Mice with mutations in Fas and Fas ligand demonstrate increased herpetic stromal keratitis following corneal infection with HSV-1

Abstract

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas(+) bone marrow-derived inflammatory cells and thus reduces the severity of HSK.

Figure 1

Defective expression of both Fas and to a lesser extent FasL, results in increased HSK following infection with HSV-1, KOS strain. Eyes of BALB/c wild-type (n=30), BALB-lpr (n=25) and BALB-gld (n=25) mice were infected with 10⁷ pfu of HSV-1, KOS strain. Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these strains of mice. Significant virus-induced corneal opacity was observed for BALB-lpr (P<0.001) at all time points when compared to BALB/c controls. BALB-gld mice displayed significantly more opacity than did BALB/c controls at days 14–35 (P<0.05–0.01). BALB-lpr displayed significantly greater neovascularization at days 11–41 (P<0.05–0.001) and BALB-gld mice had greater neovascularization at days 14–22 (P<0.05–0.01) than did BALB/c controls.

Figure 2

Defective expression of Fas but not FasL, results in increased blepharitis following infection with HSV-1, KOS strain. Eyes of BALB/c wild-type (n=20), BALB-lpr (n=20) and BALB-gld (n=20) mice were infected with 10⁷ pfu of HSV-1, KOS strain and blepharitis measured. Significant virus-induced blepharitis was observed for BALB-lpr (P<0.01) at days 14 and 21 when compared to both BALB/c controls and BALB-gld mice.

Figure 3

Defective expression of both Fas and to a lesser extent FasL, results in increased HSK following infection of C57BL/6 (B6) mice with HSV-1, McKrae strain. Eyes of B6 wild-type (n=25), B6-lpr (n=22) and B6-gld (n=23) mice were infected with 2×10⁶ pfu of HSV-1, McKrae strain. Corneal opacity (A) and corneal neovascularization (B) were measured and compared between these strains of mice. Significant virus-induced corneal opacity was observed for B6-lpr (P<0.01–0.001) at Days 11–30 when compared to B6 controls. B6-gld mice displayed significantly more opacity than did B6 controls at days 14–30 (P<0.05–0.01). B6-lpr displayed significantly greater neovascularization at days 11–44 (P<0.05–0.001) and B6-gld mice had greater neovascularization at days 14–44 (P<0.05–0.01) than did B6 controls.

Figure 4

Increased disease seen in mice carrying the lpr mutation is associated with the genotype of the animals bone marrow derived cells. Chimeric mice were constructed by irradiating either BALB/c or BALB-lpr mice and reconstituting them with bone marrow cells (2×10⁷) from either BALB-lpr and BALB/c respectively. Eyes of chimeric mice were then infected with 10⁷ pfu of KOS strain of HSV-1 with 20 mice in each group. BALB/c mice reconstituted with BALB-lpr bone marrow cells displayed increased disease at weeks 2–4 (P<0.05–0.005).

Figure 5

Inflammatory infiltrate from BALB/c, BALB-lpr, and BALB-gld mice does not indicate strain-specific differences. HSV-infected corneas were removed at days 17 and 23 from mice with severe HSK disease and disaggregated into single-cell suspensions and stained with anti-CD45, CD4, CD8α, Gr-1, CD11b, CD11c, and F4/80 mAb. Cells were analyzed by flow cytometry. Data represents 4 to 6 corneas per group. No significant differences were seen for any particular determination between strains of mice tested.

Figure 6

Defective expression of Fas and FasL, does not increase the magnitude of corneal viral shedding but does prolong shedding. Eyes of BALB/c wild-type (n=30), BALB-lpr (n=25) and BALB-gld (n=25) mice were infected with 10⁷ pfu of HSV-1, KOS strain and corneas were swabbed on the indicated days and then tittered. No significant differences were seen in the magnitude of viral shedding. Results displayed are means ± S.E.M. for each of the groups of mice indicated.

Figure 7

Defective expression of Fas and FasL, does not increase the magnitude of viral replication in infected trigeminal ganglia. Eyes of BALB/c wild-type (n=7 mice/time point), BALB-lpr (n=5 mice/time point) and BALB-gld (n=5 mice/time point) mice were infected with 10⁷ pfu of HSV-1, KOS strain and trigeminal ganglia removed and viral titers determined. No significant differences in viral growth were seen between the mouse strains being compared. Results displayed are means ± S.E.M. for each of the groups of mice indicated.

Figure 8

Defective expression of Fas and FasL results in reduced mortality following infection with HSV-1, McKrae strain. Eyes of C57BL/6 wild-type (n= 40 mice), B6-lpr (n= 40 mice) and B6-gld (n= 35 mice) were infected with 5×10⁶ pfu of HSV-1, McKrae strain and observed for mortality. Data displayed were compiled from two independent studies. Mortality for both B6-lpr and B6-gld were significantly less than that for B6 wild-type mice (p<0.05).