory in adulthood.

    PubMed

    Madar, Ravit; Rotter, Aviva; Waldman Ben-Asher, Hiba; Mughal, Mohamed R; Arumugam, Thiruma V; Wood, W H; Becker, K G; Mattson, Mark P; Okun, Eitan

    2015-08-01

    Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth. PMID:26021559

  19. Postnatal TLR2 activation impairs learning and memory in adulthood

    PubMed Central

    Madar, Ravit; Rotter, Aviva; Ben-Asher, Hiba Waldman; Mughal, Mohamed R.; Arumugam, Thiruma V.; Wood, WH; Becker, KG; Mattson, Mark P.; Okun, Eitan

    2015-01-01

    Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth. PMID:26021559

  20. The evolutionary history of TLR4 polymorphisms in Europe.

    PubMed

    Plantinga, Theo S; Ioana, Mihai; Alonso, Santos; Izagirre, Neskuts; Hervella, Montserrat; Joosten, Leo A B; van der Meer, Jos W M; de la Rúa, Concepcion; Netea, Mihai G

    2012-01-01

    Infections exert important evolutionary pressures shaping the human genome, especially on genes involved in host defense. A crucial step for host defense is recognition of pathogens by pattern recognition receptors on innate immune cells, among which Toll-like receptor 4 (TLR4) is one of the best known. Genetic variation in TLR4 (Asp299Gly, Thr399Ile) has been recently described. Haplotype frequencies of these polymorphisms differ among African, Asian and European populations, suggesting evolutionary pressures exerted by local infections. The TLR4 299Gly/399Ile haplotype, characteristic mainly of European populations, has relatively high frequency in the Iberian peninsula. This region is also described as refuge area during the last glacial maximum 20,000 years ago, from which repopulation of Europe took place. We speculate that a genetic bottleneck in the Iberian peninsula could have promoted the increased frequency of this haplotype by genetic drift. This hypothesis is supported by three arguments: (1) the West-East gradient of prevalence in the haplotype among European populations; (2) ancient DNA from Neolithic burials in the Iberian peninsula, dated 6,600-4,500 years before present, confirmed the relatively high frequency of this haplotype in the region, and (3) no functional differences between this haplotype and wild-type TLR4 have been found. In contrast, the disappearance of the 299Gly/399Thr haplotype in Europe is most likely due to negative selection due to sepsis. In conclusion, differences in distribution of TLR4 polymorphisms Asp299Gly and Thr399Ile in European populations are most likely due to a combination of population migration events combined with selection due to sepsis. PMID:21968286

  1. Relationship between TLR4 and MCP2 expression levels and habitual abortion.

    PubMed

    Li, X P; Song, L N; Tian, L P; Zhang, Y S

    2016-01-01

    Habitual abortion is associated with the altered expression of multiple genes. This study was carried out to investigate the relationship between expression of Toll-like receptor 4 (TLR4) and monocyte chemotactic protein 2 (MCP2 or CCL8) and habitual abortion. This was done by detecting and comparing their relative expression in peripheral blood and placental villi of patients and healthy fertile women. Based on our previous research, 85 subjects with habitual abortion (study group) and 40 healthy fertile women (control group), who were admitted to our hospital between June 2013 and December 2014, were enrolled in this study. After these subjects signed written informed consent, peripheral blood samples and villous tissues were collected, from which the total RNA was extracted. The expression of TLR4 and MCP2 was detected with quantitative reverse transcription-polymerase chain reaction, using GAPDH as a reference control. The expression of TLR4 and MCP2 in the peripheral blood and villous tissues of the study group was significantly higher than that of the control group (P < 0.05). A positive correlation was also observed between the changes in expression levels of TLR4 and MCP2. In conclusion, TLR4 and MCP2 expression correlated with the occurrence of habitual abortion. Detecting expression changes in TLR4 and MCP2 in the peripheral blood is a feasible method for predicting the occurrence of abortion in women of child-bearing age. PMID:27173235

  2. Association of Toll-like receptor 2 Arg753Gln and Toll-like receptor 1 Ile602Ser single-nucleotide polymorphisms with leptospirosis in an Argentine population.

    PubMed

    Cédola, Maia; Chiani, Yosena; Pretre, Gabriela; Alberdi, Lucrecia; Vanasco, Bibiana; Gómez, Ricardo M

    2015-06-01

    Toll-like receptor 2 (TLR2), a member of the Toll-like receptor family, plays an important role in the recognition of and subsequent immune response activation against leptospirosis in humans. The genetic polymorphism in TLR2 of an arginine to glutamine substitution at residue 753 (Arg753Gln) has been associated with a negative influence on TLR2 function, which may, in turn, determine the innate host response to Leptospira spp. This bacterium signals through TLR2/TLR1 heterodimers in human cells. The aim of the present study was to investigate the Arg753Gln single-nucleotide polymorphism (SNP) of the TLR2 gene, and the isoleucine to serine transversion at position 602 (Ile602Ser) of the TLR1 gene (previously associated with Lyme disease), in leptospirosis patients compared to healthy controls, carrying out a retrospective case/control study. The TLR2 polymorphism adenine (A) allele was observed in 7.3% of leptospirosis patients but was not found in the control group, whereas the guanine (G) allele of the TLR1 polymorphism was found in 63.6% of patients and 41.6% of controls. Susceptibility to leptospirosis disease was increased 10.57-fold for carriers of the TLR2 G/A genotype (P=0.0493) and 3.85-fold for carriers of the TLR1 G/G genotype (P=0.0428). Furthermore, the risk of developing hepatic insufficiency and jaundice was increased 18.86- and 27.60-fold for TLR2 G/A carriers, respectively. Similarly, the risk of developing jaundice was increased 12.67-fold for TLR1 G allele carriers (G/G and T/G genotypes). In conclusion, the present data suggest that the TLR2 Arg753Gln and TLR1 Ile602Ser SNPs influence the risk of developing leptospirosis and its severity. PMID:25784560

  3. Identification and characteristic analysis of TLR28: A novel member of the TLR1 family in teleost.

    PubMed

    Wang, Yanjin; Li, Jinrui; Han, Jingjing; Shu, Chang; Xu, Tianjun

    2016-09-01

    Toll-like receptors (TLRs) play a critical role in the innate immune response of fish to recognize microorganisms. Fish TLRs have significant variety and distinct features. This study focuses on a novel TLR member that belongs to the TLR1 family and was first discovered in miiuy croaker (designated as TLR28, mmiTLR28). In phylogenetic analysis, the mmiTLR28 clustered in the TLR1 family. Further characteristic analysis showed a high homology with TLR2 despite some differences between them. The predicted tertiary structure of mmiTLR28 possesses a hydrophobic pocket in the ectodomain region. Expression analysis showed the high expression level in the liver of miiuy croaker. Further functional experiments on the liver after Vibrio anguillarum, Staphylococcus aureus, lipopolysaccharides (LPS), and poly (I:C) stimulation showed significant upregulation; these results indicate the potential role of mmiTLR28 in immune response. For LPS stimulation in miiuy croaker leukocytes, mmiTLR28 also displayed significant upregulation. The discovery of mmiTLR28 will enrich the information on TLR family; the functional experiments have shown the role of mmiTLR28 in immunity. The results of this study lay the foundation for future research on fish immune systems. PMID:27155354

  4. Mycoplasma gallisepticum lipid associated membrane proteins up-regulate inflammatory genes in chicken tracheal epithelial cells via TLR-2 ligation through an NF-κB dependent pathway.

    PubMed

    Majumder, Sanjukta; Zappulla, Frank; Silbart, Lawrence K

    2014-01-01

    Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (R(low)) or a non-virulent (R(high)) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, R(low) exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2-3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either R(low) or R(high) exposure. Taken together we conclude that LAMPs isolated from both R(high) and R(low) induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway. PMID

  5. mTOR regulates TLR-induced c-fos and Th1 responses to HBV and HCV vaccines.

    PubMed

    He, Li; Zang, Aiping; Du, Min; Ma, Dapeng; Yuan, Chuanping; Zhou, Chun; Mu, Jing; Shi, Huanjing; Li, Dapeng; Huang, Xulin; Deng, Qiang; Xiao, Jianhua; Yan, Huimin; Hui, Lijian; Lan, Ke; Xiong, Sidong; Li, Xiaoxia; Huang, Zhong; Xiao, Hui

    2015-06-01

    Although IL-12 plays a critical role in priming Th1 and cytotoxic T lymphocyte (CTL) responses, Toll-like receptor (TLR) signaling only induces low amounts of IL-12 in dendritic cells and macrophages, implying the existence of stringent regulatory mechanisms. In this study, we sought to uncover the mechanisms underlying TLR-induced IL-12 expression and the Th1 response. By systemic screening, we identified a number of protein kinases involved in the regulation of TLRinduced IL-12 expression. In particular, PI3K, ERK, and mTOR play critical roles in the TLR-induced Th1 response by regulating IL-12 and IL-10 production in innate immune cells. Moreover, we identified c-fos as a key molecule that mediates mTOR-regulated IL-12 and IL-10 expression in TLR signaling. Mechanistically, mTOR plays a crucial role in c-fos expression, thereby modulating NFκB binding to promoters of IL-12 and IL-10. By controlling the expression of a special innate gene program, mTOR can specifically regulate the TLR-induced T cell response in vivo. Furthermore, blockade of mTOR by rapamycin efficiently boosted TLR-induced antigen-specific T and B cell responses to HBV and HCV vaccines. Taken together, these results reveal a novel mechanism through which mTOR regulates TLR-induced IL-12 and IL-10 production, contributing new insights for strategies to improve vaccine efficacy. PMID:26122641

  6. HMGB1-Driven Inflammation and Intimal Hyperplasia After Arterial Injury Involves Cell-Specific Actions Mediated by TLR4

    PubMed Central

    Cai, Jingjing; Yuan, Hong; Wang, Qingde; Yang, Huan; Al-Abed, Yousef; Hua, Zhong; Wang, Jiemei; Chen, Dandan; Wu, Jinze; Lu, Ben; Pribis, John P.; Jiang, Weihong; Yang, Kan; Hackam, David J.; Tracey, Kevin J.; Billiar, Timothy R.; Chen, Alex F.

    2016-01-01

    Objective Endoluminal vascular interventions such as angioplasty initiate a sterile inflammatory response resulting from local tissue damage. This response drives the development of intimal hyperplasia (IH) that, in turn, can lead to arterial occlusion. We hypothesized that the ubiquitous nuclear protein and damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), is one of the endogenous mediators that activates processes leading to IH after endoluminal injury to the arterial wall. The aim of this study is to investigate whether approaches that reduce the levels of HMGB1 or inhibit its activity suppresses IH after arterial injury. Approach and Results Here, we show that HMGB1 regulates IH in a mouse carotid wire injury model. Induced genetic deletion or neutralization of HMGB1 prevents IH, monocyte recruitment, and smooth muscle cell growth factor production after endoluminal carotid artery injury. A specific inhibitor of HMGB1 myeloid differentiation factor 2–toll-like receptor 4 (TLR4) interaction, P5779, also significantly inhibits IH. HMGB1 deletion is mimicked in this model by global deletion of TLR4 and partially replicated by myeloid-specific deletion of TLR4 but not TLR2 or receptor for advanced glycation endproducts deletion. The specific HMGB1 isoform known to activate TLR4 signaling (disulfide HMGB1) stimulates smooth muscle cell to migrate and produce monocyte chemotactic protein 1/CCL2) via TLR4. Macrophages produce smooth muscle cell mitogens in response to disulfide HMGB1 also in a TLR4/myeloid differentiation primary response gene (88)/Trif-dependent manner. Conclusions These findings place HMGB1 and its receptor, TLR4 as critical regulators of the events that drive the inflammation leading to IH after endoluminal arterial injury and identify this pathway as a possible therapeutic target to limit IH to attenuate damage-associated molecular pattern molecule–mediated vascular inflammatory responses. PMID:26515416

  7. Molecular cloning and characterization of a Toll receptor gene from Macrobrachium rosenbergii.

    PubMed

    Srisuk, Chutima; Longyant, Siwaporn; Senapin, Saengchan; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

    2014-02-01

    Toll receptors are cell surface molecules acting as pattern recognition receptors (PRRs) that have been implicated in the signaling pathway of innate immune responses. In this study, the full-length cDNA of a Toll receptor gene of Macrobrachium rosenbergii, designated MrToll, was successfully isolated using designed degenerate primers and the rapid amplification of cDNA ends (RACE). The MrToll gene sequence contained an open reading frame (ORF) of 2799 nucleotides encoding a protein of 932 amino acid residues. The protein contained distinct structural motifs of the Toll-like receptor (TLR) family, including an extracellular domain containing 15 leucine-rich repeats (LRRs), a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/interleukin-1R (TIR) domain of 139 residues. Phylogenetic analysis revealed that MrToll and Toll receptor of Marsupenaeus japonicus (MjToll) evolved closely. However, the MrToll ORF demonstrated only 48-49% identity with shrimp Toll1, suggesting that MrToll isolated from a palaemonid shrimp might belong to a novel class of Toll receptors in shrimp. The transcripts of the MrToll gene were constitutively expressed in various tissues, with high levels in hemocytes, the stomach and muscle. A reverse transcriptase PCR assay demonstrated that the expression patterns of MrToll were distinctly modulated after Aeromonas caviae stimulation, with significant enhancement at 3-12 h post-challenge and a decline to basal levels at 24 h post-challenge. In addition, when MrToll-silenced shrimp were challenged with A. caviae, there was a significant increase in mortality and bacterial CFU counts. These results suggest that MrToll might be involved in host innate defense, especially against the pathogen A. caviae. PMID:24398262

  8. Episodic Positive Selection in the Evolution of Avian Toll-Like Receptor Innate Immunity Genes

    PubMed Central

    Grueber, Catherine E.; Wallis, Graham P.; Jamieson, Ian G.

    2014-01-01

    Toll-like receptors (TLRs) are a family of conserved pattern-recognition molecules responsible for initiating innate and acquired immune responses. Because they play a key role in host defence, these genes have received increasing interest in the evolutionary and population genetics literature, as their variation represents a potential target of adaptive evolution. However, the role of pathogen-mediated selection (i.e. episodic positive selection) in the evolution of these genes remains poorly known and has not been examined outside of mammals. A recent increase in the number of bird species for which TLR sequences are available has enabled us to examine the selective processes that have influenced evolution of the 10 known avian TLR genes. Specifically, we tested for episodic positive selection to identify codons that experience purifying selection for the majority of their evolution, interspersed with bursts of positive selection that may occur only in restricted lineages. We included up to 23 species per gene (mean = 16.0) and observed that, although purifying selection was evident, an average of 4.5% of codons experienced episodic positive selection across all loci. For four genes in which sequence coverage traversed both the extracellular leucine-rich repeat region (LRR) and transmembrane/intracellular domains of the proteins, increased positive selection was observed at the extracellular domain, consistent with theoretical predictions. Our results provide evidence that episodic positive selection has played an important role in the evolution of most avian TLRs, consistent with the role of these loci in pathogen recognition and a mechanism of host-pathogen coevolution. PMID:24595315

  9. Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists

    PubMed Central

    Birmachu, Woubalem; Gleason, Raymond M; Bulbulian, Barbara J; Riter, Christie L; Vasilakos, John P; Lipson, Kenneth E; Nikolsky, Yuri

    2007-01-01

    Background Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011. Results Global gene expression was evaluated using the Affymetrix U133A GeneChip® and selected genes were confirmed using real time TaqMan® RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNα subtypes, IFNα2, α5, α6, α8, α1/13, α10, α14, α16, α17, α21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro

  10. The role of TLR2 in the acute inflammatory response induced by Bothrops atrox snake venom.

    PubMed

    Moreira, Vanessa; Teixeira, Catarina; Borges da Silva, Henrique; D'Império Lima, Maria Regina; Dos-Santos, Maria Cristina

    2016-08-01

    Envenomation by snakes of the species Bothrops atrox induces local and systemic effects. Local effects include drastic tissue damage and a marked inflammatory response as a result of the synthesis and release of a variety of protein and lipid mediators. Toll-like receptor (TLR) signaling pathways can play an important role in this response, leading to synthesis of these inflammatory mediators. This study investigated the influence of TLR2 on the acute inflammatory response induced by Bothrops atrox venom. Wild-type C57BL/6 mice (WT) and TLR2 gene knockout mice (TLR2(-/-)) were injected with Bothrops atrox venom (BaV), and the following responses to the venom were assessed in peritoneal exudate: leukocyte accumulation; release of mediators, including CCL-2, IL-10, IL-1β, IL-6 and LTB4; protein expression of COX-1 and COX-2; and quantification of their products PGE2 and TXA2. After injection with BaV, the TLR2(-/-) mice (TLR2(-/-)BaV) had higher levels of IL-6 and CCL-2 than WT animals kept under the same conditions (WTBaV), together with an accumulation of polymorphonuclear leukocytes (PMNs), inhibition of IL-1β and LTB4 and reduced mononuclear leukocyte influx. However, no significant differences in COX-2 protein expression or PGE2, TXA2 and IL-10 production between the TLR2(-/-)BaV and WTBav animals were observed. Together, these results indicate that the signaling pathway activated by TLR2 acts by modulating the induced inflammatory response to BaV through the direct action of venom-associated molecular patterns (VAMPs) or indirectly by forming damage-associated molecular patterns (DAMPs) and that this may have important therapeutic implications. PMID:27109323

  11. Reprogramming of Murine Macrophages through TLR2 Confers Viral Resistance via TRAF3-Mediated, Enhanced Interferon Production

    PubMed Central

    Perkins, Darren J.; Polumuri, Swamy K.; Pennini, Meghan E.; Lai, Wendy; Xie, Ping; Vogel, Stefanie N.

