Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37: effects on the production of inflammatory mediators by mouse macrophages.

Abstract

The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopolysaccharides are a major component of the bacterial biofilm with a well-documented impact on adherence of bacteria to host cells. However, their immunoregulatory properties are unknown. The aim of this study was to examine the immunostimulatory potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS on the production of inflammatory mediators by mouse peritoneal macrophages and compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at concentrations higher than those of LPS, stimulated production of both pro-inflammatory (TNF-α, IL-6, IL-12) and anti-inflammatory (IL-10) cytokines. Interestingly, analysis of the balance of TNF-α/IL-10 production showed a potential pro-inflammatory effect of EPS. Furthermore, our data demonstrate that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by reduced production of TNF-α after restimulation with either LPS or EPS ('cross-tolerance'). By contrast, EPS could make cells tolerant only to subsequent stimulation by the same stimulus. We also examined the relationship between TNF-α production and activation of mitogen-activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF-α production induced by both stimuli to the same extent. In conclusion, these data demonstrate that EPS can effectively stimulate production of inflammatory mediators by macrophages in vitro. However, to predict whether EPS could be clinically useful as an immunomodulatory agent, further in vivo studies with highly purified EPS are necessary.

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ORIGINAL ARTICLE
Immunoregulatory potential of exopolysaccharide from
Lactobacillus rhamnosus KL37. Effects on the production
of inflammatory mediators by mouse macrophages
Marta Ciszek-Lenda?, Bernadeta Nowak?, Małgorzata S´ro ´ttek?, Andrzej Gamian?and
Janusz Marcinkiewicz*,?
*Chair of Immunology, Jagiellonian University Medical College, Cracow, Poland and ?Institute of Immunology and Experimental
Therapy, Polish Academy of Science, Wroclaw, Poland
Probiotics are live bacteria exhibiting health-promoting
activities. Recently, an increasing number of clinical studies
report that probiotics may be useful in the prevention⁄treat-
ment of atopic diseases and gastrointestinal infections in
children (Vaarala 2003; Shida & Nanno 2008). The com-
monly used probiotics in human medicine are various species
of lactic acid bacteria including different strains of Lactoba-
cillus rhamnosus (L. rhamnosus) (Marcone et al. 2008;
Prescott et al. 2008; Szyman ´ski et al. 2008).
It has been postulated that the observed clinical effects of
probiotic bacteria depend on their immunoregulatory prop-
erties (e.g. anti-inflammatory properties) and are strain
specific (Christensen et al. 2002; Matsumoto et al. 2005).
However, probiotic lactic acid bacteria have been shown to
INTERNATIONAL
JOURNAL OF
EXPERIMENTAL
PATHOLOGY
doi: 10.1111/j.1365-2613.2011.00788.x
Received for publication: 8 February
2011
Accepted for publication: 20 July
2011
Correspondence:
Prof. Janusz Marcinkiewicz
Chair of Immunology
Jagiellonian University Medical
College
18 Czysta Street
31-121 Cracow
Poland
Tel/Fax:. +48126339431
E-mail: mmmarcin@cyf-kr.edu.pl
Summary
The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli
including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopoly-
saccharides are a major component of the bacterial biofilm with a well-documented
impact on adherence of bacteria to host cells. However, their immunoregulatory pro-
perties are unknown. The aim of this study was to examine the immunostimulatory
potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS
on the production of inflammatory mediators by mouse peritoneal macrophages and
compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at con-
centrations higher than those of LPS, stimulated production of both pro-inflamma-
tory (TNF-a, IL-6, IL-12) and anti-inflammatory (IL-10) cytokines. Interestingly,
analysis of the balance of TNF-a⁄IL-10 production showed a potential pro-inflam-
matory effect of EPS. Furthermore, our data demonstrate that exposure of macro-
phages to LPS induced a state of hyporesponsiveness, as indicated by reduced
production of TNF-a after restimulation with either LPS or EPS (‘cross-tolerance’).
By contrast, EPS could make cells tolerant only to subsequent stimulation by the same
stimulus. We also examined the relationship between TNF-a production and activa-
tion of mitogen-activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of
macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF-a
production induced by both stimuli to the same extent. In conclusion, these data dem-
onstrate that EPS can effectively stimulate production of inflammatory mediators by
macrophages in vitro. However, to predict whether EPS could be clinically useful as
an immunomodulatory agent, further in vivo studies with highly purified EPS are
necessary.
Keywords
cytokines, exopolysaccharides, Lactobacillus rhamnosus, macrophages, probiotics
Int. J. Exp. Path. (2011), 92, 382–391
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stimulate both pro-inflammatory (Th1) and anti-inflamma-
tory responses (Th2, Treg) (Matsuguchi et al. 2003; Mo-
hamadzadeh et al. 2005). Therefore, the selection of the
correct probiotic bacterial strain seems to be crucial to
achieve the desired therapeutic effect.
