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

Plasmacytoid dendritic cells (pDCs) are specialized innate immune cells which play a pivotal role in antiviral immunity by producing large quantities of type I interferons (IFNs) upon sensing nucleic acids via Toll-like receptor 9 (TLR9). Synthetic oligodeoxynucleotides (ODNs) such as CpGA and CpGB, both containing unmethylated CpG motifs, are commonly used experimental TLR9 agonists. Interestingly, CpGA and CpGB elicit markedly different responses in pDCs – CpGA induces robust IFN-α production, whereas CpGB does not. The mechanistic basis underlying this ligand-specific functional divergence has remained unclear. Here, we identify PIEZO1, a mechanosensory ion channel, as a critical determinant of ligand-specific IFN responses in human primary pDCs. We demonstrate that unlike CpGB, CpGA self-associates into larger aggregates that generate membrane tension during cellular uptake, leading to activation of PIEZO1. This activation triggers localized calcium influx and cytoskeletal remodeling, resulting in the formation of local F-actin structures that retain CpGA within early endosomes enabling sustained IRF7 nuclear translocation and robust type I IFN production. Disruption of PIEZO1 or actin polymerization abrogates CpGA-induced IFN production, while pharmacological activation of Piezo1 enhances IFN production in response to CpGB. Thus, these findings uncover a previously unrecognized biophysical checkpoint in nucleic acid sensing, wherein membrane tension is transduced via PIEZO1 into spatially controlled TLR9 signaling. Overall, our study establishes PIEZO1 mechanosensing at the plasma membrane as a key regulatory event in nucleic acid-induced immunity, with different functional outcomes based on the cargo structure, opening potential new avenues for modulating type I IFN responses in infection and autoimmunity.