Regulatory t cell induction promoter, therapeutic or prophylactic agent for autoimmune disease, therapeutic or prophylactic agent for allergic disease, food composition for preventing autoimmune disease, food composition for preventing allergic disease, food composition for improving allergic state, food composition for maintaining healthy immune function, method for treating or preventing autoimmune disease or allergic disease, and method for screening for substance capable of promoting induction of regulatory t cell

Abstract

The present invention addresses the problem of providing a regulatory T cell induction promoter containing D-sorbose and / or D-allose as an active ingredient.　The problem is solved by providing a regulatory T cell induction promoter containing D-sorbose and / or D-allose as an active ingredient.

Description

Agents for promoting induction of regulatory T cells, agents for treating or preventing autoimmune diseases, agents for treating or preventing allergic diseases, food compositions for preventing autoimmune diseases, food compositions for preventing allergic diseases, food compositions for improving allergic conditions, food compositions for maintaining healthy immune function, methods for treating or preventing autoimmune diseases or allergic diseases, and methods for screening for substances that promote induction of regulatory T cells.

[0001] The disclosure in the present application relates to an agent for promoting the induction of regulatory T cells, an agent for treating or preventing an autoimmune disease, an agent for treating or preventing an allergic disease, a food composition for preventing an autoimmune disease, a food composition for preventing an allergic disease, a food composition for improving an allergic condition, a food composition for maintaining healthy immune function, a method for treating or preventing an autoimmune disease or an allergic disease, and a method for screening for a substance that promotes the induction of regulatory T cells.

[0002] T cells are a type of white blood cell called lymphocytes that circulate in the blood, and are an important cell group that can be considered the command center of the immune response, a mechanism that protects the body from foreign substances. The immune response is important for eliminating harmful pathogens, but because it involves an inflammatory response, if it is not terminated in a timely manner, it will attack the body's own body. For this reason, the immune system is equipped with various mechanisms to suppress and control the immune response.

[0003] For example, at the end of an immune response, most antigen-specific immune cells undergo apoptosis, a type of programmed cell death, and regulatory T cells (also known as Tregs) are activated to suppress immune responses. Regulatory T cells also play an important role in maintaining immune cells that recognize the body's own proteins (self-antigens) in an inactive state (immune tolerance). T cell differentiation and function are controlled by intracellular energy metabolism. Glycolysis, in particular, is important for the fate selection of effector T cells or regulatory T cells.

[0004] Monosaccharides, the smallest functional unit of sugar, exist in nature in various forms, such as sugar chains, glycoproteins, and nucleic acids, and are fundamental to life activities. Rare sugars, on the other hand, are defined as monosaccharides and their derivatives that are found in small amounts in nature. While there are approximately 50 types, they are thought to account for only about 0.1% of the total sugars in nature. These rare sugars have a common sweetness, but because they are difficult to use as an energy source, they have been called "leftover sugars."

[0005] However, in recent years, reports have begun to suggest that monosaccharides, including rare sugars, have physiological effects. For example, it has been reported that the monosaccharide D-mannose suppresses autoimmune diseases (see Non-Patent Document 1) through the secretion of inhibitory cytokines such as IL-10 and TGF-β, and through the induction of regulatory T cells, which are known to suppress immune responses by inhibiting the maturation of dendritic cells through cell-cell interactions mediated by CTLA-4.

[0006] Sakaguchi S. et al. , “Regulatory T cells and immunity tolerance”, Cell. 2008;133:775-787. Zhang D. et al. , “D-mannose induces regulation T cells and suppresses immunopathology”, Nat Med. 2017;23:1036-1045.

[0007] As described above, it has been reported that D-mannose suppresses autoimmune diseases through the induction of regulatory T cells. However, there are various mechanisms by which substances administered to the body affect cells. Therefore, it is desirable to explore substances other than D-mannose that induce regulatory T cells.

[0008] The present invention has been disclosed to solve the above-mentioned problems. As a result of extensive research, the present inventors have found that the rare sugars D-sorbose and D-allose promote the induction of regulatory T cells.

[0009] That is, an object of the disclosure of the present application is to provide an agent for promoting the induction of regulatory T cells, which contains D-sorbose and / or D-allose as an active ingredient. Also, an object of the disclosure of the present application is to provide an agent for treating or preventing autoimmune diseases, an agent for treating or preventing allergic diseases, a food composition for preventing autoimmune diseases, a food composition for preventing allergic diseases, a food composition for improving allergic conditions, a food composition for maintaining healthy immune function, a method for treating or preventing autoimmune diseases or allergic diseases, and a method for screening for substances that promote the induction of regulatory T cells.

[0010] (1) An agent for promoting the induction of regulatory T cells, comprising D-sorbose and / or D-allose as an active ingredient. (2) An agent for promoting the induction of regulatory T cells according to (1) above, which contains only D-sorbose among the active ingredients. (3) An agent for promoting the induction of regulatory T cells according to (1) above, which contains only D-allose among the active ingredients. (4) An agent for promoting the induction of regulatory T cells according to (1) above, wherein the D-sorbose is a D-sorbose derivative, and the D-allose is a D-allose derivative. (5) An agent for promoting the induction of regulatory T cells according to any one of (1) to (4) above, which is used for the treatment or prevention of autoimmune diseases or allergic diseases via the induction of regulatory T cells. (6) An agent for promoting the induction of regulatory T cells according to any one of (1) to (4) above, wherein regulatory T cells are induced by inhibition of the hexamine pathway, which is an intracellular sugar metabolic pathway. (7) The agent for promoting the induction of regulatory T cells according to any one of (1) to (4) above, which is used as an ingredient of a pharmaceutical composition. (8) The agent for promoting the induction of regulatory T cells according to any one of (1) to (4) above, which is used as an ingredient of a food composition. (9) An agent for treating or preventing an autoimmune disease, containing at least one selected from the group consisting of D-sorbose, a D-sorbose derivative, D-allose, and a D-allose derivative as an active ingredient. (10) The agent for treating or preventing an autoimmune disease according to (9) above, wherein the autoimmune disease is type 1 diabetes. (11) An agent for treating or preventing an allergic disease, containing at least one selected from the group consisting of D-sorbose, a D-sorbose derivative, D-allose, and a D-allose derivative as an active ingredient. (12) A food composition for preventing an autoimmune disease, containing at least one selected from the group consisting of D-sorbose, a D-sorbose derivative, D-allose, and a D-allose derivative as an active ingredient. (13) A food composition for preventing allergic diseases, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives.(14) A food composition for improving an allergic condition, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose, and D-allose derivatives. (15) A food composition for maintaining healthy immune function, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose, and D-allose derivatives. (16) A method for treating or preventing an autoimmune disease or an allergic disease, comprising the step of administering to an animal (excluding humans) an effective amount of at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose, and D-allose derivatives, which has the effect of promoting the induction of regulatory T cells. (17) A screening method for a substance that promotes the induction of regulatory T cells, comprising: (a) a step of contacting a test substance with regulatory T cells; (b) a step of measuring the induction rate of regulatory T cells contacted with the test substance and comparing it with the induction rate of regulatory T cells in a control group that is not contacted with the test substance; and (c) a step of selecting a test substance that promotes the induction of regulatory T cells based on the comparison result of (b).

[0011] The induction of regulatory T cells is promoted by the agent for promoting the induction of regulatory T cells disclosed in the present application, which contains D-sorbose and / or D-allose as an active ingredient. D-Sorbose and / or D-allose can also be used in therapeutic or preventive agents for autoimmune diseases, therapeutic or preventive agents for allergic diseases, food compositions for preventing autoimmune diseases, food compositions for preventing allergic diseases, food compositions for improving allergic conditions, food compositions for maintaining healthy immune function, and methods for treating or preventing autoimmune diseases or allergic diseases. Furthermore, the method for screening for substances that promote the induction of regulatory T cells disclosed in the present application is expected to lead to the discovery of substances other than D-sorbose and / or D-allose that promote the induction of regulatory T cells.

[0012] FIG. 1 shows the results of ex vivo evaluation of the Treg induction ability of various rare sugars. FIG. 2 shows the results of examining the biological effects of long-term, continuous oral administration of D-sorbose. FIG. 3 shows the results of evaluating the Treg induction ability of D-sorbose using C57BL / 6J mice in a steady state. FIG. 4 shows the results of recovering splenocytes from mice previously administered D-sorbose and stimulating them with PMA / ionomycin to mimic an immune-activated state. FIG. 5 shows the in vivo evaluation of Treg induction ability and its effects using NOD / ShiJcl mice, a spontaneous autoimmune diabetes model. FIG. 6 shows that D-sorbose prevents the onset of autoimmune diabetes. FIG. 7 shows an analysis of the proportion of Tregs in various tissues to immunologically confirm the diabetes prevention effect of D-sorbose. FIG. 8 shows the analysis of the proportion of Tregs in various tissues to immunologically confirm the preventive effect of D-sorbose on the onset of diabetes. FIG. 9 shows the results of evaluating the difference in sensitivity to D-sorbose between Tregs and other T cells using cell viability. FIG. 10 shows that D-sorbose induces Tregs by inhibiting intracellular glucose metabolism. FIG. 11 shows the results of evaluating the Treg induction ability using an inhibitor of an enzyme that catalyzes the metabolic pathway branching off from glucose-6-phosphate in glucose metabolism. FIG. 12 shows the results of examining the biological effects of long-term oral administration of D-allose. FIG. 13 shows the analysis of the proportion of Tregs in medial lymph nodes to confirm that D-sorbose prevents the onset of OVA-induced allergic bronchitis. FIG. 14 shows the results of evaluating the lung to confirm that D-sorbose prevents the onset of OVA-induced allergic bronchitis.

[0013] The regulatory T cell induction promoter (hereinafter, simply referred to as "induction promoter") disclosed in the present application will be described in detail below. Note that in this specification, numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the technical field.

[0014] (Embodiments of Agent for Promoting Induction of Regulatory T Cells) The agent for promoting induction of regulatory T cells contains D-sorbose and / or D-allose as an active ingredient.

[0015] D-sorbose is an epimer at the C4 position of D-fructose, and is industrially produced from galactitol, which is produced by reducing the natural monosaccharide D-galactose, via D-tagatose using D-tagatose isomerase. The structural formula of D-sorbose is shown in formula (1).

[0016] Although there are few reports on the physiological effects of D-sorbose, it has been reported that it inhibits sucrase activity in the small intestine and suppresses blood glucose elevation and insulin secretion (Oku T. et al., "D-sorbose inhibits disaccharidase activity and demonstrates suppressive action on postprandial blood levels of glucose and insulin in the rat", Nutr Res. 2014; 34: 961-967).

[0017] D-sorbose is approximately 70% as sweet as sucrose (common sugar). When orally ingested, 25% of D-sorbose is absorbed in the intestinal tract, 24% is fermented by intestinal bacteria, and the remaining 51% is excreted in the feces. However, it has been reported that the 25% absorbed in the intestinal tract is also excreted in urine (Kitagawa Machiko et al., "Evaluation of Absorption and Fermentation Utilization of D-Mannose, D-Sorbose, and D-Allose in Healthy Adults," Luminacoid Research: Journal of the Japanese Society of Dietary Fiber. 2018; 22: 75-82). Therefore, it is thought that D-sorbose is not utilized as an energy source.

[0018] D-allose is an epimer at the 3-position of glucose. The structural formula of D-allose is shown in formula (2).

[0019] D-allose is known to suppress increased glucose metabolism in tumor cells by increasing thioredoxin interacting protein (TXNIP) and negatively regulating the expression of glucose transporter 1 (GLUT1) (Yamaguchi F. et al., "Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1", Int J Oncol. 2008;32:377-385).

[0020] Commercially available D-sorbose and D-allose can be used.

[0021] D-sorbose and D-allose may be derivatives. In this specification, "D-sorbose derivative" means a substance that can be metabolized in vivo to liberate "D-sorbose." Furthermore, "D-allose derivative" means a substance that can be metabolized in vivo to liberate "D-allose." "D-sorbose derivatives" and "D-allose derivatives" are metabolized in vivo to become "D-sorbose" and "D-allose," respectively. Therefore, although the "D-sorbose derivatives" and "D-allose derivatives" contained in the induction promoter are different substances from "D-sorbose" and "D-allose," respectively, they exert the same effects as "D-sorbose" and "D-allose" after being administered to a living body.

[0022] There are no particular limitations on the D-sorbose derivatives as long as they can be metabolized in vivo to liberate D-sorbose, and there are no particular limitations on the D-allose derivatives as long as they can be metabolized in vivo to liberate D-allose.

[0023] Non-limiting examples of D-sorbose derivatives and D-allose derivatives include the following: (i) a polymer in which two or more, preferably two to six, and more preferably two to four, D-sorbose units are polymerized, or a polymer in which two or more, preferably two to six, and more preferably two to four, D-allose units are polymerized. (ii) a sugar adduct in which one or more, preferably one to six, and more preferably one to four, glucose units are polymerized to D-sorbose, or a sugar adduct in which one or more, preferably one to six, and more preferably one to four, glucose units are polymerized to D-allose.

[0024] The polymer (i) in which two or more D-sorbose units are polymerized or the polymer in which two or more D-allose units are polymerized may contain other components as long as the structural unit contains D-sorbose or D-allose as the main component, respectively, but a polymer containing only D-sorbose or only D-allose as the structural unit is more preferred.

[0025] Specific examples of the D-sorbose sugar adducts of (ii) include monoglucosyl-α-D-sorbose, α-maltosyl-α-D-sorbose, α-maltotriosyl-α-D-sorbose, and α-maltotetraosyl-α-D-sorbose. Specific examples of the D-allose sugar adducts of (ii) include monoglucosyl-α-D-allose, α-maltosyl-α-D-allose, α-maltotriosyl-α-D-allose, and α-maltotetraosyl-α-D-allose.

[0026] In the following description, except in the Examples, unless otherwise specified, the term "D-sorbose" means "D-sorbose" and "D-sorbose derivatives." Similarly, unless otherwise specified, the term "D-allose" means "D-allose" and "D-allose derivatives." In the Examples, "D-sorbose" and "D-allose" refer to the monosaccharides D-sorbose and D-allose, respectively.

[0027] Dosage forms of the induction promoter according to the embodiments include, for example, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, soft and hard gelatin capsules, suppositories, eye drops, nasal drops, patches, sterile injectable solutions, and sterile sealed powders.

[0028] The induction promoter according to the embodiment may also contain a carrier, excipient, or diluent. Examples include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, phosphate-buffered saline (PBS), syrup, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate, and mineral oil. Furthermore, lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners, or flavoring agents may also be added. Furthermore, when the induction promoter according to the embodiment is administered by injection, it may be combined with known drugs such as water-soluble hyaluronic acid, steroids, anesthetics, and anti-inflammatory agents, depending on the injection site.

[0029] Rare sugars are a field that has not been extensively studied to date. The dosage of the induction promoter according to the embodiment (the dosage of D-sorbose and / or D-allose) can be determined appropriately through safety experiments using animals. Examples of the lower limit of D-sorbose per kg / day include, but are not limited to, 1 mg or more, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 6 mg or more, 7 mg or more, 8 mg or more, 9 mg or more, and 10 mg or more. Examples of the upper limit of D-sorbose per kg / day include 50 mg or less, 45 mg or less, 40 mg or less, 35 mg or less, 30 mg or less, 25 mg or less, 20 mg or less, and 15 mg or less. Furthermore, by combining any of the exemplified numerical values, the dosage may be expressed within a predetermined range, for example, 1 mg to 50 mg / kg / day, 3 mg to 40 mg / kg / day, 5 mg to 30 mg / kg / day, 7 mg to 20 mg / kg / day, etc.

[0030] Examples of the lower limit of D-allose per kg / day include 3 mg or more, 4 mg or more, 5 mg or more, 6 mg or more, 7 mg or more, 8 mg or more, 9 mg or more, 10 mg or more, 11 mg or more, and 12 mg or more. Examples of the upper limit of D-allose per kg / day include 50 mg or less, 45 mg or less, 40 mg or less, 35 mg or less, 30 mg or less, 25 mg or less, 20 mg or less, and 15 mg or less. Furthermore, by combining any of the exemplified numerical values, the dosage may be expressed within a predetermined range, such as 3 mg to 50 mg / kg / day, 5 mg to 40 mg / kg / day, 7 mg to 30 mg / kg / day, or 10 mg to 20 mg / kg / day.

[0031] In the above dosages, the dosages of "D-sorbose derivatives" and "D-allose derivatives" refer to the amounts converted into the amounts of D-sorbose and D-allose moieties in the respective derivatives.

[0032] As shown in the Examples below, D-sorbose prevented the onset of autoimmune diabetes through the induction of differentiation into Treg. Furthermore, D-sorbose inhibits intracellular glucose metabolism, and it was shown that inhibition of this metabolic pathway with existing inhibitors also induced differentiation into Treg, similar to D-sorbose.

