INFECTION AND IMMUNITY, Jan. 2008, p. 189–197
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 1
Mechanisms of Dexamethasone-Mediated Inhibition of Toll-Like
Receptor Signaling Induced by Neisseria meningitidis and
Trine H. Mogensen,1* Randi S. Berg,1,2Søren R. Paludan,2and Lars Østergaard1
Department of Infectious Diseases, Skejby Hospital, Aarhus, Denmark,1and Institute of Medical Microbiology and Immunology,
University of Aarhus, Aarhus, Denmark2
Received 21 June 2007/Returned for modification 29 July 2007/Accepted 6 October 2007
Excessive inflammation contributes to the pathogenesis of bacterial meningitis, which remains a serious
disease despite treatment with antibiotics. Therefore, anti-inflammatory drugs have important therapeutic
potential, and clinical trials have revealed that early treatment with dexamethasone significantly reduces
mortality and morbidity from bacterial meningitis. Here we investigate the molecular mechanisms behind the
inhibitory effect of dexamethasone upon the inflammatory responses evoked by Neisseria meningitidis and
Streptococcus pneumoniae, two of the major causes of bacterial meningitis. The inflammatory cytokine response
was dependent on Toll-like receptor signaling and was strongly inhibited by dexamethasone. Activation of the
NF-?B pathway was targeted at several levels, including inhibition of I?B phosphorylation and NF-?B
DNA-binding activity as well as upregulation of I?B? synthesis. Our data also revealed that the timing of
steroid treatment relative to infection was important for achieving strong inhibition, particularly in response
to S. pneumoniae. Altogether, we describe important targets of dexamethasone in the inflammatory responses
evoked by N. meningitidis and S. pneumoniae, which may contribute to our understanding of the clinical effect
and the importance of timing with respect to corticosteroid treatment during bacterial meningitis.
Bacterial meningitis is a severe acute infectious disease
which remains a serious condition, with considerable mortality
and morbidity worldwide. Among the most frequent causes of
bacterial meningitis are Streptococcus pneumoniae and Neisse-
ria meningitidis. It has been well established for decades that
immunopathology, i.e., the exaggerated activation of the host’s
immune response induced by bacteria or their products, plays
a major role in the pathogenesis of bacterial infection in the
central nervous system (9). This view is underscored by the fact
that mortality from bacterial meningitis, and from pneumococ-
cal meningitis in particular, has not declined significantly for
many years, even in the presence of appropriate antibiotic
treatment. Large clinical trials have demonstrated that early
treatment with glucocorticoids significantly reduces mortality
and morbidity from bacterial meningitis in children and, as
more recent reports have demonstrated, also in adults (11, 33,
40, 44). These results have led to the recommendation of
introducing adjuvant glucocorticoids together with antibiotics
for the early treatment of bacterial meningitis (44).
When pathogens enter the central nervous system, they rep-
licate and, in this process, expose microbial material to host
cells, which subsequently trigger an inflammatory response.
This response is mediated by cytokines such as tumor necrosis
factor alpha (TNF-?), interleukin-1 (IL-1), IL-6, IL-8, and
prostaglandins produced by macrophage-equivalent brain
cells, including astrocytes and microglia, as well as cerebral
capillary endothelial cells (13, 40). The resultant leukocyte
recruitment, meningeal inflammation, and increased perme-
ability of the blood-brain barrier, if not orchestrated and reg-
ulated tightly, may cause cerebral edema, increased intracranial
pressure, and neuronal injury (9).
The innate inflammatory immune response is of crucial im-
portance for the early containment of infection but, at the
same time, has the potential to result in immunopathology.
The final outcome of infection therefore depends on an intri-
cate balance between the pathogen and the host response. One
of the central components of the innate immune system is the
family of Toll-like receptors (TLRs). These pattern recogni-
tion receptors recognize evolutionarily conserved pathogen-
associated molecular patterns present on most types of micro-
organisms (19). Once TLRs are activated, they signal to the
host the presence of infection and trigger signaling cascades
leading to antimicrobial and inflammatory responses involving
both innate and adaptive immunity (19).
TLR ligand engagement results in intracellular signal trans-
duction, including activation of nuclear factor ?B (NF-?B) and
mitogen-activated protein kinases (MAPKs). The TLR-acti-
vated signaling pathways proceed through adaptor proteins
(most importantly MyD88) and lead to activation of the
MAPKs and the inhibitory ?B (I?B) protein kinase (IKK)
complex. IKK in turn phosphorylates I?B and targets it for
degradation, hence liberating NF-?B, which migrates to the
nucleus and activates transcription of target genes (2, 12, 20,
Glucocorticoids are widely used due to their potent anti-
inflammatory and immunosuppressive effects. However, the
molecular mechanisms behind these effects are very complex
and still not fully understood, although several targets have
been identified. Since glucocorticoids are known to interfere
with many signaling pathways and molecules involved in TLR
* Corresponding author. Mailing address: Department of Infectious
Diseases, Skejby Hospital, Brendstrupgaardsvej, DK-8200 Aarhus N,
Denmark. Phone: 45 89498499. Fax: 45 89498310. E-mail: trine.mogensen
?Published ahead of print on 15 October 2007.
