PROJECT SUMMARY/ABSTRACT Lipid metabolism, including lipid synthesis and degradation, is essential for the immune system and cellular functions. Lipid droplets (LDs) are major lipid storage organelles that modulate macrophage function and are a potential therapeutic target. The molecular mechanisms that link LDs and macrophage functions are still poorly understood. Systemic lupus erythematosus (SLE) is an autoimmune disease in which efferocytosis, or clearance of apoptotic cells, by macrophages is impaired. Current medications for SLE rely on nonspecific immunosuppressive drugs with severe toxicities, and new targeted approaches are necessary. Two ancient innate molecules, high mobility group box 1 (HMGB1) and C1q, have unique features in SLE. In humans, C1q deficiency is highly associated with SLE. Conversely, HMGB1, which functions to block efferocytosis, is significantly elevated in the serum of approximately one third of patients with SLE. We identified an interaction between C1q and HMGB1, demonstrating that exposure to both proteins polarizes monocytes to an anti- inflammatory (M2-like) phenotype. We also showed that exposure to HMGB1 plus C1q enhances LD quantity in M2-like macrophages compared with macrophages exposed to HMGB1 alone, which exhibit an M1-like phenotype. We replicated our findings in alternatively activated (M2) macrophages differentiated from human monocytes. The M2 macrophages had significantly more LDs than classically activated (M1) macrophages. In addition, M2 and M2-like macrophages showed better engulfment of apoptotic cells than M1 and M1-like macrophages. Based on these observations, we hypothesize that LDs carrying specific lipids and proteins in M2 and M2-like macrophages support efferocytosis function in response to exposure to apoptotic cells. In this proposal, we describe experiments to (i) characterize LDs in macrophage subsets, and (ii) determine if we can change macrophage functionality by manipulating the content of LDs. In Aim 1, we will characterize LDs using biochemical and histologic approaches based on quantitative mass spectrometry, lipidomics, proteomics, and high-resolution imaging. In Aim 2, we will define how LDs from M2 macrophages are associated with efferocytosis and determine if targeted LD modulation either in quantity or composition can shift macrophage function. Collectively, we expect this work to uncover the connection between LDs and macrophage function and to reveal a mechanism by which HMGB1 and C1q promote immune tolerance in macrophages. If our hypotheses are correct, we will continue these studies to help pinpoint new therapeutic targets in SLE based on the manipulation of LDs and macrophage function.