JP6178088B2 - M2 macrophage differentiation inducer - Google Patents

Description

  The present invention relates to a differentiation inducer for macrophages containing lactic acid bacteria, a pharmaceutical composition containing the lactic acid bacteria or the inducer, a nutritional or pharmaceutical preparation, a food containing lactic acid bacteria, and a method for inducing differentiation of macrophages using lactic acid bacteria, And a method for producing the nutritional or pharmaceutical preparation.

  Macrophages are phagocytic cells that play an important role in the immune system and inflammation. The symbol can be expressed as Mφ. First, monocytes differentiate from bone marrow cells, which circulate in the blood and are recruited to tissues throughout the body, and finally differentiate into macrophages. Macrophages have a subset of M1 and M2.

  M1 macrophages are activated by LPS and IFN-γ and secrete high levels of IL-12 and low levels of IL-10. M1 macrophages are mainly responsible for Th1-dependent immune responses (cellular immunity) and inflammatory responses. M2 macrophages are activated by IL-4 and IL-13 and produce high levels of IL-10 and low levels of IL-12. M2 macrophages express genes such as arginase-1 and chitinase-like protein Ym1, and are mainly involved in repairing damage and suppressing inflammation. Thus, M1 macrophages and M2 macrophages play different roles in vivo, and the balance between M1 macrophages and M2 macrophages is important for the normal functioning of inflammation-induced inflammation and damage repair by external stimuli.

  Thus, an imbalance in the balance between M1 macrophages and M2 macrophages can cause various diseases or disorders. IL-4 dependent M2 macrophages produced by eosinophils are present in the adipose tissue. However, in a diet-induced obesity model, there is a report that adipose tissue macrophages change from M2 polarity to M1 type pro-inflammatory state and contribute to insulin resistance (Non-patent Document 1). In addition, there is a report that M2 macrophages shift to M1 type as obesity progresses and can be involved in the development of type 2 diabetes associated with obesity (Non-patent Document 2).

  On the other hand, Patent Literature 1 describes lactic acid bacteria that suppress intestinal barrier breakdown. However, there are various factors for intestinal barrier failure, and the relationship between the failure of the intestinal barrier function and M1 and M2 macrophages is not necessarily clear. Patent Document 1 describes that lipoteichoic acid, which is a lipid component of the disclosed lactic acid bacteria, prevents intestinal barrier function breakdown (paragraph [0008]).

Habil et al. Reported that probiotic bacterial strains modulate macrophage cytokine production (Non-patent Document 3). According to pages 285-286 of Non-Patent Document 3, the effect of heat-sterilized Lactobacillus rhamnosus GG strain (NCIMB 8824) on TNFα production ability, IL-6 production ability, and NFκB production ability of already differentiated M1 or M2 macrophages (Pages 287-289, Figures 1-3). As shown in the summary and figure footnotes, the M1 and M2 macrophages used are CD14 high and CD14 low THP-1-NFκB reporter monocytes, phorbol 12-myristate 13-acetate , or 1, It was prepared by differentiating with 25- (OH) ₂ vitamin D3. What is reported in this document is only the effect of probiotic preparations on the cytokine profile of differentiated M1 and M2 macrophages.

Japanese Patent No. 5046684 (JP 2008-212006)

Lumeng et al., J Clin Invest. 2007 Jan; 117 (1): 175-84 Satoh et al., Diabetes Care. 2010; 33 (1) Habil N et al., Beneficial Microbes, vol. 2, pp. 283-293, (2011)

  When macrophages differentiate as described above, various diseases can occur due to imbalance in the M1 / M2 balance. Differentiation into M2 macrophages may be induced by IL-4, IL-13, or a fluorine compound analog of vitamin D3, but these substances are expensive, have other physiological effects, or are foods There is a problem such as being unsuitable for addition to the skin. Therefore, there is a need for a safe and simple method for inducing differentiation into M2 macrophages to correct the M1 / M2 balance imbalance for the prevention and treatment of various diseases.

