Plasmacytoid dendritic cell (PDC) activator and composition containing same as active ingredient

Abstract

The present invention addresses the problem of providing a novel means for activating plasmacytoid dendritic cells (pDC). This pDC activator contains: bacterial cells of spore-forming bacteria; or a component, content, or a secretion thereof, or a culture composition thereof. The spore-forming bacterium is preferably a bacterium belonging to the genus Bacillus, the genus Weizmannia or the genus Heyndrickxia such as Heyndrickxia coagulans strain SANK 702558 or a derivative strain thereof.

Description

Plasmacytoid dendritic cell (pDC) activator and composition containing same as active ingredient

[0001] The present invention relates to the use of spore-forming bacteria, such as spore-forming lactic acid bacteria, etc. On the other hand, the present invention also relates to an agent for activating plasmacytoid dendritic cells (sometimes referred to as "pDC" in this specification) and a composition containing the same as an active ingredient.

[0002] The Heyndrickxia coagulans SANK70258 strain is a spore-forming lactic acid bacterium strain with high lactic acid production capacity that was isolated from green malt in 1949. A food preparation containing the Heyndrickxia coagulans SANK70258 strain (trade name "Lacris") has been on the market for over 50 years, from 1966 to the present day. Heindrickxia coagulans was previously named Bacillus coagulans, but due to a reorganization of the Bacillus genus, it was renamed Weizmannia coagulans in 2020, and then renamed Heindrickxia coagulans in 2022.

[0003] Regarding uses of spore-forming lactic acid bacteria (Heindrixia coagulans), for example, the following prior art documents can be mentioned.

[0004] Patent Document 1 describes "a lactic acid bacteria preparation that promotes in vivo production of IgA and IFN-γ, comprising a strain belonging to the spore-forming lactic acid bacterium Weizmannia coagulans that promotes IgA and IFN-γ production, or a mutant, disruption, culture, extract, or processed product thereof." IgA is an antibody secreted mainly from Peyer's patch cells in the intestinal tract, and IFN-γ is a type 2 interferon secreted mainly from NK cells.

[0005] Patent Document 2 describes a method for enhancing an immune response to a microbial pathogen in a subject, the method comprising: identifying a subject infected with the microbial pathogen; and administering to the subject an immune response-enhancing amount of a composition comprising viable Bacillus coagulans bacteria, a nonviable fragment of the bacteria, or a nonviable extracellular product of the bacteria. Patent Document 2 also describes that the "enhancing of the immune response" may include "increasing cytokine production or increasing immune cell migration to the site of infection" or "increasing cytokine production, activating the immune surveillance aspect of polymorphonuclear leukocytes (PMNs), increasing immune cell chemotaxis, activating natural killer (NK) cells, or increasing monocyte phagocytosis," and that the "cytokine" may include "interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), or interferon-γ (IFN-γ)." Patent Document 2 also describes that "inactivated or killed" Bacillus coagulans and "purified cell wall fractions" may be used.

[0006] Non-Patent Document 1 describes that feeding pigs Bacillus coagulans increases the expression levels of various proteins and genes in the intestinal tract. However, while Non-Patent Document 1 describes that the composition of the intestinal microbiota changes and that IFN-α is included among the many genes whose expression levels increase, it does not describe or suggest that activation of plasmacytoid dendritic cells (pDCs) increases the expression level of the IFN-α gene. It is well known that type 1 interferons such as IFN-α are typically produced by macrophages, neutrophils, dendritic cells, B cells, and the like in response to many viruses and several pathogens.

[0007] Non-Patent Document 2 describes that feeding Bacillus coagulans to broilers significantly increases the levels of IFN-α, Toll-like receptor (TLR3), and melanoma differentiation-associated protein 5 (MDA5) in the duodenum. However, although Non-Patent Document 2 mentions that the intestinal mucosal barrier and immune function are strengthened, it does not describe or suggest that the level of IFN-α increases due to the activation of plasmacytoid dendritic cells (pDC).

[0008] On the other hand, plasmacytoid dendritic cells (pDCs) are cells involved in the innate immune system, which is responsible for the primary response against bacterial and viral infections, and are known to be the main in vivo producers of type I interferons such as IFN-α and IFN-β. For example, IFN-α acts on NK cells, killer T cells, helper T cells, B cells, etc., and activates all immune cells, thereby enabling comprehensive enhancement of antiviral activity. There is demand for products that can enhance antiviral activity, etc. through the action of the innate immune system, as so-called probiotics, not only as foods and beverages to be ingested by humans, but also as additives to be ingested by livestock (for example, by mixing with feed), and research and development into these products is ongoing.

[0009] With regard to pDC activation, for example, the following prior art documents can be mentioned.

[0010] Patent Document 3 describes "an IFN production inducer comprising, as an active ingredient, lactic acid bacteria or a culture or processed product thereof capable of activating plasmacytoid dendritic cells (pDCs) and inducing IFN production." Patent Document 3 lists "preferably lactic acid bacteria, more preferably lactic acid bacteria belonging to the genera Lactococcus, Leuconostoc, Pediococcus, or Streptococcus," such as "Lactococcus lactis subsp. lactis" (strain JCM5805, strain JCM20101, etc.) as "lactic acid bacteria strains capable of activating plasmacytoid dendritic cells (pDCs) and inducing IFN production." As is clear from the scientific names, the "lactic acid bacterium" described in Patent Document 3 is a lactic acid bacterium of a different genus from the "spore-forming lactic acid bacteria" such as Heindrixia coagulans described in Patent Document 1. Furthermore, Patent Document 3 lists "type I IFN" (IFN-α, IFN-β) and "type III IFN" (IFN-λ) as "IFN."

