Composition for activating plasmacytoid dendritic cells and immunostimulator

Abstract

To provide a composition for activating plasmacytoid dentritic cells, which does not require expensive culture equipment for its active ingredient (Tetragenococcus lactic acid bacteria), hardly requires labor and cost in production, is a safe food material, and further activates plasmacytoid dentritic cells.SOLUTION: The composition for activating the plasmacytoid dentritic cells contains at least one kind selected from Tetragenococcus lactic acid bacteria activating the plasmacytoid dentritic cells and cultures of the lactic acid bacteria.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a plasmacytoid dendritic cell activation composition and an immunostimulant. More specifically, the present invention relates to a plasmacytoid dendritic cell activation composition and an immunostimulant that can activate plasmacytoid dendritic cells, do not require expensive culture equipment, are less time-consuming and costly to produce, and contain a safe food material (lactic acid bacteria) as an active ingredient. [Background technology]

[0002] Macrophages, natural killer cells, and dendritic cells are known as cells of the innate immune system, among which dendritic cells are powerful and important constituent cells of the innate immune system.

[0003] One type of dendritic cell known as plasmacytoid dendritic cell (pDC) activates various immune cells such as natural killer cells (NK cells), B cells, and T cells, and acts as a command center for immune cells. Plasmacytoid dendritic cells are also called plasmacytoid dendritic cells.

[0004] These plasmacytoid dendritic cells are thought to be extremely important cells in terms of biological defense, such as immune function and antiviral infection protection, and research is being conducted to act on plasmacytoid dendritic cells to enhance immune function and increase virus defense activity.

[0005] A plasmacytoid dendritic cell inducer containing lactic acid bacteria belonging to Lactobacillus helveticus as an active ingredient has been reported to act on plasmacytoid dendritic cells (to induce an increase in plasmacytoid dendritic cells) (see Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-031109 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the plasmacytoid dendritic cell inducer described in Patent Document 1 contains Lactobacillus helveticus as its active ingredient, but this Lactobacillus helveticus requires culture equipment that can be completely sterilized, and great care must be taken to prevent contamination with other bacteria. This makes it difficult to work at the manufacturing site, and there are problems with the effort and cost involved during production.

[0008] Therefore, there is a need for the development of a composition for activating plasmacytoid dendritic cells that can activate plasmacytoid dendritic cells, does not require expensive culture equipment, is less time-consuming and costly to produce, and contains a safe food material (lactic acid bacteria) as an active ingredient.

[0009] The present invention was developed by discovering that Tetragenococcus lactic acid bacteria activate plasmacytoid dendritic cells, and that Tetragenococcus lactic acid bacteria do not require expensive culture equipment, are less time-consuming and costly to produce, and are also a safe food ingredient. [Means for solving the problem]

[0010] According to the present invention, the following plasmacytoid composition for activating dendritic cells and immunostimulatory agent are provided.

[0011] [1] A composition for activating plasmacytoid dendritic cells, comprising at least one selected from Tetragenococcus lactic acid bacteria that activate plasmacytoid dendritic cells and cultures of the lactic acid bacteria.

[0012] [2] The plasmacytoid dendritic cell activation composition according to [1], wherein the Tetragenococcus lactic acid bacteria is Tetragenococcus halophilus.

[0013] [3] The composition for activating plasmacytoid dendritic cells according to [1], wherein the Tetragenococcus lactic acid bacteria are lactic acid bacteria under accession number NITE BP-02318.

[0014] [4] An immunostimulating agent containing the plasmacytoid composition for dendritic cell activation according to any one of [1] to [3] above. [Effects of the Invention]

[0015] The composition for activating plasmacytoid dendritic cells of the present invention does not require expensive culture equipment for its active ingredient (Tetragenococcus lactic acid bacteria), is less time-consuming and costly to produce, is a safe food ingredient, and furthermore, has the effect of activating plasmacytoid dendritic cells.