As a result of diligent studies to solve the above-mentioned problems, the present inventors have surprisingly found that L. rhamnosus OLL2838 (Accession No. NITE P-313) has an M2 macrophage differentiation-inducing action and completed the present invention. I let you. That is, the present invention is as follows.
[1] An agent for inducing differentiation of M2 macrophages comprising Lactobacillus rhamnosus as an active ingredient.
[2] The differentiation-inducing agent according to [1], wherein Lactobacillus rhamnosus is L. rhamnosus specified by accession number NITE P-313.
[3] A pharmaceutical composition for treating or preventing a disease associated with an imbalance in M1 / M2 balance of macrophages, comprising the M2 macrophage differentiation inducer according to [1] or [2].
[4] A nutritional or pharmaceutical preparation for treating or preventing a disease associated with an imbalance in macrophage M1 / M2 balance, comprising the M2 macrophage differentiation inducer according to [1] or [2].
[5] A pharmaceutical composition for treating or preventing a disease associated with an imbalance in M1 / M2 balance of macrophages, comprising Lactobacillus rhamnosus as an active ingredient.
[6] The pharmaceutical composition according to [5], wherein Lactobacillus rhamnosus is L. rhamnosus identified by the accession number NITE P-313.
[7] The pharmaceutical composition according to [3], [5] or [6], wherein the disease associated with an imbalance in macrophage M1 / M2 balance is a disease selected from the group consisting of diabetes and arteriosclerosis. .
[8] The nutritional or pharmaceutical preparation according to [4], wherein the disease associated with an imbalance in macrophage M1 / M2 balance is a disease selected from the group consisting of diabetes and arteriosclerosis.
[9] A macrophage M1 / M2 balance regulator or a macrophage M1 / M2 balance food composition comprising Lactobacillus rhamnosus as an active ingredient.
[10] The adjusting agent or food composition according to [9], wherein Lactobacillus rhamnosus is L. rhamnosus specified by the accession number NITE P-313.
[11] A method for inducing differentiation of M2 macrophages in vitro, comprising a step of using L. rhamnosus.
[12] The differentiation induction method according to [11], wherein Lactobacillus rhamnosus is L. rhamnosus specified by the accession number NITE P-313.
[13] A method for producing a nutritional or pharmaceutical preparation for treating or preventing a disease associated with an imbalance in M1 / M2 balance of macrophages, comprising a step of blending L. rhamnosus .
[14] The production method according to [13], wherein the disease associated with an imbalance in M1 / M2 balance of macrophages is a disease selected from the group consisting of diabetes and arteriosclerosis.
[15] The production method according to [13] or [14], wherein Lactobacillus rhamnosus is L. rhamnosus specified by the accession number NITE P-313.

  When the differentiation inducer according to the present invention is used, differentiation into M2 macrophages can be induced. This can also reduce, alleviate, treat or prevent diseases and symptoms associated with M1 / M2 imbalance.

The results of analyzing the amount of IL-10 produced by adding heat-sterilized L. rhamnosus (HK-Lr) according to the present invention and various factors to bone marrow-derived M-CSF-dependent macrophages by ELISA are shown. Error bars in the figure represent standard errors. In the figure, ** indicates a significant difference compared to None (no addition) (** p <0.01), and $$ indicates a significant difference compared to IL-4 addition ($ $ P <0.01), ## indicates that there is a significant difference compared with IFN-γ addition (## P <0.01). Results of qPCR analysis of IL-10 / β-actin ratio when L. rhamnosus (HK-Lr) and various factors according to the present invention were added to bone marrow-derived M-CSF-dependent macrophages Indicates. Error bars in the figure represent standard errors. In the figure, ** indicates a significant difference compared to None (no addition) (** p <0.01), and $$ indicates a significant difference compared to IL-4 addition ($ $ P <0.01), ++ indicates that there is a significant difference compared to the addition of heat sterilized L. rhamnosus (HK-Lr) (++ P <0.01), ## compared with the addition of IFN-γ Indicates that there is a significant difference (## P <0.01). Results of qPCR analysis of CD206 / β-actin ratio when L. rhamnosus (HK-Lr) heat-sterilized according to the present invention and various factors are added to bone marrow-derived M-CSF-dependent macrophages . Error bars in the figure represent standard errors. In the figure, ** indicates a significant difference compared to None (no addition) (** p <0.01), and $$ indicates a significant difference compared to IL-4 addition ($ $ P <0.01), ++ indicates that there is a significant difference compared to the addition of heat sterilized L. rhamnosus (HK-Lr) (++ P <0.01). Results of qPCR analysis of TNF-α / β-actin ratio when bone marrow-derived M-CSF-dependent macrophages were added with heat-sterilized L. rhamnosus (HK-Lr) according to the present invention or various factors Indicates. Error bars in the figure represent standard errors. In the figure, ** indicates a significant difference compared to None (no addition) (** p <0.01), and $$ indicates a significant difference compared to IL-4 addition ($ $ P <0.01), ## indicates that there is a significant difference compared with IFN-γ addition (## P <0.01).

