Monocyte–red blood cell crosstalk supports clearance and heme metabolism in sickle cell anemia

Background: Monocytes can interact with erythroid cells and contribute to macrophage pools under anemic stress, thereby supporting the erythropoietic niche. In sickle cell anemia (SCA), extensive hemolysis and ineffective erythropoiesis may expose circulating monocytes to abnormal red blood cells (RBCs), potentially altering their phenotype and function to accommodate the higher erythropoietic response and iron demand.

Methods and results: We characterized circulating monocytes from SCA patients at steady state using flow cytometry and in vitro phagocytosis assays. Intracellular RBC material was detected in all circulating monocyte subsets from SCA patients, indicating active erythrophagocytosis. Mechanistically, SCA monocytes displayed upregulated VCAM-1 and reduced SIRP-α expression, favoring RBC binding and internalization even when CD47 expression on RBCs was preserved. Following RBC engulfment, monocytes upregulated heme oxygenase-1 (HO-1) and ferroportin (FPN), consistent with enhanced heme degradation and iron export, and expressed higher levels of CD206, suggesting a regulatory phenotype. In vitro assays confirmed that both sickle RBCs and SCA monocytes synergistically promoted erythrophagocytosis.

Conclusion: Circulating monocytes from SCA patients undergo phenotypic and functional reprogramming upon interaction with sickled RBCs, acquiring features reminiscent of erythroblastic island macrophages. These findings highlight a previously underappreciated role for monocytes in RBC clearance, heme metabolism, and iron recycling in SCA, with potential implications for inflammation and disease progression.

1 Introduction

Erythroid cells remain closely associated with macrophages throughout several stages of their development, particularly because erythropoiesis occurs within specialized niches known as erythroblastic islands (EBI) (1, 2). These niches consist of erythroid cells at various stages of maturation organized around a central macrophage. The functions of this central macrophage include supplying iron for hemoglobin synthesis, promoting proliferation and survival of erythroid precursors, and engulfing extruded nuclei during terminal differentiation. In addition, macrophages in the liver and splenic red pulp are responsible for the phagocytosis of damaged or senescent red blood cells (RBC) (3–9).

Monocytes also have the capacity to differentiate and contribute to the macrophage pool, particularly under anemic conditions (10), and appear to establish close interaction with erythroid cells even prior to their differentiation into macrophages. For instance, monocytes can induce erythropoiesis, repress gamma globin synthesis, and contribute to the clearance of damaged or senescent RBCs from circulation (11–13). One of the main functions of tissue-resident macrophages is the uptake of heme from the complex haptoglobin-hemoglobin via the receptor CD163 (14). Circulating monocytes express CD163 at much lower levels than resident macrophages and are therefore not the primary cells responsible for hemoglobin clearance. Nevertheless, in inflammatory contexts, CD163 can be upregulated on monocytes, conferring them a limited scavenging capacity (15–18).

Sickle cell anemia (SCA) is a hereditary disorder caused by a point mutation in the sixth codon of the β-globin gene, resulting in the substitution of valine for glutamic acid. Consequently, hemoglobin A (HbA) is replaced with hemoglobin S (HbS), which is prone to polymerization under low oxygen conditions. HbS polymerization alters the morphology of erythroid cells, converting them from flexible, biconcave discs into rigid, sickle-shaped cells, thereby reducing deformability and causing membrane damage (19–21).

In SCA, approximately 40% of erythroid cells die between the polychromatic and orthochromatic erythroblast stages, overloading EBI macrophages due to the increased burden of apoptotic cells (22, 23). Kupffer cells are likewise affected, as the heightened erythrophagocytosis triggered by membrane damage in sickled RBCs leads to hyperplasia and hepatomegaly (9). Monocytes, in turn, have been reported to participate in the clearance of RBCs adherent to the endothelium of SCA patients at steady-state and during vaso-occlusive crises. They also display phenotypic adaptations to meet the increased iron demand and are activated by hemolysis and the consequent erythropoietic response (13, 24, 25). The specific role of monocytes in SCA and the influence of disease features in their function has been investigated through single-cell and functional studies in recent years (26–29).

With this in mind, we sought to characterize the phenotypic and functional properties of circulating monocytes in SCA patients, with particular emphasis on their ability to interact with and engulf RBCs, and to evaluate the impact of this interaction on iron metabolism and monocyte activation status, suggested by the expression of key markers. Our data demonstrated that circulating monocytes from these patients exhibit phenotypic and functional alterations consistent with active interaction with erythroid cells. These findings support the hypothesis that, in SCA, circulating monocytes undergo functional reprogramming upon contact with abnormal erythrocytes, acquiring features reminiscent of macrophages within EBIs.