    2013-01-01

    The cell surface/endosomal Toll-like Receptors (TLRs) are instrumental in initiating immune responses to both bacteria and viruses. With the exception of TLR2, all TLRs and cytosolic RIG-I-like receptors (RLRs) with known virus-derived ligands induce type I interferons (IFNs) in macrophages or dendritic cells. Herein, we report that prior ligation of TLR2, an event previously shown to induce “homo” or “hetero” tolerance, strongly “primes” macrophages for increased Type I IFN production in response to subsequent TLR/RLR signaling. This occurs by increasing activation of the transcription factor, IFN Regulatory Factor-3 (IRF-3) that, in turn, leads to enhanced induction of IFN-β, while expression of other pro-inflammatory genes are suppressed (tolerized). In vitro or in vivo “priming” of murine macrophages with TLR2 ligands increase virus-mediated IFN induction and resistance to infection. This priming effect of TLR2 is mediated by the selective upregulation of the K63 ubiquitin ligase, TRAF3. Thus, we provide a mechanistic explanation for the observed antiviral actions of MyD88-dependent TLR2 and further define the role of TRAF3 in viral innate immunity. PMID:23853595

  12. A novel crosstalk between TLR4- and NOD2-mediated signaling in the regulation of intestinal inflammation.

    PubMed

    Kim, Hajeong; Zhao, Quanju; Zheng, Hua; Li, Xin; Zhang, Tuo; Ma, Xiaojing

    2015-01-01

    Although Toll-like receptor 4 (TLR4)- and nucleotide-binding oligomerization domain 2 (NOD2)-mediated signaling mechanisms have been extensively studied individually, the crosstalk between them in the regulation of intestinal mucosal defense and tissue homeostasis has been underappreciated. Here, we uncover some novel activities of NOD2 by gene expression profiling revealing the global nature of the cross-regulation between TLR4- and NOD2-mediated signaling. Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies. This balancing act is mediated through receptor-interacting serine/threonine kinase 2, and the transcriptional regulator CCAAT/enhancer-binding protein α (C/EBPα) via its serine 248 phosphorylation by Protein Kinase C. Mice deficient in C/EBPα in the hematopoietic compartment are highly susceptible to chemically induced experimental colitis in an IL-12-dependent manner. Additionally, in contrast to the dogma, we find that the major Crohn's disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense. To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases. PMID:26153766

  13. MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers

    PubMed Central

    Franzenburg, Sören; Fraune, Sebastian; Künzel, Sven; Baines, John F.; Domazet-Lošo, Tomislav; Bosch, Thomas C. G.

    2012-01-01

    Toll-like receptor (TLR) signaling is one of the most important signaling cascades of the innate immune system of vertebrates. Studies in invertebrates have focused on the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, and there is little information regarding the evolutionary origin and ancestral function of TLR signaling. In Drosophila, members of the Toll-like receptor family are involved in both embryonic development and innate immunity. In C. elegans, a clear immune function of the TLR homolog TOL-1 is controversial and central components of vertebrate TLR signaling including the key adapter protein myeloid differentiation primary response gene 88 (MyD88) and the transcription factor NF-κB are not present. In basal metazoans such as the cnidarians Hydra magnipapillata and Nematostella vectensis, all components of the vertebrate TLR signaling cascade are present, but their role in immunity is unknown. Here, we use a MyD88 loss-of-function approach in Hydra to demonstrate that recognition of bacteria is an ancestral function of TLR signaling and that this process contributes to both host-mediated recolonization by commensal bacteria as well as to defense against bacterial pathogens. PMID:23112184

  14. A novel crosstalk between TLR4- and NOD2-mediated signaling in the regulation of intestinal inflammation

    PubMed Central

    Kim, Hajeong; Zhao, Quanju; Zheng, Hua; Li, Xin; Zhang, Tuo; Ma, Xiaojing

    2015-01-01

    Although Toll-like receptor 4 (TLR4)- and nucleotide-binding oligomerization domain 2 (NOD2)-mediated signaling mechanisms have been extensively studied individually, the crosstalk between them in the regulation of intestinal mucosal defense and tissue homeostasis has been underappreciated. Here, we uncover some novel activities of NOD2 by gene expression profiling revealing the global nature of the cross-regulation between TLR4- and NOD2-mediated signaling. Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies. This balancing act is mediated through receptor-interacting serine/threonine kinase 2, and the transcriptional regulator CCAAT/enhancer-binding protein α (C/EBPα) via its serine 248 phosphorylation by Protein Kinase C. Mice deficient in C/EBPα in the hematopoietic compartment are highly susceptible to chemically induced experimental colitis in an IL-12-dependent manner. Additionally, in contrast to the dogma, we find that the major Crohn’s disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense. To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases. PMID:26153766

  15. The effects of oral and enteric Campylobacter concisus strains on expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells.

    PubMed

    Ismail, Yazan; Lee, Hoyul; Riordan, Stephen M; Grimm, Michael C; Zhang, Li

    2013-01-01

    Campylobacter concisus, a Gram-negative bacterium that colonizes the human oral cavity, has been shown to be associated with inflammatory bowel diseases (IBD). The effects of different C. concisus strains on intestinal epithelial expression of Toll like receptors (TLR) have not been investigated. This study examined the effects of C. concisus strains isolated from patients with IBD and controls on expression of TLR4, its co-receptor myeloid differentiation factor (MD)-2; TLR2, TLR5, cyclooxygenase-2 (COX-2) and interleukin (IL)-8 in HT-29 cells.Fourteen oral and enteric C. concisus strains isolated from patients with IBD and healthy controls were co-incubated with HT-29 cells. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells in response to C. concisus infection was examined by Western blot, flow cytometry analysis and immunofluorescent staining visualized by confocal microscope. Production of IL-8 was evaluated by enzyme-linked immunosorbent assay.Both oral and enteric C. concisus strains upregulated expression of TLR4 in HT-29 cells. The levels of glycosylated TLR4 (Gly-TLR4) and surface TLR4 induced by C. concisus strains isolated from patients with IBD were significantly higher than those induced by C. concisus strains isolated from the healthy controls. Four C. concisus strains isolated from patients with IBD induced more than two-fold increase of surface expression of MD-2. C. concisus did not affect expression of TLR2 and TLR5. All C. concisus strains induced production of IL-8 and COX-2 in HT-29 cells.This study shows that some C. concisus strains, most from patients with IBD, upregulate surface expression of TLR4 and MD-2 in HT-29 cells. These data suggest that a potential role of specific C. concisus strains in modulating the intestinal epithelial responses to bacterial LPS needs to be investigated. PMID:23437263

  16. Influence of Intron II microsatellite polymorphism in human toll-like receptor 2 gene in leprosy.

    PubMed

    Suryadevara, Naveen Chandra; Neela, Venkata Sanjeev Kumar; Devalraju, Kamakshi Prudhula; Jain, Suman; SivaSai, Krovvidi S R; Valluri, Vijaya Lakshmi; Jonnalagada, Subbanna; Anandaraj, M P J S

    2013-08-01

    Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy. PMID:23619473

  17. Down-Regulation of TLR and JAK/STAT Pathway Genes Is Associated with Diffuse Cutaneous Leishmaniasis: A Gene Expression Analysis in NK Cells from Patients Infected with Leishmania mexicana

    PubMed Central

    Castillo-Fernández, Juan E.; Miranda-Ortíz, Haydee; Fernández-López, Juan C.; Becker, Ingeborg; Rangel-Escareño, Claudia

    2016-01-01

    An important NK-cell inhibition with reduced TNF-α, IFN-γ and TLR2 expression had previously been identified in patients with diffuse cutaneous leishmaniasis (DCL) infected with Leishmania mexicana. In an attempt to pinpoint alterations in the signaling pathways responsible for the NK-cell dysfunction in patients with DCL, this study aimed at identifying differences in the NK-cell response towards Leishmania mexicana lipophosphoglycan (LPG) between patients with localized and diffuse cutaneous leishmaniasis through gene expression profiling. Our results indicate that important genes involved in the innate immune response to Leishmania are down-regulated in NK cells from DCL patients, particularly TLR and JAK/STAT signaling pathways. This down-regulation showed to be independent of LPG stimulation. The study sheds new light for understanding the mechanisms that undermine the correct effector functions of NK cells in patients with diffuse cutaneous leishmaniasis contributing to a better understanding of the pathobiology of leishmaniasis. PMID:27031998

  18. Btk inhibition treats TLR7/IFN driven murine lupus.

    PubMed

    Bender, Andrew T; Pereira, Albertina; Fu, Kai; Samy, Eileen; Wu, Yin; Liu-Bujalski, Lesley; Caldwell, Richard; Chen, Yi-Ying; Tian, Hui; Morandi, Federica; Head, Jared; Koehler, Ursula; Genest, Melinda; Okitsu, Shinji L; Xu, Daigen; Grenningloh, Roland

    2016-03-01

    Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling. PMID:26821304

  19. Impaired TLR5 Functionality Is Associated with Survival in Melioidosis

    PubMed Central

    Chantratita, Narisara; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Myers, Nicolle D.; Emond, Mary J.; Wurfel, Mark M.; Hawn, Thomas R.; Peacock, Sharon J.; Skerrett, Shawn J.

    2013-01-01

    Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR51174C>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney–293 cells transfected with TLR5 and found that B. pseudomallei induced TLR51174C- but not TLR51174T-dependent activation of NF-κB. We tested the association of TLR51174C>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR51174C>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08–0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19–0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR51174C>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR51174T compared with carriers of TLR51174C. B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR51174T. We conclude that the hypofunctional genetic variant TLR51174C>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis. PMID:23447684

  20. Impaired TLR5 functionality is associated with survival in melioidosis.

    PubMed

    West, T Eoin; Chantratita, Narisara; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Myers, Nicolle D; Emond, Mary J; Wurfel, Mark M; Hawn, Thomas R; Peacock, Sharon J; Skerrett, Shawn J

    2013-04-01

    Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis. PMID:23447684

  1. Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis

    PubMed Central

    Dheer, Rishu; Santaolalla, Rebeca; Davies, Julie M.; Lang, Jessica K.; Phillips, Matthew C.; Pastorini, Cristhine; Vazquez-Pertejo, Maria T.

    2016-01-01

    Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the host's susceptibility to colitis. PMID:26755160

  2. Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis.

    PubMed

    Dheer, Rishu; Santaolalla, Rebeca; Davies, Julie M; Lang, Jessica K; Phillips, Matthew C; Pastorini, Cristhine; Vazquez-Pertejo, Maria T; Abreu, Maria T

    2016-03-01

    Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the host's susceptibility to colitis. PMID:26755160

  3. Toll-like Receptors of the Ascidian Ciona intestinalis

    PubMed Central

    Sasaki, Naoko; Ogasawara, Michio; Sekiguchi, Toshio; Kusumoto, Shoichi; Satake, Honoo

    2009-01-01

    Key transmembrane proteins in the innate immune system, Toll-like receptors (TLRs), have been suggested to occur in the genome of non-mammalian organisms including invertebrates. However, authentic invertebrate TLRs have been neither structurally nor functionally investigated. In this paper, we originally present the structures, localization, ligand recognition, activities, and inflammatory cytokine production of all TLRs of the ascidian Ciona intestinalis, designated as Ci-TLR1 and Ci-TLR2. The amino acid sequence of Ci-TLR1 and Ci-TLR2 were found to possess unique structural organization with moderate sequence similarity to functionally characterized vertebrate TLRs. ci-tlr1 and ci-tlr2 genes were expressed predominantly in the stomach and intestine as well as in hemocytes. Ci-TLR1 and Ci-TLR2 expressed in HEK293 cells, unlike vertebrate TLRs, were localized to both the plasma membrane and endosomes. Intriguingly, both Ci-TLR1 and Ci-TLR2 stimulate NF-κB induction in response to multiple pathogenic ligands such as double-stranded RNA, and bacterial cell wall components that are differentially recognized by respective vertebrate TLRs, revealing that Ci-TLRs recognize broader pathogen-associated molecular patterns than vertebrate TLRs. The Ci-TLR-stimulating pathogenic ligands also induced the expression of Ci-TNFα in the intestine and stomach where Ci-TLRs are expressed. These results provide evidence that the TLR-triggered innate immune systems are essentially conserved in ascidians, and that Ci-TLRs possess “hybrid” biological and immunological functions, compared with vertebrate TLRs. Moreover, it is presumed that chordate TLR ancestors also acquired the Ci-TLR-like multiple cellular localization and pathogen-associated molecular pattern recognition. PMID:19651780

  4. Heterogeneity of Host TLR2 Stimulation by Staphylocoocus aureus Isolates

    PubMed Central

    Hilmi, Dina; Parcina, Marijo; Stollewerk, Daniel; Ostrop, Jenny; Josten, Michaele; Meilaender, Alina; Zaehringer, Ulrich; Wichelhaus, Thomas A.; Bierbaum, Gabriele; Heeg, Klaus; Wolz, Christiane; Bekeredjian-Ding, Isabelle

    2014-01-01

    High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2) are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106). Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling. PMID:24810614

  5. Engineering AAV receptor footprints for gene therapy.

    PubMed

    Madigan, Victoria J; Asokan, Aravind

    2016-06-01

    Adeno-associated viruses (AAV) are currently at the forefront of human gene therapy clinical trials as recombinant vectors. Significant progress has been made in elucidating the structure, biology and tropisms of different naturally occurring AAV isolates in the past decade. In particular, a spectrum of AAV capsid interactions with host receptors have been identified and characterized. These studies have enabled a better understanding of key determinants of AAV cell recognition and entry in different hosts. This knowledge is now being applied toward engineering new, lab-derived AAV capsids with favorable transduction profiles. The current review conveys a structural perspective of capsid-glycan interactions and provides a roadmap for generating synthetic strains by engineering AAV receptor footprints. PMID:27262111

  6. Targeting TLR2 for Vaccine Development

    PubMed Central

    2014-01-01

    Novel and more effective immunization strategies against many animal diseases may profit from the current knowledge on the modulation of specific immunity through stimulation of innate immune receptors. Toll-like receptor (TLR)2-targeting formulations, such as synthetic lipopeptides and antigens expressed in fusion with lipoproteins, have been shown to have built-in adjuvant properties and to be effective at inducing cellular and humoral immune mechanisms in different animal species. However, contradictory data has arisen concerning the profile of the immune response elicited. The benefits of targeting TLR2 for vaccine development are thus still debatable and more studies are needed to rationally explore its characteristics. Here, we resume the main features of TLR2 and TLR2-induced immune responses, focusing on what has been reported for veterinary animals. PMID:25057505

  7. Pattern-Recognition Receptors and Gastric Cancer

    PubMed Central

    Castaño-Rodríguez, Natalia; Kaakoush, Nadeem O.; Mitchell, Hazel M.

    2014-01-01

    Chronic inflammation has been associated with an increased risk of several human malignancies, a classic example being gastric adenocarcinoma (GC). Development of GC is known to result from infection of the gastric mucosa by Helicobacter pylori, which initially induces acute inflammation and, in a subset of patients, progresses over time to chronic inflammation, gastric atrophy, intestinal metaplasia, dysplasia, and finally intestinal-type GC. Germ-line encoded receptors known as pattern-recognition receptors (PRRs) are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses. Given that H. pylori is initially targeted by PRRs, it is conceivable that dysfunction within genes of this arm of the immune system could modulate the host response against H. pylori infection, and subsequently influence the emergence of GC. Current evidence suggests that Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR5, and TLR9), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2, and NLRP3), a C-type lectin receptor (DC-SIGN), and retinoic acid-inducible gene (RIG)-I-like receptors (RIG-I and MDA-5), are involved in both the recognition of H. pylori and gastric carcinogenesis. In addition, polymorphisms in genes involved in the TLR (TLR1, TLR2, TLR4, TLR5, TLR9, and CD14) and NLR (NOD1, NOD2, NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) signaling pathways have been shown to modulate the risk of H. pylori infection, gastric precancerous lesions, and/or GC. Further, the modulation of PRRs has been suggested to suppress H. pylori-induced inflammation and enhance GC cell apoptosis, highlighting their potential relevance in GC therapeutics. In this review, we present current advances in our understanding of the role of the TLR and NLR signaling pathways in the pathogenesis of GC, address the involvement of other recently identified PRRs in GC, and discuss the potential implications of PRRs in GC immunotherapy

  8. TLR Ligands Induce Antiviral Responses in Chicken Macrophages

    PubMed Central

    Barjesteh, Neda; Behboudi, Shahriar; Brisbin, Jennifer T.; Villanueva, Alexander Ian; Nagy, Éva; Sharif, Shayan

    2014-01-01

    Chicken macrophages express several receptors for recognition of pathogens, including Toll-like receptors (TLRs). TLRs bind to pathogen-associated molecular patterns (PAMPs) derived from bacterial or viral pathogens leading to the activation of macrophages. Macrophages play a critical role in immunity against viruses, including influenza viruses. The present study was designed to test the hypothesis that treatment of chicken macrophages with TLR ligands reduces avian influenza replication. Furthermore, we sought to study the expression of some of the key mediators involved in the TLR-mediated antiviral responses of macrophages. Chicken macrophages were treated with the TLR2, 3, 4, 7 and 21 ligands, Pam3CSK4, poly(I:C), LPS, R848 and CpG ODN, respectively, at different doses and time points pre- and post-H4N6 avian influenza virus (AIV) infection. The results revealed that pre-treatment of macrophages with Pam3CSK4, LPS and CpG ODN reduced the replication of AIV in chicken macrophages. In addition, the relative expression of genes involved in inflammatory and antiviral responses were quantified at 3, 8 and 18 hours post-treatment with the TLR2, 4 and 21 ligands. Pam3CSK4, LPS and CpG ODN increased the expression of interleukin (IL)-1β, interferon (IFN)-γ, IFN-β and interferon regulatory factor (IFR) 7. The expression of these genes correlated with the reduction of viral replication in macrophages. These results shed light on the process of immunity to AIV in chickens. PMID:25165812

