KR102225939B1 - Composition for preventing or treating immunometabolic diseases comprising Bacteroides vulgates - Google Patents

Abstract

The novel strain of the present invention Bacteroides vulgatus SNUG40005 strain is essential for maintaining the anti-obesity bacterium Akkermansia muciniphila, and forms and maintains a healthy microbial community, thereby preventing obesity and immune metabolic dysfunction. Show ability. In addition, Bacteroides vulgatus SNUG40005 strain is capable of reducing IL-12 receptor expression and IL-12 receptor-expressing ILC cells, as well as regulatory T cells and type-2 congenital lymphoid cell immunity, as well as intestinal immunity. Since it entails rapid recovery of the microbial structure and metabolic product profile, it can be used as a composition for preventing, treating or improving metabolic diseases including obesity.

Description

A novel Bacteroides vulgatus strain and a composition for preventing or treating immune and metabolic diseases using the same as an active ingredient {Composition for preventing or treating immunometabolic diseases comprising Bacteroides vulgates}

The present invention relates to the Bacteroides vulgatus (Bacteroides vulgatus) SNUG40005 strain (Accession No. KCTC 13276BP) having an effect of preventing, treating or improving metabolic diseases and uses thereof.

Gut dysbiosis interferes with immune homeostasis and promotes the development of various inflammatory and metabolic diseases. Changes in obesity-related microbes lead to an increase in intestinal permeability and circulating endotoxin levels, leading to a low degree of systemic inflammation. However, it is still unclear how members of the microbial community interact with each other and how they interact with the host immune system to prevent or protect obesity.

Obesity disturbs metabolism and immune homeostasis, as well as the balance of intestinal microbiota. It has been reported that the gut microbiota modified in obesity has an important link between metabolism and immune dysfunction. For example, insulin resistance is induced by toxins in the serum absorbed from the'leaky gut' and increases systemic inflammation. Certain bacterial classes (texa) (eg, Akkermansia muciniphila) have been shown to modulate these symptoms by reducing endotoxemia and promoting the accumulation of regulatory T cells in adipose tissue. However, no bacterial taxa have been reported associated with an adaptive immune response in obesity and weight control.

In an inconsistent twin analysis for obesity, we identified Bacteroides vulgatus (Bvul) as a major symbiotic species associated with reduced clinical indicators and an altered immune correlation of obesity. It was confirmed that the mice administered Bvul repeatedly showed the benefits shown to humans by reducing diet-induced weight gain, glucose intolerance, and adipose tissue inflammation. These advantages entail dramatic restoration of the structure and metabolite profile of the gut microbiota, as well as regulatory T cell and type-2 innate lymphoid cell immunity. According to the relationship between Bvul and IL-21 in humans, mice treated with Bvul showed a decrease in IL-21 receptor expression and IL-21 receptor expressing ILC cells in mesenteric lymph nodes. In addition, Bvul was essential for maintaining the anti-obesity bacterium Akkermansia muciniphila. These results imply the ability of the symbiotic Bacteroides species to prevent obesity and immune metabolic dysfunction by forming and maintaining a healthy microbial community.

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Olga Castaner, et al., "The Gut Microbiome Profile in Obesity: A Systematic Review," International Journal of Endocrinology, Volume 2018 (March 22, 2018)

The present inventors identified the strain Bacteroides vulgatus (Bvul) of the present invention as a major symbiotic species associated with a reduced clinical indicator and an immune correlation of altered obesity. Mice administered Bvul have repeatedly demonstrated the benefits seen in humans by reducing diet-induced weight gain, glucose intolerance, and adipose tissue inflammation. This advantage was accompanied by a dramatic restoration of the structure and metabolite profile of the gut microbiota, as well as regulatory T cell and type-2 innate lymphoid cell immunity. According to the relationship between Bvul and IL-21 in humans, mice treated with Bvul showed a decrease in IL-21 receptor expression and IL-21 receptor expressing ILC cells in mesenteric lymph nodes. In addition, Bvul was essential for maintaining the anti-obesity bacterium Akkermansia muciniphila. Through these results, the ability of the symbiotic Bacteroides species to prevent obesity and immune metabolic dysfunction by forming and maintaining a healthy microbial community was confirmed, and the present invention was completed.

