INFECTION AND IMMUNITY, Feb. 2010, p. 865–871 Vol. 78, No. 20019-9567/10/$12.00 doi:10.1128/IAI.01110-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Toll-Like Receptor Stimulation Enhances Phagocytosis andIntracellular Killing of Nonencapsulated and Encapsulated

Streptococcus pneumoniae by Murine Microglia�†Sandra Ribes,1 Sandra Ebert,1 Tommy Regen,2 Amit Agarwal,3 Simone C. Tauber,1‡ Dirk Czesnik,4

Annette Spreer,1 Stephanie Bunkowski,1 Helmut Eiffert,5 Uwe-Karsten Hanisch,2Sven Hammerschmidt,6 and Roland Nau1,7*

Department of Neurology,1 Institute of Neuropathology,2 Department of Neurophysiology and Cellular Biophysics,4 and Department ofMedical Microbiology,5 University of Gottingen, Gottingen 37075, Germany; Department of Neurogenetics, Max-Planck Institute of

Experimental Medicine, Gottingen 37075, Germany3; Institute for Genetics and Functional Genomics, Department ofGenetics of Microorganisms, Ernst-Moritz-Arndt-University, Greifswald, Germany6; and Department of Geriatrics,

Evangelisches Krankenhaus Gottingen-Weende, Gottingen 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 expressedin microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses againstStreptococcus 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, confirmingmicroglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteriathan 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 killingwas also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest thatmicroglial 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 organismswith low pathogenicity in the immunocompetent host (34, 37).Moreover, the distribution of the pathogens also differs fromthe immunocompetent host and depends on the nature of theimmune defect. Patients with a decrease in B-lymphocyte func-tion or with a loss of splenic function have an increased risk ofmeningitis caused by encapsulated bacteria, while patients withan 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 toCNS infections probably is a decreased local immune defense(33).

CNS infections not only are more frequent but also areassociated 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 typicalclinical 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 morphologicaltransformation as well as functional changes, such as the pro-duction of proinflammatory cytokines, chemokines, and reac-tive oxygen species, enhanced phagocytic activity, and antigenpresentation (15, 39). This immune reaction cannot eliminatehigh amounts of pneumococci from the CSF but does preventor minimize the invasion of these pathogens into the braintissue, thereby limiting tissue destruction and neuronal injury.

TLR2, -4, and -9 contribute to the recognition and responseto Streptococcus pneumoniae in the CNS (31). A deficiency ofTLR2, -4, or -9 or of the coreceptor CD14, which is necessaryfor TLR4 signaling increases the susceptibility of mice to S.pneumoniae (1, 11, 12, 40).

Here, we hypothesized that activation of the innate immuneresponse in microglia could increase the resistance of the braintissue against CNS pneumococcal infections (14). This may beof particular interest in immunocompromised patients, whoseoutcome after S. pneumoniae meningitis is worse than that ofimmunocompetent individuals (9, 44). The aim of the presentstudy was to investigate whether the stimulation of microgliaby respective PAMPs can increase their ability to phagocytoseand kill intracellular nonencapsulated and encapsulated S.

* Corresponding author. Mailing address: Department of Geriatrics,Evang. Krankenhaus Gottingen-Weende, An der Lutter 24, D-37075Gottingen, Germany. Phone: 49 551 5034-1560. Fax: 49 551 5034-1562.E-mail: rnau@gwdg.de.

† Supplemental material for this article may be found at http://iai.asm.org/.

‡ Present address: Department of Neurology, RWTH University,52062 Aachen, Germany.

� Published ahead of print on 23 November 2009.

865

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

pneumoniae strains, thereby protecting the brain during men-ingitis. Moreover, by using an encapsulated and a nonencap-sulated pneumococcal strain, we assessed the protective effectof the capsule against phagocytosis by microglial cells.

