WO2010076851A1 - Therapeutic/prophylactic agent for infections which relies on regulation of il-17a/il-17f - Google Patents

Abstract

Disclosed are an IL-17F-knockout mouse and an IL-17A/IL-17F-double knockout mouse.  The function of IL-17F in vivo can be revealed using the mice.  Specifically disclosed is an agent for protecting from infections, which comprises (i) an IL-17F protein or (ii) a substance capable of binding to an IL-17 receptor A (IL-17RA) or an IL-17 receptor C (IL-17RC) to exhibit an equivalent function to that of IL-17F as an active ingredient.

Description

Infectious disease treatment / prevention drug controlled by IL-17A / 薬 IL-17F

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. Recently, 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-β 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. Consistent with this finding, 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). Overexpression of 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).

In mice, experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), and inflammatory bowel disease (in IL-23-IL-17A signaling rather than IL-12-IFN-γ signaling) Some evidence has been identified that is involved in the development of autoimmune diseases such as IBD) and allergic diseases such as contact hypersensitivity (CHS) and delayed hypersensitivity (DTH) (Non-patent Documents 1 and 18). In recent studies, 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). In addition, 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. In addition, since 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. Furthermore, 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.

That is, according to the present invention, the following inventions are provided.
(1) 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.
(3) 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.
(4) 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.
(10) Use of the non-human mammal according to any one of (4) to (9) for screening for an infectious disease therapeutic / prophylactic agent.
(11) When the 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. In particular, unlike IL-17A, it is possible to activate only the beneficial infection-protective ability without inducing a harmful inflammatory response. In the present invention, it was clarified that 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. Furthermore, 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-γ 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-γ 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. (D) Arthritis incidence and severity score in Il1rn ^{− / −} mice. Left: Il1rn ^{− / −} background Il17f ^{+ / +} , Il17f ^+/− and Il17f ^{− / −} mice. Right: Il1rn ^{− / −} background Il17a ^{+ / +} Il17f ^{+ / +} , Il17a ^+/− Il17f ^+/− and Il17a ^{− / −} Il17f ^{− / −} mice (n = 15-22 / group). *, p <0.05 and **, p <0.01 for Il17f ^{+ / +} or Il17a ^{+ / +} Il17f ^{+ / +} mice as measured using the χ ² test. (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. (A) Weight and macroscopic morphology of submandibular LN of BALB / cA mice (n = 3-8 / group) aged 12-16 weeks. (B) Immunoglobulin titer in serum of 8-10 week old mice (n = 6-8 / group). Similar results were observed in the C57BL / 6J background. (C) Il17a ^{− / −} Il17f ^{− / −} pathology of mucosal tissue around the nose and mouth (H & E, upper, 40 ×; lower, 120 ×). Data represent 4 mice in each group. (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. (E) Representative plate showing bacterial colonies recovered from skin mucosal tissue of 12-16 week old BALB / cA mice. (F) CFU of Staphylococcus aureus in homogenate of mucocutaneous tissue of 12-16 week old mice. Data was collected from 3 independent experiments. (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. * P <0.05, ** p <0.05 and *** p <0.05 versus WT. 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. (A) 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). Data represent results collected from 2 or 3 independent experiments. (E, F) Histopathology (E) and intestinal crypt length (F) at the distal colon site 14 days after oral infection (H & E, 40 ×). Data was collected from 3 separate experiments. * P <0.05, ** p <0.01 and *** p <0.001 for wild type (WT) mice. IL-17F and IL-17A are required for the induction of β-defensin expression during C. rodentium infection. (A) Expression of inflammatory mediators in the colon 14 days after infection with C. rodentium was measured using semi-quantitative RT-PCR. (B) The expression of antimicrobial peptides in the colon 14 days after infection with C. rodentium was measured using real-time RT-PCR. RNA samples were collected from 6-8 mice in each group. All data represent 3 independent experiments. 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-γ expression in colonic PMA and ionomycin-stimulated lymphocytes of uninfected mice (C) or mice (D) 14 days after infection with C. rodentium Profiles. (E, F) Real-time RT-PCR for IL-17A and IL-17F mRNA expression in colon and MLN of C57BL / 6J wild type (WT) and Rag2 ^-/- mice 7 days after infection with C. rodentium Analyzed using (E) (n = 5-6 / group). Expression in wild type (WT) colon was taken as 1. Expression of these cytokines in the colon and MLN of Rag2 ^{− / −} mice was measured as a percentage of expression in wild type (WT) mice (F). (G) Whole colon of uninfected wild type (WT), Rag2 ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice were cultured for 24 hours in the presence or absence of IL-23 20 ng / ml. The concentration of IL-17A or IL-17F in the supernatant was measured by ELISA and normalized to the total protein content of each sample (n = 5-8 / group). Similar results were observed in CB-17SCID