A composition for the treatment of autoimmune diseases containing Lactobacillus aspergillus or extracellular vesicles derived therefrom as an active ingredient.

Abstract

The present invention relates to a composition comprising Lactobacillus sakei or extracellular vesicles derived therefrom.The present invention also relates to a novel Lactobacillus sakei LBML6 strain.The Lactobacillus sakei of the present invention is a strain that has blood TNFα and IgG suppressive activity, and increases the ratio of tolDC cells and Tregs cells, and can be used in various applications such as prevention, improvement and / or treatment of autoimmune diseases in humans or animals, as well as intestinal regulation.

Description

Detailed description of the invention

[0001] [Technical Field] The present invention relates to a composition for the prevention, improvement, or treatment of autoimmune diseases, comprising Lactobacillus aspergillus, its culture, lysate, extract, or fermentate, or extracellular vesicles isolated therefrom, as an active ingredient.

[0002] [Background technology] Autoimmune diseases are illnesses in which the body's immune system attacks itself. They typically develop over long periods, cause chronically persistent symptoms, and generally lead to permanent organ damage. In reality, there are very few cures. Although much has been learned about autoimmune diseases, the exact mechanisms of their development, the nature of autoantigens, and regulatory genetic factors remain unclear. Autoimmune diseases are broadly classified into organ-specific diseases and systemic diseases.

[0003] Organ-specific autoimmune diseases (SADs) arise from an immune response to organ-specific antigens and can occur in almost any organ of the body. Systemic autoimmune diseases, on the other hand, do not involve an immune response to specific cells but rather to antigens expressed throughout the body. These systemic autoimmune diseases can also selectively cause disease in specific organs.

[0004] Examples of autoimmune diseases include asthma, atopic dermatitis, psoriatic dermatitis, allergic rhinitis, allergic conjunctivitis, and urticaria, which are known to cause clinical symptoms by damaging and abnormalizing bodily organs through allergic reactions. They also include rheumatoid arthritis and lupus (Vaughan JH. Med Times 1969;97:187-204), which are known to develop due to a hypersensitive reaction (including autoantibody reactions) to the body's own proteins (autoantigens such as immunoglobulins, collagen, and DNA). These diseases, such as lupus and rheumatoid arthritis, are known to induce damage and inflammation in specific organs through antigen-specific IgG antibodies that react with the body's own antigens (autoantigens) due to a hypersensitive reaction to autoantigens present in the human body; therefore, they are classified as autoimmune diseases.

[0005] Currently, the direct causes of autoimmune diseases are not clearly understood, and various therapeutic agents such as steroids, non-steroidal anti-inflammatory drugs (NSAIDs), and immunosuppressants are used to treat them. However, these therapeutic agents do not provide a fundamental cure, and their use is limited due to various side effects. In addition, some chemotherapeutic drugs have drawbacks, such as insufficient efficacy against autoimmune diseases that have already developed.

[0006] Therefore, there is a current need for the development of autoimmune disease treatments that have few therapeutic side effects, are effective in relieving inflammatory symptoms and pain, and are easy to take.

[0007] The matters described above as background technology are intended solely to enhance understanding of the background of the present invention and should not be understood as constituting prior art well known to those with ordinary skill in the art.

[0008] [Summary of the Invention] [Problems the invention aims to solve] The inventors have strived to find a strain of Lactobacillus that can be applied to various autoimmune diseases, including rheumatoid arthritis. As a result, they have confirmed that Lactobacillus asakei or its culture has preventive or therapeutic effects on autoimmune diseases by suppressing pro-inflammatory factors that cause various autoimmune diseases or by increasing the proportion of immunosuppressive cells, and have completed the present invention.

[0009] Therefore, the object of the present invention is to provide a composition for the prevention, improvement, or treatment of autoimmune diseases, comprising Lactobacillus sakei, its culture, crushed product, extract, or fermented product; or extracellular vesicle derived from Lactobacillus sakei; as an active ingredient.

[0010] Another object of the present invention is to provide a method for preventing, improving or treating an autoimmune disease, comprising administering a therapeutically effective amount of Lactobacillus aspergillus, its culture, lysate, extract or fermentate; or extracellular vesicle derived from Lactobacillus aspergillus; to a subject in need thereof.

[0011] A further object of the present invention is to provide Lactobacillus aspera, its cultures, crushed products, extracts, or fermented products for therapeutic use.

[0012] A further object of the present invention is to provide therapeutic uses for extracellular endoplasmic reticulum derived from Lactobacillus aspergillus, its cultures, lysates, extracts, or ferments.

