Biomedical subjects

Shizuo Akira

Publications and source records attributed to Shizuo Akira.

At least 73 records · Page 4Linked to original sources

PubMed2006

Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2.

The epidermis, which covers the surface of all mammals, serves as a front line of defense against the invasion of pathogenic microbes and acts as a crucial site for innate immune responses. Various antimicrobial molecules are expressed not only on the surfaces of monocytes but also on epithelial cells. beta-Defensins, a family of antimicrobial peptides, are produced by several types of epithelial cells, including keratinocytes. However, the induction pathways for beta-defensins in keratinocytes are not fully understood. We hypothesized that bacterial components would trigger the expression of beta-defensins in keratinocytes through a toll-like receptor (TLR)-MyD88 signaling pathway that plays important roles in innate immunity. Production of TNF-alpha and IL-1 alpha following stimulation with lipopolysaccharide or bacterial lipopeptides was completely abolished in TLR2&TLR4-doubly deficient keratinocytes and in MyD88-deficient keratinocytes. Expression of murine beta-defensin was upregulated by bacterial lipopeptides in wild-type keratinocytes, while it was attenuated in TLR2-deficient keratinocytes. To evaluate the in vivo role of TLRs in keratinocytes, we inoculated Staphylococcus aureus into the tail skin from TLR2-deficient mice that had been grafted on the dorsal skin of syngeneic mice. The grafted skin from TLR2-deficient mice resulted in erosion. These studies strongly suggest that the TLR2-MyD88-dependent pathway in keratinocytes is essential for antimicrobial activity in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2006

IC31, a novel adjuvant signaling via TLR9, induces potent cellular and humoral immune responses.

IC31, the combination of a novel immunostimulatory oligodeoxynucleotide containing deoxy-Inosine/deoxy-Cytosine (ODN1a) and the antimicrobial peptide KLKL(5)KLK, represents a promising novel adjuvant signaling via the TLR9/MyD88-dependent pathway of the innate immune system. In mice, IC31 induces potent peptide-specific type 1 cellular immune responses, as well as mainly type 1 dominated protein-specific cellular and humoral immune responses. In addition, cytotoxic T lymphocytes were induced, able to kill efficiently target cells in vivo. Activation of murine dendritic cells by IC31 induced efficiently proliferation of naïve CD4(+) TCR transgenic T cells (DO.11.10) as well as their differentiation into IFN-gamma- and IL-4-producing T cells in vitro.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction.

TRIF-related adaptor molecule (TRAM) is the fourth Toll/IL-1 resistance domain-containing adaptor to be described that participates in Toll-like receptor (TLR) signaling. TRAM functions exclusively in the TLR4 pathway. Here we show by confocal microscopy that TRAM is localized in the plasma membrane and the Golgi apparatus, where it colocalizes with TLR4. Membrane localization of TRAM is the result of myristoylation because mutation of a predicted myristoylation site in TRAM (TRAM-G2A) brought about dissociation of TRAM from the membrane and its relocation to the cytosol. Further, TRAM, but not TRAM-G2A, was radiolabeled with [3H]myristate in vivo. Unlike wild-type TRAM, overexpression of TRAM-G2A failed to elicit either IFN regulatory factor 3 or NF-kappaB signaling. Moreover, TRAM-G2A was unable to reconstitute LPS responses in bone marrow-derived macrophages from TRAM-deficient mice. These observations provide clear evidence that the myristoylation of TRAM targets it to the plasma membrane, where it is essential for LPS responses through the TLR4 signal transduction pathway, and suggest a hitherto unappreciated manner in which LPS responses can be regulated.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Toll-like receptor function and signaling.

