Biomedical subjects
T Kaisho
Publications and source records attributed to T Kaisho.
Endotoxin-induced maturation of MyD88-deficient dendritic cells.
LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.
Essential role of nuclear factor (NF)-kappaB-inducing kinase and inhibitor of kappaB (IkappaB) kinase alpha in NF-kappaB activation through lymphotoxin beta receptor, but not through tumor necrosis factor receptor I.
Both nuclear factor (NF)-kappaB-inducing kinase (NIK) and inhibitor of kappaB (IkappaB) kinase (IKK) have been implicated as essential components for NF-kappaB activation in response to many external stimuli. However, the exact roles of NIK and IKKalpha in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKalpha in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin beta receptor (LTbetaR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IkappaBalpha in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTbetaR stimulation induced NF-kappaB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKalpha-deficient mice. Consistent with the essential role of IKKalpha in LTbetaR signaling, we found that development of Peyer's patches was defective in IKKalpha-deficient mice. These results demonstrate that both NIK and IKKalpha are essential for the induction of NF-kappaB through LTbetaR, whereas the NIK-IKKalpha pathway is dispensable in TNFR-I signaling.
IkappaB kinase alpha is essential for mature B cell development and function.
IkappaB kinase (IKK) alpha and beta phosphorylate IkappaB proteins and activate the transcription factor, nuclear factor (NF)-kappaB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKalpha is involved in skin and limb morphogenesis, whereas IKKbeta is essential for cytokine signaling. To elucidate in vivo roles of IKKalpha in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKalpha-deficient fetal liver cells. The mature B cell population was decreased in IKKalpha(-/-) chimeras. IKKalpha(-/-) chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKalpha(-/-) B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-kappaB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKalpha(-/-) chimeras. Taken together, these results demonstrate that IKKalpha is critically involved in the prevention of cell death and functional development of mature B cells.
T cell-specific loss of Pten leads to defects in central and peripheral tolerance.
PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.
Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice.
Based on recent findings in myeloid differentiation factor 88 (MyD88)- and Toll-like receptor (TLR)-knockout mice, Tsuneyasu Kaisho and Shizuo Akira discuss the roles of TLRs and MyD88 in dendritic cell (DC) maturation and cytokine production. Lipopolysaccharide binds TLR4 and can induce DC maturation in the absence of MyD88, whereas CpG DNA binds TLR9 and induces DC maturation in a MyD88-dependent manner.
Toll-like receptors: critical proteins linking innate and acquired immunity.
Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R--associated protein kinase and tumor necrosis factor receptor--activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.
Toll-like receptors and their signaling mechanism in innate immunity.
In Drosophila the Toll family, a group of transmembrane proteins, plays crucial roles in the host defense against invading pathogens. Mammalian species also conserve this system as the Toll-like receptor (TLR) family, which includes more than 10 members that have been identified so far. Both the Toll and TLR families recognize various kinds of microorganisms through pathogen-associated molecular patterns. Mammalian TLRs are expressed on macrophages and dendritic cells and mediate the signal for cytokine release or upregulation of costimulatory molecules. These activities cooperatively generate host defense mechanisms. Recently, gene targeting experiments, including ours, have contributed much to clarifying not only the function but also the signaling mechanism of TLRs. TLR2 is essential for recognizing lipopeptides and lipoproteins from several microorganisms and also peptidoglycans derived from gram-positive bacteria. TLR4 recognizes lipopolysaccharides and lipoteichoic acids from gram-negative and- positive bacteria, respectively. Furthermore, TLR9 is critical for recognizing bacterial DNAs. Thus, TLRs distinguish various immunostimulatory molecular patterns. Although TLR9 can produce similar biological responses, studies with mutant mice lacking a TLR-associating protein, MyD88, showed that TLR signaling is differentially regulated among TLR family members. Here, we describe recent progress in elucidating the function and signaling mechanisms of the TLR family.
In vitro and in vivo antibacterial activities of TAK-083, an agent for treatment of Helicobacter pylori infection.