  9. Investigation of Toll-Like Receptor-2 (2258G/A) and Interferon Gamma (+874T/A) Gene Polymorphisms among Infertile Women with Female Genital Tuberculosis

    PubMed Central

    Bhanothu, Venkanna; Lakshmi, Vemu; Theophilus, Jane P.; Rozati, Roya; Badhini, Prabhakar; Vijayalaxmi, Boda

    2015-01-01

    Background Toll-like receptor 2 (TLR2) and interferon-gamma (IFN-γ) coordinate with a diverse array of cellular programs through the transcriptional regulation of immunologically relevant genes and play an important role in immune system, reproductive physiology and basic pathology. Alterations in the functions of TLR2 2258G (guanine)/ A, IFN-γ (+874T/A) and signalling molecules that result from polymorphisms are often associated with susceptibility or resistance, which may, in turn, establish the innate host response to various infectious diseases. Presently, we proposed to investigate the risk of common single nucleotide polymorphism (SNP) of TLR2 and IFN-γ genes, for their effect on infertility in women with female genital tuberculosis (FGTB) and healthy women as controls. Methodology/Principal Findings Genotyping of TLR2 and IFN-γ gene polymorphisms was performed by amplification refractory mutation system multi-gene/multi-primer polymerase chain reaction followed by restriction fragment length polymorphism in 175 FGTB patients and 100 healthy control women (HCW). The TLR2 polymorphism [adenine (A) allele] was observed in 57.7 and 58.0% of FGTB patients and HCW, respectively. The IFN-γ (+874T/A) polymorphism (A allele) was significant in 74.3 and 71.0% of FGTB patients and HCW, respectively, while the odds ratios for the AA and TA genotypes for predisposition of FGTB were found to be 0.304 and 1.650 in HCW, respectively. The SNP of TLR2 was not associated with FGTB but the SNP of IFN-γ was found to be associated with mycobacteria infections and to induce infertility. Conclusions/Significance At present, we hypothesize that infertile women with FGTB and HCW without tuberculosis (TB) have identical frequency of TLR variants, which may be adequate in the production of IFN-γ in response to Mycobacterium tuberculosis infections. Thus, the study appears to be the first of its kind reporting a mutation in the IFN-γ gene [+874 T (thymine) to A] responsible for

  10. TLR5, a novel mediator of innate immunity-induced osteoclastogenesis and bone loss.

    PubMed

    Kassem, Ali; Henning, Petra; Kindlund, Bert; Lindholm, Catharina; Lerner, Ulf H

    2015-11-01

    Accumulating evidence points to the importance of the innate immune system in inflammation-induced bone loss in infectious and autoimmune diseases. TLRs are well known for being activated by ligands expressed by bacteria, viruses, and fungi. Recent findings indicate that also endogenous ligands in inflammatory processes are important, one being a TLR5 agonist present in synovial fluid from patients with rheumatoid arthritis (RA). We found that activation of TLR5 by its specific ligand, flagellin, caused robust osteoclast formation and bone loss in cultured mouse neonatal parietal bones dependent on increased receptor activator of NF-κB ligand (RANKL):osteoprotegerin ratio, with half-maximal stimulation at 0.01 μg/ml. Flagellin enhanced Rankl mRNA in isolated osteoblasts by a myeloid differentiation primary response gene 88 and NF-κB-dependent mechanism. Injection of flagellin locally over skull bones in 5-wk-old mice resulted in increased mRNA expression of Rankl and osteoclastic genes, robust osteoclast formation, and bone loss. The effects in vitro and in vivo were absent in Tlr5(-/-) mice. These data show that TLR5 is a novel activator of RANKL and osteoclast formation and, therefore, a potential key factor in inflammation-induced bone erosions in diseases like RA, reactive arthritis, and periodontitis. TLR5 might be a promising novel treatment target for prevention of inflammatory bone loss. PMID:26207027

  11. Widespread ectopic expression of olfactory receptor genes

    PubMed Central

    Feldmesser, Ester; Olender, Tsviya; Khen, Miriam; Yanai, Itai; Ophir, Ron; Lancet, Doron

    2006-01-01

    Background Olfactory receptors (ORs) are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information. PMID:16716209

  12. TLR9 and IRF3 Cooperate to Induce a Systemic Inflammatory Response in Mice Injected With Liposome:DNA

    PubMed Central

    Walker, Wendy E; Booth, Carmen J; Goldstein, Daniel R

    2010-01-01

    Liposome:DNA is a promising gene therapy vector. However, this vector can elicit a systemic inflammatory response syndrome (SIRS). Prior reports indicate that liposome:DNA vectors activate Toll-like receptor (TLR)9. We hypothesized that liposome:DNA vectors also activate the cytosolic DNA-sensing pathway, which signals via interferon (IFN) regulatory factor (IRF)3. To test this, we treated dendritic cells (DCs) with liposome:DNA in vitro and found that IRF3 was phosphorylated independent of TLR9. To test the contribution of this pathway in vivo, we injected a liposome:DNA vector into wild-type (WT), TLR9-knockout (KO), IRF3-KO, and TLR9-IRF3-double-KO (DKO) mice. WT mice exhibited a systemic inflammatory response, evidenced by elevations in serum cytokines, serum enzyme changes indicating organ damage, hypothermia, and mortality. The cytokine response was reduced in TLR9-KO, IRF3-KO, and TLR9-IRF3-DKO mice and all three groups survived. We found that IFN-γ-KO mice that receive liposome:DNA had a reduced cytokine response and 100% survival. CD11c+ and NK1.1+ cells produced IFN-γ and depleting CD11c+ cells reduced the cytokine response in mice injected with liposome:DNA. These findings may facilitate the development of immunologically inert gene therapy vectors and may provide general insight into the mechanisms of SIRS. PMID:20145605

  13. Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis

    PubMed Central

    Abdollahi-Roodsaz, Shahla; Joosten, Leo A.B.; Koenders, Marije I.; Devesa, Isabel; Roelofs, Mieke F.; Radstake, Timothy R.D.J.; Heuvelmans-Jacobs, Marleen; Akira, Shizuo; Nicklin, Martin J.H.; Ribeiro-Dias, Fátima; van den Berg, Wim B.

    2007-01-01

    TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist–knockout (IL1rn–/–) mice, which spontaneously develop an autoimmune T cell–mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn–/–Tlr2–/– mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-γ production by T cells. IL1rn–/–Tlr4–/– mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis. PMID:18060042

  14. Toll-like receptor 8 deletion accelerates autoimmunity in a mouse model of lupus through a Toll-like receptor 7-dependent mechanism

    PubMed Central

    Tran, Ngoc Lan; Manzin-Lorenzi, Céline; Santiago-Raber, Marie-Laure

    2015-01-01

    Systemic lupus erythematosus is an autoimmune disorder characterized by increased levels of lymphocyte activation, antigen presentation by dendritic cells, and the formation of autoantibodies. This leads to immune complex-mediated glomerulonephritis. Toll-like receptor 7 (T7) and TLR9 localize to the endosomal compartment and play important roles in the generation of autoantibodies against nuclear components, as they recognize RNA and DNA, respectively. In contrast, very little is known about endogenous TLR8 activation in mice. We therefore tested whether TLR8 could affect autoimmune responses in a murine model of lupus. We introduced a Tlr8 null mutation into C57BL/6 mice congenic for the Nba2 (NZB autoimmunity 2) locus and bearing the Yaa (Y-linked autoimmune acceleration) mutation containing a tlr8 duplicated gene, and monitored disease development, autoantibody production, and glomerulonephritis-associated mortality. Cellular responses were investigated in female Nba2.TLR8−/− mice bearing no copy of tlr8. The TLR8 deficiency accelerated disease progression and mortality, increased the number of circulating antibodies and activated monocytes, and heightened cellular responses to TLR7 ligation. TLR8-deficient antigen-presenting cells exhibited increased levels of MHC class II expression. The ability of dendritic cells to present antigens to allogeneic T cells after TLR7 ligation was also improved by TLR8 deficiency. TLR8 deletion accelerated autoimmunity in lupus-prone mice in response to TLR7 activation. Antigen-presenting cell function seemed to play a key role in mediating the effects of TLR8 deficiency. PMID:25424423

  15. Selective Toll-Like Receptor Expression in Human Fetal Lung

    PubMed Central

    Petrikin, Joshua E; Gaedigk, Roger; Leeder, J Steven; Truog, William E

    2010-01-01

    Toll-like receptors (TLRs) are critical components of the innate immune system, acting as pattern recognition molecules and triggering an inflammatory response. TLR associated gene products are of interest in modulating inflammatory related pulmonary diseases of the neonate. The ontogeny of TLR related genes in human fetal lung has not been previously described and could elucidate additional functions and identify strategies for attenuating the effects of fetal inflammation. We examined the expression of 84 TLR related genes on 23 human fetal lung samples from three groups with estimated ages of 60 (57-59d), 90 (89-91d), and 130 (117-154d) days. Using a false detection rate algorithm, we identified 32 genes displaying developmental regulation with TLR2 having the greatest up-regulation of TLR genes (9.2 fold increase) and TLR4 unchanged. We confirmed the TLR2 up-regulation by examining an additional 133 fetal lung tissue samples with a fluorogenic polymerase chain reaction assay (TaqMan®) and found an exponential best-fit curve over the time studied. The best-fit curve predicts a 6.1 fold increase from 60d to 130d. We conclude that TLR2 is developmentally expressed from the early pseudoglandular stage of lung development to the canalicular stage. PMID:20581745

  16. Association of NOD1, CXCL16, STAT6 and TLR4 gene polymorphisms with Malaysian patients with Crohn’s disease

    PubMed Central

    Chua, Kek Heng; Ng, Jin Guan; Ng, Ching Ching; Hilmi, Ida; Goh, Khean Lee

    2016-01-01

    Crohn’s disease (CD) is a prominent type of inflammatory bowel disease (IBD) that can affect any part of the gastrointestinal tract. CD is known to have higher prevalence in the Western countries, but the number of cases has been increasing in the past decades in Asia, including Malaysia. Therefore, there is a need to investigate the underlining causes of CD that may shed light on its prevention and treatment. In this study, genetic polymorphisms in NOD1 (rs2075820), CXCL16 (rs2277680), STAT6 (rs324015) and TLR4 (rs4986791) genes were examined in a total of 335 individuals (85 CD patients and 250 healthy controls) with PCR-RFLP approach. There was no significant association observed between NOD1 rs2075820 and STAT6 rs324015 with the onset of CD in the studied cohort. However, the G allele of CXCL16 rs2277680 was found to have a weak association with CD patients (P = 0.0482; OR = 1.4310). The TLR4 rs4986791 was also significantly associated to CD. Both the homozygous C genotype (P = 0.0029; OR = 0.3611) and C allele (P = 0.0069; OR = 0.4369) were observed to confer protection against CD. On the other hand, the heterozygous C/T genotype was a risk genotype (P = 0.0015; OR = 3.1392). Further ethnic-stratified analysis showed that the significant associations in CXCL16 rs2277680 and TLR4 rs4986791 were accounted by the Malay cohort. In conclusion, the present study reported two CD-predisposing loci in the Malay CD patients. However, these loci were not associated to the onset of CD in Chinese and Indian patients. PMID:27069792

  17. C-reactive protein gene and Toll-like receptor 4 gene polymorphisms can relate to the development of psoriatic arthritis.

    PubMed

    Akbal, Ayla; Oğuz, Sevilay; Gökmen, Ferhat; Bilim, Serhat; Reşorlu, Hatice; Sılan, Fatma; Uludağ, Ahmet

    2015-02-01

    We aimed to determine in psoriatic arthritis (PsA) patients the Toll-like receptor (TLR) 4 and C-reactive gene (CRP) polymorphisms and allele frequency and to investigate the relationship between clinical parameters and gene polymorphisms. We enrolled in this study 31 PsA and 41 healthy control subjects. PsA diagnosis was according to CASPAR criteria. Bath ankylosing spondylitis diseases activity index, Maastricht ankylosing spondylitis enthesitis score, and Bath ankylosing spondylitis functional index were measured. C, A, and T alleles of CRP and A and G alleles of TLR 4 were determined using the analysis of melting curves after real-time PCR. CRP A, C, and T allele frequency in controls was 26.8, 73.2, and 36.6%, respectively. In the PsA patient group, A, C, and T allele frequency was 9.7, 87.1, and 12.9%, respectively. Between control and PsA groups, there was a significant difference in A, C, and T allele frequency (P = 0.008, 0.038, and 0.001, respectively). The frequency of CRP gene polymorphisms (CA, AA, CT, TA, and TT alleles) in the control group was 56.1% and in the PsA group was 22.6%. There was a significant difference between the two groups (P = 0.004). The absence of a CRP gene polymorphism was a risk factor for PsA (odds ratio 4.3, 95% CI; 1.5-12.4, P = 0.005). TLR gene haploid frequency was investigated, and all control subjects had the wild-type AA allele. PsA patient GA allele frequency was 6.5%. There was no significant difference between the two groups (P = 0.182). GA mutant allele frequency was related to PsA (odds ratio 7.03, 95% CI; 0.32-151.9, P = 0.214). We have shown that CRP gene polymorphisms are higher in control subjects than PsA patients, and TLR 4 gene polymorphisms were found to be related to PsA. PMID:24696367

  18. Toll-Like Receptor 4 Mutant and Null Mice Retain Morphine-Induced Tolerance, Hyperalgesia, and Physical Dependence

    PubMed Central

    Mattioli, Theresa Alexandra; Leduc-Pessah, Heather; Skelhorne-Gross, Graham; Nicol, Christopher J. B.; Milne, Brian; Trang, Tuan; Cahill, Catherine M.

    2014-01-01

    The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (−) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence. PMID:24824631

  19. The influence of genetic polymorphisms in TLR4 and TIRAP, and their expression levels in peripheral blood, on susceptibility to sepsis

    PubMed Central

    ZHANG, JIANPING; YANG, JINGPING; XU, XIYUAN; LIANG, LIANGSHEN; SUN, HAIXIA; LIU, GUOHUA; ZHANG, LIHONG; SU, YUN

    2016-01-01

    The present study aimed to investigate whether genetic polymorphisms in the Toll-like receptor (TLR)-4 and Toll/interleukin-1 receptor (TIR)-associated protein (TIRAP) genes, and/or their expression levels, influence the susceptibility of a patient to sepsis. A total of 106 patients with sepsis were divided into two groups on the basis of their acute physiology and chronic health evaluation (APACHE) II scores: i) Sepsis group A (APACHE II <20) and ii) Sepsis group B (APACHE II >20). In addition, 100 healthy volunteers were enrolled into the control group. Polymerase chain reaction-restriction fragment length polymorphism assay was used to detect the following genetic polymorphisms: The Ser180Leu allele of the TIRAP gene and the Asp299Gly and Thr399I1e alleles of the TLR4 gene. Furthermore, the protein expression levels of TLR4 and TIRAP were analyzed using an enzyme-linked immunosorbent assay. Genetic polymorphisms were not detected for the TLR4 and TIRAP genes; however, the protein expression levels of TLR4 and TIRAP differed significantly between the control, sepsis A and sepsis B groups (P<0.01). An APACHE II score of 20 was used as a baseline in order to differentiate sepsis severity. Pearson analysis demonstrated that TLR4 and TIRAP protein expression levels were positively correlated with sepsis severity (r=0.931 and 0.972; P<0.05), and TLR4 protein expression levels were positively correlated with those of TIRAP (r=0.936; P<0.05). The results of the present study suggested that the protein expression levels of, but not genetic polymorphisms in, TLR4 and TIRAP were associated with the severity of sepsis. PMID:26889229

  20. IL-1R/TLR2 through MyD88 Divergently Modulates Osteoclastogenesis through Regulation of Nuclear Factor of Activated T Cells c1 (NFATc1) and B Lymphocyte-induced Maturation Protein-1 (Blimp1).

    PubMed

    Chen, Zhihong; Su, Lingkai; Xu, Qingan; Katz, Jenny; Michalek, Suzanne M; Fan, Mingwen; Feng, Xu; Zhang, Ping

    2015-12-11

    Toll-like receptors (TLR) and the receptor for interleukin-1 (IL-1R) signaling play an important role in bacteria-mediated bone loss diseases including periodontitis, rheumatoid arthritis, and osteomyelitis. Recent studies have shown that TLR ligands inhibit the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from un-committed osteoclast precursors, whereas IL-1 potentiates RANKL-induced osteoclast formation. However, IL-1R and TLR belong to the same IL-1R/TLR superfamily, and activate similar intracellular signaling pathways. Here, we investigate the molecular mechanisms underlying the distinct effects of IL-1 and Porphyromonas gingivalis lipopolysaccharide (LPS-PG) on RANKL-induced osteoclast formation. Our results show that LPS-PG and IL-1 differentially regulate RANKL-induced activation of osteoclast genes encoding Car2, Ctsk, MMP9, and TRAP, as well as expression of NFATc1, a master transcription factor of osteoclastogenesis. Regulation of osteoclast genes and NFATc1 by LPS-PG and IL-1 is dependent on MyD88, an important signaling adaptor for both TLR and IL-1R family members. Furthermore, LPS-PG and IL-1 differentially regulate RANKL-costimulatory receptor OSCAR (osteoclast-associated receptor) expression and Ca(2+) oscillations induced by RANKL. Moreover, LPS-PG completely abrogates RANKL-induced gene expression of B lymphocyte-induced maturation protein-1 (Blimp1), a global transcriptional repressor of anti-osteoclastogenic genes encoding Bcl6, IRF8, and MafB. However, IL-1 enhances RANKL-induced blimp1 gene expression but suppresses the gene expression of bcl6, irf8, and mafb. Our study reveals the involvement of multiple signaling molecules in the differential regulation of RANKL-induced osteoclastogenesis by TLR2 and IL-1 signaling. Understanding the signaling cross-talk among TLR, IL-1R, and RANK is critical for identifying therapeutic strategies to control bacteria-mediated bone loss. PMID:26483549

  1. TLR-signaling Networks

    PubMed Central

    Brown, J.; Wang, H.; Hajishengallis, G.N.; Martin, M.