Bacteroides spp is an early colonizing bacteria in the mammalian gut and is known to form and regulate the host's immune system and metabolic activity. The capsular polysaccharide of B. fragilis regulates regulatory T cells and prevents inflammatory diseases including inflammatory bowel disease, autoimmune disease and CNS demyelinating disease. Bacteroides species also regulate host physiology through metabolite production. For example, the results of genomic analysis of B. thetaiotaomicron indicate host niche and metabolic potential to produce bacterial metabolites that use a variety of dietary nutrients and can be reused by the host. The effects of Bacteroides spp. also include the restoration of microbial intestinal imbalances during the pathogenesis of obesity. However, immunological and metabolic changes induced by Bvul as an intestinal symbiont have not yet been reported, particularly with respect to obesity.

In order to solve the above problems, the present invention provides a Bacteroides vulgatus (Bacteroides vulgatus) SNUG40005 strain.

In addition, the strain can reduce diet-induced weight gain, glucose intolerance or adipose tissue inflammation.

The strain can reduce IL-12 receptor expression and IL-12 receptor-expressing ILC cells.

In addition, the strain can maintain the anti-obesity bacteria Akkermansia muciniphila (Akkermansia muciniphila) strain.

The present invention provides a pharmaceutical composition for the treatment or prevention of metabolic diseases using Bacteroides vulgatus SNUG40005 strain as an active ingredient.

The metabolic disease may be any one of obesity, diabetes, hyperlipidemia, hypercholesterolosis, arteriosclerosis, fatty liver or cardiovascular disease.

The present invention provides a health functional food for improving metabolic diseases using Bacteroides vulgatus SNUG40005 strain as an active ingredient.

The metabolic disease may be obesity, diabetes, hyperlipidemia, hypercholesterolosis, arteriosclerosis, fatty liver or cardiovascular disease.

The novel strain of the present invention Bacteroides vulgatus SNUG40005 strain is essential for maintaining the anti-obesity bacterium Akkermansia muciniphila, and forms and maintains a healthy microbial community, thereby preventing obesity and immune metabolic dysfunction. Show ability. In addition, Bacteroides vulgatus SNUG40005 strain is capable of reducing IL-12 receptor expression and IL-12 receptor-expressing ILC cells, as well as regulatory T cells and type-2 congenital lymphoid cell immunity, as well as intestinal immunity. Since it entails rapid recovery of the microbial structure and metabolic product profile, it can be used as a composition for preventing, treating or improving metabolic diseases including obesity.

