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The Journal of Immunology, 2006, 177: 583-592.
Copyright © 2006 by The American Association of Immunologists

A Mechanism for Neurodegeneration Induced by Group B Streptococci through Activation of the TLR2/MyD88 Pathway in Microglia1

Seija Lehnardt2,*,§, Philipp Henneke2,||,#, Egil Lien#, Dennis L. Kasper{dagger}, Joseph J. Volpe{ddagger}, Ingo Bechmann§, Robert Nitsch§, Joerg R. Weber, Douglas T. Golenbock2,# and Timothy Vartanian2,3,*

* Department of Neurology, Beth Israel Deaconess Medical Center, Center for Neurodegeneration and Repair, and the Program in Neuroscience, {dagger} Channing Laboratory, Brigham and Women’s Hospital, and {ddagger} Department of Neurology, Children’s Hospital, Harvard Medical School, Boston, MA 02115; § Center for Anatomy, Institute of Cell Biology and Neurobiology, and Department of Neurology, Charité-Universitaetsmedizin Berlin, Berlin, Germany; || Division of Pediatric Infectious Diseases, Children’s Hospital, Albert-Ludwigs-University, Freiburg, Germany; and # Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Group B Streptococcus (GBS) is a major cause of bacterial meningitis and neurological morbidity in newborn infants. The cellular and molecular mechanisms by which this common organism causes CNS injury are unknown. We show that both heat-inactivated whole GBS and a secreted proteinaceous factor from GBS (GBS-F) induce neuronal apoptosis via the activation of murine microglia through a TLR2-dependent and MyD88-dependent pathway in vitro. Microglia, astrocytes, and oligodendrocytes, but not neurons, express TLR2. GBS as well as GBS-F induce the synthesis of NO in microglia derived from wild-type but not TLR2–/– or MyD88–/– mice. Neuronal death in neuronal cultures complemented with wild-type microglia is NO-dependent. We show for the first time a TLR-mediated mechanism of neuronal injury induced by a clinically relevant bacterium. This study demonstrates a causal molecular relationship between infection with GBS, activation of the innate immune system in the CNS through TLR2, and neurodegeneration. We suggest that this process contributes substantially to the serious morbidity associated with neonatal GBS meningitis and may provide a potential therapeutic target.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The group B Streptococcus (GBS)4 bacteria or Streptococcus agalactiae is considered the leading etiologic agent of neonatal sepsis in the Western world. The current incidence in the neonatal period in the United States is 1.8 cases per 1000 live births, with a mortality rate of 6% (1). In addition, GBS is the third most frequent cause of bacterial meningitis in the United States (2, 3). In newborns GBS accounts for ~50% of all cases of meningitis (4). It is well established that bacterial meningitis is associated with numerous neurological sequelae, including cognitive impairment, seizures, and motor handicaps, in up to 52% of the survivors (5, 6).

Resident cells of the innate immune system of any organ serve as the first line of defense against an invading organism. Recognition of pathogens is achieved in part through the germline encoded cell surface TLRs.

To date, 13 TLR orthologs, of which 10 are expressed in humans, have been identified. TLRs recognize invariant molecular structures associated with pathogens (7). These microbial motifs include LPS, viral DNA, and unmethylated DNA that is rich in CpG motifs (8). Intracellular signaling events downstream of TLRs are of striking complexity. The best-characterized pathway involves the intracellular proteins MyD88, IL-1R-associated kinases 1–4, and TNFR-associated factor 6; these molecules ultimately result in the activation of the transcriptional factor NF-{kappa}B (9). All of the TLRs, save TLR3, use this pathway to some extent. MyD88 is an adapter protein that serves to bridge TLRs to the downstream signaling elements. MyD88-independent signaling pathways also exist. Three additional adapter molecules, TIR domain-containing adapter inducing IFN-beta (TRIF, also known as TICAM-1), TIR domain-containing adapter protein (TIRAP)/MyD88-adapter-like (Mal), and TRIF-related adapter molecule (TRAM) are suggested to play a critical role in MyD88-dependent (TIRAP/Mal) and MyD88-independent (TRIF, TRAM) signaling pathways (10, 11, 12, 13).

TLR2 has been characterized as an immune receptor with an extraordinarily large repertoire of ligands. Indeed, all classes of microorganisms tested to date have been found to activate TLR2. Several Gram-positive bacteria, as well as the bacterial cell wall components peptidoglycan and lipoteichoic acid (LTA), signal through TLR2 (14, 15, 16, 17, 18, 19). The cytoplasmic domain of TLR2 dimerizes with either TLR1 or TLR6, resulting in the generation of a signal that ultimately results in the production of cytokines (20). It has recently been shown that TLR2 mRNA is constitutively expressed in the CNS, particularly in the choroid plexus (21).

