TIMI 2129 No.

of Pages 16

Trends in
Microbiology
Review

Gut microbiome-mediated mechanisms for

reducing cholesterol levels: implications for
ameliorating cardiovascular disease
Baolei Jia, 1,* Yuanqiang Zou, 2 Xiao Han, 3 Jin-Woo Bae, 4,5,6 and Che Ok Jeon 1,
*

Cardiovascular disease (CVD) is a health problem worldwide, and elevated cho- Highlights
lesterol levels are a key risk factor for the disease. Dysbiotic gut microbiota has Short-chain fatty acids and secondary
been shown to be associated with CVD development. However, the beneficial bile acids can decrease cholesterol levels
by regulating cholesterol metabolism.
effects of healthy microbiota in decreasing cholesterol levels have not been
summarized. Herein, we begin by discussing the potential mechanisms by which Microbial bile salt hydrolases can in-
the gut microbiota reduces cholesterol levels. We further sketch the application crease cholesterol disposal rates by
of probiotics from the genera Lactobacillus and Bifidobacterium in reducing accelerating its conversion to bile acids.

cholesterol levels in clinical studies. Finally, we present the cholesterol-lowering

Uncultured bacteria from the human gut
function of beneficial commensal microbes, such as Akkermansia and Bacteroides can transform cholesterol to coprostanol.
spp., as these microbes have potential to be the next-generation probiotics
(NGPs). The information reviewed in this paper will help people to understand Probiotics from the genera Lactobacillus
and Bifidobacterium are efficient re-
how the gut microbiome might alter cholesterol metabolism and enable the devel- ducers of cholesterol levels in clinical
opment of NGPs to prevent and treat CVD. studies.

Several candidate next-generation

CVD, cholesterol, and the gut microbiome probiotics, including Akkermansia
muciniphila, Bacteroides spp.,
CVD is a life-threatening condition and a leading cause of death worldwide [1]. An elevated
Clostridium spp., Christensenella
cholesterol level is considered a risk factor, biomarker, and predictor of CVD, as cholesterol minuta, Eubacterium spp., and
deposited in blood vessel walls can reduce or block the flow of blood and transport of oxygen [2]. Faecalibacterium prausnitzii, have
Hypercholesterolemia (see Glossary) has consistently been associated with an increased risk of been shown to decrease cholesterol
levels in human or animal studies.
developing CVD, in the form of atherosclerosis, heart failure, or hypertension; it also adversely
contributes to other diseases, such as diabetes, liver diseases, and Alzheimer’s disease [3]. In mam-
1
mals, cholesterol is mainly synthesized in the hepatocytes and intestine and can also be obtained Department of Life Science, Chung-Ang
University, Seoul 06974, Republic of Korea
from dietary components; hepatocytes secrete one gram of cholesterol, and the human diet 2
BGI-Shenzhen, Shenzhen 518083, China
provides approximately 0.4 g per day [4]. Cholesterol from the sources is packed in lipoproteins, 3
College of Biological Science and
primarily absorbed in the duodenum and proximal jejunum, and transported to various organs and Engineering, Fuzhou University, Fuzhou
350108, China
tissues via circulation. Based on density, lipoproteins are classified into four types: high-density lipo- 4
Department of Biology, Kyung Hee
proteins (HDLs) and non-HDLs, divided into very-low-, intermediate-, and low-density lipoproteins University, Seoul 02447, Republic of Korea
5
(VLDLs, IDLs, and LDLs). Non-HDLs transport lipids from the liver to the peripheral tissues. In con- Department of Biomedical and
Pharmaceutical Sciences, Kyung Hee
trast, HDLs transfer cholesterol from peripheral tissues to the liver, a process known as reverse cho-
University, Seoul 02447, Republic of Korea
lesterol transport (RCT). As the density of lipoproteins is related to the transport of cholesterol, HDL 6
Department of Life and
cholesterol has been considered to have a protective effect on atherosclerosis development, whereas Nanopharmaceutical Sciences, Kyung
Hee University, Seoul 02447, Republic of
LDL cholesterol is harmful [5]. In clinical trials, statins have been used to inhibit cholesterol synthesis in
Korea
hepatocytes and to lower serum cholesterol levels. Ezetimibe, an inhibitor of intestinal cholesterol
absorption, has also been widely used to reduce cholesterol levels [6]. Thus, regulating cholesterol
metabolism is a practical and effective strategy for reducing cholesterol levels.

*Correspondence:
Advanced meta-omics studies in recent decades have opened new avenues for understanding baoleijia@cau.ac.kr (B. Jia) and
how the human microbiome can improve human health. The link between the microbiome and cojeon@cau.ac.kr (C.O. Jeon).

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© 2022 Elsevier Ltd. All rights reserved.
 Trends in Microbiology

