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(Journal of Leukocyte Biology. 2002;71:538-544.)
© 2002 by Society for Leukocyte Biology

Toll-like receptor 9 mediates CpG-DNA signaling

Tsung-Hsien Chuang, Jongdae Lee, Lois Kline, John C. Mathison and Richard J. Ulevitch

Department of Immunology, The Scripps Research Institute, La Jolla, California

Correspondence: Tsung-Hsien Chuang, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: chuang{at}scripps.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Among the bacterial products known to activate the innate immune ‘1system is bacterial DNA. This activity resides within the nonmethylated CpG motifs of the DNA and is recapitulated using appropriate synthetic CpG containing oligodeoxynucleotides (CpG-ODN). TLR9-deficient mice were shown to exhibit a nonresponsive phenotype-to-bacterial DNA and CpG-ODN. Here, we describe a model system to further characterize CpG-ODN and TLR9 interactions using ectopically expressed TLR9 in HEK293 cells. Expression of TLR9 confers cellular responsiveness to CpG-ODN but not to the other bacterial products. Previous studies identified species-specific CpG-containing sequences; here, we show that expression of murine TLR9 favors responses to CpG-ODN motifs specific to mouse cells, and expression of human TLR9 favors CpG-ODN known to preferentially activate human cells. Response patterns to various CpG-ODN motifs were parallel when cells containing an ectopically expressed TLR9 and endogenous receptor were compared. Here, we also show that TLR9 acts at the cell surface and engages an intracellular signaling pathway that includes MyD88, IRAK, and TRAF6.

Key Words: innate immunity • NF-{kappa}B • IFN-{gamma} • MyD88


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of innate immunity is closely linked to host defense and secondary adaptive immune responses. Members of the Toll-like receptor (TLR) family are essential components in this process [1 2 3 ]. Ten TLRs (TLR1–10) have been identified in mammalian systems; the current paradigm is that individual TLRs have distinct ligands [3 , 4 ]. TLR4 is a receptor for the lipopolysaccharide (LPS) from Gram-negative bacteria, TLR2 controls cellular responsiveness to a variety of bacterial cell-wall components including lipoteichoic acid, peptidoglycan, and bacterial outer-membrane lipoproteins, and TLR5 mediates bacterial flagellin-induced cell activations [5 6 7 8 9 10 11 12 13 14 ]. Members of the TLR family have some common structural features including an extracellular domain consisting of a signal peptide, multiple leucine-rich repeats, and a cysteine-rich domain, followed by a transmembrane region and a cytoplasmic Toll/interleukin (IL)-1-receptor (TIR) domain. In general, the signaling pathway used by TIR domain-containing proteins includes MyD88, IL-1 receptor-associate kinase (IRAK), and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) [15 16 17 ].