As the M2 macrophage differentiation inducing agent according to the present invention, lactic acid bacteria (including treated cells thereof) can be used. In this specification, lactic acid bacteria refers to bacteria that assimilate glucose to produce lactic acid, and are physiologically gram-positive cocci or bacilli, have no motility, no spore-forming ability, and are catalase-negative. It has the following properties. Lactic acid bacteria can be said to be extremely safe microorganisms such as those used for food fermentation and constituting the intestinal bacterial flora of mammals. The lactic acid bacteria can be used in the present invention, Lactobacillus spp, Lactococcus spp, Leuconostoc spp, Pediococcus spp, Streptococcus spp, W e issella genus, Tetragenococcus genus, Oenococcus spp, Enterococcus spp, Vagococcus sp, Carnobacterium spp, Melissococcus genus, Trichococcus Genus, Atopobium genus. Preferably, as the lactic acid bacteria that can be used in the present invention, Lactobacillus genus, especially Lactobacillus rhamnosus species, more preferably Lactobacillus rhamnosus specified by the accession number NITE P-313 (Lactobacillus rhamnosus), but is not limited to this. In addition, if it retains M2 macrophage differentiation-inducing activity, a mutant or transformed strain of Lactobacillus rhamnosus specified by accession number NITE P-313, or Lactobacillus rhamnosus specified by accession number NITE P-313 Strains derived from can also be used.

Lactobacillus identified by accession number NITE P-313 (Lactobacillus) rhamnosus) was previously isolated as a strain that contributes to the recovery of intestinal barrier function (see Patent Document 1). The deposit information is described below.
(1) Depositary institution name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: Postal code 292-0818 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture
Phone number 0438-20-5580
(3) Accession number: NITE P-313
(4) Display for identification: Lactobacillus rhamnosus OLL2838
(5) Date of deposit: February 14, 2007.

  Lactobacillus rhamnosus identified by accession number NITE P-313 is a Gram-positive bacilli, and the colony form when anaerobically cultured on BL agar is round, white, smooth and conical Raise into a shape. Physiological characteristics include homolactic fermentation, growth at 15 ° C., rhamnose, ribose, glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, trehalose, melittose, mannitol, sorbitol.

  The M2 macrophage differentiation-inducing agent and the pharmaceutical composition, nutritional or pharmaceutical preparation or food composition containing the same according to the present invention may contain the above-mentioned lactic acid bacteria in various forms. For example, the lactic acid bacteria may be live or dead bacteria, lactic acid fermented products (lactic acid bacteria beverages or foods, sour milk, yogurt, etc.), microbial cells or microbial cell fragments, microbial cell processed products (crushed material, crushed material, Cell lysate and the like). Thus, when referred to as an M2 macrophage differentiation inducer containing lactic acid bacteria, this includes live bacteria, dead bacteria, fermented products, bacterial cells, bacterial cell fragments, and processed bacterial cells as described above.

