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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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Related in: MedlinePlus

Functional classification of DEGs after the stimulation of RAW264.7 macrophage-like cells with BoNT/A (1 or 5 nM).Each bar indicates the absolute number of down- or up-regulated genes identified within each functional class.
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pone.0120840.g002: Functional classification of DEGs after the stimulation of RAW264.7 macrophage-like cells with BoNT/A (1 or 5 nM).Each bar indicates the absolute number of down- or up-regulated genes identified within each functional class.

Mentions: DEGs between 0 h and other times following BoNT/A treatment of the macrophages were analyzed using the Panther database to identify altered biological processes (Fig 2). In cells stimulated with both 1 and 5 nM BoNT/A, Panther classification of the 233 DEGs showed that a broad range of major functional processes was affected. A marked effect was found in biological processes involving signal transduction and immunity/defense, in which 30.9% of DEGs (72 of 233 genes) were affected. Considering the clinical characteristics of BoNT/A, we were interested in determining if the DEGs were associated with neuronal activities, intracellular protein trafficking, and muscle contraction. Compared to cells treated with 1 nM BoNT/A, cells treated with 5 nM BoNT/A showed changes in nine biological processes that were over-represented including homeostasis, protein targeting and localization, nitrogen metabolism, blood circulation and gas exchange, sensory perception, electron transport, carbohydrate metabolism, sulfur metabolism, and non-vertebrate processes. Table A and B in S1 File list the DEGs based on grouping according to their known or proposed biological function.


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)

Functional classification of DEGs after the stimulation of RAW264.7 macrophage-like cells with BoNT/A (1 or 5 nM).Each bar indicates the absolute number of down- or up-regulated genes identified within each functional class.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120840.g002: Functional classification of DEGs after the stimulation of RAW264.7 macrophage-like cells with BoNT/A (1 or 5 nM).Each bar indicates the absolute number of down- or up-regulated genes identified within each functional class.
Mentions: DEGs between 0 h and other times following BoNT/A treatment of the macrophages were analyzed using the Panther database to identify altered biological processes (Fig 2). In cells stimulated with both 1 and 5 nM BoNT/A, Panther classification of the 233 DEGs showed that a broad range of major functional processes was affected. A marked effect was found in biological processes involving signal transduction and immunity/defense, in which 30.9% of DEGs (72 of 233 genes) were affected. Considering the clinical characteristics of BoNT/A, we were interested in determining if the DEGs were associated with neuronal activities, intracellular protein trafficking, and muscle contraction. Compared to cells treated with 1 nM BoNT/A, cells treated with 5 nM BoNT/A showed changes in nine biological processes that were over-represented including homeostasis, protein targeting and localization, nitrogen metabolism, blood circulation and gas exchange, sensory perception, electron transport, carbohydrate metabolism, sulfur metabolism, and non-vertebrate processes. Table A and B in S1 File list the DEGs based on grouping according to their known or proposed biological function.

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