KR101215670B1 - Composition for preventing or treating immune disease comprising Grim19 as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of immune diseases containing Grim19 as an active ingredient, and more particularly, the present invention provides a composition for the prevention or treatment of immune diseases, containing Grim19 protein as an active ingredient, an immunosuppressive composition, A method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells using Grim19 protein and a method for activating regulatory T cells. Grim19 according to the present invention is excellent in inhibiting the differentiation of cytotoxic Th17 cells that produce and secrete inflammatory cytokines, immunomodulation having the characteristics of inhibiting the function of abnormally activated immune cells and controlling the inflammatory response T cell (Treg) has an excellent activating effect, so it can be usefully used as a pharmaceutical composition or immunosuppressant that can prevent or treat immune diseases such as autoimmune diseases, inflammatory diseases and transplant rejection diseases caused by abnormal regulation of immune response. Can be.

Description

Composition for preventing or treating immune disease comprising Grim19 as an active ingredient

The present invention relates to a pharmaceutical use of Grim19 used to prevent or treat immune diseases caused by an abnormal immune response, and more particularly, to a composition for preventing or treating immune diseases containing Grim19 as an active ingredient. .

T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other so that they are not activated with each other.

Therefore, most of the immune diseases can be attributed to the imbalance between these two immune cells, for example, abnormally increased activity of Th1 cells can cause autoimmune diseases, and abnormally increased activity of Th2 cells It is known that immune diseases occur due to hypersensitivity reactions.

Meanwhile, according to a recent study on the differentiation of Th1 cells, the existence of a new group of immunoregulatory T cells (Tregs), which can control Th1 cell activity, is known, and studies on the treatment of immune diseases using the same are emerging. In addition, since Treg cells inhibit the function of abnormally activated immune cells to control the inflammatory response, many experiments have been reported to treat immune diseases through the action of increasing the activity of Treg cells.

In addition to the Treg cells, another group made during the differentiation is Th17 cells, which are known to be formed through a process similar to the differentiation of Treg cells during the differentiation of undifferentiated T cells. That is, differentiation of Treg cells and Th17 cells is performed in the presence of TGF-β in common but does not require IL-6 in Treg cells, whereas IL-6 is present in combination with TGF-β in Th17 cells. Differentiate in situations In addition, differentiated Th17 cells are characterized by the secretion of IL-17.

Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression. Therefore, in the case of autoimmune diseases which are not controlled by Treg cells among autoimmune diseases, development of therapeutic agents for autoimmune diseases that target the inhibition of Th17 cell activity has been highlighted.

Currently used immunosuppressive drugs are immunosuppressants that block signal transduction pathways in T cells. These immunosuppressive agents are toxic, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, and nausea. There is a problem that side effects such as renal failure occur.

In addition to the treatment of immune diseases by inhibiting the activation of T cells, a treatment for controlling the amount of cytokines secreted from immune cells and a therapeutic method using antibodies targeting cytokines secreted from immune cells have been developed. There is. However, this method has to take a long time to apply to the patients after the actual clinical trials, the method using the antibody has a problem that too much cost in the antibody manufacturing process.

Therefore, there is a need for the development of a new immune disease treatment agent that is inexpensive and has excellent therapeutic effect.

Accordingly, an object of the present invention is to provide a composition for the prevention or treatment of immune diseases containing Grim19 protein as an active ingredient, which can effectively treat immune diseases caused by an abnormal immune response.

Another object of the present invention is to reduce the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating the undifferentiated T cells with an expression vector comprising a Grim19 protein or a polynucleotide encoding said protein. Or to provide a method for suppressing.

Another object of the present invention is to provide a regulatory T cell comprising the step of treating a regulatory T cell (Treg) with an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein in vitro. To provide a way to activate it.

Furthermore, another object of the present invention is to provide an immunosuppressive composition containing an expression vector containing a Grim19 protein or a polynucleotide encoding the protein as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of immune diseases containing Grim19 protein as an active ingredient.

In one embodiment of the present invention, the Grim19 protein may have an amino acid sequence represented by SEQ ID NO: 1.

In one embodiment of the invention, the Grim19 protein can reduce or inhibit the differentiation of undifferentiated T cells into Th17 cells.

In one embodiment of the invention, the Grim19 protein may promote or increase the activity or amplification of Regulatory T cells (Treg).

In one embodiment of the present invention, the immune disease is an autoimmune disease; Inflammatory disease; And a transplant rejection disease of cells, tissues or organs.

In one embodiment of the present invention, the autoimmune disease or inflammatory disease is autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, scleroderma, myasthenia gravis , Polymyositis / skin myositis, Hashimoto's disease, autoimmune cytopenia, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus.

In addition, the present invention provides a method for reducing the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating the undifferentiated T cells with an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein. It provides a method of suppressing.

In one embodiment of the present invention, the reduction or inhibition of differentiation of undifferentiated T cells into Th17 cells may be achieved through inhibition of production of IL-17 cytokines.

In addition, the present invention activates a regulatory T cell comprising the step of treating a regulatory T cell (Treg) with an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein in vitro. It provides a method to make it.

In one embodiment of the present invention, activated regulatory T cells may have increased expression of Foxp3 and increased production of IL-10 cytokines.

The present invention also provides a composition for the prevention or treatment of immune diseases comprising a promoter and an expression vector comprising a polynucleotide encoding Grim19 operably linked thereto as an active ingredient.

In one embodiment of the present invention, the polynucleotide may have a nucleotide sequence represented by SEQ ID NO: 12.

Furthermore, the present invention provides an immunosuppressive composition containing an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein as an active ingredient.

Grim19 according to the present invention is excellent in inhibiting the differentiation of cytotoxic Th17 cells that produce and secrete inflammatory cytokines, immunomodulation having the characteristics of inhibiting the function of abnormally activated immune cells and controlling the inflammatory response It is effective as a pharmaceutical composition or immunosuppressant that can prevent or treat immune diseases such as autoimmune disease, inflammatory disease and transplant rejection disease caused by abnormal regulation of immune response because of its excellent effect of enhancing T cell (Treg) activity. Can be used.

