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EMBO reports 5, 2, 172–177 (2004)
doi:10.1038/sj.embor.7400078
AOP Published online: 23 January 2004
Toll-like receptors differentially induce nucleosome remodelling at the IL-12p40 promoter
Inka Albrecht1, 2, Thomas Tapmeier1, 2, Stefan Zimmermann1, Markus Frey1, Klaus Heeg1 & Alexander Dalpke1
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1 Institute of Medical Microbiology and Hygiene,
Philipps-University Marburg, Pilgrimstein 2, D-35037 Marburg,
Germany
2 These authors contributed equally to this work
To whom correspondence should be addressed
Klaus Heeg Tel: +49 6421 286 6453; Fax: +49 6421 286 6420;
E-mail: heeg@med.uni-marburg.de
Received 13 August 2003; Accepted 5 December 2003; Published online 23 January 2004.
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Abstract
Toll-like receptors (TLRs) mediate recognition of microbial
components. Despite activation of a shared set of signal transduction
molecules, the biological effects of certain TLR agonists differ considerably.
In macrophages and dendritic cells, stimulation by the prototypical stimuli
CpG-DNA (TLR9), lipopolysaccharide (LPS; TLR4) and lipoteichoic acid (LTA;
TLR2) resulted in striking differences in expression of IL-12. However, these
stimuli induced similar amounts of the common proinflammatory cytokine
TNF . Surprisingly, an IL-12p40 promoter reporter construct was activated
equally by CpG-DNA, LPS and LTA. Examinations of the chromatin structure of the
endogenous IL-12p40 promoter revealed that nucleosome remodelling contributed
to differential IL-12 induction. Upon stimulation, nucleosome architecture was
changed to provide increased access to the IL-12p40 promoter. In dendritic
cells, a differential induction of nucleosome remodelling at the IL-12p40
promoter was observed upon triggering with different TLR agonists. These
results identify nucleosome remodelling as an additional restriction point in
differential TLR signalling.
EMBO reports 5, 2, 172–177 (2004)
doi:10.1038/sj.embor.7400078
AOP Published online: 23 January 2004
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Introduction
Detection of pathogens through the innate immune system critically
involves Toll-like receptors (TLRs). TLRs recognize major bacterial components
such as lipopolysaccharide (LPS), lipoteichoic acid (LTA) or bacterial CpG-DNA.
Receptor activation by different TLR agonists leads to the activation of a
shared signalling pathway. However, the biological effects of different TLR
agonists are quite diverse, suggesting additional TLR-specific signalling
mechanisms.
An example of specific immunostimulation is the peculiar capacity of
TLR9 to induce TH1 immune responses. Interleukin-12 (IL-12) is one
of the most important cytokines of innate immunity that shapes the subsequent
adaptive immune response and especially influences
TH1/TH2 commitment. IL-12 is composed of two subunits
(p35, p40) of which p40 is highly regulated in antigen-presenting cells (APCs).
It has been revealed that IL-12 gene expression is tightly controlled at the
transcriptional level. Control elements within the IL-12p40 promoter have been
identified by reporter gene assays (Murphy et al,
1995; Plevy et al, 1997). In this
respect, a Rel/NF B element was found to be most important for promoter
activity stimulated by IFN and LPS. In addition, proteins belonging to
the IFN regulatory factor (IRF) family, such as IFN consensus sequence binding
protein (ICSBP) and IRF-1, have a significant role in regulating IL-12
expression (Salkowski et al, 1999;
Wang et al, 2000).
Recently, an additional mechanism of regulation has been reported. It
has been shown that the endogenous IL-12p40 promoter is assembled in four
tightly positioned nucleosomes. Nucleosomes are the higher-order structure of
chromatin and represent an obstacle to the binding of transcription factors.
Nucleosome 1 within the p40 gene promoter encompasses the critical Rel and
C/EBP elements. Upon cellular activation, chromatin modifications take place
and in the case of the IL-12p40 promoter this results in remodelling of the
structure of the first nucleosome (Weinmann et al,
1999). As a consequence, the locus is more accessible and now allows
binding of the respective transcription factors. The results of footprinting
technology also corroborate these findings (Becker et
al, 2001). However, the impact of IL-12p40 nucleosome remodelling
on the outcome of various TLR-dependent stimuli is unknown.
