Induction and characterization of endotoxin tolerance in equine peripheral blood mononuclear cells in vitro

• Main Author: Frellstedt, Linda
• Other Authors: Biomedical and Veterinary Sciences
• Format: Others
• Published: Virginia Tech 2014
• Subjects: Endotoxin; endotoxin tolerance; interleukin-12; interleukin-10; equine
• Online Access: http://hdl.handle.net/10919/44307; http://scholar.lib.vt.edu/theses/available/etd-08192010-172631/

Endotoxemia is responsible for severe illness in horses. Individuals can become unresponsive to the endotoxin molecule after an initial exposure; this phenomenon has been called developing a state of â endotoxin toleranceâ (ET). ET has been induced in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMCs) in vitro, and to describe the cytokine profile which is associated with the ET. <p> Blood was collected from 6 healthy horses and PBMCs were isolated. ET was induced by culturing cells with three concentrations of endotoxin given to induce ET, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. <p> ET was induced in all cells (n=6) exposed to the 2-step endotoxin challenge. In PBMCs treated with 1.0 ng/ml of endotoxin followed by challenge with 10 ng/ml of endotoxin, the relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge compared with positive control cells. <p> This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMCs. A marked suppression of IL-12 production is associated with ET. The production of IL-10 was not altered in ET in our model. === Master of Science