    2011-01-01

    Toll-like receptors play a critical role in innate immunity by detecting invading pathogens. The ability of TLRs to engage different intracellular signaling molecules and cross-talk with other regulatory pathways is an important factor in shaping the type, magnitude, and duration of the inflammatory response. The present review will cover the fundamental signaling pathways utilized by TLRs and how these pathways regulate the innate immune response to pathogens. Abbreviations: TLR, Toll-like receptor; PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; LPS, lipopolysaccharide; APC, antigen-presenting cell; IL, interleukin; TIR, Toll/IL-1R homology; MyD88, myeloid differentiation factor 88; IFN, interferon; TRIF, TIR-domain-containing adapter-inducing interferon-β; IRAK, IL-1R-associated kinase; TAK1, TGF-β-activated kinase; TAB1, TAK1-binding protein; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B-cells; MAPK, mitogen-activated protein kinase; NLR, NOD-like receptors; LRR, leucine-rich repeats; DC, dendritic cell; PI3K, phosphoinositide 3-kinases; GSK3, glycogen synthase kinase-3; mTOR, mammalian target of rapamycin; DAF, decay-accelerating factor; IKK, IκB kinase; IRF, interferon regulatory factors; TBK1, TANK-binding kinase 1; CARD, caspase activation and recruitment domain; PYD, pyrin N-terminal homology domain; ATF, activating transcription factor; and PTEN, phosphatase and tensin homolog. PMID:20940366

  2. Toll-Like Receptor 4 Signaling Augments Transforming Growth Factor-β Responses

    PubMed Central

    Bhattacharyya, Swati; Kelley, Kathleen; Melichian, Denisa S.; Tamaki, Zenshiro; Fang, Feng; Su, Yunyun; Feng, Gilbert; Pope, Richard M.; Budinger, G.R. Scott; Mutlu, Gökhan M.; Lafyatis, Robert; Radstake, Timothy; Feghali-Bostwick, Carol; Varga, John

    2014-01-01

    Because recent studies implicate Toll-like receptors (TLRs) in the pathogenesis of fibrosis, we sought to investigate the in vitro and in vivo role and mechanism of TLR4-mediated fibroblast responses in fibrogenesis. We found that TLR4 was constitutively expressed, and accumulation of endogenous TLR4 ligands significantly elevated, in lesional skin and lung tissues from patients with scleroderma. Activation of TLR4 signaling in explanted fibroblasts resulted in enhanced collagen synthesis and increased expression of multiple genes involved in tissue remodeling and extracellular matrix homeostasis. Moreover, TLR4 dramatically enhanced the sensitivity of fibroblasts to the stimulatory effect of transforming growth factor-β1. These profibrotic responses were abrogated by both genetic and pharmacological disruption of TLR4 signaling in vitro, and skin fibrosis induced by bleomycin in vivo was attenuated in mice harboring a mutated TLR4. Activation of TLR4 in fibroblasts augmented the intensity of canonical Smad signaling, and was accompanied by suppression of anti-fibrotic microRNA expression. Together, these results suggest a novel model to account for persistent fibrogenesis in scleroderma, in which activation of fibroblast TLR4 signaling, triggered by damage-associated endogenous TLR4 ligands, results in augmented transforming growth factor-β1 sensitivity with increased matrix production and progressive connective tissue remodeling. Under these conditions, fibroblast TLR4 serves as the switch for converting self-limited tissue repair into intractable fibrosis. PMID:23141927

  3. Different dimerisation mode for TLR4 upon endosomal acidification?

    PubMed Central

    Gangloff, Monique

    2012-01-01

    TLR4 is unique among pathogen-recognition receptors in that it initiates different pathways in different cellular locations. Binding of a bridging factor, Mal, allows recruitment of an adapter protein, MyD88, at the plasma membrane, which leads to the production of proinflammatory cytokines. Upon internalization, TLR4 uses a different bridging factor, TRAM, to activate a MyD88-independent pathway that results in type I interferon expression. Interestingly, both Mal and TRAM are localised initially at the plasma membrane. In this Opinion, I suggest a possible mechanism by which endosomal acidification triggers the differential adaptor usage of TLR4. I discuss the evidence of the pH sensitivity of TLR4 and propose a new dimerisation mode for TLR4 based on the crystal structure of the related receptor TLR3 bound to its ligand, double-stranded RNA. PMID:22196451

  4. Different dimerisation mode for TLR4 upon endosomal acidification?

    PubMed

    Gangloff, Monique

    2012-03-01

    TLR4 is unique among pathogen-recognition receptors in that it initiates different pathways in different cellular locations. Binding of a bridging factor, Mal, allows recruitment of an adapter protein, MyD88, at the plasma membrane, which leads to the production of proinflammatory cytokines. Upon internalization, TLR4 uses a different bridging factor, TRAM, to activate a MyD88-independent pathway that results in type I interferon expression. Interestingly, both Mal and TRAM are localised initially at the plasma membrane. In this Opinion, I suggest a possible mechanism by which endosomal acidification triggers the differential adaptor usage of TLR4. I discuss the evidence of the pH sensitivity of TLR4 and propose a new dimerisation mode for TLR4 based on the crystal structure of the related receptor TLR3 bound to its ligand, double-stranded RNA. PMID:22196451

  5. Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation

    PubMed Central

    Boehm, Olaf; El Aissati, Sakina; Foltz, Fabian; Goelz, Lina; Goertz, David; Kebir, Sied; Weisheit, Christina; Wolf, Michael; Meyer, Rainer; Baumgarten, Georg

    2015-01-01

    Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4-/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4-/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4-/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory

  6. Unilateral Partial Nephrectomy with Warm Ischemia Results in Acute Hypoxia Inducible Factor 1-Alpha (HIF-1α) and Toll-Like Receptor 4 (TLR4) Overexpression in a Porcine Model

    PubMed Central

    Zhang, Zhiyong; Haimovich, Beatrice; Kwon, Young Suk; Lu, Tyler; Fyfe-Kirschner, Billie; Olweny, Ephrem Odoy

    2016-01-01

    Purpose Ischemia/reperfusion (I/R) during partial nephrectomy (PN) contributes to acute kidney injury (AKI), which is inaccurately assessed using existent clinical markers of renal function. We evaluated I/R-related changes in expression in hypoxia inducible factor 1α (HIF-1α) and toll-like receptor 4 (TLR4), within kidney tissue and peripheral blood leukocytes (PBL) in a porcine model of PN. Materials and Methods Three adult pigs each underwent unilateral renal hilar cross clamping for 180 min followed by a 15 min reperfusion. The contralateral kidney served as control. Biopsies of clamped kidneys were obtained at baseline (time 0), every 60 min during the hypoxic phase, and post-reperfusion. Control kidneys were biopsied once at 180 min. Peripheral blood was sampled at time 0, every 30 min during the hypoxic phase, and post-reperfusion. HIF-1α and TLR4 expression in kidney tissue and PBL were analyzed by Western blotting. I/R-related histological changes were assessed. Results Expression of HIF-1α in clamped kidneys and PBL was below detection level at baseline, rising to detectable levels after 60 min of hypoxia, and continuing to rise throughout the hypoxic and reperfusion phases. Expression of TLR-4 in clamped kidneys followed a similar trend with initial detection after 30–60 min of hypoxia. Control kidneys exhibited no change in HIF-1α or TLR-4 expression. I/R-related histologic changes were minimal, primarily mild tubular dilatation. Conclusions In a porcine model of PN, HIF-1α and TLR4 exhibited robust, I/R-related increases in expression in kidney tissue and PBL. Further studies investigating these molecules as potential markers of AKI are warranted. PMID:27149666

  7. A coding IRAK2 protein variant compromises Toll-like receptor (TLR) signaling and is associated with colorectal cancer survival.

    PubMed

    Wang, Hui; Flannery, Sinead M; Dickhöfer, Sabine; Huhn, Stefanie; George, Julie; Kubarenko, Andriy V; Lascorz, Jesus; Bevier, Melanie; Willemsen, Joschka; Pichulik, Tica; Schafmayer, Clemens; Binder, Marco; Manoury, Bénédicte; Paludan, Søren R; Alarcon-Riquelme, Marta; Bowie, Andrew G; Försti, Asta; Weber, Alexander N R

    2014-08-15

    Within innate immune signaling pathways, interleukin-1 receptor-associated kinases (IRAKs) fulfill key roles downstream of multiple Toll-like receptors and the interleukin-1 receptor. Although human IRAK4 deficiency was shown to lead to severe immunodeficiency in response to pyogenic bacterial infection during childhood, little is known about the role of human IRAK2. We here identified a non-synonymous IRAK2 variant, rs35060588 (coding R214G), as hypofunctional in terms of NF-κB signaling and Toll-like receptor-mediated cytokine induction. This was due to reduced ubiquitination of TRAF6, a key step in signal transduction. IRAK2 rs35060588 occurs in 3-9% of individuals in different ethnic groups, and our studies suggested a genetic association of rs35060588 with colorectal cancer survival. This for the first time implicates human IRAK2 in a human disease and highlights the R214G IRAK2 variant as a potential novel and broadly applicable biomarker for disease or as a therapeutic intervention point. PMID:24973222

  8. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

    PubMed

    Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

    2015-11-16

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. PMID:26481685

  9. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis

    PubMed Central

    Zambirinis, Constantinos P.; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H.; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D.; Tuveson, David

    2015-01-01

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. PMID:26481685

  10. TLR9 signaling through NF-κB/RELA and STAT3 promotes tumor-propagating potential of prostate cancer cells

    PubMed Central

    Moreira, Dayson; Zhang, Qifang; Hossain, Dewan Md S.; Nechaev, Sergey; Li, Haiqing; Kowolik, Claudia M.; D'Apuzzo, Massimo; Forman, Stephen; Jones, Jeremy; Pal, Sumanta K.; Kortylewski, Marcin

    2015-01-01

    Prostate cancer progression was associated with tumorigenic signaling activated by proinflammatory mediators. However, the etiology of these events remains elusive. Here, we demonstrate that triggering of the innate immune receptor, Toll-like Receptor 9 (TLR9), in androgen-independent prostate cancer cells initiates signaling cascade leading to increased tumor growth and progression. Using limited dilution/serial transplantation experiments, we show that TLR9 is essential for prostate cancer cells' potential to propagate and self-renew in vivo. Furthermore, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of LNCaP- and PC3-derived prostate cancer cell variants. Genome-wide transcriptional analysis of prostate cancer cells isolated from xenotransplanted TLR9-positive and -negative tumors revealed a unique gene expression signature, with prominent upregulation of inflammation- and stem cell-related markers. TLR9 signaling orchestrated expression of critical stem cell-related genes such as NKX3.1, KLF-4, BMI-1 and COL1A1, at both mRNA and protein levels. Our further analysis identified that TLR9-induced NF-κB/RELA and STAT3 transcription factors co-regulated NKX3.1 and KLF4 gene expression by directly binding to both promoters. Finally, we demonstrated the feasibility of using TLR9-targeted siRNA delivery to block RELA- and STAT3-dependent prostate cancer cell self-renewal in vivo. The intratumoral administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates inhibited growth of established prostate tumors and reduced clonogenic potential of cancer cells. Overcoming cancer cell self-renewal and tumor-propagating potential by targeted inhibition of TLR9 signaling can provide therapeutic strategy for late-stage prostate cancer patients. PMID:26046794

  11. TLR9 signaling through NF-κB/RELA and STAT3 promotes tumor-propagating potential of prostate cancer cells.

    PubMed

    Moreira, Dayson; Zhang, Qifang; Hossain, Dewan Md S; Nechaev, Sergey; Li, Haiqing; Kowolik, Claudia M; D'Apuzzo, Massimo; Forman, Stephen; Jones, Jeremy; Pal, Sumanta K; Kortylewski, Marcin

    2015-07-10

    Prostate cancer progression was associated with tumorigenic signaling activated by proinflammatory mediators. However, the etiology of these events remains elusive. Here, we demonstrate that triggering of the innate immune receptor, Toll-like Receptor 9 (TLR9), in androgen-independent prostate cancer cells initiates signaling cascade leading to increased tumor growth and progression. Using limited dilution/serial transplantation experiments, we show that TLR9 is essential for prostate cancer cells' potential to propagate and self-renew in vivo. Furthermore, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of LNCaP- and PC3-derived prostate cancer cell variants. Genome-wide transcriptional analysis of prostate cancer cells isolated from xenotransplanted TLR9-positive and -negative tumors revealed a unique gene expression signature, with prominent upregulation of inflammation- and stem cell-related markers. TLR9 signaling orchestrated expression of critical stem cell-related genes such as NKX3.1, KLF-4, BMI-1 and COL1A1, at both mRNA and protein levels. Our further analysis identified that TLR9-induced NF-κB/RELA and STAT3 transcription factors co-regulated NKX3.1 and KLF4 gene expression by directly binding to both promoters. Finally, we demonstrated the feasibility of using TLR9-targeted siRNA delivery to block RELA- and STAT3-dependent prostate cancer cell self-renewal in vivo. The intratumoral administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates inhibited growth of established prostate tumors and reduced clonogenic potential of cancer cells. Overcoming cancer cell self-renewal and tumor-propagating potential by targeted inhibition of TLR9 signaling can provide therapeutic strategy for late-stage prostate cancer patients. PMID:26046794

  12. Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection

    PubMed Central

    Chen, Jincheng; Ng, Mary Mah-Lee; Chu, Justin Jang Hann

    2015-01-01

    Dengue virus (DV) infection is the most prevalent mosquito-borne viral disease and its manifestation has been shown to be contributed in part by the host immune responses. In this study, pathogen recognition receptors, Toll-like receptor (TLR) 2 and TLR6 were found to be up-regulated in DV-infected human PBMC using immunofluorescence staining, flow cytometry and Western blot analyses. Using ELISA, IL-6 and TNF-α, cytokines downstream of TLR2 and TLR6 signaling pathways were also found to be up-regulated in DV-infected PBMC. IL-6 and TNF-α production by PBMC were reduced when TLR2 and TLR6 were blocked using TLR2 and TLR6 neutralizing antibodies during DV infection. These results suggested that signaling pathways of TLR2 and TLR6 were activated during DV infection and its activation contributed to IL-6 and TNF-α production. DV NS1 protein was found to significantly increase the production of IL-6 and TNF-α when added to PBMC. The amount of IL-6 and TNF-α stimulated by DV NS1 protein was reduced when TLR2 and TLR6 were blocked, suggesting that DV NS1 protein is the viral protein responsible for the activation of TLR2 and TLR6 during DV infection. Secreted alkaline phosphatase (SEAP) reporter assay was used to further confirm activation of TLR2 and TLR6 by DV NS1 protein. In addition, DV-infected and DV NS1 protein-treated TLR6-/- mice have higher survivability compared to DV-infected and DV NS1 protein-treated wild-type mice. Hence, activation of TLR6 via DV NS1 protein could potentially play an important role in the immunopathogenesis of DV infection. PMID:26226614

  13. TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

    PubMed Central

    Paradowska, Edyta; Jabłońska, Agnieszka; Studzińska, Mirosława; Skowrońska, Katarzyna; Suski, Patrycja; Wiśniewska-Ligier, Małgorzata; Woźniakowska-Gęsicka, Teresa; Nowakowska, Dorota; Gaj, Zuzanna; Wilczyński, Jan; Leśnikowski, Zbigniew J.

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease. PMID:27105145

  14. Blood-stage malaria of Plasmodium chabaudi induces differential Tlr expression in the liver of susceptible and vaccination-protected Balb/c mice.