2 Materials and methods

2.1 Sample collection

Patients with SCA were recruited at Blood Center of the University of Campinas (UNICAMP). The exclusion criteria were transfusions, hospitalizations, and pain crisis for at least 3 months prior to blood collection. All patients were prescribed hydroxyurea; six of them were unable to access the medication in the three months preceding sample collection. The control group consisted of healthy individuals (HC), blood donors in the same institution, matched by age and sex with the patients. Ethical approval was obtained from the UNICAMP Human Research Ethics Committee (protocol number CAAE: 88768318.6.0000.5404), and all participants provided written informed consent. Demographic characteristics are detailed in Table 1.

Table 1

Table 1. Main demographic, hematological, and laboratorial characteristic of sickle cell anemia patients’ population.

2.2 Peripheral blood mononuclear cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from 20mL of whole blood by density gradient separation using Ficoll-Hypaque (Cytiva). The blood was diluted with sterile phosphate-buffered saline (PBS), carefully layered on top of Ficoll-Hypaque, and centrifuged at 1500 rpm for 30 minutes at room temperature (without acceleration or brake). The PBMC layer was collected and washed twice with PBS. The remaining RBCs were lysed with chilled lysis buffer (0.155 M NH₄Cl and 0.0010 M KHCO₃) at 4 °C for 15 minutes. Following an additional wash, the cells were resuspended and quantified.

2.3 Monocyte analyses

The phenotypic characterization of circulating monocytes was performed via flow cytometry (FC) using the Cytoflex equipment (Beckman Coulter). Cell populations were initially gated based on forward and side scatter (FSC × SSC) parameters, followed by the exclusion of doublets using SSC-A × SSC-H. Based on CD14 and CD16 expressions, monocytes were categorized into three subsets: classical (C-MC, CD14⁺⁺CD16^-), intermediate (I-MC, CD14⁺⁺CD16⁺), and non-classical (NC-MC, CD14⁺CD16⁺). The expression of target molecules was assessed using fluorescent antibodies. Antibody details are provided in Supplementary Table 1. The mean fluorescence intensity (MFI) of the different markers were accessed in each monocyte subset.

The RBCs phagocytosis was evaluated following the protocol described by Liu et al. (25). Monocytes were stained with anti-CD14 and anti-CD16 antibodies, fixed, and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences, 554714) according to the manufacturer instructions before intracellular staining with an anti-glycophorin A antibody. The data was analyzed using FlowJo software (BD Biosciences).

2.4 In vitro phagocytosis assay

The methodology described by Haschka et al. (13) was adapted as follows: monocytes were isolated from PMBCs using anti-CD14 magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions and allowed to adhere overnight. RBCs from patients or HCs were isolated from peripheral blood, labeled with the membrane dye PKH26 (Sigma), and co-cultured with total monocytes for 2 hours to allow phagocytosis (5×10⁶ cells RBCs and 5×10⁵ monocytes per well). Subsequently, cells were collected, and remaining RBCs were lysed with chilled lysis buffer containing NH₄Cl 0.155M and KHCO₃ 0.0010M at 4°C for 15 minutes, to clear the adherent cells. Monocyte were gated in the singlet population and PKH26 fluorescence was evaluated by MFI and percentage. Ferroportin (FPN) expression on the cell membrane was assessed using an anti-FPN primary antibody followed by incubation with a fluorescent secondary antibody. Intracellular expression of heme oxygenase-1 (HO-1) was evaluated by staining with an anti-HO-1 antibody after fixation and permeabilization using the Cytofix/Cytoperm kit.

2.5 Statistical analyses

Statistical analyses were performed using Prism 5.1 software (GraphPad). P-values ≤0.05 were considered statistically significant. Results are expressed as mean ± standard deviation. Statistical significance was determined using a two-tailed paired t-test or the Mann-Whitney test, as indicated in the figure legends.

3 Results

3.1 Circulating monocytes contribute to the phagocytosis of RBCs in SCA patients

Flow cytometry analysis of circulating monocyte subsets in the present cohort showed an increased proportion of intermediate monocytes (CD14⁺CD16⁺) and reduced classical monocytes (CD14⁺⁺CD16^-) in SCA patients compared to healthy controls (HC) (Figure 1A). It was consistent with a previous study that demonstrated a similar shift in monocyte subsets among patients under hydroxyurea (HU) therapy (30). Since monocytes from SCA patients have previously been reported to participate in RBCs phagocytosis (20), we evaluated their involvement in this process in our cohort by assessing the intracellular presence of glycophorin A, a membrane glycoprotein of erythroid cells, within circulating monocytes. While monocytes from HC displayed minimal basal internalization of RBCs, those from SCA patients exhibited markedly higher percentages and mean fluorescence intensity (MFI) of intracellular glycophorin A (Figures 1B, C). These findings corroborate previous reports, reinforcing the role of these monocytes in the clearance of erythroid cells from circulation. When analyzing the proportion of glycophorin A⁺ cells across monocyte subsets, all three subsets were involved, with I-MC displaying the highest percentage of RBCs uptake (Figure 1D), and C-MC from SCA patients showing significantly increased glycophorin A MFI compared to HC (Figure 1D).

