KR20240001620A - Composition for preventing, treating, or alleviating asthma comprising expression stimulators of hsa-miR-4517 and Method for diagnosing asthma using vesicles derived from Micrococcus luteus - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or improving asthma containing an hsa-miR-4517 expression promoter and a method for diagnosing asthma using Micrococcus luteus-derived vesicles (MlEV). It was confirmed that the amount of vesicles was lower in asthma patients compared to normal controls, and Micrococcus luteus-derived vesicles decreased IL-1β production and NLRP3 expression in monocytes by increasing the expression of hsa-miR-4517 in airway epithelial cells. It was confirmed that it suppresses neutrophilic airway inflammation by suppressing innate lymphoid cells 3 (ILC3) that produce IL-17. Accordingly, it is expected that the Micrococcus luteus-derived vesicle according to the present invention can be usefully used in asthma diagnosis methods and compositions for preventing, treating, or improving asthma.

Description

Composition for preventing, treating, or alleviating asthma comprising expression stimulators of hsa-miR-4517 and method for diagnosing asthma using vesicles derived from Micrococcus luteus {Composition for preventing, treating, or alleviating asthma comprising expression stimulators of hsa-miR-4517 and Method for diagnosing asthma using vesicles derived from Micrococcus luteus}

The present invention relates to a composition for preventing, treating, or improving asthma containing an hsa-miR-4517 expression promoter and a method for diagnosing asthma using Micrococcus luteus-derived vesicles.

Asthma is a multifactorial disease characterized by chronic airway inflammation with complex interactions between genetic and environmental factors. For decades, allergen exposure has been widely accepted to induce adaptive immunity in asthma development. In particular, eosinophils have been recognized as key effector cells contributing to persistent airway inflammation. Nevertheless, current treatments targeting Th2-driven immune responses have limitations as 5-10% of asthma patients suffer from uncontrolled conditions. Neutrophilic asthma (NA) is one of the severe asthma phenotypes associated with innate immune responses induced by viruses or bacteria. Neutrophils play a central role in host defense, but their massive infiltration into the airways causes frequent asthma exacerbations and steroid resistance.

It is known that bacterial burden contributes to the pathogenesis of NA, suggesting the importance of the microbiota (or microbiome) in the innate immune system of the respiratory tract. In particular, airway epithelial cells (AECs) are important in initiating neutrophil recruitment to the airways by releasing IL-8 in response to invading microorganisms. Additionally, various innate immune cells such as dendritic cells, monocytes, and innate lymphoid cells (ILCs) can influence the homeostasis of the airway microbiota. Additionally, the pathological role of ILC3 is highlighted as a major source of IL-17 and IL-22 to enhance airway inflammation and remodeling in asthma. Additionally, secretion of these cytokines can be amplified by IL-1β-dependent interactions with monocytes or macrophages. Therefore, manipulation or modulation of the microbial community may be applied to modulate innate immune responses in NA.

Meanwhile, the number of microorganisms living in the human body reaches 100 trillion, which is 10 times more than human cells, and the number of genes in microorganisms is known to be more than 100 times that of humans. Microbiota (microbiota or microbiome) refers to the microbial community including bacteria, archaea, and eukaryotes that exist in a given habitat, and the intestinal microflora plays an important role in human physiology. It is known to have a significant impact on human health and disease through interactions with human cells. Bacteria that live in our body secrete nanometer-sized vesicles to exchange information such as genes and proteins with other cells. The mucous membrane forms a physical barrier that prevents particles larger than 200 nanometers (nm) from passing through, so bacteria that live in the mucosa cannot pass through the mucosa. However, vesicles derived from bacteria are usually less than 100 nanometers in size, so they are relatively easy to pass through. It freely passes through mucous membranes and is absorbed into our body.

Extracellular vesicles (EVs) are membrane particles containing various molecules such as lipids, proteins, and nucleic acids, and are released from all living cells in physiological and pathological states. In particular, the role of bacterial EVs in regulating host immunity in various diseases has been highlighted. Additionally, recent metagenomic analyzes have revealed the importance of bacterial EVs found in serum or urine as new biomarkers.

Micrococcus luteus bacteria are Gram-positive bacteria belonging to the Micrococcus genus and are widely distributed in nature, such as water, dust, and soil. This bacterium is known to produce riboflavin when growing in toxic organic pollutants such as pyridine and to absorb ultraviolet rays through the lutein pigment. In addition, this bacteria has been isolated from dairy products and beer, grows even in dry or high-salt environments, and does not form spores, but is known to survive for a long period of time even at refrigerated temperatures such as refrigerators.

The present inventors confirmed the effect of Micrococcus luteus-derived vesicles on asthma by confirming the effect on the expression of miRNA in airway epithelial cells, but nothing is yet known about the effect of Micrococcus luteus-derived vesicles.

Korean Patent No. 10-1923969

The present inventors evaluated the relative amount of EV-specific antibodies in the serum of asthma patients according to their inflammatory phenotype and investigated the effect of Micrococcus luteus -derived EVs (MlEV) on airway immune responses in vitro or ex vivo. We investigated and attempted to prove the importance of MlEV in patients with neutrophilic asthma by clearly confirming the effect of MlEV in controlling neutrophilic airway inflammation in mice.

Accordingly, the purpose of the present invention is to provide a composition for preventing, treating, or improving asthma, which contains an hsa-miR-4517 expression promoter as an active ingredient.

Another object of the present invention is to provide an information provision method for diagnosing asthma by measuring the amount of EV-specific IgG4 derived from Micrococcus luteus or the expression level of hsa-miR-4517.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,

Provided is a pharmaceutical composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .

In addition, the present invention provides an inhalant composition for preventing or treating asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,

Provided is an inhalant composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .

In one embodiment of the present invention, the hsa-miR-4517 expression promoter may increase hsa-miR-4517 expression in airway epithelial cells (AEC), but is not limited thereto.

In another embodiment of the present invention, the hsa-miR-4517 expression promoter may inhibit neutrophilic airway inflammation, but is not limited thereto.

As another embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm, but are not limited thereto.

As another embodiment of the present invention, the vesicles may be naturally secreted or artificially produced by Micrococcus luteus, but are not limited thereto.

The present invention includes the steps of (a) measuring the level of vesicle-specific immunoglobulin G4 (IgG4) derived from Micrococcus luteus from a biological sample; and

(b) It provides a method of providing information for the diagnosis of asthma, including the step of classifying it as asthma when the measured level of IgG4 is reduced compared to a normal person.

In one embodiment of the present invention, (c) if the level of IgG4 measured in step (b) is reduced compared to normal people and reduced compared to eosinophilic asthma patients, it may further include the step of classifying it as neutrophilic asthma. may, but is not limited to this.

