Calcium-dependent redox signaling connects mitochondrial remodeling with PAD-associated NETosis during respiratory mycoplasma infection.

Neutrophil extracellular trap (NET) formation is controlled by redox signaling and mitochondrial stress, but the connection between pathogen-induced Ca2+ influx, mitochondrial remodeling, and PAD-associated chromatin execution remains insufficiently defined. Using Mycoplasma gallisepticum (MG) as a model of respiratory mycoplasma infection, we examined how pathogen-activated redox signaling modulates NET formation and the subsequent fate of extracellular DNA. In neutrophils, MG promoted NET formation, although visible trap deposition was partly obscured by MG-associated nuclease activity. Early proteomic analysis indicated enrichment of calcium signaling, ROS-related pathways, autophagy/mitophagy, lysosome/phagosome programs, and MAPK-linked responses. Mechanistically, MG-induced NETosis was mediated through involved a Ca2+/ROS-associated program, in which mitochondrial dysfunction and mitophagy-related remodeling facilitated PAD3 nuclear redistribution, histone citrullination, and extracellular DNA release. In agreement with a supportive rather than exclusive function, mitophagy activation enhanced NET-associated responses, whereas mitophagy inhibition weakened but did not completely prevent MG-induced NET release. MG-associated nuclease activity digested extracellular traps, enabled MG to acquire DNA signals derived from digested trap structures, and lowered NET-dependent inflammatory activation in recipient macrophages and epithelial cells. In vivo, MG infection caused local NET-related responses and systemic neutrophil priming, linked to mainly associated with ROS/MAPK activation rather than prolonged mitophagy-related alterations. MG-Escherichia coli co-infection intensified inflammatory pathology, whereas DNase I produced partial protection. These results support a context-dependent model in which MG stimulates Ca2+-dependent redox signaling and mitochondrial remodeling, thereby contributing to PAD-associated NETosis, while pathogen nuclease activity modifies extracellular NET DNA fate and downstream inflammatory pathology.