WO2019203625A1 - Novel bacteroides vulgatus strain and composition, for preventing, relieving or treating immune and metabolic diseases, having same as active ingredient - Google Patents

Novel bacteroides vulgatus strain and composition, for preventing, relieving or treating immune and metabolic diseases, having same as active ingredient Download PDF

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WO2019203625A1
WO2019203625A1 PCT/KR2019/004790 KR2019004790W WO2019203625A1 WO 2019203625 A1 WO2019203625 A1 WO 2019203625A1 KR 2019004790 W KR2019004790 W KR 2019004790W WO 2019203625 A1 WO2019203625 A1 WO 2019203625A1
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bvul
obesity
bacteroides vulgatus
snug40005
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WO2019203625A8 (en
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고광표
유현주
윤성현
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서울대학교산학협력단
주식회사 고바이오랩
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to the bacteroid Bacteroides vulgatus SNUG40005 strain (Accession No. KCTC 13276BP) and its use having the effect of preventing, treating or ameliorating metabolic diseases.
  • Gut dysbiosis interferes with immune homeostasis and promotes the development of various inflammatory and metabolic diseases. Changes in obesity-associated microorganisms lead to increased intestinal permeability and circulating endotoxin levels, leading to low grade of systemic inflammation. However, it is still unclear how the microbial community members interact with each other and with the host immune system to prevent or protect obesity.
  • Intestinal microorganisms modified in obesity have been reported to have an important link between metabolism and immune dysfunction.
  • insulin resistance is induced by serum toxins absorbed in the 'leaky gut' and increases systemic inflammation.
  • Certain bacterial texas eg, Akkermansia muciniphila
  • no bacterial taxa associated with adaptive immune responses in obesity and weight control have been reported.
  • Bacteroides vulgatus (Bvul) as the major symbiosis associated with reduced clinical markers and immune correlates of altered obesity. Mice receiving Bvul have been shown to repeatedly show the benefits to humans by reducing dietary induced weight gain, glucose intolerance and adipose tissue inflammation. These benefits involve dramatic recovery of structural and metabolite profiles of gut microorganisms as well as regulatory T cell and type 2 innate lymphoid cell (ILC) immunity. According to the relationship between Bvul and IL-21 in humans, Bvul treated mice showed a decrease in IL-21 receptor expression and IL-21 receptor expression ILC in mesenteric lymph nodes. Bvul was also essential for maintaining the anti-obese bacteria Akkermansia muciniphila . These results indicate the ability of symbiotic Bacteroides species to prevent obesity and immune metabolic dysfunction by forming and maintaining healthy microbial communities.
  • Bvul Bacteroides vulgatus
  • Mice administered Bvul have repeatedly demonstrated the benefits to humans by reducing dietary induced weight gain, glucose intolerance and adipose tissue inflammation. These benefits have accompanied dramatic recovery of regulatory T cell and type 2 ILC immunity, as well as the structure and metabolite profiles of the intestinal microorganisms. According to the relationship between Bvul and IL-21 in humans, Bvul treated mice showed a decrease in IL-21 receptor expression and IL-21 receptor expression ILC in mesenteric lymph nodes.
  • Bvul was also essential for maintaining the anti-obese bacteria Akkermansia muciniphila . These results confirmed the ability of symbiotic bacteroids ( Bacteroides ) species to prevent obesity and immune metabolic dysfunction by forming and maintaining a healthy microbial community, and completed the present invention.
  • symbiotic bacteroids Bacteroides
  • Bacterial species are early colonizing bacteria in the mammalian gut and are known to form and regulate the host's immune system and metabolic activity.
  • B. ragilis capsular polysaccharides regulate regulatory T cells and prevent inflammatory diseases including inflammatory bowel disease, autoimmune diseases and CNS demyelinating diseases.
  • Bacteroid species also regulate host physiology through metabolite production. For example, genome analysis of B. hetaiotaomicron reveals the potential for host niche and metabolism to utilize various dietary nutrients and produce bacterial metabolites that the host can reuse.
  • the effects of bacteroid species also include the restoration of microbial intestinal imbalances during the pathogenesis of obesity.
  • immunological and metabolic changes induced by Bvul as intestinal commensals have not yet been reported, particularly in relation to obesity.
  • the present invention provides a bacteroid Bacteroides vulgatus SNUG40005 strain.
  • the strain may reduce diet-induced weight gain, glucose intolerance or adipose tissue inflammation.
  • the strain can reduce IL-12 receptor expression and ILC expressing IL-12 receptor.
  • the strain can maintain the anti-obesity bacterium Akkermansia muciniphila strain.
  • the present invention provides a pharmaceutical composition for treating or preventing metabolic diseases using the bacteroid Bacteroides vulgatus SNUG40005 strain as an active ingredient.
  • the metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
  • the present invention provides a health functional food for improving metabolic disease comprising the bacteroid Bacteroides vulgatus SNUG40005 strain as an active ingredient.
  • the metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
  • the present invention also provides a method of preventing, ameliorating or treating metabolic diseases by administering to a subject in need thereof an effective amount of a Bacteroides vulgatus SNUG40005 strain.
  • the present invention also provides the use of a bacteroid Bacteroides vulgatus SNUG40005 strain for the preparation of a composition for preventing, ameliorating or treating a metabolic disease.
  • the metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
  • the subject may mean an animal that is expected to exert the effect according to the bacteroid Bacteroides vulgatus SNUG40005 strain by administering a composition comprising the strain or strain according to the present invention, the animal is a dog, a monkey , Goat, pig, mouse or human.
  • the prophylaxis may mean any action of inhibiting or delaying the progression of metabolic disease by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.
  • the treatment or improvement may refer to any action in which a symptom of a metabolic disease is improved or advantageously changed by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.
  • the administration may mean providing a subject with a composition comprising any of the strains or strains according to the present invention as an active ingredient in any suitable manner.
  • the novel strain Bacteroides vulgatus SNUG40005 of the present invention is essential for maintaining the anti-obese bacterium Akkermansia muciniphila , and is a symbiotic bacteroid species that prevents obesity and immune metabolic dysfunction by forming and maintaining healthy microbial communities. Indicates the ability.
  • the Bacteroides vulgatus SNUG40005 strain can reduce IL-12 receptor expression and ILC expressing IL-12 receptors, as well as regulatory T cells and type 2 ILC immunity, as well as the structure and metabolism of intestinal microorganisms. Since it involves a rapid recovery of the product profile, it can be used as a composition for the prevention, treatment or improvement of metabolic diseases including obesity.
  • Figure 1 shows the results of experiments on the relationship between obesity status and immune profile and Bvul abundance in mismatched MZ twins.
  • (a) is an experimental summary of the study, and
  • (b) and (c) are results of differences in the amount of major genera and serum IL-12 levels in twins with inconsistent waist circumference.
  • (d) and (e) are for significant changes in Bvul analyzed by multivariate association with a linear model (MaAsLin) describing waist circumference and serum IL-21 levels.
  • Waist_low and Waist_high mean that the difference in waist circumference of the mismatched twins is less than 4 cm and more than 4 cm, respectively.
  • P means greater than 0.28 pg / ml
  • N means less than 0.28 pg / ml in serum.
  • (a) is a hierarchical clustering result of the correlation between serum cytokine levels and intestinal bacterial phyla in Korean discordant twins. Heatmaps were created based on correlation coefficients. The tree is illustrated using the Euclidean method and split into two arms for the cytokine profile.
  • (b) to (d) are the results of PICRUSt analysis of potential functional properties of the intestinal microorganisms.
  • FIG. 3 is an experimental result of the Bvul SNUG40005 strain that reduces obesity and glucose tolerance in HFD-induced obese mice.
  • (a) is a diagram of a phenotype of a mouse.
  • (b) is a diagram of a weight measurement value for each group.
  • (c) is a diagram of fasting blood glucose level measurement values by group.
  • (d) is a diagram of the results of the intraperitoneal glucose tolerance test (IPGTT).
  • IPGTT intraperitoneal glucose tolerance test
  • e is a plot of IPGTT AUC values of C57Bl6 female mice treated with Bvul for 16 weeks.
  • (f) is a diagram of the results for the weight of gonad adipose tissue by group.
  • (g) is a diagram showing the results of measuring the size of adipocytes in each group.
  • (h) is the result of measuring changes in gene expression for low grade inflammation in gonad adipose tissue for 18 weeks after administration of Bvul. Measurements are expressed as mean ⁇ SD, and measurements with different superscripts appear to be quite different according to post hoc ANOVA one-way statistical analysis. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, and **** P ⁇ 0.0001.
  • FIG 4 is a diagram of the inflammatory control effect (IL-10 / THF- ⁇ ratio in differentiated THP-1 macrophages) of bacteroid strains isolated from mouse and human fecal samples. IL-10 / THF- ⁇ ratios were calculated after treatment with 10 different strains of bacteroid species, P. goldsteinii , and Akkermansia muciniphila .
  • (b) is a scanning microscope diagram of Bacteroides vulgatus SNUG40005.
  • Bvul SNUG40005 recovers A. muciniphila depleted in obesity and regulates intestinal microorganisms and metabolites
  • (b) are heatmap results of 25 major texas based on different dietary groups in the cecum.
  • (c) is the classification result of HFD and Bvul treatment by bacterial abundance. For the top 15 bacteria in each group.
  • (d) is for the sPLS-DA plot based on the quantitative value of 80 cecal metabolites.
  • (e) shows 10 representative metabolites most affected by HFD and Bvul treatment.
  • (f) is a PCoA result based on the top 10 bacteria with Bray-Curtis dissimilarity and major cecal metabolites. Arrows indicate strong significant correlations, points represent cecal microorganisms at the genus level, and color gradients indicate the weight of the mouse.
  • FIG. 6 (a) is the PCoA results using unweighted UniFrac metrics
  • (b) is a result of analysis of abundance of microbial community based on administration time (fecal microbeads) (Pre: 1 week before Bvul administration; Day 0: Start of dosing after 1 week of antibiotic treatment; Day 5, Wk 9, Wk 18: 5 days, 9 weeks, and 18 weeks after Bvul administration).
  • (c) is the result of the shift of representative microbial taxa (texa).
  • (d) is the result of classification of HFD feeding and Bvul treatment by microbial abundance.
  • (e) is the result of functional classification of HFD feeding and Bvul treatment by KEGG pathway analysis using PICRUSt.
  • (f) shows the abundance measurement results for cholate in each group.
  • FIG. 7 shows Bvul SNUG40005 inducing regulatory T cell (Treg) and IL-21-independent ILC2 responses in HFD-induced obesity, (a) expression of Il21 and Il21r in MLN, (b ) Is for the expression results of Foxp3 and Il10 in MLN. (c) and (d) shows flow cytometric analysis of CD4 + Foxp3 + Treg cells in MLN, (e) and (f) shows flow cytometric analysis of whole ST2 + ILC in MLN, (g) shows IL-21 + ILC in MLN , Flow cytometric analysis of IL-21 + ST2 ( ⁇ ) ILC, and IL-21 + ST2 (+) ILC.
  • intestinal microbial composition and serum cytokine profiles using 16s rRNA gene profiling and multiplex cytokine analysis on fecal and blood samples of 30 twins The correlation with Th1, Th2, Th17, Tregs, and 20 cytokines associated with inflammation was examined (see FIG. 1A).
  • Hierarchical cluster analysis revealed that certain microbial populations, including bacteroids and Oscillospira , negatively correlate with levels of Th17-related inflammatory cytokines such as IL-17A, IL-21, CCL20, IL-27, and TNF- ⁇ . lost.
  • Analysis of body mass index (BMI) as a quantitative measure of obesity revealed intestinal microbial compositions distinguished from identical discrepancies to obesity.
  • serum cytokine profiles did not differ significantly between twin samples with inconsistent BMI (FIG. 2A).
  • the abundance of Bifidobacterium spp. was lower in twins (4 cm or more) than twins (Fig. 1b).
  • Bvul type ATCC 8482 A total of 182 bacteroid strains isolated from humans and mice, including Bvul type ATCC 8482, were screened for in vitro immunomodulatory activity. As a marker, obesity is well known to cause metabolic inflammation, so pro-inflammatory and anti-inflammatory cytokine production of THP-1 cell derived macrophages has been measured.
  • Bvul SNUG40005 (hereinafter referred to as Bvul) was selected based on immunogenicity leading to high levels of IL-10 / TNF- ⁇ ratio (FIG. 3). This strain was selected for administration to test mice daily for 18 weeks (10 9 cfu / mouse).
  • mice Conventional colonization studies using gnotobiotic mice have reported that the same species of bacteroid species cannot replace already clustered strains due to shared niche in the gut.
  • the inventors first administered with an antimicrobial agent (ampicillin (A), vancomycin (V), neomycin (N), metronidazole (M), streptomycin, penicillin, kanamycin, and AVNM) for 1 week. Based on the results, the mice were pretreated with ampicillin for 1 week to reduce the existing Bvul (Bvul SNUG40005) strain, and promoted the colonization of Bvul isolated from humans in the mouse intestine.
  • an antimicrobial agent ampicillin (A), vancomycin (V), neomycin (N), metronidazole (M), streptomycin, penicillin, kanamycin, and AVNM
  • mice fed HFD significantly changed the microbial community
  • FIG. 5A The microbial structures of the NDB and HFDB groups showed similarities for 18 weeks after Bvul administration (FIG. 5A).
  • Time course analysis of the microbial community (FIG. 6A) and its ⁇ -diversity (FIG. 5B) showed that the ampicillin pretreatment effect prior to the Bvul community was partially overcome after 18 weeks.
  • the composition of the major bacterial taxa in mice fed HFD was significantly changed by Bvul treatment. The abundance of A.
  • muciniphila was significantly reduced by HFD but rapidly recovered by Bvul treatment (FIGS. 5B and 6C).
  • A. muciniphila is a mucus-resident bacterium that affects metabolic profiles and glucose homeostasis by improving intestinal barrier dysfunction and inflammation in metabolic diseases.
  • Oscillospira showed the opposite pattern to A. muciniphila , increased in the HFD group, decreased rapidly in the HFDB group, and increased in the NDB group compared to the NB group. Streptococcus and Eubacterium also showed the opposite pattern. Streptococcus was rarely found in the intestines of mice, but Bvul treatment increased the amount.
  • Eubacterium was abundant in mouse viscera, but HFD and Bvul treatments reduced the amount. Interestingly, the abundance of OTUs representing the administered species did not show a significant difference between the groups (FIG. 6C) and explained the strain specific effects on Bvul treatment and microbeads on obesity. Natural mouse-community Bvul strains were ineffective during the course of obesity. In addition, the amount of A. muciniphila was reduced by Bvul treatment in non-obese mice fed low-fat diet. As a result of functional microbiome analysis, microbial metabolic changes were most evident in the NDB group than in the HFDB group (FIG. 6E). This result suggests that the remodeling of Bvul-induced microbial structures depends on the host's obesity status and leads to unique microbial community profiles.
  • BCAAs such as Leucine and Isoleucine (FIG. 5E) caused by Bvul and A. muciniphila may explain the reduction of inflammation in adipose tissue of the HFDB group.
  • serum BCAA levels were increased in the obese group, and BCAA may be a biomarker linking metabolites and intestinal microorganisms.
