In Vitro Production of Neutrophils Extracellular Traps Is Affected by the Lactational Stage of Dairy Cows

Abstract

We aimed to research the neutrophil extracellular traps (NETs) and reactive oxygen species (ROS) formation capacity of polymorphonuclear cells (PMN) during different lactational stages of Holstein cows. We also aimed to validate a model which could mimic infection and inflammation in vitro by adding increasing concentrations of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) to PMN suspensions isolated from nulliparous heifers and evaluate their capacity to produce NETs and ROS. In 3 replicates, we collected blood from nulliparous heifers (n = 3), cows at the end of gestation (n = 3), early postpartum (n = 3) and in mid-lactation (n = 3) in which PMN were isolated. The production of ROS in PMN were assessed using the 2’,7’-Dichlorofluorescein diacetate method, while the SYTOX Orange and Quant-iT™ PicoGreen dsDNA ultra-sensitive nucleic fluorescent acid staining methods were applied in order to quantitatively analyze the formation of NETs. Statistical analyses were performed via linear regression models using the replicate as a random. ROS values of PMN harvested from peripartum cows were 1.3 times increased compared with those in nulliparous heifers (p < 0.01). Compared with nulliparous heifers, the production of NETs by PMN isolated from mid-lactation and postpartum cows was 2.1 and 2.5 times higher (p < 0.01), respectively. In 3 replicates, in vitro stimulation of PMN isolated from nulliparous heifers (n = 3) with LPS linearly increased the production of ROS and NETs (R2 = 0.96 and 0.86, respectively). Similarly, when PMN isolated from nulliparous heifers were stimulated with PMA, a linear increase in the production of ROS (R2 = 0.99) and NETs (R2 = 0.78) was observed. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic inflammation and study fundamental aspects of the production of NETs and ROS in vitro.

The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows

“…In vitro models of inflammation have been effectively developed using 100 μg/mL LPS for 1 h or 80 μg/mL LPS for 3 h [ 10 , 21 ]. To ensure the reliability of our model, cell viability measurements were employed to eliminate the interference of deceased cells.…”

“…The EDTA tubes were used for PMNs isolation. PMNs isolation was conducted following the method outlined by Xie et al [ 10 ]. Cell viability was assessed using Trypan blue solution, and Giemsa staining was employed to evaluate cell purity [ 28 ].…”

“…The Quant-iT™ PicoGreen dsDNA Assay kit was employed to quantify the NETs level according to the method outlined by Xie et al [ 10 ]. Briefly, PMNs were diluted with RPMI 1640 medium, and 200 µL containing 6 × 10 5 cells was subsequently inoculated into 96-well plates and cultured with 0, 20, 40, 60, and 80 μg/mL LPS for 1 and 3 h at 37 °C and 5% CO 2 .…”

“…Previous studies have shown that LPS can stimulate the release of NETs in a concentration-dependent manner [ 10 , 11 , 12 ]. However, the LPS concentration and duration of exposure showed variability across different in vitro studies [ 13 , 14 , 15 , 16 ].…”

“…These differences may be attributed to inherent variations in the enzymatic function of PMNs granules among distinct species [ 17 ]. Remarkably, the process of NETs formation coincides with the generation of ROS, therefore, the measurement of ROS levels may serve as an indirect approach to evaluate NETs production in vitro [ 10 , 14 ]. For research purposes, the quantification of NETs-associated proteins, including citrullinated histone H3 (Cit-H3), peptidyl arginine deiminase 4 (PAD4), histone3 (H3), and cathepsin G (CG) can be used as NETs-generated indicators [ 18 , 19 ].…”