Central Role for MyD88 in the Responses of

Microglia to Pathogen-Associated Molecular

This information is current as
of April 14, 2022.
Patterns
Nilufer Esen and Tammy Kielian
J Immunol 2006; 176:6802-6811; ;
doi: 10.4049/jimmunol.176.11.6802
http://www.jimmunol.org/content/176/11/6802

Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022

References This article cites 76 articles, 21 of which you can access for free at:
http://www.jimmunol.org/content/176/11/6802.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision
• No Triage! Every submission reviewed by practicing scientists
• Fast Publication! 4 weeks from acceptance to publication
*average

Subscription Information about subscribing to The Journal of Immunology is online at:

http://jimmunol.org/subscription
Permissions Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
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.
 The Journal of Immunology

Central Role for MyD88 in the Responses of Microglia to

Pathogen-Associated Molecular Patterns1

Nilufer Esen and Tammy Kielian2

Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microor-
ganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3,
use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in
response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, sug-
gesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S.
aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from
MyD88 knockout (KO) and wild-type mice. The induction of TNF-␣, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and
peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in
MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and

Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022

lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated
expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88
KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms
for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways. The
Journal of Immunology, 2006, 176: 6802– 6811.

M icroglia, the resident innate immune effector cells of
the CNS parenchyma, represent a first line of defense
against invading CNS pathogens (1). As such, micro-
glia are armed with an extensive repertoire of pattern recognition
receptors (PRRs)3 including TLRs that are likely pivotal for max-
imizing pathogen detection (2). To date, the TLR family includes
a total of 11 receptors that are responsible for the recognition of
highly conserved structural motifs that are essential for pathogen
survival and are conserved across broad subclasses of microorgan-
isms (3, 4). These conserved microbial structural motifs are re-
ferred to as pathogen-associated molecular patterns (PAMPs), and
at least one agonist has been identified for each TLR with the
exception of TLR10. TLR2 recognizes the widest array of PAMPs
in heterodimeric complexes with either TLR1 or TLR6, including
lipoproteins and peptidoglycan (PGN) that are components of all
bacteria possessing cell walls as well as lipoteichoic acid (LTA)
Department of Neurobiology and Developmental Sciences, University of Arkansas
for Medical Sciences, Little Rock, AR 72205
Received for publication December 14, 2005. Accepted for publication March
22, 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 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
Disorders and Stroke-supported core facility at University of Arkansas for Medical
Sciences (P30 NS047546).
2
Address correspondence and reprint requests to Dr. Tammy Kielian, Department of
Neurobiology and Developmental Sciences, University of Arkansas for Medical Sci-
ences, 4301 West Markham Street, Slot 846, Little Rock, AR 72205. E-mail address:
KielianTammyL@uams.edu
3
Abbreviations used in this paper: PRR, pattern recognition receptor; PAMP, patho-
gen-associated molecular pattern; PGN, peptidoglycan; LTA, lipoteichoic acid; KO,
knockout; WT, wild type; TIR, TLR/IL-1R; IRAK, IL-1R-associated kinase; qRT-
PCR, quantitative real-time RT-PCR; PTX3, pentraxin-3; LOX-1, lectin-like oxidized
low density lipoprotein receptor-1; NOD2, nucleotide-binding oligomerization do-
main 2; NOS, NO synthase; iNOS, inducible NOS; MDP, muramyl dipeptide; TRIF,
TIR domain-containing adaptor inducing IFN-␤; IRF-3, IFN regulatory factor-3.
from the cell wall of Gram-positive bacteria (4, 5). Our laboratory
is interested in defining the signals responsible for microglial ac-
tivation and subsequent induction of proinflammatory mediator ex-
pression in response to the Gram-positive bacterium Staphylococ-
cus aureus, a prevalent CNS pathogen and frequent etiologic agent
of brain abscess in humans (6, 7). Understanding the mechanisms
by which Gram-positive bacteria engage CNS glia may lead to the
development of novel therapeutic strategies aimed at attenuating
inappropriate innate immune activation that has been associated
with several CNS infectious diseases including bacterial meningi-
tis and brain abscess (8 –11).
Recent studies have established that microglia constitutively ex-
press TLR2 (12–18) (reviewed in Ref. 2). With regard to TLR2
Ags, as defined by studies investigating the functional importance
of TLR2 in macrophage activation, microglia are capable of rec-
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
activation, our group was the first to report that this PRR plays an
essential role in enabling PGN recognition using primary micro-
glia isolated from TLR2 knockout (KO) and wild-type (WT) mice
(18). Interestingly, microglial responses to the Gram-positive
pathogen S. aureus were found to be largely TLR2 independent,
sponse to intact bacteria equivalently expressed in both TLR2 KO
and WT cells, suggesting that microglia use alternative PRRs for
S. aureus recognition (18). One possible candidate is TLR9 which
recognizes nonmethylated CpG motifs that are prevalent in bacte-
rial DNA (5). In addition, it is plausible that TLR1 and TLR6, both
of which are expressed by microglia (13, 16), are sufficient to
account for the residual proinflammatory mediator production ob-
served in TLR2 KO microglia following bacterial stimulation (18).
However, although several studies have defined the array of TLRs
expressed on microglia (12, 16, 22, 23), a comprehensive assess-
ment of the functional relevance of TLRs in enabling microglia to

