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Bacteroides vulgatus and Bacteroides dorei Reduce Gut Microbial Lipopolysaccharide Production and Inhibit Atherosclerosis

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.118.033714Circulation. 2018;138:2486–2498

Abstract

Background:

It is increasingly recognized that gut microbiota play a pivotal role in the development of atherosclerotic cardiovascular disease. Previously, we have reported that the abundance of genus Bacteroides is lower in patients with coronary artery disease (CAD) than in patients without CAD with coronary risk factors or in healthy volunteers. However, it remains unclear which and how specific gut bacteria contribute to the progression of atherosclerosis.

Methods:

We recruited patients with CAD patients and controls without CAD with coronary risk factors. We then compared gut microbial composition using 16S ribosomal RNA gene sequencing in fecal samples to detect species with differential abundance between 2 groups. Subsequently, we used atherosclerosis-prone mice to study the mechanisms underlying the relationship between such species and atherosclerosis.

Results:

Human fecal 16S ribosomal RNA gene sequencing revealed a significantly lower abundance of Bacteroides vulgatus and Bacteroides dorei in patients with CAD. This significant differential abundance was confirmed by quantitative polymerase chain reaction. Gavage with live B. vulgatus and B. dorei attenuated atherosclerotic lesion formation in atherosclerosis-prone mice, markedly ameliorating endotoxemia followed by decreasing gut microbial lipopolysaccharide production, effectively suppressing proinflammatory immune responses. Furthermore, fecal lipopolysaccharide levels in patients with CAD were significantly higher and negatively correlated with the abundance of B. vulgatus and B. dorei.

Conclusions:

Our translational research findings identify a previously unknown link between specific gut bacteria and atherosclerosis. Treatment with live B. vulgatus and B. dorei may help prevent CAD.

Clinical Trial Registration:

URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000018051. Unique identifier: UMIN000015703.

Clinical Perspective

What Is New?

  • Analysis of gut microbial profile in patients with coronary artery disease (CAD) shows a relative depletion of Bacteroidesvulgatus and Bacteroidesdorei compared with controls without CAD with coronary risk factors.

  • Gavage with live B. vulgatus and B. dorei decreases fecal and plasma lipopolysaccharide levels and protects against atherosclerosis in apolipoprotein E–deficient mice.

  • Fecal lipopolysaccharide levels in patients with CAD were significantly higher compared with controls without CAD.

What Are the Clinical Implications?

  • Bacteroides treatment may serve as a novel and attractive therapeutic strategy for suppressing lipopolysaccharide-induced inflammatory response in CAD.

Introduction

The increasing recognition of gut microbiota and its role in host metabolism and immunity has promoted an unprecedented interest in developing gut microbiota-related diagnostic and therapeutic targets for many diseases. Next-generation sequencing techniques and multiomics approaches have dramatically expanded our knowledge of the microbial world. Increasing evidence has suggested a strong relationship between gut microbiota and the progression of cardiovascular diseases, and we previously reported such a relationship with coronary artery disease (CAD) for the first time.1,2 Trimethylamine (TMA) and TMA N-oxide, which are gut microbiota metabolites of dietary phosphatidylcholine, are known to be associated with cardiovascular disease and with the atherosclerosis process in particular.3,4 The metagenome-wide association study showed that gut microbial enzymes producing TMA were enriched in patients with CAD compared with healthy controls.5 This finding supports the encouraging prospect that preventing CAD is feasible through gut microbiota modulation. However, a recent clinical trial has shown contradictory results indicating that fish consumption drastically increases circulating TMA N-oxide levels, highlighting the substantial limitations in our current understanding of the relationship between diet and gut microbiota governing TMA N-oxide production.6

The genus Bacteroides includes some of the predominant gut bacteria in humans and is known to have an important role in maintaining a healthy gut ecosystem.7 Individuals classified as enterotype 3, which is characterized by low levels of Bacteroides,8 have a higher incidence of symptomatic atherosclerosis.1,9 Moreover, Bacteroides abundance was found to be decreased in patients with atherosclerotic ischemic stroke and transient ischemic attack.10 In line with these observations, our previous study using terminal-restriction fragment length polymorphism analysis confirmed that the abundance of Bacteroides is lower in CAD patients than in non-CAD patients with coronary risk factors or in healthy volunteers.1,2 Taken together, these findings strongly suggest a relationship between Bacteroides and CAD. However, it remains unclear which specific Bacteroides species are involved and what their exact role is in CAD.11

CAD remains the leading cause of morbidity and mortality worldwide, despite the widespread use of statin therapy in the last decade.12 The goal of our present study was to identify elements of the gut microbiota that may serve as targets in novel, inexpensive therapeutic strategies for preventing CAD. To achieve this goal, we first performed 16S ribosomal RNA (rRNA) gene sequencingto compare the gut flora in patients with or without CAD, and we found that the abundance of Bacteroides vulgatus and Bacteroides dorei was decreased in patients with CAD. Subsequently, we performed a series of analyses in atherosclerosis-prone mice to clarify the underlyingmechanisms linking these Bacteroides species to atherosclerosis.

Methods

The data that support the findings of this study are available from the corresponding author on reasonable request.The sequence data have been deposited with links to BioProject accession No. PRJDB6472 in the DNA Data Bank of Japan BioProject database (http://trace.ddbj.nig.ac.jp/bioproject/index_e.html).