Figure 1

Figure 1. Phagocytosis of erythroid cells by circulating monocytes in sickle cell anemia patients (SCA). (A) Flow cytometry analysis of circulating monocytes (CD14⁺) from PBMC of healthy controls (HC) and SCA patients subdivided into the main subsets: C-MC: classical monocytes; I-MC: intermediate monocytes; NC-MC: non-classical monocytes. Percentage (B) mean fluorescence intensity (MFI) (C) of glycophorin A in total circulating monocytes. (D) Percentage and MFI of glycophorin A in each monocyte subset. HC, n = 13; SCA, n = 15.

3.2 Mechanisms involved in erythrophagocytosis by circulating monocytes from SCA patients

Erythrophagocytosis by macrophages involves cell attachment mediated by adhesion molecules and the interaction between SIRP-α and CD47. SIRP-α, expressed on macrophages and monocytes, recognize CD47 on healthy RBCs. The loss of CD47 on senescent or damaged RBCs, or the downregulation of SIRP-α on phagocytes, results in the clearance of RBCs from circulation (31). To explore potential mechanisms influencing the engulfment of erythroid cells by circulating monocytes in SCA patients, we analyzed the expression of VCAM-1 and SIRP-α on monocytes, as well as CD47 on RBCs. VCAM-1, a major adhesion molecule mediating interactions between erythroid cells and monocytes, was upregulated on I-MC from SCA patients compared to HC (Figure 2A), suggesting an enhanced ability of these monocytes to interact with and adhere to circulating RBCs. Furthermore, all monocyte subsets from SCA patients exhibited reduced SIRP-α expression compared to their HC counterparts, although the reduction was statistically significant only in NC-MC (Figure 2B). These correlative findings suggest an impaired capacity to recognize CD47 on RBCs, potentially promoting their phagocytosis even when CD47 is normally expressed on sickle RBCs (Figure 2C).

Figure 2

Figure 2. Phenotypic characterization of circulating monocytes in SCA patients. Mean fluorescence intensity (MFI) of VCAM-1 (A), SIRP-α (B) and CD206 (E) on healthy controls (HC) and SCA monocyte subsets: C-MC: classical monocytes; I-MC: intermediate monocytes; NC-MC: non-classical monocytes. (C) MFI of CD47 in RBCs. (D) Percentage of HO-1⁺ monocytes. HC, n = 13; SCA, n = 15.

In line with the increased uptake of RBCs, all monocyte subsets from SCA patients displayed upregulated expression of heme oxygenase-1 (HO-1), with statistical significance for C-MC and I-MC and a similar trend for NC-MC (Figure 2D), indicating an enhanced capacity to metabolize heme derived from RBC engulfment. Moreover, both I-MC and NC-MC from SCA patients exhibited higher CD206 expression compared to HC, suggesting a shift toward a more regulatory phenotype (Figure 2E).

3.3 Influence of monocytes and RBCs on phagocytosis and iron metabolism in SCA

To investigate the interaction between monocytes and RBCs, we performed an in vitro phagocytosis assay by incubating monocytes separated by CD14-selective magnetic beads and PKH26-labeled RBCs from either HC or SCA patients, followed by quantification of PKH26 signal within monocytes after lysis of adherent RBCs. After co-culture, monocytes from both groups interacted significantly more with SCA RBCs than with HC RBCs, as evidenced by higher percentage of PKH26⁺ cells and increased PKH26 MFI (Figures 3A, B), highlighting the role of sickle RBCs in promoting their engulfment. In addition, monocytes from SCA patients interacted to a greater number of RBCs compared to HC monocytes, both when incubated with HC RBCs and with SCA RBCs (Figures 3A, B). These findings indicate that both RBCs and monocytes from SCA patients contribute to enhancing this interaction, thereby facilitating RBC clearance, as previously described.

Figure 3

Figure 3. Erythrophagocytosis assay of circulating monocytes. RBCs from healthy controls and SCA patients were PKH26-labeled and incubated with allogeneic monocytes either from HC (HC monocytes) or from SCA patients (SCA monocytes) for 2 h. The graphs show percentage (A) and mean fluorescence intensity (MFI) (B) of PKH26 in total monocytes. HC, n = 6; SCA, n = 6.

To investigate the impact of RBCs engulfment by circulating monocytes, we assessed the expression of HO-1 and ferroportin (FPN), two key proteins involved in heme and iron metabolism. When comparing monocytes that had internalized SCA RBCs (PKH26⁺) with those that had not participated in the phagocytosis/interaction process (PKH26^-), we found that HO-1 and FPN expression were increased in both HC and SCA monocytes following contact with RBCs (Figures 4A-D). These findings suggest that circulating monocytes internalize RBCs, thereby promoting heme degradation and iron export, possibly as a compensatory mechanism for the increase of intracellular load.

