Probiotic Bacteria Produce Conjugated Linoleic Acid
Locally in the Gut That Targets Macrophage PPAR c to
Josep Bassaganya-Riera1*, Monica Viladomiu1, Mireia Pedragosa1, Claudio De Simone2, Adria Carbo1,
Rustem Shaykhutdinov4, Christian Jobin3, Janelle C. Arthur3, Benjamin A. Corl6, Hans Vogel4, Martin
Storr4,5, Raquel Hontecillas1
1Nutritional Immunology and Molecular Medicine Laboratory, Center for Modeling Immunity to Enteric Pathogens, Virginia Bioinformatics Institute, Virginia Tech,
Blacksburg, Virginia, United States of America, 2Experimental Medicine, L’Aquila University, L’Aquila, Italy, 3Department of Medicine, Division of Gastroenterology and
Hepatology and Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America,
4Department of Biological Sciences, Metabolomics Research Centre, and Department of Medicine, Division of Gastroenterology, University of Calgary, Alberta, Canada,
5Department of Medicine, Division of Gastroenterology, University of Munich, Munich, Germany, 6Department of Dairy Science, Virginia Tech, Blacksburg, Virginia,
United States of America
Background: Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side
effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce
immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-
inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-
inflammatory efficacy of probiotic bacteria using a mouse model of colitis.
Methodology/Principal Findings: The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were
investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and
flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina
propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to
determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment
ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic
CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood.
VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR c in myeloid
cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.
Conclusions/Significance: Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the
colon that targets myeloid cell PPAR c to suppress colitis.
Citation: Bassaganya-Riera J, Viladomiu M, Pedragosa M, De Simone C, Carbo A, et al. (2012) Probiotic Bacteria Produce Conjugated Linoleic Acid Locally in the
Gut That Targets Macrophage PPAR c to Suppress Colitis. PLoS ONE 7(2): e31238. doi:10.1371/journal.pone.0031238
Editor: Markus M. Heimesaat, Charite ´, Campus Benjamin Franklin, Germany
Received December 5, 2011; Accepted January 5, 2012; Published February 21, 2012
Copyright: ? 2012 Bassaganya-Riera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: No current external funding sources for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com.
Inflammatory bowel disease (IBD) is an immune-mediated illness
of unknown cause characterized by a chronic and uncontrolled
inflammation of the intestinal mucosa . Its two main clinical
manifestations are Crohn’s disease (CD) and ulcerative colitis (UC).
Current therapies ameliorate IBD by inducing and maintaining
clinical remission, but cannot be considered for the long-term
management of the disease due to their significant adverse side
effects . Thus, exploring novel therapeutic and preventive
approaches for IBD is novel and important.
The adult human gut contains about 100 trillion microbial
organisms . Changes in the composition of the gut microbiome
can modulate the induction of regulatory versus effector immune
responses. Therefore, manipulating the gut bacterial composition
and local metabolite production by using probiotic bacteria has been
explored as a promising avenue for therapeutic intervention against
IBD. However, the mechanisms of action underlying the immuno-
regulatory effects of probiotics and their key metabolites in the gut
mucosaare incompletelyunderstood. Gut microbial populations can
synthesize a wide range of lipid molecules that vary in chemical
structure from short chain fatty acids (SCFA) such as butyrate,
acetate and propionate to polyunsaturated fatty acids (PUFA)
involved in regulating apoptosis and the immune response [2,4,5,6],
including conjugated linoleic acid (CLA) and conjugated linolenic
acid (CLNA) isomers [7,8]. Interestingly, CLA, punicic acid and
eleostearic acid have received some attention in the treatment of
colitis and colitis-associated cancer [2,9,10,11,12,13,14].
PLoS ONE | www.plosone.org1 February 2012 | Volume 7 | Issue 2 | e31238
Bifidobacterium breve strains, a B. bifidum strain and a B.
pseudolongum strain, produce CLA and CLNA isomers in vitro from
linoleic acid or alpha-linoleic acid, respectively . However,
there is no in vivo evidence of production of CLA by probiotic
bacteria in the gut. CLA-producing bacterial strains are found in a
probiotic mixture known as VSL#3.
