*3.7. MPO Is Detected on the Surface of Amoebic Trophozoites Early after Contact with Neutrophils and Its Activity Is Required for Trophozoite-Induced NETosis*

The above results point out to the possibility of ROS transfer between the two cells in very early stages of contact. As ROS were not detected in neutrophils stimulated with trophozoites at a 1:20 ratio, but since MPO activity was always detected independent of the trophozoite:neutrophil ratio assayed, we explored the possibility that MPO was transferred from the neutrophil to the amoeba. As shown in Figure 8A, anti-MPO antibodies started to react with the surface of trophozoites after 5 min, covering all amoeba surfaces after 10 min of contact with neutrophils, suggesting that MPO was rapidly transferred from neutrophils to amoebas. The fluorescence detected was not due to autofluorescence or unspecific binding of the secondary antibody, as shown in trophozoites alone (Figure 8A).

To determine if the MPO activity in the surface of amoebic trophozoites is involved in NETosis, isoluminol (which does not cross the cell membrane) was used instead of luminol to scavenge MPO-derived HClO. In contrast to luminol, isoluminol reduced NETosis triggered by amoebic trophozoites at all ratios tested, showing significant differences with respect to the control (Figure 8B). Isoluminol also reduced PMA- and A23187-induced NETosis; however, NETosis was not completely abolished.

**Figure 8.** Extracellular MPO activity is required for NETosis induced by *E. histolytica*. (**A**) Neutrophils (2 <sup>×</sup> <sup>10</sup>5) were co-cultured with 1 <sup>×</sup> 104 *E. histolytica* trophozoites during 5 or 10 min. Cells were fixed and immunofluorescence was performed using anti-MPO antibody followed by anti-mouse IgG-FITC secondary antibody. DNA was counterstained with DAPI. Amoebas alone were used as a control. Trophozoites are indicated by doted white lines. Images were taken at <sup>100</sup><sup>×</sup> magnification. Scale bar 100 <sup>μ</sup>m. (**B**) Neutrophils (1 <sup>×</sup> 105) were pretreated with 50, 100 and 200 <sup>μ</sup>M isoluminol (Iso) 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 after 4 h. NET amount is expressed in fluorescence relative units (FRU). Values are means ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.
