KR100913406B1 - Compositions for preventing or treating th1-mediated immune diseases - Google Patents

Abstract

A composition for preventing and treating Th-1 mediated immune diseases, which contains probiotics strain as an active ingredient is provided to reduce the Th1 immune response by inducing hypo-responsiveness without inducing apoptosis of T-cell and treat Th1 cell-mediated diseases. A pharmaceutical composition and a food composition for treating Th1-mediated immune diseases comprise a probiotics mixture strain as active ingredients. The probiotics mixture strain comprises Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidium, and Streptococus thermophilus. The probiotics mixture strain induces the formation of CD4^+Foxp3^+Tregs, and increases the inhibitor activation of naturally accrring Tregs(CD4^+CD25^+). The Th1-mediated immune diseases are inflammatory diseases, transplantation rejection or autoimmune disease. The Th1 cell synthesizes IL-2(interleukin 2) and IFN-gamma(interferon-gamma) but has the cytokine expression pattern that IL-4, IL-5, IL-10 and IL-13 are not synthesized.

Description

Compositions for Preventing or Treating Tｈ１-Mediated Immune Diseases

The present invention relates to a composition for the prevention or treatment of Th1-mediated immune diseases, including probiotics as an active ingredient.

There are many benefits of gastrointestinal tract microorganisms to host physiological aspects, including, for example, metabolic activity, trophic effects and inhibition of pathogen overgrowth in the gastrointestinal tract (Guarner, 2003; Rautava, 2005). Several microorganisms cause a variety of phenomena in the gastrointestinal tract, which have a detrimental or beneficial effect on the mucosal immune system. Probiotics can be defined as nonpathogenic microorganisms having beneficial effects on the physiological aspects of the host or on health (Marteau, 2006). Lactobacilli and Bifidobacteria groups are the most representative microorganisms with anti-inflammatory properties (Braat, 2004). For example, probiotic microbial strains show protective efficacy against colitis (Marteau, 2000; Hoentjen, 2003), inflammatory bowel disease (Foligne, 2005; Kennedy, 2001) and local allergies (Viljanen, 2005). Various knockout transformation and adaptive transfer animal models for inflammatory bowel disease (IBD) have been developed and tested for the therapeutic potential of probiotics. For example, Lactobacillus and Bifidobacterium species have been shown to be effective in reducing inflammation (McCarthy, 2003). Increased expression of IL-10 and IL-4 resulted in Lactobacillus Casei was observed in mice fed. In contrast, some strains of probiotic strains significantly induced the expression of Th1 type cytokines (IL-2 and IL-12), especially Lactobacillus. acidophilus did this. However, the problem of which probiotic strains alone or in combination should be used and what kind of strain is desired for a particular disease condition has not been solved yet (Shanahan, 2003). Combinations of probiotic strains used in the treatment of certain diseases should be well selected.

There has been much research on the therapeutic efficacy of probiotics, but their mechanism of regulation of the immune system is not well understood yet.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors have sought to develop probiotics that can prevent or treat diseases, disorders or conditions caused by Th1 immune responses, such as inflammatory diseases. As a result, certain Bifidobacterium and / or lactobacillus, in particular mixtures of probiotics, induce the formation of CD4 ⁺ Foxp3 ⁺ Tregs, which are immunoregulatory / inhibiting T-cells, and naturally occurring Tregs (CD4 ⁺ By increasing the inhibitory activity of CD25 ⁺ ) to induce hypo-responsiveness of T cells to reduce the Th1 immune response, the present invention was confirmed by showing excellent therapeutic efficacy in inflammatory animal models. It was completed.

Accordingly, it is an object of the present invention to provide a composition for preventing or treating a Th1-mediated immune disease.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for the prevention or treatment of Th1-mediated immune disease comprising Bifidobacterium bifidium or Lactobacillus reuteri as an active ingredient.

The inventors have sought to develop probiotics that can prevent or treat diseases, disorders or conditions caused by Th1 immune responses, such as inflammatory diseases. As a result, certain Bifidobacterium and / or lactobacillus, in particular mixtures of probiotics, induce the formation of CD4 ⁺ Foxp3 ⁺ Tregs and inhibit the inhibitory activity of naturally occurring Tregs (CD4 ⁺ CD25 ⁺ ). Increasing T cells induced hypo-responsiveness to reduce the Th1 immune response, and it was confirmed that it exhibits excellent therapeutic efficacy in inflammatory animal models.

The composition of the present invention is Bifidobacterium bifidium ( Bifidobacterium bifidium ) or Lactobacillus reuteri ) as an active ingredient. The two microorganisms have not yet been identified as being effective in the treatment of inflammatory diseases and have been selected by the inventors from many microbial species.

According to a preferred embodiment of the present invention, the composition of the present invention is Lactobacillus Axidophilus ( Lactobacillus) acidophilus ), Streptococcus thermophilus ) and Lactobacillus casei , at least one microorganism selected from the group consisting of:

More preferably, the composition of the present invention is at least three selected from the group consisting of Bifidobacterium biphidium, Lactobacillus leutheria, Lactobacillus axidophyllus, Streptococcus thermophilus and Lactobacillus cascai And even more preferably at least four microorganisms.

When the composition of the present invention contains a total of three microorganisms as an active ingredient, the composition of the present invention preferably includes Lactobacillus rutheli, Lactobacillus axidophyllus, and Lactobacillus cascai.

When the composition of the present invention contains a total of four microorganisms as an active ingredient, the composition of the present invention includes bifidobacterium biphydium, lactobacillus rutheri, lactobacillus axidophyllus, and lactobacillus casei. desirable.

According to the most preferred embodiment of the present invention, the composition of the present invention is a total of five microorganisms, including Bifidobacterium biphydium, Lactobacillus rutheri, Lactobacillus axidophyllus, Streptococcus thermophilus and Lactobacillus casei It includes.

The prophylactic or therapeutic composition for Th1-mediated immune disease of the present invention may be prepared as a pharmaceutical composition, in which case it includes a pharmaceutically acceptable carrier in addition to the above components as an active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention is preferably administered orally.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-2000 mg / kg body weight per day.

The pharmaceutical composition of the present invention is prepared in unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by one of ordinary skill in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

On the other hand, the prophylactic or therapeutic composition for Th1-mediated immune disease of the present invention may be prepared from food, in particular functional food compositions. Functional food compositions of the present invention include ingredients that are commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the extract of hawthorn as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used. Given the ease of access to food, the food of the present invention is very useful for the prevention or treatment of Th1-mediated immune diseases such as inflammatory diseases.

The compositions of the present invention can be used for the prevention or treatment of various Th1-mediated immune diseases, diseases or conditions.

As used herein, the term “Th1-mediated immune disease” refers to cytokines produced by the production and / or activity of Th1 cells, such as IL-1β, IL-2, IL-12, IL-17, IFN-γ or TNF-α or the like is a disease involved.

