KR20140091904A - Composition for treatment or prevention of autoimmune diseases comprising HtrA2 - Google Patents

Abstract

The present invention relates to a composition containing high temperature requirement protein A2 (HtrA2) as an active ingredient for treatment or prevention of autoimmune diseases. According to the present invention, the HtrA2 has an effect for effectively treating autoimmune diseases induced by abnormally regulating various immune reactions and inflammation reactions since the HtrA2 reduces the expression of STAT3 which is a transcription factor of a Th17 cell, suppresses the expression of inflammatory cytokine, and regulates the excessive immune reaction by promoting the activity of a regulatory T cell (Treg).

Description

[0001] The present invention relates to a composition for preventing or treating an autoimmune disease comprising an HtrA2 protein as an active ingredient,

The present invention relates to a composition for preventing or treating an autoimmune disease comprising an HtrA2 protein as an active ingredient.

Immunity is one of the biological self-protection systems for all external polymeric substances (antigens) that enter or are injected into living tissue. A major component of the immune system is the lymphocyte, which is made up of the bone marrow and circulates along the blood to the lymphatic tissues and organs, mainly lymph nodes, spleen, and tonsil. As cells involved in the immune response, B cells proliferate rapidly when stimulated by appropriate antigens to form clones that produce specific antibodies (immunoglobulins) that neutralize the antigen, and antibodies produced by B cells are circulating in body fluids While performing humoral immunity. In addition, T cells are generated from the thymus and migrate to lymphoid tissue, which is responsible for cell-mediated immunity that directly attacks the antigen.

On the other hand, one of the most important characteristics of all normal individuals is that they do not react harmlessly to the antigenic substances constituting the self, whereas many non-self antigens recognize it, I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

If a problem arises in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs, thereby attacking the tissue of the self, resulting in multiple sclerosis, type 1 diabetes, rheumatoid arthritis, Autoimmune diseases such as Hashimoto's thyroiditis will occur, as well as immune rejection reaction will occur after the same procedure as transplantation.

Specifically, the autoimmune disease is a disease caused by an adverse reaction to a self-cell, and therefore, a drug that suppresses autoimmune function is mainly used for treatment. However, because of the high incidence of side effects, it is difficult to continue to use it and it does not prevent recurrence sufficiently. In the case of multiple sclerosis, beta interferon is used but the side effects are less expensive, the cost of treatment is expensive, there is inconvenience that the injection should be taken throughout life and the effect of preventing the recurrence is minimal. In addition, a method of inhibiting intercellular interaction by administering an antibody to a CD40 ligand has been used but is not so successful. There are many other immunotherapies, but none has been recognized as a way to show definite therapeutic efficacy.

Therefore, there is a need to study a therapeutic agent that can effectively treat autoimmune diseases and is harmless to the human body and has no side effects.

European Patent No. 01669089 European Patent No. 01709202

Therefore, the inventors of the present invention have found that HtrA2 protein decreases STAT3 expression, inhibits the expression of inflammatory cytokines, and regulates T regulatory T cells (Tregs), while studying a new therapeutic method for effectively preventing and treating autoimmune diseases. The present inventors have completed the present invention by confirming that it is possible to effectively treat autoimmune diseases.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising HtrA2 (High temperature requirement protein A2) as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases, which comprises a polynucleotide encoding HtrA2 (High temperature requirement protein A2) protein as an active ingredient.

Further, the present invention provides an immunosuppressive agent comprising the above composition as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease comprising HtrA2 (High temperature requirement protein A2) as an active ingredient.

In one embodiment of the present invention, the high temperature requirement protein A2 (HtrA2) protein may have the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the High temperature requirement protein A2 (HtrA2) protein may be one that decreases STAT3 expression and activity.

In one embodiment of the present invention, the high temperature requirement protein A2 (HtrA2) protein promotes or increases the activity of regulatory T cells (Tregs) and decreases the total IgG and IgM concentrations in the blood .

In one embodiment of the present invention, the HtrA2 protein may be one that reduces or inhibits the expression of IL-1b, IL-6 and IL-17, which are inflammatory cytokines.

In one embodiment of the present invention, the HtrA2 protein may inhibit osteoclast differentiation.

In one embodiment of the present invention, the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease.

The present invention also provides a pharmaceutical composition for the prevention or treatment of an autoimmune disease comprising, as an active ingredient, a polynucleotide encoding HtrA2 (High temperature requirement protein A2) protein.

