WO2010076851A1 - Agent thérapeutique/prophylactique pour infections fonctionnant par régulation de il-17a/il-17f - Google Patents

Agent thérapeutique/prophylactique pour infections fonctionnant par régulation de il-17a/il-17f Download PDF

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WO2010076851A1
WO2010076851A1 PCT/JP2009/007341 JP2009007341W WO2010076851A1 WO 2010076851 A1 WO2010076851 A1 WO 2010076851A1 JP 2009007341 W JP2009007341 W JP 2009007341W WO 2010076851 A1 WO2010076851 A1 WO 2010076851A1
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mice
cells
il17f
infection
il17a
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岩倉洋一郎
石亀晴道
角田茂
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国立大学法人 東京大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine

Definitions

  • the present invention relates to a non-human mammal having a deletion and / or insertion mutation at the IL-17F locus on a chromosome useful as an infectious disease model animal, and a screening for an infectious disease treatment / prevention drug using the non-human mammal.
  • the present invention relates to a method, an infection protective agent containing IL-17F protein or the like as an active ingredient, and a method for screening an infectious disease treatment / prevention drug using the expression level of IL-17F or IL-23 as an index.
  • Naive CD4 + T cells are classified into multiple helper T cell subsets, including Th1 and Th2 cells, based on cytokine production characteristics and effector functions.
  • Th17 cells that preferentially produce IL-17A, IL-17F, IL-21 and IL-22 have been identified in mice (Non-patent Documents 1 and 2).
  • Differentiation of Th17 cells is induced by TGF- ⁇ and IL-6 (Non-Patent Documents 3 to 5) or IL-21 (Non-Patent Documents 6 to 8), and is promoted by synchronized activities of IL-1 and TNF. (Non-patent document 9).
  • IL-23 is required for Th17 cell proliferation, survival and effector function, and IL-17A and IL-17F production is promoted by this T cell subset (Non-patent Documents 5 and 8).
  • IL-17F and IL-17A are highly homologous proteins belonging to the 1L-17 protein family encoded by genes that are close to each other in both humans and mice (Non-Patent Documents 9 to 11).
  • IL-17A and IL-17F have been reported to be able to bind to the same receptor complex consisting of IL-17RA and IL-17RC (Non-patent Documents 12 and 13), and these cytokines have similar biological properties. It has been suggested to have a function.
  • IL-17A and IL-17F both induce the production of antimicrobial peptides (defensins), cytokines (IL-6, G-CSF, GM-CSF) and chemokines (CXCL1, CXCL2, CXCL5) It promotes granulocyte production and neutrophil recruitment (Non-Patent Documents 9 to 11).
  • IL-17F or IL-17A in the lung increases proinflammatory cytokine and chemokine expression and causes inflammation with neutrophil infiltration (Non-Patent Documents 14 to 17).
  • Non-patent Documents 1 and 18 autoimmune diseases such as IBD
  • CHS contact hypersensitivity
  • DTH delayed hypersensitivity
  • Th17 cells are also involved in host defense against infection because antigen-presenting cells stimulated with microbial products such as LPS, peptidoglycan and zymosan produce large amounts of IL-23 and cause Th17 cell development It has been suggested (Non-Patent Documents 19 to 21).
  • Il17ra ⁇ / ⁇ mice and / or Il23a ⁇ / ⁇ mice can be identified as Klebsiella pneumoniae in the lung (Non-patent Document 22) and Citrobacter rodentium in the intestine (Non-patent Documents 4 and 13). Sensitive to However, the relative involvement of IL-17A and IL-17F in autoimmune and allergic diseases and host defense processes remains unclear.
  • IL-17F had the highest homology with IL-17A in the IL-17 family, and was thought to be involved in the development of inflammatory diseases because it shared receptors with IL-17A.
  • the role of IL-17F in vivo is almost unknown.
  • the genes encoding IL-17A and IL-17F are located in the vicinity of the same chromosome, in order to produce double-deficient mice of IL-17A and IL-17F, two consecutive times in ES cells It was technically difficult to require homologous recombination.
  • An object of the present invention is to prepare IL-17F -deficient mice and IL-17A and IL-17F double-deficient mice and to elucidate the function of IL-17F in vivo using these mice.
  • another object of the present invention is to provide a therapeutic agent for infectious diseases by controlling IL-17F function.
  • the present inventors have intensively studied to solve the above problems, and for the purpose of discriminating differences in functions of IL-17F and IL-17A in the host defense mechanism against immune response and bacterial infection, IL-17F (Il17f -/- ) Or IL-17A and IL-17F (Il17a -/- Il17f -/- ) mice were generated and these were treated with Il17a -/- mice (Nakae et al., Immunity 17, 375-387 , 2002) showed that IL-17A and IL-17F play different roles in the development of immune responses against T cell mediated inflammation and bacterial infection.
  • the present invention has been completed based on these findings.
  • the following inventions Binds to (i) IL-17F protein, or (ii) IL-17 receptor A (IL-17RA) or IL-17 receptor C (IL-17RC) and has the same function as IL-17F
  • An infection protective agent containing the indicated substance as an active ingredient (2) The infection protective agent according to (1), which is used to protect against microbial infection in the mucous membrane.
  • Infectious disease treatment comprising administering a test substance to IL-17F-producing cells and selecting a test substance that increases the expression level of IL-17F or IL-23 in the IL-17F-producing cells as a candidate substance -Screening methods for prophylactic drugs.
  • a non-human mammal having a deletion and / or insertion mutation at the IL-17F locus on the chromosome (5) A non-human mammal having a deletion and / or insertion mutation at both the IL-17A locus and the IL-17F locus on the chromosome. (6) The non-human mammal according to (5), wherein susceptibility to opportunistic infection is increased. (7) The non-human mammal according to any one of (4) to (6), which is used as an infectious disease model animal. (8) The non-human mammal according to any one of (4) to (7), which is a rodent. (9) The non-human mammal according to any one of (4) to (8), which is a mouse.
  • test substance is administered to the non-human mammal according to any one of (4) to (9), the degree of infection susceptibility in the non-human mammal is measured, and the test substance is not administered;
  • a method for screening an infectious disease treatment / prevention drug comprising selecting, as a candidate substance, a test substance that reduces infection susceptibility.
  • Infection protective ability can be promoted by specifically expressing IL-17F in epithelial cells.
  • IL-17F unlike IL-17A, it is possible to activate only the beneficial infection-protective ability without inducing a harmful inflammatory response.
  • IL-17A producing cells and IL-17F producing cells are different, and it was shown that IL-17F can be artificially induced in non-lymphocytes of the intestinal tract.
  • IL-17F-deficient mice and IL-17A / IL-17F double-deficient masses were produced for the first time in the present invention.
  • the knockout non-human mammal of the present invention can be used to elucidate the role of IL-17A / IL-17F in vivo and its molecular mechanism, to analyze the structure of the IL-17 receptor and to analyze the ligand properties, IL-17A / IL It is useful for the development of a specific induction method for -17F.
  • IL-17F contributes to the development of spontaneous autoimmune arthritis in Il1rn ⁇ / ⁇ mice.
  • A Profiles of intracellular IL-17F, IL-17A and IFN- ⁇ expression in LN cells of wild type (WT) and arthritic Il1rn ⁇ / ⁇ mice stimulated with PMA and ionomycin in vitro.
