Bifidobacterium bifidum strains and polysaccharides derived from the strains

Abstract

The present invention relates to a Bifidobacterium bifidum that induces regulatory T cells (Treg), a polysaccharide derived from Bifidobacterium bifidum, and a probiotic strain that produces polysaccharides; and more specifically to a polysaccharide containing β-1-6-glucan as an effective ingredient, a probiotic strain that produces β-1-6-glucan, a food containing the polysaccharide or strain as an effective ingredient for alleviating immune diseases or inflammatory diseases, a therapeutic agent containing the polysaccharide or strain as an effective ingredient for alleviating immune diseases or inflammatory diseases, a method for preparing induced regulatory T cells (iTreg) by treating with the polysaccharide or strain, and a cell therapy product for preventing or treating immune diseases or inflammatory diseases, comprising induced regulatory T cells prepared by the method.

Description

Technical Field

[0001] The present invention relates to a Bifidobacterium bifidum that induces regulatory T (Treg) cells, a polysaccharide derived from Bifidobacterium bifidum, and a probiotic strain that produces the polysaccharide; and more specifically to a polysaccharide containing β-1,6-glucan as an active ingredient, a probiotic strain that produces β-1,6-glucan, a food or therapeutic agent containing the polysaccharide or the strain as an active ingredient, a method for producing induced regulatory T (iTreg) cells by treating with the polysaccharide or the strain, and a cell therapeutic agent for preventing or treating immune diseases or inflammatory diseases containing the induced regulatory T cells produced by the method. Background Art

[0002] In the gastrointestinal tract of mammals, many bacterial species that constitute "microbiome" are symbiotic. The change of microbiome is closely related to various diseases (such as allergic diseases and autoimmune disorders and gastrointestinal inflammatory disorders). Compared with mice without specific pathogens (SPF), germ-free (GF) mice show abnormal responses and immune responses to lymphoid tissue formation (Macpherson and Harris, Nature Reviews Immunology 4: 478-485, 2004), and these phenomena can be overcome by introducing symbiotic microorganisms (Mazmanian et al., Cell 122: 107-118, 2005). Intestinal microbes regulate the development and differentiation of specific lineages of CD4 T cells, such as T helper 17 (Th17) cells or regulatory T (Treg) cells (Ivanov and Honda, Cell hostµbe 12:496-508, 2012; Wu and Wu, Gut microbes 3:4-14, 2012) (Atarashi et al., Science 331:337-341, 2011; Macpherson and Harris, Nature Reviews Immunology 4:478-485, 2004; Round and Mazmanian, Nature Reviews Immunology 9:313-323, 2009). Treg cells are CD4 + A subgroup of T cells with suppressive function, characterized by the expression of the transcription factor Foxp3 (S. Hori et al., Science 299: 1057-1061, 2003). Treg cells are usually formed in the thymus (nTreg), but can also be found in normal CD4 + Occurs near cells (pTreg).

[0003] Recently, it has been shown that each species of microbial communities can form the immune components of the host. In other words, Treg and Th17 cells interact to regulate the host's immune response to various antigens. For example, segmented filamentous bacteria (SFB) strongly induce Th17 cells in the small intestine, and the Th17 cells promote systemic autoimmunity and play an important role in the host's resistance to intestinal pathogens (Gaboriau-Routhiau et al., Immunity 31: 677-689, 2009; Ivanov et al., Cell 139: 485-498, 2009; Wu et al., Immunity 32: 815-827, 2010). In contrast, it is known that some strains constituting intestinal flora and some probiotic strains can affect the differentiation, accumulation and function of Treg cells in mouse colon (Tanoue, T et al., Nature reviews Immunology 16: 295-309, 2016). In the digestive lamina propria (LP), especially in the large intestine, more CD4+ T cells expressing the transcription factor Foxp3 (forkhead box P3) were found compared to other organs. + Regulatory T cells (Treg) (Atarashi et al., Science 331:337-341, 2011). Several studies have shown that mucosal Foxp3 +The number and function of Treg are affected by the presence of some intestinal bacteria. A mixture of 17 strains belonging to Clostridium IV, XIVa and XVIII clusters improves differentiation, accumulation and function of Treg cells in mouse colon (Atarashi et al., Nature 500:232-236,2013). Mice treated with probiotic bacteria (i.e., Bifidobacterium and Lactobacillus) every day show an inflammatory state of change by inducing Treg (Di Giacinto et al., The Journal of Immunology 174:3237-3246,2005; Karimi et al., American journal of respiratory and critical care medicine 179:186-193,2009; Lyons et al., Clinical & Experimental Allergy 40:811-819,2010). Human commensal Bacteroides fragilis colonized mice enhanced the production of Th1 and IL-10 of Treg cells (Round and Mazmanian, PNAS 107: 12204-12209, 2010; Telesford et al., Gut microbes 6: 234-242, 2015). Previous studies have shown that IRT5, as a mixture of five probiotic bacteria, can induce Treg cells and inhibit various immune diseases (Chae et al., PloS One 7, e52119, 2012; Kwon et al., Clinical Immunology 146: 217-227, 2013; Kwon et al., PNAS 107: 2159-2164, 2010). However, the basic molecular mechanism of Treg cell production is still unclear. Various studies have shown that metabolites or cell wall components produced by bacteria can promote the differentiation of Treg cells. For example, butyrate has been reported to be the main effector molecule for inducing colonic Treg cells by Clostridium (Furusawa et al., Nature 504:446-450, 2013). Polysaccharide A (PSA), as a zwitterionic polysaccharide of Bacteroides fragilis, has been identified as an important immunomodulator for inducing IL-10 for Treg production (Mazmanian et al., Cell 122:107-118, 2005; Ochoa-Reparaz et al., The Journal of Immunology 185:4101-4108, 2010). Giving insufficient main microorganisms to patients can repair the imbalance of imbalanced microorganisms. For example, it is reported that transferring fecal microbiota (FMT) from healthy donors to patients has a beneficial effect on gastrointestinal diseases. However, due to safety issues, FMT has not yet been approved as a general treatment method.In addition, it is reported that the intake of sufficient amounts of safe bacteria including probiotics can alleviate various immune diseases. However, beneficial microorganisms may vary depending on the patient's immune status. Some patients need to suppress overactivated immune responses (i.e., allergies or autoimmune diseases), while some patients need to strengthen the immune system (i.e., cancer or viral infection). For example, when Th17 inducible probiotic bacteria Bifidobacterium were given to rheumatoid arthritis animal models, arthritis symptoms worsened (Tze Guan Tan, 113 (50): E8141-E8150, 2016). Therefore, identifying beneficial microorganisms and discovering the mechanism of their effectors has important therapeutic significance.

[0004] Various hyperimmune diseases, such as autoimmune and allergic diseases, can be regulated by enhancing Treg cells in vivo. In the present invention, by screening probiotic candidate strains for the purpose of improving Treg cells induced by stable microorganisms, Bifidobacterium bifidum PRI1 was selected as the best candidate. The ability of this bacterium to induce the production of food antigens or symbiotic-induced regulatory T cells (iTreg cells) was identified using a germ-free mouse (GF mouse) system. Effector molecules derived from Bifidobacterium bifidum PRI1 include butyrate and acetate as metabolites. In addition, according to the present invention, cell surface β-glucan / galactan polysaccharide (CSGG) derived from Bifidobacterium bifidum PRI1 functionally inhibits intestinal inflammation.

[0005] Therefore, the inventors have made extensive efforts to discover the mechanism of inducing Treg cells with the potential to treat immunoregulatory diseases and inflammatory immune diseases. As a result, the inventors found that the cell surface β-glucan / galactan polysaccharide (CSGG) of the selected probiotic bacteria is the main effector molecule that can induce the production of iTreg cells, and confirmed that the iTreg cells induced by CSGG can inhibit colitis, thereby completing the present invention.

[0006] Public Content

[0007] Technical issues

[0008] The object of the present invention is to provide a polysaccharide comprising β-1,6-glucan inducing regulatory T (Treg) cells as an active ingredient, and a probiotic strain producing the polysaccharide.

[0009] Another object of the present invention is to provide a composition for immunomodulation, comprising the polysaccharide or strain as an active ingredient.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing, treating or alleviating immune diseases or inflammatory diseases, wherein the pharmaceutical composition comprises the polysaccharide or strain as an active ingredient.

[0011] Another object of the present invention is to provide a method for preventing or treating immune diseases or inflammatory diseases, which comprises administering a composition comprising the polysaccharide or strain as an active ingredient.

[0012] Another object of the present invention is to provide use of a composition for preventing or treating immune diseases or inflammatory diseases, wherein the composition comprises the polysaccharide or strain as an active ingredient.

[0013] Another object of the present invention is to provide an immunosuppressant or immunotherapeutic agent comprising the polysaccharide or strain as an active ingredient.

[0014] Another object of the present invention is to provide a method for producing induced regulatory T (iTreg) cells using the polysaccharide or strain.

[0015] Another object of the present invention is to provide a cell therapeutic agent for preventing, treating or alleviating immune diseases or inflammatory diseases, comprising the induced regulatory T (iTreg) cells produced by the method as an active ingredient.

[0016] Another object of the present invention is to provide a method for preventing or treating an immune disease or an inflammatory disease, the method comprising administering a cell therapeutic agent comprising the induced regulatory T cells (iTregs) produced by the method as an active ingredient.

[0017] Another object of the present invention is to provide use of a cell therapeutic agent in preventing or treating an immune disease or an inflammatory disease, wherein the cell therapeutic agent comprises the induced regulatory T cells (iTreg) produced by the method as an active ingredient.

[0018] Technical Solution

[0019] In order to achieve the above object, the present invention provides a probiotic strain that produces β-1,6-glucan.

[0020] The present invention also provides a polysaccharide comprising β-1,6-glucan as an active ingredient.

[0021] The present invention also provides a composition for immunomodulation, which comprises the polysaccharide or strain as an active ingredient.

[0022] The present invention also provides a pharmaceutical composition and a therapeutic agent for preventing or treating immune diseases or inflammatory diseases, wherein the pharmaceutical composition and the therapeutic agent comprise the polysaccharide or the strain as an active ingredient.

[0023] The present invention also provides food comprising the polysaccharide or the strain as an active ingredient.

[0024] The present invention also provides a method for preventing or treating immune diseases or inflammatory diseases, which comprises administering a composition comprising the polysaccharide or strain as an active ingredient.

[0025] The present invention also provides use of the composition in preventing or alleviating immune diseases or inflammatory diseases, wherein the composition comprises the polysaccharide or strain as an active ingredient.

[0026] The present invention also provides an immunosuppressant or immunotherapeutic agent comprising the polysaccharide or strain as an active ingredient.

[0027] The present invention also provides a method for producing induced regulatory T (iTreg) cells, the method comprising: treating dendritic cells (DC) with the polysaccharide or strain to obtain regulatory dendritic cells (rDC); and + T cells were co-cultured to induce regulatory T (Treg) cells.

[0028] The present invention also provides a therapeutic agent for preventing, treating or alleviating immune diseases or inflammatory diseases, which comprises the induced regulatory T (iTreg) cells produced by the method as an active ingredient.

[0029] The present invention also provides a method for preventing or treating an immune disease or an inflammatory disease, the method comprising administering a cell therapeutic agent comprising the induced regulatory T (iTreg) cells produced by the method as an active ingredient.

[0030] The present invention also provides use of a cell therapeutic agent in preventing or treating an immune disease or an inflammatory disease, wherein the cell therapeutic agent comprises the induced regulatory T (iTreg) cells produced by the method as an active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 demonstrated an increase in the Treg population in the colonic lamina propria caused by monocolonization with Bifidobacterium bifidum. Figure 1 A to 1C show CD4 Treg cells in the colon of GF mice or in the colon of mice colonized with Lpa or Bb (cLP). + FOXP3 + Cells, CTLA4 + and IL10 + Flow cytometry and percentage analysis results. Figure 1D to 1F show the absolute number of different types of Treg cells in the colon of GF mice colonized with bacteria and representative flow cytometry graphs and signals. The numbers in the quadrants represent the cell percentage, and the circles in the graphs represent individual mice corresponding to each parameter, the data represent 3 to 5 independent experiments (n ≥ 3 mice), and all graphs show the mean ± SEM, and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t test).

[0032] Figure 2 Results confirming that Bifidobacterium bifidum PRI1 is a Treg-inducing bacterium are shown. Figure 2 A and 2B show representative IL-10 / IL-12 cytokines in the candidate probiotic strain Bifidobacterium bifidum PRI1 (Bb) selected as a bacterial strain to promote Treg induction ( Figure 2 A) and Foxp3 expression ( Figure 2 B) Ratios in which each bacterial strain with the indicated colony forming unit (cfu) titer was incubated with total mesenteric lymph node (mLN) cells for 72 h and then analyzed for cytokine levels or Foxp3 expression by ELISA or flow cytometry. Figure 2 C shows the localization analysis of Bifidobacterium bifidum PRI1 (Bb) in the intestinal interstitium of GF mice by HISTO-FISH staining using a DNA-Cy5 probe (EUB338, red) 3 weeks after colonization. Figure 2 D shows the cecal images (left) and cecal weights (right) of GF mice, Bb-monocolonized mice (Bb), and SPF mice. Figure 2 E shows the results of measuring butyrate and acetate levels in the cecum and feces of GF mice or Bb-monocolonized mice (Bb). Figure 2 F shows the expression of CD4 in the colonic lamina propria (cLP) of GF mice, mice colonized with SFB or Lpa, and SPF mice. + Foxp3 + Flow cytometry graphs of T cells. Numbers in quadrants indicate cell percentages, circles in graphs indicate individual mice corresponding to each parameter, data represent 3 to 5 independent experiments (n ≥ 3 mice) (Bifidobacterium bifidum: Bb; Lactobacillus acidophilus: Lac; Lactobacillus casei: Lca; Lactobacillus reuteri: Lre; Lactobacillus paracasei: Lpa; segmented filamentous bacteria: SFB), and all graphs show mean ± SEM, and *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t test).

