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Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

Kim YJ, Kim JH, Lee KJ, Choi MM, Kim YH, Rhie GE, Yoo CK, Cha K, Shin NR - PLoS ONE (2015)

Bottom Line: Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells.Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK).As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

View Article: PubMed Central - PubMed

Affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Cheongju, Korea.

ABSTRACT
Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

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Dose-dependent production of NO (A), TNFα (B), and IL-6 (C) in BoNT/A-treated RAW264.7 cells.Cells were incubated with BoNT/A (0, 1, 2, 5, and 10 nM) or 1 μg/ml LPS as a positive control for 24 h at 37°C. Culture supernatants were collected, and the levels of NO, TNFα, and IL-6 were measured. Values are the mean ± SD of three replicates for each group.
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pone.0120840.g003: Dose-dependent production of NO (A), TNFα (B), and IL-6 (C) in BoNT/A-treated RAW264.7 cells.Cells were incubated with BoNT/A (0, 1, 2, 5, and 10 nM) or 1 μg/ml LPS as a positive control for 24 h at 37°C. Culture supernatants were collected, and the levels of NO, TNFα, and IL-6 were measured. Values are the mean ± SD of three replicates for each group.

Mentions: The inflammatory response of RAW264.7 macrophages to various concentrations of BoNT/A (ranging from 0 to 10 nM) was investigated. Following exposure to BoNT/A, RAW264.7 cells expressed increased levels of TNFα and NO in a dose-dependent manner (Fig 3A and 3B). IL-6 was detected only when stimulated with more than 5 nM BoNT/A (Fig 3C). However, IL-1β and IL-12 were not detected with the highest concentration of BoNT/A tested (10 nM). No cytotoxicity was observed with any of the BoNT/A concentrations examined.


Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

Kim YJ, Kim JH, Lee KJ, Choi MM, Kim YH, Rhie GE, Yoo CK, Cha K, Shin NR - PLoS ONE (2015)

Dose-dependent production of NO (A), TNFα (B), and IL-6 (C) in BoNT/A-treated RAW264.7 cells.Cells were incubated with BoNT/A (0, 1, 2, 5, and 10 nM) or 1 μg/ml LPS as a positive control for 24 h at 37°C. Culture supernatants were collected, and the levels of NO, TNFα, and IL-6 were measured. Values are the mean ± SD of three replicates for each group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390353&req=5

pone.0120840.g003: Dose-dependent production of NO (A), TNFα (B), and IL-6 (C) in BoNT/A-treated RAW264.7 cells.Cells were incubated with BoNT/A (0, 1, 2, 5, and 10 nM) or 1 μg/ml LPS as a positive control for 24 h at 37°C. Culture supernatants were collected, and the levels of NO, TNFα, and IL-6 were measured. Values are the mean ± SD of three replicates for each group.
Mentions: The inflammatory response of RAW264.7 macrophages to various concentrations of BoNT/A (ranging from 0 to 10 nM) was investigated. Following exposure to BoNT/A, RAW264.7 cells expressed increased levels of TNFα and NO in a dose-dependent manner (Fig 3A and 3B). IL-6 was detected only when stimulated with more than 5 nM BoNT/A (Fig 3C). However, IL-1β and IL-12 were not detected with the highest concentration of BoNT/A tested (10 nM). No cytotoxicity was observed with any of the BoNT/A concentrations examined.

Bottom Line: Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells.Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK).As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

View Article: PubMed Central - PubMed

Affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Cheongju, Korea.

ABSTRACT
Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

Show MeSH
Related in: MedlinePlus