CVD is one of the most widely studied [7]. A study using hypercholesterolemic mice revealed that Glossary
intestinal microbiota depletion by antibiotics resulted in elevated cholesterol uptake and synthe- Atherosclerosis: a disease,
sis in hepatic cells. The same study also showed that intestinal microbiota transplantation to mice characterized by hardening or thickening
of the arteries, which is caused by the
from humans with elevated plasma cholesterol levels induced increased cholesterol levels [8].
deposition of cholesterol, fat, and other
This suggests that the gut microbiota could regulate cholesterol metabolism and that it is feasible plaque-forming elements in the inner
to reduce cholesterol levels by interventions involving the gut microbiota, such as fecal microbiota lining of an artery.
transplantation, antibiotics, probiotics, and prebiotics. Hypercholesterolemia: high blood
cholesterol levels. Environmental (diet,
weight, and stress) and genetic factors
Probiotics have been defined by the International Scientific Association for Probiotics and contribute to hypercholesterolemia.
Prebiotics in 2014 as ‘live microorganisms that, when administered in adequate amounts, confer Leptin: a peptide hormone secreted by
a health benefit on the host’ [9]. Probiotics are broadly accepted by the public and are fat cells. Leptin regulates food intake,
energy balance, and neuroendocrine
used as functional foods or supplements; certain probiotics incorporating Lactobacillus and
functions.
Bifidobacterium can be used to lower cholesterol levels in the host [10]. Over the past two Lipoproteins: particles composed of
decades, next-generation sequencing (NGS) has enabled a dramatic expansion in the range proteins, cholesteryl esters, and
of microorganisms known to have potential benefits for host health. These microorganisms, triglycerides that are used for the
transport and absorption of lipids in the
which have been identified due to these NGS developments, are sometimes termed NGPs and body.
are also referred to as live biotherapeutic products (LBPs) [11]. NGPs have been defined as ‘live Meta-omics: the characterization and
commensal microorganisms, identified upon comparative microbiota analyses, that when admin- quantification of the biological molecules
istered in adequate amounts, confer a host health benefit’ [12,13]. They are mainly from the genera inhabiting a particular ecological niche. It
includes metagenomics (total DNA),
Akkermansia, Bacteroides, and Faecalibacterium. The association of the gut microbiota with CVD metatranscriptomics (gene expression),
and cholesterol metabolism has been discussed in previous review articles (Box 1) [7,14]. How- metaproteomics (the entire protein
ever, the underlying mechanisms and medicinal effects of gut microbes and their metabolites in collection), and metabolomics (small
molecules).
cholesterol level reduction have not yet been reviewed in detail. Herein, we summarize what is
Microbiome: all the microorganisms
known about the molecular mechanisms of the microbiota in reducing cholesterol levels, and and their genetic material in a specific
then describe the cholesterol-lowering function of NGPs. The purpose of this review is to provide niche.
mechanistic information that the research community can apply to guide the discovery, usage, Microbiota: all the microorganisms in a
specific niche.
and development of NGPs as therapies for CVD.
Next-generation sequencing (NGS):
large-scale and high-speed DNA or RNA
Cholesterol-lowering mechanisms of the gut microbiota and NGPs sequencing technologies. This term is
As noted in Box 1, the gut microbiota and its metabolites can be beneficial in terms of decreasing intended to distinguish this method from
the chain-termination method.
the CVD risk using cholesterol-lowering effects; the metabolites are mainly short-chain fatty
Prebiotics: compounds in food, such
acids (SCFAs) and secondary bile acids (BAs). The metabolites may originate from candidate as oligosaccharide carbohydrates,
NGPs, such as Akkermansia muciniphila, Bacteroides spp., Clostridium spp., Christensenella which assist in the growth and/or activity
minuta, Eubacterium spp., and Faecalibacterium prausnitzii. The mechanisms by which each of of healthy microorganisms, thereby
improving the health of the host.
Probiotics: live microorganisms
Box 1. The link between the gut microbiome and CVD (bacteria and yeasts) that benefit the
host when consumed in adequate
CVD is associated with lifestyle, diet, and the gut microbiome [14]. Dysbiosis of the gut microbiota has been observed in
amounts.
several types of CVD, such as heart failure, atherosclerotic CVD, hypertension, ischemic attack, heart attack, and stroke
Short-chain fatty acids (SCFAs):
[98]. For example, heart failure has been linked to a decreased abundance of E. rectale and F. prausnitzii as well as an
fatty acids with one to six carbon atoms.
increased abundance of Escherichia coli, Klebsiella pneumoniae, and Streptococcus viridans [99]. At the molecular level,
SCFAs are the dominant and most
alterations in the metabolites produced by the gut microbiota are more closely related to CVD than changes at the
important metabolites produced by gut
taxonomic level [100]. Among these metabolites, trimethyl-N-oxide (TMAO), short-chain fatty acids (SCFAs), and bile acids
microbiota.
(BAs) levels are significantly associated with CVD. Elevated TMAO levels increase the risk of coronary artery disease (CAD)
by decreasing RCT, altering cholesterol and BA metabolism, activating inflammatory signaling, accelerating foam cell
formation, and causing hyperactivation of platelets [101]. Given these damaging effects of TMAO, inhibition of its produc-
tion by 3,3-dimethyl-1-butanol has been explored for treating atherosclerosis in a preclinical study [102]. In contrast,
SCFAs produced by the gut microbiota have beneficial effects on cardiometabolic health and prevent CAD development
by regulating metabolic and immune homeostasis, as well as maintaining gut barrier function [103]. Finally, a complex BA
pool has also been linked to CVD risk. Cell- and animal-based studies have shown that hydrophobic secondary BAs
appear to have a cardiotoxic effect, whereas hydrophilic BAs are cardioprotective [104]. These data indicate that TMAO
worsens CVD, while SCFAs and BAs could contribute to its treatment. Hence, summarizing the cholesterol-lowering
mechanisms of SCFAs, BAs, and other molecules may contribute to extending the collection of microbes able to decrease
cholesterol levels, and enable the development of novel and clinically useful microorganisms.

2 Trends in Microbiology, Month 2022, Vol. xx, No. xx

Trends in Microbiology

these strains benefits the host in terms of cholesterol reduction may differ and include processes
such as regulation of cholesterol absorption and metabolism via metabolites, inhibition of choles-
terol synthesis in the liver, acceleration of cholesterol consumption, conversion of cholesterol to
coprostanol, and direct incorporation of cholesterol. In the following section, we review the
mechanisms by which microbial metabolites and enzymes decrease cholesterol levels, and the
microbes performing these actions.

Mechanisms by which SCFAs decrease cholesterol levels

SCFAs (mainly acetate, propionate, and butyrate) are the dominant and most important metab-
olites produced by gut microbiota in the colon. Acetate and butyrate produced by Streptococcus
sp. and Clostridium cluster XIVa sp. are also detected in the small intestine [15]. In a study of
hamsters fed a high-cholesterol diet, supplementation with three SCFAs significantly reduced
the total cholesterol (TC) levels by 24%, 18%, and 17%, indicating that SCFAs are effective mol-
ecules for decreasing cholesterol levels [16]. F. prausnitzii, Eubacterium rectale, Eubacterium
hallii, Roseburia intestinalis, and Ruminococcus bromii are the main species responsible for buty-
rate production in the human gut. Other bacteria, such as strains of Butyricimonas,
Coprococcus, Dysosmobacter, and Subdoligranulum, can also produce butyrate in the human
gut [17,18]. Bacteroides spp. are also important producers of SCFAs in the gut, and a positive
association between the abundance of Bacteroides and fecal levels of SCFAs has been shown
[19]. Clostridium butyricum can also produce a high amount of butyrate, along with acetate
and propionate [20]. Taken together, these reports reveal the ability of a wide range of gut
microbes to produce SCFAs, and thus potentially decrease cholesterol levels.

