ORAL COMPOSITION FOR ACTIVATING PLASMACYTOID DENDRITIC CELLS (pDCs)

Abstract

To provide an oral composition for effectively activating plasmacytoid dendritic cells (pDCs).SOLUTION: An oral composition for activating plasmacytoid dendritic cells (pDCs), comprising Lacticaseibacillus rhamnosus CRL1505, wherein the composition is preferably intended for immune activation. The oral composition for activating pDCs is considered to: regulate both innate immunity and acquired immunity; act on an overall immune function, maintain a normal immune function, and maintain good physical conditions. The present invention also provides an oral composition for immune activation characterised in that it comprises Lacticaseibacillus rhamnosus CRL1505, wherein the immune activation is intended for symptoms such as nasal congestion.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an oral composition for activating pDCs. [Background technology]

[0002] Dendritic cells (DCs) play an important role in the innate immune detection of pathogens and the subsequent activation of adaptive immune responses. Dendritic cells are broadly classified into myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). mDCs are well known for their ability to recognize foreign substances such as pathogens and transmit information about them to T cells, thereby activating foreign substance-specific immunity. On the other hand, pDCs are the main producers of type I interferon (IFN), which has antiviral activity, and play an important role in the body's antiviral defense.

[0003] Representative examples of type I IFNs include IFN-α, and their induction requires stimulation of Toll-like receptors (TLRs), specifically endosomal TLRs such as TLR9. pDCs highly express TLR7 and TLR9, which recognize viral nucleic acids, and upon recognition of viral infection, release large amounts of type I IFNs, including IFN-α and IFN-β. Type I IFNs activate the 2-5A system, a mechanism that inhibits viral replication, exerting a direct antiviral effect. Furthermore, pDCs can activate adaptive immune systems, including CD8+ T cells, CD4+ T cells, and B cells, which recognize and eliminate specific viruses from the body. Furthermore, type I IFNs produced by pDCs are essential factors for activating NK cells, which are part of the innate immune system, and regulate the early response to infection. Thus, pDCs play an essential role in regulating immune responses during both the early and late stages of viral infection.

[0004] It is known that certain lactic acid bacteria activate pDCs to produce IFN-α (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-073314 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an effective oral composition for activating pDCs. [Means for solving the problem]

[0007] The present inventors discovered that Lacticaseibacillus rhamnosus CRL1505 is effective in activating pDCs, and completed the present invention. Therefore, the present invention provides an oral composition for activating pDCs, which comprises Lacticaseibacillus rhamnosus CRL1505. The present invention also provides an oral composition for immunostimulation, characterized by containing Lacticaseibacillus rhamnosus CRL1505, wherein immunostimulation improves at least one symptom selected from "nasal congestion," "scratchy throat," "phlegm accumulation or chest tightness caused by phlegm," "hoarseness," "fuzzy head," "nausea or heartburn," "arthralgia," "muscle pain," "abdominal pain," "sensation of ear fullness or difficulty hearing," "lymph node pain," "fever," "fatigue," "chills," "loss of appetite," "dizziness or unsteadiness," "unspecified complaint," "malaise," and "sweating." [Effects of the Invention]

[0008] The present invention provides an oral composition for activating pDCs, which contains a specific lactic acid bacteria strain, and thereby activates pDCs and contributes to immunostimulation. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing the results of IFN-α gene expression evaluated in Example 1-1 and the like. [Figure 2] FIG. 2 is a graph showing the results of IFN-α gene expression evaluated in Example 2-1 and the like. [Figure 3] FIG. 3 is a graph showing the results of IFN-α gene expression evaluated in Example 3-1 and the like. [Figure 4] FIG. 4 is a graph showing the results of IFN-α gene expression evaluated in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below. The present invention is an oral composition for activating plasmacytoid dendritic cells (pDCs), which contains Lacticaseibacillus rhamnosus CRL1505 (hereinafter also referred to as "CRL1505") as an active ingredient.

[0011] Lacticaseibacillus rhamnosus CRL1505 used in the present invention is a lactic acid bacterium that can activate pDCs and promote IFN production by pDCs. This lactic acid bacterium strain was called "Lactobacillus rhamnosus CRL1505" before the reclassification published in 2020. The name after the reclassification published in 2020 is as above.

[0012] CRL1505 used in the present invention can exert an immunostimulatory effect on the living body when orally ingested. The above-mentioned lactic acid bacteria strain can be a commercially available product. CRL1505 has been deposited at the German Collection of Microorganisms and Cell Cultures (Deposit number DSM 29673).

[0013] The oral composition for activating pDCs of the present invention can induce Type I IFN (type I interferon) in pDCs. Type I IFN refers to cytokines that are effective against viral infections and includes IFN-α, IFN-β, etc. The oral composition for activating pDCs of the present invention activates pDCs. Activated pDCs produce IFNs such as Type I IFN. The oral composition for activating pDCs of the present invention is particularly potent at inducing Type I IFN production, particularly the production of Type I IFN-α. Type I IFNs such as IFN-α are known to activate NK cells and eliminate infected cells. IFN-α also induces the maturation of B cells into plasma cells (cells differentiated from B cells) and the production of immunoglobulins. Type I IFNs such as IFN-α also activate CD8+ T cells, killer T cells that play a central role in the innate immune system, and specifically kill infected cells and cancer cells. Furthermore, the oral composition for activating pDCs of the present invention activates pDCs and causes them to express molecules such as HLA-DR. HLA-DR is known to bind to peptides derived from antigenic proteins ingested from extracellular sources by phagocytosis and present antigens to naive helper T cells (CD4+ T cells). As a result, T cells are activated and proliferate, activating adaptive immunity. As described above, the oral composition for activating pDCs of the present invention can activate the entire body's immune functions, including the innate immune system and adaptive immune system, through pDC activation, while also suppressing excessive immune responses and maintaining a balance in immune responses in the body. Note that cells other than pDCs also exist that have functions such as expressing MHC II molecules or CD80 / 86 molecules. Furthermore, cells other than pDCs also exist that produce IFN-α. However, in the present invention, activating pDCs has the advantage that a single type of dendritic cell can exert the function of promoting multiple types of immune expression, thereby enabling an immune response to the invasion of pathogens such as viruses in a short period of time.

