JP2012019768A - Jmjd3 gene-modified non-human mammal, jmjd3 gene-modified bone marrow chimera non-human mammal, and utilization thereof - Google Patents

Abstract

Jmjd3 gene-modified non-human useful for screening for substances that control differentiation into M2 macrophages, screening for therapeutic agents for M2 macrophage-related diseases such as response to parasitic infection, wound healing, allergy, cancer, arteriosclerosis, etc. Provided are screening methods for mammals, Jmjd3 gene-modified bone marrow chimeric non-human mammals, and substances that control differentiation of Jmjd3 gene-modified bone marrow cells, Jmjd3 gene-modified macrophages, and M2 macrophages derived from these animals.
A Jmjd3 gene-modified non-human mammal in which a Jmjd3 gene in the genome is modified.
[Selection figure] None

Description

  The present invention relates to a non-human mammal having a modified Jmjd3 gene, a bone marrow chimeric non-human mammal having a modified Jmjd3 gene, and use thereof.

  Innate immunity is a biological defense mechanism that plays an important role in the initial recognition of infectious pathogens such as bacteria, viruses, and parasites, the subsequent induction of inflammatory responses, and the induction of acquired immunity. In recent years, it has become clear that innate immunity is also important in arteriosclerosis, antitumor immunity and the like. Macrophages, which are the cells responsible for the core of innate immunity, express pattern recognition receptors (PRRs) that recognize structures (PAMPs) unique to pathogens, and activation signals are transmitted through these PRRs. The Toll-like receptor (TLR) family functions as PRRs that recognize PAMPs (lipopolysaccharides, lipoproteins, nucleic acids, etc.) of various pathogens, and is one of the most well-studied members of the PRPs family It is. TLRs play an important role in eliciting natural responses to infection by various pathogens that result in the production of inflammatory mediators including inflammatory cytokines, chemokines, interferons and the like.

  In the formation of macrophages, it has been found that the production of inflammatory cytokines in infection and autoimmune diseases is important for pathogenesis. In addition, it is known that macrophages functionally differentiate into M1 macrophages and M2 macrophages in response to infection with microorganisms and the like.

  M1 macrophages are important for bacterial and viral infection responses. M2 macrophages are macrophages also called selectively activated macrophages and are involved in the development of parasitic infection responses, wound healing, cancer and arteriosclerosis. M2 macrophages are selective activation markers arginase 1 (Arg1), chitinase-like Ym1 (Chi313), found in inflammation zone 1 (also known as Fizzl, Retnla), mannose receptor (also known as MR, CD206) Is characterized by high expression of chemokines such as CCL17.

  As described above, M2 macrophages are involved in the parasitic infection response, wound healing, and progression of cancer and arteriosclerosis. Therefore, if M2 macrophage differentiation can be regulated, prevention or improvement of diseases or symptoms involving M2 macrophages is possible. Alternatively, it is considered effective for treatment. It has been reported that M2 macrophages are induced when macrophages are stimulated with interleukin 4 (IL4) or interleukin 13 (IL13) (Non-patent Documents 1 and 2). However, the mechanism of differentiation into M2 macrophages has not yet been clarified. For this reason, an evaluation system such as a screening method for substances that regulate differentiation into macrophages M2 has not yet been developed.

Mantovani, A. et al., Trends Immunol 23, 549-555 (2002). Gordon, S., Nat Rev Immunol 3, 23-35 (2003).

  In view of the above problems, the present invention is useful for screening for substances that control differentiation into M2 macrophages, screening for therapeutic agents for M2 macrophage-related diseases such as parasitic infection response, wound healing, allergy, cancer, arteriosclerosis, and the like. Jmjd3 gene-modified non-human mammals, Jmjd3 gene-modified bone marrow chimeric non-human mammals, Jmjd3 gene-modified bone marrow cells derived from these animals, Jmjd3 gene-modified macrophages, screening methods for substances that control differentiation of M2 macrophages, etc. The issue is to provide.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems, prepared Jmjd3 gene-modified mice and Jmjd3 gene-modified bone marrow chimeric mice, and obtained these findings by analyzing these mice. The Jmjd3 gene homo-modified mouse prepared by the present inventors (homock-out mouse: Jmjd3 ^{− / −} mouse) died immediately after birth due to a lung injury. Hepatocyte transplantation was performed to prepare a bone marrow chimeric mouse having Jmjd3 gene homo-modified bone marrow cells, and Jmjd3 gene homo-modified bone marrow-derived cells (Jmjd3 gene homo-modified macrophages) were obtained. When the role of Jmjd3 protein in the obtained Jmjd3 gene homo-modified macrophage (Jmjd3-deficient macrophage) was examined, the following findings were obtained. In the present specification, a protein encoded by the Jmjd3 gene is also referred to as a Jmjd3 protein.
(i) No abnormalities were observed in Jmjd3-deficient macrophages in differentiation into M1 macrophages and antibacterial response.
(ii) Jmjd3-deficient macrophages were found to have significant obstacles to differentiation into parasite Nippostrongylus brasiliensis and parasite component chitin stimulation to M2 macrophages. In addition, the differentiation of macrophages derived from Jmjd3 gene homo-modified bone marrow into M2 macrophages was also impaired, and this was recovered by expressing the histone demethylase region having the enzyme activity of Jmjd3 protein in macrophages.
(iii) As a result of ChIP-Seq analysis, it was revealed that Jmjd3 protein controls histone demethylation in the promoter region of IRF4 gene.
(iv) IRF4-deficient mice were also impaired in differentiation into M2 macrophages, similar to Jmjd3-deficient mice.
(v) From the above results, it was revealed that Jmjd3 protein is an essential molecule for differentiation into M2 macrophages and antiparasitic response. Jmjd3 protein has an enzyme region that demethylates 27th lysine (H3K27) of histone H3 (amino acid residues 1141-1164). This enzyme activity is important for differentiation into M2 macrophages. It became clear that there was. It was also revealed that Jmjd3 protein regulates gene expression of IFN regulatory factor 4 (IRF4), and that expression of IRF4 is important for differentiation into M2 macrophages.
(vi) M2 macrophages are known to be involved in various diseases such as parasite infection response, wound healing, cancer progression, arteriosclerosis, and metabolic syndrome. Therefore, Jmjd3 gene-modified non-human mammal, Jmjd3 gene-modified bone marrow chimeric non-human mammal and Jmjd3 gene-modified macrophage are useful models in M2 macrophage-related disease research.
For example, screening for substances that activate Jmjd3 protein or Jmjd3 protein target molecule using Jmjd3 gene-modified animals (monitoring the expression of M2 macrophage-related molecules), parasitic infection response, allergic response, promotion of wound healing It is possible to develop therapeutic drugs that are useful for In addition, a drug that suppresses demethylase activity of Jmjd3 protein suppresses differentiation into M2 macrophages, and is useful for suppression of cancer metastasis, arteriosclerosis, and the like.

The present invention has been completed based on the above findings, and provides the following inventions.
Item 1. A Jmjd3 gene-modified non-human mammal, wherein the Jmjd3 gene in the genome is modified.
Item 2. Item 2. The non-human mammal according to Item 1, which is a Jmjd3 gene homo-modified animal in which both alleles in the Jmjd3 gene are inactivated.
Item 3. A Jmjd3 gene-modified bone marrow chimeric non-human mammal having the Jmjd3 gene homo-modified bone marrow cells derived from the non-human mammal of Item 2 in the bone marrow.
Item 4. Item 4. The non-human mammal according to any one of Items 1 to 3, wherein the non-human mammal is a mouse or a rat.
Item 5. Item 5. The non-human mammal according to any one of Items 1 to 4, wherein the non-human mammal is a mouse.
Item 6. Item 6. A Jmjd3 gene-modified bone marrow cell derived from the non-human mammal according to any one of Items 1 to 5.
Item 7. Item 7. A non-human mammal according to any one of Items 1 to 5 or a bone marrow cell-derived Jmjd3 gene-modified macrophage according to Item 6.
Item 8. (I) a step of administering a candidate substance to the non-human mammal according to any one of Items 1 to 5, (II) (a) expression of M2 macrophages and / or markers of M2 macrophages in the non-human mammals, Or (b) monitoring the activity of the Jmjd3 protein and / or Jmjd3 protein target molecule, and (III) (a) M2 macrophages and / or M2 macrophages in step (II) as compared to the case where no candidate substance is administered Or (b) selecting a candidate substance in which increased activity of the Jmjd3 protein and / or Jmjd3 protein target molecule is detected, and promoting the differentiation into M2 macrophages Agent screening method.
Item 9. (I) a step of bringing a candidate substance into contact with the Jmjd3 gene-modified macrophage according to Item 7, (II) (a) differentiation of the Jmjd3 gene-modified macrophage into M2 macrophage, (b) expression of a marker of M2 macrophage, or (c ) Monitoring the activity of the Jmjd3 protein and / or Jmjd3 protein target molecule in Jmjd3 gene-modified macrophages, and (III) differentiation into M2 macrophages in step (II) compared to the case where no candidate substance is contacted And (b) selecting a candidate substance in which increased expression of a marker of M2 macrophage is detected, or (c) increased activity of a target molecule of Jmjd3 protein and / or Jmjd3 protein is selected. A screening method for an agent for promoting differentiation into M2 macrophages.
Item 10. (I) a step of bringing a candidate substance into contact with the Jmjd3 gene and / or Jmjd3 protein in vitro or in a non-human mammal, and (II) a step of monitoring changes in Jmjd3 gene expression and / or demethylase activity of the Jmjd3 protein. And (III) selecting a candidate substance in which a change in Jmjd3 gene expression and / or demethylase activity is detected in step (II) as compared with the case where the candidate substance is not contacted, A screening method for a substance that controls differentiation of M2 macrophages.

  According to the present invention, a useful model animal or the like in M2 macrophage-related disease research is provided. According to the present invention, since a substance involved in the control of M2 macrophages can be screened, prevention of diseases or symptoms involving M2 macrophages such as parasitic infection response, wound healing, allergy, cancer, arteriosclerosis, metabolic syndrome, Drugs and the like effective for improvement or treatment can be developed.

