PROJECT SUMMARY Adipose tissue inflammation is a known link between obesity and metabolic dysfunction and is associated with accumulation of adipose tissue macrophages (ATM). However, the interplay between ATMs and obese tissue inflammation and tissue expansion is still not well understood. Unbiased techniques such as RNA-sequencing of whole tissue or single cells have been extremely useful in characterizing adipose tissue cellular changes with obesity, though these methods lose anatomical context. The fine detail of tissue architecture and ATMs within their anatomical niche is a critical aspect of understanding their function in vivo, but methods such as immunolabeling can only detect a small subset of known markers and structures. Here I propose to use the recently available method of spatial transcriptomics (STX) to capture gene expression signatures across adipose tissue, retaining anatomical context of cell signatures and near single cell resolution. My preliminary data validate the feasibility of this approach for adipose tissue and identify lysosomal signatures in scavenger receptor high (SRhi) ATMs as a high priority target for investigation in obesity and diabetes. In conjunction with STX, I will perform single cell transcriptomics to delineate representative ATM and adipocyte signatures, which I will use to generate spatial maps of ATM-adipocyte niches during obesity. My overall goal is to map SRhi and SRlo ATM niches within adipose tissue in metabolic dysfunction. My central hypothesis is that dysfunctional lysosomal processing during SRlo ATM clearance of dying adipocytes precedes internal lipid accumulation and aggregation into proinflammatory crown-like structures (CLS). To examine this hypothesis, I will investigate ATMs and adipocytes in STX and single cell transcriptomics data and ATM lysosomal function in a time course of murine obesity. My research aims are: (1) Identify ATM niches and their lysosomal signatures in obese adipose tissue. I will examine the hypothesis that the anatomical niche of SRlo ATMs promotes lysosomal stress during interface with apoptotic adipocytes prior to CLS formation. Single cell transcriptomics will enable identification of prominent ATM subsets across the time course, and I will map these signatures to specific anatomical locations within adipose tissue. (2) Determine lysosomal function, lipid accumulation, and proinflammatory cytokine production in SRhi and SRlo ATMs. I will examine the hypothesis with increased duration of obesity, lysosomal stress in SRlo ATMs associates with lipid accumulation and production of proinflammatory mediators prior to development of insulin resistance. To determine lysosomal signatures and function, lipid-laden vs. lipid-lo ATMs from the time course will be examined through bulk RNA-sequencing, lysosomal staining and quantification, and assays to identify mediators of lysosomal stress and lipid accumulation. The proposed work develops a new line of my research focusi...