Composition for activating plasmacytoid dendritic cells

Abstract

The present invention addresses the problem of providing a novel material that activates plasmacytoid dendritic cells and promotes the production of type-I IFN, thereby making it possible to maintain, improve, or enhance an immune function.　Provided is a composition for activating plasmacytoid dendritic cells, the composition containing, as an active ingredient, bacterial cells of Lactobacillus pargasseri and / or Lactobacillus gasseri. Also provided is the composition for activating plasmacytoid dendritic cells that has an effect of promoting the production of IFNα in plasmacytoid dendritic cells. Further provided are a pharmaceutical product, a food or beverage, and a feed for activating plasmacytoid dendritic cells that contain the composition for activating plasmacytoid dendritic cells.

Description

Plasmacytoid composition for activating dendritic cells

[0001] The present invention relates to a composition for activating plasmacytoid dendritic cells, which contains Lactobacillus paragasseri and / or Lactobacillus gasseri cells as an active ingredient.

[0002] The immune system is a biological defense mechanism found in many animals, responsible for eliminating pathogens such as bacteria and viruses that invade from the outside, as well as tumor cells that develop within the body. The immune system consists of two systems: the innate immune system and the adaptive immune system, which are responsible for the body's defense functions. The innate immune system is a system that is born with the body, while the adaptive immune system is an immune system that develops after birth in response to stimulation by foreign substances. When foreign substances such as bacteria or viruses enter the body, macrophages, NK cells, and dendritic cells, which are responsible for the innate immune system, first work to eliminate the foreign substances. Dendritic cells then transmit information about the foreign substance to T cells and B cells, thereby inducing CD8+ T cells that specifically attack the foreign substances and antibody production, thereby activating the adaptive immune system.

[0003] As such, dendritic cells are responsible for the innate immune system and also participate in the adaptive immune system, functioning as a command center for the entire immune system. There are various types of dendritic cells, but they can be broadly classified into classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). Classical dendritic cells have strong antigen-presenting ability and therefore play an important role in inducing the adaptive immune system. On the other hand, plasmacytoid dendritic cells, while having weaker antigen-presenting ability, are key cells for maintaining homeostasis of the immune system, as they regulate not only the immunostimulatory system, which aims to eliminate foreign substances, but also the immunoregulatory system, which suppresses excessive immune responses.

[0004] One of the characteristics of plasmacytoid dendritic cells in eliminating foreign substances is that they produce large amounts of type I interferon (hereinafter sometimes referred to as IFN) during viral infection. Type I IFN is a cytokine that induces the production of antiviral factors naturally contained in the body to inhibit viral proliferation, and plays a role in preventing the spread of viral infection throughout the tissues.

[0005] In this reaction, TLR7 and TLR9, which are highly expressed in plasmacytoid dendritic cells, recognize viral RNA or DNA, strongly stimulating the production of type I IFN. Therefore, type I IFN inducers that stimulate TLR7 or TLR9 as antiviral agents have been widely studied. The IFN production inducer described in Patent Document 1 contains lactic acid bacteria as an active ingredient. This patent document describes activating plasmacytoid dendritic cells to induce IFN production, but the only lactic acid bacterium disclosed in this patent document that activates plasmacytoid dendritic cells is Lactococcus lactis, and it has been shown that Lactobacillus rhamnosus lactic acid bacteria do not have this activating effect.

[0006] Non-Patent Document 1 reports that the proportion of plasmacytoid dendritic cells increases in the intestinal mucosal lamina propria of mice administered Lactobacillus gasseri OLL2809, but increasing the proportion of plasmacytoid dendritic cells and activating plasmacytoid dendritic cells are fundamentally different things. That is, there is no disclosure of Lactobacillus gasseri or the activation effect of Lactobacillus gasseri on plasmacytoid dendritic cells. In other words, the solution provided by the present application is neither disclosed nor suggested in any of the documents.

[0007] Republished Publication No. 2012-91081

[0008] A. Aoki-Yoshida, K. Yamada, S. Hachimura, T. Sashihara, S. Ikegami, M. Shimizu, M. Totsuka. 2016. Enhancement of oral tolerance induction in DO11.10 mice by Lactobacillus gasseri OLL2809 via increase of effector regulatory T cells. PLOS ONE. 11:e0158643.

[0009] An object of the present invention is to provide a novel material that can maintain, improve or strengthen immune function by activating plasmacytoid dendritic cells and promoting type I IFN production.