FIG. 1 is a schematic diagram of the mouse Jmjd3 gene (top), targeting vector (center), and target allele. FIG. 2a is a diagram showing the results of Southern blot analysis of pups obtained from heterozygous outcrossing, and FIG. 2b shows wild-type (WT) and Jmjd stimulated with LPS (1 μg / ml) for a certain period of time. ^-/- It is a figure which shows the RT-PCR analysis result of RNA derived from MEFs. FIGS. 3 (a) and 3 (b) are diagrams showing H & E staining of lung sections of wild-type mice (WT) (FIG. 3 (a)) and Jmjd3 ^{− / −} mice (FIG. 3 (b)). FIGS. 4a and 4b show stimulation of endogenous peritoneal exudate cells (PEC) from wild type mice (WT) and Jmjd3 ^{− / −} BM chimeric mice with each TLR ligand to produce TNF (FIG. 4a) and IL-6. FIG. 4c and FIG. 4d show the results of measurement of the production (FIG. 4b) of thioglycolic acid-induced PECs derived from wild type (WT) and Jmjd3 ^{− / −} BM chimeric mice with each TLR ligand. The results of TNF production (FIG. 4c) and IL-6 production (FIG. 4d) measured by ELISA are shown. FIG. 5 is taken from WT and Jmjd3 ^{− / −} chimeric mice 3 days after treatment with no stimulation (FIG. 5 (a)), thioglycolic acid (FIG. 5 (b)) or Listeria monocytogenes (FIG. 5 (c)) It is a figure which shows the result of having analyzed the macrophage in the peritoneal exudate cell (PEC) which carried out. Figures 6 (a)-(d) show no stimulation (Figure 6 (a)), WT treated with thioglycolic acid (Figure 6 (b)) or Listeria monocytogenes (Figures 6 (c) and 6 (d)). FIG. 4 shows the results of examining the number of F4 / 80 ⁺ CD11b ⁺ macrophages in PEC collected from Jmjd3 ^{− / −} chimeric mice. FIGS. 7 (a)-(d) show TNF (FIG. 7 (a)), IFN-γ (FIG. 7 (b)), IL-6 in sera of WT and Jmjd3 ^{− / −} chimeric mice infected with Listeria monocytogenes. (FIG. 7 (c)) and IL-12p40 (FIG. 7 (d)) concentrations. FIGS. 8 (a)-(d) show TNF (FIG. 8 (a)), iNOS (FIG. 8 (b)), IL-6 (FIG. 8 (c)) and PEC of mice infected with Listeria monocytogenes. It is a figure which shows the result of having investigated the expression of IL-12p40 (FIG.8 (d)) by Q-PCR analysis. FIGS. 9 (a) to (d) show CD11b and F4 / 80 of PEC collected from WT and Jmjd3 ^{− / −} chimeric mice two days after injection of chitin into the peritoneal cavity (FIGS. 9 (a) and 9 (b)). ) Or CD11b and Siglec-F (FIG. 9 (c) and FIG. 9 (d)), and shows the results of analysis by flow cytometry. 9 (a) and 9 (c) are WT, and FIGS. 9 (b) and 9 (d) are Jmjd3 ^{− / −} chimeras. Figures 10a and b show the number of F4 / 80 ⁺ CD11b ⁺ macrophages (Figure 10a) and Siglec-F ⁺ neutrophils in PEC taken from WT and Jmjd3 ^{− / −} chimeric mice 2 days after chitin injection into the peritoneum FIG. 10 is a diagram showing the number of (FIG. 10b). FIGS. 11 (a) and (b) are diagrams showing the results of Diff-Quick staining of PEC cell types derived from chitin-treated mice with cytospin smears (FIG. 11 (a): wild type (WT), FIG. 11 (b): Jmjd3 ^{− / −} ). FIG. 12 is a view showing the results of analyzing the level of surface MR expression of peritoneal F4 / 80 ⁺ CD11b ⁺ macrophages derived from chitin-treated mice by flow cytometry. FIGS. 13 (a) to (e) show arginase 1 (FIG. 13 (a)), Ym1 (FIG. 13 (b)), Fizz1 (FIG. 13 (c) in total RNA prepared from PEC 48 hours after chitin administration. )), MR (FIG. 13 (d)) and iNOS (FIG. 13 (e)) mRNA expression by Q-PCR. Figures 14 (a) and (b) show BAL fluid extracted from WT and Jmjd3 ^-/- fetal liver chimeric mice 13 days after Nb infection, and macrophages and eosinophils were measured on cytospin smears stained with Diff-quik. FIG. 14 (a): wild type (WT), FIG. 14 (b): Jmjd3 ^{− / −} chimeric mouse). FIGS. 15 (a) and (b) show macrophages in BAL derived from WT (n = 7) and Jmjd3 ^{− / −} (n = 7) chimeric mice 5 and 13 days after Nb infection (FIG. 15 (a)). ) And the total cell number of eosinophils (FIG. 15 (b)). FIGS. 16 (a) to (h) show arginase 1, Ym1, Fizz1, and 5% of total RNA prepared from infected and non-infected WT and infected and non-infected Jmjd3 ^{− / −} chimeric mice, respectively, 5 days after Nb infection. It is a figure which shows the expression level of mRNA which codes each protein of MR, iNOS, L-4, IL-13, and eotaxin (FIG. 16 (a): Arginase 1, FIG. 16 (b): Ym1, FIG. ): Fizz1, FIG. 16 (d): MR, FIG. 16 (e): iNOS, FIG. 16 (f): IL-4, FIG. 16 (g): IL-13, FIG. 16 (h): eotaxin). FIG. 17 shows intracellular IL-4 and IFN− in T cells when hilar lymph node cells collected from WT and Jmjd3 ^{− / −} chimeric mice 9 days after Nb infection were stimulated with CD3 and CD28 and stained with antibodies. It is a figure which shows the typical result of (gamma) dyeing | staining. FIG. 18 is a diagram showing the number of IL-4 producing cells in the hilar 9 days after Nb infection. Figures 19a-d stimulate WT and Jmjd3 ^-/- GM-BMM with specific TLR ligands and TNF (Figure 19a and 19c) and IL-6 (Figures 19b and 19d) in the culture supernatant by ELISA. It is a figure which shows the result of having measured. 20 (a)-(e) show the expression of mRNA encoding each protein of arginase 1, Ym1, Fizz, MR and IL-13 in total RNA prepared from WT and Jmjd3 ^{− / −} M-BMM. FIG. 20 shows results determined by PCR analysis (FIG. 20 (a): arginase 1, FIG. 20 (b): Ym1, FIG. 20 (c): Fizz1, FIG. 20 (d): MR and FIG. 20 (e ): IL-13). Figures 21 (a) and (b) show arginase 1 (Figure 21 (a)) and Fizz1 in total RNA prepared from WT and Jmjd3 ^-/- M-BMM stimulated with IL-4 (10 ng / ml). FIG. 22 is a diagram showing the results of determining the expression of (FIG. 21 (b)) by Q-PCR analysis. FIG. 22 is derived from Jmjd3 ^{− / −} BM cells expressing the C-terminal part of JMJD3 containing the JmjC domain (aa 1141-1641) or its iron binding deficient mutant (ΔFe ²⁺ binding deficient mutant) (A1388H). It is a figure which shows the result of having analyzed the expression of jmjd3 and Bactin in prepared M-BMM by RT-PCR. FIG. 23 (a)-(c) shows Jmjd3 ^{− / −} BM infected with a retrovirus expressing wild type JMJD3 (aa 1141-1641) or its iron binding deficient mutant (ΔFe ²⁺ binding deficient mutant). FIG. 24 shows the results of Q-PCR analysis of the expression of arginase 1 (FIG. 23 (a)), Ym1 (FIG. 23 (b)) and MR (FIG. 23 (c)) mRNA in M-BMMs generated from CHO . FIGS. 24 (a) and (b) are diagrams showing the results of subjecting a chromatin solution derived from WT and Jmjd3 ^{− / −} BMM to ChIP analysis using an anti-H3K27me3 antibody (FIG. 24 (a): WT, FIG. 24 (b): Jmjd3 ^{− / −} ). FIG. 25 shows the expression level of Irf4 in Jmjd3 ^{− / −} M-BMM reconstituted with wild-type JMJD3 (aa 1141-1641) or a retrovirus having an iron binding deficiency mutant thereof (ΔFe ²⁺ binding deficiency mutant). It is a figure which shows the result determined by Q-PCR analysis. FIGS. 26 (a) to (f) are diagrams showing the results of Q-PCR examining the expression of genes encoding M2 marker and JMJD3 in BM cells derived from WT and Irf4 ^{− / −} mice (FIG. 26). (a): Arginase 1, FIG. 26 (b): Ym1, FIG. 26 (c): Fizz1, FIG. 26 (d): MR, FIG. 26 (e): IL-13, FIG. 26 (f): JMJD3). FIG. 27 shows the number of M-BMM cells obtained from WT and Irf4 ^{− / −} mice. FIGS. 28 (a) and 28 (b) are the results of analyzing the recruitment of macrophages and eosinophils in PECs obtained from WT and Irf4 ^{− / −} mice administered intraperitoneally with chitin (FIG. 28 (a): WT, FIG. 28 (b): Irf4 ^{− / −} mice). FIGS. 29 (a) and (b) are the numbers of chitin-induced macrophages and chitin-induced eosinophils in PEC (FIG. 29 (a): chitin-induced macrophages, FIG. 29 (b): chitin-induced eosinophils). . FIG. 30 shows MR expression in macrophages during chitin-induced PEC. FIGS. 31 (a)-(d) show the results of subjecting RNA prepared from PEC obtained from chitin-treated WT and Irf4 ^{− / −} mice to Q-PCR analysis for expression of specific M2 marker genes. FIG. 31 (a): arginase 1, FIG. 31 (b): Ym1, FIG. 31 (c): Fizz1, FIG. 31 (d): MR). 32 (a) to (f) show the results of determining the expression of a specific gene by Q-PCR analysis in Jmjd3 ^{− / −} M-BMM in which IRF4 is ectopically expressed using a retrovirus. FIG. 32 (a): Arginase 1, FIG. 32 (b): Ym1, FIG. 32 (c): Fizz1, FIG. 32 (d): MR, FIG. 32 (e): JMJD3, FIG. 32 (f) : IRF4).

Hereinafter, the present invention will be described in detail.
The Jmjd3 gene modified non-human mammal of the present invention is a non-human mammal in which the Jmjd3 gene in the genome is modified.
The Jmjd3 gene is a gene widely present in mammals such as humans, dogs, rats, and mice, and encodes a protein having an enzyme region (Jmjd3 protein) that demethylates histone H3 lysine 27 residue (H3K27). It has been known. For example, the mouse Jmjd3 gene is registered with NCBI under the ID number NM_001017426. The rat Jmjd3 gene is registered with NCBI under ID number NM_001108829. The human Jmjd3 gene is registered with NCBI under the ID number NM_001080424. The amino acid sequence of mouse Jmjd3 protein is registered with NCBI under ID number NP_001017426.

  “Jmjd3 gene has been modified” means that part or all of the DNA region of Jmjd3 gene has been modified. A part or all of the DNA region of the Jmjd3 gene is modified means that a base has been added to a part of the genomic DNA of the Jmjd3 gene, or part or all of the genomic DNA of the Jmjd3 gene has been deleted or replaced. Or inversion in a specific range region. The Jmjd3 gene may be modified as long as the Jmjd3 gene is inactivated or deleted. In order to inactivate the Jmjd3 gene, two or more methods of addition, deletion or substitution may be used for the Jmjd3 gene. “Inactivation” means that the modified expression product of Jmjd3 gene does not function or does not exist.

  In the present invention, “addition” of a base to genomic DNA is a function of an expression product of Jmjd3 gene in which one or more bases are inserted into the genomic DNA sequence of Jmjd3 gene and DNA other than the Jmjd3 gene is inserted. It means not to exist or not to exist. “Deletion” of genomic DNA means that the expression product of Jmjd3 gene does not function or does not exist by deleting part or all of the genome of Jmjd3 gene. `` Replacement '' of genomic DNA means that part or all of the genome of the Jmjd3 gene gene is replaced with a separate sequence that is not related to the Jmjd3 gene so that the expression product of the Jmjd3 gene does not function or does not exist. Say.

  The non-human mammal of the present invention may be a Jmjd3 gene hetero-modified animal in which one of the alleles in the Jmjd3 gene is inactivated, or a Jmjd3 gene homo-modified animal in which both alleles in the Jmjd3 gene are inactivated. There may be. Preferably, it is a Jmjd3 gene homo-modified animal in which both alleles in the Jmjd3 gene are inactivated. As described later, homozygous genetically modified animals can be obtained by crossing one or more generations of Jmjd3 gene heteromodified animals.

  The kind of the non-human mammal of the present invention is not particularly limited, and examples thereof include mouse, rat, rabbit, guinea pig, dog, pig, sheep, goat and the like. Among these, a mouse or a rat is preferable because it is easy to handle and easy to reproduce, and a mouse is particularly preferable.

  The method for producing the Jmjd3 gene-modified non-human mammal of the present invention is not particularly limited, and can be produced by a known method for producing knockout animals using embryonic stem (ES) cells. For example, a step of introducing a gene into an ES cell using a targeting vector to modify the Jmjd3 gene, a step of producing a Jmjd3 gene hetero-modified animal derived from the ES cell, and mating the Jmjd3 gene hetero-modified animal for one generation or more Thus, there is a method including a step of obtaining a Jmjd3 gene homo-modified animal in which both alleles in the Jmjd3 gene are inactivated. This method is an example of the method for producing the Jmjd3 gene-modified non-human mammal of the present invention, and the method for producing the non-human mammal of the present invention is not limited to the following method. Moreover, a modification and / or a change can be appropriately added to the method shown below as necessary.

  In the method using ES cells, first, a targeting vector is prepared in vitro using gene recombination technology, and the Jmjd3 gene is destroyed by introducing the vector into ES cells. Among the ES cells into which the gene has been introduced, those in which the Jmjd3 gene has been disrupted are selected by a method such as Southern blotting. Next, the ES cell from which the Jmjd3 gene is disrupted is transferred into a blastocyst derived from a normal non-human mammal, and the resulting chimeric animal is mated with a normal animal to produce an ES cell-derived non-human mammal. be able to. This ES cell-derived non-human mammal is a Jmjd3 gene hetero-modified animal in which one of the alleles in the Jmjd3 gene is inactivated. Then, by crossing one or more generations of this Jmjd3 gene hetero-modified animal, a Jmjd3 gene homo-modified non-human mammal in which both Jmjd3 genes of a pair of chromosomes are modified can be obtained.

A method for producing the Jmjd3 gene-modified non-human mammal of the present invention by the above method is specifically described below.
(1) Preparation of targeting vector First, a DNA fragment containing a site to be mutated in the Jmjd3 gene of a mutant animal to be prepared is obtained from a DNA library. The DNA fragment is used to construct a targeting vector for homologous recombination of ES cells. For this DNA fragment, it is preferable to isolate and use DNA having the same sequence as the ES cell to be prepared so that recombination occurs more efficiently when homologous recombination is performed. For example, when a mouse is used as an animal into which mutations are introduced, a mouse DNA fragment capable of deleting the Jmjd3 gene is prepared from an ES cell-derived genomic DNA library.

  As a method for modifying the Jmjd3 gene, a normal site-specific gene mutation introduction method can be used. For example, the target mutation can be introduced using a method such as BAC recombineering (see Lee, E.C. et al. Genomics, vol. 73, p. 56-65 (2001)).