In one embodiment of the present invention, the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease.

In one embodiment of the present invention, the polynucleotide may have the nucleotide sequence shown in SEQ ID NO: 2.

In one embodiment of the present invention, the polynucleotide may be contained in an expression vector.

In one embodiment of the present invention, the expression vector may be a plasmid or a viral vector.

Further, the present invention provides an immunosuppressant comprising the above composition as an active ingredient.

The high temperature requirement protein A2 (HtrA2) protein according to the present invention reduces the expression of STAT3, which is a transcription factor of Th17 cells, inhibits the expression of inflammatory cytokines, and regulates the activity of regulatory T cells (Treg) And thus, it is possible to effectively treat an autoimmune disease caused by abnormalities of inflammation and various immune responses.

FIG. 1 shows the results of measuring STAT3 expression by staining immunostaining with STAT3 in spleen tissues of Wildtype and HtrA2 mutant mice.
FIG. 2 shows the results of immunofluorescence analysis of the expression of STAT3 and markers of T cells, B cells, and dendritic cells in wild-type (wildtype) and HtrA2 mutant mouse spleen.
Figure 3 shows the results of flow cytometry of Treg (CD4 + CD25 + Foxp3 +) cell populations in wild-type and HtrA2 variant mouse spleen.
FIG. 4 shows the results obtained by realtime-PCR of Blump-1 and antibody markers IgG and IgM, which are transcription factors of plasma cells capable of producing antibodies in wild-type and HtrA2 variant mouse spleen cells, The result is confirmed.
FIG. 5 shows the results of realtime-PCR measurement of gene expression levels in cells isolated from the spleen from the inflammatory cytokines Il-1b and IL-6.
6 shows the results of TRAP staining of osteoclast differentiation in bone marrow of HtrA2 mutant mouse and HtrA2 heteromouse.
FIG. 7 shows the results of real-time PCR for the expression of IL-17, an inflammatory cytokine according to HtrA2 expression.
Fig. 8 shows the result of induction of an animal model through mutant mouse or mutant expression vector of HtrA2.

The present invention is based on the fact that the HtrA2 protein reduces the expression of STAT3, inhibits the expression of inflammatory cytokines, and promotes the activity of regulatory T cells (Tregs), thereby effectively treating autoimmune diseases Therefore, the present invention is characterized in that it provides a composition for preventing or treating autoimmune diseases comprising an HtrA2 protein as an active ingredient.

When initial STAT3-related factors were discussed, PIAS3 and SOCS3 (suppressor of cytokine signaling 3) were suggested as STAT3 inhibitory feedback. SOCS protein inhibits JAKs and inhibits ligand-induced responses, PIAS protein is known to have an activity of inhibiting phosphorylation of STAT3 (Starr R, Hilton DJ. Negative regulation of the JAK / STAT pathway. Bioessays 1999; 21: 4752).

In addition, HtrA2 (high temperature requirement protein A2) was first identified as a mitochondrial serine protease, a protein that interacts with Mxi2. HtrA2 is composed of the mitochondrial targeting sequence (MTS), transmembrane domain (TM), protease domain, and PDZ domain, and is present in the intermembrane space of mitochondria. Bacteria HtrA acts as a chaperone at physiological temperature (37 ° C) and is likely to act as a chaperon or protease. Recently, when cell apoptotic stimuli such as UV, staurosporine and the like are given, HtrA2 is released into the cytoplasm together with Smac / DIABLO and cytochrome c from mitochondria to inhibit the action of inhibitor of apoptosis protein (IAP) -Dependent apoptosis, and several studies have shown that HtrA2 is one of the regulatory factors playing a central role in the apoptotic pathway. In addition, linkage analysis and mutation screening confirmed that serine 276 was mutated to cysteine in the HtrA2 gene of mnd2 (motor neuron degeneration 2) mouse. Due to the loss of serine protease function of HtrA2, mnd2 mice were more susceptible to spleen and thymus The size of the body is small, the mobility is decreased, and the average life span is about 40 days. This indicates that HtrA2 is effective in the prevention and treatment of degenerative brain diseases.

However, there is no information on the relationship between the HtrA2 protein and autoimmune diseases and the mechanism of their treatment.

Therefore, in the present invention, it was firstly found that HtrA2 protein can be used for prevention or treatment of autoimmune diseases.