  • B Profiles of intracellular IL-17F, IL-17A and IFN- ⁇ expression in cells of ankle joints of wild type (WT) and arthritic Il1rn ⁇ / ⁇ mice stimulated with PMA and ionomycin.
  • C Expression of IL-17A and IL-17F mRNA in arthritic Il1rn ⁇ / ⁇ mouse joints.
  • E Wild type (WT), Il17a + / + Il17f + / + Illrn -/- , Il17f -/- I1lrn -/- , and Il17a -/- Il1rn -/- mouse LN cells stimulated with PMA and ionomycin Intracellular IL-17A expression.
  • Data represent 2 (C, E) or 3 (A, B) independent experiments. Increased susceptibility of Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice to opportunistic S. aureus infection.
  • (B) Immunoglobulin titer in serum of 8-10 week old mice (n 6-8 / group). Similar results were observed in the C57BL / 6J background.
  • (D) BALB / cA background Il17a -/- Il17f -/- mice (n 10 / group) submandibular LN with or without oral antibiotics between 4 and 8 weeks of age Weight.
  • (G) Survival rate of mice after iv injection with S. aureus 1 ⁇ 10 7 CFU (n 11 / group). Data represent two independent experiments.
  • (H) Staphylococcus aureus CFU (n 4 / group) in kidney homogenate collected 3 days after iv injection of S. aureus 1 ⁇ 10 7 CFU. Data represent two independent experiments.
  • IL-17F and IL-17A are required for protection against C. rodentium infection.
  • Wild-type (WT), Il17f -/- , Il17a -/- and Il17a -/- Il17f -/- mice were orally infected with C. rodentium 2 x 10 8 CFU and the colon and spleen were postinfected It was collected at the time.
  • CFU of C. rodentium in colon homogenates (n 10-16 / group). Data represent results collected from 2 or 3 independent experiments.
  • B Visualization of C. rodentium at the distal colon site 14 days after oral infection (up, 40 ⁇ , down, 120 ⁇ ). Data represent 4-6 mice in each group.
  • C, D Colon weight (C) and spleen weight (D) after oral infection as shown in (A).
  • IL-17F and IL-17A are produced by different cells.
  • A IL-17A and IL-17F mRNA expression in the colon, small intestine and peripheral LN of wild type (WT) mice was analyzed using real-time RT-PCR. Expression in LN cells was taken as 1.
  • B IL-17A and IL-17F expression in mouse colon 7 and 14 days after infection with C. rodentium was measured using real-time RT-PCR. The RNA sample is a collection of samples of 4-6 mice in each group. Expression in uninfected wild type (WT) mice was taken as 1.
  • C, D Intracellular IL-17F, IL-17A and IFN- ⁇ expression in colonic PMA and ionomycin-stimulated lymphocytes of uninfected mice (C) or mice (D) 14 days after infection with C. rodentium Profiles.
  • H, I Wild type (WT) and Rag2 ⁇ / ⁇ mouse splenocytes (5 ⁇ 10 5 cells) (H) or MLN (1.5 ⁇ 10 5 cells) (I), LPS 5 ⁇ g / ml and IL ⁇ 23 Cultured for 72 hours in a 24-well plate or 48-well plate, respectively, in the presence or absence of 20 ng / ml, and the amounts of IL-17A and IL-17F in the culture supernatant were measured using ELISA.
  • J Colon of epithelial (CD45 ⁇ and high FSC / SSC, gates R1 and R3) cells and intraepithelial immune cells (CD45 + , gate R2) in uninfected wild type (WT) mice using flow cytometry. And the expression of IL-17F and IL-17A was examined using RT-PCR.
  • K Expression of IL-17A and IL-17F in mouse colonic epithelial cell lines (CMT93 or Colon26) was examined using RT-PCR. Data represent 2 (A, B, K) or 3 (CJ) independent experiments. IL-17RA and IL-17RC show different tissue distributions.
  • (E) C57BL / 6J mouse CD4 + T cells obtained by MACS sorting were stimulated with IL-17A or IL-17F 5 to 250 ng / ml for 48 hours, and CCL2 in the culture supernatant was bio-Plex suspension array system Measured using (Bio-Rad).
  • G, H, I Colonic epithelial cell line (CMT93) (G), peritoneal macrophages (H) of C3H / HeJ mice, or CD4 + T cells (I) of C57BL / 6J mice, IL-17A or IL- Stimulate for 24 hours (G, H) or 48 hours (I) individually with 17F 50 ng / ml or in combination with IL-17A and IL-17F 50 ng / ml.
  • GM-CSF, CCL3 or CXCL1 were measured using a Bio-Plex suspension array system (Bio-Rad). ND, not detected. * P ⁇ 0.05, ** p ⁇ 0.01 and *** p ⁇ 0.001 for medium only.
  • Il17f -/- and Il17a -/- Il17f -/- mice are generated by replacing exon 2 and 3 of the Il17f gene with hygromycin B phosphotransferase (hph) resistance gene using Il17a +/- ES cells did.
  • hph hygromycin B phosphotransferase
  • the diphtheria toxin A (DT) gene was ligated to the 5 ′ end of the genomic fragment for negative selection.
  • the outer homologous region shown in the targeting allele was used as a genomic probe for Southern blot analysis.
  • Southern blot analysis for target clone screening was performed using BamHI.
  • B Correct targeting of the Il17f locus was confirmed by genomic Southern blot analysis. Endogenous (9.5 kb) and / or mutations using 3 ′ probes and BamHI digested tail DNA from Il17f WT (+ / +), heterozygous (+/-) and mutated (-/-) littermates A band of (7.2 kb) was detected.
  • C Expression of IL-17A and IL-17F mRNA.
  • A Proliferative response of LN cells 48 hours after mitogen stimulation.
  • B Proliferative response of splenic T cells 48 hours after stimulation with plates coated with anti-CD3 mAb.
  • C The amount of IFN- ⁇ in the culture supernatant of (B) was quantified using ELISA.
  • D Production of IL-17A by DO11.10 and Il17f ⁇ / ⁇ DO11.10 LN cells cultured with OVA peptide ⁇ IL-23 for 3 days.
  • E Wild type of C57BL / 6J background cultured for 72 hours in the presence of anti-CD3 and anti-CD28 monoclonal antibodies and TGF- ⁇ , IL-6, anti-IL-4 monoclonal antibody and anti-IFN- ⁇ monoclonal antibody ( WT), IL-17F and IL-17A intracellular expression profiles in CD4 + T cells from Il17f ⁇ / ⁇ , Il17a ⁇ / ⁇ and Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice.
  • AC All data represent results from at least two independent experiments. IL-17A but not IL-17F is required for the development of EAE.
  • mice were immunized intradermally with chicken collagen type II emulsified with CFA at several sites on the tail base of mice.
  • A, B Shows the incidence (A) and severity (B) of CIA (16-17 mice per group). * P ⁇ 0.05 for WT mice was determined using the ⁇ 2 test.
  • IL-17A is involved in the development of DTH.
  • A Intracellular IL-17F, IL-17A and IFN- ⁇ profiles in mBSA stimulated LN cells. Wild type (WT) mice were immunized with mBSA / CFA and 10 days after immunization, LN cells were cultured for 72 hours in the presence or absence of 50 ⁇ g / ml mBSA.
  • (C) mBSA-specific LN cell proliferation response induced by culturing with 50 ⁇ g / ml mBSA for 72 hours as in (A) (n 5 / group).
