Inducer of cells for treating inflammatory bowel disease, pharmaceutical composition for treating inflammatory bowel disease, and method for producing cells for treating inflammatory bowel disease

Abstract

To provide cell inducers and pharmaceutical compositions for inflammatory bowel disease treatment, which are applicable for a novel treatment agent for inflammatory bowel disease, and to provide methods for manufacturing a cell for inflammatory bowel disease treatment.SOLUTION: Provided is a cell inducer for inflammatory bowel disease treatment containing the membrane fraction component of Bacteroides bacteria. Provided is a pharmaceutical composition for inflammatory bowel disease treatment containing the inducer and a pharmaceutically acceptable carrier. Provided is a method for manufacturing a cell for inflammatory bowel disease treatment comprising: inducing the differentiation of a hematopoietic cell by contacting the hematopoietic cell with the inducer; and acquiring a differentiated cell from the hematopoietic cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an inducer of cells for treating inflammatory bowel disease, a pharmaceutical composition for treating inflammatory bowel disease, and a method for producing cells for treating inflammatory bowel disease. [Background technology]

[0002] In recent years, it has become clear that intestinal bacteria are involved in many diseases (Non-Patent Document 1), and there have been several reports on the involvement of intestinal bacteria in hematopoiesis. Mice in which intestinal bacteria were reduced by long-term administration of antibiotics showed various effects such as erythrocyte reduction, thrombocytosis, and lymphocyte reduction in peripheral blood, and not only atrophy of the thymus and spleen but also suppression of bone marrow hematopoiesis were observed (Non-Patent Document 2). The reason for this is that no change was observed in myeloid cells, but the proliferation of hematopoietic cells was suppressed and the number of cells was significantly reduced. Since these changes are reactions dependent on interferon signals, which are known as immune control factors, it is thought that immune activation occurs constantly by intestinal bacteria even under physiological conditions, and hematopoiesis is controlled through the interferon signals induced at that time. Similarly, it has been reported that a decrease in hematopoietic cells in the bone marrow was observed in germ-free mice that do not have intestinal bacteria, and that peptidoglycan (PGN), one of the bacterial components, is thought to be the causative molecule (Non-Patent Document 3). Furthermore, it has been reported that CX3CR1-positive cells exist in bone marrow as cells that detect the influx of enterobacteria into the bone marrow, detect the stimulation of enterobacteria-derived molecules in a Toll-like receptor-dependent manner, and produce humoral molecules such as IL-1β, IL-6, and TNF-α to control the proliferation of hematopoietic cells and the differentiation of myeloid lineages (Non-Patent Document 4). In addition, Tet-2 gene mutations cause blood cancer due to abnormal bone marrow proliferation in humans (Non-Patent Document 5), and it has been reported that a specific enterobacteria signal contributes to the onset of the disease (Non-Patent Document 6).

[0003] However, the effect of intestinal bacteria on hematopoiesis in inflammatory diseases such as intestinal inflammation, which has been increasing in recent years, remains unclear.

[0004] Inflammatory bowel disease is a disease that causes inflammation in the intestinal tract. Inflammatory bowel disease is mainly classified into ulcerative colitis and Crohn's disease. Both ulcerative colitis and Crohn's disease are intractable chronic diseases. In recent years, the number of patients with inflammatory bowel disease has been increasing, and effective treatment methods are required. The relationship between inflammatory bowel disease and intestinal bacteria has attracted attention, and a treatment method using probiotics has been proposed (for example, Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2016-517425 [Non-patent literature]