Figure 4

Figure 4. Expression of heme oxygenase-1 (HO-1) and ferroportin (FPN) after engulfment of sickle cell anemia patients red blood cells. Percentage of HO-1⁺ (A) and ferroportin (FPN⁺) (B) among PKH26⁺ and PKH26^- monocytes from healthy controls (HC monocytes) and SCA patients (SCA monocytes); Mean of fluorescence intensity (MFI) of HO-1 (C) and FPN (D) among PKH26⁺ and PKH26^- monocytes. HC, n = 6; SCA, n = 6.

4 Discussion

In the present study, we characterized circulating monocytes from patients with SCA and identified key phenotypic and functional alterations associated with their interaction with erythroid cells. Our findings highlight a dynamic interplay between circulating monocytes and sickled RBCs, potentially contributing to systemic changes in both inflammation and iron homeostasis in SCA. While monocytes from HC do not engulf RBCs normally in the circulation of these individuals, our ex vivo analyses revealed the presence of RBCs material in monocytes from SCA patients, consistent with RBC binding and internalization, as previously described (20).

VCAM-1 expressed by macrophages plays a key role in adhesion to RBCs through binding to α4β1 integrin (29). We observed that circulating monocytes from SCA patients expressed higher levels of VCAM-1, suggesting one mechanism by which monocytes interact with RBCs in this context. Moreover, SIRP-α, an important inhibitor of erythrophagocytosis, was reduced in monocytes, mainly in NC-MC, from SCA patients. Given that all monocyte subsets interact with or contained intracellular remnants of erythroid cells, and that no correlation was seen between SIRP-α and glycophorin A MFI (data not shown), our findings suggest that SIRP-α, although relevant, is neither the sole nor the dominant regulator of this process. Nevertheless, reduced SIRP-α expression may imply that these monocytes are more actively engaged in RBC clearance, due to impaired recognition of CD47. Supporting this view, Pan and colleagues (48) demonstrated increased phagocytic activity in monocytes and neutrophils from Sirp-α knock-out mice (22), while Londino et al. showed that loss of SIRP-α enhances inflammatory signaling (32). Indeed, the SIRP-α-CD47 axis seems to be particularly critical under inflammatory conditions. Bian and colleagues reported that blocking this interaction in mice under physiological conditions had no impact on macrophage phagocytosis; however, during inflammation it resulted in severe anemia (33), emphasizing how inflammation modulates this pathway. In our cohort of hydroxyurea (HU)-treated patients, sickle RBCs exhibited CD47 expression comparable to normal RBCs, indicating that CD47 loss does not account for RBCs interaction. However, since CD47 levels increase with HU therapy (34), reduced CD47 expression in untreated patients may contribute to the enhanced phagocytosis of sickle RBCs by circulating monocytes. Additional functional assays targeting SIRP-α/CD47 and VCAM-1/α4β1 integrin would further clarify and quantify the involvement of SIRP-α and VCAM-1 in the process.

Our data demonstrated that monocytes from SCA patients upregulated intracellular HO-1 expression, suggesting a response to heme from phagocytosed RBCs or RBC microvesicles (35–38). This finding further supports the internalization of part of sickled RBCs or RBCs microvesicles by circulating monocytes, consistent with previous reports showing increased of HO-1 expression following phagocytosis (25). Macrophages with enhanced phagocytic and scavenging activity, such as the central macrophages within EBIs, have been shown to express high levels of CD206, a marker of alternatively activated cells (39–41). In line with this, our cohort revealed that enhanced RBC uptake was associated with increased CD206 expression in SCA monocytes, particularly within I-MC and NC-MC subsets.

Our in vitro assays indicates that both monocytes and RBCs from SCA patients contribute to enhanced erythrophagocytosis by circulating monocytes, suggesting that alterations in the surface molecules of both cell types, or structural changes in sickled RBCs, account for their interaction and subsequent internalization. In contrast to previous findings, which indicated that activated endothelial cells are required for sickle RBCs uptake by monocytes in vitro (25), our results show that co-culture of monocytes with RBCs alone was sufficient to induce their interaction and RBCs internalization. The upregulation of HO-1 in PKH26⁺ monocytes provides additional evidence of RBC engulfment. However, we acknowledge the limitations of our assays in proving complete internalization of all RBCs, then we cannot exclude the possibility that RBC remnants, such as microvesicles, were internalized, or that some adherent cells were detected. The fact that the patients in this study were receiving HU therapy may also account for these differences, as HU has been reported to alter monocyte phenotype and inflammatory capacity (30). Moreover, the induction of HO-1 and FPN, the only known iron exporter (42, 43), in PKH26⁺ monocytes indicates their capacity to metabolize RBC content, degrading hemoglobin-derived heme and releasing the resulting iron. It is well established that hemoglobin can activate the transcription of both HO-1 and FPN, and that their co-expression facilitates iron recycling by macrophages (44). Notably, the increase in FPN observed as early as 2 hours after the initial contact with RBCs indicates that monocytes are promptly capable of releasing iron. We were not able to detect iron in the supernatant, possibly due to the low number of phagocytosing monocytes in culture. Thus, a definitive proof of iron export and a time-course analysis would be valuable in future studies to further refine these observations.