Recruitment of Patients With CAD and Controls

All participants provided written informed consent on enrollment, and the study was conducted according to the guidelines of the Declaration of Helsinki. This study was approved by the Ethics Committee of Kobe University (approval No. 1595) and registered with the University hospital Medical Information Network Clinical Trials Registry (trial registration No. UMIN000015703).

Thirty patients with CAD and 30 controls without CAD with coronary risk factors were recruited at Kobe University Hospital between October 2014 and July 2015. The sample size was calculated using R software (power=0.9, significance level=0.05, mean difference=6, SD=7; n=30 per group.). The group with CAD included patients with stable angina pectoris and old myocardial infarction with preserved left ventricular ejection fraction (>40%) who underwent percutaneous coronary intervention or coronary artery bypass graft surgery ≥6 months before the present study. Patients with acute coronary syndrome were excluded. Single- or multivessel disease was defined in terms of the number of major coronary vessels demonstrating >75% stenosis on diagnostic coronary angiography.

Thirty patients without CAD with coronary risk factors, such as hypertension, diabetes mellitus, or dyslipidemia, but without present or past history of coronary or other vascular diseases, were recruited, and age and sex were matched in the control group. All patients without CAD were hospitalized to treat supraventricular tachycardia such as atrial fibrillation and atrial flutter, hypertension, or diabetes mellitus. History of coronary or other vascular disease was defined as documented vascular disease, symptoms indicating angina pectoris, abnormality on electrocardiography indicating old myocardial infarction or angina pectoris, or abnormality on chest x-ray film.

Patients with systemic disease, including hepatic disease, renal disease (serum creatinine levels >2.0 mg/dL), collagen disease, or malignancy, were excluded from the study. Patients treated with antibiotics were also excluded. Diabetes mellitus was defined as glycohemoglobin levels >6.5% (per the indications of the National Glycohemoglobin Standardization Program), use of oral antihyperglycemic, or use of insulin therapy. Hypertension was defined as blood pressure >140/90 mm Hg or use of antihypertensive drugs. Dyslipidemia was defined as low-density lipoprotein cholesterol levels >140 mg/dL, triglyceride levels >150 mg/dL, or use of antidyslipidemic drugs per the guidelines issued by the Japan Atherosclerosis Society.13

DNA Extraction, 16S rRNA Gene Simplification, and Pyrosequencing (Human Fecal Samples)

Fecal samples were collected while participants were hospitalized and thus consuming a hospital diet for >1 day. DNA extractions from human fecal samples were performed by Nihon Gene Research Laboratories Inc according to a previously established procedure.14–16

Parts of the 16S rRNA genes (the V3–V4 region, corresponding to positions 342–806 in the Escherichia coli numbering system) were polymerase chain reaction–amplified using our nondegenerate universal primer set for 342F and 806R. A detailed description of the primer set and polymerase chain reaction conditions is available elsewhere.17 After addition of the sequencing adapters, the amplicons were sequenced (250-bp paired end) at Takara Bio Inc using an Illumina MiSeq platform (Illumina Inc) according to the manufacturer’s protocol.

To create the bacterial composition matrix, we used USEARCH version 10.0.240.18 Our previous protocol was also used to select high-quality 16S rRNA gene amplicon sequences generated using Trimmomatic19 version 0.33 with the parameters LEADING:17 TRAILING:17 AVGQUAL:25 MINLEN:100. The remaining reads were processed using the -fastq_mergepairs command of USEARCH, with default parameters. Next, we removed sequences without the primer region using Tagcleaner20 version 0.16, with parameters -tag5 CTACGGGGGGCAGCAG -mm5 3 -tag3 AGATACCCCGGTAGTCC -mm3 3 -nomatch 3. After the removal of the primer, sequences with N were removed using an in-house python script. To remove PhiX reads, we used the -filter_phix command of USEARCH. Finally, we removed short sequences using the USEARCH command -sort_by_length, with the parameter -minseqlength 300. Finally, we generated operational taxonomic unit (OTU) tables using UPARSE algorithms (-fastx_uniquecomand and otu_cluster command with the parameter -minsize 1).21 The representative sequences of each OTU were annotated to bacterial genus using RDP Classifier version 2.12, with a bootstrap value ≥0.5.22 Moreover, we annotated each representative sequence of each OTU to the reference database SILVA Living Tree Project version 12323 using BLASTN version 2.2.25, with identity threshold ≥97% and coverage ≥80%.

Predictive Functional Profiling of Gut Microbiota Based on 16S rRNA Gene Sequences

OTUs were constructed using the raw data reads from fecal samples. To join 2 paired-end reads, we used fastq-join software with default options. The chimeric sequences were deleted with usearch6.1.24 OTUs at the 97% similarity threshold were selected with the Green genes database in the QIIME 1.8.0 pipeline.25 Functional analysis of gut microbiota based on the KEGG Orthology database (Kyoto Encyclopedia of Genes and Genomes)26 was performed using PICRUSt 1.1.1.27 Identification of KEGG modules with a significant difference in mean relative expression was performed using Statistical Analysis of Metagenomic Profiles (STAMP 2.1.3) software.28 The lipopolysaccharide (LPS) biosynthesis pathway map (map00540) was cited from http://www.kegg.jp/kegg/kegg1.html26 with permission.

