Toxin inhibition of antimicrobial factors induced by Bacillus anthracis peptidoglycan in human blood

Abstract

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.

Fig 1

AT-mediated suppression of cytokines/chemokines in BaPGN-treated WBCs after 6 h. Fresh human WBCs were treated with BaPGN (10 μg/ml) for 6 h in the presence or absence of ET (25 pmol EF and 25 pmol PA), LT (25 pmol LF and 25 pmol PA), or AT (25 pmol EF, 25 pmol LF, and 25 pmol PA). (A and B) Antibody array depicting cytokines/chemokines secreted by WBCs under each condition. The array shown is representative of 3 experiments. Circled 1, GRO-α; circled 2, CXCL10; circled 3, MCP1. (C and D) Relative abundances of the cytokines/chemokines induced by BaPGN and modulated by ET and LT, as determined by densitometry (n = 3). The error bars indicate the standard errors of the mean (SEM). The asterisks indicate a significant (P < 0.05) decrease compared to BaPGN alone.

Fig 2

Immunosuppressive effects of AT on BaPGN-treated human monocytes. Fresh human WBCs were treated with BaPGN, ET, or LT alone or BaPGN plus ET or LT, as for Fig. 1. To identify the major cell types, the WBCs were stained with antibodies to cell surface markers: CD14 (monocytes), CD16b (neutrophils), CD19 (B lymphocytes), and CD3 (T lymphocytes). The cells were then stained for intracellular cytokines/chemokines, and flow cytometry was used to analyze cytokine/chemokine production for each cell type. (A) Representative contour plots showing cytokine (IL-1β and TNF-α) and chemokine (IL-8 and MIP-1α) staining intensities (horizontal axis) in monocytes. The numbers in the upper right corners indicate the percentages of cells expressing the relevant surface marker (CD14) as well as the cytokine or chemokine. (B and C) Intracellular levels of cytokines IL-1α, IL-1β, IL-6, and TNF-α (B) and chemokines GRO-α, IL-8, MIP-1α, and MIP-1β (C) in monocytes, quantified as the MFI. The data shown are the averages of three separate experiments, each using WBCs from a different donor. The error bars indicate SEM. The asterisks indicate a significant (P < 0.05) decrease in MFI compared to BaPGN treatment alone.

Fig 3

Immunosuppressive effects of AT on BaPGN-treated human neutrophils. Fresh human WBCs were treated with BaPGN, ET, or LT alone or BaPGN plus ET or LT, as for Fig. 1, and then stained with antibodies to intracellular cytokines/chemokines and to cell surface markers: CD14 (monocytes), CD16b (neutrophils), CD19 (B lymphocytes), and CD3 (T lymphocytes). Cytokine/chemokine profiles for each cell type were then analyzed by flow cytometry. (A) Representative contour plots showing the staining intensities of cytokines (IL-1β and TNF-α) and chemokines (IL-8 and MIP-1α) in neutrophils. The numbers in the upper right corners indicate the percentages of cells in the samples that stained positive for the relevant cytokine or chemokine. (B and C) Intracellular levels of cytokines IL-1α, IL-1β, IL-6, and TNF-α (B) and chemokines GRO-α, IL-8, MIP-1α, and MIP-1β (C) in neutrophils, quantified as MFI. The data shown are the averages of three separate experiments, each using WBCs from a different donor. The error bars indicate SEM. The asterisks indicate a significant (P < 0.05) decrease compared to treatment with BaPGN alone.