[0033] Autoimmune diseases and allergic diseases caused by excessive activation of the immune system in the body are increasing year by year. However, treatment for these diseases mainly involves symptomatic treatment with immunosuppressants and antihistamines, and many cases show resistance to these treatments, so the establishment of new treatment methods is desired. Therefore, the induction promoter disclosed in the present application induces Tregs, which are an immune tolerance mechanism inherent in the body, and can therefore be used as a new treatment for these hyperimmune diseases.

[0034] Examples of autoimmune diseases that can be treated or prevented by using the induction promoter disclosed in the present application include rheumatoid arthritis (anti-CCP antibody, MMP-3, rheumatoid factor), systemic lupus erythematosus (anti-ds-DNA antibody, anti-Sm antibody, antinuclear antibody, etc.), systemic sclerosis (anti-centromere antibody, anti-Scl-70 / topoisomerase I antibody, anti-RNA polymerase III antibody, antinuclear antibody, etc.), polymyositis / dermatomyositis (anti-Jo-1 antibody, anti-ARS antibody, etc.), and rheumatoid arthritis (anti-inflammatory drug, anti ... antibodies, etc.), mixed connective tissue disease (anti-U1-RNP antibody), Sjogren's syndrome (anti-SS-A antibody, anti-SS-B antibody, antinuclear antibody, etc.), antiphospholipid syndrome (lupus anticoagulant, anticardiolipin antibody, etc.), microscopic polyangiitis (MPO-ANCA), granulomatosis with polyangiitis (PR3-ANCA), eosinophilic granulomatosis with polyangiitis (MPO-ANCA), and crescentic glomerulonephritis (MPO-ANCA).

[0035] In addition, examples of organ-specific autoimmune diseases include Graves' disease (anti-thyroid receptor antibodies, etc.), Hashimoto's disease (anti-thyroglobulin antibodies, anti-peroxidase antibodies, anti-thyroid microsome antibodies, etc.), autoimmune hepatitis (anti-smooth muscle antibodies, antinuclear antibodies, etc.), primary biliary cholangitis (anti-mitochondrial antibodies, antinuclear antibodies, elevated IgM levels, etc.), autoimmune pancreatitis (anti-nuclear antibodies, elevated IgG levels, etc.), type I diabetes (anti-islet of Langerhans antibodies, etc.), myasthenia gravis (anti-acetylcholine receptor antibodies), Guillain-Barre syndrome (anti-ganglioside antibodies), multiple sclerosis (anti-aquaporin 4 antibodies, etc.), Goodpasture's syndrome (anti-GBM antibodies), autoimmune hemolytic anemia (anti-erythrocyte antibodies, etc.), megaloblastic anemia (anti-intrinsic factor antibodies, etc.), and idiopathic thrombocytopenic purpura (anti-platelet antibodies).

[0036] Other conditions such as primary sclerosing cholangitis, polyarteritis nodosa, Takayasu's arteritis, giant cell arteritis, polymyalgia rheumatica, adult Still's disease, Behçet's disease, sarcoidosis, ulcerative colitis, and Crohn's disease are also thought to be autoimmune diseases, although no specific autoantibodies have been found for these conditions.

[0037] The involvement of Tregs in autoimmune diseases in general is described in detail in Tara Fiyouzi et al., "Enhancing Regulatory T Cells to Treat Inflammatory and Autoimmune Diseases", Int. J. Mol. Sci. 2023, 24(9), 7797; https: / / doi.org / 10.3390 / ijms24097797. Therefore, D-sorbose and / or D-allose, which promote the induction of Tregs, are useful for autoimmune diseases in general.

[0038] Furthermore, allergic diseases that can be treated or prevented using the induction promoter disclosed in the present application include bronchial asthma, atopic dermatitis, allergic rhinitis, hay fever, food allergies, allergic conjunctivitis, and the like.

[0039] The involvement of Tregs in asthma and airway allergies is described in Jing Zhang et al., "Regulatory T Cells, a Viable Target Against Airway Allergic Inflammatory Responses in Asthma," Front. Immunol., 10 June 2022, Sec. T Cell Biology, Volume 13-2022 | https: / / doi.org / 10.3389 / fimmu.2022.902318.

[0040] The involvement of Tregs in food allergies is described in Guirong Liu, et al., "The Role of Regulatory T Cells in Epicutaneous Immunotherapy for Food Allergy", Front. Immunol., 08 July 2021, Sec. T Cell Biology, Volume 12-2021 | https: / / doi.org / 10.3389 / fimmu.2021.660974.

[0041] The involvement of Treg in atopy is described in Masutaka Furue, et al., "Aryl Hydrocarbon Receptor in Atopic Dermatitis and Psoriasis", Int. J. Mol. Sci. 2019, 20(21), 5424; https: / / doi.org / 10.3390 / ijms20215424.

[0042] The involvement of Treg in hay fever is described in Iris Bellinghausen, et al., "Current Strategies to Modulate Regulatory T Cell Activity in Allergic Inflammation", Front. Immunol., 26 May 2022, Sec. Vaccines and Molecular Therapeutics, Volume 13-2022 | https: / / doi.org / 10.3389 / fimmu.2022.912529.

[0043] Therefore, D-sorbose and / or D-allose, which promote the induction of Tregs, are useful for allergic diseases in general.

[0044] The induction promoter disclosed in the present application can induce regulatory T cells in, for example, a local area in an immune-activated state (e.g., a local area where inflammation is occurring), such as the nose, eyes, throat, oral cavity, pancreas, respiratory tract (trachea, lungs, etc.), skin, digestive tract (esophagus, stomach, duodenum, small intestine, large intestine), brain, liver, gallbladder, spleen, kidney, thymus, thyroid, adrenal gland, muscle, lymphatic vessels, blood vessels, heart, testis, uterus, ovaries, prostate, etc.

[0045] The induction promoter according to the embodiment can be used as an ingredient of a pharmaceutical composition or a food composition.

[0046] First, a case where the induction promoter according to the embodiment is used as a component of a food composition will be described. As long as the induction promoter according to the embodiment is included as a component of the food composition, there are no particular limitations on the other components of the food composition.

[0047] The food composition may be in any form, such as a solid, gel, or liquid. Examples include fermented dairy products such as lactic acid bacteria drinks, various processed foods and beverages, dry powders, tablets, capsules, and granules, as well as various beverages, yogurt, liquid foods, jellies, candies, retort pouch foods, tablet confections, cookies, castella cakes, bread, biscuits, and chocolate. The food composition may also contain additives such as sugars, proteins, lipids, vitamins, minerals, flavors, carbohydrates, sweeteners, fragrances, colorants, texture improvers, or mixtures thereof to improve nutritional balance and flavor. The term "food composition" also encompasses drinking water.

[0048] The intake period of the food composition is not particularly limited, but from the viewpoint of prevention or progression inhibition, long-term intake is preferable. Examples of such intake periods include, but are not limited to, one week or more, two weeks or more, three weeks or more, one month or more, three months or more, six months or more, and one year or more. The lower limit of the D-sorbose content (mass%) in the food composition is, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, and 10% or more. Meanwhile, the upper limit of the D-sorbose content (mass%) in the food composition is, for example, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, and 20% or less. Furthermore, by combining any of the exemplified numerical values, the content may be expressed within a predetermined range, such as 1% to 30%, 3% to 25%, or 5% to 20%.

[0049] Examples of lower limits for the D-allose content (% by mass) in a food composition include 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, and 10% or more. On the other hand, examples of upper limits for the D-allose content (% by mass) in a food composition include 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, and 10% or less. Furthermore, by combining any of the above-listed numerical values, the content may be expressed within a predetermined range, such as 1% to 20%, 3% to 15%, or 5% to 10%.

[0050] Next, a case where the induction promoter according to the embodiment is used as an ingredient of a pharmaceutical composition will be described. As long as the induction promoter according to the embodiment is contained as an ingredient of the pharmaceutical composition, there are no particular limitations on the other ingredients of the pharmaceutical composition. The pharmaceutical composition containing the induction promoter according to the embodiment can be provided as a pharmaceutical composition having an efficacy of treating or preventing autoimmune diseases or allergic diseases via the induction of regulatory T cells, or as a pharmaceutical composition with added efficacy.