[0011] Patent Document 4 describes "an immunostimulatory composition containing at least one active ingredient selected from the group consisting of cells, cell components, cultures, and processed products thereof of lactic acid bacteria of the genus Pediococcus, and having the effect of activating innate and adaptive immunity via plasmacytoid dendritic cells." Patent Document 4 lists "Pediococcus acidilactici" and "Pediococcus pentosaceus" as "lactic acid bacteria of the genus Pediococcus," and states that "the extent of the effect of lactic acid bacteria of the genus Pediococcus to promote the production of IFN-α and IL-12 by pDC was significantly greater than that of Lactococcus lactis subsp. lactis described in Patent Document 1 (Note: Patent Document 3 in this specification)." Furthermore, Patent Document 4 cites "the action of promoting the production of interferon-α and interleukin-12" as an "action of activating natural immunity and adaptive immunity."

[0012] JP 2023-023981 A International Publication WO2010 / 127132 (corresponding to JP 2012-525428 A, Patent Nos. 5836264, 6133937, and 6419253) International Publication WO2012 / 091081 (corresponding to Patent Nos. 5950827, 6170190, and 6598824) JP 2019-201590 A (corresponding to Patent No. 6796299)

[0013] Tal Wu et al., Beneficial Impact and Molecular Mechanism of Bacillus coagulans on Piglets' Intestine, Int. J. Mol. Sci. 2018, 19(7), 2084; https: / / doi.org / 10.3390 / ijms19072084Xu L. et al., Bacillus Coagulans Enhance the Immune Function of the Intestinal Mucosa of Yellow Broilers, Bras. Cienc. Avic. 19 (01), Jan-Mar 2017, https: / / doi.org / 10.1590 / 1806-9061-2015-0180

[0014] An objective of the present invention is to provide a new means for activating plasmacytoid dendritic cells (pDCs).

[0015] In another aspect, an object of the present invention is to provide a new use of spore-forming lactic acid bacteria or other spore-forming bacteria.

[0016] The present inventors have discovered that spore-forming lactic acid bacteria (spore-forming bacteria) such as Heyndrickxia coagulans have the ability to activate plasmacytoid dendritic cells (pDCs), for example, by further enhancing the expression levels of TLR7, CD304, and the like during viral infection and inducing increased production of type 1 interleukins such as IFN-α, thereby completing the present invention.

[0017] That is, in one aspect, the present invention provides the following inventions. [Item 1] An activator of plasmacytoid dendritic cells (hereinafter referred to as "pDC"), characterized by containing spore-forming bacteria cells or components, contents, or secretions thereof, or a culture composition of spore-forming bacteria. [Item 2] The pDC activator of Item 1, wherein the activation of pDCs includes enhancing production of type 1 interferon by pDCs. [Item 3] The pDC activator of Item 2, wherein the type 1 interferon comprises at least one selected from the group consisting of IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-υ. [Item 4] The pDC activator of Item 1, wherein the spore-forming bacteria are bacteria of the genus Bacillus, Weizmannia, or Heyndrickxia. [Item 5] The pDC activator according to Item 4, wherein the bacterium of the genus Heindrixia is Heindrixia coagulans. [Item 6] The pDC activator according to Item 5, wherein the Heindrixia coagulans is Heindrixia coagulans strain SANK70258 or a derivative thereof. [Item 7] The pDC activator according to Item 1, wherein the spore-forming bacteria are viable. [Item 8] The pDC activator according to Item 5, wherein the Heindrixia coagulans bacteria are viable. [Item 9] An oral composition for activating pDCs in a host, comprising the pDC activator according to any one of Items 1 to 8 as an active ingredient. [Item 10] The oral composition according to Item 9, which is a food, beverage, food and beverage additive, supplement, or feed additive. [Item 11] The effective cell count of the spore-forming bacteria is 1 x 10 １０ Item 10. The oral composition of item 9, wherein the number of infected cells is less than or equal to CFU.

[0018] In addition, in light of the spirit of the present invention and the detailed description of the inventions set forth in this specification, a person skilled in the art may convert the above inventions and other inventions described herein into inventions of other categories that are compatible with patent practice, or may redefine them as inventions that combine the constituent elements of each invention.

[0019] For example, the invention of [Item 1] above can be translated into "a bacterial cell of a spore-forming bacterium (excluding food-poisoning bacteria), or a component, content, or secretion thereof (or an agent containing the bacterial cell), for use in (or to be used in) a method for activating plasmacytoid dendritic cells (pDCs)" or "use of a bacterial cell of a spore-forming bacterium (excluding food-poisoning bacteria), or a component, content, or secretion thereof (or an agent containing the bacterial cell), in a method for activating plasmacytoid dendritic cells (pDCs)." Furthermore, the invention of [Item 9] above can be translated into "a method for activating pDCs in a subject (host, human or non-human animal), comprising orally ingesting (administering) a composition containing an effective amount of the pDC activator of [Item 1]," "use of the pDC activator of [Item 1] for use in (or for use in) activating pDCs in a subject's body (or for a pDC activation method)," "use of the pDC activator of [Item 1] for (or in) producing an oral composition for (or to be used in) activating pDCs in a subject's body (or for a pDC activation method)," or "use of the pDC activator of [Item 1] for (or in) producing an oral composition for (or to be used in) activating pDCs in a subject's body (or for a pDC activation method)." Furthermore, the items described in each item can be combined in any desired manner. For example, in the invention of [Item 9] above, based on the constituent features of [Item 2] which is dependent on [Item 1], it is possible to define "an oral composition for enhancing production of type 1 interferon by pDC in the body of a host, characterized by containing the pDC activator described in [Item 2] as an active ingredient."