Mammals sense pathogen invasion through pattern-recognition receptors. A group of transmembrane proteins, Toll-like receptors (TLRs), play critical roles as pattern-recognition receptors. They are mainly expressed on antigen-presenting cells, such as macrophages or dendritic cells, and their signaling activates antigen-presenting cells to provoke innate immunity and to establish adaptive immunity. Each TLR has common effects, such as inflammatory cytokine induction or upregulation of costimulatory molecule expression, but also has its specific function, exemplified by type I IFN-inducing ability. These immunoadjuvant effects are not only critical in antimicrobial immunity but are also involved in manifestations of autoimmunity. Furthermore, some TLR agonists are now promising therapeutic tools for various immune disorders, including allergy. Therefore understanding molecular mechanisms on TLRs should be quite useful in the development of therapeutic maneuvers against allergy and autoimmune diseases.

Animals↗

PubMed2006

Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance.

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

Animals↗

PubMed2006

Pathogen recognition and innate immunity.

Microorganisms that invade a vertebrate host are initially recognized by the innate immune system through germline-encoded pattern-recognition receptors (PRRs). Several classes of PRRs, including Toll-like receptors and cytoplasmic receptors, recognize distinct microbial components and directly activate immune cells. Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of overlapping and unique genes involved in the inflammatory and immune responses. New insights into innate immunity are changing the way we think about pathogenesis and the treatment of infectious diseases, allergy, and autoimmunity.

Adaptation, Physiological↗

PubMed2006

Macrophage/cancer cell interactions mediate hormone resistance by a nuclear receptor derepression pathway.

Defining the precise molecular strategies that coordinate patterns of transcriptional responses to specific signals is central for understanding normal development and homeostasis as well as the pathogenesis of hormone-dependent cancers. Here we report specific prostate cancer cell/macrophage interactions that mediate a switch in function of selective androgen receptor antagonists/modulators (SARMs) from repression to activation in vivo. This is based on an evolutionarily conserved receptor N-terminal L/HX7LL motif, selectively present in sex steroid receptors, that causes recruitment of TAB2 as a component of an N-CoR corepressor complex. TAB2 acts as a sensor for inflammatory signals by serving as a molecular beacon for recruitment of MEKK1, which in turn mediates dismissal of the N-CoR/HDAC complex and permits derepression of androgen and estrogen receptor target genes. Surprisingly, this conserved sensor strategy may have arisen to mediate reversal of sex steroid-dependent repression of a limited cohort of target genes in response to inflammatory signals, linking inflammatory and nuclear receptor ligand responses to essential reproductive functions.

Amino Acid Sequence↗

PubMed2006

Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity.

The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-yr history of use in >400 million people globally. Despite this efficacy, there is presently no information about the immunological mechanisms by which YF-17D acts. Here, we present data that suggest that YF-17D activates multiple Toll-like receptors (TLRs) on dendritic cells (DCs) to elicit a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs 2, 7, 8, and 9 to elicit the proinflammatory cytokines interleukin (IL)-12p40, IL-6, and interferon-alpha. Interestingly, the resulting adaptive immune responses are characterized by a mixed T helper cell (Th)1/Th2 cytokine profile and antigen-specific CD8+ T cells. Furthermore, distinct TLRs appear to differentially control the Th1/Th2 balance; thus, whilst MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D. Together, these data enhance our understanding of the molecular mechanism of action of YF-17D, and highlight the potential of vaccination strategies that use combinations of different TLR ligands to stimulate polyvalent immune responses.

Adaptor Proteins, Signal Transducing↗

PubMed2006

TLR1- and TLR6-independent recognition of bacterial lipopeptides.

