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  • Development of an in vitro macrophage screening system on the immunomodulating effects of feed components.

Development of an in vitro macrophage screening system on the immunomodulating effects of feed components.

Journal of animal science and biotechnology (2020-09-05)
S E Sivinski, L K Mamedova, R A Rusk, C C Elrod, T H Swartz, J M McGill, B J Bradford
ABSTRACT

While feed components capable of modulating the immune system are highly sought after and marketed, often little evidence is available to support functional immune response claims. Thus, a high-throughput in vitro cell screening system was developed to test these compounds for innate immune signaling effects, using Saccharomyces cerevisiae and its cell wall components in addition to lauric acid and its esters as models in two separate experiments. This screening system utilized RAW 264.7 murine macrophages to assess live S. cerevisiae cells and S. cerevisiae-derived cell wall components β-glucan, mannan, and zymosan (a crude cell wall preparation containing both β-glucan and mannan). D-mannose was also evaluated as the monomer of mannan. We also examined the effect of a saturated fatty acid (C12:0, lauric acid) and its esters (methyl laurate and glycerol monolaurate) on innate immune cell activation and cellular metabolism. RAW cells were transfected with a vector that drives expression of alkaline phosphatase upon promoter activation of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB), a major inflammatory/immune transcription factor. RAW cells were incubated with 0.01, 0.1 or 1 mg/mL of yeast compounds alone or RAW cells were challenged with LPS and then incubated with yeast compounds. In a separate experiment, RAW cells were incubated with 0, 0.5, 2.5, 12.5, 62.5, and 312.5 μmol/L of lauric acid, methyl laurate, or glycerol monolaurate alone, or RAW cells were challenged with LPS and then incubated with fatty acid treatments. Treatment with zymosan or β-glucan alone induced NFκB activation in a dose-dependent manner, whereas treatment with D-mannose, mannan, or live S. cerevisiae cells did not. Post-treatment with mannan after an LPS challenge decreased NFκB activation, suggesting that this treatment may ameliorate LPS-induced inflammation. Slight increases in NFκB activation were found when fatty acid treatments were applied in the absence of LPS, yet substantial reductions in NFκB activation were seen when treatments were applied following an LPS challenge. Overall, this cell screening system using RAW macrophages was effective, high-throughput, and sensitive to feed components combined with LPS challenges, indicating modulation of innate immune signaling in vitro.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
D-(+)-Mannose, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Resazurin sodium salt, powder, BioReagent
Sigma-Aldrich
Mannan from Saccharomyces cerevisiae, prepared by alkaline extraction
Sigma-Aldrich
Yeast from Saccharomyces cerevisiae, Type II
Sigma-Aldrich
Zymosan A from Saccharomyces cerevisiae, for inducing inflamatory response
Supelco
Lauric Acid, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Lipopolysaccharides from Escherichia coli O111:B4, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
L-(−)-Glucose, ≥99%