NET formation as innate effector mechanism against Trypanosoma brucei brucei stages and new insights on activated PMN by metazoan parasites

Abstract

Polymorphonuclear neutrophils (PMN) have been described as the most abundant leukocyte in lymph and bloodstream, being rapidly recruited from circulation to sites of infection to fight against foreign pathogens. PMN reacts against protozoan and metazoan parasites by different effector mechanisms, including the release of immunomodulatory molecules (i. e. cytokines and chemokines), phagocytosis, production of reactive oxygen species (ROS), and release of neutrophil extracellular traps (NETs). NETs have been described as a delicate extracellular structure formed by decondensed chromatin and adorned with antimicrobial components, such as myeloperoxidase (MPO), neutrophil elastase (NE), lactoferrin, calprotein, LL37, pentraxin, proteinase 3 or cathepsin G. In this sense, several protozoan and metazoan parasite species have been described as inducers of NETs, including Besnoitia besnoiti, Neospora caninum and Dirofilaria immitis, among others. A close interaction between members of these groups were here studied, specifically in the case of the bloodstream parasites Trypanosoma brucei brucei and Angiostrongylus vasorum. In this sense, the main findings in the current work are: Co-culture of bovine PMN with T. b. brucei stages, i. e. procyclic trypomastigotes and bloodstream metacyclic trypomastigotes, resulted in strong NETs release and PMN activation, independent of the time-point and parasitic stage. A fast and sustained increase in time of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) was achieved, when live T. b. brucei stages were confronted with bovine PMN, suggesting that ROS production might be caused as a response against the parasite. NET structures where visualized by SEM pictures, laser scanning confocal microscopy and live cell 3D-holotomographic microscopy analyzes, unveiling the co-localization of DNA with antimicrobial molecules present in PMN nucleus and granules, such as histones (H1, H2A/H2B, H3, H4) and neutrophil elastase, confirming the classical components of these extracellular defense structures. Moreover, morphological changes in expansion of nuclear area from bovine PMN were observed, as a consequence of chromatin decondensation. Analysis of fluorescence microscopy-derived images was here performed, applying a semi-automatic quantification of NETs formation called ‘DNA area and NETosis analysis’ (DANA). Assessment of different phenotypes of NETs was performed, proving that the formation of aggNETs was the most bundant phenotype present, when bovine PMN where stimulated with T. b. brucei procyclic trypomastigotes and metacyclic trypomastigotes, independent of the timepoint and PMN : parasite ratio. Regarding the participation of purinergic receptors in activation of bovine PMN and aggNETs formation, the purinergic inhibitors NF449 (antagonist of P2X1 receptor) and MRS2578 (antagonist of P2Y6 receptor) were tested. In this context, NF449 at 100 μM and 10 μM unveiled the inhibitory effect, proving a specific role for the P2X1 in T. b. brucei-mediated NETosis. Thus, the effect of MRS2578 at 10 μM was confirmed, verifying a role of the P2Y6 in this process. These results were achieved by confirming the inhibition of aggNETs formation with images analyzes and measurements of OCR in bovine PMN. Canine PMN were stimulated with vital A. vasorum L3, observing changes in the NAE of the cells and NETs formation, being sprNETs the most abundant phenotype. Interestingly, after stimulation with A. vasorum soluble antigen (AvAg), activation of canine aortic endothelial cells (CAEC) was confirmed, by measurements of classical adhesion molecules, including Pselectin, E-selectin, VCAM-1 and ICAM-1. In this line, host endothelial cell damage triggered by B. besnoiti tachyzoites isolated NETs and H2A was demonstrated, confirming NETs-derived adverse effects against BUVEC-monolayers infected with B. besnoiti, under physiological flow conditions. Remarkably, experiments with colostrum PMN from bitches were here performed, observing that canine colostral PMN are able to cast NET against vital and dead N. caninum tachyzoites. Even so, the ability of colostral PMN to perform its phagocytic function in neonates was also here achieved. Lastly, experiments of bovine PMN against S. scabiei stages (adults, nymphs, larvae and eggs) and soluble S. scabiei antigen ScAg) were performed, proving a slight attachment of PMN to the exoskeleton of S. scabiei stages. However, after stimulation of bovine PMN with 10 μM ScAg, a weak NETs production was achieved. Notably, phagocytosis, ROS production and Ca++ fluxes were observed, after stimulation with 10 μM ScAg. Overall, presented data evidence that not all parasites can induce NETosis and therefore demanding future work on modulation or evasion strategies of this innate effector process by parasites.