[0010] The present inventors conducted extensive research to find a material with excellent plasmacytoid dendritic cell activation activity, and discovered that both Lactobacillus paragasseri cells and Lactobacillus gasseri cells have potent plasmacytoid dendritic cell activation activity, leading to the completion of the present invention. Specifically, the present invention includes the following features. <1> A composition for activating plasmacytoid dendritic cells, containing Lactobacillus paragasseri and / or Lactobacillus gasseri cells as active ingredients. <2> The plasmacytoid dendritic cell activation composition according to <1>, which has the effect of promoting interferon-α production in plasmacytoid dendritic cells. <3> The plasmacytoid composition for dendritic cell activation according to <1>, wherein the Lactobacillus paragasseri is Lactobacillus paragasseri SBT2055 (FERM BP-10953), Lactobacillus paragasseri SBT2056 (FERM BP-11038), or Lactobacillus paragasseri SBT1703 (FERM P-17785). <4> The Lactobacillus gasseri is Lactobacillus gasseri SBT0274 (FERM BP-11039), or Lactobacillus gasseri JCM1131. Ｔ<5> A food or drink for activating plasmacytoid dendritic cells, comprising the plasmacytoid composition for activating dendritic cells according to any one of <1> to <4>. <6> A pharmaceutical product for activating plasmacytoid dendritic cells, comprising the plasmacytoid composition for activating dendritic cells according to any one of <1> to <4>. <7> A feed for activating plasmacytoid dendritic cells, comprising the plasmacytoid composition for activating dendritic cells according to any one of <1> to <4>. <8> Use of Lactobacillus paragasseri and / or Lactobacillus gasseri cells for the production of a plasmacytoid composition for activating dendritic cells. <9> A method for activating plasmacytoid dendritic cells in a subject in need thereof, the method comprising a step of administering or having a subject ingest a composition containing an effective amount of Lactobacillus paragasseri and / or Lactobacillus gasseri cells to the subject. <10> The method for activating plasmacytoid dendritic cells according to <9>, which is a method for maintaining, improving, or strengthening immune function.

[0011] According to the present invention, by ingesting Lactobacillus paragasseri and / or Lactobacillus gasseri cells into the body, plasmacytoid dendritic cells can be activated, and as a result, maintenance, improvement, or strengthening of immune function can be expected.

[0012] 1 is a graph showing the expression levels of CD86 and HLA-DR in plasmacytoid dendritic cells when Lactobacillus paragasseri SBT2055 cells of the present invention are added to human peripheral blood mononuclear cells (PBMC). 2 is a graph showing the expression levels of CD86 in plasmacytoid dendritic cells when Lactobacillus paragasseri SBT2055, SBT2056, and SBT1703 cells of the present invention are added to human peripheral blood mononuclear cells (PBMC). 3 is a graph showing the expression levels of CD86 in plasmacytoid dendritic cells when Lactobacillus paragasseri SBT2055 cells of the present invention, Lactobacillus gasseri SBT0274, and JCM1131 cells of the present invention are added to human peripheral blood mononuclear cells (PBMC). Ｔ1 is a graph showing the expression levels of CD86 and HLA-DR in plasmacytoid dendritic cells when Lactobacillus paragasseri SBT2055 cells of the present invention were added to PBMCs. 2 is a graph showing the IFN-α concentration in the culture supernatant when ssRNA was not added, and the right graph shows the case when ssRNA was added.

[0013] The present invention provides a novel plasmacytoid composition for activating dendritic cells. The plasmacytoid composition for activating dendritic cells of the present invention, which contains Lactobacillus paragasseri and / or Lactobacillus gasseri cells as an active ingredient, and various uses of the composition are described in detail below.