  Lactic acid bacteria can be cultured using an appropriate medium known in the art. Examples of media include lactic acid bacteria selection media such as MRS media (de Man JC, Rogosa M. and Sharpe M. Elisabeth (1960) Appl. Bact. 23. 130-135) and lactic acid bacteria culture media. Medium is used. That is, any medium can be used as long as it contains a nitrogen source, an inorganic substance and other nutrients in addition to the main carbon source. As the carbon source, lactose, glucose, sucrose, fructose, starch hydrolyzate, waste molasses and the like can be used according to the assimilation ability of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, whey protein hydrolyzate, and soy protein hydrolyzate can be used. In addition, meat extract, fish extract, yeast extract and the like are used as growth promoters. As the microbial cells, not only the microbial cells obtained from the lactic acid bacterium culture solution cultured using such a medium, but also the lactic acid bacterium culture solution after completion of the culture can be used as it is or as a concentrated product of the culture solution. The lactic acid bacterium may be a living bacterium, a dead bacterium, or a mixture thereof, and can be used as a alive cell, a wet cell, or a dry cell (which can be obtained by freeze drying, spray drying, vacuum drying, drum drying, or the like). Dead bacteria can be obtained by heat treatment, pressure treatment, heat pressure treatment, ultraviolet irradiation, or the like. In this specification, heat treatment may be used synonymously with heat sterilization. Cell bodies, culture solutions, and concentrates obtained regardless of whether they are live or dead, may be suspended again in an appropriate medium and used as a suspension. Examples of the medium include a culture solution, water, and physiological saline.

  In the present invention, the active ingredient lactic acid bacteria may be in the form of a fermented product. Examples of fermented products include lactic acid bacteria beverages, lactic acid bacteria foods, sour milk, fermented milk, and yogurt. Moreover, in this invention, a lactic acid bacteria processed material can also be used as lactic acid bacteria which are active ingredients. Lactic acid bacteria treated products include lactic acid bacteria, lactic acid bacteria-containing materials, fermented milk concentrates, pasted products, dried products, etc. And pulverized pulverized product. In addition to products manufactured as lactic acid fermented foods, the food composition can be applied to various foods manufactured in advance, lactic acid bacteria according to the present invention, fermented lactic acid bacteria, processed microbial cells, macrophage M1 / M2 balance regulator, nutrition Or a pharmaceutical preparation or a pharmaceutical composition may be added.

  In the present invention, to induce differentiation of M2 macrophages or to promote differentiation induction, when undifferentiated macrophages derived from bone marrow mononuclear cells further differentiate into M1 or M2 macrophages, lactic acid bacteria according to the present invention, induction of differentiation of M2 macrophages M1 / M2 ratio when the lactic acid bacteria according to the present invention, the M2 macrophage differentiation inducing agent or the composition containing the same is allowed to act is compared with the M1 / M2 ratio when the agent or the composition containing the same is not acted Smaller (that is, the proportion of M2 in the entire system increases). Therefore, the term “inducing differentiation into M2 macrophages” includes suppression of differentiation into M1 macrophages.

  The lactic acid bacterium, the M2 macrophage differentiation inducer or the composition containing the same according to the present invention can be used for treatment or prevention of a disease associated with an imbalance in macrophage M1 / M2 balance. Moreover, the M1 / M2 balance adjusting agent or food composition containing lactic acid bacteria according to the present invention can be used for adjusting the M1 / M2 balance of macrophages.

  Here, the M1 / M2 balance of macrophages means that M1 macrophages and M2 macrophages were balanced so that a series of mechanisms ranging from inflammatory responses to external stimuli to damage repair in the living body function normally. It exists in a ratio. Normal M1 / M2 balance of macrophages can be determined based on M1 / M2 abundance ratio in healthy individuals or populations of healthy individuals, using conventional statistical techniques when determining on a population basis Can do. When the M1 / M2 balance is not achieved, deviates from the normal range, or is abnormal, this is referred to as an M1 / M2 balance imbalance or an M1 / M2 balance collapse. Furthermore, the M1 / M2 ratio changes when the living body receives external stimuli (including pro-inflammatory stimuli), but if the M1 excess / M2 excess exceeds the change range in healthy subjects, or temporarily Even if it takes a long time to return to normal balance after M1 excess / M2 underestimation or it takes a long time to return, it is included in “M1 / M2 balance imbalance” and “M1 / M2 balance collapse”. To do. Therefore, in the present specification, the imbalance of the M1 / M2 balance not only refers to the case where M1 macrophages are excessively present compared to M2 or M2 macrophages are excessively small compared to M1, When the M1 / M2 ratio fluctuates in the subject when the subject receives external stimuli (including pro-inflammatory stimuli), the M1 / M2 ratio fluctuates in the subject tends to M1 excess, or temporarily This includes the case where the recovery to the original M1 / M2 ratio from the typical inclination to M1 excess is not shown for a long time.

