RESUMEN
LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow-derived macrophages from TLR2-/- mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-ß without affecting cell surface expression of TLR4. LTA did not suppress IL-1ß-induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.
Asunto(s)
Lipopolisacáridos , Receptor Toll-Like 2 , Animales , Ratones , Humanos , Receptor Toll-Like 2/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/metabolismo , Ácidos Teicoicos/farmacologíaRESUMEN
BACKGROUND: Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. OBJECTIVES: We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. METHODS: After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM + HDM or vehicle and analysed. RESULTS: In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM + HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. CONCLUSIONS & CLINICAL RELEVANCE: TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident in the in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.
Asunto(s)
Asma/inmunología , Inmunidad Innata , Linfocitos/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Mezclas Complejas/química , Mezclas Complejas/toxicidad , Femenino , Interleucina-33/inmunología , Interleucina-33/farmacología , Pulmón/inmunología , Pulmón/patología , Linfocitos/patología , Ratones , Ratones Noqueados , Pyroglyphidae/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Receptor Toll-Like 2/genéticaRESUMEN
Assays using lysate reagents prepared from horseshoe crab hemocyte extract (limulus amoebocyte lysate, LAL) are commonly and widely used to detect and measure endotoxin in parenteral drugs and medical devices. However, lysate reagents suffer from lot-to-lot variations leading to possible fluctuations in testing. Also, this continued usage of lysate reagents leads to the possible decline of the horseshoe crab population. Recently, a new recombinant chromogenic reagent, PyroSmart, consisting of three recombinant factors was introduced to the market. There are now three recombinant products; two with recombinant factor C reagents and PyroSmart with the complete recombinant LAL system. We evaluated the applicability of the reagent to the harmonized bacterial endotoxins test in the United States, European and Japanese pharmacopeias. The recombinant product showed equivalent potency of thirteen endotoxins from different bacterial strains to conventional chromogenic lysate reagents as long as their assay modes are identical. All analytical characteristics or assay parameters of the reagent satisfied the acceptance criteria which are set for the use for the bacterial endotoxins test filed in the pharmacopeias. All of 109 parenteral drugs tested can be measured with PyroSmart within respective maximum allowable dilutions. The lot-to-lot variation in recovery of endotoxin added in the parenteral drugs for PyroSmart was equal to or less than those of six limulus lysate reagents. In conclusion, the present study suggests that the recombinant reagent, PyroSmart, provide a good alternative to the LAL reagents with better lot-to-lot variation.
Asunto(s)
Proteínas Bacterianas/análisis , Endotoxinas/análisis , Indicadores y Reactivos/química , BioensayoRESUMEN
The signal transducer and activator of transcription 1 (STAT1), a pivotal transcription factor in Janus kinase (JAK)-STAT signaling, regulates the expression of a wide range of immune-related genes, including interferon (IFN) regulatory factor 1 (IRF1). In this study, we found that IRF1 could induce STAT1 phosphorylation and in turn STAT1 activation. When IRF1 was transiently expressed in HEK293 cells, STAT1 phosphorylated at Y701, dimerized and bound to an oligonucleotide containing a gamma-activated sequence (GAS) derived from the IRF1 promoter. IRF1 expression also induced GAS-dependent promoter reporter activity, and phosphorylation of JAK1, a kinase upstream of STAT1. Although no direct interaction between IRF1 and STAT1 was observed, the transactivation domain of IRF1 was required for IRF1-mediated STAT1 activation, indicating the involvement of gene product(s) regulated by IRF1. Moreover, supernatants from cells expressing IRF1 induced phosphorylation of STAT1 and JAK1, and subsequent GAS binding by STAT1 that could not be blocked by treatment with antibodies against IFN-ß or IFN-γ. IFN-γ-induced STAT1 phosphorylation persisted for up to 30 h following stimulation of HEK293, but declined in IRF1-deficient HEK293 cells. IRF1-promoter activity induced by IFN-γ was also reduced in IRF1-deficient HEK293 cells, which could be rescued by complementation with IRF1. Together these results indicate that IRF1 promotes DNA binding of STAT1, which can in turn participate in a positive feedback loop of JAK-STAT signaling.
