Immunomodulation agent

Abstract

This immunomodulation agent contains yeast deposited to the National Institute of Technology and Evaluation under international deposition number NITE BP-04012. Preferably, the immunomodulation agent is for use in modulation of cytokine production. More preferably, the immunomodulation agent modulates the production of a cytokine in macrophage cells or dendritic cells. Further preferably, the immunomodulation agent modulates immunity via TLR2. The cytokine is preferably at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α.

Description

immunomodulators

[0001] The present invention relates to yeast-based immunomodulators.

[0002] Macrophages and dendritic cells are cells that act on the front lines of innate immunity. In addition to phagocytosis, macrophages are responsible for transmitting antigen information to T cells, which act as commanders. Dendritic cells are antigen-presenting cells (cells that convey substances that are judged to be foreign to other cells) and also have the ability to phagocytose. Macrophages and dendritic cells produce various cytokines when stimulated by pathogens, etc., and this is widely known to be important in immune responses.

[0003] For example, bacterial components (e.g., lipopolysaccharide (LPS)) activate TLRs (toll-like receptors) present on the surface of macrophages and dendritic cells, resulting in the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, initiating inflammation. IL-8 is also a pro-inflammatory cytokine, and in addition to inducing the migration of neutrophils and other granulocytes toward the site of infection, it also plays a role in inducing phagocytosis.

[0004] On the other hand, IL-10 and IL-12 are also produced by macrophage cells stimulated with LPS or the like, and these cytokines are known to have both immunostimulatory and anti-inflammatory properties. For example, IL-10 exhibits immunostimulatory properties, including stimulating the proliferation of thymocytes treated with IL-2 and IL-4, increasing the survival rate of B cells, and stimulating MHC class II expression. On the other hand, IL-10 can suppress immune responses by inhibiting the expression of IL-1α, IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF in activated monocytes and activated macrophages, and also suppresses the production of interferon-γ (IFN-γ) by NK cells.

[0005] IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits encoded by two separate genes, IL-12A and IL-12B, respectively. The main functions of IL-12 are to induce interferon-γ production in NK cells and T cells and to enhance the activity of NK cells and cytotoxic T cells. It is also known to act on naive T cells (Th0) to induce their differentiation into Th1 cells, promote interferon-γ production via Th1 cells, and preferentially activate cellular immunity. IL-12 is also said to activate cellular immunity by preferentially activating Th1 cells and suppressing Th2 cells via IgE suppression, etc. For this reason, IL-12 has attracted attention for its application in the treatment of Th2-driven immune disorders such as allergies, and in the treatment of cancer and infectious diseases by activating cellular immune function.

[0006] As described above, TLRs play a central role in the recognition of various pathogens and the activation of the immune system. Furthermore, among TLRs, TLR2 is expressed on the surface of specific cells such as macrophages and plays a fundamental role in the recognition of pathogens and the activation of innate immunity.

[0007] It has been known that yeast can be used as an immunostimulant (see Patent Documents 1 and 2).

[0008] JP 2004-107281 A JP 2006-75039 A

[0009] The immunomodulatory function is the function of regulating the immune state to a favorable state. An excellent immunomodulatory function can achieve immunoenhancing and / or immunosuppressive effects in order to maintain the immune state in a favorable state. There is a demand for immunomodulators with such excellent immunomodulatory effects.

[0010] As a result of extensive research, the present inventors have discovered that a specific strain of bacteria has excellent immunomodulatory activity, and have completed the present invention. The present invention is based on the above findings and provides an immunomodulator deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04012.

[0011] According to the present invention, an immunoregulatory agent having excellent immunomodulatory activity can be provided. The immunoregulatory agent of the present invention can exert excellent immunoenhancing and / or immunosuppressive activity.

[0012] FIG. 1 is a graph showing the results of evaluating the gene expression of various cytokines in macrophage-like cells in response to a specific yeast powder. FIG. 2 is a graph showing the results of evaluating the gene expression of various cytokines in macrophage-like cells in response to a specific yeast extract. FIG. 3 is a graph showing the results of evaluating the gene expression of IL-6 in dendritic cell-like cells in response to a specific yeast extract. FIG. 4 is a graph showing the results of evaluating the amount of cytokines produced in macrophage-like cells in response to a specific yeast powder. FIG. 5 is a graph showing the results of evaluating the amount of cytokines produced in macrophage-like cells in response to a specific yeast extract. FIG. 6 is a graph showing the results of evaluating the gene expression of various cytokines in macrophage-like cells in the presence of LPS in response to a specific yeast powder. FIG. 7 is a graph showing the results of evaluating the gene expression of various cytokines in macrophage-like cells in the presence of LPS in response to a specific yeast extract. FIG. 8 is a graph showing the results of evaluating the amount of cytokines produced in macrophage-like cells in the presence of LPS in response to a specific yeast powder. Figure 9 is a graph showing the results of evaluating the amount of various cytokines produced in macrophage-like cells in the presence of LPS by a specific yeast extract. Figure 10 is a graph showing the results of evaluating the amount of TLR protein expression in genetically modified HEK293 cells by a specific yeast powder. Figure 11 is a graph showing the results of evaluating the amount of TLR protein expression in genetically modified HEK293 cells by a specific yeast extract.

