*3.5. MPO Activity Is Detected Early during Neutrophil-Amoeba Interaction*

Trophozoites induced a rapid MPO activity, detected by luminol, that is independent of the ratio amoeba:neutrophil tested. This activity is detectable in the first minutes of interaction, reaching the maximum value at 20 min (Figure 6A–D). Then, the luminescence signal gradually decreases until it disappears after 90 min. A23187 also induced a similar kinetic of MPO activity that reached the maximum value at 10 min and then, decreased drastically until it disappeared after 100 min (Figure 6E). On the other hand, PMA induced a slower MPO activity that gradually increased during the first 20 min, then reached a steady state for 25 min (Figure 6F). Afterwards, the activity increased and newly reached the maximum value at 100 min, and then decreased to completely disappear at 200 min. It is important to notice that amoebas did not exhibit any MPO activity (Figure 6G).

**Figure 6.** MPO activity is detected during interaction between neutrophils with *E. histolytica*. Neutrophils (1 <sup>×</sup> 105) were culture in RPMI-1640 medium supplemented with 5%FBS and luminol (200 μM). Cells were stimulated with viable *E. histolytica* trophozoites at ratios of 1:100 (**A**), 1:50 (**B**), 1:20 (**C**) and 1:10 (**D**), as well as 50 nM PMA (**E**), 10 μM A23187 (**F**). Amoebas alone (1 <sup>×</sup> <sup>10</sup>4) also were tested (**G**). Black line represents MPO activity (as luminescence relative units, LRU) after stimulation and dotted line represents MPO activity on neutrophils in the absence of stimuli (same for all plots). Values are means of three independent experiments.

#### *3.6. MPO Activity Is Required for NETosis Induced by E. histolytica*

Luminol, a scavenger of MPO-derived HClO, decreased NET release triggered by *E. histolytica* trophozoites in a dose-dependent manner, suggesting that HClO is involved in this process (Figure 7A). Similar results were observed in PMA and A23187-induced NETosis. When we performed this assay using different trophozoite:neutrophil ratios, luminol (200 μM) decreased NET release induced by trophozoites at 1:50, 1:20 and 1:10 ratios; nevertheless, no differences were observed at a 1:100 ratio (Figure 7B).

To confirm that this effect was due to the reduction of MPO-derived HClO from neutrophils but not a reduction of ROS from amoebas, we pretreated trophozoites with luminol for 1.5 h and then used them to induce NETosis. Surprisingly, a decrease in NET release was observed in a similar way to in the previous experiment (Figure 7C). Therefore, we estimated ROS in luminol-pretreated trophozoites and found that, unlike neutrophils, luminol strikingly caused a dose-dependent increase of ROS in amoebas, as compared to the vehicle DMSO (Figure 7D). Viability of trophozoites was not affected by the treatment with luminol throughout the experiment (Supplementary Figure S1). Taken together, these data suggest that MPO activity from neutrophils and ROS from amoebas is produced early during the contact of the two cells, which is necessary for NETosis.

**Figure 7.** MPO activity is required for NETosis induced by *E. histolytica* trophozoites. (**A**) Neutrophils (1 <sup>×</sup> <sup>10</sup>5) were culture in RPMI-1640 medium supplemented with 5% FBS, 500 nM SYTOX® Green and luminol (Lum, 50, 100 or 200 μM or DMSO). Cells were stimulated with PMA (50 nM), A23187 (10μM) or 5 <sup>×</sup> <sup>10</sup><sup>3</sup> luminol-pretreated trophozoites (according with concentration present in the medium). Fluorescence was read after 4 h. (**B**) Neutrophils (1 <sup>×</sup> 105) were pretreated with 50, 100 and 200 μM luminol or DMSO during 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. (**C**) Neutrophils (1 <sup>×</sup> 105) were cultured in RPMI-1640 medium supplemented with 5% FBS, 500 nM SYTOX® Green and luminol (200 μM or DMSO). Cells were stimulated with trophozoites pretreated with luminol (200 μM or the vehicle DMSO) at ratios 1:100, 1:50, 1:20 or 1:10. Fluorescence was read at 4 h. (**D**) Amoebic trophozoites were treated with DMSO or luminol 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> <sup>10</sup><sup>5</sup> trophozoites were placed and fluorescence was read. For (**A**–**D**) the NET or ROS amount are expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments. \* *p* < 0.01, \*\* *p* < 0.001, # *p* < 0.0001 with respect to the control.
