JP6645737B2 - Biomarkers for autoimmune diseases - Google Patents

Description

  The present invention relates to a biomarker capable of accurately evaluating an autoimmune disease.

  Autoimmune diseases have heretofore been understood as diseases caused by injuries of tissues or organs caused by autoreactive T cells or autoantibodies accompanying breakdown of autoimmune tolerance. Innate immunity is an innate immune system of a living body, and is composed of neutrophils, macrophages, natural killer (NK) cells, and the like. However, in recent years, as understanding of the innate immune system has advanced, it has been suggested that disruption of the immune response in the innate immune system may be the cause or exacerbating factor of autoimmune diseases.

  As substances that stimulate the innate immune system, for example, pathogen-derived inflammatory substances (PAMPs: Pathogen-Associated Molecular Patterns) and autologous inflammatory substances (DAMPs: Damage-Associated Molecular Patterns) are known. PAMPs are substances that are not present in humans and the like but are present universally in microorganisms. Phagocytic cells (macrophages, neutrophils), dendritic cells, and the like are detected by pattern recognition receptors (PRRs) to eliminate microorganisms. For the reaction. DAMPs are also released from cells damaged by trauma, apoptosis, necrosis, etc., and activate innate immunity.

  Stimulation with PAMPs and DAMPs promotes the production of inflammatory cytokines. It also damages host cells and mitochondria present in the cells, which causes mitochondrial degradation and cell death, and promotes release of mitochondrial DNA (mtDNA) into the cytoplasm and extracellularly (in blood). Further, it is known that this mtDNA becomes DAMPs and further activates immune cells (for example, see FIG. 1). For example, patients with multiple trauma often have pneumonia or systemic inflammatory response syndrome (SIRS), which is caused by mitochondria (or cells) damaged by the trauma releasing mtDNA, which is further immunized as DAMPs. Because it acts on cells, it is thought to cause pneumonia and systemic inflammatory response syndrome (SIRS) that seemingly unrelated to trauma (for example, see Non-Patent Document 1). The present inventors have focused on abnormal immune responses resulting from disruption of mitochondrial homeostasis that functions as a platform for such an innate immune system, and have been studying the onset mechanism of autoimmune diseases.

  Generally, diagnosis of an autoimmune disease is made by a specialist based on measurement of clinical symptoms and autoantibody titer. The disease state is measured by measuring inflammatory markers such as C-reactive protein (CRP) and blood sedimentation, and the severity is diagnosed based on the degree of the involved organ lesion. CRP is a protein that appears in the blood when an inflammatory response or tissue destruction occurs in the body, and the amount of CRP produced usually correlates with the intensity of the inflammatory response. However, there are also autoimmune diseases in which disease activity and CRP are not linked, such as Behcet's disease. Therefore, a biomarker that can accurately evaluate an autoimmune disease and that can be easily measured has been demanded.

Qin Zhang et. al. , Nature, vol. 464, 4 Mach 2010, pg. 104-107

  An object of the present invention is to provide a biomarker that can accurately evaluate an autoimmune disease. The present invention also provides a diagnostic kit including a reagent capable of detecting the biomarker, a diagnostic method using the biomarker, and an agent for preventing or treating an autoimmune disease.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the serum of an autoimmune disease patient contains about 50 to 500 times as much mtDNA as a healthy person. Furthermore, the present inventors have found that this mtDNA is localized in extracellular membrane vesicles consisting of exosomes in serum and microvesicles called ectosomes, particularly exosomes (vesicles). The present invention has been completed as a result of further studies based on these findings.

That is, the present invention provides the following embodiments.
Item 1. A biomarker for an autoimmune disease comprising mitochondrial DNA contained in a biological body fluid.
Item 2. Item 2. The biomarker according to Item 1, wherein mitochondrial DNA is contained in extracellular membrane vesicles in a biological fluid.
Item 3. Item 3. The biomarker according to Item 1 or 2, wherein mitochondrial DNA is contained in exosomes in a biological fluid.
Item 4. The autoimmune disease comprises Behcet's disease, rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease (MCTD), Crohn's disease and ulcerative colitis. The biomarker according to any one of Items 1 to 3, which is any one selected from the group.
Item 5. The biomarker according to any one of Items 1 to 4, wherein the autoimmune disease is Behcet's disease.
Item 6. A diagnostic kit for an autoimmune disease, comprising a reagent capable of detecting mitochondrial DNA.
Item 7. The reagent capable of detecting the mitochondrial DNA includes cytochrome b, COXI, COXII, COXIII, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 2, NADH dehydrogenase subunit 3, NADH dehydrogenase subunit 4, NADH dehydrogenase subunit 4L, and NADH dehydrogenase. Item 7. The diagnostic kit according to item 6, which is a reagent capable of detecting at least one selected from the group consisting of subunit 5, NADH dehydrogenase subunit 6, ATPase sixth subunit and ATPase eighth subunit.
Item 8. The autoimmune disease comprises Behcet's disease, rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease (MCTD), Crohn's disease and ulcerative colitis. Item 8. The diagnostic kit for an autoimmune disease according to Item 6 or 7, which is any one selected from the group.
Item 9. Item 9. The diagnostic kit for an autoimmune disease according to any one of Items 6 to 8, wherein the autoimmune disease is Behcet's disease.
Item 10. A method for detecting the presence or absence of an autoimmune disease including the following steps:
(I) obtaining a body fluid sample from a test animal;
(Ii) a step of measuring mitochondrial DNA in the body fluid sample; and (iii) a step of detecting the presence or absence of an autoimmune disease based on the result of the step (ii).
Item 11. The step (iii) is performed using the result of the step (ii) obtained for the test animal as an index, as compared with the result of the step (ii) obtained for the normal control, as an index of an increase in the amount of mitochondrial DNA. Item 13. The detection method according to Item 10.
Item 12. The autoimmune disease comprises Behcet's disease, rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease (MCTD), Crohn's disease and ulcerative colitis. Item 12. The method for detecting an autoimmune disease according to Item 10 or 11, which is any one selected from the group.
Item 13. An agent for preventing or treating an autoimmune disease, comprising as an active ingredient a substance that suppresses secretion of mitochondrial DNA into body fluids or suppresses the function of mitochondrial DNA.
Item 14. Use of a substance that suppresses secretion of mitochondrial DNA into body fluids or suppresses the function of mitochondrial DNA for the manufacture of a prophylactic or therapeutic agent for an autoimmune disease.
Item 15. A substance used to prevent or treat an autoimmune disease, which suppresses secretion of mitochondrial DNA into body fluids or suppresses the function of mitochondrial DNA.

  The present invention makes it possible to accurately evaluate an autoimmune disease by using, as a biomarker, mtDNA which is stably present in a biological fluid, particularly an extracellular membrane vesicle, particularly an exosome fraction of the biological fluid, in an autoimmune disease patient. I do. Since mtDNA can also be a cause of autoimmune diseases, it can directly reflect the pathology of autoimmune diseases. Therefore, according to the biomarker of the present invention having such characteristics, it is difficult to evaluate or diagnose a disease state because a symptom and a biochemical index are not linked in the conventional diagnosis or disease state evaluation method. Can also accurately evaluate the presence or absence and severity of the disease. And the result obtained based on the biomarker of the present invention contributes to optimization of a treatment method according to each patient's disease state. Further, according to the present invention, a diagnostic kit for an autoimmune disease containing the biomarker can be provided. Furthermore, since mtDNA in a biological fluid can cause etiology of an autoimmune disease, targeting it can provide a drug effective for preventing or treating an autoimmune disease.