  • mice were sensitized intraperitoneally with OVA in alum and administered aerosolized OVA. Twenty-four hours after the last OVA inhalation, BAL fluid was collected and BAL cells were counted. Data show pooled results from two independent experiments (10-19 mice per group).
  • B Total number of cells in BAL fluid. Il17f + / + , Il17f +/ ⁇ and Il17f ⁇ / ⁇ DO11.10 mice were treated with aerosolized OVA or PBS for 4 days without sensitization. BAL fluid was collected and BAL cells were counted 24 hours after the last OVA or PBS inhalation. Data show pooled results from two independent experiments (12-14 mice per group).
  • mice Mono, monocytes / macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.
  • IL-17F is produced by innate immune cells in response to IL-23.
  • A C57BL / 6J wild type (WT) and Rag2 ⁇ / ⁇ mouse splenocytes (1 ⁇ 10 6 to 5 ⁇ 10 4 cells) in the presence or absence of 0.01 to 100 ng / ml IL-23 Then, the cells were cultured in a 96-well plate for 72 hours, and the amounts of IL-17A and IL-17F in the culture supernatant were measured using ELISA.
  • B The whole colon of BALB / cA wild type (WT) and CB-17 SCID mice was cultured for 24 hours in the presence or absence of 20 ng / ml IL-23.
  • C BALB / cA wild type (WT) and CB-17 SCID mouse splenocytes (5 ⁇ 10 5 cells) in 24-well plates in the presence or absence of 5 measured using ELISA Cultured.
  • D Flow cytometry of CD11c and CD11b expression on splenocytes from BALB / cA WT and CB-17 SCID mice.
  • E Flow cytometry of expression of CD11c, CD11b and DX5 on MACS sorted CD11c + or CD11b + splenocytes from CB-17 SCID mice.
  • F Flow cytometry of CD11c, CD11b, DX5, Gr-1, F4 / 80 and B220 expression on MACS sorted CD11c and CD11b negative cell fractions derived from CB-17 SCID splenocytes.
  • A, B, C Peritoneal macrophages from C3H / HeJ mice (A), CD4 + T cells from C57BL / 6J mice (B), or colonic epithelial cell line (CMT93) (C), 50 ng / ml Stimulated for 24 hours (A, B) or 48 hours (C) with IL-17A or IL-17F individually or in combination with 50 ng / ml IL-17A and IL-17F, and the indicated cytokines in the culture supernatant And the amount of chemokine was measured by using a Bio-Plex suspension array system (Bio-Rad). ND, not detected. * P ⁇ 0.05, ** p ⁇ 0.01 and *** p ⁇ 0.001 for medium alone. All data represent 3 independent experiments.
  • the present invention relates to a non-human mammal having a deletion and / or insertion mutation in the IL-17F locus on the chromosome, and the IL-17A locus and the IL-17F locus on the chromosome. Relates to a non-human mammal having a deletion and / or insertion mutation in both.
  • the IL-17F gene is inactivated by having a deletion and / or insertion mutation at the IL-17F locus on the chromosome, and has a function of expressing IL-17F. It is lost.
  • Non-human mammals with deletion and / or insertion mutations at both the IL-17A and IL-17F loci on the chromosome have increased susceptibility to opportunistic infections such as S. aureus. It has the feature of being.
  • the knockout non-human mammal of the present invention described above can be used as an infectious disease model animal.
  • non-human mammals examples include rodents such as mice, hamsters, guinea pigs, rats, rabbits, dogs, cats, goats, sheep, cows, pigs, monkeys, etc.
  • Rodents such as mice, hamsters, guinea pigs, rats, rabbits and the like are preferable from the viewpoint of easy growth and use, and among these, mice are most preferable.
  • a non-human mammal having a deletion and / or insertion mutation at the IL-17F locus on the chromosome, and a deletion and / or insertion mutation at both the IL-17A locus and the IL-17F locus on the chromosome can be produced by introducing a mutation into a target gene (IL-17F gene) by embryonic stem cells (ES cells) by homologous gene recombination.
  • IL-17F gene embryonic stem cells
  • ES cells embryonic stem cells
  • a predetermined region within the IL-17F gene is selected as a drug selection marker gene (for example, hygromycin B phosphotransferase in the following examples)
  • a drug selection marker gene for example, hygromycin B phosphotransferase in the following examples
  • a transgene is constructed using a targeting vector (homologous recombination vector) replaced with (hph) resistance gene) and introduced into mouse embryonic stem cells (ES cells) by electroporation or the like.
  • Il17a +/ ⁇ Il17f +/ ⁇ ES cells may be obtained by introducing the targeting vector for knockout of the IL-17F gene described above.
  • ES cells that have undergone homologous recombination with the drug are selected by PCR and Southern blotting, etc., and ES cells that have undergone homologous recombination with fertilized mice are mixed by the collective chimera method to obtain chimeric mice, Finally, an IL-17F gene knockout mouse can be produced from a chimeric chimeric mouse in which ES cells have differentiated into germ cells in the individual chimeric mouse.
  • a targeting vector for homologous recombination of embryonic stem cells (ES cells) is constructed using genomic DNA of IL-17F gene.
  • Genomic DNA should be isolated from the same animal species (more preferably from the same strain) as the animal species from which the ES cells are to be produced so that recombination occurs more efficiently when homologous recombination is performed. Is preferred. For example, when obtaining a genomic clone of mouse IL-17F gene, it can be screened from a mouse genomic library using a cDNA probe of IL-17F gene derived from mouse.
  • a targeting vector containing the disabled IL-17F gene Using a targeting vector containing the disabled IL-17F gene, homologous recombination is performed on the genomic DNA of the IL-17F gene in ES cells.
  • an IL-17F gene whose function has been disabled an IL-17F gene whose partial region has been deleted, or an IL-17F gene whose function has been disabled by introducing an insertion mutation can be used. .
  • a selection marker gene for example, drug resistance gene
  • a part of the isolated genomic DNA of IL-17F gene is deleted by restriction enzyme treatment, and a selectable marker gene (for example, drug resistance gene) in which a promoter is linked instead of the deleted DNA region
  • a targeting vector can be prepared by inserting.
  • a promoter having strong expression activity in ES cells phosphoglycerate kinase-1 (PGK-1) promoter, elongation factor 2 (EF-2) promoter, MC-1 promoter and the like can be used.
  • the promoter activity is greatly influenced by the locus to be targeted and its DNA region, in general, the stronger the promoter, the better. Therefore, the PGK-1 promoter is particularly preferably used.
  • a selectable marker gene a neomycin resistance gene, a hygromycin B phosphotransferase gene, a thymidine kinase gene, a diphtheria toxin A gene fragment, a lacZ gene, etc. can be used depending on the purpose for negative selection.
  • Selectable marker genes linked to the promoter include, for example, neomycin resistance gene cassette (neo cassette, provided as pKJ2), hygromycin B phosphotransferase gene cassette (hph cassette), thymidine kinase gene cassette (tk cassette, provided as pMCtk), diphtheria toxin
  • selectable marker genes that are commercially available while inserted into a plasmid, such as A gene fragment cassette (donated as DT-A cassette, pMCDT-A or pMC1DT-A), lacZ gene cassette (donated as pBSlacZ) Also good.
  • the targeting vector may be constructed using any selection marker among the selection markers linked to these promoters.