STAT3 was stained with immunofluorescence in spleen tissues of Wildtype and HtrA2 mutant mice, and STAT3 expression level was measured. As a result, p-STAT3 tyr705 and STAT3 Ser727 expression was more expressed in the spleen of HtrA2 mutant mice than in the wild type, and the expression of STAT3 was increased when the HtrA2 mutant was generated (see FIG. 1).

In another example, the expression of T3, B, Dendritic cell markers and STAT3 in wildtype and HtrA2 variant mouse spleen was confirmed by immunofluorescence. As a result, T cells, B cells, dendritic cells were densely packed in the important follicle, and STAT3 was hyperactivated (see FIG. 2).

In addition, the HtrA2 protein reduces the activity of STAT3, a transcription factor of h17 cells, promotes the activity of regulatory T cells (Treg), and suppresses the concentration of total IgG and IgM in the blood, (See Figs. 3 and 4).

According to another embodiment of the present invention, when the expression levels of IL-1b, IL-6 and IL-17, which are inflammatory cytokines, in the cells isolated from the spleen were examined by realtime-PCR, the HtrA2 protein of the present invention showed an inflammatory cytokine IL-1b, IL-6, and IL-17 (see FIGS. 5 and 7).

Furthermore, the inventors of the present invention conducted an osteoclast differentiation experiment in the bone marrow of HtrA2 mutant mouse and HtrA2 heterozygous mouse to examine whether the HtrA2 protein of the present invention is effective for the treatment of arthritis, which is an autoimmune disease. HtrA2 (see Fig. 6).

The present inventors also found that when arthritis was induced by injection of an HtrA2 mutant mouse or a mutant expression vector, arthritis was further intensified (see FIG. 8) in order to examine how the effect of HtrA2 is inhibited in inducing arthritis, .

Therefore, based on the facts confirmed through one embodiment of the present invention together with the known facts, it has been found that the HtrA2 protein of the present invention can treat autoimmune diseases such as immune diseases.

In the present invention, the immunological disease refers to a disease in which components of the mammalian immune system cause, mediate, or contribute to a pathological condition of a 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 diseases include, but are not limited to, autoimmune diseases.

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 problems arise in inducing or maintaining such self-tolerance, an immune response to self-antigen occurs, thereby attacking the tissue of self. The disease caused by this process is called autoimmune disease .

The autoimmune diseases to which the composition can be applied in the present invention include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease have.

Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating autoimmune diseases containing HtrA2 protein as an active ingredient, wherein the HtrA2 protein contained in the pharmaceutical composition of the present invention has substantially the same physiological activity as the protein Lt; / RTI > HtrA2 proteins having substantially equivalent physiological activity include the above proteins and their functional equivalents and functional derivatives.

The above-mentioned "functional equivalent" means a modified amino acid sequence in which some or all of the native protein amino acid is substituted, or a part of the amino acid is deleted or added, and has substantially the same physiological activity as the native HtrA2 protein. "Functional derivative" means a protein that has been modified to increase or decrease the physicochemical properties of the HtrA2 protein and has substantially the same physiological activity as the native HtrA2 protein.

Preferably, the HtrA2 protein refers to a protein having the amino acid sequence of SEQ ID NO: 1.

In the present invention, the term " treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term " treatment ", as used herein, refers to an act of treating when " treating " is defined as described above.

The pharmaceutical compositions for the prevention or treatment of autoimmune diseases according to the present invention may comprise a pharmaceutically effective amount of the HtrA2 protein alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of autoimmune diseases.

The pharmaceutically effective amount of the HtrA2 protein according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of the autoimmune disease symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term " pharmaceutically acceptable " as used herein 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 humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

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 autoimmune disease according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may vary depending on the administration route, , Body weight and severity of the patient, and the composition for preventing or treating autoimmune disease according to the present invention may be selected according to various factors such as known compounds having an effect of preventing, ameliorating or treating symptoms of autoimmune diseases May be administered concurrently.

Further, the present invention can provide a pharmaceutical composition for preventing or treating an autoimmune disease comprising a polynucleotide encoding an HtrA2 protein as an active ingredient.

The polynucleotide encoding the HtrA2 protein may comprise DNA or RNA, and preferably has the DNA sequence shown in SEQ ID NO: 2.

Here, the polynucleotide may be introduced into an expression vector such as a plasmid or a viral vector by a known method, and then the expression vector may be expressed in an expression form by infection or transduction by various methods known in the art Can be introduced into cells.