  • FIG. 7B Bvul treatment in HFD feed mice induced a significant increase in both Foxp3 and Il10 expression in colon and MLN, suggesting that Bvul treatment changed the composition of immune cells.
  • Tregs and ILCs were analyzed in MLN to analyze immune cell profiles. Consistent with the increased expression of Foxp3 and Il10 , Tregs increased in MLN after Bvul treatment (FIG. 7C and FIG. 7D).
  • the ILC composition was affected by Bvul (FIGS. 7E and 7F).
  • Type 2 ILC (ILC2) expressing IL-33 receptor (ST2) was reduced by HFD feeding, but Bvul treatment restored the ILC2 population.
  • ILC exists in a tissue dependent manner and is regulated by symbiotic bacteria. In particular, adipose tissue contains ILC2, which is associated with insulin resistance and the obesity phenotype.
  • Faecalibacterium prausnitzii induces Treg accumulation in the intestine and protects against autoimmune diseases.
  • Some bacteroid species eg B. fragilis
  • Bacterial strains that induce Treg and ILC responses in obesity-induced modified immune systems and reduce inflammatory responses in adipose tissue have not been reported.
  • CD4 + T cell responses induced by other bacteroid species exhibit unique patterns depending on the host's physiological and immunological conditions.
  • IL-21 + Tregs and ILCs were analyzed to address IL-21 effects on ILC2 and Tregs cells.
  • Bvul treatment did not increase IL-21 receptor-expressing ILC in MLN.
  • HFD increased IL-21 receptor + ILC but ILC2 did not respond to IL-21 (FIG. 7G).
  • IL-21 receptor expression for ST2 (-) ILC was reduced by Bvul treatment.
  • the maturation and development of ILC is regulated by IL-1 ⁇ , IL-12, IL-18, IL-23, IL-25 and IL-33.
  • uniscript based transcriptome analysis showed that type 1 ILC was IL-21 reactive.
  • the ST2 (-) ILC responsible for IL-21 signaling may be ILC1.
  • Bvul has a negative correlation with obesity and serum IL-21 levels.
  • Bvul induces Treg and ILC responses in the intestinal and lymph nodes of obese mice, resulting in a novel immunomodulatory effect on obesity.
  • Bvul reconstructs intestinal microorganisms and microbial metabolism in a way unique to obese and obese people. A more comprehensive understanding of the interactions between Bvul and A. muciniphila observed in this study may lead to a better understanding of complex host-microbial interactions and the development of microbial therapeutics.
  • DNA in human and mouse feces was extracted using MoBio Power Soil DNA Isolation Kit (MoBio, Solana Beach, Calif., USA) and Qiagen Faststool DNA Extraction Kit (Qiagen, Valencia, Calif., USA) according to the manufacturer's protocol.
  • the nucleic acid solution was stored at -70 ° C until use.
  • the 27-F / 534R primer set was used to amplify the V1-V3 region of the 16S rRNA gene and pyrosequence on the 454 Life Sciences FLX Titanium platform (Roche, Indianapolis, IN, USA).
  • V4 region of the 16S rRNA gene was amplified using Illumina adapted universal primer set 515F / 806R and purified using MoBio UltraClean PCR Clean-Up Kit (MO BIO Laboratories, Carlsbad, Calif., USA). . Quantities of PCR amplification results were double checked using a KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, Mass., USA) followed by Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, Calif., USA). Samples were pooled and sequenced on a MiSeq platform using a 2 ⁇ 300 bp reagent kit (Illumina, San Diego, Calif., USA).
  • OTU tables generated through the pipeline are further filtered to include OTUs: (i) found in at least two samples. (ii) 100 reads or more, and (iii) classified as prokaryotic.
  • OTU was selected using a closed-referenced OUT picking to the gg_13_5 Greengenes database32 via the QIIME pipeline. Representative sequences were sorted using PyNAST34 and taxonomy assigned using the Ribosomal Database Project Classifier. The chimeric sequence was verified using the ChimeraSlayer algorithm and filtered before generating the OTU table.
  • Serum cytokine levels (TNF- ⁇ , TNF- ⁇ , IL-6, IL-1 ⁇ , CCL20 / MIP3a, IFN- ⁇ , IL-2, IL-12 (p70), IL-4, IL-5, IL- 13, IL-17A, IL-21, IL-22, IL-23, IL-10, IL-27, IL-28a, IL-31, and IL-33), waist circumference MaAsLin (twins as random variables, Host factor as covariate). The calculation of microbial abundance and ordination analysis was performed using the R package phyloseq36.
  • Population variation was analyzed by principal coordinate analysis (PCoA) using unweighted UniFrac distance for time series analysis in mouse feces and Bray-Curtis mismatch for clusters in caecum samples.
  • PCoA principal coordinate analysis
  • PERMANOVA used the 'adonis' feature of the vegan package to determine the difference between ND vs NDB and HFD vs HFDB.
  • Random forest classification was performed with 10-fold cross-validation using the R package random Forest38 to identify the bacteria that caused the changes observed between diets. Human data were modified and adjusted for age and twins before comparison. The magnitude of the effect on group differences is determined by dividing the Z score by the square root of N (observation number).
  • a total of 182 bacteroid strains were isolated from fresh feces of healthy Korean volunteers (30) between 19 and 40 years of age. The type of strain obtained from ATCC was used to compare the immunomodulatory activity as a control for the isolated strain. All 182 bacteroid species strains, mainly strains consisting of Bvul strains, were incubated anaerobicly with chocolate agar for 24 hours at 37 ° C. Bacterial cells were collected and centrifuged at 13,000 ⁇ g for 5 minutes to treat THP-1 cells.
  • Human monocyte cell line THP-1 was obtained from KCTC (Daejeon, Korea) and differentiated into macrophages by treatment with 10 ng / ml PMA for 48 hours at 37 ° C. in medium without serum under 5% CO 2 atmosphere. Bacterial cell pellets were added to differentiated THP-1 cells for 24 hours and cellless supernatants were collected and stored at ⁇ 80 ° C. prior to analysis. IL-10 and TNF- ⁇ levels were analyzed with an ELISA kit (BD Biosciences).
  • Intake and body weight were measured once a week. Stool samples were collected at five time points (before antibiotic treatment, before Bvul administration, 5 days after administration, 9 weeks, 18 weeks). At week 16, an intraperitoneal glucose tolerance test was performed. Mice were sacrificed at 18 weeks and tissues (MLN, liver, colon, cecum and gonad fat) were collected and stored at -80 ° C before analysis. Blood samples were obtained by cardiac puncture under isoflurane anesthesia. Thirty minutes after blood coagulation at RT, serum was separated by centrifugation (2,000 ⁇ g, 10 minutes, 4 ° C.). The supernatant (serum) was transferred to a 1.5 mL tube and stored at -80 ° C until use. Experimental procedures were reviewed and approved by the Institutional Animal Care and Usage Committee.
  • A. muciniphila in HFD-induced obese mice brain heart infution agar (BD Difco, USA) supplemented with A. muciniphila ATCC BAA-835 (Akk) supplemented with 0.5% mucin (Sigma, USA) at 37 ° C. Incubated for 48 hours in anaerobic conditions and microorganisms were collected in a sterile loop (1 mm in diameter). The collected cells were washed with sterile PBS supplemented with 0.5% cysteine and centrifuged at 10,000 ⁇ g for 5 minutes. The bacterial pellet was resuspended at 10 9 cfu / 200 ⁇ l with PBS supplemented with 0.5% cysteine and 20% glycerol.
  • the prepared bacterial solution was stored at -80 ° C until use.
  • the Akk strain was prepared daily by washing the stored stock solution with 0.5% cysteine PBS and then administered by oral gavage at 5 ⁇ 10 8 cfu per mouse. The weight of each mouse was measured once a week during the test period, then the mice were anesthetized, and MLN and colon were collected to measure the expression of target biomarkers ( Il21, Il21r, Foxp3 and Il10 ).
  • MLN and terminal colon were homogenized with 5 mm stainless steel beads (Qiagen, Hilden, Germany) for 5-8 minutes at 30 Hz.
  • Total RNA was isolated using an Easy-spin Total RNA Extraction Kit (Intron, Seoul, Korea).
  • CDNA was synthesized using the High Capacity RNA to cDNA kit (Applied Biosystems, Carlsbad, Calif., USA). All qPCR reactions were performed using the Rotor-Gene SYBR Green PCR kit (Qiagen) using the Rotor-Gene Q (Qiagen) according to the manufacturer's instructions. Relative RNA amounts were determined by the 2- ⁇ Ct method using Gapdh as the reference transscript.
  • Samples were prepared for NMR based metabolic analysis with slight modifications according to Lamichhane's method. Briefly, pancreatic samples (about 130 mg) were mixed with 600 ⁇ l of DDW, vortexed for 30 seconds, and homogenized by a tissue homogenizer. 60 ⁇ l of deuterium oxide (D 2 O), 1 mM Al containing 0.025 mg / mL 3- (trimethylsilyl) propionic acid-d4 sodium salt (TSP) after centrifugation (14,000 ⁇ g, 4 ° C.) for 10 minutes. 60 ⁇ l of dozol, 60 ⁇ l of 2 mM NaN 3 and 120 ⁇ l of 0.5M KH 2 PO 4 were added to 300 ⁇ l of the supernatant as a solvent.
  • D 2 O deuterium oxide
  • TSP trimethylsilyl propionic acid-d4 sodium salt
  • MLN and gonad adipose tissue were ground and digested with 1.6 mg / mL collagenase type 4 (Worthington, Lakewood, NJ) and 0.1% DNase I (fraction IX; Sigma) at 37 ° C. for 1 hour.
  • Whole cells were treated to lyse red blood cells and then stained for FACS analysis. Single cell suspensions were preincubated with anti-Fc ⁇ R blocking monoclonal antibody (2.4G2) and washed before staining.
  • Mouse immune cells were stained with the following antibodies: phycoerythrin-Texas red-conjugated anti-human CD45 (MHCD4517; Invitrogen); fluorescein isothiocyanate-conjugated anti-CD3 (anti-CD3; UCHT1 (555332); BD Biosciences); Anti-CD19 (H1B19 (302206), BioLegend); Anti-CD11b (ICRF44 (11-0118-42); eBioscience); Anti-CD14 (HCD14 (325604); eBioscience) or anti-Fc ⁇ R1 (MAR-1 (134306), BioLegend), Alexa Fluor 647-conjugated anti-CD127 (eBioRDR5 (51-1278-73); eBioscience).

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Abstract

Bacteroides vulgatus strain SNUG40005 which is a novel strain of the present invention is essential for maintaining anti-obesity bacteria Akkermansia muciniphila and has the function of symbiotic Bacteroides sp. which prevents obesity and immune and metabolic dysfunction by means of forming and maintaining a healthy microbial community. Bacteroides vulgatus strain SNUG40005 can reduce the IL-12 receptor expression and innate lymphoid cells enabling the expression of the IL-12 receptor, allows quick recovery of the structure of intestinal organisms and a metabolite profile as well as regulatory T cell and type 2 innate lymphoid cell immunity, and thus can be utilized as a composition for preventing, treating or relieving metabolic diseases comprising obesity.

Description

신규한 박테로이드 불가투스 균주 및 이를 유효 성분으로 하는 면역 및 대사성 질환 예방, 개선 또는 치료용 조성물Novel bacteroidal vulgaris strain and composition for preventing, improving or treating immune and metabolic diseases using the same
관련 출원(들)과의 상호 인용Cross Citation with Related Application (s)
본 출원은 2018년 4월 19일자 한국특허출원 제10-2018-0045772호에 기초한 우선권의 이익을 주장하며， 해당 한국 특허 출원의 문헌들에 개시된 모든 내용은 본 명세서의 일부로서 포함된다.This application claims the benefit of priority based on Korean Patent Application No. 10-2018-0045772 dated April 19, 2018, and all the contents disclosed in the documents of that Korean patent application are incorporated as part of this specification.
본 발명은 대사성 질환 예방, 치료 또는 개선 효능을 가진 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주(기탁번호 KCTC 13276BP) 및 이의 용도에 대한 것이다.The present invention relates to the bacteroid Bacteroides vulgatus SNUG40005 strain (Accession No. KCTC 13276BP) and its use having the effect of preventing, treating or ameliorating metabolic diseases.
장세균총 이상(Gut dysbiosis)은 면역 항상성을 방해하고 다양한 염증성 및 대사성 질환의 발달을 촉진한다. 비만 관련 미생물의 변화는 장 투과성 및 순환 내독소 수준의 증가로 이어져 낮은 등급의 전신성 염증을 유발한다. 그러나 미생물 군집 구성원이 서로 어떻게 상호 작용하는지 그리고 숙주 면역계와 상호작용하여 비만을 예방하거나 보호하는지는 아직 불분명하다.Gut dysbiosis interferes with immune homeostasis and promotes the development of various inflammatory and metabolic diseases. Changes in obesity-associated microorganisms lead to increased intestinal permeability and circulating endotoxin levels, leading to low grade of systemic inflammation. However, it is still unclear how the microbial community members interact with each other and with the host immune system to prevent or protect obesity.
비만은 신진 대사와 면역 항상성뿐만 아니라 장 미생물 군락의 균형을 교란한다. 비만에서 변형된 장 미생물은 신진 대사와 면역기능장애 사이의 중요한 연관성이 있는 것으로 보고되고 있다. 예를 들어, 인슐린 저항성은 'leaky gut'에서 흡수된 혈청 내 독소에 의해 유도되고 전신성 염증을 증가시킨다. 특정 박테리아 분류(texa) (예, 아커멘시아 뮤시니필리아(Akkermansia muciniphila))는 내독소혈증(endotoxemia)을 감소시키고 지방 조직에서 조절 T 세포의 축적을 촉진시킴으로써 상기 증상을 조절하는 것으로 밝혀졌다. 그러나 비만 및 체중 조절에서 적응(adaptive) 면역 반응과 관련된 박테리아 분류군은 보고된 적이 없다. Obesity disturbs the balance of intestinal microflora as well as metabolism and immune homeostasis. Intestinal microorganisms modified in obesity have been reported to have an important link between metabolism and immune dysfunction. For example, insulin resistance is induced by serum toxins absorbed in the 'leaky gut' and increases systemic inflammation. Certain bacterial texas (eg, Akkermansia muciniphila ) have been found to modulate these symptoms by reducing endotoxemia and promoting the accumulation of regulatory T cells in adipose tissue. However, no bacterial taxa associated with adaptive immune responses in obesity and weight control have been reported.
