1-Methyl-tryptophan can interfere with TLR signaling in dendritic cells independently of IDO activity.

Abstract

The compound 1-methyl-tryptophan (1-MT) is a competitive inhibitor of IDO that can break tolerance and induce fetus, graft, and tumor rejection. Because of its broad effect on immune-related mechanisms, the direct action of 1-MT on human monocyte-derived dendritic cells (DC) was analyzed. It is shown here that the effect of 1-MT on DC is dependent on the maturation pathway. Although 1-MT had no effect on DC stimulated by the TLR3 ligand poly(I:C), it strongly enhanced the Th1 profile of DC stimulated with TLR2/1 or TLR2/6 ligands. Drastic changes in the function of DC stimulated by the TLR4 ligand LPS were induced by 1-MT. These cells could still activate allogeneic and syngeneic T cells but stimulation yielded T cells secreting IL-5 and IL-13 rather than IFN-gamma. This action of 1-MT correlated with an increased phosphorylation of p38 and ERK MAPKs and sustained activation of the transcription factor c-Fos. Inhibiting p38 and ERK phosphorylation with synthetic inhibitors blocked the effect of 1-MT on LPS-stimulated DC. Thus, 1-MT can modulate DC function depending on the maturation signal and independently of its action on IDO. This is consistent with previous observations and will help further understanding the mechanisms of DC polarization.

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1-methyl-tryptophan can interfere with TLR signaling in dendritic cells
independently of IDO activity1
Running title: 1-MT can modify dendritic cell functions
Sophie Agaugué*, Laure Perrin-Cocon*, Frédéric Coutant*, Patrice André* and Vincent
Lotteau*2
* Institut National de la Santé et de la Recherche Médicale U503, IFR 128 BioSciences Lyon-
Gerland, 21 Avenue Tony Garnier, 69007 Lyon, France.
Corresponding author:
Dr Vincent Lotteau
INSERM U503
21 Avenue Tony Garnier
69007, Lyon, France
Tel : +33 437 28 24 12
Fax : +33 437 28 23 41
E-mail : lotteau@cervi-lyon.inserm.fr
Key words: Dendritic cells - Protein kinases - Th1/Th2 cells - Toll-like receptor - 1-methyl-
tryptophan
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Abstract
1-methyl-tryptophan (1-MT) is a competitive inhibitor of indoleamine 2,3-dioxygenase that
can break tolerance and induce fetus, graft and tumor rejection. Because of its broad effect on
immune-related mechanisms, the direct action of 1-MT on human monocyte-derived dendritic
cells (DC) was analyzed. It is shown here that 1-MT effect on DC is dependent on the
maturation pathway. 1-MT had no effect on DC stimulated by the TLR3 ligand polyI:C but
strongly enhanced the Th1 profile of DC stimulated with TLR2/1 or TLR2/6 ligands. 1-MT
induced drastic changes in the function of DC stimulated by the TLR4 ligand LPS. These
cells could still activate allogeneic and syngeneic T cells but stimulation yielded T cells
secreting IL-5 and IL-13 rather than IFNγ. This action of 1-MT correlated with an increased
phosphorylation of p38 and ERK MAP-kinases and sustained activation of the transcription
factor c-Fos. Inhibiting p38 and ERK phosphorylation with synthetic inhibitors blocked the
effect of 1-MT on LPS-stimulated DC. Thus, 1-MT can modulate DC function depending on
the maturation signal and independently of its action on IDO. This is consistent with previous
observations and will help further understanding the mechanisms of DC polarization.
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Introduction
1-methyl-tryptophan3 (1-MT) is a competitive inhibitor of indoleamine 2,3-dioxygenase
(IDO) which is a rate limiting enzyme in the catabolism of tryptophan (Trp). IDO converts the
amino acid L-Trp to kynurenine and further catabolites (1, 2). Trp metabolism plays a major
role in the control of propagation of various intracellular pathogens like Toxoplasma gondii,
Chlamydia psitacci, human cytomegalovirus and different cocci. It is an essential amino acid
for the growth of pathogens and Trp shortage induced by IDO activity can inhibit their
replication (3-7). Trp depletion can block the proliferation of various cell types including
tumor cells (8-10).
