S100A9 interacts with a dynamic region on CD14 to activate Toll-like receptor 4.

S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates inflammatory pathways via Toll-like receptor 4 (TLR4). This activity plays important homeostatic roles in tissue repair, but can also contribute to inflammatory diseases. The mechanism of activation is unknown. Here, we follow up on a previous observation that the protein CD14 is an important co-receptor that enables S100A9 to activate TLR4. Using cell-based functional assays and a combination of mutations and pharmocological perturbations, we found that CD14 must be membrane bound to potentiate TLR4 activation by S100A9. Additionally, S100A9 is sensitive to inhibitors of pathways downstream of TLR4 internalization. Together, this suggests that S100A9 induces activity via CD14-dependent internalization of TLR4. We then used mutagenesis, structural modeling, and