Biomedical subjects

Ken J Ishii

Publications and source records attributed to Ken J Ishii.

At least 19 recordsLinked to original sources

PubMed2025

An adjuvant database for preclinical evaluation of vaccines and immunotherapeutics.

Adjuvants are immunostimulators used to enhance vaccine efficacy against infectious diseases. However, current methods for evaluating their efficacy and safety are limited, hindering large-scale screening. To address this, we developed a prototype Adjuvant Database (ADB) containing transcriptome data, generated using the same protocols as the widely used Open TG-GATEs (OTG) toxicogenomics database, covering 25 adjuvants across multiple species, organs, time points, and doses. This enabled cross-database integration of ADB and OTG. Transcriptomic patterns successfully distinguished each adjuvant regardless of organs or species. Using both databases, we built machine learning models to predict adjuvanticity and hepatotoxicity. Notably, we identified colchicine's adjuvant activity and FK565's liver toxicity through data-driven analysis. Overall, ADB combined with OTG offers a framework for transcriptomics-based, data-driven screening of adjuvant candidates.

Animals↗

PubMed2006

Cutting Edge: Pivotal function of Ubc13 in thymocyte TCR signaling.

The Ubc13 E2 ubiquitin-conjugating enzyme is essential for BCR-, TLR-, and IL-1 receptor (IL-1R)-mediated immune responses. Although Ubc13-deficient mice show defects in BCR-, TLR/IL-1R-, or CD40-mediated activation of mitogen-activated protein kinases, the function of Ubc13 in TCR-mediated signaling and responses remains uncertain. To address this, we here generated T cell-specific conditional Ubc13-deficient mice. The frequency of T lymphocytes was severely reduced in spleens from Ubc13-deficient mice. Moreover, Ubc13-deficient thymocytes displayed defective proliferation in response to anti-CD3/CD28 or PMA/ionophore stimulation. Regarding the signal transduction, although NF-kappaB activation was modestly affected, PMA/ionophore-induced activation of Jnk and p38 was profoundly impaired in Ubc13-deficient thymocytes. In addition, PMA/ionophore-mediated ubiquitination of NF-kappaB essential modulator (NEMO)/IkappaB kinase gamma (IKKgamma) and phosphorylation of TGF-beta-activated kinase 1 (TAK1) were nearly abolished in Ubc13-deficient thymocytes. Thus, Ubc13 plays an important role in thymocyte TCR-mediated signaling and immune responses.

Animals↗

PubMed2006

Pathological role of Toll-like receptor signaling in cerebral malaria.

Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8+, CCR5+ T cells and CD11c+ dendritic cells, including CD11c+, NK1.1+ and B220+ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Innate immune recognition of, and regulation by, DNA.

DNA in microbes or host cells is normally sequestered from the immune system, and therefore inert, but becomes an active immunostimulatory molecule during infection or tissue damage. Recent evidence suggests that Toll-like receptor (TLR)9, currently the only known immune sensor for DNA, recognizes more diverse elements in its ligand than initially thought, and must cooperate with additional host factors to provoke an optimal innate immune response in the physiological environment. Moreover, the innate immune system possesses a TLR9-independent, as-yet-undefined intracellular recognition machinery of double-stranded DNA that induces type I interferons through distinct signaling pathways. TLR9-dependent and TLR9-independent immune recognition of DNA might play crucial roles in DNA-associated protective immunity and in pathological autoimmunity.

Animals↗

PubMed2006

Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling.

The Ubc13 E2 ubiquitin-conjugating enzyme is key in the process of 'tagging' target proteins with lysine 63-linked polyubiquitin chains, which are essential for the transmission of immune receptor signals culminating in activation of the transcription factor NF-kappaB. Here we demonstrate that conditional ablation of Ubc13 resulted in defective B cell development and in impaired B cell and macrophage activation. In response to all tested stimuli except tumor necrosis factor, Ubc13-deficient cells showed almost normal NF-kappaB activation but considerably impaired activation of mitogen-activated protein kinase. Ubc13-induced activation of mitogen-activated protein kinase required, at least in part, ubiquitination of the adaptor protein IKKgamma. These results show that Ubc13 is key in the mammalian immune response.

Animals↗

PubMed2006

Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells.

Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 is triggered by bacterial flagellin. Here we generated Tlr5-/- mice and assessed TLR5 function in vivo. Unlike other TLRs, TLR5 was not expressed on conventional dendritic cells or macrophages. In contrast, TLR5 was expressed mainly on intestinal CD11c+ lamina propria cells (LPCs). CD11c+ LPCs detected pathogenic bacteria and secreted proinflammatory cytokines in a TLR5-dependent way. However, CD11c+ LPCs do not express TLR4 and did not secrete proinflammatory cytokines after exposure to a commensal bacterium. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5-/- mice. These data suggest that CD11c+ LPCs, via TLR5, detect and are used by pathogenic bacteria in the intestinal lumen.

Animals↗

PubMed2006

Essential role of IPS-1 in innate immune responses against RNA viruses.

IFN-beta promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1-deficient mice showed severe defects in both RIG-I- and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus-induced interferon regulatory factor-3 and nuclear factor kappaB activation was also impaired in IPS-1-deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.

The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.

Animals↗

PubMed2006

Contribution of interferon-beta to the immune activation induced by double-stranded DNA.