[0013] Another object of the present invention is to provide a method for producing compositions for the prevention, improvement, or treatment of autoimmune diseases.

[0014] Another object of the present invention is to provide a Lactobacillus sakei LBML6 strain deposited under accession number KCCM13011P.

[0015] Other objects and advantages of the present invention will become clearer from the detailed description of the invention, claims and drawings below.

[0016] [Means for solving the problem] According to one aspect of the present invention, the present invention provides a composition for the prevention, improvement, or treatment of autoimmune diseases, comprising Lactobacillus asylum, its culture, crushed product, extract, or fermented product; or extracellular vesicle derived from Lactobacillus asylum; as an active ingredient.

[0017] The Lactobacillus sakei strains available for use in this invention are not limited, but preferably include Lactobacillus sakei LBML6 strain deposited with the Korea Microbial Conservation Center under accession number KCCM13011P, Lactobacillus sakei WIKIM31 strain deposited with accession number KCCM12654P, and Lactobacillus sakei WIKIM0109 strain deposited with the Biological Resource Center under accession number KCTC13818BP, with Lactobacillus sakei LBML6 strain being the most preferred.

[0018] According to another aspect of the present invention, the present invention provides the Lactobacillus sakei LBML6 strain deposited under accession number KCCM13011P.

[0019] Lactobacillus assimilates contained in the compositions according to the present invention can exist as live or dead cells, and can also exist in a dried or freeze-dried form. Suitable forms of lactic acid bacteria and formulation methods for inclusion in various compositions are well known to those skilled in the art. For example, Lactobacillus assimilates can be formulated in the form of a culture obtained by culturing in a known liquid or solid medium, a fermented product obtained by culturing the strain together with additional components, an extract obtained by extracting the strain with an organic solvent, or a lysate (or crushed product) obtained by dissolving, crushing, or homogenizing the cell membrane of the strain, but is not limited thereto.

[0020] In one specific example, the composition may contain a Lactobacillus sakei strain that exists as live or dead bacteria.

[0021] In other specific examples, the composition may be a composition comprising a culture, crushed product, extract, or fermented product of Lactobacillus aspergillus strain.

[0022] In other specific examples, the composition may be a composition comprising extracellular vesicles derived from Lactobacillus aspera, its culture, lysate, extract, or fermentation.

[0023] Extracellular vesicles (EVs) enable the exchange of substances (proteins, lipids, genetic materials) between cells and act as mediators for transmitting signals physiologically / pathologically. Extracellular vesicles are broadly classified into exosomes and microvesicles. Exosomes vary in size depending on their biological origin and are intraluminal vesicles generated when the endosomal membrane invaginates during the maturation of multi-vesicular endosomes, and are secreted when the multi-vesicular endosome binds to the cell surface. Microvesicles are vesicles with a size of 10 - 1000 nm, formed by the protrusion and separation of the plasma membrane to the outside and secreted extracellularly. Each cell produces extracellular vesicles differently depending on its physiological state and secretes extracellular vesicles with a specific lipid / protein / nucleic acid composition (I. Zeuk (2019). Considerations on the cell biology of extracellular vesicles. BRIC View 2019 - R03).

[0024] As used herein, the term "extracellular vesicles" is used to include the aforementioned exosomes and microvesicles.

[0025] The exosomes or extracellular vesicles have various diameters ranging from about 1 to 1,000 nm, preferably having a diameter of 10 - 1,000 nm, more preferably 10 - 800 nm, and most preferably 20 - 600 nm.

[0026] The exosomes or extracellular vesicles contained in the composition of the present invention are abundantly present in the culture broth of Lactobacillus sakei (for example, culture supernatant).

[0027] The exosomes or extracellular vesicles are present in large quantities in Lactobacillus aspergillus cultures (e.g., culture supernatant), and the separated and purified exosomes or extracellular vesicles can be used as a therapeutic agent themselves, or cultures, lysates, extracts, or fermentations containing large quantities of the exosomes or extracellular vesicles can be used as a therapeutic agent.

[0028] In this specification, the term "isolation" includes not only the process of selectively obtaining a target substance (e.g., exosomes) from a biological sample (e.g., a Lactobacillus saccharis culture) (positive isolation), but also the process of selectively removing impurities other than the target substance (negative isolation). Therefore, the term "isolation" may be used interchangeably with "obtain," "extract," and "purify." In this specification, the process for separating exosomes or extracellular vesicles may be any method commonly used in the industry, including, but not limited to, the use of the aforementioned commercialized exosome isolation kits (e.g., EXO-BB, ExoQuick®-ULTRA, ExoQuick®-TC, Capturem® Exosome Isolation Kit, Total Exosome Isolation Kit, ExoTrap® Exosome Isolation Spin Column Kit, Exo2DTM, etc.), separation based on differences in specific gravity between components in solution (e.g., centrifugation), separation based on size (e.g., ultrafiltration or vacuum filtration), and separation based on affinity to a specific substrate (e.g., affinity chromatography), as well as any method commonly used in the industry for separation based on the inherent properties of the target substance in a heterogeneous sample.

