WO2015194525A1 - Trigger factor of chronic fatty inflammatory disease - Google Patents

Trigger factor of chronic fatty inflammatory disease Download PDF

Info

Publication number
WO2015194525A1
WO2015194525A1 PCT/JP2015/067248 JP2015067248W WO2015194525A1 WO 2015194525 A1 WO2015194525 A1 WO 2015194525A1 JP 2015067248 W JP2015067248 W JP 2015067248W WO 2015194525 A1 WO2015194525 A1 WO 2015194525A1
Authority
WO
WIPO (PCT)
Prior art keywords
chronic
onset
inflammatory disease
fat
gene
Prior art date
Application number
PCT/JP2015/067248
Other languages
French (fr)
Japanese (ja)
Inventor
優 石井
下村　伊一郎
船橋　徹
法一 前田
Original Assignee
国立大学法人大阪大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人大阪大学 filed Critical 国立大学法人大阪大学
Priority to JP2016529350A priority Critical patent/JP6558645B2/en
Publication of WO2015194525A1 publication Critical patent/WO2015194525A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a trigger factor for fat chronic inflammatory disease, a fatty chronic inflammatory disease onset inhibitor, and a test method for fat chronic inflammatory disease.
  • Inflammation is a symptom in which an immune response is activated when a living body receives some harmful stimulus, thereby appearing in the living body.
  • the types of inflammation include acute inflammation with neutrophil-based cell infiltration and fibrin deposition as well as slow tissue destruction and tissue construction due to infiltration of lymphocytes, plasma cells, macrophages, and tissue growth. It is classified as chronic inflammation with modification. Inflammation is an important biodefense function, which is essential, but unnecessary or excessive (long-term) inflammatory reactions can exacerbate or cause new diseases.
  • chronic inflammation is a pathological condition common to lifestyle-related diseases such as obesity, metabolic syndrome, arteriosclerotic disease, and cancer, and is considered to be an important factor in Alzheimer's disease, renal failure, cirrhosis, and the like.
  • Acute inflammation causes inflammatory reactions such as fever, swelling, pain, and redness, but chronic inflammation can hardly be confirmed.
  • acute inflammation is often reversible and heals without restructuring the tissue, whereas chronic inflammation is caused by tissue fibrosis or inflammation cells such as macrophages. Entering, the cells of the tissue are enlarged and proliferated, the structure changes, causing dysfunction.
  • chronic inflammation has been considered as the etiology of many diseases, such as type II diabetes.
  • S100A8 / A9 is known to be associated with chronic inflammation (Patent Document 1, Non-Patent Document 1).
  • Patent Document 1 Non-Patent Document 1
  • these documents do not show the relationship between chronic inflammation and diabetes.
  • ethyl palmitate was continuously administered intravenously to mice, expression of S100A8 and S100A9 in epididymal adipose tissue was observed 4 hours after administration, and macrophage accumulation was observed 12 hours later.
  • Non-patent Document 2 It has been reported that S100A8 and S100A9 have been shown to contribute to the induction of macrophages into adipose tissue by palmitic acid (Non-patent Document 2). However, the animal model produced in Non-Patent Document 2 cannot be said to be a model showing chronic inflammation of adipose tissue.
  • the present invention aims to provide a trigger factor for the onset of fat chronic inflammatory disease, and provides an agent for suppressing the onset of fatty chronic inflammatory disease characterized by suppressing the trigger factor.
  • Another object of the present invention is to provide a method for predicting the onset of chronic fat inflammatory disease, characterized by measuring the expression level of the trigger factor.
  • the present inventors have succeeded in capturing the initial phenomenon of chronic inflammation by visualizing the chronification process of inflammation using a biological tissue imaging experimental system developed independently. Furthermore, S100A8 was identified as the first trigger factor for inducing this initial phenomenon, and the present invention was completed.
  • this invention consists of the following.
  • Trigger factor for the development of chronic fat inflammatory disease comprising S100A8.
  • 2. The trigger factor for the onset of chronic fat inflammatory disease according to item 1, wherein the trigger factor is a macrophage migration ability stimulating factor in adipose tissue.
  • 3. The trigger factor for the onset of chronic fat inflammatory disease according to item 1 above, wherein the trigger factor is an expression inducer of inflammatory cytokines and / or chemokines. 4).
  • An agent for inhibiting the onset of chronic fat inflammatory disease comprising as an active ingredient a substance that targets the trigger factor for the onset of chronic fat inflammatory disease according to any one of items 1 to 3. 5. 5. 5.
  • a test method for predicting the onset of fat chronic inflammatory disease or measuring the degree of progression of fat chronic inflammation characterized by measuring the expression level of S100A8 or S100A8 gene in a specimen. 9. In addition to the measurement of the expression level of S100A8 or S100A8 gene in a specimen, the expression level of at least one protein or gene of S100A9, inflammatory cytokine and / or chemokine is further measured. Inspection method described. 10. 11. The examination method according to item 10 above, wherein the inflammatory cytokine and / or chemokine is at least one selected from CCL2 / MCP-1, CCL3, IL-1 ⁇ , TNF- ⁇ , SAA3, CXCL1, CXCL5 and HMGB1. 11.
  • the expression level of S100A8 or S100A8 gene exceeds the level of healthy people, and the expression level of at least one protein or gene of S100A9, inflammatory cytokine and / or chemokine is at the level of healthy people 10.
  • S100A8 By collecting adipocytes at the time of high fat diet loading and measuring the molecular expression level, among various chemokines / cytokines and immune activators, only S100A8 increased its expression from 1 week of high fat diet loading. It was confirmed that Then, by measuring the expression level of S100A8, a test for predicting the onset of chronic fat inflammatory disease or measuring the degree of progression of fat chronic inflammation can be performed. In particular, morphological changes such as adipocyte hypertrophy and the expression of other inflammatory cytokines and chemokines such as S100A8 are observed at an early stage compared to S100A9.
  • the progression to fat chronic inflammatory diseases such as diabetes, hyperlipidemia, arteriosclerosis, etc. can be suppressed by dealing early.
  • Administration of a factor that suppresses S100A8, for example, an anti-S100A8 antibody can suppress immune cell migration and suppress the onset of chronic fatty inflammation.
  • FIG. 1 It is a result figure which shows the body weight of each mouse
  • Reference Example 1 It is a result figure which shows the blood glucose level of each mouse
  • Reference Example 1 It is a result figure which shows the fat cell diameter of each mouse
  • Reference Example 1 It is a result figure which shows the macrophage migration ability of each mouse
  • FIG. 6 is a diagram showing the results of confirming the expression levels of S100A8, S100A9, HMGB1, CCL2 / MCP-1, CCL3, and IL-1 ⁇ as alarmins, which are molecules that induce inflammatory responses, for each mouse.
  • S100A8 which is a molecule
  • CCL2 / MCP-1 about each mouse
  • S100A8 which is a molecule
  • Reference Example 2 It is a figure which shows the result of having confirmed the effect
  • Example 1 It is a figure which shows the result of having confirmed the effect
  • Example 2 It is a figure which shows the result of having confirmed the effect
  • Example 2 It is a figure which shows the result of having confirmed the effect
  • Example 4 It is a figure which shows the result of having confirmed the influence which acts on CCL2 / MCP-1, SAA3, CXCL1, and CXCL5 gene expression. (Example 3) It is a figure which confirmed about the LysM EGFP mouse
  • Example 4 It is a figure which confirmed about the LysM EGFP mouse
  • S100A8 is an early trigger of chronic inflammation in lifestyle-related diseases, and by suppressing this action, chronic and sustained inflammation is prevented, and it is a breakthrough that suppresses the onset of obesity and abnormal lipid metabolism. It can be a treatment method.
  • the present invention provides a trigger factor for the onset of chronic fat inflammatory disease comprising S100A8.
  • S100A8 the amino acid sequence of S100A8 and the DNA sequence encoding it are disclosed, for example, in Hum Genet (2002) 111: 310-313.
  • the chronic fat inflammatory disease includes the basic pathological conditions of chronic fat inflammation and metabolic syndrome.
  • obese adipose tissue not only changes in adipocytes themselves with adipocyte hypertrophy, but also angiogenesis, increase in extracellular matrix, infiltration and qualitative changes in immune cells such as macrophages, neutrophils, and T cells Inflammation changes due to.
  • specific examples of such fat chronic inflammatory diseases include diabetes, hyperlipidemia, arteriosclerosis and the like.
  • Adipocyte hypertrophy is observed in the early stages of obesity.
  • TNF- ⁇ In enlarged fat cells, TNF- ⁇ , CCL2 (chemokine [CC motif] ligand 2) / MCP-1 (Monocyte Chemoattractant Protein-1), IL-1, Production of inflammatory cytokines such as IL-6, IL-4, IL-10, and HMGB-1 is increased, and production of anti-inflammatory cytokines such as adiponectin is decreased.
  • CCL2 chemokine [CC motif] ligand 2
  • MCP-1 Monocyte Chemoattractant Protein-1
  • IL-1 Production of inflammatory cytokines such as IL-6, IL-4, IL-10, and HMGB-1 is increased, and production of anti-inflammatory cytokines such as adiponectin is decreased.
  • M1 macrophage classically activated macrophage
  • M2 macrophage alternatively activated macrophage
  • the type of macrophages of adipose tissue that is stimulated by S100A8 is not particularly limited, and examples thereof include M1 macrophages that are inflammatory macrophages.
  • Examples of inflammatory cytokines and / or chemokines in adipose tissue whose expression is enhanced by S100A8 in the present invention include those belonging to alarmin or DAMPs (danger-associated molecular patterns), CCL2 / MCP-1, CCL3, IL-1 ⁇ and TNF- ⁇ can be mentioned. Furthermore, serum amyloid A3 (SAA3), CXCL1 (chemokine (C-X-C-motif) ligand 1) and CXCL5 can be mentioned.
  • SAA3 serum amyloid A3
  • CXCL1 chemokine (C-X-C-motif) ligand 1
  • CXCL5 can be mentioned.
  • the trigger factor for the onset of chronic fatty inflammatory disease of the present invention also has a function as a macrophage migration ability stimulating factor and inflammatory cytokine and / or chemokine expression inducing factor in the adipose tissue as described above. Also included are macrophage migration-stimulating factors and a pro-inflammatory factor and / or chemokine expression inducer in adipose tissue consisting of S100A8.
  • the present invention further extends to a fat chronic inflammatory disease onset inhibitor that targets a trigger factor for the development of fat chronic inflammatory disease.
  • the agent for suppressing the onset of chronic fat inflammatory disease refers to a drug that prevents the onset of fat chronic inflammation or fat chronic inflammatory disease, prevents the progression of the disease, or treats it.
  • a substance that inhibits the function of S100A8 or the expression of the S100A8 gene can be mentioned. Examples thereof include an antibody against S100A8 and a nucleic acid that inhibits expression of the S100A8 gene, such as siRNA.
  • the substance may be a low molecular compound in addition to a high molecular compound such as a protein, peptide, or nucleic acid substance.
  • the pharmacologically acceptable carrier can be included in the fat chronic inflammatory disease onset inhibitor of the present invention.
  • the pharmacologically acceptable carrier used in the fat chronic inflammatory disease onset inhibitor include excipients, disintegrants or disintegrants, binders, lubricants, coating agents, dyes, diluents, Examples include bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, and adhesives.
  • the agent for inhibiting the onset of chronic fatty inflammatory disease of the present invention may be administered locally or systemically.
  • Formulations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous diluents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic ester compositions such as ethyl oleate, which are suitable for injection.
  • Aqueous carriers may include water, alcoholic aqueous solutions, emulsions, suspensions, saline and buffered media.
  • Parenteral carriers may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactic acid and binding oil.
  • Intravenous carriers may include, for example, fluid supplements, nutrients and electrolytes (eg, those based on Ringer's dextrose).
  • the fat chronic inflammatory disease onset inhibitor of the present invention may further contain a preservative and other additives such as antimicrobial compounds, antioxidants, chelating agents and inert gases.
  • the present invention further extends to a test method for predicting the onset of fat chronic inflammatory disease or measuring the progression of fat chronic inflammation, characterized by measuring the expression level of S100A8 or S100A8 gene in a specimen.
  • S100A8 is a trigger factor for the onset of fat chronic inflammatory disease
  • S100A8 or S100A8 gene the expression level of S100A8 or S100A8 gene
  • the onset of fat chronic inflammatory disease can be predicted.
  • S100A8 triggers the onset of chronic fat inflammatory disease
  • S100A9 is expressed with a delay.
  • other inflammatory cytokines and / or chemokines except S100A8 are also expressed late.
  • a test for measuring the progression of fat chronic inflammation can be performed by measuring the expression level of the S100A8 or S100A8 gene in the specimen.
  • the degree of progression of fat chronic inflammation can be measured by measuring the expression level of the S100A8 or S100A8 gene in the specimen.
  • the expression level of at least one S100A9, inflammatory cytokine and / or chemokine protein or gene among adipose tissue S100A9, inflammatory cytokine and / or chemokine in the specimen is measured. By doing so, the progress of fat chronic inflammation can be measured.
  • the expression level of the S100A8 or S100A8 gene exceeds the level of a healthy person, and at least one of the S100A9, inflammatory cytokine and / or chemokine protein of S100A9, inflammatory cytokine and / or chemokine
  • the progression of fat chronic inflammation can be measured by confirming that the gene expression level is at the level of a healthy person.
  • the inflammatory cytokine and / or chemokine of adipose tissue is alarmin or DAMPs, and examples of alarmin or DAMPs include CCL2 / MCP-1, CCL3, IL-1 ⁇ , TNF- ⁇ , SAA3, CXCL1, CXCL5, HMGB1, etc. Can be mentioned.
  • the expression level of the S100A8 or S100A8 gene exceeds the level of a healthy person, and at least one of S100A9, inflammatory cytokine and / or chemokine protein or gene among S100A9, inflammatory cytokine and / or chemokine
  • an inhibitor of the onset of chronic fat inflammatory disease consisting of a substance that inhibits the function of S100A8 or the expression of the S100A8 gene to patients with the progression of chronic fat inflammation whose expression level is normal Can prevent the onset or progression of fat chronic inflammation.
  • the test specimen is not particularly limited as long as it is a specimen capable of measuring the gene expression level.
  • the expression level of the S100A8 gene can be measured by methods such as RT-PCR, quantitative PCR, Northern blot, ELISA, Western blotting, in situ hybridization, immunohistochemical staining, and the like.
  • the expression level of the reporter gene depends on the type of reporter gene, it can be measured by fluorescence intensity, luminescence intensity, radioactivity intensity, and the like.
  • RAW264.7 cells were cultured using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% cochlear fetal serum (FCS) and 1% penicillin / streptomycin (P / S).
  • DMEM Dulbecco's modified Eagle medium
  • FCS cochlear fetal serum
  • P / S penicillin / streptomycin
  • 3T3-L1 cells were cultured in DMEM supplemented with 10% FCS, 1% P / S and 0.5 mM mM 1-methyl-3-isobutylxanthine, 1M dexamethasone and 5 mg / mL insulin. After culturing for 48 hours, the maintenance medium used was DMEM supplemented with 10% FCS and 1% P / S.
  • Example 3 Effect of human or mouse S100A8 on adipocytes
  • Mouse fibroblast-derived established adipocytes (3T3-L1) were transformed into human or mouse genetically modified S100A8 (Giotto Biotech) 10 ⁇ g / mL, and endogenous.
  • PolyB polymyxin B
  • Example 4 Changes in immune cell dynamics after administration of S100A8 antibody
  • An adipocyte tissue was imaged on a LysM EGFP mouse on the fifth day after feeding the HF / HS diet. Prior to imaging, BODIPY staining was performed for adipose tissue and unlabeled Q-dots was administered intravenously for the vessel wall. LysM EGFP positive cells, which are myelomonocytic immune cells, were stained green. After anti-S100A8 antibody or IgG isotype as a control was administered to adipocytes, the kinetics of LysM EGFP positive cells was observed.
  • S100A8 is a trigger factor for the development of chronic fat inflammatory disease.
  • S100A8 is a trigger factor for the development of chronic fat inflammatory disease.
  • morphological changes such as adipocyte hypertrophy and the expression of other inflammatory cytokines and chemokines such as S100A8 are observed at an early stage compared to S100A9.
  • a factor that suppresses S100A8 for example, an anti-S100A8 antibody, can suppress immune cell migration and suppress the onset of chronic fatty inflammation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Analytical Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • Mycology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Provided is a trigger factor for the onset of chronic fatty inflammatory disease. Also provided is an agent for suppressing the onset of chronic fatty inflammatory disease, characterized by suppressing this trigger factor. Additionally provided is a method for predicting the onset of chronic fatty inflammatory disease, characterized by measuring the expression level of this trigger factor. Testing is performed to predict the onset of chronic fatty inflammatory disease or measure the advance of chronic fatty inflammation by measuring the expression level of S100A8 as the earliest trigger factor inducing the earliest phenomenon of chronic inflammation. Expression of S100A8 is found particularly at the stage of morphological changes such as adipocyte hypertrophy and at an earlier stage than the expression of other inflammatory cytokines and chemokines, for example, S100A9. Administration of a factor suppressing S100A8, for example, an anti-S100A8 antibody, can suppress the migration of immune cells and suppress the onset of chronic fatty inflammation.