[0033] Figure 3 The effects of monocolonization with Bifidobacterium bifidum on the generation and phenotype of Treg cells are demonstrated. Figure 3 A shows CD4+ T cells in the small intestinal lamina propria (siLP), mLN, pLN, and spleen of SPF mice, GF mice, or mice monocolonized with Bb, SFB, or Lpa. + Foxp3 + The frequency of T cells. Figure 3 B and 3C show CD4 in cLPs or siLPs from mice monocolonized with the corresponding bacteria. + CD103 + Foxp3 + ( Figure 3 B) and CD4 + CD44 hi CD62L lo Foxp3 + T cells ( Figure 3 C) Flow cytometry graphs and frequencies, where the graphs and frequencies are expressed as percentages. The numbers in the quadrants represent the cell percentages, the circles in the graphs represent individual mice corresponding to each parameter, the data represent 3 to 5 independent experiments (n ≥ 3 mice), and all graphs show mean ± SEM, and *p < 0.05, **p < 0.01 (Student's t test).

[0034] Figure 4 Increases in Treg populations in other lymphoid organs resulting from monocolonization with B. bifidum are shown, where cells were isolated from lymphoid organs of GF mice or mice at 3 weeks after monocolonization with Bb or Lpa. Figure 4 A and 4B show Helios in the indicated lymphoid tissues - and Nrp1 - Frequency of Treg population. Figure 4 C and 4D show Helios from the indicated organs from GF mice or mice monocolonized with Bb or Lpa. - Foxp3 + cell( Figure 4 C) or RORγt + Helios - Foxp3 + Treg cells Figure 4 D) Flow cytometry graphs and frequencies, where the graphs and frequencies are expressed as percentages. The numbers in the quadrants represent the cell percentages, the circles in the graphs represent individual mice corresponding to each parameter, the data represent 3 to 5 independent experiments (n ≥ 3 mice), and all graphs show mean ± SEM, and *p < 0.05, **p < 0.01 (Student's t test).

[0035] Figure 5 showed that monocolonization with Bifidobacterium bifidum promoted the new growth of pTreg cells. Figure 5A shows the experimental strategy used to analyze the de novo generation of pTreg cells after monocolonization with Bb. Figure 5 B and 5C show the potential for + Foxp3 GFP Reporter mice sorted naive CD4 + Foxp3 - Results obtained after T cells were transferred into GF mice and monocolonized with Bb mice for 3 weeks, as measured by cLP ( Figure 5 B) and siLP( Figure 5 C) GFP expression in the cells to analyze Foxp3 + Treg population. Figure 5 D shows Foxp3 measured by GFP expression in the indicated lymphoid organs. + Results of the frequency of Treg population. Figure 5 E shows that the + Foxp3 GFP Reporter mice sorted naive CD4 + Foxp3 - T cells were transferred into SPF mice and fed with mock (PBS) or Bb (5x10 8 cfu) for 3 weeks, Foxp3 + Results of Treg cell analysis. Data represent 3 to 5 independent experiments (n≥3 mice), and all graphs show mean±SEM, and *p<0.05, **p<0.01 (Student's t test).

[0036] Figure 6 The induction of microbiota-reactive Treg cells by dietary antigens as well as colonization with Bifidobacterium bifidum was demonstrated. Figure 6 A and 6B show the expression of Thy1.1 + Foxp3 GFP CTV-labeled naive CD4 + OT-II + Foxp3 - After T cells were adopted into GF mice or GF mice colonized for 14 days and then fed with OVA (20 mg) every other day for 7 days, the donor (Thy1 + cLP CD4 of OT-II cells + Foxp3 + The results of the group analysis, Figure 6 A and 6B show the FACS plots and frequencies of Treg cells generated in vivo, respectively. Figure 6 C to 6E shows that the Tg CD45.1 +Foxp3 GFP Mouse sorted naive CD4 + CBir + Foxp3 - T cells adoptively transferred into SPF Rag1 - / - The recipients were then fed Bb or PBS daily until the end of the experiment, and body weight, colon length, and histopathological changes were measured and analyzed (C, D, and E, respectively). Figure 6 F to 6G show CBir in the cLP of mock- or Bb-colonized mice + CD45.1 + Foxp3 + Treg cells Figure 6 F) and IFN-γ + cell( Figure 6 G) Flow cytometry plots and frequencies. Figure 6 H shows the CD4 + Results of relative proliferation analysis of Treg cells corresponding to the indicated antigens by FACS 3 days after cells were labeled with CTV and then cultured in the presence of fecal antigens from GF mice and mice colonized with Bb or Lpa together with spleen APCs from which T cells had been depleted. Figure 6 I shows the results of comparing the frequency distribution of 43 intrinsic α chains and 61 intrinsic β chains in the TCR-CDR3 region in Tregs of colon, mLN, and spleen of Bb-monocolonized mice and GF mice. The numbers in the quadrants indicate the percentage of cells, the circles in the graphs indicate the individual mice corresponding to each parameter, the data represent 3 to 5 independent experiments (n ≥ 3 mice), and all bars show mean ± SEM, and *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t test).

[0037] Figure 7 The induction of microbiota-reactive Treg cells by dietary antigens as well as colonization with Bifidobacterium bifidum was demonstrated. Figure 7 A shows the experimental strategy used to identify OVA-specific pTreg cells induced in response to dietary antigen (OVA-specific OTIIT cells) in GF mice or Bb-colonized GF mice. Figure 7 B shows the results of the study to identify Rag1 derived from cells fed PBS or Bb. - / - Experimental strategy for microbiota-specific pTreg cells (flagellin-specific CBir T cells) in SPF mice. Figure 7 C shows Rag1 in the presence of PBS or Bb - / - CD4 in cLP of SPF mice into which CBir naive T cells were transferred +Foxp3 - or CD4 + Foxp3 + Analysis of RORγ, GATA3, and T-bet populations of T cells. Figure 7 D shows the experimental strategy used to determine Bb-induced Treg expansion in the presence of a combination of fecal antigens from mice monocolonized with Bb or Lpa or GF mice. Figure 7 E shows total colonic CD4 + Cells were labeled with CTV and then cultured in the presence of a combination of fecal antigens from GF or mice colonized with Bb or Lpa alone together with spleen cells (APC) from which T cells have been removed for 3 days, and the results of relative proliferation analysis of Treg cells corresponding to the indicated antigens by FACS. Data represent 3 independent experiments (n≥3 mice), and all graphs show mean ± SEM, and *p<0.05, **p<0.01 (Student's t test).

[0038] Figure 8 The effect of monocolonization with Bifidobacterium bifidum on the TCR repertoire of Treg cells is demonstrated. Figure 8 A shows the results comparing the Shannon diversity of α and β chains of the Treg cell repertoires obtained from different organs (colon, MLN, and spleen) of GF mice or Bb-monocolonized mice. Figure 8 B shows a heat map of α and β chains showing the frequency of the top 85 TCR-CDR3 regions in colonic Treg cells of Bb monocolonized mice and GF mice, with CDR3 regions of “common” markers (28 peptides) indicating the presence of read counts in each group and hue indicating the relative frequency at which a given TCR was found. Figure 8 C shows the frequency distribution of typical TCR-CDR3α regions represented by a bidirectional bar graph: 85 in the large intestine, 1469 in the MLN, and 1381 in the spleen, and Figure 8 D shows the frequency distribution of typical TCR-CDR3β regions represented by a bidirectional bar graph: 118 in colon, 1724 in MLN, and 1381 in spleen.

[0039] Fig. 9 demonstrated that cell surface β-glucan / galactan polysaccharide (CSGG) of Bifidobacterium bifidum mediates Treg cell induction. Fig. 9 A shows the structure of the cell surface β-1,6-glucan (CSβG) polysaccharide of Bifidobacterium bifidum; Glcp: glucopyranose. Fig. 9 B shows the effect of β-1,6-glucan treatment on CSGG-induced iTreg cells. Fig. 9 C shows the population and phenotype of Treg cells in GF mice or GF mice colonized with Bb or Lpa or subjected to intraperitoneal injections of CSGG (100 μg / dose) every other day for 3 weeks. Fig. 9 D shows the confirmation of the expression of naive CD4 + human CD4 + CD25 + Foxp3 + Results of Treg induction. Fig. 9 E shows the expression of CD11c cells treated with mock or CSGG + RNA-seq analysis of DCs. Fig. 9 F shows the expression of naive CD4 T cells derived from mice pretreated with mock or CSGG. + T cells and CD11c + DCs along with suboptimal iTreg production, where CD4 + Foxp3 + Treg cell population. Fig. 9 G shows that in cultured naive CD4 + T cells together with wild-type or TLR2-deficient CD11c T cells pretreated with mock or CSGG + Results of measuring TGFβ1 levels in culture supernatants obtained after DC induction. Data represent at least 3 independent experiments, and all bar graphs show mean±SEM, and *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).

[0040] Fig.10 demonstrated that Bifidobacterium bifidum promoted Treg cell induction through a DC-dependent mechanism. Fig.10 A shows the quantification of relative mRNA expression of normalized cytokines to Hprt in whole colon of GF mice or Bb monocolonized mice. Fig.10 B to 10D show cLP-DC (MHCII + CD11c + CD11b + CD103 + F4 / 80 - )'s relative mRNA expression levels. Fig.10 E shows the experimental strategy for in vitro induction of Tregs in a DC:T co-culture system. Fig.10 F shows the response to splenic CD11c + DC and various amounts of Bb (cfu) titration, Foxp3+ Dose-dependent induction and survival of T cells. Fig.10 G shows the expression of CD11c in spleen pretreated with Bb or Lpa under suboptimal Treg induction conditions (anti-CD3: 0.1 μg / ml; TGF-β: 0.1 ng / ml; IL-2: 100 units / ml). + After DCs were co-cultured with naive CD4+ T cells for 3 days, the results of Foxp3+ Treg cells in living cells were analyzed, and IL-10 secretion was measured by ELISA analysis of culture supernatants. Data represent 3 to 5 independent experiments (n ≥ 3 mice), all graphs show mean ± SEM, *p < 0.05, **p < 0.01 (Student t test), and CD4 + Foxp3 + Representative flow cytometry and mean frequency of Treg cells.

[0041] Fig.11 Results showing the identification of β-glucan / galactan polysaccharide (CSGG) as a core effector molecule of Treg cells induced by Bifidobacterium bifidum are presented. Fig.11 A shows the CD11c of spleen pretreated with a portion of Bifidobacterium bifidum under suboptimal Treg induction conditions. + DC and naive CD4 + Results of FACS analysis of Foxp3+ Treg cells 3 days after co-culture of T cells. Fig.11 B shows the CD11c in spleen pretreated with the indicated amount of total cell surface polysaccharide (tCSPS) + DC and naive CD4 + After 3 days of co-culture with T cells, Foxp3 + Results of Treg cell analysis. Fig.11 C shows the structures of neutral (CSGG) or negatively charged polysaccharides (PGβG) isolated from total cell surface polysaccharides (tCSPS) of Bifidobacterium bifidum (Glcp: glucopyranose, Galp: galactopyranose, Galf: furanose glucose, Gro: glycerol; n = repeating unit, the relative abundance of each polysaccharide is indicated in % (mol / mol)). Fig.11 D shows a comparison of CD4 between neutral (CSGG; 5 μg / ml) and negatively charged (PGβG: 100 μg / ml) polysaccharides. + Foxp3 + Results of Treg induction activity (compared with tCSPS (100 μg / ml)). Fig.11E shows the effects of β-1,6-glucanase, β-1,4-galactanase, and β-1,4-glucanase treatment on the induction of CSGG-induced iTreg cells (CSGG; 50 μg / ml). Fig.11 F shows the treatment of naive CD4 Tregs with CSG (50 μg / ml) without DCs under suboptimal Treg induction conditions (anti-CD3: 0.1 μg / ml; α-CD28: 0.1 μg / ml; TGF-β: 0.1 ng / ml; IL-2: 100 units / ml). + Results obtained after T cells. Data represent 3 to 5 independent experiments (n ≥ 3 mice), and all graphs show mean ± SEM, and *p < 0.05, **p < 0.01 (Student t test);

[0042] Fig.12 We demonstrated enhanced Treg cell induction by CSGG both in vitro and in vivo. Fig.12 A shows the generation of the in vitro CD4+Foxp3+ population after co-culture of naive CD4+ T cells with CD11c+ DCs pretreated with bacteria or CSGG under suboptimal Treg induction conditions. Fig.12 B shows the CD4+Foxp3+ population in GF mice or mice monocolonized by intraperitoneal injection of Bb, Lpa or CSGG (100 μg / dose) into GF mice every other day for 3 weeks. Fig.12 C and 12D show the results of analyzing the frequency of the indicated Treg cell populations in lymphoid organs after intraperitoneal injection of CSGG (100 μg / dose) or PBS into GF mice every other day for 3 weeks. The data represent 3 to 5 independent experiments (n≥3 mice), and all graphs show mean ± SEM, and *p<0.05, **p<0.01, ***p<0.001 (Student's t test).

[0043] Fig.13 We demonstrated that CSGG promoted the induction of iTregs through the generation of TLR2-dependent regulatory DCs. Fig.13 A shows the CD11c cells pretreated with mock or CSGG + DC and naive CD4 + The results of cytokine levels were confirmed in the culture supernatant obtained after T cell co-culture. Fig.13 B shows the effect of anti-TGFβAb treatment on in vitro CD4+Foxp3+ Treg induction after Bb or CSGG treatment. Fig.13 C shows that IL-10 - / - KO mice or wild-type CD11c + DCs and wild-type naive CD4+ The outcome of iTreg induction was measured 3 days after T cell co-culture. Fig.13 D shows the results of analyzing RNA-seq data to determine the expression of genes encoding TLR subtypes after CSGG treatment. Fig.13 E shows that under suboptimal iTreg generation conditions, naive CD4 + CD11c T cells from mice pretreated with mock or CSGG + After 3 days of DC co-culture, CD4 + Foxp3 + Results of Treg cell induction. Fig.13 F shows the expression of wild-type CD11c + DCs or TLR2-deficient CD11c pretreated with mock or CSGG + DC and naive CD4 + After T cell co-culture, the results of IL-10 and IFNγ cytokine levels in the culture supernatant were measured. Fig.13 G shows pretreatment of CD11c with mock or CSGG in the presence of blocking antibodies against C-type lectins + DCs then associate the cells with naive CD4 + After co-culturing T cells together, the results of iTreg induction were examined by flow cytometry. Fig.13 H shows the results of the experiment on Dectin1 pretreated with wild type or CSGG. - / - Dectin2 - / - CD11c KO mice + DC and naive CD4 + Three days after T cells were co-cultured together, the results of iTreg induction were measured. Data are representative of at least 3 independent experiments, and all bar graphs show mean ± SEM, and *p < 0.05 (Student's t-test).