mice. (H, I) Wild type (WT) and Rag2 ^{− / −} mouse splenocytes (5 × 10 ⁵ cells) (H) or MLN (1.5 × 10 ⁵ cells) (I), LPS 5 μg / 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. (A, B) 129 / Ola × C57BL / 6J Wild-type (WT) mice (A) and IL-17RA and IL-17RC expression in tissues of different cell lines (B) were measured by real-time RT-PCR. (C) The expression of IL-17RA, IL-17RC and Act1 in different cell populations obtained by MACS sorting was measured using RT-PCR. (D) Peritoneal macrophages were stimulated with IL-17A or IL-17F 5 to 250 ng / ml or LPS 10 to 100 ng / ml for 24 hours, and the amount of IL-6 in the culture supernatant was measured using ELISA. (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). (F) Lipocalin 2 in a colonic epithelial cell line (CMT93) stimulated for 6 hours with IL-17A or IL-17F individually or in combination with IL-17A and IL-17F 50-250 ng / ml and β-defensin 3 expression was measured using real-time RT-PCR. (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. All data represent 3 independent experiments. Generation of Il17f ^-/- and Il17a ^-/- Il17f ^-/- mice. Il17f ^-/- mice 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. (A) Structure of mouse Il17f locus (wild type (WT) allele), IL-17F targeting construct, and predicted mutant Il17f gene (target allele). Exons are represented by boxes. Exons 2 and 3 of the Il17f gene were replaced with hph resistant genes. 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. RNA was isolated from splenocytes stimulated with ConA for 48 hours, and the mRNA levels of IL-17A and IL-17F were measured using RT-PCR. IL-17F is not required for Th17 differentiation in vitro. (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-γ 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-β, IL-6, anti-IL-4 monoclonal antibody and anti-IFN-γ 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. (A) Clinical score during MOG-induced EAE in wild type (WT), Il17f ^{− / −} , Il17a ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice (n = 12-18 per group). * P <0.05 for WT mice was determined using the Mann-Whitney U test. Data show pooled results from two independent experiments. (B) Histopathology of the lumbar spinal cord of mice 42 days after MOG immunization (H & E, 120 ×). Data represent 4-6 mice per group. (C) MOG-specific proliferative response of LN cells (n = 5-8 / group). Mice were immunized with MOG. Ten days after immunization, LN cells were cultured for 72 hours in the presence or absence of 50 μg / ml MOG peptide and [ ³ H] thymidine incorporation was measured. * P <0.05 for WT cell culture in the presence of MOG peptide. (D) The amount of IL-17A in the culture supernatant shown in (C) was quantified using ELISA. (E) Intracellular IL-17A and IL-17F profiles in CD4 ⁺ T cells stimulated with MOG peptide as in (C). Data are representative of two (C, D, E) independent experiments. IL-17F is not required for the development of CIA. On days 0 and 21, 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 χ ² test. IL-17A is involved in the development of DTH. (A) Intracellular IL-17F, IL-17A and IFN-γ 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 μg / ml mBSA. (B) Increased footpad thickness during mBSA-induced DTH response in wild type (WT), Il17f ^{− / −} , Il17a ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice (13-17 mice per group) . ** p <0.01 vs WT mice. Data show pooled results from two independent experiments. (C) mBSA-specific LN cell proliferation response induced by culturing with 50 μg / ml mBSA for 72 hours as in (A) (n = 5 / group). (D) Profiles of intracellular IFN-γ and IL-17A in mBSA-stimulated CD4 ⁺ T cells using the culture conditions described in (A). (E) Amount of methylated BSA-specific Ig in the mouse-derived serum used in (B). One week after mBSA administration, serum was collected and mBSA-specific Ab levels were measured by ELISA (n = 13-17 / group). ** p <0.01 vs WT mice. Data represent 2 (A) or 3 (C, D) independent experiments. IL-17F is not essential for CHS response. Ear swelling after administration of low dose (0.1%) or high dose (3.0%) TNCB was measured 24 hours after the second dose (6-10 mice per group). IL-17F is not essential for OVA / alum-induced eosinophilic and OVA-induced neutrophilic lung inflammation. (A) Number of cells in BAL fluid. 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). Mono, monocytes / macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils. The amount of C. rodentium-specific antibody in the serum of mice 14 days after oral infection. Mice were orally infected with C. rodentium (2 × 10 ⁸ CFU) as shown in FIG. After 14 days, serum was collected and the amount of C. rodentium specific Ig in the serum was measured using ELISA. Data shows pooled results from two separate experiments. * P <0.05, ** p <0.01 and *** p <0.001 for wild type (WT) mice. 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. The concentration of IL-17A or IL-17F in the supernatant was measured by ELISA and normalized to the total protein content for each sample (n = 5 / group). Similar results were observed in Rag2 ^{− / −} mice. (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. (G) Spleen cells from CB-17 SCID mice were stained with microbead-conjugated anti-mouse CD11c and CD11b and positive and negative cell fractions (5 × 10 ⁵ cells) in the presence of 20 ng / ml IL-23 or The cells were cultured in a 24-well plate for 72 hours in the absence, and the amounts of IL-17A and IL-17F in the culture supernatant were measured by ELISA. Data represent 2 (A, G) or 3 (BF) independent experiments. Different regulation of cytokine and chemokine production by IL-17A or IL-17F in macrophages, T cells and colon EC. (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.

