Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye

Abstract

Recent outbreaks of Ebola and Zika have highlighted the possibility that viruses may cause enduring infections in tissues like the eye, including the neural retina, which have been considered immune privileged. Whether this is a peculiarity of exotic viruses remains unclear, since the impact of more common viral infections on neural compartments has not been examined, especially in immunocompetent hosts. Cytomegalovirus is a common, universally distributed pathogen, generally innocuous in healthy individuals. Whether in immunocompetent hosts cytomegalovirus can access the eye, and reside there indefinitely, was unknown. Using the well-established murine cytomegalovirus infection model, we show that systemic infection of immunocompetent hosts results in broad ocular infection, chronic inflammation and establishment of a latent viral pool in the eye. Infection leads to infiltration and accumulation of anti-viral CD8+ T cells in the eye, and to the development of tissue resident memory T cells that localize to the eye, including the retina. These findings identify the eye as an unexpected reservoir for cytomegalovirus, and suggest that common viruses may target this organ more frequently than appreciated. Notably, they also highlight that infection triggers sustained inflammatory responses in the eye, including the neural retina.

Fig 1. Pathological changes in the eye after systemic MCMV infection.

(A) SD-OCT image of the anterior segment of the eye from an uninfected mouse. (B) Cross-sections of the anterior chamber from mice infected with MCMV at the indicated times pi. Cellular deposits (white arrows), vessel dilation and iris thickening (yellow arrows). (C) A representative image demonstrating iris bombé, and the formation of synechia (yellow arrow). (D) The frequency of mice displaying the indicated pathological features following MCMV infection was quantified where n = 10 mice for each time point from at least 3 independent experiments. ND = not detected. (E) SD-OCT fundus images of the retina at the indicated times pi. Black arrows indicate retinal vessel enlargement (dilation and calibre variation). (F) Vessel diameter was measured at the indicated times pi with mean ± standard error of the mean (SEM) plotted, where n≥8 eyes (* P = 0.035; *** P = 0.0006).

Fig 2. MCMV infection causes inflammation in the iris and retina.

(A) Schematic diagram with the major compartments of the eye labelled. (B) Sections of the anterior chamber at the indicated times pi were stained with haematoxylin and eosin. Thickening of the iris was evident at day 5. Cellular infiltrates and keratic precipitates were present after infection (black arrows). Synechia (adherence of the iris to the cornea) was frequently observed (red arrows). (C) Haematoxylin and eosin stained sections of the retina show normal structure at day 5 pi. Enlarged vessels (two tailed arrow head), folds in the retina (asterisk) and infiltrating cells were present (black arrows) at day 10pi. At day 25 pi infiltrating cells within the retina (black arrow heads) and the vitreous (red arrow heads) were evident. Scale bar: 100 μm. Results are representative of those from 5 mice per time-point.

Fig 3. MCMV infects and replicates within the eye after systemic infection.

(A) Mice were infected with MCMV-mCherry, and injected with fluorescein prior to in vivo imaging. The iris was assessed using a Micron IV imaging microscope with the vessels staining green and red fluorescence indicating MCMV-infected cells. (B) The indicated organs were removed from MCMV infected mice at day 5 pi, homogenized, and MCMV titres determined by plaque assay. The dashed line represents the limit of detection for individual organs. Mean ± SEM are plotted, where n≥5. (C) Whole-mounts of the iris were prepared at various times pi and stained with antibodies directed against MHC-II (red) and the viral antigen IE1 (green). Scale bar: 200 μm (D) The percentage of mice with detectable MCMV in the iris at various times pi is shown where n≥ 9 for each time point. ND = not detected.

Fig 4. Defining the compartments of the eye infected by MCMV.

