Toxoplasma gondii-induced neutrophil extracellular traps are relevant to glycolysis, TLR2, and TLR4 MAPK signaling pathway in goats.

Toxoplasma gondii (T. gondii) exhibits a significantly high prevalence of infection in goats, leading to adverse consequences such as abortion and stillbirth in ewes, thereby posing a substantial challenge to the goat farming industry. Neutrophil extracellular traps (NETs) have been shown to capture T. gondii in goats; however, the precise mechanisms underlying NET release in goats remain poorly understood. Therefore, the aim of our research was to elucidate the involved mechanism. We assessed the cytotoxicity of T. gondii on neutrophils using CCK-8 assay, visualized the structure of T. gondii-induced goat NETs through immunofluorescence, quantified ROS release during T. gondii-induced NET formation using fluorescence microplate analysis, and employed inhibitors targeting TLR 2, TLR4, NADPH oxidase, ERK1/2, and P38 MAPK signaling pathways as well as glycolysis to dissect the mechanisms underlying T. gondii-induced NET release. Within 1 h, T. gondii did not exhibit significant cytotoxicity towards neutrophils in our findings. The formation of typical NET structures induced by T. gondii involved DNA, citrullinated histone 3 (citH3), and neutrophil elastase (NE). Additionally, T. gondii significantly stimulated the release of NETs in goats. The process was accompanied by the production of reactive oxygen species (ROS) mediated through NADPH oxidase, p38, and ERK