[0016] The immunostimulant of the present invention contains the plasmacytoid dendritic cell activation composition of the present invention, and therefore does not require expensive culture equipment for its active ingredient (Tetragenococcus lactic acid bacteria), is less time-consuming and costly to produce, is a safe food ingredient, and furthermore, has the effect of activating plasmacytoid dendritic cells. DETAILED DESCRIPTION OF THE INVENTION

[0017] The following describes embodiments of the present invention, but the present invention is not limited to the following embodiments. In other words, it should be understood that modifications and improvements to the following embodiments, based on the ordinary knowledge of a person skilled in the art, as long as they do not deviate from the spirit of the present invention, also fall within the scope of the present invention.

[0018] (1) Plasmacytoid composition for dendritic cell activation: The composition for activating plasmacytoid dendritic cells of the present invention contains at least one species selected from Tetragenococcus lactic acid bacteria that activate plasmacytoid dendritic cells and cultures of these lactic acid bacteria.

[0019] This composition for activating plasmacytoid dendritic cells does not require expensive cultivation equipment for its active ingredient (Tetragenococcus lactic acid bacteria), is less time-consuming and costly to produce, is a safe food ingredient, and activates plasmacytoid dendritic cells.

[0020] Here, when ordinary lactic acid bacteria (lactic acid bacteria that do not have salt tolerance, etc.) are commercially produced, concentration and purification of the bacterial cells and treatment of the culture medium (for example, sterilization using an autoclave or similar) are required. Furthermore, during cultivation, an environment must be maintained that prevents the introduction of contaminating bacteria such as miscellaneous bacteria, which increases the effort and cost of production. On the other hand, the Tetragenococcus lactic acid bacteria, which are the active ingredients of the plasmacytoid dendritic cell activation composition of the present invention, are salt-tolerant and can therefore be cultivated in high-salt environments where contaminating bacteria have difficulty growing, making cultivation easy under conditions that suppress the growth of contaminating bacteria.

[0021] (1-1) Tetragenococcus lactic acid bacteria and cultures of these lactic acid bacteria: Tetragenococcus lactic acid bacteria are salt-tolerant lactic acid bacteria. "Having salt tolerance" means that they have the property of being able to be cultured in a medium with a high salt concentration (specifically, a salt concentration of 11 w / v% or more).

[0022] As described above, the Tetragenococcus lactic acid bacteria are salt-tolerant lactic acid bacteria, making them easy to cultivate. Furthermore, cultivation is even easier when the growth rate is 30 times or more when cultured in a medium with a salt concentration of 12 w / v%. The "growth rate" refers to the value calculated by the formula: "number of bacteria after 20 hours of culture (cfu / ml) / initial number of bacteria (cfu / ml)."

[0023] The Tetragenococcus lactic acid bacteria can be, for example, those isolated during the brewing process of miso (particularly rice miso). Specific examples of salt-tolerant lactic acid bacteria isolated during the brewing process of rice miso include the trade name "Zoka Lactic Acid Bacteria LTK-1" (manufactured by Ichibiki Co., Ltd.).

[0024] Furthermore, lactic acid bacteria "isolated during the miso brewing process" refers to salt-tolerant lactic acid bacteria that are established in the "kura," "muro," and "oke" (wood vats) used in the miso brewing process. Furthermore, it refers to lactic acid bacteria that can grow from the preparation of miso to the maturation process. These lactic acid bacteria "isolated during the miso brewing process" can also be called salt-tolerant lactic acid bacteria contained in miso (i.e., miso lactic acid bacteria), or, in other words, salt-tolerant lactic acid bacteria derived from miso (i.e., salt-tolerant lactic acid bacteria originating from miso). In the present invention, lactic acid bacteria "isolated during the miso brewing process" are not limited to those isolated during the miso brewing process, but also include those isolated and preserved during the miso brewing process and subsequently cultured.

[0025] As mentioned above, the lactic acid bacteria of the genus Tetragenococcus are not particularly limited as long as they activate plasmacytoid dendritic cells, but Tetragenococcus halophilus is a good example. In particular, the lactic acid bacteria of the genus Tetragenococcus under accession number NITE BP-02318 can be mentioned. The lactic acid bacteria of the genus Tetragenococcus under accession number NITE BP-02318 is known under the trade name "Zoka Lactic Acid Bacteria LTK-1" (manufactured by Ichibiki Co., Ltd.).

