RESUMEN
Intracellular pathogens like Toxoplasma gondii often target proteins and pathways critical for host cell survival and stress response. Molecular chaperones encoded by the evolutionary conserved Heat shock proteins (Hsps) maintain proteostasis and are vital to cell survival following exposure to any form of stress. A key protein of this family is Hsp70, an ATP-driven molecular chaperone, which is stress inducible and often indiscernible in normal cells. Role of this protein with respect to intracellular survival and multiplication of protozoan parasite like T. gondii remains to be examined. We find that T. gondii infection upregulates expression of host Hsp70. Hsp70 selective inhibitor 2-phenylethynesulfonamide (PES) attenuates intracellular T. gondii multiplication. Biotinylated PES confirms selective interaction of this small molecule inhibitor with Hsp70. We show that PES acts by disrupting Hsp70 chaperone function which leads to dysregulation of host autophagy. Silencing of host Hsp70 underscores its importance for intracellular multiplication of T. gondii, however, attenuation achieved using PES is not completely attributable to host Hsp70 indicating the presence of other intracellular targets of PES in infected host cells. We find that PES is also able to target T. gondii Hsp70 homologue which was shown using PES binding assay. Detailed molecular docking analysis substantiates PES targeting of TgHsp70 in addition to host Hsp70. While establishing the importance of protein quality control in infection, this study brings to the fore a unique opportunity of dual targeting of host and parasite Hsp70 demonstrating how structural conservation of these proteins may be exploited for therapeutic design.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Espacio Intracelular/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular , Células Endoteliales/parasitología , Proteínas HSP70 de Choque Térmico/genética , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Ratones , Microglía/parasitología , Simulación del Acoplamiento Molecular , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/parasitología , Sulfonamidas/farmacología , Toxoplasmosis/parasitología , TransfecciónRESUMEN
PURPOSE: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity. MATERIALS AND METHODS: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice. RESULTS: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed. CONCLUSION: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.
Asunto(s)
Apoptosis/efectos de los fármacos , Nanopartículas del Metal/química , NADPH Oxidasa 4/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/parasitología , Plata/farmacología , Toxoplasmosis/patología , Animales , Autofagia/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Fase G1/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Fosforilación/efectos de los fármacosAsunto(s)
Infecciones Parasitarias del Ojo/diagnóstico , Miasis/diagnóstico , Enfermedades de la Retina/diagnóstico , Epitelio Pigmentado de la Retina/parasitología , Trastornos de la Visión/diagnóstico , Infecciones Parasitarias del Ojo/parasitología , Infecciones Parasitarias del Ojo/fisiopatología , Femenino , Angiografía con Fluoresceína , Humanos , Persona de Mediana Edad , Miasis/parasitología , Miasis/fisiopatología , Miopía Degenerativa , Enfermedades de la Retina/parasitología , Enfermedades de la Retina/fisiopatología , Epitelio Pigmentado de la Retina/fisiopatología , Tomografía de Coherencia Óptica , Trastornos de la Visión/parasitología , Trastornos de la Visión/fisiopatología , Agudeza Visual/fisiología , Vitrectomía , Hemorragia Vítrea/cirugíaRESUMEN
Purpose: Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Methods: Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Results: Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Conclusions: Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.
