INFECTION AND IMMUNITY, Feb. 2010, p. 865–871
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 2
Toll-Like Receptor Stimulation Enhances Phagocytosis and
Intracellular Killing of Nonencapsulated and Encapsulated
Streptococcus pneumoniae by Murine Microglia?†
Sandra Ribes,1Sandra Ebert,1Tommy Regen,2Amit Agarwal,3Simone C. Tauber,1‡ Dirk Czesnik,4
Annette Spreer,1Stephanie Bunkowski,1Helmut Eiffert,5Uwe-Karsten Hanisch,2
Sven Hammerschmidt,6and Roland Nau1,7*
Department of Neurology,1Institute of Neuropathology,2Department of Neurophysiology and Cellular Biophysics,4and Department of
Medical Microbiology,5University of Go ¨ttingen, Go ¨ttingen 37075, Germany; Department of Neurogenetics, Max-Planck Institute of
Experimental Medicine, Go ¨ttingen 37075, Germany3; Institute for Genetics and Functional Genomics, Department of
Genetics of Microorganisms, Ernst-Moritz-Arndt-University, Greifswald, Germany6; and Department of Geriatrics,
Evangelisches Krankenhaus Go ¨ttingen-Weende, Go ¨ttingen 37075, Germany7
Received 1 October 2009/Returned for modification 1 November 2009/Accepted 14 November 2009
Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed
in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against
Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period.
Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam3CSK4), TLR4 (lipopolysaccharide),
and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2)
or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5
(RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming
microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria
than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton,
blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing
was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that
microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.
Immunocompromised patients have a higher risk of devel-
oping bacterial infections in the central nervous system (CNS)
(34, 37, 42). The list of the pathogens includes many organisms
with low pathogenicity in the immunocompetent host (34, 37).
Moreover, the distribution of the pathogens also differs from
the immunocompetent host and depends on the nature of the
immune defect. Patients with a decrease in B-lymphocyte func-
tion or with a loss of splenic function have an increased risk of
meningitis caused by encapsulated bacteria, while patients with
an impaired T-lymphocyte-macrophage system are more sus-
ceptible to CNS infections caused by intracellular pathogens
(7, 42). One additional cause of this increased susceptibility to
CNS infections probably is a decreased local immune defense
CNS infections not only are more frequent but also are
associated with higher mortality rates and more severe long-
term sequelae in immunocompromised than in immunocom-
petent individuals (9, 17, 34, 44). Polymicrobial infections, mul-
tiple organ system presentation, and the absence of typical
clinical manifestations subsequent to the host’s diminished in-
flammatory response are challenging aspects in the manage-
ment of these infections (34, 37, 42).
The brain tissue shows a well-organized innate immune re-
action in response to bacteria in the cerebrospinal fluid (CSF)
(3, 21). Microglial cells, the resident phagocytes of the CNS,
express Toll-like receptors (TLRs) that identify pathogen-as-
sociated molecular patterns (PAMPs) (41). The receptor-li-
gand interactions activate microglia to undergo morphological
transformation as well as functional changes, such as the pro-
duction of proinflammatory cytokines, chemokines, and reac-
tive oxygen species, enhanced phagocytic activity, and antigen
presentation (15, 39). This immune reaction cannot eliminate
high amounts of pneumococci from the CSF but does prevent
or minimize the invasion of these pathogens into the brain
tissue, thereby limiting tissue destruction and neuronal injury.
TLR2, -4, and -9 contribute to the recognition and response
to Streptococcus pneumoniae in the CNS (31). A deficiency of
TLR2, -4, or -9 or of the coreceptor CD14, which is necessary
for TLR4 signaling increases the susceptibility of mice to S.
pneumoniae (1, 11, 12, 40).
Here, we hypothesized that activation of the innate immune
response in microglia could increase the resistance of the brain
tissue against CNS pneumococcal infections (14). This may be
of particular interest in immunocompromised patients, whose
outcome after S. pneumoniae meningitis is worse than that of
immunocompetent individuals (9, 44). The aim of the present
study was to investigate whether the stimulation of microglia
by respective PAMPs can increase their ability to phagocytose
and kill intracellular nonencapsulated and encapsulated S.
* Corresponding author. Mailing address: Department of Geriatrics,
Evang. Krankenhaus Go ¨ttingen-Weende, An der Lutter 24, D-37075
Go ¨ttingen, Germany. Phone: 49 551 5034-1560. Fax: 49 551 5034-1562.
† Supplemental material for this article may be found at http://iai
‡ Present address: Department of Neurology, RWTH University,
52062 Aachen, Germany.
?Published ahead of print on 23 November 2009.
pneumoniae strains, thereby protecting the brain during men-
ingitis. Moreover, by using an encapsulated and a nonencap-
sulated pneumococcal strain, we assessed the protective effect
of the capsule against phagocytosis by microglial cells.
MATERIALS AND METHODS
Primary mouse microglial cell cultures. Primary cultures of microglial cells
were prepared from the brains of newborn C57/BL6N mice (1 to 3 days) as
previously described (10, 36). Microglial cells were isolated by shaking at 200
times/min for 30 min, and the cells in the supernatant were replated in 96-well
plates (for phagocytosis assay) and in 24-well plates (for intracellular survival
assay) at a density of 50,000 to 65,000 cells/well. In addition, microglia were
plated on poly-L-lysine-coated coverslips in 12-well plates for subsequent staining
and confocal microscopy at the same number of cells/well.
Microglial stimulation with TLR agonists. Cells seeded into 24- and 96-well
plates were exposed to one of the different TLR agonists for 24 h. Tripalmitoyl-S-
glyceryl-cysteine (Pam3CSK4; molecular mass, 910.5 Da; EMC Microcollections,
Tu ¨bingen, Germany), endotoxin (lipopolysaccharide [LPS] from Escherichia coli
serotype O26:B6; Sigma, Taufkirchen, Germany), and CpG oligodesoxynucleotide
(ODN) 1668 (TCC ATG ACG TTC CTG ATG CT; molecular mass, 6,383 Da;
TIB Molbiol, Berlin, Germany) were used as specific ligands of TLR1/2, -4, and
-9. A control group with unstimulated microglial cells was included in all exper-
iments. TLR agonists were used at the lowest concentrations inducing maximum
stimulation of microglial cells in terms of NO release (10): Pam3CSK4was tested
at 0.1 ?g/ml (0.1 ?M), LPS was tested at 0.01 ?g/ml (1 nM), and CpG was tested
at 1 ?g/ml (150 nM).
Supernatants from stimulated microglial cultures and unstimulated controls
were collected after 24 h of incubation and stored frozen at ?80°C until mea-
surement of the cytokine and chemokine levels. Microglial cells were assayed for
phagocytosis or intracellular survival by quantitative plating of intracellular bac-
teria or used for staining and subsequent confocal microscopy.
Cytokine and chemokine release. Interleukin-6 (IL-6) and CCL5 (RANTES
[regulated upon activation normal T-cell expressed and secreted]) were chosen
as representatives of the inducible spectrum of microglial cytokines and chemo-
kines (15). DuoSet ELISA development kits (R&D Systems, Wiesbaden, Ger-
many) were used for their measurement. The color reaction was measured at 450
nm on a microplate reader (Bio-Rad, Munich, Germany). The total protein content
was determined by using the MicroBCA protein assay (Pierce, Rockford, IL).
Bacterial strains, culture conditions, and protein purification. Streptococcus
pneumoniae strains D39 (encapsulated, serotype 2) and its nonencapsulated
derivative R6 were used in phagocytosis and intracellular survival assays. Pneu-
mococcal strains were grown in a medium consisting of Dulbecco modified Eagle
medium with Glutamax I (DMEM; Gibco, Karlsruhe, Germany) supplemented
with 10% heat-inactivated fetal calf serum (FCS).