  • ES cells For example, mouse ES cells can be used. Examples of mouse-derived ES cells include, but are not particularly limited to, TT2 cells, AB-1 cells, J1 cells, R1 cells, and E14.1 cells.
  • a targeting vector in which the IL-17F gene has a non-functional sequence is introduced into ES cells. Then, the genomic DNA sequence of the target IL-17F gene in the ES cell is replaced by homologous recombination with the DNA sequence of the disabled IL-17F gene in the targeting vector. Homologous recombination can be generated stochastically using the homology between the IL-17F gene genomic DNA sequence and the sequence of the unmodified portion in the targeting vector.
  • an electroporation method As a method for introducing the targeting vector into ES cells, an electroporation method, a calcium phosphate method, a DEAE-dextran method, or the like can be used. From the viewpoint of efficiency and ease of work, electroporation is preferred.
  • the obtained recombinant ES cells are plated on an appropriate feeder cell layer and screened for homologous recombinants using a selection marker introduced into ES cells by homologous recombination in an ES culture solution. Selective culture can be performed. Depending on the type of the selectable marker gene, for example, neomycin (G418, GIBCO BRL) or hygromycin B (Calbiochem) can be contained in the culture medium. For example, colonies that survive on days 6-8 of selective culture can be picked as resistant clones. After picking up resistant clones and growing them, further screening can be performed to see if the target IL-17F gene is targeted. For example, whether the target IL-17F gene is targeted at the DNA level can be confirmed by PCR, Southern blot hybridization, or the like.
  • ES cells obtained by homologous recombination are transplanted into the 8-cell stage or blastocyst embryo.
  • a chimeric animal can be produced by transplanting the embryo into which the ES cell has been transplanted into the uterus of a pseudopregnant temporary parent and giving birth.
  • Examples of the method for transplanting ES cells into the embryo include a micromanipulation method and an aggregation method.
  • mice for example, female mice subjected to superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) are mated with male mice.
  • embryos are collected from the uterus on day 2.5 after fertilization when using an 8-cell stage embryo, and on day 3.5 after fertilization when using a blastocyst.
  • the embryos thus collected can be injected in vitro with ES cells that have undergone homologous recombination using a targeting vector to produce a chimeric embryo.
  • a pseudopregnant female mouse for use as a foster parent can be obtained by mating a normal-period female mouse with a male mouse castrated by vagina ligation or the like.
  • a chimeric animal can be produced by transplanting the chimeric embryo produced by the above method into the uterus to the produced pseudopregnant mouse, and allowing it to become pregnant and give birth. It is desirable to create female mice that collect fertilized eggs and pseudopregnant mice that become foster mothers from a group of female mice in the same sexual cycle in order to ensure that implantation and pregnancy of the chimeric embryo occur. .
  • mice male mice derived from embryos transplanted with ES cells are selected. After the male embryonic mouse derived from the selected ES cell transplanted embryo matures, this mouse is mated with a female mouse of a pure mouse strain, and the ES cell-derived hair color appears in the next-generation offspring. It can be confirmed that it has been introduced into the germ line of mice. In order to confirm that the ES cell has been introduced into the germ line, various traits can be used as an index, but it is desirable to use a coat color in consideration of ease of confirmation. In mice, the fur color of agouti, black, ocher (cinnamon), chocolate (chocolate) and white (albino) is known.
  • a mouse strain to be mated with the chimeric mouse can be appropriately selected in consideration of the origin strain.
  • an individual in which a target gene is deleted can be obtained by selecting an animal into which a recombinant ES cell transplanted into an embryo is introduced into the germ line and breeding the chimeric animal.
  • IL-17F gene-deficient homozygous mice can be obtained.
  • mice used for backcrossing can be appropriately selected by those skilled in the art. Examples of mice that can be used include, but are not limited to, BALB / c, C57BL / 6, DBA / 1, ICR, and the like.
  • the above-described knockout non-human mammal of the present invention can be used to screen a therapeutic / prophylactic agent for infectious diseases.
  • the test substance is administered to the above-described non-human mammal of the present invention, the degree of infection susceptibility in the non-human mammal is measured, and the infection susceptibility is reduced compared to the case where the test substance is not administered.
  • a test substance as a candidate substance, it is possible to screen for an infectious disease treatment / prevention drug.
  • the measurement of the degree of infection susceptibility is not particularly limited, and can be performed, for example, by the method described in (8) Infection with Staphylococcus aureus in the method (I) in the following Examples or a method analogous thereto.
  • test substance used in the screening method of the present invention examples include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. These compounds may be novel compounds or known compounds.
  • a library containing a large number of molecules such as a peptide library or a compound library can also be used as a test substance.
  • test substance for example, oral administration, intravenous injection and the like are used.
  • the dosage of the test substance can be appropriately selected according to the administration method, the nature of the test substance, and the like.
  • the substance obtained by using the screening method of the present invention is a substance selected from the above-mentioned test substances, and has a therapeutic / preventive effect against infectious diseases, and therefore can be used as a therapeutic / prophylactic agent for infectious diseases. . Furthermore, a compound derived from the substance obtained by the above screening can also be used as a medicament.
  • Infection protective agent In the present invention, pathogenic E. coli (C. rodentium) infection in IL-17F knockout mice and opportunistic infection by S. aureus in IL-17A / IL-17F double knockout mice It was shown that the sensitivity to increased. That is, this finding is equivalent to IL-17F by binding to (i) IL-17F protein, or (ii) IL-17 receptor A (IL-17RA) or IL-17 receptor C (IL-17RC). A substance exhibiting the above function indicates that it is effective as an anti-infection agent. Therefore, according to the present invention, IL-17F binds to (i) IL-17F protein, or (ii) IL-17 receptor A (IL-17RA) or IL-17 receptor C (IL-17RC). An infection protective agent containing a substance exhibiting a function equivalent to that as an active ingredient is provided.
  • Opportunistic infections are caused by bacteria, viruses, protozoa, etc. such as Gram-negative bacteria (E. coli, Klebsiella, Pseudomonas aeruginosa, etc.), Gram-positive bacteria (S. aureus, Streptococcus pneumoniae, Streptococcus, Enterococcus, etc.)
  • the anti-infective agent of the invention can also be applied to protect these opportunistic infectious agents. Among these, it can be preferably used particularly for S. aureus and E. coli.
  • the IL-17F of the present invention is used for the purpose of protecting opportunistic infections in these patients.
  • (Or Il-17F-like substance) can be used.
  • IL-17F of the present invention can improve mucosal immunity, in addition to opportunistic infection protection, for example, when used in combination with a mucosal vaccine, when improving systemic immunity via mucosal immunity promotion,
  • the anti-infection agent of the present invention can also be used when promoting mucosal immunity, such as when it is desired to promote protection against intestinal infection bacteria.
  • IL-17F of the present invention can also be referred to as a mucosal immunity promoter or mucosal immunity activator. Therefore, the infection-protecting agent of the present invention can be widely used for protecting mucosal microbial infection.
  • the mucous membrane include respiratory (tracheal, lung, etc.) mucosa and digestive tract (intestinal tract, stomach, etc.) mucous membrane.
  • IL-17F As a substance that binds to IL-17 receptor A (IL-17RA) or IL-17 receptor C (IL-17RC) and exhibits a function equivalent to IL-17F, a partial peptide of IL-17F protein, or IL Examples include, but are not limited to, a protein consisting of an amino acid sequence substantially identical to -17F protein or an amino acid sequence having high homology, or a partial peptide thereof.
