KR20230008619A - Composition for treating autoimmune disease comprising lactobacillus sakei or extracellular vesicle derived therefrom as an active ingredient - Google Patents

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 according to the present invention is a strain which has TNFα and IgG inhibitory activity in blood and in which the ratio of tolDC cells to Tregs cells increases, thereby being used for various applications such as the prevention, alleviation, and/or treatment of autoimmune diseases of humans or animals, and intestinal regulation.

Description

Composition for treating autoimmune disease comprising lactobacillus sakei or extracellular vesicles derived therefrom as an active ingredient}

The present invention relates to a composition for preventing, improving or treating autoimmune diseases comprising Lactobacillus sakeai , its culture, lysate, extract or fermented product or extracellular vesicles isolated therefrom as an active ingredient.

An autoimmune disease is a disease that occurs when the body's immune function attacks itself. It is a common example that it is formed over a long period of time, the symptoms persist chronically, and it usually causes permanent damage to organs, and there is almost no cure. reality is that there is no Over a long period of time, knowledge about autoimmune diseases has advanced a lot, but the precise mechanism of their formation, identity of autoantigens, and regulatory genetic factors are still unclear. Autoimmune diseases can be broadly classified into organ-specific diseases and systemic diseases.

Organ-specific autoimmune diseases are caused by an immune response to organ-specific antigens and can occur in almost all organs of the body. Systemic autoimmune diseases are caused by an immune response to antigens expressed throughout the body, rather than an immune response to a specific cell. These systemic autoimmune diseases can also selectively cause diseases in specific organs.

Examples of autoimmune diseases include asthma, atopic dermatitis, psoriatic dermatitis, allergic rhinitis, allergic conjunctivitis, urticaria, and the like, which are known to cause damage and abnormality to body organs due to allergic reactions and cause clinical symptoms. In addition, rheumatoid arthritis and lupus, which are known to cause diseases by showing hypersensitivity reactions (including autoantibody reactions) to their own proteins (autoantigens such as immunoglobulin, collagen, and DNA) (Vaughan JH. Med Times 1969;97 :187-204) and the like. The lupus and rheumatoid arthritis, etc., cause damage and inflammation to specific organs by antigen-specific IgG antibodies that react with antigens (autoantigens) present in the human body due to hypersensitivity to self-proteins present in the human body. It is known that it does, and it is classified as an autoimmune disease accordingly.

To date, the direct cause of autoimmune disease has not yet been clearly identified, and various therapeutic agents such as steroids, non-steroidal anti-inflammatory drugs, and immunosuppressants have been used for its treatment. However, these treatments do not exhibit fundamental therapeutic effects and their use is limited due to various side effects. In addition, some chemotherapeutic agents have drawbacks such as lack of efficacy against already occurring autoimmune diseases.

Therefore, there is a demand for the development of an autoimmune disease treatment that is effective in alleviating inflammatory symptoms and pain while having fewer side effects and is easy to take.

The matters described as the above background art are only for improving understanding of the background of the present invention, and should not be taken as an admission that they correspond to prior art already known to those skilled in the art.

The present inventors have tried to find lactic acid bacteria strains applicable to various types of autoimmune diseases including rheumatoid arthritis. As a result, by confirming that Lactobacillus sakei or its culture inhibits pro-inflammatory factors that cause various autoimmune diseases or increases the ratio of immunosuppressive cells to prevent or treat autoimmune diseases, completed the present invention.

Therefore, an object of the present invention is Lactobacillus sakei ( Lactobacillus sakei ), its culture, lysate, extract or fermented product; It is to provide a composition for preventing, improving or treating autoimmune diseases comprising as an active ingredient; or an extracellular vesicle derived from the Lactobacillus sakei .

Another object of the present invention is a therapeutically effective amount of Lactobacillus sakeai , its culture, lysate, extract or fermented product; It is to provide a method for preventing, improving or treating autoimmune diseases comprising administering to a subject in need thereof; or an extracellular vesicle derived from the Lactobacillus sakei.

