Summary: Project 3 Our Program Project has unveiled key roles for macrophage metabolism, depot-, cue-, and time-dependent molecular re-programming and intraorgan trafficking in the pathogenesis of cardiometabolic dysfunction. In each metabolic setting, including the atherosclerotic plaque, obese adipose tissue and liver, the composition of the tissue-specific niche, such as excess lipid content, and recruitment and trafficking of infiltrating bone marrow- derived immune cells, which deliver signals to activate endogenous signaling pathways in resident immune cells (e.g., adipose tissue macrophages or liver Kupffer cells), defines the breadth of possible consequences. Project 3 studies reveal novel, complex roles for the receptor for advanced glycation end products (RAGE; gene name Ager) and its cytoplasmic domain binding partner, DIAPH1, in parenchymal vs. immune cell dysfunctions. Project 3 key discoveries during Cycle 1 of the Program Project include: (1) deletion of Ager or Diaph1 in myeloid cells significantly increases insulin resistance without further increasing body mass in high fat diet-fed mice; (2) RAGE/DIAPH1 contributes to regulation of hepatic lipid metabolism; (3) macrophage RAGE contributes to regulation of Interferon Regulatory Factor 7 (IRF7); IRF7 bridges lipid metabolism and inflammation in macrophages; and (4) in mice fed a non-alcoholic steatohepatitis (NASH)-inducing diet, myeloid deletion of Ager or novel small molecule antagonists of RAGE/DIAPH1 imparts complex consequences on steatosis and fibrosis. These considerations lead us to hypothesize that RAGE/DIAPH1 contributes to regulation of macrophage metabolism; molecular re-programming in response to tissue- and cue-specific stimuli; and macrophage intra- and interorgan communications in cardiometabolic dysfunction. We will pursue three specific aims: Aim 1 will test the hypothesis that DIAPH1 contributes to atherosclerosis through intra- and interorgan regulation of lipid metabolism and inflammation; AIM 2 test the hypothesis that RAGE/DIAPH1/IRF7 uncouples liver steatosis and fibrosis in NASH through regulation of lipid metabolism and dynamic reprogramming of infiltrating Mɸs and resident Kupffer cells; and AIM 3 will test the hypothesis that RAGE/DIAPH1 contributes to cardiometabolic disease through interorgan communications. Project 3, with Projects 1-2, will identify the depot-, cue- and temporal-mediating mechanisms of cardiometabolic dysfunction, driven by macrophages and, critically, their interactions with parenchymal and non-parenchymal niche-specific cells. Fortified by complementary examinations in human tissues and transcriptome databases, we will employ state-of-the-art RNA sequencing, coupled with strategically-utilized spatial transcriptomics, to generate and “visualize” a comprehensive map of the putative interactome and the upstream transcriptional regulators that regulate intra- and interorgan cross- talk in cardiometabolic disorders. This work and the ...