The plasmid expression vector is a method of delivering plasmid DNA directly to human cells using an approved FDA-approved gene transfer method that can be used in humans (Nabel, E. G., et al., Science, 249: 1285-1288, 1990). Unlike virus vectors, plasmid DNA has the advantage of being homogeneously purified. As the plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. Examples include, but are not limited to pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291) and pVL1392 (PharMingen).

A plasmid expression vector comprising a polynucleotide according to the present invention may be prepared by a method known in the art such as, but not limited to, transient transfection, microinjection, transduction, , Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection ), Electroporation, gene gun, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267 : 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).

The vector capable of expressing HtrA2 can be administered by a known method. For example, topically, parenterally, orally, nasally, intravenously, intramuscularly, subcutaneously, or by any other suitable means.

Accordingly, the present invention provides a pharmaceutical agent for the prevention or treatment of autoimmune diseases, which comprises a composition containing an expression vector comprising a polynucleotide encoding an HtrA2 protein as an active ingredient, An immunosuppressive agent comprising the polynucleotide encoding the polypeptide of the present invention as an active ingredient.

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.

HtrA2 On by STAT3  Confirmation of expression

STAT3, which is known as a transcription factor of Th17 cells, was stained with immunofluorescence in spleen tissues of Wildtype and HtrA2 mutant mice and the expression of STAT3 was examined in wild-type HtrA2 mutant mice. The HtrA2 variant mouse (Stock number 004608) was purchased from the Jaxson lab and is a mouse with a chromosomal mutation in the mnd2 gene located in the mouse chromosome 6.

As a result, the expression of p-STAT3 tyr705 and ser727 was more expressed in the spleen of HtrA2 mutant mice than in the wild type, and the expression of STAT3, a transcription factor of Th17 cells, was increased when the HtrA2 mutant was generated (See Fig. 1).

HtrA2 T cells, B cells, Dendritic cell  And STAT3  Confirmation of expression

In wildtype and HtrA2 variant mouse spleen, markers of T cells, B cells, dendritic cells and STAT3 expression were confirmed by immunofluorescence in tissues for observation.

As a result, the number of immune cells such as CD4, B220 and CD11C increased and the expression of STAT3 was also increased in HtrA2 mutant mice. As a result, T cells, B cells, and dendritic cells were densely packed inside the follicle, which is important for antibody production, and STAT3 was hyperactivated (see FIG. 2).

HtrA2 Mutant  In the spleen of mice Foxp3  Expression Treg  Cell analysis

Cells of Treg (CD4 + CD25 + Foxp3 +) that have the ability to suppress activated immune cells such as effector T cells in cells isolated from the spleen of 38-day old wildtype and HtrA2 mutant mice Were confirmed by flow cytometry.

As a result, there was no difference in Lymphocyte gate, but it was confirmed that in CD4 + cell group, HtrA2 mutant mouse was 13% higher than wild type, and 5.74% of Treg was about 4% lower than wild type (see FIG. 3 ).

HtrA2 Analysis of Antibody Production Capacity by

AID and Blimp-1, which are transcription factors of plasma cells capable of producing antibodies in cells isolated from the spleen of wild-type and HtrA2 mutant mice of 38 days old, and IgG and IgM, PCR.

As a result, the levels of IgG and IgM as antibody markers increased in HtrA2 mutant mice compared with wild type mice. It was also found that AID and Blimp-1, which are important transcription factors of plasma cells for antibody production, also increased in HtrA2 mutant mice compared with wild type mice (See FIG. 4).

HtrA2 Of inflammatory cytokine expression by abnormal morphology

IL-1b and TNF-a were increased in HtrA2 mutant mice by real-time PCR analysis of the expression levels of IL-1b and IL-6, which are known as typical inflammatory cytokines, in spleen cells. As a result, it was found that IL-1b, TNF-a, which is important for inflammation induction, can cause inflammation in the abnormal form of HtrA2 (see FIG. 5).

HtrA2 Of osteoclast differentiation

To investigate whether HtrA2 is involved in osteoclast differentiation associated with arthritis, osteoclast differentiation experiments were performed in the bone marrow of HtrA2 mutant mice and HtrA2 heterozygous mice.

