TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway.

Abstract

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (TIR) domain-containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.

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TLRs discriminate among molecular patterns associated with microbial
components. TLRs are abundantly expressed on professional antigen-
presenting cells such as macrophages and dendritic cells, and serve as an
important link between the innate and adaptive immune responses1,2.
So far, genes encoding ten TLRs (TLR1–TLR10) have been found in the
human genome, and each TLR recognizes specific pathogen-associated
patterns. TLR1 recognizes triacylated lipoprotein3. TLR2 is essential for
recognition of peptidoglycan (PGN), the main Gram-positive bacterial
cell wall component4. TLR3 is involved in recognition of double-
stranded RNA, which is generated in the life cycle of RNA viruses5.
TLR4 is a receptor for lipopolysaccharide (LPS), a Gram-negative cell
wall component6,7. TLR5 recognizes flagellin, a component of bacterial
flagella8. TLR6 is required for recognition of diacylated lipoprotein9,
whereas TLR7 is crucial for recognition of imidazoquinoline, an antivi-
ral synthetic compound, and its derivative, R-848 (ref. 10). TLR9 is a
receptor for bacterial unmethylated CpG DNA11.
Intracellular signaling pathways of TLRs are elicited from the TIR
domain, which is conserved among the cytoplasmic regions of TLRs. A
cytoplasmic molecule, MyD88, contains a TIR domain and a death
domain. The death domain of MyD88 is required for interaction with
other death domain–containing molecules such as IRAK-1 and IRAK-
4 (refs. 12–14). The TIR domain is reportedly required for the forma-
tion of dimers with other TIR domain–containing receptors or
adaptors. Indeed, MyD88-deficient mice show neither splenocyte pro-
liferation nor production of proinflammatory cytokines in response to
all TLR ligands and interleukin 1 (IL-1), indicating that MyD88 is
essential for the immune responses through all TLRs and the IL-1
receptor (IL-1R)15. However, a TLR3 ligand, poly(I:C), and the TLR4
ligand, LPS, still stimulate the expression of certain genes, such as the
gene encoding interferon-β (IFN-β), in MyD88-deficient mice.
Induction of IFN-β expression leads to maturation of dendritic cells
and subsequent expression of IFN-inducible genes16–18. These obser-
vations indicated that TLR signaling is composed of at least two path-
ways: a MyD88-dependent pathway that leads to the production of
proinflammatory cytokines, and a MyD88-independent pathway asso-
ciated with the induction of IFN-inducible genes and maturation of
dendritic cells. Moreover, the specificity of the MyD88-dependent sig-
naling pathways through all TLRs is provided by TIRAP, the second
TIR domain–containing adaptor discovered19,20. TIRAP-deficient
mice show defects in activation of the MyD88-dependent signaling
pathway through TLR2 and TLR4, but not other TLRs21,22.
Although the precise molecular mechanisms of the MyD88-inde-
pendent signaling pathway are unknown, identification of another TIR
domain–containing molecule, TRIF23,24(also called TICAM-1), and
genetic evidence from mice carrying a mutation in this gene demon-
strated that TRIF is crucial in the MyD88-independent signaling path-
way shared by TLR3 and TLR4 (refs. 25,26). Furthermore, two
noncanonical IκB kinases (IKKs), IKK-i (also called IKKε) and TBK1
(also called T2K), interact with TRIF, activate IRF-3 and, finally, lead to
IFN-βinduction27,28.
Two more TIR domain–containing adaptors have been identified in
the human genome. The physiological function of the first, SARM (for
sterile alpha motif (SAM) and Armadillo motif (ARM) domain–
containing protein), in TLR–IL-1R signaling remains unclear29,30.
1Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, and 2ERATO, Japan Science and Technology Corporation, 3-1 Yamada-oka,
Suita Osaka 565-0871, Japan. 3RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Correspondence should be addressed to S.A. (sakira@biken.osaka-u.ac.jp).
Published online 12 October 2003; doi:10.1038/ni986
TRAM is specifically involved in the Toll-like receptor
4–mediated MyD88-independent signaling pathway
Masahiro Yamamoto1, Shintaro Sato2, Hiroaki Hemmi1, Satoshi Uematsu1, Katsuaki Hoshino3,
Tsuneyasu Kaisho1,3, Osamu Takeuchi1, Kiyoshi Takeda1,2& Shizuo Akira1,2
Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated
by Toll–interleukin 1 receptor (TIR) domain–containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor
that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3
and TLR4. Here we have identified a fourth TIR domain–containing adaptor, TRIF-related adaptor molecule (TRAM), and
analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response
to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-β production
and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-
independent component of TLR4 signaling.