비만에 대해 불일치하는 쌍둥이 분석에서 본 발명자는 박테로이드 불가투스 (Bacteroides vulgatus, Bvul)를 감소된 임상 지표 및 변경된 비만의 면역 상관성과 관련있는 주요 공생종으로 확인했다. Bvul을 투여한 마우스는 식이 유발성 체중 증가, 포도당 불내성 및 지방 조직 염증을 감소시킴으로써 인간에게 나타나는 이점을 반복적으로 나타내고 있음을 확인하였다. 이러한 이점은 조절성 T 세포 및 타입 2 선천성 림프성 세포(innnate lymphoid cell, ILC) 면역뿐만 아니라 장내 미생물의 구조 및 대사 산물 프로파일의 극적인 회복을 수반한다. 사람에서 Bvul과 IL-21 사이의 관계에 따라, Bvul를 처리한 마우스는 장간막 림프절에서 IL-21 수용체 발현 및 IL-21 수용체 발현 ILC의 감소를 나타냈다. 또한 Bvul은 항비만 박테리아 Akkermansia muciniphila를 유지하는 데 필수적이었다. 이러한 결과는 건강한 미생물 군집을 형성하고 유지함으로써 비만과 면역대사성 기능 장애를 예방하는 공생 박테로이드(Bacteroides) 종의 능력을 의미한다.In an inconsistent twin analysis of obesity, we identified Bacteroides vulgatus (Bvul) as the major symbiosis associated with reduced clinical markers and immune correlates of altered obesity. Mice receiving Bvul have been shown to repeatedly show the benefits to humans by reducing dietary induced weight gain, glucose intolerance and adipose tissue inflammation. These benefits involve dramatic recovery of structural and metabolite profiles of gut microorganisms as well as regulatory T cell and type 2 innate lymphoid cell (ILC) immunity. According to the relationship between Bvul and IL-21 in humans, Bvul treated mice showed a decrease in IL-21 receptor expression and IL-21 receptor expression ILC in mesenteric lymph nodes. Bvul was also essential for maintaining the anti-obese bacteria Akkermansia muciniphila . These results indicate the ability of symbiotic Bacteroides species to prevent obesity and immune metabolic dysfunction by forming and maintaining healthy microbial communities.
본 발명자는 본 발명의 박테로이드 불가투스(Bacteroides vulgatus, Bvul)균주를 감소된 임상 지표 및 변경된 비만의 면역 상관성과 관련 있는 주요 공생종으로 확인했다. Bvul을 투여한 마우스는 식이 유발성 체중 증가, 포도당 불내성 및 지방 조직 염증을 감소시킴으로써 인간에게 나타나는 이점을 반복적으로 나타냈다. 이러한 이점은 조절성 T 세포 및 타입 2 ILC 면역 뿐만 아니라 장내 미생물의 구조 및 대사 산물 프로파일의 극적인 회복을 수반하였다. 사람에서 Bvul과 IL-21 사이의 관계에 따라, Bvul를 처리한 마우스는 장간막 림프절에서 IL-21 수용체 발현 및 IL-21 수용체 발현 ILC의 감소를 나타냈다. 또한 Bvul은 항비만 박테리아 Akkermansia muciniphila를 유지하는 데 필수적이었다. 이러한 결과를 통해 건강한 미생물 군집을 형성하고 유지함으로써 비만과 면역대사성 기능 장애를 예방하는 공생 박테로이드(Bacteroides) 종의 능력을 확인하였고, 본 발명을 완성하였다.We have identified the bacteroid Bacteroides vulgatus (Bvul) strain of the present invention as a major commensal species associated with reduced clinical markers and immune correlates of altered obesity. Mice administered Bvul have repeatedly demonstrated the benefits to humans by reducing dietary induced weight gain, glucose intolerance and adipose tissue inflammation. These benefits have accompanied dramatic recovery of regulatory T cell and type 2 ILC immunity, as well as the structure and metabolite profiles of the intestinal microorganisms. According to the relationship between Bvul and IL-21 in humans, Bvul treated mice showed a decrease in IL-21 receptor expression and IL-21 receptor expression ILC in mesenteric lymph nodes. Bvul was also essential for maintaining the anti-obese bacteria Akkermansia muciniphila . These results confirmed the ability of symbiotic bacteroids ( Bacteroides ) species to prevent obesity and immune metabolic dysfunction by forming and maintaining a healthy microbial community, and completed the present invention.
박테로이드 종은 포유류 장내에서의 조기 군집 박테리아(early colonizing bacteria)이고, 숙주의 면역계와 대사 활동을 형성하고 조절하는 것으로 알려져 있다. B. ragilis의 피막 다당체는 조절 T 세포를 조절하고 염증성 장질환, 자가 면역 질환 및 CNS 탈수초성 질환을 포함하는 염증성 질환을 예방한다. 박테로이드 종은 또한 대사 산물 생산을 통해 숙주 생리를 조절한다. 예를 들어, B. hetaiotaomicron의 게놈분석 결과는 다양한 식이 영양분을 이용하고, 숙주가 재사용 할 수 있는 박테리아 대사 산물을 생산할 수 있는 host niche 및 신진 대사 가능성을 나타낸다. 박테로이드 종의 효과 또한 비만의 발병기전 중 미생물 장내 불균형의 복원을 포함한다. 그러나, 장내 공생체로서 Bvul에 의해 유도된 면역학적 및 대사적 변화는, 특히 비만과 관련하여 아직 보고된 바 없다.Bacterial species are early colonizing bacteria in the mammalian gut and are known to form and regulate the host's immune system and metabolic activity. B. ragilis capsular polysaccharides regulate regulatory T cells and prevent inflammatory diseases including inflammatory bowel disease, autoimmune diseases and CNS demyelinating diseases. Bacteroid species also regulate host physiology through metabolite production. For example, genome analysis of B. hetaiotaomicron reveals the potential for host niche and metabolism to utilize various dietary nutrients and produce bacterial metabolites that the host can reuse. The effects of bacteroid species also include the restoration of microbial intestinal imbalances during the pathogenesis of obesity. However, immunological and metabolic changes induced by Bvul as intestinal commensals have not yet been reported, particularly in relation to obesity.
상기 과제를 해결하기 위해, 본 발명은 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 제공한다.In order to solve the above problems, the present invention provides a bacteroid Bacteroides vulgatus SNUG40005 strain.
또한, 상기 균주는 식이-유발성 체중 증가, 포도당 불내성 또는 지방 조직 염증을 감소시킬 수 있다.In addition, the strain may reduce diet-induced weight gain, glucose intolerance or adipose tissue inflammation.
상기 균주는 IL-12 수용체 발현 및 IL-12 수용체를 발현하는 ILC를 감소시킬 수 있다. The strain can reduce IL-12 receptor expression and ILC expressing IL-12 receptor.
또한, 상기 균주는 항-비만 박테리아 아커멘시아 뮤시니필리아(Akkermansia muciniphila) 균주를 유지시킬 수 있다.In addition, the strain can maintain the anti-obesity bacterium Akkermansia muciniphila strain.
본 발명은 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 유효성분으로 하는 대사성 질환 치료 또는 예방용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for treating or preventing metabolic diseases using the bacteroid Bacteroides vulgatus SNUG40005 strain as an active ingredient.
상기 대사성 질환은 비만, 당뇨병, 고지혈증, 고콜레스테롤증, 동맥경화증, 지방간 또는 심혈관질환일 수 있다.The metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
본 발명은 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 유효성분으로 하는 대사성 질환 개선용 건강기능성 식품을 제공한다.The present invention provides a health functional food for improving metabolic disease comprising the bacteroid Bacteroides vulgatus SNUG40005 strain as an active ingredient.
상기 대사성 질환은 비만, 당뇨병, 고지혈증, 고콜레스테롤증, 동맥경화증, 지방간 또는 심혈관질환일 수 있다.The metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
또한, 본 발명은 유효한 양의 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 이것이 필요한 개체에게 투여하여 대사성 질환을 예방, 개선 또는 치료하는 방법을 제공한다.The present invention also provides a method of preventing, ameliorating or treating metabolic diseases by administering to a subject in need thereof an effective amount of a Bacteroides vulgatus SNUG40005 strain.
또한, 본 발명은 대사성 질환을 예방, 개선 또는 치료하기 위한 조성물의 제조를 위한 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주의 용도를 제공한다.The present invention also provides the use of a bacteroid Bacteroides vulgatus SNUG40005 strain for the preparation of a composition for preventing, ameliorating or treating a metabolic disease.
상기 대사성 질환은 비만, 당뇨병, 고지혈증, 고콜레스테롤증, 동맥경화증, 지방간 또는 심혈관질환일 수 있다.The metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
상기 개체는 본 발명에 따른 균주 또는 균주를 유효성분으로 포함하는 조성물을 투여하여 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주에 따른 효과 발휘를 기대하는 동물을 의미할 수 있고, 상기 동물은 개, 원숭이, 염소, 돼지, 쥐 또는 인간일 수 있다.The subject may mean an animal that is expected to exert the effect according to the bacteroid Bacteroides vulgatus SNUG40005 strain by administering a composition comprising the strain or strain according to the present invention, the animal is a dog, a monkey , Goat, pig, mouse or human.
상기 예방은 본 발명에 따른 균주 또는 균주를 유효성분으로 포함하는 조성물의 투여로 대사성 질환의 진행을 억제하거나 지연시키는 모든 행위를 의미할 수 있다.The prophylaxis may mean any action of inhibiting or delaying the progression of metabolic disease by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.
상기 치료 또는 개선은 본 발명에 따른 균주 또는 균주를 유효성분으로 포함하는 조성물의 투여로 대사성 질환의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미할 수 있다.The treatment or improvement may refer to any action in which a symptom of a metabolic disease is improved or advantageously changed by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.
상기 투여는 임의의 적절한 방법으로 개체에게 소정의 본 발명에 따른 균주 또는 균주를 유효성분으로 포함하는 조성물을 제공하는 것을 의미할 수 있다.The administration may mean providing a subject with a composition comprising any of the strains or strains according to the present invention as an active ingredient in any suitable manner.
본 발명의 신규한 균주 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주는 항비만 박테리아 Akkermansia muciniphila를 유지하는 데 필수적이며, 건강한 미생물 군집을 형성하고 유지함으로써 비만과 면역대사성 기능 장애를 예방하는 공생 박테로이드 종의 능력을 나타낸다. 또한, 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주는 IL-12 수용체 발현 및 IL-12 수용체를 발현하는 ILC를 감소시킬 수 있으며, 조절성 T 세포 및 타입 2 ILC 면역뿐만 아니라 장내 미생물의 구조 및 대사 산물 프로파일의 빠른 회복을 수반하므로, 비만을 포함한 대사성 질환의 예방, 치료 또는 개선용 조성물로 활용될 수 있다.The novel strain Bacteroides vulgatus SNUG40005 of the present invention is essential for maintaining the anti-obese bacterium Akkermansia muciniphila , and is a symbiotic bacteroid species that prevents obesity and immune metabolic dysfunction by forming and maintaining healthy microbial communities. Indicates the ability. In addition, the Bacteroides vulgatus SNUG40005 strain can reduce IL-12 receptor expression and ILC expressing IL-12 receptors, as well as regulatory T cells and type 2 ILC immunity, as well as the structure and metabolism of intestinal microorganisms. Since it involves a rapid recovery of the product profile, it can be used as a composition for the prevention, treatment or improvement of metabolic diseases including obesity.
도 1은 불일치 MZ 쌍둥이에서 비만상태 및 면역 프로파일과 Bvul 존재량(abundance)과의 상관관계를 실험한 결과이다. (a)는 본 연구의 실험 개요이며, (b) 및 (c)는 허리둘레가 불일치하는 쌍둥이에서 주요 속(genera)의 존재량의 차이값과 혈청내 IL-12 수준에 대한 결과이다. (d) 및 (e)는 허리 둘레와 혈청 IL-21 수준을 설명하는 선형 모델(MaAsLin)과의 다변수 연관성에 의해 분석된 Bvul의 유의미한 변화값에 대한 것이다. Waist_low와 Waist_high는 불일치 쌍둥이의 허리 둘레의 차이가 각각 4 cm 미만과 및 4 cm 이상인 것을 의미한다. IL-21의 경우 P는 0.28 pg/ml 이상, N은 혈청에서 0.28 pg/ml 미만인 것을 의미한다.Figure 1 shows the results of experiments on the relationship between obesity status and immune profile and Bvul abundance in mismatched MZ twins. (a) is an experimental summary of the study, and (b) and (c) are results of differences in the amount of major genera and serum IL-12 levels in twins with inconsistent waist circumference. (d) and (e) are for significant changes in Bvul analyzed by multivariate association with a linear model (MaAsLin) describing waist circumference and serum IL-21 levels. Waist_low and Waist_high mean that the difference in waist circumference of the mismatched twins is less than 4 cm and more than 4 cm, respectively. For IL-21 P means greater than 0.28 pg / ml, N means less than 0.28 pg / ml in serum.
도 2에서 (a)는 한국인 불일치 쌍둥이에서 혈청 사이토카인의 수준과 장내 박테리아 문(phyla) 사이의 상관관계에 대한 계층적 클러스터링(clustering) 결과이다. 히트맵(heatmap)은 상관 계수를 기반으로 작성되었다. 트리(tree)는 Euclidean 방법을 사용하여 도시화하였으며, 사이토카인 프로파일을 위한 두 개의 가지(arms)로 분리된다. (b) 내지 (d)는 장내 미생물의 잠재적인 기능적 특성을 PICRUSt를 통해 분석한 결과이다.In FIG. 2, (a) is a hierarchical clustering result of the correlation between serum cytokine levels and intestinal bacterial phyla in Korean discordant twins. Heatmaps were created based on correlation coefficients. The tree is illustrated using the Euclidean method and split into two arms for the cytokine profile. (b) to (d) are the results of PICRUSt analysis of potential functional properties of the intestinal microorganisms.
도 3은 HFD-유도 비만 마우스에서 비만 상태 및 포도당 내성을 감소시키는 Bvul SNUG40005 균주에 대한 실험결과이다. (a)는 마우스의 표현형에 대한 도면이다. (b)는 그룹별 체중 측정값에 대한 도면이다. (c)는 그룹별 단식 혈중 포도당 레벨 측정값에 대한 도면이다. (d)는 IPGTT(intraperitoneal glucose tolerance test) 결과값에 대한 도면이다. (e)는 16주 동안 Bvul로 처리(treatment)된 C57Bl6 암컷 마우스의 IPGTT AUC값에 대한 도면이다. (f)는 그룹별 생식선 지방 조직 중량에 대한 결과값에 대한 도면이다. (g)는 그룹별 지방세포의 크기를 측정한 결과에 대한 도면이다. (h)는 Bvul를 투여(administration)한 후 18주 동안 생식선 지방 조직에서 낮은 등급 염증에 대한 유전자 발현 변화를 측정한 결과이다. 측정값은 평균±SD 로 표시되고, 서로 다른 위첨자를 가진 측정값은 post hoc ANOVA one-way 통계 분석에 따라 상당히 상이한 것으로 나타난다. *P<0.05, **P<0.01, ***P<0.001, 및 ****P<0.0001. ND: 대조군 식이 마우스(10% 지방, n=10). NDB: 매일 Bvul이 투여된 대조군 식이 마우스(n=9). HFD: HFD 마우스 (60% 지방, n=10). HFDB: 매일 Bvul이 투여된 HFD 마우스 (n=9).3 is an experimental result of the Bvul SNUG40005 strain that reduces obesity and glucose tolerance in HFD-induced obese mice. (a) is a diagram of a phenotype of a mouse. (b) is a diagram of a weight measurement value for each group. (c) is a diagram of fasting blood glucose level measurement values by group. (d) is a diagram of the results of the intraperitoneal glucose tolerance test (IPGTT). (e) is a plot of IPGTT AUC values of C57Bl6 female mice treated with Bvul for 16 weeks. (f) is a diagram of the results for the weight of gonad adipose tissue by group. (g) is a diagram showing the results of measuring the size of adipocytes in each group. (h) is the result of measuring changes in gene expression for low grade inflammation in gonad adipose tissue for 18 weeks after administration of Bvul. Measurements are expressed as mean ± SD, and measurements with different superscripts appear to be quite different according to post hoc ANOVA one-way statistical analysis. * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001. ND: control diet mice (10% fat, n = 10). NDB: Control dietary mice (n = 9) administered Bvul daily. HFD: HFD mice (60% fat, n = 10). HFDB: HFD mice administered Bvul daily (n = 9).
