Abstract
The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.
Keywords:
Bacillus anthracis; Macrophages; Nitric oxide; Platelet activating factor receptor (PAFR); Poly-γ-d-glutamic acid (PGA) capsule.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antibodies, Neutralizing / pharmacology
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Bacillus / chemistry*
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Bacillus / immunology
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Bacillus anthracis / chemistry
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Bacillus anthracis / immunology
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Bacterial Capsules / chemistry
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Bacterial Capsules / immunology
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Cell Line
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Dose-Response Relationship, Immunologic
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Enzyme Inhibitors / pharmacology
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Female
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Gene Expression Regulation
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Macrophages / cytology
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Macrophages / drug effects
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Macrophages / immunology
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Mice
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Mice, Inbred C57BL
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Mitogen-Activated Protein Kinases / genetics
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Mitogen-Activated Protein Kinases / immunology
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NF-kappa B / genetics
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NF-kappa B / immunology
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Nitric Oxide / agonists*
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Nitric Oxide / biosynthesis
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Nitric Oxide Synthase Type II / genetics
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Nitric Oxide Synthase Type II / immunology*
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Platelet Membrane Glycoproteins / antagonists & inhibitors
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Platelet Membrane Glycoproteins / genetics
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Platelet Membrane Glycoproteins / immunology*
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Polyglutamic Acid / analogs & derivatives*
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Polyglutamic Acid / isolation & purification
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Polyglutamic Acid / pharmacology
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Primary Cell Culture
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RNA, Small Interfering / genetics
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RNA, Small Interfering / immunology
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Receptors, G-Protein-Coupled / antagonists & inhibitors
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Receptors, G-Protein-Coupled / genetics
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Receptors, G-Protein-Coupled / immunology*
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STAT1 Transcription Factor / genetics
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STAT1 Transcription Factor / immunology
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Signal Transduction
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Toll-Like Receptor 2 / antagonists & inhibitors
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Toll-Like Receptor 2 / deficiency
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Toll-Like Receptor 2 / genetics
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Toll-Like Receptor 2 / immunology*
Substances
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Antibodies, Neutralizing
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Enzyme Inhibitors
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NF-kappa B
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Platelet Membrane Glycoproteins
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RNA, Small Interfering
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Receptors, G-Protein-Coupled
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STAT1 Transcription Factor
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Stat1 protein, mouse
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Tlr2 protein, mouse
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Toll-Like Receptor 2
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platelet activating factor receptor
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poly(gamma-glutamic acid)
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Polyglutamic Acid
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Nitric Oxide
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Nitric Oxide Synthase Type II
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Nos2 protein, mouse
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Mitogen-Activated Protein Kinases