It has previously been reported that TLR2, TLR6, and MyD88 are involved in the inflammatory response to GBS both in vitro and in vivo (22, 23). TLRs are of significant interest with respect to GBS-induced neurodegeneration because we previously demonstrated that activation of TLR4 by LPS induces oligodendrocyte and neuronal injury in vitro. The interaction of LPS with TLR4 can convert a subthreshold hypoxic-ischemic CNS injury into irreversible injury in vivo (24, 25). In this report, we define a cellular and molecular relationship between GBS and neurodegeneration. We describe how GBS induces neuronal injury only in the presence of microglia using two functionally distinct preparations of GBS: bacterial cell walls and a released heat-labile factor that has been designated GBS-F. Experiments in MyD88 and TLR2 knockout mice indicate that this microglial activation and neuronal injury induced by GBS cell walls and GBS-F require these signal transduction molecules. Glial cells, such as microglia, oligodendrocytes, and astrocytes, but not neurons, express TLR2. Furthermore, only wild-type, but not TLR2–/– or MyD88–/– microglia produce NO, which we found to be largely responsible for GBS-induced neuronal injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

MyD88–/– and TLR2–/– mice were generously provided by Dr. S. Akira (Department of Host Defense, Osaka University, Osaka, Japan). C.C3H-Tlr4Lps-d (lpsd), BALB/cJ, and C57BL/6J mice were purchased from The Jackson Laboratory. Sprague-Dawley rats were purchased from Taconic Farms. All animal experiments were conducted in accordance with the guidelines of the Harvard Medical School animal facility and were approved by the local ethics committee.

Generation of heat-inactivated GBS, GBS-F, and LTA

Heat-inactivated GBS and GBS-F were prepared as previously described (22). GBS-F was further concentrated by size exclusion chromatography and lyophilization. GBS LTA was prepared as previously described (26). Concentrations of the bacterial challenge are stated as equivalent of CFU corresponding to the number of CFU of living GBS.

Primary cultures of purified cortical neurons

Primary cultures of purified cortical neurons were generated from forebrains of embryonic day 17 (E17) mice, as previously described (25). Briefly, cortices were dissociated by trituration with papain (Worthington) in EBSS (Invitrogen Life Technologies) for 5 min at 37°C. Subsequently, cells were resuspended in 0.25% trypsin inhibitor and 0.25% BSA (both obtained from Sigma-Aldrich) in EBSS, and incubated at 37°C for 5 min. Cells were pelleted by centrifugation at 1000 x g for 5 min. The cell concentration was adjusted to 1 x 106 cells/ml in MEM with GlutaMAX medium (Invitrogen Life Technologies) supplemented with 10% FBS and penicillin/streptomycin. A total of 1 x 106 cells were plated onto poly-D-lysine-coated glass slides (BD Biosciences) and were maintained in humidified 5% CO2/95% air at 37°C. Immediate immunostaining revealed 90–95% purity for neurons.

Primary cultures of purified microglia, oligodendrocytes, and astrocytes

Purified glial cells were generated from forebrains either of 2-day-old Sprague-Dawley rats or of E17 mice as previously described (27). Briefly, brain tissue was dissociated with trypsin (Invitrogen Life Technologies) for 20 min at 37°C. After mechanical dissociation, cells were plated in 75-cm2 culture flasks in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS and penicillin/streptomycin. After 1 wk in culture, mixed glial cultures were shaken for 30 min at 180 rpm. The supernatant containing >95% microglia was plated on poly-D-lysine-coated (BD Bioscience) glass coverslips. Microglia were maintained in DMEM with 10% FBS. Oligodendrocytes were isolated from the remaining adherent cells by shaking for 12 h at 180 rpm. After this second shake, supernatant was plated on tissue culture flasks for 1 h in the presence of leucine methylester and passed through 20-µm and then 10-µm mesh filters, removing most of the remaining astrocytes and microglia. Purified oligodendrocytes were plated on poly-D-lysine-coated coverglass in serum-free DMEM with 0.05% BSA, N2 supplements, platelet-derived growth factor-AA (10 ng/ml), and beta-fibroblast growth factor (10 ng/ml). After removal of oligodendrocytes by the second shake, astrocytes remained as the sole cells of the initial glial cell culture.

Measurement of nitrite

GBS- and GBS-F-induced production of NO in purified microglia was analyzed by measuring the stable end product nitrite in culture supernatants. The amount of NO was determined by using the colorimetric Griess reaction (Sigma-Aldrich) as previously described (28). The inducible NO synthase (iNOS) inhibitor aminoguanidine was also obtained from Sigma-Aldrich.