Hereinafter, the present invention will be specifically described.
(1) Knockout non-human mammal 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. In the knockout non-human mammal of the present invention, 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.

Examples of non-human mammals that can be used 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 A non-human mammal can be produced by introducing a mutation into a target gene (IL-17F gene) by embryonic stem cells (ES cells) by homologous gene recombination. For example, when an IL-17F gene knockout mouse is prepared, a mouse IL-17F gene is first isolated from a mouse genomic library. A predetermined region within the IL-17F gene (for example, the region containing the second and third exons of the Il17f gene in the following examples) is selected as 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. Here, in order to create a non-human mammal having a deletion and / or insertion mutation in both the IL-17A locus and the IL-17F locus on the chromosome, against Il17a ^+/- ES cells, Il17a ^+/− Il17f ^+/− ES cells may be obtained by introducing the targeting vector for knockout of the IL-17F gene described above. Next, 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.

Hereinafter, a method for producing a knockout non-human mammal will be described.
(Isolation of IL-17F gene)
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.

(Construction of targeting vector)
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. As 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. .

From the viewpoint of facilitating screening of homologous recombinants, it is preferable to incorporate a selection marker gene (for example, drug resistance gene) into the targeting vector. Specifically, 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. As 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. Since 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. As 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 Using 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.

(Homologous recombination with targeting vector)
Using the targeting vector prepared above, homologous recombination is performed in cells. In this gene targeting method, it is preferable to use 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. In the present invention, 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.

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.

(Screening for homologous recombinants)
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.

(Production of knockout non-human mammals)
Recombinant 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. In the case of 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. Thereafter, early 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. .

From these chimeric 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. As described above, 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. By crossing the obtained IL-17F gene-deficient heterozygous mice, IL-17F gene-deficient homozygous mice can be obtained.

In order to obtain an animal with the desired genetic composition, it is preferable to perform backcrossing with the same strain for about 4 to 8 generations. A mouse 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.

(2) Screening method using knockout non-human mammal The above-described knockout non-human mammal of the present invention can be used to screen a therapeutic / prophylactic agent for infectious diseases. Specifically, 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. By selecting 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.

Examples of the test substance used in the screening method of the present invention 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.

As a method for administering the test substance to the non-human mammal of the present invention, 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.

(3) 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. Since opportunistic infections are likely to occur in AIDS patients, leukemia patients, and patients undergoing treatment with immune depression such as anticancer drugs and steroid drugs, 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. In addition, since 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. From this point, 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. Here, examples of the mucous membrane include respiratory (tracheal, lung, etc.) mucosa and digestive tract (intestinal tract, stomach, etc.) mucous membrane.

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.

(i) 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. As 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. In addition, also when using as a mucosal immunity promoter etc., it can determine suitably in the range of said description, such as an administration route and dosage.

(4) Infectious Disease Treatment / Prevention Screening Method In the present invention, 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. In addition, 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.
That is, according to the present invention, 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.

