The role of neutrophils against Pythium insidiosum

Abstract

Pythium insidiosum (P. insidiosum) is an oomycetes fungus-like microorganism causing pythiosis infection in humans. The zoospores (infective stage) of this pathogen contaminate aquatic environments such as moist soil and swampy area, which is a natural habitiat. Humans are infected with P. insidiosum via cutaneous route which is the main entry of the zoospores. Recent studies have shown that patients with pythiosis shows high morbidity and mortality rates particularly those with hematologic diseases. As neutrophils are the first line of immune defense during fungal infections, this study therefore investigated the activity of neutrophils in response to P. insidiosum. The neutrophil killing activity, phagocytosis, and neutrophil extracellular trap (NET) formation were determined. Zoospores from six different strains of P. insidiosum were randomly selected and incubated with isolated healthy neutrophils (n=6). �The results showed that human neutrophils significantly decreased number of zoospores as observed in both colony counts on blood agar and live/dead cell staining (p < 0.001). Phagocytosis measured by flow cytometry showed only two strains of zoospores (pHrodo-labeled) were significantly phagocytosed by neutrophils (p < 0.01). In contrast, all six strains of heat-killed zoospores significantly induced NETs (p < 0.001) detected by immunofluorescence staining and picogreen assay. Our findings suggests that human neutrophils produce NET formation as a main mechanism rather than phagocytosis in response to P. insidiosum.