Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that focal sites of inflammation around dying adipocytes, so-called crown-like structures, exhibit a unique microenvironment for macrophage proliferation. Interestingly, locally proliferating macrophages were not classically activated (M1), but they exhibited a rather alternatively activated (M2) immune phenotype. In this study, we established organotypic cell cultures of AT explants to study the impact of cytokine treatment on local ATM proliferation, without the bias of early monocyte recruitment. We show that exposure of AT to Th2 cytokines, such as IL-4, IL-13, and GM-CSF, stimulates ATM proliferation, whereas Th1 cytokines, such as TNF-α, inhibit local ATM proliferation. Furthermore, AT from obese mice exhibits an increased sensitivity to IL-4 stimulation, indicated by an increased phosphorylation of STAT6. In line with this, gene expression of the IL-4 receptor (Il4ra) and its ligand IL-13 are elevated in AT of obese C57BL/6 mice. Most importantly, Il4ra expression and susceptibility to IL-4 or IL-13 treatment depend on IL-6 signaling, which seems to be the underlying mechanism of local ATM proliferation in obesity. We conclude that IL-6 acts as a Th2 cytokine in obesity by stimulating M2 polarization and local ATM proliferation, presumably due to upregulation of the IL-4 receptor α.

A chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.

Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA sequencing (snRNA-seq) analyses largely uncovered overlapping or similar cell-type landscapes. We hypothesized that adipocyte subtypes, differentiation trajectories and/or intercellular communication patterns could illuminate this depot similarity–difference gap. For this, we performed snRNA-seq of human subcutaneous or visceral adipose tissues (five or ten samples, respectively). Of 27,665 adipocyte nuclei in both depots, most were ‘classical’, namely enriched in lipid metabolism pathways. However, we also observed ‘nonclassical’ adipocyte subtypes, enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization or angiogenesis or ribosomal and mitochondrial processes. Pseudo-temporal analysis showed a developmental trajectory from adipose progenitor cells to classical adipocytes via nonclassical adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Last, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation and intercellular communication patterns to human fat depot differences.

Obesity is frequently associated with a chronic low-grade inflammation within adipose tissue (AT). Although classical signs of inflammation are missing in AT inflammation, there is a significant increase in macrophages and, to a lesser extent, other immune cells, such as T cells, B cells, mast cells, and neutrophils. The spatial and temporal activation of these cells as well as their accumulation in the AT seem to be tightly linked to so-called crown-like structures (CLS). CLS are accumulations of adipose tissue macrophages (ATMs) around dead adipocytes and are thought to reflect a scavenger response. At present, data on the life cycle of CLS are missing. To better understand the cellular events underlying AT inflammation, we developed an approach that allows long-term imaging of ATMs, adipocytes, and CLS within live AT explants. We tested three putative reporter mouse lines for myeloid cells in regard to their suitability for live imaging. Thereby, we identified ATMs from CSF1R-eGFP mice to exhibit the most robust expression of eGFP. AT explants from these mice allowed stable live imaging for more than 7 days without significant phototoxicity. Long-term imaging thus revealed the accumulation of ATMs around dying adipocytes, migration of ATMs within AT, and also the degradation of the lipid remnants of perishing adipocytes. The observed behavior of ATMs in the context of AT inflammation is in line with previous studies but for the first time provides data on the specific behavior of individual ATMs and on the life cycle of CLS with unprecedented spatiotemporal resolution.

Obesity is associated with chronic low-grade inflammation of visceral adipose tissue (AT) characterized by an increasing number of AT macrophages (ATMs) and linked to type 2 diabetes. AT inflammation is histologically indicated by the formation of so-called crown-like structures, as ATMs accumulate around dying adipocytes, and the occurrence of multinucleated giant cells (MGCs). However, to date, the function of MGCs in obesity is unknown. Therefore, the aim of this study was to characterize MGCs in AT and unravel the function of these cells. We demonstrated that MGCs occurred in obese patients and after 24 weeks of a high-fat diet in mice, accompanying signs of AT inflammation and then representing ∼3% of ATMs in mice. Mechanistically, we found evidence that adipocyte death triggered MGC formation. Most importantly, MGCs in obese AT had a higher capacity to phagocytize oversized particles, such as adipocytes, as shown by live imaging of AT, 45-µm bead uptake ex vivo, and higher lipid content in vivo. Finally, we showed that interleukin-4 treatment was sufficient to increase the number of MGCs in AT, whereas other factors may be more important for endogenous MGC formation in vivo. Most importantly, our data suggest that MGCs are specialized for clearance of dead adipocytes in obesity.