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

Abstract

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.

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

“…This is opposite to the idea that E. histolytica actively destroy neutrophils by inducing them to execute apoptosis . Previously, we did not find evidence for apoptosis in neutrophils interacting with E. histolytica trophozoites, as shown by the lack of phosphatidylserine expression, or a decrease in CD16b expression on the membrane of neutrophils. Now, cell imaging analysis confirms that neutrophils not actively engaging amoebas remain alive (Figs.…”

“…Because peroxiredoxin could degrade ROS released from immune cells, it has been proposed that this enzyme is involved in protecting amoebas from the respiratory burst of neutrophils . However, there is no clear evidence that amoebas are directly damaged by ROS and more importantly, neither E. histolytica nor E. dispar (Fig. ) were capable of inducing ROS production by neutrophils.…”

“…The total area of all fluorescent particles was used to estimate the amount of NET formation, considering that neutrophils losing their membrane integrity by necrosis become SYTOX Green‐positive, but their DNA remains condensed, and that unstimulated neutrophils have an area of 62 ± 6.1 μm 2 . Thus, only particles with a fluorescent DNA area larger than 70 μm 2 were considered NET . In addition, because only neutrophils in contact with amoebas release DNA, and the DNA fibers formed by neutrophils after interacting with E. histolytica are indeed NET (Fig.…”

“…NET formation was quantified by detecting DNA release spectrophotometrically with the DNA‐binding dye SYTOX ® Green as previously described . Briefly, neutrophils were resuspended at 1 × 10 6 cell/ml in RPMI‐1640 medium (Gibco ® ), containing 500 nM SYTOX ® Green.…”

“…Recently, we have found that E. histolytica trophozoites are capable of inducing NET formation . This response is triggered by an unknown receptor, nevertheless we were able to determine that E. histolytica induce a signaling pathway that involves Raf/MEK/ERK, and it is independent of protein kinase C and reactive oxygen species (ROS) …”