Another object of the present invention is to provide a therapeutic use (for use in therapy) of Lactobacillus sakeai , its culture, lysate, extract or fermented product.

Another object of the present invention is to provide a therapeutic use of extracellular vesicles derived from Lactobacillus sakei , its culture, lysate, extract or fermented product.

Another object of the present invention is to provide a method for preparing a composition for preventing, improving or treating autoimmune diseases.

Another object of the present invention is to provide a Lactobacillus sakei LBML6 strain deposited with accession number KCCM13011P.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention Lactobacillus Sakeai , its culture, lysate, extract or fermented product; Or it provides a composition for preventing, improving or treating autoimmune diseases comprising as an active ingredient; extracellular vesicles derived from (derived) the Lactobacillus sakei .

The Lactobacillus sakeai strain usable in the present invention is not limited, but preferably, the Lactobacillus sakeai LBML6 strain deposited with the Korea Microorganism Conservation Center under the accession number KCCM13011P or the Lactobacillus sakeai WIKIM31 strain and organism deposited under the accession number KCCM12654P It includes the Lactobacillus sakeai WIKIM0109 strain deposited with the Resource Center under accession number KCTC13818BP, most preferably the Lactobacillus sakeai LBML6 strain.

According to another aspect of the present invention, the present invention provides a Lactobacillus Sakeai LBML6 strain deposited with accession number KCCM13011P.

Lactobacillus sakei included in the composition according to the present invention may exist as live cells or dead cells, and may also exist in a dried or lyophilized form. Types of lactic acid bacteria suitable for inclusion in various compositions and formulation methods are well known to those skilled in the art. For example, Lactobacillus sakeai is a culture obtained by culturing in a known liquid medium or solid medium, a fermentation product obtained by culturing the strain and additional components together, or an extract obtained by extracting the strain with an organic solvent; It may be formulated in the form of a lysate (or lysate) obtained by dissolving the cell membrane of the strain, or crushing or homogenizing, but is not limited thereto.

In one embodiment, the composition may be a composition containing a strain of Lactobacillus sakei present as live or dead bacteria.

In another embodiment, the composition may be a composition comprising a culture, lysate, extract or fermented product of a strain of Lactobacillus sakei .

In another embodiment, the composition may be a composition comprising extracellular vesicles derived from Lactobacillus sakei , its culture, lysate, extract or fermentation product.

Extracellular vesicles (EVs) enable the exchange of substances (proteins, lipids, genetic materials) between cells and function as physiological/pathological signal transduction mediators. Extracellular vesicles are largely classified into exosomes and microvesicles. Exosomes vary in size depending on the origin of the organism, and are intraluminal vesicles created by the endosome membrane coming in during the maturation of multi-vesicular endosomes. It is secreted when it binds to the cell surface. Microvesicles are vesicles with a size of 10-1000 nm, and the plasma membrane protrudes to the outside and is separated and secreted out of the cell. Each cell produces extracellular vesicles differently depending on the physiological state, and secretes extracellular vesicles with specific lipid/protein/nucleic acid composition (Lee Jae-wook (2019). Illumination of the cell biology of extracellular endoplasmic reticulum. BRIC View 2019-R03 ).

In the present specification, the term "extracellular vesicles" is used to mean including the exosomes and microvesicles.

The exosomes or extracellular vesicles have diameters varying within the range of about 1-1,000 nm, preferably 10-1,000 nm, more preferably 10-800 nm, most preferably 20-600 nm. .

Exosomes or extracellular vesicles included in the composition of the present invention are included in a large amount in the Lactobacillus sakei culture medium (eg, culture supernatant).

The exosomes or extracellular vesicles are contained in a large amount in the Lactobacillus sakei culture (eg, culture supernatant), and the purified exosomes or extracellular vesicles themselves are used as a therapeutic agent, or the exosomes Alternatively, a culture, lysate, extract, or fermented product containing a large amount of extracellular vesicles may be used as a therapeutic agent.

