Taraktsoglou Maria, Szalabska Urszula, Magee David A, Browne John A, Sweeney Torres, Gormley Eamonn, MacHugh David E
Animal Genomics Laboratory, UCD School of Agriculture, Food Science and Veterinary Medicine, UCD College of Life Sciences, University College Dublin, Belfield, Dublin 4, Ireland.
Vet Immunol Immunopathol. 2011 Mar 15;140(1-2):130-9. doi: 10.1016/j.vetimm.2010.12.002. Epub 2010 Dec 10.
The involvement of Toll-like receptors (TLRs) and other immune signalling genes during challenge of bovine macrophages with bacterial products derived from disease-causing bacteria in cattle was investigated. An in vitro cell culture model of bovine monocyte derived macrophages (MDM) was established and these cells were exposed to purified protein derivative (PPD-b) derived from Mycobacterium bovis and to lipopolysaccharide (LPS) derived from Escherichia coli. Following 24h incubation, total RNA was extracted and expression of immune related genes was determined by real time quantitative reverse transcription PCR (qRT-PCR). Expression of a selection of genes spanning the TLR-2 and TLR-4 pathways, from the initial activation of the receptors to the production of pro-inflammatory cytokines and chemokines was determined. Results from repeat experiments using MDM from seven different age-matched dairy cattle showed that PPD-b treatment caused significant up-regulation of the TLR2 and TLR4 genes and the expression profile of TLR adaptor molecules suggested that this signalling is MYD88-dependent. Conversely, LPS caused significant up-regulation of TLR4 via a MYD88-independent signalling pathway. Significant up-regulation of genes involved with NF-κB signalling was also detected in PPD-b- and LPS-treated samples accompanied by the expression of pro-inflammatory cytokine (TNF, IL1B, IL6) and chemokine genes (IL8, CCL5, CCL3). Overall, LPS challenge resulted in a more marked up-regulation of immune-related genes. Furthermore, the magnitude fold-change difference in gene expression suggests, at least in part, that bovine macrophages produce IFN-γ as a result of LPS challenge.
研究了在用源自牛致病细菌的细菌产物攻击牛巨噬细胞期间Toll样受体（TLR）和其他免疫信号基因的参与情况。建立了牛单核细胞衍生巨噬细胞（MDM）的体外细胞培养模型，并将这些细胞暴露于源自牛分枝杆菌的纯化蛋白衍生物（PPD-b）和源自大肠杆菌的脂多糖（LPS）。孵育24小时后，提取总RNA，并通过实时定量逆转录PCR（qRT-PCR）测定免疫相关基因的表达。测定了一系列跨越TLR-2和TLR-4途径的基因的表达，从受体的初始激活到促炎细胞因子和趋化因子的产生。使用来自七头不同年龄匹配奶牛的MDM进行重复实验的结果表明，PPD-b处理导致TLR2和TLR4基因显著上调，TLR衔接分子的表达谱表明该信号传导是MYD88依赖性的。相反，LPS通过MYD88非依赖性信号通路导致TLR4显著上调。在PPD-b和LPS处理的样品中也检测到与NF-κB信号传导相关的基因显著上调，同时伴有促炎细胞因子（TNF、IL1B、IL6）和趋化因子基因（IL8、CCL5、CCL3）的表达。总体而言，LPS攻击导致免疫相关基因的上调更为明显。此外，基因表达的倍数变化差异至少部分表明，牛巨噬细胞因LPS攻击而产生IFN-γ。
