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Bacterial Teichoic Acids Reverse
Predominant IL-12 Production Induced by
Certain Lactobacillus Strains into
Predominant IL-10 Production via
TLR2-Dependent ERK Activation in
Macrophages
Rumi Kaji, Junko Kiyoshima-Shibata, Masato Nagaoka,
Masanobu Nanno and Kan Shida
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Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 184:3505-3513; Prepublished online 26
February 2010;
doi: 10.4049/jimmunol.0901569
http://www.jimmunol.org/content/184/7/3505
The Journal of Immunology
Bacterial Teichoic Acids Reverse Predominant IL-12
Production Induced by Certain Lactobacillus Strains into
Predominant IL-10 Production via TLR2-Dependent ERK
Activation in Macrophages
Rumi Kaji, Junko Kiyoshima-Shibata, Masato Nagaoka, Masanobu Nanno, and
Kan Shida
L
actobacilli are widely used in many foods as probiotics,
defined as, “Live microorganisms which, when administered in adequate amounts confer a health benefit on the
host” (1), and they have become the focus of much research due to
their potential immunoregulatory activities. An increasing number
of immunoregulatory effects of lactobacilli, including augmentation
of NK cell activity, enhancement of IgA production, promotion of
dendritic cell maturation and regulatory T cell differentiation, and
downregulation of inflammation, have been reported (2, 3).
One of the well-researched immunoregulatory functions of
probiotics is the induction of cytokine production. In particular, the
induction of IL-10 and IL-12 production by probiotics has been
studied intensively, because the balance of IL-10/IL-12 secreted by
macrophages and dendritic cells in response to microbes is crucial
for determination of the direction of the immune response. IL-10 is
an anti-inflammatory cytokine and is expected to improve chronic
inflammation, such as that of inflammatory bowel diseases and
autoimmune diseases (4). IL-10 downregulates phagocytic and
Yakult Central Institute for Microbiological Research, Tokyo, Japan
Received for publication June 30, 2009. Accepted for publication January 20, 2010.
Address correspondence and reprint requests to Dr. Kan Shida, Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650,
Japan. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: ICW, intact cell wall; LTA, lipoteichoic acid; MDP,
muramyl dipeptide; PGN, peptidoglycan; PSPG, polysaccharide–peptidoglycan complex; TAPG, teichoic acid–peptidoglycan complex; WT, wild-type; WTA, cell wall
teichoic acid.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901569
T cell functions, including the production of proinflammatory
cytokines, such as IL-12, TNF-a, and IFN-g (5), that control inflammatory responses. IL-10 promotes the development of regulatory T cells for the control of excessive immune responses (6). In
contrast, IL-12 is an important mediator of cell-mediated immunity and is expected to augment the natural immune defense
against infections and cancers (7). IL-12 stimulates T cells to
secrete IFN-g, promotes Th1 cell development, and, directly or
indirectly, augments the cytotoxic activity of NK cells and macrophages. IL-12 also suppresses redundant Th2 cell responses for
the control of allergy (8). Because of their widespread immunological effects, much attention has been paid to the ability of
probiotics to induce IL-10 and IL-12 production in view of the
many potential benefits of induction of these cytokines, such as
defense against inflammatory bowel diseases, autoimmune diseases, allergies, infections, and cancers. Therefore, clarification of
the mechanism of IL-10 and IL-12 production by macrophages
and dendritic cells in response to probiotics is expected to lead to
a greater understanding of their immunoregulatory activities and
thus to contribute to a more effective utilization of probiotics in
health maintenance and disease prevention.
There have been few reports concerning the active bacterial
components of probiotics that induce IL-10 and IL-12 production.
Unique oligodeoxynucleotides with or without the immunostimulatory CpG motif have been identified as components of Lactobacillus rhamnosus and L. gasseri that are active in IL-12 induction (9,
10). In contrast, we recently showed that the insoluble intact cell
wall, but not the soluble one, of L. casei was required for the potent
induction of IL-12 production by macrophages, suggesting that
recognition of the three-dimensional structure of the cell wall of
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The cytokine response of macrophages to probiotic lactobacilli varies between strains, and the balance of IL-10/IL-12 production is
crucial for determination of the direction of the immune response. To clarify the mechanism whereby Lactobacillus strains
differentially induce production of IL-10 and IL-12, we examined the potential relationship between cytokine production and
MAPK activation. In mouse peritoneal macrophages, Lactobacillus plantarum potently induced IL-10 but weakly induced IL-12
production, whereas L. casei potently induced IL-12 but weakly induced IL-10 production. Kinetic analysis of the activation of
ERK, p38, and JNK showed that L. plantarum induced a more rapid and intense activation of MAPKs, especially of ERK, than L.
casei. A selective blockade of ERK activation induced by L. plantarum resulted in a decrease in IL-10 production and a simultaneous increase in IL-12 production. Interestingly, when macrophages were stimulated with a combination of L. plantarum and L.
casei, IL-10 production was induced synergistically. We identified cell wall teichoic acid and lipoteichoic acid as key factors for
triggering the synergistic induction of IL-10 production, although these teichoic acids alone only weakly induced IL-10 production. The effect of these teichoic acids on IL-10 production was mediated by TLR2-dependent ERK activation. Our data
demonstrate that activation of the ERK pathway is critical for determination of the balance of the IL-10/IL-12 response of
macrophages to lactobacilli and that predominant IL-12 production induced by certain lactobacilli such as L. casei can be
converted into predominant IL-10 production when stimulated in the presence of teichoic acids. The Journal of Immunology,
2010, 184: 3505–3513.