1-MT has been initially used to block the immune privilege of placenta (11). 1-MT treatment
of pregnant mice induced the rejection of the allogeneic fetus by breaking the tolerance of
maternal T lymphocytes for the fetus. Maternal T cell tolerance appeared to rely on IDO-
expressing cells at the maternal-fetal interface that deprive the local microenvironment in Trp
and inhibit T cell proliferation. It is believed that 1-MT restores the local concentration of Trp
in the placenta, allowing T cell activation. 1-MT can also break tolerance for tumor cells (9,
10). Mice receiving 1-MT before injection of tumor cells developed tumors later than control
mice and mice receiving 1-MT during tumor growth developed tumors more slowly than
control mice. The inhibition of IDO activity in tumor cells leads to an enhanced CTL activity
against tumor cells and a reduced tumor growth. It has also been shown that long-term
survival of pancreatic islet allografts induced by CTLA4-Ig is abrogated by 1-MT treatment
of recipient mice (12). Overall, in vivo, 1-MT seems to enhance T cell alloreactivity and T
cell responses against tumor Ag, allograft Ag and auto Ag (13-17). From these in vivo
studies, it was proposed that IDO could have an immunoregulatory function. In vitro studies
have focused primarily on the main activators of the immune system, DC and macrophages
that can express IDO. The addition of 1-MT in cocultures of T or NK cells with APC or tumor
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cells maintains high concentrations of Trp and improve T cell and NK cell proliferation.
Moreover, by blocking IDO, 1-MT inhibits the production of Trp catabolites like kynurenine
that have been shown to reduce T cell and NK cell proliferation (18-24).
The maturation of DC can be induced by many bacterial components which are recognized by
different TLR. LPS from Gram- bacteria are recognized by TLR4, components of Gram+
bacteria and bacterial lipopeptides are recognized by TLR2/TLR1 or TLR2/TLR6, and
polyI:C (pIC) mimicking double-stranded RNA is recognized by TLR3. TLR use various
adaptors activating different signaling pathways. Briefly, all TLR except TLR3 can use the
MyD88 adaptor leading to NFκB activation. TLR3 uses the TRIF adaptor that activates the
transcription factor IRF-3 and NFκB. TLR4 can also activate a MyD88-independent pathway
involving the adaptors TRAM and TRIF, triggering IRF3 activation and NFκB stimulation
(25).
The polarization of T cells is dependent on various factors including the origin of DC, their
degree of maturation and kinetic of activation (26-30). Myeloid DC, like monocyte derived-
DC, prime mainly Th1 cells secreting large amounts of IFNγ but little IL-4, IL-5 and IL-10,
whereas plasmacytoid DC can activate Th1 or Th2 cells that secrete large amounts of IL-4,
IL-5 and IL-10 but little IFNγ, depending on the activation signal and the infectious agent. At
least three distinct functional subsets of DC have been reported according to the final outcome
of maturation. Cells with high costimulatory capacity and IL-12 production would promote
Th1 responses, cells with high costimulatory capacity but low IL-12 production would drive
Th2 differentiation and cells with low costimulatory capacity and IL-12 production would
give rise to tolerogenic Th cells. Semi-mature DC with high expression of costimulatory
molecules and low secretion of pro-inflammatory cytokines have also been described and may
stimulate regulatory T cells (31). Lanzavecchia et al. also proposed the DC exhaustion model
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where Th1-polarized DC are first generated whereas the same cells analyzed at later time
points of maturation stop secreting IL-12 and prime Th2 and non polarized cells (28).
IDO can be induced in vitro or in vivo by various agents like cytokines (IFNγ, TNFα),
CD40L, CTLA4-Ig, influenza virus or bacterial LPS (32-37). Several subsets of IDO-
expressing DC have been described. CD11c+ murine DC express IDO protein but enzyme
activity is only detected in the CD8+ subset (38). In mice, plasmacytoid DC that express IDO
can inhibit T cell responses (39, 40). A particular subset of human myeloid DC expressing
CCR6, CD123 and a constitutively active form of IDO is deficient for T cell stimulation and
may thus play a central role in tolerance (21). Another group has shown that human
monocyte-derived DC expressing active IDO after IFNγ stimulation do not suppress T cell
proliferation (41). Therefore, further studies will be necessary to describe the different
functions of human DC expressing IDO (42). Moreover, some studies suggest that IDO is
necessary for DC activation (43).