VSL#3 has demonstrates efficacy in patients with ulcerative
colitis [16,17,18] and pouchitis , and in animal models of colitis
. VSL#3 probiotic bacteria are claimed to regulate intestinal
microbial balance by synthesizing antibacterial substances like
lantibiotics , and other bacteriocins-like compounds ,
competing with pathogens by preventing their adherence to
intestinal epithelial cells [23,24], suppressing gut inflammation by
up-regulating anti-inflammatory cytokines, like IL-10 , favoring
an expansion of mucosal regulatory cells during ileal pouchitis ,
and down-regulating LPS-driven production of IL-8, TNF-a and
IFN-c . In contrast the the reported anti-inflammatory actions,
a recent study demonstrates that pre-treatment with VSL#3 of
mice in a SAMP ileitis model prevents intestinal inflammation
through a mechanism involving stimulation of epithelial TNF-a,
activation of NF-kB and restitution of normal barrier function ,
thereby suggesting immunostimulatory effects at the epithelial layer.
Therefore, there is a lack of comprehensive understanding of the
mechanisms of action underlying the protective effects of probiotics.
Thisstudy aims to investigate the mechanisms of immunoregulation
of gut probiotic bacteria in mice by focusing on their ability to
produce anti-inflammatory metabolites and influence mucosal
Materials and Methods
Animal procedures and experimental diets
C57BL6 mice were used for DSS colitis (n=60) studies. In a
follow up study, we also used macrophage-specific PPAR c
conditional knockout mice with a Cre recombinase targeted to the
LysM-Cre promoter (LysM-Cre+) and control LysM-Cre2 (wild-
type phenotype) mice in a C57BL/6J background (n=60). For
each experiment, mice were fed purified AIN-93G rodent diets
(Table S1) with or without 1% CLA for 24 days prior the
induction of colitis (n=60). The CLA supplement administered
orally contained a 50:50 mixture of the cis-9, trans-11 CLA and
trans-10, cis-12 isomers (Clarinol, Loders Croklaan BV). All
experimental procedures were approved by the Institutional
Animal Care and Use Committee.
All experimental procedures were approved by the Virginia
Tech Institutional Animal Care and Use Committee (IACUC) and
met or exceeded requirements of the Public Health Service/
National Institutes of Health and the Animal Welfare Act.
Oral treatment with probiotic bacteria
Mice received 0.5 mL of the VSL#3 probiotic solution daily by
orogastric gavage using a ball tip gavage needle. The probiotic
solution was freshly prepared daily in sterile conditions with a final
concentration of 0,0072 g VSL#3/mL, corresponding to 1.26109
bacteria per mouse/day, in phosphate buffered saline (PBS) at
pH 7.1. VSL#3 is a commercial mixture composed of four strains
of lactobacilli (Lactobacillus casei, L. plantarum, L. bulgaricus, and L.
acidophilus), three strains of bifidobacteria (Bifidobacterium longum, B.
breve, and B. infantis) and Streptococcus thermophilus. This dose was
selected because probiotic concentrations ranging between 108
and 109cfu/mouse/day are sufficient to efficiently colonize the
intestinal mucosa of rodent [28,29]. Further, this dose is
biologically relevant since it is based on a daily intake of about
3,600 billion bacteria for an adult human weighing 70 kg.
DSS challenge for assessment of colitis and colorectal
To induce colitis, mice were challenged with 2.5% dextran
sodium sulfate (DSS), 36,000–44,000 molecular weight (ICN
Biomedicals, Aurora, OH) in the drinking water for 7 days. Disease
activity indices and rectal bleeding scores were calculated using a
modification of a previously published compounded score .
Mice were euthanized on day 7 of the DSS challenge.
Colonic sections were fixed in 10% buffered neutral formalin,
later embedded in paraffin, and then sectioned (6 mm) and stained
in an Olympus microscope (Olympus America Inc., Dulles, VA).