Results
Differential induction of IL-12 by CpG-DNA, LPS or
LTA
In order to examine differences in the cytokine response patterns of
different TLRs, we tested the capacity of prototypical TLR ligands to induce
IL-12 in dendritic cells. For comparison, we also determined TNF, which
has been described as a target gene induced through the common MyD88-dependent
pathway for most of the TLR ligands described so far. In Fig
1, it is shown that CpG-DNA, LPS and LTA induced TNF in a
dose-dependent way. However, CpG-DNA was far more effective in the equivalent
secretion of IL-12p70 as compared to LPS. Moreover, LTA was not capable of
inducing significant amounts of IL-12. Similar results were also observed with
peritoneal macrophages and RAW264.7 macrophages, albeit with lower IL-12
induction (data not shown). For the following experiments, we decided to use
concentrations of the different TLR stimuli that induced similar amounts of
TNF. Differences in IL-12 protein secretion were also reflected by
different IL-12p40 mRNA induction as examined by quantitative reverse
transcription (RT)-PCR (data not shown).
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Figure 1
Differential induction of IL-12p70 by TLR-dependent stimuli.
Bone-marrow-derived dendritic cells were stimulated with CpG-DNA, LPS or LTA as
indicated for 24 h. TNF and IL-12p70 were determined in supernatants by
ELISA.
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Comparable activation of IL-12p40 reporter
constructs
To examine whether the observed differences in p40 induction in
response to different TLR agonists are due to differential activation of
required transcription factors, we made use of p40 reporter gene assays.
Surprisingly, although RAW264.7 macrophages only showed minor IL-12p40 protein
secretion after stimulation with LTA, a luciferase reporter plasmid of the
IL-12p40 promoter (-703/+54) was efficiently induced by LTA in
transient transfection assays (Fig 2A). Moreover, no
major differences in the activation of this reporter by CpG-DNA, LPS and LTA
were observed. Similar results were obtained when cells with a stably
integrated construct were used (Fig 2B). Again, all three
stimuli were capable of activating the reporter construct. In contrast, only
CpG-DNA induced secretion of endogenous IL-12p40 protein in the same cells
(Fig 2C). Thus, the endogenous IL-12p40 promoter locus
must be controlled by additional mechanisms.
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Figure 2
IL-12p40 promoter activation by TLR agonists. (A) RAW264.7
cells were transiently transfected with an IL-12p40 promoter reporter plasmid.
After 24 h, cells were stimulated with 100 nM CpG-DNA, 100 ng/ml LPS or 30
 g/ml LTA for 8 h. Subsequently, reporter gene activation was measured and
is expressed relative to nonstimulated cells. Three independent transfections
were performed. (B) RAW264.7 cells stably expressing the IL-12p40
promoter reporter plasmid were stimulated as above for the indicated time
periods. (C) The same cells as in (B) were assayed for endogenous
IL-12p40 protein after 24 h of stimulation.
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Equal activation of common transcription factors
For further insight into differences in IL-12p40 regulation after
TLR triggering, we investigated the activation of transcription factors with a
known role in IL-12p40 induction. First, CpG-DNA, LPS and LTA were equally
effective in inducing an NFB-dependent luciferase reporter plasmid
(Fig 3A). With respect to MAP kinase activation, we only
observed differences in JNK activation, with LPS being more effective and LTA
showing nearly no activation (Fig 3B). However, these
differences did not correlate with differential capacity to induce IL-12p40.
There were no differences in the total amounts of the respective kinases (data
not shown). IRF1 has also been reported to contribute to IL-12p40 secretion.
Using IRF1-deficient mice, we observed a dramatic decrease in IL-12p40
secretion and also diminished TNF production (Fig 3C).
However, the differences in p40 secretion upon CpG-DNA, LPS or LTA stimulation
were still observed, albeit in an attenuated way. Finally, ICSBP induction was
determined by quantitative RT-PCR, yet no differences between CpG-DNA, LPS and
LTA triggering were obvious (Fig 3D).
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Figure 3
Signal transduction by CpG-ODN, LPS and LTA. (A) RAW264.7
cells were transiently transfected with an NFB-dependent reporter
plasmid. Cells were stimulated with 100 nM CpG-ODN, 100 ng/ml LPS or 30
g/ml LTA for 7 h, and luciferase induction was determined. (B)
RAW264.7 cells were stimulated as above for 15 min. Similar amounts of cellular
extracts were blotted with phosphospecific antibodies for p38, JNK or ERK MAP
kinase. (C) Peritoneal macrophages from
IRF1-/- and IRF1+/- mice
were stimulated as above. Supernatants were assayed for TNF and
IL-12p40. (D) RAW264.7 macrophages were stimulated as above for the
indicated time periods. ICSBP mRNA expression was measured by quantitative
RT-PCR.
