Composition for activating innate immunity

Abstract

The present invention addresses the problem of providing a composition for activating innate immunity that is capable of activating innate immunity and exhibits excellent stability and safety, as well as foods and beverages, pharmaceuticals, and feeds for activating innate immunity that contain this composition.　Provided are a composition for activating innate immunity that is characterized by containing Limosilactobacillus mucosae as an active ingredient, as well as foods and beverages, pharmaceuticals, and feeds for activating innate immunity that are characterized by containing this composition.

Description

Composition for activating innate immunity

[0001] The present invention relates to foods, pharmaceuticals, and feeds containing a composition for activating innate immunity.

[0002] Innate immunity is a form of immune response that organisms inherently possess and is a biological defense mechanism possessed by many animals from mammals to invertebrates. Innate immunity is initiated by immune cells such as dendritic cells and macrophages responding to pathogens and foreign substances that invade from the outside. In addition, innate immunity is also involved in the activation of subsequent acquired immunity and promotes the efficiency of the immune response.

[0003] There are various signal transduction pathways in the innate immune response, which are responsible for the initial defense reaction against invading microorganisms. The Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway induced by cytokines is an evolutionarily widely conserved signal transduction pathway that causes transcriptional regulation of DNA and functions in multiple physiological processes such as hematopoiesis, differentiation, metabolism, and immune regulation. The JAK-STAT pathway is also an important signal pathway in immune function and is related to the activation of innate immune pathways such as the control of wound repair and the activation of neutrophils and macrophages. On the other hand, abnormalities in the JAK-STAT pathway have been reported to be related to autoimmune diseases such as rheumatoid arthritis and Crohn's disease (Casanova et al. 2012).

[0004] The human gut is home to 40 trillion bacteria of 1,000 different species, forming a complex symbiotic relationship. Lactic acid bacteria, widely known as beneficial gut bacteria, have been reported to have numerous beneficial effects on physiological functions, including immunity. For example, interferon-λ inducers containing Lactobacillus gasseri (Japanese Patent Publication No. 2017-197486) and antimicrobial peptide production promoting compositions containing Lactobacillus helveticus (Japanese Patent Publication No. 2024-79724) have been reported. Furthermore, there are numerous reports on innate immune activators using probiotics (Japanese Patent Publication Nos. 6505018, 6594911, 6675703, 6935866, 7369976, and 7425429).

[0005] Regarding the effects of rimosiractobacillus mucosae on immune function, for example, it has been reported that pre-administration of rimosiractobacillus mucosae to piglets receiving intraperitoneal injections of LPS improves intestinal health through its anti-inflammatory effect (Li et al. 2023), and that administration of rimosiractobacillus mucosae suppresses inflammation in mice in which inflammation was induced by Escherichia coli K1 strain (Kim et al. 2020).

[0006] Thus, the anti-inflammatory effects, or immunosuppressive effects, of Rimosiractobacillus mucosae have been reported in multiple studies. However, none of the literature discloses or suggests that Rimosiractobacillus mucosae has the effect of activating innate immunity.

[0007] As medicines with anti-inflammatory effects, mainly steroidal anti-inflammatory drugs such as dexamethasone and non-steroidal anti-inflammatory drugs such as indomethacin are known. For example, dexamethasone is used to treat inflammation such as thyroiditis, rheumatic carditis, systemic vasculitis, polyarteritis nodosa, polymyositis, asthmatic bronchitis, localized enteritis, fulminant hepatitis, diffuse interstitial pneumonia, encephalomyelitis, encephalitis, ankylosing spondylitis, eczema / dermatitis, atopic dermatitis, uveitis, chorioretinitis, optic neuritis, conjunctivitis, keratitis, acute and chronic otitis media, allergic rhinitis, laryngitis, and glossitis (Nichi-Iko Pharmaceutical Co., Ltd., Decadron Tablets 0.5 mg / Decadron Tablets 4 mg, Package Insert No. 2454002F1183_3_16), but it is not used for infection prevention where activation of innate immunity is effective. Furthermore, indomethacin is used to treat osteoarthritis, periarthritis of the shoulder, tendinitis, tenosynovitis, epicondylitis (tennis elbow, etc.), muscle pain, and post-traumatic swelling and pain, as well as to relieve pain and inflammation (Sawai Pharmaceutical Co., Ltd., Indomethacin Cream 1% "Sawai", Package Insert No. 2649719N1114_1_05), but it is not used for infection prevention where activation of innate immunity is effective. Moreover, the same package insert states, "9. Precautions concerning patients with specific backgrounds / 9.1 Patients with complications or a history of illness / 9.1.2 Patients with skin infections / When used for inflammation accompanied by infection, use in combination with an appropriate antibacterial or antifungal agent, observe carefully, and use with caution. There is a risk of masking skin infections."

[0008] Drosophila melanogaster (hereinafter referred to as Drosophila), a model organism used in various fields of biology, lacks an adaptive immune system, unlike vertebrates, and is therefore widely used in the study of the molecular mechanisms of innate immunity. TLRs, which are important factors in the major innate immune response of mammals, were discovered as homologs of the Toll receptor, whose antifungal function was demonstrated in Drosophila (Medzhitov et al. 1997). Homology between Drosophila and mammals has also been shown in other innate immune pathways, such as the Imd pathway, the major pathway of intestinal immunity in Drosophila, and the human TNFR pathway and JAK-STAT pathway, due to the high similarity of their respective components (Myllymaki et al. 2014, Myllymaki and Ramet 2014).

[0009] Furthermore, the evaluation of immunomodulatory agents using Drosophila melanogaster is also being actively conducted. For example, it has been shown that extracts of Anthopanax senticosus improve the survival rate of Drosophila melanogaster during pathogenic bacterial infection and promote the expression of antimicrobial peptide genes (Li et al. 2013), and extracts of Rhoudiola crenulata increase the expression of antimicrobial peptide genes and reduce epithelial cell death during pathogenic bacterial infection (Zhu et al. 2014). From these findings, Drosophila melanogaster is considered a suitable model system for verifying innate immune activity.

[0010] Japanese Patent Publication No. 2017-197486, Japanese Patent Publication No. 2024-79724, Japanese Patent Publication No. 6505018, Japanese Patent Publication No. 6594911, Japanese Patent Publication No. 6675703, Japanese Patent Publication No. 6935866, Japanese Patent Publication No. 7369976, Japanese Patent Publication No. 7425429

[0011] Jean-Laurent Casanova, Steven M Holland, Luigi D Notarangelo. Inborn errors of human JAKs and STATs. Immunity. 2012 Apr 20;36(4):515-28Jingjing Li, Shengkai Feng, Yu Pi, Xianren Jiang, Xilong Li, Zutao Zhou, Xiangdong Liu, Hong Wei, Shiyu Tao Limosilactobacillus johnsoni and Limosilactobacillus mucosae and Their Extracellular Vesicles Alleviate Gut Inflammatory Injury by Mediating Macrophage Polarization in a Lipopolysaccharide-Challenged Piglet Model. J Nutr. 2023 Aug;153(8):2497-2511.Jeon-Kyung Kim, Kyung-Eon Lee, Sang-Ah Lee, Hyo-Min Jang, Dong-Hyun Kim Interplay Between Human Gut Bacteria Escherichia coli and Lactobacillus mucosae in the Occurrence of Neuropsychiatric Disorders in Mice. Front Immunol. 2020 Feb 25:11:273.R Medzhitov, P Preston-Hurlburt, C A Janeway Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997 Jul 24;388(6640):394-7. Henna Myllymaki, Susanna Valanne, Mika Ramet. The Drosophila imd signaling pathway. J Immunol. 2014 Apr 15;192(8):3455-62. H Myllymaki, M Ramet. JAK / STAT pathway in Drosophila immunity. Scand J Immunol. 2014 Jun;79(6):377-85. Wenjia Li, Qiuxiang Luo, Li Hua Jin. Acanthopanax senticosus extracts have a protective effect on Drosophila gut immunity. J Ethnopharmacol. 2013 Mar 7;146(1):257-63. Caixia Zhu, Fachun Guan, Chao Wang, Li Hua Jin. The protective effects of Rhodiola renulate extracts on Drosophila melanogaster gut immunity induced by bacteria and SDS toxicity. Phytother Res. 2014 Dec; 28(12):1861-6. Ekengren S, Tryselius Y, Dushay MS, Liu G, Steiner H, Hultmark D. A human stress response in Drosophila. Curr Biol. 2001 May 1;11(9):714-8. ;

[0012] The present invention aims to provide a composition containing Limosilactobacillus mucosae for activating innate immunity. Furthermore, the present invention aims to provide a food, pharmaceutical, and feed for activating innate immunity containing the said composition.