signaling, it has been hypothesized that TLR signaling path-
ways may be important targets for glucocorticoid action and
may explain many of their anti-inflammatory and immunosup-
pressive effects (32). A number of levels at which glucocorti-
coids can exert their multiple anti-inflammatory effects have
been identified, including direct interaction with the transcrip-
tional machinery, interference with upstream signal transduc-
tion, and modulation of RNA stability (32). Briefly, glucocor-
ticoids bind specifically to the intracellular glucocorticoid
receptor ?, thereby promoting dissociation from heat shock
protein 90 and subsequent translocation to the nucleus, where
this ligand-activated transcription factor can activate the ex-
pression of genes with anti-inflammatory effects, including li-
pocortin, IL-1 receptor antagonist, IL-10, and I?B? genes (1,
4), through binding to glucocorticoid response elements (6).
Another major anti-inflammatory mechanism is glucocorti-
coid-mediated repression of a whole array of genes encoding
proinflammatory mediators, such as cytokines, chemokines,
and leukocyte adhesion molecules (32). This effect is mainly
achieved through direct protein-protein interactions between
the glucocorticoid-glucocorticoid receptor complex and the
transcription factors NF-?B and AP-1, thereby reducing or
preventing interaction with the essential coactivator CBP/p300,
resulting in inhibition of their transactivating potential. At the
level of signal transduction, glucocorticoids can inhibit up-
stream proinflammatory signaling through both the NF-?B and
MAPK pathways (32). Dexamethasone has been demonstrated
to inhibit activation of the MAPKs extracellular signal-regulated
kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), and p38 by a
mechanism involving upregulation and decreased degradation of
MAPK phosphatase 1, thereby preventing phosphorylation and
activation of these MAPKs (8, 15, 25, 43), and the NF-?B path-
way seems also to be targeted at a level upstream of I?B degra-
Here we investigate the effect and mechanism of action of
dexamethasone on the inflammatory responses evoked by N.
meningitidis and S. pneumoniae, two of the main causes of
MATERIALS AND METHODS
Cell culture. Peripheral blood mononuclear cells (PBMCs) were isolated from
blood obtained from healthy adult donors by Isopaque-Ficoll separation. The
blood was diluted, laid on top of Ficoll-Paque (Amersham Biosciences), and
centrifuged at 600 ? g for 30 min at room temperature. The PBMC-containing
interphase was isolated, and the cells were washed in phosphate-buffered saline
containing 100 ?g of heparin per ml. Subsequently, the cells were centrifuged at
200 ? g for 15 min at room temperature and resuspended in RPMI 1640 medium
containing 5% heat-inactivated fetal calf serum (FCS). The cells were seeded in
96-well tissue culture plates at a density of 2.0 ? 105cells per well and left
overnight to settle before further treatment. The human monocytic cell line
THP-1 was grown in RPMI 1640 medium supplemented with 10% FCS and
antibiotics. For experiments, cells were seeded in 96- and 6-well tissue culture
plates at densities of 2.0 ? 105and 4.0 ? 106cells per well, respectively, and left
for 2 h to settle before further treatment. The RAW 264.7-derived cell lines
RAW TNF-? 3? untranslated region (UTR) AU? and RAW TNF-? 3? UTR
AU? (16, 36, 46) were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% FCS, antibiotics, and 500 ?g/ml G418 (Roche, Basel,
Switzerland). For experiments, the cells were seeded in six-well tissue culture
plates at a density of 2 ? 105cells/well and left for 4 h before further treatment.
The cells stably contain a reporter system where chloramphenicol acetyltrans-
ferase (CAT) mRNA is expressed constitutively. In one of the cell lines, the 3?
UTR of the CAT-encoding mRNA was taken from wild-type TNF-? (RAW
TNF-? 3? UTR AU?), whereas in the other cell line, the 3? UTR was again
taken from TNF-?, but the AU-rich region (AUR) was mutated (RAW TNF-?
3? UTR AU?). The idea is that any observed differences in CAT protein levels
can be ascribed to the AUR in the 3? UTR, which is a major regulator of mRNA
Bacteria and reagents. The bacteria used were the N. meningitidis strain
NGO93 and the S. pneumoniae strain SK1013. The bacteria were grown over-
night in brain heart infusion broth with 10% Levinthal broth (Statens Serum
Institute, Copenhagen, Denmark), reaching a concentration of 18.0 ? 108?
2.2 ? 108bacteria per ml, as determined in a Thoma counting chamber.
Pam3CSK4, lipopolysaccharide (LPS; ultrapure from Escherichia coli O111:B4),
and oligodeoxynucleotide (ODN) M362 were all obtained from Invivogen (San
Diego, CA). TNF-? was purchased from R&D Systems. The MyD88 ho-
modimerization inhibitory peptide was obtained from Imgenex (San Diego, CA).
Dexamethasone was obtained from Pharmacia (Uppsala, Sweden), and cyclo-
heximide was obtained from Sigma-Aldrich (St. Louis, MO).