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

The Journal of Immunology
recognize PAMPs has not yet been performed. Therefore, the cur-
rent study was designed to directly investigate the functional im-
portance of multiple TLRs in mediating microglial activation
through the use of primary microglia isolated from MyD88 KO
and WT mice, where MyD88 represents a common adaptor protein
required for signaling of all TLRs identified to date, with the ex-
ception of TLR3.
Upon ligand binding, the intracellular portion of TLRs, referred
to as the Toll/IL-1R (TIR) domain due to its similarity with the
cytoplasmic domain of the IL-1R, initiates the TLR signaling cas-
cade (24, 25). The TIR domain is highly conserved among all
TLRs (with the exception of TLR3) and mediates its downstream
signaling effects by interacting with the TIR-containing common
adaptor protein MyD88. Interestingly, in addition to TLRs, both
the IL-1 and IL-18 receptors also use MyD88 to transduce activa-
tion signals emanating from receptor ligation by their respective
cytokines (26). Association of the TIR domain of MyD88 with that
of TLRs recruits the serine/threonine kinase IL-1R-associated ki-
nase (IRAK). Subsequently, IRAK is activated by autophosphor-
ylation and dissociates from the receptor complex to interact with
TNFR-associated factor-6. Consequently, the association of
TNFR-associated factor-6 with IRAK leads to the activation of
two distinct signaling pathways, namely the NF-␬B and MAPK
pathways. Both the NF-␬B and MAPK pathways are capable of
regulating the expression of numerous immune response genes in-
cluding proinflammatory cytokines and chemokines (27). Several
groups have demonstrated the importance of MyD88 in signaling
through numerous TLRs either in MyD88 knockout mice (26, 28 –
31) or through the expression of a mutant MyD88 protein that no
longer is capable of transducing an activation signal (32, 33).
However, the role of MyD88, and as an extension all TLRs, in
enabling microglia to recognize and respond to PAMPs of clinical
importance to CNS infectious diseases has not yet been examined.
To determine the functional importance of multiple TLRs in S.
aureus-dependent microglial activation, we evaluated the role of
MyD88-dependent pathways using primary microglia isolated
from MyD88 KO and WT mice.
Materials and Methods
Primary microglia cell culture and reagents
MyD88 gene KO mice (originally from Dr. S. Akira, Osaka University,
Suita, Osaka, Japan) (26) were purchased from the Centre de La Recherche
Scientifique and have been previously backcrossed with C57BL/6 mice for
over 10 generations (29, 34). Primary microglia were isolated from MyD88
KO or WT C57BL/6 pups (Harlan Laboratories; 1– 8 days of age) as pre-
viously described (35). The purity of microglial cultures was evaluated by
immunohistochemical staining using Abs against CD11b (BD Pharmingen)
and glial fibrillary acidic protein (DakoCytomation). The purity of primary
microglial cultures was routinely ⬎95%.
Heat-inactivated S. aureus (strain RN6390, provided by Dr. A. Cheung,
Dartmouth Medical School, Hanover, NH) was prepared as previously de-
scribed (13). PGN derived from S. aureus was obtained from Fluka and
Invivogen and Escherichia coli O11:B1 LPS was from List Biological
Laboratories. Heat-inactivated S. aureus was used in these studies rather
than live organisms to control the standard dosing of bacteria within and
between individual experiments. Live organisms exhibit an extremely rapid
doubling time (i.e., 5–10 min) that can quickly lead to culture overgrowth,
medium exhaustion, and subsequent microglial death. Therefore, we
elected to use heat-inactivated bacteria throughout the course of our studies
to avoid these potentially confounding effects. All non-LPS reagents and
culture medium were verified to have endotoxin levels ⬍0.03 endotoxin
units/ml as determined by Limulus amebocyte lysate assay (Associates of
Cape Cod). In addition, a phosphate assay (Pierce) was performed on PGN
stocks to ensure reagent purity since commercial PGN preparations have
been reported to be contaminated with LTA and bacterial DNA (36),
whereas PGN does not contain phosphate residues. The phosphate content
of the PGN preparation used in this study was below the limit of detection,
indicating that it was not contaminated with alternative microbial products.
6803
Unless otherwise indicated, heat-inactivated S. aureus (107 CFU/ml), PGN
(10 ␮g/ml), and LPS (100 ng/ml) were added to microglial cultures at the
final concentrations indicated, representing doses that were previously de-
termined to be optimal for inducing maximal proinflammatory cytokine
expression in microglia without any evidence of cytotoxicity (18, 35). In
these studies, we used a multiplicity of infection of S. aureus to microglia
of 50:1. In our in vivo model of brain abscess, bacterial titers reach 106- 107
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
S. aureus localization in the brain parenchyma, and the fact that bacteria
double in number every 5–10 min, the ratios of S. aureus:microglia used in
our study are likely an underrepresentation of the actual pathogen burden
that microglia would encounter in vivo.
ELISA
Comparisons in cytokine and chemokine expression between MyD88 WT
and KO microglia were performed using standard sandwich ELISA kits
according to the manufacturer’s instructions (OptEIA mouse TNF-␣, IL-12
p40, IL-1␤, and MCP-1/CCL2; BD Pharmingen; DuoSet mouse MIP-2/
CXCL2; R&D Systems). The level of sensitivity for all ELISAs was 15.6
pg/ml.
Cell viability assays
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
To confirm that the observed changes in inflammatory mediator expression
between MyD88 WT and KO microglia were not due to differences in cell
seeding densities or potential confounding proliferative effects, a standard
MTT assay based on the mitochondrial conversion of MTT into formazan
crystals was performed as previously described (35).
Quantitative real-time RT-PCR (qRT-PCR)
Total RNA from MyD88 WT and KO microglia was isolated using the
TRIzol reagent and treated with DNase1 (both from Invitrogen Life Tech-
nologies) before use in qRT-PCR studies. The experimental procedure was
performed as previously described (18). Briefly, TLR2 and GAPDH prim-
ers and TAMRA TaqMan probes were designed as previously described
(18, 38) and synthesized by Applied Biosystems. ABI Assays-on-Demand
TaqMan kits were used to examine microglial IL-23 p19, TLR9, pen-
traxin-3 (PTX3), lectin-like oxidized low density lipoprotein receptor-1
(LOX-1), and nucleotide-binding oligomerization domain 2 (NOD2) ex-
pression. Comparisons in gene expression between MyD88 WT and KO
primary microglia were calculated after normalizing cycle thresholds
against the “housekeeping” gene GAPDH and are presented as the fold-
induction (2⫺⌬⌬Ct, where Ct is the cycle threshold) value relative to un-
stimulated microglia.
Protein extraction and Western blotting
Protein extracts were prepared and quantitated from both MyD88 WT and
KO microglia as previously described (35). The effects of heat-inactivated
S. aureus, PGN, and LPS on microglial TLR2, MyD88, and inducible NO
synthase (iNOS) protein expression were evaluated by Western blot anal-
ysis. Blots were probed using goat anti-mouse TLR2 (R&D Systems), rab-
bit anti-mouse MyD88 (eBioscience), or rabbit anti-mouse NOS2 (iNOS;
Santa Cruz Biotechnology) Abs followed by the appropriate IgG-HRP con-
jugated secondary Abs (Jackson ImmunoResearch Laboratories). Blots
were stripped and reprobed with a rabbit anti-actin polyclonal Ab (Sigma-
Aldrich) for normalization. Blots were developed using the Immobilon
Western substrate (Millipore) and visualized by exposure to x-ray film.
Statistics
Significant differences between experimental groups were determined by
the Student t test at the 95% confidence interval using Sigma Stat (SPSS
Science).
Results
Both Gram-positive and -negative bacterial stimuli enhance
MyD88 expression in primary microglia
The signal transduction pathways emanating from all of the TLRs
identified to date, with the exception of TLR3, use the common
adaptor protein MyD88 (28, 39, 40). Recently, we have shown that
both S. aureus and its cell wall component PGN significantly in-
creased TLR2 expression at both the mRNA and protein levels
(18). In the present study, the effects of S. aureus and PGN as well
as the Gram-negative bacterial cell wall product LPS, on MyD88
6804
protein expression in primary microglia was evaluated by Western
blotting. As shown in Fig. 1, all three bacterial stimuli were found
to enhance microglial MyD88 protein levels. As we had recently
reported, TLR2 protein expression was elevated in response to S.
aureus, PGN, and LPS in WT microglia (18, 38) (Fig. 2). Inter-
estingly, compared with WT microglia, constitutive TLR2 expres-
sion was extremely low and not induced in response to either S.
aureus or PGN treatment in MyD88 KO cells (Fig. 2). In contrast,
LPS stimulation was capable of inducing TLR2 expression in
MyD88 KO microglia (Fig. 2). Collectively, these results suggest
that MyD88 is pivotal for the induction of TLR2 protein expres-
sion in response to both S. aureus and PGN, which may serve to
potentate microglial responses to Gram-positive pathogens
through receptor up-regulation. In contrast, the induction of TLR2
expression in response to LPS occurs via a MyD88-independent
pathway possibly via alternative adaptor proteins such as TIR do-
main-containing adaptor protein and TIR domain-containing adap-
tor inducing IFN-␤ (TRIF).
MyD88 regulates LPS-induced iNOS expression
NO is a highly reactive free radical that performs a wide variety of
cellular functions in addition to serving as an effective cytotoxic
agent used by immune cells to inactivate pathogens (41). It is
produced by any one of three enzyme isoforms termed NO syn-
thases (NOS). Endothelial NOS and neuronal NOS occur consti-
tutively and mediate vasodilation and neurosignaling, respectively.
iNOS was first demonstrated in monocytes but is now known to be
expressed in a variety of cell types, including microglia (42). iNOS
expression is stimulated in glia by a variety of inflammatory cy-
tokines and bacterial products including LPS. Although recent
studies have implicated TLR4 in mediating the LPS-dependent
production of reactive oxygen species in microglia (43), the ques-
tion of whether MyD88-independent pathways are also involved
and the role of MyD88 in mediating NO expression in response to
Gram-positive bacterial stimuli has not yet been investigated.
MyD88-dependent signals, presumably delivered via TLR4 en-
gagement, were found to be pivotal for iNOS induction in LPS-
stimulated microglia as reflected by a significant attenuation in
iNOS levels in MyD88 KO microglia (Fig. 3B). However, the
residual iNOS expression detected in MyD88 KO cells indicated
that MyD88-independent pathways are also involved. Interest-
ingly, we could not detect iNOS protein expression in either S.
FIGURE 1. MyD88 is induced in primary microglia in response to both
Gram-positive and -negative bacterial stimuli. MyD88 KO and WT pri-
mary microglia were stimulated with either 107 heat-inactivated S. aureus,
10 ␮g/ml PGN, or 100 ng/ml LPS for 24 h, whereupon protein extracts
from whole cell lysates (40 ␮g/sample) were evaluated for MyD88 expres-
sion by Western blotting as described in Materials and Methods. Following
analysis, blots were stripped and reprobed with an Ab specific for ␤-actin
to verify uniformity in gel loading. Results are representative of three in-
dependent experiments.
MyD88 IN MICROGLIA
FIGURE 2. LPS-induced TLR2 expression is regulated by a MyD88-
independent pathway. MyD88 KO and WT primary microglia were stim-
ulated with either 107 heat-inactivated S. aureus, 10 ␮g/ml PGN, or 100
ng/ml LPS for 24 h, whereupon protein extracts from whole cell lysates (40
␮g/sample) were evaluated for TLR2 expression by Western blotting as
described in Materials and Methods. Following analysis, blots were
stripped and reprobed with an Ab specific for ␤-actin to verify uniformity
in gel loading. Results are representative of three independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
aureus- or PGN-stimulated microglia suggesting that these bacte-
rial stimuli are poor inducers of iNOS in primary microglia (Fig.
3B). The inability to detect iNOS protein expression in response to
S. aureus and PGN by Western blots may have resulted from lim-
itations in the sensitivity of this technique. Therefore, we per-
formed highly sensitive qRT-PCR analysis and were able to dem-
onstrate that both S. aureus and PGN were capable of inducing
iNOS mRNA expression in WT microglia. However, compared
with LPS, the induction levels were very low, ⬃20- to 30-fold less
in S. aureus or PGN-stimulated cells compared with the former
(Fig. 3A). In addition, iNOS mRNA levels in response to both S.
aureus and PGN were significantly attenuated in MyD88 KO mi-
croglia compared with WT cells (Fig. 3A). Therefore, the reason(s)
that iNOS protein could not be detected in primary microglia in
response to S. aureus or PGN could be due to either sensitivity
issues with Western blots and/or the fact that alterations in mRNA
levels do not always necessarily extend to differences in protein
expression.
MyD88-dependent signals are pivotal for the induction of
proinflammatory mediator release from S. aureus- and PGN-
stimulated microglia
Recently, we reported that TLR2 plays an important role in mi-
croglial recognition of PGN, whereas responses to intact S. aureus
were primarily TLR2 independent because TLR2 ablation did not
completely inhibit proinflammatory mediator production from S.
aureus-stimulated microglia (18). Similar findings have also been
reported in macrophages; however, macrophage activation was
completely attenuated in response to S. aureus treatment in
MyD88 KO cells, suggesting an important role for alternative
TLRs (30, 31, 44). In the present study, an essential role for TLRs
in S. aureus-dependent microglial activation was demonstrated.
Specifically, TNF-␣ production was completely abolished in
MyD88 KO microglia treated with PGN and minimal cytokine
expression was detected in response to intact S. aureus only at the
highest dose examined (Fig. 4, A and B). In contrast, microglial
TNF-␣ production in response to LPS was found to be mediated by
both MyD88-dependent and -independent pathways (Fig. 4C).
Similar results were obtained for IL-1␤ (data not shown). Exam-
ination of chemokine expression in MyD88 KO and WT microglia
revealed a similar requirement for MyD88 in response to S. aureus
The Journal of Immunology
FIGURE 3. Role of MyD88-dependent signals in regulating iNOS ex-
pression. A, MyD88 KO and WT microglia were seeded at 2 ⫻ 106 cells/
well in 6-well plates and incubated overnight. The following day, cells
were stimulated with 107 heat-inactivated S. aureus, 10 ␮g/ml PGN, or 100
ng/ml LPS for 6 or 24 h, whereupon total RNA was isolated and examined
for iNOS expression by qRT-PCR as described in Materials and Methods.
Gene expression levels were calculated after normalizing iNOS signals
against GAPDH and are presented in relative mRNA expression units
(mean ⫹ SEM of three independent experiments). Significant differences
in iNOS mRNA expression between unstimulated (data not shown) vs
PAMP-treated microglia are denoted with asterisks (ⴱ, p ⬍ 0.05), whereas
significant differences between MyD88 KO vs WT microglia are denoted
with a hatched sign (#, p ⬍ 0.05). B, MyD88 KO and WT primary mi-
croglia were stimulated with either 107 heat-inactivated S. aureus, 10
␮g/ml PGN, or 100 ng/ml LPS for 24 h, whereupon protein extracts from
whole cell lysates (40 ␮g/sample) were evaluated for iNOS expression by
Western blotting as described in Materials and Methods. Following anal-
ysis, blots were stripped and reprobed with an Ab specific for ␤-actin to
verify uniformity in gel loading. Results are representative of three inde-
pendent experiments.
or PGN. Specifically, production of the potent neutrophil chemoat-
tractant MIP-2/CXCL2 was completely attenuated in response to
either S. aureus or PGN in MyD88 KO microglia compared with
WT cells (Fig. 5A). In addition, MyD88 KO microglia produced
significantly lower levels of MIP-2/CXCL2 in response to LPS
compared with WT cells (Fig. 5A). Similar findings were observed
for the monocyte/lymphocyte chemoattractant MCP-1/CCL2 (data
not shown). Importantly, the concentrations of all bacterial stimuli
used in this study did not adversely affect microglial cell viability,
indicating that the attenuated responses of MyD88 KO microglia
were not due to overt cell death or random differences in cell-
seeding density (Fig. 4D).
Recent studies from our laboratory have demonstrated that, un-
like other proinflammatory cytokines examined, IL-12 p40 release
was significantly increased in both S. aureus-stimulated TLR2 and
CD14 KO microglia compared with WT cells (18, 38). Based on
these results we had suggested that TLR2 signaling, potentially in
cooperation with CD14, regulates IL-12 p40 release by inducing
suppressor cytokines and/or activating a direct suppressor path-
6805
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
FIGURE 4. MyD88 is essential in mediating microglial activation in
response to S. aureus and PGN. MyD88 KO and WT microglia were
seeded at 2 ⫻ 105 cells/well in 96-well plates and incubated overnight. The
following day, cells were exposed to various concentrations of heat-inac-
tivated S. aureus (A), PGN (B), or LPS (C) for 24 h, whereupon condi-
tioned supernatants were collected and analyzed for TNF-␣ protein expres-
sion by ELISA. Results are presented as the amount of TNF-␣ (nanogram)
per milliliter of culture supernatant (mean ⫾ SD). Microglial cell viability
was assessed using a standard MTT assay and the raw OD570 absorbance
values are reported (D; mean ⫾ SD). Significant differences in TNF-␣
expression between unstimulated vs PAMP-treated microglia are denoted
with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001), whereas significant differences
between MyD88 KO vs WT microglia are denoted with a hatched sign (##,
p ⬍ 0.001). Results are representative of three independent experiments.
way. Recently, several reports have demonstrated the presence of
numerous negative regulators of TLR signaling, most of which
target the MyD88-dependent signaling cascade (45, 46). It is en-
visioned that the multimolecular complex formed by MyD88 fol-
lowing TLR signaling is an important target for some negative
regulators and consequently affects the fine tuning of TLR re-
sponses (45). Indeed, in the present study we found that S. aureus
stimulation was not capable of inducing IL-12 p40 production in
MyD88KO microglia, similar to what was observed with PGN
(Fig. 5B). IL-12 p40 production was also attenuated in MyD88 KO
microglia in response to LPS (Fig. 5B). Therefore, it appears as if
S. aureus activation of microglia induces a MyD88-dependent neg-
ative regulatory signal(s) that acts to attenuate the expression of
select proinflammatory mediators such as IL-12 p40.
Biologically active IL-12 consists of two subunits, p35 and p40,
the latter of which is promiscuous in its ability to interact with
other members of the IL-12 cytokine family (47). One such ex-
ample is IL-23, which is composed of a unique p19 subunit and the
common p40 subunit shared with IL-12 (48, 49). Although IL-12
and IL-23 share some overlapping activities in terms of their abil-
ity to impact T cell activation, IL-23 is unique in its ability to
induce memory T cell proliferation (49, 50). Microglia have been
6806 MyD88 IN MICROGLIA

