CA-074Me protection against anthrax lethal toxin

Abstract

Anthrax lethal toxin (LT) activates the NLRP1b (NALP1b) inflammasome and caspase-1 in macrophages from certain inbred mouse strains, but the mechanism by which this occurs is poorly understood. We report here that similar to several NLRP3 (NALP3, cryopyrin)-activating stimuli, LT activation of the NLRP1b inflammasome involves lysosomal membrane permeabilization (LMP) and subsequent cytoplasmic cathepsin B activity. CA-074Me, a potent cathepsin B inhibitor, protects LT-sensitive macrophages from cell death and prevents the activation of caspase-1. RNA interference knockdown of cathepsin B expression, however, cannot prevent LT-mediated cell death, suggesting that CA-074Me may also act on other cellular proteases released during LMP. CA-074Me appears to function downstream of LT translocation to the cytosol (as assessed by mitogen-activated protein kinase kinase cleavage), K(+) effluxes, and proteasome activity. The initial increase in cytoplasmic activity of cathepsin B occurs at the same time or shortly before caspase-1 activation but precedes a larger-scale lysosomal destabilization correlated closely with cytolysis. We present results suggesting that LMP may be involved in the activation of the NLRP1b inflammasome.

FIG. 1.

CA-074Me treatment protects against LT-induced macrophage death. (A) RAW 264.7 cells and (B) BALB/cJ BMDMs were incubated with the cathepsin B inhibitor CA-074Me or the vehicle for 2 h at 37°C. Cells were then treated with LT (1 μg/ml) (equal concentrations of PA and LF). Cell viability was accessed after 2.5 h, and the percent viability was calculated relative to control CA-074Me-treated cells.

FIG. 2.

CA-074Me delays but does not prevent MEK cleavage by LF. BALB/cJ BMDMs were primed with LPS (1 μg/ml) for 2 h, pretreated with either 500 μM CA-074Me or vehicle for 10 min, and then treated with LT (1 μg/ml) for various amounts of time. Western blotting was performed with antibodies against the N termini of MEK1, MEK2, and MEK3.

FIG. 3.

CA-074Me blocks activation of caspase-1 by LT. LPS-primed BALB/cJ BMDMs (LPS, 1 μg/ml, 2 h) were pretreated with either 500 μM CA-074Me or vehicle for 10 min, followed by LT (1 μg/ml) for various amounts of time. Western blots with antibodies for the p10 subunit of activated caspase-1 (A) and IL-1β (B) are shown. (C) LPS-primed RAW 264.7 cell sucrose lysates were incubated with active recombinant caspase-1 in the presence of the indicated inhibitors for 2 h and then subjected to Western blotting with an IL-1β antibody to monitor IL-1β cleavage.

FIG. 4.

Saponin-mediated differential permeabilization of plasma and lysosomal membranes. BALB/cJ BMDMs were treated with a range of saponin concentrations in PBS for 10 min on ice. LDH activity and cathepsin B activity of the supernatant were measured in parallel assays. An asterisk indicates the optimum concentration of saponin (125 μg/ml) that results in minimal baseline cathepsin B activity (minimal lysosomal permeabilization) and maximum released LDH activity (maximum cytoplasmic release).

FIG. 5.

LT induces an increase in cytoplasmic cathepsin B activity. BALB/cJ BMDMs were first treated with Leu-Leu-OMe (1 mM) for 30 min (A and B), LT (2 μg/ml) for 60 min (C and D), or left untreated (identified by “NT”). LDH activity and cathepsin B activity after saponin permeabilization of plasma membranes was measured as described in Materials and Methods. Panels B and D show the cathepsin B activity (right panel) and LDH activity (left panel) at the ideal saponin permeabilization concentration (125 μg/ml) in cells treated with Leu-Leu-OMe and LT, respectively. (E) In a similar experiment, BALB/cJ BMDMs were treated with LT (2 μg/ml) for either 50 or 60 min and subjected to a similar analysis of cytoplasmic cathepsin B (right panel) and LDH activities (left panel). An asterisk indicates statistically significant differences (P < 0.005; two-tailed Student t test). No statistically significant differences were noted between all other treatments.

FIG. 6.

LT induces late large-scale lysosomal membrane permeabilization. BALB/cJ BMDMs were stained with LysoTracker Red (100 nM, 30 min) and the same field was imaged every 5 min after LT treatment (2 μg/ml), with or without CA-074Me pretreatment (500 μM, 10 min). NT represents untreated cells stained with the dye. The scale bars represent 50 μm.

FIG. 7.

LT-induced increases in cytoplasmic cathepsin B activity and other known LT-dependent events. BALB/cJ BMDMs were pretreated with lactacystin (10 μM) for 10 min (A) or heat shocked at 45°C for 2 h (B). The cells were then treated with LT (2 μg/ml) for 60 min or left untreated (NT). LDH activity and cathepsin B activity after saponin permeabilization of plasma membranes was measured as described in Materials and Methods. Cathepsin B activities (right panels) and LDH activities (left panels) are indicated at the ideal saponin permeabilization concentration (125 μg/ml). Lactacystin- and heat shock-treated samples treated with toxin were not statistically different for cathepsin B activity from those not treated with toxin (P values are 0.0455 [A] and 0.0473 [B]).

FIG. 8.

The caspase inhibitor Boc-d-CMK acts as a cathepsin B inhibitor. (A) Boc-d-CMK (80 μM, 60 min)-pretreated BALB/cJ BMDMs were left untreated or were treated with LT for 60 min (2 μg/ml). Cytoplasmic cathepsin B activity was assessed after saponin permeabilization as described in Materials and Methods for a range of saponin concentrations. Cathepsin B activity inhibition by Boc-d-CMK after permeabilization at 125 μg of saponin/ml is shown in the inset.

FIG. 9.

Cathepsin B alone may not be necessary for LT-dependent cell death. RAW 264.7 (A) and BALB/cJ BMDMs (B) were nucleofected with cathepsin B siRNA. At 40 h postnucleofection cells were treated with a range of LT concentrations for 105 min (RAW 264.7 cells) (A) or 75 min (BALB/cJ BMDMs) (B) prior to accessing cell viability by the MTT assay. Insets indicate cathepsin B Western blots run in parallel to the cytotoxicity assays. Blots were reprobed with a XIAP antibody to confirm equal loading.