Asunto(s)
Neutrófilos/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Toxoplasmosis Ocular/inmunología , Adulto , Línea Celular , Ensayos de Migración de Leucocitos , Movimiento Celular/fisiología , Quimiocinas/metabolismo , Técnicas de Cocultivo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Activación Neutrófila/fisiología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/parasitología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Toxoplasma/fisiologíaRESUMEN
Omega-3 polyunsaturated fatty acids (ω3-PUFAs) have potential protective activity in a variety of infectious diseases, but their actions and underlying mechanisms in Toxoplasma gondii infection remain poorly understood. Here, we report that docosahexaenoic acid (DHA) robustly induced autophagy in murine bone marrow-derived macrophages (BMDMs). Treatment of T. gondii-infected macrophages with DHA resulted in colocalization of Toxoplasma parasitophorous vacuoles with autophagosomes and reduced intracellular survival of T. gondii. The autophagic and anti-Toxoplasma effects induced by DHA were mediated by AMP-activated protein kinase (AMPK) signaling. Importantly, BMDMs isolated from Fat-1 transgenic mice, a well-known animal model capable of synthesizing ω3-PUFAs from ω6-PUFAs, showed increased activation of autophagy and AMPK, leading to reduced intracellular survival of T. gondii when compared with wild-type BMDMs. Moreover, Fat-1 transgenic mice exhibited lower cyst burden in the brain following infection with the avirulent strain ME49 than wild-type mice. Collectively, our results revealed mechanisms by which endogenous ω3-PUFAs and DHA control T. gondii infection and suggest that ω3-PUFAs might serve as therapeutic candidate to prevent toxoplasmosis and infection with other intracellular protozoan parasites.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Antiparasitarios/farmacología , Autofagia/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Macrófagos/efectos de los fármacos , Toxoplasma/efectos de los fármacos , Toxoplasmosis Animal/prevención & control , Toxoplasmosis Cerebral/prevención & control , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/parasitología , Encéfalo/patología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Activación Enzimática , Humanos , Macrófagos/enzimología , Macrófagos/parasitología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/parasitología , Transducción de Señal , Toxoplasma/patogenicidad , Toxoplasmosis Animal/enzimología , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/patología , Toxoplasmosis Cerebral/enzimología , Toxoplasmosis Cerebral/parasitología , Toxoplasmosis Cerebral/patologíaRESUMEN
Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.
Asunto(s)
Antiprotozoarios/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/parasitología , Factores Inmunológicos/farmacología , Compuestos Onio/farmacología , Especies Reactivas de Oxígeno/metabolismo , Toxoplasma/crecimiento & desarrollo , Antiprotozoarios/toxicidad , Línea Celular , Células Epiteliales/fisiología , Humanos , Factores Inmunológicos/toxicidad , Compuestos Onio/toxicidad , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/parasitología , Toxoplasma/efectos de los fármacosRESUMEN
Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome-with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)-but very limited changes in the small RNA transcriptome-totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.
Asunto(s)
Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/parasitología , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Toxoplasmosis Ocular/genética , Toxoplasmosis Ocular/inmunología , Anciano , Cadáver , Técnicas de Cultivo de Célula , Línea Celular , Separación Celular , Perfilación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Humanos , Fenómenos Inmunogenéticos , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , RNA-Seq , Epitelio Pigmentado de la Retina/citología , Toxoplasmosis Ocular/parasitologíaRESUMEN
Diffuse unilateral subacute neuroretinitis (DUSN) is caused by a subretinal live and mobile nematode. Acute phase: Patients usually present with severe pain, decreased vision, vitritis/papillitis, and tracks of grayish-white lesions-and a live nematode. Late phase: Arterial narrowing, optic atrophy, diffuse disruption of the retinal pigment epithelium (RPE), with severe visual loss.
Asunto(s)
Infecciones Parasitarias del Ojo/fisiopatología , Infecciones por Nematodos/fisiopatología , Retinitis/fisiopatología , Humanos , Epitelio Pigmentado de la Retina/parasitología , Retinitis/parasitologíaRESUMEN
Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2ß, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.