[0029] According to a preferred embodiment of the present invention, the autoimmune disease is selected from the group consisting of atopic dermatitis, psoriatic dermatitis, alopecia areata, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, Crohn's disease, inflammatory bowel disease, ankylosing spondylitis, lupus, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder, tendinitis, and multiple sclerosis.

[0030] The Lactobacillus aspergillus, its culture, lysate, extract, or fermentate of the present invention; or the extracellular vesicles derived from the Lactobacillus aspergillus, its culture, lysate, extract, or fermentate of the present invention can be effectively used for the prevention or treatment of autoimmune diseases by suppressing pro-inflammatory factors (e.g., TNFα and autoantibodies such as collagen antigen-specific IgG) that cause various autoimmune diseases, or by increasing the proportion of immunosuppressive cells (e.g., tolDCs and Tregs).

[0031] According to a preferred embodiment of the present invention, the composition is a pharmaceutical composition.

[0032] According to another aspect of the present invention, the present invention provides a method for preventing, improving or treating an autoimmune disease, comprising administering a therapeutically effective amount of Lactobacillus aspergillus, its culture, lysate, extract or fermentate; or extracellular vesicles derived from said Lactobacillus aspergillus, its culture, lysate, extract or fermentate; to a target body requiring such treatment.

[0033] According to yet another aspect of the present invention, the present invention provides for therapeutic use of Lactobacillus saccharis, its cultures, crushed products, extracts, or fermented products.

[0034] According to yet another aspect of the present invention, the present invention provides for use in therapy extracellular endoplasmic reticulum derived from Lactobacillus aspergillus, its culture, lysate, extract, or fermentation.

[0035] According to a preferred embodiment of the present invention, the therapeutic use is for the prevention, improvement, or treatment of autoimmune diseases.

[0036] The term "subject" as used herein refers to a mammal that is the subject of treatment, observation, or experimentation, and may preferably be a human or animal requiring prevention, improvement, and / or treatment of an autoimmune disease.

[0037] The pharmaceutical composition according to the present invention can be administered orally or parenterally.

[0038] When administered parenterally, for example, intravenous injection, transdermal injection, subcutaneous injection, intramuscular injection, intravitreal injection, eye drop administration, intracerebral injection, intrathecal injection, intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisional injection, intracoronary injection, intracranial injection, intradural injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection It can be administered via injection, intraperitoneal injection, intrathoracic injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection, local injection, and intraperitoneal injection.

[0039] The pharmaceutical compositions of the present invention may include a pharmaceutically acceptable carrier. In this specification, the term "pharmaceutically acceptable carrier" means a carrier or diluent that does not significantly irritate a living organism and does not inhibit the biological activity and properties of the administered component. Pharmaceutically acceptable carriers in this specification can be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and may be formulated into an injectable dosage form suitable for injection into tissue or organs by adding other common additives such as antioxidants, buffers, and bacteriostatic agents as needed. Alternatively, the dosage form may be an isotonic sterile solution, or optionally a dry formulation (particularly a lyophilized formulation) that can be made into an injectable solution by adding sterile water or physiological saline. Furthermore, target organ-specific antibodies or other ligands may be conjugated to the carrier to enable specific action on the target organ. Suitable formulations known in the art can be those disclosed in the literature (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA).

[0040] Preferably, the composition of the present invention may further contain fillers, excipients, disintegrants, binders, and lubricants. The composition of the present invention can also be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.

[0041] In this specification, “administration” means introducing the composition of the present invention into a target body by a suitable method, and the administration route of the composition of the present invention may be by various routes, oral or parenteral, as long as it can reach the target tissue.

[0042] For example, the composition of the present invention may be administered by intramuscular or intraperitoneal injection during clinical administration.

[0043] For injection, the formulation may preferably be in a pharmacologically suitable buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. For mucosal administration, a non-permeable formulation that conforms to the barrier being passed through is used. Such non-permeable formulations are generally known in the art.

