Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation.

Amoebiasis is an infection of global importance, caused by the eukaryotic parasite Entamoeba histolytica. Pathogenic E. histolytica is associated worldwide with over a million cases of amoebic dysentery, colitis, and amoebic liver abscess. In contrast, the nonpathogenic Entamoeba dispar does not cause these diseases, although it is commonly found in the same areas as pathogenic amoeba. Entamoeba histolytica infection is usually associated with infiltrating neutrophils. These neutrophils appear to play a defensive role against this parasite, by mechanisms not completely understood. Recently, our group reported that neutrophil extracellular traps (NET) are produced in response to E. histolytica trophozoites. But, there is no information on whether nonpathogenic E. dispar can also induce NET formation. In this report, we explored the possibility that E. dispar leads to NET formation. Neutrophils were stimulated by E. histolytica trophozoites or by E. dispar trophozoites, and NET formation was assessed by video microscopy. NET induced by E. histolytica were important for trapping and killing amoebas. In contrast, E. dispar did not induce NET formation in any condition. Also E. dispar did not induce neutrophil degranulation or reactive oxygen species production. In addition, E. histolytica-induced NET formation required alive amoebas and it was inhibited by galactose, N-acetylgalactosamine, and lactose. These data show that only alive pathogenic E. histolytica activates neutrophils to produce NET, and suggest that recognition of the parasite involves a carbohydrate with an axial HO- group at carbon 4 of a hexose.

Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation.

Amoebiasis, the disease caused by Entamoeba histolytica is the third leading cause of human deaths among parasite infections. E. histolytica was reported associated with around 100 million cases of amoebic dysentery, colitis and amoebic liver abscess that lead to almost 50,000 fatalities worldwide in 2010. E. histolytica infection is associated with the induction of inflammation characterized by a large number of infiltrating neutrophils. These neutrophils have been implicated in defense against this parasite, by mechanisms not completely described. The neutrophil antimicrobial mechanisms include phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Recently, our group reported that NETs are also produced in response to E. histolytica trophozoites. But, the mechanism for NETs induction remains unknown. In this report we explored the possibility that E. histolytica leads to NETs formation via a signaling pathway similar to the pathways activated by PMA or the Fc receptor FcγRIIIb. Neutrophils were stimulated by E. histolytica trophozoites and the effect of various pharmacological inhibitors on amoeba-induced NETs formation was assessed. Selective inhibitors of Raf, MEK, and NF-κB prevented E. histolytica-induced NET formation. In contrast, inhibitors of PKC, TAK1, and NADPH-oxidase did not block E. histolytica-induced NETs formation. E. histolytica induced phosphorylation of ERK in a Raf and MEK dependent manner. These data show that E. histolytica activates a signaling pathway to induce NETs formation, that involves Raf/MEK/ERK, but it is independent of PKC, TAK1, and reactive oxygen species (ROS). Thus, amoebas activate neutrophils via a different pathway from the pathways activated by PMA or the IgG receptor FcγRIIIb.

Entamoeba histolytica Trophozoites Induce a Rapid Non-classical NETosis Mechanism Independent of NOX2-Derived Reactive Oxygen Species and PAD4 Activity.

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.