FIGURE 5. The loss of MyD88 results in diminished MIP-2 and IL-12

p40 expression. Primary microglia from MyD88 KO and WT mice were
seeded at 2 ⫻ 105 cells/well in 96-well plates and incubated overnight. The
following day, cells were exposed to either 107 heat-inactivated S. aureus,

Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022

10 ␮g/ml PGN, or 100 ng/ml LPS for 24 h, whereupon conditioned su-
pernatants were collected and analyzed for MIP-2 (A) and IL-12 p40 (B)
protein expression by ELISA. Results are presented as the amount of cy-
tokine (nanograms) per milliliter of culture supernatant (mean ⫾ SD). Sig-
nificant differences in cytokine expression between unstimulated vs
PAMP-treated microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.001), whereas significant differences between MyD88 KO vs WT micro-
glia are denoted with a hatched sign (##, p ⬍ 0.001). Results are repre-
sentative of three independent experiments.

reported to express IL-23 in response to IFN-␥ and LPS stimula-

tion (51, 52); however, the role of MyD88-dependent signals in
regulating IL-23 levels has not yet been examined. To extend our
analysis of cytokines associated with adaptive immunity, we eval-
uated the expression of IL-23 p19 mRNA in response to intact S.
aureus, PGN, and LPS in MyD88 KO and WT microglia using
qRT-PCR. All three bacterial stimuli were capable of eliciting
IL-23 p19 expression in WT microglia; however, the induction of
IL-23 p19 in response to S. aureus did not reach statistical signif-
icance (Fig. 6). Although there was no induction of IL-23 p19
mRNA expression in MyD88 KO microglia in response to S. au-
reus, the differences between S. aureus-treated WT and KO cells
was statistically insignificant (Fig. 6A). Conversely, MyD88-de-
FIGURE 6. MyD88 is involved in IL-23 p19 mRNA expression.
pendent signals were found to be important for IL-23 p19 induc- MyD88 KO and WT microglia were seeded at 2 ⫻ 106 cells/well in 6-well
tion in response to both PGN and LPS (Fig. 6, B and C). However, plates and incubated overnight. The following day, cells were stimulated
LPS was still capable of significantly augmenting IL-23 p19 with either 107 heat-inactivated S. aureus (A), 10 ␮g/ml PGN (B), or 100
mRNA expression in MyD88 KO microglia, suggesting that this ng/ml LPS (C) for 6, 12, or 24 h, whereupon total RNA was isolated and
cytokine is also influenced by MyD88-independent pathway(s). examined for IL-23 p19 expression by qRT-PCR as described in Materials
Collectively, these data demonstrate that MyD88 is essential for and Methods. Gene expression levels were calculated after normalizing
signaling the production of proinflammatory mediators in response IL-23 p19 signals against GAPDH and are presented in relative mRNA
to both S. aureus and PGN and as such, implicates the involvement expression units (mean ⫾ SEM of three independent experiments). Sig-
of multiple TLRs in mediating maximal microglial activation in nificant differences in IL-23 p19 mRNA expression between unstimulated
vs PAMP-treated microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
response to these Gram-positive stimuli. In contrast, LPS-induced
0.001), whereas significant differences between MyD88 KO vs WT micro-
microglial activation occurs via both MyD88-dependent and -in- glia are denoted with a hatched sign (##, p ⬍ 0.001).
dependent pathways.