[0044] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0045] In this specification, “effective dose” means the amount necessary to delay or completely halt the onset or progression of a specific disease to be treated, and the effective dose of Lactobacillus asylum contained in the pharmaceutically active composition of the present invention means the amount required to achieve the preventive, ameliorative, or therapeutic effect of an autoimmune disease. Accordingly, the effective dose may be adjusted by various factors, including the type of disease, the severity of the disease, the types and amounts of other components contained in the composition, and the patient’s age, weight, general health, sex and diet, administration time, route of administration, duration of treatment, and drugs used concurrently. It will be apparent to those skilled in the art that an appropriate total daily dose can be determined by the treating physician within the correct range of medical judgment.

[0046] For the purposes of the present invention, it is preferable to individually determine the specific therapeutically effective dose for a particular patient based on various factors, including the type and degree of response to be achieved, the specific composition including whether other formulations are used if applicable, the patient's age, weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, duration of treatment, drugs used in combination with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical field.

[0047] In this specification, “treatment” means an approach to obtain a beneficial or favorable clinical outcome. For the purposes of the present invention, beneficial or favorable clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease extent, stabilization of the disease state (i.e., no worsening), delay or reduction in disease progression, improvement or temporary relief and reduction (partially or entirely) of the disease state, and detection or undetectability. “Treatment” may also mean extending survival compared to the survival rate expected when no treatment is provided. “Treatment” refers to both therapeutic treatment and preventive or preventive measures. These treatments include treatment required for a disorder that has already occurred, as well as for the disorder that is prevented. To “alleviate” a disease means that the extent of the disease state and / or unfavorable clinical signs are reduced and / or the time course of progression is delayed or extended compared to when no treatment is provided.

[0048] According to a preferred embodiment of the present invention, the composition of the present invention is a food composition.

[0049] Lactobacillus aspergillus, its culture, crushed product, extract, or fermented product, or extracellular endoplasmic reticulum derived from Lactobacillus aspergillus, its culture, crushed product, extract, or fermented product, may be subject to all of the above-described provisions as is.

[0050] When the composition of the present invention is used as a food composition, the food composition may include the form of a health functional food, seasoning, beverage, bar, etc. Furthermore, a food composition containing the strain as an active ingredient may include beverages such as fermented milk.Therefore, the present invention provides a lactic acid bacteria starter for food fermentation consisting of Lactobacillus asakei or its culture.

[0051] The food composition of the present invention may be manufactured using, in addition to the above-mentioned active ingredients, food-grade and physiologically acceptable auxiliary agents, such as excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, smoothing agents, or flavoring agents.

[0052] The aforementioned food composition can be suitably formulated as a food composition for administration by including one or more food-grade acceptable carriers in addition to the above-mentioned active ingredients.

[0053] For example, for formulation in the form of tablets or capsules, the active ingredient may be bound to an orally non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. Appropriate binders, lubricants, disintegrants, and colorants may also be included in the mixture as desired or required. Appropriate binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, and xanthan gum. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers that are sterile and suitable for biological use include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added, and the compositions may be formulated as injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0054] The food composition according to the present invention can be added to various foods. Examples of foods to which the composition of the present invention can be added include beverages, vitamin complexes, and health supplements.

[0055] The food composition of the present invention may contain ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates mentioned above include monosaccharides, such as glucose and fructose; disaccharides, such as maltose, sucrose, and oligosaccharides; and polysaccharides, such as dextrin and cyclodextrin, which are common sugars, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings, natural flavorings [thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavorings (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is produced as a drink and beverage, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts.

[0056] According to yet another aspect of the present invention, the present invention provides a feed additive or feed containing as an active ingredient Lactobacillus asie, its culture, crushed product, extract or fermented product; or extracellular vesicles derived from said Lactobacillus asie, its culture, crushed product, extract or fermented product.

[0057] When used as a feed additive, the composition may be manufactured as a highly concentrated liquid of 20-90%, or in powder or granular form. The feed additive may further contain one or more of the following: organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polymerized phosphate); and natural antioxidants such as polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. When used as feed, the composition may be formulated in the usual feed form and may also contain the usual feed components.

[0058] The aforementioned feed additives and feeds may further contain grains, such as ground or crushed wheat, oats, barley, corn, and rice; plant-based protein feeds, such as feeds mainly composed of rapeseed, beans, and sunflowers; animal-based protein feeds, such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products, such as dried components consisting of various milk powders and whey powders, and may also further contain nutritional supplements, digestive and absorption enhancers, growth promoters, and the like.

[0059] The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additive can be easily administered to animals as a top dressing, mixed directly into animal feed, or in an oral dosage form separate from the feed. When the feed additive is administered separately from animal feed, it can be prepared in combination with a food-grade acceptable carrier, as is well known in the art, to produce an immediate-release or sustained-release dosage form. Such carriers may be solid or liquid, such as corn starch, lactose, sucrose, bean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of tablets, capsules, powders, lozenges, sugar-containing tablets, or a mildly acidic top dressing. When a liquid carrier is used, the feed additive may be in the form of gelatin capsules, syrups, suspensions, emulsions, or solutions.

