Download of the TLR2/MyD88 Pathway in Microglia by Group B Streptococci

A Mechanism for Neurodegeneration Induced
by Group B Streptococci through Activation
of the TLR2/MyD88 Pathway in Microglia
This information is current as
of June 12, 2017.
Seija Lehnardt, Philipp Henneke, Egil Lien, Dennis L.
Kasper, Joseph J. Volpe, Ingo Bechmann, Robert Nitsch,
Joerg R. Weber, Douglas T. Golenbock and Timothy
Vartanian
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The Journal of Immunology is published twice each month by
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1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 177:583-592; ;
doi: 10.4049/jimmunol.177.1.583
http://www.jimmunol.org/content/177/1/583
The Journal of Immunology
A Mechanism for Neurodegeneration Induced by Group B
Streptococci through Activation of the TLR2/MyD88 Pathway
in Microglia1
Seija Lehnardt,2*§ Philipp Henneke,2储# Egil Lien,# Dennis L. Kasper,† Joseph J. Volpe,‡
Ingo Bechmann,§ Robert Nitsch,§ Joerg R. Weber,¶ Douglas T. Golenbock,2# and
Timothy Vartanian2,3*
T
he 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. Recog-
*Department of Neurology, Beth Israel Deaconess Medical Center, Center for Neurodegeneration and Repair, and the Program in Neuroscience, †Channing Laboratory,
Brigham and Women’s Hospital, and ‡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; 储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
Received for publication November 17, 2005. Accepted for publication April
11, 2006.
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.).
2
S.L., P.H., D.T.G., and T.V. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Timothy Vartanian, Harvard
Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address:
[email protected]
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.
Copyright © 2006 by The American Association of Immunologists, Inc.
nition 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-␬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-␤ (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 –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 –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
0022-1767/06/$02.00
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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
TLR2ⴚ/ⴚ or MyD88ⴚ/ⴚ 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. The Journal of Immunology, 2006, 177: 583–592.
584
GBS INDUCE NEURONAL INJURY VIA TLR2/MyD88 PATHWAY
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
MyD88⫺/⫺ and TLR2⫺/⫺ mice were generously provided by Dr. S. Akira
(Department of Host Defense, Osaka University, Osaka, Japan). C.C3HTlr4Lps-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.
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 ⫻60) 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.
Primary cultures of purified cortical neurons
Results
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 ⫻ g for 5 min. The cell
concentration was adjusted to 1 ⫻ 106 cells/ml in MEM with GlutaMAX
medium (Invitrogen Life Technologies) supplemented with 10% FBS and
penicillin/streptomycin. A total of 1 ⫻ 106 cells were plated onto poly-Dlysine-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.
GBS and GBS-F induce neuronal death via microglia
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-␮m and then 10-␮m 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 ␤-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.
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 ␮g/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).
LTA is a well-defined constituent of the cell wall of Grampositive 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 ␮g/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
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Animals
Measurement of nitrite
The Journal of Immunology
585
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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
␮g/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 ␮m. Quantitation of MAP2⫹ neurons in purified and microgliaenriched cultures in the presence or absence of 108 CFU GBS/ml (B) or 2.5 ␮g/ml GBS-F (D). Purified neurons cocultured with microglia were treated
with GBS LTA in doses from 0.001–20 ␮g/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 ␮g/ml GBS LTA for 36 h is shown. Scale bar,
50 ␮m. 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).
586
GBS INDUCE NEURONAL INJURY VIA TLR2/MyD88 PATHWAY
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 ␮g/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 ␮m.
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
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 ␮g/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.
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
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 ␮m.
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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.
The Journal of Immunology
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.
GBS- and GBS-F-induced NO production is dependent on a
functional MyD88/TLR2 pathway in microglia and is a main
cause of neurotoxicity
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 ␮g/ml
GBS-F compared with control conditions and reached a peak with
108 CFU GBS/ml or 2.5 ␮g/ml. In contrast, in microglia derived
from MyD88⫺/⫺ mice secretion of NO was only marginally increased with 108 CFU GBS/ml or 2.5 ␮g/ml GBS-F. Peaks in the
latter microglia required 109 CFU GBS/ml or 25 ␮g/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 ␮g/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 ␮M), 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
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 ␮g/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.
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We next investigated the mechanism by which GBS- and GBS-Ftreated 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 domaincontaining adapters) triggers the activation of NF-␬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.
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
587
588
GBS INDUCE NEURONAL INJURY VIA TLR2/MyD88 PATHWAY
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FIGURE 5. (Legend continues)
The Journal of Immunology
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 ␮g/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.
TLR4 functions as the signal-transducing receptor for LPS (46),
which is a major component of the outer membrane of Gramnegative 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-␣, IL-1,
and IL-6 (46 – 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
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-␣, 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
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 ␮g/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 ␮m. (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 ␮g/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.
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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 ␮g/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.
589
590
GBS INDUCE NEURONAL INJURY VIA TLR2/MyD88 PATHWAY
with respect to TLR2/6-dependent activation of macrophages and
TLR2-dependent activation of microglia. Moreover, GBS-F is
clearly distinct from GBS-␤-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 ␣-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
The authors have no financial conflict of interest.
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GBS LTA is an attractive candidate molecule to account for both
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TLR multimer (63, 64). It is likely that GBS-F contains several
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