1 is a result of an experiment of correlation between obesity status and immune profile and Bvul abundance in mismatched MZ twins. (a) is the outline of the experiment in this study, and (b) (c) is the difference in the abundance of major genera and the result of the serum IL-12 level in twins with mismatched waist circumference. (d)(e) The significant changes in Bvul are for the significant changes in Bvul analyzed by multivariate association with a linear model (MaAsLin) that explains the waist circumference and serum IL-21 levels. Waist_low and Waist_high mean that the difference between the waist circumference of discordant twins is less than 4 cm and more than 4 cm, respectively. For IL-21, P means more than 0.28 pg/ml and N means less than 0.28 pg/ml in serum.
In FIG. 2 (a) is a hierarchical clustering result of the correlation between serum cytokine levels and intestinal bacterial phyla in Korean discordant twins. The heatmap was created based on the correlation coefficient. The tree was plotted using the Euclidean method and separated into two arms for cytokine profiles. (b)(c) shows the results of analysis of potential functional traits of intestinal microbes through PICRUSt.
3 is an experimental result of Bvul SNUG 40005 strain reducing obesity status and glucose tolerance in HFD-induced obese mice. (a) It is a diagram of the mouse phenotype. (b) It is a diagram of the weight measurement value for each group. (c) A diagram of the measured values of fasting blood glucose levels for each group. (d) A diagram of the IPGTT results. (e) A diagram of the IPGTT AUC values of C57Bl6 female mice treated with Bvul for 16 weeks. (f) It is a diagram of the result value for the weight of gonadal adipose tissue by group. (g) It is a diagram of the result of measuring the size of adipocytes by group. (h) It is the result of measuring the gene expression change for low-grade inflammation in the gonadal tissue for 18 weeks after treatment with Bvul. The measurements are expressed as mean±SD, and measurements with different superscripts appear to be quite different according to the 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). ND/B: Control diet mice injected with Bvul every day (n=9). HFD: HFD mice (60% fat, n=10). HFD/B: HFD mice injected with Bvul every day (n=9).
Figure 4 (a) It is a diagram for the inflammation control effect (IL-10/THF-a ratio in the differentiated THP-1 macrophage) of the bacterial strains isolated from mouse and human fecal samples. The IL-10/THF-a ratio was calculated after treatment with 10 different strains of Bacteroides spp., P. goldsteinii , and Akkermansia muciniphila. (b) This is a scanning microscope drawing of Bacteroides vulgatus SNUG 40005.
Figure 5 is for Bvul SNUG 40005 that recovers A. muciniphila depleted from obesity and regulates intestinal microorganisms and metabolites, (a) is unweighted Results for PCoA using UniFrac metrics and (b) are heat map results of 25 major categories (texa) based on different dietary groups in the cecum. (c) The classification result of HFD and Bvul treatment by the amount of bacteria present. This is for the top 15 bacteria in each group. (d) It is about sPLS-DA plot based on the quantitative value of 80 cecal metabolites. (f) PCoA results based on the top 10 bacteria with Bray-Curtis dissimilarity and major cecal metabolites. The arrow indicates a strong significant correlation, the cecal microbe at the level of the point, and the color gradient indicates the weight of the mouse.
In Figure 6, (a) is the result of PCoA using unweighted UniFrac metrics, and (b) is the result of analysis of the abundance of microbial community based on administration time (fecal microempty) (Pre: 1 week before Bvul administration; Day 0: administration start point after 1 week of antibiotic treatment; Day 5, Wk 9, Wk 18: after 5 days, 9 weeks, and 18 weeks of Bvul administration). (c) It is the result of the shift value of the representative microbial classification (texa). (d) is the classification result of HFD administration and Bvul treatment by the amount of microorganism present. (e) is the functional classification result of HFD administration and Bvul treatment by KEGG pathway analysis using PICRUSt. (f) is the measurement result of cholate in each group, and (g) is the measurement result of the abundance of N-acetylglucosamine metabolite in each group. (ND: mice on a control diet (10% fat, n) =10).NDB: mice on a control diet administered Bvul daily (n=9).HFD: mice on a HFD (60% fat, n=10).HFDB: mice on a HFD administered Bvul daily (n=9) .)
7 is for Bvul SNUG 40005, which induces Treg and IL-21-independent ILC2 responses in HFD-induced obesity. In MLN, (a) 1121 and 1121r and (b) Foxp3 and 1110 expression results, MLN In (c,d) CD4 + Foxp3 + Treg cells, (e, f) whole ST2 + ILC cells (g) IL-21 + ILC cells, IL-21 + ST2 (-) ILC cells and -21 + in IL MLN ST2 (+) flow cytometric analysis of ILC cells, (h) CD11c + CD206 + M2 polar macrophages and (i) M2 / M1 macrophage ratio in reproductive adipose tissue ( .* P <0.05, * * P <0.01, *** P <0.001, and **** P <0.0001.ND: mice on a control diet (10% fat, n=10).NDB: mice on a control diet administered Bvul daily (n =9).HFD: mice on a HFD (60% fat, n=10).HFDB: mice on a HFD administered Bvul daily (n=9).HFD_Akk: mice on a HFD administered Akk daily for 15 weeks (n= 10)).

To investigate key bacteria involved in human metabolic and immune profiles, intestinal microbial composition and serum cytokine profiles (16s rRNA gene profiling and multiplex cytokine analysis on fecal and blood samples of 30 twins) were used. The correlation with Th1, Th2, Th17, Tregs and 20 cytokines related to inflammation) was investigated (see Fig. 1A).

Hierarchical cluster analysis revealed that certain microbial taxa, including Bacteroides and Oscillospira, negatively correlated with the levels of Th17-related inflammatory cytokines such as IL-17A, IL-21, CCL20, IL-27 and TNF-α. . As a result of analyzing using the body mass index (BMI) as a quantitative measure of obesity, a distinct intestinal microbial composition was found in twins who were inconsistent with obesity. However, there was no significant difference in the serum cytokine profile between the twin samples with inconsistent BMI (FIG. 2A ). The inventors investigated MZ twins with significant differences in waist circumference as an indicator of obesity. This variable is due to better capturing the correlation between the gut microbial texa and biomarkers associated with obesity and inflammation. Bacteroides spp., Faecalibacterium sp. And Bifidobacterium spp were lower in twins with a larger waist circumference (4 cm or more) than in other twins (see FIG. 1B).

The general taxa associated with the serum cytokine profile and obesity-related variables was Bacteroides spp., which is associated with low serum levels of IL-21 (Fig. 1c). Target species of the genus Bacteroides, which showed significantly different abundances between twins with inconsistent individual clinical variables and serum cytokine levels, were multivariately correlated with a linear model (adjusted according to age and sex, as well as random variables of twins, MaAsLin). Reanalyzed by OUT (operational taxonomic unit) representing Bacteroides vulgatus (Bvul) was significantly associated with obesity and serum immunity profiles (FIGS. 1D and 1E ). Functional microbiome analysis of stool samples showed significant differences between the high and low waist groups in fatty acid and amino acid metabolism pathways, especially branched chain amino acid (BCAA) degradation pathways. In addition, aromatic amino acids and lipid metabolism pathways differed between the high level (P) and low level (N) groups of IL-21 (FIGS. 2B and 2C ).