도 4에서 (a)는 마우스 및 인간 분변 샘플로부터 분리된 박테로이드 균주의 염증 조절 효과(분화된 THP-1 마크로파지에서의 IL-10/THF-α 비율)에 대한 도면이다. IL-10/THF-α 비율은 박테로이드 종, P. goldsteinii, 및 Akkermansia muciniphila의 10개의 서로 다른 균주로 처리한 후 계산되었다. (b)는 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005에 대한 주사현미경 도면이다.In Figure 4 (a) is a diagram of the inflammatory control effect (IL-10 / THF-α ratio in differentiated THP-1 macrophages) of bacteroid strains isolated from mouse and human fecal samples. IL-10 / THF-α ratios were calculated after treatment with 10 different strains of bacteroid species, P. goldsteinii , and Akkermansia muciniphila . (b) is a scanning microscope diagram of Bacteroides vulgatus SNUG40005.
도 5는 비만에서 고갈된 A. muciniphila를 회복시키고, 장내 미생물 및 대사 산물을 조절하는 Bvul SNUG40005에 대한 것으로, (a)는 비가중 UniFrac metrics를 사용한 PCoA에 대한 결과값이고, (b)는 맹장에서 서로 다른 식이 그룹에 근거한 25개 주요 분류(texa)의 히트맵 결과이다. (c)는 박테리아 존재량에 의한 HFD 및 Bvul 처리의 분류 결과이다. 각 그룹간 상위 15의 박테리아에 대한 것이다. (d)는 80 맹장 대사 산물의 정량값에 근거한 sPLS-DA plot에 대한 것이다. (e)는 HFD 및 Bvul 처리로 가장 영향을 많이 받는 대표적인 10가지 대사 산물을 나타낸 것이다. (f)는 Bray-Curtis dissimilarity와 주요 맹장 대사 산물을 갖는 상위 10의 박테리아에 근거한 PCoA 결과이다. 화살표는 강력한 유의미한 상관 관계를 의미하며, 포인트는 속 수준에서 맹장 미생물을 의미하고, 색 구배는 마우스의 체중을 표시한다.5 is for Bvul SNUG40005, which recovers A. muciniphila depleted in obesity and regulates intestinal microorganisms and metabolites, (a) unweighted Results for PCoA using UniFrac metrics, and (b) are heatmap results of 25 major texas based on different dietary groups in the cecum. (c) is the classification result of HFD and Bvul treatment by bacterial abundance. For the top 15 bacteria in each group. (d) is for the sPLS-DA plot based on the quantitative value of 80 cecal metabolites. (e) shows 10 representative metabolites most affected by HFD and Bvul treatment. (f) is a PCoA result based on the top 10 bacteria with Bray-Curtis dissimilarity and major cecal metabolites. Arrows indicate strong significant correlations, points represent cecal microorganisms at the genus level, and color gradients indicate the weight of the mouse.
도 6에서 (a)는 비가중 UniFrac metrics를 사용한 PCoA 결과이며, (b)는 투여(administration) 시간(분변 마이크로비옴)에 근거한 미생물 군집의 존재량 분석 결과이다(Pre: Bvul 투여 1주 전; Day 0: 항생제 처리 1주 후 투여 시작 시점; Day 5, Wk 9, Wk 18: Bvul 투여 5일 후, 9주 후, 및 18주 후). (c)는 대표적인 미생물 분류(texa)의 이동값에 대한 결과이다. (d)는 미생물 존재량에 의한 HFD 급여(feeding) 및 Bvul 처리의 분류 결과이다. (e)는 PICRUSt.를 사용한 KEGG pathway 분석에 의한 HFD 급여 및 Bvul 처리의 기능적 분류 결과이다. (f)는 각 그룹에서 cholate에 대한 존재량 측정 결과이다. (g)는 각 그룹에서 N-아세틸글루코사민(N-acetylglucosamine) 대사 산물에 대한 존재량 측정 결과이다. ND: 대조군 식이 마우스(10% 지방, n=10). NDB: 매일 Bvul이 투여된 대조군 식이 마우스(n=9). HFD: HFD 마우스 (60% 지방, n=10). HFDB: 매일 Bvul이 투여된 HFD 마우스(n=9).In Figure 6 (a) is the PCoA results using unweighted UniFrac metrics, (b) is a result of analysis of abundance of microbial community based on administration time (fecal microbeads) (Pre: 1 week before Bvul administration; Day 0: Start of dosing after 1 week of antibiotic treatment; Day 5, Wk 9, Wk 18: 5 days, 9 weeks, and 18 weeks after Bvul administration). (c) is the result of the shift of representative microbial taxa (texa). (d) is the result of classification of HFD feeding and Bvul treatment by microbial abundance. (e) is the result of functional classification of HFD feeding and Bvul treatment by KEGG pathway analysis using PICRUSt. (f) shows the abundance measurement results for cholate in each group. (g) shows the abundance measurement results for N-acetylglucosamine metabolites in each group. ND: control diet mice (10% fat, n = 10). NDB: Control dietary mice (n = 9) administered Bvul daily. HFD: HFD mice (60% fat, n = 10). HFDB: HFD mice administered Bvul daily (n = 9).
도 7은 HFD-유도 비만에서 조절 T 세포(Regulatory T cell, Treg) 및 IL-21-비의존적 ILC2 반응을 유도하는 Bvul SNUG40005에 대한 것으로, (a)는 MLN에서 Il21 및 Il21r의 발현, (b)는 MLN에서 Foxp3 및 Il10의 발현 결과에 대한 것이다. (c) 및 (d)는 MLN에서 CD4+ Foxp3+ Treg 세포의 Flow cytometric 분석결과, (e) 및 (f)는 MLN에서 전체 ST2+ ILC의 Flow cytometric 분석결과, (g)는 MLN에서 IL-21+ ILC, IL-21+ ST2(-) ILC, 및 IL-21+ ST2(+) ILC의 Flow cytometric 분석결과이다. (h)는 CD11c+ CD206+ M2 극성 대식세포에 대한 것이고, (i)는 생식선 지방 조직에서의 M2/M1 대식세포 비율에 대한 것이다. *P<0.05, **P<0.01, ***P<0.001, 및 ****P<0.0001. ND: 대조군 식이 마우스(10% 지방, n=10). NDB: 매일 Bvul이 투여된 대조군 식이 마우스(n=9). HFD: HFD 마우스 (60% 지방, n=10). HFDB: 매일 Bvul이 투여된 HFD 마우스(n=9). HFD_Akk: 15주 동안 매일 Akk가 투여된 HFD 마우스(n=10).FIG. 7 shows Bvul SNUG40005 inducing regulatory T cell (Treg) and IL-21-independent ILC2 responses in HFD-induced obesity, (a) expression of Il21 and Il21r in MLN, (b ) Is for the expression results of Foxp3 and Il10 in MLN. (c) and (d) shows flow cytometric analysis of CD4 + Foxp3 + Treg cells in MLN, (e) and (f) shows flow cytometric analysis of whole ST2 + ILC in MLN, (g) shows IL-21 + ILC in MLN , Flow cytometric analysis of IL-21 + ST2 (−) ILC, and IL-21 + ST2 (+) ILC. (h) is for CD11c + CD206 + M2 polar macrophages and (i) is for M2 / M1 macrophages ratio in gonad adipose tissue. * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001. ND: control diet mice (10% fat, n = 10). NDB: Control dietary mice (n = 9) administered Bvul daily. HFD: HFD mice (60% fat, n = 10). HFDB: HFD mice administered Bvul daily (n = 9). HFD_Akk: HFD mice administered Akk daily for 15 weeks (n = 10).
인간의 대사 및 면역 프로필과 관련된 핵심 박테리아를 조사하기 위해, 16s rRNA 유전자 프로파일링과 30 쌍둥이의 분변과 혈액 샘플에 대한 멀티플렉스 사이토카인 분석을 이용하여 장내 미생물 조성물(composition)과 혈청 사이토카인 프로파일(Th1, Th2, Th17, Treg 및 염증과 관련된 20개의 사이토카인)과의 상관 관계를 조사하였다(도 1a 참조).To investigate key bacteria associated with human metabolic and immune profiles, intestinal microbial composition and serum cytokine profiles using 16s rRNA gene profiling and multiplex cytokine analysis on fecal and blood samples of 30 twins The correlation with Th1, Th2, Th17, Tregs, and 20 cytokines associated with inflammation) was examined (see FIG. 1A).
계층적 클러스터 분석 결과 박테로이드와 Oscillospira를 포함한 특정 미생물 분류군은 IL-17A, IL-21, CCL20, IL-27 및 TNF-α와 같은 Th17 관련 염증성 사이토카인의 수준과 음의 상관 관계가 있음이 밝혀졌다. 체질량지수(BMI)를 비만의 양적 척도로 사용하여 분석한 결과, 비만에 대해 불일치하는 쌍둥이에서 구분되는 장내 미생물 조성물이 발견되었다. 그러나 혈청 사이토카인 프로파일은 BMI가 일치하지 않는 쌍둥이 샘플간에 유의미한 차이가 없었다(도 2a). 본 발명자는 비만의 지표로서 허리 둘레의 유의미한 차이가 있는 MZ 쌍둥이에 대해 조사했다. 이 변수는 장내 미생물 분류(gut microbial texa)와 비만 및 염증과 관련된 바이오마커와의 상관관계를 더 잘 포착했기 때문이다. 박테로이드 종, Faecalibacterium sp. 및 Bifidobacterium spp.의 존재량(abundance)은 쌍둥이에서 허리 둘레가 더 큰(4 cm 이상) 쌍둥이가 다른 쌍둥이보다 낮았다(도 1b 참조).Hierarchical cluster analysis revealed that certain microbial populations, including bacteroids and Oscillospira , negatively correlate with levels of Th17-related inflammatory cytokines such as IL-17A, IL-21, CCL20, IL-27, and TNF-α. lost. Analysis of body mass index (BMI) as a quantitative measure of obesity revealed intestinal microbial compositions distinguished from identical discrepancies to obesity. However, serum cytokine profiles did not differ significantly between twin samples with inconsistent BMI (FIG. 2A). We investigated MZ twins with significant differences in waist circumference as an indicator of obesity. This variable is due to better capture of the correlation between gut microbial texa and biomarkers associated with obesity and inflammation. Bacteroid species, Faecalibacterium sp. And the abundance of Bifidobacterium spp. Was lower in twins (4 cm or more) than twins (Fig. 1b).
혈청 사이토카인 프로파일 및 비만관련변수와 관련된 일반적인 분류군(texa)은 박테로이드 종이었으며, IL-21의 낮은 혈청 수준과 관련이 있다(도 1c). 개별 임상 변수 및 혈청 사이토카인 수준이 불일치하는 쌍둥이 사이에 유의하게 다른 존재량을 보인 박테로이드 속의 표적 종은 선형 모델(쌍둥이의 무작위 변수뿐만 아니라 연령 및 성별에 따라 조정됨, MaAsLin)과의 다변수연관성에 의해 재분석되었다. 박테로이드 불가투스(Bacteroides vulgatus, Bvul)를 대표하는 OUT(operational taxonomic unit)는 비만 및 혈청 면역 프로필과 유의미한 관련이 있었다(도 1d 및 1e). 대변 샘플의 기능적 마이크로비옴 분석(functional microbiome analysis)을 실시한 결과, 지방산과 아미노산 대사 경로, 특히 분지 사슬 아미노산(Branched chain amino acid, BCAA) 분해 경로가 고 허리 둘레와 저 허리 둘레 그룹간에 유의미한 차이를 보였다. 또한 방향족 아미노산과 지질 대사 경로는 IL-21의 높은 수준(P)과 낮은 수준(N)의 그룹간에 차이가 있었다(도 2b 내지 도 2d).A common taxa associated with serum cytokine profiles and obesity related variables was the bacteroid species, which was associated with low serum levels of IL-21 (FIG. 1C). Target species in the genus bacteroids with significantly different abundances between individual clinical variables and twins with inconsistent serum cytokine levels were multivariate with the linear model (adjusted by age and gender as well as by random variables in twins). Reanalyzed by association. The operational taxonomic unit (OUT), representing Bacteroides vulgatus (Bvul), was significantly associated with obesity and serum immune profiles (FIGS. 1D and 1E). Functional microbiome analysis of stool samples showed significant differences in fatty acid and amino acid metabolic pathways, especially branched chain amino acid (BCAA) degradation pathways between the high waist and low waist groups. . Aromatic amino acid and lipid metabolic pathways also differed between groups of high (P) and low (N) levels of IL-21 (FIGS. 2B-2D).
면역 및 인간으로부터 얻은 장내 미생물 프로파일은 Bvul과 IL-21에 의한 면역반응이 비만에 대해 공통적인 상호작용 요소임을 나타낸다. 이 가설을 더 연구하기 위해 본 발명자는 고지방식(HFD, 60% 지방)과 대조 저지방식이(10% 지방)를 섭취한 C57BL/6J 암컷 마우스에서 Bvul 투여 효과를 조사했다. 숙주 생리학에 대한 장내 미생물의 영향이 박테리아 균주에 따라 다르다는 것을 감안하여, 본 발명자는 처음에는 관련 없는 건강한 성인 한국인에게서 Bvul의 여러 균주를 분리했다. Bvul은 마우스 내장에서 매우 풍부한 공생물이기 때문에 마우스 배설물에서도 균주가 분리된다. 서로 다른 지방 함량(10%, 45%, 60%)의 3가지 유형의 사료를 급여한 마우스 그룹은 Bvul 종을 분리하여 대사 잠재력에 대한 식이의 영향을 결정하는데 사용되었다. Bvul type ATCC 8482를 포함하여 인간 및 마우스로부터 분리된 총 182 종의 박테로이드 균주를 인비트로(in vitro) 면역 조절 활성에 대해 스크리닝 하였다. 마커로, 비만은 대사성 염증을 일으키는 것으로 잘 알려져 있으므로, THP-1 세포 유래 대식세포의 전염증성(pro-inflammatory) 및 항염증성(anti-inflammatory) 사이토카인 생성이 측정되었다. Bvul SNUG40005(이하 Bvul이라 칭함)는 높은 수준의 IL-10/TNF-α 비율을 유도하는 면역원성에 근거하여 선택되었다(도 3). 이 균주를 18주 동안 매일 시험 마우스에 투여하기 위해 선택하였다(109 cfu/마우스).Immunity and gut microbial profiles obtained from humans indicate that immune responses by Bvul and IL-21 are a common interacting factor for obesity. To further study this hypothesis, we examined the effects of Bvul administration in C57BL / 6J female mice fed high fat diet (HFD, 60% fat) and control low fat diet (10% fat). Given that the effect of intestinal microorganisms on host physiology varies with bacterial strains, we initially isolated several strains of Bvul from unrelated healthy adult Koreans. Bvul is a very abundant symbiosis in mouse intestines, so strains are isolated from mouse feces. Groups of mice fed three types of feed of different fat contents (10%, 45%, 60%) were used to isolate Bvul species to determine the effect of diet on metabolic potential. A total of 182 bacteroid strains isolated from humans and mice, including Bvul type ATCC 8482, were screened for in vitro immunomodulatory activity. As a marker, obesity is well known to cause metabolic inflammation, so pro-inflammatory and anti-inflammatory cytokine production of THP-1 cell derived macrophages has been measured. Bvul SNUG40005 (hereinafter referred to as Bvul) was selected based on immunogenicity leading to high levels of IL-10 / TNF-α ratio (FIG. 3). This strain was selected for administration to test mice daily for 18 weeks (10 9 cfu / mouse).
gnotobiotic 마우스를 사용한 종래의 군집 연구는 장내에서 공유된 niche로 인하여 박테로이드 종의 동일한 종이 이미 군집화된 균주를 대체할 수 없다고 보고되었다. 본 발명자는 먼저 항균제(암피실린(A), 반코마이신(V), 네오마이신(N), 메트로니다졸(M), 스트렙토마이신, 페니실린, 카나마이신, 및 AVNM의 혼합물)로 1주일간 투여하였다. 결과를 바탕으로 기존 Bvul(Bvul SNUG40005) 균주를 감소시키기 위해 암피실린으로 1주일간 마우스를 전처리하였고, 마우스 장내에서 인간에서 분리된 Bvul의 군집화를 촉진하였다.Conventional colonization studies using gnotobiotic mice have reported that the same species of bacteroid species cannot replace already clustered strains due to shared niche in the gut. The inventors first administered with an antimicrobial agent (ampicillin (A), vancomycin (V), neomycin (N), metronidazole (M), streptomycin, penicillin, kanamycin, and AVNM) for 1 week. Based on the results, the mice were pretreated with ampicillin for 1 week to reduce the existing Bvul (Bvul SNUG40005) strain, and promoted the colonization of Bvul isolated from humans in the mouse intestine.
