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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 induced phosphorylation of MAPK molecules in RAW264.7 cells.A and B, production of NO (A) and TNFα (B) following stimulation with BoNT/A (1 nM) in RAW264.7 cells pretreated with specific inhibitors of each pathway. RAW264.7 cells (5.0 × 105 cells/ml) were incubated with 20 μM SAPK/JNK inhibitor (SP), 20 μM ERK inhibitor PD98059 (PD), 20 μM p38 inhibitor (SB), 20 μM NF-κB inhibitor (SN50), or their combinations for 1 h at 37°C and then stimulated with 1 nM BoNT/A for 24 h. The levels of NO (A) and TNFα (B) production were assessed in the supernatants. C, western blot of phosphorylation of the signaling molecules in BoNT/A-stimulated RAW264.7 cells. The cells were incubated with 1 nM BoNT/A for 0 to 60 min. The proteins from the cells were used to detect phosphorylated or total forms of NF-κB or the three MAPK molecules. Representative results of three independent experiments are shown.
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pone.0120840.g005: BoNT/A induced phosphorylation of MAPK molecules in RAW264.7 cells.A and B, production of NO (A) and TNFα (B) following stimulation with BoNT/A (1 nM) in RAW264.7 cells pretreated with specific inhibitors of each pathway. RAW264.7 cells (5.0 × 105 cells/ml) were incubated with 20 μM SAPK/JNK inhibitor (SP), 20 μM ERK inhibitor PD98059 (PD), 20 μM p38 inhibitor (SB), 20 μM NF-κB inhibitor (SN50), or their combinations for 1 h at 37°C and then stimulated with 1 nM BoNT/A for 24 h. The levels of NO (A) and TNFα (B) production were assessed in the supernatants. C, western blot of phosphorylation of the signaling molecules in BoNT/A-stimulated RAW264.7 cells. The cells were incubated with 1 nM BoNT/A for 0 to 60 min. The proteins from the cells were used to detect phosphorylated or total forms of NF-κB or the three MAPK molecules. Representative results of three independent experiments are shown.

Mentions: We also examined the TLR-mediated intracellular signaling pathways in BoNT/A-stimulated RAW264.7 cells. The pathways were analyzed with a blocking test using inhibitors of NF-κB and three MAPK molecules including ERK1/2, JNK, and p38 MAPK. RAW264.7 cells were treated with the inhibitor prior to stimulation with 1 nM BoNT/A for 24 h, and then the concentration of NO and TNFα was determined in the supernatants. The p38 inhibitor and the JNK inhibitor (SP600125) each significantly reduced BoNT/A-induced NO and TNFα production in RAW264.7 cells (p<0.05). The ERK inhibitor effectively suppressed TNFα production in BoNT/A-stimulated RAW264.7 cells (p<0.05) but only slightly decreased NO production. The combination of p38, JNK, and ERK inhibitors completely blocked NO and TNFα production. However, the NF-κB inhibitor did not block NO or TNFα production in BoNT/A-stimulated RAW264.7 cells (Fig 5). These results were confirmed by examining phosphorylation of the signaling molecules. Activation of MAPKs is dependent on phosphorylation by their respective upstream MAP kinases. Thus, MAPK phosphorylation was analyzed with western blot analysis using phospho-specific antibodies. RAW264.7 cells were treated with 1 nM BoNT/A for 0 to 60 min. As shown in Fig 5, phosphorylation of all three MAPKs occurred within 20 min of BoNT/A stimulation and peaked at 30 min. These results indicate that BoNT/A-stimulated RAW264.7 cells induce NO and TNFα production through TLR2-mediated signal transduction via activation of ERK, JNK, and p38.


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 induced phosphorylation of MAPK molecules in RAW264.7 cells.A and B, production of NO (A) and TNFα (B) following stimulation with BoNT/A (1 nM) in RAW264.7 cells pretreated with specific inhibitors of each pathway. RAW264.7 cells (5.0 × 105 cells/ml) were incubated with 20 μM SAPK/JNK inhibitor (SP), 20 μM ERK inhibitor PD98059 (PD), 20 μM p38 inhibitor (SB), 20 μM NF-κB inhibitor (SN50), or their combinations for 1 h at 37°C and then stimulated with 1 nM BoNT/A for 24 h. The levels of NO (A) and TNFα (B) production were assessed in the supernatants. C, western blot of phosphorylation of the signaling molecules in BoNT/A-stimulated RAW264.7 cells. The cells were incubated with 1 nM BoNT/A for 0 to 60 min. The proteins from the cells were used to detect phosphorylated or total forms of NF-κB or the three MAPK molecules. Representative results of three independent experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390353&req=5

pone.0120840.g005: BoNT/A induced phosphorylation of MAPK molecules in RAW264.7 cells.A and B, production of NO (A) and TNFα (B) following stimulation with BoNT/A (1 nM) in RAW264.7 cells pretreated with specific inhibitors of each pathway. RAW264.7 cells (5.0 × 105 cells/ml) were incubated with 20 μM SAPK/JNK inhibitor (SP), 20 μM ERK inhibitor PD98059 (PD), 20 μM p38 inhibitor (SB), 20 μM NF-κB inhibitor (SN50), or their combinations for 1 h at 37°C and then stimulated with 1 nM BoNT/A for 24 h. The levels of NO (A) and TNFα (B) production were assessed in the supernatants. C, western blot of phosphorylation of the signaling molecules in BoNT/A-stimulated RAW264.7 cells. The cells were incubated with 1 nM BoNT/A for 0 to 60 min. The proteins from the cells were used to detect phosphorylated or total forms of NF-κB or the three MAPK molecules. Representative results of three independent experiments are shown.
Mentions: We also examined the TLR-mediated intracellular signaling pathways in BoNT/A-stimulated RAW264.7 cells. The pathways were analyzed with a blocking test using inhibitors of NF-κB and three MAPK molecules including ERK1/2, JNK, and p38 MAPK. RAW264.7 cells were treated with the inhibitor prior to stimulation with 1 nM BoNT/A for 24 h, and then the concentration of NO and TNFα was determined in the supernatants. The p38 inhibitor and the JNK inhibitor (SP600125) each significantly reduced BoNT/A-induced NO and TNFα production in RAW264.7 cells (p<0.05). The ERK inhibitor effectively suppressed TNFα production in BoNT/A-stimulated RAW264.7 cells (p<0.05) but only slightly decreased NO production. The combination of p38, JNK, and ERK inhibitors completely blocked NO and TNFα production. However, the NF-κB inhibitor did not block NO or TNFα production in BoNT/A-stimulated RAW264.7 cells (Fig 5). These results were confirmed by examining phosphorylation of the signaling molecules. Activation of MAPKs is dependent on phosphorylation by their respective upstream MAP kinases. Thus, MAPK phosphorylation was analyzed with western blot analysis using phospho-specific antibodies. RAW264.7 cells were treated with 1 nM BoNT/A for 0 to 60 min. As shown in Fig 5, phosphorylation of all three MAPKs occurred within 20 min of BoNT/A stimulation and peaked at 30 min. These results indicate that BoNT/A-stimulated RAW264.7 cells induce NO and TNFα production through TLR2-mediated signal transduction via activation of ERK, JNK, and p38.

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