Animals

All apolipoprotein E–deficient mice were on C57BL/6 background. All mice were housed in a specific pathogen-free animal facility at the Kobe University Institute. The animals were fed normal chow (CLEA) and water ad libitum under a strict 12-hour light/dark cycle. Six-week-old mice were randomly divided into 3 treatment groups: mice in the control group were gavaged with culture medium, those in the live Bacteroides group were gavaged with live B. vulgatus and B. dorei, and those in the heat-killed Bacteroides group were gavaged with heat-killed B. vulgatus and B. dorei at a dose of 2.5×109 cfu/100 μL of B. vulgatus and B. dorei 5 times per week. All experiments were performed according to the Guidelines for Animal Experiments in effect at Kobe University School of Medicine (guideline No. P160701).

Culture and Preparation of B. vulgatus and B. dorei

B. vulgatus (No. 8482; American Type Culture Collection) and B. dorei (No. 17855; Deutsche Sammlung von Mikroorganismen) were cultured anaerobically in Difco-reinforced clostridial medium (No. 218081; BD Bioscience) at 37°C. An anaerobic chamber (Coy Laboratory Products) containing 10% CO2, 10% H2, and 80% N2 was used for all anaerobic microbiology steps. B. vulgatus and B. dorei were heat-killed at 121°C (treatment duration, 15 minutes). The success of the heat treatment was confirmed by the absence of growth of plated heat-killed bacteria.

Preparation of the Fecal Supernatant

The same human fecal samples were used for LPS measurement and 16S rRNA gene sequencing. Mouse fecal samples were collected from 16-week-old controls and Bacteroides-treated mice at 24 hours after the last oral gavage. Fecal supernatant was obtained according to a previously described protocol with some modifications.29,30 Briefly, the fecal samples were suspended in sterile PBS to a concentration of 1 g per 10 mL (human) or 50 mg per 500 μL (mouse) and vortexed mildly to avoid disruption of bacterial cells. After centrifugation for 15 minutes at 3000 rpm, the supernatant was collected, sterilized by filtration through a 0.45-μm filter followed by refiltration through a 0.22-μm filter, inactivated for 15 minutes at 90°C, and stored at −80°C.

Analysis of LPS Levels in Plasma and Fecal Supernatant

The plasma and fecal LPS levels were determined using a limulus amebocyte lysate assay (No. K50-643J; Lonza Inc) according to the manufacturer’s instructions. The plasma was diluted 10-fold, and the fecal supernatant was diluted 10 000-fold in pyrogen-free water and inactivated for 15 minutes at 90°C. LPS measurements were performed in pyrogen-free glass tubes, Eppendorf tubes, and plates.

Statistical Analysis

Statistical analyses were performed using R software, version 3.1.0, JMP version 10 (SAS Institute), and Prism version 7.0 (GraphPad Software). The Shapiro-Wilk test was used to determine whether the data were normally distributed. Results were expressed as mean±SEM or mean±SD for normally distributed data, and median ± interquartile range (25th to 75th percentiles) for nonnormally distributed data. The significance of differences between 2 groups was evaluated using the 2-tailed Student’s t test for normally distributed data or Mann-Whitney U test for nonnormally distributed data. The Fisher exact test or χ2 test was used to compare categorical variables. For all tests, a value of P<0.05 was considered to indicate statistical significance. To discover the strength and direction of a link between 2 parameters, Spearman’s rank correlation coefficient was calculated. One-way ANOVA was used to detect significant differences among 3 groups. The q values were calculated using the Benjamini-Hochberg method to adjust the P values for multiple comparisons. The clustering of data from 30 patients with CAD and 30 controls was performed at the genus level, as described in detail elsewhere.31 The Shannon-Wiener index was calculated using the vegan package for R software. Principal component analysis was performed using JMP.

Results

Gut Microbial Profile in Patients With CAD

We recruited 30 patients with CAD and 30 age- and sex-matched controls without CAD with coronary risk factors, such as hypertension, diabetes mellitus, or dyslipidemia. We then performed a detailed comparison of gut microbial profile using 16S rRNA gene sequencing in fecal samples. The baseline characteristics of the participants are shown in Table 1. The α diversity, Bacteroidetes/Firmicutes ratio, and Gram-positive to Gram-negative strain ratio did not differ significantly between the 2 groups (Figure 1A and 1C). To analyze the gut microbial profiles of the study participants, the samples were clustered into 3 clusters at the genus level, according to the procedure followed in a previous report (Figure 1D).8,31 Each cluster was characterized by a high abundance of a specific genus as follows: Bacteroides in cluster 1, Prevotella in cluster 2, and Faecalibacterium, Ruminococcus, or Bifidobacterium in cluster 3 (Figure 1E). The controls without CAD were significantly more likely to be categorized into cluster 1, whereas few patients with CAD were categorized into this cluster (Figure 1F). A comprehensive comparison between the patients with CAD and controls in terms of the abundance of genera with an average relative abundance >0.1% is provided in Table I in the online-only Data Supplement. We found that the relative abundance of the genus Bacteroides was visibly lower in patients with CAD compared with controls (Figure 1G). The associations of gut microbial genera with clinical indices are shown in Figure I in the online-only Data Supplement. Principal coordinate analysis showed that the 2 groups differed in the abundance of major species of the gut microbiome, as patients with CAD showed a relative depletion of B. vulgatus and B. dorei and enrichment in Faecalibacterium prausnitzii and Prevotella copri (Figure 1H). Considering that previous reports have shown a lower abundance of the genus Bacteroides in patients with atherosclerosis,1,9,10 we focus on the Bacteroides species, B. vulgatus and B. dorei. Because these 2 species share similar 16S rRNA sequencing patterns,32 we were not able to distinguish them using our methodology. These species have been implicated to have an anti-inflammatory response33 and were the 2 most abundant species within the genus Bacteroides in 16S rRNA gene sequencing analysis (Figure 1I). We thus designed a mouse model involving the gavage of atherosclerosis-prone mice with B. vulgatus and B. dorei to confirm the causal relationship between these species and atherosclerosis and to clarify its underlying mechanisms. We also confirmed the significantly decreased abundance of B. vulgatus and B. dorei in patients with CAD by quantitative polymerase chain reaction (Figure 1J and Table II in the online-only Data Supplement).

Table 1. Baseline Characteristics and Laboratory Data of the Study Population

VariablesControls Without CAD (n=30)Patients With CAD (n=30)
Age, y62.9±6.863.6±7.2
Sex, male23 (77)27 (90)
BMI, kg/m224.8±4.125.1±2.8
Hospital stay, d13.5±12.19.1±6.2
Blood pressure, mm Hg
 Systolic123.1±18.1122.2±12.9
 Diastolic68.9±10.567.7±8.4
Laboratory data
 AST, U/L22.9±5.627.8±13.7
 ALT, U/L23.2±11.627.3±17.1
 BUN, mg/dL16.2±4.414.9±3.7
 Creatinine, mg/dL0.94±0.260.87±0.16
 T-Cho, mg/dL183.1±34.9162.2±27.7*
 HDL-C, mg/dL52.4±13.850.8±19.2
 LDL-C, mg/dL113.8±35.791.9±26.1
 TG, mg/dL145.6±73.6150.3±75.7
 HbA1c, NGSP, %6.55±1.306.35±0.86
 CRP, mg/dL0.15±0.200.09±0.09
Fecal SCFAs, nmoL/µg
 Acetic acid54.74±22.0549.54±24.19
 Propionic acid19.95±12.5016.35±8.85
 Iso-butyric acid1.65±1.241.99±1.93
N-butyric acid9.76±7.099.16±7.77
 Iso-valeric acid1.10±0.971.46±1.49
N-valeric acid1.91±1.441.90±1.24
 Formic acid2.15±6.331.04±1.92
History of smoking21 (70)23 (77)
Current smoker4 (13)4 (13)
Past history
 Diabetes mellitus12 (40)11 (37)
 Dyslipidemia18 (60)28 (93)
 Hypertension23 (77)26 (87)
Medications
 ACE-I/ARB17 (57)16 (53)
 Antidiabetic11 (37)9 (30)
 Anticoagulant or antiplatelet17 (57)30 (100)
 β-Blocker9 (30)16 (53)
 Calcium channel blocker14 (47)19 (63)
 PPI/H2 blocker13 (43)29 (97)
 Statin12 (40)27 (90)

Data are shown as mean±SD or n (%). The averages of continuous variables were compared using the two-tailed Student’s t test. Fisher exact test was used to compare the proportions of categorical variables between groups. A 2-sided value of P<0.05 was considered statistically significant.

ACE-I indicates angiotensin-converting enzyme inhibitor; ALT, alanine aminotransferase; ARB, angiotensin receptor blocker; AST, aspartate aminotransferase; BMI, body mass index; BUN, blood urea nitrogen; CAD, coronary artery disease; CRP, C-reactive protein; H2 blocker, histamine H2-receptor antagonist; HbA1c, glycohemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; NGSP, National Glycohemoglobin Standardization Program; PPI, proton pump inhibitor; SCFA, short-chain fatty acid; T-Cho, total cholesterol; and TG, triglycerides.

*P<0.05.

P<0.01.

P<0.001.

Figure 1.

Figure 1. Gut microbial alteration in patients with or without coronary artery disease (CAD). The V3–V4 region of the bacterial 16S ribosomal RNA was sequenced in fecal samples from 30 control patients (without CAD but with risk factors) and 30 patients with CAD. A, The Shannon-Wiener index at the family and genus levels. B, Bacteroidetes to Firmicutes (BF) ratio. C, Ratio of Gram-positive (GP) to Gram-negative (GN) strains. D, Participants were classified into 3 clusters based on genus-level abundance in the gut microbiota. E, Distribution of each major genus across clusters. One-way ANOVA was used to compare the cross-cluster distribution for each genus. F, Distribution of controls and patients with CAD in each cluster. The χ2 test was used to compare the 3 clusters. G, Relative abundance of genus Bacteroides (percentage of total gut microbiota) in controls and patients with CAD. H, Principal coordinate analysis at the species level was performed to compare the distribution of species in gut microbiota. Only species with larger weight on principal coordinate analysis are shown. I, Relative abundance of Bacteroides species (percentage of total genus Bacteroides) in controls and patients with CAD. J, Relative abundance of B. vulgatus and B. dorei (percentage of total gut microbiota) was assessed by quantitative polymerase chain reaction. B. dorei indicates Bacteroides dorei; B. vulgatus, Bacteroides vulgatus; F. prausnitzii, Faecalibacterium prausnitzii; P. copri, Prevotella copri; and PC, principal component. The Mann-Whitney U test was used to determine statistical significance between the 2 groups (A through C, G, J). Data are shown as median±interquartile range (25th to 75th percentile) (A through C, E, G, J).*P<0.05; †P<0.01; ‡P<0.001.

Gavage With Live B. vulgatus and B. dorei Attenuated the Formation of Atherosclerotic Plaque

To determine the effect of B. vulgatus and B. dorei on the development of atherosclerosis, 6-week-old female apolipoprotein E–deficient mice were treated with live B. vulgatus and B. dorei by oral gavage 5 times per week for 10 weeks. Control mice received only a vehicle (culture medium) according to the same protocol. At 16 weeks of age, the mice were euthanized, and several analyses were performed to evaluate atherosclerosis. Compared with control mice, mice gavaged with live Bacteroides showed significantly reduced lesion size in the aortic root and en face analysis of thoracoabdominal aortas (Figure 2A and 2B) without significant difference in body weight (Figure 2F), plasma cholesterol levels (Figure 2G), or plasma glucose levels (data not shown). Furthermore, immunohistochemical staining followed by morphometric analysis of atherosclerotic lesions in the aortic sinus revealed a marked reduction in macrophage and CD4+ T-cell accumulation in mice gavaged with live Bacteroides compared with controls (Figure 2C and 2D). Subsequently, we performed quantitative reverse transcription–polymerase chain reaction assays to evaluate the mRNA expression of immune cell markers and chemokines/chemokine receptors in mouse atherosclerotic aortas. The mRNA expression of several proatherogenic immune cell markers and chemokines/chemokine receptors was decreased in mice gavaged with live Bacteroides (Figure 2E). These results suggested that replenishment of live B. vulgatus and B. dorei by oral gavage reduced plaque inflammation, attenuating atherosclerotic lesion formation.

Figure 2.

Figure 2. Live B. vulgatus and B. dorei attenuate atherosclerotic lesions by ameliorating endotoxemia. Six-week-old female apolipoprotein E–deficient- mice treated with live B. vulgatus and B. dorei or vehicle (control) were killed at 16 weeks of age. A, Representative photomicrographs of oil red O staining and quantitative analysis of atherosclerotic lesion area in the aortic sinus (8 to 9 samples per group). The black bar represents 200 μm. B, Representative photomicrographs of oil red O staining and quantitative analysis of atherosclerotic plaque area in the aortas (7 to 9 samples per group). The white bar represents 1.5 mm. C, Representative fluorescent staining of macrophages and quantitative analysis of MOMA-2–positive staining in the aortic sinus (7 samples per group). The black bar represents 100 μm. D, Representative sections and quantitative analysis of CD4+ T cells in the aortic sinus (8 to 9 samples per group). The black bar represents 50 μm. E, mRNA levels in the atherosclerotic aorta. Data were normalized to the housekeeping Gapdh gene. Relative expression levels (in mice treated with live Bacteroides relative to that in control mice) are shown in a heat map (4 to 5 samples per group). F, Body weight changes over the course of the experimental period (8 to 9 samples per group). G, Comparison of plasma lipid profiles (8 to 9 samples per group). H, Plasma LPS levels quantified via limulus amebocyte lysate assay (5 samples per group). I, Plasma cytokine levels quantified via cytokine bead array (4 to 5 samples per group). B. dorei indicates Bacteroides dorei; B. vulgatus, Bacteroides vulgatus; EU, endotoxin unit; HDL, high-density lipoprotein cholesterol; IL, interleukin; IFN, interferon; LB, treated with live B. vulgatus and B. dorei; LDL, low-density lipoprotein cholesterol; LPS, lipopolysaccharide; T-Cho, total cholesterol; TG, triglycerides; and TNF, tumor necrosis factor. The 2-tailed Student’s t test was used (A through D, F through I). Data are shown as mean±SEM (A through D, F through I). *P<0.05.

Live Bacteroides Treatment Ameliorated Endotoxemia and Systemic Inflammation

Endotoxemia is known to cause systemic inflammation leading to the disruption of innate and adaptive immunity and the development of atherosclerosis.34–36 Indeed, compared with control mice, Bacteroides-treated mice had significantly lower plasma levels of LPS (Figure 2H) and lower plasma levels of proatherogenic cytokines, such as interleukin (IL)–2, IL-4, IL-6, IL-17A, interferon-γ, and tumor necrosis factor–α (Figure 2I). Because LPS stimulates cells through TLR4 and upregulates costimulatory molecules on antigen-presenting cells,37 we further examined the source of these cytokines via flow cytometry assay of splenocytes. We found that the percentages of CD11bhighF4/80high macrophages, CD11bhighLy6Ghigh neutrophils, and CD11chigh dendritic cells were not significantly different between the 2 groups (Figure IIA in the online-only Data Supplement), but mice gavaged with Bacteroides exhibited decreased expression of major histocompatibility complex class II and costimulatory molecule CD86, as well as increased expression of the coinhibitory molecules programmed death ligand–1 and programmed death ligand–2 on splenic CD11chigh dendritic cells (Figure IIB in the online-only Data Supplement). Consistent with the higher phenotypic tolerogenicity of dendritic cells, the number of CD4+ T cells was also lower in live Bacteroides-treated mice (Figure IIC in the online-only Data Supplement). Mice gavaged with live Bacteroides had lower numbers of effector CD44highCD62Llow CD4+ T cells and a significantly higher proportion of CD4+CD25+Foxp3+ regulatory T cells, with higher levels of intracellular CTLA4 (Figure IID and IIE in the online-only Data Supplement), indicating that immune balance was shifted toward suppression. Taken together, these data suggest that the dampening of systemic innate immune-cell activation and Th1-driven inflammation involved in the pathogenesis of atherosclerosis was caused by the live Bacteroides treatment–induced reduction in plasma LPS concentration.

