JP2018188436A - Follicular regulatory t cell increasing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for increasing follicular regulatory T-cell (Tfr cell) with high safety, for example, Tfr cell increasing agents administered to a subject in which follicular helper T cells (Tfh cell) in blood are excessively increased.SOLUTION: A lactic acid bacterium belonging to Lactobacillus, for example, Lactobacillus paracasei MCC1849 (NITE BP-01633) is contained as an active ingredient in medicines, foods and drinks, food and drink compositions, and feed and the like which are used for increasing Tfr cells.SELECTED DRAWING: None

Description

  The present invention relates to a follicular regulatory T cell increasing agent and use thereof.

  Immunity is an advanced biological defense mechanism for excluding foreign substances (antigens) such as bacteria and viruses that try to enter the living body. Antibodies produced by B cells, a type of immune cell, are proteins that recognize and bind to antigens, and are one of the defense mechanisms that efficiently eliminate foreign substances. Autoimmune diseases, on the other hand, cause symptoms when immunity for recognizing and eliminating foreign substances reacts excessively to normal cells and tissues of one's own. It is known that one of the causes is that a large amount of autoantibodies are produced. In order for B cells to produce antibodies that specifically recognize antigens, helper T cells (Th cells) characterized by CD4 positivity play a major role. For antibody production from B cells, B cells that produce antibodies with high affinity for antigens are selected at a place called the germinal center in the lymphoid follicle, which is a localized region of B cells. Known to be important. Selection of B cells producing such high affinity antibodies involves the involvement of follicular helper T cells (Tfh cells: Follicular Helper T cells) and follicular regulatory T cells (Tfr cells: Follicular Regulatory Helper T Cells). (Non-Patent Document 1).

  Tfr cells are a type of Th cell present in lymph node follicles that express CXCR5, PD-1 (Programmed Cell Death 1) on the cell surface and express Foxp3 in the nucleus. It is a cell. Normally, T cells are localized in the T cell region in the immune tissue, and are present in a different location from the follicle where B cells are localized. However, when an antigen invades, some of the activated T cells become Tfh cells and move to the germinal center where many B cells are localized, which matures the B cells and promotes the production of antigen-specific antibodies. To do. Tfr cells are induced to differentiate from regulatory T cells (Treg cells) that have differentiated in the thymus. For B cells and Tfh cells, in addition to cytokines such as IL-10 and TGF-β, costimulation such as PD-1 and ICOS Mathematical or functional control is achieved through molecular interactions. When Tfr cells are deficient or decrease in number, Tfh cells increase excessively and increase B cells producing immature antibodies with low antigen specificity (Non-patent Document 2). Therefore, for the germinal center reaction to produce high-quality antibodies specific for antigens from B cells, it is essential to control the number or function of B cells and Tfh cells by Tfr cells, which has a high affinity for antigens. The numerical or functional action of Tfr cells is important for the efficient induction of antibodies.

  A decrease in the number or function of Tfr cells is known to be involved in many autoimmune diseases as a result of excessive increases in Tfh cells. In patients with autoimmune diseases such as myasthenia gravis, such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and Sjogren's syndrome, it has been reported that Tfh cells in the blood are excessively increased and the germinal center reaction is enhanced. (Non-patent Document 3). In SLE patients, excessive production of Tfh cells and germinal center B cells is observed, and the number of Tfh cells in peripheral blood is strongly correlated with the amount of anti-double-stranded DNA antibodies that cause SLE (non-patented). Reference 4). There is a need for a means to improve the function of Tfr cells in those who have a reduced number and function of these Tfr cells, but no safe and effective means have yet been provided.

  In recent years, lactic acid bacteria, mold, yeast, and the like have been reported as food ingredients that regulate the immune function of living bodies. So far, some lactic acid bacteria have been reported to regulate immune cells such as Th1, Th2 and Tfh cells (Non-patent Document 5, Patent Document 1), but food components containing lactic acid bacteria induce Tfr cells. It has not been reported to increase the cell number.

Japanese Unexamined Patent Publication No. 2016-113378

Vanderleyden, I. et al., Arthritis Res Ther. (2014) 16 (5): 471 Linterman, M.A., Nat Med. (2011) 17: 975-982 Yangyang, Z., Int Immunol. (2016) 28 (4): 173-9 Simpson, N. et al., Arthritis Rheum. (2010) 62: 234-244 Shida, K. et al. Gut. Microbes. (2011) 2: 109-114

  An object of the present invention is to provide a highly safe means for increasing Tfr cells, for example, a Tfr cell increasing agent to be administered to a subject in which Tfh cells in blood are excessively increased.

