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*
Department of Host Defense, Research Institute for Microbial Diseases and
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka University, Osaka, Japan;
Department of Tumor Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan;
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan; and
¶ Immunobiology Research Group, Gesellschaft für Biotechnologische Forsechung, Braunscheweig, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, IL-1
, and chemokines (9). The
cellular response by LPS occurs through interaction of LPS with a
circulating LPS-binding protein and CD14, a glycosylphosphatidyl
inositol-linked surface receptor, and subsequent activation of TLR4.
TLR4 has been genetically identified as a signaling molecule essential
for the responses to LPS (10). Furthermore, mice with
targeted disruption of the TLR4 gene are LPS unresponsive
(11). In contrast to TLR4-deficient mice, TLR2-deficient
mice are LPS responsive, but are unresponsive to Staphylococcus
aureus peptidoglycans and macrophage-activating lipopeptide-2 kDa
(MALP-2) derived from Mycoplasma fermentans, indicating the
different roles of the TLR family in the recognition of different PAMPs
(12, 13).
TLRs consist of two major domains characterized by extracellular
leucine-rich repeats and an intracellular region belonging to the IL-1R
family (14). Therefore, it is considered that TLRs use the
same signaling components as those in the IL-1R. Ligation of IL-1 to
the cell-surface receptor results in recruiting an adapter molecule
MyD88 to the receptor (15, 16, 17). A serine/threonine kinase
IL-1R-associated kinase (IRAK) is subsequently recruited,
becomes phosphorylated, dissociates from the receptor complex, and
associates with TNFR-associated factor (TRAF) 6 (18, 19).
This subsequently leads to the activation of two different pathways
involving the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated
protein (MAP) kinase family and the Rel family transcription factor
NF-
B. Our previous studies with MyD88-deficient mice showed that
this molecule is indispensable for the responses to IL-1, IL-1-related
cytokine IL-18, LPS, and MALP-2, demonstrating that MyD88 functions as
a general adaptor molecule for the IL-1R/TLR family (13, 20, 21). However, the difference between TLR2 and TLR4
signalings has been suggested. In MyD88-deficient macrophages,
production of IL-1, TNF-, and IL-6 in response to LPS and MALP-2
was completely impaired. However, LPS stimulation of MyD88-deficient
macrophages activates NF-B and JNK/p38, although this activation is
delayed when compared with wild-type (21). In contrast,
NF-B activation by MALP-2 stimulation is completely abolished in
MyD88-deficient macrophages (13). These results suggested
the existence of MyD88-independent pathway(s) that lead to NF-B and
JNK/p38 activation in TLR4 signaling.
In the present study, we demonstrate that activation of IFN regulatory factor (IRF) 3, as well as induction of IFN-inducible genes, is regulated by the MyD88- and TRAF6-independent pathway in TLR4 signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli-type synthetic lipid A (compound
506) was purchased from Dai-ichi Pure Chemicals (Tokyo, Japan; Ref.
21). MALP-2 was synthesized as described previously
(13). MyD88-deficient and TLR4-deficient mice were
generated and maintained as described previously (11, 20).
Age-matched mice were used for all experiments. C3H/HeN and C3H/HeJ
mice were obtained from SLC (Shizuoka, Japan). Peritoneal macrophages
were purified 4 days after 2 ml of 4.0% thioglycollate injection of
mice. Ab against mouse IRF-3 was as described previously
(22). Anti-IB, anti-CREB-binding protein
(CBP), anti-p300, and anti-JNK1 Abs were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IRAK1 Ab was
provided by Hayashibara Biochemical Laboratories (Okayama,
Japan). Pyrrolidine dithiocarbamate (PDTC) was also
purchased from Calbiochem (San Diego, CA). Recombinant TNF- and
IL-1 were purchased from Genzyme (Cambridge, MA).
Preparation of mouse embryonic fibroblasts (EFs)
E13.5 embryos were dissected, cut into small pieces, and soaked for 30 min in 5 ml 0.25% trypsin EDTA at room temperature with shaking and then inactivated with DMEM supplemented with 10% FBS. The cells were then suspended by pipetting, plated on two 10-cm dishes per embryo, and cultured in DMEM supplemented with 10% FBS. Cells cultured during days 3–5 were used for experiments.
Subtractive hybridization
Subtraction was performed essentially according to the manufacturer’s instructions for the PCR-select subtraction kit (Clontech Laboratories, Palo Alto, CA) (23). In brief, 1 x 108 peritoneal macrophages from MyD88-deficient mice were stimulated with 1.0 µg/ml lipid A for 4 h. Cytoplasmic RNA was extracted by guanidine isothiocyanate-cecium chloride gradient centrifugation. Poly(A) + RNA was purified using oligo-dT30 latex beads (Takara Shuzo, Kyoto, Japan). cDNAs were synthesized from 2.0 µg of poly(A) + RNA from lipid A-stimulated cells for tester and 2.0 µg of unstimulated cells for driver. Following RsaI digestion, adoptors 1 and 2R were ligated to the tester. Tester cDNA was hybridized with excess driver cDNA. After hybridization, differential cDNAs were selectively amplified by suppression PCR. Nested PCR products were ligated into a pGEM-T vector (Promega, Madison, WI). A subtracted library was constructed and independent clones were amplified by colony PCR. Differential screening against 500 clones was performed according to the manufacturer’s instructions (Clontech Laboratories).
