Composition for inducing proliferation or accumulation of regulatory cells

ES 2 809 232 T3

DESCRIPTION

Composition to induce proliferation or accumulation of regulatory cells

Technical field

This document describes a composition that has an effect to induce proliferation or accumulation of regulatory T cells, and which comprises, as an active principle, bacteria belonging to the genus Clostridium, a physiologically active substance derived from bacteria, bacterial spores, or Similar.

A method for inducing regulatory T cell proliferation or accumulation is also described, as well as a method for inhibiting such proliferation or accumulation. Also described is a vaccine composition containing at least one strain of bacteria belonging to the genus Clostridium or a spore of bacteria, as well as a method for treating or preventing at least one disease or a selected state of infectious diseases and autoimmune diseases by administering of the vaccine composition to an individual in need of it.

Also described is a method for selecting a compound that promotes the proliferation or accumulation of regulatory T cells, as well as a non-human mammal that is used in this method, and in which a reporter gene is expressed under the control of the expression of the gene. of IL-10.

Background of the technique

Hundreds of species of commensal microorganisms are found in the gastrointestinal tracts of mammals, interacting intimately with the host's immune systems. Some research results using aseptic animals (GF, germ-free) have shown that commensal microorganisms exert a great influence on the development of mucosal immune systems such as histogenesis of Peyer's patches (PP) and isolated lymphatic follicles (ILF). ), secretion of antimicrobial peptides from the epithelium and accumulation of single lymphocytes in mucosal tissues, including plasma single lymphocytes that produce immunoglobulin A, intraepithelial lymphocytes, IL-17 (Th 17) producing CD4 positive T cells, and IL-22 producing NK lymphocyte-like cells (non-patent documents 1 to 7). Consequently, the presence of intestinal bacteria enhances the protective functions of the mucous membranes, endowing the hosts with robust immune responses against pathogenic microbes that invade the organisms. On the other hand, the mucosal immune systems maintain the lack of response to food antigens and harmless microbes (non-patent document 3). For this reason, an abnormality in the regulation of interference between commensal bacteria and an immune system (intestinal dysbiosis) can lead to an overly robust immune response to environmental antigens, causing inflammatory bowel disease (IBD) (documents no. Patent 8-10).

Some recent study results have shown that individual commensal bacteria control the differentiation of their specific immune cells in the mucosal immune system. For example, Bacteroides fragilis, which is a commensal bacterium in humans, specifically induces a systemic Th1 cell response and an IL-10-producing T cell response in the mucosa in mice, and plays a role in host protection against infection. colitis, which would otherwise be caused by a pathogen (non-patent document 3). Segmented filamentous bacteria, which are intestinal commensal bacteria in mice, have been shown to induce a Th17 cell response in the mucosa and thus enhance resistance against infection of the host's gastrointestinal tracts with a pathogen (documents no. Patent 11-13). Furthermore, short chain fatty acids derived from various commensal bacteria are known to suppress intestinal inflammation (non-patent document 14). Furthermore, it is presumed that the presence of some species of gut microbiota exerts a great influence on the differentiation of regulatory T cells (hereinafter referred to as "Treg cells") that maintain the homeostasis of the immune system.

Meanwhile, the regulatory T cells that have been identified as a subset that suppress immunity are CD4 T cells.⁺ in which a Foxp3 transcription factor is expressed, and are known to play an important role in the maintenance of immunological homeostasis (non-patent documents 8, 9, 15 and 16). Furthermore, Foxp3-expressing cells are known to be present in large numbers, especially in the colon, and only Treg cells present locally in the colon consistently express IL-10, which is an immunosuppressive cytokine, at a high level (non-patent document 17). Animals that have CD4 cells are also known to⁺ Foxp3⁺, from which IL-10 is specifically eliminated, develop inflammatory bowel disease (non-patent document 18).

Therefore, if the mechanism of induction of IL-10 producing Treg cells in the colon at a high level is elucidated, immunosuppression can be enhanced, which in turn can be applied to the treatment of autoimmune diseases such as inflammatory bowel disease. as well as organ transplantation.

However, the mechanisms of how large numbers of Treg cells become present in the colon and how

ES 2 809 232 T3 Treg cells produce IL-10 in the colon at a high level still remains unclear. Furthermore, it remains unclear which species of bacteria that make up the intestinal commensal bacterial flora exert the influence on the induction of regulatory T cells.

List of references

Non-patent bibliography

[NPL 1] JJ Zebra, "Am J Clin Nutr", May 1999, 69, 1046S

[NPL 2] AJ Macpherson, NL Harris, "Nat Rev Immunol," June 2004, 4, 478

[NPL 3] JL Round, SK Mazmanian, "Nat Rev Immunol," May 2009, 9, 313

[NPL 4] D. Bouskra et al., "Nature", November 27, 2008, 456, 507

[NPL 5] K. Atarashi et al., "Nature", October 9, 2008, 455, 808

[NPL 6] Ivanov, II et al., "Cell Host Microbe", October 16, 2008, 4, 337

[NPL 7] SL Sanos et al., "Nat Immunol", January 2009, 10, 83

[NPL 8] MA Curotto de Lafaille, JJ Lafaille, "Immunity", May 2009, 30, 626

[NPL 9] MJ Barnes, F. Powrie, "Immunity," September 18, 2009, 31, 401

[NPL 10] WS Garrett et al., "Cell," October 5, 2007, 131, 33

[NPL 11] Ivanov, II et al., "Cell", October 30, 2009, 139, 485.

[NPL 12] V. Gaboriau-Routhiau et al., "Immunity", October 16, 2009, 31, 677

[NPL 13] NH Salzman et al., "Nat Immunol", 11, 76.

[NPL 14] KM Maslowski et al., "Nature," October 29, 2009, 461, 1282

[NPL 15] LF Lu, A. Rudensky, "Genes Dev", January 1, 2009, 23, 1270

[NPL 16] S. Sakaguchi, T. Yamaguchi, T. Nomura, M. Ono, "Cell", May 30, 2008, 133, 775

[NPL 17] CL Maynard et al., "Nat Immunol", September 2007, 8, 931

[NPL 18] YP Rubtsov et al., "Immunity", April 2008, 28, 546

GABORIAU-ROUTHIAU et al. (2009), Immunity 31: 677 describe segmented filamentous bacteria (FBS) that do not belong to the genus Clostridium, and are in fact phylogenetically distant from them.

SOKOL et al. (2008-10-28) "Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients", Proc Natl Acad Sci USA 105 (43): 16731-16736, propose Faecalibacterium prausnitzii (belonging to the group Clostridium IV) as a candidate probiotic agent in the treatment of Crohn's disease (CD).

KARIMI et al. (2009), "Lactobacillus reuteri-induced regulatory T cells protect against and allergic airway response in mice" American Journal of Respiratory and Critical Care Medicine 17 (3): 186-193, describe that Lactobacillus reuteri induces Foxp3 regulatory T cells⁺ which have the potential to protect against and response of allergic airways in mice.

EP 1 955 706 A1 refers to a composition containing alpha interferon and natural bile pigment Cphocyanin for treating autoimmune diseases, allergy and cancer by inducing regulatory T cells.

ITOH K et al. (1985) "Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice." Laboratory Animals 19 (2): 111-118, disclose the characterization of 115 Clostridium strains accumulated from 3 separate isolates from the feces of 1 mouse with limited flora (LF) produced by inoculating aseptic mice with chloroform-treated feces of conventional mice, and the effect was studied. on the size of the cecum when associated with aseptic mice.

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BAKKEN (2009) Anaerobe 15: 285-289 refers to fecal bacteriotherapy as a treatment for recurrent Clostridium difficile infection.

Summary of the invention

The invention is defined in the claims, and provides a pharmaceutical composition for use in a method of suppressing the immunity of a subject to prevent or treat an autoimmune disease or an allergic disease by inducing proliferation or accumulation of positive regulatory T cells. for the Foxp3 transcription factor, the composition comprising, as active principle, bacteria belonging to the genus Clostridium, wherein the bacteria induce said proliferation or accumulation of positive regulatory T cells for the Foxp3 transcription factor. Other aspects of the invention are also defined in the claims.

The present inventors have discovered that a chloroform-treated fraction and a spore-forming fraction of a fecal sample obtained from a mammal induces the accumulation of regulatory T cells (Treg cells) in the colon. Furthermore, the present inventors have discovered that bacteria belonging to the genus Clostridium induce proliferation or accumulation of regulatory T cells in the colon. The present inventors have also discovered that the regulatory T cells induced by these bacteria suppress the proliferation of effector T cells. Furthermore, the present inventors have also discovered that the colonization of bacteria belonging to the genus Clostridium and the resulting proliferation or accumulation of Treg cells regulate local and systemic immune responses.

From these findings, the present inventors have discovered that the use of bacteria belonging to the genus Clostridium, spores thereof or a physiologically active substance derived therefrom allows to induce the proliferation or accumulation of regulatory T cells (Treg cells), and also suppress immune functions.

Advantageous effects

The compositions described herein, which contain as active ingredient bacteria belonging to the genus Clostridium or a physiologically active substance derived from bacteria, serve as an excellent composition for inducing the proliferation or accumulation of regulatory T cells (Treg cells). Immunity in a living organism can be suppressed, according to the claims, through the administration of the composition of the present invention as defined in the claims as a pharmaceutical product or by ingesting the composition as a food or a drink. Accordingly, the composition of the present invention can be used to prevent or treat autoimmune diseases or allergic diseases, as defined in the claims. It is also disclosed to suppress immune rejection in organ transplantation or the like. Furthermore, if a food or drink, such as a health food, comprises the composition of the present invention, healthy individuals can ingest the composition easily and routinely. As a result, it is possible to induce the proliferation or accumulation of regulatory T cells and thus enhance the immune functions, as defined in the claims.

Brief description of the drawings

[Figure 1] Figure 1 is a schematic diagram showing a production method of an Il10 mouse^Venus.

[Figure 2] Figure 2 is a diagram showing the results of Southern blotting performed for analysis as to whether Il10 mice^Venus whether or not they have an Il10 allele^Venus.

[Figure 3] Figure 3 is a FACS dot plot diagram showing the results obtained when Venus positive cells and Venus negative cells from Il10 mice were sorted.^Venus.

[Figure 4] Figure 4 is a graph showing the results obtained when the amounts of IL-10 mRNA expressed in Venus-positive cells and Venus-negative cells from Il10 mice were analyzed.^Venusby real-time RT-PCR.

[Figure 5] Figure 5 is a graph showing the change in Foxp3 cell ratio⁺ in CD4 lymphocytes⁺ of SPF mice.

[Figure 6] Figure 6 shows FACS dot plot diagrams showing Foxp3 cell ratios analysis results⁺ in CD4 lymphocytes⁺ isolated from the small intestine, colon, and peripheral lymph nodes of GF mice and SPF mice.

[Figure 7] Figure 7 is a graph showing the results of Foxp3 cell ratios analysis⁺ in CD4 lymphocytes⁺ isolated from the small intestine, colon, and peripheral lymph nodes of GF mice and SPF mice.

[Figure 8] Figure 8 shows graphs showing the results of the analysis of CD4 cell numbers⁺ Foxp3⁺

ES 2 809 232 T3 isolated from the small intestine, colon and peripheral lymph nodes of GF mice and SPF mice.

[Figure 9] Figure 9 is a graph diagram showing the results of the analysis of the Venus cell ratios⁺ on CD4 cells⁺ in various tissues of SPF mice treated with antibiotics.

[Figure 10] Figure 10 shows FACS dot plot diagrams showing Foxp3 cell ratio analysis results⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of the colon of GF mice to which a fecal suspension of SPF mice was administered.

[Figure 11] Figure 11 is a graph showing the results of Foxp3 cell ratios analysis⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of the colon and the lamina propria of the small intestine of GF mice to which a fecal suspension of SPF mice was administered.

[Figure 12] Figure 12 is a graph showing the results of Foxp3 cell ratio analysis⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of mice deficient in ILF, PP and colon plaques.

[Figure 13] Figure 13 shows FACS dot plot diagrams showing Foxp3 cell ratios analysis results⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of the colon of GF mice given specific commensal bacteria.

[Figure 14] Figure 14 shows graphs showing the results of Foxp3 cell ratios analysis⁺in CD4 lymphocytes⁺ isolated from the lamina propria of the colon of GF mice given specific commensal bacteria.

[Figure 15] Figure 15 is a graph showing the results of the analysis of the IFN-y cell ratios⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of the colon of mice in which specific commensal bacteria were colonized.

[Figure 16] Figure 16 is a graph showing the results of the analysis of the IL-17 cell ratios⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of the colon of mice in which specific commensal bacteria were colonized.

[Figure 17] Figure 17 is a graph showing the results of Foxp3 cell ratios analysis⁺ in CD4 lymphocytes⁺ isolated from the colon of SPF mouse types each being deficient in a pathogen-associated molecular pattern recognition receptor-associated factor.

[Figure 18] Figure 18 is a graph showing the results of Foxp3 cell ratios analysis⁺ in CD4 lymphocytes⁺ isolated from the lamina propria of the colon of Myd88 mice^-/- in which Clostridium was colonized.

[Figure 19] Figure 19 shows FACS dot plot diagrams showing Venus cell ratio analysis results⁺ in lymphocytes isolated from various tissues of Il10 mice^Venus.

[Figure 20] Figure 20 is a FACS dot plot diagram showing the results of analysis of the expression of a β chain of the T cell receptor on cell surfaces of lymphocytes isolated from the lamina propria of the colon of Il10 mice.^Venus.

[Figure 21] Figure 21 shows FACS dot plot diagrams showing the results of analysis of IL-17, IL-4 and IFN-γ expression in lymphocytes isolated from the lamina propria of the colon of Il10 mice.^Venus.

[Figure 22] Figure 22 shows graphs showing the results of the analysis of the amounts of IL10, CTLA4, Foxp3 and GITR mRNA expressed in Foxp3 cells^- CD4⁺ spleen, CD4 cells⁺ Foxp3⁺ spleen, Venus cells⁺of the lamina propria of the colon and Venus cells⁺ of the lamina propria of the small intestine.