FIG. 1 is a schematic diagram showing an inflammatory cascade involving mitochondria. It is the schematic which shows the secretion and metabolic pathway of exosome. It is a graph which shows DNA concentration in serum and mtDNA value of an autoimmune disease patient. In FIG. 3, “SLE” indicates systemic lupus erythematosus, “SSc” indicates systemic scleroderma, and “PM / DM” indicates polymyositis / dermatomyositis. It is a graph which shows the expression level of mtDNA in the serum of a patient with an autoimmune disease. In FIG. 4, "BD" indicates Behcet's disease, "RA" indicates rheumatoid arthritis, "SLE" indicates systemic lupus erythematosus, and "Cont" indicates healthy subjects. It is a graph which shows the DNA concentration in serum and mtDNA value in a Behcet's disease patient and a control group (healthy person). FIG. 3 shows serum mtDNA values in Behcet's disease patients having each clinical characteristic. (a) shows a comparison between a case not exhibiting clinical symptoms of active uveitis (Non) and a case presenting clinical symptoms of active uveitis (Uveitis). b) shows a comparison between a non-treated group treated with only colchicine (Non), a group treated with an immunosuppressant (Conv), and a group treated with an anti-TNF-α antibody (Mab). (A) is a graph showing that most of mtDNA present in serum is localized in extracellular membrane vesicles, particularly in exosomes. (B) is a graph showing that mtDNA is more localized in the exosome fraction of a Behcet's disease patient than in the control group. (C) shows that DNA fragments are localized in MVB of monocytes derived from peripheral blood of Behcet's disease patient. It is a schematic diagram showing the hypothesis that mtDNA released into the cytoplasm by mitochondrial stress is taken up by exosomes and released outside the cell. It is a photograph showing that exosomes and mitochondria co-localize due to mitochondrial stress. It is a graph which shows that the amount of mtDNA in exosome increased by mitochondrial stress. The vertical axis in FIG. 10 indicates the relative expression level of mtDNA in exosomes. (a) is a graph showing the results obtained by stimulating monocytes, lymphocytes, and neutrophils obtained from a Behcet's disease patient to evaluate the enhanced mtDNA release. In a, “Non” is no stimulation, “ATP” is stimulation by ATP, “Nig” is stimulation by Nigericin, “Pam” is stimulation by Pam3CSK4, “PMA / IONO” is stimulation by PMA and ionomycin, “fMLP” is stimulation 3 shows stimulation by fMLP. b is a graph showing the results obtained by stimulating CD14-positive monocytes derived from Behcet's disease patients and CD14-positive monocytes derived from healthy subjects to evaluate the enhancement of mtDNA release. In b, “ATP” indicates stimulation by ATP, “Nig” indicates stimulation by Nigericin, and “Pam” indicates stimulation by Pam3CSK4. Panel a shows the results obtained by stimulating CD14-positive monocytes derived from Behcet's disease patient and measuring the expression of lipid-bound LC3 and Atg5 / Atg12 complex by Western blotting. Panel b shows the results obtained by stimulating CD14-positive monocytes derived from Behcet's disease patient and observing the formation of reactive oxygen species (ROS) -producing mitochondria and puncta formation of LC3 using a confocal microscope. c shows the results of observing CD14-positive monocytes stimulated with PMA and Rotenone using a transmission electron microscope. FIG. 4 is a graph showing that exosomes and mtDNA from Behcet's disease (BD) patients enhance neutrophil migration activity. It is a figure which shows the typical example of the result of the time plus observation which tracked the time-dependent movement change of a neutrophil. It is a graph which shows that the neutrophil migration activity by the exosome derived from a patient with Behcet's disease is mediated by a G protein-coupled receptor. It is a figure that production of inflammatory cytokines was induced in mouse and human-derived macrophages or dendritic cells by exosomes derived from Behcet's disease patients. It is a figure showing that induction of inflammatory cytokine production by exosomes derived from Behcet's disease patients is TLR9-dependent. It is a figure showing that induction of IL-1β production by exosomes derived from Behcet's disease patients is inflammasome-dependent. It is a figure showing that exosomes derived from Behcet's disease patients induced infiltration of Ly6C + Ly6G + neutrophils and Ly6C + Ly6G- inflammatory monocytes. FIG. 2 shows that a part of infiltration of neutrophils and inflammatory monocytes by exosomes derived from Behcet's disease patients is TLR9-dependent. It is a figure that a part of infiltration of neutrophils and inflammatory monocytes by exosomes derived from Behcet's disease patients is dependent on NLRP3 inflammasome and IL-1. (a) shows the results of evaluating the induction effect of IL-17-producing cells by exosomes derived from Behcet's disease patients (the expression levels of IL-17-related genes by RT-PCR). b is a diagram showing the results of evaluating the induction effect of IL-17-producing cells by exosomes derived from Behcet's disease patients (in vivo differentiation induction of IL-17-producing cells). (c) shows the results of a test performed on TLR dependence of induction of differentiation of IL-17-producing cells by exosomes derived from Behcet's disease patients using TLR9-deficient mice and MyD88-deficient mice. d is a diagram showing that induction of differentiation of IL-17-producing cells by exosomes derived from Behcet's disease patient is dependent on IL-1 and IL-23. (a) shows that administration of exosomes derived from a patient with Behcet's disease resulted in extensive infiltration of inflammatory cells around the retina, and destruction of the retinal structure and formation of ulcers and granulation were observed. (b) shows that mice administered with exosomes derived from Behcet's disease patients showed an increase in histological severity score. In the figures, the abbreviation "BD" indicates a Behcet's disease patient or a sample derived from a Behcet's disease patient, and the abbreviation "Cont" indicates a healthy person or a sample derived from a healthy person.

Biomarker The biomarker for an autoinflammatory disease of the present invention is characterized by comprising mitochondrial DNA (mtDNA) contained in a biological fluid. In the present invention, mtDNA is used as an index for evaluating an autoimmune disease based on the fact that mtDNA is present at a high concentration in a biological fluid of an autoimmune disease patient (eg, serum, urine, and cerebrospinal fluid).

  mtDNA is circular double-stranded DNA present in the matrix of mitochondria, an organelle. mtDNA includes cytochrome b, cytochrome c oxidase (COX) subunits I, II, III, II; NADH dehydrogenase (NADH) subunits 1, 2, 3, 4, 4L, 5, 6; ATP synthase (ATPase) ) The sixth and eighth subunits are coded. Detection of mtDNA as a biomarker of the present invention can be performed by detecting DNA of these genes encoded by mtDNA. Any one of the specific genes may be detected as a biomarker for an autoimmune disease, or two or more of them may be detected in combination and used for evaluating an autoimmune disease. For example, COXIII, NADH dehydrogenase, cytochrome b and the like can be suitably used for detecting mtDNA which is a biomarker of the present invention.

  Further, since the above-mentioned series of proteins are encoded on circular mtDNA, the behavior of mtDNA can be accurately detected using any of the DNAs encoding the proteins as an index.

  The origin of the biological body fluid is not particularly limited, and may be any animal. More specifically, human and non-human mammals can be mentioned. Examples of mammals other than humans include rodents such as mice, rats, hamsters, and guinea pigs, and experimental animals such as rabbits; domestic animals such as pigs, cows, goats, horses, and sheep; pets such as dogs and cats; Primates, such as orangutans and chimpanzees. In the present invention, the biological fluid is preferably derived from a primate, more preferably a human.

  In the present invention, the biological body fluid is not particularly limited as long as it is a liquid that can be collected in the living body and contains mtDNA. Specific examples of the biological body fluid include bodily fluids such as whole blood, serum, plasma, urine, cerebrospinal fluid, saliva, and alveolar lavage fluid, and preferred examples include serum, urine, and cerebrospinal fluid. Further, from the viewpoint of low invasiveness at the time of collection, serum and urine are more preferable examples.

  For example, when the biological fluid is serum in the present invention, it is obtained by removing blood cells and blood coagulation factors such as fibrinogen (factor I), prothrombin (factor II), factor V, and factor VIII from blood (whole blood). . The serum can be obtained according to a method adopted in clinical tests and the like, and is not particularly limited. For example, a supernatant obtained after allowing blood to stand, or a supernatant obtained by subjecting blood to centrifugation Can be obtained as Prior to the detection of the biomarker of the present invention, if necessary, pretreatment using a filter or a column may be performed to remove impurities such as serum proteins. Serum proteins include, for example, albumin, transferrin, haptoglobin, transthyretin, α1 antitrypsin, α2 macroglobulin, α1-acid glycoprotein, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) , Complement C3, apolipoprotein AI, apolipoprotein AII, and the like.

  Further, when the biological fluid is urine, for example, by using urine in the early morning, errors due to the measurement time are corrected, and it is expected that the test results are stabilized and the correlation with the disease state is improved (that is, the false positive rate is reduced). You.

  As the biomarker of the present invention, it is more preferable to use mtDNA localized in the extracellular membrane vesicle fraction in a biological body fluid. Although the extracellular membrane vesicle contains exosomes and microvesicles called ectosomes, it is more preferable to use mtDNA localized in the exosome fraction as the biomarker of the present invention. Exosomes are vesicles composed of a lipid bilayer membrane with a diameter of 50 to 200 nm, contain mRNA, microRNA, and various proteins present in the cytoplasm, and have various biological activities depending on donor cells. It is known. Exosomes bud toward secretory vesicles evolving from late endosomes, forming a multivesicular body (MVB) and being transported to the cell surface. Then, the exosome, which is the content, is released outside the cell by MVB being fused with the cell membrane. In addition to being transported to the cell surface, MVB is also transported to lysosome, where it fuses with lysosome and its contents are degraded (for the above, see FIG. 2).

  The mtDNA exists in such an extracellular membrane vesicle having a lipid bilayer membrane, in particular, in an exosome, so that it is protected from degradation by an extracellular DNAse (DNase) and stably exists. it is conceivable that. Therefore, by using mtDNA present in exosomes as a biomarker, it is possible to more accurately evaluate an autoimmune disease. In addition, exosomes are known to be contained in serum and urine in large amounts. By isolating exosomes from serum or urine and measuring mtDNA contained therein, exosomes can be minimally invasive or non-invasive. The possibility of establishing an invasive evaluation system is opened.

  The method for separating exosomes is not particularly limited as long as a sample that can be used as a sample for detecting mtDNA is obtained, and can be appropriately selected from conventionally known methods. Although exosomes can be separated by ultracentrifugation, exosomes can be separated more easily using a commercially available kit. Specifically, ExoQuickTM Exosome precipitation solution, Exoquick-TC, etc. (Both are manufactured by System Biosciences).

  The detection of mtDNA is carried out by preparing a polynucleotide from the biological fluid or the exosome fraction and performing an amplification reaction using primers as a template or a hybridization reaction using a probe with the polynucleotide. The polynucleotide to be prepared is preferably DNA from the viewpoint of stability and accuracy of evaluation. Preparation of a polynucleotide from a biological body fluid or an exosome fraction can be carried out using a known method as appropriate, and examples thereof include a method by phenol extraction and ethanol precipitation, and a method using glass beads. More simply, it can be prepared using a commercially available DNA extraction reagent or DNA extraction kit. For example, a DNA extractor kit specifically includes DNA Extractor SP Kit (manufactured by Wako Pure Chemical Industries, Ltd.).

  Detection of DNA corresponding to the above-described protein encoded by mtDNA can be appropriately selected from conventionally known methods. For detection, usually, a polynucleotide serving as a probe or an oligonucleotide serving as a primer is used to measure or amplify the expression level. Examples of the primer include an oligonucleotide having a specific sequence capable of specifically hybridizing to at least a part of a target gene and amplifying the gene. In addition, examples of the probe include a polynucleotide having a unique sequence that specifically hybridizes to at least a part of a target gene.

  These primers or probes can be designed and synthesized according to a conventionally known method based on the acquisition of sequence information of a target gene using programs and databases such as BLAST and FASTA. The base sequence length of the primer is usually 16 to 32 bp, preferably 18 to 30 bp, more preferably 20 to 24 bp. For example, specific examples of the primer set for specifically amplifying the COXIII gene include the primer sets represented by SEQ ID NOS: 1 and 2 used in Examples described later. Further, as a primer set for specifically amplifying the NADH gene, specifically, primers represented by SEQ ID NOs: 3 and 4 used in Examples described later are exemplified. Furthermore, specific examples of primers for specifically amplifying the cytochrome b gene include 5′-ATGACCCCAAATACGCAAAAT-3 ′ (forward primer: SEQ ID NO: 5); 5′-CGAAGTTTTCATCATGCGGAG-3 ′ (reverse primer: SEQ ID NO: 6) Is exemplified.