As used herein, the term “Th1 cell” refers to a subset of helper T cell lymphocytes that are specified in terms of gene expression, protein secretion, and functional activity. For example, Th1 cells exhibit cytokine expression patterns that synthesize IL-2 and IFN-γ but not IL-4, IL-5, IL-10 and IL-13. Th1 cells are involved in cell-mediated immune responses, organ-specific autoimmune diseases and delayed hypersensitivity to various intracellular pathogens.

The Th1-mediated immune disease to which the composition of the present invention is applied is not particularly limited and is preferably applied to transplant rejection, autoimmune disease or inflammatory disease. More preferably, the Th1-mediated immune disease to which the composition of the present invention is applied is an autoimmune disease or inflammatory disease, and even more preferably colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes and rheumatoid arthritis.

Th1-mediated autoimmune diseases to which the composition of the present invention is applied include colitis, inflammatory bowel disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, reactive arthritis, osteoarthritis, psoriasis, scleroderma, porosity, atherosclerosis , Myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, nervous-borelia, tuberculosis, sarcoidosis, lupus, discoid Lupus, alumni lupus, lupus nephritis, systemic lupus erythematosus, asthma, macular degeneration, uveitis, irritable bowel syndrome, Croix's disease, Shogran's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgia encephalomyelitis, muscular dystrophy Including but not limited to lateral sclerosis, Parkin's disease, multiple sclerosis, autism spectrum disorder, attention deficit disorder and attention deficit hyperactivity disorder It is not.

The composition of the present invention induces the formation of CD4 ⁺ Foxp3 ⁺ Tregs, increases the inhibitory activity of naturally occurring Tregs (CD4 ⁺ CD25 ⁺ ) and induces hypo-responsiveness of T cells to induce Th1 immunity. By reducing the response, it is effective in treating Th1 cell-mediated diseases. In addition, the composition of the present invention does not induce apoptosis of T cells in reducing the Th1 immune response, and can provide a safe medicine or food because it uses a microbial strain that is harmless to the human body.

The features and advantages of the present invention are summarized as follows:

(Iii) The composition of the present invention is a probiotic containing a microorganism as an active ingredient, and exhibits very good efficacy in treating Th-1 mediated immune diseases.

(Ii) the composition of the present invention induces the formation of CD4 ⁺ Foxp3 ⁺ Tregs and increases the inhibitory activity of naturally occurring Tregs (CD4 ⁺ CD25 ⁺ ) to induce hypo-responsiveness of T cells Thereby reducing the Th1 immune response to treat Th1 cell-mediated diseases.

(Iii) Since the composition of the present invention does not induce apoptosis of T cells in reducing the Th1 immune response, safety of the human body is excellent.

(Iii) Since the composition of the present invention uses a microbial strain that is harmless to the human body, a safe medicine or food can be provided.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Experimental Methods

Mouse and Muline IBD Of AD  Disease model

Balb / c J mice (6-8 weeks old) were purchased from SLC company (Japan) and maintained under sterile conditions in an animal kennel at GIST. All animal experiments were approved by the GIST Animal Breeding Society.

To induce colitis, mice were anesthetized with 1 mg TNBS in 50% ethanol, in which case a catheter inserted into the rectum was used; 100 μl TNBS-ethanol mixture was carefully injected into the colon. One day after colitis induction, groups of mice were fed the same volume of control PBS or probiotic mixture for 6-7 days. To analyze the effect of the probiotic combination, mice were divided into seven groups after colitis induction and PBS, each of five strains and a mixture of five strains were administered for 6 days. Symptoms of colitis were evaluated by mortality, weight loss and microscopic observation.

To induce atopic dermatitis, mice were divided into three groups and the ear surface was stripped three times with surgical tape (Nichiban, Tokyo, Japan). After 30 minutes, 20 ml 1% DNCB (Sigma) dissolved in acetone / olive oil solution (acetone: olive oil = 1: 3) was painted on the surface of each ear. Four days after DNCB painting, the cells were re-patined with 20 ml of 10 mg / ml mite extract (Yonsei University, Seoul, South Korea). These DNCB and tick antigen paintings were performed once a week. Tape stripping and DNCB / tick extract painting continued for 4 weeks. Two weeks after the induction process, IgE levels were checked by performing eye breathing to determine whether atopic dermatitis was induced. After induction of atopic dermatitis was confirmed, probiotics and PBS were orally administered to each experimental group (Pro and Cont group) every day until the 4 week induction process. Ear thickness and clinical score were measured immediately before tape stripping and ear thickness and clinical score were measured 24 hours after DNCB and tick application. In the non-AD group, only tape stripping and PBS painting were performed.

Probiotic  Strain

The probiotic mixture used in the experiment is Bifidobacterium bifidium , 1 X 10 ⁹ CFUs), Lactobacillus acidophilus , 1 X 10 ⁹ CFUs), Streptococcus thermophilus , 1 X 10 ⁹ CFUs), Lactobacillus leuteri , 1 X 10 ⁹ CFUs) and Lactobacillus 5 × 10 ⁹ cfu / 100 μl of live bacteria containing casei , 1 × 10 ⁹ CFUs). In the atopy model, 5 x 10 ⁹ cfu / 100 μl of each strain was administered to mice to confirm the effect of each single strain.

Histological observation

In IBD cases, whole intestines were isolated from mice and evaluated according to conventional methods (Ameho et al. 1997). For histological scoring, the whole field was immobilized in 4% paraformaldehyde in PBS and embedded in paraffin. Paraffin sections were stained with H & E and scored according to previous methods (Marceau et al. 2004).

For atopic dermatitis cases, previous methods (Matsuoka et al. 2003) evaluated clinical conditions and symptoms for each mouse. After sacrifice, the ears were cut, fixed in 4% paraformaldehyde (Sigma) for 16 hours, and embedded in paraffin. Tissues were prepared 3-10 mm and stained with H & E. The total number of infiltrating cells stained by H & E in the dermis was measured according to known methods (Matsuoka et al. 2003). Based on histological samples, total infiltrating cells were determined. 3-5 sections from each group were counted with a 40 × objective and a 10 × alternative lens with reticle (0.22 mm by 0.22 mm image obtained). Data is expressed as number of cells per square milliliter. The calculated cell number is the average of five independent counting.