The antibacterial activity of TAK-083 was tested against 54 clinical isolates of Helicobacter pylori and was compared with those of amoxicillin, clarithromycin, and metronidazole. The growth-inhibitory activity of TAK-083 was more potent than that of amoxicillin, clarithromycin, or metronidazole (the MICs at which 90% of the strains are inhibited were 0.031, 0.125, 64, and 8 microg/ml, respectively). The antibacterial activity of TAK-083 was highly selective against H. pylori; there was a >30-fold difference between the concentration of TAK-083 required to inhibit the growth of H. pylori and that required to inhibit the growth of common aerobic and anaerobic bacteria. Exposure of H. pylori strains to TAK-083 at the MIC or at a greater concentration resulted in an extensive loss of viability. When four H. pylori strains were successively subcultured in the medium containing subinhibitory concentrations of TAK-083, no significant change in the MICs of this compound was observed. TAK-083 strongly inhibited the formation of tryptophanyl-tRNA in H. pylori while exhibiting little effect on the same system in eukaryotes. TAK-083 was efficacious in the treatment of gastric infection caused by H. pylori in Mongolian gerbils. The results presented here indicate that TAK-083 is a promising candidate for the treatment of H. pylori infection.
A Toll-like receptor recognizes bacterial DNA.
DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
Cutting edge: essential role of phospholipase C-gamma 2 in B cell development and function.
Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-gamma 2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-gamma 2 in murine lymphopoiesis, the PLC-gamma 2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-gamma 2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-gamma 2 is a critical component of BCR signaling pathways and is required to promote B cell development.
[Critical roles of the CCAAT/enhancer-binding protein family].
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Critical roles of Toll-like receptors in host defense.
Drosophila Toll is involved not only in dorsoventral patterning of embryos but also in immune responses to microbial infection. Several Toll-like receptors (TLRs) have also been identified in mammals. They are expressed on macrophages or dendritic cells (DCs), which are essential sentinels for innate immunity. These cells utilize TLRs as a recognition and signal transducing receptor for microbial molecular components. The most characterized mammalian TLR, TLR4, is a receptor for lipopolysaccharides (LPS). TLR2 recognizes other components, such as peptideglycans (PGN). This recognition, called pattern recognition, is essential for the establishment of innate immunity, which is the basis for host defense. In this article, we review recent findings about this expanding receptor family.
The role of Toll-like receptors and MyD88 in innate immune responses.
Toll-like receptors (TLRs) are phylogenetically conserved receptors that recognize pathogen associated molecular patterns (PAMPS). We previously generated mice lacking TLR2 and TLR4 and showed the differential role of TLR2 and TLR4 in microbial recognition. TLR4 functions as the transmembrane component of the lipopolysaccharide (LPS) receptor, while TLR2 recognizes peptidoglycan from Gram-positive bacteria and lipoprotein. We also generated mice lacking MyD88, an adaptor involved in IL-1R/TLR signalings. The responses to a variety of bacterial components were completely abrogated in MyD88-deficient cells. However, unlike the signaling mediated by other bacterial components such as lipoprotein and bacterial DNA, activation of NF-kappaB and MAP kinases was induced in response to LPS even in the absence of MyD88, which indicates the existence of a MyD88-independent pathway. We have recently found that the MyD88-independent pathway is involved in LPS-induced maturation of dendritic cells (DCs).
Impairment of natural killer cytotoxic activity and interferon gamma production in CCAAT/enhancer binding protein gamma-deficient mice.
We have investigated in vivo roles of CCAAT/enhancer binding protein gamma (C/EBPgamma) by gene targeting. C/EBPgamma-deficient (C/EBPgamma(2/-)) mice showed a high mortality rate within 48 h after birth. To analyze the roles of C/EBPgamma in lymphoid lineage cells, bone marrow chimeras were established. C/EBPgamma(2/-) chimeras showed normal T and B cell development. However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells. In addition, the ability of C/EBPgamma(-/-) chimera splenocytes to produce interferon (IFN)-gamma in response to IL-12 and/or IL-18 was markedly impaired. NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPgamma(2/-) newborn spleen cells. However, they also showed lower cytotoxic activity and IFN-gamma production when stimulated with IL-12 plus IL-18 than control NK cells, as observed in C/EBPgamma(2/-) chimera splenocytes. In conclusion, our study reveals that C/EBPgamma is a critical transcription factor involved in the functional maturation of NK cells.
A novel LPS-inducible C-type lectin is a transcriptional target of NF-IL6 in macrophages.
