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J. Microbiol. Biotechnol. (2013), 23(4), 518–526http://dx.doi.org/10.4014/jmb.1205.05018First published online January 19, 2013pISSN 1017-7825 eISSN 1738-8872
Effect of Probiotics Lactobacillus and Bifidobacterium on Gut-DerivedLipopolysaccharides and Inflammatory Cytokines: An In Vitro Study Usinga Human Colonic Microbiota Model
Rodes, Laetitia1, Afshan Khan
1, Arghya Paul
1, Michael Coussa-Charley
1, Daniel Marinescu
1,
Catherine Tomaro-Duchesneau1, Wei Shao
1, Imen Kahouli
1,2, and Satya Prakash
1*
1Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells andOrgans Research Centre, Faculty of Medicine, McGill University, Quebec, H3A 2B4, Canada2Department of Experimental Medicine, McGill University, Quebec, H3A 2B4, Canada
Received: May 9, 2012 / Revised: November 28, 2012 / Accepted: December 2, 2012
Gut-derived lipopolysaccharides (LPS) are critical to
the development and progression of chronic low-grade
inflammation and metabolic diseases. In this study, the
effects of probiotics Lactobacillus and Bifidobacterium on
gut-derived lipopolysaccharide and inflammatory cytokine
concentrations were evaluated using a human colonic
microbiota model. Lactobacillus reuteri, L. rhamnosus, L.
plantarum, Bifidobacterium animalis, B. bifidum, B. longum,
and B. longum subsp. infantis were identified from the
literature for their anti-inflammatory potential. Each
bacterial culture was administered daily to a human colonic
microbiota model during 14 days. Colonic lipopolysaccharides,
and Gram-positive and negative bacteria were quantified.
RAW 264.7 macrophage cells were stimulated with
supernatant from the human colonic microbiota model.
Concentrations of TNF-α, IL-1β, and IL-4 cytokines
were measured. Lipopolysaccharide concentrations were
significantly reduced with the administration of B. bifidum
(-46.45 ± 5.65%), L. rhamnosus (-30.40 ± 5.08%), B. longum
(-42.50 ± 1.28%), and B. longum subsp. infantis (-68.85 ±
5.32%) (p < 0.05). Cell counts of Gram-negative and
positive bacteria were distinctly affected by the probiotic
administered. There was a probiotic strain-specific effect on
immunomodulatory responses of RAW 264.7 macrophage
cells. B. longum subsp. infantis demonstrated higher
capacities to reduce TNF-α concentrations (-69.41 ± 2.78%;
p < 0.05) and to increase IL-4 concentrations (+16.50 ± 0.59%;
p < 0.05). Colonic lipopolysaccharides were significantly
correlated with TNF-α and IL-1β concentrations (p < 0.05).
These findings suggest that specific probiotic bacteria,
such as B. longum subsp. infantis, might decrease colonic
lipopolysaccharide concentrations, which might reduce
the proinflammatory tone. This study has noteworthy
applications in the field of biotherapeutics for the prevention
and/or treatment of inflammatory and metabolic diseases.
Key words: Lactobacilli, bifidobacteria, probiotics, cytokine,
lipopolysaccharide, inflammation
Gut-derived lipopolysaccharides (LPS) are critical to the
development and/or progression of a variety of human
conditions, such as chronic low-grade inflammation [5],
metabolic diseases [5, 6, 8], and liver diseases [1, 34]. LPS
are potent immunomodulatory components derived from
the cell wall of Gram-negative bacteria [28], which can
translocate to the blood circulation [28]. The immune
recognition of LPS is mediated through the cluster of
differentiation-14 / toll-like receptor-4 receptor complex in
epithelial cells of the intestine and immune cell types,
predominantly macrophages, B cells, and dendritic cells
[12, 28, 43, 45]. An intracellular signaling cascade is
subsequently promoted, leading to the secretion of
proinflammatory cytokines, such as interleukin (IL)-4, IL-
1β and tumor necrosis factor (TNF)-α [5, 33]. IL-4, IL-1β,
and TNF-α have all been linked with the initiation and/or
maintenance of metabolic diseases [9].