  • IL-17F protein or (ii) a substance that binds to IL-17 receptor A (IL-17RA) or IL-17 receptor C (IL-17RC) and exhibits a function equivalent to that of IL-17F It can be administered orally or parenterally after being dissolved or suspended in a suitable sterile vehicle.
  • a parenteral route of administration for example, systemic administration such as intravenous, intraarterial, intramuscular, intraperitoneal, intratracheal, etc., or local administration may be used.
  • the dose of the infection protective agent of the present invention varies depending on the route of administration, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc.
  • the amount ranges from about 0.0001 to about 10 mg / kg, preferably from about 0.001 to about 1 mg / kg, and can be administered once or divided into several times.
  • it can determine suitably in the range of said description, such as an administration route and dosage.
  • IL-17F knockout mice pathogenic Escherichia coli (C. rodentium) infection is used in IL-17F knockout mice, and S. aureus is used in IL-17A / IL-17F double knockout mice. S. aureus) has been shown to increase susceptibility to opportunistic infections.
  • IL-17F production has been found to be stimulated by IL-23. That is, based on these findings, a test substance that can administer a test substance to IL-17F-producing cells and increase the expression level of IL-17F or IL-23 in the IL-17F-producing cells is used to treat infections. It turns out to be a candidate substance for preventive drugs.
  • a test substance is administered to IL-17F-producing cells, and a test substance that increases the expression level of IL-17F or IL-23 in the IL-17F-producing cells is selected as a candidate substance.
  • a method for screening an infectious disease treatment / prevention drug is provided.
  • IL-17F producing cells epithelial cells, innate immune cells, Th17 cells and the like can be used.
  • test substance the same substance as described in the screening method using the (2) knockout non-human mammal in the present specification can be used.
  • the expression level of IL-17F or IL-23 in IL-17F-producing cells can be measured by a conventional method by methods known to those skilled in the art, such as RT-PCR method or Northern blot method.
  • the screening method shown in the present embodiment can be used as a screening method for a mucosal immunity promoter as well as a screening method for infectious disease treatment / prevention drugs.
  • the nucleotide sequence of the mouse IL-17F gene (NCBI accession number: Genebank NM — 145856.2) is shown in SEQ ID NO: 54, and the amino acid sequence is shown in SEQ ID NO: 55.
  • the nucleotide sequence of the human IL-17F gene (NCBI accession number: AF384857.1) is shown in SEQ ID NO: 56, and the amino acid sequence is shown in SEQ ID NO: 57.
  • the following examples further illustrate the present invention, but the scope of the present invention is not limited by the examples.
  • Il17f -/- Il17rn -/- and Il17a -/- Il17f -/- Il1rn -/- mice are made by crossing Il17f -/- and Il17a -/- Il17f -/- mice with Il1rn -/- mice Then, 8 generations were backcrossed to BALB / cA mice (Horai, R., et al. (2004) J. Clin. Invest. 114, 1603-1611).
  • Rag2 ⁇ / ⁇ mice were obtained from the Central Laboratory for Experimental Animals.
  • C3H / HeJ and C3H / HeN or CB-17SCID mice were purchased from Japan SLC or Japan Marie, respectively. All mice were maintained under specific pathogen-free conditions in an environmentally controlled breeding room at the Human Disease Model Research Center (University of Tokyo Institute of Medical Science). Experiments were performed according to laboratory ethical guidelines for animal experiments and safety guidelines for genetic manipulation experiments.
  • Thyl.2 + , CD4 + , B220 + , CDllc + , CD1lb + cells were respectively anti-mouse Thyl.2, CD4, B220, CD1Ic and CD11b monoclonal antibodies (Miltenyi Biotec ) And according to the manufacturer's instructions and then isolated from the spleen using autoMACS (Miltenyi Biotec).
  • peritoneal macrophages elicited by thioglycolic acid peritoneal cells were collected by injecting mice intraperitoneally with 2 ml of 4% thioglycolic acid (Nissui) and washing with PBS 4 days after injection. .
  • CMT39 cells, peritoneal macrophages or CD4 + T cells were treated with recombinant mouse IL-17A or IL-17F (R & D systems) 5 to 250 ng / ml for 6 to 48 hours to determine the amount of cytokines, chemokines and antimicrobial peptides .
  • CCL2 ⁇ , IL-1 ⁇ , IL-9, IL-10, IL-12 / 23 p40, IL-12 p70, IL-13, G-CSF, GM-CSF, IFN- ⁇ , CXCL1 in the culture supernatant The amounts of CCL2, CCL3, CCL4 and CCL5 were measured using the Bio-Plex system (Bio-Rad) according to the manufacturer's instructions. The amount of C. rodentium specific Ig in the serum was measured as previously reported (Bry, L., et al. (2004) J. Immunol. 172, 433-441).
  • the cells were fixed with 4% paraformaldehyde. After washing with a permeation buffer (a staining buffer containing 0.1% saponin [Sigma]), the cells were treated with PE anti-mouse IFN- ⁇ monoclonal antibody (XMG1.2, BD Pharmingen), PE anti-mouse IL-17A monoclonal antibody (TC11 -18H10, BD Pharmingen) or goat anti-mouse IL-17F polyclonal antibody (AF2057 or BAF2057, R & D systems).
  • PE anti-mouse IFN- ⁇ monoclonal antibody XMG1.2, BD Pharmingen
  • PE anti-mouse IL-17A monoclonal antibody TC11 -18H10, BD Pharmingen
  • goat anti-mouse IL-17F polyclonal antibody AF2057 or BAF2057, R & D systems.
  • Alexa Fluor 488 anti-goat IgG A-11055; Invitrogen
  • PE anti-goat IgG Santa Cruz
  • FITC-streptavidin BD Pharmingen
  • the targeting vector is a phosphoglycerate kinase (PGK) 1 promoter containing a 2.1 kb genomic fragment containing the second and third exons of the Il17f gene. It was constructed by replacement with a 2.5 kb DNA fragment encoding the hygromycin B phosphotransferase (hph) resistance gene under the control of. The diphtheria toxin A (DT) gene under the control of the MC1 promoter was ligated to the 5 ′ end of the targeting vector for negative selection.
  • PGK phosphoglycerate kinase
  • Il17a +/- , Il17f +/- or Il17a +/- Il17f +/- ES cells (E14.1) were obtained from Il17a +/- ES cells electroporated with targeting vectors, and chimeric mice were (Nakae, S., et al. (2002) Immunity 17, 375-387.).
  • Il17f ⁇ / ⁇ DO11.10 transgenic mice were generated by crossing Il17f ⁇ / ⁇ with DO11.10 mice, a gift of Dr. Dennis Y. Loh (Washington University School of Medicine). The genotyping of Il17a ⁇ / ⁇ mice was performed as previously reported (Nakae, S., et al. (2002) Immunity 17, 375-387.).
  • PCR primers were used for genotyping IL-17F: Primer 1: 5'-TGG TAC TGC ATC AAA GTG ACA GTC-3 '(SEQ ID NO: 51); Primer 2: 5'-AAG GGT TCA GAG TCT GCG CTG CTC-3 ′ (SEQ ID NO: 52); Primer 3: 5′-GGA AGA TAG CAG GCA TGC TGG-3 ′ (SEQ ID NO: 53). Primers 1 and 2 were used to detect the wild type (WT) allele (800 bp) and primers 1 and 3 were used to detect the mutant allele (500 bp).