RNA  detach, cDNA  Synthetic, quantitative real time PCR  And standard PCR

Spleen and mesenteric lymph nodes were obtained from each experimental group, from which total RNA was obtained using TRIzol reagent (Molecular Research Center). Reverse transcription was performed using reverse transcriptase (Promega) and oligo (dT) primers. The synthesized cDNA was amplified by real-time PCR, in which case the following primers were used: murine L-32 (forward 5'-GCC CAA GAT CGT CAA AAA GA-3 'and reverse 5'-ATT GTG GAC CAG GAA CTT GC-3 '), IL-2 (forward 5'-CCT GAG CAG GAT GGA GAA TTA CA-3' and reverse 5'-TCC AGA ACA TGC CGC AGA G-3 '). IL-4 (Forward 5'-ACA GGA GAA GGG ACG CCA T-3 'and Reverse 5'-GAA GCC CTA CAG ACG AGC TCA-3'), IL-5 (Forward 5'-AGC ACA GTG GTG AAA GAG AC-3 'and Reverse 5'-TCC AAT GCA TAG CTG GTG ATT T3'), IL-10 (Forward 5'-ATA ACT GCA CCC ACT TCC CA-3 'and Reverse 5'-TCA TTT CCG ATA AGG CTT GG-3 '), IL-12 p40 (Forward 5'-GGA AGC ACG GCA GCA GAA TA-3' and Reverse 5'- AAC TTG AGG GAG AAG TAG GAA TGG-3 '), IL-13 5'-GCA ACA TCA CAC AGG ACC AGA-3 'and 5'-GTC AGG GAA TCC AGG GCT AC-3'), IL-17 (5'-TTC ATC TGT GTC TCT GAT GCT-3 ') And reverse 5'-TTG ACC TTC ACA TTC TGG AG-3 ') IFN-g (forward 5'-TCA AGT GGC ATA GAT GTG GAA GAA-3' and reverse 5'-TGG CTC TGC AGG ATT TTC ATG-3 '), TNF-α (forward 5'-CAT CTT CTC AAA ATT CGA GTG ACA A-3' and reverse 5'-TGG GAG TAG ACA AGG TAC AAC CC-3 '), TGF-β (forward 5' -GAA GGC AGA GTT CAG GGT CTT-3 'and reverse 5'-GGT TCC TGT CTT TGT GGT GAA-3') and Foxp3 (forward 5'-CCC ATC CCC AGG AGT CTT G-3 'and reverse 5'- CCA TGA CT A GGG GCA CTG TA-3 ').

B220 ⁺  B cells and CD4 ⁺  Isolation of T Cells

T cells were isolated from the spleen and mesenteric lymph nodes of each experimental group. CD4 ⁺ T cell isolation beads (Miltenyi Biotech Cat No. 130-049-201) and columns (Miltenyi Biotech Cat No. 130-042-401), or mouse CD4 beads (Dynal Cat No.114.45) and DETACHaBEAD (Dynal Cat No 124.06) was used to isolate CD4 ⁺ T cells. To separate CD4 ⁺ CD25 ⁺ and CD4 ⁺ CD25 ⁻ T cells, CD4 ⁺ T cells were labeled with biotin-conjugated rat anti-mouse CD25 (BD Pharmigen; Cat No. 553069) and labeled buffer (PBS pH 7.2, 2 mM EDTA) with streptavidin microbeads (Miltenyi Biotech; Cat No. 130-048-101). In addition, CD4 ⁺ CD25 ⁺ regulatory T cell separation kit (Miltenyi Biotech; Cat No. 130-091-041) was used. In order to remove the B220 ⁺ B cells, B220 ⁺ B cells isolated beads: was used (Miltenyi Biotech Cat No. 139-049-501).

Cell culture and stimulation

All isolated B and T cells were treated with 10% fetal bovine serum (Hyclone), 3 mM L-glutamine (Sigma), 10 mM HEPEs (Sigma), 100 U / ml penicillin (Sigma) and 100 U / ml streptomycin (Sigma). ) And DMEM (Invitrogen) supplemented with 0.05 mM 2-β-mercaptoethanol (Sigma). For cytokine profile analysis, cells were treated with PMA (20 nM) and ionomycin (2 μM) for 4 hours. For proliferation analysis, isolated T cells were stimulated for 72 hours with anti-CD3 (1 μg) or anti-CD3 and anti-CD28 (1 μg).

In vitro T cell and B cell proliferation assay

CD4 ⁺ T cells were resuspended in 100 μl T cell medium and washed with 1 μg / ml anti-CD3 (BD Pharmigen; Cat No.553058) and 1 μg / ml soluble anti-CD28 (BD Pharmigen; Cat No.553294). Stimulated. For B cells, the cells were resuspended in 100 µl of T cell medium containing 10 µg / ml LPS (Sigma) for stimulation. Cells were incubated in 96-well plates for 56-72 hours in T cell medium containing 10% heat-inactivated FBS (Hyclone). 0.5 μCi [H ³ ] -thymidine (NEN) was then added to each well and incubated for an additional 16 hours. Cells were then collected and the [H ³ ] -thymidine uptake was measured by liquid scintillation counting.

By suppressor T-cells Effector  T-cell activity inhibition test

MLN CD4 ⁺ CD25 ⁺ T cells (suppressor cells) were isolated from PBS or probiotic administered mice and plated on 1 mg / ml anti-CD3 coated plates and then CD4 ⁺ CD25 ⁻ isolated from wild type Balb c / J mice. Co-cultures with various proportions of T cells (effector cells). Three sets of independent experiments were conducted for each sample and incubated for 72 hours. After 56-72 hours of incubation, 0.5 μCi [H ³ ] -thymidine (NEN) was added to each well and incubated for an additional 6 hours. Cells were then collected and the [H ³ ] -thymidine uptake was measured by liquid scintillation counting.

Flow cytometry

To measure apoptosis populations, 1 × 10 ⁶ CD4 ⁺ T cells were resuspended in 1 ml of 1 × Annexin V binding buffer (BD bioscience, Cat No. 556454). 100 μl of suspended cells (1 × 10 ⁵ cells) were transferred to 5 ml culture tubes and 5 μl of Annexin V-PE and 7-ADD were added and reacted at 25 ° C. for 15 minutes in dark conditions. 400 μl of 1 × Binding Buffer was then added to each tube and immediately analyzed by FACS. CD4 ⁺ CD25 ⁺ T cells were stained for CD4 ⁺ T cell population in order to measure the anti -CD4-FITC (BD Bioscience Cat No.553047 ) and anti -CD25-PE (BD Bioscience Cat No. 553866). For Foxp3 detection, cells were permeabilized with Foxp3 fixation / permeation buffer (eBioscience Cat No; 00-5523-00) and stained with anti-Foxp3-PE (eBioscience; Cat No; 12-5773). In all FACS experiments, purified rat IgG2b isotype (eBioscience; Cat No; 14-4031-81) was used as a control and IgG2b positive population was less than 0.2%.

A unique edition ( lamina propria : LP ) And ear CD4 ⁺  Isolation of T Cells

The ears and intestines of the mice were cut and transferred directly to the dish containing RPMI medium. Ears and intestines were cut into 1-2 cm sections, washed with PRMI medium, and gently stirred for 20 minutes at room temperature in a flask containing 25 ml of PBS containing 5% FBS and 1.0 mM EDTA. The sections were crushed and then transferred to a 50-ml centrifuge tube containing 15 ml of serum-free RPMI medium and stirred vigorously twice for 15 seconds. The tissues were then transferred to five T flasks containing 10 ml of 0.5 mg / ml collagenase type V (Sigma) and reacted with stirring at 37 ° C. for 1 hour. After the reaction, the cell suspension was washed with PBS and used as LP total cells or ear total cells. LP CD4 ⁺ T cells were further isolated according to the conventional CD4 ⁺ T cell separation method.