Interestingly, beneficial (anti-inflammatory) effects have
been achieved not only using live bacteria, but also using
dead bacteria, bacterial cell-wall components (peptydogly-
can, lipoteichoic acids) and bacterial DNA (Dalpke et al.
2002; Rachmilewitz et al. 2002; Matsuguchi et al. 2003).
Zhang et al. (2005) reported that both live and heat-killed
L. rhamnosus GG may ameliorate inflammation by decreas-
ing TNF-a-induced IL-8 production by epithelial Caco-2
cells. Moreover, lipoteichoic acid (LTA), a major component
of the cell wall of lactobacilli, activates macrophages and
dendritic cells through TLR2, in a strain-specific manner
(Matsuguchi et al. 2003). The great structural diversity in
the LTAs derived from different bacteria may give rise to a
variety of immunoregulatory properties.
Much less is known about the immunoregulatory potential
of lactobacilli-derived exopolysaccharides (EPS). Exopolysac-
charides are a key component of the biofilm matrix of many
biofilm-forming bacteria, including Lactobacillus species
(Watnick & Kolter 2000; Laws et al. 2001; Ciszek-Lenda
2011), although biofilms contain a whole variety of proteins,
glycoproteins, glycolipids and extracellular DNA (Flemming
et al. 2007).
Nevertheless, it is well established that EPS plays an
important role in bacteria immune evasion, resistance
towards antibacterial agents and bacterial adhesion proper-
ties (Vuong et al. 2004; Ruas-Madiedo et al. 2006).
More recently, it has been reported that EPS-producing
probiotics significantly attenuate experimental colitis, in a
dose-dependent manner (Sengu ¨l et al. 2006). However, the
target(s) and the presence of EPS-specific receptor(s) are still
not established, and it has proved difficult to define the com-
mon biological properties of lactic acid bacteria EPS because
of its extraordinary structural diversity.
In a previous study, we have shown that exopolysaccha-
ride-producingLactobacillus
L. johnsonii 142, L. animalis 148) induce cytokine produc-
tion by macrophages in a strain-specific manner (Mar-
cinkiewicz et al. 2007). In preliminary studies, crude EPS
isolated from L. rhamnosus KL37 also ameliorated the
development of collagen-induced arthritis in mice. Bacterial
exocellular polymeric substances may therefore exert differ-
ent immunoregulatory effects.
The main aim of this study was to further evaluate the
immunoregulatory potential of EPS derived from the high-
exopolysaccharide producer, L. rhamnosus KL37. We have
examined the stimulatory effects of EPS on the release of
inflammatory mediators by thioglycollate-induced mouse
peritoneal macrophages in vitro, a well-studied model of the
in vivo inflammatory response (Shnyra et al. 1998; Kaji
et al. 2010). The immunoregulatory potential of EPS was
compared with that of Lipopolysaccharide (LPS), to estab-
strains(L.reuteri 115,
lish whether EPS may become good candidates for clinical
use as the active components of probiotic bacteria.
Materials and methods
Mice
Inbred CBA⁄J mice (8–12 weeks of age, 18–22 g) were main-
tained in the Animal Breeding Unit, Department of Immunol-
ogy, Jagiellonian University Medical College, Cracow. All
mice were housed in the laboratory room with water and
standard diet ad libitum. The authors were granted permis-
sion by the Local Ethics Committee to use mice in this study.
EPS isolation
Exopolysaccharide was obtained from L. rhamnosus KL37
strain. The strain was isolated from the faeces of the human
newborns and then stored at )70 ?C in MRS broth supple-
mented with 10% glycerol. Bacteria were cultivated in sup-
plemented MRS liquid broth (Oxoid, Cambridge, UK) under
anaerobic conditions at 37 ?C for 48 h. Cells were harvested
by centrifugation at 7300 g (4 ?C, 30 min) and washed twice
with phosphate buffer solution (PBS). Bacterial mass was sus-
pended in water (10 ml) and sonicated three times for 5 min,
in an ice bath. After centrifugation at 4000 g (30 min, 4 ?C),
the supernatant was centrifuged twice at 16,300 g at 4 ?C
for 1 h and then precipitated with five volumes of cold etha-
nol ()20 ?C, overnight). The precipitated material was recov-
ered by centrifugation at 16,300 g, 4 ?C for 20 min and
freeze-dried. Purification of EPS was performed by gel filtra-
tion on a column in TSK HW-50 (1.6 · 100 cm) in 0.05 M
aqueous pyridine acetate buffer (pH 5.6). The eluate was
monitored with a Knauer differential refractometer. The first
fraction, which eluted in the void volume, contained EPS and
was the subject of the present investigation.