MATERIALS AND METHODS

Primary mouse microglial cell cultures. Primary cultures of microglial cellswere prepared from the brains of newborn C57/BL6N mice (1 to 3 days) aspreviously described (10, 36). Microglial cells were isolated by shaking at 200times/min for 30 min, and the cells in the supernatant were replated in 96-wellplates (for phagocytosis assay) and in 24-well plates (for intracellular survivalassay) at a density of 50,000 to 65,000 cells/well. In addition, microglia wereplated on poly-L-lysine-coated coverslips in 12-well plates for subsequent stainingand confocal microscopy at the same number of cells/well.

Microglial stimulation with TLR agonists. Cells seeded into 24- and 96-wellplates were exposed to one of the different TLR agonists for 24 h. Tripalmitoyl-S-glyceryl-cysteine (Pam3CSK4; molecular mass, 910.5 Da; EMC Microcollections,Tubingen, Germany), endotoxin (lipopolysaccharide [LPS] from Escherichia coliserotype 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 maximumstimulation of microglial cells in terms of NO release (10): Pam3CSK4 was testedat 0.1 �g/ml (0.1 �M), LPS was tested at 0.01 �g/ml (1 nM), and CpG was testedat 1 �g/ml (150 nM).

Supernatants from stimulated microglial cultures and unstimulated controlswere 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 forphagocytosis 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 chosenas 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 450nm on a microplate reader (Bio-Rad, Munich, Germany). The total protein contentwas determined by using the MicroBCA protein assay (Pierce, Rockford, IL).

Bacterial strains, culture conditions, and protein purification. Streptococcuspneumoniae strains D39 (encapsulated, serotype 2) and its nonencapsulatedderivative R6 were used in phagocytosis and intracellular survival assays. Pneu-mococcal strains were grown in a medium consisting of Dulbecco modified Eaglemedium with Glutamax I (DMEM; Gibco, Karlsruhe, Germany) supplementedwith 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 strainwas 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 grownin 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) weregenerated by transformation of pneumococci with plasmid pMV158GFP (29).

The bacterial inoculum was determined for each assay by quantitative platingon sheep blood agar plates.

Phagocytosis and intracellular survival assay. After 24 h of stimulation withone TLR agonist, microglial cells were exposed to either S. pneumoniae D39 orR6 (with a ratio of approximately 50 bacteria per phagocyte). Phagocytosis wasleft to proceed for 30 or 90 min at 37°C and 5% CO2. For phagocytosis inhibitionstudies cytochalasin D (final concentration, 10 �M; Sigma-Aldrich, St. Louis,MO) was added to the cell monolayers 30 min prior to the addition of bacteriaand remained present throughout the experiment (36). After bacterial exposure,cells were incubated for 1 h in culture medium containing gentamicin (finalconcentration, 200 �g/ml; Sigma-Aldrich). After gentamicin incubation, the cellmonolayers were washed and lysed with distilled water. The intracellular bacteriawere enumerated by quantitative plating of serial dilutions of the lysates onsheep blood agar plates. The limit of detection was 10 CFU/well. Each protocolwas performed at least three times in independent experiments. During thephagocytosis assay, extracellular bacterial replication and gentamicin activitywere checked (36).

To monitor intracellular survival and replication inside microglia, cells wereallowed to phagocytose bacteria for 30 min. Thereafter, cells were washed and

incubated in culture medium containing gentamicin (200 �g/ml) for 2 h. Atvarious times (30, 60, 90, and 120 min), the monolayers were washed and lysedwith distilled water, and the amounts of intracellular viable bacteria were quan-titatively determined.

Staining and confocal laser imaging of microglia. Scanning laser confocalmicroscopy was used to confirm intracellular localization of the encapsulatedD39gfp and the nonencapsulated D39gfp�cps pneumococcal strain after coin-cubation with microglia. Cells plated on coverslips in 12-well plates were exposedto one of the different TLR agonists for 24 h. Thereafter, the cell monolayerswere 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 wasadded (see above). After 1 h of incubation with gentamicin, cells were washedand 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 opticalsections in Z-plane were acquired at intervals of 0.6 �m. Stacks of images wereprocessed by using ImageJ (version 1.43f). In order to illustrate the intracellularlocalization of fluorescent bacteria, the z-planes (XZ and YZ) of the images weredepicted as orthogonal views. For better visualization of the fluorescent bacteria,three-dimensional (3D) videos were generated by using the ImageJ plugin 3DViewer (by Benjamin Schmid) and are presented in the supplemental material(Fig. S1 to S6).