[0014] (Lactobacillus paragasseri and / or Lactobacillus gasseri) The present invention uses Lactobacillus paragasseri and / or Lactobacillus gasseri cells as an active ingredient, which exhibit plasmacytoid dendritic cell activation effects when orally ingested. Preferred examples of Lactobacillus paragasseri include Lactobacillus paragasseri SBT2055 (Accession No. FERM BP-10953), Lactobacillus paragasseri SBT2056 (Accession No. FERM BP-11038), and Lactobacillus paragasseri SBT1703 (Accession No. FERM P-17785). Lactobacillus gasseri is preferably Lactobacillus gasseri SBT0274 (accession number FERM BP-11039) or Lactobacillus gasseri JCM1131. Ｔ Furthermore, strains substantially equivalent to the above strains can be mentioned. Ｔis a publicly available strain in the Japan Collection of Microorganisms (JCM), established by the Microbial Materials Development Division of the RIKEN BioResource Research Center (RIKEN BRC). A substantially equivalent strain is one that exhibits plasmacytoid dendritic cell activation effects upon oral ingestion, and whose 16S rRNA gene base sequence has 98% or more, preferably 99% or more, and more preferably 100% homology with the 16S rRNA gene base sequence of the above-mentioned strains of Lactobacillus paragasseri or Lactobacillus gasseri, and preferably has the same mycological properties. Furthermore, as long as the effects of the present invention are not impaired, the strain may be one bred from the above-mentioned strains or substantially equivalent strains by mutation, genetic recombination, selection of natural mutants, or the like. Lactobacillus paragasseri SBT2055, Lactobacillus paragasseri SBT2056, and Lactobacillus paragasseri SBT1703 are strains whose species have been reclassified through a revision of classification, and are identical to the strains described in previous literature as Lactobacillus gasseri SBT2055, Lactobacillus gasseri SBT2056, and Lactobacillus gasseri SBT1703. In addition, Lactobacillus paragasseri or Lactobacillus gasseri strains exhibiting plasmacytoid dendritic cell activation effects used in the present invention can also be obtained by screening strains exhibiting plasmacytoid dendritic cell activation effects under the desired conditions of use using the evaluation method described below.

[0015] (Preparation of active ingredient) The Lactobacillus paragasseri or Lactobacillus gasseri cells, which are the active ingredient of the present invention, can be obtained by culturing Lactobacillus paragasseri or Lactobacillus gasseri according to a conventional method for culturing lactic acid bacteria, and separating them from the resulting culture by a collection means such as centrifugation. The Lactobacillus paragasseri or Lactobacillus gasseri cells may be not only purely isolated cells, but also cells containing cells such as cultures and suspensions, as well as dried products, concentrates, pastes, etc. of these. The cells may be live or killed cells, preferably live cells. In addition, the cell culture may be a culture obtained by culturing Lactobacillus paragasseri or Lactobacillus gasseri in a general lactic acid bacteria medium, or cells contained in dairy products such as cheese, fermented milk, and lactic acid bacteria drinks prepared with Lactobacillus paragasseri or Lactobacillus gasseri. In addition, one type of Lactobacillus paragasseri or Lactobacillus gasseri may be used, or two or more types may be used.In addition, one or more types of Lactobacillus paragasseri and one or more types of Lactobacillus gasseri may be used.

[0016] (Plasmacytoid Composition for Activating Dendritic Cells) The plasmacytoid composition for activating dendritic cells of the present invention may contain only the active ingredient, Lactobacillus paragasseri and / or Lactobacillus gasseri cells, or other ingredients as long as they do not interfere with the plasmacytoid dendritic cell activation activity. Examples of other ingredients include oral food and drink compositions, oral pharmaceutical compositions, and feed compositions. Furthermore, the composition may contain probiotics such as other lactic acid bacteria as long as they do not interfere with the plasmacytoid dendritic cell activation activity.

[0017] (Plasmacytoid Dendritic Cell Activation Food and Drink) The form of a food and drink, which is one embodiment of the plasmacytoid dendritic cell activation composition of the present invention, will now be described. The plasmacytoid dendritic cell activation food and drink of the present invention may be in any form as long as it does not interfere with the dendritic cell activation effect of plasmacytoid. The plasmacytoid dendritic cell activation food and drink of the present invention can be produced by adding the above-mentioned active ingredient to a known food and drink. Examples of known food and drink include processed foods such as beverages, bread, snacks, cakes, pudding drinks, noodles, and sausages, as well as various types of powdered milk, infant foods, and supplements. Fermented foods such as fermented milk, cheese, and lactic acid bacteria drinks prepared using Lactobacillus paragasseri and / or Lactobacillus gasseri, which have plasmacytoid dendritic cell activation activity, may also be used. The food and drink of the present invention also includes foods for specified health uses, foods with functional claims, and foods with nutrient functions.

[0018] (Plasmacytoid dendritic cell activation pharmaceutical product, plasmacytoid dendritic cell activation animal feed) The following describes the forms of pharmaceutical products and animal feed, which are embodiments of the plasmacytoid dendritic cell activation composition of the present invention. When preparing pharmaceutical products, excipients, stabilizers, flavoring agents, etc. permitted for pharmaceutical formulations can be appropriately mixed and used, and formulations can also be made by mixing excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and any other drugs within a range that does not impair the effects of the present invention. Dosage forms include tablets, capsules, granules, powders, dusts, syrups, and the like, which are administered orally. When the composition is included in animal feed, similar effects can be expected from animals that ingest it.