  A targeting vector is prepared by inserting a DNA fragment containing the modified Jmjd3 gene into the vector. The targeting vector preferably contains, as essential elements, a chromosomal DNA fragment into which a mutation has been introduced, a DNA fragment encoding a selection marker, a promoter for controlling transcription thereof, and a selection marker expression unit including a terminator. The chromosomal DNA fragment into which the mutation is introduced is a part necessary for causing homologous recombination in the ES cell, and the chromosomal DNA fragments before and after the part into which the mutation has been introduced are necessary. That is, the targeting vector has a DNA fragment different from the original chromosomal DNA only in the mutated base.

As the DNA fragment encoding the selectable marker, a drug resistance gene, a reporter gene and the like are preferable. Examples of drug resistance genes include neomycin phosphotransferase II (nptII) gene (neomycin resistance gene), hygromycin phosphotransferase (hpt) gene and the like, and reporter genes include, for example, green fluorescent protein (GFP) gene, β -Galactosidase (lacZ) gene, chloramphenicol acetyltransferase (cat) gene, etc. are mentioned. Herpes simplex virus thymidine kinase and the like are also preferably used as a selection marker.
As the promoter, CAG promoter, phosphoglycerate kinase-1 (PGK-1) promoter, elongation factor 2 (EF-2) promoter, MC-1 promoter and the like can be used.

  The vector serving as the basic skeleton of the targeting vector into which the DNA fragment containing the modified Jmjd3 gene is introduced is not particularly limited as long as it can self-replicate in a cell to be transformed (for example, DH5a). For example, commercially available pBluescript (manufactured by Stratagene), pZErO 1.1 (Invitrogen), pGEM-1 (Promega) and the like can be used.

(2) Introduction of vector into ES cells and selection of gene-introduced ES cells After introducing the prepared targeting vector into ES cells, the ES cells are cultured, and the appearing colonies are collected. As the ES cell, any of already established cell lines and newly established cell lines can be used. For gene introduction into ES cells, methods such as calcium phosphate coprecipitation method, electroporation method, lipofection method, retrovirus infection method, aggregation method, microinjection method, and particle gun method can be adopted. The electroporation method is preferable in that the cells can be treated.

  In ES cells into which a transgene has been incorporated, the Jmjd3 gene is usually destroyed by screening chromosomal DNA isolated from colonies obtained by culturing single cells on feeder cells by Southern hybridization or PCR. You can choose what you want. Alternatively, selection may be performed by using a vector containing a selection marker such as a drug resistance gene as described above.

(3) Production of transgenic non-human mammal
By transferring the ES cell in which the destruction of the Jmjd3 gene is confirmed into a blastocyst derived from the same kind of non-human mammal, the ES cell is incorporated into the cell mass of the host embryo to form a chimeric embryo. A chimeric transgenic animal can be obtained by transplanting this chimeric embryo to a foster parent and generating and growing it. For example, when a mouse is used, a blastocyst is separately taken out from a pregnant mouse, and the selected ES cell is transferred, and then introduced into the uterus of a female mouse subjected to pseudopregnancy. Then, choose a chimera from among the children born from temporary parents. In the case of a chimeric animal, for example, when an ES cell (recombinant ES cell) into which a targeting vector has been introduced is introduced into a blastocyst derived from another strain that has a distinct coat color from the strain from which the ES cell is derived Can be selected by hair color. For example, in the case of mice, C57BL / 6 is different from 129 mice in that various loci that have black hair color and can be used as markers for ES cells derived from 129 strains having agouti coat color. It is preferable to use a blastocyst such as a mouse. Thereby, the chimera mouse can judge the chimera rate from the hair color. A heterozygous animal with a modified Jmjd3 gene is obtained by mating a chimeric animal with a normal animal, extracting genomic DNA from the tail of a born pup, etc., and selecting a mutation introduced by the PCR method be able to.

  As a specific selection method, a part of the tail of the resulting pup is collected and chromosomal DNA is extracted. Using the extracted chromosomal DNA as a substrate, PCR is performed using two oligodeoxynucleotides having primers designed to sandwich the mutation site of the Jmjd3 gene as primers. Perform agarose gel electrophoresis of the PCR product, confirm the band containing mutations by hybridization using oligodeoxynucleotides with the presence or absence of bands, mobility of the bands on the gel, and base sequences containing mutation sites, etc. Whether or not the extracted chromosomal DNA contains the modified Jmjd3 gene can be examined. The nucleotide sequence of the oligonucleotide used for the PCR primer may be any as long as it can detect the modified Jmjd3 gene. By selecting an animal having the modified Jmjd3 gene based on the PCR result, an animal having the modified Jmjd3 gene heterozygously can be obtained.

  By crossing two or more generations of Jmjd3 gene hetero-modified animals, a Jmjd3 gene homo-modified non-human mammal in which both alleles in the Jmjd3 gene are inactivated can be obtained.

  When the Jmjd3 gene is deficient in homology, it is lethal, but normal cells can be obtained by transplanting cells or tissues collected from Jmjd3 gene homo-modified non-human mammals at the embryo or fetal stage into other non-human mammals. Alternatively, a Jmjd3 gene-modified chimeric non-human mammal in which part or all of the tissue is replaced with Jmjd3 gene homo-modified cells can be produced. For example, liver cells collected from Jmjd3 gene homo-modified non-human mammals at the embryonic or fetal stage are transplanted into other non-human mammals in vivo, so that some or all of the bone marrow cells are non-Jmjd3 gene homo-modified. A Jmjd3 gene-modified bone marrow chimeric non-human mammal substituted with bone marrow cells derived from a human mammal can be produced.

A Jmjd3 gene-modified bone marrow chimeric non-human mammal having a Jmjd3 gene homo-modified bone marrow cell derived from the non-human mammal in the bone marrow is also one aspect of the present invention. Such a bone marrow chimeric non-human mammal has Jmjd3 gene homo-modified bone marrow cells (bone marrow cells lacking the Jmjd3 gene) in the bone marrow.
Examples of non-human mammals include mice, rats, rabbits, guinea pigs, dogs, pigs, sheep, and goats described above. Among these, a mouse or a rat is preferable, and a mouse is most preferable.

  A Jmjd3 gene-modified bone marrow chimeric non-human mammal can be prepared by performing fetal hepatocyte transplantation using hepatocytes collected from a Jmjd3 gene homo-modified non-human mammal at the embryonic or fetal stage. For example, IkappaB kinase alpha is essential for mature B cell development and function., Kaisho T, Takeda K, Tsujimura T, Kawai T, Nomura F, Terada N, Akira S., J Exp Med. 2001 Feb 19; 193 (4): 417-26.

  The bone marrow chimeric non-human mammal was obtained, for example, as a recipient (animal to be transplanted), a surface marker expressed on the blood cell surface of a donor (animal collecting fetal liver cells), This can be easily confirmed by using animals that express different surface markers. For example, after the completion of fetal hepatocyte transplantation (bone marrow transplantation), the expression rate of the recipient-derived surface marker and the donor-derived surface marker in the blood collected from the recipient is stained with a specific antibody for each marker, and the flow By analyzing using cytometry, cells derived from recipients or donors can be distinguished. It can be confirmed that a bone marrow chimeric non-human mammal was obtained by detecting donor-derived blood cells.

  For example, in the case of a mouse, when performing fetal hepatocyte transplantation (bone marrow transplantation), the recipient uses a mouse that expresses CD45.1 as a surface marker for blood cells, and a donor (a mouse that collects fetal hepatocytes) It is preferable to use a mouse expressing CD45.2 as a surface marker. In this case, after completion of fetal hepatocyte transplantation (bone marrow transplantation), the ratio of CD45.1 and CD45.2 expression in blood is stained with specific antibodies for each marker and analyzed using flow cytometry. Can distinguish recipient-derived or donor-derived cells. In practice, bone marrow-derived cells can be almost completely replaced by donors by irradiating the recipient with 10 Gy of gamma rays.

  The Jmjd3 gene-modified non-human mammal of the present invention is defective in the Jmjd3 gene, which is a molecule essential for differentiation into M2 macrophages. Moreover, since the Jmjd3 gene-modified bone marrow chimeric non-human mammal of the present invention has Jmjd3 gene-modified bone marrow cells, it produces Jmjd3 gene-modified blood cells such as macrophages (Jmjd3 gene-modified macrophages) in which the Jmjd3 gene has been modified. For this reason, these non-human mammals are useful model animals for elucidating the function of Jmjd3 protein at the individual level in differentiation into M2 macrophages, the mechanism of action thereof, and the study of symptoms or diseases associated with M2 macrophages. .

  For example, various candidate substances are administered to the Jmjd3 gene-modified non-human mammal or Jmjd3 gene-modified bone marrow chimeric non-human mammal of the present invention, and M2 macrophages and / or M2 macrophages in the non-administered group animals or the control substance-administered animals. By comparing the expression of the marker or the change in activity of the Jmjd3 protein and / or Jmjd3 protein target molecule, a substance useful for the control of M2 macrophages can be evaluated. As a representative example of evaluation, screening of candidate substances can be mentioned. Such (I) the step of administering a candidate substance to the non-human mammal of the present invention, (II) (a) expression of M2 macrophage and / or M2 macrophage marker in the non-human mammal, or (b) Jmjd3 Monitoring the activity of the protein and / or Jmjd3 protein target molecule, and (III) the expression of M2 macrophages and / or markers of M2 macrophages in step (II) as compared to when no candidate substance is administered A screening method comprising the step of selecting a candidate substance in which an increase is detected or (b) an activity increase in a Jmjd3 protein target molecule and / or a Jmjd3 protein target molecule is detected is also one aspect of the present invention.

  The expression of M2 macrophages can usually be confirmed by the expression of mannose receptor on the cell surface. Moreover, the expression of M2 macrophages, that is, the differentiation of macrophages into M2 macrophages can usually be detected by quantitative PCR or flow cytometry. Markers for M2 macrophages include, for example, marker molecules known to be highly expressed in the expression of M2 macrophages, such as arginase 1, chitinase-like Ym1 (Chi313), also known as found (Fizzl, Retnla in inflammation zone 1 One or more of mannose receptor (also known as MR, CD206), inducible NO synthase (iNOS), interleukin 4 (IL4), interleukin 13 (IL13), eotaxin 2, etc. It is. The expression of the marker may be determined by examining the expression level of the gene of these markers, such as mRNA. The method for measuring gene expression is not particularly limited. For example, it can be performed by quantitative PCR. PCR primers and kits for measuring these gene expressions are commercially available, and gene expression can also be measured using the commercially available products.

  Examples of the activity of the Jmjd3 protein include the demethylase activity of the 27th lysine of histone 3 (H3K27). The demethylase activity of Jmjd3 protein can usually be confirmed by measuring the intracellular level of trimethyl H3K27. The intracellular amount of trimethyl H3K27 can be measured, for example, by chromatin immunoprecipitation-sequencing (ChIP-seq) and analysis of the data as described in the Examples. In the screening method, the expression level of the Jmjd3 gene (for example, the expression level of mRNA of the Jmjd3 gene) can also be measured as the activity of the Jmjd3 protein. The expression level of the gene can be measured by a known method, for example, quantitative PCR.

Examples of the Jmjd3 protein target molecule include genes and proteins that are activated or inactivated by the Jmjd3 protein. The Jmjd3 protein target molecule is preferably a gene of IFN regulatory factor 4 (IRF4), a C / ebpb (CCAAT / enhancer binding protein β) gene, or the like.
For example, the activity of the gene of IFN regulatory factor 4 (IRF4) can be confirmed by measuring the expression level of the gene (mRNA or the like) or the activity of the protein encoded by the gene. The expression level of the gene can be measured by a known method, for example, quantitative PCR. The activity of IFN regulatory factor 4 (IRF4) and C / ebpb can be measured by measuring the binding with a DNA probe.

  The screening method is suitably used as a screening method for an agent for promoting differentiation of M2 macrophages. For example, when a certain candidate substance is administered, compared to the case where the candidate substance is not administered, (a) increased expression of M2 macrophage and / or M2 macrophage marker, and (b) Jmjd3 protein and / or Jmjd3 protein When either or both of (a) and (b) of increased activity of the target molecule are detected, the candidate substance used can be selected as an agent for promoting differentiation into M2 macrophages.

  M2 macrophages are involved in resistance to parasitic infection, promotion of wound healing, arteriosclerosis, antiallergy, tumor formation and the like. Substances that promote differentiation into M2 macrophages are useful for promoting parasitic infection response, promoting wound healing, preventing or treating allergies, and the like. In contrast, substances that inhibit M2 macrophage differentiation may have an antitumor effect. Therefore, the screening method is preferably used as a pharmaceutical screening method for parasitic infection, wound healing, allergy, and the like.