[0020] Therefore, in another aspect, the present invention provides, for example, the following inventions. [Item 1A] A method for activating plasmacytoid dendritic cells (hereinafter referred to as "pDCs") in a subject, comprising orally administering to the subject cells of a spore-forming bacterium or a component, content, or secretion thereof, or a culture composition of a spore-forming bacterium. [Item 2A] The method for activating pDCs according to Item 1A, wherein the activation of pDCs comprises enhancing production of type 1 interferon by pDCs. [Item 3A] The method for activating pDCs according to Item 2A, wherein the type 1 interferon comprises at least one selected from the group consisting of IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-υ. [Item 4A] The method for activating pDCs according to Item 1A, wherein the spore-forming bacterium is a bacterium belonging to the genus Bacillus, Weizmannia, or Heyndrickxia. [Item 5A] The method for activating pDCs according to Item 4A, wherein the bacterium of the genus Heindrixia is Heindrixia coagulans. [Item 6A] The method for activating pDCs according to Item 5A, wherein the Heindrixia coagulans is Heindrixia coagulans SANK70258 strain or a derivative thereof. [Item 7A] The method for activating pDCs according to Item 1A, wherein the spore-forming bacteria are viable bacteria. [Item 8A] The method for activating pDCs according to Item 5A, wherein the Heindrixia coagulans bacteria are viable bacteria. [Item 9A] The method for activating pDCs according to Item 1A, wherein the spore-forming bacteria or their components, contents or secretions, or a culture composition of the spore-forming bacteria is in the form of a food, beverage, food and beverage additive, supplement, or feed additive. [Item 10A] The effective cell count of the spore-forming bacteria is 1 x 10 １０ The method for activating pDCs according to Item 1A, wherein the number of pDCs is CFU or less.

[0021] In yet another aspect, the present invention provides, for example, the following inventions. [Item 1B] Use of spore-forming bacteria, or a component, content, or secretion thereof, or a culture composition of spore-forming bacteria, for the manufacture of an oral composition for use in activating plasmacytoid dendritic cells (hereinafter referred to as "pDC") in the body of a subject. [Item 2B] The use according to Item 1B, wherein the activation of pDC comprises enhancing the production of type 1 interferon by pDC. [Item 3B] The use according to Item 2B, wherein the type 1 interferon comprises at least one selected from the group consisting of IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-υ. [Item 4B] The use according to Item 1B, wherein the spore-forming bacteria is a bacterium belonging to the genus Bacillus, Weizmannia, or Heyndrickxia. [Item 5B] The use according to Item 4B, wherein the bacterium of the genus Heindrixia is Heindrixia coagulans. [Item 6B] The use according to Item 5B, wherein the Heindrixia coagulans is Heindrixia coagulans strain SANK70258 or a derivative thereof. [Item 7B] The use according to Item 1, wherein the spore-forming bacteria are viable bacteria. [Item 8B] The use according to Item 5, wherein the Heindrixia coagulans bacteria are viable bacteria. [Item 9B] The use according to Item 1, wherein the orally ingestible composition is in the form of a food, beverage, food and beverage additive, supplement, or feed additive. [Item 10B] The effective cell count of the spore-forming bacteria is 1 x 10 １０ Item 1. The use according to Item 1, wherein the number of infected cells is less than CFU.

[0022] In the present invention, by utilizing spore-forming bacterial cells or their components, contents, or secretions, and culture compositions, it is possible to activate plasmacytoid dendritic cells (pDCs), for example, by further enhancing the expression levels of TLR7, CD304, and the like during viral infection, and thereby induce enhanced production of type 1 interferons such as IFN-α. This invention makes it possible to use spore-forming bacterial cells and the like as pDC activators in vivo and ex vivo, for example, to produce and use oral compositions containing a pDC activator as an active ingredient for activating pDCs in the host's body. The effects of this invention are expected to help strengthen the host's innate immune system to prevent infectious diseases caused by viruses, bacteria, and the like.

[0023] Fig. 1 shows the quantitative results of IFN-α gene expression (mRNA) in peripheral blood mononuclear cells after co-culture with influenza virus before (0W) and after (8W) intervention for the Heindrixia coagulans SANK70258 strain (HC) intervention group ("HC", gray bars on the left) and the placebo group ("Placebo", white bars on the right), as measured in Example 1. Fig. 2 shows the quantitative results of TLR7 gene expression (mRNA) in peripheral blood mononuclear cells after co-culture with influenza virus before (0W) and after (8W) intervention for the HC intervention group ("HC", gray bars on the left) and the placebo group ("Placebo", white bars on the right), as measured in Example 1. Figure 3 shows the quantitative results of CD304 gene expression (mRNA) in peripheral blood mononuclear cells after co-culture with influenza virus before (0W) and after (8W) intervention for the HC intervention group ("HC", gray bar on the left) and the placebo group ("Placebo", white bar on the right), measured in Example 1. Figure 4 shows the quantitative results (MFI) of CD86 expression (protein) in pDCs treated with HC and seven other types of lactic acid bacteria, measured in Example 2. The numbers on the horizontal axis correspond to the following lactic acid bacteria (the same applies to Figures 5 to 9). 1; Lactobacillus antri JCM15950, 2; L. sakei subsp. sakei JCM1157, 3; L. buchneri JCM1068, 4; L. gasseri JCM1131, 5; L. plantarum subsp. plantarum JCM1149, 6; Leuconostoc mesenteroides subsp. cremoris JCM16943, 7; L. mesenteroides subsp. cremoris JCM6124. Figure 5 shows the quantitative results (MFI) of the expression level (protein) of MHC class I in pDCs treated with HCs and seven other species of lactic acid bacteria, as measured in Example 2. Figure 6 shows the quantitative results (MFI) of the expression level (protein) of MHC class II in pDCs treated with HCs and seven other species of lactic acid bacteria, as measured in Example 2.Figure 7 shows the quantitative results (MFI) of CD86 expression (protein) in cDCs treated with HC and seven other types of lactic acid bacteria, as measured in Example 2. Figure 8 shows the quantitative results (MFI) of MHC class I expression (protein) in cDCs treated with HC and seven other types of lactic acid bacteria, as measured in Example 2. Figure 9 shows the quantitative results (MFI) of MHC class II expression (protein) in cDCs treated with HC and seven other types of lactic acid bacteria, as measured in Example 2.