[0019] (Intake Amount) In order for the plasmacytoid of the present invention to exert its dendritic cell activation effect, it is desirable for adults to ingest 1 to 1,000 mg / day in terms of lactic acid bacteria body weight. When preparing a plasmacytoid dendritic cell activation food or drink containing Lactobacillus paragasseri or Lactobacillus gasseri of the present invention, the content of Lactobacillus paragasseri or Lactobacillus gasseri is not particularly limited, and may be adjusted appropriately depending on the ease of production, the preferred daily dose, etc. For example, when the form is liquid, 1 x 10 ５ cells / ml~1×10 １０ It is preferable to set the concentration to 1 x 10 cells / ml, and in the case of solids, ５ cells / g~1×10 １０ When two or more kinds are used in combination, the total amount is adjusted to the above amount.

[0020] The plasmacytoid dendritic cell activation composition of the present invention can promote the production of type I IFNs, such as IFN-α, by activating plasmacytoid dendritic cells. Therefore, due to its action, it can also be used as a type I IFN production-promoting composition or an IFNα production-promoting composition. In such cases, the plasmacytoid dendritic cell activation composition should be read as a type I IFN production-promoting composition or an IFNα production-promoting composition in the above-mentioned preparation method, intake amount, food, beverage, pharmaceutical, and animal feed. Furthermore, since the promotion of IFN production is particularly pronounced when stimulated by a virus, the composition can be preferably used as a type I IFN production-promoting composition or an IFNα production-promoting composition administered to humans or animals susceptible to viral infection. Another aspect of the present invention is a method for activating plasmacytoid dendritic cells in a subject in need thereof, comprising the step of administering or ingesting a composition containing an effective amount of Lactobacillus paragasseri and / or Lactobacillus gasseri cells to the subject in need thereof. The present invention also relates to a method for maintaining, improving, or strengthening immune function by activating the plasmacytoid dendritic cells.Furthermore, the present invention also relates to a method for promoting type I IFN production or IFNα production in a subject in need thereof, which comprises a step of administering or ingesting to the subject a composition containing an effective amount of Lactobacillus paragasseri and / or Lactobacillus gasseri cells.

[0021] (Evaluation Method) The effect of the plasmacytoid dendritic cell activation composition of the present invention is evaluated, for example, by the method described below. The composition is added to an immune cell population, and after allowing it to act for a predetermined period of time, the expression levels of cell surface activation markers for plasmacytoid dendritic cells contained in the immune cell population are measured. The plasmacytoid dendritic cell activation effect of the present invention can be evaluated if the expression levels of the activation markers are increased compared to when the composition is not added. Various cells can be used for the immune cell population, but human-derived peripheral blood mononuclear cells (PBMCs) are preferred. Furthermore, the activation markers to be measured are not limited, but CD86, HLA-DR, CD40, and CD80 are preferred, with CD86 and HLA-DR being particularly preferred. The effect of the plasmacytoid dendritic cell activation composition of the present invention can also be evaluated by collecting PBMCs from the blood of a human who has ingested the composition for a certain period of time and measuring the expression levels of cell surface activation markers for plasmacytoid dendritic cells contained in the PBMCs in the same manner as described above. In this case, the effects of the present invention can be evaluated as being observed when the expression level of the activation marker is increased in a person who has taken the composition compared to a person who has not taken the composition.

[0022] (Subjects for Administration or Ingestion) The subjects for administration or ingestion of the plasmacytoid dendritic cell activation composition of the present invention are humans in need of plasmacytoid dendritic cell activation. Examples include humans with weakened immune function, humans infected with a virus, humans with cold symptoms, or humans susceptible to these symptoms, as well as healthy humans who wish to prevent these symptoms. Cold symptoms include runny nose, stuffy nose, sneezing, sore throat, hoarseness, headache, fatigue, and chills. Humans with weakened immune function can be identified, for example, by whether the salivary IgA secretion rate or IgA concentration in blood or saliva is lower than the average value for healthy individuals. Non-human animals in similar conditions are also subjects for administration or ingestion.

[0023] Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.

[0024] [Test Example 1] Confirmation of the activation effect of Lactobacillus paragasseri on plasmacytoid dendritic cells (in vitro) 1. Preparation of Lactobacillus paragasseri cells A preparation example of Lactobacillus paragasseri SBT2055 (accession number FERM BP-10953) cells used in this test is shown below. Lactobacillus paragasseri was cultured in 100 mL of MRS liquid medium at 37 ° C for 16 hours, and then the cells were collected by centrifugation (8,000 × g, 4 ° C, 10 minutes), washed twice with saline and once with sterilized MilliQ water, and freeze-dried to obtain Lactobacillus paragasseri cells, which were used in the following test examples and the formulation examples described below.