[Figure 23] Figure 23 shows FACS dot plot diagrams showing the results of analysis of CD4, Foxp3 and Venus expression in the lamina propria of the small intestine and the lamina propria of the colon of Il10 mice.^Venus GF and Il10 mice^Venus SPF.

[Figure 24] Figure 24 shows FACS dot plot diagrams showing results of analysis of CD4 cell expression of Venus and Foxp3 in various tissues of SPF Il10 mice^Venus.

[Figure 25] Figure 25 shows FACS dot plot diagrams showing the results of analysis of Foxp3 and Venus expression in Il10 mice^Venus in which specific commensal bacteria were colonized.

[Figure 26] Figure 26 is a graph showing the results of the analysis of Foxp3 and / or Venus expression of CD4 cells⁺ in the small intestine of Il10 mice^Venus in which specific commensal bacteria were colonized.

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[Figure 27] Figure 27 is a graph showing the results of the analysis of Foxp3 and / or Venus expression of CD4 cells⁺ in the colon of Il10 mice^Venus in which specific commensal bacteria were colonized.

[Figure 28] Figure 28 is a graph diagram showing the results of the analysis of the Venus cell ratios⁺ on CD4 cells⁺ isolated from various tissues of Il10 mice^Venus treated with antibiotics.

[Figure 29] Figure 29 is a graph showing the results of the analysis of immunoregulatory functions of CD4 cells⁺ Venus⁺ of the lamina propria of the colon of Il10 mice^Venus GF in which the genus Clostridium was colonized, CD4 cells⁺ Venus⁺ of the lamina propria of the colon of Il10 mice^Venus SPF and CD4 cells⁺ GFP⁺ spleen of Foxp3 indicator mice^eGFP

[Figure 30] Figure 30 is a graph showing the results obtained when SPF B6 mice were treated with polymyxin B or vancomycin for 4 weeks, and then analyzed for Foxp3 cell ratio⁺ in the CD4 cell cluster⁺.

[Figure 31] Figure 31 is a graph showing the results obtained when chloroform-treated feces derived from SPF mice were orally administered to GF mice, and then analyzed to determine the Foxp3 cell ratio⁺ in the CD4 cell cluster⁺.

[Figure 32] Figure 32 is a graph showing the general results of flow cytometric analysis of Helios expression in LP lymphocytes in the thymus or colon of SPF mice, GF mice, Lactobacillus colonized mice, or mice colonized with Clostridium.

[Figure 33] Figure 33 shows graphical diagrams showing representative results of flow cytometric analysis of CD4 expression, Foxp3 expression, and Helios expression in LP lymphocytes in the thymus or colon of SPF mice. , GF mice, Lactobacillus colonized mice or Clostridium colonized mice.

[Figure 34] Figure 34 is a graph showing the results obtained when whole colons derived from GF mice, Lactobacillus colonized mice or Clostridium colonized mice were cultured, and the culture supernatants thereof were analyzed to determine the concentration. of TGF-P1 by ELISA.

[Figure 35] Figure 35 is a graph showing the results obtained when intestinal epithelial cells (IEC) derived from GF mice or Clostridium colonized mice were cultured, and the culture supernatants thereof were analyzed to determine the concentration of TGF -P1 by ELISA.

[Figure 36] Figure 36 is a graph showing the results obtained when CD4 T cells were cultured⁺ of the spleen together with an anti-CD3 antibody and with an IEC culture supernatant isolated from GF mice or mice colonized with 46 bacterial strains of the genus Clostridium (Clost.) in the presence or absence of an anti-TGF-p antibody, and the T cells on day 5 of culture and analyzed for Foxp3 expression by real-time RT-PCR.

[Figure 37] Figure 37 is a graph showing the results obtained when C57BL / 6 GF mice were inoculated orally with 46 bacterial strains of the genus Clostridium (Clost.) Or three bacterial strains of the genus Lactobacillus (Lacto.), And IECs were collected three weeks after inoculation and analyzed for relative mRNA expression level of the MMP2 gene by real-time RT-PCR.

[Figure 38] Figure 38 is a graph showing the results obtained when C57BL / 6 GF mice were inoculated orally with 46 bacterial strains of the genus Clostridium (Clost.) Or three bacterial strains of the genus Lactobacillus (Lacto.), And IECs were collected three weeks after inoculation and analyzed for relative mRNA expression level of the MMP9 gene by real-time RT-PCR.

[Figure 39] Figure 39 is a graph showing the results obtained when C57BL / 6 GF mice were inoculated orally with 46 bacterial strains of the genus Clostridium (Clost.) Or three bacterial strains of the genus Lactobacillus (Lacto.), And IECs were collected three weeks after inoculation and analyzed for relative mRNA expression level of the MMP13 gene by real-time RT-PCR.

[Figure 40] Figure 40 is a graph showing the results obtained when C57BL / 6 GF mice were inoculated orally with 46 bacterial strains of the genus Clostridium (Clost.) Or three bacterial strains of the genus Lactobacillus (Lacto.), And IECs were collected three weeks after inoculation and analyzed for relative expression level of IDO gene mRNA by real-time RT-PCR.

[Figure 41] Figure 41 is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were treated with 2% DSS, observed and measured to determine loss body weight, stool hardness and bleeding for six days, and then evaluated numerically.

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[Figure 42] Figure 42 is a photograph showing the state of colons collected on day 6 after control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were treated with DSS 2 %.

[Figure 43] Figure 43 shows photomicrographs showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were treated with 2% DSS, and colons were collected from them. on day 6 and analyzed histologically by HE staining.

[Figure 44] Figure 44 is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were sensitized with oxazolone, and the interior of each rectum was subsequently treated with a solution of 1% oxazolone / 50% ethanol, and body weight loss was measured.

[Figure 45] Figure 45 shows photomicrographs showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were sensitized with oxazolone, and the interior of each rectum was subsequently treated with a 1% oxazolone / 50% ethanol solution, and the colons obtained by the treatment were histologically analyzed by HE staining.

[Figure 46] Figure 46 is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were immunized by administering ovalbumin (OVA) absorbed with alum twice in a interval of 2 weeks, and the sera were collected therefrom and analyzed for the concentration of OVA-specific IgE in these sera by ELISA.

[Figure 47] Figure 47 is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were immunized by administering OVA absorbed with alum twice at an interval 2 weeks old, and spleen cells were harvested and analyzed for IL-4 production from these spleen cells by restimulation with OVA in vitro.

[Figure 48] Figure 48 is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF + Clost.) Were immunized by administering OVA absorbed with alum twice at an interval 2 weeks old, and spleen cells were harvested and analyzed for IL-10 production from these spleen cells by in vitro OVA restimulation.

[Figure 49] Figure 49 is a phylogenetic tree constructed by the neighbor joining method with the resulting sequences of the 41 Clostridium strains and those of known bacteria obtained from the Genbank database using Mega software.

[Figure 50] Figure 50 are histograms showing Foxp3 expression by selected CD4 cells from gF mice (aseptic mouse # 1 and # 2) or GF mice colonized with three Clostridium strains belonging to group IV (mouse with 3 Clost strains # 1 and # 2).

[Figure 51] Figure 51 are histograms showing Foxp3 expression by CD4 positive lymphocytes from gF mice (GF) or GF mice gavaged with chloroform-treated human stools (GF + chlorine.)

[Figure 52] Figure 52 is a graph showing Foxp3 expression by CD4-positive lymphocytes from GF mice (GF) or GF mice gavaged with chloroform-treated human stools (GF + chlorine.)

[Figure 53] Figure 53 is a graph showing the amounts of Clostridium and Bacteroides in faeces of mice gavaged with chloroform-treated human faeces.

Description <Composition that induces proliferation or accumulation of regulatory T cells>

This document describes a composition that induces the proliferation or accumulation of regulatory T cells, the composition comprising, as active principle, at least one substance selected from the group consisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and (c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.

Herein, "regulatory T cells" means T cells that have a function of suppressing a

ES 2 809 232 T3 abnormal or excessive immune response, and which play a role in immune tolerance. The regulatory T cells of the invention are CD4 positive T cells positive for the transcription factor Foxp3. However, other regulatory T cells also include Foxp3 transcription factor negative regulatory T cells which are CD4 positive T cells that produce IL-10.

The meaning of "induces the proliferation or accumulation of regulatory T cells" in the present invention includes an effect of inducing the differentiation of immature T cells into regulatory T cells, the differentiation of which leads to the proliferation or accumulation of regulatory T cells. Furthermore, the meaning of "induces proliferation or accumulation of regulatory T cells" in the present invention includes in vivo effects, in vitro effects, and ex vivo effects. Therefore, all of the following effects are included: an effect of induction of proliferation or in vivo accumulation of regulatory T cells through the administration or ingestion of the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria; an effect of inducing proliferation or accumulation of cultured regulatory T cells by causing bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria to act on the cultured regulatory T cells; and an effect of induction of proliferation or accumulation of regulatory T cells that are collected from a living organism and that subsequently intend to enter a living organism, such as the organism from which they were obtained or another organism, causing the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from bacteria act on regulatory T cells. The effect of inducing proliferation or accumulation of regulatory T cells can be evaluated, for example, as follows. Specifically, the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria is administered orally to an experimental animal such as an aseptic mouse, then the CD4-positive cells are isolated in the colon and the ratio of regulatory T cells contained in CD4 positive cells by flow cytometry (see example 7).

Regulatory T cells whose proliferation or accumulation is induced by the composition of the present invention are positive regulatory T cells for the transcription factor Foxp3.

The "bacteria belonging to the genus Clostridium", which are the active principle in the composition of the present invention, are not particularly limited as long as the bacteria have the effect of inducing proliferation or accumulation of regulatory T cells. The bacteria preferably belong to group XIVa or group IV. One strain of the bacteria alone can be used for the composition of the present invention, but two or more strains of the bacteria can be used together for the composition of the present invention. The use of multiple strains of bacteria belonging to group XIVa or group IV in combination can achieve an excellent effect on regulatory T cells. In addition to bacteria belonging to these groups, bacteria belonging to other groups (eg bacteria belonging to group III) can also be used in combination. If more than one strain of bacteria is used (for example, one or more strains belonging to group XIVa, one or more strains belonging to group IV, one or more strains belonging to a group other than group XIVa or group IV, such as one or more strains belonging to group III), the type and number of strains used can vary widely. The type and number to be used can be determined based on a variety of factors (e.g., the desired effect, such as induction or inhibition of proliferation or accumulation of regulatory T cells; the disease or condition to be treated, prevented or be reduced in severity; the age or gender of the recipient). The strains can be present in a single composition, in which case they will be consumed or ingested together, or they can be present in more than one composition (for example, each can be in a separate composition), in which case they can be consumed individually. or the compositions can be combined and the resulting combination (combined compositions) consumed or ingested. Any number or combination of strains that is effective can be administered (for example, any number from one to 200, such as 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, and any number in between). In certain embodiments of the present invention, a combination of some or all of the 46 strains described in the document (Itoh, K., and Mitsuoka, T. Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice. Lab. Animals 19: 111-118 (1985)). For example, at least one, two or more, three, three or more, four, four or more, five, five or more, six, six or more, or any other number of the 46 described strains can be used, including the 46 strains. They can be used in combination with each other and in combination with strains not described in the cited reference (for example, in combination with one or more strains belonging to group III). Note that the group of "bacteria belonging to the genus Clostridium" can be identified, for example, as follows. Specifically, bacteria belonging to the genus Clostridium are classified by PCR using a set of primers consisting of SEQ ID NO: 64 and 65 (for Clostridium spp. Belonging to group XIVa) or a set of primers consisting of SEQ ID NO : 66 and 67 (for Clostridium spp. Belonging to group IV) (see example 18). Furthermore, bacteria belonging to the genus Clostridium are classified by sequencing the amplified 16S rRNA gene using a set of primers consisting of SEQ ID NO: 19 and 20 (see example 7).

Viable cells of bacteria belonging to the genus Clostridium can be used for the composition of the present invention, and inactivated cells thereof can also be used for the composition. Furthermore, from the point of view of heat stability, resistance to treatment with antibiotics and the like, and long storage period, bacteria belonging to the genus Clostridium are preferably in the spore form.

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The meaning of the "physiologically active substance derived from bacteria belonging to the genus Clostridium" of the present invention includes substances contained in the bacteria, secretion products of the bacteria and metabolites of the bacteria. Such a physiologically active substance can be identified by purification of an active component of the bacteria, a culture supernatant thereof, or the contents of the intestinal tract in the intestinal tract of a mouse in which only bacteria belonging to the genus Clostridium are colonized. by a known purification method.

The active ingredient "spore-forming fraction of a fecal sample obtained from a mammal" in a composition described herein is not particularly limited, as long as the fraction includes spore-forming bacteria present in the feces of a mammal, and it has the effect of inducing the proliferation or accumulation of regulatory T cells.

The active ingredient "chloroform-treated fraction of a fecal sample obtained from a mammal" in the composition described herein is not particularly limited, as long as the fraction is obtained by treating the feces of a mammal with chloroform (for example , 3% chloroform), and has the effect of inducing proliferation or accumulation of regulatory T cells.

Note that "mammal" herein includes humans, mice, rats, cattle, horses, pigs, sheep, monkeys, dogs, and cats.

Meanwhile, when the "spore-forming fraction of a fecal sample obtained from a mammal" or the "chloroform-treated fraction of a fecal sample obtained from a mammal" is cultured in a medium, the substances contained in the bacteria, the secretion products of the bacteria and metabolites of the bacteria are released from the bacteria and the like contained in the fraction. The meaning of the active principle "fraction culture supernatant" in the composition described herein includes such substances, secretion products and metabolites. The culture supernatant is not particularly limited, as long as the culture supernatant has the effect of inducing proliferation or accumulation of regulatory T cells. Examples of the culture supernatant include a protein fraction from the culture supernatant, a polysaccharide fraction from the culture supernatant, a lipid fraction from the culture supernatant, and a low molecular weight metabolite fraction from the culture supernatant.

