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Joao M. Furtado, Arpita S. Bharadwaj, Antoinette S. Olivas, Liam M. Ashander, Yuzhen Pan, Justine R. Smith; Migration of Toxoplasma gondii-Infected Dendritic Cells across Simulated Human Retinal Endothelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2781.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular toxoplasmosis, caused by the protozoan parasite, Toxoplasma gondii, is a leading cause of posterior uveitis worldwide. T. gondii reaches the retina via the bloodstream. Our previous research showed that parasites cross human retinal endothelium unassisted (ARVO Abstract #5865, 2011). The present study evaluated the possibility that parasites might also transmigrate retinal endothelium inside dendritic cells (DC).
CD14+ monocytes were isolated from peripheral blood of healthy humans using a RosetteSep Kit (Stemcell), and cultured for 7 days in RPMI-1640 + 10% FBS + L-glutamine (200mM), with GM-CSF (100ng/ml) and IL-4 (12.5ng/ml) at 37°C to generate DC. Purity and phenotype of DC were assessed by cell surface expression of CD14, CD11c, CD80, CD86 and HLA-DR, as determined by flow cytometry. Retinal endothelial transmigration assays were performed in transwells (0.3cm2, 3µm pores) seeded with human retinal endothelial cells, and incubated 4 days in MCDB-131 + 10% FBS + EGM-2 at 37°C. DC were infected with YFP-tagged T. gondii tachyzoites (RH strain, gift of Boris Striepen, PhD) for 6 hours (MOI=3:1), and separated from extracellular tachyzoites by centrifugation. Infected and uninfected DC were applied to upper chambers of transwells (5x105 cells/well) and incubated for 18 hours at 37°C. Migrated DC in lower chambers were quantified by hematocytometer. Rate of diffusion of Texas Red-conjugated 70 KDa dextran between chambers was measured to assess intactness of endothelial monolayers.
The purity of monocyte-derived DC was 77-93%. Over 90% of cells demonstrated high expression of HLA-DR and CD11c. 29% of cells expressed CD80 alone and 45% expressed both CD80 and CD86. Less than 5% of cells expressed CD14. Approximately 70% of DC were infected using the stated protocol, as determined by fluorescence microscopy. Per plaque assay, parasite viability was greater than 30% at the time of the assay, consistent with published reports. Both uninfected and T. gondii-infected human DC transmigrated human retinal endothelial monolayers. The DC infected with T. gondii tachyzoites exhibited an augmented transmigratory ability compared to uninfected DC (mean±SEM: 21080±1710 versus 11250±1788 migrated DC; p<0.01; t-test; n=6 wells; representative of 3 independent experiments).
This study demonstrates the ability of human DC to transmigrate a simulated human retinal endothelium. Furthermore, our findings show that T. gondii-infected dendritic cells are hypermotile. Taken together with our previous studies, we conclude that T. gondii tachyzoites may cross human retinal endothelium both in leukocyte taxis and unassisted, to initiate ocular toxoplasmosis.
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