Polarized type 1 cytokine response and cell-mediated immunity determine genetic resistance to mousepox

Abstract

Ectromelia virus (ECTV), a natural mouse pathogen and an orthopoxvirus, has been used to investigate the correlation between polarized type 1 or type 2 immune responses and resistance to disease in poxvirus infections by using well defined resistant and susceptible mouse strains. Our data show that distinct differences exist in the cytokine profiles expressed in resistant and susceptible mice infected with ECTV. Resistant C57BL/6 mice generate a type 1 cytokine response [IFN-gamma, IL-2, and tumor necrosis factor (TNF)], within the first few days of infection, which is associated with strong cytotoxic T lymphocyte response (CTL) and recovery from ECTV infection. Susceptible strains of mice (BALB/c and A/J) on the other hand generate a type 2 cytokine response (IL-4 but little or no IFN-gamma and IL-2), which is associated with a weak or an absent CTL response, resulting in uncontrolled virus replication and death. Although deletion of IL-4 function alone did not change the outcome of infection in susceptible mice, the loss of IFN-gamma function in resistant mice abrogated natural killer (NK) cell and CTL effector functions resulting in fulminant disease and 100% mortality. Therefore, a clear link exists between the early production of specific type 1 cytokines, in particular, IFN-gamma, the nature of the cellular immune response, and disease outcome in this virus model. This finding in the mousepox model raises the possibility that inappropriate cytokine responses may result in increased susceptibility to smallpox in humans.

Fig. 1.

ECTV titers in resistant and susceptible mice. Groups of C57BL/6, BALB/c, and A/J mice infected with ECTV were killed on the days indicated. Virus titers in PLN (A), spleen (B), and liver (C) of individual animals were determined. BALB/c and A/J mice did not survive beyond day 7. Virus titers are expressed as mean log₁₀ PFU per g of tissue ± SEM from five mice per group. The limit of detection is 2 log₁₀ PFU (indicated by the dotted line).

Fig. 2.

NK cell and ECTV-specific CTL activity. Groups of five C57BL/6, BALB/c, and A/J mice were infected with ECTV. Splenic NK cell activity (A) was measured by using ⁵¹Cr-labeled YAC-1 targets. Data shown are mean ± SEM of lysis values from five individual mice for each group at effector-to-target-cell ratio of 20:1. Anti-ECTV-specific CTL responses in the PLN (B) and spleen (C) were measured by using ⁵¹Cr release assay. Data shown are mean ± SEM of lysis values (lysis of virus-infected syngeneic targets minus lysis of uninfected targets) of pooled PLN from five mice (B) and from five individual spleens (C) at an effector-to-target-cell ratio of 20:1. The results are representative of two separate experiments.

Fig. 3.

ECTV-specific Ab response. Sera were collected from groups of five mice at days indicated p.i. and levels of ECTV-specific IgM (A) and IgG (B) determined for individual animals by ELISA at a serum dilution of 1:200. Data presented are expressed as mean ± SD of one of three separate animal experiments.

Fig. 4.

Kinetics of cytokine production in resistant and susceptible mice. C57BL/6(A and C) and BALB/c(B and D) mice were inoculated with ECTV, and groups of three mice were killed on the days indicated. PLN (A and B) and spleen (C and D) from each mouse were sectioned, stained for cytokine-producing cells, and positive cells were counted. Data presented are expressed as means of three mice from each group.

Fig. 5.

Effect of cytokine neutralization on NK cell and ECTV-specific CTL responses. Groups of three C57BL/6 mice were infected with ECTV and untreated or treated with neutralizing mAbs against murine IL-2, TNF, or IFN-γ. Mice were killed at day 6 p.i., and NK cell activity (A) and anti-ECTV-specific CTL responses (B) were measured as for Fig. 2. The results are expressed as mean ± SEM (these fall within the symbols for most data points) of one of two separate experiments.