1 is a graph showing the results of measuring the secretion amount of IL-17 cytokines produced under Th17 differentiation conditions after treatment with siRNA Grim19 to CD4 + T cells isolated in one embodiment of the present invention.
Figure 2 is a graph showing the result of measuring the secretion amount of IL-17 cytokines produced after treatment with each concentration of Grim19 (1, 10, 50ng / ml) to CD4 + T cells isolated in one embodiment of the present invention to be.
Figure 3 shows the results of comparing the expression level of Foxp3 in cells after RT-PCR after treatment with the concentration of 1 and 10ng / ml Grim19 to CD4 + T cells isolated in one embodiment of the present invention It is a graph.
FIG. 4 shows a recombinant vector (pcDNA3.1-mGrim19) prepared by cutting pcDNA3.1 + and Grim19 cDNA clone into restriction enzymes BamH1 and Xho 1, respectively, and inserting Grim19 cDNA into pcDNA3.1 + in one embodiment of the present invention. It is shown.
Figure 5 is a graph showing the results of measuring the expression level of IL-17 and Foxp3 through the flow cytometry (FACs stain) after transducing mouse CD4 + T cells by preparing a Grim19 expression vector in one embodiment of the present invention to be.
Figure 6 is a graph showing the results of evaluating arthritis index according to the date by treating the Grim19 expression vector in the arthritis mouse biological model in one embodiment of the present invention as a control group (MOCK) It was made.
FIG. 7 shows the results obtained by treating the joints of arthritis-induced mice in one embodiment of the present invention and then staining with hematoxylin and eosin (H & E), Safranin O, and Toluidine blue, and assessing the arthritis index of joint tissue. As a graph shows, CIA refers to a group of arthritis mice not treated with Grim19 protein.
8 is a graph showing an analysis of IgG2a antibody using enzyme-linked immunosorbent adsorption (ELISA) method by separating serum from blood of a group of arthritis-induced mice in one embodiment of the present invention.
9 is an embodiment of the present invention treated with arthritis-induced mice Grim19 expression vector by concentration and then the joints were collected by immunochemical staining method inflammatory cytokines IL-1b, IL-6, IL-17, TNF -a, RANK, VEGF stained and analyzed by light microscopy.
Figure 10 is treated with Grim19 protein by concentration in arthritis induced mice in one embodiment of the present invention and observed the expression of IL-17 / Th17 and Foxp3 + Treg cells expressed in the spleen using flow cytometry (FACs stain) The results and the expression of the gene expression of IL-17 and Foxp3 were observed by real-time PCR.
Figure 11 shows the results of staining by treating the spleen tissue induced arthritis-induced mouse spleen tissue treated with Grim19 protein in one embodiment of the present invention using confocal microscopy.

The present invention first identified that the Grim19 protein has an effect of preventing or treating immune diseases through an immunomodulatory action, and thus the present invention provides a composition for preventing or treating immune diseases containing Grim19 protein as an active ingredient. It has its features.

The present inventors have focused on the Grim19 gene in order to develop a new immunomodulator for the treatment of immune diseases. The Grim19 (gene associated with retinoid-IFN-induced mortality 19) has been found to be a gene associated with cell death, and more recently yeast- It has been known as a partner to interact with STAT3 through 2-hybrid screening (Zhang J, Yang J, Roy SK, Tininini S, Hu J, Bromberg JF.The cell death regulator Grim-19 is an inhibitor of signal transducer and activator of transcription 3. Proc Natl Acad Sci USA . 2003; 100: 93429347). When early STAT3 related factors were discussed, PIAS3 and suppressor of cytokine signaling 3 (SOCS3) have been cited as inhibitory feedback of STAT3, and SOCS proteins have the activity of inhibiting ligand-induced responses by inhibiting JAKs, The PIAS protein is known to have activity that inhibits the phosphorylation of STAT3 (Starr R, Hilton DJ. Negative regulation of the JAK / STAT pathway. Bioessays 1999; 21: 4752). Grim19 is also known to play a role in inhibiting STAT3-induced transcription. Interestingly, Grim19, unlike SOCS3 and PIAS3, does not inhibit the phosphorylation of tyrosin and serine residues or interfere with DNA binding (Chung CD). , Liao J, Liu B, Rao X, Jay P, Berta P. Specific inhibition of Stat3 signal transduction by PIAS 3. Science. 1997; 278: 18031805). On the other hand, Grim19 binds to the trans activation domain (TAD) of STAT3 and inhibits STAT3 phosphorylation and translocation into the nucleus when it inhibits STAT3-related transcription (Dhananjaya V. Kalvakolanu. The Grims: a new interface between cell death regulation and interferon / retinoid induced growth suppression.Cytokine & Growth Factor Reviews. 2004; 15: 169194).

On the other hand, STAT (Signal transducers and activators of transcription) is a signal transduction and transcription regulator protein, is activated by extracellular stimulation of cytokines, hormones, growth factors, etc., phosphorylation of tyrosine residues, dimers by the interaction of the SH2 domain A dimer is formed that enters the nucleus and binds to a specific promoter. The signaling system of these STAT proteins can be inhibited by dephosphorylation and protein degradation.

In particular, recently, active forms of STAT1, STAT3 and STAT5 have been found in various carcinomas. STAT3 is not only a hematological cancer such as leukemia, but also breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer and prostate cancer. It is active in various solid cancers and has become an important anticancer target (Hua Yu and Richard Jove, Nature Review Cancer ., 2004, 8, 945).

In addition, STAT3 activity has been known to inhibit apoptosis, induce angiogenesis and induce immune evasion (Wang T. et al., Nature Medicine ., 2004, 10, 48). Therefore, inhibition of STAT3 activity is effective in controlling tumors by a complex anti-cancer mechanism, and since STAT3 protein is involved in various intracellular functions as well as tumors, its inhibitor discovery can be developed as an immunosuppressive agent.

In general, the immune system controls specific immune responses to autoantigens in a normal state, and also suppresses immune responses to external antigens. For example, a pregnant woman's response to an unborn baby and a chronically infected microorganism Reaction. These phenomena are known to be induced by clonal deletion, clone anergy and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen specific immunotolerance can be induced. Investigating some patients who accidentally acquired immunotolerance against transplanted antigens or experimentally induced animal models showed that all three of these mechanisms are involved in immunological tolerance. Is attracting attention as an important cell involved in controlling almost all immune responses of living body such as autoimmune, tumoral immunity, infectious immune response as well as transplantation immune response.