Gavage With Live Bacteroides Alters Gut Microbial LPS Production

We next examined the gut microbial composition. 16S rRNA gene sequencing of mouse fecal samples revealed changes in gut microbiota composition in response to gavage with live B. vulgatus and B. dorei (Figure IIIA and IIIB in the online-only Data Supplement). The Bacteroidetes/Firmicutes ratio, Gram-positive to Gram-negative strain ratio, and fecal water content did not differ significantly among the 3 groups Figure IIIC, IIID, and IIIG in the online-only Data Supplement). Copy number of 16S rDNA per gram feces was significantly increased in mice treated with live Bacteroides compared with control mice (Figure IIIE in the online-only Data Supplement). The abundance of B. vulgatus and B. dorei was considerably higher in feces from live Bacteroides-gavaged mice (Figure IIIF in the online-only Data Supplement). It is important to note that the prediction of bacterial gene functions based on 16S rRNA gene sequences using PICRUSt27 revealed some functional diversity among the 3 groups in accordance with the difference in gut microbiota composition (Figure IIIH in the online-only Data Supplement). Although the expression of genes related to TMA synthesis (CutC, CutD) had no difference among the groups, the expression of genes involved in LPS biosynthesis, especially LpxA and LpxD, which are involved in lipid A biosynthesis and constitute the essential acyltransferases of the lipid A structure (Figure 3B),38,39 was significantly decreased in mice gavaged with live Bacteroides (Figure 3A and 3C, Figure IV in the online-only Data Supplement). Therefore, we measured fecal LPS levels as an indicator of colon LPS concentrations produced by gut microbiota. It is surprising to note that the fecal LPS levels were dramatically lower in mice gavaged with live Bacteroides than in control mice (Figure 3D). Thus, endotoxemia was attenuated in live Bacteroides-gavaged mice (Figure 2H). The fecal LPS levels as well as atherosclerotic plaque in aortic sinus (P=0.74) and plasma LPS levels (P=0.93) in heat-killed Bacteroides gavaged mice were not decreased compared with control mice (P=0.92) (6 samples per group).

Figure 3.

Figure 3. Alteration in gut microbial lipopolysaccharide (LPS) biosynthesis after treatment with live Bacteroides. A, LPS biosynthesis in 16-week-old apolipoprotein E–deficient mice treated with live Bacteroides or vehicle (control) according to 16S ribosomal RNA sequencing data analyzed using PICRUSt. For each gene involved in lipid A biosynthesis, its relative expression (in mice treated with live Bacteroides relative to that in control mice) is plotted on a heat map (6 samples per group). Genes not involved in LPS biosynthesis based on the KEGG Orthology database (Kyoto Encyclopedia of Genes and Genomes) were labeled N/A. B, Lipid A structure of Escherichia coli. LpxA and LpxD constitute the essential, early acyltransferases of lipid A biosynthesis. C, Expression of genes involved in LPS biosynthesis as predicted using PICRUSt (6 samples per group). D, Fecal LPS levels from 3 independent experiments quantified via limulus amebocyte lysate assay. The 2-tailed Student’s t test was used to detect significant differences between the 2 groups (A, C, and D). EU indicates endotoxin unit; and LB, treated with live Bacteroides vulgatus and Bacteroides dorei. Data are expressed as mean±SEM (C, D). *P<0.05; ‡P<0.001. LPS biosynthesis pathway map (map00540) is cited from http://www.kegg.jp/kegg/kegg1.html with permission. The lipid A structure was obtained from a previous article with permission.33

Live Bacteroides Treatment Strengthened Tight Junction Formation In Vivoand In Vitro

Given that leaky gut followed by alteration in gut microbiota composition promotes endotoxemia and atherosclerosis,35 we next investigated whether live Bacteroides treatment had an effect on intestinal tight junction permeability. Mice gavaged with live Bacteroides showed lower intestinal permeability to fluorescein isothiocyanate dextran (Figure VA in the online-only Data Supplement) and significantly higher mRNA expression of tight junction gene Zo1 (Figure VB and Table III in the online-only Data Supplement), reflected in higher mean fluorescence intensity of ZO-1 in the colon (Figure VC in the online-only Data Supplement). Because LPS increases intestinal permeability in a TLR4-dependent manner,40,41 we stimulated HT29 human colorectal adenocarcinoma cells, which showed responsiveness to LPS through TLR4, with fecal supernatant from mice. Consistent with in vivo results, we observed significantly higher expression levels of ZO-1 (mRNA and protein) in HT29 cells stimulated with fecal supernatant from live Bacteroides-treated mice than in cells stimulated with fecal supernatant from control mice (Figure VD and VE in the online-only Data Supplement). These results suggest that gut microbiota–induced changes in colon LPS concentrations may directly affect tight junction–mediated paracellular permeability.

Live Bacteroides Suppressed Intestinal Immune Response

We previously reported that atherosclerosis can be prevented by modulation of intestinal immunity.42,43 Mice gavaged with live Bacteroides had substantially lower mRNA expression of Cd11c, Cd80, and proinflammatory cytokines in the colon. In addition, the mRNA expression of Ccr7, which is critically important for migration of antigen-presenting cells in the intestinal lamina propria to the mesenteric lymph nodes (MLNs),44 was significantly lower in the colon of mice gavaged with Bacteroides (Figure VIA and VIB, Table III in the online-only Data Supplement). Flow-cytometric analysis of MLNs showed that Bacteroides-gavaged mice had significantly lower abundance of CD11chigh cells, as well as significantly reduced expression of TLR4, major histocompatibility complex class II, and CD80 on CD11chigh cells, whereas the number of CD4+ T cells and several markers on CD4+ T cells did not significantly change (Figure VIC and VIF in the online-only Data Supplement). Cytokine mRNA expression in MLNs tended to be lower in mice treated with live Bacteroides (Figure VIG and Table III in the online-only Data Supplement). Further, we obtained TLR4-knockdown RAW cells by small-interfering RNA transfection. We found that, on transfection with control small-interfering RNA, the secretion of proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor-α was markedly lower in RAW cells stimulated with fecal supernatant from live Bacteroides-treated mice than in RAW cells treated with fecal supernatant from control mice. RAW cells transfected with Tlr4 small-interfering RNA secreted significantly lower levels of proinflammatory cytokines than RAW cells transfected with control small-interfering RNA (Figure VIH in the online-only Data Supplement). Taken together, these findings suggest that colonic LPS concentration regulates colon inflammation via LPS/TLR4-dependent signaling.

Fecal LPS Levels Were Increased in Patients With CAD

Finally, to further investigate the relationship between CAD incidence and gut microbial production of LPS in humans, we predicted gut microbial function and measured fecal LPS levels in the same samples used for 16S rRNA gene sequencing. Although the expression of LpxA and LpxD did not differ between the patients with CAD and controls, the expression of LpxM, which yields hexa-acylated lipid A (a strong TLR4 agonist), tended to be higher in the group with CAD (Figure 4A and 4B). Notably, fecal LPS levels in patients with CAD were significantly higher than controls (Figure 4C). It is interesting to note that the abundance of genus Bacteroides tended to be negatively correlated with fecal LPS levels (r=−0.227, P=0.0838). The plot with fecal LPS levels and the abundance of B. vulgatus and B. dorei in patients with CAD (red dots) revealed a significant negative correlation between 2 parameters (r=−0.541, P=0.002) (Figure 4D). These results strongly support the idea that B. vulgatus and B. dorei regulate gut microbial production of LPS and that their activity can influence the progression of atherosclerosis in patients with CAD.

Figure 4.

Figure 4. Gut microbial lipopolysaccharide (LPS) production in patients with or without CAD. Gut microbial function and fecal LPS levels in the same samples used for 16S rRNA gene sequencing. A, The expression of genes involved in lipid A biosynthesis according to 16S rRNA sequencing data analyzed using PICRUSt. For each gene involved in lipid A biosynthesis, its relative expression (in CAD relative to that in control) is plotted on a heat map (30 samples per group). Genes not involved in lipid A biosynthesis based on the KEGG Orthology database (Kyoto Encyclopedia of Genes and Genomes) were labeled N/A. B, The expression of genes involved in LPS biosynthesis according to 16S rRNA sequencing data analyzed using PICRUSt (30 samples per group). The 2-tailed Student’s t test was used to detect significant differences between the 2 groups. C, Fecal LPS levels were quantified via limulus amebocyte lysate assay. The Mann-Whitney U test was used to determine significance. Data are shown as median±interquartile range (25th to 75th percentile). D, Spearman’s rank correlation coefficient was calculated between fecal LPS levels and relative abundance of Bacteroides vulgatus (B. vulgatus) and Bacteroides dorei (B. dorei). CAD indicates coronary artery disease. *P<0.05. LPS biosynthesis pathway map (map00540) is cited from http://www.kegg.jp/kegg/kegg1.html with permission.

Discussion

Growing evidence suggests that gut microbiota plays a key role in the development of atherosclerosis. Although the latest metagenome-wide association study has already provided a characterization of the fecal microbiota profile in patients with CAD,5 the clinical impact of these descriptive data remains unclear because a specific gut microbiota-based target to prevent CAD has yet to emerge. Here, we found that the abundance of B. vulgatus and B. dorei is lower in the gut microbiome of patients with CAD and that oral gavage with live B. vulgatus and B. dorei can decrease the fecal and plasma LPS concentrations and protect mice against atherosclerosis (Figure VII in the online-only Data Supplement).

B. vulgatus and B. dorei are the dominant species of genus Bacteroides in human gut microbiota. It is important to note that the LPS compounds penta- and tetra-acylated lipid A of these Bacteroides species are structurally distinct from the hexa-acylated LPS of E.scherichiacoli and elicit reduced TLR4 responses.33 For this reason, we originally hypothesized that these antiinflammatory effects of B. vulgatus and B. dorei LPS would suppress the immune response and protect against atherosclerosis after gavage with live B. vulgatus and B. dorei. In the present study, we reported that gavage with B. vulgatus and B. dorei dramatically decreased colon LPS concentrations after gut microbial alterations and confirmed the protective effect related to the suppression of immune response in mice. Moreover, we found that fecal LPS levels were higher in patients with lower abundance of B. vulgatus and B. dorei. This finding suggests that the abundance of B. vulgatus and B. dorei has a direct impact on microbial LPS synthesis in the human gut. Considering that LPS content per cell is known to vary substantially even within the same strain because bacteria can adapt LPS production according to environmental conditions,45 we believe that B. vulgatus and B. dorei might improve the enteric environment, providing gut bacteria with good living conditions and having a beneficial effect on bacterial LPS production.