The green fluorescent protein (GFP)-expressing strains D39gfp and its non-
encapsulated derivative D39gfp?cps were used for confocal microscopy to con-
firm the intracellular location of bacteria in microglial cells. The D39gfp strain
was grown in a medium consisting of DMEM supplemented with 10% heat-
inactivated FCS and 0.5 ?g of tetracycline/ml. The D39gfp?cps strain was grown
in DMEM supplemented with 10% heat-inactivated FCS, 0.5 ?g of tetracycline/
ml, and 50 ?g of kanamycin/ml. GFP-expressing D39 and D39?cps (35) were
generated by transformation of pneumococci with plasmid pMV158GFP (29).
The bacterial inoculum was determined for each assay by quantitative plating
on sheep blood agar plates.
Phagocytosis and intracellular survival assay. After 24 h of stimulation with
one TLR agonist, microglial cells were exposed to either S. pneumoniae D39 or
R6 (with a ratio of approximately 50 bacteria per phagocyte). Phagocytosis was
left to proceed for 30 or 90 min at 37°C and 5% CO2. For phagocytosis inhibition
studies cytochalasin D (final concentration, 10 ?M; Sigma-Aldrich, St. Louis,
MO) was added to the cell monolayers 30 min prior to the addition of bacteria
and remained present throughout the experiment (36). After bacterial exposure,
cells were incubated for 1 h in culture medium containing gentamicin (final
concentration, 200 ?g/ml; Sigma-Aldrich). After gentamicin incubation, the cell
monolayers were washed and lysed with distilled water. The intracellular bacteria
were enumerated by quantitative plating of serial dilutions of the lysates on
sheep blood agar plates. The limit of detection was 10 CFU/well. Each protocol
was performed at least three times in independent experiments. During the
phagocytosis assay, extracellular bacterial replication and gentamicin activity
were checked (36).
To monitor intracellular survival and replication inside microglia, cells were
allowed to phagocytose bacteria for 30 min. Thereafter, cells were washed and
incubated in culture medium containing gentamicin (200 ?g/ml) for 2 h. At
various times (30, 60, 90, and 120 min), the monolayers were washed and lysed
with distilled water, and the amounts of intracellular viable bacteria were quan-
Staining and confocal laser imaging of microglia. Scanning laser confocal
microscopy was used to confirm intracellular localization of the encapsulated
D39gfp and the nonencapsulated D39gfp?cps pneumococcal strain after coin-
cubation with microglia. Cells plated on coverslips in 12-well plates were exposed
to one of the different TLR agonists for 24 h. Thereafter, the cell monolayers
were washed and then incubated with a Vybrant DiI cell-labeling solution
(VybrantCell labeling solution kit; Molecular Probes, Leiden, The Netherlands)
for 3 min at 37°C according to the manufacturer’s instructions. Subsequently,
cells were washed twice with warm phosphate-buffered saline (PBS), and bacte-
ria were added for 30 min. For phagocytosis inhibition studies cytochalasin D was
added (see above). After 1 h of incubation with gentamicin, cells were washed
and fixed in 4% formaldehyde in PBS. The cells were imaged by using a laser-
scanning confocal microscope (Zeiss LSM 510 Meta). DiI and GFP S. pneu-
moniae strains were sequentially excited at 488 and 543 nm. Series of optical
sections in Z-plane were acquired at intervals of 0.6 ?m. Stacks of images were
processed by using ImageJ (version 1.43f). In order to illustrate the intracellular
localization of fluorescent bacteria, the z-planes (XZ and YZ) of the images were
depicted as orthogonal views. For better visualization of the fluorescent bacteria,
three-dimensional (3D) videos were generated by using the ImageJ plugin 3D
Viewer (by Benjamin Schmid) and are presented in the supplemental material
(Fig. S1 to S6).
Statistical analysis. Prism software (GraphPad Software, San Diego, CA) was
used to perform statistical analyses and graphical presentation. Analysis of vari-
ance (ANOVA), followed by Bonferroni’s multiple comparison test, was used to
compare enzyme-linked immunosorbent assay (ELISA) data among all groups.
The data from the phagocytosis and intracellular survival assays were not nor-
mally distributed and were analyzed by using the Kruskal-Wallis test, followed by
Dunn’s multiple comparison test to correct for repeated testing. A P value of ? 0.05
was considered significant.
