Neutrophil-activation-regulating composition containing saccharification bacteria

Abstract

[Problem] To provide a neutrophil-activation-regulating composition having the dual functions of promoting and / or suppressing neutrophil extracellular trap formation. [Solution] The present disclosure was completed after confirming that saccharification bacteria (particularly Bacillus subtilis) can promote and / or suppress neutrophil extracellular traps (NETs) formation by neutrophil-like cells.

Description

Neutrophil activation-regulating composition containing saccharifying bacteria

[0001] The present disclosure relates to a composition for modulating neutrophil activation comprising saccharifying bacteria.

[0002] There are many types of immune cells, but neutrophils account for the majority of immune cells in human peripheral blood. Neutrophils defend the body against foreign microorganisms in various ways. One of these methods is neutrophil extracellular traps (NETs) (Non-Patent Document 1). NETs are a phenomenon in which neutrophils release a meshwork of DNA and proteins extracellularly to capture microorganisms during microbial infection. This is an innate immune function that prevents localized infection from spreading to the whole body. NETs formation is characterized by the breakdown of the neutrophil nuclear membrane, followed by chromatin expansion and cell membrane rupture. This series of cell death processes is called NETosis. Meanwhile, NETosis has recently been reported to occur in various inflammatory diseases other than microbial infection. For example, it has been suggested that NETs formation may induce excessive inflammation in respiratory diseases such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF); thrombotic diseases such as antiphospholipid syndrome (APS), stroke, and thrombotic microangiopathy (TMA); autoimmune diseases such as systemic lupus erythematosus (SLE), antineutrophil cytoplasmic antibody vasculitis (AAV), and rheumatoid arthritis (RA); cancer; and sepsis (Non-Patent Document 2). Furthermore, the release of antibacterial substances, such as histones, myeloperoxidase, and elastase, into the host's blood or tissues can cause damage to host tissues and cells. As described above, NETs formation or NETosis is a natural immune function that possesses both host defense and inflammation exacerbation, and appropriate control is desirable.

[0003] Several substances have been reported to regulate neutrophil activation. For example, a neutrophil activation regulator containing a thrombin-like enzyme as an active ingredient, which inhibits neutrophil degranulation, Mac-1 expression, and NETs formation, has been reported (Patent Document 1). Histidine-rich glycoproteins, which are synthesized in the liver and present in plasma and are known to be involved in regulating the coagulation-fibrinolysis system and angiogenesis, have also been reported to be neutrophil activation regulators (Patent Document 2). Furthermore, many pathogenic microorganisms have been reported to induce NETs and activate neutrophils (Non-Patent Document 3). Lacticaseibacillus rhamnosus GG, a type of probiotic lactic acid bacteria, is known to suppress NETs (Non-Patent Document 4). When evaluating the effect on NETs formation in vitro, neutrophil-like HL-60 cells, differentiated from the human leukemia cell line HL-60, are often used as a neutrophil model (Non-Patent Document 5).

[0004] Patent No. 7100854 Patent No. 5807937

[0005] Brinkmann V et al:Neutrophil extracellular traps kill bacteria. Science. 303:1532-1535, 2004Sollberger G et al:Neutrophil Extracellular Traps: The Biology of Chromatin Externalization. Dev.Cell. 44(5):542-553, 2018Mitsios A et al:NETopathies? Unraveling the dark side of old diseases through neutrophils.Front. Immunol. 7(678):678, 2017Vong L et al: Probiotic Lactobacillus rhamnosus Inhibits the Formation of Neutrophil Extracellular Traps. J. Immunol. 192(4):1870-1877, 2014Manda-Handzlik A et al: The influence of differentiating agents HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps. Immunol. Cell Biol.96(4):413-425, 2018

[0006] As described above, NETs formation or NETosis development is a natural immune function that has two aspects: host defense and inflammation exacerbation, and it is desirable to appropriately control it. However, there have been no reports of substances that can control NETs in both directions, promoting and suppressing them. Therefore, an objective of the present disclosure is to provide a composition for regulating neutrophil activation that has the functions of both promoting and / or suppressing NETs formation.