Immunity and intestinal 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, the present inventors investigated the effect of Bvul administration in C57BL/6J female mice fed a high fat diet (HFD, 60% fat) and a control low fat diet (10% fat). Given that the effect of intestinal microflora on host physiology varies with bacterial strains, the present inventors initially isolated several strains of Bvul from unrelated healthy adult Koreans. Because Bvul is a very abundant symbiotic in mouse gut, the strain is also isolated from mouse feces. Groups of mice fed three types of diets with different fat content (10%, 45%, 60%) were used to isolate Bvul species and determine the effect of diet on metabolic potential. A total of 182 Bacteroides strains isolated from humans and mice, including Bvul type ATCC 8482, were screened for immunomodulatory activity in vitro. As a marker, since obesity is well known to cause metabolic inflammation, pro-inflammatory and anti-inflammatory cytokine production of THP-1 cell-derived macrophages was measured. Bvul SNUG 40005 (hereinafter referred to as Bvul) was selected based on the immunogenicity that induces a high level 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).

Conventional cluster studies using gnotobiotic mice have reported that the same species of Bacteroides spp cannot replace already clustered strains due to the niche shared in the intestine. The present inventors were first administered with an antimicrobial agent (a mixture of ampicillin (A), vancomycin (V), neomycin (N), metronidazole (M), streptomycin, penicillin, kanamycin, and AVNM) for 1 week. Based on the results, we pretreated mice for 1 week with ampicillin to reduce the existing Bvul strain, and promoted the colonization of Bvul isolated from humans in the mouse intestine.

Starting from 5 weeks after Bvul administration, the body weights of the HFD-fed and Bvul-treated groups (HFDB) were significantly lower than those of the group fed HFD but no Bvul administration (HFD). After 18 weeks, the average body weight of the HFDB group was about 30% lower than the average body weight of the HFD group (FIGS. 3A and 3B ). The weight of vocal cord fat was 40% lower (Fig. 3f), and the size of adipocytes in the NDB and HFDB groups was smaller than that of 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, even in the normal diet group, Bvul treatment suppressed the increase in blood sugar. This dramatic effect of Bvul treatment on obesity-induced metabolic changes in rats is consistent with a negative correlation between Bvul and the human obesity phenotype. In this study, the inventors demonstrated the anti-obesity effect of Bvul for the first time in both humans and mice, and confirmed the ability to modulate the visceral microbiome and immune system. These results are consistent with previous studies of other Bacteroides 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.

To investigate the changes induced by Bvul in intestinal microflora and metabolism, microbial and metabolite analyzes were performed on cecal and fecal samples from mice fed HFD. As a result, HFD significantly changed the microbial community (FIG. 5A). The microbial structures of the NDB and HFDB groups showed similarity 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 effect of ampicillin pretreatment 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 was rapidly recovered by Bvul treatment (FIGS. 5B and 6C ). A. muciniphila is a muco-dwelling bacterium that affects metabolic profile and glucose homeostasis by improving intestinal barrier dysfunction and inflammation in metabolic diseases.

Other taxa such as Oscillospira and Streptococcus were also affected by the administration of HFD and Bvul. Oscillospira showed the opposite pattern to A. muciniphila, increased in HFD group, rapidly decreased in HFDB group, and increased in NDB group compared to NB group. Streptococcus and Eubacterium also showed opposite patterns. Streptococcus was rarely found in the intestines of rats, but Bvul treatment increased the amount.

Eubacterium was abundant in the intestines of rats, but treatment with HFD and Bvul reduced the amount. Interestingly, the abundance of OTU representing the administered species did not show a significant difference between the groups (Fig. 6c), and for obesity. The strain-specific effects on Bvul treatment and microempty were described. The natural mouse-column Bvul strain was ineffective during obesity progression. In addition, the amount of A. muciniphila was reduced by Bvul treatment in non-obese mice fed a low-fat diet. As a result of functional microbial analysis, changes in microbial metabolism were most evident in the NDB group than in the HFDB group (Fig. 6e). These results indicate that the remodeling of the microbial structure induced by Bvul is dependent on the obesity state of the host and leads to a unique microbial community profile. In the pathogenesis of chronic diseases, bacterial interaction with the microbial ecosystem is considered to be an important factor. The concept of microbiome therapy based on synergism and antagonism in complex microbiota has recently emerged. The present inventor's research is on the function of Bvul to restore the imbalance of the intestinal flora and restore host physiology through such a microbial remodeling method.

