JP6679021B2 - Psoriatic arthritis model animal and pustular psoriasis model animal - Google Patents

Description

  The present invention relates to an arthritic psoriasis model animal and a pustular psoriasis model animal. More specifically, the present invention relates to a method for producing an arthritic psoriasis model animal, a method for producing a pustular psoriasis model animal, a screening method using an arthritic psoriasis model animal, or a pustular psoriasis model animal.

  Psoriasis is roughly divided into five types: psoriasis vulgaris, psoriasis arthritis (psoriatic arthritis), guttate psoriasis, erythroderma psoriasis, and pustular psoriasis. Arthritic psoriasis, psoriatic erythroderma, and pustular psoriasis are sometimes difficult to treat, and research for the development of therapeutic methods is vigorously pursued. According to previous studies, most pustular psoriasis is attributed to a genetic predisposition to recessive inheritance of the IL36RN gene encoding an interleukin 36 receptor antagonist (hereinafter sometimes abbreviated as “IL-36RN”). It has been clarified (see Non-Patent Document 1).

  An IL36RN gene knockout mouse has been produced (see Non-Patent Document 2), and dermatitis is caused by overexpressing IL-36 in the mouse or by externally applying imiquimod, which is a ligand (agonist) of Toll-like receptor 7. It was reported (see Non-Patent Documents 2 and 3). However, this model does not cause clinical (gross) pustules and is used in various experiments as a model mouse for psoriasis vulgaris.

  Patients with pustular psoriasis often have arthritic psoriasis. On the other hand, the expression of IL-36 is observed in the joints of patients with psoriatic arthritis (see Non-Patent Document 4). In addition, cases of pustular psoriasis with IL36RN gene mutation (recessive) that are associated with arthritic psoriasis have been reported. On the other hand, based on the results of experiments using various arthritis models including the collagen-induced arthritis (CIA) model, reports denying the involvement of IL-36 in arthritis have been accumulated (for example, see Non-Patent Document 5). ), "IL-36 is not involved in arthritis" is a dogma.

Sugiura K. et al. J Invest Dermatol. 2013 Nov; 133 (11): 2514-21. Blumberg H. et al., J. Ex. Med. 2007 Oct; 204 (11): 2603-14. Tortola L. et al., J Clin Invest. 2012 Nov; 122 (11): 3965-76. Frey S. et al., Ann Rheum Dis. 2013 Sep; 72 (9): 1569-74. Damien Dietrich & Cem Gabay Nat Rev Rheumatol. 2014 Nov; 10 (11): 639-40.

  Arthritic psoriasis and pustular psoriasis are refractory, and there is a strong demand for new treatments. In order to establish an effective treatment method, it is desired to create a model animal that reproduces these pathological conditions. If a model animal that reproduces the pathophysiology of arthritic psoriasis / pustular psoriasis can be constructed, for example, a screening system useful for the search for therapeutic agents can be constructed, and aimed at establishing a therapeutic method for arthritic psoriasis and pustular psoriasis The research advances greatly. Therefore, an object of the present invention is to provide a model animal (psoriasis model animal) that reproduces the pathological condition of arthritic psoriasis or the pathological condition of pustular psoriasis.

  In order to solve the above-mentioned problems, the inventors of the present invention have conducted extensive studies focusing on the IL36RN gene. Fighting is often carried out when multiple mice (males) are kept in one cage, but in the case of IL36RN gene-deficient mice (males), arthritis is caused by fighting (wild-type mouse or clown-like ichthyosis model). No such phenomenon was reported in mice (ABCA12 gene knockout mouse), and a surprising phenomenon was observed. Regarding this phenomenon that can be said to overturn the above-mentioned theory that "IL-36 is not involved in arthritis", it was considered that innate immunity might be activated from infection associated with trauma and arthritis might have occurred, and research was advanced. It was Specifically, focusing on Toll-like receptor 4 (TLR4) involved in innate immunity, its ligand, lipopolysaccharide (LPS), was subcutaneously administered to the hind legs of IL36RN gene-deficient mice. As a result, marked swelling / thickness (induction of adhesitis) was observed as compared with the control (wild type) group. That is, the pathology of arthritic psoriasis was successfully induced by administering a TLR4 ligand to IL36RN gene-deficient mice.