  Detection of mtDNA can be performed based on an amplification product obtained by a gene amplification reaction using a primer as described above or a hybrid product obtained by a hybridization reaction using a probe.

  For the amplification of the target gene, a conventionally known method can be employed, and is not particularly limited. Examples thereof include amplification of DNA / RNA by polymerase chain reaction (PCR), and more specifically, RT-PCR, Examples include nested PCR, real-time PCR, competitive PCR, TaqMan PCR, and Direct PCR. In addition, the LAMP (Loop-mediated isomerized Amplification) method, the ICAN (Isothermal and Chimeric primer-initialized Amplification of Nucleic acids) method, the RCA (Rolling method) or the RCA (Rolling method) may be used.

The detection of the amplification product is not particularly limited as long as it is a method capable of determining whether or not the polynucleotide is the desired polynucleotide.For example, it is confirmed whether or not a polynucleotide of a predetermined size has been amplified by agarose gel electrophoresis. be able to. In addition, it can be detected by labeling deoxynucleotide triphosphate (dNTP) incorporated in the amplification product in the course of the amplification reaction and measuring the labeling substance of dNTP incorporated in the amplification product. Examples of the label include fluorescent substances such as fluorescein (FITC), sulforhodamine (SR), and tetramethylrhodamine (TRITC); luminescent substances such as luciferin; and radioisotopes such as ³² P, ³⁵ S, and ¹²¹ I. . Alternatively, quantitative detection may be performed by an intercalator method using Syber Green or the like.

  The length of the nucleotide sequence of the probe used for detecting mtDNA is usually 20 to 250 bp, preferably 20 to 100 bp, and more preferably 20 to 50 bp. The probe that hybridizes to the polynucleotide may be any probe that hybridizes to at least a part of the polynucleotide and can be detected, and may not have a completely complementary sequence.

  In the hybridization reaction using the above-mentioned probe, a condition under which the probe specifically hybridizes to the target polynucleotide (a stringent condition) based on conventionally known conditions, for example, DNA having 90% or more homology Conditions under which DNAs hybridize with each other and DNAs with lower homology do not hybridize can be appropriately set. Stringent conditions include, for example, washing at a temperature of 40 ° C. to 70 ° C. with a sodium concentration of 150 to 900 mM and a pH of 6 to 8. More specifically, 50 ° C., 2 × SSC (300 mM NaCl, 30 mM Citric acid) and 0.1% by volume SDS for washing.

If necessary, a probe may be used as a fluorescent substance such as fluorescein (FITC), sulforhodamine (SR) or tetramethylrhodamine (TRITC); a luminescent substance such as luciferin; a radioactive isotope such as ³² P, ³⁵ S, ¹²¹ I or the like. Body; enzymes such as alkaline phosphatase and horseradish peroxidase; and may be labeled using a labeling substance such as biotin. After the hybridization reaction, these target substances are detected according to a conventionally known method based on the type of each labeling substance, whereby a hybridization product can be detected.

  In an animal (preferably a human) suffering from an autoimmune disease, the amount of mtDNA contained in a biological fluid (or in an exosome), which is a biomarker of the present invention, is significantly increased as compared to a normal control group. In the present invention, when the amount of mtDNA contained in the biological fluid is larger than that in the normal control group, the amount of mtDNA in the serum of the test animal is preferably 10 times or more, more preferably 50 times or more, of the normal control group, Particularly preferably, it refers to the case of 100 times or more. Here, the normal control group refers to a healthy test animal (preferably a human) that does not suffer from an autoinflammatory disease and has no inflammatory symptoms.

  In the present invention, an autoimmune disease means that the immune system malfunctions, abnormal antibodies called autoantibodies and self-reactive immune cells are produced, and a specific cell or tissue of itself is recognized as a foreign substance and attacked. This is a group of diseases that occur when the disease occurs. In addition, an autoimmune disease is a systemic disease that causes inflammation and tissue damage due to an immune reaction to the self and involves fever, pain, organ damage, joint destruction, and the like. Specific examples of autoimmune diseases include rheumatoid arthritis (RA), polymyositis / dermatomyositis (PM / DM), systemic lupus erythematosus (SLC), systemic scleroderma (SSc), Sjogren's syndrome, mixed connective tissue So-called collagen diseases such as disease (MCTD); Behcet's disease, Crohn's disease, ulcerative colitis, familial Mediterranean fever (FMF), Cryopyin-related periodic fever syndrome (CAPS), TNF receptor-related periodic fever syndrome (TRAPS), Mevalonate So-called autoinflammatory syndromes such as Kinase-related periodic fever syndrome (MAPS: also known as high IgG syndrome), periodic fever with aphthous stomatitis, pharyngitis, and lymphadenopathy (PFAPA), Blaau syndrome (also known as juvenile sarcoidosis); Castle Mann's disease and the like are exemplified. Autoimmune diseases to which the biomarkers of the present invention are applied preferably include collagen disease and autoinflammatory syndrome, and more specifically, Behcet's disease, rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, and polymorphism Myositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease (MCTD), Crohn's disease, ulcerative colitis. More preferably, the autoimmune disease to which the biomarker of the present invention is applied is an autoinflammatory syndrome, more specifically, Behcet's disease.

  Although it is not desired to limit the present invention, it is presumed that abnormalities occur in the inflammatory cascade involving mitochondria shown in FIG. 1, which results in the development of autoimmune diseases (particularly autoinflammatory diseases). You.

  That is, bacterial components such as bacteria (PAMPs) and host-derived components (DAMPs) released by cell injury promote the production of mitochondrial-derived reactive oxygen species (mROS), which activates the inflammasome to cause IL- Promotes the production of inflammatory cytokines such as 1β and IL-18. In addition, mitochondria are damaged by mROS and release their contents into the cytoplasm. When they go out of the cell due to apoptosis or necrosis, they act as DAMPs and further enhance inflammation. Injured mitochondria are isolated and degraded (mitofazi) by autophagy, thereby suppressing inflammation. However, if a defect is observed in the inflammatory cascade involving mitochondria, it is considered that excessive inflammation is caused or the inflammation does not converge and is prolonged (see FIG. 1 for the above). Mitochondria contain mtDNA encoding information such as mitochondrial proteins. Thus, when mitochondria are damaged, mtDNA is released along with other contents.

  As described above, the present invention is characterized in that an autoimmune disease is evaluated by using, as a biomarker, mtDNA released into a biological fluid that directly reflects abnormalities of the inflammatory cascade. Therefore, according to the biomarker of the present invention, it is possible to accurately evaluate a disease for which a biomarker that accurately reflects a disease state has not been found conventionally. Among the aforementioned specific autoimmune diseases, in particular, for Behcet's disease, etc., in the inflammatory markers used in the conventional evaluation methods, the actual disease state and the behavior of the substance used as the marker are not necessarily linked. And accurate evaluation was difficult. Therefore, Behcet's disease is exemplified as a particularly suitable target disease to which the biomarker of the present invention is applied.

  In addition, among patients with Behcet's disease, in patients presenting with clinical symptoms of active uveitis, the amount of mtDNA in biological fluids tends to be high, and the degree of symptoms should be diagnosed according to the amount of mtDNA in biological fluids. Can also. Further, among patients with Behcet's disease, in difficult treatment cases requiring TNF-α antibody therapy, the amount of mtDNA in a biological body fluid tends to increase, and the selection of treatment intensity and prognosis according to the amount of mtDNA in a biological body fluid. It is also possible to make a guess.

Diagnostic Kit The diagnostic kit for an autoimmune disease of the present invention contains a reagent capable of detecting the mtDNA. As a reagent capable of detecting mtDNA, a primer that specifically amplifies the gene or gene fragment, if the detection of mtDNA is performed based on the gene or gene fragment encoded by mtDNA, Alternatively, a probe that specifically hybridizes to a gene fragment is included as a reagent. Specific examples of the reagents contained in the kit of the present invention include the primers shown in SEQ ID NOs: 1 to 6. These primers and probes are as described in the above “biomarker”.

  Furthermore, the reagent capable of detecting mtDNA may contain a buffer, a salt, a stabilizer, a preservative, and the like, and may be formulated according to a conventionally known method. In addition, the diagnostic kit of the present invention includes, in addition to the reagents, a labeling substance, a labeling substance detection agent, a reaction diluent, a standard antibody, a buffer, a lysis agent, which may be required for detecting mtDNA. , A detergent, a reaction stopping solution, a control sample, and the like.

  In the kit of the present invention, for example, a probe for detecting mtDNA can be used by immobilizing it on an insolubilized carrier. Therefore, the diagnostic kit of the present invention may also include an insolubilized carrier. The material of the insolubilizing carrier is not particularly limited as long as it does not hinder the detection of mtDNA.For example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, fluororesin, cross-linked dextran, polysaccharide, paper, silicon, glass, Metal, agarose and the like can be exemplified. Further, two or more of these materials may be used in combination. The shape of the insolubilized carrier may be any shape such as a microplate, a tray, a sphere, a fiber, a bar, a disk, a container, a cell, and a test tube.

  For example, by immobilizing a probe capable of specifically hybridizing with the gene encoded by mtDNA on the above-described insolubilized carrier, a DNA chip used for detecting an autoimmune disease can be obtained. The probe can be immobilized on the insolubilized carrier according to a conventionally known method. Further, mtDNA can be semi-quantitatively detected by immobilizing probes for mtDNA at different concentrations on a carrier at equal intervals and hybridizing with mtDNA.

Evaluation Method Based on mtDNA Amount in Biological Fluid The present invention provides a method for detecting the presence or absence of an autoimmune disease, which includes the following steps, using mtDNA in a biological fluid, which is the aforementioned biomarker.
(I) obtaining a body fluid sample from a test animal;
(Ii) a step of measuring mitochondrial DNA in the body fluid sample; and (iii) a step of detecting the presence or absence of an autoimmune disease based on the result of the step (ii).

  The preparation of the body fluid sample from the test animal and the measurement of mtDNA in the body fluid sample described in the steps (i) and (ii) can be performed according to the description in the above-mentioned “biomarker” column. Further, in the detection step described in the step (iii), the amount of mitochondrial DNA is increased by comparing the result of the step (ii) obtained for the test animal with the result of the step (ii) obtained for the normal control. Can be used as an index. Here, the normal control group is as described in the “biomarker” column.

