Downloaded from on May 21, 2020 by guest · 8 Hanisch, 2 Sven Hammerschmidt, 6 and Roland Nau 1,7 * 9 ... 24 Toll-like receptors (TLRs) are crucial pattern recognition rec eptors

Toll-like receptor stimulation enhances phagocytosis intracellular killing of 1

nonencapsulated and encapsulated Streptococcus pneumoniae by murine microglia 2

3

Running title: TLRs agonists increase S. pneumoniae phagocytosis by microglia 4

5

Sandra Ribes,1 Sandra Ebert,

1 Tommy Regen,

2 Amit Agarwal,

3 Simone C. Tauber,

1,# 6

Dirk Czesnik,4 Annette Spreer,

1 Stephanie Bunkowski,

1 Helmut Eiffert,

5 Uwe-Karsten 7

Hanisch,2 Sven Hammerschmidt,

6 and Roland Nau

1,7 *

8

9

Department of Neurology,1

Institute of Neuropathology,2

Department of 10

Neurophysiology and Cellular Biophysics,4

Department of Medical Microbiology, 11

University of Göttingen, Göttingen 37075, Germany;5

Department of Neurogenetics, 12

Max-Planck Institute of Experimental Medicine, Göttingen 37075, Germany3; Institute 13

for Genetics and Functional Genomics, Department of Genetics of Microorganisms, 14

Ernst-Moritz-Arndt-University, Greifswald, Germany6; and Department of Geriatrics, 15

Evangelisches Krankenhaus Göttingen-Weende, Göttingen 37075, Germany7 16

#Present address: Department of Neurology, RWTH University, 52062 Aachen, 17

Germany. 18

*Corresponding author. Mailing address: Department of Geriatrics, Evang. 19

Krankenhaus Göttingen-Weende e.V., An der Lutter 24, D-37075 Göttingen, Germany. 20

Phone: +49 551 5034-1560; fax: +49 551 5034-1514 21

E-mail: [email protected] 22

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01110-09 IAI Accepts, published online ahead of print on 23 November 2009

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Abstract 23

Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity 24

that are expressed in microglia, the resident macrophages of the brain. TLR2, 4 and 9 25

are important in the responses against Streptococcus pneumoniae, the most frequent 26

agent causing bacterial meningitis beyond the neonatal period. Murine microglial 27

cultures were stimulated with

agonists for TLR1/2 (Pam3CSK4), TLR4 28

(lipopolysaccharide) and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed 29

to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. 30

pneumoniae. After stimulation, levels of interleukin 6 and CCL5 (Regulated upon 31

Activation Normal T cell Expressed and Secreted, RANTES) were increased 32

confirming microglial activation. The TLR1/2, 4 and 9 agonist-stimulated microglia 33

ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of 34

cytochalasin D, an inhibitor of actin polymerizaton, blocked > 90% of phagocytosis. 35

Along with an increased phagocytic activity, the intracellular bacterial killing was also 36

increased in TLR-stimulated cells in comparison to unstimulated cells. Together our 37

data suggest that microglial stimulation by these TLRs may increase the resistance of 38

the brain against pneumococcal infections. 39


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INTRODUCTION 40

Immunocompromised patients have a higher risk of developing bacterial infections in 41

the central nervous system (CNS) (34, 37, 42). The list of the pathogens includes many 42

organisms with low pathogenicity in the immunocompetent host (34, 37). Moreover, the 43

distribution of the pathogens also differs from the immunocompetent host and depends 44

on the nature of the immune defect. Patients with a decrease in B-lymphocyte function 45

or with a loss of splenic function have an increased risk of meningitis caused by 46

encapsulated bacteria while patients with an impaired T-lymphocyte-macrophage 47

system are more susceptible to CNS infections caused by intracellular pathogens (7, 48

42). One additional cause of this increased susceptibility to CNS infections probably is 49

a decreased local immune defense (33). 50

CNS infections not only are more frequent but also are associated with higher mortality 51

rates and more severe long-term sequelae in immunocompromised than in 52

immunocompetent individuals (9, 17, 34, 44). Polymicrobial infections, multiple organ 53

system presentation and the absence of typical clinical manifestations subsequent to the 54

host’s diminished inflammatory response are challenging aspects in the management of 55

these infections (34, 37, 42). 56

The brain tissue shows a well-organized innate immune reaction in response to bacteria 57

in the cerebrospinal fluid (CSF) (3, 21). Microglial cells, the resident phagocytes of the 58