Statistical analysis. Prism software (GraphPad Software, San Diego, CA) wasused to perform statistical analyses and graphical presentation. Analysis of vari-ance (ANOVA), followed by Bonferroni’s multiple comparison test, was used tocompare 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 byDunn’s multiple comparison test to correct for repeated testing. A P value of � 0.05was considered significant.

RESULTS

TLR agonists stimulated microglia and induced cytokineand chemokine release. In order to confirm effective microglialstimulation by the different TLR agonists, we determined theinduction of representative cytokines and chemokines such asIL-6 and CCL5 (Fig. 1). Microglial cells remained viable after24 h of exposure to these agonists (35). In all experiments, agroup of unstimulated cells was included for comparison.

The supernatants of unstimulated microglia were devoid ofmeasurable amounts of IL-6 and CCL5. Microglial cells incu-bated with the individual TLR agonists released much higheramounts of IL-6 and CCL5 than did unstimulated cells (P �0.05).

Confocal laser imaging confirmed the intracellular localiza-tion of encapsulated and nonencapsulated pneumococci. Con-focal microscopy confirmed the intracellular localization of theencapsulated D39gfp and the nonencapsulated D39gfp�cps S.pneumoniae strains within microglial cells. Bacteria expressingGFP and microglia with their cell membrane labeled by redVybrant DiI were simultaneously visualized in two fluorescentchannels, as depicted in the reconstructed images of the z-sections (Fig. 2). The animated 3D isosurface reconstructionsare 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 andR6 pneumococcal strains was compared quantitatively after 30and 90 min of incubation with bacteria in unstimulated cultures

866 RIBES ET AL. INFECT. IMMUN.

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

(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 phagocyticactivity of microglia. Treatment with 1 �g of CpG/ml resultedin an increased uptake of both D39 and R6 strains at 30 and 90min of exposure (P � 0.001). After stimulation with 0.1 �g ofPam3CSK4/ml, the ingestion of the encapsulated D39 strainwas increased at 90 min (P � 0.05), while phagocytosis of thenonencapsulated R6 strain was enhanced at 30 and 90 min(P � 0.001). Treatment with 0.01 �g of LPS/ml enhanced theingestion 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 foundthat TLR1/2- and TLR9-stimulated cells phagocytosed compa-rable numbers of bacteria (P � 0.05 at 30 and 90 min). Incontrast, LPS-stimulated cells ingested lower numbers of bothencapsulated D39 (P � 0.05 at 90 min versus TLR9-treatedcells) and nonencapsulated R6 strains (P � 0.05 at 30 minversus TLR1/2- and TLR9-treated cells).

The phagocytic rates were different for both strains: theuptake of the nonencapsulated R6 strain was approximately 10times more rapid than the internalization of the encapsulatedD39 strain.

The internalization of both pneumococcal strains by micro-glia occurred via phagocytosis. Cytochalasin D blocked theuptake of S. pneumoniae D39 and R6 strains by �90% in

unstimulated and TLR-stimulated cells, as it was revealed in30-min phagocytosis inhibition studies.

The extracellular concentration of both pneumococcalstrains 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 gentamicintreatment, the number of extracellular bacteria was below thelevel of detection in all experiments.

TLR stimulation increased the intracellular killing of S.pneumoniae D39 and R6 by microglia. Next, we studiedwhether in TLR-stimulated microglial cells the increase of thephagocytic activity was accompanied by a higher intracellularkilling of the ingested bacteria (Fig. 4).

The absolute amounts of killed S. pneumoniae D39 (calcu-lated as the difference between the medians of intracellularbacteria at 30 and 120 min) were higher in TLR-stimulatedmicroglia than in unstimulated cells (Fig. 4A). The time courseof intracellular killing of S. pneumoniae R6 strain was similarto that of the encapsulated strain (Fig. 4B).

