Proteomic Signature Reveals Modulation of Human Macrophage Polarization and Functions Under Differing Environmental Oxygen Conditions

Abstract

Macrophages are innate immune cells which can react to a large number of environmental stimuli thanks to a high degree of plasticity. These cells are involved in a variety of tissue functions in homeostasis, and they play essential roles in pathological contexts. Macrophages' activation state, which determines their functional orientation, is strongly influenced by the cellular environment. A large body of macrophage literature is devoted to better defining polarizations from a molecular viewpoint. It is now accepted that a multidimensional model of polarization is needed to grasp the broad phenotype repertoire controlled by environmental signals. The study presented here aimed, among other goals, to provide a molecular signature of various polarizations in human macrophages at the protein level to better define the different macrophage activation states. To study the proteome in human monocyte-derived macrophages as a function of their polarization state, we used a label-free quantification approach on in-gel fractionated and LysC/Trypsin digested proteins. In total, 5102 proteins were identified and quantified for all polarization states. New polarization-specific markers were identified and validated. Because oxygen tension is an important environmental parameter in tissues, we explored how environmental oxygen tension, at either atmospheric composition (18.6% O) or "tissue normoxia" (3% O), affected our classification of macrophage polarization. The comparative results revealed new polarization-specific makers which suggest that environmental oxygen levels should be taken into account when characterizing macrophage activation states. The proteomic screen revealed various polarization-specific proteins and oxygen sensors in human macrophages. One example is arachidonate 15-lipoxygenase (ALOX15), an IL4/IL13 polarization-specific protein, which was upregulated under low oxygen conditions and is associated with an increase in the rate of phagocytosis of apoptotic cells. These results illustrate the need to consider physicochemical parameters like oxygen level when studying macrophage polarization, so as to correctly assess their functions in tissue.

Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

“…Hence, it is crucial to determine the most suitable differentiation conditions when using these cells as model system, as this can significantly impact their response to various innate immune stimuli. Quantitative high-resolution mass spectrometry-based proteomic approaches have been widely employed to investigate the proteomes of monocytes and macrophages as well as altered cellular proteomes and complex cellular/biological mechanisms in several biological conditions (21-23). However, to date, no studies have directly compared the proteome changes of the PMA-mediated differentiation process.…”

Decoding communication patterns of the innate immune system by quantitative proteomics

“…Phenotype plasticity of Mϕs is associated with a broad spectrum of activation states or polarizations, focusing on pro‐ and anti‐inflammatory states and classical (M1) versus alternative activation (M2a) . In this study, quantitative proteomics measured protein abundance profiles of 5,102 proteins from Mϕs at different polarization states mediated by high (18.6%) and low (3%) oxygen conditions . The results showed polarization‐specific markers (e.g., CD14, CD40, CD74, CD163, CD206, and CD274) and suggested an impact of oxygen on rates of phagocytosis of apoptotic cells.…”

Hypoxia Drives Dihydropyrimidine Dehydrogenase Expression in Macrophages and Confers Chemoresistance in Colorectal Cancer

“…To order to understand the involvement of TAM in chemoresistance in CRC, we designed the present study to incorporate oxygen concentration as a key environmental parameter. We had previously reported that the oxygen disponibility in macrophages' environment greatly influences their immune functions such as their ability to clear apoptotic cells (Court et al, 2017). As colon tissues are naturally exposed to levels of oxygen that are usually lower than 5% O2 (Keeley and Mann, 2018) with values that could reach even lower (<1% O2) in tumors, oxygen appeared as a fundamental parameter to understand macrophage involvement in chemoresistance.…”

“…Interestingly, TAMs increase hypoxia in tumor tissues, which is susceptible to favoring chemoresistance in return (Jeong et al, 2019). We also know that hypoxia affects macrophage biology (Court et al, 2017) and could mediate resistance to anticancer treatment and cancer relapse (Henze and Mazzone, 2016). Based on the abundance of macrophages in CRC, we hypothesized that hypoxia could directly modulate macrophage involvement in 5-FU resistance.…”