Spatio-temporal regulation of ligand trafficking and TLR9 activation by PIEZO1 in human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are innate immune cells that produce type I interferons (IFNs) upon sensing nucleic acids via Toll-like receptor 9 (TLR9). Synthetic oligodeoxynucleotides CpGA and CpGB are widely used TLR9 agonists, yet only CpGA robustly induces IFN-α in pDCs. In contrast, CpGB drives much less IFN production. The mechanism underlying this ligand-specific response is not known. Here, we identify PIEZO1, a mechanosensory ion channel, as a regulator for this ligand-specific response. We show that CpGA, unlike CpGB, self-associates into large aggregates that generate membrane tension during cellular uptake, activating PIEZO1. This triggers calcium influx and localized F-actin assembly, retaining CpGA in early endosomes to sustain IRF7 activation and IFN production. PIEZO1 deficiency or inhibition abolishes CpGA-induced IFN responses, while PIEZO1 activation enhances IFN production by CpGB. Our findings reveal a hitherto unknown biophysical checkpoint in TLR9 signaling, where PIEZO1 translates membrane tension into spatially controlled TLR9 signaling. This study uncovers a novel role for mechanosensing in nucleic acid immunity, with implications for modulating IFN responses in infections and autoimmunity.