Role of MyD88 on the expression of alternative PRRs in
microglia
Previous studies from our laboratory have revealed that both S.
aureus and PGN modulate the expression of alternative PRRs in
microglia, including the phagocytic receptors LOX-1 and PTX3 as
well as TLR9 (18, 38). Because LOX-1 and PTX3 are considered
phagocytic receptors and can directly bind pathogens (53–55), they
may be responsible for S. aureus uptake by microglia. Although in
general, engagement of phagocytic receptors does not directly lead
to cytokine induction, these receptors are capable of influencing
the activity of cytokine signaling receptors through receptor cross-
talk. Therefore, we investigated the potential involvement of
MyD88-dependent pathways on the modulation of phagocytic
PRRs. The induction of microglial LOX-1 expression in response
to all bacterial stimuli was time dependent, with maximal effects
observed at 6-h poststimulation (Fig. 7). MyD88 was found to play
The Journal of Immunology
FIGURE 7. Microglial LOX-1 expression is partially regulated by
MyD88. MyD88 KO and WT microglia were seeded at 2 ⫻ 106 cells/well
in 6-well plates and incubated overnight. The following day, cells were
stimulated with either 107 heat-inactivated S. aureus (A), 10 ␮g/ml PGN
(B), or 100 ng/ml LPS (C) for 6, 12, or 24 h, whereupon total RNA was
isolated and examined for LOX-1 expression by qRT-PCR as described in
Materials and Methods. Gene expression levels were calculated after nor-
malizing LOX-1 signals against GAPDH and are presented in relative
mRNA expression units (mean ⫾ SEM of three independent experiments).
Significant differences in LOX-1 mRNA expression between unstimulated
vs PAMP-treated microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.001), whereas significant differences between MyD88 KO vs WT micro-
glia are denoted with a hatched sign (#, p ⬍ 0.05; ##, p ⬍ 0.001).
an important role in regulating microglial LOX-1 expression in
response to S. aureus and PGN as evident by a reduction in LOX-1
levels in MyD88 KO cells; however, MyD88 did not appear to
modulate microglial LOX-1 induction in response to LPS (Fig. 7).
A similar requirement for MyD88-dependent signaling on the in-
duction of PTX3 expression was demonstrated by the failure of
MyD88 KO microglia to up-regulate PTX3 levels (Fig. 8). TLR9
6807
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
FIGURE 8. The expression of PTX3 is regulated by MyD88 in primary
microglia. MyD88 KO and WT microglia were seeded at 2 ⫻ 106 cells/
well in 6-well plates and incubated overnight. The following day, cells
were stimulated with either 107 heat-inactivated S. aureus (A), 10 ␮g/ml
PGN (B), or 100 ng/ml LPS (C) for 6, 12, or 24 h, whereupon total RNA
was isolated and examined for PTX3 expression by qRT-PCR as described
in Materials and Methods. Gene expression levels were calculated after
normalizing PTX3 signals against GAPDH and are presented in relative
mRNA expression units (mean ⫾ SEM of three independent experiments).
Significant differences in PTX3 mRNA expression between unstimulated
vs PAMP-treated microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.001), whereas significant differences between MyD88 KO vs WT micro-
glia are denoted with a hatched sign (#, p ⬍ 0.05; ##, p ⬍ 0.001).
recognizes bacterial DNA which contains high levels of unmeth-
ylated CpG motifs and this receptor uses a MyD88-dependent sig-
naling pathway. Similar to what we have recently reported in pri-
mary microglia (18), TLR9 expression was attenuated in response
to all three microbial stimuli examined; however, the inhibition re-
sulting from S. aureus treatment did not reach statistical significance
6808
(Fig. 9A). Interestingly, the level of PGN- and LPS-induced suppres-
sion of TLR9 expression was significantly less in MyD88 KO micro-
glia compared with WT cells (Fig. 9, B and C).
To expand our analysis of alternative PRRs in microglia, we
evaluated changes in the expression of NOD2 in response to bac-
terial stimuli in MyD88 KO and WT microglia. NOD2 is a mem-
FIGURE 9. The loss of MyD88 results in differential expression of
TLR9 mRNA expression in response to S. aureus, PGN, or LPS. MyD88
KO and WT microglia were seeded at 2 ⫻ 106 cells/well in 6-well plates
and incubated overnight. The following day, cells were stimulated with
either 107 heat-inactivated S. aureus (A), 10 ␮g/ml PGN (B), or 100 ng/ml
LPS (C) for 6, 12, or 24 h, whereupon total RNA was isolated and exam-
ined for TLR9 expression by qRT-PCR as described in Materials and
Methods. Gene expression levels were calculated after normalizing TLR9
signals against GAPDH and are presented in relative mRNA expression
units (mean ⫾ SEM of three independent experiments). Significant differ-
ences in TLR9 mRNA expression between unstimulated vs PAMP-treated
microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001), whereas
significant differences between MyD88 KO vs WT microglia are denoted
with a hatched sign (#, p ⬍ 0.05; ##, p ⬍ 0.001).
MyD88 IN MICROGLIA
ber of the nucleotide-binding oligomerization domain family of
proteins and considered a PRR because it recognizes the bacterial
product muramyl dipeptide (MDP), a degradation product of bac-
terial PGN (56, 57). A recent study has proposed that NOD2 could
represent a negative regulator of TLR2-dependent signaling (58).
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-
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
fects on CNS integrity.
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-
fected, HEK293 cells. However, in a recent study, Dziarski et al.
(62) re-evaluated the effects of highly purified PGN on TLR2-
transfected HEK293 cells and found that polymeric PGN is a po-
tent TLR2 ligand, whereas muramidase treatment dramatically re-
duced the responsiveness of cells to PGN. Therefore, the authors
suggested that various purification procedures could damage the
cross-linked TLR2-activating structure of PGN which is required
for TLR2, but not NOD activation. Our previous and current find-
ings support the premise that PGN represents a TLR2 agonist in
microglia, because we demonstrated that neither TLR2 nor MyD88
KO microglia were responsive to PGN (18). Importantly, the com-
mercial PGN preparations used in this study were not found to
contain detectable phosphate levels, indicating that any differences
The Journal of Immunology
FIGURE 10. NOD2 mRNA is expressed in primary microglia. MyD88
KO and WT microglia were seeded at 2 ⫻ 106 cells/well in 6-well plates
and incubated overnight. The following day, cells were stimulated with
either 107 heat-inactivated S. aureus (A), 10 ␮g/ml PGN (B), or 100 ng/ml
LPS (C) for 6, 12, or 24 h, whereupon total RNA was isolated and exam-
ined for NOD2 expression by qRT-PCR as described in Materials and
Methods. Gene expression levels were calculated after normalizing NOD2
signals against GADPH and are presented in relative mRNA expression
units (mean ⫾ SEM of three independent experiments). Significant differ-
ences in NOD2 mRNA expression between unstimulated vs PAMP-treated
microglia are denoted with asterisks (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001), whereas
significant differences between MyD88 KO vs WT microglia are denoted
with a hatched sign (#, p ⬍ 0.05).
observed between MyD88 KO and WT microglia throughout this
study cannot be explained by effects of contaminating LTA or
DNA, which may be present in various commercial PGN prepa-
rations (36).
In our previous studies, we demonstrated that microglial proin-
flammatory mediator production was not attenuated in either TLR2
or CD14 KO microglia following S. aureus stimulation, suggesting
6809
the activation of alternative receptors (18, 38). However, in the
present work we have reported that cytokine/chemokine produc-
tion in MyD88 KO microglia was nearly abolished in response to
intact S. aureus. This finding directly implicates the functional
involvement of additional TLRs due to the absence of this key
