Ex vivo observation of Pythium insidiosum-antigen treated neutrophils on three Pythium insidiosum strains isolated from vascular pythiosis patients.

The mechanisms of Pythium insidiosum-antigen (PIA) immunotherapy activating a patient's immune system are unknown. We evaluated the interleukin-8 (IL-8) serum levels during P. insidiosum infection and after vaccination with PIA in vascular pythiosis cases. Furthermore, we studied the anti-P. insidiosum activity of neutrophils stimulated with various concentrations of PIA ex vivo in 3 strains of P. insidiosum isolated from vascular pythiosis patients. IL-8 serum levels were evaluated using the ELISA technique. We assessed the effect of PIA-stimulated neutrophils on the viability of zoospores using MTT assay, visualized neutrophil extracellular trap (NET) formation via microscopy, and measured the levels of double-stranded DNA (dsDNA) using PicoGreen dsDNA quantitation assay in 3 strains of P. insidiosum isolated from vascular pythiosis patients. Serum levels of IL-8 gradually lowered from the early to the end phases of vaccination with PIA among the surviving group of vascular pythiosis cases. Neutrophils stimulated with 0.01 µg/ml PIA reduced zoospore viability significantly compared to PIA-unstimulated neutrophils for strain 1 and strain 3 (p < .05). Neutrophils stimulated with 0.01, 0.1, 1, and 10 µg/ml PIA exhibited significantly lower zoospore viability than PIA-unstimulated neutrophils for strain 2 (p < .05). IL-8 can be used as a biomarker for monitoring vascular pythiosis cases treated with the PIA vaccine. Also, anti-P. insidiosum activity of PIA-stimulated neutrophils was probably due to the disruption of cellular activity in zoospores rather than the mechanisms based on the formation of NETs.

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.