Liam M. Ashander

Toxoplasma gondii Tachyzoites Cross Retinal Endothelium Assisted by Intercellular Adhesion Molecule-1 In Vitro

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.

Migration of Toxoplasma gondii–Infected Dendritic Cells across Human Retinal Vascular Endothelium

PURPOSE Toxoplasma gondii, the parasite responsible for ocular toxoplasmosis, accesses the retina from the bloodstream. We investigated the dendritic cell as a potential taxi for T. gondii tachyzoites moving across the human retinal endothelium, and examined the participation of adhesion molecules and chemokines in this process. METHODS CD14-positive monocytes were isolated from human peripheral blood by antibody-mediated cell enrichment, and cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate dendritic cells. Transmigration assays were performed over 18 hours in transwells seeded with human retinal endothelial cells and using dendritic cells exposed to laboratory or natural strains of T. gondii tachyzoites. Parasites were tagged with yellow fluorescent protein to verify infection. In some experiments, endothelial monolayers were preincubated with antibody directed against adhesion molecules, or chemokine was added to lower chambers of transwells. RESULTS Human monocyte-derived dendritic cell preparations infected with laboratory or natural strain T. gondii tachyzoites transmigrated in larger numbers across simulated human retinal endothelium than uninfected dendritic cells (P ≤ 0.0004 in 5 of 6 experiments). Antibody blockade of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and activated leukocyte cell adhesion molecule (ALCAM) inhibited transmigration (P ≤ 0.007), and CCL21 or CXCL10 increased transmigration (P ≤ 0.031). CONCLUSIONS Transmigration of human dendritic cells across retinal endothelium is increased following infection with T. gondii. Movement may be impacted by locally produced chemokines and is mediated in part by ICAM-1, VCAM-1, and ALCAM. These findings have implications for development of novel therapeutics aimed at preventing retinal infection by T. gondii.

Toxoplasma gondii migration within and infection of human retina.

Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5×107 parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110–250 live tachyzoites; 64.5–95.2% (mean  = 79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p≤0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.

Retinal Pigment Epithelial Cells are a Potential Reservoir for Ebola Virus in the Human Eye

Purpose Success of Ebola virus (EBOV) as a human pathogen relates at the molecular level primarily to blockade the host cell type I interferon (IFN) antiviral response. Most individuals who survive Ebola virus disease (EVD) develop a chronic disease syndrome: approximately one-quarter of survivors suffer from uveitis, which has been associated with presence of EBOV within the eye. Clinical observations of post-Ebola uveitis indicate involvement of retinal pigment epithelial cells. Methods We inoculated ARPE-19 human retinal pigment epithelial cells with EBOV, and followed course of infection by immunocytochemistry and measurement of titer in culture supernatant. To interrogate transcriptional responses of infected cells, we combined RNA sequencing with in silico pathway, gene ontology, transcription factor binding site, and network analyses. We measured infection-induced changes of selected transcripts by reverse transcription-quantitative polymerase chain reaction. Results Human retinal pigment epithelial cells were permissive to infection with EBOV, and supported viral replication and release of virus in high titer. Unexpectedly, 28% of 560 upregulated transcripts in EBOV-infected cells were type I IFN responsive, indicating a robust type I IFN response. Following EBOV infection, cells continued to express multiple immunomodulatory molecules linked to ocular immune privilege. Conclusions Human retinal pigment epithelial cells may serve as an intraocular reservoir for EBOV, and the molecular response of infected cells may contribute to the persistence of live EBOV within the human eye. Translational Relevance This bedside-to-bench research links ophthalmic findings in survivors of EVD who suffer from uveitis with interactions between retinal pigment epithelial cells and EBOV.

Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study

Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1) mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α), and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (si)RNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.

Molecular Signals Involved in Human B Cell Migration into the Retina: In Vitro Investigation of ICAM-1, VCAM-1, and CXCL13

ABSTRACT Purpose: B cells participate in diverse retinal immunopathologies. Endothelial adhesion molecules and chemokines direct leukocyte trafficking. We examined the involvement of three molecular signals in retinal transendothelial migration of human B cells: ICAM-1, VCAM-1, and CXCL13. Methods: Peripheral blood B cells were isolated by negative selection. Migration was studied in transwells populated with human retinal endothelial monolayers, using antibody to block ICAM-1 or VCAM-1. Retinal expression of CXCL13 was investigated. Results: B cells crossed retinal endothelium. ICAM-1 blockade significantly reduced migration when results for all subjects were combined, and for a majority when results were analyzed by individual. This effect was irrespective of the presence or absence of CXCL13, although CXCL13 increased migration. CXCL13 was detected in neural retina and retinal pigment epithelium. Endothelial cells of some retinal vessels presented CXCL13 protein. Conclusion: ICAM-1 blockade may be an effective treatment in some patients with retinal diseases that involve B cells.

Ubiquitin carboxyl-terminal esterase L1 promotes proliferation of human choroidal and retinal endothelial cells

PurposeWe aimed: (1) to establish endothelial expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in human choroid and retina and; (2) to investigate a role for UCHL1 in basic processes involved in intraocular neovascularization. DesignControlled translational experimental study. MethodsEthanol-fixed human choroid and retina (n = 3 eyes) were indirectly immunostained with rabbit anti-human UCHL1 antibody. Endothelial proliferation and migration assays were performed using cultured human choroidal and retinal endothelial cells (n = 6 isolates/assay). Cells were transfected with UCHL1-targeted or non-targeted small interfering (si)RNA and a commercially available transfection system, and used 48 hours later in experiments. Cell proliferation was evaluated using an assay in which cellular DNA was fluorescently tagged for quantification by microplate reader. Cell migration was examined in an assay that involved counting the number of endothelial cells moving across a perforated membrane. Transcript silencing was verified by Western blot for all assays. ResultsImmunohistochemistry confirmed expression of UCHL1 by endothelium in human choroid and retina in vivo. UCHL1-specific knockdown resulted in significantly less proliferation (p < 0.0001) for 3 human choroidal endothelial isolates and 3 human retinal endothelial isolates, and significantly less migration (p ⩽ 0.016) for 2 of 3 human choroidal endothelial isolates and 1 of 3 human retinal endothelial isolates. ConclusionsOur results suggest that UCHL1 may be involved in choroidal and retinal endothelial proliferation in most persons, and endothelial migration in some persons. UCHL1 may be a suitable target for a new treatment of intraocular neovascularisation.

Selection of reference genes for studies of human retinal endothelial cell gene expression by reverse transcription-quantitative real-time polymerase chain reaction

Background Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Methods Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) less than 0.2 and coefficient of variation (CV-value) less than 0.1. Results Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11-21 (41-78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. Conclusions We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.

Molecular Responses of Human Retinal Cells to Infection with Dengue Virus

Recent clinical reports indicate that infection with dengue virus (DENV) commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin-β1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

Effect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells: Nox1/Nox4 blockade in retinal endothelial cells

Over‐production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS‐generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans.