Regulation of Primary Herpes Stromal Keratitis (HSK) by Human Apolipoprotein E (ApoE) Alleles 3 and 4 in the Mouse Eye

Abstract

Purpose:: To determine if primary HSK is associated with a specific human ApoE allele.

Methods:: Transgenic mice expressing the human ApoE ε3 or ApoE ε4 gene were used to determine if primary HSK is influenced by apoE in an isoform-specific manner. Controls were C57Bl/6 mice with ApoE-null background. The virus used was HSV-1 strain KOS-GFP. Viral replication and clearance were monitored through corneal GFP expression and HSV-1 shedding in tears. Corneas were assessed by slit lamp examination for opacity and neovascularization. At 0, 7, 14 and 21 days post-infection (dpi), groups of mice were sacrificed and corneas, TG, and brain harvested. Tissues were analyzed for viral load by quantitative PCR and immunofluorescence (IF) staining. Tissues were analyzed for expression of ApoE and toll-like receptor-9 (TLR-9) for RNA by RT-PCR and for protein levels by Western blot and immunohistochemistry.

Results:: ApoE ε4 mice had delayed clearance of HSV-1 from cornea compared to ApoE ε3 mice, as determined by plaque assay of tears. Human ApoE had no allele-specific role in viral replication kinetics, as determined by corneal GFP expression. The ApoE ε4 allele ameliorated HSK: (i) ε4 mice had earlier onset (9 dpi) of HSK compared to ε3 (15 dpi), and (ii) primary HSK severity (opacity) and neovascularization were significantly higher in ε4 mice compared to ε3 mice. Quantitative PCR of HSV-1 DNA load and IF staining of infected tissues yielded no significant differences between ε3 and ε4 mice. Ocular HSV-1 infection resulted in significant upregulation of host factors, ApoE and TLR-9, in ε4 mice vs ε3 mice, as analyzed for RNA and protein levels.

Conclusions:: Human ApoE allele ε4 is a susceptibility gene involved in the pathogenesis of primary HSK. However, viral factors in this model do not appear to contribute to HSK severity. The human ApoE ε4 allele could modulate primary HSK, in part, through the TLR-9 pathway which is a component of innate immunity. This is the first report to implicate a human gene as a risk factor in the regulation of HSK.

Keywords: herpes simplex virus • keratitis • transgenics/knock-outs