In previous in vitro experiments, 1-MT was added in cocultures of DC with T cells to study
the role of IDO on T cell proliferation and activation (18, 19, 24). Since DC maturation is a
crucial step to control immune responses, we analyzed the direct effect of 1-MT on the
maturation of human monocyte-derived DC. It is shown that 1-MT affected differentially the
function of DC depending on the quality of the maturation signal. In the presence of 1-MT,
pIC-stimulated DC maintained their capacity to induce a Th1 response while DC stimulated
with TLR2 ligands had an increased ability to stimulate IFNγ secretion by T cells. In contrast,
1-MT on TLR4-stimulated DC reoriented DC toward a Th2 function, a process involving both
ERK and p38-MAPK. Interestingly, all these effects of 1-MT were not correlated to the
inhibition of IDO activity.
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Materials and methods
Generation and treatment of DC
PBMC were isolated from human peripheral blood of healthy donors by standard density
gradient centrifugation on Ficoll-Hypaque. Mononuclear cells were separated from PBL by
centrifugation on a 50% Percoll solution (Amersham Biosciences, Uppsala, Sweden).
Monocytes were purified by immunomagnetic depletion (Dynal, Oslo, Norway) using a
cocktail of monoclonal Ab anti-CD19 (4G7 hybridoma), anti-CD3 (OKT3, ATCC, Rockville,
MD, USA) and anti-CD56 (NKH1, Beckman Coulter, Fullerton, CA, USA). Monocytes
(purity > 90%) were differentiated to immature DC (iDC) during 7 days with 40 ng/ml human
rGM-CSF and 250 U/ml human rIL-4 in RPMI 1640 (Abcys, Paris, France) supplemented
with 2 mM glutamine, 10 mM Hepes, 40 ng/ml gentamycin (Life Technologies, Rockville,
MD, USA) and 10% FCS. Differentiating monocytes were treated at day 5 with 1 mM 1-
methyl-DL-tryptophan or 2,5 mM of Trp or 60 µM of kynurenine (Sigma-Aldrich, St
Quentin-Fallavier, France) and at day 6 with 1 µg/ml LPS (Escherichia coli, serotype
0127:B8, Sigma-Aldrich), 10 µg/ml polyI:C (pIC - Amersham Biosciences), 10 µg/ml
peptidoglycan (PGN) of Staphylococcus aureus (Sigma-Aldrich) or 10 µg/ml of Pam3CSK4
(Pam - Axxora, San Diego, CA). All cells and supernatants were collected at day 7. Control
mature DC (mDC) were obtained by adding TLR ligands at day 6 for 24 h. When indicated,
40 µM PD98059, an inhibitor of MEK1/2 (Biomol, Plymouth Meeting, PA, USA), or 25 µM
SB203580, an inhibitor of p38-MAPK (Biomol), were added 30 min before 1-MT treatment.
All DC were more than 95% pure as assessed by CD14 and CD1a labeling.
Phenotype
Phenotype was analyzed on a FACScalibur (BD Biosciences, Le Pont de Claix, France) using
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FITC-conjugated anti-CD14, -HLA-DR, -CD80, -CD54, and PE-conjugated anti-CD1a,
-CD86, -CD83 and -CD40 (Beckman Coulter).
Cytokine assay
Culture supernatants were stored at -80°C. IL-6, IL-10, IL-1β, TNFα and IL-13 levels were
determined using cytokine-specific ELISA kits (Endogen, Woburn, MA, USA). IL-12 p40
and p70 were assayed using ELISA kits from Biosource (Camarillo, CA, USA). IL-2, IL-4,
IL-5, IL-10 and IFNγ were determined using the human Th1-Th2 cytokine CBA kit I (BD
Biosciences).
