Macrophages can polarize towards opposing functional states with distinct effector functions in different diseases. While macrophage subsets likely exist in vivo along a continuum of these functional subsets, in vitro polar opposites can be distinguished, such as M1-type (IFN-?/LPS-induced) or M2a-type (IL-4- induced) macrophages. Distinct triggers can also induce other subsets of M2-type macrophages with specific functions. M1-type macrophages are considered to have antitumorigenic and proinflammatory functions, while M2-type macrophages are considered proangiogenic and protumorigenic. Particularly M2-type macrophages have been shown to promote pathologic angiogenesis in many disease conditions, including cancer, neovascular age-related macular degeneration (AMD) or wound healing disorders. Thus, defining the key molecular mechanisms that differentially regulate M1- versus M2-type polarization and identifying druggable targets to therapeutically shift polarization from the M2-type to the M1-type has important translational impact. In order to obtain a global unbiased and comprehensive characterization of the signaling mechanisms that lead to M1- or M2-type macrophage polarization we have used quantitative proteomic and phosphoproteomic analyses of macrophages during early time-courses after induction of M1- versus M2a-type polarization. Moreover, we conducted chemical inhibitor screens to define specific signaling pathways that when inhibited can block M2a-type polarization of macrophages. These approaches identified a critical role of Raf/MEK/ERK signaling for promoting M2a-type polarization while inhibiting M1-type polarization. Notably, MEK and pan-Raf inhibitors blocked M2a-type polarization of macrophages, but promoted M1-type polarization. We discovered that during M1-type macrophage polarization MEK2 activity is actively suppressed through selective phosphorylation of MEK2 T394 by ERK1 that suppresses the kinase activity. Thus, our novel data strongly suggest that inhibition of MEK signaling in macrophages can block M2-type and promote M1-type polarization. In order to test this hypothesis we have now generated mice with inactivation of MEK1 and/or MEK2 specifically in macrophages. We will determine how lack of MEK signaling specifically in macrophages affects their ability to undergo M1- versus M2-type polarization. In direct comparison to these findings we will also test how specific MEK inhibitors affect macrophage polarization, which will allow us to distinguish potential off- target effects of these inhibitors. Next, we will test whether macrophage-specific inactivation of MEK1 and/or MEK2 inhibits M2-type polarization and pathologic angiogenesis in a wound healing assay and a model for neovascular AMD. We will compare the outcomes with groups of mice treated with MEK inhibitors, allowing us to distinguish effects of MEK inhibition in macrophages from those in other cells. Our data provide a strong scientific premise for therapeutically targeting MEK signaling in macrophages and our proposed experiments have an important clinical relevance for various diseases that are promoted by M2-type macrophages.
Our goal is to define the cellular signaling mechanisms that are critical for polarization of macrophages towards the M1- versus the M2-type and to establish targeted treatment strategies that shift the polarization from M2- type to M1-type by targeting Raf/MEK/ERK signaling in various disease conditions during which M2-type polarization of macrophages can have detrimental effects. We will target MEK signaling genetically specifically in macrophages or pharmacologically in all cells in in vivo animal models of wound healing and neovascular age-related macular degeneration that show a strong M2-type polarization. Our findings are likely to provide the foundation for novel treatments in a variety of disease conditions that are promoted by polarized M2-type macrophages, such as cancer, abnormal wound healing diseases or neovascular age-related macular degeneration.