In addition, substantial evidence has demonstrated that
gut dysbiosis [25, 34, 46] might be responsible for increased
endotoxemia [37], which further induces chronic low-grade
inflammation [5, 33] and participates in the pathophysiology
*Corresponding authorPhone: +1-514-398-3676; Fax: +1-514-398-7461;E-mail: [email protected]
519 Rodes et al.
of metabolic [5, 6, 8] and liver [1, 34] diseases. The
manipulation of the gut microbiota composition to reduce
endotoxemia and prevent and/or treat human diseases has
already been performed [6-8, 17, 18, 40, 50]. Studies using
antibiotics to selectively eliminate Gram-negative bacteria
have reported significant decreases in cecal and plasma
LPS concentrations [6, 17, 18]. Strategies based on the use
of prebiotics have demonstrated normalized low-grade
inflammation and improved glucose tolerance. Those effects
were associated with increased gut bifidobacterial content [7,
8]. Interestingly, recent studies have suggested that probiotic
supplements might reduce endotoxemia and chronic low-
grade inflammation [40, 50]. However, to our knowledge,
there have been no previous studies examining the effect
of probiotics Lactobacillus and Bifidobacterium on gut-
derived LPS and inflammatory cytokines.
The most widely used probiotic bacteria belong to
Bifidobacterium and Lactobacillus spp. Those species have
been largely investigated for their health promoting properties
in human diseases where gut dysbiosis is inferred to play a
pathogenic role. They are assumed to re-equilibrate the gut
microbiota composition towards health promoting bacterial
populations [40, 50]. Among many hypothesized mechanisms
of action, Bifidobacteria and Lactobacilli have been
extensively studied for their immunomodulatory activities
[13-15, 19, 22, 24, 27, 29, 30, 32, 35, 36, 39, 41, 47-49].
Bacterial cells and components interact with a wide variety
of cells present in the intestines, such as epithelial, dentritic,
and macrophage cells, which further induce the secretion
of pro- and anti-inflammatory cytokines [4, 10, 11, 24, 27,
29, 39, 49]. Frequently, in vitro studies are performed using
bacterial cultures exposed to intestinal cell lines, such as
intestinal epithelial cells-6 [4], Caco-2 cells [4], and RAW
264.7 macrophage cells [10, 11]. Those studies are useful
to investigate the immunomodulatory properties of bacterial
preparations from a pure bacterial culture. However, they
neglect the effects induced in the existing gut microbiota.
In this study, we investigated the effect of probiotics
Lactobacillus and Bifidobacterium on colonic LPS and
inflammatory cytokine concentrations, using an established
model of human colonic microbiota [38] and RAW 264.7
macrophage cell lines.
MATERIALS AND METHODS
Bacterial Cultures and Growth Conditions
Probiotic bacteria tested were identified from the literature for their
anti-inflammatory potential (Table 1). Bacterial strains tested included
L. reuteri NCIMB 701359, L. rhamnosus ATCC 53103, L. plantarum
ATCC 14917, B. animalis ATCC 25527, B. bifidum ATCC 29521,
B. longum ATCC 15707, and B. longum subsp. infantis ATCC
Table 1. List of probiotic strains of Lactobacillus and Bifidobacterium tested in this study.
Bacterial species Strains screened in this study Literature on anti-inflammatory potential
Lactobacillus reuteri NCIMB 701359 [22, 29, 30, 49]
Bifidobacterium bifidum NCIMB 702715 [13, 24]
Lactobacillus rhamnosus ATCC 53103 [15, 19, 36]
Bifidobacterium animalis NCIMB 702242 [27, 39, 47]
Lactobacillus plantarum NCIMB 11974 [32, 41, 48, 49]
Bifidobacterium longum NCIMB 702259 [13, 49]
Bifidobacterium longum subsp. infantis NCIMB 702205 [14, 35]
Fig. 1. Schematic representation of the experimental design:Administration of probiotics Lactobacillus and Bifidobacteriumin a human colonic microbiota model and quantification ofcolonic lipopolysaccharides, and Gram-negative/positive bacterialcell counts and inflammatory cytokines. A human colonic microbiota model was set up. After inoculation with
fresh human feces, a model stabilization period of 14 days was performed.