Description

脂肪慢性炎症疾患のトリガー因子Trigger factors for fat chronic inflammatory disease
　本発明は、脂肪慢性炎症疾患のトリガー因子、脂肪性慢性炎症疾患発症抑制剤及び脂肪慢性炎症疾患の検査方法に関する。 The present invention relates to a trigger factor for fat chronic inflammatory disease, a fatty chronic inflammatory disease onset inhibitor, and a test method for fat chronic inflammatory disease.
　炎症とは、生体が何らかの有害な刺激を受けたときに免疫応答が働き、それによって生体に出現した症候である。炎症の種類としては、好中球を主体とする急激な細胞浸潤とフィブリンの析出を伴う急性炎症と、リンパ球や形質細胞、マクロファージの浸潤、組織の増加などにより緩徐な組織破壊、組織構築の改変を伴う慢性炎症などに分類される。炎症は重要な生体防御機能の１つで、なくてはならないものであるが、不必要な、または過剰（長期）な炎症反応は、疾患を増悪させたり新たな疾患を引き起こしたりする。例えば慢性炎症は、肥満、メタボリックシンドローム、動脈硬化性疾患などの生活習慣病、およびがんに共通する病態で、アルツハイマー病、腎不全、肝硬変などでも重要な因子と考えられている。急性炎症では発熱、腫れ、痛み、赤みなどの炎症反応が生じるが、慢性炎症ではほとんど確認できない。しかしながら、予後については、急性炎症が多くの場合は可逆的であり、組織の再構築にまでは至らずに治癒するのに対し、慢性炎症は、組織が繊維化したり、マクロファージなどの炎症細胞が入り、組織の細胞が肥大・増殖して構造が変わり、機能障害を引き起こしたりする。特に、慢性炎症が多くの疾病の病因と考えられるようになり、例えばII型糖尿病もその代表的なものといえる。 Inflammation is a symptom in which an immune response is activated when a living body receives some harmful stimulus, thereby appearing in the living body. The types of inflammation include acute inflammation with neutrophil-based cell infiltration and fibrin deposition as well as slow tissue destruction and tissue construction due to infiltration of lymphocytes, plasma cells, macrophages, and tissue growth. It is classified as chronic inflammation with modification. Inflammation is an important biodefense function, which is essential, but unnecessary or excessive (long-term) inflammatory reactions can exacerbate or cause new diseases. For example, chronic inflammation is a pathological condition common to lifestyle-related diseases such as obesity, metabolic syndrome, arteriosclerotic disease, and cancer, and is considered to be an important factor in Alzheimer's disease, renal failure, cirrhosis, and the like. Acute inflammation causes inflammatory reactions such as fever, swelling, pain, and redness, but chronic inflammation can hardly be confirmed. However, with regard to the prognosis, acute inflammation is often reversible and heals without restructuring the tissue, whereas chronic inflammation is caused by tissue fibrosis or inflammation cells such as macrophages. Entering, the cells of the tissue are enlarged and proliferated, the structure changes, causing dysfunction. In particular, chronic inflammation has been considered as the etiology of many diseases, such as type II diabetes.
　近年、肥満や高脂血症などの生活習慣病の基礎病態として、脂肪組織の慢性炎症が注目されている。慢性状態の脂肪組織では、脂肪細胞が肥大化しており、中には肥大化しすぎて細胞死を起こしたものが観察される。その周囲にはマクロファージを始めとして様々な免疫細胞が集積しており、これらが種々のサイトカインを放出することで脂肪細胞の傷害や全身性の代謝異常を誘導することでさらに病状を悪化させている。高脂肪食を負荷させる肥満の動物モデルにおいても、低いレベルの炎症が慢性に持続的に進行していく様子が観察される。これまでの研究により、この脂肪組織の慢性炎症において重要な役割を果たす種々の免疫・炎症細胞やケモカイン・サイトカインなどが同定されてきたが、この慢性炎症の契機が何によって引き起こされるのか、その「最初のトリガー」については謎のままであった。 In recent years, chronic inflammation of adipose tissue has attracted attention as a basic condition of lifestyle-related diseases such as obesity and hyperlipidemia. In the adipose tissue in a chronic state, fat cells are enlarged, and some of them are observed to be too enlarged and cause cell death. A variety of immune cells, including macrophages, accumulate around it, which release various cytokines to further aggravate the pathology by inducing fat cell damage and systemic metabolic abnormalities. . Even in obese animal models loaded with a high fat diet, it is observed that low levels of inflammation continue to progress chronically. Various studies have identified various immunity / inflammatory cells, chemokines, and cytokines that play an important role in this chronic inflammation of adipose tissue. What is the cause of this chronic inflammation? The “first trigger” remained a mystery.
　S100タンパク質は、現在までに20種類のサブファミリーが確認されている。細胞内におけるシグナル伝達だけでなく、細胞外に分泌され機能するといわれており、これらS100タンパク質ファミリーの機能は、複雑で多岐に渡ると考えられており、未解明の部分が多く残されている。上記S100タンパク質ファミリーのうちS100A8/A9は、慢性炎症に関係していることは公知である（特許文献１、非特許文献１）。しかしながら、これらの文献では慢性炎症と糖尿病との関係については示されていない。一方、マウスにパルミチン酸エチルを持続的に静脈投与した際に、投与４時間で精巣上体脂肪組織でのS100A8、S100A9発現が認められ、これに遅れて１２時間でマクロファージの集積が認められ、パルミチン酸による脂肪組織へのマクロファージ誘導にS100A8、S100A9が寄与していることが示されたことが報告されている（非特許文献２）。しかしながら、非特許文献２において作製された動物モデルは脂肪組織の慢性炎症を示すモデルとはいいがたい。 20 20 types of subfamilies have been confirmed so far. It is said that not only intracellular signal transduction but also extracellular functions are secreted and functioned. The functions of these S100 protein families are considered to be complex and diverse, and many unexplained parts remain. Of the S100 protein family, S100A8 / A9 is known to be associated with chronic inflammation (Patent Document 1, Non-Patent Document 1). However, these documents do not show the relationship between chronic inflammation and diabetes. On the other hand, when ethyl palmitate was continuously administered intravenously to mice, expression of S100A8 and S100A9 in epididymal adipose tissue was observed 4 hours after administration, and macrophage accumulation was observed 12 hours later. It has been reported that S100A8 and S100A9 have been shown to contribute to the induction of macrophages into adipose tissue by palmitic acid (Non-patent Document 2). However, the animal model produced in Non-Patent Document 2 cannot be said to be a model showing chronic inflammation of adipose tissue.
特開2011-47932号公開公報JP 2011-47932 A Publication
　本発明は、脂肪慢性炎症疾患発症のトリガー因子を提供することを課題とし、当該トリガー因子を抑制することを特徴とする脂肪性慢性炎症疾患発症抑制剤を提供する。また、当該トリガー因子の発現量を測定することを特徴とする、脂肪慢性炎症疾患発症の予測方法を提供することを課題とする。 The present invention aims to provide a trigger factor for the onset of fat chronic inflammatory disease, and provides an agent for suppressing the onset of fatty chronic inflammatory disease characterized by suppressing the trigger factor. Another object of the present invention is to provide a method for predicting the onset of chronic fat inflammatory disease, characterized by measuring the expression level of the trigger factor.
　本発明者らは上記課題を解決するために、独自に開発した脂肪組織の生体イメージング実験系を用いて炎症の慢性化過程を可視化することで、慢性炎症の最初期現象を捉えることに成功し、さらにこの初期現象を誘導する最初のトリガー因子としてS100A8を同定し、本発明を完成した。 In order to solve the above problems, the present inventors have succeeded in capturing the initial phenomenon of chronic inflammation by visualizing the chronification process of inflammation using a biological tissue imaging experimental system developed independently. Furthermore, S100A8 was identified as the first trigger factor for inducing this initial phenomenon, and the present invention was completed.
　すなわち、本発明は以下よりなる。
１．S100A8からなる脂肪慢性炎症疾患発症のトリガー因子。
２．トリガー因子が、脂肪組織内のマクロファージ遊走能刺激因子である、前項１に記載の脂肪慢性炎症疾患発症のトリガー因子。
３．トリガー因子が、炎症性サイトカインおよび／またはケモカインの発現誘導因子である、前項１に記載の脂肪慢性炎症疾患発症のトリガー因子。
４．前項１～３のいずれかに記載の脂肪慢性炎症疾患発症のトリガー因子を標的とする物質を有効成分として含む脂肪慢性炎症疾患発症抑制剤。
５．脂肪慢性炎症疾患発症のトリガー因子を標的とする物質が、抗S100A8抗体である、前項４に記載の脂肪慢性炎症疾患発症抑制剤。
６．脂肪慢性炎症疾患発症のトリガー因子を標的とする物質が、S100A8の発現を抑制する核酸である、前項４に記載の脂肪慢性炎症疾患発症抑制剤。
７．S100A8の発現を抑制する核酸が、S100A8遺伝子のsiRNAである、前項６に記載の脂肪慢性炎症疾患発症抑制剤。
８．検体におけるS100A8またはS100A8遺伝子の発現量を測定することを特徴とする、脂肪慢性炎症疾患発症の予測、あるいは脂肪慢性炎症の進行度を測定するための検査方法。
９．検体におけるS100A8またはS100A8遺伝子の発現量の測定に加えて、更にS100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一種以上のタンパク質または遺伝子の発現量を測定することを特徴とする、前項８に記載の検査方法。
１０．炎症性サイトカインおよび／またはケモカインが、CCL2/MCP-1、CCL3、IL-1α、TNF-α、SAA3、CXCL1、CXCL5およびHMGB1から選択される少なくとも一種である、前項１０に記載の検査方法。
１１．S100A8またはS100A8遺伝子の発現量が健常人のレベルを超えて発現し、S100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一つ以上のタンパク質または遺伝子の発現量が健常人のレベルであることを確認することを特徴とする、前項９に記載の検査方法。 That is, this invention consists of the following.
1. Trigger factor for the development of chronic fat inflammatory disease comprising S100A8.
2. 2. The trigger factor for the onset of chronic fat inflammatory disease according to item 1, wherein the trigger factor is a macrophage migration ability stimulating factor in adipose tissue.
3. 2. The trigger factor for the onset of chronic fat inflammatory disease according to item 1 above, wherein the trigger factor is an expression inducer of inflammatory cytokines and / or chemokines.
4). 4. An agent for inhibiting the onset of chronic fat inflammatory disease, comprising as an active ingredient a substance that targets the trigger factor for the onset of chronic fat inflammatory disease according to any one of items 1 to 3.
5. 5. The fat chronic inflammatory disease onset inhibitor according to item 4 above, wherein the substance that targets the trigger factor for the onset of fat chronic inflammatory disease is an anti-S100A8 antibody.