Although macrophages exert the greatest impact on erythropoiesis and RBCs clearance (45), monocytes also emerge as contributors of erythroid proliferation and survival being found near specific hematopoietic niches in the bone marrow (11). Depletion of CD169⁺ macrophages under homeostatic conditions reduced the number of erythroblasts in the bone marrow, although it did not result in anemia, possibly due to impaired erythrophagocytosis. In contrast, depletion of CD169⁺ macrophages markedly impaired erythropoietic recovery following hemolytic anemia or acute blood loss (3), suggesting that macrophage represent a potential therapeutic target in erythropoietic disorders. Considering the similarities between EBI macrophages and SCA monocytes, we hypothesize that circulating monocytes could enhance erythropoietic turnover induced by anemia, as iron export may support hemoglobin synthesis in newly formed erythroblasts. Moreover, monocytes may assist macrophages in the clearance of RBCs, thereby preventing intravascular hemolysis and the subsequent release of free hemoglobin and heme that trigger chronic inflammation. Depletion of monocytes in animal model of SCA may help to elucidate their contribution to disease pathophysiology.

The main limitation of this study is that most patients were undergoing HU therapy. The remaining patients were prescribed HU but experienced difficulties in obtaining the medication for the three months prior to sample collection. Although these patients did not differ from the overall group, future comparisons between treated and untreated patients will be critical, given the impact of HU on cell phenotypes, monocyte subset distribution, and their direct influence on inflammation and hemolysis (30). HU is also known to modulate the expression of various adhesion molecules on RBCs and may also change SIRP-α expression. Previous studies have demonstrated that HU reduces the expression of α4β1, CD36, VLA-4, and ICAM-4 in erythroid cells from patients with SCA (34, 46, 47). Nevertheless, despite these effects, HU treatment does not fully restore cellular profiles to those of healthy individuals. Future studies including additional patients and independent cohorts will be important to further strengthen and validate the findings presented here.

Our findings offer novel insights into the role of monocytes in RBCs clearance and iron recycling in SCA and establish a foundation for future studies aiming at addressing key questions raised by our results. These include the impact of HU treatment, the potential for monocyte adhesion to erythroid cells at different developmental stages, and the molecular mechanisms that regulate erythrophagocytosis. Collectively, our data highlight circulating monocytes and their interaction to erythroid cells as possible targets for future mechanistic and translational investigations.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The studies involving humans were approved by UNICAMP Human Research Ethics Committee (protocol number CAAE: 88768318.6.0000.5404). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Author contributions

MB: Formal Analysis, Writing – review & editing, Methodology, Conceptualization, Data curation, Writing – original draft, Investigation. MS: Writing – review & editing, Methodology. IP: Methodology, Writing – review & editing. DL: Methodology, Writing – review & editing. DA: Writing – review & editing. CL: Writing – review & editing. SS: Investigation, Writing – review & editing. RS-C: Data curation, Supervision, Writing – review & editing, Conceptualization, Writing – original draft, Formal Analysis. FC: Supervision, Writing – original draft, Writing – review & editing, Conceptualization.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by the São Paulo Research Foundation (FAPESP) (grants to FFC, 2014/00984-3 and 2019/18886-1, a grant to RS-C, 2019/09704-7, and a grant for flow cytometry 2022/06227-6) and by the National Council for Scientific and Technological Development (CNPq), Brazil (grant to MDB, 140594/2019-1).

Acknowledgments

The authors thank Irene Pereira de Freitas and Nádia Ghinelli Amôr for the technical assistance with flow cytometry experiments and Ana Luisa Bortoluzo de Lorenzo for administrative support. We also thank FAPESP and CNPq for funding support.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2025.1699306/full#supplementary-material

Supplementary Table 1 | List of antibodies used for flow cytometry analysis.