As IL-17F producing cells, epithelial cells, innate immune cells, Th17 cells and the like can be used. As the 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.
In addition, 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.

(I) Method (1) Mouse Il17f ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice were prepared as shown in FIG. Il17a ^-/- (Nakae, S., et al., (2002) Immunity 17, 375-387), Il17f ^-/- and Il17a ^-/- Il17f ^-/- mice, or C57BL with 129 / Ola x C57BL / 6J background / 6J mice (Japan SLC) were used for 8 generations or BALB / cA mice (Clea Japan) were backcrossed for 4 generations. Mice's sex and age (2-3 months old) were matched in all experimental groups. 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 Claire, 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.

(2) Isolation of cells 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). To isolate 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. .

(3) Cell culture Mouse T cell line (BW5147), B cell line (X5563), macrophage cell line (RAW264) and colonic epithelial cell line (CMT93 or Colon26) were cultured in RPMI1640 (Sigma) containing 10% FBS. did. The mouse dendritic cell line (DC2.4) was cultured in RPMI 1640 containing 10% FBS, HEPES and nonessential amino acids (GIBCO). 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 .

(4) Measurement of cytokines, chemokines and antigen-specific Ig IFN-γ, IL-6 (OptEIA ™ kit, BD Pharmingen), IL-17A and IL-17F (DuoSet ELISA kit, R & D systems) in the culture supernatant ) Was measured using an ELISA according to the manufacturer's instructions. IL-1α, IL-1β, IL-9, IL-10, IL-12 / 23 p40, IL-12 p70, IL-13, G-CSF, GM-CSF, IFN-γ, 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).

(5) Flow cytometry Cells were stimulated with PMA (Sigma) 50 ng / ml, ionomycin (Sigma) 500 ng / ml and monensin (Sigma) 1 μM for 5 hours. Intracellular cytokine staining was performed as previously reported (Komiyama, Y., et al. (2006) J. Immunol. 177, 566-573). Cells are treated with anti-mouse CD16 / CD32 monoclonal antibody (2.4G2) in staining buffer (HBSS containing 2% FCS and 0.1% sodium azide) to block FcR binding and then APC-anti Stained with CD4 monoclonal antibody (Gk1.5, BioLegend). After washing, 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-γ 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). For secondary staining, Alexa Fluor 488 anti-goat IgG (A-11055; Invitrogen), PE anti-goat IgG (Santa Cruz) or FITC-streptavidin (BD Pharmingen) was used. Cells were analyzed with a FACSCalibur system (Becton Dickinson) and data were analyzed with FlowJo software (Tree Star).

(6) Real-time RT-PCR
Total RNA was extracted using Sepasol reagent (Nacalai Tesque) according to the manufacturer's instructions. RNA was denatured in the presence of oligo dT primers and then reverse transcribed using a high efficiency cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time RT-PCR was performed using the SYBR Green qPCR kit (Invitrogen) and iCycler system (Bio-Rad) using the primer sets (SEQ ID NOs: 1 to 50) described in Table 1 below.

(7) Clinical Evaluation of Arthritis The onset of arthritis in Il1rn ^{− / −} mice was monitored by gross evaluation as previously reported (Horai, R., et al. (2004) J. Clin. Invest. 114, 1603-1611 ). The incidence of arthritis in each foot was graded as follows by macroscopic evaluation. 0, no change, 1, mild swelling, 2, obvious joint swelling, 3, severe joint swelling and tonicity change.

(8) Infection with Staphylococcus aureus S. aureus 834 was prepared as previously reported (Nakane, A., et al. (1995) Infect. Immun. 63, 1165-1172). Bacteria were cultured on tryptic soy agar (Difco) at 37 ° C. for 12 hours, inoculated into tryptic soy broth (Difco) and incubated for an additional 12 hours. The cells were collected by centrifugation and resuspended in PBS. The concentration of resuspended cells was adjusted by spectrophotometry at 550 nm. Mice were intravenously infected with 200 μl of a solution containing 1 × 10 ⁷ CFU of live S. aureus cells dissolved in PBS. To measure the bacterial load in infected tissues, kidneys were homogenized and diluted 10-fold with sterile PBS. Bacterial CFU was measured by seeding the respective dilutions on tryptic soy agar after culturing at 37 ° C. for 12 hours.