    PubMed

    Al-Quraishy, Saleh; Dkhil, Mohamed A; Alomar, Suliman; Abdel-Baki, Abdel Azeem S; Delic, Denis; Wunderlich, Frank; Araúzo-Bravo, Marcos J

    2016-05-01

    Protective vaccination induces self-healing of otherwise lethal blood-stage infections of Plasmodium chabaudi malaria. Here, we investigate mRNA expression patterns of all 12 members of the Toll-like receptor (Tlr) gene family in the liver, a major effector organ against blood-stage malaria, during lethal and vaccination-induced self-healing infections of P. chabaudi in female Balb/c mice. Gene expression microarrays reveal that all 12 Tlr genes are constitutively expressed, though at varying levels, and specifically respond to infection. Protective vaccination does not affect constitutive expression of any of the 12 Tlr genes but leads to differential expression (p < 0.05) of seven Tlrs (1, 2, 4, 7, 8, 12, and 13) in response to malaria. Quantitative PCR substantiates differential expression at p < 0.01. There is an increased expression of Tlr2 by approximately five-fold on day 1 post-infection (p.i.) and Tlr1 by approximately threefold on day 4 p.i.. At peak parasitemia on day 8 p.i., none of the 12 Tlrs display any differential expression. After peak parasitemia, towards the end of the crisis phase on day 11 p.i., expression of Tlrs 1, 4, and 12 is increased by approximately four-, two-, and three-fold, respectively, and that of Tlr7 is decreased by approximately two-fold. Collectively, our data suggest that though all 12 members of the Tlr gene family are specifically responsive to malaria in the liver, not only Tlr2 at the early stage of infection but also the Tlrs 1, 4, 7, and 12 towards the end of crisis phase are critical for vaccination-induced resolution and survival of otherwise lethal blood-stage malaria. PMID:26809341

  15. IFNα enhances the production of IL-6 by human neutrophils activated via TLR8

    PubMed Central

    Zimmermann, Maili; Arruda-Silva, Fabio; Bianchetto-Aguilera, Francisco; Finotti, Giulia; Calzetti, Federica; Scapini, Patrizia; Lunardi, Claudio; Cassatella, Marco A.; Tamassia, Nicola

    2016-01-01

    Recently, we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. In this study, we demonstrate that IFNα, a cytokine that modulates the early innate immune responses toward viral and bacterial infections, potently enhances the production of IL-6 in neutrophils stimulated with R848, a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848, but, rather, it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine, in an autocrine manner, leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPβ transcription factor to the IL-6 promoter. Moreover, we show that neutrophils from SLE patients with active disease state, hence displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether, data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils, which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases. PMID:26790609

  16. TLR1 Variant H305L Associated with Protection from Pulmonary Tuberculosis

    PubMed Central

    Meyer, Christian G.; Reiling, Norbert; Ehmen, Christa; Ruge, Gerd; Owusu-Dabo, Ellis; Horstmann, Rolf D.; Thye, Thorsten

    2016-01-01

    Toll like receptors (TLR) are key elements of the innate immune response and involved in the recognition of pathogens. To test common and rare TLR variants involved in susceptibility or resistance to infection with Mycobacterium tuberculosis we screened the exons of the genes encoding TLR 1, 2, 4, and the adaptor molecule TIRAP in more than 4500 tuberculosis (TB) cases and controls from Ghana. The analysis yielded 109 variants with possible functional impact, including 101 non-synonymous variants, three stop-variants, and five indels. Association analyses yielded a significant result for the TLR1 variant rs3923647, conferring strong protection against TB (Odds ratio [OR] 0.21, CI confidence interval [CI] 0.05–0.6, Pnominal 1 x 10−3) when applying a recessive model of inheritance. Replication analyses with an additional 3370 Ghanaian cases and control samples, and with data from a recent TB study of 533 African-Americans confirmed the protective effect and resulted in a combined OR of 0.19, with a nominal P value of 2.2 x 10−5, and a corrected P value of 4.1 x 10−4. The SNP is located near the binding pocket of TLR1 and causes an amino acid exchange from histidine to leucine at position 305. The observed effect may, therefore, be attributable to structural changes in the recognition site of the TLR1 molecule, allowing to bind those mycobacterial ligands which preferentially may induce a protective immune response. This is supported by the analysis of BCG-stimulated peripheral blood mononuclear cells, showing increased induction of the proinflammatory cytokine IFN-γ in carriers of the mutant TLR1 rs3923647 TT genotype, compared to the IFN-γ levels of individuals with the AT and AA genotypes. PMID:27214039

  17. Distinct TLR-mediated cytokine production and immunoglobulin secretion in human newborn naïve B cells.

    PubMed

    Pettengill, Matthew A; van Haren, Simon D; Li, Ning; Dowling, David J; Bergelson, Ilana; Jans, Jop; Ferwerda, Gerben; Levy, Ofer

    2016-08-01

    Neonatal innate immunity is distinct from that of adults, which may contribute to increased susceptibility to infection and limit vaccine responses. B cells play critical roles in protection from infection and detect PAMPs via TLRs, that, when co-activated with CD40, can drive B-cell proliferation and Ab production. We characterized the expression of TLRs in circulating B cells from newborns and adults, and evaluated TLR- and CD40-mediated naïve B-cell class-switch recombination (CSR) and cytokine production. Gene expression levels of most TLRs was similar between newborn and adult B cells, except that newborn naïve B cells expressed more TLR9 than adult naïve B cells. Neonatal naïve B cells demonstrated impaired TLR2- and TLR7- but enhanced TLR9-mediated cytokine production. Significantly fewer newborn naïve B cells underwent CSR to produce IgG, an impairment also noted with IL-21 stimulation. Additionally, co-stimulation via CD40 and TLRs induced greater cytokine production in adult B cells. Thus, while newborn naïve B cells demonstrate adult-level expression of TLRs and CD40, the responses to stimulation of these receptors are distinct. Relatively high expression of TLR9 and impaired CD40-mediated Ig secretion contributes to distinct innate and adaptive immunity of human newborns and may inform novel approaches to early-life immunization. PMID:27252169

  18. Androgen receptor gene polymorphism in zebra species

    PubMed Central

    Ito, Hideyuki; Langenhorst, Tanya; Ogden, Rob; Inoue-Murayama, Miho

    2015-01-01

    Androgen receptor genes (AR) have been found to have associations with reproductive development, behavioral traits, and disorders in humans. However, the influence of similar genetic effects on the behavior of other animals is scarce. We examined the loci AR glutamine repeat (ARQ) in 44 Grevy's zebras, 23 plains zebras, and three mountain zebras, and compared them with those of domesticated horses. We observed polymorphism among zebra species and between zebra and horse. As androgens such as testosterone influence aggressiveness, AR polymorphism among equid species may be associated with differences in levels of aggression and tameness. Our findings indicate that it would be useful to conduct further studies focusing on the potential association between AR and personality traits, and to understand domestication of equid species. PMID:26236645

  19. Molecular characterization and functional analysis of Toll-like receptor 3 gene in orange-spotted grouper (Epinephelus coioides).

    PubMed

    Lin, Kebing; Ge, Hui; Lin, Qi; Wu, Jianshao; He, Libin; Fang, Qiongshan; Zhou, Chen; Sun, Minqiu; Huang, Zhongchi

    2013-09-15

    Toll-like receptor 3 (TLR3) plays an important role in activating innate immune responses during viral infection. In this report, TLR3 (EcTLR3) was characterized and analyzed for the first time in Epinephelus coioides. The full-length EcTLR3 cDNA is predicted to encode a 909 amino acid polypeptide that contains a signal peptide sequence, 18 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time PCR revealed that the EcTLR3 mRNA was much more abundant in the liver than in other immune organs, and that the expression levels were very low in hemocyte and muscle. During development of the grouper, the levels of EcTLR3 transcripts increased with age, with very low expression levels at the early stages of development. EcTLR3 mRNA levels were examined in the liver at different times after treatment with polyriboinosinic polyribocytidylic acid (Poly I:C), and in nervous necrosis virus (NNV)-infected larval groupers. The results suggested that EcTLR3 plays an important role in a fish's defense against viral infection. PMID:23792060

  20. Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages

    PubMed Central

    Bosmann, Markus; Haggadone, Mikel D.; Hemmila, Mark R.; Zetoune, Firas S.; Sarma, J. Vidya; Ward, Peter A.

    2012-01-01

    There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27 (p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. Here, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (Poly I:C) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LPS-binding protein (LBP) completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28) producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)+F4/80+C5aR+ phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor, rather than the C5L2 receptor. C5aR−/− mice after endotoxemia displayed higher plasma levels of IL-27(p28) when compared to C57BL/6J mice. C5a did not affect the release of IL-27(p28) or frequency of IL-27(p28)+F4/80+ macrophages after engagement of TLR3. Mechanistically, LPS activated both NFκB and the PI3K-Akt pathways, whereas C5a activated only the PI3K-Akt pathway. Engagement of PI3K-Akt was inhibitory for IL-27(p28) production, since PI3K-Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K-Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, for TLR3-mediated release of IL-27(p28), the PI3K-Akt pathway was not involved. These data provide new evidence on how complement activation may selectively interfere with production of T cell regulatory cytokines by antigen-presenting cells in the varying contexts of either bacterial (TLR4-pathway) or viral (TLR3-pathway) infection. PMID:22491257

  1. Genetic differences in ChTLR15 gene polymorphism and expression involved in Salmonella enterica natural and artificial infection respectively, of Chinese native chicken breeds, with a focus on sexual dimorphism.

    PubMed

    Hu, Y; Chen, W W; Liu, H X; Shan, Y J; Zhu, C H; Li, H F; Zou, J M

    2016-02-01

    Chicken Toll-like receptor 15 (ChTLR15) has been shown to participate in immune activation in response to various pathogens and in the innate defence against infection. Two genetically distinct Chinese breeds of chicken (Qinyuan Partridge and Baier breeds) were used to study the correlation between ChTLR15 single nucleotide polymorphisms and the natural infection status of salmonella in hens, and also to examine genetic and sex-specific effects on ChTLR15 mRNA expression in heterophils and spleen during acute infection with Salmonella enterica serovar Enteritidis (SE) from 1 to 10 days after experimental infection. Three single-nucleotide polymorphisms (G168A, C726T and A1166G) in a single exon of ChTLR15 were identified in the two breeds, but only C726T showed a significant association with salmonella infection. Compared with layer-type Baier chicks, meat-type Qingyuan chicks showed a higher tolerance for capture stress and (SE) infection, as measured, respectively, by the modified body weight of chicks in the control group and in the infection group. Meanwhile, ChTLR15 down-regulation in heterophils and up-regulation in spleen were involved in the response to pathogenic SE colonization during the acute infection period. These significant genetic effects in females led to greater differences in both innate and adaptive immune responses than those exhibited in males. These results suggest that genetics, time and gender play important roles in the modulation of ChTLR15 mRNA level elicited by the SE-mediated immune response differentially in the two genetically distinct breeds, with a focus on sexual dimorphism. PMID:26488442

  2. TLR7 is required for optimal immune defense against bacterial infection in tongue sole (Cynoglossus semilaevis).

    PubMed

    Li, Xue-peng; Sun, Li

    2015-11-01

    In mammals as well as in teleost, toll-like receptor 7 (TLR7) is known to be involved in antiviral immunity by recognizing viral RNA. However, the antibacterial potential of fish TLR7 is unclear. In this study, we analyzed the TLR7 of tongue sole (Cynoglossus semilaevis), CsTLR7, and examined its potential involvement in antibacterial immunity. CsTLR7 is composed of 1052 amino acid residues and shares 64.0%-75.9% overall sequence identities with known teleost TLR7. CsTLR7 possesses a toll/interleukin-1 receptor domain and six leucine-rich repeats. Constitutive expression of CsTLR7 occurred in relatively high levels in kidney, spleen and liver. Bacterial infection upregulated CsTLR7 expression, whereas viral infection downregulated CsTLR7 expression. Knockdown of CsTLR7 significantly enhanced bacterial dissemination in the tissues of tongue sole. Treatment of tongue sole with the imidazoquinoline compound R848 (TLR7 activator) and the endosomal acidification inhibitor chloroquine (TLR7 inhibitor) caused enhanced and reduced resistance against bacterial infection respectively. These results indicate that CsTLR7 plays an essential role in the antibacterial immunity of tongue sole. PMID:26327112

  3. Alternative TLRs are stimulated by bacterial ligand to induce TLR2-unresponsive colon cell response.

    PubMed

    Mukherjee, Subhadeep; Biswas, Ratna; Biswas, Tapas

    2013-08-01

    Although pathogenic bacteria penetrate colonic cells causing infection, the role of its surface molecules serving as key Toll-like receptor (TLR) ligands and triggering response remains unexplored. We show that TLR2-ligand porin up-regulated TLR4 on HT-29 cells, which the TLR4-ligand LPS could not. TLR1 that co-express with TLR2 got stimulated with TLR4. Besides the two TLRs, MD-2 was expressed revealing that the TLR4 co-receptor is not exclusive for LPS signaling. SARM-1 that mostly down-regulates TLR-signaling, demonstrated central role in signaling by engaging IRF-3 and NF-κB for cell activity. Porin induced type 1 chemokines particularly MCP-3, while porin-stimulated HT-29 culture supernatant displayed PBMC migration, collectively suggesting that the chemokines influence colon and immune cell cross-talk. In TLR2 down-regulated HT-29 cells, we found TLR1 and TLR4 as substitute TLRs to identify porin and orchestrate signaling. Thus, TLR replacement for PAMP recognition demonstrates specificity of ligand·TLR association can compromise and is a necessary alternative for successful execution of immune responses. PMID:23665374

  4. Toll-like receptor activation of XBP1 regulates innate immune responses in macrophages

    PubMed Central

    Martinon, Fabio; Chen, Xi; Lee, Ann-Hwee; Glimcher, Laurie H.

    2011-01-01

    Sensors of pathogens, such as Toll-like receptors (TLRs), detect microbes to activate transcriptional programs that orchestrate adaptive responses to specific insults. Here we report that TLR4 and TLR2 specifically activated the endoplasmic reticulum (ER)-stress sensor kinase IRE1α and its downstream target, the transcription factor XBP1. Previously described XBP1 ER stress target genes were not induced by TLR signaling. Instead, TLR-activated XBP1 was required for optimal and sustained production of proinflammatory cytokines in macrophages. Consistent with this finding, IRE1α activation by ER-stress synergized with TLR activation for cytokine production. Moreover, XBP1 deficiency markedly increased bacterial burden in animals infected with the TLR2-activating human pathogen Francisella tularensis. Our findings uncover an unsuspected critical new function for the XBP1 transcription factor in mammalian host defenses. PMID:20351694

  5. Identification and characterization of three TLR1 subfamily members from the orange-spotted grouper, Epinephelus coioides.

    PubMed

    Li, Yan-Wei; Xu, Dong-Dong; Li, Xia; Mo, Ze-Quan; Luo, Xiao-Chun; Li, An-Xing; Dan, Xue-Ming

    2016-08-01

    Toll-like receptors (TLRs), which play important roles in host defense against pathogen infection, are the most intensively studied pattern recognition receptors (PRRs). In this study, we identified three novel TLR1 subfamily members, including TLR1 (EcTLR1b), TLR2 (EcTLR2b) and TLR14 (EcTLR14), from the orange-spotted grouper (Epinephelus coioides). EcTLR1b and EcTLR2b displayed low sequence identity with the previously reported grouper TLR1 (EcTLR1a) and TLR2 (EcTLR2a), respectively. The open reading frames (ORFs) of EcTLR1b, EcTLR2b and EcTLR14 contain 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively. All three TLRs have leucine-rich repeat (LRR) domains (including an LRR-NT (except for EcTLR1b), several LRR motifs and an LRR-CT), a trans-membrane region and a Toll/interleukin-1 receptor (TIR) domain. The TIR domains of the three TLRs exhibited conserved boxes, namely box1, box2 and box3, and their 3D models were similar to those of human TLR1 or TLR2. Sequence alignment demonstrated that the TIR domains of the three TLRs shared higher sequence identity with those of other species than the full-length receptors. Phylogenetic analysis indicated that EcTLR1s and EcTLR2s are characterized by their differing evolutionary status, whereas EcTLR14 was found to be in the same group as other piscine TLR14/18s. The three TLRs were ubiquitously expressed in seven tested tissues of healthy groupers, although their expression profiles were different. Post Cryptocaryon irritans infection, TLR1s expression was up-regulated in the gills. The expression of TLR2b was mainly increased in the spleen, but decreased in the gills, which was similar to the expression pattern of TLR2a post C. irritans infection. Unlike EcTLR1b and EcTLR2b, however, the grouper TLR14 transcript was substantially induced in both tissues post challenge. These findings may be helpful in understanding the innate immune mechanism of host

  6. Identification of TLR2 and TLR4-induced microRNAs in human mesenchymal stem cells and their possible roles in regulating TLR signals

    PubMed Central

    WANG, XINGBING; ZHU, YUNXIA; XU, BO; WANG, JING; LIU, XIN

    2016-01-01

    Toll-like receptors (TLRs) are expressed in human bone marrow-derived mesenchymal stromal cells (BM-MSCs), and the activation of TLRs is important in proliferation, differentiation, migration and hematopoiesis-supporting functions of BM-MSCs. However, the molecular mechanisms underlying these processes remain to be elucidated. MicroRNAs (miRNAs) are involved in various biological functions by mediating mRNA degradation or inhibiting translation of target genes. The present study aimed to identify whether TLRs regulate the expression of miRNAs in BM-MSCs and elucidate the regulatory roles of miRNAs. Illumina high-throughput sequencing was used to profile miRNAs expressed in BM-MSCs stimulated with TLR2 agonist, PAM3CSK4 (PM) or TLR4 agonist, lipopolysaccharides (LPS). A marked expression change upon PM or LPS treatment was observed for 164 known miRNAs and six novel miRNAs that were identified. The expression of six novel miRNAs and 40 randomly selected known miRNAs was further validated by reverse transcription-quantitative polymerase chain reaction. In addition, bioinformatic methods were used to predict the potential target genes of the abundant known miRNAs. The gene ontology analysis demonstrated that predicted targets were enriched in the regulation of signal transduction, cellular processes and macromolecule metabolic processes. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that these potential targets were involved in numerous important pathways, predominantly including mitogen-activated protein kinase, phosphati-dylinositol-4,5-bisphosphate 3-kinase-Akt, neurotrophin and cancer-associated signaling pathways. The present study aimed to identify the global expression change of miRNAs in BM-MSCs stimulated with LPS and PM, providing the opportunity to further elucidate the roles of miRNAs in mediating TLR signals to regulate the functions of BM-MSCs. PMID:27121537

  7. Identification and characterization of toll-like receptors (TLRs) in the Chinese tree shrew (Tupaia belangeri chinensis).