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Previous in vitro analysis of the other, TRAM (also called TIRP)31,
indicates that its ectopic expression activates NF-κB, as does MyD88,
TIRAP and TRIF. However, it does not activate the promoter for the
gene encoding IFN-β, unlike TRIF. Dominant negative mutants of this
protein inhibit the NF-κB activation through IL-1R, but not through
TLRs, indicating that TRAM is a specific adaptor protein in the IL-1R-
mediated MyD88-dependent signaling pathway. However, the func-
tion of TRAM in vivo remains to be clarified.
Here we report the generation of TRAM-deficient mice and detailed
analysis of TLR–IL-1R signaling in these mutant mice. TRAM-
deficient mice showed normal responses to ligands for TLR2, TLR7, TLR9
and IL-1β, but defective macrophage responses to the TLR4 ligand in
cytokine production and B cell activation. Furthermore, activation of the
TLR4-mediated MyD88-independent, but not MyD88-dependent, sig-
naling cascade was abolished in TRAM-deficient mice. Although this
phenotype was reminiscent of that of TRIF-deficient mice, which lack
activation of the MyD88-independent pathway in both TLR3 and TLR4
signaling, TRAM-deficient mice showed a normal response to the TLR3
ligand. These results indicate that TRAM is an adaptor molecule that pro-
vides specificity for the MyD88-independent pathway of TLR4 signaling.
RESULTS
Identification of TRAM
The TIR domain–containing adaptor TRIF was identified by a database
search analysis23(Fig. 1a). During this analysis, we identified another
TIR domain–containing molecule. The TIR domain of this molecule
showed greater homology with the TIR domain of TRIF than with that
of MyD88 or TIRAP (data not shown). The gene encoding has an open
reading frame of 708 base pairs (bp) that encodes 235 amino acids.
Ectopic expression of TRAM in 293 cells activated luciferase reporters
containing the NF-κB–dependent promoter or the promoter for the
gene encoding IFN-β, albeit at low amounts compared with that of
MyD88- and TRIF-mediated activation, respectively (Fig. 1b,c). Thus,
in vitro studies indicated that TRAM is involved in TLR–IL-1R signal-
ing pathways.
Generation of TRAM-deficient mice
To elucidate the physiological function of TRAM, we generated TRAM-
deficient mice by gene targeting. The gene encoding mouse TRAM
consists of one exon. We constructed the targeting vector to replace the
entire exon with a neomycin-resistance gene cassette (Fig. 1d). We
Figure 1 Cloning and characterization of human TRAM and targeted
disruption of the gene encoding mouse TRAM. (a) Comparison of the
structures of human TRAM, TRIF, MyD88 and TIRAP. Domains were
determined with the BLAST program. Lengths are indicated in amino acids
(aa). DD, death domain; TIR, TIR domain. (b,c) 293 cells were transiently
transfected with 1 µg TRAM, MyD88, TRIF or empty vector (–), plus 0.1 µg
NF-κB reporter (b) or the IFN-β promoter luciferase reporter (c). Then, 24 h
after transfection, luciferase activity was measured. Numbers over each
column indicate the ‘fold induction’, which for empty vector was normalized
to onefold. (d) The structures of the gene encoding TRAM (top), the targeting
vector (middle) and the predicted disrupted gene (bottom). Gray box, coding
exon. B, BamHI. (e) Southern blot analysis of offspring from the
heterozygote intercrosses. Genomic DNA was extracted from mouse tail
tissue, digested with BamHI, separated by electrophoresis and hybridized
with the radiolabeled probe shown in d. Southern blot analysis detected a
single 7.6-kb band for wild-type mice (+/+), a 2.4-kb band for homozygous
TRAM-deficient mice (–/–) and both bands for heterozygous mice (+/–).