adaptor molecule. Intact S. aureus presents microglia with a com-
plex milieu of Ags including lipoproteins, PGN, and DNA-con-
taining nonmethylated CpG motifs that serve as agonists for TLR1
and TLR6 (lipoproteins and PGN) and TLR9 (CpG DNA) (2).
Both TLR1 and TLR6 form functional heterodimers with TLR2,
which dictate ligand specificity to various bacterial lipoproteins
(63– 65). In addition, upon phagocytosis, endosomal degradation
of pathogens leads to the exposure of bacterial DNA that can di-
rectly trigger activation signals via TLR9, which is an intracellular
PRR (3, 66 – 68). We found that the ability of MyD88 KO and WT
microglia to phagocytize S. aureus was equivalent (N. Esen and T.
Kielian, unpublished observations), indicating that the failure of
MyD88-deficient cells to produce proinflammatory mediators was
not due to impaired access to bacterial Ags. Evaluation of TLR9
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
expression revealed that this TLR was regulated by both MyD88-
dependent and -independent pathways, which was dependent on
the type of stimulus used. Specifically, TLR9 levels were signifi-
cantly different in PGN- and LPS-stimulated MyD88 KO micro-
glia compared with WT cells, whereas TLR9 levels were equiva-
lent in S. aureus-activated MyD88 KO and WT microglia.
Recently, it has been reported that various forms of CpG DNA can
activate dendritic cells differentially via TLR9-dependent and -in-
dependent signals (68). Specifically, conventional CpG DNA ac-
tivated dendritic cells via a MyD88-dependent manner whereas a
distinct CpG DNA, where polyG stretches are located at the 5⬘ and
3⬘ ends of a central CpG motif, induced IFN-␣ through IFN reg-
ulatory factor-3 (IRF-3) which did not require MyD88. Therefore,
heterogeneity in the chemical composition of bacterial DNA may
lead to activation of microglia via MyD88-dependent and -inde-
pendent mechanisms; however, this possibility remains specula-
tive. Finally, although every effort was made to ensure that endo-
toxin contamination of non-LPS reagents was not a confounding
factor in our study, we cannot completely rule out the presence of
minor LPS contaminants that were below the detection limit of our
Limulus amebocyte lysate assay. Minor LPS contaminants may
explain the low levels of residual cytokine expression observed in
MyD88 KO microglia in response to S. aureus. Another possibility
is that intact S. aureus may stimulate additional, as of yet uniden-
tified, cytokine signaling PRRs that function independently of
MyD88. It should be noted that there was no residual cytokine
expression in PGN-treated MyD88 KO microglia, suggesting that
this reagent did not contain trace amounts of LPS that could func-
tion via a MyD88-independent manner.
Unlike proinflammatory cytokine production, which was nearly
completely attenuated in MyD88 KO microglia in response to both
S. aureus and PGN, the expression of alternative PRRs was not as
affected by the loss of this TLR adaptor protein. For example,
LOX-1 which has been reported to serve as a phagocytic receptor
for S. aureus (55), was significantly increased in both MyD88 KO
and WT microglia. In addition, the repeating carbohydrate moi-
eties of PGN also make it a potential target for lectin-like mole-
cules such as LOX-1 (69). Although it was evident, to a limited
extent, that MyD88-dependent pathways were involved in regu-
lating LOX-1 induction following PGN stimulation, in the major-
ity of treatment conditions, microglial LOX-1 expression was
equivalent in MyD88 KO and WT cells. These findings are similar
to what we have previously reported in TLR2 KO microglia (18).
The role of LOX-1 in microglial phagocytosis has not yet been
6810
examined; however, it is possible that LOX-1 is involved in bac-
terial uptake and subsequent processing, providing ligands to en-
gage TLR9 to augment antibacterial immunity. Additional studies
examining the functional significance of LOX-1 in microglial re-
sponses to bacteria are needed to assess its role in phagocytosis.
We also examined the role of MyD88 in regulating the expression
of the soluble PRR PTX3 in microglia. PTX3 expression was sig-
nificantly enhanced following stimulation with either, S. aureus,
PGN, or LPS in WT cells; however, PTX3 levels in MyD88 KO
microglia were similar to baseline, indicating that MyD88-depen-
dent signal(s) are critical for inducing the expression of this sol-
uble PRR. This finding is in agreement with our previous report
demonstrating that CD14-dependent signals, likely in cooperation
with TLR2, are essential for maximal PTX3 expression in PGN-
activated microglia (18, 38).
NOD2 is an intracellular PRR that recognizes MDP moieties
that are released as a result of hydrolytic or lysosomal digestion of
PGN (56, 57). The interaction of NOD2 with MDP leads to NF-␬B
activation and subsequent proinflammatory gene expression (70).
One interesting finding that surfaced during the course of these
studies was the ability of all three bacterial stimuli examined to
induce NOD2 expression in MyD88 KO and WT microglia. To our
knowledge, this is the first report demonstrating the expression of
NOD2 in primary microglia. We also observed that NOD2 levels
were attenuated in response to both S. aureus and PGN, but not
LPS, in MyD88 KO microglia, suggesting a role for MyD88-de-
pendent signals in regulating the expression of this intracellular
PRR. It is intriguing that NOD2 expression was found to be in-
fluenced, in part, by MyD88 in response to stimuli that harbor
NOD2 ligands upon intercellular processing (i.e., PGN from the
cell wall of intact S. aureus and PGN itself). The functional im-
portance of NOD2 in mediating microglial activation and its role
in the context of the myriad of PRRs expressed by microglia re-
main to be determined.
LPS is a well-established TLR4 ligand and activates macro-
phages and dendritic cells via both MyD88-dependent and -inde-
pendent pathways (3, 71, 72). Specifically, the induction of clas-
sical proinflammatory cytokines (i.e., TNF-␣, IL-1␤) by LPS is
mediated via MyD88-dependent signals, whereas IFN-inducible
genes such as iNOS, IFN-inducible protein 10, and IFN␣␤ are
regulated via MyD88-independent pathways (29, 73). These IFN-
inducible genes are controlled by the activation of two adaptor
proteins, TIR domain-containing adaptor protein and TRIF, that
act downstream to activate IRF-3 through Tank-binding kinase 1
and I␬B kinase (74, 75). One interesting observation in the current
study was the finding that LPS-induced proinflammatory cytokine
expression was still evident in MyD88 KO microglia and although
levels were significantly less compared with WT cells residual
cytokine expression was still significantly elevated compared with
unstimulated microglia. This finding differs from a study by Kawai
et al. (29) where LPS-induced proinflammatory cytokine expres-
sion was abolished in MyD88 KO macrophages. The reason(s)
responsible for these discrepancies are not known; however, our
results suggest that proinflammatory cytokine production in mi-
croglia may be influenced by MyD88-independent signals, a find-
ing which may be cell type specific. A recent study provides ad-
ditional evidence to support our findings where LPS-stimulation
was found to induce several chemokine and cytokine genes such as
MIP-1␣, MIP-1␤, MIP-2, and TNF-␣ in MyD88-deficient cells as
evaluated by microarray analysis (76). In addition, this study dem-
onstrated that the TRIF-dependent pathway activated TNF-␣ pro-
duction and secretion via a NF-␬B-independent manner and sug-
gested that secreted TNF-␣ acts in an autocrine manner to induce
delayed NF-␬B activation. Because depletion of IRF-3 was found
MyD88 IN MICROGLIA
to impair NF-␬B activation, IRF-3 has been implicated as a me-
diator of TNF-␣ activation via a MyD88-independent pathway.
In conclusion, our results indicate that microglial proinflamma-
tory mediator release induced by the Gram-positive stimuli S. au-
reus and PGN occurs via a MyD88-dependent pathway and in-
