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.
Highlights
Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is widely recognized as the most poisonous substance in nature
The array data were processed with the Illumina BeadStudio software program, with data filtering by detecting a p value
In k-means clustering, most of the genes that overlapped with 1 and 5 nM Botulinum neurotoxin type A (BoNT/A) stimulation (58 of 60 genes) showed similar patterns of expression according to the duration of treatment, and more than 96% (55 genes) of the 57 commonly up-regulated genes reached a peak within 4 h after BoNT/A stimulation
Summary
Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is widely recognized as the most poisonous substance in nature. TLR2-Mediated Inflammation by Botulinum Toxin A more rarely, F, are known to be responsible for natural botulism in humans. The threat of botulinum toxin used as a biological weapon [3] that would cause inhalational botulism has been identified [4]. Botulinum toxin is a dichain polypeptide that consists of a 100-kDa heavy chain joined by a single disulfide bond to a 50-kDa light chain [5]. While heavy chain of the toxin plays dual roles of receptor binding and translocation, the light chain of the toxin is a zinc endopeptidase that blocks acetylcholine-containing vesicles from fusing with the presynaptic terminal membrane of the motor neuron. The actions of the light chain and their effects on presynaptic vesicles result in flaccid muscle paralysis [6]
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