International Immunology

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International Immunology, Vol. 13, No. 7, 933-940, July 2001
© 2001 Japanese Society for Immunology

Discrimination of bacterial lipoproteins by Toll-like receptor 6

Osamu Takeuchi^1,2, Taro Kawai^1,2, Peter F. Mühlradt³, Michael Morr³, Justin D. Radolf⁴, Arturo Zychlinsky⁵, Kiyoshi Takeda^1,2 and Shizuo Akira^{1,2 1} Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, and
² Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corp., 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
³ Immunobiology Research Group, Gesellschaft für Biotechnologische Forschung, Mascheroderweg 1, 38124 Braunschweig, Germany
⁴ Center for Microbial Pathogenesis, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA
⁵ Skirball Institute and Department of Microbiology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

Correspondence to: S. Akira

	Abstract

Top Abstract Introduction Methods Results and discussion References

 
Bacterial lipoproteins (BLP) trigger immune responses via Toll-like receptor 2 (TLR2) and their immunostimulatory properties are attributed to the presence of a lipoylated N-terminus. Most BLP are triacylated at the N-terminus cysteine residue, but mycoplasmal macrophage-activating lipopeptide-2 kD (MALP-2) is only diacylated. Here we show that TLR6-deficient (TLR6^–/–) cells are unresponsive to MALP-2 but retain their normal responses to lipopeptides of other bacterial origins. Reconstitution experiments in TLR2^–/– TLR6^–/– embryonic fibroblasts reveal that co-expression of TLR2 and TLR6 is absolutely required for MALP-2 responsiveness. Taken together, these results show that TLR6 recognizes MALP-2 cooperatively with TLR2, and appears to discriminate between the N-terminal lipoylated structures of MALP-2 and lipopeptides derived from other bacteria.

Keywords: cytokine, inflammatory mediator, knockout, macrophage, monocyte, transgenic

	Introduction

Top Abstract Introduction Methods Results and discussion References

 
The innate immune system senses invading pathogens by germline-encoded pattern recognition receptors (1). Recent studies have revealed that the Toll-like receptors (TLR), homologues of the Drosophila Toll protein, play an important role in recognizing microorganisms (2). TLR are type I transmembrane receptors characterized by the presence of extracellular leucine-rich repeat (LRR) motifs and a cytoplasmic Toll/IL-1R (TIR) homology domain, which is required for the signaling leading to activation of the transcription factor NF-B (3). Nuclear translocation of NF-B induces the transcription of proinflammatory cytokine genes, which leads to the establishment of acquired immunity. Among the nine reported TLR family members (3–6), TLR2, TLR4 and TLR9 have been implicated in the recognition of differential bacterial components. TLR2 recognizes peptidoglycan (PGN), lipoproteins from various microorganisms, lipoarabinomannan and zymosan (7–14). In contrast, TLR4 and TLR9 are essential for the responses to lipopolysaccharide (LPS) and bacterial DNA, respectively (15–17). However, the roles of the remaining TLR family members have yet to be fully elucidated.

We previously reported the molecular cloning of TLR6, which is closely related to TLR1 and TLR2. In the present study, we generated TLR6^–/– mice by gene targeting in order to investigate the in vivo function of TLR6. Macrophages from TLR6^–/– mice did not respond to mycoplasmal lipopeptide, i.e. diacylated mycoplasmal macrophage-activating lipopeptide-2 kD (MALP-2), although they showed normal responses to triacylated lipopeptides derived from other bacteria. In contrast, we observed that TLR2^–/– cells failed to recognize either type of lipopeptide, suggesting that TLR6 cooperates with TLR2 in the recognition of MALP-2 and is responsible for discriminating among different TLR2 ligands.