In another embodiment of the present invention, the step of measuring the level of IgG4 includes Western blot, ELISA (enzyme linked immunosorbent assay), ImmunoCAP, RIA (Radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS) and protein chips, but is not limited thereto.

In addition, the present invention includes the steps of (a) measuring the expression level of hsa-miR-4517 from a biological sample; and

(b) It provides a method of providing information for the diagnosis of asthma, including the step of classifying it as asthma when the measured expression level of hsa-miR-4517 is reduced compared to normal people.

In one embodiment of the present invention, the step of measuring the expression level of hsa-miR-4517 is polymerase chain reaction (polymerase chain reaction, PCR), reverse transcription polymerase chain reaction (RT-PCR). , Competitive RT-PCR, Real-time PCR, quantitative PCR, quantitative real-time PCR, RNase protection assay (RPA), Southern blotting One or more methods selected from the group consisting of blotting, Northern blotting, and DNA chip may be used, but are not limited thereto.

In another embodiment of the present invention, hsa-miR-4517 may be expressed in airway epithelial cells (AEC), but is not limited thereto.

As another embodiment of the present invention, the biological sample may be one or more selected from the group consisting of blood, plasma, serum, bone marrow, saliva, urine, and feces, but is not limited thereto.

As another embodiment of the present invention, the asthma may be neutrophilic asthma or eosinophilic asthma, but is not limited thereto.

In addition, the present invention provides a method for treating or improving asthma, comprising the step of administering a composition containing an hsa-miR-4517 expression promoter as an active ingredient to an individual in need thereof, wherein the hsa-miR-4517 expression promoter is a micro A method is provided, characterized in that the vesicle is derived from Micrococcus luteus .

In addition, the present invention provides a use for the prevention, treatment, or improvement of asthma of a composition containing an hsa-miR-4517 expression promoter as an active ingredient, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus. It provides a use characterized in that.

In addition, the present invention relates to the use of an hsa-miR-4517 expression promoter for the production of a drug for preventing, treating, or improving asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus . Provides features and uses.

The present inventors confirmed that the amount of Micrococcus luteus-derived vesicles (MlEV) was lower in asthma patients compared to normal controls, and that Micrococcus luteus-derived vesicles increased the expression of hsa-miR-4517 in airway epithelial cells. It was confirmed that neutrophilic airway inflammation was suppressed by reducing IL-1β production and NLRP3 expression in monocytes and suppressing innate lymphoid cells 3 (ILC3) that produce IL-17. Accordingly, it is expected that the Micrococcus luteus-derived vesicle according to the present invention can be usefully used in asthma diagnosis methods and compositions for preventing, treating, or improving asthma.

Figure 1a is a diagram showing the levels of MlEV- and EcEV-specific IgG1 in the serum of asthma patients according to one embodiment of the present invention.
Figure 1b is a diagram showing the levels of MlEV- and EcEV-specific IgG4 in the serum of asthma patients according to one embodiment of the present invention.
Figure 1c is a diagram confirming the correlation between MlEV- and EcEV-specific IgG4 in serum and baseline FEV1 (%) in asthma patients according to one embodiment of the present invention.
Figure 2a is a diagram showing the shape and size of MlEV according to an embodiment of the present invention (scale bar = 100 nm).
Figure 2b is a diagram showing protein profiles identified according to cellular components or biological processes in MlEV according to one embodiment of the present invention.
Figure 2c is a diagram confirming the level of IL-8 by MlEV treatment in airway epithelial cells according to one embodiment of the present invention.
Figure 2d is a diagram confirming the phosphorylation of p65 by MlEV treatment in airway epithelial cells according to one embodiment of the present invention.
Figure 2e is a diagram showing the RNA composition in airway epithelial cells according to one embodiment of the present invention.
Figure 2f is a diagram confirming the expression of miRNA in airway epithelial cells according to an embodiment of the present invention through microarray analysis.
Figure 3a is a diagram showing the results of observing EVs derived from airway epithelial cells according to one embodiment of the present invention (scale bar = 50 nm).
Figure 3b is a diagram confirming the expression of EV markers in airway epithelial cell culture medium (CM), cells, and cell-derived vesicles (EV) according to one embodiment of the present invention.
Figure 3c is a diagram confirming the protein pattern in airway epithelial cell culture medium (CM), cells (Cell), and cell-derived vesicles (EV) according to one embodiment of the present invention.
Figure 3D is a diagram showing the relative expression of miRNA in EVs derived from airway epithelial cells according to one embodiment of the present invention.
Figure 3e is a diagram confirming IL-1β production and NLRP3 expression in peripheral monocytes of asthma patients according to one embodiment of the present invention.
Figure 4a is an image (scale bar = 200 nm) of EVs derived from airway epithelial cells in the plasma of an asthma patient according to an embodiment of the present invention and a view confirming the expression of EV markers (ALIX and TSG101) and epithelial cell marker (EpCAM) am.
Figure 4b is a diagram confirming the protein profile in plasma and EV in plasma of an asthma patient according to an embodiment of the present invention.
Figure 4c is a diagram confirming the expression of hsa-miR-4517 in EVs in the plasma of an asthma patient according to an embodiment of the present invention.
Figure 4D is a diagram confirming the level of IL-22 in the plasma of an asthma patient according to an embodiment of the present invention.
Figure 4e is a diagram confirming the correlation between the expression level of hsa-miR-4517 in EVs in plasma and the level of IL-22 in plasma of asthma patients according to an embodiment of the present invention.
Figure 4f is a diagram confirming the expression of GATA-3, ROR-γt, and IL-22 in innate lymphoid cells (ILCs) of asthma patients according to one embodiment of the present invention.
Figure 4g is a diagram confirming the decrease in IL-22 expression by MlEV treatment in the ILC of an asthma patient according to an embodiment of the present invention.
Figure 5a is a diagram confirming the results of histological analysis of lung tissue by MlEV treatment in LPS-induced asthma mice according to an embodiment of the present invention (scale bar = 100 μm).
Figure 5b is a diagram confirming the number of neutrophils, IL-1β, and IL-17 levels by MlEV treatment in LPS-induced asthma mice according to one embodiment of the present invention.
Figure 5c is a diagram confirming the expression of NLRP3, T-bet, and ROR-γt by MlEV treatment in LPS-induced asthma mice according to one embodiment of the present invention.
Figure 5d is a diagram confirming the ratio of NCR-ILC3 producing IL-17 in LPS-induced asthma mice according to an embodiment of the present invention by flow cytometry.
Figure 6 is a diagram illustrating the level of MlEV-specific IgG4 and the mechanism of action of MlEV in asthma patients according to an embodiment of the present invention.