Bacterial DNA is a potent stimulus to immune cells. This activity of DNA is assigned to sequence motifs containing unmethylated CpG dideoxynucleotides. This feature provides a major distinction between bacterial and mammalian host DNA [18 19 20 21 ]. Synthetic CpG containing oligodeoxynucleotide (CpG-ODN) mimics the stimulatory effect of bacterial DNA. The CpG-DNA (both bacterial DNA and CpG-ODN) induces B-cell proliferation and activates cells of the myeloid lineage including dendritic cells [22 23 24 25 26 27 28 29 ]. Injection of CpG-DNA in vivo evokes immune responses in mice without the serious pathophysiological changes that follow injection of LPS, suggesting their potential uses in immunotherapy of human diseases [30 , 31 ]. A molecular understanding of cellular recognition of CpG-DNA is only now beginning to emerge [18 , 20 , 32 ]. Herein, we provide data supporting the concept that the species-dependent and sequence-dependent responses to various CpG-ODN reside within TLR9 and that TLR9 functions as a cell-surface protein and uses intracellular signaling cascades that include MyD88, IRAK, and TRAF6.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
LPS (Re595) was isolated from Salmonella Minnesota R595, and heat-killed Straphylococcus aureus (HKSA) was prepared as described [33 , 34 ]. Lipoteichoic acid (LTA) from Bacillus subtillis (BS) was purchased from Sigma Chemical Co. (St. Louis, MO). hTNF-{alpha} and hIL-1ß were purchased from PeproTech (Rocky Hill, NJ). A palmitylated synthetic peptide analog of bacterial lipoprotein Pam3Cys was purchased from Boehringer Mannheim (Indianapolis, IN). Soluble peptidoglycan (sPGN) was obtained from Dr. R. Dziarski (Indiana University, Bloomington). Bacterial lipoprotein LipL32 was isolated from Leptospira interrogans as described [35 ]. Lipoprotein OspA isolated from Borrelia burgdorferi was obtained from Dr. T. Sellati (University of Connecticut, Storrs). CpG-ODNs, including phosphorothioate-modified mCpG, mCpG.1-mCpG.5, hCpG, non-CpG, and the phosphodiester mCpG.0, were purchased from Research Genetics (Huntsville, AL). The sequences of mCpG (TCC,ATG,ACGx,TTC,CTG,ACGx,TT), hCpG (TCGx,TCGx,TTT,TGT,CGxT,TTT,GTC,GxTT), and non-CpG (TCC,ATG,AGC,TTC,CTG,AGC,TT) were selected from previously published work of Krieg and co-workers [18 ].

Expression plasmids
Expression vectors hTLR2/pFlag.CMV1 and {Delta}MyD88/pRK7 (aa 152–296) were gifts from Tularik (South San Francisco, CA). Expression vectors for truncated {Delta}IRAK1 (aa 1–215), {Delta}TRAF6 (aa 289–522), and {Delta}TRAF2 (aa 87–501) were constructed by subcloning cDNAs encoding the truncated proteins into a pRK5 mammalian expression vector. These cDNAs were polymerase chain reaction (PCR)-amplified from cDNA encoding full-length IRAK1, TRAF6, and TRAF2, respectively. Expression vectors for hTLR9, mTLR9, mTLR9{Delta}1, and mTLR9{Delta}2 were constructed by PCR amplification from the cDNA sequence corresponding to the beginning of the mature peptide to the 3' end, as indicated and subcloned into a pFlagCMV1 vector (Sigma Chemical Co.). This vector contains sequences encoding a preprotrypsin signal peptide followed by a Flag epitope tag. The inserted cDNAs were fused in-frame after the Flag epitope tag. All cDNA constructs were confirmed by DNA sequencing. Plasmids were isolated with a Qiagen (Valencia, CA) Endo-free Maxi-prep kit.

Cloning mTLR9
Several expressed sequence tag (EST) cDNAs (accession numbers AA273731, AA197442, AA162495, and AA451215) encoding partial mTLR9 cDNA sequence were identified in a search of the DNA databases of NCBI with a Blast program. These cDNAs cover the 3'-end sequence of the mTLR9. A rapid amplification of cDNA ends (RACE) method was used to clone cDNA containing the 5' end of this mTLR9 from a cDNA library, which was prepared from mouse-spleen polyA+ mRNA using a SmartTM RACE cDNA amplification kit (Clontech, Palo Alto, CA). The RACE products were subcloned into a T/A cloning vector (Invitrogen, La Jolla, CA) and sequenced. Based on the cDNA sequences from the RACE product and the EST cDNA, primers corresponding to the sequences at both ends of the mTLR9 were designed. The full-length mTLR9 was PCR-amplified from a mouse-spleen first-strand cDNA library. This library was synthesized from polyA+ mRNA using a SuperScriptTM preamplification kit (Gibco BRL, Gaithersburg, MD). The amplified, full-length cDNA was subcloned into a T/A cloning vector and sequenced. The cDNA sequence for mTLR9 is in GenBank under accession number AF314224.