The composition of the present invention can be in the form of a pharmaceutical composition, a food or a drink (which can also be a feed), or a reagent used for an animal model experiment, the pharmaceutical composition, the food or the drink having , and the reagent the effect of inducing proliferation or accumulation of regulatory T cells. An example herein revealed that regulatory T cells (Treg cells) induced by bacteria or the like belonging to the genus Clostridium suppressed the proliferation of effector T cells. Accordingly, the composition of the present invention can be suitably used as defined in the claims as a composition having an immunosuppressive effect. The immunosuppressive effect can be evaluated, for example, in the following way. Specifically, regulatory T cells isolated from an experimental animal, such as a mouse, orally administered the composition of the present invention are caused to act on effector T cells (CD4 cells⁺ CD25^-) isolated from the spleen, and then their proliferation capacity is measured using the intake amount of [³H] -thymidine as an index (see example 14).

The composition of the present invention can be used, for example, as claimed, as a pharmaceutical composition to prevent or treat an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis or Hashimoto's disease. , or an allergic disease, such as pollinosis or asthma. A pharmaceutical composition for suppressing rejection in an organ transplant or the like is also disclosed; a food or drink to improve immune functions; or a reagent for suppressing effector T cell proliferation or function.

Autoimmune diseases, allergic diseases, and organ transplant rejection, and the like, include inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, type I diabetes, multiple sclerosis, disease graft versus host after bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, insulin-dependent diabetes mellitus, thyroiditis ,. asthma, psoriasis, dermatitis, scleroderma, atopic dermatitis, graft versus host disease, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome , Wegener's granulomatosis, Henoch-Schoenlejn purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, Toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, type I polyglandular deficiency syndrome and syndrome of type II polyglandular deficiency, Schmidt syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, chlamydia, spondyloarthropathy arthropathy associated with Yersinia and Salmonella, disease

ES 2 809 232 T3 atheromatous / arteriosclerosis, atopic allergy, food allergies, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, dermatosis with linear IgA deposits, autoimmune hemolytic anemia, hemolytic anemia with positive Coombs test, pernicious anemia Juvenile pernicious anemia, myalgic encephalitis / Royal Free disease, chronic mucocutaneous candidosis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiency disease syndrome, diseases related to acquired immunodeficiency, hepatitis C, common variable immunodeficiency (common variable hypogammaglobulinemia), dilated cardiomyopathy, pulmonary fibrosis, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, interstitial lung disease associated with connective tissue disease, lung disease associated with mixed connective tissue disease, interstitial lung disease associated with systemic sclerosis, interstitial lung disease associated with rheumatoid arthritis, lung disease associated with systemic lupus erythematosus, lung disease associated with dermatomyositis / polymyositis, lung disease associated with Sjogren's disease, lung disease associated with ankylosing spondylitis, diffuse vasculitic lung disease, pulmonary disease associated with hemosiderosis, drug-induced lung disease, radiation fibrosis, obliterative bronchiolitis, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, interstitial lung disease after infection, gouty arthritis, autoimmune hepatitis, type l autoimmune hepatitis (lupoid or classic autoimmune hepatitis ), autoimmune hepatitis type 2 (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, Type B insulin resistance with acanthosis pigmentosa, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthritis, primary sclerosing cholangitis, idiopathic leukocytopenia, autoimmune neutropenia, NOS kidney disease, glomerulonephritis, vasculitis microscopic kidney disease, discoid lupus erythematosus, idiopathic male infertility or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis , Takayasu disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, Autoimmune goiter hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergies), anaphylaxis, pet allergies, latex allergies, allergies, drug allergies, rhinoconunctivitis Eosinophilic esophagitis, hypereosinophilic syndrome, eosinophilic gastroenteritis, cutaneous lupus erythematosus, eosinophilic esophagitis, hypereosinophilic syndrome and eosinophilic gastroenteritis.

Pharmaceutical preparations can be formulated from the composition of the present invention by known drug formulation methods. For example, the composition of the present invention can be used orally or parenterally in the forms of capsules, tablets, lozenges, liquids, powders, granules, fine granules, film-coated preparations, microgranules, troches, sublingual preparations, chewing gums, preparations. mouthwash, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal absorption systems, lotions, inhalations, aerosols, injections, suppositories, and the like.

To formulate these preparations, the composition of the present invention can be used in appropriate combination with pharmacologically acceptable or acceptable carriers for a food or drink, specifically, with sterile water, physiological saline solution, vegetable oil, solvent, a base material, an emulsifier , a suspending agent, a surfactant, a stabilizer, a flavoring agent, an aromatic compound, an excipient, a vehicle, a preservative, a binder, a diluent, a tonicity adjusting agent, a sedative, a bulking agent, a disintegrating agent, a buffering agent, a coating agent, a lubricant, a colorant, a sweetener, a thickening agent, a flavor corrector, a solubilizer, other additives, or the like.

Meanwhile, to formulate a pharmaceutical preparation thereof, and particularly to formulate a pharmaceutical preparation for oral administration, it is preferable to use in combination a composition that allows the effective administration of the composition of the present invention to the colon, from the point of view to more effectively induce the proliferation or accumulation of regulatory T cells in the colon.

Such a composition or method that allows administration to the colon is not particularly limited, and known compositions or methods may be employed as appropriate. Examples thereof include pH sensitive compositions, more specifically enteric polymers that release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach. When a pH-sensitive composition is used to formulate the pharmaceutical preparation, the pH-sensitive composition is preferably a polymer whose pH threshold for decomposition of the composition is 6.8 to 7.5. Such a range of numerical values ​​is in the range in which the pH shifts towards the alkaline side in the distal part of the stomach and is therefore a suitable range for use in administration to the colon.

Furthermore, another example of the composition that allows administration to the colon is a composition that ensures administration to the colon by delaying the release of the contents by approximately 3 to 5 hours, which corresponds to the transit time of the small intestine. In an exemplary formulation of a pharmaceutical preparation using the composition to delay release, a hydrogel is used as a shell. The hydrogel hydrates and swells after

ES 2 809 232 T3 contact with the gastrointestinal fluid, so that the content is effectively released. In addition, sustained release dosage units include drug-containing compositions having a material that selectively coats or coats a drug. Examples of such a selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water soluble polymers and / or enzyme degradable polymers. The preferred coating material for effectively delaying release is not particularly limited, and examples thereof include cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid polymers and copolymers, such as methacrylic acid polymers and copolymers, and polymers and vinyl copolymers, such as polyvinylpyrrolidone.

Examples of the composition that allows administration to the colon further include bioadhesive compositions that specifically adhere to the mucous membrane of the colon (eg, a polymer described in US Patent Specification No. 6,368,586), and compositions in which a protease inhibitor is incorporated to particularly protect a biopharmaceutical preparation in the gastrointestinal tracts against decomposition due to protease activity.

An example of a system that allows administration to the colon is a system for administering a composition to the colon by means of a pressure change in such a way that the contents are released using the pressure change caused by the generation of gas in bacterial fermentation in the distal part of the stomach. Such a system is not particularly limited, and a more specific example thereof is a capsule having the contents dispersed in a suppository base and which is coated with a hydrophobic polymer (eg ethyl cellulose).

Another example of the system that allows administration to the colon is a system for administering a composition to the colon, the system being specifically broken down by an enzyme (for example, a carbohydrate hydrolase or a carbohydrate reductase) present in the colon. . Such a system is not particularly limited, and more specific examples thereof include systems using food components such as non-starch polysaccharides, amylose, xanthan gum, and azo polymers.

When used as a pharmaceutical composition, the composition of the present invention can be used in combination with a known pharmaceutical composition for use in immunosuppression. A known pharmaceutical composition of this type is not particularly limited, and may be at least one therapeutic composition selected from the group consisting of corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporine A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, antileukotrienes, anticholinergic drugs for rhinitis, anticholinergic decongestants, mast cell stabilizers, monoclonal anti-IgE antibodies, vaccines (preferably vaccines used for vaccination in which the amount of allergen is gradually increased), and combinations thereof. It is preferable to use these therapeutic compositions in combination with the composition of the present invention.

When the composition of the present invention is used as a food or a drink, the food or the drink can be, for example, a health food, a functional food, a specified health food, a food supplement, a patient food. or a feed. The food or drink of the present invention can be ingested in the forms of the compositions as described above, and can also be ingested in the forms of various foods and beverages. Specific examples of the foods and beverages include various beverages such as juices, soft drinks, tea drinks, beverage preparations, gelatin beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice, noodle, bread, and pasta food products; pasta products such as fish hams, sausages, seafood pasta products; sterilized bagged products such as curries, foods seasoned with thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy drinks, ice cream, cheeses, and yogurts; fermented products such as fermented soybeans, yogurts, fermented drinks, and pickles; bean products; various confectionery such as Western confectionery including muffins, cookies, and the like, Japanese confectionery including steamed bean jam buns, red soy soft jellies, and the like, candies, gum, gummies, cold desserts including jellies , puddings and frozen desserts; instant foods such as instant soups and instant soy soups; food for microwave cooking; and the like. Furthermore, examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies. The composition of the present invention can be used for animals including humans. Animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but animals are not limit them.

Without wishing to be bound by theory, individuals in whom the relative abundance of bacteria belonging to the Firmicutes group (the group to which Clostridium groups IV and XlVa belong) is higher, gain more body weight than individuals in which the abundance Relative of bacteria belonging to the Bacteroidetes group is higher. Accordingly, the composition of the present disclosure is capable of conditioning nutrient absorption and improving feeding efficiency. From such a standpoint, the composition of the present disclosure can be used to

ES 2 809 232 T3 to promote body weight gain, or for a feed with good feeding efficiency.

Furthermore, the addition of the composition of the present disclosure to an antibiotic-free feed allows to increase the body weight of a subject who ingests the feed to a level equal to or greater than that achieved by feed containing antibiotics, and also allows to reduce bacteria pathogens in the stomach to a level equal to that achieved by feed containing typical antibiotics. Accordingly, the composition of the present disclosure can be used for a feed that does not need the addition of antibiotics.

Furthermore, unlike conventional bacteria (Lactobacillus and Bifidobacterium) in commercial use which are not easy to incorporate into livestock production, the composition of the present invention in spore form can be easily microgranulated, pulverized or mixed with a feed, and It can also be added to drinking water.

The feeding of such a feed using the composition described herein is not particularly limited, and the feed can be fed to a subject at regular intervals selectively, or it can be fed for a certain period (for example, in its birth, during weaning or when the subject to be fed relocates or is sent).

Furthermore, from the point of view described above, the composition of the present disclosure can preferably be used for malnourished humans. In other words, also when the subject ingesting the composition is a human, the composition can be used to promote body weight gain and enhance energy absorption from food.

The food or drink can be manufactured by a manufacturing technique that is well known in the technical field. One or more components (eg a nutrient) that are effective in enhancing an immune function through an immunosuppressive effect may be added to the food or drink. Furthermore, the food or drink may be combined with another component or other functional food having a function other than the function of enhancing an immune function to thereby serve as a multifunctional food or drink.

Furthermore, the composition described herein can be incorporated into foods that require a processing step that can destroy ordinary probiotic strains. Specifically, most probiotic strains that can be used commercially cannot be incorporated into foods that need to be processed by any one of heat treatment, long-term storage, freeze treatment, mechanical stress treatment, and high pressure treatment (e.g., extrusion or profiling). On the other hand, due to an advantageous spore-forming nature, the composition of the present invention can be easily incorporated into such processed foods.

For example, the spore compositions can survive even in dry food, and can remain alive even after ingestion. Likewise, the composition described herein can withstand low temperature sterilization procedures, typically procedures at a temperature ranging from 70 ° C to the boiling point, both inclusive. Therefore, the composition described herein can be incorporated into all kinds of dairy products. Furthermore, the composition described herein can withstand long-term storage of many years; high temperature processing such as baking and boiling; low temperature processing such as freezing and cold storage; and high pressure treatments such as extrusion and profiling.

Foods that need to be processed under such harsh conditions are not particularly limited, and examples thereof include foods that need to be processed in a microwave oven to be edible (for example, oatmeal), foods that need to be baked to be edible (for example, muffins), foods that need to undergo a high-temperature sterilization treatment for a short period of time to be edible (for example, milk) and foods that need to be heated to be drinkable (for example, hot tea).

When the composition is administered or ingested, the amount thereof for administration or ingestion is selected as appropriate depending on the age, body weight, symptoms, health problems of the subject, the kind of composition (a pharmaceutical product , a food or a drink, or the like), and the like. For example, the amount per administration or ingestion is generally 0.01 mg / kg of body weight to 100 mg / kg of body weight, and preferably 1 mg / kg of body weight to 10 mg / kg of body weight. The present disclosure also includes a method for suppressing the immunity of a subject, the method being characterized in that the bacteria belonging to the genus Clostridium or the physiologically active substance derived from the bacteria is administered into or ingested by the subject as described above.

A product of the composition of the present invention (a pharmaceutical product, a food or a drink or a reagent) or a manual thereof may be provided with a note indicating that the product can be used to suppress immunity (including a note indicating that the product has an immunosuppressive effect and a note indicating that the product has an effect of suppressing the proliferation or function of effector T cells). In this case, the "provision to the product or the manual thereof with the note" means that the note is provided to a main body, container, container, or the like of the product, or the note is provided to a manual, a prospectus of

ES 2 809 232 T3 packaging, a brochure or other printed materials, which disclose information about the product.

<Method to induce proliferation or accumulation of regulatory T cells>

As described above, and as will be shown in the examples, administration of the composition described herein to an individual enables the proliferation or accumulation of regulatory T cells to be induced in the individual. Thus, the present disclosure includes a method for inducing the proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering, to the individual, at least one substance selected from the group consisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and (c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.

Note that the "individual" in the present disclosure is not particularly limited, and examples thereof include humans, various kinds of livestock, poultry, pets, experimental animals, and the like. The "individual" can be in a healthy state or a sick state.

Furthermore, as will be shown in Example 5 to be described later, Gram positive commensal bacteria play major roles in the proliferation or accumulation of regulatory T cells. Accordingly, the present disclosure includes a method of inducing the proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering an antibiotic against Gram negative bacteria to the individual.

