A phagocytosis promoter for macrophages, an activator of plasmacytoid dendritic cells, and an activator of myeloid dendritic cells

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 panisapium M1 strain (NITE BP-03106)). [Background technology]

[0002] Lactic acid bacteria have attracted attention as a substance that activates the immune system (immunostimulatory). It is also known that immune activation occurs through the activation of immune cells. The immune system can be broadly divided into innate immunity, which is primarily comprised of macrophages, dendritic cells, and NK cells, and shows a non-specific initial response to antigens, and adaptive immunity, which is primarily comprised of lymphocytes and shows an antigen-specific response.

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

[0004] Furthermore, Patent Document 2 discloses a method for activating plasmacytoid dendritic cells (pDCs) 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 panisapium M1 strain (NITE BP-03106) isolated from the intestines of adult queen bees. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2022-108860 [Patent Document 2] Japanese Patent Application Publication No. 2017-201984 [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 agent for promoting phagocytosis of macrophages, an activator of plasmacytoid dendritic cells (pDC), and an activator of myeloid dendritic cells (mDC). [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 panisapium 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 panisapium M1 strain (NITE BP-03106) as the active ingredient. [2] An activator of plasmacytoid dendritic cells (pDCs) containing Lactobacillus panisapium M1 strain (NITE BP-03106) as an active ingredient. [3] An activator of myeloid dendritic cells (mDCs) containing Lactobacillus panisapium M1 strain (NITE BP-03106) as the 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 pharmaceutical product, or a quasi-drug. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a novel agent for promoting phagocytosis of macrophages, an activator of plasmacytoid dendritic cells (pDC), and an activator of myeloid dendritic cells (mDC). [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing the phagocytosis rate and mean fluorescence intensity of macrophages as a result of addition of Lactobacillus panisapium M1 strain. [Figure 2] 1 is a graph showing the mean fluorescence intensity of dendritic cell activation markers in pDCs and mDCs after addition of Lactobacillus panisapium strain M1. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0014] The pDC activator according to this embodiment contains the Lactobacillus panisapium M1 strain as an active ingredient. That is, the pDC activator according to this embodiment contains the Lactobacillus panisapium M1 strain as an active ingredient, and is therefore capable of activating pDCs.

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

[0016] Specifically, activation of pDCs or mDCs may mean, for example, improving the ability of pDCs or mDCs to present antigens to T cells.

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

[0018] The M1 strain as an active ingredient may be the bacterial cells themselves or a treated bacterial cell product. The bacterial cells may be live or killed. The treated bacterial cells may be bacteria that have been treated, for example, by heating, pasting, drying (lyophilization, vacuum drying, spray drying, etc.), freezing, lysing, crushing, extraction, etc. Crushing can be carried out, for example, by ultrasonication. A combination of treatments may also be used. The treated bacterial cell product may be the supernatant after deproteinization of the treated bacterial cell product, or the supernatant from which the solids have been removed from a bacterial cell culture or fermentation product. The M1 strain may be isolated bacterial cells, or a fermentation product or culture of the bacterial cells.

[0019] The M1 strain can be cultured according to conventional methods. There are no particular limitations on the medium, so long as it allows the bacterium to be cultured, and natural, synthetic, or semi-synthetic media can be used. The medium may contain a nitrogen source and a carbon source. Nitrogen sources include meat extract, peptone, casein, yeast extract, gluten, soybean flour, soybean hydrolysate, and amino acids, while carbon sources include glucose, lactose, fructose, inositol, sorbitol, starch syrup, starch, koji (malted rice), wheat bran, bacasu, and molasses. Other ingredients that may be added include minerals (e.g., ammonium sulfate, potassium phosphate, magnesium chloride, salt, calcium carbonate, iron, manganese, and molybdenum), various vitamins, and the like.

[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 with aeration and shaking or aeration and stirring. The pH of the medium may be, for example, 4.0 to 9.0, and preferably 6.0 to 8.0. As a culture method, for example, the cells may be inoculated into MRS medium and cultured at 30°C for 48 hours.

[0021] When a macrophage phagocytosis promoter, pDC activator, or mDC activator contains the 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, or a drink containing lactic acid bacteria.

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

[0023] The content of the M1 strain in a macrophage phagocytosis-promoting agent, 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, based on the total amount of the agent. Furthermore, the content of the M1 strain in a macrophage phagocytosis-promoting agent, 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, based on the total amount of the agent.

[0024] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present invention may further contain other ingredients in addition to the active ingredients described above. Examples of other ingredients include pharmaceutically 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, flavorings, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, and humectants).

[0025] The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment may be a food composition, pharmaceutical, or quasi-drug. The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment can be used as a food composition, pharmaceutical, or quasi-drug itself, or can be used as an ingredient in a food composition, pharmaceutical, or quasi-drug. A food composition, pharmaceutical, or 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-promoting agent, pDC activator, or mDC activator according to the present embodiments 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.), capsule, pill, powder (pulverized agent), fine granules, granules, liquid, suspension, emulsion, syrup, paste, or injection (including a case where the agent is mixed with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion to prepare a liquid at the time of use). These various formulations can be prepared, for example, by mixing the active ingredient with other ingredients as necessary and shaping the mixture into the above-mentioned dosage form.

