Therapeutic Use of Soluble Fas Ligand Ameliorates Acute and Recurrent Herpetic Stromal Keratitis in Mice

Abstract

Purpose: The present study was designed to test the therapeutic value of soluble FasL (sFasL) in an acute model of herpetic stromal keratitis (HSK) and, more importantly, a recurrent model of HSK using BALB/c, BALB-lpr, and National Institutes of Health (NIH) mice.

Methods: Mice were infected either acutely with the KOS strain of herpes simplex virus 1 (HSV-1) or latently with the McKrae strain of HSV-1. Acutely infected mice as well as ultraviolet-B (UV-B) reactivated mice (recurrent infection) were treated with sFasL, or soluble TNF-related apoptosis inducing ligand (sTRAIL), or BSA daily or 3 times/wk by using either a combination of subconjunctival injection and topical ointment, or with topical ointment alone. These mice then were evaluated for corneal opacity and neovascularization for 6 weeks.

Results: Following acute and recurrent HSV-1 infection, wild-type BALB/c mice treated with sFasL displayed significantly reduced incidence of corneal opacity and neovascularization compared to the control animals. However, BALB-lpr mice, which are deficient in Fas+ inflammatory cells, displayed no such differences in ocular disease, as expected. Latently infected NIH mice treated with sFasL displayed similar results. Flow cytometric analysis revealed that the corneal inflammatory infiltrate in those treated with sFasL was significantly less than in sTRAIL- or BSA-treated mice. Furthermore, corneas from sFasL-treated mice displayed relatively more cells undergoing apoptosis.

Conclusions: This study provides evidence that sFasL treatment has potential therapeutic benefit in reducing inflammatory infiltrate and neovascularization in primary and recurrent forms of HSK, and that it does so by augmenting the restriction of Fas+ inflammatory cells mediated by membrane FasL.

Figure 1

BALB/c mice treated with sFasL following infection display significantly less corneal disease than mice treated with sTRAIL. Eyes of BALB/c were infected with 10⁷ pfu of HSV-1, KOS strain. Following infection, mice were treated with sFasL, sTRAIL, or BSA by a combination of topical application and subconjunctival injection. Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatment groups of mice. The incidence of mice experiencing opacity scores greater than 1 was compared between these groups. Results are a combination of two independent studies with a total of 30 mice for each group. Significantly less virus-induced corneal opacity was observed for BALB mice treated with sFasL from 2 to 5 weeks following reactivation (*P < 0.01–0.001).

Figure 2

BALB-lpr mice treated with sFasL following infection do not display differences in corneal disease than mice treated with sTRAIL. Eyes of BALB-lpr were infected with 10⁷ pfu HSV-1, KOS strain. Following infection, mice were treated with sFasL, sTRAIL, or BSA by a combination of topical application and subconjunctival injection. Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these different treatment groups of mice. The incidence of mice experiencing opacity scores greater than 1 was compared between these groups. Results are a combination of two independent studies with a total of 30 to 35 mice for each group. These data did not show any significant differences between the treatment groups with BALB-lpr mice.

Figure 3

BALB/c mice treated with sFasL following UV-B–induced reactivation displayed significantly less corneal disease than mice treated with sTRAIL. Eyes of BALB/c treated were infected with 10⁶ pfu of HSV-1, McKrae strain, and allowed to establish latency. Six weeks following infection, the eyes of BALB/c were irradiated with UV-B to reactivate the latent infection. These mice were separated into three groups, in which they were treated with sFasL (n = 20), sTRAIL (n = 20), or BSA (n = 20) daily beginning on the day following reactivation. Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these strains of mice. Significantly less virus-induced corneal opacity and neovascularization was observed for BALB/c mice treated with sFasL from 3 to 5 weeks after infection (*P < 0.01–0.001).

Figure 4

BALB-lpr mice treated with sFasL following UV-B–induced reactivation displayed corneal disease that was indistinguishable from mice treated with sTRAIL. Eyes of BALB-lpr were infected with 10⁶ pfu of HSV-1, McKrae strain and allowed to establish latency. Six weeks following infection, the eyes of BALB/c were irradiated with UV-B to reactivate the latent infection. These mice were separated into three groups, in which they were treated with sFasL (n = 20), sTRAIL (n = 20), or BSA (n = 20) mice. Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these strains of mice. No statistical significance was seen between any of the treatment groups.

Figure 5

Treatment of NIH inbred mice with sFasL significantly reduced corneal disease following UV-B–induced reactivation. Eyes of NIH mice were infected with 10⁶ pfu of HSV-1, McKrae strain. Six weeks following infection mice were irradiated with UV-B to reactivate the latent infection. Mice were separated into three treatment groups, sFasL (n = 15), sTRAIL (n = 15), and BSA (n = 15). Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these groups of mice. Mice receiving topical treatment of sFasL displayed significantly less virus-induced corneal opacity at 2 to 5 weeks and less neovascularization at 2, 3, and 5 weeks following reactivation (*P < 0.01–0.001).

Figure 6

Histologic sections of corneas indicate that (A) treatment with sFasL results in reduced numbers of inflammatory cells when compared to (B) corneas that were treated with BSA. Hematoxylin and eosin stain of corneal sections are from mice 15 days after reactivation. Original magnification: 20.

Figure 7

Immunohistochemical staining of sections from corneas treated with sFasL display a much higher percentage of CD45+ cells that also express the apoptotic active caspase 3 marker. Corneal sections were removed from mice 15 days following UV-B reactivation that were either (A) sFasL treated (original magnification: ×40) or (B) received control treatment (BSA, original magnification: ×40). These sections then were stained with rat anti-mouse CD45 + Alexa Fluor 594 goat anti-rat IgG (red) and rabbit anti-mouse active caspase 3 + Alexa Fluor 488 goat anti-rabbit IgG (green). DAPI (4′,6-diamidino-2-phenylendole, blue) was used to counterstain the nuclei. Arrows refer to cells that coexpress CD45 and caspase 3.

Figure 8

As expected, mice treated with sFasL had significantly reduced inflammatory cell infiltrates. Mice treated with sFasL (n = 10) or sTRAIL (n = 10) were killed 17 days following reactivation and single cells isolated from their corneas. These cells then were characterized for neutrophil, macrophage, and T-cell markers. Cell numbers for each of the subsets of cells (A) and percent of CD45⁺ cells of these subsets of cells (B) were performed. Mice treated with sFasL had significantly fewer cells for all of the subsets evaluated (*P < 0.02-0.001) and a lower percentage of neutrophils (*P < 0.01).