Microbial Pathogenesis 138 (2020) 103827

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Both gut microbiota and cytokines act to atherosclerosis in ApoE−/− mice T

a,1 a,1 a b c d
Qiuxia Liu , Yuchuan Li , Xue Song , Jing Wang , Zebao He , Jiansheng Zhu ,
Huazhong Chend, Jing Yuane, Xue Zhanga, Haiyin Jianga, Sheng Zhangf,∗∗, Bing Ruana,∗
a
The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, College of Medicine, Zhejiang University, Hangzhou, China
b
The First Affiliated Hospital, Department of Cardiology, College of Medicine, Zhejiang University, Hangzhou, China
c
Department of Infectious Diseases, Taizhou Enze Medical Center (Group) Enze Hospital, Taizhou, China
d
Department of Infectious Diseases, Affiliated Taizhou Hospital of Wenzhou Medical University, Linhai, China
e
The Third People's Hospital of Shenzhen, Shenzhen, China
f
Institute of Biotechnology, Cornell University, Ithaca, NY, United States

A R T I C LE I N FO A B S T R A C T

These authors take responsibility for all aspects Background: Several studies have suggested a role for the gut microbiome and cytokines in atherosclerosis de-
of the reliability and freedom from bias of the velopment, but combined analyses of the changes of the gut microbiota and cytokines have not been explored
data presented and their discussed previously.
interpretation. Methods: We treated ApoE−/− and wild-type mice with a high-fat diet for 12 weeks. The gut microbiome and
cytokine composition were analyzed using 16S ribosomal DNA sequencing and RayBio Quantibody Arrays, re-
Keywords: spectively. GO and KEGG analysis were performed to rationalize the potential mechanisms involved in the
Atherosclerosis
process of atherosclerosis.
Gut microbiota
Inflammation
Results: Gut bacterial characteristics in ApoE−/− mice were clearly separated and 21 gut bacterial clades were
Cytokines detected by the LEfSe analysis showing significant differences during the development of atherosclerosis. The
relative abundance of Verrucomicrobia, Bacteroidaceae, Bacteroides, and Akkermansia showed significant positive
correlations with serum total cholesterol, triglyceride (TG), high-density lipoprotein (HDL) and low-density li-
poprotein (LDL). Additionally, the relative abundance of Ruminococcaceae was positive with the level of HDL and
the abundance of Rikenellaceae showed a negative relationship with the level of TG and LDL. Thirteen differ-
entially expressed proteins were identified with P-value < 0.05. CXCL5, FGF2, and E-Selectin were significantly
negatively associated with Akkermansia and Verrucomicrobia. Additionally, CXCL5 was significantly negatively
correlated with Bacteroides and Bacteroidaceae. Three "cellular component" subcategories, 24 ″molecular func-
tion" subcategories, 752 ″biological process" subcategories and 29 statistically remarkable KEGG pathway ca-
tegories were identified.
Conclusions: Gut microbiota changes of the mice having atherosclerosis and their relationship with the in-
flammatory status could be one of the major etiological mechanisms underlying atherosclerosis.

1. Introduction targeting inflammatory pathways in immune cells potentially reduce

Atherosclerosis and resulting cardiovascular disease are still the
leading cause of death and loss of productive life years worldwide,
though considerable advances in prevention, diagnosis and therapy
have been achieved [1]. Atherosclerosis is considered a chronic in-
flammatory disease, encompassing both innate and adaptive immunity.
Increasing evidence has suggested that the cytokines are involved in all
stages of atherosclerosis and have a profound influence on the patho-
genesis of this disease [2]. Furthermore, inhibitors and antibodies
atherosclerotic disease [3–5].
Trillions of bacteria live in the mammalian intestine and interact
with both the adaptive and innate immune systems which contributee
to the development of atherosclerosis [6]. Recent studies have shown
that the aberrant immune response linked to gut microbial dysbiosis,
which is often accompanied by abnormal production of inflammatory
cytokines [7–9]. Studies have demonstrated that the microbiota is as-
sociated with atherosclerosis and CAD in humans by promoting plaque
development via activation of the immune system [10,11]. In a recent

∗
Corresponding author.
∗∗
Corresponding author.
E-mail address: ruanbing@zju.edu.cn (B. Ruan).
1
These authors have contributed equally to this work.