There are five main pathways by which SCFAs can decrease cholesterol levels in the host
(Figure 1). Firstly, SCFAs can inhibit cholesterol synthesis by downregulating genes encoding
enzymes involved in the synthesis of cholesterol from acetyl coenzyme A (Figure 1A). The synthesis
of cholesterol via this pathway is activated by the transcription factor sterol regulatory element-
binding protein 2 (SREBP2); the process can be divided into four steps and requires more than
22 enzymatic reactions within the liver and intestines [3]. Gene expression assays in human
enterocytes revealed that nine genes involved in cholesterol biosynthesis are downregulated
in response to propanoate and butyrate, including that for 3-hydroxy-3-methylglutaryl-CoA
reductase (HMGCR), a rate-determining enzyme in the synthesis process [21]. Animal-based
experiments have shown that ingestion of an SCFA mixture decreased plasma cholesterol levels
in mice by reducing cholesterol synthesis rates in both the liver and small intestine [22]. Notably,
acetate has been shown to downregulate Srebp2 expression and decrease serum cholesterol
levels in hamsters and rats [23].

SCFAs can enhance the conversion of cholesterol to BAs, thereby reducing the cholesterol levels
(Figure 1B). Cholesterol 7α-hydroxylase (CYP7A1) is the only rate-limiting enzyme in the conver-
sion of cholesterol to BAs. An animal-based experiment demonstrated that administration of an
SCFA mixture to hamsters increased the expression level of Cyp7a1 in the liver [16]. Upregulation
of Cyp7a1 by SCFAs leads to an increase in the conversion of cholesterol to BAs and the intake
of cholesterol from the blood to the liver without altering cholesterol absorption, thus increasing
the fecal excretion of BAs and leading to a decrease in plasma cholesterol concentrations [16].
Furthermore, the administration of butyrate to high-fat diet (HFD)-fed mice can increase the
expression of Cyp7a1 and protect against atherosclerosis [24].

SCFAs can inhibit intestinal cholesterol assimilation by regulating cholesterol transporter expres-
sion (Figure 1C). In intestinal cells, Niemann–Pick C1-like 1 (NPC1L1) abundantly expressed in
duodenum mediates cholesterol absorption, while the ATP-binding cassette transporters G5

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 Trends in Microbiology

Cholesterol

O
NPC1L1 ABCG5/8
Cholic acid
HO OH
O
OH ABCA1
HO OH Cholesterol
HO OH
OH Chenodeoxycholic acid
CYP7A1
HO
HO
Cholesterol
7-Hydroxycholesterol

Enterocyte Macrophage cell

(B) (C) (D)

SREBP2
HO HO
O O (A)
O
OH
HO Acetate
(E)
OH
S
O HMGCR
Cholesterol Butyrate
CoA S Propionate
OH Fibers
Acetyl-CoA CoA Mevalonic acid
HMG-CoA
GPR41
Adipocyte
Leptin

Upregulation Downregulation Activation

Trends in Microbiology

Figure 1. Proposed mechanisms by which short-chain fatty acids (SCFAs) reduce cholesterol levels. (A) SCFAs produced by fermentation of fiber by gut
microbes decrease the expression of genes in the cholesterol synthesis pathway via SREBP2 regulation. (B) SCFAs also increase the expression of CYP7A1, which
accelerates the transformation of cholesterol to BAs. (C) SCFAs also mediate cholesterol absorption in intestinal cells through upregulation of ABCG5/8 expression and
downregulation of NPC1L1 expression. (D) In macrophage cells, SCFAs increase the expression of ABCA1 to enhance cholesterol efflux. (E) SCFAs also activate
GPR41 in adipocytes to produce leptin, which further suppresses the expression of SREBP2. Abbreviations: ABCA1, ATP-binding cassette transporter A1; ABCG5/8,
ATP-binding cassette transporters G5 and G8; BA, bile acid; CYP7A1, cholesterol 7α-hydroxylase; GPR41, G protein-coupled receptor 41. NPC1L1, Niemann–Pick
C1-like 1; SREBP2, sterol regulatory element-binding protein 2.

and G8 (ABCG5/8) can form heterodimers to cause efflux of cholesterol [25]. Incubation of
Caco-2 cells, a human intestinal cell line, with butyrate decreased the expression of NPC1L1,
whereas the mRNA levels of ABCG5/8 in the cells increased [26]. An animal-based experiment
showed that exogenous propionate decreased Npc1l1 expression, thereby attenuating athero-
sclerosis, in HFD-fed mice. Oral supplementation with propionate to humans also significantly
reduced LDL cholesterol, TC, and non-HDL cholesterol levels in subjects with increased baseline
LDL cholesterol levels [27]. Furthermore, butyrate decreases Npc1l1 expression and increases
Abcg5/8 expression in the intestinal tissues of mice, resulting in the inhibition of intestinal choles-
terol absorption and prevention of atherosclerotic development [28].

Additionally, SCFAs can influence cholesterol levels in macrophages by accelerating cholesterol

export via the regulation of ATP-binding cassette transporter A1 (ABCA1), which transports
cholesterol out of peripheral macrophages and the liver (Figure 1D). Upon taking up excess
LDL, macrophages can form foam cells, which are filled with fat droplets and contribute to
CVD [29]. In vivo, in vitro, and ex vivo experiments have shown that butyrate significantly promotes
the expression of Abca1 and increases the efflux of cholesterol from macrophages, thereby
preventing HFD-induced atherosclerosis in mice [24].

