Title: Increased erythrophagocytosis induces ferroptosis in macrophages and alters the immune response to subsequent stimuli
Description: Red blood cell (RBC) transfusions are associated with adverse effects, such as an increased risk of bacterial infection. In preparing RBCs for transfusion, donor RBCs are refrigerator stored for extended periods of time, during which they undergo oxidative damage, ultimately leading to their rapid post-transfusion clearance from the circulation. Macrophages play important roles in recycling iron derived from the clearance of RBCs. They are also a critically important component of host defense, protecting against invading pathogens. However, the effects on macrophage biology of acutely ingesting large numbers of RBCs are not completely understood. To investigate this issue, we used a mouse model of RBC transfusion and clearance, which mimics the clinical setting. In this model, transfusions of refrigerator storage-damaged (i.e., “old”) RBCs led to increased erythrophagocytosis by splenic red pulp macrophages (RPMs). This robust erythrophagocytosis induced ferroptosis, an iron-dependent form of cell death, in RPMs.

This was accompanied by increases in reactive oxygen species and lipid peroxidation in vivo, which were reduced by treatment in vitro with ferrostatin-1, a ferroptosis inhibitor. Old RBC transfusions also induced RPM-dependent chemokine expression by splenic Ly6Chi monocytes, which signaled Ly6Chi monocyte migration from bone marrow to spleen, where these cells subsequently differentiated into RPMs. The combination of cell division among remaining splenic RPMs, along with the influx of bone marrow-derived Ly6Chi monocytes, suggests that, following RPM depletion induced by robust erythrophagocytosis, there is a coordinated effort to restore homeostasis of the RPM population by local self-maintenance and contributions from circulating monocytes. However, the effects on the overall functioning of the splenic Ly6Chi monocytes and remaining RPMs are unclear, especially their responses to subsequent immune challenges. In a mouse model of RBC storage and transfusion, we found that, following a transfusion of old RBCs, macrophages were less capable of phagocytosing a subsequent particle stimulus, such as bacteria (i.e., Escherichia coli and Staphylococcus aureus) or additional old RBCs.

However, splenic Ly6Chi monocytes became activated in a specific timeframe following the initial old RBC transfusion, thereby increasing their phagocytic capacity. Nonetheless, despite contributions from activated splenic Ly6Chi monocytes, RPM function was indispensable for clearing S. aureus; this functional impairment may make the transfusion recipient susceptible to S. aureus sepsis. In conclusion, these findings may be clinically relevant to pathological conditions that can arise as a result of increased erythrophagocytosis, such as transfusion-related immunomodulation and impaired host immunity.