[0007] As a result of extensive research to solve the above-mentioned problems, the inventors of the present invention confirmed that saccharifying bacteria (particularly Bacillus subtilis) can promote and / or inhibit the formation of NETs in neutrophil-like cells, and thus completed the present disclosure.

[0008] The present disclosure is as follows: 1. A composition for regulating neutrophil activation, comprising saccharifying bacteria. 2. The composition according to item 1, wherein the saccharifying bacteria is a bacterium of the genus Bacillus. 3. The composition according to item 1, wherein the saccharifying bacteria is a Bacillus subtilis species. 4. The composition according to item 1, wherein the saccharifying bacteria is Bacillus subtilis TO-A. 5. The composition according to item 3 or 4, wherein the regulation of neutrophil activation is promotion and / or inhibition of neutrophil NETs formation. 6. The composition according to item 1, wherein the regulation is promotion. 7. A therapeutic agent for a disease caused by insufficient neutrophil activation, comprising the composition according to item 6. 8. The composition according to item 1, wherein the regulation is inhibition. 9. A therapeutic agent for a disease caused by excessive neutrophil activation, comprising the composition according to item 8. 10. A method for regulating neutrophil activity, comprising the step of administering the composition according to any one of items 1 to 4 to a mammal other than a human. 11. 11. A method for treating a disease caused by excessive neutrophil activation, comprising the step of administering saccharifying bacteria to a subject. 12. A method for treating a disease caused by insufficient neutrophil activation, comprising the step of administering saccharifying bacteria to a subject. 13. Use of saccharifying bacteria in the production of a composition for regulating neutrophil activation. 14. Use of a composition for regulating neutrophil activation comprising saccharifying bacteria.

[0009] The compositions of the present disclosure can promote and / or inhibit NETs formation in neutrophils.

[0010] Neutrophil-like HL-60 cells are shown. NETs were released from neutrophil-like HL-60 cells after treatment with the NETs inducer phorbol 12-myristate 13-acetate (PMA) for 3 hours. DNA was digested with DNase I after treatment with PMA for 3 hours. NETs were released from neutrophil-like HL-60 cells treated with Bacillus subtilis TO-A strain after treatment with PMA for 3 hours.

[0011] (Subject of the Present Disclosure) The present disclosure relates to a composition for regulating neutrophil activation, a therapeutic agent for diseases caused by insufficient neutrophil activation, a therapeutic agent for diseases caused by excessive neutrophil activation, and a method for regulating neutrophil activity. The present disclosure is described in detail below. The compositions of the present disclosure can be formulated into powders, tablets, granules, capsules, liquids, etc. by known methods, including, but not limited to, carriers such as starch, lactose, soy protein, excipients, binders, disintegrants, lubricants, stabilizers, and suspending agents. Furthermore, the present disclosure relates to prescription drugs, over-the-counter drugs, quasi-drugs, veterinary drugs, feed, feed additives, foods for specified health uses, foods with nutrient functions, foods with functional claims, nutritional drinks, and the like, which contain the compositions. Furthermore, when preferred numerical ranges (e.g., ranges of content and weight-average molecular weight) are described in stages in this specification, the respective lower and upper limits can be independently combined. For example, in the description "preferably 10 to 100, more preferably 20 to 90," the "preferable lower limit: 10" and the "more preferable upper limit: 90" can be combined to form "10 to 90."

[0012] (Neutrophil Activation) In the present disclosure, "neutrophil activation" refers to the action (phenomenon) of neutrophils caused by stimulation with a neutrophil-activating factor, including NETs formation, degranulation, reactive oxygen production, Mac-1 expression, transendothelial migration, and tissue infiltration. In the present disclosure, "regulating neutrophil activation" particularly refers to promoting and / or inhibiting NETs formation, but does not exclude the other phenomena mentioned above. Note that "NETs formation" refers to the formation of a meshwork structure consisting of DNA and proteins due to the expansion of chromatin and rupture of the cell membrane following the breakdown of the neutrophil nuclear membrane.