DISCUSSION

Streptococcus pneumoniae is an important cause of bacterialmeningitis causing death in ca. 25% of the cases and long-termneurological sequelae in up to one-third of the survivors (9, 17,38, 44). Proinflammatory and directly cytotoxic pneumococcalproducts (such as pneumococcal cell wall products, pneumo-lysin, and bacterial DNA) contribute to neuronal injury in S.pneumoniae meningitis.

Microglial cells are the major constituents of innate immu-nity within the CNS (20). Parenchymal microglia, as well asmeningeal and perivascular macrophages, which become acti-vated by bacterial products are critically involved in protectingthe brain from infection (30, 33). On the one hand, microglialcells 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, lackof MyD88 expression in the CNS compartment led to elevatedintracerebral S. aureus burdens despite the presence of immu-nocompetent bone marrow-derived cells (14). On the otherhand, activated microglial cells can be toxic to surroundingneurons by releasing, e.g., nitric oxide, glutamate, TNF-�, andIL-1�. The diminished inflammatory response decreased hear-ing loss in pneumococcal meningitis in MyD88-deficient mice,and neuronal injury caused by group B streptococci dependedon the presence of TLR2 and MyD88 (18, 22). Thus, activationof microglia during infections seems to be a double-edgedsword. The innate immune response can protect neurons bypreventing the entry of pathogens into the brain, but its dys-regulation can also be harmful for neuronal integrity and cancause neuronal injury (6, 16, 20, 22, 28). Deeper understandingof the roles for TLRs in resident CNS glia and infiltratingimmune cells will provide insights into how the immune re-sponse to bacterial infection can be tailored to achieve effectivepathogen destruction without inducing excessive bystanderdamage of surrounding brain parenchyma (13, 26).

In this context, we focused our research on the phagocytosisof microglia activated by TLR stimulation. We hypothesizedthat the activation of the TLR system in microglial cells by

FIG. 1. (A) IL-6 and (B) CCL5 (RANTES) concentrations in the super-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 plus10% FCS (unstim). The data are shown as means � the standard deviation(SD) (n � 13 wells/group from three independent experiments). The datawere analyzed by using ANOVA, followed by Bonferroni’s multiple compar-ison test (�, P � 0.05; ��, P � 0.01; ���, P � 0.001).

VOL. 78, 2010 TLR AGONISTS INCREASE S. PNEUMONIAE PHAGOCYTOSIS 867

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

FIG. 2. Phagocytosis of the encapsulated D39gfp (A to C) and the nonencapsulated D39gfp�cps (D to F) S. pneumoniae strains by murinemicroglial cells after 30 min of bacterial exposure. Internal and external cell membranes were stained with red Vybrant DiI prior to the additionof 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 andy-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 andF) The addition of cytochalasin D (final concentration, 10 �M) blocked the phagocytosis of S. pneumoniae strains by CpG-stimulated microglialcells. Scale bars are shown in panel A, 5 �m (x-y plane) and 2 �m (x-z and y-z projected planes).

868 RIBES ET AL. INFECT. IMMUN.

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

agonist stimulation may enhance their phagocytic activity,thereby enabling them to protect the brain in pneumococcalCNS infections in patients with an impaired immune system.

The release of cytokines and chemokines in the CSF duringpneumococcal meningitis has been analyzed. IL-6 is one of themajor 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, chemokineproduction is rapidly upregulated upon activation by stimulisuch as bacteria or inflammatory mediators (24, 32). An up-

regulation of the expression of CCL2, CCL5, and CXCL2chemokines was observed in lungs, blood, and brain tissue afterintranasal inoculation of S. pneumoniae strains (serotypes 2, 4,and 6A) in mice (25). In the present study, when microgliawere exposed to a TLR1/2, -4, or -9 ligand for 24 h, the releaseof IL-6 and CCL5 was strongly increased, confirming micro-glial activation.