Total in serum IgE  And tick specific IgE Measure

Two to four weeks after AD induction, blood was obtained from the tail vein or heart. Total serum IgE levels were obtained with an ELISA kit (BD Biosciences). Briefly, serum was diluted 200-fold to measure IgE and serum 250-fold diluted with 0.1 M sodium carbonate (pH 9.5) for use as primary antibody. The detection antibody was initially diluted 500-fold in diluent (PBS containing 10% of FBS), and streptavidin-conjugated horseradish peroxidase (HRP) was added to the detection antibody diluent at a ratio of 250: 1 as a detection antibody. For detection of tick specific IgE, 100 μl of 10 μg / ml mite extract was encoded into a 96-well plate and reported overnight at 4 ° C. After the antigen solution was discarded, each well was blocked with blocking solution for 1 hour at room temperature, 100 ml of 10-fold diluted sample was added, and the rest of the procedure was carried out according to the manufacturer's protocol. Three wells were prepared for each antigen and serum. Concentration and OD values are the average of each well and serum. The absorbance value of the antigen well was obtained by subtracting the absorbance value of the non-antigen well as the OD value of the antigen.

Total in serum IgG  And tick specific IgG Measure

To determine serum IgG levels, serum was analyzed with a mouse IgG ELISA Quantitation Kit (BETHYL). Briefly, the obtained serum was diluted 2000-fold and diluted 100-fold with 0.05 M carbonate-bicarbonate (pH 9.5) for use as a capturing antibody. HRP-conjugate detection antibody was diluted 20000 times with diluent (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, 1% BSA at pH 8.0). The method for the evaluation of tick specific IgG is similar to the method for detecting specific IgE, and serum was diluted 200-fold. Three wells were prepared for each antigen and serum. Concentration and OD values are the average of each well and serum. The absorbance value of the antigen well was obtained by subtracting the absorbance value of the non-antigen well as the OD value of the antigen.

Within the organization Foxp3 Immunohistochemical staining for detection of

Paraffin sections (3 mm) of mouse colon and ears were used for immunohistochemical staining. Immunohistochemical staining was performed using anti-rat Foxp3 (eBioscience; Cat No: 14-5773) and anti-rabbit Foxp3 (Santa Cruz; Cat No: sc-28705). For counter staining, propidium iodide (Molecular Probes; Cat No: P21493) and Hoeschst34580 (Molecular Probes; Cat No: H21486) were used. After staining, images of tissue sections were obtained by Fluoview microscopy (Olympus).

adaptability transfer  Experiment

30 days after oral administration of PBS or probiotics, mice were sacrificed and MLN CD4 ⁺ T cells were isolated from each group. Prior to transferring the cells, 5 × 10 ⁶ CD4 ⁺ T cells were labeled with 5 mM CFSE at 37 ° C. for 15 minutes and transferred to AD induced mice by intraperitoneal injection. Transferring was performed five times according to the same procedure. Twenty four hours after the last adaptive transfer procedure, mice were sacrificed and ears removed to analyze regulatory T cell migration according to immunohistochemical staining methods.

Evaluation of Clinical Score

For AD, the clinical conditions and symptoms of each mouse were assessed with a slight modification of the known method (Matsuoka et al. 2003a). Simply zero (invisible), one (mild), two (usually) and three (severe) scoring was performed by three independent observers, with dryess, erosion / Excoriation / erosion, edema and erythema were scored. Average values were taken for each group.

Rheumatism  arthritis( RA Treatment efficacy experiment

1. Preparation of Collagen-induced Arthritis Animal Model

Collagen induced arthritis (CIA) animal models were prepared by injecting collagen, which is believed to be the cause of rheumatoid arthritis, as follows. 4 mg of collagen type II collagen (Sigma) was mixed in 1 ml of 100 mM acetic acid and dissolved at 4 ° C. for 1 day. It was mixed and emulsified in 1 ml of Freund's complete adjuvant (DIFCO) containing 4 mg / ml of Mycobacterium tuberculosis and 150 μl (300) in the tail region of 6-8 week old Lewis rats. [Mu] g) was immunized by intradermal injection. On day 7 after the first immunization, 4 mg of collagen type II collagen of chicken was mixed with 1 ml of 100 mM acetic acid and dissolved for 1 day at 4 ° C., and then emulsified by mixing with 1 ml of Freund's incomplete adjuvant (DIFCO). I was. 150 μl (300 μg) of the emulsion was injected intradermally into the tail to induce a secondary immunity (boosting) reaction. After 1-2 weeks after the second immunization, erythema or swelling, which is a symptom of arthritis, began to appear gradually. .

2. Analysis of therapeutic efficacy in animal models of arthritis

Arthritis-induced rats were divided into three groups, and in the control group, PBS, MTX group, methotrexate, which is conventionally used as a RA treatment agent, and Probiotics group, the changes appearing after oral administration of a mixture of five strains of the present invention three times a week Was observed. The clinical score of arthritis was calculated as shown in Table 1 below.

Scoring system score The progress of the disease 0 Erythema and swelling do not appear One Erythema and slight swelling only in the midfoot or ankle joint 2 Erythema and slight swelling from the ankle joint to the middle of the foot 3 Erythema and moderate swelling from ankle joint to metatarsal joint 4 Erythema and severe swelling, including ankle, foot and toe

In addition, H & E staining observation and quantitative real-time PCR were performed according to the above-described experimental method.

Statistical analysis

For statistical analysis, a two-tailed Student's t-test was conducted and evaluated as being significant when P <0.05 ( ^* <0.05, ^** <0.005, ^*** <0.001).

Experiment result

Probiotic  Oral administration In mesenteric lymph nodes CD4 ⁺ Foxp3 ⁺  Regulatory T cells Population  Increases

We selected five probiotic strains known or not known to have anti-inflammatory efficacy: Bifidobacterium bifidium , BB), Lactobacillus acidophilus (LA), Streptococcus thermophilus (ST), Lactobacillus reuteri , LR) and Lactobacillus casei , LC). To elucidate the mechanism for the anti-inflammatory function of probiotics, we analyzed the relationship between T cells and probiotics. Thirty days after PBS or probiotics administration, mesenteric lymph nodes (MLN) CD4 ⁺ T cells were isolated and examined for Foxp3 ⁺ population by flow cytometry (FACS) (FIGS. 1A-1B). Compared to PBS (8.5%) treated mice (Cont), Foxp3 ⁺ population increased in LA (11.54%), LC (10.4%), LR (11.36%) and BB (10.82%) groups, and ST group ( 7.48%).

We then investigated whether administration of a probiotic mixture consisting of several strains could induce a synergic increase in Foxp3 ⁺ population: 3 strains, LA / LC / LR; 4 strains, LA / LC / LR / BB and 5 strains, LA / LC / LR / BB / ST. After 30 days of administration of three different probiotic mixtures, MLN CD4 ⁺ Foxp3 ⁺ populations were analyzed in each group (FIG. 1C). For probiotic mixtures of three strains, Foxp3 ⁺ population increased significantly, and for probiotic mixtures of four or five strains, Foxp3 ⁺ population increased significantly (four strains 14.57% And 5 strains 15.97%).

The experimental results show that administration of probiotics increases Foxp3 ⁺ regulatory T cell population in MLN, and this increase is specific to the type of strain, and a combination of suitable strains can synergistically increase the immunomodulatory capacity by probiotics. Prove that there is.

