Trypanosoma brucei brucei Induces Polymorphonuclear Neutrophil Activation and Neutrophil Extracellular Traps Release

Abstract

Trypanosoma brucei brucei trypomastigotes are classical blood parasites of cattle, these stages might become potential targets for circulating polymorphonuclear neutrophils (PMN). We here investigated NETs extrusion and related oxygen consumption in bovine PMN exposed to motile T. b. brucei trypomastigotes in vitro. Parasite exposure induced PMN activation as detected by enhanced oxygen consumption rates (OCR), extracellular acidification rates (ECAR), and production of total and extracellular reactive oxygen species (ROS). Scanning electron microscopy (SEM) showed that co-cultivation of bovine PMN with motile trypomastigotes resulted in NETs formation within 120 min of exposure. T. b. brucei-induced NETs were confirmed by confocal microscopy demonstrating co-localization of extruded DNA with neutrophil elastase (NE) and nuclear histones. Immunofluorescence analyses demonstrated that trypomastigotes induced different phenotypes of NETs in bovine PMN, such as aggregated NETs (aggNETs), spread NETs (sprNETs), and diffuse NETs (diffNETs) with aggNETs being the most abundant ones. Furthermore, live cell 3D-holotomographic microscopy unveiled detailed morphological changes during the NETotic process. Quantification of T. b. brucei-induced NETs formation was estimated by DNA and nuclear area analysis (DANA) and confirmed enhanced NETs formation in response to trypomastigote stages. Formation of NETs does not result in a decrease of T. b. brucei viability, but a decrease of 26% in the number of motile parasites. Referring the involved signaling pathways, trypomastigoteinduced NETs formation seems to be purinergic-dependent, since inhibition via NF449 treatment resulted in a significant reduction of T. b. brucei-triggered DNA extrusion. Overall, future studies will have to analyze whether the formation of aggNETs indeed plays a role in the outcome of clinical disease and bovine African trypanosomiasisrelated immunopathological disorders, such as increased intravascular coagulopathy and vascular permeability, often reported to occur in this disease.

Canine Angiostrongylus vasorum-Induced Early Innate Immune Reactions Based on NETs Formation and Canine Vascular Endothelial Cell Activation In Vitro

“…Consistently, typical NET-associated components were here confirmed for A. vasorum L3-induced NETs by demonstrating co-localization of NE and histones on DNA-rich extracellular fibres being released from canine PMN. In line with other reports on parasites triggering NETosis [ 8 , 27 , 30 , 58 ], the induction of different NET phenotypes was here observed. In principle, all types ( spr NETs, diff NETs, agg NETs) were detected upon contact of canine PMN with A. vasorum L3; however, the most abundant were spr NETs, which is consistent with findings on D. immitis -triggered NETosis [ 30 ].…”

“…Despite these efforts of escape, PMN successfully captured several larvae as visualized by SEM; nevertheless, future studies are required to confirm the importance of this observation in vivo. Current observations mainly indicated suicidal NETs formation, which corresponds well to previous NET-related reports on motile protozoan and metazoan parasites [ 8 , 27 , 30 , 38 , 62 ].…”

“…In the current study, an increase in NAE in A. vasorum L3-confronted canine PMN confirmed the parasite-triggered induction of NETotic cells and these data corresponded well to observer-based microscopic observations of NETs. Worth noting, DANA has already been successfully performed before in human, murine and bovine systems [ 25 , 27 , 63 , 64 ], with the current report representing the first application for canine PMN.…”

“…NET phenotypes were quantified by immunofluorescence microscopy as previously described elsewhere [ 27 ]. Therefore, five randomly taken pictures were analyzed by manual counting, based on morphological and morphometric characteristics for each phenotype of canine NETs, as previously described [ 8 , 30 ].…”

“…NETs are delicate extracellular structures formed by decondensed chromatin, mainly via PAD4-mediated citrullination, and adorned with antimicrobial components, such as myeloperoxidase (MPO), neutrophil elastase (NE), lactoferrin, calprotein, LL37, pentraxin, proteinase 3 or cathepsin G [ 21 , 24 , 25 ]. Meanwhile, different NET phenotypes were reported, including diffuse ( diff NETs), spread ( spr NETs), aggregated ( agg NETs), cell free and anchored NETs [ 26 , 27 , 28 ]. diff NETs consist of globular and compact forms with sizes of 15–20 nm diameter whilst spr NETs are smooth and elongated web-like structures with extremely thin fibers of 15–17 nm diameter [ 29 , 30 ].…”