Exogenous interleukin-12 protects against lethal infection with coxsackievirus B4

Abstract

Infections with the group B coxsackieviruses either can be asymptomatic or can lead to debilitating chronic diseases. To elucidate the mechanism by which these viruses cause chronic disease, we developed a mouse model of chronic pancreatitis by using a virulent variant of coxsackievirus B4, CVB4-V. Infection with CVB4-V results in an early, severe pancreatitis, which can lead to mortality or progress to chronic pancreatitis. Chronic pancreatitis, in this model, is due to immunopathological mechanisms. We investigated whether interleukin-12 (IL-12) could modulate the outcome of CVB4-V infection. Eighty-five percent of the infected mice treated with 500 ng of IL-12 survived, whereas all untreated mice succumbed. To understand the mechanism underlying the beneficial effect of IL-12, we investigated the role of gamma interferon (IFN-gamma). Three lines of evidence suggest that the protective effect of IL-12 is due to IFN-gamma. First, administration of IL-12 increased the production of endogenous IFN-gamma in CVB4-V-infected mice. Both NK and NKT cells were identified as the source of IFN-gamma. Second, IFN-gamma knockout mice treated with IL-12 succumbed to infection with CVB4-V. Third, wild-type mice treated with IFN-gamma survived infection with CVB4-V. Due to the antiviral effects of IFN-gamma, we examined whether IL-12 treatment affected viral replication. Administration of IL-12 did not decrease viral replication in the pancreas, but it did prevent extensive tissue damage and the subsequent development of chronic pancreatitis. The data suggest that IL-12 treatment during CVB4-V infection is able to suppress the immunopathological mechanisms that lead to chronic disease.

FIG. 1.

Survival of CVB4-V-infected, IL-12-treated mice. Infected mice treated with 500 ng of IL-12 (dashed line) or vehicle alone (solid line) were monitored daily for 2 weeks. Mice that became moribund were euthanized immediately. Each group contained 20 to 21 mice. Treatment with 500 ng of IL-12 significantly (P < 0.001) increased the survival of CVB4-V-infected mice.

FIG. 2.

Viral replication in the pancreatic tissues of untreated and IL-12-treated mice. Pancreata harvested from CVB4-V-infected, IL-12-treated mice (○) and from infected, untreated mice (•) were assayed for viral infectivity by plaque assay. Organs from four mice were analyzed at each time point. Mean values and standard deviations are shown.

FIG. 3.

Histopathology of pancreatic tissues from CVB4-V-infected mice treated with either IL-12 or IFN-γ. Pancreata were harvested at 14 days postinfection, processed for routine histology, and stained with hematoxylin and eosin. (A) Uninfected; (B) CVB4-V-infected; (C) CVB4-V-infected and treated with 500 ng of IL-12; (D) CVB4-V-infected and treated with 100 ng of IFN-γ on multiple days (0.5, 1, and 2) after infection. Labeling: A, acini; I, islet of Langerhans; L, lymphocytic infiltrate; AN, acinar cell necrosis. Magnifications: ×234 for panels A, B, and C; ×117 for panel D.

FIG. 4.

IFN-γ production in CVB4-V-infected mice treated with IL-12. Organs and sera were collected from groups (n = 3) of IL-12-treated (○) or untreated (•) mice at various times after infection and assayed for IFN-γ by ELISA. Uninfected mice (▪) were included as controls. (A) Serum; (B) spleen; (C) pancreas; (D) heart. Each datum point represents the result for one mouse. The mean value for each group is shown. Statistically significant differences (P < 0.05) between IL-12-treated and untreated mice are denoted by asterisks.

FIG. 5.

Administration of IL-12 increases IFN-γ production by NK and NKT cells. Intracellular IFN-γ staining of splenocytes from IL-12-treated and CVB4-V-infected, CVB4-V-infected, and uninfected mice. (A) Representative histograms of IFN-γ staining (thick lines) NK cells and background staining (thin lines). Each panel represents data from one mouse. (B) Summary of IFN-γ staining NK cells, NKT cells, and T cells from CVB4-V-infected, IL-12-treated mice, CVB4-V-infected mice, and uninfected mice (n = 6 for each group). Theresults are expressed as the percentage of each cell population staining for IFN-γ minus the background staining. Mean values and standard deviations are shown. Statistically significant differences (P < 0.01) between IL-12-treated and untreated mice are denoted by asterisks.

FIG. 6.

Cytokine treatment of CVB4-V-infected mice. (A) Administration of IL-12 to CVB4-V-infected GKO mice was not protective. Each group contained seven mice. Curves: dashed line, 500 ng of IL-12; solid line, vehicle alone. (B) Administration of IFN-γ to CVB4-V-infected, BALB/c mice was protective. Each group contained four to six mice. IFN-γ treatment significantly (P < 0.05) increased survival of CVB4-V-infected mice. Curves: dotted line, 50 ng of IFN-γ; dashed line, 100 ng of IFN-γ; solid line, vehicle alone.

FIG. 7.

Model of IL-12-induced suppression of immunopathology during CVB4-V infection. Administration of IL-12 (or IFN-γ) early in infection prevents the development (or functioning) of effector CD8 T cells involved in immunopathology.