Probiotic  Dosing CD4 ⁺  T cell and B220 ⁺  B cell Hyporesponsiveness  Induce

Based on the experimental results for the Foxp3 ⁺ regulatory T cell population described above, the anti-inflammatory efficacy and mechanism were analyzed using the five probiotic mixtures (Pro) that exhibited the best efficacy. The function of effector T cells was investigated by measuring cytokine expression and proliferation rate in the spleen (SP) and mesenteric lymph nodes (MLN) (FIGS. 2A-2B). After probiotics mixture (Pro) or PBS as a control (Cont) was administered to normal Balb / c mice for 30 days, CD4 ⁺ T cells of spleen (SP) and mesenteric lymph nodes (MLN) were isolated and PMA / ionoma Stimulated with cin (P + I).

Transcript levels of Th1-type effector cytokines (IL-2, IFN-γ, TNF-α) and Th2 type cytokines (IL-4, IL-10) were measured by real-time PCR (RT-PCR). Surprisingly, probiotics reduced the levels of all cytokines (p <0.005, FIG. 2A). The results of this experiment indicate that the probiotics of the present invention can induce CD4 ⁺ T cell hyporesponsiveness. It was investigated whether probiotics inhibit T cell proliferation CD4 ⁺ T cells isolated from probiotic-administered and PBS-administered mice were stimulated with or without anti-CD3 / CD28 and then unstimulated CD4 ⁺ T The stimulation index was calculated by comparing the level of [H ³ ] -thymidine binding (cpm) in the cells As shown in Figure 2b, the probiotics group (Pro) split less than the control (Cont) in both SP and MLN. It became.

Next, the present inventors examined the function of effector B cells by measuring cytokine expression and proliferation rate in the spleen (SP) and mesenteric lymph nodes (MLN) (Fig. 2C-2D). Briefly, normal Balb / c mice were administered PBS or probiotics for 30 days, then B220 ⁺ B cells were isolated from SP or MLN and stimulated with P + I to express cytokines and stimulate LPS to stimulate proliferation rate. Stimulated. Probiotic administration significantly reduced all cytokines (Th1 and Th2 cytokines) in B220 ⁺ B cells of the spleen (SP) and mesenteric lymph nodes (MLN) compared to the PBS-administered group. In addition, probiotics inhibited B cell proliferation for foreign LPS stimulation, which was observed in both SP (stimulation index: 14.4 vs 62.45) and MLN (stimulation index: 29.92 vs 62.98).

According to the above experimental results, it can be seen that the probiotics of the present invention induce CD4 ⁺ T cells and B220 ⁺ B cell low reactivity in mucosal and systemic sites.

Probiotics CD4 ⁺ CD25 ^-  From T cells Foxp3 ⁺ CD4 ⁺  Induces the formation of T cells

Mechanisms of peripheral T cell tolerance include T cell apoptosis (Wells et al. 1999). Whether or not the low reactivity induced by probiotics is associated with T / B cell apoptosis was analyzed using Annexxin V and 7-ADD (FIG. 3). CD4 ⁺ T cells and B220 ⁺ B cells from cells obtained from MLN and SP of the Pro and Cont groups were stained with Annexxin V and 7-ADD and analyzed for apoptosis population (Annexin V ⁺ / 7-ADD ⁺ ) by flow cytometry. . Apoptosis was not increased (FIGS. 3A and 3B); Annexin V ⁺ / 7-ADD ⁺ ) The same proportion of double positive MLN T and B cells was identified in the Pro and Cont groups. Interestingly, it was observed that the proportion of double positive cells in the Pro group was lower than the Cont group, indicating that the treatment of probiotics has an anti-apoptotic effect on splenic T and B cells (FIGS. 3A and 3B). This experimental finding demonstrates that T and B cell low reactivity by probiotics is not induced by apoptosis. Immune hyporeactivity is mediated by immune-regulating cytokines such as IL-10 and TGF-β (Zeller et al. 1999; Li et al. 2006) or Tregs (Sakaguchi et al. 2001). However, the inventors have found that probiotics do not increase TGF-β levels and confirm that they reduce IL-10 levels. Foxp3 is a marker of Treg cells (Sakaguchi 2005) and because it directly inhibits effector cell functions such as T cells, B cells and other immune cells (Grossman et al. 2004; Lim et al. 2005; Sakaguchi 2005) The inventors focused their experiments on immune low reactivity mediated by regulatory T cells (Tregs). In the previous experimental results (FIG. 1), we found that T and B cell immune-low reactivity was expressed in the spleen as well as MLN, thereby analyzing the extent of Foxp3 mRNA expression in both tissues treated with probiotics (FIG. 4A). Interestingly, probiotics increased Foxp3 expression of MLN T cells at mRNA and protein levels (p <0.005) but did not have a pronounced effect on splenic CD4 ⁺ T cells (FIG. 4). We also tested whether Tregs (CD4 ⁺ Foxp3 ⁺ ) produced by probiotics are derived from CD4 ⁺ CD25 ⁺ or CD4 ⁺ CD25 ⁻ cells. Because CD4 ⁺ CD25 ⁺ is a marker for spontaneous Tregs (nTregs) of Foxp3 high expression (Stephens and Mason 2000; Jordan et al. 2001; Bluestone and Abbas 2003), we found that MLN CD4 ⁺ T of the Pro and Cont groups CD4 ⁺ CD25 ⁺ double positive populations were measured in cells, but no significant difference was found between the two groups of CD4 ⁺ CD25 ⁺ Trep populations (Pro: 5.29% vs Cont: 5.59%) (FIG. 5). Since CD4 ⁺ CD25 ^- Foxp3 ⁺ T cells also have Treg function (Bluestone and Abbas 2003), it was tested whether probiotics increased the number of CD4 ⁺ CD25 ^- Foxp3 ⁺ Tregs. Interestingly, the probiotics are in the CD4 ⁺ CD25 ⁺ T cells did not CD4 ⁺ CD25 ^- T cells increased in the population of Foxp3 ⁺ cells, up to five times (Fig. 4c). Although there was no difference between Foxp3 populations observed in CD4 ⁺ CD25 ⁺ T cells between groups, we compared the Treg effector function between the two populations. Since Tregs induce hypo-responsiveness in target T cells through cell-to-cell contact, inhibition of CD4 ⁺ CD25 ⁺ Tregs was compared from the Pro and Cont groups through inhibition studies. CD4 ⁺ CD25 ⁻ target T cells from normal mice were co-cultured with inhibitor CD4 ⁺ CD25 ⁺ T cells of Pro or Cont group MLN and stimulated with anti-CD3. Inhibition by CD4 ⁺ CD25 ⁺ T cells was analyzed by assessing T cell proliferation by varying the ratio of Treg: target cells. Proliferation levels of target cells alone (CD4 ⁺ CD25 ⁻ ) were considered 100%. As can be seen in Figure 4d, CD4 ⁺ CD25 ⁺ Tregs of the Pro group showed higher inhibitory activity than Tregs of the Cont group (p * <0.05). These experimental results show that the probiotics of the present invention not only stimulate the production of Foxp3 ⁺ Tregs from the pool of CD4 + CD25 ⁻ cells but also enhance their function as inhibitors of CD4 ⁺ CD25 ⁺ Tregs.