As used herein, the term "isolation" refers to a process of selectively obtaining a desired substance (eg, exosome) within a biological sample (eg, Lactobacillus sakei culture) (positive isolation) as well as It includes all processes of selectively removing impurities other than the target material (negative isolation). Therefore, the term "separation" can be used in the same sense as "obtain", "extract", and "purify". In the present specification, in the process of separating exosomes or extracellular vesicles, all methods commonly used in the art may be used without limitation, for example, the commercially available exosome separation kit (eg, EXO-BB , ExoQuick ^{® -ULTRA, ExoQuick ® -TC} , Capturem ^TM Exosome Isolation Kit, Total Exosome Isolation Kit, ExoTrap ^TM Exosome Isolation Spin Column Kit, Exo2DTM, etc.) in heterogeneous samples, including, but not limited to, separation based on size (e.g. ultrafiltration or vacuum filters), separation based on affinity for a particular substrate (e.g. affinity chromatography), As a separation method based on the inherent physical properties of the target material, all methods commonly used in the art may be used without limitation.

According to a preferred embodiment of the present invention, the autoimmune disease is atopic dermatitis, psoriatic dermatitis, alopecia areata, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, Crohn's disease, inflammatory colitis, ankylosing spondylitis, lupus , psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, and multiple sclerosis.

Lactobacillus sakei of the present invention, its culture, lysate, extract or fermented product; Or the extracellular vesicles derived from the Lactobacillus sakeai , its culture, lysate, extract or fermented product are pro-inflammatory factors that cause various autoimmune diseases (eg, TNFα and Autoantibody collagen antigen-specific IgG, etc.) It can be effectively used for preventing or treating autoimmune diseases by suppressing or increasing the ratio of immune suppressor cells (eg, tolDC and Tregs).

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

According to another aspect of the present invention, the present invention provides a therapeutically effective amount of Lactobacillus sakeai , a culture, lysate, extract or fermented product thereof; Or the Lactobacillus sakeai , its culture, lysate, extract, or extracellular vesicles derived from fermentation thereof; to a subject in need thereof to prevent, improve or treat an autoimmune disease comprising administering to provide a method.

According to another aspect of the present invention, the present invention provides a therapeutic use (for use in therapy) of Lactobacillus sakei , its culture, lysate, extract or fermented product.

According to another aspect of the present invention, the present invention provides for use in therapy of extracellular vesicles derived from Lactobacillus sakeai , cultures, lysates, extracts or fermentations thereof.

According to a preferred embodiment of the present invention, the therapeutic use is for preventing, ameliorating or treating autoimmune diseases.

As used herein, “subject” refers to a mammal that is a subject of treatment, observation, or experimentation, and may preferably be a human or animal that requires prevention, improvement, and/or treatment of an autoimmune disease.

The pharmaceutical composition according to the present invention may be administered orally or parenterally.

In the case of parenteral administration, such as intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intravitreal injection, eye drop administration, intracerebroventricular injection, intrathecal injection (intrathecal injection), intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection ( intracerebral injection), intracisternal injection, intracoronary injection, intracranial injection, intrathecal injection, epidural injection, intrahippocampal injection ), intranasal injection, intraosseous injection, intraperitoneal injection, intrathoracic injection, intraspinal injection, intrathoracic injection, Intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection bconjunctival injection), intratumoral injection, local injection, and intraperitoneal injection.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means a carrier or diluent that does not significantly stimulate living organisms and does not inhibit the biological activity and properties of the administered component. The pharmaceutically acceptable carrier in the present specification may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or a mixture of one or more of these components, If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added, and formulated into an injection suitable for injection into tissues or organs. In addition, it may be formulated as an isotonic sterile solution or, in some cases, as a dry preparation (particularly a freeze-dried preparation) that can be an injectable solution by adding sterile water or physiological saline. In addition, a target organ-specific antibody or other ligand may be used in combination with the carrier so as to specifically act on the target organ. Suitable formulations known in the art may be used those disclosed in the literature (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA).

