Activators for plasmacytoid dendritic cells and myeloid dendritic cells

Abstract

This invention provides an activator for plasmacytoid dendritic cells (pDCs) using lactic acid bacteria. It also provides an activator for myeloid dendritic cells (mDCs) using lactic acid bacteria. [Solution] By including Apiractobacillus kunky BPS402 (FERM BP-11439) as an active ingredient, it is possible to provide an activator for plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs).

Description

Technical Field

[0001] The present invention relates to an activator of plasmacytoid dendritic cells (pDC) and an activator of myeloid dendritic cells (mDC). Specifically, it relates to an activator of plasmacytoid dendritic cells and an activator of myeloid dendritic cells, which contains Apilactobacillus kunkeei BPS402 (FERM BP-11439) as an active ingredient.

Background Art

[0002] Immunity is broadly classified into "innate immunity" and "acquired immunity". Innate immunity shows an initial response non-specifically to antigens and is a mechanism for sensing and removing pathogens and self-cells that have invaded the body through receptors on the cell surface. Innate immunity is mainly carried out by phagocytic cells such as neutrophils, macrophages, and dendritic cells. Acquired immunity shows a response specifically to antigens and is a mechanism for memorizing pathogen information and effectively eliminating it when encountering the memorized pathogen. Acquired immunity is mainly carried out by lymphocytes such as T cells and B cells.

[0003] Patent Document 1 describes that Lactobacillus kunkeei BPS402 promotes the phagocytic ability of macrophages. Also, Patent Document 2 discloses that Lactobacillus kunkeei BPS402 has an IgA production promoting effect. Patent Document 3 discloses a method for activating plasmacytoid dendritic cells (pDC) by Lactococcus lactis JCM5805. [[ID=1,8]]

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

[0005] The present invention aims to provide novel activators for plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs). [Means for solving the problem]

[0006] As a result of diligent research to achieve the above objective, the inventors discovered that Apiractobacillus kunkie BPS402 has activating effects on plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs), thus completing the present invention.

[0007] In other words, the present invention relates to, for example, the following inventions. [1] A plasmacytoid dendritic cell activator containing Apiractobacillus kunky BPS402 (FERM BP-11439) as the active ingredient. [2] A myeloid dendritic cell activator containing Apiractobacillus kunky BPS402 (FERM BP-11439) as the active ingredient. [3] An immunostimulant comprising the activator described in [1] or [2]. [4] A food composition, pharmaceutical product, or quasi-drug, as described in any of [1] to [3]. [Effects of the Invention]

[0008] The present invention provides novel activators for plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs). [Brief explanation of the drawing]

[0009] [Figure 1] (a) is a graph showing the average fluorescence intensity of MHC-II in pDCs, and (b) is a graph showing the average fluorescence intensity of CD86 in pDCs. [Figure 2] (a) is a graph showing the average fluorescence intensity of CD80 in pDCs, and (b) is a graph showing the average fluorescence intensity of CD40 in pDCs. [Figure 3] (a) is a graph showing the average fluorescence intensity of MHC-II in mDCs, and (b) is a graph showing the average fluorescence intensity of CD86 in mDCs. [Figure 4] (a) is a graph showing the average fluorescence intensity of mDC CD80, and (b) is a graph showing the average fluorescence intensity of mDC CD40. [Modes for carrying out the invention]

[0010] The embodiments for carrying out the present invention will be described in detail below. 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." In the present invention, Apilactobacillus kunkeei BPS402 is also referred to as "BPS402." In addition, what was previously called Lactobacillus kunkeei has been renamed Apilactobacillus kunkeei due to reclassification.

[0011] The pDC activator according to this embodiment contains Apiractobacillus kunky BPS402 as an active ingredient. Because the pDC activator according to this embodiment contains Apiractobacillus kunky BPS402 as an active ingredient, it can activate pDCs.

[0012] The mDC activator according to this embodiment contains Apiractobacillus kunky BPS402 as an active ingredient. Because the mDC activator according to this embodiment contains Apiractobacillus kunky BPS402 as an active ingredient, it can activate mDCs.

[0013] In this specification, activation of pDCs or mDCs may specifically mean, for example, an improvement in the ability of pDCs or mDCs to present antigens to T cells.

[0014] Macrophages take in pathogens and debris and use intracellular enzymes to break down foreign substances. IgA is secreted by plasma cells differentiated from B cells and neutralizes toxins and viruses. Dendritic cells, on the other hand, induce their own maturation by capturing and processing antigens. Mature dendritic cells migrate to lymph nodes and present antigen information to naive T cells, thereby developing antigen-specific adaptive immunity. In other words, dendritic cells bridge the gap between innate and adaptive immunity. Therefore, dendritic cells differ in function from IgA and macrophages in terms of "information transmission." While macrophages and IgA play the main roles in innate and adaptive immunity, respectively, dendritic cells play an important role in both.