Asunto(s)
Expresión Génica , Interacciones Huésped-Parásitos/genética , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina/parasitología , Transducción de Señal , Serina-Treonina Quinasas TOR , Toxoplasma/patogenicidad , Toxoplasmosis/genética , Toxoplasmosis/parasitología , Animales , Células Cultivadas , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Like most intracellular pathogens, the apicomplexan parasites Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum scavenge metabolites from their host cells. Recruitment of the Golgi complex to the vicinity of the parasitophorous vacuole (PV) is likely to aid in this process. In this work, we comparatively assessed B. besnoiti, T. gondii and N. caninum infected human retinal pigmented epithelial (hTERT-RPE-1) cells at 24â¯h post-infection and used antibodies to confirm Golgi ribbon compaction in B. besnoiti, and Golgi ribbon dispersion in T. gondii, while no alteration in Golgi morphology was seen in N. caninum infected cells. In either case, the Golgi stacks of infected cells contained both cis- (GM130) and trans- (TGN46) Golgi proteins. The localization of Rab9A, an important regulator of endosomal trafficking, was also studied. GFP-tagged Rab9A was recruited to the vicinity of the PV of all three parasites. Toxoplasma-infected cells exhibited increased expression of Rab9A in comparison to non-infected cells. However, Rab9A expression levels remained unaltered upon infection with N. caninum and B. besnoiti tachyzoites. In contrast to Rab9A, a GFP-tagged dominant negative mutant form of Rab9A (Rab9A DN), was not recruited to the PV, and the expression of Rab9A DN did not affect host cell invasion nor replication by all three parasites. Thus, B. besnoiti, T. gondii and N. caninum show similarities but also differences in how they affect constituents of the endosomal/secretory pathways.
Asunto(s)
Coccidiosis/metabolismo , Aparato de Golgi/metabolismo , Neospora , Toxoplasmosis/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Autoantígenos/inmunología , Western Blotting , Línea Celular , Coccidiosis/enzimología , Endosomas/parasitología , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/inmunología , Aparato de Golgi/ultraestructura , Humanos , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía de Interferencia , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/parasitología , Toxoplasmosis/enzimología , Red trans-Golgi/inmunología , Red trans-Golgi/metabolismo , Red trans-Golgi/ultraestructuraRESUMEN
Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.
Asunto(s)
Barrera Hematorretinal , Quinasa 1 de Adhesión Focal/inmunología , Monocitos , Comunicación Paracrina/inmunología , Transducción de Señal/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Barrera Hematorretinal/inmunología , Barrera Hematorretinal/parasitología , Línea Celular , Humanos , Interleucina-8/inmunología , Monocitos/inmunología , Monocitos/parasitología , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/parasitología , Proteína de la Zonula Occludens-1/inmunologíaRESUMEN
Posterior ophthalmomyiasis interna is a rare, potentially devastating infestation of the posterior segment by fly larvae. The authors report the first demonstration of spectral-domain optical coherence tomography (SD-OCT) (Spectralis; Heidelberg Engineering, Heidelberg, Germany), wide-field angiography (Optos, Dunfermline, Scotland) and photography, and fundus autofluorescence with temporal progression during a period of 6 months. A 12-year-old white female presented with acute, painless vision loss with hand motions visual acuity. No larva was visible, so she was treated with oral ivermectin. Visual acuity improved to 20/80. OCT demonstrated hyporeflective spaces of the outer retina and retinal pigment epithelium, which resolved during 1-month period with improved ellipsoid layer by 6 months. Fundus autofluorescence demonstrated linear hypoautofluorescent tracks. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:682-685.].