(f) Cell lysates prepared from macrophages were immunoprecipitated and
immunoblotted with anti-TRAM. Bottom, the same lysates blotted with anti-
actin to monitor protein expression. WT, wild-type; KO, TRAM-deficient.
a
b
d
c
e
f
Figure 2 IL-1-induced responses in TRAM-
deficient cells. (a) Embryonic fibroblasts from
wild-type and TRAM-deficient mice were
stimulated with 100 ng/ml of IL-1β. Supernatants
were collected 24 h later for IL-6 analysis by
ELISA. Data represent means ± s.d. of triplicate
samples. N.D., not detected. (b) Embryonic
fibroblasts were stimulated with 100 ng/ml of
IL-1β (time, above lanes). Nuclear extracts were
prepared, and NF-κB DNA-binding activity was
determined by electrophoretic mobility shift assay
with an NF-κB-specific probe. (c) JNK activation
was also determined by immunoblot of cell
extracts with anti-phospho-JNK (p-JNK).
WT, wild-type; KO, TRAM-deficient.
abc
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microinjected two correctly targeted embryonic stem cell clones into
C57BL/6 blastocysts to generate chimeric mice. We crossed male
chimeric mice with female C57BL/6 mice, and monitored transmission
of the mutated allele by Southern blot analysis (Fig. 1e). We then inter-
bred heterozygous mice to produce offspring carrying a null mutation
of the gene encoding TRAM. TRAM-deficient mice were born at the
expected mendelian ratio and showed no developmental abnormali-
ties. To confirm the disruption of the gene encoding TRAM, we ana-
lyzed total RNA from wild-type and TRAM-deficient lung fibroblast
cells by RNA blot. We found no transcripts for TRAM in RNA from
TRAM-deficient mice (data not shown). Immunoprecipitation analy-
sis of macrophages further confirmed that disruption of the gene
encoding TRAM abolished expression of TRAM protein (Fig. 1f).
TRAM is not involved in IL-1R signaling
As a previous in vitro study showed that TRAM (also known as TIRP)
is involved in the IL-1R-mediated MyD88-dependent pathway31, we
first tested whether IL-1R signaling was impaired in TRAM-deficient
cells. We measured production of IL-6 by mouse embryonic fibrob-
lasts from wild-type and TRAM-deficient mice in response to IL-1βby
enzyme-linked immunosorbent assay (ELISA; Fig. 2a). IL-1β-induced
IL-6 production was normal in TRAM-deficient mouse embryonic
fibroblasts. We next analyzed activation of NF-κB and the kinase JNK1
in response to IL-1β (Fig. 2b,c). IL-1β stimulation activated NF-κB
and JNK1 activation equivalently in both wild-type and TRAM-defi-
cient mouse embryonic fibroblasts, demonstrating that TRAM is not
involved in the IL-1R-mediated signaling pathway.
TLR4-mediated cytokine production and B cell activation
We next analyzed the production of proinflammatory cytokines such
as tumor necrosis factor (TNF) and IL-6 by peritoneal macrophages
in response to various TLR ligands, including PGN, LPS, R-848 and
CpG DNA. Wild-type and TRAM-deficient macrophages produced
similar amounts of IL-6 and TNF when stimulated with PGN, R-848
or CpG DNA. However, LPS-induced production was reduced in
TRAM-deficient cells (Fig. 3a). Wild-type cells produced TNF, IL-6
and p40 subunit of IL-12 in response to LPS stimulation in a dose-
dependent way (Fig. 3b). In contrast, LPS-induced production in
TRAM-deficient cells was impaired. Thus, TRAM-deficient mice
showed defective responses in terms of LPS-induced cytokine pro-
duction. Next, we analyzed splenocyte proliferation in response to
stimulation with LPS, R-848 or CpG DNA. Proliferation of wild-type
and TRAM-deficient splenocytes in response to R-848 and CpG
DNA was similar. However, TRAM-deficient splenocytes showed
defective proliferation after LPS stimulation (Fig. 3c). We next ana-
lyzed surface expression of CD69, CD86 and major histocompatibil-
ity complex class II molecules on B220-positive splenocytes by flow
cytometry after stimulation with LPS or antibody to IgM (anti-IgM).
Both wild-type and TRAM-deficient B220-positive cells showed
similar CD69, CD86 and major histocompatibility complex class II
up-regulation in response to anti-IgM (Fig. 3d). However, LPS-
induced up-regulation of these molecules was perturbed in TRAM-
deficient cells. Therefore, these data indicated that TLR4-mediated,
but not other TLR-mediated, responses were impaired in TRAM-
deficient mice.