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TLRs differentially reduce nucleosome remodelling
To test the hypothesis that differences in chromatin regulation
contribute to the differential capacity of various TLR agonists to induce
IL-12p40, we focused on inducible changes in nucleosomal architecture. To
measure nucleosome remodelling, we made use of a restriction enzyme
accessibility assay combined with modified ligation-mediated PCR (LM-PCR)
(Fig 4A). With this approach, we were able to show that
in dendritic cells the promoter is not accessible under starting conditions.
However, upon stimulation, restriction enzyme accessibility increases, and
differences between stimulation of CpG-DNA, LPS and LTA can be observed (Fig 4B). Although all of the stimuli were able to induce
nucleosome remodelling, CpG-DNA induced more prominent changes, which
corroborates the observed differences in IL-12p40 protein secretion. Using the
same assay with stimulated macrophages, we only observed a remodelling upon
challenge with CpG-DNA (supplementary fig
S1 online).
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Figure 4
Differential nucleosome remodelling at the IL-12p40 promoter in
response to TLR-dependent stimuli. (A) Schematic drawing of the
structure of the IL-12p40 promoter. Half arrows indicate primer sets for two
ChART PCRs. One is spanning the restriction enzyme recognition site for
MseI, and the second amplifies outside this region. Straight lines
indicate positions of the TaqMan probes for both these primer sets. In
addition, the IL-12p40 promoter-specific primer used in the LM-PCR is indicated
(arrow). (B) Bone-marrow-derived dendritic cells were examined for
changes in nucleosome structure by restriction enzyme accessibility followed by
LM-PCR. Cells were stimulated with 100 nM CpG-ODN, 100 ng/ml LPS or 10 g/ml
LTA for 8 h. Arrows indicate the amplicons either generated by accessibility
for MseI or XbaI as a normalization. Positive controls were
obtained by digestion of purified DNA with MseI (negative image).
(C) Bone-marrow-derived dendritic cells were stimulated as above for 2
h, and nuclear extracts were tested for restriction enzyme accessibility by
ChART assay. Remodelling is plotted in percentage of nonstimulated cells.
(D) RAW264.7 cells with a stably integrated IL-12p40 reporter plasmid
were tested for MseI accessibility by modified ChART assay. To this were
added reverse primers that specifically hybridized either within the endogenous
IL-12p40 gene or the luciferase reporter gene. Nuclear extracts of
nonstimulated cells (nucl. extract) were treated with MseI and
accessibility was measured as normalized expression of the specific amplicons
in relation to the normalization PCR. As controls, genomic DNA (gDNA) was left
untreated or completely digested (pos).
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To strengthen the evidence and to verify the observed differences in
TLR-mediated IL-12p40 promoter remodelling, we next measured remodelling
quantitatively. For this, we made use of a restriction enzyme accessibility
assay quantified by real-time PCR (published as ChART). Three quantitative PCRs
were set up (Fig 4A). The first PCR product spanned the
expected cleavage site. A second PCR amplified a region outside the cleavage
site within the promoter and finally an amplicon of the housekeeping gene
 -actin was determined. The last two PCRs were used for normalization of
possible differences during the DNA preparation.
Using this assay, we were able to quantify changes in nucleosome
remodelling at the IL-12p40 promoter upon TLR triggering. Normalized changes in
the amounts of the reporter PCR were measured and decreases were expressed as
remodelling in comparison to nonstimulated cells. A complete loss of the PCR
amplicon indicated full accessibility and thus 100% remodelling. In
dendritic cells, nonstimulated cells showed similar or only slightly higher
threshold values in the PCR reaction as with uncut genomic DNA. Thus, under
starting conditions, the promoter was indeed nearly completely protected.
However, upon stimulation, CpG-DNA induced a remodelling of about 74%,
with LPS (49%) and LTA (22%) again being less effective (Fig 4C). Remodelling was dependent on the presence of the CpG
motif, as control DNA did not induce nucleosome remodelling (supplementary fig S2 online). No significant
remodelling was observed within the upstream nucleosome 2 (supplementary fig S3 online). Finally, the RAW264.7
macrophages with the stably integrated IL-12p40 reporter construct were assayed
for restriction enzyme accessibility. We were able to show that indeed the
endogenous locus is completely protected, whereas the integrated reporter
plasmid is largely accessible (sixfold decrease upon restriction enzyme
digestion) under nonstimulated conditions (Fig 4D).
Discussion
So far, cellular activation by different TLR agonists has been
reported to induce and activate a set of shared intracellular signal
transduction molecules (O'Neill, 2002).