Immunofluorescence microscopy

Cells were fixed and immunostained as previously described (25). To identify neurons, astrocytes, and oligodendrocytes, the Abs used were, respectively, microtubule-associated protein (MAP2), glial fibrillary acidic protein (GFAP) (both obtained from Chemicon International), and oligodendrocyte type-4 (O4) (American Type Culture Collection). Microglia were stained with IB4-Alexa (Molecular Probes). Nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI; Molecular Probes). The mouse TLR2 Ab was engineered as previously described (29) and obtained from eBioscience.

The Chinese hamster ovary (CHO)-K1 fibroblast-derived cell lines CHO/EL1 (Elam-luc) and CHO/EL1/moTLR2Flag (Elam-luc) used as negative and positive control for TLR2 immunostainings have been previously described (30, 31). TUNEL staining of CNS cultures was conducted using the In Situ Death Detection kit, (TMR red; Roche), following the instruction manual.

Statistical analysis

GBS-, GBS-F-, or LTA-treated cell cultures were stained with an Ab directed against MAP2 or NeuN, with IB4 and with DAPI. Surviving MAP2+ cells were counted manually. For each experiment, triplicate wells were analyzed. Six different fields (at magnification x60) of each culture well were counted. Mean values and SD were calculated from these 18 values. Relative neuronal viability was determined by quantifying NeuN+ or MAP2+ cells. Numbers of NeuN+ or MAP2+ cells under control conditions were set to 100% and all other neuronal numbers are displayed relative to control numbers. The number of independently conducted experiments is indicated in the representative figures. Data are expressed as mean ± SD. Statistical analysis was performed with SigmaStat (version 2.03; SPSS) using the Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GBS and GBS-F induce neuronal death via microglia

To investigate the effect of GBS and GBS-F on neurons, we generated purified neuronal and microglial cultures from cortices of E17 wild-type mice. Neurons were incubated alone or in coculture with microglia and subsequently treated with 108 CFU GBS/ml or 2.5 µg/ml GBS-F for 36 h. The cells were stained with the Ab MAP2 and with the isolectin IB4 to identify neurons and microglia, respectively, and the nuclei were labeled with DAPI (Fig. 1, A and C). In neuronal cultures supplemented with microglia, both GBS and GBS-F induced a significant reduction in numbers of neurons. Also, treatment with GBS or GBS-F resulted in a diminishment of the numbers of microglia compared with control conditions. In contrast, purified neurons alone were not affected by treatment with GBS or GBS-F. The loss of cells was quantified by examining the number of MAP2+ cells per square millimeter. This analysis demonstrated that GBS and GBS-F induced a 15.5-fold reduction and a 19.8-fold reduction in neuronal numbers, respectively, compared with control conditions (Fig. 1, B and D). Furthermore, GBS induced a 6.5-fold reduction in microglial numbers after 36 h compared with control conditions. Time-dependence experiments showed that neuronal numbers decreased significantly after 18 h at the earliest, whereas microglial numbers decreased significantly after 32 h at the earliest (data not shown).


Figure 1
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  		FIGURE 1. GBS and GBS-F induce neuronal cell death that is dependent on the presence of microglia. Cortical neurons and microglia were prepared from E17 C57BL/6J mouse brains. Purified neurons and neurons in combination with microglia were incubated with either 108 CFU GBS/ml (A) or 2.5 µg/ml GBS-F (C). Parallel control cultures were incubated with PBS. After 36 h, cultures were fixed and immunostained with both MAP2 and IB4 to mark neurons and microglia, respectively. All nuclei were stained with DAPI. Scale bar, 50 µm. Quantitation of MAP2+ neurons in purified and microglia-enriched cultures in the presence or absence of 108 CFU GBS/ml (B) or 2.5 µg/ml GBS-F (D). Purified neurons cocultured with microglia were treated with GBS LTA in doses from 0.001–20 µg/ml for 12–72 h. Parallel control cultures were incubated with PBS. Cells were then fixed and stained with an Ab directed against MAP2, with IB4, and with DAPI. E, A representative picture of cells treated with 20 µg/ml GBS LTA for 36 h is shown. Scale bar, 50 µm. F, Data are presented as a percentage of untreated controls. Experiments were performed six times. Results are presented as the mean ± SD. p < 0.001 (Student’s t test).

 
LTA is a well-defined constituent of the cell wall of Gram-positive bacteria (32). To assess the role of LTA in GBS-induced neuronal injury, purified neurons cocultured with microglia were treated with GBS LTA in doses from 0.001–20 µg/ml for 12–72 h. Cells were then fixed and stained with an Ab directed against MAP2, with IB4 and with DAPI (Fig. 1E). GBS LTA did not induce neuronal cell death or neuronal injury during the whole period of observation. Statistical analysis assessing relative neuronal viability after 36 h under the conditions described confirmed these results (Fig. 1F).

