Induction of NETs by <i>L</i>. <i>mexicana</i> promastigotes.

<p>(A) Sorted mouse BM neutrophils were adhered on poly-L-lysin coverslips and incubated for 4 hours with <i>L</i>. <i>mexicana</i>-DsRed metacyclic promastigotes at a 5:1 parasite-cell MOI in absence or presence of DNase 10μg/mL. Samples were fixed, stained for MPO and DNA (DAPI) and analyzed by confocal microscopy. As controls, neutrophils were incubated with 100ng/mL PMA or medium and similarly analyzed. (B) Higher magnification of <i>L</i>. <i>mexicana-</i>DsRed-induced NETs. (C) BM neutrophils were incubated with <i>L</i>. <i>mexicana</i>-luciferase promastigotes at a 5:1 parasite-cell MOI in absence or presence of 10μg/mL DNase. After 4 hours parasite survival was determined in each group by dividing the luciferase relative light units (RLUs) obtained in presence of neutrophils from that obtained in the absence of neutrophils. (D) RR3-16-treated, 1A8 PMN-depleted C57BL/6 and <i>Genista</i> mice were inoculated i.d. with 10⁶ metacyclic <i>L</i>. <i>mexicana</i>. Ears were collected 24h later and histology performed on 4 micron sections and inflammatory cell recruitment determined by hematoxylin and eosin. (E) Ear sections were stained with polyclonal anti-MPO Ab and DNA stained with PI. Arrowheads point to neutrophil DNA fibers and arrows to parasite DNA. (F) Frequency of neutrophils producing NETs/section as determined by confocal microscopy. Data presented as mean ± SEM for n ≥ 4/group. Results representative of 3 or more independent experiments. Scale bars: 10 micron. * p<0.05 ** p<0.01, ns: non significant.