Developing an Adipocyte-Macrophage Coculture Model to Improve Assessment of Environmental Toxin-Induced Metabolic Syndrome

Introduction: : The NIH and FDA are both pushing for the development of better human-based in vitro models of human disease as an alternative to animal testing. To this end, our group has been systematically building capabilities to model human adipose tissue in vitro. Adipose tissue is an incredibly important tissue in our body whose function and dysfunction is linked to many diseases including diabetes, autoimmune diseases, and cancer. Adipose tissue is comprised of many cell types including adipocytes, adipose mesenchymal stem cells (MSCs), and resident immune cells such as macrophages that collectively dictate the health and function of the tissue. It is important to consider all these cells in a model of adipose tissue because the pathological expansion of fat is not just due to alterations in adipocytes, but it also due to activation of adipose tissue macrophages that results in inhibition of adipogenesis. Here, we took a stepwise approach to developing models of adipose tissue that increase in complexity to reveal how in vitro model complexity impacts our ability to reveal adipose response to environmental triggers known to be linked to metabolic disorders. We show that the model can be used to ask important questions about how exposure to forever chemicals such as polychlorinated biphenyls (PCBs) impacts the development of metabolic dysfunction. Our findings highlight the pivotal role of macrophages in mediating adipose tissue dysfunction following PCB exposure.

Materials and

Methods: : Human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and differentiated into macrophages using M-CSF over six days. Macrophages were subsequently polarized for two days toward M1 (IFNγ + LPS), M2a (IL-4), or M2c (dexamethasone) phenotypes, followed by exposure to PCBs (Aroclor 1254, 10μM) for two days. After polarization and/or PCB exposure, the macrophages were either directly cocultured with adipocyte organoids or used to produce conditioned media.
Adipose mesenchymal stem cells (MSCs) were isolated from the stromal vascular fraction of human surgical discard adipose tissue. Organoids containing 10,000 MSCs were formed using either ultra-low attachment round bottom plates or agarose micromolds. These organoids were differentiated into adipocyte organoids using adipogenic differentiation media with or without supplementation of macrophage conditioned media for 14 days.
For coculture experiments, adipocyte organoids in micromolds were transferred to wells containing differentiated and polarized macrophages cultured within agarose punchouts. The two cell types were cocultured for two days before media was collected to assess changes in adipokine release. To assess the impact of macrophages on adipogenesis, secreted factors from macrophage conditioned media were incorporated throughout the entirety of adipocyte differentiation before assessing lipid accumulation and adipokine secretion.

Results, Conclusions, and Discussions:: Compared to explant adipose tissue, adipocyte-only organoid models lack key adipokines, including MCP-1, IL-8, leptin, and IFN-γ. Here, we demonstrate that coculturing M1 or M2c macrophages with the adipocyte organoids restores MCP-1, IL-8, and IFN-γ secretion to levels comparable to explant tissue, yielding a model that is more physiologically relevant. A key advantage of this system compared to macrophages embedded within adipocyte organoids is the ability to further separate the adipocytes and macrophages to analyze the directionalities of the cell-cell interactions. To test how macrophage secreted factors impact adipocyte differentiation, we separated out the macrophage culture and collected macrophage conditioned media from M1 and M2c macrophages. Then, we applied it to differentiating mesenchymal stem cells (MSCs) before assessing markers of adipogenesis such as adipokine levels and lipid accumulation. Both M1 and M2a macrophages inhibited lipid accumulation and altered adipokine secretion, specifically decreased adiponectin and increased IL-8, suggesting that macrophage-derived factors alone can influence adipocyte development (Figure 1B). Applying these models to environmental toxicology, we found that PCB-exposed macrophages further suppressed adipogenesis and amplified inflammatory signaling, leading to greater reductions in adiponectin and increased IL-8 levels (Figure 1C). Together, these results underscore the critical role of macrophages in mediating toxicant-induced adipose dysfunction and highlight the value of incorporating immune components into in vitro adipose models.

Acknowledgements and/or References (Optional):: This study was supported by the NIEHS P42ES013661 (J.A.A. and A.J.K.). R.M.B.-.B was supported by the NIGMS T32GM139776, and NIEHS F30ES035622. The TPC was supported by NCI P30CA086862 and the University of Iowa Carver College of Medicine.