Increased sensitivity to staphylococcal enterotoxin B following adenoviral infection

Abstract

Staphylococcal enterotoxin B induces toxic shock and is a major virulence factor of staphylococcal diseases. We examined the effects of systemic adenoviral infection on responses to staphylococcal enterotoxin B in a murine model. We found that adenoviral infection markedly increases the severity of liver injury following exposure to staphylococcal enterotoxin B without d-galactosamine sensitization. In adenovirus-infected mice, staphylococcal enterotoxin B triggered a more profound hypothermia and increased apoptosis in the liver. Consistent with these observations, we also found that adenoviral infection primed for an increased production of gamma interferon in vivo and in vitro following stimulation with staphylococcal enterotoxin B. Gamma-interferon-knockout mice did not show increased sensitivity to staphylococcal enterotoxin B following adenoviral infection. These data suggest that a preexisting viral infection primes mice for subsequent staphylococcal enterotoxin B exposure, possibly via a gamma-interferon-mediated mechanism.

FIG. 1.

Adenoviral infection increases severity of responses to SEB. (a) DBA/2 mice were infected with human adenovirus (serotype 5, 10⁶ PFU/mouse) via the intravenous route for 7 days. Control and adenovirus-infected mice were challenged with 125 μg/mouse of SEB. Body temperature was measured using an infrared veterinary thermometer. The hypothermia was significantly more profound in adenovirus-infected mice than in control mice at 4 h after SEB treatment (marked with *, P < 0.01, n = 13). (b) Control and adenovirus-infected DBA/2 mice were euthanized at 4 h and 8 h after SEB exposure to collect blood samples. Serum cytokines were measured with ELISA. IFN-γ was significantly higher in adenovirus-infected mice than in control mice at 4 and 8 h after SEB treatment (*, compared to the controls at the same time points, P < 0.01, n = 4). (c) IFN-γ-deficient mice (GKO) on a BALB/c background and wild-type BALB/c mice were infected with human adenovirus (serotype 5, 10⁶ PFU/mouse) via the intravenous route for 7 days. Control and adenovirus-infected mice were challenged with 125 μg/mouse of SEB. Adenovirus-infected BALB/c mice had significantly lower body temperatures from 2 to 8 h after SEB treatment than did other groups at the same time points (marked with *, P < 0.05, n = 4 mice in each group). The kinetics of body temperature is shown in two panels for clarity, but all groups were treated in parallel in the same experiment.

FIG. 2.

Effects of adenoviral infection on T cells in spleen and liver. (a) Spleens from control and adenovirus-infected DBA/2 mice are shown. (b) Flow cytometry analysis of SEB-responsive T cells in the splenocyte populations. Splenocytes from control and adenovirus-infected mice were analyzed after staining with antibodies recognizing Vβ8 TCR (FITC labeled) and CD4 or CD8 (PE labeled). The data are representative of three independent experiments. (c) Flow cytometry analysis of liver mononuclear cells isolated from control and adenovirus-infected mice was performed as in panel b.

FIG. 3.

Production of IFN-γ and IL-2 in splenocyte cultures after in vitro stimulation with SEB. Splenocytes from control and adenovirus-infected mice were stimulated with SEB for 24 h. Cytokines were measured by ELISA. (a) Splenocytes from adenovirus-infected mice produce significantly higher amounts of IFN-γ after 24 h of stimulation with SEB (*, P < 0.05; the data are means ± SEM from seven independent experiments). (b) Splenocytes from adenovirus-infected mice produce moderately lower amounts of IL-2 after 24 h of stimulation with SEB (*, P < 0.01; the data are means ± SEM from seven independent experiments). (c) Depletion of CD4⁺ T cells with magnetic beads prior to stimulation with SEB eliminates IFN-γ production by splenocytes from adenovirus-infected mice. Depletion of CD8⁺ T cells also decreases the levels of IFN-γ. Representative data from two independent experiments are shown (means ± SEM from triplicate cultures).

FIG. 4.

Increased IFN-γ signaling in the livers of adenovirus-infected mice following SEB exposure. Phosphorylation of STAT1 (a) and total levels of STAT1 (b) and NOS2 (c) at 4 h after SEB treatment of DBA/2 mice were evaluated by immunoblot analysis of liver lysates. Densitometry data (means ± SEM of four individual mice per group) are shown. The signal intensities of phospho-STAT1, total STAT1, and NOS2 were significantly higher in the group treated with adenovirus and SEB (*, P < 0.05, compared to the group treated with SEB alone). The signal intensity of total STAT1 was significantly higher in the adenovirus-infected group (#, P < 0.01, compared to control group).Representative scans are shown below each densitometry chart. (d) Phosphorylation of STAT1 and total levels of STAT1 and NOS2 at 4 h after SEB treatment of BALB/c and GKO mice were evaluated by immunoblot analyses. Two mice per group were used to perform immunoblot analyses in individual lysates and shown in two scans for each protein. Equal loading was confirmed by Ponceau S staining of the blots (not shown).

FIG. 5.

SEB exposure increases caspase activity in the liver in adenovirus-infected DBA/2 mice. (a) Caspase activity in the liver lysates was measured using a fluorogenic substrate, Ac-DEVD-AMC, which can be cleaved by caspase 3 or 7. The fluorescence intensity was normalized to protein concentration (relative light units [RLU] per μg protein). Caspase activity was significantly increased in the livers of adenovirus-infected mice at 4 h after SEB treatment and was significantly higher than in the livers of mice treated with SEB alone (n = 5 mice). (b) Cleavage of PARP was evaluated by immunoblot analysis. Densitometry data (means ± SEM of four individual mice per group) are shown. A representative scan is shown. The signal intensity of cleaved PARP (89 kDa) was significantly higher in the group treated with adenovirus and SEB (*, P < 0.01, compared to the group treated with SEB alone). Equal loading was confirmed by Ponceau S staining of the blots (not shown).

FIG. 6.

Adenoviral infection sensitizes mice to liver injury induced by SEB. (a) Serum samples were obtained from control (C) and adenovirus-infected (Ad) mice exposed to SEB for 24 h or left untreated. Serum levels of ALT (expressed in specific units per liter [U/L]) significantly increased in adenovirus-infected mice at 24 h after SEB and were significantly higher than in mice treated with SEB alone (n = 4). (b) TUNEL assay for apoptosis in the liver of control and adenovirus-infected mice at 8 h after SEB exposure. Cryosections were prepared and analyzed from four mice from each group. Representative photomicrographs are shown.