	Methods

Top Abstract Introduction Methods Results and discussion References

 
Generation of TLR6^–/– mice
A mouse genomic clone of TLR6 was obtained from the 129/Sv genomic library. A fragment from this clone including one exon was subcloned into pBluescript (Stratagene, La Jolla, CA), and was characterized by restriction enzyme mapping and DNA sequencing. A targeting vector was constructed consisting of the neomycin resistance gene flanked by 5 kb of the 5' genomic region of TLR6 and 1.1 kb of the 3' region. This construct was designed to remove a 1.7 kb region of the genomic TLR6 sequence that encodes a portion of the extracellular domain, the transmembrane domain and the cytoplasmic domain of TLR6, and replace it with the neomycin resistance gene. An HSV-tk cassette was introduced at the 5' end of the vector to select against non-homologous recombination. The linearized targeting vector was introduced by electroporation into E14.1 embryonic stem (ES) cells. Clones resistant to G418 and gancyclovir were screened by PCR for homologous recombination, and confirmed by Southern blot analysis using the probe shown in Fig. 1(A). Homologous recombination was confirmed in four out of 126 ES clones resistant to G418 and gancyclovir. Chimeric mice were generated by microinjection of the targeted ES clones into C57BL/6 blastocysts. Male chimeric mice were bred to C57BL/6 females to produce heterozygous mice. Heterozygous mice were then interbred to obtain homozygotes. TLR6^–/– mice and their wild-type littermates from these intercrosses were used for experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Targeted disruption of the TLR6 locus. (A) The mouse TLR6 locus and the targeting vector. Filled boxes denote the coding exon. Restriction enzymes: B, BamHI; E, EcoRI. (B) Southern blotting of genomic DNA. Mouse genomic DNA was extracted from mouse tails, digested with EcoRI and hybridized with radiolabeled probe A. The 2.5 kb wild-type and the 3.2 kb targeted alleles are indicated by arrows. (C) Northern blot analysis of thioglycollate-elicited peritoneal macrophages. Total RNA was extracted, electrophoresed, transferred to nylon membranes, and hybridized with cDNA probes for TLR6 and TLR2. The same membrane was rehybridized with a GAPDH cDNA. (D) Responsiveness of TLR6–/– macrophages to LPS and PGN. Thioglycollate-elicited peritoneal macrophages (5x104) from wild-type and TLR6–/– mice were stimulated with S. minnesota Re595 LPS or S. aureus PGN for 24 h. Concentrations of TNF- in the culture supernatants were measured by ELISA. The results are shown as the mean ± SEM of triplicate wells.

 
Reagents, mice and cells
LPS from Salmonella minnesota Re595 and PGN derived from Staphylococcus aureus were purchased from Sigma (St Louis, MO). MALP-2, lipopeptides from Borrelia burgdorferi (OspAL and OspCL) and Treponema pallidum (17L and 47L) were synthesized as described (14,18,19). Synthetic Pam₃CSK₄ was obtained from Boehringer Mannheim (Mannheim, Germany). IFN- was obtained from Genzyme (Cambridge, MA). TLR2^–/– mice were generated by gene targeting as described previously (7).

Peritoneal macrophages were collected 3 days after i.p. injection of 2 ml of 4% thioglycollate and cultured in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Rockville, MD). Murine embryonic fibroblasts (MEF) were prepared from day 13.5 embryos and cultured in DMEM supplemented with 10% fetal bovine serum (Life Technologies). All experiments were done at passage 3 and passage 5.

ELISA
Concentrations of tumor necrosis factor (TNF)- and IL-12 in the culture supernatants were measured by ELISA (Genzyme, Minneapolis, MN). Production of NO was measured by the Greiss method using the NO₂/NO₃ Assay Kit-C (Dojindo, Kumamoto, Japan).

Electrophoretic mobility shift assay (EMSA)
Peritoneal macrophages (2x10⁶) were stimulated with MALP-2 or Pam₃CSK₄ as described previously (20). Nuclear extracts were incubated with a probe specific for the NF-B DNA binding site, subjected to gel shift assay and visualized by autoradiography as described previously (20).

