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The Journal of Immunology, 2007, 178, 7110 -7119
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Central Leucine-Rich Repeat Region of Chicken TLR16 Dictates Unique Ligand Specificity and Species-Specific Interaction with TLR21

A. Marijke Keestra, Marcel R. de Zoete, Rémon A. M. H. van Aubel and Jos P. M. van Putten2

Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-{kappa}B activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys (Pam3CSK4), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6–16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system is designed to rapidly respond to a broad range of environmental danger signals. These signals are picked up by evolutionary conserved biological sensors that are present at the cell surface or associated with intracellular compartments. One class of innate sensor molecules are the TLRs (1). All TLRs have a similar architecture and consist of an extracellular domain that contains an array of leucine rich repeat (LRR)3 motifs, a single transmembrane (TM) domain, and a cytoplasmic Toll/IL-1 receptor (TIR) domain (reviewed in Refs. 1 and 2). Activation of the receptors results in the recruitment of cytoplasmic adaptor molecules such as MyD88, TIRAP, TRIF, or TRAM to the TIR domain and initiation of a complex signaling cascade that regulates the transcription of inflammatory and immunomodulatory genes (1). The gene products (e.g., cytokines, chemokines, NO, and anti-microbial peptides) act as the immediate first line of defense against invading microbes and direct the activity the adaptive immune system.

To date, >10 different TLRs have been identified in humans and mice that sense distinct molecular patterns including bacterial lipopeptides, LPS, flagellin, and different forms of RNA and DNA (2). However, evidence is accumulating that TLR signaling often does not follow a simple ligand-receptor interaction; several TLRs only function as part of a larger complex that confers a further degree of specificity. For example, LPS only signals via TLR4 complexed to MD2 and is most effective in the presence of lipid scavenger CD14 (3). Similarly, the signaling of distinct lipopeptides via TLR2 requires heterodimerization with TLR1 or TLR6 and is also more efficient in the presence of CD14 and/or CD36 (4, 5, 6, 7, 8, 9). Colocalization of both the TLR2/1 and hTLR2/6 complexes with a number of other cell surface receptors further suggests that they may be part of an even larger TLR signaling complex (10).

One particularly instrumental approach to deciphering the molecular interactions and constraints to the formation of the TLR complex has been the comparison of the function of homologous TLRs in different species. TLRs are highly conserved during evolution, with homologues identified in a variety of species including Drosophila, fish, plants, and mammalian species (11, 12, 13). The power to exploit these differences to dissect TLR function is illustrated by the different responses noted for the human and murine TLR4/MD2 complexes to distinct forms of lipid A (14, 15) and Taxol (16). Other documented species differences in TLR function include the differential responses of human and murine TLR9 and TLR2 complexes to defined CpG-DNA motifs and trilauroylated peptides, respectively (17, 18, 19).

In the present study, we investigated the function of TLRs from the avian species. The chicken genome encodes a number of putative TLRs (chTLR) that share homology with mammalian TLRs, including two TLR2 isoforms (chTLR2 type 1 and type 2), chTLR3, chTLR4, chTLR5, chTLR7, and a disrupted chTLR8 (20, 21, 22, 23, 24, 25, 26). In addition, two TLRs (chTLR15 (27) and chTLR21) that appear specific for the avian species and one TLR (here designated as chTLR16) that formed an evolutionary cluster with mammalian TLR1, TLR6, and TLR10 are present. Functional analysis of the chTLR repertoire has thus far only been performed for chTLR2 type 1 and type 2 (20). When expressed in human HEK293 cells, these receptors, which differ mainly in the composition at their corresponding LRR8–14 regions, conferred a 3-fold (type 1) to 10-fold (type 2) increase in NF-{kappa}B activity to the mammalian TLR2 ligand S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys (MALP-2) as well as to LPS (20).

In this study, we report that chTLR16 bears the ligand specificities of mammalian TLR1 and TLR6 in a single receptor when expressed in conjunction with chTLR2t2 (2). The formation of functional signaling complexes between the human and chicken TLR2 and its coreceptor(s) was species specific. Chimeras of human TLRs (hTLRs) and chTLRs located the incompatibilities in the formation of functional TLR complexes and indicated the ligand-specific domains critical for the observed dual ligand specificity of the chTLR16/chTLR2t2 complex compared with the hTLR2 complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

The HeLa 57A cell line, stably transfected with a NF-{kappa}B luciferase reporter construct (28), was generously provided by Dr. R. T. Hay (Institute of Biomolecular Sciences, University of St. Andrews, St. Andrews, Scotland, U.K.). Cells were routinely propagated in 25-cm2 tissue culture flasks (Corning) in DMEM with 10% FCS at 37°C in 5% CO2 atmosphere. NSCU chicken macrophages (29) were kindly provided by Dr. J. Kramer (Animal Sciences Group, Lelystad, The Netherlands) and maintained in RPMI 1640 with 5% FCS. HEK293 cells (provided by Dr. B. van der Burg, Hubrecht Laboratory, Utrecht, The Netherlands) were maintained in DMEM with 10% FCS. The synthetic diacylated lipopeptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the synthetic tri-acylated lipoprotein tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys (Pam3CSK4) were purchased from InvivoGen.