C-type lectins serve multiple functions through recognizing carbohydrate chains. Here we report a novel C-type lectin, macrophage-inducible C-type lectin (Mincle), as a downstream target of NF-IL6 in macrophages. NF-IL6 belongs to the CCAAT/enhancer binding protein (C/EBP) of transcription factors and plays a crucial role in activated macrophages. However, what particular genes are regulated by NF-IL6 has been poorly defined in macrophages. Identification of downstream targets is required to elucidate the function of NF-IL6 in more detail. To identify downstream genes of NF-IL6, we screened a subtraction library constructed from wild-type and NF-IL6-deficient peritoneal macrophages and isolated Mincle that exhibits the highest homology to the members of group II C-type lectins. Mincle mRNA expression was strongly induced in response to several inflammatory stimuli, such as LPS, TNF-alpha, IL-6, and IFN-gamma in wild-type macrophages. In contrast, NF-IL6-deficient macrophages displayed a much lower level of Mincle mRNA induction following treatment with these inflammatory reagents. The mouse Mincle proximal promoter region contains an indispensable NF-IL6 binding element, demonstrating that Mincle is a direct target of NF-IL6. The Mincle gene locus was mapped at 0.6 centiMorgans proximal to CD4 on mouse chromosome 6.
Activating mutation in the catalytic domain of c-kit elicits hematopoietic transformation by receptor self-association not at the ligand-induced dimerization site.
The c-kit receptor tyrosine kinase (KIT) is constitutively activated by naturally occurring mutations in either the juxtamembrane domain or the kinase domain. Although the juxtamembrane domain mutations led to ligand-independent KIT dimerization, the kinase domain mutations (Asp814 --> Val or Tyr) did not. In an effort to determine if the kinase domain mutant could transfer oncogenic signaling without receptor dimerization, we have constructed the truncated types of c-kitWild and c-kitTyr814 cDNAs (c-kitDel-Wild and c-kitDel-Tyr814 cDNAs, respectively), in which ligand-binding and ligand-induced dimerization domains were deleted. When c-kitDel-Wild and c-kitDel-Tyr814 genes were introduced into a murine interleukin-3 (IL-3)-dependent cell line Ba/F3, KITDel-Tyr814 was constitutively phosphorylated on tyrosine and activated, whereas KITDel-Wild was not. In addition, Ba/F3 cells expressing KITDel-Tyr814 (Ba/F3(Del-Tyr814)) grew in suspension culture without the addition of exogenous growth factor, whereas Ba/F3 cells expressing KITDel-Wild (Ba/F3(Del-Wild)) required IL-3 for growth. The factor-independent growth of Ba/F3(Del-Tyr814) cells was virtually abrogated by coexpression of KITW42 that is a dominant-negative form of KIT, but not by that of KITWild, suggesting that KITDel-Tyr814 may not function as a monomer but may require receptor dimerization for inducing factor-independent growth. Furthermore, KITDel-Tyr814 was found to be coimmunoprecipitated with KITWild or KITW42 by an ACK2 monoclonal antibody directed against the extracellular domain of KIT. Moreover, KITW42 was constitutively associated with a chimeric FMS/KITTyr814 receptor containing the ligand-binding and receptor dimerization domain of c-fms receptor (FMS) fused to the transmembrane and cytoplasmic domain of KITTyr814, but not with a chimeric FMS/KITWild receptor even after stimulation with FMS-ligand. These results suggest that constitutively activating mutation of c-kit at the Asp814 codon may cause a conformation change that leads to receptor self-association not in the extracellular domain and that the receptor self-association of the Asp814 mutant may be important for activation of downstream effectors that are required for factor-independent growth and tumorigenicity.
Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils.
We have generated mice with a cell type-specific disruption of the Stat3 gene in macrophages and neutrophils. The mutant mice are highly susceptible to endotoxin shock with increased production of inflammatory cytokines such as TNF alpha, IL-1, IFN gamma, and IL-6. Endotoxin-induced production of inflammatory cytokines is augmented because the suppressive effects of IL-10 on inflammatory cytokine production from macrophages and neutrophils are completely abolished. The mice show a polarized immune response toward the Th1 type and develop chronic enterocolitis with age. Taken together, Stat3 plays a critical role in deactivation of macrophages and neutrophils mainly exerted by IL-10.