[0013] The present invention will be described below based on preferred embodiments. The present invention is an immunomodulator comprising a yeast deposited with the National Institute of Technology and Evaluation (NIET) under international deposit number NITE BP-04012. This specific yeast was deposited with the National Institute of Technology and Evaluation (NIET) under deposit number NITE P-04012 on November 14, 2023, and transferred to international deposit on September 11, 2024. The yeast deposited with the National Institute of Technology and Evaluation (NIET) under international deposit number NITE BP-04012 (hereinafter also referred to as "specific yeast") is known by the scientific name Saccharomyces cerevisiae. Its scientific properties are as follows: 1. Scientific properties: It forms white to pale yellow colonies in a nutrient medium containing a carbon source and a nitrogen source. Under an optical microscope, budding growth is observed as a division morphology. It is possible to distinguish between mother and daughter cells. 2. Taxonomic position: Yeast: Edible yeast (genus Saccharomyces) 3. Culture conditions (1) Name of medium: YPD medium (2) Composition of medium: 10g of yeast extract, 20g of peptone, 20g of glucose per 1000ml of medium

[0014] (3) pH of the culture medium: 4 to 7 (optimum pH: 5 to 6) (4) Sterilization conditions of the culture medium: 121°C for 10 minutes (5) Culture temperature: 30°C (6) Culture period: 2 to 3 days (7) Oxygen requirement: facultative anaerobic

[0015] 4. Storage conditions: Can be stored by freezing. (1) Freezing conditions: -80°C (2) Protecting agent: 10-20% glycerin aqueous solution (optimum 15%) (3) Recovery rate after freezing: 40-80% in 2 years

[0016] 5. Conditions for survival test (1) Restoration of microorganisms: 30°C (2) Inoculation, cultivation, and confirmation method: The same conditions as for cultivation.

[0017] As described above, the specific yeast of the present invention is preferably cultured in a YPD medium at a pH of 4 to 7, preferably at a pH of 5 to 6. The culture may be carried out under aerobic conditions, such as in the air.

[0018] The yeast of the present invention may take the form of yeast cell walls, yeast contents, or yeast cells themselves. Yeast cell walls are fractions insoluble in aqueous liquid media such as water, and are obtained by removing contents soluble in aqueous liquid media, such as proteins, carbohydrates, amino acids, nucleic acids, and organic acids, from yeast cells. The method for preparing yeast cell walls is not particularly limited. For example, yeast cells may be crushed or disrupted to allow the water-soluble contents to be eluted, followed by solid-liquid separation to obtain the water-insoluble fraction, which may then be dried. Examples of methods for crushing or disrupting yeast cells include physical disruption methods such as ultrasonication, grinding using a bead mill, and pressurized liquid shearing using a French press, as well as chemical disruption methods using surfactants or lytic enzymes such as cell wall-degrading enzymes. Any drying method, such as freeze-drying or spray-drying, can be used. Physical disruption is preferred as a method for enabling the contents of yeast cells to be eluted into an aqueous liquid medium. Examples of aqueous liquid media include water, ethanol, and mixtures thereof, with water being preferred. When the aqueous liquid medium is water, the water may contain components other than water and may have a water content of more than 50% by mass, preferably 60% by mass or more.

[0019] Furthermore, the yeast content can be obtained by disrupting yeast cells to allow the content to be dissolved in an aqueous medium, and then extracting the resulting disrupted material with an aqueous medium. Instead of disrupting yeast cells, the yeast cell walls may be dissolved with a cell wall-degrading enzyme to allow the content to be dissolved in an aqueous medium. The yeast content is also referred to as yeast extract. The yeast extract may be a solid dried by any method. In the present invention, among the yeast cell walls and yeast content, the use of a yeast component containing the yeast content is preferred because of its excellent immunomodulatory activity, specifically, its immunoenhancing and immunosuppressive activity. Examples of yeast components containing the yeast content include the yeast content itself, as well as a mixture obtained by disrupting or destroying yeast cells, in which the yeast cell walls and yeast content are mixed. By contacting such a mixture with an aqueous liquid and performing solid-liquid separation, the yeast extract can be separated from the cell walls. The proportion of yeast extract among the yeast components is preferably 10% by mass or more, more preferably 20% by mass or more, even more preferably 50% by mass or more, and particularly preferably 60% by mass or more.

[0020] There are no strict limitations on the dosage when using the specific yeast, which is the active ingredient of the immunomodulator of the present invention. Since the effects obtained vary depending on various modes of use, such as the subject and the applicable disease, it is desirable to set the dosage appropriately. However, the preferred dosage for the specific yeast is 10 mg to 100 g, more preferably 160 mg to 4 g, of cell weight per day. The agent of the present invention can be taken continuously, and can be taken continuously for one week or more, two weeks or more, or four weeks or more.

