Figure 1.

Neutrophil extracellular traps (NETs) impair fully differentiation of monocytes into dendritic cells. Adhered monocytes were incubated or not with NETs-enriched supernatants, which were treated or not with DNase, or filtered for 18 h. IL-4 and GM-CSF were added to the cultures (as indicated), which were maintained at 37°C/5%CO₂. After 5 days, cells were harvested and stained for CD1a (B,C) and CD14 (D,E) and analyzed on a FACSCalibur flow cytometer. (A) Contour plots of one representative experiment showing the gate strategy. (B–E) The column Mon means that the adhered monocytes were not treated with NETs nor IL-4/GM-CSF. Digested NET (dNET) means supernatants enriched in NET pretreated with DNase (10 U/mL) 30 min before addition to monocytes. fNET means NET-free supernatants filtered through a 0.22 µm pore filter. PF-La and lipopolysaccharide (LPS) mean supernatants enriched in NET produced by paraformaldehyde killed parasites or LPS-activated neutrophils, respectively. Results of at least six independent experiments are shown as mean ± SEM. (B) ****, ***, **P < 0.01. (C) ***, **P < 0.01; *P < 0.05. (D) ****, **P < 0.01; *P < 0.05. (E) ****P < 0.01; *P < 0.05. (F) Adhered monocytes were incubated or not with different concentrations of NETs-enriched supernatants and the percentage of CD1a⁺ cells were analyzed as in panels (A–E). Results of three independent experiments are shown as mean ± SEM. *P < 0.05 related to monocytes only incubated with IL-4/GM-CSF (first column). (G) Adhered monocytes were incubated with NETs-enriched supernatants for 4 h at 37°C/5%CO₂. Cells were fixed and stained for DNA (blue) with 4′,6-diamidino-2-phenylindole and with anti-elastase followed by Texas red-labeled secondary antibody. Left image shows the overlay of DNA and elastase staining and right image shows the overlay of DNA and elastase staining with differential interference contrast image. (H) Monocytes were pretreated with cytochalasin D for 30 min before the addition of NETs and the percentage of CD1a⁺ cells were analyzed as in panels (A–E). Results of four independent experiments are shown as mean ± SEM. *P < 0.01 related to monocytes only incubated with IL-4/GM-CSF (first column).