Background: Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activated the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1 (IL-1).
Methods: We investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA.
Results: Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4-/- but not in TLR2-/- BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 were also necessary for PGA-induced inflammatory responses. Furthermore, PGA-enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), were responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88-/- BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggested that immune responses induced by PGA occurred via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.
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