Asunto(s)
ADN/metabolismo , Factor 1 Regulador del Interferón/metabolismo , Factor de Transcripción STAT1/metabolismo , Comunicación Autocrina , Línea Celular , ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Factor 1 Regulador del Interferón/química , Factor 1 Regulador del Interferón/genética , Quinasas Janus/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
Body and excrement extracts from Dermatophagoides farinae were used to study stimulation of Toll-like receptors (TLRs). The excrement extract stimulated nuclear factor (NF)-κB-dependent reporter activity to an extent similar to lipopolysaccharide (LPS) in a mouse macrophage cell line, J774A.1, but the activity of the body extract was negligible. The excrement extract also activated NF-κB in HEK293 cells expressing TLR1/TLR2, TLR2/TLR6 and CD14/TLR4/MD-2, whereas no activation was observed in cells expressing TLR3, TLR5, TLR7, TLR8 or TLR9. Although the excrement extract required co-expression of CD14, TLR4 and MD-2 in HEK293 cells to activate NF-κB, efficient activation was still observed in I-13.35 cells, a bone-marrow macrophage cell line established from LPS-hypo-responsive C3H/HeJ mice. The excrement extract activated NF-κB in HEK293 cells expressing TLR2 alone, but the activation was significantly increased by co-expression of CD14. Polymyxin B inhibited CD14/TLR4/MD-2- and CD14/TLR2-mediated activation of NF-κB but not the activation in I-13.35 cells. These results indicate that CD14/TLR4/MD-2-dependent and CD14/TLR2-dependent mechanisms are involved in the activation of NF-κB by the excrement extract of D. farinae and suggest that the extract also contains substances that activate NF-κB through non-TLR-mediated mechanisms.
Asunto(s)
Dermatophagoides farinae , Hipersensibilidad/metabolismo , Macrófagos/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Antibacterianos/farmacología , Línea Celular , Células HEK293 , Humanos , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Polimixina B/farmacologíaRESUMEN
Eosinophils are typical effector cells associated with type 2 immune responses and play key roles in the pathogenesis of allergic diseases. These cells are activated by various stimuli, such as cytokines, chemokines, and growth factors, but the regulatory mechanisms of eosinophil effector functions remain unclear. Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR), a transmembrane protein belonging to the tumor necrosis factor (TNF) receptor superfamily, is a well-known regulatory molecule for T cell activation. Here, we show that GITR is also constitutively expressed on eosinophils and functions as a costimulatory molecule for these cells. Although degranulation was unaffected by GITR engagement of murine bone marrow-derived eosinophils, secretion of inflammatory cytokines such as interleukin (IL)-4, IL-6, and IL-13 from IL-33-activated bone marrow-derived eosinophils was augmented by anti-mouse GITR agonistic antibody (DTA-1). In conclusion, our results provide a new regulatory pathway of cytokine secretion from eosinophils in which GITR functions as a costimulatory molecule.
Asunto(s)
Eosinófilos , Glucocorticoides , Animales , Ratones , Eosinófilos/metabolismo , Proteína Relacionada con TNFR Inducida por Glucocorticoide , Receptores del Factor de Necrosis Tumoral , Citocinas/metabolismo , Factores de Necrosis Tumoral , Factores de TranscripciónRESUMEN
TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys63-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKß, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu544, Glu587, Glu706) of IRAK-1 but not its catalytic site (Asp340). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys48- and Lys63-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys63-linked polyubiquitination on IRAK-1 is reported to compete with Lys48-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Regulación hacia Abajo , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Inhibidores de Proteasoma , Factor 6 Asociado a Receptor de TNF/genética , Receptores Toll-Like/genética , Ubiquitina/metabolismoRESUMEN
Strategies for the prevention of multiorgan dysfunction (MOD) in acetaminophen (APAP)-induced acute liver failure (ALF) are an unmet need. Our study tested the hypothesis that sterile inflammation induced by APAP in a mouse model would activate toll-like receptor 4 (TLR4) in the liver and extrahepatic organs and lead to the progression of ALF and MOD and that the administration of the novel TLR4 antagonist STM28 (a peptide formed of 17 amino-acids) would prevent liver injury and associated MOD. ALF and, subsequently, MOD were induced in TLR4-knockout (KO) mice (B6.B10ScN-Tlr4 (lpsdel) /JthJ) and wild-type (WT) mice (C57BL/6) with APAP (500 mg/kg). A second set of experiments was conducted to evaluate the effects of a pretreatment with a novel TLR4 antagonist, STM28, on APAP-induced MOD in CD1 mice. Animals were sacrificed at the coma stage, and plasma, peripheral blood cells, liver, kidneys, and brain were collected. Biochemistry values and cytokines were measured. Liver and kidneys were studied histologically and were stained for TLR4 and activated Kupffer cells, and the expression of nuclear factor kappa B-p65 was quantified with western blotting. Brain water was measured in the frontal cortex. After APAP administration, TLR4-KO (NFkBp65) mice were relatively protected from liver necrosis and end-organ dysfunction and had significantly better survival than WT controls (P < 0.01). STM28 attenuated liver injury and necrosis, reduced creatinine levels, and delayed the time to a coma significantly. The increases in cytokines in the plasma and liver, including TLR4 expression and the activation of Kupffer cells, after APAP administration were reduced significantly in the STM28-treated animals. The increased number of circulating myeloid cells was reduced significantly after STM28 treatment. In conclusion, these data provide evidence for an important role of the TLR4 antagonist in the prevention of the progression of APAP-induced ALF and MOD.
