A single point mutation in the gene that encodes the beta globin chain results in the production of abnormal sickle hemoglobin (HbS) and sickle cell disease (SCD) [1]. SCD is encountered as homozygous sickle cell disease (HbSS), termed sickle cell anemia (SCA), or in compound heterozygosity with another hemoglobin gene mutation (e.g., HbC disease or beta thalassemia) [2], where the polymerization of HbS, when deoxygenated, and the consequent formation of hemoglobin fibers causes the sickling of red blood cells (RBCs), which is the primary cause of SCD pathophysiology [3]. RBC sickling incurs multiple disease mechanisms that ultimately trigger vaso-occlusion in blood vessels and end-organ damage [4]. The clinical complications of SCD are many and varied, and they can be classified as chronic (e.g., chronic pain, chronic kidney disease, sickle retinopathy, avascular necrosis) or acute (e.g., painful vaso-occlusive episodes, acute chest syndrome, stroke) in nature ^[4][5][4,5]. Of all the clinical complications of SCD, painful vaso-occlusive episodes and anemia, caused principally by continuous hemolysis, are considered the most characteristic of SCD [6].

Erythroid production is directly correlated to EBI number; thus, the EBI microenvironment is crucial for events of erythropoiesis. Macrophages present in these structures play several key regulatory roles in the process via both contact- and soluble factor-dependent mechanisms. These cells are able to induce erythroblast proliferation and survival, provide iron for hemoglobin synthesis, and engulf the nucleus expelled during erythroblast enucleation ^[7][8][9][83,84,85].

Although EBI macrophages are not a homogeneous population, they display an M2-like phenotype, with the expression of CD206 and CD169, and they exhibit an anti-inflammatory profile ^[7][10][83,86]. These anti-inflammatory macrophages express CD163, which scavenges the hemoglobin–haptoglobin complex besides acting as an adhesion receptor to erythroblasts, although its ligand is still not known ^[11][87]. The island’s integrity is achieved by the attachment of macrophages to erythroblasts, which is mediated by several adhesion molecules, such as VCAM-1, α_V integrin, ephrin-B2, and Emp expressed by EBI macrophages, which in turn bind to α4β1 integrin, ICAM-4, EphB4, and Emp, expressed by erythroblasts, respectively. Each molecule of these sets of receptors plays a role in the EBI structure, as demonstrated by mice models. The pharmacological inhibition of the interaction between either α4β1 integrin with VCAM-1 or ephrin-B2 with EphB4, as well as the depletion of ICAM-4, in mice results in lower numbers of EBI and the dissociation of erythroblasts from macrophages ^[12][13][14][88,89,90]; furthermore, the depletion of Emp is reportedly lethal due to severe anemia in mice fetuses ^[15][91]. Additionally, a secreted form of ICAM-4, ICAM-4S, was found to be released by orthochromatic erythroblasts and reticulocytes; this secreted molecule competes with the membrane-bound molecule by binding to α_V integrin on macrophages ^[16][92], potentially weakening reticulocyte–macrophage interactions and releasing them from the EBI niche toward the sinusoids.

These molecule sets seem to be heterogeneously distributed among EBI macrophages throughout the bone marrow ^[17][93], which might represent phenotypic and functional differences of EBIs located at distinct zones. For instance, macrophages scattered through the bone marrow are adhered to proerythroblasts and early erythroblasts, whereas macrophages close to sinusoids are attached to cells in the late stages of differentiation, such as polychromatic and orthochromatic erythroblasts ^[18][94]. It is not known, though, whether the macrophages are either different cells or cells in a distinct stage of maturation that migrate toward sinusoids upon erythropoiesis progression. Furthermore, some adhesion molecules have distinct levels of contributions to steady-state and stress erythropoiesis, indicating heterogeneity in the expression of surface molecules between EBI macrophages from physiological (bone marrow) and stress (spleen and liver) tissues. Mutations in the α4 integrin gene inhibit stress erythropoiesis, as illustrated by the impairment of recovery after anemia induced by hemolysis, but these mutations have little effect on steady-state erythropoiesis ^[19][95]. Additionally, it has been shown that the hematopoietic niche of SCD patients is extremely altered, with changes in the number and frequency of different cell progenitors and with modified EBI structure ^[20][96], which is possibly due to changes in adhesion protein expression.

It has been demonstrated that circulating monocytes, particularly the intermediate CD14^highCD16⁺ cells, are able to boost the differentiation of CD34⁺ cells to erythrocytes in vitro in a contact-independent manner, increasing expansion and reducing the death of CD34⁺ cells before they enter the erythroblast stage. Like EBI macrophages, these monocytes express CD206, CD163 and CD169, enabling them to interact with erythroid cells and, in the presence of glucocorticoid, promote erythropoiesis and engulf the nucleus that is expelled during the erythroid differentiation process ^[10][21][86,97]. In SCD, where the number of circulating monocytes is elevated, these cells might play an important role in stress erythropoiesis since the spleen is frequently dysfunctional. In fact, patrolling monocytes from SCD patients were seen to phagocyte circulating RBCs ^[22][98]. Whether the iron recycled by those monocytes can be used for the synthesis of hemoglobin by the circulating erythroblasts present in SCD patients is a question to be investigated.