Introducing double-stranded DNA (dsDNA) into the cytoplasm of macrophages and dendritic cells triggers the activation of these professional antigen-presenting cells (APCs). This process is characterized by the up-regulation of costimulatory molecules and the production of various cytokines, chemokines, and antibacterial/viral factors. Current findings indicate that interferon-beta (IFN-beta) plays a key role in the stimulatory cascade triggered by dsDNA. Both immune and non-immune cells respond to intracytoplasmic dsDNA by up-regulating IFN-beta) expression, a process that reduces host susceptibility to infection. The immune activation induced by dsDNA is independent of MyD88, TRIF and DNA-PKcs, indicating that a Toll-like receptor-independent mechanism underlies the cellular activation mediated by intracytoplasmic dsDNA.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Toll-like receptor adaptor molecules enhance DNA-raised adaptive immune responses against influenza and tumors through activation of innate immunity.

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.

Adaptor Proteins, Signal Transducing↗

PubMed2006

'Toll' gates for future immunotherapy.

Toll-like receptors (TLRs) are evolutionary conserved transmembrane proteins that recognize a unique pattern of molecules derived from pathogens or damaged cells, triggering robust but defined innate immune responses. TLR-mediated innate and/or adaptive immune responses play an important role in a variety of diseases including infectious diseases, sepsis, autoimmune diseases, allergy, and atherosclerosis. Each TLR displays a differential expression pattern, intracellular localization and signaling pathway, resulting in distinct immune responses. A variety of new TLR ligands including agonists (e.g. urinary Tamm-Horsfall glycoprotein as a TLR4 ligand, siRNA as TLR3 or 7 ligand, Plasmodium falciparum Hemozoin as a TLR9 ligand, Profilin-like protein in Toxoplasma gondii as a TLR11 ligand) and antagonists (G-rich oligodeoxynucleotides as antagonist for TLR9) have been identified, and some of other TLR ligands are already under clinical trials. The manipulation or intervention of TLR-mediated immune responses is a possible multiple 'Toll' gate for future developments of immunotherapies.

Immunotherapy↗

PubMed2005

Innate immune recognition of nucleic acids: beyond toll-like receptors.

During infection or tissue damage, the innate immune system detects and responds to nucleic acids released from pathogens or damaged host cells. Accumulating evidence has showed that specific sequences, modifications or structures of nucleic acids influence their immunomodulatory activities. Resulting innate immune modulations are regulated by Toll-like receptor (TLR)-dependent or -independent signaling pathways. The first step in host defense against foreign or unwelcome self nucleic acids may play important roles in immune responses against infectious organisms, as well as in clearance of unnecessary tissues, which may be linked to autoimmune diseases and possibly to other immunological disorders. Elucidating mechanisms of innate immune activation by nucleic acids will help future development of more efficient or safer nucleic acid-based immunotherapies and gene therapies.

CpG Islands↗

PubMed2005

A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA.

The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-beta through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kappaB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-beta and NF-kappaB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9.

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9(-/-) mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9(-/-), but absent in MyD88(-/-) mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-alpha and IFN-gamma, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.

Animals↗

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

Effect of plasmid backbone modification by different human CpG motifs on the immunogenicity of DNA vaccine vectors.

DNA vaccines, in general, have been found to be poorly immunogenic in nonhuman primates and humans as compared with mice. As the immunogenicity of DNA plasmids relies, to a large extent, on the presence of CpG motifs as built in adjuvants, we addressed the issue of poor immunogenicity by inserting recently identified CpG oligonucleotides (ODN) optimal for human (K-type or D-type CpG ODN) into the backbone of plasmid VR1020. We found that plasmid DNA containing K-type CpG motifs or D-type CpG motifs significantly enhanced the up-regulation of surface molecules and production of interleukin-6 from human peripheral blood mononuclear cells (PBMC) and stimulated monocytes to develop into functionally mature dendritic cells (DC) compared with unmodified plasmid. Monocyte maturation into DC was through plasmacytoid DC present in the culture. It is interesting that the K-type CpG motif-modified plasmid stimulated significant levels of interferon (IFN)-gamma and IFN-alpha from human PBMC. Immunization of mice with D-type CpG motif-modified plasmid, encoding Plasmodium falciparum surface protein 25, yielded enhanced antigen-specific antibodies. Taken together, these results suggest that insertion of immunomodulatory human CpG motifs into plasmid DNA can improve immunogenicity of DNA vaccines.

Animals↗

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

CpG RNA: identification of novel single-stranded RNA that stimulates human CD14+CD11c+ monocytes.

Synthetic immunostimulatory nucleic acids such as CpG DNA are being harnessed therapeutically as vaccine adjuvants, anticancer or antiallergic agents. Efforts to identify nucleic acid-based agents capable of more specifically modulating the immune system are being developed. The current study identifies a novel class of single-stranded oligoribonucleotides (ORN) containing unmethylated CpG motifs and a poly(G) run at the 3' end (CpG ORN) that directly stimulate human CD14+CD11c+ monocytes but not dendritic cells or B cells. CpG ORN activate NF-kappaB and p38 MAPK, resulting in IL-6 and IL-12 production and costimulatory molecule up-regulation but not IFNalpha. Methylation of cytosine at the 5' portion in core CpG motif abrogates such activation. TLR3, 7, 8, or 9 alone did not confer response to CpG ORN, in contrast to previously reported respective nucleic acid ligands. These data suggest that CpG ORN represent a novel class of synthetic immunostimulatory nucleic acids with distinct target cells, receptors, and functions from that of previously known immunomodulatory nucleic acids.

3' Untranslated Regions↗