[0051] The pharmaceutical composition can be administered locally or systemically. The administration route is not particularly limited, and may be either oral or parenteral (e.g., ophthalmic or nasal). In addition, the pharmaceutical composition may contain, in addition to the active ingredient lactoferrin, a pharmacologically acceptable carrier depending on the administration route. Examples of the carrier include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, pigments, diluents, bases, solubilizers or solubilizers, isotonicity agents, pH adjusters, stabilizers, propellants, and adhesives.

[0052] In addition to the induction promoter according to the embodiment, other components contained in the pharmaceutical composition include, for example, immunosuppressants, immune checkpoint inhibitors, allergy treatment drugs, antibody preparations, etc. Examples of immunosuppressants include, but are not limited to, cyclosporine, mizoribine, cyclophosphamide, azathioprine, tacrolimus, mycophenolate mofetil, etc. Furthermore, examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, durvalumab, avelumab, atezolizumab, cemiplimab, ipilimumab, tremelimumab, etc.

[0053] Antiallergy drugs include, but are not limited to, Allegion epinastine 1994, Ebastel ebastine 1996, Zyrtec cetirizine 1998, Talion bepotastine 2000, Allegra fexofenadine 2001, Allelock olopatadine 2001, Claritin loratadine 2002, Xyzal levocetirizine 2010, Bilanoabilastine 2016, Desalex desloratadine 2016, and Rupafin rupatadine 2017.

[0054] Examples of antibody preparations include, but are not limited to, omalizumab, an anti-IgE antibody, mepolizumab, an anti-IL-5 antibody, benralizumab, an anti-IL-5 receptor antibody, dupilumab, an anti-IL-4 / 13 receptor antibody, nemolizumab, an IL-31 receptor antibody, and tezepelumab, an anti-TSLP antibody.

[0055] The dosage of the pharmaceutical composition varies depending on the patient's weight, age, etc., and is not particularly limited. The dosage may be set appropriately by a physician. For example, the dosage may be set so that the amount of induction promoter contained in the pharmaceutical composition is the same as the dosage of the induction promoter described above (the dosage of D-sorbose and / or D-allose), without being limited thereto.

[0056] (Embodiments of the therapeutic or preventive agent for autoimmune diseases and embodiments of the therapeutic or preventive agent for allergic diseases) As described above, the induction promoter disclosed in the present application can be used for the treatment or prevention of autoimmune diseases or allergic diseases through the induction of regulatory T cells. In other words, D-sorbose and / or D-allose can be used as a therapeutic or preventive agent for autoimmune diseases or allergic diseases through the induction of regulatory T cells. In this specification, "treatment" refers to reversing, alleviating, inhibiting, or preventing the progression of autoimmune diseases and allergic diseases, disorders and symptoms caused by such diseases, or one or more signs of such disorders or symptoms. It also includes alleviating symptoms and improving quality of life. "Prevention" refers to suppressing the onset of symptoms.

[0057] The therapeutic or preventive agent for autoimmune diseases according to the embodiments can be obtained by replacing the "agent for inducing regulatory T cells" described in the above "Embodiment of an agent for inducing regulatory T cells" with "agent for treating or preventing autoimmune diseases." Similarly, the therapeutic or preventive agent for allergic diseases according to the embodiments can be obtained by replacing the "agent for inducing regulatory T cells" described in the above "Embodiment of an agent for inducing regulatory T cells" with "agent for treating or preventing allergic diseases." Therefore, to avoid repetition, detailed descriptions of the embodiments of the therapeutic or preventive agent for autoimmune diseases and the embodiments of the therapeutic or preventive agent for allergic diseases will be omitted.

[0058] (Embodiments of Food Compositions for Preventing Autoimmune Diseases, Embodiments of Food Compositions for Preventing Allergic Diseases, Embodiments of Food Compositions for Improving Allergic Conditions, and Embodiments of Food Compositions for Maintaining Healthy Immune Function) As described above, the induction promoters disclosed in the present application can be used for the prevention of autoimmune diseases or allergic diseases through the induction of regulatory T cells. In other words, by adding D-sorbose and / or D-allose to food, they can be used as food compositions for the prevention of autoimmune diseases or allergic diseases. The meaning of "prevention" is the same as that described above in "Embodiments of Agents for the Treatment or Prevention of Autoimmune Diseases and Agents for the Treatment or Prevention of Allergic Diseases."

[0059] The food composition for preventing autoimmune diseases and the food composition for preventing allergic diseases according to the embodiments are the same as the description in the above section "Embodiments of an Agent for Promoting Induction of Regulatory T Cells," which states, "We will explain the case where the induction promoter according to the embodiments is used as an ingredient of a food composition." In other words, the part explaining that the induction promoter is included as an ingredient of the food composition can be read as a food composition for preventing autoimmune diseases or a food composition for preventing allergic diseases that contains D-sorbose and / or D-allose as active ingredients. Therefore, detailed descriptions of the embodiments of the food composition for preventing autoimmune diseases and the embodiments of the food composition for preventing allergic diseases will be omitted.

[0060] The food composition for improving an allergic condition according to the embodiment and the food composition for maintaining a healthy immune function according to the embodiment are the same as the description in the above section "Embodiments of an Agent for Promoting the Induction of Regulatory T Cells," which states, "We will explain the case where the induction promoter according to the embodiment is used as an ingredient of a food composition." In other words, the part explaining that the induction promoter is included as an ingredient of the food composition can be read as a food composition for improving an allergic condition or a food composition for maintaining a healthy immune function that contains D-sorbose and / or D-allose as active ingredients. Therefore, detailed descriptions of the embodiments of the food composition for improving an allergic condition and the embodiments of the food composition for maintaining a healthy immune function will be omitted.

[0061] In this specification, the term "allergic condition" is not particularly limited as long as it refers to a physical disorder caused by allergies, and includes a condition that does not constitute a disease (for example, a condition before becoming a disease). More specific examples of allergic conditions include local discomfort (for example, in the nose, eyes, throat, skin, respiratory system, or digestive tract) due to allergies, itching or roughness, nasal congestion, sneezing, watery eyes, and increased redness of the eyes.

[0062] Furthermore, the food composition according to the present invention can be used to maintain healthy immune function. As used herein, "maintaining healthy immune function" refers to suppressing excessive immune responses (e.g., a state in which the immune system is activated in a local area where inflammation is occurring) to bring the body closer to a healthy state.

[0063] (Embodiments of methods for treating or preventing autoimmune diseases or allergic diseases) Next, methods for treating or preventing autoimmune diseases or allergic diseases (hereinafter, sometimes simply referred to as "therapeutic / preventive methods") according to embodiments will be described.

[0064] The therapeutic and preventive methods include administering to an animal (excluding humans) an effective amount of D-sorbose and / or D-allose, which has the effect of promoting the induction of regulatory T cells. The effective amount is not particularly limited as long as it is an amount that exerts a therapeutic or preventive effect, and may be set, for example, to be the same as the dosage of the induction promoter (dosage of D-sorbose and / or D-allose) described above in (Embodiments of the agent for promoting the induction of regulatory T cells).

[0065] Animals include humans (however, humans are excluded in countries where therapeutic interventions for humans are excluded from patent protection) and other mammals. Examples of mammals include mice, rats, rabbits, hamsters, sheep, monkeys, pigs, cats, dogs, cows, and horses. Furthermore, test organisms that are the subject of contact, ingestion, or administration of a test substance include human or animal individuals, tissues, and cells (including cultured cells). Examples of cells include, but are not limited to, cell lines derived from mammals such as humans, mice, and rats, or cell lines derived from these cell lines, primary cells derived from various tissues, stem cells, ES cells, and iPS cells.

[0066] (Method for screening for a substance that promotes the induction of regulatory T cells) In the examples described below, it was confirmed that D-sorbose and D-allose promote the induction of regulatory T cells, and this experimental system can also be used to screen for a substance that promotes the induction of regulatory T cells. The method for screening for a substance that promotes the induction of regulatory T cells according to the embodiment includes the steps of: (a) contacting a test substance with regulatory T cells; (b) measuring the induction rate of regulatory T cells contacted with the test substance and comparing it with the induction rate of regulatory T cells in a control group not contacted with the test substance; and (c) selecting a test substance that promotes the induction of regulatory T cells based on the comparison results of step (b).

[0067] The test substance is not particularly limited as long as it has the potential to promote the induction of regulatory T cells. Examples of test substances include, but are not limited to, nucleic acids, antibodies, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, and plasma. The test substance may be a novel substance or a known substance. These test substances may form salts, and salts of the test substance with physiologically acceptable acids or bases are used.

