The human antibacterial factor APOL3 couples lysosomal damage to mitochondrial DNA efflux and type I IFN induction.

Lysosomal damage is an endogenous danger signal, but its significance for innate immunity and the specific signaling pathways it engages remain unclear. Here, we uncover an immune-inducible pathway that connects lysosomal damage to mitochondrial DNA (mtDNA) efflux and type I IFN production. We find that transient lysosomal damage elicits sub-lethal mitochondrial outer membrane permeabilization (MOMP) via BAK/BAX macropores; however, the inner mitochondrial membrane (IMM) maintains a barrier against wholesale mtDNA release. Priming with type II IFN (IFN-γ) induced the antibacterial factor APOL3, which, upon sensing lysosomal damage, targets mitochondria undergoing MOMP to selectively permeabilize the IMM, enhance mtDNA release, and potentiate downstream cGAS signaling. Biochemical and cellular reconstitution revealed that, analogous to its bactericidal detergent-like mechanism, APOL3 permeabilized the IMM by solubilizing cardiolipin. Our findings illustrate how cells enlist an antibacterial protein to expedite the breakdown of endosymbiosis and facilitate a heightened response to injury and infection.