As used herein, the "antibiotic against Gram negative bacteria" is not particularly limited, and examples thereof include aminoglycoside antibiotics (amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin and paromomycin), cephalosporin antibiotics (cefaclor, cefamandol, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditorene, cefoperazone, cefotaxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone and cefoxotin), sulfonamides, ampicillin, and streptomycin. Without wishing to be bound by theory, the "antibiotic against Gram negative bacteria" according to the present invention is preferably one that reduces Gram negative bacteria and contributes to the colonization of Gram positive bacteria.

In addition, a prebiotic composition such as almond skin, inulin, oligofructose, raffinose, lactulose, pectin, hemicellulose (such as xyloglucan and alpha-glucans), amylopectin and resistant starch that do not break down in the upper part of the gastrointestinal tract and promote the growth of gut microbes in the intestinal tract, as well as growth factors such as acetyl-CoA, biotin, beet molasses, and yeast extracts, They contribute to the proliferation of bacteria belonging to the genus Clostridium. Accordingly, the present disclosure includes a method of inducing the proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering, to the individual, at least one substance selected from the group consisting of these substances.

Meanwhile, in a "method for inducing the proliferation or accumulation of regulatory T cells", the composition of the present disclosure, the "antibiotic against Gram negative bacteria" described above and the "prebiotic composition or growth factor" described above can used in combination. Such combined use is not particularly limited, and examples of the combined use are as follows: the "antibiotic against Gram negative bacteria" is administered to an individual beforehand, and then the composition of the present invention is administered; the "antibiotic against Gram negative bacteria" and the composition of the present invention are administered simultaneously to an individual; the "prebiotic composition or growth factor" is administered to an individual beforehand, and then the composition of the present invention is administered; the "prebiotic composition or growth factor" and the composition of the present invention are administered simultaneously to an individual; the composition of the present invention, the "antibiotic against Gram negative bacteria" and the "prebiotic composition or growth factor" are administered to an individual simultaneously or individually at any appropriate time.

In addition, a therapeutic composition can be administered to an individual together with at least one substance selected from the group consisting of the composition of the present disclosure, the "antibiotic against Gram negative bacteria" and the "prebiotic composition or growth factor".

Such a therapeutic composition is not particularly limited, and may be at least one therapeutic composition selected from the group consisting of corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporine A, mercaptopurine, azathiopurine, prednisone, methotrexate. , antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, antileukotrienes, anticholinergic drugs for rhinitis, anticholinergic decongestants, mast cell stabilizers, anti-IgE antibodies

ES 2 809 232 T3 monoclonal vaccines (preferably vaccines used for vaccination in which the amount of allergen is gradually increased), and combinations thereof. It is preferable to use these therapeutic compositions in combination with the substance described above.

Furthermore, there is no particular limitation imposed on the combined use of the therapeutic composition with at least one substance selected from the group consisting of the composition of the present disclosure, the "antibiotic against Gram negative bacteria" and the "prebiotic composition or the factor of growth". For example, the "a substance" and the therapeutic composition are administered orally or parenterally to an individual simultaneously or individually at any appropriate time.

Furthermore, in the "method for inducing proliferation or accumulation of regulatory T cells" described above, it can be determined whether or not the administration of the composition described herein or the like actually induces proliferation or accumulation of regulatory T cells, using as index, increase or boost of at least one selected from the group consisting of the number of regulatory T cells, the ratio of regulatory T cells in the group of colon T cells, a regulatory T cell function and the expression of a regulatory T cell marker. It is preferable to use a measure selected from the group consisting of enhancement of IL-10 expression, enhancement of CTLA4 expression, enhancement of IDO expression, and suppression of IL-4 expression expression, as an index of induction of proliferation or accumulation of regulatory T cells.

Note that examples of a method for detecting such expression include Northern blotting, RTPCR, and dot blotting for the detection of gene expression at the transcriptional level; and ELISA, radioimmunoassay, immunoblotting, immunoprecipitation and flow cytometry for the detection of gene expression at the translational level.

Meanwhile, a sample used to measure such an index is not particularly limited, and examples thereof include blood sampled from an individual and tissue parts obtained in a biopsy.

<Method for predicting the response of an individual to the composition of the present invention and / or the prognosis of an individual>

This document describes a method in which the absolute amount or ratio of bacteria belonging to the genus Clostridium in a microbiota of an individual is determined, and when the ratio or absolute value of bacteria belonging to the genus Clostridium is reduced by comparison with a reference value obtained by making a similar determination in an individual with a typical state of health, the individual is determined to be possibly sensitive to the composition described herein.

Also described is a method for predicting a subject's response to a substance and / or a subject's prognosis. The method comprises measuring the percentage or absolute amounts of Clostridium groups IV and XIV in the subject's microbiota and comparing them with a reference value of the same measurements in a healthy prototype subject, in which an absolute amount or a level of Decreased percentage of Clostridium groups IV and / or XIV indicates that the subject may respond favorably to the compositions described herein.

The method may further comprise measuring the composition of the subject's microbiota after administration of the substance, wherein an increase in the percentage or absolute number of Clostridium spp. belonging to groups IV, XIV after administration of the compositions of the present invention with respect to before administration is a positive indicator of enhanced immunosuppression (or immunoregulation). Measurement of the composition of a subject's microbiota can be performed with techniques known in the art, such as 16S rRNA sequencing.

Note that, in these methods, the substance is at least one substance selected from the group consisting of the following (a) through (e):

(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction;

(c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction;

(d) an antibiotic against Gram negative bacteria; and (e) at least one substance selected from the group consisting of almond skin, inulin, oligofructose, raffinose, lactulose, pectin, hemicellulose (such as xyloglucan and alpha-glucans), amylopectin, acetyl-CoA, biotin, beet molasses. , yeast extracts and resistant starch.

ES 2 809 232 T3 <Method to inhibit proliferation or accumulation of regulatory T cells>

As will be shown in Example 5 to be described later, Gram positive commensal bacteria play major roles in the proliferation or accumulation of regulatory T cells. Accordingly, the present disclosure also includes a method of inhibiting the proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering an antibiotic against Gram positive bacteria to the individual.

As used herein, the term "antibiotic against Gram-positive bacteria" is not particularly limited, and examples thereof include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefachlor, cefamandole, cefoxitin, cefprozil and ceftobiprole); fluoroquinolone antibiotics (Cipro, Levaquin, Floxin, Tequina, Avelox, and Norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin, and meropenem).

As described above, the term "individual" is not particularly limited, and examples thereof include humans, various kinds of livestock, poultry, pets, experimental animals, and the like. The "individual" can be in a healthy state or a sick state. Such a diseased state is not particularly limited, and examples thereof include states of being subjected to immunotherapy against cancer and suffering from an infectious disease.

Furthermore, as another mode of the "method of inhibiting the proliferation or accumulation of regulatory T cells", the present disclosure includes a method of inhibiting the proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering, to the individual , any one of an antibody, an antibody fragment and a peptide, which are against an antigen that is at least one substance selected from the group consisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and (c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.

<Vaccine composition and method of treating or preventing an infectious disease or an autoimmune disease using the vaccine composition>

As described above, and as will be shown in Example 15 to be described later, the induction of Treg cells in the colon by Clostridium plays an important role in local and systemic immune responses. Accordingly, the present disclosure includes a "vaccine composition comprising at least one substance selected from the group consisting of the following (a) to (c): (a) bacteria belonging to the genus Clostridium; (b) a bacterial spore in a spore-forming fraction of a fecal sample obtained from a mammal; and (c) bacteria in a chloroform-treated fraction of a fecal sample obtained from a mammal ", and a" method of treating, assisting in the treatment, reducing the severity of, or preventing at least one disease selected from infectious diseases and autoimmune diseases. in an individual, the method comprising administering the vaccine composition to the individual ".

Note that such "autoimmune diseases" are not particularly limited, and examples thereof include those described as the "specific examples of target diseases" in <Composition having effect of inducing proliferation or accumulation of regulatory T cells>. The "infectious diseases" are also not particularly limited, and examples thereof include infectious diseases associated with the "infectious pathogens" described as the "examples of infectious pathogens" in <Composition having effect of inducing proliferation or accumulation of cells Regulatory T>.

<Method of selecting a compound that has activity to promote the proliferation or accumulation of regulatory T cells>

A method for selecting a compound having an activity to promote the proliferation or accumulation of regulatory T cells is also described, the method comprising:

(1) prepare a test substance from at least one substance selected from the group consisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the

ES 2 809 232 T3 bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and (c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.

(2) preparing non-human mammals in which a reporter gene is to be expressed under the control of IL-10 gene expression;

(3) bringing the test substance into contact with the non-human mammal;

(4) after contact with the test substance, detect cells expressing the reporter gene in a cluster of CD4 cells⁺ Foxp3⁺ of the non-human mammal, and determine the number of cells in the CD4 cell pool⁺ Foxp3⁺expressing the reporter gene or a ratio of cells in the CD4 cell cluster⁺ Foxp3⁺ expressing the reporter gene relative to cells in the CD4 cell cluster⁺ Foxp3⁺ that do not express the reporter gene;

(5) detect cells expressing the reporter gene in a cluster of CD4 cells⁺ Foxp3⁺ from the non-human mammal that has not been in contact with the test substance, and determine the number of cells in the cD4 cell group⁺Foxp3⁺ expressing the reporter gene or a ratio of cells in the CD4 cell cluster⁺ Foxp3⁺ expressing the reporter gene relative to cells in the CD4 cell cluster⁺ Foxp3⁺ that do not express the reporter gene; and (6) compare the absolute numbers or the ratios determined in steps (4) with the number or the ratio determined in (5), and determine, when the number or the ratio determined in (4) is greater than that determined in (5), that the test substance is a compound that promotes the proliferation or accumulation of Treg cells.

The term "test substance", as used herein, is not particularly limited, as long as the test substance is a substance prepared from at least one substance selected from the group consisting of the substances (a) a (c). Examples of the test substance include proteins, polysaccharides, lipids and nucleic acids that are derived from at least one substance selected from the group consisting of substances (a) to (c) described above.

The term "non-human mammal in which a reporter gene is to be expressed under the control of IL-10 gene expression", as used herein, is not particularly limited, as long as the non-human mammal is a non-human mammal having a reporter gene whose expression is controlled by an expression control region of the IL-10 gene (eg, a promoter or enhancer). Examples of such a reporter gene include genes encoding fluorescent proteins (eg, GFP) and genes encoding luciferase. As the "non-human mammal in which a reporter gene is to be expressed under the control of IL-10 gene expression" according to the present invention, an Il10 mouse can preferably be used.^Venus which will be shown later in the examples.

The term "contact", as used herein, is not particularly limited, and examples thereof include administration of the test substance to the non-human mammal orally or parenterally (eg, intraperitoneal injection or injection intravenous).

Also described is a non-human mammal that is used for the method, and in which the reporter gene is to be expressed under the control of the expression of the IL-10 gene.

In addition, this document describes a method to isolate, from a sample of bacteria belonging to the genus Clostridium, a compound that has an activity to promote the proliferation or accumulation of regulatory T cells, the method comprising the following steps (1 ) to (3):

(1) preparing a genomic DNA from the sample of bacteria belonging to the genus Clostridium;

(2) insert genomic DNA into a cloning system, and prepare a library derived from the sample of bacteria belonging to the genus Clostridium; and (3) isolating a compound that has an activity to promote the proliferation or accumulation of regulatory T cells, by using the library obtained in step (2).

In such steps, the methods for preparation and isolation are not particularly limited, and known techniques for an in vitro or in vivo system can be used as appropriate. Furthermore, the compound isolated by this method is not particularly limited, and examples thereof include nucleic acids (for example, a DNA, an mRNA and an rRNA) derived from bacteria belonging to the genus Clostridium, as well as polypeptides and proteins derived from the bacteria belonging to the genus Clostridium.

ES 2 809 232 T3

A method for determining the composition of a microbiota in an individual is also described, wherein the increase in the ratio or the absolute number of bacteria belonging to the genus Clostridium after the administration of the composition of the present invention to the individual with respect to the ratio or absolute number before administration is used as an index of increased immunosuppression. In such a method, the method for determining the composition of the microbiota is not particularly limited, and known techniques (eg, 16S rRNA sequencing) can be used as appropriate.

Also described is a method for measuring Treg cell differentiation, wherein the increase in Treg cell differentiation in an individual after administration of the composition described herein to the individual relative to that before administration is used as an index of increased immunosuppression (or immunoregulation).

Furthermore, the composition described herein can also be administered to an individual under antibiotic treatment. The timing of administration is not particularly limited, and the composition can be administered, for example, before or simultaneously with antibiotic treatment. Meanwhile, the composition is preferably administered in the form of spores from the viewpoint of resistance to antibiotic treatment.

Furthermore, in a preferred mode of such administration, the composition is administered, for example, after or simultaneously with the administration of an antibiotic against Gram positive bacteria. Note that such a "Gram positive bacterial antibiotic" is not particularly limited, and examples thereof include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefachlor, cefamandole, cefoxitin, cefprozil and ceftobiprole); fluoroquinolone antibiotics (Cipro, Levaquin, Floxin, Tequina, Avelox, and Norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem / cilastatin, and meropenem).

Meanwhile, in another preferred mode of such administration, the composition is administered, for example, after (or simultaneously with) a treatment using vancomycin, metronidazole, linezolid, ramaplanin or fidaxomicin.

Examples

Hereinafter, the present invention is more specifically described based on the examples. However, the present invention is defined in the claims.

Note that the mice used in the examples were prepared or produced in the following manner. In the following description, reference may be made to mice with "SPF" or "GF" attached behind them. These "SPF" and "GF" indicate that the mice were kept in the absence of specific pathogenic bacteria (specific pathogen free, SPF) and that the mice were kept under aseptic conditions (GF, germ-free), respectively.

