Phagocytotic promoter of macrophage, activator of plasmacytoid dendritic cell, and activator of myeloid dendritic cell

Abstract

To provide a phagocytotic promotor of macrophage by a lactic acid bacterium, an activator of a plasmacytoid dendritic cell (pDC) by a lactic acid bacterium, and an activator of a myeloid dendritic cell (mDC) by a lactic acid bacterium.SOLUTION: A phagocytotic promotor of macrophage, an activator of a plasmacytoid dendritic cell (pDC), an activator of a myeloid dendritic cell (mDC) can be provided by including Lactobacillus panisapium M1 strain (NITE BP-03106) as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a macrophage phagocytosis promoter, a plasmacytoid dendritic cell (pDC) activator, and a myeloid dendritic cell (mDC) activator. Specifically, the present invention relates to a macrophage phagocytosis promoter, a plasmacytoid dendritic cell activator, and a myeloid dendritic cell activator using queen bee-derived lactic acid bacteria (Lactobacillus vanisapium M1 strain (NITE BP-03106)). [Background technology]

[0002] Lactic acid bacteria are attracting attention as a substance that activates the immune system (immunostimulatory). It is also known that immune activation occurs when immune cells are activated. The immune system is broadly divided into innate immunity, which is mainly comprised of macrophages, dendritic cells, and NK cells and shows an early response in a non-specific manner to antigens, and adaptive immunity, which is mainly comprised of lymphocytes and shows an antigen-specific response.

[0003] Methods for activating macrophages using lactic acid bacteria have been studied. For example, Patent Document 1 discloses that lactic acid bacteria isolated from pollen called Lactobacillus kunkeii BPS402 has the ability to activate macrophages.

[0004] Furthermore, Patent Document 2 discloses a method for activating plasmacytoid dendritic cells (pDC) using lactic acid bacteria.

[0005] Furthermore, Patent Document 3 discloses a method for converting 10-hydroxy-2-decenoic acid contained in royal jelly into 10-hydroxydecanoic acid using a lactic acid bacterium called Lactobacillus panicipium M1 strain (NITE BP-03106) isolated from the intestine of an adult queen bee. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2022-108860 [Patent Document 2] JP 2017-201984 A [Patent Document 3] International Publication No. 2021 / 033726 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel macrophage phagocytosis promoter, a novel plasmacytoid dendritic cell (pDC) activator, and a novel myeloid dendritic cell (mDC) activator. [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered that Lactobacillus vanisapium M1 strain (NITE BP-03106) has the effect of promoting phagocytosis by macrophages, activating plasmacytoid dendritic cells (pDC), and activating myeloid dendritic cells (mDC), and thus completed the present invention.

[0009] That is, the present invention relates to, for example, the following inventions. [1] A macrophage phagocytosis promoter containing Lactobacillus vanaspium M1 strain (NITE BP-03106) as the active ingredient. [2] An activator of plasmacytoid dendritic cells (pDCs) containing Lactobacillus vanisapium M1 strain (NITE BP-03106) as an active ingredient. [3] An activator of myeloid dendritic cells (mDCs) containing Lactobacillus vanisapium M1 strain (NITE BP-03106) as an active ingredient. [4] An immunostimulant comprising the agent according to any one of [1] to [3]. [5] The agent according to any one of [1] to [4], which is a food composition, a medicine or a quasi-drug. Effect of the Invention

[0010] The present invention can provide a novel macrophage phagocytosis promoter, a novel plasmacytoid dendritic cell (pDC) activator, and a novel myeloid dendritic cell (mDC) activator. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing the phagocytosis rate and average fluorescence intensity of macrophages upon addition of Lactobacillus vanisapium M1 strain. [Diagram 2] 1 is a graph showing the average fluorescence intensity of dendritic cell activation markers in pDCs and mDCs after addition of Lactobacillus vanisapium M1 strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, the embodiment for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiment. In the present invention, plasmacytoid dendritic cells are also referred to as "pDC", and myeloid dendritic cells are also referred to as "mDC". In addition, in the present invention, Lactobacillus panisapium M1 strain is also referred to as "M1 strain".

[0013] The macrophage phagocytosis promoter according to the present embodiment contains Lactobacillus panisapium M1 strain as an active ingredient. That is, the macrophage phagocytosis promoter according to the present embodiment contains Lactobacillus panisapium M1 strain as an active ingredient, and therefore can promote phagocytosis by macrophages.