Mixed Lymphocyte Reaction
T lymphocytes were purified after Ficoll-Hypaque and Percoll gradient centrifugation by
immunomagnetic depletion using a cocktail of monoclonal Ab anti-CD19 (4G7), anti-CD56
(NKH1), anti-CD16 (3G8), anti-CD14 (RMO52) and anti-glycophorin A (11E4B7.6)
(Beckman Coulter). T lymphocytes were more than 95% pure as assessed by CD3 labeling.
Primary MLR were conducted in 96-well flat-bottom culture plates. DC recovered at day 7
were extensively washed and resuspended in complete RPMI 1640 / 10% FCS. Cells were
cocultured in triplicates with 2.105 allogeneic T cells in 200 µl at DC/T cell ratios ranging
from 1/10 to 1/40. Supernatants were recovered at indicated time points for IL-2, IL-4, IL-5,
IL-10, IL-13 and IFNγ measurement.
T cell response against tetanus toxin
DC were treated as for MLR and autologous CD3 T cells were purified as described above
from frozen PBL. DC recovered at day 7 were extensively washed and resuspended in
complete RPMI 1640 / 10% FCS. Cells were cocultured in triplicates with 2.105 allogeneic T
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cells in 200 µl at 1/20 DC/T cell ratio. 25 µg/ml purified tetanus neurotoxin (kindly provided
by Dr Villiers, INSERM U548, CEA Grenoble, France) was then added to cocultures.
Supernatants were recovered after 5 days of coculture for IL-2, IL-4, IL-5, IL-10, IL-13 and
IFNγ measurement. Tetanus neurotoxin has no effect on DC or T cells alone (data not shown).
Intracellular staining of cytokines
MLR were conducted for 5 days and T cells were expanded for 7 days with 25 U/ml rhIL-2
(Biosource), washed and restimulated with 10 ng/ml PMA (Sigma-Aldrich) and 1 µg/ml
ionomycin (VWR International, Fontenay-sous-Bois, France) for 5 h. 10 ng/ml Brefeldin A
(Sigma-Aldrich) was added during the last 2 h. Cells were fixed and permeabilized using
Cytofix/Cytoperm kit (BD Biosciences). Intracellular staining was performed using FITC-
labeled anti-IFNγ monoclonal Ab and PE-labeled anti-IL-5 and IL-13 monoclonal Ab (BD
Biosciences).
IDO expression and activity
Total RNA was extracted from cells collected at day 7 using RNeasy Mini kit (Qiagen,
Courtaboeuf, France). 100 ng of total RNA was reverse transcribed using the thermoscript
RT-PCR system (Life Technologies). Primers used for PCR amplification are: 5’-
GCTTTCACACAGGCGTCATA-3’ and 5’-GGTCATGGAGATGTCCGTAA-3’ for IDO,
and 5’-GGAGGTGTAATGGACGTTA-3’ and 5’-CTGAGACTCCTTGCCATAG-3’ for S12.
The amplified products were analyzed by gel electrophoresis (691 bp for IDO and 311 bp for
S12).
Trp is converted by IDO to N-formylkynurenine which is further catabolized to kynurenine.
Quantification of kynurenine in supernatants thus reflects IDO activity. Kynurenine was
measured in fresh supernatants of DC collected at day 7 as described previously (44). Briefly,
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100 µl of 30% TCA was added to 200 µl of supernatant and vortexed. After centrifugation,
125 µl of supernatant was incubated with 125 µl of Ehrlich reagent (p-
dimethylaminobenzaldehyde; Sigma-Aldrich) in a microtiter plate for 10 min at room
temperature. Optical density was measured at 490 nm. Values were referred to a standard
curve with defined kynurenine concentrations (0-120 µM, Sigma-Aldrich) and normalized to
106 cells.
Phosphorylation of p38-MAPK, ERK and c-Fos
For studies on ERK and p38 phosphorylation, 2.106 differentiating monocytes were treated at
day 5 with 1 mM 1-MT and collected at day 6. Cells were extensively washed and starved for
2 h in complete RPMI 1640 medium without serum. Cells were treated with the different TLR
ligands for 5, 10, 15, 30 or 45 min. Cells were washed twice with cold PBS and pellets were
lyzed in RIPA buffer containing 1 mM PMSF and 1% protease inhibitors. Phosphorylated and
total ERK and p38 MAPK were quantified by specific ELISA (Assay Designs Inc, Ann
Arbor, MI, USA).
