Exploring the Crosstalk Between Iron Homeostasis and Antioxidant Systems in Ferroptosis Regulation within Immunosuppressive Macrophages

Ferroptosis is a recently identified form of cell death driven by iron-dependent lipid peroxidation (LPO). Recent studies link ferroptosis to both classical and alternative activation in macrophages depending on the model and the context. Our research group has identified a major accumulation of immunosuppressive bone marrow-derived macrophages (BMDMs) within the tumor microenvironment of Glioblastoma, in particular in the core hypoxic area. These cells are characterized by a strong activation of iron metabolism, showing a pronounced expression of Heme-Oxygenase 1 (HO-1), a key enzyme in heme catabolism. Notably, inhibition of this enzyme by ZnPPIX fully abolished the immunosuppressive activity of macrophages, leading to the complete restoration of T cell proliferation in both BMDMs and in vitro-model of immunosuppressive macrophages (MΦ). Given the central role of macrophages in iron handling, we aimed to explore their susceptibility to ferroptosis and whether ferroptosis modulation affected their immunosuppressive activity. Moreover, given the HO-1’s central role in heme catabolism and intracellular iron levels, we investigated its role in the ferroptosis process, both in normoxia and in hypoxia. First, macrophages were treated with RSL3, a ferroptosis inducer that inhibits GPX4, the key enzyme involved in ferroptosis defence, and lipid peroxidation (LPO) levels, an early hallmark of ferroptosis, were measured by flow cytometry. Cell viability and LPO measurements conducted under normoxic and hypoxic conditions revealed that hypoxic macrophages are more susceptible to GPX4 blockade. To study the role of HO-1 in the ferroptosis process, we administered ZnPPIX to ferroptotic macrophages and detected a notable abrogation of LPO. This result suggested that ZnPPIX treatment has a protective effect on ferroptosis induction. To elucidate the mechanisms involved in ZnPPIX-mediated ferroptosis resistance, we performed an analysis of the presence of HO-1 by immunoblotting and evaluated its enzymatic activity. To further explore and study signatures associated with ferroptosis regulation, we performed transcriptomic analysis using RNA-seq on untreated and ZnPPIX-treated MΦ, revealing upregulation of genes conferring resistance to ferroptosis. Then, the immunosuppressive capacity of macrophages was assessed through a T cell proliferation assay. Results confirmed the loss of immune suppression mediated ZnPPIX as previously described by our group. At the same time, a rescue in T cell proliferation was observed also with ferroptotic macrophages. Further studies will investigate the mechanisms of ferroptosis impact on macrophages function. Additional experiments are planned to clarify the role of HO-1 in ferroptosis onset and immunosuppression by testing alternative HO-1 inhibitors and inducers.