RESUMEN
The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria-induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-3 is linked to NF-κB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by enzyme-linked immunosorbent assay for chemokines. NF-κB activation was determined using a luciferase reporter gene assay and chromatin-immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by M. catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro-inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF-κB pathway via Syk and the CARD9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM-like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Antígeno Carcinoembrionario/metabolismo , Granulocitos/fisiología , Moraxella catarrhalis/fisiología , Infecciones por Moraxellaceae/microbiología , Quinasa Syk/metabolismo , Degranulación de la Célula , Quimiocinas/metabolismo , Granulocitos/microbiología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Estallido Respiratorio , Transducción de SeñalRESUMEN
The association of the miRNA-146a polymorphism rs2910164 with atherosclerosis and restenosis was investigated. We found no association with atherosclerosis; however, we found a negative association for the G/C (P = 0.007) and a positive association for the C/C genotype with the risk of restenosis, which is the main drawback for cardiac surgery.
Asunto(s)
Reestenosis Coronaria/genética , Estudios de Asociación Genética , MicroARNs/genética , Polimorfismo Genético , Adulto , Anciano , Alelos , Estudios de Casos y Controles , Enfermedad Coronaria/genética , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oportunidad RelativaRESUMEN
BACKGROUND: Vitamin D mediates immunomodulatory functions, and beneficial functions in allergic diseases have been suggested. Vitamin D receptor gene (VDR) polymorphisms are known but have not been studied in patients with atopic dermatitis (AD). OBJECTIVES: To investigate the frequency of four common VDR gene polymorphisms in patients with AD, and their potential functional relevance. METHODS: In this case-control study, 265 patients with AD [n=142 severe AD, Scoring AD index (SCORAD) > 40; n=123 moderate AD, SCORAD 15-40] and 265 healthy controls were genotyped for four common VDR gene polymorphisms by restriction fragment length polymorphism analysis. The VDR haplotype sequences were analysed in silico. Baseline and activation-induced gene expression of VDR and the vitamin D metabolizing enzyme CYP24A1 were analysed in monocytes of homozygous VDR haplotype carriers by quantitative reverse transcription-polymerase chain reaction. RESULTS: In patients with severe AD, the VDR BsmI (rs1544410) G allele, ApaI (rs7975232) C allele and TaqI (rs731236) T alleles were over-represented compared with healthy controls. These single nucleotide polymorphisms (SNP) were tightly linked, and the VDR haplotype GCT was correlated with severe AD and complementary AAC with protection from AD. The VDR haplotype GCT region is evolutionarily conserved. The VDR FokI (rs2228570) SNP was not associated with AD. Baseline VDR expression in monocytes and short-term activation were haplotype independent. CONCLUSION: A specific VDR haplotype is more frequent in patients with severe AD. These data indicate that VDR contributes to the control of AD, e.g. by regulation of the epidermal barrier function and/or local immune response.
Asunto(s)
Dermatitis Atópica/genética , Polimorfismo de Nucleótido Simple/genética , Receptores de Calcitriol/genética , Adulto , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Estudios de Casos y Controles , Haplotipos/genética , Homocigoto , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease with a multifactorial pathogenesis and increasing incidence in the Western world. A genetically determined defective function of pattern recognition receptors such as toll-like receptors (TLRs) has been proposed as a candidate mechanism in the pathogenesis of AD. AIM: To study the impact of genetic predisposition of five genes encoding for pattern recognition-related molecules for the phenotype of AD. METHODS: We examined nine different single-nucleotide polymorphism (SNP) frequencies in the genes encoding TLR1, -2, -4, -9 and the adapter molecule TIRAP by PCR with subsequent melting curve analysis in a case/control cohort of 136 adult AD patients and 129 age and gender matched non-atopic, healthy individuals. TLR2-expression and -function in cells from genotyped individuals were analysed. RESULTS: For the SNPs examined, similar genotype frequencies were found in both groups. In a subgroup of patients suffering from severe AD (SCORAD >50), a significantly increased representation of the A-allele in position -16934 of the tlr2 gene was present (P = 0.004). Constitutive tlr2 mRNA expression in peripheral monocytes was independent of this tlr2 promoter SNP. Stimulation assays indicated that IL-6, but not TNF-alpha secretion following TLR2 stimulation is reduced in homozygous tlr2-16934-A allele carriers. CONCLUSION: These data indicate that TLR2 is relevant for the phenotype of severe AD in adults.
Asunto(s)
Dermatitis Atópica/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Receptor Toll-Like 2/genética , Adulto , Estudios de Casos y Controles , Células Cultivadas , Dermatitis Atópica/inmunología , Dermatitis Atópica/fisiopatología , Femenino , Genotipo , Humanos , Interleucina-6/metabolismo , Masculino , Monocitos/inmunología , Monocitos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS: We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS: This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.
Asunto(s)
Predisposición Genética a la Enfermedad , Infecciones por VIH/genética , Receptor Toll-Like 8/genética , Adulto , Línea Celular , Progresión de la Enfermedad , Exones , Femenino , Regulación de la Expresión Génica/fisiología , Genotipo , Humanos , Masculino , Persona de Mediana Edad , FN-kappa B/genética , FN-kappa B/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: In the course of inflammatory bowel diseases (IBD) and acute murine ileitis following peroral Toxoplasma gondii infection, commensal Escherichia coli accumulate at inflamed mucosal sites and aggravate small intestinal immunopathology. AIM: To unravel the molecular mechanisms by which commensal E coli exacerbate ileitis. METHODS: Ileitis was investigated in mice that lack Toll-like receptors (TLR) 2 or 4, specific for bacterial lipoproteins (LP) or lipopolysaccharide (LPS), respectively. Gnotobiotic mice, in which any cultivable gut bacteria were eradicated by antibiotic treatment, were used to study the role of LPS in ileitis. RESULTS: Microbiological analyses revealed that E coli increase in the inflamed ileum. TLR4(-/-), but not TLR2(-/-), mice displayed reduced mortality and small intestinal immunopathology. Decreased interferon (IFN)-gamma and nitric oxide (NO) levels in the inflamed terminal ileum of TLR4(-/-) mice indicated that TLR4 signalling aggravates ileitis via local mediator release from immune cells. E coli strains isolated from the inflamed ileum activated cultured mouse macrophages and induced TLR4-dependent nuclear factor kappaB activation and NO production in human embryonic kidney 293 cells and in peritoneal macrophages, respectively. Most strikingly, in contrast with wild-type mice, gnotobiotic TLR4(-/-) mice were protected from induction of ileitis by treatment with purified E coli lipid A or colonisation with live E coli. Finally, prophylactic treatment with the LPS scavenger polymyxin B ameliorated T gondii-induced ileitis. CONCLUSION: These findings highlight the innate immune system as a key player in T gondii-induced ileal immunopathology. Treatment with LPS or TLR4 antagonists may represent a novel strategy for prophylaxis and/or therapy of small intestinal inflammation in IBD.