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SCFAs have also been proposed to affect cholesterol levels through signaling pathways
mediated by G protein-coupled receptors 41 and 43 (GPR41/43) (Figure 1E). In adipocytes,
SCFA–GPR interactions stimulate the production of leptin [30], a signaling molecule that
decreases hepatic lipogenesis and cholesterol synthesis by suppressing Srebp2 and expression
of cholesterogenic genes, which further decreases cholesterol levels [31]. Acetate administration
to pigs has been shown to decrease TC and increase leptin concentrations [32], suggesting that
SCFAs could affect cholesterol metabolism through leptin signaling.

Together, the five mechanisms by which SCFAs affect cholesterol metabolism reveal that there are
diverse means by which SCFA-producing bacteria can be beneficial to the health of the host.
Furthermore, SCFA-producing activity can consequently be used as a guide for the discovery
and development of NGPs.

Mechanisms by which gut microbial bile salt hydrolases decrease cholesterol levels
The conversion of cholesterol to BAs accounts for ~90% of the daily output of cholesterol and is
the most significant process in cholesterol catabolism. The two primary BAs, cholic acid (CA) and
chenodeoxycholic acid (CDCA), are the main metabolites produced by the BA synthesis
pathways in the liver [33]. CA and CDCA are conjugated to either taurine or glycine in the liver
and released into the duodenum to facilitate lipid metabolism (Figure 2). Conjugated BAs are
further hydrolyzed by microbial bile salt hydrolases (BSHs) in the small and large intestines to
produce amino acids and primary BAs, which initiate BA conversion in the gut [34,35]. BSHs
can decrease cholesterol levels in the host via two mechanisms: (i) micelle formation in the
small intestine, and (ii) increased BA excretion via feces (Figure 2). In the small intestine, BAs
form micelles with cholesterol, fatty acids, and monoglycerides. The unconjugated BAs produced
by BSH are more hydrophobic and less soluble than conjugated BAs, which decreases their
passive influx into enterocytes. Hence, increased BSH activity may reduce the absorption of
dietary cholesterol and increase BA excretion through feces, resulting in a cholesterol-lowering
effect [36]. Increased BA excretion can further reduce the size of the circulating pool of BAs in
the gut. These BAs may be replaced via increased de novo BA synthesis from cholesterol, result-
ing in further reduction in host cholesterol levels [37].

BSH activity is a critical criterion for probiotic screening [34,38]. Animal experiments have
shown that BSHs from these species may have contrasting effects on host health. For example,
high-level expression of BSHs from Lactobacillus salivarius (belonging to Firmicutes) in mice
significantly reduces plasma cholesterol and host weight gain [39]. In contrast, mice colonized
by BSH-producing Bacteroides thetaiotaomicron gained more weight on an HFD than mice
colonized with bacteria lacking the relevant hydrolases [40]. The conflicting impact of the BSHs
from L. salivarius and B. thetaiotaomicron may be due to differences in colonization of the strains.
L. salivarius is likely to colonize the small intestine to influence lipid metabolism [39], while
B. thetaiotaomicron is more likely to colonize the cecum to affect the BA pool and related
signaling [40]. These data suggest that BSH-encoding probiotics from Firmicutes, such as
Lactobacillus, are promising candidates for the reduction of cholesterol levels.

Cholesterol-lowering function of secondary BAs

Secondary BAs are formed from primary BAs via microbe-catalyzed dehydroxylation, epimerization,
and/or dehydrogenation reactions in the large intestine [41] (Figure 3). Deoxycholic acid (DCA) and
lithocholic acid (LCA) are the dominant secondary BAs, accounting for 34 ± 16% and 26 ± 10%
of the BA component of human feces, respectively. The third most abundant component of gut
BA consists of 3β-hydroxy(iso)-BAs (27 ± 18%), and ursodeoxycholic acid (UDCA, 5.5 ± 9.3%)
is also present [42]. Furthermore, iso-, oxo-, allo-, oxoallo-, and isoallo-LCA are enriched in the

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Bile acid (90-95%) reabsorption and transport to liver

H
H O
H R
T/G
H H
H
H H Cholesterol
HO H H OH Bile acid pool
HO
Conjugated bile acids O O O
H O R R R
R T/G OH OH
OH
H Stored in the gallbladder Dehydroxylation
H and excreted to the Bile salt Dehydrogenation
Bile acids hydrolases Epimerization
H H
intestinal tract HO OH HO OH HO
OH
HO Conjugated bile acids Primary bile acids Secondary bile acids
Bile acid pool

Bile acid excretion

(0.3-0.5g/d)

Trends in Microbiology

Figure 2. The process of cholesterol conversion to bile acids (BAs) and the function of bile salt hydrolases (BSHs) in the process. In the liver, cholesterol
can be converted into primary BAs. These are conjugated with either taurine or glycine and transported to the small intestine, where conjugated BAs are hydrolyzed to
produce primary BAs. The primary BAs can be further converted to secondary BAs by microbial catalyzed reactions. During the transformation from cholesterol to
secondary BAs, microbial BSH catalysis is the key step. Increasing the abundance of BSHs in the gut accelerates BA excretion, which hastens the entire process of
conversion of cholesterol to BAs. Abbreviation: CYP7A1, cholesterol 7α-hydroxylase.

guts of Japanese centenarians [43], suggesting that BAs are strongly related to human health and
longevity. The large and complex BA pool has been linked to various processes through its interac-
tions with, and activation of, various receptors, mainly the nuclear farnesoid X receptor (FXR) and
Takeda G protein-coupled receptor (TGR5) localized in the cell membrane [44].