  In addition, examples of test animals include humans and mammals other than humans. Examples of mammals other than humans include rodents such as mice, rats, hamsters, and guinea pigs, and experimental animals such as rabbits; domestic animals such as pigs, cows, goats, horses, and sheep; pets such as dogs and cats; Primates, such as orangutans and chimpanzees. In the present invention, the test animal is preferably a primate, and more preferably a human.

  Further, the evaluation based on the biomarkers of the present invention, in addition to detecting the presence or absence of an autoimmune disease, a method for predicting susceptibility to an autoimmune disease, a method for detecting the severity, a method for predicting a prognosis, It can be used for screening methods and differential diagnosis of autoimmune diseases.

  When assessing susceptibility to an autoimmune disease based on the biomarkers of the present invention (method for predicting susceptibility to an autoimmune disease), the test is performed in the same manner as in the method for detecting the presence or absence of the autoimmune disease. Based on the result of obtaining a body fluid sample of an animal and measuring mitochondrial DNA in the body fluid sample, based on a criterion that the subject is more susceptible to an autoimmune disease when the amount of the mitochondrial DNA is larger than that in a normal control group. Predict susceptibility.

  When the severity of an autoimmune disease is evaluated based on the biomarkers of the present invention (method for detecting the severity of an autoimmune disease), the same method as in the method for detecting the presence or absence of the autoimmune disease can be used. A body fluid sample is obtained, and based on the result of measuring mitochondrial DNA in the body fluid sample, when the amount of the mitochondrial DNA is larger than that in the normal control group, the severity of the autoimmune disease is determined based on the criterion that the autoimmune disease is severe. Detection is performed. Alternatively, if the biomarker value detected from the test animal is higher than the standard value of the biomarker for the disease, the severity is detected based on the criterion that the disease is severe.

  When the prognosis of an autoimmune disease is evaluated based on the biomarker of the present invention (a method of predicting the prognosis of an autoimmune disease), a body fluid sample of a test animal is used in the same manner as in the method of detecting the presence or absence of the autoimmune disease. And, based on the result of measuring mitochondrial DNA in the body fluid sample, predicting the prognosis based on the criterion that the prognosis of the autoimmune disease is poor when the amount of the mitochondrial DNA is larger than that in the normal control group. Is performed. Alternatively, when the biomarker value detected from the test animal is higher than the standard value for the disease, the prognosis is evaluated based on the criterion that the prognosis of the disease is poor.

  When screening a therapeutic agent effective for an autoimmune disease based on the biomarker of the present invention, a step of administering a subject to a test animal suffering from the autoimmune disease, and a step of obtaining a body fluid sample from the test animal Measuring the amount of mitochondrial DNA in the body fluid; if the amount of mitochondrial DNA is smaller than that of a test animal suffering from an autoimmune disease to which the test substance has not been administered, the test substance is effective for the autoimmune disease. This is performed by sequentially performing the steps of selecting as a suitable therapeutic agent.

  When performing a differential diagnosis in an autoimmune disease based on the biomarkers of the present invention, that is, when distinguishing similar diseases, a body fluid sample of a test animal is obtained in the same manner as in the method for detecting the presence or absence of the autoimmune disease. Based on the result of measuring mitochondrial DNA in the body fluid sample, if the amount of the mitochondrial DNA is larger than the standard value of the biomarker in the disease serving as a control, it is said that the patient has a specific autoimmune disease. A distinction is made from similar diseases based on criteria.

  In each method, a correlation diagram is prepared in advance for the amount of mtDNA in serum and the presence or absence of autoimmune disease, severity, susceptibility, prognosis, etc., and the amount of mtDNA in serum in the test animal is determined. The evaluation may be performed by applying to the correlation diagram.

  Diseases can be evaluated at the individual level based on these evaluation methods, and treatment methods can be optimized according to individual disease states.

The present invention provides a preventive or therapeutic agent for an autoimmune disease, comprising as an active ingredient a substance that suppresses secretion of mitochondrial DNA into a body fluid or suppresses the function of mitochondrial DNA.

  Here, suppressing the secretion of mitochondrial DNA into body fluids includes, for example, suppressing the incorporation of mtDNA into exosomes, suppressing the release of exosomes from donor cells, and controlling the exosomes in the cell membrane of acceptor cells. It refers to suppressing the incorporation by fusion or endocytosis. In addition, suppressing the function of mitochondrial DNA refers to directly or indirectly inhibiting the induction of an inflammatory reaction by mtDNA, for example, mtDNA is cut by hydrolysis or the like, and exosomes containing mtDNA are destroyed. And signal pathways activated by mtDNA (eg, inhibition of signal pathways via G protein-coupled receptors or IL-1 receptors, Toll-like receptors or inflammasomes).

  Examples of the active ingredient of the agent for preventing or treating an autoimmune disease of the present invention include DNA degrading enzymes, active oxygen chelators, antioxidants, G protein-coupled receptor inhibitors, anti-IL-1β antibodies, and anti-IL-1 Receptor antibodies, Toll-like receptor antagonists and the like are exemplified. Furthermore, useful substances include antisense DNA, nucleic acid aptamers, chelating agents for mROS, and inhibitors for the synthesis of exosome membranes, which selectively hybridize to mtDNA and suppress the induction of inflammatory reaction by mtDNA.

  The dose of the prophylactic or therapeutic agent of the present invention is not particularly limited as long as it is an effective amount for treating an autoimmune disease, and is appropriately set based on the age, weight, symptoms, and the like of the patient.

  In addition, the prophylactic or therapeutic agent of the present invention includes conventionally known carriers, excipients, suspending agents, dispersants, pH adjusters, antioxidants, preservatives, and the like, as long as the effects of the active ingredients are not impaired. May be included. In addition, the prophylactic or therapeutic agent of the present invention is carried on a carrier such as nano-robot, which is designed to selectively open capsules on a cell-selective basis, or a nano-particle that selectively binds to mtDNA or exosomes, and transported to target cells. You may.

  The administration form of the prophylactic or therapeutic agent of the present invention can be appropriately selected from conventionally known ones according to the age, symptom, and type of disease of the patient. Examples thereof include intravenous administration, intraarterial administration, oral administration, and tissue administration. Examples include internal administration, transdermal administration, transmucosal administration, and airway administration. Further, the formulation of the prophylactic or therapeutic agent of the present invention is not limited, and may be appropriately selected depending on the dosage form employed. For example, injections, drops, oral preparations, eye drops, inhalants, ointments, lotions Agents and the like.

  The disease to which the preventive or therapeutic agent of the present invention is applied is an autoimmune disease, and specific examples thereof are as described above. Autoimmune diseases to which the preventive or therapeutic agent of the present invention is applied preferably include collagen disease and autoinflammatory syndrome, and more specifically, Behcet's disease, rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma Disease, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease (MCTD), Crohn's disease, ulcerative colitis, more preferably autoinflammatory syndrome, and more specifically Behcet's disease Is mentioned.

  Hereinafter, the present invention will be described with reference to Test Examples, but the present invention is not limited to these Test Examples.

1. Sample Preparation All tests were performed with the approval of the Osaka University Graduate School of Medicine Institutional Review Board. As samples, diagnostic criteria by the Japan Ministry of Health, Labor and Welfare Behcet's disease research group, diagnostic criteria for systemic lupus erythematosus (SLE) by the American College of Rheumatology, revised diagnostic criteria for rheumatoid arthritis (RA) by the American College of Rheumatology, systemic strength by the Japanese Ministry of Health, Labor and Welfare research team Serum collected from patients diagnosed based on the diagnostic criteria for dermatosis (SSc) and polymyositis / dermatomyositis (PM / DM) by the Ministry of Health and Welfare specific disease research group was used. Mitochondrial DNA used in the following test examples and comparative test examples was collected from 36 Behcet's disease patients, 22 SLE patients, 28 RA patients, 14 SSc patients, and 8 PM / DM patients. Was used. As controls, those collected from 10 healthy volunteers (sometimes referred to as healthy subjects or controls) were used. However, Reference Test Example 1 was evaluated using exosomes collected from 75 Behcet's disease patients and 9 healthy volunteers. The blood cell fraction used in the following test examples was evaluated using those collected from 6 Behcet's disease patients and 6 healthy volunteers.

2. Separation of Serum DNA Serum DNA was prepared from serum (100 μl) using DNA Extractor SP Kit (manufactured by Wako Pure Chemical Industries, Ltd.) according to the attached protocol. Specifically, 100 μl of a serum sample was digested with 5 μl of a protease (56 ° C., 10 minutes), and treated with 300 μl of a sodium iodide solution to remove proteins from the serum. The DNA was precipitated using 100% by volume of ethanol, and the supernatant was removed. The resulting DNA pellet was purified by adding 70% by volume of ethanol. The obtained serum DNA was resuspended in DNase-free water (20 μl).

3. Real-time PCR
Using SYBR Premix Ex Taq (Perfect Real Time) (manufactured by Takara Bio Inc.), according to the attached protocol, ABI PRISM 7700 (Life Technologies Japan), an intercalator by real-time PCR using SYBR Green I using a SYBR Green I method. . The PCR conditions are as follows.
Stage 1 (1 cycle): 95 ° C. for 30 seconds;
Stage 2 (40 cycles): 95 ° C. for 5 seconds, 60 ° C. for 30 seconds;
Stage 3 (1 cycle): 95 ° C for 15 seconds, 60 ° C for 1 minute, 95 ° C for 15 seconds

  In order to quantify the mtDNA concentration, a real-time standard curve was prepared using purified DNA derived from a whole cell lysate of peripheral blood mononuclear cells (PBMC) of a healthy subject. Further, a sample in which no PCR product was produced even after the completion of 40 cycles of the PCR reaction (that is, a sample in which fluorescence emission by SYBR Green was not recognized) was determined to be “not detectable”.