[0068] When selecting a test substance, a threshold value for the induction rate of the control group may be determined as necessary, and a substance that shows an induction rate equal to or greater than the threshold value may be selected.

[0069] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application.

[0070] Example 1 The materials and experimental methods used in Example 1 of the present application are described below. (1) Experimental Animals C57BL / 6J or C57BL / 6N wild-type male mice were purchased from Charles River Japan, and NOD / ShiJcl female mice were purchased from CLEA Japan. All mice were acclimatized to the breeding environment for at least one week and used in experiments at 6-11 weeks of age. Mice were housed in a specific pathogen-free environment, with free movement within dedicated cages (3 mice / cage) and free access to food and water. The temperature in the breeding room was maintained at 23±3°C and humidity at 50±10%, with a 12-hour light-dark cycle (lights on at 8:00 AM).

[0071] (2) Reagents Roswell Park Memorial Institute medium (RPMI-1640 medium) was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and supplemented with 10% FBS (HyClone; Cytiva, Tokyo, Japan), 100 U / mL penicillin, and 100 mg / mL streptomycin (Sigma-Aldrich, St. Louis, MO) to prepare complete RPMI medium.

[0072] Of the monosaccharides shown in Table 1 below, D-glucose was purchased from Nacalai Tesque (Kyoto, Japan), and D-mannose, D-allose, D-psicose, D-tagatose, and D-sorbose were provided by Matsutani Chemical Industry Co., Ltd. (Hyogo, Japan).

[0073]

[0074] D-glucose, D-mannose, D-allose, D-psicose, D-tagatose, and D-sorbose were dissolved in complete RPMI medium and adjusted to 500 mM. D-Sorbose for oral administration to mice was dissolved in distilled water to a concentration of 1.1 M or 0.28 M.

[0075] The carbohydrate metabolites, glucose-6-phosphate monosodium, ribose-5-phosphate sodium, and lactate, were purchased from Nacalai Tesque, DL-malate and monopotassium phosphoenolpyruvate from Fujifilm Wako Pure Chemical Industries, DL-glyceraldehyde-3-phosphate from Cayman Chemical (Ann Arbor, MI), and UDP-N-acetyl-D-glucosamine and disodium salt from Calbiotech (El Cajon, CA). All metabolites were dissolved in distilled water and used as standard solutions for intracellular metabolite measurements.

[0076] The glucose metabolism inhibitor KAN0438757 (KAN) was purchased from Selleck Biotech (Tokyo, Japan), 6-Aminonicotinamide (6-AN) was purchased from Cayman Chemical, and 6-Diazo-5-oxo-L-norleucine (DON) was purchased from Fujifilm Wako Pure Chemical Industries.

[0077] The inhibitors were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and then diluted with complete RPMI medium to a final DMSO concentration of 0.033% or less in the cell culture medium.

[0078] (3) Cell isolation from mouse tissues Tissues collected from mice were treated as follows to isolate lymphocytes. The spleen was crushed and then soaked in a pH 7.3 hemolysis buffer (NH ４ Cl: 8.26g, NaHCO ３ : 1.19 g, EDTA-2Na: 37.8 mg in 100 mL Milli-Q water) was added and incubated on ice for 3 minutes to perform hemolysis. The mixture was then filtered through a 70 mm filter to obtain a splenocyte suspension. Peripheral blood was collected in a tube containing ethylenediamine-N,N,N',N'-tetraacetic acid-2Na (EDTA-2Na; 0.5 mg, Dojindo Laboratories, Kumamoto, Japan) and subjected to hemolysis in a pH 7.3 hemolysis buffer in the same manner as for the spleen. Lymph nodes were mashed and then filtered through a 70 mm filter.

[0079] (4) Differentiation culture of mouse Treg CD3 ＋ T cells were isolated from splenocytes of 8-week-old C57BL / 6J male mice by magnetic cell sorting (MACS; Pan T Cell Isolation Kit II mouse, Miltenyi Biotec, Tokyo, Japan). ＋ T cells were seeded on the flat bottom of a 96-well plate (2 × 10 ５ cells / well) and incubated with anti-CD3 / CD28 (beads:cells = 1:5, Dynabeads) in the presence of 25 mM of various monosaccharides. Ｒ Mouse T-Activator CD3 / CD28, Thermo Fisher Scientific, Waltham, MA) and rIL-2 (30 IU / mL, Recombinant Human IL-2, Bio-techne, Minneapolis, MN) at 37°C, 5% CO ２ The cells were cultured under the following conditions. Complete RPMI medium was added to the control group instead of the monosaccharide. The cells were restimulated on day 3 of culture, and the cells collected on day 6 of culture were analyzed by fluorescence-activated cell sorting (FACS).

[0080] (5) Flow Cytometry Analysis (FACS) Foxp3 nuclear staining was performed using a fixation / permeabilization buffer solution (Foxp3 / Transcription Factor Staining Buffer Set, eBioscience; Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Briefly, cells were subjected to FC receptor blocking with anti-CD16 / CD32 antibody (Clone: ​​93, BioLegend, San Diego, CA), and then subjected to dead cell staining with ethidium bromide (Sigma-Aldrich). Cell surface staining was then performed with anti-CD3 antibody (PE, Clone: ​​17A2, BioLegend) and anti-CD4 antibody (FITC, Clone: ​​GK1.5, BioLegend). Cells were then fixed and permeabilized, and intranuclear staining was performed with anti-Foxp3 biotin antibody (Clone: ​​FJL-16s, BioLegend) and streptavidin (APC, BioLegend). CD8 cell surface staining was also performed in the same manner. Briefly, cells were stained for FC receptors with anti-CD16 / CD32 antibodies, stained on the cell surface with anti-CD8a antibodies (FITC, Clone: ​​53-6.7, BioLegend), and then stained for dead cells with 7-AAD Viability Staining Solution (BioLegend). Stained cells were analyzed using FACS-Calibur (BD, Franklin Lakes, NJ) or Galios (Beckman Coulter, Brea, CA). Data were analyzed using FlowJo ver. 9.9.5 or Kaluza ver. 1.1.

[0081] (6a) D-sorbose safety test Six-week-old C57BL / 6J male mice were given 1.1 M or 0.28 M D-sorbose solutions in ad libitum drinking water instead of drinking water. Distilled water was administered to the control group. During the administration period, body weight, water intake, and food intake were measured every 2–3 days, and spleens and peripheral blood samples were collected 4 weeks after the start of administration. Peripheral blood was centrifuged (1400 × g, 24°C, 10 min), and serum was obtained by collecting the supernatant. Serum was stored at −80°C until biochemical analysis.

[0082] (6b) Safety test of D-allose Six-week-old C57BL / 6J male mice were given 0.28 M or 0.56 M D-allose solutions ad libitum instead of drinking water. Distilled water was administered to the control group. During the administration period, body weight, water intake, and food intake were measured every 2-3 days.

[0083] (7) Biochemical analysis of mouse serum components Total protein (TP), alanine aminotransferase (ALT), creatinine (Cre), and glucose (Glu) in the serum were measured using an automatic biochemical analyzer (JCA-BM9130, JEOL, Tokyo, Japan).

[0084] (8) Evaluation of IL-10 production ability Splenocytes were stimulated by the following procedure. Splenocytes isolated from the spleens of C57BL / 6J mice that had been given 0.28 M D-sorbose solution in their drinking water ad libitum for 6 weeks from the age of 6 weeks were seeded (5 × 10) on the flat bottom of a 96-well plate. ５ The cells were cultured at 37°C in 5% CO. The cells were then stimulated with Phorbol 12-myristate 13-acetate (PMA; 50 ng / mL, Sigma-Aldrich) and ionomycin (500 ng / mL, Sigma-Aldrich). ２ The cells were cultured under these conditions. Splenocytes from mice administered distilled water were used as a control group. 72 hours after stimulation, the cell growth morphology was photographed and the culture supernatant was collected. The amount of IL-10 produced in the culture supernatant was measured using a Mouse IL-10 ELISA kit (Life Technologies; Thermo Fisher Scientific).

