RESUMO
Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (<30 min) in both B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis.
Assuntos
Proteínas Quinases Ativadas por AMP , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Armadilhas Extracelulares , Neutrófilos , Sarcocystidae , Transdução de Sinais , Animais , Bovinos , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Armadilhas Extracelulares/metabolismo , Sarcocystidae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Coccidiose/parasitologia , Coccidiose/veterinária , Coccidiose/imunologia , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/imunologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
M onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2-3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.
Assuntos
Doenças dos Bovinos , Coccidiose , Eimeria , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/parasitologia , Coccidiose/veterinária , Microscopia , Oocistos , EsporozoítosRESUMO
Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced ß-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.
Assuntos
Envelhecimento/fisiologia , Doenças dos Bovinos/parasitologia , Coccidiose/veterinária , Células Endoteliais/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Animais , Bovinos , Coccidiose/parasitologia , Eimeria/fisiologia , Masculino , FenótipoRESUMO
Besnoitia 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.
Assuntos
Autofagia , Doenças dos Bovinos/imunologia , Coccidiose/veterinária , Neutrófilos/imunologia , Sarcocystidae/fisiologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/imunologia , Coccidiose/parasitologia , Feminino , FrançaRESUMO
Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24-30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that-in terms of host cell cycle modulation-infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti-infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii-related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti-infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.
Assuntos
Doenças dos Bovinos/fisiopatologia , Coccidiose/veterinária , Pontos de Checagem da Fase G2 do Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Sarcocystidae/fisiologia , Animais , Apoptose , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/parasitologia , Coccidiose/fisiopatologia , Células Endoteliais/citologia , Células Endoteliais/parasitologiaRESUMO
Toxoplasma gondii is an obligate intracellular parasite that modulates a broad range of host cell functions to guarantee its intracellular development and replication. T. gondii includes three classical clonal lineages exhibiting different degrees of virulence. Regarding the genetic diversity of T. gondii circulating in Europe, type II strains and, to a lesser extent, type III strains are the dominant populations, both in humans and animals. Infections with the type I strain led to widespread parasite dissemination and death in mice, while type III is considered avirulent. Previously, we demonstrated that primary endothelial cells infected with the T. gondii RH strain (haplotype I) were arrested in the G2/M-phase transition, triggering cytokinesis failure and chromosome missegregation. Since T. gondii haplotypes differ in their virulence, we here studied whether T. gondii-driven host cell cycle perturbation is strain-dependent. Primary endothelial cells were infected with T. gondii Me49 (type II strain) or NED (type III strain), and their growth kinetics were compared up to cell lysis (6-30 h p. i.). In this study, only slight differences in the onset of full proliferation were observed, and developmental data in principle matched those of the RH strain. FACS-based DNA quantification to estimate cell proportions experiencing different cell cycle phases (G0/1-, S-, and G2/M-phase) revealed that Me49 and NED strains both arrested the host cell cycle in the S-phase. Cyclins A2 and B1 as key molecules of S- and M-phase were not changed by Me49 infection, while NED infection induced cyclin B1 upregulation. To analyze parasite-driven alterations during mitosis, we demonstrated that both Me49 and NED infections led to impaired host cellular chromosome segregation and irregular centriole overduplication. Moreover, in line with the RH strain, both strains boosted the proportion of binucleated cells within infected endothelial cell layers, thereby indicating enhanced cytokinesis failure. Taken together, we demonstrate that all parasite-driven host cell cycle arrest, chromosome missegregation, and binucleated phenotypes are T. gondii-specific but strain independent.
RESUMO
BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.
Assuntos
Toxoplasma , Toxoplasmose , Humanos , Animais , Bovinos , Toxoplasma/fisiologia , Citocinese , Ciclina B1/genética , Ciclina B1/metabolismo , Segregação de Cromossomos , Toxoplasmose/parasitologiaRESUMO
Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.
Assuntos
Toxoplasma , Toxoplasmose , Humanos , Animais , Toxoplasmose/parasitologia , DNA , Dano ao DNA , Instabilidade Cromossômica , Recombinação Homóloga , Proteínas Mutadas de Ataxia Telangiectasia/genéticaRESUMO
Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on Toxoplasma gondii-triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in T. gondii-induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during T. gondii-induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable T. gondii tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.