Northern blot analysis
Total RNA was extracted using the TRIzol reagent (Life
Technologies, Gaithersburg, MD). Total RNA was electrophoresed,
transferred to a nylon membrane, and hybridized with cDNA probes as
described previously (20). cDNA probes specific for
IFN-
-inducible protein 10 (IP-10), glucocorticoid attenuated
response gene (GARG) 16, and immune-responsive gene 1 (IRG1)
were obtained from the subtractive screening. Probes for IL-6 and
TNF- were described previously (21). The cyclooxygenase
(COX)-2 probe was amplified by RT-PCR from LPS-stimulated mouse
peritoneal macrophages.
EMSA
The nuclear extracts of peritoneal macrophages (5 x
105) were purified after lipid A or MALP-2
stimulation as described previously (20). The
extracts were incubated with a specific probe for the
IFN-stimulated regulatory element (ISRE;
5'-GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3') found in the
ISG15 gene (24) or NF-B
(5'-ATCAGGGACTTTCCGCTGGGGACTTTCC-3') DNA-binding site,
electrophoresed, and visualized by autoradiography as described
previously (20).
Immunostaining of cells
Peritoneal macrophages seeded on glass plates at a density of 20,000 cells/ml were stimulated with 1.0 µg/ml lipid A for 4 h. The cells were washed twice with PBS before being incubated in a mixture of 3.0% paraformaldehyde and 0.3% Triton X-100 in PBS for 5 min to simultaneously fix and permeabilize, and then incubated in 3.0% paraformaldehyde for 20 min. The cells were washed three times in PBS and, following blocking with 3.0% BSA, then were washed in PBS for 60 min. The cells were incubated with anti-IRF-3 Ab for 60 min. They then were washed three times in PBS and incubated with biotinylated anti-rabbit Ig Ab (Vector Laboratories, Burlingame, CA) for 30 min. After washing, the cells were incubated with avidin-FITC (BD PharMingen, San Diego, CA) and 0.5 µg of 4',6-diamidino-2-phenylindole (Wako, Osaka, Japan) for an additional 30 min. The glass plates were washed four times in PBS, and drained. Microscopy analysis was conducted under the conditions of fluorescent light.
Western blot analysis
The nuclear extracts were separated on SDS-PAGE, transferred onto a nitrocellulose filter membrane, and incubated with the blocking buffer containing 5.0% skim milk. The filter was incubated with the indicated Ab before being washed three times in TBST and then incubated with anti-rabbit peroxidase-conjugated secondary Ab (Amersham Pharmacia Biotech, Chalfont, U.K.). After further washing with TBST, peroxidase activity was detected by using the ECL system (DuPont Pharmaceuticals, Boston, MA).
Reporter assay
EF cells (1 x 105) seeded on a
6-well plate were transiently cotransfected with 1.0 µg reporter gene
plasmid (pNF-B Luc) together with 0.1 µg of pRL-SV40 by
lipofection. After 24 h of transfection, cells were stimulated
with 1.0 µg/ml lipid A, 30 ng/ml MALP-2, 10 ng/ml IL-1, or 10
ng/ml TNF- for 8 h. The relative NF-B activity was
determined and normalized on the basis of sea pansy luciferase activity
as described previously (20).
In vitro kinase assay
EF cells stimulated with 100 ng/ml lipid A were lysed and immunoprecipitated with anti-IRAK Ab, then the kinase activity was measured by in vitro kinase assay as described previously (21).
The cell lysates were immunoprecipitated with anti-JNK1 Ab, then an in vitro kinase assay was performed using GST-c-Jun as the substrate as described previously (20).
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Results |
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To search for genes activated upon LPS stimulation in a
MyD88-independent manner, we prepared a cDNA library from
MyD88-deficient peritoneal macrophages with or without lipid A
stimulation and performed the suppression-subtractive hybridization.