<Mice>

C57BL / 6, Balb / c and IQI mice maintained under SPF or GF conditions were purchased from Sankyo Labo Service Corporation, Inc. (Japan), JAPAN SLC, INC. (Japan), CLEA JAPAN, Inc. (Japan) or Jackson Laboratory (USA). GF mice and gnotobiotic mice were raised and kept within the gnotobiotic facility of the University of Tokyo, Yakult Central Institute for Microbiological Research, or Sankyo Labo Service Corporation, Inc.Myd88 mice^-/-, Rip2^-/- and Card9^-/- they were produced as described in non-patent documents 1 to 3, and were backcrossed for 8 generations or more, so that a C57BL / 6 gene pool was achieved. Foxp3 mice^eGFP were purchased from Jackson Laboratory.

<Mice Il10^Venus>

To form a bicistronic locus encoding both Il10 and Venus under the control of an Il10 promoter, a targeting construct was first created. Specifically, a cassette was inserted (IRES-Venus-SV40 polyA signal cassette, referred to in non-patent document 4), which was made from an internal ribosome entry site (IRES), a protein fluorescent yellow (Venus) and an SV40 polyA signal (SV40 polyA) and that was arranged after the neomycin resistant one (neo), between a stop codon and a polyA signal (exon 5) of an Il10 gene. Next, the targeting construct obtained was used to cause homologous recombination with the region of the Il10 gene in the genome of mice. Therefore, Il10 mice were produced^Venusthat had Il10 alleles^Venus (referenced in figure 1). Note that, in Figure 1, "tk" represents a gene encoding thymidine kinase, "neo" represents the neomycin resistant gene, and "BamH1" represents a restriction enzyme cleavage site BamH1.

Genomic DNA was extracted from the Il10 mice^Venus, treated with BamH1 and Southern blotted using a probe shown in Figure 1. Figure 2 shows the results obtained. I know

ES 2 809 232 T3 detected wild type and Il10 alleles^Venus as bands having sizes of 19 kb and 5.5 kb, respectively. Therefore, as is evident from the results shown in Figure 2, the homologous recombination shown in Figure 1 was found to occur in the genome of Il10 mice.^Venus.

In addition, CD4 cells were sorted⁺ Venus- or CD4 cells⁺ Venus⁺ in the lamina propria of the colon of Il10 mice^Venus by using a FACSAria device. Real-time RT-PCR was then carried out on an ABI 7300 system by a method to be described later, to determine the amount of IL-10 mRNA expressed. Figures 3 and 4 show the results obtained. As evident from the results shown in Figures 3 and 4, it was found that since IL-10 mRNA growth was detected only in CD4 cells⁺ Venus⁺, IL-10 mRNA expression in Il10 mice^Venus it was correctly reflected in Venus's expression. Note that the aseptic states of such Il10 mice^Venus were established at the Central Institute for Experimental Animals (Kawasaki, Japan). Il10 mice^Venus in aseptic states they were maintained in vinyl insulators at Sankyo Labo Service Corporation, Inc. (Tokyo, Japan), and were used in the following examples.

Meanwhile, the experiments and analyzes in the examples were carried out as follows.

<Method for the colonization of mice with bacteria and their analysis>

According to the description in non-patent documents 5 and 6, mice were produced in which SFB or Clostridium were colonized. The contents of the cecum or feces of the gnotobiotic mice obtained were dissolved in sterile water or an anaerobic dilution solution. The contents of the cecum or dissolved feces as they were or after chloroform treatment were administered orally to GF mice. Three Lactobacillus strains and 16 Bacteroides strains were cultured separately from each other on EG or BL agar medium anaerobically. Cultured bacteria were harvested, suspended in anaerobic TS broth, and forcibly administered orally to GF mice. Bacterial colonization status in mice was assessed by microscopic observation performed on a fecal microgranule smear preparation.

<Cell separation and flow cytometry>

In order to isolate lymphocytes from the lamina propria of the colon and the lamina propria of the small intestine, the small intestine and colon were harvested and opened longitudinally. They were then washed to remove fecal content and the like from inside them. Subsequently, the small intestine and colon were shaken in HBSS containing 5 mM EDTA at 37 ° C for 20 minutes. After removal of the epithelium and adipose tissue, the intestinal tissues were cut into small pieces. RPMI 1640 (4% fetal bovine serum (FBS), 1 mg / ml collagenase D, 0.5 mg / ml dispase, and 40 pg / ml DNase I were added to the small pieces (all of these were manufactured by Roche Diagnostics KK)), and the mixture was stirred in a water bath kept at 37 ° C for 1 hour. The delayed tissues were washed with HBSS containing 5 mM EDTA, and resuspended in 5 ml of 40% Percoll (GE Healthcare). The suspension was overlaid on 2.5 ml of 80% Percoll in a 15 ml Falcon tube. Centrifugation was then carried out at room temperature and 2000 rpm for 20 minutes to carry out cell separation by Percoll density gradient centrifugation. Cells were harvested at the contact surface and used as lamina propria lymphocytes. Harvested cells were suspended in a staining buffer (PBS, 2% FBS, 2 mM EDTA, and 0.09% NaNs) and stained using a labeled anti-CD4 antibody (RM4-5, BD Biosciences). with PE or PE-Cy7. After CD4 staining, cells were stained Foxp3 using the Cytofix / Cytoperm Plus kit with Golgistop (BD Biosciences) or Foxp3 staining buffer set (eBioscience), as well as an anti-Foxp3 antibody (FjK -16s, eBioscience) tagged with Alexa647. Flow cytometry was performed using a FACSCanto II device, and data was analyzed using FlowJo software (TreeStar Inc.). Cell sorting was performed using a FACSAria device.

<Real-time RT-PCR>

From an RNA prepared using the RNeasy Mini kit (Qiagen), a cDNA was synthesized using an mMv reverse transcriptase (Promega KK). The cDNA obtained was analyzed by real-time RT-PCR using the Power SYBR Green PCR master mix (Applied Biosystems) and the ABI 7300 real-time PCR system (Applied Biosystems), or real-time RT-PCR using the reagent. SYBR Premix Ex Taq (TAKARA) and a Light Cycler 480 device. For each sample, the value obtained was normalized with respect to the amount of GAPDH. A set of primers were designed using Primer Express version 3.0 (Applied Biosystems), and those that showed 90% or greater sequence identity at initial evaluation were selected. The set of primers used was the following:

Foxp3

5'-GGCAATAGTTCCTTCCCAGAGTT-3 '(SEQ ID NO: 1)

5'-GGGTCGCATATTGTGGTACTTG-3 '(SEQ ID NO: 2)

ES 2 809 232 T3

CTLA4

5'-CCTTTTGTAGCCCTGCTCACTCT-3 '(SEQ ID NO: 3)

5'-GGGTCACCTGTATGGCTTCAG-3 '(SEQ ID NO: 4)

GITR

5'-TCAGTGCAAGATCTGCAAGCA-3 '(SEQ ID NO: 5)

5'-ACACCGGAAGCCAAACACA-3 '(SEQ ID NO: 6)

IL-10

5'-GATTTTAATAAGCTCCAAGACCAAGGT-3 '(SEQ ID NO: 7)

5'-CTTCTATGCAGTTGATGAAGATGTCAA-3 '(SEQ ID NO: 8)

GAPDH

5'-CCTCGTCCCGTAGACAAAATG-3 '(SEQ ID NO: 9)

5'-TCTCCACTTTGCCACTGCAA-3 '(SEQ ID NO: 10)

Mmp2

5'-GGACATTGTCTTTGATGGCA-3 '(SEQ ID NO: 11)

5'-CTTGTCACGTGGTGTCACTG-3 '(SEQ ID NO: 12)

Mmp9

5'-TCTCTGGACGTCAAATGTGG-3 '(SEQ ID NO: 13)

5'-GCTGAACAGCAGAGCCTTC-3 '(SEQ ID NO: 14)

Mmp13

5'-AGGTCTGGATCACTCCAAGG-3 '(SEQ ID NO: 15)

5'-TCGCCTGGACCATAAAGAA-3 '(SEQ ID NO: 16)

Idol

5'-AGAGGATGCGTGACTTTGTG-3 '(SEQ ID NO: 17)

5'-ATACAGCAGACCTTCTGGCA-3 '(SEQ ID NO: 18).

<Preparation and culture of epithelial cells of the large intestine (IEC)>

First, the colon was harvested, opened lengthwise, and rinsed with PBS. Subsequently, the colon was treated with 1 mM dithiothreitol (DTT) at 37 ° C for 30 minutes on a shaker, and then vortexed for one minute to alter epithelial integrity. Released IECs were collected and suspended in 5 ml of 20% Percoll. The suspension was overlaid on 2.5 ml of 80% Percoll in a 15 ml Falcon tube. The tube was then centrifuged at 25 ° C and 780 g for 20 minutes to carry out cell separation by Percoll density gradient centrifugation. Cells were harvested at the contact surface and used as colon IEC (purity: 90% or greater, viability: 95%). The IECs thus collected were suspended in RPMI containing 10% FBS, and cultured 1x10⁵ IEC cells in a 24-well plate for 24 hours. After that, the culture supernatant was collected and measured for the level of active TGF-P1 by ELISA (Promega).

Meanwhile, to culture T cells in vitro, 1.5x105 CD4 T cells were cultured.⁺ MACS purified spleen cells in each well of a 96-well round bottom plate, together with 50% conditioned medium in which IEC isolated from GF mice or Clostridium colonized mice were grown, and with 25 ng / ml hIL -2 (Peprotech), in the presence or absence of 25 pg / ml of an anti-TGF-β (R&amp;D) antibody. Note that 10 pg / ml of an anti-CD3 antibody and an anti-CD28 antibody (BD Bioscience) bound to the round bottom plate. After a culture of 5 days, the CD4 T cells were harvested.⁺ and were subjected to real-time PCR.

ES 2 809 232 T3 <Experimental colitis model>

A fecal suspension of Clostridium colonized mice was administered orally to C57BL / 6 mice (2 weeks old) and grown in a standard environment for six weeks.

To prepare a model of DSS-induced colitis, 2% (w / v) DSS (reagent grade, DSS salt, molecular weight = 36 to 50 kD, manufactured by MP Biomedicals) was administered, along with potable water, mice for six days.

Meanwhile, to prepare a model of oxazolone-induced colitis, mice were previously sensitized by transdermal application to mice of 150 µl of a 3% solution of oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one , Sigma-Aldrich) / 100% ethanol. Five days thereafter, 150 µl of a 1% oxazolone / 50% ethanol solution was injected intrarectally back into the previously sensitized mice with light anesthesia. Note that intrarectal administration was carried out using a 3.5 F catheter.

Each mouse was tested daily for body weight, occult blood, naked-eye bleeding (gross blood), and stool hardness. In addition, the percentage of body weight loss, intestinal bleeding (no bleeding, occult blood (hemoccult +) or bleeding visible to the naked eye) and hard stool (normal stool, semi-liquid stool or diarrhea) were numerically evaluated and calculated the disease activity index (DAI) as described in “S. Wirtz, C. Neufert, B. Weigmann, MF Neurath, Nat Protoc 2, 541 (2007) ".

<OVA-specific IgE reaction>

BALB / c SPF mice were inoculated with a fecal suspension of Clostridium colonized mice (2 weeks old) and grown in a conventional environment. Mice (at their ages of 4 weeks and 6 weeks) were then injected intraperitoneally with 1 pg of OVA (Grade V, Sigma) and 2 mg of alum (Thermo Scientific), 0.2 ml in total. Sera were collected weekly from the mice from the root of their tails and OVA-specific IgE was measured by ELISA (Chondrex). Then, at their ages of 8 weeks, spleen cells were harvested, inoculated into a 96-well plate at 1x10 6 cells per well, and stimulated with OVA (100 pg / ml) for three days. After that, the culture supernatant was collected and measured for IL-4 and IL-10 levels by ELISA (R&amp;D).

<Statistical analysis>

The difference between the control and experimental groups was evaluated by Student's t test.

(Example 1)

First, it was investigated whether the accumulation of regulatory T cells (Treg cells) in the lamina propria of the colon was dependent or not on commensal bacteria. Specifically, lymphocytes were isolated from the peripheral lymph nodes (pLN) of Balb / c mice reared in the absence of specific pathogenic bacteria (SPF) or from the lamina propria of the colon or small intestine (SI) of mice. CD4 and Foxp3 were stained by antibodies. The Foxp3 cell ratio was then analyzed⁺ in CD4 lymphocytes⁺ by flow cytometry. Figure 5 shows the results obtained. As evident from the results shown in Figure 5, it was found that Foxp3 Treg cells⁺ they were present at a high frequency in the lamina propria of the gastrointestinal tracts, especially in the lamina propria of the colon, of mice kept in an environment free of specific pathogenic microorganisms (SPF). Furthermore, it was also found that the number of Foxp3 Treg cells⁺ in the lamina propria of the colon increased gradually up to three months after birth, while the number of Foxp3 Treg cells⁺ in peripheral lymph nodes it was basically constant from the time of two weeks after birth.

(Example 2)

Next, it was investigated whether or not the temporary accumulation of Treg cells in the colon as found in Example 1 had a relationship with the colonization of the intestinal commensal microbiota. Specifically, CD4 expression and Foxp3 expression were analyzed in lymphocytes isolated from the small intestine, colon, and peripheral lymph nodes of mice reared in an aseptic (GF) or SPF environment (8 weeks old: Balb / c mice, IQI mice and C57BL / 6 mice). Similar results were obtained in three or more independent experiments. Figures 6 and 7 show the results obtained. Note that in Figure 7, each white circle represents the absolute number of CD4 cells.⁺ Foxp3⁺ in an individual mouse, and the error bars represent the standard deviations (SD).

Furthermore, lymphocytes were collected from the lamina propria of SPF mice and GF mice (Balb / c mice or C57BL / 6 mice). CD4 and Foxp3 were stained with antibodies. The lymphocytes of the lamina propria were then analyzed by FACS. Figure 8 shows the results obtained. Note that, in Figure 8, each white circle represents the number

ES 2 809 232 T3 absolute CD4 cells⁺ Foxp3⁺ in an individual mouse, ** indicates that "P &lt;0.001" and * indicates that "P &lt;0.01".