[0024] In this specification, unless otherwise specified, the terms "pDC activator of the present invention," "oral composition of the present invention," "pDC activation method of the present invention," "use of the present invention," etc. are not limited to the pDC activator, oral composition, pDC activation method, use, etc. described in the "Claims" or "Summary of the Invention," but comprehensively refer to the pDC activator, oral composition, pDC activation method, use, etc. in any embodiment disclosed by the description in this specification.

[0025] - pDC activator - The plasmacytoid dendritic cell (pDC) activator of the present invention contains spore-forming bacteria cells or components, contents, or secretions thereof (sometimes referred to as "bacterial cells, etc." in this specification), or a culture composition of spore-forming bacteria.

[0026] In the present invention, "activation" of pDCs means that the expression levels at the gene level (mRNA) or protein level of pDC intracellular Toll-like receptors (TLRs) and / or cell surface markers and / or products secreted extracellularly by pDCs are statistically significantly (significance level 5%) or tend to be significantly (significance level 10%) higher after application of the pDC activating agent of the present invention than before application.

[0027] Examples of intracellular TLRs in pDC include TLR7 and TLR9, and examples of cell surface markers for pDC include CD123, CD303, and CD304. Products secreted extracellularly by pDC include type 1 interferons (IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-υ, etc.) and type 3 interferons (IFN-λ, etc.). In the present invention, activation of pDC preferably increases the expression level of at least type 1 interferon, particularly IFN-α, at least at the genetic level upon viral infection, and more preferably increases the expression levels of CD304 and / or TLR7 at least at the genetic level.

[0028] The expression levels of pDC cell surface markers and / or products secreted extracellularly by pDC at the gene (mRNA) or protein level can be quantified by common measurement methods. The expression levels at the gene level can be quantitatively measured, for example, by real-time PCR. For more specific procedures for these measurement methods, please refer to the Examples described below.

[0029] "Spore-forming bacteria" refers to bacteria that have the ability to form spores when the surrounding environment deteriorates. Spore-forming bacteria include bacteria known as food poisoning bacteria, such as Bacillus cereus, Clostridium perfringens, and Clostridium botulinum, but such food poisoning bacteria are excluded from the spore-forming bacteria of the present invention. Examples of spore-forming bacteria of the present invention include bacteria of the genus Bacillus, Weizmannia, and Heyndrickxia. Any one type (species, strain) of spore-forming bacteria may be used alone, or two or more types may be used in combination in any ratio.

[0030] A specific example of a bacterium belonging to the genus Heindrixia is Heindrixia coagulans, known as a spore-forming lactic acid bacterium. Several strains of Heindrixia coagulans are known, including, for example, strain P-22, strain lilac-01, strain SIM-7 DSM14043, strain C101, strain NBRC12583, strain GBI-1, strain GBI-20, strain GBI-30, strain GBI-40, and derivatives (mutants) thereof. In the present invention, an appropriate strain can be selected and used in consideration of the effects to be achieved. In one embodiment of the present invention, Heindrixia coagulans SANK70258 or a derivative thereof is a preferred spore-forming bacterium not only in terms of the effects of the present invention, but also in terms of supply stability (high environmental tolerance, mass production possible by aerobic culture), and ease of acquisition.

[0031] Other specific examples of spore-forming bacteria include Bacillus subtilis (natto bacteria). Several strains of Bacillus subtilis are known, and an appropriate one can be selected and used in consideration of the effects of the present invention.

[0032] The spore-forming bacterial cells may be vegetative cells, spores, or a mixture thereof. The spore-forming bacterial cells may be live or dead (inactive), or may be processed products such as heat-dried products, freeze-dried products, or crushed products. The spore-forming bacterial cells are preferably live, and live bacteria can be processed into processed products such as heat-dried products or freeze-dried products. Being live bacteria, effects can be expected not only through the constituent components of the bacterial cells but also through metabolic products of the spore-forming bacteria, and proliferation in the intestinal tract can also be expected. Since spore-forming bacteria form spores, they are resistant to digestive enzymes such as gastric acid and bile acid and exhibit high survival rates in the intestines, so the effects of being live bacteria are particularly significant.

[0033] Examples of components of spore-forming bacteria include cytoplasmic components and cell wall components of spore-forming bacteria, and each component purified from the bacterial body (fraction) can be used. The contents of spore-forming bacteria refer to nucleic acids, proteins, etc. contained in the cytoplasm of spore-forming bacteria, and can be recovered, for example, from the cytoplasmic components. Examples of secretions of spore-forming bacteria include membrane vesicles (MVs) containing the components and contents of spore-forming bacteria, and MVs can be recovered from the culture medium (culture supernatant).

[0034] The pDC activator of the present invention may consist of the bacterial cells of spore-forming bacteria themselves (e.g., a purified product containing the bacterial cells or one or more of their components, contents, or secretions), or may be a formulation of the bacterial cells (e.g., a powder of the bacterial cells or one or more of their components, contents, or secretions using an excipient such as lactose or starch).Furthermore, the pDC activator of the present invention may consist of a culture composition of spore-forming bacteria (usually a mixture of the bacterial cells and their secretions), or may be a formulation of the culture composition.