The primers used for mitochondrial DNA detection are shown below.
(Human cytochrome C oxidase subunit III: COXIII)
Forward primer (SEQ ID NO: 1):
5'-ATGACCCACCAATCCACATGC-3 '
Reverse primer (SEQ ID NO: 2):
5′-ATCACATGGCTAGGCCGGGAG-3 ′
(Human NADH dehydrogenase: NADH)
Forward primer (SEQ ID NO: 3):
5'-ATACCATCATGCCAACCTCCT-3 '
Reverse primer (SEQ ID NO: 4):
5'-GGGCCTTTGCGGTAGTTGTAT-3 '

4. Exosome separation (separation using a commercial kit)
Exosomes were separated from plasma (100 μl) using ExoQuick ™ Exomesome precipitation solution (manufactured by System Biosciences) according to the attached protocol. Specifically, the serum (100 μl) and the ExoQuick solution (25 μl) were mixed gently, cooled for 30 minutes, and then subjected to centrifugation at 1500 × g for 30 minutes. Thereafter, the supernatant was completely removed to obtain a pellet (extracellular membrane vesicle containing the exosome fraction). The supernatant was used as a non-exosome fraction in Test Example 3 below. This pellet was resuspended in 100 μl of PBS solution to obtain an exosome suspension for use in the following test examples. From the obtained exosome suspension, the above-mentioned 2. MtDNA was isolated according to the method described in (1).

(Separation using ultracentrifugation)
Exosomes were isolated by ultracentrifugation. Specifically, after 1.5 ml of serum was centrifuged at 2000 × g for 10 minutes to remove cell fragments, the supernatant was centrifuged at 10,000 × g at 4 ° C. for 30 minutes to remove cell debris. . Thereafter, the supernatant was filtered through a 0.22 μm filter, and the filtrate was further centrifuged at 110,000 × g at 4 ° C. for 2.5 hours. The obtained pellet was dissolved in 9 ml of PBS, layered on a 30% sucrose solution, and further subjected to centrifugation at 100,000 × g at 4 ° C. for 2.5 hours. The sucrose solution containing the exosome was recovered and centrifuged at 100,000 × g at 4 ° C. for 2.5 hours to obtain a pellet composed of the exosome. This pellet was suspended in a PBS solution to obtain a highly pure exosome suspension for use in the following test examples.

5. ELISA
4. The exosome suspension prepared in 1) was suspended in an exosome binding buffer (Exomesome Binding Buffer) of ExoELISA Kit of System Bioscience to be diluted 5000 times. This suspension (100 μl) was coated on a 96-well plastic plate for 16 hours at 37 ° C. (solid phase immobilization). After blocking with 5% by volume BSA (solvent: PBS), the cells were incubated with 0.5 μg / ml anti-human CD9 monoclonal antibody at 37 ° C. for 2 hours, and thereafter, anti-rabbit IgG labeled with HRP (horseradish peroxidase). (100 μl at 10,000-fold dilution) was added and incubated at room temperature for 30 minutes. Human CD9 is known as a marker protein present on exosomes. 100 μl of a subrate reagent kit from R & D was added as a substrate, and the luminescence due to the enzymatic reaction was detected at 425 nm using ARVO manufactured by perkinelmer.

6. Isolation of blood cell fraction With respect to the peripheral blood fraction, 30 ml of blood was collected from the subject, separated into mononuclear cells and polynuclear cells using polymorphprep (Axis-Sheid) according to the attached recommended protocol, and subjected to multinuclear cell fractionation. Was evaluated as a neutrophil. From the mononuclear cell fraction, CD14-positive monocytes were further isolated using an anti-CD14 antibody with magnetic beads (Miltenyi Biotec). The remaining monocyte fraction was evaluated as the lymphocyte fraction.

7. Regarding statistical mtDNA, a relative value obtained based on a calibration curve was logarithmically processed, and then statistically examined by student's t-test. Regarding exosomes, the relative values obtained based on the calibration curve were statistically examined by student's t-test. Other studies on inflammatory reactions in vivo and in vitro were statistically performed by Mann-Whitney U-test.

[Test Example 1: Relationship between serum mitochondrial DNA level and autoimmune disease]
Serum DNA concentrations in patients with Behcet's disease, SLE patients, SSc patients and PM / DM patients with autoimmune diseases were determined using the suspension obtained by the method described in “2. The measurement was performed using a total meter (NanoDrop: manufactured by Thermo), and the serum mtDNA value was measured according to the method described in the above “3. Real-time PCR”. The results are shown in FIGS.

  As shown in FIGS. 3 and 4, the median serum DNA concentration was similar between the autoimmune disease patient and the control group, whereas the serum mtDNA value was higher for both COXIII and NADH. The value was clearly higher in patients with immune diseases. As shown in the right column of FIG. 3, the serum of an autoimmune disease patient contained about 50 to 500 times as much mtDNA as a healthy person.

[Test Example 2: Elevation of mitochondrial DNA in serum in patients with Behcet's disease]
The concentration of serum DNA collected from patients with Behcet's disease or healthy volunteers (control group) and the amount of mtDNA in serum were measured. The measurement of the serum DNA concentration and the serum mtDNA value was performed in the same manner as in Test Example 1. FIG. 5 shows the results.

  As shown in FIG. 5, the median serum DNA value between Behcet's disease patients and the control group was similar, whereas the serum mtDNA value was significant for both COXIII and NADH. Patients showed significantly higher values. That is, among autoimmune diseases, it was shown that the mtDNA level in serum was more remarkably increased in patients with Behcet's disease, which is one of the autoinflammatory diseases.

[Test Example 3: Serum mtDNA value and clinical characteristics in Behcet's disease patient]
The clinical symptoms of patients with Behcet's disease were investigated using medical records, and serum mtDNA levels and clinical characteristics were examined by a retrospective study. As shown in FIG. 6 a), among patients with Behcet's disease, a high mtDNA value was shown in a case (Uveitis) showing clinical symptoms of active uveitis at the time of examination. In addition, patients were classified into a non-treated group treated with only colchicine (Non), a group treated with a standard immunosuppressant such as steroid (Conv), and a group treated with an anti-TNF-α antibody (Mab). When the values were compared, an increase in serum mtDNA value was observed in the group to which the anti-TNF-α antibody was administered, as shown in FIG. This suggests that serum mtDNA may be high in the acute activity period and in difficult-to-treat cases, and may be useful for monitoring disease activity of Behcet's disease and predicting prognosis.

[Test Example 4: Localization of mitochondrial DNA in serum of Behcet's disease patient]
In order to evaluate in which fraction of serum mtDNA is localized in Behcet's disease patient, serum collected from a Behcet's disease patient or a control group was used, and "4. Isolation of exosomes (separation using a commercial kit) , An exosome fraction and a non-exosome fraction were obtained, respectively. For each fraction, COXIII and NADH were detected according to the method described in “3. Real-time PCR”. The results are shown in FIGS. 7 (a) and (b). The unit of the vertical axis in FIG. 7A is%, and is given by (amount of mtDNA contained in each fraction) / (amount of mtDNA contained in serum (sum of both fractions)) × 100. The relative expression level shown on the vertical axis in FIG. 7 (b) is based on a DNA solution obtained by diluting a DNA extract obtained from PBMC (peripheral blood mononuclear cell) of a healthy person five times by six steps as a template. A calibration curve is created by performing PCR as shown in FIG. In addition, the localization of DNA fragments in MVB of monocytes derived from peripheral blood of Behcet's disease patients was examined by immunoelectron microscopic analysis using an anti-DNA antibody. The cyclic structure shown in FIG. 7 (c) indicates MVB, and vesicles therein indicate exosomes. In addition, the black point at the end of the arrow indicates a DNA fragment stained with the anti-DNA antibody, and the white bar is 500 nm.

  FIG. 7 (a) showed that mtDNA in serum was localized in extracellular membrane vesicles represented by exosomes. In addition, FIG. 7 (b) shows that mtDNA present in extracellular membrane vesicles typified by exosomes, not mtDNA present free in blood, is higher in Behcet's disease patients. . FIG. 7 (c) shows that DNA fragments are contained in vesicles in the MVB of peripheral blood-derived monocytes, and that DNA fragments that appear to be mtDNA are present in exosomes. It was also confirmed at the electron microscope level. According to the findings so far, free mtDNA alone is thought to act as DAMPs (for example, see Non-Patent Document 1). However, the above results indicated that high levels of mtDNA localized in exosomes, rather than free mtDNA released with cell injury, are associated with these diseases.

[Reference Test Example 1]
It was determined whether the amount of the exosome fraction in the serum of a Behcet's disease patient was elevated as compared to the control group (healthy volunteers). Using serum collected from a Behcet's disease patient or a control group, CD9 protein was detected according to the method described in “5. ELISA”. Table 1 shows the results. The relative expression level is expressed as a relative value based on a calibration curve prepared using a solution obtained by diluting the exosome suspension obtained from the serum of a healthy individual three times in ten steps in ten steps.

  Table 1 shows that the exosome levels do not differ between healthy subjects and Behcet's disease patients.

[Test Example 5: Incorporation of mtDNA by exosome]
From the above Test Examples 1 to 4, it was revealed that mtDNA in serum (more specifically, in exosomes) shows abnormally high values in patients with Behcet's disease. Therefore, the present inventors have established a hypothesis that mtDNA released into the cytoplasm due to mitochondrial stress may be released outside the cell by being taken into exosomes (for example, see FIG. 8). Was considered.

(5-1) Changes in intracellular localization of mitochondria and exosomes by stimulation of PAMPs and DAMPs Mitochondria-specific GFP expression in THP-1 cells (ATCC Cat. No. TIB-202), a human monocyte cell line. The day after transfection of mito-GFP vector (manufactured by invitrogen) expressing 2 × 10 ⁴ cells was seeded on a glass bottom dish (manufactured by IWAKI), and 10% FCS-containing cells were added for differentiation into macrophage-like cells. 100 nM PMA (phorbol myristate acetate) was added to the RPMI1640 medium and cultured for 3 days. Thereafter, 5 mM ATP and 15 μM Nigericin were added, and stimulation was performed for 30 minutes. Here, ATP acts as DAMPs, and nigericin acts as PAMPs. Thereafter, the cells were fixed with 4% by volume paraformaldehyde (PFA). Then, blocking was performed with 30% by volume NGS (Normal Goat Serum), and a 100-fold diluted anti-CD63 antibody (manufactured by abcam), a marker of exosome, and a 1000-fold diluted solution of a fluorescently labeled anti-rabbit IgG antibody (manufactured by invitrogen) were used. And observed with a confocal laser microscope. In addition, in order to examine whether the change in intracellular localization is due to mitochondrial-derived reactive oxygen species (mROS), THP-1 cells previously treated for 1 hour with 500 μM of mito-TEMPO (manufactured by Alexis) which is a chelating agent for mROS But a similar experiment was performed. FIG. 9 shows the results.

