Method for predicting phases of inflammatory exacerbation and remission in autoinflammatory diseases

Abstract

To provide a biomarker and a method for predicting the phases of inflammatory exacerbation and remission in autoinflammatory diseases.SOLUTION: A prediction method includes the step for measuring, as indicators, the quantity of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for predicting the inflammatory aggravation phase and remission phase in an autoinflammatory disease, a test kit, and a method for screening agents for relieving autoinflammatory diseases.

[0002] Autoinflammatory diseases are classified as a type of primary immunodeficiency disorder and are characterized by systemic chronic inflammation and do not have an autoimmune cause.

[0003] There are several known pathological conditions for autoinflammatory diseases, but they often involve repeated periods of exacerbation of inflammation, during which inflammatory symptoms such as persistent fever last for hours to weeks, followed by periods of relief in which the inflammatory symptoms subside.

[0004] For example, in familial Mediterranean fever, a type of autoinflammatory disease, inflammatory symptoms such as fever are repeated at intervals of 12 to 72 hours (approximately 1 to 3 days). Inflammatory symptoms, also known as fever attacks, have no fixed cycle and are unstable, and may occur without any particular trigger or may be triggered by physical or mental conditions such as stress.

[0005] In relapsing rheumatoid arthritis, pain attacks accompanied by inflammation in the joints last for several hours to several days and then disappear. Such pain attacks occur irregularly in multiple joints (Non-Patent Document 1).

[0006] Furthermore, in TRAPS (TNF Receptor-Associated Periodic Syndrome), a type of autoinflammatory disease, fever attacks last for three days to several weeks. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sanmarti et al. Palindromic Rheumatism: Justa Pre-rheumatoid Stage or Something Else? Front. Med. 8:657983. doi: 10.3389 / fmed.2021.657983 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, each autoinflammatory disease has an exacerbation phase during which inflammation persists and a remission phase during which inflammation disappears. However, for patients in the exacerbation phase, the anxiety of not knowing how long the inflammatory symptoms will persist is one of the factors that reduces their quality of life (QOL).

[0009] However, the period of inflammation in autoinflammatory diseases is irregular and has no fixed cycle, and no biomarker or method for predicting the cycle has been reported. For example, if a biomarker capable of predicting the period of inflammation in autoinflammatory diseases were found, it would be useful because it would enable appropriate treatment to be started before or at the early stage of the period of inflammation. [Means for solving the problem]

[0010] In view of the above problems, the present inventors conducted intensive research and discovered that it is possible to predict the inflammation aggravation phase and remission phase in autoinflammatory diseases by evaluating the amount of a specific protein or the expression level of a specific gene in a biological sample as an indicator, thereby completing the present invention.

[0011] That is, the present invention provides the methods described below. [1] The method includes a step of measuring, in vitro, the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B, as an index; A method for predicting the inflammatory aggravation phase and remission phase in an autoinflammatory disease.

[0012] [2] The method according to [1], wherein it is determined that there is a high possibility that the autoinflammatory disease is heading toward a remission phase when the following changes are confirmed in the index: Increase in the amount of at least one protein selected from the group consisting of IL-1β protein and wild-type ASC protein; and / or Enhancement of the expression level of at least one gene selected from the group consisting of wild-type ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B.

[0013] [3] The method according to [1] or [2], further comprising a step of measuring rs8056505 (A→G) SNP in a biological sample.

[0014] [4] The method according to any one of [1] to [3], wherein the biological sample is serum, blood cells, saliva, or urine.

[0015] [5] The method according to any one of [1] to [4], wherein the autoinflammatory disease is a disease in which inflammasomes are involved in the pathogenesis.

[0016] [6] The method according to [5], wherein the disease in which the inflammasome is involved in the pathogenesis is relapsing rheumatism, periodic fever syndrome, Mediterranean fever, gout, Behcet's disease, VEXAS syndrome, SAPHO syndrome, palmoplantar pustulosis, pustular psoriasis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, atherosclerosis, myocardial infarction, cerebral infarction, diabetes, obesity, hypertension, asthma, allergic rhinitis, inflammatory bowel disease, non-alcoholic fatty liver disease (NASH), non-alcoholic steatohepatitis (NASH), infectious disease, or myelodysplastic syndrome.

[0017] [7] The method according to any of [1] to [6], wherein the improvement period is associated with the suppression of inflammasome.

[0018] [8] The present invention also provides the following test kits. A test kit for predicting the worsening and remission phases of inflammation in an autoinflammatory disease, comprising a means for measuring in vitro the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample as an indicator.

[0019] [9] The test kit according to [8], wherein the means for measuring the amount of the protein as an index is an antibody specific to the protein.