[0035] The pDC activator may contain, as needed, oligosaccharides that can be assimilated by the spore-forming bacteria (e.g., galactooligosaccharides, fructooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, mannooligosaccharides, cyclodextrin, inulin, guar gum hydrolysate, pectin, glucomannan, sodium alginate, resistant starch, acacia dietary fiber), salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts), amino acids (e.g., lysine, methionine, threonine, tryptophan, valine), organic acids (e.g., tannic acid), vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, biotin, folic acid, ascorbic acid).

[0036] Although the effects of pDC activation according to the present invention are essentially achieved by the cells of spore-forming bacteria, fungi other than spore-forming bacteria that exhibit pDC activation activity may also be incorporated into the pDC activator of the present invention in order to enhance the effects of the present invention or to achieve additional effects different from those of the present invention. Examples of other fungi include lactic acid bacteria other than Heyndrickxia coagulans, butyric acid-producing bacteria, bifidobacteria, koji mold, yeast, etc.

[0037] Examples of lactic acid bacteria other than Heindrixia coagulans include bacteria of the genus Lactobacillus or Lactococcus. Examples of bacteria of the genus Lactobacillus include Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus gasseri, and Lactobacillus plantarum. Examples of bacteria of the genus Lactococcus include Lactococcus lactis, Lactococcus plantarum, Lactococcus garvieae, and Lactococcus raffinolactis.

[0038] When the pDC activator of the present invention contains bacteria other than Heindrixia coagulans, the proportion of Heindrixia coagulans to the total amount of bacteria is typically 1% by weight or more, preferably 10% by weight or more, and preferably 50% by weight or more, with the upper limit being typically 100% by weight.

[0039] (Uses) The pDC activator of the present invention can be ingested alone as a food such as a supplement, or can be ingested by blending or adding it to other foods, beverages, feed, etc. Alternatively, the pDC activator of the present invention may be prepared in advance and then blended into the oral composition of the present invention as part of the raw materials for producing the oral composition.

[0040] However, the uses of the pDC activator of the present invention are not limited to those described above, and the pDC activator can be used for various purposes related to the effect of activating pDCs. For example, one embodiment of the pDC activator of the present invention is one that is used to activate pDCs by contacting with pDCs (e.g., adding to a culture medium) ex vivo in humans or other animals or in a culture environment in vitro, rather than by oral ingestion to activate pDCs in the host (human or other animal).

[0041] The pDCs activated by the pDC activator of the present invention may be pDCs possessed by or derived from humans, or may be pDCs from non-human animals such as livestock, pets, and laboratory animals, for example, monkeys, horses, cows, sheep, pigs, dogs, cats, rabbits, ferrets, guinea pigs, gerbils, hamsters, rats, and mice.

[0042] (Method for producing pDC activator) The pDC activator of the present invention can be produced by a method similar to that used for general products (formulations) containing spore-forming bacteria. For example, Mitsubishi Chemical Corporation's "Lacris (registered trademark)-S," "Lacris (registered trademark)-S Granules," "Lacris (registered trademark)-15," and "Lacris (registered trademark)-10 for feed" are products formulated with spore-forming lactic acid bacteria (Heindrixia coagulans), and can also be used as the pDC activator of the present invention. In one embodiment of the present invention, the pDC activator can be produced or formulated by a method similar to that used for the above-mentioned products.

[0043] - Oral Composition - The "oral composition" (composition for oral ingestion) of the present invention is an oral composition that contains the "pDC activator" of the present invention as an active ingredient and is used to activate pDCs in the host's body.

[0044] The "oral composition" is not particularly limited as long as it can contain a pDC activator and can exert a predetermined effect when ingested. Specific examples of oral compositions include foods, beverages, food and beverage additives, supplements, and feed additives. Foods, beverages, food and feed additives, and supplements refer to products intended for human ingestion, while feed additives refer to products intended for ingestion by animals other than humans (particularly mammals, such as livestock, pets, and laboratory animals).

[0045] Examples of "food" include bread, cereals, sweets, fermented foods (cheese, pickles, solid fermented milk (set type yogurt), paste-like fermented milk (soft type yogurt), etc.).

[0046] Examples of "drinks" include water, soft drinks, milk drinks, liquid fermented milk (drink-type yogurt), lactic acid bacteria drinks, alcoholic drinks, drinks prepared from powders, and the like.

[0047] An "additive" for food or beverage is a product that can be in the form of powder, granules, paste, or the like, and is intended to be added to existing foods or beverages (which do not contain the pDC activator of the present invention), and examples thereof include dressings and sprinkles.

[0048] A "supplement" is a product intended to be ingested directly, and can take the form (dosage form) of, for example, tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. Furthermore, to protect the active ingredient, a pDC activator, from gastric acid and allow it to act in the intestine, it may be in the form of an enteric coating with different solubility at different pH levels.

[0049] "Feed additives" are products intended to be added to conventional feed and can take the form of, for example, tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. Examples of "conventional feed" include fresh grass, hay, green forage crops (e.g., green corn), grains (e.g., corn, milo, barley, oats, rice, foxtail millet, barnyard millet, millet, sorghum, etc.), grain by-products (e.g., rice bran, bran), root vegetables, straw, and oilseed meal (e.g., peanut meal, cottonseed meal, sunflower meal, rapeseed meal, sesame meal, flaxseed meal). The above-mentioned feed additives in tablet or other form can be blended and mixed with such conventional feed for use.