For studies on c-Fos phosphorylation, 2.106 differentiating monocytes were treated at day 5
with 1 mM 1-MT and collected at day 6. Cells were extensively washed and resuspended in
RPMI / 0,3% delipidated BSA. Cells were treated with the different TLR ligands for 1, 2, 4 or
6 hours. Cells were then washed twice with cold PBS and pellets were lyzed in RIPA buffer
containing 1 mM PMSF and 1% protease inhibitors. Phosphorylated c-Fos was quantified by
a chemoluminescent ELISA (Endogen) and normalized to the amount of protein determined
with the microBCA assay kit (Pierce, Rockford, IL, USA).
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Results
The effect of 1-MT on DC is dependent on the maturation signal
To test the action of 1-MT on DC maturation, DC were treated with 1-MT 24 h before TLR
stimulation. LPS was used as a prototype of TLR4 ligand, pIC for TLR3 stimulation, and
PGN or Pam as ligands of the heterodimers TLR2/TLR6 or TLR2/TLR1 respectively. DC
were then analyzed for their ability to stimulate allogeneic T cells in MLR and cytokines
released were measured in supernatants after 2 to 5 days of coculture. Under these
experimental conditions, 1-MT was present before and during the induction of DC maturation
but not in the cocultures with allogeneic T cells.
Viability of DC was not affected by the various treatments and addition of 1-MT to iDC had
no effect on cytokine secretion in MLR (data not shown and table I). Treatment of DC with 1-
MT 24 h before the addition of the maturation agent increases the capacity of DC to stimulate
IL-2 secretion by T cells (table I). DC stimulated with TLR ligands were good inducers of
IFNγ secretion although DC treated with pIC or PGN were less efficient than DC treated with
LPS or Pam (table I). 1-MT pre-treatment of pIC-activated DC had no effect on IFNγ, IL-5
and IL-13 secretion by T cells. IFNγ production in MLR was enhanced by 1-MT when DC
were activated with either PGN or Pam while IL-5 was not modified and IL-13 was slightly
increased (table I). In contrast, 1-MT pre-treatment of LPS-activated DC yielded cells with a
reduced ability to stimulate IFNγ production by T cells (table I). DC treated with 1-MT and
LPS had an increased ability to induce IL-5 and IL-13 production by T cells in MLR,
compared to LPS-treated DC.
Thus the effect of 1-MT on functional maturation of DC is dependent on the TLR
triggered on DC.
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1-MT induces a Th2 polarization of DC matured with LPS
1-MT-treated DC activated by LPS had a reduced ability to induce IFNγ secretion by
allogeneic T cells but stimulated IL-5 and IL-13 secretion (table I). This could result from a
shift in DC function or from a rapid exhaustion of the Th1 potential of DC. The kinetic of
secretion of IFNγ, IL-5 and IL-13 was thus analyzed. In MLR with control LPS-treated DC,
reasonable amount of IFNγ could be detected at day 2 of coculture while IL-5 and IL-13
began to be detectable at day 3. Cytokine concentration in the supernatants progressively
increased until day 5 (Fig. 1A). The kinetic of IFNγ production was not modified by 1-MT
pretreatment of DC although the quantity of cytokine released was drastically reduced. The
kinetic of IL-5 and IL-13 secretion was not modified either and confirmed the strong
induction of these cytokines by 1-MT pretreatment. Although IL-5 and IL-13 were detected
later than IFNγ, these secretions did not follow a first peak of IFNγ secretion, indicating that
the Th2 function of T cells was not the result of a Th1 exhaustion.
T cell populations activated in these MLR were further analyzed by intracellular staining. As
expected, control LPS-treated DC predominantly stimulated Th1 cells producing IFNγ (58%
of IFNγ+ T cells) (Fig. 1B). 19% of these T cells also produced IL-13. Only 5% of T cells
presented a Th2 profile with production of IL-13 without IFNγ. No IL-5 producing T cells
were detected under these experimental conditions. DC pre-treated with 1-MT before LPS
activated an increased number of T cells producing IL-13 without IFNγ (36% versus 5% in
control MLR) while only 30% of T cells produced IFNγ (versus 58% in control MLR). 5% of
T cells synthesized IL-5. These data indicate that 1-MT treatment induced a shift in the
function of LPS-stimulated DC toward a Th2-type function.