Assuntos
Armadilhas Extracelulares , Toxoplasma , Humanos , Bovinos , Animais , Proteínas de Ciclo Celular , Neutrófilos , Ciclo Celular , Receptores Proteína Tirosina Quinases , Acetato de TetradecanoilforbolRESUMO
Introduction: Infections with the apicomplexan obligate intracellular parasite Cryptosporidium parvum lead to cryptosporidiosis-a worldwide zoonotic infection. C. parvum is one of the most common diarrheal pathogens in young calves, which are the main reservoir of the pathogen. Cryptosporidiosis leads to severe economic losses in the calf industry and being a major contributor to diarrhea morbidity and mortality in children. Polymorphonuclear neutrophils (PMN) are part of the innate immune system. Their effector mechanisms directed against invasive parasites include phagocytosis, production of antimicrobial molecules as well as the formation of so-called neutrophil extracellular traps (NETs). Like other leukocytes of the innate immune system, PMN are thus able to release chromatin fibers enriched with antimicrobial granular molecules extracellularly thereby immobilizing and partially killing invasive bacteria, viruses, fungi and parasites. Methods: In vitro interactions of neonatal bovine PMN and C. parvum-oocysts and sporozoites were illustrated microscopically via scanning electron microscopy- and live cell imaging 3D holotomographic microscopy analyses. C. parvum-triggered NETosis was quantified via extracellular DNA measurements as well as verified via detection of NET-typical molecules [histones, neutrophil elastase (NE)] through immunofluorescence microscopy analysis. To verify the role of ATP in neonatal-derived NETosis, inhibition experiments were performed with NF449 (purinergic receptor antagonist with high specificity to P2X1 receptor). Results and discussion: Using immunofluorescence- and SEM-based analyses, we demonstrate here for the first time that neonate bovine PMN are capable of forming NETs against C. parvum-sporozoites and oocysts, thus as a stage-independent cell death process. Our data further showed that C. parvum strongly induces suicidal neonatal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite ensnarement and hampered sporozoite excystation, but also via direct exposure to NETs-associated toxic components.
RESUMO
ß Amyloid, present in senile plaques, has been related largely to neuronal loss in the brain of patients with Alzheimer's disease. However, how neurons respond to ß amyloid insults is still poorly understood. Here we show that ß amyloid increases somatostatin and cortistatin gene expression mainly through an increase in histone 3 lysine 4 methylation (H3K4me3), a modification associated with transcriptional activation. Somatostatin and cortistatin partially decreased ß amyloid toxicity in primary cortical neurons in culture. Thus we suggest that neurons respond to ß amyloid insults by releasing somatostatin and cortistatin, which will act as a protective agent against ß amyloid toxicity. Our results suggest a relevant function for both neuropeptides against ß amyloid toxicity, providing new insights into Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Epigenômica , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Somatostatina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Interações Medicamentosas , Embrião de Mamíferos , Quinase 3 da Glicogênio Sintase/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Fosforilação/efeitos dos fármacos , Serina/metabolismo , Somatostatina/genética , Somatostatina/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
Apicomplexan parasites are considered as defective in cholesterol synthesis. Consequently, they need to scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating host cellular de-novo biosynthesis. Given that Eimeria bovis macromeront formation in bovine lymphatic endothelial host cells in vivo is a highly cholesterol-demanding process, we here examined host parasite interactions based on host cellular uptake of different low-density lipoprotein (LDL) types, i.e., of non-modified (LDL), oxidized (oxLDL), and acetylated LDL (acLDL). Furthermore, the expression of lipoprotein-oxidized receptor 1 (LOX-1), which mediates acLDL and oxLDL internalization, was monitored throughout first merogony, in vitro and ex vivo. Moreover, the effects of inhibitors blocking exogenous sterol uptake or intracellular transport were studied during E. bovis macromeront formation in vitro. Hence, E. bovis-infected primary bovine umbilical vein endothelial cells (BUVEC) were treated with inhibitors of sterol uptake (ezetimibe, poly-C, poly-I, sucrose) and of intracellular sterol transport and release from endosomes (progesterone, U18666A). As a read-out system, the size and number of macromeronts as well as merozoite I production were estimated. Overall, the internalization of all LDL modifications (LDL, oxLDL, acLDL) was observed in E. bovis-infected BUVEC but to different extents. Supplementation with oxLDL and acLDL at lower concentrations (5 and 10 µg/ml, respectively) resulted in a slight increase of both macromeront numbers and size; however, at higher concentrations (25-50 µg/ml), merozoite I production was diminished. LOX-1 expression was enhanced in E. bovis-infected BUVEC, especially toward the end of merogony. As an interesting finding, ezetimibe treatments led to a highly significant blockage of macromeront development and merozoite I production confirming the relevance of sterol uptake for intracellular parasite development. Less prominent effects were induced by non-specific inhibition of LDL internalization via sucrose, poly-I, and poly-C. In addition, blockage of cholesterol transport via progesterone and U18666A treatments resulted in significant inhibition of parasite development. Overall, current data underline the relevance of exogenous sterol uptake and intracellular cholesterol transport for adequate E. bovis macromeront development, unfolding new perspectives for novel drug targets against E. bovis.