6). 5. The agent for inhibiting the onset of chronic fat inflammatory disease according to item 4, wherein the substance targeting the trigger factor for the onset of chronic fat inflammatory disease is a nucleic acid that suppresses the expression of S100A8.
7). 7. The agent for inhibiting the onset of chronic fatty inflammatory disease according to item 6, wherein the nucleic acid that suppresses the expression of S100A8 is a siRNA of the S100A8 gene.
8). A test method for predicting the onset of fat chronic inflammatory disease or measuring the degree of progression of fat chronic inflammation, characterized by measuring the expression level of S100A8 or S100A8 gene in a specimen.
9. In addition to the measurement of the expression level of S100A8 or S100A8 gene in a specimen, the expression level of at least one protein or gene of S100A9, inflammatory cytokine and / or chemokine is further measured. Inspection method described.
10. 11. The examination method according to item 10 above, wherein the inflammatory cytokine and / or chemokine is at least one selected from CCL2 / MCP-1, CCL3, IL-1α, TNF-α, SAA3, CXCL1, CXCL5 and HMGB1.
11. The expression level of S100A8 or S100A8 gene exceeds the level of healthy people, and the expression level of at least one protein or gene of S100A9, inflammatory cytokine and / or chemokine is at the level of healthy people 10. The inspection method according to item 9, wherein the inspection is performed.
　高脂肪食負荷時の脂肪細胞を回収し、分子発現量を測定することにより、種々のケモカイン・サイトカインや免疫活性化因子の中で、S100A8のみが高脂肪食負荷１週から発現が亢進していることが確認された。そして、S100A8発現量を測定することにより、脂肪慢性炎症疾患発症の予測やあるいは脂肪慢性炎症の進行度を測定するための検査を行うことができる。S100A8は、特に脂肪細胞肥大などの形態的変化や、他の炎症性サイトカイン、ケモカインの発現、例えばS100A9に比べて初期の段階で発現が認められる。そこで、ある程度のS100A8発現量を認めた時点で、早期に対処することで、例えば糖尿病・高脂血症・動脈硬化等の脂肪慢性炎症疾患への進行を抑制することができる。S100A8を抑制する因子、例えば抗S100A8抗体の投与により、免疫細胞の遊走を抑制し、脂肪慢性炎症の発症を抑制することができる。 By collecting adipocytes at the time of high fat diet loading and measuring the molecular expression level, among various chemokines / cytokines and immune activators, only S100A8 increased its expression from 1 week of high fat diet loading. It was confirmed that Then, by measuring the expression level of S100A8, a test for predicting the onset of chronic fat inflammatory disease or measuring the degree of progression of fat chronic inflammation can be performed. In particular, morphological changes such as adipocyte hypertrophy and the expression of other inflammatory cytokines and chemokines such as S100A8 are observed at an early stage compared to S100A9. Thus, when a certain amount of S100A8 expression is recognized, the progression to fat chronic inflammatory diseases such as diabetes, hyperlipidemia, arteriosclerosis, etc. can be suppressed by dealing early. Administration of a factor that suppresses S100A8, for example, an anti-S100A8 antibody, can suppress immune cell migration and suppress the onset of chronic fatty inflammation.
本発明の参考例、実施例で使用するLys M-EGFPトランスジェニック（LysMEGFP）マウスについて、正常／固形（NC）食または高脂肪／高ショ糖（HF/HS）食の摂食スケジュールを示す図である。（参考例１）The feeding schedule of a normal / solid (NC) diet or a high fat / high sucrose (HF / HS) diet is shown for the Lys M-EGFP transgenic (LysM EGFP ) mice used in the Reference Examples and Examples of the present invention. FIG. (Reference Example 1) 各マウスの体重を示す結果図である。（参考例１）It is a result figure which shows the body weight of each mouse | mouth. (Reference Example 1) 各マウスの血中グルコース量を示す結果図である。（参考例１）It is a result figure which shows the blood glucose level of each mouse | mouth. (Reference Example 1) 各マウスの脂肪細胞径を示す結果図である。（参考例１）It is a result figure which shows the fat cell diameter of each mouse | mouth. (Reference Example 1) 各マウスのマクロファージ遊走能を示す結果図である。（参考例１）It is a result figure which shows the macrophage migration ability of each mouse | mouth. (Reference Example 1) 各マウスについて、炎症反応を誘導する分子であるアラーミンとしてのS100A8、S100A9、HMGB1、CCL2/MCP-1、CCL3、IL-1αの発現量を確認した結果図である。（参考例１）FIG. 6 is a diagram showing the results of confirming the expression levels of S100A8, S100A9, HMGB1, CCL2 / MCP-1, CCL3, and IL-1α as alarmins, which are molecules that induce inflammatory responses, for each mouse. (Reference Example 1) 各マウスについて、炎症応答を誘導する分子であるS100A8とCCL2/MCP-1の発現量を確認した結果図である。（参考例２）It is a figure which confirmed the expression level of S100A8 which is a molecule | numerator which induces an inflammatory response, and CCL2 / MCP-1 about each mouse | mouth. (Reference Example 2) マクロファージ株化細胞（RAW264.7細胞）に及ぼすS100A8の作用を確認した結果を示す図である。RAW264.7細胞の遊走能を確認した結果図である。（実施例１）It is a figure which shows the result of having confirmed the effect | action of S100A8 on a macrophage cell line (RAW264.7 cell). It is the result figure which confirmed the migration ability of RAW264.7 cell. (Example 1) RAW264.7細胞に及ぼすS100A8の作用を確認した結果を示す図である。アラーミンとしてのTNF-αまたはCCL2/MCP-1の各遺伝子の発現量を測定した結果図である。（実施例２）It is a figure which shows the result of having confirmed the effect | action of S100A8 on RAW264.7 cell. It is a result figure which measured the expression level of each gene of TNF- (alpha) or CCL2 / MCP-1 as an alarmin. (Example 2) マウス線維芽細胞由来株化脂肪細胞（3T3-L1細胞）に及ぼすS100A8の作用を確認した結果を示す図である。CCL2/MCP-1遺伝子の発現量を測定した結果図である。（実施例２）It is a figure which shows the result of having confirmed the effect | action of S100A8 exerted on a mouse | mouth fibroblast derived cell line fat cell (3T3-L1 cell). It is a result figure which measured the expression level of CCL2 / MCP-1 gene. (Example 2) 3T3-L1細胞に及ぼすヒトまたはマウスのS100A8の作用を確認した結果を示す図である。CCL2/MCP-1、SAA3、CXCL1およびCXCL5各遺伝子発現に及ぼす影響を確認した結果を示す図である。（実施例３）It is a figure which shows the result of having confirmed the effect | action of human or mouse | mouth S100A8 on 3T3-L1 cell. It is a figure which shows the result of having confirmed the influence which acts on CCL2 / MCP-1, SAA3, CXCL1, and CXCL5 gene expression. (Example 3) LysMEGFPマウスについて、S100A8抗体投与後の免疫細胞動態変化について確認した結果図である。（実施例４）It is a figure which confirmed about the LysM EGFP mouse | mouth about the immune cell dynamics change after S100A8 antibody administration. Example 4
　本発明を説明するにあたり、本発明を完成するに至った経緯をまず詳述する。これまでの組織学的解析では、高脂肪食を負荷する肥満モデル動物において、負荷後８週経過後において慢性炎症現象を観察できたが、負荷後１週後などの初期段階では明らかな組織変化をとらえることはできなかった。本発明者は、独自に確立した脂肪組織の蛍光生体イメージング実験系を用いて、脂肪の慢性炎症が進行していく過程を、時系列を追って詳細に解析することに成功した。この結果、高脂肪食負荷１週間後において、すでに脂肪組織内でのマクロファージの動態が有意に亢進していることを明らかにした。定常状態では脂肪組織内のマクロファージは殆ど動かないが、負荷後たった１週経過した時点で、マクロファージの可動性が著しく亢進していることが確認された。さらに、高脂肪負荷時の脂肪組織を回収し、分子発現を測定することにより、種々のケモカイン・サイトカインや免疫活性化因子の中で、S100A8のみが負荷１週間から発現が亢進していることを確認した。本発明者はさらに、生体イメージング系を用いてS100A8が脂肪組織内のマクロファージの遊走能を刺激すること、また脂肪細胞やマクロファージに作用し、その他の炎症性サイトカインやケモカインの発現を誘導し、炎症のトリガーとして作用することを明らかにした。これらのことから、S100A8は生活習慣病における慢性炎症の初期トリガーであり、この作用を抑えることにより炎症の慢性化・持続化を防ぎ、肥満や脂質代謝異常などの発症を抑制する画期的な治療方法となりうる。 In describing the present invention, the background to the completion of the present invention will be described in detail first. In previous histological analyses, chronic inflammatory phenomena could be observed in obese model animals loaded with a high-fat diet 8 weeks after loading, but obvious tissue changes were observed at an early stage such as 1 week after loading. I couldn't catch. The present inventor succeeded in analyzing the process of chronic fat inflammation in a time series in detail using a fluorescent tissue imaging experimental system for adipose tissue that has been independently established. As a result, it was clarified that the dynamics of macrophages in the adipose tissue was significantly enhanced already after 1 week of high fat diet loading. In a steady state, macrophages in the adipose tissue hardly move, but it was confirmed that the mobility of macrophages was remarkably enhanced when only one week passed after loading. Furthermore, by collecting adipose tissue under high-fat load and measuring molecular expression, among various chemokine cytokines and immune activators, only S100A8 increased its expression from 1 week of load. confirmed. The inventor further uses a biological imaging system to stimulate the migration of macrophages in adipose tissue by S100A8, act on adipocytes and macrophages, induce the expression of other inflammatory cytokines and chemokines, It was clarified that it acts as a trigger. From these facts, S100A8 is an early trigger of chronic inflammation in lifestyle-related diseases, and by suppressing this action, chronic and sustained inflammation is prevented, and it is a breakthrough that suppresses the onset of obesity and abnormal lipid metabolism. It can be a treatment method.