TLR agonists stimulated microglia and induced cytokine
and chemokine release. In order to confirm effective microglial
stimulation by the different TLR agonists, we determined the
induction of representative cytokines and chemokines such as
IL-6 and CCL5 (Fig. 1). Microglial cells remained viable after
24 h of exposure to these agonists (35). In all experiments, a
group of unstimulated cells was included for comparison.
The supernatants of unstimulated microglia were devoid of
measurable amounts of IL-6 and CCL5. Microglial cells incu-
bated with the individual TLR agonists released much higher
amounts of IL-6 and CCL5 than did unstimulated cells (P ?
Confocal laser imaging confirmed the intracellular localiza-
tion of encapsulated and nonencapsulated pneumococci. Con-
focal microscopy confirmed the intracellular localization of the
encapsulated D39gfp and the nonencapsulated D39gfp?cps S.
pneumoniae strains within microglial cells. Bacteria expressing
GFP and microglia with their cell membrane labeled by red
Vybrant DiI were simultaneously visualized in two fluorescent
channels, as depicted in the reconstructed images of the z-
sections (Fig. 2). The animated 3D isosurface reconstructions
are provided as separate figures in the supplemental material.
The addition of cytochalasin D prior to the exposure to bac-
teria inhibited the internalization of pneumococcal strains
(Fig. 2C and F).
TLR stimulation increased the phagocytosis of S. pneu-
moniae D39 and R6 by microglia. The phagocytosis of D39 and
R6 pneumococcal strains was compared quantitatively after 30
and 90 min of incubation with bacteria in unstimulated cultures
866 RIBES ET AL.INFECT. IMMUN.
(control group) and in microglia that were previously stimu-
lated with the TLR1/2, TLR4, or TLR9 agonist (Fig. 3).
Although unstimulated cells ingested bacteria at a low rate,
stimulation with one TLR agonist increased the phagocytic
activity of microglia. Treatment with 1 ?g of CpG/ml resulted
in an increased uptake of both D39 and R6 strains at 30 and 90
min of exposure (P ? 0.001). After stimulation with 0.1 ?g of
Pam3CSK4/ml, the ingestion of the encapsulated D39 strain
was increased at 90 min (P ? 0.05), while phagocytosis of the
nonencapsulated R6 strain was enhanced at 30 and 90 min
(P ? 0.001). Treatment with 0.01 ?g of LPS/ml enhanced the
ingestion of the R6 strain at 90 min (P ? 0.05).
When we compared the amounts of phagocytosed pneumo-
cocci among the different TLR-stimulated groups, we found
that TLR1/2- and TLR9-stimulated cells phagocytosed compa-
rable numbers of bacteria (P ? 0.05 at 30 and 90 min). In
contrast, LPS-stimulated cells ingested lower numbers of both
encapsulated D39 (P ? 0.05 at 90 min versus TLR9-treated
cells) and nonencapsulated R6 strains (P ? 0.05 at 30 min
versus TLR1/2- and TLR9-treated cells).
The phagocytic rates were different for both strains: the
uptake of the nonencapsulated R6 strain was approximately 10
times more rapid than the internalization of the encapsulated
The internalization of both pneumococcal strains by micro-
glia occurred via phagocytosis. Cytochalasin D blocked the
uptake of S. pneumoniae D39 and R6 strains by ?90% in
unstimulated and TLR-stimulated cells, as it was revealed in
30-min phagocytosis inhibition studies.
The extracellular concentration of both pneumococcal
strains did not significantly differ throughout 90 min of incu-
bation either in experiments studying phagocytosis or in exper-
iments with phagocytosis inhibitors. After 1 h of gentamicin
treatment, the number of extracellular bacteria was below the
level of detection in all experiments.
TLR stimulation increased the intracellular killing of S.
pneumoniae D39 and R6 by microglia. Next, we studied
whether in TLR-stimulated microglial cells the increase of the
phagocytic activity was accompanied by a higher intracellular
killing of the ingested bacteria (Fig. 4).