  Although the present invention is not limited to a specific mechanism of action, in one embodiment, the lactic acid bacteria according to the present invention, a regulator or food containing the same, an M2 macrophage differentiation inducing agent, or a composition containing the same, By inducing differentiation into M2 macrophages, or by adjusting the M1 / M2 balance, the M1 / M2 balance imbalance is corrected, or the disrupted M1 / M2 balance is restored to normal, and thus the M1 of the macrophages Can treat or prevent diseases associated with imbalance in the / M2 balance. In this specification, to correct the M1 / M2 balance imbalance and to restore the broken M1 / M2 balance to normal, the M1 / M2 balance imbalance and the balance imbalance are normal M1 / M2 balanced. It means returning to a state where it is in contact or promoting it. Adjusting the M1 / M2 balance not only corrects the M1 / M2 balance imbalance, restores the broken M1 / M2 balance to normal, but also maintains the M1 / M2 balance. It also includes.

  Diseases associated with imbalance in macrophage M1 / M2 balance include diabetes, eg type 2 diabetes due to increased insulin resistance, arteriosclerosis, eg arteriosclerosis associated with inflammation, arterial atherosclerosis, atherosclerosis Etc. In diet-induced obesity model animals, it has been reported that adipose tissue macrophages change from M2 polarity to M1 type pro-inflammatory state and contribute to insulin resistance (Non-patent Document 1). In addition, there is a report that M2 macrophages shift to M1 type as obesity progresses and can be involved in the development of type 2 diabetes associated with obesity (Non-patent Document 2). Therefore, those skilled in the art understand that type 2 diabetes can be treated or prevented if differentiation into M2 macrophages can be induced in such an obesity model. Arterial atherosclerosis, atherosclerosis, is a complex inflammation of lipid abnormalities and inflammation, but macrophages activated by IFN-γ, etc. take up low density lipoprotein (LDL) cholesterol and become foam cells. It develops by inducing inflammation. Therefore, those skilled in the art will understand that arteriosclerosis can be prevented or prevented from progressing if differentiation into M2 macrophages can be induced in such potential arteriosclerosis. Furthermore, it was reported that imbalance to M1 promotes recurrent encephalomyelitis (Mult Scler. 2011 Jan; 17 (1): 2-15), and M1 polarized macrophages cause pathogenesis of multiple sclerosis (Int J Pharm. 2011 Sep 20; 416 (2): 499-506) is also reported. Those skilled in the art understand from these reports that inducing differentiation into M2 macrophages and restoring the M1 / M2 balance can lead to the treatment and prevention of these diseases. Thus, the lactic acid bacteria, M2 macrophage differentiation inducer or pharmaceutical composition, nutritional or pharmaceutical preparation or food composition containing the same according to the present invention can be used for diabetes prevention, arteriosclerosis prevention and the like.

  In the present invention, the M2 macrophage differentiation inducing agent is prepared so as to contain as an active ingredient the lactic acid bacterium cells of the present invention (regardless of viable and dead bacteria, and also mixtures thereof). In addition to the use of the microbial cell as an active ingredient alone, it includes the addition of necessary excipients and the like as appropriate, and the use as nutritional or pharmaceutical preparations, pharmaceutical compositions, and food compositions. . Examples of such a composition include, but are not limited to, various dosage forms and supplements such as tablets and granules, capsules, injections and solutions, dry syrups, powders, and syrups. In addition, if desired, other active substances such as anti-inflammatory agents, anti-analgesic agents, vitamins, appropriate excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, Coating agents, adjuvants, preservatives, fragrances, coloring agents and the like can also be added.