[0025] 2. Test Method Cryopreserved human PBMC (Peripheral Blood Mononuclear Cell) (iQ BIOSCIENCES) was thawed in a water bath at 37 ° C., washed with a medium containing RPMI1640 (11875-093: Gibco) supplemented with FBS (final conc. 10%) (Gibco) and Penicillin-Streptomycin (final conc. 100 U-100 μg / mL) (15140-122: Life Technologies), and then seeded on a plate. Lactobacillus paragasseri cells were added to the seeded cells to a final concentration of 10 μg / mL, and the cells were incubated at 5% CO ２The plates were cultured at 37°C for 24 hours in an incubator. After culturing, the cell-containing medium was collected from the plate, and the cells were collected by centrifugation (350 x g, 4°C, 5 minutes) and washed with 1 mL of PBS. After washing, Fixable Viability Stain 780 (stock solution) (BD: 565388) was added and the cells were left to stand at room temperature in the dark for 15 minutes to stain dead cells. After standing, the cells were washed twice with Cell Staining buffer (Biolegend: 420201), and Human TruStain FcX™ (Fc Receptor blocking Solution) (Biolegend: 422302) was added and the cells were left to stand at room temperature in the dark for 10 minutes. Thereafter, fluorescently labeled antibodies (FITC anti-human Lineage Cocktail (CD3, CD14, CD16, CD19, CD20, CD56) (Sony), BV421 Mouse Anti-Human CD123, APC Mouse Anti-Human CD86, PE-Cy™7 Mouse Anti-Human CD11c, PE Mouse Anti-Human HLA-DR (BD)) were added, and the cell surface was stained for 20 minutes on ice in the dark. After staining, the cells were washed with Cell Staining Buffer, fixed in Cell Fixation Buffer (Biolegend: 420801) in the dark at room temperature for 20 minutes, washed again with Cell Staining Buffer, resuspended, and subjected to fluorescence-activated cell sorting (FACS) analysis. Among the live cells detected by FACS, a group of cells that were Lineage-, HLA-DR+, CD123+, and CD11c- were gated as plasmacytoid dendritic cells, and CD86 and HLA-DR expression levels were measured as fluorescence values ​​to evaluate plasmacytoid dendritic cell activity. The mean fluorescence intensity was used as the measurement value. A control group without sample was used, and the test was performed with n=3.

[0026] 3. Test Results As a result of the evaluation, the CD86 and HLA-DR fluorescence levels of plasmacytoid dendritic cells significantly increased at the level where Lactobacillus paragasseri cells (SBT2055 in the figure) were added (Figure 1). These results suggest that Lactobacillus paragasseri cells activate plasmacytoid dendritic cells.

[0027] [Test Example 2] Confirmation of the activation effect of Lactobacillus paragasseri on plasmacytoid dendritic cells (in vitro) In addition to the Lactobacillus paragasseri SBT2055 cells used in Test Example 1, the CD86 expression levels of plasmacytoid dendritic cells were also evaluated for Lactobacillus paragasseri SBT2056 (FERM BP-11038) cells and SBT1703 (FERM P-17785) cells. The preparation method and test method for the cells were the same as in Test Example 1. As a result of the evaluation, even at the level where Lactobacillus paragasseri cells SBT2056 and SBT1703 were added, the CD86 fluorescence level of plasmacytoid dendritic cells significantly increased (Figure 2). This result suggests that Lactobacillus paragasseri cells activate plasmacytoid dendritic cells.

[0028] [Test Example 3] Confirmation of the activation effect of Lactobacillus gasseri on plasmacytoid dendritic cells (in vitro) In addition to the Lactobacillus paragasseri SBT2055 cells used in Test Example 1, Lactobacillus gasseri SBT0274 (FERM BP-11039), Lactobacillus gasseri JCM1131 Ｔ The same test was also carried out on the bacterial cells. The method for preparing the bacterial cells and the test method were the same as in Test Example 1. As a result of the evaluation, the Lactobacillus gasseri bacterial cells SBT0274 and JCM1131 Ｔ Even at the level where the bacteria were added, the CD86 and HLA-DR fluorescence levels of plasmacytoid dendritic cells were significantly increased (Figure 3).This result suggests that Lactobacillus gasseri bacteria activate plasmacytoid dendritic cells.