Figure 3 Impaired TLR4-mediated cytokine
production and B cell activation in TRAM-
deficient mice. (a) Peritoneal macrophages from
TRIF-deficient mice or wild-type mice were left
unstimulated (Med) or were stimulated with
10 µg/ml of PGN, 100 ng/ml of LPS, 100 nM
R-848 or 1 µM CpG DNA in the presence of
30 ng/ml of IFN-γ. Supernatants were collected
24 h later for TNF (left) and IL-6 (right) analysis
by ELISA. Data represent means ± s.d. of
triplicate samples. N.D., not detected.
(b) Peritoneal macrophages were stimulated for
24 h with LPS (concentrations, horizontal axes)
in the presence of 30 ng/ml of IFN-γ, and
TNF (left), IL-6 (middle) and IL-12 p40 (right)
in supernatants was measured by ELISA. Data
represent means ± s.d. of triplicate samples.
(c) Proliferation of splenocytes stimulated with
LPS, R-848 or CpG DNA. Splenocytes were
cultured for 24 h with LPS, R-848 or CpG DNA
(concentrations, horizontal axes; wedges indicate
increasing concentrations). Samples were pulsed
with [3H]thymidine (1 µCi) for the last 12 h, then
[3H]thymidine incorporation was measured with
a scintillation counter. (d) Splenic B220-positive
cells were cultured with 10 µg/ml of LPS or
10 µg/ml of anti-IgM (α-IgM). After 36 h of
culture, cells were collected and stained with
biotin-conjugated anti-CD69, anti-CD86
or anti-I-Abfollowed by phycoerythrin-conjugated
streptavidin. WT, wild-type; KO, TRAM-deficient;
Med, medium.
a
b
c
d
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TLR4-mediated MyD88-independent responses
TLR3 and TLR4 ligands induce IFN-β production and subsequent
expression of IFN-inducible genes in a MyD88-independent way5,17,18.
The impaired TLR4-mediated cytokine production and B cell activation
in TRAM-deficient mice prompted us to investigate whether the MyD88-
independent responses were also impaired in these mice. We analyzed
expression of the gene encoding IFN-βand IFN-inducible genes in both
wild-type and TRAM-deficient mice by RNA blot. LPS stimulated
expression of the gene encoding IFN-βand IFN-inducible genes, includ-
ing Ifit2 (encoding ISG54), Cxcl10 (encoding IP-10) and Ccl5 (encoding
RANTES), in wild-type peritoneal macrophages. However, LPS-induced
expression of these genes was impaired in TRAM-deficient cells (Fig. 4a).
As for TLR3 responses, both wild-type and TRAM-deficient peritoneal
macrophages showed similar amounts of gene expression after poly(I:C)
stimulation (Fig. 4b). These data indicated that TRAM deficiency
affected the MyD88-independent responses mediated by TLR4.
TLR4-mediated MyD88-independent signal transduction
LPS induction of Cxcl10 mRNA is dependent on signal transducer
and activator of transcription 1 (STAT-1), as demonstrated with
STAT-1-deficient mice, which show a reduction in Cxcl10 mRNA
expression in LPS-stimulated macrophages compared with that of
wild-type cells32–34. In wild-type macrophages, the tyrosine residue at
position 701 of STAT-1 was phosphorylated after 2 h of LPS stimula-
tion. However, this typrosine remained unphosphorylated in TRAM-
deficient macrophages (Fig. 5a). In contrast, poly(I:C) stimulation
resulted in similar degrees of STAT-1 phosphorylation in both wild-
type and TRAM-deficient cells (Fig. 5b). Given that STAT-1 phospho-
rylation is IFN-β dependent34, we investigated the activation of
signaling molecules required for IFN-β production. The transcrip-
tion factor IRF-3 is essential for LPS- and poly(I:C)-mediated IFN-β
production35,36. Indeed, native PAGE analysis showed LPS-induced
formation of IRF-3 homodimers in wild-type cells. However, LPS-
induced IRF-3 activation was abolished in cells from TRAM-deficient
mice (Fig. 5c). In contrast, TRAM-deficient cells showed amounts of
IRF-3 dimer formation similar to those of wild-type cells after
poly(I:C) stimulation (Fig. 5d). As LPS-induced STAT-1 phosphory-
lation and IRF-3 activation are MyD88 independent, these data indi-
cated that TLR4-mediated, but not TLR3-mediated, activation of the
MyD88-independent signaling pathway was impaired in TRAM-defi-
cient mice. In addition to IRF-3 activation, TLR4-mediated MyD88-
independent signaling leads to late-phase NF-κB activation, which is
evident in MyD88-deficient mice16,21,22. In contrast to wild-type
macrophages, MyD88-deficient macrophages did not show NF-κB
activation 10 min after LPS stimulation. However, we found NF-κB
activation at 20 min or at later time points in MyD88-deficient
macrophages21,22. Therefore, LPS-induced NF-κB activation is
probably biphasic, and the early-phase NF-κB activation is MyD88
ab
c
d
ef
g
Figure 4 Defects in TLR4-mediated MyD88-independent responses in
TRAM-deficient mice. Peritoneal macrophages were stimulated with
100 ng/ml of LPS (a) or 50 µg/ml of poly(I:C) (b). Total RNA (10 µg) was
extracted and analyzed by RNA blot for expression of Ifnb (IFN-β), Ifit2
(ISG54), Cxcl10 (IP-10), Ccl5 (RANTES) and Actb (β-actin). Stimulation
times are above each lane. WT, wild-type; KO, TRAM-deficient.