[0021] The specific yeast of the present invention can be ingested by oral ingestion, transdermal ingestion, transmucosal ingestion, enteral ingestion, etc., with oral ingestion being particularly preferred.

[0022] The immunomodulators of the present invention can be used as pharmaceuticals, quasi-drugs, or food and beverage products for animals, including humans, or for the production thereof. The immunomodulators of the present invention can be administered or ingested directly by animals, including humans, as pharmaceuticals, quasi-drugs, or food and beverage products, or can be added or blended into food and beverage products or animal feeds such as pet food to be used as immunomodulatory food and beverage products or animal feeds. In the latter case, the method of adding or blending the specific yeast into food and beverage products or animal feeds is not particularly limited. For example, the specific yeast can be directly blended into raw materials or ingredients before the production of the food and beverage products or animal feeds, added during the production process of the food and beverage products or animal feeds, or added to the produced food and beverage products or animal feeds. The term "food and beverage products" refers to products that can be consumed by humans as food, and includes general foods and beverage products, including so-called health foods, as well as health functional foods such as foods for specified health uses and foods with nutrient functions specified under the Ministry of Health, Labor, and Welfare's Health Function Food System, and supplements. The term "animal feed" refers to products given as food to non-human animals (animals kept by humans), such as livestock, poultry, and farmed fish, and includes, for example, livestock feed, pet food, etc.

[0023] When the immunomodulator of the present invention is used as a pharmaceutical or quasi-drug, it may contain the specific yeast as the active ingredient alone, or may further contain a pharmaceutically acceptable carrier, or may further contain other active ingredients or pharmacological ingredients to the extent that the immunomodulatory effect of the specific yeast is not impaired. Examples of such carriers include excipients, coating agents, binders, fillers, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, disinfectants, flavorings, and odor enhancers.

[0024] When the agent of the present invention is used as a pharmaceutical or quasi-drug, it can be administered in any dosage form. The dosage form may be oral or parenteral. For example, oral dosage forms include solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, as well as liquid dosage forms such as elixirs, syrups, and suspensions. Parenteral dosage forms include injections, infusions, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, and patches. Among these, oral dosage forms are preferred. The amount of specific yeast in the agent of the present invention may be any amount that can serve as an active ingredient, and the specific yeast may account for 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, or 70% by mass or more of the solid content of the agent. Note that the solid content here refers to the total amount excluding the solvent, and examples of the solvent include water, ethanol, and organic solvents commonly used as solvents.

[0025] When the agent of the present invention is used as a food or beverage, it may contain the specific yeast as an active ingredient alone, or may further contain various additives used in the production of foods and beverages, as long as the immunomodulatory effect of the specific yeast is not impaired. Examples of such additives include various oils and fats, herbal medicines, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, taste components, acidulants such as organic acids, stabilizers, flavors, colorants, fragrances, etc.

[0026] Examples of foods and beverages include oral preparations (gum, candy, etc.), processed seafood paste products such as kamaboko (fish cake) and chikuwa (fish cake), livestock products such as sausages and ham, bread, Western confectionery, Japanese confectionery, noodles such as fresh noodles, Chinese noodles, boiled noodles, and buckwheat, seasonings such as sauces, soy sauce, dressings, sugar, honey, powdered sugar, and starch syrup, spices such as curry powder, mustard powder, and pepper powder, jam, marmalade, chocolate spread, pickles, greens, furikake (rice seasoning), and processed vegetables and fruits such as various canned and bottled vegetables and fruits, dairy products such as cheese, butter, and yogurt, beverages such as miso soup, soup, fruit juice, vegetable juice, whey drinks, soft drinks, and alcoholic beverages, as well as other general foods and beverages such as health foods.

[0027] The present invention encompasses a package comprising a package and the immunomodulatory agent of the present invention, or a food, drink, or animal feed containing the same, contained in the package. The package is not particularly limited in shape or material, as long as it can contain the agent, food, drink, or animal feed of the present invention and can print ingredient information thereon. Examples of the shape of the package include a box or a bag. Examples of the material of the package include paper, plastic, woven fabric, and metal. The package clearly displays various information, such as the content of a specific yeast in the agent, food, drink, or animal feed of the present invention contained in the package. The method of presenting information in such a package is not particularly limited, and for example, 1) it may be printed on the outer or inner surface of the package, 2) the immunomodulatory uses may be printed on a printing medium such as printing paper contained inside the package together with the food, drink, or animal feed, or 3) a QR code (registered trademark) containing information that allows access to an internet URL or a site introducing the agent, food, drink, animal feed, etc. of the present invention may be printed on the package or the printing medium contained therein, and the information may be presented by accessing the URL.

