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. 2017 May 15;198(10):3989-3998.
doi: 10.4049/jimmunol.1601525. Epub 2017 Mar 20.

Interference of the T Cell and Antigen-Presenting Cell Costimulatory Pathway Using CTLA4-Ig (Abatacept) Prevents Staphylococcal Enterotoxin B Pathology

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Free PMC article

Interference of the T Cell and Antigen-Presenting Cell Costimulatory Pathway Using CTLA4-Ig (Abatacept) Prevents Staphylococcal Enterotoxin B Pathology

Sarah J C Whitfield et al. J Immunol. .
Free PMC article

Abstract

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds the receptors in the APC/T cell synapse and causes increased proliferation of T cells and a cytokine storm syndrome in vivo. Exposure to the toxin can be lethal and cause significant pathology in humans. The lack of effective therapies for SEB exposure remains an area of concern, particularly in scenarios of acute mass casualties. We hypothesized that blockade of the T cell costimulatory signal by the CTLA4-Ig synthetic protein (abatacept) could prevent SEB-dependent pathology. In this article, we demonstrate mice treated with a single dose of abatacept 8 h post SEB exposure had reduced pathology compared with control SEB-exposed mice. SEB-exposed mice showed significant reductions in body weight between days 4 and 9, whereas mice exposed to SEB and also treated with abatacept showed no weight loss for the duration of the study, suggesting therapeutic mitigation of SEB-induced morbidity. Histopathology and magnetic resonance imaging demonstrated that SEB mediated lung damage and edema, which were absent after treatment with abatacept. Analysis of plasma and lung tissues from SEB-exposed mice treated with abatacept demonstrated significantly lower levels of IL-6 and IFN-γ (p < 0.0001), which is likely to have resulted in less pathology. In addition, exposure of human and mouse PBMCs to SEB in vitro showed a significant reduction in levels of IL-2 (p < 0.0001) after treatment with abatacept, indicating that T cell proliferation is the main target for intervention. Our findings demonstrate that abatacept is a robust and potentially credible drug to prevent toxic effects from SEB exposure.