The screening identified several genes that are induced in
MyD88-deficient macrophages upon stimulation with lipid A. Sequence
analysis of these clones revealed that most were identical to the gene
encoding IP-10, a family of CXC chemokines. Other than
IP-10, GARG16 and IRG1 were obtained
by this screening. These genes were reported to be induced upon
stimulation with IFN (25, 26, 27, 28, 29, 30). Consistent with our
previous study with MyD88-deficient mice, neither gene coding for
TNF-, IL-6 nor IL-1 was obtained in this screening. We therefore
examined the expression of IP-10, GARG16, and
IRG1 in response to lipid A in wild-type, MyD88- and
TLR4-deficient macrophages. Thioglycollate-elicited peritoneal
macrophages were treated with 1 µg/ml lipid A for 4 or 8 h and
expression of these genes was examined by Northern blot analysis. As
shown in Fig. 1
A, mRNAs for
IP-10, GARG16, and IRG1 were
significantly induced in response to lipid A in both wild-type and
MyD88-deficient macrophages. In contrast, induction of these genes was
not observed in TLR4-deficient macrophages. These results indicate that
MyD88 is dispensable for lipid A-induced expression of these genes,
although TLR4 is essential to these inductions. In contrast, lipid A
induction of the COX-2 gene was completely abolished in
MyD88-deficient macrophages.
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B),
raising the possibility that the MyD88-independent pathway could be
transmitted from TLR4. In addition, stimulation with Taxol resulted in
the delayed activation of NF-B in MyD88-deficient macrophages as did
LPS (Fig. 1C).
We next used MALP-2 that activates macrophages via TLR2 and
investigated its ability to induce the IP-10 gene. As shown
in Fig. 1D, stimulation with lipid A induced strong
expression of IP-10 mRNA in both wild-type and MyD88-deficient
macrophages, whereas the IP-10 gene was only slightly
induced in response to MALP-2 and its induction is MyD88-dependent. In
contrast, IL-6 mRNA was induced in wild-type macrophages after MALP-2
stimulation to the same extent as lipid A stimulation.
However, IL-6 mRNA induction was dramatically decreased in
lipid A-stimulated cells and completely abolished in MALP-2-stimulated
cells from MyD88-deficient mice. These results demonstrate that
IP-10 gene expression is regulated mainly by TLR4-dependent
and MyD88-independent pathways.
Induction of ISRE-binding activity in response to lipid A in MyD88-deficient mice
Previous reports indicated that expression of IP-10 is regulated
in part by the IRF family of transcription factors, and the promoter
region of the IP-10 gene contains typical ISRE and
NF-B-binding motifs recognized by IRFs and NF-B, respectively
(25, 34). Therefore, we investigated the ISRE-binding
activity after lipid A stimulation in macrophages. Nuclear extracts of
peritoneal macrophages treated with lipid A or MALP-2 were subjected to
EMSA using ISRE found in the IFN-stimulated gene 15 (ISG15)
promoter as a probe. As shown in Fig. 2A, ISRE binding was clearly
induced after 120 min of stimulation with lipid A. In agreement with
Northern blot analysis of IP-10 expression, MALP-2 had no ability to
induce ISRE-binding activity. However, NF-B was significantly
activated in the nuclear extract of cells stimulated with either lipid
A or MALP-2. These results strongly suggest a possible involvement of
ISRE-binding proteins in the induction of IP-10 in TLR4, but not TLR2,
signaling. We next tested whether MyD88 is required for ISRE binding
after lipid A stimulation. As shown in Fig. 2B, ISRE binding
was also induced in MyD88-deficient macrophages as in the case of
wild-type macrophages, suggesting that ISRE activation after lipid A
stimulation is MyD88-independent. We next examined lipid A-induced
NF-B and ISRE-binding activation in C3H/HeJ mice that are
unresponsive to LPS due to a point mutation of proline at 712 to
histidine residue in the cytoplasmic region of TLR4 (10, 11). As shown in Fig. 2C, lipid A-induced NF-B and
ISRE-binding activation were completely abolished in C3H/HeJ
macrophages, whereas the activity was induced in C3H/HeN macrophages.
The possibility that our C3H/HeJ mice might have a defect in NF-B
activation was ruled out because MALP-2-induced NF-B activation was
comparable between C3H/HeN and HeJ macrophages (Fig. 2D). In
addition, it was reported that LPS or lipid A-induced IL-6 and IP-10
expression was abolished in C3H/HeJ macrophages (32).
These results demonstrate that the proline residue at 712 is critical
for activation of both NF-B and ISRE in TLR4 signaling and for
induction of IP-10 as well as of proinflammatory cytokines.