In addition, preferably, the composition of the present invention may further include fillers, excipients, disintegrants, binders, lubricants, and the like. In addition, the composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

As used herein, "administration" means introducing the composition of the present invention to a subject by any suitable method, and the route of administration of the composition of the present invention is administration through various routes, oral or parenteral, as long as it can reach the target tissue. It can be.

For example, the composition of the present invention can be administered by intramuscular or intraperitoneal injection in clinical administration.

For injection, it may preferably be formulated in a pharmacologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, non-penetrating agents suitable for the barrier to be passed are used in the formulation. Such impermeable agents are generally known in the art.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

As used herein, "effective amount" means an amount necessary to delay or completely stop the onset or progression of a specific disease to be treated, and an effective amount of Lactobacillus sakeai included in the pharmaceutical composition of the present invention is an autoimmune It means the amount required to achieve the effect of preventing, improving or treating a disease. Therefore, the effective amount depends on the type of disease, the severity of the disease, the type and amount of other components contained in the composition, and the patient's age, weight, general health condition, sex and diet, administration time, administration route, treatment period, concurrent use It can be adjusted according to various factors including the drug used. It is obvious to those skilled in the art that an appropriate total daily amount can be determined by a treating physician within the scope of sound medical judgment.

For purposes of this invention, a specific therapeutically effective amount for a particular patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general health condition, and gender. And it is preferable to apply differently according to various factors including diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with the specific composition, and similar factors well known in the medical field.

As used herein, "treatment" refers to an approach for obtaining beneficial or desirable clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, disease state improvement or palliation and relief (partial or total), detectable or undetectable. "Treatment" can also mean prolonging the survival rate compared to the expected survival rate if not receiving the treatment. “Treatment” refers to both therapeutic treatment and prophylactic or prophylactic measures. The treatments include treatment required for disorders that have already occurred as well as disorders that are prevented. “Relieving” a disease means that the extent of the disease state and/or undesirable clinical signs are reduced and/or the time course of the disease is slowed or lengthened, compared to no treatment. do.

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

Lactobacillus Sakeai contained in the food composition of the present invention, its culture, lysate, extract or fermented product; Alternatively, Lactobacillus sakei , its culture, lysate, extract, or extracellular vesicles derived from fermented product may be applied as described above.

When the composition of the present invention is used as a food composition, the food composition may include a health functional food or seasoning, beverage, bar, or the like. In addition, the food composition containing the strain as an active ingredient may include beverages such as fermented milk. Accordingly, the present invention provides a lactic acid bacteria starter for food fermentation comprising Lactobacillus sakeai or a culture thereof.

The food composition of the present invention may be prepared using food-compatible and physiologically acceptable adjuvants in addition to the above active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, and lubricants. agents or flavoring agents may be used.

For administration, the food composition may be preferably formulated as a food composition by including at least one food chemically acceptable carrier in addition to the above-described active ingredients.

For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable 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, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. ates, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. For compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The food composition according to the present invention can be added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, health supplements, and the like.

The food composition of the present invention may include ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is made into drinks and beverages, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included.

According to another aspect of the present invention, the present invention is Lactobacillus Sakeai , its culture, lysate, extract or fermented product; Or the Lactobacillus sakeai , its culture, lysate, extract or extracellular vesicles derived from fermentation; provides a feed additive or feed containing as an active ingredient.

When used as a feed additive, the composition may be at a high concentration of 20 to 90% or prepared in powder or granular form. The feed additives are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, and rosemary. extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like, or any one or more natural antioxidants may be further included. When used as a feed, the composition may be formulated in a conventional feed form, and may include a common feed component together.

The feed additives and feeds include grains such as pulverized or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean, and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; It may further include sugar and dairy products, for example, dry ingredients composed of various powdered milk and whey powder, etc., and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered to animals as a top dressing, or directly mixed with animal feed, or in an oral formulation separate from feed. When the feed additive is administered separately from animal feed, as is well known in the art, it can be prepared as an immediate release or sustained release formulation by combining it with an edible carrier acceptable for food science. Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be a gelatin soft capsule, or a syrup, suspension, emulsion, or solution formulation.