(9) C. rodentium infection C. rodentium infection was performed as previously reported (Nagai, T., et al. (2005) J. Biol. Chem. 280, 2998-3011.). Briefly, 129 / Ola × C57BL / 6J mice were inoculated with 200 μl of bacterial suspension (2 × 10 ⁸ CFU / head) by oral gavage. For colony formation assays, colons were harvested, homogenized, and sterile diluted homogenates were seeded on MacConkey agar (Difco). For histochemical analysis, colons were fixed with 4% paraformaldehyde in PBS overnight at 4 ° C. and frozen in tissue freezing medium (Leica Jung). Frozen sections were prepared and stained with anti-C. Rodentium serum as previously reported (Nagai, T., et al. (2005) J. Biol. Chem. 280, 2998-3011).

(10) Statistics Unless otherwise indicated, all results are expressed as mean and SEM. Independent student t-test, Mann-Whitney U test or χ ² test was used to statistically analyze the results. Differences were considered significant when p <0.05.

(11) Generation of Il17f ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice 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. 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.). The following 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).

(12) T cell differentiation in In vitro Spleen CD4 ⁺ T cells were isolated by positive selection using autoMACS (Miltenyi Biotech). Th17 cells are 1μg / ml of anti-CD3 monoclonal antibody purified from the culture supernatant of 145.2C11 cells, 1μg / ml of anti-CD28 monoclonal antibody (37.51; eBioscience), anti-mouse IFN-γ (XMG1.2, kindly provided by Dr. Yoshimoto 10 μg / ml, anti-mouse IL-4 (11B11, generously granted by Dr. Yoshimoto) 10 μg / ml, recombinant human TGF-β (PeproTech) 1 ng / ml and recombinant mouse IL-6 (R & D systems) CD4 ⁺ cells were induced by culturing for 3 days in the presence of 20 ng / ml. To measure IL-23-induced IL-17A production, lymph node cells from DO11.10 mice were present in the presence of 0.1 μM OVA _323-339 peptide and 0.1-10 ng / ml recombinant mouse IL-23 (R & D systems). Cultured.

(13) CHS
2,4,6-Trinitrochlorobenzene (TNCB; Tokyo Kasei) -induced CHS was performed as previously reported (Nakae, S., et al. (2002) Immunity 17, 375-387.). Briefly, the skin of the abdomen of 129 / Ola x C57BL / 6J background mice was sensitized with 25 μl of low (0.3%) or high (3%) TNCB dissolved in acetone and olive oil mixture (4: 1). Made. Five days later, 25 μl of 1% TNCB was administered to the ear. Mice were euthanized 24 hours after the second dose, ear tissue discs were removed and weighed. Ear swelling was calculated as follows: Increased ear swelling (mg) = weight of ear administered (mg) −weight of ear treated with vehicle (mg).

(14) DTH
Methyl-BSA-induced DTH was examined as previously reported (Nakae, S., et al. (2002) Immunity 17, 375-387.). Briefly, C57BL / 6J background mice were sensitized subcutaneously at the base of the tail with mBSA (Sigma) 1.25 mg / ml containing complete Freund's adjuvant (CFA; Difco). Seven days after the sensitization, 200 μg / 20 μl of mBSA was administered to one footpad of the mouse, and the same amount of PBS was injected into another footpad. The swelling of the footpad was measured with a caliper with a dial, and the results were calculated as follows.
Footpad swelling degree (mg) = thickness of footpad injected with mBSA (mg)-thickness of footpad injected with PBS (mg)

(15) 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 μg 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

(16) CIA
CIA was examined as previously reported (Nakae et al., 2003). Briefly, C57BL / 6J background mice were challenged with 100 μl 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

(17) Ovalbumin-induced pulmonary inflammation Mice were sensitized intraperitoneally with OVA / alum 100 μg / 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. For 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.

(18) Isolation of 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 . To isolate colon lymphocytes, including intraepithelial and lamina propria 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.

(19) 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.

(20) Microbiological analysis Specimens were collected from Il17a ^{− / −} Il17f ^{− / −} mice and incubated at 37 ° C. for 2 days on blood agar plates (Eiken Chemical) or egg yolk mannitol salt medium (Eiken Chemical). Multiple colonies were randomly selected from each plate and bacteria were identified using IDtestSP-18 (Nissui Pharmaceutical). To detect opportunistic bacterial colonization, skin and mucosal tissues around the nose and mouth were collected from BALB / cA mice. These tissues were homogenized and diluted 10-fold with sterile PBS. Bacterial CFU values were measured by seeding each dilution on tryptic soy agar (Difco) after culturing at 37 ° C. for 12 hours.

