A new mechanism for NLRP3 inflammasome activation has been identified, involving mitochondrial DNA (mtDNA) synthesis triggered by Toll-like receptor (TLR) engagement. The study shows that mtDNA synthesis is crucial for NLRP3 signaling, with the MyD88 and TRIF adaptors playing key roles in activating IRF1, which in turn promotes the expression of CMPK2, a rate-limiting enzyme for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments, which then associate with the NLRP3 inflammasome complex and are required for its activation. The study also demonstrates that macrophage priming enhances mtDNA synthesis and the production of oxidized mtDNA, which is essential for NLRP3 inflammasome activation. LPS induces mtDNA replication in macrophages, which is dependent on TLR signaling and the activity of CMPK2. The study further shows that IRF1 controls mtDNA replication and NLRP3 activation, while CMPK2 is essential for mtDNA synthesis and NLRP3 inflammasome activation. The findings suggest that mtDNA synthesis is a critical component of NLRP3 inflammasome activation, and that targeting this pathway could provide new therapeutic approaches for NLRP3-related diseases. The study also highlights the importance of mtDNA in the activation of the NLRP3 inflammasome and the role of CMPK2 in this process. The results indicate that the synthesis of mtDNA is a key step in the activation of the NLRP3 inflammasome, and that this process is regulated by a complex network of signaling pathways involving TLRs, IRF1, and CMPK2. The study provides new insights into the mechanisms underlying NLRP3 inflammasome activation and highlights the potential for developing targeted therapies for NLRP3-related diseases.A new mechanism for NLRP3 inflammasome activation has been identified, involving mitochondrial DNA (mtDNA) synthesis triggered by Toll-like receptor (TLR) engagement. The study shows that mtDNA synthesis is crucial for NLRP3 signaling, with the MyD88 and TRIF adaptors playing key roles in activating IRF1, which in turn promotes the expression of CMPK2, a rate-limiting enzyme for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments, which then associate with the NLRP3 inflammasome complex and are required for its activation. The study also demonstrates that macrophage priming enhances mtDNA synthesis and the production of oxidized mtDNA, which is essential for NLRP3 inflammasome activation. LPS induces mtDNA replication in macrophages, which is dependent on TLR signaling and the activity of CMPK2. The study further shows that IRF1 controls mtDNA replication and NLRP3 activation, while CMPK2 is essential for mtDNA synthesis and NLRP3 inflammasome activation. The findings suggest that mtDNA synthesis is a critical component of NLRP3 inflammasome activation, and that targeting this pathway could provide new therapeutic approaches for NLRP3-related diseases. The study also highlights the importance of mtDNA in the activation of the NLRP3 inflammasome and the role of CMPK2 in this process. The results indicate that the synthesis of mtDNA is a key step in the activation of the NLRP3 inflammasome, and that this process is regulated by a complex network of signaling pathways involving TLRs, IRF1, and CMPK2. The study provides new insights into the mechanisms underlying NLRP3 inflammasome activation and highlights the potential for developing targeted therapies for NLRP3-related diseases.