[0060] Furthermore, the feed additive and feed may contain auxiliary agents, such as preservatives, stabilizers, wetting agents or emulsifiers, and solution accelerators. The feed additive may be used by immersion, spraying, or mixing and adding it to animal feed.

[0061] The feed or feed additive of the present invention can be applied to a wide range of animal diets, including those of mammals, poultry, and fish.

[0062] As the mammals, it can be used for pigs, cows, sheep, goats, laboratory rodents, and in addition to laboratory rodents, for pets (e.g., dogs, cats), etc. As the poultry, it can also be used for chickens, turkeys, ducks, geese, pheasants, and quails, etc. As the fish, it can be used for salmon, etc., but is not limited thereto.

[0063] The feed or feed additive of the present invention is applied to animal diets and can be used for animal growth, immune enhancement, etc.

[0064] The amount of Lactobacillus sakei strain contained in the composition according to the present invention may be about 10 6 ~10 12 cfu / ml, for example, 10 7 ~10 11 cfu / ml, 10 8 ~10 10 cfu / ml. When administering the strain, it is preferably administered in the viable state, and can be administered after being killed or in an attenuated state before ingestion. Also, when producing using a culture supernatant, etc., a sterilization process by heat treatment can be further performed. The amount of the strain necessary to have the minimum efficacy and the degree of daily ingestion may vary depending on the body or health condition of the ingestor, but generally, it may be about 10 6 ~10 12 cfu / ml, for example, 10 7 ~10 11 cfu / ml, 10 8 ~10 10 cfu / ml.

[0065] According to still another aspect of the present invention, the present invention provides a method for producing a composition for preventing, improving, or treating an autoimmune disease, including the following steps: (a) preparing a Lactobacillus sakei strain; and (b) culturing the strain in a culture medium.

[0066] According to a preferred embodiment of the present invention, the production method further comprises the step of separating extracellular vesicles derived from the Lactobacillus aspergillus strain from the culture medium.

[0067] According to yet another aspect of the present invention, the present invention provides a method for producing a composition for the prevention, improvement, or treatment of an autoimmune disease, comprising the step of preparing extracellular vesicles derived from Lactobacillus aspergillus strain.

[0068] [Effects of the invention] The features and advantages of this invention can be summarized as follows: (i) The present invention provides a composition for the prevention, improvement, or treatment of autoimmune diseases, comprising as an active ingredient Lactobacillus aspergillus, its culture, crushed product, extract, or fermented product; or extracellular endoplasmic reticulum derived from said Lactobacillus aspergillus, its culture, crushed product, extract, or fermented product.

[0069] (ii) The Lactobacillus sakei or extracellular endoplasmic reticulum contained in the composition of the present invention can be effectively used for the prevention or treatment of autoimmune diseases by suppressing pro-inflammatory factors (e.g., TNFα and autoantibody collagen antigen-specific IgG) that cause various autoimmune diseases, or by increasing the proportion of immunosuppressive cells (e.g., tolDC and Tregs).

[0070] [Brief explanation of the drawing] [Figure 1] This graph shows the arthritis score and incidence rate after 17 weeks of oral administration of the Lactobacillus sakei LBML6 strain to a collagen-induced arthritis mouse model.

[0071] [Figure 2] This shows the results of measuring the concentration of TNFα, a proinflammatory cytokine, in blood samples taken from experimental animals.

[0072] [Figure 3] This shows the results of measuring the concentration of collagen antigen-specific autoantibody IgG in blood collected from experimental animals.

[0073] [Figure 4] This shows the results of measuring the percentage of tolerogenic dendritic cells (tolDCs) that suppress immunity in the intestinal lymph nodes.

[0074] [Figure 5] This shows the results of measuring the percentage of regulatory T cells (Tregs) that suppress immunity in the intestinal lymph nodes.

[0075] [Figure 6] Shows the size of extracellular organisms (EVs) isolated from Lactobacillus sakei LBML6 strain.

[0076] [Figure 7] Shows the concentration of EVs isolated from Lactobacillus sakei LBML6 strain.

[0077] [Figure 8] This shows the results of confirming the expression of K10, a cleavage cell division marker, during EV treatment of Lactobacillus sakei LBML6 strain or cells isolated from it.

[0078] [Figure 9] This shows the results of confirming the expression of involucrin, a cell division marker for skin cells, during EV treatment of the Lactobacillus sakei LBML6 strain or cells isolated from it.