[0020]

[10] The present invention also provides the following screening methods. A step of in vitro acting a candidate substance on cells derived from a patient with an autoinflammatory disease and / or cells derived from human monocytes; measuring, in the cells, the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, and EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B as an index; A method for screening whether or not the candidate substance is an agent for alleviating an autoinflammatory disease. Effect of the Invention

[0021] According to the present invention, by measuring in vitro the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B as an index, it becomes possible to predict the inflammation aggravation phase and remission phase in an autoinflammatory disease. [Brief description of the drawings]

[0022] [Figure 1]FIG. 1 is a schematic diagram of the splicing cycle involved in predicting cyclical inflammation exacerbation or remission in autoinflammatory diseases. [Diagram 2] FIG. 2 shows the results of ASC gene mutation analysis in Test Example 1. [Diagram 3] FIG. 3 shows the results of the exon trapping assay in Test Example 2. [Figure 4] FIG. 4 shows the results of the volcano plot for Test Examples 3 and 4. [Diagram 5] FIG. 5 shows genes in Test Examples 3 and 4 for which fluctuations were confirmed in the volcano plot. [Figure 6] FIG. 6 shows DEGs in Test Examples 3 and 4 whose fluctuations were confirmed in the volcano plot. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] [Method for predicting the inflammatory aggravation and remission periods in autoinflammatory diseases] In one embodiment, the present invention relates to a method for the detection of IL-1β protein and ASC protein in a biological sample, comprising measuring, in vitro, the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B as an index; Provided is a method for predicting the inflammatory aggravation phase and remission phase in an autoinflammatory disease.

[0024] As used herein, the term "autoinflammatory disease" refers to a disease classified as a type of primary immunodeficiency disease, characterized by chronic inflammation occurring systemically or locally, and not caused by an autoimmune disease.

[0025] Autoinflammatory diseases are distinguished from autoimmune diseases and do not include diseases driven by autoreactive T lymphocytes or autoantibodies.

[0026] The type of autoinflammatory disease is not particularly limited, and includes specific diseases known as autoinflammatory diseases. The type of autoinflammatory disease is not limited to diseases in which a relationship between genetic abnormalities and pathology is recognized, and also includes diseases that are suspected to be autoinflammatory diseases based on their pathology. From the viewpoint of significantly achieving the effects of the present invention, the autoinflammatory disease may include, but is not limited to, diseases that cause an inflammation aggravation phase and a remission phase. In the inflammation aggravation phase, fever, pain, etc. caused by inflammation may occur for at least several hours, at least several days, or at least several weeks. In addition, in the inflammation remission phase, fever, pain, etc. caused by inflammation may disappear or be alleviated.

[0027] Examples of autoinflammatory diseases include relapsing rheumatism, periodic fever syndrome, Mediterranean fever, gout, Behcet's disease, VEXAS syndrome, SAPHO syndrome, palmoplantar pustulosis, pustular psoriasis, multiple sclerosis, Alzheimer's dementia, Parkinson's disease, atherosclerosis, myocardial infarction, cerebral infarction, diabetes, obesity, hypertension, asthma, allergic rhinitis, inflammatory bowel disease, nonalcoholic fatty liver disease (NASH), nonalcoholic steatohepatitis (NASH), infectious diseases, myelodysplastic syndrome, hyper-IgD syndrome, Muckle-Wells syndrome, familial cold autoinflammatory syndrome, chronic infantile neurological cutaneous arthritis syndrome, neonatal onset multiorgan inflammatory disease, NLRC4-MAS, PLAID (PLCγ2 associated antibody deficiency and immune dysregulation), APLAID (autoinflammation and PLCγ2 associated antibody deficiency and immune dysregulation), TRAPS (TNF Receptor-Associated Antibody Deficiency and Immune Dysregulation), and inflammatory bowel disease (IGD). Periodic Syndrome), suppurative sterile arthritis-pyoderma gangrenosum-acne syndrome (PAPA syndrome), Blau syndrome, ADAM17 deficiency, chronic recurrent multifocal osteomyelitis and congenital dyserythropoietic anemia (Majeed syndrome), DIRA (Deficiency of the Interleukin 1 Receptor Antagonist), DITRA (IL-36 receptor antagonist deficiency), SLC29A3 mutation, CAMPS (CARD14 mediated psoriasis), cherubism, CANDLE (chronic atypical neutrophilic dermatitis with lipodystrophy), COPA (Coatamer protein complex,subunit alpha) deficiency, cryopyrin-associated periodic fever syndrome (CAPS), NLRP12-associated periodic fever syndrome (NAPS12), Nakajo-Nishimura syndrome / JMP syndrome / CANDLE syndrome, phospholipase Cγ2-associated antibody deficiency immunopathology (PLAID), HOIL-1 deficiency, periodic fever-aphthous stomatitis-pharyngitis-cervical lymphadenitis syndrome (PFAPA), systemic juvenile idiopathic arthritis, adult-onset Still's disease, Crohn's disease, asbestosis, Schnitzler syndrome, etc.

[0028] Among these autoinflammatory diseases, the present invention is preferably used for, for example, diseases in which inflammasomes are involved in the pathogenesis. As shown in the Examples below, the present inventors have confirmed that deletion of exon 2 in apoptosis-associated speck-like protein containing a CARD (also referred to as ASC in this specification) activates inflammasomes and enhances the production of IL-1β during the inflammation aggravation phase. Furthermore, an increase in the amount of at least one protein selected from the group consisting of IL-1β protein and wild-type ASC protein, and / or an increase in the amount of a specific gene group (ASC (e.g., wild-type ASC), MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C ... When the expression level of at least one gene selected from the group consisting of RD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B is confirmed, it leads to the suppression of inflammasomes, and the inflammation exacerbation phase of autoinflammatory diseases tends to turn into a remission phase. In addition, in the inflammation remission phase, the production of IL-1β is reduced under the influence of wild-type ASC, and ASC lacking exon 2 (exon 2-deficient ASC) increases relative to wild-type ASC, which may lead to the activation of inflammasomes again (see cycle model in Figure 1). Thus, in diseases in which inflammasomes are involved in the pathogenesis, it has been suggested that the transition to a remission phase of inflammation in autoinflammatory diseases in particular is brought about by the production of IL-1β protein and wild-type ASC protein, as well as increased expression levels of the specific genes mentioned above.