  • WT wild type
  • primers 1 and 3 were used to detect the mutant allele (500 bp).
  • Th17 cells are 1 ⁇ g / ml of anti-CD3 monoclonal antibody purified from the culture supernatant of 145.2C11 cells, 1 ⁇ g / ml of anti-CD28 monoclonal antibody (37.51; eBioscience), anti-mouse IFN- ⁇ (XMG1.2, kindly provided by Dr. Yoshimoto 10 ⁇ g / ml, anti-mouse IL-4 (11B11, generously granted by Dr.
  • EAE MOG-induced EAE was examined as previously reported ( Komiyama, Y., et al. (2006) J. Immunol. 177, 566-573.). Briefly, 129 / Ola x C57BL / 6J mice were treated with 300 ⁇ g MOG35-55 peptide emulsified with CFA containing 5 mg / ml of heated Mycobacterium tuberculosis H37RA (Difco) on day 0 On the 7th day, the other side was immunized subcutaneously. Pertussis toxin (Alexis) (200 ng) was injected intravenously on days 0 and 2. After the first immunization, EAE severity was monitored and graded from 0 to 5: 0, no disease, 1, tail dragging, 2, hindlimb weakness, 3, hindlimb paralysis, 4, hindlimb And forelimb paralysis, 5, morbidity and death
  • CIA CIA was examined as previously reported (Nakae et al., 2003). Briefly, C57BL / 6J background mice were challenged with 100 ⁇ l of CFA-emulsified avian IIC (Sigma) 1 mg / ml and heat-killed Mycobacterium tuberculosis H37RA (Difco) 5 mg / ml at several sites at the base of the tail. Vaccinated. Twenty-one days after the first immunization, the mice were again administered intradermally with collagen / CFA near the location of the first injection. The onset of arthritis was determined by macroscopic evaluation. The swelling and redness of each joint was examined and the severity graded from 0 to 3 for each foot: 0, no change, 1, mild swelling of the footpad and / or redness of the footpad, 2, joint Obvious swelling, 3, severe joint swelling and tonic change
  • Ovalbumin-induced pulmonary inflammation Mice were sensitized intraperitoneally with OVA / alum 100 ⁇ g / ml on days 0 and 12, and OVA (1% in PBS) for 20 minutes on days 21, 22 and 23 was administered to the respiratory tract by nebulization with and the lung inflammation in BALF was assessed 24 hours after the last OVA administration.
  • OVA / PBS-induced lung inflammation Il17f + / + , Il17f +/- or Il17f -/- mice mated with DO11.10 mice were administered daily for 4 days and OVA (1% in PBS) for 20 minutes did.
  • colonic epithelial cells To isolate colonic epithelial cells, the colon was dissected longitudinally and washed to remove feces. The colon was then cut into short pieces, transferred to a 50 ml conical tube and incubated at 37 ° C. for 60 minutes with slow shaking in RPMI 1640 containing 2% FCS and EDTA 2.5 mM. The cell suspension was passed through a strainer. To purify colonic epithelial cells, cells were stained with FITC anti CD45mAb (BD Pharmingen), CD45 - , high FSC / SSC populations The Institute of Medical Science, were separated on a FACSAria system (Becton Dickinson) in FACS Core Laboratory .
  • CD45mAb BD Pharmingen
  • colon lymphocytes including intraepithelial and lamina limbal lymphocytes
  • the colon is minced and transferred to a 50 ml conical tube and gently shaken in RPMI 1640 containing 2% FCS and type VIII collagenase (Sigma) 200 U / ml Incubated for 60 minutes at 37 ° C. Suspended cells were passed through a strainer. These cells were resuspended in 5 ml of 80% fraction of a 40:80 Percoll gradient (Amersham Biosciences) and this solution was placed under 5 ml of 40% fraction in a 15 ml conical tube. Percoll gradient separation was performed by centrifugation at 2200 rpm for 20 minutes at room temperature. Colon lymphocytes gathered in the middle layer of the Percoll gradient.
  • Colon organ culture Three pieces of colon (0.5 cm x 0.5 cm) were washed with cold PBS supplemented with penicillin and streptomycin (GIBCO). These fragments were cultured in serum-free RPMI1640 medium supplemented with penicillin and streptomycin in the presence or absence of IL-23 (R & B systems) 20 ng / ml. After 24 hours, the supernatant was centrifuged and a constant volume was stored at -80 ° C until analysis.
  • IL-17F contributes to the development of arthritis in IL-1 receptor antagonist-deficient (Il1rn ⁇ / ⁇ ) mice.
  • Il17a ⁇ / ⁇ , Il17f ⁇ / ⁇ and Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice were generated (FIG. 7). These mice were born healthy at the expected Mendelian ratio, were fertile, and did not show gross phenotypic abnormalities, including lymphocyte-like cell populations.
  • IL-17A production is normal in Il17f ⁇ / ⁇ mice
  • IL-17A and IL-17F are TGF- ⁇ and IL ⁇ It was not required for 6-induced Th17 cell differentiation (FIG. 8).
  • IL-17A plays an important role in the spontaneous development of arthritis in Il1rn ⁇ / ⁇ mice (Nakae, S., et al. (2003b) Proc. Natl. Acad. Sci. USA 100, 5986-5990.). Arthritis Il1rn ⁇ / ⁇ LN cells have more IL-17F-producing cells than wild-type (WT) LN cells, which also produce IL-17A, which is also true for IFN- ⁇ -producing cells ( Horai, R., et al. (2004) J. Clin. Invest. 114, 1603-1611.) (FIG. 1A).
  • IL-17A + IL-17F + cell count and IL-17A and IL-17F mRNA expression were also enhanced in LN cells from arthritic Il1rn ⁇ / ⁇ mice (FIGS. 1B and 1C).
  • the onset of arthritis is observed in Il17f -/- Il1rn -/- mice compared to the littermate Il17f + / + Il1rn -/- and Il17f +/- Il1rn -/- controls during the 30-week observation period. Although significant, suppression was partial (FIG. 1D).
  • Table 2 Disease incidence (number of diseased mice / total number of mice in group), mortality (number of dead mice / total number of mice in group), start date (mean day of clinical disease start ⁇ SEM), and disease severity (Maximum disease score median ⁇ SEM) is shown for mice of all genotypes. a; Only mice that developed clinical signs of disease were analyzed.
  • Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice show increased susceptibility to opportunistic infections by S. aureus.
  • WT wild type
  • Il17f -/- or Il17a -/- mice This effect was observed in various genetic backgrounds including C57BL / 6J, BALB / cA and 129 / OlaXC57BL / 6J strains (FIG. 2A).
  • IgM titers were similar between Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice and wild type (WT) mice.
  • mice When cultured the mucocutaneous tissue homogenates from these mice, wild-type (WT), Il17f - / - and IL17A - / - as compared to the case of a sample from a mouse, Il17a - / - Il17f - / - mice Since more bacteria were observed in the homogenate (FIGS. 2E and 2F), both IL-17A and IL-17F are crucial to protect mice from mucocutaneous S. aureus infection It was suggested. To investigate whether IL-17A and IL-17F play a role in systemic infection with S. aureus, mice were administered S. aureus by intravenous injection.