In addition, the feed additives and feeds may contain auxiliary agents, for example, preservatives, stabilizers, wetting agents or emulsifying agents, solution accelerators, and the like. The feed additive may be used by adding it to the animal's feed by soaking, spraying or mixing.

The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish.

As the mammal, it can be used for pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (eg dogs, cats), etc., and as the poultry, chickens, turkeys, ducks, geese, pheasants, And it can also be used for quail, etc., and can be used for trout as the fish, but is not limited thereto.

The feed or feed additive of the present invention can be applied to animal diet and used for animal growth, immunity enhancement, and the like.

The amount of Lactobacillus sakei strain included in the composition according to the present invention may be about 10 ⁶ to 10 ¹² cfu / ml, for example, 10 ⁷ to 10 ¹¹ cfu / ml, 10 ⁸ to 10 ¹⁰ cfu/ml. When administering a strain, it is preferable to administer it in a viable state, and it can be killed before ingestion or administered in an attenuation state. In addition, when manufacturing using a culture supernatant, etc., a sterilization process through a heat treatment process may be additionally performed. The amount of strain required to have the minimum efficacy and the daily intake level may vary depending on the body or health condition of the consumer, but may generally be about 10 ⁶ to 10 ¹² cfu/ml, for example, 10 ⁷ to 10 ¹¹ cfu/ml , 10 ⁸ to 10 ¹⁰ cfu/ml.

According to another aspect of the present invention, the present invention provides a method for preparing a composition for preventing, improving or treating autoimmune diseases, comprising the following steps:

(a) preparing a Lactobacillus Sakeai strain; and

(b) culturing the strain in a culture medium.

According to a preferred embodiment of the present invention, the preparation method further comprises the step of separating extracellular vesicles derived from the Lactobacillus sakeai strain from the culture medium.

According to another aspect of the present invention, the present invention provides a method for preparing a composition for preventing, improving or treating autoimmune diseases, comprising the step of preparing extracellular vesicles derived from the Lactobacillus sakeai strain.

The features and advantages of the present invention are summarized as follows:

(i) the present invention Lactobacillus Sakeai , its culture, lysate, extract or fermented product; Or the Lactobacillus sakeai , its culture, lysate, extract or extracellular vesicles derived from fermentation; to provide a composition for preventing, improving or treating autoimmune diseases comprising as an active ingredient.

(ii) Lactobacillus sakei or extracellular vesicles contained in the composition of the present invention inhibits pro-inflammatory factors (eg, TNFα and Autoantibody collagen antigen-specific IgG, etc.) that cause various autoimmune diseases, or It can be effectively used for preventing or treating autoimmune diseases by increasing the ratio of suppressor cells (eg, tolDC and Tregs).

1 shows the arthritic score and the incidence rate for 7 weeks after oral administration of the Lactobacillus sakei LBML6 strain to a collagen-induced arthritis mouse model.
2 is a result of measuring the concentration of TNFα, a proinflammatory cytokine, in the blood of experimental animals.
3 is a result of measuring the concentration of Autoantibody collagen antigen-specific IgG in the blood of experimental animals.
Figure 4 is a result of measuring the ratio of tolerogenic dendritic cells (tolDC) cells that suppress immunity in the intestinal lymph nodes.
5 is a result of measuring the ratio of immunosuppressive Regulatory T Cells (Tregs) cells in the intestinal lymph nodes.
Figure 6 shows the size of EVs isolated from Lactobacillus Sakeai LBML6 strain.
Figure 7 shows the concentration of EVs isolated from Lactobacillus Sakeai LBML6 strain.
8 is a result of confirming the expression of K10, a mitosis marker of skin cells, when the Lactobacillus Sakeai LBML6 strain or an EV isolated therefrom was treated.
9 is a result confirming the expression of Invorucrin, a division marker of skin cells, when the Lactobacillus Sakeai LBML6 strain or an EV isolated therefrom was treated.
10 is a result of confirming whether or not the activity of CD4 T cells is inhibited upon treatment with the Lactobacillus Sakeai LBML6 strain or EVs isolated therefrom.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Materials and Methods