Asunto(s)
Escherichia coli/patogenicidad , Ileítis/inmunología , Lipopolisacáridos/inmunología , Receptor Toll-Like 4/inmunología , Toxoplasmosis/inmunología , Animales , Antibacterianos/uso terapéutico , Traslocación Bacteriana/inmunología , Células Cultivadas , Recuento de Colonia Microbiana , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Vida Libre de Gérmenes , Ileítis/tratamiento farmacológico , Ileítis/microbiología , Ileítis/parasitología , Íleon/microbiología , Ratones , Ratones Endogámicos C57BL , Polimixina B/uso terapéutico , Transducción de Señal/inmunología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 4/deficienciaRESUMEN
Toll-like receptors (TLRs) are key components of the innate immune system that trigger antimicrobial host defense responses. The aim of the present study was to analyze the effects of probiotic Escherichia coli Nissle strain 1917 in experimental colitis induced in TLR-2 and TLR-4 knockout mice. Colitis was induced in wild-type (wt), TLR-2 knockout, and TLR-4 knockout mice via administration of 5% dextran sodium sulfate (DSS). Mice were treated with either 0.9% NaCl or 10(7) E. coli Nissle 1917 twice daily, followed by the determination of disease activity, mucosal damage, and cytokine secretion. wt and TLR-2 knockout mice exposed to DSS developed acute colitis, whereas TLR-4 knockout mice developed significantly less inflammation. In wt mice, but not TLR-2 or TLR-4 knockout mice, E. coli Nissle 1917 ameliorated colitis and decreased proinflammatory cytokine secretion. In TLR-2 knockout mice a selective reduction of gamma interferon secretion was observed after E. coli Nissle 1917 treatment. In TLR-4 knockout mice, cytokine secretion was almost undetectable and not modulated by E. coli Nissle 1917, indicating that TLR-4 knockout mice do not develop colitis similar to the wt mice. Coculture of E. coli Nissle 1917 and human T cells increased TLR-2 and TLR-4 protein expression in T cells and increased NF-kappaB activity via TLR-2 and TLR-4. In conclusion, our data provide evidence that E. coli Nissle 1917 ameliorates experimental induced colitis in mice via TLR-2- and TLR-4-dependent pathways.
Asunto(s)
Colitis Ulcerosa/microbiología , Colitis Ulcerosa/prevención & control , Escherichia coli/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/fisiología , Animales , Línea Celular , Colitis Ulcerosa/inmunología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Probióticos , Transducción de Señal/genética , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
Genetic host factors play a substantial role in susceptibility to and severity of malaria, which continues to cause at least one million deaths per year. Recently, members of the toll-like receptor (TLR) family have been shown to be involved in recognition of the etiologic organism Plasmodium falciparum: The glycosylphosphatidylinisitol anchor induces signaling in host cells via TLR-2 and -4, while hemozoin-induced immune activation involves TLR-9. Binding of microbial ligands to the respective TLRs triggers the release of pro-inflammatory cytokines via the TLR/IL-1 receptor (TIR) domain and may contribute to the host response, including pro-inflammatory cytokine induction and malarial fever. In a case-control study among 870 Ghanaian children, we examined the influence of TLR-2, -4, and -9 polymorphisms in susceptibility to severe malaria. TLR-2 variants common in Caucasians and Asians were completely absent. However, we found a new, rare mutation (Leu658Pro), which impairs signaling via TLR-2. We failed to detect any polymorphisms within the TLR-9/interleukin-1 receptor domain. Two frequent TLR-9 promoter polymorphisms did not show a clear association with malaria severity. In contrast, the TLR-4-Asp299Gly variant occurred at a high rate of 17.6% in healthy controls, and was even more frequent in severe malaria patients (24.1%, p<0.05). Likewise, TLR-4-Thr399Ile was seen in 2.4% of healthy children and in 6.2% of patients (p=0.02). TLR-4-Asp299Gly and TLR-4-Thr399Ile conferred an 1.5- and 2.6-fold increased risk of severe malaria, respectively. These findings suggest TLR4-mediated responses to malaria in vivo and TLR-4 polymorphisms to be associated with disease manifestation. However some gray areas also suggest the scope for further improvements.
