Unraveling Macrophage Polarization: Deciphering Heme Signaling Dynamics after Hemolysis for Precision Inflammation Modulation

Abstract

Hemolysis, the premature death of red blood cells (RBCs), is a common pathology in various diseases. Macrophages are essential in clearing damaged RBCs, mitigating RBC-derived hemoglobin toxicity, and recycling heme iron. Moreover, macrophages play a central role in the regulation of immune responses. While macrophages are recognized for their homeostatic and immune-regulatory functions, the impact of hemolytic stress on their immune phenotype and functions has been unclear. In this work, we first characterized three mouse models of hereditary hemolytic anemia and identified mice with spherocytosis as suitable to investigate the in vivo effect of hemolysis on macrophage polarization. Using single-cell RNA sequencing, we identified a hemolysis-induced expansion of liver macrophages with a distinct Hmox1high, Marcohigh, and MHCIIlow gene expression phenotype in these mice. We termed these macrophages erythrophagocytes and demonstrated that heme-activated signaling through transcription factor NRF2 was the essential phenotype driver. Moreover, heme-NRF2 signaling exhibited a strong anti-inflammatory effect in vivo, protecting against macrophage-driven inflammatory liver diseases. Subsequently, we have investigated tissue selective reprogramming of liver macrophages through heme-NRF2 signaling as a therapeutic paradigm, addressing immune-related adverse effects in the context of cancer immunotherapy. The efficacy of agonistic anti-CD40 antibodies in cancer immunotherapy has so far been hindered by macrophage-driven liver toxicity. We established a treatment protocol using recombinant Ter-119 antibody to direct RBCs toward erythrophagocytosis by liver macrophages. This treatment triggered heme-NRF2 macrophage polarization and mitigated liver damage without compromising systemic immune response and anti-tumor effectiveness. Identifying a novel macrophage phenotype and the therapeutic potential of the heme-NRF2 pathway pave the way for innovative approaches in addressing macrophage-driven inflammatory liver diseases.