Cells
Peritoneal mouse macrophages (M/) were induced by intra-
peritoneal injection of 2.0 ml of thioglycollate or paraffin oil
(both Sigma, St. Louis, MO, USA). Cells were collected 72 h
later by washing out the peritoneal cavity with 5 ml of
DPBS (Dubelco’s phosphate buffer solution) containing
5 U heparin⁄ml (Polfa, Warsaw, Poland). Cells were centri-
fuged, and red blood cells were lysed by osmotic shock using
lysing buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM
EDTA). Osmolarity was restored by addition of 2 · concen-
trated PBS. For each experiment, at least three mice were
used as donors of peritoneal macrophages.
Cell culture and treatment
Macrophages (M/) were cultured in 24-well flat-bottom cell
culture plates at 5 · 105per well in RPMI 1640 medium
(JR Scientific Inc., Woodland, CA, USA) supplemented with
5% FBS, and gentamicin 50 mg⁄ml (Krka, Novo Mesto,
L. rhamnosus exopolysaccharide and inflammation
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Slovenia), at 37 ?C in an atmosphere of 5% CO2. After 1 h,
non-adherent cells were removed and the adherent cells (M/)
were stimulated with indicated concentrations of EPS (1–
100 lg⁄ml), LPS (0.0001–0.1 lg⁄ml) (Sigma-Aldrich, Stein-
ham, Germany) or 107cfu⁄ml of whole heat-killed bacteria,
L. rhamnosus KL37 or Escherichia coli 0111:B4. After
24 h, culture supernatants were collected and frozen at
)80 ?C until used. All groups were investigated in dupli-
cates, if not stated otherwise.
Flow cytometric analysis of thioglycollate-induced
peritoneal exudate cells
Macrophages (M/) induced by intraperitoneal injection of
thioglycollate were harvested as described earlier, washed
with PBS containing 2% FBS and 0.02% sodium azide and
stained with allophyocyanin-conjugated anti-mouse F4⁄80
monoclonal antibody (eBioscience, Frankfurt, Germany) in
combination with: PE-conjugated anti-mouse CD11b (BD
Pharmingen, San Diego, CA, USA) and biotin-conjugated
anti-mouse Gr1 (BioLegend, San Diego, CA, USA). Non-spe-
cific binding of antibodies was blocked by 2.4G2 monoclo-
nal antibody (BD Pharmingen). Isotype-matched controls (all
from BD Pharmingen) were included. Cells were incubated
with antibodies 40 min, at 4 ?C in the dark, washed twice
and suspended in PBS containing 2% FBS and 0.05%
sodium azide. For the detection of biotinylated antibodies
incubation with FITC-conjugated streptavidin (BD Pharmin-
gen) followed for 30 min, at 4 ?C in the dark. To exclude
dead cells, propidium iodide (PI; Sigma-Aldrich) was added
just before analysis. Cells were analysed on Becton Dickin-
son FACSCalibur with CellQuest Pro Software (BD Bio-
sciences, San Jose, CA, USA).
Cell culture and priming effect.
induced priming effect, macrophages were incubated in med-
ium with increasing concentrations of EPS (0.1–30 lg⁄ml)
or LPS (0.00001–0.01 lg⁄ml) for 6 h. Cells then were
washed with PBS, and new medium was added before the
second stimulation with either EPS (30 lg⁄ml) or LPS
(0.1 lg⁄ml). After further 18 h, culture supernatants were
collected and frozen at )80 ?C until used. The cytokine pro-
duction was determined by ELISA.
To study polysaccharide-
Cell culture and inhibitors of MAP kinases.
cell signalling induced by EPS, M/ were precultured with
SB 203580, the inhibitor of MAP kinase p38 and PD 98059,
the inhibitor of Erk-MEK1⁄2 kinase (both Calbiochem, NY,
USA), at concentrations 10 and 20 lM, respectively, 30 min
before stimulationwith
(100 lg⁄ml). After 20 h, culture supernatants were collected
and frozen at )80 ?C until used.
To investigate
LPS(0.1 lg⁄ml) orEPS
Cytokines determination
Cytokine concentrations in culture supernatants were mea-
sured using sandwich ELISA as described previously (Mar-
cinkiewicz et al. 2007). For IL-6, IL-10 and IL-12 microtiter
plates (Corning, NY, USA) were coated overnight with rat
antibodies against a mouse cytokine (capture antibody). For
IL-6, rat anti-mouse IL-6 and biotinylated rat anti-mouse IL-
6 (both BD Pharmingen) mAbs were used as capture and
detecting antibodies. Recombinant mouse IL-6 (PeproTech,
Rocky Hill, New York, USA) was used as a standard. For IL-
10, rat anti-mouse IL-10 and biotinylated rat anti-mouse IL-
10 mAbs were used as capture and detecting antibodies.