One. Lactobacillus sakeai LBML6( Lactobacillus sakei LBML6) Preparation of strains

On June 15, 2021, the Lactobacillus sakei LBML6 ( Lactobacillus sakei LBML6) strain deposited with the Korea Microbial Conservation Center under the accession number of KCCM13011P was inoculated with 0.1% in 30 ml of MRS liquid medium and incubated at 30 ° C. for 18 hours. After culturing, the cells were centrifuged at 3500 rpm for 10 minutes, and the cells were washed three times with a PBS solution and then the remaining media components were removed.

2. Preparation of experimental animals

This test was performed to evaluate the effect of the test substance after making a rheumatoid arthritis model, one of the autoimmune diseases, using Bovine collagen in DBA-1J Mice.

3. Administration of test substance

Preparation of the test substance administered in Examples was prepared by weighing an appropriate amount of the test substance and then diluting it in an excipient. The composition of the test group and dosage settings are shown in Table 1 below.

army gender number of animals
(number of animals) Whether or not it causes arthritis administration substance dose amount
(μL/head) Normal M 5 N PBS 200 CIA M 10 Y PBS 200 CIA+ strain M 10 Y LBML6 200

The test substance was orally administered at 200 μL/head, 5 times/week for 7 weeks, and only physiological saline was administered as a control group. For oral administration, the animals were fixed by fixation of the skin on the cervical region and administered directly into the stomach using a sonde for oral administration.

As for the test group, Normal (Control; PBS), CIA (arthritis-inducing + PBS), and CIA + strain (arthritis-inducing + strain of the present invention) were treated to set the composition and dosage of the test group.

4. Isolation of EVs

Lactobacillus Sakeai (LBML6) was inoculated with 0.1% in 30 ml of MRS broth and cultured at 30° C. for 18 hours. After incubation, the first centrifugation at 3500 rpm, 4 ℃ for 10 minutes, followed by the second centrifugation at 10,000 xg, 4 ℃ for 20 minutes, the culture supernatant was recovered, and the culture supernatant was filtered with a final 0.22 μm filter to remove the cells. recovered. The culture supernatant was mixed with equal amounts of 1 M NaCl solution in which 16% PEG6000 was dissolved, reacted at 4°C for 15 hours, and then centrifuged at 10,000 xg at 4°C for 20 minutes to obtain an EV pellet. The obtained EV pellet was resuspension with 0.5 M NaCl solution in which 5% PEG6000 was dissolved, washed, and centrifuged at 11,000 rpm and 4℃ for 20 minutes. ) derived EVs were isolated. The size (FIG. 6) and concentration (FIG. 7) of the isolated EVs were measured using ZetaView (Particle Metrix GmbH).

5. Differentiation of immature dendritic cells from bone marrow

Ficoll gradient method (Hlozkova, K., Starkova, J. Assessment) from bone marrow cells (Orient Bio, Balb/c, female 6wk) of mouse femur and tibia of the Metabolic Profile of Primary Leukemia Cells. The isolated monocytes were prepared in a 6 well plate at a concentration of 2 x 10 ⁶ /well, and GM-CSF 20 ng/ml, IL-4 in a medium (RPMI) containing Fetal Bovine Serum (FBS). 10 ng/ml were treated together. After that, it was cultured for 6 days, and when 3 days had elapsed, it was replaced with fresh medium and cytokines to obtain differentiated immature dendritic cells.

6. Differentiation of mature dendritic cells

In order to differentiate immature DCs obtained above into mature DCs, positive control LPS was treated for 2 hours to induce differentiation and activity into mature DCs. EVs derived from Bacillus sakeai (LBML6) and Lactobacillus sakeai (LBML6) were co-cultured for 24 hours at MOIs of 1 and 10, respectively.