[0013] (Therapeutic Agent for Diseases Caused by Excessive Neutrophil Activation) In the present disclosure, a "therapeutic agent for diseases caused by excessive neutrophil activation" refers to a therapeutic agent that can be used to treat diseases caused by damage to tissues and organs due to neutrophils forming more NETs than necessary for host defense or by excessive production of reactive oxygen species, cytokines, antimicrobial peptides, or proteolytic enzymes. Specific diseases include respiratory diseases such as acute lung injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and cystic fibrosis; thrombotic diseases such as antiphospholipid syndrome, stroke, and thrombotic microangiopathy; autoimmune diseases such as systemic lupus erythematosus, antineutrophil cytoplasmic antibody vasculitis, and rheumatoid arthritis; cancer; and sepsis. Furthermore, the target population for administration includes patients with the above-described diseases and patients in need of prevention of the diseases.

[0014] (Therapeutic Agent for Diseases Caused by Insufficient Neutrophil Activation) In the present disclosure, a "therapeutic agent for diseases caused by insufficient neutrophil activation" refers to a therapeutic agent that can be used to treat diseases caused by insufficient neutrophil NETs formation, production of reactive oxygen species, cytokines, antimicrobial peptides, or protein differentiation enzymes when a living body is infected with pathogenic microorganisms such as bacteria, viruses, fungi, mycoplasma, or parasites. Specific diseases include bacterial infections caused by Streptococcus aureus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, pathogenic Escherichia coli, and Pseudomonas aeruginosa, viral infections such as influenza, viral pneumonia, viral hepatitis, viral gastroenteritis, cytomegalovirus infection, and Japanese encephalitis, fungal infections such as aspergillosis and candidiasis, and parasitic infections such as schistosomiasis japonicum, phalariosis, and ascariasis. Furthermore, the target population for administration includes patients with the above-described diseases and patients in need of prevention of such diseases.

[0015] (Composition of the Present Disclosure) The composition of the present disclosure is a composition containing saccharifying bacteria as an essential component. The saccharifying bacteria (Amylolytic Bacillus) are preferably Bacillus genus, more preferably Bacillus subtilis, Bacillus mesentericus, or Bacillus polyfermenticus species, and even more preferably Bacillus subtilis TO-A. Furthermore, Bacillus genus is a spore-forming bacterium, a gram-positive bacillus that is obligately aerobic (partially facultatively anaerobic). When spore-forming bacteria are placed in poor environmental conditions, such as poor nutrition and temperature, or come into contact with a compound that is toxic to the bacteria, spores are formed within the bacterial cells. For this reason, spores are sometimes referred to as a durable or dormant form of the bacteria. When spores are placed in an environment suitable for bacterial growth or under stressful conditions such as heat shock, the spores germinate into vegetative or vegetative cells, which are bacterial cells with normal growth and metabolic capabilities. When saccharifying spores enter the body, they germinate into different numbers of vegetative cells depending on the environment, and then exert their regulatory function of neutrophil activation. The above bacteria may be live or killed, but live bacteria are preferred. The bacteria may also be processed products or extraction residues, such as sonicated products, homogenized products, methanol extraction residues, or ethyl acetate extraction residues. Additionally, the compositions of the present disclosure may be in the form of powders, granules, orally disintegrating tablets, plain tablets, coated tablets, capsules, suspensions, or liquids. The compositions of the present disclosure are available from Toa Pharmaceutical Co., Ltd. (see http: / / www.toabio.co.jp / medicalitem). Bacillus subtilis TO-A is available as a strain having a unique accession number, FERM BP-07462, from the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation. Additionally, the compositions of the present disclosure can be formulated by blending, in addition to saccharifying bacteria, known carriers such as starch, lactose, soy protein, pharmaceutically or food-acceptable excipients, protective agents, buffers, isotonicity agents, flavoring agents, colorants, binders, disintegrants, lubricants, stabilizers, suspending agents, coating agents, and the like.

[0016] (Content of saccharifying bacteria in the composition of the present disclosure) Based on the results of Example 2 below, the composition of the present disclosure can be made into a "neutrophil activation-promoting composition" by reducing the content of saccharifying bacteria (particularly, trophozoites) in the composition, and can be made into a "neutrophil activation-inhibiting composition" by increasing the content. That is, the composition of the present disclosure can promote neutrophil activation when the activation state of neutrophils is low (when there are few activated neutrophils). On the other hand, when the activation state of neutrophils is too high (when there are too many activated neutrophils), it can inhibit neutrophil activation. For example, the content of saccharifying bacteria in the composition of the present disclosure (particularly, the content in an aqueous solution, and further, the content in a cell suspension) can be set as follows: Bacterial content of neutrophil activation-regulating composition: 1.0x10 4 / mL to 1.0x10 11 / mL, or 5.0x10 4 / mL to 1.0x10 10 / mL, more preferably 1.0x10 5 / mL to 6.0x10 8 / mL. Bacterial content of the neutrophil activation-promoting composition: 1.0x10 4 / mL to 2.0x10 6 / mL, 1.0x10 4 / mL to 2.1x10 6 / mL, 1.0x10 6 / mL to 2.2x10 6 / mL, 1.0x10 4 / mL to 2.3x10 6 / mL or 1.0x10 4 / mL to 2.4x10 6 / mL, preferably 5.0x10 4 / mL to 2.0x10 6 / mL, 5.0x10 4 / mL to 2.1x10 6 / mL, 5.0x10 4 / mL to 2.2x10 6 / mL, 5.0x10 4 / mL to 2.3x10 6 / mL, 5.0x10 4 / mL to 2.4x106 / mL, 1.0x10 4 / mL to 1.5x10 6 / mL, 1.0x10 4 / mL to 1.6x10 6 / mL, 1.0x10 4 / mL to 1.7x10 6 / mL, 1.0x10 4 / mL to 1.8x10 6 / mL or 1.0x10 4 / mL to 1.9x10 6 / mL, 5.0x10 4 / mL to 1.4x10 6 / mL, 5.0x10 4 / mL to 1.3 x 10 6 / mL, 5.0x10 4 / mL to 1.2x10 6 / mL, 5.0x10 4 / mL to 1.1x10 6 / mL, more preferably 1.0x10 5 / mL to 1.1x10 6 / mL, 1.0x10 5 / mL to 1.2x10 6 / mL, 1.0x10 5 / mL to 1.3 x 10 6 / mL, 1.0x10 5 / mL to 1.4x10 6 / mL, 1.0x10 5 / mL to 1.5x10 6 / mL, 1.0x10 5 / mL to 1.6x10 6 / mL, 1.0x10 5 / mL to 1.7x10 6 / mL, 1.0x10 5 / mL to 1.8x10 6 / mL, 1.0x10 5 / mL to 1.9x10 6 / mL, 1.0x10 5 / mL to 2.0x10 6 / mL, 1.0x10 5 / mL to 2.1x10 6 / mL, 1.0x10 5 / mL to 2.2x10 6 / mL, 1.0x105 / mL to 2.3x10 6 / mL or 1.0x10 5 / mL to 2.4x10 6 / mL. Bacterial content of the neutrophil activation inhibitory composition: 2.5x10 6 / mL or more, 5.0x10 6 / mL or more, 1.0x10 7 / mL or more, 2.0x10 7 / mL or more, 5.0x10 7 / mL or more, 1.0x10 8 / mL or more, 1.0x10 9 / mL or more, 1.0x10 10 / mL or more or 1.0x10 11 / mL or higher, with an upper limit of 1.0x10 12 / mL or less, 1.0x10 15 / mL or less or 1.0x10 17 / mL or less. The above content is the content in an aqueous solution (cell suspension), so a person skilled in the art can calculate the amount converted into a powder, granules, orally disintegrating tablets, plain tablets, coated tablets, capsules, or liquid formulation as appropriate. For example, when the composition of the present disclosure is a powder, granules, orally disintegrating tablets, plain tablets, coated tablets, capsules, or liquid formulation, the content of saccharifying bacteria in the formulation when converted into a solution is 1.0 x 10 4 / mL to 2.4x10 6 / mL range.