  In order to solve the above problems, the present inventors have eagerly searched for a factor capable of inducing Tfr cells from food materials, and found that intake of lactic acid bacteria belonging to the genus Lactobacillus increases Tfr cells. The invention has been completed.

That is, the present invention provides a follicular regulatory T cell (Tfr cell) increasing agent comprising a lactic acid bacterium belonging to the genus Lactobacillus as an active ingredient.
The Tfr cell increasing agent has a preferred embodiment in which the lactic acid bacterium is Lactobacillus paracasei.
Moreover, the said Tfr cell increasing agent makes it a preferable aspect that the said lactic acid bacteria are Lactobacillus paracasei MCC1849 (NITE BP-01633).
Further, the Tfr cell increasing agent is preferably administered to a subject in which follicular helper T cells (Tfh cells) in blood are excessively increased.
Moreover, the said Tfr cell increasing agent makes it a preferable aspect that the said lactic acid bacteria are the living bacteria of lactic acid bacteria, and / or the dead bacteria of lactic acid bacteria.
Furthermore, this invention provides the food-drinks composition for follicular control T cell increase which uses the lactic acid bacteria which belong to the Lactobacillus genus as an active ingredient.

  The present invention provides a means for effectively increasing Tfr cells. The Tfr cell increasing agent of the present invention contains lactic acid bacteria belonging to the genus Lactobacillus as an active ingredient, has high safety, and is useful as a pharmaceutical product, food and drink, food and drink composition, feed, and the like.

FIG. 3 is a graph showing the ratio (%) of Tfr cells in the flow cytometry of Example 2.

Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.
The Tfr cell increasing agent of the present invention contains lactic acid bacteria as an active ingredient. Examples of lactic acid bacteria include Lactobacillus genus, Leuconostoc genus, Streptococcus genus, Pediococcus genus, Melissococcus genus, Enterococcus (Enterococcus) genus, Trichococcus), Lactococcus, Carnobacterium,
Vagococcus genus, Tetragenococcus genus, Atopobium genus, Weissella genus, Oenococcus genus, Abiotrophia genus, Desmesia genus (Desemzia) Paralactobacillus genus, Granulicatella genus, Alkalibacterium genus, Orsenella genus, Isobacrum genus, Marinilactibacillus genus, Atopostipes genus, ovum Lactum Lactic acid bacteria belonging to the genus, Pilibacter genus, Fructobacillus genus, Lacticigemium genus, Bavariicoccus genus, Bifidobacterium genus, etc. are preferred, and Lactobacillus genus is more preferred

Examples of the lactic acid bacteria belonging to the genus Lactobacillus include, for example, Lactobacillus paracasei, Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alba, Lactobacillus alactus Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apodemi, Lactobacillus aquaticus, Lactobacillus aviaries, Lactobacillus bifermentans, Lactobacillus bobalius, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capilatus, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus ceti, Lactobacillus coleohominis , Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus cru storum, Lactobacillus curvatus, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus delbrueckii subsp.delbrueckii, Lactobacillus delbrueckii subsp.indicus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus dextrinicus, obas
farciminis, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus graminis, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus hordei, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus kimchii, Lactobacillus kisonensis, Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus lindneri , Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mindensis, Lactobacillu s mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus nodensis, Lactobacillus oligofermentans, Lactobacillus oris, Lactobacillus otakiensis, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri, Lactobacillus paralimentarius , Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus rapi, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus satsumensis, Lactobacill
us secaliphilus, Lactobacillus senmaizukei, Lactobacillus sharpeae, Lactobacillus siliginis, Lactobacillus spicheri, Lactobacillus suebicus, Lactobacillus sunkii, Lactobacillus susicola, Lactobacillus taiwanensis, Lactobacillus thailandensis, Lactobacillus tucceti, Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vini , Lactobacillus vitulinus, Lactobacillus zeae, Lactobacillus zymae, etc. are preferable, and Lactobacillus paracasei MCC1849 (NITE BP-01633) (hereinafter sometimes abbreviated as “MCC1849”), and bacteria substantially equivalent to MCC1849 More preferred. Hereinafter, in this specification, a lactic acid bacterium belonging to the genus Lactobacillus may be simply referred to as a lactic acid bacterium.
MCC1849 is a strain isolated using human feces as a separation source. The bacteriological properties of this strain are shown in Example 1 below. On June 6, 2013, the strain was transferred to NITE P-01633 in the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Room 2-5-8 Kazusa, Kazusa, Kisarazu 292-0818, Japan). And was transferred to an international deposit based on the Budapest Treaty on December 19, 2013, and a deposit number of NITE BP-01633 was assigned.