[0028] As will be described in the Examples below, the specific yeast has an excellent effect of regulating cytokine production from cells such as macrophage cells or dendritic cells. The macrophage cells and dendritic cells are preferably derived from mammals. Examples of mammals include non-human mammals such as humans, rats, mice, rabbits, cows, pigs, dogs, cats, sheep, and monkeys. Macrophage cells and dendritic cells mature and are capable of performing their original functions, such as phagocytic function and antigen-presenting function.

[0029] For example, as shown in Examples 1 to 5 below, the specific yeast of the present invention promotes cytokine production by administering it to macrophage cells or dendritic cells, for example, macrophage cells or dendritic cells that have been differentiated and have not yet received much stimulation by antigens. For example, the yeast effectively enhances gene expression and promotes the production of at least one cytokine selected from interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), and TNF-α.

[0030] IL-1β, IL-6, IL-8, and TNF-α are pro-inflammatory cytokines. IL-1β is a potent pro-inflammatory cytokine. IL-1β stimulates CD4 ＋ It stimulates cells to differentiate into Th17 cells. TNF-α plays an important role in inflammatory responses both locally and in the blood circulation. TNF-α not only induces the expression of vascular endothelial cells, but also enhances leukocyte adhesion molecules that stimulate immune cell infiltration, promoting lymphocyte infiltration into the site of infection. IL-6 is important in inducing the differentiation of B cells into antibody-producing cells (plasma cells). IL-8 induces chemotaxis of neutrophils and other granulocytes to the site of infection, and once they arrive, IL-8 further induces phagocytosis.

[0031] In addition to suppressing immune responses, IL-10 also exhibits immunostimulatory properties, including stimulating the proliferation of IL-2 and IL-4 treated thymocytes, increasing B cell survival, and stimulating MHC class II expression.

[0032] Furthermore, IL-12 induces the production of interferon-γ in natural killer cells (NK cells) and T cells, enhancing the activity of NK cells and cytotoxic T cells. The promotion of IL-12 production is evident from the promotion of the production of its subunit, IL-12A.

[0033] Based on the above, the specific yeast of the present invention has the effect of promoting the production of the various cytokines described above, thereby activating T cells, B cells, NK cells, etc., and the immune response caused by the action of the various cytokines makes it possible to protect the living body from pathogens, viruses, etc.

[0034] On the other hand, the specific yeast of the present invention can exhibit immunosuppressive effects against macrophage cells and dendritic cells that have already been stimulated and activated by an antigen (e.g., lipopolysaccharide (LPS)). For example, in macrophage cells, it can suppress the production of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. LPS is an antigen that can strongly induce the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. However, excessive production of these pro-inflammatory cytokines can cause tissue damage and has been associated with various inflammatory diseases, such as rheumatoid arthritis and osteoarthritis, Alzheimer's disease, adult respiratory distress syndrome, allergies, asthma, eating disorders, atherosclerosis, muscle contusion, and cancer. Therefore, these diseases can be prevented by using the specific yeast of the present invention.

[0035] LPS is also known to have endotoxic activity, and TNF-α and IL-1 produced by macrophage cells in response to large amounts of LPS are known to increase intravascular blood coagulation and vascular permeability, causing a drop in blood pressure and peripheral circulatory failure, leading to a state of shock (endotoxic shock). It is also known that when IL-1, IL-6, TNF-α, and other factors produced by various cells due to the action of LPS are transported to the brain via the bloodstream, prostaglandin E2 is produced by brain cells, which acts on the pituitary gland and hypothalamus, which have a thermoregulatory function, causing fever. Therefore, the use of the specific yeast of the present invention may potentially prevent or ameliorate these intravascular shock effects and fever.

[0036] Furthermore, it is known that when fat cells become too large due to obesity, they die, and immune cells such as macrophages that phagocytose them gather in visceral fat tissue, causing inflammation. This inflammation is thought to affect the entire body and lead to lifestyle-related diseases such as diabetes. Therefore, by using the specific yeast of the present invention, diseases such as inflammation caused by obesity can be prevented.

[0037] Furthermore, the specific yeast of the present invention can exhibit the effect of increasing IL-12 in macrophage cells and dendritic cells that have already been stimulated and activated by an antigen (e.g., LPS, etc.). Increasing IL-12 production in macrophage cells and dendritic cells is thought to lead to enhanced expression of IL-10 in T cells, thereby leading to anti-inflammatory effects. Furthermore, IL-12 has the effect of suppressing IgE production, which is known to suppress Th2-type immune responses, and is therefore thought to lead to suppression of Th2-type immune responses.

[0038] Th2-type immune abnormalities are thought to be associated with allergic diseases (atopic dermatitis), myasthenia gravis, chronic active hepatitis (HBsAg(-)), membranous glomerulonephritis, pemphigus vulgaris, hyperthyroidism or hypothyroidism, sarcoidosis, etc. Furthermore, they are believed to be linked to rheumatoid arthritis, SLE, urticaria, bronchial asthma, etc. Therefore, these diseases can be prevented by using the specific yeast of the present invention.

