Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury.

Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties.

Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4.

Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

Manwani:Novartis: Consultancy; Pfizer: Consultancy; Global Blood Therapeutics: Consultancy. Vinchi:Vifor (International) Ltd: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; PharmaNutra S.p.A: Research Funding.