(557a) Activating Innate Immunity Through Toll-Like Receptors: Mechanism Of Signal Induction Is Elucidated By Quantifying Receptor-Ligand Complex Formation

The innate immune response provides the first line of defense against invading pathogens and helps to determine the nature of the adaptive immune response generated. An important component of this early response is initiated by a family of germline-encoded transmembrane biosensors, the Toll-Like Receptors (TLRs). Signaling through TLRs triggers inflammation and protects the body against some infections, but excess TLR signaling can be harmful, as is the case with systemic septic shock. Although TLRs are known to initiate signaling in response to specific pathogen-associated molecular patterns, the mechanism by which this occurs remains unknown. In particular, it is not known whether these receptor-ligand interactions are specific, forming defined complexes, or whether the interaction instead induces non-specific aggregation. Identifying the mechanism by which signaling is initiated would inform the design of agonists and antagonists for therapeutically modulating TLR signaling.

We have defined the mechanism of activation for one member of the TLR family, TLR3, which signals in response to a characteristic viral product - double-stranded RNA (dsRNA). Using biochemical analyses with purified TLR3 protein, we demonstrated that dsRNA binds to a site on TLR3 in a specific manner. This binding was quantified using surface plasmon resonance and showed that the affinity is a function of both dsRNA length and the pH at which the interaction occurs. Importantly, high affinity binding appeared to require the formation of complexes stabilized by protein-protein interactions. More specifically, dsRNA was found to bind pairs of TLR3, even though the protein was monomeric in the absence of its ligand. Finally, cellular assays were used to show that the formation of these complexes initiates TLR3 signaling, and the minimal signaling unit is a 2:1 complex of TLR3 with dsRNA.