[0039] As shown in Examples 10 and 11 below, the specific yeast of the present invention selectively enhances the expression of TLR2 on the cell surface, whether it is a mixture of cell wall and contents or the contents. TLR2 is expressed and functions not only in cells such as dendritic cells and macrophages that control innate immunity, but also in T cells, which play a central role in adaptive immunity, and has been reported to play an important role in inducing infection immunity, anti-tumor immunity, and autoimmune disease. Therefore, it is believed that the cytokine production regulation function of the specific yeast of the present invention is mediated by TLR2.

[0040] The immunomodulators of the present invention are expected to provide immunomodulatory effects suited to the physical condition at the time of administration. For example, administration during fatigue or after exercise is believed to provide an immunoenhancing effect that suppresses viral and bacterial infections during times of immunosuppression. Therefore, the immunomodulators of the present invention can also be used as immunoenhancing agents. Furthermore, administration of the immunomodulators of the present invention during colds or to obese or elderly individuals is expected to exert immunosuppressive effects, such as anti-inflammatory effects, thereby alleviating inflammation caused by colds, chronic inflammation, and autoimmune diseases. Therefore, the immunomodulators of the present invention can also be used as anti-inflammatory or immunosuppressive agents. Whether an immunoenhancing or immunosuppressive effect is exerted in the same individual may depend on the level of inflammation in that individual, for example, the level or condition of inflammation at the site of administration. Methods for assessing the level or condition of inflammation in mammals such as humans include, for example, inflammatory markers in the blood.

[0041] Generally, microbial strains previously reported to have immune-related effects were known to have only one of immunosuppressive and immunoenhancing effects, whereas the specific yeast of the present invention has been found to have both immunoenhancing and immunosuppressive effects. Such specific yeast of the present invention may exert an effect appropriate to the physical condition of the consumer. The immunomodulator of the present invention is believed to have the effect of promoting immunity in states where immune function is weakened, and of lowering immunity in states where immune function is strengthened. Such immunomodulators of the present invention are believed to be able to suppress immunity without weakening immunity. Furthermore, they are expected to control abnormalities in both weakened and strengthened immune states in individuals, preventing pathological conditions from occurring or restoring existing pathological conditions to normal. Therefore, even if the body's immune state changes when continuously ingested, they may be able to exert an appropriate effect, making them excellent immunomodulators.

[0042] For example, the present invention provides the following: [1] An immunomodulator comprising the yeast deposited at the National Institute of Technology and Evaluation (NIET) under International Deposit Number NITE BP-04012. [2] The immunomodulator according to [1], which is used for regulating cytokine production. [3] The immunomodulator according to [2], which regulates cytokine production in macrophage cells or dendritic cells. [4] The immunomodulator according to any one of [1] to [3], which regulates immunity via TLR2. [5] The immunomodulator according to any one of [2] to [3], wherein the cytokine is at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α. [6] The immunomodulator according to any one of [1] to [5], which is used for immunoenhancing and immunosuppressing. [7] The immunomodulator according to [6], which is used to exert an immunoenhancing or immunosuppressive effect depending on the state or degree of inflammation in the body. [8] The immunomodulator according to any one of [1] to [7], which is used to enhance the production of at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α. [9] The immunomodulator according to any one of [1] to [7], which is used to suppress the production of at least one selected from IL-1β, IL-6, and TNF-α under inflammation, or to promote the production of IL-12 under inflammation.

[10] The immunomodulator according to any one of [1] to [7], which is used to regulate the production of at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α in macrophages, or to regulate IL-6 production in dendritic cells.

[11] The immunomodulator according to any one of [1] to

[10] , comprising a cell wall component of the yeast.

[12] The immunomodulator according to any one of [1] to

[10] , comprising the contents of the yeast.

[13] An immunomodulatory food or drink comprising the immunomodulator according to any one of [1] to

[12] .

[14] Use of the yeast deposited at the National Institute of Technology and Evaluation under International Deposit Number NITE BP-04012 for the production of an immunomodulator.

[0043] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. In the following tests, macrophage-like cells were used as a model of macrophage cells, and dendritic cell-like cells were used as a model of dendritic cells. Macrophage cells and dendritic cells in the present invention include the macrophage-like cells and dendritic cell-like cells described below, respectively.

[0044] <Cultivation and drying of yeast> A yeast strain (international deposit number NITE BP-04012) was cultured by the following method. First, an inoculum stored under the above conditions was collected, and the inoculum was inoculated onto a YPD agar medium by streaking. The agar medium was cultured in air at 30°C for 3 days, and then multiple colonies that appeared on the agar medium were cultured under the above conditions. After culturing, the yeast was dried by spray drying.

[0045] <Preparation of Yeast Powder> 150 mg of the dried powder obtained in the above <Cultivation and Drying of Yeast> was suspended in 1.5 mL of sterilized water and placed in a tube. The cell walls were crushed using a bead cell crusher (Tommy Medico, Micro Smash™ MS-100R), and then freeze-dried to obtain a crushed product with the contents eluted. 10 mg of the resulting solid was suspended in 10 mL of each test medium to obtain a yeast powder with the yeast contents eluted (concentration in the medium: 1 w / v %; hereinafter, sometimes simply referred to as "yeast powder"). The obtained yeast powder contained both yeast cell walls and yeast extract, with the yeast extract accounting for approximately 60% by mass.