  • Il17f -/- , Il17a -/- and Il17a -/- Il17f -/- mice had substantially more bacteria detected at each time point after infection than in wild-type (WT) mice
  • Bacterial load in mutant mice decreased by day 21 and returned to wild-type (WT) levels by day 28 after infection in all genotype mice.
  • colonic bacterial counts were similar among Il17f ⁇ / ⁇ , Il17a ⁇ / ⁇ and Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice.
  • IL-17F and IL-17A are required for ⁇ -defensin expression in the colon.
  • the antibacterial mechanism induced by IL-17A and IL-17F was analyzed. (Mundy, R., et al., (2005) Cell. Microbiol. 7, 1697-1706.) Serum levels of C. rodentium-specific IgG increased in all mutant mice (FIG. 14), suggesting that the humoral immune response against C. rodentium is not involved in delayed bacterial clearance in Il17f ⁇ / ⁇ , Il17a ⁇ / ⁇ and Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice.
  • IL-17F and IL-17A are produced by different cells in the colon.
  • IL-17A mRNA is more highly expressed in the small intestine than in the colon (Ivanov, II, et al. (2006) Cell 126, 1121-1133.).
  • IL-17F mRNA expression in the colon was higher than in the small intestine (FIG. 5A).
  • WT wild type mice
  • IL-17A mRNA expression was unaffected by IL-17F deficiency and vice versa.
  • IL-17A and IL-17F producing cells were found in colon lymphocytes of uninfected wild type (WT) mice (FIG. 5C)
  • the population of IL-17F producing cells that also produce IL-17A is: Increased in infected wild type (WT) mice (FIG. 5D).
  • WT uninfected wild type mice
  • FIG. 5D the coordinated production of IL-17A and IL-17F by LN cells after the onset of DTH, EAE or arthritis
  • IL-17A and IL-17F may be produced by different cells in the colon because the kinetics of induction differ between the two molecules and IL-17F producing cells were rarely found in colon lymphocytes .
  • the mRNA expression of these molecules was examined in the colons of recombinant activating gene-2 deficient (Rag2 ⁇ / ⁇ ) mice in the absence of T and B cells.
  • IL-17A mRNA expression in mesenteric LN (MLN) was much higher than in colon 7 days after C. rodentium infection in wild type (WT) mice (FIG. 5E).
  • WT wild type mice
  • IL-17A mRNA levels are markedly reduced in Rag2 ⁇ / ⁇ mice (approximately 20% of wild type (WT)) (FIGS.
  • Th17 cells are the major producers of IL-17A. It was suggested that there is. In contrast, the amount of IL-17F expression was only reduced by about 50% in the MLN of these mice (FIGS. 5E and 5F). IL-17A mRNA expression was also significantly reduced in the colons of Rag2 ⁇ / ⁇ mice, whereas IL-17F mRNA expression was similar between wild-type (WT) and Rag2 ⁇ / ⁇ mice (FIG. 5E and 5F). Furthermore, IL-17F production in whole colon culture supernatants from Rag2 ⁇ / ⁇ mice was increased by treatment with IL-23, but IL-17A production was not increased.
  • IL-17A and IL-17F production are both induced in whole colon culture supernatants from wild type (WT) mice, and IL-17F can also be produced by non-T and non-B cells.
  • WT wild type mice
  • IL-17F can also be produced by non-T and non-B cells.
  • FIG. 5G Next, it was investigated which cells produce IL-17F in response to IL-23.
  • stimulation with IL-23 increased IL-17F production in splenocytes or MLNs derived from Rag2 ⁇ / ⁇ or CB-17 SCID mice compared to wild type (WT) mice. Only a small amount of IL-17A was produced in these cells (FIGS. 5H, 5I and FIG. 16).
  • CD11b low DX5 + CD11c ⁇ Gr1 ⁇ B220 ⁇ F4 / 80 ⁇ Gr1 ⁇ cells tend to mainly produce IL-17F after stimulation with IL-23 (FIG. 15).
  • IL-17F is expressed in lung epithelial cells (EC) (Suzuki, S., et al. (2007) Int. Arch. Allergy Immunol. 143 (Suppl 1), 89-94.) Whether it was expressed in EC was also examined. IL-17F mRNA was detected in CD45-colon ECs obtained by sorting by FACS from infected wild type (WT) mice, but IL-17A mRNA was not detected. This result is in contrast to CD45 + intraepithelial immune cells and ConA stimulated splenocytes in which both IL-17A and IL-17F were detected (FIG. 5J).
  • EC lung epithelial cells
  • IL-17F mRNA was expressed in the mouse colon EC system, but IL-17A was not expressed (FIG. 5K). These results indicate that, in response to C. rodentium infection, IL-17F is produced by non-T non-B innate immune cells and colon EC in addition to infiltrating lymphocytes.
  • IL-17RC is strongly expressed in colonic epithelial cells.
  • Two receptor molecules, IL-17RA and IL-17RC have been reported to bind to IL-17A and IL-17F (Toy, D., et al. (2006) J. Immunol. 177, 36- 39 .; Zheng, Y., et al. (2008) Nat. Med. 14, 282-289.).
  • the binding affinities of IL-17A and IL-17F for these receptors are different (Hymowitz, SG, et al., (2001) EMBO J. 20, 5332-5341 .; Kuestner, RE, et al., (2007) J.
  • IL-17RA mRNA was strongly expressed in lymphoid tissues such as thymus, spleen and LN (FIG. 6A). .
  • IL-17RC mRNA was abundantly expressed in non-hematopoietic tissues such as colon, small intestine and lung (FIG. 6A).
  • T cells and macrophage cells expressed more IL-17RA mRNA than the colon EC line, whereas colon EC expressed more IL-17RC mRNA than the T cells (Fig. 6B).
  • IL-17RA or Act1 mRNA is constitutively expressed in Thyl.2 + cells, B220 + cells, CD11c + cells, CD11b + cells, peritoneal macrophages and colon epithelial cells (CMT93), whereas IL-17RC mRNA is It was also found to be detected only in peritoneal macrophages and colon EC (FIG. 6C). That is, the tissue distribution of these receptors is significantly different, and IL-17RC is preferentially expressed in colon EC.
  • IL-17F can transmit signals to T cells, peritoneal macrophages or colon epithelial cells.
  • IL-17A can induce IL-6 by peritoneal macrophages, CCL2 by CD4 + T cells, or lipocalin 2 and ⁇ -defensin 3 by colonic epithelial cells (CMT93) in a dose-dependent manner (FIGS. 6D to 6F).
  • 50 ng / ml IL-17A was found to be sufficient to induce multiple cytokines and chemokines in these cells (FIGS. 6D-6J and FIG. 16).
  • IL-17F treatment in colonic epithelial cells induced the majority of the inflammatory mediators examined, but IL-17F activity was only slightly compared to IL-17A activity. (Figs. 6F, 6G and Fig. 16).
  • IL-17A also induced the production of multiple cytokines and chemokines in CDC4 + T cells, but not IL-17F. No synergistic effect was observed between IL-17A and IL-17F (FIGS. 6F, 6G and FIG. 16). These results indicate that IL-17A and IL-17F can induce the expression of cytokines and antimicrobial peptides in a manner that differs specifically to the cell type.