[0079] [Figure 10] This shows the results of confirming whether the activity of CD4 T cells is suppressed when treated with Lactobacillus sakei LBML6 strain or EV cells isolated from it.

[0080] [Modes for carrying out the invention] The present invention will be described in more detail below using examples. These examples are merely for the purpose of illustrating the present invention in more detail, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited to these examples, as is the essence of the invention.

[0081] Examples Materials and methods 1. Preparation of the Lactobacillus sakei LBML6 strain The Lactobacillus sakei LBML6 strain, deposited with the Korea Microbial Conservation Center on June 15, 2021, under deposit number KCCM13011P, was inoculated at 0.1% in 30 ml of MRS liquid medium and cultured at 30°C for 18 hours. After culturing, the cells were centrifuged at 3500 rpm for 10 minutes, washed three times with PBS solution, and then the remaining medium components were removed to obtain the final product.

[0082] 2. Preparation of experimental animals This study was conducted to evaluate the effects of the test substance after creating a rheumatoid arthritis model, one of the autoimmune diseases, using bovine collagen in DBA-1J mice.

[0083] 3. Administration of the test substance The test substances for administration in the examples were prepared by weighing an appropriate amount of the test substance and then diluting it with an excipient. The composition of the test groups and the dosage settings are as shown in Table 1 below.

[0084] [Table 1]

[0085] Each test substance was administered orally at a dose of 200 μL per animal, 5 times per week for 7 weeks, while the control group received only physiological saline. Oral administration was performed by fixing the animals with a neck-and-back skin fixation method and directly administering the substance into the stomach using an oral administration tube.

[0086] The test groups were configured and dosages were determined by treating the following groups: Normal (Control; PBS), CIA (arthritis-induced + PBS), and CIA + strain (arthritis-induced + the strain of the present invention).

[0087] 4. Separation of EVs Lactobacillus sakei (LBML6) was inoculated at 0.1% in 30 ml of MRS liquid medium and cultured at 30°C for 18 hours. After culturing, primary centrifugation was performed at 3500 rpm at 4°C for 10 minutes, followed by secondary centrifugation at 10,000 x g at 4°C for 20 minutes to collect the culture supernatant. The supernatant was then filtered through a 0.22 μm filter to remove the bacterial cells. The culture supernatant and an equal volume of 1 M NaCl solution containing 16% PEG6000 were mixed and reacted at 4°C for 15 hours. After centrifugation at 10,000 x g at 4°C for 20 minutes, an EV pellet was obtained. The obtained EV pellet was resuspended in 0.5 M NaCl solution containing 5% PEG6000, washed, and then centrifuged at 11,000 rpm at 4°C for 20 minutes. The pellet was then resuspended in PBS to finally isolate EVs derived from Lactobacillus sakei (LBML6). The separated EVs were measured for size (Figure 6) and concentration (Figure 7) using ZetaView (Particle Metrix GmbH).

[0088] 5. Differentiation of immature dendritic cells from bone marrow. Monocytes were isolated from bone marrow cells (Orient Bio, Balb / c, female, 6 weeks old) of mouse femurs and tibias by centrifugation using the Ficoll gradient method (Hlozkova, K., Starkova, J. Assessment of the Metabolic Profile of Primary Leukemia Cells. J.Vis.Exp.(141), e58426, doi:10.3791 / 58426(2018)). The isolated monocytes were divided into 2 × 10⁶ cells. 6 Cells were prepared in a 6-well plate at a concentration of / well, and treated with GM-CSF 20 ng / ml and IL-4 10 ng / ml in a culture medium containing fetal bovine serum (FBS) (RPMI). After that, the cells were cultured for 6 days, and after 3 days, the medium was replaced with fresh medium and cytokines to obtain differentiated immature dendritic cells.

[0089] 6. Differentiation of mature dendritic cells To differentiate the immature dendritic cells (Immature DCs) obtained above into mature dendritic cells (Mature DCs), they were treated with a positive control group LPS for 2 hours to induce differentiation and activity into mature dendritic cells. Subsequently, Lactobacillus aspergillus (LBML6), a secondary stimulus source, and Lactobacillus aspergillus (LBML6)-derived EVs were co-cultured at MOI 1 and 10 for 24 hours, respectively.

[0090] 7.T cell co-culture CD3+ T cells stained with CFSE (Invitrogen) and isolated from mouse spleens were co-cultured for 3 days in a 1:1 ratio with Lactobacillus saccharis (LBML6) and dendritic cells cultured for 24 hours. To evaluate whether T cell activity was suppressed by tolDC, the degree of T cell division (CFSE) and the amount of secreted cytokines (IFNr, IL-17) were confirmed using a CBA assay kit (BD Th1 / 2 / 17CBA kit). The culture medium from the co-culture was stored and used for application to an in vitro model of psoriasis.

