Anthrax lethal toxin impairs CD1d-mediated antigen presentation by targeting the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway

Abstract

Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway.

FIG. 1.

Anthrax lethal toxin inhibits CD1d-mediated antigen presentation in an ERK/12-dependent manner. (A and B) LMTK-CD1d1 cells (A) or BMDCs (B) were treated with vehicle, PA, LF, or LT as indicated, washed, fixed, and cocultured with the indicated NKT cell hybridomas. IL-2 production was measured by ELISA. *, P < 0.001 compared to vehicle. (C) BMDCs were treated with vehicle or LT for 24 h, washed, fixed, and cocultured with primary NKT cells for 48 h. Supernatants were harvested to measure IFN-γ. *, P < 0.001 compared to vehicle treatment. (D) LMTK-CD1d1 cells were treated with vehicle or LT as described above, and the levels of phospho- and total ERK1/2 were analyzed by Western blotting. Lanes: 1, vehicle; 2, PA (1 μg/ml); 3, LF (1 μg/ml); 4, PA (1 μg/ml) plus LF (0.1 μg/ml); 5, PA (1 μg/ml) plus LF (0.5 μg/ml); 6, PA (1 μg/ml) plus LF (0.5 μg/ml). *, P < 0.001 compared to vehicle. (E) LMTK-CD1d1 cells were treated with vehicle or the ERK1/2 pathway-specific inhibitor U0126 (1, 2, 5, 10, or 20 μM) for 24 h, washed, fixed, and cocultured with the indicated NKT cell hybridomas. *, P < 0.001 compared to vehicle. Error bars indicate standard deviations.

FIG. 2.

Anthrax lethal toxin impairs MHC class II-mediated antigen presentation. (A) TA3 cells were treated with vehicle or LT in the presence of HEL, fixed, and cocultured with the 3A9 T cell hybridoma. *, P < 0.001 compared to vehicle. (B) L-CD1d1-DR4 cells were treated with vehicle or LT in the presence of HSA, fixed, and then cocultured with the 17.9 T cell hybridoma. *, P < 0.001 compared to vehicle. (C) BMDCs from C3H/HeN mice were treated with vehicle, PA, LF, or LT in the presence of HEL; fixed; and cocultured as described above. The amount of IL-2 in the supernatant was measured by ELISA. *, P < 0.001 compared to vehicle. Error bars indicate standard deviations.

FIG. 3.

Inhibition of ERK1/2 impairs MHC class II-mediated antigen presentation. (A) TA3 cells were treated with vehicle or the indicated concentrations of U0126 in the presence of HEL, fixed, and cocultured with the 3A9 T cell hybridoma. *, P < 0.001 compared to vehicle. (B) L-CD1d1-DR4 cells were treated with vehicle or the indicated concentrations of U0126 in the presence of HSA, fixed, and then cocultured with the 17.9 T cell hybridoma. *, P < 0.001 compared to vehicle. (C) BMDCs from C3H mice were treated with vehicle or the indicated concentrations of U0126 in the presence of HEL, fixed, and cocultured as described above. The amount of IL-2 in the supernatant was measured by ELISA. *, P < 0.001 compared to vehicle. Error bars indicate standard deviations.

FIG. 4.

Treatment with LT alters the intracellular localization of CD1d. LMTK-CD1d1 cells were treated with vehicle, PA (1 μg/ml), LF (1 μg/ml), LT (PA plus LF at 1 μg/ml each), or U0126 (10 μM) as described in the legend to Fig. 1. The cells were then washed, fixed, stained for CD1d (green) and LAMP-1 (red), and analyzed by confocal microscopy (A), and six random fields were chosen to calculate the percent colocalization (B). *, P < 0.001 compared to vehicle alone. Error bars indicate standard deviations.