[0085] (9) Non-obese autoimmune diabetes (NOD) model. The NOD / ShiJcl model is a spontaneous autoimmune diabetes model characterized by hyperglycemia and hyperglycemia due to T cell-dependent insulitis caused by various factors, including negative selection disorders in the thymus due to MHC-II mutations. Onset of the disease begins around 18 weeks of age, with a cumulative incidence of 60% by 35 weeks of age (provided by CLEA Japan). Experimental endpoints were set at 17 weeks of age, representing the early stage of the disease, and 30 weeks of age, representing the end stage of the disease. From 7.5 weeks of age, 1.1 M D-sorbose solution was administered ad libitum in drinking water, while the control group received distilled water. During the administration period, blood samples were taken every 3 days, and weight, water intake, and food intake were measured. Serum was separated from peripheral blood using the procedure described in (6) above, and blood glucose levels were measured by a microplate assay using a glucose kit (GLU Reagent L, Sysmex, Hyogo, Japan). Sustained hyperglycemia (blood glucose levels of 200 mg / dL or higher recorded for six or more consecutive days) was evaluated as the onset of autoimmune diabetes. Peripheral blood was collected into tubes containing EDTA-2Na at 15 weeks of age, and peripheral blood, spleen, pancreas, and pancreatic lymph nodes were collected at 17 and 30 weeks of age.

[0086] (10) Preparation of pathological specimens The pancreas of mice was divided into gastric, splenic, and duodenal lobes, and pancreases were collected from each site. The duodenal lobe, which contains the most islets, was fixed in 10% neutral buffered formalin, wrapped in a 100 μm mesh, and embedded in paraffin.

[0087] (11) Histological Evaluation Multiple 3 mm thick sections were prepared from the duodenal lobe of the mouse pancreas at 50 μm intervals. The prepared sections were stained with hematoxylin and eosin (HE), and lymphocyte infiltration into the pancreatic islets of Langerhans was scored as follows: 0: No lymphocytes observed. 1: Lymphocytes surround the islets but do not infiltrate. 2: Infiltration observed in less than 25% of the islets. 3: Infiltration observed in 25-50% of the islets. 4: Infiltration observed in 50% or more of the islets. 5: Infiltration observed in 100% of the islets, or the islets have disappeared, leaving only remnants. Thirty islets from each individual were scored, and the proportion of each grade was analyzed.

[0088] (12) Immunohistochemical Staining 1-μm-thick paraffin sections were prepared from the duodenal lobe of mouse pancreas and subjected to immunohistochemical staining for Foxp3. The primary antibody used was rabbit anti-Foxp3 antibody (ab215206, Abcam, Cambridge, England). After deparaffinization and dehydration, the sections were heat-treated at 121°C for 20 minutes in Histofine antigen retrieval solution (pH 9.0) (Nichirei Biosciences, Tokyo, Japan). To remove endogenous peroxidase activity, the sections were immersed in a 0.3% hydrogen peroxide-containing methanol solution for 30 minutes. After blocking nonspecific binding with 1% BSA, the sections were incubated with the primary antibody overnight at 4°C. The sections were incubated with a peroxidase polymer-conjugated secondary antibody (ImmPRESS Reagent anti-rabbit IgG, Vector Laboratories, Newark, CA) for 30 minutes at room temperature, followed by a substrate solution of 3,3'-diaminobenzidine tetrahydrochloride (DAB; Dako, Santa Clara, CA). Sections were then counterstained with Mayer's hematoxylin.

[0089] (13) Measurement of cell viability. Cells were isolated from the spleens of 11-week-old C57BL / 6N male mice using MACS (Pan T Cell Isolation Kit II mouse, CD4 ＋ CD25 ＋ Tregs (CD4) were isolated using a Regulatory T Cell Isolation Kit (Miltenyi Biotec). ＋ CD25 ＋ ) and non-Treg (CD4 ＋ CD25 － Each cell was seeded (1 × 10) on the flat bottom of a 96-well plate. ５ The cells were then cultured at 37°C in 5% CO. The cells were then stimulated with anti-CD3 / CD28 (beads:cells = 1:5) and rIL-2 (30 IU / mL) in the presence of 25 mM monosaccharide. ２ The cells were cultured under the conditions described above. After 24 and 72 hours of stimulation, the cells were collected, and dead cells were stained with trypan blue stain (Fujifilm Wako Pure Chemical Industries, Ltd.), and the cell viability was measured.

[0090] (14) Measurement of Intracellular Glucose Metabolites Mouse T lymphoma cells (EL-4 cells) were purchased from the JCRB Cell Bank (NIBIOHN; National Institutes of Biomedical Innovation, Health and Nutrition) and cultured in complete RPMI medium at 37°C and 5% CO ２ The cells were subcultured in suspension under the conditions described above. The cells were collected together with the old medium, and the cell number was counted using trypan blue staining solution. ６ The cells were seeded again and fresh medium was added. After the cell proliferation rate stabilized, they were used for experiments. Metabolic products were extracted from the cells as follows. EL-4 cells were seeded (4 x 10) on the flat bottom of a 96-well plate. ５ cells / well) at 37°C, 5% CO ２ After pre-incubation for 24 hours to stabilize the metabolism, D-sorbose was added to a final concentration of 25 mM, and the cells were cultured at 37°C and 5% CO ２ The cells were cultured again under the same conditions. Complete RPMI medium was added to the control group instead of D-sorbose. Six hours after addition, the cells were harvested and washed three times with PBS (800 × g, 4 °C, 5 min) to completely remove the medium. Distilled water was added to the cell pellet and mixed well, and the cells were disrupted using an ultrasonic homogenizer (VP-050N, Taitec, Aichi, Japan). An equal volume of methanol was added to the distilled water, and the mixture was centrifuged (20,000 × g, 4 °C, 10 min) to collect the supernatant. The supernatant was stored at -80 °C until analysis. 30 μL of 10 μM 2-morpholinoethanesulfonic acid and 200 μL of the supernatant dissolved on ice were mixed, evaporated to dryness under reduced pressure, redissolved in 30 μL of water, and then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS).

[0091] (15) High-Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS) To comprehensively investigate the dynamics of glucose metabolism, a total of seven metabolites (see (2) above) were measured, taking into account various branched metabolic pathways. An LCMS-8060 mass spectrometer (Shimadzu Corporation, Kyoto, Japan) was used with a Nexcera HPLC system. HPLC was performed using an Inert Sustain PFP column (150 mm x 2.1 mm, 1.9 mm, GL Sciences, Tokyo, Japan) at 40 °C, with 0.1% formic acid aqueous solution as the initial mobile phase. Cell extract (1 μL) was injected, and metabolites were separated using a 3-minute mobile phase gradient of 0.1% formic acid in acetonitrile (Fujifilm Wako Pure Chemical Industries) increasing to 25% at a flow rate of 300 μL / min. The total analysis time was 6 min. Data was acquired using LabSolutions software ver. 2.2, and the peak areas of metabolites were determined. The conversion efficiency was calculated from the quantitative values ​​of metabolites and used as the metabolic enzyme activity.

[0092] (16) Induction of Treg differentiation by inhibiting glucose metabolism To examine the effect of intracellular metabolism on Treg differentiation, Treg differentiation culture was performed using glucose metabolism inhibitors. ＋ T cells were isolated from the spleens of 9-week-old C57BL / 6N male mice by MACS. ＋ T cells were seeded on the flat bottom of a 96-well plate (2 × 10 ５ The cells were then cultured at 37°C in 5% CO. The cells were then stimulated with anti-CD3 / CD28 (beads:cells = 1:2) and rIL-2 (30 IU / mL) in the presence of various inhibitors. ２ The cells were cultured under the conditions shown in Table 1. The final concentration of inhibitors was set at approximately 10 times the IC50 of each inhibitor, and KAN (2 μM), 6-AT (5 μM), and DON (2 μM) were used. To the group without inhibitors, complete RPMI medium containing DMSO at the same concentration as the solvent for the inhibitors was added. The cells were restimulated on day 3 of culture, and the cells collected on day 6 of culture were analyzed by FACS.

[0093] (17) Statistical Processing GraphPad Prism software version 6 was used for statistical analysis. All data were expressed as mean ± standard deviation. Unpaired Student's t test was used to test for significant differences between two groups, and one-way ANOVA or two-way ANOVA with Saidak's multiple comparison test or Dunnett's multiple comparison test was used to test for significant differences between three or more groups. P < 0.05 was determined to be statistically significant.

[0094] The results of the experiment in Example 1 carried out using the above-described experimental method will be described below.

[0095] (1) Ex vivo evaluation of the Treg induction ability of various rare sugars Figure 1 shows the results of analysis by flow cytometry. T cells are a type of lymphocyte, and precursor cells produced in the bone marrow are differentiated and matured after selection in the thymus. T cells (CD3 ＋ CD4, CD8, and other proteins that are specifically expressed on the cell surface are used as indicators to classify them into several types. ＋ T cells play a role in supporting immune responses and are also called helper T cells. ＋ T cells have the ability to kill cells infected with viruses and tumor cells, and are called killer T cells. ＋ CD4 ＋ Foxp3 ＋ The cells were defined as Tregs (see Hori S. et al., "Control of regulatory T cell development by the transcription factor Foxp3", Science, 2003; 299: 1057-1061).