[0091] 8. Preparation of a psoriasis model using human keratinocytes 4 x 10 5 Hacat cells (CLS) were dispensed into 6 wells. Upon cell attachment, the cells were treated with CaCl2 (Sigma) for 4 hours, and further treated with imiquimod (IMQ, Calbiochem) to induce cell inflammation. After 24 hours, the culture medium used in 5 experiments was treated in each well, and after 24 hours, the cells were harvested and isolated to RNA for gene expression measurement.

[0092] 9. Gene expression measurement (qPCR) For gene expression measurement in each cell, cellular RNA was synthesized into cDNA using reverse thranscriptase and dNTPs from a cDNA synthesis kit (Thermo). This was then amplified using each gene's primer (macrogen).

[0093] Experimental results Example 1. Measurement of arthritis incidence and index in a collagen-induced arthritis mouse model. Arthritis score measurement: Immediately before administration of the test substance, and three times a week during the study period after administration, the degree of arthritis was scored according to the criteria shown in Table 2 below. The scores for all four limbs were totaled, with a maximum score of 16. The scoring table for each condition is as follows:

[0094] [Table 2]

[0095] Figure 1 shows the arthritis score and incidence rate after 17 weeks of oral administration of the Lactobacillus sakei LBML6 strain to a collagen-induced arthritis mouse model.

[0096] As a result, the highest incidence rate and arthritis index were observed in the collagen-induced arthritis control group, and a significant reduction in the incidence rate and arthritis index was confirmed in the Lactobacillus sakei LBML6 strain-treated group.

[0097] Example 2. Measurement of blood TNFα and collagen antigen-specific IgG concentrations Blood samples were collected from experimental animals, and the concentrations of the inflammatory cytokine TNFα and the autoantibody collagen antigen-specific IgG were measured. Figure 2 shows the results of the TNFα concentration measurement, and Figure 3 shows the results of the IgG and collagen antigen-specific IgG concentration measurement.

[0098] As shown in Figures 2 and 3, it was confirmed that the production of TNFα and IgG in the blood was promoted when arthritis was induced compared to the normal group, and that the concentrations of TNFα and IgG decreased when the Lactobacillus sakei LBML6 strain was administered to experimental animals.

[0099] Example 3. Measurement of immunosuppressive cells in intestinal lymph nodes The proportions of tolerogenic dendritic cells (tolDCs), which suppress the immune system, and regulatory T cells (Tregs) were examined in the intestinal lymph nodes. Figure 4 shows the results of measuring the proportion of tolDC cells, and Figure 5 shows the results of measuring the proportion of Tregs cells.

[0100] As shown in Figures 4 and 5, we confirmed that the proportion of immunosuppressive tolDC cells and Tregs cells in the intestinal lymph nodes increased when the Lactobacillus sakei LBML6 strain was administered, compared to the normal group and the arthritis-induced control group.

[0101] Example 4. Psoriasis relief efficacy of LBML6 Treating HacaT cells with CaCl2 and IMQ can induce cell division and inflammation, thereby inducing the formation of the stratum corneum. When the expression of K10 (Figure 8) and involucrin (Figure 9), which are markers of skin cell division, was examined, gene expression was found to be significantly reduced in culture media induced by LBML6 (MOI 1) and LBML6 EV, thus confirming that skin cell division is suppressed.

[0102] Example 5. Efficacy to suppress CD4 T cell activity Increased CD4 T cell division is known to activate T cells and participate in various inflammatory responses. To investigate whether tolDCs induced by Lactobacillus sakei-derived EVs can directly suppress CD4 T cell activity, Lactobacillus sakei (LBML6) and tolDCs induced by Lactobacillus sakei-derived EVs were co-cultured with CFSE-stained CD4 T cells in a 1:1 ratio for 3 days. After 3 days, the co-cultured cells were harvested and the degree of CFSE reduction was measured.

[0103] T cells co-cultured with dendritic cells treated with LPS showed approximately 90% cell division. However, T cells co-cultured with dendritic cells treated with Lactobacillus sakei-derived EV cells showed approximately 60% suppressed cell division. This suggests that tolDCs induced by Lactobacillus sakei-derived EV cells can directly suppress CD4 T cell activity and reduce the immune response.