In vitro kinase assay and Western blotting
JNK kinase activity was assayed as described (20). In brief, thioglycollate-induced peritoneal macrophages (1x10⁶) were treated with MALP-2 or Pam₃CSK₄ for the indicated period, cells were solubilized in lysis buffer (20 mM Tris–HCl, pH 7.4, 137 mM NaCl, 5 mM EDTA and Triton X-100) and the lysates subjected to immunoprecipitation with anti-JNK1 antibody (Santa Cruz, Santa Cruz, CA) followed by in vitro kinase assay. The same lysates were fractionated by SDS–PAGE and transferred to nitrocellulose membrane. The membrane was blotted with anti-JNK1 and visualized using the enhanced chemiluminescence system (DuPont, Boston, MA).

Plasmids
Human TLR6 tagged with Myc at the C-terminus was generated by PCR and ligated into the expression plasmid pEF-BOS. The transmembrane and the cytoplasmic portions of TLR2 (amino acids 584–784) were fused to the extracellular domain of Fas (amino acids 1–169), then cloned into pEF-BOS. Human TLR2 and TLR4 were generated by PCR and cloned into the pCMV-FLAG vector (Stratagene).

C-terminal truncation variants of human TLR2 (amino acids 1–643) and TLR6 (amino acids 1–644) were generated by PCR. A TLR2/TLR6 chimera was generated by fusing the extracellular domain of TLR2 (amino acids 1–583) to the transmembrane and the cytoplasmic portion of TLR6 (amino acids 578–796). The reciprocal construct, a TLR6/TLR2 chimera, was generated by joining the extracellular domain of TLR6 (amino acids 1–577) to the transmembrane and cytoplasmic domains of TLR2 (amino acids 584–784). The pMX/IRES-GFP vector (pMIG) was constructed by inserting polioma virus IRES and EGFP into the pMX vector (21). Variants of pMIG containing TLR2, TLR2, TLR6, TLR6, the TLR2/TLR6 chimera or the TLR6/TLR2 chimera were constructed by inserting the respective cDNAs in front of IRES in pMIG.

Luciferase assay
Human embryonic kidney 293 cells were transiently transfected with 1.0 µg pNF-B Luc reporter plasmid (Stratagene) together with the indicated expression vectors by lipofection (Mirus, Madison, WI). Cells were lysed after 36 h of transfection, and relative NF-B activity was determined and normalized based on sea-pansy luciferase activity (Promega, Madison, WI) as described previously (20).

Retroviral infection
The Plat-E packaging cell line (22) was transfected with retroviral vectors using the Lipofectamine reagent (Life Technologies). The viral supernatant was harvested 48 h after transfection and used to infect MEF with 10 µg/ml Polybrene (Sigma). The MEF were incubated with retroviral solution for 24 h, further cultured with fresh medium for 48 h, collected and analyzed for green fluorescent protein (GFP) fluorescence, and 3x10⁴ infected cells were plated onto 24-well plates. After 24 h, the cells were treated with MALP-2 (10 ng/ml) or Pam₃CSK₄ (100 ng/ml) for 18 h. The concentration of IL-6 in the supernatants was determined by ELISA (Endogen, Boston, MA). The transfection efficiencies of GFP were ~40–60%.

	Results and discussion

Top Abstract Introduction Methods Results and discussion References

 
To investigate the functional role of TLR6, we generated TLR6^–/– mice by gene targeting in E14.1 ES cells. The targeting vector was designed to replace the exon which encodes a portion of the extracellular domain, the transmembrane and the cytoplasmic domain of TLR6 with the neomycin resistance gene (Fig. 1A). Three heterozygous ES cell lines containing a mutant TLR6 allele were microinjected into C57BL/6 blastocysts. Two lines of these chimeric mice successfully transmitted the disrupted TLR6 gene through the germline (Fig. 1B). TLR6^–/– mice were produced in Mendelian ratios from crosses among heterozygous mice. The mice were healthy, fertile and did not show any obvious abnormalities for the first 6 months. To confirm the TLR6 mutation indeed had inactivated the TLR6 gene, we performed Northern blot analysis. TLR6 mRNA was not detected in peritoneal macrophages from TLR6^–/– mice, and expression of TLR2 was unaltered between wild-type and TLR6^–/– macrophages (Fig. 1C).