In particular, immunoregulatory T lymphocytes (Tregs), which have recently been discovered, can be divided into natural Treg and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, Is immunosuppressed from the time when it is newly produced in the thymus, and is present in a frequency of 5 to 10% of the peripheral CD4 + T lymphocytes of normal individuals. The mechanism of immunosuppression of this cell is not yet known, but it has recently been discovered that the expression control factor of the gene, Foxp3, plays an important role in the differentiation and activity of the cell. In addition, peripheral natural T cells can be differentiated into cells that exhibit immunosuppressive effects upon stimulation of autologous or external antigens under certain circumstances, which are called adaptive or inducible Tregs and secrete IL-10. These include Tr1, Th3 and CD8 Ts that secrete TGF-β.

In addition, as described in the prior art, T cells also differentiate into Th17 cells through differentiation in addition to Treg cells. Th17 cells are made in the presence of TGF-β in common with Treg cells, but in the case of Treg cells, IL While it does not require -6, Th17 cells are characterized by differentiating in the presence of IL-6 with TGF-β and secreting IL-17.

In addition, Th17 cells are characterized by having cytotoxicity that maximizes the signal of the inflammatory response to accelerate disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the ways to treat immune diseases.

In this regard, the present inventors examined whether Grim19 of the present invention has an effect of treating immune disease by inhibiting the differentiation from T cells to pathological Th17 cells. According to one embodiment of the present invention, the human peripheral blood nucleus SiRNA Grim19 was treated on CD4 + T cells isolated from cells to knock down the Grim19 gene in the cell to inhibit expression, and then the conditions required for differentiation into Th17 cells, ie, cytokines necessary for differentiation into Th17 cells (eg, After culturing in the presence of IL-6, IL-21 or IL-23), the amount of IL-17, a cytokine produced in Th17 cells, was measured. As a result, when the siRNA Grim19 was introduced into the cell, the production amount of IL-17 was increased compared to the control group that did not introduce the siRNA Grim19 into the cell (see Fig. 1).

In addition, according to another embodiment of the present invention, after treatment with Grim19 for each concentration of CD4 + T cells isolated in the present invention, and then cultured in Th17 cell differentiation conditions, and then measuring the production of IL-17, The higher the concentration of Grim19 was shown to decrease the production of IL-17 (see Figure 2).

Therefore, the present inventors have found that the Grim19 protein according to the present invention acts to inhibit or reduce the differentiation of undifferentiated T cells into Th17 cells. It was found that they inhibit differentiation into Th17 cells.

Therefore, the Grim19 protein of the present invention is characterized by preventing or treating immune diseases through the action of reducing or inhibiting the differentiation of undifferentiated T cells into pathological Th17 cells.

In addition, the present inventors investigated whether Grim19 having an inhibitory effect on the production of IL-17 through STAT3 also affects Treg cells, which are immunoregulatory T cells. According to one embodiment of the present invention, after treatment of undifferentiated T cells with Grim19 After culturing under conditions differentiating to Th17 cells, the amount of IL-10 cytokine secreted from Treg cells and the expression level of Foxp3 gene were observed. -10 cytokine production was increased, Foxp3 gene expression was increased compared to the control group, IL-10 cytokine production and Foxp3 gene expression was increased proportionally as the concentration of Grim19 treated (See FIG. 3).

In one embodiment of the present invention, the present inventors, in order to determine the activation level of regulatory T cells (Treg) by Grim19, the expression level of Foxp3 gene and IL-10 cytokine production amount according to the treatment of Grim19 Foxp3 is mainly present in regulatory T cells derived from the thymus and is a transcriptional factor present in cells with CD4 + CD25 + labeling antigen. Its function is that of recognizing antigens against T cells expressing Foxp3, T cells that have low reactivity to the antigen and can potentially cause autoimmunity among CD4 + CD25- T cells that do not express Foxp3 differentiated from the thymus Has a role as a suppressor T cell that inhibits IL-2 production and cell division. In addition, Foxp3 stimulates not only IL-2 but also IL-4, which is influenced by NFAT, which is a transcription factor, in CD25- T cells through regulatory T cells expressing Foxp3 and cell-cell contact with them, Lt; RTI ID = 0.0 > IFN-gamma. ≪ / RTI > Therefore, in the case of T cells expressing Foxp3 having such a function, it is applied to the treatment of immune diseases through the action of inhibiting or regulating the immune response, and furthermore, the CD4 T cells expressing Foxp3 present in humans. To apply self-antigen specific T cell clones to cell therapy by increasing their number through high concentration of IL-2 cytokine and combination treatment with anti-CD3 and anti-CD28 antibodies There have been attempts.

Therefore, the present inventors confirm that the expression of Foxp3 is increased in regulatory T cells (Treg) when Grim19 is treated through the results of one embodiment according to the present invention, and IL, a cytokine secreted from Tregs By confirming that the secretion amount of -10 was increased, it was found that Grim19 of the present invention can be used to prevent or treat immune diseases by activating or amplifying Regulatory T cells (Treg).

Therefore, Grim19 protein of the present invention is characterized by preventing or treating immune diseases through the action of promoting or increasing the activity or amplification of Regulatory T cells (Treg).

In the present invention, the activity means that all the mechanisms of the regulatory T cells (Treg), ie, Treg cells including both natural and adaptive Treg cells, are promoted or promoted in vivo. In other words, the immunomodulatory action such as an immunosuppressive reaction is promoted or promoted so that the immune response in vivo is maintained in a normal state.

In addition, the amplification (expansion) refers to the differentiation and proliferation of undifferentiated T cells into regulatory T cells, 'differentiation' is a phenomenon that the structure or function of each other during the division and growth of cells, that is, the organism Cells or tissues are changed in form or function in order to perform a given task, and 'proliferation' refers to the division of cells and homogeneous ones, which usually increase the number of cells in the body of a multicellular organism. It says to go.

Therefore, the present invention can provide a composition for the prevention or treatment of immune diseases containing the Grim19 protein as an active ingredient, wherein the Grim19 according to the present invention may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 1.