FXR is abundantly present in the intestine, liver, and kidney, and its activation regulates choles-
terol metabolism via diverse pathways (Figure 3). Animal experiments have shown that activation
of FXR substantially prevents atherosclerosis progression via a cholesterol-lowering effect [45].
The binding affinity of the BAs that mediate FXR stimulation can be ranked from strongest to
weakest as follows: CDCA > DCA > LCA > CA. Firstly, FXR activation can decrease SREBP-2
and HMGCR protein levels in the mouse liver [46] (Figure 3A). Secondly, FXR regulates choles-
terol efflux and excretion by regulating ABCG5/8 expression in enterocytes (Figure 3B). In a
study on cholesterol excretion in mice, FXR activation in the intestine with an FXR agonist
(PX20606) was shown to induce upregulation of Abcg5/8, resulting in cholesterol efflux into the
lumen and excretion of approximately 60% of TC [47]. Furthermore, FXR activation in hepatic
cells by obeticholic acid (OCA), an FXR agonist, inhibited intestinal cholesterol absorption and
increased fecal cholesterol excretion [47]. Another study found that CDCA-activated FXR can
induce the upregulation of Abcg5/8 in the intestine via irisin signaling, which increases the fecal
excretion of cholesterol [48]. Thirdly, activation of hepatic FXR by OCA increases Abca1
expression and macrophage RCT, a process known to be atheroprotective [49] (Figure 3C).
Together, these mechanisms of action lead to reduced synthesis and decreased absorbance,
and enhanced disposal of cholesterol in feces.

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In addition to activating FXR, BAs can also bind TGR5, which is extensively expressed in the
intestine, spleen, gallbladder, and placenta, but not in hepatocytes [50]. LCA shows the highest affin-
ity to TGR5, followed by conjugated and unconjugated DCA, CDCA, and CA; stimulation of TGR5
leads to activation of adenylyl cyclase, which produces cAMP from ATP and induces glucagon-like
peptide-1 (GLP-1) secretion [50] (Figure 3D). GLP-1 is an intestine-derived peptide hormone secreted
by L cells, which are specialized enteroendocrine cells found in the intestinal tract and pancreas. The
GLP-1 secretion resulting from TGR5 activation by secondary BAs increases RCT levels and Abca1
expression, and reduces lipid loading and macrophage inflammation, resulting in marked attenuation
of atherosclerosis in mice [51]. Additionally, the activation of GLP-1 receptors by agonists has been
shown to decrease total and LDL cholesterol levels in patients with type 2 diabetes (T2D) [52],
implying that GLP-1 signaling from TGR5 activation is a potential pathway for regulating cholesterol
metabolism in the host. There is also a further signaling pathway in which TGR5 is activated by cholic
acid-7-sulfate, which is produced by hydroxysteroid sulfotransferase 2A (SULT2A) in the liver and
then transported to the intestinal lumen, while the expression of SULT2A is induced via activation
of the hepatic vitamin D receptor by LCA [53]. Sulfation of BAs catalyzed by SULT2A increases
solubility, limits reabsorption, and promotes elimination of BAs with stool. Sulfation of BAs also
prevents cholesterol solubilization by increasing the BA-micellar critical concentration [54]. These
data indicate that TGR5 can be activated in several ways and that the TGR5 signaling pathway
that produces GLP-1 can be used to reduce cholesterol levels.

Regarding the microbial origins of BAs, the main secondary BAs, DCA and LCA, are produced by
the Clostridium spp. Clostridium scindens, Clostridium hiranonis, and Clostridium hylemonae; no
probiotic function of these bacteria has yet been reported [44]. However, the metabolism of BAs
in the gut is performed by multiple species. It requires the products from one bacterial species be-
fore another can utilize the resulting product as a substrate (similar to an ‘assembly line’). This
(cometabolic) process is a complex method by which BAs are metabolized. For example, DCA
and LCA can be further transformed by gut bacteria. Parabacteroides distasonis can transform
LCA to 3-oxo-delta-4-LCA. Several Bacteroidetes strains, including Bacteroides dorei,
Bacteroides vulgatus, Parabacteroides merdae, and Odoribacter laneus can produce isoallo-
LCA from 3-oxo-delta-4-LCA [43,55]. Isoallo-LCA produced by the species can bind to nuclear
receptor 4A1 (NR4A1, also called Nur77) [55], whose activation in the liver can suppress the
expression of SREBP2 and HMGCR to regulate hepatic cholesterol synthesis and restrict athero-
sclerosis progression [56]. These data imply that NGPs of these species may act via mechanisms
other than SCFAs and BSHs to decrease host cholesterol levels.

Transformation of cholesterol to coprostanol as a microbial cholesterol-lowering mechanism

Cholesterol is mainly absorbed in the small intestine but its absorption percentage ranges from
29% to 80% in humans [57]. Unabsorbed cholesterol can be transformed into coprostanol by
the commensal bacteria, leading to a decreased concentration in the cholesterol pool and a
lower serum cholesterol level [58]. Coprostanol is a cis derivative of cholesterol that is only weakly
absorbed by intestinal cells. The transformation from cholesterol to coprostanol occurs mainly in
the large intestine while coprostanol can also be detected from the first half of the small intestine
[59], implying that different microorganisms inhabiting the small or the large intestine may convert
cholesterol to coprostanol. There are three microbial pathways for this conversion, operating
indirectly via catalysis of intermediate products, indirectly via isomerization, and directly via
reduction (Figure 4). B. dorei D8, isolated from human feces, has previously been shown to
catalyze the conversion with 4-cholesten-3-one and coprostanone as intermediates [60].
Eubacterium coprostanoligenes, isolated from a hog sewage lagoon, but not from human
feces, also uses this pathway. Administration of E. coprostanoligenes to mice transiently
decreased the plasma blood cholesterol concentration; however, the strain could not colonize

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SREBP2
HO HO
O O

(A) O
OH
HO
OH
CDCA>DCA>LCA>CA S Cholesterol
DCA FXR O HMGCR
LCA CoA S
CA isoalloLCA OH
UDCA NR4A1 Acetyl-CoA CoA Mevalonic acid
CDCA iso- HMG-CoA
O
oxo-... LCA HO
VDR OH

HO OS4
CA7S

LCA>DCA>CDCA>CA

(B) (C) (D)