Probiotics TNBS The development of inflammatory bowel disease Relax

Probiotics of the present invention in animal models of inflammatory bowel disease (IBD) because probiotics administration in normal mice increases CD4 ⁺ Foxp3 ⁺ Treg populations and downregulates effector cytokines and proliferation, leading to CD4 ⁺ T cell hyporesponsiveness. We investigated whether can treat or relieve colitis. We also investigated whether the same dose of each individual strain contained in the probiotic mixture exhibits therapeutic efficacy. TNBS-induced colitis is a well-defined animal model for Th1-mediated IBD, similar to human Crohn's disease (Jurjus et al. 2004). Prior to inducing IBD, mice were subjected to the same colony forming unit (CFU), or PBS, of various probiotics, including five, four or three strain mixtures and a single strain (LA, LC, LR, BB and ST) daily in mice. Administration was performed for 20 days, followed by rectal injection of TNBS into Balb / c mice of each group to induce colitis (FIG. 6).

Efficacy on the development of colitis was assessed by viability (Figures 7b, 8a), body weight (Figures 7a, 8b), overall appearance of the colon (Figure 7c) and histology (Figure 7d). Interestingly, only probiotic mixtures comprising three, four and five strains significantly reduced mortality, single strains did not exhibit this efficacy, and preventive efficacy was consistent with mortality results (FIGS. 7A and 8B). . In addition, the inventors have found that a probiotic mixture comprising five strains shows the best colitis prevention efficacy. Probiotic mixtures comprising five strains greatly reduced inflammation of the colon (FIGS. 7C and 7D). Weight loss and mortality were in good agreement with the overall shape assessment of the intestine (FIG. 7C) and the colon tissue fragment assessment (FIG. 7D).

To further elucidate the prophylactic efficacy of the probiotic mixtures of the present invention against IBD, the expression levels of proinflammatory cytokines of CD4 ⁺ T cells and B220 ⁺ B cells in probiotic or PBS treated MLNs were examined after IBD induction. . Expression of proinflammatory cytokines such as IL-17, IFN-γ and TNF-α was significantly reduced in MLN CD4 ⁺ T cells by probiotic treatment (FIG. 7E). In addition, the expression of proinflammatory cytokines such as IL-10, IFN-γ and TNF-α in B220 ⁺ B cells was significantly reduced by probiotic treatment (FIG. 7F). These experimental results demonstrate that the probiotic mixtures of the present invention comprising five strains show the best prophylactic efficacy by greatly reducing proinflammatory cytokines in the IBD animal model.

Probiotics Rheumatism  arthritis( RA Therapeutic efficacy for

As can be seen in Figure 15a, oral administration of the probiotics of the present invention shows a superior RA therapeutic efficacy than the conventional therapeutic MTX. In addition, in histological observation (H & E staining) of the joint site (Fig. 15b), it can be seen that a large number of lymphocytes infiltrated into the tissue in the control CIA animal model. Lymphocytes are stained with hematoxylin and observed as blue spots, and surrounding joint tissues are stained with eosin and observed in red. On the other hand, lymphocyte infiltration is hardly seen in the group administered with the composition of the present invention and MTX-administered group. Therefore, it can be seen that the composition of the present invention inhibits the immune inflammatory response induced by lymphocytes very efficiently.

Finally, CD4 ⁺ T cells were isolated from the joints of the three groups for molecular mechanisms, and the amount of cytokines expressed therein was confirmed by quantitative real-time PCR (FIG. 15C). Oral administration of probiotics was found to inhibit the expression of inflammatory cytokines a little more than MTX, and it was confirmed that Foxp3, a regulatory T cell marker, also increased significantly by mechanisms such as IBD and AD.

Probiotics  The development of atopic dermatitis Relax

For IBD, a typical T cell mediated immune disease, it has been demonstrated that the probiotics of the present invention have very good prophylactic efficacy, and therefore, the present inventors further demonstrate that the probiotics of the present invention are directed against atopic dermatitis (AD), a B cell mediated immune disease. The degree of therapeutic efficacy was investigated. Two weeks prior to dosing, the murine AD model was constructed using house dust mite extracts and DNCB, followed by oral administration of probiotics (five strain mixtures; 1 × 10 ⁹ CFU) or PBS to the AD mouse model for two weeks. Induction of AD continued. During the induction process, ear thickness and clinical scores were measured twice a week 24 hours after AD induction (FIGS. 9C and 9D).

During AD construction, ear thickness was almost the same in the Pro and Cont groups, but a significant difference was observed when probiotics were administered for 2 weeks (FIG. 9C). In the Pro group, the increase in ear thickness was significantly decreased (p <0.02) after 7 days of treatment, and the thickness of the ear was gradually decreased by continuous administration of probiotics for 4 weeks. The Cont group continued to increase ear thickness (FIG. 9C). Consistent with the experimental results for ear thickness, the clinical score was also significantly reduced in the Pro group (FIG. 9D). We examined the therapeutic efficacy of each single strain or mixture of three and four strains included in the probiotic mixture (FIG. 10). Contrary to the results of the IBD experiment, some probiotic strains (LA, LC, and LR) showed some therapeutic efficacy by reducing the increase in ear thickness and decreasing the clinical score, and BB and ST showed poor therapeutic efficacy. (FIG. 10A, 10B). Probiotic mixtures containing five strains showed the best therapeutic efficacy.

To elucidate the mechanism of treatment of probiotics in detail, after finally inducing AD, mice were sacrificed, ears were removed from the Cont and Pro groups and histological analysis was performed (FIG. 9E). Compared to the w / o induction group, ear edema and tissue destruction were easily observed in the Cont group, but this phenomenon was significantly reduced in the probiotic group (FIG. 9E). The same pattern was also shown for the invaded cell number (FIG. 9F). Compared to w / o induced ears, Cont ears showed 10-fold invasive cell counts (w / o induced; 238 ± 37 cells / mm ² vs Cont; 2630 ± 139 cells / mm ² , p <0.001) This severe invasion was significantly reduced in the Pro ears (1423 ± 115 cells / mm ² , p <0.005) (FIG. 9F). In addition, microscopic observations showed significantly increased invasive granulocytes, particularly eosinophils, in the Cont ear section and significantly reduced eosinophil count in the Pro ear section.

Since IgE overproduction is one of the characteristics of AD (Gao et al. 2004), we investigated whether probiotic treatment can regulate IgE levels. Serum was collected on days 14 and 31 from both groups to analyze total serum IgG and IgE levels in blood samples (FIG. 9G). Over time, total IgG increased slightly in both groups, but the IgE concentration (41 ± 29 ng / ml) in serum at Day 31 of Pro was nearly three times that of Cont (149 ± 51 ng / ml). Reduced values were shown.

