Introduction
Granulocyte–macrophage colony-stimulating factor (GM-CSF), a significant myelopoietic growth factor and pro-inflammatory cytokine, has been shown to be upregulated and has attracted increasing interest as a therapeutic target for many inflammatory diseases, including coronavirus disease 2019 (COVID-19; [
1‐
3]). In general, GM-CSF is barely detectable in the peripheral blood of healthy people, and GM-CSF plays a minor role in homeostatic myelopoiesis, as evidenced by the fact that GM-CSF knock-out mice have a virtually normal lifespan and have less dramatic alterations in the basal myeloid system [
4,
5]. However, severe GM-CSF deficiency causes pulmonary alveolar proteinosis (PAP), a life-threatening interstitial lung disease in which dysfunctional alveolar macrophages cannot clear surfactant [
6,
7]. For erythropoiesis, human GM-CSF stimulates primitive and definitive erythropoiesis in mouse embryos expressing human GM-CSF receptors [
8]. Additionally, GM-CSF levels increased in sickle cell disease (SCD), leading to downregulation of fetal hemoglobin expression [
9]. Importantly, interleukin-6 (IL-6), interferon-γ (IFN-γ) and GM-CSF were notably unregulated in the mouse model of anemia of inflammation (AI) induced by heat-killed Brucella abortus [
10,
11]. Despite these studies, the role of GM-CSF per se in adult human and mouse erythropoiesis remains unclear.
Erythropoiesis is a process by which hematopoietic stem cells (HSCs) proliferate and differentiate via multiple distinct developmental stages, to eventually generate mature red blood cells (RBCs; [
12‐
14]). The process occurs at the erythroblastic island (EBI), which is composed of a central macrophage surrounded by developing erythroid cells [
15]. The EBI, first described by
Marcel Bessis in 1958 [
15], was functionally validated by Narla Mohandas et al. [
16], who found significantly lower numbers in hyper-transfused rat bone marrow (BM; [
16]). Recently, studies indicated that EBI macrophages promote erythropoiesis by directly interacting with erythroblasts, secreting growth factors, phagocytosing senescent RBCs, providing iron, and finally engulfing the nucleus enucleated by erythroblasts [
13,
17,
18]. Many hematopoietic growth factors regulate erythropoiesis by affecting the function of EBI macrophages. Erythropoietin (EPO) acts on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis [
17,
19]; Granulocyte colony-stimulating factor (G-CSF) blocks medullary erythropoiesis by depleting EBI macrophages in mouse BM [
20,
21]. These functional studies strongly suggest that hematopoietic growth factors can regulate erythropoiesis by affecting the roles of EBI macrophages.
In previous studies, both G-CSF and GM-CSF were used to mobilize HSCs from the BM into the blood in order to harvest large quantities of HSCs for subsequent transplantation in humans [
22,
23]. Both G-CSF and GM-CSF cause HSCs mobilization by perturbing HSC niches in the BM. To define these mechanisms, studies have shown that G-CSF results in downregulation of the cell adhesion molecule vascular cell adhesion molecule-1(VCAM-1) and the chemokine CXC-motif ligand-12(CXCL12; [
24,
25]). These two molecules are both essential to HSC retention within the BM. Additionally, studies have also shown that the effect of G-CSF on HSC niches is mediated in part by a subpopulation of BM macrophages [
26]. G-CSF also causes a significant loss of BM macrophages expressing VCAM-1, CD169, and ER-HR3 and blocks medullary erythropoiesis in BM [
20]. Furthermore, using imaging flowcytometry (IFC),
Joshua Tay et al
. found that G-CSF reduces EBI frequency in the BM by more than 100 times [
21]. In contrast to the many studies of G-CSF’s role in regulating erythropoiesis, the precise role of GM-CSF is less well understood.
In the present study, we demonstrate that GM-CSF significantly decreases human EBI formation in vitro. Bioinformatics analysis of RNA sequencing (RNA-seq) on GM-CSF-treated and control EBI macrophages further confirmed the impaired EBI formation, as evidenced by decreased adhesion molecule expression of CD163. In particular, GM-CSF injection into mice also significantly decreases BM erythroblasts as well as EBI numbers by decreasing both the number of EBI macrophages and adhesion molecule expression of CD163 and of Vcam1. Our study presents novel data that GM-CSF impairs erythropoiesis by disturbing EBI formation and proposes targeting EBI macrophages as a potential treatment option for AI.
