Neospora caninum triggers the release of heterophil extracellular traps in chickens

Neospora caninum (N. caninum) has been recognized as a pathogen responsible for neosporosis in chicken. Heterophils, which are a crucial part of the chicken's innate immune response, employ heterophil extracellular traps (HETs) as a principal defense strategy against invading pathogens. However, whether N. caninum can trigger the release of HETs remains unclear. This study presents the first evidence that N. caninum can induce the release of HETs in chickens and delves into the mechanisms that regulate this phenomenon. The impact of N. caninum on heterophil activity was evaluated using the CCK-8 assay. N. caninum- induced HETs were characterized using scanning electron microscopy and immunofluorescence. The release of N. caninum-induced HETs was quantified using a PicoGreen-based fluorescence microplate assay. Our findings revealed that heterophil activity remained unaffected by N. caninum infection. N. caninum induced thicknesses, reminiscent of HET-like structures, which primarily consisted of a DNA skeleton, citrullinated histone (citH3), and elastin. Furthermore, an increase in reactive oxygen species (ROS) production was detected during the release of N. caninum-induced HETs, a process that was found to be dependent on ROS-mediated activation, NADPH oxidase activity, glycolysis, toll-like receptor 2/4 (TLR2/4) and MAPK signaling pathways. N. caninum induced-HETs release was shown to be ROS-dependent, involving the activation of NADPH oxidase, ERK_1/2, glycolysis, p38 MAPK, and toll-like receptor signaling pathways. In conclusion, this study is the first to elucidate the release mechanism of N. caninum-induced HETs in chicken, offering significant insights into the mechanisms underlying immune innate system against N. caninum infection.