[0050] Oral compositions may be either functional foods (products with functional claims) that can be labeled under national legal systems (products with functional claims) or general foods (products without functional claims). Functional foods include foods for specified health uses (foods recognized based on scientific evidence as beneficial for maintaining and promoting health and permitted to be labeled as such; the claimed effects and safety are reviewed by the government and approved by the Commissioner of the Consumer Affairs Agency for each food), foods with nutrient functions (foods containing specific amounts of scientifically confirmed nutrients, such as vitamins and minerals, that can be used to supplement or complement these nutrients in dietary supplements, and which can be labeled as functional according to government-specified expressions), and foods with functional claims (foods labeled with scientific evidence by the business operator, for which information on safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale). General foods include so-called health foods (no legal definition, all foods other than pharmaceuticals that are taken orally and marketed with claims of specific health benefits and consumed in the hope of achieving such benefits). The above categories of "health functional foods" and "general foods" may include any of the "foods," "beverages," "supplements," and "additives" described in this specification as embodiments of oral compositions.

[0051] In one embodiment of the present invention, the oral composition can claim functionality based on the effects of the pDC activator (spore-forming bacteria) contained as an active ingredient. Such functionality may be directly or indirectly displayed on the oral composition. Examples of direct display include inscription on tangible objects such as product packaging, containers, labels, and tags. Examples of indirect display include advertising and promotional activities via locations or means such as websites, storefronts, exhibitions, signs, bulletin boards, newspapers, magazines, television, radio, mail, and email.

[0052] The oral composition of the present invention may contain, in addition to the pDC activator, basic ingredients contained in common foods, beverages, food and beverage additives, supplements, and feed additives. On the other hand, when the pDC activator itself is formulated (e.g., Mitsubishi Chemical Corporation's product "Lacris"), such a formulated pDC activator can be considered as the oral composition of the present invention.

[0053] (Method for Producing Oral Composition) The oral composition of the present invention can be produced in a manner similar to that of a general oral composition (food composition, beverage composition, etc.), except that it includes a process of adding the pDC activator of the present invention (cells of spore-forming bacteria or components thereof, contents or secretions, or a culture composition of spore-forming bacteria). For example, a production method can be employed in which, while following a general method for producing an oral composition, the pDC activator is also added as one of the ingredients at an appropriate stage in the process of blending the ingredients of the oral composition. The pDC activator can be prepared in advance as described above and stored until the oral composition is produced.

[0054] The content of the pDC activator in the oral composition is not particularly limited, as long as the effect of activating pDC in the host's body by ingestion of the oral composition is achieved. Such a content can be determined taking into consideration the amount of active ingredient per meal or per day, the weight of the oral composition to be ingested, and the form and manufacturing method of the oral composition, as appropriate.

[0055] For example, when the oral composition contains live spore-forming bacteria, the effective number of live bacteria is 1 x 10 per day. ９ Preferably, the number of CFU or less is 1 x 10 １０ The effective number of viable spore-forming bacteria contained in the oral composition is preferably as high as possible, and is preferably, for example, 1 x 10 CFU or less per day. ３ CFU or more, preferably 1 x 10 ４ CFU or more, more preferably 1 x 10 ５ CFU or more, more preferably 1 x 10 ６ CFU or more, particularly preferably 1 x 10 ７ The oral composition can be a product in which the effective bacterial count (content) of spore-forming bacteria in the oral composition and the daily intake amount of the oral composition (number of intakes per day x intake amount per serving) are designed so that a subject can ingest live spore-forming bacteria within this range as the effective bacterial count. An appropriate range of the effective bacterial count of spore-forming bacteria can be set based on the results of non-clinical or clinical tests conducted on humans or non-human animals. The upper and lower limits of the effective bacterial count of these live bacteria can be combined in any desired manner.

[0056] When the oral composition contains killed (inactive) spore-forming bacteria, the effective number of the killed bacteria is 1 x 10 per day. ９ cells or less, and 1 x 10 １０ The effective number of killed spore-forming bacteria contained in the oral composition is preferably as high as possible, and is preferably, for example, 1 x 10 per day. ３ cells or more, preferably 1 x 10 ４ cells or more, more preferably 1 x 10 ５ cells or more, more preferably 1 x 10 ６ cells or more, particularly preferably 1 × 10 ７ These upper and lower limits of the effective number of killed bacteria can be combined in any manner.

[0057] When the oral composition is a supplement containing live spore-forming bacteria, the effective number of live bacteria per tablet is 1 x 10 per day when multiplied by the number of tablets of the supplement taken per day. ９ The amount is preferably 1 x 10 CFU or less. １０ The more effective number of live spore-forming bacteria per tablet contained in the supplement is, the more preferable it is. When multiplied by the number of tablets of the supplement taken per day, it is, for example, 1 × 10 ３ CFU or more, preferably 1 x 10 ４ CFU or more, more preferably 1 x 10 ５ CFU or more, more preferably 1 x 10 ６ CFU or more, particularly preferably 1 x 10 ７ The upper and lower limits of the effective number of live bacteria can be arbitrarily combined.

[0058] When the oral composition is a supplement containing killed (inactive) spore-forming bacteria, the effective number of the killed bacteria per tablet is 1 x 10 per day when multiplied by the number of tablets of the supplement taken per day. ９ The amount is preferably 1 x 10 １０ Furthermore, the more effective number of killed spore-forming bacteria per tablet contained in the supplement is, the more preferable it is, and when multiplied by the number of tablets of the supplement taken per day, it is, for example, 1 × 10 ３ cells or more, preferably 1 x 10 ４ cells or more, more preferably 1 x 10 ５ cells or more, more preferably 1 x 10 ６ cells or more, particularly preferably 1 × 10 ７ These upper and lower limits of the effective number of killed bacteria can be combined in any manner.