(A) Haematoxylin and eosin stained section of an uninfected mouse eye with the major compartments labelled. Scale bar: 500 μm. (B) Eyes were removed from MCMV-infected mice at day 5 pi, components of the eye dissected and whole-mounts prepared. The samples were stained with antibodies against MHC-II (red) and IE1 (green—for viral antigen) and microscopy performed. Scale bar: 200 μm. Results are representative of at least 5 independent experiments with n≥20. (C) Mice were infected with MCMV-LacZ and eyes removed at day 5 pi. Frozen tissue sections were prepared and stained with x-gal to identify virally infected cells (blue) and counterstained with neutral red to identify cell nuclei. Scale bar: 50 μm.

Fig 5. Characterisation of cells infected by MCMV within the iris.

(A) Wholemounts of the iris were prepared at 24 hr pi and stained with antibodies to CD31 (red) and the IE1 viral antigen (green). Iris whole-mounts at day 2 pi were stained with anti-IE1 (green) and (B) anti-CD31 or (C) anti-PDGFRβ antibodies. Blue = DAPI nuclear stain. (D) Wholemounts of the iris at day 5 pi were stained with anti-IE1 (green) along with antibodies directed against the indicated cell surface markers (red). Anti-IE1 detects a nuclear antigen. Scale bar: 50 μm. Results are representative of at least 3 independent experiments with n≥9.

Fig 6. Leukocytes and lymphocytes, including T cells, infiltrate the iris after systemic MCMV infection.

Whole- mounts of the iris at the indicated time points pi were prepared and stained with (A) anti-MHC-II or (B) anti-CD45 antibodies. (C) Mice were infected with MCMV, and at the indicated times pi single cell preparations of the iris prepared. Iris samples were pooled from 2–5 mice. The resulting pooled samples were stained with lineage specific antibodies and analysed by flow cytometry. The percentage of cells in specific populations are graphed as mean ± SEM where samples represent n≥10 mice for each time point from 3 independent experiments. (D) Iris whole-mounts from mice at day 25 pi were stained with collagen IV (green) and CD4 (left panel) or CD8 (right panel) antibodies (red). Scale bar: 100 μm.

Fig 7. Leukocytes and lymphocytes, including T cells, infiltrate and accumulate in the retina after systemic MCMV infection.

Retinal sections were stained with (A) anti-MHC-II or (B) anti-CD45 antibodies. (C) Mice were infected with MCMV and at the indicated times pi the retina isolated. Single cell preparations from individual retina samples were stained with lineage specific antibodies and analysed by flow cytometry. The percentages of cells in specific populations are graphed as mean ± SEM where n≥10 mice for each time point from 3 independent experiments. (D) Retinal whole-mounts at the indicated times pi were stained with collagen IV (green) anti-CD45 (red) antibodies. Scale bars: 100 μm.

Fig 8. Virus-specific CD8⁺ T cells infiltrate and accumulate in the iris and retina after systemic MCMV infection and form tissue resident memory (T_RM) populations.

Mice were infected with MCMV, and at the indicated times pi single cell preparations were prepared and stained for T cell markers. (A) The number of CD8⁺ T cells, CD8⁺ IE1⁺ antiviral T cells and CD8⁺ IE1⁺ CD103^+/- CD69^+/- T cells are graphed as mean ± SEM for (A) the iris and (B) the retina. Samples represent n = 6 for uninfected mice, and n = 15 for MCMV infected mice for each time point from 3 independent experiments. Iris samples were pooled from 5 mice.

Fig 9. MCMV establishes a latent infection in ocular tissue.

(A) At the indicated times pi iris whole-mounts were stained with anti-CD45 antibodies. (B) A representative image of the cellular monolayer of iris explant cultures established from an uninfected mouse (left panel) or a latently infected mouse (right panel). The culture established from a latently infected mouse shows foci of viral replication and cytomegalic cells characteristic of MCMV infection. Scale bar: 200 μm. (C) Percentage of cultures where MCMV reactivation was detected. (n = 23 mice). The table on the right shows the frequency of viral reactivation in specific compartments of the eye from individual mice.