Colons were scored for leukocyte infiltration, epithelial erosion and
RNA isolation and real-time polymerase chain reaction of
Total RNA from colon was isolated using the Qiagen RNA
isolation kit (Qiagen) according to the manufacturer’s instructions,
and then was used to generate the cDNA template using the
iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) and real-time
RT-PCR was performed as previously described .
Spleen and mesenteric lymph nodes (MLN) were excised and
single-cell suspensions of tissues were resuspended in PBS and
enumerated with the Coulter Counter (Beckman Coulter, Full-
erton, CA). Colon samples were processed for lamina propria
lymphocyte (LPL) isolation. Specifically, cells (36106cells/well)
were seeded into 96 well-plates, centrifuged at 4uC at 3000 rpm
for 4 minutes, and washed with PBS containing 5% serum and
0,09% sodium azide (FACS buffer). Cells were then incubated for
macrophage assessment with fluorochrome-conjugated primary
antibodies to T cell and macrophage markers.
DNA extraction, T-RFLP and community composition
from feces analysis
Fecal samples from between 50–200 mg were resuspended in
lysis buffer containing 20 mg/mL lysozyme and incubated for
30 minutes at 37uC to extract the DNA. Proteinase K to 350 mg/
mL and 10% Sodium dodecyl sulfate was added for further lysis.
Samples were homogenized using a bead beater and 0.1 mm
zieconium beads (BioSpec Products, Bartlesville, OK), and then
processed using a DNA extraction kit (DNeasy; Qiagen, Chats-
worth, CA). The DNA extracted from each sample was used to
amplify the 16S ribosomal RNA gene by polymerase chain
reaction (PCR) using fluorescently labeled primers (forward primer
8F FAM 59-AGAGTTTGATCCTGGCTCAG-39 and reverse
primer 1492R Hex 59-GGTTACCTTGTTACGACTT-39). Am-
plification products were purified using a Qiagen purification kit,
and digested with HhaI, RsaI and MspI restriction enzymes to
generate terminal restriction fragments (TRFs) of varying size. All
data shown are from HhaI digested samples; results from RsaI and
MspI digested samples gave equivalent results. The TRFs were
then processed by capillary electrophoresis on the ABI 3100
genetic analyzer and size, area, and height were obtained. The size
of each TRF corresponds to a different bacterium or bacterial
group due to polymorphisms in the 16S rRNA gene. Size and
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abundance data were obtained from Genemapper and compiled
into a data matrix using Sequentix software (Sequentix, Germany).
These data were standardized (individual TRF peak height as a
proportion of total TRF peak heights within that sample),
transformed by square root, and compiled into a Bray Curtis
similarity matrix using PRIMER v. 6 (Primer-e, Ivybridge, UK).
To test for differences in global community composition, TRF
data were subjected to hierarchical cluster followed by analysis of
similarity (ANOSIM). Biodiversity of each simple was assessed by
Margalef’s test for richness and Shannon-Weiner diversity index;
groups were compared by ANOVA followed by Tukey’s test.
Similarity percentages (SIMPER) were used to determine which
TRFs contribute most to similarity or dissimilarity within a group.
Fatty acid analysis from plasma samples and colonic
Plasma and colonic contents fatty acid methyl esters were
analyzed by gas chromatography (Agilent 6890 N GC) using a
CP-Sil 88 capillary column (100 m60.25 mm i.d. with 0.2 mm
thickness; Varian, Inc., Palo Alto, CA). The conditions were as
follows: the oven temperature was initially set at 70uC, then
increased at 8uC/min to 110uC, then increased at 5uC/min to
170uC and held for 10 min, then increased 4uC/min to 225uC
and held for 15 min. The inlet and flame-ionization detector
temperatures were 250uC, the split ratio was 100:1, and a 1 mL
injection volume was used. Hydrogen was used as the carrier gas
and constantly flowed through the column at 1 mL/min. The
hydrogen gas flow to the detector was 25 mL/min, airflow was
400 mL/min, and the flow of nitrogen makeup gas was 40 mL/
min. Fatty acid peaks were identified by using pure methyl ester
standards (Nu-Check Prep Inc.).