Upon TLR stimulation, reactive microglia develop a phago-cytic phenotype to engulf and kill microbes. In contrast tocytokine 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 murinemicroglial 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 controlgroup of unstimulated cells was included in all experiments. After stimulation, cells were washed and bacteria were added for different times (30and 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 independentexperiments). 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] Streptococcuspneumoniae [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 ofintracellular bacteria was quantified by plating at several postinfection times for up to 120 min. For each group, intracellular killing is expressedas 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 PHAGOCYTOSIS 869

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ricidal profiles of activated microglia have been explored lessthoroughly. Our group has recently reported that TLR1/2, -4,and -9 agonists can increase the ability of murine microglialcells to phagocytose and kill intracellularly located Escherichiacoli strains (36). The present data demonstrate that microgliacan also phagocytose and kill Gram-positive bacteria whichhave a thicker cell wall and that stimulation of TLRs canincrease their phagocytic and bactericidal activity. This appliesfor both nonencapsulated apathogenic and encapsulated patho-genic pneumococci. Stimulation with either a TLR1/2, -4, or -9agonist 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 theeffect caused via TLR1/2 or TLR4. Similarly, phagocytosis andkilling of live S. pneumoniae were found to be impaired inalveolar and bone marrow-derived macrophages from TLR9-deficient mice (1) and in blood-derived polymorphonuclearleukocytes from TLR2-deficient mice (23).

Once bacteria have been phagocytosed, they are incorpo-rated into phagolysosomes and exposed to reactive oxygenspecies that eventually will result in bacterial lysis. The intra-cellular killing of S. pneumoniae by microglial cells was morerapid than that of E. coli studied in the same experimentalsetting (36). For this reason, the number of viable intracellularbacteria determined after 90 min of phagocytosis was lowerthan the concentration of viable intracellular bacteria detectedafter 30 min.

The presence of the polysaccharide capsule is an importantvirulence factor of pneumococci because it decreases bacterialuptake into microglia by more than 10 times (Fig. 3). In addi-tion, we showed that the internalization of pneumococcalstrains by murine microglia requires intact actin filaments sincethis process was blocked by �90% by cytochalasin D (Fig. 2).Not only the phagocytic but also the bactericidal activities ofreactive 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 killedbacteria in TLR-stimulated than in unstimulated microglia,i.e., TLR stimulation clearly increased the efficacy of microgliain neutralizing the internalized S. pneumoniae (Fig. 4).

An intact TLR signaling through the pathway organized byMyD88 appears to be necessary to protect the brain tissueagainst invading microorganisms. A poor outcome because ofhigh bacterial counts in the CNS and severe bacteremia wasobserved 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 bacterialspread into the bloodstream, and increased mortality (2).These findings illustrate the importance of an intact innateimmune system to efficiently limit the spread of S. pneumoniae.

Stimulation of the TLR system is a potential target for thedevelopment of new therapies in multiple diseases (45). Sev-eral TLR agonists are currently at different stages of clinicaltrials (4). The TLR7 agonist imiquimod has been successfullyused and approved for the treatment of warts associated withhuman papillomavirus and is in a second phase trial as atherapeutic 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 goodresults (8). One of the most interesting clinical trials with CPG7909 has been recently completed and aimed at comparing theimmune responses after TLR9-boostered pneumococcal vaccinationin human immunodeficiency virus-infected adults (www.clinicaltrials.gov/ct2/show/NCT00562939?termTLR9&rank3).

Therefore, the agonists used in the present study or relatedcompounds could be of value as adjuvants to improve theefficiency of the local immune system of the CNS against bac-teria. In the pharmacological administration of TLR agonistsas adjuvants, the dose, timing, and duration of the immuno-therapy, as well as the route of administration, have to beselected not only to maximize the benefit of the enhancementof the immune response but also to restrict an excessive in-duced response that might lead to autoimmune diseases orincreased neuronal injury (4).

One clear advantage of using TLR agonists as adjuvants forthe 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 preferablebecause of their higher penetration across the BBB (4). The entryof LPS into the central nervous compartments is minimal (5).

In conclusion, stimulation of TLRs increases phagocytosis ofGram-positive S. pneumoniae by microglia. Stimulation of theTLR system may be a therapeutic approach to protect thebrain from invading pathogens. Further studies in immuno-compromised mice are in progress in order to assess whetherthe resistance of the brain against infections can be increasedby priming microglial cells with TLR agonists.