[0029] The biological sample is not particularly limited, so long as it allows evaluation of the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein, and / or the expression level of the above-mentioned specific gene group.

[0030] Biological samples include, for example, ascites; blood; blood cells (blood cells such as monocytes); body fluids; bone marrow or components thereof (e.g., hematopoietic cells, bone marrow adipose tissue, interstitial cells, etc.); cerebrospinal fluid (CSF); feces; hair; immune infiltrates; lymph; peritoneal fluid; plasma; saliva; skin or components thereof (e.g., hair follicles, etc.); sputum; surgically obtained tissue; tissue scraped or swabbed from the skin or mucous membranes (e.g., nasal, oral or vaginal); biopsy samples; urine; washings or lavages such as ductal lavage or bronchoalveolar lavage; other body fluids, tissues, secretions and / or drainages, etc. Biological samples may also include immune cells, such as NK cells and / or macrophages, which are found in a number of tissues and organs, including the spleen and lymph nodes.

[0031] Among these biological samples, from the viewpoint of significantly exhibiting the effects of the present invention, for example, blood, blood cells, saliva, feces, or urine is preferred, and serum, blood cells, saliva, or urine is more preferred.

[0032] The method for evaluating the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein, and / or the expression level of the above-mentioned specific gene group is not particularly limited and can be performed by known methods.

[0033] Methods for evaluating protein amount include, for example, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip, but are not limited to these.

[0034] Among these methods, for example, Western blot or ELISA is preferably used to evaluate the amount of at least one protein selected from the group consisting of IL-1β and ASC protein, and ELISA is more preferably used. The amount of IL-1β protein can be evaluated using a known anti-IL-1β antibody or an ELISA kit for quantifying IL-1β.

[0035] Furthermore, when evaluating the amount of an intracellular protein such as the ASC protein, known cell disruption means, protein purification means, protein detection means, and the like can be used in combination as necessary.

[0036] Methods for evaluating gene expression levels include, but are not limited to, RNA sequencing, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time RT-PCR, real time quantitative RT-PCR, RNase protection method, Northern blotting, and gene chips (microarrays).

[0037] Among these methods, for evaluating the expression levels of the above-mentioned specific gene group, for example, RNA sequencing or real time polymerase reaction (real time RT-PCR) is preferable, and RNA sequencing is more preferable.

[0038] For the above specific gene groups, information such as sequence information and identification numbers can be obtained from publicly known databases, for example, NCBI (URL: https: / / www.ncbi.nlm.nih.gov / ).

[0039] For the specific gene groups listed above, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, and LOC10 Any combination of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, or twenty-seven types of 7984935, MIR6721, RNVU1-14, MIR760, and SNORD19B may be measured, or all 27 types may be measured.

[0040] The name of each gene, its corresponding INSD accession number, Refseq (Reference Sequence) and accession number are summarized in the table below. The nucleotide sequence of each gene is identified by the sequence indicated by the accession number registered in the corresponding database.

[0041] [Table 1]

[0042] Of the above specific gene groups, from the viewpoint of significantly exhibiting the effects of the present invention, it is preferable to evaluate at least one selected from the group consisting of, for example, ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, RNVU1-3, and RNVU1-14, From the viewpoint of enhanced gene expression in THP-1 cells stimulated with IL-1β, it is more preferable to evaluate at least one selected from the group consisting of wild-type ASC, exon 2-deficient ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, and EEF1A1.

[0043] In another embodiment, from the viewpoint of significantly exhibiting the effects of the present invention, it is preferable that the above-mentioned specific gene group includes, for example, MASCRNA, which is a splicing-related gene.

[0044] In another embodiment, from the viewpoint of significantly exhibiting the effects of the present invention, it is preferable that the specific gene group includes, for example, RNVU1-3 (RNA, variant U1 small nuclear 3) and / or RNVU1-14 (RNA, variant U1 small nuclear 14) from the above-mentioned specific gene group.

[0045] When an increase in the amount of IL-1β protein and / or an increase in the expression levels of the above-mentioned specific genes is confirmed in longitudinal evaluation of the same subject, it can be determined that there is a high possibility that the autoinflammatory disease is heading toward a remission phase.

[0046] In one embodiment, an increase in the amount of at least one protein selected from the group consisting of IL-1β protein and wild-type ASC protein, compared to a previous assessment, when two or more assessments are performed in the same subject, the assessments being at least 24 hours apart; and / or Enhancement of the expression level of at least one gene selected from the group consisting of wild-type ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B. When this is confirmed, it can be determined that there is a high possibility that the autoinflammatory disease will progress to a remission phase.

[0047] In one embodiment, an increase in the amount of at least one protein selected from the group consisting of IL-1β protein and wild-type ASC protein, compared to a previous assessment, when two or more assessments are performed in the same subject, the assessments being at least 24 hours apart; Decreased amount of exon 2 deleted ASC protein; and / or Enhancement of the expression level of at least one gene selected from the group consisting of wild-type ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B. When this is confirmed, it can be determined that there is a high possibility that the autoinflammatory disease will progress to a remission phase.