To investigate metabolic changes due to HFD and Bvul treatment, and to evaluate the importance of A. muciniphila to mediate these changes, functional prediction of the Gut microbiome 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, treatment with Bvul 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 alter both taxa and functional microbiome during obese people. However, the metabolite profiles of NDB and HFDB mice were similar, so Bvul had a greater effect on metabolites than diet. The metabolism modified by Bvul treatment is also related to the differentiation of immune cells and systemic immunity. The low concentration of BCAA by Bvul and A. muciniphila (Figure 5e) may explain the reduction of inflammation in the 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 biological marker linking metabolites and intestinal microbes. In addition, according to a co-housing study, the diet-induced phenotype was recovered due to the invasion of Bacteroidales from skinny to obese constitution. In our analysis, the NMDS plot shows the obesity-related phenotype (weight, high valine, and cholate) and the obesity-related microbial classification (Oscillospira sp.) and the anti-obesity-related bacterial classification (Bacteroides sp.Bvul and Akkermansia sp.) (Fig.5f). These results show that Bvul and A. muciniphila are interdependent in obese individuals and help to balance host metabolic homeostasis.

In order to confirm the negative correlation between Bvul and Bvul and IL-21, the present inventors analyzed representative immune cells regulating obesity such as regulatory T cells (Tregs) and innate lymphocytes (ILCs) using twin cohort data. . First, we analyzed the effect of Bvul on M1/M2 macrophage polarization in gonadal adipocytes, but there was no significant increase in M2 polarization. However, as a result of the expression analysis of Il21 and Il21r, it was confirmed that IL-21 receptors were mainly decreased by Bvul in mesenteric lymph nodes (MLN), and that intestinal-related lymphocyte tissues reflected changes in the microbial group in the colon (Fig. 7a). Moreover, Bvul treatment in HFD feed mice induced a significant increase in both Foxp3 and Il10 expression in the colon and MLN, suggesting that Bvul treatment changed the composition of immune cells (Fig. 7b). Tregs and ILCs were analyzed in MLNs to analyze the immune cell profile. Consistent with the increased expression of Foxp3 and Il10, Tregs increased in MLN after Bvul treatment (Figs. 7c and d). Interestingly, the ILC composition was affected by Bvul (Figs. 7e and f). Type 2 ILCs (ILC2s) expressing the IL-33 receptor (ST2) were reduced by HFD feeding, but Bvul treatment restored the ILC2 population. ILCs exist in a tissue-dependent manner and are regulated by symbiotic bacteria. In particular, adipose tissue contains ILC2, which is associated with insulin resistance and obesity phenotypes.

Previous studies have shown that the establishment of SFB induces a Th17 response in the intestine and prevents infectious diseases. Faecalibacterium prausnitzii protects against autoimmune diseases by inducing Treg accumulation in the intestine. Some Bacteroides species (eg, B. fragilis) and their cellular components have been reported to cause Treg expansion and protective effects against various autoimmune diseases. To our knowledge, bacterial strains that induce Treg and ILC responses in obesity-induced modified immune systems and reduce inflammatory responses in adipose tissue have not yet been reported. CD4 + T cell responses induced by different Bacteroides species exhibit unique patterns depending on the physiological and immunological conditions of the host.

IL-21 + Tregs and ILC cells were analyzed to address the effects of IL-21 on ILC2 and Treg cells. Bvul treatment did not increase IL-21 receptor-expressing ILC in MLN. HFD increased IL-21 receptor + ILCs, but ILC2 cells did not respond to IL-21 (Fig. 7g). In particular, IL-21 receptor expression for ST2 (-) ILCs 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, single cell-based transcriptome analysis revealed that type 1 ILCs were IL-21-responsive30. Therefore, the ST2 (-) ILC responsible for IL-21 signaling may be ILC1. We also analyzed IL-21 receptor expression in Tregs, but couldn't find any differences (data not shown). Overall, Bvul regulated the ILC population in a way that decreased IL21 expression, decreased IL-21 responsive ILC1/3 cells, and increased the ILC2 subset in MLNs 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. In this study, the ILC subset profile found in MLN reflected systemic immunological changes as a target of Bvul, and indicated the role of intestinal microbes in regulating immune dysfunction during obesity in an ILC-dependent manner.