Construction of expression plasmids

ChTLR2t1 (GenBank accession no. AB050005), chTLR2t2 (accession no. AB046533) (both lacking their own signal peptide), and full-length chTLR16 (accession no. EF413646) were PCR amplified from the chromosomal DNA of NCSU chicken macrophages using Pfu polymerase (Promega) and the gene-specific primers listed in Table I. PCR products were purified, digested with the appropriate restriction enzymes (underlined in Table I), and ligated into the pFLAG-CMV1 mammalian expression vector (Sigma-Aldrich) to yield pFLAG-chTLR2t1, pFLAG-chTLR2t2, and pFLAG-chTLR16, respectively. Full-length human TLR1 (accession no. U88540), hTLR2 (GenBank accession no. BC033756), hTLR6 (GenBank accession no. AB020807), and hCD14 (GenBank accession no. NM_000591) were PCR amplified from the chromosomal DNA of HEK293 cells and cloned into pTracer-CMV2 (Invitrogen Life Technologies), yielding pTracer-hTLR1, pTracer-hTLR2, pTracer-hTLR6, and pTracer-hCD14. The chimeric TLR receptor genes chTLR16-ecto/hTLR1-TIR, chTLR16-ecto/hTLR6-TIR, hTLR1-ecto/chTLR16-TIR, and hTLR6-ecto/chTLR16-TIR (designated as chimera 1–4, respectively; ecto denotes ectodomain) were engineered by overlap extension PCR (30). cDNA encoding the respective extracellular domains was PCR amplified and fused at the highly conserved last cysteine of the juxtamembrane cysteine-rich (LRR-CT) domain (asterisk in Fig. 1) to the desired TM and cytoplasmic domain using primers with species-specific overhangs (Table I). Chimera A, in which the LRR9–12 of hTLR6 was replaced with the corresponding region of chTLR16, and chimera B, in which the LRR6–16 of hTLR6 was replaced with LRR6–16 of chTLR16, were constructed by overlap extension PCR with the primers listed in Table I. In these experiments, the (three) DNA fragments needed for the construction of one chimera were amplified by PCR, gel purified, mixed, and fused in one PCR. All chimeras were cloned into the pFLAG vector. Nucleotide sequences of all constructs were verified by DNA sequencing with an ABI Prism 310 genetic analyzer (Applied Biosystems).


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  		Table I. Primers used in this study to construct recombinant TLR proteinsa

 

Figure 1
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  		FIGURE 1. Sequence characteristics of chTLR16. The predicted protein sequence of chTLR16 was aligned with the hTLR1, hTLR6, and hTLR10 proteins using the ClustalW program. Conserved amino acids and synonymous substitutions are shadowed in black and gray, respectively. The cytoplasmic (TIR) domain, the TM region, the extracellular domain with the conserved cysteine-rich CT region (LRR-CT), and LRR1–19 are indicated below the sequence. SigP is the signal peptide. The asterisk at position 588 marks the conserved cysteine residue at which position the human and chicken TLR domains were fused to construct chimeric receptors. Note that LRR10 of chTLR16 contains an extended stretch of 12 aa that is absent in the human receptors.

 
Transient transfection

HeLa 57A cells were grown in 24-well tissue culture plates in DMEM with 10% FCS until 70% confluence was reached (~24 h). Then, cells were transiently transfected in DMEM without FCS using FuGENE 6 (Roche Diagnostics) at a lipid to DNA ratio of 3 to 1. Plasmids carrying the desired inserts were added at concentrations of 125 ng/plasmid. Variable amounts of empty vector were included to equalize the total amount of transfected plasmid DNA (500 ng) added to the cells. In all transfections, the pTK-LacZ vector was used for normalization of transfection efficiency. After 4 h of incubation (37°C) of the cells in the presence of the added DNA, the medium was replaced with fresh medium containing DMEM with 10% FCS. Functional assays were performed 24 h posttransfection.