　上記の知見より、本発明ではS100A8からなる脂肪慢性炎症疾患発症のトリガー因子を提供する。ここで、S100A8のアミノ酸配列およびそれをコードするDNA配列は、例えばHum Genet (2002) 111:310-313に開示されている。 Based on the above findings, the present invention provides a trigger factor for the onset of chronic fat inflammatory disease comprising S100A8. Here, the amino acid sequence of S100A8 and the DNA sequence encoding it are disclosed, for example, in Hum Genet (2002) 111: 310-313.
　本明細書において、脂肪慢性炎症疾患とは、脂肪慢性炎症およびメタボリック症候群の基盤病態が挙げられる。肥満の脂肪組織では、脂肪細胞の肥大化に伴う脂肪細胞自身の変化のみならず、血管新生や細胞外基質の増加、マクロファージや好中球、Ｔ細胞などの免疫担当細胞の浸潤や質的変化による炎症変化が認められる。このような脂肪慢性炎症疾患の具体例としては、例えば糖尿病、高脂血症、動脈硬化などが挙げられる。脂肪細胞の肥大化は、肥満の早期から認められ、肥大化した脂肪細胞ではTNF-α、CCL2（chemokine [C-C motif] ligand 2）/MCP-1（Monocyte Chemoattractant Protein-1）やIL-1、IL-6、IL-4、IL-10、HMGB-1、などの炎症性サイトカインの産生が亢進し、アディポネクチンなどの抗炎症性サイトカインの産生が減少する。 In the present specification, the chronic fat inflammatory disease includes the basic pathological conditions of chronic fat inflammation and metabolic syndrome. In obese adipose tissue, not only changes in adipocytes themselves with adipocyte hypertrophy, but also angiogenesis, increase in extracellular matrix, infiltration and qualitative changes in immune cells such as macrophages, neutrophils, and T cells Inflammation changes due to. Specific examples of such fat chronic inflammatory diseases include diabetes, hyperlipidemia, arteriosclerosis and the like. Adipocyte hypertrophy is observed in the early stages of obesity. In enlarged fat cells, TNF-α, CCL2 (chemokine [CC motif] ligand 2) / MCP-1 (Monocyte Chemoattractant Protein-1), IL-1, Production of inflammatory cytokines such as IL-6, IL-4, IL-10, and HMGB-1 is increased, and production of anti-inflammatory cytokines such as adiponectin is decreased.
　肥満における脂肪組織の炎症の決定的な特徴は、脂肪組織の拡大とマクロファージ浸潤の著しい増加であると考えられる。マクロファージは、自然免疫において中心的な役割を果たしており、細菌やウイルス感染の際に活性化し、それらを排除する。また、アレルギー応答、脂肪代謝、創傷治癒および癌の転移・浸潤等にも寄与している可能性が近年の研究から明らかとなりつつある。これらの病態はそれぞれ異なったマクロファージによって担われていると考えられており、前者の病原体の感染の際に活性化する細胞集団をM1マクロファージ（classically activated macrophage）、一方で後者の病気の際に活性化する細胞集団をM2マクロファージ（alternatively activated macrophage）という。本発明においてS100A8により刺激を受ける脂肪組織のマクロファージの種類は特に限定されないが、例えば炎症性マクロファージであるM1マクロファージが挙げられる。 It is thought that the decisive feature of inflammation of adipose tissue in obesity is the expansion of adipose tissue and a marked increase in macrophage infiltration. Macrophages play a central role in innate immunity and are activated and eliminated during bacterial and viral infections. The possibility of contributing to allergic response, fat metabolism, wound healing and cancer metastasis / invasion is becoming clear from recent studies. These pathologies are thought to be carried by different macrophages, and the cell population activated upon infection with the former pathogen is called M1 macrophage (classically activated macrophage), whereas it is active during the latter disease. The cell population that transforms is called M2 macrophage (alternatively activated macrophage). In the present invention, the type of macrophages of adipose tissue that is stimulated by S100A8 is not particularly limited, and examples thereof include M1 macrophages that are inflammatory macrophages.
　本発明においてS100A8により発現が亢進する脂肪組織の炎症性サイトカインおよび／またはケモカインとしては、アラーミン（alarmin）またはDAMPs（danger-associated molecular patterns）に属するものが挙げられ、CCL2/MCP-1、CCL3、IL-1α、TNF-αが挙げられる。さらには、serum amyloid A3 (SAA3)、CXCL1（chemokine (C-X-C motif) ligand 1）やCXCL5が挙げられる。 Examples of inflammatory cytokines and / or chemokines in adipose tissue whose expression is enhanced by S100A8 in the present invention include those belonging to alarmin or DAMPs (danger-associated molecular patterns), CCL2 / MCP-1, CCL3, IL-1α and TNF-α can be mentioned. Furthermore, serum amyloid A3 (SAA3), CXCL1 (chemokine (C-X-C-motif) ligand 1) and CXCL5 can be mentioned.
　本発明の脂肪慢性炎症疾患発症のトリガー因子は、上述のような脂肪組織内のマクロファージ遊走能刺激因子や炎症性サイトカインおよび／またはケモカインの発現誘導因子としての機能も有しており、本発明はS100A8からなる脂肪組織内のマクロファージ遊走能刺激因子および炎症性サイトカインおよび／またはケモカインの発現誘導因子も含まれる。 The trigger factor for the onset of chronic fatty inflammatory disease of the present invention also has a function as a macrophage migration ability stimulating factor and inflammatory cytokine and / or chemokine expression inducing factor in the adipose tissue as described above. Also included are macrophage migration-stimulating factors and a pro-inflammatory factor and / or chemokine expression inducer in adipose tissue consisting of S100A8.
　本発明は、さらに脂肪慢性炎症疾患発症のトリガー因子を標的とする脂肪慢性炎症疾患発症抑制剤にも及ぶ。脂肪慢性炎症疾患発症抑制剤とは、脂肪慢性炎症又は脂肪慢性炎症疾患の発症を予防したり、進行を防いだり、治療したりする薬剤のことをいう。具体的には、S100A8の機能を阻害したり、S100A8遺伝子の発現を阻害する物質が挙げられる。例えばS100A8に対する抗体やS100A8遺伝子の発現を阻害する核酸、例えばsiRNAが挙げられる。また、このような作用を有する物質であれば、タンパク質、ペプチド、核酸物質等の高分子化合物の他、低分子化合物であってもよい。 The present invention further extends to a fat chronic inflammatory disease onset inhibitor that targets a trigger factor for the development of fat chronic inflammatory disease. The agent for suppressing the onset of chronic fat inflammatory disease refers to a drug that prevents the onset of fat chronic inflammation or fat chronic inflammatory disease, prevents the progression of the disease, or treats it. Specifically, a substance that inhibits the function of S100A8 or the expression of the S100A8 gene can be mentioned. Examples thereof include an antibody against S100A8 and a nucleic acid that inhibits expression of the S100A8 gene, such as siRNA. Further, as long as the substance has such an action, it may be a low molecular compound in addition to a high molecular compound such as a protein, peptide, or nucleic acid substance.
　本発明の脂肪慢性炎症疾患発症抑制剤には、製薬学的に許容しうる担体を含めることができる。係る脂肪慢性炎症疾患発症抑制剤に用いられる薬理学的に許容しうる担体としては、例えば、賦形剤、崩壊剤若しくは崩壊補助剤、結合剤、滑沢剤、コーティング剤、色素、希釈剤、基剤、溶解剤若しくは溶解補助剤、等張化剤、ｐＨ調節剤、安定化剤、噴射剤、および粘着剤等を挙げることができる。本発明の脂肪慢性炎症疾患発症抑制剤は、局所的に投与しても全身的に投与してもよい。非経口投与用の製剤は、滅菌した水性の、または非水性の溶液、懸濁液および乳濁液を含んでいてもよい。非水性希釈剤の例として、プロピレングリコール、ポリエチレングリコール、植物油、例えば、オリーブ油および有機エステル組成物、例えば、エチルオレエートであり、これらは注射用に適している。水性担体には、水、アルコール性水性溶液、乳濁液、懸濁液、食塩水および緩衝化媒体が含まれていてもよい。非経口的担体には、塩化ナトリウム溶液、リンゲルデキストロース、デキストロースおよび塩化ナトリウム、リンゲル乳酸および結合油が含まれていてもよい。静脈内担体には、例えば、液体用補充物、栄養および電解質（例えば、リンゲルデキストロースに基づくもの）が含まれていてもよい。本発明の脂肪慢性炎症疾患発症抑制剤はさらに、保存剤および他の添加剤、例えば，抗微生物化合物、抗酸化剤、キレート剤および不活性ガスなどを含むことができる。 The pharmacologically acceptable carrier can be included in the fat chronic inflammatory disease onset inhibitor of the present invention. Examples of the pharmacologically acceptable carrier used in the fat chronic inflammatory disease onset inhibitor include excipients, disintegrants or disintegrants, binders, lubricants, coating agents, dyes, diluents, Examples include bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, and adhesives. The agent for inhibiting the onset of chronic fatty inflammatory disease of the present invention may be administered locally or systemically. Formulations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous diluents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic ester compositions such as ethyl oleate, which are suitable for injection. Aqueous carriers may include water, alcoholic aqueous solutions, emulsions, suspensions, saline and buffered media. Parenteral carriers may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactic acid and binding oil. Intravenous carriers may include, for example, fluid supplements, nutrients and electrolytes (eg, those based on Ringer's dextrose). The fat chronic inflammatory disease onset inhibitor of the present invention may further contain a preservative and other additives such as antimicrobial compounds, antioxidants, chelating agents and inert gases.