ACKNOWLEDGMENTS

This study was supported by the European Union (grant CAREPNEUMO), the Else Kroner-Fresenius-Stiftung (R.N. and A.S.) andthe SFB/TR43 (U.-K.H.). S.R. was the recipient of a fellowship fromthe Departament d’Educacio i Universitats de la Generalitat deCatalunya.

This work is dedicated to Viktor Papiol.

REFERENCES

1. Albiger, B., S. Dahlberg, A. Sandgren, F. Wartha, K. Beiter, H. Katsuragi, S.Akira, S. Normark, and B. Henriques-Normark. 2007. Toll-like receptor 9acts at an early stage in host defense against pneumococcal infection. CellMicrobiol. 9:633–644.

2. Albiger, B., A. Sandgren, H. Katsuragi, U. Meyer-Hoffert, K. Beiter, F.Wartha, M. Hornef, S. Normark, and B. H. Normark. 2005. Myeloid differ-entiation factor 88-dependent signaling controls bacterial growth duringcolonization and systemic pneumococcal disease in mice. Cell Microbiol.7:1603–1615.

3. Aravalli, R. N., P. K. Peterson, and J. R. Lokensgard. 2007. Toll-like recep-tors in defense and damage of the central nervous system. J. NeuroimmunePharmacol. 2:297–312.

4. Averett, D. R., S. P. Fletcher, W. Li, S. E. Webber, and J. R. Appleman. 2007.The pharmacology of endosomal TLR agonists in viral disease. Biochem.Soc. Trans. 35:1468–1472.

5. Banks, W. A., and S. M. Robinson. 2010. Minimal penetration of lipopoly-saccharide across the murine blood-brain barrier. Brain Behav. Immun.24:102–109.

6. Chao, C. C., S. Hu, T. W. Molitor, E. G. Shaskan, and P. K. Peterson. 1992.Activated microglia mediate neuronal cell injury via a nitric oxide mecha-nism. J. Immunol. 149:2736–2741.

7. Cunha, B. A. 2001. Central nervous system infections in the compromisedhost: a diagnostic approach. Infect. Dis. Clin. N. Am. 15:567–590.

8. Daubenberger, C. A. 2007. TLR9 agonists as adjuvants for prophylactic andtherapeutic vaccines. Curr. Opin. Mol. Ther. 9:45–52.

9. Durand, M. L., S. B. Calderwood, D. J. Weber, S. I. Miller, F. S. Southwick,V. S. Caviness, Jr., and M. N. Swartz. 1993. Acute bacterial meningitis inadults: a review of 493 episodes. N. Engl. J. Med. 328:21–28.

870 RIBES ET AL. INFECT. IMMUN.

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

10. Ebert, S., J. Gerber, S. Bader, F. Muhlhauser, K. Brechtel, T. J. Mitchell,and R. Nau. 2005. Dose-dependent activation of microglial cells by Toll-likereceptor agonists alone and in combination. J. Neuroimmunol. 159:87–96.

11. Echchannaoui, H., K. Frei, M. Letiembre, R. M. Strieter, Y. Adachi, and R.Landmann. 2005. CD14 deficiency leads to increased MIP-2 production,CXCR2 expression, neutrophil transmigration, and early death in pneumo-coccal infection. J. Leukoc. Biol. 78:705–715.

12. Echchannaoui, H., K. Frei, C. Schnell, S. L. Leib, W. Zimmerli, and R.Landmann. 2002. Toll-like receptor 2-deficient mice are highly susceptible toStreptococcus pneumoniae meningitis because of reduced bacterial clearingand enhanced inflammation. J. Infect. Dis. 186:798–806.

13. Esen, N., and T. Kielian. 2009. Toll-like receptors in brain abscess. Curr.Top. Microbiol. Immunol. 336:41–61.

14. Garg, S., J. R. Nichols, N. Esen, S. Liu, N. K. Phulwani, M. M. Syed, W. H.Wood, Y. Zhang, K. G. Becker, A. Aldrich, and T. Kielian. 2009. MyD88expression by CNS-resident cells is pivotal for eliciting protective immunityin brain abscesses. ASN Neuro 1 pii:e00007. doi:10.1042/AN20090004.