ab
Ifnd
Ifit2
Cxcl10
Ccl5
Actb
Ifnd
Ifit2
Cxcl10
Ccl5
Actb
Figure 5 TRAM-deficient cells lack TLR4-
mediated MyD88-independent signal transduction.
(a–d) Peritoneal macrophages were stimulated with
1 µg/ml of LPS (a,c) and lung fibroblasts were
stimulated with 50 µg/ml of poly(I:C) (b,d).
(a,b) STAT-1 activation determined by immunoblot
of cell extracts with anti-phospho-STAT-1 (p-STAT-
1α/β). (c,d) Cell lysates were prepared and
separated by native PAGE, and monomeric (arrows)
and dimeric (arrowheads) forms of IRF-3 were
detected by immunoblot. (e,f) Peritoneal
macrophages were stimulated with 100 ng/ml
LPS (e) and lung fibroblasts were stimulated with
150 µg/ml poly(I:C) (f). Nuclear extracts were
prepared, and NF-κB DNA-binding activity was
determined by electrophoretic mobility shift assay
with an NF-κB-specific probe. (e) LPS-induced JNK activation was also determined by immunoblot with anti-phospho-JNK (bottom; p-JNK). (g) Lysates from
macrophages stimulated with LPS were immunoprecipitated with anti-IRAK-1. The kinase activity of IRAK-1 was measured by in vitro kinase assay (top; Auto,
auto-phosphorylation). The same lysates were blotted with anti-IRAK-1 (bottom). Stimulation times are above lanes. WT, wild-type; KO, TRAM-deficient.
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dependent, whereas the late-phase activation is MyD88 independent.
Wild-type cells showed steady amounts of NF-κB activation even
after 60 and 120 min of LPS stimulation. In TRAM-deficient cells,
LPS-induced NF-κB activation occurred normally at early time
points (10 and 20 min) but gradually faded at later time points
(60 and 120 min), indicating that the late-phase NF-κB activation,
which is characteristic of TLR4-mediated MyD88-independent sig-
naling, was impaired in TRAM-deficient mice (Fig. 5e). In contrast,
poly(I:C)-induced NF-κB activation was normal in TRAM-deficient
cells (Fig. 5f). LPS stimulates activation of the kinase MAPK as well as
NF-κB in a biphasic way. Even at 40 min after LPS stimulation, phos-
phorylated JNK was still present in wild-type cells. TRAM-deficient
cells showed similar amounts of JNK activation at early time points
(10 and 20 min). However, at 40 min, phosphorylated JNK was not
detected in TRAM-deficient cells, indicating TRAM deficiency abol-
ished the LPS-induced late-phase MAPK activation (Fig. 5e).