  A bacterium substantially equivalent to MCC1849 is a lactic acid bacterium belonging to Lactobacillus paracasei and having a Tfr cell increasing action comparable to that of MCC1849. In addition, a substantially equivalent bacterium further has a homology of 98% or more, preferably 99% or more, more preferably 100%, with the base sequence of the 16S rRNA gene of MCC1849. And preferably has the same bacteriological properties as MCC1849. Furthermore, MCC1849 or a bacterium substantially equivalent thereto may be a strain bred from such a bacterium by mutation treatment, gene recombination, selection of a natural mutant strain, etc., as long as the effects of the present invention are not impaired. Good.

  Lactic acid bacteria such as MCC1849 can be easily grown by culturing them. The method for culturing is not particularly limited as long as lactic acid bacteria can proliferate, and the method usually used for culturing lactic acid bacteria can be appropriately modified as necessary. For example, the culture temperature may be 25 to 50 ° C, and preferably 35 to 42 ° C. The culture may be performed under anaerobic conditions or anaerobic conditions, but is preferably performed under anaerobic conditions. For example, the culture can be performed while anaerobic gas such as carbon dioxide gas is aerated. Moreover, you may culture | cultivate on microaerobic conditions, such as liquid stationary culture.

  The medium for culturing lactic acid bacteria is not particularly limited, and a medium usually used for culturing lactic acid bacteria can be appropriately modified as necessary. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolyzate, and molasses can be used according to the utilization. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean powder, defatted soybean meal, meat extract, yeast extract and the like may also be used. Moreover, as the prepared medium, for example, an MRS medium can be preferably used.

As the lactic acid bacterium, after culturing, the obtained culture may be used as it is, diluted or concentrated and used, or microbial cells recovered from the culture may be used. In addition, various additional operations such as heating and freeze-drying can be performed after culturing as long as the effects of the present invention are not impaired. The additional operation is preferably one in which viability of viable bacteria is high. In addition, as shown in the Examples, the heat-sterilized bacterial cells of lactic acid bacteria have a Tfr cell increasing action. Therefore, even if it is a living microbe, it is thought that Tfr cell increase action is shown similarly. That is, in the Tfr cell increasing agent of the present invention, the lactic acid bacteria may be live or dead, or both live and dead. The dead bacteria may be a crushed material of dead cells.

The above lactic acid bacteria can be used as an active ingredient of a Tfr cell increasing agent. In the present invention, “Tfr cell increase” means an increase in the number of Tfr cells or an increase in the proportion of Tfr cells in the lymphocyte fraction. Tfr cells increased if the number of Tfr cells increased or the percentage of Tfr cells in the lymphocyte fraction increased in subjects who took the Tfr cell augmentation agent compared to when they did not. I can say that.
A composition containing a lactic acid bacterium such as MCC1849 or the Tfr cell increasing agent of the present invention containing the lactic acid bacterium as an active ingredient can be widely used as a medicine, food or drink, food or drink composition, or feed. For example, a drug for increasing Tfr cells, a food / beverage composition for increasing Tfr cells, or a feed for increasing Tfr cells, which contains lactic acid bacteria belonging to the genus Lactobacillus as an active ingredient, can be provided.

  The Tfr cell increasing agent of the present invention is not particularly limited as long as it contains lactic acid bacteria. For the Tfr cell increasing agent of the present invention, lactic acid bacteria may be used as they are, or a liquid or solid pharmaceutical carrier that is physiologically acceptable may be blended into a pharmaceutical preparation.

  The dosage form of the Tfr cell increasing agent of the present invention is not particularly limited, and specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups, suppositories, injections , Ointments, patches, eye drops, nasal drops and the like. In formulation, addition of excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, or solvents for injections that are commonly used as pharmaceutical carriers Agents can be used.

