Nucleic Acids Exert a Sequence-independent Cooperative Effect on Sequence-dependent Activation of Toll-like Receptor 9

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect. Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect. The mammalian innate immune system initiates conserved responses against an array of microbial challenges by targeting biomolecules that are conserved within microbes but largely absent from the host. These pathogen-associated molecular patterns (PAMPs) 2The abbreviations used are: PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; CMV, cytomegalovirus; ODN, oligodeoxynucleotide; PTO, phosphothioate; PD, phosphodiester; PBMC, peripheral blood mononuclear cell; PNGase F, peptide N-glycosidase F; LBD, ligand-binding domain. are recognized by germ line-encoded, pattern recognition receptors that include the Toll-like receptors (TLRs) (1Hallman M. Raömet M. Ezekowitz R.A. Pediatr. Res. 2001; 50: 315-321Crossref PubMed Scopus (115) Google Scholar). TLRs are highly conserved type I integral membrane proteins that sample PAMPs in both the extracellular and intracellular compartments (2Ahmad-Nejad P. Hacker H. Rutz M. Bauer S. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: 1958-1968Crossref PubMed Scopus (633) Google Scholar). The ligand-binding regions of the TLRs consist primarily of repeating elements of a leucine-rich repeat motif (3Bell J.K. Mullen G.E. Leifer C.A. Mazzoni A. Davies D.R. Segal D.M. Trends Immunol. 2003; 24: 528-533Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). This motif is present in a large number of eukaryotic proteins that often share the unifying characteristic of mediating biomolecular interactions (4Kobe B. Kajava A.V. Curr. Opin. Struct. Biol. 2001; 11: 725-732Crossref PubMed Scopus (1297) Google Scholar). Indeed, the primary function of the leucine-rich repeat motif appears to be to provide an adaptable structural matrix for biomolecular interactions, the versatility of which is exemplified by the vast and structurally diverse ligands that are recognized by different TLRs as follows: microbial DNA (TLR9), lipopolysaccharides (TLR4), and bacterial flagella (TLR5) (5Janssens S. Beyaert R. Clin. Microbiol. Rev. 2003; 16: 637-646Crossref PubMed Scopus (453) Google Scholar, 6Kirk P. Bazan J.F. Immunity. 2005; 23: 347-350Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). There is considerable evidence that microbial DNA serves as the physiological ligand for TLR9. Most convincingly, TLR9 knock-out mice are unresponsive to bacterial DNA but can be made responsive through expression of TLR9 (7Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sajo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5391) Google Scholar). Foreign and host DNA appear to be discriminated on the basis of unmethylated cytosine-phosphate-guanine (CpG) dinucleotide sequences, which are frequent within microbial DNA but largely absent from host genetic material (8Bird A.P. Nature. 1986; 321: 209-213Crossref PubMed Scopus (3032) Google Scholar). The ability for CpG motifs, in the context of bacterial DNA or synthetic oligodeoxynucleotides (ODN) analogs, to activate innate immune responses is well documented (9Krieg A.M. Yi A.K. Matson S. Waldschmidt T.J. Bishop G.A. Teasdale R. Koretzky G.A. Klinman D.M. Nature. 1995; 374: 546-549Crossref PubMed Scopus (3106) Google Scholar, 10Wagner H. Adv. Immunol. 1999; 73: 329-368Crossref PubMed Google Scholar). Although the CpG dinucleotide represents the minimum recognition element, TLR9 undergoes preferential activation, in a manner, by higher order mice are responsive to a whereas human and are activated by Leifer C.A. S. M. Klinman D.M. J. Immunol. 2001; PubMed Scopus Google Scholar, S. Hacker H. S. Akira S. Wagner H. S. A. 2001; PubMed Scopus Google Scholar). The ability for of to mediate responses is to the ligand of from is direct evidence to this The of and of nucleic acids TLR9 an for have the of of the of with A. R. S. 2003; PubMed Scopus Google Scholar). There have human the of the of the innate immune system to mediate against as as and as in the of D.M. Rev. Immunol. PubMed Scopus Google Scholar, M. K. T. Opin. 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TLR9 has considerable for its in the of innate immune responses as well as for the ability to its responses through nucleic of TLR9 are for a of as immune and the of this of with to as is and to the structural that TLR9 ligands to the of To investigations of TLR9 ligand binding have by with the purification of of the receptor for of the of ligand binding have from cell stimulation of this is that the of physiological responses to a to the of within the to conclusions on as ligand is on the of TLR9 activation as follows: the of the receptor in binding the absolute for CpG motifs within nucleic acids to mediate ligand binding and the ability of TLR9 to higher order motifs in a species-specific the of the on receptor and TLR9 ligand binding is influenced by higher order to that have recently for we report for both the purification of the soluble ligand-binding of human TLR9 as well as a high that of nucleic binding by the we are to address of Although has that innate immune responses can be by DNA (9Krieg A.M. 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This to that the of TLR9 the of ligand with this we also observe that the of TLR9 ligand in a system that of the receptor or is highly with the physiological of the TLR9 its the receptor ability to and investigations of immune cell responses the of activation of TLR9 has well The of activation is with the that motifs are in microbial host genetic a structural by which the innate immune system can PAMPs from a structurally which activation of the innate has to sequence-specific binding by in vitro binding have reached contradictory conclusions with respect to the ability of the receptor to with nucleic acids in a sequence-dependent M. J. T. P. Wagner H. Bauer S. Eur. J. Immunol. PubMed Scopus Google Scholar, S. J. A.M. B. J. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. T. Scopus Google Scholar, K. Rutz M. B. J. T. A. Bauer S. Wagner H. Eur. J. Immunol. PubMed Scopus Google Scholar, R. Scopus Google Scholar, M. Rev. Immunol. PubMed Scopus Google Scholar, M. J. 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