Figures

FIGURE 1.
FIGURE 1.
Murine splenocyte proliferation and cytokine assay. Isolated splenocytes were treated with PBS (negative control), SEB (positive control), or SEB and abatacept (n = 5). Proliferation assays were performed. Abatacept-treated cells were normalized against the negative control and the positive SEB control (A) (0 and 100%, respectively). Supernatants were analyzed for IL-1β (B), IL-2 (C), and CCL2 (D) production. Repeated-measures ANOVA showed that the effect of treatment was significant for proliferation, F(6, 24) = 7.141, p = 0.0002; IL-1β, F(6, 24) = 3.948, p = 0.0069; IL-2, F(6, 24) = 5.831, p = 0.0007; and CCL2, F(6, 24) = 3.988, p = 0.0065. Means of five replicates and SEM are represented. Post hoc analysis using Dunnett multiple comparison are indicated. *p < 0.05, **p < 0.01, ***p < 0.001. ns, p > 0.05.
FIGURE 2.
FIGURE 2.
Murine model of SEB with abatacept treatment. Kaplan–Meier plot of SEB mouse survival exposed to 0.25 and 0.5 μg SEB per gram of mouse weight (A) and weight change after abatacept treatment at 3 (B) or 8 h (C) postexposure. Significant differences in survival were observed between the control PBS group and mice receiving 0.5 μg/g SEB (χ2 = 24.24, p < 0.0001) or 0.25 μg/g SEB (χ2 = 5.52, p = 0.0188). For weight change, graphs represent mean with error bars for 95% CI. Clinical signs of mice receiving PBS (D), SEB (E), or SEB followed by abatacept treatment (F) at 8 h are shown. Graphs represent the percentage of mice with severe, moderate, mild, or no clinical signs plotted for each time point. Statistical differences are summarized as: *p < 0.05, ****p < 0.0001.
FIGURE 3.
FIGURE 3.
Lung pathology scores and organ to body percent weight of lung. Lungs were removed and scored for visible signs of damage (A) and histopathology scores (B), and subsequently weighed to determine the lung-to-body weight ratio (C). Scores ranged from 1 (normal) to 4 (very severe lung pathology/75–100% inflammatory cell infiltrate), as defined in the Materials and Methods. Lung visual pathology, cell infiltrate scores, and lung-to-body weight ratio were analyzed by Kruskal–Wallis test with post hoc Dunn multiple comparison test. Visual pathology scores significance for treatment were: day 3, p = 0.0002; day 6, p < 0.0001; and day 14, p = 0.0169. Cell infiltrate scores significance for treatment were: day 3, p < 0.0001; day 6, p < 0.0001; and day 14, p = 0.0029. Lung-to-body weight ratio had significant differences in treatment: day 3, p < 0.0001; day 6, p < 0.0001; and day 14, p = 0.0059. Graphs for lung pathology, histopathology scores, and lung-to-body weight ratio represent median and range. Post hoc analysis using Dunnett multiple comparison are indicated. *p < 0.05, **p < 0.01, ***p < 0.001. ns, p > 0.05.
FIGURE 4.
FIGURE 4.
Histopathology and MRI of lungs after SEB intoxication in mice. Tissue sections were stained using H&E, at a nominal 5-μm thickness before assessment [SEB treated (A, D, and E); PBS-control (B); and SEB after abatacept therapy (C and F)]. White boxes highlight air space within the lung, and white arrows indicate inflammatory cell infiltrate; SEB lung pathology is predominantly characterized by an acute alveolar inflammatory cell infiltrate. Scale bar, 100 μm. Mice were sacrificed on days 3, and whole-body imagery was performed on SEB-treated (G), PBS-control (H), or SEB mice treated with abatacept at 8 h (I). White boxes indicate lung cavity, whereas gray shading within white boxes indicates the presence of fluid.
FIGURE 5.
FIGURE 5.
Plasma concentrations of cytokines. Plasma concentrations of IFN-γ (A), IL-6 (B), IL-1β (C), IL-5 (D), TNF-α (E), and IL-2 (F) were measured for six animals in the SEB negative control, PBS positive control, and SEB-treated groups at 3, 6, and 14 d post SEB exposure. Two-way ANOVA analysis was performed for cytokines with post hoc Bonferroni multiple comparison test. Significance for treatment was: IFN-γ, F(2, 46) = 34.75, p < 0.0001; IL-6, F(2, 46) = 9.61, p = 0.0003; IL-1β, F(2, 46) = 3.88, p = 0.0277; IL-5, F(2, 46) = 1.87, p = 0.1652; TNF-α, F(2, 46) = 346.15, p < 0.0001; and IL-2, F(2, 46) = 7.66, p = 0.0013. Significance for time was: IFN-γ, F(2, 46) = 44.37, p < 0.0001; IL-6, F(2, 46) = 22.23, p < 0.0001; IL-1β, F(2, 46) = 5.19, p = 0.0093; IL-5, F(2, 46) = 5.32, p = 0.0084; TNF-α, F(2, 46) = 8.28, p = 0.0008; and IL-2, F(2, 46) = 1.41, p = 0.254. Graphs depict mean cytokine values with SEM. Post hoc Bonferroni multiple comparison tests are summarized as: *p < 0.05, **p < 0.01, ****p < 0.0001.
FIGURE 6.
FIGURE 6.
Lung tissue extract concentrations of IFN-γ, IL-6, CCL2, IL-1β, IL-5, and TNF-α. Lung tissue extract concentrations of IFN-γ (A), IL-6 (B), CCL2 (C), IL-1β (D), IL-5 (E), and TNF-α (F) were measured. One-way ANOVA analysis was performed for cytokines with post hoc Dunnett multiple comparison test. Significance for treatment was: IFN-γ, F(2, 15) = 10.04, p = 0.0017; IL-6, F(2, 15) = 14.95, p = 0.0003; CCL2, F(2, 15) = 13.16, p = 0.0005; IL-1β, F(2, 15) = 3.729, p = 0.0485; IL-5, F(2, 15) = 6.120, p = 0.0114; and TNF-α, F(2, 15) = 10.29, p = 0.0015. Significant difference graphs depict mean cytokine values with SEM. Post hoc Dunnett multiple comparison test are summarized as: *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 7.
FIGURE 7.
Human PBMC stimulation assay for intracellular and secreted cytokine expression. Human PBMCs isolated from volunteers and treated with SEB in combination with PBS (control), abatacept (1 and 10 μg/ml of treatment), or ipilimumab (1 and 10 μg/ml of CTLA4 pathway antagonist). Intracellular cytokine production was determined by flow cytometry for TNF-α, IL-2, and IFN-γ (n = 3 per group). Representative gating strategy (A) and pooled data are shown (B). Supernatants were analyzed by ELISA to determine secretion TNF-α, IL-2, and IFN-γ (n = 4 per group) (C). Two-way ANOVA with Dunnett multiple comparison test. Significance for intracellular cytokine treatment was F(2, 6) = 3.25, p = 0.6805 and for ELISA was F(2, 9) = 14.31, p = 0.0016. Bar graphs represent median 95% CI. Post hoc Dunnett multiple comparison test are summarized as: *p < 0.05, ****p < 0.0001.

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