[0014] In particular, as described below, CRL1505 is thought to have effective immunoregulatory ability because it can effectively promote the expression of IFN-α in pDCs isolated from cells derived from intestinal Peyer's patches, peripheral blood, and bone marrow.

[0015] Furthermore, as shown in the evaluation results described below, CRL1505 induces the expression of the Mx1 gene in human peripheral blood mononuclear cells (PBMCs). Human PBMCs are composed of various blood cells, including T cells, B cells, NK cells (natural killer cells), monocytes, and dendritic cells. The expression of the Mx1 gene is not directly induced by viruses, but rather by type I IFNs such as IFN-α, and is known as an IFN-α marker. The Mx1 gene is also known to confer resistance to viruses such as influenza virus, and mice lacking the Mx1 gene are known to have reduced viral resistance. For example, the Mx1 gene is believed to inhibit the replication of viruses such as influenza virus, vesicular stomatitis virus, and yellow fever virus. (Ciancanelli MJ, Abel L, Zhang SY, Casanova JL. Host genetics of severe influenza: from mouse Mx1 to human IRF7. Curr Opin Immunol 2016; 38: 109-20, and Holzinger D, Jorns C, Stertz S, Boisson-Dupuis S, Thimme R, Weidmann M. et al. Induction of MxA gene expression by influenza A virus requires type I or type III interferon signaling. See Journal of virology 2007; 81: 7776-85) Therefore, it is thought that CRL1505 enhances the expression of IFN-α in pDCs, thereby enhancing the expression of the Mx1 gene and increasing resistance to various viruses in individuals.

[0016] The oral composition for pDC activation of the present invention contains a culture of CRL1505. The term "culture" refers to live cells, killed cells, disrupted live or killed cells, freeze-dried live or killed cells, disrupted freeze-dried cells, culture medium, culture medium extract, etc., and also includes parts of lactic acid bacteria and processed products of lactic acid bacteria. Here, processed lactic acid bacteria include, for example, lactic acid bacteria treated with enzymes or heat, or products recovered from such treated products by ethanol precipitation. Furthermore, lactic acid bacterial cultures also contain DNA or RNA of the above lactic acid bacteria. It is believed that the DNA or RNA of the above lactic acid bacteria can activate pDCs and induce IFN production. Killed CRL1505 is preferred for use in the oral composition for pDC activation due to its superior pDC activation ability. Among killed bacteria, heat-killed bacteria (killed bacteria sterilized by heat) are particularly preferred due to their superior pDC activation ability. Furthermore, in the present production method, it is preferable not to perform a disruption treatment of the bacterial cells, as this will result in a stable effect of activating pDCs, and it is particularly preferable not to perform a disruption or extraction treatment.

[0017] Lactic acid bacteria can be cultured by a known method using a known medium. Examples of the medium that can be used include MRS medium, GAM medium, and LM17 medium. The medium may be used by adding inorganic salts, vitamins, amino acids, antibiotics, serum, etc. as appropriate. The culture may be carried out at 25 to 40°C for several hours to several days.

[0018] The pDC activity of CRL1505 of the present invention can be confirmed, for example, by collecting pDCs from the blood after oral administration and observing that the decrease in HLA-DR expression is suppressed.

[0019] Oral ingestion of the oral composition for pDC activation of the present invention can achieve immunostimulation. For example, oral ingestion of the oral composition for pDC activation of the present invention can suppress one or more symptoms selected from "stuffy nose," "scratchy throat," "phlegm accumulation or chest tightness caused by phlegm," "hoarseness," "fuzzy head," "nausea or heartburn," "joint pain," "muscle pain," "abdominal pain," "ear fullness or difficulty hearing," "lymph node pain," "fever," "fatigue," "chills," "loss of appetite," "dizziness or unsteadiness," "unspecified complaint," "malaise," and "sweating." The present invention also provides oral compositions for immunostimulation that ameliorate such symptoms. "Sore throat" is a type of discomfort in the throat and includes the sensations of "a scratchy throat" and "a tickly throat." "Chest tightness due to phlegm accumulation or its associated pressure" refers to the feeling of phlegm building up in the throat, causing a blocked throat, or shortness of breath due to phlegm accumulation. "Hoarseness of voice" is also called "hoarseness of voice" and encompasses conditions such as a hoarse voice, being unable to speak, difficulty speaking, a raspy voice, or a weak voice. "Feeling dizzy" encompasses states such as not being able to think clearly, feeling foggy, feeling heavy, or not being able to think properly. "Muscle soreness" includes both exercise-associated and non-exercise-associated muscle soreness. "Ear congestion" refers to the feeling that something is stuck in the ear or that the ear is blocked. "Difficulty hearing" refers to the feeling that it is somehow difficult to hear sounds, as if you are hearing sounds through a thin membrane. "Indefinite symptoms" refers to "feeling unwell for some reason," including subjective symptoms such as feeling sluggish or fatigued. It does not matter whether or not you have seen a doctor or whether the cause has been identified. "Sweating" includes thermal sweating and emotional sweating. As will be shown in the Examples below, the oral composition for activating pDCs of the present invention is particularly useful for immunostimulation, as it can effectively alleviate and / or prevent each of the above symptoms.

[0020] The oral composition for activating pDC of the present invention can be used in mammals such as humans, dogs, and cats, and is particularly preferable for humans in that it has an excellent effect of promoting IFN-α expression and is particularly expected to have an antiviral effect.

[0021] The oral composition for activating pDCs of the present invention is preferably taken orally continuously, for example, preferably for two or more consecutive weeks, more preferably for four or more consecutive weeks, and particularly preferably for eight or more consecutive weeks. The number of times of intake per week is more preferably one or more times.

[0022] The oral composition for activating pDCs of the present invention can be used as a pharmaceutical that activates pDCs and enhances immune activity in the body. This pharmaceutical can be used as a preventive or therapeutic agent for cancer (kidney cancer, blood cancer, brain cancer, skin cancer, etc.), viral infections (coronavirus, influenza virus, HTLV-I, hepatitis B, hepatitis C, vesicular stomatitis virus, etc.), bacterial infections (plague, tuberculosis, diphtheria, cholera, bacterial shigellosis, enterohemorrhagic Escherichia coli infection, typhoid fever, etc.), and autoimmune diseases (collagen diseases (rheumatoid arthritis, systemic lupus erythematosus, etc.), polymyositis, polyangiitis, etc.). The pharmaceutical is particularly useful as a preventive or therapeutic agent for viral infections.