Cell culture, nuclear factor-{kappa}B (NF-{kappa}B) reporter assay and flow cytometry analysis
Human embryonic kidney 293 (HEK293) cells and the murine cell line, RAW264.7, were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum. The HEK293 cells were plated in six-well plates and transfected on the following day by Lipofectamine 2000 (Gibco BRL) with indicated amounts of expression vectors plus 0.1 µg endothelial leukocyte adhesion molecule 1 (ELAM-1) luciferase-reporter plasmid and 0.1 µg ß-galactosidase plasmid for normalization. The RAW264.7 cells were transfected by Superfect (Qiagen) with the indicated amount of expression vectors plus 1 µg ELAM-1 luciferase-reporter plasmid and 1 µg ß-galactosidase plasmid. Twenty-four hours later, the cells were treated with indicated agonists for an indicated time period. The cells were lysed, and luciferase activity was determined by using reagents from Promega Corp. (Madison, WI). Relative luciferase activities were calculated as folds of induction compared with unstimulated vector control. The data presented are the mean ± SE (n=3). To demonstrate that the mTLR9 was expressed on the cell surface, 5 x 105 HEK293 cells expressing TLR9 were dislodged with phosphate-buffered saline (PBS) plus 2 mM diethylenediaminetetraacetate (EDTA) and washed with PBS, maintaining all solutions and cells at 4°C. The cells were stained with anti-Flag fluorescein isothiocyanate (FITC)-conjugated antibody (Sigma Chemical Co.) for 30 min at 4°C and were washed with ice-cold PBS followed by flow cytometric analysis.

Immunoprecipitation and Western blot analysis
HEK293 cells were transfected with indicated cDNAs. Twenty-four hours after transfection, cells were washed once with PBS and lysed in buffer containing of 50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 µg/ml aprotinin. Cell lysates were centrifuged, and nuclei were removed. The lysates were incubated with anti-Flag monoclonal antibody (mAb) M2 (Sigma Chemical Co.) for 4 h at 4°C. Immune complexes were precipitated by the addition of protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). After extensive washing with lysis buffer, precipitated complexes were solubilized by boiling in SDS-PAGE sample buffer fractionated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blotted with anti-Flag mAb. After washes, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG; Jackson Immuno Research Laboratories, West Grove, PA). The reactive bands were visualized with the ECL+Plus Western blotting detection reagents (Amersham Pharmacia Biotech).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TLR9 mediates CpG-DNA-induced NF-{kappa}B activation
We have isolated cDNA encoding human TLR9 (hTLR9) [36 ] and murine TLR9 (mTRL9). hTLR9 and mTLR9 encode proteins of 1032 amino acid residues; there is a 77% identity to each other. In contrast, TLR9 is about 28% identical to TLR2 and TLR4. TLR9 contains all the structural features characteristic of other TLR family members (Fig. 1 ). These are (from amino to carboxyl terminus) signal peptide, multiple leucine-rich repeats, a cysteine-rich domain, a transmembrane region, and a TIR domain. The number and distribution of leucine-rich repeats in the ectodomain of mTLR9 and hTLR9 are not identical. There are 16 and 18 leucine-rich repeats in the ectodomains of mTLR9 and hTLR9, respectively. In mTLR9, the 4th, 7th, and 11th leucine-rich repeats found in hTLR9 are not present. In contrast, the 16th leucine-rich repeat in mTLR9 was absent in hTLR9 (Fig. 1) . The consequence of expression of hTLR9 or mTLR9 on cell activation by CpG-ODN or other potential activators of innate immunity was investigated in HEK293 cells. We transiently cotransfected these cells with a mammalian expression vector for m- or hTLR9 together with a luciferase-reporter gene driven by an NF-{kappa}B-dependent E-selectin (ELAM-1) promoter [37 ]. Three different CpG-ODNs used in these experiments were selected based on findings demonstrated by others and include those shown to be more selective for murine or human cells (here termed mCpG and hCpG, respectively) [18 ]. The mCpG contains GACGTT hexamer motifs, and hCpG contains GTCGTT motifs. The sequence of the control ODN (non-CpG) is identical to the sequence for mCpG except that the CpG dideoxynucleotides are reversed. In transfected HEK293 cells expressing mTRL9, NF-{kappa}B activity was stimulated by mCpG but only marginally induced by hCpG and not induced by the control ODN. Moreover, a panel of other microbial products failed to induce activation (Figs. 2A and 3A). The other stimuli included Re595 LPS, LTA, sPGN, HKSA, lipoprotein LipL32 isolated from L. interrogans, OspA isolated from B. burgdorferi, and a palmitylated synthetic-peptide analog of bacterial lipoprotein Pam3Cys (Fig. 2 A). In contrast, hTLR2-transfected HEK293 cells responded to sPGN, HKSA, LipL32, OspA, and Pam3Cys but not to the CpG-ODNs (Fig. 2B) . HEK293 cells expressing hTLR9 responded to the hCpG, weakly to mCpG, and not to the control ODN (Fig. 2C) .



