The study investigates the role of mitochondrial DNA (mtDNA) synthesis in NLRP3 inflammasome activation. It is found that mtDNA synthesis, induced by Toll-like receptor (TLR) engagement, is crucial for NLRP3 signaling. TLRs signal through MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, an enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments, which associate with the NLRP3 inflammasome complex and are required for its activation. The study also shows that IRF1 controls mtDNA replication and NLRP3 activation, and that CMPK2 catalytic activity is essential for NLRP3 inflammasome activation. These findings provide new insights into the mechanism of NLRP3 inflammasome activation and suggest potential therapeutic targets for NLRP3-related diseases.The study investigates the role of mitochondrial DNA (mtDNA) synthesis in NLRP3 inflammasome activation. It is found that mtDNA synthesis, induced by Toll-like receptor (TLR) engagement, is crucial for NLRP3 signaling. TLRs signal through MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, an enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments, which associate with the NLRP3 inflammasome complex and are required for its activation. The study also shows that IRF1 controls mtDNA replication and NLRP3 activation, and that CMPK2 catalytic activity is essential for NLRP3 inflammasome activation. These findings provide new insights into the mechanism of NLRP3 inflammasome activation and suggest potential therapeutic targets for NLRP3-related diseases.