CNS, express Toll-like receptors (TLRs) that identify pathogen-associated molecular 59

patterns (PAMPs) (41). The receptor-ligand interactions activate microglia to undergo 60

morphological transformation as well as functional changes, such as production of pro-61

inflammatory cytokines, chemokines and reactive oxygen species, enhanced phagocytic 62

activity and antigen presentation (15, 39). This immune reaction cannot eliminate high 63


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amounts of pneumococci from the CSF, but prevents or minimizes the invasion of these 64

pathogens into the brain tissue thereby limiting tissue destruction and neuronal injury. 65

TLR2, 4 and 9 contribute to the recognition and response to S. pneumoniae in the CNS 66

(31). A deficiency of TLR2, 4 or 9 or of the co-receptor CD14 which is necessary for 67

TLR4 signaling increases the susceptibility of mice to S. pneumoniae (1, 11, 12, 40). 68

Here, we hypothesized that activation of the innate immune response in microglia could 69

increase the resistance of the brain tissue against CNS pneumococcal infections (14). 70

This may be of particular interest in immunocompromised patients whose outcome after 71

S. pneumoniae meningitis is worse than that of immunocompetent individuals (9, 44). 72

The aim of this study was to investigate whether stimulation of microglia by respective 73

PAMPs can increase their ability to phagocytose and to kill intracellular both 74

nonencapsulated and encapsulated S. pneumoniae strains thereby protecting the brain 75

during meningitis. Moreover, by using an encapsulated and a nonencapsulated 76

pneumococcal strain, we assessed the protective effect of the capsule against 77

phagocytosis by microglial cells. 78

79

MATERIAL AND METHODS 80

Primary mouse microglial cell cultures 81

Primary cultures of microglial cells were prepared from brains of newborn C57/BL6N 82

mice (1–3 days) as previously described (10, 36). Microglial cells were isolated by 83

shaking 200x/min for 30 min and the cells in the supernatant were replated in 96-well 84

plates (for phagocytosis assay) and in 24-well plates (for intracellular survival assay) at 85

a density of 50,000-65,000 cells/well. Additionally, microglia were plated on poly-L-86

lysine-coated cover slips in 12-well plates for subsequent staining and confocal 87

microscopy at the same number of cells/well.

88


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Microglial stimulation with TLR agonists 89

Cells seeded into 24-well and 96-well plates were exposed to one of the different TLR 90

agonists for 24 h. Tripalmitoyl-S-glyceryl-cysteine (Pam3CSK4; molecular mass: 910.5 91

Da; EMC Microcollections, Tübingen, Germany), endotoxin (LPS from Escherichia 92

coli Serotype 026:B6; Sigma, Taufkirchen, Germany) and CpG oligodesoxynucleotide 93

(ODN) 1668 (TCC ATG ACG TTC CTG ATG CT; molecular mass: 6383 Da) from 94

TIB Molbiol (Berlin, Germany) were used as specific ligand of TLR1/2, 4 and 9. A 95

control group with unstimulated microglial cells was included in all experiments. TLR 96

agonists were used at the lowest concentrations inducing maximum stimulation of 97

microglial cells in terms of NO release (10): Pam3CSK4 was tested at 0.1 µg/ml (0.1 98

µM); LPS at 0.01 µg/ml (1 nM); and CpG at 1 µg/ml (150 nM). 99

Supernatants from stimulated microglial cultures and unstimulated controls were 100

collected after 24 h of incubation and stored frozen at −80 °C until measurement of 101

cytokine and chemokine levels. Microglial cells were assayed for phagocytosis or 102

intracellular survival by quantitative plating of intracellular bacteria or used for staining 103

and subsequent confocal microscopy. 104

Cyto- and Chemokine release 105

Interleukin-6 (IL-6) and CCL5 (Regulated upon Activation Normal T cell Expressed 106

and Secreted, RANTES) were chosen as representatives of the inducible spectrum of 107

microglial cyto- and chemokines (15). DuoSet ELISA Development Kits (R&D 108

Systems, Wiesbaden, Germany) were used for their measurement. The colour reaction 109

was measured at 450 nm on a microplate reader (Bio-Rad, Munich, Germany). Total 110

protein content was determined using the MicroBCA protein assay (Pierce, Rockford, 111