Activation of the MyD88-dependent signaling pathway causes IRAK-
1 activation, as indicated by autophosphorylation and degradation
(Fig. 5g). TRAM-deficient macrophages showed LPS-induced IRAK-
1 activation similar to that of wild-type cells, indicating that activa-
tion of TLR4-mediated MyD88-dependent signaling was intact in
TRAM-deficient mice. These observations show that TRAM is essen-
tial specifically for the TLR4-mediated MyD88-independent signal-
ing pathway (Fig. 6)
DISCUSSION
Here we analyzed the function of TRAM in the TLR–IL-1R signaling
pathways in vivo. TRAM-deficient mice showed defects in cytokine
production, splenocyte proliferation and up-regulation of surface
molecules in response to the TLR4 ligand, but not to other TLR lig-
ands. Furthermore, TLR4-mediated, but not TLR3-mediated, expres-
sion of the gene encoding IFN-β and IFN-inducible genes was
inhibited in TRAM-deficient mice. In intracellular signal transduc-
tion, LPS-induced autophosphorylation of IRAK-1 and the early
phase of NF-κB activation were intact in TRAM-deficient mice.
However, we noted no activation of IRF-3 and a defect in the late phase
of NF-κB activation in response to LPS, but not poly(I:C), in TRAM-
deficient cells. Given that these events are features of the MyD88-inde-
pendent signaling pathway, we propose that TRAM specifically
mediates the MyD88-independent pathway of TLR4 signaling.
In TRAM-deficient mice, TLR4-mediated activation of the MyD88-
dependent pathway, which is characterized by autophosphorylation of
IRAK-1 and early-phase NF-κB activation, was similar to that of wild-
type cells. However, TLR4-mediated production of proinflammatory
cytokines was impaired. Similarly, TLR4-mediated production of
proinflammatory cytokines was reduced in mice lacking TRIF, which
is essential for TLR4- and TLR3-mediated MyD88-independent path-
way. As MyD88-deficient mice showed similar phenotype, activation
of the MyD88-independent pathway is apparently required for induc-
tion of proinflammatory cytokines25,26. Therefore, in TLR4 signaling,
activation of both the MyD88-dependent and the MyD88-indepen-
dent (TRAM–TRIF-dependent) pathways is required for proinflam-
matory cytokine production and B cell activation. However, in the
signaling pathways of TLR2, TLR5 and TLR9, none of which activate
the MyD88-independent pathway, activation of the MyD88-depen-
dent pathway alone is sufficient to induce proinflammatory
cytokines8,34,37,38. Therefore, at present, it remains unclear why TLR4
signaling requires activation of both the MyD88-dependent and the
TRIF-dependent pathways to induce proinflammatory cytokines.
Individual TLRs provoke distinct cellular responses, as indicated by
the presence of the MyD88-independent pathways in TLR3 and TLR4
signaling. Even in the MyD88-dependent pathways, different TLRs
induce distinct types of gene expression. For example, TLR7 and TLR9
signaling induce type I IFN production39, whereas TLR1, TLR2 and
TLR6 ligands do not. Therefore, there must be some mechanisms to
provide specificity to these signaling pathways. It is possible that other
TIR domain–containing adaptor molecules like TIRAP are involved in
MyD88-dependent TLR signals, such as TLR5, TLR7 and TLR9. In this
case, the only remaining candidate is SARM29,30. The C-terminal por-
tion of SARM contains the TIR domain in addition to the SAM and
ARM domains. At present, neither in vitro nor in vivo analysis has
shown whether SARM is involved in the TLR–IL-1R signaling path-
ways. Alternatively, it is possible that MyD88-dependent TLR5, TLR7,
TLR9 or IL-1R signaling does not require such TIR domain–containing
adaptor molecules in addition to MyD88. In this case, signaling mole-
cules acting together with MyD88 should exist to generate the different
cellular responses of these TLRs. These molecules may include the
Pellino family of proteins, which was originally identified in Drosophila
melanogaster as a molecule that is associated with Pelle, a Drosophila
homolog of IRAK40. The Pellino family consists of three members at
present. Pellino1 seems to activate NF-κB in the IL-1R signaling path-
way through the integration of the IL-1R–IRAK1–IRAK4–TRAF6
complex41. Pellino2 and Pellino3 promote activation of the kinase JNK
in the TLR-IL-1R signaling pathway42–44. Although the physiological
functions of the Pellino family of proteins remain to be determined, it is
possible that these proteins differentially take part in distinct TLR or
IL-1R signaling pathways to diversify the cellular responses.