3506
TEICHOIC ACID-INDUCED ERK ACTIVATION AND IL-10 PRODUCTION
Materials and Methods
Reagents and culture medium
N-Acetylmuramidase SG and benzon nuclease were purchased from Seikagaku Kogyo (Tokyo, Japan) and Merck (Darmstadt, Germany), respectively. DNase, RNase, trypsin, and pronase were obtained from Roche
Diagnostic Systems (Somerville, NJ). U0126, a MEK1/2 inhibitor,
SB203580, a p38 inhibitor, and SP600125, a JNK inhibitor, were purchased from Calbiochem (San Diego, CA). Lipoteichoic acid (LTA) from
Staphylococcus aureus was obtained from InvivoGen (San Diego, CA).
RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with
10% heat-inactivated FCS, 100 U/ml penicillin, 100 mg/ml streptomycin,
and 0.05 mM 2-ME was used to culture macrophages.
Bacteria
The probiotic strain, L. casei Shirota (YIT 9029), was originally isolated
and maintained at the Yakult Central Institute. L. johnsonii JCM 2012T and
L. plantarum ATCC 14917T were obtained from the Japan Collection of
Microorganisms (Wako, Japan) and the American Type Culture Collection
(Manassas, VA), respectively. These bacteria were cultured for 20 h at 37˚
C in Lactobacilli-de Man, Rogosa, and Sharpe broth (Difco, Detroit, MI),
collected by centrifugation, and washed several times with sterile distilled
water. The bacteria were killed by heating at 100˚C for 30 min and lyophilized. To prepare FITC-labeled lactobacilli, heat-killed lactobacilli
were suspended at a concentration of 5 mg/ml in 50 mM carbonate buffer
(pH 9.6), reacted with FITC isomer I (4.5 mg/ml; Dojindo Laboratory,
Kumamoto, Japan) at 37˚C for 60 min, and then washed with sterile PBS.
Preparation of bacterial cell components
Bacterial cell components were prepared from heat-killed L. plantarum and
L. casei, and the preparation steps are summarized in Supplemental Figs. 1
and 2. Preparation of polysaccharide–peptidoglycan complex (PSPG) and
protoplast was described previously (25). Briefly, heat-killed lactobacilli
were exhaustively digested with N-acetylmuramidase. After centrifugation,
the precipitate was washed with distilled water and then collected as the
protoplast. The supernatant was treated with DNase, RNase, and trypsin.
The digested supernatant was dialyzed against distilled water and used as
the PSPG fraction. The preparation isolated from L. plantarum is described
as the teichoic acid–peptidoglycan complex (TAPG) in this study.
Intact cell wall (ICW) was prepared by the method of Sekine et al. (26)
as described previously (25). Briefly, heat-killed L. casei or L. plantarum
was treated with 0.3% SDS and then washed with acetone. The cells were
treated with pronase and delipidated by successive refluxing with methanol, methanol–chloroform–water (1:1:1), and methanol–chloroform (1:1).
The preparation was treated with benzon nuclease and pronase. The insoluble material was washed with distilled water and then used as the ICW
preparation. To prepare intact peptidoglycan (PGN), which was obtained
by release of the polysaccharide moiety from the linkage region of ICW,
the ICW was treated with 47% hydrogen fluoride at 4˚C for 20 h (27). The
material was washed with distilled water and then used as the PGN.
Isolation of LTA was described previously (25). Briefly, the protoplast
fraction prepared from L. casei was broken by sonication. The sonicated
material was extracted with hot phenol and treated with benzon nuclease.
LTA was purified by column chromatography over Macro-Prep High Q
(Bio-Rad, Hercules, CA) and Octyl-Sepharose CL-4B (Pharmacia Biochem, Uppsala, Sweden) columns.
Isolation of peritoneal macrophages
Peritoneal macrophages were harvested 4 d after i.p. injection of 2 ml 4%
thioglycollate broth (Difco). Cells were washed with HBSS containing 10
mM HEPES and cultured in RPMI 1640.
Flow cytometric analysis of phagocytosis
Peritoneal macrophages (6 3 105 cells) were cultured with FITC-labeled
lactobacilli (10 mg/ml) in 1.2 ml RPMI 1640 in a 24-well culture plate for
various time periods (1–24 h). Macrophages were dislodged by treatment
with 10 mM EDTA/PBS for 10 min and washed with 3 mM EDTA/PBS.
Analysis was performed on an EPICS Altra flow cytometer with Expo32
software (Beckman Coulter, Fullerton, CA).
Microscopic analysis of macrophage lysis of phagocytosed
lactobacilli
Intracellular digestion of lactobacilli by macrophages was analyzed as
described previously (11). Briefly, peritoneal macrophages (2 3 105
cells) growing on round, 12-mm, collagen type I-coated cover glasses
(Asahi Techno Glass, Funabashi, Japan), were cultured with heat-killed
lactobacilli (10 mg/ml) in a 24-well culture plate in 1.2 ml RPMI 1640
for 24 h. The macrophages were then fixed with methanol and stained
with Giemsa. Lysis of the lactobacilli by macrophages was observed by
light microscopy.
Animals
Female BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan)
and used for preparation of macrophages, unless otherwise specified. Male
TLR2-deficient mice with a BALB/c genetic background were obtained
from Oriental Bioservice (Kyoto, Japan), and male BALB/c mice were
used as controls. Animals were used at 8–12 wk of age. Experiments were
performed in accordance with the guidelines for the care and use of laboratory animals set by the Yakult Central Institute (Tokyo, Japan).