[0017] (Subjects for Administration of the Composition of the Present Disclosure) The subjects for administration of the composition of the present disclosure are not particularly limited, but include organisms with diseases caused by insufficient neutrophil activation, or organisms with diseases caused by excessive neutrophil activation, more preferably vertebrates with a central nervous system, and even more preferably humans, mammals including livestock, chickens, seafood, and pets. In the case of humans, this includes both healthy individuals and patients. In addition, since compositions containing the strain have been safely used as oral pharmaceuticals for approximately 60 years by people from infants to the elderly, it is possible to target humans of all ages.

[0018] (Method of Administration of the Composition of the Present Disclosure) The dosage, frequency of administration, and administration interval of the administration method of the present disclosure are not particularly limited and are appropriately selected depending on factors such as the purpose of prevention (particularly prevention of recurrence) and / or clinical treatment, the type of disease, the patient's weight, age, and severity of the disease. For example, the following dosage forms can be used, but are not particularly limited. For powders (daily dose), 75 to 150 mg of saccharified bacterial powder is desirable, and for orally disintegrating tablets or plain tablets (daily dose), 30 to 60 mg of saccharified bacterial powder is desirable. Mixtures containing these bacteria, processed products, or extract residues are preferably administered in the form of powders, granules, orally disintegrating tablets, plain tablets, coated tablets, capsules, or liquids, and are administered once or twice a day or multiple times a day (morning, noon, evening). While daily administration is preferred, administration once every few days or several times every other week is also possible. Furthermore, the saccharifying bacteria contained in the composition of the present disclosure are preferably present in the form of dormant spores to maintain the stability of the formulation. The recommended daily intake for humans is 10 ３ ~10 ２０ Individual bacterial cells (particularly spores), preferably 10 ５ ~10 １５ Individual bacterial cells (particularly spores), more preferably 10 ６ ~10 １３ The saccharifying bacteria germinate into different numbers of vegetative bodies (particularly spores) according to different in vivo environments, as described in Example 1, and exert the function of regulating neutrophil activation.

[0019] (Method for Regulating Neutrophil Activity) The "method for regulating neutrophil activity" of the present disclosure includes a step of administering the composition of the present disclosure to a mammal, including humans, livestock, and pets. Specific administration methods include, in the case of a composition containing a bacterium, dispersing the composition in an appropriate excipient and then administering it orally, rectally, enterally, intravaginally, or topically; in the case of a composition containing a treated or extracted residue of the bacterium, diluting the composition to an appropriate concentration and then administering it orally, rectally, enterally, intravaginally, or topically; intramuscularly, subcutaneously, intradermally, intraperitoneally, sublingually, nasal mucosally, transdermally, inhaling, or topically administering the composition to an organ and / or tissue suffering from a disease caused by insufficient or excessive activation of neutrophils.

[0020] (Use of saccharifying bacteria for producing a composition for regulating neutrophil activation) One embodiment of the present disclosure also relates to the use of the saccharifying bacteria described above for producing a composition for regulating neutrophil activation.

[0021] (Use of a Composition for Regulating Neutrophil Activation Comprising Saccharifying Bacteria) One embodiment of the present disclosure also relates to the use of a composition for regulating neutrophil activation comprising the above-described saccharifying bacteria.

[0022] The present disclosure will be described in detail below using specific examples, but the present disclosure is not limited to these examples.