[0013] The inventors of this invention, after diligent research to solve the above problem, discovered that Rimosiractobacillus mucosae, particularly Rimosiractobacillus mucosae, has an innate immune-activating effect, and thus completed the present invention. In other words, the present invention is based on the discovery of a novel use for the above microorganism that has not been reported to date. That is, the present invention has the following configuration.

[0014] [Aspect 1] A composition for activating innate immunity, comprising Limosilactobacillus mucosae. [Aspect 2] The composition according to aspect 1, characterized in that the activation of innate immunity is due to activation of the JAK-STAT pathway. [Aspect 3] The composition according to aspect 1 or 2, characterized in that the Limosilactobacillus mucosae species is Limosilactobacillus mucosae strain SBT10028 (NITE BP-03275), strain SBT2958 (NITE P-02803), strain SBT10038 (NITE P-03283), or strain SBT10043 (NITE BP-03187). [Aspect 4] The composition according to any one of aspects 1 to 3, which is not for anti-inflammatory purposes. [Aspect 5] Food and beverage for activating natural immunity, characterized by containing the composition according to any one of aspects 1 to 3. [Aspect 6] A pharmaceutical for activating innate immunity, characterized by comprising the composition described in any one of Aspects 1 to 3. [Aspect 7] A feed for activating innate immunity, characterized by comprising the composition described in any one of Aspects 1 to 3. [Aspect 8] A food or beverage, pharmaceutical for activating innate immunity, or feed for activating innate immunity, as described in any one of Aspects 5 to 7, which is not for anti-inflammatory purposes. [Aspect 9] A pharmaceutical according to Aspect 8, wherein the activation of innate immunity is for the treatment, prevention, and improvement of infectious diseases. [Aspect 10] A composition for activating innate immunity, as described in Aspects 1 to 4, a food or beverage for activating innate immunity, as described in Aspect 5 or 8, or a feed for activating innate immunity, as described in Aspect 7 or 8, for use in non-therapeutic purposes.

[0015] According to the present invention, the present invention can provide a composition for activating innate immunity that can activate innate immunity, as well as food and beverages, pharmaceuticals, and animal feed for activating innate immunity that contain the composition.

[0016] This shows the survival rate after infection with highly virulent bacteria following administration of rimosiractobacillus mucosaepre. *** indicates p < 0.001 by log-rank test. This shows the results of quantitative PCR of the TotA gene when rimosiractobacillus mucosae was administered to adult Drosophila melanogaster. *** indicates p < 0.001 by Student's t-test. This shows the results of quantitative PCR of the TotA gene when rimosiractobacillus mucosae was administered to Drosophila embryo-derived cells. *** indicates p < 0.001 by Student's t-test.

[0017] The following describes in detail embodiments for carrying out the present invention (hereinafter simply referred to as "these embodiments"). These embodiments are illustrative for explaining the present invention and are not intended to limit the present invention to the following content. The present invention can be implemented by modifying it as appropriate within the scope of its gist.

[0018] (Composition for Activating Innate Immunity) The composition for activating innate immunity of the present invention is characterized by containing Limosilactobacillus mucosae as an active ingredient. In the present invention, Limosilactobacillus mucosae refers to bacteria belonging to the Limosilactobacillus mucosae genus. That is, it refers to a bacterial strain in which the homology of the base sequence of the 16S ribosomal RNA gene to the Limosilactobacillus mucosae reference strain JCM12515 is 97% or more, more preferably 98% or more, and even more preferably 99% or more. Any source of isolation is acceptable, but more preferably derived from the human intestinal tract. Rimosilactobacillus mucosae is a bacterium that was previously classified under the genus Lactobacillus, but was reclassified into the genus Rimosilactobacillus in 2020 in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) in accordance with the rules of the International Committee for Nomenclature of Prokaryotes (ICSP) (ICNP).

[0019] The strain of Limosilactobacillus mucosae used as the active ingredient in this invention is not particularly limited as long as it is a strain that has an innate immune-activating effect. Preferred examples include Limosilactobacillus mucosae strains SBT10028, SBT2958, SBT10038, or SBT10043. Limosilactobacillus mucosae may be used alone or in combination of two or more strains. It can also be combined with other lactic acid bacteria.