Cytokine production by macrophages
Peritoneal macrophages (1 3 105 cells) were plated in triplicate in a 96well culture plate and cultured with heat-killed lactobacilli (1–30 mg/ml)
or bacterial components (1–10 mg/ml) in 200 ml RPMI 1640 for 24 h,
unless indicated otherwise. In some experiments, the cells were pretreated
with U0126 (2.5 mM), SB203580 (5 mM), or SP600125 (10 mM) for 30
min before stimulation with lactobacilli or bacterial components. The
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certain Lactobacillus strains is required for the production of IL-12
(11). Little information is available concerning the components that
are active in the induction of IL-10 production. It has been shown
that genomic DNA, isolated from bifidobacteria or lactobacilli,
could induce IL-10 production by human PBMCs (12). Other reports have shown that soluble or insoluble cell preparations, obtained from centrifugation of sonicated bifidobacteria, were
responsible for induction of IL-10 production (13, 14). Further
studies are required to clarify the active components of probiotics
that induce these cytokines.
Cytokine production is regulated by intracellular signal transduction pathways that are activated following recognition of
microorganisms or microbial components via receptors, such as
TLRs, that exist on the cell and phagosomal membranes of
phagocytes. One of the crucial signal transduction events that
control cytokine production is activation of MAPKs, including
ERK, JNK, and p38 (15). Furthermore, several reports have shown
that activation of the ERK pathway following the recognition of
cell components via TLRs is important for determination of the
balance of IL-10/IL-12 production. ERK activation, induced by
recognition of Pam3Cys, LPS, and CpG DNA via TLR2, TLR4,
and TLR9, respectively, preferentially induces IL-10 production
and downregulates IL-12 production (16–19). It also has been
shown that defective activation of ERK is responsible for elevated
production of IL-12 by macrophages in response to Helicobacter
hepaticus (20). As for the cytokine response of phagocytes to
probiotic bacteria, the roles of MAPKs, especially ERK, have not
yet been fully elucidated, although some reports showed the importance of the MAPK pathways in the regulation of cytokine
production in response to probiotics as well as other bacteria (21–
24). Little is known regarding the receptors and active components
of probiotics involved in MAPK activation.
In this report, we aimed to clarify the mechanism of IL-10 and IL12 production induced by lactobacilli by studying MAPK activation
in mouse peritoneal macrophages following recognition of three
different strains of lactobacilli: L. plantarum, which potently induces IL-10 but weakly induces IL-12 production, L. casei, which
potently induces IL-12 but weakly induces IL-10 production, and L.
johnsonii, which weakly induces the production of both IL-10 and
IL-12. Additionally, we investigated the bacterial cell components
and their receptors that are involved in MAPK activation. We report
that ERK activation induced by TLR2 recognition of teichoic acids
plays a key role in determination of the balance of IL-10/IL-12
production by macrophages in response to lactobacilli.
The Journal of Immunology
3507
supernatants were harvested, and IL-10 and IL-12 levels were analyzed
by ELISA.
Expression of cytokine mRNA in macrophages
Peritoneal macrophages (1.2 3 106 cells) were cultured in a 12-well
culture plate with heat-killed lactobacilli (10 mg/ml) in 2.4 ml RPMI
1640 for various time periods (2–24 h). In some experiments, U0126 (2.5
mM) was added to the cultures 30 min before the stimulation with L.
plantarum, or a rat anti-mouse IL-10 mAb (clone JES5-2A5, 10 mg/ml;
BD Pharmingen, San Diego, CA) was added at the beginning of the
stimulation. Total RNA was extracted from the cells using the RNAqueous kit (Ambion, Austin, TX), and reverse transcription was performed
using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed
using TaqMan Universal PCR Master Mix and TaqMan Gene Expression
Assays for IL-10, IL-12 p35, and IL-12p40 expression (Applied Biosystems) in an ABI 7500 Real-Time PCR System (Applied Biosystems).
The GAPDH gene was used as an endogenous control to normalize the
expression of cytokine genes.
Cytokine determination by ELISA
Western blot analysis
Peritoneal macrophages (1 3 106) were stimulated with heat-killed
lactobacilli (10 mg/ml) or bacterial components (3 mg/ml) in 2 ml RPMI
1640 in a 24-well culture plate for various time periods (0.17–24 h). The
cells were lysed with 1% NP-40 in 25 mM Tris-HCl buffer (pH 7.5)
supplemented with 150 mM NaCl, 10% glycerol, 1% protease inhibitor
mixture (Sigma-Aldrich), 1 mM Na3VO4, and 50 mM NaF. The cell
lysates were treated with SDS sample buffer containing 4.5% SDS, 125
mM Tris, 20% glycerine, 0.02% bromphenol blue, and 10% 2-ME,
boiled for 5 min, separated by SDS-PAGE, and then transferred onto
a polyvinylidene difluoride membrane (Pall, East Hills, NY). After being
blocked for 1 h with 4% BSA in 20 mM Tris-HCl buffer (pH 7.5) supplemented with 0.1% NaCl, 50 mM NaF, and 0.005% protease inhibitor
mixture, the membranes were blotted with anti-phosphorylated ERK,
anti-phosphorylated p38, anti-phosphorylated JNK, and anti-ERK polyclonal Abs (Cell Signaling Technology, Beverly, MA) overnight at 4˚C.
The membranes were incubated with a secondary HRP-conjugated antirabbit IgG Ab (Cell Signaling Technology) for 1 h. Proteins were visualized using ECL Plus Western Blotting Detection Reagents (GE
Healthcare, Buckinghamshire, U.K.) according to the manufacturer’s
instructions.