Bvul 투여 후 5주부터 시작하여, HFD-급여 및 Bvul-처리 그룹(HFDB)의 체중은, HFD를 급여하지만 Bvul 투여가 없는 그룹(HFD)의 체중보다 유의하게 낮았다. 18주 후에, HFDB 그룹의 평균 체중은 HFD 그룹의 평균 체중보다 약 30% 낮았다(도 3a 및 도 3b). 생식선 지방의 무게는 40% 낮았고(도 3f), NDB와 HFDB 그룹이 ND와 HFD 그룹보다 지방 세포 크기가 작았다(도 3g). 비만에 의해 유도된 지방 조직에서의 낮은 등급 염증은 또한 Bvul 처리에 의해 약화되었다(도 3h). intraperitoneal glucose tolerance test(IPGTT)로 측정한 공복 혈당(P<0.01, 도 3c)과 포도당 내성(P<0.0001, 도 3d; P<0.001, 도 3e)은 HFD 그룹이 HFDB 그룹보다 더 많았다. 특히 대조군 식이 그룹에서도 Bvul 처리는 혈당 증가를 억제했다. 마우스의 비만 유발 대사 변화에 대한 Bvul 처리의 이러한 극적인 효과는 Bvul과 인간의 비만 표현형 사이의 음의 상관 관계와 일치한다. 이 연구에서 본 발명자는 인간과 마우스 모두에서 Bvul의 항비만 효과를 처음으로 확인했으며 내장 마이크로비옴과 면역 체계를 조절하는 능력을 확인했다. 이 결과는 HFD 유발성 비만 마우스에서 비만을 예방하거나 신진대사 및 면역 기능 이상을 개선하는 것으로 밝혀진 B. acidifaciens 및 B. uniformis와 같은 다른 박테로이드 종에 대한 이전 연구와 일치한다.Starting at 5 weeks after Bvul administration, body weights in the HFD- and Bvul-treated groups (HFDB) were significantly lower than those in the HFD-fed but not Bvul-administered groups (HFD). After 18 weeks, the mean body weight of the HFDB group was about 30% lower than the mean body weight of the HFD group (FIGS. 3A and 3B). The gonad fat weight was 40% lower (FIG. 3F) and the NDB and HFDB groups had smaller fat cell sizes than the ND and HFD groups (FIG. 3G). Low grade inflammation in adipose tissue induced by obesity was also attenuated by Bvul treatment (FIG. 3H). Fasting blood glucose (P <0.01, FIG. 3C) and glucose tolerance (P <0.0001, FIG. 3D; P <0.001, FIG. 3E) measured by the intraperitoneal glucose tolerance test (IPGTT) were higher in the HFD group than in the HFDB group. In particular, Bvul treatment inhibited blood glucose increase in the control diet group. This dramatic effect of Bvul treatment on obesity-induced metabolic changes in mice is consistent with the negative correlation between Bvul and the human obesity phenotype. In this study, we identified for the first time the anti-obesity effect of Bvul in both humans and mice, and the ability to modulate the visceral microbiome and immune system. This result is consistent with previous studies on other bacteroid species, such as B. acidifaciens and B. uniformis , which have been shown to prevent obesity or improve metabolic and immune dysfunction in HFD-induced obese mice.
장내 미생물 및 신진 대사에서 Bvul에 의해 유도된 변화를 조사하기 위해, HFD가 급여된 마우스의 맹장 및 대변 샘플에 대한 미생물 및 대사 산물 분석을 수행하였다. 그 결과, HFD는 미생물 군집을 크게 변화시켰다(도 5a). NDB와 HFDB 그룹의 미생물 구조는 Bvul 투여 후 18주 동안 유사성을 보였다(도 5a). 미생물 군집(도 6a)과 그것의 α-다양성(도 5b)의 시간 경과 분석은 Bvul 군집 이전의 암피실린 전처리 효과가 부분적으로 18주 후에 극복되었음을 보여주었다. HFD를 급여한 마우스의 주요 박테리아 분류군의 구성은 Bvul 처리에 의해 현저하게 변화되었다. A. muciniphila의 존재량(abundance)은 HFD에 의해 유의하게 감소되었지만 Bvul 처리에 의해 급격히 회복되었다(도 5b 및 도 6c). A. muciniphila는 대사성 질환에서 장의 장벽 기능장애 및 염증을 개선하여 대사 프로파일과 포도당 항상성에 영향을 주는 점액-거주 박테리아이다.To investigate Bvul-induced changes in intestinal microorganisms and metabolism, microbial and metabolite analyzes were performed on cecum and fecal samples of mice fed HFD. As a result, HFD significantly changed the microbial community (FIG. 5A). The microbial structures of the NDB and HFDB groups showed similarities for 18 weeks after Bvul administration (FIG. 5A). Time course analysis of the microbial community (FIG. 6A) and its α-diversity (FIG. 5B) showed that the ampicillin pretreatment effect prior to the Bvul community was partially overcome after 18 weeks. The composition of the major bacterial taxa in mice fed HFD was significantly changed by Bvul treatment. The abundance of A. muciniphila was significantly reduced by HFD but rapidly recovered by Bvul treatment (FIGS. 5B and 6C). A. muciniphila is a mucus-resident bacterium that affects metabolic profiles and glucose homeostasis by improving intestinal barrier dysfunction and inflammation in metabolic diseases.
Oscillospira와 Streptococcus와 같은 다른 분류군도 HFD와 Bvul의 투여로 인하여 영향을 받았다. Oscillospira는 A. muciniphila와 정반대의 양상을 보였으며 HFD 군에서 증가하였고 HFDB 군에서 급격히 감소하였고, NDB군에서 NB군과 비교하여 증가하였다. Streptococcus와 Eubacterium도 반대의 패턴을 보였다. Streptococcus는 마우스의 내장에서는 거의 발견되지 않았지만, Bvul 처리는 그 양을 증가시켰다.Other taxa such as Oscillospira and Streptococcus were also affected by HFD and Bvul. Oscillospira showed the opposite pattern to A. muciniphila , increased in the HFD group, decreased rapidly in the HFDB group, and increased in the NDB group compared to the NB group. Streptococcus and Eubacterium also showed the opposite pattern. Streptococcus was rarely found in the intestines of mice, but Bvul treatment increased the amount.
Eubacterium은 마우스의 내장에 풍부했지만, HFD와 Bvul 처리는 그 양을 감소시켰다. 흥미롭게도, 투여된 종을 나타내는 OTU의 존재량(abundance)은 그룹들(도 6c) 사이에 유의미한 차이를 보이지 않았으며, 비만에 대한 Bvul 처리와 마이크로비옴에 대한 균주 특이적 영향을 설명하였다. 자연 마우스-군집 Bvul 균주는 비만이 진행되는 동안 효과가 없었다. 또한, 저지방식이를 급여한 비-비만 마우스에서 A. muciniphila의 양은 Bvul 처리에 의해 감소되었다. 기능적 마이크로비옴 분석 결과, 미생물 대사 변화가 HFDB 군보다 NDB 군에서 가장 분명하게 나타났다(도 6e). 이 결과는 Bvul로 유도된 미생물 구조의 리모델링은 숙주의 비만 상태에 의존적이고, 독특한 미생물 군집 프로파일로 이어짐을 의미한다. 만성 질환의 발병 기전에 있어 박테리아의 미생물 생태계와 상호 작용이 중요한 요소로 여겨진다. 복잡한 microbiota에서 synergism과 antagonism에 기반한 마이크로비옴 치료법의 개념이 최근에 나타났다. 본 발명자의 연구는 이러한 미생물 리모델링 방식을 통하여 장내 세균총 불균형을 회복시키고 숙주 생리를 회복시키는 Bvul의 기능에 대한 것이다. Eubacterium was abundant in mouse viscera, but HFD and Bvul treatments reduced the amount. Interestingly, the abundance of OTUs representing the administered species did not show a significant difference between the groups (FIG. 6C) and explained the strain specific effects on Bvul treatment and microbeads on obesity. Natural mouse-community Bvul strains were ineffective during the course of obesity. In addition, the amount of A. muciniphila was reduced by Bvul treatment in non-obese mice fed low-fat diet. As a result of functional microbiome analysis, microbial metabolic changes were most evident in the NDB group than in the HFDB group (FIG. 6E). This result suggests that the remodeling of Bvul-induced microbial structures depends on the host's obesity status and leads to unique microbial community profiles. In the pathogenesis of chronic disease, interaction with bacteria's microbial ecosystem is considered to be an important factor. The concept of microbiome therapies based on synergism and antagonism in complex microbiota has recently emerged. The inventor's research is on Bvul's ability to restore intestinal flora total imbalance and restore host physiology through this microbial remodeling approach.
HFD 및 Bvul 처리로 인한 대사 변화를 조사하고 이러한 변화를 매개하는 A. muciniphila의 중요성을 평가하기 위해 장내 마이크로비옴의 기능적 예측을 수행하고 KEGG 경로와 미생물 분류군 간의 상관 관계를 분석하였다(도 6e). HFD는 대부분의 대사 경로에서 대표적인 분류군의 역할을 감소시켰다. 그러나 Bvul 처리는 A. muciniphila와 Streptococcus ssp.의 대사 기능을 회복시키거나 향상시켰다. sPLS-DA 플롯은 미생물 군집 프로파일(도 5a)과 일치하는 명확한 대사 산물 패턴을 보였다(도 5d). Bvul 처리는 taxa와 기능성 마이크로비옴 모두를 비만인 동안 변화시키는 것으로 밝혀졌다. 그러나 NDB와 HFDB 마우스의 대사 산물 프로파일은 유사하여 Bvul은 식이보다 대사 산물에 더 큰 영향을 미쳤다. Bvul 처리로 인해 변형된 대사는 또한 면역 세포의 분화 및 전신 면역과 관련성이 있다. Bvul과 A. muciniphila에 의해 초래되는 류신(Leucine), 이소류신(Isoleucine)등 BCAA의 낮은 농도(도 5e)는 HFDB 그룹의 지방 조직에서 염증의 감소를 설명할 수 있다. 사람 피검자의 미생물 및 대사 물질 분석 결과, 비만 군에서 혈청 BCAA 수치가 증가하였고, BCAA는 대사 산물과 장내 미생물을 연결하는 바이오마커일 수 있다. 또한 co-housing 연구에 따르면 Bacteroidales들이 마른 체질에서 비만 체질로의 침입으로 인해 식이 유도된 표현형이 회복되었다. 본 발명자의 분석에서 NMDS 플롯은 비만 관련 표현형(체중, 고 발린 및 콜레이트)과 비만 관련 미생물 분류(Oscillospira sp.) 및 항비만 관련 박테리아 분류(박테로이드 종, Bvul 및 Akkermansia sp.)(도 5f) 사이에서 모두 음의 상관관계를 나타냈다. 이 결과는 Bvul과 A. muciniphila가 비만 개체에서 상호 의존적이며 호스트의 대사 항상성을 균형 있게 유지하는데 도움이 된다는 것을 보여준다.To investigate metabolic changes due to HFD and Bvul treatment and to assess the importance of A. muciniphila that mediates these changes, functional prediction of intestinal microbiomes was performed and the correlation between the KEGG pathway and the microbial taxa was analyzed (FIG. 6E). HFD reduced the role of representative taxa in most metabolic pathways. However, Bvul treatment restored or improved the metabolic function of A. muciniphila and Streptococcus ssp. The sPLS-DA plot showed a clear metabolite pattern consistent with the microbial community profile (FIG. 5A) (FIG. 5D). Bvul treatment has been shown to change both taxa and functional microbiomes during obesity. However, the metabolite profiles of NDB and HFDB mice were similar, indicating that Bvul had a greater effect on metabolites than diet. Metabolism modified due to Bvul treatment is also associated with differentiation and systemic immunity of immune cells. Low concentrations of BCAAs such as Leucine and Isoleucine (FIG. 5E) caused by Bvul and A. muciniphila may explain the reduction of inflammation in adipose tissue of the HFDB group. As a result of analysis of microorganisms and metabolites in human subjects, serum BCAA levels were increased in the obese group, and BCAA may be a biomarker linking metabolites and intestinal microorganisms. In addition, co-housing studies have shown that Bacteroidales has recovered from its diet-induced phenotype due to invasion from dry to obese constitution. In our analysis, the NMDS plots are characterized by the obesity related phenotype (weight, high valine and cholate) and the obesity related microbial classification ( Oscillospira sp.) And the anti-obesity related bacterial classification (bacterial species, Bvul and Akkermansia sp.) (FIG. 5F). All showed a negative correlation. These results show that Bvul and A. muciniphila are interdependent in obese individuals and help to balance host metabolic homeostasis.