[0014] The pDC activator according to the present embodiment contains Lactobacillus panisapium M1 strain as an active ingredient. That is, the pDC activator according to the present embodiment contains Lactobacillus panisapium M1 strain as an active ingredient, and therefore can activate pDC.

[0015] The mDC activator according to the present embodiment contains Lactobacillus panisapium M1 strain as an active ingredient. That is, the mDC activator according to the present embodiment contains Lactobacillus panisapium M1 strain as an active ingredient, and therefore can activate mDC.

[0016] Specifically, activation of pDC or mDC may refer to, for example, improvement of the ability of pDC or mDC to present antigen to T cells.

[0017] [Lactobacillus vanisapium M1 strain] The Lactobacillus panisapium M1 strain has been deposited under the accession number NITE BP-03106 at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on January 16, 2020, and is available.

[0018] The M1 strain as an active ingredient may be the bacterial cell itself or a treated bacterial cell product. The bacterial cell may be a live bacterial cell or a killed bacterial cell. The treated bacterial cell product may be a bacterial cell that has been subjected to a process such as heating, pasting, drying (freeze drying, vacuum drying, spray drying, etc.), freezing, lysis, crushing, extraction, etc. Crushing can be performed, for example, by ultrasonication. A combination of multiple types of treatments may be performed. The treated bacterial cell product may be a supernatant obtained after a protein removal process is performed on the treated bacterial cell product, or a supernatant obtained by removing solids from a bacterial cell culture and fermentation product. The M1 strain may be an isolated bacterial cell, or a fermentation product or culture of the bacterial cell.

[0019] The M1 strain can be cultured according to a conventional method. There is no particular limitation on the medium as long as the bacterium can be cultured in the medium, and a natural medium, a synthetic medium, a semi-synthetic medium, etc. can be used. The medium can contain a nitrogen source and a carbon source. Examples of the nitrogen source include meat extract, peptone, casein, yeast extract, gluten, soy flour, soy hydrolysate, and amino acids, and examples of the carbon source include glucose, lactose, fructose, inositol, sorbitol, starch syrup, starch, koji soup, bran, bagasse, and molasses. In addition, inorganic substances (e.g., ammonium sulfate, potassium phosphate, magnesium chloride, salt, calcium carbonate, iron, manganese, and molybdenum), various vitamins, and the like can be added.

[0020] The culture temperature may be, for example, 4 to 45°C, or may be 25 to 40°C, 28 to 37°C, or 30 to 33°C. The culture may be performed by aeration shaking or aeration stirring. The pH of the medium may be, for example, 4.0 to 9.0, and is preferably 6.0 to 8.0. As a culture method, for example, the cells are inoculated into MRS medium and cultured at 30°C for 48 hours.

[0021] When the macrophage phagocytosis promoter, pDC activator, or mDC activator contains an M1 strain as an active ingredient, the macrophage phagocytosis promoter, pDC activator, or mDC activator may be, for example, a fermented product containing the M1 strain, a lactic acid bacteria drink, a drink containing lactic acid bacteria, etc.

[0022] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment can be used at a dose of 1 mg to 50 mg per day, preferably 3 mg to 30 mg, and more preferably 5 mg to 15 mg, in terms of the solid content of the M1 strain, for an adult weighing 60 kg. The dose can be appropriately set within the above range depending on factors such as the health condition of the person taking it, the method of administration, and the combination with other agents.

[0023] The content of the M1 strain in the macrophage phagocytosis promoter, pDC activator, or mDC activator may be 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.02% by mass or more, 0.03% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 1% by mass or more, or 10% by mass or more in terms of solid content relative to the total amount of the agent. Also, the content of the M1 strain in the macrophage phagocytosis promoter, pDC activator, or mDC activator may be 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.1% by mass or less, 0.05% by mass or less, 0.03% by mass or less, or 0.01% by mass or less in terms of solid content relative to the total amount of the agent.

[0024] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment may further contain other ingredients in addition to the above-mentioned active ingredients. Examples of other ingredients include pharma- ceutically acceptable ingredients (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents, and antioxidants) and food acceptable ingredients (e.g., minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavors, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, and wetting agents).