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  		Figure 1. Architecture analysis of mouse and human TLR9. This analysis was performed by a SMART architecture research computer program (http://smart.EMBL-heidelberg.de/). SP, Signal peptide; LRRs, leucine-rich repeats; TM, transmembrane domain; TIR, Toll/IL-1R cytoplasmic domain. The scale shows amino acid residue numbers. The mTLR9 cDNA sequence was submitted to GenBank under accession number AF314224; hTLR9 is under accession number AF245704.

 


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  		Figure 2. TLR9 mediates CpG-ODN-induced NF-{kappa}B activation. HEK293 cells were transfected with 0.2 µg mTLR9 (A, D), 0.2 µg hTLR2 (B), or 0.2 µg hTLR9 (C) expression vector plus ELAM luciferase-reporter plasmid. Twenty-four hours later, the transfected cells were treated with 10 ng/ml each hIL-1 and hTNF-{alpha}, 5 µM each mCpG, hCpG, and non-CpG, 200 ng/ml LPS(Re595), 2 µg/ml each LTA and PGN, 2 x 106 bacteria/ml HKSA, 1 µg/ml each LipL32 lipoprotein from L. interrogans, OspA lipoprotein from B. burgdorferi, and Pam3Cys (A–C), or 1 µg/ml chloroquine plus 5 µM mCpG (D) for 6 h. The cells were washed and lysed, and relative luciferase activities in each sample were determined as described in Materials and Methods.

 
NF-{kappa}B activation induced by mCpG was blocked by pretreatment of cells with chloroquine (Fig. 2D) . This is consistent with the previously described inhibitory effects of chloroquine on CpG-ODN-induced activation in macrophages, dendritic cells, and B cells [38 ]. Using explanted cells, others have shown that the optimal concentration of CpG-ODN varied from submicromolar to micromolar [18 , 20 ]. Herein, the mCpG concentration required for a maximal NF-{kappa}B activation in HEK293 cells expressing mTLR9 was observed at about 3 µM CpG-ODN (Fig. 3 A). This concentration is similar to that required for the optimal stimulation of RAW264.7 cells using the same luciferase reporter system (Fig. 3B) .



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  		Figure 3. Concentrations of CpG-ODNs required for the NF-{kappa}B activation. (A) HEK293 cells were transfected with 0.2 µg mTLR9 expression vector plus ELAM luciferase-reporter plasmid. (B) RAW 246.7 cells were transfected with ELAM luciferase-reporter plasmid. Twenty-four hours later, the transfected cells were treated with different concentrations of mCpG, hCpG, and non-CpG as indicated. Six hours later, the cells were washed and lysed, and relative luciferase activities in each sample were determined.