Confocal laser microscopy

HeLa 57A cells were grown on glass coverslips in 24-well tissue culture plates in DMEM with 10% FCS until 70% confluence (~24 h) and transiently transfected with 500 ng of pFLAG-chTLR2t2, pFLAG-chimera A, or pFLAG-chimera B. After 24 h, the cells were washed with PBS, fixed (30 min) with 2% paraformaldehyde, and permeabilized (15 min) with 0.2% Triton-X-100. After blocking with 2% BSA in PBS (1 h), the cells were incubated (1 h) with an anti-Flag M2 Ab (1/2000 dilution; Sigma-Aldrich) and subsequently with Alexa Fluor 488 goat anti-mouse IgG (1/500 dilution, Invitrogen Life Technologies). Stained cells were embedded in FluorSave (Calbiochem) and viewed in a Leica TCS SP confocal laser-scanning microscope.

Luciferase assays

TLR signaling was assessed using the NF-{kappa}B-luciferase reporter system. Cells were stimulated with the appropriate ligands for 5 h, washed three times with 0.5 ml of PBS, and immediately lysed in 0.2 ml of reporter lysis buffer (Promega). Firefly luciferase activity was measured with a luciferase assay system (Promega) using a luminometer (TD-20/20, Turner Designs). For normalization of the efficiency of transfection, luciferase values were adjusted to beta-galactosidase values as determined with the beta-galactosidase assay (Promega). Results were expressed in relative light units (RLU) and represent the mean ± SEM values of at least three independent experiments. Statistical significance was confirmed by a paired ratio t test. A two-tailed p < 0.05 was taken to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cloning and sequence analysis of chTLR16

An inspection of the chicken genome sequence revealed one putative TLR with similarity to the mammalian TLR1, TLR6, and TLR10 group (21, 22, 23). To assess the function of this putative TLR (designated here as chTLR16), we cloned the chTLR16 gene as well as chTLR2t1 and chTLR2t2 genes from the chicken NCSU macrophage cell line. The obtained sequences of chTLR2t1 and chTLR2t2 were identical to those published in the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov). The sequence of chTLR16 was identical to the released chTLR1/6/10 homologue (GenBank accession no. AB109401) except for its C-terminal end, resulting in mature chTLR16 of 804 aa.

Comparative analysis of chTLR16 and the known human TLR receptors at the protein level revealed that the putative chTLR16 protein closely resembled hTLR1, hTLR6, and hTLR10 with amino acid similarity values of ~83% for the predicted cytoplasmic (TIR) and TM domain of chTLR16 and ~60% for the extracellular domain (Table II). Structural analysis showed that chTLR16 carried a proline residue at position 688, previously implicated in hTLR1 and hTLR6 function (31, 32), and a glycine residue at position 689. This amino acid has been suggested to be critical for TLR signaling and the interaction of hTLR1 with hTLR2 (33). The ectodomain of chTLR16 contained the juxtamembrane cysteine-rich (LRR-CT) region implicated in the regulation of TLR signaling (34) along with 19 consecutive LRR/LRR-like motifs as found for hTLR1, hTLR6, and hTLR10 (Fig. 1). The most notable difference between the chicken and human receptors was located in LRR10 of chTLR16. This region contained an extra 12-amino acid stretch that was absent in hTLR1, hTLR6, and hTLR10 (Fig. 1). A prediction of putative N-glycosylation sites using the NetNGlyc 1.0 (www.cbs.dtu.dk/services/NetNGlyc) and ScanProsite (expasy.org/tools/scanprosite) algorithms revealed five asparagine residues in the TLR16 ectodomain (Asn33, Asn78, Asn204, Asn366, and Asn589), which is less than that predicted for hTLR1 (six sites), hTLR6 (nine sites), and hTLR10 (nine sites). Thus, the sites and degree of glycosylation may be different between the chicken and human receptors.


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  		Table II. Amino acid sequence similarity (%) of the TIR domain, extracellular region (ectodomain), and full putative chTLR16 protein with the corresponding regions of the various human TLR1–10 receptorsa

 
Functional activity of coexpressed chTLR16 and chTLR2

For mammalian species the function of TLR10 is still unknown. However, TLR1 and TLR6, in conjunction with TLR2, respond to synthetic triacylated or diacylated lipopeptides Pam3CSK4 and FSL-1, respectively (4, 5, 6). Therefore, we focused on these ligands to investigate the function of chTLR16. Functional TLR assays were performed with HeLa 57A cells. These cells are unresponsive to Pam3CSK4 and FSL-1 as determined with the NF-{kappa}B reporter assay. Transfection of the cells with chTLR16, chTLR2t1, or chTLR2t2 followed by stimulation with Pam3CSK4 or FSL-1 did not stimulate NF-{kappa}B-luciferase activity (Fig. 2, A and B). To assess whether chTLR16 and chTLR2 may form a functional unit reminiscent of the mammalian TLR1/TLR2 and TLR6/TLR2 heterodimers, we next tested HeLa 57A cells expressing chTLR16 in combination with either chTLR2t1 or chTLR2t2. NF-{kappa}B reporter assays after FSL-1 or Pam3CSK4 stimulation of these cells showed that the combination of chTLR16 and chTLR2t1 did not yield a functional complex (Fig. 2, A and B). However, cells expressing chTLR16 as well as chTLR2t2 strongly responded to both the hTLR1/hTLR2- and hTLR6/hTLR2-specific ligands, Pam3CSK4 (Fig. 2A) and FSL-1 (Fig. 2B).