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IRF-3 is reported to bind ISRE and induce expression of many genes
containing this motif including RANTES (35). IRF-3 is also
known to be phosphorylated in response to viral infection and
DNA-damaging agents (36). Phosphorylated IRF-3
translocates into the nucleus to induce gene expression by associating
with coactivator p300/CBP (22, 37). Therefore, we next
tested whether or not IRF-3 is activated by TLR4 signaling. As shown in
Fig. 3A, lipid A-induced
ISRE-binding complex in EMSA was supershifted by adding specific Ab
against IRF-3 in both wild-type and MyD88-deficient macrophages,
indicating that IRF-3 is responsible for ISRE binding in response to
lipid A in a MyD88-independent manner. The anti-IRF-3 Ab did not
cross-react with mouse IRF-1, IRF-2, IRF-7, and latent cytosolic
transcription factor(ISGF3) (data not shown). In addition, the
levels of IRF-3 protein expression were not altered by
stimulation with LPS in both wild-type and MyD88-deficient macrophages,
suggesting that IRF-3 is activated posttranslationally in response to
lipid A (data not shown). We also investigated whether or not signaling
molecules other than IRF-3 are also involved in ISRE binding after
lipid A stimulation. However, we could not detect any supershifted band
in EMSA in the extracts incubated with Abs against ISGF3, IRF-4,
IRF-7, STAT1, STAT2, STAT3, and phosphotyrosine (data not shown).
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B, IRF-3 was normally localized in the cytoplasms
in both wild-type and MyD88-deficient macrophages. Following lipid A
stimulation, IRF-3 protein accumulated in the nuclei in both
macrophages.
To confirm further, the nuclear extract of lipid A-stimulated
macrophages from wild-type and MyD88-deficient mice was immunoblotted
with anti-IRF-3 Ab. As shown in Fig. 3C, IRF-3 protein
was detected in the nuclear extracts of cells after 2 h of
stimulation with lipid A in both wild-type and MyD88-deficient cells,
confirming that IRF-3 translocated into the nucleus in response to
lipid A.
Phosphorylation of IRF-3 is known to be required for its
transcriptional activation. Peritoneal macrophages from wild-type and
MyD88-deficient mice were stimulated with lipid A, and mobility shift
of the IRF-3 protein on SDS-PAGE was studied by western blot analysis
with anti-IRF-3 Ab. As shown in Fig. 3D, IRF-3 protein
migrated more slowly on SDS-PAGE in response to lipid A in both
wild-type and MyD88-deficient macrophages. Phosphatase treatment of the
lysates reduced this mobility shift, indicating that this shift was due
to phosphorylation (data not shown).
To investigate whether coactivators CBP and p300 are involved in the
ISRE-binding complex, we incubated the nuclear extract of lipid
A-stimulated cells with anti-CBP Ab and/or anti-p300 Ab before
they were subjected to EMSA analysis. The ISRE-binding complex was
diminished by adding Abs to the coactivators CBP and p300 (Fig. 3E), indicating that the mixture of CBP/p300 is involved in
this complex.
Activation of NF-B is required for induction of the
IP-10 gene
Our previous study showed that lipid A-stimulation of
MyD88-deficient macrophages resulted in the induction of
NF-B-binding activity with delayed kinetics in EMSA
(21). We next examined whether NF-B translocation is
accompanied by IB degradation. Peritoneal macrophages from wild-type
and MyD88-deficient mice were incubated with lipid A, then the cell
extracts were prepared and used for Western blot analysis with
anti-IB Ab. As shown in Fig. 4A. IB degraded in 10
min and reappeared 30 min after stimulation. In contrast, the
degradation of IB was observed with the delayed kinetics in
MyD88-deficient macrophages consistent with the kinetics of NF-B
activation. Degradation of IB was also delayed in MyD88-deficient
cells (data not shown). However, it is not clear whether NF-B in
MyD88-deficient mice possesses a transcriptional activity. Therefore,
we determined by reporter gene assay whether NF-B activation in
MyD88-deficient mice is functional or not. EF cells derived from
MyD88-deficient mice also exhibited the induction of the
IP-10 gene and the delayed NF-B DNA-binding in response
to lipid A as seen in macrophages (Fig. 5C). Therefore, we used these
cells to perform the reporter gene assay. EF cells were transiently
transfected with NF-B reporter gene plasmid, and then stimulated
with lipid A, MALP-2, TNF-, and IL-1. As shown in Fig. 4B, NF-B-driven reporter gene expression was stimulated
in response to lipid A in both wild-type and MyD88-deficient cells. In
contrast, activation of NF-B was not observed in MyD88-deficient
cells in response to MALP-2 and IL-1, being consistent with the
results obtained from EMSA. TNF--stimulated NF-B activation was
comparable between wild-type and MyD88-deficient cells. These results
indicate that lipid A-mediated NF-B activation in MyD88-deficient
macrophages is also functional. It was shown that there are typical
ISRE and adjacent NF-B-binding sites in the promoter region of the
IP-10 gene, and both sites were required for induction of
IP-10 (34). We next used PDTC, a potent inhibitor for
NF-B, to investigate its affect on induction of the IP-10
gene. Peritoneal macrophages were pretreated with PDTC for 1 h and
stimulated with lipid A for 4 h. As shown in Fig. 4C,
induction of IP-10 mRNA was impaired by treatment with PDTC in both
wild-type and MyD88-deficient macrophages. Whereas this reagent
decreased lipid A-induced NF-B activation in EMSA, it did not affect
the nuclear translocation of IRF-3 (Fig. 4D), indicating
that lipid A-induced IRF-3 activation occurs independent of NF-B
translocation. Taken together, these results indicate that the delayed
activation of NF-B observed in MyD88-deficient cells is functional
and suggest that NF-B activation is required for IP-10
gene induction in response to lipid A.