7. T cell co-culture

CFSE (Invitrogen) stained CD3+ T cells isolated from mouse spleen were co-cultured with Lactobacillus sakeai (LBML6) and dendritic cells cultured for 24 hours at a 1:1 ratio for 3 days. To evaluate whether T cell activity was suppressed by tolDC, T cell division was measured (CFSE) and secreted cytokine production (IFNr, IL-17) was measured using CBA assay kit (BD Th1/2/17 CBA kit). It was confirmed using In addition, the culture medium from the co-culture was used after storage for application to an in vitro model of psoriasis.

8. Preparation of psoriasis model of human keratinocytes

4x10 ⁵ Hacat cells (CLS) were seeded into 6 wells. When the cells were attached, CaCl ₂ (Sigma) was treated for 4 hours and imiquimod (IMQ, Calbiochem) was additionally treated to induce cell inflammation. After 24 hours, the culture medium performed in the 5 experiments was processed into each well, and after 24 hours, the cells were harvested and RNA isolated for gene expression measurement.

9. Gene expression measurement (qPCR)

To measure gene expression in each cell, cell RNA was synthesized into cDNA using reverse thranscriptase and dNTP from a cDNA synthesis kit (Thermo). It was amplified using each primer (Macrogen) of the gene.

Experiment result

Example 1. Measurement of arthritis incidence and index in collagen-induced arthritis mouse model

Arthritis score measurement: The degree of arthritis was scored according to the criteria shown in Table 2 below, immediately before and after administration of the test substance, three times a week during the test period. The scores of the limbs were summed and the highest score was 16 points. The score table according to status is as follows.

score state 0 No edema or swelling One Mild swelling and redness localized to the joint 2 Mild swelling and redness from the joint to the carpal and tarsal bones 3 Moderate swelling and redness from the joint to the carpal and tarsal bones 4 Overall, there is swelling and redness, and joint stiffness appears.

Figure 1 shows the arthritic score and the incidence rate during 7 weeks of orally administering the Lactobacillus sakei LBML6 strain to a collagen-induced arthritis mouse model.

As a result, the highest incidence rate and arthritis index appeared in the collagen-induced arthritis-induced control group, and it was confirmed that the incidence rate and arthritis index decreased significantly in the Lactobacillus sakei LBML6 strain-treated group.

Example 2. Measurement of TNFα and Collagen Antigen-specific IgG Concentrations in Blood

The blood of the experimental animals was collected to measure the concentrations of proinflammatory cytokines TNFα and autoantibody collagen antigen-specific IgG in the blood. Figure 2 is the result of measuring the concentration of TNFα, Figure 3 is the result of measuring the concentration of IgG and collagen antigen-specific IgG.

As shown in Figures 2 and 3, it can be seen that the production of TNFα and IgG in the blood is promoted when arthritis is induced compared to the normal group, and when the Lactobacillus sakei LBML6 strain is administered to the experimental animals, TNFα And it was confirmed that the concentration of IgG decreased.

Example 3. Intestinal lymph node immunosuppressive cell measurement

The ratio of immunosuppressive tolerogenic dendritic cells (tolDC) and regulatory T cells (Tregs) in the intestinal lymph nodes was confirmed. 4 is a result of measuring the ratio of tolDC cells, and FIG. 5 is a result of measuring the ratio of Tregs cells.

As shown in Figures 4 and 5, when the Lactobacillus sakei LBML6 ( Lactobacillus sakei LBML6) strain was administered compared to the normal group and the arthritis-induced control group, the increase in the ratio of tolDC cells and Tregs cells that suppress immunity in the intestinal lymph nodes Confirmed.

Example 4. Psoriasis relief efficacy of LBML6

When Hacat cells are treated with CaCl ₂ and IMQ, cell division and inflammation can be induced to induce stratum corneum. When the expression of K10 (FIG. 8) and Invorucrin (FIG. 9), which are mitosis markers of skin cells, were confirmed, gene expression was significantly reduced in the culture medium induced from LBML6 (MOI 1) and LBML6 EV. It was confirmed that cleavage was inhibited.