[0025] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment may be a food composition, a medicine, or a quasi-drug. The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment can be used as a food composition, a medicine, or a quasi-drug itself, and can also be used as an ingredient in a food composition, a medicine, or a quasi-drug. A food composition, a medicine, or a quasi-drug containing a macrophage phagocytosis promoter, pDC activator, or mDC activator as an ingredient can be produced, for example, by adding the macrophage phagocytosis promoter, pDC activator, or mDC activator to an intermediate product in the production process of these products.

[0026] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment may be in any form, such as a solid, liquid, or paste, and may be in the form of a tablet (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (powdered drugs), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections (including the case where the liquid is prepared by mixing with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion at the time of use), etc. These various preparations can be prepared, for example, by mixing the active ingredient with other ingredients as necessary and forming the mixture into the above-mentioned dosage form.

[0027] When used as a food composition or as one component of a food composition, the food composition is preferably one in which the tertiary function of the food, i.e., the function of regulating physical condition, is emphasized. Examples of products in which the tertiary function of the food is emphasized include health foods, functional foods, nutritional functional foods, nutritional supplements, supplements, and foods for specified health uses.

[0028] Examples of food compositions include beverages such as soft drinks such as coffee, juice, and tea drinks, milk drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, and alcoholic beverages such as sake, Western liquor, fruit liquor, and honey wine; spreads such as custard cream; pastes such as fruit paste; Western sweets such as chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, and puddings; Japanese sweets such as daifuku, mochi, manju, castella, anmitsu, and yokan; frozen desserts such as ice cream, popsicles, and sorbet; cooked foods such as curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, and jam; and seasonings such as dressings, furikake, umami seasonings, and soup bases.

[0029] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment is preferably ingested into the body. The administration mode may be oral administration or parenteral administration. The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment may be administered once a day, or may be administered in multiple doses, such as twice a day or three times a day. EXAMPLES

[0030] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0031] According to the following method, we investigated the effects of Lactobacillus vanisapium M1 strain (NITE BP-03106) isolated from the intestine of adult queen bees on the phagocytic activity of macrophages, activation of plasmacytoid dendritic cells (pDC), and activation of myeloid dendritic cells (mDC).

[0032] Example 1: Evaluation of macrophage phagocytic activity The macrophage activation ability of the test substance was evaluated using phagocytosis as an index. In summary, macrophages were cultured in a culture medium containing the test substance for 24 hours, and then fluorescently labeled Calboxylate latex beads were added and incubated for 1 hour. The cells were collected and subjected to analysis by a flow cytometer. The details are as follows.

[0033] The mouse macrophage J774.1 cell line purchased from the JCRB Cell Bank was used in the study. The J774.1 cell line is one of the cell lines commonly used in macrophage phagocytosis studies.

[0034] The negative control was the culture medium used to dissolve the test substance. The culture medium was a mixture of 500 mL of RPMI medium (Nacalai, #30264-85), 55.5 mL of inactivated FBS, and 5.5 mL of penicillin-streptomycin-glutamine (100x). The inactivated FBS was prepared by heating FBS at 56°C for 30 minutes, dispensing it, and storing it at -20°C.

[0035] [Test substance] The test substance used was Lactobacillus panisapium M1 strain (NITE BP-03106). Details are as follows. The Lactobacillus panisapium M1 strain (NITE BP-03106) was prepared by heat-treating live bacteria at 80°C or higher for 20 minutes, freeze-drying, and drying to obtain a powder. The powder was dissolved in culture medium to a concentration of 10 mg / mL, diluted to concentrations of 1, 5, and 10 μg / mL, and then ultrasonicated for 30 minutes before use.

[0036] [Preculture] J774.1 cells were subcultured in RPMI medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured in 10 cm dishes, and 6.08 × 10 5 The cells were subcultured at 1000 cells / mL in a 5% CO2 incubator set at 37°C.

[0037] [Calboxylate latex beads suspension] 5 μL of Calboxylate latex beads (Sigma, #L3030) were dissolved in 995 μL of RPMI medium and used in the test.

[0038] [Preparation of Hoechest staining solution] 1 μL of Hoechest 33342 (Invitrogen, #H3570) was diluted with 2000 μL of PBS to prepare a staining solution.