On the other hand, in view of the relationship between pustular psoriasis and arthritic psoriasis, we believe that IL36RN gene-deficient mice may be used for the production of pustular psoriasis model, and we administered LPS subcutaneously to the back of IL36RN gene-deficient mice. , The progress was observed. The treated mice surprisingly developed pustules in just a few days. That is, the pathology of pustular psoriasis was also successfully induced by administering TLR4 ligand to IL36RN gene-deficient mice. Remarkably, pustule formation was also observed in wild-type mice by the same treatment. This fact means that a screening system useful for developing a therapeutic drug for pustular psoriasis can be constructed not only by using the IL36RN gene-deficient mouse but also by using a wild-type mouse.
The following inventions are based on the above achievements and consideration.
[1] A method for producing a psoriasis model animal, which comprises the step of administering a Toll-like receptor 4 ligand to a transgenic non-human animal deficient in the IL36RN gene encoding an interleukin 36 receptor antagonist.
[2] The production method according to [1], wherein the transgenic non-human animal lacks a homozygous IL36RN gene encoding an interleukin 36 receptor antagonist.
[3] The production method according to [1] or [2], wherein the transgenic non-human animal is a rodent.
[4] The production method according to [3], wherein the rodent is a mouse.
[5] The production method according to any one of [1] to [4], wherein the ligand is lipopolysaccharide.
[6] The production method according to any one of [1] to [5], wherein the ligand is administered near the joint of the transgenic non-human animal.
[7] The production method according to [6], wherein the administration site of the ligand is the foot.
[8] The production method according to any one of [1] to [5], wherein the ligand is subcutaneously or intradermally administered to the transgenic non-human animal.
[9] The production method according to [8], wherein the administration site of the ligand is the back or abdomen.
[10] A psoriasis model animal obtained by the production method according to any one of [1] to [5] [11] [6] or a pathological condition of psoriatic arthritis obtained by the production method of [7] Model animal with psoriasis.
[12] A psoriasis model animal exhibiting the pathological condition of pustular psoriasis obtained by the method according to [8] or [9].
[13] A method for screening a drug for psoriasis, which comprises the following (1) and (2):
(1) a step of administering a test substance to a psoriasis model animal obtained by the production method according to any one of [1] to [12];
(2) A step of determining the preventive or therapeutic effect of the test substance.
[14] The screening method according to [13], wherein the administration of the test substance in step (1) is performed before the administration of the Toll-like receptor 4 ligand for producing the psoriasis model animal.
[15] The screening method according to [13] or [14], wherein the determination in step (2) is performed based on the comparison with the control group.
[16] The screening method according to any one of [13] to [15], wherein the drug for psoriasis is a drug for preventing or treating arthritic psoriasis, or for preventing or treating pustular psoriasis.
[17] The screening method according to any one of [13] to [15], wherein the drug for psoriasis is a drug for preventing or treating psoriasis vulgaris.
[18] A method for screening a drug for preventing or treating pustular psoriasis, which comprises the following (i) and (ii):
(I) administering to the wild-type mouse a ligand for Toll-like receptor 4 and a test substance;
(Ii) A step of determining the preventive or therapeutic effect of the test substance.

IL36RN (Il1f5) gene knockout mouse plasmid and target site used. Results of induction experiment of arthritis. For each dose of lipopolysaccharide (LPS), the state of the hindpaw was compared 6 hours after the third administration (day 2) between the wild-type mouse (WT) and the IL36RN (Il1f5) gene homo-deficient mouse. Induction of arthritis in IL36RN (Il1f5) gene homo-deficient mice. The thickness of the hind paw was measured 6 hours after the third administration (day 2). Induction of pustules in mice homozygous for IL36RN (Il1f5) gene. The state of the back on day 2 (upper row) and day 4 (lower row) when LPS 50 μg was administered is shown in comparison between wild-type mice (WT) and IL36RN (Il1f5) gene homo-deficient mice. The therapeutic effect of anti-IL-1b antibody. To the IL36RN (Il1f5) gene homo-deficient mouse, 60 μg of anti-IL-1b antibody was administered one day before the administration of LPS, and its effect was examined. The upper row shows the state of the back on the second day. The lower row shows the state of the back on the 5th day.

