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.