In addition, the Grim19 protein of the present invention may be a functional equivalent to the polypeptide having an amino acid sequence represented by SEQ ID NO: 1. The term 'functional equivalent' refers to a sequence homology with at least 60%, preferably 70%, more preferably 80% or more of the amino acid sequence represented by SEQ ID NO. 1 as a result of the addition, substitution or deletion of amino acids. A polypeptide exhibiting substantially homogeneous activity with Grim19 of the present invention. Here, "substantially homogeneous activity" refers to the activity of Grim19 described above. Such functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted or added. Substitution of amino acids may preferably be conservative substitutions, examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located at portions not directly involved in the activity of Grim19 of the present invention. The functional equivalent may also include polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of Grim19 and its physiological activity. For example, fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activities for altering the stability, storage, volatility or solubility of the polypeptide of the present invention may be included.

Grim19 protein can also be obtained using genetic recombination techniques. When using recombinant technology, a polynucleotide encoding Grim19 protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured so that the Grim19 protein is expressed, and the Grim19 protein is recovered from the host cell. It can be obtained by the process. The protein may be expressed in a selected host cell and then subjected to conventional biochemical separation techniques such as treatment with a protein precipitant (salting-out method), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like. In order to separate proteins having high purity, they are used in combination.

Therefore, the present invention can provide a composition for the prevention or treatment of immune diseases comprising a vector comprising a promoter and a polynucleotide encoding Grim19 operably linked thereto.

In the present invention, the polynucleotide encoding Grim19 may be isolated or artificially modified in nature, and the base sequence encoding the Grim19 protein may be modified by substitution, deletion or insertion of one or more nucleic acid bases. The protein expressed by the modification should not contain significant changes in its biological functionality. Such modifications include modifications to heterologous homologous genes.

Therefore, the polynucleotide encoding the Grim19 protein is preferably characterized in that it is represented by the nucleotide sequence of SEQ ID NO: 12, but is not limited to the above polynucleotide, that is, at least 70%, preferably at least 80% More preferably, it is represented by the base sequence which has 90% or more homology.

Preferably, the polynucleotide encoding the Grim19 protein of the invention may be provided in the form of a recombinant expression vector operably linked to the vector expressing it. Expression vectors comprising Grim19 polynucleotides include, but are not limited to, plasmids, phages, cosmids, viral vectors or other mediators known in the art. The vector may be self-replicating or integrated into the host DNA.

Polynucleotides encoding the Grim19 protein may be combined with expression control sequences such as promoter / enhancer sequences and other sequences necessary for transcription, translation or processing. Regulatory sequences include tissue-specific regulatory and / or inducible sequences as well as direct constitutive expression of nucleotides. The design of the expression vector may be determined by factors such as the host cell to be transfected, the level of expression desired, and the like.

As the expression vector expressing the Grim19 protein included in the composition of the present invention, a non-viral vector or a viral vector may be used. Viral vectors include, for example, replication defective retroviruses, adenoviruses, adenovirus-associated viruses, and the like. The viral vector must meet the following criteria: (1) it should be able to infect the desired cell, so that a viral vector with an appropriate host range has to be selected; (2) And (3) the vector must be safe for the host. Other viral vectors that can be used for gene transfer into cells include murine leukemia virus (MLV), JC, SV40, polyoma, Epstein-Barr virus papilloma virus, vaccinia, poliovirus, herpes virus, sindbis virus, lenti Viruses, and other human and animal viruses.

The expression vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells Can be introduced into cells by the method of (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988). .

In the present invention, the polynucleotide encoding the Grim19 protein as an active ingredient means that the polynucleotide itself encoding the Grim19 protein is included as an active ingredient, and the expression vector into which the polynucleotide encoding the Grim19 protein is inserted. Means to include as an active ingredient.

On the other hand, the present inventors investigated whether Grim19 has an inhibitory effect and an immunostimulating effect on Th17, a pathological cell in vivo.

First, it was examined whether overexpression of Grim19 affects Th17 / IL-17 and Foxp3 + Treg. According to one embodiment of the present invention, the plasmid vector, pcDNA3.1-mGrim19, was inserted by inserting Grim19 cDNA into pcDNA3.1 +. 4), transfected mouse CD4 + T cells and analyzed for Th17 / IL-17 cells and Foxp3 + Treg cells using flow cytometry (FACs). Th17 / IL-17 expressing cells when Grim19 was overexpressed. Was suppressed while the expression of Foxp3 + Tregs was significantly increased (see FIG. 5).

In addition, according to another embodiment of the present invention, rheumatoid arthritis occurs as a major protein component of cartilage, bones, and tendons, which are transformed into destructive anti-immune weapons that weaken the body's immune system, resulting in chronic inflammation of joints. Grim19 treatment was performed to evaluate the arthritis index in mice treated with arthritis with type II collagen (CII) protein, which is well known to promote Grim19. Arthritis development and arthritis were inhibited in the group treated with the vector, the inhibitory activity was found to be dependent on the Grim19 vector concentration (see Fig. 6), and in the joint tissues, the degree of destruction of the joint was increased in the group treated with the Grim19 vector. Not only was it significantly suppressed, but the infiltration of inflammatory cells was also shown to be inhibited (see Figure 7).

The present inventors investigated whether Grim19 of the present invention has an effect of inhibiting the production of inflammatory autoantibodies. According to one embodiment of the present invention, blood is collected after 3 weeks after inducing arthritis in a mouse biomodel. After isolation, the expression level of inflammatory antibody (IgG2a) in serum was measured. In the mice treated with Grim19, the expression of IgG2a in serum was suppressed and the expression of CII-IgG2a specific to type 2 collagen, an arthritis autoantigen, was detected. It has also been shown to be inhibited (see FIG. 8).

Thus, the present inventors have found that the Grim19 protein acts to inhibit inflammatory antibodies in vivo in an arthritis animal model, and selectively inhibits inflammatory autoantibodies specific for the type 2 collagen protein antigen. And it was found.

In addition, the present inventors investigated whether the Grim19 protein of the present invention has an effect of inhibiting inflammatory cells in joints and antigen-specific autoantibodies in serum. According to one embodiment of the present invention, arthritis induced mouse living bodies Osteoarthritis is treated because it contains the inflammatory cytokines IL-1b, IL-6, IL-17, TNF-a, and various TNF receptor-associated factor (TRAF) -binding sites, by treating the joints of the model and using immunochemical staining. Receptor Activator of NF-κB (RANK), which regulates the differentiation, function or survival of cells, and Vascular Endothelial Growth Factor (VEGF), which is known to be involved in the growth and migration of vascular endothelial cells, were stained and analyzed by light microscopy. As a result, in the mouse group treated with Grim19 protein, inflammatory cells, cells expressing RANK and VEGF, were found to be significantly inhibited (see FIG. 9).

