Role ofAIM in development ofatherosclerosis
Role of AIM in development of atherosclerosis
Macrophages that clear circulating lipids from the bloodstream can transform into plaque forming foam cells if they survive long enough. We pursue discovery of regulatory mechanism that control macrophage viability with the goal of identifying novel ways to prevent atherogenesis and treat atherosclerosis.
Over the past year the increasing interest in understanding the activities of the Apoptosis Inhibitor in Macrophages (AIM) pro-survival protein has been reflected in a number of high profile publications from labs around the world. The key recent discovery is that, despite being regarded as a predominantly secreted circulating protein, AIM also functions intracellularly to define the inflammatory state of T helper cells. This finding allowed us to refine our original hypothesis. We now think that AIM also plays a role in controlling the pathogenic fate of human macrophages by changing the availability of endogenous ligands for several key transcription factors. Testing this hypothesis is the focus of Specific Aim 3 of the project. Using CRISPR-Cas9 technology, we have generated a complete AIM deletion in a human macrophage-like cell line. By testing the response of these mutant cells to inflammatory stimuli, such as the bacterial outer cell membrane component LPS, we were able to obtain preliminary data that indeed confirms the notion that AIM defines both the starting point and the magnitude of the resulting cytokine response. Moreover, the AIM deletion cell line now allows us to move further towards our Specific Aim 2 by testing the differences in function of AIM variants found in humans. We have generated 5 recombinant AIM variants that we are reintroducing into our deletion cell line to test the extent to which they restore the default inflammatory response profile of macrophages. In addition, in 2016 we established a collaboration with researchers from the NTNU Department of Public Health that oversee the analysis of the HUNT data. From this collaboration we will get information about the genetic variation in AIM found in the Norwegian population. This will allow us to pursue Specific Aim 1 of the project which is to determine whether AIM variants found in the Norwegian population are associated with cardiovascular disease.
Role of AIM in development of atherosclerosis
Macrophage viability following ingestion of modified lipids is one of the key determinants of foam cell formation and subsequent atherogenesis. We pursue discovery of regulatory mechanisms that control macrophage viability with the goal of identifying novel ways to predict and treat atherosclerosis.
Apoptosis Inhibitor in Macrophages (AIM) has been recently characterized by us and others as a multifunctional protein that controls several aspects of macrophage viability. As such it contributes significantly to atherogenesis as was demonstrated by resistance to plaque development in mouse models that were deleted for AIM. Because this and other crucial observations were made in mouse models, our main goal in the initial phase of the project is testing to what extent these findings are applicable to human cells. To achieve this in the report period we developed an ex vivo human macrophage model maintained in the absence of exogenous AIM that is normally present in human serum used for the culturing of primary cells. This now allows us to control the magnitude and extent of macrophage exposure to AIM by reintroduction of recombinant protein. To date we have used our newly developed system to make two key observations that impact the future course of our project: 1. Human macrophages, unlike their murine counterparts, produce low levels of endogenous AIM. We were able to induce human AIM expression by stimulating macrophages with LXR/RXR ligands. Nevertheless the induced levels of AIM were still significantly below the levels found in mouse macrophages. This would indicate that in humans resident macrophages are not the primary source of circulating AIM. We will expand our analysis to other cell types, with the CD105+ bone marrow endothelial cells marrow being our primary candidate based on bioinformatic analysis of human tissue arrays. 2. Human macrophages have distinct properties depending on the mode of their differentiation. Depending on the cytokine used for ex vivo differentiation of human monocytes into macrophages the resulting cells have distinct inflammatory profiles. GM-CSF produces classical pro-inflammatory M1 macrophages and M-CSF induces development of alternatively activated M2 cells. We have found that in M1 macrophages inflammatory stimuli induce AIM expression while in M2 cells similar treatment represses AIM expression. Based on this finding we now have to determine which class of tissue macrophages is normally found in human vasculature and atherosclerotic plaque in order to use a relevant ex vivo model for our analysis The work carried out to date has provided us with the experimental framework to analyze AIM function at the molecular level. We will use this framework for systematic testing of the functional significance the AIM polymorphic variants that are found in human population, which is the main goal of our project.