Type I Interferon Signaling Is Critical During the Innate Immune Response to HSV-1 Retinal Infection

Abstract

Purpose: Acute retinal necrosis (ARN) is a herpesvirus infection of the retina with blinding complications. In this study, we sought to create a reproducible mouse model of ARN that mimics human disease to better understand innate immunity within the retina during virus infection.

Methods: C57Bl/6J wild type (WT) and type I interferon receptor-deficient (IFNAR-/-) mice were infected with varying amounts of herpes simplex virus type 1 (HSV-1) via subretinal injection. Viral titers, optical coherence tomography (OCT) and fundus photography, the development of encephalitis, and ocular histopathology were scored and compared between groups of WT and IFNAR-/- mice.

Results: The retina of WT mice could be readily infected with HSV-1 via subretinal injection resulting in retinal whitening and full-thickness necrosis as determined by in vivo imaging and histopathology. In IFNAR-/- mice, HSV-1-induced retinal pathology was significantly worse when compared with WT mice, and viral titers were significantly elevated within two days after infection and persisted to day 5 after infection within the retina. These results were also observed in the brain where there were significantly higher viral titers and frequency of encephalitis in IFNAR-/- when compared to WT mice.

Conclusions: Collectively, these findings show that our new mouse model of ARN mimics human disease and can be used to study innate immunity within the retina. We conclude that type I interferons are critical in containing HSV-1 locally within retinal tissues and prohibiting spread into the brain.

Figure 1.

HSV-1 dose curve in WT mice. WT mice were infected with increasing concentrations of HSV-1 [100PFUs (green line), 1000 PFUs (red line), or 10,000 PFUs (blue line)] and probability of death or developing severe encephalitis recorded five days pi. Graph represents two independent experiments for a total of three to six mice per group (n = 3–6/group). **P < 0.05.

Figure 2.

Retinal whitening, full-thickness necrosis, and replicating virus within WT mice five days pi. WT mice were subretinally injected with PBS or 1000 PFUs of HSV-1. At five days pi, fundus photography (a, c) and OCT imaging (b, d) were performed. HSV-1-infected mice showed retinal whitening (c, green dotted line), full-thickness necrosis (blue arrow) with overlying vitritis (yellow arrow) not seen in PBS controls. A small, residual subretinal bleb was occasionally seen five days after injection (red arrow). Retinal whitening appeared similar to that of the peripheral retina of a patient with HSV-1 PCR-proven ARN (green arrow, e). Five days pi, retinal tissue was evaluated for HSV-1 by plaque assay (f). Graph represents log₁₀ PFUs/retina ± SEM of three independent experiments, n = 4-5/group. **P < 0.01. INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 3.

HSV-1 infection of the retina results in local tissue destruction. WT mice were subretinally injected with PBS or 1,000 PFUs of HSV-1. At five days pi, tissue was isolated, fixed, sectioned, and H&E stained. WT PBS controls retained good retinal architecture (a). Retinas from WT mice infected with HSV-1 showed intraretinal hemorrhages (red arrow), thinning of the inner and outer nuclear layers, overlying vitreous lymphocytes, and microglia within areas of tissue necrosis (b). Ocular pathology was then compared using a previously validated histology scoring system for retinitis (c). (d, e) Fluorescent imaging was then performed five days pi in WT mice. Images represent two independent experiments of two to four eyes/group. **P < 0.01. INL, inner nuclear layer; ONL, outer nuclear layer; White star, area of HSV-1 antigen expression without colocalization; orange arrows, HSV-1 and Iba-1 colocalization; yellow arrows, Iba-1 positivity without HSV-1 expression; white inset, digital magnification of affected area. White scale bars are equal to 75 µm.

Figure 4.

Loss of IFN signaling results in comprised viral containment by day 2 pi and persists. WT and IFNAR^−/− mice were subretinally injected with 1000 PFUs of HSV-1. At two and five days pi, retinal tissue was isolated and evaluated by plaque assay. Graph represents the mean log₁₀ PFUs/retina ± SEM of three independent experiments of six to nine retinas/group. **P < 0.01; D2, day 2; D5, day 5; ns, not significant.

Figure 5.

Loss of IFN signaling results in worse pathology. WT and IFNAR^−/− mice were subretinally injected with PBS or 1000 PFUs of HSV-1. Five days pi, eyes were isolated, fixed, and H&E stained. (a-h) Top images are ×25 of area of black box in magnification ×10 images (bottom). WT and IFNAR^−/− PBS controls showed normal retinal architecture (a, b and e, f, respectively) five days pi injection. WT mice show focal areas of infiltration of microglia, changes in retinal architecture and overlying vitreous inflammation in areas of necrosis (c, d). IFNAR^−/− eyes had gross disorganization of the retina, intraretinal hemorrhages (yellow asterisk), and dense overlying vitritis in nearly all areas of the retina (g, h). Pathology was scored five days pi among these groups (i). Results represent two independent experiments of one to three eyes per group for a total of two to six eyes/group. Western blot analysis of apoptosis and necrosis markers within the retina of WT and IFNAR^−/− mice was then performed, and results represent two independent experiments (j). **P < 0.01; INL, inner nuclear layer; ONL, outer nuclear layer. White scale bars: 75 µm.

Figure 6.

Loss of IFN signaling results in chemokine expression changes. WT and IFNAR^−/− mice were subretinally injected with PBS or 1000 PFUs of HSV-1. Five days pi, retinas were isolated, and chemokine concentrations were compared (a-k). Results represent three independent experiments of one to four eyes per group for a total of two to eight eyes/group and are plotted as the mean concentration (pg/mg) ± standard error of the mean. **P < 0.01.

Figure 7.

HSV-1 dissemination into the brain to cause encephalitis. WT and IFNAR^−/− mice were subretinally injected with 1000 PFUs of HSV-1. At two and five days pi, brain tissue was isolated and evaluated by plaque assay. Graphs represent three independent experiments of the log₁₀ PFUs/retina ± SEM (six to nine mice/group). **P < 0.01; D2, two days after infection; D5, five days after infection.