Recombinant mouse IL-10 was used as a standard (all reagents
from BD Pharmingen). For IL-12p40, rat anti-mouse IL-12
(p40rp70) (BD Pharmingen) mAb and biotinylated rat anti-
mouse IL-12(p40) (Endogen, Woburn, MA, USA) mAb were
used as capture and detecting antibodies. Recombinant mouse
IL-12 (Genzyme, Cambridge, UK) was used as a standard. For
TNF-a, hamster anti-mouse⁄rat TNF-a and biotinylated rab-
bit anti-mouse⁄rat TNF-a (both BD Pharmingen) mAbs were
used as capture and detecting antibodies. Recombinant mouse
TNF-a (Sigma-Aldrich) was used as a standard. After blocking
the plates with 3% skimmed milk (4% albumin for IL-10) for
2 h, standards and tested supernatants were added and incu-
bated overnight. Finally, biotinylated antibodies against the
same cytokine were added for 1 h. The ELISA was developed
using horseradish peroxidase conjugated with streptavidin
(Vector, Burlingame, CA, USA) followed with o-phenylenedi-
amine and H2O2 (both Sigma-Aldrich). The reaction was
stopped with 3 M H2SO4. The optical density of each sample
was measured at 492 nm in a microplate reader; 0.05%
Tween-20 in phosphate buffer was used as a washing solution.
Nitrite (NO?
2) determination
Nitric oxide, quantified by the accumulation of nitrite as a
stable end product, was determined by a microplate assay
(Ding et al. 1988). Briefly, 100 ll of sample supernatants
were incubated with an equal volume of Griess reagent [1%
sulphanilamide in 2 M HCl (Sigma-Aldrich) and 0.1% N-1-
naphthylenediamine dihydrochloride
(POCH, Gliwice, Poland)] at room temperature for 10 min.
The absorbance at 550 nm was measured with a microplate
reader. Nitrite concentration was calculated from a sodium
nitrite standard curve.
indeionizedwater
PGE2determination
PGE2concentration in supernatants was determined by Pros-
taglandin E2Monoclonal EIA kit (Cayman Chemical, Ann
Arbor, MI, USA) according to the manufacturer’s instruction.
Statistical analysis
Statistical significance of differences between groups was
analysed using one-way anova, followed, if significant, by
an LSD test for post hoc comparison. Results are expressed
as mean ± SEM values. A P-value <0.05 was considered sta-
tistically significant. Analysis was performed using Graphpad
Prism v. 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).
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Results
The stimulatory effect of EPS isolated from
L. rhamnosus KL37 and whole bacterial cells on cytokine
production by peritoneal macrophages
Previously, we have shown that various strains of lactobacil-
li effectively stimulate the production of inflammatory medi-
atorsfromoil-inducedmouse
(Marcinkiewicz et al. 2007). All these bacteria are strong
producers of EPS. In this study, to examine immunostimula-
tory potential of EPS, mouse peritoneal macrophages were
cultured either with EPS derived from L. rhamnosus KL37
or with the whole killed bacteria cells and cytokine produc-
tion was analysed. The effect was compared with the effect
of killed E. coli bacteria and LPS. As shown in Table 1,
both pro-inflammatory (TNF-a, IL-6, IL-12) and anti-inflam-
matory (IL-10) cytokines were released from oil-induced
macrophages in response to dead L. rhamnosus KL37 bacte-
ria. In contrast, EPS derived from these bacteria was less
effective than whole bacteria or LPS. In addition, the bal-
ance of macrophage TNF-a⁄IL-10 and IL-12⁄IL-10 produc-
tion induced by EPS differs from that induced by whole
bacteria (see Table 1). Interestingly, EPS induced more TNF-
a and IL-12 than IL-10, suggesting its pro-inflammatory
(Th1-type) immunoregulatory potential.
In subsequent experiments, we used thioglycollate-induced
mouse peritoneal macrophages as more appropriate for our
experimental model. As examined by flow cytometry, more
than 75% of these cells express were F4⁄80, which is a mar-
peritonealmacrophages
ker of mature macrophages, 75% were CD11b+ and 18%
were Gr1+. Moreover, analysis of cytokine production in
response to EPS showed that thioglycollate-induced perito-
neal macrophages were significantly more effective than oil-
induced macrophages (see Table 2).