In addition, according to another embodiment of the present invention, in order to determine whether the Grim19 protein has the effect of simultaneously regulating IL-17 and Foxp3 gene, Grim19 is treated in a mouse biomodel, and expressed in spleen cells IL-17 / FACs staining was performed to observe the expression of Th17 and Foxp3 + Treg cells. Real-Time PCR was performed to investigate the expression of IL-17 and Foxp3 at the gene level. Foxp3 + Tregs were activated in the mouse group, and the same result was observed in gene expression, and gene expression of IL-17 was reduced in spleen cells (see FIG. 10).

Therefore, through the above results, the present inventors can inhibit the infiltration of inflammatory cells by the Grim19 protein according to the present invention in arthritic joints, and inhibit the invasion of cells expressing inflammatory cytokines, angiogenesis factors, and articular destructive factors, IL It was found that gene expression of -17 and Foxp3 could be regulated simultaneously.

Furthermore, the present inventors have an effect of inhibiting IL-17 through STAT3 of the Grim19 protein according to the present invention, and thus, Foxp3 / Treg and IL-17 / Th17 are affected when the Grim19 protein is inhibited. According to one embodiment of the present invention, staining cells expressing Foxp3 and IL-17 in mouse spleen tissue of the Grim19-treated arthritis mice group and confirmed by confocal microscopy, the Grim19 protein IL-17 + cells were inhibited in expression in treated arthritis mice, whereas Foxp3 + Treg cells were found to increase in expression (see FIG. 11).

Therefore, the Grim19 protein of the present invention regulates the expression of Foxp3 and IL-17 genes and regulates Foxp3 + Tregs and Th17 / IL-17 in splenic cells in arthritis mice, thereby controlling autoimmune arthritis. Furthermore, Grim19 protein was found to be effective in controlling both inflammatory and autoimmune diseases.

Therefore, the Grim19 protein according to the present invention can reduce or inhibit Th17 cells, which are pathological cells, while preventing or treating immune diseases through the action of promoting or enhancing the activity or amplification of regulatory T cells.

In the present invention, the immune disease means a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to the pathology of the mammal. In addition, the stimulation or discontinuation of the immune response may include all diseases that have a compensatory effect on the progress of the disease, and the present invention may include diseases caused by an irritable immune response. Examples of such immune disorders include, but are not limited to, autoimmune diseases; Inflammatory disease; And transplant rejection diseases of cells, tissues or organs.

In addition, one of the most important properties of all normal individuals is that they do not react negatively to the antigenic material that constitutes the self, while non-self antigens are recognized and reacted to remove I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when there is a problem in inducing or maintaining such self-tolerance, an immune response occurs to autoantigens, which causes the attack of one's own tissue. The disease caused by this process is called an autoimmune disease. .

Inflammatory diseases are also referred to as TNF-α (tumor necrosis factor-α) and IL-1 (interleukin- 1), refers to a disease caused by inflammatory substances (inflammatory cytokines) such as IL-6, prostagladin, luecotriene or nitric oxide (NO).

On the other hand, in order to successfully transplant the organ, the recipient's immune rejection response to the transplanted cells and organs must be overcome. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) A reaction occurs. Immunosuppressive agents have been used to reduce these transplant immune rejection responses. A common goal of these immunosuppressive agents is to inhibit T cell-mediated immune responses to grafts. Recently, regulatory T cells have been used to suppress immune responses, Methods to treat refractory diseases have been attempted.

In addition, the type of autoimmune disease or inflammatory disease in the present invention is not limited thereto, but autoimmune hepatitis, chronic rheumatoid arthritis, insulin-dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, skin Sclerosis, myasthenia gravis, multiple myositis / skin myositis, Hashimoto's disease, autoimmune cytopenia, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus.

Therefore, the composition according to the present invention can be used as a pharmaceutical composition that can prevent or treat immune diseases.

The term 'treatment', unless stated otherwise, means to reverse, alleviate, inhibit, or prevent the progression or alleviation of the disease or condition to which the term applies, or one or more symptoms of the disease or condition. As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above. Thus, "treatment" or "therapy" of an immune disease in a mammal may include one or more of the following:

(1) inhibiting the growth of the immune system, i.e., inhibiting its development,

(2) preventing the spread of immune diseases, i.e., preventing metastasis,

(3) alleviation of immune diseases.

(4) preventing the recurrence of immune disorders, and

(5) palliating symptoms of immune disorders

The composition for preventing or treating immune diseases according to the present invention may include a pharmaceutically effective amount of Grim19 protein alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of an immune disease.

The pharmaceutically effective amount of Grim19 protein according to the present invention is 0.5-100 mg / day / kg body weight, preferably 0.5-5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed according to the degree of symptoms of immune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

Also, the pharmaceutically acceptable hereinabove refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating immune diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by the route of administration, age, sex, It may be appropriately selected according to various factors such as the weight and the severity of the patient, and the composition for preventing or treating an immune disease according to the present invention is combined with a known compound having the effect of preventing, ameliorating or treating the symptoms of an immune disease. May be administered.

Accordingly, the present invention can provide a medicament for the prevention or treatment of immune diseases, including a composition comprising a Grim19 protein or an expression vector comprising the polynucleotide encoding the protein as an active ingredient, and the present invention further provides a Grim19 protein or An immunosuppressive composition containing an expression vector containing a polynucleotide encoding the protein as an active ingredient may be provided.

In addition, the present invention provides for the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising the step of treating the undifferentiated T cells with an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein. It is possible to provide a method of reducing or inhibiting, wherein the reduction or inhibition of differentiation of the undifferentiated T cells into Th17 cells may be achieved through inhibition of production of IL-17 cytokines.

In addition, the present invention provides a regulatory T cell comprising the step of treating a regulatory T cell (Treg) with an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein in vitro. It is possible to provide a method for activating the regulatory T cells activated according to the present invention may increase the expression of Foxp3, the production of IL-10 cytokines may be increased.

The method of reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, and the method of activating regulatory T cells, according to the present invention, comprises a Grim19 protein or a polynucleotide encoding said protein. The method of treating an expression vector comprising a cell may be to directly treat the Grim19 protein in a medium for culturing the cell, or an expression vector comprising a gene encoding a Grim19 protein using a method known in the art. By introducing into the cell, the Grim19 gene can be expressed in the cell.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Th17 In a cell Grim19 of IL -17 Production Inhibitory Activity Assay

In order to determine whether Grim19 is effective in treating diseases caused by abnormal immune response, such as autoimmune diseases, the present inventors secreted IL-17 cytokines by Grim19 in Th17 cells associated with autoimmune diseases. The change was measured. That is, for this purpose, Th 17 cells were obtained from differentiation from CD4 + T cells, and CD4 + T cells were collected from a normal person using a heparin-treated syringe, and then blood cells were obtained from peripheral blood mononuclear cells (PBMC). ) Was separated by centrifugal gradient using Ficoll-PaqueTM. The isolated PBMC was reacted with anti-human CD4 ⁺ T cell biotin-Ab cocktail for 15 minutes at 4 ° C. and then with anti-human CD4 biotin microbeads at 4 ° C. for 15 minutes, washed with MACs buffer, and then autoMACs CD4 ⁺ T cells were isolated by a negative fraction. Isolated CD4 ⁺ T cells were washed with PBS and cell cultures containing 10% fetal calf serum, penicillin (100 U / mL) and streptomycin (100 g / mL) inactivated at 55 ° C. for 30 minutes (RPMI1640 medium). , Gibco BRL, USA). Thereafter, differentiation was induced from the isolated CD4 ⁺ T cells to Th 17 cells. The differentiation into Th 17 cells was determined by antigen donor cells irradiated at 5 × 10 ⁵ and 5000 rad of CD4 ⁺ T cells isolated from human PBMC. APC) 5 × 10 ⁵ cells were dispensed into 24-well plates coated with 1 μg / mL of anti-CD3, 1 μg / mL of anti-CD28 antibody, 20 ng / mL of IL-6, 20 ng of IL-1β Differentiation was induced by Th17 cells by treatment with / mL, IL-23 20 ng / ml, anti-IL-4 10ng / ml and anti-IFNr 10ng / ml.

<1-1> si RNA Grim19 On by IL -17 Production Inhibition Activity Measurement

SiRNA Grim19 was used to investigate whether Grim19 is active in inhibiting the secretion of IL-17 cytokines in Th17 cells induced differentiation from CD4 + T cells as described above, ie, CD4 + T cells into Th17 cells. Before inducing cell differentiation, isolated CD4 ⁺ T cells 5 × 10 ⁵ were incubated in serum-free RPMI medium, and siRNA Grim19 was commercially treated with siRNA Grim19 on the cells for 48 hours according to the method of use thereof. Krim19 gene knocked down. Then, the amount of IL-17 cytokines in the culture medium and then cultured in a cytokine environment, that is, the above-described cytokines are 5 × 10 ⁵ cell number of APC and Th17 cells irradiated at 5000 rad to differentiate their containing medium for 48 hours. Was measured. In this case, a group treated with a transduction reagent without siRNA Grim19 was used as a control. The measurement of the produced IL-17 cytokine was collected by collecting the supernatant of the culture medium in which the cells were cultured to investigate the level of IL-17 expression using human IL-17 and sandwich ELISA. React overnight at 4 ° C. with 2 μg / mL of IL-17, block nonspecific binding with blocking solution (1% BSA / PBST), and then serially dilute recombinant IL-17 for standard measurement. The cell culture supernatant was added and reacted at room temperature for 2 hours. Thereafter, the biotinylated anti-IL-17 was reacted at room temperature for 2 hours, washed four times after the reaction, and then diluted by adding an ExtraAvidin-Alkaline Phosphatase conjugate and reacted at room temperature for 2 hours, followed by PNPP / The DEA solution was added and developed to measure absorbance at 405 nm.

As a result, as shown in FIG. 1, IL-17 cytokine production was increased when knocking down the Grim19 gene with siRNA Grim19, compared to the control group.

<1-2> Grim19  By treatment IL -17 Production Inhibition Activity Measurement

When knocking down the Grim19 gene in Th17 cells based on the results of Example <1-1>, it was confirmed that the production of IL-17 cytokines in the cells increased compared to the control group, so as to confirm more clearly. This time, the cells were treated with Grim19 and the production of IL-17 cytokines was measured in the same manner as in <1-1>.

As a result, as shown in Figure 2, Grim19 was shown to reduce the production of IL-17 cytokines in the cell, the degree of reduction of IL-17 was shown to be proportional to the concentration of Grim19 treated.

Therefore, the present inventors found that Grim19, an inhibitor of STAT3, has an effect of inhibiting the production of IL-17 cytokines. In addition, in Th17 cells recognized as important cells for inflammation-related diseases by neutrophil activity and the production of inflammatory cytokines and chemokines, Grim19 inhibits the production of IL-17 cytokines through STAT3. It could be expected to be able to prevent or treat autoimmune diseases.

< Example  2>

Treg In a cell Grim19 Impact analysis

Controlled T lymphocytes, or Treg (regulatory T lymphocytes) cells, are known to play a key role in the maintenance of immune tolerance (Nat. Rev. Immunol. 2009, 7: 305) and are produced primarily in the thymus and are transcription factors Expression of Foxp3 is required and Foxp3 is known to play a role in inhibiting the production of inflammatory cytokines. In addition, IL-10-producing cells in Treg cells are called Tr1, which is known to play a role in eliminating Tp function and tissue inflammation without Foxp3 expression. The present inventors analyzed the production of IL-10 and the expression level of Foxp3 in Grim19-treated cells undergoing Th17 differentiation.

First, the expression of Foxp3 was examined by reverse transcriptase reaction, i.e. after treatment with Grim19 at 0 ng / ml, 1 ng / ml and 10 ng / ml for the cells under Th17 differentiation conditions, Each RNA was extracted according to a method known in the art, and then polymerase chain reaction was performed using primers capable of amplifying human Foxp3 and a control β-actin, and a total of 25 μl of the reaction compound for RT-PCR was used. The composition of the solution was 2.5 μl of 10 × reaction buffer, 0.5 mM dNTP and each primer pair having the sequence shown in Table 1 below, and amplification was performed in a denaturation step using a Dual-bay Thermal cycler system instrument. The reaction was repeated 25 times at 94 ° C. for 30 seconds, at the annealing step at 60 ° C. for 30 seconds, and at the stretching step for 72 ° C. for 30 seconds. In addition, the production analysis of IL-10 was treated with Grim19 for each concentration of cells as described above, and then measured the degree of IL-10 cytokine production in the same manner as in <1-1>.