The absolute amounts of killed S. pneumoniae D39 (calcu-
lated as the difference between the medians of intracellular
bacteria at 30 and 120 min) were higher in TLR-stimulated
microglia than in unstimulated cells (Fig. 4A). The time course
of intracellular killing of S. pneumoniae R6 strain was similar
to that of the encapsulated strain (Fig. 4B).
Streptococcus pneumoniae is an important cause of bacterial
meningitis causing death in ca. 25% of the cases and long-term
neurological sequelae in up to one-third of the survivors (9, 17,
38, 44). Proinflammatory and directly cytotoxic pneumococcal
products (such as pneumococcal cell wall products, pneumo-
lysin, and bacterial DNA) contribute to neuronal injury in S.
Microglial cells are the major constituents of innate immu-
nity within the CNS (20). Parenchymal microglia, as well as
meningeal and perivascular macrophages, which become acti-
vated by bacterial products are critically involved in protecting
the brain from infection (30, 33). On the one hand, microglial
cells can exert protective effects by phagocytosis of both patho-
gens and injured cells, and by mediating repair mechanisms
(20, 28). When MyD88 bone marrow chimeric mice were stud-
ied after intracerebral injection of Staphylococcus aureus, lack
of MyD88 expression in the CNS compartment led to elevated
intracerebral S. aureus burdens despite the presence of immu-
nocompetent bone marrow-derived cells (14). On the other
hand, activated microglial cells can be toxic to surrounding
neurons by releasing, e.g., nitric oxide, glutamate, TNF-?, and
IL-1?. The diminished inflammatory response decreased hear-
ing loss in pneumococcal meningitis in MyD88-deficient mice,
and neuronal injury caused by group B streptococci depended
on the presence of TLR2 and MyD88 (18, 22). Thus, activation
of microglia during infections seems to be a double-edged
sword. The innate immune response can protect neurons by
preventing the entry of pathogens into the brain, but its dys-
regulation can also be harmful for neuronal integrity and can
cause neuronal injury (6, 16, 20, 22, 28). Deeper understanding
of the roles for TLRs in resident CNS glia and infiltrating
immune cells will provide insights into how the immune re-
sponse to bacterial infection can be tailored to achieve effective
pathogen destruction without inducing excessive bystander
damage of surrounding brain parenchyma (13, 26).
In this context, we focused our research on the phagocytosis
of microglia activated by TLR stimulation. We hypothesized
that the activation of the TLR system in microglial cells by
FIG. 1. (A)IL-6and(B)CCL5(RANTES)concentrationsinthesuper-
natants of microglia after 24 h of stimulation with 0.1 ?g of Pam3CSK4/ml
(P3C), 0.01 ?g of LPS/ml, 1 ?g of bacterial CpG DNA/ml, or DMEM plus
10% FCS (unstim). The data are shown as means ? the standard deviation
(SD) (n ? 13 wells/group from three independent experiments). The data
were analyzed by using ANOVA, followed by Bonferroni’s multiple compar-
ison test (?, P ? 0.05; ??, P ? 0.01; ???, P ? 0.001).
VOL. 78, 2010TLR AGONISTS INCREASE S. PNEUMONIAE PHAGOCYTOSIS 867
FIG. 2. Phagocytosis of the encapsulated D39gfp (A to C) and the nonencapsulated D39gfp?cps (D to F) S. pneumoniae strains by murine
microglial cells after 30 min of bacterial exposure. Internal and external cell membranes were stained with red Vybrant DiI prior to the addition
of bacteria. Confocal images of microglial cells ingesting green fluorescent S. pneumoniae are shown in the x-y plane, as well as two z-axis (x-z and
y-z) cuts through (A and D) unstimulated cells and through microglia stimulated for 24 h with (B and E) 1 ?g of bacterial CpG DNA/ml. (C and
F) The addition of cytochalasin D (final concentration, 10 ?M) blocked the phagocytosis of S. pneumoniae strains by CpG-stimulated microglial
cells. Scale bars are shown in panel A, 5 ?m (x-y plane) and 2 ?m (x-z and y-z projected planes).
