Reactive Species from Two-Signal Activated Macrophages Interfere with Their Oxygen Consumption Measurements

Metabolic modulation of macrophage activation has emerged as a promising strategy lately in immunotherapeutics. However, macrophages have a broad spectrum of functions and thus, understanding the exact metabolic changes that drive a particular immune response, is of major importance. In our previous work, we have reported a key role of nitric oxide (NO^●) in two(2)-signal activated macrophages [M(2-signals)]. Further characterization using metabolic analysis in intact cells, showed that the basal and maximal respiration levels of M(2-signals) were comparable, with cells being unresponsive to the injections-inducd mitochondrial stress. Here, we show that excessive NO^● secretion by the M(2-signals) macrophages, interferes with the oxygen (O₂) consumption measurements on cells using the seahorse metabolic analyzer. This is attributed mainly to the consumption of ambient oxygen by NO^● to form NO₂⁻ and/or NO₃⁻ but also to the reduction of O₂ to superoxide anion (O₂^●−) by stray electrons from the electron transport chain, leading to the formation of peroxynitrite (ONOO⁻). We found that reactive species-donors in the absence of cells, produce comparable oxygen consumption rates (OCR) with M(2-signals) macrophages. Furthermore, inhibition of NO^● production, partly recovered the respiration of activated macrophages, while external addition of NO^● in non-activated macrophages downregulated their OCR levels. Our findings are crucial for the accurate metabolic characterization of cells, especially in cases where reactive nitrogen or oxygen species are produced in excess. View Full-Text