Purification of RNA and RT-PCR. Total RNA was extracted with TRIzol
(Invitrogen, Carlsbad, CA) according to the recommendations of the manufac-
turer. Briefly, cells were lysed in TRIzol, and chloroform was added, followed by
phase separation by centrifugation. RNA was precipitated with isopropanol and
pelleted by centrifugation. Pellets were washed with 80% ethanol and redissolved
in RNase-free water. For cDNA generation, 1 ?g of RNA was subjected to
reverse transcription (RT) with oligo(dT) as a primer and with Expand reverse
transcriptase (both from Roche). Prior to RT-PCR, RNA was treated with
DNase I (Ambion, Austin, TX) to remove any contaminating DNA, the absence
of which was confirmed in control experiments in which the reverse transcriptase
enzyme was omitted (data not shown). The cDNA was amplified by PCR using
the following primers: for IL-8, 5?-TTG TGA GGA CAT GTG GAA GC-3?
(forward) and 5?-ACA CAG CTG GCA ATG ACA AG-3? (reverse); for I?B?,
5?-CGG AAT TCC AGG CGG CCG AGC GCC CC-3? (forward) and 5?-GGG
GTA CCT CAT AAC GTC AGA CGC TG-3? (reverse); and for ?-actin, 5?-CCA
ACC GTG AAA AGA TGA CC-3? (forward) and 5?-GCA GTA ATC TCC TTC
TGC ATC C-3? (reverse). The primers were obtained from DNA Technology
Preparation of whole-cell extracts. To assay for phosphorylation of I?B?, p38,
and JNK, cells were seeded in six-well plates as described above and treated with
bacteria as specified in the text. At different time points poststimulation, cells
were lysed using a Bio-Plex cell lysis kit (Bio-Rad, Hercules, CA) according to
the recommendations of the manufacturer. Briefly, the cells were washed with 3
ml cell wash buffer per well and treated with 1 ml lysing solution supplemented
with phenylmethylsulfonyl fluoride, followed by incubation for 20 min at 4°C.
The suspension was centrifuged at 4,500 ? g for 20 min at 4°C, and supernatants
were harvested as whole-cell extracts.
Preparation of nuclear extracts. To isolate nuclear proteins, we used a nuclear
extraction kit (Active Motif, Carlsbad, CA). Briefly, cells were washed twice with
ice-cold phosphate-buffered saline supplemented with phosphatase inhibitors,
scraped off the plate, and spun down (2,000 ? g for 1 min) before resuspension
in 250 ?l 1? hypotonic buffer for 15 min on ice. Twenty-five microliters of the
supplied detergent was added, and the mixture was vortexed for 10 s and cen-
trifuged at 14,000 ? g for 30 s. The supernatants were removed, and 25 ?l of
complete lysis buffer was added to the nuclei, which were incubated for 30 min
at 4°C with rocking. The samples were vortexed and centrifuged at 14,000 ? g for
10 min at 4°C. Supernatants containing nuclear proteins were harvested and
transferred to new prechilled tubes.
NF-?B DNA-binding activity. Enzyme-linked immunosorbent assay (ELISA)-
based measurement of the DNA-binding activity of the nuclear NF-?B subunit
p65 was performed according to the manufacturer’s protocol (Active Motif,
Carlsbad, CA). Briefly, 5 ?g of nuclear extract was used per sample in duplicate
in a 96-well plate precoated with a consensus oligonucleotide for NF-?B (5?-G
GGACTTTCC-3?). After washing of wells to remove nonspecific binding, a
specific antibody to the NF-?B subunit p65 was added. After antibody binding,
the plate was washed again before adding a horseradish peroxidase-conjugated
secondary antibody. The peroxidase substrate was added, and the colorimetric
change was measured as the optical density at 450 nm.
Luminex technology for detection of cytokines and phosphoproteins. Cytokine
expression and phosphoprotein levels were measured using Luminex technology
and Luminex kits purchased from Bio-Rad (total I?B?, phospho-I?B?, p38,
JNK, and STAT2) and Biosource (Camarillo, CA) (human IL-8, IL-6, and
TNF-?). Briefly, the filter plates were washed with assay buffer, and freshly
vortexed antibody-conjugated beads were added to each well. The plate was
washed with assay buffer, and samples and standards were added. For detection
of cytokines, samples were diluted between 4 and 10 times in growth medium,
and for detection of phosphoproteins, 45 ?g of protein in 50 ?l lysis buffer was
used per sample. After a brief shake (30 s at 1,100 rpm), the plate was incubated
with shaking (300 rpm) either at room temperature in the dark for 45 min to 2 h
190MOGENSEN ET AL.INFECT. IMMUN.
(cytokine assay) or overnight at 4°C (phosphoprotein assay). After a wash step,
a detection antibody was added to each well, and the plate was shaken (300 rpm)
and incubated at room temperature in the dark for 45 min to 2 h (cytokine and
phosphoprotein assays). Subsequently, the plate was washed and incubated for
10 min with 50 ?l of a streptavidin-phycoerythrin solution, with shaking (30 s at
1,100 rpm and 10 min at 300 rpm). Finally, the plate was washed, 125 ?l of assay
buffer was added to each well, and the plate was shaken for 10 s at 1,100 rpm and
read immediately on a Bio-Plex reader.