[0023] (Materials and Methods) Example 2 below was carried out using the following materials and methods. In this example, Bacillus subtilis TO-A was used as the saccharifying bacterium, and neutrophil-like HL-60 cells were used as a neutrophil model.

[0024] [Maintenance culture of HL-60 cells] Undifferentiated HL-60 cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM medium) containing 20% ​​(v / v) FBS under humid conditions at 37°C, 5% CO2. Untreated cell culture flasks were used as culture vessels. [Induction of differentiation of neutrophil-like HL-60 cells] The HL-60 cell concentration was 1 x 10 5 After adjusting the concentration to viable cells / mL, dimethyl sulfoxide (DMSO) was added to IMDM medium to a final concentration of 1.25% (v / v), and the mixture was cultured for 5 days at 37°C, 5% CO2, and humidified. [Preparation of Bacterial Suspension] Bacillus subtilis TO-A colonies grown on brain heart infusion agar were suspended in brain heart infusion liquid medium and cultured overnight with shaking at 37°C and 100 rpm. The culture was diluted appropriately with the same medium and further cultured with shaking. After the logarithmic growth phase of the Bacillus subtilis TO-A culture was washed three times with Hanks' Balanced Salt Solution (HBSS), a vegetative suspension of Bacillus subtilis TO-A was prepared in HBSS containing 25 mM HEPES, and used in Example 2.

[0025] (Confirmation of the Function of Saccharifying Bacteria to Regulate Neutrophil Activation) In this example, it was confirmed whether saccharifying bacteria can promote and / or suppress NETs formation in neutrophils.

[0026] [NET induction] The neutrophil-like HL-60 cells were harvested using HBSS buffer containing 25 mM HEPES, and the viable cell count was measured using the trypan blue method. 6 Next, 50 μL of the cell suspension was added to each well of a 96-well plate, and the total number of neutrophil-like HL-60 cells in each well was adjusted to 1.0 x 10 5 The logarithmic growth phase Bacillus subtilis TO-A suspension was measured using a particle counter analyzer CDA-1000 (Sysmex Corporation), and the number of particles was measured at 2.0 x 10 using HBSS buffer containing 25 mM HEPES. 7 A Bacillus subtilis TO-A suspension was prepared at a concentration of 1.0x10 cells / mL. Next, 0, 0.5, 5.0, 12.5, 25.0, or 50.0 μL of Bacillus subtilis TO-A was added to wells of a 96-well plate containing neutrophil-like HL-60 cells at a multiplier (MOI) of 0, 0.1, 1, 2.5, 5, or 10 Bacillus subtilis TO-A per neutrophil-like HL-60 cell, respectively. Various amounts of HBSS buffer containing 25 mM HEPES were added to each well to adjust the total volume of each well to 100 μL. This adjustment was made so that the concentration of neutrophil-like HL-60 cells in each well was 1.0x10. 6 cells / mL, and the Bacillus subtilis TO-A suspension concentration was 0, 0.1, 1, 2.5, 5, or 10 x 1.0x10 6 This is a state where the number of cells / mL is 37. o C. After co-culture under humid conditions for 1 hour, PMA was added to a concentration of 10 nM and the cells were further cultured for 3 to 4 hours.

[0027] [Quantification of NET Release] NETs were induced with 10 nM PMA in a co-culture cell suspension. DNase I was added to a concentration of 0.4 U / mL and incubated at room temperature for 15 minutes to partially digest the NETs. The DNase I digestion reaction was then stopped by adding EGTA to a concentration of 2.5 mM. After centrifugation, the supernatant was used as a sample for quantitative analysis. Anti-MPO antibody (475915-1MLCN, Merck) diluted 500-fold in carbonate-bicarbonate buffer (pH 9.5) was added to a Nunc-Immuno Plate CII (430341, Thermo Fisher Scientific) (hereinafter referred to as the plate) and incubated overnight at 4°C. The plate was washed three times with PBS containing 0.05% (v / v) Tween 20 (hereinafter referred to as the wash buffer). The solution was replaced with PBS containing 2% (w / v) BSA (hereafter referred to as blocking buffer) and incubated at room temperature for 1 hour to further coat the plate. The plate was washed three times with wash buffer. Samples diluted to the desired concentration in blocking buffer were added and incubated at room temperature for 1 hour. The plate was washed three times with wash buffer. Peroxidase-conjugated anti-DNA antibody (clone MCA-33, 11544675001, Merck) diluted 320-fold in blocking buffer was added and incubated at room temperature for 1 hour. The plate was washed three times with wash buffer. A color reaction was performed using a TMB Peroxidase EIA Substrate Kit (#1721066, Biorad). The reaction was stopped by adding 1N sulfuric acid solution. The absorbance at 450 nm was measured, and this was used to determine the amount of NETs released into the culture supernatant.

