Percorrer por autor "Hermosilla, Carlos"
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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.
- Besnoitia besnoiti tachyzoite replication in bovine primary endothelial cells relies on host Niemann–Pick type C protein 1 for cholesterol acquisitionPublication . Larrazabal, Camilo; Hermosilla, Carlos; Taubert, Anja; Silva, Liliana M. R.Besnoitia besnoiti is a cyst-forming apicomplexan parasite and the causal agent of bovine besnoitiosis. During early phase of infection, tachyzoites replicate within host endothelial cells in a host cell cholesterol-dependent process. By applying U18666A treatments, we here evaluated the role of Niemann–Pick type C protein 1 (NPC1) in both, intracellular B. besnoiti replication and host cellular cholesterol distribution. Additionally, B. besnoiti-driven changes in NPC1 gene transcription were studied by qPCR. Overall, U18666A treatments significantly reduced B. besnoiti proliferation and induced cholesterol accumulation in host cytoplasmic dense vesicles. However, NPC1 gene transcription was not affected by B. besnoiti infection.
- Fasciola hepatica soluble antigens (FhAg) induce ovine PMN innate immune reactions and NET formation in vitro and in vivoPublication . Muñoz-Caro, Tamara; Gómez-Ceruti, Marcela; Silva, Liliana M. R.; Gutiérrez-Expósito, Daniel; Wagner, Henrik; Taubert, Anja; Hermosilla, CarlosFasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.
- Interaction of chicken heterophils and Eimeria tenella results in different phenotypes of heterophil extracellular traps (HETs)Publication . Rentería-Solís, Zaida; Silva, Liliana M. R.; Grochow, Thomas; Zhang, Runhui; Nguyen-Ho-Bao, Tran; Daugschies, Arwid; Taubert, Anja; Conejeros, Iván; Hermosilla, CarlosChicken coccidiosis causes annual losses exceeding GBP 10 billion globally. The most pathogenic species for domestic fowls including Eimeria tenella, E. acervulina, and E. maxima, can lead to gastrointestinal issues ranging from mild to fatal. In this study, stages of E. tenella and freshly isolated chicken heterophils were co-cultured for 180 min. These interactions were analyzed using live 3D holotomographic and confocal microscopy. We observed that E. tenella stages were entrapped by heterophils and heterophil extracellular traps (HETs). Notably, different HET phenotypes, specifically sprHETs and aggHETs, were induced regardless of the stage. Furthermore, the quantification of extracellular DNA release from co-cultures of heterophils and sporozoites (ratio 1:1) for 180 min demonstrated a significantly higher release (p = 0.04) compared to negative controls. In conclusion, research on the chicken innate immune system, particularly fowl-derived HETs, remains limited. More detailed investigations are needed, such as exploring the time-dependent triggering of HETs, to establish a standard incubation time for this pathogen defense mechanism. This will enhance our understanding of its role in parasite survival or death during HET confrontation.