From day 0, Lactobacillus or Bifidobacterium probiotic strains were
administered daily to the human colonic microbiota model. A control
fermentation system was performed without administration of probiotic
bacteria. Statistical analysis was analyzed to determine the effect of time
vs. day 0. On days 0, 7, and 14, samples were extracted from the colonic
model for quantification of colonic LPS and Gram-negative/positive
bacterial concentrations. RAW 264.7 macrophage cells were stimulated
with bacterial supernatant extracted from the colonic model. Positive and
negative controls consisted of RAW 264.7 cells exposed to, respectively,
2 µg/ml LPS, and DMEM supplemented with 10% FBS. Concentrations of
TNF-α, IL-1β, and IL-4 cytokines were measured.
PROBIOTICS, LIPOPOLYSACCHARIDES, AND INFLAMMATION 520
15697. L. reuteri NCIMB 701359 was purchased from NCIMB
(Aberdeen, Scotland). The other bacterial cultures were purchased
from Cedarlane (Burlington, Canada). A 1% (v/v) inoculum of each
bacterial strain was passaged daily in de Man, Rogosa, and Sharpe
(MRS) broth (Fisher Scientific, Ottawa, Canada) at 37oC in 5%
CO2. Bacterial cell viability was determined on MRS agar triplicate
plates at 3 different dilutions. For Lactobacilli enumeration, agar
plates were incubated for 24 h at 37oC in 5% CO2. For Bifidobacteria
enumeration, agar plates were supplemented with 0.05% (w/v) cysteine
(Fisher Scientific) and incubated in anaerobic jars with anaerobe
atmosphere-generating bags (Oxoid, Nepean, Canada) for 48 h at 37oC.
The Human Colonic Microbiota Model
A semi-continuous human colonic microbiota fermentation system
was operated, as described previously [38]. The system was operated
with a retention time of 144 h. Following the fermentation system
start-up, a stabilization period of 14 days was performed to allow
the bacterial communities to stabilize in order to be representative of
the gut microbiota. On day 0, administration of pure cultures of
Bifidobacterium or Lactobacillus strains was initiated, where 5 × 108 CFU
was administered daily to the human colonic microbiota model during
14 days. A control fermentation system was performed without
daily administration of probiotics. A schematic representation of the
experimental procedure is given in Fig. 1.
Quantification of Gram-Positive and Gram-Negative Bacteria
Gram-negative and positive bacteria were quantified by plate
counting. On days 0, 7, and 14, samples were extracted from the
human proximal colonic microbiota model and serially diluted in
physiological solution (8.5 g/l NaCl). Bacterial cell viability was
determined on selective agar triplicate plates at 3 different dilutions.
McConkey agar (Becton Dickinson, Mississauga, Canada) was used
for the quantification of Gram-negative bacteria. Phenyl ethyl
alcohol agar (Becton Dickinson) was used for the quantification of
Gram-positive bacteria. The agar media were supplemented with
0.05% (w/v) cysteine for the quantification of anaerobes. The agar
plates were incubated for 24 h at 37oC in 5% CO2 for aerobes.
Anaerobic incubation was performed in anaerobic jars with anaerobe
atmosphere-generating bags for 48 h at 37oC. Total Gram-positive
and Gram-negative bacterial cell counts were calculated as the sum
of, respectively, the anaerobic and aerobic Gram positive and negative
bacterial cell counts.
Lipopolysaccharides Quantification
Samples from the human colonic microbiota model were extracted
on days 0, 7, and 14 and instantly frozen at -20oC in glass vials.
LPS were quantified using the QCL 1000, a commercially available
quantitative chromogenic Limulus amebocyte lysate test from Lonza
(Cedarlane). Samples were assayed at different dilutions against a
standard curve of LPS concentrations. Kit instructions were followed
to pursue the Limulus amebocyte lysate assay procedure.
RAW 264.7 Macrophage Cells Maintenance
RAW 264.7 cells were obtained from Cedarlane. The cells were grown
and maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Invitrogen, Carlsbad, Canada) supplemented with 10% fetal bovine
serum (FBS) from Invitrogen. Cell cultures were performed at 5%
CO2 in a humidified atmosphere at 37oC. Cells were grown to 95%
to 100% confluency and passaged every 3-4 days.
