Submitted to: Molecular Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2009
Publication Date: 8/1/2009
Citation: Rath, N.C., Kannan, L., Liyanage, R., Lay, J.O. 2009. Evaluation of beta defensin 2 production by chicken heterophils using direct MALDI mass spectrometry. Molecular Immunology. 46(15)3151-3156. Interpretive Summary: White blood cells fight against disease causing organisms secreting many factors. One of such factor called beta defensin 2 (BD2) induces leakage in bacterial membrane. We wanted to measure BD2 produced by chicken white blood cell type called heterophils, artificially stimulating them with various bacterial and viral products. We grew chicken heterophils with different microbial products and estimated BD2 secreted into culture medium by its chemical modification and comparing with their non stimulated counterparts using an instrument called mass spectrometer. Our results show that many bacterial products significantly increased BD production.
Technical Abstract: Beta defensins (BD) are cysteine rich, cationic antimicrobial peptides (AMP) produced mainly by epithelial and myeloid cells such as neutrophils. In birds, the equivalent of neutrophils, heterophils produce avian beta defensins (AvBD) of which AvBD2 is the major isoform. Heterophils recognize pathogens/products, through a series of pattern recognition receptors called toll-like receptors (TLR) which when activated, may or may not influence AvBD2 production. This work represents the first report of TLR modulation of AvBD2 expression in chickens. To measure the effect of TLR activation on AvBD2 production, heterophils were cultured with different TLR agonists for 6 h. Modulation of AvBD2 levels by TLR activation was measured using direct MALDI mass spectrometry without stable isotopic labeling or chromatographic separation. Chemical modification of the conditioned media was performed using reduction/alkylation with dithiothreitol/iodoacetamide to distinguish TLR treated AvBD2 (reduced/alkylated) from controls (non reduced). Changes in corrected ion intensity ratios were assumed to reflect AvBD2 modulation in heterophils upon activation with different TLR agonists. In general, TLR agonists increased AvBD2 production, with LPS showing the greatest induction and CpG-ODN showing little or no effect. These data provide evidence that direct MALDI-MS coupled with reduction/alkylation may provide a rapid relative quantitative approach to the measurement of agonist-induced differential expression of AvBD2.