Between the orthochromatic erythroblast and reticulocyte stages of differentiation, erythroid cells expel the nucleus and most of their organelles, which are then cleared by EBI macrophages, promoting erythroid maturation. Indeed, the depletion of EBIs macrophages in mice impaired the elimination of mitochondria, resulting in reduced mature RBCs ^[23][99]. The extruded nuclei were shown to be recognized by their externalized phosphatidylserine (PS) and reduction in PS recognition, as occurs in MFG-E8 mutant mice that display impaired clearance of nuclei from erythroblasts ^[8][84]. Notably, the retention of mitochondria has been detected in both reticulocytes and RBCs in SCD patients ^[24][100]. The mitochondria in mature RBCs have been shown to be metabolically competent and were associated with high levels of ROS in SCD. In addition, mitochondria-containing RBCs are able to activate immune responses in vitro, indicating that the retention of mitochondria may play a role in the complications of SCD ^[25][101]. Together with the presence of circulating nucleated RBCs, the retention of mitochondria in RBCs suggests a defect in the elimination process by EBI macrophages in addition to the weakness in the macrophage–erythroblast interaction. The digestion of the nuclei and mitochondrial DNA collected from erythroid cells is essential for the survival of EBI macrophages and is performed by DNase II, whose absence in the KO mice model causes the death of the macrophages due to the accumulation of intracellular non-degraded DNA, resulting in lethal impairment in erythropoiesis and anemia ^[26][102].

Erythropoiesis is supported by the iron provision of EBI macrophages for hemoglobin synthesis. Macrophages recycle iron from erythrophagocytosis and the clearance of hemoglobin/Hp and heme/Hpx complexes through HMOX-1 activity, which is up-regulated in SCD and catabolizes heme to equimolar amounts of iron, biliverdin and carbon monoxide (CO). The role of HMOX-1 in stress erythropoiesis is essential, as a drop in its activity impairs the maturation of erythroid cells ^[27][28][103,104]. It has been demonstrated that besides iron, CO can support RBC maturation, at least in cultures of the erythroid cell lineage K562 ^[29][105]. Recycled iron can have one of two destinations, either stored as ferritin or exported by ferroportin and transported bound to transferrin (Trf) ^[30][106]. Erythroblasts can internalize the iron/Trf complex through their Trf receptor (CD71). In addition, macrophages have been reported to secrete ferritin in macrophage/erythroblast cocultures, which is then captured by erythroblasts ^[31][107]. The systemic control of iron load in SCD is complex, as hepcidin is secreted in response to iron and inflammation, whereas it is down-regulated by hypoxia and erythropoiesis. Additionally, recent findings have shown that despite anemic conditions, treatment with a ferroportin inhibitor improved some aspects of SCD pathology by restricting iron to RBCs and consequently reducing the HbS concentration ^[32][108]. Thus, a better understanding of iron metabolism, specifically in the EBI niche, is still needed and would provide insights for novel therapeutic approaches.

KLF1 (Kruppel Like Factor 1) is the transcription factor that best defines EBI macrophages. It is responsible for up-regulation of DNase II and the expression of VCAM-1, CD163, CD169, enabling the macrophage to promote erythroid development ^[33][34][35][109,110,111]. In the absence of KLF1 expression, mice have reduced DNase II and die of severe anemia ^[36][37][112,113]. KLF1 also regulates the expression of IL-33, which is a cytokine that promotes the maturation of RBCs ^[35][111]. Additionally, IL-33 differentiates monocytes into macrophages involved in erythrophagocytosis and iron recycling ^[38][114] and has been shown to be elevated in SCD patients ^[39][115]. Other soluble factors found to be secreted by EBI macrophages are insulin-like growth factor (IGF1) and IL-18 ^[33][109]. IGF1 was shown to increase erythropoiesis by binding to the IGF1R expressed by erythroblasts in several development stages ^[40][116], whereas the possible role of IL-18 in erythropoiesis has still not been investigated, even though the expression of IL-18R has been detected in erythroid lineage cells and the production of IL-18 may be stimulated in SCD by heme-activated inflammasome formation ^[41][117]. Although the presence of systemic levels of these factors in SCD has been investigated, the contribution of EBI macrophages to the production and the dynamics of these mechanisms specifically in the EBI niche in SCD patients still needs clarification.

Depletion of CD169⁺ macrophages in mice models of polycythemia vera and β-thalassemia, which both display activated stress erythropoiesis, was able to reverse some aspects of the diseases pathology, such as the defect in erythroid development and lifespan as well as splenomegaly ^[7][42][83,118], revealing EBI macrophages as possible targets for future therapies for stress and ineffective erythropoiesis in disorders such as SCD. The secreted factors induced in the EBI niche should be investigated as an option to enhance the proliferation and maturation of RBCs, whereas the signaling molecules that are modulated upon stress erythropoiesis are a potential target for pharmaceutical approaches. Furthermore, uncovering the mechanisms by which EBI macrophages influence the switch to HbF production in erythroid cells would shed light on other options for HbF-inducing treatments to improve the clinical manifestations of SCD patients.