These data demonstrate that GBS and GBS-F induce neuronal injury and cell death and indicate that this neurotoxicity is not cell autonomous but rather requires the presence of microglia.

GBS- and GBS-F-induced neuronal injury shows apoptotic characteristics in the presence of microglia

Neuronal apoptosis is induced after various brain insults, including infection with GBS (33, 34). Furthermore, in experimental pneumococcal meningitis, apoptosis is the major mechanism leading to neuronal loss (35, 36). To determine whether the observed neuronal death after treatment of neuronal/microglial cultures with GBS and GBS-F (Fig. 1) was due to apoptosis, we analyzed neurons under the conditions described using TUNEL assay after incubation with GBS (Fig. 2A) and GBS-F (Fig. 2B) for 16 h. Treatment of purified neuronal cultures enriched with microglia with 108 CFU GBS/ml or 2.5 µg/ml GBS-F resulted in the appearance of TUNEL staining, indicating that apoptosis had occurred. In contrast, neuronal cultures without microglia similarly treated with GBS and GBS-F were TUNEL negative, confirming the finding that GBS- and GBS-F-induced neuronal death is dependent on the presence of microglia. As a positive control for TUNEL staining, neurons cocultured with microglia were incubated with 0.1 M camptothecin, a DNA topoisomerase-I inhibitor. In addition, neurons from wild-type mice alone were incubated with GBS and 0.1 M camptothecin.


Figure 2
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  		FIGURE 2. Neurons in microglia-enriched cultures undergo TUNEL-positive cell death after treatment with GBS or GBS-F. Cortical neurons and microglia were prepared from C57BL/6J mouse forebrains. Purified neurons alone as well as neurons cocultured with purified microglia were incubated with either 108 CFU GBS/ml (A) or 2.5 µg/ml GBS-F (B). As a positive control for TUNEL staining, neurons cocultured with microglia were incubated with 0.1 M camptothecin. In addition, neurons from wild-type mice alone were incubated with GBS and 0.1 M camptothecin. After 16 h, cultures were fixed and analyzed by a TUNEL assay. Similar results were obtained in three experiments. Scale bar, 50 µm.

 
TLR2 is expressed in microglia, astrocytes, and oligodendrocytes, but not in neurons

TLR2 plays a critical role in the recognition of Gram-positive bacteria, mycobacteria, and lipopeptides (14, 30, 37, 38). TLR2 mRNA is constitutively expressed in the mouse brain, particularly in the choroid plexus (21). TLR2 mRNA expression in mouse brains has been shown to be increased during pneumococcal infection (39). Expression of TLR2 in microglia has been shown by PCR and Western blot techniques (40, 41). To investigate the presence and localization of TLR2 protein in various CNS cells, primary cultures of microglia, oligodendrocytes, astrocytes, and cortical neurons were fixed and immunostained with TLR2 Ab (Fig. 3). Simultaneously, cells were stained either with IB4 or with the Abs O4, GFAP, or MAP2 to mark microglia, oligodendrocytes, astrocytes, or neurons, respectively. CHO/EL1 cells served as a negative control, whereas CHO/EL1/moTLR2 cells were used as a positive control for TLR2 staining. In parallel, nuclei were detected by DAPI staining. After incubation with the relevant secondary Abs, cells were analyzed by immunofluorescence microscopy. Microglia, astrocytes, and oligodendrocytes revealed intense labeling with the TLR2 Ab. In contrast, neurons did not show labeling with the TLR2 Ab. To test whether neurons express TLR2 after treatment with GBS, purified cortical neurons were incubated with 108 CFU GBS/ml for 6, 12, 24, and 48 h. Subsequently, TLR2 expression was analyzed by immunofluorescence. No TLR2 staining was observed throughout the whole period of observation (data not shown). These data indicate that glial cells express TLR2, whereas neurons do not.


Figure 3
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  		FIGURE 3. Microglia, astrocytes, and oligodendrocytes express TLR2, whereas neurons do not. Enriched cultures of microglia, astrocytes, oligodendrocytes, and neurons were studied for expressing TLR2 by incubating the cells with a mAb raised against mouse TLR2. Cells were double immunostained for IB4, GFAP, O4, and MAP2, respectively. Intense immunofluorescence for TLR2 was observed in microglial, astrocyte, and oligodendrocyte cultures, whereas neurons showed no labeling with the Ab. CHO cells served as a negative control whereas CHO/moTLR2 cells were used as a positive control. Experiments were performed three times. Scale bar, 100 µm.