[0048] On the other hand, a decrease in IL-1β protein compared to the previous assessment in two or more assessments in the same subject, with an interval of at least 24 hours between each assessment; Increased amount of exon 2 deleted ASC protein; and / or A decrease in the expression level of at least one gene selected from the group consisting of wild-type ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B. When this is confirmed, it can be determined that there is a high possibility that the autoinflammatory disease will progress to an exacerbation phase.

[0049] In one embodiment, when two or more evaluations are performed on the same subject with an interval of at least 24 hours or more, the change in the protein amount of the protein and / or the change in the expression level of the specific gene group described above compared to the previous evaluation value is, for example, at least 1%, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, When the above-mentioned change in the protein amount and / or the expression level of the specific gene group is confirmed to be 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc., the above-mentioned judgment of an autoinflammatory disease can be made. Although not limited thereto, when the same subject is evaluated two or more times with an interval of at least 24 hours or more, the above-mentioned judgment of an autoinflammatory disease can be made when the above-mentioned change in the protein amount and / or the change in the expression level of the specific gene group is confirmed to be preferably at least 5% or more compared to the previous evaluation value.

[0050] In addition to the above, the method may include a step of measuring rs8056505 (A→G) SNP (single nucleotide polymorphism) in a biological sample. The single nucleotide polymorphism is preferably confirmed to be present in the 5' untranslated region of the ASC gene of the subject.

[0051] As shown in the Examples below, the present inventors have found that when the 5' untranslated region of the ASC gene has rs8056505 (A → G) SNP, it can induce deletion of exon 2 in ASC. Therefore, it is presumed that when the 5' untranslated region of the ASC gene has rs8056505 (A → G) SNP, deletion of exon 2 in ASC is likely to occur, inflammasome is activated, and the cycle model shown in FIG. 1 is more likely to occur.

[0052] [Test kit for predicting the progression and remission of inflammation in autoinflammatory diseases] In another embodiment, the present invention provides a test kit for predicting the exacerbation and remission phases of inflammation in an autoinflammatory disease, comprising a means for measuring in vitro the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample as an index.

[0053] As described above, by measuring the amount of at least one protein selected from the group consisting of IL-1β protein, wild-type ASC protein, and exon 2-deficient ASC protein in a biological sample as an indicator, it is possible to predict, in the cycle model shown in Figure 1, whether the subject who provided the biological sample will transition from an inflammatory exacerbation phase to a remission phase of inflammation in an autoinflammatory disease, or from a remission phase to an exacerbation phase of inflammation in an autoinflammatory disease.

[0054] For example, when the test kit is used to measure the amount of at least one protein selected from the group consisting of IL-1β protein, wild-type ASC protein, and exon 2-deficient ASC protein in a biological sample over time, if it is evaluated that the amount of IL-1β protein and / or the amount of wild-type ASC protein is tending to increase and / or the amount of exon 2-deficient ASC protein is tending to decrease, it is possible to predict that an autoinflammatory disease will transition from an inflammatory exacerbation phase to a remission phase.

[0055] For example, when the test kit is used to measure the amount of at least one protein selected from the group consisting of IL-1β protein, wild-type ASC protein, and exon 2-deficient ASC protein in a biological sample over time, if it is evaluated that the amount of IL-1β protein and / or the amount of wild-type ASC protein is tending to decrease and / or the amount of exon 2-deficient ASC protein is tending to increase, it is possible to predict that an autoinflammatory disease will transition from a remission phase to an exacerbation phase.

[0056] In one embodiment, when two or more evaluations are performed on the same subject with an interval of at least 24 hours, the above prediction of autoinflammatory disease can be made when an increase in the amount of IL-1β protein is confirmed, for example, at least 1%, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc., compared to the previous evaluation value. Although not limited thereto, when two or more evaluations are performed on the same subject with an interval of at least 24 hours or more, the above prediction of autoinflammatory disease can be made when an increase in the amount of IL-1β protein is confirmed, preferably by at least 5%, compared to the previous evaluation value.

[0057] The test kit preferably contains an antibody capable of measuring the amount of the protein. In addition to the antibody, the kit may contain one or more reagents necessary for carrying out the method of the present invention, a detection device or its components, and / or instructions describing the procedure for carrying out the method of the present invention. The types of reagents, the contents of the instructions, and other components included in the kit of the present invention may be appropriately determined depending on the type of specific immunoassay method to be used, etc.

[0058] When evaluating the amount of an intracellular protein, such as ASC protein, the method may include a combination of reagents required for known cell disruption means, protein purification means, and protein detection means, as necessary.

[0059] Examples of immunoassay methods include, but are not limited to, various known immunoassay methods, such as ELISA (enzyme-linked immunosorbent assay) using a microtiter plate carrying an antibody; latex particle agglutination assay using latex particles (e.g., polystyrene latex particles, etc.) carrying an antibody; immunochromatography using a membrane carrying an antibody; a sandwich assay using a detection antibody labeled with colored particles or particles having color-developing ability, an enzyme or a fluorescent substance, etc., and a capture antibody immobilized on a solid phase carrier such as a magnetic particle; ELISA using a single antibody; and a biosensor method.

[0060] For this test kit, the above description of [Method for predicting the inflammatory aggravation phase and remission phase in autoinflammatory disease] may be referred to as appropriate.

[0061] [Method for screening agents for alleviating autoinflammatory diseases] In another embodiment, the present invention relates to a method for the treatment of autoinflammatory disease, comprising the steps of: exposing a candidate substance to cells derived from a patient with an autoinflammatory disease and / or cells derived from a human monocyte in vitro; measuring, in the cells, the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, and EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B as an index; A method for screening whether the candidate substance is an agent for alleviating an autoinflammatory disease is provided.

