*Article* **New Insights on NETosis Induced by** *Entamoeba histolytica***: Dependence on ROS from Amoebas and Extracellular MPO Activity**

**César Díaz-Godínez 1, Joshue Fabián Jorge-Rosas 1, Mario Néquiz 2, Santiago Martínez-Calvillo <sup>3</sup> , Juan P. Laclette 1, Carlos Rosales 1,\* and Julio C. Carrero 1,\***


**Abstract:** NETosis is a neutrophil process involving sequential steps from pathogen detection to the release of DNA harboring antimicrobial proteins, including the central generation of NADPH oxidase dependent or independent ROS. Previously, we reported that NETosis triggered by *Entamoeba histolytica* trophozoites is independent of NADPH oxidase activity in neutrophils, but dependent on the viability of the parasites and no ROS source was identified. Here, we explored the possibility that *E. histolytica* trophozoites serve as the ROS source for NETosis. NET quantitation was performed using SYTOX® Green assay in the presence of selective inhibitors and scavengers. We observed that respiratory burst in neutrophils was inhibited by trophozoites in a dose dependent manner. Mitochondrial ROS was not also necessary, as the mitochondrial scavenger mitoTEMPO did not affect the process. Surprisingly, ROS-deficient amoebas obtained by pre-treatment with pyrocatechol were less likely to induce NETs. Additionally, we detected the presence of MPO on the cell surface of trophozoites after the interaction with neutrophils and found that luminol and isoluminol, intracellular and extracellular scavengers for MPO derived ROS reduced the amount of NET triggered by amoebas. These data suggest that ROS generated by trophozoites and processed by the extracellular MPO during the contact with neutrophils are required for *E. histolytica* induced NETosis.

**Keywords:** *Entamoeba histolytica*; neutrophil extracellular traps (NETs); neutrophils; reactive oxygen species (ROS); myeloperoxidase