To determine the statistical significance of the model, analysis of
variance (ANOVA) was performed using the general linear model
procedure of Statistical Analysis Software (SAS), and probability
value (P),0.05 was considered to be significant. When the model
was significant, ANOVA was followed by Fisher’s Protected Least
Significant Difference multiple comparison method.
VSL#3 and CLA ameliorate disease activity, gross
pathology and histopathology, and colonic gene
expression in mice with DSS colitis
VSL#3 and CLA treatment decreased disease activity scores
associated with DSS colitis dramatically in comparison to control
mice (Figure 1A). Overall, VSL#3 probiotic bacteria treatment was
more effective than CLA in decreasing inflammation and reducing
disease activity (Figure 1A). In line with these clinical findings,
VSL#3 and CLA significantly ameliorated gross pathology incolon
(Figure 1B), MLN and spleen (data not shown), colonic histopa-
thology such as leukocyte infiltration (Figure 1C) and mucosal
thickening (Figure 1D), and colonic mRNA expression such as
TNF-a (Figure 1E) and MCP-1 (Figure 1F) in comparison to
untreated control mice with DSS colitis.
The loss of PPAR c in macrophages abrogates the
protective effect of VSL#3 in DSS colitis
The percentages of CCR2 and TNF-a expressing macrophag-
es in MLN were lower in CLA and VSL#3 treated mice with
DSS colitis in comparison to the control group (Figure 2 A&B).
To further characterize the role of macrophages and PPAR c on
the regulation of DSS colitis in mice, we designed a follow up
experiment using a loss-of-function approach (i.e., conditional
PPAR c knockout mice). Our data indicate that the protective
effect of VSL#3 in clinical disease and gross pathology was
abrogated in macrophage-specific PPAR c null mice (PPAR c flfl
Cre+) with DSS colitis. Histopathological examination of colons
recovered from control PPAR c-expressing mice (PPAR c flfl
Cre2) with DSS colitis revealed increased mucosal thickness and
leukocytic infiltration (Figure 3A) when compared to CLA or
VSL#3-treated mice (Figure 3 B&C). Conversely, treatment with
CLA or VSL#3 did not protect macrophage-specific PPAR c
null mice from multifocal leukocytic infiltration and thickening of
the colonic mucosa following DSS challenge, although it
protected PPAR c flfl Cre2 mice (Figure 3 D–F). At the
molecular level, VSL#3 decreased TLR-4 levels when compared
to the untreated control PPAR c-expressing mice (Figure 4A).
VSL#3 probiotic bacteria and CLA reduced the numbers
mouse genotype compared with the untreated control group
VSL#3 and CLA decrease gut microbial diversity in a
manner consistent with improved histological correlates
The fecal microbiota of control, CLA and VSL#3 treated mice
was assessed daily 30 days after feeding (day 0 of DSS) using
terminal restriction fragment length polymorphism (TRFLP)
analysis. Microbial community composition was compared between
the treatment groups using Analysis of similarities (ANOSIM).
Depicted in multidimensional scaling plots (Figure 5A), ANOSIM
revealed the fecal microflora composition from CLA- and VSL#3-
treated mice differed significantly from that of control mice
(Figure 5A). More specifically, for ANOSIM comparisons: ctrl vs.
VSL#3, R=0.340, P,0.001; ctrl vs. CLA, R=0.378, P,0.005;
CLA vs. VSL#3, no difference, R=0.087, P=0.112. Moreover,
microbial communities clustered by day 7 DSS histology score
(circle size) (Figure 5A). To directly assess microbial diversity within
each community, we calculated Margalef’s richness and Shannon-
Weiner diversity indices for each sample. VSL#3 and CLA treated
mice exhibited significantly lower microbial diversity compared to
control fed mice (VSL#3 or CLA vs. control, P,0.05). We used
Similarity Percentage analysis (SIMPER) to determine which
bacterial groups, represented as terminal restriction fragments
(TRFs), best define a particular treatment. Interestingly, VSL#3
and CLA treated mice shared an enrichment and predominance of
TRF H-116 (Figure 5C), which was significantly less abundant in
control mice (VSL#3 or CLA vs. control, P,0.05 by t test).