[0062] As used herein, the "candidate substance" is not particularly limited, and examples thereof include nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, and natural extracts.

[0063] The cells on which the candidate substance is to act may be cells derived from a patient with an autoinflammatory disease, or may be cells derived from human monocytes.

[0064] Examples of human monocyte-derived cells include THP-1 cells (JCRB cell bank JCRB0112) and U937 cells (JCRB cell bank JCRB9021).

[0065] For example, an increase in the amount of at least one protein selected from the group consisting of IL-1β protein and wild-type ASC protein in a cell treated with a candidate substance; Decreased amount of exon 2 deleted ASC protein; and / or Enhancement of the expression level of at least one gene selected from the group consisting of wild-type ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B. When this is confirmed in a time-course evaluation, the substance can be determined to be capable of inducing a remission phase in an autoinflammatory disease.

[0066] Although not limited thereto, the above judgment of the candidate substance can be made when two or more evaluations are performed at intervals of at least 24 hours and a change in the amount of the protein and / or the expression level of the specific gene group is confirmed, preferably by at least 5%, compared to the previous evaluation value.

[0067] The candidate substances selected by the above-mentioned judgment can be used as agents for alleviating autoinflammatory diseases.

[0068] The above-mentioned relief agent can be added to or mixed with medicines, foods, beverages, etc., and used as such. Alternatively, it can be used as a medicine, food, beverage, etc. as it is. The above-mentioned relief agent can be used as a food or beverage that explicitly or implicitly indicates that it has a function of preventing or improving fever or pain symptoms caused by inflammation due to an autoinflammatory disease, i.e., a health food, a functional food, a food for sick people, and a food for specified health uses. Even if the above-mentioned functionality is not explicitly or implicitly indicated, it can be used as a so-called doctor's supplement recommended or presented by a doctor in an internal medicine department, a surgery department, a veterinary hospital, etc. in a hospital and / or clinic.

[0069] Pharmaceuticals, health foods, functional foods, foods for the sick, and foods for specified health uses can be used in various formulation forms, such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candies, etc.), liquid preparations (syrups, suspensions), and liquid foods.

[0070] For example, tablets can be prepared by mixing the selected candidate substance in powder form with a pharma- ceutically acceptable carrier component (such as an excipient) and compressing the mixture, or tablets can be prepared by pouring the mixture into a mold. Tablets can also be sugar-coated to form sugar-coated tablets. Furthermore, tablets can be single-layer tablets or layered tablets such as double-layer tablets.

[0071] Powdered granules such as granules may be prepared by various granulation methods (such as extrusion granulation, grinding granulation, dry compaction granulation, fluidized bed granulation, rolling granulation, and high-speed stirring granulation), and tablets can be prepared by an appropriate combination of the above-mentioned granulation methods and tableting methods (wet tableting, direct tableting), etc.

[0072] Capsules can be prepared by filling a capsule (soft or hard capsule) with a powdered agent (powder, granules, etc.) by a conventional method.

[0073] The liquid preparation can be prepared by dissolving or dispersing each component in an aqueous medium (purified water, purified water containing ethanol, etc.) which is a carrier component, filtering or sterilizing the mixture as necessary, filling the mixture into a predetermined container, and sterilizing the mixture. The preferred dosage form of the solid preparation of the present invention is a capsule or tablet, and a soft capsule (soft capsule) is more preferred.

[0074] Soft capsules have a smooth surface and are easy to swallow, and are therefore preferred by users. Typical methods for producing soft capsules include the plate method, the rotary method, and the seamless method.

[0075] In the rotary method (punching method), a sheet-like capsule shell sandwiches the flowing filling contents and forms a capsule shape along the holes of a rotating cylindrical die. On the other hand, in the seamless method (dropping method), the capsule shell composition and contents are simultaneously discharged from multiple concentric nozzles to form a seamless capsule shape.

[0076] The base material for the soft capsule shell is not particularly limited, but starch, pullulan, cellulose, polyvinyl alcohol, gelatin, succinated gelatin, etc. can be used, with starch, gelatin, and succinated gelatin being preferred, and gelatin and succinated gelatin being more preferred. These may be used alone or in combination of two or more kinds.

[0077] The above preparations can also be manufactured as liquid beverages such as soups, juices, fruit juice drinks, milk, milk beverages, whey beverages, lactic acid bacteria beverages, tea beverages, alcoholic beverages, coffee beverages, carbonated beverages, soft drinks, water beverages, cocoa beverages, jelly-like beverages, sports drinks, and diet drinks; semi-solid foods such as pudding and yogurt; noodles, confectioneries, spreads, and the like.

[0078] When preparing it as a food, various food additives may be blended in. Examples of food additives include antioxidants, colorants, flavorings, seasonings, sweeteners, acidulants, pH adjusters, quality stabilizers, preservatives, etc.

[0079] When preparing a pharmaceutical product, the selected candidate substance, which is an active ingredient, is prepared as a formulation containing preferably a pharma- ceutical acceptable carrier. A pharma- ceutical acceptable carrier generally refers to an inert, non-toxic, solid or liquid filler, diluent, or encapsulating material that does not react with the active ingredient, such as water, ethanol, polyols, appropriate mixtures thereof, and solvents or dispersion media such as vegetable oils.

