Researchers are attempting to uncover the basics of how fat tissue is structured and, specifically, inflammation associated with obesity, in the hopes of unlocking the connection between the accumulation of fat and poor health outcomes. Now, a new study by researchers at the University of Michigan revealed previously unrecognized immune cell types and interactions within adipose tissue using single-cell analysis of gene expression combined with spatial transcriptomics.

The findings are published in JCI Insight in an article titled, “A lipid-associated macrophage lineage rewires the spatial landscape of adipose tissue in early obesity.”

“Adipose tissue macrophage (ATM) infiltration is associated with adipose tissue dysfunction and insulin resistance in mice and humans,” wrote the researchers. “Recent single-cell data highlight increased ATM heterogeneity in obesity but do not provide a spatial context for ATM phenotype dynamics. We integrated single-cell RNA-Seq, spatial transcriptomics, and imaging of murine adipose tissue in a time course study of diet-induced obesity. Overall, proinflammatory immune cells were predominant in early obesity, whereas nonresident antiinflammatory ATMs predominated in chronic obesity.”

Studying fat is easier said than done. In tissues that are organized into defined layers for example the spinal cord or the brain “it’s easier to do sanity checks with your data and identify this or that layer as a particular cell type and know that it should be expressing genes X, Y, and Z,” said Lindsey Muir, PhD, a research assistant professor in the department of computational medicine and bioinformatics.

“With adipose, it’s a lot more challenging because the cell types are distributed evenly throughout the tissue, without defined cell layers.” In obesity, fat cells, or adipocytes, expand and can reach a limit that ultimately causes cell death and leads to inflammation.

The research team fed mice a high-fat diet over the course of 14 weeks, collected fat tissue, and then used single-cell and spatial analyses to produce a readout of all the mRNAs present in the sample. Using clustering in the single-cell data, they were able to group cells whose genetic makeup more closely resembled each other than other groups or the overall sample.

“We knew going in that macrophages would likely have multiple subtypes… what surprised us were the number that came out that were highly different from each other and coming up at different times and becoming more dominant over time.”

They identified five types, which they named Mac1, 2, 3, 4, and 5. Mac1 were resident to the tissue in both lean mice on a normal diet and obese mice. Mac2 and Mac3, which were identified by their pro-inflammatory genes, peaked after eight weeks of the high-fat diet.

However, as the high-fat diet progressed to 14 weeks, Mac4 and Mac5 cells, which had low pro-inflammatory gene expression, predominated, while pro-inflammatory Mac2 and Mac3 cells decreased.

“The thinking in the field has been that the type of macrophages that accumulate in obesity are promoting an inflammatory state. Based on these data there’s a lot more to the story,” said Muir.

Her hypothesis is that Mac4 and Mac5 are the lipid associated macrophages (LAMs) described in her own prior work and by other researchers and may be a sign of the body attempting to quell a damaging level of inflammation from pro-inflammatory macrophages and dying adipocytes.

Next, the researchers used spatial transcriptomics to analyze fat tissue. The study examined these images looking for tell-tale markers called crown-like structures—structures that are associated with insulin resistance.

“Once crown-like structures appear, it takes them a long time to go away and their appearance indicates tissue dysfunction,” noted Muir. “Using image processing, we identified based on the density of these regions what was likely to be a crown-like structure and then went in to verify that we could see them visually,” said Muir. These structures had gene expression indicating the presence of Mac4 and Mac5 LAMs.

With more insight into the cellular makeup and spatial organization of fat tissue in the context of obesity, the next step, Muir said, is to examine the signaling processes and proteins associated with the development of LAMs and metabolic disorders.

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