  Examples of candidate substances in the present invention include nucleic acids, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammals (eg, mice, rats, Pig, cow, sheep, monkey, human etc.) tissue extract, plasma and the like. The candidate substance may be a novel substance or a known substance. These candidate substances may form salts, and as salts of candidate substances, salts with physiologically acceptable acids and bases are used.

  As a method for administering a candidate substance to a non-human mammal, for example, oral administration, intravenous injection, application, subcutaneous administration, intradermal administration, intraperitoneal administration, etc. are used, and the method is appropriately selected according to the properties of the candidate substance. Can do. The dosage of the candidate substance can be appropriately selected according to the administration method, the nature of the candidate substance, and the like.

A cell derived from a Jmjd3 gene-modified non-human mammal and a Jmjd3 gene-modified bone marrow chimeric non-human mammal is a cell in which the Jmjd3 gene is modified, such as spleen cells, bone marrow cells, and bone marrow cells or spleen cells Blood cells and the like are suitably used for research on M2 macrophage-related diseases.
Such a bone marrow cell derived from the non-human mammal and having a modified Jmjd3 gene is also one aspect of the present invention.

  Bone marrow cells derived from a Jmjd3 gene-modified non-human mammal can be obtained, for example, by collecting bone marrow cells from the Jmjd3 gene-modified bone marrow chimeric non-human mammal. The method of collecting bone marrow cells from a Jmjd3 gene-modified bone marrow chimeric non-human mammal is not particularly limited, and can be performed by collecting bone marrow fluid from the non-mammal by a conventional method. By culturing the collected Jmjd3 gene-modified bone marrow cells in an appropriate medium, Jmjd3 gene-modified macrophages can be induced from the bone marrow cells. Such bone marrow cell-derived Jmjd3 gene-modified macrophages are also encompassed by the present invention. The Jmjd3 gene-modified macrophage is preferably a Jmjd3 gene homo-modified macrophage. The modification of the Jmjd3 gene in macrophages can be easily confirmed by performing PCR using a known method, for example, a gene extracted from macrophages.

  The method of inducing Jmjd3 gene-modified macrophages from bone marrow cells in vitro is not particularly limited, but is usually Sequential control of Toll-like receptor-dependent responses by IRAK1 and IRAK2., Kawagoe T, Sato S, Matsushita K, Kato H, Matsui K, Kumagai Y, Saitoh T, Kawai T, Takeuchi O, Akira S., Nat Immunol. 2008 Jun; 9 (6): 684-91.

Jmjd3 gene-modified macrophages can also be obtained directly from the above-mentioned Jmjd3 gene-modified non-human mammal and Jmjd3 gene-modified bone marrow chimeric non-human mammal.
When the macrophages are collected from Jmjd3 gene-modified non-human mammals and Jmjd3 gene-modified bone marrow chimeric non-human mammals, they can usually be easily collected from peritoneal lavage or spleen cells. A Jmjd3 gene-modified bone marrow chimeric non-human mammal or a Jmjd3 gene-modified macrophage derived from a Jmjd3 gene-modified non-human mammal is also one aspect of the present invention.

  For example, inducing Jmjd3 gene-modified macrophages by culturing spleen cells collected from Jmjd3 gene-modified bone marrow chimeric non-human mammals or Jmjd3 gene homo-modified non-human mammals at the embryonic or fetal stage under appropriate conditions Can do. Induction of spleen cells into macrophages can be induced, for example, by culturing spleen cells in the presence of M-CSF for 5 days.

  Cells such as the above-mentioned Jmjd3 gene-modified macrophages are also useful for elucidation of the function of Jmjd3 protein in differentiation into M2 macrophages, its action mechanism, etc., various studies relating to symptoms or diseases associated with M2 macrophages, screening systems, and the like.

  For example, when a candidate substance is brought into contact with a Jmjd3 gene-modified macrophage, differentiation of the Jmjd3 gene-modified macrophage into M2 macrophage, expression of a marker of M2 macrophage, or Jmjd3 gene, compared to a case where the candidate substance is not contacted When one or more of the activities of the Jmjd3 protein or the Jmjd3 protein target molecule in the modified macrophage is increased, the candidate substance used can be selected as an agent for promoting differentiation into M2 macrophages. (I) a step of bringing a candidate substance into contact with Jmjd3 gene-modified macrophages, (II) (a) differentiation of Jmjd3 gene-modified macrophages into M2 macrophages, (b) expression of markers of M2 macrophages, or (c) The step of monitoring the activity of the Jmjd3 protein and / or Jmjd3 protein target molecule in the Jmjd3 gene-modified macrophages, and (III) compared with the case where the candidate substance is not contacted, M2 macrophages comprising the step of: (b) selecting a candidate substance in which increased expression of a marker of M2 macrophage is detected or (c) increased activity of a target molecule of Jmjd3 protein and / or Jmjd3 protein is detected Another method for screening for a differentiation promoting agent is one of the present invention. By such a screening method, a differentiation promoting agent for M2 macrophages useful as a therapeutic agent for parasitic infection, wound, allergy, etc. can be screened.

  Differentiation of Jmjd3 gene-modified macrophages into M2 macrophages can usually be detected by quantitative PCR or flow cytometry. M2 macrophage markers and Jmjd3 protein target molecules are the same as described above.

  Examples of the method of bringing the candidate substance into contact with the Jmjd3 gene-modified macrophage include a method of administering the candidate substance to the non-human mammal, a method of adding the candidate substance to the Jmjd3 gene-modified macrophage in vitro, and the like. In the screening method described above, (a) differentiation induction into M2 macrophages, (b) increased expression of markers of M2 macrophages, and (c) Jmjd3 compared to the case where the candidate substances are not contacted by contact with the candidate substances A candidate substance in which any one or more of the increased activity of the Jmjd3 protein and / or Jmjd3 protein target molecule in the genetically modified macrophage is detected may be selected as a differentiation promoting agent for M2 macrophage. A candidate substance is not specifically limited, The candidate substance etc. which were mentioned above can be used.

Since Jmjd3 protein is an essential molecule for differentiation of M2 macrophages, a substance that controls differentiation into M2 macrophages can be screened using Jmjd3 protein or Jmjd3 gene.
(I) a step of bringing a candidate substance into contact with the Jmjd3 gene and / or Jmjd3 protein in vitro or in a non-human mammal, and (II) a step of monitoring changes in Jmjd3 gene expression and / or demethylase activity of the Jmjd3 protein. And (III) differentiation of M2 macrophages comprising the step of selecting a candidate substance in which a change in Jmjd3 gene expression and / or demethylase activity is detected in step (II) as compared with the case where no candidate substance is contacted A method for screening a substance that regulates is also one aspect of the present invention.

For example, when a candidate substance is brought into contact with the Jmjd3 gene and / or Jmjd3 protein, either or both of the expression of the Jmjd3 gene and the demethylase activity of the Jmjd3 protein are compared with the case where the candidate substance is not contacted. Can be selected, the candidate substance used can be selected as an inhibitor of differentiation into M2 macrophages.
A substance having an action of suppressing differentiation of M2 macrophages is useful for suppression of cancer metastasis, prevention, improvement or treatment of diseases or symptoms such as arteriosclerosis and metabolic syndrome.

  Further, when a certain candidate substance is brought into contact with Jmjd3 gene expression and / or Jmjd3 protein, compared with the case where the candidate substance is not brought into contact, either of the Jmjd3 gene expression and demethylase activity of Jmjd3 protein or When both are elevated, the candidate substance used can be selected as an agent for promoting differentiation into M2 macrophages.

  Examples of the method of bringing the candidate substance into contact with the Jmjd3 gene or Jmjd3 protein include a method of administering the candidate substance to a non-human mammal, a method of adding the candidate substance to the Jmjd3 gene and / or Jmjd3 protein in vitro, and the like.

  The non-human mammal used in the screening method using the Jmjd3 protein or Jmjd3 gene is preferably a non-human mammal having the Jmjd3 gene and / or Jmjd3 protein, and a normal non-human mammal can be used. Preferably, it is a rat or a mouse, more preferably a mouse.

The Jmjd3 protein used in the in vitro method can be obtained by isolation and purification from a mammal. Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay., Matsushita K, Takeuchi O, Standley DM, Kumagai Y, Kawagoe T, Miyake T, Satoh T, Kato H, Tsujimura T, Nakamura H, Akira S. , Nature. 2009 Apr 30; 458 (7242): 1185-90. A cell having a Jmjd3 protein can be obtained by introducing the Jmjd3 gene introduced into the vector into a host cell and expressing it. Purification of the Jmjd3 protein can be performed by a known method.
The method for measuring the expression of Jmjd3 protein is not particularly limited, and for example, it can be measured by a Western blot method.

By using the non-human mammal of the present invention, the Jmjd3 gene-modified macrophage derived from the non-human mammal, a screening method, and the like, a substance that controls differentiation into M2 macrophages can be selected. Substances that promote differentiation into M2 macrophages are useful for parasitic infection response, wound healing promotion, allergy prevention or treatment, and the like. Substances that suppress Jmjd3 gene expression and / or demethylase activity of Jmjd3 protein to suppress M2 macrophage differentiation are useful for suppressing metastasis of cancer (eg, colon cancer, lung cancer, etc.), and for treating arteriosclerosis, etc. It is.
The substance selected by the screening method can be mixed with other components to obtain a medicine or the like.

I. experimental method
(i) Preparation of Jmjd3 ^-/- mice (Jmjd3 gene homo-modified mice)
The jmjd3 gene was isolated from genomic DNA extracted from ES cells (GSI-I) by polymerase chain reaction (PCR). The targeting vector is constructed by replacing the 4 kb fragment encoding the ORF of Jmjd3 (exon 14-21 including the exon encoding JmjC region) using the neomycin resistance gene cassette (neo), and is simply derived from the PGK promoter. Herpesvirus thymidine kinase was inserted into the genomic fragment for negative selection.

After transfecting the targeting vector into ES cells, both G418 and ganciclovir resistant colonies were selected and screened by polymerase chain reaction (PCR). Furthermore, genetic recombination was confirmed by Southern blotting. These homologous recombination clones were individually microinjected into blastocysts generated from C57BL / 6 mice and transferred to pseudopregnant females. Male chimeric mice were bred to C57BL / 6 female mice to transfer the mutant allele to the germline. Then, the prepared Jmjd3 ^+/− mice were cross-crossed to produce Jmjd3 ^{− / −} mice. All of these animal experiments were conducted with the permission of the Animal Experiment Committee of the Institute for Microbial Diseases. In addition, all the Jmjd3 genes in the examples are mouse Jmjd3 genes.

(ii) mice, cells and reagents
Irf4 ^{− / −} mice were generated by the method described in previous studies (Honma, K. et al. Proc Natl Acad Sci USA 102, 16001-16006 (2005)). BM-induced macrophages are generated in RPMI1640 medium containing 10% FCS, 50 μM 2-mercaptoethanol and 10 ng / ml GM-CSF (Peprotech) or 10 ng / ml M-CSF (Peprotech). It was. Pam ₃ CSK ₄ and R-848 were previously described (Pam was Takeuchi O et al., J Immunol. 2002 Jul 1; 169 (1): 10-4, R-848 was Hemmi H et al., Nat Immunol 2002 Feb; 3 (2): 196-200). LPS (Salmonella minnesota Re595) was purchased from Sigma. CpG-DNA (ODN1668) was synthesized by Invitrogen.

(iii) Preparation of BM (bone marrow cell) chimeric mouse Fetal hepatocyte transplantation was performed to prepare a BM chimeric mouse. When performing fetal liver transplantation, the recipient (transplanted side) uses a mouse that expresses CD45.1 as a surface marker for blood cells, and the donor (the mouse from which hepatocytes are collected) uses CD45. Mice expressing 2 were used.
Fetal hepatocytes were prepared from wild type and Jmjd3 ^{− / −} embryos (e15.5). The prepared cell suspension was injected intravenously into CD45.1 C57BL / 6 mice that received a lethal dose of radiation (10 Gy gamma radiation). The resulting chimeric mice were given drinking water containing neomycin and ampicillin for 4 weeks. Mice were examined for at least 8 weeks after reconstitution.
The replacement of the marrow cells of the chimeric mouse with Jmjd3 ^-/- embryonic Jmjd3 ^-/- bone marrow cells indicates the proportion of CD45.1 and CD45.2 expression in the blood after the completion of fetal hepatocyte transplantation. Cells from recipients or donors were distinguished by staining with specific antibodies to the markers and analysis using flow cytometry. In practice, bone marrow-derived cells could be almost completely replaced with donor-derived cells by transplanting fetal liver cells after irradiating the recipient with 10 Gy of gamma rays. As a result, spleen cells derived from chimeric mice were 90% or more CD45.2 positive.