Figure 2
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  		FIGURE 2. NF-{kappa}B-activation in TLR-transfected HeLa 57A cells. HeLa 57A cells transfected with chTLR2t1, chTLR2t2, chTLR16, or combinations of these chicken receptors (A and B) or hTLR1, hTLR6, hTLR2, or combinations of these human receptors (C and D) were either not stimulated (open bars) or stimulated (5 h) with Pam3CSK4 (100 ng/ml) (A and C) or FSL-1 (100 ng/ml) (B and D), after which NF-{kappa}B luciferase activity was measured. Values are in RLU and are the mean ± SEM of at least three experiments. *, p < 0.05.

 
To ensure that the apparent dual ligand specificity of the chicken receptor transfected cells was mediated by the chTLR16/chTLR2t2 combination, the experiments were repeated with cells transfected with hTLR2 in combination with either hTLR1 or hTLR6. Pam3CSK4 stimulation of these cells yielded a potent response for the hTLR1/hTLR2 expressing cells, but not for the hTLR6/hTLR2 receptor combination (Fig. 2C), consistent with the reported ligand specificity of the human hTLR1/TLR2 receptor complex (5, 6). Similarly, FSL-1 caused a strong NF-{kappa}B activation in hTLR6/hTLR2-expressing cells, whereas no increase in response was observed for hTLR1/hTLR2-transfected cells compared with cells transfected with hTLR2 alone (Fig. 2D). The latter cells moderately responded to FSL-1, although RT-PCR analysis did not reveal endogenous hTLR6 mRNA expression (data not shown). Overall, our results indicate that chTLR16 and chTLR2t2 can form a functional unit and that this complex covers the ligand specificities of both hTLR1 and hTLR6.

Effect of CD14 on the function of the chTLR16/chTLR2t2 receptor complex

Membrane and soluble forms of the myeloid receptor CD14 play a key role in the delivery of lipid A toward the mammalian TLR4/MD2 complex (3). CD14 also facilitates the capturing of lipopeptides and their association with the hTLR2 complex, enhancing the cellular NF-{kappa}B activation particularly at low ligand concentrations (7, 8). To further characterize the species specificity of this effect, we assessed the effect of CD14 on the chTLR16/chTLR2t2-mediated NF-{kappa}B activation. In this study, we used human CD14 as the chicken CD14 receptor has not yet been identified. Immunological analysis using anti-human CD14 Abs suggests that CD14 is present on chicken cells (35, 36, 37, 38). The expression of human CD14 into HeLa 57A cells did not yield responsiveness toward Pam3CSK4 or FSL-1 (data not shown). In contrast, the expression of CD14 in conjunction with chTLR16 and chTLR2t2 rendered the cells highly sensitive to both Pam3CSK4 and FSL-1, with up to 100-fold increases in NF-{kappa}B activation at submaximal concentrations of ligand (Fig. 3, A and B). Control experiments using hTLR1/hTLR2 and hTLR6/hTLR2 transfectants showed that the effects of CD14 were comparable with those obtained with the chicken TLR receptors (Fig. 3, C and D). These data collectively indicate that human CD14 acts in concert with chicken TLRs and enhances the sensitivity of the chicken TLR response toward lipoproteins. Of note, the presence of CD14 did not result in a noticeable Pam3CSK4 or FSL-1 response in chTLR16/chTLR2 type 1 transfected cells (data not shown), indicating that the NF-{kappa}B activation required chTLR2 type 2.


Figure 3
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  		FIGURE 3. Effect of hCD14 on the function of the chTLR2t2/chTLR16 complex as measured by NF-{kappa}B-luciferase activity. CD14-negative ({circ}) and CD14-positive (•) HeLa 57A cells expressing chTLR2t2/chTLR16 (A and B) or their human homologues hTLR2/hTLR1 or hTLR2/hTLR6 (C and D) were stimulated (5 h) with the indicated concentrations of Pam3CSK4 or FSL-1. Values are in RLU and are the mean ± SEM of at least three experiments. *, p < 0.05.

