Common environmental toxicants induce phenotypic plasticity of human monocyte-derived macrophages: Implications for cell therapy.

Introduction: : Interest in macrophages as a cell therapy is growing rapidly for the treatment of stroke, cardiomyopathy, critical limb ischemia and other applications. These therapies rely on in vitro polarization of macrophages toward inflammatory (M1) or anti-inflammatory (M2) phenotypes before administration into the patient. Despite effective in vitro polarization, these macrophages retain significant plasticity and may adapt to environmental cues post-transplantation. In this study, we investigated how in vitro polarized human macrophages respond to a mixture of common environmental toxicants, Aroclor 1254. Aroclor 1254 is a mixture of polychlorinated biphenyls (PCBs) that were manufactured and used heavily in building materials and electrical equipment globally until its production was banned in the late 1970s. As a forever chemical, signatures of Aroclor 1254 are still commonly found in buildings, food, and importantly, humans. Given its ubiquity, it is important to assess how such exposures may influence macrophage function and the efficacy of macrophage therapies.

Materials and

Methods: : Primary human monocytes were isolated from leukocyte reduction cones and purified using a monocyte negative selection kit. The monocytes were then differentiated into macrophages using M-CSF and then polarized to M1 (IFN+LPS), M2a (IL-4), or M2c (Dexamethasone) macrophages. After in vitro polarization, each type of macrophage was challenged with exposure to either vehicle (DMSO) or 10 uM of Aroclor 1254 for 48 hours. Macrophages were then analyzed for changes to surface marker expression, cytokine production, and metabolic profile using a SCENITH assay.

Results, Conclusions, and Discussions:: Our results show that acute exposure of in vitro polarized macrophages to Aroclor 1254 enhanced the inflammatory phenotype of M1 macrophages by increasing the secretion of inflammatory cytokines IL-8, TNF, and CCL17 and decreasing secretion of anti-inflammatory IL-10. Interestingly, Aroclor 1254 not only heightened M1 macrophages but also shifted M2 macrophages from an anti-inflammatory state towards a more inflammatory state. Both M2a and M2c macrophages exposed to Aroclor 1254 had significantly decreased levels of IL-10, CD163, and CD206 (anti-inflammatory markers) while production of inflammatory cytokines, such as IL-8, increased dramatically (Figure 1A). Furthermore, when we analyzed the metabolic profile of M2 macrophages after exposure to Aroclor 1254, we found they were more dependent on glycolytic processes (Figure 1B) and less able to utilize fatty acids or amino acids as fuel sources (Figure 1C), both indicative of an inflammatory metabolic state. Taken together, these results demonstrate that Aroclor 1254 promotes an inflammatory immunometabolic shift in macrophages, even after polarization. This underscores the vulnerability of macrophage therapies to environmental exposures and highlights the need to consider post-transplantation environments when developing and deploying cell-based immunotherapies.

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.