(iv) Quantitative PCR analysis Total RNA was isolated using TRIzol (Invitrogen), and reverse transcription was performed using ReverTra Ace (Toyobo) according to the instruction manual. For quantitative PCR, the cDNA fragments were amplified with a real-time PCR master mix (Toyobo), and fluorescence from the Taqman probe for each cytokine was detected with a 7500 real-time PCR system. In order to determine the relative induction of cytokine mRNA in response to various stimuli, the mRNA expression level of each gene was normalized to the expression level of 18sRNA. The experiment was repeated at least twice.

(v) Immunoplot analysis
M-BMM (mouse bone marrow cell-derived macrophages) was cultured in a medium for 4 hours without using M-CSF (Protech), recovered, and transferred to a plate again. M-BMM was stimulated with M-CSF for a certain period of time, using Lysis buffer (20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA and 1% NP40) containing complete mini (Roche). Dissolved. Cell lysates were separated by standard SDS-PAGE and analyzed by immunoblotting. Antibodies against the following proteins were used.
Cell signaling / phosphorylated ERK (# 9101), phosphorylated ATK (# 9271), phosphorylated p38 (# 9211), and AKT (# 9272), Santa Cruz / p38 (# C-20), ERK (# K- 23), and β-actin (# C-11)

(vi) Flow cytometry Antibodies for flow cytometry were purchased from BD and eBioscience. The cell suspension was prepared by sieving and gently and gently pipetting. Cells were washed with ice-cold FACS buffer (2% FCS, 2 mM EDTA in PBS), then incubated with each antibody for 15 minutes and washed twice with FACS buffer. Intracellular cytokines were stained using Cytofix / Cytoperm and Fixation / Permeabilization according to the instructions for use. Data was acquired using a FACS Calibur flow cytometer (BD) and analyzed using FlowJo (Three Star).

(vii) Generation of expression plasmid for Jmjd3
Jmjd3 cDNA (corresponding to aa1141-1641) was obtained from a mouse cDNA library by PCR, and a point mutation resulting in A1388H conversion in the JmjC domain was performed by site-directed mutagenesis (Stratagene Stratagene). Introduced. Full length or mutated Jmjd3 cDNA was cloned into the pMRX-irespuro vector for retrovirus production (Saitoh, T. et al. J Biol Chem 278, 36005-36012 (2003)).

(viii) Retroviral transduction BM (bone marrow) cells were isolated from Jmjd3 ^{− / −} mice injected intraperitoneally with 5 mg of 5-fluorouracil (Nacalai Tesque) 4 days ago. Cells were treated with stem cell medium (15% FCS, 10 mM sodium pyruvate, 2 μML-glutamine, 50 μM β-mercaptoethanol, 100 U / ml penicillin, 100 g / ml streptomycin, 100 ng / ml stem cell factor, 10 ng / ml IL-6 And RPMI supplemented with 10 ng / ml IL-3). And 48 hours later, two days later, these cells were transduced with retroviral supernatant (added with stem cell factor, IL-6, IL-3, and 10 ng / ml polybrene). Viruses were generated using PlatE packaged cells transfected with various plasmids. After the second transduction, the cells were washed and macrophage growth medium (10% FCS, 50 μM β-mercaptoethanol, 100 U / ml penicillin, 100 g / ml streptomycin, and 20 ng / ml M-CSF was added. RPMI 1640 medium). After 3.5 days of culture, the cells were washed once more and macrophage growth medium with 2.5 μg / ml puromycin (Invitrogen) was added. After changing the medium, the cells were cultured for 2 days and subjected to analysis.

(ix) Chitin administration Chitin (Sigma) was washed 3 times in PBS and then sonicated with UR-20P (Tomy) for 30 minutes on ice. After filtration through a 100 μM cell filter, chitin was diluted with 50 ml PBS. Approximately 800 ng of chitin solution was injected intraperitoneally and PEC was collected 2 days after administration.

(x) Response experiment to Brazilian worm (N. brasiliensis) infection 8 weeks after fetal liver metastasis, wild type (WT) and Jmjd3 ^-/- fetal liver chimeric mice were 300 third-stage larvae N. brasiliensis (Nb) Was inoculated subcutaneously. Five days after infection, mice inoculated with Nb were sacrificed, perfused with PBS, and total RNA from the lung was extracted. RNA was subjected to quantitative PCR for analysis of specific gene expression. Nine days after infection, hilar lymph nodes were collected, single cell suspensions were made, and the number of cells was counted. Lymph node cells were stimulated with CD3 and CD28 antibodies. These were stained with CD4, treated with cytofix (BD), and stained with anti-IL-4 and IFN-γ antibodies. The cells were then analyzed by flow cytometry. BAL was performed 5 and 13 days after Nb infection, and macrophages and eosinophils were counted on cytospin smears stained with Diff-quik (Baxter Healthcare).

(xi) Microarray analysis
Total RNA was isolated from wild-type and Jmjd3 ^{− / −} M-BMM using TRLzol RNA isolation kit (Invitrogen), and further purified using RNeasy kit (Qiagen Qiagen). Biotinylated cDNA was synthesized from 100 ng of total RNA using the Ovation Biotin RNA amplification and labeling system (Nugen Nugen) according to the manufacturer's protocol. The product was purified using the DyeEx 2.0 spin kit (Qiagen QIAGEN), fragmented and hybridized to the Affymetrix mouse expression array A430. 2.0 microarray chip according to the manufacturer's protocol (Affymetrix). It was. The Affymetrix mouse genome 430 2.0 microarray chip was stained, washed and scanned according to the instructions for use. Robust multichip average (RMA) expression values were calculated using R and Bioconductor Affi packages. For each probe, the change in expression between the wild type and Jmjd3 ^{− / −} samples was defined as the difference in log2 values relative to the wild type and Jmjd3 ^{− / −} M-BMM. Genes were assigned values for their corresponding probes. If the gene is associated with many probes, an average value was taken.

(xii) Chromatin immunoprecipitation-sequencing (ChIP-seq)
M-BMM (mouse bone marrow cell-derived macrophages) was generated and 5 × 10 ⁶ macrophages were fixed with 10% formaldehyde for histones that directly cross-linked to DNA. Cells were washed twice with ice-cold PBS containing complete mini and lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (PH = 8.1)) was added. After collecting the cells in a conical tube, ultrasonic waves (Tomy UR-20P) were applied to obtain a DNA fragment. The fragment length had a peak between 150 and 300 bps. Antibody against histone H3K27me3 (Millipore 07-449) was pre-bound overnight in 80 μl Dynabeads (Veritas) in PBS / BSA 0.5%. The beads were then added to the lysate and incubated overnight. Beads are once with high salt immune complex wash buffer (Millipore) and low salt immune complex wash buffer (Millipore), 7 times with LiCl immune complex wash buffer (Millipore), Then, it was washed once with TE containing 50 mM NaCl. DNA was eluted using elution buffer (1% SDS 10 mM EDTA, 50 mM Tris-HCl (pH = 8.1)) and cross-linked by incubating at 65 ° C. overnight. DNA was purified by ethanol precipitation using Etachinmate (Nippon Gene) after treatment with RNase A and Proteinase K. The yield was ˜100 ng / 5 × 10 ⁶ macrophages. 200 ng of DNA was used for the fragment library. P1 and P2 adapter ligation reactions and all subsequent procedures were performed according to the SOLiD3 plus system fragment library construction protocol. Template bead generation for each library was performed according to the full scale protocol of the SOLiD 3 Plus system template bead production guide. Each sample was placed in six places on the slide with a target bead density of 160-200K beads / panel. High-throughput sequencing was performed using the SOLiD 3 Plus system and analyzed for 50 bp reads.

(xiii) Analysis of chromatin immunoprecipitation-sequencing (ChIP-Seq) data For each ChIP-Seq dataset, specifically mapped ChIP tags (tags) with up to 4 mismatches are further analyzed Invented for. The tag locations were moved in units of +100 bps from their 5 ′ ends to reflect the center of the original DNA fragment corresponding to each tag.

1. Global H3K27 methylation in different regions of genes within M-BMM
To obtain an overall view of the H3K27me3 distribution, the transcribed region (UCSC database (Rhead, B. et al., Nucleic Acids Res 38, D613-619 (2010)), http://genome.ucsc.edu/ Genomic-wide July 2007 mm9 RefSeq (based on mouse gene annotation), and tags mapped to upstream and downstream regions (30 kb each) were collected. The upstream and downstream were each divided into 30 bins per kb, and the transcription region was divided into 50 bins of the same size. The ChIP-Seq tags mapped for each bin were counted in both wild type and Jmjd3 ^{− / −} M-BMM. The ratio of tag counts in the control data that did not anti-immunoprecipitate with the sample was calculated for each bin.

2. Correlation between ChIP-Seq data and gene expression data For each gene X, the number of tags mapped within the transcribed region was determined for each of the ChIP-Seq experimental sample and the control sample (Cmod (x) and Ccontrol (x) ).
The intensity of histone modification is calculated as follows.

  In the above formula, Nmod and Ncontrol are the total counts of tags specifically mapped in the mouse genome. The correlation between histone modification and gene expression was evaluated by Pearson's correlation.

  The 16,090 RefSeq genes were classified into 20 bins by classifying the genes according to their expression changes. The average intensity of modification was calculated for each bin.

3. Assessment of H3K27me3 status in individual genes Key tag peaks were defined using tag count thresholds based on the estimated false discovery rate (FDR) of 1e-6. FDR was determined for each data set separately according to the following method. The gene sequence was divided into bins of 1 kb size in steps of 500 bps. The distribution of actual tag counts per bin was obtained with each data set, and the distribution of random tag counts was evaluated by random mapping the same number as in the actual data for the entire gene.

From these two distributions, the FDR is calculated for each tag count p as follows:
FDRp = Rp / Op
Here, Rp represents the proportion of random peaks with ≧ P tags, and Op represents the proportion of observed (actual) peaks with ≧ P tags. The threshold for significant tag peaks was set as the lowest tag count with an FDR ≦ 1e-6. This resulted in a threshold tag count of 18 for wild type and Jmjd3 ^{− / −} M-BMM H3K27me3. Any bin with a tag count equal to or higher than these thresholds was considered significant.

Genes were classified based on the presence or absence of a significant tag peak for H3K27me3 in wild-type and Jmjd3 ^{− / −} M-BMM in the region around their transcription start positions. For each RefSeq gene, the location of the TSS could be obtained from the UCSC database, and the -5 kb to 1 kb region was divided into 1 kb bins in steps of 500 bps. The tag count for each bin was counted. The genes were divided into the following three classes.
Class 1 genes have one or more significant peaks in the wild type.
Class 2 genes have no significant peaks in both wild type and Jmjd3 ^{− / −} .
Class 3 genes have one or more significant peaks in Jmjd3 ^{− / −} but not in the wild type.

(xiv) Statistics Statistical significance was calculated by Student's t-test (two-sided).

II. Results
(i) Jmjd3 ^{- / -} to study the functional role of JMJD3 (Jmjd3 protein) in the preparation immune response in mice, Jmjd3 ^{- / -} mice were prepared (FIGS. 1 and 2a). FIG. 1 is a schematic diagram of the mouse Jmjd3 gene, targeting vector, and target allele. The targeting vector was designed to replace exon21 containing the exon14 to JmjC H3K27 demethylase domain with the neomycin resistance gene B.BglII, as shown in FIG. FIG. 2a shows the results of Southern blot analysis of offspring obtained from outbred heterozygotes. Genomic DNA was extracted from MEFs, cleaved with Bgl II, separated by electrophoresis, and hybridized with a probe labeled with the radioisotope shown in FIG. Southern blots detected a 7.0 kb band in wild type (+ / +), a 5.2 kb band in homozygous (-/-), and both bands in heterozygous (+/-) mice. Reverse transcription (RT) -PCR analysis showed that Jmjd3 expression was lost in fetal fibroblasts of Jmjd3 ^{− / −} mice (FIG. 2b). FIG. 2b is a diagram showing RT-PCR analysis results of RNA derived from wild type (WT) and Jmjd3 ^{− / −} MEFs stimulated with LPS (1 μg / ml) for a certain period of time. The RNA was subjected to RT-PCR analysis to examine the expression of Jmjd3 mRNA. The expression of the β-actin gene was analyzed using the same RNA. Jmjd3 ^{− / −} mice showed perinatal death and no mature Jmjd3 ^{− / −} mice were obtained (Table 1). Table 1 shows the genotyping of surviving newborns obtained from outbred heterozygotes.