IL, USA). 112

Bacterial strains, culture conditions, and protein purification 113


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Streptococcus pneumoniae strains D39 (encapsulated, serotype 2) and its 114

nonencapsulated derivative R6 were used in phagocytosis and intracellular survival 115

assays. Pneumococcal strains were grown in a medium consisting of Dulbecco modified 116

Eagle medium with Glutamax I (DMEM, Gibco, Karlsruhe, Germany) supplemented 117

with 10% heat-inactivated fetal calf serum (FCS). 118

The GFP-expressing strains D39gfp and its nonencapsulated derivative D39gfp∆cps 119

were used for confocal microscopy to confirm intracellular location of bacteria in 120

microglial cells. The D39gfp strain was grown in a medium consisting of DMEM 121

supplemented with 10% heat-inactivated FCS and 0.5 µg/ml tetracycline. The 122

D39gfp∆cps strain was grown in DMEM supplemented with 10% heat-inactivated FCS, 123

0.5 µg/ml tetracycline and 50 µg/ml kanamycin. GFP-expressing D39 and D39∆cps (35) 124

were generated by transformation of pneumococci with plasmid pMV158GFP (29). 125

The bacterial inoculum was determined for each assay by quantitative plating on sheep 126

blood agar plates. 127

Phagocytosis and intracellular survival assay 128

After 24 h of stimulation with one TLR agonist, microglial cells were exposed to either 129

S. pneumoniae D39 or R6 (with a ratio of approximately 50 bacteria per phagocyte). 130

Phagocytosis was left to proceed for 30 or 90 min at 37ºC and 5% CO2. For 131

phagocytosis inhibition studies cytochalasin D (final concentration, 10 µM, Sigma-132

Aldrich, St. Louis, MO) was added to the cell monolayers 30 min prior to the addition 133

of bacteria and remained present throughout the experiment (36). After bacterial 134

exposure, cells were incubated for 1 h in culture medium containing gentamicin (final 135

concentration, 200 µg/ml; Sigma-Aldrich, St. Louis, MO). After gentamicin incubation, 136

cell monolayers were washed and lysed with distilled water. The intracellular bacteria 137

were enumerated by quantitative plating of serial dilutions of the lysates on sheep blood 138


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agar plates. The limit of detection was 10 CFU/well. Each protocol was performed at 139

least three times in independent experiments. During the phagocytosis assay, 140

extracellular bacterial replication and gentamicin activity were checked (36). 141

To monitor intracellular survival and replication inside microglia, cells were allowed to 142

phagocytose bacteria for 30 min. Thereafter, cells were washed and incubated in culture 143

medium containing gentamicin (200 µg/ml) for 2 h. At various times (30, 60, 90 and 144

120 min), the monolayers were washed, lysed with distilled water and the amounts of 145

intracellular viable bacteria were quantitatively determined. 146

Staining and confocal laser imaging of microglia 147

Scanning laser confocal microscopy was used to confirm intracellular localization of the 148

encapsulated D39gfp and the nonencapsulated D39gfp∆cps pneumococcal strain after 149

co-incubation with microglia. Cells plated on coverslips in 12-well plates were exposed 150

to one of the different TLR agonists for 24 h. Thereafter, the cell monolayers were 151

washed and then incubated with Vybrant DiI cell-labeling solution (VybrantCell 152

labeling solution kit; Molecular Probes, Leiden, The Netherlands) for 3 min at 37°C 153

according to the manufacturer's instructions. Subsequently, cells were washed twice 154

with warm PBS, and bacteria were added for 30 min. For phagocytosis inhibition 155

studies cytochalasin D was added (see above). After 1 h of incubation with gentamicin, 156

cells were washed and fixed in 4% formaldehyde in PBS. Cells were imaged using a 157

laser-scanning confocal microscope (Zeiss LSM 510 meta). DiI and GFP S.pneumoniae 158

strains were sequentially excited at 488 and 543 nm. Series of optical sections in Z-159

plane were acquired at intervals of 0.6 µm. Stacks of images were processed using 160

ImageJ (version 1.43f). In order to illustrate the intracellular localization of fluorescent 161

bacteria, the z-planes (XZ and YZ) of the images were depicted as orthogonal views. 162

For better visualization of the fluorescent bacteria 3D videos were generated using the 163


