Percorrer por autor "Zhou, Ershun"
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- Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosisPublication . Zhou, Ershun; Silva, Liliana M. R.; Conejeros, Iván; Velásquez, Zahady D.; Hirz, Manuela; Gärtner, Ulrich; Jacquiet, Philippe; Taubert, Anja; Hermosilla, CarlosBesnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.
- Cellular immune responses of bovine polymorphonuclear neutrophils to Calicophoron daubneyiPublication . Silva, Liliana M. R.; López-Osorio, Sara; Peixoto, Raquel; Zhou, Ershun; Espinosa, Gabriel; Gärtner, Ulrich; Taubert, Anja; Conejeros, Iván; Hermosilla, CarlosCalicophoron daubneyi infections have increased in Europe, being more frequent than fasciolosis in some areas. Infection occurs once definitive hosts ingest encysted metacercariae present on vegetation. Following excystation, juvenile flukes penetrate the small intestinal mucosa and migrate into the rumen where adults mature. Throughout the somatic migration, juveniles come across different microenvironments and tissues and encounter host leukocytes. Besides phagocytosis, production of reactive oxygen species (ROS) and degranulation, polymorphonuclear neutrophils also cast neutrophil extracellular traps (NETs), which can entrap several parasite species, including the closely related liver fluke Fasciola hepatica. In this study, we analyzed whether in vitro exposure of bovine neutrophils to C. daubneyi antigen (CdAg) and eggs triggered neutrophils activation and NET formation. Results on scanning electron microscopy (SEM) and immunofluorescence analyses show weak formation of short spread NETs upon CdAg stimulation, corroborated by increased extracellular DNA measurements. Likewise, early NETosis was confirmed via nuclear area expansion assays. Bovine neutrophil stimulation with CdAg 100 µg/mL concentration led to a significant increase in oxygen consumption rates (p = 0.0152) and extracellular acidification rates (p = 0.0022), while lower concentrations of CdAg (10 µg/mL) failed to induce neutrophil activation, suggesting a dose dependent response. Both intra- and extracellular ROS production was not affected by any CdAg concentration here studied. Bovine neutrophil total adenosine triphosphate concentration significantly decreased after exposure to CdAg 100 µg/mL, in line to the observed with the positive control (phorbol myristate acetate/ionomycin). In summary, C. daubneyi activates bovine neutrophils with rather weak responses, which might suggest that the release of C. daubneyi-specific molecules (i.e. excretory-secretory antigens, proteases, or nucleases) could interfere with neutrophil-related effector mechanisms. Further ex vivo analyses will clarify if such mechanisms are also involved in pathogenesis of paramphistomosis by demonstrating neutrophil recruitment into affected intestinal mucosa.