In addition, allergen-specific increases in IgG and IgE concentrations were investigated, since down-regulation of allergen-specific IgE responses is important in determining the efficacy of a medicament or therapy in the treatment of allergic diseases (Till et al. 2004; Larche et al. 2006). Tick extracts as allergens were encoded on plates followed by ELISA (FIG. 9H). Tick specific serum IgE concentrations in the Pro group were reduced by up to 2 fold compared to the Cont group (P <0.05) (FIG. 9H). In addition, serum IgE concentrations were compared in single, three, four and five strain treatment groups (FIG. 10C). Serum IgE concentrations were slightly decreased in the LA and LC treated groups and significantly decreased in the three, four and five strain treated groups. The greatest reduction was observed in the 5 strain treated groups.

Finally, lymph nodes of the Cont and Pro groups (DLN; cervical lymph node, secondary lymph node and brachial lymph node) CD4 ⁺ T cells were isolated, and in AD, IL-2, IL-4, IL-5, IL-10, IL-13 And expression patterns of pathogenic cytokines such as IFN-γ (FIG. 9I). Probiotic administration significantly down-regulated pathogenic cytokine expression such as IL-4, IL-5, IL-10 and IL-13 in DLN CD4 ⁺ T cells (FIG. 9I). In conclusion, the experimental data show that the five-probiotic mixtures exhibit the most potent therapeutic efficacy by reducing serum total IgE and tick specific IgE concentrations and reducing the expression of pathogenic cytokines.

Probiotics  Dosing is in a disease state MLN Foxp3 ⁺ CD4 ⁺ Adjustability  Specifically increases T cells

Since an increase in Foxp3 ⁺ CD4 ⁺ regulatory T cell population was observed in MLN of probiotic administered w / o diseased mice, the present inventors have found that the therapeutic efficacy of probiotics has been shown to affect Foxp3 ⁺ CD4 ⁺ regulatory T cell population in inflammatory disease states. It was investigated whether it was associated with an increase (FIG. 6). To demonstrate this, after administration of PBS or probiotics in IBD, DLN and MLN CD4 ⁺ T cells were isolated and examined for Foxp3 ⁺ population by FACS (FIG. 11A). For DLN CD4 ⁺ T cells, there was little difference in Foxp3 ⁺ population between the Cont and Pro groups (Cont; 6.1% vs Pro; 5.9%), but for MLN CD4 ⁺ T there was a significant difference (Cont; 6.4%). vs Pro; 10.2%) (FIG. 11A). In addition, the Pro group showed increased Foxp3 ⁺ population for MLN CD4 ⁺ T cells in AD state, but not for DLN CD4 ⁺ T cells (FIG. 11B).

The effect of probiotic administration on the production of CD4 ⁺ Foxp3 ⁺ Tregs was analyzed. Recent studies show that APC subsets are associated with the production of regulatory T cells (Kuwana 2002; Mellor and Munn 2004; Yamazaki et al. 2006; Mahnke et al. 2007). Thus, we investigated whether skewing of APCs for tolerance is involved in the generation of Tregs. APCs (non-CD4 ⁺ / CD8 ⁺ ) were prepared in the colon and ear of Pro and Cont diseased mice and expression of co-stimulatory molecules and indole amine 2,3-dioxygenase (IDO) was measured. APCs expressing IDO regulate T cell activity and induce T cell tolerance (Mellor and Munn 2004). There was little difference in the levels of B7.1, B7.2, B7DC, or ICOSL. Interestingly, ex vivo IDO expression was increased (p <0.0003) in Pro group APCs (FIG. 12). In conclusion, the data show that administration of probiotics produces MLN Foxp3 ⁺ regulatory T cells even in disease states and that IDO-expressing dendritic cells are produced by Tregs.

Upregulated at the inflammation site CD4 ⁺ Foxp3 ⁺  T cells lead to suppression of inflammatory diseases

Because Tregs can be recirculated through lymph node tissue and enter the inflammatory site (Iellem et al. 2001; Szanya et al. 2002; Huehn and Hamann 2005a; Maul et al. 2005), we believe that the therapeutic efficacy of probiotics is an inflammation site. Whether or not it is associated with the enrichment of CD4 ⁺ Foxp3 ⁺ Treg cells in (colon and ear) was investigated. In the IBD system, lamina propria (LP) CD4 ⁺ T cells were isolated from the colon and Foxp3 protein concentration was measured by FACS. CD4 ⁺ Foxp3 ⁺ T cells were compared in healthy mice, control (Cont) mice and probiotic-treated (Pro) colitis mice. LP CD4 ⁺ Foxp3 ⁺ T cells showed lower levels in colitis mice (14%) than healthy mice (22%) (FIG. 13A). Surprisingly, the Pro group (31%) showed at least two-fold CD4 ⁺ Foxp3 ⁺ T cell levels compared to the Cont group (14%). In addition, enrichment of Foxp3 ⁺ CD4 ⁺ regulatory T cells in inflammatory colon of the Pro group was also confirmed in immunohistochemistry (IHC) (FIG. 13B). According to the IHC experiment, IBD induction slightly reduced Foxp3 ⁺ cell number at the colitis site compared to w / o induction conditions, but administration of probiotics significantly increased Foxp3 ⁺ cell number at the colitis site (FIG. 13B).

Consistent with the results of the IBD model, ear CD4 ⁺ T cells were also isolated from w / o induction, Cont and Pro groups in the AD system, and then analyzed for Foxp3 ⁺ population via FACs. 13C and 13D. Similar to IBD, AD induction resulted in a slight decrease in Foxp3 ⁺ CD4 ⁺ regulatory T cells in ear tissue (w / o induction; 7.4% vs Cont; 4.6%) (FIG. 13C). Interestingly, the ear CD4 ⁺ T cells (33%) of the Pro group showed 8 fold increased levels compared to the Cont group (4.6%) and 5 fold increased compared to the w / o induction group (7.4%). The results of this experiment were also confirmed by IHC (FIG. 13D). In conclusion, the experimental data demonstrate that the enrichment of Foxp3 ⁺ CD4 ⁺ regulatory T cells at the inflammation site (colon and ear) is the main mechanism of inhibition of inflammation by probiotics.

Probiotics  By administration Adjustability  T cell On trafficking  For chemical inducers ( chemoattractants ) In increasing and inflamed position Treg In vivo move of

It is well known that various regulatory cells settle in inflammatory sites and tune the imbalance of immune-always (Brandtzaeg and Johansen 2005; Huehn and Hamann 2005b; Kim 2006). The concept of homing and inhibition (Huehn and Hamann 2005b) is well studied in the field of regulatory T cells (Brandtzaeg and Johansen 2005; Huehn and Hamann 2005b; Kim 2006). In addition, our previous experiments clearly show the in vivo production of MLN regulatory T cells in normal and diseased states, and detected Foxp3 ⁺ regulatory T cells largely enriched in the inflammatory colon and ear region. Thus, the present inventors hypothesized that in vivo generated regulatory T cells migrate to the site of inflammation to maintain immunosupplement. To demonstrate this hypothesis, the change in chemokine expression at the inflammation site was examined using the AD model, which showed that the increase in regulatory T cells at the inflammation site was noticed by the treatment of probiotics in the AD model compared to the IBD model. Because.

