MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling

Abstract

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.

FIG. 1.

Recognition of HKBa by TLR2-expressing HEK293 cells. HEK293 cells were transfected in triplicate wells and then exposed 48 h after transfection to buffer control, PGN (10 μg/ml), PAM₃CSK₄ (100 ng/ml), LPS (100 ng/ml or 1 μg/ml), or HKBa, as indicated. Cell lysates were collected 6 h postchallenge, and firefly luciferase activity was measured after addition of luciferin reagent. Results are expressed as the fold increase in NF-κB reporter gene (luciferase) activity in response to the given stimulus compared to response to the buffer control. (A) TLR2-expressing cells (black bars) compared to empty vector-expressing control cells (white bars). (B) TLR4-expressing cells (black bars) compared to empty vector-expressing control cells (white bars). The effect of adding polymyxin B (30 μg/ml) to TLR2- or TLR4-expressing cells stimulated with a given agonist is as indicated (gray bars); nt, not tested. In panel B, purified LPS at 1 μg/ml was tested without polymyxin B to demonstrate the higher concentration required to achieve a >5-fold increase in luciferase activity. For all data shown, 5 to 17 separate experiments were performed using triplicate samples in each experiment. Data are plotted as the mean fold increase value ± standard error of the mean. Statistical significance was determined using Student's t test analysis. Mean values of fold increase in luciferase activity for TLR2- or TLR4-expressing cells were compared to those of the empty vector-expressing cells in response to the same stimulus. *, P < 0.05; **, P < 0.01.

FIG. 2.

HKBa stimulation of TNF-α production in C3H/HeN, C3H/HeJ, C57BL/6J, TLR2^−/−, and MyD88^−/− BMDMs. BMDMs were challenged with HKBa or with the control ligand, PAM₃CSK₄ (100 ng/ml) or LPS (100 ng/ml), as indicated. Cells were incubated for 6 h, and cell-free supernatants were collected and analyzed for TNF-α by enzyme-linked immunosorbent assay. (A) C3H/HeN BMDM TNF-α results (black bars) compared to C3H/HeJ BMDMs (white bars). Six separate experiments were performed using triplicate samples in each experiment. (B) C57BL/6J control strain BMDM TNF-α results (black bars) compared to TLR2^−/− BMDMs (white bars). Four separate experiments were performed using triplicate samples in each experiment. (C) C57BL/6J control strain BMDM TNF-α results (black bars) compared to MyD88^−/− BMDMs (white bars). Two separate experiments were performed using six replicate samples in each experiment. In all of these experiments, cells were viable (>95% survival) as measured by CCK-8 assay. Data are plotted as the mean value ± standard error of the mean. Statistical significance was determined using Student's t test analysis. In all cases, mean TNF-α values for a given stimulus were compared to those generated by buffer control-treated cells. **, P < 0.01. nd, not detected.

FIG. 3.

Survival of A/J, C57BL/6J, TLR2^−/−, C3H/HeOuJ, C3H/HeJ, and MyD88^−/− mice challenged with the B. anthracis Sterne strain. Mice were exposed to aerosolized spores prepared from B. anthracis strain 7702 as described in Materials and Methods. The survival of groups of mice with a retained dose after challenge between 1 × 10⁶ and 5 × 10⁶ spores from two independent experiments is shown. (A) Challenge performed with A/J (circles), C57BL/6J (squares), or TLR2^−/− (X's) mice. (B) Challenge performed with A/J (circles), C3H/HeOuJ (squares), and C3H/HeJ (X's) mice. (C) Challenge performed with A/J (circles), C57BL/6J (squares), or MyD88^−/− (X's) mice. For each strain, the cumulative mortality of two independent challenges is represented in the graph (n = 20 animals).