Molecular Responses of Human Retinal Cells to Infection with Dengue Virus

Abstract

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.

Figure 1

Infection of human retinal pigment epithelial cells with DENV: viral strain = Mon601; multiplicity of infection = 1; evaluated time points postinoculation = 6, 24, 48, and 72 hours (hr). (a) Graphs showing expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) transcripts in DENV-infected retinal pigment epithelial cells versus mock-infected cells. Reference genes were glyceraldehyde-3-phosphate dehydrogenase and TATA-binding protein. Bars represent mean relative expression, with error bars showing standard deviation. n = 3 cultures/condition. Data were analyzed by two-tailed Student's t-test. (b) DENV-infected and mock-infected retinal pigment epithelial cells viewed by light microscopy. Original magnification = 100x. (c) DENV- and mock-infected retinal pigment epithelial cells immunolabeled to detect double-stranded RNA (dsRNA) and DENV antigen (Ag). Alexa Fluor 555 (red) and Alexa Fluor 488 (green) with Hoechst 33342 nuclear counterstain (blue). Original magnification: 630x. (d) Graphs of DENV RNA copy number for DENV-infected retinal pigment epithelial monolayers and plaque-forming units (pfu) for culture supernatant collected from infected cells. n = 3 cultures/condition. Bars represent mean DENV RNA copy number (relative to peptidylprolyl isomerase A (PPIA)) or pfu/mL, with error bars showing standard deviation.

Figure 2

Infection of human retinal endothelial cells with DENV: viral strain = Mon601; multiplicity of infection = 1; evaluated time points postinoculation = 6, 24, 48, and 72 hours (hr). (a) Graphs showing relative expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) transcripts in DENV-infected endothelial cells versus mock-infected cells. Reference genes were glyceraldehyde-3-phosphate dehydrogenase and TATA-binding protein. Bars represent mean relative expression, with error bars showing standard deviation. n = 3 cultures/condition. Data were analyzed by two-tailed Student's t-test. (b) DENV-infected and mock-infected endothelial cells viewed by light microscopy. Original magnification = 100x. (c) DENV- and mock-infected endothelial cells immunolabeled to detect double-stranded RNA (dsRNA) and DENV antigen (Ag). Alexa Fluor 555 (red) and Alexa Fluor 488 (green) and with Hoechst 33342 nuclear counterstain (blue). Original magnification: 630x. (d) Graphs of copy number of DENV RNA for DENV-infected endothelial monolayers and plaque-forming units (pfu) for culture supernatant collected from infected cells. n = 3 cultures/condition. Bars represent DENV RNA copy number (relative to cellular peptidylprolyl isomerase A (PPIA)) or mean pfu/mL, with error bars showing standard deviation.

Figure 3

Infection of primary human retinal pigment epithelial cells and primary human retinal endothelial cells with DENV: viral strain = Mon601; multiplicity of infection = 1; evaluated time point postinoculation = 48 hours. (a) DENV- and mock-infected retinal pigment epithelial cells and (b) DENV- and mock-infected retinal endothelial cells immunolabeled to detect double-stranded RNA (dsRNA) and DENV antigen (Ag). Alexa Fluor 555 (red) and Alexa Fluor 488 (green) with Hoechst 33342 nuclear counterstain (blue). Original magnification: 630x.

Figure 4

Antiviral type I interferon (IFN) response of human retinal pigment epithelial cells and human retinal endothelial cells infected with DENV: viral strain = Mon601; multiplicity of infection = 1; evaluated time points postinoculation = 24, 48, and 72 hours (hr). (a) and (b) Graphs showing IFN-β protein concentration in culture supernatant of (a) DENV-infected retinal pigment epithelial cells and (b) DENV-infected endothelial cells. Bars represent mean protein concentration, with error bars showing standard deviation. n = 3 cultures/condition. No IFN-β protein was detected in mock-infected cells. ND = not detectable. (c) and (d) Graphs showing relative transcript expression for IFN-stimulated gene products in (c) DENV-infected versus mock-infected retinal pigment epithelial cells and (d) DENV-infected versus mock-infected endothelial cells. Reference genes were glyceraldehyde-3-phosphate dehydrogenase and TATA-binding protein. Bars represent mean relative expression, with error bars showing standard deviation. n = 3 cultures/condition. Data were analyzed by two-tailed Student's t-test. IFITM1 = IFN-induced transmembrane protein 1; EIF2AK2 = eukaryotic translation initiation factor 2-alpha kinase 2; RSAD2 = radical SAM domain-containing 2 (also known as viperin); ISG15 = IFN-stimulated gene 15.

Figure 5

Cell adhesion molecule expression by human retinal endothelial cells infected with DENV: viral strain = Mon601; multiplicity of infection = 1; evaluated time points postinoculation = 24, 48, and 72 hours (hr). (a) Graphs showing relative transcript expression for adhesion molecules in DENV-infected versus mock-infected endothelial cells. Reference genes were glyceraldehyde-3-phosphate dehydrogenase and TATA-binding protein. Bars represent mean relative expression, with error bars showing standard deviation. n = 3 cultures/condition. Data were analyzed by two-tailed Student's t-test. (b) Graph showing relative expression of VCAM-1 protein on the surface of DENV-infected versus mock-infected endothelial cells. Each pair of circles joined by a line represents the mean expression for DENV-infected versus mock-infected endothelial cells for one of 3 independent experiments. n = 8 cultures/condition. Data were analyzed by one-tailed paired Student's t-test. ICAM-1 = intercellular adhesion molecule, 1; VCAM-1 = vascular cell adhesion molecule 1; ALCAM = activated leukocyte cell adhesion molecule.

Figure 6

Effect of DENV infection on barrier function of human retinal pigment epithelial cells and human retinal endothelial cells: viral strain = Mon601; multiplicity of infection = 1; evaluated up to 60 hours (hr) postinoculation. (a) and (d) Plots of electrical resistance across (a) DENV-infected retinal pigment epithelial monolayers and (d) DENV-infected endothelial monolayers versus mock-infected monolayers, measured at hourly intervals and expressed as transcellular resistance by xCELLigence. Lines indicate mean electrical resistance: blue lines represent DENV-infected monolayers and red lines represent mock-infected monolayers. Error bars indicate standard error of mean. n = 4 monolayers/condition. Arrowheads mark time of inoculation and 24 and 48 hours postinoculation. Drop in resistance immediately after inoculation with virus is due to removal of plate from xCELLigence for inoculation procedure. (b) DENV- and mock-infected retinal pigment epithelial cells immunolabeled to detect double-stranded RNA (dsRNA) and filamentous- (F-) actin. Alexa Fluor 555 (red) and Alexa Fluor 488 (green) with Hoechst 33342 nuclear counterstain (blue). Images are merged z-stacks. Original magnification: 630x. (c) and (e) Graphs showing relative transcript expression for junctional molecules in (c) DENV-infected retinal pigment epithelial cells and (e) DENV-infected endothelial cells versus mock-infected cells. Reference genes were glyceraldehyde-3-phosphate dehydrogenase and TATA-binding protein. Bars represent mean relative expression, with error bars showing standard deviation. n = 3 cultures/condition. Data were analyzed by two-tailed Student's t-test. ZO-1 = zonula occludens 1; CLDN5 = claudin 5; OCLN = occludin; JAM-3 = junctional adhesion molecule 3; CTNNB1 = catenin-β1. CLDN5 and VE-cadherin were not detected in epithelial cells.