Bacterial cell walls contain lipoproteins/peptides, which are strong modulators of the innate immune system. Triacylated lipopeptides are assumed to be recognized by TLR2/TLR1-, whereas diacylated lipopeptides use TLR2/TLR6 heteromers for signaling. Following our initial discovery of TLR6-independent diacylated lipopeptides, we could now characterize di- and triacylated lipopeptides (e.g. Pam(2)C-SK(4), Pam(3)C-GNNDESNISFKEK), which have stimulatory activity in TLR1- and in TLR6-deficient mice. Furthermore, for the first time, we present triacylated lipopeptides with short length ester-bound fatty acids (like PamOct(2)C-SSNASK(4)), which induce no response in TLR1-deficient cells. No differences in the phosphorylation of MAP kinases by lipopeptide analogs having different TLR2-coreceptor usage were observed. Blocking experiments indicated that different TLR2 heteromers recognize their specific lipopeptide ligands independently from each other. In summary, a triacylation pattern is necessary but not sufficient to render a lipopeptide TLR1-dependent, and a diacylation pattern is necessary but not sufficient to render a lipopeptide TLR6-dependent. Contrary to the current model, distinct lipopeptides are recognized by TLR2 in a TLR1- and TLR6-independent manner.

Animals↗

PubMed2006

TAK1 is a component of the Epstein-Barr virus LMP1 complex and is essential for activation of JNK but not of NF-kappaB.

Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-kappaB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-kappaB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-kappaB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IkappaB kinase gamma is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-kappaB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-kappaB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-kappaB activation in LMP1 signaling.

Animals↗

PubMed2006

Toll-like receptor 2 controls expansion and function of regulatory T cells.

Tregs play a central role in the suppression of immune reactions and prevention of autoimmune responses harmful to the host. During acute infection, however, Tregs might hinder effector T cell activity directed toward the elimination of the pathogenic challenge. Pathogen recognition receptors from the TLR family expressed by innate immune cells are crucial for the generation of effective immunity. We have recently shown the CD4CD25 Treg subset in TLR2 mice to be significantly reduced in number compared with WT littermate control mice, indicating a link between Tregs and TLR2. Here, we report that the TLR2 ligand Pam3Cys, but not LPS (TLR4) or CpG (TLR9), directly acts on purified Tregs in a MyD88-dependent fashion. Moreover, when combined with TCR stimulation, TLR2 triggering augmented Treg proliferation in vitro and in vivo and resulted in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting Tregs. Importantly, WT Tregs adoptively transferred into TLR2 mice were neutralized by systemic administration of TLR2 ligand during the acute phase of a Candida albicans infection, resulting in a 100-fold reduced C. albicans outgrowth. This demonstrates that in vivo TLR2 also controls the function of Tregs and establishes a direct link between TLRs and the control of immune responses through Tregs.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappaB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.

Adaptor Proteins, Signal Transducing↗

PubMed2006

MyD88 signaling controls autoimmune myocarditis induction.

BACKGROUND: Experimental autoimmune myocarditis (EAM) is a CD4+ T-cell-mediated mouse model of postviral cardiomyopathy. Activation of interleukin-1 type 1 and Toll-like receptors that share the common downstream adaptor molecule MyD88 is required for disease induction. The specific role of MyD88 in myocarditis, however, is not known. METHODS AND RESULTS: In contrast to control littermates, MyD88(-/-) mice were protected from myocarditis after immunization with alpha-myosin heavy chain-derived peptide (MyHC-alpha) and complete Freund's adjuvant. Disease resistance of MyD88(-/-) mice resulted from impaired expansion of heart-specific CD4+ T cells after immunization. Intrinsic defects of MyD88(-/-) CD4+ T cells were excluded. In contrast, MyD88(-/-) but not MyD88(+/+) primary antigen presenting dendritic cells (DCs) were defective in their capacity to prime CD4+ T cells. This defect mainly resulted from the inability of MyD88(-/-) DCs to release tumor necrosis factor-alpha. The critical role of MyD88 signaling in DCs in the peripheral lymphatic compartments was finally proven by repetitive injection of activated, MyHC-alpha-loaded MyD88(+/+) DCs that fully restored T-cell expansion and myocarditis in MyD88(-/-) mice. CONCLUSIONS: Autoimmune myocarditis induction depends on MyD88 signaling in self-antigen presenting cells in the peripheral compartments. We conclude that MyD88 might become a target for prevention of heart-specific autoimmunity and cardiomyopathy.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor.

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.

Animals↗

PubMed2006

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages.