Results
Characteristic patterns of macrophage IL-10 and IL-12
production induced by Lactobacillus strains
To analyze the effect of Lactobacillus strains on macrophage
cytokine production, mouse peritoneal macrophages were cultured with various concentrations of L. plantarum, L. casei, or L.
johnsonii for 24 h, and IL-10 and IL-12p70 concentrations in the
supernatants were measured by ELISA. L. plantarum induced
high levels of IL-10 in a dose-dependent manner but relatively
low levels of IL-12, which had an optimal dose of 3 mg/ml (Fig.
1A). In contrast, L. casei induced high levels of IL-12 and lower
levels of IL-10 in a dose-dependent manner, whereas L. johnsonii induced very low levels of both IL-10 and IL-12. We examined the kinetics of cytokine production. When macrophages
were stimulated with 10 mg/ml lactobacilli, L. plantarum induced earlier production of IL-10 and IL-12 than L. casei (Fig.
1B). The level of IL-12 induced by L. plantarum increased until
12 h and continued to be of a similar degree until 24 h, whereas
that induced by L. casei continued to increase until the end of
the culture.
FIGURE 1. Cytokine production by macrophages stimulated with lactobacilli. A, Peritoneal macrophages were cultured with various concentrations (1–30 mg/ml) of L. plantarum (Lp), L. casei (Lc), or L. johnsonii
(Lj) for 24 h, and the levels of IL-10 and IL-12p70 in supernatants were
determined by ELISA. Data are means 6 SD of triplicate cultures. Experiments were repeated three times with similar results. The dash represents medium only. B, Peritoneal macrophages were cultured with 10 mg/
ml lactobacilli for 2, 4, 8, 12, and 24 h, and the cytokine levels in supernatants were determined. Experiments were repeated twice with similar
results.
The effect of these strains on macrophage expression of cytokine
mRNAs was also examined. L. plantarum potently, and L. casei
moderately, induced IL-10 mRNA expression, and that induced by
L. plantarum increased earlier than that induced by L. casei (Fig.
2). L. plantarum also induced potent and rapid expression of IL12p35 and IL-12p40 mRNA, whereas IL-12p35 mRNA expression
was only moderately induced by L. casei and L. johnsonii. IL12p40 mRNA expression was only weakly induced by L. johnsonii. In contrast, L. casei induced potent expression of IL-12p40;
however, this expression increased at a later time than that induced
by L. plantarum.
Phagocytosis and lysis of lactobacilli by macrophages
To determine the phagocytosis of lactobacilli by macrophages, peritoneal macrophages were cultured with FITC-labeled L. plantarum, L.
casei, or L. johnsonii, and bacterial uptake was analyzed by flow
cytometry. The time course of phagocytosis varied depending on
the Lactobacillus strain (Fig. 3A). Over 50% of macrophages had
phagocytosed L. plantarum within 2 h, and ∼50% of macrophages
had phagocytosed L. johnsonii by 4 h. However, phagocytosis of L.
casei by 50% of the macrophages required a period of 16 h.
To analyze the macrophage intracellular digestion of lactobacilli, macrophages were cultured with lactobacilli for 24 h, stained
with Giemsa, and then observed under a light microscope. Phagocytosed L. casei cells maintained their cell morphology during the
culture period, whereas L. johnsonii cells lost the morphology and
only their digested fragments were observed (Fig. 3B). L. plantarum
showed an intermediate pattern of digestion between L. casei and L.
johnsonii. Some of the L. plantarum cells that were phagocytosed by
macrophages retained their cell morphology, whereas others appeared to be digested.
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Cytokine concentrations in culture supernatants were assayed by sandwich
ELISA. IL-10 levels were determined using a rat anti-mouse IL-10 mAb
(clone JES5-SXC1) as the capture Ab and a biotinylated rat anti-mouse IL10 mAb (clone JES5-2A5) as the detection Ab. To determine IL-12p70
levels, rat anti-mouse IL-12p35 (clone 9A5) and biotinylated rat anti-mouse
IL-12p40 (clone C17.8) mAbs were used. All of these Abs and recombinant
cytokines were purchased from BD Pharmingen.
3508
TEICHOIC ACID-INDUCED ERK ACTIVATION AND IL-10 PRODUCTION
culture, following which intense phosphorylation was maintained
over 24 h. The time course of p38 and JNK activation induced by
the lactobacilli showed a similar tendency to that of ERK activation. The kinetics of MAPK phosphorylation induced by each
Lactobacillus strain appeared to be related to the time course of
their phagocytosis by macrophages.
Effects of MAPK inhibitors on cytokine production
Activation of MAPK pathways by lactobacilli
Macrophages were stimulated with L. plantarum, L. casei, or L.
johnsonii, and the activation profiles of ERK, p38, and JNK were
compared. L. plantarum induced intense phosphorylation of ERK
and JNK, whereas L. casei and L. johnsonii induced weaker
phosphorylation of ERK and JNK (Fig. 4). All three lactobacilli
induced comparable levels of p38 phosphorylation.
ERK activation induced by L. plantarum peaked 1 h after
stimulation, whereas that induced by L. johnsonii peaked between
4 and 8 h after stimulation. The latest induced peak of ERK activation was that induced by L. casei, which peaked after 8 h of
L. plantarum synergizes with L. casei to enhance IL-10
production
FIGURE 3. Phagocytosis and lysis of lactobacilli by macrophages. A,
Peritoneal macrophages were cultured with FITC-labeled L. plantarum
(Lp, 10 mg/ml), L. casei (Lc), or L. johnsonii (Lj) for 1, 2, 4, 8, 16, and 24
h, and bacterial uptake was analyzed by flow cytometry. The percentage of
FITC-positive cells is shown. Experiments were repeated twice with
similar results. B, Peritoneal macrophages were cultured with L. plantarum
(10 mg/ml), L. casei, or L. johnsonii for 24 h and stained with Giemsa.