[0020] Rimosilactobacillus mucosae strains SBT10028, SBT2958, SBT10038, and SBT10043 are deposited with the Patent Microorganism Depository Center (NPMD) of the National Institute of Biotechnology, National Institute of Product Evaluation, and are available from that institution. The accession numbers are shown in Table 1.

[0021] Rimosilactobacillus mucosae can be cultured according to standard methods. Various culture media can be used, including milk media, media containing milk components, and semi-synthetic media that do not contain milk components. Examples of such media include reduced skim milk media.

[0022] The Rimosilactobacillus mucosae contained in the innate immunity activating composition of the present invention can be used as the active ingredient of the present invention after being isolated from the obtained culture by means of bacterial collection such as centrifugation. Alternatively, the bacterial cells may be subjected to some treatment, such as concentration, drying, or freeze-drying, or they may be killed by heat drying, and the treatment method is not particularly limited.

[0023] Furthermore, not only purely isolated bacterial cells can be used, but also cultures, suspensions, other bacterial cell-containing materials, and cytoplasm and cell wall fractions obtained by treating bacterial cells with enzymes or physical means. Examples of culture forms include not only cultures prepared using synthetic media such as MRS medium (manufactured by DIFCO) and reduced skim milk medium, which are commonly used for culturing lactic acid bacteria, but also dairy products such as cheese, fermented milk, and lactic acid bacteria beverages, but are not particularly limited.

[0024] (Activation of Innate Immunity) In this invention, "innate immunity" refers to the body's immune response mechanism, which is initiated when immune cells such as dendritic cells and macrophages respond to pathogens and foreign substances invading from the outside, and which activates various signal transduction cascades to produce antiviral cytokines, chemokines, etc., and eliminate pathogens. In other words, innate immunity plays an important role in protecting the body from various infectious substances that can induce serious diseases.

[0025] In this invention, "innate immunity activation" refers to the enhancement of intestinal immune function by activating the signaling pathways of the innate immune system, thereby demonstrating effectiveness in the treatment, prevention, and improvement of infections associated with abnormalities in intestinal immunity caused by pathogenic microbial infection. In this case, the innate immune system signaling pathways that are activated are not limited, but it is preferable that they are via the JAK-STAT pathway.

[0026] In this invention, innate immune activity can be evaluated using Drosophila melanogaster. Because its innate immune response signaling pathway is homologous to that of mammals, Drosophila melanogaster is a useful model organism for evaluating innate immune activity. Innate immune activation ability was evaluated by measuring the survival rate after infection with highly virulent bacteria, as specifically described in the examples.

[0027] (Foods and Beverages, Pharmaceuticals and Feeds) The innate immunity activating composition of the present invention can be added to foods and beverages, pharmaceuticals, feeds, etc. Furthermore, foods and beverages, pharmaceuticals and feeds containing the innate immunity activating composition of the present invention may also contain various auxiliary ingredients, as long as they do not impair the effects of the present invention. Examples of foods and beverages include general foods, functional foods, nutritional supplements, and foods for specified health uses. The form of the innate immunity activating composition of the present invention is not particularly limited, but examples include powders, granules, tablets, capsules, oral solutions, etc.

[0028] Although the present invention has been described above using embodiments, it goes without saying that the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be obvious to those skilled in the art that various modifications or improvements can be made to the above embodiments. Furthermore, it is clear from the claims that such modified or improved forms may also be included in the technical scope of the present invention.

[0029] Examples and test examples are shown below to illustrate the present invention in detail, but these are for illustrative purposes only and the present invention is not limited in any way by them.

[0030] The Drosophila melanogaster used in this example were female adults 7 to 10 days after emergence from the standard strain w1118. Before the experiment, the flies were subcultured for at least three generations in standard medium containing 50 μg / mL of tetracycline to eliminate enteric bacteria and intracellular parasitic bacteria. Autoclaved standard medium was used for the infection experiments. Rimosilactobacillus mucosae strains SBT10028, SBT2958, SBT10038, and SBT10043 were used.

