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Hemolytic disorders are associated with recurrent events of intra- and extra-vascular hemolysis. Under these conditions, circulating heme levels and erythrophagocytosis are increased resulting in heme and iron accumulation in macrophages of the reticuloendothelial system (RES). The heme scavenger Hemopexin limits heme accumulation in RES macrophages in heme-overloaded Hx-null mice, a model mimicking heme release in hemolytic events. As a consequence cell activation as well as cytokine and ROS production is reduced.

Here we investigated, both in vitro and in vivo, whether heme and/or iron affect macrophage activation and polarization (e.g. M1 inflammatory vs M2 anti-inflammatory macrophages) and, whether this is prevented by heme/iron scavengers.

We characterized bone marrow-derived macrophages treated with aged RBCs (Red blood cells), heme or iron and analyzed the expression of iron-related genes and cytokines, ROS production and polarization markers. Additionally, we evaluated the effect of co-treatment with the heme scavenger Hemopexin or the iron chelator Desferrioxamine. Macrophage polarization was further assessed in vivo, in mice injected with heme or iron-dextran as well as in a mouse model of sickle cell disease, which is hallmarked by enhanced macrophage heme/iron retention. Finally the effect of Hemopexin administration on macrophage polarization was evaluated in vivo.

Macrophages treated with RBC, heme or iron increase mRNA expression of HO-1, L-Ferritin and FPN and decrease TfR1 mRNA levels. This correlates with an increase of M1 markers (MHCII/CD86/TNFa) and a decrease of M2 markers (CD206/Arg1), indicating that heme and iron drive macrophage polarization towards a M1-like pro-inflammatory phenotype. Interestingly, heme/iron treatment further potentiates M1 polarization and shifts M2 macrophages to the M1 phenotype. Similarly, heme and iron affect macrophage plasticity in vivo, promoting macrophage polarization towards a M1 phenotype. These effects are reversible by the co-treatment with Hemopexin or Desferrioxamine both in vitro and in vivo. Additionally, the treatment with heme together with the anti-oxidant N-acetyl-cystein or the TLR4 inhibitor, TAK-242, rescued the up-regulation of some M1 markers and the down-regulation of some M2 markers, likely suggesting that both oxidative stress (induced by both heme and iron) and the activation of the TLR4 pathway (activated by heme) are responsible for heme-induced M1 polarization of macrophages. Interestingly, hepatic macrophages from sickle mice show an enhanced M1-like phenotype, correlating with increased iron accumulation in these cells and Hemopexin depletion. The administration of Hemopexin to sickle mice rescued the expression of M1 markers in liver macrophages to levels similar to control mice, indicating that heme scavenging reduces the pro-inflammatory state of macrophages, by reducing cell heme-iron loading.

Here we show that heme and iron induce the polarization of macrophages towards the M1 pro-inflammatory phenotype. This finding is of pathophysiological relevance in disorders associated with heme and iron loading in RES macrophages, such as sickle cell disease. We expect that the heme/iron-induced increase in cytokine production and ROS will significantly contribute to the enhanced inflammatory status of some hemolytic diseases. The administration of anti-oxidant and hemoglobin/heme scavenger could be beneficial to counteract the heme-induce pro-inflammatory status of macrophages in these disorders.