Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro

Abstract

During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory response of PMNL from cows in early and mid-lactation in vitro. Twenty Holstein cows in early (n=10; days in milk=17±3.1) and mid-lactation (n=10; days in milk=168±14.8) were used for this study. Jugular blood was analyzed for serum concentrations of nonesterified fatty acids, β-hydroxybutyrate, and glucose. Polymorphonuclear leukocytes were isolated and diluted using RPMI (basal glucose concentration was 7.2 mM) to different concentrations of PMNL/mL for phagocytosis, chemotaxis, gene expression, and medium analyses. Working solutions of glucose (0 or 4 mM of d-glucose) and lipopolysaccharide (0 or 50μg/mL) were added and tubes were incubated for 120 min at 37°C. Media were analyzed for concentrations of glucose and tumor necrosis factor-α (TNF-α). Data were analyzed in a randomized block (stage of lactation) design. Challenge with lipopolysaccharide increased the expression of the genes encoding for nuclear factor kappa B (NFKB1), IL-10 (IL10), IL1B, IL6, IL8, TNF-α (TNFA), glucose transporter 3 (SLC2A3), and the concentration of TNF-α in medium (147.3 vs. 72.5 pg/mL for lipopolysaccharide and control, respectively). Main effect of stage of lactation was minimal where the expression of IL10 increased for cows in early compared with cows in mid-lactation. After lipopolysaccharide challenge, cows in early lactation experienced more marked increases in the expression of IL6, TNFA, and IL8 when compared with cows in mid-lactation. Glucose supplementation had minimal effects on gene expression where glucose supplementation increased the expression of lysozyme (LYZ). Glucose supplementation increased PMNL phagocytosis but did not alter chemotaxis, morphology, or concentration of TNF-α in the medium. Under the conditions of the experiment, stage of lactation had minimal effects on PMNL response to glucose supply where only the expression of NFKB1 and the production of TNF-α were greater for cows in mid-lactation when compared with early lactation. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain results for this study. Future studies investigating the effect of glucose supply on bovine PMNL function in vivo and how this may be altered by stage of lactation are warranted.

Short communication: Amino acid supplementation and stage of lactation alter apparent utilization of nutrients by blood neutrophils from lactating dairy cows in vitro

“…A lack of stage of lactation effect was also observed in the expression of genes coding for inflammatory PMN receptors, signaling factors, cytokines, enzymes, and receptors involved in glucose and AA metabolism (for a complete list of genes and their effects see Supplemental Table S4; http://dx.doi.org/10.3168/jds.2015-10086). In a recent paper (Garcia et al, 2015b) we also observed that stage of lactation did not alter PMN function and minimal differences in gene expression were observed after glucose supplementation.…”

“…Thirteen primiparous and 5 multiparous (≥2 lactations) Holstein cows in early (n = 9; DIM = 17 ± 3.2; 34.0 kg of milk/d) and mid lactation (n = 9; DIM = 165 ± 14.4; 38.6 kg of milk/d) were used for this study. Details regarding animal selection, housing, management, isolation of PMN, and analysis are described in Garcia et al (2015b). Jugular blood (~150 mL) was collected from cows in early and mid lactation.…”

“…For gene expression and media analyses only, we transferred tubes to a sterile hood where LPS (50 μg/mL final concentration; L055; Sigma Aldrich Co.) or no LPS (i.e., PBS) was added. Lipopolysaccharide was used based on previous experiments (Revelo and Waldron, 2012;Garcia et al, 2015b). The addition of 4 mM of a pool of AA were supplemented in the AAS treatment at ~2 times that of physiological concentration (Meijer et al, 1995) and equivalent to doses used in our laboratory (Garcia et al, 2015b).…”

“…Lipopolysaccharide was used based on previous experiments (Revelo and Waldron, 2012;Garcia et al, 2015b). The addition of 4 mM of a pool of AA were supplemented in the AAS treatment at ~2 times that of physiological concentration (Meijer et al, 1995) and equivalent to doses used in our laboratory (Garcia et al, 2015b). Final concentrations of AA were 3.1 and 7.1 mM for the CTL and AAS groups, respectively (details of individual AA are presented in Supplemental Table S1; http://dx.doi.…”

“…After incubation, we analyzed PMN for gene expression. Isolation of RNA, quality evaluation, cDNA synthesis, primer selection and design sequence, efficiency of dilution, and real-time quantitative PCR analysis were as previously described (Garcia et al, 2015b). Additional primer sequence and efficiency of dilution curves for BCKDHB is presented in Supplemental Tables S2 and S3, respectively (http:// dx.doi.org/10.3168/jds.2015-10086).…”