Fig. 5

Visualization of NETs by fluorescence staining of DNA, H3Cit and MPO. Purified peripheral blood neutrophils pre-treated with or without the antagonist (Boc) or the agonist (LXA4) were exposed to L. infantum promastigotes for 5 h at 37 °C and immunostained for DNA (blue), H3Cit (red) and MPO (green). DNA: DNA backbone is stained with DAPI. H3Cit: citrullinated histone H3 is visualized with a red fluorescence label. MPO: myeloperoxidase is detected with a green fluorescence-labeled specific antibody. Merge: merged image showing fluorescence staining of DNA, H3Cit and MPO. a Control group of untreated neutrophils; b Boc-pretreated neutrophils stimulated by L. infantum promastigotes; c Untreated neutrophils stimulated by L. infantum promastigotes obviously showing DNA net-like fibers and releasing H3Cit and MPO; d Untreated neutrophils stimulated by LXA4 expressing H3Cit and MPO; e LXA4-pretreated neutrophils stimulated by promastigotes obviously exhibiting DNA net-like fibers and releasing H3Cit and MPO; f Boc-pretreated neutrophils stimulated by LXA4 showing intact cell structure. NETs positive for DNA, H3Cit and MPO were visible in Boc-untreated and lipoxin A4 pre-treated neutrophils stimulated by promastigotes. However, most cells in the Boc-primed neutrophil samples showed intact and smaller nuclei; this means that Boc had an inhibitory effect on NET extrusion via its antagonistic effects on the lipoxin A4 receptor. Scale-bars: 100 µm