[0044] Fig.14 The intestinal inflammation inhibitory function of CSGG-induced iTreg cells was demonstrated. Fig.14 A to 14C show that in the + Foxp3 GFP Naive CD4 isolated from reporter mice + Foxp3 - T cells were adoptively and co-transferred into RAG1 in the indicated combinations with iTreg cells - / - Results were obtained in the recipients, where body weight, colon length, and pathological changes in colon tissue were measured. Fig.14D shows the results of analyzing the Foxp3 stability of Treg cells transferred from cLPs at the end of the experiment, where CD4 + Foxp3 GFP+ The purity of the cells was 98% or higher. Fig.14 E to 14G shows that in + Foxp3 GFP Reporter mouse isolated CD4 + Foxp3 - Adoptive transfer of T cells into RAG1 - / - Results were obtained after intraperitoneal administration of PBS or CSGG (100 μg / ml) to mice, where body weight, colon length, and pathological changes in colon tissue were measured. Fig.14 H shows the results of analyzing IFNγ production by effector T cells in cLPs at the end of the experiment. Data represent at least 3 independent experiments, and all bar graphs show mean ± SEM, and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t test).

[0045] Fig.15 The intestinal inflammation inhibitory function of CSGG-induced iTreg cells was demonstrated. Fig.15 A shows the expression of Thy1.1 in naive responders to CTV markers. + CD4 + Foxp3 - Results obtained after T cell sorting and co-culture, in which CD45.1 + CD4 + Foxp3 + iTreg cells were derived from spleen cells (CD45.2 labeled cells were removed) + Purified APCs treated with mock or CSGG were then isolated from T cells of mice and the proliferation of responding T cells was analyzed by flow cytometry. Fig.15 B shows adoptively transferred RAG1 suppressed by nTreg and iTreg cells induced in vitro by mock, Bb, or CSGG - / - Colon length in colitis. Fig.15 C shows the induction of RAG1 by adoptively transferred naive T cells by intraperitoneal administration of mock (PBS) or CSGG (100 μg / dose). - / - Colon length in colitis. Fig.15 D shows adoptively transferred RAG1 in response to mock or csGG treatment - / - CD4 in mLN of mice + CD45.1 + Foxp3 +Treg population. Data are representative of at least 2 independent experiments, and all bar graphs show mean ± SEM, and *p < 0.05 (Student's t-test). DETAILED DESCRIPTION

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0047] The present invention shows that the human commensal bacterium Bifidobacterium bifidum PRI1 (B. bifidum) can enhance the functional activity of naive CD4 T cells to suppress inflammatory colitis Foxp3 + Differentiation of iTreg cells. Cell surface-derived β-glucan / galactan polysaccharide (CSGG) is an active molecule that encompasses the entire bacterial function in inducing iTreg cells. The mechanism of action of CSGG is mediated by the induction of the TLR2 / IL-10 regulatory pathway in dendritic cells (DCs).

[0048] Since the dysbiosis of commensal microorganisms is closely related to the development of many diseases, the promotion of Treg cells in vivo by Treg-inducing microorganisms can achieve the control of various inflammatory diseases. Gut microorganisms play an important role in maintaining immune homeostasis, and this gut microbial dysbiosis can cause the immune suppressive Foxp3 + Inflammatory diseases associated with impaired T regulatory (Treg) cell function. Although some commensal microorganisms are known to enhance the induction of Treg cells (Tanoue, T et al., Nature reviews Immunology 16: 295-309, 2016), the "effector molecules" that control this process (i.e., the molecular mechanisms, antigen specificity, and target cells of Treg cells induced by bacteria) are still unknown.

[0049] Therefore, in the present invention, a candidate strain of probiotics that is generally recognized as safe (GRAS) is selected, and Bifidobacterium bifidum PRI1 (Bifidobacterium bifidum) with multiple TCR specificities for food antigens or commensal bacteria is selected as the best candidate for Treg-inducing bacteria. Bifidobacterium bifidum PRI1 was deposited in the Korean Collection of Type Cultures on May 19, 2017 (Deposit Number: KCTC13270BP). In addition, it has been confirmed that Bifidobacterium bifidum and its cell surface-derived β-glucan / galactan polysaccharide (CSGG) are the main components for inducing Treg, and specifically, CSGG efficiently reproduces the activity of the entire bacterium and partially mediates regulatory dendritic cells through a TLR2-dependent mechanism, and acts in both mice and humans, thereby inducing Treg cells. Treg cells induced by Bifidobacterium bifidum or by purified CSGG show stable and powerful therapeutic or inhibitory abilities against colitis.

[0050] Therefore, the present invention relates to a strain producing β-1,6-glucan.

[0051] In addition, in one aspect, the present invention relates to a polysaccharide comprising β-1,6-glucan as an active ingredient.

[0052] In the present invention, the polysaccharide may further include β-1,4-galactan, β-1,6-galactan or β-galactofuranosan, and these polysaccharides may be used alone. Specifically, the polysaccharide may be a complex with β-1,6-glucan as the main component, or may be a single substance.

[0053] In addition, β-1,6-glucan, β-1,4-galactan, β-1,6-galactan and β-galactofuranosan may be contained in a molar ratio of 5-50:2-15:2-15:1-5 relative to the total polysaccharide, more preferably in a molar ratio of 10-20:3-10:3-10:2-4, and most preferably in a molar ratio of 18:5:5:2, but the present invention is not limited thereto. The polysaccharide may include 5-50wt% of β-1,6-glucan, 2-15wt% of β-1,4-galactan, 2-15wt% of β-1,6-galactan and 1-5wt% of β-galactofuranosan relative to a total value of 100wt%. Preferably, the polysaccharide may include 10-20wt% of β-1,6-glucan, 3-wt% of β-1,4-galactan, 3-10wt% of β-1,6-galactan and 2-4wt% of β-galactofuranan relative to 100wt% total value, and each polysaccharide may be included in an amount of 18wt%, 5wt%, 5wt% and 2wt% relative to 100wt% total value.

[0054] In the present invention, the polysaccharide preferably has a molecular weight of about 3-5 kDa, more preferably 4 kDa, but is not limited thereto as long as it is any mixture of various molecular weights of 100 kDa or less.

[0055] In the present invention, the charge of the polysaccharide may be neutral.

[0056] In the present invention, polysaccharides can induce regulatory T (Treg) cells, and regulatory T (Treg) cells are preferably CD4 + Foxp3 + Treg cells, but not limited thereto.

[0057] The induction of regulatory T (Treg) cells can be mediated by dendritic cells (DC). In addition, regulatory T cells (Treg) can increase the induction of IL-10 and TGF-β production.

[0058] In the present invention, the polysaccharide is preferably derived from a Bifidobacterium bifidum strain, more preferably from a strain having a base sequence at least 99% homologous to Bifidobacterium bifidum PRI1, and most preferably from Bifidobacterium bifidum PRI1 (KCTC13279BP) strain, but is not limited thereto.

[0059] In the present invention, the strain having a base sequence having at least 99% homology to Bifidobacterium bifidum PRI1 may be Bifidobacterium bifidum A8 or Bifidobacterium bifidum LMG11582.

[0060] In the present invention, the polysaccharide preferably has anti-inflammatory or immune function regulating activity, and more preferably has immunosuppressive activity, but is not limited thereto. If people take probiotics incorrectly, people with intestinal trauma (such as intestinal bleeding, etc.) may have side effects, and in such cases, a therapeutic effect can be achieved when polysaccharides are administered.

[0061] In another aspect, the present invention relates to a strain producing β-1,6-glucan polysaccharide.

[0062] In the present invention, the strain is preferably Bifidobacterium bifidum, but is not limited thereto, as long as the strain produces CSGG polysaccharide. In addition, in the present invention, Bifidobacterium bifidum preferably has a base sequence having at least 99% homology with Bifidobacterium bifidum PRI1, and is more preferably Bifidobacterium bifidum PRI1 (KCTC13279BP), but is not limited thereto.

[0063] In the present invention, the strain may further produce β-1,4-galactan, β-1,6-galactan or β-galactofuranan, but is not limited thereto.

[0064] In the present invention, the strain can induce regulatory T cells (Treg), and the regulatory T (Treg) cells may preferably be CD4 + Foxp3 + Treg cells, but not limited thereto. In addition, the strain can increase the induction of IL-10 and TGF-β production.

[0065] The induction of regulatory T (Treg) cells can be mediated by DCs.

[0066] In the present invention, the strain preferably has anti-inflammatory or immune function regulating activity, and more preferably has an activity of suppressing hyperimmunity, but is not limited thereto.

[0067] Specifically, the strain produces CSGG polysaccharide, thus inducing antigen-specific Tregs by various antigens (e.g., food, other intestinal bacteria, or bacteria-derived substances), and thus the strain can be used for the treatment of inflammatory or immune diseases.

[0068] In another aspect, the present invention relates to a composition for immunomodulation, comprising β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide as an active ingredient.

[0069] In the present invention, the polysaccharide may further include β-1,4-galactan, β-1,6-galactan or β-galactofuranosan, but is not limited thereto.

[0070] In the present invention, the polysaccharide or strain can induce regulatory T (Treg) cells, and the regulatory T (Treg) cells are preferably CD4 + Foxp3 + Treg cells, but not limited thereto. In addition, polysaccharides or strains can increase the induction of IL-10 and TGF-β production.

[0071] The induction of regulatory T (Treg) cells can be mediated by DCs.

[0072] As used herein, the term "immunomodulation" refers to alleviating immune imbalance in the blood and maintaining immune homeostasis. Maintaining immune homeostasis refers to a state of balancing immune tolerance as a mechanism of suppressing immunity and immunity promoting immune response, and maintaining this state is an essential factor for treating immune diseases, especially autoimmune diseases.

[0073] In another aspect, the present invention relates to an immunosuppressant or immunotherapeutic agent comprising β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide as an active ingredient.

[0074] As used herein, the term "immunosuppressant" refers to various substances used to reduce or block the host's ability to produce antibodies (humoral immune response) or to elicit a cellular immune response to the action of an antigen.

[0075] In addition, the term "immunotherapy" of the present invention refers to a treatment for resisting a disease by inducing activation or suppression of the body's immune system. The most representative immunotherapy is cancer immunotherapy, which induces immune activity and kills cancer cells, and for diseases induced by abnormal immune responses such as autoimmune diseases, inflammatory bowel disease (IBD) and allergies, immunotherapy can be performed by immunosuppressants.

[0076] Therefore, in the present invention, "immunosuppressant" and "immunotherapeutic agent" can be used with the same meaning.

[0077] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating an immune disease or an inflammatory disease, comprising β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide as an active ingredient.

[0078] In the present invention, the polysaccharide may further include β-1,4-galactan, β-1,6-galactan or β-galactofuranosan, but is not limited thereto.

[0079] In the present invention, the polysaccharide or strain can induce regulatory T (Treg) cells, and the regulatory T (Treg) cells are preferably CD4 + Foxp3 + Treg cells, but not limited thereto. In addition, polysaccharides or strains can increase the induction of IL-10 and TGF-β production.

[0080] The induction of regulatory T (Treg) cells can be mediated by DCs.

[0081] In addition, the immunomodulatory composition can be used as a pharmaceutical composition for the purpose of regulating immune activity and preventing, improving or treating immune diseases. In this regard, the amount and form thereof can be appropriately adjusted according to the purpose of use.

[0082] The composition for immunomodulation can exhibit immunostimulatory and hyperimmunosuppressive effects. Immunostimulatory effects are effects that increase the expression of TNF-α in macrophages or enhance the proliferation capacity of splenocytes to promote immunity. Hyperimmunosuppressive effects are effects that inhibit hyperimmunity by inhibiting the overexpression of lymphocytes caused by nonspecific stimulants in splenocytes.

[0083] The "immune disease" prevented or treated by the composition according to the present invention can be any one selected from the following: dermatitis, allergy, rhinitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, peritendinitis, type 1 diabetes, scleroderma, neurodegenerative diseases, type 2 diabetes, silicosis, atherosclerosis, vitiligo, conjunctivitis and autoimmune diseases, but is not limited thereto.

[0084] The "autoimmune disease" prevented or treated by the composition according to the present invention can be any one selected from the following: rheumatoid arthritis, systemic sclerosis, atopic dermatitis, psoriasis, asthma, Guillain-Barré syndrome, myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, polyarteritis nodosa, temporal arteritis, childhood diabetes, alopecia areata, pemphigus, aphthous stomatitis, Crohn's disease, and Behcet's disease, but is not limited thereto.

[0085] The "inflammatory diseases" prevented or treated by the composition according to the present invention collectively refer to diseases with inflammation as the main lesion, and may be any one selected from the following: swelling, allergy, asthma, conjunctivitis, periodontitis, rhinitis, tympanitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, myasthenia gravis and multiple sclerosis, but are not limited thereto.

[0086] As used herein, the term "prevention" refers to all effects of suppressing or delaying the onset of immune diseases or inflammatory diseases by administering the pharmaceutical composition according to the present invention.

[0087] As used herein, the term "treatment" refers to all actions that improve or beneficially alter the symptoms of immune diseases or inflammatory diseases by administering the pharmaceutical composition according to the present invention.

[0088] The pharmaceutical composition according to the present invention exhibits the immunostimulatory effect of the active ingredient, or the preventive or therapeutic and anti-inflammatory effects on various immune diseases through hyperimmunosuppressive effects.