Histological examination reveals that the alveolar cell wall is thickened in the tissue and that the airspace is hardly observed in the new lung with Jmjd3 ^-/- (Fig. 3 (b)), which is observed in Jmjd3 ^-/- mice. This suggested that a postnatal lethal phenotype might be present due to early development of lung tissue. 3 (a) and 3 (b) are diagrams showing H & E staining of lung parts of wild type (WT) and Jmjd3 ^{− / −} mice, respectively. Data are typical of the lungs of 3 mice. To analyze the role of Jmjd3 ^{− / −} in hematopoietic cells, fetal hepatocytes were obtained from Jmjd3 ^{− / −} e15.5 embryos, and BM chimeric mice (hereinafter referred to as Jmjd3 ^{− / −} chimera or Jmjd3 ^{− / −} BM chimera). Also called). Analysis by flow cytometry revealed that in the spleen, T cells, B cells, standard dendritic cells (DCs), plasmacytoid dendritic cells, natural killer cells, neutrophils, F4 / 80 ⁺ CD11b ⁺ macrophages and Ly6c ^high CD11b ⁺ The proportion of inflammatory monocytes was found to be comparable between wild type and Jmjd3 ^{− / −} chimeras. The proliferative response of splenic T and B cells to mitogen and antigen receptor stimulation was also altered. Further, splenic T cells cultured under Th1 and Th2 conditions produced IFN-γ and IL-4 to the same extent in response to T cell receptor (TCR) stimulation. These results indicate that Jmjd3-deficient T cells have the ability to differentiate into Th1 and Th2 cells in response to cytokines.

(ii) Jmjd3 is not essential for M1 macrophage activation and mobilization in vivo.
Since Jmjd3 is a TLR-inducible gene in macrophages, first, lipopeptide (Pam ₃ CSK ₄ ; activated TLR2), lipopolysaccharide (LPS; TLR4), imidazoquinoline analog (R-848; TLR7) and CpG motif Endogenous and thioglycolic acid-induced peritoneal exudate cells (PECs) produced cytokines against TLR ligands containing oligonucleotides with CpG-DNA (TLR9). TNF and IL-6 production in response to TLR ligands did not change between wild-type and endogenous and thioglycolic acid-induced peritoneal exudate cells (PEC) (FIGS. 4a and 4b). Peritoneal exudate cells (PEC) induced by peptone treatment from wild-type and Jmjd3 ^{− / −} chimeras also produced TNF and IL-6 to the same extent upon TLR ligand stimulation. In FIGS. 4a to 4d, “med” is a control to which no TLR ligand was added, and “ND” means that no cytokine production was detected. Moreover, black is WT and white is Jmjd3 ^{− / −} .

Flow cytometry reveals that the ratio of CD11b ⁺ F4 / 80 ⁺ Gr1 ^- macrophages in endogenous and thioglycolic acid-induced peritoneal exudate cells (PEC) does not change between wild type and Jmjd3 ^-/- chimeras (Fig. 4c). The total number of resident (endogenous) macrophages, thioglycolic acid-induced macrophages and peptone-induced macrophages was unchanged with Jmjd3 ^{− / −} chimeras (FIG. 4c).

Thus, the role of JMJD3 in differentiation into M1 macrophages in response to Listeria monocytogenes infection was examined. When Listeria monocytogenes is inoculated intraperitoneally, the production of cytokines, TNF, IL-6, IFN-γ and IL-12p40, which promote inflammatory responses in serum, is similar in wild-type and Jmjd3 ^{− / −} chimeras (FIGS. 7 (a) to (d)). FIGS. 7 (a) to (d) show that WT and Jmjd3 ^{− / −} chimeras were infected with Listeria monocytogenes, and TNF, IL-6, IFN-γ and IL-12p40 concentrations in serum were determined by ELISA after a certain period of time. It is a result. FIG. 7 (a) shows TNF, FIG. 7 (b) shows IFN-γ, FIG. 7 (c) shows IL-6, and FIG. 7 (d) shows IL-12. In FIG. 7 (a) to FIG. 7 (d), the black square is WT, and the white circle is Jmjd3 ^{− / −} chimera.

F4 / 80 ⁺ CD11b ⁺ Gr1 ^{+ macrophages, F4 / 80 int CD11b + Gr1} high neutrophils and F4 / 80 ^- CD11b ^int B220 ⁺ and ^{F4 / 80 - CD11b - B220 +} B cells were prepared from Listeria-infected mice Observed in peritoneal exudate cells (PEC). The number of macrophages recruited to the abdominal cavity was similar in the wild type and Jmjd3 ^{− / −} chimeras (FIGS. 5 (a) to (c) and FIGS. 6 (a) to (d)). FIG. 5 shows PEC from WT and Jmjd3 ^{− / −} chimeras 3 days after treatment with no stimulation (FIG. 5 (a)), thioglycolic acid (FIG. 5 (b)) or Listeria monocytogenes (FIG. 5 (c)). It is a figure which shows the result of having extract | collected and then dye | staining a cell with anti- CD11b and F4 / 80 antibody, and analyzing by flow cytometry. 5 (a) shows resident macrophages, FIG. 5 (b) shows thioglycolic acid-induced macrophages, FIG. 5 (c) shows macrophages 24 hours after infection with Listeria monocytogenes, and FIG. 5 (d) shows after infection with Listeria monocytogenes. Macrophages at 48 hours.
Figures 6 (a)-(d) show no stimulation (Figure 6 (a)), treated with thioglycolic acid (Figure 6 (b)) or Listeria (Figures 6 (c) and 6 (d)) 3 It is a figure which shows the result of having investigated the number of F4 / 80 ⁺ CD11b ⁺ macrophage in PEC extract | collected from WT and Jmjd3 ^{− / −} chimera every day. 6 (a) is a resident macrophage, FIG. 6 (b) is a thioglycolic acid-induced macrophage, FIG. 6 (c) is a macrophage 24 hours after Listeria monocytogenes infection, and FIG. 6 (d) is Macrophages 48 hours after infection with Listeria monocytogenes.

Furthermore, the expression of genes encoding TNF, IL6, IL12p40 and iNOS (inducible NO synthase) genes in peritoneal exudate cells (PEC) was equally enhanced (FIGS. 8 (a) to (d)). ). FIGS. 8A to 8D are diagrams showing the results of examining the results of examining the expression of Tnf, Il6, Il12b, and Nos2 in PEC of mice infected with Listeria monocytogenes by Q-PCR analysis. 8A shows TNF, FIG. 8B shows iNOS, FIG. 8C shows IL-6, and FIG. 8D shows IL-12p40.
To summarize the results of FIGS. 4-8, these data indicate that JMJD3 (Jmjd3 protein) is not involved in the generation and mobilization of M1 macrophages in response to inflammatory agents and bacterial infections in vivo. It suggests that. The results in FIGS. 4a, 4b, and 7-8 are representative examples of two independent tests, and the results in FIGS. 4c, 4d, and 5-6 are representative examples of four independent tests. is there. Error bars in FIGS. 4A to 4D and FIGS. 6 to 8 are standard deviations.

(iii) The main role of Jmjd3 in differentiation to M2 in response to in vivo chitin administration Chitin is a polymerized sugar and a constituent of parasites, arthropods and molds (Bowman, SM & Free, SJ, Bioessays 28, 799-808 (2006)). Chitin administration has been shown to attract macrophages with M2 properties at the site of administration, which is also important for subsequent eosinophil recruitment (Reese, TA et al., Nature 447, 92 -96 (2007) and Kreider, T. et al., Curr Opin Immunol 19, 448-453 (2007)). Indeed, intraperitoneal administration of chitin wilded F4 / 80 ⁺ CD11b ⁺ macrophages (Figures 9 (a) and 9 (b)), Siglec-F ⁺ CCR3 ⁺ CD4 ^- eosinophils and CD11b ^int B220 ⁺ B cells. Type mice were mobilized 48 hours later (FIGS. 9 (c) and 9 (d)). In contrast, the recruitment of eosinophils and B cells was greatly impaired in the Jmjd3 ^{− / −} chimera (FIGS. 9 (c), 9 (d) and 10b). Although the number of chitin-induced F4 / 80 ⁺ CD11b ⁺ macrophages was similar (Figure 10a), MR (mannose receptor, also known as CD206) expression is greatly impaired in Jmjd3 ^-/- chimeras (Figure 12). Chitin-induced macrophages, although not eosinophils or B cells, are characteristic of M2 macrophages: Arginase-1 (Arginase-1), Ym1 (chitinase-like Ym1 (Chi313)), Fizz1 (in inflammation zone 1) found, also known as Retnla)) and expressed high levels of mRNA encoding MR. Expression of genes encoding arginase-1, Ym1, Fizz1 and MR is significantly lower in chitin-induced peritoneal exudate cells (PEC) obtained from Jmjd3 ^{− / −} chimeras compared to wild type controls, whereas Thus, the expression of the gene encoding iNOS was not changed (FIGS. 13 (a) to (e)). It should be noted that eosinophils circulating in the blood were observed to the same extent in wild-type and Jmjd3 ^{− / −} chimeric mice, and the growth of eosinophils depends on the lack of Jmjd3 ^{− / −} Suggests that it is not significantly impaired (data not shown). Based on these, Jmjd3 is important for differentiation into M2 macrophages in response to chitin administration.

FIG. 9 shows CD11b and F4 / 80 (FIG. 9 (a) and FIG. 9 (b)) or CD11b and Siglec− PEC collected from WT and Jmjd3 ^{− / −} chimeric mice two days after injection of chitin into the peritoneum. FIG. 10 shows the results of staining with F (FIG. 9 (c) and FIG. 9 (d)) and analysis by flow cytometry. 9 (a) and 9 (c) are wild type (WT), and FIG. 9 (b) and FIG. 9 (d) are Jmjd3 ^{− / −} .
In FIG. 13, * p <0.05 and ** p <0.01 (two-tailed Student's t-test). The results shown in FIGS. 9-13 are representative of 5 (FIGS. 9-10), 2 (FIG. 11) or 3 (FIGS. 12 and 13) independent studies. Error bars in FIG. 10a, FIG. 10b and FIGS. 13 (a) to (e) are standard deviations.

(iv) Major role of Jmjd3 in pulmonary pathology in response to Nippostrongylus brasiliensis infection in mice Next, we investigated whether JMJD3 contributes to the response to parasitic infections in vivo. A N. brasiliensis (Nb) infection model was used, which induces a strong type 2 immune response in the lungs. Bronchoalveolar lavage (BAL) staining 5 or 13 days after infection revealed that macrophages were recruited to the lung to a similar extent in wild type and Jmjd3 ^{− / −} BM chimeras (FIG. 14 (a ): Wild type mouse, FIG. 14 (b): Jmjd3 ^{− / −} chimeric mouse). Fig. 14 (a) and Fig. 14 (b) show BAL fluid extracted from WT and Jmjd3 ^-/- chimeric mice 13 days after Nb infection, and on cytospin smell stained with macrophages and eosinophils with Diff-quik. FIG. 14 (a): Wild type (WT), FIG. 14 (b): Jmjd3 ^{− / −} chimeric mouse). However, eosinophil recruitment was greatly impaired in Jmjd3 ^{− / −} chimeric mice (FIGS. 15 (a)-(b)). In FIGS. 15 (a) to 15 (b), black is WT and white is Jmjd3 ^{− / −} chimera.

To examine the nature of the recruited macrophages, RNA was extracted from lung tissue 5 days after Nb infection. Expression of M2 markers such as arginase-1, Ym1, Fizz1, and MR was hardly induced in the Jmjd3 ^{− / −} chimera (FIGS. 16 (a) to (h)). In FIG. 16, the square is the control (black is the wild type (WT), white is the Jmjd3 ^-/- chimera), the circle is the NB-infected mouse (black is the wild type (WT), white is the Jmjd3 ^-/- chimera) is there. Furthermore, induction of genes encoding chemokines that recruit eosinophils and eotaxin 2 as well as cytokines that induce Th2, IL-4 and IL-13 were greatly impaired in Jmjd3 ^{− / −} chimeric mice ( FIG. 16). FIG. 16 (a)-(h) shows mRNA encoding a specific protein in total RNA prepared from infected and non-infected WT and infected and non-infected Jmjd3 ^{− / −} chimeras 5 days after Nb inoculation. It is a figure which shows the expression level of. Figure 16 (a) is arginase 1, Figure 16 (b) is Ym1, Figure 16 (c) is Fizz1, Figure 16 (d) is MR, Figure 16 (e) is iNOS, Figure 16 (f) is IL-4 FIG. 16 (g) shows the expression level of mRNA encoding IL-13, and FIG. 16 (h) shows the expression level of mRNA encoding eotaxin.
14 and 15 are representative results of two tests using 4 mice per group, and FIG. 16 is a result of one test using 7 mice per group. is there. Error bars in FIG. 15 are standard deviations.