Since we anticipated that TLR6 plays a role in the recognition of bacterial components, we first examined the response of mutant TLR6^–/– peritoneal macrophages to LPS, PGN and CpG oligonucleotide. Peritoneal macrophages derived from wild-type and TLR6^–/– mice were cultured with various concentrations of LPS or S. aureus PGN for 24 h, and the production of TNF- was measured. Peritoneal macrophages from TLR6^–/– mice produced TNF- in response to LPS in a dose-dependent manner to the same extent as those from wild-type mice (Fig. 1D). Induction of TNF- production by S. aureus PGN was slightly reduced in TLR6^–/– macrophages compared with wild-type macrophages, but was still significant (Fig. 1D). TNF- production in response to an oligonucleotide with the CpG motif was normal in TLR6^–/– macrophages (data not shown).

Bacterial lipoproteins (BLP) are produced by many different species of bacterial pathogens and are known to induce cytokine production in macrophages (23). All lipoproteins contain a lipoylated N-terminal amino acid residue, most often N-acyl-S-diacylglyceryl cysteine (24). In contrast, the cysteine residue of macrophage-activating lipopeptide-2kD (MALP-2) from Mycoplasma fermentans is only S-diacylated (25). Synthetic lipoprotein analogs (sBLP), such as tripalmitoyl cysteinyl (Pam₃Cys) lipopeptide Pam₃CSK₄ and dipalmitoyl MALP-2, have been shown to mimic the proinflammatory properties of BLP (Fig. 2A) (11,14). A monoacylated lipopeptide failed to induce TNF- production even in wild-type macrophages (data not shown), indicating that the type of lipid moiety determines activation of host macrophages. We have previously shown that induction of TNF- and NO production by MALP-2 was abrogated in TLR2^–/– macrophages (17). It was further reported that Pam₃CSK₄ activates cells via TLR2 in vitro (11). Therefore, we next analyzed the responses of TLR6^–/– and TLR2^–/– cells to the synthetic lipopeptides, MALP-2 and Pam₃CSK₄. Peritoneal macrophages from wild-type, TLR6^–/– and TLR2^–/– mice were treated with increasing doses of MALP-2. Whereas wild-type macrophages secreted TNF- in a dose-dependent manner, TLR2^–/– and TLR6^–/– macrophages produced no detectable amount of TNF- in response to MALP-2 (Fig. 2B). When cells were treated with MALP-2 together with IFN-, wild-type macrophages produced NO and IL-12, but TLR2^–/– and TLR6^–/– cells did not produce detectable amounts of either (Fig. 2C and D). These results indicate that both TLR2 and TLR6 are required for MALP-2-induced cytokine production. We then tested stimulation by the synthetic tripalmitoyl lipopeptide Pam₃CSK₄. We found that Pam₃CSK₄ stimulated production of TNF-, NO and IL-12 at similar levels in wild-type and TLR6^–/– macrophages, but failed to stimulate production in TLR2^–/– macrophages (Fig. 2E–G). We next examined the synthetic analogs of B. burgdorferi and T. pallidum lipopeptides, which also possess a tripalmitoyl-S-glyceryl-cysteine moiety at the N-terminus (18,19). Consistent with the results using Pam₃CSK₄, production of TNF- in response to spirochetal lipopeptides was similar between wild-type and TLR6^–/– macrophages (Fig. 2H), whereas the responses of TLR2^–/– cells to these lipopeptides were completely abrogated. These data indicate that TLR2 is essential for cytokine production in response to both diacylated and triacylated lipopeptides, whereas TLR6 is required only for production in response to diacylated lipopeptides. To further investigate whether TLR6 or TLR2 are involved in the recognition of mycoplasma components, we examined the responsiveness of these mutant macrophages to heat-killed mycoplasma. Both TLR6^–/– and TLR2^–/– macrophages showed impaired TNF- production in response to heat-killed mycoplasma compared to wild-type cells (Fig. 2I), indicating TLR2 and TLR6 play a central role in recognizing mycoplasma.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Responsiveness of macrophages to BLP. (A) Structures of synthetic BLP, MALP-2 and Pam3CSK4. (B–G) Cytokine production in response to MALP-2 or Pam3CSK4 in TLR6–/– TLR2–/– macrophages. Peritoneal macrophages (5x104) were stimulated with the indicated concentrations of MALP-2 (B–D) or Pam3CSK4 (E–G) in the absence (B and E) or presence (C, D and G) of 30 U/ml IFN- for 24 h. Concentrations of TNF- (B and E), NO (C and F) and IL-12 (D and G) in culture supernatants were measured. (H) Responses of macrophages to spirochetal lipopeptides. Peritoneal macrophages were stimulated with 15 µg/ml B. burgdorferi lipopeptides (OspAL and OspCL) or T. pallidum lipopeptides (17L and 47L) and TNF- production measured. (I) Requirement of TLR2 and TLR6 in mycoplasma recognition. Peritoneal macrophages were cultured with heat-killed M. fermentans clone II-29/1 and their TNF- production analyzed. The results are shown as the mean ± SEM of triplicate wells.