As a result, TRAP staining confirmed that osteoclast differentiation was further promoted in HtrA2 mutant mice than in HtrA2 heterozygous mice. The real-time PCR confirmed osteoclast marker TRAP as a cDNA obtained from osteoclast, HtrA2 mutant mice, indicating that HtrA2 affects osteoclast differentiation (see FIG. 6).

HtrA2 Expression of Inflammatory Cytokines by Expression Changes

In order to confirm the expression of IL-17, which is an inflammatory cytokine according to the expression of HtrA2, the present inventors transplanted HTRA2 overexpressing vector and mock vector into T cell lymphoma cell line LBRM cells, 17 expression was measured by real time PCR.

As a result, when HtrA2 was overexpressed, the expression of IL-17 decreased and when it was inhibited with a chemical inhibitor, IL-17 increased, indicating that HtrA2 regulated IL-17 expression Reference).

HtrA2 of Mutant mice and Mutant  Analysis of the effect on arthritis

In order to determine what results in arthritis when HtrA2 function is inhibited, the present inventors measured the arthritic index by injecting HtrA2 variant mouse or mutant expression vector.

As a result, it was confirmed that the effect of treatment of arthritis was lost when the HtrA2 mutant was generated and the CIA arthritis index was increased in the mice that did not function, thereby inhibiting HtrA2 function (see FIG. 8).

