Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 21
Filter
Add more filters










Publication year range
1.
iScience ; 26(3): 106081, 2023 Mar 17.
Article in English | MEDLINE | ID: mdl-36843852

ABSTRACT

Mycobacterium infection gives rise to granulomas predominantly composed of inflammatory M1-like macrophages, with bacteria-permissive M2 macrophages also detected in deep granulomas. Our histological analysis of Mycobacterium bovis bacillus Calmette-Guerin-elicited granulomas in guinea pigs revealed that S100A9-expressing neutrophils bordered a unique M2 niche within the inner circle of concentrically multilayered granulomas. We evaluated the effect of S100A9 on macrophage M2 polarization based on guinea pig studies. S100A9-deficient mouse neutrophils abrogated M2 polarization, which was critically dependent on COX-2 signaling in neutrophils. Mechanistic evidence suggested that nuclear S100A9 interacts with C/EBPß, which cooperatively activates the Cox-2 promoter and amplifies prostaglandin E2 production, followed by M2 polarization in proximal macrophages. Because the M2 populations in guinea pig granulomas were abolished via treatment with celecoxib, a selective COX-2 inhibitor, we propose the S100A9/Cox-2 axis as a major pathway driving M2 niche formation in granulomas.

2.
J Biol Chem ; 298(7): 102100, 2022 07.
Article in English | MEDLINE | ID: mdl-35667438

ABSTRACT

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.


Subject(s)
HLA-A24 Antigen , HLA-C Antigens , Lipopeptides , Amino Acids/chemistry , HLA-A24 Antigen/chemistry , HLA-C Antigens/chemistry , Histocompatibility Antigens Class I/chemistry , Humans , Ligands , Protein Binding
3.
Antiviral Res ; 199: 105267, 2022 03.
Article in English | MEDLINE | ID: mdl-35227759

ABSTRACT

The central role of Ebola virus (EBOV) VP40 in nascent virion assembly and budding from infected host cells makes it an important therapeutic target. The mechanism of dimerization, following oligomerization of VP40 leading to the production of virus-like particles (VLP) has never been investigated for the development of therapeutic candidates against Ebola disease. Molecular dynamics-based computational screening targeted VP40 dimer with 40,000,000 compounds selected 374 compounds. A novel in vitro screening assay selected two compounds, NUSU#1 and NUSU#2. Conventional VLP assays consistently showed that both compounds inhibited EBOV VP40-mediated VLP production. Intriguingly, NUSU#1 inhibited the VP40-mediated VLP production in other ebolavirus species and the Marburg virus, but did not inhibit Lassa virus Z-mediated VLP production. These results strongly suggested that the selected compounds are potential lead drug candidates against Filovirus disease via disruption of VP40-mediated particle production.


Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Marburgvirus , Ebolavirus/chemistry , Humans , Viral Matrix Proteins/chemistry , Virus Release
4.
Cell Struct Funct ; 45(2): 155-163, 2020 Dec 25.
Article in English | MEDLINE | ID: mdl-33191384

ABSTRACT

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z'-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl)-pyrido[4',3':4,5]thieno[2,3-d]pyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly.Key words: Arenavirus, Förster resonance energy transfer, anti-viral drugs, Z protein.


Subject(s)
Antiviral Agents , Arenavirus/physiology , Biosensing Techniques , Fluorescence Resonance Energy Transfer , Viral Proteins/metabolism , Virus Assembly/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , HEK293 Cells , HeLa Cells , Humans
5.
Int Immunol ; 32(12): 805-810, 2020 11 23.
Article in English | MEDLINE | ID: mdl-32720986

