Bacillus anthracis capsule activates caspase-1 and induces interleukin-1beta release from differentiated THP-1 and human monocyte-derived dendritic cells

Abstract

The poly-gamma-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infection. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Recently, efforts have been made to include PGA as a component of anthrax vaccine; however, the innate immune response of PGA itself has been poorly investigated. In this study, we characterized the innate immune response elicited by PGA in the human monocytic cell line THP-1, which was differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and human monocyte-derived dendritic cells (hMoDCs). PGA capsules were isolated from the culture supernatant of either the pXO1-cured strain of B. anthracis H9401 or B. licheniformis ATCC 9945a. PGA treatment of differentiated THP-1 cells and hMoDCs led to the specific extracellular release of interleukin-1beta (IL-1beta) in a dose-dependent manner. Evaluation of IL-1beta processing by Western blotting revealed that cleaved IL-1beta increased in THP-1 cells and hMoDCs after PGA treatment. Enhanced processing of IL-1beta directly correlated with increased activation of its upstream regulator, caspase-1, also known as IL-1beta-converting enzyme (ICE). The extracellular release of IL-1beta in response to PGA was ICE dependent, since the administration of an ICE inhibitor prior to PGA treatment blocked induction of IL-1beta. These results demonstrate that B. anthracis PGA elicits IL-1beta production through activation of ICE in PMA-differentiated THP-1 cells and hMoDCs, suggesting the potential for PGA as a therapeutic target for anthrax.

FIG. 1.

Dose-dependent cytokine induction in THP-1 cells treated with PGA. Differentiated THP-1 cells were treated with various concentrations (0, 10, 20, 40, 80, and 100 μg/ml) of PGA from the pXO1-cured strain of B. anthracis H9401 (A) or from B. licheniformis ATCC 9945a (B). Extracellular levels of IL-1β were measured in triplicate by ELISA after 24 or 48 h of treatment. Each bar represents the average concentration from triplicate ELISAs. Bars represent the interassay standard deviations.

FIG. 2.

Dose-dependent cytokine induction in hMoDCs treated with PGA. hMoDCs were treated with various concentrations (0, 10, 20, 40, 80, and 100 μg/ml) of PGA from the pXO1-cured strain of B. anthracis H9401 (A) or from B. licheniformis ATCC 9945a (B). Extracellular levels of IL-1β were measured in triplicate by ELISA after 24 or 48 h of treatment. Each bar represents the average concentration from triplicate ELISAs. Bars represent the interassay standard deviations.

FIG. 3.

Processing of IL-1β, activation of ICE, and dose-dependent inhibition of IL-1β production by an ICE inhibitor in PGA-treated THP-1 cells. Cultures of THP-1 cells were treated with a fixed dose of PGA (100 μg/ml) from the pXO1-cured strain of B. anthracis H9401 (A and C) or from B. licheniformis ATCC 9945a (B and D) for increasing periods as indicated. Protein lysates generated from these cultures were analyzed by Western blotting (50 μg of total protein per lane) for the presence of cleaved IL-1β (17 kDa) (A and B, upper panels), the activated cleavage product of ICE, p20 (C and D, upper panels), and β-actin (A, B, C, and D, lower panels). Shown are the results of one representative experiment from three separate experiments. For the inhibitor experiments, cultures of THP-1 cells were pretreated with or without increasing concentrations of Z-WEHD-FMK. After 30 min of pretreatment, cultures were treated with or without 100 μg of PGA/ml from the pXO1-cured strain of B. anthracis H9401 (E) or from B. licheniformis ATCC 9945a (F) for 48 h. Values represent the average extracellular cytokine concentrations of IL-1β measured in triplicate by ELISA. Bars indicate the interassay standard deviations.

FIG. 4.

Processing of IL-1β, activation of ICE, and dose-dependent inhibition of IL-1β production by an ICE inhibitor in PGA-treated hMoDCs. hMoDCs were treated with a fixed dose of PGA (100 μg/ml) from the pXO1-cured strain of B. anthracis H9401 (A and C) or from B. licheniformis ATCC 9945a (B and D) for increasing periods as indicated. Protein lysates generated from these cultures were analyzed by Western blotting (50 μg of total protein per lane) for the presence of cleaved IL-1β (17 kDa) (A and B, upper panels), the activated cleavage product of ICE, p20 (C and D, upper panels), and β-actin (A, B, C, and D, lower panels). Shown are the results of one representative experiment from three separate experiments. For the inhibitor experiments, cultures of hMoDCs were pretreated with or without increasing concentrations of Z-WEHD-FMK. After 30 min of pretreatment, cultures were treated with or without 100 μg of PGA/ml from the pXO1-cured strain of B. anthracis H9401 (E) or from B. licheniformis ATCC 9945a (F) for 48 h. Values represent the average extracellular cytokine concentrations of IL-1β measured in triplicate by ELISA. Bars indicate the interassay standard deviations.