Asunto(s)
Inmunidad Innata/genética , Malaria Falciparum/inmunología , Polimorfismo de Nucleótido Simple/inmunología , Receptor Toll-Like 4/genética , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Ghana , Humanos , Lactante , Malaria Falciparum/genética , Masculino , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunologíaRESUMEN
Periodontitis is an inflammatory disease affecting the connective tissue surrounding the teeth leading to tooth loss. Pathogens associated with periodontitis interact with Toll-like receptors (TLRs) to induce cytokines causing and aggravating disease. We screened 197 individuals suffering from generalized periodontitis for the presence of Asp299Gly and Thr399Ile of TLR-4 as well as Arg753Gln of TLR-2 in comparison to matched controls. Single-nucleotide polymorphisms (SNPs) of TLR-4 were elevated among patients (odd's ratio 3.650, 95% CI 1.573-8.467, P < or = 0.0001), while no difference was observed for TLR-2. TLR-4 SNPs were correlated with chronic periodontitis (odd's ratio 5.562, 95% CI 2.199-14.04, P < or = 0.0001), but not with aggressive periodontitis. This observation was confirmed employing a group of periodontally healthy probands over 60 years of age. These data demonstrate that genetic variants of TLR-4 may act as risk factors for the development of generalized chronic periodontitis in humans.
Asunto(s)
Sustitución de Aminoácidos/genética , Periodontitis/genética , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 4/genética , Adulto , Anciano , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
This article describes the development and application of new didactic methods for use in computer-assisted teaching and learning systems for training doctors and dentists. Taking the Meducase project as an example, didactic models and their technological implementation are explained, together with the limitations of imparting knowledge with the "new media". In addition, legal concepts for a progressive, pragmatic, and innovative distribution of knowledge to undergraduate students are presented. In conclusion, potential and visions for the wide use of electronic learning in the German and European universities in the future are discussed. Self-directed learning (SDL) is a key component in both undergraduate education and lifelong learning for medical practitioners. E-learning can already be used to promote SDL at undergraduate level. The Meducase project uses self-directed, constructive, case- and problem-oriented learning within a learning platform for medical and dental students. In the long run, e-learning programs can only be successful in education if there is consistent analysis and implementation of value-added factors and the development and use of media-didactic concepts matched to electronic learning. The use of innovative forms of licensing - open source licenses for software and similar licenses for content - facilitates continuous, free access to these programs for all students and teachers. These legal concepts offer the possibility of innovative knowledge distribution, quality assurance and standardization across specializations, university departments, and possibly even national borders.
Asunto(s)
Instrucción por Computador , Educación en Odontología/métodos , Tecnología Educacional , Internet , Enseñanza/métodos , Educación Médica/métodos , Alemania , Humanos , Propiedad Intelectual , Internet/legislación & jurisprudencia , Aprendizaje Basado en Problemas , Programas de Autoevaluación , Programas Informáticos/economía , Programas Informáticos/legislación & jurisprudenciaRESUMEN
Toll-like receptors (TLRs) are key elements in the innate immune response, functioning as pattern-recognition receptors for the detection and response to endotoxins and other microbial ligands. Inflammatory cytokines play an important role in the activation of the hypothalamic-pituitary-adrenal HPA axis during inflammation and sepsis. The newly recognized major role of TLR2 and TLR4 and the adrenal stress response during critical illnesses such as inflammation and sepsis demand comprehensive analysis of their interactions. Therefore, we analyzed TLR2 and TLR4 expression in human adrenal glands. Western blot analysis demonstrated the expression of TLR2 and TLR4 in the human adrenocortical cell line NCI-H295. Immunohistochemical analysis of normal human adrenal glands revealed TLR2 and TLR4 expression in the adrenal cortex, but not in the adrenal medulla. Considering the crucial role of the HPA axis and the innate immune response during acute sepsis or septic shock, elucidating the functional interaction of these systems should be of great clinical relevance.