volves the activity of multiple TLRs. In addition, the expression of
additional phagocytic PRRs, including LOX-1 and PTX3, is reg-
ulated, in part, by MyD88-dependent signals. In contrast, LPS-
dependent microglial activation is regulated by both MyD88-de-
pendent and -independent mechanisms. Moreover, we report the
novel expression of the intracellular PRR, NOD2, in microglia and
find that this receptor is augmented in response to Gram-positive
bacteria and its cell wall product PGN. We are currently extending
these studies to examine the role of MyD88 in S. aureus-dependent
microglial activation in vivo using the experimental brain abscess
model developed in our laboratory, where microglial responses
will be quantified in FACS-purified populations from MyD88 WT
and KO mice. Collectively, these results reveal the importance of
MyD88, and by extension, multiple TLRs, in mediating microglial
activation in response to a wide array of PAMPs. However, it is
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
apparent that alternative PRRs also play a role in enabling maxi-
mal microglial responses to pathogens, which is not unexpected
because bacteria such as S. aureus have the potential to elicit dev-
astating consequences in a tissue that has limited regenerative ca-
pacity such as the CNS.
Acknowledgments
We thank Patrick Mayes for excellent technical assistance.
Disclosures
The authors have no financial conflict of interest.
References
1. Aloisi, F. 2001. Immune function of microglia. Glia 36: 165–179.
2. Kielian, T. 2006. Toll-like receptors (TLR) in central nervous system glial in-
flammation and homeostasis. J. Neurosci. Res. 83: 711–730.
3. Kaisho, T., and S. Akira. 2004. Pleiotropic function of Toll-like receptors. Mi-
crobes Infect. 6: 1388 –1394.
4. Kopp, E., and R. Medzhitov. 2003. Recognition of microbial infection by Toll-
like receptors. Curr. Opin. Immunol. 15: 396 – 401.
5. Takeda, K., and S. Akira. 2004. TLR signaling pathways. Semin. Immunol.
16: 3–9.
6. Mathisen, G. E., and J. P. Johnson. 1997. Brain abscess. Clin. Infect. Dis. 25:
763–779; 780 –761.
7. Townsend, G. C., and W. M. Scheld. 1998. Infections of the central nervous
system. Adv. Intern. Med. 43: 403– 447.
8. Koedel, U., W. M. Scheld, and H. W. Pfister. 2002. Pathogenesis and pathophys-
iology of pneumococcal meningitis. Lancet Infect. Dis. 2: 721–736.
9. Scheld, W. M., U. Koedel, B. Nathan, and H. W. Pfister. 2002. Pathophysiology
of bacterial meningitis: mechanism(s) of neuronal injury. J. Infect. Dis.
186(Suppl. 2): S225–S233.
10. Baldwin, A. C., and T. Kielian. 2004. Persistent immune activation associated
with a mouse model of Staphylococcus aureus-induced experimental brain ab-
scess. J. Neuroimmunol. 151: 24 –32.
11. Kielian, T. 2004. Immunopathogenesis of brain abscess. J. Neuroinflammation
1: 16.
12. Bsibsi, M., R. Ravid, D. Gveric, and J. M. van Noort. 2002. Broad expression of
Toll-like receptors in the human central nervous system. J. Neuropathol. Exp.
Neurol. 61: 1013–1021.
13. Kielian, T., P. Mayes, and M. Kielian. 2002. Characterization of microglial re-
sponses to Staphylococcus aureus: effects on cytokine, costimulatory molecule,
and Toll-like receptor expression. J. Neuroimmunol. 130: 86 –99.
14. Laflamme, N., G. Soucy, and S. Rivest. 2001. Circulating cell wall components
derived from Gram-negative, not Gram-positive, bacteria cause a profound in-
duction of the gene-encoding Toll-like receptor 2 in the CNS. J. Neurochem. 79:
648 – 657.
15. Laflamme, N., H. Echchannaoui, R. Landmann, and S. Rivest. 2003. Cooperation
between Toll-like receptor 2 and 4 in the brain of mice challenged with cell wall
components derived from Gram-negative and Gram-positive bacteria. Eur. J. Im-
munol. 33: 1127–1138.
16. Olson, J. K., and S. D. Miller. 2004. Microglia initiate central nervous system
innate and adaptive immune responses through multiple TLRs. J. Immunol. 173:
3916 –3924.
17. Rasley, A., J. Anguita, and I. Marriott. 2002. Borrelia burgdorferi induces inflam-
matory mediator production by murine microglia. J. Neuroimmunol. 130: 22–31.
18. Kielian, T., N. Esen, and E. D. Bearden. 2005. Toll-like receptor 2 (TLR2) is
The Journal of Immunology
pivotal for recognition of S. aureus peptidoglycan but not intact bacteria by
microglia. Glia 49: 567–576.
19. Ebert, S., J. Gerber, S. Bader, F. Muhlhauser, K. Brechtel, T. J. Mitchell, and
R. Nau. 2005. Dose-dependent activation of microglial cells by Toll-like receptor
agonists alone and in combination. J. Neuroimmunol. 159: 87–96.
20. Chien, H. F., K. Y. Yeh, Y. F. Jiang-Shieh, I. H. Wei, C. Y. Chang, M. L. Chang,
and C. H. Wu. 2005. Signal transduction pathways of nitric oxide release in
primary microglial culture challenged with Gram-positive bacterial constituent,
lipoteichoic acid. Neuroscience 133: 423– 436.
21. Jung, D. Y., H. Lee, B. Y. Jung, J. Ock, M. S. Lee, W. H. Lee, and K. Suk. 2005.
TLR4, but not TLR2, signals autoregulatory apoptosis of cultured microglia: a
critical role of IFN-␤ as a decision maker. J. Immunol. 174: 6467– 6476.
22. Lehnardt, S., L. Massillon, P. Follett, F. E. Jensen, R. Ratan, P. A. Rosenberg,
J. J. Volpe, and T. Vartanian. 2003. Activation of innate immunity in the CNS
triggers neurodegeneration through a Toll-like receptor 4-dependent pathway.
Proc. Natl. Acad. Sci. USA 100: 8514 – 8519.
23. Lehnardt, S., C. Lachance, S. Patrizi, S. Lefebvre, P. L. Follett, F. E. Jensen,
P. A. Rosenberg, J. J. Volpe, and T. Vartanian. 2002. The Toll-like receptor
TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the
CNS. J. Neurosci. 22: 2478 –2486.
24. Akira, S. 2003. Toll-like receptor signaling. J. Biol. Chem. 278: 38105–38108.
25. Akira, S., and S. Sato. 2003. Toll-like receptors and their signaling mechanisms.
Scand. J. Infect. Dis. 35: 555–562.
26. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami,
K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results
in loss of IL-1- and IL-18-mediated function. Immunity 9: 143–150.
27. Hanada, T., and A. Yoshimura. 2002. Regulation of cytokine signaling and in-
flammation. Cytokine Growth Factor Rev. 13: 413– 421.
28. Medzhitov, R., P. Preston-Hurlburt, E. Kopp, A. Stadlen, C. Chen, S. Ghosh, and
C. A. Janeway, Jr. 1998. MyD88 is an adaptor protein in the hToll/IL-1 receptor
family signaling pathways. Mol. Cell 2: 253–258.
29. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira. 1999. Unresponsive-
ness of MyD88-deficient mice to endotoxin. Immunity 11: 115–122.
30. Takeuchi, O., K. Takeda, K. Hoshino, O. Adachi, T. Ogawa, and S. Akira. 2000.
Cellular responses to bacterial cell wall components are mediated through
MyD88-dependent signaling cascades. Int. Immunol. 12: 113–117.
31. Takeuchi, O., K. Hoshino, and S. Akira. 2000. Cutting edge: TLR2-deficient and
MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection.
J. Immunol. 165: 5392–5396.
32. Underhill, D. M., A. Ozinsky, K. D. Smith, and A. Aderem. 1999. Toll-like
receptor-2 mediates mycobacteria-induced proinflammatory signaling in macro-
phages. Proc. Natl. Acad. Sci. USA 96: 14459 –14463.
33. Underhill, D., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson, M. Bassetti,
and A. Aderem. 1999. The Toll-like receptor 2 is recruited to macrophage phago-
somes and discriminates between pathogens. Nature 401: 811– 815.
34. Fremond, C. M., V. Yeremeev, D. M. Nicolle, M. Jacobs, V. F. Quesniaux, and
B. Ryffel. 2004. Fatal Mycobacterium tuberculosis infection despite adaptive
immune response in the absence of MyD88. J. Clin. Invest. 114: 1790 –1799.
35. Kielian, T., M. McMahon, E. D. Bearden, A. C. Baldwin, P. D. Drew, and
N. Esen. 2004. S. aureus-dependent microglial activation is selectively attenuated
by the cyclopentenone prostaglandin 15-deoxy-␦12,14-prostaglandin J2 (15d-
PGJ2). J. Neurochem. 90: 1163–1172.
36. Travassos, L. H., S. E. Girardin, D. J. Philpott, D. Blanot, M. A. Nahori, C. Werts,
and I. G. Boneca. 2004. Toll-like receptor 2-dependent bacterial sensing does not
occur via peptidoglycan recognition. EMBO Rep. 5: 1000 –1006.