In addition, lymphocytes were isolated from the lamina propria of the colon, the lamina propria of the small intestine (SI), Peyer's patches (PP) and the mesenteric lymph nodes (MLN) of mice (C57BL / 6 SPF mice) at those who were given oral antibiotics with water for eight weeks. CD4 and Foxp3 were stained with antibodies. The lymphocytes were then analyzed by FACS. Similar results were obtained in two or more independent experiments. Figure 9 shows the results obtained (the ratio of Foxp3 cells⁺ on CD4 cells⁺ of an individual mouse). Note that the following antibiotics were used in combination as described in the following document:

ampicillin (A; 500 mg / l, Sigma) vancomycin (V; 500 mg / l, NACALAI TESQUE, INC.) metronidazole (M; 1 g / l, NACALAI TESQUE, INC.) neomycin (N; 1 g / l, NACALAI TESQUE, INC.)

Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, R. Medzhitov, Cell 118, 229 (July 23, 2004)

Fagarasan et al., Science 298, 1424 (November 15, 2002).

In figure 9, each white circle represents the absolute number of CD4 cells⁺ Foxp3⁺ in an individual mouse, each horizontal bar represents the average value of the absolute numbers, * indicates that "P &lt;0.01" and "AVMN" represents the classes of antibiotics administered using the first letters of the antibiotics.

As evident from the results shown in Figures 6 to 9, the frequencies and absolute numbers of CD4 cells⁺ Foxp3⁺ in the small intestine and peripheral lymph nodes of GF mice they were equal to or greater than those of SPF mice (see Figures 6 to 8). Furthermore, the numbers of Treg cells in the lamina propria of the small intestine, Peyer's patches, and mesenteric lymph nodes of SPF mice orally administered antibiotics for eight weeks were equal to or greater than those of mice. SPF (see figure 9). Meanwhile, the number of CD4 cells⁺ Foxp3⁺ in the lamina propria of the colon of GF mice it was significantly decreased compared to that of SPF mice (see Figures 6 and 7). This decrease was commonly observed among mice from different gene pools (Balb / c, IQI, and C57BL / 6), as well as among mice raised in different animal facilities (see Figure 7 for data regarding the different gene pools, the data regarding mice reared in the different animal facilities are not shown in the drawings). Furthermore, the number of Treg cells in the lamina propria of the colon of the C57BL / 6 SPF mice administered the antibiotics was also shown to be significantly decreased (see Figure 9).

(Example 3)

Next, it was directly verified whether the decrease in the number of Treg cells in the lamina propria of the colon of the GF mice shown in example 2 was attributed or not to the absence of microbiota. Specifically, a fecal suspension of B6 SPF mice purchased from Jackson Laboratory was orally administered to IQI GF mice (conventionalization). Three weeks after administration, lymphocytes were isolated from the lamina propria of the colon and the expression of Foxp3 was analyzed in CD4 lymphocytes.⁺. Figures 10 and 11 show the results obtained. Note that each white circle in Figure 11 represents the absolute number of CD4 cells.⁺ Foxp3⁺ in an individual mouse, the error bars represent the standard deviations (SD), * indicates that "P &lt;0.01" in the Student's t test and ** indicates that "P &lt;0.001". As is evident from the results shown in Figures 10 and 11, the number of Treg cells in the lamina propria of the small intestine did not change. However, the number of Treg cells in the lamina propria of the colon increased significantly. Therefore, the interaction between the host and the microbes was shown to play an important role in the accumulation of Foxp3 Treg cells.⁺ in the lamina propria of the colon, whereas the accumulation of Treg cells in the lamina propria of the small intestine had a different mechanism.

(Example 4)

Next, the relationship between the lymphatic tissues associated with the gut of mice and the number of Foxp3 cells was investigated.⁺ in the lamina propria of the colon of mice according to the method described in MN Kweon et al., J Immunol 174, 4365 (April 1, 2005). Specifically, 100 μg of a recombinant extracellular domain protein (a fusion protein (LTpR-Ig) between a lymphotoxin β receptor (LTpR) and a human IgG1 Fc region was injected intraperitoneally, see Honda et al., J Exp Med 193, 621 (March 5, 2001)) in pregnant C57BL / 6 mice 14 days after conception. The fetuses obtained from such mice were again injected intraperitoneally with LTβR-Ig, so that mice were produced from which isolated lymphatic follicles (ILF), Peyer's patches (PP), and colon plaques (CP) were completely removed. ). The Foxp3 cell ratios were then analyzed⁺ in cells

ES 2 809 232 T3

CD4⁺ in the lamina propria of the colon of mice treated with LTpR-Ig and of mice treated with rat IgG (control) by FACS. Figure 12 shows the results obtained. Note that, in Figure 12, each white circle represents the Foxp3 cell ratio⁺ in an individual mouse and the error bars represent the standard deviations. As is evident from the results shown in Figure 12, it was found that the ratio of Foxp3 cells⁺ in the lamina propria of the colon of mice deficient in isolated lymphatic follicles, Peyer's patches and colon plaques (mice treated with LTpR-Ig) increased significantly. Therefore, it was suggested that the decrease in the number of Treg cells in the lamina propria of the colon of GF mice and mice treated with antibiotics was due to the transmission of specific signals that promote the accumulation of Treg cells in the lamina propria of the colon. and the one caused by gut microbes did not occur, rather than simply due to a side effect of disordered gut-associated lymphatic tissues.

(Example 5)

To investigate whether or not the specific intestinal flora induced the accumulation of colon Treg cells, vancomycin was administered as an antibiotic against Gram positive bacteria or polymyxin B as an antibiotic against Gram negative bacteria to SPF mice (4 weeks old) for four weeks and was analyzed to determine the Foxp3 cell ratio⁺ in the CD4 cell cluster⁺ ([%] of Foxp3⁺ on CD4). Figure 30 shows the results obtained. Note that, in Figure 30, "SPF" indicates the result of SPF mice (control), "poly B" indicates the result of SPF mice administered polymyxin B and "Vanco." indicates the result of SPF mice administered vancomycin. Meanwhile, * indicates that "P &lt;0.01".

As is evident from the results shown in Figure 30, the number of Treg cells in the colon of mice administered vancomycin was markedly decreased compared to that of the control. In contrast, no influence was observed on the number of Treg cells in mice administered polymyxin B. Those facts suggested that Gram-positive commensal bacteria played a major role in the accumulation of Treg cells.

(Example 6)

A recent report has suggested that spore-forming bacteria play an important role in the gut's T-cell response (see V. Gaboriau-Routhiau et al., Immunity 31,677 (October 16, 2009)). In this regard, fecal microorganisms (spore formation fraction) resistant to treatment with 3% chloroform were administered orally to GF mice, which were then analyzed to determine the ratio of Foxp3 cells⁺in the CD4 cell cluster⁺ ([%] of Foxp3⁺ on CD4). Figure 31 shows the results obtained. Note that, in Figure 31, "GF" indicates the result of GF mice and "+ chlorine" indicates the result of GF mice given chloroform-treated feces. Meanwhile, ** indicates that "P &lt;0.001".

As is evident from the results shown in Figure 31, three weeks after the administration of the chloroform-treated feces, the number of Treg cells in the administered mice increased markedly to the same level as that of SPF mice. and GF mice to which untreated feces were forcibly administered (see Figures 7 and 11).

Therefore, taking into account the results shown in Example 5 in combination, it was revealed that the specific components of the endogenous microbiota had a high probability of belonging to the Gram positive group and that the spore formation fraction played an important role in induction of Treg cells.

(Example 7)

Next, the gut microbiota species that induced the accumulation of Treg cells in the colon were identified as suggested in Examples 4 to 6. Specifically, segmented filamentous bacteria (FBS), 16 Bacteroides strains, were administered orally. spp. (Bactero. (6 strains of B. vulgatus, 7 of group 1 of B. acidifaciens and 3 of group 2 of B. acidifaciens)), 3 strains of Lactobacillus (Lacto. (L. acidophilus, L. fermentum and L. murinum)) and 46 strains of Clostridium spp. (Clost., See “Itoh, K., and Mitsuoka, T. Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice. Lab. Animals 19: 111-118 (1985 )) ”), Or microbiota collected from mice (SPF) reared in a conventional environment to Balb / c GF mice or IQI GF mice. The mice were kept in vinyl insulators for three weeks. CD4 cells were then isolated from the colon and small intestine of these mice. Treg cell numbers in the colon and small intestine were analyzed by flow cytometry.

Figure 13 shows graphs of FACS dots obtained when an acquisition window was established in CD4 cells.⁺ of Balb / c mice. Figure 14 shows the ratio of Foxp3 cells⁺ on CD4 cells⁺ of each mouse.

Note that bacteria belonging to the genus Clostridium are classified by 16S rRNA gene sequencing as follows. Specifically, bacteria 16S rRNA genes were amplified by PCR using specific primer pairs of 16S rRNA genes: 5-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 19) and 5'-ATTACCGCGGCKGCTG-3' (SEQ ID NO : 20) (see T. Aebischer et al., Vaccination prevents

ES 2 809 232 T3

Helicobacter pilori-induced alterations of the gastric flora in mice. FEMS Immunol. Med. Microbiol. 46,221-229 (2006)). The 1.5 kb PCR product was then introduced into the pCR-Blunt vector. The inserts were sequenced and aligned using the ClustalW software program. The resulting sequences of 16S rRNA genes derived from strain 1-41 of 46 strains of Clostridium spp. are shown in SEQ ID NO: 21-61. The phylogenetic tree that was built by the neighbor joining method with the resulting sequences of the 41 Clostridium strains and those of known bacteria obtained from the Genbank database using the Mega software is shown in figure 49.

As evident from the results shown in Figures 13 and 14, no effect on the number of Treg cells in the colon was observed in GF mice colonized with segmented filamentous bacteria (FBS) (see Figure 14). Furthermore, mice in which the cocktail of three Lactobacillus strains was colonized gave similar results (see Figure 14). On the other hand, it was shown that the accumulation of Foxp3 cells⁺ in the lamina propria of the colon it was strongly induced in mice in which 46 strains of Clostridium spp. were colonized. Importantly, such accumulation was fostered independently of the gene pools of the mice, and led to an increase in numbers similar to that of SPF mice despite the fact that only a single genus gut microbiota was colonized. It was also shown that Clostridium colonization did not change the number of Treg cells in the lamina propria of the small intestine (see Figure 14). Note that when all 16 Bactericides spp. Strains were colonized, the number of Treg cells in the colon increased significantly. However, the degree of the increase varied as a function of the gene pool of the mice in which the bacteria were colonized (see Figures 13 and 14).

(Example 8)

Next, CD4 expression, Foxp3 expression, and Helios expression in thymus and colon LP lymphocytes of SPF mice, GF mice, Lactobacillus-colonized mice, and Clostridium-colonized mice were analyzed by flow cytometry.

Figures 32 and 33 show the results obtained. Note that, in Figures 32 and 33, "GF" or "aseptic" indicates the results of the GF mice, "SPF" indicates the results of the SPF mice, "Lacto." indicates the results of mice colonized with Lactobacillus and "Clost." indicates the results of mice colonized with Clostridium. In figure 32, the vertical axis represents the Helios cell ratio^- in the Foxp3 cell group⁺ ([%] of Helios^- on Foxp3⁺) and ** indicates that "P &lt;0.001".

As is evident from the results shown in Figures 32 and 33, the majority of Foxp3 cells⁺found in SPF mice or mice colonized with Clostridium did not express Helios. Note that Helios is a transcription factor known to be expressed in time-derived natural Treg cells (see AM Thornton et al., J Immunol 184, 3433 (April 1, 2010)). Accordingly, it was suggested that the majority of Treg cells in SPF mice and Clostridium colonized mice were Treg cells induced in peripheral portions, ie, so-called iTreg cells.

(Example 9)

Next, it was investigated whether or not the colonization of Clostridium or the like influenced other T cells. Specifically, SFB, 16 strains of Bacteroides spp. (Bactero.), 46 strains of Clostridium spp. (Clost.) Or microbiota collected from mice reared in a conventional environment (SPF) in IQI GF mice. Three weeks later, lymphocytes were isolated from the lamina propria of the colon of these mice, and stimulated with PMA (50 ng / ml) and ionomycin (1 pg / ml) for four hours in the presence of Golgistop (BD Bioscience). After providing stimulation, intracellular cytokines were stained using an anti-IL-17 antibody from PE (TC11-18H10) and an anti-IFN-g antibody from FITC (BD Bioscience) according to the manual for a Cytofix / Cytoperm kit (BD Bioscience). The ratio of IFN-y cells was then analyzed.⁺ or IL-17 cells⁺ on CD4 leukocytes⁺ by flow cytometry. Figures 15 and 16 show the results obtained. Note that, in Figures 15 and 16, each white circle represents the absolute number of CD4 cells.⁺IFN-y⁺ or the absolute number of CD4 cells⁺ IL-17⁺ in each individual mouse and the error bars represent the standard deviations (SD). As is evident from the results shown in Figures 15 and 16, Clostridium colonization did not influence Th1 cells (CD4 cells⁺ IFN-y⁺) in the colon and caused only a slight increase in Th17 cells (cD4 cells⁺ IL-17 +). Accordingly, it was suggested that the genus Clostridium was a genus of bacteria that specifically induced Treg cells.

(Example 10)

46 strains of Clostridium spp. influence the accumulation of CD8 intraepithelial lymphocytes (IEL)⁺of the intestinal tract in the colon. Accordingly, it is conceivable that Clostridium regulates the immune system in various aspects, and that Clostridium exhibits a remarkable ability to induce and maintain Treg cells especially in the colon, as described above. Furthermore, a class of cytokines, transforming growth factor-β (TGF-β), is known to play an important role in the regulation of Treg cell generation.