 
Species-specificity of TLR2 complex formation

The unique dual ligand specificity of the chTLR16/chTLR2t2 receptor combination compared with the human TLR1/TLR2 and TLR6/TLR2 signaling complexes led us to examine the species specificity of the interaction between chicken and human TLR2 with the various TLR2 coreceptors. For this purpose, HeLa 57A cells were first transfected with hTLR2 and hCD14 in combination with chTLR16. Exposure of these cells to either Pam3CSK4 (Fig. 4C) or FSL-1 (Fig. 4D) did not result in NF-{kappa}B activation, indicating that chTLR16 cannot functionally interact with hTLR2. Similarly, cells that expressed chTLR2t2 and hCD14 (or chTLR2t1, data not shown) in combination with either hTLR1 or hTLR6 did not form a functional complex when challenged with Pam3CSK4 or FSL-1 (Fig. 4, A and B). In all cases, the combined expression of the receptors from the homologous species caused strong NF-{kappa}B activation (Fig. 4). These data strongly suggest that the functional interplay between the various chicken and human receptors is highly species-specific despite their similar architecture at the amino acid level and their ability to activate NF-{kappa}B in response to Pam3CSK4 and FSL-1.


Figure 4
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  		FIGURE 4. Species specificity of the functional TLR2 complex as measured by NF-{kappa}B-luciferase activity. HeLa 57A cells carrying empty vector (Ctrl), chTLR16, hTLR1, or hTLR6 in combination with hCD14 and either chTLR2t2 (A and B) or hTLR2 (C and D) were either not stimulated ({square}) or stimulated (5 h) with Pam3CSK4 (100 ng/ml) (A and C) or FSL-1 (100 ng/ml) (B and D). Data are in RLU and are the mean ± SEM of at least three experiments. *, p < 0.05.

 
Role of the TM and cytoplasmic TLR domains in species-specific TLR2 interactions

The formation of a functional hTLR1/hTLR2 complex requires interactions with both the TIR domain and the extracellular domains (39). To identify which TLR domain(s) of the human and chicken TLR2 complex are incompatible and thus cause the observed species specificity, we engineered a set of chimeric receptors. First, the ectodomain of chicken TLR16 (aa 1–588) was fused to the TM/TIR region of human TLR1 (aa 578–786) and TLR6 (aa 583–796) (Fig. 1), yielding chimera 1 and 2, respectively. Expression of each of these chimeric receptors in conjunction with human TLR2 and CD14 followed by a challenge with Pam3CSK4 and FSL-1 demonstrated that the fusion of the hTLR1 or hTLR6 TM/TIR region to the chTLR16 ectodomain did not restore the functional interaction with hTLR2 (Fig. 5, D and E). The reciprocal experiment in which cells carried chimeric receptors with the ectodomains of human TLR1 and TLR6 fused to the TM/TIR region of chTLR16 (chimera 3 and 4) in combination with chTLR2t2 and CD14 did not cause NF-{kappa}B activation in response to the TLR ligands either (Fig. 5, A and B). Thus, the exchange of the TM/TIR region of the chicken or human coreceptors was not sufficient to overcome the species-specific formation of a functional signaling complex with TLR2.


Figure 5
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  		FIGURE 5. Role of TIR domains in the species-specific formation of a functional TLR2 complex as measured by NF-{kappa}B-luciferase activity. HeLa 57A cells carrying hCD14 and either chTLR2t2 (A and B) or hTLR2 (D and E) in combination with empty vector (Ctrl), hTLR1, hTLR6, chTLR16, chimera (chim)1, chimera 2, chimera 3, or chimera 4 were either not stimulated ({square}) or stimulated (5 h) with Pam3CSK4 (100 ng/ml) (A and D) or FSL-1 (100 ng/ml) (B and E). Values are in RLU and are the mean ± SEM of at least three experiments. *, p < 0.05. The composition of the used TLR receptor combinations and their functionality are depicted in panels C and F with human TLR domains in black and chicken TLR domains in gray. The numbers (16, 1, and 6) indicate the origin of the respective regions (chTLR16, hTLR1, or hTLR6, respectively).