Quantification of Inflammatory Cytokines Secreted
On days 0, 7, and 14, samples were removed from the human
colonic microbiota model and centrifuged for 20 min at 2,000 ×g
and 4oC. The supernatant, containing the free LPS, was collected.
RAW 264.7 cells adjusted to a density of 5 × 105 cells/ml were
exposed for 24 h to the bacterial supernatant. The positive control
consisted of RAW 264.7 macrophage cells stimulated with 2 µg/ml
LPS from Escherichia coli (Sigma-Aldrich, Oakville, Canada). The
negative control consisted of RAW 264.7 macrophage cells exposed
to DMEM supplemented with 10% FBS. After 24 h of stimulation,
the culture supernatant was collected and stored at -20oC until
cytokines analysis. The induction of TNF-α, IL-1β, and IL-4 cytokines
was assayed using commercial mouse ELISA kits (eBioscience, San
Diego, USA). Kit instructions were followed to pursue the solid-
phase enzyme-linked immunoassay procedure.
Statistical Analysis
Values are reported as the mean ± SD of triplicates. All data were
analyzed using Prism software (Prism, Version 5.0). The D’Agostino
and Pearson normality test was performed to assess the Gaussian
distribution of the data. Bartlett’s test was performed to assess the
homogeneity of variances. Statistical analysis was analyzed using
one-way ANOVA followed by Bonferroni post-hoc tests or Kruskal-
Wallis test followed by Dunn’s post hoc tests. Correlations were
performed using Spearman’s rank correlation. Value of p < 0.05 were
considered significant.
RESULTS
Effects of Probiotics Lactobacillus and Bifidobacterium
on Colonic Lipopolysaccharides Concentrations
Colonic LPS concentrations in the human colonic microbiota
model were determined before and after 7 and 14 days
of daily administration of probiotics Lactobacillus and
Bifidobacterium (Table 2). L. reuteri, B. bifidum, L.
rhamnosus, B. longum, and B. longum subsp. infantis
reduced LPS concentrations in a time-dependent manner.
Reductions in LPS concentrations on day 14 averaged
23.81 ± 3.18% with L. reuteri (1.80 × 104 ± 2.92 × 103 EU/ml
on day 0 vs. 1.37 × 104 ± 2.55 × 103 EU/ml on day 14),
46.45 ± 5.65% with B. bifidum (1.80 × 104 ± 1.42 × 103 EU/ml
on day 0 vs. 9.63 × 103 ± 1.06 × 103 EU/ml on day 14),
30.40 ± 5.08% with L. rhamnosus (1.92 × 104 ± 1.59 ×
103 EU/ml on day 0 vs. 1.33 × 104 ± 3.50 × 102 EU/ml on
day 14), 42.50 ± 1.28% with B. longum (1.86 × 104 ± 1.87
× 103 EU/ml on day 0 vs. 1.07 × 104 ± 1.29 × 103 EU/ml on
day 14), and 68.85 ± 5.32% with B. longum subsp. infantis
(2.09 × 104 ± 2.03 × 103 EU/ml on day 0 vs. 6.50 × 103 ±
5.30 × 102 EU/ml on day 14). The effect was significant
with B. bifidum, L. rhamnosus, B. longum, and B. longum
subsp. infantis (p < 0.05). Conversely, L. plantarum
administration significantly increased LPS concentrations
by 28.84 ± 4.44% on day 7 (1.79 × 104 ± 8.46 × 102 EU/ml
on day 0 vs. 2.31 × 104 ± 3.18 × 103 EU/ml on day 7)
(p < 0.05). The effect was not maintained after 14 days of
daily L. plantarum administration.