[0089] The pharmaceutical composition may contain suitable carriers, excipients and diluents commonly used in pharmaceutical compositions in addition to β-1,6-glucan, β-1,4-galactan, β-1,6-galactan and β-galactofuranosan (CSGG) as active ingredients or the Bifidobacterium bifidum strain producing the polysaccharides.

[0090] The carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. When formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants are usually used.

[0091] The pharmaceutical composition according to the present invention can be used by preparing it into various forms according to conventional methods. Suitable preparations include oral preparations such as tablets, pills, powders, granules, sugar-coated tablets, hard or soft capsules, solutions, suspensions or emulsions; injections; aerosols, etc.; external preparations; suppositories; and sterile injections, but are not limited thereto.

[0092] Pharmaceutical compositions according to the present invention can be prepared into suitable formulations using pharmaceutically inert organic or inorganic carriers. That is, when formulations are in the form of tablets, coated tablets, sugar-coated tablets and hard capsules, organic or inorganic carriers may include lactose, sucrose, starch or its derivatives, talcum, calcium carbonate, gelatin or stearic acid or its salts. In addition, when formulations are in the form of soft capsules, organic or inorganic carriers may include vegetable oils, waxes, fats and semisolid and liquid polyols. In addition, when formulations are in the form of solutions or syrups, organic or inorganic carriers may include water, polyols, glycerine, vegetable oils etc.

[0093] In addition to the above-mentioned carriers, the pharmaceutical composition according to the present invention may also include preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, osmotic pressure control agents, antioxidants and the like.

[0094] The pharmaceutical composition according to the present invention is given with a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to the amount that is enough to treat the disease by the reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined according to factors including the patient's disease type, the severity of the disease, the activity of the drug, the sensitivity to the drug, the administration time, the route of administration, the excretion rate, the treatment time, and the drug administered together, as well as other factors well known in the medical field. The pharmaceutical composition according to the present invention can be given as a single therapeutic agent or in combination with other therapeutic agents, can be given in sequence or simultaneously with conventional therapeutic agents, and can be given in single doses or multiple doses. Importantly, considering all the factors described above, the pharmaceutical composition is given in a minimum amount without side effects to maximize the effect, which can be easily determined by those of ordinary skill in the art.

[0095] The composition according to the present invention can be administered in combination with immune-related proteins, particularly autoimmune-related proteins or allergy-related proteins. Specific examples of the protein include autoantigens involved in autoimmune diseases. For example, the protein may include autoantigens involved in rheumatoid arthritis, such as heat shock proteins (HSP), citrullinated filaggrin, glucose-6-phosphate isomerase, p205, collagen, etc.; autoantigens involved in type I diabetes, such as insulin, zinc transporter 8 protein (ZnT8), pancreatic and duodenal homeobox 1 (PDX1), chromogranin A (CHGA), and islet amyloid polypeptide (IAPP); and autoantigens involved in myasthenia gravis, such as acetylcholine receptors. In addition to all types of autoantigens known in autoimmune diseases, proteins may also include various allergens known to cause food allergies, such as peanuts, milk, eggs, tree nuts, beans, crustaceans (such as shrimp, etc.), fish-derived materials, etc.

[0096] The pharmaceutical composition of the present invention can be administered to a subject by various routes. The pharmaceutical composition can be administered, for example, by subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, or intraventricular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as the active ingredient together with various relevant factors (e.g., the disease to be treated, the route of administration, the age, sex and weight of the patient, the severity of the disease).

[0097] The administration method of the pharmaceutical composition according to the present invention can be easily selected according to the dosage form, and the pharmaceutical composition can be administered orally or parenterally. The dosage can vary according to the age, sex and weight of the patient, the severity of the disease and the route of administration.

[0098] In another aspect, the present invention relates to a therapeutic agent for preventing or treating an immune disease or an inflammatory disease, comprising β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide as an active ingredient.

[0099] In another aspect, the present invention relates to a method for preventing or treating an immune disease or an inflammatory disease, comprising administering a composition comprising β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide as an active ingredient.

[0100] In the present invention, the polysaccharide may further include β-1,4-galactan, β-1,6-galactan or β-galactofuranosan.

[0101] In another aspect, the present invention relates to use of a composition for preventing or treating an immune disease or an inflammatory disease, wherein the composition comprises β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide as an active ingredient.

[0102] In the present invention, the polysaccharide may further include β-1,4-galactan, β-1,6-galactan or β-galactofuranosan.

[0103] The pharmaceutical composition according to the present invention not only provides excellent immunoenhancing and hyperimmunosuppressive effects, but also has low toxicity and side effects, so that the pharmaceutical composition can be safely used even for long-term administration for the purpose of treating or preventing immune diseases.

[0104] The Bifidobacterium bifidum or CSGG according to the present invention can induce various antigen-specific Treg cells in vivo and thus can be used as an oral resistance adjuvant.

[0105] In another aspect, the present invention relates to a food comprising β-1,6-glucan polysaccharide or a strain of Bifidobacterium bifidum producing the polysaccharide as an active ingredient.

[0106] In the present invention, the polysaccharide may further produce β-1,4-galactan, β-1,6-galactan or β-galactofuranan, but is not limited thereto.

[0107] In the present invention, the polysaccharide or strain can induce regulatory T (Treg) cells, and the regulatory T (Treg) cells can preferably be CD4 + Foxp3 + Treg cells, but not limited thereto.

[0108] The induction of regulatory T (Treg) cells can be mediated by DCs and can induce the production of IL-10 and TGF-β.

[0109] As used herein, the term "food" includes all foods in the general sense, such as meat, sausages, bread, chocolate, candy, snacks, candy desserts, pizza, ramen, other noodles, gelatin, dairy products (including ice cream), various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, health foods, etc.

[0110] The term "health food" refers to food that has an effective health maintenance or promotion effect compared to ordinary food, and "health supplement food" refers to food used for health supplement purposes. In some cases, the terms "health functional food", "health food" and "health supplement food" are used interchangeably.

[0111] The food composition may further include a carrier that is physiologically acceptable, and the type of the carrier is not particularly limited and may be any carrier generally used in the art.

[0112] In addition, the composition may include other ingredients commonly used in food compositions to improve smell, taste, vision, etc. For example, the composition may include vitamins A, C, D, E, B1, B2, B6 and B12, niacin, biotin, folic acid, pantothenic acid, etc. The composition may also include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. In addition, the composition may include amino acids, such as lysine, tryptophan, cysteine, valine, etc.

[0113] In addition, the composition may include food additives, such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and advanced bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar color, etc.), color fixatives (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, etc.), sweeteners (glucose, sodium cyclamate, saccharin sodium, etc.), spices (vanillin, lactone, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), enhancers, emulsifiers, thickeners (starch adhesives), film-forming agents, gum bases, defoaming agents, solvents, conditioners, etc. The additives may be selected according to the type of food and may be used in appropriate amounts.

[0114] The composition according to the present invention, in addition to CSGG polysaccharide or a strain producing the polysaccharide, may also contain a dietetic acceptable food supplement additive, and may be used in combination with other foods or food ingredients, and may be used appropriately according to a general method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health care or treatment).

[0115] In another aspect, the present invention relates to a method for producing induced regulatory T (iTreg) cells, comprising the steps of: (a) treating dendritic cells (DC) with β-1,6-glucan polysaccharide or a Bifidobacterium bifidum strain producing the polysaccharide to obtain regulatory dendritic cells (rDC); and (b) combining rDC with CD4 + T cells were co-cultured to induce regulatory T (Treg) cells.

[0116] In the present invention, regulatory T (Treg) cells can be CD4 + Foxp3 + Treg cells. In addition, regulatory T (Treg) cells can increase the production of IL-10 and TGF-β.

[0117] In the present invention, step (b) may be to induce regulatory T (Treg) cells by stimulation with anti-CD3 antibodies, IL-2 and TGF-β.

[0118] In the present invention, dendritic cells and CD4 + T cells may be derived from peripheral blood mononuclear cells (PBMC), but are not limited thereto.

[0119] In another aspect, the present invention relates to a cell therapeutic agent for preventing or treating an immune disease or an inflammatory disease, comprising the induced regulatory T cells (iTregs) produced by the method as an active ingredient.

[0120] In another aspect, the present invention relates to a method for preventing or treating an immune disease or an inflammatory disease, the method comprising administering a cell therapeutic agent comprising, as an active ingredient, regulatory T (iTreg) cells produced by the method.

[0121] In another aspect, the present invention relates to use of a cell therapeutic agent in preventing or treating an immune disease or an inflammatory disease, wherein the cell therapeutic agent comprises the induced regulatory T (iTreg) cells produced by the method as an active ingredient.

[0122] In the present invention, regulatory T (Treg) cells can be CD4 + Foxp3 + Treg cells. In addition, regulatory T (Treg) cells can increase the production of IL-10 and TGF-β.

[0123] In the present invention, the immune disease or inflammatory disease is preferably inflammatory colitis, but is not limited thereto.

[0124] Bifidobacterium bifidum is one of the main bacterial members of the intestinal microflora of breast-fed infants, and the lack of this bacterium is related to allergies, asthma and autoimmune diseases in children. Some commercial probiotic products contain Bifidobacterium bifidum, but the efficacy is unclear, and some Bifidobacterium species may have harmful effects, such as worsening the progression of rheumatoid arthritis (PNAS, 113 (50): E8141-E8150, 2016). Therefore, it can be seen that some components of symbiotic bacteria have beneficial effects on the immune system. The present invention proves that CSGG purified from Bifidobacterium bifidum PRI1 is very effective in stimulating Treg cells and can inhibit the progression of colitis, so the immunomodulatory effect of bacterial cell wall polysaccharides is considered to be particularly useful for selecting probiotic bacteria for treating immune and allergic diseases.

[0125] By vaccinating SPF mice with Bifidobacterium bifidum or colonizing GF mice with it, naive CD4 + Cells differentiate into Nrp1 - Foxp3 + 、Helios - Foxp3 + Nrp1- Helios - Foxp3 + Peripheral induced Treg (iTreg) cells expressed high levels of IL-10 and CTLA4.

[0126] In the present invention, two parallel mechanisms are proposed by which Bifidobacterium bifidum can induce pTreg production in vivo.

[0127] First, increasing butyrate levels by single colonization with Bifidobacterium bifidum can indirectly promote the generation of Tregs by directly targeting T cells or by inducing a regulatory DC phenotype (Y. Furusawa et al., Nature 504: 446-450, 2013; N. Arpaia et al., Nature 504: 451-455, 2013). That is, the molecular mechanism of Bifidobacterium bifidum stimulating the induction of colonic Treg cells can be mediated by at least two types of effector molecules-metabolites (butyrate) and cell surface polysaccharides (CSGG). Previous studies have shown that among short-chain fatty acids (SCFA), butyrate, a fermentation product produced by symbiotic microorganisms, is one of the common effector molecules of iTreg inducers. Administration of butyrate-producing bacteria (Clostridium butyricum; Atarashi et al., Nature 500:232-236, 2014) or butylated starch to SPF mice can lead to an increase in colonic Treg cells by increasing histone H3 acetylation in the enhancer and promoter regions of the Foxp3 locus (Yukihiro Furusawa et al., Nature 504:446-450, 2013). However, it is unclear whether substances directly derived from intestinal microorganisms can induce CD4 + Foxp3 + Treg cells.

[0128] In one embodiment of the present invention, the cecal size of GF mice was normalized by colonization with Bifidobacterium bifidum, and the levels of butyrate in the cecum and feces were found to be significantly increased. In addition to butyrate, cell surface-derived β-glucan / galactan polysaccharides (CSGG) were identified as key effector molecules that mimic the Treg-inducing activity of all bacteria. The negatively charged polysaccharide PGβG (phosphoglycero-β-galactofuran) is another polysaccharide isolated from the cell surface and does not have Treg-inducing activity. PGβG enhances Th1 / Th17 responses. The chemical structure of PGβG is very similar to lipoteichoic acid (LTA) of Bifidobacterium bifidum subspecies pennsylvanicum DSM20239 (Fisher W., Eur. J. Biochem. 165 (3): 639-646, 1987). LTA of Gram-positive bacteria generally has pro-inflammatory activity, and when LTA is eliminated, the anti-inflammatory ability of probiotic lactic acid bacteria is enhanced (Sara Lebeer, Trends in Microbiology, January 2012, Vol. 20, No. 1). These results indicate that the structure of bacterial polysaccharides derived from the same bacteria can differentially modulate immune responses, and therefore, it is important to elucidate the relationship between the structure and function of polysaccharides derived from commensal bacteria.

[0129] Secondly, CSGG derived from the cell surface of Bifidobacterium bifidum induces Treg cells through a DC-dependent mechanism (VKRaker et al., Frontiers in immunology 6:569, 2015). DC plays an important role in the generation of iTreg induced by CSGG and Bifidobacterium bifidum. Bifidobacterium bifidum and CSGG (which are its effector molecules) can induce DC phenotype into regulatory DC (rDC) through the TLR / IL-10 pathway. Colonization with Bifidobacterium bifidum or treatment with CSGG can increase the expression of whole colon and cLP-DC (MHCII + CD11c + CD11b + CD103 + F4 / 80 -The expression levels of IL-10, TGF-β, GM-CSF and other Treg inducing factors in CDF. Among various Toll receptors (TLRs), TLR2 of DC plays an important role in the detection and transmission of CSGG-induced resistance signals, and it is shown that TLR2 knockout DCs are important defects in the generation of Treg cells induced by CSGG (about 35% of the Treg cell population compared to TLR2 DCs). Other studies have also shown that TLR2 of CD4 T cells (Round JL, 2011 Science) or DCs (Suryasarathi Dasgupta, Cell host and microbes, 2014) plays an important role in recognizing bacterial-derived polysaccharides to produce IL-10. In addition to TLR2, other receptors may also be involved in the generation of iTreg induced by CSGG, because TLR2KO DCs still induce Treg cells. CLR may not be involved, because C-type lectin receptor (CLR) inhibitors do not reduce the generation of Treg induced by CSGG. Further research is needed to determine which receptors or their combinations are involved in the recognition of CSGG and the transmission of resistance signals to T cells. TGF-β and IL-10 can be key effector molecules for differentiation of Treg cells by CSGG-TLR2 signaling in DC. Adding TGF-β blocking antibodies can completely eliminate the Treg cells induced by CSGG. Immunomodulatory cytokine IL-10 plays an important role in the production of Treg cells induced by CSGG. TLR2 KO DC produces much lower levels of IL-10 and shows reduced activity in induced Treg cells. These results show that IL-10 is the main regulator of immunoregulation in various immune responses (Lochner et al., The Journal of experimental medicine 205:1381-1393,2008).

