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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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BoNT/A suppressed the phosphorylation of MAPKs in LPS-stimulated RAW264.7 macrophages.RAW264.7 cells were pretreated with 1 nM BoNT/A for 24 h, and then stimulated with 1 μg/ml LPS for 24 h. The cellular proteins were used to detect phosphorylated or total forms of the three MAPKs, ERK1/2, JNK1/2, and p38. Representative results of three independent experiments are shown.
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pone.0120840.g008: BoNT/A suppressed the phosphorylation of MAPKs in LPS-stimulated RAW264.7 macrophages.RAW264.7 cells were pretreated with 1 nM BoNT/A for 24 h, and then stimulated with 1 μg/ml LPS for 24 h. The cellular proteins were used to detect phosphorylated or total forms of the three MAPKs, ERK1/2, JNK1/2, and p38. Representative results of three independent experiments are shown.

Mentions: MAPKs (including ERK, JNK/SAPK, and p38) are important regulators of iNOS-NO expression by IFNγ and LPS [27]. Thus, MAPKs are likely to be associated with the anti-inflammatory effects of BoNT/A in LPS-stimulated RAW264.7 cells. RAW264.7 cells were stimulated with 1 μg/ml LPS for 15 min with or without 1 nM BoNT/A, and the cell lysates were then used to examine the phosphorylation of MAPKs with western blotting. LPS stimulation strongly induced the phosphorylation of ERK, JNK, and p38 in RAW264.7 cells (Fig 8). However, BoNT/A significantly suppressed the phosphorylation of these three MAPK molecules, whereas the non-phosphorylated forms of these MAPKs remained unchanged. Untreated RAW264.7 cells expressed basal levels of ERK, JNK, and p38. These results indicate that signal transduction by MAPKs may be effectively blocked by BoNT/A in activated macrophages.


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)

BoNT/A suppressed the phosphorylation of MAPKs in LPS-stimulated RAW264.7 macrophages.RAW264.7 cells were pretreated with 1 nM BoNT/A for 24 h, and then stimulated with 1 μg/ml LPS for 24 h. The cellular proteins were used to detect phosphorylated or total forms of the three MAPKs, ERK1/2, JNK1/2, and p38. Representative results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120840.g008: BoNT/A suppressed the phosphorylation of MAPKs in LPS-stimulated RAW264.7 macrophages.RAW264.7 cells were pretreated with 1 nM BoNT/A for 24 h, and then stimulated with 1 μg/ml LPS for 24 h. The cellular proteins were used to detect phosphorylated or total forms of the three MAPKs, ERK1/2, JNK1/2, and p38. Representative results of three independent experiments are shown.
Mentions: MAPKs (including ERK, JNK/SAPK, and p38) are important regulators of iNOS-NO expression by IFNγ and LPS [27]. Thus, MAPKs are likely to be associated with the anti-inflammatory effects of BoNT/A in LPS-stimulated RAW264.7 cells. RAW264.7 cells were stimulated with 1 μg/ml LPS for 15 min with or without 1 nM BoNT/A, and the cell lysates were then used to examine the phosphorylation of MAPKs with western blotting. LPS stimulation strongly induced the phosphorylation of ERK, JNK, and p38 in RAW264.7 cells (Fig 8). However, BoNT/A significantly suppressed the phosphorylation of these three MAPK molecules, whereas the non-phosphorylated forms of these MAPKs remained unchanged. Untreated RAW264.7 cells expressed basal levels of ERK, JNK, and p38. These results indicate that signal transduction by MAPKs may be effectively blocked by BoNT/A in activated macrophages.

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