(II) Results (1) IL-17F contributes to the development of arthritis in IL-1 receptor antagonist-deficient (Il1rn ^{− / −} ) mice.
In order to elucidate the difference in function of IL-17F and IL-17A in the immune system, 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. Proliferative response and IFN-γ production are normal in Il17a ^{− / −} Il17f ^{− / −} mice, IL-17A production is normal in Il17f ^{− / −} mice, IL-17A and IL-17F are TGF-β 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-γ-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). Compared with Il17f ^{− / −} Il1rn ^{− / −} mice, the development of arthritis was markedly suppressed in Il17a ^{− / −} Il17f ^{− / −} Il1rn ^{− / −} mice (FIG. 1D). There was no change in IL-17A + T cell populations in LN from Il17a ^{+ / +} Il17f ^{+ / +} Il1rn ^{− / −} and Il17f ^{− / −} Il1rn ^{− / −} mice (FIG. 1E).

Similarly, neutrophilic airways induced by OVA in EAE, CIA, DTH, 2,4,6-trinitrochlorobenzene (TNCB) -induced CHS, DO11.10 mice, where IL-17A plays an important role Inflammation (Komiyama, Y., et al. (2006) J. Immunol. 177, 566-573 .; Nakae, S., et al. (2002) Immunity 17, 375-387 .: Nakae, S., et al. (2003a ) J. Immunol. 171, 6173-6177 .; Nakae, S., et al. (2007) Blood 109, 3640-3648.) Were also normally expressed in Il17f ^{− / −} mice (FIGS. 9-13 and Tables). 2). These results indicate that IL-17A plays a major role in T cell-dependent autoimmune and allergic responses, but IL-17F contributes little if any to these responses.

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.

(2) Il17a ^{− / −} Il17f ^{− / −} mice show increased susceptibility to opportunistic infections by S. aureus.
We found that the submandibular LN of Il17a ^-/- Il17f ^-/- mice expands with age, but not wild type (WT), Il17f ^-/- or Il17a ^-/- mice. This effect was observed in various genetic backgrounds including C57BL / 6J, BALB / cA and 129 / OlaXC57BL / 6J strains (FIG. 2A). At 8-10 weeks of age, IgM titers were similar between Il17a ^{− / −} Il17f ^{− / −} mice and wild type (WT) mice. However, total IgG, IgG1, IgG2a and IgG2b titers, IL17A compared to wild-type (WT) mice ^{- / -} Il17f ^{- / -} in mouse increased 2-4 ^{fold, Il17a - / - Il17f - /} - mice IgG3 titer was decreased in (FIG. 2B). Interestingly, Il17a ^{− / −} Il17f ^{− / −} mice developed mucocutaneous abscesses around the nose and mouth (FIG. 2C). Histological analysis revealed a fibrin-encapsulated abscess and significant leukocyte infiltration, especially in the mucosal skin tissue of Il17a ^{− / −} Il17f ^{− / −} mice.

Increased LN, subcutaneous abscess formation and increased antibody production suggest that Il17a ^{− / −} Il17f ^{− / −} mice may have responded to infection. In support of this idea, treatment with antibiotics suppressed LN expansion under the mandible of Il17a ^{− / −} Il17f ^{− / −} mice (FIG. 2D). Next, an attempt was made to collect infectious microorganisms from mucocutaneous tissue around the nose and mouth of Il17a ^{− / −} Il17f ^{− / −} mice. An opportunistic staphylococcus aureus was recovered from the affected tissues of these 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. However, there were no differences in survival and the number of bacteria recovered from the kidney after 72 hours between wild type (WT) and Il17a ^{− / −} Il17f ^{− / −} mice (FIGS. 2G and 2H). These results suggest that both IL-17A and IL-17F play an important role in protecting against local but not systemic infections against S. aureus.