https://doi.org/10.1016/j.micpath.2019.103827
Received 11 September 2019; Received in revised form 29 October 2019; Accepted 29 October 2019
Available online 01 November 2019
0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Q. Liu, et al.
study, germ-free ApoE−/− mice showed reduced systemic inflamma-
tion and decreased atherosclerotic lesion formation compared with
conventionally fed ApoE−/− mice [12]. These findings strongly sug-
gest relationship between the gut microbiota, inflammation and
atherogenesis. Nevertheless, to date, little is known about the roles of
various cytokines and microbial communities in process of atherogen-
esis.
Therefore, the goal of our present study was to characterize the
combined profiles of microbiomes and cytokines between ApoE−/−
mice (a widely used mouse model for atherosclerosis) [13] and wile
type (WT) mice that were fed with high-fat diet (HFD). To do so, 16S
ribosomal RNA sequencing and RayBio Quantibody Arrays were used to
identify the microbiomes, and inflammation cytokines of fecal samples
and serum, respectively. In addition, the correlations between micro-
biome composition and inflammatory cytokines were analyzed and
discussed. Furthermore, GO and KEGG analysis were performed to ra-
tionalize the potential mechanisms involved in the process of athero-
sclerosis.
2. Materials and methods
2.1. Animals
Mice (8 weeks, male) WT C57BL/6J and ApoE−/− on C57BL/6J
background were purchased from Beijing Huafukang Bioscience Co.
(Beijing, China). All mice were housed in a specific pathogen-free an-
imal facility at the Zhejiang University Animal Center, with free access
to water and HFD (MD12015, Medicine Ltd.) under a strict 12-h light
cycle. ApoE−/− mice supplied with HFD for 12 weeks constructed
atherosclerosis models successfully as reported before(Wang et al.).
During the period, body weight was measured once a week. All the
treatment protocols were approved by the Zhejiang University Animal
Care Committee according to the Institutional Guidelines for Animal
Experiments (NO.ZJU2009101007Y).
2.2. Serum lipid and cytokine measurements
After 12 weeks of HFD feeding, all mice were sacrificed. Whole
blood was collected from mice eyes in an anti-coagulant-free test tube
and centrifuged to obtain serum that was aliquoted and stored at
−80 °C until use for analysis. The levels of serum total cholesterol
(TCho), triglyceride (TG), high-density lipoprotein (HDL) and low-
density lipoprotein (LDL) were measured using the appropriate assay
kits (Nanjing Jiancheng Bioengineering Institute, China). Serum cyto-
kines levels were detected with a Mouse Cytokine Antibody Array Kit
(AMM-CYT-G1000, RayBiotech, Norcross, GA, USA). All experiments
were performed according to the manufacturer's instruction. Briefly,
after blocking, the chips were incubated at room temperature (RT) for
2 h with 100 μL of serum samples. The chips were then washed and
incubated for 2 h with biotinylated antibodies at RT. The chips were
washed again and incubated for 1 h with labeled-streptavidin in the
dark at RT. After additional washes, the signals were visualized using
the InnoScan 300 Microarray Scanner, and the data were extracted
using Axon GenePix software.
2.3. Fecal DNA extraction, 16S ribosomal DNA (16S rDNA) sequencing
Fecal samples were collected every weeks and immediately stored at
−80 °C before further analysis. About 0.5 g fecal sample was used for
DNA extraction with QIAamp Fast DNA Stool Mini kit (Qiagen) ac-
cording to the manufacturer's protocols. The 16S rDNA sequencing was
performed on the Illumina platform at Novogene Bioinformatics
Technology Co. The 16S rDNA genes of distinct regions (V3–V4) were
amplified using specific primerses:341F-CCTAYGGGRBGCASCAG;
806R-GGACTACNNGGGTATCTAAT. All PCR reactions were carried out