[0006] [Non-Patent Document 1] Kenya Honda, et al., Nature. The Microbiota in Adaptive Immune Homeostasis and Disease. Nature, 2016 Jul 7;535(7610):75-84. [Non-Patent Document 2] Kamilla S. Josefsdottir, et al., Antibiotics impair murine hematopoiesis by depleting the intestinal microbiota. BLOOD, 2017 Feb 9;129(6):729-739 [Non-Patent Document 3] Chiaki Iwamura, et al., Sensing of the microbiota by NOD1 in mesenchymal stromal cells regulates murine hematopoiesis. BLOOD, 2017 Jan 12;129(2):171-176 [Non-Patent Document 4] Seungwon Lee, et al., Bone marrow CX3CR1+ mononuclear cells relay a systemic microbiota signal to control hematopoietic progenitors in mice. BLOOD, 2019 Oct 17;134(16):1312-1322 [Non-Patent Document 5] Olivier Kosmider, et al., TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDSs). BLOOD, 2009 Oct 8;114(15):3285-3291 [Non-Patent Document 6] Marlies Meisel, et al., Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host. Nature, 2018 May;557(7706):580-584. Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, the effect of intestinal bacteria on hematopoiesis in inflammatory bowel disease has not been clarified at all. In addition, the intestinal flora is influenced by various factors, and even if a probiotic preparation such as that described in Patent Document 1 is taken, the desired intestinal flora is often not formed, and the desired therapeutic effect is often not achieved or is insufficient. Anti-inflammatory agents, immunomodulators, etc. are used as therapeutic agents for inflammatory bowel disease, but the effects of each are limited. Therefore, there is a demand for the development of a new IBD therapeutic agent.

[0008] Therefore, an objective of the present invention is to provide an inducer of therapeutic cells for inflammatory bowel disease, which can be used as a novel therapeutic agent for inflammatory bowel disease, a pharmaceutical composition containing the inducer, and a method for producing the therapeutic cells for inflammatory bowel disease. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A cell inducer for treating inflammatory bowel disease, containing membrane fraction components of Bacteroides bacteria. [2] A pharmaceutical composition for treating inflammatory bowel disease, comprising the inducer according to [1] and a pharma- ceutical acceptable carrier. [3] A method for producing cells for treating inflammatory bowel disease, comprising the steps of: contacting hematopoietic cells with the inducer described in [1] to induce differentiation of the hematopoietic cells; and obtaining cells differentiated from the hematopoietic cells. Effect of the Invention

[0010] According to the present invention, there are provided an inducer of therapeutic cells for inflammatory bowel disease, which can be used as a novel therapeutic agent for inflammatory bowel disease, a pharmaceutical composition containing the inducer, and a method for producing the therapeutic cells for inflammatory bowel disease. [Brief description of the drawings]

[0011] [Figure 1] Wild-type mice and mice lacking innate immune signals were administered dextran sulfate sodium (DSS) to induce enteritis, and FACS analysis of bone marrow cells and mesenteric lymph node cells was performed. [Diagram 2] FIG. 1 shows the results of counting LT-HSC, ST-HSC, and MPP2 in mesenteric lymph node cells based on the FACS analysis. [Diagram 3] The results of administering a Bacteroides membrane fraction to wild-type mice and mice lacking innate immune signals and performing FACS analysis of mesenteric lymph node cells are shown in Fig. 1. MPP and Gr-1 positive cells were counted by FACS analysis. [Figure 4A]Photographs show the intestines excised from DSS-administered wild-type mice (inducing enteritis) administered with anti-Gr-1 antibody or control antibody, and from water-administered wild-type mice administered with anti-Gr-1 antibody or control antibody. [Figure 4B] The results of measuring the lengths of the intestines excised from DSS-administered wild-type mice (inducing enteritis) administered with anti-Gr-1 antibody or control antibody, and from water-administered wild-type mice administered with anti-Gr-1 antibody or control antibody are shown. [Diagram 5] The graph shows changes in body weight during the test period in DSS-administered wild-type mice (inducing enteritis) administered with anti-Gr-1 antibody or control antibody, and in water-administered wild-type mice administered with anti-Gr-1 antibody or control antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] [Cell inducer for treating inflammatory bowel disease] In one embodiment, the present invention provides an inducer for cells for treating inflammatory bowel disease, comprising a membrane fraction component of a Bacteroides bacterium.

[0013] The inflammatory bowel disease is not particularly limited as long as it is a disease that causes inflammation in the intestinal tract. Representative inflammatory bowel diseases include ulcerative colitis and Crohn's disease. "Therapeutic cells for inflammatory bowel disease" (hereinafter, simply referred to as "therapeutic cells") refer to cells that contribute to the repair of intestinal tissue damaged by inflammatory bowel disease. Therapeutic cells are cells that are differentiated or induced to become hematopoietic cells. The differentiation or induction of therapeutic cells from hematopoietic cells is promoted by the inducer of the present embodiment.