[0059] The supplement may contain ingredients other than spore-forming bacteria, such as starch, dextrin, lactose, calcium carbonate, cellulose, silicon compounds, etc. as excipients, etc. The content of ingredients other than spore-forming bacteria, such as excipients, can be appropriately adjusted depending on the embodiment of the supplement.

[0060] When the oral composition is a beverage containing live spore-forming bacteria, the effective number of live bacteria in the beverage is 1 x 10 １１ CFU / L or less is preferred, and 1 x 10 １０ The effective number of viable spore-forming bacteria contained in the beverage is preferably as high as possible, for example, 1 × 10 ４ CFU / L or more, more preferably 1 x 10 ５ CFU / L or more, more preferably 1 x 10 ６ CFU / L or more, particularly preferably 1 x 10 ７ CFU / L. The upper and lower limits of the effective number of live bacteria can be combined in any desired manner.

[0061] When the oral composition is a beverage containing killed (inactive) spore-forming bacteria, the effective number of the killed bacteria in the beverage is 1 x 10 １１ cells / L or less, and preferably 1 x 10 １０ The effective number of killed spore-forming bacteria contained in the beverage is preferably as high as possible, for example, 1 × 10 ４ cells / L or more, more preferably 1 x 10 ５ cells / L or more, more preferably 1 x 10 ６ cells / L or more, particularly preferably 1 x 10 ７ These upper and lower limits of the effective number of killed bacteria can be combined arbitrarily.

[0062] Example 1 Ability of HCs to Activate pDCs In Vitro 1. Subject Selection Criteria Healthy men and women aged 20 to 65 years who are prone to catching colds (having low salivary s-IgA and having suffered from an upper respiratory tract infection in the winter within the past two years) were selected as subjects.

[0063] 2. Test method: Except for one subject who voluntarily dropped out, all subjects received 1 billion (1 x 10 9 The group that took capsules containing HC for 8 weeks was designated the HC group (39 subjects), and the group that took capsules not containing HC in the same manner was designated the placebo group (40 subjects).

[0064] 3. Sorting of peripheral blood mononuclear cells (PBMCs) Peripheral blood samples were collected from the subjects before and after the 8-week ingestion period, and PBMCs were separated from the blood using Ficoll-Paque PLUS (Cytiva). The separated PBMCs were washed with PBS, immersed in a Cell Banker, and frozen at -80°C.

[0065] 4. Inactivated influenza virus exposure culture experiment RPMI 1640 medium supplemented with 1 mM sodium pyruvate, 2.5 mM HEPES, 100 U / mL penicillin-streptomycin, 50 μM 2-mercaptoethanol, and 10% fetal bovine serum was used as the cell culture medium. Cryopreserved PBMCs were thawed in a 37°C water bath and then cultured at a density of 1.0 × 10 in cell culture medium. ６ The solution was adjusted to 1.0 μg / mL, and 0.2 mL of the solution was seeded into a 96-well cell culture plate. Inactivated influenza virus (Influenza A H1N1 Virus Antigen; HyTest Ltd.) was added to the solution to a concentration of 1.0 μg / mL. The incubation temperature was 5% CO ２ The cells were cultured in an incubator for 24 hours. After the culture, the cells were immersed in RNA-later and then cryopreserved at -80°C.

[0066] 5. Gene expression analysis Total RNA was extracted from the cultured cells using NucleoSpin (R)RNA was extracted using RNA XS (MACHEREY-NAGEL GmbH & Co. KG). cDNA was synthesized from the total RNA solution using reverse transcriptase (PrimeScript RT Reagent Kit, TAKARA). This was used as a template for real-time PCR analysis using Rotor-Gene Q (Qiagen) and the primers and TaqMan probes listed in Table 1. The Universal Probe Library No. listed in Table 1 is the set number sold by Roche Diagnostics. Using the β-actin gene as a reference, relative mRNA expression levels were calculated using the ΔΔCt method. CD304 gene expression levels were relatively low in all samples, with the average Ct value exceeding 40 in the pre-intake samples in particular, resulting in significant variability in the measurement results. Therefore, to select appropriate samples for this study, CD304 gene expression levels were analyzed in samples with Ct values ​​below 40. A Mann-Whitney U test was used to compare between groups before and after the 8-week intake period.

[0067]

[0068] 6. Results The results for gene expression levels of IFN-α, TLR7, and CD304 are shown in Figures 1 to 3, respectively. Pre-intake is shown as 0W, and post-intake is shown as 8W. No significant differences were observed between the groups at 0W. However, at 8W, the HC group showed significantly higher gene expression levels for IFN-α and TLR7, and a significant trend toward higher gene expression levels for CD304 compared to the placebo group. These results suggest that pDCs are activated by HC ingestion.

[0069] Example 2: Activation ability of each lactic acid bacteria on mouse-derived DCs <Experimental method> Bone marrow cells were collected from the femur of C57BL / 6 male mice according to a standard method and subjected to red blood cell removal treatment. The obtained bone marrow cells were then added to RPMI medium (GIBCO or Fujifilm Wako) containing 10% FCS, 100 μM β-mercaptoethanol, 10 mM HEPES, 1 mM pyruvic acid, 10 mM non-essential amino acid solution, 100 U / mL penicillin, and 100 mg / mL streptomycin at a concentration of 5 × 10 ５To the obtained cell suspension, Flt-3L (manufactured by BioLegend) was added as a pDC-inducing cytokine at a final concentration of 100 ng / ml, and the cells were incubated for 1 hour at 25°C for 1 hour with CO ２ Incubator at 37°C and 5% CO ２ On the 8th day of cultivation, a suspension of gamma-ray-sterilized Heindrixia coagulans SANK70258 strain (HC) cell powder suspended in phosphate-buffered saline was added to the medium so that the cell concentration in the medium became 10 μg / mL, and the medium was cultivated under the above conditions for 24 hours. The cultured cells were immunostained using anti-CD11c PECy7 and anti-PDCA-1 APC for gating plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs), respectively, and activation markers were anti-CD86-FITC (BioLegend), anti-MHC class I-PE (PE anti-mouse H-2) (BioLegend), and anti-MHC class II (PerCP anti-mouse IA / IE)-PerCP (BioLegend). Fluorescence was detected using a flow cytometer (MACS Quant Analyzer; Miltenyi Biotec). Data were analyzed using Flowjo (Tomy Digital Biology, Tokyo, Japan). Note that the embodiment targeting cDCs is shown as a reference example.