Example 5. CD4 T cell activity inhibitory efficacy

It is known that increased division of CD4 T cells activates T cells and is involved in various inflammatory responses. To examine whether tolDCs induced by Lactobacillus sakei-derived EVs can directly inhibit the activity of CD4 T cells, tolDCs induced by Lactobacillus sakei (LBML6) and Lactobacillus sakeai-derived EVs were stained with CFSE. CD4 T cells were co-cultured at a ratio of 1:1 for 3 days. After 3 days, the co-cultured cells were harvested and the degree of CFSE reduction was measured.

T cells co-cultured with LPS-treated dendritic cells showed nearly 90% division. However, it was confirmed that about 60% of division was suppressed in T cells co-cultured with dendritic cells treated with Lactobacillus sakeai-derived EV. This means that tolDC induced by EVs derived from Lactobacillus sakei can reduce the immune response by directly suppressing the activity of CD4 T cells.

Taking the above results together, it can be confirmed that Lactobacillus sakeai (LBML6) and Lactobacillus sakeai (LBML6)-derived EV suppress the activity of CD4 T cells through the reduction of DC activity, thereby reducing the immune response. Therefore, various autoimmune diseases including rheumatoid arthritis can be controlled.

Although the embodiments of the present invention have been described above, those skilled in the art can add, change, delete, or add components within the scope not departing from the spirit of the present invention described in the claims. The present invention can be variously modified and changed by the like, and this will also be said to be included within the scope of the present invention.

Korea Microbial Conservation Center (Overseas) KCCM13011P 20210615 Korea Microbial Conservation Center (Overseas) KCCM12654P 20200114 Korea Research Institute of Bioscience and Biotechnology KCTC13818BP 20190307

Claims (12)

Lactobacillus sakei , its culture, lysate, extract or fermented product; Or a composition for preventing, improving or treating autoimmune diseases comprising as an active ingredient; extracellular vesicles derived from (derived) the Lactobacillus sakei .
The composition according to claim 1, wherein the composition is a pharmaceutical composition or a food composition.
The method of claim 1, wherein the autoimmune disease is atopic dermatitis, psoriatic dermatitis, alopecia areata, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, Crohn's disease, ankylosing spondylitis, lupus, psoriatic arthritis, osteoarthritis , A composition characterized in that it is selected from the group consisting of rheumatoid arthritis, shoulder joint inflammation, tendonitis and multiple sclerosis.
The composition according to claim 1, wherein the Lactobacillus sakei , its culture, lysate, extract or fermented product contains extracellular vesicles of the Lactobacillus sakei .
The composition according to claim 1 or 4, wherein the diameter of the extracellular vesicles is 10 to 1,000 nm.
The composition according to claim 1 or 4, wherein the Lactobacillus sakeai is a strain of Lactobacillus sakeai LBML6 deposited under accession number KCCM13011P.
a therapeutically effective amount of Lactobacillus sakeai , a culture, lysate, extract or ferment thereof; or an extracellular vesicle derived from the Lactobacillus sakeai ; a method for preventing, improving or treating an autoimmune disease comprising administering to a subject in need thereof.
Lactobacillus sakeai , for use in therapy of its culture, lysate, extract or fermentation.
Therapeutic use of extracellular vesicles derived from Lactobacillus sakeai , cultures, lysates, extracts or fermentations thereof.
Method for producing a composition for preventing, improving or treating autoimmune diseases comprising the following steps:
(a) preparing a Lactobacillus Sakeai strain; and
(b) culturing the strain in a culture medium.
A method for preparing a composition for preventing, improving or treating autoimmune diseases comprising the step of preparing extracellular vesicles derived from the Lactobacillus Sakeai strain.
Lactobacillus sakei LBML6 ( Lactobacillus sakei LBML6) strain deposited with accession number KCCM13011P.

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