References

1. Bessis M. Erythroblastic Island, functional unity of bone marrow. Rev Hematol. (1958) 13:8–11.

PubMed Abstract | Google Scholar

2. Chasis JA and Mohandas N. Erythroblastic islands: Niches for erythropoiesis. Blood. (2008) 112:470–8. doi: 10.1182/blood-2008-03-077883

PubMed Abstract | Crossref Full Text | Google Scholar

3. Chow A, Huggins M, Ahmed J, Hashimoto D, Lucas D, Kunisaki Y, et al. CD169 + macrophages provide a niche promoting erythropoiesis under homeostasis and stress. Nat Med. (2013) 19:429–36. doi: 10.1038/nm.3057

PubMed Abstract | Crossref Full Text | Google Scholar

4. Yoshida H, Kawane K, Koike M, Mori Y, Uchiyama Y, and Nagata S. Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. Nature. (2005) 437:754–8. doi: 10.1038/nature03964

PubMed Abstract | Crossref Full Text | Google Scholar

5. Sadahira Y, Yasuda T, Yoshino T, Manabe T, and Takeishi T. Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis Abstract: Erythropoiesis occurs in the presence of erythropoietin ( EPO ) without m. Jounal Leukocyte Biol. (2000) 68:464–70. doi: 10.1189/jlb.68.4.464

Crossref Full Text | Google Scholar

6. Hanspal M and Hanspal JS. The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: A 30-kD heparin-binding protein is involved in this contact. Blood. (1994) 84:3494–504. doi: 10.1182/blood.V84.10.3494.3494

PubMed Abstract | Crossref Full Text | Google Scholar

7. Morris L, Crockerf PR, Fraser I, Hill M, and Gordon S. Expression of a divalent cation-dependent erythroblast adhesion receptor by stromal macrophages from murine bone marrow. J Cell Sci. (1991) 99:141–7. doi: 10.1242/jcs.99.1.141

PubMed Abstract | Crossref Full Text | Google Scholar

8. Jacobsen RN, Perkins AC, and Levesque JP. Macrophages and regulation of erythropoiesis. Vol. 22, Current Opinion in Hematology. Lippincott Williams Wilkins;. (2015) 22(3):212–9. doi: 10.1097/MOH.0000000000000131

PubMed Abstract | Crossref Full Text | Google Scholar

9. Gurkan E, Ergun Y, Zorludemir S, Baslamisli F, and Koçak R. Liver involvement in sickle cell disease. Turkish J Gastroenterology. (2005) 16:194–8.

PubMed Abstract | Google Scholar

10. Liao C, Prabhu KS, and Paulson RF. Monocyte-derived macrophages expand the murine stress erythropoietic niche during the recovery from anemia. Blood. (2018) 132:1–4. doi: 10.1182/blood-2018-06-856831

PubMed Abstract | Crossref Full Text | Google Scholar

11. Heideveld E, Masiello F, Marra M, Esteghamat F, Yağcı N, von Lindern M, et al. CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield. Haematologica. (2015) 100:1396–406. doi: 10.3324/haematol.2015.125492

PubMed Abstract | Crossref Full Text | Google Scholar

12. Heshusius S, Heideveld E, von Lindern M, and van den Akker E. CD14+ monocytes repress gamma globin expression at early stages of erythropoiesis. Sci Rep. (2021) 11. doi: 10.1038/s41598-021-81060-7

PubMed Abstract | Crossref Full Text | Google Scholar

13. Haschka D, Petzer V, Kocher F, Tschurtschenthaler C, Schaefer B, Seifert M, et al. Classical and intermediate monocytes scavenge non-transferrin-bound iron and damaged erythrocytes. JCI Insight. (2019) 4:1–23. doi: 10.1172/jci.insight.98867

PubMed Abstract | Crossref Full Text | Google Scholar

14. Pradhan P, Vijayan V, Gueler F, and Immenschuh S. Interplay of heme with macrophages in homeostasis and inflammation. Int J Mol Sci. (2020) 21(3):740. doi: 10.3390/ijms21030740

PubMed Abstract | Crossref Full Text | Google Scholar

15. Greco R, Demartini C, Zanaboni AM, Tumelero E, Persico A, Candeloro E, et al. Cd163 as a potential biomarker of monocyte activation in ischemic stroke patients. Int J Mol Sci. (2021) 22(13):6712. doi: 10.3390/ijms22136712

PubMed Abstract | Crossref Full Text | Google Scholar

16. Liu Q, Ou Q, Chen H, Gao Y, Liu Y, Xu Y, et al. Differential expression and predictive value of monocyte scavenger receptor CD163 in populations with different tuberculosis infection statuses. BMC Infect Dis. (2019) 19:1006. doi: 10.1186/s12879-019-4525-y

PubMed Abstract | Crossref Full Text | Google Scholar

17. Kaminski TW, Sivanantham A, Mozhenkova A, Smith A, Ungalara R, Dubey RK, et al. Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease. Am J Physiol Cell Physiol. (2024) 327:C423–37. doi: 10.1152/ajpcell.00386.2023

PubMed Abstract | Crossref Full Text | Google Scholar

18. Li XH, Du N, Xu G, Zhang P, Dang R, Jiang Y, et al. Expression of CD206 and CD163 on intermediate CD14++CD16+ monocytes are increased in hemorrhagic fever with renal syndrome and are correlated with disease severity. Virus Res. (2018) 253:92–102. doi: 10.1016/j.virusres.2018.05.021