The content of lactic acid bacteria in the Tfr cell increasing agent of the present invention is appropriately set depending on the dosage form, usage, age of subject, sex, type of disease, degree of disease, and other conditions, but usually 1 × 10 Preferably it is in the range of ⁶ to 1 × 10 ¹² cfu / g or 1 × 10 ⁶ to 1 × 10 ¹² cfu / ml, 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / g or 1 × 10 ⁷ to 1 More preferably, it is within the range of × 10 ¹¹ cfu / ml. When the lactic acid bacterium is dead, cfu / g or cfu / ml can be replaced with individual cells / g or individual cells / ml.

  Moreover, unless the effect of this invention is impaired, you may use together the Tfr cell increasing agent of this invention, the Tfr cell increasing agent which uses components other than lactic acid bacteria as an active ingredient, an immunostimulant, etc.

  The administration time of the Tfr cell increasing agent of the present invention is not particularly limited, and the administration time can be appropriately selected according to the treatment method for the target disease. It may be administered prophylactically or used for maintenance therapy. The dosage form is preferably determined according to the dosage form, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. In any case, the Tfr cell increasing agent of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.

The Tfr cell-increasing agent of the present invention is administered to a subject effective for suppressing an excessive increase in Tfh cells, for example, a subject effective for reducing autoantibodies and immature antibodies. The subject is, for example, a human. These subjects are expected to control follicular helper T cells or antibody-producing cells by increasing follicular regulatory T cells and to regulate excessive enhancement of antibody production function. Further, the Tfr cell increasing agent of the present invention prevents diseases or symptoms caused by an increase in autoantibodies due to excessive increase in Tfh cells, such as systemic lupus erythematosus, rheumatoid arthritis, Graves' disease, Hashimoto's disease, and Sjogren's syndrome, It can be used to prevent, treat or ameliorate a disease or condition that can be prevented, treated or ameliorated by treating or ameliorating or increasing Tfr cells.
Here, the state where “the Tfh cells are excessively increased” preferably means that the number of Tfh cells in the blood is excessively increased relative to the healthy subject, specifically, in the blood of the healthy subject. It is more preferable that the number of Tfh cells is increased by 2 times or more. Examples of blood include, but are not limited to, peripheral blood.
Although Lactobacillus paracasei is known to have IL-12 production-inducing ability (International Publication No. 2012/133827), the Tfr cell increasing agent of the present invention aims to promote IL-12 production. It is preferable to exclude applications. Lactobacillus paracasei is known to have a Tfh cell-increasing action (Patent Document 1), but the Tfr cell-increasing agent of the present invention preferably excludes applications intended to increase Tfh cells.

  Another aspect of the present invention is the use of lactic acid bacteria in the manufacture of a Tfr cell augmentation agent. In addition, another aspect of the present invention is a method for preventing, treating, or improving a disease that can be prevented, treated, or ameliorated by an increase in Tfr cells, comprising the step of administering a lactic acid bacterium or a Tfr cell increasing agent of the present invention to an application subject. It is.

  Lactic acid bacteria such as MCC1849 of the present invention can be used as a food or drink or a food or drink composition (hereinafter sometimes referred to as “food or drink”). In addition, the food or drink or the food or drink composition containing the Tfr cell increasing agent is not particularly limited as long as it contains lactic acid bacteria. Examples of the food and drink include soft drinks, carbonated drinks, nutrition drinks, fruit juice drinks, and lactic acid bacteria drinks. Beverages (including concentrated concentrates and powders for preparation of these beverages); ice confectionery such as ice cream, sorbet, shaved ice; candy, chewing gum, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, and Pastries such as baked goods; dairy products such as processed milk, milk drinks, fermented milk, drink yogurt, and butter; bread; enteral nutrition, liquid food, milk for childcare, sports drinks; other functional foods Is done. The food and drink may be a supplement, for example, a tablet-like supplement. In the case of a supplement, lactic acid bacteria can be ingested without being influenced by other foods with respect to the daily meal amount and calorie intake.

Such foods and drinks can be produced by adding lactic acid bacteria to raw materials such as foods and drinks, and can be produced in the same manner as normal foods and drinks except that lactic acid bacteria are added. The addition of lactic acid bacteria may be performed at any stage of the production process of food and drink. Moreover, food-drinks etc. may be manufactured through the fermentation process by the added lactic acid bacteria. Examples of such foods and beverages include lactic acid bacteria beverages and fermented milk.
As a raw material for food or beverage, a raw material used for a normal beverage or food can be used. The manufactured food or drink can be taken orally.