Asunto(s)
Corteza Suprarrenal/metabolismo , Médula Suprarrenal/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Humanos , Inmunohistoquímica , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
MD-2 is an accessory protein of the Toll-like receptor (TLR)-4, necessary for assembling a receptor complex to sense low quantities of lipopolysaccharide in order to subsequently trigger innate immune responses. MD-2 and TLR-4 are expressed on a variety of immunocompetent cells. Mutations within the TLR-4 gene have been shown to attenuate immune responses against lipopolysaccharide in mice. In humans, a TLR-4 polymorphism has been associated with a higher risk for developing severe Gram-negative sepsis and with a lower risk for atherosclerosis. Since MD-2 is an essential part of the lipopolysaccharide receptor complex, we screened 20 patients that underwent surgical cancer therapy for novel MD-2 mutations by a single-strand conformation polymorphism technique. In one patient we found an A --> G substitution at position 103, resulting in an amino-acid exchange from Thr 35 to Ala. Reporter gene assays revealed that this mutation resulted in a reduced lipopolysaccharide-induced signaling. The patient displayed an uneventful postoperative course, with the exception of slightly decreased TNF-alpha levels after in vitro stimulation with LPS as compared to wt patients. Genotyping of a further 41 patients by a newly developed Lightcycler/FRET method failed to detect any additional polymorphism carriers, indicating that this is a rare mutation.
Asunto(s)
Antígenos de Superficie/genética , Lipopolisacáridos/inmunología , Mutación , Transducción de Señal/inmunología , Alanina/genética , Antígenos de Superficie/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Antígeno 96 de los Linfocitos , Glicoproteínas de Membrana/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Receptores de Superficie Celular/metabolismo , Temperatura , Treonina/genética , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Toll-like receptor (TLR) 2 is a member of the vertebrate protein family of TLRs that has been studied in substantial detail over the last years. The extracellular domain of the type I receptor molecule TLR2 contains 18 to 20 leucine rich repeat (LRR) and LRR like motives. The intracellular domain of TLR2 contains a Toll/IL-1 receptor/resistance protein typical TIR domain. After the first implication of TLR4 in immunity thereinafter followed by the discovery of the lipopolysaccharide signal transducer function of TLR4, TLR2 was the first of ten mammalian TLRs proven to be directly involved in recognition of pathogen associated molecular patterns (PAMPs). Among the TLR2 specific agonists are microbial products representing broad groups of species such as Gram-positive and Gram-negative bacteria, as well as mycobacteria, spirochetes, and mycoplasm. PAMP induced phagosomal localization of TLR2 and TLR2 dependent apoptosis have been shown. Complex formation with other molecules involved in pattern recognition such as CD14, MD2, TLR1, and TLR6 has been implicated for TLR2. Surprisingly even proteinaceous host material such as heat shock protein (HSP) 60 has been demonstrated to activate cells through TLR2. Thus, TLR2 may be a sensor and inductor of specific defense processes, including oxidative stress and cellular necrosis initially spurred by microbial compounds. Here we summarize the current knowledge on the structure and function of TLR2, which is far from being complete. Detailed understanding of the biology of TLR2 will probably contribute to the characterization of a number of infectious diseases and potentially help in the development of novel intervention strategies.
Asunto(s)
Proteínas de Drosophila , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Secuencia de Aminoácidos , Animales , Drosophila melanogaster , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Receptores de Superficie Celular/genética , Transducción de Señal , Receptor Toll-Like 1 , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptores Toll-Like , VertebradosRESUMEN
Lipopolysaccharide-binding protein (LBP), an acute-phase protein recognizing lipopolysaccharide (LPS), catalyzes in low concentrations its transfer to the cellular LPS receptor consisting of CD14 and Toll-like receptor-4. It has recently been shown that high concentrations of recombinant LBP can protect mice in a peritonitis model from the lethal effects of LPS. To determine whether in humans the acute-phase rise of LBP concentrations can inhibit LPS binding to monocytes and induction of proinflammatory cytokines, LBP concentrations were analyzed in 63 patients meeting the American College of Chest Physicians/Society of Critical Care Medicine criteria of severe sepsis or septic shock and the ability of these sera to modulate LPS effects in vitro was assessed employing different assays. Transfer of fluorescein isothiocyanate-labeled LPS to human monocytes was assessed by a fluorescence-activated cell sorter-based method, and activation of monocytes was investigated by measuring LPS-induced tumor necrosis factor-alpha secretion in the presence of the sera. Anti-LBP antibodies and recombinant human LBP were instrumental for depletion and reconstitution of acute-phase sera and subsequent assessment of their modulating effects on LPS activity. Sera of patients with severe sepsis/septic shock exhibited a diminished LPS transfer activity and LPS-induced tumor necrosis factor-alpha secretion as compared with sera from healthy controls. LBP depletion of sepsis sera and addition of rhLBP resulting in concentrations found in severe sepsis confirmed that LBP was the major serum component responsible for the observed effects. In summary, the inhibition of LPS effects by high concentrations of LBP in acute-phase serum, as described here, may represent a novel defense mechanism of the host in severe sepsis and during bacterial infections.
