WO2006070860A1

WO2006070860A1 - Novel screening method for inflammatory cytokine inhibitor

Info

Publication number: WO2006070860A1
Application number: PCT/JP2005/024043
Authority: WO
Inventors: Tadatsugu Taniguchi; Akinori Takaoka; Tak W. Mak; Kenya Honda
Original assignee: The University Of Tokyo
Priority date: 2004-12-28
Filing date: 2005-12-28
Publication date: 2006-07-06

Abstract

It is intended to provide a novel screening method for an inflammatory cytokine inhibitor. Transcription factor IRF-5, whose relationship with IFN induction is known but whose function is hardly known, was focused and its function was elucidated. As a result, it was elucidated that IRF-5 is an important transcription factor for inflammatory cytokine induction by TLR activation. More specifically, it was elucidated that IRF-5 is associated with MyD88 and TRAF6 and activated by these molecules, and the TLR activation induces the nuclear transport of IRF-5 and activates the transcription of a cytokine gene. Further, it was elucidated that IRF-4 binds to MyD88 competitively with IRF-5 and the induction of inflammatory cytokine is inhibited. Based on these findings, a screening method for an inflammatory cytokine inhibitor with a focus on IRF-5 or IRF-4 was constructed.

Description

Specification

Novel screening method for inflammatory site force-in inhibitor

Technical field

[0001] 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.

Background art

[0002] Toll-like receptors (TLRs) recognize conserved microbial structures called pathogen-related molecular patterns and form the core of innate and adaptive immunity. So far, there are 10 known TLRs in humans. Pathogen-related molecular patterns recognized by TLR include gram-negative bacterial outer membrane components lipopolysaccharide (LPS), peptide daricans, and flajulin, a protein present in motile bacteria. LPS is a ligand for TLR4, peptidoglycan is a ligand for TLR2, and furadulin is a ligand for TLR5. TLR9 recognizes CpGDNA, and TLR3 recognizes double-stranded RNA and poly (I: C) functionally similar to double-stranded RNA. In addition, TLR ligands other than pathogenic molecules are known, and imidazoquinoline derivatives are TLR7 ligands. Thus, TLR is activated by various ligands.

[0003] 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. In most cases, 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). These MyD88-interacting molecules that associate with MyD88 and are involved in 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-κB and AP-1 transcription factors (Non-patent Documents 1, 2, 5, 6). ). The fact that 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. In other words, substances that regulate TLR signal transmission are expected to be useful in the treatment and prevention of diseases involving cytodynamic force-in. However, since 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

., Ghosh, S. & Janeway, C. A "Jr. (1998) Mol. Cell 2, 253-258.

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.

Gene 237, 1-14 (1999).

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)

Disclosure of the invention

Problems to be solved by the invention [0005] 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.

Means for solving the problem

[0006] The present inventors made extensive efforts to solve the above problems, and focused on the Interferon regulatory fact or (IRF) family. IRF is a transcription factor, and its ability to contribute to the regulation of the immune system is also attracting much attention (Non-patent Documents 7-9). IRF-5, a member of the IRF family, has received a lot of attention in relation to the induction of type I interferon (IFN) gene, but its function is almost unknown at the moment (Non-patent Document 10). .

[0007] 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-α (TNF-α). That 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 "Λ 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 .

[0008] Based on the above-mentioned novel findings, the present inventors further examined the possibility of involvement of other IRF families in the TLR-MyD88 signaling pathway. Analysis of the interaction of IRF-4 and IRF-8 with MyD-88 revealed that IRF-4 interacts with IRF-8. Interestingly, the analysis of the region of MyD88 that interacts with IRF-4 suggests that IR F-4 competes with IRF-5, so we are competing with IRF-4 and IRF-5. Further investigation was conducted on the possibility of this. As a result, IRF-4 competitively binds to the binding of IRF-5 to MyD88, inhibits IRF-5 nuclear translocation, and negatively regulates the induction of inflammatory site force-in by TLR signaling It became clear that it functions as a factor. Based on the above-described series of studies by the present inventors, IRF-5 was found to be an immune For the first time, it became clear that it would be a new target for the development of treatments for patients. 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.

(D Screening method for inflammatory site force-in inhibitor using as a marker the IRF-5 activity in a test substance when the test substance is brought into contact with an IRF-5-expressing cell,

(2) A screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (d):

(a) A step of bringing a test substance into contact with an IRF-5 expressing cell carrying a reporter gene operably linked downstream of a promoter region containing an ISRE sequence

(b) contacting the TRF ligand or virus with the IRF-5 expressing cells

(c) detecting the reporter activity of the IRF-5 expressing cell

(d) a step of selecting a test substance whose reporter activity due to the test substance contact shows a lower V value than the non-test substance non-contact control;

(3) The screening method according to (1) or (2) above, wherein the IRF-5-expressing cell is a cell that expresses endogenous IRF-5,

(4) IRF-5-expressing cell force The screening method according to (1) or (2) above, which is a cell expressing IRF-5 introduced exogenously,

(5) Using TFSEARCH (ht tp: 〃 www.cbrc.jp/research/db/TFSEARCHJ.html), an application that predicts transcription factor binding sites on DNA, an inflammatory site containing a promoter region Among the sequences obtained by searching for transcription factor binding regions in the entire region of the force-in gene, by setting the scole to 50 points or more, ISRE sequences, IRF-1 binding sequences and Z or IRF-2 The screening method according to any one of (2) to (4) above, wherein a sequence detected as a binding sequence is used as an ISRE sequence,

(6) ISRE sequence ability SEQ ID NO: 7 to SEQ ID NO: 58 Any one of the strengths The screening method according to (2) to (5) above,

(7) TLR is any one of TLR3, TLR4, TLR5, TLR7, TLR8, TLR9. The screening method according to any one of (1) to (8) above,

(8) A screening method for an inflammatory site force-in inhibitor using as a marker the amount of IRF-5 expression in a cell when IRF-5-expressing cells are contacted with a test substance,

(9) The screening method according to (8) above, comprising the following steps (a) to (c):

(a) A step of bringing a test substance into contact with an IRF-5 expressing cell

(b) a step of measuring IRF-5 mRNA in the IRF-5-expressing cells

(c) The amount of mRNA due to contact with the test substance is smaller than the amount of mRNA in the non-test substance contact control!

(10) The screening method according to (8) above, comprising the following steps (a) to (c):

(b) Quantifying the IRF-5 protein in the IRF-5 expressing cells

(c) Quantity of protein due to contact with test substance Smaller than the amount of protein for non-test substance contact control!

(11) Inflammatory site force using as an index the ability to inhibit the binding of MyD88 to IRF-5 by the test substance when MyD88 is contacted with IRF-5 in the presence of the test substance In inhibitor screening method,

(12) A screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (c):

(a) 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

(b) 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

(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

(13) A screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (d):

(a) A step of bringing a test substance into contact with cells expressing MyD88 protein and IRF-5 protein

(b) a step of separating a fraction containing MyD88 protein or a fraction containing IRF-5 from a cell lysate of a cell strain expressing the MyD88 protein and IRF-5 protein.

(c) Detecting the binding of MyD88 protein to IRF-5 protein in the separated fraction

(d) the step of selecting a test substance that decreases compared to the binding force between the MyD88 protein and the IRF-5 protein compared to a non-test substance contact control;

(14) 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,

(15) The screening method according to (14) above, comprising the following steps (a) to (c):

(a) A step of bringing a test substance into contact with an IRF-4 expressing cell

(b) measuring IRF-4 mRNA in the IRF-4 expressing cells

(16) The screening method according to (14) above, comprising the following steps (a) to (c):

(b) quantifying the IRF-4 protein in the IRF-4 expressing cells

(c) The amount of protein due to the contact of the test substance is smaller than the amount of the protein in the non-test substance contact control! / ヽ The process of selecting the test substance,

(17) Inflammatory site force in is any of IL-1, IL-6, IL-8, IL-12, IL-18, TNF-α, IFN-γ. The screening method according to any one of (1) to (16) above,

(18) Using the method described in any one of (1) to (17) above, a method for screening a therapeutic agent for a disease associated with inflammatory site force-in, (19) MyD88-IRF-5 binding inhibitor comprising any of the following (a) to (e)

(a) an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 60 or 62

(b) an isolated polynucleotide encoding a polypeptide in which one or more amino acids of SEQ ID NO: 60 or 62 are deleted, appended, substituted, Z or inserted.