본 발명자는 Bvul과 Bvul 및 IL-21 사이의 음의 상관관계를 확인하기 위해, 조절 T 세포(Treg)와 선천성 림프성 세포(ILC)와 같은 비만을 조절하는 대표적인 면역 세포를 트윈 코호트 데이터로 분석하였다. 첫째, Bvul이 생식선 지방 세포에서 M1/M2 대식세포 분극화에 미치는 영향을 분석하였는데, M2 분극화에서 유의미한 증가를 보이지 않았다. 그러나 Il21 및 Il21r의 발현 분석 결과, 장간막 림프절(MLN)에서 Bvul에 의해 IL-21 수용체가 주로 감소하고, 장 관련 림프구 조직이 결장 내 미생물 군에서 변화를 반영한다고 확인되었다(도 7a). 더욱이, HFD 사료 마우스에서 Bvul 처리는 결장 및 MLN에서 Foxp3 및 Il10 발현 모두의 유의미한 증가를 유도하여 Bvul 처리가 면역 세포의 조성을 변화시켰음을 제시한다(도 7b). 면역 세포 프로파일을 분석하기 위해 MLN에서 Treg와 ILC를 분석하였다. Foxp3과 Il10의 증가된 발현과 일치하여 Treg는 Bvul 처리 후 MLN에서 증가했다(도 7c 및 도 7d). 흥미롭게도 ILC 조성은 Bvul의 영향을 받았다(도 7e 및 도 7f). IL-33 수용체(ST2)를 발현하는 타입 2 ILC(ILC2)는 HFD 급여에 의해 감소되었지만, Bvul 처리는 ILC2 집단을 회복시켰다. ILC는 조직에 의존적인 방식으로 존재하며 공생균에 의해 조절된다. 특히, 지방 조직에는 인슐린 저항성 및 비만 표현형과 관련된 ILC2가 포함되어 있다.To determine the negative correlation between Bvul and Bvul and IL-21, we analyzed representative immune cells, such as regulatory T cells (Treg) and congenital lymphoid cells (ILC), with twin cohort data. It was. First, we analyzed the effect of Bvul on M1 / M2 macrophage polarization in gonad fat cells, but there was no significant increase in M2 polarization. However, expression analysis of Il21 and Il21r revealed that IL-21 receptor is mainly reduced by Bvul in mesenteric lymph nodes (MLN), and that intestinal related lymphocyte tissue reflects changes in colonic microflora (FIG. 7A). Moreover, Bvul treatment in HFD feed mice induced a significant increase in both Foxp3 and Il10 expression in colon and MLN, suggesting that Bvul treatment changed the composition of immune cells (FIG. 7B). Tregs and ILCs were analyzed in MLN to analyze immune cell profiles. Consistent with the increased expression of Foxp3 and Il10 , Tregs increased in MLN after Bvul treatment (FIG. 7C and FIG. 7D). Interestingly, the ILC composition was affected by Bvul (FIGS. 7E and 7F). Type 2 ILC (ILC2) expressing IL-33 receptor (ST2) was reduced by HFD feeding, but Bvul treatment restored the ILC2 population. ILC exists in a tissue dependent manner and is regulated by symbiotic bacteria. In particular, adipose tissue contains ILC2, which is associated with insulin resistance and the obesity phenotype.
이전의 연구에 따르면 SFB의 정착은 장에서 Th17 반응을 유도하고 감염성 질환을 예방한다. Faecalibacterium prausnitzii는 장에서 Treg 축적을 유도하고 자가 면역 질환으로부터 보호한다. 일부 박테로이드 종(예, B. fragilis)과 그 세포 구성 요소는 Treg의 확대와 다양한 자가 면역 질환에 대한 보호 효과를 유발한다고 보고되어 왔다. 비만 유도된 변형된 면역 체계에서 Treg와 ILC 반응을 유도하고 지방 조직에서의 염증 반응을 감소시키는 박테리아 균주는 아직 보고되지 않았다. 다른 박테로이드 종에 의해 유발된 CD4+ T 세포 반응은 숙주의 생리학적 상태 및 면역학적 상황에 따라 독특한 패턴을 나타낸다.Previous studies have shown that the establishment of SFB induces a Th17 response in the gut and prevents infectious diseases. Faecalibacterium prausnitzii induces Treg accumulation in the intestine and protects against autoimmune diseases. Some bacteroid species (eg B. fragilis ) and their cellular components have been reported to cause widening of Tregs and protective effects against various autoimmune diseases. Bacterial strains that induce Treg and ILC responses in obesity-induced modified immune systems and reduce inflammatory responses in adipose tissue have not been reported. CD4 + T cell responses induced by other bacteroid species exhibit unique patterns depending on the host's physiological and immunological conditions.
ILC2와 Treg 세포에 대한 IL-21 효과를 다루기 위해 IL-21+ Treg와 ILC를 분석했다. Bvul 처리는 MLN에서 IL-21 수용체-발현 ILC를 증가시키지 않았다. HFD는 IL-21 수용체+ ILC를 증가시켰지만 ILC2는 IL-21에 반응하지 않았다(도 7g). 특히, ST2(-) ILC에 대한 IL-21 수용체 발현은 Bvul 처리에 의해 감소되었다. ILC의 성숙과 발달은 IL-1β, IL-12, IL-18, IL-23, IL-25 및 IL-33에 의해 조절된다. 그러나 단세포 기반의 transcriptome 분석에서 타입 1 ILC가 IL-21 반응성으로 나타났다. 따라서 IL-21 신호 전달을 담당하는 ST2(-) ILC는 ILC1일 수 있다. 또한 Treg에서 IL-21 수용체 발현을 분석했으나 차이점을 발견할 수 없었다(데이터는 표시되지 않음). 전체적으로 Bvul은 Il21r 발현을 감소시켰고 IL-21 반응성 ILC1/3을 감소시키고 비만 마우스의 MLN에서 ILC2 subset를 증가시키는 방식으로 ILC 집단을 조절하였다. 이것은 인간 연구에서 보여준 비만 표현형에서 Bvul과 IL-21 사이의 관계의 역할을 설명하는 잠재적 메커니즘을 나타낸다. 내장 microbiota의 해부학적인 제한에도 불구하고, 전신 면역 반응은 ILC에 의한 조절을 통해 특정 미생물 제제에 의해 유도된다. 본 연구에서 MLN에서 발견된 ILC subset 프로파일은 Bvul의 표적으로서 전신적인 면역학적 변화를 반영하였으며 ILC 의존적인 방식으로 비만인 동안 면역기능 장애를 조절하는 데 장내 미생물의 역할을 나타낸다.IL-21 + Tregs and ILCs were analyzed to address IL-21 effects on ILC2 and Tregs cells. Bvul treatment did not increase IL-21 receptor-expressing ILC in MLN. HFD increased IL-21 receptor + ILC but ILC2 did not respond to IL-21 (FIG. 7G). In particular, IL-21 receptor expression for ST2 (-) ILC was reduced by Bvul treatment. The maturation and development of ILC is regulated by IL-1β, IL-12, IL-18, IL-23, IL-25 and IL-33. However, uniscript based transcriptome analysis showed that type 1 ILC was IL-21 reactive. Thus, the ST2 (-) ILC responsible for IL-21 signaling may be ILC1. We also analyzed IL-21 receptor expression in Tregs, but found no difference (data not shown). Overall, Bvul regulated the ILC population by decreasing Il21r expression, decreasing IL-21 reactive ILC1 / 3 and increasing the ILC2 subset in MLN of obese mice. This represents a potential mechanism explaining the role of the relationship between Bvul and IL-21 in the obesity phenotype shown in human studies. Despite the anatomical limitations of the visceral microbiota, systemic immune responses are induced by specific microbial agents through regulation by ILC. The ILC subset profiles found in MLN in this study reflect systemic immunological changes as targets of Bvul and indicate the role of gut microorganisms in controlling immune dysfunction during obesity in an ILC dependent manner.
Bvul에 의해 유도된 면역학적 변화가 A. muciniphila의 증가된 양에 기인한 것인지를 결정하기 위해, 본 발명자는 60% HFD를 급여받고 A. muciniphila 처리된 C57BL/6J 마우스를 조사했다. 이들 마우스에서 Foxp3과 Il10의 발현은 MLN에서 증가하지 않았다. 또한, Bvul 처리에 의해 MLN에서 감소된 Il21r의 발현은 A. muciniphila 처리 마우스의 MLN에서 유의하게 증가하였으며, Bvul 및 A. muciniphila 처리가 상이한 면역학적 효과를 초래하고, 면역 조절 활성 Bvul은 A. muciniphila의 존재량을 증가시키는 능력과 무관하다.To determine if the immunological changes induced by Bvul were due to increased amounts of A. muciniphila, we examined C57BL / 6J mice fed 60% HFD and treated with A. muciniphila . The expression of Foxp3 and Il10 in these mice did not increase in MLN. In addition, the expression of Il21r decreased in MLN by Bvul treatment was significantly increased in MLN of A. muciniphila treated mice, and Bvul and A. muciniphila treatment resulted in different immunological effects, and the immunomodulatory activity Bvul is A. muciniphila. It is independent of the ability to increase the amount of
Bvul 처리된 마우스에 대한 본 발명자의 발명은 인간 Bvul 균주가 식이요법으로 유발된 비만에서 다양한 변화를 이끌어 내고 있음을 보여주는 몇 가지 증거를 기술하고 있다. 첫째, 인간 마이크로비옴 면역 프로파일링 연구 결과와 마찬가지로 Bvul은 비만과 혈청 IL-21 수준과 음의 상관관계가 있다. 둘째, Bvul은 비만 마우스의 장과 림프절에서 Treg와 ILC 반응을 유도하여 비만에 대한 새로운 면역 조절 효과를 나타낸다. 셋째, Bvul은 비만 및 비만인에게 독특한 방법으로 장내 미생물 및 미생물 대사를 재구성한다. 본 연구에서 관찰된 Bvul과 A. muciniphila 사이의 상호 작용에 대한 보다 포괄적인 이해는 복잡한 숙주-미생물 상호 작용을 더 잘 이해하고 미생물 치료제 개발로 이어질 수 있다.The inventors' invention on Bvul treated mice describes some evidence showing that human Bvul strains elicit various changes in diet-induced obesity. First, as with the results of human microbiome immune profiling studies, Bvul has a negative correlation with obesity and serum IL-21 levels. Second, Bvul induces Treg and ILC responses in the intestinal and lymph nodes of obese mice, resulting in a novel immunomodulatory effect on obesity. Third, Bvul reconstructs intestinal microorganisms and microbial metabolism in a way unique to obese and obese people. A more comprehensive understanding of the interactions between Bvul and A. muciniphila observed in this study may lead to a better understanding of complex host-microbial interactions and the development of microbial therapeutics.
실험예 Experimental Example
1) 인간 연구 집단 및 표본 수집(Human study population and sample collection)1) Human study population and sample collection
2005년 11월부터 2009년 1월까지 한국 게놈 전염병 연구의 일환으로 건강한 쌍둥이 연구에서 총 30명의 피험자를 모집하였다. 피험자의 평균 연령은 40.13±2.74세였다. 참가자들은 허리의 차이가 4 cm 이상 불일치하는 일란성 쌍둥이였다. 허리 둘레는 갈비뼈의 최 외측 테두리와 최상부 외측 장골 크레스트의 중간에서 측정되었다. 대변 샘플은 가정용 냉동고에 저장한 후 병원에 배달하여 -80℃에서 보관하였다. 혈액 샘플을 응고시키고 2000×g에서 20분간 원심 분리하고 상등액을 분석할 때까지 -80℃에서 보관하였다. 피실험자들은 이 연구에 참여하기 위한 서면 동의서를 제공했다. 인간을 대상으로 한 모든 실험은 질병 통제 센터와 서울대학교 임상 시험위원회(제144-2011-07-11호)의 승인을 받았다. 역학 및 임상 바이오마커(연령, 성별, 체중, 신장, BMI, 허리 둘레 및 허리 둘레, 알부민, AST, rGTP, dbp, sbp, 공복시 인슐린, 공복 혈당, HbA1c, 총 콜레스테롤, 트리글리세리드, HDL 콜레스테롤, LDL 콜레스테롤, hsCRP, 크레아티닌 및 요산)을 측정하여 분석하였다. From November 2005 to January 2009, a total of 30 subjects were recruited from healthy twin studies as part of the Korean Genome Epidemic. The mean age of the subjects was 40.13 ± 2.74 years. Participants were identical twins with a difference in waist difference of more than 4 cm. Waist circumference was measured midway between the outermost rim of the rib and the uppermost iliac crest. Stool samples were stored in a home freezer and delivered to a hospital and stored at -80 ° C. Blood samples were coagulated and centrifuged at 2000 × g for 20 minutes and stored at −80 ° C. until supernatant was analyzed. Subjects provided written informed consent to participate in this study. All experiments in humans were approved by the Centers for Disease Control and the Clinical Trial Committee of Seoul National University (No. 144-2011-07-11). Epidemiological and clinical biomarkers (age, gender, weight, height, BMI, waist and waist circumference, albumin, AST, rGTP, dbp, sbp, fasting insulin, fasting blood sugar, HbA1c, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol , hsCRP, creatinine and uric acid) were measured and analyzed.
2) DNA 추출 및 16S rRNA 시퀀싱2) DNA Extraction and 16S rRNA Sequencing
제조사의 프로토콜에 따라 MoBio Power Soil DNA Isolation Kit(MoBio, Solana Beach, CA, USA)와 Qiagen Faststool DNA 추출 키트(Qiagen, Valencia, CA, USA)를 사용하여 인간 및 마우스 분변 내 DNA를 추출하였다. 핵산 용액은 사용할 때까지 -70℃에서 보관하였다. 인간 분변 유래 DNA의 경우, 27F/534R 프라이머 세트를 사용하여 16S rRNA 유전자의 V1-V3 영역을 증폭시키고 454 Life Sciences FLX Titanium 플랫폼(Roche, Indianapolis, IN, USA)에서 파이로시퀀싱 하였다. 마우스 분변 유래 DNA의 경우 16S rRNA 유전자의 V4 영역을 Illumina 어댑티드 유니버설 프라이머 세트 515F/806R을 사용하여 증폭하고 MoBio UltraClean PCR Clean-Up Kit(MO BIO Laboratories, Carlsbad, CA, USA)를 사용하여 정제하였다. KAPA 라이브러리 정량 키트(KAPA Biosystems, Wilmington, MA, USA)를 사용 후 Quant-iT PicoGreen dsDNA 분석 키트(Life Technologies, Carlsbad, CA, USA)를 사용하여 PCR 증폭 결과물의 양을 이중 확인하였다. 샘플을 2×300 bp 시약 키트(Illumina, San Diego, CA, USA)를 사용하여 MiSeq 플랫폼에서 풀링하고 시퀀싱 하였다.DNA in human and mouse feces was extracted using MoBio Power Soil DNA Isolation Kit (MoBio, Solana Beach, Calif., USA) and Qiagen Faststool DNA Extraction Kit (Qiagen, Valencia, Calif., USA) according to the manufacturer's protocol. The nucleic acid solution was stored at -70 ° C until use. For human fecal-derived DNA, the 27-F / 534R primer set was used to amplify the V1-V3 region of the 16S rRNA gene and pyrosequence on the 454 Life Sciences FLX Titanium platform (Roche, Indianapolis, IN, USA). For mouse fecal-derived DNA, the V4 region of the 16S rRNA gene was amplified using Illumina adapted universal primer set 515F / 806R and purified using MoBio UltraClean PCR Clean-Up Kit (MO BIO Laboratories, Carlsbad, Calif., USA). . Quantities of PCR amplification results were double checked using a KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, Mass., USA) followed by Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, Calif., USA). Samples were pooled and sequenced on a MiSeq platform using a 2 × 300 bp reagent kit (Illumina, San Diego, Calif., USA).