Measurement of CAT levels. Cells were seeded and treated as described above
for the appropriate amount of time. For cell lysis and measurement of CAT
levels, a CAT ELISA kit from Roche (Basel, Switzerland) was used.
Statistical analysis. The data are presented as means ? standard errors of the
means (SEM). The statistical significance was estimated with the Wilcoxon rank
sum test. P values of ?0.05 were considered statistically significant.
Dexamethasone inhibits IL-8 production induced by N. men-
ingitidis and S. pneumoniae. In human PBMCs, N. meningitidis
and S. pneumoniae trigger a potent inflammatory response
involving several cytokines and chemokines (30). In an initial
set of experiments, we examined the ability of the two bacterial
species to induce the expression of cytokines. PBMCs were
treated with increasing doses of N. meningitidis and S. pneu-
moniae, and accumulation of IL-8 in the culture supernatants
was measured. Both bacteria strongly induced expression of
the cytokine, with N. meningitidis being slightly more potent
(Fig. 1A). In order to investigate the effect of dexamethasone
on proinflammatory cytokine production induced in the pres-
ence of these bacteria, PBMCs were seeded in culture and
treated as indicated in Fig. 1B to D. Both bacteria and the
purified TLR ligands (LPS for TLR4, Pam3Csk4for TLR2, and
ODN M362 for TLR9) induced IL-8, IL-6, and TNF-?, all of
which were strongly inhibited by dexamethasone (P ? 0.05 for
all stimuli). The inhibitory activity was not due to cytotoxic
FIG. 1. Dexamethasone inhibits induction of proinflammatory cytokines by N. meningitidis and S. pneumoniae. (A) PBMCs were seeded and
treated for 20 h with N. meningitidis or S. pneumoniae at increasing doses, from 9 ? 104bacteria/ml to 9 ? 107bacteria/ml. Supernatants were
harvested, and IL-8 levels were measured by Luminex technology. (B to D) PBMCs were treated with 1 ?M dexamethasone 1 h before stimulation
of the cells with LPS (100 ng/ml), Pam3Csk4(200 ng/ml), ODN M362 (1 ?M), or 9 ? 107bacteria/ml of N. meningitidis (N.m.) or S. pneumoniae
(S.p.). Twenty hours later, supernatants were harvested and cytokine levels were measured by Luminex technology. (E and F) PBMCs and THP-1
cells were treated with dexamethasone at doses from 1 nM to 1.0 ?M 1 h prior to stimulation with 9 ? 107bacteria/ml of N. meningitidis or S.
pneumoniae or with LPS (100 ng/ml). Twenty hours later, supernatants were harvested and cytokine levels were measured by Luminex technology.
The data are shown as means for triplicate cultures ? SEM. (G) Total RNA was harvested from THP-1 cells pretreated with dexamethasone (1
?M) for 1 h and treated with bacteria (9 ? 107bacteria/ml) for 5 h. IL-8 and ?-actin mRNAs were detected by RT-PCR. Similar results were
obtained in two or three independent experiments. UT, untreated cells.
VOL. 76, 2008DEXAMETHASONE AND TOLL-LIKE RECEPTOR SIGNALING 191
effects of the steroid, as assessed by trypan blue staining (data
not shown), and was also specific, since the ability of alpha
interferon to phosphorylate STAT2 was not modified by pre-
treatment with dexamethasone (data not shown).
Next, we preincubated PBMCs and the human monocytic
cell line THP-1 with different doses of dexamethasone 30 min
before the addition of N. meningitidis and S. pneumoniae. After
20 h of incubation, the supernatants were analyzed for IL-8. As
shown in Fig. 1E and F, both N. meningitidis and S. pneumoniae
induced large amounts of IL-8, which was inhibited in a dose-
dependent manner by dexamethasone. For N. meningitidis and
LPS, the cytokine response was inhibited significantly by all
concentrations of dexamethasone used (1 nM to 1 ?M),
whereas for S. pneumoniae significant inhibition was seen when
dexamethasone was present at concentrations from 10 nM to 1
?M. Finally, we examined IL-8 mRNA expression in THP-1
cells and found that dexamethasone inhibited expression of the
cytokine, although it exerted a less pronounced effect on S.
pneumoniae-induced IL-8 than on that induced by N. meningitidis
N. meningitidis and S. pneumoniae induce cytokine expres-
sion through a TLR-dependent mechanism. We previously in-
vestigated the pattern of TLR activation by bacteria causing men-
ingitis and found that N. meningitidis activates TLR2, TLR4, and
TLR9, whereas S. pneumoniae activates TLR2 and TLR9 (30).
