Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

Obesity is strongly associated with various diseases, including type 2 diabetes mellitus, cardiovascular disease, and cancer. While body mass index (BMI) is a common metric, metabolically healthy obese individuals highlight that adipose tissue (AT) mass alone isn't sufficient to predict these comorbidities. AT dysfunction, characterized by adipocyte hypertrophy, immune cell infiltration, and increased pro-inflammatory cytokines, plays a crucial role. The increase in immune cells, primarily ATMs, is a hallmark of chronic low-grade inflammation in obese AT. ATMs are broadly classified into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. While obesity was initially thought to increase M1 ATMs, recent research points to a metabolically activated ATM phenotype, characterized by increased lysosomal biogenesis and lipid metabolism. This activation is linked to the digestion of apoptotic adipocytes within CLS, forming lipid-laden foam cells and contributing to inflammation. The origin of these pro-inflammatory ATMs—whether from recruited monocytes or resident M2 polarization—remains unclear. Obesity is associated with increased adipocyte turnover, but the in vivo degradation process is poorly understood. This study aimed to investigate the immune response following adipocyte death using a novel laser injury model to induce controlled cell death and observe subsequent macrophage interactions.

Existing literature establishes a strong link between obesity and chronic low-grade inflammation within adipose tissue (AT). This inflammation is characterized by the accumulation of ATMs around dead or dying adipocytes, forming crown-like structures (CLS). Studies have shown that obesity leads to an increase in the number of CLS and a shift in the ATM population toward a pro-inflammatory M1 phenotype. However, the exact mechanisms driving this inflammatory process remain unclear, particularly the role of adipocyte death itself and the contribution of recruited monocytes versus resident ATMs. Previous research suggested that the pro-inflammatory ATMs in CLS are primarily recruited from blood monocytes. However, others have posited that M1 polarization of resident M2 ATMs also contributes to the inflammation. The in vivo mechanisms of adipocyte degradation are not fully understood, despite extensive research on adipocyte differentiation. This study aimed to address the knowledge gap by investigating the role of adipocyte death in triggering the local inflammatory response.

The researchers employed several methods to investigate the immune response following adipocyte death. First, they used an established live-imaging approach to analyze ATM activity around lipid droplets of varying sizes in AT explants from high-fat diet (HFD)-fed mice. This allowed them to categorize lipid handling by ATMs into three distinct processes: classical efferocytosis, fragmentation, and CLS formation. They measured the size of lipid droplets associated with each process to determine a threshold size for efficient efferocytosis. Secondly, they developed a laser injury model to induce precise, targeted cell death of individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). This allowed for high spatiotemporal resolution live-imaging of ATM-adipocyte interactions and CLS formation following adipocyte death. The researchers performed post-hoc immunostaining for M1 and M2 markers to analyze the activation state of ATMs in newly formed CLS. To verify the findings from the laser injury model, they also stained AT from lean mice to identify physiologically occurring CLS. They performed RNA sequencing on ATMs sorted based on CD11c expression 48 hours post-laser injury to investigate gene expression changes. Finally, they conducted parabiosis experiments using wild-type and Acta1GFP/+ reporter mice to determine the involvement of monocyte recruitment in CLS formation and inflammation. They analyzed AT from wild-type parabiotic partners for GFP-positive ATMs to track recruited cells from the bloodstream. The researchers also stained naturally occurring CLS in lean parabiotic mice for M1 markers to assess monocyte contribution to CLS formation.

The study revealed a critical threshold size of approximately 25 µm for efficient efferocytosis of lipid remnants by ATMs. Lipid droplets larger than this threshold, representing dead adipocytes, predominantly resulted in CLS formation. The laser injury model demonstrated that adipocyte death alone, even in lean mice, was sufficient to trigger CLS formation, with approximately 60% of laser-injured adipocytes developing CLS within 60 hours. ATMs within CLS exhibited a predominantly M1 phenotype, expressing high levels of CD11c, CD86, and CD9, while interstitial ATMs showed primarily M2 markers. RNA sequencing of ATMs sorted based on CD11c expression revealed increased expression of genes related to antigen presentation, oxidative phosphorylation, lysosomal biogenesis, and M1 polarization in CD11c-high ATMs, indicating metabolic activation. Parabiosis experiments showed minimal recruitment of monocytes to CLS formation, suggesting that the pro-inflammatory response is primarily driven by resident ATMs. The observed metabolic activation was distinct from classical M1 activation, characterized by increased oxidative phosphorylation and lipid metabolism.

The findings directly link adipocyte death to the initiation of local inflammation in AT, even under homeostatic conditions. The critical size threshold for efferocytosis highlights how adipocyte hypertrophy, a hallmark of obesity, contributes to inefficient clearance of dead adipocytes and subsequent inflammation. The study challenges the traditional view of monocyte recruitment as the primary driver of AT inflammation, demonstrating that resident ATMs play a central role in the response to adipocyte death. The identification of a unique metabolically activated ATM phenotype in CLS provides a nuanced understanding of AT inflammation in obesity, differing from the classic M1/M2 paradigm. The findings suggest that strategies to protect adipocytes from death could be crucial for preventing or treating obesity-related inflammation.

This study provides compelling evidence that adipocyte death, irrespective of the cause, is a critical trigger for CLS formation and the subsequent metabolic activation and pro-inflammatory response of resident ATMs. The size-dependent nature of efferocytosis underscores the contribution of adipocyte hypertrophy to AT inflammation. Future research should focus on developing strategies that prevent or accelerate adipocyte clearance, potentially mitigating the chronic inflammation associated with obesity and related metabolic diseases.

The laser injury model, while providing precise control over adipocyte death, might not perfectly mimic all aspects of physiological adipocyte death. The study primarily focused on lean mice; further investigation is needed to determine if the findings extend to obese models and how obesity-associated factors might modify the response. The RNA sequencing analysis was conducted at a specific time point; longitudinal studies would provide a more comprehensive understanding of gene expression changes over time. Finally, the parabiosis experiments, while suggesting minimal monocyte contribution, did not exclude the possibility of some recruitment over longer timeframes.