Asunto(s)
Infecciones Parasitarias del Ojo/diagnóstico , Miasis/diagnóstico , Segmento Posterior del Ojo/patología , Epitelio Pigmentado de la Retina/patología , Animales , Niño , Infecciones Parasitarias del Ojo/parasitología , Femenino , Angiografía con Fluoresceína , Fondo de Ojo , Humanos , Larva , Fotograbar , Segmento Posterior del Ojo/parasitología , Epitelio Pigmentado de la Retina/parasitología , Tomografía de Coherencia Óptica/métodos , Agudeza VisualRESUMEN
Ocular toxoplasmosis is the most frequent cause of uveitis, leading to partial or total loss of vision, with the retina the main affected structure. The cells of the retinal pigment epithelium (RPE) play an important role in the physiology of the retina and formation of the blood-retinal barrier. Several pathogens induce barrier dysfunction by altering tight junction (TJ) integrity. Here, we analysed the effect of infection by Toxoplasma gondii on TJ integrity in ARPE-19 cells. Loss of TJ integrity was demonstrated in T. gondii-infected ARPE-19 cells, causing increase in paracellular permeability and disturbance of the barrier function of the RPE. Confocal microscopy also revealed alteration in the TJ protein occludin induced by T. gondii infection. Disruption of junctional complex was also evidenced by scanning and transmission electron microscopy. Cell-cell contact loss was noticed in the early stages of infection by T. gondii with the visualization of small to moderate intercellular spaces. Large gaps were mostly observed with the progression of the infection. Thus, our data suggest that the alterations induced by T. gondii in the structural organization of the RPE may contribute to retinal injury evidenced by ocular toxoplasmosis.
Asunto(s)
Barrera Hematorretinal/fisiología , Epitelio Pigmentado de la Retina/parasitología , Uniones Estrechas/fisiología , Toxoplasma/fisiología , Toxoplasmosis Ocular/fisiopatología , Animales , Barrera Hematorretinal/ultraestructura , Células Cultivadas , Impedancia Eléctrica , Femenino , Humanos , Ratones , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Epitelio Pigmentado de la Retina/fisiopatología , Epitelio Pigmentado de la Retina/ultraestructura , Uniones Estrechas/ultraestructura , Toxoplasma/ultraestructura , Toxoplasmosis Ocular/patologíaRESUMEN
This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of l-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting l-Phe(D8) from host cells as soon as it invades the cell. l-Phe(D8) from the host cell completely replaces the l-Phe within T. gondii tachyzoites 7-9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5-1.6 × 10(4) molecules/s. On the other hand, extracellular tachyzoites were not able to consume l-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Marcaje Isotópico , Fenilalanina/metabolismo , Epitelio Pigmentado de la Retina , Toxoplasma/fisiología , Toxoplasmosis/metabolismo , Línea Celular , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/parasitología , Espectrometría RamanRESUMEN
A 52-yr-old male was referred for progressive visual loss in the left eye. The decimal best-corrected visual acuity (BCVA) was 0.01. Fundus examination revealed diffuse retinal pigment epithelial degeneration, focal yellow-white, infiltrative subretinal lesion with fuzzy border and a live nematode within the retina. Diffuse unilateral subacute neuroretinitis (DUSN) was diagnosed and the direct laser photocoagulation was performed to destroy the live nematode. During eight months after treatment, BCVA gradually improved to 0.2 along with the gradual restoration of outer retinal layers on SD-OCT. We report on the first case of DUSN in Korea. DUSN should be included in the differential diagnosis of unexplained unilateral visual loss in otherwise healthy subjects.
Asunto(s)
Infecciones Parasitarias del Ojo/terapia , Nematodos/patogenicidad , Epitelio Pigmentado de la Retina/parasitología , Retinitis/parasitología , Retinitis/terapia , Animales , Ceguera/diagnóstico , Ceguera/parasitología , Infecciones Parasitarias del Ojo/diagnóstico , Infecciones Parasitarias del Ojo/parasitología , Fondo de Ojo , Humanos , Terapia por Láser/métodos , Fotocoagulación/métodos , Masculino , Persona de Mediana Edad , República de Corea , Epitelio Pigmentado de la Retina/patología , Retinitis/diagnóstico , Agudeza VisualRESUMEN
PURPOSE: The aim of this study was to evaluate the expression of the protein annexin A1 (ANXA1), a potent endogenous regulator of the inflammatory process, in ocular toxoplasmosis. METHODS: C57BL/6 female mice were infected using intravitreal injections of either 10(6) tachyzoites of Toxoplasma gondii (RH strain; T. gondii) or PBS only (control groups). After 24, 48, and 72 h, animals were sacrificed and their eyes were harvested for histopathological, immunohistochemical, and ultrastructural immunocytochemical analysis of ANXA1. Human retinal pigment epithelial (RPE) cells (ARPE-19) were infected in vitro with T. gondii and collected after 60, 120, 240 min, and 24 h. RESULTS: Compared with non-infected eyes, an intense inflammatory response was observed in the anterior (24 h after infection) and posterior segments (72 h after infection) of the infected eye, characterized by neutrophil infiltration and by the presence of tachyzoites and their consequent destruction along with disorganization of normal retina architecture and RPE vacuolization. T. gondii infection was associated with a significant increase of ANXA1 expression in the neutrophils at 24, 48, and 72 h, and in the RPE at 48 and 72 h. In vitro studies confirmed an upregulation of ANXA1 levels in RPE cells, after 60 and 120 min of infection with T. gondii. CONCLUSIONS: The positive modulation of endogenous ANXA1 in the inflammatory and RPE cells during T. gondii infection suggests that this protein may serve as a therapeutic target in ocular toxoplasmosis.