[0104] Based on these results, it can be confirmed that Lactobacillus sakei (LBML6) and Lactobacillus sakei (LBML6)-derived extracellular derivatives suppress CD4 T cell activity by reducing dendritic cell (DC) activity, thereby reducing the immune response. Therefore, they can regulate various autoimmune diseases, including rheumatoid arthritis.

[0105] Although embodiments of the present invention have been described above, any person with ordinary skill in the relevant art can modify and change the present invention in various ways, such as by adding, changing, deleting, or adding components, without departing from the spirit of the invention as described in the claims, and these modifications are also considered to fall within the scope of the rights of the present invention.

[0106] [Accession Number] Depository name: Korea Microbial Conservation Center (overseas) Accession number: KCCM13011P Date of acceptance: 20210615 Depository name: Korea Microbial Conservation Center (overseas) Accession number: KCCM12654P Date of acceptance: 20200114 Depository name: Korea Institute of Biotechnology Accession number: KCTC13818BP Date of acceptance: 20190307

[0107] [Table 3]

[0108] [Table 4]

[0109] [Table 5] [Brief explanation of the drawing]

[0110] [Figure 1] This graph shows the arthritis score and incidence rate after 17 weeks of oral administration of the Lactobacillus sakei LBML6 strain to a collagen-induced arthritis mouse model. [Figure 2] This is the result of measuring the concentration of TNFα, a proinflammatory cytokine, in the blood of experimental animals. [Figure 3] This is the result of measuring the concentration of collagen antigen-specific IgG autoantibody in blood collected from experimental animals. [Figure 4] This is the result of measuring the percentage of tolerogenic dendritic cells (tolDCs) that suppress the immune system in the intestinal lymph nodes. [Figure 5] This is the result of measuring the proportion of regulatory T cells (Tregs) that suppress immunity in the intestinal lymph nodes. [Figure 6] This shows the size of extracellular organisms (EVs) isolated from Lactobacillus sakei LBML6 strain. [Figure 7] This shows the concentration of extracellular viable cells (EVs) isolated from Lactobacillus sakei LBML6 strain. [Figure 8] This is the result of confirming the expression of K10, a cleavage cell division marker, during EV treatment of Lactobacillus sakei LBML6 strain or cells isolated from it. [Figure 9] This is the result of confirming the expression of involucrin, a cell division marker for skin cells, during EV treatment of the Lactobacillus sakei LBML6 strain or a strain isolated from it. [Figure 10] This study confirmed whether the activity of CD4 T cells was suppressed during EV treatment with Lactobacillus sakei LBML6 strain or cells isolated from it.

Claims

1. A composition for the prevention, improvement, or treatment of autoimmune diseases, comprising as an active ingredient Lactobacillus sakei; or a culture thereof; or extracellular vesicles derived from said Lactobacillus sakei; The aforementioned Lactobacillus sakei is the Lactobacillus sakei LBML6 strain deposited under accession number KCCM13011P. The autoimmune disease is selected from the group consisting of psoriatic dermatitis, psoriatic arthritis, osteoarthritis, and rheumatoid arthritis, and is a composition for the prevention, improvement, or treatment of an autoimmune disease.

2. The composition according to claim 1, characterized in that the composition is a pharmaceutical composition or a food composition.

3. The composition according to claim 1, characterized in that the Lactobacillus sakei or its culture contains the extracellular vesicle of Lactobacillus sakei.

4. The composition according to claim 1 or 3, characterized in that the diameter of the extracellular endoplasmic reticulum is 10 to 1,000 nm.

5. A method for producing a composition for the prevention, improvement, or treatment of autoimmune diseases, comprising the following steps: (a) A step of preparing a Lactobacillus sakei strain, wherein the Lactobacillus sakei strain is the Lactobacillus sakei LBML6 strain deposited under accession number KCCM13011P; and (b) The step of culturing the bacterial strain in a culture medium; The method for producing a composition for the prevention, improvement, or treatment of an autoimmune disease, wherein the autoimmune disease is selected from the group consisting of psoriatic dermatitis, psoriatic arthritis, osteoarthritis, and rheumatoid arthritis.

6. A method for producing a composition for the prevention, improvement, or treatment of an autoimmune disease, comprising the step of preparing extracellular vesicles derived from Lactobacillus aspergillus strain, The aforementioned Lactobacillus sakei strain is the Lactobacillus sakei LBML6 strain deposited under accession number KCCM13011P. The method for producing a composition for the prevention, improvement, or treatment of an autoimmune disease, wherein the autoimmune disease is selected from the group consisting of psoriatic dermatitis, psoriatic arthritis, osteoarthritis, and rheumatoid arthritis.

7. The Lactobacillus sakei LBML6 strain was deposited under accession number KCCM13011P.

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