Primer sequence primer Primer sequence SEQ ID NO: Foxp3 sense  5-ATGCCTCCTCTTCTTCCTTGA-3 2 Foxp3 antisense  5-TGAGAAGGGCAGGGCACAAT-3 3 beta-actin sense  5-GGACTTCGAGCAAGAGATGG-3 4 beta-actin antisense  5-TGTGTTGGCGTACAGGTCTTTG-3 5

As a result, as shown in FIG. 3, the expression of Foxp3 was gradually increased in concentration-dependent manner by the treatment of Grim19, and the production of IL-10 was also increased in concentration-dependent manner.

Therefore, through the above results, the present inventors confirmed that Grim19 inhibits the action of STAT3 and inhibits cell differentiation to Th17, whereas Grim19 has an activity of increasing expression of Foxp3 and production of IL-10. By regulating the immune system by regulating cells, it was found that the symptoms of inflammation or autoimmune disease can be improved, prevented or treated.

< Example  3>

Th17 Of IL With -17 suppression Foxp3 + Treg  Increase effect analysis

Grim19 overexpression vectors were first constructed to investigate the effect and function of Grim19 when activated and differentiated by STAT3 transcription factors to produce etiological Th17 cells. That is, as shown in FIG. 4, the Grim19 cDNA clone (purchased from 21C Human Gene Bank) was cut with restriction enzymes BamH1 and Xho 1 and inserted into pcDNA3.1 + (promega) to insert Grim19 cDNA. The recombinant vector (pcDNA3.1-mGrim19) was prepared, E. coli was transformed with the recombinant vector, plasmid DNA was isolated by plasmid extraction kit (Qiagan), and the nucleotide sequence of the recombinant gene was analyzed. The recombinant plasmid vector with the correct sequence was selected and cultured to obtain a large amount of plasmid vector DNA.

Subsequently, the recombinant vector capable of overexpressing Grim19 in the cell was transfected into mouse CD4 + T cells isolated using MACs bead, and then differentiated into Th17 differentiation conditions one day later, and then Th17 / IL-17 cells and Foxp3 + Treg cells. Analysis was performed using flow cytometers (FACs). In this case, a group transduced with a mock vector was used as a control instead of the recombinant vector. In addition, in order to identify cells expressing IL-10, IFNr, IL-17, each fluorescent cytokine antibody (anti-body) with a fluorescent treatment to the cells, and at the same time a CD4 antibody having a different fluorescence than the cytokine antibody After binding to the cells, each cytokine-expressing cell was analyzed using a flow cytometer.

As a result, as shown in FIG. 5, Th17 / IL-17 cells were suppressed while Grim19 was overexpressed, whereas the expression of Foxp3 + Tregs was greatly increased, and the cells expressing Th1 / IFNr were There was no significant difference from the control group.

Therefore, Grim19 of the present invention was able to specifically inhibit Th17 / IL-17, a pathogen cell, Grim19 was able to induce Tregs even under Th17 differentiation conditions.

< Example  4>

Grim19  Effect of Arthritis Inhibition on Treatment

To determine whether Grim19 actually inhibits arthritis, we investigated the arthritis inhibitory effect of Grim19 treatment in the arthritis biomodel. Induced arthritis by intradermal injection of collagen type 2 into DBA1 / J mice At 8 days after induction, 50 μg and 100 μg of the recombinant DNA vector (Grim19 overexpressed recombinant vector) prepared in Example 3 were intravenously injected using a hydrodynamic injection technique, and a MOCK vector was injected as a control. On the 14th and 18th day after inducing arthritis, MOCK vector, 50μg and 100μg Grim19 recombinant vector were injected into leg joint muscle by electroporation. Arthritis index was evaluated after arthritis induction, and the index was evaluated from 1 to 4 points.

As a result of evaluating the arthritis index of each group, as shown in FIG. 6, the arthritis index in the mouse group injected with the recombinant vector overexpressing Grim19 was significantly reduced compared to the control group injected with the MOCK vector. The inhibitory effect of arthritis increased with increasing recombinant vector concentration of injected Grim19. In addition, the incidence of arthritis was suppressed in the group of mice treated with the Grim19 vector, and the group treated with 100 μg of the Grim19 vector showed more marked inhibition of arthritis than the control group.

< Example  5>

Inhibition of Arthritis Destruction and Inflammation Inhibition in Arthritis Biomodels

In order to confirm that Grim19 can inhibit the occurrence of arthritis through the <Example 4> and to determine whether such inhibition is caused by the destruction of the joint in the joint tissue and inflammatory cells, the joints of the mice of each group were collected and 10% Fixed in neutral buffered formalin and bone demineralized with EDTA. After embedding in paraffin, the joint tissue was made into 7 μm thick sections and attached to the slides. Before proceeding with the basic dyeing, xylene was used to pass the deparaffinization process, and ethanol was functioned from high to low concentrations. The staining process was hematoxylin and eosin staining, and Safranin O and Toluidine blue methods were used to detect proteoglycans in cartilage.

As a result of evaluating the joint tissues of each group, as shown in Fig. 7, arthritis as well as arthritis index in the mouse group injected with the Grim19 vector was significantly inhibited the degree of joint destruction, in particular the infiltration of inflammatory cells is suppressed Yes was observed.

Therefore, the present inventors have found that Grim19 according to the present invention can not only suppress the occurrence of arthritis, but also inhibit the destruction of the joint and the infiltration of inflammatory cells in the joint.

< Example  6>

Inhibition analysis of inflammatory autoantibody production

In order to determine whether inflammatory autoantibodies were inhibited by serum Grim19 in the arthritis biomodel, arthritis was induced in the mouse biomodel in the same manner as in <Example 4>. Three weeks after the induction of arthritis, blood was collected from each mouse group, and serum was separated using a centrifuge. The expression level of inflammatory antibody (IgG2a) in serum was measured using a mouse IgG2a ELISA kit provided by Bethyl.

As a result, as shown in FIG. 8, in the mouse group injected with Grim19, expression of IgG2a in serum was significantly suppressed. In addition, it was shown that the expression of CII-IgG2a specific to collagen type 2, which is an arthritis autoantigen, was significantly suppressed.

