Exploring the Role of Heme Oxygenase-1 in Modulating the Immunosuppressive Function of Tumor-Associated Macrophages

Tumor-associated macrophages (TAMs) are key players among immune cells infiltrating the microenvironment of various tumors, where they contribute significantly to creating an immunosuppressive environment that allows cancer cells to escape immune surveillance and elimination. Recent studies from our research group on glioblastoma (GBM) have highlighted extensive tumor infiltration by bone marrow-derived macrophages (BMDMs), which exhibit potent immunosuppressive properties. A strong correlation has been identified between the immunosuppressive phenotype of such myeloid cells and their commitment to an iron-recycling phenotype, characterized by the overexpression of the rate-limiting enzyme in heme catabolism, namely heme oxygenase-1 (HO-1). Indeed, treatment of macrophages with ZnPPIX, an inhibitor of HO-1 enzymatic activity, effectively restores T cell proliferation in vitro and reduces the expression of downstream markers associated with immunosuppressive traits. Studies on its enzymatic activity have identified HO-1 as a protein associated with the Endoplasmic Reticulum (ER). Nevertheless, recent research in diverse cell types, such as human cancer cells and murine fibroblasts, indicates that it can translocate to the nucleus, where it exerts non-canonical functions. Moreover, preliminary findings from our group suggest that both the HO-1 protein and its inhibitor ZnPPIX are found in the macrophages’ nuclei. Based on these premises, my thesis project aims to investigate the subcellular localization of HO-1 and its non-canonical role in TAMs. We established in vitro models of both pro- and anti-inflammatory macrophages derived from blood monocytes of healthy donors. Our findings indicate a decrease in the expression of anti-inflammatory markers at the mRNA and protein levels in pro-inflammatory macrophages. In addition, we assessed the functional activity of both macrophages by performing an in vitro T cell proliferation assay: anti-inflammatory macrophages exert an immunosuppressive behavior by negatively affecting T cell proliferation, while pro-inflammatory macrophages did not exhibit a significant impact on T cell proliferation. We observed a differential HO-1 expression in total protein extracts between in vitro-derived pro-inflammatory and anti-inflammatory macrophages. In addition, we confirmed the nuclear localization of HO-1 and observed distinct patterns of HO-1 subcellular distribution across the different macrophage models. We speculate that nuclear localization of HO-1 protein may serve to regulate genes that promote immunosuppressive behavior in TAMs. To further explore this hypothesis, we are setting up a chromatin immunoprecipitation (ChIP) experiment tailored to in vitro-derived macrophages to identify HO-1 target genes. By understanding the molecular mechanisms underlying HO-1’s nuclear function, we aim to develop novel therapeutic strategies targeting HO-1 to reprogram TAMs and enhance anti-tumor immunity.