    PubMed

    Yu, Dandan; Wu, Yong; Xu, Ling; Fan, Yu; Peng, Li; Xu, Min; Yao, Yong-Gang

    2016-07-01

    In mammals, the toll-like receptors (TLRs) play a major role in initiating innate immune responses against pathogens. Comparison of the TLRs in different mammals may help in understanding the TLR-mediated responses and developing of animal models and efficient therapeutic measures for infectious diseases. The Chinese tree shrew (Tupaia belangeri chinensis), a small mammal with a close relationship to primates, is a viable experimental animal for studying viral and bacterial infections. In this study, we characterized the TLRs genes (tTLRs) in the Chinese tree shrew and identified 13 putative TLRs, which are orthologs of mammalian TLR1-TLR9 and TLR11-TLR13, and TLR10 was a pseudogene in tree shrew. Positive selection analyses using the Maximum likelihood (ML) method showed that tTLR8 and tTLR9 were under positive selection, which might be associated with the adaptation to the pathogen challenge. The mRNA expression levels of tTLRs presented an overall low and tissue-specific pattern, and were significantly upregulated upon Hepatitis C virus (HCV) infection. tTLR4 and tTLR9 underwent alternative splicing, which leads to different transcripts. Phylogenetic analysis and TLR structure prediction indicated that tTLRs were evolutionarily conserved, which might reflect an ancient mechanism and structure in the innate immune response system. Taken together, TLRs had both conserved and unique features in the Chinese tree shrew. PMID:26923770

  8. Differential regulation of TLR4 expression in human B cells and monocytes

    PubMed Central

    Ganley-Leal, Lisa M.; Liang, YanMei; Jagannathan-Bogdan, Madhumita; Farraye, Francis A.; Nikolajczyk, Barbara S.

    2010-01-01

    Toll-like receptor 4 (TLR4) is an innate immune receptor that is constitutively and inducibly activated in monocytes. Although TLR4 is expressed at very low levels on human B cells from healthy individuals, recent reports showed that TLR4 expression and function is elevated in B cells from inflammatory disease patients. New data showed that TLR4 expression on B cell is increased upon stimulation through surface Igμ and CD40 in combination with IL-4. In contrast, monocyte stimulation through CD40 and IL-4 receptors decreased TLR4 surface expression. Analysis of molecular signatures of TLR4 activation in stimulated B cells suggested that TLR4 is regulated by different mechanisms in B cells compared to monocytes. PU.1 and interferon regulatory factor association with the TLR4 promoter are sufficient for TLR4 transcription, but are not sufficient for surface TLR4 expression on B cells. In contrast, the PU.1/IRF combination is sufficient for surface TLR4 expression on monocytes. These data identify mechanisms that can activate B cell TLR4 expression in inflammatory disease patients, and demonstrate that B cells have additional layers of TLR4 regulation absent in monocytes. PMID:20956019

  9. Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation.

    PubMed

    Rebl, Alexander; Rebl, Henrike; Liu, Shuzhen; Goldammer, Tom; Seyfert, Hans-Martin

    2011-01-01

    Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout (Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-κB and CEBP factors as crucial for promoter activation. Overexpression of NF-κB p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-κB and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms. PMID:20813127

  10. Toll-Like Receptor 4 Is an Essential Upstream Regulator of On-Time Parturition and Perinatal Viability in Mice.

    PubMed

    Wahid, Hanan H; Dorian, Camilla L; Chin, Peck Yin; Hutchinson, Mark R; Rice, Kenner C; Olson, David M; Moldenhauer, Lachlan M; Robertson, Sarah A

    2015-10-01

    An inflammatory response is instrumental in the physiological process of parturition but the upstream signals initiating inflammation are undefined. Because endogenous ligands for Toll-like receptor 4 (TLR4) are released in late gestation, we hypothesized that on-time labor requires TLR4 signaling, to trigger a cytokine and leukocyte response and accelerate the parturition cascade. In pregnant TLR4-deficient (Tlr4-/-) mice, average gestation length was extended by 13 hours and increased perinatal mortality was seen compared with wild-type controls. Quantification of cytokine and uterine activation gene expression showed that late gestation induction of Il1b, Il6, Il12b, and Tnf expression seen in control placenta and fetal membranes was disrupted in Tlr4-/- mice, and accompanied by a transient delay in expression of uterine activation genes, including prostaglandin F receptor, oxytocin receptor, and connexin-43. Leukocyte populations were altered before birth in TLR4-deficient females, with fewer neutrophils and macrophages in the placenta, and fewer dendritic cells and more regulatory T cells in the myometrium. Administration of TLR4 ligand lipopolysaccharide to pregnant wild-type mice induced cytokine expression and fetal loss, whereas Tlr4-/- pregnancies were protected. The small molecule TLR4 antagonist (+)-naloxone increased mean duration of gestation by 16 hours in wild-type mice. Collectively, these data demonstrate that TLR4 is a key upstream regulator of the inflammatory response acting to drive uterine activation and control the timing of labor. Because causal pathways for term and preterm labor converge with TLR4, interventions to manipulate TLR4 signaling may have therapeutic utility for women at risk of preterm labor, or in postterm pregnancy. PMID:26151355

  11. Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-gamma and regulatory cytokine IL-10 expression in chicken monocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Toll-like receptors (TLRs) are the pattern recognition receptors of the innate immune system for various conserved pathogen-associated molecular motifs. The chicken TLR3 and TLR21 (avian equivalent to mammalian TLR9) recognize poly I:C (viral double-stranded RNA) and CpG-ODN (a CpG-motif containing...

  12. Saturated fatty acids activate TLR-mediated pro-inflammatory signaling pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report (ATVB 11:1944, 2009) suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for conjugating f...

  13. Macrophages activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression

    PubMed Central

    Blich, Miry; Golan, Amnon; Arvatz, Gil; Sebbag, Anat; Shafat, Itay; Sabo, Edmond; Cohen-Kaplan, Victoria; Petcherski, Sirouch; Avniel-Polak, Shani; Eitan, Amnon; Hammerman, Haim; Aronson, Doron; Axelman, Elena; Ilan, Neta; Nussbaum, Gabriel; Vlodavsky, Israel

    2012-01-01

    Objective Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Results/Methods Highly purified heparanase was added to mouse peritoneal macrophages (MPM) and macrophage-like J774 cells and the levels of TNFα, MMP-9, IL-1, and MCP-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll like receptor (TLR)-2 and -4 knockout mice (KO) were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction (MI), stable angina (SA), and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with SA or acute MI. Addition or over expression of heparanase variants resulted in marked increase in TNFα, MMP-9, IL-1 and MCP-1 levels. MPM harvested from TLR-2 or TLR-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute MI, compared to patients with SA and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared to specimens of stable plaque and controls. Conclusion Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression towards vulnerability. PMID:23162016

  14. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  15. Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

    PubMed Central

    Yazji, Ibrahim; Sodhi, Chhinder P.; Lee, Elizabeth K.; Good, Misty; Egan, Charlotte E.; Afrazi, Amin; Neal, Matthew D.; Jia, Hongpeng; Lin, Joyce; Branca, Maria F.; Prindle, Thomas; Richardson, Ward M.; Ozolek, John; Billiar, Timothy R.; Binion, David G.; Gladwin, Mark T.; Hackam, David J.

    2013-01-01

    Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS−/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate—a precursor for enteral generation of nitrite and nitric oxide—and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate–nitrite–NO signaling. PMID:23650378

  16. Bioinformatics analysis of the structural and evolutionary characteristics for toll-like receptor 15

    PubMed Central

    Wang, Jinlan; Chang, Fen

    2016-01-01

    Toll-like receptors (TLRs) play important role in the innate immune system. TLR15 is reported to have a unique role in defense against pathogens, but its structural and evolution characterizations are still poorly understood. In this study, we identified 57 completed TLR15 genes from avian and reptilian genomes. TLR15 clustered into an individual clade and was closely related to family 1 on the phylogenetic tree. Unlike the TLRs in family 1 with the broken asparagine ladders in the middle, TLR15 ectodomain had an intact asparagine ladder that is critical to maintain the overall shape of ectodomain. The conservation analysis found that TLR15 ectodomain had a highly evolutionarily conserved region on the convex surface of LRR11 module, which is probably involved in TLR15 activation process. Furthermore, the protein–protein docking analysis indicated that TLR15 TIR domains have the potential to form homodimers, the predicted interaction interface of TIR dimer was formed mainly by residues from the BB-loops and αC-helixes. Although TLR15 mainly underwent purifying selection, we detected 27 sites under positive selection for TLR15, 24 of which are located on its ectodomain. Our observations suggest the structural features of TLR15 which may be relevant to its function, but which requires further experimental validation. PMID:27257554

  17. Bioinformatics analysis of the structural and evolutionary characteristics for toll-like receptor 15.

    PubMed

    Wang, Jinlan; Zhang, Zheng; Chang, Fen; Yin, Deling

    2016-01-01

    Toll-like receptors (TLRs) play important role in the innate immune system. TLR15 is reported to have a unique role in defense against pathogens, but its structural and evolution characterizations are still poorly understood. In this study, we identified 57 completed TLR15 genes from avian and reptilian genomes. TLR15 clustered into an individual clade and was closely related to family 1 on the phylogenetic tree. Unlike the TLRs in family 1 with the broken asparagine ladders in the middle, TLR15 ectodomain had an intact asparagine ladder that is critical to maintain the overall shape of ectodomain. The conservation analysis found that TLR15 ectodomain had a highly evolutionarily conserved region on the convex surface of LRR11 module, which is probably involved in TLR15 activation process. Furthermore, the protein-protein docking analysis indicated that TLR15 TIR domains have the potential to form homodimers, the predicted interaction interface of TIR dimer was formed mainly by residues from the BB-loops and αC-helixes. Although TLR15 mainly underwent purifying selection, we detected 27 sites under positive selection for TLR15, 24 of which are located on its ectodomain. Our observations suggest the structural features of TLR15 which may be relevant to its function, but which requires further experimental validation. PMID:27257554

  18. Toll-like Receptor Polymorphisms Are Associated with Increased Neurosyphilis Risk

    PubMed Central

    Marra, Christina M.; Sahi, Sharon K.; Tantalo, Lauren C.; Ho, Emily L.; Dunaway, Shelia B.; Jones, Trudy; Hawn, Thomas R.

    2015-01-01

    Background Single nucleotide polymorphisms (SNPs) in Toll-like receptors (TLR) 1, 2, and 6 impair cell signaling in response to spirochetal lipoproteins. We investigated whether common SNPs in TLR1, 2, or 6 were associated with laboratory- or clinically-defined neurosyphilis. Methods Polymorphisms in the genes for TLR1 (a T->G mutation at position 1805), TLR2 (a G->A mutation at position 2258), and TLR6 (a C->T mutation at position 745) were sought in 456 Caucasian patients with syphilis. Laboratory-defined neurosyphilis included a reactive CSF-Venereal Disease Research Laboratory test. Clinically-defined neurosyphilis included new vision or hearing loss. Controls had CSF white blood cells ≤5/ul, nonreactive CSF-VDRL, and no vision or hearing loss. Results Overall, 26.2% of patients had laboratory-defined and 36.2% had clinically-defined neurosyphilis. Compared to controls, patients with any of the 3 SNPs were more likely to have laboratory-defined neurosyphilis. Those with TLR2 or TLR6 SNPs were more likely to have clinically-defined neurosyphilis. These associations were independent of serum rapid plasma reagin titer. Conclusions A common TLR1 polymorphism is associated with an increased risk of laboratory-defined neurosyphilis and common TLR2 and TLR6 polymorphisms are associated with an increased risk of both laboratory- and clinically-defined neurosyphilis. These data suggest that host factors impact the natural history of syphilis. PMID:24922103

  19. Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

    PubMed

    Sinha, Siddhartha S; Cameron, Jody; Brooks, James C; Leifer, Cynthia A

    2016-08-15

    TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation. PMID:27421483

  20. Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

  1. Methamphetamine inhibits Toll-like receptor 9-mediated anti-HIV activity in macrophages.

    PubMed

    Cen, Ping; Ye, Li; Su, Qi-Jian; Wang, Xu; Li, Jie-Liang; Lin, Xin-Qin; Liang, Hao; Ho, Wen-Zhe

    2013-08-01

    Toll-like receptor 9 (TLR9) is one of the key sensors that recognize viral infection/replication in the host cells. Studies have demonstrated that methamphetamine (METH) dysregulated host cell innate immunity and facilitated HIV infection of macrophages. In this study, we present new evidence that METH suppressed TLR9-mediated anti-HIV activity in macrophages. Activation of TLR9 by its agonist CpG-ODN 2216 inhibits HIV replication, which was demonstrated by increased expression of TLR9, interferon (IFN)-α, IFN regulatory factor-7 (IRF-7), myeloid differentiation factor 88 (MyD88), and myxovirus resistance gene A (MxA) in macrophages. However, METH treatment of macrophages greatly compromised the TLR9 signaling-mediated anti-HIV effect and inhibited the expression of TLR9 downstream signaling factors. Dopamine D1 receptor (D1R) antagonists (SCH23390) could block METH-mediated inhibition of anti-HIV activity of TLR9 signaling. Investigation of the underlying mechanisms of the METH action showed that METH treatment selectively down-regulated the expression of TLR9 on macrophages, whereas it had little effect on the expression of other TLRs. Collectively, our results provide further evidence that METH suppresses host cell innate immunity against HIV infection by down-regulating TLR9 expression and its signaling-mediated antiviral effect in macrophages. PMID:23751096

  2. Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

    PubMed

    Imbesi, M; Yildiz, S; Dirim Arslan, A; Sharma, R; Manev, H; Uz, T

    2009-01-23

    Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning. PMID:19017537

  3. Identification of chemosensory receptor genes from vertebrate genomes.

    PubMed

    Niimura, Yoshihito

    2013-01-01

    Chemical senses are essential for the survival of animals. In vertebrates, mainly three different types of receptors, olfactory receptors (ORs), vomeronasal receptors type 1 (V1Rs), and vomeronasal receptors type 2 (V2Rs), are responsible for the detection of chemicals in the environment. Mouse or rat genomes contain >1,000 OR genes, forming the largest multigene family in vertebrates, and have >100 V1R and V2R genes as well. Recent advancement in genome sequencing enabled us to computationally identify nearly complete repertories of OR, V1R, and V2R genes from various organisms, revealing that the numbers of these genes are highly variable among different organisms depending on each species' living environment. Here I would explain bioinformatic methods to identify the entire repertoires of OR, V1R, and V2R genes from vertebrate genome sequences. PMID:24014356

  4. TLR2 Activation Inhibits Embryonic Neural Progenitor Cell Proliferation

    PubMed Central

    Okun, Eitan; Griffioen, Kathleen J.; Gen-Son, Tae; Lee, Jong-Hwan; Roberts, Nicholas J.; Mughal, Mohamed R.; Hutchison, Emmette; Cheng, Aiwu; Arumugam, Thiruma V.; Lathia, Justin D.; van Praag, Henriette; Mattson, Mark P.

    2010-01-01

    Toll-like receptors (TLRs) play essential roles in innate immunity, and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain, where they mediate responses to infection, stress and injury. To address the possibility that TLR2 heterodimer activation could affect progenitor cells in the developing brain, we analyzed the expression of TLR2 throughout the mouse cortical development, and assessed the role of TLR2 heterodimer activation in neural progenitor cell (NPC) proliferation. TLR2 mRNA and protein was expressed in the cortex in embryonic and early postnatal stages of development, and in cultured cortical NPC. While NPC from TLR2-deficient and wild type embryos had the same proliferative capacity, TLR2 activation by the synthetic bacterial lipopeptides Pam3CSK4 and FSL1, or low molecular weight hyaluronan, an endogenous ligand for TLR2, inhibited neurosphere formation in vitro. Intracerebral in utero administration of TLR2 ligands resulted in ventricular dysgenesis characterized by increased ventricle size, reduced proliferative area around the ventricles, increased cell density, an increase in PH3+ cells and a decrease in BrdU+ cells in the sub-ventricular zone. Our findings indicate that loss of TLR2 does not result in defects in cerebral development. However, TLR2 is expressed and functional in the developing telencephalon from early embryonic stages and infectious agent-related activation of TLR2 inhibits NPC proliferation. TLR2–mediated inhibition of NPC proliferation may therefore be a mechanism by which infection, ischemia and inflammation adversely affect brain development. PMID:20456021

  5. Evidence for adaptation of porcine Toll-like receptors.

    PubMed

    Darfour-Oduro, Kwame A; Megens, Hendrik-Jan; Roca, Alfred; Groenen, Martien A M; Schook, Lawrence B

    2016-03-01

    Naturally endemic infectious diseases provide selective pressures for pig populations. Toll-like receptors (TLRs) represent the first line of immune defense against pathogens and are likely to play a crucial adaptive role for pig populations. This study was done to determine whether wild and domestic pig populations representing diverse global environments demonstrate local TLR adaptation. The genomic sequence encoding the ectodomain, responsible for interacting with pathogen ligands of bacterial (TLR1, TLR2 and TLR6) and viral (TLR3, TLR7 and TLR8) receptors, was obtained. Mitochondrial D-loop region sequences were obtained and a phylogenetic analysis using these sequences revealed a clear separation of animals into Asian (n = 27) and European (n = 40) clades. The TLR sequences were then analyzed for population-specific positive selection signatures within wild boars and domesticated pig populations derived from Asian and European clades. Using within-population and between-population tests for positive selection, a TLR2-derived variant 376A (126Thr), estimated to have arisen in 163,000 years ago with a frequency of 83.33% within European wild boars, 98.00% within domestic pig breeds of European origin, 40.00% within Asian wild boars, and 11.36% within Asian domestic pigs, was identified to be under positive selection in pigs of European origin. The variant is located within the N terminal domain of the TLR2 protein 3D crystal structure and could affect ligand binding. This study suggests the TLR2 gene contributing to responses to bacterial pathogens has been crucial in adaptation of pigs to pathogens. PMID:26701185

  6. Dopamine receptor genes: new tools for molecular psychiatry.

    PubMed Central

    Niznik, H B; Van Tol, H H

    1992-01-01

    For over a decade it has been generally assumed that all the pharmacological and biochemical actions of dopamine within the central nervous system and periphery were mediated by two distinct dopamine receptors. These receptors, termed D1 and D2, were defined as those coupled to the stimulation or inhibition of adenylate cyclase, respectively, and by their selectivity and avidity for various drugs and compounds. The concept that two dopamine receptors were sufficient to account for all the effects mediated by dopamine was an oversimplification. Recent molecular biological studies have identified five distinct genes which encode at least eight functional dopamine receptors. The members of the expanded dopamine receptor family, however, can still be codifed by way of the original D1 and D2 receptor dichotomy. These include two genes encoding dopamine D1-like receptors (D1 [D1A]/D5 [D1B]) and three genes encoding D2-like receptors (D2/D3/D4). We review here our recent work on the cloning and characterization of some of the members of the dopamine receptor gene family (D1, D2, D4, D5), their relationship to neuropsychiatric disorders and their potential role in antipsychotic drug action. Images Fig. 1 PMID:1450188

  7. The TLR7/8 ligand resiquimod targets monocyte-derived dendritic cell differentiation via TLR8 and augments functional dendritic cell generation.