In one experimental example of the present invention, the amount of Micrococcus luteus-derived vesicle (MlEV)-specific IgG subclass was confirmed in patients with neutrophilic asthma. As a result, the level of MlEV-specific IgG4 was found to be lower in asthma patients compared to normal controls. In particular, it was confirmed that IgG4 levels were lower in neutrophilic asthma (NA) patients compared to eosinophilic asthma (EA) patients. In addition, the level of MlEV-specific IgG4 was positively correlated with the baseline forced expiratory volume in 1 second (FEV1 (%)), indicating that the prevalence of bacterial EV is related to the decline in lung function in asthma patients. It was found that there may be (see Experimental Example 1).

In another experimental example of the present invention, the effect of MlEV on the pathophysiological conditions of airway epithelial cells was confirmed, and it was confirmed that MlEV reduces IL-8 production in airway epithelial cells and inhibits phosphorylation of p65 in cells. In addition, MlEV was confirmed to restore 7 up-regulated and 6 down-regulated miRNAs to their original state in airway epithelial cells treated with LPS (see Experimental Example 2).

In another experimental example of the present invention, the function of miRNA on IL-1β-producing monocytes was confirmed. As a result, hsa-miR-3065-5p, hsa-miR-1272, and and the expression of hsa-miR-4517 increased, and in particular, hsa-miR-4517 was confirmed to significantly reduce IL-1β production and NLRP3 expression in monocytes stimulated with LPS (see Experimental Example 3).

In another experimental example of the present invention, the correlation between the inflammatory phenotype of asthma and hsa-miR-4517 in plasma-EV was confirmed, and the results showed that hsa-miR-4517 expression in plasma-EV was lower in asthma patients compared to normal controls, and IL Plasma levels of -22 were higher in NA patients than in EA patients, and expression of hsa-miR-4517 was negatively correlated with plasma levels of IL-22. Additionally, NA patients display more increased ROR-γt and IL-22 expression in innate lymphoid cells (ILCs), and when ILCs were cultured with monocytes in the presence of LPS, the levels of IL-22 increased significantly with hsa-miR-4517 It was confirmed that it was reduced by (see Experimental Example 4).

In another experimental example of the present invention, the role of MlEV on airway inflammation in neutrophilic asthma mice was confirmed. As a result, when LPS-induced asthma mice were treated with MlEV, the number of neutrophils and the production of IL-1β and IL-17 in BALF were increased. was reduced, the expression of NLRP3, T-bet, and ROR-γt was suppressed in the lung tissue, and the proportion of IL-17-producing NCR-ILC3 was lower, indicating that MlEV treatment induced immunity in the lung tissue of asthmatic mice. It was confirmed that not only cell invasion but also immune response was significantly reduced (see Experimental Example 5).

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,

Provided is a pharmaceutical composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .

In the present invention, the hsa-miR-4517 expression promoter can increase hsa-miR-4517 expression in airway epithelial cells (AEC) and suppress neutrophilic airway inflammation, but is not limited thereto.

In the present invention, “expression” refers to the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA, and translation of this mRNA into polypeptide(s).

In the present invention, “hsa-miR-4517 expression promoter” means promoting the expression of hsa-miR-4517 in airway epithelial cells, reducing IL-1β production and NLRP3 expression in monocytes, and congenital lymph that produces IL-17. It refers to any substance that can inhibit cell 3 (ILC3), and there is no limitation on its type, but according to the present invention, the hsa-miR-4517 expression promoter may be a vesicle derived from Micrococcus luteus .

In the present invention, “extracellular vesicle” refers to a nano-sized membrane structure secreted from various cells, and in the present invention, it is naturally secreted from cells, artificially produced, or contained in urine or blood. A general term for all membrane-based structures. These vesicles can be used interchangeably with extracellular vesicles, extracellular vesicles, extracellular vesicles, exosomes, nanovesicles, nanovesicles, microvesicles, microvesicles, etc. Vesicles contain various types of proteins, DNA, mRNA, micro RNA, etc. derived from cells. The vesicles are extracted from urine or blood by centrifugation, ultracentrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical disintegration, chemical treatment, filtration by filter, and gel filtration chromatography. It can be separated using one or more methods selected from the group consisting of , free-flow electrophoresis, and capillary electrophoresis. Additionally, processes such as washing to remove impurities and concentrating the obtained vesicles may be additionally included.

In the present invention, the vesicles have an average diameter of 10 to 200 nm, 10 to 190 nm, 10 to 180 nm, 10 to 170 nm, 10 to 160 nm, 10 to 150 nm, 10 to 140 nm, and 10 to 130 nm. , 10 to 120 nm, 10 to 110 nm, 10 to 100 nm, 10 to 90 nm, 10 to 80 nm, 10 to 70 nm, 20 to 200 nm, 20 to 180 nm, 20 to 160 nm, 20 to 140 nm , 20 to 120 nm, 20 to 100 nm, or 20 to 80 nm, but is not limited thereto.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners may be used in the syrup according to the present invention, and, if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75(S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating asthma, comprising vesicles derived from Micrococcus luteus and hsa-miR-4517 as active ingredients.

In addition, the present invention provides an inhalant composition for preventing or treating asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,

Provided is an inhalant composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .

In the inhalant composition of the present invention, the active ingredient can be added as is to the inhalant or used together with other ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or treatment).

Inhalation agents for parenteral administration include aerosols, powders for inhalation, or liquids for inhalation. These inhalation liquids may be dissolved or suspended in water or another suitable medium for use. These inhalants are manufactured according to known methods. For example, in the case of liquid preparations for inhalation, preservatives (benzalkonium chloride, parabens, etc.), colorants, buffering agents (sodium phosphate, sodium acetate, etc.), isotonic agents (sodium chloride, concentrated glycerin, etc.), thickeners (carboxyvinyl polymer, etc.), absorption agents, etc. It is prepared by appropriately selecting accelerators, etc. according to need.

In the case of powder for inhalation, lubricants (stearic acid and its salts, etc.), binders (starch, dextrin, etc.), excipients (lactose, cellulose, etc.), colorants, preservatives (benzalkonium chloride, parabens, etc.), absorption accelerators, etc. are required. It is appropriately selected and prepared according to the following.

The inhalant composition can be administered through an inhalant device, which is a device capable of delivering the composition to an individual's lung tissue, such as an inhaler, nebulizer, or ventilator. When administering a liquid medication for inhalation, a nebulizer (atomizer, nebulizer) is usually used, and when administering a powder medication for inhalation, an inhalation dispenser for powder medication is usually used.

Additionally, the present invention provides an inhalant composition for preventing or treating asthma, comprising vesicles derived from Micrococcus luteus and hsa-miR-4517 as active ingredients.

In addition, the present invention provides a food composition for preventing or improving asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,

Provided is a food composition for preventing or improving asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .

In the present invention, the food composition may be a health functional food composition, but is not limited thereto.

When using the Micrococcus luteus-derived vesicles of the present invention as a food additive, the Micrococcus luteus-derived vesicles can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when producing food or beverages, the Micrococcus luteus-derived vesicle of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, These include alcoholic beverages and vitamin complexes, and include all health functional foods in the conventional sense.

The health drink composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of natural carbohydrates is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain carbonating agents used in carbonated drinks. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

Additionally, the present invention provides a food composition for preventing or improving asthma, comprising vesicles derived from Micrococcus luteus and hsa-miR-4517 as active ingredients.

In addition, the present invention provides a quasi-drug composition for preventing or improving asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,

Provided is a quasi-drug composition for preventing or improving asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .

In the present invention, "quasi-drugs" refers to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act, Quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

When the composition of the present invention is included in a quasi-drug for the purpose of preventing or improving asthma, the composition can be included as is or used together with other quasi-drug ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

The quasi-drug of the present invention may contain various bases and additives necessary for formulation depending on its formulation, and the types and amounts of these ingredients can be easily selected by a person skilled in the art.

The quasi-drug composition of the present invention includes, for example, disinfectant cleaners, detergents, kitchen cleaners, cleaning detergents, wet tissues, detergents, soaps, hand washes, humidifier fillers, masks, ointments, filter fillers, and temporary inhalation of air or oxygen directly or indirectly. It can be manufactured in a formulation selected from the group consisting of portable products that supply air or oxygen, but is not limited thereto.

In addition, the present invention provides a quasi-drug composition for preventing or improving asthma, comprising vesicles derived from Micrococcus luteus and hsa-miR-4517 as active ingredients.

Additionally, the present invention provides a composition for delivering an asthma treatment drug, comprising vesicles derived from Micrococcus luteus as an active ingredient.

In the present invention, “drug delivery” refers to any means or act of loading and delivering a drug for treating asthma into a Micrococcus luteus-derived vesicle according to the present invention in order to deliver the drug to a specific organ, tissue, cell, or organelle. means.

In the present invention, there is no limitation to the type of drug for treating asthma, and hsa-miR-4517 may be included.

In addition, the present invention includes the steps of (a) measuring the level of vesicle-specific immunoglobulin G4 (IgG4) derived from Micrococcus luteus from a biological sample; and

(b) It provides a method of providing information for the diagnosis of asthma, including the step of classifying it as asthma when the measured level of IgG4 is reduced compared to a normal person.

In the present invention, the step (c) of classifying as neutrophilic asthma may be further included if the level of IgG4 measured in step (b) is decreased compared to normal people and decreased compared to eosinophilic asthma patients. It is not limited to this.

In the present invention, “asthma” is a disease of the ‘bronchial tubes’, which are passages connected to the lungs. When exposed to a specific causative agent, the bronchial tubes become severely narrowed due to inflammation of the bronchial tubes, causing coughing, wheezing (wheezing sound when breathing), A disorder of the lungs characterized by recurrent episodes of shortness of breath and chest tightness, whatever the cause (endogenous, exogenous, or both; allergic or non-allergic), characterized by changes in lung airflow associated with airway constriction. refers to a disease. The term asthma may be used with one or more adjectives indicating the cause. In the present invention, the asthma may be neutrophilic asthma or eosinophilic asthma, but is not limited thereto.

In the present invention, "measurement of the level of IgG4" is a process of confirming the presence or production level of allergen-specific IgG4 antibodies against Micrococcus luteus-derived vesicles in a biological sample in order to predict the possibility of asthma. It can be determined by checking the amount of IgG4 using specifically binding antigens, antibodies, peptides, aptamers, etc. Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay), ImmunoCAP, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket. (rocket) There are immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), and protein chip. Any method that can measure the amount of IgG4 is not limited thereto.

In the present invention, “diagnosis” in a broad sense means judging the actual condition of the patient's disease in all aspects. The contents of the judgment include the name of the disease, etiology, type, severity, detailed condition of the disease, presence or absence of complications, and prognosis. In the present invention, diagnosis refers to determining whether asthma has occurred and the level of asthma.

In addition, the present invention includes the steps of (a) measuring the expression level of hsa-miR-4517 from a biological sample; and

(b) It provides a method of providing information for the diagnosis of asthma, including the step of classifying it as asthma when the measured expression level of hsa-miR-4517 is reduced compared to normal people.

In the present invention, “measuring the expression level of hsa-miR-4517” is a process of confirming the presence or expression level of hsa-miR-4517 in a biological sample to predict the possibility of asthma. This can be determined by checking the expression level of hsa-miR-4517 using primers, probes, etc. that specifically bind to. Analysis methods for this include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), and real-time polymerase. Consisting of chain reaction (Real-time PCR, quantitative PCR, quantitative real-time PCR), RNase protection assay (RPA), Southern blotting, Northern blotting, and DNA chip. One or more methods selected from the group may be used, but the method is not limited thereto.

In the present invention, the hsa-miR-4517 may include the base sequence of NCBI Reference Sequence: NR_039742.1, but is not limited thereto.

In the present invention, “biological sample” refers to a sample that can be obtained non-invasively from a subject and includes IgG4 or expresses hsa-miR-4517, preferably It may be blood, plasma, serum, bone marrow, tissue, cells, saliva, sputum, urine, feces, etc., more preferably blood, serum, plasma, etc., or a sample capable of measuring the amount of IgG4 or hsa-miR-4517. If so, it is not limited to this.

In the present invention, hsa-miR-4517 may be expressed in airway epithelial cells (AEC), but is not limited thereto.

Additionally, the present invention provides a composition or kit for diagnosing asthma, including a material for measuring the level of vesicle-specific IgG4 derived from Micrococcus luteus .

The substance may be an antigen, antibody, peptide, or aptamer that specifically binds to Micrococcus luteus-derived vesicle-specific IgG4, but is not limited thereto.

Additionally, the present invention provides a composition or kit for diagnosing asthma, including a substance for measuring the expression level of hsa-miR-4517.

The material may be a primer or probe that specifically binds to hsa-miR-4517, but is not limited thereto.

In the present invention, the “kit” can predict asthma by including a material that measures the level of vesicle-specific IgG4 derived from Micrococcus luteus or a material that measures the expression level of hsa-miR-4517. It means a tool that allows In addition to the above substances, the kit of the present invention may include other components, compositions, solutions, devices, etc. commonly required for detection methods. At this time, the substance for measuring the level of Micrococcus luteus-derived vesicle-specific IgG4 or the substance for measuring the expression level of hsa-miR-4517 can be applied at any number of times, one or more times, and each substance is applied. There is no limitation, and the application of each material may proceed simultaneously or at a microscopic level.

