The Concentration and Duration of Lipopolysaccharide Stimulation Produce Different Cytokine Responses in an Ex Vivo Whole Blood Model in Horses

Lipopolysaccharide (LPS) is frequently used in equine research to model clinical endotoxemia; however, there is no standardized protocol for inducing cytokine production in equine whole blood. To address this knowledge gap, the goal of this study was to compare the cytokine responses elicited by three different LPS stimulation protocols. Whole blood was collected from six healthy horses (aged 5-30 years; mixed breeds and genders) from the North Carolina State University teaching herd (IACUC #23-412). Sixty milliliters of heparinized blood were aseptically drawn and divided into 15 mL aliquots. Samples were stimulated with LPS at concentrations of 100 ng/mL, 1000 ng/mL, or using a two-hit model (500 ng/mL initially and again at 1.5 h). Incubation occurred at 37 °C on an orbital shaker for time points ranging from 1.5 h to 24 h. Cytokine concentrations were measured using the Cornell Equine Cytokine and Chemokine Panel and were compared to non-stimulated controls. LPS stimulation induced the production of TNF-α, IL-1β, IL-10, CCL5, and CCL11 in a time- and concentration-dependent manner. Notably, reliable and robust cytokine responses were observed only after 12 h of stimulation with either 1000 ng/mL or the two-hit 500/500 ng/mL protocol. These findings suggest that both the concentration and duration of LPS exposure significantly influence cytokine expression in equine whole blood. Therefore, the optimal stimulation protocol may vary depending on the specific cytokine of interest, and careful consideration of these variables is essential for designing reproducible and physiologically relevant ex vivo models of equine endotoxemia.