  • IL-17A is essential for the development of DTH, CHS, EAE, CIA and arthritis in Il1rn ⁇ / ⁇ mice, whereas IL-17F is not essential for the induction of these responses, It was shown that it has no effect on the effect of IL-17A on these disorders.
  • IL-17A and IL-17F are produced simultaneously by Th17 cells and bind to the same receptor, but IL-17F is less active in these immune responses compared to IL-17A. It shows that it does not have.
  • the cytokine-inducing activity of IL-17F from macrophages or T cells is much lower than IL-17A.
  • IL-17A enhances immune responses by activating T cell stimulation (Nakae, S., et al. (2002) Immunity 17, 375-387 .; Nakae, S., et al. (2003b) Proc. Natl Acad. Sci. USA 100, 5986-5990.), Macrophages (Da Silva, CA, et al. (2008) J. Immunol. 181, 4279-4286 .; Jovanovic, DV, et al. (1998) J. Immunol. 160, 3513-3521.) And dendritic cells (A Antonysamy, MA, et al. (1999) Immunol. 162, 577-584 .; Coury, F., et al. (2008) Nat. Med.
  • Inflammation is induced by inducing cytokines from various cell types, including). Therefore, there is a possibility that this cytokine does not act in allergic and autoimmune responses due to the low cytokine-inducing activity of IL-17F on immune cells.
  • Il17a -/- Il17f -/- mice were shown to be susceptible to opportunistic infections with Staphylococcus aureus, indicating that IL-17A and IL-17F are important for host defense against this bacterium .
  • IL-17A and IL-17F complement each other here because Il17f ⁇ / ⁇ and Il17a ⁇ / ⁇ mice show normal sensitivity to S. aureus.
  • IL-17A and IL-17F have been shown to be involved in the response to C. rodentium. Bacterial burden in the colon after infection with C.
  • rodentium showed a similar increase in Il17f -/- , Il17a -/- and Il17a -/- Il17f -/- mice, with only one IL-17 protein It was suggested that only deficiency would make them completely susceptible to C. rodentium infection. In particular, splenomegaly and colon hypertrophy associated with severe colon inflammation are more prominent in Il17f -/- than in Il17a -/- , and IL-17F protects colon epithelial cells from the pathogenic effects of this bacterium. It was shown to be more important than IL-17A. The result that both IL-17A and IL-17F are required for protection against C. rodentium is not the case for S. aureus, where either IL-17A or IL-17F is sufficient for protection. Clearly in contrast, suggesting a different defense mechanism against S. aureus and C. rodentium infection.
  • ⁇ -Defensin production was found to be impaired in the infected colon of Il17a -/- and Il17f -/- mice, and only one of IL-17A or IL-17F stimulated ⁇ -defensin production from EC
  • both IL-17A and IL-17F have been shown to be required for the induction of these molecules in vivo (Kao, CY, et al. (2004) J. Immunol. 173, 3482-3491. Liang, SC, et al. (2006) J. Exp. Med. 203, 2271-2279.). Since ⁇ -defensins play an important role in the immune response against these pathogens (LeBlanc, PM, et al.
  • IL-17A and IL-17F were directed against C. rodentium, since the production of C. rodentium specific antibodies in Il17f ⁇ / ⁇ , Il17a ⁇ / ⁇ and Il17a ⁇ / ⁇ Il17f ⁇ / ⁇ mice was normal. It is not necessary for the acquired immune response.
  • IL-17A and IL-17F producing cells in the colon are different, IL-17F is mainly produced by colon EC and innate immune cells, while most of IL-17A is likely to be Th17 cells It was found to be produced by Rag2-dependent cells. Furthermore, the data in this example show that IL-17A production is significantly induced after bacterial infection, whereas the induction of IL-17F is less pronounced in the infected colon. These results indicate that colonic EC and / or IL-17F from innate immune cells induce antimicrobial peptides in EC, resulting in protection against early bacterial invasion and spread. The distinct effects of IL-17A and IL-17F and the obvious synergy between these two molecules in defensin induction also explains why these two cytokines do not complement in C. rodentium infection in the colon Can be explained.
  • Il17ra -/- mice were reported to show ulcerative syndrome around the mouth and eyes mucosa by staphylococcal colonization (Schwarzenberger, P., et al., (2002) J. Cell. Biochem. Suppl 38, 88-95.). This phenotype is very similar to that seen in Il17a -/- Il17f -/- mice, suggesting that IL-17RA is involved in both IL-17A and IL-17F signaling The However, it was found that IL-17RC is strongly expressed in colonic epithelial cells, while IL-17RA is preferentially expressed in immune cells such as macrophages and T cells.
  • IL-17F The binding affinity of IL-17F to IL-17RA is much lower than IL-17A (Hymowitz, SG, et al. (2001) EMBO J. 20, 5332-5341 .; Wright, JF, et al., (2008). J. Immunol. 181, 2799-2805.), Since only IL-17F binds to IL-17RC in mice (Kuestner, RE, et al. (2007) J. Immunol. 179, 5462-5473.) The use of these receptors for 17A and IL-17F appears to be different.
  • IL-17A and IL-17F differ in colon epithelial cells, macrophages and T cells, and both IL-17A and IL-17F are neutrophil chemoattractants and ⁇ -Defensin could be induced, but only IL-17A was shown to be able to efficiently induce cytokines in macrophages and T cells.
  • IL-17RA and IL-17RC can also form homodimers (Kramer, JM, et al. (2006) J. Immunol.
  • IL-17RA is an IL-25 signaling Interacts with IL-17RB (Rickel, EA, et al. (2008) J. Immunol. 181, 4299-4310.). Therefore, in addition to the difference in production cells, the distribution of cell-type specific IL-17 receptors and the binding affinity of IL-17A and IL-17F to these receptors It is important for both host and host defense responses and can explain whether only IL-17F is involved in epithelial cells in innate immune responses.

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Abstract

L'invention porte sur une souris knockout pour IL-17F et une souris double knockout pour IL-17A/IL-17F. Il est possible de mettre à jour la fonction de IL-17F in vivo à l'aide des souris. L'invention porte spécifiquement sur un agent de protection contre les infections, lequel agent comprend (i) une protéine IL-17F ou (ii) une substance apte à se lier à un récepteur A de IL-17 (IL-17RA) ou un récepteur C de IL-17 (IL-17RC) pour présenter une fonction équivalente à celle de IL-17F en tant que principe actif.
PCT/JP2009/007341 2008-12-30 2009-12-28 Agent thérapeutique/prophylactique pour infections fonctionnant par régulation de il-17a/il-17f WO2010076851A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAAK S. ET AL.: "IL-17A and IL-17F do not contribute vitally to autoimmune neuro- inflammation in mice", JOURNAL OF CLINICAL INVESTIGATION, vol. 119, no. 1, 2009, pages 61 - 69, Retrieved from the Internet <URL:http://www.jci.org/articles/view/35997> [retrieved on 20081215] *
ISHIGAME H. ET AL.: "The Roles of Interleukin (IL)-17A and IL-17F in the Development of Inflammatory Responses", CYTOKINE, vol. 39, no. 1, 2007, pages 17 - 18, 62 *
LIANG S.C. ET AL.: "Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 203, no. 10, 2006, pages 2271 - 2279 *
WU Q. ET AL.: "IL-23-dependent IL-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory Mycoplasma pneumoniae infection", MICROBES AND INFECTION, vol. 9, no. 1, 2007, pages 78 - 86 *

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