[0023] The oral composition for activating pDCs of the present invention may be in any form, including, for example, tablets, capsules, powders, granules, liquids, particles, rods, plates, blocks, rounds, pastes, creams, caplets, gels, chewable tablets, and sticks.

[0024] The oral composition for activating pDC may contain an excipient, a lubricant, a fluidizing agent, a coloring agent, and the like. Examples of excipients include starch or derivatives thereof such as starch, pregelatinized starch, partially pregelatinized starch, and starch hydrolysates, crystalline cellulose, powdered cellulose, sugar alcohol, lactose, brewer's yeast, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, refined sucrose, light anhydrous silicic acid, calcium silicate, titanium oxide, precipitated calcium carbonate, maltose, and maltose syrup. As the lubricant, calcium stearate and sucrose fatty acid esters are preferably used from the viewpoint of moldability and storage stability. A fluidizing agent is used to improve the fluidity of mixed powders or granules. There are no particular limitations on the fluidizing agents that can be used in the present invention, and examples include silicon dioxide, aluminum silicate, magnesium aluminosilicate, calcium phosphate, magnesium carbonate, magnesium oxide, etc.

[0025] Furthermore, the oral composition for pDC activation of the present invention can be incorporated into food and beverages, and by incorporating it into food and beverages, the food and beverages can be used as food and beverages for pDC activation. The types of food and beverages that can be used are not particularly limited as long as they do not inhibit the active ingredients for pDC activation. Examples of such foods and beverages include milk and dairy products; beverages such as soft drinks, fruit juice drinks, dairy drinks, alcoholic drinks, sports drinks, and energy drinks; seasonings; alcoholic beverages; processed agricultural and forestry products; confectioneries and breads; grain flour and noodles; processed seafood products; processed livestock products; oils and fats; prepared frozen foods; retort foods; instant foods; and food ingredients.

[0026] In the present invention, from the viewpoint of facilitating continuous intake for the purpose of improving immunity, it is preferably used as an oral composition such as a supplement, food or drink. The supplement may be in the form of, for example, a tablet, capsule, powder, granule, or liquid, but from the viewpoint of enabling more efficient intake of CRL1505, a tablet, capsule, or granule form is preferred, with a tablet or granule form being particularly preferred.

[0027] The food and drink products of the present invention include health food and drink products, foods for specified health use, foods with functional claims, nutritional functional food and drink products, health supplementary food and drink products, etc. These food and drink products may be labeled to indicate activation of pDC. Specifically, for example, advertising and promotion using expressions such as "acts on pDC (plasmacytoid dendritic cells) and helps maintain the immune function of healthy people" and "assists the function of pDC (plasmacytoid dendritic cells) and helps maintain the immune function of healthy people" are possible, and these advertising and promotion can be regarded as the implementation of the present invention.

[0028] From the perspective of continuously ingesting and activating human pDC to activate the immune system, the number of CRL1505 bacterial cells contained in the oral composition of the present invention is preferably 1×10 3 or more in terms of the number of bacteria in the daily intake of the composition, more preferably 1×10 5 or more, and even more preferably 1×10 7 or more. 7 From the perspective of continuously ingesting and activating human pDC to activate the immune system, the number of CRL1505 bacterial cells contained in the oral composition of the present invention is preferably 1×10 Also, the upper limit of the CRL1505 is not particularly limited, but since there may be a possibility that the expected effect cannot be obtained according to the intake amount, it may be 1×10 14 or less, further 1×10 12 or less, 1×10 12 d>or less. 12 10 From the perspective of continuously ingesting and activating human pDC to activate the immune system, the number of CRL1505 bacterial cells contained in the oral composition of the present invention is preferably 1×10 The number of bacterial cells can be measured by the method described below.

[0029] <Measurement method of CRL1505 bacterial cell count> [Preparation of sample solution] (1) After weighing 0.1 g of the composition (hereinafter referred to as the specimen), add phosphate buffered saline with an emulsifier so that the specimen is diluted 100-fold, and mix to obtain a mixed solution. The specimen to be weighed should be pretreated optimally according to the specimen, for example, if it is a tablet, it should be powdered with a mortar and used. The emulsifier is selected to be able to suppress the aggregation of CRL15o5 bacterial cells. (2) Next, homogenize the bacterial cells in the mixed solution by vortex mixer treatment and ultrasonic treatment to obtain a sample stock solution. The conditions for vortex mixer treatment and ultrasonic treatment are set such that the lactic acid bacterial cells are not destroyed and the bacterial cells are separated from impurities and the like. (3) Confirm by microscopic observation that there is no aggregation of lactic acid bacteria in the sample solution. (4) The sample stock solution is further diluted with the emulsifier-added phosphate buffered saline used in (1) to prepare a measurement sample. The dilution is adjusted so that the number of bacteria per image is approximately 50 to 100 in the DAPI staining method described below.

[0030] [DAPI staining method] (1) Filter 1 mL of the sample solution to be measured through a 0.2 μm membrane filter. (2) The filtered sample is reacted with 1 mL of DAPI solution for 1 minute, and then filtered in the same manner. (3) Images of the lactic acid bacteria captured on the membrane filter are taken using an epifluorescence microscope at a magnification of 400x, and 25 fields of view are taken evenly across the entire filtration area of ​​the membrane filter to reduce variations in measurement location. (4) Count the number of lactic acid bacteria in the captured image and calculate the average number of lactic acid bacteria per image. For chains of bacteria, count the number of individual bacteria that make up the chain. Also, count only bacteria that maintain their shape, excluding crushed bacteria. (5) Calculate the number of bacteria in the sample intake per day using the following formula. Number of bacteria (per daily intake) = Average number of bacteria per image × Area of ​​the filtration range of the membrane filter ÷ Area of ​​one image × Dilution ratio ÷ Filtration volume × Daily intake of the sample

[0031] From the viewpoint of immunostimulation, the CRL1505 contained in the oral composition of the present invention is contained in an amount of preferably 0.01 to 90 mass %, more preferably 0.1 to 50 mass %, and even more preferably 1 to 25 mass %, of the solid content of the oral composition of the present invention. [Example]

[0032] The present invention will be described below based on examples, but the present invention is not limited to the following examples.