The dose-dependent effect of EPS on cytokine release
from peritoneal macrophages
In vitro, thioglycollate-induced peritoneal macrophages spon-
taneously produce negligible amounts of TNF-a, IL-6, IL-
12p40 and undetectable amounts of IL-10. Upon in vitro
stimulation of these macrophages with EPS, a substantial
release of both pro- and anti-inflammatory cytokines was
observed (Figure 1). EPS stimulated the release of cytokines
in a dose-dependent manner. At concentrations above
3 lg⁄ml, EPS induced a massive release of cytokines (>10-
fold increase). At lower concentrations (0.01–1 lg⁄ml), EPS
had no effect on cytokine production (data not shown). In
response to EPS, macrophages produced much more pro-
inflammatory cytokines (TNF-a, IL-6) than anti-inflamma-
tory cytokines (IL-10). The ratio of TNF-a⁄IL-10 was above
30:1, indicating a pro-inflammatory pattern of cytokines
secreted by macrophages incubated with EPS.
Kinetics of EPS-induced cytokine release from
macrophages in vitro
To examine the kinetics of cytokine production by macro-
phages, the cells were incubated with EPS for different
Table1 The stimulatory effect of EPS isolated from Lactobacillus rhamnosus KL37 and the whole bacterial cells on cytokine produc-
tion by peritoneal macrophages
TNF-a?
IL-6?
IL-10?
IL-12p40?
TNF-a vs IL-10?
IL-12p40?vs IL-10
Escherichia coli
LPS
L. rhamnosus
EPS
Untreated
1413 ± 655**
1085 ± 241**
2107 ± 562***
469 ± 119*
167 ± 22
29,181 ± 5593***
23,195 ± 1749***
15,659 ± 56***
11,685 ± 2068***
2073 ± 308
857 ± 88***
104 ± 8***
1231 ± 396***
77 ± 22*
9 ± 4
3652 ± 847***
1820 ± 328 **
1006 ± 426***
1243 ± 233**
385 ± 55
1.6:1
10:1
1.7:1
6:1
18:1
4:1
17:1
0.8:1
16:1
43:1
Cytokines were analysed by ELISA in supernatants collected from 24 h cultures of oil-induced peritoneal macrophages (5 · 105per well) stim-
ulated with exopolysaccharides (EPS) (100 lg⁄ml), Lipopolysaccharide (LPS) (1 lg⁄ml) or killed bacteria (107cfu per well). Data are mean ±
SEM values of three independent experiments.*P < 0.05, **P < 0.005, ***P < 0.001, treated vs. untreated macrophages.
?Level of cytokines is expressed in pg⁄ml.
?The balance was calculated from data shown in this table.
Table2 Cytokine production in response to EPS by peritoneal macrophages
TNF-a*
IL-6*
IL-10*
IL-12p40*
Thio-induced macrophages
Oil-induced macrophages
3587 ± 547
318 ± 74
21302 ± 1081
6135 ± 1526
101 ± 37
31 ± 8
2057 ± 217
869 ± 207
Cytokines were analysed by ELISA in supernatants collected from 24 h cultures of oil-induced or thioglycollate-induced peritoneal macrophages
(5 · 105per well) stimulated with exopolysaccharides (EPS) at concentration of 30 lg⁄ml. Data are mean ± SEM values of three independent
experiments.
*Level of cytokines is expressed in pg⁄ml.
L. rhamnosus exopolysaccharide and inflammation
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(a)
(b)
(c)
(d)
Figure 1 Dose-dependent effect of exopolysaccharides (EPS) on cytokine secretion from peritoneal macrophages. TNF-a (a), IL-6 (b),
IL-12p40 (c) and IL-10 (d) were analysed by ELISA in supernatants collected from 24 h cultures of peritoneal macrophages (5 · 105
per well) stimulated with indicated concentrations of EPS. Data are mean ± SEM values of three independent experiments.
*P < 0.05, **P < 0.005, ***P < 0.001, EPS-treated vs. untreated macrophages.
(a)(b)
(c) (d)
Figure 2 Kinetics of the EPS-induced secretion of cytokines. Mouse peritoneal macrophages (5 · 105per well) were stimulated with
exopolysaccharides (EPS) (30 lg⁄ml). The amounts of TNF-a (a), IL-6 (b), IL-12p40 (c) and IL-10 (d) in culture supernatants were
estimated after 6, 24 or 48 h of incubation with EPS. Data are mean ± SEM values of three independent experiments.
