Figure 4.

NET release against F. pedrosoi hyphae infection is a mechanism independent of TLR-2 and TLR-4 and responsible for hyphae killing. (A) WT neutrophils purified by magnetic beads were resuspended in media containing 5 µM styox green dye in resting condition (dashed lines; negative control) or incubated with F. pedrosoi hyphae or conidia. Florescence was recorded by a plate reader for every 30 min up to 3 h. DNA release (NETotic index) shows NETosis over hyphae but not conidia infection (A). After 180 min, the neutrophils incubated with hyphae were fixed with 4% (v/v) PFA for 15 min and analyzed by immunofluorescence microscopy (D e E). Using TLR-2KO (B) and TLR-4KO (C) neutrophils, we verified that NET release is a mechanism independent from TLR-2KO and TLR-4KO. To confirm that NETs kill F. pedrosoi hyphae (F) but not conidia (G), WT-purified neutrophils were incubated with F. pedrosoi conidia or hyphae in the presence or absence of DNase. After 2 h, the cells were lysed with distilled water and seeded in Sabouraud agar at 37°C for 5 days. Data are expressed as mean ± SEM; n = 5, two-way ANOVA with Bonferroni’s posttest. ***p < 0.001.