[0028] [Results] PMA was used to induce NET formation in neutrophil-like HL-60 cells. Preparations were prepared using the t-butyl alcohol freeze-drying method and observed under a tabletop transmission electron microscope. Neutrophil-like HL-60 cells treated with DMSO alone exhibited spherical cells with surface protrusions (Fig. 1A). In contrast, neutrophil-like HL-60 cells treated with 10 nM PMA exhibited cells adhering to the bottom of the cells on an amorphous thin layer. Amorphous filamentous structures were also observed around the cells (Fig. 1B). Furthermore, after 15 minutes of incubation with 2 U / ml DNase I, the adhering cells remained visible, but the surrounding filamentous structures were no longer visible (Fig. 1C). Addition of PMA induced neutrophil-like HL-60 cells to burst, and DNA-containing filamentous structures were dispersed around the burst cells. Next, neutrophil-like HL-60 cells were co-cultured with 0, 0.1, 1, 2.5, 5, or 10 Bacillus subtilis TO-A cells at a multiplier of infection (MOI) of 0.1 to 1 (0.1 to 1x10). 6 When PMA-stimulated neutrophil-like HL-60 cells were co-cultured with Bacillus subtilis TO-A at an MOI of 2.5–10 (2.5–10 × 10 cells / mL), the amount of NETs released from the cells was increased compared to when PMA was not co-cultured with Bacillus subtilis TO-A. 6 When cells were co-cultured with Bacillus subtilis TO-A (at a concentration of 100 cells / mL), the amount of NETs was reduced compared to when cells were not co-cultured with Bacillus subtilis TO-A (Figure 2). These results suggest that the addition of a low ratio (low concentration) of Bacillus subtilis TO-A to neutrophil-like HL-60 cells, a model for neutrophils, promoted NET formation, whereas a high ratio (high concentration) inhibited NET formation. These results demonstrate that saccharifying bacteria (especially Bacillus subtilis) can modulate neutrophil activation (promote and / or inhibit activation) by promoting and / or inhibiting NET formation in neutrophils.

[0029] A composition for regulating neutrophil activation containing saccharifying bacteria can be provided.

Claims

1. A composition for regulating neutrophil activation, comprising a saccharifying bacterium.

2. The composition according to claim 1, wherein the saccharifying bacterium is a bacterium belonging to the genus Bacillus.

3. The composition according to claim 1, wherein the saccharifying bacterium is of the species Bacillus subtilis.

4. The composition according to claim 1, wherein the saccharifying bacterium is Bacillus subtilis TO-A.

5. The composition according to claim 3 or 4, wherein the regulation of neutrophil activation is the promotion and / or inhibition of the formation of NETs of neutrophils.

6. The composition according to claim 1, wherein the regulation is promotion.

7. A therapeutic agent for a disease caused by insufficient neutrophil activation, comprising the composition according to claim 6.

8. The composition according to claim 1, wherein the regulation is inhibition.

9. A therapeutic agent for a disease caused by excessive neutrophil activation, comprising the composition according to claim 8.

10. A method for regulating neutrophil activity, comprising the step of administering the composition according to any one of claims 1 to 4 to mammals other than humans.

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