[0130] In addition, colonization with B. bifidum or administration of CSGG not only increased RORγt + Foxp3 + Treg cells also increased CD103 + , indicating that CSGG treatment can induce intestinal Treg cells (B. Yang et al., Mucosal immunology 9: 444-457, 2016; E. Sefik et al., Science 349: 993-997, 2015). Colonization of Bifidobacterium bifidum can induce food and plant reactive Treg cells, thereby inhibiting not only food poisoning but also plant dysregulation. Therefore, it can be considered that RORγt induced by Bifidobacterium bifidum or CSGG + Foxp3 +Treg cells belong to the highly suppressive gut-specific effector Tregs (M. Esposito et al., The Journal of Immunology 185:7467-7473, 2010; DM Tartar et al., The Journal of Immunology 184:3377-3385, 2010).

[0131] Compared with the major immunomodulatory molecule capsular polysaccharide A (PSA) of the commensal Gram-negative bacterium Bacteroides fragilis, CSGG has very different characteristics (Neeraj K. Surana et al., Immunol Rev. 245 (1): 13-26, 2012). Both CSGG and PSA require TLR2 / IL-10 signaling to induce Treg cells, but the characteristics of Treg cells are very different. PSA or outer membrane vesicles (OMV) of Bacteroides fragilis mainly induce IL-10 high regulatory T cells, but do not induce Foxp3 + Treg cells (Cell Host & Microbe 124:509-520, 2012; Cell, 122(1):107-18, 2005; PNAS. 107(27):12204-9, 2010) and no CD4 + Foxp3 + Direct evidence for the generation of Treg cells. PSA also induces high levels of IFN-γ 高 Th1 production (Round et al., Science 332:974-977, 2011; Shen et al., Cell host μbe 12:509-520, 2012). PSA-induced Treg cells have phenotypes such as Foxp3-IL-10 高 IFNγ 高 (JL Sound et al., Science 332:974-977, 2011; Y. Shen et al., Cell host 12:509-520, 2012), but most CSGG-induced Treg cells showed Foxp3 + IL-10 高 IFNγ 低 PSA promotes IL-12 secretion by activating antigen presenting cells and induces activation of dendritic cells rather than inducing resistance (Cell, 122(1):107-18, 2005). CSGG induces Nrp1-Helios-Foxp3 that produces high levels of IL-10 and CTLA4 + and RORγt + Foxp3+ Treg cells.

[0132] In one embodiment of the present invention, Treg-inducing activity was compared between B. fragilis and B. bifidum PRI1 in vitro and in vivo in GF mice. + Treg cells did not increase, but CD4 + IFNγ + IL-10 高 Cytokine analysis by flow cytometry and ELISA showed high expression levels of IL-10 and IFN-γ in the B. fragilis-treated group. In addition, B. bifidum induced Foxp3 + iTreg cells, while Bacteroides fragilis has the ability to induce IL-10 高 Furthermore, there is no direct evidence that administration of PSA or Bacteroides fragilis can enhance CD4 + Foxp3 + Treg cells.

[0133] Furthermore, under suboptimal Treg induction conditions, PSA-treated DCs predominantly induced Foxp3 - IFNγ + IL-10 高 cells, while CSGG-treated DCs produced Foxp3 + IFNγ - IL-10 高Cells. PSA cannot induce antigen-specific Treg cells because it is processed and presented to T cells bound to MHCII molecules (BACoBb et al., Cellular microbiology 7: 1398-1403, 2005). In contrast, CSGG is difficult to load onto MHCII, so it can induce a variety of antigen-specific Treg cells that respond to food antigens or commensal bacteria. These functional differences may be mediated by differences in chemical structure and mechanism. PSA has a unique zwitterionic pattern that alternately contains positively charged and negatively charged sugar residues. Depending on such charge and size, PSA has different activities in T cell stimulation. The average molecular weight of natural PSA is about 130kDa, and its very small fragments (5000Da fragments) are inactive, but it is reported that the 17.1kDa fragment (about 22 repeating units) has the function of inhibiting colitis (Kalka-Moll WM, et al. J Immunol. 164: 719-724, 2000). PSA is absorbed into DC through TLR-2, cut appropriately, and then combined with MHCII molecules to interact with T cells that recognize PSA, thereby inducing the differentiation of specific helper T cells (Cobb BA et al., Cell Microbiol.7:1398-1403, 2005). In this case, Treg induced by PSA, i.e., Tr1 immunoregulatory cells, are specific to PSA, and this phenomenon has limitations on future antigen-specific Treg cell induction. CSGG is a neutral polysaccharide, averaging about 4000Da, and unlike PSA, it mainly works by regulating DC characteristics rather than directly acting on T cells. This difference in mechanism of action indicates the possibility of inducing various antigen-specific Treg cells by CSGG treatment. That is, when the processed PSA is loaded onto MHC II, only PSA-specific Treg cells can be produced. On the other hand, CSGG causes rDC by inducing phenotypic changes in DC rather than by endocytosis-mediated processing and loading onto MHC II.

[0134] In one embodiment of the present invention, when CSGG is administered to GF mice, CD4 + Nrp - Foxp3 + Treg cells were significantly enhanced. Thus, B. bifidum or CSGG treated with various antigens can induce antigen-specific Treg cells, such as food antigen-specific or commensal bacterial antigen-specific Treg cells. An interesting result is that Bb colonization or CSGG administration enhanced RORγt in the large intestine. + Foxp3 +In addition, compared with mock iTregs, iBregs induced with Bb or CSGG had higher RORγt, which is able to suppress intestinal inflammation. + Foxp3 + Treg cell population. Compared with the simulated iTreg cells of the colitis model, iTreg cells induced by Bb or CSGG showed sustained Foxp3 expression in CNS2 of the Foxp3 locus and had a higher demethylation status (50% to 60%). Therefore, it is believed that RORγt induced by Bb or CSGG + Foxp3 + Treg cells belong to intestinal specific effector Treg (Esposito et al., J Immunology 185:7467-7473, 2010; Tartar et al., J Immunology 184:3377-3385, 2010; BHYang et al., Mucosal Immunology 9:444-457, 2016). By studying the role of innate immune cells in this process, we can further study whether Bb or CSGG treatment enhances RORγt + Foxp3 + Basic mechanisms of Treg cells.

[0135] The present invention proves that Bifidobacterium bifidum PRI1 and its effector molecule CSGG promote the production of iTreg cells. Bifidobacterium bifidum belongs to actinomycetes, has been specifically identified in the intestinal tract of people, and has been shown to be one of the important bacterial members of the visceral microbiome of breast-fed infants (Turroni et al., Applied and environmental microbiology 75: 1534-1545, 2009). Compared with natural childbirth, cesarean section leads to a lack of Bifidobacterium, which is associated with allergies, asthma and autoimmune diseases in children. Compared with healthy controls, the proportion of Bifidobacterium bifidum in the feces of IBD patients is smaller (Wang et al., Journal of clinical microbiology 52: 398-406, 2014; Verma et al., Journal of clinical microbiology 48: 4279-428, 2010). Among various types of symbiotic bacteria, Bifidobacterium genus generally has stronger anti-inflammatory properties than Lactobacillus genus in its ability to regulate host responses. However, the immunomodulatory effect of commensal bacteria is strain-specific, and the identification of functional strains remains a great challenge. For example, in the same Bacteroides fragilis, strains producing lipopolysaccharide increase inflammatory signals and worsen disease progression (Walter J. Lukiw et al., Front Microbiol 7: 1544, 2016). On the other hand, strains producing PSA are known to inhibit inflammatory diseases. Therefore, it is very important to determine how each identical species has different immunological properties. As a method for identifying strain-specific immunomodulatory specificity, there is a method for identifying bacterial-derived substances and their immunological characteristics and using these substances as functional markers. For example, butyrate can be a marker for Treg-inducible bacteria because butyrate promotes the differentiation of mesenteric Treg cells (Yukihiro Furusawa et al., Nature 504: 446-450, 2013; Liao HY et al., Scientific reports, 6: 20481, 2016). Another method is to identify strains with high genetic homology to known strains by comparative genomic analysis (C. Preston Neff, Cell Host & Microbe, 20 (4): 535-547, 2016). In the present invention, among the Bifidobacterium bifidum strains, A8 (99.97%) and LMG11582 (99.98%) were found to have the highest base similarity and orthologous genes involved in CSGG synthesis. IPLA20015 and A8 strains are known to act as Treg-enhanced bacteria in human PBMC culture systems.As a result of in vitro comparison of Treg inducing activity, the A8 strain showed similar activity to the present strain, but ATCC 29521, which has low base homology (98.9%) and lacks some orthologous genes associated with CSGG synthesis, exhibited much lower Treg inducing activity.

[0136] In the present invention, it is confirmed that the administration of Bifidobacterium bifidum or CSGG can be used as an oral resistance adjuvant, thereby inducing various antigen-specific Treg cells in vivo. In addition, Bifidobacterium bifidum or CSGG treatment induces rDC without interfering with antigen presentation in MHC II. Therefore, it is confirmed that when Bifidobacterium bifidum or CSGG is administered in combination with autoimmune or allergy-related proteins, antigen-specific Treg cells are induced, thereby inhibiting disease progression. Specific examples of the protein include autoantigens involved in autoimmune diseases. For example, the protein may include autoantigens involved in rheumatoid arthritis, such as heat shock proteins (HSPs), citrullinated filamentous proteins, glucose-6-phosphate isomerase, p205, collagen, etc.; autoantigens involved in type I diabetes, such as insulin, zinc transporter 8 protein (ZnT8), pancreatic and duodenal homeobox 1 (PDX1), chromogranin A (CHGA), and islet amyloid polypeptide (IAPP); and autoantigens involved in myasthenia gravis, such as acetylcholine receptors. In addition to all types of autoantigens known in autoimmune diseases, proteins can also include various allergens known to cause food allergies, such as peanuts, milk, eggs, tree nuts, beans, crustaceans (such as shrimp, etc.), fish-derived substances, etc.

[0137] The present invention relates to a cell surface β-glucan / galactan (CSGG) polysaccharide capable of treating hyperimmune diseases by inducing or generating regulatory T (Treg) cells, and a Bifidobacterium bifidum PRI1 expressing the cell surface β-glucan / galactan (CSGG) polysaccharide. The cell surface β-glucan / galactan (CSGG) polysaccharide expressed by Bifidobacterium bifidum PRI1 is different from the polysaccharides of well-known probiotic bacteria, and CSGG has been found to be an inducing factor for the CD4 + Foxp3 +It is an important substance for the formation of Treg cells, and exhibits the effect of treating inflammatory bowel disease or hyperimmune disease by the mechanism of producing TLR2-mediated DC. In the present invention, the specific structural formula of cell surface β-glucan / galactan (CSGG) polysaccharide expressed by Bifidobacterium bifidum PRI1 was identified, and it was discovered for the first time that CSGG is the main agonist for inducing regulatory T (Treg) cells. The structure of the β-D-glucan of CSGG of the present invention is β-1,6-glucan, which has a function completely different from the 2-substituted-(1,3)-β-D-glucan of the known β-D-glucan structure. Although the known β-D-glucan has a prebiotic function, the β-1,6-glucan of the present invention itself has an immunomodulatory function. In addition, polysaccharides such as polysaccharide A (PSA) do not have the ability to induce antigen-specific Tregs other than PSA-specific Tregs, while strains producing CSGG are able to induce Tregs against substances derived from various antigens (i.e., food, other intestinal bacteria, and bacteria themselves), so PSA and CSGG are completely different in their structure and mechanism. It is difficult to summarize polysaccharides on the surface of cells as a single substance, but the present invention has identified how structural differences are related to function. That is, CSGG itself is a substance produced by the bacteria of the present invention and is an active indicator that can represent the function of the bacteria themselves that induce Tregs.

[0138] CSGG can serve as a new functional marker for Treg-type bacteria, and CSGG or CSGG-producing Bifidobacterium bifidum can be used as resistance-inducing adjuvants to develop specific immunotherapies for autoimmune and allergic diseases.

[0139] Example

[0140] Hereinafter, the present invention will be described in more detail with reference to the following examples. It is clear to those skilled in the art that these examples are provided only to explain the present invention in more detail, and are not intended to limit the scope of the present invention according to the gist of the present invention.

[0141] Materials and methods

[0142] 1. Mice and Bacterial Strains

[0143] 1-1: Mouse

[0144] Mice were managed in the animal facility of the POSTECH Biotechnology Center, and all experimental procedures were approved by the POSTECH Institutional Animal Care and Use Committee (IACUC). Germ-free C57BL / 6 (B6) mice were obtained from Dr. Andrew Macpherson (University of Bern, Switzerland) and David Artis (then at the University of Pennsylvania, currently at Cornell University, USA) and established by POSTECH and maintained in sterile flexible membrane isolators (Class BiologicalClean Ltd., USA). GF status was monitored monthly by cecal contents. Foxp3-eGFP, Tlr2 - / - 、Tlr4 - / - 、Tlr6 - / - , and MyD88 - / - Animals were purchased from Jackson Laboratory. C57BL / 6-CD45a(Ly5a)-Rag1 was obtained by Tagonic exchange procedure. - / - TCR OT-II (Rag1 - / - OT-II TCR transformants) and Rag1 - / - Mice. CBir mice were obtained from O. Elsona (University of Alabama at Birmingham, Birmingham, AL 35294, USA; Yingzi Cong, PNAS, 19256-19261, doi: 10.1073 / pnas.0812681106). Mice aged 6 to 12 weeks were used according to sex and age.