1. Method for producing psoriasis model animal (first aspect of the invention)
A first aspect of the present invention relates to a method for producing an animal showing a pathological condition of psoriasis, that is, a psoriasis model animal. In the production method of the present invention, “a step of administering a ligand of Toll-like receptor 4 to a transgenic non-human animal deficient in IL36RN gene encoding an interleukin 36 receptor antagonist” is performed.

  In the production method of the present invention, first, an animal having a predetermined characteristic (referred to as “treatment animal” in the present specification) is prepared and a treatment for inducing psoriasis is performed. The therapeutic animal used in the present invention comprises a transgenic non-human animal deficient in the IL36RN gene encoding an interleukin 36 receptor antagonist (hereinafter, also referred to as “IL36RN gene deficient animal”). The animal species of the IL36RN gene-deficient animal is not particularly limited, and includes mouse, rat, guinea pig, hamster, rabbit, dog, cat, sheep, pig, cow, horse and the like. Preferably mouse (e.g. 129, 129P3, 129S1, A, AHe, AKR, B57BL, C57BL / 6N, C57BL / 6J, C57BL / 6Ei, C57BL / 10, C57BR / cd, BALB / c, BALB / cBy, B6C3F1, B6D2F1). , B6Albino, CB-17 SCID, C3H, CBA / J, CBA / N, CBA / Ca, CD2F1, db, DBA / 1, DBA / 2, HRS / J, C57L / J, NZB, NZW, RIIIS / J, Rodents such as ICR, NC, NOD, NOD SCID, NSG, ob, SHO, SJL, SWR etc.) and rats (eg BN, CD (SD), Wistar, F344, LEW etc.) Yes, most preferably mouse.

  The IL36RN gene-deficient animal preferably lacks the IL36RN gene homozygously. When an IL36RN gene-deficient animal having this characteristic is used, the pathological condition of psoriasis can be easily induced. The messenger RNA of the IL36RN gene has been reported to be induced in psoriatic lesions. As an example of the sequence of IL36RN gene, the sequence of mouse Il1f5 gene (corresponding to IL36RN gene), which is registered in a public database, is SEQ ID NO: 1 (ACCESSION: NM_001146087, DEFINITION: Mus musculus interleukin 1 family, member 5 ( delta) (Il1f5), transcript variant 1, mRNA), and the sequence of rat Il36rn gene is shown in SEQ ID NO: 2 (ACCESSION: NM_001107814, DEFINITION: Rattus norvegicus interleukin 36 receptor antagonist (Il36rn), mRNA).

  An IL36RN gene-deficient animal can be produced, for example, by using gene targeting. Gene targeting is a technique for modifying a genome by utilizing homologous recombination of genes. By utilizing gene targeting, a gene-modified animal lacking a specific gene (knock-out animal), a gene-modified animal into which a specific gene has been introduced (knock-in animal), etc. can be produced. The method for producing genetically modified animals (knockout animals, knockin animals) using gene targeting is roughly classified into (a) construction of a targeting vector and (b) introduction of the targeting vector into ES cells, taking a mouse as an example. And identification of homologous recombinants, (c) injection of homologous recombinants into blast cysts, (d) embryo transfer into uterus of foster mother and delivery (chimeric offspring mice), (e) chimeric offspring and wild-type mice (F) generation of heterozygous mice, and (f) F1 generation mating (production of F2 generation homozygous mice). A gene-modified animal can be obtained by the same procedure when other rodents such as rat are used. Incidentally, for the method for producing a genetically modified animal by gene targeting, for example, Joyner, AL: Gene Targeting (IRL press), Capeccchi, MR, Science, 244, 1288-1292 (1989), Hua Gu, et al., Science, 265 , 103-106 (1994), McHugh, TJ et al., Cell, 87, 1339-1349 (1996), Shibata, H. et al., Science, 278, 120-123 (1997). Mice in which the IL36RN gene is homozygously deleted have been produced by gene targeting using mouse ES cells (see, for example, Non-Patent Documents 2 and 3).