In the present invention, the kit includes a container; directions; And it may include a material for measuring the level of the Micrococcus luteus-derived vesicle-specific IgG4 or a material for measuring the expression level of hsa-miR-4517. The container may serve to package the material, and may also serve to store and secure the material. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partially or entirely made of plastic, glass, paper, or foil. , wax, etc. The container may be equipped with a completely or partially removable closure that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also provide access to the contents by needle. A stopper can be installed. The kit may include an external package, and the external package may include instructions for use of the components.

In addition, the present invention provides a method for treating or improving asthma, comprising the step of administering a composition containing an hsa-miR-4517 expression promoter as an active ingredient to an individual in need thereof, wherein the hsa-miR-4517 expression promoter is a micro A method is provided, characterized in that the vesicle is derived from Micrococcus luteus .

In addition, the present invention provides a method for treating or improving asthma, comprising administering a composition containing vesicles derived from Micrococcus luteus and hsa-miR-4517 as active ingredients to an individual in need thereof. .

In addition, the present invention provides a use for the prevention, treatment, or improvement of asthma of a composition containing an hsa-miR-4517 expression promoter as an active ingredient, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus. It provides a use characterized in that.

In addition, the present invention provides the use of a composition containing Micrococcus luteus -derived vesicles and hsa-miR-4517 as active ingredients for preventing, treating, or improving asthma.

In addition, the present invention relates to the use of an hsa-miR-4517 expression promoter for the production of a drug for preventing, treating, or improving asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus . Provides features and uses.

Additionally, the present invention provides the use of vesicles derived from Micrococcus luteus and hsa-miR-4517 for the production of a medicament for preventing, treating, or ameliorating asthma.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

In the present invention, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

Below, preferred examples and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples and experimental examples.

[Example]

Example 1. Patient Recruitment and Clinical Parameters

The experiment for the present invention was approved by the Ajou University Hospital Institutional Research Ethics Review Committee (AJIRB → BMR → SUR → 15 → 498), and all experimental subjects submitted written consent at the time of recruitment. We recruited 40 healthy controls (HC) and 45 asthma patients diagnosed by an allergist based on their clinical history of recurrent cough, dyspnea, recurrent wheeze, chest tightness, and signs of airway obstruction. Severe asthma was defined according to GINA guidelines. Additionally, asthma was classified into EA and NA according to eosinophils and neutrophils in sputum (cutoff value: 3% and 65%, respectively). Atopy was defined as one or more positive results in a skin prick test using commonly inhaled allergens (Bencard, Bradford, UK). Serum total IgE was measured using the ImmunoCAP system (ThermoFisher Scientific, Waltham, CA, USA), and serum levels of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were determined by the manufacturer. Measurements were made using each ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the recommendations.

Example 2. Bacterial culture and extracellular vesicle (EV) isolation

Micrococcus luteus or Escherichia coli (Korea Microbiological Culture Center) were cultured on edible LP medium (MD Healthcare Inc., Seoul) under aerobic conditions until the optical density at 600 nm reached 1.5, respectively. , Korea) or LB Broth (ThermoFisher Scientific). To isolate extracellular vesicles (EVs), the culture medium was centrifuged at 10,000 g for 20 minutes and the supernatant was filtered through a 0.45 μm vacuum filter. The filtrate was concentrated using QuixStand (GE Healthcare, Little Chalfont, UK) and then filtered through a 0.22 μm bottle-top filter (Sigma-Aldrich, St Louis, MO, USA). The filtrate was pelleted by ultracentrifugation in a 45-Ti rotor (Beckman Coulter, Fullerton, CA, USA) at 150,000 g for 2 hours at 4°C, and the final pellet was resuspended in phosphate-buffered saline (PBS). and stored at -80°C.

Example 3. EV Characterization and Identification

EV morphology was observed using a JEM1011 microscope (JEOL, Akishima, Japan). Additionally, EV size was measured using Zetasizer Nano S (Malvern Instruments, Malvern, UK). To identify EV proteins, dried samples were analyzed as previously reported (Proteomics Clin Appl. 2017;11(1-2)). That is, the trypsin-digested peptide was loaded on a trap column filled with resin and eluted using a linear gradient from 5% to 40% solvent B (acetonitrile (ACN) containing 0.1% formic acid). The eluted peptide was separated on an analytical column filled with resin and sprayed on a nano ESI source using an electronic spray. The Q Exactive mass spectrometer was operated to select the most abundant precursor ions using a survey scan, and survey MS scans were acquired using an Orbitrap analyzer.

Example 4. Evaluation of EV-specific IgG subclasses in human serum

Levels of Micrococcus luteus-derived EV (MlEV)- or Escherichia coli-derived EV (EcEV)-specific IgG subclasses were measured in human serum. Each EV was coated at a concentration of 100 ng/mL on a 96-well plate (Thermo Fisher Scientific) overnight at 4 °C, and the plate was washed twice with PBS and incubated with 1% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. blocked. After washing it three times, the plate was added with each serum of the test subject and incubated for 2 hours. Then, peroxidase-conjugated anti-human IgG1 or IgG4 antibody (Sigma-Aldrich) was incubated for 1 hour. The reaction was induced with 3,3`,5,5`-tetramethylbenzidine (BD Biosciences, San Diego, CA, USA) and stopped using stop solution, and the intensity was measured using a microplate reader with a wavelength of 450 nm. It was evaluated using .

Example 5. Airway epithelial cell (AEC) stimulation and AEC-EV isolation

A549 cells, human airway epithelial cells (American Type Culture Collection, Manassas, VA, USA), were purchased and cultured in RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (ThermoFisher Scientific). Then, A549 cells were treated with 1 μM dexamethasone (Dex; Sigma-Aldrich) or 1 μg/mL MlEV in the presence of 10 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich) for 24 hours. IL-8 levels in culture supernatants were measured using an ELISA kit (R&D Systems), and intracellular protein expression was measured using phospho-p65 (Cell Signaling Technology, Danvers, MA, USA), p65 (Abcam, Cambridge, UK), and It was evaluated using antibodies such as actin (Santa Cruz, Dallas, TX, USA). Airway epithelial cell (AEC)-EVs were isolated in the same manner as bacterial EV extraction, and these EVs were labeled with ALG-2 interacting protein X (ALIX; Santa Cruz) and tumor susceptibility 101 (TSG101; This was confirmed using EV markers including Santa Cruz). Additionally, EV protein patterns were analyzed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Example 6. Microarray analysis for library generation

Adapter ligation, reverse transcription, PCR amplification, and pooled gel purification were performed to generate the library. RNA 3' adapters have been specifically modified to target microRNAs and other small RNAs with a 3' hydroxyl group due to enzymatic cleavage by Dicer or other RNA processing enzymes. The adapter was ligated to each end of the RNA molecule and single-stranded cDNA was generated using the RT reaction. The cDNA was then amplified by PCR using a common primer and a primer containing one of the 48 index sequences, and the index sequence was introduced in the PCR step to isolate the index from the RNA ligation reaction. This design allows the index to be read using a second read and significantly reduces bias compared to designs that include the index within the first read.