Despite similar signal transduction mechanisms, different TLR agonists can be
distinguished in their biological effects and this could be due in part to
TLR-specific signalling pathways, which have recently been described (Yamamoto et al, 2003). Of various TLRs, the specific
capacity of immunostimulatory CpG-DNA to activate TH1-dominated
immunity is impressive (Chu et al,
1997).
The present study extends these findings by using a comparative
approach to study the prototypical ligands of TLR9, TLR4 and TLR2. In
particular, we normalized the different compounds on their ability to induce
TNF, a common MyD88-dependent target gene. Indeed, all of the stimuli
were able to induce this cytokine in a similar way. However, they differed
markedly in their ability to induce IL-12p40. CpG-oligodeoxynucleotide (ODN)
proved to be the strongest inducer of p40 expression as compared to LPS or LTA.
This correlates with an earlier report (Cowdery et al,
1999) that showed a stronger increase of IL-12p40 mRNA and protein
after stimulation with CpG-DNA than with LPS. Moreover, a recent report also
demonstrates the inability of LTA to induce IL-12 in human cells (Hermann et al, 2002). The data clearly support the
concept of TLR-specific response profiles that enable the innate immune system
to generate pathogen-specific activation patterns.
IL-12 is apparently regulated differentially by various TLR agonists.
The promoter of the IL-12p40 gene has been shown to be located in specific
nucleosomes, with nucleosome 1 containing relevant transcription factor binding
sites (Weinmann et al, 1999). Upon
stimulation with LPS, increased accessibility of the promoter was reported, and
thus nucleosome remodelling seems to be an additional mechanism regulating
IL-12 induction. Here, we report that differences in IL-12p40 nucleosome
remodelling occur upon stimulation with various TLR stimuli, and this
correlates with differences in TLR-induced gene expression of IL-12. Although
differences in remodelling induced by TLR4 and TLR9 were only moderate, the
effects on IL-12 secretion were more prominent. Thus, it could be that a
certain threshold level of remodelling is necessary for robust gene
transcription. By now, further genes have been shown to be additionally
regulated by nucleosome remodelling (Rao et al,
2001; Holloway et al, 2003).
Moreover, the existence of immediately accessible genes as well as delayed
accessible promoters has been demonstrated, which suggests that changes in
chromatin architecture contribute to inducible inflammatory gene regulation
(Saccani et al, 2001). It is of general
interest that reporter gene assays, which are valuable tools for the
examination of gene regulation, did not reflect physiological regulatory
mechanisms. Thus, RAW264.7 macrophages which secrete IL-12p40 in low levels
only in response to CpG-DNA showed an equal activation of an IL-12p40 reporter
construct in response to all of the tested TLR agonists. Corroborating these
findings, we found that the integrated reporter plasmid was constitutively
accessible, whereas the endogenous IL-12p40 locus was protected.
An interesting observation was recently provided by Lomvardas & Thanos (2002), who were able to alter
artificially the nucleosome position in the IFN- promoter. Thereby, they
changed the kinetics of IFN- gene induction and furthermore they changed
the specificity of induction in HeLa cells.
Now, the unresolved question is the nature of the stimulus that
induces nucleosome remodelling. TLRs trigger an association with adaptor
molecules of the MyD88 family. Recently, TICAM/TRIF has been shown to be
another such adaptor in TLR4 signalling. It induces IFN--dependent genes,
thus providing a new signal quality (Oshiumi et al,
2003). However, the observed differences in IL-12 signalling were
observed quantitatively, and thus the exclusive activation of specific adaptors
seems to be unlikely. In this respect, spatio-temporal components in TLR
signalling could have a role. It is possible that the kinetics of activation of
transcription factors differ between various TLR agonists. Perhaps the
composition of NFB dimers changes over time and this affects the
activation of different genes. Indeed, TLR9 signalling differs from other TLRs
by the fact that activation occurs within the endosome where TLR9 is expressed
(Ahmad-Nejad et al, 2002).
With respect to the mode of activation of nucleosome remodelling, it
has been shown that c-rel is not involved despite the requirement for c-rel for
transcriptional activation (Weinmann et al,
2001). In addition, C/EBP proteins are also insufficient for
nucleosome remodelling. Moreover, our results make it unlikely that different
dimers of NFB induce nucleosome remodelling, because of the similar
activation of an NFB reporter. Also, neither IRF1 nor ICSBP seems to be
responsible for differential induction of IL-12p40. As binding of NFB to
its binding site in the promoter seems to be hindered by nucleosome 1, another
mechanism must precede and induce remodelling, thus permitting access of
further transcription factors.