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B and induces IP-10 gene
expression in a MyD88- and TRAF6-independent manner
We next tested whether or not TRAF6 is also involved in a
MyD88-independent pathway. EF cells derived from wild-type,
MyD88-deficient, TRAF6-deficient, and MyD88/TRAF6-doubly deficient mice
were stimulated with lipid A or IL-1 for 4 h and subjected to
the expression of IP-10 and IL-6. Induction of IL-6 in response to
lipid A or IL-1 was impaired in MyD88-, TRAF6-, and
MyD88/TRAF6-deficient cells, whereas IP-10 was significantly induced in
response to lipid A in a MyD88/TRAF6-independent manner (Fig. 5A). In contrast, IL-1 had no ability to induce IP-10 in
wild-type cells (Fig. 5A).
We next investigated activation of NF-B in response to lipid A or
IL-1. As shown in Fig. 5B, NF-B was activated in
response to lipid A in cells lacking MyD88, TRAF6, and MyD88/TRAF6. In
contrast, stimulation with IL-1 failed to activate NF-B in these
cells. Because lipid A-induced NF-B activation was delayed in
MyD88-deficient macrophages, we next performed time-course analysis
using EF cells. As shown in Fig. 5C, NF-B was activated
at 10 min of stimulation in wild-type cells but not in cells lacking
MyD88, TRAF6, and MyD88/TRAF6. At 60 min stimulation, NF-B was
activated in all types of cells. Furthermore, activation of JNK in
response to lipid A was also delayed in MyD88-, TRAF6-, and
MyD88/TRAF6-deficient EF cells (Fig. 5D). The activation of
other MAP kinases, such as extracellular signal-related kinase and p38,
was also delayed in these cells (data not shown). We next investigated
the activation of IRAK in response to lipid A. Consistent with our
previous report using macrophages, activation of IRAK was completely
abolished in MyD88-deficient EF cells. Surprisingly, IRAK activation
was also impaired in TRAF6- and MyD88/TRAF6-deficient cells (Fig. 5E). This result indicates that activation of IRAK is
dependent on both MyD88 and TRAF6 in EF cells. Taken together, the
delayed activation of NF-B and MAP kinases and the expression of
IP-10 in response to lipid A occur through MyD88- and TRAF6-independent
mechanisms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and TNF- in response
to LPS. Nevertheless, activation of NF-B and MAP kinases was
observed in LPS-stimulated MyD88-deficient cells with the delayed
kinetics. For the present, the nature and role of the MyD88-independent
pathway are not well understood. In this study, we demonstrated that
LPS activates IRF-3 and induces expression of a subset of LPS-inducible
genes in a MyD88-independent manner.
IRF-3 was originally identified as a member of the IRF family that
binds to the ISRE of the ISG15 (24, 38). The IRF-3 protein
is ubiquitously present in a variety of tissues and phosphorylated in
response to viral infection, dsRNA treatment, or DNA-damaging agents
(22, 36, 37, 39). Phosphorylated IRF-3 then translocates
to the nucleus, associates with the p300/CBP coactivator, and binds to
the ISRE, which results in induction of several IFN-regulated genes.
Recently, it has been reported that virus-induced IP-10 induction is
dependent on IRF-3 and ISGF3 (40). It has been shown that
IRF-3 also translocates to the nucleus upon stimulation with LPS in a
human astrocytoma cell line (41). In the present study, we
demonstrated that IRF-3 translocates to the nucleus in response to LPS
in MyD88-deficient macrophages as well as wild-type macrophages.
MyD88-deficient macrophages responded to LPS to induce IFN-regulated
genes to similar extents as those of wild-type macrophages, indicating
that LPS activation of IRF-3 is MyD88-independent. In contrast,
induction of IL-6, IL-1, and TNF- mRNA in response to LPS was
dramatically reduced in MyD88-deficient macrophages. Furthermore, lipid
A-induction of COX-2 mRNA was completely abolished in MyD88-deficient
macrophages. Thus, it appears that LPS activates at least two signaling
pathways to induce different subsets of genes; the MyD88-dependent
pathway regulates expression of IL-6, IL-1, TNF-, and COX-2,
whereas the MyD88-independent pathway regulates expression of
IFN-regulated genes such as IP-10, GARG16, and
IRG-1, possibly through coordinate action of IRF-3 and
NF-B. Although we used the ISG15 ISRE in all of our IRF-3 binding
studies, we recognize that this does not imply that IRF-3 will bind to
ISREs derived from the three genes identified by subtractive
hybridization. Because our present study does not show any direct
involvement of IRF-3 in LPS-induced IP-10,
GARG16, or IRG-1 gene expression, further study
will be required to ascertain this point.
