Interferon regulatory factor 3-dependent pathways are critical for control of herpes simplex virus type 1 central nervous system infection

Abstract

The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via interferon (IFN) regulatory factor 3 (IRF-3)-dependent pathways. The absence of IRF-3 would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-3(-/-) mice following intravenous HSV type 1 (HSV-1) challenge (K. Honda, H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi, Nature 434:772-777, 2005). In contrast, the present study demonstrated that IRF-3(-/-) mice are significantly more susceptible to HSV infection via the corneal and intracranial routes. Following corneal infection with 2 x 10(6) PFU of HSV-1 strain McKrae, 50% of wild-type mice survived, compared to 10% of IRF-3-deficient mice. Significantly increased viral replication and inflammatory cytokine production were observed in brain tissues of IRF-3(-/-) mice compared to control mice, with a concomitant deficit in production of both IFN-beta and IFN-alpha. These data demonstrate a critical role for IRF-3 in control of central nervous system infection following HSV-1 challenge. Furthermore, this work underscores the necessity to evaluate multiple routes of infection and animal models in order to fully determine the role of host resistance factors in pathogenesis.

FIG. 1.

Loss of IRF-3 has minimal impact on HSV-1 replication in peripheral tissues following cornea infection. WT and IRF-3^−/− mice were infected with 2 × 10⁶ PFU HSV-1 McKrae per eye. Cornea swabs and trigeminal ganglia were harvested and titers determined at the specified days. The graphs represent averages and standard deviations for several mice at each time point from two independent experiments (n ≥ 6).

FIG. 2.

IRF-3^−/− mice have decreased survival and increased viral replication in brain tissues following HSV-1 McKrae cornea infection. (A) Survival plot following infection of WT and IRF-3^−/− mice with 2 × 10⁶ PFU HSV-1 McKrae per eye. Survival experiments were conducted independently of the other experiments and represent the sum of multiple experiments (n = 21). (B and C) Brains and brain stems were harvested and titers determined at the specified days following infection of WT and IRF-3^−/− mice with 2 × 10⁶ PFU HSV-1 McKrae per eye. The graphs represent the averages and standard deviations for several mice from two independent experiments (n ≥ 5). The dotted line represents the limit of detection for this assay. **, P < 0.01.

FIG. 3.

IRF-3^−/− mice have reduced survival and increased viral titers in the brain following HSV-1 intracranial infection. (A) Survival plot of IRF-3^−/− and WT mice following intracranial (i.c.) infection with 100 PFU HSV-1 17. Survival experiments were conducted independent of the other experiments and represents the sum of two experiments (n ≥ 31). (B) Viral titers in whole brain tissue harvested at the specified days. Data represent the averages and standard deviations for several mice from two independent experiments (n ≥ 9). The dotted line represents the limit of detection for this assay. *, P < 0.05; **, P < 0.01.

FIG. 4.

IRF-3^−/− brain sections have increased antigen staining following intracranial HSV-1 infection. Following i.c. infection with 100 PFU HSV-1 strain 17, brains were harvested on days 3 and 5 postinfection, formalin fixed, sectioned sagitally, and stained with a polyclonal anti-HSV antibody. Sections were divided into five regions (olfactory bulb, central brain, mid-brain cerebellum, and pons/medulla/brain stem) and scored as either positive or negative for HSV antigen staining in a masked fashion. (A) Following scoring, total antigen-positive regions were then divided by total sections counted in order to calculate a percentage of antigen-positive regions for day 3 and day 5. (B and C) Representative anti-HSV-1 peroxidase staining images from the central brain regions of WT and IRF-3^−/− mice. *, P < 0.05; **, P < 0.01.

FIG. 5.

IRF-3^−/− mice show increased inflammatory cytokine production following intracranial infection with HSV-1. Following i.c. infection with 100 PFU HSV-1 17, brains were harvested on days 3 and 5 postinfection, processed, and assayed via a bead-based cytokine assay (BioPlex; Bio-Rad). The results shown are the averages and standard deviations for four to six mouse brains per group per time point. Statistical calculations were based on infected WT and infected IRF-3^−/− mice. *, P < 0.05.

FIG. 6.

IRF-3^−/− mice show increased inflammatory cytokine production following peripheral infection. Following ocular infection with 1 × 10⁶ PFU HSV-1 McKrae per eye, brains were harvested on days 3 and 5 postinfection, processed, and assayed via a bead-based cytokine assay (BioPlex; Bio-Rad). The results shown are the averages and standard deviations for four to six mouse brain stems per group per time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 7.

IRF-3^−/− mice have a deficit in type I IFN production following intracranial infection with HSV-1. WT and IRF-3^−/− mice were infected i.c. with 1 × 10⁶ PFU HSV-1 strain 17. (A and B) Whole brain tissue was harvested at the specified times, processed, and analyzed for IFN-β (A) and IFN-α (B) by ELISA (PBL Laboratories). The results shown represent the averages and standard deviations for 10 to 14 mice per group per time point from two separate experiments. (C) Following infection with 100 PFU HSV-1 strain 17, brain tissue, excluding brain stem and olfactory bulb, was harvested for RNA at 18 h postinfection. Samples were assayed by real-time RT-PCR, and results are expressed as fold expression over that for mock-infected samples. The results shown are the average fold expression and standard deviation from six or seven mice per group per time point from two separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.