ABSTRACT

The covalent conjugation of a 14-carbon fatty acid (myristic acid) to the N-terminal Gly residue, termed N-myristoylation, occurs in some viral proteins to dictate their pathological function. This protein lipidation reaction, however, is monitored by host cytotoxic T lymphocytes that are capable of recognizing N-terminal lipopeptide fragments in the context of major histocompatibility complex (MHC) class I molecules. In a rhesus model of human AIDS, for example, the classical MHC class I allomorph, Mamu-B*05104, was shown to bind SIV Nef-derived 4-mer lipopeptides (myristic acid-Gly-Gly-Ala-Ile; C14nef4) and present them to the CD8+ T-cell line, SN45. These lipopeptides accommodated in MHC class I molecules expose much shorter peptide chains than conventional MHC class I-presented 8-10-mer peptides, and the molecular mechanisms by which αß T-cell receptors (TCRs) recognize lipopeptides currently remain unclear. An X-ray crystallographic analysis of the SN45 TCR α and ß heterodimer in a form that was co-crystallized with the C14nef4-bound Mamu-B*05104 complex indicated that the amide group of the N-myristoylated glycine residue offered a primary T-cell epitope by establishing a sole hydrogen bond between its nitrogen atom and the side chain of Glu at position 101 of CDR3ß. Accordingly, the Glu to Ala mutation at this position resulted in the loss of lipopeptide recognition. On the other hand, TCRs were positioned remotely from the peptide portion of C14nef4, and strong interactions were not observed. Thus, these observations provide novel structural insights into lipopeptide recognition by TCRs, which contrast sharply with the general molecular principle of peptide recognition.


Subject(s)
Histocompatibility Antigens Class I/chemistry , Lipopeptides/chemistry , Receptors, Antigen, T-Cell/chemistry , Crystallography, X-Ray , Histocompatibility Antigens Class I/immunology , Humans , Lipopeptides/immunology , Models, Molecular , Protein Conformation , Receptors, Antigen, T-Cell/immunology
6.
J Biol Chem ; 295(20): 6983-6991, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32269076

ABSTRACT

Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N-myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N-myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands.


Subject(s)
Histocompatibility Antigens Class I/chemistry , Lysophospholipids/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Histocompatibility Antigens Class I/immunology , Lipoylation/immunology , Lysophospholipids/immunology , Macaca mulatta , Peptides/chemistry , Peptides/immunology , Protein Structure, Quaternary , nef Gene Products, Human Immunodeficiency Virus/chemistry , nef Gene Products, Human Immunodeficiency Virus/immunology
7.
Blood Adv ; 3(13): 1989-2002, 2019 07 09.
Article in English | MEDLINE | ID: mdl-31270081

ABSTRACT

Signal transducer and activator of transcription 3 (STAT3) exists in 2 alternatively spliced isoforms, STAT3α and STAT3ß. Although truncated STAT3ß was originally postulated to act as a dominant-negative form of STAT3α, it has been shown to have various STAT3α-independent regulatory functions. Recently, STAT3ß gained attention as a powerful antitumorigenic molecule in cancer. Deregulated STAT3 signaling is often found in acute myeloid leukemia (AML); however, the role of STAT3ß in AML remains elusive. Therefore, we analyzed the STAT3ß/α messenger RNA (mRNA) expression ratio in AML patients, where we observed that a higher STAT3ß/α mRNA ratio correlated with a favorable prognosis and increased overall survival. To gain better understanding of the function of STAT3ß in AML, we engineered a transgenic mouse allowing for balanced Stat3ß expression. Transgenic Stat3ß expression resulted in decelerated disease progression and extended survival in PTEN- and MLL-AF9-dependent AML mouse models. Our findings further suggest that the antitumorigenic function of STAT3ß depends on the tumor-intrinsic regulation of a small set of significantly up- and downregulated genes, identified via RNA sequencing. In conclusion, we demonstrate that STAT3ß plays an essential tumor-suppressive role in AML.