  The food / beverage products of the present invention include raw materials for the production of food / beverage products, and food additives, etc., which are added to the food / beverage products after the production process or production. For example, the lactic acid bacteria of the present invention can be used as a starter for producing fermented milk. Moreover, the lactic acid bacteria of this invention can also be added later to the manufactured fermented milk.

The content of live bacteria or killed bacteria of lactic acid bacteria in foods and drinks and the like is appropriately set depending on the mode of the food and drinks and the like, but usually 1 × 10 ⁶ to 1 × 10 ¹² cfu / g (cells) / G) or 1 × 10 ⁶ to 1 × 10 ¹² cfu / ml (cells / ml), preferably 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / g (cells / g) or 1 × More preferably, it is in the range of 10 ⁷ to 1 × 10 ¹¹ cfu / ml (cells / ml).

  The food / beverage products or food / beverage product compositions containing a lactic acid bacterium such as MCC1849 or a Tfr cell increasing agent of the present invention can be used for various applications utilizing effects such as Tfr cell increase and immune function improvement. That is, a Tfr cell increasing agent and an immune function improving agent that are in the form of a food or drink or a food or drink composition can be provided.

  Food / beverage products or food / beverage product compositions containing a lactic acid bacterium such as MCC1849 of the present invention or a Tfr cell increasing agent are to be sold as food / beverage products or food / beverage product compositions for which Tfr cell increase and immune function improvement applications are displayed. Can do. In addition, such a food or drink or a food or drink composition can be labeled as “for increasing Tfr cells” or “for improving immune function”. In addition, it is needless to say that words other than the above can be used as long as the wording expresses an effect that is secondarily caused by an increase in Tfr cells.

  The “display” means all acts for informing the consumer of the use, and if it is a display that can recall and analogize the use, the purpose of the display, the content of the display, the display Regardless of the object and medium to be processed, all fall under the “display” of the present invention. However, it is preferable to display in such an expression that the consumer can directly recognize the application. Specifically, the act of describing the above-mentioned use on the product or the product packaging related to the food or drink of the present invention, the product or the product packaging on which the above-mentioned use is described is transferred, for delivery, transfer or delivery Display and import activities, advertisements regarding products, price lists or transaction documents to display or distribute the above uses, or to describe the above uses in information containing these contents (such as the Internet) Actions provided by the method can be exemplified, and in particular, display on advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POPs, and other documents is preferable.

  In addition, the display is preferably a display permitted by the government or the like (for example, a display which is approved based on various systems determined by the government and is performed in a mode based on such approval). For example, indications such as health foods, functional foods, enteral nutritional foods, special-purpose foods, functional nutritional foods, quasi-drugs, and other indications approved by the Ministry of Health, Labor and Welfare or the Consumer Affairs Agency For example, foods for specified health use, functional display foods, and labels approved by a similar system can be exemplified. Examples of the latter include a display as a food for specified health use, a display as a food for specified specific health use, a display that affects the structure and function of the body, a display for reducing disease risk, and the like. In more detail, the indication as food for specified health (especially the indication of the use of health) stipulated in the Enforcement Regulation of the Health Promotion Act (Ministry of Health, Labor and Welfare Ordinance No. 86 of April 30, 2003), and Similar displays can be exemplified.

  Examples of feed containing lactic acid bacteria such as MCC1849 of the present invention or Tfr cell increasing agent include pet food, livestock feed, and fish feed. Such a feed is produced by mixing lactic acid bacteria with a common feed such as cereals, potatoes, potatoes, fish meal, bone meal, fats and oils, skim milk powder, whey, mineral feed, yeasts, etc. be able to. Moreover, a feed may be manufactured through the fermentation process by the added lactic acid bacteria like silage, for example. The produced feed can be orally administered to general mammals, livestock, fish farms, pets and the like.

The content of live or dead bacteria of lactic acid bacteria in the feed is appropriately set depending on the mode of the feed and the administration target, but is 1 × 10 ⁶ to 1 × 10 ¹² cfu / g (cells / g) or 1 × 10 ⁶ to It is preferably within the range of 1 × 10 ¹² cfu / ml (cells / ml), 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / g (cells / g) or 1 × 10 ⁷ to 1 × 10 ¹¹ cfu / More preferably in the range of ml (cells / ml).