  As a result of the study by the present inventors, it was revealed that an IL36RN gene-deficient animal can exhibit psoriasis symptoms by administration of a Toll-like receptor 4 ligand (hereinafter referred to as “TLR4 ligand”). The manufacturing method of the present invention utilizes this knowledge. That is, the pathological state of psoriasis is induced by administering a TLR4 ligand to a treatment animal (IL36RN gene-deficient animal). Without being bound by theory, when TLR4 ligand is administered to treatment animals, IL-36α (Il-1f6), IL-36β (Il-1f8), IL-36γ (Il -1f9) is induced, and the signal of inflammation through the IL-36 receptor of epidermal keratinocytes and inflammatory cells is enhanced, resulting in the manifestation of psoriasis. Examples of the TLR4 gand include, but are not limited to, R-type or S-type lipopolysaccharide (LPS), monophosphoryl lipid A, lipid A (a component of LPS), Heat-killed Bacteria, paclitaxel (Paclitaxel). , LPS peptide, Fetuin-A or hyaluronic acid is used. These substances are commercially available and are easily available. For example, in the case of lipopolysaccharide, those provided by Sigma (for example, product number L3024), those provided by InvivoGen (for example, product number tlrl-eblps), those provided by Wako (for example, product number 125-05181), etc. Can be used. Two or more TLR4 ligands may be used in combination.

  In one aspect (first aspect) of the present invention, a TLR4 ligand is administered in the vicinity of the joint of a treatment animal to induce the pathological condition of arthritic psoriasis. Arthritic psoriasis is also called psoriatic arthritis.In addition to the various symptoms of psoriasis vulgaris (erythema that makes the skin red and swelling, small scales like Casabuta, and scales that peel off like dandruff), It is a type of psoriasis that is accompanied by inflammation, strength, and deformity of joints throughout the body and is accompanied by pain. In Japan, patients with arthritic psoriasis make up 6-8% of all psoriasis patients.

  Administration of TLR4 ligand near the joints of treated animals induces adherent inflammation and swelling and thickening are observed. The TLR4 ligand is administered by subcutaneous injection, intramuscular injection or the like to the foot (forefoot or hindpaw), preferably the sole of the foot, for reasons such as easy treatment and easy observation of symptoms. The dose of TLR4 ligand is adjusted to be an amount sufficient to induce the symptoms of arthritic psoriasis. Specifically, for example, the treatment animal used in the present invention is a mouse, and when administering lipopolysaccharide as a TLR4 ligand, for example, the dose per administration is 40 ng to 100 μg, preferably 100 ng to 50 μg, More preferably, it is 300 ng to 3 μg. If the dose is too small, a sufficient induction effect cannot be obtained. On the contrary, if the dose is too large, unnecessary irritation is applied, which is not preferable.

  Typically, the adherent part is administered by two or more administrations (for example, 2 to 20 times, preferably 2 to 10 times) at time intervals (for example, half a day to 3 days, preferably 1 to 2 days). Induces flames.

  In another aspect (second aspect) of the present invention, the TLR4 ligand is administered subcutaneously or intradermally to the treated animal to induce the pathological condition of pustular psoriasis. Pustular psoriasis is characterized by skin lesions associated with pus (genesis), generalized ones with systemic symptoms such as fever, and severe cases with the risk of death. In Japan, pustular psoriasis patients make up about 1% of all psoriasis patients.

  By administering the TLR4 ligand subcutaneously or intradermally to the treated animals, pustule formation is observed. A TLR4 ligand is administered to the back or abdomen for reasons such as easy treatment and easy observation of symptoms. Preferably, the back is the administration site. Usually, prior to the administration of TLR4 ligand, the hair at the administration site and its periphery is removed. The dose of TLR4 ligand is set to be an amount sufficient to produce pustules. Specifically, for example, the treatment animal used in the present invention is a mouse, and when administering lipopolysaccharide as a TLR4 ligand, for example, the dose per administration is 50 ng to 200 μg, preferably 100 ng to 100 μg, It is more preferably 1 μg to 20 μg. If the dose is too small, a sufficient induction effect cannot be obtained. On the contrary, if the dose is too large, unnecessary irritation is applied, which is not preferable.

