Toll–Like Receptor 4 (TLR4) Mediated Cell Survival Promotes HSV–1 Induced Murine ARN

Abstract

Abstract: : Purpose: To investigate the role of TLR4 in the pathogenesis of HSV–1 induced acute retinal necrosis (ARN). Methods: The anterior chamber of the right eye of TLR4^–/– and control BALB/cJ mice was inoculated with HSV–1 (KOS, 2 × 10⁴ PFU/eye) or with an equivalent volume of Vero cell extract. On day 5, 7, 8 and 9 p.i., uninoculated eyes were enucleated from experimental and control mice (day 7 only) and were stored at –80°C for virus titration or immunohistochemistry. RPE cells were isolated from TLR4^–/– and BALB/c mice and their identity was confirmed by RPE65 staining. Cultured RPE cells were infected with HSV–1 at an MOI of 5, harvested at 4, 8 and 16h p.i., and immunohistochemical staining for TUNEL assay and HSV–1 antigen, FACS staining for Annexin V and HSV–1 antigen was performed. A one–step HSV–1 growth experiment (0, 2, 4, 6, 8, 12, 24h p.i.) was also performed on RPE cells from TLR4^–/– and BALB/cJ mice. Results:In wild type BALB/cJ control mice, ARN was observed in the contralateral eyes of HSV–1 infected mice beginning on d7 p.i. with progressive retinal involvment on d8 and d9 p.i. In contrast, in the contralateral eyes of TLR4^–/– mice, ARN was observed later, on d9 p.i. and the disease was less severe compared with that in the wild type mice. There were more TUNEL+ cells in the contralateral retina of TLR4^–/– mice than in control mice and most of the TUNEL+ cells were not HSV–1 infected. No virus was recovered from contralateral eyes of TLR4^–/– mice at d7 and d8 p.i. However, in control mice, virus titers ranged from 2.45 × 10⁵ to 1.26 × 10⁶ PFU/ml at d7 and 8 p.i. respectively. One step growth curves showed that starting at 8h p.i. and continuing to the end of the experiment, the titer of virus recovered from TLR4^–/– RPE cells was significantly lower than the virus titers recovered from wild type BALB/cJ RPE at all time points. There was no significant difference in virus recovery between the TLR4^–/– and the wild type inoculum after 1h of adsorption. Flow cytometric analysis using Annexin V and anti–HSV–1 antibody showed significantly higher numbers of Annexin V+, HSV–1+ cells in HSV–1 infected TLR4^–/– RPE than in HSV–1 infected BALB/cJ RPE at 8 and 16h p.i. Conclusions:TLR4 may play a role in the pathogenesis of HSV–1 ARN by allowing virus spread in synaptically connected neurons and preventing apoptosis.

Keywords: retina • herpes simplex virus • apoptosis/cell death