    PubMed

    Hackstein, Holger; Knoche, Angela; Nockher, Angelika; Poeling, Jochen; Kubin, Thomas; Jurk, Marion; Vollmer, Jörg; Bein, Gregor

    2011-01-01

    Imidazoquinolone compounds, such as resiquimod are Toll-like receptor (TLR) 7/8 ligands representing novel immune response modifiers undergoing clinical testing. Resiquimod has been reported to modulate conventional human monocyte-derived DC (moDC) differentiation, but the role of TLR7 and TLR8 is unclear. We directly dissected the TLR7- and TLR8-dependency by employing selective TLR7 ligands and resiquimod-coculture experiments with inhibitory oligonucleotides (iODN) suppressing TLR7, TLR7+8 or TLR7+8+9. Selective TLR7 ligands did not affect conventional moDC differentiation as analyzed by CD14/CD1a expression. iODN experiments confirmed that resiquimod's effects during DC differentiation were antagonized only with TLR8 iODNs. Direct comparison of resiquimod DC with TLR7- and control-DC revealed significantly higher T-cell costimulatory molecule and MHC class II expression. Resiquimod DC promoted significantly stronger allogeneic T-cell proliferation and stronger naïve CD4(+) T-cell proliferation. These results indicate the relevance of TLR8 for human monocyte-derived DC differentiation and maturation and may be relevant for clinical trials employing resiquimod. PMID:21889130

  8. The Scavenger Receptor SREC-I Cooperates with Toll-Like Receptors to Trigger Inflammatory Innate Immune Responses

    PubMed Central

    Murshid, Ayesha; Borges, Thiago J.; Lang, Benjamin J.; Calderwood, Stuart K.

    2016-01-01

    Scavenger receptor expressed by endothelial cell-I (SREC-I) is a class F scavenger receptor expressed by immune cells with a significant role in CD8+- and CD4+-mediated T cell immunity. This receptor can also modulate the function of toll-like receptors (TLRs), which play essential roles in innate immunity. Earlier, it was found that human monocyte/macrophage THP1 cells and bone marrow-derived macrophages from mice exhibited increased responses to polyinosine–polycytidylic acid (poly I:C, PIC) and CpG (unmethylated) DNA and enhanced production of inflammatory cytokines with overexpressed SREC-I. Our data also showed that intracellular/endocytic TLR3 and TLR9 could directly interact with SREC-I in the presence of their respective ligands. We also observed that the internalized ligand along with TLR3/TLR9 colocalized in the endosome in macrophages and THP-1 cells overexpressing these receptors. In the absence of these ligands, there was no detectable colocalization between the SREC-I and endocytic TLRs. Earlier, it was shown that SREC-I stimulated double-stranded RNA/CpGDNA-mediated TLR3/TLR9 activation of the innate immune response by triggering signaling through the NF-κB, IRF3, and MAP kinase pathways leading to transcription of cytokine genes. We also established that SREC-I can associate with plasma membrane TLRs, such as TLR2 and TLR4. We demonstrated that SREC-I–TLR4 signals more efficiently from lipid microdomain in which lipopolysaccharide (LPS) can associate with SREC-I–TLR4 complex. We also proved that SREC-I is an alternate receptor for LPS capable of internalizing the complex and for endocytic TLR ligands as well. This binding activated endocytic TLR-mediated downstream cytokine production in THP1 cells and macrophages. Finally, SREC-I could also form complexes with TLR2 and induce the release of cytokines in the presence of bacterial, viral, and fungal ligands. PMID:27379091

  9. Sex Differences in monocytes and TLR4 associated immune responses; implications for systemic lupus erythematosus (SLE)

    PubMed Central

    Jiang, Wei; Gilkeson, Gary

    2014-01-01

    It has been shown that TLR7 and TLR9 signaling play a role in SLE pathogenesis. Our recent study revealed that estrogen receptor α knockout mice have impaired inflammatory responses to TLR3, TLR4, TLR7 and TLR9 ligand stimulation in DCs, B cells and whole spleen cells. These findings indicate that estrogen receptor mediated signaling may impact universal TLR responsiveness. Whether estrogen has a direct or indirect effect on TLR responsiveness by immune cells is not clear. There is evidence of a role of TLR4 in SLE disease pathogenesis, such as the kidney damage, the induction of CD40 and autoantibodies, the suppression of regulatory T cells, and the role of pro-inflammatory cytokines (e.g., IL-6, IL-1β, TNF-α) in SLE pathogenesis that can be induced by TLR4-mediated monocyte activation, suggesting that TLR4 and TLR4 responsiveness are also important for SLE disease. This review will focus on TLR4 responses and monocytes, which are understudied in systemic autoimmune diseases such as SLE. PMID:25309746

  10. Multifaceted contribution of the TLR4-activated IRF5 transcription factor in systemic sclerosis.

    PubMed

    Saigusa, Ryosuke; Asano, Yoshihide; Taniguchi, Takashi; Yamashita, Takashi; Ichimura, Yohei; Takahashi, Takehiro; Toyama, Tetsuo; Yoshizaki, Ayumi; Sugawara, Koji; Tsuruta, Daisuke; Taniguchi, Tadatsugu; Sato, Shinichi

    2015-12-01

    Systemic sclerosis (SSc) is a multisystem autoimmune disorder with clinical manifestations resulting from tissue fibrosis and extensive vasculopathy. A potential disease susceptibility gene for SSc is IFN regulatory factor 5 (IRF5), whose SNP is associated with milder clinical manifestations; however, the underlying mechanisms of this association remain elusive. In this study we examined IRF5-deficient (Irf5(-/-)) mice in the bleomycin-treated SSc murine model. We show that dermal and pulmonary fibrosis induced by bleomycin is attenuated in Irf5(-/-) mice. Interestingly, we find that multiple SSc-associated events, such as fibroblast activation, inflammatory cell infiltration, endothelial-to-mesenchymal transition, vascular destabilization, Th2/Th17 skewed immune polarization, and B-cell activation, are suppressed in these mice. We further provide evidence that IRF5, activated by Toll-like receptor 4 (TLR4), binds to the promoters of various key genes involved in SSc disease pathology. These observations are congruent with the high level of expression of IRF5, TLR4, and potential endogenous TLR4 ligands in SSc skin lesions. Our study sheds light on the TLR4-IRF5 pathway in the pathology of SSc with clinical implications of targeting the IRF5 pathways in the suppression of disease development. PMID:26598674

  11. The TLR7 7926A>G polymorphism is associated with susceptibility to systemic lupus erythematosus.

    PubMed

    Tian, Jing; Ma, Yan; Li, Jing; Cen, Han; Wang, De-Guang; Feng, Chen-Chen; Li, Ruo-Jie; Leng, Rui-Xue; Pan, Hai-Feng; Ye, Dong-Qing

    2012-07-01

    Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder that predominantly affects women of childbearing age, with a female-to-male ratio of approximately 9:1. Previous findings indicated that male cases of SLE were associated with Klinefelter's syndrome (47, XXY), whereas females with Turner's syndrome (45, X0) did not contract SLE. Additionally, duplicated Toll-like receptor 7 (TLR7) was found to promote lupus-like disease. Consequently, the aim of this study was to evaluate whether the TLR7 gene served as a genetic marker for the development of SLE. A case-control study was performed on one tag single nucleotide polymorphism TLR7 rs1634323 in a population with 507 SLE patients and 513 healthy controls. Genotyping was determined by the TaqMan genotyping assay using the ABI 7300 real-time reverse transcription polymerase chain reaction system. The results showed a significantly elevated risk of SLE with the rs1634323 AG genotype in females (P = 0.040, OR = 1.897, 95% CI 1.031-3.491), whereas a similar association was not replicated in males (P = 0.303, OR = 0.338, 95% CI 0.043-2.656). In a subgroup analysis by clinical manifestation of lupus nephritis, no significant differences were found. These findings indicate that the TLR7 gene rs1634323 polymorphism may contribute to SLE susceptibility in females. PMID:22505023

  12. Multifaceted contribution of the TLR4-activated IRF5 transcription factor in systemic sclerosis

    PubMed Central

    Saigusa, Ryosuke; Asano, Yoshihide; Taniguchi, Takashi; Yamashita, Takashi; Ichimura, Yohei; Takahashi, Takehiro; Toyama, Tetsuo; Yoshizaki, Ayumi; Sugawara, Koji; Tsuruta, Daisuke; Taniguchi, Tadatsugu; Sato, Shinichi

    2015-01-01

    Systemic sclerosis (SSc) is a multisystem autoimmune disorder with clinical manifestations resulting from tissue fibrosis and extensive vasculopathy. A potential disease susceptibility gene for SSc is IFN regulatory factor 5 (IRF5), whose SNP is associated with milder clinical manifestations; however, the underlying mechanisms of this association remain elusive. In this study we examined IRF5-deficient (Irf5−/−) mice in the bleomycin-treated SSc murine model. We show that dermal and pulmonary fibrosis induced by bleomycin is attenuated in Irf5−/− mice. Interestingly, we find that multiple SSc-associated events, such as fibroblast activation, inflammatory cell infiltration, endothelial-to-mesenchymal transition, vascular destabilization, Th2/Th17 skewed immune polarization, and B-cell activation, are suppressed in these mice. We further provide evidence that IRF5, activated by Toll-like receptor 4 (TLR4), binds to the promoters of various key genes involved in SSc disease pathology. These observations are congruent with the high level of expression of IRF5, TLR4, and potential endogenous TLR4 ligands in SSc skin lesions. Our study sheds light on the TLR4-IRF5 pathway in the pathology of SSc with clinical implications of targeting the IRF5 pathways in the suppression of disease development. PMID:26598674

  13. Multigenic Control of Measles Vaccine Immunity Mediated by Polymorphisms in Measles Receptor, Innate Pathway, and Cytokine Genes

    PubMed Central

    Kennedy, Richard B.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; O’Byrne, Megan; Jacobson, Robert M.; Pankratz, V. Shane; Poland, Gregory A.

    2012-01-01

    Measles infection and vaccine response are complex biological processes that involve both viral and host genetic factors. We have previously investigated the influence of genetic polymorphisms on vaccine immune response, including measles vaccines, and have shown that polymorphisms in HLA, cytokine, cytokine receptor, and innate immune response genes are associated with variation in vaccine response but do not account for all of the inter-individual variance seen in vaccinated populations. In the current study we report the findings of a multigenic analysis of measles vaccine immunity, indicating a role for the measles virus receptor CD46, innate pattern-recognition receptors (DDX58, TLR2, 4, 5,7 and 8) and intracellular signaling intermediates (MAP3K7, NFKBIA), and key antiviral molecules (VISA, OAS2, MX1, PKR) as well as cytokines (IFNA1, IL4, IL6, IL8, IL12B) and cytokine receptor genes (IL2RB, IL6R, IL8RA) in the genetic control of both humoral and cellular immune responses. This multivariate approach provided additional insights into the genetic control of measles vaccine responses over and above the information gained by our previous univariate SNP association analyses. PMID:22265947

  14. Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4

    PubMed Central

    Allam, Ramanjaneyulu; Scherbaum, Christina Rebecca; Darisipudi, Murthy Narayana; Mulay, Shrikant R.; Hägele, Holger; Lichtnekert, Julia; Hagemann, Jan Henrik; Rupanagudi, Khader Valli; Ryu, Mi; Schwarzenberger, Claudia; Hohenstein, Bernd; Hugo, Christian; Uhl, Bernd; Reichel, Christoph A.; Krombach, Fritz; Monestier, Marc; Liapis, Helen; Moreth, Kristin; Schaefer, Liliana

    2012-01-01

    In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI. PMID:22677551

  15. Adenovirus receptors and their implications in gene delivery

    PubMed Central

    Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  16. Altered Molecular Expression of the TLR4/NF-κB Signaling Pathway in Mammary Tissue of Chinese Holstein Cattle with Mastitis

    PubMed Central

    Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

    2015-01-01

    Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis. PMID:25706977

  17. TLR2 and TLR4 mediated host immune responses in major infectious diseases: a review.

    PubMed

    Mukherjee, Suprabhat; Karmakar, Subhajit; Babu, Santi Prasad Sinha

    2016-01-01

    During the course of evolution, multicellular organisms have been orchestrated with an efficient and versatile immune system to counteract diverse group of pathogenic organisms. Pathogen recognition is considered as the most critical step behind eliciting adequate immune response during an infection. Hitherto Toll-like receptors (TLRs), especially the surface ones viz. TLR2 and TLR4 have gained immense importance due to their extreme ability of identifying distinct molecular patterns from invading pathogens. These pattern recognition receptors (PRRs) not only act as innate sensor but also shape and bridge innate and adaptive immune responses. In addition, they also play a pivotal role in regulating the balance between Th1 and Th2 type of response essential for the survivability of the host. In this work, major achievements rather findings made on the typical signalling and immunopathological attributes of TLR2 and TLR4 mediated host response against the major infectious diseases have been reviewed. Infectious diseases like tuberculosis, trypanosomiasis, malaria, and filariasis are still posing myriad threat to mankind. Furthermore, increasing resistance of the causative organisms against available therapeutics is also an emerging problem. Thus, stimulation of host immune response with TLR2 and TLR4 agonist can be the option of choice to treat such diseases in future. PMID:26775799

  18. Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids

    PubMed Central

    Jiménez-Dalmaroni, Maximiliano J; Radcliffe, Catherine M; Harvey, David J; Wormald, Mark R; Verdino, Petra; Ainge, Gary D; Larsen, David S; Painter, Gavin F; Ulevitch, Richard; Beutler, Bruce; Rudd, Pauline M; Dwek, Raymond A; Wilson, Ian A

    2015-01-01

    Toll-like receptors (TLRs) are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system, and its biochemical as well as ligand binding properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid (LTA) from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chain containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED. The design of novel inhibitors of TLR2, based on their ability to bind to TLR2 but not activate the TLR2 signaling pathway, may lead to the development of novel treatments for septic shock caused by Gram- positive bacteria. PMID:24591200

  19. Raftlin Is Involved in the Nucleocapture Complex to Induce Poly(I:C)-mediated TLR3 Activation*

    PubMed Central

    Watanabe, Ayako; Tatematsu, Megumi; Saeki, Kazuko; Shibata, Sachiko; Shime, Hiroaki; Yoshimura, Akihiko; Obuse, Chikashi; Seya, Tsukasa; Matsumoto, Misako

    2011-01-01

    The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-β production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3. PMID:21266579

  20. TLR2 and TLR4 in autoimmune diseases: a comprehensive review.

    PubMed

    Liu, Yu; Yin, Heng; Zhao, Ming; Lu, Qianjin

    2014-10-01

    Autoimmune diseases are immune disorders characterized by T cell hyperactivity and B cell overstimulation leading to overproduction of autoantibodies. Although the pathogenesis of various autoimmune diseases remains to be elucidated, environmental factors have been thought to contribute to the initiation and maintenance of auto-respond inflammation. Toll-like receptors (TLRs) are pattern recognition receptors belonging to innate immunity that recognize and defend invading microorganisms. Besides these exogenous pathogen-associated molecular patterns, TLRs can also bind with damage-associated molecular patterns produced under strike or by tissue damage or cells apoptosis. It is believed that TLRs build a bridge between innate immunity and autoimmunity. There are five adaptors to TLRs including MyD88, TRIF, TIRAP/MAL, TRAM, and SARM. Upon activation, TLRs recruit specific adaptors to initiate the downstream signaling pathways leading to the production of inflammatory cytokines and chemokines. Under certain circumstances, ligation of TLRs drives to aberrant activation and unrestricted inflammatory responses, thereby contributing to the perpetuation of inflammation in autoimmune diseases. In the past, most studies focused on the intracellular TLRs, such as TLR3, TLR7, and TLR9, but recent studies reveal that cell surface TLRs, especially TLR2 and TLR4, also play an essential role in the development of autoimmune diseases and afford multiple therapeutic targets. In this review, we summarized the biological characteristics, signaling mechanisms of TLR2/4, the negative regulators of TLR2/4 pathway, and the pivotal function of TLR2/4 in the pathogenesis of autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, psoriasis, multiple sclerosis, and autoimmune diabetes. PMID:24352680

  1. Multiple human D sub 5 dopamine receptor genes: A functional receptor and two pseudogenes

    SciTech Connect

    Grandy, D.K.; Yuan Zhang; Bouvier, C.; Qunyong Zhou; Johnson, R.A.; Allen, L.; Buck, K.; Bunzow, J.R.; Salon, J.; Civelli, O. )

    1991-10-15

    Three genes closely related to the D{sub 1} dopamine receptor were identified in the human genome. One of the genes lacks introns and encodes a functional human dopamine receptor, D{sub 5}, whose deduced amino acid sequence is 49% identical to that of the human D{sub 1} receptor. Compared with the human D{sub 1} dopamine receptor, the D{sub 5} receptor displayed a higher affinity for dopamine and was able to stimulate a biphasic rather than a monophasic intracellular accumulation of cAMP. Neither of the other two genes was able to direct the synthesis of a receptor. nucleotide sequence analysis revealed that these two genes are 98% identical to each other and 95% identical to the D{sub 5} sequence. Relative to the D{sub 5} sequence, both contain insertions and deletions that result in several in-frame termination codons. Premature termination of translation is the most likely explanation for the failure of these genes to produce receptors in COS-7 and 293 cells even though their messages are transcribed. The authors conclude that the two are pseudogenes. Blot hybridization experiments performed on rat genomic DNA suggest that there is one D{sub 5} gene in this species and that the pseudogenes may be the result of a relatively recent evolutionary event.

