Arachidonic acid enhances mitophagy and decreases inflammatory response in primary macrophages

Macrophages are actively involved in recognition, capturing, and destruction of foreign pathogens, as well as removal of cellular debris. The most important role of macrophages is to initiate and regulate the inflammatory response: they synthesize and secrete a wide range of proinflammatory cytokines that activate other immune cells and promote the development of inflammation. The functional state of macrophages directly depends on mitochondrial activities, both as energy suppliers, and as key participants in signaling pathways associated with production of reactive oxygen species and inflammasome activation. Mitochondrial dysfunction may lead to excessive macrophage activation and chronic inflammation, typical of diseases like atherosclerosis and metabolic disorders. Damaged mitochondria release components such as mtDNA and cardiolipin, potentially triggering autoimmune responses. To prevent these events, the cells are capable of mitophagy, a selective autophagy process that removes dysfunctional mitochondria via the lysosomal pathway. Polyunsaturated fatty acids are known to influence inflammation and mitochondrial function, including mitophagy. Arachidonic acid, a precursor of prostaglandins and leukotrienes, modulates immune responses, but its role in mitophagy remains unclear. The aim of this study was to investigate whether arachidonic acid affects mitophagy and the proinflammatory response of human macrophages. Primary monocytes were isolated from whole blood of healthy donors and differentiated into macrophages over 5 days. The cells were treated with 20 μM arachidonic acid for 24 hours, followed by 1 μg/mL LPS stimulation for another 24 hours. Cytokine secretion (TNF, IL-6, IL-8, CCL2) was measured by ELISA technique. Mitophagy was assessed using confocal microscopy by evaluating co-localization of mitochondrial and lysosomal dyes. The results showed that arachidonic acid enhanced mitophagy and reduced secretion of TNF, IL6, and CCL2 in response to LPS. These findings suggest that activation of mitophagy may contribute to the anti-inflammatory effects of arachidonic acid in macrophages.