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1] Mycological properties of MCC1849
MCC1849 was isolated from a human stool sample. Table 1 shows the cell morphology, motility, sporulation, Gram staining, catalase, gas production from glucose, and sugar fermentability as the bacteriological properties of the bacterium. The fermentability of sugar was examined using a bacterial identification kit API 50CH (Sysmex Biomelieu). The bacterial solution after overnight culture is inoculated into a medium containing each substrate according to the method described in the manual attached to the kit, and cultured in a 37 ° C. incubator. Fermentation properties were evaluated.
These mycological properties are shown in Table 1. From this result, MCC1849 was determined to be Lactobacillus paracasei.

From the analysis of the 16S rRNA gene base sequence of MCC1849, MCC1849 was determined to be Lactobacillus paracasei or Lactobacillus casei. Lactobacillus paracasei and Lactobacillus casei are closely related species, and it has been shown that the homology of the 16S rRNA gene base sequence between the standard strains is 99% or more (Huang CH, and Lee FL., Antonie Van Leeuwenhoek, 2011, 99 (2): 319-27). The genomic sequence of MCC1849 was determined, and the homology was confirmed by mapping the lead as a reference genome of Lactobacillus paracasei standard strain (ATCC25302) or Lactobacillus casei standard strain (ATCC393).

As a result, MCC1849 had 85% homology with the Lactobacillus paracasei standard strain, whereas it had 45% homology with the Lactobacillus casei standard strain.
Therefore, MCC1849 was confirmed to be Lactobacillus paracasei.

[Example 2] Increase of Tfr cells by MCC1849 (1) Preparation of heat sterilized MCC1849
Cells cultured for 16 hours in MRS (de Man Rogasa Sharpe) medium (Difco) for MCC1849 were washed twice with PBS, then once with distilled water, suspended in distilled water, and heated at 100 ° C for 30 minutes. Processed. After the heat treatment, the cells were washed twice with distilled water, suspended in distilled water, freeze-dried, and MCC1849 heat-sterilized bodies were obtained.

(2) Preparation of dendritic cells (DC)
After suspending 475 μL of MACS buffer and 25 μL of CD11c MicroBeads, mouse (Miltenyi Biotech) per 1 × 10 ⁸ cells in Peyer's patch (PP) cells derived from BALB / cA Jcl mice (obtained from CLEA Japan) Then, the mixture was allowed to stand on ice for 15 min, and MACS buffer was added and centrifuged (4 ° C., 400 G, 5 min) to remove CD11c Beads together with the supernatant. Thereafter, the cells were resuspended in MACS buffer, and the cell suspension was passed through a MACS LS separation column (Miltenyi Biotech) according to the attached protocol for the first purification. This crudely purified cell was passed through a MACS MS separation column (Miltenyi Biotech) to perform the second purification, thereby purifying CD11c ⁺ cells. This was used as PP DC.

(3) Preparation of CD4 ⁺ T cells
Suspend DO11.10 mouse spleen (SPL) cells by adding 45 μL of MACS buffer and 5 μL of CD4 MicroBeads, mouse (Miltenyi Biotech) per 1 × 10 ⁷ cells, and then let stand for 15 min on ice. Then, MACS buffer was added and centrifuged (4 ° C., 400 G, 5 min) to remove the CD4 MicroBeads together with the supernatant. The Then cells were purified CD4 ⁺ cells through the cell suspension MACS LS separation columns (Miltenyi Biotec). This was used as a CD4 ⁺ T cell.

(4) Preparation of B cells
BALB / c mouse-derived SPL cells are suspended by adding 2500 μL of MACS buffer and 5 μL of biotinylated rat anti-mouse IgM antibody per 1 × 10 ⁸ cells, then left on ice for 15 min, and MACS buffer is added. And centrifuged (4 ° C., 400 G, 5 min) to remove the biotinylated rat anti-mouse IgM antibody together with the supernatant. Then add 980 μL of MACS buffer and 20 μL of Streptavidin MicroBeads, mouse (Miltenyi Biotech) per 1 × 10 ⁸ cells, suspend, then let stand for 15 min on ice, add MACS buffer and centrifuge (4 ° C , 400 G, 5 min), and the supernatant was removed for Streptavidin MicroBeads. Then cells were purified IgM ⁺ cells through the cell suspension MACS LS separation columns (Miltenyi Biotec). This was used as SPL B cells.