TLR4 expression increases after LPS exposure in a dose-dependent manner, and TLR-mediated dendritic cell activation and maturation upregulate major histocompatibility complex and costimulatory molecules, as well as cytokine production and T cell activation.37 We demonstrated that live Bacteroides treatment reduced TLR4 expression and activation and maturation markers, such as major histocompatibility complex class II, CD80, and CD86, on CD11chigh dendritic cells in gut-draining MLNs. Similar phenotypic changes along with increased expression of PD-L1 and PD-L2 in dendritic cells were observed in the spleen. Although we could not fully prove the causality, we considered that augmentation of these tolerogenic dendritic cells’ phenotype in MLNs or spleen was suspected to migrate into the blood and was closely associated with attenuated atherosclerotic lesion formation as previously reported.42,43,46 In addition, given the involvement of TLR4 and chemokine receptors in sterile inflammation, it is intriguing to assume that decreased circulating LPS levels after live Bacteroides treatment may reduce the expression of TLR4, CCR7, CCL17, and CCL21 in the aorta, resulting in less macrophage and CD4+ T cell accumulation in atherosclerotic plaque.

We also showed that live Bacteroides treatment strengthened the gut barrier. Although LPS from E. coli increases intestinal permeability in a TLR4-dependent manner,40,41 the impact of colon LPS concentrations on intestinal permeability has not been fully investigated to date. We show for the first time that ZO-1 expression in the colon was significantly higher in mice treated with live Bacteroides and significantly higher expression levels of ZO-1 in HT29 cells stimulated with fecal supernatant from live Bacteroides-treated mice. Although we did not elucidate the molecular mechanisms underlying the regulation of ZO-1, our study provides the first evidence that colon LPS concentrations produced by gut microbiota contribute to the gut barrier strength in vivo and in vitro.

From a clinical perspective, our results pave the way for further studies investigating gut microbial LPS production in the prevention and management of CAD. Despite the growing understanding of gut microbiota in patients with CAD, it is difficult to conclude the definition of dysbiosis in atherosclerosis because each gut microbial profile depends on age, sex, local food or lifestyle, and many other factors. Our findings suggest that fecal LPS levels might serve as a novel dysbiosis marker, although the type and immunogenicity of LPS could not be determined at this point. Given that gut microbiota–derived LPS and systemic endotoxemia are involved in the onset and progression of not only atherosclerosis but also many prevalent disorders, such as inflammatory bowel disease, obesity and related metabolic diseases, and nonalcoholic steatohepatitis,47–49 our findings suggest that Bacteroides treatment may serve as a novel and attractive therapeutic strategy for suppressing inflammatory response in such diseases. A previous article has already reported that B. vulgatus protected against E. coli–induced colitis in gnotobiotic IL-2–deficient mice.50

The present study had several limitations that should be considered when interpreting the results. First, the gut microbiome was analyzed by 16s rRNA sequencing analysis. It has low phylogenetic power at the species level and poor discriminatory power for some genera. In addition, the gut microbial analysis in patients with CAD was a single-center study with a relatively small number of patients. Moreover, because we did not perform coronary angiography for all the controls without CAD, there might have been individuals with subclinical CAD in the control group. These limitations imply that we did not fully elucidate the characteristics of gut microbial composition in CAD; however, 16S rRNA sequencing analysis helped to seek candidate and translate to animal experiments. Second, we quantified the LPS levels via the limulus amebocyte lysate assay. Although the limulus amebocyte lysate assay is the gold standard assay for LPS measurements, it could not measure LPS immunogenicity and the impact of Bacteroides on the structure and function of LPS. Third, we did not completely elucidate the mechanisms of atherosclerosis protection by lower LPS concentrations in mice. Besides, the relative abundances of most of the dominant gut microbiota in mice and human are quite different. Further study is warranted to bolster our data and identify the causal link between fecal LPS concentrations and atherosclerotic plaque formation. Fourth, we performed gavage only with B. vulgatus and B. dorei. More effective strains for preventing CAD may exist.

In summary, we identified the gut bacterial species involved in human CAD and used a mouse model to clarify the causal relationship between B. vulgatus and B. dorei and atherosclerosis.

Acknowledgments

We are indebted to the study participants and grateful to the medical staff for their co-operation with collecting the fecal samples. We thank Tetsuya Hara, Shigeyasu Tsuda, Toshihiko Oshita, Mitsumasa Okano, Koichi Watanabe, Emi Ichiyanagi, Yukiko Takeuchi, and Emiko Yoshida for their excellent technical support. We also acknowledge Takahiro Kodama for creating the graphical abstract and Toshitaka Odamaki for technical discussions.

Footnotes

*Drs Yoshida and Emoto contributed equally.

Sources of Funding, see page 2497

The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCULATIONAHA.118.033714.

https://www.ahajournals.org/journal/circ

Tomoya Yamashita, MD, PhD, Department of Internal Medicine, Division of Cardiovascular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 6500017, Japan. Email

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