Therefore, we hypothesized that bacterially induced IL-8 produc-
tion was TLR dependent. To test this, we inhibited TLR signal-
ing, using a cell-permeative MyD88 inhibitory peptide or a con-
trol peptide. MyD88 exists as a homodimer when recruited to
activated TLRs. The inhibitor peptide contains a sequence from
the MyD88 TIR homodimerization domain. MyD88 monomers
bind to this inhibitor peptide, thereby blocking MyD88 ho-
modimerization. The cells were treated with the peptide 24 h
prior to stimulation, and supernatants were harvested at 20 h
poststimulation for measurement of IL-8. As shown in Fig. 2, the
ability of both N. meningitidis and S. pneumoniae to induce IL-8
production was strongly inhibited in the presence of the MyD88
inhibitory peptide in both PBMCs (Fig. 2A) and THP-1 cells (Fig.
2B). The specificity of the MyD88 inhibitory peptide was shown
by its ability to inhibit IL-8 induction by LPS but not by TNF-?,
the latter of which signals independently of TLRs and MyD88
(26). Thus, the two bacteria induced cytokine expression largely
through a TLR-dependent mechanism.
Kinetics of dexamethasone-mediated inhibition of TLR sig-
naling. In clinical trials of dexamethasone treatment of pa-
tients with bacterial meningitis, timing has proved to be im-
portant for achievement of significant clinical effects (11). In
order to examine if the action of dexamethasone was also
dependent on the time of addition relative to the addition of
bacteria in our cell culture system and to gain further insight
into the mechanism of action of glucocorticoids, we examined
the consequences of dexamethasone added prior to, concom-
itant with, or following stimulation of PBMCs or THP-1 cells
with relevant bacteria (Fig. 3). N. meningitidis potently induced
IL-8 in both cellular systems, and this response was completely
abrogated when dexamethasone was added 2 h prior to or
concomitant with bacteria and partially inhibited when the
steroid was added at later time points. The inhibition was
highly significant even when dexamethasone was added at 7 h
postinfection. S. pneumoniae-induced IL-8 production was
strongly inhibited when dexamethasone was added between 2 h
FIG. 2. N. meningitidis and S. pneumoniae induce inflammatory
cytokine expression through TLRs. PBMCs (A) or THP-1 cells
(B) were seeded and treated with control peptide (Con pep.) or
MyD88 inhibitory peptide (100 ?M; MyD88 pep.) for 24 h before
stimulation for 20 h with 9 ? 107bacteria/ml of N. meningitidis (N.m.)
or S. pneumoniae (S.p.), 100 ng/ml of LPS, or 25 ng/ml of TNF-?.
Levels of IL-8 in the supernatants were measured by Luminex tech-
nology. The data are shown as means for triplicate cultures ? SEM.
Similar results were obtained in two independent experiments.
FIG. 3. N. meningitidis and S. pneumoniae display differential sen-
sitivity to timing of dexamethasone treatment with respect to induction
of cytokine expression. PBMCs (A) or THP-1 cells (B) were treated
with 1.0 ?M of dexamethasone at the indicated time points before
stimulation with 9 ? 107bacteria/ml of N. meningitidis (N.m.) or S.
pneumoniae (S.p.). At 20 h poststimulation, supernatants were har-
vested and levels of IL-8 were determined. The data are shown as
means for triplicate cultures ? SEM. Similar results were obtained in
two to four independent experiments.
192 MOGENSEN ET AL.INFECT. IMMUN.
before and 2 h after the bacteria were added. However, in
contrast to the pattern observed with N. meningitidis, dexa-
methasone was no longer able to mediate significant inhibition
of IL-8 production if it was added at later time points. These
results indicate that in the case of N. meningitidis, dexameth-
asone seems to have a maximal effect when it can interfere with
signaling at early time points but also has some potency when
included several hours after bacterial infection. With respect to
S. pneumoniae, dexamethasone similarly works at early time
points, fully inhibiting the IL-8 response, but does not have any
effect when added later than 2 h after treatment with bacteria.
Dexamethasone inhibits N. meningitidis- and S. pneumoniae-
induced TLR signaling by interfering with upstream signal
transduction. The data described above strongly suggested
that dexamethasone interferes with the bacterium-induced
proinflammatory response at an upstream level relatively soon
after bacterial challenge. Glucocorticoids are known to inter-
fere with several signaling pathways at different levels (1, 5, 7,
17, 37, 43). In subsequent experiments, we focused on the
transcription factor NF-?B, which we reasoned may be acti-
vated by these bacteria and also could represent relevant tar-
gets for dexamethasone action. THP-1 cells pretreated with
dexamethasone for 2 h were incubated for 2 h in the presence
of N. meningitidis or S. pneumoniae, and whole-cell extracts
were isolated and analyzed by Luminex technology to deter-
mine the levels of total and phosphorylated I?B?. As shown in
Fig. 4A, N. meningitidis and S. pneumoniae induced three- to
fourfold increases in I?B? phosphorylation. Although dexa-
methasone significantly inhibited the responses induced by
both bacteria, it exerted the strongest effect on N. meningitidis-
induced I?B? phosphorylation. Following I?B? phosphoryla-
tion, the inhibitory protein is ubiquitinated and degraded by
the proteasome pathway. Accordingly, total levels of I?B? in
cells decreased in response to treatment with bacteria or LPS
(Fig. 4B), and this was largely prevented if cells had been
pretreated with dexamethasone. Interestingly, we also ob-
served that dexamethasone treatment alone led to a significant
and reproducible induction of I?B? mRNA and protein (Fig.