37. Kielian, T., B. Barry, and W. F. Hickey. 2001. CXC chemokine receptor-2 li-
gands are required for neutrophil-mediated host defense in experimental brain
abscesses. J. Immunol. 166: 4634 – 4643.
38. Esen, N., and T. Kielian. 2005. Recognition of Staphylococcus aureus-derived
peptidoglycan (PGN) but not intact bacteria is mediated by CD14 in microglia.
J. Neuroimmunol. 170: 93–104.
39. Wesche, H., W. J. Henzel, W. Shillinglaw, S. Li, and Z. Cao. 1997. MyD88: an
adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7: 837– 847.
40. Burns, K., F. Martinon, C. Esslinger, H. Pahl, P. Schneider, J. L. Bodmer,
F. Di Marco, L. French, and J. Tschopp. 1998. MyD88, an adapter protein in-
volved in interleukin-1 signaling. J. Biol. Chem. 273: 12203–12209.
41. MacMicking, J., Q. W. Xie, and C. Nathan. 1997. Nitric oxide and macrophage
function. Annu. Rev. Immunol. 15: 323–350.
42. Lee, S. C., and C. F. Brosnan. 1996. Cytokine regulation of iNOS expression in
human glial cells. Methods 10: 31–37.
43. Qin, L., G. Li, X. Qian, Y. Liu, X. Wu, B. Liu, J. S. Hong, and M. L. Block. 2005.
Interactive role of the Toll-like receptor 4 and reactive oxygen species in LPS-
induced microglia activation. Glia 52: 78 – 84.
44. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, and
S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative
and Gram-positive bacterial cell wall components. Immunity 11: 443– 451.
45. Liew, F. Y., D. Xu, E. K. Brint, and L. A. O’Neill. 2005. Negative regulation of
Toll-like receptor-mediated immune responses. Nat. Rev. Immunol. 5: 446 – 458.
46. Negishi, H., Y. Ohba, H. Yanai, A. Takaoka, K. Honma, K. Yui, T. Matsuyama,
T. Taniguchi, and K. Honda. 2005. Negative regulation of Toll-like receptor
signaling by IRF-4. Proc. Natl. Acad. Sci. USA 102: 15989 –15994.
47. Langrish, C. L., B. S. McKenzie, N. J. Wilson, R. de Waal Malefyt,
R. A. Kastelein, and D. J. Cua. 2004. IL-12 and IL-23: master regulators of innate
and adaptive immunity. Immunol. Rev. 202: 96 –105.
48. Watford, W. T., M. Moriguchi, A. Morinobu, and J. J. O’Shea. 2003. The biology
of IL-12: coordinating innate and adaptive immune responses. Cytokine Growth
Factor Rev. 14: 361–368.
6811
49. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega,
N. Yu, J. Wang, K. Singh, et al. 2000. Novel p19 protein engages IL-12p40 to
form a cytokine, IL-23, with biological activities similar as well as distinct from
IL-12. Immunity 13: 715–725.
50. Lankford, C. S., and D. M. Frucht. 2003. A unique role for IL-23 in promoting
cellular immunity. J. Leukocyte Biol. 73: 49 –56.
51. Li, J., B. Gran, G. X. Zhang, E. S. Ventura, I. Siglienti, A. Rostami, and
M. Kamoun. 2003. Differential expression and regulation of IL-23 and IL-12
subunits and receptors in adult mouse microglia. J. Neurol. Sci. 215: 95–103.
52. Sonobe, Y., I. Yawata, J. Kawanokuchi, H. Takeuchi, T. Mizuno, and
A. Suzumura. 2005. Production of IL-27 and other IL-12 family cytokines by
microglia and their subpopulations. Brain Res. 1040: 202–207.
53. Garlanda, C., E. Hirsch, S. Bozza, A. Salustri, M. De Acetis, R. Nota, A. Maccagno,
F. Riva, B. Bottazzi, G. Peri, et al. 2002. Non-redundant role of the long pentraxin
PTX3 in anti-fungal innate immune response. Nature 420: 182–186.
54. Mantovani, A., C. Garlanda, and B. Bottazzi. 2003. Pentraxin 3, a non-redundant
soluble pattern recognition receptor involved in innate immunity. Vaccine
21(Suppl. 2): S43–S47.
55. Shimaoka, T., N. Kume, M. Minami, K. Hayashida, T. Sawamura, T. Kita, and
S. Yonehara. 2001. LOX-1 supports adhesion of Gram-positive and Gram-neg-
ative bacteria. J. Immunol. 166: 5108 –5114.
56. Netea, M. G., G. Ferwerda, D. J. de Jong, T. Jansen, L. Jacobs, M. Kramer,
T. H. Naber, J. P. Drenth, S. E. Girardin, B. J. Kullberg, et al. 2005. Nucleotide-
binding oligomerization domain-2 modulates specific TLR pathways for the in-
duction of cytokine release. J. Immunol. 174: 6518 – 6523.
57. Girardin, S. E., I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas,
Downloaded from http://www.jimmunol.org/ by guest on April 14, 2022
D. J. Philpott, and P. J. Sansonetti. 2003. Nod2 is a general sensor of peptidoglycan
through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278: 8869 – 8872.
58. Watanabe, T., A. Kitani, P. J. Murray, and W. Strober. 2004. NOD2 is a negative
regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat. Im-
munol. 5: 800 – 808.
59. Sugawara, I., H. Yamada, S. Mizuno, K. Takeda, and S. Akira. 2003. Mycobac-
terial infection in MyD88-deficient mice. Microbiol. Immunol. 47: 841– 847.
60. Koedel, U., T. Rupprecht, B. Angele, J. Heesemann, H. Wagner, H. W. Pfister,
and C. J. Kirschning. 2004. MyD88 is required for mounting a robust host im-
mune response to Streptococcus pneumoniae in the CNS. Brain 127: 1437–1445.
61. Henneke, P., O. Takeuchi, R. Malley, E. Lien, R. R. Ingalls, M. W. Freeman,
T. Mayadas, V. Nizet, S. Akira, D. L. Kasper, and D. T. Golenbock. 2002.
Cellular activation, phagocytosis, and bactericidal activity against group B Strep-
tococcus involve parallel myeloid differentiation factor 88-dependent and inde-
pendent signaling pathways. J. Immunol. 169: 3970 –3977.
62. Dziarski, R., and D. Gupta. 2005. Staphylococcus aureus peptidoglycan is a Toll-
like receptor 2 activator: a reevaluation. Infect. Immun. 73: 5212–5216.
63. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong,
R. L. Modlin, and S. Akira. 2002. Cutting edge: role of Toll-like receptor 1 in
mediating immune response to microbial lipoproteins. J. Immunol. 169: 10 –14.
64. Takeuchi, O., T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky,
K. Takeda, and S. Akira. 2001. Discrimination of bacterial lipoproteins by Toll-
like receptor 6. Int. Immunol. 13: 933–940.
65. Omueti, K. O., J. M. Beyer, C. M. Johnson, E. A. Lyle, and R. I. Tapping. 2005.
Domain exchange between human Toll-like receptors 1 and 6 reveals a region
required for lipopeptide discrimination. J. Biol. Chem. 280: 36616 –36625.
66. Akira, S., and H. Hemmi. 2003. Recognition of pathogen-associated molecular
patterns by TLR family. Immunol. Lett. 85: 85–95.
67. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto,
K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor
recognizes bacterial DNA. Nature 408: 740 –745.
68. Hemmi, H., T. Kaisho, K. Takeda, and S. Akira. 2003. The roles of Toll-like
receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the
effects of two distinct CpG DNAs on dendritic cell subsets. J. Immunol. 170:
3059 –3064.
69. Gordon, S. 2002. Pattern recognition receptors: doubling up for the innate im-
mune response. Cell 111: 927–930.
70. Inohara, N., M. Chamaillard, C. McDonald, and G. Nunez. 2004. NOD-LRR
proteins: role in host-microbial interactions and inflammatory disease. Annu. Rev.
Biochem. 74: 355– 83
71. Akira, S., M. Yamamoto, and K. Takeda. 2003. Role of adapters in Toll-like
receptor signalling. Biochem. Soc. Trans. 31: 637– 642.
72. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Im-
munol. 21: 335–376.
73. Lee, J. Y., C. A. Lowell, D. G. Lemay, H. S. Youn, S. H. Rhee, K. H. Sohn,
B. Jang, J. Ye, J. H. Chung, and D. H. Hwang. 2005. The regulation of the
expression of inducible nitric oxide synthase by Src-family tyrosine kinases me-
diated through MyD88-independent signaling pathways of Toll-like receptor 4.
Biochem. Pharmacol. 70: 1231–1240.
74. Akira, S., and K. Hoshino. 2003. Myeloid differentiation factor 88-dependent and
-independent pathways in toll-like receptor signaling. J. Infect. Dis. 187(Suppl.
2): S356 –S363.
75. Kawai, T., O. Takeuchi, T. Fujita, J. Inoue, P. F. Muhlradt, S. Sato, K. Hoshino,
and S. Akira. 2001. Lipopolysaccharide stimulates the MyD88-independent path-
way and results in activation of IFN-regulatory factor 3 and the expression of a
subset of lipopolysaccharide-inducible genes. J. Immunol. 167: 5887–5894.
76. Covert, M. W., T. H. Leung, J. E. Gaston, and D. Baltimore. 2005. Achieving
stability of lipopolysaccharide-induced NF-␬B activation. Science 309:
1854 –1857.