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

Panagiotis F Christopoulos,Mantas Grigalavicius,Theodossis A Theodossiou,Kristian Berg,Alexandre Corthay

doi:10.3390/antiox10071149

Panagiotis F Christopoulos, Mantas Grigalavicius + Show 3 more

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https://doi.org/10.3390/antiox10071149

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Abstract

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.

Highlights

Immunometabolism has, as of late, emerged as a highly promising research field, in various immune-related human pathologies including cancer [1]
Similar to the Warburg effect in cancer cells, it has been reported that some immune cells like T cells and/or macrophages exhibit a downregulated oxidative phosporylation (OXPHOS) and rely on glycolysis following their activation [3,4]
We aimed to investigate the role of NO and O2 − in oxygen consumption, during metabolic assays in intact cells

Summary

Introduction

Immunometabolism has, as of late, emerged as a highly promising research field, in various immune-related human pathologies including cancer [1]. In contrast to most immune cells, macrophages have a diverse role in cancer, as well as in autoimmune diseases progression or regression, depending on their activation status (M1 or M2). Metabolic manipulation of macrophages has been suggested to hold a great potential for clinical applications [2]. Given the complexity of the field and the fact that immune cells perform non-immune related functions, understanding the exact metabolic changes that drive a particular immune response is of major importance. Similar to the Warburg effect in cancer cells, it has been reported that some immune cells like T cells and/or macrophages exhibit a downregulated oxidative phosporylation (OXPHOS) and rely on glycolysis following their activation [3,4]. A hallmark of classically activated murine macrophages (M1) is the production of nitric oxide (NO ) [5]

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