4B and C). The final steps in NF-?B activation are transloca-
tion to the nucleus, binding to specific sites in gene promoters,
and activation of NF-?B-driven gene transcription. To inves-
tigate the effect of dexamethasone on the DNA-binding activ-
ity of NF-?B, we also harvested nuclear extracts from cells
pretreated with dexamethasone and stimulated with bacteria
for 2 h and subsequently measured DNA binding of the NF-?B
subunit p65. As shown in Fig. 4D, both bacterial species in-
duced the DNA-binding activity of p65, and as seen at the level
of P-I?B?, dexamethasone inhibited S. pneumoniae p65 DNA
binding less efficiently than that of N. meningitidis.
Taken together, the two different bacteria induced I?B phos-
phorylation followed by I?B degradation and NF-?B activation,
and dexamethasone inhibited N. meningitidis-induced signaling
more potently than that induced by S. pneumoniae, as already
observed at the level of IL-8 mRNA synthesis (Fig. 1G).
Effect of dexamethasone on bacterially induced cytokine
production is independent of de novo protein synthesis. The
findings above suggested that dexamethasone inhibits signaling
to NF-?B both by inducing synthesis of I?B? and by preventing
phosphorylation of this inhibitory protein. To examine if inhi-
bition of I?B? phosphorylation was dependent on de novo
protein synthesis or if dexamethasone was mediating this func-
tion independently of its activity as a transcription factor, we
treated cells with the protein synthesis inhibitor cycloheximide
and subsequently added dexamethasone, followed by bacteria
or LPS. Total cell lysates were isolated after 2 h of incubation
with bacteria, and the levels of phosphorylated I?B? were
determined. As also shown in Fig. 4A, we found that N. men-
ingitidis, S. pneumoniae, and LPS all induced I?B? phosphor-
ylation, which was significantly inhibited by dexamethasone
(Fig. 5). Importantly, the ability of dexamethasone to prevent
phosphorylation of I?B? was also observed in cells treated with
cycloheximide, thus suggesting that dexamethasone exerted
FIG. 4. Dexamethasone inhibits cellular signaling induced by bac-
teria at the upstream level. THP-1 cells were treated with 1.0 ?M of
dexamethasone 1 h before stimulation with 9 ? 107bacteria/ml of N.
meningitidis (N.m.) or S. pneumoniae (S.p.). At 2 h poststimulation, the
cells were lysed and whole-cell and nuclear extracts were harvested. (A
and B) Levels of total and phosphorylated I?B? in whole-cell extracts
were measured by Luminex technology. (C) Total RNA was harvested
from THP-1 cells either left untreated (UT) or incubated for 4 h in the
presence of 1.0 ?M dexamethasone. I?B? and ?-actin mRNAs were
amplified by RT-PCR and visualized by ethidium bromide staining of
agarose gels. (D) Nuclear extracts were analyzed for NF-?B p65 DNA-
binding activity. The data are shown as means for duplicate cultures ?
SEM. Similar results were obtained in two or three independent ex-
VOL. 76, 2008DEXAMETHASONE AND TOLL-LIKE RECEPTOR SIGNALING 193
this function independently of the induction of de novo protein
Bacterial infection enhances mRNA stability, which is not
affected by dexamethasone. In addition to being regulated at
the transcriptional level, many proinflammatory mediators are
regulated at the level of mRNA stability through AURs in the
3? UTR of the mRNA (22). We wanted to examine if the
ability of S. pneumoniae and N. meningitidis to stimulate ex-
pression of proinflammatory cytokines involves stabilization of
mRNA and if this is counteracted by glucocorticoids. For this
purpose, we used a previously reported system with two cell
lines derived from the macrophage-like cell line RAW264.7
(16, 36, 46). These cells were treated with dexamethasone 2 h
prior to incubation with bacteria, and cellular lysates were
prepared at 20 h postinfection. As shown in Fig. 6, the addition
of either bacterium led to an increase in the levels of CAT,
with S. pneumoniae being far more potent than N. meningitidis.
The ability of bacteria to stabilize TNF-? mRNA was largely
AUR dependent, although a minor stabilizing activity of S.
pneumoniae remained in the RAW TNF-? 3? UTR AU? cells.
Importantly, dexamethasone did not affect CAT expression,
regardless of which other stimuli were given. In separate ex-
periments, we found that dexamethasone did work in the cel-
lular system used, since LPS-induced expression of IL-6 and
TNF-? was strongly inhibited (data not shown). Thus, N. men-
ingitidis and, in particular, S. pneumoniae trigger AUR-depen-
dent stabilization of mRNAs, and this activity is not counter-
acted by dexamethasone.
The molecular mechanisms of dexamethasone action have
been studied extensively, and dexamethasone is known to in-
terfere with proinflammatory signal transduction, gene expres-
sion, and protein synthesis at various levels (32). However, the
precise targets of dexamethasone with respect to TLR-medi-
ated signaling and cytokine production are less well defined.