Intracellular lysis of bacteria was observed by light microscopy.
Our data showed that activation of the ERK pathway by lactobacilli may favor IL-10 production and that L. plantarum is
a potent activator of this pathway. Therefore, we hypothesized
that the potent ERK activation induced by L. plantarum may
modulate the balance of macrophage IL-10/IL-12 production
induced by other lactobacilli when macrophages are stimulated
with a combination of L. plantarum and these other lactobacilli.
To test this possibility, macrophages were cultured with L. casei
or L. johnsonii in the presence of L. plantarum, and cytokine
production was analyzed. L. plantarum synergized with L. casei
to enhance IL-10 production and simultaneously inhibited the
IL-12 production that can be potently induced by L. casei in the
absence of L. plantarum (Fig. 6). Addition of a neutralization Ab
against IL-10 to the cultures diminished the inhibitory effect of
L. plantarum on secretion of IL-12 induced by L. casei, suggesting that the increased production of IL-10 induced by L.
plantarum played a role in the inhibition of IL-12 secretion
(Supplemental Fig. 3). The synergistic induction of IL-10 production was more clearly observed when low doses (1 and 3 mg/
ml) of L. plantarum were added. Individual treatment with these
low doses of L. plantarum was not sufficient to induce effective
IL-10 production. In contrast, L. plantarum did not synergize
with L. johnsonii or with latex beads for IL-10 production. This
result suggests that L. plantarum induces potent activation of
ERK, which may synergize with certain stimuli derived specifically from L. casei to enhance IL-10 production.
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FIGURE 2. Expression of cytokine mRNA in macrophages stimulated
with lactobacilli. Peritoneal macrophages were cultured with L. plantarum
(Lp, 10 mg/ml), L. casei (Lc), or L. johnsonii (Lj) for 2, 4, 8, 16, and 24 h,
and the expression of IL-10, IL-12p35, and IL-12p40 mRNAs in the cells
was analyzed by quantitative RT-PCR. The relative expression of cytokine
mRNA was normalized to GAPDH. Data are means 6 SD of three independent experiments.
To investigate the functional role of ERK, p38, and JNK pathways
in cytokine production, macrophages were pretreated with a selective inhibitor of the ERK (U0126), p38 (SB203580), or JNK
(SP600125) pathway, prior to induction of cytokine production by
stimulation with L. plantarum, L. casei, or L. johnsonii. As shown
in Fig. 5A, IL-10 secretion induced by all three strains dramatically declined in the presence of inhibitors of the ERK, p38, and
JNK pathways, indicating that all three pathways are important for
IL-10 production.
In contrast, the potent IL-12 production induced by L. casei
was markedly suppressed in the presence of SB203580 and
SP600125, but only slightly suppressed by U0126. Intriguingly,
the weak production of IL-12 that was induced by L. plantarum,
but not that induced by L. johnsonii, was dramatically increased
by the addition of U0126. Next, we examined the effects of
U0126 on the expression of IL-12 mRNAs induced by L. plantarum. Inhibition of the ERK pathway both enhanced the peak of
IL-12p40 mRNA expression at 8 h and diminished the decrease
of its expression at 16 h, whereas neutralization of IL-10 with
a specific Ab only reduced the decrease at 16 h, suggesting that
activation of the ERK pathway inhibited IL-12p40 mRNA expression through IL-10-dependent and IL-10-independent
mechanisms (Fig. 5B). The data obtained using selective inhibitors of MAPK pathways suggest that activation of both p38
and JNK pathways is required for both IL-10 and IL-12 production, whereas the ERK pathway is critical for determination
of the balance of IL-10/IL-12 production.
The Journal of Immunology
3509
FIGURE 4. Activation of MAPKs in macrophages stimulated with lactobacilli. Peritoneal
macrophages were cultured with L. plantarum
(Lp, 10 mg/ml), L. casei (Lc), or L. johnsonii
(Lj). Whole-cell lysates were prepared at the
indicated time points and analyzed by immunoblotting using specific Abs to phosphorylated
p38, JNK, and ERK and to total ERK. Experiments were repeated three times with similar
results. M represents unstimulated macrophages.
Bacterial components that include teichoic acid induce ERK
activation and promote synergistic induction of IL-10
production with L. casei
ERK plays a critical role in synergistic production of IL-10
induced by teichoic acid
The results shown in Fig. 7 suggested that WTA extending from
PGN of L. plantarum, and LTA extending from the plasma membrane of L. plantarum and L. casei, are responsible for the synergistic production of IL-10. Thus, we tested the ability of LTA,
purified from L. casei, and TAPG from L. plantarum, which is
composed of WTA and a small residue of PGN, to synergize with
L. casei in the induction of IL-10 production. The ability of LTA
purified from nonlactobacillus bacterium S. aureus was also tested
in this assay. All of these teichoic acids synergized with L. casei for
enhancement of the production of IL-10 and for simultaneous
decrease in IL-12 production, although, when treated alone, they
induced very little IL-10 production (Fig. 8). Addition of the selective inhibitor of the ERK pathway abrogated the synergistic
effect of these teichoic acids, indicating that teichoic acid-induced
ERK activation plays a critical role in the synergism with L. casei
for the production of IL-10. The effect of teichoic acids on IL-10
FIGURE 5. Effects of inhibitors specific for MAPK pathways on cytokine production. A, Peritoneal macrophages were pretreated with U0126
(ERK inhibitor, 2.5 mM), SB203580 (p38 inhibitor, 5 mM), or SP600125
(JNK inhibitor, 10 mM) for 30 min and then stimulated with L. plantarum
(Lp, 10 mg/ml), L. casei (Lc), or L. johnsonii (Lj) for 24 h. The levels of
IL-10 and IL-12p70 in supernatants were determined by ELISA. Data are
means 6 SD of triplicate cultures. Experiments were repeated four times
with similar results. B, Peritoneal macrophages were cultured with L.
plantarum in the presence of U0126 or an anti-IL-10 mAb (10 mg/ml) for
2, 4, 8, 16, and 24 h, and the expression of IL-12p35 and IL-12p40 mRNAs
in the cells was analyzed by quantitative RT-PCR. The relative expression
of cytokine mRNA was normalized to GAPDH. Experiments were repeated twice with similar results.