[0026] The Tetragenococcus lactic acid bacteria with accession number NITE BP-02318 was deposited at the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation on August 3, 2016 (domestic deposit date), and was transferred to international deposit under the Budapest Treaty on September 6, 2017.

[0027] The Tetragenococcus lactic acid bacteria may be killed or live.

[0028] There is no particular limit to the intake concentration (effective concentration when ingested) of Tetragenococcus lactic acid bacteria, but it can be 1.25 mg / day or more, with the upper limit being approximately 1,250 mg / day. In other words, similar to regular lactic acid bacteria, approximately 5 billion (equivalent to approximately 1.25 mg) to 5 trillion (equivalent to approximately 1.25 g) of bacteria can be ingested per day. For example, it can be taken orally before or after meals as a medicine or supplement.

[0029] (1-2) Cultivation method of Tetragenococcus lactic acid bacteria: There are no particular limitations on the culture conditions for Tetragenococcus lactic acid bacteria, but they can be cultured, for example, in a medium with a salt concentration of 11 to 18 w / v%, preferably 11 to 16 w / v%, and particularly preferably 12 to 14 w / v%. Note that "w / v%" means (mass (g) / volume (100 mL))%.

[0030] Cultivation under these conditions makes it difficult for other bacteria (contaminants such as miscellaneous bacteria) to grow, while Tetragenococcus lactic acid bacteria can be cultivated, allowing for easy and efficient cultivation of Tetragenococcus lactic acid bacteria as a primary agent. Furthermore, culturing at a salt concentration within the above range prevents the proliferation of non-halotolerant contaminants while allowing the proliferation rate to be faster than that of salt-tolerant bacteria (halotolerant contaminants), such as halotolerant Staphylococcus bacteria. In other words, even with respect to halotolerant contaminants, the culture can be terminated and the product can be harvested before the proliferation of the contaminants. Cultivation in a medium with a salt concentration of less than 11 w / v% tends to facilitate the proliferation of contaminants, making it difficult to ensure a sufficient yield. Furthermore, culturing in a medium with a salt concentration of more than 18 w / v% makes it more difficult for contaminants to grow, but the required cultivation period to obtain the required yield is prolonged, potentially resulting in the proliferation of halotolerant contaminants.

[0031] As described above, when cultured in a medium with the above salt concentration, Tetragenococcus lactic acid bacteria can be cultured efficiently and in large quantities even in a simple open culture device (which can be sterilized and kept warm) without using special equipment such as a closed sterile culture device. This culture may be used in the plasmacytoid composition for dendritic cell activation.

[0032] The culture temperature is preferably 20 to 40° C., more preferably 28 to 37° C. The culture time is about 24 to 120 hours, and the culture may be stirred during culture. The pH of the medium is preferably 5 to 9, more preferably 6 to 7.

[0033] The medium used may contain a nitrogen source and a carbon source.

[0034] The nitrogen source is not particularly limited and examples thereof include soy sauce, miso, meat extract, peptone, gluten, casein, yeast extract, amino acids, etc. The carbon source is also not particularly limited and examples thereof include glucose, koji digestion liquid, rice saccharification liquid, sucrose, starch, powdered sugar, glycerin, etc. Furthermore, in addition to the nitrogen source and carbon source, the culture medium may contain inorganic substances such as inorganic salts such as sodium acetate, magnesium, manganese, and iron, and may also contain vitamins.

[0035] Lactic acid bacteria of the genus Tetragenococcus can be prepared by sterilization or other treatments after cultivation. Specifically, after cultivation is completed, medium components including salt are removed by centrifugation or other means, followed by washing and purification. If necessary, heat sterilization is performed, and then the bacteria are dried and concentrated by freeze-drying, vacuum drying, hot air drying or other means. In this way, lactic acid bacteria of the genus Tetragenococcus can be prepared after cultivation.

