Immune cells, particularly macrophages, are major regulators of lipid metabolism of parenchymal cells in various tissues. It has been shown that secretion of TNF-? by tissue-resident macrophages activates lipolysis in white adipose tissue (WAT) and lipogenesis in the liver. However, the molecular mechanisms underlying the macrophage crosstalk, leading to cellular remodeling in WAT upon diet-induced obesity (DIO) and metaflammation, remain not fully understood. 3D culture systems, such as organoids and spheroids, have recently emerged as a powerful platform to model cell-to-cell interactions and disease ex vivo. In this project, we will employ advanced, immune-competent models, consisting of primary murine- and human-derived WAT spheroids supplemented with macrophages, to mechanistically dissect how adipocyte functions are controlled by immune cells and vice versa, under physiological and obese conditions. First, we will identify novel soluble mediators, mediating the interaction between adipose tissue macrophages and adipocytes during metaflammation. To achieve these goals, complex adipocyte-macrophage co-cultures from WAT of control diet (CD)- and high-fat diet (HFD)-fed animals in combination with imaging, flow cytometry, and multi-omics approaches, such as proteomics and transcriptomics, will be employed. Complementary analyses will be performed using human WAT spheroids in co-culture with induced pluripotent stem cells (iPSC)-derived macrophages. In parallel, we will apply these immune-competent WAT spheroids co-culture models to examine the influence of WAT macrophages on adipocyte lipid composition and triglyceride cycling by mass spectrometry-based lipidomics. Further, we will study the exchange of lipid material between adipocytes and macrophages. Taken together, we will provide a powerful and versatile ex vivo tool to broaden our understanding on adipocyte-macrophage crosstalk in WAT during homeostasis and metaflammation and uncover new insights into the energetic importance of triglyceride cycling and the regulatory role of immune cells in metabolic processes.

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