[0033] [Examples 1-1, 1-2 and Comparative Example 1] To investigate the effects of CRL1505 intake in healthy mice, an animal study was conducted using mice.

[0034] <Test Method> (1) Test substance As the test substance, heat-killed CRL1505 cells (sold by Sacco Systems, Inc.; the same applies to the following examples) that had not been subjected to disruption or extraction were used. (2)Animals (2-1) Animals used Male C57BL / 6J mice (Japan SLC) were used. They arrived at 7 weeks of age and were allowed to acclimate for one week before being used in the study at 8 weeks of age (body weight 16-19 g). (2-2) Animal care Each mouse was housed in a polycarbonate cage (manufactured by CLEA Japan, 172mm x 240mm x 129mm). The light period was from 8:00 to 20:00, with a 12-hour light-dark cycle. During the acclimation period, the mice were fed MF powdered feed (manufactured by Oriental Yeast Co., Ltd.), and during the test period, they were given free access to homemade feed and tap water. (3) Administration of the test substance CRL1505 was mixed into the MF powdered feed at 0.1% by mass (Example 1-1 (low dose)) and 1% by mass (Example 1-2 (high dose)). No CRL1505 was mixed into the control. These powdered feeds were placed in feeders and placed in each cage, and the mice were fed daily.

[0035] (4) Evaluation of immune activation (4-1) Sample collection On day 14 of the experiment, intestinal Peyer's patches were excised from the mice under isoflurane anesthesia. (4-2) Sample pretreatment Intestinal-derived Peyer's patches were minced in HBSS (balanced salt solution containing 5% FBS and 1 mg / mL collagenase IV) and cultured at 37°C for 1 hour with shaking. The intestinal-derived Peyer's patch solution was passed through a 70 μm strainer and centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was removed, and the tissue suspension resuspended in 37% Percoll was gently layered on top of 70% Percoll. After centrifugation at 800 × g for 20 minutes at 4°C, the interphase was collected. 2 mM EDTA-0.5% FBS-PBS was added, mixed, and centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was removed, and the same process was repeated. The tissue was resuspended in 2 mM EDTA-0.5% FBS-PBS and pDCs were isolated using the Plasmacytoid Dendritic Cell Isolation Kit for mouse (Milteny Biotec). (4-3) Analysis of gene expression levels RNA was isolated from pDCs isolated from intestinal Peyer's patches using an RNeasy Mini Kit (QIAGEN), and quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (Takara). Gene expression levels were analyzed using IFN-α primers (QIAGEN). Additionally, as an endogenous control, Actb (also known as β-actin) primers (QIAGEN) were used to measure the gene expression level of Actb, and the ratio of IFN-α gene expression level to Actb gene expression level was calculated. The relative value of the IFN-α gene expression level ratio, with the control value set at 1, was calculated, and the average value was calculated.

[0036] <Test Results> The results of the obtained IFN-α gene expression levels are shown in Figure 1. Compared with the control (Comparative Example 1), a significant increase in the IFN-α expression level was observed in the CRL1505 low-dose group (Example 1-1) and the CRL1505 high-dose group (Example 1-2). CRL1505 promoted IFN-α production in Peyer's patches in mice, suggesting that CRL1505 activates pDCs.

[0037] [Examples 2-1 to 2-3, Reference Example 1 and Comparative Example 2] In this study, we evaluated the gene expression level of type I interferon α (hereinafter referred to as IFN-α) in pDCs isolated from mouse bone marrow-derived dendritic cells.

[0038] <Test Method> (1) Test substance The test substance used was heat-killed CRL1505 cells that had not been disrupted or extracted. As a positive control (Reference Example 1), a TLR9 ligand, CpG-A ODN 2336 (hereinafter referred to as ODN2336) (manufactured by Eurofins Genomics) was used. (2)Cell culture (2-1) Cell Murine bone marrow-derived dendritic cells were used. (2-2)Culture Bone marrow mononuclear cells were collected from the bone marrow of the femur and tibia of 10-week-old male C57BL / 6J mice (Japan SLC) and cultured for 8 days in a 5% CO2 incubator at 37°C using 100 μM 2-mercaptoethanol, 100 ng / mL Flt3 ligand, and 10% FBS-RPMI (hereinafter also referred to as "differentiation induction medium"). The medium was changed every 3 to 4 days, and the cells were induced to become bone marrow-derived dendritic cells (hereinafter also referred to as "BMDCs"). On the 8th day of culture, pDCs were isolated using the EasySep™ Mouse Plasmacytoid DC Isolation Kit (STEMCELL) and seeded onto 96-well U-bottom plates. The seeding density was 7.0 × 10 3 The number of cells / well was 100.

[0039] (3) Evaluation of pDC activation ability (3-1) Preparation of test substance solution CRL1505 and ODN2336 were diluted in differentiation-inducing medium containing 0.2 mg / mL kanamycin sulfate and 0.5 μg / mL amphotericin B. CRL1505 was diluted at 7.0 × 10 3 cells / well (Example 2-1), 7.0 × 10 4cells / well (Example 2-2), 7.0 × 10 6 The cells were prepared at 100 μL / well (Examples 2-3). ODN2336 was prepared at 7 μg / mL (Reference Example 1). All concentrations are final concentrations. As a control (Comparative Example 2), a differentiation-inducing medium containing 0.2 mg / mL kanamycin sulfate and 0.5 μg / mL amphotericin B was used. In each example, the liquid volume per well was 100 μL. (3-2) Addition of test substance The test substance solution prepared above was added to the wells seeded with the cells, and the cells were cultured in a 5% CO 2 incubator at 37° C. for 20 hours.