  Typically, pustules are removed by two or more administrations (for example, 2 to 20 times, preferably 3 to 10 times) at time intervals (for example, half to 3 days, preferably 1 to 2 days). Induce formation. The administration schedule of lipopolysaccharide, in which 1 μg to 20 μg (preferably 3 μg to 10 μg) is administered once a day, is important as a condition capable of inducing pustules in IL36RN gene-deficient mice, in comparison with wild-type mice. Is. In addition, as shown in Examples described later, it was revealed by the study of the present inventors that, depending on the conditions, pustules are caused by administration of TLR4 ligand even in wild-type mice (see Examples).

2. Use of psoriasis model animal (second aspect of the invention)
The second aspect of the present invention relates to the use of the psoriasis model animal obtained by the production method of the present invention (hereinafter, also referred to as “the psoriasis model animal of the present invention”). As a typical application, a method for screening a substance effective for preventing or treating (ameliorating) psoriasis, that is, a drug for psoriasis is provided. The following steps are performed in the screening method of the present invention. In the present specification, the term "medicament for psoriasis" refers not only to drugs used for the purpose of improving or curing the symptoms of patients who have psoriasis, but also to those who may develop psoriasis. On the other hand, it is also used as a term that includes a drug used prophylactically (including the purpose of preventing recurrence). Thus, the drug selected by using the screening method of the present invention can be used for the purpose of preventing or treating (ameliorating) psoriasis.
(1) Step of administering a test substance to a psoriasis model animal obtained by the production method of the present invention (2) Step of determining a preventive or therapeutic effect of the test substance

  In step (1), a test substance is administered to the psoriasis model animal of the present invention. Examples of the test substance administration method include application to the skin, intracutaneous, subcutaneous, intravenous, intraarterial, intramuscular or intraperitoneal injection, oral administration, spray administration, nasal administration, and transtracheal administration. .

  The timing of administration of the test substance (timing of administration) is not particularly limited. For example, the test substance is administered before, at the same time as, or after administration of the TLR4 ligand for producing a psoriasis model animal. The test substance may be administered multiple times. In that case, the dose for each administration may be the same or different.

  As test substances, organic compounds of various molecular sizes (nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosyl glycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, etc.) ) Or an inorganic compound can be used. The test substance may be of natural origin or synthetic. In the latter case, an efficient screening system can be constructed by using, for example, a combinatorial synthesis method. An existing drug (eg, anti-cytokine antibody, anti-rheumatic drug, steroid drug) may be used as the test substance. In addition, cell extracts, culture supernatants and the like may be used as test substances.

In step (2) following step (1), the preventive or therapeutic effect of the test substance is determined. A test substance determined to have a preventive or therapeutic effect is a potential drug candidate. When an arthritic psoriasis model animal is used as a psoriasis model animal (that is, when the production method of the first aspect of administering a TLR4 ligand in the vicinity of a joint to induce adherent part inflammation is applied), Judgment is performed using an evaluation index according to the pathological condition of arthritic psoriasis, and a substance effective for prevention or treatment of arthritic psoriasis is screened. Examples of specific indicators include the presence or degree of swelling / thickness, the time until swelling / thickness is observed, the amount of inflammatory cytokines, the ratio of various inflammatory cells to the total inflammatory cells in the lesion and regional lymph nodes, Or the number of various inflammatory cells and the weight of the spleen. By macroscopic observation, microscopic observation of tissue (pathological tissue), measurement of joint thickness, immunological detection, flow cytometry, radiography, CT, MRI, immunohistochemistry, ELISA, real-time PCR, weight measurement, etc. , It is possible to evaluate using these indicators. The determination may be made by using two or more indexes in combination. Usually, the more reliable the determination result is, the more the number of adopted indexes. Specific examples of the judgment criteria are shown below.
(i) If swelling / thickness is no longer observed or the degree of swelling / thickness is reduced by administration of the test substance (if swelling / thickness is suppressed or ameliorated), preventive effect or treatment of the test substance Determined to be effective.
(ii) If administration of the test substance delays the time when swelling / thickness is observed, it is determined that the test substance has a preventive effect.