 
Role of the TLR ectodomain in species-specific TLR2 interactions

To decipher the role of the TLR ectodomain in the species-specific TLR2 signaling, the chimeric receptors with the ectodomains of hTLR1 or hTLR6 fused to the chicken TLR16 TM/TIR region (chimeras 3 and 4) were expressed in HeLa 57A cells together with hTLR2 and CD14. Challenge of these cells with Pam3CSK4 or FSL-1 clearly induced NF-{kappa}B activation (Fig. 6, D and E). Similarly, cells expressing the chicken TLR16 ectodomain fused to the hTLR1 or hTLR6 TM/TIR fragments (chimeras 1 and 2) in combination with chTLR2t2 and CD14 gained responsiveness toward the TLR2 ligands (Fig. 6, A and B). Overall, it was noted that chimeras that contained a chicken TM/TIR region yielded more powerful responses than chimeras carrying human TM/TIR domains (Fig. 6, chimeras 3 and 4 vs chimeras 1 and 2) despite the fact that the receptors were expressed in a human background with human TLR adaptor molecules. Together, our results show that the chimeric receptors were functional and strongly suggest that the ectodomains rather than the TM/TIR domains dictate the species-specific functional interaction between TLR2 and its coreceptors.


Figure 6
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  		FIGURE 6. Importance of the TLR ectodomain in the species-specific functional TLR interactions as measured by NF-{kappa}B-luciferase activity. HeLa 57A cells expressing hCD14 and either chTLR2t2 (A and B) or hTLR2 (D and E) in combination with empty vector (Ctrl), hTLR1, hTLR6, chTLR16, chimera (chim) 1, chimera 2, chimera 3, or chimera 4 were either not stimulated ({square}) or stimulated (5 h) with the TLR2 ligand Pam3CSK4 (100 ng/ml) (A and D) or FSL-1 (100 and 1000 ng/ml) (B and E). Values represent RLU and are the mean ± SEM of at least three experiments. *, p < 0.05. The composition of the used TLR receptor combinations and their functionality are depicted in C and F with human TLR domains in black and chicken TLR domains in gray. The numbers (16, 1, and 6) indicate the origin of the respective regions (chTLR16, hTLR1, or hTLR6, respectively).

 
Transfer of the chTLR16 ligand and species specificity toward hTLR6

The successful engineering of functional complexes consisting of TLR2 and the various chimeric TLR2 coreceptors also enabled us to further dissect the molecular basis of the difference in ligand specificity of the human and chicken TLR2 signaling complexes. The finding that the chTLR2t2/chimera 1 and 2 receptor combinations responded toward both Pam3CSK4 and FSL-1 (Fig. 6, A and B) indicated that the dual ligand specificity of the chicken TLR2 complex was located in the ectodomain of chTLR16. To further define the region involved in the (differential) recognition of Pam3CSK4 and FSL-1, we engineered a novel pair of chimeric receptors in which distinct LRR regions of hTLR6 were replaced by the corresponding LRR region of chTLR16. The functionality of these receptors was then assessed by the gain of reactivity toward Pam3CSK4 in chTLR2t2- and CD14-expressing cells. Because the LRR10 of chTLR16 contains an extra 12-aa stretch compared with human TLR1, TLR6, and TLR10 (Fig. 1), we first replaced LRR9–12 (chimera A). As shown in Fig. 7A, transfer of this region was not sufficient to introduce Pam3CSK4 ligand specificity into hTLR6. However, replacement of LRR6–16 (chimera B) did render the cells responsive to Pam3CSK4 (Fig. 7A). The cells carrying chimera B were also still responsive to FSL-1 (Fig. 7B). Because chimera A and B are expressed at comparable levels in HeLa 57A cells as evidenced by confocal laser microscopy (data not shown), these data indicate that the region LRR6–16 is critical for the dual ligand specificity. This result also implies that transfer of chLRR6–16 into hTLR6 is sufficient to establish species-specific interaction of the human receptor with chTLR2t2. Cells that expressed either chimeras A or B in combination with hTLR2 and CD14 did not respond to either Pam3CSK4 or FSL-1 (Fig. 7, D and E), indicating that the replacement of LRR9–12 in hTLR6 with the corresponding regions of chTLR16 already caused a loss of the ability to form a functional hTLR6/hTLR2 complex.