 
GBS- and GBS-F-induced NO production is dependent on a functional MyD88/TLR2 pathway in microglia and is a main cause of neurotoxicity

We next investigated the mechanism by which GBS- and GBS-F-treated microglia lead to neuronal death. Several possibilities, based on studies by others, were worthy of consideration. Activation of TLRs leads to recruitment of the adapter molecule MyD88 (42). Engagement of MyD88 (as well as the other TIR domain-containing adapters) triggers the activation of NF-{kappa}B (15, 43). GBS and GBS-F induce NO production in murine macrophages (22). Reactive oxygen species, including NO, contribute to neuronal death (44, 45). Indeed, this mechanism of cell death has been proposed to occur in an infant rat model of GBS meningitis (33).

To determine whether microglia respond to GBS and GBS-F by releasing NO, we incubated purified microglia derived from C57BL/6J, MyD88–/–, or TLR2–/– mice with either GBS or GBS-F at various concentrations (Fig. 4, A and B) and for various times (Fig. 4, C and D). Subsequently, culture supernatants were analyzed for NO by the colorimetric Griess reaction.


Figure 4
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  		FIGURE 4. GBS and GBS-F induce NO production in microglia through an MyD88- and TLR2-dependent pathway. Purified microglia derived from C57BL/6J, MyD88–/–, and TLR2–/– mice were incubated for 48 h with increasing concentrations of GBS (A) or GBS-F (B), or treated with either 108 CFU GBS/ml (C) or 2.5 µg/ml GBS-F (D) for various incubation times. The amount of nitrite in the culture supernatants was determined using the Griess reaction. E, Cortical neurons and microglia were prepared from C57BL/6J mouse forebrains. Cocultures were incubated with either 108 CFU GBS/ml alone or in combination with the iNOS inhibitor aminoguanidine (AG). After 36 h, cultures were fixed and immunostained with NeuN and DAPI. Data are presented as a percentage of untreated controls and as mean ± SD. p < 0.001 (Student’s t test). Experiments were performed three times with similar results.

 
Purified microglia derived from C57BL/6J mice responded to both GBS and GBS-F with production of NO, whereas no such response was seen in microglia derived from TLR2–/– or MyD88–/– mice. The dose dependence of the NO response to GBS or GBS-F was analyzed in microglia from C57BL/6J, TLR2–/–, and MyD88–/– mice after incubation for 72 h. The production of NO in microglia derived from wild-type mice was significantly increased with 105 CFU GBS/ml or 0.25 µg/ml GBS-F compared with control conditions and reached a peak with 108 CFU GBS/ml or 2.5 µg/ml. In contrast, in microglia derived from MyD88–/– mice secretion of NO was only marginally increased with 108 CFU GBS/ml or 2.5 µg/ml GBS-F. Peaks in the latter microglia required 109 CFU GBS/ml or 25 µg/ml GBS-F, and were four and 13 times lower, respectively, than those observed in wild-type-derived microglia. Microglia generated from TLR2–/– mice did not show increased NO production after treatment with the indicated concentrations of GBS or GBS-F.

The time course of NO secretion observed in microglia from wild-type mice revealed a significant increase after 3 h incubation with 108 CFU GBS/ml. Treatment with 2.5 µg/ml GBS-F resulted in an increase in NO production after 12 h. In contrast, in microglia generated from MyD88–/– mice, an elevation of NO production after treatment with GBS or GBS-F was not seen until after 72 h, and the level of NO concentration was approximately four times lower when compared with wild-type mice. Microglia derived from TLR2–/– mice did not significantly respond to treatment with GBS or GBS-F throughout the whole round of observation.

To determine the contribution of NO to GBS-induced neurotoxicity wild-type cortical neurons cocultured with microglia derived from wild-type mice were treated either with 108 CFU/ml GBS alone or in combination with the iNOS inhibitor aminoguanidine (200 µM), starting 1 h before stimulation with GBS, for 36 h (Fig. 4E). Cells were then stained for NeuN and DAPI. Whereas in neuronal cultures supplemented with microglia, GBS induced a reduction in the number of neurons to 10% compared with control cultures, the number of neurons in cultures pretreated with aminoguanidine was only slightly diminished to ~90% of the control cell count. This analysis demonstrated that blockade of iNOS protected neurons from microglial GBS-induced cell toxicity. As described, not only neurons but also microglia undergo cell death after treatment with GBS. Although GBS-induced microglial apoptosis is delayed as compared with neuronal apoptosis, we cannot rule out that microglia undergoing cell death may release other yet unidentified molecules besides NO that contribute to neuronal cell death after treatment with GBS.

To investigate whether microglia produce NO in response to GBS LTA, we incubated purified microglia with GBS LTA in doses from 0.001–20 µg/ml for 12–72 h. Whole GBS served as a positive control. Cell supernatants were then analyzed by the Griess reaction. In contrast to whole GBS, incubation with GBS LTA did not increase the content of NO in the supernatant of microglia compared with control conditions throughout the whole round of observation (data not shown).

