Fig 1.
E. histolytica trophozoites induce the formation of human neutrophil extracellular traps.
A) Human neutrophils isolated by positive selection from peripheral heparinized blood were incubated with E. histolytica HM1:IMSS trophozoites (ratio 1 amoeba to 20 neutrophils) and the release of NETs monitored with 7AAD stain at 5, 15, 30 and 60 mins. Spider web-like fibers are observed as rapid as 5 min of incubation and increase in number and density over time. The networks were initially seen projected out of neutrophils toward amoebas (5 and 15 mins), and later gradually increase in number until completely cover the trophozoites that seem to be snared in the mesh (30 and 60 mins). Neutrophils in the absence of amoebas and incubated for 60 min are shown stained with Giemsa or 7ADD.B) Incubation of isolated human neutrophils with formaldehyde-fixed trophozoites did not induce NETs at 15 and 60 min. In A and B, trophozoites location is indicated by white head-arrows; magnification 40X. C) Incubation of isolated human neutrophils with fresh trophozoites whole extract induces scarce NETs at 15 and 60 min. B and C were stained with Hoechst.
Fig 2.
Trophozoites induced NETs are unable to inhibit parasite growth despite containing anti-microbial cathelicidin LL-37.
A) Growth kinetic of trophozoites co-incubated with NETs for the indicated times (5 to 60 min) and thereafter cultured in fresh TYI-S-33 medium. Live trophozoites were counted every 24 h under light microscope using Trypan blue. Data shown at each time is the mean ± SD of three independent assays. B) Immunofluorescence assay upon NETs induced by amoebas at 15 and 60 min (left panels) using anti-LL-37 antibody and an anti-rabbit IgG conjugated to FITC (right panels). Magnification 40X.
Fig 3.
Interaction and growth of E. histolytica trophozoites with neutrophil extracellular traps.
NETs induced with PMA were incubated 1 h with amoeba trophozoites. Top: A-D, Sytox Green; E and F, Hoechst 33342. A, B at 10X and C-F at 40X objectives at the fluorescence microscope. A and C, phase contrast fields corresponding to B and D, respectively. F, florescence; E, merges phase contrast with fluorescence. Arrow tips, E. histolytica trophozoites. Bottom: after amoeba-NET interaction, trophozoites were growth in TYI-S-33 culture medium during 72 h and cells harvested were counted under a light microscope. Controls, trophozoites cultured at the same conditions in the absence of NETs. Amoeba growth after interaction with NETs and EGTA or EGTA + E-64 was significantly different from controls without NETs (p <0.001). No other significant difference (p <0.05) was observed compared to the control without NETs in the absence of other compounds.
Fig 4.
Induction of neutrophil extracellular traps by different concentrations of lipopeptidophosphoglycan from E. histolytica.
Human neutrophils were incubated for 2 h at 37°C with 0 to 15 ng/μL of purified EhLPPG. A, C, E and G, phase contrast fields of B, D, F, and H, respectively. Sytox Green observed at 40X objective in the fluorescence microscope.
Fig 5.
Kinetics of NET induction by lipopeptidophosphoglycan isolated from E. histolytica.
Induction of neutrophil extracellular traps by 10 ng/μL of EhLPPG during the times indicated. Control, neutrophils incubated at the same conditions for 2 h in the absence of EhLPPG. Hoechst 33342 stain and confocal microscope observation, bars 10 μm.
Fig 6.
Localization of LL-37 in NET induced by LPPG from E. histolytica.
Neutrophils or NET induced by EhLPPG were stained by an anti-LL-37 antibody and an anti-rabbit IgG conjugated to Alexa594 (red). Control PMN, polymorphonuclear leucocytes with only the secondary antibody. PMN, PMN stained with anti-LL-37 and the anti-rabbit IgG conjugated to Alexa594. NET, PMN incubated with LPPG showing the release of NET and colocalization with LL-37. Hoechst 33342 stain and confocal microscope observation, bars 10 μm.
Fig 7.
Development of amoebic liver abscesses (ALA) on hamsters by amoebas pre-treated with NETs.
Virulent E. histolytica trophozoited (1x106) were incubated with 500 μg DNA NET solution (PMA-preformed) or PBS for 1 h at 37°C, and then injected in the portal vein of hamsters. Untreated trophozoites were used as control of infectivity. Seven days post-challenge, animals were sacrificed, livers escinded and analyzed. A) Table showing the infection rate and weight average of infected livers. B) Upper: Distribution and magnitud of ALA on the liver of the 5 animals of each group. Uninfected livers of groups PBS and NETs are inside ovals. Bottom: Representative histological analysis of ALA from each group stained with PAS. Magnification 20X. Arrows: trophozoites; N: necrosis; Ii: Inflammatory infiltrate, mainly neutrophils.