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Toxoplasma gondii tachyzoites cross retinal endothelium assisted by intercellular adhesion molecule-1 in vitro.

Furtado JM, Bharadwaj AS, Chipps TJ, Pan Y, Ashander LM, Smith JR - Immunol. Cell Biol. (2012)

Bottom Line: Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains.Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye.In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health and Science University, Portland, OR 97239, USA.

ABSTRACT
Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

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Toxoplasma gondii tachyzoites transmigrate a simulated human retinal vascular endothelium(A) Graph showing number of tachyzoites recovered from lower chambers of transwells divided by a human retinal endothelial monolayer cultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = 44%). Representative of two independent experiments.(B) Graph showing dextran permeability of transwells presented in A, as well as dextran permeability of transwells incubated in parallel, but without tachyzoites, and containing endothelial monolayers or type I collagen alone. There was no significant difference in dextran permeability of transwells containing endothelial monolayers and incubated with live tachyzoites (live), heat-killed tachyzoites (heat-killed) or without tachyzoites (EC) (p > 0.05), but a highly significant difference between dextran permeability of these wells and wells containing membranes that were coated with collagen I alone (collagen) (***p < 0.001) (n=3–6 wells). ANOVA with post-hoc Tukey tests. Dextran permeability was determined in all experiments.(C) Analysis of CD144 expression on human retinal endothelial cells cultured at confluence for 3 days, i.e., at least one day less than endothelial cells plated in transwells. Cells were gated on the basis of forward and side scatter to exclude debris and dead cells, and plotted for CD31 and CD144 expression. Numbers indicate percentage of gated cells expressing CD31 and/or CD144. Representative of two independent experiments. (D) Graph showing results of the same transmigration assay, conducted with three natural isolates (i.e., GT-1, TgCatBR2 and GPHT) in place of the RH laboratory strain (n=6 wells, parasite viability = GT-1, 58%; TgCatBR2, 27%; GPHT, 23%). Data were obtained in three independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.
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Figure 1: Toxoplasma gondii tachyzoites transmigrate a simulated human retinal vascular endothelium(A) Graph showing number of tachyzoites recovered from lower chambers of transwells divided by a human retinal endothelial monolayer cultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = 44%). Representative of two independent experiments.(B) Graph showing dextran permeability of transwells presented in A, as well as dextran permeability of transwells incubated in parallel, but without tachyzoites, and containing endothelial monolayers or type I collagen alone. There was no significant difference in dextran permeability of transwells containing endothelial monolayers and incubated with live tachyzoites (live), heat-killed tachyzoites (heat-killed) or without tachyzoites (EC) (p > 0.05), but a highly significant difference between dextran permeability of these wells and wells containing membranes that were coated with collagen I alone (collagen) (***p < 0.001) (n=3–6 wells). ANOVA with post-hoc Tukey tests. Dextran permeability was determined in all experiments.(C) Analysis of CD144 expression on human retinal endothelial cells cultured at confluence for 3 days, i.e., at least one day less than endothelial cells plated in transwells. Cells were gated on the basis of forward and side scatter to exclude debris and dead cells, and plotted for CD31 and CD144 expression. Numbers indicate percentage of gated cells expressing CD31 and/or CD144. Representative of two independent experiments. (D) Graph showing results of the same transmigration assay, conducted with three natural isolates (i.e., GT-1, TgCatBR2 and GPHT) in place of the RH laboratory strain (n=6 wells, parasite viability = GT-1, 58%; TgCatBR2, 27%; GPHT, 23%). Data were obtained in three independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.

Mentions: We observed migration of live T. gondii tachyzoites through a simulated human retinal vascular endothelium in a transwell system over a 4-hour period (Figure 1A). Smaller numbers of heat-killed tachyzoites, loaded into upper chambers of control transwells, were also recovered from lower chambers at this time. However, there was no significant difference (p > 0.05) in permeability to high molecular weight dextran for endothelial monolayers incubated with live, heat-killed or no tachyzoites, and permeability under these conditions remained significantly less (p < 0.001) than permeability of wells containing membranes coated with collagen alone (Figure 1B), suggesting that the endothelial monolayer remained intact for the duration of the experiment. Universal expression of CD144 (VE-cadherin) by CD31-positive retinal endothelial cells after extended confluent culture (Figure 1C), also was consistent with the formation of intercellular junctions across the monolayers. To address the possibility that transendothelial movement was peculiar to RH strain tachyzoites, we performed the same assay, but separately substituted one of three different natural parasite isolates for the clonal strain. The same result was obtained in this series of experiments (Figure 1D).


Toxoplasma gondii tachyzoites cross retinal endothelium assisted by intercellular adhesion molecule-1 in vitro.

