Molecular genetic assessment of chicken macrophage innate immunity: toll-like receptors, mechanisms of action, and kinetic transcriptome profile

摘要

Understanding the genetic regulation of host response governing disease resistance mechanisms is of primary importance for improving animal health and food safety. Many of the biological characteristics of the chicken make it an ideal organism for studies in immunology, evolution, agriculture, medicine and comparative functional analyses. Different cell lines or primary cells from the immune system generate different immune responses even though they are induced with the same stimulant, suggesting that it is important to dissect the immune system to understand the mechanisms that are shaping the host response. Therefore, we used chicken macrophages as a model to study these mechanisms. This dissertation involves a comprehensive set of experimental and bioinformatic studies designed to deepen the current knowledge of chicken macrophage innate immune responses. In the first study, we determined the role of Toll-like receptor 15 (TLR15) and its downstream effector responses in the chicken macrophage HD11 cell line. TLR15 is an avian-specific pattern recognition receptor (PRR) of unknown specificity that is structurally different from other TLRs in the arrangement of the leucine rich region (LRR). TLR15, in collaboration with, TLR2 and TLR21 can respond to CpG oligonucleotides (ODN) and is able to rapidly distinguish different types of CpG-ODNs, as does mammalian TLR9. Using RNA interference technology, we demonstrated that the MyD88 adaptor molecule is required to have upregulation of of Interleukin 1 beta (IL1B) after induction of TLR15 gene expression with CpG-ODNs. In a second , the effect of Salmonella typhimurium-798 (ST-798)-derived endotoxin on kinetic mRNA profiles of chicken macrophages HD11 cells at 1, 2, 4 and 8 hours post-stimulation (hps) was tested using Affymetrix GeneChip chicken genome arrays containing 38535 probesets. By comparison with non-stimulated cells, the greatest gene expression response was observed at 4 hps. Comparative analysis of gene networks from the microarray data indicated that 10% of the differentially expressed genes were involved in the response to Salmonella endotoxin. Additionally, this work demonstrated a consistent signature set of four up-regulated genes at all tested time points: IL1BB, IL8, NFKBIA, and CCL4. The differentially expressed genes obtained from microarray analysis were categorized by function with emphasis on inflammatory response. The examination of chicken macrophage immune response on a global scale and the investigation of regulatory mechanisms of innate response are the main objectives of this dissertation. Our findings hold great potential to expand possibilities for production of better pharmaceuticals, and identification of beneficial genetic selection markers, by defining immune response pathways.