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ImageJ plugin 3D Viewer (by Benjamin Schmid) and are provided as supplemental 164

material (Fig. S1 to Fig. S6). 165

Statistical Analysis 166

GraphPad Prism Software (GraphPad Software, San Diego, CA, USA) was used to 167

perform statistical analyses and graphical presentation. ANOVA followed by 168

Bonferroni's multiple comparison test was used to compare ELISA data among all 169

groups. Data from the phagocytosis and intracellular survival assays were not normally 170

distributed and analyzed by Kruskal-Wallis test followed by Dunn's multiple 171

comparison test to correct for repeated testing. A P value of <0.05 was considered 172

significant. 173

174

RESULTS 175

TLR agonists stimulated microglia and induced cytokine and chemokine release. 176

In order to confirm effective microglial stimulation by the different TLR agonists, we 177

determined the induction of representative cyto- and chemokines such as IL-6 and 178

CCL5 (Fig. 1). Microglial cells remained viable after 24 h of exposure to these agonists 179

(35). In all experiments, a group of unstimulated cells was included for comparison. 180

The supernatants of unstimulated microglia were devoid of measurable amounts of IL-6 181

and CCL5. Microglial cells incubated with the individual TLR agonists released much 182

higher amounts of IL-6 and CCL5 than unstimulated cells (P < 0.05). 183

Confocal laser imaging confirmed the intracellular localization of encapsulated 184

and nonencapsulated pneumococci. 185

Confocal microscopy confirmed the intracellular localization of the encapsulated 186

D39gfp and the nonencapsulated D39gfp∆cps S. pneumoniae strains within microglial 187

cells. Bacteria expressing the green fluorescent protein GFP and microglia with their 188


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cell membrane labeled by red Vybrant DiI were simultaneously visualized in two 189

fluorescent channels, as depicted in the reconstructed images of the z-section (Fig. 2A 190

to F). The animated 3D isosurface reconstructions are provided as separate figures in the 191

supplemental material. The addition of cytochalasin D prior to the exposure to bacteria 192

inhibited the internalization of pneumococcal strains (Fig. 2C and 2F). 193

TLR stimulation increased the phagocytosis of S. pneumoniae D39 and R6 by 194

microglia. 195

The phagocytosis of D39 and R6 pneumococcal strains was compared quantitatively 196

after 30 and 90 min of incubation with bacteria in unstimulated cultures (control group) 197

and in microglia that were previously stimulated with the TLR1/2, TLR4 or TLR9 198

agonists (Fig. 3). 199

While unstimulated cells ingested bacteria at a low rate, stimulation with one TLR 200

agonist increased the phagocytic activity of microglia. Treatment with 1 µg/ml CpG 201

resulted in an increased uptake of both D39 and R6 strains at 30 and 90 min of exposure 202

(P < 0.001). After stimulation with 0.1 µg/ml Pam3CSK4, the ingestion of the 203

encapsulated D39 strain was increased at 90 min (P < 0.05) while phagocytosis of the 204

nonencapsulated R6 strain was enhanced at 30 and 90 min (P < 0.001). Treatment with 205

0.01 µg/ml LPS enhanced the ingestion of the R6 strain at 90 min (P < 0.05). 206

When we compared the amounts of phagocytosed pneumococci among the different 207

TLR-stimulated groups, we found that TLR1/2- and TLR9-stimulated cells 208

phagocytosed comparable numbers of bacteria (P > 0.05 at 30 and 90 min). In contrast, 209

LPS-stimulated cells ingested lower numbers of both encapsulated D39 (P < 0.05 at 90 210

min vs TLR9-treated cells) and nonencapsulated R6 strains (P < 0.05 at 30 min vs 211

TLR1/2- and TLR9-treated cells). 212


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The phagocytic rates were different for both strains: the uptake of the nonencapsulated 213

R6 strain was approximately 10 times more rapid than the internalization of the 214

encapsulated D39 strain. 215

The internalization of both pneumococcal strains by microglia occurred via 216

phagocytosis. Cytochalasin D blocked the uptake of S. pneumoniae D39 and R6 strains 217

by > 90% in unstimulated and TLR-stimulated cells, as it was revealed in 30 min-218

phagocytosis inhibition studies. 219

The extracellular concentration of both pneumococcal strains did not significantly differ 220

throughout 90 min of incubation neither in experiments studying phagocytosis nor in 221

experiments with phagocytosis inhibitors. After 1 h of gentamicin treatment the number 222

of extracellular bacteria was below the level of detection in all experiments. 223

