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Abstract
Tolllike receptors (TLRs) recognize conserved molecular motifs of microbes and elicit inflammatory and immune responses through specific intracellular adaptor molecules, particularly MyD88 and TRIF. Initiation of inflammatory responses is an important component of the host response to microbial pathogens and for the development of immunity. The first two studies reporting this dissertation project established the basis for the subsequent three studies. Firstly, an optimal source of lipopolysaccharide-binding protein (LBP) for eliciting responses by equine monocytes to lipopolysaccharide (LPS) was identified. Secondly, twenty-nine equine primer pairs to measure gene expression by real-time quantitative RT-PCR in equine monocytes were optimized and validated. The remaining three studies investigated the response of equine monocytes to different TLR agonists and a specific TLR4 antagonist. Based on the results of the initial study, pooled commercial and autologous equine sera are optimal sources of LBP activity for studies elucidating the effects of LPS on equine monocytes. A reliable set of equine primer pairs were used to determine that similar gene expression profiles were induced by the TLR4 (E. coli LPS) and TLR2 (Pam3CSK4) ligands that signal via MyD88. In contrast, the TLR3 (Poly I:C) ligand that signals through TRIF induced significantly higher expression of interferon-, IFN- inducible protein 10 and CCL5 than either the TLR2 or TLR4 ligands. Natural and synthetic lipid A compounds derived from E. coli strongly induced signaling through the MyD88-dependent pathway. Furthermore, the presence of the KDO moiety, resulted in significantly greater expression of inflammatory cytokines than did lipid A compounds lacking KDO. Differences in the length of the fatty acid chains attached to the lipid A backbone induced different levels of biological responses from equine monocytes. Finally, the second-generation synthetic lipid A analagoue, E5564, induced minimal pro-inflammatory effects in equine whole blood or monocytes. Most importantly, E5564 inhibited LPS-induced expression of procoagulant activity and tumor necrosis factor-alpha production and mRNA expression of TNF-alpha, interleukin-1 (IL-1) and IL-10.