[0080] Pharmaceuticals are administered orally or parenterally, for example, into the oral cavity, the digestive tract, or the nasal cavity. Oral preparations include solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candy, etc.) and liquid preparations (syrups, suspensions, inhalants). Parenteral preparations include eye drops, drops, nasal drops, and injections. The formulation form is not limited, but is preferably an orally disintegrating tablet, a chewable tablet, a candy, a granule, a powder, or a liquid from the viewpoint of significantly achieving the effects of the present invention.

[0081] The pharmaceutical product may further contain additives commonly used in the pharmaceutical field. Such additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavoring agents, etc., and can be used appropriately as needed. In order to achieve sustained release so as to act for a long time, the product may also be coated with a known retardant, etc. The pharmaceutical composition may further contain other additives or drugs, such as antacids and gastric mucosa protectants, as needed.

[0082] The pharmaceutical agent may be applied in the form of oral administration, internal administration, etc. The pharmaceutical agent may be used therapeutically or non-therapeutically.

[0083] The daily oral intake or administration amount of the selected candidate substance for an adult can be determined appropriately depending on the individual's condition, body weight, sex, age, activity of the material, intake or administration route, intake or administration schedule, formulation form, or other factors.

[0084] The daily oral intake or dosage of the relief agent for an adult may be divided into 1 to 6 capsules, 1 to 4 capsules, 1 to 3 capsules, or 1 to 2 capsules depending on the dosage form.

[0085] The above-mentioned relief agent can be taken or administered once or several times a day, usually 1 to 6 times a day, 1 to 3 times a day, 1 to 2 times a day, or at any period and interval, but once a day is preferable. EXAMPLES

[0086] Next, the present invention will be specifically described with reference to examples and test examples, but the present invention is not limited to the following examples and test examples.

[0087] [Test Example 1. ASC gene mutation analysis] Genomic DNA from three patients with relapsing rheumatoid arthritis was isolated from peripheral blood using the PAXgene Blood DNA Kit (QIAGEN). Genomic ASC DNA fragments were obtained as five separate fragments by PCR.

[0088] The names of the primers used to amplify each fragment and their base sequences are as follows: (Fragment 1) hPYCARD3313F forward: 5'-TTCAGCAGGGTAGCAGAGC-3' (SEQ ID NO: 1) hPYCARD4445R reverse: 5'-CCGAAACCTTGCCAGATGCT-3' (SEQ ID NO: 2)

[0089] (Fragment 2) hPYCARD4129F forward: 5'-ACAGTTAAGGTCTCCGATGC-3' (SEQ ID NO: 3) hPYCARD4927R reverse: 5'-TTACTACACCCTTGGTCCCCT-3' (SEQ ID NO: 4)

[0090] (Fragment 3) hPYCARD4770F forward: 5'-CAAGCCCAGAGACAAGCAGG-3' (SEQ ID NO: 5) hPYCARD5767R reverse: 5'-CAGGGTGTGGGTTGGTGG-3' (SEQ ID NO: 6)

[0091] (Fragment 4) hPYCARD5634F forward: 5'-AGGCCTCCACACCCAG-3' (SEQ ID NO: 7) hPYCARD6627R reverse: 5'-ATGTGGAAGGTAGGGGAGC-3' (SEQ ID NO: 8)

[0092] (Fragment 5) hPYCARD6339F forward: 5'-CTATGCTTCAGAGGCCACCC-3' (SEQ ID NO: 9) hPYCARD7854R reverse: 5'-GGGAGGACAAGTAGGAGGT-3' (SEQ ID NO: 10)

[0093] The numbers in the primer names indicate the 5' positions to which the primers anneal according to NCBI accession number: NG_029446. The PCR products obtained were purified using a QIAquick PCR Purification Kit (QIAGEN) and used for sequence analysis. The results are shown in FIG. 2.

[0094] As shown in Figure 2 , a heterozygous SNP (rs8056505 A / G) was identified in the 5′-UTR (888 bp upstream from the 1st ATG) in all three patients ( Figure 2 ).

[0095] [Test Example 2. Exon Trapping Assay] To confirm the effect of the rs8056505 SNP on ASC splicing, we performed exon trapping assays. The wild-type ASC rs8056505 A allele and the mutant ASC rs8056505 G allele were amplified by PCR from genomic DNA derived from peripheral blood mononuclear cells of relapsing RA patients containing the heterozygous rs8056505 A→G single nucleotide polymorphism (SNP).

[0096] The names of the primers used for amplification and their base sequences are as follows: hPYCARD4129F _Fusion forward: 5'-GGGATCACCAGAATTCACAGTTAAGGTCTCCGATGC-3' (SEQ ID NO: 11) hPYCARD6480R_Fusion reverse: 5'-TCGAGCTCCAGAATTCCTTTAGGGAGCACTGGAGGA-3' (SEQ ID NO: 12)

[0097] The resulting PCR product was inserted into EcoRI-digested pSPL3 exon trap vector (Thermo Fisher Scientific, Waltham, MA, USA) using the In-Fusion HD Cloning Kit (Takara Bio).

[0098] THP-1 cells obtained from the JCRB Cell Bank were cultured at 1.5 × 10 cells / mL in RPMI 1640 medium (Fujifilm Wako) supplemented with 100 U / mL penicillin (Thermo Fisher Scientific), 100 μg / mL streptomycin (Thermo Fisher Scientific), and 10% Equa FETAL. 6 Cells were seeded at a density of 1000 cells / mL and incubated overnight at 37°C.