3) 미생물 분석3) microbiological analysis
인간 서열 데이터는 Broad Institute에서 개발된 ABC 파이프라인에 의해 처리되었다. 파이프라인을 통해 생성된 OTU 테이블은 추가로 필터링되어 OTU를 포함한다: (i) 최소 2 개의 샘플에서 발견되고. (ii) 100 reads 이상이고, (iii) 원핵으로 분류됨. 마우스 시퀀스 데이터의 경우 OTU는 QIIME 파이프 라인을 통해 gg_13_5 Greengenes database32에 대한 폐쇄적으로 레퍼런스된 OTU 피킹(closed-referenced OUT picking)를 사용하여 선택되었다. 대표적인 서열을 PyNAST34를 사용하여 정렬하고 Ribosomal Database Project Classifier를 사용하여 분류학적 할당을 진행하였다. 키메릭 시퀀스는 ChimeraSlayer 알고리즘을 사용하여 확인되었고 OTU 테이블 생성전에 필터링되었다. 혈청 사이토카인 수준(TNF-α, TNF-β, IL-6, IL-1β, CCL20/MIP3a, IFN-γ, IL-2, IL-12(p70), IL-4, IL-5, IL-13, IL-17A, IL-21, IL-22, IL-23, IL-10, IL-27, IL-28a, IL-31, 및 IL-33), 허리 둘레를 MaAsLin(쌍둥이을 무작위 변수로, 호스트 인자를 공변량으로 사용)을 사용한 다변량 분석으로 분석하였다. 미생물 풍부도 및 안수 분석의 계산은 R 패키지 phyloseq36을 사용하여 수행되었다. 집단 변이는 마우스 분변에서 시계열 분석을 위한 비가중 UniFrac 거리 및 맹장 샘플에서 클러스터에 대한 Bray-Curtis 불일치를 사용하여 주요 좌표 분석(PCoA)에 의해 분석되었다. PERMANOVA는 비건(vegan) 패키지의 'adonis'기능을 사용하여 ND 대 NDB와 HFD 대 HFDB의 차이를 결정했다. 다이어트간에 관찰된, 변화를 유발하는 박테리아를 확인하기 위해 R 패키지 random Forest38을 사용하여 10배의 교차 검증을 통해 랜덤 포레스트 분류를 수행했다. 인간 데이터는 비교하기 전에 나이와 쌍둥이를 변형하고 조정하였다. 그룹 차이에 대한 효과 크기는 Z 점수를 N(관측 수)의 제곱근으로 나눔으로써 결정된다.Human sequence data was processed by the ABC pipeline developed at the Broad Institute. OTU tables generated through the pipeline are further filtered to include OTUs: (i) found in at least two samples. (ii) 100 reads or more, and (iii) classified as prokaryotic. For mouse sequence data, OTU was selected using a closed-referenced OUT picking to the gg_13_5 Greengenes database32 via the QIIME pipeline. Representative sequences were sorted using PyNAST34 and taxonomy assigned using the Ribosomal Database Project Classifier. The chimeric sequence was verified using the ChimeraSlayer algorithm and filtered before generating the OTU table. Serum cytokine levels (TNF-α, TNF-β, IL-6, IL-1β, CCL20 / MIP3a, IFN-γ, IL-2, IL-12 (p70), IL-4, IL-5, IL- 13, IL-17A, IL-21, IL-22, IL-23, IL-10, IL-27, IL-28a, IL-31, and IL-33), waist circumference MaAsLin (twins as random variables, Host factor as covariate). The calculation of microbial abundance and ordination analysis was performed using the R package phyloseq36. Population variation was analyzed by principal coordinate analysis (PCoA) using unweighted UniFrac distance for time series analysis in mouse feces and Bray-Curtis mismatch for clusters in caecum samples. PERMANOVA used the 'adonis' feature of the vegan package to determine the difference between ND vs NDB and HFD vs HFDB. Random forest classification was performed with 10-fold cross-validation using the R package random Forest38 to identify the bacteria that caused the changes observed between diets. Human data were modified and adjusted for age and twins before comparison. The magnitude of the effect on group differences is determined by dividing the Z score by the square root of N (observation number).
4) 장내 마이크로비옴의 기능적 추론 4) Functional Inference of Intestinal Microbiomes
다양한 식이군 간의 기능적 차이는 PICRUSt-1.0.0 (http://picrust.github.com)을 사용하여 16S-rRNA 기반 서열로부터 결정되었다. 닫힌 참조로 OTU를 피킹한 후에는 OTU가 인간 및 마우스 데이터의 경우 샘플당 2,500 및 30,000 시퀀스로 희박하였다. 신진대사 경로는 Kruskal-Wallis test와 Benjamini-Hochberg false discovery rate(FDR) 보정을 사용하여 식이 요법 그룹에 따라 비교되었다.Functional differences between the various dietary groups were determined from 16S-rRNA based sequences using PICRUSt-1.0.0 (http://picrust.github.com). After picking the OTU with closed reference, the OTU was sparse at 2,500 and 30,000 sequences per sample for human and mouse data. Metabolic pathways were compared by dietary group using the Kruskal-Wallis test and the Benjamini-Hochberg false discovery rate (FDR) correction.
5) 박테로이드 균주의 분리 및 배양5) Isolation and Culture of Bacterial Strains
19 세에서 40 세 사이의 건강한 한국인 지원자 (30 명)의 신선한 대변에서 총 182 개의 박테로이드 균주가 분리되었다. ATCC로부터 얻은 타입 균주를 사용하여 면역 조절 활성을 단리 된 균주에 대한 대조군으로 비교 하였다. 모든 182 박테로이드 종 균주, 주로 Bvul 균주로 구성된 균주를 37℃에서 24시간 동안 혐기성으로 초콜릿 한천 배양 하였다. 박테리아 세포를 모으고 13,000×g에서 5분 동안 원심 분리하여 THP-1 세포를 처리하였다.A total of 182 bacteroid strains were isolated from fresh feces of healthy Korean volunteers (30) between 19 and 40 years of age. The type of strain obtained from ATCC was used to compare the immunomodulatory activity as a control for the isolated strain. All 182 bacteroid species strains, mainly strains consisting of Bvul strains, were incubated anaerobicly with chocolate agar for 24 hours at 37 ° C. Bacterial cells were collected and centrifuged at 13,000 × g for 5 minutes to treat THP-1 cells.
6) 박테로이드 균주의 면역 조절 활성에 대한 스크리닝6) Screening for immunomodulatory activity of bacteroid strains
인간 단구 세포주 THP-1은 KCTC(Daejeon, Korea)로부터 얻어졌고, 5% CO2 대기 하에서 혈청이 없는 배지에서 37℃에서 48시간 동안 10 ng/ml PMA로 처리하여 대식세포로 분화되었다. 박테리아 세포 펠릿을 24 시간 동안 분화된 THP-1 세포에 첨가하고 세포 없는 상등액을 수집하여 분석 전에 -80℃에서 저장하였다. IL-10 및 TNF-α 수준을 ELISA 키트(BD Biosciences)로 분석하였다.Human monocyte cell line THP-1 was obtained from KCTC (Daejeon, Korea) and differentiated into macrophages by treatment with 10 ng / ml PMA for 48 hours at 37 ° C. in medium without serum under 5% CO 2 atmosphere. Bacterial cell pellets were added to differentiated THP-1 cells for 24 hours and cellless supernatants were collected and stored at −80 ° C. prior to analysis. IL-10 and TNF-α levels were analyzed with an ELISA kit (BD Biosciences).
7) 마우스 연구를 위한 실험 디자인7) Experimental Design for Mouse Research
Central Lab Animal Inc.(한국)에서 암컷 C57BL/6J 마우스(6 주령)를 얻었고 서울 대학교 의과 대학 실험 동물 연구소에서 SPF(specific pathogen-free) 조건으로 유지하였다. 1주간의 적응 후, 마우스를 박테로이드 종에 의한 집락을 줄이기 위해 일주일 동안 식수에서 암피실린(ampicillin, 1 g/L)으로 처리하고, 네 그룹으로 나누었다. 마우스에 대조군 식이(ND와 NDB, 10% 지방; n=19, Research Diets 사-D12450K) 또는 HFD(HFD와 HFDB, 60% 지방, n=19, Research Diets 사-D12492) 중 한 가지를 급여했다. 매일 준비한 Bvul 균주를 마우스 당 109 cfu의 경구 위관 영양법(NDB 및 HFDB 군, 각각 n=9)으로 투여하였다. 섭취량과 체중을 일주일에 한 번 측정했다. 대변 샘플은 5개 시점(항생제 처리전, Bvul 투여 전, 투여 후 5 일, 9 주, 18 주)에서 수집되었다. 16 주에 복막 내 당부하 검사가 수행되었다. 18주에 마우스를 희생시켰고, 조직(MLN, 간, 결장, 맹장 및 생식선 지방)을 수집하고 분석하기 전에 -80℃에서 보관했다. 혈액 샘플은 isoflurane 마취 하에 심장 천공에 의해 얻어졌다. RT에서 혈액 응고 후 30분, 혈청을 원심 분리(2,000×g, 10분, 4℃)로 분리하였다. 상등액(혈청)을 1.5 mL 튜브에 옮기고 사용할 때까지 -80℃에서 보관했다. 실험 절차는 Institutional Animal Care and Usage Committee에 의해 검토되고 승인되었다.Female C57BL / 6J mice (6 weeks old) were obtained from Central Lab Animal Inc. (Korea) and maintained under SPF (specific pathogen-free) conditions at the Laboratory Animal Laboratory of Seoul National University Medical School. After a week of adaptation, mice were treated with ampicillin (1 g / L) in drinking water for one week to reduce colonization by bacteroid species and divided into four groups. Mice received either control diet (ND and NDB, 10% fat; n = 19, Research Diets Inc.-D12450K) or HFD (HFD and HFDB, 60% fat, n = 19, Research Diets Inc.-D12492) . The daily prepared Bvul strain was administered by oral gavage of 10 9 cfu per mouse (NDB and HFDB group, n = 9 respectively). Intake and body weight were measured once a week. Stool samples were collected at five time points (before antibiotic treatment, before Bvul administration, 5 days after administration, 9 weeks, 18 weeks). At week 16, an intraperitoneal glucose tolerance test was performed. Mice were sacrificed at 18 weeks and tissues (MLN, liver, colon, cecum and gonad fat) were collected and stored at -80 ° C before analysis. Blood samples were obtained by cardiac puncture under isoflurane anesthesia. Thirty minutes after blood coagulation at RT, serum was separated by centrifugation (2,000 × g, 10 minutes, 4 ° C.). The supernatant (serum) was transferred to a 1.5 mL tube and stored at -80 ° C until use. Experimental procedures were reviewed and approved by the Institutional Animal Care and Usage Committee.
　HFD로 유도한 비만 마우스의 A. muciniphila 치료를 위해 A. muciniphila ATCC BAA-835(Akk)를 37℃에서 0.5% mucin(Sigma, USA)을 보충한 brain heart infution 한천(BD Difco, USA)을 엄격한 혐기성 조건에서 48시간 동안 배양하고, 미생물을 멸균 루프(직경 1 mm)로 수집하였다. 수집된 세포를 0.5% 시스테인이 보충된 멸균 PBS로 세척하고 10,000×g에서 5분간 원심 분리 하였다. 박테리아 펠릿을 0.5% 시스테인 및 20% 글리세롤을 보충한 PBS로 109 cfu/200 μl로 재현탁시켰다. 준비된 박테리아 용액을 사용할 때까지 -80℃에서 보관하였다. 6주된 수컷 C57BL/6J 마우스를 서울대학교 실험 동물 연구소(Institute for Experimental Animals)에서 SPF로 얻었고 유지하였다. 마우스는 새로운 환경에 적응하기 위해 개입하지 않고 1주일 동안 모으고 15주 동안 HFD(HFD 및 HFD-Akk, 60% 지방, n=20, Research Diets 사-D12492)를 먹였다. Akk 균주는 저장된 스톡 용액을 0.5% 시스테인 PBS로 세척하여 매일 제조한 다음 마우스 당 5×108 cfu의 구강 위관 영양법으로 투여하였다. 각 마우스의 체중을 시험 기간 중 일주일에 1회 측정한 후 마우스를 마취시키고, MLN 및 결장을 수집하여 표적 바이오마커(Il21, Il21r, Foxp3 및 Il10)의 발현을 측정하였다. To treat A. muciniphila in HFD-induced obese mice, brain heart infution agar (BD Difco, USA) supplemented with A. muciniphila ATCC BAA-835 (Akk) supplemented with 0.5% mucin (Sigma, USA) at 37 ° C. Incubated for 48 hours in anaerobic conditions and microorganisms were collected in a sterile loop (1 mm in diameter). The collected cells were washed with sterile PBS supplemented with 0.5% cysteine and centrifuged at 10,000 × g for 5 minutes. The bacterial pellet was resuspended at 10 9 cfu / 200 μl with PBS supplemented with 0.5% cysteine and 20% glycerol. The prepared bacterial solution was stored at -80 ° C until use. Six week old male C57BL / 6J mice were obtained and maintained with SPF at the Institute for Experimental Animals in Seoul National University. Mice were pooled for one week without intervention to adapt to the new environment and fed HFD (HFD and HFD-Akk, 60% fat, n = 20, Research Diets Inc.-D12492) for 15 weeks. The Akk strain was prepared daily by washing the stored stock solution with 0.5% cysteine PBS and then administered by oral gavage at 5 × 10 8 cfu per mouse. The weight of each mouse was measured once a week during the test period, then the mice were anesthetized, and MLN and colon were collected to measure the expression of target biomarkers ( Il21, Il21r, Foxp3 and Il10 ).
8) 마우스 조직에서의 바이오 마커 측정8) Biomarker Measurements in Mouse Tissues
MLN과 말단 결장을 5 mm 스테인레스 스틸 비드(Qiagen, Hilden, Germany)로 30 Hz에서 5~8분 동안 균질화시켰다. Easy-spin Total RNA 추출 키트(Intron, Seoul, Korea)를 사용하여 총 RNA를 분리하였다. High Capacity RNA to cDNA 키트(Applied Biosystems, Carlsbad, CA, USA)를 사용하여 cDNA를 합성하였다. 모든 qPCR 반응은 Rotor-Gene SYBR Green PCR kit(Qiagen)를 Rotor-Gene Q(Qiagen)를 사용하여 제조사의 지침에 따라 수행하였다. 상대 RNA 양은 Gapdh를 레퍼런스 트렌스크립트로 사용하여 2-ΔΔCt 방법으로 결정하였다.MLN and terminal colon were homogenized with 5 mm stainless steel beads (Qiagen, Hilden, Germany) for 5-8 minutes at 30 Hz. Total RNA was isolated using an Easy-spin Total RNA Extraction Kit (Intron, Seoul, Korea). CDNA was synthesized using the High Capacity RNA to cDNA kit (Applied Biosystems, Carlsbad, Calif., USA). All qPCR reactions were performed using the Rotor-Gene SYBR Green PCR kit (Qiagen) using the Rotor-Gene Q (Qiagen) according to the manufacturer's instructions. Relative RNA amounts were determined by the 2-ΔΔCt method using Gapdh as the reference transscript.