[0096] As is clear from Figures 1A and 1B, compared with the D-glucose group, the rare sugars D-sorbose and D-allose groups showed a significantly higher CD4 ＋ Foxp3 on cells ＋The proportion of Tregs showed an increasing trend. It is known that the transcription factor FoxP3 is the master regulator of Tregs. ＋ Foxp3 on cells ＋ A higher proportion of Tregs indicates that differentiation into Tregs is more strongly induced. The results shown in Figures 1A and 1B were comparable to those of the D-mannose group, which is already known to have Treg inducibility. These results indicate that the rare sugars D-sorbose and D-allose, like D-mannose, acted directly on T cells and induced their differentiation into Tregs.

[0097] On the other hand, as is clear from Figures 1C and 1D, the D-mannose group showed a significantly higher CD8 ＋ The number of CD8 T cells was significantly reduced, but no significant changes were observed in the D-sorbose and D-allose groups. ＋ T cells (killer T cells) play an important role in antiviral responses and antitumor activity, and are also the main cells in the hyperimmune response to self-antigens. Therefore, D-mannose is effective in increasing Tregs and CD8 ＋ While depletion of T cells is expected to have a synergistic suppressive effect on excessive immune responses such as autoimmune diseases, long-term administration may lead to vulnerability to viral infection and tumor growth. In other words, D-sorbose and D-allose have the same effect as D-mannose in inducing differentiation into Tregs, but they have a lower effect on CD8 ＋ D-mannose has a different effect from D-mannose in that it does not deplete T cells. By using the system used in this example and administering a test substance instead of D-sorbose and D-allose, it is possible to screen for substances that promote the induction of regulatory T cells.

[0098] (2) Effects of Long-Term Oral Administration of D-sorbose on the Body The safety of D-sorbose, which induced Treg differentiation ex vivo, was evaluated in vivo. The results are shown in Figure 2. As shown in Figure 2A, the mice in the 1.1 M D-sorbose solution group experienced a rapid weight loss within a few days of the start of administration, but gradually recovered thereafter, reaching a level equivalent to that of the distilled water-administered group (control group) by two weeks after the start of administration. During the administration period, no physical findings such as diarrhea were observed in the D-sorbose group (not shown), and although water and food intakes were continuously reduced, no direct cause of weight loss was identified. These decreases in body weight, water intake, and food intake were ameliorated by lowering the D-sorbose administration concentration to 0.28 M.

[0099] Furthermore, as shown in Figure 2B, the D-sorbose group showed no pathological or significant changes in serum total protein (TP), alanine aminotransferase (ALT), creatinine (Cre), and glucose (Glu) levels compared to the control group, but serum glucose levels tended to increase. These results confirmed that D-sorbose administration ensures basic safety in terms of liver function, kidney function, and nutritional status, although it causes initial weight loss.

[0100] (3) Induction of Tregs by D-sorbose requires an immune-activated state. The Treg induction ability of D-sorbose was evaluated using C57BL / 6J mice under steady state conditions. The results are shown in Figure 3. As is clear from Figure 3, the D-sorbose group showed no significant change in the proportion of Tregs in the spleen compared to the control group. This result contradicts the ex vivo effects of D-sorbose, suggesting that Treg induction by D-sorbose under steady state conditions is very insensitive.

[0101] Next, to mimic an immune-activated state, splenocytes from mice previously administered D-sorbose were collected and stimulated with PMA / ionomycin. The results are shown in Figure 4. As is clear from Figure 4A (scale bar: 100 μm), the formation of numerous small cell colonies was observed in the D-sorbose group 72 hours after stimulation compared with the control group. Furthermore, as shown in Figure 4B, the amount of IL-10 produced in the D-sorbose group tended to increase compared with the control group.

[0102] These results suggest that D-sorbose-induced Treg induction is less sensitive under steady-state conditions, but may be effective under conditions of immune activation (inflammatory background). In particular, the proinflammatory cytokine IL-2 is important for Treg differentiation and maturation, and is more important for inducible Tregs (iTregs) than for endogenous Tregs (nTregs). These findings suggest that IL-2 supplied under inflammatory conditions is necessary for Treg induction by D-sorbose, and that D-sorbose promotes differentiation into iTregs. Furthermore, because IL-2 is produced in large quantities when the immune system is about to go out of control, even daily intake of IL-2 as a health food (e.g., a food for specified health uses or a food with functional claims) is expected to have fewer side effects, as it functions only when abnormalities occur.

[0103] (4) D-sorbose Prevents the Development of Autoimmune Diabetes: Because the inflammatory background is important for D-sorbose's Treg induction, we evaluated its in vivo Treg induction potential and its effects using NOD / ShiJcl mice, a spontaneous autoimmune diabetes model. As shown in Figure 5, during the administration period, changes in body weight (Figure 5A), feeding status (Figures 5B and 5C), and serum total protein, ALT, and creatinine levels (Figure 5D) were similar to those observed in steady-state C57BL / 6J mice, confirming that D-sorbose can be safely administered to NOD / ShiJcl mice.

[0104] As shown in Figure 6A, some individuals in the control group developed diabetic hyperglycemia, while those in the D-sorbose group maintained normal blood glucose levels until the end of the disease. Supporting this, as shown in Figures 6B and 6C, the D-sorbose group showed reduced lymphocyte infiltration into pancreatic islets (indicated by black arrows in Figure 6C; scale bar in Figure 6C is 100 μm) at the end of the disease compared with the control group, and many normal islets remained.

[0105] Next, to immunologically confirm the preventive effect of D-sorbose on the onset of diabetes, the proportion of Tregs in each tissue was analyzed. As shown in Figures 7A and 7B, compared with the control group, the D-sorbose group showed a significantly higher CD3+ Treg count in the pancreatic lymph nodes at the early stage of the disease (17 weeks of age, 10 weeks after D-sorbose administration). ＋ The number of T cells tended to decrease, and as shown in Figures 7C-7F, the percentage of Tregs in peripheral blood (Figures 7C, D) and pancreatic lymph nodes (Figures 7E, F) significantly increased. On the other hand, as shown in Figures 8A and 8B, no significant change was observed in the percentage of Tregs in the spleen.

[0106] Furthermore, as shown in Figure 8C (scale bar: 100 μm), in the D-sorbose group, Foxp3 was detected in the islet-infiltrating lymphocyte population at the early stage of the disease (17 weeks of age, 10 weeks after D-sorbose administration). ＋ These results suggest that D-sorbose induces Treg differentiation in local lymph nodes, where T cells differentiate, at the early stage of the onset of the disease, and exerts a functional suppressive effect at the local site of inflammation.

[0107] (5) D-sorbose does not affect the survival rate of T cell subsets. As shown in Figures 7E and 7F, D-sorbose increases the proportion of Tregs at the site of inflammation. However, as shown in Figures 7A and 7B, D-sorbose does not affect the survival rate of CD3 ＋There was a tendency for T cells to decrease. This result is not due to the induction of Tregs by D-sorbose, but rather it is possible that the relative proportion of Tregs increased simply due to a decrease in T cells other than Tregs. Therefore, the difference in sensitivity to D-sorbose between Tregs and other T cells was evaluated by cell viability. The results are shown in Figure 9. The D-sorbose group showed similar cell viability to the D-glucose group, regardless of the culture time or T cell population. These results suggest that the increase in Tregs by D-sorbose is not a relative increase, but rather due to the induction of differentiation from naive T cells.

[0108] (6) D-sorbose induces Tregs by inhibiting intracellular glucose metabolism. To clarify the effect of D-sorbose on intracellular glucose metabolism, intracellular metabolites were measured. As shown in Figures 10A and 10B, the D-sorbose group showed a significant increase in hexose-6-phosphate, the initial glycolytic pathway, compared with the control group (no monosaccharides added). However, the metabolic enzyme activities of the metabolic pathways branching from D-sorbose to glyceraldehyde-3-phosphate, ribose-5-phosphate in the pentose phosphate pathway (PPP), and UDP-GluNAc in the hexosamine pathway (HP) were significantly decreased.