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Anexina A1/genética , Segmento Anterior del Ojo/inmunología , Células Epiteliales/inmunología , Segmento Posterior del Ojo/inmunología , Epitelio Pigmentado de la Retina/inmunología , Toxoplasmosis Animal/inmunología , Toxoplasmosis Ocular/veterinaria , Animales , Anexina A1/metabolismo , Segmento Anterior del Ojo/parasitología , Segmento Anterior del Ojo/patología , Células Epiteliales/parasitología , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/inmunología , Segmento Posterior del Ojo/parasitología , Segmento Posterior del Ojo/patología , Epitelio Pigmentado de la Retina/parasitología , Epitelio Pigmentado de la Retina/patología , Toxoplasma/inmunología , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/patología , Toxoplasmosis Ocular/inmunología , Toxoplasmosis Ocular/parasitología , Toxoplasmosis Ocular/patologíaRESUMEN
One of the manifestations of leishmaniases is eye injuries which main characteristics are the injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. We investigated Leishmania amazonensis-RPE cells relationship and its impact in laminin and fibronectin production. Using RPE cell (ARPE-19), we demonstrated that L. amazonensis adhere to these cells in the first hour of infection, whereas parasite internalization was only observed after 6 h. Seventy-two hours after infection, vacuoles with parasites debris were observed intracellularly, and no parasite were observed intra- or extracellularly at the 96 h, suggesting that Leishmania can infect ARPE-19 cells although this cells are able to clear the infection. Fibronectin and laminin were associated with L. amazonensis-ARPE-19 interaction. Confocal analysis showed no substantial alterations in fibronectin presence in ARPE-19-infected or ARPE-19-noninfected cells, whereas laminin levels increased three times 10 h after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis-ARPE-19 infection induces increased production of laminin in the beginning of infection which may facilitate parasite-host cell interactions.
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Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Proteínas de la Matriz Extracelular/metabolismo , Leishmania mexicana/patogenicidad , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/parasitología , Adhesión Celular , Línea Celular , Humanos , Factores de Tiempo , Vacuolas/parasitologíaRESUMEN
PURPOSE: Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, can lead to severe visual impairment. T. gondii inhibits or delays programmed cell death caused by various apoptotic triggers; however, the mechanisms involved in the T. gondii-induced suppression of apoptosis in retinal cells have not been analysed in detail. METHODS: We investigated the role of T. gondii infection in H(2)O(2) -induced apoptosis in human retinal pigment epithelial cells (ARPE-19) by monitoring the activities of apoptosis-regulating molecules and mitogen-activated protein kinases (MAPKs), including p38 MAPK. We also examined the gene downstream from p38 MAPK. RESULTS: T. gondii infection significantly inhibited the cellular toxicity of H(2)O(2) (500 µm) and increased cell viability in a multiplicity of infection (MOI)-dependent manner by reducing DNA fragmentation and reactive oxygen species (ROS) generation in ARPE-19 cells. Western blot analysis also showed that T. gondii infection prevented the host cell expression of pro-apoptotic factors, such as Bad and Bax, and the activation of caspase-3. Infection with T. gondii increased the expression of the anti-apoptotic factor Bcl-2 in ARPE-19 cells under oxidative stress. In accordance with these findings, Toxoplasma infection was protective enough to suppress the phosphorylation of p38 MAPK following H(2)O(2) treatment. Exposure to H(2)O(2) increased the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells, and its expression was significantly inhibited in H(2)O(2) -treated infected cells. CONCLUSION: The protective function of T. gondii infection against ROS-induced apoptosis results from changes in the expression of apoptotic molecules and the downregulation of stress-induced intracellular signalling.