Cholesterol
ABCG5/8

TGR5
ABCA1 Cholesterol

cAMP
PKA
GLP-1
FXR

FXR

GLP-1
Enterocyte Macrophage cell L-cell

Reverse cholesterol transport

Upregulation Downregulation Activation

Trends in Microbiology

Figure 3. Secondary bile acids (BAs) reduce cholesterol levels via several pathways. (A) The BAs produced by the gut microbiota (CA, CDCA, DCA, LCA, isoallo-
LCA, and UDCA) activate nuclear receptors FXR, NR4A1, and VDR in the liver. The affinity of different BAs to FXR is ranked CDCA > DCA > LCA > CA. Activation of FXR by
these BAs and NR4A1 by isoallo-LCA suppresses the expression of SREBP2 and the key enzymes involved in cholesterol synthesis in the liver. Activation of VDR by LCA
increases the production of CA7S, which is transported to the intestine, binds TGR5 in L cells, and induces the secretion of GLP-1 via cAMP-PKA signaling. (B,C) FXR
activation in intestinal cells and macrophages increases cholesterol excretion by increasing the expression of the transporters ABCG5/8 and ABCA1, which mediate
the excretion of sterols. (D) LCA, conjugated/unconjugated DCA, CDCA, and CA can activate TGR5 directly within L cells, thus stimulating the secretion of GLP-1.
Abbreviations: ABCA1, ATP-binding cassette transporter A1; ABCG5/8, ATP-binding cassette transporters G5 and G8; CA, cholic acid; CA7S, cholic acid-7-sulfate;
CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; FXR, farnesoid X receptor; GLP-1, glucagon-like peptide-1; LCA, lithocholic acid; NR4A1, nuclear receptor
4A1; SREBP2, sterol regulatory element-binding protein 2; TGR5, Takeda G protein-coupled receptor; UDCA, ursodeoxycholic acid; VDR, vitamin D receptor.

the intestines of the same mice [61]. Furthermore, Eubacterium ATCC 21408, isolated from rat
feces, may isomerize cholesterol to allocholesterol and reduce its molecules to coprostanol [62].
In addition to the two indirect pathways mentioned, a direct pathway has been proposed, wherein
the 5,6-double bond in the cholesterol structure is reduced to produce coprostanol without the
formation of an intermediate [63]; however, there is no report of any bacterium utilizing this reaction.

Based on activity assays and multiomics analyses, the intestinal sterol metabolism A gene (IsmA)
from E. coprostanoligenes encodes a microbial cholesterol dehydrogenase that catalyzes the
transformation of cholesterol to 4-cholesten-3-one and coprostanone to coprostanol. It is
currently the only known enzyme that converts cholesterol to coprostanol [64]. Unfortunately,
the bacteria encoding IsmA homologs from the human gut are currently unable to be cultured

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O O

4-Cholesten-3-one Coprostanone

IsmA IsmA

HO HO
Cholesterol Coprostanol

HO

Allocholesterol

Trends in Microbiology

Figure 4. The gut microbiota-catalyzed transformation of cholesterol to coprostanol. Three proposed pathways
for the transformation of cholesterol to coprostanol are shown, including indirect catalysis via 4-cholesten-3-one and
coprostanone (blue), indirect via allocholesterol (green), and direct reduction of cholesterol (orange). The confirmed
reactions are shown as a unbroken line. The hypothesized reactions are shown as a broken line. The intestinal sterol
metabolism A gene (IsmA) has been identified from Eubacterium coprostanoligenes and unculturable bacteria found in the
human gut, while other enzymes and mechanisms for these pathways remain unknown.

and have only been identified from metagenomic species. Genomic sequence comparison
showed that the metagenomic species were different from E. coprostanoligenes but similar to
Clostridium leptum, F. prausnitzii, and R. bromii. Additionally, the average relative abundance of
IsmA-encoding species is quite low (1.4%) and the percentage of samples containing the species
varied from 37% to 92% in human cohorts. However, the abundance of IsmA is negatively asso-
ciated with serum cholesterol levels in humans, and ismA+ encoders have more coprostanol and
less fecal cholesterol than non-encoders, as determined by paired metagenomic−metabolomic
analysis [64]. These data suggest that gut bacteria harboring IsmA are a potential gold mine
and may be useful as probiotics to reduce host cholesterol levels and CVD risk.

Adsorption and absorption of cholesterol by bacteria

Bacteria can also adsorb cholesterol on the cellular surface or incorporate cholesterol into the
cellular membrane without undergoing transformation. The assimilated cholesterol could be

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 Trends in Microbiology

eliminated through feces with bacteria. Lactobacilli strains such as Lactobacillus gasseri and
Lactobacillus bulgaricus can bind cholesterol onto cellular surfaces and remove cholesterol
from cultural media. The peptidoglycan and exopolysaccharides secreted by bacteria may
account for the adsorbed cholesterol [65]. More studies on cholesterol uptake from the medium
have focused on the incorporation of cholesterol into bacterial membranes, which has been
widely reported in strains from lactobacilli and bifidobacteria [66]. The incorporation of cholesterol
is strain-specific and is dependent on bacterial growth [65,66]. A successful example is
Bifidobacterium longum SPM1207, which was isolated from the feces of healthy Koreans, and
was able to assimilate cholesterol in vitro. By treating rats fed a hypercholesterolemic diet with
the strain, it could significantly reduce TC and LDL cholesterol concentrations in serum [67].
However, the detailed changes in the gut caused by the bacteria are still unknown, and further
studies should be performed to determine if incorporation of cholesterol is the key mechanism
to decrease cholesterol levels in the host.

Clinical relevance of conventional probiotics for decreasing cholesterol levels

Several bacterial strains have been investigated as candidates for decreasing CVD risk by reduc-
ing total and LDL cholesterol levels. The best-studied probiotics of this type in humans belong to
the genera Lactobacillus and Bifidobacterium [68]. A double-blind, randomized study with parallel
controls involving 114 hypercholesterolemic adults showed that consuming yogurt containing
microencapsulated Lactobacillus reuteri NCIMB 30242 could decrease total, LDL, and non-HDL
cholesterol levels by approximately 4.8%, 8.9%, and 6.0%, respectively [69]. Furthermore, a
randomized double-blind controlled study in hypercholesterolemic patients showed that the
consumption of mixed Lactobacillus acidophilus and Bifidobacterium bifidum could reduce total,
HDL, and LDL cholesterol levels by 15.8%, 11.9%, and 15.9%, respectively [70]. Meta-analyses
of randomized controlled trials involving human participants between 2000 and 2018 also showed
that probiotic consumption affects the cholesterol profile and may decrease TC levels to within the
range of 6.4 mg/dl to 13.3 mg/dl (dl: deciliter) [71]. Taken together, these studies present probiotics
as strong candidates for decreasing cholesterol levels to improve host health.