Materials and methods
Antibodies and mice
All antibodies related to this study are listed in Additional file
9: Table S1. Wild type (WT) mice that are 8–12 weeks old are on the C57BL/6 background and are maintained at the Experimental Animal Center of Zhengzhou University. GM-CSF (300 μg/kg, Do-D1 or D0-D3) was injected into sex-matched male and female mice to study the functional role in vivo. Clodronate-loaded liposomes injection experiment was performed as previous described [
27].
Blood parameter analysis
About 30–50 μl of peripheral blood was collected through the orbital vein using an Eppendorf tube containing 1 μl of 0.5 M ethylenediaminetetraacetate (EDTA; Fisher) after anesthesia. Blood was diluted 1:10 in phosphate balanced saline (PBS) and analyzed via an advia120 hematology analyzer. The RBC numbers, hemoglobin, hematocrit (HCT), and reticulocytes of mice before and after GM-CSF injection were analyzed using GraphPad Prism 9.0 software.
Preparation of single cells for flowcytometry
Under terminal anesthesia using isoflurane, mice were killed via cervical dislocation. The BM and spleen (SP) were collected and processed for single-cell preparation for flowcytometry. The single cell suspensions were prepared as previously described [
17]. In brief, BM cells were flushed with PBS + 2%FBS + 2 mM EDTA, and SP cells were smashed in PBS + 2%FBS + 2 mM EDTA. Then, the cell suspensions were washed with PBS plus 2% fetal bovine serum (FBS) and 2 mM EDTA, centrifugated at 300 g for 10 min and gently pushed through a 70 µm cell strainer. The single cells suspension was then used for flowcytometry staining and analyses.
Flowcytometry staining and analyses
For burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E) staining, 5 × 106 cells were blocked with 50 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with BV-421-Lin (1μL/5 × 106cells), BV-421-CD41(1μL/5 × 106cells), PE-Cy7-CD34(1μL/5 × 106cells), PE-Cy7-CD16/32(1μL/5 × 106 cells), APC-Cy7-Scal1(1μL/5 × 106 cells), APC-CD117(1μL/5 × 106 cells), and Percp-CD71(1μL/5 × 106 cells) for 30 min on ice in the dark. For murine erythroblast staining, 3 × 106cells were blocked with 25 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with anti-CD11b-APC-Cy7 (0.1 µg/106cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-CD45-APC-Cy7 (0.1 µg/106cells), anti-Ter119-PE (0.5 µg/106cells) or FITC-Ter119(0.5 µg/106cells), and anti-CD44-APC (0.2 µg/106cells) for 30 min on ice in the dark. For macrophage staining, 5 × 106 cells were blocked with 50 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with anti-CD11b-APC-Cy7 (0.1 µg/106cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-AF647-F4/80 (5 µg/106 cells), anti-Percpcy5.5-CD106 (0.3 µg/106 cells), anti-FITC-CD169 (4 µg/106 cells), anti-Percp-CD163 (0.2 µg/106 cells), anti-FITC-Timd4 (0.25 µg/106 cells), anti-PE-Cy7-Mertk (0.5 µg/106 cells), anti-PE-Cy7Axl (0.125 µg/106 cells), anti-PE-Cy7-MHC-II (0.5μL/5 × 106 cells), anti-FITC-CD206 (1μL/5 × 106 cells), anti-Percpcy5.5-CD14 (1μL/5 × 106 cells), anti-FITC-CD86 (1μL/5 × 106 cells), and anti-PE-Cy7-CD80 (1μL/5 × 106 cells) for 30 min on ice in the dark. After staining, the cells were washed once with PBS plus 0.5%BSA and 2 mM EDTA. DAPI were used to gate out dead cells. Then, cells were resuspended with PBS plus 0.5%BSA and 2 mM EDTA, and run on a BD Air III (BD bioscience). Flow Jo software (BD) was used to analyze the data.