The finding that MyD88-deficient mice, but not TLR4-deficient and
C3H/HeJ mice, could activate both NF-B and IRF-3 as well as the
IP-10 gene in response to LPS or Taxol indicates that the
MyD88-independent pathway originates from the cytoplasmic portion of
TLR4. In contrast, TLR2-dependent stimuli, such as MALP-2, failed to
activate IRF-3 and induce IP-10, which is consistent with our previous
finding that MALP-2-mediated activation of NF-B and MAP kinases is
completely abolished in MyD88-deficient cells. Consistent with our
result, Hirschfeld et al. (42) recently showed that
macrophage stimulation by Porphyromonas gingivalis LPS
preparation, a potent TLR2 ligand, resulted in diminished IP-10
expression compared with stimulation by E. coli LPS. Taken
together, these findings show that TLR2 activates NF-B and MAP
kinases only through the MyD88-dependent pathway as is the case with
the IL-1R family, whereas TLR4 activates NF-B and MAP kinases
through MyD88-dependent and -independent pathways (Fig. 6).
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and IP-10, was
diminished in CD11b/CD18-deficient macrophages . These results suggest
that there are different mechanisms in the regulation of the these
genes in response to LPS. The genes affected by CD11b/CD18 were induced
by the MyD88-dependent pathway, implying that the MyD88-dependent
pathway may be modulated by CD11b/CD18.
How does LPS activate NF-B, MAP kinases, and IRF-3 in a
MyD88-independent manner? TRAF6 is a candidate that participates in the
MyD88-independent pathway as it has the ability to activate NF-B and
MAP kinases by associating IRAK, and TRAF6-deficient cells are
defective in activating NF-B by IL-1 (17, 44, 45).
However, induction of IP-10 and activation of NF-B and JNK were
observed in response to lipid A but not IL-1 in TRAF6-deficient
fibroblast cells, as seen in MyD88-deficient cells. Furthermore,
MyD88/TRAF6-doubly deficient cells also responded to lipid A by
inducing the IP-10 gene and activating NF-B and JNK.
Taken together, TRAF6 is dispensable for LPS-mediated induction of
IP-10 and activation of NF-B and the MyD88-independent pathway in
TLR4 signaling integrates at a level downstream of TRAF6, ultimately
leading to the activation of NF-B and MAP kinases (Fig. 6). Previous
reports have indicated that TRAF6 acts downstream to IRAK (15, 16). However, we showed in this study that lipid A-induced
activation of IRAK was completely abolished in TRAF6-deficient mice.
This observation demonstrates that IRAK activation completely depends
on both MyD88 and TRAF6.
Other than TRAF6, LPS is known to activate a number of signaling
molecules that include protein kinase C, src-type tyrosine
kinases, small G protein, PI3K, Akt, TAK1, and double-stranded
RNA-dependent protein kinase, all of which are reported to have
abilities to activate NF-B (46, 47, 48, 49, 50, 51, 52). It is possible
that these molecules are involved in the MyD88-independent pathway
leading to NF-B and IRF-3 activation in response to LPS.
In summary, we showed in this study that IRF-3 can be activated in
response to LPS in a MyD88-independent manner. The MyD88-dependent
pathway regulates expression of genes encoding inflammatory cytokines,
whereas the MyD88-independent pathway regulates a subset of
LPS-inducible genes that have been previously reported to be
IFN-inducible. In contrast, TLR2 failed to induce the IP-10
gene. Given that TLR4, but not TLR2, also activates NF-B in a
MyD88-independent mechanism, it is possible that TLR4 specifically uses
an unknown molecule other than MyD88 for its signaling to both NF-B
and IRF-3. Future research on identification of such a molecule may
clarify its precise mechanism on the signaling cascade triggered by the
TLR family, especially TLR4.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shizuo Akira, Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka Suita, Osaka 565-0871, Japan. E-mail address: sakira{at}biken.osaka-u.ac.jp
3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; IRAK, IL-1R-associated kinase; TRAF, TNFR-associated factor; MAP, mitogen-activated protein; MALP-2, macrophage-activating lipopeptide-2 kDa; JNK, c-Jun N-terminal kinase; IRF, IFN regulatory factor; CBP, CREB-binding protein; PDTC, pyrrolidine dithiocarbamate; EF, embryonic fibroblast; ISRE, IFN-stimulated regulatory element; COX, cyclooxygenase; IP-10, IFN--inducible protein 10; GARG, glucocorticoid attenuated response gene; IRG1, immune-responsive gene 1; ISGF3, latent cytosolic transcription factor.
Received for publication December 18, 2000. Accepted for publication September 7, 2001.
