Project Summary Inflammation is essential for tissue repair and removal of harmful pathogens and molecules , but if activated inappropriately, the innate immune system triggers autoinflammatory conditions. Macrophages play a key role in inflammation and have tremendous impact on human health, both positive and negative, yet many questions about these cells remain unanswered. They are heterogeneous and adopt a spectrum of activation states, both as distinct tissue-resident macrophages and in circulation as monocytes. The drivers of these activation states remain elusive. Metabolism plays a role, with macrophages undergoing metabolic changes during pro- inflammatory activation, including switching from oxidative phosphorylation to glycolysis. However, whether metabolism or other intracellular processes alone can dictate activation states remains unclear and is critical to understanding a host of human diseases associated with inappropriate inflammation. The current proposed research program addresses this question and significantly expands our previous work by analyzing these processes in different organ systems (including the liver and the intestine). We are leveraging the unique advantages zebrafish provide for exquisite genetic manipulations, high throughput screening, and in vivo imaging to dissect the complex relationship between intracellular processes and macrophage activation. The discovery of an inactivating mutation in a NOD-like receptor, nlrc3l, that causes inappropriate macrophage activation and loss of microglia led us to create a new genetic screen in zebrafish that yielded several novel macrophage mutations that we will investigate. These new mutations cause inappropriate macrophage activation, disrupt microglia development or both. We are investigating whether nlrc3l and associated genes as well as new genes we identify from the screen regulate macrophage activation by altering metabolic and other intracellular processes. We are taking an integrated approach at multiple levels—using differential transcriptomics, proteomics, and metabolomics, high power in vivo single cellular and whole-body imaging, CRISPR based genetic engineering, protein-protein interactions, and cross-species experimentations—to address these questions. Furthermore, our previous results raise critical new questions regarding how activation states are regulated to ensure normal tissue macrophage development, and whether common mechanisms exist to control macrophage activation across multiple organs. Our research program will expand to address these key areas in vivo using a diverse set of tissue macrophages (microglia, intestinal macrophages, and Kupffer cells). These studies aim to provide necessary insights into macrophage differentiation, plasticity, and potential in a disease-relevant context, and address how seemingly disparate functions (development and activation regulation) are encoded in the molecular program governing macrophages. In summary, decipheri...