*3.4. Amoebas Derived ROS Are Required for NETosis*

Based on the above results, we evaluated the effect of reducing the amount of ROS in viable trophozoites on their capability to induce NETosis. First, we demonstrated that the pretreatment of *E. histolytica* trophozoites with the ROS scavenger pyrocatechol (50 to 200 μM) for 1.5 h resulted in a significant and dose-dependent reduction of ROS levels. Moreover, this effect persisted during the 4 h period that NETosis assay lasted (Figure 4A). The result was confirmed in H2DCFDA-stained trophozoites. As shown in Figure 4B, the amoebic trophozoites exhibited intense green florescence in basal conditions (high ROS levels), whereas the pyrocatechol-treated amoebas exhibited a dose-dependent decrease of green fluorescence denoting a reduction of trophozoite-derived ROS. Many trophozoites pretreated with pyrocatechol 200 μM were virtually non-fluorescent (Figure 4B, lower panel, white arrows). The treatments with pyrocatechol and luminol did not affect the viability of trophozoites (Supplementary Figure S1).

Once we had demonstrated the reduction of ROS in pyrocatechol-pretreated trophozoites, we then carried out NETosis assays with these amoebas. It is noteworthy that ROS-reduced trophozoites induced a reduced NET amount compared with trophozoites pretreated with the vehicle DMSO, and the reduction was statistically significant when pyrocatechol at 100 and 200 μM was used (Figure 5A). As expected, pyrocatechol present in the culture media abolished NET release by PMA at any concentration, but it did not affect NET release by A23187 when used at doses under 100 μM. Surprisingly, A23187-induced NETosis, which is NADPH-ROS independent, was reduced with 200 μM of pyrocatechol (Figure 5A). Involvement of amoeba-derived ROS in NETosis became more evident when lower ratios of parasites per neutrophil were tested. As shown in Figure 5B, trophozoites pretreated with pyrocatechol induced less NET release than DMSO-treated parasites in a dose-dependent manner. Moreover, pretreated amoebas lost the ability to induce NE-Tosis at lower ratios (1:50 and 1:100). It is noteworthy that catalase added to the culture media was unable to prevent NETosis induced by untreated trophozoites (Supplementary Figure S2).

**Figure 4.** Pyrocatechol reduces ROS generation in viable *E. histolytica* trophozoites. Amoebic trophozoites were treated with DMSO or pyrocatechol (Pyro) at 50, 100 and 200 μM for 30 min and then H2DCFDA (100 μM) was added. Cells were incubated for another hour and after treatment, trophozoites were resuspended in RPMI-1640 medium supplemented with 5% FBS. A total of 1 <sup>×</sup> 105 trophozoites were placed and fluorescence was read every hour during 4 h (**A**) or were fixed and counterstained with DAPI for visualization under fluorescence microscopy (**B**). NET amount is expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments. # *p* < 0.01 with respect to the control.

**Figure 5.** ROS-reduced trophozoites failed to induce NETosis on human neutrophils. (**A**) Neutrophils (1 <sup>×</sup> <sup>10</sup>5) were cultured in RPMI-1640 medium supplemented with 5% FBS, 500 nM SYTOX® Green and pyrocatechol (Pyro, 50, 100 or 200 <sup>μ</sup>M or DMSO). Cells were stimulated with PMA (50 nM), A23187 (10 <sup>μ</sup>M) or 5 <sup>×</sup> 103 pyrocatechol-pretreated trophozoites (according to the concentration present in the medium). Fluorescence was read after 4 h. (**B**) Neutrophils (1 <sup>×</sup> 105) were cultured in RPMI-1640 medium supplemented with 5% FBS, 500 nM SYTOX® Green and pyrocatechol (200 μM or DMSO). Cells were stimulated with trophozoites pretreated with pyrocatechol (200 μM or the vehicle DMSO) at ratios 1:100, 1:50, 1:20 or 1:10. Fluorescence was read after 4 h. NET amount is expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments. \* *p* < 0.01, \*\* *p* < 0.0001, # *p* < 0.05 with respect to the control.
