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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 used TLR2 for NO (A) and TNFα (B) production in the RAW264.7 mouse macrophage cell line.The levels of NO (A) and TNFα (B) were assessed in RAW264.7 cells stimulated with BoNT/A after preincubation with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml). TLR2, TLR4, IgG1, and IgG2a indicate the counterpart antigen against which the respective antibody was used to pretreat RAW264.7 cells. Mouse anti-IgG1 or rat anti-IgG2a was used as a negative control. Anti-TLR2 had a significant (p<0.05) inhibitory effect on both NO and TNFα production by RAW264.7 cells stimulated with BoNT/A but anti-TLR4 had no noticeable effect.
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pone.0120840.g004: BoNT/A used TLR2 for NO (A) and TNFα (B) production in the RAW264.7 mouse macrophage cell line.The levels of NO (A) and TNFα (B) were assessed in RAW264.7 cells stimulated with BoNT/A after preincubation with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml). TLR2, TLR4, IgG1, and IgG2a indicate the counterpart antigen against which the respective antibody was used to pretreat RAW264.7 cells. Mouse anti-IgG1 or rat anti-IgG2a was used as a negative control. Anti-TLR2 had a significant (p<0.05) inhibitory effect on both NO and TNFα production by RAW264.7 cells stimulated with BoNT/A but anti-TLR4 had no noticeable effect.

Mentions: TLRs are a set of innate immune receptors that recognize structures common to many different pathogens. TLR-mediated stimulation induces production of pro-inflammatory and immune-related cytokines. Thus, we hypothesized that RAW264.7 cells would utilize TLR in response to BoNT/A, resulting in induction of NO and TNFα. To investigate the functional involvement of TLR in BoNT/A-induced NO and TNFα responses by RAW264.7 cells, the cells were incubated with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml) before stimulation with BoNT/A. Compared to the negative control that was incubated with mouse anti-IgG1 or rat anti-IgG2a, anti-TLR2 almost completely blocked both NO (Fig 4A left) and TNFα (Fig 4B left) production from BoNT/A-stimulated RAW264.7 cells (p<0.05), whereas anti-TLR4 had no noticeable effect on NO (Fig 4A right) and TNFα (Fig 4B right) production. These results demonstrate that TLR2, but not TLR4, is essential for the induction of NO and TNFα by murine macrophages 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)

BoNT/A used TLR2 for NO (A) and TNFα (B) production in the RAW264.7 mouse macrophage cell line.The levels of NO (A) and TNFα (B) were assessed in RAW264.7 cells stimulated with BoNT/A after preincubation with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml). TLR2, TLR4, IgG1, and IgG2a indicate the counterpart antigen against which the respective antibody was used to pretreat RAW264.7 cells. Mouse anti-IgG1 or rat anti-IgG2a was used as a negative control. Anti-TLR2 had a significant (p<0.05) inhibitory effect on both NO and TNFα production by RAW264.7 cells stimulated with BoNT/A but anti-TLR4 had no noticeable effect.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120840.g004: BoNT/A used TLR2 for NO (A) and TNFα (B) production in the RAW264.7 mouse macrophage cell line.The levels of NO (A) and TNFα (B) were assessed in RAW264.7 cells stimulated with BoNT/A after preincubation with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml). TLR2, TLR4, IgG1, and IgG2a indicate the counterpart antigen against which the respective antibody was used to pretreat RAW264.7 cells. Mouse anti-IgG1 or rat anti-IgG2a was used as a negative control. Anti-TLR2 had a significant (p<0.05) inhibitory effect on both NO and TNFα production by RAW264.7 cells stimulated with BoNT/A but anti-TLR4 had no noticeable effect.
Mentions: TLRs are a set of innate immune receptors that recognize structures common to many different pathogens. TLR-mediated stimulation induces production of pro-inflammatory and immune-related cytokines. Thus, we hypothesized that RAW264.7 cells would utilize TLR in response to BoNT/A, resulting in induction of NO and TNFα. To investigate the functional involvement of TLR in BoNT/A-induced NO and TNFα responses by RAW264.7 cells, the cells were incubated with polyclonal anti-TLR2 (50 μg/ml) or anti-TLR4 (20 μg/ml) before stimulation with BoNT/A. Compared to the negative control that was incubated with mouse anti-IgG1 or rat anti-IgG2a, anti-TLR2 almost completely blocked both NO (Fig 4A left) and TNFα (Fig 4B left) production from BoNT/A-stimulated RAW264.7 cells (p<0.05), whereas anti-TLR4 had no noticeable effect on NO (Fig 4A right) and TNFα (Fig 4B right) production. These results demonstrate that TLR2, but not TLR4, is essential for the induction of NO and TNFα by murine macrophages 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