*3.2. NETosis Induced by E. histolytica Trophozoites Is Independent of Neutrophil's ROS*

As shown in Figure 2A, NADPH oxidase inhibitor apocynin significantly reduced NE-Tosis induced by PMA, whereas NETosis induced by A23187 was not affected, as expected. Amoebas-induced NETosis was not affected by apocynin at any trophozoite:neutrophil ratio tested, suggesting that this process is certainly independent of ROS from neutrophils NADPH oxidase (Figure 2A). Then, we evaluated general production of ROS by neutrophils at the different ratios and found that although the respiratory burst was completely suppressed at a 1:20 ratio, as we previously reported, the neutrophils produce ROS at 1:100 and 1:50 ratios (Figure 2B). Taken together, the data strongly suggest that amoeba-induced NETosis is independent of ROS from neutrophils.

**Figure 1.** *Entamoeba histolytica* induces NETosis on human neutrophils in a dose-dependent manner. (**A**) Human neutrophils (1 <sup>×</sup> <sup>10</sup>5) were cultured with *E. histolytica* trophozoites at ratios amoeba:neutrophil of 1:100, 1:50, 1:20 and 1:10 in RPMI-1640 medium added with 5% FBS and 500 nM SYTOX® Green. PMA (50 nM) and A23187 (10 μM) were used as positive controls of NETosis. Finally, fluorescence was read at 4 h. NETs amount is expressed in fluorescence relative units (FRU). Values are means <sup>±</sup> SD of three independent experiments. \* *<sup>p</sup>* < 0.05, \*\* *<sup>p</sup>* < 0.001, # *<sup>p</sup>* < 0.001 respect to control. (**B**) Neutrophils (2 <sup>×</sup> 105) were stimulated with PMA (50 nM), A23187 (10 <sup>μ</sup>M) or *E. histolytica* trophozoites (1 <sup>×</sup> 104) during 4 h. After fixation, cells were marked using anti-MPO primary antibody followed by anti-mouse IgG-FITC secondary antibody. DNA was counterstained with DAPI. Images were taken at 40<sup>×</sup> magnification. Scale bar 100 <sup>μ</sup>m. (**C**) Neutrophils (2 <sup>×</sup> <sup>10</sup>5) were co-cultured with 1 <sup>×</sup> <sup>10</sup><sup>4</sup> *E. histolytica* trophozoites for 4 h. Cells were fixed and immunofluorescence was performed using anti-NE and or anti-acetylated histone H4 primary antibodies followed by anti-mouse IgG-FITC (for NE) or anti-rabbit-TRITC (for histone) secondary antibodies. DNA was counterstained with DAPI. Images were taken at 100× magnification. Scale bar: 50 μm.

**Figure 2.** NETosis induced by *E. histolytica* occurs independently of NADPH oxidase. (**A**) Neutrophils (1 <sup>×</sup> 105) were pretreated with 400 <sup>μ</sup>M apocynin (Apo) or DMSO for 30 min. Posteriorly, cells were transferred to RPMI-1640 medium added with 5% FBS and 500 nM SYTOX® Green and then stimulated with PMA (50 nM), A23187 (10 μM) or *E. histolytica* trophozoites at ratios of 1:100, 1:50, 1:20 and 1:10. Fluorescence was read at 4 h. (**B**) H2DCFDA-pretreated neutrophils (1 <sup>×</sup> <sup>10</sup>5) were culture in RPMI-1640 medium supplemented with 5% FBS and then stimulated with PMA (50 nM), A23187 (10 μM) or *E. histolytica* trophozoites at ratios 1:100, 1:50 or 1:20. Fluorescence was read at 1 h. NET and ROS amounts are expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments. \* *p*< 0.0001, # *p* < 0.001 with respect to control.