ES 2 809 232 T3

In this regard, it was examined whether or not the colonization of Clostridium provided a TGFβ-rich environment in the colon. Specifically, first, the entire colon of GF mice, Clostridium-colonized mice, and Lactobacillus-colonized mice were cultured for 24 hours, and the culture supernatants thereof were measured to determine the concentration of active TGF-β (TGF- β1) by ELISA (the number of mice analyzed was four per group). Figure 34 shows the results obtained. Note that, in Figure 34, "GF" indicates the result of GF mice, "Clost." indicates the result of mice colonized with Clostridium and "Lacto." indicates the result of mice colonized with Lactobacillus. Meanwhile, * indicates that "P &lt;0.02", and ** indicates that "P &lt;0.001".

As is evident from the results shown in Figure 34, the amount of TGF-β produced in the colon of Clostridium-colonized mice was significantly greater than that of GF mice and Lactobacillus-colonized mice.

Next, intestinal epithelial cells (IEC) from GF mice and mice colonized with Clostridium were cultured for 24 hours, and the culture supernatants thereof were measured to determine the concentration of active TGFβ (TGF-β1) by ELISA (the number of mice analyzed was four per group). Figure 35 shows the results obtained. Note that, in Figure 35, "GF" indicates the result of GF and "Clost mice." indicates the result of mice colonized with Clostridium. Meanwhile, ** indicates that "P &lt;0.001".

As is evident from the results shown in Figure 35, GF-β was detected in the culture supernatant of IECs isolated from Clostridium-colonized mice, while TGF-β was not detected in the culture supernatant of the IEC isolated from GF mice.

Then, as described above, CD4 T cells were cultured.⁺ spleen for five days together with a 50% conditioned medium in which IEC isolated from GF mice or mice colonized with Clostridium, and with the anti-CD3 antibody, were cultured in the presence or absence of an anti-TGF-β antibody. T cells were then harvested and analyzed for Foxp3 expression by real time RT-PCR. Figure 36 shows the results obtained. Note that, in Figure 36, "Medium" indicates the result of a medium in which no cells were cultured, "GF" indicates the result of the conditioned medium in which IEC of GF mice were cultured, "Clost." indicates the result of the conditioned medium in which IEC of mice colonized with Clostridium and "Clost. + αTGFβ ”indicates the result of the conditioned medium to which the anti-TGF-β antibody was added and in which IEC of mice colonized with Clostridium were cultured. Meanwhile, ** indicates that "P &lt;0.001".

As evident from the results shown in Figure 36, when the culture supernatant of IEC derived from Clostridium-colonized mice was added to CD4 T cells⁺ spleen, differentiation into Foxp3-expressing cells was accelerated. Meanwhile, differentiation in Treg cells was inhibited by anti-TGF-β antibody.

In addition, the expression of MMP2, MMP9 and MMP13, which are thought to contribute to the activation of latent TGFβ, was investigated. The expression of indolamine 2,3-dioxygenase (IDO), which is thought to be involved in the induction of Treg cells, was also investigated. Specifically, 46 bacterial strains of the genus Clostridium (Clost.) Or three bacterial strains of the genus Lactobacillus (Lacto.) Were administered orally to aseptic C57BL / 6 mice. Three weeks after administration, the IECs were collected and analyzed for relative mRNA expression levels of the MMP2, MMP9, MMP13, and IDO genes by real-time RT-PCR (the number of mice tested was three per group). Figures 37 to 40 show the results obtained. Note that, in Figures 37 to 40, "GF # 1 to 3" indicates the results from GF mice, "Clost. Nos. 1 to 3 "indicate the results of mice colonized with Clostridium and" Lacto. Nos. 1 to 3 "indicate the results of mice colonized with Lactobacillus.

For the relationship between latent TGF-β activation and the MMPs described above, see D'Angelo et al., J. Biol. Chem. 276, 11347-11353, 2001; Heidinger et al., Biol. Chem. 387, 69-78, 2006; Yu et al., Genes Dev. I4, 163176, 2000. For the relationship between IDO and Treg cell induction, see G. Matteoli et al., Gut 59, 595 (May 2010).

As evident from the results shown in Figures 37 to 39, consistent with the TGF-β production described above, the transcription products of the genes encoding MMP2, MMP9 and MMP13 were expressed at higher levels. in IEC derived from Clostridium-colonized mice than from GF mice and Lactobacillus-colonized mice.

Furthermore, as is evident from the results shown in Figure 40, IDO was expressed only in Clostridium colonized mice.

Consequently, it was revealed that Clostridium activated IECs and led to the production of TGF-β and other molecules that induce Treg cells in the colon.

(Example 11)

ES 2 809 232 T3

Next, it was investigated whether the accumulation of Treg cells induced by Clostridium colonization was dependent or not on the transmission of signals by pathogen-associated molecular pattern recognition receptors. Specifically, the numbers of Treg cells in the lamina propria of the colon of each of the Myd88 SPF mice were examined.^-/- (deficient in Myd88 (signaling adapter for Toll-like receptor)), Rip2^-/- (poor in Rip2 (NOD receiver adapter)) and Card9^-/- (Deficient in Card9 (essential signal transmission factor for Dectin-1 signal transmission)). Furthermore, the colonization of Clostridium spp. in Myd88 mice^-/- GF, and the change in the number of Treg cells was investigated. Figures 17 and 18 show the results obtained. As is evident from the results shown in Figures 17 and 18, the number of Treg cells from each class of SPF mice deficient in pathogen-associated molecular pattern recognition receptor associated factors did not change from that of wild-type mice from the same litter, which served as a control. Furthermore, it was also found that when Clostridium spp. in Myd88-deficient GF mice, accumulation of Treg cells was induced in the lamina propria of the colon. Consequently, it has been suggested that the mechanism of induction of the accumulation of Treg cells in the lamina propria of the colon is not based on the activation of the recognition pathway for molecular patterns associated with major pathogens, as is elicited by most bacteria, but rather specific commensal bacteria species.

(Example 12)

Foxp3 Treg cells are known to⁺ of the intestinal tract exert some immunosuppressive functions through the production of IL-10 (see non-patent document 9). Meanwhile, it is known that animals that have CD4 cells⁺ Foxp3⁺ of which IL-10 is specifically removed develop inflammatory bowel disease (see non-patent document 18). In this regard, first, the expression of IL-10 in lymphocytes of various tissues was examined. Specifically, lymphocytes were isolated from various tissues of SPF Il10 mice.^Venus, and CD4 expression and Venus expression were analyzed by flow cytometry. Figure 19 shows the results obtained. Note that each numerical value in Figure 19 represents the ratio of cells within one of the corresponding regions divided into four.

In addition, lymphocytes were isolated from the lamina propria of the colon of Il10 mice.^Venus, and T cell receptor (TCRP) β chain expression was detected on cell surfaces by FACS. Figure 20 shows the results obtained (FACS dot plots obtained when an acquisition window was established in CD4 cells⁺). Note that each numerical value in Figure 20 represents the ratio of cells within one of the corresponding regions divided into four.

In addition, lymphocytes were isolated from the lamina propria of the colon of Il10 mice.^Venus. Lymphocytes were stimulated with PMA (50 ng / ml) and ionomycin (1 pg / ml) for four hours in the presence of Golgistop (BD Bioscience). Then, after providing stimulation, intracellular cytokines were stained using a PE anti-IL-17 antibody, an APC anti-IL-4 antibody (11B11) and a FITC anti-IFN-g antibody (BD Bioscience) according to the manual for a Cytofix / Cytoperm kit (BD Bioscience). Figure 21 shows the results obtained (FACS dot plots obtained when an acquisition window was established in CD4 cells⁺).

Note that each numerical value in Figure 21 represents the ratio of cells within one of the corresponding regions divided into four.

In addition, CD4 cells were isolated⁺ Foxp3⁺ and CD4 cells⁺ Foxp3- from the spleen (Spl) of Foxp3 reporter mice^eGFP, and Venus cells were isolated⁺ of the lamina propria of the colon and the lamina propria of the small intestine (SI) of jl10ve mice^wildebeests. The cells obtained were then analyzed for the expression of predetermined genes. Gene expression was analyzed by real-time RT-PCR using a Power SYBR Green PCR master mix (Applied Biosystems) and an ABI 7300 real-time PCR system (Applied Biosystems). In this case, the value for each cell was normalized to the amount of GAPDH. Figure 22 shows the results obtained. Note that, in Figure 22, the error bars represent the standard deviations.

As is evident from the results shown in Figures 19 to 22, hardly any Venus cells were detected.⁺ (IL-10 producing cells) in the lymph nodes of the cervix (peripheral lymph nodes), thymus, peripheral blood, lung and liver of mice maintained under SPF conditions. Meanwhile, in the spleen, Peyer's patches and mesenteric lymph nodes thereof, slightly Venus cells were detected⁺ (see figure 19). On the other hand, many Venus cells were found⁺ in the lymphocytes of the lamina propria of the small intestine and the lamina propria of the colon. Also, most Venus cells⁺ in the intestines they were positive for CD4, and also positive for the T cell receptor (TCRβ) chain (see Figures 19 and 20). Furthermore, it was found that CD4 T cells⁺ Venus⁺ expressed Foxp3 and other Treg cell-associated factors, such as a cytotoxic T lymphocyte antigen (CTLA-4) and a glucocorticoid-induced TNFR-associated protein (GITR), although CD4 T cells⁺ Venus⁺ they did not show any of the Th2 (IL-4 producers) and Th17 (IL-17 producers) phenotypes (see Figures 21 and 22). Furthermore, it was shown that the expression level of CTLA-4 in Venus cells⁺ intestine was greater than that of Treg GFP cells⁺ spleen isolated from Foxp3 reporter mice^eGFP (see figure 22).

ES 2 809 232 T3 (Example 13)

Venus cells⁺ can be classified into at least two subsets, namely Venus double positive (DP) Treg cells⁺ Foxp3⁺ and Venus Treg cells⁺ Foxp3- based on intracellular Foxp3 expression. The cells of the latter subset correspond to type 1 (Tr1) regulatory T cells (see non-patent documents 8 and 9). In this regard, Venus cells were investigated⁺ (IL-10 producing cells) observed in Example 8 for Foxp3 expression. Specifically, the expression of CD4, Foxp3 and Venus in the lamina propria of the colon and the lamina propria of the small intestine of Il10 mice was analyzed.^Venus maintained under GF or SPF conditions by FACS, and Venus cell numbers were compared⁺ in the lamina propria of the intestinal tract among Il10 mice^Venus SPF and GF. Figure 23 shows the results obtained (dot graphs obtained when an acquisition window was established in CD4 cells⁺).

In addition, the intracellular expression of Venus and Foxp3 in CD4 cells was analyzed in various tissues of Il10 mice.^VenusSPF by flow cytometry. Figure 24 shows the results obtained (dot plots obtained when an acquisition window was established in CD4 cells⁺). Note that each numerical value in Figure 24 represents the ratio of cells within one of the corresponding regions divided into four.

Furthermore, in order to investigate whether or not the presence of commensal bacteria influenced the expression of IL-10 in regulatory cells in the gastrointestinal tracts, Il10 mice were prepared.^Venus aseptic (GF). The colonization of predetermined species of bacteria was then induced in the Il10 mice.^VenusGF obtained. Three weeks after the bacteria species were colonized, a group of CD4 + cells (V⁺F^-, Venus cells⁺Foxp3^-; V⁺F⁺, Venus cells⁺ Foxp3⁺; and V^-F⁺, Venus cells^- Foxp3⁺) in which Foxp3 and / or Venus were expressed in the colon and small intestine by flow cytometry. Figure 25 shows dot plots obtained when an acquisition window was established in CD4 cells.⁺ colon, and Figures 26 and 27 show the ratios in the CD4 cell group⁺ of each mouse. Note that each numerical value in Figure 25 represents the ratio of cells within one of the corresponding regions divided into four. Meanwhile, the error bars in Figures 26 and 27 represent standard deviations, * indicates that "P &lt;0.02" and ** indicates that "P &lt;0.001".

In addition, in order to check whether the presence of commensal bacteria influenced or not the expression of IL-10 in regulatory cells in the gastrointestinal tracts, antibiotics with water were given orally to five or six Il10 mice.^Venus per group for 10 weeks. The following antibiotics were used in combination:

ampicillin (A; 500 mg / l, Sigma) vancomycin (V; 500 mg / l, NACALAI TESQUE, INC.) metronidazole (M; 1 g / l, NACALAI TESQUE, INC.) neomycin (N; 1 g / l, NACALAI TESQUE, INC.)

CD4 and Foxp3 lymphocytes from the colon lamina propria, small intestine lamina propria (SI), mesenteric lymph nodes (MLN), and Peyer's patches (PP) were then stained with antibodies and analyzed by FACS. The results were obtained from two or more independent experiments that provided similar results. Figure 28 shows the results obtained (the ratio of Venus cells⁺ on CD4 cells⁺ in each sample). Note that each white circle in Figure 28 represents an individual sample, each horizontal bar represents an average value, * indicates that "P &lt;0.02" and "AVMN" represents the classes of antibiotics administered using the first letter of the antibiotics. .

As is evident from the results shown in Figures 23 and 24, the lamina propria of the small intestine was shown to be rich in Venus cells.⁺ Foxp3^-, specifically, Tr1-type cells, and that Treg DP Venus cells⁺ Foxp3⁺ they were present at a high frequency in the colon of SPF mice (see Figures 23 and 24). On the contrary, although sufficient numbers of Foxp3 cells were also observed⁺ in other tissues, Venus expression was not observed in almost any of the cells (see Figure 24).

Furthermore, as is evident from the results shown in Figures 23 and 25-28, it was shown that all the Venus regulatory T cell fractions⁺ Foxp3^-, Venus⁺ Foxp3⁺ and venus^- Foxp3⁺ in the colon they decreased significantly under GF conditions (Figures 23 and 26 to 27). In addition, a similar decrease was also observed in Venus cells.⁺ in Il10 mice^Venus SPF treated with antibiotics (see figure 28).