(c) an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 59 or 61

(d) an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 59 or 61

(e) an isolated polypeptide encoded by any of the polynucleotides of (a) to (d) above,

(20) A therapeutic agent for a disease associated with inflammatory site force-in, comprising the MyD88-IRF-5 binding inhibitor described in (19) above.

Brief Description of Drawings

[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. These results show a single 6.7 kb band for wild type mice ( ^{+ / +} ), a 4.4 kb band for homozygous mice (- ^{/ _} ), and heterozygous ( ^+/- ) mice. Both show both. c, RNA blot analysis of MEF-derived RNA stimulated by c, pol y (l: C). Wild-type ( ^{+ / +} ) mice, heterozygous ( ^+/- ) and homozygous (-) MRF from IRF-5 mutant mice with poly (I: C) (200 / z gZml) for 3 hours And stimulated for 6 hours. Total 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. a, Ability to simulate normal DC (cDC) derived from the spleen of wild type (WT) mice (black bars) or IRF-5— ^Λ mice (open bars) (medium only), or 1.0 Β Stimulated for 24 hours with type CpG ODN (CpG-B) or CpG-A ODN (CpG-A), and measured the concentrations of IL-6, IL-12p40 and TNF-a in the culture supernatant by ELISA It is a graph. 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— ^Λ 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. c, spleen macrophages from WT mice (black bars) or IRF-5— ^Λ mice (open bars) with CpG-A ODN (1.0 μΜ) or CpG-Β ODN (1.0 μΜ) 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— ^Λ 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-γ, and TNF-α, IL- 6 is a graph showing the concentrations of 6 and IL-12p40 measured by ELISA. Data are representative of two independent experiments. ND, not detected.

[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- ^Λ 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 IκBζ 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. YFP and CFP fluorescence images of RAW264.7 (upper figures) and HEK293T (lower figures), and FRET images. Cells expressing YFP-IRF-5 and CFP-MyD88 were collected using an inverted fluorescence microscope equipped with a cooled CCD camera. FRETC (corrected FRET) was determined as described previously (Honda, K., Mizutani, T. & Taniguchi, T. Negative regulation of IFN-a / β signaling by IFN regulatory factor 2 for homeostatic developmen t of Dendritic cells. Proc. Natl. Acad. Sci. USA. 101, 2416-2421 (2004).) ₀ CFP and YFP fluorescence images, composite image and FRETC (displayed in pseudo color mode) from left to right drawings, respectively. An image is shown. b, Intermolecular FRET analysis for binding of three types of IRF to MyD88. The FRETC of HEK293T cells co-expressing one of the indicated YFP-labeled IRF and CFP-MyD88 was calculated, and the FRETCZCFP value was plotted as a histogram. c, shows results of co-immunoprecipitation of IRF-5 or IRF-3 with MyD88 (upper figures) or TRAF6 (lower figures). PCAGGS-HAIRF-5 for HEK293T cells Alternatively, pEF-HA-IRF-3 was transfected with pCXN2-FLAG-MyD88, and the cell lysate was subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting using anti-HA antibody. After stripping, immunoblotting of FLAG-labeled MyD88 was then performed with anti-FLAG antibody. 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). To examine TLR9-dependent activation of ISRE by IRF-5, 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. a, 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 μΜ) for the specified period. Cells were fractionated and 30 μg 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. The Using Raw 264.7 cells transiently expressing HA-tagged IRF-5, IRF-5 against endogenous ISRE in IL-12p40 gene after 6 hours stimulation with CpG-B ODN (l μ Μ) The bond was examined (left drawing). 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-κB activation in the absence of IRF-5. Spleen B cells from WT, IRF-5 ^{__ / _} and MyD88— ^Λ mice were stimulated by CpG-B ODN (0.3 M) for the specified period. The activity of NF-κB was evaluated by EMSA. Activation of NF-κB 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- ^Λ splenic B cells and treated for the specified period. The activity of ρ38 and JNK was analyzed using phospho-specific antibodies. An immunoblotting method using antibodies against ρ38 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. a, CpGB ODN (5 nmol) with D-galactosamine (D-GalN) (20 mg) in age-matched wild-type (n = 5) and IRF-5 chi (n = 4) mice Were injected intraperitoneally. Survival was observed for 4 days. b. Serum of mice injected with CpG-B ODN was collected 1 hour and 3 hours after injection. Serum concentrations of TNF-a, IL-12p40 and IL-6 were measured by ELISA. ND., Not detected. The results show the mean sd of serum samples from 3 mice. FIG. 5cd is a diagram following FIG. 5ab. c, wild-type-matched the number of age against (n = 4) and IRF- 5 Ji (n = 3) mice, LPS and (0.7 _i ug) were injected intraperitoneally with D-GalN (8mg). Survival was observed for 4 days. As described in d and b, 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 μg 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. RT-PCR for IRF-5 mRNA expression after cells were stimulated with CpG-B 1.0 (1.0 / ζ Μ), LPS (lOngZml) and poly (I: C) (100 g Zml) for 2 hours Minutes served for prayer. c, Table showing hematopoietic cell population in IRF-5 chi mice. Cells from designated tissues of heterozygous and homozygous mice and PBL (peripheral blood leukocytes) were analyzed for IRF-5 alleles by flow cytometry (BD biosciences) to calculate the relative percentage of each cell population . IRF-5 — ^{/ _} mice showed normal cellularity with respect to thymus, spleen, lymph nodes and bone marrow, which resembled that of heterozygous mice.

FIG. 7 is a diagram showing induction of IFN-a mRNA and 1FN-β mRNA by poly (I: C) in splenocytes. Spleen cells from heterozygous and homozygous mice were stimulated with poly (I: C) (200 μg / ml) for 6.5 hours and total RNA was pan-IFN-a (a4 and non-α4) or It was subjected to semi-quantitative RT-PCR analysis using any of IFN-ι8. No genomic DNA was found in any cDNA obtained by RT reaction. In relation to this result, induction of IFN-α in pDC and cDC from IRF-5— 'mice in response to infection with VSV (vesicular stomatitis virus) or HSV (herpes simplex virus) was observed normally. (Η. Υ., Unpublished data).

FIG. 8 is a graph showing IL-6 induction by CpG ODN stimulation in splenic B cells. Prepare sputum cells from the spleen of wild-type mice (WT; black bars) or IRF-5— ^Λ mice (open bars) or leave them unstimulated or CpG-A ODN (1.0 _i u M) or Stimulated with CpG-B ODN (l.O ^ M). After 24 hours, 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. Total RNA obtained from spleen macrophages of WT mice and IRF-5— mice was subjected to real-time RT-PCR. There is no significant difference between WT macrophages and IRF-5-macrophages for all TLR5 mRNA expression levels examined. This is consistent with the fact that NF-κBZMAPK activation and IFN-α induction (both require MyD88, TRAF6 and IRAK) occur normally in IRF-5-deficient cells. b, (Left) A graph showing the results of treating splenic B cells derived from WT mice or IRF-5 Chi mice with a specified amount of CpG-B (RPMI in 10% FCS). After 24 hours, the cells were pulsed with [ ³ H] thymidine for 12 hours, and the radioactivity uptake was measured with a j8-scintillation counter. (Right drawing) This is a graph showing the results of treating spleen B cells derived from WT mice or 1RF-5- ^Λ mice with 1.0 μΜ CpG-B (RPMI in 10% FCS). After a specified period, cells were pulsed with [ ³ H] thymidine for 12 hours, and radioactivity uptake was measured with a j8 scintillation counter.

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-. d, Cell viability of macrophages after TLR stimulation or X-ray irradiation. Spleen macrophages derived from WT and IRF-5— 'mice were stimulated by CpG-B (1.0 μ μ), LPS (lOngZml) or X-ray irradiation (5.0 Gy). Viability was assessed 24 hours later by Probidium iodide (PI) staining

[Fig. 10] ISRE signs in mouse IL-6, IL-12p40, TNF-a and IκΒζ 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 Ικκζ.

[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). (D) Cell lysates that have also been prepared with HEK293T cells that transiently express the combination of FLAG-MyD88 and HA-IRF were immunoprecipitated (IP) using anti-FLAG antibodies and anti-HA or It was subjected to immunoblot (IB) analysis using anti-FLAG antibody. WCL, whole cell extract.

FIG. 13 shows competition between IRF-4 and IRF-5 for MyD88 interaction. (a) Schematic representation of MyD88 truncation mutant. +, Positive interaction with IRF;-, no interaction (assessment by immunoprecipitation assay). (B) In HEK293T cells, each FLAG-MyD88 mutant was co-expressed with HA-IRF-4 (left) or HA-IRF-5 (right) and subjected to co-immunoprecipitation analysis. (c) For HEK293T cells, a certain amount of HA-IRF-5 expression vector (1.0 g) or HA-IRF-7 expression vector (2.0 μg), FLAG-MyD88 expression vector (1 g), and 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. (a) Schematic of IRF-4 truncation mutant. DBD, DNA binding domain; AD, active domain; RD, regulatory domain. (B) Confocal image of HEK293T cells that transiently express YFP-IRF-4, YFP-IRF-4ΔDBD or YFP-IRF-4ΔRD. (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. 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. (f) It is a figure which shows the influence of IRF-4 with respect to the nuclear translocation of IRF-5 induced by ODN1668 stimulation. RAW264.7 cells expressing YFP-IRF-5 alone or both YFP-IRF-5 and RFP-IRF-4 were placed on a Timelabs microscope and images were collected at 1 minute intervals. Cells were stimulated with 1 μM ODN1668 or left untreated and incubated for up to 60 minutes in the presence of lOngZml leptomycin. Nuclear region normalization YFP intensity was plotted against time.

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-γ. The concentrations of IL-12p40, IL-6 and TNF-α in the culture supernatant were measured by ELIS. The results shown are the average of three measurements (Shi SD). (C) Spleen pDCs prepared from wild-type mice and Irf4 − ^{/ _} mice were treated with ODN-D19 or poly (U) at 24 hours Stimulated for a while. IFN-α concentration was measured by ELISA. (D) It is a graph which shows the induction | guidance | derivation of mRNA of the gene induced | guided | derived by proinflammatory site force in, chemo force in, and NF-κB. Resident peritoneal macrophages from wild-type mice, Irf4 or IriS mice were stimulated by ODN1668 for the specified period. Total RNA was prepared and subjected to quantitative real-time RT-PCR analysis of designated genes.

FIG. 16 shows cell type-specific contributions of IRF-4 and IRF-5 systems. (a) BMM from wild-type mice, IriS — ^{/ _} or Irf4 — ^{/ _} mice was stimulated with the specified stimulus in the presence of IFN-γ. The 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-4ΔDBD expression vectors were transiently transfected into RAW264.7 cells by electroporation. OD cells after 12 hours

Stimulated for the period specified by N1668. Total RNA was prepared and subjected to quantitative real-time RT-PCR analysis.

FIG. 17 shows the role of IRF-4 in vivo. ODN1668 (10 nmol) and D-GalN (20 mg) were injected intraperitoneally into age-matched wild-type mice (n = 7) and Irf4- ^Λ mice (n = 4). (A) Serum concentrations of IL-6, IL-12p40 and TNF-α were measured by ELISA. Results shown are the average of serum samples (SD). (B) The survival of these mice was observed for 15 hours.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The present invention relates to a novel method for screening for an inflammatory site force-in inhibitor using IRF-5. As described above, for the first time, the present inventors have revealed that IRF-5 plays an essential role in inducing inflammatory site force-in via the TLR signal pathway. With this new knowledge, the present inventors have also found a new screening method for an inflammatory site force-in inhibitor using IRF-5.

[0011] 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. In the present invention, “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.

[0012] 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. It is known that 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. Many examples of surface antigens specific to various cells are already known (see, for example, Shujunsha Cell Engineering Separate Volume Book Book “New Edition Handbook of Adhesion Molecules”). 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. Specifically, 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. In the case of dendritic cells, cDC and plasmacytoid dendritic cells (pDC) can be selected using B220 and CDllc as indicators. In the case of macrophages, the above organ or tissue force can also be prepared using an anti-CD1 lb antibody.

[0013] When cells expressing IRF-5 introduced exogenously are used, desired cells can be prepared by introducing the isolated IRF-5 gene into appropriate cells. As an example of the IRF-5 gene, 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 amino acid sequence of mouse IRF-5 SEQ ID NO: 4 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. Alternatively, 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.

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. For example, 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. For example, 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.”. It should be noted that the above 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.

[0015] 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. And high homology. 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. USA 90: 5873-5877, 1993 by Karlin and Altschul. ) Can be determined. Based on this algorithm, a program called BLASTX has been developed (Altschule al. J. Mol. Biol. 215: 403-410, 1990). When the amino acid sequence is analyzed by BLASTX, the parameter 1 is, for example, score = 50 and wordlength = 3. When using BLAST and Gapped B LAST programs, use the default parameters of each program. Specific methods for these analysis methods are known (http: 〃 www.ncbi.nlm.nih.gov.).

Furthermore, even 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. For example, 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. Can be used. In 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. Usually, it is within 10% of the total number of amino acids, preferably within 3% of the total number of amino acids, more preferably within 1% of the total number of amino acids. Such a polynucleotide can be prepared by a person skilled in the art by a well-known technique. For example, 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. Alternatively, 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.

[0017] 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.

[0018] 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. For example, 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. As long as it is a mammal-derived cell or cell line, vectors based on various viruses such as adenovirus, papillomavirus, papovavirus, and retrovirus can be used.

[0019] 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. It is desirable to express TLR, MyD88 and Z or TRAF6 as the “IRF-5 expressing cell” used in the method of the present invention. The expressed TLR, MyD88 and TRAF6 may be endogenous TLR, MyD88 and TRAF6 or an artificially introduced exogenous TLR, MyD88 and TRAF6. 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

Homo NM.003265

Mouse NM— 126166

TLR4

Human variant 1 hires—138554

variant2 NMichi 138556

variant3 NMichi 003266

variant4 NMichi 138557

Mouse Advisor— 021297

TLR5

Human NM— 003268

mouse AF186107

TLR7

Human NM— 016562

Mouse NM— 133211

TLR8

Human variant 1 NM— 016610

variant2 NMichi 138636

Mouse NM— 133212

T and R9

Human variantA NM— 017442

variantB NMichi 138688

Mouse NM one 031178

MyD88

Human NM.002468 (Amino acid sequence NP—002459)

Mouse NM— 010851 (amino acid sequence NP_034981) TRAF6 Human variant 1 NM— 145803 (amino acid sequence NP— 665802.1)

variant2 NM— 004620 (amino acid sequence NP— 004611.1)

Mouse NM.009424 (Amino acid sequence NP_033450.2)

[0020] 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. -5 ISRE sequence, considered to be a binding site, was shown to be included in the inflammatory site force in gene. Therefore, 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.

[0021] 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. As such 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. When searching for a transcription factor binding region on the entire inflammatory site force-in gene region, including the region, 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. As specific ISRE sequences that can be used in the method of the present invention, the sequences described in SEQ ID NOs: to 58 can be exemplified.

[0022] The reporter gene can be selected from known reporter genes that are suitable for the conditions such as the detection method. For example, 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.

[0023] 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.

[0024] The above-described screening method using reporter gene activity as an index can include, for example, the following steps:

(b) contacting the TRF ligand or virus with the IRF-5 expressing cells

(c) detecting the reporter activity of the IRF-5 expressing cell

(d) A step of selecting a test substance that shows a value that is lower in the reporter activity due to the contact of the test substance than in the non-test substance contact control.

[0025] The above-mentioned "step of contacting a test substance with an IRF-5-expressing cell holding a reporter gene operably linked downstream of a promoter region containing an ISRE sequence" refers to TLR signal transduction in the cell. Is a step for bringing the member constituting the sample into contact with the test substance. In the screening method of the present invention, the type of test substance is not particularly limited. Examples of 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. Further, the 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.

[0026] 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. For the TLR ligand used in this method, use 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. Similarly, TLR4 is LPS, TLR5 is flagellin, TLR7 and TLR8 are synthetic. Various 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. The above ligands and viruses are examples, and usable ligands and viruses are not limited to these.

[0027] The above-mentioned "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.

[0028] In the above-mentioned "step of selecting a test substance in which the reporter activity by contact with the test substance shows a lower value than the control without contact with the test substance", the reporter activity obtained as described above is used as an index. And selecting a test substance that can be an inflammatory site force-in inhibitor. 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.

[0029] 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.

[0030] 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.

(b) Quantifying the IRF-5 protein in the IRF-5 expressing cells

[0031] When measuring the amount of mRNA, 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.

(b) a step of measuring IRF-5 mRNA in the IRF-5-expressing cells

(c) Amount of mRNA due to contact with test substance Less than the amount of mRNA in non-test substance contact control!

[0032] 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" A screening method for inflammatory site force-in inhibitors using the ability as an index. In the TRL signal pathway, IRF-5 forms a complex with MyD88 and TRAF6 and interacts with it to exert its function. IRF-4 binds MyD88 to IRF-5. Competitively inhibits the expression of inflammatory site force-in. 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.

In the present invention, “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.

[0034] 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. As an example of MyD88 that can be used in the method of the present invention, 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, and the amino acid sequence is It is shown in SEQ ID NO: 66, but is not limited thereto. Even 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.

[0035] The method for detecting the binding between MyD88 and IRF-5 in situ includes, for example, the following steps.

(a) 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;

(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.

[0036] As a method including the steps (a) to (c), a method using analysis by FRET can be shown. In step (a) above, cell MyD88 and IRF-5 can be detected by FRET. Te in the process of labeling. FRET (Fluorescence resonance energy transfer) is a phenomenon in which excitation energy is transferred from one fluorescent molecular force to another fluorescent molecule. In the present invention, the “donor fluorescent protein” is a fluorescent protein that gives energy to another fluorescent protein (acceptor fluorescent protein) in FRET, and 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. As examples of fluorescent proteins, BFP, GFP, CFP, YFP, DsRed, AmCyan, ZsYellow, ZsGreen, etc. are actually used. 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. For example, 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.

[0037] In the step (b), 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. What is the specific method of FRET analysis above? J, Honda, K., Mizutani, T. & Tanigucm, T. 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.

[0038] As a result of the above analysis, if the FRET force in the presence of the test substance is less attenuated or disappears than FRET in the absence of the test substance, the test substance inhibits the binding between MyD88 and IRF-5. it seems to do. Therefore, the FRET power in the presence of the test substance The test substance attenuated or disappears more than the FRET in the absence of the test substance It is considered to be a substance that suppresses the expression of ins.

[0039] In addition, when detecting in vitro the ability to inhibit the binding between MyD88 and IRF-5 by a test substance, it can be carried out by a method including the following steps, for example.

(a) contacting a test substance with cells expressing MyD88 protein and IRF-5 protein;

(b) a step of separating the cell lysate of the cell force expressing the MyD88 protein and the IRF-5 protein, the fraction containing the MyD88 protein or the fraction containing the IRF-5,

(c) detecting the binding of MyD88 protein to IRF-5 protein in the separated fraction;

(d) A step of selecting a test substance that decreases the binding force between the MyD88 protein and the IRF-5 protein as compared to a non-test substance contact control.

[0040] 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.

[0041] After contacting the test substance and the "cells expressing MyD88 protein and IRF-5 protein" in the step (a), from the cell lysate prepared from the cells in the step (b) Separate fractions containing MyD88 protein or IRF-5. Separation of a fraction containing MyD88 protein or IRF-5 can be performed using, for example, an anti-MyD88 antibody or an anti-IRF-5 antibody. When cells expressing a fusion protein with a known labeled peptide such as FRAG or HA are used as the exogenous MyD88 protein and IRF-5 protein, an antibody against the labeled peptide such as anti-FRAG antibody or anti-HA antibody is used. It can also be used to separate a fraction containing the desired protein. For example, 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.

[0042] In the 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. Indicates that Alternatively, 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.

[0043] 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.

(a) contacting MyD88 protein with IRF-5 protein in the presence of a test substance;

(b) a step of detecting the binding activity between MyD88 protein and IRF-5 protein in the presence of the test substance;

(c) Compare the binding activity of MyD88 protein and IRF-5 protein in the presence of the test substance with the binding activity of MyD88 protein and IRF protein in the absence of the test substance. A step of selecting a test substance that exhibits a lower binding activity than its activity.

[0044] 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). In the case of BIACORE, in 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. In step (b) above, the sensor chip is irradiated with light and the change in the refractive index of the reflected light is monitored. Thus, the binding activity between the ligand and the analyte can be known. For test substances that reduce or eliminate the binding between the ligand and the analyte, the use and operation of BIACORE can be found on the BIACOR E website (http: 〃 www.biacore.co.jp/).

[0045] 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.

[0046] 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.

(b) quantifying the IRF-4 protein in the IRF-4 expressing cells

[0047] When measuring the amount of mRNA, 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. .

(b) measuring IRF-4 mRNA in the IRF-4 expressing cells

[0048] As described above, 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-α, IFN-γ You can choose from.

[0049] 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. As a substance, IRF-4 protein is provided. Although 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.

[0050] As an example of IRF-4 in the present invention, the human IRF-4 cDNA sequence is SEQ ID NO: 59, the amino acid sequence is SEQ ID NO: 60, and the mouse IRF-4 cDNA sequence is SEQ ID NO: 61. The amino acid sequence is shown in SEQ ID NO: 62. In the mouse IRF-4 amino acid sequence (SEQ ID NO: 62), positions 21 and 127 are the DNA binding domain, positions 200 and 267 are the active domain, and positions 268 to 450 are the regulatory domains. . IRF-4 protein can be prepared by known methods of IRF-5. For example, 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. In addition, 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.

[0051] 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. For preparation of these IRF-4s, 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.

[0052] In addition to the IRF-4 protein, the present invention also provides the IRF-4 gene as an inflammatory site force-in inhibitor. By administering the IRF-4 gene to patients with increased inflammatory site force-in expression, the IRF-4 gene is expressed in the patient's body, so a sustained inflammatory site force-in inhibitory effect is expected. it can.

[0053] 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. For example, 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.

[0054] When 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. 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.

[0055] As described above, 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. In addition to the invention described above, based on the novel findings of the present inventors, 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.

[0056] It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

Example

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

(Example 1) Function of IRF-5 in gene induction by TLR activity

(1 1) Materials and methods

[Production of IRF- ⁵ Chi-Mouse]

[0058] 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.

[Other mice and reagents]

[0059] 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. [0060] 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).

[Preparation of rod cells, macrophages and B cells]

[0061] 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.

[Measurement of site force in production]

[0062] 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.

[0063] The concentrations of IL-12p40, IL-6, TNF-a and IFN-a in the culture supernatant were measured by solid-phase enzyme immunoassay (ELISA). ELISA kits for mouse IL-12p40, IL-6 and TNF-α were obtained from R & D Systems. Seven ELISA kits for mouse IFN-α were purchased from PBL Biomedical Laboratories.

CRNA analysis]

[0064] RNA extraction and polymerase chain reaction (RT-PCR) analysis with reverse transcription have been previously described. T'7 ko U'akaoka, A. et al. Integration of interferon- / β signal ling to p53 responses in tumour suppression and antiviral defense (Nature 424, 516—523 (2003)) o Quantitative real-time RT-PCR analysis , LightCycler and SYBRGreen system (Roche). Data are standard for 13-actin expression in each sample. Normalized. Primers for j8-actin, IL-6, TNF-α, and IL-12p40 are based on previous literature [7aKaguchi, b. et al. Essential role of IRF-3 in lipopolysaccharide- inducedinterferon-β gene expression and endotoxin shock. Biochem. Biophys. Res. Commun. 306, 860-866 (2003)). The primers used for RT-PCR are specifically as follows.

IRF-5: 5- AATACCCCACCACCTTTTGA-3 (SEQ ID NO: 5) (sense)

5- TTGAGATCCGGGTTTGAGAT-3 (SEQ ID NO: 6) (antisense)

Ι κ Β ζ: 5- ATCCGAAGCAACAAGCAGAA-3 (SEQ ID NO: 67) (sense)

5-CACGAAGTGAGAAGGCAACA-3 (SEQ ID NO: 68) (antisense)

TLR3: 5- TTAGAGTCCAACGGCTTAGAT-3 (SEQ ID NO: 69) (sense)

5-AACGGATTGAAGCGCATA-3 (SEQ ID NO: 70) (antisense) TLR4: 5-GAGCCGGAAGGTTATTGTGGT-3 (SEQ ID NO: 71) (sense)

5- CCTCTGCTGTTTGCTCAGGAT-3 (SEQ ID NO: 72) (antisense) TLR5: 5-AAGTTCCGGGGAATCTGTTT-3 (SEQ ID NO: 73) (sense)

5- GCATAGCCTGAGCCTGTTTC-3 (SEQ ID NO: 74) (antisense)

(Renshaw, M. et al. Cutting edge: impaired Γοΐΐ- like receptor expression and functi on in aging. J. Immunol. 169, 4697-4701 (2002))

TLR7: 5—GTTCTTGACCTTGGCACTA— 3 (SEQ ID NO: 75) (sense;)

5- CCGTGCATATTCATCGTA-3 (SEQ ID NO: 76) (antisense) TLR9: 5-ATGGACGGGAACTGCTACTACA-3 (SEQ ID NO: 77) (sense)

5-GACCTTGGAACCAGGAAGAGTT-3 (SEQ ID NO: 78) (antisense)

[Plasmid and gene transfer]

[0065] For 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).

[0066] Mouse IRF-5 cDNA was obtained by RT-PCR of total MEF-derived RNA and cloned into a pCRII (Stratagene) vector.

[0067] To create a 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 / β signaling to p53 responses in tumour suppression and antiviral defence. Nature 424, 516 -523 (2003)).

[0068] For ChIP assembly, 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)!

[Fluorescence microscopy]

[0069] Cells were cultured on glass bottom 35mm tissue culture dishes (MATSUNAMI GLASS) and imaged by fluorescence microscopy as described previously (Honda, K., Mizutani, T. & Taniguchi, T. Negative regulation of IFN— a / β signaling by IFN regulatory factor 2 ror nomeo static development of dendritic cells. Proc. Natl. Acad. Sci. USA. 101, 2416-2421 (2004)) o

[0070] FRET analysis was performed as previously described (Honda, K., Mizutani, T. & Taniguchi, Τ. Negative regulation or irN-a / β signaling oy IFN regulatory factor 2 for home ostatic development of dendritic Proc. Natl. Acad. Sci. USA. 101, 2416-2421 (2004)) o

[Immunoprecipitation and immunoblotting]

[0071] Cell solubilization, immunoprecipitation and immunoblotting were performed as described in previous literature.

(Takaoka, A. et al. Integration or interreron—a / β signaling to p53 responses in tumour suppression and antiviral defense. Nature 424, 516-523 (2003)).

[0072] Antibodies against the following proteins were purchased: HA (Roche), FLAG (Sigma- Aldrich Co

.), Phospho-SAPK / JNK, SAPK / JNK, Phospho-p38 MAPK and p38 MAPK (Cell Signaling Technology). [Nuclear Western Blotting and Electrophoretic Mobility Shift Assay (EMSA)] These assays were performed as previously described (see also the Supplemental Information section).

[0073] Mouse embryonic fibroblast (MEF) was prepared according to standard procedures (Takaoka, A. et al. Integration or interferon- / β 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 — Κ B— mediated up— r egulation of Bcl-χ 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.

[0074] EMSA for NF-κΒ 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)).

[Chromatin Immunoprecipitation Atsey (ChIP)]

[0075] ChIP Atsey was performed as described in the previous literature (Takaoka, A. et al. Integration of interferon-a / β 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.

[0076] From the purified DNA, the following primers for detecting a sequence (nucleotide numbers -67 to -55) containing IL-12p40-ISRE:

5-ACCCCGAAGTCATTTCCTCT-3 (SEQ ID NO: 79) (sense)

5- ACCCACTGTTCCTTCTGCT-3 (SEQ ID NO: 80) (antisense),

And 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):

5-GATGCAACGTTGGAAAGGA-3 (SEQ ID NO: 81) (sense)

5-TTCAACAGCATAAGGCCAAG-3 (SEQ ID NO: 82) (antisense)

Analyzed by PCR amplification.

[0077] The region of the IFN-β promoter (nucleotide numbers -74 to +48) contains the following primers: 5 -GGGAGAACTGAAAGTGGGAAA-3 '(SEQ ID NO: 83) (sense)

5-ACCTGCAAGATGAGGCAAAG-3 (SEQ ID NO: 84) (antisense)

Was amplified by using

[0078] PCR products were visualized by bromide modified gel.

[Reporter Atssey]

[0079] On HEK293T cells plated on 12-well plates, using FuGene reagent (Roche), with 50 ng of reporter plasmid p-55ClBLuc and the specified amount of expression plasmid for IRF-5, MyD88 and TRAF6 A transient simultaneous transfection was performed. For CpG-B ODN stimulation experiments, 50 ng reporter plasmid and lOOng pCAGGS-HA-IRF-5 were transferred to HEK293 cells expressing TLR9 (InvivoGen), followed by CpG-B ODN stimulation. For 12 hours. 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 / β signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells.Proc. Natl. Acad. Sci. USA. 101, 2416-2421 (2004)

) o

[0080] Reporter plasmid used in this assembly: p-55ClBLuc 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).

[Reagents for flow cytometry analysis]

[0081] 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.

(1-2) IRF-5 gene-deficient mice

[0082] Transcription factors of the IRF family have attracted much attention because they contribute to the regulation of the immune system. 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. Of note, 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.

[0083] To elucidate whether 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).

[0084] The mice developed normally and were strong with no apparent difference in the hematopoietic cell population (Fig. 6c).

(1-3) Involvement of IRF-5 in TLR signaling

[0085] Two TLR9 ligands that have been extensively studied, CpG-B oligonucleotide (CpG-B ODN, also known as 1668 or K-type ODN) and CpG-A ODN (D 19 (Also referred to as D-type ODN), and 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.

[0086] 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-α in the culture supernatant were measured by ELISA. The supernatant was also measured for IFN-o; induction. In addition, plasmacytoid DC (pDC) derived from the spleen of WT mice or IRF-5 − ^{/ _} mice was also examined in the same manner as cDC.

[0087] Induction of IL-6, IL-12, and TN F-a in cDCs stimulated with CpG-B ODN or CpG-A ODN was compared to wild-type (WT) cDC in IRF-5 — ^{/ _} cDC (Fig. 2a). A similar reduction in cytoforce-in production was also observed in pDCs from IRF-5 chi mice (Figure 2b;). In contrast, the IFN-α levels observed only in pDC stimulated by CpG-A ODN Induction was normally enhanced in IRF-5 ^{_ /} —pDC, indicating that IRF-5 is not involved in induction of type I IFN (see Figure 2b; Figure 7) ).

[0088] Next, splenic macrophages of WT mice or IRF-5 chi mice were treated with CpG-A ODN (1.0 μΜ) or CpG-Β ODN dO ^ M) in the presence of IFN-γ 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-α in the culture supernatant were measured by ELISA as described above. To observe cytoforce-in production upon stimulation by other TLR ligands, leave splenic macrophages of WT mice or IRF-5 ^{__ /} — mice unstimulated, or use various TLR ligands such as TLR 3 (polyinosine -Polycytidylic acid [poly (I: C)]; 100 gZml), TLR4 (lipopolysaccharide [LPS]; 10 ngZml), TLR5 (flagellin lO / z gZml) and 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-γ After stimulation with NF-a) for 24 hours, the concentrations of TNF-a, IL-6 and IL-12p40 in the culture supernatant were measured by ELISA in the same manner as described above.

[0089] Induction of IL-6, IL-12 and TNF-a in splenic macrophages and IL-6 in B cells by CpG-B ODN stimulation was also impaired in cells derived from IRF-5- ^{/ _} mice (Figure 2c and Figure 8a, b). Interestingly, the failure of cytoforce-in induction can cause spleen mouth phages to bind to other TLR ligands, namely TLR3 ligand (polyinosine-polycytidylic acid [poly (I: C)]), TLR4 ligand (lipopolysaccharide [LPS]) It was also observed when stimulated with TLR5 ligand (flagellin) and TLR7 (8) ligand (polyuridylic acid [poly (U)]) (Fig. 2d). In the absence of IRF-5, TLR mRNA expression remained unaffected, and B cell proliferation and induction of immunoregulatory cell surface molecules such as CD40 and CD86 in B cells and cDC were normal. (Fig. 9a, b, c). Furthermore, the lack of IRF-5 did not affect macrophage viability after TLR stimulation (Fig. 9d). Therefore, IRF-5 is selectively involved in the induction of pro-inflammatory site force-in induced by these TLRs, and probably seems to be the same for all other TLRs!

[0090] The induction of mRNA of the pro-inflammatory site force-in was analyzed. Spleen macrophages were prepared from WT and IRF-5 mice and stimulated with CpG-B ODN, LPS or poly (I: C) under the same conditions as described in d. Extract total RNA at specified time points, Subsequently, it was subjected to quantitative RT-PCR analysis. In addition, splenic macrophages were stimulated with the designated PAMP, and ΙκΒζ mRNA expression was monitored in the same manner as in the case of the above-mentioned proinflammatory site force-in.

[0091] Quantitative RT-PCR analysis revealed that this induction was impaired at the mRNA level, suggesting the transcriptional activity of these site-force-in genes by IRF-5 (Fig. 2e) _{0 The} site force S, which is considered the IRF-5 binding site, which is actually named the interferon response element (ISRE), was identified in these site force-in genes (Fig. 10). All of these genes are NF-κB binding sites, as well as transcription factors activated by stress-activated protein kinase (SAPK) Z JNK and p38 MAP kinases, which are also activated by TLR signaling. Since it further includes a binding site, we speculate that IRF-5 acts in concert with these transcription factors to activate these genes. We also speculate that the residual mRNA induction observed in some IRF-5 cells is a reflection of differences in the contribution of these transcription factors and depends on the cell type or stimulus. . Since the present inventors discovered four ISRE candidates in the promoter of IκBζ (Fig. 10), we investigated RNA induction in macrophages stimulated by various sputum, and this induction was absent in IRF-5. We found that it was significantly reduced below, suggesting that this gene is another target for IRF-5 (Figure 2f). I _K B zeta the force has been shown to regulate TLR signaling ^{s (Yamamoto, M. et al.} Regulation of Toll / IL-1- receptor- m ediated gene expression by the inducible nuclear protein I κ B ζ Nature 430, 218—22 (2004)), our in vitro data (Figure 2a-e) and in vivo data (see below) for IRF-5 chi mice are inflammatory compared to those in I κ B ζ-deficient mice. It differs in the point of guidance of site power in. For this reason, although interesting, the role of IRF-5 for I κ 依存 ζ dependent events needs further investigation.

(1-4) Examination of the relationship between IRF-5 and MyD88-TRAF6 signaling pathway

[0092] To gain insight into the mechanism of TRF-induced IRF-5 activity, 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. First, turn IRF-5 or IRF-3 yellow Labeled with fluorescent protein (YFP) (hereinafter referred to as YFP-IRF-5 and YFP-IRF-3) is expressed in mouse macrophage RAW264.7 cell line and human fetal kidney 293T (HEK293T) cell line, These cells were subjected to fluorescence microscopy analysis. As shown in Figure 3a, 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)

[0093] Furthermore, regarding the formation of an intermolecular complex between IRF-5 and MyD88 and TRAF6, 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

[0094] 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. These observations suggest that IRF-7, which also interacts with these molecules in pDC and is activated by them to induce type I IFN, and the MyD88-TRAF6 pathway diverges into these two types The activation of a class of IRF molecules, which in turn, activates different classes of site force-in genes, suggesting a unique mechanism. (1-5) Relationship between IRF-5 and TLR stimulation

[0095] We investigated how IRF-5 behaves in response to TLR stimulation by expressing YFP-IRF-5 in RAW264.7 cells. When stimulated with CpG-B ODN or LPS, a significant proportion of YF P-IRF-5 can be detected in the nucleus, which suggests that activation of TLR9 and TLR4 causes nuclear translocation of IRF-5 (Fig. 4a, Fig. L la, b). On the other hand, CFP-MyD88 expressed in these cells in the same manner was not detected in the nucleus after stimulation with CpG-B ODN (Fig. 4a). Furthermore, IRF-5 (HA-IRF-5) labeled with hemadalchun was expressed in mouse fetal fibroblasts (MEF) .After CpG-B ODN stimulation, HA-IRF-5 became WT MEF nucleus. However, it was not detected in MyD88— 'MEF, indicating that the nuclear translocation of IRF-5 induced by force TLR9 is actually MyD88-dependent (Fig. 4b). It is currently unknown how IRF-5 is activated by the TLR_MyD88 pathway, and this seems to be an interesting topic to be studied in the future.

[0096] In addition, 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). In order to investigate whether or not it binds, 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. Differential regulation of the IL -12 p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharid e. J. Immunol. 162, 6770-6775 (1999)). As shown in Figure 4c (left), a PCR-amplified DNA band corresponding to IL-12p40-ISRE is selective in the anti-HA antibody immunoprecipitates of cells stimulated with CpG-B ODN for 6 hours. Detected. The DNA band corresponding to the IFN- | 8 promoter is not amplified in this immunoprecipitate (Figure 4c, right), and this observation is due to the fact that the IFN-jS gene is not induced by CpG-B ODN in these cells. Match (data not shown). Overall, these results suggest that TLR9 signaling causes a MyD88-dependent activity of IRF-5, which in turn induces transcription of the IL_12p40 gene. There are many ISREs inside the IL-6 and TNF-a genes In view of this (FIG. 10), we speculated that these genes would be similarly activated by IRF-5 through one or more of these ISREs. Further analysis is needed to confirm that the ISRE is actually activated by IRF-5.

[0097] TLR-dependent activation of NF-κB, (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 μΜ) for the specified period. Activation of NF-κΒ was evaluated by EM SA. As a result, it was found that the activity of NF-κΒ, ρ38, and JNK by CpG-B ODN was almost normal in IRF-5— 'cells (Fig. 4d. Proposed the presence of a transduction transcription processor (CTTP), in which MyD88 forms a multimolecular complex with IRF_7, TRAF6 and IRAK4, which subsequently leads to an extracellular signal that induces a downstream gene induction program. Proc. `` Process '' properly (Honda, K., Mizutani, T. & Taniguchi, T. Negative regulation of IFN- I j8 signaling by IFN regulatory factor 2 for homeostatic development of dendritic ce lis. Acad. Sci. USA. 101, 2416-2421 (2004)) ₀ This study suggests that it is another component of the IRF-5 force SCTTP complex. The difference in the contribution of IRF-5 and IRF-7 in the null transmission results in a sufficient TLR response. To ensure that these and other transcription factors are activated, 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.

(16) Examination of the role of IRF-5 in vivo

[0098] To investigate the role of IRF-5 in vivo, lethal shock induced by CpG-B ODN or LPS was examined in D-galactosamine (D-GalN) -sensitized mice. CpGB ODN (5 nmol) was injected intraperitoneally with D-galactosamine (D-GalN) (20 mg) in age-matched wild-type (n = 5) and IRF-5 chi (n = 4) mice. 4 days regarding survival Observed for a while. In addition, 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. L PS ^ was intraperitoneally injected with LPS (0.7 g) with D-GalN (8 mg) for age-matched wild-type (n = 4) and IRF-5 chi (n = 3) mice. The animals were injected and observed for survival for 4 days. Similarly to D-GalN, serum from mice injected with LPS was collected 1 hour and 2 hours after injection.

[0099] As reported in ij (Sparwasser, T. et al. Macrophages sense pathogens v ia DNA motifs: induction of tumor necrosis factor- -mediated shock. Eur. J. Imm unol. 27, 1671- 1679 (1997)), WT mice died within 10 hours after CpG-B ODN administration, and this was accompanied by a marked increase in serum IL-6, IL-12 and TNF-α levels. IRF-5- ^{/ _} mice survived, and the induction of these cytoforce-ins in serum was markedly suppressed (Fig. 5a, b). In addition, IRF-5- ^{/ _} mice also showed resistance to LPS-induced endotoxin shock (Fig. 5c, d). These results are consistent with the above in vitro data, and together, they show the essential role of IRF-5 in the activity of TLR9 and TLR4, and probably the same for all other TLRs. It seems that it seems. As expected from these data, preliminary results indicate that IRF-5 chi mice exhibit impaired Thl-type immune response! / Sound (data not shown).

[0100] The present investigation by the present inventors sheds light on the elucidation of the mechanism of the gene induction program downstream of TLR signaling. In fact, detailed studies have been conducted on transcription factors activated by NF-κB and MAP kinase, and TRF-3 and IRF-7 are also involved in the activity of type I interferon system. Our results differ from IRF-3 and IRF-7, where IRF-5 is generally downstream of the TLR-MyD88 signaling pathway and is a gene for inflammatory site force-in by various pathogen-derived molecules ( Probably other genes are also involved as master transcription factors in the activity of other genes. Thus, our results suggest that IRF-5 is a candidate for a new target for therapeutic intervention aimed at suppressing adverse immune responses.

(Example 2) IRF-4 in TLR signaling

(2-1) Materials and methods [mouse]

[0101] C57BLZ6 mice were purchased from CLEA Japan, Osaka. The production of IriS— ^Λ mice and Irf4— mice has been described in the literature (Takaoka, A., Yanai, H., Kondo, S., Duncan, G., Negishi, H., Mizutani, T., Kano, S., Honda, K., Ohba, Y., Mak, TW & Taniguchi, T. (2005) Nature 434, 243-249, and Mittrucker, HW, Matsuyama, T., Grossman, A., Kundig, TM, Potter, J ., Shahinian, A., Wakeham, A., Patterson, B., Ohashi, PS & Mak, TW (1997) Science 275, 540-543).

[Plasmid construction]

[0102] Hemagglutinin (HA) labeled mice IRF-3, IRF-5 and IRF-7, FLAG labeled full length mice MyD88 and a series of deletion mutants of MyD88, cyan fluorescent protein (CFP) labeled MyD88 and yellow Expression vectors related to fluorescent protein (YFP) -labeled IRF-5 are available in literature (Mit trucker, HW, Matsuyama, T., urossman, A., Kundig, TM, Potter, J., Shahinian,

A., Wakeham, A., Patterson, B., Ohashi, PS & Mak, TW (1997) Science 275, 5 40-543, and Takaoka, A., Yanai, H., Kondo, S., Duncan, G ., Negishi, H., Mizuta ni, T., Kano, S., Honda, K., Ohba, Y., Mak, TW & Taniguchi, T. (2005) Nature 4 34, 243-249) ing. Full-length mice IRF-4, IRF-8 and IRF-9, and DNA binding domain deletion mutant IRF-4 (IRF-4 Δ DBD) and regulatory domain deletion mutant IR F-4 (IRF-4 A RD ) Fragments were obtained by RT-PCR and cloned individually into pCAGGS-HA, pCAGGS-YFP or pCAGGS-red fluorescent protein vectors (Mittrucker, H.

W., Matsuyama, T., Grossman, A., Kundig, TM, Potter, J., bhaninian, A., Wakeh am, A., Patterson, B., Ohashi, PS & Mak, TW (1997) Science 275 , 540-543, and Takaoka, A., Yanai, H., Kondo, S., Duncan, G., Negishi, H., Mizutani, T., Kan o, S., Honda, K., Ohba, Y., Mak, TW & Taniguchi, T. (2005) Nature 434, 243-24 9). FLAG-tagged expression vector for TRAF6 was donated by J. Inoue (University of Tokyo)

C reagent]

[0103] Unmethylated CpG-oligodeoxynucleotide (CpG-ODN), 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. Lipopolysaccharide (LPS), polyuridylic acid [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.

[Imaging]

[0104] 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. Timelabs image collection, fluorescence resonance energy transfer (FRET) analysis and correction FRET (FRET ^C ) calculations are available in the literature (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).

[Immunoprecipitation and immunoblotting]

[0105] Immunoprecipitation and immunoblotting were performed in the literature (Honda, K., Yanai, H., Mizutani, T., Negis hi, H., Shimada, N., Suzuki, N., Ohba'Y., Takaoka, A , Yeh, WC & Taniguchi, T. (2004) Proc. Natl. Acad. Sci. USA 101, 15416-15421). Anti-HA and anti-FLAG monoclonal antibodies were purchased from Sigma.

[Reporter Atssey]

[0106] 100 ng of 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. Sexuality (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).

[Measurement of site force in production]

Resident 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). For obtaining bone marrow derived macrophages (BMM), 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 μg) 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 Ν1668, 3 ^ Μ ODN-D19, 1 μg / ml LPS and 5 μg Zml poly (U) (complexed with DOTAP ) Or 10 μg Zml peptide darlicans for the specified period. When specified, 30 units Zml of IFN-γ (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-α was purchased from PBL Biomedical Laboratories (Piscataway, NJ). For 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 β-actin in each sample. The same primers as in Example 1 were used for IL-12p40, IL-6, TNF-a and β-actin. The following primers were used for IRF-4, IκΒζ, FLI CE inhibitory protein (FLIP), vascular cell adhesion molecule-1 (VCAM-1) and macrophage inflammatory protein (MIP) 2.

IRF-4 5 -GCCCAACAAGCTAGAAAG-3 '(SEQ ID NO: 85) and

5 '-TCTCTGAGGGTCTGGAAACT-3' (SEQ ID NO: 86);

Ι κ ζ ζ 5 '-TTGGTGACTTTGGGTGCT-3' (SEQ ID NO: 87) and

5'- TGACATCAGCCCCACATTT-3 '(SEQ ID NO: 88); FLIP 5 '-ACAGAGTGAGGCGGTTTGAC-3' (SEQ ID NO: 89) and

5 '-GCTCTCACCAATCTCCATCAG-3' (SEQ ID NO: 90); VCAM-1 5 '-CCGTCATTGAGGATATTGG-3' (SEQ ID NO: 91) and

5 '-TCATTGTCACAGCACCAC-3' (SEQ ID NO: 92);

MIP2 5 '-GTGCCTCGCTGTCTGAGAGTT-3' (SEQ ID NO: 93) and

5 AGTTCCCAACTCACCCTCTCC-3 '(SEQ ID NO: 94)

(2-2) Interaction between IRF-4 and MyD88

[0108] According to Example 1 above, 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. In addition, 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. First, IRF-4 and IRF-8 labeled with YFP (referred to as YFP-IRF-4 and YFP-IRF-8) are expressed in HEK293T cells together with CFP-labeled MyD88 (CFP-MyD88). The cells were subjected to fluorescence microscope analysis. As shown in Figure 12a, 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. In contrast, YFP-IRF-8 did not coexist with CFP-MyD88. In addition, the FR ETc image shows that there is a high energy transfer from CFP-MyD88 to YFP-IRF-4. This is not seen in YFP-IRF-8, which indicates that MyD88 and IRF- 4 were shown to be in direct contact with each other (Figures 12b and c). Consistent with this, when co-expressed in HEK29 3T cells, HA-tagged IRF-4 (HAIRF-4) co-immunoprecipitated with FLAG-tagged MyD88 (FLA G-MyD88), but with HA-IRF-8 Did not coprecipitate (Fig. 12d). IRF-9, another IRF member, known to be involved in IFN signaling, has shown no signs of interaction in these assays (Figures 12c and d).

(2-3) MyD88 region interacting with IRF-4

[0109] Next, a deletion mutant of MyD88 labeled with a FLAG epitope was prepared. Thus, 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. As shown in Figure 13b, FLAG-MyD88 (A 1-151) and FLA G-MyD88 (Δ 173-296) interact with HA-IRF-4 FLAG-MyD88 (Δ 1-193) And FLAG-MyD88 (A 60-296) did not interact. Interestingly, similar results were obtained when the inventors analyzed the region of FLAG_MyD88 that interacts with HA-IRF-5 (Fig. 13b), indicating that IRF-4 and IRF-5 It was suggested that they compete with each other for interaction with the central region of MyD88 (intermediate domain and part of the TollZlL-1 receptor domain).

(2-4) Competition between IRF-4 and IRF-5 for MyD88 interaction

[0110] Because 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. For HEK293T cells, transfer a certain amount of HA-IRF-5 or HA-IRF-7 expression vector, FLAG-MyD88 expression vector, and gradually increase the amount of HA-IRF-4 expression vector, then IRF_MyD88 The interaction was monitored by co-immunoprecipitation analysis. In parallel, HA-IRF-3 (Honda, K., Yanai, H., Mizutani, T., Negishi,,., Shimada, Ν., Suzuki, Ν., Ohba, which does not interact with MyD88 Υ, Takaoka, A., Yeh, WC & Tanig uchi, T. (2004) Proc. Natl. Acad. Sci. USA 101, 15416-15421) were also included as controls. As shown in Figure 13c, IRF-4 inhibited the interaction between IRF-5 and MyD88 in a dose-dependent manner (upper), but IRF-4 expression was reduced between IRF-7 and MyD88. It exerted no influence on the interaction (downward).

(2-5) Inhibition of IRF-5-dependent gene induction by IRF-4

[0111] As one approach to further explore the significance of IRF-4, which selectively competes with IRF-5 for MyD88 interaction, we next deleted one of the regulatory domains (IRF -4 Δ RD), the other lacks a DNA-binding domain! /, (IRF-4 Δ DBD), and two types of IRF-4 mutants labeled with YFP (each YFP-IRF- 4ΔRD and YFP-IRF-4ΔDBD; FIG. 14a) were prepared. V, in contrast to YFP-IRF-4 and YFP-IRF-4ΔDBD, which are also predominantly expressed in the nucleus, YFP-IRF-4ΔRD was mainly expressed in the cytoplasm (FIG. 14b). Both these IRF-4 variants still interacted with MyD88, as shown in Figure 14c.

[0112] 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 Δ DBD mutants (Fig. 14d), suggesting that IRF-4 is less potent than IRF-5 in the cytoplasm. It was confirmed that the inhibitory effect was exhibited. As IRF-4 is expected since it did not compete with IRF-7 in about the MyD88 (FIG. 13c), MyD88-IRF of IRF-4 expression IFN-beta-flop opening motor _(pl25 -L _uc) No effect on -7 dependent activation (Figure 14).

[0113] We also observed the effect of IRF-4 expression on nuclear translocation of YFP-labeled IRF-5 in RAW264.7 cells, in response to TLR9 activation by CpG-ODN, designated ODN1668. investigated. As shown in Figure 14f, a significant percentage of YFP-IRF-5 is detected in the nucleus after ODN1668 stimulation, and this nuclear translocation is suppressed when IRF-4 is co-expressed. It was.

(2-6) Hypersensitivity to TLR stimulation in IRF4-deficient macrophages

[0114] 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. We have various 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) We investigated the induction of IRF-4 mRNA in peritoneal macrophages derived from wild-type mice. As shown in Figure 15a, IRF-4 mRNA is induced by these stimuli, which increases the level of IRF-4-expression level after TLR stimulation, when IRF-4 is negatively fed. This suggests the possibility of acting as a regulator. Interestingly, Irf4 — ^{/ _} peritoneal macaque phages are IL-3, IL-12p40 and TNF-α at levels 2-3 times higher than wild type macrophages when stimulated with various TLR stimuli. (Figure 15b). In contrast, IFN-α 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-α, 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.

[0115] Residual peritoneal macrophages from wild-type mice, Irf4 — ^{/ _} or IriS—'stimulated with ODN16 68, and mRNA of genes induced by pro-inflammatory site force-in, chemokines and NF-κB The induction of was analyzed. It is noteworthy that, by quantitative RT-PCR analysis, Irf4- ^Λ macrophage showed an mRNA induction profile that mirrored IriS— macrophages upon ODN1668 stimulation. Induction of mRNA by TLR9 is dependent on IRF-5 (IL-12p40, IL-6 and MIP2), the induction level increased in the absence of IRF-4, but IRF-5 was induced by mRNA. mRNA induction level (IB, FLIP, and VC AM- 1) in Irf4- / ^_ macro port phage for other genes not required for remained the same as in wild-type macrophages (FIG. 15d). The latter gene is known to be dependent on the NF-κB transcription factor, and our results indicate that, at least under our experimental conditions, the TLR-NF-κB pathway is It means not affected by -4. (2-7) Cell type-specific effects of IRF-5 and IRF-4

[0116] 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.

[0117] As an approach to further evaluate the negative regulation of TLR signaling by IRF-4, 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. As shown in FIG. 16b, induction of IL-6 mRNA and IL-12p40 mRNA was remarkably suppressed in IRF-4 expressing cells. Consistent with the reporter assay above, it is noteworthy that the induction of these site force in genes in RAW264.7 cells was also suppressed by the expression of IRF-4A DBD (FIG. 14d). On the other hand, the induction of Iκζζ mRNA (Fig. 15d), which is independent of IRF-5 and dependent on NF-κB, was observed between IRF-4 expressing cells and IRF-4 ΔDBD expressing cells. However, it occurred normally (Fig. 16b). Overall, these results support the view that IRF-4 functions as a negative regulator by selectively competing with IRF-5.

(2-8) In vivo role of IRF-4 in TLR signaling

[0118] To evaluate the in vivo function of IRF-4, ODN1668 was intravenously administered to wild-type mice and Irf4- ^{/ _} mice. As shown in Figure 17a, 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- ^Λ 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.

Industrial applicability According to the present invention, 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. In addition, 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.

Claims

The scope of the claims

[1] A screening method for an inflammatory site force-in inhibitor using as a marker the IRF-5 activity in an IRF-5-expressing cell brought into contact with a test substance.

[2] A screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (d):

(b) contacting the TRF ligand or virus with the IRF-5 expressing cells

(c) detecting the reporter activity of the IRF-5 expressing cell

[3] The screening method according to claim 1 or 2, wherein the IRF-5-expressing cells are cells that express endogenous IRF-5.

[4] The screening method according to claim 1 or claim 2, wherein the IRF-5-expressing cells are cells expressing IRF-5 introduced exogenously.

[5] "TFSEARCH" (http: /), an application that predicts transcription factor binding sites on DNA

/www.cbrc.jp/research/db/TFSEARCHJ.html) is used to search for transcription factor binding regions on the entire inflammatory site force-in gene region including the promoter region. Among sequences obtained by setting to ISRE sequence, IRF-1 binding sequence and

The screening method according to any one of claims 2 to 4, wherein a sequence detected as a Z or IRF-2 binding sequence is used as an ISRE sequence.

[6] The screening method according to claim 2, wherein the ISRE sequence is a sequence of SEQ ID NO: 7 to SEQ ID NO: 58, or one or more of them.

[7] TLR force TLR3, TLR4, TLR5, TLR7, TLR8, TLR9 !, one or more TLRs

The screening method according to any one of claims 1 to 8.

[8] A screening method for an inflammatory site force-in inhibitor using, as an index, the amount of IRF-5 expressed in an IRF-5-expressing cell when the test substance is brought into contact with the test substance.

[9] The screening method according to claim 8, comprising the following steps (a) to (c):

(a) A step of bringing a test substance into contact with an IRF-5 expressing cell (b) a step of measuring IRF-5 mRNA in the IRF-5-expressing cells

[10] The screening method according to claim 8, comprising the following steps (a) to (c):

(b) Quantifying the IRF-5 protein in the IRF-5 expressing cells

[11] An inflammatory site force-in inhibitor that uses the test substance's ability to inhibit the binding of MyD 88 and IRF-5 as an index when MyD88 is contacted with IRF-5 in the presence of the test substance. Screening method.

[12] A screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (c):

(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

[13] A screening method for an inflammatory site force-in inhibitor comprising the following steps (a) and (d):

(b) a step of separating a fraction containing MyD88 protein or a fraction containing IRF-5 from a cell lysate of a cell strain expressing the MyD88 protein and IRF-5 protein (c) Detecting the binding of MyD88 protein to IRF-5 protein in the separated fraction

(d) A step of selecting a test substance that decreases the binding force between the MyD88 protein and the IRF-5 protein compared to a non-test substance contact control.

[14] A screening method for an inflammatory site force-in inhibitor using, as an index, the expression level of IRF-4 in an IRF-4-expressing cell when the test substance is brought into contact with the test substance.

[15] The screening method according to claim 14, comprising the following steps (a) to (c):

(b) measuring IRF-4 mRNA in the IRF-4 expressing cells

[16] The screening method according to claim 14, comprising the following steps (a) to (c):

(b) quantifying the IRF-4 protein in the IRF-4 expressing cells

[17] Inflammatory 'inflammatory site force in is IL-1, IL-6, IL-8, IL-12, IL-18, TNF-α, IFN-γ. The screening method according to claim 1, wherein:

[18] A screening method for a therapeutic agent for a disease involving inflammatory site force-in, using the method according to any one of claims 1 to 17.

[19] A MyD88-IRF-5 binding inhibitor comprising any of the following (a) to (e):

(c) an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 59 or 61 (d) an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 59 or 61

(e) an isolated polypeptide encoded by any of the polynucleotides (a) to (d) above

A therapeutic agent for a disease involving inflammatory site force-in, comprising the MyD88-IRF-5 binding inhibitor according to claim 19.