Abstract
Neutrophil extracellular traps (NETs) are DNA fibers decorated with histones and antimicrobial proteins from cytoplasmic granules released into the extracellular space in a process denominated NETosis. The molecular pathways involved in NETosis have not been completely understood. Classical NETosis mechanisms involve the neutrophil elastase (NE) translocation to nucleus due to the generation of reactive oxygen species (ROS) by NADPH oxidase (NOX2) or the peptidyl arginine deiminase 4 (PAD4) activation in response to an increase in extracellular calcium influx; both mechanisms result in DNA decondensation. Previously, we reported that trophozoites and lipopeptidophosphoglycan from Entamoeba histolytica trigger NET release in human neutrophils. Here, we demonstrated in a quantitative manner that NETs were rapidly form upon treatment with amoebic trophozoites and involved both nuclear and mitochondrial DNA (mtDNA). NETs formation depended on amoeba viability as heat-inactivated or paraformaldehyde-fixed amoebas were not able to induce NETs. Interestingly, ROS were not detected in neutrophils during their interaction with amoebas, which could explain why NOX2 inhibition using apocynin did not affect this NETosis. Surprisingly, whereas calcium chelation reduced NET release induced by amoebas, PAD4 inhibition by GSK484 failed to block DNA extrusion but, as expected, abolished NETosis induced by the calcium ionophore A23187. Additionally, NE translocation to the nucleus and serine-protease activity were necessary for NET release caused by amoeba. These data support the idea that E. histolytica trophozoites trigger NETosis by a rapid non-classical mechanism and that different mechanisms of NETs release exist depending on the stimuli used.
Highlights
Neutrophils are the most abundant leucocytes in the mammal peripheral blood
Mitochondrial DNA NETosis (Yousefi et al, 2009) has been reported in neutrophils which were primed with granulocyte/macrophage-colony stimulatory factor (GM-CSF) and stimulated with lipopolysaccharide (LPS) or complement fraction C5a; this form of Neutrophil extracellular traps (NETs) release was named vital NETosis because only mitochondrial DNA (mtDNA) is extruded without compromise cell viability of neutrophils, in contrast with the lethal nuclear NETosis
In the neutrophil/amoeba co-culture, DNA extracellular fiber were detected by DAPI staining and these structures co-localized with proteins associated with NETs such as neutrophil elastase (NE), MPO and histone H4 (Figure 1B)
Summary
Neutrophils are the most abundant leucocytes in the mammal peripheral blood. Neutrophils have different strategies to combat pathogens including phagocytosis, degranulation and production of neutrophil extracellular traps (NETs). Calcium ionophores ionomycin and A23187 appear to induce NETosis independently of NOX2derived ROS; they depend on peptidyl arginine deiminase 4 (PAD4) activity to promote DNA decondensation by changing positive charged arginine residues to none charged citrulline residues in histones (Wang et al, 2009). This mechanism occurs due to an increase in extracellular calcium influx that activates PAD4 (which has four sites for calcium binding), and theoretically any molecule with the capacity to form pores in the cytoplasmic membrane of neutrophils (e.g., bacterial toxins or complement) are able to induce NETosis through this pathway (Konig and Andrade, 2016). Mitochondrial DNA (mtDNA) NETosis (Yousefi et al, 2009) has been reported in neutrophils which were primed with granulocyte/macrophage-colony stimulatory factor (GM-CSF) and stimulated with lipopolysaccharide (LPS) or complement fraction C5a; this form of NETs release was named vital NETosis because only mtDNA is extruded without compromise cell viability of neutrophils, in contrast with the lethal nuclear NETosis
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