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periods of time. The concentration of cytokines was mea-
sured in supernatants collected after 6, 24 and 48 h. The
cytokine release from macrophages stimulated with EPS
was time dependent (Figure 2). Rapid release of TNF-a
was observed during the first 6 h of the culture. The level
of TNF-a slightly decreased after 24 h and declined signifi-
cantly after 48 h of stimulation with EPS (Figure 2a). IL-6
production was maximal at 24 h and decreased after 48 h
(Figure 2b). A similar pattern of cytokine secretion was
seen for IL-10 (Figure 2d). Interestingly, when superna-
tants were removed after 6 h and replaced with the cul-
ture medium without EPS, neither TNF-a nor IL-10 was
detectable in supernatants collected after 24 h (data not
shown). In contrast, the level of IL-12p40 induced by EPS
continued to increase until the end of the culture (Fig-
ure 2c).
(a)
(b)
(c)
(d)
Figure3 Comparison of EPS and lipopolysaccharide (LPS) capacity to induce the production of inflammatory mediators. Mouse peri-
toneal macrophages (5 · 105per well) were stimulated with indicated concentrations of either EPS (grey bars) or LPS (black bars).
After 24 h, supernatants were collected and the amounts of TNF-a (a), IL-10 (b), NO?
described in Methods. Data are mean ± SEM values of three independent experiments. *P < 0.05; **P < 0.005, EPS-treated vs.
untreated macrophages.
2(c) and PGE2(d) were determined as
(a)
(b)
Figure 4 Priming effect of LPS and EPS on TNF-a (a) and IL-10 (b) production by peritoneal macrophages re-stimulated with LPS.
The cells (5 · 105per well) were preincubated for 6 h with indicated concentrations of EPS and LPS, washed twice and re-stimulated
with LPS (0.1 lg⁄ml). After 18 h, supernatants were collected and the amounts of cytokines were estimated by ELISA. Data are
mean ± SEM values of three independent experiments. Control group (white bars) – not-primed macrophages, re-stimulated only with
LPS. *P < 0.05 control macrophages vs. macrophages primed with LPS 0.01 and 0.001 lg⁄ml.
L. rhamnosus exopolysaccharide and inflammation
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The relative potency of EPS and LPS in induction of pro-
inflammatory mediators
Lipopolysaccharide, the main component of cell walls of
Gram-negative bacteria, is known to be a strong inducer of
inflammatory mediators from immune cells (Erroi et al.
1993; Zhang et al. 2008). To examine whether EPS has
comparable immunoregulatory properties, peritoneal macro-
phages were stimulated with EPS or LPS. As shown in Fig-
ure 3, LPS is a much stronger inducer of cytokines and
inflammatory mediators than EPS. LPS, even at a concentra-
tion of 0.01 lg⁄ml, induced substantial amounts of all
mediators tested (TNF-a, IL-10, NO?
trast, EPS required 100-fold higher concentrations (at least
3 lg⁄ml). EPS induced the production of all cytokines in a
dose-dependent manner. LPS induced the production of IL-
10 and NO in a dose-dependent fashion, while production
of TNF-a was maximal at 0.01 lg⁄ml and declined at
higher concentrations. Importantly, the balance of pro-
⁄anti-inflammatory cytokines induced by EPS was different
from that induced by LPS. Namely, the ratio of TNF-a⁄IL-
10 was approximately 5:1 for the highest concentration of
LPS used. On the other hand, regardless of the EPS concen-
tration, the ratio of TNF-a⁄IL-10 was always above 30:1.
These data suggest pro-inflammatory (‘Th1 type’) properties
of crude EPS isolated from L. rhamnosus KL37.
2and PGE2). In con-
The priming effect of EPS and LPS on cytokine
production by peritoneal macrophages
It is well known that priming (pretreatment) of macrophages
with very low doses of LPS attenuates the effect of sub-
sequent LPS stimulation (Zhang & Morrison 1993; Randow
et al. 1995). To examine the ability of EPS to induce a simi-
lar effect, macrophages were preincubated with either LPS
(0.00001–0.01 lg⁄ml) or EPS (0.01–30 lg⁄ml) for 6 h and
then were re-stimulated with LPS (0.1 lg⁄ml) (Figure 4) or
EPS (30 lg⁄ml, the effective stimulatory concentration of
EPS) (Figure 5). Priming of macrophages with LPS at a con-
centration of 0.01 or 0.001 lg⁄ml resulted in significantly
diminished production of TNF-a and increased production
of IL-10 in response to LPS re-stimulation. In contrast, pre-
treatment of macrophages with EPS did not alter the cyto-
kine production (TNF-a, IL-10) induced by subsequent
stimulation with LPS (Figure 4).
We also tested the effect of LPS and EPS priming on the
secretion of cytokines after re-stimulation with EPS. As
shown in Figure 5, a statistically significant reduction in
TNF-a production was observed in macrophages pretreated
with LPS, even when used at very low concentrations. Inter-
estingly, macrophage priming with EPS also reduced TNF-a
production after subsequent stimulation with EPS (Fig-
ure 5a). In contrast, no significant changes between the
levels of IL-10 produced by macrophages pretreated with
(a) (b)
Figure 5 Priming effect of LPS and EPS on TNF-a (a) and IL-10 (b) production by peritoneal macrophages re-stimulated with EPS.