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The effect of 1-MT can also be observed in a recall response
Since allogeneic MLR does not reflect an antigen-specific response, we examined the effect
of 1-MT treatment of DC in an autologous response to tetanus neurotoxin. As shown in figure
2, the results found in allogeneic MLR can be observed in an antigen specific response.
Without antigen, DC induced basal secretions of cytokines by T cells and 1-MT pre-treatment
of LPS-activated DC yielded cells with a reduced ability to stimulate IFNγ secretion by T
cells, but with increased capacity to induce IL-2 and IL-5. These basal secretions are due to
the presentation of FCS antigens to T cells. When antigen is added, all secretions are
increased and the impact of 1-MT pre-treatment on LPS-stimulated DC is more striking,
inducing a strong reduction of DC ability to stimulate IFNγ secretion by T cells. This
treatment also tends to increase the ability of DC to induce IL-13 secretion by T cells, but this
is not as obvious as for IL-5 or IL-2.
These results indicate that 1-MT modulates the capacity of mature DC to stimulate memory
responses, suggesting that this molecule could have an impact in vivo on immune responses to
pathogens.
Effect of 1-MT on phenotypic maturation and cytokine secretion by DC
DC stimulated by LPS, pIC or PGN showed a classic phenotype of mDC with a strong
induction of CD86 and CD40 (Fig. 3) as well as CD80, CD83 and HLA-DR (data not shown).
Pam was a weaker inducer of phenotypic maturation of DC. 1-MT pre-treatment had no effect
on phenotypic maturation induced by pIC or Pam. Obvious effects of 1-MT were observed
when DC maturation was induced by PGN and LPS. Indeed, for PGN- or LPS-matured DC,
the expression of maturation markers was reduced by 1-MT pre-treatment although these
markers were still expressed at high level (Fig. 3). An intermediate expression was also
observed for CD80, CD83, CD54, HLA-DR (data not shown). All cells were CD14- CD1a+
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whatever the treatment and 1-MT alone did not affect the phenotype of iDC (Fig. 3E and data
not shown).
Cytokine secretion of DC treated or not with 1-MT and stimulated with the different TLR
ligands was then examined. pIC and Pam induced weak cytokine secretions and 1-MT pre-
treatment had only minor effects on these secretions (table II). As expected, LPS and PGN
were strong inducers of all the cytokines tested, except for IL-12p70 which was only induced
by LPS. 1-MT pre-treatment of LPS or PGN-stimulated DC was characterized by a strong
reduction in secretion of IL-6, IL-10, IL-12p70 and TNFα (table II). In all conditions, cells
remained negative for IFNγ and IL-1β secretion and DC viability was not modified (data not
shown). 1-MT alone did not affect the basal level of cytokine secretion by iDC (table II).
The overall data indicate that 1-MT pre-treatment of DC can interfere with the phenotypic
maturation and the cytokine secretion depending on TLR signaling.
Inhibition of IDO activity is not sufficient to change DC polarization
IDO mRNA was weakly detected in iDC while LPS, pIC and PGN strongly increased its
transcription. Pam was a weak inducer of IDO transcription (Fig. 4A). Treating DC with 1-
MT did not affect the induction of mRNA by the different TLR ligands (data not shown).
Increased expression of IDO by LPS and pIC treatment correlated with an enhanced
enzymatic activity that was measured by the production of the Trp catabolite kynurenine in
culture supernatants (Fig. 4B). Pretreatment of DC with 1-MT before activation with LPS or
pIC blocked the induction of IDO activity which remained at basal level. Induction of IDO
activity by Pam and PGN was weak and may not be significant, especially in regards of the
lack of inhibition by 1-MT following PGN stimulation. No IFNγ secretion was detected in DC
culture supernatants, confirming that IDO induction can be IFNγ-independent.