Additionally, there was a negative correlation between the
abundance of this bacterial group and histology score (Spearman
R=20.525, P,0.005, Figure 5D).
Plasma metabolomic analyses
Metabolic changes in plasma of control, CLA and VSL#3
treated mice induced by the DSS challenge were established using
an O-PLS-DA strategy, comparing1H NMR profiled metabolite
concentrations obtained from DSS challenge and no challenge
mice. Three O-PLS-DA models were built for control, CLA and
VSL#3-treated mice. The model summary statistics, explained
variance of X (R2X), DSS challenge status class variation (R2Y)
together with cross-validated predictive abilities (Q2Y) are
presented in Table S2. A clear discrimination was achieved
between metabolic profiles of DSS challenge and no challenge
mice fed control diet, as evidenced by high Q2Y value.
Discrimination between two groups of mice with and without
DSS colitis and treated with CLA was not so clear, and almost no
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difference was found in metabolic profiles of plasma samples from
mice treated with VSL#3 (Figure S1). The degree of differential
abundance of individual metabolites is also summarized in Table
S2 for the predictive component distinguishing DSS challenge
samples from no challenge samples.
VSL#3 treatment increases CLA concentrations in the
Fatty acid analyses of plasma and colonic content specimens
showed an increase in the percentages of cis-9, trans-11 CLA in
colonic contents for both CLA and VSL#3 treated mice (Figure 6).
Figure 1. VSL#3 and conjugated linoleic acid (CLA) ameliorate disease activity. (A), colonic gross pathology (B), leukocyte infiltration (C),
mucosal thickening (D), and down-modulate colonic expression of tumor necrosis factor (TNF-a, E) and monocyte chemoattractant protein 1 (MCP-1,
F) in mice with DSS colitis. Data are represented as mean 6 standard error. Statistically significant differences (P,0.05) when compared to control
(CONT) mice are depicted with an asterisk.
Figure 2. VSL#3 and conjugated linoleic acid (CLA) modulate immune cell subsets in mesenteric lymph nodes from wild type mice
were immunophenotyped to identify CCR2+ + (A) and TNF-a+ + (B) macrophage subsets by flow cytometry. Data are represented as mean
6 standard error. Statistically significant differences (P,0.05) when compared to control (CONT) mice are depicted with an asterisk.
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However, CLA levels were only increased in the plasma of those
mice fed the CLA-supplemented diet (Figure 6), suggesting that
the CLA is produced locally by gut microbes in colons of VSL#3-
treated mice but cannot be absorbed or distributed systemically.
Manipulation of gut microflora with probiotic bacteria can
regulate gut homeostasis and barrier function in part through
production of bacterial metabolites . While SCFA are well-
established bacterial derived metabolites with effector functions in
the gut mucosa, probiotic bacteria can also produce longer chain
fatty acids, including CLA, a compound that suppresses
inflammation by activating PPAR c . CLA ameliorates
experimental IBD in mice and pigs and CD in humans
[2,4,11,33], and other PPAR c agonists have shown clinical
efficacy against human UC [34,35]. Interestingly, VSL#3 also
exerts protective effects in patients with ulcerative colitis
[16,17,18]. We investigated whether VSL#3 suppresses intestinal
inflammation by altering colonic microbial diversity and enhanc-
ing microbial CLA production locally that in turn activates PPAR
c in macrophages.
Mice treated with VSL#3 or CLA showed improved DSS
colitis that was associated with lower percentages of inflammatory
macrophages expressing CCR2 (the receptor for MCP-1) and
TNF-a in MLN. Also, both CLA and VSL#3 suppressed colonic
mRNA expression of TNF-a and MCP-1, indicating that the
protective effect against DSS colitis may be due to a decrease in
macrophage recruitment. Of note, dietary CLA recapitulated the
effects of VSL#3 administration in mice with DSS colitis,
suggesting similarities in the mechanism of action underlying the
anti-inflammatory efficacy of CLA and probiotic bacteria.