15. Hanisch, U. K. 2002. Microglia as a source and target of cytokines. Glia40:140–155.

16. Iliev, A. I., A. K. Stringaris, R. Nau, and H. Neumann. 2004. Neuronal injurymediated via stimulation of microglial Toll-like receptor-9 (TLR9). FASEBJ. 18:412–414.

17. Kastenbauer, S., and H. W. Pfister. 2003. Pneumococcal meningitis in adults:spectrum of complications and prognostic factors in a series of 87 cases.Brain 126:1015–1025.

18. Klein, M., C. Schmidt, S. Kastenauer, R. Paul, C. J. Kirschning, H. Wagner,B. Popp, H. W. Pfister, and U. Koedel. 2008. MyD88-dependent immuneresponse contributes to hearing loss in experimental pneumococcal menin-gitis. J. Infect. Dis. 195:1189–1193.

19. Koedel, U., T. Rupprecht, B. Angele, J. Heesemann, H. Wagner, H. W.Pfister, and C. J. Kirschning. 2004. MyD88 is required for mounting a robusthost immune response to Streptococcus pneumoniae in the CNS. Brain 127:1437–1445.

20. Kreutzberg, G. W. 1996. Microglia: a sensor for pathological events in theCNS. Trends Neurosci. 19:312–318.

21. Laflamme, N., and S. Rivest. 1999. Effects of systemic immunogenic insultsand circulating proinflammatory cytokines on the transcription of the inhib-itory factor B alpha within specific cellular populations of the rat brain.J. Neurochem. 73:309–321.

22. Lehnardt, S., P. Henneke, E. Lien, D. L. Kasper, J. J. Volpe, I. Bechmann,R. Nitsch, J. R. Weber, D. T. Golenbock, and T. Vartanian. 2006. A mech-anism for neurodegeneration induced by group B streptococci through ac-tivation of the TLR2/MyD88 pathway in microglia. J. Immunol. 177:583–592.

23. Letiembre, M., H. Echchannaoui, P. Bachmann, F. Ferracin, C. Nieto, M.Espinosa, and R. Landmann. 2005. Toll-like receptor 2 deficiency delayspneumococcal phagocytosis and impairs oxidative killing by granulocytes.Infect. Immun. 73:8397–8401.

24. Lokensgard, J. R., S. Hu, E. M. van Fenema, W. S. Sheng, and P. K.Peterson. 2000. Effect of thalidomide on chemokine production by humanmicroglia. J. Infect. Dis. 182:983–987.

25. Mahdi, L. K., A. D. Ogunniyi, K. S. LeMessurier, and J. C. Paton. 2008.Pneumococcal virulence gene expression and host cytokine profiles duringpathogenesis of invasive disease. Infect. Immun. 76:646–657.

26. Mariani, M. M., and T. Kielian. 2009. Microglia in infectious diseases of thecentral nervous system. J. Neuroimmune Pharmacol. 4:448–461.

27. Mark, K. E., L. Corey, T. C. Meng, A. S. Magaret, M. L. Huang, S. Selke,H. B. Slade, S. K. Tyring, T. Warren, S. L. Sacks, P. Leone, V. A. Bergland,and A. Wald. 2007. Topical resiquimod 0.01% gel decreases herpes simplexvirus type 2 genital shedding: a randomized, controlled trial. J. Infect. Dis.195:1324–1331.

28. Nguyen, M. D., J. P. Julien, and S. Rivest. 2002. Innate immunity: themissing link in neuroprotection and neurodegeneration? Nat. Rev. Neurosci.3:216–227.

29. Nieto, C., and M. Espinosa. 2003. Construction of the mobilizable plasmidpMV158GFP, a derivative of pMV158 that carries the gene encoding thegreen fluorescent protein. Plasmid 49:281–285.

30. Nimmerjahn, A., F. Kirchhoff, and F. Helmchen. 2005. Resting microglialcells are highly dynamic surveillants of brain parenchyma in vivo. Science308:1314–1318.