  In the present invention, the food composition means a processed food to which the lactic acid bacteria according to the present invention (live bacteria, dead bacteria), bacterial cell components, or a differentiation inducer containing the same is added. This includes foods obtained using the lactic acid bacteria according to the present invention, as well as food compositions in which the lactic acid bacteria content of the present invention is increased to the same or higher than when obtained in a normal process. The food composition includes existing foods such as milk, yogurt, cheese, fermented milk, tofu, porridge, kuzuyu, tea and fruit juice, bread, biscuits, crackers, pizza crust, formula milk, Includes foods made from various edible materials such as liquid foods, foods for the sick, infant milk powders, infant milk powders (including powdered milk), and nutritional foods. Or not only those with added gas components, but also various kinds of proteins (whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, lactoferrin, soy protein, egg protein, meat Animal and plant proteins such as proteins, plant proteins such as lecithin, soybean protein, etc., various carbohydrates (monosaccharides such as glucose, fructose, rhamnose, disaccharides such as sucrose, polyhydric alcohols such as xylitol and glycerin, dextrin, Processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), polysaccharides such as dietary fiber), various lipids (lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterification) Animal oils such as oil, soybean oil, oarm oil, safflower oil, corn oil, rapeseed oil, these fractionated oils, vegetable oils such as hydrogenated oil, transesterified oil, unsaturated fatty acids, etc.), various vitamins (Vitamin A, carotenoids, vitamin B group, vitamin C, vitamin D group, vitamin E, bi Min K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid, etc.) and various minerals (calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, etc.) Also, food compositions prepared by mixing various nutrients such as organic acids (malic acid, citric acid, lactic acid, tartaric acid, etc.) in any proportions, and adding a gelling agent to the mixture as it is or further adding them to adjust the viscosity It is done. The food composition according to the present invention includes so-called food for specified health use. This specifically displays foods and health indications that enable the indication of efficacy based on the action of the present invention, such as diabetes prevention, arteriosclerosis prevention, prevention or improvement of diseases related to imbalance in M1 / M2 balance of macrophages It is a food that is publicly permitted and a functional nutritional food.

A person skilled in the art can appropriately set the intake (or dose) of lactic acid bacteria according to the present invention. The amount of intake varies depending on the age, sex, weight, symptom, or purpose of use of the subject. For example, as a single intake in an adult human, 1 × 10 ⁴ to 1 × 10 ¹⁵ cells, preferably 1 × Although it can be used by 10 < ⁷ > -1 * 10 < ¹³ > piece, it is not limited to this. As long as it is dead, it can be used at 1 × 10 ⁴ to 10 ¹⁶ , preferably 1 × 10 ⁷ to 1 × 10 ¹⁴ , but is not limited thereto. The cell concentration depends on the mode of use, but it may be, for example, 0.001 to 100% (w / w), preferably 0.01 to 100% (w / w), more preferably 0.1 to 100% (w / w). it can. Moreover, when using as a lactic-acid-bacteria fermented material, a microbial cell processed material, etc., Preferably it converts into a microbial cell and uses as said range.

  The intake of lactic acid bacteria can be appropriately increased or decreased depending on the age, sex, weight, symptom, and intended use (treatment, prevention) of the subject, and can be adjusted as appropriate depending on the type and intake of the pharmaceutical composition or food composition. Examples of the route of intake include oral administration, tube administration, enteral administration, and the like. The subject of ingestion or administration is not limited to humans but may include pets and livestock, for example, mammals such as dogs, cats, monkeys, cows, pigs, horses.

  The pharmaceutical composition, nutritional or pharmaceutical preparation or food composition according to the present invention has a disease associated with an imbalance in macrophage M1 / M2 balance or is capable of treating, improving, Can be used for mitigation, mitigation, prevention. Ingestion is preferably before meals, after meals, and between meals in the case of pharmaceuticals, and in the case of food compositions as one item of meal or as a material during meals, and during meals as supplements, dietary supplements, etc. can do. Furthermore, the differentiation inducer according to the present invention can also be used when it is desired to induce differentiation of cells into M2 macrophages in an in vitro experimental system.