868RIBES ET AL.INFECT. IMMUN.
agonist stimulation may enhance their phagocytic activity,
thereby enabling them to protect the brain in pneumococcal
CNS infections in patients with an impaired immune system.
The release of cytokines and chemokines in the CSF during
pneumococcal meningitis has been analyzed. IL-6 is one of the
major early response cytokines that can trigger an inflamma-
tory cascade in pneumococcal meningitis (15). In many resi-
dent cells, such as microglial cells and astrocytes, chemokine
production is rapidly upregulated upon activation by stimuli
such as bacteria or inflammatory mediators (24, 32). An up-
regulation of the expression of CCL2, CCL5, and CXCL2
chemokines was observed in lungs, blood, and brain tissue after
intranasal inoculation of S. pneumoniae strains (serotypes 2, 4,
and 6A) in mice (25). In the present study, when microglia
were exposed to a TLR1/2, -4, or -9 ligand for 24 h, the release
of IL-6 and CCL5 was strongly increased, confirming micro-
Upon TLR stimulation, reactive microglia develop a phago-
cytic phenotype to engulf and kill microbes. In contrast to
cytokine and chemokine induction, the phagocytic and bacte-
FIG. 3. Phagocytosis of the encapsulated D39 (A) and the nonencapsulated R6 (B) Streptococcus pneumoniae (Spn) strains by murine
microglial cells after 24 h of stimulation with TLR agonists: Pam3CSK4(P3C, 0.1 ?g/ml), LPS (0.01 ?g/ml), or CpG DNA (1 ?g/ml). A control
group of unstimulated cells was included in all experiments. After stimulation, cells were washed and bacteria were added for different times (30
and 90 min). After addition of gentamicin (200 ?g/ml), the number of ingested bacteria was determined by quantitative plating of the cell lysates.
The data are shown as CFU of recovered bacteria per well (median, 75% interquartile range) (n ? 10 wells/group obtained from four independent
experiments). Statistical analysis was performed by using the Kruskal-Wallis test, followed by Dunn’s multiple-comparison test (?, P ? 0.05; and
???, P ? 0.001 versus the control group; #, P ? 0.05; and ##, P ? 0.01 versus the LPS-treated group).
FIG. 4. Time course of the number of live intracellular pneumococci (encapsulated D39 [A] and nonencapsulated R6 [B] Streptococcus
pneumoniae [Spn]) detected within microglial cells after 24 h of stimulation with the TLR agonists Pam3CSK4(P3C, 0.1 ?g/ml), LPS (0.01 ?g/ml),
or CpG DNA (1 ?g/ml). Monolayers were washed and allowed to ingest bacteria for 30 min. Then, gentamicin was added, and the amount of
intracellular bacteria was quantified by plating at several postinfection times for up to 120 min. For each group, intracellular killing is expressed
as the number of recovered bacteria (median) at the different time points (n ? 6 wells/group obtained from three independent experiments).
VOL. 78, 2010 TLR AGONISTS INCREASE S. PNEUMONIAE PHAGOCYTOSIS869
ricidal profiles of activated microglia have been explored less
thoroughly. Our group has recently reported that TLR1/2, -4,
and -9 agonists can increase the ability of murine microglial
cells to phagocytose and kill intracellularly located Escherichia
coli strains (36). The present data demonstrate that microglia
can also phagocytose and kill Gram-positive bacteria which
have a thicker cell wall and that stimulation of TLRs can
increase their phagocytic and bactericidal activity. This applies
for both nonencapsulated apathogenic and encapsulated patho-
genic pneumococci. Stimulation with either a TLR1/2, -4, or -9
agonist significantly increased the ability of microglia to phago-
cytose pneumococci. From our data, the effect of the stimula-
tion through the TLR9 system was clearly greater than the
effect caused via TLR1/2 or TLR4. Similarly, phagocytosis and
killing of live S. pneumoniae were found to be impaired in
alveolar and bone marrow-derived macrophages from TLR9-
deficient mice (1) and in blood-derived polymorphonuclear
leukocytes from TLR2-deficient mice (23).