PubMed Abstract | Crossref Full Text | Google Scholar

19. Kato GJ, Piel FB, Reid CD, Gaston MH, Ohene-Frempong K, Krishnamurti L, et al. Sickle cell disease. Nat Rev Dis Primers. (2018) 4:1–22. doi: 10.1038/nrdp.2018.10

PubMed Abstract | Crossref Full Text | Google Scholar

20. Linus Pauling HAI, Singer SJ, and Wells IC. Sickle cell anemia, a molecular disease. Science. (1949) 110:543–8. doi: 10.1126/science.110.2865.543

PubMed Abstract | Crossref Full Text | Google Scholar

21. Ware RE, de Montalembert M, Tshilolo L, and Abboud MR. Sickle cell disease. Lancet. (2017) 390:311–23. doi: 10.1016/S0140-6736(17)30193-9

PubMed Abstract | Crossref Full Text | Google Scholar

22. Brewin J, El Hoss S, Strouboulis J, and Rees D. A novel index to evaluate ineffective erythropoiesis in hematological diseases offers insights into sickle cell disease. Haematologica. (2022) 107(1):338–41. doi: 10.3324/haematol.2021.279623

PubMed Abstract | Crossref Full Text | Google Scholar

23. El Nemer W, Godard A, and El Hoss S. Ineffective erythropoiesis in sickle cell disease: New insights and future implications. Curr Opin Hematol. (2021) 28:171–6. doi: 10.1097/MOH.0000000000000642

PubMed Abstract | Crossref Full Text | Google Scholar

24. Wongtong N, Jones S, Deng Y, Cai J, and Ataga KI. Monocytosis is associated with hemolysis in sickle cell disease. Hematol (United Kingdom). (2015) 20:593–7. doi: 10.1179/1607845415Y.0000000011

PubMed Abstract | Crossref Full Text | Google Scholar

25. Liu Y, Zhong H, Bao W, Mendelson A, An X, Shi P, et al. Patrolling monocytes scavenge endothelial-adherent sickle RBCs: A novel mechanism of inhibition of vaso-occlusion in SCD. Blood. (2019) 134:579–90. doi: 10.1182/blood.2019000172

PubMed Abstract | Crossref Full Text | Google Scholar

26. Allali S, Rignault-Bricard R, De Montalembert M, Taylor M, Bouceba T, Hermine O, et al. HbS promotes TLR4-mediated monocyte activation and proinflammatory cytokine production in sickle cell disease. Blood. (2022) 140(18):1972–82. doi: 10.1182/blood.2021014894

PubMed Abstract | Crossref Full Text | Google Scholar

27. Liu Y, Su S, Shayo S, Bao W, Pal M, Dou K, et al. Hemolysis dictates monocyte differentiation via two distinct pathways in sickle cell disease vaso-occlusion. J Clin Invest. (2023) 133(18):e172087. doi: 10.1172/JCI172087

PubMed Abstract | Crossref Full Text | Google Scholar

28. Allali S, Maciel TT, Hermine O, and De Montalembert M. Innate immune cells, major protagonists of sickle cell disease pathophysiology. Haematologica. (2020) 105(2):273–83. doi: 10.3324/haematol.2019.229989

PubMed Abstract | Crossref Full Text | Google Scholar

29. Tozatto-Maio K, Rós FA, Weinlich R, and Rocha V. Inflammatory pathways and anti-inflammatory therapies in sickle cell disease. HemaSphere. (2024) 8(12):e70032. doi: 10.1002/hem3.70032

PubMed Abstract | Crossref Full Text | Google Scholar

30. Guarda CC, Silveira-Mattos PSM, Yahouédéhou SCMA, Santiago RP, Aleluia MM, Figueiredo CVB, et al. Hydroxyurea alters circulating monocyte subsets and dampens its inflammatory potential in sickle cell anemia patients. Sci Rep. (2019) 9(1):14829. doi: 10.1038/s41598-019-51339-x

PubMed Abstract | Crossref Full Text | Google Scholar

31. Matozaki T, Murata Y, Okazawa H, and Ohnishi H. Functions and molecular mechanisms of the CD47-SIRPα signalling pathway. Trends Cell Biol. (2009) 19(2):72–80. doi: 10.1016/j.tcb.2008.12.001

PubMed Abstract | Crossref Full Text | Google Scholar

32. Londino JD, Gulick D, Isenberg JS, and Mallampalli RK. Cleavage of signal regulatory protein α (sirpα) enhances inflammatory signaling. J Biol Chem. (2015) 290:31113–25. doi: 10.1074/jbc.M115.682914

PubMed Abstract | Crossref Full Text | Google Scholar

33. Bian Z, Shi L, Guo YL, Lv Z, Tang C, Niu S, et al. Cd47-Sirp α interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells. Proc Natl Acad Sci. (2016) 113(37):E5434–43. doi: 10.1073/pnas.1521069113