Finally, we investigated the ability of purified cortical neurons to produce NO after treatment with GBS. Purified neurons were treated with 107, 108, and 109 CFU GBS/ml for 12, 24, 48, and 72 h. Microglia treated with GBS served as a positive control. Cell supernatants were then analyzed by the Griess reaction. No increase of NO in the supernatant of neurons compared with control conditions was observed throughout the whole period of observation (data not shown).

In summary, GBS and GBS-F induce microglial NO secretion, which requires a functional TLR2/MyD88 pathway. Furthermore, we have identified NO as a main effector of GBS-induced neurotoxicity.

GBS- and GBS-F-induced neuronal death is dependent on a functional MyD88 and TLR2 pathway in microglia

To determine whether GBS- and GBS-F-induced neurotoxicity requires functional MyD88 and TLR2, we purified microglia from C57BL/6J, MyD88–/–, and TLR2–/– mice and added these cells to purified cortical neurons prepared from wild-type mice. Cultures were then treated with 108 CFU GBS/ml or 2.5 µg/ml GBS-F (Fig. 5, A and C). Immunostaining with MAP2 showed that treatment of neurons cocultured with MyD88–/– or TLR2–/– microglia did not affect neuronal survival. In contrast, neurons supplemented with microglia derived from mice of the wild-type strain C57BL/6J suffered cell death. Statistical analysis of the number of MAP2+ neurons per square millimeter confirmed these results (Fig. 5, B and D). In parallel, neurons from wild-type mice cocultured with microglia derived from either MyD88–/– or TLR2–/– mice were incubated with 0.1 M camptothecin to confirm the specificity of the resistance to GBS- and GBS-F-induced neuronal injury. In addition, neurons from wild-type mice alone were incubated with GBS and 0.1 M camptothecin (Fig. 5, A and C). Subsequent MAP2, IB4, and DAPI staining demonstrated neuronal injury and cell loss in both cultures.


Figure 5
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  		FIGURE 5. GBS and GBS-F induce neuronal death dependent on an MyD88- and TLR2-dependent pathway in microglia. Cortical neurons were prepared from C57BL/6J mouse forebrains and supplemented with purified microglia from C57BL/6J or MyD88–/– mice (A and C) or TLR2–/– mice (E and G). Cells were incubated with either 108 CFU GBS/ml (A and E) or 2.5 µg/ml GBS-F (C and G). After 36 h, cultures were fixed and stained with both MAP2 Ab and IB4 to mark neurons and microglia, respectively. All nuclei were stained with DAPI. Scale bar, 50 µm. (B, D, F, and H) Quantification of MAP2-positive neurons in purified C57BL/6J, MyD88–/–, or TLR2–/– microglia-enriched cultures in the presence or absence of either 108 CFU GBS/ml or 2.5 µg/ml GBS-F. Purified microglia from lpsd and wild-type mice were added to cortical neurons prepared from wild-type mice. Cultures were treated with 108 CFU GBS/ml (I). After 36 h, cultures were fixed and stained with both MAP2 Ab and IB4 to mark neurons and microglia, respectively. All nuclei were stained with DAPI. MAP2+ neurons were quantified. Results are presented as mean ± SD. p < 0.001 (Student’s t test). Experiments were performed three times with similar results.

 
TLR4 functions as the signal-transducing receptor for LPS (46), which is a major component of the outer membrane of Gram-negative bacteria. To determine whether GBS-induced neurotoxicity requires functional TLR4, we purified microglia from lpsd (C.C3H-Tlr4Lps-d) and BALB/cJ (wild-type) mice and added these cells to purified cortical neurons prepared from wild-type mice. The lpsd mutation originated from the C3H/HeJ mouse and was introduced into the BALB/cJ background by backcrossing C3H/HeJ mice to BALB/cJ mice. The resulting lpsd (C.C3H-Tlr4Lps-d) mice were used in our experiments. The tlr4 mutant mouse C3H/HeJ is characterized by hyporesponsiveness to LPS as a consequence of a functionally defective TLR4 membrane protein due the point mutation that interferes with LPS-induced signaling. Macrophages from this strain fail to induce inflammatory cytokines such as TNF-{alpha}, IL-1, and IL-6 (46, 47, 48). Cultures were treated with 108 CFU GBS/ml for 36 h (Fig. 5I). Immunostaining with MAP2 showed that treatment of neurons cocultured with microglia derived from lpsd mice suffered cell death to a similar extent as neurons supplemented with microglia derived from the wild-type strain. Quantitative analysis of the number of MAP2+ neurons per square millimeter confirmed these results. Thus, GBS-induced neuronal injury is not dependent on a functional TLR4 pathway in microglia. Taken together, these findings show that GBS- and GBS-F-induced neuronal death requires a functional TLR2/MyD88 pathway in microglia.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
GBS remains the single most common cause of bacterial meningitis in the first year of life. Moreover, GBS has become the third most common cause of bacterial meningitis overall since the general implementation of Haemophilus influenzae type b immunization (49).