TLR stimulation increased the intracellular killing of S. pneumoniae D39 and R6 224

by microglia. 225

Next we studied whether in TLR-stimulated microglial cells the increase of the 226

phagocytic activity was accompanied by a higher intracellular killing of the ingested 227

bacteria (Fig. 4). 228

The absolute amounts of killed S. pneumoniae D39 (calculated as the difference 229

between the medians of intracellular bacteria at 30 and 120 min) were higher in TLR-230

stimulated microglia than in unstimulated cells (Fig. 4A). The time course of 231

intracellular killing of S. pneumoniae R6 strain was similar to that of the encapsulated 232

strain (Fig. 4B). 233

234

DISCUSSION 235

Streptococus pneumoniae is an important cause of bacterial meningitis causing death in 236

approximately 25% of the cases and long-term neurological sequelae in up to one-third 237


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of the survivors (9, 17, 39, 44). Pro-inflammatory and directly cytotoxic pneumococcal 238

products (such as pneumococcal cell-wall products, pneumolysin, and bacterial DNA) 239

contribute to neuronal injury in S. pneumoniae meningitis. 240

Microglial cells are the major constituents of innate immunity within the CNS (20). 241

Parenchymal microglia as well as meningeal and perivascular macrophages which 242

become activated by bacterial products are critically involved in protecting the brain 243

from infection (30, 33). On the one hand, microglial cells can exert protective effects by 244

phagocytosis of both pathogens and injured cells, and by mediating repair mechanisms 245

(20, 28). When MyD88 bone marrow chimeric mice were studied after intracerebral 246

injection of Staphylococcus aureus, lack of MyD88 expression in the CNS compartment 247

led to elevated intracrebral S. aureus burdens despite the presence of immunocompetent 248

bone marrow-derived cells (14). On the other hand, activated microglial cells can be 249

toxic to surrounding neurons by releasing e.g. nitric oxide, glutamate, TNFα, and IL-250

1beta. The diminished inflammatory response decreased hearing loss in pneumococcal 251

meningitis in MyD88-deficient mice, and neuronal injury caused by group B 252

streptococci depended on the presence of TLR2 and MyD88 (18, 22). Thus, activation 253

of microglia during infections seems to be a double-edged sword. The innate immune 254

response can protect neurons by preventing the entry of pathogens into the brain but its 255

dysregulation can also be harmful for neuronal integrity and can cause neuronal injury 256

(6, 16, 20, 22, 28). Deeper understanding of the roles for TLRs in resident CNS glia and 257

infiltrating immune cells will provide insights into how the immune response to 258

bacterial infection can be tailored to achieve effective pathogen destruction without 259

inducing excessive bystander damage of surrounding brain parenchyma (13, 26). 260

In this context, we focused our research on the phagocytosis of microglia activated by 261

TLR stimulation. We hypothesized that the activation of the TLR system in microglial 262


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cells by agonist stimulation may enhance their phagocytic activity, thereby enabling 263

them to protect the brain in pneumococcal CNS infections in patients with an impaired 264

immune system. 265

The release of cyto-/chemokines in the CSF during pneumococcal meningitis has been 266

analyzed. Interleukin 6 (IL-6) is one of the major early response cytokines that can 267

trigger an inflammatory cascade in pneumococcal meningitis (15). In many resident 268

cells such as microglial cells and astrocytes, chemokine production is rapidly up-269

regulated upon activation by stimuli such as bacteria or inflammatory mediators (24, 270

32). An up-regulation of the expression of CCL2, CCL5, and CXCL2 chemokines was 271

observed in lungs, blood and brain tissue after intranasal inoculation of S. pneumoniae 272

strains (serotypes 2, 4 and 6A) in mice (25). In our study, when microglia were exposed 273

to a TLR1/2, 4 or 9 ligand for 24 h, the release of IL-6 and CCL5 was strongly 274

increased confirming microglial activation. 275

Upon TLR stimulation reactive microglia develop a phagocytic phenotype to engulf and 276

kill microbes. In contrast to cyto-/chemokine induction, the phagocytic and bactericidal 277

profiles of activated microglia have been explored less thoroughly. Our group has 278

recently reported that TLR1/2, 4 and 9 agonists can increase the ability of murine 279

microglial cells to phagocytose and kill intracellularly located Escherichia coli strains 280

