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M icroglia, the resident innate immune effector cells of from the cell wall of Gram-positive bacteria (4, 5). Our laboratory
the CNS parenchyma, represent a first line of defense is interested in defining the signals responsible for microglial ac-
against invading CNS pathogens (1). As such, micro- tivation and subsequent induction of proinflammatory mediator ex-
glia are armed with an extensive repertoire of pattern recognition pression in response to the Gram-positive bacterium Staphylococ-
receptors (PRRs)3 including TLRs that are likely pivotal for max- cus aureus, a prevalent CNS pathogen and frequent etiologic agent
imizing pathogen detection (2). To date, the TLR family includes of brain abscess in humans (6, 7). Understanding the mechanisms
a total of 11 receptors that are responsible for the recognition of by which Gram-positive bacteria engage CNS glia may lead to the
highly conserved structural motifs that are essential for pathogen development of novel therapeutic strategies aimed at attenuating
survival and are conserved across broad subclasses of microorgan- inappropriate innate immune activation that has been associated
isms (3, 4). These conserved microbial structural motifs are re- with several CNS infectious diseases including bacterial meningi-
ferred to as pathogen-associated molecular patterns (PAMPs), and tis and brain abscess (8 –11).
at least one agonist has been identified for each TLR with the Recent studies have established that microglia constitutively ex-
exception of TLR10. TLR2 recognizes the widest array of PAMPs press TLR2 (12–18) (reviewed in Ref. 2). With regard to TLR2
in heterodimeric complexes with either TLR1 or TLR6, including
Ags, as defined by studies investigating the functional importance
lipoproteins and peptidoglycan (PGN) that are components of all
of TLR2 in macrophage activation, microglia are capable of rec-
bacteria possessing cell walls as well as lipoteichoic acid (LTA)
ognizing numerous TLR2 ligands including PGN, LTA, and tri-
palmitoyl-S-glycerylcysteine (Pam3Cys) (13, 16, 18 –21). Con-
cerning the functional importance of TLR2 in microglial
Department of Neurobiology and Developmental Sciences, University of Arkansas
for Medical Sciences, Little Rock, AR 72205 activation, our group was the first to report that this PRR plays an
Received for publication December 14, 2005. Accepted for publication March essential role in enabling PGN recognition using primary micro-
22, 2006. glia isolated from TLR2 knockout (KO) and wild-type (WT) mice
The costs of publication of this article were defrayed in part by the payment of page (18). Interestingly, microglial responses to the Gram-positive
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
pathogen S. aureus were found to be largely TLR2 independent,
1 with the majority of proinflammatory mediators examined in re-
This work was supported by the National Institutes of Health National Institute of
Mental Health (RO1 MH65297) to T.K. and the National Institute of Neurological sponse to intact bacteria equivalently expressed in both TLR2 KO
Disorders and Stroke-supported core facility at University of Arkansas for Medical and WT cells, suggesting that microglia use alternative PRRs for
Sciences (P30 NS047546).
2
S. aureus recognition (18). One possible candidate is TLR9 which
Address correspondence and reprint requests to Dr. Tammy Kielian, Department of
Neurobiology and Developmental Sciences, University of Arkansas for Medical Sci- recognizes nonmethylated CpG motifs that are prevalent in bacte-
ences, 4301 West Markham Street, Slot 846, Little Rock, AR 72205. E-mail address: rial DNA (5). In addition, it is plausible that TLR1 and TLR6, both
KielianTammyL@uams.edu of which are expressed by microglia (13, 16), are sufficient to
3
Abbreviations used in this paper: PRR, pattern recognition receptor; PAMP, patho- account for the residual proinflammatory mediator production ob-
gen-associated molecular pattern; PGN, peptidoglycan; LTA, lipoteichoic acid; KO,
knockout; WT, wild type; TIR, TLR/IL-1R; IRAK, IL-1R-associated kinase; qRT- served in TLR2 KO microglia following bacterial stimulation (18).
PCR, quantitative real-time RT-PCR; PTX3, pentraxin-3; LOX-1, lectin-like oxidized However, although several studies have defined the array of TLRs
low density lipoprotein receptor-1; NOD2, nucleotide-binding oligomerization do-
main 2; NOS, NO synthase; iNOS, inducible NOS; MDP, muramyl dipeptide; TRIF, expressed on microglia (12, 16, 22, 23), a comprehensive assess-
TIR domain-containing adaptor inducing IFN-; IRF-3, IFN regulatory factor-3. ment of the functional relevance of TLRs in enabling microglia to
recognize PAMPs has not yet been performed. Therefore, the cur- Unless otherwise indicated, heat-inactivated S. aureus (107 CFU/ml), PGN
rent study was designed to directly investigate the functional im- (10 g/ml), and LPS (100 ng/ml) were added to microglial cultures at the
portance of multiple TLRs in mediating microglial activation final concentrations indicated, representing doses that were previously de-
termined to be optimal for inducing maximal proinflammatory cytokine
through the use of primary microglia isolated from MyD88 KO expression in microglia without any evidence of cytotoxicity (18, 35). In
and WT mice, where MyD88 represents a common adaptor protein these studies, we used a multiplicity of infection of S. aureus to microglia
required for signaling of all TLRs identified to date, with the ex- of 50:1. In our in vivo model of brain abscess, bacterial titers reach 106- 107
ception of TLR3. CFU within the first 24 h following infection and continue to peak until day
5 (10, 37). When one considers the focal nature of brain abscess, and hence
Upon ligand binding, the intracellular portion of TLRs, referred S. aureus localization in the brain parenchyma, and the fact that bacteria
to as the Toll/IL-1R (TIR) domain due to its similarity with the double in number every 5–10 min, the ratios of S. aureus:microglia used in
cytoplasmic domain of the IL-1R, initiates the TLR signaling cas- our study are likely an underrepresentation of the actual pathogen burden
cade (24, 25). The TIR domain is highly conserved among all that microglia would encounter in vivo.
