In vitro methionine supplementation during lipopolysaccharide stimulation modulates immunometabolic gene network expression in isolated polymorphonuclear cells from lactating Holstein cows.

Methionine (Met) is one of the 2 most limiting amino acids for milk production in dairy cow diets. The accepted "ideal" ratio of lysine (Lys) to Met (L:M) when formulating diets is 3:1. However, blood from cows fed corn silage-based diets without supplemental rumen-protected Met averages approximately 3.6:1 L:M. Recent in vivo research on cattle immunonutrition has revealed that the immune system could benefit from greater Met supply. To study more closely the effects of different L:M ratios, blood polymorphonuclear cells (PMN) were isolated from 5 Holstein cows in mid-lactation (238 ± 20 d postpartum, 33.8 ± 3.8 kg of milk/d; mean ± SD). The PMN were incubated at 3 different levels of L:M (3.6:1, 2.9:1, or 2.4:1) and stimulated with lipopolysaccharide (LPS) at either 0 or 50 μg/mL for 2 h at 37°C. Target genes were associated with cytokines, pathogen recognition, nuclear receptors, killing mechanisms, and Met and glutathione metabolism. Data were subjected to ANOVA using PROC MIXED in SAS, with L:M, LPS, and their interaction as fixed effects. Stimulation with LPS upregulated genes related to cytokines (IL1B, TNF, IL10 and IL6) and nuclear receptors, including nuclear factor kappa B (NFKB1) and glucocorticoid receptor (NR3C1), and downregulated the mRNA abundance of chemokine receptor 1 (CXCR1), lysozyme (LYZ) and glutathione reductase (GSR). A linear decrease was observed in the mRNA abundance of TNF when L:M was decreased. A similar response was observed for interleukin-1 receptor-associated kinase 1 (IRAK1) and NFKB1 abundance in cells stimulated with LPS (linear effect). A linear increase of LYZ mRNA expression as L:M decreased was detected in unstimulated cells. Furthermore, a decrease in L:M led to a linear decrease of superoxide dismutase 1 (SOD1) mRNA abundance in cells challenged with LPS. Overall, LPS challenge triggered the activation of isolated PMN from mid-lactation cows. However, data suggest the use of a shorter incubation time to capture the peak response and not the resolution of the inflammatory response as in the present study. Our results indicate a possible involvement of Met in modulating PMN inflammatory and oxidative stress status and in helping the resolution of inflammation after initial stimulation. The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).