 
TLR9 distinguishes different CpG motifs
Here, we confirm the results of others with respect to sequence and species specificity for CpG-ODN ([18 ] and Figure 2A and 2C ). Thus, our data confirm our contention that TLR9 is likely to be involved in the recognition of specific CpG-ODN sequences. We further investigated whether the base sequence in the hexamer-CpG motif required for optimal mTLR9 stimulation also reflects previous findings determined in systems using B lymphocytes and natural killer (NK) cells [39 40 41 ]. The GACGTT has been identified as one of the best motifs for mouse-cell stimulation. Further, it was shown that CpG dideoxynucleotides preceded by a C or followed by a G are inhibitory rather than stimulatory. For positions two bases ahead or after the CpG dideoxynucleotides, a purine at the 5' side and a pyrimidine at 3' generally lead to more stimulatory activity [18 , 39 40 41 ]. To decide if these effects are determined by TLR9, we synthesized several CpG-ODNs and compared their activity with the mCpG using mTRL9-HEK293 cells. These CpG-ODNs were mCpG.1 with a C at one position ahead of the CpG, mCpG.2 with a G at one position after the CpG, mCpG.3 with a pyrimidine at two positions ahead of the CpG, mCpG.4 with a purine at two positions after the CpG, and mCpG.5, which had an AGCGxTT motif instead of the GACGxTT motif but still followed the rule for deriving an optimized CpG motif. The mCpG.1 and mCpG.2 ODNs (5 µM) failed to activate mTLR9-293 cells (Fig. 4 A ) but acted as inhibitors of mCpG-ODN (5 µM)-induced NF-{kappa}B activation (Fig. 4B) . This effect was selective for TLR9 but not the common signaling components downstream of TLRs, because neither blocked IL-1-induced NF-{kappa}B activation in HEK293 cells (Fig. 4B) . In other control experiments not shown, they also failed to inhibit NF-{kappa}B activation induced by HKSA or bacterial lipoprotein. The mCpG.3 and mCpG.4 were activating ligands, although they were less active than mCpG and mCpG.5 (Fig. 4A) . These data further support our contention that the results observed in explanted cells and/or cell lines demonstrated by others reflect a key role for TLR9 in CpG-ODN-induced cell activation [39 40 41 ].



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  		Figure 4. Activation of mTLR9 overexpressed cells by CpG-ODNs containing a different CpG motif and backbone. HEK293 cells were transfected with 0.2 µg mTLR9 expression vector plus ELAM luciferase-reporter plasmid for 24 h. (A) The transfected cells were treated with 5 µM mCpG, mCpG.0, mCpG.1, mCpG.2, mCpG.3, mCpG.4, and mCpG.5. (B) The transfected cells were treated with 10 ng/ml IL-1 (squares), 5 µM phosphorothioate-modified mCpG (circles), and phosphodiester mCpG.0 (triangles) plus different concentrations of inhibitory mCpG.1 (closed symbols) or mCpG.2 (open symbols), as indicated. Six hours later, the cells were washed and lysed, and relative luciferase activities in each sample were determined. The nucleotide sequences of these CpG-ODNs are shown below the figures. The CpG dinucleotide in each CpG-ODN and the nucleotide base varied from the mCpG are underlined. The mCpG.0 contains a native phosphodiester backbone with nucleotide sequence identical to mCpG. All the other CpG-ODNs are phosphorothioate-modified.

 
We also compared the activation of mTLR9 by CpG-ODNs containing phosphodiester or phosphorothioate backbones. Others have shown the phosphorothioated CpG-ODNs to be significantly more active than the corresponding phosphodiester CpG-ODNs because of the increased stability of the phosphorothioated modification and a higher cell-uptake rate presumably as a result of a higher cell-surface binding capability [42 43 44 ]. Here, we synthesized a mCpG.0 containing a native phosphodiester backbone with a nucleotide sequence identical to the mCpG synthesized with a phosphorothioated backbone. We failed to observe marked differences between the two ODNs using the mTLR9-293 cell system (Fig. 4A) . We speculated that the differences were minimal because of the short timeframe of the assay. However, the potency differences between mCpG and mCpG.0 were observed when we used mCpG.1 and mCpG.2 as inhibitors in the TLR9-293 cells. The mCpG-induced NF-{kappa}B activation decreased nearly linearly by increasing concentrations of mCpG.1 and mCpG.2. In contrast, mCpG.0-induced activation was blocked more effectively (Fig. 4B) . This result suggests that the mTLR9 may bind phosphorothioated CpG-ODNs more effectively and may account for the higher cell uptake rate observed by others [42 43 44 ]. In totality, the data provided here show that activity of a CpG motif toward TLR9 is determined by the backbone and specific base sequence.