[0040] (3-3) Measurement of IFN-α gene expression level RNA was extracted from mouse BMDC-derived pDCs cultured for 20 hours using an RNeasy Mini Kit (QIAGEN), and quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (Takara). IFN-α gene expression levels were measured using IFN-α primers (QIAGEN). Furthermore, as an endogenous control, Rn18s gene expression levels were measured using Rn18s primers (QIAGEN), and the ratio of IFN-α gene expression levels to Rn18s gene expression levels was calculated. The relative values ​​of the IFN-α gene expression levels were calculated by setting the value of the control (Comparative Example 2) to 1, and the average values ​​were calculated.

[0041] <Test Results> The results are shown in Figure 2. Compared with the control (Comparative Example 2), the IFN-α gene expression level was significantly increased in the CRL1505 group (Examples 2-1, 2-2, and 2-3) and the ODN2336 group (Reference Example 1). Increased expression of the IFN-α gene was observed in the CRL1505 groups (Examples 2-1, 2-2, and 2-3), suggesting that CRL1505 directly activates mouse BMDC-derived pDCs.

[0042] [Examples 3-1 to 3-2, Reference Example 2, Comparative Example 3] In this study, to evaluate the ability of CRL1505 to activate pDCs, the gene expression level of IFN-α in human peripheral blood mononuclear cells, including pDCs, was assessed.

[0043] <Test Method> (1) Test substance The test substance used was heat-killed CRL1505 cells that had not been disrupted or extracted. As a positive control, CpG-A ODN 2336 (hereinafter referred to as ODN2336) (manufactured by Eurofins Genomics), a TLR9 ligand, was used. (2)Cell culture (2-1) Cell Human peripheral blood mononuclear cells (hereinafter also referred to as "PBMCs"; manufactured by iQ Biosciences) were used. (2-2)Culture Human PBMCs were seeded in 96-well U-bottom plates using 10 ng / mL IL-3-10% FBS-RPMI1640 medium and pre-cultured for 24 hours in a 37°C, 5% CO2 incubator. The seeding density was 6.0 × 10 5 The number of cells / well was 100.

[0044] (3) Evaluation of pDC activation ability (3-1) Preparation of test substance solution CRL1505 and ODN2336 were diluted in 10 ng / mL IL-3-10% FBS-RPMI1640 medium. CRL1505 was diluted at 1.0 × 10 4 cells / well (Example 3-1) and 1.0 × 10 7 The cells were prepared at 1000 cells / well (Example 3-2). ODN2336 was prepared at 10 μg / mL (Reference Example 2). All concentrations are final concentrations. As a control (Comparative Example 3), 10 ng / mL IL-3-10% FBS-RPMI1640 medium was used. In each example, the liquid volume per well was 200 μL. (3-2) Addition of test substance The test substance solution prepared above was added to the wells seeded with the cells, and the cells were cultured in a 5% CO 2 incubator at 37° C. for 48 hours.

[0045] (3-3) Measurement of IFN-α gene expression level RNA was isolated from human PBMCs cultured for 48 hours using an RNeasy Mini Kit (QIAGEN), and quantitative real-time PCR was performed using a One Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (Takara). IFN-α gene expression levels were measured using IFN-α primers (QIAGEN). Furthermore, as an endogenous control, GAPDH gene expression levels were measured using GAPDH primers (QIAGEN), and the ratio of IFN-α gene expression levels to GAPDH gene expression levels was calculated. The relative values ​​of the IFN-α gene expression levels were calculated by setting the value of the control (Comparative Example 3) to 1, and the average values ​​were calculated.

[0046] <Test Results> The results are shown in Figure 3. Compared with the control (Comparative Example 3), the expression level of the IFN-α gene was significantly increased in the CRL1505 group (Examples 3-1 and 3-2) and the ODN2336 group (Reference Example 2). Increased expression of the IFN-α gene was observed in the CRL1505 group (Examples 3-1 and 3-2), suggesting that CRL1505 directly activates pDCs in human PBMCs.

[0047] [Example 4, Reference Example 3 and Comparative Example 4] In this study, to evaluate the pDC activation ability of CRL1505, the gene expression level of IFN-α in pDCs isolated from human BMDCs was assessed.

[0048] <Test Method> (1) Test substance The test substance used was heat-killed CRL1505 cells that had not been disrupted or extracted. As a positive control, CpG-A ODN 2336 (hereinafter referred to as ODN2336) (manufactured by Eurofins Genomics), a TLR9 ligand, was used. (2)Cell culture (2-1) Cell Human bone marrow mononuclear cells (LONZA) were used. (2-2)Culture Human bone marrow mononuclear cells (manufactured by LONZA) were used and cultured for 7 days in a 5% CO2 incubator at 37°C using 100 ng / mL Flt3 ligand-10% FBS-DMEM (hereinafter also referred to as "differentiation induction medium"). The medium was changed once every 3 days to induce BMDCs. On the 7th day of culture, pDCs were isolated using the EasySep™ Human Plasmacytoid DC Enrichment Kit (manufactured by STEMCELL) and seeded onto 96-well U-bottom plates. The seeding density was 1.0 x 10 4 The number of cells / well was 100.

[0049] (3) Evaluation of pDC activation ability (3-1) Preparation of test substance solution CRL1505 and ODN2336 were diluted in differentiation-inducing medium containing 0.2 mg / mL kanamycin sulfate and 0.5 μg / mL amphotericin B. CRL1505 was diluted at 1.0 × 10 7 The cells were prepared at 100 μL / well (Example 4). ODN2336 was prepared at 10 μg / mL (Reference Example 3). All concentrations are final concentrations. As a control (Comparative Example 4), a differentiation-inducing medium containing 0.2 mg / mL kanamycin sulfate and 0.5 μg / mL amphotericin B was used. In each example, the liquid volume per well was 100 μL. (3-2) Addition of test substance The test substance solution prepared above was added to the wells seeded with the cells, and the cells were cultured in a 5% CO 2 incubator at 37° C. for 24 hours.

[0050] (3-3) Measurement of IFN-α gene expression level RNA was extracted from human BMDC-derived pDCs cultured for 24 hours using an RNeasy Mini Kit (QIAGEN), and quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (Takara). IFN-α gene expression levels were measured using IFN-α primers (Eurofins Genomics). Furthermore, as an endogenous control, GAPDH gene expression levels were measured using GAPDH primers (QIAGEN), and the ratio of IFN-α gene expression levels to GAPDH gene expression levels was calculated. The relative values ​​of the IFN-α gene expression levels were calculated by setting the value of the control (Comparative Example 4) to 1, and the average was calculated.