When a pustular psoriasis model animal is used as a psoriasis model animal (that is, when the method for producing the second embodiment in which a TLR4 ligand is subcutaneously or intradermally administered to induce a pustule is applied), pustular psoriasis Judgment is performed using an evaluation index according to the pathological condition, and a substance effective for the prevention or treatment of pustular psoriasis is screened. Examples of specific indicators include the presence or extent of pus, the time until pus is observed, the presence or extent of erythema, the time until erythema is observed, the amount of inflammatory cytokines, the inflammation of lesions and regional lymph nodes. It is the ratio of various inflammatory cells to the whole cells, or the number of various inflammatory cells and the weight of the spleen. By macroscopic observation, microscopic observation of tissue (pathological tissue), measurement of size of lesion (pus, erythema) site, immunological detection, flow cytometry, immunohistochemistry, ELISA, real-time PCR, weight measurement, etc. Evaluation using indicators is possible. The determination may be made by using two or more indexes in combination. Usually, the more reliable the determination result is, the more the number of adopted indexes. Specific examples of the judgment criteria are shown below.
(i) If pus formation is not observed or the degree of pus is reduced by administration of the test substance, it is determined that the test substance has a preventive or therapeutic effect.
(ii) If administration of the test substance delays the time when pus formation is observed, it is determined that the test substance has a preventive effect.
(iii) If the formation of erythema is not observed or the degree of erythema is reduced by the administration of the test substance, it is determined that the test substance has a preventive or therapeutic effect.
(iv) If administration of the test substance delays the time when erythema formation is observed, it is determined that the test substance has a preventive effect.

  By the way, there is a report that IL-36 is involved in psoriasis vulgaris as in pustular psoriasis (Non-patent Document 3 above). In the present application, it is shown that IL-36 plays an important role in the pathogenesis of psoriatic arthritis (see the section "Means for Solving Problems"). In view of these reports and results, the screening method using the pustular psoriasis model animal or the arthritic psoriasis model animal as the psoriasis model animal is also useful as a means for searching for an effective substance for the prevention or treatment of psoriasis vulgaris. Can be said. Therefore, it is also envisaged to utilize the screening method characterized by the above steps (1) and (2) as a means for identifying a drug for the prevention or treatment of psoriasis vulgaris.

On the other hand, as mentioned above, pustule formation was observed when TLR4 ligand was administered to wild-type mice (see also Examples). In view of this fact, the present application also provides a method for screening a drug for the prevention or treatment of pustular psoriasis, which comprises the following steps (i) and (ii).
(I) a step of administering a Toll-like receptor 4 ligand to a wild-type mouse and administering a test substance (ii) a step of determining the preventive or therapeutic effect of the test substance

  In the screening method of the present invention, preferably, a model animal group to which the test substance is administered (test group) and a model animal group (control group) under the same conditions except that the test substance is not administered are prepared. Then, the test group and the control group are compared with respect to the adopted index, and the determination in step (2) is performed based on the result. As a result of the comparison, for example, if the test group has milder symptoms than the control group, or if the test group has a slower onset or formation of symptoms than the control group, significant relief of symptoms is observed in the test group. In some cases, if the test group shows a therapeutic or prophylactic effect, it can be determined that the test substance is a strong drug candidate. By thus comparing the group to which the test substance is administered (test group) and the group not to be administered (control group), the efficacy of the test substance can be easily determined with high reliability. Incidentally, the prepared multiple model animals are first divided into a test group and a control group, and TLR4 ligand may be administered to each group to induce the pathology, or to the prepared multiple model animals. The TLR4 ligand may be administered to induce the pathological state, and then the test group and the control group may be divided.