 
BLP have been shown to activate both the NF-B and JNK signaling pathways (17,18). We have previously shown that MALP-2-mediated activation of NF-B and JNK was abrogated in TLR2^–/– macrophages (17). Therefore, we examined the role of TLR6 in the activation of these intracellular signaling pathways. In wild-type macrophages, NF-B DNA binding activity was induced following stimulation with MALP-2, but was abolished in both TLR6^–/– and TLR2^–/– macrophages (Fig. 3A). NF-B activity was also induced by Pam₃CSK₄ in both wild-type and TLR6^–/– macrophages (Fig. 3B). JNK was activated by both Pam₃CSK₄ and MALP-2 in wild-type macrophages, but was activated only by Pam₃CSK₄ in TLR6^–/– cells. Both lipopeptides failed to activate JNK in TLR2^–/– macrophages (Fig. 3C and D). These results demonstrate that MALP-2-induced signal transduction requires both TLR2 and TLR6, while Pam₃CSK₄-induced signal transduction requires TLR2 but not TLR6.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Activation of signaling cascades via TLR6–/– and TLR2–/–. (A and B) EMSA using an NF-B-specific probe. Peritoneal macrophages were stimulated with 0.3 ng/ml MALP-2 (A) or 1 ng/ml Pam3CSK4 (B) for the indicated periods. Nuclear extracts were prepared and NF-B DNA binding activity was determined by EMSA. Arrows indicate induced NF-B complex. (C and D) JNK in vitro kinase assay. Peritoneal macrophages were stimulated with 0.3 ng/ml of MALP-2 (C) or 1 ng/ml Pam3CSK4 (D) for the indicated periods. Cell lysates were prepared and immunoprecipitated with anti-JNK1 antibody. JNK kinase activity was measured by in vitro kinase assay using GST-c-Jun as the substrate. Auto, autophosphorylation. WB, Western blotting. (E) Co-expression of TLR2 and TLR6 resulted in NF-B reporter gene activation. HEK 293 cells were transiently transfected with an NF-B-regulated luciferase reporter construct, together with the indicated TLR expression vectors. Dual luciferase assay was performed 36 h after transfection.

 
To address whether the combination of TLR2 and TLR6 plays any specific role in activation of intracellular signaling cascades, human embryonic kidney 293 cells were transiently transfected with various combinations of TLR2, TLR4 and TLR6 expression vectors, together with an NF-B-dependent luciferase reporter plasmid. Interestingly, co-expression of TLR2 and TLR6 resulted in synergistic activation of the luciferase reporter gene in the absence of any ligand. In contrast, TLR4 did not show any synergistic effect on basal reporter expression when co-expressed with either TLR2 or TLR6 (Fig. 3E). This data suggests that TLR2 and TLR6 may specifically cooperate to activate signaling cascades. To address whether the cytoplasmic portions of TLR2 and TLR6 are responsible for synergistic NF-B activation, 293 cells were transiently transfected with chimeric constructs consisting of the extracellular domain of Fas fused to the transmembrane and the cytoplasmic domains of TLR2 (Fas/TLR2) or TLR6 (Fas/TLR6) (14,23). Co-expression of these chimeric constructs resulted in synergistic reporter gene activation (Fig. 3E), suggesting that aggregation of the TIR domains of TLR2 and TLR6 is sufficient for the triggering of the signaling cascade.