In summary, we have identified an essential adaptor that specifically
mediates the MyD88-independent pathway of TLR4 signaling. Precise
analysis of the involvement of TRAM in the LPS response of TRAM-
deficient mice will provide a new insight into the relationship between
TLR4-mediated MyD88-independent pathways and diseases caused
by Gram-negative bacterial infection, such as septic shock.
METHODS
Plasmids. The ELAM-1 promoter–derived luciferase reporter plasmid (NF-κB
luciferase reporter) was a gift from D.T. Golenbock (University of Massachusetts).
Figure 6 Participation of TIR domain–containing adaptor molecules in
TLR3 and TLR4 signaling pathways. Four TIR domain–containing adaptors,
MyD88, TIRAP, TRIF and TRAM, have been identified. MyD88 and TIRAP
take part in TLR4-mediated proinflammatory cytokine production and B cell
activation, but not in induction of the gene encoding IFN-β and IFN-
inducible genes, the so-called MyD88-independent pathway17,18,21,22. TRIF
is essential in the MyD88-independent pathway shared by TLR3 and TLR4
(refs. 25,26). TRAM specifically participates in the MyD88-independent
pathway of TLR4, but not TLR3, signaling.
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The mouse IFN-β promoter luciferase reporter was generated by PCR as
described45. Flag-tagged human or mouse TRAM was cloned into the pEF-BOS
vector. Expression vectors for TRIF and MyD88 were constructed as described23.
Luciferase reporter assay. For luciferase assays, 293 cells were transiently trans-
fected with reporter plasmids plus expression vectors. The luciferase activity of
total cell lysates was measured with the Dual-Luciferase Reporter Assay System
(Promega) as described23. The Renillaluciferase reporter gene (50 ng) was used
as an internal control.
Generation of TRIF-deficient mice. The gene encoding TRAM was isolated
from genomic DNA extracted from embryonic stem cells (E14.1) by PCR
with TaKaRa LA Taq (TaKaRa). The targeting vector was constructed by
replacement of a 1.0-kb fragment encoding the entire TRAM open reading
frame with a neomycin-resistance gene cassette (neor), and a gene encoding
herpes simplex virus thymidine kinase driven by a phosphoglycerate kinase
promoter was inserted into the genomic fragment for negative selection
(Fig. 1d). After the targeting vector was transfected into embryonic stem
cells, G418 and gancyclovir double-resistant colonies were selected and
screened by PCR and Southern blot analysis. Homologous recombinants
were microinjected into C57BL/6 female mice, and heterozygous F1 progeny
mice were intercrossed to obtain TRAM-deficient mice. TRAM-deficient
mice and their wild-type littermates from these intercrosses were used for
experiments. All animal experiments were done with the approval of the
Animal Research Committee of the Research Institute for Microbial Diseases
(Osaka University).
Reagents. R-848 was provided by the Pharmaceuticals and Biotechnology
Laboratory of Japan Energy Corporation. CpG oligodeoxynucleotides were
prepared as described11. LPS from Salmonella minnesotaRe 595 (prepared by a
phenol–chloroform–petroleum ether extraction procedure), PGN from
Staphylococcus aureus and poly (I:C) were purchased from Sigma, Fluka and
Amersham, respectively. Anti-phospho-JNK and anti-phospho-STAT-1 were
purchased from Cell Signaling Technology. Anti-JNK1, anti-STAT-1 and anti-
actin were from Santa Cruz Biotechnology. Polyclonal anti-TRAM was raised
against amino acids 219–232 of mouse TRAM. This antibody specifically rec-
ognizes human and mouse TRAM in vitro (Supplementary Fig. 1 online).
Polyclonal anti-IRF-3 and anti-IRAK-1 were as described25. Anti-IgM was
purchased from Jackson ImmunoResearch Laboratory. Fluorescein isothio-
cyanate–labeled streptavidin (554060), phycoerythrin-conjugated anti-B220
(553090), biotin-conjugated anti-CD69 (553235), anti-CD86 (553690) and
anti-I-Ab(06252D) were from Pharmingen.
Measurement of proinflammatory cytokine concentrations. Thioglycollate-
elicited peritoneal macrophages were cultured in 96-well plates (5 × 104cells
per well) with PGN, LPS, R-848 or CpG DNA. The concentrations of TNF, IL-6
and IL-12 p40 in the culture supernatant were measured by ELISA according to
the manufacturer’s instructions (Genzyme for TNF-α and IL-12 p40; R&D
Systems for IL-6).
