Red Blood Cell Transfusions Shape Macrophages Towards an Anti-Inflammatory Functional Phenotype and Impair the Immune Response to Infectious Cues

Reticulo-endothelial macrophages are central for the regulation of iron homeostasis, thanks to their ability to recycle red blood cell (RBC)-derived iron. Beside this role, macrophages are key innate immune cells which exhibit remarkable functional plasticity. These two roles are tightly interconnected: on the one hand, macrophage polarization dictates the expression of iron-regulated genes and determine cell iron handling; on the other, iron availability affects immune effector functions. However, it is still unclear how different iron sources affect macrophage plasticity. Here we investigated the phenotypic switching of macrophages induced by fresh RBCs compared to hemolytic RBCs, heme and different iron formulations.

Repeated transfusions of fresh RBCs in mice result in macrophage iron overload and cause extensive macrophage cell death and monocyte recruitment in liver and spleen. After transfusion, macrophages acquire an M2-like anti-inflammatory phenotype, hallmarked by elevated expression of M2 markers (CD206, Arg-1, Ym1) and anti-inflammatory cytokines (IL-10, IL-4). Conversely, M1 markers (MHCII, CD86) and pro-inflammatory cytokines (IL-6, IL1b) are markedly decreased. These results indicate that transfusion-induced erythrophagocytosis likely suppresses the inflammatory response. A clear predisposition to infections has been observed in chronically transfused individuals, suggesting a detrimental impact of transfusion practice on the immune system. Therefore we analyzed whether transfusional iron overload impairs macrophage response to infectious cues. Our in vivo experiments demonstrate that transfusions shape macrophages towards an M2-like phenotype in a more pronounced way after LPS stimulation, highlighting a novel adverse anti-inflammatory effect of transfusions upon infection. Macrophages show a drastic reduction in M1 marker expression, which is reflected by decreased levels of circulating inflammatory cytokines and increased levels of anti-inflammatory cytokines. The pathophysiological relevance of our findings is currently under investigation in a model of transfused myelodysplastic (MDS) mice.

Despite resulting in cell iron overload similarly to transfusions, treatment of mice with free heme and iron polarizes macrophages towards an M1-like pro-inflammatory phenotype. Interestingly, the M1 polarization potential of the different iron formulations (Fe-carboxymaltose/-dextrane/-nitrate) closely reflects their capacity to readily release free iron in the circulation. The administration of the heme scavenger hemopexin and the iron carrier transferrin or the chelator deferoxamine strongly attenuates the ability of heme and iron to induce M1 polarization. The pathophysiological relevance of heme/iron-triggered M1 polarization was confirmed in a mouse model of sickle cell disease (SCD), hallmarked by hemolytic RBCs and elevated circulating heme and iron, as well as in a model of lung cancer, characterized by hemolytic RBC extravasation and iron-loaded macrophages. Hemopexin administration to sickle mice significantly reduces the pro-inflammatory profile of macrophages, thus reducing the chronic inflammation state associated with SCD. Conversely, iron delivery in the tumor microenvironment via iron-oxide nanoparticles elicits pro-inflammatory macrophages with tumor killing properties, thus delaying tumor growth.

Collectively, these results suggest that iron, in its different forms, dynamically determines macrophage polarization and function. Here we have shown that the source and route of iron acquisition have a key role in shaping macrophage phenotype, independently of the ability to produce cell iron overload. Our observations indicate that transfusions dampen macrophage immune effector functions by inducing their switching towards an anti-inflammatory phenotype, which is unlikely able to properly fight infections. The weak pro-inflammatory response of the macrophage might contribute to the increased propensity of transfused patients to develop infections, having potential implications for conditions associated with chronic transfusions (MDS, thalassemia, SCD). Importantly, these findings support the concept that macrophage plasticity can be manipulated for therapeutic purposes by applying different iron sources or scavengers/chelators according to the desired beneficial effect.