[0051] <Test Results> The results are shown in Figure 4. Compared with the control (Comparative Example 4), the expression level of the IFN-α gene was significantly increased in the CRL1505 group (Example 4) and the ODN2336 group (Reference Example 3). Increased expression of the IFN-α gene was observed in the CRL1505 group, suggesting that CRL1505 directly activates human BMDC-derived pDCs.

[0052] [Example 5, Comparative Example 5] Focusing on the effects of CRL1505 on the immune system, we conducted a placebo-controlled, randomized, double-blind, parallel-group comparative study in healthy adult men and women to evaluate pDC activation, which is important for maintaining immune function, and the occurrence of cold-like symptoms as an indicator of maintaining good health. <Method for assessing the presence or absence of cold-like symptoms> (1) Test food The test food (Example 5) was prepared by mixing heat-killed Lactobacillus rhamnosus CRL1505 cells (CRL1505) with powdered reduced maltose syrup, powdered cellulose, calcium stearate, and fine silicon dioxide, and then compressing the mixture into tablets. The control food (Comparative Example 5) was prepared by replacing the CRL1505 in the test food with powdered reduced maltose syrup to make it indistinguishable from the test food in appearance. Both the test food and the control food were individually packaged in plain aluminum foil, containing the recommended daily intake of one tablet (0.25 g / tablet), ensuring blinding for the study subjects and interventionists. The calorie and nutritional values ​​per recommended daily intake (one tablet) of the test food are shown in Table 1.

[0053] [Table 1]

[0054] The number of CRL1505 bacteria contained in the test food was 880 million per recommended daily intake (1 tablet). (2) Subjects The subjects were 80 people (40 men and 40 women) aged 20 years or older and under 65 years old. Exclusion criteria for subject selection included the following: Those who suffer from, are undergoing treatment for, or have a history of serious diseases such as diabetes, kidney / liver disease, heart disease, thyroid disease, adrenal gland disease, or other metabolic diseases; those with chronic diseases and regular medication use; those with digestive diseases or a history of digestive surgery that affect digestion and absorption; those who have not experienced any symptoms of upper respiratory tract infection in the last two winters; those who suffer from hay fever or allergic rhinitis and are taking medication; those who cannot stop taking supplements or health foods (including functional foods) that may affect the immune system; those who consume foods containing lactic acid bacteria or bifidobacteria more than twice a week; those who have a drinking habit of more than approximately 60g of pure alcohol per day or those who cannot abstain from alcohol during the exam period; smokers; those who have a habit of intense exercise such as running or soccer;

[0055] (3) Test method This study was conducted as a placebo-controlled, randomized, double-blind, parallel-group comparative study (allocation ratio: 1:1) for a total of 10 weeks, consisting of a pre-observation period (2 weeks) and an intake period (8 weeks). Participants were allocated using a stratified block randomization method (block size: 4) with gender, age, and pDC activity as adjustment factors. A controller not directly involved in the study assigned the two groups to the test food group and the control food group. During the test period, subjects were instructed to take one tablet of the test food (test food in the test food group, control food in the control food group) once a day with water or lukewarm water for eight consecutive weeks. (4) Inspection items pDC activity was assessed by measuring the expression levels of pDC activity markers, HLA-DR and CD86, in peripheral blood mononuclear cells (PBMCs) at three time points: a pre-intake test (hereinafter also referred to as "pre-intake"), a 4-week post-intake test (hereinafter also referred to as "4-week post-intake"), and an 8-week post-intake test (hereinafter also referred to as "8-week post-intake"). PBMCs were isolated from blood and stained with CD123, CD304, HLA-DR, and CD86 antibodies, followed by flow cytometry analysis. CD123- and CD304-positive cells were defined as pDCs, and the mean fluorescence intensity (MFI) of HLA-DR and CD86 was calculated.

[0056] Regarding the physical condition questionnaire, questionnaires for local and general symptoms were created and evaluated. Subjects were asked to record in a daily diary the following subjective symptoms during the study: runny nose, stuffy nose, sneezing, cough, sore throat (pain, irritation), phlegm buildup and resulting chest tightness, hoarseness, headache, lightheadedness, nausea, heartburn, joint pain, muscle pain, abdominal discomfort (abdominal pain, diarrhea), ear congestion, difficulty hearing, and pain in the lymph nodes. Subjects were also asked to record the following subjective symptoms during the study: fever, fatigue, chills, loss of appetite, dizziness, lightheadedness, palpitations, shortness of breath, general feeling unwell, lethargy, and sweating. Symptoms were chosen from five levels: (1) severe symptoms, (2) moderate symptoms, (3) slight symptoms, (4) very slight symptoms, and (5) no symptoms at all. In addition, the subjects were given a food diary and a research subject diary, and were asked to fill out the following survey items every day from two weeks before the start date of intake throughout the intake period. Survey items: (1) Test food intake status, (2) Changes in lifestyle, (3) Visits to the hospital, (4) Menstruation, (5) Bowel movements, (6) Medication use, (7) Dietary content Regarding safety, adverse events were assessed through physical examinations, blood tests, urine tests, study subject diaries, and physician interviews.

[0057] (5) Statistical analysis The primary endpoint was pDC activity, and the secondary endpoint was a questionnaire on health. The analysis population was the per-protocol set (PPS). For pDC activity, a repeated-measures two-way analysis of variance was performed. If an interaction between group and time point was observed, the actual measured values ​​and the change from pre-intake were compared between groups using an unpaired t-test. For the health questionnaire, (1) severe symptoms, (2) moderate symptoms, (3) slight symptoms, and (4) very slight symptoms were defined as "symptoms present," and (5) no symptoms at all were defined as "no symptoms." The cumulative number of days with symptoms during the intake period for each group was calculated, and inter-group comparisons were performed using a chi-square test. The significance level for all tests was 5%, and statistical analysis was performed using statistical software (IBM: SPSS Statistics 28). Participant characteristics were presented as mean ± standard deviation, and other statistical data were presented as mean ± standard error.