521 Rodes et al.
Effects of Probiotics Lactobacillus and Bifidobacterium
on Colonic Gram-Negative and -Positive Bacterial Cell
Counts
Cell counts of Gram-negative bacteria (LPS-producing
bacteria) (Fig. 2A) and Gram-positive bacteria (non-LPS
producing bacteria) (Fig. 2B) in the human colonic
microbiota model were determined before and after 7 and
14 days of daily administration of probiotics Lactobacillus
and Bifidobacterium. There was a significant increase in
the Gram-negative bacterial cell count on day 7 with L.
reuteri (+21.78 ± 1.56%; 6.12 ± 0.07 log CFU/ml on day 0
vs. 7.46 ± 0.12 log CFU/ml on day 7; p < 0.05) and L.
plantarum (+7.71 ± 1.06%; 6.18 ± 0.05 log CFU/ml on day
0 vs. 6.66 ± 0.09 log CFU/ml on day 7; p < 0.05). The effect
was not significant on day 14. Conversely, there were
significant reductions on day 14 in Gram-negative bacteria
with B. longum (-7.28 ± 1.88%; 6.11 ± 0.06 log CFU/ml
on day 0 vs. 5.67 ± 0.06 log CFU/ml on day 14; p < 0.05)
and B. longum subsp. infantis (-9.55 ± 1.51%; 6.14 ±
0.06 log CFU/ml on day 0 vs. 5.55 ± 0.05 log CFU/ml on
day 14; p < 0.05). L. reuteri administration was associated
with a significant increase in total Gram-positive bacteria
on day 7 (+16.85 ± 3.64%; 6.47 ± 0.21 log CFU/ml on day
0 vs. 7.56 ± 0.01 log CFU/ml on day 7; p < 0.05). B.
animalis administration induced a significant reduction in
Gram-positive bacteria on day 14 (-18.37 ± 0.01%; 6.56 ±
0.11 log CFU/ml on day 0 vs. 5.36 ± 0.07 log CFU/ml on
day 14; p < 0.05). Colonic LPS concentrations were not
correlated with Gram-negative and -positive bacterial cell
counts.
Effects of Probiotics Lactobacillus and Bifidobacterium
on TNF-α, IL-1β, and IL-4 Cytokine Concentrations
To investigate the immunmodulatory effects of probiotics
Lactobacillus and Bifidobacterium, RAW 264.7 macrophage
cells were stimulated with bacterial supernatant (containing
most of the free LPS) sampled from the colonic model before
and after 7 and 14 days of daily probiotics administration.
The TNF-α (Fig. 3A), IL-1β (Fig. 3B), and IL-4 (Fig. 3C)
concentrations secreted by positive control (2 µg/ml LPS)
were significantly increased compared with the negative
control (DMEM supplemented with 10% FBS) (p < 0.05).
Fig. 2. Effects of probiotics Lactobacillus and Bifidobacteriumon colonic (A) Gram-negative and (B) Gram-positive bacterialcell counts in an in vitro human colonic model. Lactobacillus and Bifidobacterium probiotic strains were administered
daily to a human colonic microbiota model. A control fermentation system
was performed without administration of Lactobacillus and Bifidobacterium
strains. On days 0, 7, and 14, colonic colonic Gram-negative and Gram-
positive bacterial cell counts were quantified (log CFU/ml). Data represent
the mean ± SD (n = 3). *
Indicates statistically significant difference (p < 0.05)
obtained by a Kruskal-Wallis test followed by Dunn’s post hoc tests.
Table 2. Effects of probiotics Lactobacillus and Bifidobacterium on colonic lipopolysaccharides concentrations in a human colonicmicrobiota model.
Bacteria administered to the colonic model
Lipopolysaccharides concentration (EU/ml)
Day 0 Day 7 Day 14
Control 2.02 × 104 ± 2.53 × 103 1.84 × 104 ± 2.38 × 103 1.87 × 104 ± 3.22 × 103
L. reuteri 1.80 × 104 ± 2.92 × 103 1.79 × 104 ± 1.34 × 103 1.37 × 104 ± 2.55 × 103
B. bifidum 1.80 × 104 ± 1.42 × 103 1.17 × 104 ± 4.15 × 102 9.63 × 103 ± 1.06 × 103 *
L. rhamnosus 1.92 × 104 ± 1.59 × 103 1.55 × 104 ± 2.45 × 103 1.33 × 104 ± 3.50 × 102 *
B. animalis 1.95 × 104 ± 7.82 × 102 1.76 × 104 ± 2.10 × 103 1.81 × 104 ± 2.63 × 103
L. plantarum 1.79 × 104 ± 9.46 × 102 2.31 × 104 ± 3.18 × 103 * 1.77 × 104 ± 1.21 × 103
B. longum 1.86 × 104 ± 1.87 × 103 1.49 × 104 ± 1.23 × 103 1.07 × 104 ± 1.29 × 103 *
B. longum subsp. infantis 2.09 × 104 ± 2.03 × 103 6.87 × 103 ± 9.89 × 102 6.50 × 103 ± 5.30 × 102 *
Lactobacillus and Bifidobacterium probiotic strains were administered daily to a human colonic microbiota model. A control fermentation system was
performed without probiotic administration. On days 0, 7, and 14, colonic LPS were quantified (EU/ml). Data represent the mean ± SD (n = 3). * Indicates
statistically significant difference (p < 0.05) obtained by a Kruskal-Wallis test followed by Dunn’s post hoc tests.