[0145] 1-2: Bacteria

[0146] Bifidobacterium bifidum PRI1 and other Bifidobacterium bifidum strains were cultured anaerobically in MRS medium (BD, Difco) supplemented with 0.1% L-cysteine, and Lactobacillus paracasei (L. paracasei) was cultured in MRS medium. For single colonization, bacteria (5 x 10 8 CFU / 200 μL) were orally administered to germ-free (GF) mice.

[0147] 2. Purification of Cell Surface Polysaccharides from Bifidobacterium bifidum

[0148] Harvest cultured Bifidobacterium bifidum PRI1 and wash twice with PBS. Purification of cell surface polysaccharides was performed using a slightly modified existing method (DL Kasper et al., Journal of bacteriology 153: 991-997, 1983). Acidic phenol (Sigma-Aldrich) treatment was performed at 68 ° C to extract the coated polysaccharide, followed by ether treatment to remove residual phenol and dialyzed in distilled water for 3 days. In order to remove nucleic acids and proteins, DNase I (Roche) and RNase (Sigma-Aldrich) were treated overnight at 37 ° C, and then treated overnight with pronase (protease from Streptomyces griseus, Sigma-Aldrich) at 37 ° C. After acetic acid treatment, centrifugation was performed to remove the precipitate. Cooled ethanol was added to precipitate the polysaccharide, which was then dialyzed in distilled water for 3 days and lyophilized. The purified polysaccharide was dissolved in water and gel filtered using an HPLC column (TSK gel G5000 PWXL, Tosho). Anion exchange chromatography (HiPrep Q FF 16 / 10, GE healthcare) was performed to further separate neutral and negatively charged polysaccharides. The concentration of polysaccharides was determined by acid phenol analysis (M. DuBois et al., Analytical chemistry 28:350-356, 1956).

[0149] 3. Bacterial Colonization Analysis by 16S rRNA Gene Analysis

[0150] To confirm monocolonization, bacterial genomic DNA was isolated from feces or cavity contents of mice using the NucleoSpin DNAStool (Macherey-Nagel).

[0151] PCR analysis was performed using a C1000 Contact Thermal Cycler (Bio Rad) and the primers in Table 1 below were used.

[0152] [Table 1]

[0153]

[0154] 4. Bacterial Colonization Assay by Fluorescence In Situ Hybridization (FISH)

[0155] After 3 weeks of monocolonization with B. bifidum PRI1, mice were sacrificed and the small intestine was divided into 3 segments: duodenum (within 1 cm to 3 cm distal to the pylorus); duodenum; jejunum (within 2 cm to 4 cm from the ligament of Treitz); and ileum (within 1 cm to 3 cm at the septal valve). 1 cm of each small intestinal section and colon were fixed using Carnoy's solution for histological analysis. Carnoy's fixative solution (3:1 methanol: glacial acetic acid) was dripped, the cell suspension was centrifuged at 500 x g for 5 minutes, and the pellet was resuspended in Carnoy's fixative solution (Choolani et al., 2007). Paraffin-embedded sections fixed with Carnoy's solution were dewaxed and hybridized with Cy5-labeled B. bifidum 16S rRNA fluorescent probe (SEQ ID NO: 5: Bbif, 5′-CCACAATCACATGCGATCATG-3′) (Dinoto et al., 2006). The resulting probes were hybridized with hybridization buffer (100 nM Tris, pH 7.2, 0.9 M NaCl, 0.1% SDS) containing 100 ng of the probe in a humidified chamber at 45°C for 16 hours. The slides were washed in 100 ml preheated (37°C) hybridization buffer for 15 minutes, washed with 10 ml preheated (37°C) washing solution (100 mM Tris, pH 7.2, 0.9 M NaCl) for 15 minutes (Smith et al., 2011), and then immunostained with 4',6-diamidino-2-phenylindole (DAPI) for DNA. The slides were washed with water and air-dried, and then images were acquired using a fluorescence microscope (IX70, Olympus, Tokyo, Japan).

[0156] 5. T cell delivery model of colitis

[0157] Naive CD4 + T cells induced colitis. iTreg cells (2×10 5 ) naive CD4 + CD62L hi CD44 lo Foxp3 GFP- T cells (>99% purity, 1×106) were transferred intravenously into Rag1 - / -The progression of colitis was monitored by weighing the mice twice a week, and the experiment was terminated when the mice lost 25% of their initial body weight. Treg cells were used as a positive control, and CD4 + Foxp3GFP + Treg cells (2×10 5 ) and naive T cells co-delivered to Rag1 - / - The severity of colitis was determined by measuring colon length and histological evaluation at the end of the experiment. Treg cells and cytokine levels were also analyzed.

[0158] 6. In vivo adoptive transfer

[0159] The sorted naive polyclonal CD4 + CD62L hi CD44 lo Foxp3 GFP- T cells (>99.5% purity) (1×10 6 ) were transferred intravenously into GF C57BL / 6 mice and a single strain was administered orally. Mice were sacrificed three weeks after adoptive transfer. To analyze the TCR specificity of Treg cells in response to dietary antigens, OT-IICD4 + CD62L hi CD44 lo Foxp3 GFP- T cell separation of naive T cells (1×10 6 ) were intravenously transferred into GF C57BL / 6 mice monocolonized with B. bifidum PRI1 for 2 weeks and fed with OVA (20 mg / dose / mouse) every other day for 7 days. To analyze the TCR specificity of Treg cells in response to commensal bacteria, sorted (>99.5% purity) naive CBir CD4 + CD62L hi CD44 lo Foxp3 GFP- T cells (1×10 6 ) were delivered intravenously into C57BL / 6 mice maintained under SPF conditions and fed with Bifidobacterium bifidum PRI1 (5 x 10 8 cfu / dose) three times a week until the experiment was completed. Mice were sacrificed three weeks after adoptive transfer.

[0160] 7. DC-dependent induction of iTreg cells in vitro

[0161] Colonic LP DC (MHCII + CD11c + CD11b + CD103+ F4 / 80 - ), total splenic DCs (tDCs; MHCII + CD11c + ), spleen CD8α + (MHCII + CD11c + CD11b - ) and spleen CD8α - (MHCII + CD11c + CD11b + ) DCs were purified by cell sorting or CD11c magnetic beads (Miltenyi Biotech). The isolated DCs were cultured in complete RPMI 1640 medium containing 10% FBS, 5% penicillin / streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, non-essential amino acids and β-ME. Splenic DCs (2 x 10 4 ) were inoculated into 96-well plates and cultured for 2 hours, followed by incubation with the indicated bacteria, differentiated cell fractions of B. bifidum PRI1, or purified polysaccharides of B. bifidum PRI1 for 10 to 12 hours. The cells were washed and incubated with naive CD4 Tregs under suboptimal Treg induction conditions (0.1 μg / ml anti-CD3, 100 U IL-2, and 0.1 ng TGFβ). + T cells (2x 10 5 ) were co-cultured for 3 days. The levels of cytokines or Treg cells were determined by ELISA and flow cytometry, respectively.

[0162] 8. Human samples for DC differentiation and iTreg cell induction via CSGG treatment

[0163] The study was approved by the Institutional Review Board (IRB) of Ajou University Hospital, South Korea (AJIRB-BMR-SMP-17-155), and peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by Ficoll-Hypaque gradient (Lymphoprep, Nycomed, Norway). Monocytes were isolated from PBMCs (CD14+>95%) by positive selection using a CD14 microbead kit (Miltenyi biotech). 5x10 cells were cultured in 24-well plates in the presence of rhIL-4 (35ng / ml) and GM-CSF (70ng / ml) (R&D Systems, UK) in RPMI-1640 medium (2mM l-glutamine, 25mM HEPES; BioWhittaker) supplemented with 10% heat-inactivated FCS and antibiotics.5 monocytes / ml for 7 days. On days 2 and 5, 0.5 ml of the medium was replaced with fresh medium containing GM-CSF and IL-4. On day 7, immature DCs were recovered and pretreated with mock or different amounts of CSGG for 14 h. Next, the cells were incubated with naive CD4 isolated from human PBMCs. + T cells were cultured for 3 days under suboptimal Treg induction conditions (0.1 μg / ml anti-CD3, 100 U IL-2 and 0.1 ng TGFβ). The level of Treg cells was determined by flow cytometry.

[0164] 9. Data availability of RNA sequences

[0165] CD11c from spleen stimulated with mock or CSGG (100 μg / ml) for 4 h + Total RNA was extracted from DC and analyzed by Ribospin TM II (GeneAll biotechnology). TM (Thermo Fisher Scientific) for RNA quantification and quality control. Library preparation was performed using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina), and RNA sequencing was performed using the NextSeq 500 Sequencing System. RNA-seq data were deposited in the Gene Expression Omnibus (NCBI) data repository under the accession number GEO: RNA-seq data: GSE98947.

[0166] 10. RNA Isolation and Real-time Quantitative RT-PCR

[0167] Total RNA isolated from colon and cLP DCs using TRIzol reagent (Invitrogen) was reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) to prepare cDNA. Quantitative real-time PCR (qRT-PCR) was performed on the synthesized cDNA using the following primer sets (Table 2) and a CHROMO4 detector (Biorad).

[0168] [Table 2]

[0169]

[0170]

[0171] All qRT-PCR procedures were performed under the same conditions for 40 cycles: 5 min at 95 °C, 30 s at 95 °C, 30 s at 62 °C, and 30 s at 72 °C. The data were normalized to the value of HPRT expression level. The results are described as the relative expression level of each gene between treatment groups.

[0172] 11. Lymphocyte Isolation and Flow Cytometry

[0173] Purification of naive CD4 from mLN, pLN, and spleen using a cell separator + T cells (purity of 98% or more). The large and small intestines of the mice were opened longitudinally, and mucus was removed by washing with PBS. The large intestine was cut into small pieces and incubated at 37°C with 10 mM EDTA, 20 mM Hepes, 1 mM sodium pyruvate, 3% FBS and Ca-free 2+ and Mg 2+ The tissue was ground into smaller pieces with a razor and incubated with shaking at 37°C for 45 minutes in RPMI 1640 medium containing 3% FBS, 20 mM Hepes, 1 mM sodium pyruvate, and 0.5 mg / ml collagenase D (Roche) and DNase I (Sigma). After enzyme treatment, the supernatant was filtered using a 100 μm cell strainer with cold PBS containing 10 mM EDTA. The cells were placed on Percoll TM(GE Healthcare) gradient (40% Percoll on top, 75% Percoll on bottom) and centrifuged at 2000rpm for 20 minutes without interruption. Cells between 40% and 75% of the layer were taken and washed twice with RPMI medium (Hyclone SH30027.01) containing 10% FBS (Hyclone), Pen / Strep, β-ME, sodium pyruvate and 2.0mM L-glutamine, and used for FACS staining. Cell suspensions were first stained live / dead using LIVE / DEAD fixable viability dye (eBioscience) or PI in PBS. Thereafter, further staining was performed in a buffer containing 1% FBS (Gibco) and EDTA (Sigma-Aldrich). Surface staining was performed for 20 minutes in PBS containing 0.2% bovine serum albumin. For intracellular transcription factor staining, cells were stained with fixation / permeabilization buffer (eBioscience) and 1x permeabilization / washing buffer (eBioscience). For intracellular cytokine analysis, purified lamina propria lymphocytes were restimulated with 500 ng / ml ionomycin (Calbiochem) and 100 ng / ml PMA (Calbiochem) using GolgiPlug (BDbiosciences, 0.5 μL / sample) at 37° C. for 4 to 5 hours. Cells were fixed in IC fixation buffer (eBioscience) and permeabilized in permeabilization / wash buffer (eBioscience) and then stained with IL-10, IL-13, IFN-γ, and IL-17A antibodies.

[0174] The following antibody clones were used: CD4 (RM4-5), CD44 (IM7), CD62L (MEL-14), CD45.1 (A20), CD45.2 (104), CD90.1 (Thy1.1) (OX-7), CD90.2 (Thy1.2) (30-H12), CD103 (2E7), Vα2 (B20.1), Foxp3 (FJK-16s), CTLA4 (UC10-4B9), Nrp1 (3E12), He lios(22F6), T-bet(4B10), RORγt(AFKJS-9), GATA-3(16E10A23), IL-10(JES5-16E3), IL-13(eBio13 A), IFN-γ(XMG1.2), IL-17A(17B7), CD11c(N418), CD11b(M1 / 70), F4 / 80(BM8), MHCII(M5 / 114.15.2).

[0175] For flow cytometry sorting, dead cells were excluded and populations were gated as follows: LP DC (CD45 + MHCII + CD11c + CD11b + CD103 + F4 / 80 - )、SP DC(MHCII + CD11c + CD11b + CD8α + MHCII + CD11c + CD11b + CD8α - ), naive CD4 + T cells (CD4 + CD45.1 + CD62L hi CD44 lo Foxp3 GFP- ), naive OT-II TCR transgenic T cells (CD4 + CD90.1 + OT-II + CD62L hi CD44 lo Foxp3 GFP- ), CBir TCR transgenic naive T cells (CD4 + CD45.1 + CD62L hi CD44 lo Foxp3 GFP- ).

[0176] Cell sorting was performed using a Moflo-XDP, and flow cytometry was performed using an LSR Fortessa flow cytometer (BD Biosciences) equipped with five lasers. Data were analyzed using FACSDiva software (BD Biosciences) and FlowJo software.

[0177] 12. TCR Repertoire Analysis Using CDR3 High-Throughput Sequencing

[0178] Sorted CD4 from GF mice or mice monocolonized with B. bifidum PRI1 for 3 weeks + Foxp3 GFP+Total RNA was extracted from Treg cells. Dead cells were excluded by FVD staining using the ISOGEN kit (Nippon Gene). Next-generation sequencing was performed using the unbiased TCR library analysis technology of Repertoire Genesis. Adaptor-ligation PCR (R. Yoshida et al., Immunogenetics 52:35-45, 2000) was performed by existing methods. The first double-stranded cDNA was synthesized with Superscript III reverse transcriptase (Invitrogen), connected with 5' adaptor oligonucleotides, and then PCR amplification was performed using primers specific to the adaptor and TCRα constant region or TCRβ constant region. After amplifying TCRα and TCRβ cDNA, an index (barcode) sequence was added using Nextera XT Index Kit v2 setA (Illumina). Sequencing was performed using the Illumina Miseq paired end platform (2×300bp). Data processing was performed using the library analysis (Repertoire Analysis) software from Repertoire Genesis. The TCR sequences were assigned using a possible dataset of reference sequences from the International ImMunoGeneTicsInformation System (IMGT) database (http: / / www.imgt.org). After removing low-scoring sequences, TCR spectral library analysis was performed using bioinformatics software developed by Repertoire Genesis (Ibaraki, Japan). Sequence reads that were different from other sequence reads were defined as unique sequence reads (USRs). The copy number of the same USR was automatically calculated by the RG software.