To further confirm that both TLR6 and TLR2 are required for the response to MALP-2, primary MEF were prepared from wild-type, TLR2^–/–, TLR6^–/– and TLR2^–/– TLR6^–/– embryos. These MEF were first treated with MALP-2 or Pam₃CSK₄ and IL-6 secretion in the culture supernatant was measured. As expected, wild-type MEF produced IL-6 upon stimulation with either lipopeptide, while TLR6^–/– MEF failed to produce IL-6 in response to MALP-2. In addition, TLR2^–/– and TLR2^–/– TLR6^–/– MEF did not respond to either lipopeptide (Fig. 4A).

View larger version (40K): [in this window] [in a new window]   Fig. 4. Requirement of both TLR2 and TLR6 in MALP-2 recognition. (A) IL-6 production from mutant MEF in response to lipopeptides. MEF (3x104) derived from wild-type, TLR6–/– TLR2–/– and TLR2–/– TLR6–/– mice were cultured with 10 ng/ml MALP-2 or 100 ng/ml Pam3CSK4 for 18 h. IL-6 production was determined by ELISA. (B) Constructs used for the retroviral infection. (C and D) TLR2–/– (C) or TLR6–/– (D) MEF were infected with retrovirus encoding the indicated expression constructs. The cells (3x104) were cultured with 10 ng/ml MALP-2 or 100 ng/ml Pam3CSK4 for 18 h and IL-6 concentration in the culture supernatant was measured. (E and F) IL-6 production from TLR2–/– TLR6–/– MEF infected with retrovirus carrying TLR2 and/or TLR6 (E) or chimeric constructs (F) in response to sBLP. The results are shown as the mean ± SEM of triplicate wells.

 
We next performed reconstitution experiments, infecting the mutant MEF with retrovirus vectors expressing human TLR2, TLR6, their mutant constructs or a control construct (Fig. 4B). TLR2^–/– MEF infected with a retrovirus vector encoding human TLR2 recovered the ability to produce IL-6 in response to MALP-2 and Pam₃CSK₄ (Fig. 4C). In contrast, expression of a C-terminal truncated version of TLR2 (TLR2) in these MEF did not restore IL-6 production in response to BLP, indicating that the cytoplasmic domain of TLR2 is critical for the induction of cytokine production. Expression of TLR6 in TLR2^–/– MEF did not confer responsiveness to BLP. In the reciprocal experiment, TLR6^–/– MEF infected with a retrovirus vector encoding human TLR6 produced IL-6 in response to MALP-2, whereas neither expression of TLR2 nor C-terminal truncated TLR6 (TLR6) conferred MALP-2 responsiveness (Fig. 4D). Next, TLR2^–/– TLR6^–/– MEF were infected with each of the retroviruses and lipopeptide-induced IL-6 production was measured. IL-6 production in response to MALP-2 was restored only in the cells co-infected with retroviruses encoding TLR2 and TLR6 (Fig. 4E). In contrast, IL-6 production in response to Pam₃CSK₄ was restored by expression of TLR2 alone. Finally, to analyze the structural basis for cooperation between TLR2 and TLR6 in ligand recognition and signaling, we generated two chimeric constructs consisting of the extracellular domain of TLR2 fused to the transmembrane and cytoplasmic domains of TLR6 (TLR2/TLR6 chimera), and its reciprocal construct (TLR6/TLR2 chimera) (Fig. 4B). Expression of either chimeric protein alone failed to confer responsiveness to MALP-2 or Pam₃CSK₄ in TLR2^–/– TLR6^–/– MEF (Fig. 4F). However, cells expressing both chimeras showed significant augmentation of IL-6 production in response to MALP-2 but not Pam₃CSK₄, suggesting that the combined action of the TLR2 and TLR6 extracellular and the cytoplasmic domains is required for MALP-2 recognition and signaling (Fig. 4F). Moreover, the finding that expression of both chimeras in TLR2^–/– TLR6^–/– MEF did not restore the responsiveness to Pam₃CSK₄ implies that some other TLR, in addition to TLR2, might also be required for Pam₃CSK₄ recognition.