[0046] <Preparation of yeast extract> 10 mg of the yeast powder (crushed material) obtained in the above <Preparation of yeast powder> was added to 10 mL of each test medium, stirred thoroughly, and then allowed to settle. The resulting supernatant was collected and used as an extract (concentration in the medium: 1 w / v %, hereinafter sometimes simply referred to as "yeast extract").

[0047] <Evaluation 1: Evaluation of cytokine production amount (gene expression, macrophage-like cells)> THP-1 cells (human monocyte-derived cells) were cultured in a growth medium (10% FBS (fetal bovine serum)-RPMI1640 medium), and after 4 days, they were cultured at 1 × 10 in a differentiation-inducing medium (10% FBS, 0.5 μg / mL PMA (phorbol 12-myristate 13-acetate)-RPMI1640 medium). ４ After 4 days of culture, differentiation into macrophage-like cells was confirmed, and the culture medium was then replaced with a test medium (10% FBS, 100 Units / mL Penicillin, 100 Units / mL Streptomycin-RPMI 1640 medium) containing the test substance at each concentration or LPS (Sigma-Aldrich) and the test substance, and the medium was incubated at 37°C, 5% CO ２ After culturing for 1 hour under these conditions, the medium was replaced with a test medium containing either the test substance or LPS and the test substance, and the cells were cultured for 5 hours. The cells were washed twice with PBS, and cDNA was recovered using a FastLane cDNA kit (QIAGEN). Real-time PCR was performed using the recovered cDNA and TBGreen® Premix Ex Taq™ II (Takara). Relative gene expression levels were calculated by comparative quantification using the ΔΔCt method against the Ct value, which is the expression level of an internal control gene. The primer sequences for each gene are shown in Table 1 below.

[0048]

[0049] <Evaluation 2: Evaluation of cytokine production amount (gene expression, dendritic cell-like cells)> THP-1 cells (human monocyte-derived cells) were cultured in a growth medium (10% FBS (fetal bovine serum)-DMEM medium), and after 2 to 3 days, they were cultured at 5 x 10 in a differentiation-inducing medium (200 ng / mL ionomycin, 100 ng / mL GM-SCF, 200 ng / mL IL-4, 20 ng / mL TNF-α-DMEM medium). ５The cells were seeded onto a 24-well plate at 0.5 mL / well. After 48 hours of culture to differentiate into dendritic cell-like cells, 3.3 μL / well of the diluted solution was added to the plate so that the test substance had the designated concentration. The cells were then incubated at 37°C in 5% CO ２ The cells were cultured for 24 hours under the conditions of 0.1% PBS (0.1% PBS, 0.1% PBS, 0.2% PBS, 0.3% PBS, 0.4% PBS, 0.5% PBS, 0.5% PBS, 0.6% PBS, 0.7% PBS, 0.8% PBS, 0.9% PBS, 0.9% PBS, 0.9% PBS, 0.1% PBS, 0.2% PBS, 0.3% PBS, 0.4% PBS, 0.5% PBS, 0.5% PBS, 0.5% PBS, 0.6% PBS, 0.7% PBS, 0.8% PBS, 0.9 ...

[0050]

[0051] <Evaluation 3: Evaluation of cytokine production amount (ELISA analysis, macrophage-like cells)> After culturing THP-1 in a proliferation medium, 6.3 × 10 ４ The cells were seeded in a 24-well plate at 0.5 mL / well. ２ After confirming that the cells had differentiated into macrophage-like cells, the medium was replaced with a test medium (10% FBS, 100 units / mL Penicillin, 100 units / mL Streptomycin-RPMI1640 medium) containing the test substance at each concentration or LPS (Nacalai Tesque) and the test substance, and the medium was incubated at 37°C, 5% CO ２After culturing for 1 hour under the above conditions, the medium was replaced with a test medium containing either the test substance or LPS and the test substance, and the cells were cultured for 24 hours. The culture supernatant from each well was transferred to a 1.5 mL tube and centrifuged at 3,000 × g for 5 minutes, and the supernatant was used for ELISA analysis. In ELISA analysis, IL-1β was measured using the IL1-β ELISA Kit, Human (ProteinTech), IL-6 was measured using the Levis® Human IL-6 ELISA Kit (Fujifilm Wako Shibayagi), IL-8 was measured using the Levis® Human IL-8 ELISA Kit (Fujifilm Wako Shibayagi), IL-10 was measured using the AuthentiKine™ IL-10 ELISA Kit, Human (ProteinTech), IL-12A was measured using the Human IL-12 ELISA Kit (R&D Systems), and TNF-α was measured using the AuthentiKine™ TNF-alpha ELISA Kit, Human (ProteinTech).