TLR9 is a cell-surface receptor and uses MyD88, IRAK, and TRAF6 for CpG-DNA signaling
Although the sequence information for TLR9 suggests that it is a membrane receptor (Fig. 1) , direct studies of its cellular localization and role as a functional signaling molecule are lacking. To provide information to establish data to support the contention that TLR9 is indeed a plasma membrane protein and functions in this context as a signaling receptor, we performed the following experiments. We first showed that mTLR9 is a cell-surface molecule by using flow cytometry analysis (Fig. 5 A ). To link this protein to cell activation, we prepared two mutants of mTLR9 containing deletions in the cytoplasmic domain. The two mutants termed mTLR9{Delta}1 and mTLR9{Delta}2 represent truncations from amino acid residues 1001 and 954, respectively (Fig. 5B , upper panel). These constructs were transiently expressed in HEK293 cells, and expression was confirmed by immunoprecipitation and Western blotting with anti-Flag antibody (Fig. 5B , bottom panel). Neither mutant supported CpG-ODN-induced NF-{kappa}B activation when compared with wild-type mTLR9 (Fig. 5B , middle panel). Recent studies have demonstrated the involvement of signaling molecules downstream of the TIR domain in CpG-ODN-induced cell activation [45 , 46 ]. These signaling molecules include MyD88, IRAK, and TRAF6. Cells from MyD88 knockout mice are unable to respond to CpG-ODN, whereas cells from TLR2- and TLR4-deficient mice are unaffected [46 ]. Moreover, CpG-ODN stimulation of JNK activation in RAW264.7 cells is blocked by overexpression of dominant-negative mutants of MyD88 and TRAF6 [45 ]. To investigate whether the mTLR9-mediated CpG-ODN activation uses a similar set of downstream transducers, we cotransfected HEK293 cells with an expression vector for mTLR9, as well as vector encoding truncations in each of these three proteins plus a control protein TRAF2: {Delta}MyD88(152–296), {Delta}IRAK(1–215), {Delta}TRAF6(289–522), and {Delta}TRAF2(87–501). Here, we show that mCpG-induced NF-{kappa}B activation was blocked by overexpression of {Delta}MyD88(152–296), {Delta}IRAK(1–215), and {Delta}TRAF6(289–522) but not by {Delta}TRAF2(87–501) (Fig. 5C) . These data indicate that mTLR9 uses the same set of signaling molecules as used by other TIRs for mediating CpG-DNA signaling.



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  		Figure 5. mTLR9 is a cell-surface signaling receptor and uses MyD88, IRAK, and TRAF6 for CpG-DNA signaling. (A) Flow cytometry analysis of the mTLR9 expression. The mTLR9 on the cell surface was stained with FITC-conjugated anti-Flag antibody and detected by flow cytometry analysis. The shaded histogram represents the parental HEK293 cells; open histogram represents cells expressed with mTLR9. (B, Upper panel) Schematic illustration of truncated mTLR9 constructs. The cDNAs were subcloned into a pFlagCMV1 vector. (B, Middle panel) Cytoplasmic signaling domain of mTLR9 is required for CpG-ODN-induced NF-{kappa}B. HEK293 cells were transfected with 0.2 µg different mTLR9 expression vectors as indicated, plus ELAM luciferase-reporter plasmid. Twenty-four hours later, the transfected cells were stimulated with 5 µM mCpG. Six hours later, the cells were washed and lysed, and relative luciferase activities in each sample were determined. (B, Bottom panel) Expression of the mTLR9 and truncated mTLR9 were confirmed by immunoprecipitation and Western blotting of the proteins with anti-Flag antibody. (C) mTLR9 uses a signaling molecule downstream of the IL-1 receptor to mediate CpG-ODN-induced NF-{kappa}B activation. HEK293 cells were transfected with 0.2 µg mTLR9 expression vector and 0.2 µg {Delta}MyD88 (152–296), {Delta}IRAK1 (1–215), {Delta}TRAF6 (289–522), or {Delta}TRAF2 (87–501), respectively, plus ELAM luciferase-reporter plasmid. Twenty-four hours later, the transfected cells were stimulated with 5 µM mCpG or 20 ng/ml TNF-{alpha} for 6 h. The cells were harvested, and relative luciferase activities in each sample were determined.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we show that ectopic expression of TLR9 in HEK293 cells confers cellular responsiveness to CpG containing ODNs. This model system provided information supporting the contention that the species-specific activity and sequence-dependent activity of CpG-ODNs reside in properties of the TLR9. We also showed that the surface expression of TLR9 results in cell activation as measured by NF-{kappa}B activation through intracellular pathways dependent on MyD88, IRAK, and TRAF6. Thus, expression of TLR9 is sufficient to render HEK293 cells responsive to various CpG-ODNs in a manner that reflects the same patterns observed using primary explants of cells and/or continuous cell lines.

