Figure 1

Fasciola hepatica antigen (FhAg)-induced neutrophil extracellular traps (NETs) formation, analysed via fluorescence microscopy, SEM and PicoGreen®-derived fluorescence intensities. Fluorescence microscopy analysis demonstrates co-localization of DNA (A, E; blue), neutrophil elastase (NE) (B, F; green) and pan-histones (H1, H2A/H2B, H3, H4) (C, G; red) of ovine PMN confronted to FhAg 100 μg/mL. Depiction of the overlay of the three channels (D, H). SEM analysis confirmed ovine NET formation observing fine spread NETs (sprNETs) after exposure to FhAg 100 μg/mL (I). As expected, PMN negative control remained mostly inactivated (J). Immunofluorescence analysis shows proportion of ovine PMN undergoing NETosis observing differences of NET formation on PMN exposed to FhAg 100 μg/mL compared to control (p = 0.022) after 120 min of incubation (K). PicoGreen®-derived fluorescence intensities analysis shows quantification of anchored NETs observing a significant increase compared with DNase control (p = 0.027) at 120 min of incubation (L) and an increase on release of cell free NETs induced by FhAg 100 μg/mL at 120 min incubation when compared to FhAg 10 μg/mL (p = 0.039) (M). As controls, PMN alone, PMN treated with saponin (1% w/v) for assessment of total DNA, PMN treated with DNase to dissolve NETs, and PMN exposed to zymosan 1 mg/mL as positive control were here used. For statistical analyses ANOVA with Kruskal–Wallis test was used.