The present invention has been described with reference to the preferred embodiments. 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.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Composition for treatment or prevention of autoimmune diseases          comprising HtrA2 <130> PN1211-515 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 458 <212> PRT <213> Unknown <220> <223> HtrA2 amino acid sequence <400> 1 Met Ala Ala Leu Lys Ala Gly Arg Gly Ala Asn Trp Ser Leu Arg Ala   1 5 10 15 Trp Arg Ala Leu Gly Gly Ile Phe Trp Arg Lys Pro Pro Leu Leu Ala              20 25 30 Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Pro Asp Ser Gln Ile          35 40 45 Trp Met Thr Tyr Gly Thr Pro Ser Leu Pro Ala Gln Val Pro Glu Gly      50 55 60 Phe Leu Ala Ser Arg Ala Asp Leu Thr Ser Arg Thr Pro Asp Leu Trp  65 70 75 80 Ala Arg Leu Asn Val Gly Thr Ser Gly Ser Ser Asp Gln Glu Ala Arg                  85 90 95 Arg Ser Pro Gly Ser Arg Arg Glu Trp Leu Ala Val Ala Val Gly             100 105 110 Ala Gly Gly Ala Val Val Leu Leu Leu Trp Gly Trp Gly Arg Gly Leu         115 120 125 Ser Thr Val Leu Ala Ala Val Pro Ala Pro Pro Thr Ser Pro Arg     130 135 140 Ser Gln Tyr Asn Phe Ile Ala Asp Val Val Glu Lys Thr Ala Pro Ala 145 150 155 160 Val Val Tyr Ile Glu Ile Leu Asp Arg His Pro Phe Ser Gly Arg Glu                 165 170 175 Val Pro Ile Ser Asn Gly Ser Gly Phe Val Val Ala Ser Asp Gly Leu             180 185 190 Ile Val Thr Asn Ala His Val Val Ala Asp Arg Arg Arg Val Val Arg Val         195 200 205 Arg Leu Pro Ser Gly Asp Thr Tyr Glu Ala Met Val Thr Ala Val Asp     210 215 220 Pro Val Ala Asp Ile Ala Thr Leu Arg Ile Gln Thr Lys Glu Pro Leu 225 230 235 240 Pro Thr Leu Pro Leu Gly Arg Ser Ala Asp Val Arg Gln Gly Glu Phe                 245 250 255 Val Val Ala Met Gly Ser Pro Phe Ala Leu Gln Asn Thr Ile Thr Ser             260 265 270 Gly Ile Val Ser Ser Ala Gln Arg Pro Ala Arg Asp Leu Gly Leu Pro         275 280 285 Gln Asn Asn Val Glu Tyr Ile Gln Thr Asp Ala Ile Asp Phe Gly     290 295 300 Asn Ser Gly Gly Pro Leu Val Asn Leu Asp Gly Glu Val Ile Gly Val 305 310 315 320 Asn Thr Met Lys Val Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp                 325 330 335 Arg Leu Arg Glu Phe Leu His Arg Gly Glu Lys Lys Asn Ser Trp Phe             340 345 350 Gly Thr Ser Gly Ser Gln Arg Arg Tyr Ile Gly Val Met Met Leu Thr         355 360 365 Leu Thr Pro Ser Ile Leu Ile Glu Leu Gln Leu Arg Glu Pro Ser Phe     370 375 380 Pro Asp Val Gln His Gly Val Leu Ile His Lys Val Ile Leu Gly Ser 385 390 395 400 Pro Ala His Arg Ala Gly Leu Arg Pro Gly Asp Val Ile Leu Ala Ile                 405 410 415 Gly Glu Lys Leu Ala Gln Asn Ala Glu Asp Val Tyr Glu Ala Val Arg             420 425 430 Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg Gly Ser Glu Thr Leu         435 440 445 Thr Leu Tyr Val Thr Pro Glu Val Thr Glu     450 455 <210> 2 <211> 1739 <212> DNA <213> Unknown <220> <223> HtrA2 nucleotide sequence <400> 2 ctggagagga gcgcgtcctg acgggactac gtttcccggc atgcgctgaa cagctaccgt 60 cgtgccctgc ttctcctaga ccgcttgccg ggttctagag gaccgcctct gagtagggcg 120 ggcgaggaga gtcgaggcgg agctgatggc tgcgctgaaa gcggggcggg gtgcaaactg 180 gagccttcgg gcatggcggg ctttgggggg catcttctgg aggaagcctc ccctgttagc 240 tcctgacctc cgggctctgc tgacgtcagg aactcctgac tctcagatct ggatgactta 300 tgggaccccc agtctcccgg cccaggtgcc tgaggggttc cttgcatcac gggcagatct 360 gccgtctagg accccggatc tctgggcacg attgaatgtg gggacttcag gctccagtga 420 ccaggaggcc cgcaggagcc ctggaagccg tcggcgcgaa tggctggcgg tggcggtggg 480 cgcaggaggt gcagtggtgc tgctgttgtg gggttggggt cggggtcttt ccacggtgct 540 ggctgctgtt cctgctccgc cacccacttc tccccggagc cagtacaatt tcatcgcaga 600 tgtggtggag aagacagccc ctgctgtggt ctatatcgag atcctagacc ggcacccttt 660 ctccggccgt gaagtcccca tctcaaacgg atcaggattc gtagtggctt cagatgggct 720 catcgttacc aacgcccacg tggtggctga tcggcgccga gtacgagtga ggctgcctag 780 cggggatact tatgaggcca tggtcacagc tgtggatccc gtagcagaca ttgccacact 840 gaggattcaa accaaggagc ctcttcccac actgcccctc ggccgctctg ctgatgtccg 900 gcaaggggag tttgttgttg ccatgggaag cccctttgca ctgcagaaca cgatcacatc 960 tggtattgtc agctctgctc agcgcccagc cagggacctg ggactccctc aaaacaacgt 1020 ggaatacatt cagaccgatg cagctattga ttttggaaat tctggtggtc ccctggttaa 1080 cctggatggg gaggtgattg gagtgaacac catgaaggtg acagctggaa tctcctttgc 1140 catcccttct gatcgcctta gggagtttct gcatcgcggg gaaaagaaaa attcctggtt 1200 tggaaccagt gggtcccagc gccgctacat tggagtgatg atgctgaccc tgactcccag 1260 catccttatt gaactacagc tccgtgagcc aagcttccct gatgttcagc atggtgtcct 1320 cattcataaa gttatcctgg gctcccccgc acacagggct ggtctgcggc ctggtgatgt 1380 gatcttggcc attggggaga aattggcaca aaatgctgaa gatgtttatg aagctgttcg 1440 aacccaatca cagctggcag tgcggatccg gcgcggatca gagacactga ccttatatgt 1500 gacccccgag gtcacagaat gaatgactgg accggcaaga gtgtgaagct cttgccctga 1560 tctcctcctt gctttcctag cctaggttct gagtgcatgt