The library was gel purified with BluePippin and verified by checking size, purity, and concentration on an Agilent Bioanalyzer. Additionally, the libraries were quantified using the KAPA Library Quantificatoin kit for the Illumina sequencing platform according to the qPCR quantification protocol guide (Kapa Biosystems, Wilmington, MA, USA). The indexed library was then submitted to an Illumina Hiseq2500 (Illumina, San Diego, CA, USA) instrument to generate 51 primary reads. Image decomposition and quality value calculation were performed using modules of the Illumina pipeline.

Example 7. Confirmation of miRNA expression

Raw reads were preprocessed to remove adapter sequences, and adapters in the raw reads were trimmed using the cutadapt program. If the sequence matched more than the first 5 bp of the 3' adapter for read 1 or the 5' adapter for read 2, it was considered the adapter sequence and was then excised from each read. Trimmed reads longer than 18 bp in length were selected for mapping reliability, and the remaining reads were then classified as non-adapter reads whose adapter sequences were not sequenced. To minimize sequence duplication for computational efficiency, the trimmed reads were clustered by sequence. Unique clusters consisted of reads with identical sequence and length, and reads aligned to human 45S pre-rRNA and mitochondrial rRNA were excluded to remove rRNA. Sequence alignment and detection of known and novel miRNAs were performed using the miRDeep2 software algorithm to identify known miRNA leads. Accordingly, rRNA-filtered reads were aligned to human mature and precursor miRNAs obtained from miRBase v22.1 using the miRDeep2 quantifier module.

The miRDeep2 algorithm is based on the miRNA biogenesis model; it aligns reads to potential hairpin structures to determine if the mapping context matches one with Dicer processing and assigns a score that indicates the probability that the hairpin is a true miRNA precursor. To predict new miRNAs, the human reference genome, release hg19, was searched in RefSeq. Bowtie (1.1.2) was then used to index the reference genome and map the rRNA filtered reads. Using miRDeep2, new miRNAs were predicted from maturity, star, and loop sequences according to the RNAfold algorithm. The RNAfold function uses the nearest-neighbor thermodynamic model to predict the minimum free energy secondary structure of an RNA sequence, sequentially aligning uniquely clustered reads to the reference genome, miRBase v22.1, and the non-coding RNA database RNAcentral release 14.0. , identify known miRNAs and other types of RNA for classification.

Example 8. Monocyte isolation and miRNA transfection

Blood from asthma patients was collected via percutaneous venous catheter sampling into vacuum vessel tubes containing acidic citrate dextrose solution (BD Biosciences). Blood was layered in Lymphoprep solution (Axis-Shield, Oslo, Norway) and then centrifuged at 800 × g for 25 min at 20°C. The layer containing peripheral blood monocytes was separated and red blood cells were removed by hypotonic lysis. Then, the cells were purified using a human monocyte isolation kit (Miltenyi Biotec, Auburn, CA, USA). For transfection of the miRNA mimic, 1 × 10 ⁶ human peripheral monocytes were seeded in a 24-well plate, and the cells were treated with 1 μg/mL LPS (Sigma-Aldrich) before transfection with the miRNA mixture. Transfections were performed using synthetic miRNA mimics such as 1272 and 1291 (Sigma-Aldrich) as well as miRNA-197-5p, 579-3p, 3065-5p, and 4517 (MyBioSource, San Diego, CA, USA). . Then, cells were transfected with 50 nM synthetic miRNA mimics using Lipofectamine™ 3000 Transfection Reagent (ThermoFisher Scientific) according to the manufacturer's instructions. After 48 hours, the transfected cells and supernatant were obtained, and the expression of intracellular NOD-like receptor pyrin domain-containing protein 3 (NOD-, LRR-, and pyrin domain-containing protein 3, NLRP3) was analyzed through Western blot analysis. observed. IL-1 levels in the supernatant were measured using an ELISA kit (R&D Systems).

Example 9. Isolation and stimulation of innate lymphoid cells (ILCs)

Innate lymphoid cells (ILCs) were purified from peripheral blood mononuclear cells using a human pan-ILC enrichment kit (STEMCELL Technologies, Vancouver, Canada). Isolated ILCs were clarified by performing flow cytometry using antibodies such as anti-lineage (BD Biosciences), anti-CD45 (BD Biosciences), and anti-CD127 (BioLegend, San Diego, CA, USA). The cells were analyzed by FACSAria III (BD Biosciences), and graphs were created using FlowJo software (Tree Star, Ashland, OR, USA). The expression of GATA-3 (Abcam), ROR-γt (Invitrogen, Waltham, MA, USA), and IL-22 (Abcam) in ILC was confirmed through Western blot analysis. Then, 1 × 10 ³ ILCs were co-colocalized with 1 × 10 ⁵ monocytes (with or without transfection of hsa-miR-4517) seeded in 48-well plates in RPMI 1640 medium supplemented with 2% fetal bovine serum. Cultured. These cells were stimulated with 1 μg of LPS for 24 hours.

Example 10. Plasma-EV isolation and characterization

Human plasma-derived EVs were purified using an Exosome Isolation kit (Microgentas Inc., Seoul, Korea), and AEC-EVs were detected using an EpCAM antibody (Abcam). Additionally, protein patterns between plasma and plasma-EV were compared through SDS-PAGE.

For miRNA analysis of EVs, total RNA was extracted using the miRNeasy kit (Qiagen, Venlo, Netherlands), and then MiniAmp Plus Termal Cycler (Applied Biosystems, Waltham, MA, USA) was used for reverse transcription and detection of miRNAs. From miRNA, cDNA was synthesized using the HBMiR Multi Assay kit (HIMBITOEK, Seongnam, Korea), and quantitative PCR was performed using QuantStudio 5 (Applied Biosystems). Relative miRNA expression was calculated using the comparative 2 ^-ΔΔCt method, which was normalized by the expression of miRNA 39-3p.