with Phusion® High-Fidelity PCR Master Mix (New England Biolabs).
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Microbial Pathogenesis 138 (2020) 103827
Sequencing libraries were generated using the TruSeq® DNA PCR-Free
Sample Preparation Kit (Illumina, USA). The library quality was as-
sessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent
Bioanalyzer 2100 system. Paired-end reads were merged using FLASH
(V1.2.7, http://ccb.jhu.edu/software/FLASH/) and Quality filtering on
the raw tags were performed under specific filtering conditions to ob-
tain the high-quality clean tags according to the QIIME (V1.9.1, http://
qiime.org/index.html). Chimera sequences in the quality-filtered tags
were detected by the UCHIME algorithm (http://www.drive5.com/
usearch/manual/uchime_algo.html) and were removed.
2.4. Gut microbiota diversity and composition analysis
Sequences analysis were performed by Uparse software (Uparse
V7.0.1001, http://drive5.com/uparse/). Sequences with ≥97% simi-
larity were assigned to the same operational taxonomic units (OTUs).
For each representative sequence, the Green Gene Database (http://
greengenes.lbl.gov/cgi-bin/nph-index.cgi) was used based on RDP
classifier (Version 2.2, http://sourceforge.net/projects/rdp-classifier/)
algorithm to annotate taxonomic information. Additionally, alpha di-
versity was applied to analyze the complexity of species diversity for
the samples. ANOISM analysis was used to evaluate differences among
the samples in species complexity. Then, for β diversity, OUT table was
used to generate unweighted UniFrac distance matrix, PCoA and Linear
regression plots and LDA Effect Size (LEfSe) was performed and dis-
played. All these indices in our samples were calculated with QIIME
(Version 1.7.0).
2.5. Gene ontology (GO) analysis and signaling pathway assignment using
the Kyoto encyclopedia of genes and genomes (KEGG)
GO and KEGG analysis were performed on the serum cytokines data.
Protein function annotation including three subtypes: biological process
(BP), molecular function (MF) and cellular component (CC) were as-
signed according to the GO database (http://go-database-sql.org/).
Signaling pathway assignments were carried out according to the KEGG
resource (https://www.genome.jp/kegg/kegg1.html).
2.6. Statistical analysis
Statistical analyses were performed using R software (Version
2.15.3) and Prism version 7.0 (GraphPad Software). For comparisons
between two independent groups, an unpaired Student's t-test (data of
normalized distribution) and Mann-Whitney test (data of non-normal-
ized distribution) were used. Spearman's correlation analysis was used
for the statistical correlation between the two parameters. All statistical
analyses were two sided, P < 0.05 was considered statistically sig-
nificant.
3. Results
3.1. Gut microbiota changes in the development of atherosclerosis
We performed 16S rDNA sequencing for 84 fecal samples (10, 10, 12
and 9 samples from ApoE−/− mice and 11, 10, 12 and 10 samples
from WT mice collected at 0, 4, 8 and 12 weeks, respectively). A total of
6,241,747 sequences were generated, with an average of 74,307 reads
per sample. For each sample, a majority of 59,491 high-quality se-
quences were obtained. The α diversity, in term of Shannon index, was
significantly lower in ApoE−/− mice at 0-week but did not statisti-
cally significantly different between two groups at 4, 8 and 12 week
(Fig. 1A). Dissimilarities in the fecal composition between two groups
among 4 times were shown in Fig. 1B at the phylum level. Principal
coordinate analysis (PCoA) (unweighted UniFrac distance metric based
on the OUT table) showed that fecal sample bacterial characteristics in
ApoE−/− mice were clearly separated during the development of
Q. Liu, et al. Microbial Pathogenesis 138 (2020) 103827