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. In these mice, the expression of Foxp3 and Il10 did not increase in MLN. In addition, the expression of Il21r, which was reduced 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 was A. muciniphila. Has nothing to do with the ability to increase the abundance of

The inventors' invention of Bvul-treated rats describes some evidence to show that human Bvul strains elicit various changes in diet-induced obesity. First, as in the results of human microbiome immune profiling studies, Bvul negatively correlated with obesity and serum IL-21 levels. Second, Bvul induces Treg and ILC responses in the intestinal and lymph nodes of obese mice, exposing new immunomodulatory effects against obesity. Third, Bvul reorganizes the gut microbiota and microbial metabolism in a way that is unique to obese and obese people. A more comprehensive understanding of the interactions between Bvul and A. muciniphila observed in this study could lead to a better understanding of complex host-microbial interactions and the development of microbial therapeutics.

Experimental example

Human study population and sample collection.

From November 2005 to January 2009, a total of 30 subjects were recruited from a healthy twin study as part of the Korean genome epidemic study. The average age of the subjects was 40.13 ± ± 2.74 years. Participants were identical twins with a disparity of more than 4 cm in the waist. Waist circumference was measured at the middle of the outermost rim of the ribs and the uppermost lateral iliac crest. Fecal samples were stored in a home freezer, delivered to the hospital, and stored at -80°C. Blood samples were coagulated, centrifuged at 2000xg for 20 minutes, and stored at -80 °C until the supernatant was analyzed. Subjects provided written informed consent to participate in this study. All human trials were approved by the Centers for Disease Control and the Clinical Trials Committee of Seoul National University (No. 144-2011-07-11). Epidemiological and clinical biomarkers (age, sex, 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.

DNA extraction and 16S rRNA sequencing.

Human and mouse genomic DNA was extracted using the MoBio Power Soil DNA Isolation Kit (MoBio, Solana Beach, CA, USA) and the Qiagen Faststool DNA Extraction Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The nucleic acid solution was stored at -70 °C until use. For human genomic DNA, a 27F/534R primer set was used to amplify the V1-V3 region of the 16S rRNA gene and pyrosequenced on the 454 Life Sciences FLX Titanium platform (Roche, Indianapolis, IN, USA). In the case of mouse DNA, the V4 region of the 16S rRNA gene was amplified using Illumina adaptive universal primer set 515F/806R and purified using MoBio UltraClean PCR Clean-Up Kit (MO BIO Laboratories, Carlsbad, CA, USA). After using the KAPA library quantification kit (KAPA Biosystems, Wilmington, MA, USA), the amount of the PCR amplification result was double-checked using the Quant-iT PicoGreen dsDNA assay kit (Life Technologies, Carlsbad, CA, USA). Samples were pooled and sequenced on the MiSeq platform using a 2 × × 300 bp reagent kit (Illumina, San Diego, CA, USA).

Microbial analysis.

Human sequence data was processed by the ABC pipeline developed by Broad Institute. The OTU table generated through the pipeline is further filtered to contain OTUs: (i) found in at least 2 samples. (ii) 100 reads or more, and (iii) classified as prokaryotic. For mouse sequence data, the OTU was selected using closed-referenced OUT picking to the gg_13_5 Greengenes database32 through the QIIME pipeline. Representative sequences were aligned using PyNAST34 and taxonomic assignment was performed using Ribosomal Database Project Classifier. The chimeric sequence was identified using the ChimeraSlayer algorithm and filtered prior to generating the OTU table. Serum cytokine levels (TNF-αα, TNF-ββ/ γIL-21, IL-22, IL-23, IL-10, IL-27, IL-28a, IL-31 and IL-33), MaAsLin waist circumference It was analyzed by multivariate analysis using (twins as random variables and host factors as covariates). Calculation of microbial abundance and ordination analysis was performed using the R package phyloseq36. Population variation was analyzed by major coordinate analysis (PCoA) using unweighted UniFrac distances for time series analysis in mouse feces and Bray-Curtis mismatch for clusters in cecal samples. PERMANOVA used the'adonis' feature of the vegan package to determine the difference between ND versus NDB and HFD versus HFDB. Random forest classification was performed through 10-fold cross-validation using the R package randomForest38 to identify the bacteria that cause changes observed between diets. Human data transformed and adjusted age and twins before comparison. The size of the effect for the group difference is determined by dividing the Z score by the square root of N (number of observations).

Functional inference of the intestinal microbiome

Functional differences between the various dietary groups were determined from 16S-rRNA based sequences using PICRUSt-1.0.0 (http://picrust.github.com). After peaking the OTU with a 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 diet group using Kruskal-Wallis test and Benjamini-Hochberg false discovery rate (FDR) correction.

Isolation and culture of Bacteroides strains.

