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The Effects Of M1 Macrophage Activation On M1-to-M2 Phenotypic Switching
Erin M. O'Brien, Kara L. Spiller, PhD.
Drexel University, Philadelphia, PA, USA.

BACKGROUND: The roles of specific macrophage phenotypes in angiogenesis are not well understood, but there is a growing body of evidence suggesting that early M1 macrophages initiate sprouting, and subsequent M2 activity stabilizes these structures. In some pathologies where angiogenesis is inhibited, M1 macrophages are insufficiently activated and also fail to switch to the M2 phenotype. Thus, in order to properly design strategies to remedy deficient angiogenesis, it is necessary to understand how M1 activation affects the switch to the M2 phenotype.
METHODS: The first part of this study was conducted using primary human monocyte-derived macrophages to test our hypothesis that M1 macrophages are primed to undergo phenotypic switching. Using unactivated M0 macrophages as a comparison, we measured M1 expression of IL-4 receptor-alpha (IL-4Rα) via flow cytometry, as IL-4 is the primary cytokine used to induce polarization to the M2 phenotype. Then, to test our hypothesis that M1 pre-polarization enhances M2 behavior, we compared M0- and M1-derived M2 macrophage gene expression of M2 and angiogenesis markers, secretion of M2-associated angiogenic proteins, and migration of endothelial cells in response to macrophage-conditioned media.
RESULTS: M1 macrophages upregulated surface expression of IL-4Rα compared to M0, suggesting they are primed to switch to the M2 phenotype. Interestingly, M2 macrophages derived from M0 (M0-M2) and M1 (M1-M2) macrophages differentially upregulated M2 markers and genes associated with angiogenesis, indicating they are divergent phenotypes. M1-M2 macrophages especially upregulated CCL17, a chemokine associated with regulatory T cell recruitment, and CXCR4, a highly angiogenic chemokine receptor. ELISA results showed that M1-M2 macrophages also secreted more CCL17 than M0-M2 did, as well as more PDGF-BB, a late-stage angiogenic protein. Finally, M1-M2 macrophage-conditioned media induced significantly more migration of endothelial cells compared to M0-M2.
CONCLUSIONS: Together, these findings suggest that M2 macrophages that have previously been M1-activated are phenotypically unique and exhibit enhanced M2 function, particularly in the context of angiogenesis. The results of these studies also indicate that promoting the M1 macrophage phenotype may rescue dysfunctional angiogenesis.


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