TLRs (with the exception of TLR3) and mediates its downstream ELISA
signaling effects by interacting with the TIR-containing common
adaptor protein MyD88. Interestingly, in addition to TLRs, both Comparisons in cytokine and chemokine expression between MyD88 WT
and KO microglia were performed using standard sandwich ELISA kits
the IL-1 and IL-18 receptors also use MyD88 to transduce activa- according to the manufacturer’s instructions (OptEIA mouse TNF-␣, IL-12
tion signals emanating from receptor ligation by their respective p40, IL-1, and MCP-1/CCL2; BD Pharmingen; DuoSet mouse MIP-2/
cytokines (26). Association of the TIR domain of MyD88 with that CXCL2; R&D Systems). The level of sensitivity for all ELISAs was 15.6
of TLRs recruits the serine/threonine kinase IL-1R-associated ki- pg/ml.
nase (IRAK). Subsequently, IRAK is activated by autophosphor- Cell viability assays
ylation and dissociates from the receptor complex to interact with
Role of MyD88 on the expression of alternative PRRs in general, engagement of phagocytic receptors does not directly lead
microglia to cytokine induction, these receptors are capable of influencing
Previous studies from our laboratory have revealed that both S. the activity of cytokine signaling receptors through receptor cross-
aureus and PGN modulate the expression of alternative PRRs in talk. Therefore, we investigated the potential involvement of
microglia, including the phagocytic receptors LOX-1 and PTX3 as MyD88-dependent pathways on the modulation of phagocytic
well as TLR9 (18, 38). Because LOX-1 and PTX3 are considered PRRs. The induction of microglial LOX-1 expression in response
phagocytic receptors and can directly bind pathogens (53–55), they to all bacterial stimuli was time dependent, with maximal effects
may be responsible for S. aureus uptake by microglia. Although in observed at 6-h poststimulation (Fig. 7). MyD88 was found to play
The Journal of Immunology 6807
(Fig. 9A). Interestingly, the level of PGN- and LPS-induced suppres- ber of the nucleotide-binding oligomerization domain family of
sion of TLR9 expression was significantly less in MyD88 KO micro- proteins and considered a PRR because it recognizes the bacterial
glia compared with WT cells (Fig. 9, B and C). product muramyl dipeptide (MDP), a degradation product of bac-
To expand our analysis of alternative PRRs in microglia, we terial PGN (56, 57). A recent study has proposed that NOD2 could
evaluated changes in the expression of NOD2 in response to bac- represent a negative regulator of TLR2-dependent signaling (58).
terial stimuli in MyD88 KO and WT microglia. NOD2 is a mem- However, to our knowledge the expression of NOD2 in microglia
has not yet been reported. All three microbial stimuli tested were
effective at augmenting microglial NOD2 expression (Fig. 10). In
addition, MyD88-dependent signal(s) were found to be important,
in part, for regulating NOD2 levels as demonstrated by the atten-
uation of NOD2 expression in MyD88 KO microglia compared
with WT cells (Fig. 10).
Collectively, these findings revealed that MyD88-dependent
signaling pathways regulate, to varying extents, the expression of
distinct PRRs including LOX-1, TLR9, PTX3, and NOD2. These
findings reveal the complexity by which PRR expression is regu-
lated in microglia. The establishment of an intricate set of signal-
ing paradigms to augment PRR expression would ensure that the
loss of a particular signaling pathway would not compromise mi-
croglial recognition of pathogens with potentially devastating ef-
Discussion
Bacterial infections of the CNS continue to occur, despite ad-
vances made in detection and therapy. In addition, the emergence
of multidrug-resistant strains of CNS pathogens, such as S. aureus,
has become a confounding factor that is magnified by the inability
of many antibiotics to reach high therapeutic levels in brain tissue.