[0099] The pSPL3 control vector, the pSPL3 vector containing the wild-type ASC rs8056505 A allele, or the pSPL3 vector containing the mutant ASC rs8056505 G allele were transfected into THP-1 cells using jetOPTIMUS in vitro DNA transfection reagent (Polyplus-transfection) according to the manufacturer's guidelines and incubated for 48 h.

[0100] After incubation, the transfected cells were washed and stimulated with 50, 100, or 200 pg / mL IL-1β (R&D Systems) and 5 or 20 μM C6-pyridinium-ceramide (Avanti Polar Lipids) for 24 hours.

[0101] Total RNA fractions from THP-1 cells were extracted using RNAiso Plus (Takara Bio) and reverse transcribed into cDNA using PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio). Expression of mRNA was confirmed by RT-PCR using a reaction mixture of 0.2 mM of each dNTP, 1× Gflex PCR Buffer (Takara Bio), 0.2 μM of each primer, and 1.25 U of Tks Gflex DNA Polymerase (Takara Bio).

[0102] The names of the primers used for amplification and their base sequences are as follows: pSPL3 SD6 forward: 5'-TCTGAGTCACCTGGACAACC-3' (SEQ ID NO: 13) pSPL3 SA2 reverse: 5'-ATCTCAGTGGTATTTGTGAGC-3' (SEQ ID NO: 14)

[0103] The primer names and their base sequences used in the second nested PCR are as follows: hPYCARD225F forward: 5'-ATGGACGCCTTGGACCTCA-3' (SEQ ID NO: 15) hPYCARD696R reverse: 5'-GGAGGTGTTGCTGGGAAGGA-3' (SEQ ID NO: 16)

[0104] As mentioned above, to confirm the effect of the rs8056505 SNP on ASC splicing, an exon trapping assay was performed, and the results are shown in Figure 3.

[0105] As shown in Figure 3, the expression of exon 2-deficient ASC was induced by the rs8056505 G allele compared to that observed with the A allele in THP-1 cells. In addition, the effect of costimulation of cells with IL-1β or ceramide was evaluated. Ceramide is one of the downstream molecules of IL-1β and functions as a splicing regulator. It was confirmed that the expression of exon 2-deficient ASC was suppressed by stimulation with 50-200 pg / mL IL-1β and 20 μM ceramide (Figure 3).

[0106] [Test Example 3. Total RNA Sequencing (RNA-seq)] THP-1 cells were cultured at 1.0 × 10 in RPMI 1640 medium (Fujifilm Wako) supplemented with 100 U / mL penicillin (Thermo Fisher Scientific), 100 μg / mL streptomycin (Thermo Fisher Scientific), and 10% Equa FETAL (Atlas Biologicals). 6 The cells were seeded at a density of 1000 cells / mL and stimulated for 24 hours at 37° C. in the presence or absence of 50 pg / mL IL-1β (R&D Systems).

[0107] Total RNA was extracted from THP-1 cells using RNAiso Plus (Takara Bio), treated with DNase I, and purified using the PureLink RNA Mini Kit (Thermo Fisher Scientific).

[0108] The purified RNA was subjected to total RNA sequencing (RNA-seq, Eurofin Genomics) using a NovaSeq 6000 high-throughput sequencer (Illumina). The size of the library insert was 200 bp, and the sequence was determined using 150 bp paired-end reads.

[0109] The assigned indexes are CTTAGGAC-CTTCACTG and ATCTGACC-CTCGACTT, and the DDBJ (DNA Databank of Japan) DRA accession number is DRA014002.

[0110] [Test Example 4. RNA-seq data analysis] RNA-seq data were analyzed as follows. First, each sample was quality checked using FastQC (a quality check tool, http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and low-quality reads were trimmed using Trimmomatic (an adapter trimming tool, http: / / www.usadellab.org / cms / ?page=trimmomatic). After trimming, a quality check was performed again to ensure that low-quality reads had been removed.

[0111] The trimmed samples were mapped using HISAT2 (http: / / daehwankimlab.github.io / hisat2 / ) and the hg38 reference genome, followed by normalization and calculation of gene expression levels using StringTie (http: / / ccb.jhu.edu / software / stringtie / ). Data were analyzed using TCC-GUI, and genes with a false discovery rate (FDR) of less than 0.05 were considered as DEGs (Differentially Expressed Genes), i.e., genes whose expression was significantly increased in the experimental group compared to the control group.

[0112] The expression of exon 2-deficient ASC was suppressed by IL-1β stimulation, suggesting that some genes affected by IL-1β may be involved in the splicing regulation of ASC.

[0113] Total RNA-seq analysis was performed to isolate genes with altered expression levels in IL-1β-stimulated THP-1 cells. The number of reads was 44,797,770 and 59,862,218, the number of called bases was 6,720 Mbp and 8,979 Mbp, and the Q30 scores were 92.18% and 92.25%, respectively, in control and IL-1β-stimulated cells.

[0114] The obtained data was processed using the analysis tools mentioned above, and gene expression levels between control and IL-1β-treated cells were compared using TCC-GUI. 131,426 transcripts were detected and plotted on a Volcano plot to confirm trends. The results are shown in Figure 4.