P-STAT5 staining
For P-STAT5 staining, the BD Transcription Factor Phospho Buffer Set (BD Cat#565575) was used. In short, the BM cells were starved for 4 h, and were stimulated with GM-CSF for 15 min. Then, the cells were fixed with TF Fix/Perm Buffer(1X) for 50 min at 4℃ and washed using 1 × TFP Perm/Wash Buffer. After fixation, cells were incubated with BD Phosflow™ Perm Buffer III, and the cells were washed once to remove Perm Buffer III using 1 × TFP Perm/Wash Buffer. Finally, cells were stained with CD11b, Gr-1, F4/80, and P-stat5 (ThermoFisher Scientific,12–9010-42) for 50 min on ice in the dark, washed using 1 × TFP Perm/Wash Buffer, and run on a BD Air III. Flow Jo software (BD) was used to analyze the data.
Engulfment staining and analysis
We followed the same method as
Jessica A. Hamerman’s group [
28]. BM single cells from both control and GM-CSF treatment mice were washed and prepared for intracellular staining with Fixation and Permeabilization buffer (BD Biosciences), washed in Perm/Wash buffer (BD Biosciences); and then stained with anti-CD11b-APC-Cy7 (0.1 µg/10
6cells), anti-Gr1-APC-Cy7 (0.1 µg/10
6cells), anti-AF647-F4/80 (5 µg/10
6 cells), and anti-FITC-Ter119 (0.5 µg/10
6cells) for 30 min on ice in the dark to detect cells that had phagocytosed RBCs. BD Air III was used to collect events and Flow Jo software (BD) was used to analyze the data.
EBI enrichment
EBIs in mouse BM were enriched and the numbers were quantified. Our EBI enrichment method matches that of previous studies [
17,
21,
29]. The protocol is as follows: (1) preparation of different concentration of density gradient solution (0%, 1.5% and 3%, Additional file
10: TableS2). (2) flush all of 4 bones (2 femur and 2 tibia) in 2 mL of 0% density gradient solution using a 1 mL syringe with a 25 G (tibia) or 23 G (femur) needle in doses of 500 μL (rapidly in order to preserve erythroblastic islands). (3) gently pipette the bone cells flushed with a 1 ml syringe (once or twice only) and filter through a 70 μm cell strainer. (4) In a 50 ml centrifuge tube first add 5 mL of 3% density gradient solution, then slowly add 5 mL of 1.5% density gradient solution along the side wall of the tube with a pipette to bring it above the 3% density gradient solution. A further 5 mL of 0% density gradient solution was slowly added with a pipette along the side wall of the tube to place it above the 1.5% density gradient solution. The bone marrow cell solution containing the erythroblastic islands was adjusted to a volume of 5 mL with 0% density gradient solution and slowly added to the tube containing the stratified density gradient solution with a pipette and placed on top of the 0% density gradient solution. (5) leave at room temperature for 30 min. Pipette the 0% and 1.5% density layers into a new 15 mL tube., and collect the 3% layer, which contains the erythroblastic islands. (6) Cytospins were performed using 3% layer from mouse BM, and the number of EBIs in each slide was quantified.
Frozen section preparation and hematoxylin and eosin (HE) staining
SP cells were obtained from both GM-CSF treated and control mice and immediately embedded into an optimal cutting temperature (OCT) compound. The tissues were kept in a − 80 ℃ freezer. Then, hematoxylin and eosin (HE) staining was performed as previously described [
30].