Assuntos
Doenças dos Bovinos , Eimeria , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Colesterol/metabolismo , Eimeria/metabolismo , Células Endoteliais/metabolismo , MerozoítosRESUMO
Neospora caninum represents a major cause of abortive disease in bovines and small ruminants worldwide. As a typical obligate intracellular apicomplexan parasite, N. caninum needs to modulate its host cell for successful replication. In the current study, we focused on parasite-driven interference with host cell cycle progression. By performing DNA content-based cell cycle phase analyses in N. caninum-infected primary bovine umbilical vein endothelial cells (BUVEC), a parasite-driven S-phase arrest was detected at both 24 and 32 h p. i., being paralleled by fewer host cells experiencing the G0/G1 cell cycle phase. When analyzing S-subphases, proliferation cell nuclear antigen (per PCNA)-based experiments showed a reduced population of BUVEC in the late S-phase. Analyses on key molecules of cell cycle regulation documented a significant alteration of cyclin A2 and cyclin B1 abundance in N. caninum-infected host endothelial cells, thereby confirming irregularities in the S-phase and S-to-G2/M-phase transition. In line with cell cycle alterations, general nuclear parameters revealed smaller nuclear sizes and morphological abnormalities of BUVEC nuclei within the N. caninum-infected host cell layer. The latter observations were also confirmed by transmission electron microscopy (TEM) and by analyses of lamin B1 as a marker of nuclear lamina, which illustrated an inhomogeneous nuclear lamin B1 distribution, nuclear foldings, and invaginations, thereby reflecting nuclear misshaping. Interestingly, the latter finding applied to both non-infected and infected host cells within parasitized BUVEC layer. Additionally, actin detection indicated alterations in the perinuclear actin cap formation since typical nucleo-transversal filaments were consistently lacking in N. caninum-infected BUVEC, as also documented by significantly decreased actin-related intensities in the perinuclear region. These data indicate that N. caninum indeed alters host cell cycle progression and severely affects the host cell nuclear phenotype in primary bovine endothelial host cells. In summary, these findings add novel data on the complex N. caninum-specific modulation of host cell and nucleus, thereby demonstrating clear differences in cell cycle progression modulation driven by other closely related apicomplexans like Toxoplasma gondii and Besnotia besnoiti.
RESUMO
[This corrects the article DOI: 10.3389/fcell.2022.946335.].
RESUMO
The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis-an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)-is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.