That is, the M2 macrophage differentiation inducer containing the lactic acid bacterium according to the present invention as an active ingredient can be used, for example, in vitro as a research reagent, and thereby can induce differentiation of undifferentiated macrophages into M2 macrophages. Thus, the present invention also provides a method for inducing differentiation of M2 macrophages, comprising the step of bringing the differentiation inducer into contact with undifferentiated macrophages in vitro. As an example, bone marrow mononuclear cells are cultured in the presence of macrophage colony stimulating factor (M-CSF) to obtain bone marrow-derived M-CSF-dependent macrophages (undifferentiated macrophages). An undifferentiated macrophage refers to a macrophage that has not yet differentiated into M1 or M2 macrophages. At this time, the dendritic cells are preferably removed from the system by magnetic cell separation or the like. Lactic acid bacteria according to the present invention can be added to the undifferentiated macrophages, and in some cases, an appropriate factor (IL-4 or the like) can be further added to induce differentiation into M2 macrophages. The lactic acid bacteria used for differentiation induction can be, for example, lactic acid bacteria 10 ⁴ to 10 ⁸ cfu, 10 ⁵ to 10 ⁷ cfu, for example 2 × 10 ⁶ cfu with respect to undifferentiated macrophage 2 × 10 ⁵ cells. Differentiation induction into M2 macrophages can be confirmed by analyzing an appropriate marker using conventional methods such as ELISA and qPCR. Examples of markers for M2 macrophages include IL-10 and CD206. Conversely, the decrease or suppression of differentiation induction into M1 macrophages can also be confirmed. In this case, examples of markers for M1 macrophages include TNF-α and IL-12.

If the above induction method is applied, it can be easily confirmed whether a certain lactic acid bacterium has an M2 macrophage differentiation inducing action. That is, a person skilled in the art can contact an undifferentiated macrophage and a lactic acid bacterium to be tested according to a conventional method, analyze a marker of M2 macrophage, and confirm whether or not there is differentiation. For example, 10 ⁴ to 10 ⁸ cfu lactic acid bacteria may be contacted per 2 × 10 ⁵ cells of undifferentiated macrophages, and then IL-10 and CD206 may be confirmed by ELISA, qPCR, or the like. A person skilled in the art can also confirm by a high-throughput assay using a 96-well plate.

  The following examples are intended for illustration only and are not intended to limit the technical scope of the present invention.

  Unless otherwise specified, the materials and reagents are commercially available, or obtained or prepared according to methods commonly used in the art and procedures in known literature.

[Preparation of lactic acid bacteria]
In this example, Lactobacillus rhamnosus (Lactobacillus) is used as the lactic acid bacterium. rhamnosus) OLL2838 (Accession Number NITE P-313) was used. The lactic acid bacteria were cultured in MRS medium for 18 hours, washed with sterilized distilled water three times, and sterilized distilled water was added to prepare a cell suspension of 1 × 10 ⁸ cells / mL. The killed bacteria sterilized by heat-treating the cells at 75 ° C. for 1 hour were used in the following experiments.

[General experiment method]
Briefly describing ELISA, a specific protein in a sample is detected and quantified by using a highly specific antigen-antibody reaction and using color development / luminescence based on an enzyme reaction as a signal.

  In brief, quantitative PCR (qPCR) is one of quantitative PCR. By measuring the polymerase chain reaction (PCR) amplification over time (real time), the template DNA can be quantified based on the amplification rate. Is what you do.

[Induction of differentiation of macrophages]
Bone marrow mononuclear cells (BMs) collected from tibia and femur bones of Balb / c mice were obtained, cultured in the presence of macrophage colony stimulating factor (M-CSF) for 6 days, and differentiated into macrophages. Specifically, non-adherent cells were collected from the collected BMs, and cultured in a flask (1 × 10 ⁶ cells / ml) in the presence of M-CSF (added at 10 ng / mL on the first and third culture days). mL) and differentiated into bone marrow-derived M-CSF-dependent macrophages (Mφ).