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-kappaB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IkappaB kinase (IKK)-alphabeta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alphabeta. These interactions augmented MyD88- and/or IKK-alphabeta-dependent signals, leading to enhancement of the NF-kappaB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappaB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

"Endogenous adjuvant" activity of the RNA components of lupus autoantigens Sm/RNP and Ro 60.

OBJECTIVE: Most lupus patients produce autoantibodies against small ribonucleoproteins such as Sm/RNP and Ro 60 (containing U1 and Y1-Y5 RNAs, respectively). We undertook this study to investigate whether the RNA components of these antigens, which contain extensive tracts of single- and double-stranded RNA, signatures of viral infection, activate innate immunity. METHODS: U1 and Y RNAs were affinity purified from K562 cells. Murine bone marrow-derived dendritic cells (DCs), human HEK 293 cells, and murine RAW264.7 cells were stimulated with U1 RNA and other known Toll-like receptor (TLR) ligands. Expression of the interferon (IFN)-inducible gene Mx1 and other genes was quantified using real-time polymerase chain reaction, and cytokine production was measured by enzyme-linked immunosorbent assay. DC maturation was assessed using flow cytometry. RESULTS: Purified U1 and Y1-Y5 RNAs and synthetic stem-loop II of U1 RNA stimulated type I IFN (IFN-I) production by cell lines and murine bone marrow-derived DCs and promoted DC maturation (CD86 expression). U1 RNA-stimulated, but not TLR-3 ligand-stimulated, IFN-I was blocked by bafilomycin A1, indicating that immunostimulation by U1 RNA requires endosomal acidification. Myeloid differentiation factor 88-deficient cells responded poorly to U1 RNA, suggesting that an endosomal TLR, probably TLR-7, mediates the stimulatory effects of U1 RNA. U1 RNA-induced IFN-I and interleukin-6 production also were protein kinase R (PKR) dependent (abrogated by 2-aminopurine and greatly reduced in PKR-/- cells). CONCLUSION: We conclude that the RNA components of the Ro 60 (Y1-Y5 RNA) and Sm/RNP (U1 RNA) small ribonucleoproteins act as endogenous adjuvants that could play a role in the pathogenesis of autoimmunity by stimulating DC maturation and IFN-I production.

Animals↗

PubMed2006

Mechanism of pathogen-specific TLR4 activation in the mucosa: fimbriae, recognition receptors and adaptor protein selection.

The mucosal host defence discriminates pathogens from commensals, and prevents infection while allowing the normal flora to persist. Paradoxically, Toll-like receptors (TLR) control the mucosal defence against pathogens, even though the TLR recognise conserved molecules like LPS, which are shared between pathogens and commensals. This study proposes a mechanism of pathogen-specific mucosal TLR4 activation, involving adhesive ligands and their host cell receptors. TLR4 signalling was activated in CD14-negative, LPS-unresponsive epithelial cells by P fimbriated, uropathogenic Escherichia coli but not by a mutant lacking fimbriae. Epithelial TLR4 signalling in vivo involved the glycosphingolipid receptors for P fimbriae and the adaptor proteins Toll/IL-1R (TIR) domain-containing adaptor inducing IFN-beta (TRIF)/TRIF-related adaptor molecule (TRAM), but myeloid differentiation protein 88 (MyD88)/TIR domain-containing adaptor protein were not required for the epithelial response. Substituting the P fimbriae with type 1 fimbriae changed TLR4 signalling from the TRIF to the MyD88 adaptor pathway. In addition, the adaptor proteins and the fimbrial type were found to influence bacterial clearance. Trif(-/-) and Tram(-/-) mice remained infected with P fimbriated E. coli but cleared the type 1 fimbriated strain, while Myd88(-/-) mice became carriers of both the P and the type 1 fimbriated bacteria. Thus, TLR4 may be engaged specifically by pathogens, when the proper cell surface receptors are engaged by virulence ligands.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO.

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions.

Adenoviridae↗