[0014] The therapeutic cells are not particularly limited as long as they are cells induced from hematopoietic cells and have a therapeutic effect on enteritis. Whether or not a cell has a therapeutic effect on enteritis can be determined, for example, by administering the cell to a subject with enteritis and confirming whether the symptoms of enteritis are suppressed, reduced, alleviated, or disappeared. For example, in the case of mice, the therapeutic cells include Gr-1 positive cells. Gr-1 (Ly6G / Ly6C) is known to be highly expressed in mouse neutrophils, monocytes, etc., and Gr-1 positive cells are considered to include these cells. In humans, molecules corresponding to Gr-1 include, for example, CD14 / 15 (Pillay et al., Cell Mol Life Sci 2013 Oct;70(20):3813-27; Claudia et al., Cancer Immunol Immunother. 2012 Aug;61(8):1155-67.; Suzanne Ostrand-Rosenberg et al., J Immunol. 2018 Jan 15;200(2):422-431.) and CD177 (Sachs et al., J Biol Chem 2007 Aug 10;282(32):23603-12.; Hickey et al., Cochrane Database Syst Rev. 2012 Sep 12;2012(9):CD001895.). Examples of human therapeutic cells include cells positive for at least one selected from the group consisting of CD14, CD15, and CD177. Human CD14 is known to be highly expressed in human monocytes, and human CD15 is known to be highly expressed in human neutrophils. It has also been reported that human CD177 is involved in the migration of human neutrophils. Therefore, human therapeutic cells may include monocytes, neutrophils, etc.

[0015] "Hematopoietic cells" are cells that can differentiate into cellular components in blood (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, erythrocytes, platelets, mast cells, dendritic cells, etc.). Hematopoietic cells include hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs). Hematopoietic stem cells may be long-term hematopoietic stem cells (LT-HSCs) or short-term hematopoietic stem cells (ST-HSCs). The organism from which the hematopoietic cells are derived is not particularly limited. The hematopoietic cells are, for example, mammalian hematopoietic cells. The mammal is not particularly limited, and examples thereof include humans and non-human mammals. The non-human mammal is not particularly limited, and examples thereof include primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, etc. The hematopoietic cells are preferably mouse or human hematopoietic cells.

[0016] The term "membrane fraction component of Bacteroides bacteria" refers to a component fractionated in the cell membrane fraction of Bacteroides bacteria. The membrane fraction component may be the membrane fraction itself, an extract of a portion of the membrane fraction, or a component isolated and purified from the membrane fraction.

[0017] The Bacteroides bacteria are not particularly limited, and examples of the Bacteroides bacteria include, but are not limited to, Bacteroides dorei, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides stercoris, Bacteroides uniformis, and Bacteroides vulgatus.

[0018] The membrane fraction of the bacterium of the genus Bacteroides can be obtained by a known method for preparing a bacterial membrane fraction. For example, the membrane fraction can be prepared as follows. For example, Bacteroides bacteria are cultured under anaerobic conditions using a medium for growing enterobacteria such as YCFA medium (JCM medium number 1130). The culture temperature is not particularly limited, but can be, for example, 20 to 40°C, and is preferably 30 to 38°C. After the culture, bacterial cells are collected from the culture liquid by centrifugation or the like. Then, the bacterial cells are disrupted to prepare a bacterial disruption liquid. The method for disrupting bacteria is not particularly limited, and a known method can be used. Examples of the method for disrupting bacteria include ultrasonic treatment, French press, osmotic shock, homogenizer, and glass bead disruption. Then, the bacterial disruption liquid is ultracentrifuged to prepare a membrane fraction. The ultracentrifugation conditions may be any conditions that allow separation of the membrane fraction, and examples include 100,000 g for 60 minutes.

[0019] The inducer of the present embodiment can be used for inducing / differentiating therapeutic cells from hematopoietic cells in vitro or in vivo. Furthermore, the inducer of the present embodiment can be used for treating inflammatory bowel disease by being contained in a pharmaceutical composition described below.

[0020] [Pharmaceutical composition] In one embodiment, the present invention provides a pharmaceutical composition for treating inflammatory bowel disease comprising the inducer of the above embodiment and a pharma- ceutically acceptable carrier.

[0021] When the inducing agent of the embodiment is administered to a subject suffering from inflammatory bowel disease, therapeutic cells are induced in the subject's body. The induced therapeutic cells repair intestinal tissue damaged by enteritis, and symptoms of enteritis are suppressed, reduced, alleviated, or eliminated. Therefore, a pharmaceutical composition containing the inducing agent of the embodiment can be used for the treatment of inflammatory bowel disease. The inflammatory bowel disease to be treated by the pharmaceutical composition of the present embodiment is not particularly limited.

[0022] The subject of application of the pharmaceutical composition of the present embodiment is not particularly limited as long as it is an animal that develops inflammatory bowel disease. For example, the pharmaceutical composition of the present embodiment can be suitably used for humans or mammals other than humans. Mammals other than humans include, but are not particularly limited to, primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, etc.

[0023] The pharmaceutical composition of this embodiment may contain at least one pharma- ceutically acceptable carrier in addition to the inducing agent of the embodiment. The term "pharma-ceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the subject of administration. The term "not substantially toxic" refers to the ingredient not showing toxicity to the subject of administration at a dose normally used. In the pharmaceutical composition of this embodiment, the pharma-ceutically acceptable carrier is a carrier that does not inhibit the therapeutic cell induction action of the inducing agent of the embodiment and does not show substantial toxicity to the subject of administration. The pharma-ceutically acceptable carrier includes any known pharma-ceutically acceptable ingredient that is typically considered to be an inactive ingredient. The pharma- ceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, flow enhancers, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizing agents, stabilizers, emulsifying agents, fillers, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweetening agents, thickening agents, buffers, coloring agents, etc. One type of pharma- ceutically acceptable carrier may be used alone, or two or more types may be used in combination.

[0024] The pharmaceutical composition of the present embodiment may contain optional ingredients in addition to the above. The optional ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without any particular limitation. The pharmaceutical composition of the present embodiment may contain active ingredients other than the inducer of the above embodiment. Examples of active ingredients include vitamins and their derivatives, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, irritation relief agents, analgesics, cell activators, plant, animal, and microbial extracts, antipruritic agents, anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensive agents, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptic agents, coronary vasodilators, herbal medicines, antipruritics, and keratin softening and peeling agents, but are not limited thereto. The other ingredients may be used alone or in combination of two or more.

[0025] The dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and may be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Examples of parenteral preparations include injections, suppositories, ointments, sprays, external liquids, nasal drops, and inhalants. Pharmaceutical compositions of these dosage forms can be formulated according to standard methods (for example, methods described in the Japanese Pharmacopoeia).

[0026] The administration route of the pharmaceutical composition of the present embodiment is not particularly limited, and can be administered orally or parenterally. The parenteral route includes all administration routes other than oral, such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, rectal, intravaginal, and intraperitoneal administration. The administration can be local or systemic.

[0027] The pharmaceutical composition of the present embodiment can be administered in a therapeutically effective amount of the inducer of the embodiment. The term "therapeutically effective amount" means an amount of a drug effective for treating or preventing a target disease. For example, the therapeutically effective amount of the inducer of the embodiment (membrane fraction component of Bacteroides bacteria) can be an amount capable of suppressing, reducing, alleviating, or eliminating the symptoms of inflammatory bowel disease. The therapeutically effective amount may be appropriately determined depending on the symptoms, weight, age, and sex of the patient, the dosage form of the pharmaceutical composition, and the administration method. For example, the pharmaceutical composition of the present embodiment can be administered in a single dose of the inducer of the embodiment (membrane fraction component of Bacteroides bacteria) in an amount of 0.01 to 1000 mg per kg of the body weight of the subject. The dose may be 0.15 to 800 mg / kg, 0.5 to 500 mg / kg, 1 to 400 mg / kg, or 1 to 300 mg / kg.

[0028] The pharmaceutical composition of the present embodiment may contain a therapeutically effective amount of the inducer (membrane fraction component of Bacteroides bacteria) of the above embodiment per unit dosage form. For example, the content of the inducer (membrane fraction component of Bacteroides bacteria) of the above embodiment in the pharmaceutical composition of the present embodiment may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.

[0029] The administration interval of the pharmaceutical composition of the present embodiment may be appropriately determined depending on the symptoms, body weight, age, sex, etc. of the subject, as well as the dosage form of the pharmaceutical composition, the administration method, etc. The administration interval may be, for example, every few hours, once a day, once every 2 to 3 days, once a week, etc.