Recent studies on the TLR family have revealed that TLR2 and TLR4 have different roles in the recognition of microbial components. In particular, TLR2 has been shown to be essential for the recognition of a wide variety of microbial components, including PGN, lipoarabinomannan, lipoproteins and zymosan (7–13). However, the molecular mechanisms by which these receptors recognize various bacterial components are poorly understood. We show here that a combination of TLR2 and TLR6 recognizes and specifically distinguishes MALP-2 from other lipoproteins. Previous studies demonstrated that the potent immunostimulatory properties of lipoproteins are entirely dependent on the presence of ester-bound fatty acids at the N-terminal cysteinyl residue (11). The molecular structures of mycoplasmal and BLP differ in that lipoproteins derived from many bacterial species contain one N-terminal and two ester-bound palmitic acid substituents, while lipoproteins from mycoplasmas do not contain this N-acyl group (24,25). Therefore, TLR6 appears to participate in discriminating the subtle differences between dipalmitoyl and tripalmitoyl cysteinyl residues (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5. A model of BLP recognition. See text for details.

 
At present, there is no direct evidence demonstrating high affinity binding of TLR to their respective biological ligands. Nevertheless, recent findings suggest that TLR may directly recognize microbial cell wall components in mammals, unlike the situation in Drosophila, where microbial infection triggers protease cascades that proteolytically process inactive ligand precursors into active forms that in turn interact with the TLR (26,27). A interesting future question is whether TLR6 alone, or TLR2 and TLR6 together, are bound directly by MALP-2.

During preparation of this manuscript, Ozinsky et al. demonstrated a cooperative effect between TLR2 and TLR6 in the recognition of Gram-positive bacteria (28,29). They showed that the overexpression of a dominant-negative TLR2 or TLR6 in RAW-TT10 cells completely inhibited TNF- production in response to PGN and heat-killed S. aureus (28). However, their results are inconsistent with ours. In our case, TLR6^–/– cells responded significantly to PGN. Moreover, we have previously shown that even TLR2^–/– macrophages produce significant levels of TNF- and IL-6 in response to heat-killed S. aureus (30). This discrepancy might be due to differences between the systems.

It is tempting to speculate that other TLR2 ligands, such as PGN and other BLP, are also selectively recognized by heterodimers formed from TLR2 and other TLR. Identification of the ligands recognized by each TLR may contribute to the development of novel therapies based on the host recognition of pathogens.

	Acknowledgments

 
We thank Drs T. Kaisho and M. Lamphier for critical reading of this manuscript, Dr A. Miyajima for providing pMIG vector, Dr T. Kitamura for providing plat-E cells, and Dr R. Medzhitov for providing TLR2 and TLR4 plasmid. We thank N. Tsuji for excellent secretarial assistance and E. Nakatani for excellent technical assistance. This work was supported by Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government, and by the Deutsche Forschungsgemeinschaft (Mu672/2-5).

	Abbreviations

 

BLP bacterial lipopeptide

EMSA electrophoretic mobility shift assay

ES embryonic stem

GFP green fluorescent protein

LPS lipopolysaccharide

MALP-2 macrophage-activating lipopepitde-2 kD

MEF murine embryonic fibroblast

PGN peptidoglycan

TLR Toll-like receptor

TNF tumor necrosis factor

	Notes

 
Transmitting editor: T. Watanabe

Received 16 March 2001, accepted 10 April 2001.

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Top Abstract Introduction Methods Results and discussion References

 

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