　本発明は、さらに検体におけるS100A8またはS100A8遺伝子の発現量を測定することを特徴とする、脂肪慢性炎症疾患発症の予測、あるいは脂肪慢性炎症の進行度を測定するための検査方法に及ぶ。本発明において、S100A8は脂肪慢性炎症疾患発症のトリガー因子となることが確認されたことにより、S100A8またはS100A8遺伝子の発現量を測定することで、脂肪慢性炎症疾患発症を予測することができる。この場合において、脂肪慢性炎症疾患発症のトリガーとなるのはS100A8であり、S100A9は遅れて発現される。また、S100A8を除く他の炎症性サイトカインおよび／またはケモカインも遅れて発現される。このことより、検体におけるS100A8またはS100A8遺伝子の発現量を測定することで、脂肪慢性炎症の進行度を測定するための検査を行うことができる。 The present invention further extends to a test method for predicting the onset of fat chronic inflammatory disease or measuring the progression of fat chronic inflammation, characterized by measuring the expression level of S100A8 or S100A8 gene in a specimen. In the present invention, it has been confirmed that S100A8 is a trigger factor for the onset of fat chronic inflammatory disease, and by measuring the expression level of S100A8 or S100A8 gene, the onset of fat chronic inflammatory disease can be predicted. In this case, S100A8 triggers the onset of chronic fat inflammatory disease, and S100A9 is expressed with a delay. In addition, other inflammatory cytokines and / or chemokines except S100A8 are also expressed late. Thus, a test for measuring the progression of fat chronic inflammation can be performed by measuring the expression level of the S100A8 or S100A8 gene in the specimen.
　脂肪慢性炎症の進行度は、検体におけるS100A8またはS100A8遺伝子の発現量を測定することにより測定することができる。S100A8またはS100A8遺伝子の発現量の他、検体における脂肪組織のS100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一つ以上のS100A9、炎症性サイトカインおよび／またはケモカインのタンパク質または遺伝子の発現量を測定することにより、脂肪慢性炎症の進行度を測定することができる。この場合において、S100A8またはS100A8遺伝子の発現量が健常人のレベルを超えて発現し、S100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一つ以上のS100A9、炎症性サイトカインおよび／またはケモカインのタンパク質または遺伝子の発現量が健常人のレベルであることを確認することで脂肪慢性炎症の進行度を測定することができる。脂肪組織の炎症性サイトカインおよび／またはケモカインが、アラーミンまたはDAMPsであり、アラーミンまたはDAMPsの例として、CCL2/MCP-1、CCL3、IL-1α、TNF-α、SAA3、 CXCL1、CXCL5、HMGB1等が挙げられる。 The degree of progression of fat chronic inflammation can be measured by measuring the expression level of the S100A8 or S100A8 gene in the specimen. In addition to the expression level of S100A8 or S100A8 gene, the expression level of at least one S100A9, inflammatory cytokine and / or chemokine protein or gene among adipose tissue S100A9, inflammatory cytokine and / or chemokine in the specimen is measured. By doing so, the progress of fat chronic inflammation can be measured. In this case, the expression level of the S100A8 or S100A8 gene exceeds the level of a healthy person, and at least one of the S100A9, inflammatory cytokine and / or chemokine protein of S100A9, inflammatory cytokine and / or chemokine Alternatively, the progression of fat chronic inflammation can be measured by confirming that the gene expression level is at the level of a healthy person. The inflammatory cytokine and / or chemokine of adipose tissue is alarmin or DAMPs, and examples of alarmin or DAMPs include CCL2 / MCP-1, CCL3, IL-1α, TNF-α, SAA3, CXCL1, CXCL5, HMGB1, etc. Can be mentioned.
　特に、S100A8またはS100A8遺伝子の発現量が健常人のレベルを超えて発現し、S100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一つ以上のS100A9、炎症性サイトカインおよび／またはケモカインのタンパク質または遺伝子の発現量が健常人のレベルである脂肪慢性炎症の進行度の患者に、S100A8の機能を阻害したり、S100A8遺伝子の発現を阻害する物質からなる脂肪慢性炎症疾患発症抑制剤を投与することで、脂肪慢性炎症の発症または進行を防ぐことができる。 In particular, the expression level of the S100A8 or S100A8 gene exceeds the level of a healthy person, and at least one of S100A9, inflammatory cytokine and / or chemokine protein or gene among S100A9, inflammatory cytokine and / or chemokine By administering an inhibitor of the onset of chronic fat inflammatory disease consisting of a substance that inhibits the function of S100A8 or the expression of the S100A8 gene to patients with the progression of chronic fat inflammation whose expression level is normal Can prevent the onset or progression of fat chronic inflammation.
　本明細書において、検査のための被検体としては遺伝子発現量を測定可能な検体であればよく、特に限定されない。また、S100A8遺伝子の発現量はRT-PCR、定量PCR、ノーザンブロット、ELISA、Western blotting、In situ hybridization、免疫組織染色などの方法により測定することができる。レポーター遺伝子の発現量はレポーター遺伝子の種類にもよるが、蛍光強度や発光強度、放射能強度などによって測定することができる。 In the present specification, the test specimen is not particularly limited as long as it is a specimen capable of measuring the gene expression level. The expression level of the S100A8 gene can be measured by methods such as RT-PCR, quantitative PCR, Northern blot, ELISA, Western blotting, in situ hybridization, immunohistochemical staining, and the like. Although the expression level of the reporter gene depends on the type of reporter gene, it can be measured by fluorescence intensity, luminescence intensity, radioactivity intensity, and the like.
　以下に、本発明を完成させるために行なった実験結果を参考例として示し、実施例において本発明をさらに具体的に説明するが、本発明はこれらに限定されるものではなく、本発明の技術的思想を逸脱しない範囲内で種々の応用が可能である。 In the following, experimental results conducted to complete the present invention will be shown as reference examples, and the present invention will be described in more detail by way of examples. However, the present invention is not limited thereto, and the technology of the present invention Various applications are possible without departing from the spirit of the invention.
（参考例１）脂肪慢性炎症のトリガー因子の確認
１）脂肪組織における可視化したリゾチームM（LysM）陽性マクロファージの遊走
　生存マウスの脂肪組織内のマクロファージの動的挙動を視覚的に解析するために、骨髄単球系統でEGFPを発現し、特に免疫細胞動態を可視化するためのLys M-EGFPトランスジェニック（LysMEGFP）マウスを用いた。肥満の過程で脂肪組織における免疫細胞の動力学を分析するために、正常／固形（NC）食を摂食させたLysMEGFPマウス（対照leanマウス）または高脂肪／高ショ糖（HF/HS）食を摂食させたLysMEGFPマウス（DIOマウス）を用いた。さらにこれらのマウスの精巣上体白色脂肪組織（WAT）を用いて各種検討を行った。食餌の投与スケジュールは図１に従った。 (Reference Example 1) Confirmation of trigger factor for fat chronic inflammation 1) Migration of lysozyme M (LysM) positive macrophages visualized in adipose tissue In order to visually analyze the dynamic behavior of macrophages in adipose tissue of surviving mice, Lys M-EGFP transgenic (LysM EGFP ) mice were used to express EGFP in a bone marrow monocyte lineage, and in particular to visualize immune cell dynamics. LysM EGFP mice (control lean mice) or high fat / high sucrose (HF / HS) fed a normal / solid (NC) diet to analyze immune cell dynamics in adipose tissue during obesity LysM EGFP mice (DIO mice) fed with food were used. Furthermore, various studies were performed using the epididymal white adipose tissue (WAT) of these mice. The dietary administration schedule followed FIG.
　上記スケジュールで摂食させた各マウスの体重および血中グルコース量を測定した。その結果、HF/HS食を与えたマウス（DIOマウス）の体重は徐々に増加し、摂食後約８週目で対照に比べて約1.5倍の体重を示した（図２）。一方、グルコース量はDIOマウスでも摂食後第４週までは目立った増加を示さなかったのに対し、摂食後第８週目で急激な増加を認めた（図３）。 The body weight and blood glucose level of each mouse fed on the above schedule were measured. As a result, the body weight of the mice fed the HF / HS diet (DIO mice) gradually increased, and showed about 1.5 times the body weight of the control at about 8 weeks after feeding (FIG. 2). On the other hand, the glucose level did not show a noticeable increase until the 4th week after feeding in the DIO mouse, whereas a sharp increase was observed in the 8th week after feeding (FIG. 3).
　上記スケジュールで摂食させた各マウスのWATに含まれる脂肪細胞のサイズを測定したところ、摂食後第１週目ではDIOマウスと対照マウスの間にほとんど差を認めなかったが、第８週目で脂肪細胞のサイズが肥大していることが確認された（図４）。一方、マクロファージについては第１週にて、明らかな数量の増大は認めないものの、遊走能の亢進が認められた（図５）。 When the size of adipocytes contained in WAT of each mouse fed on the above schedule was measured, there was almost no difference between DIO mice and control mice in the first week after feeding, but in the eighth week. Thus, it was confirmed that the size of fat cells was enlarged (FIG. 4). On the other hand, in macrophages, although no apparent increase in quantity was observed in the first week, an increase in migration ability was observed (FIG. 5).
２）脂肪慢性炎症初期応答遺伝子発現の確認
　前記WATにおける、マクロファージ遊走に伴う脂肪慢性炎症初期遺伝子の発現量を測定した。その結果、炎症応答を誘導する分子であるアラーミンの１つであるS100A8のmRNAが、HF/HS食摂食後１週目で大幅に上昇していることが確認された。他のアラーミンであるS100A9やHMGB1はほとんど上昇していないことが確認された。一方、CCL2/MCP-1、CCL3、IL-1αはHF/HS食摂食後１週目では殆ど上昇しないが、既報の通り摂食後８週目では上昇していることが確認された（図６）。各因子のmRNA量は、PCRの手法により測定した。 2) Confirmation of fat chronic inflammation early response gene expression The expression level of fat chronic inflammation early gene accompanying macrophage migration in the WAT was measured. As a result, it was confirmed that the mRNA of S100A8, which is one of the alarmins that are molecules that induce inflammatory response, was significantly increased in the first week after feeding the HF / HS diet. It was confirmed that other alarmins such as S100A9 and HMGB1 hardly increased. On the other hand, CCL2 / MCP-1, CCL3, and IL-1α hardly increased in the first week after feeding the HF / HS diet, but were confirmed to increase in the eighth week after feeding as previously reported (FIG. 6). ). The amount of mRNA of each factor was measured by the PCR method.
（参考例２）S100A8が脂肪慢性炎症のトリガーとなることの確認実験