Since dexamethasone is widely used clinically to inhibit or
dampen inflammation, whether it is triggered by infectious
microorganisms or autoimmunity, and since TLRs play impor-
tant roles in inducing such inflammation, we were interested in
studying the molecular targets of dexamethasone-mediated in-
hibition of inflammation triggered by TLRs. In this study, we
therefore investigated how dexamethasone inhibits proinflam-
matory signaling induced by live N. meningitidis and S. pneu-
moniae, two leading causes of bacterial meningitis, as large
clinical trials have demonstrated the beneficial effect of early
treatment with dexamethasone (11).
It has been proposed that TLR signaling pathways are key
targets for the anti-inflammatory and immunosuppressive ef-
fects of glucocorticoids (32, 34, 35). Furthermore, LPS-induced
inflammation has been demonstrated to be inhibited by glu-
cocorticoids (27). We and others have previously demonstrated
that S. pneumoniae activates TLRs 2 and 9 (3, 30, 47), whereas
N. meningitidis signals through TLRs 2, 4, and 9 (18, 28, 30, 48).
In our experimental system, we found that the vast majority of
IL-8 produced in the presence of bacteria could be inhibited
when TLR signaling through MyD88 was blocked. Therefore,
the bacterially induced cytokine production observed was
largely mediated through TLRs. However, we cannot formally
exclude that the residual IL-8 measured in the presence of the
MyD88 inhibitor may be due to TLR-independent pathways
activated by the bacteria, which has indeed been described for
S. pneumoniae (23). The nature of potential TLR-independent
(or at least MyD88-independent) signaling mechanisms trig-
gered by bacteria remains unknown.
Aiming at understanding the molecular targets of dexameth-
asone in bacterially induced, TLR-mediated proinflammatory
signaling, we studied the diverse levels upon which dexameth-
asone might potentially act. First, we found that both I?B?
phosphorylation and degradation as well as NF-?B DNA-bind-
ing activity were potently inhibited by dexamethasone. The
finding that I?B? phosphorylation was affected strongly sug-
FIG. 5. Dexamethasone inhibits cellular signaling induced by bac-
teria, independent of de novo protein synthesis. THP-1 cells were
treated with cycloheximide (20 ?g/ml; CHX) 30 min before receiving
1.0 ?M of dexamethasone. One hour later, the cells were stimulated
with 9 ? 107bacteria/ml of N. meningitidis (N.m.) or S. pneumoniae
(S.p.) for 2 h, and whole-cell extracts were isolated. Phosphorylation of
I?B? was measured by Luminex technology. The data are shown as
means for duplicate cultures ? SEM. Similar results were obtained in
two independent experiments.
FIG. 6. TNF-? mRNA stability is increased by N. meningitidis and
S. pneumoniae through an AUR-dependent mechanism, which is not
affected by dexamethasone. RAW TNF-? 3? UTR AU? and RAW
TNF-? 3? UTR AU? cells were seeded and left for 4 h before the
addition of 1 ?M dexamethasone. Two hours later, 9 ? 107bacteria/ml
of N. meningitidis (N.m.) or S. pneumoniae (S.p.) were added to the
cells as indicated. Twenty hours later, the cells were lysed and CAT
levels were measured by ELISA. The data are shown as means ? SEM.
Similar results were obtained in three independent experiments.
194MOGENSEN ET AL.INFECT. IMMUN.
gests a target upstream of this molecule. This might be IKK as
well as other kinases or adaptor molecules involved in up-
stream TLR-mediated signaling (21). A previously described
mechanism of direct interaction of glucocorticoids with p65
may also be involved (1), but our experimental setup does not
allow us to determine the relative contributions of direct p65
inhibition and inhibition of I?B phosphorylation. Further-
more, we identified a separate mechanism by which dexameth-
asone itself induced increases in the levels of total cellular
I?B? mRNA and protein, which ultimately may result in bind-
ing and inhibition of free transcriptionally active nuclear or
cytoplasmic p65. For different experimental systems, similar
mechanisms have been reported, including glucocorticoid-in-
duced I?B? synthesis and upregulation of I?B? mRNA ex-
pression in the brain (5, 37). Finally, we observed that regard-
less of whether I?B? phosphorylation, NF-?B DNA binding,
or IL-8 production was measured, dexamethasone seemed to
exert a more powerful effect on N. meningitidis-induced activ-
ities than on those triggered by S. pneumoniae.
JNK and p38, both belonging to the family of MAPKs, are
prominent targets of dexamethasone as well (7, 17, 41, 43), but
since the THP-1 cell line did not activate the MAPKs in re-
sponse to either N. meningitidis or S. pneumoniae, the question
of a possible effect of dexamethasone on these MAPKs could
not be addressed.