gggtagagga ggagtcagtg 1620 aacctgcgga gggcaagtcc ctctaaccgc tgcatcagtc ctgggctccg aagaacacat 1680 tttatataaa ataaaattat acctagcaac atgttacagt caaaaaaaaa aaaaaaaaa 1739 <210> 3 <211> 458 <212> PRT <213> Unknown <220> <223> HtrA2 mutation amino acid sequence <400> 3 Met Ala Ala Leu Lys Ala Gly Arg Gly Ala Asn Trp Ser Leu Arg Ala   1 5 10 15 Trp Arg Ala Leu Gly Gly Ile Phe Trp Arg Lys Pro Pro Leu Leu Ala              20 25 30 Pro Asp Leu Arg Ala Leu Leu Thr Ser Gly Thr Pro Asp Ser Gln Ile          35 40 45 Trp Met Thr Tyr Gly Thr Pro Ser Leu Pro Ala Gln Val Pro Glu Gly      50 55 60 Phe Leu Ala Ser Arg Ala Asp Leu Thr Ser Arg Thr Pro Asp Leu Trp  65 70 75 80 Ala Arg Leu Asn Val Gly Thr Ser Gly Ser Ser Asp Gln Glu Ala Arg                  85 90 95 Arg Ser Pro Gly Ser Arg Arg Glu Trp Leu Ala Val Ala Val Gly             100 105 110 Ala Gly Gly Ala Val Val Leu Leu Leu Trp Gly Trp Gly Arg Gly Leu         115 120 125 Ser Thr Val Leu Ala Ala Val Pro Ala Pro Pro Thr Ser Pro Arg     130 135 140 Ser Gln Tyr Asn Phe Ile Ala Asp Val Val Glu Lys Thr Ala Pro Ala 145 150 155 160 Val Val Tyr Ile Glu Ile Leu Asp Arg His Pro Phe Ser Gly Arg Glu                 165 170 175 Val Pro Ile Ser Asn Gly Ser Gly Phe Val Val Ala Ser Asp Gly Leu             180 185 190 Ile Val Thr Asn Ala His Val Val Ala Asp Arg Arg Arg Val Val Arg Val         195 200 205 Arg Leu Pro Ser Gly Asp Thr Tyr Glu Ala Met Val Thr Ala Val Asp     210 215 220 Pro Val Ala Asp Ile Ala Thr Leu Arg Ile Gln Thr Lys Glu Pro Leu 225 230 235 240 Pro Thr Leu Pro Leu Gly Arg Ser Ala Asp Val Arg Gln Gly Glu Phe                 245 250 255 Val Val Ala Met Gly Ser Pro Phe Ala Leu Gln Asn Thr Ile Thr Ser             260 265 270 Gly Ile Val Ser Ser Ala Gln Arg Pro Ala Arg Asp Leu Gly Leu Pro         275 280 285 Gln Asn Asn Val Glu Tyr Ile Gln Thr Asp Ala Ile Asp Phe Gly     290 295 300 Asn Ala Gly Gly Pro Leu Val Asn Leu Asp Gly Glu Val Ile Gly Val 305 310 315 320 Asn Thr Met Lys Val Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp                 325 330 335 Arg Leu Arg Glu Phe Leu His Arg Gly Glu Lys Lys Asn Ser Trp Phe             340 345 350 Gly Thr Ser Gly Ser Gln Arg Arg Tyr Ile Gly Val Met Met Leu Thr         355 360 365 Leu Thr Pro Ser Ile Leu Ile Glu Leu Gln Leu Arg Glu Pro Ser Phe     370 375 380 Pro Asp Val Gln His Gly Val Leu Ile His Lys Val Ile Leu Gly Ser 385 390 395 400 Pro Ala His Arg Ala Gly Leu Arg Pro Gly Asp Val Ile Leu Ala Ile                 405 410 415 Gly Glu Lys Leu Ala Gln Asn Ala Glu Asp Val Tyr Glu Ala Val Arg             420 425 430 Thr Gln Ser Gln Leu Ala Val Arg Ile Arg Arg Gly Ser Glu Thr Leu         435 440 445 Thr Leu Tyr Val Thr Pro Glu Val Thr Glu     450 455 <210> 4 <211> 1739 <212> DNA <213> Unknown <220> <223> HtrA2 mutation nucleotide sequence <400> 4 ctggagagga gcgcgtcctg acgggactac gtttcccggc atgcgctgaa cagctaccgt 60 cgtgccctgc ttctcctaga ccgcttgccg ggttctagag gaccgcctct gagtagggcg 120 ggcgaggaga gtcgaggcgg agctgatggc tgcgctgaaa gcggggcggg gtgcaaactg 180 gagccttcgg gcatggcggg ctttgggggg catcttctgg aggaagcctc ccctgttagc 240 tcctgacctc cgggctctgc tgacgtcagg aactcctgac tctcagatct ggatgactta 300 tgggaccccc agtctcccgg cccaggtgcc tgaggggttc cttgcatcac gggcagatct 360 gccgtctagg accccggatc tctgggcacg attgaatgtg gggacttcag gctccagtga 420 ccaggaggcc cgcaggagcc ctggaagccg tcggcgcgaa tggctggcgg tggcggtggg 480 cgcaggaggt gcagtggtgc tgctgttgtg gggttggggt cggggtcttt ccacggtgct 540 ggctgctgtt cctgctccgc cacccacttc tccccggagc cagtacaatt tcatcgcaga 600 tgtggtggag aagacagccc ctgctgtggt ctatatcgag atcctagacc ggcacccttt 660 ctccggccgt gaagtcccca tctcaaacgg atcaggattc gtagtggctt cagatgggct 720 catcgttacc aacgcccacg tggtggctga tcggcgccga gtacgagtga ggctgcctag 780 cggggatact tatgaggcca tggtcacagc tgtggatccc gtagcagaca ttgccacact 840 gaggattcaa accaaggagc ctcttcccac actgcccctc ggccgctctg ctgatgtccg 900 gcaaggggag tttgttgttg ccatgggaag cccctttgca ctgcagaaca cgatcacatc 960 tggtattgtc agctctgctc agcgcccagc cagggacctg ggactccctc aaaacaacgt 1020 ggaatacatt cagaccgatg cagctattga ttttggaaat gccggtggtc ccctggttaa 1080 cctggatggg gaggtgattg gagtgaacac catgaaggtg acagctggaa tctcctttgc 1140 catcccttct gatcgcctta gggagtttct gcatcgcggg gaaaagaaaa attcctggtt 1200 tggaaccagt gggtcccagc gccgctacat tggagtgatg atgctgaccc tgactcccag 1260 catccttatt gaactacagc tccgtgagcc aagcttccct gatgttcagc atggtgtcct 1320 cattcataaa gttatcctgg gctcccccgc acacagggct ggtctgcggc ctggtgatgt 1380 gatcttggcc attggggaga aattggcaca aaatgctgaa gatgtttatg aagctgttcg 1440 aacccaatca cagctggcag tgcggatccg gcgcggatca gagacactga ccttatatgt 1500 gacccccgag gtcacagaat gaatgactgg accggcaaga gtgtgaagct cttgccctga 1560 tctcctcctt gctttcctag cctaggttct gagtgcatgt gggtagagga ggagtcagtg 1620 aacctgcgga gggcaagtcc ctctaaccgc tgcatcagtc ctgggctccg aagaacacat 1680 tttatataaa ataaaattat acctagcaac atgttacagt caaaaaaaaa aaaaaaaaa 1739

