Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1β, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFβ) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.
The ATP-binding Cassette Transporter G1 (ABCG1) promotes cholesterol efflux from cells and regulates intracellular cholesterol homeostasis. Recent studies have shown that in mice in the absence of ABCG1, macrophages shift from a tumor-promoting M2 phenotype to a tumor-fighting M1 phenotype within the tumor and suppress bladder cancer growth in vivo. Two important aspects of this effect are still unknown: (1) whether ABCG1-would also impact polarization of human macrophages, and (2) the underlying molecular mechanism of this effect. Here, we show that in human monocyte-derived macrophages the inhibition of ABCG1 by siRNA significantly increased the expression of the M1 markers IDO1, CXCL10, CD64 and TNF after LPS/IFNγ (M1) stimulation. In contrast, after IL-4 (M2) stimulation the expression of the M2 markers MRC1, TGM2 and CD163 was significantly reduced. These data indicate that ABCG1-deficiency promotes the polarization of macrophage to an M1 phenotype in human macrophages. To address the molecular mechanism, we analysed cell signalling in bone marrow-derived macrophages from Abcg1−/− mice. These macrophages displayed reduced levels of Akt activation after stimulation with LPS/IFNγ or IL4. In addition, while Abcg1−/− macrophages stimulated with LPS/IFNγ produced more TNF compared to WT macrophages, after treatment with an Akt activator the TNF-production of Abcg1−/− and WT macrophages was comparable. These data suggest that the M1 bias of Abcg1−/− macrophages is mediated through the Akt signalling pathway. In summary, these findings deepen our mechanistic understanding of the M1/M2 switch in macrophages and suggest that ABCG1 could be a potential new target to modulate macrophage polarization for cancer immunotherapy.
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