　参考例１と同手法により図１のスケジュールでHF/HS食を与えたマウス（DIOマウス）とNC食を与えたマウス（対照）のWATにおけるS100A8遺伝子の相対的発現量を測定した。その結果、DIOマウスでは摂食後第１週目でS100A8遺伝子発現が上昇し始めたことが確認された（図７）。一方、CCL2/MCP-1について確認したところ、DIOマウスでは摂食後第４週目でCCL2/MCP-1遺伝子の発現がやや上昇していたが、第８週目では対照とDIOマウスでのCCL2/MCP-1遺伝子の発現量が逆転していた。このことより、脂肪慢性炎症の初期にはS100A8遺伝子が発現することが確認された（図７）。 (Reference example 2) Confirmation experiment that S100A8 triggers chronic fat inflammation Mice (DIO mouse) fed with HF / HS diet and mouse fed with NC diet according to the schedule shown in Fig. 1 by the same method as in reference example 1 ( The relative expression level of S100A8 gene in WAT of control) was measured. As a result, it was confirmed that S100A8 gene expression began to increase in the first week after feeding in DIO mice (FIG. 7). On the other hand, when CCL2 / MCP-1 was confirmed, the expression of CCL2 / MCP-1 gene was slightly elevated in the 4th week after feeding in DIO mice, but in the 8th week, CCL2 in the control and DIO mice The expression level of / MCP-1 gene was reversed. From this, it was confirmed that the S100A8 gene was expressed in the early stage of fat chronic inflammation (FIG. 7).
（実施例１）マクロファージに及ぼすS100A8の影響（走化能）
　マクロファージ株化細胞であるRAW264.7細胞に及ぼすS100A8の作用を確認した。
　EZ- Taxiscan（GEヘルスケアバイオサイエンス社）により、in vitroで遺伝子組換えS100A8（Giotto Biotech社）を導入したRAW264.7細胞の遊走能を確認した。RAW264.7細胞を下方のチャンバーにおき、上部のチャンバーは1μg/mLまたは10μg/mLの S100A8を含む培地で満たし、RAW264.7細胞の走化能を確認した（n=6）。その結果、S100A8濃度に依存して、RAW264.7細胞の走化能が増すことが確認された（図８）。 (Example 1) Effect of S100A8 on macrophages (chemotaxis)
The effect of S100A8 on RAW264.7 cells, a macrophage cell line, was confirmed.
EZ-Taxiscan (GE Healthcare Bioscience) confirmed the migration ability of RAW264.7 cells into which transgenic S100A8 (Giotto Biotech) was introduced in vitro. RAW264.7 cells were placed in the lower chamber, and the upper chamber was filled with medium containing 1 μg / mL or 10 μg / mL S100A8 to confirm the chemotaxis of RAW264.7 cells (n = 6). As a result, it was confirmed that the chemotaxis ability of RAW264.7 cells increased depending on the S100A8 concentration (FIG. 8).
　RAW264.7細胞の培養は、Dulbecco's modified Eagle medium (DMEM) に10% 牛胎児血清(FCS) および 1% ペニシリン・ストレプトマイシン (P/S)を添加したものを用いた。 RAW264.7 cells were cultured using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% cochlear fetal serum (FCS) and 1% penicillin / streptomycin (P / S).
（実施例２）マクロファージおよび脂肪細胞に及ぼすS100A8の影響
　株化マクロファージ細胞（RAW264.7）またはマウス線維芽細胞由来株化脂肪細胞（3T3-L1細胞）を1μg/mL若しくは10μg/mLの遺伝子組換えS100A8（Giotto Biotech社）、または1 ng/mL若しくは10ng/mLのLipopolysaccharide（LPS）を含み、25μg/mLのポリミキシンB（PolyB：Sigma-Aldrich）を含む培地で培養したときのTNF-αおよびCCL2/MCP-1の発現に及ぼす影響を確認した（n=4）。その結果、10μg/mLの S100A8を含む培地で培養した場合に、マクロファージにおいてTNF-αおよびCCL2/MCP-1の各遺伝子発現が上昇した。しかし、LPSを含む培地で培養した場合は、マクロファージでは、わずかに各遺伝子の発現上昇が認められたのみであった（図９、１０）。一方、脂肪細胞におけるCCL2/MCP-1遺伝子については、S100A8（10μg/mL）またはLPS（10ng/mL）を含む培地で培養したところ、わずかに発現増加が認められた。これにより、S100A8は炎症反応のトリガーとなり得ると考えられた。 (Example 2) Effect of S100A8 on macrophages and adipocytes 1 μg / mL or 10 μg / mL gene sets of established macrophage cells (RAW264.7) or mouse fibroblast-derived adipocytes (3T3-L1 cells) TNF-α when cultured in medium containing 25 μg / mL polymyxin B (PolyB: Sigma-Aldrich) containing 1 ng / mL or 10 ng / mL Lipopolysaccharide (LPS) and S100A8 (Giotto Biotech) The effect on the expression of CCL2 / MCP-1 was confirmed (n = 4). As a result, when cultured in a medium containing 10 μg / mL S100A8, the expression of each gene of TNF-α and CCL2 / MCP-1 increased in macrophages. However, when cultured in a medium containing LPS, only a slight increase in the expression of each gene was observed in macrophages (FIGS. 9 and 10). On the other hand, expression of the CCL2 / MCP-1 gene in adipocytes was slightly increased when cultured in a medium containing S100A8 (10 μg / mL) or LPS (10 ng / mL). This suggested that S100A8 could trigger an inflammatory response.
　3T3-L1細胞の培養は、DMEM に10% FCS、1% P/Sおよび0.5 mM 1-メチル-3-イソブチルキサンチン、1 M デキサメタゾンおよび5 g/mL インスリンを添加したものを用いた。48時間培養後、維持培地として、DMEM に10% FCS、1% P/Sを添加したものを用いた。 3T3-L1 cells were cultured in DMEM supplemented with 10% FCS, 1% P / S and 0.5 mM mM 1-methyl-3-isobutylxanthine, 1M dexamethasone and 5 mg / mL insulin. After culturing for 48 hours, the maintenance medium used was DMEM supplemented with 10% FCS and 1% P / S.
（実施例３）脂肪細胞に及ぼすヒトまたはマウスS100A8の影響
　マウス線維芽細胞由来株化脂肪細胞（3T3-L1）をヒトまたはマウスの遺伝子組換えS100A8（Giotto Biotech社）10μg/mL、および内在性のLPSの効果を抑えるための25μg/mLのポリミキシンB（PolyB）を含む培地で培養したとき、抗S100A8抗体（A8）および非特異的抗体（IgG）を加えたときのCCL2/MCP-1、CCL3、SAA3、CXCL1およびCXCL5の発現に及ぼす影響を確認した（n=4）。この結果、抗S100A8抗体を添加することにより、遺伝子組み換えS100A8タンパク質添加によるMCP-1やCXCL1の発現亢進を抑制することができた（図１１）。これより、この抗S100A8抗体がマウスおよびヒトのS100A8の機能を中和することが分かった。 (Example 3) Effect of human or mouse S100A8 on adipocytes Mouse fibroblast-derived established adipocytes (3T3-L1) were transformed into human or mouse genetically modified S100A8 (Giotto Biotech) 10 μg / mL, and endogenous. CCL2 / MCP-1 when anti-S100A8 antibody (A8) and non-specific antibody (IgG) are added when cultured in medium containing 25 μg / mL polymyxin B (PolyB) to suppress the effect of LPS The effect on the expression of CCL3, SAA3, CXCL1 and CXCL5 was confirmed (n = 4). As a result, by adding the anti-S100A8 antibody, it was possible to suppress the increased expression of MCP-1 and CXCL1 due to the addition of the recombinant S100A8 protein (FIG. 11). From this, it was found that this anti-S100A8 antibody neutralizes the function of mouse and human S100A8.
（実施例４）S100A8抗体投与後の免疫細胞動態変化について
　HF/HS食を摂食後5日目のLysMEGFPマウスについて、脂肪細胞組織をイメージングした。イメージング前に脂肪組織のためにBODIPY染色し、血管壁のために非標識 Q-dotsを静脈投与した。骨髄単球性免疫細胞であるLysMEGFP陽性細胞は緑色染色された。脂肪細胞に抗S100A8抗体、または対照としてのIgG isotypeを投与した後、LysMEGFP陽性細胞の動態を観察した。細胞動態は、IMARISソフトウェア（Bitplane社）を使用し、それぞれ0～30分、30～60分、60～90分、90～120分、120～150分、各30分間隔の5つのセクションに分割してLysMEGFP陽性細胞を定量化して測定した（n=4）。抗S100A8抗体の投与により、細胞動態の抑制が認められた（図１２）。この結果より、脂肪慢性炎症疾患発症のトリガー因子であるS100A8を抑制することで、炎症に関連する細胞の動態を抑制することができ、脂肪慢性炎症疾患の発症を抑制できると考えられた。 (Example 4) Changes in immune cell dynamics after administration of S100A8 antibody An adipocyte tissue was imaged on a LysM EGFP mouse on the fifth day after feeding the HF / HS diet. Prior to imaging, BODIPY staining was performed for adipose tissue and unlabeled Q-dots was administered intravenously for the vessel wall. LysM EGFP positive cells, which are myelomonocytic immune cells, were stained green. After anti-S100A8 antibody or IgG isotype as a control was administered to adipocytes, the kinetics of LysM EGFP positive cells was observed. Cell dynamics are divided into 5 sections, each using 0 to 30 minutes, 30 to 60 minutes, 60 to 90 minutes, 90 to 120 minutes, 120 to 150 minutes, and 30 minute intervals using IMARIS software (Bitplane). Then, LysM EGFP positive cells were quantified and measured (n = 4). Suppression of cell dynamics was observed by administration of anti-S100A8 antibody (FIG. 12). From these results, it was considered that by suppressing S100A8, which is a trigger factor for the onset of fat chronic inflammatory disease, cell dynamics related to inflammation can be suppressed, and onset of chronic fat inflammatory disease can be suppressed.
　以上詳述したように、S100A8は脂肪慢性炎症疾患発症のトリガー因子であることが確認された。S100A8発現量を測定することにより、脂肪慢性炎症疾患発症の予測、あるいは脂肪慢性炎症の進行度を測定するための検査を行うことができる。S100A8は、特に脂肪細胞肥大などの形態的変化や、他の炎症性サイトカイン、ケモカインの発現、例えばS100A9に比べて初期の段階で発現が認められる。そこで、ある程度のS100A8発現量を認めた時点で、早期に対処することで、例えば糖尿病・高脂血症・動脈硬化等の脂肪慢性炎症疾患への進行を抑制することができる。S100A8を抑制する因子、例えば抗S100A8抗体の投与により、免疫細胞の遊走を抑制し、脂肪慢性炎症の発症を抑制することができる。 As detailed above, it was confirmed that S100A8 is a trigger factor for the development of chronic fat inflammatory disease. By measuring the expression level of S100A8, it is possible to conduct a test for predicting the onset of chronic fat inflammatory disease or measuring the progression of fat chronic inflammation. In particular, morphological changes such as adipocyte hypertrophy and the expression of other inflammatory cytokines and chemokines such as S100A8 are observed at an early stage compared to S100A9. Thus, when a certain amount of S100A8 expression is recognized, the progression to fat chronic inflammatory diseases such as diabetes, hyperlipidemia, arteriosclerosis, etc. can be suppressed by dealing early. Administration of a factor that suppresses S100A8, for example, an anti-S100A8 antibody, can suppress immune cell migration and suppress the onset of chronic fatty inflammation.

