Monocytes are key regulators of inflammation and innate immunity. They obtain energy through two pathways: glycolysis and oxidative phosphorylation. Their metabolism is closely linked to the regulation of the phenotypes of the subsequent developmental stages of monocytes, M1 and M2 macrophages. M1 macrophages primarily utilize glycolysis for energy production, while M2 macrophages primarily rely on oxidative phosphorylation. Activation of monocytes with bacterial lipopolysaccharide (LPS) shifts the phenotype towards the M1 state. Using the Agilent Seahorse XFe24 analyzer, we quantified the level of glycolytic activity by measuring the extracellular acidification rate (ECAR) in real-time. We found that peripheral blood monocytes exhibit an increased ECAR after LPS activation, which is consistent with the results of other studies showing a metabolic shift in activated monocytes towards glycolysis. It is known that parasympathetic activation inhibits the activation and release of inflammatory cytokines from macrophages in the spleen, thereby limiting excessive inflammatory responses. We hypothesize that acetylcholine, the main neurotransmitter of the parasympathetic nervous system, reduces the glycolytic metabolic activity of LPS-activated monocytes via α7-nicotinic acetylcholine receptors.
In our master's thesis, we used experiments with the Seahorse XFe24 analyzer to test whether incubation of activated monocytes with the non-selective cholinergic agent acetylcholine inhibits their glycolytic activity. Using specific cholinergic antagonists, we investigated whether this inhibition is related to the effect of acetylcholine on α7-nicotinic acetylcholine receptors. Due to the presence of neutrophils in the isolated monocyte sample, we also determined their metabolic activity to evaluate their impact on the results in our monocyte isolate. Our experimental data show that acetylcholine weakly inhibited the increase in glycolytic activity of monocytes after LPS activation. This effect was only prevented by α bungarotoxin, an antagonist of α7-nicotinic acetylcholine receptors, indicating that acetylcholine inhibits the metabolic activity of activated monocytes primarily through these receptors. We did not observe a statistically significant effect of LPS activation on the mitochondrial respiration of monocytes. In experiments on the neutrophil isolate, we found that LPS significantly increased the levels of both glycolytic activity and oxidative phosphorylation, with the cholinergic influence on these metabolic changes being negligible.
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