Example 11. Mouse experiment and evaluation

All animal studies were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC-2016-0022). To induce neutrophilic asthma, 6-week-old male C57BL/6 mice (Orient BIO, Seongnam, Korea) were administered 75 μg of ovalbumin (OVA; Sigma-Aldrich) and 10 μg of LPS for sensitization at 0, 1; Treated intranasally on days 2, and 7. Then, each sensitized mouse was treated intranasally with 20 μL of PBS containing 50 μg of OVA for testing on days 14, 15, 16, 17, and 18. During testing, mice were treated intranasally with 0.2 mg/mouse Dex or 1 μg/mouse MlEVs simultaneously with OVA injection. To perform histological analysis, lung tissue was cut into 4 μm thick pieces, embedded in paraffin blocks, stained with H&E, and observed using Image J (National Institutes of Health, Bethesda, MD, USA). To measure the number of immune cells in bronchoalveolar lavage fluid (BALF), Diff-quick staining (Dade Behring, Dudingen, Switzerland) was performed, and IL-1β and IL-17 levels in BALF were measured using ELISA kits (R&D Systems). and quantified. Additionally, the expression of NLRP3 (Cell Signaling Technology), T-bet (Abcam), and ROR-γt (Invitrogen) was observed in lung tissue. Flow cytometric analysis of NCR-ILC3 was performed using the following antibodies: anti-lineage, anti-ST2, anti-CD45, anti-CD127, anti-c-Kit, and anti-NKp46 (all from BioLegend).

Example 12. Statistical analysis

All statistical analyzes were performed using IBM SPSS software version 26.0 (IBM Corporation, Armonk, NY, USA), and differences between the two groups were analyzed using Student's t-test. Data from multiple groups were compared using Bonferroni's post hoc test and one-way ANOVA. Correlation was expressed as the Pearson correlation coefficient r , and graphs were generated using GraphPad Prism 8.0 software (GraphPad Inc., San Diego, CA, USA).

[Experimental example]

Experimental Example 1. Confirmation of the amount of MlEV in neutrophilic asthma patients

The clinical characteristics of the test subjects are shown in Table 1 below.

Variables HCs
(n = 40) Asthmatics
(n = 45) P value EA
(n = 25) N.A.
(n = 20) P value Age(y) 41.1 ± 16.1 44.0 ± 17.6 .782 38.2 ± 16.6 44.8 ±15.0 .180 Female sex (%) 47.5 55.6 .458 56.0 55.0 .947 Atopy (%) 47.5 55.6 .173 72.0 50.0 .130 Severe asthma (%) N.D. 62.2 N.D. 16.0 10.0 .556 Baseline FEV1 (%) 106.3 ± 12.9 13.3 .004 95.8 ± 19.1 93.2 ± 15.7 .632 PC20 (mg/mL) N.D. 94.7 ± 17.5 N.D. 5.2 ± 6.3 5.7 ± 2.9 .759 Sputum Eos (%) N.D. 11.6 ± 16.5 N.D. 19.4 ± 18.1 0.8 ± 0.9 .001 Sputum Neu (%) N.D. 54.5 ± 30.1 N.D. 34.3 ± 24.7 79.8 ± 11.0 .001 Total IgE (IU/mL) 102.4 ± 93.1 511.6 ± 527.2 .001 544.6 ± 544.8 248.3 ± 269.1 .025 ECP (ng/mL) 38.6 ± 52.8 412.9 ± 470.5 .003 211.9 ± 230.8 42.6 ± 66.7 .002 MPO (ng/mL) 198.2 ± 89.1 273.8 ± 109.8 .019 223.6 ± 83.9 339.8 ± 184.6 .045

(HC: healthy control, EA: eosinophilic asthma, NA: neutrophilic asthma, FEV1: forced expiratory volume in 1s, methacholine PC20: decreases FEV1 by 20% Stimulating concentration of methacholine required to stimulate, Eos: eosinophils, Neu: neutrophils, IgE: immunoglobulin E, ECP: eosinophil cationic protein, MPO: myeloid peroxidase (myeloperoxidase), ND: no data)

Patients with asthma had lower baseline FEV1 (%) but higher total eosinophil count, IgE, ECP, and MPO (all P < 0.05) compared to healthy controls (HC). Among patients, baseline FEV1 (%) was not significantly different between neutrophilic asthma (NA) and eosinophilic asthma (EA), but levels of MPO were higher in NA, whereas levels of total eosinophil count, IgE, and ECP were higher in EA. It was high.

As a result of checking the level of MlEV- or EcEV-specific IgG1 in serum, there was no significant difference in all groups, as shown in Figure 1a. On the other hand, in the case of MlEV-specific IgG4, as shown in Figure 1b, NA patients showed lower levels of MlEV-specific IgG4 than EA patients, but showed the highest level of EcEV-specific IgG4 among the groups. In addition, as shown in Figure 1c, the level of MlEV-specific IgG4, but not EcEV-specific IgG4, was positively correlated with baseline FEV ₁ (%), indicating that the prevalence of bacterial EVs in asthma patients This indicates that it may be related to decreased lung function.

Experimental Example 2. Effect of MlEV on pathophysiological conditions of AEC

When M. luteus was cultured and its EVs were isolated, as shown in Figure 2a, MlEVs showed a spherical lipid bilayer morphology with an average diameter of 62.16 ± 14.70 nm, and as shown in Figure 2b, these MlEVs had a cellular composition. It contained various proteins related to elements and biological processes.

To determine whether MlEV can modulate the immune response triggered by AEC, A549 cells were treated without or with MlEV in the presence of LPS. As a result, as shown in Figure 2c, it was confirmed that MlEV significantly reduced IL-8 production in cells. Additionally, as shown in Figure 2d, phosphorylation of intracellular p65 was confirmed to be significantly inhibited by MlEV treatment.

When examining the RNA composition, miRNAs were highly expressed in the cells, as shown in Figure 2e, and in particular, as shown in Figure 2f, MlEVs expressed seven miRNAs (hsa-miR-15b-3p, hsa-miR-15b-3p, hsa-miR-3141, hsa-miR-4785, hsa-miR-5100, hsa-miR-196b-3p, hsa-miR18b-5p, hsa-miR-409-5p) and six downregulated miRNAs (hsa-miR-409-5p). (miR-3065-5p, hsa-miR-1291, hsa-miR-1272, hsa-miR-197-5p, hsa-miR-579-3p, hsa-miR-4517) were restored to their original state.

Experimental Example 3. Confirmation of the function of miRNA on IL-1β producing monocytes

Since various miRNAs are known to be contained in EVs, in the present invention, AEC-derived EVs were isolated in physiological or pathological conditions. As a result, as shown in Figure 3a, similar to bacterial EVs, A549 cells released spherical lipid bilayer EVs.

Additionally, as shown in Figure 3b, mammalian EVs strongly expressed ALIX, whereas A549 cells expressed TSG101 and actin, but expression of EV markers including ALIX and TSG101 in cell culture supernatants was very weak. The protein pattern in EV is shown in Figure 3c.

