In vitro effects of Escherichia coli lipopolysaccharide on the function and gene expression of neutrophils isolated from the blood of dairy cows.

The objectives of this study were to investigate the effect of Escherichia coli lipopolysaccharide (LPS) on the function of bovine neutrophils (PMNL) collected from mid lactation cows and determine the differential effects of LPS on gene expression of PMNL purified from early and mid lactation cows. The PMNL from mid lactation cows (187±13 d postpartum) were incubated with 0, 1, 25, and 50 µg/mL of LPS for 120 min, and the generation of reactive oxygen species (ROS), PMNL extracellular traps (NET), chemotaxis, and killing of Staphylococcus aureus were determined. Incubation of PMNL with 25 µg/mL of LPS increased intracellular ROS by 79% in mitogen-stimulated PMNL. Addition of 50 µg/mL of LPS enhanced intracellular ROS by nonstimulated and stimulated PMNL by 184 and 154%, respectively. Nonstimulated PMNL incubated with 25 and 50 µg/mL of LPS had a 105% increase in NET. Addition of LPS had no effect on subsequent PMNL chemotaxis or killing of Staph. aureus. To examine the effect of LPS on the expression of genes involved in PMNL function and cytokine production, mRNA was purified from PMNL isolated from mid lactation (146±2 postpartum; n=10) and early lactation cows (7 d postpartum; n=10), after a 120-min incubation with 0 or 50 µg/mL of LPS. Amounts of interleukin-8 (IL-8), tumor necrosis factor (TNF), bactericidal/permeability-increasing protein (BPI), myeloperoxidase (MPO), superoxide dismutase 2 (SOD2), NADPH oxidase 4 (NOX4), Cytochrome b-245, α polypeptide (CYBA), histone H2A/1 (H2A/1), and histone H2B-like (H2B) mRNA were determined relative to that of ß-actin by real-time quantitative PCR. Regardless of stage of lactation, PMNL incubated with 50 µg/mL of LPS had 537 and 45% higher mRNA contents of IL-8 and SOD2 compared with 0 µg/mL LPS, respectively. In addition, LPS augmented the expression of TNF, BPI, and CYBA (2,908, 59, and 158% compared with controls, respectively) only in PMNL from mid lactation cows. Addition of LPS did not affect mRNA levels of MPO, NOX4, H2A/1, or H2B. Independent of LPS treatment, PMNL from mid lactation cows had 99% higher mRNA contents of IL-8 compared with PMNL from early lactation cows. The PMNL from early lactation cows had a 634% increase in MPO mRNA expression relative to that from mid lactation cows. These results support that LPS directly stimulates PMNL to produce ROS and express NET. In addition, LPS enhances the generation of ROS by PMNL in response to other stimuli and increases the expression of genes encoding inflammatory mediators and enzymes involved in the production of ROS. Finally, reduced PMNL gene expression of IL-8 (regardless of LPS activation), TNF, CYBA, and BPI (upon stimulation with LPS) in early lactation may elucidate several mechanisms by which PMNL may become immune-incompetent during this period.

Effects of in vitro insulin and 2,4-thiazolidinedione on the function of neutrophils harvested from blood of cows in different physiological states.

Neutrophils [polymorphonuclear neutrophilic leukocytes (PMNL)] were isolated from 26 Holstein cows in different physiological states (12+/-1.7 d prepartum, n=8; 7+/-0 d postpartum, n=9; 253+/-25.2 d postpartum, n=9) and incubated in vitro for 120 min in a factorial arrangement of treatments with 0, 1.5, or 15 ng/mL of bovine insulin and 0 or 300 microg/mL of the peroxisome proliferator-activated receptor-gamma ligand 2,4-thiazolidinedione (TZD). Following the incubations, PMNL functional assays were performed to determine treatment effects on proxies for total, extracellular, and intracellular generation of reactive oxygen species (ROS), neutrophil extracellular trap formation, and phagocytic killing abilities. The ROS production of PMNL collected from cows at 7 d postpartum was reduced compared with that of PMNL from midlactation and prepartum cows, but neutrophil extracellular trap expression was 23 and 36% greater in PMNL from prepartum cows compared with that in PMNL from midlactation and postpartum cows, respectively. Insulin had no effect on PMNL functional assay results. In contrast, TZD inhibited a measurement of total ROS production by 89%, increased extracellular superoxide generation by 43%, but had no effect on the intracellular ROS measured. Interestingly, TZD did not alter the ability of the PMNL to release neutrophil extracellular traps and engulf or kill Staphylococcus aureus. These findings suggest a possible anti-inflammatory effect of TZD that may result in reduced extracellular oxidative damage with maintenance of PMNL antimicrobial activity.