WO2006070860A1 - Nouveau procede de criblage pour l'inhibiteur de la cytokine inflammatoire - Google Patents

Nouveau procede de criblage pour l'inhibiteur de la cytokine inflammatoire Download PDF

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WO2006070860A1
WO2006070860A1 PCT/JP2005/024043 JP2005024043W WO2006070860A1 WO 2006070860 A1 WO2006070860 A1 WO 2006070860A1 JP 2005024043 W JP2005024043 W JP 2005024043W WO 2006070860 A1 WO2006070860 A1 WO 2006070860A1
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irf
test substance
protein
myd88
cells
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PCT/JP2005/024043
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English (en)
Japanese (ja)
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Tadatsugu Taniguchi
Akinori Takaoka
Tak W. Mak
Kenya Honda
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The University Of Tokyo
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to the use of IRF-4 and IRF-5 in the suppression of inflammatory site force-in, and specifically to a novel screening method for an inflammatory site force-in inhibitor.
  • TLRs Toll-like receptors
  • TLRs Toll-like receptors
  • LPS lipopolysaccharide
  • peptide daricans a protein present in motile bacteria.
  • flajulin a protein present in motile bacteria.
  • LPS is a ligand for TLR4
  • peptidoglycan is a ligand for TLR2
  • furadulin is a ligand for TLR5.
  • TLR9 recognizes CpGDNA
  • TLR3 recognizes double-stranded RNA and poly (I: C) functionally similar to double-stranded RNA.
  • TLR ligands other than pathogenic molecules are known, and imidazoquinoline derivatives are TLR7 ligands.
  • TLR is activated by various ligands.
  • TLR activation is central to the regulation and organization of the innate and adaptive immune systems against pathogens (Non-Patent Documents 1 and 2).
  • a typical effect caused by TLR signaling is the induction of various site force-ins and chemokines, including various inflammatory site force-ins.
  • the induction of cytoforce-ins and chemokines depends on a common molecular pathway linked by an adapter protein called MyD88 (1-3).
  • MyD88 contains a structural region called the death domain. The force death domain allows MyD88 force S to associate with further signaling molecules (Non-Patent Documents 3 and 4).
  • TLR signaling include IL-1 receptor-associated kinase 1 (IRAKI) and IRAK4 (both of which contain the death domain) and the adapter molecule TNF Receptor associated factor (TRAF) 6 is included, and these signal transduction molecules are important for the activity of NF- ⁇ B and AP-1 transcription factors (Non-patent Documents 1, 2, 5, 6). ).
  • IRAKI IL-1 receptor-associated kinase 1
  • IRAK4 both of which contain the death domain
  • TNF Receptor associated factor 6 TNF Receptor associated factor 6
  • TLR signaling is involved in the induction of site force-in is thought to have the potential to suppress excessive immune responses by controlling TLR signaling.
  • substances that regulate TLR signal transmission are expected to be useful in the treatment and prevention of diseases involving cytodynamic force-in.
  • TLR signaling is not well understood, TLR signaling is not fully activated as a target for therapeutic agents for diseases related to cytoforce-in.
  • Non-patent literature l Janeway, CA A "Jr., & Medzhitov, R. (2002) Annu. Rev. Immunol. 20, 197-216.
  • Non-Patent Document 2 Akira, S. & Takeda, K. (2004) Nat. Rev. Immunol. 4, 499-511.
  • Non-Patent Document 3 Medzhitov, R., Preston- Hurlburt, P., Kopp, E., Stadlen, A., Chen, C
  • Non-Patent Document 4 Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S. & Cao, Z. (1997) I mmunity 7, 837-847.
  • Non-Patent Document 5 Suzuki, N "Suzuki, S. & Yeh, W. C. (2002) Trends Immunol. 23, 503- 506.
  • Non-Patent Document 6 Janssens, S. & Beyaert, R. (2003) Mol. Cell 11, 293-302.
  • Non-Patent Document 7 Mamane, Y. et al. Interferon regulatory factors: the next generation.
  • Non-Patent Document 8 Tanigucm, T., Ogasawara, K., Takaoka, A. & Tanaka, N. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19, 623—6 55 (2001) .
  • Non-Patent Document 9 Levy, DE, Marie, I. & Prakash, A. Ringing the interferon alarm: diff erential regulation of gene expression at the interface between innate and adaptive i mmunity. Curr. Opin. Immunol. 15, 52-58 (2003).
  • Non-Patent Document 10 Barnes, B., Lubyova, B. & Pitha, P. M. On the role of IRF in host d efense. J. Interferon Cytokine Res. 22, 59—71 (2002)
  • the present invention has been made in view of the above situation, and the problem to be solved by the present invention is to provide a novel screening method for an inflammatory site force-in inhibitor.
  • IRF Interferon regulatory fact
  • the present inventors stimulated hematopoietic cells derived from IRF-5 gene-deficient mice (IRF-5 — / _ mice) and wild-type mice with various TLR ligands, and IRF-5 "'mice Hematopoietic cells have been shown to impair the induction of inflammation-induced site force-in such as interleukin-6 (IL-6), IL-12, and tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • IRF-5 — / _ mice IRF-5 gene-deficient mice
  • TLR ligands IRF-5 "'mice Hematopoietic cells have been shown to impair the induction of inflammation-induced site force-in such as interleukin-6 (IL-6), IL-12, and tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • IL-6 interleukin-6
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IRF-5 interacts with and is activated by MyD88 and TRA F6, and that TLR activity causes nuclear translocation of IRF-5 and activates transcription of the site force-in gene IRF-5 " ⁇ mice always showed resistance to lethal shock induced by unmethylated DNA or lipopolysaccharide (LPS), which is associated with serum levels of pro-inflammatory site force-in. Correlate with significant decline. Thus, the inventors have revealed that IRF-5 is a new major downstream regulator of the TLR_MyD88 signaling pathway and a potential target for therapeutic intervention to suppress adverse immune responses .
  • LPS lipopolysaccharide
  • IRF-5 was found to be an immune
  • a substance that suppresses the activity or expression of IRF-5 such as IRF-4, negatively regulates the expression of inflammatory site force-in and can be a novel therapeutic agent for immune diseases. That is, the present invention relates to the use of IRF-4 and IRF-5 in the control of inflammatory site force-in expression, and more specifically, provides the following inventions.
  • TLR is any one of TLR3, TLR4, TLR5, TLR7, TLR8, TLR9.
  • a screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (c):
  • the donor fluorescent protein gene and the acceptor fluorescent protein gene are linked to either the MyD88 gene or the IRF-5 gene, and the fluorescent protein gene-MyD88 gene construct and the fluorescent protein gene-IRF-5 The process of introducing the gene construct into the cell
  • the cells placed in the presence of the test substance are irradiated with an excitation wavelength peculiar to the donor fluorescent protein to excite the donor fluorescent protein, and based on the fluorescence intensity based on the donor fluorescent protein and the acceptor fluorescent protein Detecting fluorescence intensity and analyzing FRET between donor fluorescence protein and acceptor fluorescence intensity protein from fluorescence intensity based on donor fluorescence protein and fluorescence intensity based on acceptor fluorescence protein
  • step (c) FRET in the presence of the test substance analyzed in step (b) above is Selecting a test substance that is attenuated or disappears from FRET
  • a screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (d):
  • a screening method for an inflammatory site force-in inhibitor using, as an index, the amount of IRF-4 expression in an IRF-4 expressing cell when the test substance is brought into contact with the test substance,
  • Inflammatory site force in is any of IL-1, IL-6, IL-8, IL-12, IL-18, TNF- ⁇ , IFN- ⁇ .
  • a therapeutic agent for a disease associated with inflammatory site force-in comprising the MyD88-IRF-5 binding inhibitor described in (19) above.
  • FIG. L A drawing for the production of IRF-5-deficient mice.
  • a Targeted disruption of the mouse IriS gene. Construction of mouse W5 gene, targeting vector and mutant allele.
  • the black frame represents Exon (2 to 9): The first (non-coding) Exon is located approximately 4.4 kb upstream of the second Exon (NCBI database session number NC 0007 2). Arrows and asterisks indicate the translation initiation ATG sequence and termination TAA sequence, respectively.
  • Restriction enzymes H, HindIII; A, Apal.
  • b DNA plot analysis using the radiolabeled probe shown in a for the genomic tail DNA digested with Hindlll.
  • RNA (10 / zg) was extracted and analyzed for IRF-5 expression by hybridization with a cDNA probe corresponding to the 278-739 fragment. Bromination after RNA transfer to membrane Um staining was included as a control (below). Note that IRF-5 mRNA is up-regulated by poly (I: C) that activates TLR3.
  • d Immunoblot analysis of spleen cell lysates from wild type ( + / + ) mice, heterozygous ( + ”) and homozygous (" _ ) IRF-5 mutant mice. Spleen lysates were incubated with an antiserum against IRF-5 peptide, PSPEDIPSDKQ (SEQ ID NO: 95), corresponding to a region in exon 6.
  • This antiserum was also examined for specificity by preincubation with an excess of IRF-5 peptide.
  • the same membrane was blotted with anti-j8-actin as a loading control (see below). Arrows indicate nonspecific bands. This figure shows that no band corresponding to the correct size (57 kDa) of IRF-5 protein is present in the spleen from IRF-5 chi mice.
  • FIG. 2a-c Diagram of pro-inflammatory site force-in and their mRNA-induced damage in response to TLR ligands in hematopoietic cells derived from IRF-5 Chi mice.
  • cDC normal DC
  • WT wild type mice
  • IRF-5— ⁇ mice open bars
  • CpG-B type CpG ODN
  • CpG-A CpG-A
  • the supernatant was also measured for IFN-a induction. Similar results were obtained in three independent experiments. Data show mean ⁇ sd. ND not detected.
  • b Stimulate plasmacytoid DCs (pDC) derived from the spleen of WT mice (black bars) or IRF-5— ⁇ mice (white bars) (medium only) or the same as described in a In this way, stimulation with CpG-B ODN or CpG-A ODN was performed, and the concentration of cytodynamic force in the culture supernatant was measured by ELISA. Similar results were obtained in three independent experiments. Data shows average sd. ND not detected.
  • spleen macrophages from WT mice black bars
  • IRF-5— ⁇ mice open bars
  • CpG-A ODN 1.0 ⁇
  • CpG- ⁇ ODN 1.0 ⁇
  • SOUml— 1 Stimulated for 24 hours in the presence (IL-12) or absence (IL-6 and TNF-a) of IFN-y, IL-6, IL-12p40 in the culture supernatant as described in a 2 is a graph showing the concentration of TNF-a measured by ELISA.
  • FIG. 2d is a diagram following FIG. 2a-c. d, WT mouse (black bar) or IRF-5— ⁇ mouse (Open bars) spleen macrophages are left unstimulated or various TLR ligands such as TLR3 (polyinosine-polycytidylic acid [poly (I: C)]; 100 g / ml), TLR 4 (lipo Polysaccharides [LPS]; 10 ngZml), TLR5 (furazirine; 10 / z gZml) and TLR7 (8) (ss-RNA; polyuridylic acid [poly (U)]; 5 / zg / ml) 1 Stimulated for 24 hours in the presence (IL-12) or absence (IL-6 and TNF-a) of IFN- ⁇ , and TNF- ⁇ , IL- 6 is a graph showing the concentrations of 6 and IL-12p40 measured by ELISA. Data are representative of two independent experiments.
  • Fig. 2ef This is a diagram following Fig. 2d.
  • e is a graph showing the induction of mRNA of pro-inflammatory site force-in.
  • Spleen macrophages were prepared by WT and IRF-5- ⁇ mouse force and stimulated with CpG-B ODN, LPS or poly (I: C) under the same conditions as described in d.
  • Total RNA was extracted at designated time points and subsequently subjected to quantitative RT-PCR analysis.
  • f is a graph showing the induction of I ⁇ B ⁇ mRNA. Splenic macrophages were stimulated with the designated PAMP and mRNA expression was monitored as described in e.
  • FIG. 3 Diagram of the interaction of IRF-5 with MyD88 and TRAF6 adapters involved in TLR signaling.
  • a Photograph showing the intracellular localization of IRF-5 and MyD88.
  • Cells expressing YFP-IRF-5 and CFP-MyD88 were collected using an inverted fluorescence microscope equipped with a cooled CCD camera.
  • FRETC corrected FRET
  • HEK293T cells were transiently transfected with pCAGGS-HA-IRF-5 or pCAGGS-HA-IR F-3 and pME-FLAG-TRAF6. The analysis was performed as described in the upper figures. The expression levels of these molecules were also determined by analyzing whole cell lysates by immunoblotting. d, Graph showing activation of IRF-5 by MyD88 and TRAF6. Luciferase reporter construct (p-55ClBLuc) (50 ng) containing multimerized ISRE alone or HERF293T cells or designated IRF-5 (20 ng), My D88 (20 ng), TRAF6 (20 ng) Transfected with expression vectors for the combined combinations (top graph).
  • HEK293 cells expressing TLR9 cDNA were used (see method section) (graph below). Cell lysates were extracted after 24 hours of transfection and subjected to luciferase assay.
  • FIG. 4ab is a diagram showing IRF-5 activation by TLR stimulation and binding to IL-12p40 promoter.
  • CpG-B is a graph showing nuclear translocation of IRF-5 by ODN stimulation. Images of Raw 264.7 cells expressing YFP-I RF-5 were collected with a time-lapse microscope and stimulated with CpG-B ODN. Images of YFP and CFP were collected every minute, and CpG-B ODN was added 5 minutes after the start of recording. The normalized fluorescence intensity of the nuclear region identified by the DIC image was plotted against time.
  • b Myp88-dependent nuclear translocation of IRF-5 in MEF stimulated with CpG-B ODN.
  • PBabe-HA-IRF-5 was transferred to WT or MyD88—MEF by retroviral gene transfer and treated with CpG-B ODN (1.0 ⁇ ) for the specified period.
  • Cells were fractionated and 30 ⁇ g of nuclear extract was analyzed for nuclear translocation of HA-labeled IRF-5 by SDS-PAGE and immunoblotting followed by stripping and USF-2 (nuclear protein), / 3-tubulin Reprobing for (cytoplasmic protein) was performed.
  • the IRF-5 expression level in the cytosolic fraction is also shown in the figure below.
  • FIG. 4c-e This is a diagram following Fig. 4ab. c, showing the results of chromatin immunoprecipitation assembly.
  • PCR was performed to detect endogenous ISR E in the gene in the immunoprecipitated chromatin fragment: lanes 1 and 2 show the results of PCR amplification using the immunoprecipitated sample with the control antibody.
  • Lanes 3 and 4 show the results of PCR amplification of the target sequence (248 bp) in the chromatin fragment immunoprecipitated with anti-HA antibody. The results of PCR amplification using primers that detect the 3-UTR of the IL-12p40 gene are also shown.
  • ChIP assembly was performed in the same manner using a primer that allows PCR amplification of the IFN-jS promoter region (right drawing).
  • the IFN-j8 gene is not induced in these RAW 264.7 cells when stimulated by either CpG-A ODN or CpG-B ODN (H. Y-, unpublished data) and therefore serves as a negative control. Also shown is PCR amplification of all input DNA in each sample (input DNA).
  • the DNA size marker is shown on the right in bp. d, NF- ⁇ B activation in the absence of IRF-5.
  • Spleen B cells from WT, IRF-5 __ / _ and MyD88— ⁇ mice were stimulated by CpG-B ODN (0.3 M) for the specified period.
  • the activity of NF- ⁇ B was evaluated by EMSA.
  • Activation of NF- ⁇ B by poly (I: C) was essentially normal in IRF-5 thiomacrophages (AT, unpublished data).
  • e MAP kinase activation in the absence of IRF-5.
  • CpG-B ODN (0.3 M) was added to WT or IRF-5- ⁇ splenic B cells and treated for the specified period.
  • the activity of ⁇ 38 and JNK was analyzed using phospho-specific antibodies. An immunoblotting method using antibodies against ⁇ 38 and JNK was used as a loading control.
  • FIG. 5ab is a graph showing the resistance of IRF-5 chimaus to lethal shock induced by CpG-B ODN or LPS.
  • D-GalN D-galactosamine
  • 5cd is a diagram following FIG. 5ab.
  • D-GalN 8mg.
  • sera from mice injected with LPS were collected 1 hour and 2 hours after injection.
  • FIG. 6 is a photograph showing the tissue distribution of IRF-5 mRNA expression.
  • Mouse multifilament and woven Northern blot (Clontech) containing 2 ⁇ g poly (A) + RNA was hybridized with mouse IRF-5 cDNA fragment.
  • b TLR9 ligand in normal rodent cells (cDC), CpG-B oligonucleotide (ODN) stimulation (left), or LPS and poly (I: C) in splenic macrophages (right), IRF -5 Graph showing up-regulation of mRNA expression.
  • CpG-B 1.0 1.0 / ⁇ ⁇
  • LPS lasZml
  • poly (I: C) 100 g Zml
  • FIG. 7 is a diagram showing induction of IFN-a mRNA and 1FN- ⁇ mRNA by poly (I: C) in splenocytes.
  • Spleen cells from heterozygous and homozygous mice were stimulated with poly (I: C) (200 ⁇ g / ml) for 6.5 hours and total RNA was pan-IFN-a (a4 and non- ⁇ 4) or It was subjected to semi-quantitative RT-PCR analysis using any of IFN- ⁇ 8. No genomic DNA was found in any cDNA obtained by RT reaction.
  • FIG. 8 is a graph showing IL-6 induction by CpG ODN stimulation in splenic B cells.
  • WT wild-type mice
  • IRF-5— ⁇ mice open bars
  • CpG-A ODN 1.0 i u M
  • CpG-B ODN l.O ⁇ M
  • IL-6 production by ELISA as described in Figure 2a.
  • Supernatants were collected for raw determination.
  • b A graph showing the induction of IL-6 mRNA by CpG ODN in splenic B cells. Induction of mRNA was monitored by quantitative RT-PC R as described in Figure 2e.
  • FIG. 9ab is a graph showing TLR mRNA expression in splenic macrophages.
  • FIG. 9cd is a diagram following FIG. 9ab.
  • c (Left) WT and IRF-5 chi spleen B cells were treated with 1.0 / z M C pG-A or CpG-B for 12 hours, or left untreated (medium).
  • Cells were collected and analyzed by flow cytometry. Histograms show MHC class II and CD86 expression levels. (Right drawing) The ability to incubate WT and IRF-5- 'mouse-derived spleen cells with 1.0 M CpG-B for 12 hours, or left untreated (medium).
  • Histograms of cells collected and analyzed by flow cytometry show CD40 expression levels in lymphocytes (cDC) gated to CD11C + B220-.
  • cDC lymphocytes
  • FIG. 10 ISRE signs in mouse IL-6, IL-12p40, TNF-a and I ⁇ genes It is a figure which shows an complement. NCBI mouse genome databases of these genes (accession numbers N1039300, S82420, Y00467 and AB050901S1 respectively) were analyzed using TRANSFAC. This figure shows ISRE candidates whose score calculated by TFSEARCH (ver.l.3) exceeded 70.0 points. Under this criterion, four ISRE candidates are found in the promoter region of ⁇ .
  • FIG. 11 Diagram of nuclear translocation of IRF-5 by LPS or CpG-B ODN stimulation.
  • a Raw 264.7 cells expressing YF P-IRF-5 were left to be stimulated by LPS or left untreated (None). YFP images were collected every minute and LPS was added 5 minutes after the start of recording. The average intensity of the nuclear region was plotted against time.
  • b Images of Raw 264.7 cells expressing YFP-IRF-5 were collected with a Timelabs microscope and left untreated (-) or stimulated with CpG-B ODN for 2 hours (+).
  • FIG. 12 shows the interaction between IRF-4 and MyD88.
  • A A photograph showing a confocal image of HEK293T cells that transiently express YFP-IRF-4 or YFP-IRF-8 together with CFP_MyD88. The arrow indicates the coexistence of IRF-4 and MyD88.
  • B and c Analysis of intermolecular FRET between CFP-MyD88 and YFP-IRF was performed using HEK293T cells. FRETc was calculated and displayed using a pseudo color image (b) or FRETcZCFP value (c).
  • FIG. 13 shows competition between IRF-4 and IRF-5 for MyD88 interaction.
  • HA-IRF-5 expression vector 1.0 g
  • HA-IRF-7 expression vector 2.0 ⁇ g
  • FLAG-MyD88 expression vector 1 g
  • the HA-IRF-4 expression vector or HA-IRF-3 expression vector (0, 0.1, 0.2, 0.5, or 1.0 g) was transferred in increasing amounts.
  • Cell lysates were immunoprecipitated (IP) using anti-FLAG antibody and subjected to immunoblot (IB) analysis using anti-HA or anti-FLAG antibody as indicated.
  • FIG. 14 shows negative regulation of MyD88-dependent IRF-5 activation by IRF-4.
  • DBD DNA binding domain
  • AD active domain
  • RD regulatory domain.
  • B Confocal image of HEK293T cells that transiently express YFP-IRF-4, YFP-IRF-4 ⁇ DBD or YFP-IRF-4 ⁇ RD.
  • C Transiently transfer the specified combination of FLAG-labeled MyD88 and HA-labeled full-length IRF-4 or a deletion mutant of IRF-4 to HEK293T cells, and perform immunoprecipitation It was used for Atssey.
  • D shows the effect of IRF-4 expression on MyD88-TRAF6-dependent IRF-5 activity.
  • HEK293T cells For HEK293T cells, p55ClB-Luc and MyD88 (25ng), TRAF6 (25ng), IRF-5 (25ng) and full-length IRF-4 or IRF-4 variants (0, 1, 5 or 15ng) The expression vectors for the designated combinations were transiently co-transferred. The luciferase activity was measured 24 hours later. (e) shows the influence of IRF-4 expression on MyD88-dependent IRF-7 activity. For HEK293T cells, transiently pl25-Luc and an expression vector for the specified combination of MyD88 (25ng), IRF-7 (25ng) and full-length IRF-4 (0, 5 or 15ng) Simultaneously transferred. Luciferase activity was measured 24 hours after the transformation.
  • FIG. 15 is a graph showing hypersensitivity to TLR stimulation in Irf_4 — / _ peritoneal macrophages.
  • A Residual peritoneal macrophages from wild type mice were stimulated for a specified period with ODN1668, LPS or poly (U). Total RNA was prepared and analyzed for IRF-4 mRNA expression by quantitative real-time RT-PCR.
  • B Resident peritoneal macrophages derived from wild-type mice or Irf4 ⁇ / _ mice were stimulated for 24 hours with the designated TLR ligand in the presence of IFN- ⁇ . The concentrations of IL-12p40, IL-6 and TNF- ⁇ in the culture supernatant were measured by ELIS.
  • FIG. 16 shows cell type-specific contributions of IRF-4 and IRF-5 systems.
  • BMM from wild-type mice, IriS — / _ or Irf4 — / _ mice was stimulated with the specified stimulus in the presence of IFN- ⁇ .
  • concentrations of IL-12p40, IL-6 and TNF-a in the culture supernatant were measured by ELISA.
  • B Control vectors, full-length IRF-4 or IRF-4 ⁇ DBD expression vectors were transiently transfected into RAW264.7 cells by electroporation. OD cells after 12 hours
  • FIG. 17 shows the role of IRF-4 in vivo.
  • A Serum concentrations of IL-6, IL-12p40 and TNF- ⁇ were measured by ELISA. Results shown are the average of serum samples (SD).
  • B The survival of these mice was observed for 15 hours.
  • the present invention relates to a novel method for screening for an inflammatory site force-in inhibitor using IRF-5.
  • IRF-5 plays an essential role in inducing inflammatory site force-in via the TLR signal pathway.
  • the present inventors have also found a new screening method for an inflammatory site force-in inhibitor using IRF-5.
  • the present invention provides, as a first aspect of the screening method described above, "Inflammatory site force-in, using IRF-5 activity in an IRF-5-expressing cell as an index when the test substance is contacted with the test substance.
  • a screening method for inhibitors is provided.
  • IRF-5-expressing cells may be any cells that express IRF-5. Even if IRF-5 is endogenous IRF-5, artificially introduced IRF-5 It may be. IRF-5 is a transcription factor of IRF family members As mentioned above, it interacts with MyD88 and TRAF6 and is involved in the induction of inflammatory site force-in.
  • the cell expressing endogenous IRF-5 used in the method of the present invention is not particularly limited as long as it is a mammalian cell capable of expressing endogenous IRF-5, but is preferably endogenous IRF. It is a mammalian cell that can express a large amount of -5. Specific examples of mammalian cells that can express a large amount of endogenous IRF-5 include blood cells, and more specific examples include B cells, T cells, macrophages, erythrocytes, rod cells, neutrophils Spheres, eosinophils, basophils and the like. Cells capable of expressing endogenous IRF-5 can also be prepared for mammalian organs or tissue forces by methods well known to those skilled in the art.
  • IRF-5 is highly expressed in blood cells, and it is considered that an organ or tissue force that contains a large amount of blood cells can efficiently prepare IRF-5. More specifically, an antibody specific for a surface antigen characteristic of a target cell is used from a mammalian organ or tissue rich in blood cells, for example, a blood system tissue such as spleen, liver, thymus, lymph gland, It can be prepared using a cell sorter (FACS), magnetic cell sorting (MACS) using micro magnetic beads, and a affinity column.
  • FACS cell sorter
  • MCS magnetic cell sorting
  • affinity column affinity column.
  • an antibody against such a surface antigen can be prepared by a well-known method, and many specific antibodies against such a surface antigen are commercially available, and commercially available antibodies can also be used.
  • mouse T cells and B cells can be prepared using beads coated with anti-CD5 antibody and anti-CD19 antibody from the mouse spleen, for example, as in Examples described later.
  • cDC and plasmacytoid dendritic cells pDC
  • the above organ or tissue force can also be prepared using an anti-CD1 lb antibody.
  • the human IRF-5 cDNA sequence is SEQ ID NO: 1
  • the amino acid sequence of human IRF-5 is SEQ ID NO: 2
  • the mouse IRF-5 cDNA sequence is SEQ ID NO: 3
  • the IRF-5 gene can be prepared by a known method. For example, it is possible to prepare a single cDNA library in which the whole or part of the sequence shown in SEQ ID NO: 1 or 3 is used as a probe and the human or mouse tissue force is prepared using the probe.
  • a primer having the sequence ability described in SEQ ID NO: 1 or 3 is also designed, and the total RNA prepared by human or mouse can be used as a kit, and can be prepared by RT-PCR using the primer. Alternatively, it can be synthesized using a nucleic acid synthesizer based on the sequence information described in SEQ ID NO: most.
  • exogenous IRF-5 The gene used for exogenous introduction (hereinafter referred to as exogenous IRF-5) is not limited to HI-HRF-5 (SEQ ID NO: 1) or mouse IRF-5 (SEQ ID NO: 3) shown above, but includes orthologs, homologs, It may be a natural IRF-5 variant.
  • the type of mammal is not particularly limited as long as it is a mammal that can be IRF-5 derived from animals other than humans and mice. In addition to mice and humans, rats, guinea pigs, hamsters and other rodents, rabbits, dogs, cats, pigs, rushes, horses, goats, hidges, donkeys, birds, chimpanzees, monkeys, etc.
  • the derived IRF-5 can be used in the method of the present invention.
  • a variant of the IRF-5 gene sequence is also an exogenous IRF-5 gene that can be used in the present invention as long as the variant has IRF-5 activity.
  • Such an IRF-5 gene is, for example, a stringent library using a whole or part of the sequence shown in SEQ ID NO: 1 or 3 as a probe, and a human or mouse tissue force prepared using the probe. It can be prepared by isolating a polynucleotide that undergoes hybridization under various conditions. Stringent hybridization conditions can be selected as appropriate by those skilled in the art.
  • a hybridization containing 25% formamide, 50% formamide under more severe conditions 4 X SSC, 50 mM Hepes pH 7.0, 10 X Denhardt's solution, 20 g / ml denatured salmon sperm DNA. Prehybridize in solution at 42 ° C, then hybridize with labeled probe at 42 ° C. Subsequent washing, for example, ⁇ 0.5xSSC, 0.1% SDS, 42 ° C '' is more severe than ⁇ lxSSC, 0.1% SDS, 37 ° C ''. Washing may be performed with “xSSC, 0.1% SDS, 65 ° C.”.
  • hybridization conditions are examples, and stringent hybridization can be performed even under conditions different from the above conditions.
  • One skilled in the art will know the probe concentration, probe length, and reaction time.
  • An appropriate stringency can be realized in consideration of other conditions such as the above.
  • the polypeptide encoded by the polynucleotide isolated using such hybridization technology is usually an IRF-5 protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4 in the amino acid sequence.
  • high homology means at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably at least 95% or more, more preferably at least 97% or more (e.g. 9 8 to 99%) sequence homology.
  • the identity of the amino acid sequence can be determined by, for example, the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci.
  • a polynucleotide in which the IRF-5 gene is artificially mutated can be used in the screening method of the present invention as long as the polynucleotide has IRF-5 activity.
  • a polynucleotide encoding a polypeptide containing one or more deletions, substitutions, insertions, and / or additions in the amino acid sequence set forth in SEQ ID NO: 2 or 4 is suitable for the screening method of the present invention.
  • the number of amino acids added, deleted, inserted, or Z and substituted is not particularly limited as long as the polynucleotide has IRF-5 activity.
  • a polynucleotide can be prepared by a person skilled in the art by a well-known technique.
  • IRF-5 gene containing artificial mutation can be prepared by site-specific or random mutation of IRF-5 gene by cassette mutagenesis or mutagenesis by PCR method. It is.
  • a polynucleotide containing a nucleotide sequence in which a mutation is introduced into the nucleotide sequence of SEQ ID NO: 3 or 3 can be synthesized by a commercially available nucleic acid synthesizer.
  • IRF-5 homologs, orthologs, natural mutants, artificial mutants obtained as described above Whether a polynucleotide such as a test polynucleotide has IRF-5 activity can be determined by introducing the test polynucleotide into a mammalian cell line expressing TLR, MyD88TRAF6, and any inflammatory site force-in. This can be determined by detecting the expression of inflammatory site force-in when the ligand is contacted. If the expression level of inflammatory site force-in is high, it can be judged that the IRF-5 activity of the test polynucleotide is high.
  • the cells into which the exogenous IRF-5 gene is introduced can be appropriately selected as a cell or cell line capable of retaining and expressing the IRF-5 gene.
  • Mammalian cells and cell lines are generally cells capable of retaining and expressing an IRF-5 gene.
  • HEK293T is a suitable example for introducing an exogenous IRF-5 gene.
  • the IRF-5 gene may be introduced directly into the cell by means of a particle gun or the like, but a vector suitable for the cell to be introduced can be appropriately selected and used for IRF-5 gene introduction.
  • vectors based on various viruses such as adenovirus, papillomavirus, papovavirus, and retrovirus can be used.
  • the "IRF_5-expressing cells" used in the method of the present invention are required to have a mechanism for activating IRF-5.
  • the function of IRF-5 in TLR signaling is exerted by the signaling force IRF-5 activated by binding to the ligand and TLR activation, and being transmitted downstream through interaction with MyD88 and TRAF6.
  • the expressed TLR, MyD88 and TRAF6 may be endogenous TLR, MyD88 and TRAF6 or an artificially introduced exogenous TLR, MyD88 and TRAF6.
  • TLR3 When artificially introducing a gene into a foreign country, without introducing all of MyD88, TRAF6, and TLR, the ability to co-express both TRAF6 and MyD88, or TLR, can be introduced.
  • IRF-5 activity can be achieved by either stimulating TLR with a TLR ligand.
  • TLRs need only express one of TLRs located upstream of the interaction between MyD88, TRAF6, and IRF-5 in TLR signaling.For example, TLR3, TLR 4, TLR5, TLR7, TLR8, TLR9 The ability to express any one or more is preferable.
  • Nucleotide sequences of various types of TLR, MyD88 and TRAF6 are known. For example, the following Accession N Available from the Public Database (NCBI) by O. TLR3
  • the IRF-5 activity used as an index in the method of the present invention refers to an activity that qualitatively or quantitatively indicates the relationship of IRF-5 with respect to TLR signaling. That is, in the method of the present invention, if IRF-5 is an event obtained as a result of TLR signal transduction, deviation can be used as an index. Examples of such an indicator include the expression level of a reporter gene linked to a promoter region containing an ISRE sequence (hereinafter also referred to as “reporter activity”). As will be described later in the Examples, the present inventors have revealed that upon activation of TLR, IRF-5 interacts with MyD88 and TR AD6 to induce inflammatory site force-in.
  • a reporter gene linked downstream of the promoter region containing the ISRE sequence is constructed, the reporter gene construct is introduced into a cell having a TLR signal transduction pathway, and the cell is contacted with a TLR ligand. It is considered that TLR is activated and a signal based on TLR activation induces expression of the reporter gene through formation of a complex composed of IRF_5, MyD88 and TRAF. That is, the reporter activity based on the reporter gene construct can be an indicator in the screening method of the present invention.
  • the ISRE (Interferon Stimulated Response Element) sequence is a DNA base sequence motif that is specifically recognized by transcription factors of the IRF family, and includes a consensus sequence: -GAAA-. ISRE sequences have been found in IFN a and IFN jS genes that are transcriptionally regulated by the IRF family. In the method of the present invention, any nucleotide sequence that can be recognized by IRF-5 can be used as an ISRE sequence. Not only known ISRE sequences but also sequences highly homologous to known ISREs can be used in the method of the present invention. It can be used as an array.
  • an ISRE sequence in the present invention a promoter using “TFSEARCH” (http: ⁇ www.cbrc.jp/research/db/TFSEARCHJ.html), which is an application for predicting a transcription factor binding site on DNA, is used.
  • a sequence obtained by setting the score to 50 points or more Examples include ISRE sequences, IRF-1 binding sequences, and sequences detected as Z or IRF-2 binding sequences.
  • SEQ ID NOs: to 58 can be exemplified.
  • the reporter gene can be selected from known reporter genes that are suitable for the conditions such as the detection method.
  • known reporter genes such as BFP, GFP, CFP, YFP, DsRed, AmCyan, ZsYellow, ZsGreen, luciferase and other fluorescent proteins, and enzymes such as 13-Darc Mouth-Durase (GUS) and alkaline phosphatase can be used. Noh.
  • a method for linking a reporter gene downstream of a promoter region containing an ISRE sequence is known. For example, it can be performed according to the method described in the examples.
  • the above-described screening method using reporter gene activity as an index can include, for example, the following steps:
  • test substance refers to TLR signal transduction in the cell.
  • test substances include low-molecular compounds such as synthetic chemical substances, nucleic acids, proteins, peptides, plant extracts, cell extracts, animal tissue extracts, and products produced by microbial fermentation, but are not limited thereto. I can't.
  • test substance is not limited to a new substance, and may be a known substance. Among known substances, it is known to have an inflammatory site force-in inhibitory action, and it is considered that compounds exist.
  • the above-mentioned "step of contacting the IRF-5-expressing cell with a TLR ligand or virus” comprises TLR This is a process for starting signal transmission.
  • TLR TLR
  • a virus that is compatible with the TLR expressed by the cells used. For example, if TLR3 is expressed in TLR3, double-stranded RNA or poly (I: C) derived from various viruses is used.
  • TLR4 is LPS
  • TLR5 is flagellin
  • TLR7 and TLR8 are synthetic.
  • RNAs such as single-stranded RNA [poly (U)], imidazoquinoline derivatives, influenza virus, human immunodeficiency virus, Newcastle disease virus, and TLR9 by CpGDN A such as ODN1668, simple herpes virus, and mouse cytomegalovirus TLR can be activated.
  • CpGDN A such as ODN1668, simple herpes virus, and mouse cytomegalovirus TLR
  • the above ligands and viruses are examples, and usable ligands and viruses are not limited to these.
  • step of detecting the reporter activity of the IRF-5-expressing cell is a step of specifying the influence of the test substance on the IRF-5 activity of the present invention.
  • Reporter activity can be measured as a measure of the fluorescence intensity and enzyme activity of the reporter gene type.
  • the reporter activity obtained as described above is used as an index.
  • selecting a test substance that can be an inflammatory site force-in inhibitor is used as an index.
  • the reporter activity when the test substance is contacted is compared with the reporter activity of the control obtained in the same manner when the test substance is not contacted. 5 It is considered to be a substance that suppresses the function and suppresses the expression of inflammatory site force-in.
  • the second aspect of the screening method according to the present invention is as follows: "Inflammatory site force-in, using the expression level of IRF-5 in an IRF-5-expressing cell as an index when the test substance is contacted with the test substance. Provides screening methods for inhibitors. " The inventor has revealed that IRF-5 is intrinsically involved in the induction of inflammatory site force-in as a member of the TRL signaling pathway. If the expression of IRF-5 decreases, the expression level of inflammatory cytokines via the TRL signaling pathway is thought to decrease. Therefore, a substance that suppresses the expression of IRF-5 can be a candidate for an inflammatory site force-in inhibitor.
  • the expression level of IRF-5 can be measured as the amount of protein or the amount of mRNA. Tampa In the case of measuring as a mass, for example, it can be measured by a known immunoassay using an anti-IRF-5 antibody.
  • the method of the present invention for measuring the expression level of IRF-5 as the amount of protein includes the following steps.
  • the nucleotide sequence of the IRF-5 gene for example, the sequence ability described in SEQ ID NO: 1 or 3 can also be measured by RT-PCR method by designing primers. Examples of such primers are SEQ ID NOs: 5 and 6.
  • the third aspect of the screening method according to the present invention is as follows: "Inhibition of binding between MyD88 and IRF-5 by a test substance when My D88 is contacted with IRF-5 in the presence of the test substance"
  • IRF-5 forms a complex with MyD88 and TRAF6 and interacts with it to exert its function.
  • IRF-4 binds MyD88 to IRF-5.
  • the method of the present invention is a method for screening a substance that inhibits the binding between MyD88 and IRF-5 (also referred to as “MyD88-IRF-5 binding inhibitor” in the present invention).
  • MyD88-IRF_5 binding inhibitor like IRF-4, can suppress the expression of inflammatory site force-in.
  • the ability to inhibit the binding between MyD88 and IRF-5 means the ability to inhibit the binding between MyD88 and IRF-5.
  • To detect the ability of the test substance to inhibit the binding of MyD88 to IRF-5 bind MyD88 to IRF-5 in the presence of the test substance and compare it to the absence of the test substance. You can see if the binding between MyD88 and IRF-5 decreases or disappears.
  • the binding between MyD88 and IRF-5 is a combination of various in situ and in vitro assays that detect protein interactions. It can be detected by intelligent methods. For example, it can be detected by immunoprecipitation, fluorescence resonance energy transfer analysis (FRET), two-hybrid method, surface plasmon analysis method, high throughput analysis using a microarray, and the like.
  • FRET fluorescence resonance energy transfer analysis
  • MyD88 is known as an adapter molecule involved in cytoforce-in or chemokine induction.
  • MyD88 usable in the method of the present invention is not limited as long as it has the ability to bind to IRF-5 and induce inflammatory site force-in.
  • the human MyD88 cDNA sequence is SEQ ID NO: 63
  • the amino acid sequence is SEQ ID NO: 64
  • the mouse MyD88 cDNA sequence is SEQ ID NO: 65
  • the amino acid sequence is It is shown in SEQ ID NO: 66, but is not limited thereto.
  • MyD88 derived from mammals other than humans and mice, homologs, natural mutants, and artificially introduced mutants have the ability to bind to IRF-5 and induce inflammatory cytokines As long as it can be used in the method of the present invention.
  • the MyD88 protein or gene can be prepared by a known method similar to that described above for the preparation of IRF-5.
  • the method for detecting the binding between MyD88 and IRF-5 in situ includes, for example, the following steps.
  • the donor fluorescent protein gene and the acceptor fluorescent protein gene are linked to either the MyD88 gene or the IRF-5 gene, and the fluorescent protein gene-MyD88 gene construct and the fluorescent protein gene-IRF-5 Introducing the gene construct into the cell;
  • the cells placed in the presence of the test substance are irradiated with an excitation wavelength peculiar to the donor fluorescent protein to excite the donor fluorescent protein, and based on the fluorescence intensity based on the donor fluorescent protein and the acceptor fluorescent protein Detecting fluorescence intensity and analyzing FRET between donor fluorescence protein and acceptor fluorescence intensity protein from fluorescence intensity based on donor fluorescence protein and fluorescence intensity based on acceptor fluorescence protein
  • step (c) A step of selecting a test substance in which FRET in the presence of the test substance analyzed in step (b) is less attenuated or disappears than FRET in the absence of the test substance.
  • step (a) a method using analysis by FRET can be shown.
  • cell MyD88 and IRF-5 can be detected by FRET.
  • Te in the process of labeling.
  • FRET Fluorescence resonance energy transfer
  • the “donor fluorescent protein” is a fluorescent protein that gives energy to another fluorescent protein (acceptor fluorescent protein) in FRET
  • the “acceptor fluorescent protein” is another fluorescent protein (donor fluorescent protein).
  • Force is also a fluorescent protein that receives energy. Donor and acceptor fluorescent proteins can also be selected from known fluorescent proteins.
  • Donor and acceptor are different fluorescent proteins, and it is necessary that there is an overlap between the donor's fluorescence spectrum and the absorption spectrum of the acceptor.
  • selection using CFP as a donor and YFP as an acceptor is one of the selection examples that satisfy the above conditions, but is not limited to this, and any combination of fluorescent proteins that satisfies the conditions of the donor and the acceptor may cause a deviation. It can be used.
  • the donor fluorescent protein is excited by irradiating the prepared cells with an excitation wavelength peculiar to the donor fluorescent protein in the presence of the test substance.
  • FRET between the donor and the acceptor is analyzed from the fluorescence intensity based on the fluorescence intensity based on the fluorescence intensity based on the fluorescence intensity based on the fluorescence intensity based on the fluorescence fluorescence based on the donor fluorescence protein and the acceptor fluorescence protein.
  • Negative regulation of IF N- a / j8 signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells Proc. Natl. Acad. Sci. USA 101, 2416-2421 (2004), Honda, K., Yanai, H., Mizutani, T., Negishi, H., Shimada, N., Suzuki, N., Ohba'Y., Takaoka, A. Yeh, WC & Taniguchi, T. (2004) Proc. Natl. Acad. Sci. USA 101, 15416-15421.
  • the MyD88 protein and IRF-5 protein expressed in the “cells expressing MyD88 protein and IRF-5 protein” used in this method may be endogenous proteins, but may be exogenous.
  • the exogenous MyD88 protein and IR F-5 protein may be fusion proteins of known labeled peptides such as FRAG and HA.
  • either anti-MyD88 antibody or anti-I RF-5 antibody (or anti-labeled peptide antibody) is added to the cell lysate, and the MyD88 protein or IRF-5 protein and antibody in the cell preparation are combined with the antigen. After binding by the antibody reaction, the protein-antibody conjugate is fractionated by centrifugation. Alternatively, the cell preparation is contacted with the immobilized antibody to adsorb MyD88 protein or IRF-5 protein. To fractionate.
  • step (c) it is detected whether the MyD88 protein and the IRF-5 protein are bound in the separated fraction.
  • the detection can be performed, for example, by bringing the anti-IRF-5 antibody, anti-MyD88 antibody, and anti-labeled peptide antibody into contact with the separated fraction.
  • the antibody may be labeled for convenience during detection. Specifically, if IRF-5 is detected as a result of contacting an anti-IRF-5 antibody with a fraction containing MyD88 protein, the result indicates that MyD88 and IRF-5 bind to each other in the fractionated fraction.
  • the anti-MyD88 antibody is brought into contact with the fraction containing the separated IRF-5 protein, and the binding between MyD88 and IRF-5 in the separated fraction is detected.
  • the binding force between MyD88 and IRF-5 detected as described above when contacting the test substance. If the binding force is less than or disappearing from the binding between MyD88 and IRF-5 detected when the test substance is not in contact
  • the test substance is a substance that inhibits the formation of a complex of MyD88 and IRF-5 and suppresses the induction of inflammatory site force in via the TRL signal pathway.
  • Another example of a method for detecting in vitro the binding inhibition ability of MyD88 and IRF-5 by a test substance is a method including the following steps.
  • the method including the steps (a) to (c) can be performed by various known methods. For example, it can be carried out by a method using a surface plasmon resonance spectrum (BIACORE).
  • BIACORE surface plasmon resonance spectrum
  • step (a) above either MyD88 protein or IRF-5 protein is immobilized on the sensor chip (ligand), and the other is applied as a solution (analyte) to the sensor chip for contact. Let The test substance is mixed with the analyte.
  • step (b) above the sensor chip is irradiated with light and the change in the refractive index of the reflected light is monitored.
  • the binding activity between the ligand and the analyte can be known.
  • the use and operation of BIACORE can be found on the BIACOR E website (http: ⁇ www.biacore.co.jp/).
  • the present invention provides a novel screening method for further inhibitors of inflammatory site force-in, using "an IRF-4 expression level in an IRF-4 expression cell when the test substance is contacted with the test substance as an index.” , A screening method for an inflammatory site force-in inhibitor. As will be described later, the present inventors have revealed that IRF-4 works to suppress inflammatory site force-in. Therefore, a substance that enhances the expression of IRF-4 is considered to act to suppress inflammatory site force-in.
  • the expression level of IRF-4 can be measured as the amount of protein or the amount of mRNA. When measuring as a protein mass, it can measure by a well-known immunoassay using an anti- IRF-4 antibody, for example.
  • the method of the present invention for measuring the expression level of IRF-4 as the amount of protein includes the following steps.
  • the nucleotide sequence of the IRF-4 gene for example, the sequence ability described in SEQ ID NO: 59 or 61 can also be measured by RT-PCR by designing primers. .
  • the various screening methods of the present invention enable the development of novel inflammatory site force-in inhibitors.
  • the inflammatory site force-in inhibitor obtained by the method of the present invention is a novel substance or a known substance, and suppresses the expression of inflammatory site force-in via the TLR signal transduction pathway. Inflammatory site force-in to be suppressed is caused by TLR signaling. For example, IL-1, IL-6, IL-8, IL-12, IL-18, TNF- ⁇ , IFN- ⁇ You can choose from.
  • the present invention suppresses the expression of inflammatory site force-in through inhibition of the binding between the inflammatory site force-in inhibitor obtained by the above-described screening method, that is, MyD88 and IRF-5.
  • the inflammatory site force-in inhibitor obtained by the above-described screening method, that is, MyD88 and IRF-5.
  • IRF-4 protein is provided.
  • IRE-4 has been known as a T cell and B cell specific member of the IRF family that plays a role in the activation and differentiation of lymphocytes
  • the present inventors have determined that the binding between MyD88 and IRF-5 is IRF- 4 is competitively inhibited, and the together and reveal in the in vitro that IRF -4 suppresses the expression of inflammatory sites force in respect stimulation with TLR ligands, wild in IRF-4- / _ mice Compared with type mice, the inflammatory site force in level was significantly increased, indicating that it died earlier. Therefore, when IRF-4 protein is administered to an individual who develops inflammatory site force-in, the binding between MyD88 and TLR-5 is competitively inhibited with TLR-5. It can be expected to suppress the expression of inflammatory site force-in.
  • the human IRF-4 cDNA sequence is SEQ ID NO: 59
  • the amino acid sequence is SEQ ID NO: 60
  • the mouse IRF-4 cDNA sequence is SEQ ID NO: 61.
  • the amino acid sequence is shown in SEQ ID NO: 62.
  • positions 21 and 127 are the DNA binding domain
  • positions 200 and 267 are the active domain
  • positions 268 to 450 are the regulatory domains.
  • IRF-4 protein can be prepared by known methods of IRF-5.
  • a probe is prepared based on the sequence described in SEQ ID NO: 59 or SEQ ID NO: 61, the target cDNA is selected from a cDNA library prepared with human or mouse hematopoietic cell force, and the cDNA is expressed in an appropriate host-expression. It can be prepared by expressing with a vector system and purifying with a affinity column using an anti-IRF-4 antibody.
  • a primer was designed from the sequence shown in SEQ ID NO: 59 or SEQ ID NO: 61, and cDNA was prepared by performing RT-PCR using total RNA prepared from human or mouse hematopoietic cells in a cage shape. IRF-4 protein can be prepared.
  • IRF-4 in the present invention is not limited to human and mouse IRF-4 represented by the above-mentioned sequences. As long as it has a function to suppress inflammatory site force-in expression, IRF-4 derived from other mammals, homologues, natural mutants, and mutants obtained by artificially mutating natural IRF-4 sequences Are also included in the IRF-4 of the present invention.
  • cDNA was prepared in the same manner as IRF-5 preparation described above using a probe or primer prepared based on the sequence shown in SEQ ID NO: 59 or SEQ ID NO: 61.
  • the desired IRF-4 protein can be prepared by selecting an appropriate host-expression vector system for expression and purification.
  • the present invention also provides the IRF-4 gene as an inflammatory site force-in inhibitor.
  • the IRF-4 gene is expressed in the patient's body, so a sustained inflammatory site force-in inhibitory effect is expected. it can.
  • the inflammatory site force-in inhibitor obtained by the screening of the present invention containing the above IRF-4 (hereinafter abbreviated as "inflammatory site force-in inhibitor of the present invention") is an inflammatory site force-in inhibitor. It is useful as a therapeutic or prophylactic agent for diseases involving ins.
  • the inflammatory cytokine inhibitor of the present invention includes endotoxin shock, anaphylactic shock, autoimmune disease, insulin-dependent diabetes mellitus, allergic disease, rheumatoid arthritis, sepsis, myasthenia gravis, systemic lupus erythematosus, scleroderma Symptom, polymyositis, nodular polyarteritis, Crohn's disease, asthma, collagen disease, inflammatory bowel disease, multiple sclerosis, various infectious diseases, malignant tumor, stroke inflammation, etc. It is considered effective in the treatment or prevention of other diseases.
  • the inflammatory site force-in inhibitor of the present invention containing IRF-4 is used as a therapeutic or prophylactic agent for the above diseases
  • the physicochemical properties of the inflammatory site force-in inhibitor, target disease In consideration of various conditions such as age, weight, etc. of the patient to be administered, make it an appropriate formulation by known formulation technology, information necessary for taking indications such as efficacy 'effect, usage' dose, contraindication 'side effects, etc. Can be used as a therapeutic or prophylactic agent.
  • pharmacologically acceptable media for formulation, pharmacologically acceptable media, bases, excipients, stabilizers, sweeteners, corrigents, binders, tonicity agents, softeners, disintegrants, solubilizers, In combination with suspending agents, emulsifiers, dispersing agents, surfactants, coating agents, preservatives, preservatives, PH regulators, solubilizers, coloring agents, fragrances, etc., powders, pills, tablets, lozenges , Capsules, solutions, ointments, creams, suppositories, poultices, injections, and the like.
  • IRF-5 is a major member of the TRL signaling pathway based on the novel findings of the present inventors. As a bar, it became clear that it was essentially involved in the regulation of inflammatory site force-in expression.
  • the steric structure of the transcription factor IRF-5 was analyzed, and based on the steric structure, IRF-5 containing low molecular weight compounds, peptides, and polynucleotides. It is possible to provide a method for designing or searching for a function-inhibiting substance. Furthermore, it is also possible to provide a method for designing or searching for a function-inhibiting substance such as a peptide that inhibits the function of IRF-5 from the amino acid sequence or gene sequence information of the transcription factor IRF-5.
  • Genomic DNA encoding the IRF-5 gene was isolated from a 129J mouse genomic library.
  • the targeting vector was constructed by replacing a 2.1 kb fragment (corresponding to ethason 2) encoding part of the DNA binding domain with a neomycin resistance gene cassette (neo).
  • This IRF-5 gene targeting vector was transfected into embryonic stem cells (E14K). Neomycin resistant colonies were selected and screened by PCR and Southern plot analysis. Two homologous recombinants were microinjected into C57BLZ6 blastocysts. This chimeric mouse was mated with a C57BLZ6 female mouse and heterozygous F1 offspring were cross-bred to obtain IRF-5 chi mice. Mice from these independent clones showed the same phenotype.
  • mice and reagents [Other mice and reagents]
  • MyD88 mice were donated by Shizuo Akira (Osaka University) Tsujiko. All mice were housed under special aseptic conditions in the animal facility of Tokyo University and used after at least 7 backcrosses with C57BLZ6 mice.
  • Poly (U) and LPS were purchased from Sigma. A complex of poly (U) and DOTAP (Roche Diagnostics) was prepared according to the manufacturer's instructions.
  • poly (l: C) was purchased from Amersham.
  • CpG-A ODN, CpG-B ODN, and all synthetic oligodeoxynucleotides were purchased from Hokkaido. Purified flagellin from L. monocytogenes was donated by Alan Aderem (Institute for Systems Biology, Seattle, USA).
  • T cells and B cells were treated with Dynabeads (Dynal) treated with lmgZml collagenase A (Roche Biochemicals) and 20 mM EDTA and coated with anti-CD5 and anti-CD19 antibodies (BD Biosciences) and anti-HgG. Negative selection was performed. B220-Z CDllc + cDC and B220 + ZCD11C-pDC were selected using FACS Diva (BD Bioscience). Spleen Mcphage was collected using a MACS column using CDl lb MicroBeads (Miltenyi Biotec) and negatively selected for splenic B cells using a B cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec). The cells were cultured in RPMI1680 (Invitrogen) supplemented with 10% urine fetal serum.
  • cDC and pDC (2 X 10 5 ml— 1 ), B cells (5 X 10 5 ml— 1 ) or macrophages (7 X 10 5 ml— 1 ) are seeded on 96-well plates and designated The cells were cultured for 24 hours under the stimulated conditions.
  • IL-12p40, IL-6, TNF-a and IFN-a were measured by solid-phase enzyme immunoassay (ELISA).
  • ELISA kits for mouse IL-12p40, IL-6 and TNF- ⁇ were obtained from R & D Systems. Seven ELISA kits for mouse IFN- ⁇ were purchased from PBL Biomedical Laboratories.
  • RNA extraction and polymerase chain reaction (RT-PCR) analysis with reverse transcription have been previously described.
  • IRF-5 5- AATACCCCACCACCTTTTGA-3 (SEQ ID NO: 5) (sense)
  • TLR3 5- TTAGAGTCCAACGGCTTAGAT-3 (SEQ ID NO: 69) (sense)
  • TLR4 5-GAGCCGGAAGGTTATTGTGGT-3 (SEQ ID NO: 71) (sense)
  • TLR5 5-AAGTTCCGGGGAATCTGTTT-3 (SEQ ID NO: 73) (sense)
  • TLR7 5—GTTCTTGACCTTGGCACTA— 3 (SEQ ID NO: 75) (sense;)
  • TLR9 5-ATGGACGGGAACTGCTACTACA-3 (SEQ ID NO: 77) (sense)
  • FLAG-MyD88 cyan fluorescent protein labeled MyD88 (CFP-MyD88), yellow fluorescent protein labeled IRF-3 (YFP-IRF-3), YFP-IRF-7 and HA-IRF3 expression vectors (See the Supplemental Information section).
  • Mouse IRF-5 cDNA was obtained by RT-PCR of total MEF-derived RNA and cloned into a pCRII (Stratagene) vector.
  • YFP-IRF-5 expression vector or HA-tagged IRF-5 expression vector use pCAG
  • the cDNA was cloned into the Xhol and Notl sites of the GS-YFP vector or pCAGGS-HA vector.
  • the pCAGGS vector and Venus (called YFP) were donated to chiro Miyazaki (Osaka University) and Satoshi Sowaki (RIKEN), respectively.
  • the HA-IRF-5 DNA fragment was excised from pCAGGS-HA-IRF-5 and cloned into the EcoRI site and Sail site of the pBabe-puro retrovirus expression vector.
  • Retroviral gene introduction into MEF was performed as described in the previous literature (Takaoka, A. et al. Integration of interfere n ⁇ a / ⁇ signaling to p53 responses in tumour suppression and antiviral defence. Nature 424, 516 -523 (2003)).
  • pCAGGS-HA-IRF-5 was transfected into RAW264.7 cells using Superfect reagent (Qiagen).
  • the expression vector for FLAG-tagged TRAF6 was donated to Junichiro Inoue (University of Tokyo)!
  • FRET analysis was performed as previously described (Honda, K., Mizutani, T. & Taniguchi, ⁇ . Negative regulation or irN-a / ⁇ signaling oy IFN regulatory factor 2 for home ostatic development of dendritic Proc. Natl. Acad. Sci. USA. 101, 2416-2421 (2004)) o
  • Antibodies against the following proteins were purchased: HA (Roche), FLAG (Sigma- Aldrich Co
  • Mouse embryonic fibroblast (MEF) was prepared according to standard procedures (Takaoka, A. et al. Integration or interferon- / ⁇ signaling to p53 resp onses in tumour suppression and antiviral defense. Nature 424, 516-523 (2003)). Fractions of Itoda and nuclear Western immunoblotting were performed as described elsewhere (Lee, HH, Dadgostar, H., Shiheng, Q., Shu, J. & Cheng, G. NF — ⁇ B— mediated up— r egulation of Bcl- ⁇ and Bfl-1 / Al is required for CD40 survival signaling in B lympho cytes. Proc. Natl. Acad. Sci. USA. 96, 9136-9141 (1999)). Anti-USF-2 and anti-j8-tubulin antibodies were obtained from Santa Cruz Biotechnologies.
  • EMSA for NF- ⁇ was performed as previously described (Matsuyama, T. et al. Targeted disruption of IRF— 1 or IRF— 2 results in abnormal type 1 1 FN gene inaucti on and aberrant lymphocyte development. Cell 75, 83-97. (1993)).
  • ChIP Atsey was performed as described in the previous literature (Takaoka, A. et al. Integration of interferon-a / ⁇ signaling to p53 responses in tumour suppression and antiviral defence. Nature 424, 516-523 ( 2003)). Specific antibodies used for immunoprecipitation were anti-HA antibody (Roche) and anti-Lck antibody (Upstate) as an isotype control.
  • a negative control primer pair that recognizes the DNA sequence of the 3'-untranslated region (UTR) of the IL-12p40 gene (from +12863 to +13906):
  • the region of the IFN- ⁇ promoter contains the following primers: 5 -GGGAGAACTGAAAGTGGGAAA-3 '(SEQ ID NO: 83) (sense)
  • Transfection force was also lysed after 24 hours and luciferase activity was measured using the DuaHuciferase reporter assay system (Promega) as previously described (Honda, K., Mizutani, T. & Taniguchi, T. N egative regulation of IFN- a / ⁇ signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells.Proc. Natl. Acad. Sci. USA. 101, 2416-2421 (2004)
  • Reporter plasmid used in this assembly contains many ISREs (Yoney ama, M. et al. Direct triggering of the type I interferon system by virus infection: ac tivation of a transcription factor complex containing IRF -3 and CBP / p300. EMBO J. 17, 1087-1095 (1998)), this is Takashi Fujita (The metropolitan institute of Medical Science).
  • Anti-CD40 conjugated with FITC and anti-H2Kb conjugated with PE, CD86, B220 and anti-CDllc conjugated with biotin were purchased from BD Pharmingen. Streptavidin
  • APC was purchased from Molecular Probes.
  • IRF-5 a member of this family, is involved in the induction of type I interferon (IFN) genes. Although it has attracted a lot of attention, its function is hardly known at this time.
  • IRF-5 mRNA is expressed at high levels in spleen cells ( Figure 6a) and is normal when activated by various TLR ligands (ie, pathogen-associated molecular patterns; PAMP). It is upregulated in cells (cDC) and macrophages ( Figure 6b).
  • IRF family members IRF-3 and IRF-7 are known to be activated toward induction of type I IFN by TLR stimulation, but IRF-5 is involved in TLR signaling Whether or not is unknown.
  • IRF-5 is involved in TLR signal transduction
  • the inventors of the present invention used standard homologous recombination in mice lacking the IRF-5 gene (IRF-5— mice). Produced by the protocol (Fig. La) and its null conjugation was confirmed by DNA and RNA blotting and immunoblot analysis (Fig. Lb, c , d).
  • mice developed normally and were strong with no apparent difference in the hematopoietic cell population (Fig. 6c).
  • CpG-B ODN also known as 1668 or K-type ODN
  • CpG-A ODN D 19 (Also referred to as D-type ODN)
  • cytokines the induction of cytokines by the activity of TLR9 in spleen-derived cDCs and plasmacytoid DCs (pDCs) of IRF-5 and wild-type (WT) mice was examined.
  • WT or IRF-5 mock stimulation of normal DC (cDC) from mouse spleen (medium only) or 1.0 M type B CpG ODN (CpG-B) or CpG- A ODN ( CpG-A) was stimulated for 24 hours, and the concentrations of IL-6, IL-12p40 and TNF- ⁇ in the culture supernatant were measured by ELISA. The supernatant was also measured for IFN-o; induction.
  • plasmacytoid DC (pDC) derived from the spleen of WT mice or IRF-5 ⁇ / _ mice was also examined in the same manner as cDC.
  • splenic macrophages of WT mice or IRF-5 chi mice were treated with CpG-A ODN (1.0 ⁇ ) or CpG- ⁇ ODN dO ⁇ M) in the presence of IFN- ⁇ in SOUml- 1 (IL- 12) or in the absence (stimulated with IL-6 and TNF-o for 24 hours, and the concentrations of IL-6, IL-12p40 and TNF- ⁇ in the culture supernatant were measured by ELISA as described above.
  • TLR 3 polyinosine -Polycytidylic acid [poly (I: C)]; 100 gZml
  • TLR4 lipopolysaccharide [LPS]; 10 ngZml
  • TLR5 fulagellin lO / z gZml
  • TLR7 (8) ss-RNA; polyuridylic acid [poly (U)]; 5 / zg / ml), in the presence (IL-12) or absence (IL-6 and T) of 301 ⁇ 1 _1 IFN- ⁇
  • concentrations of TNF-a, IL-6 and IL-12p40 in the culture supernatant were measured by ELISA in the same
  • TLR3 ligand polyinosine-polycytidylic acid [poly (I: C)]
  • TLR4 ligand lipopolysaccharide [LPS]
  • IRF-5 which is present in the cytoplasm, was then converted to MyD88 and tumor necrosis factor receptor-related factor 6 ( TRAF6) (both of which play a crucial role in all TLR-induced signaling) and whether they form intermolecular complexes.
  • TRAF6 tumor necrosis factor receptor-related factor 6
  • YFP-IRF-5 and YFP-IRF-3 are expressed in mouse macrophage RAW264.7 cell line and human fetal kidney 293T (HEK293T) cell line, These cells were subjected to fluorescence microscopy analysis.
  • IRF-5 was expressed in the cytoplasm and a significant percentage of YFP-IRF-5 was consistent with MyD88 labeled with cyan fluorescent protein (CFP_MyD88). Consistent with this, fluorescence resonance energy transfer (FRET) was selectively observed between co-expressed YFP-IRF-5 and CFP-MyD88, indicating that they are in direct contact with each other. Indicated ( Figure 3a, b)
  • pCAGGS-HAIRF-5 or pEF-HA-IRF-3 was combined with pCXN2-FLAG-MyD88 on HEK293T cells. This transfection was performed, and the cell lysate was subjected to immunoprecipitation with an anti-FLAG antibody, followed by immunoblotting using an anti-HA antibody. After stripping, immunoblotting of FLAG-labeled MyD 88 was then performed with anti-FLAG antibody.
  • HEK293T cells were transiently transfected with pCAGGS-HA-IRF-5 or pCAGGS-HA-IRF-3 and pME-FLAG-TRAF6.
  • the expression levels of these molecules were also determined by analyzing whole cell lysates by immunoblotting. Result, the force IRF-3 when coexpressed with IRF-5 a MyD88 or TRA F 62 in HEK293T cells these were co-immunoprecipitation this happen such ChikaraTsuta ( Figure 3 c) 0
  • the present inventors next examined MyD88-dependent and TRAF6-dependent activation of ISRE sequences by IRF-5 in HEK293T cells using a p-55ClBLuc reporter gene containing a large number of ISREs. As shown in Figure 3d, weak activation of the reporter gene by expression of IRF-5 alone is dramatically enhanced by either co-expression with MyD88 and TRAF6 or CpG-B ODN stimulation This confirmed that IRF-5 was fully integrated into the MyD88-TRAF6 signal transduction pathway.
  • IRF-5 (HA-IRF-5) labeled with hemadalchun was expressed in mouse fetal fibroblasts (MEF) .
  • MEF mouse fetal fibroblasts
  • IRF-5 is one of the cytoforce-in genes, ie, the IL-12p40 gene containing a standard ISRE element within the promoter (IL-12p40-ISRE; -67 to -55 to the transcription start site) (See Fig. 10).
  • IL-12p40-ISRE a standard ISRE element within the promoter
  • chromatin immunoprecipitation (ChIP) assay was also performed by introducing HA-IRF-5 into RAW264.7 cells. These cells also respond to Cp GB ODN, leading to the induction of these site forces (Cowdery, JS, Boerth, NJ, Nonan, LA, Myung, PS & Koretzky,. A.
  • TLR-dependent activation of NF- ⁇ B, (SAPK) ZJNK kinase and p38 MAP kinase is dependent on the canonical MyD88-TRAF6 pathway 25 . Since IRF-5 interacts with both MyD88 and TRAF6, we examined whether lack of IRF-5 affects the activation of these pathways. Spleen B cells from WT, IRF-5 and MyD88— mice were stimulated with Cp GB ODN (0.3 ⁇ ) for the specified period. Activation of NF- ⁇ was evaluated by EM SA. As a result, it was found that the activity of NF- ⁇ , ⁇ 38, and JNK by CpG-B ODN was almost normal in IRF-5— 'cells (Fig.
  • TLR-MyD88 The difference in the contribution of IRF-5 and IRF-7 in the null transmission results in a sufficient TLR response.
  • the TLR-MyD88 signal is successfully processed by intracellular CTTP complexes, for example, by spatial and temporal regulation. It is suggested that a unique mechanism works and that a variety of transcription factors activated downstream of MyD88 by TLR9 may also occur with other TLRs.
  • mice D-galactosamine (D-GalN) -sensitized mice.
  • serum of mice injected with CpG-B ODN was collected 1 hour and 3 hours after injection, and the serum concentrations of TNF-a, IL-12p40 and IL-6 were measured by ELISA.
  • C57BLZ6 mice were purchased from CLEA Japan, Osaka.
  • the production of IriS— ⁇ mice and Irf4— mice has been described in the literature (Takaoka, A., Yanai, H., Kondo, S., Duncan, G., Negishi, H., Mizutani, T., Kano, S., Hyundai, K., Ohba, Y., Mak, TW & Taniguchi, T.
  • FFP cyan fluorescent protein
  • YFP fluorescent protein
  • CpG-ODN Unmethylated CpG-oligodeoxynucleotide
  • ODN1668 13) and 0 DN-D19 (14) are literature: Honda, K., Yanai, H "Negishi, H., Asagiri, M., Sato, M., Mizu It was synthesized as described in tani, T., Shimada, N., Ohba, Y., Takaoka, A., Yoshida, N. & Taniguchi, T. (2005) Nature 434, 772-777.
  • LPS Lipopolysaccharide
  • poly (U)] lebutomycin B and D-galactosamine were purchased from Sigma.
  • Poly (U) is based on the above reference (Honda, K., Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizutani, T., Shimada, N., Ohba, Y., Takaoka, A complex with DOTAP (Roche Diagnostics) was developed as described in A., Yoshida, N. & Taniguchi, T. (2005) Nature 43 4, 772-77). Peptide darican was purchased from Fluka.
  • the cells are cultured on a glass bottom 35 mm tissue culture dish (Matsunami Glass, Osaka), and an expression vector for a fusion protein labeled with a fluorescent protein is used as a FuGENE 6 reagent (Roche Diagnostics) or SuperFect transfer. Transfection was carried out using an exci- tion reagent (Qiagen, Valencia, CA). Confocal microscopy analysis was performed using an Olympus FV-1000 confocal microscope. Two-color images were collected in sequential acquisition mode to avoid cross excitation.
  • reporter plasmid [P55C1B-Luc or pl25-Luc], along with expression plasmids for IRF, MyD88 and TRAF6, on human embryonic kidney (HEK) 293T cells seeded on 24 well plates Transiently transferred using 6 reagents (Roche Diagnostics). The total amount of DNA was kept constant by supplementing the empty vector [pcDNA3.1 (Invitrogen)]. Transfer cells 24 hours later and collect luciferase activity.
  • peritoneal macrophages were obtained by peritoneal lavage.
  • lipDC (8220 ⁇ 011 ⁇ ° mediate cell) is available in the literature ( Honda ,, Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizuta ni, T "Shimada, N., Ohba, Y”) Takaoka, A., Yoshida, N. & Taniguchi, T. (2005) Nature 434, 772-777).
  • BMM bone marrow derived macrophages
  • bone marrow cells were cultured with 20 ng Zml M-CSF (Genzyme) for 6 days.
  • RAW 264.7 cells (5 x 10 7 Zml) transfected with full-length or mutant IRF-4 were prepared by electroporation of an expression vector (3 ⁇ g) using the Nucleofector apparatus (Amaxa, Gaithersburg, MD) did. Cells are plated on 96-well plates at 2 x 10 5 cells / ml, 0.3 M OD ⁇ 1668, 3 ⁇ ⁇ ODN-D19, 1 ⁇ g / ml LPS and 5 ⁇ g Zml poly (U) (complexed with DOTAP ) Or 10 ⁇ g Zml peptide darlicans for the specified period. When specified, 30 units Zml of IFN- ⁇ (Genzyme) was added to the medium.
  • the concentrations of IL-12p40, IL-6 and TNF-a in the culture supernatant were determined using an ELISA kit (R & D Systems).
  • An ELISA kit for mouse IFN- ⁇ was purchased from PBL Biomedical Laboratories (Piscataway, NJ).
  • RNA analysis total RNA was extracted using Sepaso RNA I Super (Nacalai Tesque, Kyoto), and quantitative real-time RT-PCR analysis was performed using LightCycler and SYBR Green system (Roche Diagnostics). Data were normalized to the level of ⁇ -actin in each sample.
  • the same primers as in Example 1 were used for IL-12p40, IL-6, TNF-a and ⁇ -actin.
  • the following primers were used for IRF-4, I ⁇ , FLI CE inhibitory protein (FLIP), vascular cell adhesion molecule-1 (VCAM-1) and macrophage inflammatory protein (MIP) 2.
  • VCAM-1 5 '-GCTCTCACCAATCTCCATCAG-3' (SEQ ID NO: 90); VCAM-1 5 '-CCGTCATTGAGGATATTGG-3' (SEQ ID NO: 91) and
  • IRF-5 and IRF-7 two members of IRF, interact with MyD88, and these IRFs are TLR-dependent in pro-inflammatory site force-in and type I IFNs, respectively. It was shown to be important for sexual induction.
  • the present inventors have revealed that IRF-7 also plays a similar role by interacting with MyD88. These findings raised the question of whether other IRF members are also involved in TLR-MyD88 signaling. Therefore, the present inventors examined the interaction between IRF-4 and IRF-8 (both expressed mainly in cells derived from the hematopoietic system) and MyD88.
  • IRF-4 and IRF-8 labeled with YFP are expressed in HEK293T cells together with CFP-labeled MyD88 (CFP-MyD88).
  • the cells were subjected to fluorescence microscope analysis.
  • YFP-IRF-4 was expressed primarily in the nucleus, but a significant proportion was also expressed in the cytoplasm and co-existed with CFP-MyD88.
  • YFP-IRF-8 did not coexist with CFP-MyD88.
  • the FR ETc image shows that there is a high energy transfer from CFP-MyD88 to YFP-IRF-4.
  • a deletion mutant of MyD88 labeled with a FLAG epitope was prepared.
  • the MyD88 region responsible for interaction with IRF-4 was examined (Fig. 13a).
  • HEK293T cells were coexpressed with HA-IRF-4 after each FLAG-MyD88 mutant was subjected to co-immunoprecipitation analysis.
  • FLAG-MyD88 (A 1-151) and FLA G-MyD88 ( ⁇ 173-296) interact with HA-IRF-4 FLAG-MyD88 ( ⁇ 1-193)
  • FLAG-MyD88 A 60-296) did not interact.
  • IRF-7 a different region of MyD88, namely the N-terminal region, interacts with IRF-7 (Honda, K., Yanai, H., Mizutani, T., Negishi, H., Shimada, N., buzuki , N., Ohba, Y., Takao ka, A., Yeh, WC & Taniguchi, T. (2004) Proc. Natl. Acad. Sci. USA 101, 15416-1 5421), IRF-4 interacts with MyD88 It seems to compete with IRF-5 but not with IRF-7. To test this possibility, we designed a competitive assembly that evaluates the binding of IRF-5 or IRF-7 to MyD88 while increasing the expression level of IRF-4.
  • HA-IRF-3 Honda, K., Yanai, H., Mizutani, T., Negishi,,., Shimada, ⁇ ., Suzuki, ⁇ ., Ohba, which does not interact with MyD88 ⁇ , Takaoka, A., Yeh, WC & Tanig uchi, T. (2004) Proc. Natl. Acad. Sci.
  • Example 1 above showed that IRF-5 was activated by MyD88 and TRAF6. That is, co-expression of IRF-5 with MyD88 and TRAF6 results in the activity of the p55ClB-Luc reporter gene containing multiple IFN response elements (ISRE). Based on the above results, the present inventors examined whether or not the ISRE activation mediated by IRF-4 and its mutant force IRF-5 is prevented. As shown in Figure 14d, reporter gene activation by co-expression of IRF_5, MyD88 and TRAF6 was suppressed in a dose-dependent manner by full-length IRF-4 expression. In addition, a similar inhibitory effect was still observed in HA-IRF-4 A RD and HA-IRF-4 ⁇ DBD mutants (Fig.
  • IRF-4 was initially identified as an IRF family T cell and B cell specific member that acts on lymphocyte activity and differentiation. -4 shows powerful functions that have not been known so far.
  • TLR ligands ODN1668 (for TLR9), LPS (for TLR4) and Single-stranded RNA [poly (U)] (to TLR7) (as shown in Example 1, all of which activate the IRF-5 pathway for pro-inflammatory site force-in production)
  • TLR7 Single-stranded RNA [poly (U)]
  • Irf4 — / _ peritoneal macaque phages are IL-3, IL-12p40 and TNF- ⁇ at levels 2-3 times higher than wild type macrophages when stimulated with various TLR stimuli.
  • Figure 15b IFN- ⁇ induction by TLR9 activation by IFN-induced CpG-ODN and ODN-D19 (14) was normal in Irf-mouse-derived pDC (FIG. 15c). This result is indispensable for induction of IFN- ⁇ , and is consistent with the conventional and the above results, indicating that it is not affected by the functional ability of IRF-7, IRF-4.
  • the present inventors also examined the induction of proinflammatory site force in BMM cultured under M-CSF. Curiously, the level of induction of proinflammatory site force-in observed in BMM in the absence of either IRF-5 or IRF-4 was normal ( Figure 16a). The reason for this difference in the need for IRF-5 in TLR signaling is currently Unclear power in terms We have obtained similar results with GM-CSF or Flt3 ligand in rodent cells cultured in vitro (data not shown). For this reason, the present inventors presume that the need for IRF-5 in TLR signaling varies depending on the cell type and Z or sorting state. These cells may have undergone in vitro sorting that differs from in vivo sorting. Whatever the mechanism, our results suggest that IRF-4 exerts an inhibitory effect on cells where the function of the MyD88-IRF_5 pathway is important.
  • IRF-4 is normally expressed at low levels (data not shown).
  • the IRF-4 expression vector was transiently transferred to the vesicles, and the induction of site force-in mRNA by ODN1668 stimulation was analyzed.
  • FIG. 16b induction of IL-6 mRNA and IL-12p40 mRNA was remarkably suppressed in IRF-4 expressing cells.
  • the induction of these site force in genes in RAW264.7 cells was also suppressed by the expression of IRF-4A DBD (FIG. 14d).
  • I ⁇ mRNA Fig.
  • Irf4 — / _ mice have a stronger inflammatory response than wild-type mice, as can be attributed to the significant increase in serum site power levels after lethal ODN1668 challenge. Indicated. In fact, Irf4- ⁇ mice died earlier (8-10 hours after injection) than wild-type control mice ( Figure 17b). In Irf4- / _ spleen cells from mice and hepatocytes induced levels of IL-12p40 mRNA and IL-6 mRNA was constantly increased (data not shown). These results strongly indicate the in vivo role of IRF-4 as a crucial negative regulator of TLR signaling.
  • a novel screening method for an inflammatory site force-in inhibitor is provided.
  • the present invention is a screening method focusing on the point that IRF-5 is involved in the expression of inflammatory site force in as a major member of the TRL signal pathway. It is clear for the first time by the present invention that IRF-5 is involved in the TRL signaling pathway, and the screening method of the present invention is an unprecedented and completely innovative screening method.
  • Inflammatory cytokine inhibitors can be therapeutic agents for immune diseases and the like. Therefore, according to the screening method of the present invention, it is possible to develop a therapeutic agent for immune diseases and the like that cannot be detected by conventional screening methods.
  • the present invention has found IRF-4 as a substance that suppresses the expression of inflammatory site force-in by inhibiting the function of IRF-5 in the TRL signal pathway.
  • IR F-4 can be a novel therapeutic agent for immune diseases and the like.

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Abstract

La présente invention concerne un nouveau procédé de criblage de la cytokine inflammatoire. Le facteur de transcription IRF-5, dont la relation avec l'induction de l'IFN est connue mais dont la fonction est à peine connue, a été ciblé et sa fonction a été élucidée. Le résultat a été qu’il a été élucidé que l'IRF-5 est un facteur de transcription important pour l'induction de la cytokine inflammatoire par l'activation de la TLR. Plus spécifiquement, il a été élucidé que l'IRF-5 est associé avec MyD88 et avecTRAF6 et qu’il est activé par ces molécules et que l'activation de la LTR induit le transport nucléaire de l'IRF-5 et qu’il est activé par ces molécules et que l'activation de la TLR induit le transport nucléaire de l'IRF-5 et active la transcription d'un gène de cytokine. En outre, il a été élucidé que l'IRF-4 se lie à MyD88 de manière compétitive avec l'IRF-5 et que l'induction de la cytokine inflammatoire est inhibée. A partir de ces résultats, un procédé de criblage pour un inhibiteur de la cytokine inflammatoire ciblé pour l'IRF-5 ou l'IRF-4 a été élaboré.
PCT/JP2005/024043 2004-12-28 2005-12-28 Nouveau procede de criblage pour l'inhibiteur de la cytokine inflammatoire WO2006070860A1 (fr)

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WO2012093258A3 (fr) * 2011-01-05 2013-01-03 Imperial Innovations Limited Traitement et criblage
WO2013121034A1 (fr) * 2012-02-17 2013-08-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés et compositions pharmaceutiques pour la réduction d'une inflammation du tissu adipeux
WO2019177099A1 (fr) * 2018-03-15 2019-09-19 株式会社マンダム Procédé d'évaluation d'échantillon d'essai
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HONDA K. ET AL.: "Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling", PROC. NATL. ACAD. SCI. USA, vol. 101, no. 43, 26 October 2004 (2004-10-26), pages 15416 - 15421, XP002999660 *
HONMA K. ET AL.: "Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS", PROC. NATL. ACAD. SCI. USA, vol. 102, no. 44, 1 November 2005 (2005-11-01), pages 16001 - 16006, XP002999665 *
KAWAI T. ET AL.: "Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6", NAT. IMMUNOL., vol. 5, no. 10, October 2004 (2004-10-01), pages 1061 - 1068, XP002999661 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008029220A (ja) * 2006-07-26 2008-02-14 Chube Univ 線虫を用いた有害物質又は有益物質の試験法、及び線虫を用いた解毒物質の取得法
WO2012093258A3 (fr) * 2011-01-05 2013-01-03 Imperial Innovations Limited Traitement et criblage
WO2013121034A1 (fr) * 2012-02-17 2013-08-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés et compositions pharmaceutiques pour la réduction d'une inflammation du tissu adipeux
WO2019177099A1 (fr) * 2018-03-15 2019-09-19 株式会社マンダム Procédé d'évaluation d'échantillon d'essai
CN111601898A (zh) * 2018-03-15 2020-08-28 株式会社漫丹 被测试样的评价方法
JPWO2019177099A1 (ja) * 2018-03-15 2020-12-17 株式会社マンダム 被験試料の評価方法
JP7027521B2 (ja) 2018-03-15 2022-03-01 株式会社マンダム 被験試料の評価方法
US11208650B2 (en) 2018-11-15 2021-12-28 Ionis Pharmaceuticals, Inc. Modulators of IRF5 expression

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