[0036] The heat sterilization is not particularly limited, but specifically, autoclave sterilization (121°C, 20 minutes) or a similar sterilization method is preferred.

[0037] (1-3) Other ingredients: The composition for activating plasmacytoid dendritic cells of the present invention may be composed solely of Tetragenococcus lactic acid bacteria (or a composition containing a culture of said lactic acid bacteria, etc.) that have the ability to activate plasmacytoid dendritic cells, but may also contain other components in addition to said Tetragenococcus lactic acid bacteria.

[0038] As other ingredients, ingredients that are appropriately blended depending on the application, such as pharmaceuticals, quasi-drugs, foods and beverages, can be used.

[0039] In the case of pharmaceuticals and quasi-drugs, other ingredients may include, for example, excipients, coating agents, binders, bulking agents, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, anti-inflammatory agents, disinfectants, flavoring agents, odor enhancers, etc. Active ingredients or pharmacological ingredients other than Tetragenococcus lactic acid bacteria may also be contained, provided that the object of the present invention is not impaired.

[0040] In the case of foods and beverages, other ingredients can include, for example, sweeteners, acidulants, carbon dioxide gas, inorganic salts, flavorings, fruit juice, vitamins, antioxidants, esters, colorants, emulsifiers, preservatives, seasonings, vegetable extracts, nectar extracts, quality stabilizers, bitterness suppressors, etc. In the case of foods and beverages, active ingredients and pharmacological ingredients other than Tetragenococcus lactic acid bacteria may also be contained, as long as they do not impair the object of the present invention.

[0041] The plasmacytoid composition for dendritic cell activation of the present invention can be ingested in any form (oral or parenteral). For example, when ingested orally, it may be in solid forms such as tablets, coated tablets, granules, powders, or capsules, or in liquid forms such as elixirs or syrups.

[0042] (2) Method for producing a plasmacytoid composition for dendritic cell activation: The plasmacytoid composition for dendritic cell activation of the present invention can be produced, for example, by a method comprising a lactic acid bacteria preparation step and a raw material preparation step. This method allows for the easy production of the plasmacytoid composition for dendritic cell activation. This method has the advantage that the Tetragenococcus lactic acid bacteria themselves are salt-tolerant, making them easy to culture.

[0043] The lactic acid bacteria preparation step is a step of preparing the above-mentioned Tetragenococcus lactic acid bacteria. The Tetragenococcus lactic acid bacteria may be prepared by obtaining a seed culture and culturing the seed culture by the above-mentioned method, or by purchasing a commercially available lactic acid bacteria (for example, the trade name "Zoka Lactic Acid Bacteria LTK-1" (manufactured by Ichibiki Co., Ltd.)).

[0044] The raw material preparation step is a step in which the Tetragenococcus lactic acid bacteria prepared in the lactic acid bacteria preparation step are mixed with other ingredients as necessary to prepare the raw material for the plasmacytoid dendritic cell activation composition.

[0045] In the case of a solid form such as a tablet, a step for forming the solid form (a forming step) may be adopted after the raw material preparation step.

[0046] (3) Intake period of Plasmacytoid dendritic cell activation composition: The plasmacytoid composition for dendritic cell activation of the present invention is preferably taken for one week or more, and more preferably for two weeks or more.

[0047] (4) Immunostimulants: The immunostimulant of the present invention contains at least one selected from Tetragenococcus lactic acid bacteria that activate plasmacytoid dendritic cells and cultures of such lactic acid bacteria. The active ingredient of this immunostimulant, Tetragenococcus lactic acid bacteria, is easy to culture and therefore easy to produce, eliminating the need for expensive production equipment and complicated manufacturing processes. It is an inexpensive, stable, and safe food material (lactic acid bacteria). Furthermore, this immunostimulant has the ability to activate plasmacytoid dendritic cells when ingested for a certain period of time.

[0048] The immunostimulant of the present invention can be produced by employing the lactic acid bacteria preparation step shown in the method for producing the plasmacytoid dendritic cell activation composition of the present invention described above, and other than this step, any conventional method for producing an immunostimulant can be appropriately adopted. [Example]

[0049] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples.