  As shown in FIG. 9, it was shown that mitochondria and exosomes were co-localized by mitochondrial stress caused by ATP and nigericin. In addition, since colocalization was no longer recognized by mito-TEMPO treatment, which is a chelating agent for mROS, colocalization of mitochondria and exosomes was caused by mROS released from mitochondria under stress. Indicated.

(5-2) Change in the amount of mtDNA in exosomes by stimulation of PAMPs or DAMPs 2 × 10 ⁶ human monocytic cell lines THP-1 were seeded in a 6-well dish, and cultured in the presence of 100 nM PMA. After culturing for 5 days, 5 mM ATP and 15 μM nigericin were added to the medium and stimulated for 60 minutes. Exosomes released into the cell supernatant were collected using Exoquick-TC (manufactured by System Bioscience), DNA was extracted from the exosome fraction, and the amount of mtDNA was examined by real-time PCR. Extraction of DNA from the exosome fraction is performed by extracting DNA from the exosome fraction prepared in “4. Exosome separation (separation using a kit)” according to the method described in “2. Separation of serum DNA”. (Manufactured by Kojun Pharmaceutical Co., Ltd.). The real-time PCR was performed according to the method described in “3. Real-time PCR”. Further, in the same manner as in the above (5-1), for the purpose of examining whether or not these changes were due to mROS, the same experiment was performed on cells that had been pretreated for 1 hour by adding 500 μM mito-TEMPO to the medium. The results are shown in FIG. The vertical axis in FIG. Similarly to the above, PCR is performed using a template obtained by serially diluting DNA derived from peripheral blood mononuclear cells derived from a healthy person as a template, and the relative expression level relative to that value.

  As shown in FIG. 10, mtDNA in exosomes was increased due to mitochondrial stress. In addition, no increase in mtDNA was observed in cells treated with mito-TEMPO, a chelating agent for mROS, indicating that mtDNA in exosomes increased due to mitochondrial stress and that they were carried by mROS. Was done.

  From the results of (5-1) and (5-2) above, when stress occurs in mitochondria, exosomes are localized near damaged mitochondria, and mtDNA released into cells is taken up by exosomes. Was suggested. It was also presumed that these were carried to the cell surface by MVB and released outside the cells as exosomes containing mtDNA. Thus, the present inventors have studied the cells that release the most mtDNA using peripheral blood obtained from a Behcet's disease patient. A CD14-positive monocyte fraction, a lymphocyte fraction, and a neutrophil fraction were isolated from peripheral blood by the method described in “Sample Preparation: Blood Cell Fraction”, and 100,000 cells were subjected to 1% type I collagen. After inducing the collagen condensation reaction by suspending in a solution and culturing at 37 ° C. for 30 minutes, 5 mM of ATP, 500 ng / ml of Pam3CSK4, 50 nM of PMA and 250 ng / ml of ionomycin or 1 nM as representative of PAMPs / DAMPs. Stimulated with fMLP for 30 minutes. Thereafter, the exosomes in the culture supernatant were isolated by Exoquick, and then the DNA in the exosomes was extracted, and the value of mtDNA was determined by qPCR.

  FIG. 11 shows the obtained results. As shown in FIG. 11a, in lymphocytes and neutrophils derived from patients with Behcet's disease, no enhanced mtDNA release was observed in any of the stimuli, but in CD14-positive monocytes, mtDNA was markedly stimulated by ATP and Pam3CSK4. Enhanced release was noted. In addition, as shown in FIG. 11b, the release of mtDNA by ATP, Nigericin and Pam3CSK4 was significantly enhanced in CD14-positive cells derived from a Behcet's disease patient as compared to CD14-positive cells derived from a healthy subject. These facts suggested that in patients with Behcet's disease, a large amount of mtDNA was released from myeloid cells represented by peripheral blood CD14-positive monocytes.

[Test Example 6: Examination of Factors of Increase in Serum mtDNA Level in Behcet's Disease Patient]
When the results of the above test examples and reference test examples are combined, in patients with Behcet's disease, a high serum mtDNA value is not due to a large amount of exosomes itself, but a large amount of mtDNA incorporated into exosomes as compared to the control. It was shown that. In view of the above, the inventors of the present invention have concluded that, regarding the reason that the amount of mtDNA in exosomes is large in Behcet's disease patients, in Behcet's disease-derived monocytes, it may be impaired in the degradation process of mitochondria damaged by PAMPs / DAMPs stimulation. Based on the hypothesis that peripheral blood monocytes were obtained from patients with Behcet's disease and healthy subjects, the results were verified by comparative study.

According to the method described in “6. Isolation of blood cell fraction”, a CD14-positive monocyte fraction was isolated from peripheral blood, and 1 × 10 ⁵ cells were seeded on a 35 mm dish and stimulated with 100 nM PMA. The cells were cultured for 16 hours. Thereafter, the cells were stimulated with 5 mM ATP and 15 μM Nigericin for 60 minutes as a representative of PAMPs / DAMPs. In addition, as a positive control, the cells were stimulated with Rapamycin (10 nM), which induces an effect equivalent to the autophagy induced by starvation, for 60 minutes. Then, the medium was replaced with complete RPMI1640 medium and cultured at 37 ° C. for 60 minutes. Using these samples, induction of autophagy was examined by Western blotting. That is, the cells were suspended in a TNE buffer containing 1% NP40 (Nonidet P-40), heat-treated at 96 ° C. for 5 minutes, and then subjected to electrophoresis. After transfer to a nitrocellulose membrane, blocking was performed with 1% skim milk. After overnight reaction with an anti-LC-3 antibody (manufactured by Cell signaling), an anti-ATG12 antibody (manufactured by Cell signaling), and an anti-β-actin antibody (manufactured by MBL), the resultant was reacted with a secondary antibody to which HRP was bound.

  Using a confocal microscope, the co-localization between the damaged mitochondria producing mROS and the autophagosome was examined. That is, the cells were stimulated with ATP, Nigericin and Rapamycin in the same manner as described above in the presence of 2 mM MitoSOX, and then replaced with complete RPMI medium. Thereafter, the cells were fixed with 4% PFA for 1 hour, washed several times with PBS containing 0.1% Triton-X, and reacted with an anti-LC3 antibody (manufactured by MBL) overnight. After washing, the cells were reacted with a secondary antibody to which Alexa-Fluor647 was bound, and observed with a confocal microscope.

  Using an electron microscope, the structures of mitochondria and other organelles under mitochondrial stress were examined. That is, CD14-positive monocytes were stimulated with Rotenone (10 μM), an inhibitor of mitochondrial respiratory chain complex I, for 6 hours. After fixing with 4% PFA for 1 hour, the cells were pre-fixed with 1% glutaraldehyde for 10 minutes, and further fixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 hour. After embedding the sample in a resin, a thin film having a thickness of 80 nm was formed and observed with a transmission electron microscope.

  FIG. 12 shows the obtained results. As shown in FIG. 12A, in CD14-positive monocytes derived from healthy subjects, ATP, Nigericin and Rapamycin stimulation induced the expression of lipid-bound LC3 (LC3 II) by Western blotting, whereas Behcet was observed. In monocyte cells derived from a diseased patient, induction of expression of LC3II by stimulation with Rapamycin was observed, but induction of expression of LC3II by stimulation with ATP and Nigericin was not observed. On the other hand, regarding the induction of expression of the Atg5 / Atg12 complex involved in the induction of lipid-bound LC3, no difference was observed between healthy individuals and Behcet's disease-derived monocytic cells.

  In addition, as shown in FIG. 12b, formation of autophagosome stained by LC3 antibody around mitochondria producing mROS was observed in monocytes derived from healthy subjects, whereas in monocytes derived from Behcet's disease, Phagosome formation was impaired. Interestingly, the formation of autophagosomes induced by Rapamycin was also observed in monocytic cells derived from Behcet's disease in the same manner as in samples derived from healthy subjects.

  Furthermore, as is clear from the electron micrograph shown in FIG. 12c, unlike monocytes derived from healthy individuals, in Behcet's disease-derived monocytes, small mitochondrial fragments exhibiting a deformed crystal structure due to the induction of mitochondrial stress. Many structures were found in the cytoplasm.

  The above results suggest that in Behcet's disease, normal autophagy induced by starvation or the like is not impaired, but that abnormal induction of mitochondria-specific autophagy (mitofazi) may be observed. It is generally known that when mitochondria are damaged by mitochondrial stress, mitochondria-specific autophagy is induced, and damaged mitochondria are degraded and processed. However, in Behcet's disease, when exposed to PAMPs or DAMPs as a result of infection or cell injury, the mitophagy is impaired, and the degradation of damaged mitochondria is stalled, resulting in increased production of ROS from mitochondria. , The release of mtDNA into the cytoplasm and the uptake of mtDNA into MVB via ROS increased, and it was speculated that they were released extracellularly as exosomes. Therefore, serum mtDNA levels are likely to be high in Behcet's disease.

[Test Example 7: Induction of inflammatory response by exosome derived from Behcet's disease patient]
The exosomes released from the donor cell are taken up by the acceptor cell by cell membrane fusion or endocytosis, and various biological activities of the donor cell are transmitted to the acceptor cell. In the immune system, acceptor cells have been genetically and epigenically controlled by the fact that the peptide / MHC complex is carried by exosomes and contributes to the spread of antigens, and mRNA and microRNA in exosomes (See, e.g., Clotilde Thery, Matias Ostrowski and Elodie Segura. Membrane vehicles as conveyors of immune responses. Nat. In addition, the results of Test Example 5 suggested that when stress was applied to mitochondria, mtDNA was incorporated into exosomes. In patients with autoimmune diseases, since mtDNA is present in exosomes, it is expected that mtDNA will not be affected by DNase and the like even if released outside the cell, and that mtDNA can be relatively stably present in serum and the like. Therefore, a hypothesis was made that "mtDNA and exosomes containing mtDNA might enhance the immune response", and the following were examined.

(7-1) Examination of the effect of patient-derived exosomes on neutrophil migration activity 2 × 10 ⁴ human neutrophils prepared from healthy subjects using a polymorphprep (manufactured by AKIS) using a concentration gradient were subjected to 0 Suspended in RPMI1640 medium containing 1% BSA, seeded on a glass bottom dish (manufactured by Matsunami), added with 70 μl of exosome or mtDNA equivalent to serum from Behcet's disease (BD) patient and a healthy subject, and added to neutrophil motility Was observed over time for 1 hour using a NIKON fluorescence microscope Biostation. Preparation of mtDNA and exosomes was performed according to the above-mentioned "2. Separation of serum DNA" and "4. Separation of exosomes", respectively. The moving speed and moving distance of neutrophils were evaluated using software MetaMorph (manufactured by Molecular Devices). FIG. 13 shows the results. FIG. 14 shows a typical example of the results of tracking the change of neutrophils over time with the addition of exosomes or mtDNA derived from healthy subjects and patients with Behcet's disease.

  As shown in FIG. 13, exosomes containing mtDNA derived from a Behcet's disease patient significantly increased neutrophil migration speed and migration distance as compared to exosomes derived from healthy subjects. In addition, mtDNA prepared from exosomes derived from Behcet's disease patients also showed enhanced neutrophil migration activity. In addition, as shown in FIG. 15, neutrophil migration activity by patient-derived exosomes was inhibited by B. pertussis toxin PTx (manufactured by SIGMA-ALDRICH) which is a G protein-coupled receptor inhibitor (containing 0.1% BSA). PTx treatment was performed at a concentration of 50 ng / ml in an RPMI medium), and the antibody was also treated with an antibody against formyl peptide receptor 1 (FPR1), a G protein-coupled receptor (anti-FPR1 antibody: manufactured by R & D). (Anti-FPR1 antibody treatment was performed at a concentration of 100 μg / ml in RPMI medium containing 0.1% BSA).

(7-2) Investigation of the effect of exosomes derived from Behcet's disease patient on inflammatory cytokine production The bone marrow of C57BL / 6 mice (Charles River) was cultured for 5 days in complete RPMI1640 medium containing 50 ng / ml M-CSF (Peprotec). Bone marrow-derived macrophages (1 × 10 ⁶ ), bone marrow-derived macrophages obtained by culturing for 7 days in DMEM medium containing 20% of L929 cell culture supernatant, 50 ng / ml GM-CSF ( R & D) containing bone marrow-derived dendritic cells (1 × 10 ⁶ ) obtained by culturing for 6 days in complete RPMI1640 medium, or isolated using polymorphprep (Axis-Sheide) based on the attached instructions To human peripheral blood-derived PBMC (1 × 10 ⁶ ) According to the method described in “4. Isolation of exosomes”, exosomes prepared by ultracentrifugation from 100 μl of serum collected from patients with Behcet's disease (BD) or healthy subjects by ultracentrifugation, or exosomes prepared from 70 μl of serum using Exoquick The exosomes were sprinkled and cultured for 24 hours, and the production of inflammatory cytokines of IL-1β, TNFα, IL-12p40 / IL-23p35, and IL-23p19 in the culture supernatant was examined by ELISA.

  Regarding ELISA, commercially available mouse IL-1β-Quantikine ELISA kit, mouse TNFα-Quantikine ELISA kit, mouse IL-12p40 / IL-23p35-QuantikineKITANki-AK19-anti-Kent-AK19-anti-kent And human IL-12p40 / IL-23p35-Quantikine ELISA kit and human IL-1β-Quantikine ELISA kit (all manufactured by R & D systems) according to the attached manual. That is, after blocking the plate attached to the kit on which the antibody against the cytokine is immobilized with 5% by volume BSA (solvent: PBS), a 2- to 5-fold dilution of the culture supernatant is added, and the mixture is incubated at room temperature. Incubated for hours. After washing the plate, the attached biotinylated antibody against the inflammatory cytokine was added, and the plate was incubated at room temperature for 2 hours and washed. Added and incubated for 30 minutes at room temperature. As a substrate, 50 μl of R & D's subsrate reagent kit was added, and the luminescence due to the enzyme reaction was detected at 425 nm using ARVO manufactured by perkinelmer. FIG. 16 shows the results.

  In order to clarify the involvement of TLR9, which is a Toll-like receptor (TLR) that recognizes DNA, in the above reaction, TLR9-deficient mice and MyD88-deficient mice, which are downstream adapter molecules (iFReC, Osaka University , Oriental bioservice contract sales). Further, in order to clarify the involvement of human TLR9 in the above reaction, a similar experiment was carried out using ODN2088 and ODN2088 control (manufactured by Addgene) of CpG oligo DNA which is an antagonist of TLR9. The results are shown in FIG.

  Furthermore, for the purpose of clarifying the involvement of NLRP3 and inflammasome, which are known to recognize mtDNA by the constituent molecules of inflammasome, in the above reaction, the same method was performed using NLRP3-deficient mice, ASC-deficient mice, and Caspase-1 deficient mice. An experiment was performed. The results are shown in FIG.

  As shown in FIG. 16, exosomes derived from Behcet's disease patients (that is, exosomes containing mtDNA) were inflammatory cytokines IL-1β, TNFα, and exosomes isolated by ultracentrifugation and Exoquick in cells derived from mice and humans. It was shown to have an activity of enhancing production of IL-12p40 / IL-23p35, IL-23p19, and the like.

  In addition, as shown in FIG. 17, cytokine production by exosomes derived from patients was partially reduced in bone marrow-derived macrophages and bone marrow-derived dendritic cells derived from TLR9-deficient mice, and almost completely reduced in cells derived from MyD88-deficient mice. Also, in PBMCs derived from human peripheral blood, inflammatory cytokine production by exosomes derived from BD patients was reduced by the TLR9 inhibitor. This suggests that BD-derived exosomes promote cytokine production via TLR not only in mice but also in humans.

  In addition, as shown in FIG. 18, in the bone marrow-derived macrophages prepared from the NLRP3-deficient mouse, the ASC-deficient mouse, and the Caspase-1 deficient mouse, the effect of enhancing the production of IL-1β by the exosome derived from the BD patient disappeared. This suggested that BD patient-derived exosomes promoted IL-1β production via NLRP3 inflammasome.

(7-3) Effect of exosomes derived from Behcet's disease patient on neutrophil infiltration in vivo exosomes derived from a Behcet's disease (BD) patient and a healthy subject prepared according to the above “4. Intraperitoneally administered to aging C57BL / 6 mice (CLEA Japan) (exosomes prepared by ultracentrifugation of 100 μl of serum and exosomes prepared with Exoquick of 70 μl of serum) 3 hours later, 5 ml of PBS was intraperitoneally administered to wash and collect cells infiltrating into the peritoneal cavity. After the solution was centrifuged, the total number of cells was counted using GUAVA (Millipore). In addition, some cells were stained with anti-Gr-1 (Ly6G) antibody (manufactured by BD Bioscience) and anti-Ly6C antibody (manufactured by BD Bioscience) diluted 100-fold, and neutrophils contained in cells infiltrated into the peritoneal cavity. (Ly6G-positive Ly6C-positive) and the ratio of inflammatory monocytes (Ly6G-negative Ly6C-positive) are analyzed by FACS (Canto II (manufactured by BD Bioscience)), and the number of neutrophils is calculated by multiplying by the total cell number. evaluated. The results are shown in FIG.

In addition, based on the results of (7-2) regarding the infiltration of neutrophils and inflammatory monocytes, in order to clarify the involvement of TLR9 and other major TLRs, TLR9 receptor-deficient mice (Osaka University iFReC natural MyD88-deficient mice involved in most TLR and IL-1 receptor signal transduction (created by Osaka University iFRec natural immunology lab, oriental bioservice consignment sales). A similar experiment was carried out. The results are shown in FIG.
Further, in order to clarify the involvement of NLRP3 inflammasome and IL-1 receptor, a similar experiment was performed using NLRP3-deficient mice and an IL-1 receptor antagonist (Millipore). The results are shown in FIG.

  As shown in FIG. 20, the number of neutrophils and inflammatory monocytes infiltrating the peritoneal cavity was significantly increased in mice to which exosomes containing mtDNA derived from a Behcet's disease patient were administered, as compared to exosomes derived from healthy subjects. Was done.

  In addition, as shown in FIG. 21, neutrophil infiltration activity by patient-derived exosomes was partially inhibited in TLR9-deficient mice, and MyD88 involved in signal pathways including other TLR receptors and IL-1 receptors. In mice deficient in TLR9, the inhibition was greater than in mice deficient in TLR9.

  In addition, as shown in FIG. 21, neutrophil infiltration activity by patient-derived exosomes decreased in NLRP3-deficient mice. In addition, administration of an IL-1 receptor antagonist inhibited neutrophil infiltration activity by patient exosomes.

  mtDNA has previously been reported to induce an inflammatory response via TLR9 and NLRP3. Based on these findings and the results of the analysis using the above-described gene-deficient mice, it was strongly suggested that mtDNA is the main molecule responsible for the inflammatory response induced by exosomes derived from Behcet's disease patients.

(7-4) Effect of exosomes derived from Behcet's disease patient on differentiation of IL-17-producing cells.
Recently, it has been shown that IL-1β and IL-23 are important for inducing differentiation into IL-17-producing cells (Sutton, CE et al. Interleukin-1 and IL-23 induce innate IL-17). production from gammadelta T cells, amplifying Th17 responses and autoimmunity.Immunity 31, 331-41 (2009), Martin, B., Hirota, K., Cua, DJ, Stockinger, B. & Veldhoen, M. Interleukin-17-producing gammadelta T cells selectively expand in response to pathogen products and environmental signals. Immunity 31, 321-30 (2009)). In addition, involvement of IL-17 producing cells has been suggested as a cause of Behcet's disease. Based on the results of the above (7-2), the present inventors have made the following investigation on the assumption that exosomes derived from Behcet's disease patients are involved in differentiation into IL-17-producing cells.