Asunto(s)
Acetaminofén/efectos adversos , Fallo Hepático Agudo/inducido químicamente , Insuficiencia Multiorgánica/fisiopatología , Receptor Toll-Like 4/fisiología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Citocinas/metabolismo , Progresión de la Enfermedad , Inflamación , Lipopolisacáridos/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptidos/química , Receptor Toll-Like 4/genéticaRESUMEN
The ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to discriminate laboratory-derived antibiotic-resistant bacterial strains of known genetic origin was examined. A computer-based cluster analysis of spectral data successfully discriminated the majority of single- as well as multiple-antibiotic-resistant Escherichia coli strains examined. Cluster analysis of Staphylococcus aureus strains with different levels of novobiocin resistance showed that as the degree of resistance increased similarity to the wild-type strain decreased. These results demonstrate that MALDI-TOF MS is capable of discriminating antibiotic-resistant bacterial strains and may have potential for differentiating bacterial strains with varying degrees of antibiotic-resistance.
Asunto(s)
Farmacorresistencia Bacteriana , Escherichia coli , Staphylococcus aureus , Antibacterianos , Análisis por Conglomerados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1-102), a serine/proline/threonine-rich ProST domain (amino acids 103-197), a central kinase domain with an activation loop (amino acids 198-522), and the C-terminal C1 and C2 domains (amino acids 523-712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585-591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants-including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.
Asunto(s)
FN-kappa B , Factor 6 Asociado a Receptor de TNF , Aminoácidos , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
We evaluated the capability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to discriminate twelve Escherichia strains: E. blattae, E. fergusonii, E. hermanii and nine E. coli, whose ribosomal RNA (rRNA) gene sequence homologies are in the range of 96-100%. Similarities obtained by MALDI-TOF MS were found to be 78-92% among the E. coli strains, and 74% between E. coli and E. fergusonii. E. blattae and E. hermanii showed only 32% similarity when compared to the other species. Thus, MALDI-TOF MS provides capability of distinguishing bacterial species or even strains possessing highly conserved rRNA gene sequences.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Secuencia de Bases , ADN Bacteriano , Escherichia coli/genética , Genes de ARNr , Espectrometría de Masas/métodos , Homología de Secuencia , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.
Asunto(s)
Caspasa 3/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Lipopolisacáridos/farmacología , Venas Umbilicales/citología , Animales , Caspasa 7/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/enzimología , Activación Enzimática/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/enzimología , L-Lactato Deshidrogenasa/metabolismo , Ratones , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Factores de TiempoRESUMEN
Urban air pollution, especially in developing countries, is a crucial environmental problem. Urban aerosols may contain various kinds of substances and induce harmful effects such as allergic diseases. Therefore, it is critical to clarify the biological effects of urban aerosols on human health. In this study, we evaluated the induction of airway inflammation in vitro and in vivo due to exposure of urban aerosols. We investigated cytokine production and nuclear factor-kappaB (NF-kappaB) activation after stimulation of macrophage cells by exposure of urban aerosols. Urban aerosols were found to induce the production of interleukin (IL)-8, tumor necrosis factor-alpha and IL-1beta on macrophage cells. In addition, we showed that NF-kappaB pathway regulated the urban aerosols-induced inflammatory cytokine response. Moreover, the intranasal administration of urban aerosols resulted in increases in the total cell number in bronchoalveolar lavage and infiltration of eosinophils in lung tissue. These results indicate that urban aerosols induce respiratory inflammation and onset of inflammatory disease due to an activation of the immune system.