Our data on the kinetics of dexamethasone-mediated inhi-
bition of cytokine production provide some additional insights
into the mechanisms of action of dexamethasone. First, there
may be several different mechanisms operating for dexametha-
sone-mediated inhibition of N. meningitidis-induced IL-8 pro-
duction, since the response was fully inhibited when dexameth-
asone was added prior to or concomitant with bacteria and
only partially inhibited at later time points. These results may
be explained by the existence of an early mechanism, involving
inhibition of upstream signaling as demonstrated, including
inhibition of I?B? phosphorylation and NF-?B DNA-binding
activity, where the presence of dexamethasone is required be-
fore or at least concomitant with bacterial challenge, and a late
mechanism, possibly involving induction or upregulation of
other cellular factors. The latter mechanism may be dependent
upon de novo protein synthesis. For instance, Imasato et al.
demonstrated that Haemophilus influenzae-induced upregula-
tion of TLR2 was enhanced by dexamethasone by a mechanism
involving upregulation of MKP-1, which in turn results in de-
phosphorylation and inactivation of p38 (17). However, in our
experimental system, the inhibitory action of dexamethasone
upon I?B? phosphorylation did not seem to be dependent
upon protein synthesis.
As suggested by our data, different inhibitory mechanisms
may exist, depending on whether inflammatory signaling is
induced by N. meningitidis or S. pneumoniae. Both bacteria
have in common that dexamethasone must be present before
or concomitant with bacterial challenge in order to have the
maximum effect, but we observed a clear difference with re-
spect to the effect at later time points, where dexamethasone
could partially inhibit IL-8 induced by meningococci, in con-
trast to the absence of any inhibitory effect exerted upon IL-8
induced by pneumococci. This idea is further supported by our
previous finding that N. meningitidis and S. pneumoniae use
distinct yet overlapping sets of TLRs, and possibly TLR-inde-
pendent signaling as well (30). Taken together, looking at I?B?
phosphorylation, NF-?B DNA-binding activity, and IL-8 pro-
duction, there is a tendency towards dexamethasone inhibiting
S. pneumoniae-induced inflammation to a lesser degree than
that for inflammation induced by N. meningitidis.
In addition to regulation at the level of transcription, control
of mRNA stability also represents an important level of regu-
lation of inflammatory gene expression (22). AURs in the 3?
UTRs of mRNAs are targeted by specific constitutive and
stimulus-regulated RNA-binding proteins capable of either
stabilizing or destabilizing the mRNA (10, 22). We found that
both N. meningitidis and S. pneumoniae stabilized TNF-?
mRNA in an AUR-dependent manner, with S. pneumoniae
being a particularly potent activator of this response. While it
has long been known that purified and synthetic TLR ligands
can stabilize mRNA through AUR elements (16, 38, 39), to
our knowledge this is the first report showing how live N.
meningitidis and S. pneumoniae interact with this important
step in the inflammatory response. Although glucocorticoids
have been reported to destabilize mRNAs for cyclooxygenase
2, monocyte chemoattractant protein 1, and inducible NO syn-
thase (24, 25, 39), we did not observe any effect of steroid
treatment on TNF-? mRNA stability, despite all these mRNAs
being regulated by AURs in the 3? UTR. This could indicate
that dexamethasone utilizes a mechanism that is not yet well
characterized to destabilize only a subset of transcripts bearing
the AUR signature.
In a large randomized clinical trial of dexamethasone treat-
ment of adults with bacterial meningitis, timing has proved to
be an important issue, since mortality and morbidity were
reduced only when dexamethasone was given prior to or con-
comitant with the initiation of antibiotic treatment (11). Fur-
thermore, subgroup analysis indicated that dexamethasone
may be particularly effective for patients with pneumococcal
meningitis (11, 29). In this study, we have identified some of
the cellular targets of dexamethasone in inflammatory signal-
ing induced by N. meningitidis and S. pneumoniae and, more
specifically, addressed the questions regarding the importance
of timing and the possible more important effect upon pneu-
Pneumococcal meningitis has the poorer prognosis with re-
gards to mortality and also carries a relatively great risk of
acquiring permanent sequelae. This may be due to the more
overwhelming nature of the S. pneumoniae-induced inflamma-
tory response, which even increases early after antibiotic treat-
ment, since penicillin enhances bacterial lysis mediated by
pneumococcal autolysin, hence liberating bacterial cell wall
components and DNA (14, 31), which activate TLRs and elicit
strong signals for enhanced inflammation. Similar mechanisms
may also be operating during meningococcal meningitis, al-
though to a lesser extent. Therefore, the early presence of
dexamethasone is required in order to prevent excessive in-
flammation, and this may be particularly important during
pneumococcal meningitis, for which our data indicate that
dexamethasone is indeed effective only at early time points.
Taken together, our results suggest that dexamethasone must
be given early to patients with bacterial meningitis in order to
inhibit TLR-dependent proinflammatory signaling and gene
expression, including inhibition at various levels of the tran-
scription factor NF-?B, as demonstrated in this study.
VOL. 76, 2008 DEXAMETHASONE AND TOLL-LIKE RECEPTOR SIGNALING195
This work was supported by grants from the LEO Pharma Research
Foundation, Kong Christian den Tiendes Fond, Beckett Fonden, and
The Danish Medical Research Council (grant 271-06-0438).
We thank Mogens Kilian for a critical reading of the manuscript and
invaluable discussions, as well as technicians Jonna Guldberg, Tove
Findahl, and Kirsten Stadel Pedersen for excellent assistance.
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Editor: A. Camilli
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