[0058] <Test Results> (1) Subjects of analysis Each subject completed the test, and 20 subjects were excluded from the analysis due to changes in lifestyle, etc., leaving 60 subjects (29 men and 31 women) for analysis. The background of the subjects is shown in Table 2.

[0059] [Table 2]

[0060] (2) HLA-DR expression level, CD86 expression level The analysis results of the expression levels of HLA-DR, a pDC activity marker, are shown in Table 3, and the analysis results of the expression levels of CD86 are shown in Table 4. [Table 3] [Table 4]

[0061] Because an interaction was observed in HLA-DR expression levels, group comparisons were performed for each test, based on the actual measured values ​​and the change from pre-intake. The test food group (Example 5) showed significantly higher changes after 4 and 8 weeks of ingestion than the control food group (Comparative Example 5). Because HLA-DR is a pDC activation marker, it is believed that pDC activity was maintained by ingestion of CRL1505. While no significant differences in CD86 were observed between groups, previous studies have not consistently shown the behavior of all pDC activation markers, and changes in any marker are linked to physical symptoms. Therefore, we cannot rule out the activation of pDCs by the oral composition of the present invention.

[0062] (3) Health questionnaire The results of the questionnaire regarding physical condition are shown in Table 5. [Table 5]

[0063] The cumulative number of days during the intake period for each of the following symptoms was significantly lower in the test food group (Example 5) compared to the control food group (Comparative Example 5): "stuffy nose," "uncomfortable throat (irritation)," "phlegm accumulation and resulting chest pressure," "hoarseness," "fuzzy head," "nausea / heartburn," "joint pain," "muscle pain," "abdominal discomfort (stomach pain)," "ear fullness / difficulty hearing," "lymph node pain," "fever," "fatigue," "chills," "loss of appetite," "dizziness / lightheadedness," "general feeling of unwellness," "malaise," and "sweating." These results suggest that CRL1505 activates pDC, thereby improving or preventing cold-like symptoms and maintaining good physical condition.

[0064] These results suggest that ingested CRL1505 directly acts on and activates pDCs, thereby regulating both innate and adaptive immunity, affecting the overall immune system, and maintaining normal immune function and overall health.

[0065] <Method for evaluating increased gene expression> As an efficacy test, increased expression of the IFN-α gene, which is an indicator of pDC activity, and the Mx1 gene, which is an indicator of antiviral activity, was confirmed by the following method.

[0066] (1) Test food The test food used was the test food used in Example 5. The control food used in Comparative Example 5 was used.

[0067] (2) Subjects The subjects were 80 healthy adult men and women (40 men and 40 women) aged 20 years or older and under 65 years old. The subjects were the same as those in the <Evaluation method for the presence or absence of cold-like symptoms>.

[0068] (3) Test method This study was conducted as a placebo-controlled, randomized, double-blind, parallel-group comparative study (allocation ratio: 1:1) for a total of 10 weeks, consisting of a pre-observation period (2 weeks) and an intake period (8 weeks). Participants were allocated using a stratified block randomization method (block size: 4) with gender, age, and pDC activity as adjustment factors. A controller not directly involved in the study assigned the two groups to the test food group and the control food group. During the test period, subjects were instructed to take one packet (one tablet) of the test food (test food for the test food group, control food for the control food group) once a day with water or lukewarm water. This method is the same as the test method (3) in <Method for evaluating the occurrence of cold-like symptoms>.

[0069] (4) Inspection method In human peripheral blood mononuclear cells (PBMCs) cultured in the presence of inactivated influenza A virus antigen, the expression levels of the IFN-α gene as an indicator of the pDC activation-inducing effect and the Mx1 gene as an indicator of the antiviral activity-inducing effect were measured by quantitative real-time PCR.

[0070] Blood samples were collected three times: a pre-intake test (hereafter referred to as pre-intake), a test four weeks after ingestion (hereafter referred to as four weeks after ingestion), and a test eight weeks after ingestion (hereafter referred to as eight weeks after ingestion), and PBMCs were prepared. PBMCs were cultured at a concentration of 1.0 × 10 in RPMI 1640 medium (SIGMA) containing 1 mM sodium pyruvate (Fujifilm Wako Pure Chemical Industries, Ltd.), 2.5 mM HEPES (Dojindo Laboratories, Ltd.), 100 U / mL penicillin-streptomycin (Nacalai Tesque, Ltd.), 50 μM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% fetal bovine serum. 6The medium was adjusted to a concentration of 1.0 μg / mL and 1 mL of each was seeded onto a 24-well plate. Inactivated influenza A virus antigen (Influenza A H1N1 Virus Antigen, HyTest, 8IN73) was added to the medium at a concentration of 1.0 μg / mL. After 24 hours of culture, the cells were harvested. Total RNA was extracted from the cells using an RNeasy Mini Kit (QIAGEN), and the gene expression levels of IFN-α and Mx1 were measured by quantitative real-time PCR using the One Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (Takara Bio).

[0071] The primers used were the IFN-α gene: Hs_IFNA1_1_SG QuantiTect Primer Assay (QIAGEN). The primers used for Mx1 were PCReady™ primers (Forward 5'-GCAAATCAAGGCACTGGAAG-3', Reverse 5'-TCGAAACATCTGTGAAAGCAAG-3') (Eurofins Genomics). The β-Actin gene was used as an internal standard for relative comparison. The primers used for the β-Actin gene were Hs_ACTB_1_SG QuantiTect primer assay (QIAGEN).

[0072] (5) Data processing and statistical analysis A repeated measures two-way analysis of variance was performed, and if an interaction between group and time point was observed, an unpaired t-test was used to compare the actual measured values ​​of each test and the change from before intake. A risk level of less than 5% was considered significant, and statistical analysis was performed using the statistical analysis software IBM SPSS Statistics 28. Missing data were treated as missing values, and statistical data were presented as mean ± standard error.