Asunto(s)
Proteínas de Fase Aguda/análisis , Proteínas Portadoras/sangre , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana , Monocitos/fisiología , Sepsis/sangre , Choque Séptico/sangre , Proteínas de Fase Aguda/metabolismo , Reacción de Fase Aguda , Adulto , Anciano , Femenino , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Lipopolisacáridos/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Recently Toll-like receptors (TLRs) have been found to be involved in cellular activation by microbial products, including lipopolysaccharide, lipoproteins, and peptidoglycan. Although for these ligands the specific transmembrane signal transducers TLR-4, TLR-2, or TLR-2 and -6 have now been identified, the molecular basis of recognition of lipoteichoic acids (LTAs) and related glycolipids has not been completely understood. In order to determine the role of TLRs in immune cell activation by these stimuli, experiments involving TLR-2-negative cell lines, TLR-expression plasmids, macrophages from TLR-4-deficient C3H/HeJ-mice, and inhibitory TLR-4/MD-2 antibodies were performed. Glycolipids from Treponema maltophilum and Treponema brennaborense, as well as highly purified LTAs from Staphylococcus aureus and Bacillus subtilis exhibited TLR-2 dependence in nuclear factor kappaB activation and cytokine induction; however, T. brennaborense additionally appeared to signal via TLR-4. Fractionation of the T. brennaborense glycolipids by hydrophobic interaction chromatography and subsequent cell stimulation experiments revealed two peaks of activity, one exhibiting TLR-2-, and a second TLR-4-dependence. Furthermore, we show involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids by dominant negative overexpression experiments. In summary, the results presented here indicate that TLR-2 is the main receptor for Treponema glycolipid and LTA-mediated inflammatory response.
Asunto(s)
Proteínas de Drosophila , Glucolípidos/metabolismo , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/fisiología , FN-kappa B/metabolismo , Receptores de Superficie Celular/fisiología , Ácidos Teicoicos/metabolismo , Treponema/metabolismo , Animales , Línea Celular , Interleucina-6/biosíntesis , Ratones , Transporte de Proteínas , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent stimulation with LPS termed LPS desensitization or tolerance. To date, it is not known whether similar mechanisms of macrophage refractoriness are induced on contact with components of Gram-positive bacteria. In the present study, we demonstrate that pretreatment with highly purified lipoteichoic acid (LTA) results in suppression of cytokine release on restimulation with LTA in vitro and in vivo in both C3H/HeN and C3H/HeJ mice, but not in macrophages from Toll-like receptor (TLR)-2-deficient mice. Furthermore, desensitization in response to LPS or LTA exposure also inhibits responses to the other stimulus ("cross-tolerance"), suggesting that signaling pathways shared by TLR2 and TLR4 are impaired during tolerance. Finally, we show that LPS- or LTA-induced cross-tolerance is not transferred to hyporesponsive cells cocultured with LPS/LTA-responsive macrophages, showing that soluble mediators do not suffice for tolerance induction in neighboring cells.