[0050] Example 1 (1) Preparation of plasmacytoid dendritic cells (pDCs): First, plasmacytoid dendritic cells were prepared as follows. Specifically, CD34+ cells derived from human umbilical cord blood (STEMCELL Technologies, model number "ST-200-0000") were prepared and cultured for two weeks in a CO2 incubator (37°C, 5% CO2, humidified) until the cell population reached approximately 90 million. After culture, the cell suspension was collected and centrifuged (300 x g, 10 minutes). The supernatant was removed, and CD304+ cells were isolated using a MACS separator (column: LS column (Miltenyi Biotec, model number "130-042-401")). Plasmacytoid dendritic cells were thus prepared.

[0051] (2) Evaluation of plasmacytoid dendritic cell activation: The plasmacytoid dendritic cells prepared as described above were used, and a composition for activating plasmacytoid dendritic cells (a sample containing Tetragenococcus lactic acid bacteria) was added to the plasmacytoid dendritic cells. Various evaluations (cell viability, IFN-α production, and surface antigen analysis by flow cytometry) were then performed.

[0052] The Plasmacytoid dendritic cell activation composition contained Tetragenococcus lactic acid bacteria and its basal medium (containing additives). Each of the concentrations shown in Tables 1 to 4 (concentrations of the Plasmacytoid dendritic cell activation composition (mg / mL)) was prepared at twice the concentration.

[0053] The basal medium and additives are specifically described below. Basal medium: StemSpan SFEM II (VERITAS) Additives: Recombinant Human Flt3-Ligand (Peprotech) Final concentration 100ng / mL Recombinant Human SCF (Peprotech) Final concentration 100ng / mL Recombinant Human TPO (Peprotech final concentration 50ng / mL Recombinant Human IL-3 (Peprotech) Final concentration 20ng / mL StemRegenin 1 (SR1) (Selleck Biotech) Final concentration 1μM Penicillin-Streptomycin solution (Nacalai tesque) final concentration 0.2%

[0054] (2-1) Measurement of cell viability (cell proliferation test): The prepared pDCs were seeded into a 96-well plate at 100,000 cells / 0.05 mL / well. Then, 0.05 mL of test substance-supplemented medium containing a plasmacytoid dendritic cell activation composition at twice the final concentration was added. The cells were then cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0055] After incubation, viable cell numbers were measured using the WST-8 method. The absorbance (450 nm, reference wavelength 630 nm) was measured using a plate reader 30 and 90 minutes after the start of the measurement, and the difference in absorbance per 60 minutes was calculated by subtracting the value 30 minutes after the start of the measurement from the value 90 minutes after the start of the measurement, and this was used as the relative cell number.

[0056] It was found that the addition of Plasmacytoid dendritic cell activation composition caused pDCs to form colonies. The absorbance measurement results are shown in Table 1. As shown in Table 1, it can be seen that Plasmacytoid dendritic cell activation composition promotes pDC cell proliferation.

[0057] [Table 1]

[0058] (2-2) Measurement of IFN-α production: pDCs were cultured in the same manner as in the cell proliferation test. The prepared pDCs were seeded onto a 96-well plate at 100,000 cells / 0.05 mL / well. Then, 0.05 mL of test substance-supplemented medium containing a plasmacytoid dendritic cell activation composition at twice the final concentration was added. The cells were then cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0059] The amount of IFN-α produced in the culture was then measured using the VeriKine Interferon α ELISA Kit, Human (PBL assay science). The absorbance at 450 nm was measured according to the kit's protocol. The culture supernatant was diluted 2-fold with the diluent buffer provided with the kit.

[0060] The results of measuring the amount of IFN-α produced in the culture supernatant are shown in Table 2. As shown in Table 2, it was confirmed that the amount of IFN-α produced increased by adding the plasmacytoid composition for dendritic cell activation.

