Arshad U, Cid de la Paz M, White H M, Cangiano L R
Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI 53706.
JDS Commun. 2024 May 10;5(6):740-744. doi: 10.3168/jdsc.2024-0565. eCollection 2024 Nov.
Dairy cows are susceptible to several health disorders throughout their lactation. Objectives were to characterize an in vitro model to study bioenergetic measures in CD4 T lymphocytes in dairy cows. Twenty-four healthy mid-lactation multiparous Holstein dairy cows were enrolled at a mean (±SD) of 234 ± 22 DIM. Cows were blocked according to DIM and blood was collected to isolate peripheral blood mononuclear cells followed by magnetic separation of CD4 T lymphocytes using bovine-specific monoclonal antibodies. The isolated CD4 T lymphocytes from each cow were split into 2 tubes and randomly assigned to incubate in an assay medium as control (CON) or with a combination of phorbol myristate acetate and ionomycin (PMA+IMY) to evaluate metabolic function under a resting and activated state. Mitochondrial and glycolytic functional kinetics were recorded in CD4 T lymphocytes based on real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) under basal conditions and in response to complex V inhibitor (oligomycin), a protonophore uncoupler (BAM 15), and complex I and complex III inhibitors (rotenone and antimycin A). The mean (±SD) viability and purity of CD4 T lymphocytes was 92.5 ± 2.9% and 95.2 ± 2.9%, respectively. The basal OCR in CD4 T lymphocytes treated with PMA+IMY was greater than CON; nevertheless, the maximal respiration rate (CON = 58.0 vs. PMA+IMY = 47.3 ± 5.7 pmol/min) and sparing respiratory capacity rate (CON = 42.0 vs. PMA+IMY = 28.7 ± 4.2 pmol/min) were decreased in activated CD4 T lymphocytes. The ECAR in CD4 T lymphocytes increased progressively over time in PMA+IMY compared with CON, which indicated an increase in aerobic glycolysis in PMA+IMY compared with CON (CON = 46.9 vs. PMA+IMY = 86.4 ± 7.0 pmol/min). Activated CD4 T lymphocytes exhibit a metabolic switch from oxidative phosphorylation to aerobic glycolysis, which may support rapid cell proliferation. The results observed in this experiment demonstrate the sensitivity of the technique to detect changes in metabolic function under different cellular conditions, providing a robust framework to study immuno-metabolism in dairy cattle.
奶牛在整个泌乳期易患多种健康疾病。本研究的目的是建立一种体外模型，用于研究奶牛CD4 T淋巴细胞的生物能量代谢指标。选取24头处于泌乳中期的健康经产荷斯坦奶牛，平均泌乳天数（±标准差）为234±22天。根据泌乳天数对奶牛进行分组，采集血液以分离外周血单核细胞，然后使用牛特异性单克隆抗体通过磁性分离法分离CD4 T淋巴细胞。将每头奶牛分离得到的CD4 T淋巴细胞分成2管，随机分配到测定培养基中作为对照（CON）或与佛波酯肉豆蔻酸酯和离子霉素（PMA+IMY）组合孵育，以评估静息和激活状态下的代谢功能。基于基础条件下以及对复合物V抑制剂（寡霉素）、质子载体解偶联剂（BAM 15）、复合物I和复合物III抑制剂（鱼藤酮和抗霉素A）的反应，实时测量氧消耗率（OCR）和细胞外酸化率（ECAR），记录CD4 T淋巴细胞中的线粒体和糖酵解功能动力学。CD4 T淋巴细胞的平均（±标准差）活力和纯度分别为92.5±2.9%和95.2±2.9%。用PMA+IMY处理的CD4 T淋巴细胞的基础OCR高于CON组；然而，激活的CD4 T淋巴细胞的最大呼吸速率（CON组=58.0，PMA+IMY组=47.3±5.7 pmol/min）和备用呼吸能力速率（CON组=42.0，PMA+IMY组=28.7±4.2 pmol/min）降低。与CON组相比，PMA+IMY处理的CD4 T淋巴细胞中的ECAR随时间逐渐增加，这表明与CON组相比，PMA+IMY处理组的有氧糖酵解增加（CON组=46.9，PMA+IMY组=86.4±7.0 pmol/min）。激活的CD4 T淋巴细胞表现出从氧化磷酸化到有氧糖酵解的代谢转换，这可能支持细胞的快速增殖。本实验观察到的结果证明了该技术在检测不同细胞条件下代谢功能变化方面的敏感性，为研究奶牛免疫代谢提供了一个有力的框架。