Assuntos
Doenças dos Bovinos , Coccidiose , Eimeria , Trifosfato de Adenosina , Animais , Bovinos , Glicólise , EsporozoítosRESUMO
Eimeria bovis is an intracellular apicomplexan parasite that causes considerable economic losses in the cattle industry worldwide. During the first merogony, E. bovis forms large macromeronts with >140,000 merozoites I in host endothelial cells. Because this is a high-energy demanding process, E. bovis exploits the host cellular metabolism to fulfill its metabolic requirements. We here analyzed the carbohydrate-related energetic metabolism of E. bovis-infected primary bovine umbilical vein endothelial cells during first merogony and showed that during the infection, E. bovis-infected culture presented considerable changes in metabolic signatures, glycolytic, and mitochondrial responses. Thus, an increase in both oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were found in E. bovis-infected host cells indicating a shift from quiescent to energetic cell status. Enhanced levels of glucose and pyruvate consumption in addition to increased lactate production, suggesting an important role of glycolysis in E. bovis-infected culture from 12 days p.i. onward. This was also tested by glycolytic inhibitors (2-DG) treatment, which reduced the macromeront development and diminished merozoite I production. As an interesting finding, we observed that 2-DG treatment boosted sporozoite egress. Referring to mitochondrial activities, intracellular ROS production was increased toward the end of merogony, and mitochondrial potential was enhanced from 12 d p. i. onward in E. bovis-infected culture. Besides, morphological alterations of membrane potential signals also indicated mitochondrial dysfunction in macromeront-carrying host endothelial culture.
Assuntos
Eimeria , Animais , Bovinos , Células Endoteliais/metabolismo , Glicólise , Merozoítos , MitocôndriasRESUMO
Coccidian parasites are obligate intracellular pathogens that affect humans and animals. Apicomplexans are defective in de novo synthesis of cholesterol, which is required for membrane biosynthesis and offspring formation. In consequence, cholesterol has to be scavenged from host cells. It is mainly taken up from extracellular sources via LDL particles; however, little is known on the role of HDL and its receptor SR-BI in this process. Here, we studied effects of the SR-BI-specific blocker BLT-1 on the development of different fast (Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti) and slow (Eimeria bovis and Eimeria arloingi) replicating coccidian species. Overall, development of all these parasites was significantly inhibited by BLT-1 treatment indicating a common SR-BI-related key mechanism in the replication process. However, SR-BI gene transcription was not affected by T. gondii, N. caninum and B. besnoiti infections. Interestingly, BLT-1 treatment of infective stages reduced invasive capacities of all fast replicating parasites paralleled by a sustained increase in cytoplasmic Ca++ levels. Moreover, BLT1-mediated blockage of SR-BI led to enhanced host cell lipid droplet abundance and neutral lipid content, thereby confirming the importance of this receptor in general lipid metabolism. Finally, the current data suggest a conserved role of SR-BI for successful coccidian infections.
RESUMO
Due to its localization in the canine blood stream, Angiostrongylus vasorum is exposed to circulating polymorphonuclear neutrophils (PMN) and the endothelial cells of vessels. NETs release of canine PMN exposed to A. vasorum infective stages (third stage larvae, L3) and early pro-inflammatory immune reactions of primary canine aortic endothelial cells (CAEC) stimulated with A. vasorum L3-derived soluble antigens (AvAg) were analyzed. Expression profiles of the pro-inflammatory adhesion molecules ICAM-1, VCAM-1, P-selectin and E-selectin were analyzed in AvAg-stimulated CAEC. Immunofluorescence analyses demonstrated that motile A. vasorum L3 triggered different NETs phenotypes, with spread NETs (sprNETs) as the most abundant. Scanning electron microscopy confirmed that the co-culture of canine PMN with A. vasorum L3 resulted in significant larval entanglement. Distinct inter-donor variations of P-selectin, E-selectin, ICAM-1 and VCAM-1 gene transcription and protein expression were observed in CAEC isolates which might contribute to the high individual variability of pathological findings in severe canine angiostrongylosis. Even though canine NETs did not result in larval killing, the entanglement of L3 might facilitate further leukocyte attraction to their surface. Since NETs have already been documented as involved in both thrombosis and endothelium damage events, we speculate that A. vasorum-triggered NETs might play a critical role in disease outcome in vivo.
RESUMO
Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20â¯h with sterile 0.02â¯M L-cysteine HCl/0.2â¯M NaHCO3 solution at 37⯰C in 100% CO2 atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2â¯h of incubation (37⯰C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.
Assuntos
Eimeria/fisiologia , Parasitologia/métodos , Animais , Bovinos , Células Endoteliais/parasitologia , Microscopia/métodos , Microscopia/veterinária , Esporozoítos/fisiologia , Veias Umbilicais/parasitologiaRESUMO
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, trypomastigote-induced 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 trypanosomiasis-related immunopathological disorders, such as increased intravascular coagulopathy and vascular permeability, often reported to occur in this disease.