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Kynurenine is the main catabolite of Trp and is involved in the inhibition of T cell
proliferation (18, 22). Since its production is inhibited by 1-MT, we asked whether addition of
kynurenine during the pre-treatment of DC with 1-MT could restore a normal phenotypic and
functional maturation induced by LPS. Figure 4C shows that kynurenine had no effect on the
allostimulatory function of LPS-stimulated DC and could not inhibit the Th2 shift induced by
1-MT. Kynurenine had no effect on phenotypic maturation and cytokine secretion of LPS-
stimulated DC (Fig. 4C and data not shown). IDO activity also results in Trp deprivation. An
excess of Trp did not mimic the effect of 1-MT on DC function, indicating that Trp
concentration did not regulate DC maturation mediated by TLR4 signaling (Fig. 4C). 1-MT is
a competitive inhibitor of Trp for IDO, however 1-MT action on LPS-stimulated DC was not
suppressed by an excess of Trp that could displace 1-MT from the enzyme (Fig. 4C).Thus all
these results strongly suggest that the effect of 1-MT on TLR signaling in DC is independent
of IDO activity.
p38-MAPK, ERK and c-Fos in DC polarization
p38-MAPK and ERK have been shown to play a role in DC maturation and in the type of T
cell response they can elicit (45-48). We thus asked whether these pathways could be
differentially engaged when DC were pre-treated with 1-MT before activation with the
different TLR ligands. The functional consequences of specific inhibitors of these two kinases
on DC maturation was therefore examined. SB203580 (SB) is a specific inhibitor of p38-
MAPK and PD98059 (PD) inhibits MEK activation thus preventing ERK phosphorylation.
Addition of SB before LPS maturation yielded DC that could not induce IFNγ secretion by
allogeneic T cells, confirming the involvement of p38-MAPK in LPS-induced maturation of
DC (Fig. 5A). Secretion of IL-5 and IL-13 by T cells was not affected and remained at its low
basal level. Allogeneic T cells cocultured with DC pre-treated with PD and LPS secreted
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similar amounts of IFNγ, IL-5 and IL-13 compared to T cells cocultured with control mDC,
suggesting that the MEK/ERK pathway is not essential in LPS-induced maturation (Fig. 5B).
The effect of both inhibitors was then investigated on DC pre-treated with 1-MT before
stimulation with LPS. As expected, in presence of SB, DC pre-treated or not by 1-MT were
not able to induce IFNγ secretion by T cells. However, in the presence of SB, 1-MT lost its
ability to generate DC that could induce IL-5 and IL-13 secretion by T cells (Fig. 5C,D).
Treating DC with both PD and 1-MT before stimulation with LPS restored their ability to
induce IFNγ secretion by T cells, suggesting that the MEK/ERK pathway is involved in the
inhibition of Th1-type responses by 1-MT (Fig. 5E). In contrast, the MEK/ERK pathway did
not seem to regulate the ability of DC to induce IL-13 and IL-5 secretion by T cells (Fig. 5F).
The data suggest that ERK and p38-MAPK can interfere with DC polarization and may be
involved in the shift of DC function induced by 1-MT in LPS-activated DC.
Therefore we looked directly at the activation of these two kinases triggered by the different
TLR ligands. Activation of p38-MAPK and ERK pathways was determined by the ratio of
phosphorylated enzyme to total enzyme by ELISA. In iDC, the treatment with 1-MT alone
does not activate ERK and p38 (Fig. 6A,B). All the TLR ligands increased the ratio of
phosphorylated p38 to total p38 although with different strength and kinetics. The pre-
treatment of DC with 1-MT did not modify the outcome of p38 activation induced by TLR
stimulation with pIC while it slightly reduced the level of activation induced by TLR2/6
stimulation without affecting the kinetics (Fig. 6A). The activation induced by Pam was
delayed when cells were pre-treated with 1-MT. The most striking differences were observed
with LPS stimulation. p38 phosphorylation still occurred at 5 minutes but was followed by a
second and more important peak at 15 minutes (Fig. 6A).
Like for p38, 1-MT pre-treatment had no effect on ERK phosphorylation following pIC
stimulation. ERK phosphorylation was reduced following PGN signaling or delayed
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