A total of 182 Bacteroides strains were isolated from fresh feces from healthy Korean volunteers (30 patients) aged 19 to 40 years. The type strain obtained from ATCC was used to compare the immunomodulatory activity as a control for the isolated strain. All 182 Bacteroides spp. The strain mainly composed of the Bvul strain was incubated with chocolate agar anaerobic at 37°C for 24 hours. The bacterial cells were collected and centrifuged at 13,000×g for 5 minutes to treat the THP-1 cells.

Screening for immunomodulatory activity of Bacteroides strains.

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 a serum-free medium under 5% CO2 atmosphere. Bacterial cell pellets were added to differentiated THP-1 cells for 24 hours, and cell-free supernatant was collected and stored at -80°C before analysis. IL-10 and TNF-α levels were analyzed with an ELISA kit (BD Biosciences).

Experimental design for mouse study.

Central Lab Animal Inc. Female C57BL/6J mice (6 weeks old) were obtained from (Korea) and maintained under SPF (specific pathogen-free) conditions at the Laboratory Animal Research Institute, Seoul National University College of Medicine. After 1 week of acclimatization, mice were treated with ampicillin (1 g/L) in drinking water for a week to reduce colonization by Bacteroides species, and divided into four groups. Mice were fed either a control diet (ND and NDB, 10% fat; n = 19, study diet -D12450K) or HFD (HFD and HFDB, 60% fat, n = 19, Research Diets-D12492). Bvul strain prepared daily was administered by oral gavage (NDB and HFDB groups, n = 9, respectively) of 109 cfu per mouse. Intake and weight were measured once a week. Fecal samples were collected at 5 time points (before antibiotic treatment, before Bvul administration, and at 5 days, 9 weeks, and 18 weeks after administration). At week 16, an intraperitoneal glucose tolerance test was performed. Mice were sacrificed at 18 weeks, and tissues (MLN, liver, colon, cecum and reproductive fat) were collected and stored at -80°C prior to analysis. Blood samples were obtained by cardiac puncture under isoflurane anesthesia. 30 minutes after blood coagulation at RT, the serum was separated by centrifugation (2,000 xg, 10 minutes, 4°C. The supernatant (serum) was transferred to a 1.5 mL tube and stored at -80°C until use. Experimental procedure is Institutional Animal It has been reviewed and approved by the Care and Usage Committee.

Brain heart infution agar (BD Difco, USA) supplemented with 0.5% mucin (Sigma, USA) supplemented with A. muciniphila ATCC BAA-835 (Akk) at 37 °C for the treatment of A. muciniphila in HFD-induced obese mice was strict. Incubated for 48 hours in anaerobic conditions, and the microorganisms were collected with a sterile loop (diameter 1 mm). The collected cells were washed with sterile PBS supplemented with 0.5% cysteine and centrifuged at 10,000 x g for 5 minutes. The bacterial pellet was resuspended in PBS supplemented with 0.5% cysteine and 20% glycerol at 10/9 cfu/200 μl of 10 min. 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, Seoul National University. Mice were pooled for 1 week without intervention to adapt to the new environment and fed HFD (HFD and HFD-Akk, 60% fat, n = 20, Research Diets-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 of 5 x 108 cfu per mouse. After measuring the body weight of each mouse once a week during the test period, the mice were anesthetized, and MLN and colon were collected to measure the expression of target biomarkers (Il21, Il21r, Foxp3, and Il10).

Biomarker measurement in mouse tissue.

MLN and distal colon were homogenized with 5-mm stainless steel beads (Qiagen, Hilden, Germany) at 30-Hz for 5-8 min. Total RNA was isolated using an Easy-spin Total RNA extraction kit (Intron, Seoul, Korea). CDNA was synthesized using a High Capacity RNA to cDNA kit (Applied Biosystems, Carlsbad, CA, USA). All qPCR reactions were performed using Rotor-Gene SYBR Green PCR kit (Qiagen) using Rotor-Gene Q (Qiagen) according to the manufacturer's instructions. Relative RNA amount was determined by the 2-ΔΔCt method using Gapdh as a reference transcript.