[0030] The pharmaceutical composition of the present embodiment may be used in combination with other pharmaceuticals, for example, other therapeutic agents for enteritis.

[0031] [Method for producing cells for treating inflammatory bowel disease] In one embodiment, the present invention provides a method for producing cells for treating inflammatory bowel disease, comprising the steps of contacting hematopoietic cells with the inducer of the embodiment to induce differentiation of the hematopoietic cells (hereinafter also referred to as "Step A"), and obtaining cells differentiated from the hematopoietic cells (hereinafter also referred to as "Step B").

[0032] <Process A> In step A, hematopoietic cells are contacted with the inducer of the above embodiment to induce differentiation of the hematopoietic cells.

[0033] The hematopoietic cells are the same as those described in the above section [Cell inducer for treating inflammatory bowel disease]. Hematopoietic cells can be obtained from blood or bone marrow fluid by known methods. For example, a method of isolating hematopoietic cells by flow cytometry using an antibody that binds to a membrane antigen (hematopoietic cell marker) specific to hematopoietic cells can be mentioned. Examples of human hematopoietic cell markers include CD34 and CD38. Examples of mouse hematopoietic cell markers include CD150 and CD48.

[0034] The method of contacting the inducer of the embodiment with hematopoietic cells is not particularly limited. For example, the inducer of the embodiment is added to a culture solution for hematopoietic cells, and the cells are cultured. The culture conditions are not particularly limited, and the cells can be cultured under generally used culture conditions for hematopoietic cells using a general culture medium for hematopoietic cells. Examples of media used for culturing hematopoietic cells include S-Clone medium (e.g., SF-O3 medium, manufactured by EIDIA), StemPro34 (manufactured by Invitrogen), Stemline (manufactured by Sigma-Aldrich), Stemline II (manufactured by Sigma-Aldrich), X-VIVO 10 (manufactured by Lonza), X-VIVO 15 (manufactured by Lonza), X-VIVO 20 (manufactured by Lonza), HPGM (manufactured by Lonza), StemSpan H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), and QBSF-60 (manufactured by Quality Biological). Culture conditions include, for example, 5% CO2 and 37°C.

[0035] By contacting hematopoietic cells with the inducing agent of the above embodiment, it is possible to induce differentiation of hematopoietic cells into therapeutic cells.

[0036] <Process B> In step B, cells differentiated from the hematopoietic cells are obtained. The method for recovering the differentiated cells is not particularly limited, and any known method can be used, for example, a method of recovering the cells by centrifuging the culture medium. Since differentiation of hematopoietic cells into therapeutic cells is induced in step A, the cells obtained in step B contain a large number of therapeutic cells. In this manner, therapeutic cells can be produced.

[0037] The cells obtained by the production method of this embodiment can be used for the treatment of inflammatory bowel disease.

[0038] [Other aspects] In one embodiment, the present invention provides use of a membrane fraction component of a Bacteroides bacterium in the manufacture of an inducer of cells for the treatment of inflammatory bowel disease. In one embodiment, the present invention provides the use of a membrane fraction component of a Bacteroides bacterium in the manufacture of a pharmaceutical composition for the treatment of inflammatory bowel disease. In one embodiment, the present invention provides a method for inducing cells for treating inflammatory bowel disease, comprising administering a membrane fraction component of a Bacteroides bacterium to a subject (e.g., a patient suffering from inflammatory bowel disease). In one embodiment, the present invention provides a method for treating inflammatory bowel disease, comprising administering a membrane fraction component of a Bacteroides bacterium to a subject (e.g., a patient suffering from inflammatory bowel disease). In one embodiment, the present invention provides a membrane fraction component of a Bacteroides bacterium for use in inducing cells for treating inflammatory bowel disease. In one embodiment, the present invention provides a membrane fraction component of a Bacteroides bacterium for use in the treatment of inflammatory bowel disease. EXAMPLES

[0039] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0040] [Materials] <Mouse> C57BL / 6 (CD45.2 + ) and B6.SJL (CD45.1 + ) mice (hereinafter also referred to as "wild-type mice" or "WT") were purchased from Japan SLC and Jackson Laboratories, respectively. Trif - / - ; Myd88 - / - mice (CD45.2 + ) (hereinafter also referred to as "natural immune signal-deficient mice" or "DKO") were purchased from Oriental BioService. All mice were housed at the Animal Resource Development Center of Kumamoto University. All experiments were approved by the Animal Experiment Committee of Kumamoto University. Natural immune signal-deficient mice lack the signal transduction molecules (Trif, Myd88) of Toll-like receptors, so signal transduction of Toll-like receptors does not occur.

[0041] [Example 1] (Increase in hematopoietic cells dependent on natural immune signals due to enteritis) <Induction of colitis> To induce acute colitis, wild-type mice and natural immune signal-deficient mice were treated with distilled water containing 2.5% dextran sulfate sodium (DSS) (35 - 50 kDa; MP Biomedicals, Tokyo, Japan) or distilled water without DSS for 7 days, and then treated with distilled water.

[0042] <FACS analysis> All antibodies were purchased from Thermo Fisher Scientific or Biolegend unless otherwise stated. Bone marrow or mesenteric lymph node cells were stained by incubation with the following biotinylated antibodies against lineage (Lin) markers: NK1.1 (PK136), CD11b (M1 / 70), Ter119 (Ter119), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8α (53-6.7), CD3ε (145-2C11), B220 (RA3-6B2), and IL-7Rα (SB / 199); and fluorescently labeled antibodies: c-Kit (2B8), Sca-1 (D7), fluorescently labeled antibodies: CD150 (TC15-12F12.2), and CD48 (HM48-1). Stained cells were analyzed using a FACS Rria III (BD Biosciences). FACS data analysis was performed using mFlowJo (BD Biosciences).

[0043] <Result> The results of the FACS analysis are shown in Figure 1. The results of counting LT-HSC, ST-HSC, and MPP2 by FACS analysis are shown in Figure 2. As shown in Figures 1 and 2, in wild-type mice, colitis induction increased MPPs in both bone marrow and mesenteric lymph nodes. In wild-type mice, colitis induction also increased LT-HSCs in bone marrow. In contrast, in innate immune signaling-deficient mice, no increase in LT-HSCs, ST-HSCs, or MPP2 was observed in either bone marrow or mesenteric lymph nodes. These results indicate that colitis-induced increase in hematopoietic cells is dependent on innate immune signals.

[0044] [Example 2] (Increase in MPP and Gr-1 positive cells due to Bacteroides) <Preparation of bacterial membrane fraction> Bacteroides (a mixture of Bacteroides dorei, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides stercoris, Bacteroides uniformis, and Bacteroides vulgatus in equal amounts) and Faecalibacterium prausnitzii were purchased from the RIKEN BioResource Center. Bacteroides was grown in GAM medium (Nissui) under anaerobic conditions in a Bactron 300 (Shellab). Faecalibacterium prausnitzii was grown in YCFA medium (JCM medium No. 1130) under anaerobic conditions. Each bacterium was recovered from each medium, centrifuged at 7,000 rpm for 5 minutes at 4°C, and the bacterial pellet was washed 3 times with PBS and resuspended in PBS. Next, the cells were sonicated on ice at maximum output at 30-second intervals using a UD-100 sonicator (TOMY). The bacterial lysate was ultracentrifuged at 100,000 g for 60 minutes at 4°C (CS 100FNX, HITACHI), and the resulting membrane fraction was resuspended in PBS to prepare a membrane fraction solution. The protein concentration was measured using a Bio-Rad Protein Assay Concentrate Dye Reagent (BioRad).

[0045] <Administration of cell membrane fraction> Wild-type mice and innate immune-deficient mice were intraperitoneally administered 15 μg of the bacterial membrane fraction solution (Bacteroides or Faecalibacterium prausnitzii) or PBS. Sixteen hours later, the intestinal mesenteric lymph nodes were removed and subjected to FACS analysis.

[0046] <FACS analysis> Mesenteric lymph node cells were stained by incubation with biotinylated antibodies against lineage (Lin) markers: NK1.1 (PK136), CD11b (M1 / 70), Ter119 (Ter119), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8α (53-6.7), CD3ε (145-2C11), B220 (RA3-6B2), and IL-7Rα (SB / 199); and fluorescently labeled antibodies: c-Kit (2B8), Sca-1 (D7), CD34 (HM34), Flt3 (A2F10), CD150 (TC15-12F12.2), CD48 (HM48-1), and CD16 / 32 (93). Cells were analyzed using a FACS Aria III (BD Biosciences). FACS data analysis was performed using mFlowJo (BD Biosciences). FACS data analysis was performed using mFlowJo (BD Biosciences).

