Abstract #M12

# M12
Determining immune-modulating components of Saccharomyces cerevisiae with RAW 264.7 murine macrophages.
Sarah E. Sivinski*1, Rachel A. Rusk1, Jodi L. McGill1, Barry J. Bradford1, 1Kansas State University, Manhattan, KS.

Feed components can modulate the immune system, but in vivo data are expensive and rare; thus, an effective screening tool for evaluating such nutrients is needed to enable informed selection of candidate immunomodulators for in vivo investigation. This study used RAW 264.7 murine macrophages as an innate immunity in vitro screening tool to identify immune-modulating properties of Saccharomyces cerevisiae and some of its components. Treatments were 0.01, 0.1, or 1 mg/mL of whole S. cerevisiae cells (WC), mannan, Zymosan (includes cell wall protein-carbohydrate complexes, mannans, and β-glucans), or d-mannose at either pH 3 or 7. A pH of 3 was used to mimic the acidic conditions of the stomach to assess potential alterations of component activity. The cells were transfected with a vector that drove expression of an alkaline phosphatase reporter gene upon activation of NFκB. Cells (n = 6 wells/treatment) were incubated with treatments for 18 h. Cell supernatants were then incubated for 2 h with alkaline phosphatase (AP) substrate media (QUANTI-Blue) to assay enzyme activity. After normalizing values across plates using unstimulated cells and LPS (1 ng/mL) stimulated cells as negative and positive controls, intra- and inter-assay CVs were 3.9% and 15.8%, respectively. The effects of treatment, pH, and dose(treatment) were evaluated by mixed models. Treatment and dose(treatment) affected AP activity (both P < 0.001) whereas pH did not have an effect (P = 0.31). WC at 1 mg/mL and mannan at 0.1 mg/mL tended to increase AP activity (both P = 0.07), whereas WC at 0.1 mg/mL, mannan and d-mannose at 1 mg/mL, and Zymosan at all 3 concentrations increased AP activity (all P < 0.001), compared with the unstimulated cell control. Zymosan stimulated a greater immune response than all other components (all P < 0.001), and WC stimulated a greater response than d-mannose (P = 0.03). Overall, this innate immunity in vitro screening method was useful for quickly determining immune-modulating properties and concentrations of S. cerevisiae components.

Key Words: bioactive nutrient, nutritional immunology, in vitro screening method