Asunto(s)
Aerosoles/efectos adversos , Contaminantes Atmosféricos/efectos adversos , Hiperreactividad Bronquial/inmunología , Citocinas/biosíntesis , Inflamación/inducido químicamente , Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Adyuvantes Inmunológicos/efectos adversos , Administración por Inhalación , Animales , Hiperreactividad Bronquial/inducido químicamente , Líquido del Lavado Bronquioalveolar , Línea Celular , Países en Desarrollo , Exposición a Riesgos Ambientales/efectos adversos , Eosinófilos/metabolismo , Femenino , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-8/biosíntesis , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Población UrbanaRESUMEN
MyD88 and IL-1R-associated kinase 1 (IRAK-1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL-1R superfamily, and it is known that expression of these proteins leads to the activation of NF-kappaB in a TNFR-associated factor 6 (TRAF6)-dependent manner. We found in this study, however, that a dominant-negative mutant of TRAF6, lacking the N-terminal RING and zinc-finger domain, did not inhibit IRAK-1-induced activation of NF-kappaB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88-induced activation. The dominant-negative mutant of TRAF6 did not affect the IRAK-1-induced activation, regardless of the expression level of IRAK-1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88-induced and IRAK-1-induced activation, and supplementation with the TRAF6 dominant-negative mutant did not restore the IRAK-1-induced activation. Expression of IRAK-1, but not MyD88, induced the oligomerization of TRAF6, and IRAK-1 and the TRAF6 dominant-negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88-induced and IRAK-induced activation of NF-kappaB and suggest that TRAF6 uses a distinctive mechanism to activate NF-kappaB depending on signals.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/fisiología , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Línea Celular , Humanos , Riñón , Interferencia de ARN , ARN Interferente Pequeño/genética , Eliminación de Secuencia , Bazo/enzimologíaRESUMEN
Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. It is a ligand for Toll-like receptor 4 (TLR4), which plays an essential role in innate immunity. Macrophages and dendritic cells exposed to LPS overproduce proinflammatory mediators, leading to septic shock. In this study, we screened for peptides that associate with TLR4 with a yeast two-hybrid screen using the human TLR4 extracellular domain as bait. A peptide (STM28) isolated from the screen inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in human and mouse macrophage cells and interacted with TLR4 in yeast and mammalian cells. STM28 showed no inhibitory effects against NF-kappaB activation induced by TLR1/2, TLR3 and TLR9 ligands in a mouse macrophage cell line, RAW 264. In addition, STM28 suppressed LPS-induced tumor necrosis factor-alpha production by differentiated THP-1 cells. Moreover, LPS-induced lethality in d-galactosamine-sensitized mice was significantly repressed by STM28 in a dose-dependent manner. These results demonstrate that STM28 selectivity inhibits TLR4-induced macrophage activation, and suggest that STM28 may have utility as a novel therapeutic agent for Gram-negative bacterial sepsis.
Asunto(s)
Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , FN-kappa B/efectos de los fármacos , Péptidos/farmacología , Receptor Toll-Like 4/metabolismo , Biotina/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Galactosamina/farmacología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Choque Séptico/inducido químicamente , Choque Séptico/tratamiento farmacológico , Choque Séptico/fisiopatología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Endocrine disrupting chemicals (EDCs) may potentially worsen infectious diseases because EDCs disturb human immune function by interfering with endocrine balance. To evaluate the influence of EDCs on the innate immune function of macrophages, we investigated the effects of 37 possible EDCs on lipopolysaccharide-induced activation of the IFN-beta promoter. Alachlor, atrazine, benomyl, bisphenol A, carbaryl, diethyl phthalate, dipropyl phthalate, kelthane, kepone, malathion, methoxychlor, octachlorostyrene, pentachlorophenol, nonyl phenol, p-octylphenol, simazine and ziram all inhibited the activation. Kepone and ziram showed strong inhibitory effects. Aldicarb, amitrole, benzophenone, butyl benzyl phthalate, 2,4-dichlorophenoxy acetic acid, dibutyl phthalate, 2,4-dichlorophenol, dicyclohexyl phthalate, diethylhexyl adipate, diethylhexyl phthalate, dihexyl phthalate, di-n-pentyl phthalate, methomyl, metribuzin, nitrofen, 4-nitrotoluene, permethrin, trifluralin, 2,4,5-trichlorophenoxyacetic acid and vinclozolin had no significant effects at 100 muM. These results indicate that some agrochemicals and resin-related chemicals may potentially inhibit macrophage function, which suggests that endocrine disruptors may influence the development of infectious diseases.
Asunto(s)
Disruptores Endocrinos/farmacología , Factores Inmunológicos/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/inmunología , Animales , Línea Celular , Expresión Génica/efectos de los fármacos , Genes Reporteros , Inmunidad Innata/efectos de los fármacos , Interferón beta/biosíntesis , Lipopolisacáridos/inmunología , Luciferasas/biosíntesis , Macrófagos/efectos de los fármacos , Ratones , Polisacáridos Bacterianos/inmunologíaRESUMEN
IRAK-4 plays an essential role in Toll-like receptor (TLR)/IL-1 receptor signaling. However, its signaling and regulation mechanisms have remained elusive. We have reported previously that stimulation of TLR2, TLR4 or TLR9, but not TLR3, leads to downregulation of IRAK-4 protein. Here, we show that expression of MyD88 leads to downregulation of endogenous as well as exogenously expressed IRAK-4 protein in HEK293 cells. Expression of TRIF did not cause IRAK-4 downregulation although it induced NF-kappaB activation. Expression of either a deletion mutant of MyD88 lacking its death domain or MyD88s, neither of which induced NF-kappaB activation, did not lead to IRAK-4 downregulation. MyD88-induced downregulation was observed in an IRAK-4 mutant lacking the kinase domain, but not in another mutant lacking the death domain. These results demonstrate that downregulation of IRAK-4 requires activation of the MyD88-dependent pathway and that the death domains of both MyD88 and IRAK-4 are important for this downregulation.
Asunto(s)
Regulación hacia Abajo , Quinasas Asociadas a Receptores de Interleucina-1/biosíntesis , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Línea Celular , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/metabolismo , Estructura Terciaria de Proteína , Eliminación de SecuenciaRESUMEN
We found that AKT1, a primary effector molecule of PI3K-AKT signaling, distinctively suppressed Toll-like receptor (TLR)-mediated MyD88-dependent and Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF)-dependent signaling by inhibiting NF-κB activation and IRF3 activity independently of its kinase activity. In AKT1 knockout RAW264.7 cells, lipopolysaccharide (LPS)-induced transcription and protein production of cytokines including IL-1ß and TNF-α (regulated by the MyD88-dependent pathway), as well as IFN-ß and RANTES (C-C motif chemokine ligand 5: CCL-5; regulated by the TRIF-dependent pathways) was enhanced compared to wild type cells. In response to LPS stimulation, AKT1 knockout cells also exhibited enhanced NF-κB and IFN-ß promoter activities, which were reduced to a level comparable to that in wild type cells by complementation with either AKT1 or its kinase-dead mutant (AKT1-KD). Expression of AKT1 or AKT1-KD similarly suppressed NF-κB and IFN-ß promoter activities induced by LPS and other TLR ligands in wild type cells. Analysis of NF-κB activation caused by transient expression of proteins involved in the MyD88-dependent pathway in TLR signaling revealed that AKT1 suppressed signaling that occurs between activation of IKKß and that of NF-κB. In contrast, AKT1 appeared to suppress the IFN-ß promoter through inhibition of IRF3 activity itself. These results demonstrate a novel, non-kinase function of AKT1 that inhibits TLR signaling, and suggest the multifunctional nature of AKT1.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , Animales , Citocinas/biosíntesis , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/metabolismo , Ligandos , Lipopolisacáridos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Células RAW 264.7RESUMEN
We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14-TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-kappaB-dependent reporter activity was not induced. A strong activation of NF-kappaB was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14-TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4-CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism.
Asunto(s)
Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/toxicidad , Antígeno 96 de los Linfocitos/fisiología , Receptor Toll-Like 4/fisiología , Proteínas de Fase Aguda/fisiología , Proteínas Portadoras/fisiología , Línea Celular , Humanos , Glicoproteínas de Membrana/fisiología , Transducción de SeñalRESUMEN
Helicobacter pylori is recognized as an etiologic agent of gastroduodenal diseases. Among toxic substances produced by H. pylori, LPS exhibits extremely low endotoxic activity as compared to the typical LPSs, such as that produced by Escherichia coli. We found that the LPS-low-responder stomach cancer cell line MKN28, which expresses Toll-like receptor 4 (TLR4) at extremely low levels, showed similar levels of interleukin-8 (IL-8) induction by H. pylori or E. coli LPS preparations. Weak IL-8 induction by H. pylori LPS preparations was suppressed by expression of a dominant negative mutant of TLR2 but not of TLR4. Data from luciferase reporter analysis indicated that cotransfection of TLR2-TLR1 or TLR2-TLR6 was required for the activation induced by H. pylori LPS preparations. In conclusion, the H. pylori LPS preparations significantly induce an inflammatory reaction via the receptor complex containing TLR2-TLR1 or TLR2-TLR6 but not that containing TLR4. The TLR2-TLR1 complex was preferentially recognized by the H. pylori LPS preparations over the TLR2-TLR6 complex. Whereas the magnitude of response to H. pylori LPS preparation was markedly less than that to E. coli LPS preparation in LPS-high-responder cells strongly expressing TLR4, it was comparable to that of E. coli LPS in low-responder cells expressing negligible amount of TLR4.