Although highly active antibiotics are available for treatment of GBS meningitis, around 50% of infants surviving GBS meningitis exhibit varying degrees of long-term neurodevelopmental sequels (6, 50). Accordingly, new therapeutic approaches are needed to prevent brain injury in bacterial meningitis.

It is widely accepted that the neuronal injury associated with bacterial meningitis results from the local inflammatory response (36) and bacterial toxins, at least in pneumococcal disease (35, 51). Several proinflammatory factors, such as TNF-{alpha}, IL-1, and IL-8, cause neuronal injury in meningitis (52). This report adds evidence to this pathophysiological model of meningitis by demonstrating that the prototypical organism GBS is capable of causing neuronal injury via a molecular mechanism that involves TLR2, MyD88, and NO.

Neurons, particularly in the hippocampal granule cell layer, undergo apoptosis during bacterial meningitis (53, 54). There is no consensus regarding the mechanism of cell death. Earlier data in an infant rat model of GBS-induced bacterial meningitis demonstrated necrotic cell death of cortical neurons 24 h after infection (33). Our studies support the concept that apoptosis is involved, as cortical neurons cocultured with microglia became TUNEL-positive and showed histological features consistent with neurodegeneration after 16 h of GBS treatment. It is important to make a distinction between necrosis and apoptosis because pharmacological therapy is far more likely to be capable of preventing an event of cell signaling leading to apoptosis than a necrotic event. Thus, antagonism of TLR2 and/or downstream signaling molecules in microglia might significantly prevent neuronal loss and associated complications of postmeningitis such as deafness and cognitive impairment.

Our data suggest that TLR2-mediated neuronal apoptosis via microglia occurs both as a result of direct interaction with the GBS cell wall as well as through recognition of factors secreted by GBS. GBS LTA is an attractive candidate molecule to account for both because it constitutes an integral component of the cell wall, occurs in the GBS supernatant as a result of cell wall remodeling, and engages TLR2 (26). Surprisingly, whereas engagement of TLR2 is essential for GBS cell wall-induced NO formation in microglia and hence for neuronal apoptosis, TLR2 does not significantly contribute to GBS cell wall-mediated cytokine formation in peritoneal macrophages from the same strain of wild-type mice (55). The reasons for this discrepancy are unknown. The redundant role of TLR2 in the activation of peritoneal macrophages by GBS cell wall is best explained by the existence of another signaling pathway, which is MyD88-dependent and therefore likely involves a TLR that has not been identified yet. The existence of this pathway is further documented by the observation that the essential TLR2-adapter (Mal) is dispensable for the GBS-induced cytokine response of peritoneal macrophages (P. Henneke, unpublished observations). It is tempting to speculate that differences in receptor expression between microglia and peritoneal macrophages account for the observed discrepancies in TLR engagement by GBS. Also, although macrophages outside the CNS interact with other immune cells, such as T and B lymphocytes, microglia may interact with astrocytes, oligodendrocytes, and neurons in a way that is not yet understood (56, 57).

We did not observe a toxic effect of GBS LTA on neurons. It remains uncertain at this stage whether GBS LTA contributes to GBS-induced neuronal injury at all. In contrast to LTA from other bacterial species such as Staphylococcus aureus and Lactococcus, GBS LTA exhibits weak inflammatory activity and appears not to be important for GBS cell wall-induced cytokine formation in peritoneal macrophages (58, 59). Alternative candidates for GBS cell wall components that ligate TLR2 are the more than thirty putatively N-terminal diacylated lipoproteins encoded in the GBS genome (<www.mrc-lmb.cam.ac.uk/genomes/dolop/>). Although synthetic bacterial lipoproteins and lipoproteins from Escherichia coli, mycoplasmatacea, and mycobacteria have been shown to induce apoptosis via TLR2 (60, 61, 62), little is known about the apoptotic potential of lipoproteins from Gram-positive bacteria.

Liberated factors from GBS (GBS-F) induce neuronal apoptosis via microglia. Both LTA and lipoproteins might contribute to this activity. Like GBS-F, GBS LTA engages TLR6 as a TLR2 coreceptor. However, purified GBS-F is ~100-fold more potent for TLR2 activity than highly purified LTA from GBS. Therefore, LTA cannot fully explain GBS-F activity in macrophages (22). Furthermore, GBS-F, but not purified LTA, induces the release of NO by microglia and causes neuronal injury in the presence of microglia. It is conceivable that, similar to LTA, GBS lipoproteins engage TLR2/6 because the diacylglycerol transferase lgt is highly conserved in Gram-positive bacteria and diacylated proteins from other bacterial classes such as Mycoplasmataceae recognize this TLR multimer (63, 64). It is likely that GBS-F contains several chemically distinct molecules despite its functional homogeneity with respect to TLR2/6-dependent activation of macrophages and TLR2-dependent activation of microglia. Moreover, GBS-F is clearly distinct from GBS-beta-hemolysin, which has previously been implicated in GBS-induced apoptosis (22, 65). Irrespective of its molecular identity, the ability of GBS-F to induce neuronal injury is likely of pathophysiological significance because of its potential ability to cause neurodegeneration in distinct areas of the brain independently of the local presence of living bacteria. The purification, biochemical characterization, and cloning of GBS-F should make this a testable hypothesis in the near future.

