SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization.

M1 macrophage polarization is modulated by the release of mitochondrial DNA (mtDNA) and induces the inflammatory immune response, which is further increased by the generation of mitochondrial reactive oxygen species (mtROS). The pyrimidine nucleotide carrier SLC25A33 is located in the mitochondrial inner membrane and is linked to mtDNA synthesis, but its role in the M1 macrophage inflammatory immune response remains unclear. Here, we elucidate the regulatory mechanisms responsible for upregulation of SLC25A33 expression during M1 macrophage polarization, SLC25A33-mediated mtROS production, and the inflammatory response. SLC25A33 expression was significantly elevated in CD14+ monocytes derived from patients with sepsis and LPS/interferon-gamma (IFN-γ)-stimulated peritoneal macrophages (PMs). SLC25A33 was upregulated by ATF4 through the MyD88-PI3K-mTORC1 pathway in LPS/IFN-γ-stimulated PMs. Furthermore, SLC25A33 increased mtDNA synthesis and the release of mtDNA into the cytosol, which was facilitated by mtROS-mediated voltage-dependent anion channel (VDAC) oligomer formation, thereby contributing to activation of the cGAS-STING inflammatory pathway. Conversely, knockdown and pyridoxal 5'-phosphate treatment, which inhibits SLC25A33 activity, decreased mtDNA release and reduced M1 macrophage polarization and associated inflammatory responses. These findings were consistent across and sepsis models, as well as in septic patients with liver abscesses. Our findings underscore the significant role of SLC25A33 in inflammation, suggesting that targeting of SLC25A33 could be a promising therapeutic strategy for the management of M1 macrophage-mediated inflammatory diseases, including sepsis.