[0029] Test Example 4 Confirmation of the Effect of Lactobacillus paragasseri on Promoting Type I IFN Production (in Vitro) 1. Test Method Cryopreserved human PBMCs (peripheral blood mononuclear cells) (iQ BIOSCIENCES) were thawed in a 37°C water bath, washed with a medium prepared by adding FBS (final conc. 10%) (Gibco) and Penicillin-Streptomycin (final conc. 100U-100µg / mL) (15140-122: Life Technologies) to RPMI1640 (11875-093: Gibco), and then seeded onto a plate. Lactobacillus paragaceri cells (final concentration 10 μg / mL) and ssRNA (ORN R-0006 (Miltenyi) / DOTAP (final concentration 0.1 μM) were added to the seeded cells, and the cells were incubated in a 5% CO atmosphere. ２ The plates were cultured in an incubator at 37°C for 24 hours. After culture, the cell-containing medium was collected from two plates, and the cells were removed by centrifugation (1,500 x g, 4°C, 5 minutes) to prepare samples for measuring IFN-α concentrations. A plate to which SBT2055 cells were added without adding ssRNA to the seeded PBMCs was also cultured in the same manner. A control without sample addition was used, and the test was performed with n = 3. IFN-α concentrations in the culture supernatants were measured by ELISA. ELISA Flex: Human IFN-α (HRP) (MABTECH: 3425-1H-20) was used for measurement, following the manufacturer's recommended protocol.

[0030] 2. Test Results As a result of the evaluation, in the absence of ssRNA, the IFN-α concentration in the culture supernatant at the control level was below the detection limit. On the other hand, at the level where Lactobacillus paragasseri cells (SBT2055 in the figure) were added, 8.4 pg / mL of IFN-α was detected (Figure 4, left). In addition, in the presence of ssRNA simulating viral stimulation, 161 pg / mL of IFN-α was detected at the control level, while 243 pg / mL of IFN-α was detected at the level where Lactobacillus paragasseri cells were added, indicating a significant increase in IFN-α production (Figure 4, right, p < 0.01). These results suggest that Lactobacillus paragasseri cells activate plasmacytoid dendritic cells, resulting in the promotion of IFN-α production. Furthermore, it was confirmed that Lactobacillus paragasseri cells exhibit an IFN-α production-promoting effect even under conditions where IFN-α production is induced due to viral infection. Therefore, the plasmacytoid dendritic cell activation composition of the present invention, which contains Lactobacillus gasseri cells as an active ingredient, is also expected to have an antiviral effect.

[0031] [Test Example 5] Study of the effect of ingestion of Lactobacillus paragaceri-containing capsules on plasmacytoid dendritic cell activity in humans 1. Subjects The subjects were Japanese men and women aged 20 to 60 years. When recruiting subjects, the study was fully explained to the candidates, and informed consent was obtained. In addition, the candidates were asked about their susceptibility to catching colds, and those who frequently caught colds were deemed suitable as subjects and given priority for recruitment. As a result, 200 subjects were recruited from 368 candidates.

[0032] 2. Test Food Two types of test food were used: a test food and a control food. The test food contained 3.3 x 10 Lactobacillus paragasseri SBT2055 bacteria per capsule. ８The bacterial powder was filled into hard capsules with starch and other ingredients to contain more than cfu, and then enteric-coated. The control food was prepared in the same manner as the test food, except that the Lactobacillus paragasseri SBT2055 bacterial powder was replaced with starch. It was confirmed that the test food and the control food were indistinguishable in appearance and flavor. The nutritional composition of the test food per 3 tablets was 3.6 kcal, protein 0.06 g, lipid 0.03 g, carbohydrate 0.8 mg, sodium 2.2 mg, and the nutritional composition of the control food per 3 tablets was 3.7 kcal, protein 0.01 g, lipid 0.03 g, carbohydrate 0.9 g, sodium 0.3 mg.