The cells (5 · 105per well) were preincubated for 6 h with indicated concentrations of EPS and LPS, washed twice and re-stimulated
with EPS (30 lg⁄ml). After 18 h, supernatants were collected and the amounts of cytokines were measured by ELISA. Data are
mean ± SEM values of three independent experiments. Control group (white bars) – not-primed macrophages, re-stimulated only with
EPS. *P < 0.05 control macrophages vs. macrophages primed with LPS 0.001 lg⁄ml, EPS 3 and10 lg⁄ml; **P < 0.005 control mac-
rophages vs. macrophages primed with LPS 0.01 lg⁄ml and EPS 30 lg⁄ml.
Figure6 Effects of inhibitors specific for MAPK pathway on
the production of TNF-a by macrophages. Peritoneal macro-
phages (5 · 105per well) were pretreated with SB 203580 (p38
inhibitor, 10 lM) or⁄and PD 98059 (Erk-MEK1⁄2 inhibitor,
20 lM) for 30 min and then stimulated with lipopolysaccharide
(LPS) (0.1 lg⁄ml) or exopolysaccharides (EPS) (100 lg⁄ml) for
24 h. The levels of TNF-a in supernatants were determined by
ELISA. Data are mean ± SEM values of three independent
experiments. Black bars – LPS treated, grey bars – EPS treated.
**P < 0.005; ***P < 0.001.
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either EPS or LPS were observed after re-stimulation with
EPS (Figure 5b).
The role of MAP kinases in the EPS-triggered cytokine
production
It is well known that MAP kinases (MAPKs), including p38
and ERK, are involved in the synthesis of cytokines in
response to LPS (Dong et al. 2002). Signalling pathways
engaged during the response to EPS remain unknown. To
investigate the functional role of p38 and ERK pathways in
EPS-induced cytokine production, macrophages were pre-
treated with selective inhibitors of the p38 (SB 203580) and
Erk-MEK1⁄2 (PD 98059) pathways, prior to stimulation
with EPS or LPS. The effect of these inhibitors on TNF-a
production is shown in Figure 6. Inhibition of either p38 or
Erk-MEK1⁄2 MAPK pathways significantly reduced TNF-a
production by macrophages stimulated with both LPS and
EPS. When the two inhibitors were used simultaneously, the
production of TNF-a was completely abolished. These data
suggest that both MAPKs, p38 and Erk-MEK1⁄2 (to less
extent), are involved in cytokine production by macrophages
stimulated with EPS.
Discussion
Lactobacillus rhamnosus is one of most commonly used bac-
teria in probiotic therapies. In clinical studies, L. rhamnosus
GG significantly reduced incidence of respiratory infections,
reduced duration of diarrhoea and ameliorated symptoms of
atopic dermatitis (Hojsak et al. 2010; Nermes et al. 2011;
Ferrie & Daley 2011). Such a wide spectrum of beneficial
clinical effects is difficult to explain without understanding
the mechanisms responsible for cross-talk between lactoba-
cilli, their secreted products and host cells. The immunostim-
ulatory properties of probiotic bacteria are of special
interest.
Lactobacilli strongly induce the production of inflamma-
tory mediators in a strain-specific manner (Christensen et al.
2002; Marcinkiewicz et al. 2007). Mouse peritoneal macro-
phages and macrophage cell lines showed a strong response
to lactobacilli species in vitro. The relative potency of differ-
ent components of L. casei on TNF-a production by
RAW264.7 macrophages was found to be protoplast > cell
wall > polysaccharide–peptidoglycan complex. Importantly,
it has been demonstrated that purified LTA, a component of
L. casei protoplast, markedly induced TNF-a production by
RAW264.7 cells (Matsuguchi et al. 2003). These data sug-
gest that LTA, at least from some Lactobacillus strains, is a
potent TLR2 ligand and a key molecule responsible for
immunostimulation by these bacteria (Lehner et al. 2001;
Kaji et al. 2010). Recently, an increasing number of reports
postulate a crucial role of biofilm components in bacteria–
host interactions (Servin & Coconnier 2003; Sengu ¨l et al.
2011). However, very little is known about immunostimula-
tory properties of EPS, the major components of lactic bac-
teria biofilm (Vu et al. 2009).
