Obesity triggers low-grade inflammation, contributing to insulin resistance, diabetes, and metabolic syndrome. Adipose tissue expansion leads to adipocyte death, hypoxia, and metabolic stress, fueling inflammation. Adipose tissue macrophages (ATMs), the most abundant immune cells in adipose tissue, play crucial roles in this obesity-associated inflammation. ATMs originate from the bone marrow and infiltrate adipose tissue during obesity development. Monocytes and macrophages are recruited via MCP-1/CCR2 signaling, triggered by the accumulation of free fatty acids (FFAs). FFAs activate TLR4 signaling, leading to inflammation via protein kinase C activation, ER stress, and ROS generation. In obese mice, ATMs produce inflammatory mediators like IL-6 and TNF-α, inducing insulin resistance. While ATMs in obese mice overexpress inflammatory markers, they also exhibit a distinct metabolically activated phenotype (MMe). This lipid-rich environment stimulates specific signaling pathways resulting in ATM activation. Although several transcriptional, epigenetic, and metabolic processes in macrophage activation are known, the exact signaling pathways and transcription factors involved in ATM activation require further investigation. Chromatin accessibility dynamics play a significant role in altering monocyte/macrophage phenotypes in response to environmental stimuli. This study aimed to profile chromatin accessibility in monocytes and ATMs from lean and obese mice to identify regulatory elements and transcription factors involved in macrophage activation during obesity, particularly focusing on the role of ETV5.

Existing literature highlights the crucial role of inflammation in obesity-related metabolic disorders. Studies have demonstrated the accumulation of macrophages in adipose tissue of obese individuals, contributing significantly to the chronic inflammatory state. The recruitment of monocytes from the blood to the adipose tissue and their differentiation into ATMs is a well-established process. Several signaling pathways, including MCP-1/CCR2 and TLR4, have been implicated in this process. The impact of free fatty acids on macrophage activation and the resulting inflammatory response have also been extensively investigated. However, a comprehensive understanding of the transcriptional regulatory mechanisms governing ATM activation in obesity remains incomplete. While some studies have identified specific regulatory elements in ATMs, the overall picture of transcriptional changes during ATM differentiation and activation remains unclear. The importance of chromatin accessibility dynamics in shaping macrophage phenotypes is acknowledged, but further research is needed to identify key regulatory elements and transcription factors responsible for ATM activation in obese individuals.

The study used C57BL/6 mice fed either a chow diet (CD) or high-fat diet (HFD) for 12-14 weeks to establish an obesity model. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed to assess glucose intolerance and insulin resistance. Peripheral blood monocytes and ATMs were isolated from both CD- and HFD-fed mice using flow cytometry. ATAC-seq was performed to profile chromatin accessibility, with data analyzed using ATAC-pipe for quality control, peak calling, and normalization. Differential accessibility regions were identified by comparing CD and HFD conditions, and unsupervised hierarchical clustering classified these regions into distinct clusters. Gene ontology (GO) analysis was used to annotate the functions of these clusters. Transcription factors (TFs) potentially binding to accessible sites were predicted. Bone marrow-derived macrophages (BMDMs) were cultured and polarized (M1, M2, MMe) in vitro. Lentivirus and siRNA were used for ETV5 overexpression and knockdown, respectively. RT-qPCR and Western blotting assessed gene and protein expression levels. RNA sequencing (RNA-seq) was performed on Raw264.7 cells with ETV5 knockdown to identify regulated genes, followed by GO and gene set enrichment analysis (GSEA). Single-cell RNA sequencing data from a public database (GSE154047) were analyzed to study ETV5 expression in various macrophage subpopulations. ELISA measured IL-6 protein levels in supernatants. Statistical significance was assessed using appropriate tests.

HFD-fed mice exhibited increased body weight, insulin resistance, and glucose intolerance, with ATMs showing a higher percentage of CD11c+ cells indicating activation. ATAC-seq revealed distinct chromatin accessibility landscapes between monocytes and ATMs from obese and lean mice. 9461 differential chromatin-accessible regions were identified and clustered into seven groups. Five clusters were enriched for functions related to immune activation. Analysis of these clusters revealed several transcription factors potentially involved in monocyte activation (SP1, NFYA, NFKB1, KLF5), monocyte-to-macrophage differentiation (JUN, BATF, BACH2, ATF3), and macrophage activation (JUN, ATF3, FOS, ETV5). ETV5 expression was significantly decreased in ATMs from HFD-fed mice and in vitro-induced MMes, but not in M1 or M2 polarized macrophages. Single-cell RNA-seq data confirmed decreased ETV5 expression in activated ATMs in vivo. Palmitate, but not glucose or insulin alone, induced ETV5 downregulation. ETV5 knockdown in Raw264.7 cells upregulated genes associated with inflammation, ER stress, and IL-6 production. GSEA showed enrichment of inflammatory response and positive regulation of IL-6 production pathways. ETV5 knockdown increased IL-6 expression in BMDMs and Raw264.7 cells, while ETV5 overexpression decreased IL-6 expression. Palmitate treatment increased IL-6 production, and ETV5 knockdown further enhanced this effect. These results consistently demonstrate that ETV5 negatively regulates IL-6 expression in macrophages.

This study identified ETV5 as a novel negative regulator of IL-6 expression in metabolically activated macrophages. The findings support the notion that ETV5 plays a critical role in controlling the inflammatory response in adipose tissue during obesity. The downregulation of ETV5 by palmitate in the obese microenvironment may contribute to the increased inflammatory response observed in obesity. These findings suggest that targeting ETV5 in macrophages could be a therapeutic strategy for obesity and related inflammatory diseases. The study also reveals other transcription factors involved in different stages of monocyte activation and differentiation, providing additional potential targets for therapeutic intervention. The differential effects of ETV5 on macrophages compared to other cell types such as hepatocytes and pancreatic beta cells highlights the cell-type specific roles of this transcription factor in metabolic regulation. Future studies should explore the precise molecular mechanisms through which ETV5 regulates macrophage activation, particularly the relationship with ER stress and the ERK/NF-κB pathways.

This study provides strong evidence for the role of ETV5 as a suppressor of adipose tissue macrophage activation in obesity. The downregulation of ETV5 by palmitic acid leads to increased IL-6 production and contributes to inflammation. Targeting ETV5 may be a potential therapeutic strategy for obesity-related diseases. Future research should focus on the precise mechanisms of ETV5 regulation in macrophages and its broader implications in other inflammatory conditions.

The study primarily used a mouse model of obesity, and the findings may not be directly translatable to humans. The in vitro experiments, while useful, might not fully capture the complexity of the in vivo environment. The study focused on a limited number of transcription factors and pathways, and other factors may contribute to ATM activation. Further research is needed to fully elucidate the role of ETV5 in macrophage activation and its potential as a therapeutic target.