Overview of NGPs and their effects on host cholesterol levels

Conventional probiotics have shown positive effects in decreasing cholesterol levels;
nevertheless, long-term gut colonization by these species in humans is still challenging [72].
However, NGP strains can efficiently colonize the gut and their beneficial effects on human
health may be mediated by mechanisms different from those of conventional probiotics. In
this section, we summarize the function of these NGP strains (in alphabetical order) in relation
to decreasing cholesterol levels.

A. muciniphila
A. muciniphila is a mucin degrader that colonizes the intestinal tract of >80% of humans. The type
strain of A. muciniphila, ATCC BAA-835, has been well studied and is a representative NGP [73].
Several studies have demonstrated the cholesterol-lowering effects of A. muciniphila. Specifically,
a study on hyperlipidemic mice found that treatment with A. muciniphila markedly reduced
fasting plasma TC levels compared to those in vehicle controls [74]. Furthermore, treatment
of hypercholesterolemic mice with live A. muciniphila decreased plasma LDL and HDL cho-
lesterol concentrations [75]. Notably, daily oral supplementation with 1010 A. muciniphila
cells for 3 months in overweight and obese humans was safe and ameliorated insulin resis-
tance [76]. Additionally, the mechanisms by which A. muciniphila decreases cholesterol
levels are diverse and differ from those of other gut microbes (Box 2). These studies suggest
that A. muciniphila has the potential to be developed as a probiotic for regulating human
cholesterol metabolism.

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Box 2. Proposed mechanisms regulating cholesterol metabolism by A. muciniphila

A. muciniphila regulates cholesterol metabolism via several pathways (Figure I). A. muciniphila can yield SCFAs that reduce cholesterol levels by degrading mucin [105].
Additionally, the administration of pasteurized A. muciniphila cells to mice with diet-induced obesity reduces plasma TC levels [106]. Furthermore, treatment of hyper-
cholesterolemic mice with live or pasteurized A. muciniphila or Amuc_1100 (an outer-membrane protein of A. muciniphila) decreases plasma LDL and HDL cholesterol
concentrations [75]. Amuc_1100 can interact with Toll-like receptor 2 (TLR2) to stimulate the activation of nuclear factor-kappa B (NF-κB) [75] (Figure IA). NF-κB can, in
turn, bind directly to the promoter region of the Niemann–Pick type C2 (Npc2) gene and initiate its transcription; the level of NPC2, a cholesterol transfer protein, is
negatively correlated with that of NPC1L1. NPC2 can also regulate cholesterol efflux by stimulating the ABCG5/8 transporter [107]. Treatment with A. muciniphila
has been shown to decrease Npc1l1 levels and inhibit cholesterol absorption in mice [108]. In addition, A. muciniphila has been shown to increase the level of
2-oleoylglycerol (2-OG), which can activate G protein-coupled receptor 119 (GPR119) and stimulate GLP-1 secretion from L cells [109] (Figure IB). Furthermore,
A. muciniphila secretes an 84 kDa protein (Amuc_0576) that interacts with intercellular adhesion molecule 2 (ICAM-2) in L cells. This interaction can further induce
GLP-1 secretion by means of interleukin-6 signaling [110], suggesting that A. muciniphila may regulate GLP-1 secretion via several pathways. The protein interactions
between bacteria and host cells suggest that A. muciniphila can regulate host cholesterol metabolism using its own distinctive mechanisms.

(A) Akk (B) Akk

2-oleoylglycerol Amuc_0576

TLR2
Amuc_1100
Cholesterol
ICAM-2
GPR119

ABCG5/8

NPC1L1

NPC2 cAMP PKA IL-6

GLP-1

NFκB NPC2

Reverse cholesterol GLP-1

Enterocyte transport L-cell

Upregulation Downregulation Activation

Trends in Microbiology

Figure I. Mechanism of cholesterol reduction by Akkermansia muciniphila. (A) In intestinal cells, A. muciniphila produces the protein Amuc_1100, which binds
the TLR2 receptor, thereby activating the NF-κB signaling pathway to initiate NPC2 expression and stimulate cholesterol efflux via ABCG5/8. (B) In the intestinal L cells,
A. muciniphila produces 2-OG and Amuc_0576, both of which stimulate the secretion of GLP-1, a peptide hormone that contributes to reverse cholesterol transport
(RCT); 2-OG stimulates the production of GLP-1 through the GPR119-cAMP-PKA signaling cascade, while Amuc_0576 induces the production of GLP-1 by
binding ICAM-2 to activate interleukin-6 (IL-6) signaling. Abbreviations: 2-OG, 2-oleoylglycerol; Akk, A. muciniphila; ABCG5/8, ATP-binding cassette transporters G5
and G8; GLP-1, glucagon-like peptide-1; GPR119, G protein-coupled receptor 119; ICAM-2, intercellular adhesion molecule 2; NF-κB, nuclear factor-kappa B;
NPC2, Niemann–Pick type C2; PKA, protein kinase A; TLR2, Toll-like receptor 2.

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Bacteroidetes spp.
Several species in the genus Bacteroides, such as Bacteroides acidifaciens, B. dorei,
Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, B. vulgatus, and Bacteroides
xylanisolvens, are considered beneficial to the host because they can produce SCFAs and other
metabolites that regulate host metabolism [19]. Among these, B. xylanisolvens DSM 23964 is a
good example of an NGP. This strain has been authorized by the European Commission as a
starter for fermented milk products [77]. A previous study showed that increasing the level of
B. xylanisolvens in the gut by feeding rats water-soluble cellulose reduced plasma cholesterol
concentrations by 22% compared with the level in control rats [78]. Additionally, oral administration
of B. uniformis CECT 7771 to HFD-fed mice was shown to reduce serum cholesterol levels by 18%
and further reduce liver cholesterol accumulation [79]. Thus, Bacteroides spp. are promising can-
didate probiotics that can contribute to decreasing cholesterol levels. However, the safety aspects
of Bacteroides spp. are the issue to be considered. For example, unresolved T2D after Roux-en-Y
gastric bypass was linked to an increase in B. fragilis and Bacteroides caecimuris [80]. The
Bacteroides2 enterotype, characterized by a high proportion of Bacteroides, has also been re-
ported to be associated with obesity [81]. Additionally, the genomes of Bacteroides spp. generally
encode several virulence factors, and B. fragilis has been reported to initiate abscess formation
[82], suggesting that the issue of NGP safety should be deliberated, and not limited to Bacteroides.