[0015] [Apiractobacillus kunkie BPS402] Apiractobacillus kunkie BPS402 was deposited on October 3, 2011, at the Patent Organism Depository Center (IPOD: 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, Japan (postal code 305-8566)) under accession number FERM P-22177 and is available for purchase. IPOD has since moved to Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture, Japan (postal code 292-0818). Furthermore, this strain has now been transferred to international depositary status under accession number FERM BP-11439. It should be noted that Apiractobacillus kunkie BPS402 is a reclassified and renamed version of Lactobacillus kunkie BPS402; the two are identical.

[0016] BPS402 as an active ingredient may be the bacterial cells themselves or processed products of the bacterial cells. The bacterial cells may be live bacterial cells or dead bacterial cells. The processed products of the bacterial cells may be those obtained by subjecting the bacteria to treatments such as heating, pasting, drying (freeze drying, vacuum drying, spray drying, etc.), freezing, lysing, disrupting, extraction, etc. Disruption can be carried out, for example, by ultrasonic waves. The treatments may be combined in multiple types. The processed product of the bacterial cells may also be the supernatant after protein removal treatment of the processed product of the bacterial cells, or the supernatant obtained by removing the solid content of the bacterial cell culture and fermentation product. BPS402 may be isolated bacterial cells, or may be a fermentation product or culture of the bacterial cells.

[0017] The culture of BPS402 can be carried out according to conventional methods. The culture medium is not particularly limited as long as the bacterium can be cultured, and natural media, synthetic media, semi-synthetic media, etc. can be used. As the culture medium, those containing a nitrogen source and a carbon source can be used. Examples of the nitrogen source include meat extract, peptone, casein, yeast extract, gluten, soybean powder, soybean hydrolyzate, amino acids, etc. Examples of the carbon source include glucose, lactose, fructose, inositol, sorbitol, maltose, starch, sake mash, bran, baker's yeast, molasses, etc. In addition, inorganic substances (e.g., ammonium sulfate, potassium phosphate, magnesium chloride, sodium chloride, calcium carbonate, iron, manganese, molybdenum), various vitamins, etc. can be added.

[0018] The culture temperature of BPS402 may be, for example, 4 to 45 °C, and may also be 25 to 40 °C, 28 to 37 °C, or 30 to 33 °C. The culture time may be, for example, 8 to 72 hours. The culture may be carried out with aeration and shaking or aeration and stirring. The pH of the culture medium may be, for example, 4.0 to 9.0, and preferably 6.0 to 8.0. Examples of the culture method include inoculating the bacterial cells into MRS medium and culturing at 30 °C for 48 hours.

[0019] The activator of pDC or the activator of mDC may be, for example, a fermentation product containing BPS402, a lactic acid bacteria beverage, a beverage containing lactic acid bacteria, etc.

[0020] The pDC activator or mDC activator according to this embodiment can be used in a dose of 1 mg to 50 mg per day for an adult weighing 60 kg, preferably 3 mg to 30 mg, and more preferably 5 mg to 15 mg, as the solid content of BPS402. The dose can be appropriately set within the above range depending on factors such as the health condition of the person taking the drug, the method of administration, and the combination with other agents.

[0021] The BPS402 content in the 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 solid content of the total amount of the agent. Alternatively, the BPS402 content in the 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 solid content of the total amount of the agent.

[0022] The pDC activator or mDC activator according to this embodiment may further contain other components in addition to the above-mentioned active ingredients. Examples of other components include pharmaceutically acceptable components (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents, antioxidants) and food-acceptable components (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, humectants).

[0023] The pDC activator or mDC activator according to this embodiment may be a food composition, a pharmaceutical, or a quasi-drug. The pDC activator or mDC activator according to this embodiment can be used as a food composition, pharmaceutical, or quasi-drug itself, or as an ingredient in a food composition, pharmaceutical, or quasi-drug. A food composition, pharmaceutical, or quasi-drug containing the pDC activator or mDC activator as one component can be manufactured, for example, by adding the pDC activator or mDC activator to an intermediate product in the manufacturing process of these products.

[0024] The pDC activator or mDC activator according to this embodiment may be in any form, such as solid, liquid, or paste, and may be in dosage form such as tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, granules, liquids, suspensions, emulsions, syrups, pastes, or injections (including cases where the solution is prepared by mixing it with distilled water or an infusion solution such as an amino acid solution or electrolyte solution at the time of use). These various formulations can be prepared, for example, by mixing the active ingredient with other ingredients as needed and molding them into the above dosage forms.

[0025] When used as a food composition or as a component of a food composition, it is preferable that the food composition emphasizes the tertiary function of food, namely, the function of regulating bodily functions. Examples of products that emphasize the tertiary function of food include health foods, foods with functional claims, foods with nutritional function claims, nutritional supplements, supplements, and foods for specified health uses.

[0026] Examples of food compositions include beverages such as soft drinks like 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 honey wine; spreads such as custard cream; pastes such as fruit paste; Western confectionery such as chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, and puddings; Japanese confectionery such as daifuku, mochi, manju, castella, anmitsu, and yokan; frozen desserts such as ice cream, ice pops, and sherbet; cooked foods such as curry, gyudon, zosui, miso soup, soup, meat sauce, pasta, pickles, and jam; and seasonings such as dressings, furikake, umami seasonings, and soup bases.