Using the gene-deletion approach, TLR9 has been shown as an essential element of responses to CpG-DNA in mouse [47 ]. However, the mechanism(s) for species-specific and sequence-dependent activities of CpG motifs were not defined, and it was unclear whether TLR9 was sufficient for CpG-DNA signaling. Simultaneously, another study has revealed that mice lacking the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are unable to respond to CpG-DNA [48 ], but the role of DNA-PK in distinction of different CpG motifs was not addressed. Their results suggested that CpG-DNA activated cytoplasmic DNA-PK, which directly phosphorylated I{kappa}B and activated NF-{kappa}B in a MyD88-independent way [48 ]. At present, the connection between TLR9 and DNA-PK is not clear. Our data show an exact parallel between the properties of ectopically expressed TLR9 and those of the endogenous CpG-DNA receptor. This supports the contention that TLR9 is the endogenous receptor as well as the first cellular protein to recognize CpG-DNA. The DNA-PK might be a necessary but not sufficient molecule downstream of TLR9 for CpG-DNA signaling, or alternatively, TLR9 and DNA-PK represent two parallel signal pathways in some cell types.

In addition to TLR9, other TLRs confer species-specific cellular responses. For example, mTLR4 and hTLR4 confer cellular responsiveness to LPS and lipid A. However, only mTLR4 mediates cell activation by a lipid A partial structure, lipid IVa; in fact, lipid IVa is an antagonist in human cells [49 , 50 ]. The structural basis of this specificity is still unclear. In the case of TLR9, a computer analysis has revealed distinct distribution patterns of leucine-rich repeats in the ectodomains of mTLR9 and hTLR9 (Fig. 1) . The CpG-DNA may have direct contact with this region of the receptor. Thus, it will be useful to determine if the sequence differences observed in the ectodomains of m- and hTLR9 account for the differences in ligand specificity in murine versus human TLR9.

TLR9 as well as TLR4 use intracellular signaling components including MyD88, IRAK, and TRAF6. However, the biological consequences initiated by these two receptors are different. For example, LPS induces synthesis of inducible nitric oxide in microphages, but CpG-DNA does not [25 ]. In human peripheral blood monocytes, LPS rapidly induces TNF-{alpha} and IL-6, but CpG-DNA stimulation of these cytokines does not occur for 18 h [51 ]. It is interesting that chloroquine and related compounds block cell activation by CpG-DNA but do not prevent activation by LPS [52 ]. The chloroquine effects suggest that an endosomal maturation process might be required for TLR9 signaling. Alternatively, some specific components in a TLR9 signaling pathway might be inhibited by chloroquine or related compounds. Clarification of the effects of chloroquine on LPS and CpG-DNA-induced cell activations might lead to an understanding of how signals downstream of TLR4 and TLR9 are differentially regulated.


   ACKNOWLEDGEMENTS
 
This work was supported by Grant-in-Aid #9960029 from the American Heart Association Western Affiliate (T-H. C.) and by grants from the National Institutes of Health, NIH AI15136 and GM28485 (R. J. U.). This is publication number 13789-IMM from the Department of Immunology, The Scripps Research Institute.

Received May 21, 2001; revised October 6, 2001; accepted October 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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