[0179] 13. Effect of TGF-β neutralization on the generation of iTreg cells in vitro

[0180] MHCII + CD11c + DC(2×10 4 ) were seeded into round-bottom 96-well plates for 2 h and then cultured with B. bifidum PRI1 or CSGG for 12 h. The cells were washed and incubated with naive CD4+ cells in the presence of 0.1 μg / ml anti-CD3 (BD ​​Bioscience), 100 U IL-2 (Peprotech) and 0.1 ng / ml TGF-β or in the absence of 15 μg / ml anti-TGF-β antibody (R&D). + T cells (2×10 5 ) co-cultured. After 3 days of culture, CD4 + Foxp3+ T cell populations.

[0181] 14. In vitro Inhibition Assay

[0182] CD45.1 + CD4 + Foxp3 GFP iTreg cells were sorted and compared with pulsed reactive cells (Thy1.1 + CD4 + Foxp3 - ) were incubated at 37°C for 10 minutes. The CTV-labeled cells were washed twice with PBS and used immediately. T cell-depleted spleen cells (1×10 5 ), CTV pulse-responsive cells (5×10 4 ), and the indicated amount of iTreg cells were mixed with 0.5 μg / ml anti-CD3 in a round-bottom 96-well plate. The cells were cultured for 4 days and their proliferation was analyzed by flow cytometry to determine the dilution of CTV intensity. The inhibition rate (%) was calculated as the total percentage of dividing cells by comparing the percentage of individual responding cells.

[0183] 15. Histology

[0184] The clinical status of experimental colitis was evaluated by histological analysis using H&E staining. The colon was collected and fixed in 10% formaldehyde. After fixation, the tissue was embedded in paraffin blocks, cut into 3 μm thickness, and stained with hematoxylin (Sigma-Aldrich) and eosin (Sigma-Aldrich).

[0185] 16. Statistical Analysis

[0186] Statistical analysis was performed using GraphPad Prism software (La Jolla, California), and unpaired Student's t-test was used to evaluate differences between the control and experimental groups. Data are expressed as mean ± SEM. For in vivo stability analysis of Treg cells and experimental colitis, two-way ANOVA was used for statistical analysis using Bonferroni multiple comparison test. P < 0.05 was considered significant.

[0187] Example 1: Screening of Treg-inducing bacteria

[0188] Enhancement of Treg cells in vivo by immunomodulatory microorganisms enables regulation of disorders associated with inflammatory disorders. In order to identify Treg-inducing bacteria in microorganisms considered safe, 200 or more probiotic strains were screened using a previously reported in vitro screening system (Kwon et al., PNAS 107: 2159-2164, 2010, Kim JE, J Funct Foods. 350-362, 2015).

[0189] Each bacterial strain was incubated with mesenteric lymph node (mLN) cells from Foxp3 reporter mice in the presence of antibiotics. The levels of Foxp3, IL-10 and IL-12 were measured to select strains with an IL-10 / IL-12 ratio > 100 and an increase in Foxp3 expression of at least 10%.

[0190] As a result, Bifidobacterium bifidum PRI1 (Bifidobacterium bifidum; Bb) was selected as the Foxp3 + Treg-inducing bacteria (see Figure 2 A and 2B), and Lactobacillus paracasei sub.tolerans 467 (L. paracasei; Lpa) was selected as a control strain that does not induce an immune response. The selected Bifidobacterium bifidum PRI1 was deposited in the Korea Type Culture Collection of the Korea Institute of Bioscience and Biotechnology on May 19, 2017 (Deposit No.: KCTC13270BP).

[0191] Example 2: Induction of Treg cells by Bifidobacterium bifidum

[0192] It was examined whether Bifidobacterium bifidum could induce Treg cells in vivo.

[0193] Segmented filamentous bacteria (SFB) were used as Th17 inducing control bacteria. By single administration of Bifidobacterium bifidum (Bb) to germ-free (GF) mice, the bacteria stably colonized the colon (see Figure 2 C), and the enlarged cecum size was normalized to a size similar to that of normal SPF mice (see Figure 2 D). This suggests the fiber digestion activity of Bb. In addition, short-chain fatty acids (SCFAs) such as butyrate and acetate are the end products of dietary fiber fermentation, and therefore, mice colonized with Bb showed no change in propionate levels, while acetate and butyrate levels in the cecum and feces were significantly increased (see Figure 2 E).

[0194] Unlike Lactobacillus paracasei (Lpa) or SPF, GF mice colonized with Bifidobacterium bifidum (Bb) for 3 weeks displayed Foxp3 expression in the colonic lamina propria (cLP). + The frequency of Treg cells increased dramatically (see Figure 1 A and 2F). In addition, Treg cells were slightly increased in other organs such as mesenteric lymph nodes (MLN), spleen, small intestinal lamina propria (siLP) and peripheral lymph nodes (LN) (see Figure 3 A). Treg cells in the colonic lamina propria (cLP) of GF mice colonized with Bifidobacterium bifidum (Bb) exhibited a significantly higher proportion of CD103 + and memory phenotype (CD62L lo CD44 hi )(See Figure 3 B and 3C), and also exhibited high levels of CTLA4 and IL-10 (see Figure 1 B and 1C).

[0195] Example 3: iTregs newly generated from Bb

[0196] Since Treg cells can be further differentiated into thymus-derived Treg (tTreg) or peripherally induced Treg (pTreg or iTreg), the origin of Treg was tracked by analyzing Helios and Nrp1 expressions in Treg cells.

[0197] In order to + Npr1 + Thymus-derived Treg (tTreg) and Helios - Npr1 - Peripherally derived Tregs (pTregs) (called "iTregs" when produced under in vitro culture conditions) were distinguished and the expression of IKAROS family transcription factors Helios and neuropilin 1 (Nrp1) was observed (JM Weiss et al., The Journal of experimental medicine 209:1723-1742, 2012; EM Shevach et al., Immunological reviews 259:88-102, 2014; JPEdwards et al., The Journal of Immunology 193:2843-2849, 2014).

[0198] As a result, in cLPs colonized with B. bifidum, Nrp1 + There was a slight increase in tTreg cells, but Nrp1 -and Helios lo pTreg cells increased even more (see Figure 1 D to 1F and Figure 4 A to 4C). In addition, most of the newly generated pTregs express the transcription factor RORgt (see Figure 1 G and 4D), and this is upregulated by interaction with microorganisms (B. Yang et al., Mucosal immunology 9:444-457, 2016; E. Sefik et al., Science 349:993-997, 2015).

[0199] Next, to confirm that the increase in Treg cells induced by Bb colonization was not an expansion of existing Treg cells but rather an expansion of naive CD4 + T cells were newly generated and adoptive transfer experiments were performed. GFP Allele-marked naive CD4 + CD45.1 + Foxp3 - CD62L hi CD44 lo T cells were adoptively transferred into GF mice monocolonized with B. bifidum to determine the peripheral origin of B. bifidum-induced Treg cells (see Figure 5 A).

[0200] As a result of donor cell analysis after 3 weeks, the majority of Foxp3 in donor cells was shown in the intestines of GF mice colonized with Bifidobacterium bifidum (cLP). + Treg cells (see Figure 4 A to 4D). In SPF mice given Bifidobacterium bifidum for 3 weeks, the induction of pTreg cells was similarly increased (see Figure 5 E) These results indicate that administration of Bifidobacterium bifidum enables the de novo production of iTregs primarily from naive CD4 T cells in the colon.

[0201] Example 4: TCR specificity analysis of pTreg cells

[0202] The TCR specificity of pTreg cells generated in the presence of B. bifidum was analyzed. We tested whether B. bifidum colonization promotes pTreg cells in response to dietary antigens, i.e., whether B. bifidum colonization can increase the generation of dietary antigen-responsive iTreg cells. CTV-labeled naive CD4 + Thy1.1 + Foxp3 -T cells were adoptively transferred into GF mice, which were then either colonized with Bb or not, and then given oral OVA protein. + Foxp3 GFP CTV-labeled OVA-specific naive CD4 + The cells were adoptively transferred into normal or B. bifidum-colonized GF mice given OVA protein (see Figure 7 A).

[0203] As a result, as previously observed (KSKim et al., Science 351:858-863, 2016), a small fraction of expanded OT-II cells in normal GF hosts upregulated Foxp3 in both siLPs and cLPs (see Figure 6 A and 6B). That is, compared with GF mice fed OVA, mice colonized with Bb showed Thy1.1 mainly in cLP. + OT-II-specific Foxp3 + There was a significant increase in Treg cells and host-derived Treg cells. Specifically, in mice colonized with B. bifidum, the efficiency of upregulation of Foxp3 by donor OT-II cells was increased 2- to 3-fold in cLPs but was unchanged in siLPs and mLNs (see Figure 6 B).

[0204] Example 5: Production of microbiota-specific pTreg cells

[0205] To determine whether colonization with B. bifidum promotes the generation of microbiota-specific pTreg cells, CBir TCR transgenic mice that recognize bacterial flagellin were used (Y. Cong et al., The Journal of Immunology 165: 2173-2182, 2000). + Foxp3 GFP Naive CD4 + Foxp3 - T cells and adoptively transferred into SPF Rag1 - / - Recipients were then provided with mock (PBS) or B. bifidum every other day for 5 weeks (see Figure 7 B).

[0206] As a result, only CBir T cells in the control SPF Rag1 - / - Recipients showed significant signs of colitis, such as progressive weight loss (see Figure 6 C), a small number of Treg cells (see Figure 6F) High proportion of CD4 + IFNγ - and RORγ -+ Foxp3 - T cells (see Figure 6 G and 7C), shortening of the colon (see Figure 6 D) and mucosal thickening (see Figure 6 E) and others. In the host injected with CBir T cells and administered in combination with Bifidobacterium bifidum, there was almost no evidence of these various pathological signs (see Figure 6 C to 6G and 7C).

[0207] Example 6: Specificity of iTreg cells to Bifidobacterium bifidum itself

[0208] To test whether the pTreg cells induced by B. bifidum are specific to B. bifidum itself, total colonic CD4 + T cells were co-cultured with spleen APCs pretreated with fecal antigens from normal GF mice and GF mice colonized with B. bifidum or L. paracasei, and after 3 days, these cells were analyzed for T cell proliferation (CTV dilution) (see Figure 7 D).

[0209] As a result, most Treg cells died under in vitro culture conditions, while Foxp3 + Treg cells maintained Foxp3 expression and proliferation when stimulated with APCs treated with B. bifidum Ag (see Figure 6 H and 7E).

[0210] In addition, the Foxp3 + The α-chains and β-chains of Treg cells were sequenced to compare the TCR specificity of GF and B. bifidum monocolonized mice.

[0211] As a result, although both Treg cells showed similar diversity patterns in α-chains and β-chains (see Figure 8 A), but colonic Treg cells from mice colonized with B. bifidum displayed a separate TCR pattern that was absent in GF controls (see Figure 6 I and 8A to 8D). Overall, these results suggest that B. bifidum induces functionally active CD4 + Foxp3 + Treg cells, which have a broad range of TCR specificities for dietary antigens and / or shared microbiota and B. bifidum itself.

[0212] Example 7: Colonization with Bifidobacterium bifidum results in the induction of rDCs and enhanced differentiation of new pTregs

[0213] It was examined whether colonization with B. bifidum induces a population of regulatory dendritic cells (rDCs) that could enhance new pTreg differentiation.

[0214] Since differentiation of naive T cells into iTreg cells requires an immunoregulatory cytokine milieu, we tested whether colonization with B. bifidum induced a tolerogenic environment in the colon. cLP-DCs (MHCII + CD11c + CD11b + CD103 + F4 / 80 - ) and total colonocytes not only significantly increased the mRNA expression of inhibitory molecules such as Il10, Csf2, Tgfβ1, ido, Ptgs2, and Pdcd1, but also significantly increased the mRNA expression of costimulatory molecules Cd86 and Cd40 (see Fig.10 A to 10D). This suggests that Bb colonization can induce regulatory dendritic cells (rDCs) that promote iTreg cell differentiation.

[0215] In addition, an in vivo simulation experiment was performed by culturing sorted cLP-DCs with Bifidobacterium bifidum for 10 to 12 h, then washing and then incubating with naive CD4 Tregs under suboptimal Treg-inducing conditions. + Foxp3 - T cells were co-cultured for 3 days. As a result of pretreatment of cLP-DCs with Bifidobacterium bifidum, the induction of iTreg cell and IL-10 production and proliferation was increased compared with the mock-treated group or the control treated with Lactobacillus paracasei (Lpa) (see Fig.10 E to 10G).

[0216] Example 8: Identification of effector molecules of Bifidobacterium bifidum

[0217] 8-1: Effector molecules of Bifidobacterium bifidum

[0218] Using an in vitro system, extensive experiments were performed to identify effector molecules derived from B. bifidum that promote iTreg differentiation. Since B. bifidum is anaerobic and barely survives during co-culture with DCs, it was tested whether certain cellular components of Bb are effector molecules with iTreg-inducing activity.

[0219] As a result, among the various fractions of cell wall, cell membrane, and cytosol, only the cell surface extract effectively induced Treg cells. Treatment of the cell surface extract with RNase, DNase, pronase or boiling at high temperature did not reduce the Treg induction activity, indicating that polysaccharides can be effector molecules. In fact, total cell surface polysaccharides (tCSPS) induced Treg cells in a dose-dependent manner (see Fig.11 A and 11B).

