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

摘要

Methionine (Met) is one of the 2 most limiting aminoacids for milk production in dairy cow diets. The accepted“ideal” ratio of lysine (Lys) to Met (L:M) whenformulating diets is 3:1. However, blood from cows fedcorn silage-based diets without supplemental rumenprotectedMet averages approximately 3.6:1 L:M. Recentin vivo research on cattle immunonutrition hasrevealed that the immune system could benefit fromgreater Met supply. To study more closely the effectsof 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 differentlevels of L:M (3.6:1, 2.9:1, or 2.4:1) and stimulatedwith lipopolysaccharide (LPS) at either 0 or 50 μg/mL for 2 h at 37°C. Target genes were associated withcytokines, pathogen recognition, nuclear receptors, killingmechanisms, and Met and glutathione metabolism.Data were subjected to ANOVA using PROC MIXEDin SAS, with L:M, LPS, and their interaction as fixedeffects. Stimulation with LPS upregulated genes relatedto cytokines (IL1B, TNF, IL10 and IL6) and nuclearreceptors, including nuclear factor kappa B (NFKB1)and glucocorticoid receptor (NR3C1), and downregulatedthe mRNA abundance of chemokine receptor 1(CXCR1), lysozyme (LYZ) and glutathione reductase(GSR). A linear decrease was observed in the mRNAabundance of TNF when L:M was decreased. A similarresponse was observed for interleukin-1 receptor-associatedkinase 1 (IRAK1) and NFKB1 abundance in cellsstimulated with LPS (linear effect). A linear increaseof LYZ mRNA expression as L:M decreased was detectedin unstimulated cells. Furthermore, a decreasein L:M led to a linear decrease of superoxide dismutase1 (SOD1) mRNA abundance in cells challenged withLPS. Overall, LPS challenge triggered the activation ofisolated PMN from mid-lactation cows. However, datasuggest the use of a shorter incubation time to capturethe peak response and not the resolution of the inflammatoryresponse as in the present study. Our resultsindicate a possible involvement of Met in modulatingPMN inflammatory and oxidative stress status andin helping the resolution of inflammation after initialstimulation.