Su, L.; Ejikemeuwa, A.; He, J.; Li, G.; Yuan, W.; Lu, W.; Wang, Y.; Zhang, Y.

Circulating cell-free DNA in inflamed tissues contains different forms of DNA, including extrachromosomal circular DNA (eccDNA). The role of DNA topology in innate immune sensing remains poorly understood. Here we show that circular DNA is a potent ligand for human plasmacytoid dendritic cells (pDCs) and that the cathelicidin host defense peptide LL37 selectively enhances circular DNA-induced activation. Across eccDNA, minicircle and synthetic circular DNA species, LL37 strikingly amplified type I interferon (IFN-I) and pro-inflammatory cytokine production, whereas linearized counterparts of identical sequence remained inactive. LL37-circular DNA complexes signaled through endosomal TLR9, as activation was abrogated by chloroquine and by the TLR9 antagonist IRS869. Mechanistically, LL37 promoted preferential retention of circular DNA in EEA1-positive early endosomes, and surface plasmon resonance revealed enhanced TLR9 binding to circular versus linear DNA, without differential LL37 binding. DNA topology rather than sequence, therefore, determines the immunostimulatory potential of cell-free DNA in pDCs. These findings define a mechanism by which LL37 and circular DNA jointly drive TLR9-dependent IFN-I responses, with implications for autoimmunity, cancer and nucleic acid therapeutics.