[0027] When used as a food composition or as a component of a food composition, the food composition preferably emphasizes the tertiary function of food, i.e., the function of regulating physical condition. Examples of products that emphasize the tertiary function of food include health foods, functional foods, foods with nutrient claims, nutritional supplements, dietary 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 wine, and mead; 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 bowls, rice 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 or parenteral. The macrophage phagocytosis promoter, pDC activator, or mDC activator according to the present embodiment may be administered once a day, or in divided doses such as twice or three times a day. [Example]

[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] The effects of Lactobacillus panisapium 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) were investigated using the following methods.

[0032] <Example 1: Evaluation of macrophage phagocytic activity> The macrophage activation ability of the test substance was evaluated using phagocytic activity as an index. Briefly, macrophages were cultured in a culture medium containing the test substance for 24 hours, after which fluorescently labeled calcium latex beads were added and incubated for 1 hour. The cells were then collected and subjected to analysis using a flow cytometer. The specific procedure is as follows.

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

[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 heat-inactivated FBS, and 5.5 mL of penicillin-streptomycin-glutamine (100x). The heat-inactivated FBS was prepared by heating FBS at 56°C for 30 minutes, aliquoting it, and storing it at -20°C.

[0035] [Test substance] The test substance used was the Lactobacillus panisapium M1 strain (NITE BP-03106). Details are as follows: The live Lactobacillus panisapium M1 strain (NITE BP-03106) was heat-treated at 80°C or higher for 20 minutes, then freeze-dried and powdered. 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 sonicated 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 a 10 cm dish, 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] Five microliters 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 pre-cultured in a 10 cm dish were treated with Accutase at 37°C for 5 minutes to detach them from the wall, and the resulting cell suspension was transferred to a 50 mL conical tube. The tube was centrifuged at 200 x g for 5 minutes at room temperature. The supernatant was discarded by decantation, and the cells were recovered 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. The cell count and viability were measured.

[0040] 2) Based on the measured cell number, 5 × 10 5 A cell suspension of 1000 cells / mL was prepared. 1000 μL of the resulting 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 containing the cells using a pipette. 1000 μL of medium or a 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 calcium latex beads was added, followed by incubation in a 37°C, 5% CO2 incubator for 1 hour.

[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 procedure 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 cytometry 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, and the phagocytosis rate and mean fluorescence intensity (MFI) were calculated. The phagocytosis rate represents the proportion of Hoechest-positive cells in the PE wavelength range where calboxylate latex beads are detected. MFI represents the PE fluorescence intensity of positive cells in the PE wavelength range 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 after addition of Lactobacillus panisapium strain M1. Figure 1(b) is a graph showing the mean fluorescence intensity after addition of Lactobacillus panisapium strain M1. It was confirmed that the phagocytosis rate and mean fluorescence intensity were increased with Lactobacillus panisapium strain M1 (NITE BP-03106) compared to the negative control. Therefore, it was confirmed that the Lactobacillus panisapium strain M1 (NITE BP-03106) used in this test has the effect of enhancing macrophage phagocytosis.

[0049] <Example 2: Evaluation of dendritic cell activation ability> The ability of Lactobacillus panisapium M1 strain (NITE BP-03106) to activate pDCs and mDCs was also examined. Briefly, bone marrow cells collected from ICR mice were differentiated, and the differentiated cells were cultured in a culture medium containing the test substance for 48 hours. After that, the whole cells were labeled with various markers related to antigen presentation to T cells by dendritic cells. The cells were then collected and subjected to analysis using a flow cytometer. The specific procedure is 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 produce a powder. The powder was dissolved at 10 mg / mL, diluted to concentrations of 1, 5, and 10 μg / 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 for differentiation. Cultures were performed in 12-well plates for 7 days. Cultures were performed in a 5% CO2 incubator set at 37°C.

[0052] [Dendritic cell evaluation test] 1) After differentiation induction in the pre-culture, the plate was removed from the incubator. The culture medium was slowly removed from the wells containing the seeded cells using a pipette. Next, 1000 μL of the test solution containing 10 μg / mL of Lactobacillus panisapium 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 pipette and the cells were suspended in 1000 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. The cells were then analyzed using a flow cytometer (MACSQuant 10, Miltenyi Biotec). B220-positive, CD11b-negative, CD11c-positive cells were designated pDCs, and B220-negative, CD11b-positive, CD11c-positive cells were designated mDCs. For each dendritic cell, the mean fluorescence intensity (MFI) of MHC-II, which plays a role in presenting antigen to T cells, and CD86, CD80, and CD40, which play a role in enhancing this signal, was determined. The test was performed six times, and the mean fluorescence intensity is shown as the mean ± standard deviation. A t-test was used to determine significant differences from the negative control group (*: 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 mean fluorescence intensity of various dendritic cell activation markers in pDCs (B220-positive, CD11b-negative, CD11c-positive cells) after addition of Lactobacillus panisapium strain M1. Figure 2(b) is a graph showing the mean fluorescence intensity of various dendritic cell activation markers in mDCs (B220-negative, CD11b-positive, CD11c-positive cells) after addition of Lactobacillus panisapium strain M1. It was confirmed that the addition of Lactobacillus panisapium strain M1 (NITE BP-03106) significantly increased the mean fluorescence intensity of MHC-II, CD40, and CD86 in pDCs, and MHC-II, CD40, CD80, and CD86 in mDCs compared to the negative control group. Therefore, it was confirmed that the Lactobacillus panisapium M1 strain (NITE BP-03106) used in this test has the effect of activating dendritic cells.

Claims

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

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

3. An activator of myeloid dendritic cells (mDCs), comprising Lactobacillus panisapium 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 pharmaceutical product or a quasi-drug.

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