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.