Furthermore, as is evident from the results shown in Figures 25 to 27, the colonization of Clostridium spp. strongly induced all Venus regulatory T cell fractions⁺ Foxp3^-, Venus⁺ Foxp3⁺and venus^- Foxp3⁺ in the colon, and the degrees of induction thereof were the same as in SPF mice (see Figures 25 and 27). Furthermore, the colonization of the three Lactobacillus strains or the colonization of FBS was found to have extremely little influence on the number of Venus cells.⁺ and / or Foxp3⁺ in the colon (see Figures 25 and 27). Furthermore, the colonization of 16 strains of Bacteroides spp. also induced Venus cells⁺, but the influence of colonization was specific to cells similar to Tr1 Venus⁺ Foxp3^- (see figures 25 and 27). On the other hand, it

ES 2 809 232 T3 found that none of the bacterial species tested significantly influenced the number of IL-10 producing cells in the lamina propria of the small intestine (see Figure 26).

Therefore, it was shown that the genus Clostridium colonized in the colon or a physiologically active substance derived from bacteria provided a signal to induce the accumulation of IL-10 regulatory T cells.⁺ in the lamina propria of the colon or the expression of IL-10 in T cells. Meanwhile, it was shown that the number of Venus cells⁺ in the small intestine it was not significantly influenced by the situation in which commensal bacteria were not present or commensal bacteria were reduced (see Figures 23 and 26 to 28) and IL-10 regulatory cells accumulated⁺ (Tr1-type cells) in the lamina propria of the small intestine independently of commensal bacteria.

(Example 14)

It was investigated whether the Venus cells⁺ induced by the genus Clostridium had or did not have an immunosuppressive function similar to that of Venus cells⁺ in the colon of SPF mice. Specifically, CD4 cells were seeded⁺ CD25^- (effector T cells, Teff cells) isolated from spleen in a flat-bottom 96-well plate at 2 x 10⁴/ well, and cultured for three days together with 2 x 10⁴ CD11c cells⁺ spleen (antigen-representing cells) subjected to irradiation treatment with 30 Gy radiation, 0.5 pg / ml of an anti-CD3 antibody and many Treg cells. In addition, for the last six hours, CD4 cells were cultured.⁺ CD25^-, to which [³H] -thymidine (1 pCi / well). Note that the Treg cells used in Example 14 were CD4 T cells.⁺ GFP⁺ isolated from the spleen of Foxp3 reporter mice^eGFP, or CD4 T cells⁺ Venus⁺ of the lamina propria of the colon of Il10 mice^VenusGF in which Clostridium spp. or Il10 mice^Venus SPF. The proliferation of cells was then determined based on the amount of [³H] -thymidine captured, and represented by a counts per minute (cpm) value.

As is evident from the results shown in Figure 29, Venus cells⁺ CD4⁺ of mice colonized by the genus Clostridium suppressed proliferation of activated CD25 T cells in vitro^- CD4⁺. The suppression activity was slightly lower than that of GFP cells⁺ isolated from Foxp3 reporter mice^eGFP, but equal to the Venus cell⁺ isolated from Il10 mice^Venus SPF. Accordingly, the genus Clostridium has been shown to induce IL-10 expressing T cells that have sufficient immunosuppressive activities and thus play a critical role in maintaining immune homeostasis in the colon.

(Example 15)

Next, the influence, on the local immune response, of the colonization of large numbers of Clostridium and the resulting proliferation of Treg cells was investigated.

<Sodium dextran sulfate (DSS) -induced colitis model>

First, the DSS-induced colitis model was prepared as described above, and the influence, in the model mice, of Clostridium inoculation and Treg cell proliferation was investigated. Specifically, control mice and Clostridium-inoculated mice were treated with 2% DSS, then observed and measured for six days to determine body weight loss, stool hardness, and bleeding, and then evaluated numerically. In addition, on day 6, the colon was harvested, dissected, and histologically analyzed by HE staining. Figures 41 to 43 show the results obtained. Note that, in Figures 41 to 43, "SPF + Clost." or “SPF + Clost. # 1 to 3 "indicate the results of C57BL / 6 mice inoculated with a fecal suspension of mice colonized with Clostridium and grown in a conventional environment for six weeks, and" SPF "or" SPF # 1 to 3 " indicate the results of C57BL / 6 mice (control mice) grown in a conventional environment for six weeks without inoculation with the fecal suspension. Furthermore, in Figure 41, the vertical axis "Disease Score" represents the disease activity index (DAI) described above, and the horizontal axis "after 2% DSS (d)" represents the days elapsed after of the initial administration of 2% DSS to the mice. Furthermore, in Figure 41, * indicates that "P &lt;0.02" and ** indicates that "P &lt;0.001". Meanwhile, regulatory dendritic cell-induced Treg cells are known to play a preventive role in a model of DSS-induced colitis (see S. Manicassamy et al., Science 329, 849 (August 13, 2010)).

As is evident from the results shown in Figures 41 to 43, colitis symptoms such as body weight loss and rectal bleeding were significantly suppressed in mice bearing a large number of Clostridium (also referred to below in herein "mice with abundant Clostridium") compared to control mice (see Figure 41). All the typical features of colon inflammation, such as colon shortening, edema and hemorrhage, were remarkably observed in the control mice compared to the Clostridium-abundant mice (see Figure 42). In addition, the histological features, such as mucosal erosion, edema, cellular infiltration, and crypt leakage, were less severe in the Clostridium abundant mice treated with DSS than in the control mice (see Figure 43).

ES 2 809 232 T3 <Oxazolone-induced colitis model>

Next, the oxazolone-induced colitis model was prepared as described above, and the influence, in the model mice, of Clostridium inoculation and Treg cell proliferation was investigated. Specifically, control mice and Clostridium inoculated mice were sensitized with oxazolone, and the interior of their rectum was subsequently treated with a 1% oxazolone / 50% ethanol solution. The loss of body weight was then observed and measured. In addition, the colon was dissected and histologically analyzed by HE staining. Figures 44 and 45 show the results obtained. Note that, in Figures 44 and 45, "SPF + Clost." indicates the results of C57BL / 6 mice (mice with abundant Clostridium) inoculated with a fecal suspension of mice colonized with Clostridium and grown in a conventional environment for six weeks, and "SPF" indicates the results of C57BL / 6 mice (mice control) grown in a conventional environment for six weeks without being inoculated with the fecal suspension. Furthermore, in Figure 44, the vertical axis "Weight (% of initial)" represents the body weight after the administration of 1% oxazolone when the body weight before the administration was taken as 100%, and the horizontal axis "After 1% oxazolone (d)" represents the days elapsed after administration of 1% oxazolone to the mice. Meanwhile, Th2-type T cells are known to be involved in oxazolone-induced colitis (see M. Boirivant, IJ Fuss, A. Chu, W. Strober, J Exp Med 188, 1929 (November 16, 1998)).

As is evident from the results shown in Figures 44 and 45, colitis continued along with the persistent loss of body weight in control mice. Meanwhile, the body weight loss of the mice with abundant Clostridium was reduced (see FIG. 44). Furthermore, it was also revealed that portions having histological diseases, such as mucosal erosion, edema, cellular infiltration and hemorrhage, were reduced in the colon of the mice with Clostridium in abundance (see FIG. 45).

(Example 16)

Next, the influence, on the systemic immune response (systemic production of IgE), of the colonization of large numbers of Clostridium and the resulting proliferation of Treg cells was investigated. Specifically, as described above, control mice and Clostridium inoculated mice were immunized by administering ovalbumin absorbed with alum (OVA) twice at an interval of 2 weeks. Sera were then collected from these mice and their OVA-specific IgE level was investigated by ELISA. In addition, spleen cells were collected from mice in each group, and IL-4 and IL-10 production was investigated by restimulation with OVA in vitro. Figures 46 to 48 show the results obtained. Note that, in Figures 46 to 48, "SPF + Clost." indicates the results of BALB / c SPF mice (mice with abundant Clostridium) inoculated with a fecal suspension of mice colonized with Clostridium and grown in a conventional environment, "SPF" indicates the results of BALB / c SPF mice (control mice ) grown in a conventional environment without being inoculated with the fecal suspension, and ** indicates that "P &lt;0.001". Meanwhile, in Figure 46, the vertical axis "OVA-specific IgE (ng / ml)" represents the concentration of OVA-specific IgE in the sera. Furthermore, in Figure 46, the horizontal axis represents the days elapsed after the initial administration of the alum-absorbed ovalbumin to the mice with abundant Clostridium or the control mice (4 weeks old), and "OVA + Alum" indicates the timing of the administration of ovalbumin absorbed with alum. In addition, in Figures 47 and 48, "OVA" on the horizontal axis indicates the results in the case in which restimulation was performed with OVA in vitro, and "-" indicates the results in the case in which restimulation was not performed. with OVA in vitro. Furthermore, in Figures 47 and 48, the vertical axes "IL-4 (pg / ml)" and "IL-10 (pg / ml)" show the concentration of IL-4 and the concentration of IL-10 in supernatants of spleen cell culture, respectively.

As is evident from the results shown in Figures 46 to 48, the level of IgE was significantly lower in the Clostridium-abundant mice than in the control mice (see Figure 46). In addition, IL-4 production was reduced by restimulation with OVA (see Figure 47) and thus IL-10 production (see Figure 48) increased in spleen cells of Clostridium mice in Abundance sensitized with OVA and alum, compared to control mice.

Therefore, taking into account the results shown in Example 15 in combination, it has been revealed that the induction of Treg cells by Clostridium in the colon plays an important role in local and systemic immune responses.

(Example 17)

Next, Balb / c GF were colonized with three strains of Clostridium belonging to group IV (strains 22, 23 and 32 listed in Figure 49). Three weeks later, Foxp3 Treg cells were analyzed⁺ colon using FACS. Figure 50 shows the results obtained. As is evident from the results shown in Figure 50, gnotobiotic mice colonized with three strains of Clostridium showed an intermediate pattern of Treg induction between GF mice and mice inoculated with all 46 strains.

(Example 18)

ES 2 809 232 T3

Next, it was investigated whether or not a spore-forming fraction (for example, one resistant to chloroform treatment) from a fecal sample obtained from humans had an effect of inducing proliferation or accumulation of regulatory T cells similar to the fraction of spore formation of the fecal sample obtained from mice.

Specifically, human stools from a healthy volunteer (Japanese, male, 29 years old) were suspended with phosphate buffered saline (PBS), mixed with chloroform (3% final concentration), and then incubated in a water bath. water with stirring for 60 min. After evaporation of chloroform by sparging with N2 gas, aliquots containing chloroform-resistant (e.g. spore-forming) fraction of human intestinal bacteria were inoculated orally into aseptic mice (GF) (IQI, 8 weeks old). Treated mice were kept in a vinyl isolator for 3 weeks. The colon was harvested and opened lengthwise, washed to remove fecal contents, and stirred in Hanks Balanced Salt Solution (HBSS) containing 5 mM EDTA for 20 min at 37 ° C. After removing the epithelial cells and adipose tissue, the colon was cut into small pieces and incubated with RPMl 1640 containing 4% fetal bovine serum, 1 mg / ml collagenase D, 0.5 mg / ml dispase and DNase I 40 pg / ml (all manufactured by Roche Diagnostics) for 1 hour at 37 ° C in a shaking water bath. The digested tissue was washed with HBSS containing 5 mM EDTA, resuspended in 5 ml of 40% Percoll (manufactured by GE Healthcare) and overlaid in 2.5 ml of 80% Percoll in a 15 ml Falcon tube. Percoll gradient separation was performed by centrifugation at 780 g for 20 min at 25 ° C. Cells were harvested from the contact surface and suspended in staining buffer containing PBS, 2% FBS, 2 mM EDTA, and 0.09% NaNs and stained for surface CD4 with phycoerythrin-labeled anti-CD4 Ab ( RM4-5, manufactured by BD Biosciences). Intracellular Foxp3 staining was performed using Alexa647-labeled antiFoxp3 Ab (FJK-16s, manufactured by eBioscience) and Foxp3 staining buffer set (manufactured by eBioscience). The percentage of Foxp3 positive cells within the CD4 positive lymphocyte population was analyzed by flow cytometry. Figures 51 and 52 show the results obtained.

Representative histograms (Figure 51) and pooled data (Figure 52) are shown in the figures for Foxp3 expression by CD4-positive lymphocytes from aseptic mice (GF) or GF mice gavaged with chloroform-treated human stools (GF + chlorine.). Also, the numbers in Figure 51 indicate the percentages of cells in the acquisition window. Each circle in Figure 52 represents an independent animal, the error bars indicate the SD and ** indicates that "P &lt;0.001".

As is evident from the results shown in Figures 51 and 52, it was also found that when the spore-forming fraction (e.g., resistant to chloroform treatment) of human intestinal bacteria was colonized in GF mice, it was induced the accumulation of Foxp3 regulatory cells (Tregs)⁺ in the lamina propria of the mouse colon.

Next, it was investigated which species of bacteria grew by gavage feeding chloroform-treated human stools.

Specifically, using a QIAamp DNA Stool mini-kit (manufactured by QIAGEN), bacterial genomic DNA was isolated from human stools of a healthy volunteer as described above (human stools) or fecal pellets from GF mice gavaged with human stools. treated with chloroform (GF + chlorine.). Quantitative PCR analysis was carried out using a Light-Cycler 480 device (manufactured by Roche). The relative amount was calculated by the ACt method and normalized to the amount of total bacteria, the dilution, and the weight of the sample. The following primer sets were used:

Total bacteria

5'-GGTGAATACGTTCCCGG-3 '(SEQ ID NO: 62) and 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 63)

Clostridium Group XIVa (Clostridium coccoides subgroup)

5'-AAATGACGGTACCTGACTAA-3 '(SEQ ID NO: 64) and 5'-CTTTGAGTTTCATTCTTGCGAA-3' (SEQ ID NO: 65)

Clostridium Group IV (Clostridium leptum)

5'-GCACAAGCAGTGGAGT-3 '(SEQ ID NO: 66) and 5'-CTTCCTCCGTTTTGTCAA-3' (SEQ ID NO: 24)

Bacteroides

5'-GAGAGGAAGGTCCCCCAC-3 '(SEQ ID NO: 67) and 5'-CGCTACTTGGCTGGTTCAG-3' (SEQ ID NO: 68).

Figure 53 shows the results obtained.

As is evident from the results shown in Figure 53, mice gavaged with chloroform-treated human stools exhibited high amounts of spore-forming bacteria,