Therefore, understanding the mechanisms by which CNS bacterial
pathogens are recognized by resident glia and infiltrating immune
cells are critical toward the development of new therapeutic mo-
dalities for infection. The role of MyD88, the central adaptor mol-
ecule essential for signaling of the TIR family of receptors, has
been examined in various systemic and CNS infections caused by
a variety Gram-positive organisms including Streptococcus pneu-
moniae and S. aureus (31, 59 – 61). However, the functional im-
portance of MyD88 in enabling the recognition of diverse bacterial
motifs by microglia has not yet been evaluated. Therefore, in the
present study, we examined the effects of S. aureus, a common
etiological agent of brain abscesses, and the bacterial cell wall
components PGN and LPS on the activation of MyD88 KO and
WT microglia. Our results revealed that MyD88 is pivotal for mi-
croglial activation and subsequent proinflammatory mediator re-
lease in response to all three bacterial stimuli (S. aureus, PGN,
and LPS).
A recent publication has raised a debate as to whether PGN
represents a true TLR2 ligand, a tenant that is supported by nearly
200 publications from various research groups. Specifically,
Travassos et al. (36) reported that PGN is not recognized by TLR2
but instead by the intracellular PRR NOD2 because purified PGN
was capable of activating NOD2-transfected, but not TLR2-trans-
FIGURE 9. The loss of MyD88 results in differential expression of fected, HEK293 cells. However, in a recent study, Dziarski et al.
TLR9 mRNA expression in response to S. aureus, PGN, or LPS. MyD88 (62) re-evaluated the effects of highly purified PGN on TLR2-
KO and WT microglia were seeded at 2 ⫻ 106 cells/well in 6-well plates transfected HEK293 cells and found that polymeric PGN is a po-
and incubated overnight. The following day, cells were stimulated with tent TLR2 ligand, whereas muramidase treatment dramatically re-
either 107 heat-inactivated S. aureus (A), 10 g/ml PGN (B), or 100 ng/ml duced the responsiveness of cells to PGN. Therefore, the authors
LPS (C) for 6, 12, or 24 h, whereupon total RNA was isolated and exam- suggested that various purification procedures could damage the
ined for TLR9 expression by qRT-PCR as described in Materials and cross-linked TLR2-activating structure of PGN which is required
Methods. Gene expression levels were calculated after normalizing TLR9
for TLR2, but not NOD activation. Our previous and current find-
signals against GAPDH and are presented in relative mRNA expression
units (mean ⫾ SEM of three independent experiments). Significant differ-
ings support the premise that PGN represents a TLR2 agonist in
ences in TLR9 mRNA expression between unstimulated vs PAMP-treated microglia, because we demonstrated that neither TLR2 nor MyD88
microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001), whereas KO microglia were responsive to PGN (18). Importantly, the com-
significant differences between MyD88 KO vs WT microglia are denoted mercial PGN preparations used in this study were not found to
with a hatched sign (#, p ⬍ 0.05; ##, p ⬍ 0.001). contain detectable phosphate levels, indicating that any differences
The Journal of Immunology 6809
examined; however, it is possible that LOX-1 is involved in bac- to impair NF-B activation, IRF-3 has been implicated as a me-
terial uptake and subsequent processing, providing ligands to en- diator of TNF-␣ activation via a MyD88-independent pathway.
gage TLR9 to augment antibacterial immunity. Additional studies In conclusion, our results indicate that microglial proinflamma-
examining the functional significance of LOX-1 in microglial re- tory mediator release induced by the Gram-positive stimuli S. au-
sponses to bacteria are needed to assess its role in phagocytosis. reus and PGN occurs via a MyD88-dependent pathway and in-
We also examined the role of MyD88 in regulating the expression volves the activity of multiple TLRs. In addition, the expression of
of the soluble PRR PTX3 in microglia. PTX3 expression was sig- additional phagocytic PRRs, including LOX-1 and PTX3, is reg-
nificantly enhanced following stimulation with either, S. aureus, ulated, in part, by MyD88-dependent signals. In contrast, LPS-
PGN, or LPS in WT cells; however, PTX3 levels in MyD88 KO dependent microglial activation is regulated by both MyD88-de-
microglia were similar to baseline, indicating that MyD88-depen- pendent and -independent mechanisms. Moreover, we report the
dent signal(s) are critical for inducing the expression of this sol- novel expression of the intracellular PRR, NOD2, in microglia and
uble PRR. This finding is in agreement with our previous report find that this receptor is augmented in response to Gram-positive
demonstrating that CD14-dependent signals, likely in cooperation bacteria and its cell wall product PGN. We are currently extending
with TLR2, are essential for maximal PTX3 expression in PGN- these studies to examine the role of MyD88 in S. aureus-dependent
activated microglia (18, 38). microglial activation in vivo using the experimental brain abscess
NOD2 is an intracellular PRR that recognizes MDP moieties model developed in our laboratory, where microglial responses
that are released as a result of hydrolytic or lysosomal digestion of will be quantified in FACS-purified populations from MyD88 WT
PGN (56, 57). The interaction of NOD2 with MDP leads to NF-B and KO mice. Collectively, these results reveal the importance of
activation and subsequent proinflammatory gene expression (70). MyD88, and by extension, multiple TLRs, in mediating microglial
One interesting finding that surfaced during the course of these activation in response to a wide array of PAMPs. However, it is
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