**1. Introduction**

Neutrophil extracellular traps (NETs) are DNA fibers associated with histones and antimicrobial proteins that are released by neutrophils into the extracellular space in a process denominated NETosis [1]. Initially, NETs were described as a strategy used for neutrophils to entrap and kill microorganisms [2–5]; nevertheless, these structures have been also related to other processes such as coagulation or complement activation, and even with autoimmune pathological processes such as erythematous systemic lupus [6–8]. Since its discovery, one of the central issues in the study of NETs has been the understanding of the mechanism associated with its release. Death of neutrophils through NETosis is not a random event; on the contrary, it is a highly regulated process involving a series of sequential steps [9–11]. Initially, neutrophils detect pathogens through receptors on cell surface such as TLRs, dectins, integrins or antibody receptors [12–15]. Then, diverse signaling

**Citation:** Díaz-Godínez, C.; Jorge-Rosas, J.F.; Néquiz, M.; Martínez-Calvillo, S.; Laclette, J.P.; Rosales, C.; Carrero, J.C. New Insights on NETosis Induced by *Entamoeba histolytica*: Dependence on ROS from Amoebas and Extracellular MPO Activity. *Antioxidants* **2021**, *10*, 974. https://doi.org/10.3390/ antiox10060974

Academic Editor: Serge Ankri

Received: 15 May 2021 Accepted: 6 June 2021 Published: 18 June 2021

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pathways are activated to drive events to lead NETotic commitment, resulting in reactive oxygen species (ROS) generation, histone processing, disassembling of a nuclear envelope and dissolution of internal membranes [16–19]. Decondensed DNA is then associated with antimicrobial proteins from cytoplasmic granules such as neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G (CG), proteinase 3 (PR3) and others [1]. Finally, cytoplasmic membrane ruptures and NETs are released [20].

Multiple mechanisms of NETosis have been identified depending on the stimulus used to trigger the process, including nuclear, mitochondrial or blebbing NETs [1,21,22]. The diverse mechanisms seem to differ from each other in the receptors involved, the signaling pathways activated or the ROS source required.Phorbol 12-myristate 13-acetate (PMA) and calcium ionophores (A23187 and ionomycin) are widely used stimuli to study NETosis; nevertheless, they have the disadvantage that they are stimuli with little biological relevance [23,24]. Despite this, the findings obtained with PMA and calcium ionophores have allowed us to decipher part of the process, mainly the critical need for ROS generation [25,26]. While ROS derived from NADPH oxidase was linked to PMA-induced NETosis [27], NETosis triggered by calcium ionophores does not require NADPH oxidase activity, although that depends partially on mitochondrial ROS (mitROS) [28]. Therefore, NETosis mechanisms have been classified into two groups: NADPH oxidase dependent or independent [29]. NAPDH-independent NETosis is not limited to the generation of mitROS as reactive nitrogen species (RNS), as well as exogenous ROS sources could also serve to trigger NET release [30,31]. In this context, it has been demonstrated that microorganisms such as *Escherichia coli* and *Mycobacterium smegmatis* produce ROS in stress conditions [32]; moreover, ROS from *Candida albicans* are responsible for triggering NET release in neutrophils from patients with chronic granulomatous disease [33].

*Entamoeba histolytica* is the protozoan responsible for causing intestinal (amoebic colitis or dysentery) and extraintestinal (amoebic liver abscess) amoebiasis in humans [34]. This parasite represents a public health problem, especially in developing countries where a prevalence of 1% to 20% in the population and up to 50% of diarrhea due to amoebiasis in infants have been reported [35–37]. Immune response activation against amoeba implicates a rapid recruitment of neutrophils to the infection site [38]; however, the exact role of these cells in amoebiasis remains unknown. Thus, some experiments suggest that neutrophils are required for clearance of the infection [39], whereas other evidence suggests that these cells could play a pathological role [40]. We previously showed that *E. histolytica* trophozoites induce a rapid NETosis in human neutrophils that is dependent on the viability of the parasite but independent of NADPH oxidase and PAD4 activities [41]. Nevertheless, a ROS source has not been identified. In this report, we investigated the source of ROS that leads to *E. histolytica* trophozoites-induced NETosis.
