Neutrophil extracellular traps and phagocytosis in Pythium insidiosum

Neutrophils are innate immune cells that play crucial roles in response to extracellular pathogens, including bacteria and fungi. Pythium insidiosum (P insidiosum) is a fungus-like pathogen that causes "pythiosis" in mammals. This study investigated in vitro function of human neutrophils against P. insidiosum. We demonstrated the killing mechanism of neutrophils when incubated with P. insidiosum zoospores (infective stage), such as phagocytosis and neutrophil extracellular traps (NETs). Healthy human neutrophils significantly reduced six strains of live zoospores isolated from different sources compared to the condition without neutrophils (p < 0.001), observed by colony count and trypan blue staining. As our results showed the killing ability of neutrophils, we further investigated the neutrophil killing mechanism when incubating with zoospores. Our study found that only two strains of heat-killed zoospores significantly induced phagocytosis (p < 0.01). Co-culture of heat-killed zoospores and neutrophils demonstrated NET formation, which was detected by immunofluorescence staining using DAPI, anti-myeloperoxidase, and anti-neutrophil elastase and quantitated under the fluorescence microscope. In addition, the level of cell-free DNA released from neutrophils (as a marker of NET production) after incubation with zoospores showed significantly increased levels when compared with unstimulated neutrophils (p < 0.001). Our findings demonstrate that neutrophils revealed the NET formation in response to P. insidiosum zoospores. This study is the first observation of the neutrophil mechanism against P. insidiosum, which could provide a better understanding of some parts of the innate immune response during pythiosis.