T cell activation in the pulmonary lymph nodes 9 days after Nb infection was examined. Nine days after Nb infection, hilar lymph node cells collected from WT and Jmjd3 ^{− / −} chimeras were stimulated with CD3 and CD28 for 4 hours and stained with anti-CD4, CD8, IL-4 and IFN-γ. FIG. 17 shows representative results of intracellular IL-4 and IFN-γ staining in T cells. The number of IL-4 producing cells in the hilar 9 days after Nb infection is shown in FIG. 18 (* p <0.05 and ** p <0.01 (two-tailed Student's t-test)). CD4 ⁺ T cells prepared from wild type lung lymph nodes expressed IL-4, but not IFN-γ, and the frequency of CD4 ⁺ T cells producing IL-4 was Jmjd3 ^-/- It is greatly impaired in lung T cells prepared from chimeric mice, suggesting that Jmjd3-mediated activation of M2 macrophages is important for Nb inducing Th2 responses in the lung (FIGS. 17 and 18). However, histological changes in the intestine are not significantly impaired in Jmjd3 ^{− / −} chimeric mice (data not shown).
17 and 18 show the results of one test using 5 mice. The error bars in FIG. 18 are standard deviations.
The results of FIGS. 14-18 clearly showed that Jmjd3 is essential for the initiation of an immune response to parasitic infection by directing differentiation into M2 macrophages in the lung rather than in the small intestine.

(v) Role of Jmjd3 on M2 macrophage production in response to M-CSF in BM cells
It has been reported that GM-CSF and M-CSF induce M1 and M2 macrophages from BM cells, respectively (Verreck, FA et al., Proc Natl Acad Sci USA 101, 4560-4565 (2004), Martinez, FO et al., J Immunol 177, 7303-7311 (2006), Fleetwood, AJ et al., J Immunol 178, 5245-5252 (2007), Fleetwood, AJ et al., J Leukoc Biol 86, 411- 421 (2009)). When GM-CSF is used to generate macrophages, the production of TNF and IL-6 in response to TLR ligands derived from adherent CD11b ⁺ macrophages (GM-BMM) is wild-type and Jmjd3 ^{− / -} it was comparable between the chimeric mice (Fig. 19a and FIG. 19b). On the other hand, IL-6 (not TNF) production in response to TLR ligand stimulation was partially impaired in Jmjd3 ^{− / −} BMM cultured in the presence of M-CSF (M-BMM) (FIG. 19c and FIG. 19). 19d). 19a-d, black is WT and white is Jmjd3 ^{− / −} chimera. Furthermore, the expression of genes encoding arginase-1, Ym1, Fizz1, MR and IL-13 was greatly impaired in Bm-BMM derived from Jmjd3 ^{− / −} chimera (FIGS. 20 (a) to (d)). This indicates that Jmjd3 is important for gene expression of M2 marker in M-BMM. There is a report showing that JMJD3 is involved in the response of macrophages to IL-4 stimulation (Ishii, M. et al., Blood 114, 3244-3254 (2009)). Nevertheless, arginase-1 and Fizz1 gene expression was comparable for IL-4 stimulation between wild-type and Jmjd3 ^{− / −} M-BMM (FIG. 21 (a): arginase 1, FIG. 21 ( b): Fizz1). In FIGS. 21 (a) to 21 (b), black is WT and white is Jmjd3 ^{− / −} chimera.

This suggests that the response to IL-4 is not impaired in Jmjd3 ^{− / −} M-BMM. Therefore, the profile of gene expression in wild type and Jmjd3 ^{− / −} M-BMM with and without LPS stimulation was examined by microarray analysis. The expression of 1371 genes was decreased more than 2-fold in unstimulated Jmjd3 ^{− / −} M-BMM compared to wild type. In addition to Arg1, Chi3l3 and Retla, chemokine genes such as Ccl1, Ccl17, Ccl22 and Ccl24, and cytokine genes such as Il2, IL3, Il4, IL5 and Il13 are greatly expressed in Jmjd3 ^{− / −} M-BMM. It was damaged. 2188-induced gene expression increased more than 2-fold in wild-type BMM in response to LPS stimulation and more than 2-fold decrease in 436 genes in LPS-stimulated Jmjd3 ^-/- M-BMM It was. For example, Il6 and Il12b expression was partially impaired in Jmjd3 ^{− / −} M-BMM, which was consistent with previous reports. Thus, Jmjd3 is important in inducing the expression of a series of genes, and some genes that are controlled by the presence of Jmjd3 are related to differentiation into M2 macrophages in M-BMM.

JMJD3 contains a domain presumed to be a tetratricopeptide repeat at the N-terminus in addition to the JmjC domain. The C-terminal part of JMJD3 containing the JmjC domain (aa 1141-1641) or its iron-binding defective mutant (A1388H) was expressed in Jmjd3 ^{− / −} BM cells by retrovirus to induce M-BMM (FIG. 22). The empty vector in FIG. 22 is a control. A1388H mutant (ΔFe ²⁺ binding-deficient mutant) has been shown to lose H3K27 demethylase activity of JMJD3 (De Santa, F. et al., Cell 130, 1083-1094 (2007)) . Preparation of total RNA from Jmjd3 ^-/- M-BMM generated from BM infected with retrovirus expressing wild-type JMJD3 (aa 1141-1641) or its iron-binding mutant (ΔFe 2 ⁺ -binding mutant) Then, the expression of marker genes (arginase 1, Ym1 and MR) was examined. The results are shown in FIGS. 23 (a) to (c). FIG. 23 (a) is arginase 1, FIG. 23 (b) is Ym1, and FIG. 23 (c) is MR. Expression of the C-terminal part of JMJD3 was sufficient to enhance M2 markers such as arginase 1, Ym1 and Fizz1, and to increase the number of observed M-BMMs (FIGS. 23 (a)-( c)). On the other hand, the expression of JMJD3 (A1388H) did not increase the number of M-BMM or the expression of the M2 marker gene. This indicates that the H3K27me3 demethylase activity of JMJD3 is the M2 marker gene in M-BMM. It was shown to be important and sufficient for the expression of
The results shown in FIGS. 19-23 are representative of 4 (FIGS. 19 (a)-(d)), 4 (FIGS. 20 and 21) or 2 (FIGS. 22 and 23) independent experiments. is there. Error bars in FIGS. 19-21 are standard deviations.

(vi) Necessity of Jmjd3 for cell cycle progression in M-BMM
Although the number of GM-BMM cells did not change, in addition to impaired expression of the M2 marker, the total number of M-BMM in the Jmjd3 ^{− / −} chimera became wild type 5 or 7 days after culturing with M-CSF. Compared to significantly lower. Uptake of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, was greatly impaired in Jmjd3 ^{− / −} M-BMM compared to wild type cells, whereas wild type and Jmjd3 ^{− /} -GM-BMM imported BrdU to the same extent. These results indicated that JMJD3 is important for controlling cell cycle progression in response to M-CSF stimulation. When mRNA expression of cell cycle regulatory protein was measured, c-Myc, c-Myb, cyclin D1 and cyclin D2 mRNA were found to be expressed in Jmjd3 ^-/- M-BMM in 5 days of M-CSF culture. It was damaged. Surface expression of the M-CSF receptor (CSF-1R) was not impaired in Jmjd3 ^{− / −} M-BMM (FIG. 5d). Furthermore, Jmjd3 deficiency did not affect the activation of intracellular signaling molecules ERK, p38 or AKT, as indicated by phosphorylation in M-BMM. This suggested that in Jmjd3 ^{− / −} M-BMM, cell growth defects were not due to low activation of MAP kinase or AKT. The expression of Jmjd3 in M-BMM is much higher than that in GM-BMM, indicating that the difference in Jmjd3 expression in M-BMM and GM-BMM determines the contribution of Jmjd3 to their proliferation I suggested. From these results, these data indicate that Jmjd3 is important for generating M-BMM but not GM-BMM by controlling cell proliferation downstream of CSF-1R signaling.

(vii) Genome-wide analysis of H3K27 trimethylation controlled by JMJD3 in M-BMM Next, by chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis, H3K27me3 in wild-type and Jmjd3 ^-/- M-BMM The distribution of the whole genome was analyzed. An overall view of the H3K27me3 distribution in the transcription regions and their upstream and downstream regions (30 kb each) was obtained (based on genome level RefSeq mouse gene annotation in the UCSC database). High levels of H3K27me3 tag were detected around TSSs in M-BMM from wild type and Jmjd3 ^{− / −} chimeras. On the other hand, H3K27me3 levels were lower in the genomic transcription locus than in the upstream and downstream regions. Of note, the H3K27me3 signal in the promoter region and downstream region was higher in Jmjd3 ^{− / −} M-BMM than in wild type cells.

Therefore, we compared the gene expression data obtained by microarray experiments with the status of H3K27 methylation. Overall, in the region close to TSS (-5 to +1 kb), the level of H3K27me3 was negatively correlated with the gene expression level in M-BMM (correlation coefficient -0.441). Then, the genes were rearranged based on the expression ratio between the wild type and Jmjd3 ^{− / −} M-BMM, and the level of H3K27me3 was examined. However, no clear relationship was found between the difference in gene expression level between wild type and Jmjd3 ^{− / −} M-BMM and the situation of H3K27me3. These data indicate that only a few genes are affected by Jmjd3 deficiency, and most loci are duplicated and regulated by UTX and / or JMJD3.

Focused on the H3K27me3 tag preference for the region proximal to TSS and the promoter region of the individual gene showing the peak of H3K27me3, as judged by the lack of overall correlation between expression changes and tag number . Genes were classified by the presence of peaks in wild-type and Jmjd3 ^{− / −} M-BMM samples, and the genome-wide set of genes was divided into three different classes depending on the status of H3K27me3. The class 1 gene was wild-type M-BMM and had a peak of H3K27me3. The class 2 gene had no H3K27me3 peak in either the wild type or Jmjd3 ^{− / −} M-BMM. Class 3 genes such as Irf4 and Tm7sf4 were characterized by the presence of the H3K27me3 peak in Jmjd3 ^{− / −} M-BMM, but no peak in wild type M-BMM. From the microarray data, the status of H3K27 demethylation and gene expression were compared, and a table of 500 genes expressed in wild type and Jmjd3 ^{− / −} M-BMM was generated (data not shown).

Hox genes such as Hoxa7, Hoxa9 and Hox11 and Bmp2 have been reported to be regulated by JMJD3 (De Santa, F. et al., Cell 130, 1083-1094 (2007)), but their loci Of H3K27 are highly trimethylated in M-BMM both in the presence and absence of Jmjd3, and therefore they were classified as class 1. Furthermore, Hox and Bmp2 gene expression levels did not decrease with Jmjd3 ^-/- M-BMM, indicating that these genes were not critically regulated by JMJD3 with M-BMM. Show. Furthermore, M2 markers such as Arg1, Chi3l3, Rentla, and Mrc1 were all found in class 2, indicating that their expression is not directly regulated by JMJD3. Therefore, we hypothesized that transcription factors directly regulated by JMJD3-mediated demethylation are responsible for macrophage differentiation. When examining genes classified into class 3, we found that Irf4 and Cebpb were in this class. In particular, the promoter region near Tss of Irf4 showed a high H3K27me3 tag in Jmjd3 ^{− / −} M-BMM, but not in the wild type.

(viii) Identification of IRF4 (IFN regulator 4) as a JMJD3 target molecule important for differentiation into M2 macrophages
The chromatin solution derived from WT and Jmjd3 ^{− / −} BMM was subjected to ChIP analysis using an anti-H3K27me3 antibody, and the resulting DNA was analyzed with a PCR primer pair that amplifies the promoter region of Irf4 (FIG. 24 (a): WT, FIG. 24 (b): Jmjd3 ^{− / −} BMM). ChIP analysis confirmed that H3K27 methylation was different in the promoter region of Irf4 gene in wild-type and Jmjd3 ^{− / −} macrophages (FIGS. 24 (a) and 24 (b)). Furthermore, when the C-terminal region of JMJD3 or a mutant thereof was expressed in Jmjd3 ^{− / −} macrophages by retrovirus, the expression of Irf4 increased depending on the demethylation activity (FIG. 25). Figure 25 shows the extraction of total RNA from Jmjd3 ^-/- M-BMM reconstituted with wild-type JMJD3 (aa 1141-1641) or its iron-binding mutant, and the level of Irf4 expression in Q-PCR. It is a figure which shows the result determined by analysis. The control is Jmjd3 ^{− / −} M-BMM reconstituted with a retrovirus introduced with an empty vector.

These results indicate that Irf4 is one of the target genes of JMJD3 in M-BMM. Therefore, the contribution of IRF4 to the expression of arginase-1, Ym1, Fizz1 and MR was examined by using Irf4 ^{− / −} mice. Specifically, BM cells derived from WT and Irf4 ^{− / −} mice were cultured for 5 days in the presence of M-CSF. Total RNA was prepared and the expression of specific M2 marker and gene encoding JMJD3 was examined by Q-PCR. 26 (a) is Arginase 1, FIG. 26 (b) is Ym1, FIG. 26 (c) is Fizz1, FIG. 26 (d) is MR, FIG. 26 (e) is IL-13, and FIG. 26 (f) is The expression levels of JMJD3 mRNA are shown. As shown in FIGS. 26 (a) to (e), the induction of M2-related genes was greatly impaired in Irf4 ^{− / −} M-BMM. On the other hand, the expression of Jmjd3 was comparable between the wild type and Irf4 ^{− / −} M-BMM (FIG. 26 (f)). Of note, the number of M-BMM cells did not decrease in Irf4 ^{− / −} mice (FIG. 27).

