Cytokine response to infection with Bacillus anthracis spores

Abstract

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming bacterium. The inhalational form of anthrax is the most severe and is associated with rapid progression of the disease and the outcome is frequently fatal. Transfer from the respiratory epithelium to regional lymph nodes appears to be an essential early step in the establishment of infection. This transfer is believed to occur by means of carriage within alveolar macrophages following phagocytosis. Therefore, the ability of B. anthracis to transit through the host macrophage or dendritic cell appears to be an early and critical step in B. anthracis pathogenesis. In this work, we examined the cytokine responses to spore infection in mouse primary peritoneal macrophages, in primary human dendritic cells, and during a spore aerosol infection model utilizing the susceptible A/J mouse strain. We demonstrated that both mouse peritoneal macrophages and human dendritic cells exhibited significant intracellular bactericidal activity during the first hours following uptake, providing the necessary time to mount a cytokine response prior to cell lysis. Strong tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) responses were seen in mouse peritoneal macrophages. In addition to TNF-alpha and IL-6, human dendritic cells produced the cytokines IL-1beta, IL-8, and IL-12. A mixture of Th1 and Th2 cytokines were detected in sera obtained from infected animals. In this study, we provide further evidence of an acute cytokine response when cells in culture and mice are infected with B. anthracis spores.

FIG. 1.

B. anthracis spore infection of mouse primary peritoneal macrophages. Macrophages were infected with B. anthracis spores prepared from B. anthracis strain 7702 as described in the text. (A) CFU inside the macrophages and in the supernatants were determined and are shown as a function of time postinfection. (B) The concentrations of 10 cytokines in the supernatants, collected from uninfected wells and infected wells at the indicated time points, were determined, and the results for TNF-α and IL-6 at 2.5, 5, and 7.5 h following infection are graphed. Three experiments were performed, and the results of a representative experiment are shown. Each value reported is the average of three samples. Error bars represent 1 standard deviation. Statistical significance was determined by Student's t test analysis. In all cases, means were compared to those of the uninfected control group. **, P < 0.01.

FIG. 2.

B. anthracis spore infection of primary human dendritic cells. Human dendritic cells were infected with B. anthracis spores prepared from B. anthracis strain 7702 as described in the text. (A) CFU inside the dendritic cells (closed squares) and in the supernatants (open circles) were determined and are shown as a function of time postinfection. (B to F) The concentrations of 10 cytokines in the supernatants collected from uninfected wells (gray bars) and infected wells (black bars) at the indicated time points are shown for TNF-α, IL-6, IL-1β, IL-8, and IL-12. Three experiments were performed, and the results of a representative experiment are shown. Each value reported is the average of three samples. Error bars represent 1 standard deviation. Statistical significance was determined by Student's t test analysis. For each cytokine, at each time point, means were compared to those of the uninfected control group. *, P < 0.05; **, P < 0.01.

FIG. 3.

Mouse aerosol challenge model. Mice were exposed to aerosols of spores prepared from B. anthracis strain 7702 as described in the text. (A) The survival of groups of mice challenged with 2 × 10³ (squares), 3 × 10⁵ (triangles), 8 × 10⁵ (diamonds), and 4 × 10⁶ (circles) spores is shown as a function of time postchallenge. (B) The survival of groups of mice challenged with between 1 × 10⁶ and 4 × 10⁶ spores from three sequential experiments is shown: 1 × 10⁶ (triangles), 3 × 10⁶ (circles), and 4 × 10⁶ (squares). Subsets of animals from the challenges presented in panel B were sacrificed on days 0, 3, and 5 postchallenge. The number of CFU present in the lungs (C) and in the spleens (D) is presented. The lower limit of detection of B. anthracis in spleen or lung was 250 CFU in each case. For panel D, values represent only positive spleen cultures as described in the Results section.

FIG. 4.

Levels of serum cytokines in B. anthracis-infected mice. A subset of mice from the three challenges presented in Fig. 3B were examined for their serum cytokine response to the infection. On days 3 and 5 postchallenge, mice were sacrificed and sera were collected. The concentration of 10 cytokines in serum from each mouse was determined. (A) The results for IFN-γ are presented. Each data point represents a single mouse. The level of IFN-γ in the sera of uninfected mice (n = 6) was below the level of detection (1 pg/ml). (B) The results for IL-6 are presented. Each data point represents a single mouse. The solid line indicates the mean of the concentration of IL-6 in the sera of uninfected mice (n = 6). The dashed lines indicate 1 standard deviation above and below the mean.

FIG. 5.

LPS-induced cytokines in uninfected and B. anthracis-infected mice. Mice were exposed by aerosol exposure to 2 × 10⁶ spores prepared from B. anthracis strain 7702 as described in the text. Uninfected (gray bars) or B. anthracis-infected (black bars) mice were treated with LPS, and sera were collected at 1, 3, and 5 h post-LPS injection. In each panel, the control groups consisted of infected and uninfected mice that were not treated with LPS. The concentration of 10 cytokines in each serum sample was determined, and the concentrations of the 6 cytokines present at levels above the level of quantitation of the assay are shown. Three experiments were performed, and the results of a representative experiment are shown. Error bars represent 1 standard deviation. Statistical significance was determined by Student's t test analysis. For each cytokine, at each time point, the mean of the uninfected, LPS-treated group was compared to that of the uninfected, untreated control group and the mean of the infected, LPS-treated group was compared to that of the infected, untreated control group. *, P < 0.05; **, P < 0.01. For each cytokine, at each time point, no statistically relevant difference was observed between the mean infected and the uninfected groups.