Ear total cells were isolated from normal, Cont and Pro groups and then chemokines known as chemoinducers for trafficking regulatory T cells (Jonuleit et al. 2000; Mantovani et al. 2000; Bystry et al. 2001; Lanzavecchia and Sallusto 2001; Mellman and Steinman 2001; Columba-Cabezas et al. 2002; Penna et al. 2002; Szanya et al. 2002). Interestingly, it was found that the expression of CCL1 and CCL22 was significantly increased in mixed ear cells of the Pro group (FIG. 14A). In addition, it was found that the expression of receptors CCR4 and CCR8 in the ear CD4 ⁺ T cells of the Pro group is increased (Fig. 14b). According to the experimental data, it can be seen that regulatory T cells are produced in MLN, migrate to the ear site, and maintain immunosuppressive properties. However, the above experimental results alone do not fully support our hypothesis about the migration of MLN CD4 ⁺ Foxp3 ⁺ T cells to inflammatory sites, because CCL1, CCL22, CCR4 and CCR8 are skin-homing, dermal leucosite antigens. ^{+ Is} often expressed in T cells and Th2 cells (Bonecchi et al. 1998; Campbell et al. 1999; Margherita Mariani 2004).

Therefore, in order to solve the above problems, the in vivo migration of regulatory T cells to the inflammatory position by probiotics treatment was investigated. After 30 days of oral administration of PBS or probiotics, MLN CD4 ⁺ T cells were isolated, labeled with CFSE, and then transferred to AD induced mice by intraperitoneal injection (FIG. 14C). After 5 adaptive transitions, CFSE ⁺ Foxp3 ⁺ population at the ear site was examined by IHC. For the negative control, the same volume of PBS was transferred. Interestingly, several CFSE ⁺ Foxp3 ⁺ cells were detected in Cont MLN CD4 ⁺ T cell metastatic ears, and the number of double positive cells was significantly increased in Pro MLN CD4 ⁺ T cell metastatic ears (FIG. 14C). In conclusion, according to the experimental results, it can be seen that oral administration of probiotics produces regulatory T cells in MLN and in vivo generated regulatory T cells migrate to the site of inflammation and contribute to maintaining immunosuppressive properties.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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1A-1C show an increase in CD4 ⁺ Foxp3 ⁺ regulatory T cell population in mesenteric lymph nodes by oral administration of probiotics. LA, LC, LR, BB and ST is Lactobacillus liquid filler's attempt (Lactobacillus each acidophilus ), Lactobacillus casei ), Lactobacillus reuteri ), Bifidobacterium bifidium ) and Streptococcus thermophilus .

2A-2D are experimental data showing inhibition of cytokine expression and induction of T / B cell low reactivity by a probiotic mixture comprising five strains. Figure 2a-2b is a result of examining the function of effector T cells by measuring the cytokine expression and proliferation rate in the spleen (SP) and mesenteric lymph nodes (MLN). Figure 2c-2d is a result of examining the function of effector B cells by measuring the cytokine expression and proliferation rate in the spleen (SP) and mesenteric lymph nodes (MLN). Data was collected from 10 mice per group. Error bars represent SD. (**) indicates p <0.005 and (***) indicates p <0.001. Data is representative of three single experiments.

3A-3B show that probiotics do not induce apoptosis. Spleen or mesenteric lymph node CD4 ⁺ T cells were isolated from healthy Balb / c mice administered for 20 days with probiotics (Pro) or PBS (Cont). Apoptosis population (Annexin V ⁺ and 7-ADD ⁺ ) was measured by FACS. Data is representative of three single experiments.

4A-4D show that probiotics comprising five strains induce the formation of Foxp3 ⁺ CD4 ⁺ T cells from CD4 ⁺ CD25 ⁻ T cells.

5 shows the results of CD4 ⁺ CD25 ⁺ double positive population analysis of MLN CD4 ⁺ T cells in control and probiotic-treated groups.

6 is a schematic diagram showing the experimental method of the TNBS-induced colitis animal model experiment.

7A-7F are experimental results showing the efficacy of the prophylactic / treatment of TNBS-induced colitis by a probiotic mixture comprising five strains.

8A-8B are experimental results showing the prevention / treatment efficacy of TNBS-induced colitis by each strain and probiotic mixture included in the probiotic mixture.

9A-9I are experimental results showing the therapeutic efficacy of atopic dermatitis with a probiotic mixture comprising five strains. w / o induction, Cont and Pro, respectively, represent atopic dermatitis non-induced groups, PBS treated atopic dermatitis animals and probiotic mixture treated atopic dermatitis animals.

10A-10C are experimental results showing the efficacy of treatment of atopic dermatitis by probiotics with the changed number and type of strains.

11A-11B are experimental results showing that administration of a probiotic mixture comprising five strains specifically increases MLN Foxp3 ⁺ CD4 ⁺ regulatory T cells in a disease state. 11A is an IBD case and FIG. 11B is an AD case.

12 shows increased expression of indole amine 2,3-dioxygenase (IDO) in AD induced mice administered with a probiotic mixture comprising five strains. C is a control group and P is a probiotic mixture administration group containing five strains. HPRT: Hypoxanthine-Guanine Phosphoribosyl Transferase; IDO: indole amine 2,3-dioxygenase; B7.1: CD80; B7.2: CD86; ICOSL: inducible costimulator ligand; PD1L: programmed death 1.

13A-13D are experimental results showing that CD4 ⁺ Foxp3 ⁺ T cells upregulated at inflammatory sites by probiotic mixtures result in inhibition of inflammatory diseases.

14A-14C are experimental results of in vivo migration of Tregs at increased inflammation and chemoattractants for the trafficking of regulatory T cells by probiotic mixture administration.

15A-15C are experimental data showing efficacy of arthritis treatment in rheumatoid arthritis-induced rats by administration of a probiotic mixture. MTX represents metatrexate and Probiotics represents a probiotic mixture comprising five strains, respectively.

Claims (17)

Lactobacillus reuteri , Lactobacillus acidophilus , Lactobacillus casei , Bifidobacterium bifidium and Streptococcus thermophyllus ( Streptococus thermous ) Food composition for improving the inflammatory disease comprising a mixed strain containing as an active ingredient. delete delete delete delete delete delete delete Lactobacillus reuteri , Lactobacillus acidophilus , Lactobacillus casei , Bifidobacterium bifidium and Streptococcus thermophyllus ( Streptococus thermous ) A pharmaceutical composition for preventing or treating inflammatory diseases, comprising a mixed strain comprising as an active ingredient. Lactobacillus reuteri , Lactobacillus acidophilus , Lactobacillus casei , Bifidobacterium bifidium and Streptococcus thermophyllus ( Streptococus thermous ) Food composition for improving the transplant rejection or autoimmune disease comprising a mixed strain containing as an active ingredient. delete delete delete delete delete delete Lactobacillus reuteri , Lactobacillus acidophilus , Lactobacillus casei , Bifidobacterium bifidium and Streptococcus thermophyllus ( Streptococus thermous ) A pharmaceutical composition comprising a mixed strain comprising as an active ingredient for preventing or treating transplant rejection or autoimmune disease.

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