[0031] (Example 1) Effect of improving the survival rate of Rimosilactobacillus mucosae during infection by highly virulent bacteria. For the infection experiment, the highly virulent Pseudomonas entomophila (hereinafter referred to as Pe) was used. After shaking overnight on LB medium at 29°C, the culture solution was centrifuged to collect the bacterial cells, and the resuspended in 5% sucrose to an OD600 = 200 was used for the infection experiment. Rimosilactobacillus mucosae was present in 1.0 × 10⁶ units in 5% sucrose. １０ The lactic acid bacteria suspension was prepared to a CFU / mL concentration. This suspension was added to autoclaved Drosophila standard medium. Drosophila were incubated in this medium at 29°C for two days to administer the lactic acid bacteria to the flies. 5% sucrose used as the suspension was used as a negative control. After pre-administration, a suspension of pe was added to the same medium, and the survival rate was observed over time.

[0032] Figure 1 shows the survival rates after administration of rimosiractobacillus mucosaepre during pathogenic bacterial infection. The control group is shown by a solid line, and the rimosiractobacillus mucosaepre-administered group is shown by a dotted line. In the control group, the survival rate gradually decreased after Pe infection, while in the rimosiractobacillus mucosaepre-administered group, the survival rate was significantly increased.

[0033] (Example 2) Expression analysis of JAK-STAT pathway output gene by administration of Rimosiractobacillus mucosae The Rimosiractobacillus mucosae suspension was added to autoclaved Drosophila standard medium. Drosophila were administered the lactic acid bacteria by rearing in this medium at 29°C for 2 days. 5% sucrose used as the suspension was used as a negative control. When administering to cultured cells, 0.5 × 10⁶ doses were added to 80% confluence Schneider 2 cells (derived from Drosophila embryos, hereafter referred to as S2 cells). ８ Limosiractobacillus mucosae at CFU / mL was added, and the mixture was incubated at 29°C for 48 hours.

[0034] Two days after administration, RNA was extracted from whole-body Drosophila (5 flies) or S2 cell (0.5 mL) suspension, and cDNA was synthesized using reverse transcriptase. Quantitative PCR was performed using the synthesized cDNA as a template according to standard procedures. The tubulin gene was used as the internal standard. The primers used are shown in Table 2.

[0035] Figure 2 shows the results of quantitative PCR of the TotA gene (Ekengrén et al. 2001), an output gene of the JAK-STAT pathway, when adult Drosophila melanogaster was administered with Rimosiractobacillus mucosae (SBT10028 strain), and Figure 3 shows the results when S2 cells were administered with the same Rimosiractobacillus mucosae. The vertical axis shows the relative value with the expression level of the TotA gene in the control group set to 1. In both adult Drosophila melanogaster and S2 cells, the expression level of the TotA gene was significantly increased in the Rimosiractobacillus mucosae-administered group compared to the control group.

[0036] This invention provides a composition containing Rimosiractobacillus mucosae as a composition having a novel innate immune-activating effect, as well as food and beverages, pharmaceuticals, and animal feed containing this composition. By ingesting the composition of this invention, an innate immune-activating effect can be exerted.

[0037] [References to deposited biological materials] Name and address of depositary institution: 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Patent Microorganism Depository Center (NPMD), National Institute of Technology and Evaluation, Biotechnology Center, Date of domestic deposit: September 15, 2020, Date of transfer to international deposit: March 23, 2021, Accession number: NITE BP-03275 Name and address of depositary institution: 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Patent Microorganism Depository Center (NPMD), National Institute of Technology and Evaluation, Biotechnology Center, Date of deposit: October 31, 2018, Accession number: NITE P-02803 Name and address of depositary institution: 292-0818 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan, Patent Microorganism Depository Center (NPMD), National Institute of Technology and Evaluation, Biotechnology Center, Date of Deposit: September 15, 2020, Accession Number: NITE P-03283 Name and Address of Depositor: 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Patent Microorganism Depository Center (NPMD), National Institute of Technology and Evaluation, Biotechnology Center, Date of Domestic Deposit: March 27, 2020, Date of Transfer to International Deposit: March 23, 2021, Accession Number: NITE BP-03187

[0038] [Supplement based on Rule 26, November 26, 2025]

Claims

1. A composition for activating innate immunity, comprising Limosilactobacillus mucosae.

2. The composition according to claim 1, characterized in that the innate immune activation is due to activation of the JAK-STAT pathway.

3. Food or beverage for activating innate immunity, characterized by comprising the composition described in claim 1 or 2.

4. A pharmaceutical product for activating innate immunity, characterized by comprising the composition described in claim 1 or 2.

5. A feed for activating innate immunity, characterized by comprising the composition described in claim 1 or 2.

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