[0073] <Test Results> (1) Subjects of analysis The study enrolled 80 subjects (40 men and 40 women), with no dropouts after randomization, and the study began with 80 subjects. No subjects dropped out during the study period, and 80 subjects completed the study. After the study was completed, some subjects were excluded due to changes in alcohol consumption, exercise, or lifestyle habits during the study period, so samples were collected from n=33 subjects in the control food group and n=27 subjects in the test food group. Data from some subjects was further excluded due to insufficient sample volume, and the final results for 45 subjects (n=25 in the control food group and n=20 in the test food group) were statistically analyzed and are shown in Table 6. When evaluating the effect of inducing pDC activation, an interaction was observed in the amount of IFN-α expression. Therefore, a comparison was made between groups for each test on the actual measured values ​​and the amount of change from before intake. The actual measured values ​​and amount of change after 8 weeks of intake were significantly higher in the test food group than in the control food group (P < 0.05). When evaluating the antiviral activity induction effect, an interaction was observed in the Mx1 expression level. Therefore, a comparison was made between groups for each test on the actual measured values ​​and the change from before intake. The actual measured values ​​and change after 8 weeks of intake were significantly higher in the test food group than in the control food group (P < 0.05). Furthermore, when the expression levels of both genes after 8 weeks of intake were compared across all subjects, including those for whom data before intake was missing, the test food group showed significantly higher levels than the control food group (control food group n=33, test food group n=27, IFN-α: P < 0.05, Mx1: P < 0.05).

[0074] [Table 6]

[0075] <Consideration> In this study, the effects of CRL1505 on pDC activity and antiviral activity were investigated in a placebo-controlled, randomized, double-blind, parallel-group comparative study in which healthy adult men and women were asked to take a CRL1505-containing food (test food) or a CRL1505-free food (control food) once daily for eight consecutive weeks. In assessing the effect of inducing pDC activation, IFN-α gene expression in PBMCs was maintained at a significantly higher level in the test food group compared to the control food group. pDCs are the major type I IFN-producing immune cells, and approximately 60% of the transcriptome of activated pDCs is type I IFN gene expression. Furthermore, activated pDCs are known to produce 200-1000 times more IFN-α than other immune cells, accounting for more than 95% of type I IFN production in PBMCs. Therefore, the significantly elevated IFN-α gene expression in PBMCs following test food ingestion is thought to be due to pDC activation.

[0076] Taking these results into consideration, CRL1505 appears to contribute to maintaining immune function in healthy individuals by activating pDCs and inducing the activation of various immune cells through the production of type I IFN. In assessing the antiviral activity induction effect, Mx1 gene expression in PBMCs was maintained at a significantly higher level in the test food group compared to the control food group. As mentioned above, Mx1 expression is not directly induced by viruses, but is induced by type I IFNs such as IFN-α, and its function is to inhibit viral replication. Therefore, the significantly elevated Mx1 gene expression in PBMCs following ingestion of the test food is thought to be due to IFN-α produced by pDCs. Furthermore, Mx1 is expressed not only in immune cells but also in non-immune cells (tissue cells) and plays an antiviral role. These findings suggest that IFN-α produced by pDCs may inhibit the spread of viral infection in the body by inducing Mx1 gene expression not only in immune cells but also in non-immune cells (tissue cells).

[0077] As described above, subjects who showed increased expression of both the IFN-α gene, the most potent indicator of pDC activation, and the Mx1 gene, an indicator of antiviral activity, were observed to maintain their physical condition (<Method for assessing the onset of cold-like symptoms>). These findings suggest that in vivo, pDCs activated by CL1505 maintain normal overall immune function by inducing activation of various immune cells via type I IFN production, thereby preventing the onset of cold-like symptoms caused by pathogen infection and alleviating these symptoms, thereby maintaining and promoting health.

[0078] <Production example> Based on the results of the Examples, the following Production Examples of the present invention are given below.

[0079] [Production Example 1-4: Granules] According to the formulation in Table 7, CRL1505 and other raw materials were mixed, and then fluidized bed granulation was performed using a granulator to produce the granules described in Production Examples 1-4. The granules described in Production Examples 1-4 can be taken daily at a dose of 3 g by dissolving in 100 ml of water, or can be taken as is without dissolving. All of the granules in Production Examples 1-4 activate pDC and are effective in immunostimulation.

[0080] [Table 7]

[0081] [Manufacturing Example 5-8: Tablets] According to the formulation in Table 8, CRL1505 and other raw materials were mixed and then tableted using a rotary tablet press to produce tablets (250 mg) of Production Example 5-8. Two tablets of Production Example 5-8 can be taken daily with water or the like. All tablets of Production Examples 5-8 activate pDC and are effective in immunostimulation.

[0082] [Table 8]

[0083] [Manufacturing Example 9-12: Hard Capsules] According to the formulation in Table 9, CRL1505 was mixed with other ingredients and coated with a coating containing gelatin or hydroxypropyl cellulose to produce hard capsules (contents: 300 mg). One capsule per day can be taken with water or other liquids. All of the hard capsules in Production Examples 9-12 activate pDCs and are effective in immunostimulation.

[0084] [Table 9]

[0085] [Manufacturing example 13-16: PET beverage] According to the formulation in Table 10, a liquid formulation containing CRL1505 and other ingredients was packed into a PET container to produce a PET beverage (500 ml). One bottle should be consumed per day. All of the PET beverages in Production Examples 13-16 activate pDCs and are effective in stimulating the immune system.

[0086] [Table 10] [Industrial Applicability]

[0087] The oral composition of the present invention is industrially useful because it enables pDC activation and is effective in immunostimulation.

Claims

1. An oral composition for activating pDC, characterized by containing Lacticaseibacillus rhamnosus CRL1505.

2. The composition according to claim 1, characterized in that it is for immunostimulation.

3. An immunostimulatory oral composition comprising Lacticaseibacillus rhamnosus CRL1505, wherein immunostimulation refers to improving at least one symptom selected from "stuffy nose," "scratchy throat," "phlegm accumulation or chest tightness caused by phlegm," "hoarseness," "fuzzy head," "nausea or heartburn," "joint pain," "muscle pain," "abdominal pain," "sensation of ear fullness or difficulty hearing," "lymph node pain," "fever," "fatigue," "chills," "loss of appetite," "dizziness or unsteadiness," "unspecified complaint," "malaise," and "sweating."

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