Therefore, the above results showed that Grim19 according to the present invention can inhibit inflammatory antibodies in vivo in an animal model of arthritis, and can selectively inhibit inflammatory autoantibodies specific for CII antigen.

< Example  7>

Inflammatory cytokines, Neovascular factor , Inhibitory Effect of Joint Destruction Factors

Whether Grim19 can specifically inhibit inflammatory cells such as IL-1β, IL-1, TNF-α, IL-17, and the cells expressing the joint destruction factor RANK and VEGF in arthritis animal models In order to find out, the mouse joint was treated in the same manner as in <Example 5>. In addition, inflammatory cytokines (IL-1b, IL-6, IL-17, TNF-a), RANK, and VEGF were stained and analyzed by optical microscopy using immunochemical staining.

As a result of observing the inhibitory effect of Grim19 on arthritis, inflammatory cells in joints and antigen-specific autoantibodies in serum, as shown in FIG. In contrast, Grim19-treated mice showed significant inhibition of inflammatory cells, cells expressing RANK and VEGF.

Therefore, these results indicate that Grim19 not only inhibits the invasion of inflammatory cells in arthritic joints but also inhibits the invasion of cells expressing inflammatory cytokines, angiogenesis factors and joint destruction factors.

< Example  8>

IL -17 and Foxp3  Simultaneous regulation of genes

To evaluate the co-regulation of IL-17 and Foxp3 genes by Grim19 in arthritis biomodels, IL-17 / Th17 and Foxp3 + Tregs expressed in spleen cells of each mouse group treated with 50 μg of Grim19 expression vector and MOCK vector Expression of cells was observed using the FACs stain method. In addition, in order to investigate the expression of IL-17 and Foxp3 gene, real-time PCR was performed according to a method known in the art, and the Intercalator used SYBR Green I (Roche Diagnostic), and the Light-Cycler instrument (Roche Diagnostic). It was analyzed using.

Primer sequence primer Primer sequence SEQ ID NO: IL-17 sense  5-CCT CAA AGC TCA GCG TGT CC-3 6 IL-17 antisense  5-GAG CTC ACT TTT GCG CCA AG-3 7 Foxp3 sense  5-GGC CCT TCT CCA GGA CAG A-3 8 Foxp3 antisense  5-GCT GAT CAT GGC TGG GTT GT-3 9 beta-actin sense  5-GAA ATC GTG CGT GAC ATC AAA G-3 10 beta-actin antisense  5-TGT AGT TTC ATG GAT GCC ACA G-3 11

As a result, as shown in FIG. 10, Grim19 selectively activated Foxp3 + Tregs in the arthritis biological model, and showed the same result in the gene expression of Foxp3. Gene expression of IL-17 was also significantly reduced in spleen cells of mice treated with Grim19.

Therefore, the present inventors found that Grim19 according to the present invention can simultaneously regulate gene expression of IL-17 and Foxp3 in vivo in an animal model of arthritis.

< Example  9>

Grim19  According to treatment Foxp3 Of Treg  And IL -17 / Th17 Effect analysis on

To investigate the expression changes of Foxp3 / Treg and IL-17 / Th17 cells according to Grim19 treatment in the arthritis biomodel, cells expressing Foxp3 and IL-17 in the mouse spleen tissues of arthritis and Grim19 treatment groups were collected. It was confirmed using a focus microscope. In other words, the mouse spleen was used to embed the Optimal Cutting Temperature compound (OCT compound), and then the tissue was rapidly cooled in liquid nitrogen, and attached to the slide at 7 μm thickness using the frozen slicer. Sections were then fixed with acetone and 10% normal goat serum was applied to block nonspecific reactions for 30 minutes. In the Treg assay, the primary antibody FITC-labeled anti-mFoxp3 Ab, PE-labeled anti-mCD4 Ab, APC-labeled anti-CD25 Ab diluted 1: 100 in PBS (pH7.5) and FITC for Th17 cell analysis. Reaction with -labeled anti-mCD4 Ab, PE-labeled anti-mIL-17 at 4 ° C. overnight, washed with PBS solution the next day, and stained tissues were analyzed by confocal microscopy.

As a result, as shown in Fig. 11, IL-17 + cells were significantly inhibited in the Grim19 treatment group, while Foxp3 + Treg cells were markedly increased.

Therefore, these results suggest that Grim19 regulates Foxp3 and IL-17 genes, and Foxp3 + Tregs and Th17 / IL-17 in the spleen of arthritic mice in vivo, thus enabling Grim19 to regulate both inflammation and immunity, furthermore autoimmune arthritis. It can be seen that the control.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Attach an electronic file to a sequence list

Claims (13)

A Grim19 protein as an active ingredient, wherein the Grim19 protein is a composition for preventing or treating immune diseases, characterized in that to promote or increase the activity or amplification of Regulatory T cells (Treg). delete delete delete The method of claim 1,
The immune disease is an autoimmune disease; Inflammatory diseases; And a transplant rejection disease of cells, tissues or organs, the composition for preventing or treating immune diseases. The method of claim 5,
The autoimmune diseases or inflammatory diseases include autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, skin sclerosis, myasthenia gravis, multiple myositis / skin myositis, Hashimoto disease , Autoimmune thrombocytopenia, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus, the composition for preventing or treating immune diseases. delete delete A method of activating regulatory T cells comprising treating a regulatory T cell (Treg) with an expression vector comprising a Grim19 protein or a polynucleotide encoding the protein in vitro. 10. The method of claim 9,
Activated regulatory T cells activate the regulatory T cells, characterized by increased expression of Foxp3 and increased production of IL-10 cytokines. An expression vector comprising a promoter and a polynucleotide encoding Grim19 operably linked thereto as an active ingredient, wherein the Grim19 protein expressed by the vector promotes the activity or amplification of Regulatory T cells (Tregs). Or increasing or preventing the composition for preventing or treating immune diseases. The method of claim 11,
The polynucleotide composition for the prevention or treatment of immune diseases, characterized in that it has a nucleotide sequence represented by SEQ ID NO: 12. An expression vector comprising a Grim19 protein or a polynucleotide encoding the protein as an active ingredient, wherein the Grim19 protein or the Grim19 protein expressed by the vector may inhibit the activity or amplification of Regulatory T cells (Tregs). Immunosuppressive composition, characterized in that to promote or increase.

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