9) 1H NMR 분광법9) 1 H NMR spectroscopy
NMR 기반의 대사 분석을 위해 샘플을 Lamichhane의 방법에 따라 약간 수정하여 제조하였다. 간략하게, 췌장 샘플(약 130 mg)을 DDW 600 μl와 혼합하고, 30초 동안 볼텍싱하고, 조직 균질화기에 의해 균질화시켰다. 10분 동안 원심 분리 (14,000×g, 4℃)한 후 0.025 mg/mL 3-(트리메틸 실릴) 프로피온산-d4 나트륨 염(TSP)을 함유하는 60 μl의 중수소산화물(D2O), 1 mM 이미다졸 60 μl, 2 mM NaN3 60 μl 및 0.5M KH2PO4 120 μl를 상등액 300 μl에 용매(lock solvent)로 첨가하였다. 혼합물을 1분 동안 볼텍싱하고 14,000×g에서 10분 동안 원심 분리하였다. 맑은 상등액을 NMR 분석을 위해 5 mm NMR 튜브(Wilmad-Lab glass, UK)로 옮겼다. 모든 1H-NMR 스펙트럼은 콜드 플로우 프로브가 장착 된 Varian 500 MHz NMR 시스템(Varian, Palo Alto, CA, USA) 분광계를 사용하여 수집되었다. 1H-NMR 스펙트럼은 water presaturation pulse sequence를 사용하여 25℃에서 수집되었다. 4초의 획득 시간과 2초의 재활용 지연을 사용하여 64개의 transient로 스펙트럼을 수집하였다. 1H NMR 신호의 임시 할당은 Chenomx NMR Suite 8.3(Chenomx, Canada)를 사용하여 Human Metabolome Database 및 이전 문헌에 따라 수행되었다. 콜라겐(collagen), 크레아틴(creatine), 페룰레이트(ferulate), 포르메이트(formate), 푸마레이트(fumarate), 글루코스(glucose), 글루타메이트(glutamate), 글리신(glycine), 글리코콜레이트(glycocholate), 구아니도 아세테이트(guanidoacetate), 하이포크산틴(hypoxanthine), 이소 부티레이트(isobutyrate), 아스코르브산(ascorbic acid), N-아세틸 글루코사민(N-acetylglucosamine), N-아세틸티로신(N-acetyltyrosine), O-포스포 콜린(O-phosphocholine), p-크레졸(p-cresol), 페닐알라닌(phenylalanine), 프롤린(proline), 프로피오네이트(propionate), 퓨트레신(putrescine), 피루베이트(pyruvate), 사르코신(sarcosine), 석시네이트(succinate), 글루타르산(glutaric acid), 트라이메틸아민(trimethylamine), 트리메틸아민 N-옥사이드(trimethylamine N-oxide), 트립토판(tryptophan), 티로신(tyrosine), 우라실(uracil), 우로카네이트(urocanate), 발레레이트(valerate), 발린(valine) 및 크산틴(xanthine)을 분석을 위해 할당하였다. 그룹 간의 차이는 분산 분석 (ANOVA)과 Fisher's post hoc test에 의해 결정되었고, P<0.05는 유의한 것으로 간주되었다. 미생물 데이터를 표준화하고 희소 부분 편미분 판별분석(sPLS-DA)을 사용하여 모델링하기 위해 MetaboAnalyst 3.0에 업로드하였다. 높은 부하 수치를 갖는 대사 산물을 평가하여 모델 내 차별 정도에 대한 각 실험 그룹의 순위를 매겼다.Samples were prepared for NMR based metabolic analysis with slight modifications according to Lamichhane's method. Briefly, pancreatic samples (about 130 mg) were mixed with 600 μl of DDW, vortexed for 30 seconds, and homogenized by a tissue homogenizer. 60 μl of deuterium oxide (D 2 O), 1 mM Al containing 0.025 mg / mL 3- (trimethylsilyl) propionic acid-d4 sodium salt (TSP) after centrifugation (14,000 × g, 4 ° C.) for 10 minutes. 60 μl of dozol, 60 μl of 2 mM NaN 3 and 120 μl of 0.5M KH 2 PO 4 were added to 300 μl of the supernatant as a solvent. The mixture was vortexed for 1 minute and centrifuged for 10 minutes at 14,000 × g. The clear supernatant was transferred to 5 mm NMR tubes (Wilmad-Lab glass, UK) for NMR analysis. All 1H-NMR spectra were collected using a Varian 500 MHz NMR system (Varian, Palo Alto, CA, USA) spectrometer equipped with a cold flow probe. 1 H-NMR spectra were collected at 25 ° C. using a water presaturation pulse sequence. Spectra were collected in 64 transients using a 4 second acquisition time and a 2 second recycle delay. Temporal assignment of 1H NMR signals was performed according to the Human Metabolome Database and previous literature using Chenomx NMR Suite 8.3 (Chenomx, Canada). Collagen, creatine, ferulate, formate, fumarate, glucose, glutamate, glycine, glycine, glycocholate, sphere Anyo acetate (guanidoacetate), hypoxanthine, isobutyrate, ascorbic acid, N-acetylglucosamine, N-acetyltyrosine, N-acetyltyrosine, O-phospho Choline (O-phosphocholine), p-cresol, phenylalanine, proline, propionate, putrescine, pyruvate, sarcosine ), Succinate, glutaric acid, trimethylamine, trimethylamine N-oxide, tryptophan, tyrosine, tyrosine, uracil, Urocanate, valerate, valine And xanthine were assigned for analysis. Differences between groups were determined by analysis of variance (ANOVA) and Fisher's post hoc test and P <0.05 was considered significant. The microbial data was uploaded to MetaboAnalyst 3.0 for standardization and modeling using rare partial partial differential discriminant analysis (sPLS-DA). Metabolites with high loading values were evaluated to rank each experimental group for the degree of discrimination in the model.
10) 조직 준비 및 유동 세포 계측법10) Tissue Preparation and Flow Cytometry
MLN과 생식선 지방 조직을 분쇄하고 37℃에서 1시간 동안 1.6 mg/mL 콜라겐 분해효소(collagenase) 제4형(Worthington, Lakewood, NJ) 및 0.1% DNase I(분획 IX; Sigma)로 소화시켰다. 전체 세포를 처리하여 적혈구를 용해시킨 다음 FACS 분석을 위해 염색하였다. 단일 세포 현탁액을 항-FcγR 차단 단클론 항체(2.4G2)와 미리 배양하고 염색 전에 세척하였다. 세포를 다음 항체로 염색하였다: fluorescein isothiocyanate-접합 항-CD19(6D5 (115506); BioLegend), 항-CD11b(M1/70 (101206), BioLegend), 항-CD11c(HL3(553801); BD Biosciences), 항-F4/80(BM8 (123108); BioLegend), 또는 항-FcεR1(MAR-1 (134306); BioLegend)(이들 FITC 접합된 항체는 계통 마커로서 사용됨), 마우스 CD45에 대한 phycoerythrin-Texas red-접합 항체(MCD4517; Invitrogen); phycoerythrin-접합 항-CD1d(1B1 (123509); BioLegend); phycoerythrin-접합 항-CD44(1M7 (103009); BioLegend); allophycocyanin-접합 항-c-Kit(2B8 (105812); BioLegend); allophycocyanin-접합 항-CD196(CCR6)(29-2L17 (129812); BioLegend); allophycocyanin-cyanine 5.5-접합 항-CD25(PC61 (551071); BD Biosciences); 알로피코시아닌-시아닌 5.5-접합 항-NKp46(29A1.4 (137609); BioLegend); 항-CD90.2(항-Thy-1.2; 53-2.1 (17-0902-81); eBioscience); Alexa Fluor 647-접합 항-CD206(MR5D3 (123010); BioLegend); Alexa Fluor 700-접합 항 Sca-1(D7 (56-5981-82), eBioscience); Alexa Fluor 700-접합 항-CD4(GK1.5 (100430); BioLegend); 및 Alexa Fluor 780-접합 항-CD11c(N418 (47-0114-82); eBioscience). 마우스 면역 세포를 다음의 항체로 염색하였다: phycoerythrin-Texas red-접합 항-인간 CD45(MHCD4517; Invitrogen); fluorescein isothiocyanate-접합 항-CD3(항-CD3; UCHT1 (555332); BD Biosciences); 항-CD19(H1B19 (302206), BioLegend); 항-CD11b (ICRF44 (11-0118-42); eBioscience); 항-CD14(HCD14 (325604); eBioscience) 또는 항-FcεR1(MAR-1 (134306), BioLegend), Alexa Fluor 647-접합 항-CD127(eBioRDR5 (51-1278-73); eBioscience).MLN and gonad adipose tissue were ground and digested with 1.6 mg / mL collagenase type 4 (Worthington, Lakewood, NJ) and 0.1% DNase I (fraction IX; Sigma) at 37 ° C. for 1 hour. Whole cells were treated to lyse red blood cells and then stained for FACS analysis. Single cell suspensions were preincubated with anti-FcγR blocking monoclonal antibody (2.4G2) and washed before staining. Cells were stained with the following antibodies: fluorescein isothiocyanate-conjugated anti-CD19 (6D5 (115506); BioLegend), anti-CD11b (M1 / 70 (101206), BioLegend), anti-CD11c (HL3 (553801); BD Biosciences) , Anti-F4 / 80 (BM8 (123108); BioLegend), or anti-FcεR1 (MAR-1 (134306); BioLegend) (these FITC conjugated antibodies are used as lineage markers), phycoerythrin-Texas red for mouse CD45 Conjugated antibody (MCD4517; Invitrogen); phycoerythrin-conjugated anti-CD1d (1B1 (123509); BioLegend); phycoerythrin-conjugated anti-CD44 (1M7 (103009); BioLegend); allophycocyanin-conjugated anti-c-Kit (2B8 (105812); BioLegend); allophycocyanin-conjugated anti-CD196 (CCR6) (29-2L17 (129812); BioLegend); allophycocyanin-cyanine 5.5-conjugated anti-CD25 (PC61 (551071); BD Biosciences); Allophycocyanin-cyanine 5.5-conjugated anti-NKp46 (29A1.4 (137609); BioLegend); Anti-CD90.2 (anti-Thy-1.2; 53-2.1 (17-0902-81); eBioscience); Alexa Fluor 647-conjugated anti-CD206 (MR5D3 (123010); BioLegend); Alexa Fluor 700-conjugated anti Sca-1 (D7 (56-5981-82), eBioscience); Alexa Fluor 700-conjugated anti-CD4 (GK1.5 (100430); BioLegend); And Alexa Fluor 780-conjugated anti-CD11c (N418 (47-0114-82); eBioscience). Mouse immune cells were stained with the following antibodies: phycoerythrin-Texas red-conjugated anti-human CD45 (MHCD4517; Invitrogen); fluorescein isothiocyanate-conjugated anti-CD3 (anti-CD3; UCHT1 (555332); BD Biosciences); Anti-CD19 (H1B19 (302206), BioLegend); Anti-CD11b (ICRF44 (11-0118-42); eBioscience); Anti-CD14 (HCD14 (325604); eBioscience) or anti-FcεR1 (MAR-1 (134306), BioLegend), Alexa Fluor 647-conjugated anti-CD127 (eBioRDR5 (51-1278-73); eBioscience).
11) 통계 분석11) Statistical Analysis
모든 데이터는 Prism 5(GraphPad Software, San Diego, CA) 또는 R 소프트웨어(버전 3.1.2)로 분석하였다. 클러스터링 분석은 R의 히트맵 패키지를 사용하여 수행되었다. 통계적 유의성은 Mann-Whitney 테스트를 사용하여 두 그룹을 비교하고 Kruskal-Wallis 테스트를 수행한 후 post-hoc Tukey의 테스트를 통해 세 개 이상의 그룹을 비교하여 테스트하였다. 그래픽적 표현에서, 데이터는 평균±표준 오차로 제시되었다. 각 box plot의 수염은 10~90 백분위 수를 나타낸다. 통계적 유의성은 *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001로 나타냈다.All data were analyzed with Prism 5 (GraphPad Software, San Diego, Calif.) Or R software (version 3.1.2). Clustering analysis was performed using R's heatmap package. Statistical significance was tested by comparing the two groups using the Mann-Whitney test, performing the Kruskal-Wallis test, and then comparing three or more groups by the post-hoc Tukey test. In the graphical representation, the data are presented as mean ± standard error. The whiskers in each box plot represent 10 to 90 percentiles. Statistical significance was indicated by * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
기탁기관명 : 한국생명공학연구원Depositary: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13276BPAccession number: KCTC13276BP
수탁일자 : 2017529Deposit Date: 2017529
Figure PCTKR2019004790-appb-I000001
Figure PCTKR2019004790-appb-I000001

Claims (9)

  1. 식이-유발성 체중 증가, 포도당 불내성 또는 지방 조직 염증을 감소시키는 것을 특징으로 하는 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주(기탁번호 KCTC 13276BP). Bacteroides vulgatus SNUG40005 strain (Accession No. KCTC 13276BP), characterized by reducing dietary-induced weight gain, glucose intolerance or adipose tissue inflammation.
  2. 제 1항에 있어서, 상기 균주는 IL-12 수용체 발현 및 IL-12 수용체를 발현하는 선천성 림프성 세포(innnate lymphoid cell)를 감소시키는 것을 특징으로 하는 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주.The bacteroid Bacteroides vulgatus SNUG40005 strain according to claim 1, wherein the strain reduces IL-12 receptor expression and innate lymphoid cells expressing IL-12 receptor.
  3. 제 1항에 있어서, 상기 균주는 항-비만 박테리아 아커멘시아 뮤시니필리아(Akkermansia muciniphila) 균주를 유지시키는 것을 특징으로 하는 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주.The method of claim 1, wherein the strain is characterized by maintaining the anti-obesity bacteria Akkermansia muciniphila strain Bacteroides vulgatus SNUG40005 strain.
  4. 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 유효성분으로 하는 대사성 질환 치료 또는 예방용 약학적 조성물. Bacteroides vulgatus (Bacteroides vulgatus ) A pharmaceutical composition for treating or preventing metabolic diseases comprising the SNUG40005 strain.
  5. 제 4항에 있어서, 상기 대사성 질환은 비만, 당뇨병, 고지혈증, 고콜레스테롤증, 동맥경화증, 지방간 또는 심혈관질환인 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 4, wherein the metabolic disease is obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
  6. 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 유효성분으로 하는 대사성 질환 개선용 건강기능성 식품. Bacteroides vulgatus (Bacteroides vulgatus ) A health functional food for improving metabolic diseases comprising the SNUG40005 strain.
  7. 제 6항에 있어서, 상기 대사성 질환은 비만, 당뇨병, 고지혈증, 고콜레스테롤증, 동맥경화증, 지방간 또는 심혈관질환인 것을 특징으로 하는 건강기능성 식품.7. The health functional food according to claim 6, wherein the metabolic disease is obesity, diabetes, hyperlipidemia, hypercholesterolemia, arteriosclerosis, fatty liver or cardiovascular disease.
  8. 유효한 양의 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주를 이것이 필요한 개체에게 투여하여 대사성 질환을 예방, 개선 또는 치료하는 방법.A method of preventing, ameliorating or treating a metabolic disease by administering to a subject in need thereof an effective amount of Bacteroides vulgatus SNUG40005 strain.
  9. 대사성 질환을 예방, 개선 또는 치료하기 위한 조성물의 제조를 위한 박테로이드 불가투스(Bacteroides vulgatus) SNUG40005 균주의 용도.Use of the bacteroid Bacteroides vulgatus SNUG40005 strain for the preparation of a composition for preventing, ameliorating or treating a metabolic disease.
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