FIGURE 6. Synergistic induction of IL-10 production induced by
a combination of L. plantarum and L. casei. Peritoneal macrophages were
cultured with L. casei (Lc, 10 mg/ml), L. johnsonii (Lj), or latex beads (La)
in the presence or absence of L. plantarum (Lp, 1–10 mg/ml) for 24 h. The
levels of IL-10 and IL-12p70 in supernatants were determined by ELISA.
Data are means 6 SD of triplicate cultures. Experiments were repeated
twice with similar results.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
To identify the active components responsible for the synergistic
induction of IL-10 production, ICW, TAPG/PSPG, PGN, and
protoplast were isolated from L. plantarum and L. casei, and their
effect on macrophage IL-10 production was assayed following
culture of the macrophages in the presence or absence of intact
L. casei cells. Only treatment with ICW from L. plantarum could
induce IL-10 production in the absence of L. casei cells. However,
ICW, TAPG, and protoplast, but not PGN, prepared from L.
plantarum could synergize with L. casei to enhance IL-10 production (Fig. 7A). Interestingly, protoplast from L. casei could also
synergize with L. casei cells in the induction of IL-10 production.
ICW and its soluble form, TAPG, from L. plantarum contain cell
wall teichoic acid (WTA), and protoplast from both L. plantarum
and L. casei contains LTA. However, no teichoic acid is present in
the other cell components that we tested. These findings suggest
that teichoic acids play a critical role in the synergism with L. casei
for the induction of IL-10 production, although teichoic acids alone
only weakly induce IL-10 production.
Because the ERK pathway plays a significant role in IL-10
production as shown in Fig. 5A, we therefore examined the effect of
the isolated cell components on ERK activation. As expected, ICW,
TAPG, and protoplast from L. plantarum and protoplast from L.
casei, all of which contain teichoic acid, induced strong phosphorylation of ERK (Fig. 7B). Unexpectedly, PGN from either L.
plantarum or L. casei, which contains no teichoic acid, also induced
phosphorylation of ERK, suggesting that ERK activation alone may
not be sufficient to induce the synergistic production of IL-10.
3510
TEICHOIC ACID-INDUCED ERK ACTIVATION AND IL-10 PRODUCTION
induced by teichoic acids in the presence of L. casei, macrophages
prepared from TLR2-deficient or wild-type mice were cultured
with combinations of teichoic acids and L. casei, and the concentrations of cytokines in the supernatants were measured. TAPG
and LTA failed to stimulate TLR2-deficient macrophages to affect
IL-10 and IL-12 production (Fig. 9A). Furthermore, neither TAPG
nor LTA induced potent phosphorylation of ERK in TLR2-deficient macrophages, although they could do so in wild-type
macrophages (Fig. 9B). In TLR4-deficient macrophages, these
teichoic acids could induce potent ERK activation to lead synergistic induction of IL-10 production with L. casei (data not
shown). Taken together, these data indicated that teichoic acids
were recognized by TLR2, activated the ERK pathway, and then
synergized with L. casei to induce IL-10 production (Fig. 10).
Discussion
production was observed when L. rhamnosus, but not L. johnsonii,
was substituted for L. casei (data not shown). We have previously
shown that L. rhamnosus is similar to L. casei but different from L.
johnsonii in that it is resistant to macrophage intracellular digestion
and can effectively induce IL-12 production (11).
TLR2 is involved in the synergistic production of IL-10 induced by
teichoic acids and L. casei
It has been shown that LTA is recognized by TLR2 (28). To verify
the involvement of TLR2 in the synergistic production of IL-10
FIGURE 8. Importance of ERK activation in teichoic acid-mediated
regulation of cytokine production. Peritoneal macrophages were pretreated
(+ERKi) or not treated (2) with U0126 (2.5 mM) for 30 min and cultured
with L. plantarum TAPG (3 mg/ml), L. casei LTA (LTAc), or S. aureus LTA
(LTAs) in the presence (+Lc) or absence of L. casei (10 mg/ml) for 24 h.
The levels of IL-10 and IL-12p70 in supernatants were determined by
ELISA. Data are means 6 SD of triplicate cultures. Experiments were
repeated twice with similar results.