[0047] <Result> Figure 3 shows the results of counting MPP and Gr-1 positive cells by FACS analysis. In wild-type mice, administration of Bacteroides membrane fraction significantly increased hematopoietic cells and Gr-1 cells. In particular, Gr-1 cells increased significantly in wild-type mice administered Bacteroides membrane fraction compared to wild-type mice administered PBS or Faecalibacterium prausnitzii membrane fraction. On the other hand, in mice with innate immunity deficiency, administration of Bacteroides membrane fraction did not result in a significant increase in MPP and Gr-1 positive cells. These results indicate that the Bacteroides membrane fraction induces the proliferation of MPPs and their differentiation into Gr-1 positive cells. In addition, in mice with innate immunity deficiency, the increase in MPPs and Gr-1 positive cells due to the Bacteroides membrane fraction was not observed, confirming that the action of the Bacteroides membrane fraction is innate immune signal-dependent. In other words, it is considered that the Bacteroides membrane fraction components bind to Toll-like receptors and induce Toll-like receptor signaling, which results in the proliferation of hematopoietic cells and their differentiation into Gr-1 positive cells.

[0048] [Example 3] [Induction of colitis] Colitis was induced in wild-type mice in the same manner as in Example 1. Body weight was measured using a weighing scale every few days from the start of the induction treatment.

[0049] [Removal of Gr-1 positive cells] On the 6th day after the start of the treatment, neutralizing antibody or control antibody was intraperitoneally administered to wild-type mice administered with double-distilled water containing DSS or double-distilled water. As the neutralizing antibody, 500 μg of anti-Gr-1 neutralizing antibody (RB6-8C5) (BioXCell, West Lebanon, NH, USA) was used. As the control antibody, an IgG2b isotype-matched antibody (BioXCell) was used.

[0050] [Measurement of intestinal tract] Two days after the administration of the antibody, the mice were sacrificed and the intestinal tract was excised. The length of the excised intestinal tract was measured with a ruler with an accuracy of 1 mm units.

[0051] [Results] Figure 4A shows a photograph of the intestinal tract. Figure 4B shows the results of measuring the length of the intestinal tract. As shown in Figures 4A and 4B, in the mice induced with colitis, the length of the intestinal tract became shorter. By administering anti-Gr-1 neutralizing antibody to the mice induced with colitis, the length of the intestinal tract became even shorter. In the mice not induced with colitis, no significant difference was confirmed in the length of the intestinal tract between those administered with anti-Gr-1 neutralizing antibody and those administered with control antibody.

[0052] Figure 5 shows the change in body weight of the mice during the test. In the mice induced with colitis, the body weight decreased significantly compared to the mice not induced with colitis. By administering anti-Gr-1 neutralizing antibody to the mice induced with colitis, the body weight further decreased. In the mice not induced with colitis, no significant difference was confirmed in the body weight between those administered with anti-Gr-1 neutralizing antibody and those administered with control antibody.

[0053] These results indicate that Gr-1 positive cells contribute to tissue repair in intestinal inflammation.

[0054] These results suggest that when enteritis occurs, hematopoietic cells are activated and induced to differentiate into Gr-1+ cells, which then contribute to the repair of intestinal tissue. [Industrial Applicability]

[0055] According to the present invention, there are provided an inducer of therapeutic cells for inflammatory bowel disease, which can be used as a novel therapeutic agent for inflammatory bowel disease, a pharmaceutical composition containing the inducer, and a method for producing the therapeutic cells for inflammatory bowel disease.

Claims

1. An inducer for treating inflammatory bowel disease, comprising a membrane fraction component of a bacteroides bacterium, The therapeutic cells are cells positive for Gr-1 or a molecule corresponding to Gr-1, The Gr-1 or Gr-1-corresponding molecule positive cell is a monocyte or a neutrophil. Inducer.

2. A pharmaceutical composition for treating inflammatory bowel disease, comprising the inducer of claim 1 and a pharma- ceutically acceptable carrier.

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