PubMed Abstract | Crossref Full Text | Google Scholar

34. Gambero S, Canalli AA, Traina F, Albuquerque DM, Saad STO, Costa FF, et al. Therapy with hydroxyurea is associated with reduced adhesion molecule gene and protein expression in sickle red cells with a concomitant reduction in adhesive properties. Eur J Haematol. (2007) 78:144–51. doi: 10.1111/j.1600-0609.2006.00788.x

PubMed Abstract | Crossref Full Text | Google Scholar

35. Ryter SW, Alam J, and Choi AMK. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiological Rev. (2006) 86(2):583–650. doi: 10.1152/physrev.00011.2005

PubMed Abstract | Crossref Full Text | Google Scholar

36. Maines MD. THE HEME OXYGENASE SYSTEM: A regulator of second messenger gases. Annu Rev Pharmacol Toxicol. (2025) 37:517–54. doi: 10.1146/annurev.pharmtox.37.1.517

PubMed Abstract | Crossref Full Text | Google Scholar

37. Tenhunen R, Marver HS, and Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA. (1968) 61(2):748–55. doi: 10.1073/pnas.61.2.748

PubMed Abstract | Crossref Full Text | Google Scholar

38. Immenschuh S, Baumgart-Vogt E, and Mueller S. Heme oxygenase-1 and iron in liver inflammation: A complex alliance. Curr Drug Targets. (2012) 11:1541–50. doi: 10.2174/1389450111009011541

PubMed Abstract | Crossref Full Text | Google Scholar

39. Jonsson A, Korsgren O, and Hedin A. Transcriptomic characterization of human pancreatic CD206- and CD206 + macrophages. Sci Rep. (2025) 15:12037. doi: 10.1038/s41598-025-96313-y

PubMed Abstract | Crossref Full Text | Google Scholar

40. Sica A and Mantovani A. Macrophage plasticity and polarization: In vivo veritas. J Clin Invest. (2012) 122(3):787–95. doi: 10.1172/JCI59643

PubMed Abstract | Crossref Full Text | Google Scholar

41. Luo Y, Shao L, Chang J, Feng W, Liu YL, Cottler-Fox MH, et al. M1 and M2 macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion. Blood Adv. (2018) 2:859–70. doi: 10.1182/bloodadvances.2018015685

PubMed Abstract | Crossref Full Text | Google Scholar

42. Donavan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, et al. Positional Cloning of zebrafish ferroportin1 identifies a consrved verebrate iron exporter. Nature. (2000) 403:776–81. doi: 10.1038/35001596

PubMed Abstract | Crossref Full Text | Google Scholar

43. Qiu L, Frazer DM, Hu M, Song R, Liu X, Qin X, et al. Mechanism and regulation of iron absorption throughout the life cycle: Mechanism and regulation of iron absorption. J Advanced Res. (2025) 77:107–18. doi: 10.1016/j.jare.2025.01.002

PubMed Abstract | Crossref Full Text | Google Scholar

44. Marro S, Chiabrando D, Messana E, Stolte J, Turco E, Tolosano E, et al. Heme controls ferroportin1 (FPN1) transcription involving Bach1, Nrf2 and a MARE/ARE sequence motif at position -7007 of the FPN1 promoter. Haematologica. (2010) 95:1261–8. doi: 10.3324/haematol.2009.020123

PubMed Abstract | Crossref Full Text | Google Scholar

45. Li W, Guo R, Song Y, and Jiang Z. Erythroblastic island macrophages shape normal erythropoiesis and drive associated disorders in erythroid hematopoietic diseases. Front Cell Dev Biol. (2021) 8:1–16. doi: 10.3389/fcell.2020.613885

PubMed Abstract | Crossref Full Text | Google Scholar

46. Cartron JP and Elion J. Erythroid adhesion molecules in sickle cell disease: Effect of hydroxyurea. Transfusion Clinique Biologique. (2008) 15:39–50. doi: 10.1016/j.tracli.2008.05.001

PubMed Abstract | Crossref Full Text | Google Scholar

47. Odièvre MH, Bony V, Benkerrou M, Lapouméroulie C, Alberti C, Ducrocq R, et al. Modulation of erythroid adhesion receptor expression by hydroxyurea in children with sickle cell disease. Haematologica. (2008) 93:502–9. doi: 10.3324/haematol.12070

PubMed Abstract | Crossref Full Text | Google Scholar

48. Pan L, Wang B, Chen M, Ma Y, Cui B, Chen Z, et al. Lack of SIRP-alpha reduces lung cancer growth in mice by promoting anti-tumour ability of macrophages and neutrophils. Cell Prolif. (2023) 56(2):e13361. doi: 10.1111/cpr.13361

PubMed Abstract | Crossref Full Text | Google Scholar