FIGURE 9. Requirement of TLR2 recognition of teichoic acids for both
synergistic production of IL-10 and ERK activation. A, Peritoneal macrophages from wild-type (WT) or TLR2-deficient (TLR2-KO) mice were
cultured with L. plantarum TAPG or L. casei LTA (3 mg/ml) in the presence
(+Lc) or absence of L. casei (10 mg/ml) for 24 h. The levels of IL-10 and IL12p70 in supernatants were determined by ELISA. Data are means 6 SD of
triplicate cultures. Experiments were repeated twice with similar results. B,
Peritoneal macrophages from wild-type or TLR2-deficient mice were cultured with the bacterial components for 0.5, 1, and 2 h. Whole-cell lysates
were prepared and analyzed by immunoblotting using specific Abs to
phosphorylated (p) and total ERK. Experiments were repeated twice with
similar results. M represents unstimulated macrophages.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 7. Effect of bacterial components on IL-10 production and ERK
activation. A, Peritoneal macrophages were cultured with ICW, PSPG/TAPG,
protoplast, or PGN (3 mg/ml) prepared from L. plantarum (p) or L. casei (c)
in the presence (+Lc) or absence of L. casei cells (10 mg/ml) for 24 h. The
levels of IL-10 in supernatants were determined by ELISA. Data are means
6 SD of triplicate cultures. Experiments were repeated twice with similar
results. The dash represents medium only or L. casei only. B, Peritoneal
macrophages were cultured with bacterial components (3 mg/ml) prepared
from L. plantarum (Lp) or L. casei (Lc) for 0.5, 1, and 2 h. Whole-cell lysates were prepared and analyzed by immunoblotting using specific Abs to
phosphorylated (p) and total ERK. Experiments were repeated twice with
similar results. M represents unstimulated macrophages.
We examined the relationship between MAPK activation and
regulation of IL-10 and IL-12 production in mouse peritoneal
macrophages in response to lactobacilli. L. plantarum, which
potently induces the production of IL-10, strongly activated ERK,
whereas L. casei, which potently induces the production of IL-12,
showed a weak ability to activate ERK. WTA, which is a characteristic cell wall component of L. plantarum, was responsible for
the strong activation of ERK induced by L. plantarum. Furthermore, specific inhibition of the ERK pathway by the addition of
U0126 converted L. plantarum into a potent inducer of IL-12
production, whereas ERK activation by the addition of WTA
converted L. casei into a potent inducer of IL-10 production.
These data suggest that activation of the ERK pathway is crucial
for determination of the balance of IL-10/IL-12 production induced by lactobacilli.
In contrast, neither stimulation of the ERK pathway in macrophages by WTA alone nor stimulation of macrophages with
a combination of WTA and L. johnsonii, which weakly induces IL12 production, could induce effective production of IL-10.
The Journal of Immunology
Moreover, addition of the ERK inhibitor could not result in the
induction of IL-12 production by L. johnsonii. Taken together, all
of these data suggest that production of IL-10 and IL-12 by
macrophages in response to lactobacilli requires the activation of
common signal transduction pathways and that additional potent
activation of ERK leads to IL-10 production but that insufficient
ERK activation leads to IL-12 production (Fig. 10). L. johnsonii
does not appear to be able to activate the common signaling
pathways required for IL-10 and IL-12 production, whereas L.
plantarum and L. casei appear to activate such signaling pathways. The difference between L. johnsonii and the other two
strains in their ability to activate the common signaling pathways
may be related to their difference in sensitivity to macrophage
intracellular digestion. Thus, we showed in Fig. 3B that L. johnsonii is susceptible to intracellular digestion, L. casei is resistant to
digestion and retains its cell morphology following ingestion, and
a subpopulation of the L. plantarum cells phagocytosed by macrophages also maintained their cell morphology. We have previously shown that the Lactobacillus cell wall structure that is
resistant to intracellular digestion is responsible for effective induction of IL-12 production by macrophages (11). We therefore
speculate that cell wall structure-derived stimuli could lead to
activation of the signaling pathways common to IL-10 and IL-12
production (Fig. 10). Further studies will be necessary to clarify
this issue.
Our data indicated that recognition of WTA and LTA by TLR2
leads to ERK activation, which is the key to regulation of the
balance of IL-10/IL-12 production by macrophages in response to
lactobacilli. Several reports have already revealed the importance
of the ERK pathway in the regulation of IL-10 and IL-12 production. Dillon et al. (18) clearly showed that Pam3Cys, which is
a ligand for TLR2, strongly induces ERK phosphorylation in
mouse dendritic cells and effectively induces IL-10 production.
That group also observed that selective blockade of the ERK
pathway results in a decrease in IL-10 production and a simultaneous increase in IL-12 production in response to Pam3Cys.
Similar results were obtained in studies using LPS and CpG DNA,
which are ligands for TLR4 and TLR9, respectively (17, 19).
Although these previous findings correspond well with our results,
an interesting difference between those findings and ours is that
Pam3Cys, LPS, and CpG DNA alone were able to induce IL-10
production, whereas WTA and LTA alone could not. These data
suggest that ERK activation is necessary, but not sufficient, for IL10 production and that the first three TLR ligands, but not the
latter two, can additionally activate other signaling pathways required for IL-10 production and also probably for IL-12 production. When these teichoic acids stimulate macrophages in the
presence of L. casei, teichoic acid-induced ERK activation could
lead to IL-10 production in collaboration with stimuli derived
from L. casei that induce IL-12 production (Fig. 10). Such signals
derived from L. casei have been suggested to be independent of
recognition by TLR2 (11), in contrast to the TLR2-dependent
signals induced by teichoic acids. Collaboration between TLR2derived signals and other receptor-derived signals in IL-10 production has also been observed in the response of dendritic cells to
yeast zymosan (29). In that case, dectin-1, a C-type lectin receptor
for b-glucans, was shown to act as the second receptor. It is of
great interest that TLR2-dependent and TLR2-independent signals
cooperatively regulate IL-10 production.