The ability of VSL#3 and CLA to modulate immune responses
and decrease inflammation was assessed by examining the
distribution of immune cell subsets at the gut mucosa and
systemically. Macrophages of VSL#3-treated mice expressed
lower levels of CD11c, a pro-inflammatory cell surface marker
implicated in monocyte adhesion to inflamed endothelial cells by
binding vascular cell adhesion molecule 1, an activation marker
Figure 3. VSL#3 and conjugated linoleic acid (CLA) ameliorate colonic histopathology of PPAR c-expressing but not macrophage-
specific PPAR c-null mice. Representative photomicrographs of colons from PPAR c-expressing (PPAR c flfl Cre2, top panels A–C) and
macrophage-specific PPAR c null (PPAR c flfl Cre+, bottom panels D–F) control untreated (A&D), CLA-treated (B&E), and VSL#3-treated (C&F) mice.
The colonic sections were excised on day 7 of DSS challenge, stored in formalin, sectioned and stained with hematoxylin and eosin staining. Panels B
and C, corresponding to PPAR c-expressing mice treated with CLA and VSL#3, show improved colitis. The beneficial effects of CLA and VSL#3 are
abrogated in colons from mice conditionally deficient in macrophage PPAR c (E&F). Arrows indicate leukocytic infiltration, asterisks indicate edema,
filled arrowheads indicate crypt hyperplasia, open arrowheads indicate epithelial erosion and ulceration, and MT denotes mucosa thickenning.
Original magnification at 1006.
Figure 4. VSL#3 and conjugated linoleic acid (CLA) regulate
colonic lamina propria lymphocytes (LPL) and colonic gene
expression. Expression of toll-like receptor-4 (TLR-4) was assessed by
quantitative real time RT-PCR (A). LP macrophages were immunophe-
notyped to identify their production of MCP-1 by intracellular staining
and flow cytometry (B). Values are means 6 SEM, n=10. Statistically
significant differences (P,0.05) when compared to Cre2, Cre+, or both
control (CONT) mice are depicted with an asterisk, two asterisks, or the
# symbol, respectively.
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for monocytes/macrophages . The percentages of CD44-
expressing monocytes in blood were also lower in PPAR c-
expressing mice treated with VSL#3 when compared to the
macrophage-specific PPAR c null mice, suggesting that VSL#3
reduces colonic inflammation by suppressing the recruitment of
monocytes into the colonic LP through a
mechanism that is dependent on the expression of PPAR c in
myeloid cells. Moreover, VSL#3 and CLA treatments were
associated with a decrease in MCP-1-expressing macrophages at
the colonic LP, indicating suppressed inflammatory capacity or
decreased number of M1 or classically activated macrophages.
Oral administration of either VSL#3 or CLA has been reported
to exert dichotomous properties ranging from anti-inflammatory
to immunostimulatory. For instance CLA can prevent colitis 
while enhancing antigen-specific responses to bacterial antigens
. VSL#3 can also prevent colitis  while enhancing innate
immunity at the epithelial barrier level . The role of these
interventions as immunostimulatory or immunosuppressive may
ultimately depend on other systemic and mucosal factors.
Understanding these systems level interactions will help provide
a comprehensive mechanistic understanding of their beneficial
effects in the gut.
Figure 5. VSL#3 and conjugated linoleic acid (CLA) alter the composition of the colonic microbial community. Terminal restriction
fragment (TRF) length polymorphism reveals that VSL#3 and CLA treatment alters luminal microbial community composition (A). Ordination plot by
multidimensional scaling of luminal microbial communities, with circle size depicting histology score (B). Shannon diversity and Margalef richness
(mean +/2 SEM); groups were compared by ANOVA plus Tukey’s test (C). SIMPER analysis; percent contribution of TRFs (top 80%) that contribute
most to similarity within each group (D). Ordination plot in panel A, with circle size depicting standardized abundance of TRF H116.
Figure 6. VSL#3 and conjugated linoleic acid (CLA) regulate plasma and colonic CLA concentrations. Cis-9, trans-11 CLA concentrations
were measured in plasma (A) and colonic contents (B) by gas chromatography. Values are means 6 SEM, n=8. Statistically significant differences
(P,0.05) when compared to control (CONT) mice are depicted with an asterisk.
