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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 the production of LPS-induced pro-inflammatory mediators in RAW264.7 cells.A and B, production of NO and proinflammatory cytokines in LPS-stimulated RAW264.7 cells with or without BoNT/A pretreatment. RAW264.7 cells were pretreated with 1 nM BoNT/A or 1 nM BoNToxoid/A for 24 h, and then stimulated with or without 1 μg/ml LPS. Culture media were collected at 24 h to measure NO (A), TNFα, IL-6, and IL-12 (B) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data are the mean ± S.D. *, p<0.05 vs. LPS alone. C and D, quantification of mRNA expression of Tnf and iNOS in LPS-stimulated RAW264.7 macrophages with or without BoNT/A pretreatment. Total RNAs were isolated, and mRNA levels of iNOS and TNFα were analyzed with qRT-PCR. GAPDH expression was used as an internal control for RT-PCR. Representative results of three independent experiments are shown.
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pone.0120840.g006: BoNT/A inhibited the production of LPS-induced pro-inflammatory mediators in RAW264.7 cells.A and B, production of NO and proinflammatory cytokines in LPS-stimulated RAW264.7 cells with or without BoNT/A pretreatment. RAW264.7 cells were pretreated with 1 nM BoNT/A or 1 nM BoNToxoid/A for 24 h, and then stimulated with or without 1 μg/ml LPS. Culture media were collected at 24 h to measure NO (A), TNFα, IL-6, and IL-12 (B) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data are the mean ± S.D. *, p<0.05 vs. LPS alone. C and D, quantification of mRNA expression of Tnf and iNOS in LPS-stimulated RAW264.7 macrophages with or without BoNT/A pretreatment. Total RNAs were isolated, and mRNA levels of iNOS and TNFα were analyzed with qRT-PCR. GAPDH expression was used as an internal control for RT-PCR. Representative results of three independent experiments are shown.

Mentions: To further investigate whether BoNT/A modulates the production of NO and proinflammatory cytokines, we treated RAW264.7 cells with 1 nM BoNT/A with or without 1 μg/ml LPS and analyzed culture supernatants for NO and cytokines. RAW264.7 cells expressed significant levels of NO, TNFα, IL-6, and IL-12 upon exposure to LPS (Fig 6A and 6B). Parallel cultures of RAW264.7 cells were exposed to BoNT/A or BoNToxoid/A. Upon exposure to BoNT/A, RAW264.7 cells induced low levels of NO compared to LPS-exposed cells. TNFα was expressed at very low levels, whereas IL-6 and IL-12 were not detected. BoNToxoid/A-treated cells produced barely detectable levels of NO and three proinflammatory cytokines. Importantly, pretreatment of RAW264.7 cells with BoNT/A and subsequent addition of LPS markedly decreased NO production and almost completely blocked the expression of TNFα, IL-6, and IL-12 from RAW264.7 cells (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 the production of LPS-induced pro-inflammatory mediators in RAW264.7 cells.A and B, production of NO and proinflammatory cytokines in LPS-stimulated RAW264.7 cells with or without BoNT/A pretreatment. RAW264.7 cells were pretreated with 1 nM BoNT/A or 1 nM BoNToxoid/A for 24 h, and then stimulated with or without 1 μg/ml LPS. Culture media were collected at 24 h to measure NO (A), TNFα, IL-6, and IL-12 (B) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data are the mean ± S.D. *, p<0.05 vs. LPS alone. C and D, quantification of mRNA expression of Tnf and iNOS in LPS-stimulated RAW264.7 macrophages with or without BoNT/A pretreatment. Total RNAs were isolated, and mRNA levels of iNOS and TNFα were analyzed with qRT-PCR. GAPDH expression was used as an internal control for RT-PCR. Representative results of three independent experiments are shown.
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Related In: Results  -  Collection

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

pone.0120840.g006: BoNT/A inhibited the production of LPS-induced pro-inflammatory mediators in RAW264.7 cells.A and B, production of NO and proinflammatory cytokines in LPS-stimulated RAW264.7 cells with or without BoNT/A pretreatment. RAW264.7 cells were pretreated with 1 nM BoNT/A or 1 nM BoNToxoid/A for 24 h, and then stimulated with or without 1 μg/ml LPS. Culture media were collected at 24 h to measure NO (A), TNFα, IL-6, and IL-12 (B) concentrations using the Griess reaction and sandwich ELISA, respectively. Three independent experiments were performed, and the data are the mean ± S.D. *, p<0.05 vs. LPS alone. C and D, quantification of mRNA expression of Tnf and iNOS in LPS-stimulated RAW264.7 macrophages with or without BoNT/A pretreatment. Total RNAs were isolated, and mRNA levels of iNOS and TNFα were analyzed with qRT-PCR. GAPDH expression was used as an internal control for RT-PCR. Representative results of three independent experiments are shown.
Mentions: To further investigate whether BoNT/A modulates the production of NO and proinflammatory cytokines, we treated RAW264.7 cells with 1 nM BoNT/A with or without 1 μg/ml LPS and analyzed culture supernatants for NO and cytokines. RAW264.7 cells expressed significant levels of NO, TNFα, IL-6, and IL-12 upon exposure to LPS (Fig 6A and 6B). Parallel cultures of RAW264.7 cells were exposed to BoNT/A or BoNToxoid/A. Upon exposure to BoNT/A, RAW264.7 cells induced low levels of NO compared to LPS-exposed cells. TNFα was expressed at very low levels, whereas IL-6 and IL-12 were not detected. BoNToxoid/A-treated cells produced barely detectable levels of NO and three proinflammatory cytokines. Importantly, pretreatment of RAW264.7 cells with BoNT/A and subsequent addition of LPS markedly decreased NO production and almost completely blocked the expression of TNFα, IL-6, and IL-12 from RAW264.7 cells (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