In this study, we addressed the issue whether EPS derived
from lactobacilli bacteria can stimulate production of
inflammatory mediators from mouse macrophages. For this
investigation, we have selected L. rhamnosus KL37–derived
EPS because the structure of EPS-KL37 is known (Lipin ´ski
et al. 2003). L. rhamnosus growing in basal minimum med-
ium may secrete massive amounts of EPS reaching maximum
concentrations of 500 lg⁄ml (Ruas-Madiedo & de los
Reyes-Gavila ´n 2005). In addition, our preliminary studies
showed its promising therapeutic effect on the development
of collagen-induced arthritis in mice.
In the present study, we have demonstrated that EPS, at
concentrations ranging 3–30 lg⁄ml, effectively induces the
production of macrophage cytokines, especially TNF-a, IL-6
and IL-12. This finding supported by analysis of the balance
of TNF-a to IL-10 suggests that EPS has an overall proin-
flammatory activity. However, its stimulatory potential was
significantly lower than that of intact bacterial cells and
lower than that of LPS. Importantly, effective concentrations
of EPS were similar to immunostimulatory concentrations of
LTA, the major TLR2 ligand of lactic bacteria (Vidal et al.
2002; Kim et al. 2007), suggesting EPS may also be active in
vivo (Vu et al. 2009). Whole bacteria were stronger inducers
of anti-inflammatory IL-10 than EPS, suggesting intact bac-
teria and EPS may have opposing effect on macrophage
polarization.
The overall, contribution of EPS to the production of
inflammatory mediators in vivo is difficult to predict. Exo-
polysaccharides as the exocellular component of bacteria
biofilm may not participate in the activation of immune cells
by bacterial cell-wall components (Vidal et al. 2002).
Indeed, EPS may even screen bacteria from the immune cells
in vivo.
In the present study, we also demonstrate some new
immunomodulatory properties of EPS such as EPS desensiti-
zation of macrophages. It is well documented that exposure
of macrophages to LPS induces a state of hyporesponsive-
ness (tolerance) to subsequent stimulation with LPS (Zhang
& Morrison 1993). Recently, it has been shown that similar
effect may be induced by LTA, the component of lactobacil-
li bacteria (Lehner et al. 2001). In the present study, we
addressed an issue whether EPS impact on macrophages
(‘macrophage priming’) can also induce desensitization of
macrophages. We have shown that pretreatment of macro-
phages with LPS results in decreased production of TNF-a
after re-stimulation with LPS. Interestingly, the production
of TNF-a was also reduced after re-stimulation with EPS. A
similar effect, named ‘cross-tolerance’, was seen after expo-
sure of mouse macrophages to LPS and subsequent stimula-
tionwithotherstimuli(Lehner
noteworthy that the LPS priming of macrophages resulted in
a decreased production of pro-inflammatory TNF-a and
enhanced production of anti-inflammatory IL-10. IL-10 may
contribute to the down-regulation of TNF-a production by
macrophages exposed to bacteria for the second time. In
contrast to LPS, EPS priming did not induce macrophage
‘cross-tolerance’. The molecular mechanisms responsible for
et al. 2001). Itis
L. rhamnosus exopolysaccharide and inflammation
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the differential activation of macrophages by LPS and EPS
have not been elucidated to date. However, some mecha-
nisms that underlie the induction of LPS tolerance have been
postulated. For example, LPS may lead to down-regulation
of TLR4 expression and reduced activation of MAPKs
(Martin et al. 2001; Dowling et al. 2008). Activation of
MAPKs is one of the crucial signal transduction events that
control cytokine production (Dong et al. 2002). Little is
known regarding active components of probiotics involved
in MAPK activation. Our results clearly indicate that inhibi-
tors of both ERK and p38 MAPKs inhibit the production of
TNF-a induced by LPS and EPS. Interestingly, IL-10 pro-
duction was also markedly reduced by MAPKs inhibitors,
whereas the production of IL-12p40 was even enhanced
(data not shown). These data are in agreement with the
recent study of Kaji et al. (2010) who showed that selective
blockade of ERK MAPK activation induced by L. plantarum
resulted in a decrease in IL-10 and a simultaneous increase
in IL-12 production. However, the molecular mechanism of
this effect is unclear.
In conclusion, several questions regarding the biological
properties of EPS still remain to be answered. Specific
receptor(s) for EPS is⁄are still not defined. Although our
present data clearly show the immunostimulatory potential
of crude EPS, to use EPS in humans, further in vivo studies
using highly purified EPS are necessary.
because of the heterogeneity of bacterial EPS, the strain-
specific biological properties of EPS have to be taken into
consideration.
Furthermore,
Acknowledgements
We are very grateful to Prof Benjamin Chain from UCL for
critical reading and valuable advices in preparation of the
final version of this manuscript. This study was supported
by the grants of Jagiellonian University College of Medicine:
No. K⁄PBP⁄000288 and No. K⁄ZDS⁄000684.
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