Preparation of lung fibroblasts. Lungs from mice were excised, washed in PBS,
cut into small pieces, agitated and digested enzymatically for 30 min at 37 °C.
The digestion buffer (10 ml/lung) was composed of a 0.25% trypsin solution
containing 400 nM EDTA. Ice-cold complete DMEM was added to the result-
ing cell suspension. After centrifugation (240g for 5 min), pellets were resus-
pended in complete medium, then cultured in dishes. Lung fibroblasts were
used 10 d after excision for each experiment.
Electrophoretic mobility shift assay. Peritoneal macrophages and lung fibrob-
lasts (1 × 106) were stimulated with 100 ng/ml of LPS and 50 µg/ml of
poly(I:C), respectively. Nuclear extracts were purified from cells and incubated
with a probe specific for NF-κB DNA-binding site, separated by electrophoresis
and visualized by autoradiography as described15.
Splenocyte proliferation assay. Splenocytes (1 × 105) were cultured in 96-well
plates for 24 h with LPS, R-848 or CpG DNA. Samples were ‘pulsed’ with 1 µCi
[3H]thymidine for the last 12 h, and then 3H uptake was measured in a
β-scintillation counter (Packard).
Immunoblot analysis and in vitro kinase assay. Peritoneal macrophages (2 ×
106), embryonic fibroblasts (1 ×106) and lung fibroblasts (1 ×106) were stimu-
lated with 100 ng/ml of LPS, 10 ng/ml of IL-1β and 50 µg/ml of poly (I:C),
respectively. The cells were then lysed in a lysis buffer containing 1.0% Nonidet-
P40, 150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 1 mM EDTA and a protease
inhibitor ‘cocktail’ (Roche Diagnostics). The cell lysates were separated by SDS-
PAGE and transferred onto a polyvinylfluoride membrane (BioRad). The
membrane was blotted with specific antibodies, and samples were visualized
with an enhanced chemiluminescence system (Perkin Elmer Life Sciences).
IRAK-1 activity in the cell lysates was measured by in vitro kinase assay as
described46.
Detection of TRAM protein by immunoprecipitation. For immunoprecipita-
tion, cell lysates were precleared for 2 h with protein G–Sepharose (Amersham
Pharmacia Biotech) and then incubated for 12 h with rotation at 4 °C with pro-
tein G–Sepharose containing 1.0 µg anti-TRAM. The immunoprecipitants
were washed four times with lysis buffer, eluted by being boiled with Laemmli
sample buffer and analyzed by immunoblot with anti-TRAM, as described
above.
Flow cytometry. Splenocytes (2 × 106) were cultured with 50 µg/ml of
poly(I:C), 10 µg/ml of LPS or 10 µg/ml of anti-IgM. After 36 h of culture, cells
were collected and stained with phycoerythrin-conjugated anti-B220 and
biotin-conjugated anti-CD69, anti-CD86 or anti-I-Abfollowed by fluorescein
isothiocyanate–conjugated streptavidin. Stained cells were analyzed by
FACSCalibur with CellQuest software (Becton Dickinson).
Native PAGE assay.Lung fibroblasts (1 ×106) and peritoneal macrophages (5 ×
106) were stimulated with 50 µg/ml of poly (I:C) and 1 µg/ml of LPS, respec-
tively, and then lysed. Cell lysates in native PAGE sample buffer (62.5mM Tris-
Cl, pH 6.8, 15% glycerol and 1% deoxycholate) were separated by native PAGE
and then immunoblotted with anti-IRF-3 as described46.
GenBank accession number. TRAM, AY232653.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
We thank D. Golenbock and H. Tomizawa for providing the NF-κB reporter
plasmid and R-848, respectively. We also thank T. Kawai, H. Sanjo and H. Kuwata
for discussions; M. Hashimoto for secretarial assistance; N. Okita and N. Iwami for
technical assistance; and P. Lee for critical reading of the manuscript. Supported
by grants from Special Coordination Funds; the Ministry of Education, Culture,
Sports, Science and Technology; Research Fellowships of the Japan Society for the
Promotion of Science for Young Scientists; The Uehara Memorial Foundation; The
Naito Foundation; and The Junior Research Associate from RIKEN.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 6 August; accepted 25 September 2003
Published online at http://www.nature.com/natureimmunology/
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