FIG. 28 (a) and FIG. 28 (b) show the results of analysis of the mobilization of macrophages and eosinophils in PECs after 48 hours post-treatment with chitin administered to WT and Irf4 ^{− / −} mice. (FIG. 28 (a): WT, FIG. 28 (b): Irf4 ^{− / −} mouse). When chitin was administered intraperitoneally, eosinophil recruitment (mobilization) but not macrophages was greatly impaired in Irf4 ^{− / −} mice (FIG. 28 (a): WT, FIG. 28 (b): Irf4 ^-/-And Figure 29). MR expression in chitin-induced peritoneal macrophages was greatly impaired in Irf4 ^{− / −} mice (FIG. 30). In addition, the expression of arginase-1, Ym1, Fizz1 and MR, which are M2 macrophage markers, was greatly impaired in chitin-induced macrophages derived from Irf4 ^{− / −} mice (FIGS. 31 (a) to (d)). These results indicate that IRF4 is important for differentiation of macrophages into M2 in M-BMM and in response to administration of chitin in vivo. Therefore, IRF4 was expressed in Jmjd3 ^{− / −} M-BMM using a retrovirus, and the expression of the M2 marker gene was examined (FIGS. 32 (a) to (f)). As a control, an empty vector was expressed in Jmjd3 ^{− / −} M-BMM. Expression of IRF4 in Jmjd3 ^-/- M-BMM did not change the expression of Jmjd3, but the expression of IRF4 encoded arginase-1, Ym1, Fizz1 and MR in Jmjd3 ^-/- M-BMM. The gene is increased (FIGS. 32 (a) to (f)). These results suggest that IRF4 contributes to the expression of the M2 marker downstream of JMJD3.

  32 (a) to (f), * p <0.05 and ** p <0.01 (two-tailed Student's t-test). The results shown in FIGS. 24-32 are representative of 2 (FIG. 24), 3 (FIGS. 26-31) or 2 (FIGS. 25 and 32) independent experiments. The error bars in FIGS. 26 and 31 are standard deviations.

III. Discussion In this study, we focused on the role of JMJD3 in macrophages that initiates antibacterial and antiparasitic responses. JMJD3 was not important for differentiation into M1 macrophages, whereas jmjd3-deficient mice did not have an appropriate M2 response to parasitic infection or chitin administration. Furthermore, BM macrophages induced by M-CSF showed obvious defects in the expression of various genes including M2 macrophage markers depending on the demethylase activity. Nevertheless, H3K27me3 levels of a subset of genes are regulated differently depending on the presence or absence of Jmjd3. Among these genes, Irf4 was found to be one of the direct targets of JMJD3-mediated demethylation. And IRF4 was found to be an important transcription factor for induction of M2 macrophage response.

Jmjd3 is a TLR-inducible gene, but Jmjd3 ^{− / −} mice showed strong M1 macrophage activation in response to Listeria monocytogenes inoculation. These results suggest that Jmjd3 is not essential for the generation and mobilization of M1 macrophages against bacterial infection. Our data show that gene expression in response to LPS stimulation is only moderately altered in Jmjd3-deficient macrophages and that JMJD3 fine-tunes transcriptional production in this case (De Santa, F. et al., EMBO J 28, 3341-3352 (2009)). TLR signaling not only promotes inflammation, but also enhances genes involved in arrest or tissue remodeling. For example, ATF3 and Zc3h12a are rapidly induced in response to TLR stimulation and suppress the production of inflammatory cytokines (Charo, IF, Cell Metab 6, 96-98 (2007) and Matsushita, K. et al., Nature 458, 1185-1190 (2009)). M2 macrophages have been shown to promote tissue remodeling as well as Th2 responses. Thus, Jmjd3 induction functions as part of a feedback mechanism that acts to repair inflammatory damage caused by TLR stimulation.

  Chitin is a component that is abundant in parasites, crustaceans, and molds, and administration of chitin strongly induces M2 macrophages. Intraperitoneal administration of chitin mobilized M2 macrophages and eosinophils in a Jmjd3 and Irf4-dependent manner. These results indicate that the JMJD3-IRF4 axis is important for differentiation into M2 macrophages against parasitic helminth infections. However, the addition of chitin in macrophage cultures did not stimulate cells that enhanced M2 marker gene expression. TLR2 was reported to mediate acute inflammation in response to chitin, but other reports reported that chitin-mediated activation of M2 macrophages was independent of MyD88 (Reese, TA et al., Nature 447, 92-96 (2007) and Da Silva et al., J Immunol 181, 4279-4286 (2008)). Currently, the mechanism by which chitin activates macrophages is not well understood. Furthermore, it is not yet clear how JMJD3 plays a unique role in the production of M2 macrophages in response to chitin and parasitic infections. Identification of chitin receptors will be vital in the near future to elucidate innate immune activation mechanisms in response to parasitic helminth infections.

Jmjd3 ^{− / −} mice showed a significant defect in the recruitment of M2 macrophages in N. brasiliensis infection. I don't know which components of Brazilian helminths activate innate immune cells, but JMJD3-mediated H3K27me3 demethylation appears to be essential for macrophage response to the parasite. Further studies will identify the role of JMJD3 in controlling infection by other parasites that are pathogenic in humans. It is well known that M2 macrophages are important for tissue remodeling in response to parasitic infections, as well as survival of cancer cells or responses to inflammatory responses (Mantovani, A. et al., Curr Opin Immunol (2010)). It is therefore interesting to explore how non-genetic regulation in macrophages is important for cancer progression or wound healing by using this mouse model.

Previous reports have shown that Jmjd3 expression is enhanced in response to IL-4 and that H3K27me3 levels decrease in response to IL-4 stimulation (Ishii, M. et al., Blood 114, 3244-3254 (2009)). M-BMM (mouse bone marrow cell-derived macrophages) or chitin-induced peritoneal macrophages showed severe defects in the expression of M2 macrophage markers in the absence of Jmjd3, whereas Jmjd3 ^{− / −} M-BMM It was possible to enhance the expression of representative genes of M2 macrophages in response to stimulation. These findings suggest that JMJD3-mediated H3K27 demethylation is important for the generation of differentiation to M2 regardless of the effects of IL-4. The same report showed that the H3K27me3 levels of various M2 marker genes are directly regulated by JMJD3, which activates transcription. However, our ChIP-Seq data demonstrated that the H3K27me3 levels of most M2 marker genes such as Arg1 (Arginase 1) did not change even in the absence of Jmjd3. Furthermore, loss of Irf4, one of the target genes of JMJD3, deficient in M2 response to chitin administration or M-CSF culture. Therefore, JMJD3 is likely to regulate differentiation into M2 macrophages by secondarily controlling the expression of a series of transcription factors.

In addition to M2 marker gene expression, Jmjd3-deficient M-BMM showed a growth defect in response to M-CSF stimulation. This is not due to the lack of normal functioning of M-CSF receptor expression or early signal molecule activation. Expression of genes involved in cell cycle progression, such as c-Myc, cyclin D1, and cyclin D2, ceased to function normally in Jmjd3 ^-/- M-BMM, but the H3K27me3 level of these genes is wild type (wild -type) and Jmjd3 ^-/- M-BMM. Furthermore, Irf4 ^{− / −} M-BMM showed no defects in the cell cycle (data not shown). Therefore, other JMJD3 target genes may be responsible for the control of M-BMM proliferation.

ChIP-Seq analysis generally revealed a slight difference in H3K27me3 levels between wild type and Jmjd3 ^{− / −} M-BMM in the gene promoter region. Nevertheless, the profile of gene expression examined by microarray analysis is significantly different between wild-type and Jmjd3 ^-/- M-BMM, and against chitin or parasitic infections in vivo Response was severely impaired in Jmjd3 ^{− / −} mice. The Hoxa and Bmp2 genes are known to be potential targets of JMJD3 (De Santa, F. et al., Cell 130, 1083-1094 (2007)), but the expression levels of these genes are There was no decrease in Jmjd3 ^{− / −} cells, and the level of H3K27me3 was similar between wild type and Jmjd3 ^{− / −} M-BMM. These results suggest that other H3K27 demethylases, such as UTX and UTY, compensate for Jmjd3 deficiency in macrophages. On the other hand, we identified IRF4 as one of the direct JMJD3-specific target transcription factors. IRF4 has been shown to be involved in plasma cell differentiation, class switch recombination in B cells, and Th2 cell differentiation (Ahyi, AN et al., J Immunol 183, 1598-1606 (2009) and Klein, U. et al., Nat Immunol 7, 773-782 (2006)). It has also been reported that IRF4 functions in regulatory T cells to regulate Th2 responses (Zheng, Y. et al., Nature 458, 351-356 (2009)). Indeed, Irf4 ^{− / −} mice have been shown to be defective in the Th2 response to Brazilian helminth infections (Honma, K. et al., Proc Natl Acad Sci USA 105, 15890-15895 (2008)). In Irf4 ^{− / −} mice, lack of induction of M2 macrophages upon chitin administration, macrophage loss in Irf4 ^{− / −} mice appears to contribute to an abnormal response to Brazilian helminth infection. In macrophages, IRF4 functions as a negative regulator of TLR signaling by associating with MyD88 (Honma, K. et al., Proc Natl Acad Sci USA 102, 16001-16006 (2005) and Negishi, H. et al., Proc Natl Acad Sci USA 102, 15989-15994 (2005)).

Jmjd3 ^{− / −} mice showed neonatal death due to poor growth of lung tissue. JMJD3 directly regulates macrophage IRF4 expression, while Irf4 ^{− / −} mice did not show stunting. Thus, JMJD3 regulates genes other than IRF4 for normal tissue development in lung tissue and we focused solely on the role of this molecule in macrophages.

  This change in the non-genetic context appears to be an important step in determining macrophage differentiation. Future development of means to specifically modulate JMJD3 demethylation activity may help to manipulate macrophages to gain antiparasite host defense and tissue repair.

Claims (10)

  A Jmjd3 gene-modified non-human mammal, wherein the Jmjd3 gene in the genome is modified.   The non-human mammal according to claim 1, which is a Jmjd3 gene homo-modified animal in which both alleles in the Jmjd3 gene are inactivated.   A Jmjd3 gene-modified bone marrow chimeric non-human mammal having the Jmjd3 gene homo-modified bone marrow cells derived from the non-human mammal according to claim 2 in bone marrow.   The non-human mammal according to any one of claims 1 to 3, wherein the non-human mammal is a mouse or a rat.   The non-human mammal according to any one of claims 1 to 4, wherein the non-human mammal is a mouse.   A Jmjd3 gene-modified bone marrow cell derived from the non-human mammal according to any one of claims 1 to 5.   The non-human mammal according to any one of claims 1 to 5, or the bone marrow cell-derived Jmjd3 gene-modified macrophage according to claim 6.   (I) a step of administering a candidate substance to the non-human mammal according to any one of claims 1 to 5, (II) (a) expression of M2 macrophages and / or markers of M2 macrophages in the non-human mammals Or (b) monitoring the activity of the Jmjd3 protein and / or Jmjd3 protein target molecule, and (III) in step (II) as compared to not administering the candidate substance (a) M2 macrophages and / or M2 Differentiation into M2 macrophages, comprising the step of selecting a candidate substance in which increased expression of a macrophage marker is detected or (b) an increased activity of a Jmjd3 protein and / or Jmjd3 protein target molecule is detected Accelerator screening method.   (I) a step of bringing a candidate substance into contact with the Jmjd3 gene-modified macrophage according to claim 7, (II) (a) differentiation of the Jmjd3 gene-modified macrophage into M2 macrophage, (b) expression of a marker of M2 macrophage, or ( c) monitoring the activity of Jmjd3 protein and / or Jmjd3 protein target molecule in Jmjd3 gene-modified macrophages, and (III) in step (II) compared to the case where no candidate substance is contacted, A step of selecting a candidate substance in which differentiation is induced, (b) an increase in the expression of a marker of M2 macrophage is detected, or (c) an increase in the activity of a target molecule of Jmjd3 protein and / or Jmjd3 protein is detected. A screening method for an agent for promoting differentiation into M2 macrophages.   (I) a step of bringing a candidate substance into contact with the Jmjd3 gene and / or Jmjd3 protein in vitro or in a non-human mammal, and (II) a step of monitoring changes in Jmjd3 gene expression and / or demethylase activity of the Jmjd3 protein. And (III) selecting a candidate substance in which a change in Jmjd3 gene expression and / or demethylase activity is detected in step (II) as compared with the case where the candidate substance is not contacted, A screening method for a substance that controls differentiation of M2 macrophages.