[0070] Seven non-spore-forming lactic acid bacteria, Lactobacillus antri JCM15950, L. sakei subsp. sakei JCM1157, L. buchneri JCM1068, L. gasseri JCM1131, L. plantarum subsp. plantarum JCM1149, Leuconostoc mesenteroides subsp. cremoris JCM16943, and L. mesenteroides subsp. cremoris JCM6124 (obtained from RIKEN BRC), were also prepared as controls and subjected to the same tests. These seven lactic acid bacteria were statically cultured in MRS medium at 30°C or 37°C for 24 to 48 hours. After harvesting, the cells were washed twice with sterile water and sterilized by gamma irradiation. The cells were then lyophilized and subjected to the tests.

[0071] <Results> Figures 4 to 6 show the expression levels of CD86, MHC class I, and MHC class II in pDC, and Figures 7 to 9 show the expression levels of CD86, MHC class I, and MHC class II in cDC. The vertical axis of the graph represents the median fluorescent intensity (MFI) value when HC and seven types of lactic acid bacteria were added, divided by the MFI value when no addition was made. In pDC, the highest expression level of CD86 was confirmed in the test group with the addition of HC, which was approximately 7 times the expression level when the seven types of lactic acid bacteria were added. The expression levels of MHC class I and MHC class II were highest in HC, followed by relatively high expression in L. mesenteroides subsp. cremoris JCM6124, L. gasseri JCM1131, and L. plantarum subsp. plantarum JCM1149. On the other hand, for cDC, CR86 was most activated by HC, but MHC class I was activated equally by L. mesenteroides subsp. cremoris JCM6124, L. gasseri JCM1131, and L. plantarum subsp. plantarum JCM1149. MHC class II was most activated by HC, while the activation of other lactic acid bacteria was approximately 50% or less of that by HC. From the above, it was shown that the degree of dendritic cell activation by lactic acid bacteria differs depending on the species of lactic acid bacteria and the type of dendritic cell (pDC or cDC), and that the particularly excellent effect of spore-forming bacteria (HC) in activating pDC is not necessarily a predictable, universal effect.

Claims

1. A plasmacytoid dendritic cell (hereinafter referred to as "pDC") activator, characterized in that it contains spore-forming bacteria cells or their components, contents or secretions, or a culture composition of spore-forming bacteria.

2. The pDC activator according to claim 1, wherein the activation of pDCs comprises enhancing the production of type 1 interferon by pDCs.

3. The pDC activator according to claim 2, wherein the type 1 interferon comprises at least one selected from the group consisting of IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-υ.

4. The pDC activator according to claim 1, wherein the spore-forming bacterium is a bacterium of the genus Bacillus, Weizmannia, or Heyndrickxia.

5. The pDC activator according to claim 4, wherein the bacterium of the genus Heindrixia is Heindrixia coagulans.

6. The pDC activator according to claim 5, wherein the Heindrixia coagulans is the Heindrixia coagulans SANK70258 strain or a derivative thereof.

7. The pDC activator according to claim 1, wherein the spore-forming bacteria are viable bacteria.

8. The pDC activator according to claim 5, wherein the Heindrixia coagulans cells are live cells.

9. An oral composition for activating pDCs in a host's body, comprising the pDC activator according to any one of claims 1 to 8 as an active ingredient.

10. The oral composition of claim 9, which is a food, beverage, food or beverage additive, supplement, or feed additive.

11. The effective number of spore-forming bacteria is 1 x 10 10 10. The oral composition of claim 9, wherein the number of CFU or less.

12. A method for activating plasmacytoid dendritic cells (hereinafter referred to as "pDC") in a subject, characterized in that it comprises orally administering to the subject spore-forming bacteria or their components, contents or secretions, or a culture composition of spore-forming bacteria.

13. The method for activating pDCs according to claim 12, wherein the activation of pDCs comprises enhancing the production of type 1 interferon by pDCs.

14. The method for activating pDCs according to claim 12, wherein the type 1 interferon comprises at least one selected from the group consisting of IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-υ.

15. The method for activating pDCs according to claim 12, wherein the spore-forming bacterium is a bacterium of the genus Bacillus, Weizmannia, or Heyndrickxia.

16. The method for activating pDCs according to claim 15, wherein the bacterium of the genus Heindrixia is Heindrixia coagulans.

17. The method for activating pDCs according to claim 16, wherein the Heindrixia coagulans is the Heindrixia coagulans SANK70258 strain or a derivative thereof.

18. The method for activating pDCs described in claim 12, wherein the spore-forming bacteria are viable bacteria.

19. The method for activating pDCs according to claim 16, wherein the Heindrixia coagulans cells are live cells.

20. The method for activating pDCs described in claim 12, wherein the spore-forming bacterium cells or their components, contents or secretions, or a culture composition of the spore-forming bacterium, is in the form of a food, beverage, food and beverage additive, supplement, or feed additive.

21. The effective number of spore-forming bacteria is 1 x 10 １０ The method for activating pDCs according to claim 12, wherein the number of cells is CFU or less.

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