Claims (11)

  1. S100A8からなる脂肪慢性炎症疾患発症のトリガー因子。 Trigger factor for the development of chronic fat inflammatory disease comprising S100A8.
  2. トリガー因子が、脂肪組織内のマクロファージ遊走能刺激因子である、請求項１に記載の脂肪慢性炎症疾患発症のトリガー因子。 The trigger factor for the onset of chronic fatty inflammatory disease according to claim 1, wherein the trigger factor is a macrophage migration ability stimulating factor in adipose tissue.
  3. トリガー因子が、炎症性サイトカインおよび／またはケモカインの発現誘導因子である、請求項１に記載の脂肪慢性炎症疾患発症のトリガー因子。 The trigger factor for the onset of chronic fatty inflammatory disease according to claim 1, wherein the trigger factor is an expression inducer of inflammatory cytokine and / or chemokine.
  4. 請求項１～３のいずれかに記載の脂肪慢性炎症疾患発症のトリガー因子を標的とする物質を有効成分として含む脂肪慢性炎症疾患発症抑制剤。 An agent for inhibiting the onset of chronic fat inflammatory disease comprising as an active ingredient a substance that targets the trigger factor for the onset of fat chronic inflammatory disease according to any one of claims 1 to 3.
  5. 脂肪慢性炎症疾患発症のトリガー因子を標的とする物質が、抗S100A8抗体である、請求項４に記載の脂肪慢性炎症疾患発症抑制剤。 The agent for inhibiting the onset of chronic fat inflammatory disease according to claim 4, wherein the substance targeting the trigger factor for the onset of chronic fat inflammatory disease is an anti-S100A8 antibody.
  6. 脂肪慢性炎症疾患発症のトリガー因子を標的とする物質が、S100A8の発現を抑制する核酸である、請求項４に記載の脂肪慢性炎症疾患発症抑制剤。 The agent for inhibiting the onset of chronic fat inflammatory disease according to claim 4, wherein the substance targeting the trigger factor for the onset of fat chronic inflammatory disease is a nucleic acid that suppresses the expression of S100A8.
  7. S100A8の発現を抑制する核酸が、S100A8遺伝子のsiRNAである、請求項６に記載の脂肪慢性炎症疾患発症抑制剤。 The agent for suppressing the onset of chronic fatty inflammatory disease according to claim 6, wherein the nucleic acid that suppresses the expression of S100A8 is a siRNA of the S100A8 gene.
  8. 検体におけるS100A8またはS100A8遺伝子の発現量を測定することを特徴とする、脂肪慢性炎症疾患発症の予測、あるいは脂肪慢性炎症の進行度を測定するための検査方法。 A test method for predicting the onset of fat chronic inflammatory disease or measuring the degree of progression of fat chronic inflammation, characterized by measuring the expression level of S100A8 or S100A8 gene in a specimen.
  9. 検体におけるS100A8またはS100A8遺伝子の発現量の測定に加えて、更にS100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一種以上のタンパク質または遺伝子の発現量を測定することを特徴とする、請求項８に記載の検査方法。 9. In addition to the measurement of the expression level of S100A8 or S100A8 gene in a specimen, the expression level of at least one protein or gene of S100A9, inflammatory cytokine and / or chemokine is further measured. Inspection method described in 1.
  10. 炎症性サイトカインおよび／またはケモカインが、CCL2/MCP-1、CCL3、IL-1α、TNF-α、SAA3、CXCL1、CXCL5およびHMGB1から選択される少なくとも一種である、請求項１０に記載の検査方法。 The test method according to claim 10, wherein the inflammatory cytokine and / or chemokine is at least one selected from CCL2 / MCP-1, CCL3, IL-1α, TNF-α, SAA3, CXCL1, CXCL5 and HMGB1.
  11. S100A8またはS100A8遺伝子の発現量が健常人のレベルを超えて発現し、S100A9、炎症性サイトカインおよび／またはケモカインのうち、少なくとも一つ以上のタンパク質または遺伝子の発現量が健常人のレベルであることを確認することを特徴とする、請求項９に記載の検査方法。 The expression level of S100A8 or S100A8 gene exceeds the level of healthy people, and the expression level of at least one protein or gene of S100A9, inflammatory cytokine and / or chemokine is at the level of healthy people The inspection method according to claim 9, wherein confirmation is performed.
PCT/JP2015/067248 2014-06-16 2015-06-16 Trigger factor of chronic fatty inflammatory disease WO2015194525A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2016529350A JP6558645B2 (en) 2014-06-16 2015-06-16 Trigger factors for fat chronic inflammatory disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014-123617 2014-06-16
JP2014123617 2014-06-16