Figure 7
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  		FIGURE 7. Identification of the LRR region critical for the dual specificity of chTLR16 ligand recognition and the species-specific formation of a functional TLR2 complex. HeLa 57A cells expressing hCD14 and either chTLR2t2 (A and B) or hTLR2 (D and E) in combination with empty vector (Ctrl), hTLR1, hTLR6, chTLR16, chimera (chim) A, or chimera B were either not stimulated (open bars) or stimulated (5 h) with the TLR2 ligands Pam3CSK4 (100 ng/ml) (A and D) or FSL-1 (100 ng/ml) (B and E). NF-{kappa}B activation was determined with the NF-{kappa}B luciferase reporter assay. Values are given in RLU and are the mean ± SEM of at least three experiments. *, p < 0.05. The composition of the used TLR receptor combinations and their functionality are depicted in C and F with human TLR domains in black and chicken TLR domains in gray. The numbers (16, 1, and 6) indicate the origin of the respective regions (chTLR16, hTLR1, or hTLR6, respectively).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Toll-like receptors are evolutionary highly conserved innate immune receptors. Yet, fine functional differences between TLR homologues of different species exist and have been instrumental in deciphering TLR biology. We applied this powerful comparative biology approach and discovered that a putative chicken TLR with unknown function (here designated as chTLR16) acts as a coreceptor of chicken TLR2 type 2. Coexpression of chTLR16 with chTLR2t2 resulted in lipopeptide-mediated NF-{kappa}B activation. In mammalian species TLR2 forms functional complexes with human and murine TLR1 and TLR6 that respond to the triacylated peptide Pam3CSK4 and the di-acylated peptide FSL-1, respectively (5, 6). The chicken TLR2t2/TLR16 complex is unique in that it responds to both Pam3CSK4 and FSL-1 (Fig. 2). This dual ligand specificity strongly suggests that, in the chicken species, the ligand specificities of hTLR1 and hTLR6 are contained within a single molecule.

The formation of a functional chTLR16/TLR2 complex was observed only for chTLR2t2 and not for chTLR2t1. Furthermore, we did not observe any significant NF-{kappa}B activation after the transfection of chTLR16 or chTLR2 alone (Fig. 2). These findings seem at variance with observations that both chTLR2 receptors moderately respond to the lipopeptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Ly (MALP-2) in the absence of a TLR coreceptor (20). The difference in results may be explained by the use of different cell systems (HeLa 57A vs HEK293 cells), as we also obtained weak (3- to 6-fold) responses at high concentrations (1 µg) of lipopeptide for both chTLR2t1- and chTLR2t2-transfected HEK293 cells (data not shown). The biological significance of these relatively weak responses seems minor when compared with the 60-fold-higher and species-specific activation of NF-{kappa}B at much lower concentrations of lipopeptide observed for HeLa cells expressing chTLR16/TLR2t2. The finding that chTLR2t2 but not chTLR2t1 formed a functional complex with chTLR16 suggests that there are strict structural constraints to the interaction between chTLR2 and its coreceptor. The two types of chTLR2 differ mainly in a ~200-aa stretch comprising LRR8–14 (20). This suggests that this region may be critically involved in the formation of a functional chTLR16/TLR2t2 complex.

Our data unequivocally demonstrate that CD14 dramatically (50- to 100-fold) enhances the sensitivity of the chicken TLR16/TLR2 type 2 complex. For the mammalian species, CD14 has been demonstrated to bind and deliver glycolipids (LPS) and lipopeptides toward the TLR4/MD2 complex (40) and TLR2/coreceptor complex (7, 8, 41, 42). In leukocytes, membrane-bound CD14 is predominantly found in microdomains (lipid rafts). The binding of lipopeptides to CD14 induces the recruitment of existing TLR1/TLR2 heterodimers to lipid rafts, resulting in the formation of a functional TLR2 signaling complex (7, 8, 10, 40). The exact mechanism as to how CD14 interacts with the TLR2 complex remains to be defined. Of note here is that the enhanced sensitivity of the chTLR16/chTLR2t2 complex was obtained with human CD14. The complete gene encoding chicken CD14 has thus far not been identified in the chicken genome, but several immunological studies suggests that the protein likely exists in this species (35, 36, 37, 38). The increased sensitivity of the chicken TLR16/TLR2t2 complex in the presence of human CD14 implies that the function of CD14 is not species specific.

A major challenge in TLR biology is to define the biological constraints that are imposed on the formation a functional TLR2 complex. Our results for the first time indicate that the interaction between TLR2 and its coreceptors exhibits species specificity. The molecular basis as to why chTLR16 did not form a functional complex with hTLR2 and, vice versa, why chTLR2 did not signal in conjunction with hTLR1 or hTLR6 was elucidated using constructed chimeric TLR coreceptors. The use of recombinant TLRs composed of receptor ectodomains of one species fused to TM/cytoplasmic receptor domains of the other species clearly showed that the extracellular TLR regions were essential for the species-specific functional interaction with TLR2. Additional chimeras in which the distinct LRR regions of hTLR6 had been substituted by the corresponding regions of chTLR16 revealed that the introduction of LRR9–12 of chTLR16 was insufficient to confer functional interaction with chTLR2t2 but that the species specificity was cotransferred with LRR6–16. These results strongly suggest that the central LRR region of the TLR coreceptor is critical for functional interaction with TLR2. Functional TLR2 signaling requires physical association of the TLR ectodomains (32), while interaction of the cytoplasmic tails is required for signaling (5, 32). Furthermore, LRR regions are implicated in protein-protein interactions (43, 44, 45), and assays with cells carrying TLR2 deletion constructs have indicated that the corresponding LRR region of TLR2 is likely involved in TLR2 complex formation (46). Our finding that this region confers species specificity to the TLR interaction underpins these observations and is in line with the hypothesis that the central LRR region of the chTLR16 physically interacts with the corresponding region of TLR2.

Interestingly, substitution of the ectodomain of human TLR6 with the corresponding region chTLR16 not only transferred the species specificity of the TLR interaction but also the dual ligand specificity. Although the exchange of LRR9–12 of hTLR6 with the corresponding region of chTLR16 was already sufficient to cause a loss of function in combination with hTLR2, the introduction of chLRR6–16 into hTLR6 resulted in a gain of reactivity to both Pam3CSK4 and FSL-1 when the receptor was coexpressed with chTLR2t2 (Fig. 7). These data strongly suggest that LRR6–16 of chTLR16 in combination with chTLR2t2 is required for the broad ligand specificity of the chicken TLR2 complex. Recent studies in which distinct domains of human TLR1 and TLR6 have been exchanged to better understand the ligand specificity of these receptors indicate that the substitution of LRR6–17 was needed to fully exchange the hTLR1 and hTLR6 ligand specificities (47). These data support the hypothesis that the central LRR region determines the ligand specificity of the TLR2 complex. At this time, it is difficult to discern whether the ligand specificity of the TLR2 signaling complex is solely determined by the coreceptor or by the combination of both receptors. Recently, human but not murine TLR2 was found to discriminate tripalmitoylated and trilauroylated peptides (17, 46). This differential recognition, which was attributed to a variation in a low-conserved region spanning LRR7 to LRR10 (17) suggests that also the composition of the TLR2 ectodomain can influence ligand specificity.

For the mammalian system, TLR2 signaling not only requires association of the ectodomains but also heterodimerization of the intracellular domains of TLR2 and TLR1 or TLR6 (39). Our experiments revealed that swapping of the various TM and cytoplasmic domains between hTLR1 or 6 and chTLR16 did not influence the functionality of the human or chicken TLR2 complex and, thus, that the assumed interaction between the intracellular TLR domains is not species specific. Furthermore, the observation that strong cellular activation was achieved with the chTLR16/chTLR2t2 complex expressed in a human cell background strongly suggests also that the association of the MyD88 adaptor molecule with the TIR domain of the chicken TLR2 complex is not species specific. The apparent limitation of the species-specific interactions within the TLR2 complex to the extracellular domain may be interesting from an evolutionary point of view. It can be imagined that TLR2 and its coreceptors may have coevolved as a complex but that, during evolution, there was a need for the mammalian species to diversify the TLR2 complex. It has been suggested that this may have resulted in duplication of the ancestral TLR coreceptor in mammals around the time of divergence from birds (~300 million years ago) and the evolvement of two receptors (TLR1 and TLR6), each with limited ligand specificity compared with chTLR16 (48). The structural changes needed to accomplish this altered ligand specificity may have led to the incompatibility of the chicken and human receptors that form the TLR2 complex discovered in the present study. The fact that this incompatibility seems limited to the extracellular receptor domains suggests that a need for the diversification of ligand recognition may have been a driving force in the evolution of the TLR2 complex. Whether the species specificity of the TLR2 complex is limited to the TLRs or also involves additional molecules that seem to participate in the function of the TLR2 signaling complex (10) awaits further investigation.


   Acknowledgments
 
Dr. R. T. Hay (University of St. Andrews, St. Andrews, Scotland) is gratefully acknowledged for providing the HeLa 57A cell line, Dr. J. Kramer (Animal Study Group, Lelystad, The Netherlands) for providing the NCSU cell line, and Dr. B. van der Burg (Hubrecht Laboratory, Utrecht, The Netherlands) for providing HEK293 cells.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work received financial support from the "Adaptation and Resistance Initiative" and ZonMw Grant 912-03-007. Back

2 Address correspondence and reprint requests to Dr. Jos P. M. van Putten, Department of Infectious Diseases and Immunology Utrecht University, Yalelaan 1, Utrecht, The Netherlands. E-mail address: j.vanputten{at}vet.uu.nl Back

3 Abbreviations used in this paper: LRR, leucine-rich repeat; chTLR, chicken TLR; ChTLR2t1/2, ChTLR2 isoform type 1 or type 2; FSL-1, S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe; hTLR, human TLR; RLU, relative light unit; Pam3CSK4, tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys; TIR, Toll/IL-1 receptor; TM, transmembrane. Back

Received for publication September 18, 2006. Accepted for publication March 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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