[0033] 3. Study Method: This study was conducted as a double-blind, randomized, placebo-controlled, parallel-group study from November 2022 to April 2023. The study method was approved by the Nihonbashi Cardiovascular Clinic Institutional Review Board (approval date: September 28, 2022, approval number: NJI-022-09-01). Prior to its implementation, this study was registered in the public database (UMIN-CTR) established by the University Hospital Medical Information Network (UMIN ID: UMIN000049183). Furthermore, the study complied with the "Declaration of Helsinki (amended at the WMA General Assembly in Fortaleza, Brazil, October 2013)" and the "Ethical Guidelines for Life Science and Medical Research Involving Human Subjects" (partially revised March 10, 2022), always taking into consideration the protection of the human rights of research subjects, and was conducted under the supervision of a physician. The subjects were randomized into two groups by a block randomization method using the allocation adjustment factors of sex, age, salivary sIgA, blood IgA, blood IgG, POMS2 (full version) TMD score at the time of screening test, whether or not they had received the influenza vaccine, and whether or not they had received the COVID-19 vaccine by the person in charge of statistical analysis. The controller then allocated the two groups to a test food intake group and a control food intake group. The allocation list was sealed by the controller and kept sealed until it was opened. The subjects assigned to each group took three tablets of either the test food or the research food once a day for 12 weeks. The daily intake of Lactobacillus paragasseri SBT2055 bacteria in the test food intake group was 1 x 10 ９ cfu or more.

[0034] 4. Evaluation Items 4-1. Plasmacytoid Dendritic Cell Activation Ability In this test, the plasmacytoid dendritic cell activation ability can be evaluated by measuring the expression levels of CD86 and HLA-DR in plasmacytoid dendritic cells in peripheral blood mononuclear cells recovered from the blood of a subject after ingesting a test food for a certain period of time, and comparing these expression levels with those of a subject after ingesting a control food for the same period of time. If a statistically significant difference is observed as a result of the comparison, the effectiveness of the plasmacytoid dendritic cell activation composition of the present invention can be demonstrated.

[0035] 4-2. Primary Evaluation Items The primary evaluation items in this study were items from a questionnaire on changes in physical condition related to cold symptoms (local and systemic symptoms). Local symptoms (runny nose, stuffy nose, sneezing, sore throat, hoarseness, cough, headache) and systemic symptoms (fatigue, chills, feverishness) shown in Table 2 were evaluated. Evaluations were made on a five-point scale (not at all, very slightly, a little, somewhat, very severe), and subjects used this questionnaire to record changes in their physical condition every day during the intake period.

[0036] 4-3. Other secondary endpoints In this study, secondary endpoints other than plasmacytoid dendritic cell activity included salivary sIgA (concentration, secretion rate, secretion amount), NK cell activity, blood IgA, blood IgG, body temperature, intestinal flora analysis, and POMS2 (full version). The results for these items are not shown here.

[0037] 5. Test Results 5-1. Plasmacytoid Dendritic Cell Activation Ability The test food intake group showed higher mean plasmacytoid dendritic cell activity (CD86 and HLA-DR) than the control food intake group, but no significant difference was observed between the groups. Therefore, a stratified analysis was conducted on 112 subjects (57 in the test food intake group and 55 in the control food intake group) with low salivary sIgA secretion rate (mean value (110.3 μg / min) or less at the start of intake) who had poor immune function. The results are shown in Table 1. According to these results, the test food intake group showed a significant (P<0.05) increase in CD86 expression, and a significant increase in HLA-DR expression (P=0.090). These results suggest that Lactobacillus paragasseri bacteria activate plasmacytoid dendritic cells, particularly in subjects with poor immune function.

[0038] 5-2. Results of the Physical Condition Change Questionnaire (Main Evaluation Item) Responses to the physical condition change questionnaire were converted into two levels: no symptoms (none at all) and symptoms (slightly present, a little, some, very severe), and the cumulative number of days for each symptom was assessed. The results are shown in Table 2. According to these results, the percentage of cumulative days for each symptom of runny nose, stuffy nose, sneezing, sore throat, hoarseness, and chills during the intake period was significantly lower in the test food intake group compared to the control food intake group. From the above, it was found that the intake of Lactobacillus paragasseri cells maintained immune function and suppressed the onset of cold symptoms.

[0039]

[0040]

[0041] (Example of incorporation into food) 100 mg of Lactobacillus paragasseri SBT2055 cells were mixed with 30 g of skim milk powder, 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of cornstarch and lactose. The mixture was packed into a stick-shaped bag to produce a stick-shaped health food for activating plasmacytoid dendritic cells of the present invention.

[0042] (Example of blending into feed) 20g of Lactobacillus paragasseri SBT2055 cells was suspended in 3980g of deionized water, heated to 40 ℃, and then stirred and mixed for 20 minutes at 3,600 rpm using a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.) to obtain a 2g / 4kg cell solution. 2kg of this cell solution was blended with 1kg of soybean meal, 1kg of skim milk powder, 0.4kg of soybean oil, 0.2kg of corn oil, 2.3kg of palm oil, 1kg of corn starch, 0.9kg of wheat flour, 0.2kg of bran, 0.5kg of vitamin mixture, 0.3kg of cellulose, and 0.2kg of mineral mixture, and then sterilized at 120 ℃ for 4 minutes to produce 10kg of plasmacytoid dendritic cell activation feed of the present invention.