[0115] As shown in Figure 4, we found 35 genes with more than 2-fold expression change and P value less than 0.05 between control and IL-1β stimulated cells (including 16 down-regulated genes and 19 up-regulated genes after IL-1β stimulation) (Table 1, Figure 2b). The results are shown in Table 2-1, Table 2-2, Table 3 and Figure 5. Table 2-1 and Table 2-2 show the down-regulated genes in the volcano plot. Table 3 shows the up-regulated genes in the volcano plot.

[0116] [Table 2-1]

[0117] [Table 2-2]

[0118] [Table 3]

[0119] Seven out of 16 (43.8%) of the downregulated genes were non-coding or microRNAs, six out of 16 (37.5%) were small nuclear RNAs such as SNORDs (small C / D box nucleolar RNAs) and scaRNAs (small Cajal body-specific RNAs), and one out of 16 (6.3%) was a transcription factor.

[0120] In contrast, 3 / 19 (15.8%) of the upregulated genes were typical splicing factors, such as SR protein modulators MASCRNA (MALAT1-associated small cytoplasmic RNA) and RNVU (U1 small RNA variant), and 12 / 19 (63.2%) were noncoding or microRNAs. Genes with FDR < 0.05 were also determined to be DEGs.

[0121] As a result, 52 genes were identified after IL-1β stimulation, including 41 downregulated genes and 11 upregulated genes (Table 4-1, Table 4-2, Table 4-3, Figure 6). Tables 4-1 and 4-2 show the downregulated DEGs, and Table 4-3 shows the upregulated DEGs.

[0122] [Table 4-1]

[0123] [Table 4-2]

[0124] [Table 4-3]

[0125] As shown in Table 4-1, Table 4-2, Table 4-3 and Figure 6, among splicing factors, SNORD141A was detected as a significantly down-regulated gene, and MASCRNA was detected as the most significantly up-regulated gene.

[0126] The long non-coding RNA MALAT1, which is the precursor of MASCRNA, has been reported as a modulator of SR splicing factors, strongly suggesting that MASCRNA upregulated by IL-1β may contribute to the expression of wild-type ASC.

[0127] Based on the above results, the inventors propose a splicing cycle that alternates between expression of wild-type ASC and expression of exon 2-deficient ASC, which is regulated by the rs8056505 G allele and a splicing regulator induced by IL-1β (Figure 1).

[0128] First, exon 2-deficient ASC is induced by the rs8056505 G allele. Second, exon 2-deficient ASC activates inflammasomes and increases IL-1β production compared to wild-type ASC. IL-1β affects the expression of splicing regulators, improving ASC splicing, and wild-type ASC then becomes dominant. Splicing repair of ASC reduces IL-1β production by wild-type ASC, weakening the interference of splicing regulators, and exon 2-deficient ASC becomes dominant again. This cycle may predict the periodic exacerbation or remission of inflammation in autoinflammatory diseases such as relapsing rheumatoid arthritis.

Claims

1. The method includes a step of measuring, in vitro, the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B, as an index; A method for predicting the inflammatory aggravation phase and remission phase in an autoinflammatory disease.

2. The method according to claim 1, wherein, when the following changes are confirmed in the index, it is determined that there is a high possibility that the autoinflammatory disease is heading toward a remission phase: Increase in the amount of at least one protein selected from the group consisting of IL-1β protein and wild-type ASC protein; and / or Enhancement of the expression level of at least one gene selected from the group consisting of wild-type ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B.

3. The method of claim 1, further comprising the step of measuring the rs8056505 (A→G) SNP in a biological sample.

4. The method of claim 1 , wherein the biological sample is serum, blood cells, saliva, or urine.

5. The method according to claim 1 , wherein the autoinflammatory disease is a disease in which inflammasomes are involved in the pathogenesis.

6. The method according to claim 5, wherein the disease in which the inflammasome is involved in the pathogenesis is relapsing rheumatism, periodic fever syndrome, Mediterranean fever, gout, Behcet's disease, VEXAS syndrome, SAPHO syndrome, palmoplantar pustulosis, pustular psoriasis, multiple sclerosis, Alzheimer's dementia, Parkinson's disease, atherosclerosis, myocardial infarction, cerebral infarction, diabetes, obesity, hypertension, asthma, allergic rhinitis, inflammatory bowel disease, nonalcoholic fatty liver disease (NASH), nonalcoholic steatohepatitis (NASH), infectious disease, or myelodysplastic syndrome.

7. The method of claim 1 , wherein the improvement period is associated with inhibition of inflammasomes.

8. A test kit for predicting the worsening and remission phases of inflammation in an autoinflammatory disease, comprising a means for measuring in vitro the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein in a biological sample as an indicator.

9. 9. The test kit according to claim 8, wherein the means for measuring the amount of the protein as an index is an antibody specific to the protein.

10. A step of in vitro acting a candidate substance on cells derived from a patient with an autoinflammatory disease and / or cells derived from a human monocyte; measuring, in the cells, the amount of at least one protein selected from the group consisting of IL-1β protein and ASC protein, and / or the expression level of at least one gene selected from the group consisting of ASC, MASCRNA, DDT, C3, CD99, RNA28SN5, RNA28SN4, RNA28SN2, RNA28SN3, RNA28SN1, RN7SL1, and EEF1A1, MIR922, MIR4775, SNORA5C, SNORD143, SNORD4B, MIR612, MIR6502, DDAH2, CXCL10, RNVU1-3, MIR5001, LOC107984935, MIR6721, RNVU1-14, MIR760, and SNORD19B as an index; A method for screening whether or not the candidate substance is an agent for alleviating an autoinflammatory disease.

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