Publications (1)

Publication Number Publication Date
WO2015194525A1 true WO2015194525A1 (en) 2015-12-23

Family

ID=54935510

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/067248 WO2015194525A1 (en) 2014-06-16 2015-06-16 Trigger factor of chronic fatty inflammatory disease

Country Status (2)

Country Link
JP (1) JP6558645B2 (en)
WO (1) WO2015194525A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011518126A (en) * 2008-03-25 2011-06-23 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン Methods for treating and screening IKKi inhibitors, and related IKKi diagnostic methods
JP2012010600A (en) * 2010-06-29 2012-01-19 Univ Of Tokushima Cultured fat cell

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011518126A (en) * 2008-03-25 2011-06-23 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン Methods for treating and screening IKKi inhibitors, and related IKKi diagnostic methods
JP2012010600A (en) * 2010-06-29 2012-01-19 Univ Of Tokushima Cultured fat cell

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ERIKO HASUMI ET AL.: "S100A8 wa Shibo Soshiki Ensho no Aratana Mediator de aru", JOURNAL OF JAPAN SOCIETY FOR THE STUDY OF OBESITY, vol. 16, 2010, pages 149 *
GUNASEKARAN MK ET AL.: "Inflammation triggers high mobility group box 1 (HMGB1) secretion in adipose tissue, a potential link to obesity", CYTOKINE, vol. 64, 9 August 2013 (2013-08-09), pages 103 - 111, XP055244942, Retrieved from the Internet <URL:http://www.sciencedirect.com/science/article/pii/S1043466613006327> *
GUZIK TJ ET AL.: "Adipocytokines - novel link between inflammation and vascular function?", JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY, vol. 57, 2006, pages 505 - 528, XP055244935 *
HASUMI E ET AL.: "S 100A8 and S 100A9 are Novel Proinflammatory Signal Mediators in Metabolic Syndrome", CIRCULATION JOURNAL, vol. 74, no. Supplement I, 2010, pages 552 *
REIGSTAD CS ET AL.: "Regulation of serum amyloid A3 (SAA3) in mouse colonic epithelium and adipose tissue by the intestinal microbiota", PLOS ONE, vol. 4, 9 June 2009 (2009-06-09), pages e5842, XP055244938, Retrieved from the Internet <URL:http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005842> *
SEKIMOTO R ET AL.: "Visualized macrophage dynamics and significance of S 100A8 in obese fat", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 112, 6 April 2015 (2015-04-06), pages E2058 - E2066, XP055244925, Retrieved from the Internet <URL:http://www.pnas.org/content/ 112/16/E2058.long> *
TOURNIAIRE F ET AL.: "Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-KB", PLOS ONE, vol. 8, 18 June 2013 (2013-06-18), pages e66515, XP055244940, Retrieved from the Internet <URL:http://www.plosone.org/article/metrics/info:doi/10.1371/journal.pone.0066515> *

Also Published As

Publication number Publication date
JP6558645B2 (en) 2019-08-14
JPWO2015194525A1 (en) 2017-06-22

Similar Documents

Publication Publication Date Title
Urban et al. The role of fat and inflammation in the pathogenesis and management of osteoarthritis
Kalfon et al. ATF3 expression in cardiomyocytes preserves homeostasis in the heart and controls peripheral glucose tolerance
Filippatos et al. Visfatin/PBEF and atherosclerosis-related diseases
Abdollahi et al. Dapagliflozin exerts anti-inflammatory effects via inhibition of LPS-induced TLR-4 overexpression and NF-κB activation in human endothelial cells and differentiated macrophages
Calvani et al. Th1 cytokines in the pathogenesis of lupus nephritis: the role of IL-18
Adya et al. Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis
Saubermann et al. Peroxisome proliferator-activated receptor gamma agonist ligands stimulate a Th2 cytokine response and prevent acute colitis
Dakin et al. Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair
Johnson et al. The primary function of gp130 signaling in osteoblasts is to maintain bone formation and strength, rather than promote osteoclast formation
Wang et al. Nrf2 protects against diabetic dysfunction of endothelial progenitor cells via regulating cell senescence
White et al. Differential roles of cardiac and leukocyte derived macrophage migration inhibitory factor in inflammatory responses and cardiac remodelling post myocardial infarction
Cai et al. Monocyte chemotactic protein 1 promotes lung cancer–induced bone resorptive lesions in vivo
US10426757B2 (en) Compositions and methods for promoting intestinal stem cell and/or non-stem progenitor cell function
Zarrouk-Mahjoub et al. Pro-and anti-inflammatory cytokines in post-infarction left ventricular remodeling
Nenicu et al. Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor-γ by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions
Traer et al. Ponatinib overcomes FGF2-mediated resistance in CML patients without kinase domain mutations
Swee et al. Matrix metalloproteinase-7 (matrilysin) controls neutrophil egress by generating chemokine gradients
Banskota et al. Salicylates ameliorate intestinal inflammation by activating macrophage AMPK
Tomcik et al. Interleukin-35 is upregulated in systemic sclerosis and its serum levels are associated with early disease
Yu et al. Interleukin‑21 promotes the development of ulcerative colitis and regulates the proliferation and secretion of follicular T helper cells in the colitides microenvironment
Salah et al. MCP-1 promotes detrimental cardiac physiology, pulmonary edema, and death in the cpk model of polycystic kidney disease
Takimoto et al. Down-regulation of inflammatory mediator synthesis and infiltration of inflammatory cells by MMP-3 in experimentally induced rat pulpitis
Li et al. Sectm1a deficiency aggravates inflammation-triggered cardiac dysfunction through disruption of LXRα signalling in macrophages
Yalcinkaya et al. Cholesterol accumulation in macrophages drives NETosis in atherosclerotic plaques via IL-1β secretion
Nogueira et al. Resistin is increased in periodontal cells and tissues: in vitro and in vivo studies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15809935

Country of ref document: EP

Kind code of ref document: A1

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
ENP Entry into the national phase

Ref document number: 2016529350

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15809935

Country of ref document: EP

Kind code of ref document: A1