(3) Host defense against C. rodentium infection requires both IL-17F and IL-17A.
Il17a ^-/- Il17f ^-/- mice were susceptible to opportunistic infections by S. aureus, so Il17f ^-/- , Il17a ^-/- and Il17a ^-/- Il17f ^-/- against experimental C. rodentium infection The sensitivity of the mice was examined. After oral infection with C. rodentium, the number of bacteria in the colon of wild type (WT) 129 / Ola × C57BL / 65 mice increased up to 14 days after infection and then decreased (FIG. 3A). Although 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. Of note, colonic bacterial counts were similar among Il17f ^{− / −} , Il17a ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice. 14 days after infection, a marked increase in the bacterial population in the distal colon of Il17f ^{− / −} , Il17a ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice compared to the distal colon of wild type (WT) mice Swelling was observed (FIG. 3B). In addition, significant enlargement of the colon and spleen was observed in Il17f ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice, but only mild swelling was detected in Il17a ^{− / −} mice (FIG. 3C and 3D). Consistent with these results, 14 days after infection, IL17A ^{- / -} as compared to the case of mice, severe inflammatory changes IL17F ^{- / -} and Il17a ^{- / -} Il17f ^{- / -} observed in the colon of mice This suggested that IL-17F plays a greater role than IL-17A in the immune response against this bacterium (FIGS. 3E and 3F). These results clearly show that both IL-I7A and IL-17F play an important role in protecting the host from C. rodentium infection.

(4) IL-17F and IL-17A are required for β-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.

Both IL-17A and IL-17F regulate innate immunity by inducing neutrophil recruitment and antimicrobial peptide production (Ouyang, W., et al. (2008) Immunity 28, 454-467.). However, compared to wild type (WT) mice, mRNA expression of neutrophil chemoattractants such as CXCL1 and CXCL2 and pro-inflammatory mediators such as IFN-γ, IL-1β, IL-6, TNF and iNOS 14 days after infection with C. rodentium, there was a similar increase in the colons of Il17f ^{− / −} , Il17a ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice (FIG. 4A). However, the expression of antimicrobial peptides such as β- defensins 1, 3 and 4 was observed on the 14th day after C. rodentium infection, in Il17f ^{− / −} , Il17a ^{− / −} and Il17a ^{− / −} Il17f ^{− / −} mice. Although significantly impaired in the colon, expression of β-defensin 2, lipocalin 2, S100A8, S100A9, Reg3β and Reg3γ was not impaired (FIG. 4B). These results indicated that both IL-17F and IL-17A are important in inducing the expression of β-defensin, which is important for host defense against C. rodentium.

(5) 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.). In contrast, IL-17F mRNA expression in the colon was higher than in the small intestine (FIG. 5A). During C. rodentium infection, IL-17A and IL-17F mRNA expression was induced in the colon of wild type (WT) mice, but the increase in IL-17A mRNA expression was greater (day 14; IL-17A: 29 times; IL-17F: 14 times) (FIG. 5B). Under these conditions, IL-17A mRNA expression was unaffected by IL-17F deficiency and vice versa. Although a small number of 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). However, in contrast to the coordinated production of IL-17A and IL-17F by LN cells after the onset of DTH, EAE or arthritis (FIGS. 1A and 11), IL-17A ⁺ IL-17F ⁻ in colon lymphocytes The percentage of cells was much greater than the percentage of IL-17A ⁻ IL-17F ⁺ cells (FIG. 5D). Although neither IL-17A ⁺ nor IL-17F ⁺ cells were observed in the Il17a ^{− / −} Il17f ^{− / −} colon lymphocyte population, IFN-γ ⁺ cell counts were observed during C. rodentium infection. There was a marked increase (Figure 5D).

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). However, IL-17A mRNA levels are markedly reduced in Rag2 ^{− / −} mice (approximately 20% of wild type (WT)) (FIGS. 5E and 5F), and in MLN, 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. Was shown (FIG. 5G). Next, it was investigated which cells produce IL-17F in response to IL-23. Interestingly, 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). Among innate immune cells, 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). Furthermore, 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.

(6) 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. Immunol 179, 5462-5473 .; Wright, JF, et al. (2008). J. Immunol. 181, 2799-2805.) The tissue distribution of these molecules was examined. As previously reported (Kuestner, RE, et al. (2007) J. Immunol. 179, 5462-5473.), IL-17RA mRNA was strongly expressed in lymphoid tissues such as thymus, spleen and LN (FIG. 6A). . On the other hand, IL-17RC mRNA was abundantly expressed in non-hematopoietic tissues such as colon, small intestine and lung (FIG. 6A). Consistent with these results, 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.