Co-culture of human “EBI-like” macrophages with late stages of erythroblasts
Erythroblasts and “EBI-like” macrophages were derived from cord blood CD34
+ cells. The detailed cultured method was same as our previously described [
12,
14,
17,
31]. Fresh CD34
+ cells were purified by CD34 MicroBeads (Miltenyi Biotec, Gladbach, Germany) from human healthy donors at Zhengzhou University. The cell culture procedure was comprised of 3 phases. In the present, two phases of cell culture were used. Composition of the base culture medium was Iscove's Modified Dulbecco's Medium (IMDM, Invitrogen), 2% human peripheral blood plasma (Stem Cell Technologies), 3% human AB serum (Atlanta Biologicals), 200 μg/mL Holo-human transferrin (Sigma Aldrich), 3 IU/mL heparin (The First Affiliated Hospital of Zhengzhou University), and 10 μg/mL insulin (The First Affiliated Hospital of Zhengzhou University). In the first phase (day 0 to day 6), CD34
+ cells at a concentration of 10
5/mL were cultured in the presence of 10 ng/mL stem cell factor (SCF, Stem Cell Technologies), 1 ng/mL IL-3(Stem Cell Technologies), and 3 IU/mL erythropoietin (The First Affiliated Hospital of Zhengzhou University). In the second phase (day 7 to day 11), IL-3 was omitted from the culture medium. Then, the D11 erythroblasts were harvested for the next step of co-culture experiment. Human “EBI-like” macrophages were also derived from CD34
+ cells. In the Day 0 to Day 7, CD34
+ cells were cultured in IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 10 ng/mL SCF, 1 ng/mL IL-3, 100 ng/mL M-CSF and 50 ng/mL FLT3, 1 × penicillin–streptomycin. At day 7, the suspensions were removed and IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 100 ng/mL M-CSF and 50 ng/mL FLT3 were added. Then, the adherent cells were cultured for another 4 days. At D11, all the adherent cells will differentiate into macrophages as our previously described [
17]. Macrophages were pretreated with 100 ng/mL GM-CSF for 24 h. Untreated or GM-CSF-pretreated macrophages were mixed with day 11 erythroblasts at a 1: 20 ratio and cultured for 12 h in an IMDM medium containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 200 µg/mL holo-human transferrin, 10 IU/ml EPO, 5 mM Mg
2+, and 5 mM Ca
2+. 1 × 10
5 cells were collected for cytospin analysis.
RNA-sequencing
RNA-seq was prepared and analyzed as previously described [
14,
17,
31,
32]. RNA was extracted from control and GM-CSF treated “EBI-like” macrophages. Approximately 100 ng of total RNA was used as input for cDNA library preparation, which was preformed using an Illumina TruSeq kit followed by sequencing using an Illumina HiSeq 4000 platform (Beijing Genomics Institute, BGI, China). For analysis of the RNA-seq data, gene read counts for gencode hg19 version 31 protein coding genes were generated using kallisto [
33]. Differential gene expression was examined with DESeq2 using a Wald test, where the log2 fold change is greater than 0.5 with a 0.05 adjusted p-value cutoff and independent filtering to remove genes with lower expression levels, as previously described [
12]. Gene set enrichment analysis (GSEA) was performed as previously described [
34]. The permutation number was set to 1000 and the permutation type was set to gene set. The raw data was uploaded to the National Omics Data Encyclopedia (NODE) database (
https://www.biosino.org/node) under accession number OEP002596.
Cell count
Absolute cell count was measured via flowcytometry using 123count eBeads™ Counting Beads (Cat#: 01–1234-42) according to the manufacturer’s protocol.
Enrichment of BM F4/80+ macrophages
Control and GM-CSF-treated mouse BM F4/80
+ macrophages were enriched via F4/80 microbeads (Miltenyi, Cat#:130–110-443). BM cells were incubated with F4/80 microbeads for 15 min on ice in the dark. The cells were washed with PBS plus 2% FBS and 2 mM EDTA, centrifugated at 300 g for 10 min, and resuspended with 2 mL PBS plus 2% FBS and 2 mM EDTA. Finally, the BM F4/80
+ macrophages were enriched by Quadro MACS following the manufacturer's instructions. More than 90% purification was achieved, as previously described [
35]. These cells were used for QRT-PCR.
QRT-PCR, cytospins and Giemsa-Wright staining
QRT-PCR, cytospins and Giemsa-Wright staining were performed as in our previous papers [
14,
17,
31,
32]. Primers of human
CD163, CD169, EMP and
αV-integrin used in the present study are the same in our previous work [
17]. The primers of mouse
IL1-β, TNF-α, iNOS, TGF-β, IL-10 and
Arg1 are listed in Additional file
11: Table S3.
Statistics
GraphPad Prism 9.0 software (GraphPad Software, Inc.) was used to perform the statistical analysis. All the experiments were replicated at least three times. All data were reported as mean ± SEM. Comparisons between different groups were performed by Student’s t test. P < 0.05 was considered to indicate a statistically significant difference.