Taken together, our data show a clear differential expression of
IL-12p40 mRNA and protein after stimulation with different TLR agonists, with
CpG-DNA inducing the strongest increase in IL-12 expression. This accords with
a more efficient chromatin remodelling of nucleosome 1 within the IL-12p40
promoter by TLR9, which is a prerequisite for subsequent transcription
initiation. Thus the data identify inducible nucleosome remodelling as an
additional restriction point in TLR signalling.
Methods
Cells and culture conditions. Bone-marrow-derived macrophages
and dendritic cells were isolated from BALB/c mice as described (Inaba et al, 1992). GM-CSF-differentiated dendritic
cells were harvested at day 9 and were positively sorted for CD11c expression
by MACS (Miltenyi BioTech, Bergisch Gladbach, Germany).
IRF1-/- mice were provided by M. Lohoff (Marburg,
Germany).
Reagents. Completely phosphorothioate-modified CpG-ODN 1668 was
purchased from TIB Molbiol (Berlin). Sequences of ODNs and primers are
available on request. LTA from Staphylococcus aureus was a kind gift
from S. Morath (Konstanz, Germany), and was shown to be TLR2 dependent
(Lehner et al, 2001). Highly purified LPS
from Salmonella minnesota was kindly provided by U. Seydel (Borstel,
Germany). Antibodies specific for phosphorylated MAP kinases were from Cell
Signaling Technology (Frankfurt, Germany). Cytokines were measured by
commercially available ELISA (Becton Dickinson, Heidelberg, Germany). mRNA
expression was quantified by real-time TaqMan PCR (Applied Biosystems, Germany)
by determining relative expression of target sequences to the endogenous
control -actin.
Reporter gene experiments. The IL-12p40 promoter (-703 to
+54 bp) coupled to a luciferase-encoding reporter gene was a kind gift
from H. Haecker (München, Germany). NFB reporter plasmid contained
six NFB-binding sites (GGGGAATTTCC). Transient transfections were
performed by electroporation of RAW264.7 cells. Stably transfected RAW264.7
cell clones were generated by co-transfection with a G418 resistance encoding
plasmid. Experiments were performed with one typical clone out of four.
Luciferase induction was measured with the LucLite-kit (Packard, Netherlands)
in a TopCountNXT.
Restriction enzyme accessibility assay. Cells (2 
106) were stimulated as indicated in the respective experiment.
Nuclei were prepared and tested for restriction enzyme accessibility
essentially as described (Weinmann et al,
1999). Digestion reaction was carried out with 25 U of MseI
for 10 min at 37°C. Subsequently, DNA was purified using the DNeasy Tissue
kit (Qiagen, Hilden, Germany).
Modified LM-PCR. LM-PCR was performed with the following
modifications (Garrity & Wold, 1992). For
normalization, 2 g of DNA from restriction enzyme accessibility assays was
additionally digested with XbaI, which cuts distal of MseI. Next,
two double-stranded linker primers that generate either an MseI or an
XbaI end were covalently linked to the sticky ends of
restriction-enzyme-digested DNA. Ligated DNA was used in a hot-start PCR with
an IL-12p40 promoter-specific primer and the universal linker primer as reverse
primers. PCR products were directly analysed in 2.5% agarose gels.
ChART. To quantify nucleosome remodelling, chromatin
accessibility measured by real-time PCR (ChART) assays was performed as
described for the IL-2 promoter (Rao et al,
2001). DNA from restriction enzyme accessibility assays was used in
three different quantitative PCR approaches using TaqMan technology. One primer
set spanned the restriction sites for MseI. A second primer set
amplified outside this region and a third primer set was for -actin
(Fig 4A). Both the latter primer sets were used for
normalization of DNA amounts, whereas the first primer set reported
accessibility of the locus. A decrease in the amount of amplicon generated by
the first set indicated increased accessibility and thus nucleosome
remodelling. Amounts of the reporter amplicon within the promoter were
normalized to the normalization amplicon within the IL-12p40 promoter or to
-actin. Subsequently, data were calculated and plotted as percentage of
nonstimulated cells.
Supplementary information is available at EMBO reports
online
(http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400078-s1.pdf).
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Acknowledgements
We thank Helene Bykow and Sandra Opper for excellent technical
support. This project was supported by the Deutsche Forschungsgemeinschaft
(He1452/2, He1452/4, Zi676/1) and the European Community
(QLK2-CT-2000-00336).
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