Next, it was examined whether IL-17F can transmit signals to T cells, peritoneal macrophages or colon epithelial cells. As a result, IL-17A can induce IL-6 by peritoneal macrophages, CCL2 by CD4 ⁺ T cells, or lipocalin 2 and β-defensin 3 by colonic epithelial cells (CMT93) in a dose-dependent manner (FIGS. 6D to 6F). And 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). Since IL-6 production was observed in IL-17A-treated peritoneal macrophages derived from C3H / HeN (LPS sensitive) and C3H / HeJ (LPS insensitive) mice, the cytokine-inducing activity of IL-17A was It is not the influence of mixed LPS (FIG. 6D). However, IL-6, CCL3 and G-CSF production was induced in peritoneal macrophages by treatment with IL-17F (50 ng / ml), whereas this IL-17F was induced by IL-17A Inflammatory mediators could not be increased (FIGS. 6D, 6H and FIG. 16). In contrast, like IL-17A, 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.

(III) Discussion In this example, 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. These observations indicate that 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. In this regard, it was found that 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. 14, 81-87. 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.

In addition, 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. Furthermore, 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.

β-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 β-defensin production from EC However, 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 β-defensins play an important role in the immune response against these pathogens (LeBlanc, PM, et al. (2008) Cell Host Microbe 3, 146-157 .; Simmons, CP, et al. (2002) J. Immunol 168, 1804-1812.), Deficiency in β-defensin production is thought to be associated with increased sensitivity of Il17a ^{− / −} and Il17f ^{− / −} mice to C. rodentium. These in vivo data suggest a possible synergy between IL-17A and IL-17F in protection against C. rodentium, but these in vitro using the colon epithelial cell line as a target Intermolecular synergies could not be observed, suggesting that the response of these cell lines may be different from colon defensin-producing cells in vivo. In vivo, no synergy between IL-17A and IL-17F in the protection against S. aureus could be observed. 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. 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. In support of these findings, the effects of 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 β -Defensin could be induced, but only IL-17A was shown to be able to efficiently induce cytokines in macrophages and T cells. These results suggest that forms of receptors other than the IL-17RA-IL-17RC heterodimeric complex are involved in the colon. In fact, IL-17RA and IL-17RC can also form homodimers (Kramer, JM, et al. (2006) J. Immunol. 176, 711-715.), 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.

Recent studies of Il22 ^{− / −} and Il17rc ^{− / −} mice that do not respond to IL-17A or IL-17F show that IL-22 expressed in response to IL-23 but not IL-17A and IL-17F Has been shown to be essential for the early host response to C. rodentium (Zheng, Y., et al. (2008) Nat. Med. 14, 282-289.). IL-22 is produced by innate immune cells such as dendritic cells during C. rodentium infection and induces expression of Reg family antibacterial proteins in colon EC (Zheng, Y., et al. (2008) Nat. Med. 14, 282-289.). These observations using Il17rc ^{− / −} mice seemingly contradictory to our findings that both IL-17A and IL-17F are involved in host defense against C. rodentium, but our data Is consistent with IL-22 involvement.

In the present invention, different involvement of IL-17F and IL-17A in allergic response and defense against bacterial infection was shown. The findings of the present invention provide an understanding of the molecular mechanisms of IL-17A and IL-17F mediated responses and are useful in the development of new therapies for allergic diseases and bacterial infections.

Claims (11)

1. (i) a substance that binds to IL-17F protein or (ii) IL-17 receptor A (IL-17RA) or IL-17 receptor C (IL-17RC) and exhibits a function equivalent to that of IL-17F Infection protective agent containing as an active ingredient.
2. The infection protective agent according to claim 1, which is used to protect against microbial infection in the mucosa.
3. A therapeutic / prophylactic agent for infectious diseases, 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 method.
4. 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. 6. The non-human mammal according to claim 5, wherein the susceptibility to opportunistic infection is increased.
7. The non-human mammal according to any one of claims 4 to 6, which is used as an infectious disease model animal.
8. The non-human mammal according to any one of claims 4 to 7, which is a rodent.
9. The non-human mammal according to any one of claims 4 to 8, which is a mouse.
10. The use of the non-human mammal according to any one of claims 4 to 9, for screening an infectious disease therapeutic / prophylactic agent.
11. The test substance is administered to the non-human mammal according to any one of claims 4 to 9, the degree of infection susceptibility in the non-human mammal is measured, and the infection susceptibility is compared with the case where the test substance is not administered. A method for screening an infectious disease treatment / prevention drug, comprising selecting a test substance that lowers the risk as a candidate substance.

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