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Microbial diversity analyses indicate that both VSL#3 and
CLA treatment decreased colonic bacterial diversity in compar-
ison to untreated control mice. VSL#3- and CLA-treated mice
showed the lowest diversity of gut bacterial population compared
to control untreated mice. In addition, the suppressed colonic
bacterial diversity observed in VSL#3 and CLA-treated mice
highly correlated with lower histological lesion scores. Our findings
coincide with the results of a previous report demonstrating that
the probiotic VSL#3 alters the composition of the intestinal
microbiota and these changes correlate with disease protection
. Additionally, we demonstrate for the first time that CLA
treatment can modulate gut microbial diversity.
Plasma metabolomic profiles of VSL#3-treated mice chal-
lenged with DSS resembled more closely those of healthy mice
than control mice with DSS. These findings are in line with results
of a recent study showing that probiotics might correct
inflammation-driven metabolic dysfunction . The ability of
VSL#3 to produce CLA in vitro has been shown in previous
studies . We provide evidence that VSL#3 treatment results in
high colonic concentrations of cis-9, trans-11 CLA. Interestingly,
in contrast to dietary CLA that results in increased plasma levels of
both cis-9, trans-11 and trans-10, cis-12 CLA, the plasma cis-9,
trans-11 CLA concentrations in VSL#3-treated mice where not
different than in control mice, indicating a local effect of
microbial-derived CLA without systemic distribution. This local
effect may be explained by the limited absorption of long-chain
fatty acids at the colonic level. While dietary CLA can be absorbed
in the small intestinal and enter the plasma pools, CLA produced
by the microbiota is not being absorbed in the large intestine but
exerts local immune modulatory and protective effects.
In conclusion, we provide novel in vivo evidence that changes in
microbial diversity and local CLA production are implicated in
PPAR c-dependent mechanisms of action underlying the anti-
inflammatory and anti-carcinogenic effects of probiotic bacteria
(Figure 7). This novel mechanistic model is supported by: results of
loss-of-function analyses illustrating the requirement of macrophage
PPAR c in mediating the full spectrum of anti-inflammatoryeffectsof
probiotic bacteria in the gut; in vivo evidence indicating a reduction of
colonic bacterial diversity with a marked predominance of TRF H-
remarkable similarities in the ability of probiotic bacteria and CLA to
modulate macrophage function at the gut mucosa.
regulate the plasma metabolome and fecal CLA concen-
trations. Histogram demonstrating changes in O-PLS-DA
VSL#3 and conjugated linoleic acid (CLA)
Figure 7. Proposed model for a mechanism of action underlying the protective effects of VSL#3 probiotic bacteria in mouse
models of gut inflammation and cancer. Colonization with VSL#3 probiotic bacteria modulate gut microbial diversity and favor local
production of conjugated linoleic acid (CLA) in the colon that targets myeloid cell peroxisome proliferator-activated receptor c (PPAR c) to suppress
Regulatory Mechanisms of Probiotic Bacteria
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coefficients of individual metabolites related to a dextran sodium Download full-text
sulfate (DSS) challenge in mice administered control (green), CLA
(red) and VSL#3 (blue) treatments (A). Positive bars illustrate
which metabolites were more abundant in DSS challenge mice;
negative ones demonstrate metabolites more abundant in mice
without DSS challenge. Confidence intervals derived from jack
knifing which do not cross zero line mean that metabolite
concentration changes are statistically significant (P,0.05).
Composition of experimental diets.
changes in mice plasma following DSS challenge.
Model summarystatisticsof metabolic
Conceived and designed the experiments: JBR RH CDS. Performed the
experiments: MV MP. Analyzed the data: MV MP JBR RH JA CJ RS
BAC HV MS AC. Contributed reagents/materials/analysis tools: JBR
CDS. Wrote the paper: JBR MV RH.
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Regulatory Mechanisms of Probiotic Bacteria
PLoS ONE | www.plosone.org8 February 2012 | Volume 7 | Issue 2 | e31238