  2. TLR2-independent induction and regulation of chronic intestinal inflammation.

    PubMed

    Boulard, Olivier; Asquith, Mark J; Powrie, Fiona; Maloy, Kevin J

    2010-02-01

    Interactions between the intestinal microflora and host innate immune receptors play a critical role in intestinal homeostasis. Several studies have shown that TLR2 can modulate inflammatory responses in the gut. TLR2 signals enhance tight junction formation and fortify the epithelial barrier, and may play a crucial role in driving acute inflammatory responses towards intestinal bacterial pathogens. In addition, TLR2 agonists can have direct effects on both Th1 cells and Treg. To define the role of TLR2 in the induction and regulation of chronic intestinal inflammation we examined the effects of TLR2 deletion on several complementary models of inflammatory bowel disease. Our results show that TLR2 signals are not required for the induction of chronic intestinal inflammation by either innate or adaptive immune responses. We further show that TLR2(-/-) mice harbor normal numbers of Foxp3(+) Treg that are able to suppress intestinal inflammation as effectively as their WT counterparts. We also did not find any intrinsic role for TLR2 for pathogenic effector T-cell responses in the gut. Thus, in contrast to their role in acute intestinal inflammation and repair, TLR2 signals may have a limited impact on the induction and regulation of chronic intestinal inflammation. PMID:19950179

  3. TLR3 Plays Significant Roles against HBV-Associated HCC

    PubMed Central

    Chen, Xiao-lan; Xu, Yu-yin; Chen, Li; Wang, Gui-lan; Shen, Yin

    2015-01-01

    Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). The role of TLR3 in hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) infections is not well understood. To investigate the ability of TLR3 in regulating HBV replication in HCC, 80 cases of human HCC were collected and their tissue microarray was made. In HCC cells, the expression and location of TLR3, hepatitis-associated virus, and interstitial immunoreactive cells were assayed with immunohistochemical staining. The apoptosis of tumor cells was also detected by TUNEL stain. Correlations between TLR3 expression and HBV infection, interstitial immunoreactive cells, and cells apoptosis in HCC were investigated. In addition, we explored whether TLR3 agonist dsRNA can inhibit HepG2.2.15 cells secreting HBV. We found that the cytoplasmic expression of TLR3 in HCC is positively related to HBsAg infection and HCC with cirrhosis and promotes interstitial immunoreactive cells infiltration and cancer cells apoptosis. In HepG2.2.15 cells, dsRNA inhibited the secretion of HBV and induced apoptosis. These results indicate that TLR3 signaling activity may be involved in immune responses against HBV in HCC. PMID:25983748

  4. Evaluation of TLR2 and 4 in Chronic Periodontitis

    PubMed Central

    Mahalingam, Arulpari; Parthasarathy, Harinath; Katamreddy, Vineela; Subbareddy, Venkat

    2016-01-01

    Introduction Periodontal disease is the major cause of adult tooth loss and is commonly characterized by a chronic inflammation caused by infection due to oral bacteria. Members of Toll-Like Receptor (TLR) family recognize conserved microbial structures, such as bacterial lipopolysaccharides and activate signalling pathways that result in immune responses against microbial infections. Aim The aim of the present study was to assess the mRNA expression of Toll-Like Receptor 2 and 4 in tissues with or without chronic periodontitis. Materials and Methods Gingival tissue samples were collected from controls (30 subjects with healthy periodontal tissues) and experimental group (30 subjects with chronic periodontitis). Total RNA was extracted and RT-PCR was done for evaluation of TLR-2 and TLR-4. Mann Whitney U-test, Pearson Chi-square Test was used for statistics. Results The results showed that there is a significant (p-value= 0.004) association between TLR-4 and the experimental group comprising of chronic periodontitis patients in comparison to the insignificant (p-value= 0.085) TLR-2 expression. Conclusion This study concludes that TLR-2 and TLR-4 expressed in the gingival tissues recognize different bacterial cell wall components thus helping us to associate its potential in diagnosing periodontal disease. Hence, in the future, these scientific findings can pave the way in using TLR as a diagnostic biomarker for periodontal disease. PMID:27504418

  5. Saturated fatty acids trigger TLR4-mediated inflammatory response.

    PubMed

    Rocha, D M; Caldas, A P; Oliveira, L L; Bressan, J; Hermsdorff, H H

    2016-01-01

    Toll-like receptors (TLR) mediate infection-induced inflammation and sterile inflammation by endogenous molecules. Among the TLR family, TLR4 is the best understood. However, while its downstream signaling pathways have been well defined, not all ligands of TLR4 are currently known. Current evidence suggests that saturated fatty acids (SFA) act as non-microbial TLR4 agonists, and trigger its inflammatory response. Thus, our present review provides a new perspective on the potential mechanism by which SFAs could modulate TLR4-induced inflammatory responses: (1) SFAs can be recognized by CD14-TLR4-MD2 complex and trigger inflammatory pathways, similar to lipopolysaccharide (LPS). (2) SFAs lead to modification of gut microbiota with an overproduction of LPS after a high-fat intake, enhancing this natural TLR4 ligand. (3) In addition, this metabolic endotoxemia leads to an oxidative stress thereby producing atherogenic lipids - oxLDL and oxidized phospholipids - which trigger CD36-TLR4-TLR6 inflammatory response. (4) Also, the high SFA consumption increases the lipemia and the mmLDL and oxLDL formation through oxidative modifications of LDL. The mmLDL, unlike oxLDL, is involved in activation of the CD14-TLR4-MD2 inflammatory pathway. Those molecules can induce TLR4 inflammatory response by MyD88-dependent and/or MyD88-independent pathways that, in turn, promotes the expression of proinflammatory transcript factors such as factor nuclear kappa B (NF-κB), which plays a crucial role in the induction of inflammatory mediators (cytokines, chemokines, or costimulatory molecules) implicated in the development and progression of many chronic diseases. PMID:26687466

  6. Lignin-rich Enzyme Lignin (LREL), a Cellulase-treated Lignin-Carbohydrate Derived from Plants, Activates Myeloid Dendritic Cells via Toll-like Receptor 4 (TLR4)

    PubMed Central

    Tsuji, Ryohei; Koizumi, Hideki; Aoki, Dan; Watanabe, Yuta; Sugihara, Yoshihiko; Matsushita, Yasuyuki; Fukushima, Kazuhiko; Fujiwara, Daisuke

    2015-01-01

    Lignin-carbohydrates, one of the major cell wall components, are believed to be the structures that form chemical linkage between lignin and cell wall polysaccharides. Due to the molecular complexity of lignin-containing substances, their isolation and the assignment of their biological activities have so far remained a difficult task. Here, we extracted two lignin-containing carbohydrates, lignin-rich enzyme lignin (LREL) and pure enzyme lignin (PEL), from barley husk and demonstrated that they act as immune stimulators of dendritic cells (DCs), which are particularly important in linking innate and adaptive immunity. Thioacidolysis, acid hydrolysis, and mild alkali hydrolysis of both LREL and PEL revealed that their immunostimulatory activities depended on the lignin structure and/or content, neutral sugar content (especially the characteristic distribution of galactose and mannose), and presence of an ester bond. Furthermore, we showed that the immunostimulatory potency of the lignin-carbohydrate depended on its molecular weight and degree of polymerization. We also demonstrated that the LREL-induced activation of DCs was mediated via TLR4. Thus, LREL-induced increases in the expression levels of several cell surface marker proteins, production of inflammatory cytokines IL-12p40 and TNF-α, and activation and nuclear translocation of transcription factors, as was observed in the WT DCs, were completely abrogated in DCs derived from the TLR4−/− mice but not in DCs derived from the TLR2−/−, TLR7−/−, and TLR9−/− mice. We further demonstrated that LRELs isolated from other plant tissues also activated DCs. These immunostimulatory activities of lignin-carbohydrates, extracted from edible plant tissues, could have potential relevance in anti-infectious immunity and vaccine adjuvants. PMID:25548274

  7. Therapeutic Effects of Treatment with Anti-TLR2 and Anti-TLR4 Monoclonal Antibodies in Polymicrobial Sepsis

    PubMed Central

    Lima, Cristiano Xavier; Souza, Danielle Gloria; Amaral, Flavio Almeida; Fagundes, Caio Tavares; Rodrigues, Irla Paula Stopa; Alves-Filho, Jose Carlos; Kosco-Vilbois, Marie; Ferlin, Walter; Shang, Limin; Elson, Greg; Teixeira, Mauro Martins

    2015-01-01

    Introduction Toll-like receptors (TLRs) play an important role in the recognition of microbial products and in host defense against infection. However, the massive release of inflammatory mediators into the bloodstream following TLR activation following sepsis is thought to contribute to disease pathogenesis. Methods Here, we evaluated the effects of preventive or therapeutic administration of monoclonal antibodies (mAbs) targeting either TLR2 or TLR4 in a model of severe polymicrobial sepsis induced by cecal ligation and puncture in mice. Results Pre-treatment with anti-TLR2 or anti-TLR4 mAb alone showed significant protection from sepsis-associated death. Protective effects were observed even when the administration of either anti-TLR2 or anti-TLR4 alone was delayed (i.e., 3 h after sepsis induction). Delayed administration of either mAb in combination with antibiotics resulted in additive protection. Conclusion Although attempts to translate preclinical findings to clinical sepsis have failed so far, our preclinical experiments strongly suggest that there is a sufficient therapeutic window within which patients with ongoing sepsis could benefit from combined antibiotic plus anti-TLR2 or anti-TLR4 mAb treatment. PMID:26147469

  8. Decreased expression of TLR7 in gastric cancer tissues and the effects of TLR7 activation on gastric cancer cells

    PubMed Central

    JIANG, JIONG; DONG, LEI; QIN, BIN; SHI, HAITAO; GUO, XIAOYAN; WANG, YAN

    2016-01-01

    The present study aimed to determine the expression of Toll-like receptor 7 (TLR7) in gastric cancer tissues and investigate the effects of its activation on gastric cancer cells. Patients with gastric cancer (n=30) and patients without gastric cancer (control; n=14) who underwent gastroscopy were enrolled in the study. Gastric cancer and cancer-adjacent tissues were obtained from the patients with gastric cancer, and normal gastric epithelial tissues were obtained from the control patients. The TLR7 mRNA and protein expressions in different tissues were investigated by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. The present study also determined the effects of TLR7 activation by the agonist imiquimod on TLR7 protein expression, proinflammatory cytokine secretion and viability in SGC-7901 gastric cancer cells. The mRNA and protein expression levels of TLR7 were significantly downregulated in gastric cancer tissues compared with cancer-adjacent and normal gastric epithelial tissues (P<0.01). Imiquimod significantly increased TLR7 protein expression levels, and promoted the secretion of proinflammatory cytokines tumor necrosis factor-α and interleukin-6 in SGC-7901 cells. Furthermore, imiquimod inhibited the proliferation of SGC-7901 cells in a dose- and time-dependent manner. Thus, the present study identified that the expression of TLR7 was decreased in gastric cancer tissues, and TLR7 activation enhanced TLR7 expression, promoted the production of proinflammatory cytokines and inhibited the growth of gastric cancer cells. PMID:27347192

  9. Differential expression of Toll-like receptor pathway genes in chicken embryo fibroblasts from chickens resistant and susceptible to Marek’s disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Toll-like receptor (TLR) signaling pathway is one of the innate immune defense mechanisms against pathogens in vertebrates and invertebrates. However, the role of TLR in non-MHC genetic resistance or susceptibility to Marek’s disease (MD) in the chicken is yet to be elucidated. Chicken embryo fi...

  10. Polymorphisms in pattern recognition receptors and their relationship to infectious disease susceptibility in pigs

    PubMed Central

    2011-01-01

    Background Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), are censoring receptors for molecules derived from bacteria, viruses, and fungi. The PRR system is a prerequisite for proper responses to pathogens, for example by cytokine production, resulting in pathogen eradication. Many cases of polymorphisms in PRR genes affecting the immune response and disease susceptibility are known in humans and mice. Methods We surveyed polymorphisms in pig genes encoding PRRs and investigated the relationship between some of the detected polymorphisms and molecular function or disease onset. Results Nonsynonymous polymorphisms abounded in pig TLR genes, particularly in the region corresponding to the ectodomains of TLRs expressed on the cell surface. Intracellular TLRs such as TLR3, TLR7, and TLR8, and other intracellular PRRs, such as the peptidoglycan receptor NOD2 and viral RNA receptors RIG-I and MDA5, also possessed nonsynonymous polymorphisms. Several of the polymorphisms influenced molecular functions such as ligand recognition. Polymorphisms in the PRR genes may be related to disease susceptibility in pigs: pigs with a particular allele of TLR2 showed an increased tendency to contract pneumonia. Conclusions We propose the possibility of pig breeding aimed at disease resistance by the selection of PRR gene alleles that affect pathogen recognition. PMID:21645307

  11. Prolactin receptor and signal transduction to milk protein genes

    SciTech Connect

    Djiane, J.; Daniel, N.; Bignon, C.

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  12. Altered Th17 Cytokine Expression in Helicobacter pylori Patients with TLR4 (D299G) Polymorphism.

    PubMed

    Bagheri, Nader; Azadegan-Dehkordi, Fatemeh; Rahimian, Ghorbanali; Hashemzadeh-Chaleshtori, Morteza; Rafieian-Kopaei, Mahmoud; Kheiri, Soleyman; Gholipour, Abolfazl; Shirzad, Hedayatollah

    2016-02-01

    Helicobacter pylori (H. pylori) is associated with gastric ulcer and gastric adenocarcinoma. Polymorphisms in the host genes coding for Toll-like receptors (TLRs) may influence the innate and adaptive immune response to the infection, affecting the susceptibility to H. pylori or the disease outcomes. However, the details and association with different polymorphism and clinical expression of infection remain unclear. A case-control study consisting of 58 patients with H. pylori infection and 44 H. pylori uninfection was conducted. Genomic DNA was extracted and genotypes of TLR4 Asp299Gly polymorphism were assessed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mucosal cytokines expression in H. pylori-infected and uninfected gastric biopsies was determined by real-time PCR. The expression of IL-6, IL-17, IL-21, IL-23 and TGF-β1 was significantly higher in patients with D299G polymorphism in TLR4. But the expression of IL-18 between patients with single-nucleotide polymorphisms (SNPs) in TLR4 and patients with the wild-type allele was not significant. In H. pylori-infected patients with gastritis, SNPs in TLR4 may alter cytokine expression toward Th17 immune response in the gastric mucosa and may have increased risk for the development of peptic ulcer. PMID:26853914

  13. TLR4 Deficiency Protects against Hepatic Fibrosis and Diethylnitrosamine-Induced Pre-Carcinogenic Liver Injury in Fibrotic Liver

    PubMed Central

    Weber, Susanne Nicole; Bohner, Annika; Dapito, Dianne H.; Schwabe, Robert F.; Lammert, Frank

    2016-01-01

    Background The development of hepatocellular carcinoma (HCC) is a common consequence of advanced liver fibrosis but the interactions between fibrogenesis and carcinogenesis are still poorly understood. Recently it has been shown that HCC promotion depends on Toll-like receptor (TLR) 4. Pre-cancerogenous events can be modelled in mice by the administration of a single dose of diethylnitrosamine (DEN), with HCC formation depending amongst others on interleukin (IL) 6 production. Mice lacking the hepatocanalicular phosphatidylcholine transporter ABCB4 develop liver fibrosis spontaneously, resemble patients with sclerosing cholangitis due to mutations of the orthologous human gene, and represent a valid model to study tumour formation in pre-injured cholestatic liver. The aim of this study was to investigate DEN-induced liver injury in TLR4-deficient mice with biliary fibrosis. Methods ABCB4-deficient mice on the FVB/NJ genetic background were crossed to two distinct genetic backgrounds (TLR4-sufficient C3H/HeN and TLR4-deficient C3H/HeJ) for more than 10 generations. The two congenic knockout and the two corresponding wild-type mouse lines were treated with a single dose of DEN for 48 hours. Phenotypic differences were assessed by measuring hepatic collagen contents, inflammatory markers (ALT, CRP, IL6) as well as hepatic apoptosis (TUNEL) and proliferation (Ki67) rates. Results Hepatic collagen accumulation is significantly reduced in ABCB4-/-:TLR4-/-double-deficient mice. After DEN challenge, apoptosis, proliferation and inflammatory markers are decreased in TLR4-deficient in comparison to TLR4-sufficient mice. When combining ABCB4 and TLR4 deficiency with DEN treatment, hepatic IL6 expression and proliferation rates are lowest in fibrotic livers from the double-deficient line. Consistent with these effects, selective digestive tract decontamination in ABCB4-/- mice also led to reduced tumor size and number after DEN. Conclusion This study demonstrates that liver

  14. Toll-like receptor 4 senses oxidative stress mediated by the oxidation of phospholipids in extracellular vesicles.

    PubMed

    Manček-Keber, Mateja; Frank-Bertoncelj, Mojca; Hafner-Bratkovič, Iva; Smole, Anže; Zorko, Mateja; Pirher, Nina; Hayer, Silvia; Kralj-Iglič, Veronika; Rozman, Blaž; Ilc, Nejc; Horvat, Simon; Jerala, Roman

    2015-06-16

    Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4) in a manner dependent on its co-receptor MD-2. EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation