Biomedical subjects

Osamu Takeuchi

Publications and source records attributed to Osamu Takeuchi.

At least 37 records · Page 2Linked to original sources

PubMed2005

Essential function for the kinase TAK1 in innate and adaptive immune responses.

Transforming growth factor-beta-activated kinase 1 (TAK1) has been linked to interleukin 1 receptor and tumor necrosis factor receptor signaling. Here we generated mouse strains with conditional expression of a Map3k7 allele encoding part of TAK1. TAK1-deficient embryonic fibroblasts demonstrated loss of responses to interleukin 1beta and tumor necrosis factor. Studies of mice with B cell-specific TAK1 deficiency showed that TAK1 was indispensable for cellular responses to Toll-like receptor ligands, CD40 and B cell receptor crosslinking. In addition, antigen-induced immune responses were considerably impaired in mice with B cell-specific TAK1 deficiency. TAK1-deficient cells failed to activate transcription factor NF-kappaB and mitogen-activated protein kinases in response to interleukin 1beta, tumor necrosis factor and Toll-like receptor ligands. However, TAK1-deficient B cells were able to activate NF-kappaB but not the kinase Jnk in response to B cell receptor stimulation. These results collectively indicate that TAK1 is key in the cellular response to a variety of stimuli.

Animals↗

PubMed2005

Microarray analysis identifies apoptosis regulatory gene expression in HCT116 cells infected with thermostable direct hemolysin-deletion mutant of Vibrio parahaemolyticus.

The thermostable direct hemolysin (TDH) is considered as a major virulence factor of Vibrio parahaemolyticus. We observed this potential in several human cancer cell lines by using the TDH-producing wild-type (RIMD2210633) as well as tdh-deletion mutant of V. parahaemolyticus and found that the deletion of tdh did not affect cytotoxicity to any of the cell lines tested. DNA fragmentation and annexin V staining showed that both wild-type and tdh-mutant trigger apoptosis in these cells. To understand the molecular basis of cell death in the absence of TDH, gene expression profile of human colon cancer cell line HCT116 infected with tdh-deletion mutant was carried out using human cDNA microarrays consisting of 33,000 known genes. In infected cells, differentially expressed genes including genes for early growth response, growth arrest and DNA damage, and activating transcription factor that affect programmed cell death pathways were detected. Interestingly, mutant strains having a deletion in type III secretion system 1 (TTSS1) failed to elicit DNA fragmentation in HCT116 cells. Our results strongly suggest that apoptosis requires functional TTSS1 and TTSS1-dependent translocation factor(s) to be associated with the host cell death, and thus pathogenesis of V. parahaemolyticus.

Annexin A5↗

PubMed2005

IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.

Type I interferons are central mediators for antiviral responses. Using high-throughput functional screening of interferon inducers, we have identified here a molecule we call interferon-beta promoter stimulator 1 (IPS-1). Overexpression of IPS-1 induced type I interferon and interferon-inducible genes through activation of IRF3, IRF7 and NF-kappaB transcription factors. TBK1 and IKKi protein kinases were required for the IPS-1-mediated interferon induction. IPS-1 contained an N-terminal CARD-like structure that mediated interaction with the CARD of RIG-I and Mda5, which are cytoplasmic RNA helicases that sense viral infection. 'Knockdown' of IPS-1 by small interfering RNA blocked interferon induction by virus infection. Thus, IPS-1 is an adaptor involved in RIG-I- and Mda5-mediated antiviral immune responses.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia.

Mitochondrial permeability transition (PT) is a phenomenon induced by high levels of matrix calcium and is characterized by the opening of the PT pore (PTP). Activation of the PTP results in loss of mitochondrial membrane potential, expansion of the matrix, and rupture of the mitochondrial outer membrane. Consequently, PT has been implicated in both apoptotic and necrotic cell death. Cyclophilin D (CypD) appears to be a critical component of the PTP. To investigate the role of CypD in cell death, we created a CypD-deficient mouse. In vitro, CypD-deficient mitochondria showed an increased capacity to retain calcium and were no longer susceptible to PT induced by the addition of calcium. CypD-deficient primary mouse embryonic fibroblasts (MEFs) were as susceptible to classical apoptotic stimuli as the WT, suggesting that CypD is not a central component of cell death in response to these specific death stimuli. However, CypD-deficient MEFs were significantly less susceptible than their WT counterparts to cell death induced by hydrogen peroxide, implicating CypD in oxidative stress-induced cell death. Importantly, CypD-deficient mice displayed a dramatic reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion, strongly supporting an essential role for CypD in an ischemic injury model in which calcium overload and oxidative stress have been implicated.

Animals↗

PubMed2005

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice.

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.

Animals↗

PubMed2005

Essential role of BAX,BAK in B cell homeostasis and prevention of autoimmune disease.

B cell homeostasis is maintained by a balance between the continual generation of new cells and their elimination. Here we show proapoptotic BCL-2 family members BAX and BAK are essential for regulating the number of B cells at both immature and mature developmental stages. BAX and BAK are critical mediators of B cell death induced by multiple stimuli. In addition, BAX- and BAK-deficient B cells display defective cell cycle progression to B cell receptor crosslinking and lipopolysaccharide, but not to CpG-DNA. Furthermore, inducible deletion of Bax and Bak in adult mice results in the development of severe autoimmune disease.

Animals↗

PubMed2005

Role of lipoteichoic acid in the phagocyte response to group B streptococcus.

Group B Streptococcus (GBS) cell walls potently activate phagocytes by a largely TLR2-independent mechanism. In contrast, the cell wall component lipoteichoic acid (LTA) from diverse Gram-positive bacterial species has been shown to engage TLR2. In this study we examined the role of LTA from GBS in phagocyte activation and the requirements for TLR-LTA interaction. Using cells from knockout mice and genetic complementation in epithelial cells we found that highly pure LTA from both GBS and Staphylococcus aureus interact with TLR2 and TLR6, but not TLR1, in contrast to previous reports. Furthermore, NF-kappaB activation by LTA required the integrity of two putative PI3K binding domains within TLR2 and was inhibited by wortmannin, indicating an essential role for PI3K in cellular activation by LTA. However, LTA from GBS proved to be a relatively weak stimulus of phagocytes containing approximately 20% of the activity observed with LTA from Staphylococcus aureus. Structural analysis by nuclear magnetic resonance spectrometry revealed important differences between LTA from GBS and S. aureus, specifically differences in glycosyl linkage, in the glycolipid anchor and a lack of N-acetylglucosamine substituents of the glycerophosphate backbone. Furthermore, GBS expressing LTA devoid of d-alanine residues, that are essential within immune activation by LTA, exhibited similar inflammatory potency as GBS with alanylated LTA. In conclusion, LTA from GBS is a TLR2/TLR6 ligand that might contribute to secreted GBS activity, but does not contribute significantly to GBS cell wall mediated macrophage activation.

Alanine↗

PubMed2005

Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction.

Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-alpha in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-alpha production was abolished in Irak-1-deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-kappaB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1-deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-alpha induction in pDCs.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Regulation of lipopolysaccharide-inducible genes by MyD88 and Toll/IL-1 domain containing adaptor inducing IFN-beta.

Macrophages recognize lipopolysaccharide (LPS) by Toll-like receptor 4 and activate inflammatory responses by inducing expression of various genes. TLR4 activates intracellular signaling pathways via TIR domain containing adaptor molecules, MyD88, and Toll/IL-1 domain containing adaptor inducing IFN-beta (TRIF). Although macrophages lacking MyD88 or TRIF showed impaired cytokine production, activation of intracellular signaling molecules still occurred in response to LPS in these cells. In the present study, we implemented cDNA microarrays to investigate the contribution of MyD88 and TRIF in gene expression induced by LPS stimulation. Whereas wild-type macrophages induced 148 genes in response to LPS, macrophages lacking both MyD88 and TRIF did not upregulate any genes in response to LPS. Surprisingly, 80 LPS-inducible genes were redundantly regulated by either MyD88 or TRIF. In contrast, proinflammatory cytokines and chemokines were critically regulated by MyD88 or TRIF alone. Genes critically regulated by MyD88 alone tend to be induced quickly after LPS stimulation and regulated by mRNA stability as well as transcription. Genes known to be induced by type I interferons were simply dependent on TRIF for their expression. Taken together, MyD88 and TRIF play both redundant and distinct roles in LPS-induced gene expression.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin.

Malaria parasites within red blood cells digest host hemoglobin into a hydrophobic heme polymer, known as hemozoin (HZ), which is subsequently released into the blood stream and then captured by and concentrated in the reticulo-endothelial system. Accumulating evidence suggests that HZ is immunologically active, but the molecular mechanism(s) through which HZ modulates the innate immune system has not been elucidated. This work demonstrates that HZ purified from Plasmodium falciparum is a novel non-DNA ligand for Toll-like receptor (TLR)9. HZ activated innate immune responses in vivo and in vitro, resulting in the production of cytokines, chemokines, and up-regulation of costimulatory molecules. Such responses were severely impaired in TLR9-/- and myeloid differentiation factor 88 (MyD88)-/-, but not in TLR2, TLR4, TLR7, or Toll/interleukin 1 receptor domain-containing adaptor-inducing interferon beta-/- mice. Synthetic HZ, which is free of the other contaminants, also activated innate immune responses in vivo in a TLR9-dependent manner. Chloroquine (CQ), an antimalarial drug, abrogated HZ-induced cytokine production. These data suggest that TLR9-mediated, MyD88-dependent, and CQ-sensitive innate immune activation by HZ may play an important role in malaria parasite-host interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Cell type-specific involvement of RIG-I in antiviral response.

Toll-like receptors (TLRs) play an important role in antiviral response by recognizing viral components. Recently, a RNA helicase, RIG-I, was also suggested to recognize viral double-stranded RNA. However, how these molecules contribute to viral recognition in vivo is poorly understood. We show by gene targeting that RIG-I is essential for induction of type I interferons (IFNs) after infection with RNA viruses in fibroblasts and conventional dendritic cells (DCs). RIG-I induces type I IFNs by activating IRF3 via IkappaB kinase-related kinases. In contrast, plasmacytoid DCs, which produce large amounts of IFN-alpha, use the TLR system rather than RIG-I for viral detection. Taken together, RIG-I and the TLR system exert antiviral responses in a cell type-specific manner.

Animals↗

PubMed2005

Involvement of toll-like receptor 2 in experimental invasive pulmonary aspergillosis.

Aspergillus fumigatus, an opportunistic fungal pathogen, causes severe and usually fatal invasive pulmonary aspergillosis in immunocompromised hosts. Interestingly, Drosophila cells lacking the Toll protein are prone to A. fumigatus infection. In the current study, we looked for the involvement of Toll-like receptor 2 (TLR2) in the recognition of A. fumigatus by analyzing the in vivo and ex vivo responses of immunocompromised TLR2(-/-) and TLR2(+/+) mice to this fungus. Upon intratracheal administration of conidia, survival and tumor necrosis factor alpha (TNF-alpha), interleukin-12, and macrophage inhibitory protein-2 alpha concentrations in the airspaces of TLR2(-/-) mice were significantly lower than those of TLR2(+/+) animals. In vitro analysis of TNF-alpha production by conidia-challenged alveolar macrophages from TLR2(-/-) revealed a significant deficiency in comparison with macrophages from TLR2(+/+) mice. Infected TLR2(-/-) mice also have a higher respiratory distress and a higher pathogen burden than TLR2(+/+) mice. These data demonstrate that TLR2 plays a significant role in the defense of the host against A. fumigatus infection.

Animals↗

PubMed2004

Toll-like receptor 2 mediates Staphylococcus aureus-induced myocardial dysfunction and cytokine production in the heart.

BACKGROUND: Staphylococcus aureus sepsis is associated with significant myocardial dysfunction. Toll-like receptor 2 (TLR2) mediates the inflammatory response to S aureus and may trigger an innate immune response in the heart. We hypothesized that a TLR2 deficiency would attenuate S aureus-induced cardiac proinflammatory mediator production and the development of cardiac dysfunction. METHODS AND RESULTS: Wild-type and TLR2-deficient (TLR2D) mice were studied. S aureus challenge significantly increased tumor necrosis factor, interleukin-1beta, and nitric oxide expression in hearts of wild-type mice. This response was significantly blunted in TLR2D mice. Hearts from TLR2D mice had impaired S aureus-induced activation of interleukin-1 receptor-associated kinase, c-Jun NH2 terminal kinase, nuclear factor-kappaB, and activator protein-1. Moreover, hearts from TLR2D mice were protected against S aureus-induced contractile dysfunction. CONCLUSIONS: These results show for the first time that TLR2 signaling contributes to the loss of myocardial contractility and cytokine production in the heart during S aureus sepsis.

Animals↗

PubMed2004

Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6.

Toll-like receptors (TLRs) are involved in the recognition of microbial pathogens. A subset of TLRs, TLR7, TLR8 and TLR9, induces antiviral responses by producing interferon-alpha (IFN-alpha). Production of IFN-alpha is dependent on the Toll-interleukin-1 receptor domain-containing adaptor MyD88. Here we show that MyD88 formed a complex with the transcription factor IRF7 but not with IRF3. The death domain of MyD88 interacted with an inhibitory domain of IRF7, and this interaction resulted in activation of the IFN-alpha-dependent promoters. Furthermore, the adaptor molecule TRAF6 also bound and activated IRF7. Ubiquitin ligase activity of TRAF6 was required for IRF7 activation. These results indicate that TLR-mediated IFN-alpha induction requires the formation of a complex consisting of MyD88, TRAF6 and IRF7 as well as TRAF6-dependent ubiquitination.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IkappaBzeta.

Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IkappaBzeta (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IkappaB family member Bcl-3 (refs 1-6). Transcription of IkappaBzeta is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IkappaBzeta is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IkappaBzeta-deficient cells show severe impairment of IL-6 production in response to a variety of TLR ligands as well as IL-1, but not in response to tumour-necrosis factor-alpha. Endogenous IkappaBzeta specifically associates with the p50 subunit of NF-kappaB, and is recruited to the NF-kappaB binding site of the IL-6 promoter on stimulation. Moreover, NF-kappaB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IkappaBzeta-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IkappaBzeta-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IkappaBzeta occurs earlier than transcription of these genes, some TLR/IL-1R-mediated responses may be regulated in a gene expression process of at least two steps that requires inducible IkappaBzeta.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection.

Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-beta gene induction. Recently, two IkappaB kinase (IKK)-related kinases, inducible IkappaB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-beta production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-beta and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i-deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-beta and IFN-inducible genes in both TLR-stimulated and virus-infected EFs.

Animals↗

PubMed2004

Lipopolysaccharide from Coxiella burnetii is involved in bacterial phagocytosis, filamentous actin reorganization, and inflammatory responses through Toll-like receptor 4.

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4(-/-) mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4(-/-) macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4(-/-) macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4(-/-) mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4(-/-) mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-gamma and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.

Actins↗

PubMed2004

Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense.

Toll-like receptors (TLR) are crucial pattern recognition receptors in innate immunity. The importance of TLR2 in host defense against Gram-positive bacteria has been suggested by the fact that this receptor recognizes major Gram-positive cell wall components, such as peptidoglycan and lipoteichoic acid. To determine the role of TLR2 in pulmonary Gram-positive infection, we first established that TLR2 is indispensable for alveolar macrophage responsiveness toward Streptococcus pneumoniae. Nonetheless, TLR2 gene-deficient mice intranasally inoculated with S. pneumoniae at doses varying from nonlethal (with complete clearance of the infection) to lethal displayed only a modestly reduced inflammatory response in their lungs and an unaltered antibacterial defense when compared with normal wild-type mice. These data suggest that TLR2 plays a limited role in the innate immune response to pneumococcal pneumonia, and that additional pattern recognition receptors likely are involved in host defense against this common respiratory pathogen.

Animals↗