Fig. 1. Gut microbial changes in the development of atherosclerosis.

Comparison between 16S rDNA sequencing data of fecal samples from ApoE−/− mice(0W, n = 10; 4W, n = 10; 8W, n = 12; 12W, n = 9) and control mice (0W,
n = 11; 4W, n = 10; 8W, n = 12; 12W, n = 10).
A, Box plots of the α-diversity (as assessed by Shannon index) based on the OTUs of ApoE−/− mice and controls. B, Relative abundance (%) at phylum level. C,
Principal coordinate analysis (PCoA) based on the OUT table separate groups from different times. The first two principal coordinates (PC1 and PC2) of weighted
UniFrac are plotted for each sample. The variance explained by the PCs is indicated in parentheses on the axes. D, Cladogram generated with LEfS analysis illustrating
significant shifts in the gut microbiota during the process of atherosclerosis. Size of yellow circle is proportionated to each taxon's mean relative abundance.

atherosclerosis. (all P < 0.001 by Anosim analysis and multi-response
permutation (MRPP)) (Fig. 1C). LEfSe analysis was used to investigate
the change among the microbial profiles during the process of athero-
sclerosis. Twenty-one gut bacterial clades were detected showing sig-
nificant differences with LDA score higher than 4 (Fig. 1D).
3.2. Different gut microbial species and their relations with serum lipids
To investigate the difference of the microbial communities between
the mice with or without atherosclerosis, the fecal samples collected at
12-weeks were used. The top 5 phyla were Bacteroidetes, Firmicutes,
Proteobacteria, Deferribacteres, and Verrucomicrobia. There was a
significantly higher relative abundance in bacteria belonging to the
phylum Verrucomicrobia in the ApoE−/− mice group compared with
that in the control group (Fig. 2A). We didn't observe any significant
difference in other types of phyla. In addition, the Bacteroidetes/Firmi-
cutes ratio (BF ratio) appeared lower in ApoE−/− mice, although
difference was not significant between the 2 groups. Further down-
stream taxonomic analysis at the family level suggested a significant
increase of the Ruminococcaceae and Bacteroidaceae in ApoE−/− mice
compared with that in the control mice. Inversely, a significant decrease
of the Rikenellaceae was observed in the ApoE−/− mice versus the
controls (Figurea 2B). At the genus level, a significant increase in the
abundance of the Bacteroides and Akkermansia were observed (Fig. 2C).

3
Q. Liu, et al. Microbial Pathogenesis 138 (2020) 103827

Fig. 2. Different abundance of gut microbiota between ApoE−/− mice and control mice are associated serum lipids.
A, Relative abundance of Top 5 phylum are shown. B–C, Relative abundance of Top 10 family and genus are shown. D, Heatmap of correlations between the
abundances of significantly different taxa and serum lipids. Spearman correlation coefficients are represented by color ranging from blue, negative correlation
(−0.5), to red, positive correlation (0.5). Significant correlations are noted by * P < 0.05 and **P < 0.01.

Next, to evaluate the association between alterations in the gut
microbiota composition and atherosclerosis-related serum lipid mar-
kers, taxa that showed significant difference were selected. Spearman
correlation coefficients were calculated between each bacterial taxa
and each of the serum levels of TG, TCho, HDL, and LDL (Fig. 2D). This
analysis revealed that the relative abundance of Verrucomicrobia, Bac-
teroidaceae, Bacteroides and Akkermansia showed significant positive
correlations with these markers (P < 0.05 or P < 0.01). Additionally,
the abundance of Ruminococcaceae was positive with the level of HDL
and the abundance of Rikenellaceae showed a negative relationship with
the level of TG and LDL (P < 0.05). These findings indicate that dif-
ferences in the gut microbiota seem associated with some major serum
lipids.
3.3. Cytokines and their correlations with the gut microbial species
as IFN-γ, IL-6, and Monocyte chemoattractant protein-1 (MCP-1) were
significantly higher (P < 0.05) in ApoE−/− mice.
To detect the specific effect, the correlations were examined be-
tween DEPs and significantly different taxa of gut microbiota (Fig. 3B).
The results showed that CXCL5, FGF2, E-Selectin, GITR, CXCL11, and
TIMP2 were significantly negatively associated with Akkermansia and
Verrucomicrobia (P < 0.05). Bacteroides and Bacteroidaceae were sig-
nificantly negatively correlated with CXCL5 and CXCL11 and positively
correlated with CCL22 (P < 0.05). Additionally, CXCL5 and CXCL11
showed a negative correlation with Ruminococcaceae(P < 0.05). A
positive correlation was observed between Rikenellaceae with FGF2 and
GITR (P < 0.05).
3.4. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes
(KEGG) pathway analysis

A different profile of cytokine composition was observed between
the mice with or without atherosclerosis. Among 96 cytokines ex-
amined, there were 13 differentially expressed proteins (DEPs) (Fig. 3A,
Supplementary Table 1). DEPs were defined as that P-value being less
than 0.05, fold change either more than 1.2 or less than 0.83, and
fluorescent value being over 150. These include cytokines that have
been shown to be involved in the development of atherosclerosis, such
To identify protein function annotation, GO analysis of DEPs was
performed. DEPs with GO terms corresponding to the "cellular com-
ponent" group fell into 3 subcategories, "molecular function" into 24
subcategories, and "biological process" into 752 subcategories (details
in Supplementary Table 2 - 4). In the "cellular component" and "mole-
cular function" categories, “side of membrane” and “cytokine receptor
binding” were the most abundant GO terms of each subcategory,

4
Q. Liu, et al.
Fig. 3. Cytokines and their relationship with the gut microbial species in mice with or without atherosclerosis. Serum samples from ApoE−/− mice (n = 6) and
control mice (n = 5) were detected to compare the cytokines.
A, Volcano plot for differentially expressed cytokines. Blue presented DEPs (p-value < 0.05 and absolute Log2 FC > 0.263). B, Heatmap of correlations between
DEP and significantly different taxa. Increasing values are translated into colors from blue (negative correlation) to red (positive correlation). Significant correlations
are noted by *P < 0.05 and **P < 0.01.
respectively. The top 3 subcategory found in the "biological process"
was “lipid biosynthetic process”, “positive regulation of establishment
of protein localization” and “regulation of cell activation”. Other major
biological processes, such as “cellular response to cytokine stimulus”
and “positive regulation of cell migration” were also found sig-
nificantly.
To clarify the biological pathways associated with the DEPs, we
mapped the 13 DEPs to canonical signaling pathways found in the
KEGG. A total of 29 statistically remarkable categories (P < 0.05) are
listed (details in Supplementary Table 5). The fluid shear stress and
atherosclerosis signaling pathway, Jak-STAT signaling pathway and
Cytokine-cytokine receptor interaction signaling pathway were identi-
fied as being statistically significant between the two groups. Other
major immune pathways, such as Chemokine signaling pathway and
Toll-like receptor signaling pathway, were also statistically changed.
4. Discussion
In this study, we investigate whether there are differences in the
combined profiles of microbiome and cytokines between ApoE−/−
mice and WT mice that were fed with a HFD to mimic atherosclerosis
development process. The result showed significant differences in gut
microbiota in mice with or without atherosclerosis. We found that the
profile of gut microbiota changed considerably between the two groups
and 21 gut bacterial clades showing significant differences during the
5
Microbial Pathogenesis 138 (2020) 103827
process of atherosclerosis. Meanwhile, a different profile of cytokines
was also observed between the two groups. In addition, we demon-
strated a significant correlation between DEPs and different microbiota
taxa. Hence, we revealed that the microbiota and cytokines changed
together in the process of mice with atherosclerosis. Furthermore, the
GO and KEGG analysis results were used to uncover the potential me-
chanisms involved in the process of atherosclerosis. These results imply
the dysbiosis between the microbiota and immune response could be
one of the major etiological mechanisms underlying atherosclerosis.
This study leads us to conclude that microbiota composition and in-
flammation act together in contribution to the development of ather-
osclerosis.
We identified alternations in gut microbiota associated with the
development of atherosclerosis. However, we didn't find the diversity of
gut microbiota decreased in ApoE−/− mice fed with HFD as the
previous study showed [14]. Consistent with a previous study [15], We
found that HFD induced atherosclerosis in ApoE−/− mice is asso-
ciated with an increased level of Firmicutes and a decreased amount of
Bacteroidetes, despite the fact that there was no significant difference
between the two groups.
The alterations of the gut microbiota may be related to the process
of atherosclerosis. Akkermansia muciniphila, a mucin-degrading bac-
terium belonging to the phylum of Verrucomicrobia [16], has been
shown involved in obesity, metabolic disorders and age [17,18]. We
found that the relative amount of Akkermansia muciniphila amount at
Q. Liu, et al.
genus level and Verrucomicrobia at phylum level were significantly
higher in ApoE−/− mice than WT mice when fed with a HFD, but Li
et al. [15] found that Western diet induced aggravation of athero-
sclerotic lesion was accompanied by a reduction of Akkermansia muci-
niphila and replenishment of Akkermansia muciniphila by daily oral ga-
vage reduced the size of atherosclerotic plaques in ApoE−/− mice.
However, the proportion of Akkermansia were reported to increase in
rats on a HFD [18].Further investigation should be conducted to
identify the precise role of Akkermansia muciniphila in atherosclerosis.
Bacteroides, a genus of Gram-negative belonging to the family of Bac-
teroidaceae, has been shown decreased in coronary artery disease(CAD)
patients with high predictor importance [19]. The relative abundance
of phylum Bacteroides corrected with plaque accumulation in mice with
atherosclerosis [20]. A previous study indicated that Bacteroides for-
sythus associated with atherosclerosis by TLR-dependent inflammatory
reactions [21]. These findings are consistent with recent evidence that
gut microbiota plays important roles in the development of athero-
sclerosis.
In the present study, we found that the serum levels of IFN-γ, IL-6,
and MCP-1 were increased in ApoE−/− mice, suggesting that proin-
flammatory cytokines have systematically involved in atherogenesis. In