Once bacteria have been phagocytosed, they are incorpo-
rated into phagolysosomes and exposed to reactive oxygen
species that eventually will result in bacterial lysis. The intra-
cellular killing of S. pneumoniae by microglial cells was more
rapid than that of E. coli studied in the same experimental
setting (36). For this reason, the number of viable intracellular
bacteria determined after 90 min of phagocytosis was lower
than the concentration of viable intracellular bacteria detected
after 30 min.
The presence of the polysaccharide capsule is an important
virulence factor of pneumococci because it decreases bacterial
uptake into microglia by more than 10 times (Fig. 3). In addi-
tion, we showed that the internalization of pneumococcal
strains by murine microglia requires intact actin filaments since
this process was blocked by ?90% by cytochalasin D (Fig. 2).
Not only the phagocytic but also the bactericidal activities of
reactive microglia depend on the stimulation of the TLR sys-
tem. In our study, plotting the intracellular bacterial concen-
tration versus time revealed higher absolute numbers of killed
bacteria in TLR-stimulated than in unstimulated microglia,
i.e., TLR stimulation clearly increased the efficacy of microglia
in neutralizing the internalized S. pneumoniae (Fig. 4).
An intact TLR signaling through the pathway organized by
MyD88 appears to be necessary to protect the brain tissue
against invading microorganisms. A poor outcome because of
high bacterial counts in the CNS and severe bacteremia was
observed in MyD88-deficient mice after intracisternal induc-
tion of pneumococcal meningitis (19). Similarly, MyD88?/?
mice showed an increased susceptibility to pneumococcal col-
onization within the upper respiratory tract, an enhanced bac-
terial proliferation in infected lung tissue, precocious bacterial
spread into the bloodstream, and increased mortality (2).
These findings illustrate the importance of an intact innate
immune system to efficiently limit the spread of S. pneumoniae.
Stimulation of the TLR system is a potential target for the
development of new therapies in multiple diseases (45). Sev-
eral TLR agonists are currently at different stages of clinical
trials (4). The TLR7 agonist imiquimod has been successfully
used and approved for the treatment of warts associated with
human papillomavirus and is in a second phase trial as a
therapeutic agent for herpes simplex virus (HSV) infections
(43). The TLR7/8 ligand resiquimod is also the subject of
clinical investigations for the treatment of HSV infections (27).
CpG DNA has been tested as a vaccine adjuvant showing good
results (8). One of the most interesting clinical trials with CPG
7909 has been recently completed and aimed at comparing the
in human immunodeficiency virus-infected adults (www.clinicaltrials
Therefore, the agonists used in the present study or related
compounds could be of value as adjuvants to improve the
efficiency of the local immune system of the CNS against bac-
teria. In the pharmacological administration of TLR agonists
as adjuvants, the dose, timing, and duration of the immuno-
therapy, as well as the route of administration, have to be
selected not only to maximize the benefit of the enhancement
of the immune response but also to restrict an excessive in-
duced response that might lead to autoimmune diseases or
increased neuronal injury (4).
One clear advantage of using TLR agonists as adjuvants for
the prophylaxis of bacterial meningitis is the low risk of devel-
opment of resistance to the compound. For microglial activa-
tion, agonists with a low molecular mass would be preferable
because of their higher penetration across the BBB (4). The entry
of LPS into the central nervous compartments is minimal (5).
In conclusion, stimulation of TLRs increases phagocytosis of
Gram-positive S. pneumoniae by microglia. Stimulation of the
TLR system may be a therapeutic approach to protect the
brain from invading pathogens. Further studies in immuno-
compromised mice are in progress in order to assess whether
the resistance of the brain against infections can be increased
by priming microglial cells with TLR agonists.
This study was supported by the European Union (grant CARE
PNEUMO), the Else Kro ¨ner-Fresenius-Stiftung (R.N. and A.S.) and
the SFB/TR43 (U.-K.H.). S.R. was the recipient of a fellowship from
the Departament d’Educacio ´ i Universitats de la Generalitat de
This work is dedicated to Viktor Papiol.
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