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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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TLR2 KO mice show decreased expression of TNFα and IL-6 cytokines upon BoNT/A stimulation.WT (black bar; n = 6) and TLR2 KO mice (white bar; n = 6) were used to isolate bone marrow monocytes. Differentiated BMDM cells and subjected to BoNT/A treatment. Culture supernatants were analyzed for TNFα (A) and IL6 (B). All data were given as means ± SD.
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pone.0120840.g009: TLR2 KO mice show decreased expression of TNFα and IL-6 cytokines upon BoNT/A stimulation.WT (black bar; n = 6) and TLR2 KO mice (white bar; n = 6) were used to isolate bone marrow monocytes. Differentiated BMDM cells and subjected to BoNT/A treatment. Culture supernatants were analyzed for TNFα (A) and IL6 (B). All data were given as means ± SD.

Mentions: To confirm the specific involvement of TLR2 in BoNT/A-induced TNFα and IL-6 responses by primary macrophage cells, the BMDM cells were generated from both of wild type and TLR2 KO mice. Compared to the negative control (0 nM) and positive control (Pam3), BoNT/A mediated TNFα (Fig 9A) and IL-6 (Fig 9B) responses were almost completely abolished in BMDM primary cells from TLR2-/- Knock out mice. These results confirm that TLR2 is specific for the induction of TNFα and IL-6 by macrophages in vivo stimulated with BoNT/A.


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)

TLR2 KO mice show decreased expression of TNFα and IL-6 cytokines upon BoNT/A stimulation.WT (black bar; n = 6) and TLR2 KO mice (white bar; n = 6) were used to isolate bone marrow monocytes. Differentiated BMDM cells and subjected to BoNT/A treatment. Culture supernatants were analyzed for TNFα (A) and IL6 (B). All data were given as means ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120840.g009: TLR2 KO mice show decreased expression of TNFα and IL-6 cytokines upon BoNT/A stimulation.WT (black bar; n = 6) and TLR2 KO mice (white bar; n = 6) were used to isolate bone marrow monocytes. Differentiated BMDM cells and subjected to BoNT/A treatment. Culture supernatants were analyzed for TNFα (A) and IL6 (B). All data were given as means ± SD.
Mentions: To confirm the specific involvement of TLR2 in BoNT/A-induced TNFα and IL-6 responses by primary macrophage cells, the BMDM cells were generated from both of wild type and TLR2 KO mice. Compared to the negative control (0 nM) and positive control (Pam3), BoNT/A mediated TNFα (Fig 9A) and IL-6 (Fig 9B) responses were almost completely abolished in BMDM primary cells from TLR2-/- Knock out mice. These results confirm that TLR2 is specific for the induction of TNFα and IL-6 by macrophages in vivo stimulated with BoNT/A.

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