[0109] Next, as shown in Figure 10B, Treg induction was assessed using inhibitors of enzymes catalyzing metabolic pathways branching off from glucose-6-phosphate. As shown in Figures 11A and 11B, compared with the control group (vehicle group), inhibition of 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 3 (PFKFB3) in the glycolytic pathway with KAN and inhibition of glucose-6-phosphate dehydrogenase (G6PD) in the PPP with 6-AN did not alter the proportion of Tregs. However, inhibition of glutamine-fructose-6-phosphate aminotransferase (GFAT) in the HP with DON significantly increased the proportion of Tregs. These results suggest that, among the various metabolic pathways thought to be inhibited by D-sorbose, inhibition of intracellular glucose metabolic pathways—glycolysis, the pentose phosphate pathway, and the hexosamine pathway—particularly inhibition of the hexosamine synthesis pathway, is involved in Treg induction.

[0110] Furthermore, it was shown that when the metabolic pathway inhibited by D-sorbose is inhibited by an existing inhibitor, differentiation into Tregs is also induced, similarly to D-sorbose. Therefore, D-sorbose can be used as an inhibitor of glycolysis, the pentose phosphate pathway, and the hexosamine pathway, particularly the hexamine pathway, and can also be used as a pharmaceutical composition for diseases that can be treated or prevented by inhibiting these pathways. Specific diseases include the autoimmune diseases and allergic diseases described in (Embodiments of the agent for promoting the induction of regulatory T cells).

[0111] (7) Effects of Long-Term Oral Administration of D-Allose on Living Organisms As with D-sorbose, the safety of D-allose was evaluated in vivo. The results are shown in Figure 12. As shown in Figures 12A to 12C, the higher the concentration of D-allose, the greater the decrease in water intake, food intake, and body weight loss. However, during the administration period, no physical findings such as diarrhea were observed in the D-allose group (not shown), and no particular health problems were observed.

[0112] Example 2 Next, an experiment was conducted to confirm whether D-sorbose is also useful for allergic diseases. The experiment was conducted using the same procedures as in Example 1, except for the following changes to the materials and experimental methods described in (1) to (17) of Example 1 above. Note that, since Example 2 is an experiment to confirm the usefulness of D-sorbose for allergic diseases, some of the materials and experimental methods described in (1) to (17) of Example 1 were not used. (1) Experimental Animals Balb / c wild-type female mice (purchased from Charles River Japan) were used for the experiment at 5 weeks of age.

[0113] (2) Reagents: D-sorbose alone was used as the monosaccharide and dissolved in distilled water to a concentration of 1.1 M. Ovalbumin (OVA; A5503; Sigma-Aldrich, St. Louis, MO, USA) was used as the inhibitor and dissolved in PBS at pH 7.4. Aluminum hydroxide (Alhydrogel adjuvant 2%; invivoGen, San Diego, CA, USA) was used as the adjuvant. OVA and aluminum hydroxide were mixed to concentrations of 25 μg and 2 mg / 200 μL, respectively.

[0114] (9) Non-obese autoimmune diabetes (NOD) model. Instead of the "non-obese autoimmune diabetes (NOD) model" of Example 1, an OVA-induced allergy (OVA) model mouse was generated by the following procedure. Balb / c mice were divided into two groups: one group that received 1.1 M D-sorbose solution in drinking water ad libitum from 5 weeks of age, and the other group that received distilled water as a control. These groups were administered with the solution until 10 weeks of age. At 6, 7, and 8 weeks of age, 200 μL of a mixture of OVA (25 μg) and aluminum hydroxide (2 mg) was administered intraperitoneally. Subsequently, starting at 9 weeks of age, OVA (100 μg / 20 μL) was administered intranasally every day for 1 week. During the administration period, blood samples were collected every 3 days, and body weight, water intake, and food intake were measured. At 10 weeks of age, peripheral blood, lungs, and mediastinal lymph nodes were collected.

[0115] (10) Preparation of Pathological Tissue Specimens Pathological tissue specimens were prepared in the same manner as in Example 1, except that lungs were used instead of the pancreas.

[0116] (11) Histological Evaluation Instead of the regular histological evaluation of insulitis in Example 1, 1 μm-thick paraffin sections were prepared from the lungs of OVA model mice and stained with HE staining to evaluate the following findings: Inflammatory cell infiltration Bronchial epithelial hyperplasia Goblet cell proliferation Bronchial smooth muscle hyperplasia Vascular and peribronchial mucosal edema

[0117] The experimental results of Example 2, which was carried out using the above-mentioned experimental method, are described below. (1) Regarding the prevention of the onset of OVA-induced allergic bronchitis by D-sorbose As described in "(4) Regarding the prevention of the onset of autoimmune diabetes by D-sorbose" in Example 1 above, administration of D-sorbose significantly increased the ability to induce Tregs in vivo. As shown in Figures 13A and 13B, in an experiment using an OVA-induced allergic bronchitis model, the proportion of Tregs in the medial lymph nodes was significantly increased in the D-sorbose group compared to the control group. Furthermore, as shown in Figure 13C (scale bar: 50 µm), in the D-sorbose group, the lung-infiltrating lymphocyte population was significantly higher than that of Foxp3. ＋ These results suggest that D-sorbose induces Treg differentiation in local lymph nodes, where T cells differentiate, and exerts a functional allergy-suppressing effect at the site of inflammation.

[0118] Further supporting these findings, as shown in Figure 14A, the D-sorbose group had reduced inflammatory cell infiltration in the lungs (scale bar: 100 µm), bronchial epithelial hyperplasia, goblet cell proliferation, bronchial smooth muscle hyperplasia, and vascular and peribronchial mucosal edema, compared with the control group. Consistent with these findings, as shown in Figure 14B, the D-sorbose group had a significantly reduced lung / body weight ratio compared with the control group.

[0119] It is known that allergic diseases develop when Th2 cells are induced. The OVA model mouse prepared in Example 2 is a Th2 cell-inducing model mouse. Therefore, the results of Example 2 confirmed that D-sorbose is useful for allergic diseases.

[0120] The agent for promoting the induction of regulatory T cells disclosed in the present application can be used for the treatment or prevention of autoimmune diseases such as autoimmune diabetes, allergic diseases, etc., and is therefore useful for medical institutions, pharmaceutical companies, and the health food industry.

Claims

1. An agent for promoting the induction of regulatory T cells, comprising D-sorbose and / or D-allose as active ingredients.

2. The agent for promoting the induction of regulatory T cells according to claim 1, which contains only D-sorbose among the active ingredients.

3. The agent for promoting the induction of regulatory T cells according to claim 1, wherein the active ingredients are solely D-allose.

4. The agent for promoting the induction of regulatory T cells according to claim 1, wherein the D-sorbose is a D-sorbose derivative, and the D-allose is a D-allose derivative.

5. The regulatory T cell induction promoter according to any one of claims 1 to 4, which is used for the treatment or prevention of an autoimmune disease or an allergic disease via the induction of regulatory T cells.

6. The agent for promoting induction of regulatory T cells according to any one of claims 1 to 4, wherein regulatory T cells are induced by inhibiting the hexamine pathway, which is an intracellular sugar metabolic pathway.

7. The agent for promoting the induction of regulatory T cells according to any one of claims 1 to 4, which is used as a component of a pharmaceutical composition.

8. The agent for promoting the induction of regulatory T cells according to any one of claims 1 to 4, which is used as an ingredient of a food composition.

9. A therapeutic or preventive agent for an autoimmune disease, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives.

10. The therapeutic or preventive agent for an autoimmune disease according to claim 9, wherein the autoimmune disease is type 1 diabetes.

11. A therapeutic or preventive agent for allergic diseases, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives.

12. A food composition for preventing an autoimmune disease, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives.

13. A food composition for preventing allergic diseases, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives.

14. A food composition for improving an allergic condition, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, a D-sorbose derivative, D-allose and a D-allose derivative.

15. A food composition for maintaining healthy immune function, comprising as an active ingredient at least one selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives.

16. A method for treating or preventing an autoimmune disease or an allergic disease, comprising the step of administering to an animal (excluding humans) an effective amount of at least one compound selected from the group consisting of D-sorbose, D-sorbose derivatives, D-allose and D-allose derivatives, which have the effect of promoting the induction of regulatory T cells.

17. A screening method for a substance that promotes the induction of regulatory T cells, comprising the steps of: (a) contacting a test substance with regulatory T cells; (b) measuring the induction rate of regulatory T cells contacted with the test substance and comparing it with the induction rate of regulatory T cells in a control group not contacted with the test substance; and (c) selecting a test substance that promotes the induction of regulatory T cells based on the comparison results of (b).

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