PROBIOTICS, LIPOPOLYSACCHARIDES, AND INFLAMMATION 522
There were major reductions in TNF-α concentration with
B. animalis (-67.11 ± 0.56%; 1174.33 ± 8.87 µg/ml on day
0 vs. 386.20 ± 6.98 µg/ml on day 14; p < 0.05), L. plantarum
(-68.01 ± 1.88%; 1133.09 ± 62.29 µg/ml on day 0 vs.
362.49 ± 2.51 µg/ml on day 14; p < 0.05), B. longum
(-65.14 ± 1.45%; 1083.17 ± 30.48 µg/ml on day 0 vs.
377.62 ± 5.69 µg/ml on day 14; p < 0.05), and B. longum
subsp. infantis (-69.41 ± 2.78%; 1142.80 ± 88.43 µg/ml
on day 0 vs. 349.55 ± 5.32 µg/ml on day 14; p < 0.05). As
for IL-1β concentrations, reductions were most extreme on
day 14 with L. reuteri (-25.40 ± 2.55%; 637.13 ± 11.56 µg/ml
on day 0 vs. 475.33 ± 17.58 µg/ml on day 14; p < 0.05), B.
bifidum (-26.96 ± 2.52%; 650.17 ± 21.75 µg/ml on day 0
vs. 474.87 ± 10.29 µg/ml on day 14; p < 0.05), and L.
rhamnosus (-21.48 ± 2.39%; 647.10 ± 5.52 µg/ml on day
0 vs. 508.11 ± 18.50 µg/ml on day 14; p < 0.05). There
was a time-dependent increase in IL-4 concentration with
the administration of L. reuteri (+30.73 ± 7.07%; 214.57 ±
1.65 µg/ml on day 0 vs. 284.55 ± 35.55 µg/ml on day 14;
p < 0.05), L. plantarum (+46.12 ± 3.88%; 207.58 ± 8.90 µg/ml
on day 0 vs. 303.32 ± 4.95 µg/ml on day 14; p < 0.05)
and B. longum subsp. infantis (+16.50 ± 0.59%; 233.05 ±
2.11 µg/ml on day 0 vs. 271.50 ± 4.38 µg/ml on day 14;
p < 0.05).
Correlations Between Colonic Lipopolysaccharides
Concentrations and Secretion of Inflammatory Cytokines
There was a strong positive correlation between colonic
LPS concentrations and TNF-α (r = 0.447, P = 0.002; Fig. 4A)
and IL-1β (r = 0.504, P = 0.002; Fig. 4B) concentrations.
Conversely, colonic LPS concentrations were negatively
Fig. 3. Effects of probiotics Lactobacillus and Bifidobacteriumon the concentration of (A) TNF-α, (B) IL-1β, and (C) IL-4inflammatory cytokines secreted in RAW 264.7 macrophage cells.Lactobacillus and Bifidobacterium probiotic strains were administered
daily to a human colonic microbiota model. A control fermentation system
was performed without administration of probiotic bacteria. RAW 264.7
macrophage cells were stimulated with bacterial supernatant extracted
from the colonic model. Positive and negative controls consisted of RAW
264.7 cells exposed to, respectively, 2 µg/ml LPS and DMEM supplemented
with 10% FBS. Concentrations of TNF-α, IL-1β, and IL-4 cytokines were
measured (pg/ml). All values are reported as the mean ± SD (n = 3).
Statistical analysis was analyzed using one-way ANOVA followed by
Bonferroni post hoc tests to determine the effect of treatments vs. control
fermentation # (p < 0.05), and time vs. day 0 * (p < 0.05).