[0052] <Evaluation 4: Evaluation of TLR receptor activation> In this evaluation, HEK293 cells were used as cells that can be highly efficiently transfected with genes. NF-κB Reporter Luciferase HEK293 Cell Line was used at 1 × 10 ４The cells were seeded onto a 96-well plate using growth medium (10% FBS (fetal bovine serum), 100 U / mL penicillin, 100 U / mL streptomycin, 100 μg / mL hygromycin B-DMEM medium) at a concentration of 100 μL / well. After 24 hours of culture, a plasmid vector carrying the TLR2 or TLR4 gene (TLR2: pRP[Exp]-Neo-CMV>hTLR2 (VectorBuilder), TLR4: pRP(Exp)-Neo-CMV>hTLR4 (VectorBuilder)) was introduced into the cells using the lipofection method. A transfection solution was prepared by mixing 100 ng of Plasmid DNA, 0.1 μL of X-tremeGENE™ HP DNA Transfection Reagent (MERCK), and 10 μL of DMEM medium per well. 10 μL of the transfection solution was added to each well, gently mixed, and the cells were cultured again. The next day, to confirm vector introduction, the cells were cultured for an additional day in test medium (10% FBS, 100 Units / mL Penicillin, 100 Units / mL Streptomycin, 200 μg / mL G418-RPMI1640 medium) containing 200 μg / mL G418. The culture supernatant in each well was replaced with test medium containing 100 μL / well of the test substance or a positive control (10 μg / mL Zymosan or 1 μg / mL LPS). The cells were then cultured for 6 hours, and intracellular luciferase activity was evaluated by measuring luminescence using the Bright-Glo™ Luciferase Assay System (Promega). After the culture was completed, 30 μL of the culture supernatant was removed from each well, and 70 μL of Luciferase Assay Reagent was added. After gentle stirring, the plate was allowed to stand for 2 minutes, and the relative luminescence value of each well was measured using a microplate reader. The relative luminescence value is the ratio to the control (when neither the test substance nor the positive control was added).

[0053] (Example 1 Increase in Cytokine Gene Expression Levels (Macrophage-Like Cells)) The yeast powder was added as a test substance to macrophage-like cells, and the above-mentioned <Evaluation 1: Evaluation of Cytokine Production Levels (Gene Expression, Macrophage-Like Cells)> was performed to determine the cytokine gene expression levels (relative values ​​to an internal standard). The obtained gene expression levels were calculated as a ratio to the cytokine gene expression level of a control measured in the same manner as above, except that no yeast powder was added. The results are shown in Figure 1. As shown in Figure 1, it was confirmed that the addition of yeast powder increased the expression levels of various cytokine genes compared to the case where no yeast powder was added.

[0054] (Example 2 Increase in Cytokine Gene Expression Levels (Macrophage-Like Cells)) The yeast extract was added as a test substance to macrophage-like cells, and the above-mentioned <Evaluation 1: Evaluation of Cytokine Production Levels (Gene Expression, Macrophage-Like Cells)> was performed to evaluate the cytokine gene expression levels (relative values ​​to an internal standard). The obtained gene expression levels were calculated as a ratio to the cytokine gene expression level of a control measured in the same manner as above, except that no yeast powder was added. The results are shown in Figure 2. As shown in Figure 2, it was confirmed that the addition of yeast extract increased the expression levels of various cytokine genes compared to the case where no yeast extract was added.

[0055] (Example 3 Increase in cytokine gene expression level (dendritic cell-like cells)) The yeast extract was added as a test substance to dendritic cell-like cells, and the cytokine gene expression level of IL-6 was evaluated according to <Evaluation 2: Evaluation of cytokine production level (gene expression, dendritic cell-like cells)>. The ratio of the cytokine gene expression level to the control, which was measured in the same manner as above except that no yeast extract was added, was calculated. The results are shown in Figure 3. As shown in Figure 3, it was confirmed that the cytokine gene expression level was also increased in dendritic cell-like cells compared to the case where no yeast extract was added.

[0056] (Example 4 Increase in cytokine production amount) The yeast powder was added as a test substance to macrophage-like cells, and <Evaluation 3: Evaluation of cytokine production amount (ELISA analysis, macrophage-like cells)> was performed to evaluate the cytokine production amount by ELISA analysis. The ratio to the cytokine gene expression amount of the control, which was measured in the same manner as above except that no yeast powder was added, was determined. The results are shown in Figure 4. As shown in Figure 4, it was confirmed that the addition of yeast powder increased the amounts of various cytokines compared to the case where no yeast powder was added.

[0057] (Example 5 Increase in Cytokine Production Amount) According to <Evaluation 3: Evaluation of Cytokine Production Amount (ELISA Analysis, Macrophage-Like Cells)>, the yeast extract was added as a test substance to macrophage-like cells, and the cytokine production amount was evaluated by ELISA analysis. The results are shown in Figure 5. As shown in Figure 5, it was confirmed that the addition of yeast extract increased the amounts of various cytokines compared to the values ​​of the control without addition.