[0061] [Table 2]

[0062] (2-3) Surface antigen analysis by flow cytometry: pDCs were cultured in the same manner as in the cell proliferation test. The prepared pDCs were seeded onto a 96-well plate at 100,000 cells / 0.05 mL / well. Then, 0.05 mL of test substance-supplemented medium containing a plasmacytoid dendritic cell activation composition at twice the final concentration was added. The cells were then cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0063] After incubation, pDCs were harvested from the plate and washed with FACS buffer (DPBS supplemented with 2% FBS). To prevent nonspecific antibody binding, the cells were treated with Fc Block solution (Cosmo Bio) at 4°C for 10 minutes, followed by treatment with antibody or isotype control at 4°C for 40 minutes in the dark. The pDCs were then washed using Laminar Wash (TOMY) and subjected to surface antigen analysis using Cytoflex S (Beckman Coulter).

[0064] This surface antigen analysis was performed using CytExpert analysis software Ver. 2.5 (Beckman Coulter). Specifically, the positivity rates and mean fluorescent intensity (MFI) of HLA-DR and CD86 in cells positive for pDC markers CD304 and CD123 were calculated.

[0065] In the surface antigen analysis, we focused on the expression levels and expression intensity of CD86 and HLA-DR induced by the Plasmacytoid composition for dendritic cell activation.

[0066] Activated pDCs have the ability to stimulate and activate T cells through antigen presentation. Expression of antigen-binding molecules, known as MHC, and costimulatory molecules is important for activated pDCs to activate T cells, and these molecules are used as indicators of pDC activation. HLA-DR is classified as an MHC class II molecule, and CD86 is one of the costimulatory molecules.

[0067] In this evaluation, the difference between the non-additive control (no addition) and the Plasmacytoid dendritic cell activation composition was tested by Dunnett's test, with P<0.05 indicating a significant difference.

[0068] (CD86 positivity rate and MFI) Table 3 shows the analysis results of the CD86 positivity rate and mean fluorescence intensity (MFI) in cells positive for CD304 and CD123, which are pDC markers.

[0069] As shown in Table 3, the addition of the Plasmacytoid dendritic cell activation composition increased the CD86 positivity rate (%) and MFI. In other words, the expression level and intensity of CD86 increased. These results demonstrate that pDCs are activated by the Plasmacytoid dendritic cell activation composition.

[0070] [Table 3]

[0071] (HLA-DR positivity rate and MFI) Next, Table 4 shows the analysis results of the HLA-DR positivity rate and mean fluorescence intensity (MFI) in CD304- and CD123-positive cells.

[0072] As shown in Table 4, the addition of the plasmacytoid composition for dendritic cell activation increased the HLA-DR positivity rate and MFI. In other words, the expression level and intensity of HLA-DR increased. These results demonstrate that pDCs are activated by the plasmacytoid composition for dendritic cell activation.

[0073] [Table 4]

[0074] As can be seen from the above results, the plasmacytoid dendritic cell activation composition of Example 1 activates plasmacytoid dendritic cells. In other words, products containing the plasmacytoid dendritic cell activation composition of Example 1 can also be used as immunostimulants. Furthermore, the active ingredient of the plasmacytoid dendritic cell activation composition (Tetragenococcus lactic acid bacteria) does not require expensive culture equipment, is less time-consuming and costly to produce, and is also a safe food ingredient. [Industrial Applicability]

[0075] The composition for activating plasmacytoid dendritic cells of the present invention and the immunostimulant of the present invention can be used to activate plasmacytoid dendritic cells. [Accession number]

[0076] Accession number: NITE BP-02318

Claims

1. A composition for activating plasmacytoid dendritic cells, comprising at least one selected from Tetragenococcus lactic acid bacteria that activate plasmacytoid dendritic cells and cultures of said lactic acid bacteria.

2. The plasmacytoid composition for activating dendritic cells according to claim 1, wherein the Tetragenococcus lactic acid bacterium is Tetragenococcus halophilus.

3. 2. The composition for activating plasmacytoid dendritic cells according to claim 1, wherein the lactic acid bacterium of the genus Tetragenococcus is a lactic acid bacterium having accession number NITE BP-02318.

4. An immunostimulator comprising the plasmacytoid dendritic cell activation composition according to any one of claims 1 to 3.

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