The production of around 2.5 million red blood cells (RBCs) per second in erythropoiesis is one of the most intense activities in the body. It continuously consumes large amounts of iron, approximately 80% of which is recycled from aged erythrocytes. Therefore, similar to the “making”, the “breaking” of red blood cells is also very rapid and represents one of the key processes in mammalian physiology. Under steady-state conditions, this important task is accomplished by specialized macrophages, mostly liver Kupffer cells (KCs) and splenic red pulp macrophages (RPMs). It relies to a large extent on the engulfment of red blood cells via so-called erythrophagocytosis. Surprisingly, we still understand little about the mechanistic details of the removal and processing of red blood cells by these specialized macrophages. We have only started to uncover the signaling pathways that imprint their identity, control their functions and enable their plasticity. Recent findings also identify other myeloid cell types capable of red blood cell removal and establish reciprocal cross-talk between the intensity of erythrophagocytosis and other cellular activities. Here, we aimed to review the multiple and emerging facets of iron recycling to illustrate how this exciting field of study is currently expanding.

Aging affects iron homeostasis, as evidenced by tissue iron loading and anemia in the elderly. Iron needs in mammals are met primarily by iron recycling from senescent red blood cells (RBCs), a task chiefly accomplished by splenic red pulp macrophages (RPMs) via erythrophagocytosis. Given that RPMs continuously process iron, their cellular functions might be susceptible to age-dependent decline, a possibility that has been unexplored to date. Here, we found that 10-11-month-old female mice exhibit iron loading in RPMs, largely attributable to a drop in iron exporter ferroportin, which diminishes their erythrophagocytosis capacity and lysosomal activity. Furthermore, we identified a loss of RPMs during aging, underlain by the combination of proteotoxic stress and iron-dependent cell death resembling ferroptosis. These impairments lead to the retention of senescent hemolytic RBCs in the spleen, and the formation of undegradable iron- and heme-rich extracellular protein aggregates, likely derived from ferroptotic RPMs. We further found that feeding mice an iron-reduced diet alleviates iron accumulation in RPMs, enhances their ability to clear erythrocytes, and reduces damage. Consequently, this diet ameliorates hemolysis of splenic RBCs and reduces the burden of protein aggregates, mildly increasing serum iron availability in aging mice. Taken together, we identified RPM collapse as an early hallmark of aging and demonstrated that dietary iron reduction improves iron turnover efficacy.

Aging affects iron homeostasis and erythropoiesis, as evidenced by tissue iron loading in rodents and common anemia in the elderly. Since red pulp macrophages (RPMs) continuously process iron, their cellular functions might be susceptible to age-dependent decline, affecting organismal iron metabolism and red blood cells (RBCs) parameters. However, little is known about the effects of aging on the functioning of RPMs. To study aging RPMs, we employed 10-11-months-old female mice that show serum iron deficiency and iron overload primarily in spleens compared to young controls. We observed that this is associated with iron loading, oxidative stress, diminished mitochondrial and lysosomal activities, and most relevantly, decreased erythrophagocytosis rate in RPMs. We uncovered that these impairments of RPMs lead to the retention of senescent RBCs in the spleen, their excessive local hemolysis, and the formation of iron- and heme-rich large extracellular protein aggregates, likely derived from damaged RBCs and RPMs. We further found that feeding mice an iron-reduced diet alleviates iron accumulation and reactive oxygen species build-up in RPMs, restores mitochondrial and lysosomal functions, and improves their ability to clear erythrocytes. Consequently, this diet improves splenic RBCs fitness, limits hemolysis and formation of iron-rich aggregates, normalizes splenic iron levels, and tends to increase serum iron availability in aging mice. Mechanistically, using readouts from aged RPMs and in vitro cultures of RPM-like cells, we show that diminished erythrophagocytic activity of RPMs can be attributed to a combination of increased iron levels, reduced expression of heme-catabolizing enzyme heme oxygenase 1 (HO-1), and endoplasmic reticulum stress. Taken together, we identified RPM dysfunction as an early hallmark of physiological aging and demonstrated that dietary iron reduction improves iron turnover efficacy.

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