(5) Co-culture of heat-killed MCC1849 cells, antigenic peptides, CpG oligonucleotides, DC, T cells and B cells
Using a 96-well flat-bottom plate (BD Falcon ^™ ), DC, T cells, and B cells in 1% 10 ⁴ cells, 2 × 10 ⁵ cells, and 2.5 × 10 ⁵ cells in 200 μL of 5% FCS-RPMI. The cell solution was added as follows. Antigen peptide (OVAp; final concentration 10 nM) corresponding to OVA 323-339 was suspended in 5% FCS-RPMI and added. Heat-killed cells were suspended in PBS at a final concentration of 100 μg / mL, and adjuvant CpG oligo DNA (CpG ODN 1668; CpG) was added at 10 nM. The cells were cultured in an environment of 37 ° C. and 5% CO ₂ .

(6) Flow cytometry Add about 1 × 10 ⁵ -2 × 10 ⁶ cells of cell suspension to a polystyrene round tube, add FACS buffer, centrifuge (4 ° C, 400 G, 5 minutes), and remove the supernatant. To wash. Hereinafter, FACS buffer was used for all washing and dilution. Anti-mouse CD16 / 32 antibody (2.4G2, BioLegend) was suspended at a concentration of 2.5 μg / mL, and Fc receptor was blocked on ice for 15 minutes. Washing was performed, and the fluorescently labeled antibody and the biotinylated antibody were diluted and suspended, and reacted on ice for 20 minutes. After washing, fluorescently labeled streptavidin was diluted and suspended, and reacted on ice for 20 minutes. When intracellular molecules were not stained, the cells were washed, washed immediately after adding propidium iodide and suspended, and FACS buffer was added to these samples and used for flow cytometry. For Foxp3 staining, Foxp3 Staining Buffer Set (eBioscience) was used and the attached protocol was followed. FACS Verse (BD Biosciences) was used for measurement, and data analysis was performed with FlowJo (Tree Star, Inc.).
The following antibodies and fluorescent reagents were used.
FITC-labeled anti-mouse CD4 antibody (H129.19, BD Pharmingen)
allophycocyanin (APC) labeled anti-mouse CD4 antibody (GK1.5, BioLegend)
PerCP-efluor710 labeled anti-mouse CD279 / PD-1 antibody (J43, ebioscience)
Biotinylated anti-mouse CD185 / CXCR5 antibody (SPRCL5, ebioscience)
Biotinylated anti-mouse CD11c antibody (An antibody described in N418, Cytotechnology (2011), vol. 63, p. 307-317, which is equivalent to a commercially available one)
Biotinylated anti-mouse CD279 / PD-1 antibody (J43, ebioscience)
PE-cy7 labeled streptavidin (BioLegend)
APC labeled streptavidin (BioLegend)
PE-labeled anti-mouse Foxp3 antibody (FJK-16s, ebioscience)
* Indicates that the statistical risk ratio between the two groups is less than 5%. In addition, Tukey HSD was used for the test of statistical significance, and p <0.05 was regarded as statistical significance.

(7) Results As shown in FIG. 1, the ratio of CD4 ⁺ CXCR5 ⁺ PD-1 ^high Foxp3 ⁺ cells (Tfr cells) was significantly increased by adding MCC1849 heat sterilized cells in the presence of CpG. In this experiment, the proportion of Tfr cells increased significantly even in the absence of CpG. In addition, the bar in a figure shows an average value.
From this result, it was shown that lactic acid bacteria Lactobacillus paracasei MCC1849 has an effect of increasing Tfr cells.

Claims (6)

  A follicular regulatory T cell increasing agent comprising a lactic acid bacterium belonging to the genus Lactobacillus as an active ingredient.   The follicular regulatory T cell increasing agent according to claim 1, wherein the lactic acid bacterium is Lactobacillus paracasei.   The follicular regulatory T cell increasing agent according to claim 2, wherein the lactic acid bacterium is Lactobacillus paracasei MCC1849 (NITE BP-01633).   The agent for increasing follicular regulatory T cells according to any one of claims 1 to 3, which is administered to a subject having excessively increased follicular helper T cells in blood.   The follicular regulatory T cell increasing agent according to any one of claims 1 to 4, wherein the lactic acid bacterium is a live bacterium of a lactic acid bacterium and / or a dead bacterium of a lactic acid bacterium.   A food and beverage composition for increasing follicular regulatory T cells, comprising a lactic acid bacterium belonging to the genus Lactobacillus as an active ingredient.

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