Subject(s)
Disease Susceptibility , Leukemia, Myeloid, Acute/etiology , STAT3 Transcription Factor/genetics , Tumor Suppressor Proteins/genetics , Animals , Biomarkers , Biopsy , Cell Line , Disease Models, Animal , Gene Expression Profiling , Gene Expression Regulation, Leukemic , Humans , Immunohistochemistry , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/mortality , Mice , Prognosis , STAT3 Transcription Factor/metabolism , Tumor Suppressor Proteins/metabolism
8.
J Immunol ; 202(12): 3349-3358, 2019 06 15.
Article in English | MEDLINE | ID: mdl-31043477

ABSTRACT

Similar to host proteins, N-myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid-modifying reaction creates a novel class of "lipopeptide" Ags targeted by host CTLs. The primate MHC class I-encoded protein, Mamu-B*098, was previously shown to bind N-myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N-myristoylated 4-mer peptide (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated glycine (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. The two central amino acid residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I-presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation.


Subject(s)
Autoantigens/metabolism , Epitopes, T-Lymphocyte/metabolism , Gene Products, nef/metabolism , Histocompatibility Antigens Class I/metabolism , Lipopeptides/metabolism , Peptides/metabolism , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigen Presentation , Autoantigens/chemistry , Autoantigens/immunology , Crystallography, X-Ray , Epitopes, T-Lymphocyte/immunology , Gene Products, nef/chemistry , Gene Products, nef/immunology , Histocompatibility Antigens Class I/genetics , Humans , Lipopeptides/chemistry , Lipopeptides/immunology , Lymphocyte Activation , Myristic Acid/chemistry , Peptides/chemistry , Peptides/immunology , Phylogeny , Primates
9.
Nat Commun ; 7: 10356, 2016 Jan 13.
Article in English | MEDLINE | ID: mdl-26758274

ABSTRACT

The covalent conjugation of a 14-carbon saturated fatty acid (myristic acid) to the amino-terminal glycine residue is critical for some viral proteins to function. This protein lipidation modification, termed N-myristoylation, is targeted by host cytotoxic T lymphocytes (CTLs) that specifically recognize N-myristoylated short peptides; however, the molecular mechanisms underlying lipopeptide antigen (Ag) presentation remain elusive. Here we show that a primate major histocompatibility complex (MHC) class I-encoded protein is capable of binding N-myristoylated 5-mer peptides and presenting them to specific CTLs. A high-resolution X-ray crystallographic analysis of the MHC class I:lipopeptide complex reveals an Ag-binding groove that is elaborately constructed to bind N-myristoylated short peptides rather than prototypic 9-mer peptides. The identification of lipopeptide-specific, MHC class I-restricted CTLs indicates that the widely accepted concept of MHC class I-mediated presentation of long peptides to CTLs may need some modifications to incorporate a novel MHC class I function of lipopeptide Ag presentation.


Subject(s)
Histocompatibility Antigens Class I/metabolism , Myristic Acid/metabolism , Receptors, Antigen, T-Cell/metabolism , Amino Acid Sequence , Animals , Crystallization , Histocompatibility Antigens Class I/chemistry , Lipoproteins/metabolism , Macaca mulatta , Molecular Sequence Data , Molecular Structure
10.
Blood Adv ; 1(3): 184-192, 2016 Dec 27.
Article in English | MEDLINE | ID: mdl-29296934

ABSTRACT

Macrophages have the potential to undergo cellular transformation into epithelioid cells, and their concentric accumulation in tissues results in the development of granulomas. Although epithelioid cells are an essential and dominant component of granulomas, other cell types have also been detected, which may contribute to the establishment of well-organized granulomas, as observed in human granulomatous diseases. We herein demonstrated that neutrophils may mediate these functions. By taking advantage of the guinea pig pulmonary granuloma model, we obtained a rat monoclonal antibody with unique reactivity to granuloma cells. This antibody, termed G213, reacted with clusters of neutrophils located in the central area of granulomas, and a biochemical analysis identified the G213-reactive antigen as S100A9, a calcium-binding protein of the S100 family, which was expressed abundantly in neutrophils. Consistent with the multifaceted functions attributed to S100A9, including its role in neutrophil extravasation and macrophage activation, the blockade of S100A9 functions with the specific inhibitor, tasquinimod, impaired the formation of organized granulomas with neutrophil cores. These results demonstrate the critical role of neutrophils and the S100A9 protein in granuloma formation. Because intragranuloma S100A9+ neutrophils were also detected in humans, these results indicate the potential of tasquinimod, a new anticancer drug candidate, for manipulating human granulomatous diseases.

11.
Oncoimmunology ; 1(7): 1027-1037, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-23170251

ABSTRACT

Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNß-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1(f/f) CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1(-/-) NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1(f/f) animals), maturation was not altered. However, NK cells derived from Ifnar1(f/f) Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1(f/f )Ncr1-iCre compared with Ifnar1(f/f) control mice. Moreover, the ability of Ifnar1(f/f) Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.

12.
Nat Immunol ; 12(1): 37-44, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21102435

ABSTRACT

The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5'-triphosphate-modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-ß and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.


Subject(s)
Avulavirus Infections/metabolism , DEAD-box RNA Helicases/metabolism , Newcastle disease virus/physiology , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae/physiology , Poly(ADP-ribose) Polymerases/metabolism , Protein Isoforms/metabolism , Avulavirus Infections/immunology , DEAD Box Protein 58 , DEAD-box RNA Helicases/immunology , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , HEK293 Cells , Humans , Immunity, Innate , Interferon Type I/genetics , Interferon Type I/metabolism , Newcastle disease virus/pathogenicity , Orthomyxoviridae/pathogenicity , Orthomyxoviridae Infections/immunology , Poly I-C/immunology , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/immunology , Protein Isoforms/genetics , Protein Isoforms/immunology , RNA, Small Interfering/genetics , RNA-Binding Proteins , Receptors, Immunologic , Signal Transduction/genetics , Signal Transduction/immunology , Virus Replication/genetics
13.
Clin Cancer Res ; 16(15): 3964-75, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20670950

ABSTRACT

PURPOSE: To develop a novel diagnostic method for the assessment of drug efficacy in chronic myeloid leukemia (CML) patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET). EXPERIMENTAL DESIGN: To develop FRET-based biosensors, we used CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and enhanced cyan fluorescent protein, so that CrkL intramolecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison with established methods including Western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells. RESULTS: After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect <1% minor drug-resistant populations within heterogeneous ones. We also noticed that this method enabled us to predict future onset of drug resistance as well as to monitor the disease status during imatinib therapy, using patient cells. CONCLUSION: In consideration of its quick and practical nature, this method is potentially a promising tool for the prediction of both current and future therapeutic responses in individual CML patients, which will be surely beneficial for both patients and clinicians.


Subject(s)
Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Fusion Proteins, bcr-abl/analysis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Adaptor Proteins, Signal Transducing/metabolism , Antineoplastic Agents/therapeutic use , Benzamides , Blotting, Western , Cell Separation , Drug Resistance, Neoplasm/drug effects , Flow Cytometry , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Luminescent Agents , Nuclear Proteins/metabolism , Phosphorylation , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Sensitivity and Specificity
14.
Exp Hematol ; 36(3): 255-64, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18207304

ABSTRACT

OBJECTIVE: Erythrocyte production is tightly regulated by cytokines, particularly erythropoietin (EPO), which affects expansion and viability of erythroid lineage cells via induction of several factors, including Bcl2-like 1 (Bcl-XL). Because type I interferon (IFN) is known to inhibit erythropoiesis, we studied mice deficient in the gene for interferon regulatory factor 2 (IRF2), which functions as a negative regulator of type I IFN signaling, in the context of erythropoiesis regulation. MATERIALS AND METHODS: We performed hematologic analyses and detected normocytic anemia in Irf2-deficient mice. RESULTS: Assessment of the maturation of erythroid progenitors in Irf2-deficient bone marrow by flow cytometry revealed a decreased number of late erythroblasts accompanied by an increased number of early erythroid progenitors. Irf2-deficient mice manifested elevated serum EPO levels, decreased Bcl-XL expression levels and enhanced apoptosis of erythroblasts, which may account for the decreased number of late erythroblasts. We further assessed the role of IRF2 in the regulation of type I IFN signaling during erythropoiesis, and found that additional homozygous mutation of IFNAR1, a subunit of type I IFN receptor complex, led to rescue of the defect of erythropoiesis in Irf2-deficient mice. CONCLUSIONS: Impaired erythropoiesis in Irf2-deficient mice results from excessive type I IFN signaling, which inhibits Bcl-XL expression in erythroid lineage cells. Our present study provides a mechanistic understanding of the potential cross-talk between type I IFN and EPO signaling pathways during erythropoiesis and may offer therapeutic insights into anemia.


Subject(s)
Anemia, Aplastic/genetics , Erythropoiesis/genetics , Interferon Regulatory Factor-2/genetics , Interferon Type I/metabolism , Signal Transduction , Anemia, Aplastic/blood , Anemia, Aplastic/pathology , Animals , Apoptosis , Bone Marrow/pathology , Cell Count , Cell Differentiation/genetics , Disease Models, Animal , Erythroblasts/pathology , Erythroid Precursor Cells/pathology , Erythropoiesis/drug effects , Flow Cytometry , Gene Expression Profiling , Hematologic Tests , Interferon Regulatory Factor-2/deficiency , Interferon Type I/pharmacology , Mice , Mice, Knockout , Mutation , Receptor, Interferon alpha-beta/genetics , Reverse Transcriptase Polymerase Chain Reaction , bcl-X Protein/genetics
15.
Nature ; 434(7036): 1035-40, 2005 Apr 21.
Article in English | MEDLINE | ID: mdl-15815647

ABSTRACT

Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity. It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88. How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88-IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88-IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.


Subject(s)
Antigens, Differentiation/metabolism , DNA-Binding Proteins/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Interferon Type I/immunology , Receptors, Immunologic/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Antigens, Differentiation/genetics , Biological Transport , Bone Marrow Cells/cytology , Cell Line , CpG Islands/genetics , DNA-Binding Proteins/genetics , Dendritic Cells/classification , Dendritic Cells/cytology , Endosomes/metabolism , Interferon Regulatory Factor-7 , Interferon Type I/metabolism , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Mice , Myeloid Differentiation Factor 88 , Receptors, Cell Surface/metabolism , Receptors, Immunologic/genetics , Time Factors , Toll-Like Receptor 9
16.
Nature ; 434(7034): 772-7, 2005 Apr 07.
Article in English | MEDLINE | ID: mdl-15800576

ABSTRACT

The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction. Using mice deficient in the Irf7 gene (Irf7-/- mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7-/- fibroblasts. Consistently, Irf7-/- mice are more vulnerable than Myd88-/- mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8+ T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.


Subject(s)
DNA-Binding Proteins/metabolism , Interferon Type I/immunology , Virus Diseases/immunology , Adaptor Proteins, Signal Transducing , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , CD8-Positive T-Lymphocytes/immunology , CpG Islands/genetics , CpG Islands/immunology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/virology , Fibroblasts , Gene Expression Regulation , Immunity, Innate/immunology , Interferon Regulatory Factor-7 , Membrane Proteins/pharmacology , Mice , Mice, Knockout , Myeloid Differentiation Factor 88 , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cell Surface/metabolism , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Toll-Like Receptor 9 , Virus Diseases/genetics
17.
Nature ; 434(7030): 243-9, 2005 Mar 10.
Article in English | MEDLINE | ID: mdl-15665823

ABSTRACT

The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity. All TLRs use the adaptor MyD88 for signalling, but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5-/- mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5-/- mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.


Subject(s)
Cytokines/genetics , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Transcription Factors/metabolism , Up-Regulation , Adaptor Proteins, Signal Transducing , Animals , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cytokines/biosynthesis , Cytokines/metabolism , Gene Deletion , Inflammation/genetics , Inflammation/metabolism , Interferon Regulatory Factors , Lipopolysaccharides/pharmacology , Mice , Myeloid Differentiation Factor 88 , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Shock, Septic/chemically induced , Shock, Septic/genetics , TNF Receptor-Associated Factor 6/metabolism , Toll-Like Receptors , Transcription Factors/deficiency , Transcription Factors/genetics
18.
Proc Natl Acad Sci U S A ; 101(43): 15416-21, 2004 Oct 26.
Article in English | MEDLINE | ID: mdl-15492225

ABSTRACT

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.


Subject(s)
Antigens, Differentiation/physiology , DNA-Binding Proteins/physiology , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Receptors, Immunologic/physiology , Signal Transduction/physiology , Transcription Factors/physiology , Transcription, Genetic , Transduction, Genetic , Adaptor Proteins, Signal Transducing , Animals , Cell Line , Energy Transfer , Fluorescence , Humans , Interferon Regulatory Factor-7 , Interferon-alpha/genetics , Mice , Myeloid Differentiation Factor 88 , Promoter Regions, Genetic , Toll-Like Receptor 7 , Toll-Like Receptor 9 , Toll-Like Receptors
19.
Nature ; 430(6999): 529-35, 2004 Jul 29.
Article in English | MEDLINE | ID: mdl-15229613

ABSTRACT

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across cell and organelle membranes. Here, we describe the crystal structure of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum, a representative member of the P-type ATPase superfamily, with an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites. In this state, the ATP analogue reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the transmembrane helices moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Calcium-Transporting ATPases/chemistry , Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/chemistry , Adenosine Triphosphate/chemistry , Animals , Binding Sites , Calcium/metabolism , Crystallography, X-Ray , Cytoplasm/metabolism , Hydrogen Bonding , Magnesium/metabolism , Models, Molecular , Phosphorylation , Protein Structure, Tertiary , Rabbits , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Water/chemistry , Water/metabolism
20.
Proc Natl Acad Sci U S A ; 101(8): 2416-21, 2004 Feb 24.
Article in English | MEDLINE | ID: mdl-14983024

ABSTRACT

The development and cooperation of distinct subsets of antigen-presenting cells, particularly dendritic cells (DCs), may be critical for maintaining homeostatic immune responses. Recently, much attention has been focused on IFN-alpha/beta, the cytokines induced en masse by virus infection or the activation of Toll-like receptors, in the context of DC activation. Here, we show that mice deficient in IFN regulatory factor 2 exhibit selective loss of CD8alpha- DCs, the so-called myeloid DCs, which is accompanied by a notable increase in CD11c-CD11bhigh other myeloid lineage cells. Such deficiency is intrinsic to the bone marrow precursors, in which the abnormal induction of IFN-alpha/beta genes causes excessive IFN signaling. The critical function of IFN regulatory factor 2 in the negative regulation of IFN-alpha/beta signaling is underscored by the observation that the deficiency is rescued by introducing an additional null mutation for the IFN receptor complex. In view of accumulating evidence of the critical role of IFN-alpha/beta signaling in DC activation, our present study offers a unique example in that the magnitude of a cytokine signal should be properly balanced in a stage-specific manner during the differentiation and activation of DCs.


Subject(s)
DNA-Binding Proteins/immunology , Dendritic Cells/immunology , Interferon-alpha/immunology , Interferon-beta/immunology , Repressor Proteins , Transcription Factors , Animals , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Flow Cytometry , Homeostasis , Immunohistochemistry , Interferon Regulatory Factor-2 , Lipopolysaccharides/toxicity , Lymphocyte Culture Test, Mixed , Mice , Mice, Knockout , Salmonella , Signal Transduction/immunology , Spleen/drug effects , Spleen/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...