[0027] The pDC activator or mDC activator according to this embodiment is preferably ingested into the body. The administration method may be oral or parenteral. The pDC activator or mDC activator according to this embodiment may be administered once a day, or divided into multiple doses, such as twice a day or three times a day. [Examples]

[0028] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0029] <Evaluation of dendritic cell activation ability> The activation ability of pDCs and mDCs by Apiractobacillus kunky BPS402 (accession number FERM BP-11439) was investigated. In summary, bone marrow cells isolated from ICR mice were differentiated, and the entire differentiated cell culture was cultured for 48 hours in a culture medium containing the test substance. The cells were then labeled with various markers involved in antigen presentation by dendritic cells to T cells. The cells were collected and subjected to flow cytometry analysis. The specifics are as follows:

[0030] [Test substance] Live Apiractobacillus kunky BPS402 bacteria were heat-treated at over 80°C for more than 1 minute, dextrin was added, and the mixture was freeze-dried and dried into a powder. The resulting powder was dissolved in RPMI1640 medium to a concentration of 10 mg / mL, diluted to a concentration of 1 μg / mL, and then sonicated for 30 minutes before being used as the test substance.

[0031] For the negative control group, RPMI1640 medium was used as the test substance. For the positive control group, Lactococcus lactis JCM5805 (available from the Microbial Materials Development Laboratory, BioResource Center, RIKEN) was used, treated in the same manner as BPS402 described above.

[0032] [Preculture] ICR mouse bone marrow cells (CosmoBio, #BMC12C) were differentiated by culturing them 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. Culturing was performed in 24-well plates for 7 days. The culture was carried out in a 5% CO2 incubator set to 37°C.

[0033] [Dendritic cell evaluation test] After differentiation induction in pre-culture, the plate was removed from the incubator. The culture medium was slowly removed from the wells containing seeded cells using a pipette. Subsequently, 500 μL of the test solution containing 1 μg / mL of Apiractobacillus kunky BPS402 was added. The plate was incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0034] After incubation, the supernatant was removed with a pipette and suspended in 1000 μL of ice-cold 2% BSA with 10 mM HEPES-containing HBSS.

[0035] The mixture was centrifuged at 600 × g for 5 minutes and suspended in 100 μL of ice-cold 2% BSA and 10 mM HEPES-containing HBSS.

[0036] For pDC and mDC gates, anti-VioBlue-CD45R (B220) (Miltenyi biotec, Cat:130-110-851) 2μL, anti-PEVio770-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 B515-CD40(Miltenyi 2 μL of 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, stained in the dark at 4°C for 15 minutes, and then centrifuged at 600 × g for 5 minutes.

[0037] The supernatant was removed, and HBSS containing 2% BSA and 10 mM HEPES was added. The mixture was then analyzed using a flow cytometer (MACSQuant 10, Miltenyi Biotec). B220-positive CD11b-negative CD11c-positive cells were designated as pDCs, and B220-negative CD11b-positive CD11c-positive cells were designated as mDCs. For each type of dendritic cell, the mean fluorescence intensity (MFI) of MHC-II, which plays a role in antigen presentation to T cells, and CD86, CD80, and CD40, which enhance this signaling, was calculated. The test was performed three times, and the mean fluorescence intensity is expressed as mean ± standard deviation. The mean of the mean fluorescence intensity was examined using a t-test to determine the significant difference from the negative control group (*: p<0.05).

[0038] The results are shown in Figures 1-4. Figures 1 and 2 are graphs showing the average fluorescence intensity of various dendritic cell activation markers in pDCs (B220-positive, CD11b-negative, CD11c-positive cells). Figures 3 and 4 are graphs showing the average fluorescence intensity of various dendritic cell activation markers in mDCs (B220-negative, CD11b-positive, CD11c-positive cells). Figures 1(a) and 3(a) show the average fluorescence intensity of MHC-II, Figures 1(b) and 3(b) show the average fluorescence intensity of CD86, Figures 2(a) and 4(a) show the average fluorescence intensity of CD80, and Figures 2(b) and 4(b) show the average fluorescence intensity of CD40.

[0039] The addition of Apiractobacillus kunky BPS402 resulted in a significant increase in the mean fluorescence intensity of MHC-II, CD40, and CD86 in pDCs, and in mDCs, compared to the negative control group. Therefore, it was confirmed that Apiractobacillus kunky BPS402 used in this study has a dendritic cell activating effect. Furthermore, when comparing the dendritic cell activating effect of Apiractobacillus kunky BPS402 with that of the positive control group (JCM5805), only CD86 in pDCs showed a slightly inferior effect, while all other markers showed comparable effects.

Claims

1. A plasmacytoid dendritic cell activator containing Apiractobacillus kunky BPS402 (FERM BP-11439) as the active ingredient.

2. An activator of myeloid dendritic cells containing Apiractobacillus kunky BPS402 (FERM BP-11439) as the active ingredient.

3. An immunostimulant comprising the activator described in claim 1 or 2.

4. The immunostimulant according to claim 3, which is a food composition, a pharmaceutical product, or a quasi-drug.

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