  Exosome derived from a Behcet's disease patient and a healthy person prepared according to the above “4. 48 hours later, 5 ml of PBS was intraperitoneally administered, and the cells infiltrating into the peritoneal cavity were washed and collected. The collected cells were separated into a non-T cell fraction and a T cell fraction with an anti-CD3 antibody with magnetic beads (Milliteney), mRNA was extracted from each cell using RNeasy (manufactured by Qiagen), and random primers and CDNA was obtained using reverse transcriptase (manufactured by Invitrogen). Using these as templates, the expression of Il23 mRNA for GAPDH in the non-T cell fraction and Il117a, Il23r and Rorc mRNA for GAPDH in the T cell fraction were determined by real-time PCR using I7700 using a TaqMan (Applied BioScience) probe. It was examined by. The results are shown in FIG.

  In addition, exosomes derived from patients with Behcet's disease and healthy subjects prepared according to the above “4. Isolation of exosomes” were intraperitoneally administered to C57BL / 6 mice (CLEA Japan, 6 to 8 weeks old) (70 μl equivalent of serum). (Exosomes prepared with Exoquick), and 72 hours later, 5 ml of PBS was intraperitoneally administered to wash and collect cells infiltrating into the peritoneal cavity. After the solution was centrifuged, the total number of cells was counted using GUAVA (Millipore). Some cells were stimulated with 50 ng / ml PMA and 250 ng / ml ionomycin for 4 hours in the presence of Golgi-stop (manufactured by BD Bioscience) for 4 hours and then diluted 100-fold with anti-CD3 antibody and anti-CD4 antibody ( After staining with BD Bioscience) and an anti-TCRγδ antibody (BD Bioscience), the cells were fixed with 4% PFA. Thereafter, cytoplasmic cytokine staining was performed with an anti-IL-17 antibody (manufactured by BD Bioscience). The percentage of IL-17-producing helper T cells (Th17) and IL-17-producing γδT cells (γδT17) contained in the cells infiltrating into the peritoneal cavity was analyzed by FACS (Canto II (manufactured by BD Bioscience)), and total The product of the number of cells and the number of Th17 and γδT17 cells were evaluated. The results are shown in FIG.

  Furthermore, based on the results of (7-2) and (7-3), the TLR dependence of induction of differentiation of IL-17-producing cells by exosomes derived from Behcet's disease patients was similarly determined using TLR9-deficient mice and MyD88-deficient mice. Study was carried out. The results are shown in FIG.

  Further, whether induction of differentiation of IL-17-producing cells by exosomes derived from Behcet's disease patients is dependent on IL-1β and IL-23, and the same as described above using an IL-1 receptor antagonist and an anti-IL-23 receptor antibody. Study was carried out. The results are shown in FIG.

  As shown in FIG. 22 a, the expression of Il17a mRNA is increased by exosomes derived from Behcet's disease patient, and the expression is related to the expression of Rorc mRNA, a transcription factor of IL-17, and the induction of differentiation of IL-17-producing cells. It has been shown that the expression of Il23 and Il23r mRNA, which is known to increase, is enhanced. In addition, as shown in FIG. 22 b, an increase in the number of Th17 cells and γδT17 cells in the abdominal cavity was observed by intraperitoneal administration of exosomes derived from a Behcet's disease patient. These results indicated that exosomes derived from Behcet's disease patients promote differentiation induction of IL-17-producing cells.

  In addition, as shown in Fig. 22c, the effect of exosomes derived from a Behcet's disease patient on IL-17-producing cells disappeared in TLR9- and MyD88-deficient mice, and was shown to be TLR-dependent. Furthermore, from experiments of administration of an IL-1 receptor antagonist and an anti-IL-23 receptor antibody, it was found that the effect of exosomes derived from a Behcet's disease patient on IL-17-producing cells is dependent on IL-1 and IL-23. It was suggested.

  These results indicated that exosomes derived from Behcet's disease patients are involved in the induction of differentiation of IL-17-producing cells. In sum, based on the results of (7-2) and (7-3), IL-23 is dependent on NLRP3 inflammasome from dendritic cells and macrophages in a TLR-dependent manner by exosomes rich in mtDNA derived from Behcet's disease patients. It was presumed that IL-1β was produced, which promoted the induction of differentiation of IL-17-producing cells.

(7-5) Effect of exosomes from Behcet's disease patient in experimental autoimmune uveitis (EAU) model.
In order to obtain Interphotoreceptor retinoid-binding protein (IRBP) -specific T cells, 200 μl of an emulsion prepared by mixing a complete adjuvant CFA (manufactured by Sigma) with an IRBP _1-20 peptide (manufactured by ANASPEC) was used for C57BL / 6 mouse. (Clea Japan) were immunized subcutaneously, and 0.2 μg of pertussis toxin was administered intraperitoneally at the same time. Twelve days later, cells were prepared from the spleen and lymph nodes, and CD4 cells were isolated using a CD4 antibody with magnetic beads (MACS). They were co-cultured with irradiated spleen cells for 72 hours in the presence of 10 μg / ml IRBP _1-20 peptide and 10 ng / ml IL-23. Negative fractions were isolated from the proliferated cells using a CD11c antibody with magnetic beads (MACS) to obtain IRBP-specific T cells. Thereafter, 1 × 10 ⁷ IRBP-specific T cells were intravenously administered to C57BL / 6 mice to induce experimental autoimmune uveitis (EAU), one of the animal models of Behcet's disease. went. To this model mouse, exosomes (exosomes adjusted with 30 μl of serum-equivalent Exoquick) from Behcet's disease (BD) patients and healthy persons prepared according to the above “4. (Days 2 and 7) Intravenous administration, mice were euthanized on day 13, and the eyes were taken out, fixed with 4% PFA, and subjected to H & E staining. The results are shown in FIG. In addition, regarding the histologic severity of uveitis, a paper (Agarwal, RK, Silver, PB & Caspi, RR Rodent models of experimental autoimmune uveitis.Methods in molecular biology (Clifton, NJ) 900, 443-69 (2012) ) Was evaluated based on the evaluation method. The results are shown in FIG.

  As shown in Fig. 23a, administration of exosomes derived from a Behcet's disease patient showed numerous infiltration of inflammatory cells around the retina, destruction of the retinal structure, and formation of ulcers and granulation. In addition, as shown in FIG. 23 b, the mice administered with exosomes derived from Behcet's disease patients showed an increase in histological severity score.

  These results indicate that exosomes derived from Behcet's disease patients also have an action of exacerbating uveitis, and suggest that exosomes rich in mtDNA derived from BD patients are involved in exacerbation of the disease.

  From the results of (7-1), (7-2), (7-3) and (7-4), the exosomes derived from Behcet's disease patients (that is, exosomes containing mtDNA) have a neutrophil migration-enhancing effect. , And a cytokine-producing action, and furthermore, it was shown to have an effect of enhancing infiltration of neutrophils and inflammatory monocytes and induction of differentiation of IL-17-producing cells in vivo. Further, it is suggested that the inflammation-inducing reaction by exosomes derived from Behcet's disease patients is mediated by G protein-coupled receptors, TLRs including TLR9, and NLRP3 inflammasome, and produced IL-1β and IL-23, etc. It has been suggested that pathological inflammatory responses are chain-induced, such as enhancing induction of differentiation of IL-17-producing cells through cytokines. Furthermore, the results of (7-5) show that exosomes derived from Behcet's disease are involved in exacerbation of experimental autoimmune uveitis, which is an animal experimental model of Behcet's disease, and are directly involved in exacerbation of the disease. It has been suggested.

(Summary)
In autoimmune disease patients, the amount of mtDNA in serum was significantly higher than in healthy subjects, indicating that this can be used as a biomarker for disease detection. Furthermore, in patients with autoimmune diseases, it was shown that extracellular membrane vesicles in serum, especially the exosome fraction, contained particularly large amounts of mtDNA. In addition, it was shown that exosomes derived from patients containing a large amount of mtDNA enhance the inflammatory response. These findings suggested a new mode of DAMPs transmission not due to necrosis or apoptosis, and enhanced immune response due to the stable presence of mtDNA by the exosome lipid bilayer without being affected by DNase or the like. Alternatively, mtDNA present in exosomes in blood was considered to be a major cause of autoimmune diseases. That is, it was shown that mtDNA contained in the exosome fraction had higher accuracy as a biomarker for an autoimmune disease, directly reflected the disease state, and was useful as a therapeutic target.

SEQ ID NO: 1 is the forward primer for COXIII.
SEQ ID NO: 2 is the reverse primer for COXIII.
SEQ ID NO: 3 is the forward primer for NADH dehydrogenase.
SEQ ID NO: 4 is a reverse primer for NADH dehydrogenase.
SEQ ID NO: 5 is a forward primer for cytochrome b.
SEQ ID NO: 6 is a reverse primer for cytochrome b.

Claims (4)

Use of mitochondrial DNA contained in serum or plasma as a biomarker for Behcet's disease ,
Use wherein the concentration of mitochondrial DNA in serum or plasma is used as an index.   The use according to claim 1, wherein the mitochondrial DNA is contained in extracellular membrane vesicles in serum or plasma.   3. Use according to claim 1 or 2, wherein the mitochondrial DNA is contained in exosomes in serum or plasma. A method for measuring mitochondrial DNA for detecting the presence or absence of Behcet's disease comprising the following steps,
Measuring mitochondrial DNA in serum or plasma obtained from the test animal as a biomarker,
The measurement of the mitochondrial DNA was performed using cytochrome b, COXI, COXII, COXIII, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 2, NADH dehydrogenase subunit 3, NADH dehydrogenase subunit 4, NADH dehydrogenase subunit 4L, and NADH dehydrogenase subunit 5. And a primer for at least one DNA selected from the group consisting of NADH dehydrogenase subunit 6, ATPase sixth subunit and ATPase eighth subunit, or a probe that hybridizes to the DNA.