Fig 4

Cell types targeted by anthrax toxin in human blood. (A to D) Fresh human WBCs were stained with antibodies against ANTXR1 (A and B) or ANTXR2 (C and D) and cell surface markers CD14 (monocytes), CD16b (neutrophils), CD19 (B lymphocytes), and CD3 (T lymphocytes). Flow cytometry was then used to analyze the expression levels of the receptors on individual cell types. The bar graphs (A and C) indicate ANTXR1 and ANTXR2 expression levels for each cell type, quantified as MFI (n = 3). Representative histograms (B and D) show ANTXR1 and ANTRX2 expression levels for monocytes and neutrophils. The results shown are representative of three different experiments. The error bars indicate SEM. *, P < 0.05 compared with monocytes. (E and F) Human peripheral blood was incubated with fluorescently labeled PA at 37°C for 1 or 2 h and then stained with antibodies recognizing CD14 (monocytes) and CD16b (neutrophils). Flow cytometry was used to analyze the intensity of PA staining on monocytes and neutrophils. A control sample was incubated with unlabeled PA for 1 h prior to the addition of labeled PA. A graph depicting the amounts of PA bound to monocytes and neutrophils, as indicated by MFI (E), and a representative histogram (F) are shown. The results shown are representative of three different experiments. The error bars indicate SEM. For each condition, the MFI for neutrophils was significantly lower than that for monocytes (P < 0.05 for each pairwise comparison). The asterisks indicate a significant (P < 0.05) decrease compared to 1 h of treatment with labeled PA.

Fig 5

Anthrax toxin suppresses the antimicrobial effects of supernatants from BaPGN-treated WBCs. WBCs were treated with BaPGN alone and in combination with AT as described in Materials and Methods for 15 h. (A) Filter-sterilized supernatants from WBCs alone (untreated control) (WBC sup), WBCs treated with BaPGN (WBC_BaPGN sup), and WBCs treated with BaPGN plus AT (WBC_BaPGN+AT sup) were then exposed to mid-log-phase B. anthracis Sterne 7702 vegetative cells. Bacterial growth curves were recorded in a Bioscreen C plate reader for at least three independent WBC supernatants, and the mean absorbances were plotted in GraphPad Prism software. BaPGN-treated (■) WBCs released factors that markedly reduced bacterial growth during the early stationary phase in comparison to the untreated control (●), which was suppressed by anthrax toxin (♢) when it was combined with BaPGN. The error bars indicate SEM. (B, C, and D) Bacterial cultures from WBC supernatants treated with BaPGN alone and BaPGN plus AT were harvested after 24 h, and images were taken using an IX51 Olympus microscope with a DP70 camera. Representative images from at least three independent experiments are shown at ×600 magnification. BaPGN-treated WBC supernatants markedly reduced the vegetative bacilli after 24 h of growth, which was restored by a combination of anthrax toxin and BaPGN, suggesting a role of AT in the suppression of antimicrobial effects of factors released by BaPGN-treated WBCs.

Fig 6

WBC-mediated antimicrobial effects are FtsX dependent and are suppressed by lethal toxin. Experiments were performed to determine the individual effects of ET and LT on the production of antimicrobial factors by human WBCs. The requirement for B. anthracis FtsX was also determined. (A) Growth of B. anthracis Sterne in the presence of supernatants from BaPGN-treated WBCs or BaPGN-treated WBCs cotreated with ET. (B) Growth of B. anthracis Sterne in the presence of supernatants from BaPGN-treated WBCs or BaPGN-treated WBCs cotreated with LT. (C) B. anthracis ΔftsX mutant growth in the presence of supernatants from BaPGN-treated WBCs. At least three independent experiments were performed with three individual donors, and the error bars represent the standard deviations from the mean of three samples. ●, WBC supernatant alone (untreated control); ■, supernatants from BaPGN-treated WBCs; ▽, supernatants from WBCs treated with BaPGN and ET; △, supernatants from WBCs treated with BaPGN and LT.

Fig 7

AT modulation of cytokine/chemokine production in BaPGN-treated WBCs at 15 h and impacts of candidate antimicrobial chemokines on B. anthracis growth. Purified human WBCs were treated with BaPGN and BaPGN plus AT for 15 h, and cytokine/chemokine profiles were assessed using an antibody array. (A) Representative antibody array highlighting BaPGN-induced factors modulated by AT. BaPGN-induced GRO-α (circled 1) and MCP1 (circled 3) were greatly suppressed by the AT treatment; in contrast, CXCL10 (circled 2) expression was slightly reduced. (B to D) Growth profile of B. anthracis in the presence of increasing concentrations of recombinant human rhGRO-α (CXCL1), CXCL10 (IP-10), and MCP1 (CCL2). The growth curves were curve fitted (nonlinear regression) using the statistics module of GraphPad Prism. The numbers in parentheses refer to the circled numbers in panel A. The array and growth curves are representative of three independent experiments.