Asunto(s)
Proteínas de Drosophila , Tolerancia Inmunológica , Mediadores de Inflamación/fisiología , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Ácidos Teicoicos/inmunología , Animales , Células Cultivadas , Técnicas de Cocultivo , Femenino , Tolerancia Inmunológica/genética , Inyecciones Intraperitoneales , Interleucina-1/fisiología , Interleucina-10/fisiología , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Comunicación Paracrina/inmunología , Receptores de Superficie Celular/genética , Solubilidad , Ácidos Teicoicos/administración & dosificación , Ácidos Teicoicos/farmacología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/fisiología , Proteínas de Drosophila , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Ligandos , Lipopolisacáridos/farmacología , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
We have shown previously that phenol/water extracts derived from two novel Treponema species, Treponema maltophilum, and Treponema brennaborense, resembling lipoteichoic acid (LTA), induce cytokines in mononuclear cells. This response was lipopolysaccharide binding-protein (LBP)-dependent and involved Toll-like receptors (TLRs). Here we show that secretion of tumor necrosis factor-alpha induced by Treponema culture supernatants and extracted LTA was paralleled by an LBP-dependent phosphorylation of mitogen-activated protein kinases (MAPKs) p42 and p44, and p38, as well as the stress-activated protein kinases c-Jun N-terminal kinases 1 and 2. Phosphorylation of p42/44 correlated with an increase of activity, and tumor necrosis factor-alpha levels were significantly reduced by addition of inhibitors of p42/44 and p38, PD 98059 and SB 203580, respectively. Treponeme LTA differed from bacterial lipopolysaccharide regarding time course of p42/44 phosphorylation, exhibiting a prolonged activation of MAPKs. Furthermore, MAPK activation and cytokine induction failed to be strictly correlated. Involvement of TLR-4 for phosphorylation of p42/44 was shown employing the neutralizing anti-murine TLR-4 antibody MTS 510. In TLR-2-negative U373 cells, the compounds studied differed regarding MAPK activation with T. maltophilum leading to a stronger activation. In summary, the data presented here show that treponeme LTA are able to activate the MAPK and stress-activated protein kinase pathway involving LBP and TLR-4.
Asunto(s)
Lipopolisacáridos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/metabolismo , Ácidos Teicoicos/farmacología , Treponema/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Immunoblotting , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Lipopolisacáridos/metabolismo , Macrófagos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación , Fosfotirosina/metabolismo , Piridinas/farmacología , Ácidos Teicoicos/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The complement system is an essential part of the innate defense, and C3 is an integral part of this powerful system. In previously identified complement C3 deficient guinea pigs only approx. 5% of the normal serum C3 level is detectable. No differences were found between in vitro C3 protein synthesis and C3 mRNA levels of cells from C3-deficient and wild-type animals and the amino acid sequences of both C3 proteins are identical as deduced from cDNA sequencing. Previously, the principal inability to form a C3 thiolester was discussed as a possible reason for this C3-deficiency. Here we report the isolation of two functionally different C3 species from the C3-deficient animals. Only one of these C3 proteins exhibits normal hemolytic activity and contains a thiolester group. The second C3 species is exclusively present in C3-deficient animals and lacks a thiolester, explaining its failure to express hemolytic activity. The presence of a second C3 species lacking a thiolester structure only in C3-deficient animals indicates that the stability of the thiolester may play a role in C3 deficiency. However further analysis of the in vitro stability of the thiolesters of C3 from normal and C3-deficient guinea pigs revealed no differences. A decreased in vivo thiolester stability might lead to the presence of C3 with and without a thiolester or alternatively the expression of two isoforms of C3 in these animals. Considering the central role of C3 in host defense, the mechanisms of C3 thiolester formation require further analysis.
Asunto(s)
Complemento C3/análisis , Compuestos de Sulfhidrilo/análisis , Animales , Biotina , Western Blotting/métodos , Carbohidratos/análisis , Radioisótopos de Carbono , Fraccionamiento Químico , Complemento C3/clasificación , Complemento C3/inmunología , Concanavalina A , Electroforesis en Gel de Poliacrilamida/métodos , Cobayas , Hemólisis , Marcaje Isotópico , Metilaminas , Ratones , Ratones Noqueados , Isoformas de Proteínas/análisis , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/inmunología , Dodecil Sulfato de SodioRESUMEN
Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-alpha synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-alpha secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.