1 H NMR spectroscopy.

Samples were prepared with slight modifications according to Lamichhane's method for NMR-based metabolic analysis. Briefly, pancreatic samples (ca. 130 mg) were mixed with 600 μL of DDW, vortexed for 30 seconds, and homogenized by a tissue homogenizer. After centrifugation (14,000 × g, 4° C.) for 10 minutes, 60 μL of deuterium oxide (D2O) containing 0.025 mg/mL 3-(trimethylsilyl) propionic acid-d4 sodium salt (TSP), 60 μL of 1 mM imidazole , 60 μL of 2 mM NaN3 and 120 μL of 0.5M KH2PO4 were added to 300 μL of the supernatant as a lock solvent. The mixture was vortexed for 1 minute and centrifuged at 14,000×g for 10 minutes. The clear supernatant was transferred to a 5 mm NMR tube (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. 1H-NMR spectra were collected at 25 using a water presaturation pulse sequence. Spectra were collected as 64 transients using a 4 second acquisition time and a 2 second recycle delay. Temporary 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, glycocholate, guanido acetate, hypoxanthine, isobutyrate, ascorbic acid, N-acetyl glucosamine, N-acetyl glucosamine, N-acetyl glucosamine , N-acetyltyrosine, O-phosphocholine, p-cresol, phenylalanine, proline, propionate, putresin, pyruvate, sarcosine, succinate, glutaric acid, triethylamine, trimethylamine N-oxide , Tryptophan, 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, with P <0.05 considered significant. Microbial data was standardized and uploaded to MetaboAnalyst 3.0 for modeling using sparse 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.

Tissue preparation and flow cytometry.

MLN and reproductive adipose tissue were crushed and digested with 1.6 mg/mL collagenase type 4 (Worthington, Lakewood, NJ) and 0.1% DNase I (fraction IX; Sigma) for 1 hour at 37°C. Whole cells were treated to lyse red blood cells and then stained for FACS analysis. The single cell suspension was pre-incubated with an anti-Fcγ-blocking monoclonal antibody (2.4G2) and washed before staining. Cells were stained with the following antibodies: anti-CD19 (6D5 (115506); BioLegend), anti-CD11b (M1/70 (101206), BioLegend anti-F4/80 (BM8 (123108); BioLegend) or anti-Fcε( Anti-FcR1) (anti-FcR1), anti-CD11c(HL3(553801)MAR-1(134306); BioLegend) (these FITC conjugated antibodies are used as lineage markers); pico against mouse CD45 (MCD4517; Invitrogen) Eritrin-Texas Red Conjugated Antibody; phycoerythrin-conjugated anti-CD1d (1B1 (123509); BioLegend); phycoerythrin-conjugated anti-CD44 (1M7 (103009); BioLegend); allophycocyanin-conjugated anti-CD1d -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-binding anti-CD4 (GK1.5 (100430); BioLegend); and Alexa Fluor 780-conjugated anti-CD11c (N418 (47-0114-82), eBioscience).Mice immune cells were stained with the following antibodies: anti -Picoerythrin-Texas red bound to human CD45 (MHCD4517; Invitrogen); anti-CD19 (H1B19 (30 2206), BioLegend), anti CD11b (ICRF44 (11-0118-42), eBioscience), anti-CD19 antibody (anti-CD3; UCHT1 (555332), BD Biosciences) BioLegend), anti-CD14 (HCD14 (325604), eBioscience) or anti-Fcε (MAR-1 (134306), BioLegend ), Alexa Fluor 647 conjugated anti-CD127 (eBioRDR5 (51-1278) -73), eBioscience).

Statistical analysis

All data were analyzed with Prism 5 (GraphPad Software, San Diego, CA) or R software (version 3.1.2). Clustering analysis was performed using R's heat map 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 through the post-hoc Tukey's test. In graphical representation, data are presented as mean ±± standard error. The whiskers in each box plot represent the 10th to 90th percentiles. Statistical significance was found to be * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001.

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13276BP

Consignment Date: 2017529

Claims (7)

Bacteroides vulgatus (Bacteroides vulgatus) SNUG40005 strain (accession number KCTC 13276BP) characterized by reducing diet-induced weight gain, glucose intolerance or adipose tissue inflammation. According to claim 1, wherein the strain is a bacteroid vulgatus (Bacteroides vulgatus ) SNUG40005 strain, characterized in that it reduces IL-12 receptor expression and IL-12 receptor-expressing ILC cells. The method of claim 1, wherein the strain maintains the abundance of the Akkermansia muciniphila strain in the intestinal microbial community. Bacteroides vulgatus SNUG40005 strain. As a pharmaceutical composition for the treatment or prevention of metabolic diseases using the Bacteroides vulgatus SNUG40005 strain as an active ingredient, having accession number KCTC 13276BP,
The metabolic disease is obesity, diabetes, hyperlipidemia, hypercholesterolosis, arteriosclerosis, fatty liver or cardiovascular disease, the composition. delete As a health functional food for improving metabolic diseases using the Bacteroides vulgatus SNUG40005 strain as an active ingredient, having accession number KCTC 13276BP,
The metabolic disease is obesity, diabetes, hyperlipidemia, hypercholesterolosis, arteriosclerosis, fatty liver or cardiovascular disease, food.

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