Susceptibility of human iris stromal cells to herpes simplex virus 1 entry

Abstract

Ocular herpes simplex virus 1 (HSV-1) infection can lead to multiple complications, including iritis, an inflammation of the iris. Here, we use human iris stroma cells as a novel in vitro model to demonstrate HSV-1 entry and the inflammatory mediators that can damage the iris. The upregulated cytokines observed in this study provide a new understanding of the intrinsic immune mechanisms that can contribute to the onset of iritis.

Fig 1

Analysis of herpes simplex virus 1 (HSV-1) entry in primary cultures of HIS cells. (A) Entry of HSV-1 into cultured HIS cells. Cultured HIS cells, along with HeLa cells, were plated in 96-well plates and inoculated with serial dilutions of a recombinant form of HSV-1 (KOS) gL86 that expresses β-galactosidase following cell entry at the indicated PFU/cell. After 6 h, the cells were washed, permeabilized, and incubated with o-nitrophenyl-β-d-galactopyranoside (ImmunoPure ONPG; Pierce) substrate for quantitation of β-galactosidase activity expressed from the input viral genome. The enzymatic activity was measured by spectrophotometer (Molecular Devices) at an optical density (OD) at 410 nm. Values in the figure were plotted as the means from three determinations (±standard deviations [SD]). (B) X-Gal staining of HSV-1 entry. Cultured HIS cells inoculated with HSV-1 (KOS) gL86 at an MOI of 0.01 (a) were 100% stained blue, indicating 100% viral infection, whereas uninfected cells (b) did not demonstrate any staining. (c) HeLa cells, with HSV-1 KOS gL86-infected cells stained blue, were used as a positive control. (C) Viral cell-to-cell spread. HIS cells were inoculated with serial dilutions of two recombinant forms of HSV-1, (KOS) 804, a mutant known to form high numbers of syncytia, and green fluorescent protein-tagged HSV-1 (K26) GFP. Confluent monolayer cultures inoculated with HSV-1 (KOS) 804 for 2 h were then washed and incubated with methyl cellulose containing media and stained at the various time points p.i. as indicated (a to e). Cells inoculated with HSV-1 (K26) GFP were subjected to fluorescence imaging (a1 to e1). Confluent monolayer cultures were stained with TRITC-conjugated phalloidin (red color), which binds to F-actin. HSV-1-infected cells showed both green fluorescence from HSV-1 capsid and red fluorescence from F-actin expression (green and red overlapping areas appear yellow). The images were taken with a fluorescence microscope at 20× magnification. (D) Standard nonquantitative RT-PCR analysis for entry receptor expression. Total RNA samples were isolated from HIS cells and converted to first-strand cDNAs and analyzed by PCR for the receptors as indicated. A housekeeping gene, GAPDH, was used as a control.

Fig 2

Inflammation in HIS cells upon HSV-1 infection. (A) A RayBio cytokine antibody array (RayBio, Norcross, GA) per the manufacturer's instructions was used to assess inflammatory markers produced in HSV-1 (KOS) 804 (MOI of 0.01) infection of an in vitro HIS cell culture at 1 day p.i.; (B) signal intensities of various inflammatory markers are plotted in uninfected versus infected cells at 1 day p.i.; (C) inflammatory markers measured at 2 days p.i. utilizing same material and methods as described above; (D) signal intensities of various inflammatory markers are plotted in uninfected versus infected cells at 2 days p.i.

Fig 3

Cytokine response following membrane fusion. (A) HIS cell fusion with HSV-1 glycoprotein-expressing Chinese hamster ovary (CHO-K1) cells. CHO-K1 cells were either transfected with an empty vector (pCDNA3.1) or plasmids expressing one or more of the glycoproteins gB, gD, or gH-gL. A luciferase-based reporter system was used to measure fusion. Relative luciferase activity was measured in relative luciferase units (RLU) (y axis). Cell fusion was measured in RLU. CHO-K1 cells expressing the empty vector were used as a negative control. The schematic shows both unsuccessful (a) and successful (b) cell fusion between CHO-K1 and HIS cells. (B) A RayBio cytokine antibody array (RayBio, Norcross, GA) per the manufacturer's instructions was used to assess inflammatory markers produced in HIS cell interactions with CHO-K1 cells in test versus negative-control cases. (C) Signal intensities of various inflammatory markers are plotted in negative-control (no fusion) versus test (fusion) cells.