Figure 5.

Neutrophil extracellular traps (NETs)-treated monocytes are less efficient in parasite killing. (A) Adhered monocytes were treated with NET for 18 h at 37°C/5%CO₂. After 18 h of incubation, CFSE-labeled promastigotes of Leishmania amazonensis were added and cocultured at 35°C/5%CO₂. Monocytes were then analyzed on a FACSCalibur flow cytometer (Becton Dickinson). CFSE⁺ cells were considered as monocytes that bound to or phagocytosed parasites. Results of six independent experiments are shown as mean ± SEM. (B,C) Adhered monocytes were treated with digested, elastase inhibitor-treated, or non-treated NETs or with L. amazonensis supernatant for 18 h at 37°C/5%CO₂. Cells were then washed and promastigotes were added to the culture in a 1 monocyte:3 parasites ratio and cocultured at 35°C/5%CO₂ overnight. After 48 h of infection, monocytes were lysed with 0.01% sodium dodecyl sulfate and Schneider medium supplemented with 10% FBS was added to the cultures to allow parasites grow. Viable and motile parasites were counted after 48 h in a Neubauer chamber. Results of five to seven independent experiments are shown as mean ± SEM. *P < 0.05. Insert shows the number of parasites in NET-treated and -untreated monocytes. In the insert graph, paired t-test analysis was performed and *P < 0.01. (C) Untreated monocytes (first column) were normalized and data are expressed as fold over the control. (C) ****, ***, **P < 0.01.