  The number of individuals included in the test group and the control group is not particularly limited. Generally, the larger the number of individuals used, the more reliable results are obtained, but handling a large number of individuals at the same time is difficult in terms of securing the individuals to be used and manipulating (including raising). Therefore, for example, the number of individuals included in each group is set to 1 to 50, preferably 2 to 30, and more preferably 5 to 20.

  When the compound selected by the screening method of the present invention has sufficient drug efficacy against the target disease (arthritic psoriasis, pustular psoriasis, or psoriasis vulgaris), the compound is directly used as an active ingredient of the drug. Can be used. On the other hand, when the compound does not have a sufficient drug effect, the compound can be used as an active ingredient of a drug for a target disease after modification by chemical modification or the like to enhance the drug effect. Of course, even when the drug has a sufficient drug effect, the same modification may be performed for the purpose of further increasing the drug effect.

  Matters not specifically mentioned in the present specification (conditions, operating methods, etc.) may follow conventional methods, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (Eedited by Frederick M. Ausubel et al., 1987), Current protocols in Immunology, John Wiley & Sons Inc, etc. can be referred to.

  With the aim of developing a therapeutic method for arthritic psoriasis and pustular psoriasis, we attempted to create a model animal that reproduces these pathological conditions.

1. Materials / Methods (1) Preparation of IL36RN (Il1f5) gene knockout mouse
A conventional Il1f5 (corresponding to human IL36RN gene) gene knockout mouse is prepared using ES cells. HK3i cells (C57BL / 6N strain) were used as ES cells. The structure of the used plasmid (targeting vector) and the target site are shown in FIG. A region (SEQ ID NO: 3) extending from a part of exon 1 (Ex1) of the Il1f5 gene to a part of exon 3 (Ex3) was replaced with Pr Neo pA (SEQ ID NO: 4).

(2) TLR4 ligand (agonist)
In this experiment, lipopolysaccharide (LPS) (product name Lipopolysaccharides from Escherichia coli O111: B4, SIGMA-ALDRICH) was used.

(3) Mouse strain C57BL / 6N strain mice were used in this experiment.

(4) Induction of arthritis / pustules In order to induce arthritis / pustules, LPS is administered near the joints of IL36RN (Il1f5) gene homo-deficient mice or to the soles of the hind legs as follows.
(Dose)
Arthritis: 20 μl at a concentration of 2 μg / ml to 2 mg / ml
Pustule: 50 μl at a concentration of 2 μg / ml to 2 mg / ml
(Administration schedule)
Arthritis: once a day for 3 days Pustules: once a day for 1-5 days

2. Results (1) Induction of arthritis Fighting multiple male mice in the same cage often occurs. Arthritis was caused by fighting in the IL36RN gene homo-deficiency mouse and hetero-deficiency mouse (this phenomenon was not observed in WT mice). As a mechanism of this phenomenon, it is considered that innate immunity is activated from infection associated with trauma and arthritis similar to arthritic psoriasis may occur, and therefore a TLR4 agonist is locally administered near the joint of IL36RN gene homo-deficient mice. This attempted to induce arthritis. First, LPS, which is a TLR4 ligand (agonist), was administered at a dose of 40 ng to 40 μg (volume 20 μl) in the vicinity of the joints of the hind legs of 8 to 12 weeks old, male wild type (WT) mice and IL36RN gene homo-deficient mice. Subcutaneous injection was performed once a day for 3 days. Then, 6 hours after the final administration, the thickness of the hindpaw was measured. As a result, swelling and thickening of the hindpaw were observed in the IL36RN gene homo-deficient mouse at a LPS of 300 ng or more, as compared with the WT mouse (FIGS. 2 and 3).

(2) Reproduction of pustule
LPS of 100 ng to 100 μg (volume 50 μl) was administered once daily for 1 to 5 days to the shaved back skin of 8 to 12 week old male wild type (WT) mice and IL36RN gene homo-deficient mice. It was injected subcutaneously and the skin condition was observed. In the 50 μg-administered group, significant erythema and pustules were observed in both WT and IL36RN gene homo-deficient mice after 5 days of administration (FIG. 4). Even in the group administered with 3 μg, marked erythema / pustules were observed in Il36RN gene-deficient mice, and erythema / pustules were also observed in the wild type, albeit slightly.

