Giardia duodenalis cysts induce bovine neutrophil extracellular trap formation

Giardia duodenalis, a globally distributed zoonotic intestinal protozoan, poses significant threats to public health and livestock industries. Neutrophils (polymorphonuclear neutrophils, PMNs) eliminate pathogens through multiple mechanisms, including neutrophil extracellular trap (NET) formation. However, the interaction between G. duodenalis cysts and bovine PMNs remains poorly understood. Here, we demonstrate that G. duodenalis cysts can trigger NET formation in bovine PMNs, as evidenced by immunofluorescence co-localization of extracellular DNA and neutrophil elastase (NE). NET formation was concentration-dependent and required signaling through NADPH oxidase, ERK, p38 MAPK, P2X1 receptor, and PAD4, as shown by specific pharmacological inhibition (DPI, U0126, SB202190, NF449, Cl-amidine; p < 0.01). Additionally, autophagy played a critical role: autophagy inhibitors (3-methyladenine, wortmannin) significantly suppressed NET formation (p < 0.0001), while the autophagy inducer rapamycin had no effect. Immunofluorescence revealed the formation of autophagosomes concurrent with NET formation upon cyst stimulation. These results suggest that extracellular protozoa such as G. duodenalis can induce NET formation via coordinated activation of autophagy and multiple signaling pathways, providing novel insights into host-parasite interactions and potential therapeutic targets.