[0039] [Phagocytosis test] 1) J774.1 cells precultured in a 10 cm dish were treated with Accutase at 37°C for 5 minutes to remove them from the wall, and the resulting cell suspension was transferred to a 50 mL conical tube. The tube was centrifuged at room temperature at 200 x g for 5 minutes. The supernatant was discarded by decantation, and the cells were collected as a pellet. After loosening the cells by tapping, 5 mL of culture medium was added, and 20 μL of the cell suspension was transferred to a 1.5 mL microtube, and the cell count and viability were measured.

[0040] 2) 5 × 10 based on the measured cell count 5 A cell suspension of 1000 cells / mL was prepared. 1000 μL of the obtained cell suspension was added to each well of a 12-well plate. The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0041] 3) After pre-culture, the plate was removed from the incubator. The culture medium was removed from the wells in which the cells were seeded using a pipette. 1000 μL of medium or 1 μg / mL M1 strain suspension was added. The plate was then cultured for 24 hours in a 37°C, 5% CO2 incubator.

[0042] 4) After incubation, the culture medium was removed and 1000 μL of culture medium containing Calboxylate latex beads was added, followed by incubation for 1 hour in a 37°C, 5% CO2 incubator.

[0043] 5) After slowly removing the supernatant with a pipette, 1000 μL of warmed PBS was added and washed. The PBS was removed with a pipette and 1000 μL of warmed PBS was added. This operation was repeated three times.

[0044] 6) After washing, the PBS was removed and 500 μL of Hoechest 33342 staining solution was added to each well and allowed to stand in the dark for 5 minutes.

[0045] 7) The staining solution was removed, the PBS was removed with a pipette, and 1000 μL of flow cytometer buffer (HBSS containing 2% BSA and 10 mM HEPES) was added.

[0046] 8) The cells were collected by pipetting and centrifuged at 600×g for 5 minutes.

[0047] 9) After centrifugation, the supernatant was removed and 100 μL of flow cytometer buffer (HBSS containing 2% BSA and 10 mM HEPES) was added. The cell suspension was analyzed using a flow cytometer (MACSQuant 10, Miltenyi Biotec). 50,000 cells were analyzed to determine the phagocytosis rate and mean fluorescence intensity (MFI). The phagocytosis rate indicates the ratio of positive cells in the PE wavelength region where Calboxylate latex beads are detected to Hoechest positive cells. MFI indicates the PE fluorescence intensity of positive cells in the PE wavelength region where Calboxylate latex beads are detected.

[0048] The results of the macrophage phagocytosis test are shown in Figure 1. Figure 1 (a) is a graph showing the macrophage phagocytosis rate by the addition of Lactobacillus panisapium M1 strain. Figure 1 (b) is a graph showing the average fluorescence intensity by the addition of Lactobacillus panisapium M1 strain. It was confirmed that the phagocytosis rate and average fluorescence intensity were increased in Lactobacillus panisapium M1 strain (NITE BP-03106) compared to the negative control (control). Therefore, it was confirmed that the Lactobacillus panisapium M1 strain (NITE BP-03106) used in this test has a macrophage phagocytosis enhancing effect.

[0049] <Example 2: Evaluation of dendritic cell activation ability> We also investigated the ability of Lactobacillus vanisapium M1 strain (NITE BP-03106) to activate pDC and mDC. In summary, bone marrow cells taken from ICR mice were differentiated, and the whole differentiated cells were cultured for 48 hours in a culture medium containing the test substance, and then labeled with various markers related to antigen presentation to T cells in dendritic cells. The cells were collected and subjected to analysis by flow cytometer. The details are as follows.

[0050] [Test substance] The test substance used was Lactobacillus panisapium M1 strain (NITE BP-03106). Details are as follows. Lactobacillus panisapium M1 strain (NITE BP-03106) was prepared by heat-treating live bacteria at 80°C or higher for 20 minutes, adding carrageenan and dextrin, and freeze-drying the mixture to obtain a powder. The powder was dissolved at 10mg / mL, diluted to concentrations of 1, 5, and 10μg / mL, and then sonicated for 30 minutes before use.

[0051] [Preculture] ICR mouse bone marrow cells (Cosmo Bio, #BMC12C) were cultured in RPMI medium containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mM HEPES, 2 μM 2-mercaptoethanol, and 100 ng / mL Flt-3L to induce differentiation. Culture was performed in a 12-well plate for 7 days. Culture was performed in a 5% CO2 incubator set at 37°C.

[0052] [Dendritic cell evaluation test] 1) After differentiation induction by pre-culture, the plate was removed from the incubator. The culture medium was slowly removed from the wells in which the cells were seeded using a pipette. Then, 1000 μL of the test solution containing 10 μg / mL Lactobacillus vanisapium M1 strain (NITE BP-03106) was added. The plate was cultured for 48 hours in a 37°C, 5% CO2 incubator.

[0053] 2) After incubation, the supernatant was removed using a Pipetman and the cells were suspended in 1,000 μL of ice-cold HBSS containing 2% BSA and 10 mM HEPES.

[0054] 3) The mixture was centrifuged at 600×g for 5 minutes and suspended in 100 μL of ice-cold HBSS containing 2% BSA and 10 mM HEPES.

[0055] 4) Anti-VioBlue-CD45R (B220) (Miltenyi biotec, Cat:130-110-851) 2μL, anti-PEVio 770-CD11c (Miltenyi biotec, Cat:130-110-840) 2μL, APC-Vio 770-CD11b (Miltenyi biotec, Cat:130-113-803) 2μL, Anti-7-AAD Staining Solution (Miltenyi biotec, Cat:130-111-568) 10μL, Anti-VioGreen-MHC Class II (Miltenyi biotec, Cat:130-112-395) 2μL, Anti-Vio(R) Bright 2 μL of B515-CD40 (Miltenyi biotec, Cat: 130-116-115), 2 μL of anti-PE-CD80 (Miltenyi biotec, Cat: 130-116-460), and 2 μL of anti-APC-CD86 (Miltenyi biotec, Cat: 130-122-130) were added, and the cells were stained in the dark at 4°C for 15 minutes, followed by centrifugation at 600 × g for 5 minutes.

[0056] 5) The supernatant was removed, and HBSS containing 2% BSA and 10 mM HEPES was added, followed by analysis using a flow cytometer (MACSQuant 10, Miltenyi Biotec). B220-positive CD11b-negative CD11c-positive cells were designated pDC, and B220-negative CD11b-positive CD11c-positive cells were designated mDC. For each dendritic cell, the mean fluorescence intensity (MFI) of MHC-II, which plays a role in presenting antigens to T cells, and CD86, CD80, and CD40, which play a role in enhancing the signal, were determined. The test was performed six times, and the mean fluorescence intensity is shown as the mean ± standard deviation. The mean values ​​of the mean fluorescence intensity were used to determine significant differences from the negative control group by t-test (*: p<0.05).

[0057] The results of the dendritic cell evaluation test are shown in Figure 2. Figure 2 (a) is a graph showing the average fluorescence intensity of various dendritic cell activation markers of pDC (B220-positive, CD11b-negative, CD11c-positive cells) by the addition of Lactobacillus panisapium M1 strain. Figure 2 (b) is a graph showing the average fluorescence intensity of various dendritic cell activation markers of mDC (B220-negative, CD11b-positive, CD11c-positive cells) by the addition of Lactobacillus panisapium M1 strain. It was confirmed that the addition of Lactobacillus panisapium M1 strain (NITE BP-03106) significantly increased the average fluorescence intensity of MHC-II, CD40, and CD86 in pDC, and MHC-II, CD40, CD80, and CD86 in mDC, compared to the negative control group. Therefore, it was confirmed that the Lactobacillus vanisapium M1 strain (NITE BP-03106) used in this test has the effect of activating dendritic cells.

Claims

1. A macrophage phagocytosis promoter comprising Lactobacillus vanisapium M1 strain (NITE BP-03106) as an active ingredient.

2. An activator for plasmacytoid dendritic cells (pDC), comprising Lactobacillus vanisapium M1 strain (NITE BP-03106) as an active ingredient.

3. An activator for myeloid dendritic cells (mDC), comprising Lactobacillus vanisapium M1 strain (NITE BP-03106) as an active ingredient.

4. An immunostimulant comprising the agent according to any one of claims 1 to 3.

5. The immunostimulant according to claim 4, which is a food composition, a medicine or a quasi-drug.

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