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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 inhibited LPS-induced production of TNFα and NO in a dose- and time-dependent manner in RAW264.7 macrophages.RAW264.7 cells were treated with 0 to 1 nM BoNT/A for 24 h (A and B) or with 1 nM BoNT/A for 0 to 24 h and then stimulated with or without 1 μg/ml LPS (C and D). Culture media were collected at 24 h to measure NO (A and C) and TNFα (B and D) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data shown are the mean ± SD.
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pone.0120840.g007: BoNT/A inhibited LPS-induced production of TNFα and NO in a dose- and time-dependent manner in RAW264.7 macrophages.RAW264.7 cells were treated with 0 to 1 nM BoNT/A for 24 h (A and B) or with 1 nM BoNT/A for 0 to 24 h and then stimulated with or without 1 μg/ml LPS (C and D). Culture media were collected at 24 h to measure NO (A and C) and TNFα (B and D) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data shown are the mean ± SD.

Mentions: Preincubating macrophages with various concentrations of BoNT/A (0 to 5 nM) over time (0 to 32 h) progressively inhibited the ability of the cells to produce NO and TNFα upon subsequent exposure to LPS. The inhibitory effect by BoNT/A occurred in a dose-dependent manner, and 2 nM and 1 nM BoNT/A was required for complete inhibition of NO and TNFα production, respectively (Fig 7A and 7B). With exposure to 1 nM BoNT/A, inhibition of NO and TNFα became apparent after 15 h of exposure and reached a maximum by 24 h (Fig 7C and 7D). However, co-incubating the cells with BoNT/A and LPS was insufficient to inhibit cytokine expression (Fig 6A and 6B).


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 inhibited LPS-induced production of TNFα and NO in a dose- and time-dependent manner in RAW264.7 macrophages.RAW264.7 cells were treated with 0 to 1 nM BoNT/A for 24 h (A and B) or with 1 nM BoNT/A for 0 to 24 h and then stimulated with or without 1 μg/ml LPS (C and D). Culture media were collected at 24 h to measure NO (A and C) and TNFα (B and D) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data shown are the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120840.g007: BoNT/A inhibited LPS-induced production of TNFα and NO in a dose- and time-dependent manner in RAW264.7 macrophages.RAW264.7 cells were treated with 0 to 1 nM BoNT/A for 24 h (A and B) or with 1 nM BoNT/A for 0 to 24 h and then stimulated with or without 1 μg/ml LPS (C and D). Culture media were collected at 24 h to measure NO (A and C) and TNFα (B and D) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data shown are the mean ± SD.
Mentions: Preincubating macrophages with various concentrations of BoNT/A (0 to 5 nM) over time (0 to 32 h) progressively inhibited the ability of the cells to produce NO and TNFα upon subsequent exposure to LPS. The inhibitory effect by BoNT/A occurred in a dose-dependent manner, and 2 nM and 1 nM BoNT/A was required for complete inhibition of NO and TNFα production, respectively (Fig 7A and 7B). With exposure to 1 nM BoNT/A, inhibition of NO and TNFα became apparent after 15 h of exposure and reached a maximum by 24 h (Fig 7C and 7D). However, co-incubating the cells with BoNT/A and LPS was insufficient to inhibit cytokine expression (Fig 6A and 6B).

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