[0043] (Example of formulation into pharmaceuticals) A liquid culture of Lactobacillus paragasseri SBT2055 was centrifuged at 4°C and 7,000 rpm for 15 minutes, followed by washing with sterile water and centrifugation three times to obtain washed bacterial cells. These washed bacterial cells were freeze-dried to obtain bacterial cell powder. One part of this bacterial cell powder was mixed with four parts of skim milk powder, and this mixed powder was tableted in 1g portions using a tableting machine in a conventional manner to prepare the Plasmacyto BR>C tablets for dendritic cell activation of the present invention.

[0044] The present invention provides a novel composition for activating plasmacytoid dendritic cells, which contains Lactobacillus paragasseri and / or Lactobacillus gasseri cells as an active ingredient, and also provides foods, beverages, pharmaceuticals, and feeds for activating plasmacytoid dendritic cells, which contain the composition.

[0045] [Reference to the deposited biological material] (1) SBT2055 A Name and address of the depository institution where the biological material was deposited: National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) B Date of deposit of the biological material at the depository institution in A March 27, 1996 (date of original deposit) February 26, 2008 (date of transfer to deposit under the Budapest Treaty due to original deposit) C Accession number assigned to the deposit by the depository institution in A FERM BP-10953 (2) SBT2056 A Name and address of the depository institution where the biological material was deposited: National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (Room 120, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) Date of deposit of the biological material at the depositary institution in A: April 22, 1986 (original deposit date) October 9, 2008 (date of transfer to a deposit under the Budapest Treaty due to the original deposit) Hi: Accession number assigned by the depositary institution in A: FERM BP-11038 (3) SBT1703 A: Name and address of the depositary institution where the biological material was deposited: Independent Administrative Institution, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) B: Date of deposit of the biological material at the depositary institution in A: March 15, 2000 Hi: Accession number assigned by the depositary institution in A: FERM P-17785 (4) SBT0274 A: Name and address of the depositary institution where the biological material was deposited: Independent Administrative Institution National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) Date of deposit of biological material at the depository institution in Thailand March 15, 2000 (original deposit date) October 9, 2008 (date of transfer to deposit under the Budapest Treaty due to original deposit) Accession number assigned to the deposit by the depository institution in Thailand: FERM BP-11039

[0046] [Rule 26 amendment 02.08.2024]

Claims

1. A composition for activating plasmacytoid dendritic cells, comprising Lactobacillus paragasseri and / or Lactobacillus gasseri cells as an active ingredient.

2. The composition for activating plasmacytoid dendritic cells according to claim 1, which has the effect of promoting the production of interferon-α in plasmacytoid dendritic cells.

3. The composition for activating plasmacytoid dendritic cells according to claim 1, wherein the Lactobacillus paragaceri is Lactobacillus paragaceri SBT2055 (FERM BP-10953), Lactobacillus paragaceri SBT2056 (FERM BP-11038) or Lactobacillus paragaceri SBT1703 (FERM P-17785).

4. The Lactobacillus gasseri is Lactobacillus gasseri SBT0274 (FERM BP-11039) or Lactobacillus gasseri JCM1131 Ｔ The composition for activating plasmacytoid dendritic cells according to claim 1, 5. A food or drink for activating plasmacytoid dendritic cells, comprising the composition for activating plasmacytoid dendritic cells according to any one of claims 1 to 4.

6. A pharmaceutical for activating plasmacytoid dendritic cells, comprising the composition for activating plasmacytoid dendritic cells according to any one of claims 1 to 4.

7. A feed for activating plasmacytoid dendritic cells, comprising the composition for activating plasmacytoid dendritic cells according to any one of claims 1 to 4.

8. Use of Lactobacillus paragasseri and / or Lactobacillus gasseri cells for the manufacture of a composition for activating plasmacytoid dendritic cells.

9. A method for activating plasmacytoid dendritic cells in a subject in need thereof, comprising a step of administering or having a subject ingest a composition containing an effective amount of Lactobacillus paragasseri and / or Lactobacillus gasseri cells.

10. The method for activating plasmacytoid dendritic cells according to claim 9, which is a method for maintaining, improving or enhancing immune function.

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