It is of great interest that there is increasing evidence for a connection of the immune system to critical aspects of the nervous system. Nicotinic receptor blockade is a highly effective therapeutic approach in animal models of septic shock, whereas {alpha}-adrenergic receptor activation may modulate the effects of systemic endotoxin (66, 67). We believe that microglia will ultimately prove to function on the interface of the immune system with the nervous system.

This work defines a deleterious role for the innate immune system in the CNS under conditions of bacterial inflammation and provides further evidence for the importance of this system in neuronal degeneration. An obvious advantage of inducing an inflammatory response in the brain is to protect the CNS from invading microorganisms. However, it is now apparent that an immune response contributes to neuronal injury in various neurodegenerative diseases by triggering an accelerated proinflammatory activity (68). Because the CNS is not readily capable of self-renewal as are many other organs, the outcome of such proinflammatory activity can be injurious. For example, robust transcriptional activation of TLR2 and CD14 has been shown in the brain in murine experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (69). It is well established that this demyelinating disease is based on chronic inflammation induced by immunological triggers. TLR2 is also induced in mice with amyotrophic lateral sclerosis (70). Moreover, we have demonstrated recently that in the mouse, hypoxia-ischemia in combination with LPS converts a subthreshold insult to severe neurodegeneration in a TLR4-dependent fashion (25). Nevertheless, little is known about the physiological significance of TLRs in the CNS, and the mechanisms controlling such potentially deleterious innate immune responses remain elusive.

In conclusion, we provide evidence that cortical neurons undergo cell death when treated with whole heat-inactivated group B streptococci or with a factor released from GBS. GBS-induced neurotoxicity is dependent on the presence of microglia expressing MyD88 and TLR2. Our data suggest a causal relationship between infection with Gram-positive bacteria, activation of the innate immune cells in the CNS, and subsequent neurodegeneration. These findings highlight the potential for the development of a specific immunotherapy directed toward modulating microglial activity as a means of preventing the neurological sequelae of bacterial meningitis.


   Acknowledgment
 
We thank Kimberly Rosegger and Eckart Schott for helpful comments preparing this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grant RG3426A2/1 from the National Multiple Sclerosis Society (to T.V.), Grant NS38475 from the National Institute of Neurologic Disorders and Stroke (to T.V.), Grant He 3127/2-1 from Deutsche Forschungsgemeinschaft (to P.H.), and Grants AI52455, R01AI057588, and GM54060 from the National Institutes of Health (to P.H. and E.L.). Back

2 S.L., P.H., D.T.G., and T.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Timothy Vartanian, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: tvartani{at}bidmc-harvard.edu Back

4 Abbreviations used in this paper: GBS, group B Streptococcus; LTA, lipoteichoic acid; CHO, Chinese hamster ovary; iNOS, inducible NO synthase; GFAP, glial fibrillary acidic protein; MAP, microtubule-associated protein; O4, oligodendrocyte type-4; DAPI, 4',6'-diamidino-2-phenylindole. Back

Received for publication November 17, 2005. Accepted for publication April 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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Toll-Like Receptor Signaling Is Critical for Wallerian Degeneration and Functional Recovery after Peripheral Nerve Injury
J. Neurosci., November 14, 2007; 27(46): 12565 - 12576.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
S. Lehnardt, J. Wennekamp, D. Freyer, C. Liedtke, C. Krueger, R. Nitsch, I. Bechmann, J. R. Weber, and P. Henneke
TLR2 and Caspase-8 Are Essential for Group B Streptococcus-Induced Apoptosis in Microglia
J. Immunol., November 1, 2007; 179(9): 6134 - 6143.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
O. Hoffmann, J. S. Braun, D. Becker, A. Halle, D. Freyer, E. Dagand, S. Lehnardt, and J. R. Weber
TLR2 Mediates Neuroinflammation and Neuronal Damage
J. Immunol., May 15, 2007; 178(10): 6476 - 6481.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
T. Kielian, N. K. Phulwani, N. Esen, M. Md. Syed, A. C. Haney, K. McCastlain, and J. Johnson
MyD88-Dependent Sign