Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1β (IL-1β) production and mtDNA release in mice.