(36). The present data demonstrate that microglia can also phagocytose and kill Gram-281

positive bacteria which have a thicker cell wall, and that stimulation of TLRs can 282

increase their phagocytic and bactericidal activity. This applies for both 283

nonencapsulated apathogenic and encapsulated pathogenic pneumococci. Stimulation 284

with either a TLR1/2, 4 or 9 agonists significantly increased the ability of microglia to 285

phagocytose pneumococci. From our data, the effect of the stimulation through the 286

TLR9 system was clearly greater than the effect caused via TLR1/2 or TLR4. Similarly, 287


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phagocytosis and killing of live S. pneumoniae were found to be impaired in alveolar 288

and bone marrow derived macrophages from TLR9-deficient mice (1) and in blood-289

derived polymorphonuclear leukocytes from TLR2-deficient mice (23). 290

Once bacteria have been phagocytosed, they are incorporated into phagolysosomes and 291

exposed to reactive oxygen species that eventually will result in bacterial lysis. The 292

intracellular killing of S. pneumoniae by microglial cells was more rapid than that of E. 293

coli, studied in the same experimental setting (36). For this reason, the number of viable 294

intracellular bacteria determined after 90 min of phagocytosis was lower than the 295

concentration of viable intracellular bacteria detected after 30 min. 296

The presence of the polysaccharide capsule is an important virulence factor of 297

pneumococci because it decreases bacterial uptake into microglia by more than ten 298

times (Figure 3). In addition, we showed that the internalization of pneumococcal 299

strains by murine microglia requires intact actin filaments since this process was 300

blocked by > 90% by cytochalasin D (Figure 2). Not only the phagocytic but also the 301

bactericidal activities of reactive microglia depend on the stimulation of the TLR 302

system. In our study, plotting the intracellular bacterial concentration versus time 303

revealed higher absolute numbers of killed bacteria in TLR-stimulated than in 304

unstimulated microglia, i.e. TLR stimulation clearly increased the efficacy of microglia 305

in neutralizing the internalized S. pneumoniae (Figure 4). 306

An intact Toll-like receptor (TLR) signaling through the pathway organized by MyD88 307

appears to be necessary to protect the brain tissue against invading microorganisms. A 308

poor outcome because of high bacterial counts in the CNS and severe bacteremia was 309

observed in MyD88-deficient mice after intracisternal induction of pneumococcal 310

meningitis (19). Similarly, MyD88-/-

mice showed an increased susceptibility to 311

pneumococcal colonization within the upper respiratory tract, an enhanced bacterial 312


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proliferation in infected lung tissue, precocious bacterial spread into the bloodstream, 313

and increased mortality (2). These findings illustrate the importance of an intact innate 314

immune system to efficiently limit the spread of S. pneumoniae. 315

Stimulation of the TLR system is a potential target for the development of new therapies 316

in multiple diseases (45). Several TLR agonists are currently at different stages of 317

clinical trials (4). The TLR7 agonist imiquimod has been successfully used and approved 318

for the treatment of warts associated to human papillomavirus and is in a second phase 319

trial as a therapeutic agent for herpes simplex virus (HSV) infections (43). The TLR7/8 320

ligand resiquimod also is the subject of clinical investigations for the treatment of HSV 321

infections (27). CpG DNA has been tested as vaccine adjuvant showing good results (8). 322

One of the most interesting clinical trials with CPG 7909 has been recently completed 323

and aimed at comparing the immune responses after TLR9-boostered pneumococcal 324

vaccination in HIV-infected adults 325

(www.clinicaltrials.gov/ct2/show/NCT00562939?term=TLR9&rank=3). 326

Therefore, the agonists used in this study or related compounds could be of value as 327

adjuvants to improve the efficiency of the local immune system of the CNS against 328

bacteria. In the parmacological administration of TLR agonists as adjuvants, the dose, 329

timing and duration of the immunotherapy as well as the route of administration have to 330

be selected to maximize the benefit of the enhancement of the immune response but also 331

to restrict an excessive induced response that might lead to autoimmune diseases or 332

increased neuronal injury (4). 333

One clear advantage of using TLR agonists as adjuvants for the prophylaxis of bacterial 334

meningitis is the low risk of development of resistance to the compound. For microglial 335

activation, agonists with a low molecular mass would be preferable because of their 336