Fig. 4. Correlations between colonic lipopolysaccharide concentrationsand (A) TNF-α, (B) IL-1β, and (C) IL-4 inflammatory cytokinesconcentrations. Lactobacillus and Bifidobacterium probiotic strains were administered
daily to a human colonic microbiota model. A control fermentation
system was performed without probiotic administration. On days 0, 7, and
14, colonic LPS were quantified (EU/ml) and RAW 264.7 macrophage
cells were stimulated with bacterial supernatant extracted from the colonic
model. The concentrations of TNF-α, IL-1β, and IL-4 cytokines were
measured (pg/ml). Correlations between colonic LPS concentrations and
inflammatory cytokine concentrations were performed using Spearman’s
rank correlation.
523 Rodes et al.
correlated with IL-4 concentrations, but the effect was not
significant (r = -0.160, P = 0.170; Fig. 4C).
DISCUSSION
Compelling evidence demonstrates that gut microbiota dysbiosis
may promote systemic chronic low-grade inflammation [5,
33], and initiate metabolic [5, 6, 8] and liver [1, 34] diseases
through a mechanism that involves increased exposure to
Gram-negative bacteria-derived LPS. Current therapies to
modulate the human gut microbiota to reduce colonic LPS
concentrations have been developed with the aim to
improve the inflammatory status [6-8, 17, 18, 50]. However,
to our knowledge, there have been no previous studies
examining the effect of probiotics Lactobacillus and
Bifidobacterium on gut-derived LPS and inflammatory
cytokines. This was the objective of this study.
The effects of L. reuteri NCIMB 701359, L. rhamnosus
ATCC 53103, L. plantarum ATCC 14917, B. animalis
ATCC 25527, B. bifidum ATCC 29521, B. longum ATCC
15707, and B. longum subsp. infantis ATCC 15697 on
colonic LPS, and TNF-α, IL-1β, and IL-4 inflammatory
cytokine concentrations were evaluated using a human
colonic microbiota model [38] and RAW 264.7 macrophage
cells. Although the model of human colonic microbiota
used in this study neglects differences in individual gut
microbiotas and diseases, it allows the experimentation of
a complex culture of the gut microbiota under well-
standardized conditions. Results demonstrated that the
administration of B. bifidum, L. rhamnosus, B. longum,
and B. longum subsp. infantis to the human proximal
colonic microbiota model significantly reduced LPS
concentrations in a time-dependent manner. No data on
intestinal or cecal LPS have been reported in probiotic
studies, which impedes further comparison. Nevertheless,
preclinical studies have already shown that Bifidobacterium
and Lactobacillus bacteria can decrease plasma LPS
concentrations [40, 50]. Other studies using antibiotics
have reported significant decreases in cecal and plasma
LPS concentrations [6, 17, 18]. Strategies based on the use
of prebiotics have shown reduced endotoxemia, improved
glucose tolerance, and normalized low-grade inflammation
[7, 8]. Since colonic bacterial LPS are derived from Gram-
negative bacteria, colonic Gram-negative and -positive
bacterial cell counts were monitored. Reductions in Gram-
negative bacterial cell counts were significant following
administration of B. animalis, B. longum, and B. longum
subsp. infantis. Surprisingly, neither the Gram-negative
nor -positive bacterial cell count was correlated with LPS
concentrations. We hypothesize that uncultivable bacteria,
which are not quantified using plate counting, might
account for the discrepancy. Molecular techniques, such as
real-time polymerase chain reaction should be used to
quantify Gram-negative/positive bacterial cell counts in
future studies [3].
Immunomodulatory properties of probiotics Lactobacillus
and Bifidobacterium were assessed using RAW 264.7
macrophage cells. Macrophages are known to play an
important role in the regulation of innate inflammatory
responses to intestinal bacteria, particularly to the bacterial
toxin LPS [16]. Macrophage recognition of bacterial
conserved elements is mediated predominantly by toll-like
receptors and nucleotide-binding oligomerization domain
molecules [28, 43]. This further triggers the production of
inflammatory cytokines and chemokines [5, 33, 43]. In
addition, macrophages, along with dendritic cells, present
foreign antigens to cells of the acquired immune system
[12, 43, 45]. Thus, both the innate and acquired immune
systems are integrated for efficient inflammatory responses
to microorganisms. It is believed that the activation of
innate immune signaling pathways by toll-like receptors
may help induce and/or maintain chronic inflammation
due to continuous exposure to LPS [23]. Previous in vitro
studies have investigated the inflammatory responses of
RAW 264.7 macrophage cells to pure bacterial cultures
[10, 11]. Those studies were useful in determining the
direct effects of a bacterial strain on macrophage activation.
