Glycolysis and mitochondrial function regulate the radical oxygen species production induced by platelet-activating...

Dairy cows undergo metabolic disturbances in the peripartum period, during which infectious inflammatory diseases and detrimental polymorphonuclear leukocytes (PMN) functions, such as radical oxygen species (ROS) production, are observed. Platelet-activating factor (PAF) is a key pro-inflammatory mediator that increases PMN ROS production. To date, the role of glycolysis and mitochondria in PAF-induced ROS production in bovine PMN has not been known. The aim of this study was to assess whether inhibition of glycolysis and disruption of mitochondrial function alter the oxidative response induced by PAF. We isolated PMN from non-pregnant Holstein Friesian heifers and pre-incubated them with 2-deoxy-d-glucose (2-DG; 2 mM, 30 min), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 5 μM, 5 min), oligomycin (10 μM, 30 min) or rotenone (10 μM, 30 min). Respiratory burst was measured by luminol-chemiluminescence assay, while mitochondrial ROS (_mtROS) were evaluated by MitoSOX probe and flow cytometry. Also, we detected the presence of mitochondria by MitoTracker Deep Red FM probe and changes in mitochondrial membrane potential (Δψ_m) were assessed by JC-1 probe and flow cytometry. We observed that all inhibitors separately were able to reduce PAF-induced ROS production. Presence of mitochondria was detected and PAF increased the Δψ_m, while CCCP reduced it. 2-DG and rotenone reduced the _mtROS production induced by PAF. CCCP did not alter the _mtROS and oligomycin administered independently increased _mtROS production. We concluded that PAF-induced ROS production is glycolysis- and mitochondria-dependent. Bovine PMN have a functional mitochondrion and PAF induced _mtROS via glycolysis and mitochondrial complex-I activity. Our results highlight an important modulation of cellular metabolism in the oxidative response induced by proinflammatory agents, which could contribute to PMN disfunction during peripartum in cattle.