Claims (13)

A pharmaceutical composition for preventing or treating an autoimmune disease comprising HtrA2 (High temperature requirement protein A2) as an active ingredient. The method according to claim 1,
Wherein the HtrA2 protein has the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein said high temperature requirement protein A2 protein (HtrA2) decreases STAT3 expression and activity. The method according to claim 1,
Wherein said HtrA2 (High temperature requirement protein A2) protein promotes or increases the activity of regulatory T cells (Treg) and reduces total IgG and IgM concentrations in the blood. A pharmaceutical composition for therapeutic use. The method according to claim 1,
The pharmaceutical composition for preventing or treating autoimmune diseases, wherein the HtrA2 (High temperature requirement protein A2) protein reduces or inhibits the expression of inflammatory cytokines IL-1b, IL-6 and IL-17. The method according to claim 1,
The pharmaceutical composition for preventing or treating autoimmune diseases, wherein the HtrA2 (High temperature requirement protein A2) protein inhibits osteoclast differentiation. The method according to claim 1,
Wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease. A pharmaceutical composition for the prophylaxis or treatment of an autoimmune disease comprising as an active ingredient a polynucleotide encoding HtrA2 (High temperature requirement protein A2) protein. 9. The method of claim 8,
Wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease. 9. The method of claim 8,
Wherein the polynucleotide has the nucleotide sequence shown in SEQ ID NO: 2. 2. A pharmaceutical composition for preventing or treating autoimmune diseases, 9. The method of claim 8,
Wherein the polynucleotide is contained in an expression vector. 12. The method of claim 11,
Wherein the expression vector is a plasmid or a viral vector. An immunosuppressant comprising the composition of claim 1 or 12 as an active ingredient.

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