[0220] tCSPS was separated by ion exclusion chromatography, followed by in-depth NMR analysis of the fractions eluting at low ionic strength.

[0221] As a result, the cell surface polysaccharides of Bifidobacterium bifidum consist of at least five species in different amounts (mol / mol) (see Fig.11 C); β-1,6-glucan (18%), β-1,4-galactan (5%), β-1,6-galactan (5%), β-galactofuranosan (2%), phosphoglycero-β-galactofuranosan (PGβG) (64%). The polysaccharide PGβG is the most abundant polysaccharide (64%), with an average molecular weight of 8000Da and negatively charged, while the remaining four polysaccharides (β-1,6-glucan, β-1,4-galactan, β-1,6-galactan and β-galactofuranosan) have similar molecular weights (average of about 4000Da) and are no longer separated separately.

[0222] Next, we tested which polysaccharide in the neutral mixture of polysaccharides or PGβG had the activity of inducing Treg cells in vitro. The results showed that only neutral polysaccharides, but not negatively charged polysaccharide PGβG, induced Treg cells (see Fig.11 D).

[0223] Therefore, the mixture of neutral polysaccharides derived from Bifidobacterium bifidum is called "cell surface β-glucan / galactan (CSGG)".

[0224] 8-2: Identification of the induction of Treg by CSGG

[0225] Among CSGG polysaccharides, cell surface β-1,6-glucan (CSβG) may be a key effector molecule, because the level of CSGG-induced iTreg cells was reduced in a dose-dependent manner after treatment with β-1,6-glucanase (see Fig. 9 A, 9B and 11E). In addition, in the absence of DCs, CSGG and naive CD4 + Co-culture of T cells failed to induce iTreg cells, indicating that CSGG induces Treg cells in a DC-dependent manner (see Fig.11 F).

[0226] Next, we tested whether CSGG could recapitulate the ability of intact bacteria to induce Treg cells in vitro and in vivo. CSGG-treated DCs induced Foxp3 as effectively as B. bifidum-treated DCs in a dose-dependent manner. + iTreg cells (see Fig.12 A). GF mice were intraperitoneally injected with CSGG (100 μg / dose) three times for 3 weeks, resulting in the induction of CD4 + Foxp3 + and Nrp1 - Rorγt + Treg cells, similar to mice colonized with Bifidobacterium bifidum (see Fig. 9 These Treg cells showed a higher proportion of CD103 compared with Treg cells from control GF mice. + and activated phenotype CD44 hi CD62L lo Foxp3 + (See Fig.12 D) Furthermore, co-culture of human DCs and naive CD4 T cells derived from PBMCs of healthy donors with CSGG induced CD25 + Foxp3 + Treg cells (see Fig. 9 D).

[0227] 8-3: CSGG synthetic gene homology and Treg induction activity

[0228] As is known, the anti-allergic effect of lactic acid bacteria depends on the strain and is affected by the expression and balance of Th1 / Th2 cytokines (Fujiwara D et al., Int Arch Allergy Immun. 135(3):205-215, 2004), and since the functional activity of probiotic bacteria is non-specific to species but strain-specific, it was tested whether all strains of Bifidobacterium bifidum can induce iTreg cells, or whether only strains having high base homology with the Bifidobacterium bifidum PRI1 strain according to the present invention have iTreg induction activity. The whole genome of the Bifidobacterium bifidum PRI1 strain according to the present invention was sequenced (GenBank accession number; CP018757), and the base homology and Treg induction activity of the expected orthologous genes related to CSGG synthesis and other Bifidobacterium bifidum genomes were compared.

[0229] As a result, the Bifidobacterium bifidum PRI1 of the present invention exhibits a high degree of homology with other Bifidobacterium bifidum strains. Specifically, strain A8 (Lopez P et al., PLOS ONE, 6(9): e24776, 2011) has similar activity to the Bifidobacterium bifidum PRI1 strain of the present invention, but exhibits defects in some orthologous genes related to CSGG synthesis and lower Treg induction activity.

[0230] Example 9: Mechanism of CSGG generating rDC

[0231] As observed in cLP-DCs of mice colonized with B. bifidum, we tested whether CSGG treatment induces a phenotypic modification of general DCs into regulatory DCs (rDCs). That is, to identify the underlying mechanism of rDC generation by CSGG, we performed a 4-hour CSGG treatment on CD11c + RNA-seq analysis was performed on DC.

[0232] As a result, CSGG treatment significantly enhanced the mRNA and protein levels of rDC-associated markers such as Ifna2, Pdcd1, Tgfβ1, Csf2, Ptgs2, Il10, and Il27 compared with mock control treatment (see Fig. 9 E and 13A).

[0233] CSGG treatment also significantly increased the protein levels of IL-10 and TGF-β, while reducing the level of IFN-γ. Compared with IL-10, TGF-β may play a key role, because the addition of anti-TGFβ neutralizing antibodies almost completely blocked the iTeg production induced by B. bifidum or CSGG treatment (see Fig.13 B and 13C). CSGG treatment selectively increased TLR2 expression (see Fig.13 D) and due to TLR2 - / - or Myd88 - / - DCs exhibited a significant reduction in CSGG-mediated iTreg induction (see Fig. 9 F, 9G and 13E), therefore, among the various pattern recognition receptors of DC, TLR2 and Myd88 may be the main molecules involved in CSGG recognition and signal transduction. TLR2-deficient DC induces a decrease in TGFβ1 and IL-10 in culture (see Fig. 9 G), but showed no change in IFNγ levels (see Fig.13 F). C-type lectin receptors (CLR) may not be involved in CSGG-mediated iTreg cell generation (see Fig.13 G and 13H).

[0234] Example 10: Function of Treg cells induced by CSGG

[0235] The functions of CSGG-induced Treg cells were examined in vitro and in vivo.

[0236] Since iTreg cells are usually inactive or unstable under inflammatory conditions, the immunosuppressive activity of iTreg cells induced by CSGG treatment and generated in vitro was tested. + CD4 + Foxp3 - Naive T cells were cultured with mock- or CSGG-treated DCs for 2 days and then sorted to purify CD45.1 + CD4 + Foxp3 + iTreg. CTV-labeled responders CD45.2 + CD4 + Foxp3 - T cells and CD45.1 + Treg cells were co-cultured at different ratios and their proliferation was then analyzed by FACS.

[0237] As a result, iTreg cells generated and sorted in the presence of CSGG-treated DCs in vitro exhibited significantly enhanced immunosuppressive capacity compared to cells cultured with mock-treated DCs (see Fig.15 A).

[0238] To test the suppressive activity of Treg cells in vivo, a T cell transfer model of colitis was used. + T cells alone or in combination with allele-marked (CD45.1 + )iTreg cells combined with Rag1 - / - In lymphopenic mice (see Fig.14 A).

[0239] As a result, co-injection of mimetic-induced iTreg cells was ineffective and failed to suppress signs of colitis. Mice given naive T cells alone also exhibited severe signs of colitis and weight loss within 3 weeks. In contrast, transfer of iTreg cells induced by Bifidobacterium bifidum or CSGG significantly reduced the development of colitis and significantly attenuated weight loss (see Fig.14 A to 14D and 15B).

[0240] Example 11: Administration of CSGG inhibits the development of colitis

[0241] To examine whether administration of CSGG itself could inhibit the development of colitis, we isolated CD45.1 + Foxp3 GFPMouse naive CD4 + Foxp3 - Adoptive transfer of T cells into SPF Rag1 - / - Host mice were then intraperitoneally administered PBS or CSGG (100 μg / dose) three times a week until the end of the experiment.

[0242] As a result, mice transferred with naive T cells and subsequently treated with PBS developed severe colitis, whereas mice given CSGG significantly reduced body weight loss and significantly decreased progression of colitis by histological analysis (see Fig.14 E to 14G and 15C). The protective effect of CSGG treatment is related to total Foxp3 + The increase in Treg cells was associated with a decrease in the frequency of IFN-γ-producing effector T cells (see Fig.14 H and 15C). These results indicate that CD4 + Foxp3 + Treg cells can inhibit the progression of inflammatory colitis.

[0243] Accession number

[0244] Name of depository organization: Korea Institute of Bioscience and Technology

[0245] Accession number: KCTC13270BP

[0246] Date of deposit: May 19, 2017

[0247] Practicality

[0248] The polysaccharides according to the present invention, such as cell surface β-glucan / galactan polysaccharide (CSGG), and CSGG-producing Bifidobacterium bifidum PRI1 (KCTC13270BP) can induce Treg cells through various antigens, so the strain, CSGG or induced Treg cells can be used to prevent or treat immune diseases or inflammatory diseases.

[0249] Although the present invention has been described in detail with reference to specific features, it is clear to those skilled in the art that this description is only for preferred embodiments and does not limit the scope of the present invention. Therefore, the true scope of the present invention will be defined by the appended claims and their equivalents.

[0250] Text files

[0251] See attached sequence listing.

[0252] Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure

[0253] International Forms

[0254] Proof of original preservation

[0255] It is issued in accordance with Rule 7.1

[0256] To: IM, Sin-Hyeog

[0257] Pohang University of Science and Technology Industry-Academic Cooperation Group

[0258] No. 77, Cheongam-ro, Nam-gu, Pohang, Gyeongsangbuk-do, South Korea 37673

[0259]

[0260] Form BP / 4 (KCTC Form 17) Single Page <110> Institute of Basic Sciences Pohang University of Science and Technology Industry-Academic Cooperation Group <120> Bifidobacterium bifidum strains and polysaccharides derived from the strains <130> PP-B2060 <150> KR 10-2017-0075079 <151> 2017-06-14 <160> 27 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EUB-f <400> 1 tcctacggga ggcagcagt 19 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> EUB-r <400> 2 ggactaccag ggtatctaat cctgtt 26 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> BibiF- (forward) <400> 3 ccacatgatc gcatgtgatt g 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> BibiF- (reverse) <400> 4 ccgaaggctt gctcccaaa 19 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Bbif <400> 5 ccacaatcac atgcgatcat g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HPRT (forward) <400> 6 ttatggacag gactgaaaga c 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HPRT (reverse) <400> 7 gctttaatgt aatccagcag gt 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10 (forward) <400> 8 ataactgcac ccacttccca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10 (reverse) <400> 9 tcatttccga taaggcttgg 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TGF-b (forward) <400> 10 ctcccgtggc ttctagtgc 19 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TGF-b (reverse) <400> 11 gccttagttt ggacaggatc tg 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-b (forward) <400> 12 caaccaacaa gtgatattct cc 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-b (reverse) <400> 13 tgccgtcttt cattacacag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Csf2 (forward) <400> 14 agggtctacg gggcaatttc 20 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Csf2 (reverse) <400> 15 ggcagtatgt ctggtagtag ctg 23 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PD-L1 (forward) <400> 16 gctccaaagg acttgtacgt g 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PD-L1 (reverse) <400> 17 tgatctgaag ggcagcattt c 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IDO (forward) <400> 18 gctttgctct accacatcca c 21 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IDO (reverse) <400> 19 caggcgctgt aacctgtgt 19 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COX2 (forward) <400> 20 tggctgcaga attgaaagcc ct 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COX2 (reverse) <400> 21 aaaggtgctc ggcttccagt at 22 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD80 (forward) <400> 22 acccccaaca taactgagtc t 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD80 (reverse) <400> 23 ttccaaccaa gagaagcgag g 21 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD86 (forward) <400> 24 tgtttccgtg gagacgcaag 20 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CD86 (reverse) <400> 25 cagctcactc aggcttatgt ttt 23 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CD40 (forward) <400> 26 ccttgcactg tgaggaga 18 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD40 (reverse) <400> 27 cttcgcttac aacgtgtgct 20

Claims

1. A polysaccharide comprising β-1,6-glucan as an active ingredient, wherein the β-1,6-glucan comprises a compound of the following formula: in, Glc is glucose, "=" is a β-1,6-glycosidic bond, "-" is an α-1,2-glycosidic bond, and n is an integer between 6 and 12. 2 . The polysaccharide according to claim 1 , further comprising β-1,4-galactan, β-1,6-galactan or β-galactofuranosan.

3. The polysaccharide according to claim 2, wherein the molar ratio of β-1,6-glucan, β-1,4-galactan, β-1,6-galactan and β-galactofuranosan is 5-50: 2-15: 2-15: 1-5. The polysaccharide according to claim 1 , wherein the polysaccharide is a mixture having a molecular weight of 100 kDa or less. The polysaccharide according to claim 1 , wherein the charge of the polysaccharide is neutral. The polysaccharide according to claim 1 , wherein the polysaccharide induces regulatory T (Treg) cells.

7. The polysaccharide according to claim 6, wherein the regulatory T (Treg) cells are CD4 + Foxp3 + Treg cells.

8. The polysaccharide according to claim 1, wherein the polysaccharide is derived from Bifidobacterium bifidum ( Bifidobacterium bifidum ). 9 . The polysaccharide according to claim 1 , wherein the β-1,6-glucan has a molecular weight of 3 kDa to 5 kDa.

10. A Bifidobacterium bifidum PRI1 strain, whose deposit number is KCTC13270BP.

11. Use of the polysaccharide according to claim 1 or the strain according to claim 10 as an active ingredient in the preparation of a medicament for preventing or treating immune diseases or inflammatory diseases.

12. A food comprising the polysaccharide according to claim 1 or the strain according to claim 10 as an active ingredient.

13. A method for producing induced regulatory T (iTreg) cells, comprising the following steps: (a) treating dendritic cells (DC) with the polysaccharide according to claim 1 or the strain according to claim 10 to obtain regulatory dendritic cells (rDC); and (b) the rDCs and CD4 + T cells were co-cultured to induce regulatory T (Treg) cells.

14. The method of claim 13, wherein the regulatory T (Treg) cells are CD4 + Foxp3 + Treg cells. 15 . The method of claim 13 , wherein in step (b), the regulatory T (Treg) cells are induced by stimulation with anti-CD3 antibodies, IL-2, and TGF-β.

16. The method according to claim 13, wherein the dendritic cells and the CD4 + T cells were derived from peripheral blood mononuclear cells (PBMCs).

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