Discussion
GM-CSF is barely detectable in the peripheral blood of healthy people but significantly increases in the presence of inflammatory conditions, such as, COVID-19, SCD, and so on [
2,
3,
9]. Inflammatory conditions such as infection, chronic inflammatory disorders, and hematological malignancies often cause anemia, also called AI [
46,
47]. Indeed, COVID-19 patients suffer a profound decline in hemoglobin levels but show an increase in the circulation of nucleated RBCs [
48]. In this study, ACE2 expression peaked during erythropoiesis and rendered erythroid progenitors vulnerable to infection by SARS-CoV-2, suggesting that SARS-CoV-2 infection directly induces stress erythropoiesis. In addition, many inflammatory cytokines and chemokines are also upregulated in SARS-CoV-2 and COVID-19 patients, including GM-CSF [
49,
50]. Importantly, GM-CSF was upregulated before all other inflammatory cytokines (IL-6, TNF and IFN-β) and chemokines (CCL2, CCL7 and CCL12) that were measured, indicating that GM-CSF might be involved in the initiation of this immunopathological process [
49]. Additionally, a murine model of AI is commonly induced by heat-killed Brucella abortus [
10]. Importantly, IL-6, and IFN-γ were significantly increased and GM-CSF was produced in substantial amounts after Brucella infection [
11]. Another study also indicated that GM-CSF is an upstream regulator of inflammatory macrophage function [
51]. However, the role of GM-CSF per se in adult human and mouse erythropoiesis is unclear.
In the present study, we tested the roles of GM-CSF in human and mouse EBI formation, finding that GM-CSF significantly decreases EBI formation both in vitro and in vivo. G-CSF also impairs EBI formation in vivo [
20,
21]. Conversely, previous studies have shown that G-CSF induces splenic erythropoiesis at the same time [
20]. Interestingly, GM-CSF does not induce splenic erythropoiesis, suggesting different roles for GM-CSF and G-CSF in erythropoiesis. As noted, the mouse model of AI featured anemia in peripheral blood [
10,
11] though, GM-CSF treatment alone does not induce severe peripheral blood anemia. Given that AI resulted in upregulation of several inflammatory cytokines [
10,
11], anemia is likely associated with the combined effect of these inflammatory factors.
GM-CSFR (CSF2R) is not expressed by late-stage erythroblasts but is highly expressed in human EBI macrophages, suggesting that GM-CSF may be unable to directly signal erythroblasts. Alternatively, the low expression of GM-CSFR in early-stage erythroblasts (FPKM < 10) may be insufficient, raising questions about the indirect effects of GM-CSF on erythropoiesis in vitro and in vivo. EBI, first described by
Marcel Bessis in 1958 [
15] is composed of a central macrophage surrounded by developing erythroid cells. EBI macrophages play significant roles in erythropoiesis, especially under stress conditions [
17,
19,
27,
45]. Using our well-established human EBI formation system [
17], we found that GM-CSF pre-treated EBI macrophages significantly impair EBI formation. Transcriptome analysis of control and GM-CSF-treated EBI macrophages by RNA-seq demonstrated that GM-CSF significantly decreases the expression of adhesion molecule CD163. Previous studies evidenced that CD163 is significant for the interaction between erythroblasts and EBI macrophages [
13,
17,
37,
39]. Significantly, CD163 promotes erythroid expansion in vitro, suggesting that it enhances the proliferation and/or survival [
37]. Hence, decreased expression of CD163 at least partially contributes to impaired human EBI formation and then the inhibited erythroid survival. In addition, GM-CSF significantly increases HLA-related genes and pro-inflammatory chemokines CCL1, CCL8 and CCL13, suggesting that GM-CSF polarizes EBI macrophages into the M1-like phenotype. The GSEA analysis indicated that the upregulated pathways include inflammatory mediator regulation of TRP channels, antigen processing and presentation, GvHD and so on. The down-regulated pathway includes phagosomes, the VEGF signaling pathway, ECM receptor interaction and so on. In conclusion, our results indicated that GM-CSF impairs erythropoiesis by affecting the functioning of EBI macrophages, highlighting the potential therapeutic target of GM-CSF in AI.