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higher penetration across the BBB (4). The entry of LPS into the central nervous 337

compartments is minimal (5). 338

In conclusion, stimulation of TLRs increases phagocytosis of Gram-positive S. 339

pneumoniae by microglia. Stimulation of the TLR system may be a therapeutic 340

approach to protect the brain from invading pathogens. Further studies in 341

immunocompromised mice are in progress in order to assess whether the resistance of 342

the brain against infections can be increased by priming microglial cells with TLR 343

agonists. 344

345

ACKNOWLEDGEMENTS 346

This work was supported by the European Union (grant CAREPNEUMO), the Else 347

Kröner-Fresenius-Stiftung (R.N.) and the SFB/TR43 (U.K.H.). S. R. was a recipient of 348

a fellowship from the “Departament d’Educació i Universitats de la Generalitat de 349

Catalunya”. 350


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FIG.1. (A) Interleukin 6 (IL-6) and (B) CCL5 (Regulated upon Activation Normal T 496

cell Expressed and Secreted, RANTES) concentrations (in pg/ml) in the supernatants of 497

microglia after 24 h of stimulation with 0.1 µg/ml Pam3CSK4 (P3C), 0.01 µg/ml LPS, 1 498

µg/ml bacterial CpG DNA or DMEM plus 10% FCS (unstim). Data are shown as means 499

± SD (n≥ 13 wells/group from three independent experiments). Data were analyzed 500

using ANOVA followed by Bonferroni's multiple comparison test (*P < 0.05; **P < 501

0.01; ***P < 0.001). 502


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FIG.2. Phagocytosis of (A-C) the encapsulated D39gfp and (D-F) the nonencapsulated 503

D39gfp∆cps S. pneumoniae strains by murine microglial cells after 30 min of bacterial 504

exposure. Internal and external cell membranes were stained with red Vybrant DiI prior 505

to the addition of bacteria. Confocal images of microglial cells ingesting green 506

fluorescent S. pneumoniae are shown in the x-y plane, as well as two z-axis (XZ and 507

YZ) cuts through (A and D) unstimulated cells, and through microglia stimulated for 24 508

h with (B and E) 1 µg/ml bacterial CpG DNA. (C and F) The addition of cytochalasin D 509

(final concentration, 10 µM) blocked the phagocytosis of S. pneumoniae strains by 510

CpG-stimulated microglial cells. Scale bars are shown in panel A, 5µm (XY plane) and 511

2µm (YZ and XZ projected planes). 512


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FIG.3. Phagocytosis of (A) the encapsulated D39 and (B) the nonencapsulated R6 513

Streptococcus pneumoniae (Spn) strains by murine microglial cells after 24 h of 514

stimulation with TLR agonists: Pam3CSK4 (P3C, 0.1 µg/ml), LPS (0.01 µg/ml), or CpG 515

DNA (1 µg/ml). A control group of unstimulated cells was included in all experiments. 516

After stimulation, cells were washed and bacteria were added for different times (30 and 517

90 min). After addition of gentamicin (200 µg/ml), the number of ingested bacteria was 518

determined by quantitative plating of the cell lysates. Data are shown as CFU of 519

recovered bacteria per well (median ± 75% interquartile range) (n≥ 10 wells/group 520

obtained from four independent experiments). Statistical analysis was performed using 521

Kruskal-Wallis test followed by Dunn's multiple comparison test (*P < 0.05 and ***P < 522

0.001 vs control group; #P < 0.05 and

##P < 0.01 vs LPS-treated group). 523


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FIG.4. Time course of the number of live intracellular pneumococci [(A) encapsulated 524

D39, and (B) nonencapsulated R6 Streptococcus pneumoniae (Spn)] detected within 525

microglial cells after 24 h of stimulation with the TLR agonists Pam3CSK4 (P3C, 0.1 526

µg/ml), LPS (0.01 µg/ml), or CpG DNA (1 µg/ml). Monolayers were washed and 527

allowed to ingest bacteria for 30 min. Then, gentamicin was added and the amount of 528

intracellular bacteria was quantified by plating at several post-infection times for up to 529

120 min. For each group, intracellular killing is expressed as the number of recovered 530

bacteria (median) at the different time points (n≥ 6 wells/group obtained from three 531

independent experiments). 532


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