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Anthrax lethal toxin suppresses chemokine production in human neutrophil NB-4 cells
Helen V Barson1, Hans Mollenkopf, Stefan H E Kaufmann
1Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Hertfordshire EN6 3QG, UK. hbarson@nibsc.ac.uk
Abstract:
Microarray analysis was used to investigate the effects of Bacillus anthracis lethal toxin (LT) on human neutrophil-like NB-4 cells to identify markers of intoxication. Genes down-regulated after a 2h LT exposure included those encoding chemokines and transcription factors. Significant decreases in the mRNA of interleukin-8, CCL20, CCL3 and CCL4, which are important chemoattractants for immune cells, were observed using real-time PCR (12.3, 4.0, 4.1 and 2.2-fold (p<0.05), respectively). The decreases were more pronounced at 4 and 8h and were LT-specific. Decreases in chemokine protein levels were evident after 24h and were sensitive to low concentrations of LT. Co-incubation with an anti-lethal factor mAb at 500 and 250ng/ml restored levels of interleukin-8 to 100% and 50%, respectively. The results reveal that LT suppresses the cellular immune response and that this assay is a useful tool for the analysis of toxin-neutralising antibody activity in a biologically relevant system.
Insights
Bacillus anthracis lethal toxin (LT) suppresses immune cell attraction by reducing chemokine gene expression in human cells. This study identifies LT-induced immune suppression and offers a method to test neutralizing antibodies.
Area of Science:
- Immunology
- Molecular Biology
- Microbiology
Background:
- Bacillus anthracis lethal toxin (LT) is a key virulence factor.
- Understanding LT's cellular effects is crucial for developing countermeasures.
- Neutrophils play a vital role in the innate immune response.
Purpose of the Study:
- To investigate the impact of LT on human neutrophil-like NB-4 cells.
- To identify gene expression changes indicative of LT intoxication.
- To evaluate the potential of this system for assessing toxin-neutralizing antibody efficacy.
Main Methods:
- Microarray analysis to screen for differentially expressed genes.
- Real-time PCR to validate mRNA level changes for specific chemokines (IL-8, CCL20, CCL3, CCL4).
- ELISA or similar assays to measure chemokine protein levels and assess antibody neutralization.
Main Results:
- LT exposure significantly down-regulated genes encoding chemokines and transcription factors.
- Real-time PCR confirmed decreased mRNA levels for IL-8, CCL20, CCL3, and CCL4, with effects intensifying over time.
- Chemokine protein levels decreased after 24h LT exposure and were sensitive to LT concentration.
- An anti-lethal factor monoclonal antibody (mAb) partially or fully restored IL-8 levels.
Conclusions:
- LT intoxication leads to the suppression of the cellular immune response by reducing chemokine production.
- The observed gene and protein expression changes serve as markers for LT intoxication.
- This experimental system provides a valuable platform for evaluating the activity of toxin-neutralizing antibodies.