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References |
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B sequence motifs controls IFN-- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter. J. Biol. Chem. 268:6677.B site but not IRF-1 or viral transcription. J. Interferon Cytokine Res. 18:987.[Medline]
is required for the induction of mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J. Immunol. 164:29.B as well as the MAP kinase cascade in the IL-1 signaling pathway. Nature 398:252.[Medline]
B in lipopolysaccharide-stimulated macrophages. FEBS Lett. 467:160.[Medline]
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 K. Yang, H. Shi, R. Qi, S. Sun, Y. Tang, B. Zhang, and C. Wang Hsp90 Regulates Activation of Interferon Regulatory Factor 3 and TBK-1 Stabilization in Sendai Virus-infected Cells Mol. Biol. Cell, March 1, 2006; 17(3): 1461 - 1471. [Abstract] [Full Text] [PDF]
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S. K. Biswas, L. Gangi, S. Paul, T. Schioppa, A. Saccani, M. Sironi, B. Bottazzi, A. Doni, B. Vincenzo, F. Pasqualini, et al. A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-{kappa}B and enhanced IRF-3/STAT1 activation) Blood, March 1, 2006; 107(5): 2112 - 2122. [Abstract] [Full Text] [PDF]
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 S. Chanchevalap, M. O. Nandan, B. B. McConnell, L. Charrier, D. Merlin, J. P. Katz, and V. W. Yang Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells Nucleic Acids Res., February 25, 2006; 34(4): 1216 - 1223. [Abstract] [Full Text] [PDF]
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M. Sanchez-Alavez, I. V. Tabarean, M. M. Behrens, and T. Bartfai Ceramide mediates the rapid phase of febrile response to IL-1beta PNAS, February 21, 2006; 103(8): 2904 - 2908. [Abstract] [Full Text] [PDF]
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J. S. Campbell, K. J. Riehle, J. T. Brooling, R. L. Bauer, C. Mitchell, and N. Fausto Proinflammatory Cytokine Production in Liver Regeneration Is Myd88-Dependent, but Independent of Cd14, Tlr2, and Tlr4 J. Immunol., February 15, 2006; 176(4): 2522 - 2528. [Abstract] [Full Text] [PDF]
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T. Mahieu, J. M. Park, H. Revets, B. Pasche, A. Lengeling, J. Staelens, A. Wullaert, I. Vanlaere, T. Hochepied, F. van Roy, et al. The wild-derived inbred mouse strain SPRET/Ei is resistant to LPS and defective in IFN-beta production PNAS, February 14, 2006; 103(7): 2292 - 2297. [Abstract] [Full Text] [PDF]
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S. H. Han, J. H. Kim, H. S. Seo, M. H. Martin, G.-H. Chung, S. M. Michalek, and M. H. Nahm Lipoteichoic Acid-Induced Nitric Oxide Production Depends on the Activation of Platelet-Activating Factor Receptor and Jak2 J. Immunol., January 1, 2006; 176(1): 573 - 579. [Abstract] [Full Text] [PDF]
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R. Mashima, K. Saeki, D. Aki, Y. Minoda, H. Takaki, T. Sanada, T. Kobayashi, H. Aburatani, Y. Yamanashi, and A. Yoshimura FLN29, a Novel Interferon- and LPS-inducible Gene Acting as a Negative Regulator of Toll-like Receptor Signaling J. Biol. Chem., December 16, 2005; 280(50): 41289 - 41297. [Abstract] [Full Text] [PDF]
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H. Yumoto, H.-H. Chou, Y. Takahashi, M. Davey, F. C. Gibson III, and C. A. Genco Sensitization of Human Aortic Endothelial Cells to Lipopolysaccharide via Regulation of Toll-Like Receptor 4 by Bacterial Fimbria-Dependent Invasion Infect. Immun., December 1, 2005; 73(12): 8050 - 8059. [Abstract] [Full Text] [PDF]
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Z. Guo, S. Garg, K. M. Hill, L. Jayashankar, M. R. Mooney, M. Hoelscher, J. M. Katz, J. M. Boss, and S. Sambhara A Distal Regulatory Region Is Required for Constitutive and IFN-{beta}-Induced Expression of Murine TLR9 Gene J. Immunol., December 1, 2005; 175(11): 7407 - 7418. [Abstract] [Full Text] [PDF]
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M. Svensson, S. Zubairi, A. Maroof, F. Kazi, M. Taniguchi, and P. M. Kaye Invariant NKT Cells Are Essential for the Regulation of Hepatic CXCL10 Gene Expression during Leishmania donovani Infection Infect. Immun., November 1, 2005; 73(11): 7541 - 7547. [Abstract] [Full Text] [PDF]
