Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

Abstract

Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica.

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

“…Then 100 μl of 120 nM PMA in PBS, or 100 μl of an E. histolytica suspension or an E. dispar suspension (1.25 × 10 5 cell/ml) was added to each well. The amoeba to neutrophil ratio was 1:20, as determined previously . Plates were incubated in 5% CO 2 at 37°C for 4 h. Next, 600 μl of 2% paraformaldehyde with 150 nM DAPI in PBS were gently added to each well.…”

“…NET function as a physical barrier where pathogens get trapped and can be eliminated extracellularly and independently of phagocytosis . 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) …”

Beneficial and Deleterious Effects of Neutrophil Extracellular Traps on Infection

“…This protozoan parasite causes amebiasis, amoebic colitis, and hepatic abscess. Since this parasite is too large to be phagocytosed, Avila et al [43] analyzed the possibility that this parasite induces the formation of NETs. These authors demonstrated that the amoeba lipopeptidophosphoglycan induces NETs in a dose-dependent manner.…”

“…Resistance to NETs exposure was also studied upon addition of a proteases inhibitor, resulting in that proteases are not responsible for trophozoite resistance to NETs. However, the use of ethylene glycolbis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a divalent anion chelant, had a deleterious efect in the growth of amoebas that were in contact with NETs, suggesting that trophozoites may have DNAse activity, responsible for its resistance to NETs [43].…”

“…Entamoeba histolytica is one of the main parasites that cause stomach diseases worldwide. It causes intestine and liver invasion, associated with the recruitment of large amounts of neutrophils at the early stages of infection [43].…”