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Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/parasitología , Toxoplasma/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Línea Celular , Supervivencia Celular , Fragmentación del ADN/efectos de los fármacos , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/fisiología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Fosforilación , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PURPOSE: Toxoplasma gondii is an intracellular protozoan parasite and the most common cause of infectious uveitis. This study was conducted to evaluate the in vitro effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in rat retinal cells infected with T. gondii. METHODS: Rat retinal cells, retinal pigment epithelial (RPE) cells, and retinal Müller glial (RMG) cells were in vitro infected with T. gondii RH strain tachyzoites. Cultured cells were stimulated with various concentrations of TNF-alpha and IFN-gamma. The effect of TNF-alpha and IFN-gamma in T. gondii invasion and replication between retinal cells was determined through two different methods: measuring [(3)H]-uracil incorporation and counting infected cells by microscopic examination. RESULTS: Infection by T. gondii was lesser within RPE cells than within RMG cells. IFN-gamma significantly inhibits [(3)H]-uracil incorporation in RMG and RPE cells (respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%, 30%, and 75% for RPE cells). TNF-alpha significantly inhibits [(3)H]-uracil incorporation in RPE cells (23% and 38% inhibition at 1 and 10 ng/mL), but not in RMG cells. These results were confirmed by confocal microscopic data. The percentage of infected cells decreased from 20% to 7% after IFN-gamma stimulation. CONCLUSIONS: Both cytokines IFN-gamma and TNF-alpha inhibited T. gondii replication in the RPE cells, whereas only IFN-gamma had an anti-Toxoplasma activity within the RMG cells. The differences in cytokine response may be the reason that RPE cells are less efficiently infected by T. gondii than are RMG cells.
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Células del Tejido Conectivo/parasitología , Interferón gamma/farmacología , Retina/citología , Epitelio Pigmentado de la Retina/parasitología , Toxoplasma/efectos de los fármacos , Toxoplasma/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Células Cultivadas , Células del Tejido Conectivo/efectos de los fármacos , Células del Tejido Conectivo/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Microscopía Confocal , Microscopía Fluorescente , Óxido Nítrico/metabolismo , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Uracilo/metabolismoRESUMEN
PURPOSE: To report three patients with ocular angiostrongyliasis who presented with a variety of clinical findings. METHOD: Retrospective, observational case series. The medical charts, photographs, and electrophysiologic records were reviewed. RESULTS: All patients presented with blurred vision and one had a history of eosinophilic meningitis. In each respective case, only one living larva was found in the anterior chamber, vitreous cavity, and subretinal space. The fundus examination revealed generalized retinal pigment epithelial alteration, subretinal tracks, retinal oedema, macular oedema, and a pale disc. Visually evoked potentials showed delayed latency time in one patient, which represented the secondary optic neuritis. Blood eosinophilia was not detected, and stool examinations did not show Angiostrongylus cantonensis larva or its egg. In both cases of surgical removal, an immature male worm was identified by the parasitologist. After treatment, the visual acuity was slightly improved in all cases. CONCLUSION: This case series illustrated the different ocular manifestations of angiostrongyliasis and that although several treatments were used, the visual outcome was not markedly improved and depended mainly on the initial visual acuity.