[0058] Example 6: Regulation of Cytokine-Producing Gene Expression Levels Macrophage-like cells were treated with 1 μg / mL LPS under inflammatory conditions as a control, and the yeast powder was added as a test substance in addition to 1 μg / mL LPS, and the cytokine gene expression levels (relative to the internal standard) were evaluated according to <Evaluation 1: Evaluation of Cytokine Production Levels (Gene Expression, Macrophage-Like Cells)>. The gene expression level ratio, where the value without addition of LPS and test substance is set to 1, is shown in Figure 6. It was confirmed that the addition of yeast powder reduced the gene expression levels of inflammatory cytokines (IL-6, TNF-α), which increased under inflammation.

[0059] Example 7: Regulation of cytokine-producing gene expression levels Macrophage-like cells were treated with inflammatory conditions in the presence of 1 μg / mL LPS as a control, and cytokine gene expression levels when yeast extract was added in addition to 1 μg / mL LPS were evaluated according to <Evaluation 1: Evaluation of cytokine production levels (gene expression, macrophage-like cells)>. Figure 7 shows the gene expression ratio, where the value when no LPS or test substance was added was set to 1. It was confirmed that the addition of yeast extract reduced the expression levels of inflammatory cytokine genes that increased under inflammation.

[0060] Example 8: Regulation of cytokine production levels Macrophage-like cells were treated with 1 μg / mL LPS under inflammation as a control, and the yeast powder was added as a test substance in addition to 1 μg / mL LPS, and cytokine production levels were evaluated by ELISA analysis in <Evaluation 3: Evaluation of cytokine production levels (ELISA analysis, macrophage-like cells)>. The results are shown in FIG. 8. As shown in FIG. 8, it was confirmed that the addition of yeast powder suppressed the production of inflammatory cytokines (IL-6, TNF-α) under inflammation.

[0061] Example 9: Regulation of cytokine production levels Macrophage-like cells were treated with 1 μg / mL LPS under inflammation as a control, and the yeast extract was added as a test substance in addition to 1 μg / mL LPS, and the cytokine production levels were evaluated by ELISA analysis in <Evaluation 3: Evaluation of cytokine production levels (ELISA analysis, macrophage-like cells)>. The results are shown in FIG. 9. As shown in FIG. 9, it was confirmed that the addition of yeast extract suppressed the production of inflammatory cytokines (IL-6, TNF-α, IL-1β) under inflammation.

[0062] (Example 10 Activation of TLR Receptors) The activity of TLR2 and TLR4 receptors when the yeast powder was added as a test substance was evaluated in <Evaluation 4: Evaluation of TLR Receptor Activation>. As positive controls (PC), Zymosan (Nova Biologicals) was used for TLR2, and LPS-EB (Nacalai Tesque) was used for TLR4. The results are shown in Figure 10. As shown in Figure 10, the addition of yeast powder increased luciferase activity mediated by TLR2, confirming that the yeast powder increases cytokine production via TLR2.

[0063] (Example 11 Activation of TLR Receptors) The activity of TLR2 and TLR4 receptors when yeast extract alone was added was evaluated in <Evaluation 4: Evaluation of TLR Receptor Activation>. As PC (positive control), Zymosan (Nova Biologicals) was used for TLR2, and LPS-EB (Nacalai Tesque) was used for TLR4. The results are shown in Figure 11. In Figure 11, it was confirmed that the addition of yeast extract increased luciferase activity mediated by TLR2, and that the yeast extract increased cytokine production via TLR2.

Claims

1. An immunomodulator comprising a yeast deposited at the National Institute of Technology and Evaluation under international deposit number NITE BP-04012.

2. The immunoregulatory agent according to claim 1, which is for regulating cytokine production.

3. The immunomodulator according to claim 2, which regulates cytokine production in macrophage cells or dendritic cells.

4. The immunoregulatory agent according to claim 1 or 2, which regulates immunity via TLR2.

5. The immunoregulator according to claim 2, wherein the cytokine is at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α.

6. The immunomodulator according to claim 1 or 2, which is for immunostimulation and immunosuppression.

7. The immunomodulator according to claim 6, which is used to exert an immunostimulating or immunosuppressive effect depending on the state or degree of inflammation in the body.

8. The immunomodulator according to claim 1 or 2, which is used to enhance the production of at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α.

9. The immunomodulator according to claim 1 or 2, which is used to suppress the production of at least one factor selected from IL-1β, IL-6 and TNF-α under inflammation, or to enhance the production of IL-12 under inflammation.

10. The immunomodulator according to claim 1 or 2, comprising a cell wall component of the yeast.

11. The immunomodulator according to claim 1 or 2, comprising the contents of said yeast.

12. An immunoregulatory food or beverage comprising the immunoregulator according to claim 1 or 2.

13. Use of a yeast deposited at the National Institute of Technology and Evaluation under international deposit number NITE BP-04012 for the production of an immunomodulator.

Images