In addition, we selected six target miRNAs and examined their expression in EVs. As shown in Figure 3d, when A549 cells were treated with MlEV in the presence of LPS, hsa-miR-3065-5p and hsa-miR-1272 were detected in EVs. , and hsa-miR-4517, the expression of three miRNAs was enhanced. Because monocytes are important in mediating immune responses in NA, we further evaluated the function of miRNAs on monocytes, and the results showed that by transfection of several miRNAs, hsa-miR-4517 was transfected with LPS, as shown in Figure 3e. It was confirmed that IL-1β production and NLRP3 expression were significantly reduced in stimulated monocytes.

Experimental Example 4. Confirmation of decreased expression of has-miR-4517 and increased ILC3 activation

AEC-derived EVs in plasma were identified using EV and epithelial cell markers, as shown in Figure 4a. The protein profile differed between plasma-EV and plasma itself, as shown in Figure 4b.

Additionally, as a result of confirming the expression of hsa-miR-4517 in plasma-EVs, the expression of hsa-miR-4517 in plasma-EVs was lower in asthma patients compared to normal controls, as shown in Figure 4c. Although there was no statistical significance, the expression of hsa-miR-4517 tended to be decreased in NA patients than in EA patients.

Plasma levels of IL-22 were significantly higher in NA patients than in EA patients, as shown in Figure 4D, and expression of hsa-miR-4517 in EVs was negatively correlated with plasma levels of IL-22, as shown in Figure 4E. showed a relationship. Additionally, when ILCs were isolated from peripheral blood mononuclear cells, NA patients showed more significantly increased ROR-γt and IL-22 expression in ILCs, as shown in Figure 4f. ROR-γt-expressing ILCs are the major source of IL-22 production, and as shown in Figure 4g, when ILCs were cultured with monocytes in the presence of LPS, the level of IL-22 was reduced by hsa-miR-4517 treatment. It has been done.

Experimental Example 5. Confirmation of inhibition of neutrophil asthma by MlEV treatment in mice

When LPS-induced asthma mice were treated with MlEV, immune responses as well as immune cell infiltration in lung tissue were significantly reduced, as shown in Figure 5A. Specifically, as shown in Figure 5B, MlEV reduced neutrophil numbers and IL-1β and IL-17 production in BALF. Although Dex attenuated this inflammatory state, MlEV was able to reduce neutrophilic airway inflammation more effectively.

Additionally, as shown in Figure 5c, the expression of NLRP3, T-bet, and ROR-γt was significantly suppressed by MlEV treatment in the lung tissue of asthmatic mice. Flow cytometry analysis showed a lower proportion of IL-17-producing NCR-ILC3 when asthmatic mice were treated with MlEV than Dex, as shown in Figure 5D. Cells were defined by lack of lineage markers, ST2, and NKp46, and expression of CD45, c-Kit, and CD127.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims (20)

A pharmaceutical composition for preventing or treating asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,
A pharmaceutical composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .
According to paragraph 1,
A pharmaceutical composition for preventing or treating asthma, wherein the asthma is neutrophilic asthma or eosinophilic asthma.
According to paragraph 1,
The hsa-miR-4517 expression promoter is a pharmaceutical composition for preventing or treating asthma, characterized in that it increases hsa-miR-4517 expression in airway epithelial cells (AEC).
According to paragraph 1,
A pharmaceutical composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter inhibits neutrophilic airway inflammation.
According to paragraph 1,
A pharmaceutical composition for preventing or treating asthma, wherein the vesicles have an average diameter of 10 to 200 nm.
According to paragraph 1,
A pharmaceutical composition for preventing or treating asthma, characterized in that the vesicles are naturally secreted or artificially produced by Micrococcus luteus.
An inhalant composition for preventing or treating asthma, comprising an hsa-miR-4517 expression promoter as an active ingredient,
An inhalant composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter is a vesicle derived from Micrococcus luteus .
In clause 7,
An inhalant composition for preventing or treating asthma, wherein the asthma is neutrophilic asthma or eosinophilic asthma.
In clause 7,
An inhalant composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter increases the expression of hsa-miR-4517 in airway epithelial cells (AEC).
In clause 7,
An inhalant composition for preventing or treating asthma, wherein the hsa-miR-4517 expression promoter inhibits neutrophilic airway inflammation.
(a) measuring the level of vesicle-specific immunoglobulin G4 (IgG4) derived from Micrococcus luteus from a biological sample; and
(b) A method of providing information for the diagnosis of asthma, including the step of classifying as asthma when the measured level of IgG4 is reduced compared to a normal person.
According to clause 11,
A method of providing information for the diagnosis of asthma, wherein the biological sample is one or more selected from the group consisting of blood, plasma, serum, bone marrow, saliva, urine, and feces.
According to clause 11,
A method of providing information for the diagnosis of asthma, characterized in that the asthma is neutrophilic asthma or eosinophilic asthma.
According to clause 11,
(c) providing information for the diagnosis of asthma, further comprising classifying it as neutrophilic asthma if the level of IgG4 measured in step (b) is decreased compared to normal people and decreased compared to eosinophilic asthma patients. method.
According to clause 11,
The step of measuring the level of IgG4 includes Western blot, ELISA (enzyme linked immunosorbent assay), ImmunoCAP, RIA (Radioimmunoassay), radioimmunodiffusion, and Ouchterlony immunity. Diffusion method, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter (FACS)) and protein chip. A method of providing information for the diagnosis of asthma, characterized in that it uses one or more methods selected from the group consisting of.
(a) measuring the expression level of hsa-miR-4517 from a biological sample; and
(b) A method of providing information for the diagnosis of asthma, including the step of classifying as asthma when the measured expression level of hsa-miR-4517 is reduced compared to normal people.
According to clause 16,
A method of providing information for the diagnosis of asthma, wherein the biological sample is one or more selected from the group consisting of blood, plasma, serum, bone marrow, saliva, urine, and feces.
According to clause 16,
A method of providing information for the diagnosis of asthma, characterized in that the asthma is neutrophilic asthma or eosinophilic asthma.
According to clause 16,
The step of measuring the expression level of hsa-miR-4517 is polymerase chain reaction (polymerase chain reaction, PCR), reverse transcription polymerase chain reaction (RT-PCR), and competitive reverse transcription polymerase reaction (Competitive reverse transcription polymerase chain reaction). RT-PCR), real-time polymerase chain reaction (Real-time PCR, quantitative PCR, quantitative real-time PCR), RNase protection assay (RPA), southern blotting, Northern blotting A method of providing information for the diagnosis of asthma, characterized in that it uses one or more methods selected from the group consisting of blotting), and DNA chips.
According to clause 16,
A method of providing information for the diagnosis of asthma, wherein the hsa-miR-4517 is expressed in airway epithelial cells (AEC).