agreement of these findings, IFN-γ has been shown to have important
effects on promoting and modifying atherosclerosis through both local
effects in the arterial wall as well as a systemic effect on lipoproteins
[22]. Previously, a prospective study of apparently healthy post-
menopausal women found that interleukin-6 (IL-6) was a significant
predictor for the risk of future cardiovascular events [23]. Studies found
that the absence of MCP-1 provides dramatic protection from athero-
sclerotic lesion formation in mice [24] and anti-MCP-1 gene therapy
may serve not only to reduce atherogenesis but also to stabilize vul-
nerable atheromatous plaques [25]. Furthermore, the significant cor-
relation between cytokines and gut microbiota taxonomies were ob-
served. CXCL5 is known to participate in the inflammatory response of
colonic epithelial cells by facilitating the recruitment of neutrophils
[26]. Evidence has shown that inhibition of CXCL5 induced a sig-
nificant macrophage foam cell accumulation in murine atherosclerotic
plaques [27] and CXCL5 may be a useful molecular marker and a
possible target for the treatment of CAD [28]. The mitogenic growth
factor FGF2 is critical for gut homeostasis during colitis and could
promote repair of damaged intestinal epithelium [29]. Studies have
suggested that FGF2 may contribute to the progression of athero-
sclerotic disease [30,31]. E-selectin as an adhesion molecules was
identified as a participant in inflammation through mediating the initial
attachment and rolling of leukocytes on the activated endothelium
[32].
In accordance with what we found, Toll-like receptors (TLRs) are
important in mediating the proinflammatory effects of microbiota. They
can recognize bacterial products such as lipopolysaccharides and pep-
tidoglycans. Genetic variants of TLR4 that confer differences in the
inflammatory response elicited by bacterial lipopolysaccharide are re-
lated to the development of atherosclerosis [33]. An increasing number
of studies have implicated TLR4, TLR3, and TLR2 directly in athero-
sclerosis [34–36]. Furthermore, increasing studies demonstrate that gut
microbiota could influence lipid metabolism and act as environment
factors triggering the development of cardiovascular disease [37,38]. In
support of this mechanism, we found that “regulation of phospholipid
metabolic process” appeared significantly different between the two
groups. Although the exact mechanism is still unknown, the gut mi-
crobiota has been shown essential for cholesterol converted into bile
acid, which is necessary for cholesterol excretion and cholesterol ab-
sorption [39]. Therefore, it is conceivable that cytokines could be in-
volved in the process where gut microbiota modulate host lipid meta-
bolism contributing to atherosclerotic disease [40].
Several limitations must be noted. First, the study does prove cor-
relations between gut microbiota and atherosclerosis, but a cause-and-
effect relationship is to be identified. Second, we showed that several
6
Microbial Pathogenesis 138 (2020) 103827
biological processes and pathways appear involved in the process of
atherogenesis. However, further study should be conducted to clarify
the details of the mechanisms. Thirdly, gut microbiota composition
varies greatly in natural populations from mice to humans. To further
understand the physiology mechanism of atherosclerosis in humans,
samples from patients should be ultimately applied.
In conclusion, we at the first time showed striking differences in gut
microbiota composition and cytokines in the progress of athero-
sclerosis. Furthermore, the interactions between the gut microbiota and
cytokines were analyzed towards interpretation the etiological me-
chanism in atherosclerosis. However, further studies need to be applied
to illustrate the mechanism of how the gut microbiota and cytokines
make a contribution to atherosclerosis. Thus, our results indicated that
gut microbiota promotes atherogenesis may partially through ampli-
fying systemic pro-inflammatory responses. The gut microbiota is a
possible indicator or a potential therapeutic strategy to predict or slow
the atherosclerotic process.
Author contributions
BR, YL, and JW designed and supervised the study. QL performed
statistical analyses and bioinformatics. QL, HJ, SZ, and RB wrote and/or
reviewed the manuscript. All authors took part in overseeing this study
and contributed their ideas on this paper.
Funding
This work was supported by The National Key Stone Basic Research
Program (973 Program) of China (Grant No. 2013CB531401), National
Human Genetic Resources Sharing Service Platform (Grant No.
2005DKA21300) and Sanming Project of Medicine in Shenzhen (Grant
No. SZSM201512005).
Data availability
The raw data supporting the conclusions of this manuscript will be
made available by the authors, without undue reservation, to any
qualified researcher.
Declaration of competing interest
All authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as
a potential conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.micpath.2019.103827.
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