Although our data suggest that it is likely that ERK activation
together with stimuli derived from L. casei lead to IL-10 production, one apparent contradiction of this hypothesis is that PGN,
which can induce ERK phosphorylation, did not induce IL-10
production even in the presence of L. casei (Fig. 7). One possible
explanation for this contradiction is the following: PGN might
promote inhibitory mechanisms in IL-10 production, in addition to
activation of the ERK pathway. Watanabe et al. (30) have revealed
that muramyl dipeptide (MDP), which is a by-product of PGN
digestion, inhibits IL-12 production by macrophages through its
recognition by nucleotide-binding oligomerization domain 2. The
same group also showed that MDP-derived stimuli inhibited IL-10
production by dendritic cells (31). We recently reported that PGN
isolated from lactobacilli including L. plantarum and L. casei
inhibits IL-12 production by macrophages through TLR2-dependent
and TLR2-independent mechanisms (32). Furthermore, we found
here that PGN and 6-O-stearoyl-MDP, which is a derivative of
MDP and is more effectively internalized into cells, inhibited the
production of IL-10 as well as IL-12 by macrophages stimulated
with a combination of L. casei and L. plantarum (Supplemental
Fig. 4). Such inhibitory stimuli might occur in macrophages that
have been cultured with a combination of PGN and L. casei.
Alternatively, ERK activation might be no more than one of the
essential factors to convert IL-12 production to IL-10 production,
and PGN, but not WTA and LTA, may lack the ability to turn on
the essential factors other than ERK activation.
A second apparent contradiction contained within our data are
that, although L. casei contains LTA as a cellular component, this
bacterium could not lead to either potent activation of ERK or
effective production of IL-10. In contrast, LTA purified from L.
casei potently activated ERK and induced IL-10 production in the
presence of L. casei cells. The following explanation is proposed
to explain this contradiction. L. casei expresses uncharged polysaccharides that are densely associated with PGN (33), which may
sterically hinder the access of TLR2 to LTA extending from the
plasma membrane through PGN (Supplemental Fig. 2). This assumption is supported by the result that the protoplast that was
prepared by removal of PGN and associated polysaccharides from
L. casei could activate ERK. However, L. plantarum has PGNassociated WTA on its cell surface (34), which is likely to be
easily recognized by TLR2 (Supplemental Fig. 1). This may be
the reason for the difference in the abilities of L. casei and L.
plantarum to induce IL-10 production.
There have been some reports showing that certain strains of lactic acid bacteria modulate cytokine production induced by other
strains. Christensen et al. (35) reported that strong IL-12 production
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FIGURE 10. Summary of possible mechanisms of regulation of IL-10
and IL-12 production by lactobacilli. i, L. casei (Lc) is phagocytosed by
macrophages and stimulates them through the cell wall structure to produce IL-12. ii, L. plantarum (Lp) potently activates the ERK pathway via
recognition of cell surface WTA by TLR2, in addition to the signals derived from the cell wall structure that would lead to IL-12 production,
thereby inducing potent IL-10 production and a simultaneous decrease of
IL-12 production. iii, When L. casei stimulates macrophages in the presence of WTA or LTA, the signals derived from L. casei cell wall structure
and the potent ERK activation induced through TLR2 cooperatively induce
potent production of IL-10.
3511
3512
TEICHOIC ACID-INDUCED ERK ACTIVATION AND IL-10 PRODUCTION
Acknowledgments
We are grateful to Dr. Satoshi Matsumoto (Yakult Central Institute) for valuable suggestions and discussions. We also gratefully acknowledge the staff
of the animal facility of the Yakult Central Institute for expertise in breeding mice.
Disclosures
All of the authors are employed by Yakult Honsha.
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1
Legends for Supplementary Figures
2
3
Supplementary Figure 1. Schematic representation of the cell surface structure of L.
4
plantarum and the preparation steps for its cell components. L. plantarum expresses
5
PGN-associated WTA, which may be easily recognized by TLR2. TAPG and protoplast
6
are prepared from heat-killed cells by hydrolysis of the glycosidic bond between
7
N-acetylmuramic acid and N-acetylglucosamine in PGN with N-acetylmuramidase. The
8
cell wall with the intact structure (ICW) is obtained by sequential treatment of
9
heat-killed cells with SDS, organic solvent, nuclease, and pronase to remove
10
membranes and molecules non-covalently associated to PGN. Intact PGN is obtained by
11
treatment of ICW with hydrogen fluoride to remove WTA which is covalently
12
associated to PGN.
13
14
Supplementary Figure 2. Schematic representation of the cell surface structure of L.
15
casei and the preparation steps for its cell components. L. casei densely expresses
16
PGN-associated uncharged polysaccharides (PS), which may sterically hinder the access
17
of TLR2 to LTA that extends from the plasma membrane. PSPG, protoplast, ICW, and
18
PGN are obtained from heat-killed cells by the same methods as described in Fig. S1.
1
19
20
Supplementary Figure 3. Inhibitory effect of IL-10 induced by L. plantarum on L.
21
casei-induced IL-12 production. Peritoneal macrophages were cultured with L. casei
22
(Lc, 10 μg/ml) alone or L. casei plus L. plantarum (Lp, 3 μg/ml) in the presence
23
(+Anti-IL10 Ab) or absence of an anti-IL-10 mAb (10 μg/ml) for 24 h. The levels of
24
IL-10 and IL-12p70 in supernatants were determined by ELISA. Data are means ± SD
25
of triplicate cultures. Experiments were repeated twice with similar results.
26
27
Supplementary Figure 4. Inhibition of cytokine production by PGN and MDP.
28
Peritoneal macrophages were cultured with PGN or 6-O-stearoyl-MDP (MDP) (3-30
29
μg/ml) in the presence (Lc+Lp) or absence of combinatory stimuli of L. casei (10
30
μg/ml) and L. plantarum (3 μg/ml) for 24 h. The levels of IL-10 and IL-12p70 in
31
supernatants were determined by ELISA. Data are means ± SD of triplicate cultures.
32
Experiments were repeated twice with similar results. -, medium only or L. casei plus L.
33
plantarum.
2