However, they disregarded the effects of a mixed culture
induced in the gut microbiota ecosystem. Soluble components,
predominantly LPS, released from bacterial cells into the
gut environment are responsible for the majority of
inflammatory cytokines secreted [4, 11, 14, 26]. Lamina
propria-resident macrophages have been shown to suppress
or have anergic responses to LPS stimuli [42, 44]. However,
studies involving the inflamed mucosa have not observed
this feature, probably due to the recruitment of blood
monocytes-derived macrophages [20, 21]. In addition, it
was recently suggested that the inflammatory profile
elicited by murine RAW 264.7 macrophage cells depends
primarily on the initial dosage of LPS challenge [31]. In
the present study, murine macrophage cells were stimulated
with bacterial supernatant, which contained the free LPS,
derived from our human colonic microbiota model. This
novel approach, which allows to investigate probiotic
immunomodulatory activities in the existing gut microbiota,
has already demonstrated accuracy and reproducibility [2].
Cells density and LPS concentration (positive control)
were based on published publications [10, 11]. TNF-α, IL-
1β, and IL-4 inflammatory cytokines were selected for
quantification because they are promoted following the
immune recognition of gut-derived LPS [5, 33]. In addition,
they have been linked with inflammatory and metabolic
diseases [9]. Results demonstrated that the exposure of
macrophage cells to bacterial supernatant and LPS solution
stimulated major secretions of TNF-α, IL-1β, and IL-4
cytokines compared with the negative control. In addition,
probiotics Lactobacillus and Bifidobacterium induced
PROBIOTICS, LIPOPOLYSACCHARIDES, AND INFLAMMATION 524
significant biological reductions in the TNF-α concentration.
The reductions were extreme with B. animalis, L. plantarum,
B. longum, and B. longum subsp. infantis. Reductions in
the IL-1β concentration were extreme with L. reuteri, L.
rhamnosus, and B. bifidum. The IL-4 concentration was
significantly increased with L. reuteri, L. plantarum, and
B. longum subsp. infantis. Overall, B. longum subsp.
infantis demonstrated a higher capacity to reduce colonic
LPS concentrations, to decrease TNF-α proinflammatory
cytokines, and to increase IL-4 anti-inflammatory cytokines
secretions. Interestingly, the colonic LPS concentrations
were positively and significantly correlated with the TNF-
α and IL-1β concentrations. We suggest that specific
probiotic strains of Lactobacillus and Bifidobacterium can
decrease colonic LPS concentrations, which might further
reduce the proinflammatory tone.
This is the first in vitro study to investigate the effects of
probiotics Lactobacillus and Bifidobacterium on colonic
LPS and inflammatory cytokine concentrations using a
human colonic microbiota model. The findings revealed that
specific probiotic strains, such as B. longum subsp. infantis,
can decrease colonic LPS concentrations, which might
further reduce the secretion of inflammatory cytokines in
macrophage cells. This study has noteworthy applications
in the field of biotherapeutics for the prevention and/or
treatment of inflammatory and metabolic diseases.
Acknowledgments
This work was supported by a research grant from the
Canadian Institutes of Health Research (CIHR) MOP No.
64308 to S. Prakash. L. Rodes and W. Shao acknowledge
the Doctoral Training Award from the Fonds de Recherche
Santé Québec (FRSQ). A. Paul acknowledges the Alexander
Graham Bell Canada Graduate Scholarship from the Natural
Sciences and Engineering Research Council (NSERC) and
the FRSQ Postdoctoral Training Award. M. Coussa-Charley
acknowledges the FRSQ Master’s award. C. Tomaro-
Duchesneau acknowledges the Alexander Graham Bell
Canada Graduate Scholarship from NSERC.
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