For this macrophage, the CD11c + dendritic cells (DC) were removed using a magnetic cell separation method (MACS (registered trademark)) using CD11c MicroBeads, and 2 × 10 ⁵ cells were subdivided into 2 × 10 ⁵ cells. The above L. rhamnosus heat-killed cells were added and cultured for 24 hours. Subsequently, 10 ng / mL of interferon-γ (IFN-γ) or interleukin-4 (IL-4) was added thereto to differentiate the macrophages into M1 macrophages or M2 macrophages, respectively. The supernatant and cells after culture were collected, and related factors and markers such as IL-10, CD206, and TNF-α were analyzed by qPCR and ELISA.

[result]
The results are shown in FIGS. The symbol “HK-Lr” represents heat-sterilized L. rhamnosus. FIG. 1 shows the results of analyzing the production amount of IL-10 by ELISA. “IL-4” in FIG. 1 is obtained by adding only interleukin-4 without adding lactic acid bacteria, but the amount of IL-10 produced is less than 300 pg / mL. In contrast, when IL-4 and heat sterilized L. rhamnosus (HK-Lr) were added, the IL-10 production amount increased to 1000 pg / mL, and differentiation induction into M2 macrophages was confirmed. “None” in FIG. 1 is the case where neither IL-4 nor IFN-γ is added, but IL-10 is significantly increased even when only HK-Lr is added, Differentiation into M2 macrophages is induced. In addition, when IFN-γ was added, a significant difference in IL-10 production was observed depending on the presence or absence of HK-Lr. Similarly, differentiation induction into M2 macrophages was observed.

  FIG. 2 shows the results of analyzing the amount of IL-10 / β-actin by qPCR. β-actin is a gene that is expressed in a certain amount in common in many tissues and cells. By analyzing the expression level of IL-10 in comparison with this, the exact increase in the expression level of IL-10 is analyzed. be able to. Compared to the case of IL-4 alone, the IL-10 / β-actin ratio was significantly increased when IL-4 + HK-Lr was added, confirming that differentiation into M2 macrophages was induced. It was.

  FIG. 3 shows the result of qPCR analysis of CD206, which is a surface antigen marker of M2 macrophages. The numerical value on the vertical axis in the figure is the CD206 / β-actin ratio. FIG. 3 also confirmed that differentiation into M2 macrophages was induced by adding IL-4 and HK-Lr.

  FIG. 4 shows the result of qPCR analysis of TNF-α, which is a marker for M1 macrophages. The numerical value on the vertical axis in the figure is the TNF-α / β-actin ratio. TNF-α expression level when IL-4 is added is not high, but when IL-4 and HK-Lr are added, TNF-α is hardly expressed and differentiation into M1 macrophages is hardly induced. It was confirmed. In addition, a certain amount of TNF-α was expressed in the presence of IFN-γ, which promotes differentiation into M1 macrophages, but L. rhamnosus (HK-Lr) that had been heat-sterilized in the presence of IFN-γ was added. The expression level of TNF-α was significantly reduced. From the above, it was confirmed that HK-Lr according to the present invention suppresses differentiation induction into M1 macrophages or induces selective differentiation into M2 macrophages.

  By using the M2 macrophage differentiation inducer according to the present invention, undifferentiated macrophages obtained from bone marrow mononuclear cells can be selectively induced to differentiate into M2 macrophages. Further, by using the composition containing the differentiation inducer according to the present invention, the M1 / M2 balance of macrophages can be adjusted, which can be used for the treatment or prevention of diseases such as diabetes and arteriosclerosis.

NITE P-313

Claims (8)

  An agent for inducing differentiation of M2 macrophages comprising Lactobacillus rhamnosus as an active ingredient.   The differentiation inducing agent according to claim 1, wherein Lactobacillus rhamnosus is L. rhamnosus specified by accession number NITE P-313.   A method for inducing differentiation of M2 macrophages in vitro, comprising a step of using L. rhamnosus.   4. The differentiation induction method according to claim 3, wherein Lactobacillus rhamnosus is L. rhamnosus identified by the accession number NITE P-313.   A composition for inducing differentiation of M2 macrophages, comprising Lactobacillus rhamnosus as an active ingredient.   6. The composition of claim 5, wherein the Lactobacillus rhamnosus is L. rhamnosus identified by accession number NITE P-313.   The composition according to claim 5 or 6, which is a food composition.   7. A composition according to claim 5 or 6 which is a nutritional preparation.

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