Furtado JM, Bharadwaj AS, Chipps TJ, Pan Y, Ashander LM, Smith JR - Immunol. Cell Biol. (2012)

Toxoplasma gondii tachyzoites transmigrate a simulated human retinal vascular endothelium(A) Graph showing number of tachyzoites recovered from lower chambers of transwells divided by a human retinal endothelial monolayer cultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = 44%). Representative of two independent experiments.(B) Graph showing dextran permeability of transwells presented in A, as well as dextran permeability of transwells incubated in parallel, but without tachyzoites, and containing endothelial monolayers or type I collagen alone. There was no significant difference in dextran permeability of transwells containing endothelial monolayers and incubated with live tachyzoites (live), heat-killed tachyzoites (heat-killed) or without tachyzoites (EC) (p > 0.05), but a highly significant difference between dextran permeability of these wells and wells containing membranes that were coated with collagen I alone (collagen) (***p < 0.001) (n=3–6 wells). ANOVA with post-hoc Tukey tests. Dextran permeability was determined in all experiments.(C) Analysis of CD144 expression on human retinal endothelial cells cultured at confluence for 3 days, i.e., at least one day less than endothelial cells plated in transwells. Cells were gated on the basis of forward and side scatter to exclude debris and dead cells, and plotted for CD31 and CD144 expression. Numbers indicate percentage of gated cells expressing CD31 and/or CD144. Representative of two independent experiments. (D) Graph showing results of the same transmigration assay, conducted with three natural isolates (i.e., GT-1, TgCatBR2 and GPHT) in place of the RH laboratory strain (n=6 wells, parasite viability = GT-1, 58%; TgCatBR2, 27%; GPHT, 23%). Data were obtained in three independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475739&req=5

Figure 1: Toxoplasma gondii tachyzoites transmigrate a simulated human retinal vascular endothelium(A) Graph showing number of tachyzoites recovered from lower chambers of transwells divided by a human retinal endothelial monolayer cultured on type 1 collagen, 4 hours after upper chambers were loaded with 1 × 106 live or heat-killed RH strain T. gondii tachyzoites (n=6 wells, parasite viability = 44%). Representative of two independent experiments.(B) Graph showing dextran permeability of transwells presented in A, as well as dextran permeability of transwells incubated in parallel, but without tachyzoites, and containing endothelial monolayers or type I collagen alone. There was no significant difference in dextran permeability of transwells containing endothelial monolayers and incubated with live tachyzoites (live), heat-killed tachyzoites (heat-killed) or without tachyzoites (EC) (p > 0.05), but a highly significant difference between dextran permeability of these wells and wells containing membranes that were coated with collagen I alone (collagen) (***p < 0.001) (n=3–6 wells). ANOVA with post-hoc Tukey tests. Dextran permeability was determined in all experiments.(C) Analysis of CD144 expression on human retinal endothelial cells cultured at confluence for 3 days, i.e., at least one day less than endothelial cells plated in transwells. Cells were gated on the basis of forward and side scatter to exclude debris and dead cells, and plotted for CD31 and CD144 expression. Numbers indicate percentage of gated cells expressing CD31 and/or CD144. Representative of two independent experiments. (D) Graph showing results of the same transmigration assay, conducted with three natural isolates (i.e., GT-1, TgCatBR2 and GPHT) in place of the RH laboratory strain (n=6 wells, parasite viability = GT-1, 58%; TgCatBR2, 27%; GPHT, 23%). Data were obtained in three independent experiments. In all graphs, bars represent mean and error bars represent standard error of mean.
Mentions: We observed migration of live T. gondii tachyzoites through a simulated human retinal vascular endothelium in a transwell system over a 4-hour period (Figure 1A). Smaller numbers of heat-killed tachyzoites, loaded into upper chambers of control transwells, were also recovered from lower chambers at this time. However, there was no significant difference (p > 0.05) in permeability to high molecular weight dextran for endothelial monolayers incubated with live, heat-killed or no tachyzoites, and permeability under these conditions remained significantly less (p < 0.001) than permeability of wells containing membranes coated with collagen alone (Figure 1B), suggesting that the endothelial monolayer remained intact for the duration of the experiment. Universal expression of CD144 (VE-cadherin) by CD31-positive retinal endothelial cells after extended confluent culture (Figure 1C), also was consistent with the formation of intercellular junctions across the monolayers. To address the possibility that transendothelial movement was peculiar to RH strain tachyzoites, we performed the same assay, but separately substituted one of three different natural parasite isolates for the clonal strain. The same result was obtained in this series of experiments (Figure 1D).

Bottom Line: Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains.Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye.In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health and Science University, Portland, OR 97239, USA.

ABSTRACT
Retinal infection is the most common clinical manifestation of toxoplasmosis. The route by which circulating Toxoplasma gondii tachyzoites cross the vascular endothelium to enter the human retina is unknown. Convincing studies using murine encephalitis models have strongly implicated leukocyte taxis as one pathway used by the parasite to access target organs. To establish whether tachyzoites might also interact directly with vascular endothelium, we populated a transwell system with human ocular endothelial cells. Human retinal endothelial monolayers permitted transmigration of tachyzoites of RH and three natural isolate strains. Antibody blockade of intercellular adhesion molecule-1 significantly reduced this migration, but did not impact tachyzoite movement across an endothelial monolayer derived from the choroid, which lies adjacent to the retina within the eye. In demonstrating that tachyzoites are capable of independent migration across human vascular endothelium in vitro, this study carries implications for the development of therapeutics aimed at preventing access of T. gondii to the retina.

Show MeSH
Related in: MedlinePlus