  On the other hand, 1 day before the administration, intraperitoneal administration of 60 μg of anti-IL-1b antibody (Biolegend: product number 503502) reduced erythema and pustules and reduced skin symptoms (FIG. 5).

  As a further experiment, when the relationship between dose and phenotype was examined, administration of LPS at 10 μg (once) resulted in predominant appearance of erythema and pustule from the next day in IL36RN gene homo-deficient mice. In addition, 3 μg of LPS was subcutaneously injected into the back every day, and erythema and pustule appeared predominantly homozygously from the second day after injection. From these results, it can be said that the administration of 3 μg to 10 μg (once a day) is a condition for inducing lesions in IL36RN gene-deficient mice, as compared with wild-type mice.

3. Consideration
We succeeded in inducing arthritis similar to arthritic psoriasis, that is, producing a model of arthritic psoriasis, by locally administering TLR4 ligand near the joint of IL36RN gene homo-deficient mouse. In addition, we succeeded in reproducing pustular lesions, that is, creating a pustular psoriasis model by locally administering TLR4 ligand to the skin of IL36RN gene homo-deficient mice. These psoriasis models are useful experimental animals for elucidating the pathological conditions of arthritic psoriasis and pustular psoriasis, or for developing therapeutic agents. On the other hand, the above experiment revealed that IL-36 is involved in the pathogenesis of arthritic psoriasis, which is different from the established theory established in the conventional arthritis model. This fact has important implications for future treatment strategies. In addition, it is also important to utilize the model animals that have been successfully produced.

  The model animal of the present invention reproduces the pathology of arthritic psoriasis or pustular psoriasis. A model animal that reproduces the pathophysiology of arthritic psoriasis can be used for the development of drugs and therapeutic methods for arthritic psoriasis, and for elucidation of the pathogenic mechanism of arthritic psoriasis. Similarly, a model animal that reproduces the pathological condition of pustular psoriasis is also useful for developing drugs and elucidating the pathogenic mechanism. Therefore, the present invention is expected to make a great contribution to the establishment of a treatment method for intractable disease arthritic psoriasis / pustular psoriasis.

  The present invention is not limited to the description of the embodiments and examples of the invention. Various modifications are also included in the present invention within a range that can be easily conceived by those skilled in the art without departing from the scope of the claims. The contents of papers, published patent publications, patent publications, and the like, which are specified in the present specification, are incorporated by reference in their entirety.

Claims (10)

A psoriasis model animal exhibiting a pathological condition of pustular psoriasis , which comprises the step of subcutaneously or intradermally administering a Toll-like receptor 4 ligand to a transgenic non-human animal deficient in the IL36RN gene encoding an interleukin 36 receptor antagonist. Of manufacturing.   The production method according to claim 1, wherein the transgenic non-human animal lacks a homozygous IL36RN gene encoding an interleukin 36 receptor antagonist.   The production method according to claim 1, wherein the transgenic non-human animal is a rodent.   The production method according to claim 3, wherein the rodent is a mouse.   The production method according to claim 1, wherein the ligand is lipopolysaccharide. It is a site of administration back or abdomen of the ligand, the method of preparation according to any one of claims 1-5. A psoriasis model animal exhibiting the pathological condition of pustular psoriasis, which is obtained by the production method according to any one of claims 1 to 6 . A method for screening a drug for the prevention or treatment of pustular psoriasis , which comprises the following (1) and (2):
(1) step of administering claims obtainable by the manufacturing method according to any one of claims 1 to 6, a test substance to psoriasis model animals exhibiting the pathology of pustular psoriasis;
(2) A step of determining the preventive or therapeutic effect of the test substance. The screening method according to claim 8 , wherein the administration of the test substance in step (1) is performed before the administration of the ligand of Toll-like receptor 4 for producing the psoriasis model animal. The screening method according to claim 8 or 9 , wherein the determination in step (2) is performed based on the comparison with the control group.

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