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C. W. Wieland, S. Florquin, N. A. Maris, K. Hoebe, B. Beutler, K. Takeda, S. Akira, and T. van der Poll The MyD88-Dependent, but Not the MyD88-Independent, Pathway of TLR4 Signaling Is Important in Clearing Nontypeable Haemophilus influenzae from the Mouse Lung J. Immunol., November 1, 2005; 175(9): 6042 - 6049. [Abstract] [Full Text] [PDF]
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P. Mandal, M. Novotny, and T. A. Hamilton Lipopolysaccharide Induces Formyl Peptide Receptor 1 Gene Expression in Macrophages and Neutrophils via Transcriptional and Posttranscriptional Mechanisms J. Immunol., November 1, 2005; 175(9): 6085 - 6091. [Abstract] [Full Text] [PDF]
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K. Honda, H. Yanai, A. Takaoka, and T. Taniguchi Regulation of the type I IFN induction: a current view Int. Immunol., November 1, 2005; 17(11): 1367 - 1378. [Abstract] [Full Text] [PDF]
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H. Qin, C. A. Wilson, S. J. Lee, X. Zhao, and E. N. Benveniste LPS induces CD40 gene expression through the activation of NF-{kappa}B and STAT-1{alpha} in macrophages and microglia Blood, November 1, 2005; 106(9): 3114 - 3122. [Abstract] [Full Text] [PDF]
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 K. Fuse, G. Chan, Y. Liu, P. Gudgeon, M. Husain, M. Chen, W.-C. Yeh, S. Akira, and P. P. Liu Myeloid Differentiation Factor-88 Plays a Crucial Role in the Pathogenesis of Coxsackievirus B3-Induced Myocarditis and Influences Type I Interferon Production Circulation, October 11, 2005; 112(15): 2276 - 2285. [Abstract] [Full Text] [PDF]
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S. Shi, A. Blumenthal, C. M. Hickey, S. Gandotra, D. Levy, and S. Ehrt Expression of Many Immunologically Important Genes in Mycobacterium tuberculosis-Infected Macrophages Is Independent of Both TLR2 and TLR4 but Dependent on IFN-{alpha}{beta} Receptor and STAT1 J. Immunol., September 1, 2005; 175(5): 3318 - 3328. [Abstract] [Full Text] [PDF]
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M. B. Drennan, B. Stijlemans, J. Van Den Abbeele, V. J. Quesniaux, M. Barkhuizen, F. Brombacher, P. De Baetselier, B. Ryffel, and S. Magez The Induction of a Type 1 Immune Response following a Trypanosoma brucei Infection Is MyD88 Dependent J. Immunol., August 15, 2005; 175(4): 2501 - 2509. [Abstract] [Full Text] [PDF]
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H. Shindou, S. Ishii, M. Yamamoto, K. Takeda, S. Akira, and T. Shimizu Priming Effect of Lipopolysaccharide on Acetyl-Coenzyme A:Lyso-Platelet-Activating Factor Acetyltransferase Is MyD88 and TRIF Independent J. Immunol., July 15, 2005; 175(2): 1177 - 1183. [Abstract] [Full Text] [PDF]
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U. M. Nagarajan, D. M. Ojcius, L. Stahl, R. G. Rank, and T. Darville Chlamydia trachomatis Induces Expression of IFN-{gamma}-Inducible Protein 10 and IFN-{beta} Independent of TLR2 and TLR4, but Largely Dependent on MyD88 J. Immunol., July 1, 2005; 175(1): 450 - 460. [Abstract] [Full Text] [PDF]
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V. U. Toshchakov, S. Basu, M. J. Fenton, and S. N. Vogel Differential Involvement of BB Loops of Toll-IL-1 Resistance (TIR) Domain-Containing Adapter Proteins in TLR4- versus TLR2-Mediated Signal Transduction J. Immunol., July 1, 2005; 175(1): 494 - 500. [Abstract] [Full Text] [PDF]
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G. Gautier, M. Humbert, F. Deauvieau, M. Scuiller, J. Hiscott, E. E.M. Bates, G. Trinchieri, C. Caux, and P. Garrone A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells J. Exp. Med., May 2, 2005; 201(9): 1435 - 1446. [Abstract] [Full Text] [PDF]
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S. M. Zughaier, S. M. Zimmer, A. Datta, R. W. Carlson, and D. S. Stephens Differential Induction of the Toll-Like Receptor 4-MyD88-Dependent and -Independent Signaling Pathways by Endotoxins Infect. Immun., May 1, 2005; 73(5): 2940 - 2950. [Abstract] [Full Text] [PDF]
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A. Schoenemeyer, B. J. Barnes, Margo. E. Mancl, E. Latz, N. Goutagny, P. M. Pitha, K. A. Fitzgerald, and D. T. Golenbock The Interferon Regulatory Factor, IRF5, Is a Central Mediator of Toll-like Receptor 7 Signaling J. Biol. Chem., April 29, 2005; 280(17): 17005 - 17012. [Abstract] [Full Text] [PDF]
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