31. Paterson, G. K., and T. J. Mitchell. 2006. Innate immunity and the pneu-mococcus. Microbiology 152:285–293.

32. Peterson, P. K., S. Hu, J. Salak-Johnson, T. W. Molitor, and C. C. Chao.1997. Differential production of and migratory response to beta chemokinesby human microglia and astrocytes. J. Infect. Dis. 175:478–481.

33. Polfliet, M. M., P. J. Zwijnenburg, A. M. van Furth, T. van der Poll, E. A.Dopp, C. Renardel de Lavalette, E. M. van Kesteren-Hendrikx, N. vanRooijen, C. D. Dijkstra, and T. K. van den Berg. 2001. Meningeal andperivascular macrophages of the central nervous system play a protectiverole during bacterial meningitis. J. Immunol. 167:4644–4650.

34. Pruitt, A. A. 1991. Central nervous system infections in cancer patients.Neurol. Clin. 9:867–888.

35. Rennemeier, C., S. Hammerschmidt, S. Niemann, S. Inamura, U. Zahringer,and B. E. Kehrel. 2007. Thrombospondin-1 promotes cellular adherence ofgram-positive pathogens via recognition of peptidoglycan. FASEB J. 21:3118–3132.

36. Ribes, S., S. Ebert, D. Czesnik, T. Regen, A. Zeug, S. Bukowski, A. Mildner,H. Eiffert, U. K. Hanisch, S. Hammerschmidt, and R. Nau. 2009. Toll-likereceptor prestimulation increases phagocytosis of Escherichia coli DH5alphaand Escherichia coli K1 strains by murine microglial cells. Infect. Immun.77:557–564.

37. Safdieh, J. E., P. A. Mead, K. A. Sepkowitz, T. E. Kiehn, and L. E. Abrey.2008. Bacterial and fungal meningitis in patients with cancer. Neurology70:943–947.

38. Schmidt, H., B. Heimann, M. Djukic, C. Mazurek, C. Fels, C. W. Wallesch,and R. Nau. 2006. Neuropsychological sequelae of bacterial and viral men-ingitis. Brain 129:333–345.

39. Smith, M. E., K. van der Maesen, and F. P. Somera. 1998. Macrophage andmicroglial responses to cytokines in vitro: phagocytic activity, proteolyticenzyme release, and free radical production. J. Neurosci. Res. 54:68–78.

40. Srivastava, A., P. Henneke, A. Visintin, S. C. Morse, V. Martin, C. Watkins,J. C. Paton, M. R. Wessels, D. T. Golenbock, and R. Malley. 2005. Theapoptotic response to pneumolysin is Toll-like receptor 4 dependent andprotects against pneumococcal disease. Infect. Immun. 73:6479–6487.

41. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev.Immunol. 21:335–376.

42. Tunkel, A. R., and W. M. Scheld. 2002. Central nervous system infection inthe immunocompromised host, p. 163–214. In R. H. Rubin, and L. S. Young(ed.), Clinical approach to infection in the compromised host, 4th ed. KluwerAcademic Press, Inc., New York, NY.

43. Tyring, S. K., I. I. Arany, M. A. Stanley, M. H. Stoler, M. A. Tomai, R. L.Miller, M. L. Owens, and M. H. Smith. 1998. Mechanism of action ofimiquimod 5% cream in the treatment of anogenital warts. Prim. CareUpdate Ob. Gyns. 5:151–152.

44. Weisfelt, M., D. van de Beek, L. Spanjaard, J. B. Reitsma, and J. de Gans.2006. Clinical features, complications, and outcome in adults with pneumo-coccal meningitis: a prospective case series. Lancet Neurol. 5:123–129.

45. Zuany-Amorim, C., J. Hastewell, and C. Walker. 2002. Toll-like receptors aspotential therapeutic targets for multiple diseases. Nat. Rev. Drug Discov.1:797–807.

Editor: A. Camilli

VOL. 78, 2010 TLR AGONISTS INCREASE S. PNEUMONIAE PHAGOCYTOSIS 871

on June 11, 2020 by guesthttp://iai.asm

.org/D

ownloaded from