Characterizing the polarization continuum of macrophage subtypes M1, M2a and M2c

Abstract

Macrophages are vital components of the inflammatory response and exhibit phenotypical plasticity through active conversion between pro- and anti-inflammatory cell subtypes, a feature which can be reproduced in ex vivo culture. We employed a multifaceted approach utilizing proteomics, flow cytometry, activity assays and live-cell microscopy imaging to characterize four cultured macrophage subtypes: unstimulated M, classically activated M1, alternatively activated M2a, and deactivated M2c macrophages. Whole cell proteomics identified a total of 5435 proteins, with >50% of these proteins exhibiting significant alterations in abundance between the different subtypes. This confirms that four distinct macrophage subtypes are induced from the same originating donor material through stimulation with specific cytokines. Additional surfaceome analysis revealed that M2c macrophages significantly upregulate pro-inflammatory markers compared to the M baseline and thus appear to be activated or primed to activate, similar to M1. Surface protein expression provided further subtype characterization, in particular distinguishing between the M2a and M2c macrophages. We next explored the re-polarization capabilities of macrophages using dexamethasone, an anti-inflammatory glucocorticoid known to induce macrophage polarization towards the M2c de-activated phenotype. We show that activated M1 macrophages treated with dexamethasone for 48-hours upregulate the levels of CD163 and CD206, markers synonymous with a phenotypical shift from M1 to M2c yet retain key surface markers and display the functional phenotype of M1 macrophages. The observed repolarization of M1 pro-inflammatory macrophages provides a potential mechanism through which dexamethasone treatment improves COVID-19 prognosis and constitutes evidence of partial repolarization along the macrophage continuum. These proteomic and functional ex vivo macrophage datasets provide a valuable resource for studying macrophage polarity and the impact of dexamethasone on macrophage phenotype and function.

Tolerogenic probiotics Lactobacillus delbrueckii and Lactobacillus rhamnosus promote anti‐inflammatory profile of macrophages‐derived monocytes of newly diagnosed patients with systemic lupus erythematosus

“…In accordance with other studies, it has been reported that M1 macrophages are characterized phenotypically by high expression of CD14, 61 CD80 (hallmark maker of M1 cells), 62–64 and HLADR 65 . In addition, M2a macrophages typically are defined by the lower expression of CD14 and higher expression of CD206 61 . M1 macrophages, expressing high levels of CD80, CD16, CD32, CD86, MHC II markers, and pro‐inflammatory cytokines, are known as a characteristic feature of chronic kidney disease 66 .…”

“…The current results confirmed that the M0, M1, and M2 macrophages had semi‐inflammatory, inflammatory, and anti‐inflammatory phenotypes, respectively (Figure 2). In accordance with other studies, it has been reported that M1 macrophages are characterized phenotypically by high expression of CD14, 61 CD80 (hallmark maker of M1 cells), 62–64 and HLADR 65 . In addition, M2a macrophages typically are defined by the lower expression of CD14 and higher expression of CD206 61 .…”

“…65 In addition, M2a macrophages typically are defined by the lower expression of CD14 and higher expression of CD206. 61 M1 macrophages, expressing high levels of CD80, CD16, CD32, CD86, MHC II markers, and proinflammatory cytokines, are known as a characteristic feature of chronic kidney disease. 66 Additionally, tumor-associated M1 macrophages (M1 TAMs) strongly express HLADR and nitric oxide synthase in inflamed tissues.…”

Distinct differences in immunological properties of equine orthobiologics revealed by functional and transcriptomic analysis using an activated macrophage readout system

“…Finally, we evaluated the ability of various OT treatments to affect macrophage polarization; recognizing that polarization is represented by a dynamic state beyond the traditional assessment of M1 vs. M2 ( 45 , 46 ). Assessment of up- or downregulation of genes associated with M1 versus M2 macrophage phenotypes.…”

Oral Excretion Kinetics of Food-Additive Silicon Dioxides and Their Effect on In Vivo Macrophage Activation

“…Inflammation responses are intricately linked to macrophage activation, involving M1 and M2 macrophage polarization [22]. M1 macrophages are polarized by external stimuli such as lipopolysaccharide (LPS) and interferon-γ (INF-γ) and express CD80 and CD86 surface proteins [23]. M1 macrophage regulates the production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β, IL-6, and IL-8, finally leading to inflammatory responses [24][25][26].…”