Within Bacteroidetes, P. distasonis and Parabacteroides goldsteinii have also been shown to
benefit host health [83,84]. Oral treatment of obese mice with P. distasonis for 4 weeks reduced
the serum TC concentration by 20.1% and simultaneously decreased the LDL cholesterol level
compared to that in the vehicle control group [83]. In another study, oral administration of
P. goldsteinii considerably reduced adipocyte hypertrophy and weight gain in HFD-fed mice [84].
However, data on the effects of P. goldsteinii on cholesterol metabolism have not been reported.

C. minuta
C. minuta belongs to the family Christensenellaceae, species of which are considered key
bacteria among those absent in obese individuals [85]. Treatment of mice with C. minuta resulted
in significant reductions in weight and adiposity gains [85]. In a study of school-age children from
Mexico, Christensenellaceae was the only family of bacteria that showed a significantly negative
correlation with TC levels [86]. Additionally, a negative association between Christensenellaceae
abundance and VLDL and a positive association between Christensenellaceae abundance and
HDL were observed in a study conducted on more than 2000 adults [87]. Furthermore,
C. minuta and Christensenella intestinihominis have been shown to remove cholesterol from culture
broth with efficiency rates of 36.6% and 54.3%, respectively [88]. These data suggest that
Christensenella spp. are effective cholesterol-lowering agents. However, the possible mechanisms
by which these strains decrease cholesterol levels are not well studied.

C. butyricum
C. butyricum has been shown to have ameliorative or protective effects against various human
diseases [20]. C. butyricum MIYAIRI, a strain isolated from the feces of a healthy human, is
produced as a commercial probiotic in China, Korea, and Japan for use in humans and domestic
animals [11]. This strain has also been authorized as a food ingredient by the European Parliament
and Council [77]. C. butyricum MIYAIRI has been shown to have cholesterol-lowering effects
in animal models. Oral administration of the strain to healthy rats (107 cells/day) accelerated
the cleavage of cholesterol side chains, while the plasma cholesterol level of rats treated with
both cholesterol and the strain (approximately 109 cells/day) was approximately 30% lower
than that of rats fed only cholesterol [89]. Furthermore, oral administration of C. butyricum
CGMCC0313.1, isolated in China, to mice with T2D reduced serum total and LDL cholesterol

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levels [90]. Considering that C. butyricum MIYAIRI has been commercialized, this strain could be Outstanding questions
recognized as a priority candidate for reducing cholesterol levels. Several of the studies cited in this
review were performed in animal
models; can the benefits seen therein
Eubacterium spp.
be translated to humans?
E. hallii and E. rectale from the genus Eubacterium have been considered potential NGPs.
Mukherjee and colleagues reported that Eubacterium spp. are negatively associated with plasma The species and metabolites in the
cholesterol levels in mice with HFD-induced atherosclerosis [91], implying that these strains have human gut microbiome are diverse
and complex; how can we understand
the potential to decrease cholesterol levels and can be developed as probiotics. E. rectale is also
the effect of the microbiome on
considered a health-promoting gut bacterium that can regulate the immune system and attenu- cholesterol metabolism from a global
ate inflammation [92]. Despite this knowledge, there are no reports of experimental research that perspective?
directly address the effect of either strain on cholesterol metabolism.
The safety aspects of NGPs need to be
considered; can scientists address any
F. prausnitzii safety issues using genetic engineering?
F. prausnitzii strains have also been used as probiotics in livestock animals [93]. A Mediterranean
Candidate NGPs are typically obligate
diet intervention for 4 weeks increased the levels of F. prausnitzii in overweight and obese humans
anaerobes; the challenge of production
and decreased TC levels [94]. Increasing the abundance of F. prausnitzii by xylo-oligosaccharide and delivery of the strains in the
prebiotics supplementation also decreased cholesterol levels in HFD-fed rats [95]. These studies absence of oxygen must be addressed.
imply that the abundance of F. prausnitzii may be associated with cholesterol levels in the host.
Furthermore, F. prausnitzii-treated mice fed HFD had a lower hepatic fat content, with decreased
levels of cholesteryl esters in the liver [96], suggesting that this strain has the potential to be used
for the amelioration of cholesterol-induced diseases.

Concluding remarks
In conclusion, high cholesterol levels are a clinically important risk factor for CVD, and microbes,
including both probiotics and gut microbiota, have been shown to have beneficial effects in terms
of decreasing cholesterol levels in the host. Accelerating advancements in bioinformatics and
omics research have provided detailed insights into the cholesterol-control mechanisms involving
microbial metabolites, enzymes, or direct catalysis of cholesterol reduction. This facilitates the
identification of new microorganisms and associated metabolites with beneficial functions [97].
This insight may potentially bridge the knowledge gap regarding the biology of an individual
and their microbiome to further pave the way from general to precision probiotics. Although
several issues should be considered and elucidated in the future (see Outstanding questions),
we envision that NGPs with more specific and efficient cholesterol-lowering functions will be
available to prevent and treat CVD.

Author contributions
B.J. and C.O.J. searched the literature and wrote the manuscript. Y.Z., X.H., and J-W.B. reviewed the manuscript.

Acknowledgments
We greatly appreciate the comments by the reviewers and editor. We also apologize to all the scientists whose work
could not be cited because of space limitations. This study was supported by the National Research Foundation
(2018R1A5A1025077 and 2021R1A2C1006436) of the Ministry of Science and ICT, Republic of Korea.

Declaration of interests
There are no interests to declare.

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