Because GM-CSF can stimulate numerous inflammatory processes and increase expression of pro-inflammatory chemokines, an anti-GM-CSF strategy might have broader effects than other immunomodulatory approaches in immunosuppressive therapy [
3]. Indeed, clinical trials showed that GM-CSF-targeted therapy was efficacious in patients with rheumatoid arthritis who were unresponsive to anti-TNF-α therapy [
52,
53]. Furthermore, clinical trials are ongoing or planned to assess the benefits of GM-CSF-targeted therapy for COVID-19 or CAR-T- or GvHD-related cytokine release syndrome (CRS; [
3,
54,
55]). Thus, our and many other studies have supported the immune overactivation of GM-CSF-based putative pathogenic roles. In summary, this data suggests that GM-CSF-targeting therapy may also be a novel option for AI.
Primitive and definitive erythropoiesis in mouse embryos requires the signal transduction of GM-CSF [
8]. However, adult BM erythropoiesis is different from fetal erythropoiesis as fetal erythropoiesis is a kind of stress erythropoiesis, while adult BM erythropoiesis is a steady-state erythropoiesis [
13,
56]. Yet the role of GM-CSF in adult BM erythropoiesis in vivo remains mysterious. Our study demonstrates that GM-CSF significantly decreases absolute erythroblast numbers and EBI formation. Regarding the potential mechanisms for this, GM-CSF significantly decreases the number of EBI macrophages as well as the surface expression of adhesion molecules CD163 and Vcam1 (but not CD169). Significantly, clodronate-loaded liposomes decreased erythroblast numbers in BM, which partially interpret the impaired erythropoiesis in mouse BM following GM-CSF administration. EPO-EPOR-JAK2-STAT5 signal transduction in macrophages enhances EBI formation [
17], and deletion of STAT5 in macrophages impair SP erythropoiesis [
19]. Meanwhile, GM-CSF also performs signal transduction through JAK2-STAT5 in macrophages [
57], and GM-CSF triggers JAK2-STAT5 signal transduction in BM macrophages as well. Thus, GM-CSF produces opposite effects on EBI macrophage-regulated erythropoiesis compared with EPO.
Phagocytosis of senescent RBCs represents another important role of EBI macrophages during erythropoiesis. In general, RBC clearance is thought to occur mainly in the spleen, where senescent RBCs are phagocytosed by splenic macrophages [
13]. Our previous study illustrated the iron recycling machine highly expressed by mouse BM EBI macrophages, suggesting the potential role of iron recycling for EBI macrophages in the mouse BM microenvironment [
17]. EPO enhances macrophage phagocytosis of apoptotic cells and also polarizes macrophages into an M2-like phenotype [
58]. However, GM-CSF-treated macrophages showed significantly decreased phagocytic capacity, and GM-CSF treatment polarized macrophages into an M1-like phenotype. Previous research has indicated that M2 macrophages have higher phagocytosis functions compared to M1 macrophages [
59]. In the present study, we found that GM-CSF treatment significantly decreased the expression of Mertk, Axl and Timd4. Importantly,
Mertk−/− mice showed significantly decreased engulfment of pyrenocytes by EBI macrophages [
60]. Macrophages from
Axl−/− mice showed decreased a 50% decrease in phagocytotic apoptotic cells [
61]. Phosphatidylserine (PS) is expressed by apoptotic cells and nuclei expelled by matured erythroblasts. Timd4 is expressed by macrophages, which bound apoptotic cells by recognizing PS. Anti-Timd4 antibodies significantly block the engulfment of apoptotic cells by macrophages [
62]. As such, Mertk, Axl, and Timd4 are significant for the phagocytosis-related function of EBI macrophages. Consistent with these findings, we discovered that the engulfment of senescent RBCs of mouse BM EBI macrophages dramatically decreases following GM-CSF treatment. This data suggests that GM-CSF treatment significantly inhibits the phagocytosis function of BM EBI macrophages by decreasing phagocytosis-associated molecule expression and polarizing macrophages into an M1-like phenotype.
Many proinflammatory cytokines (e.g., TNF-α, IFN-γ, IL-1β) are known to inhibit steady-state BM erythropoiesis [
63]. Heat-killed Brucella abortus are commonly used to induce AI in mouse models and have been reported to simultaneously decrease steady-state erythropoiesis in BM and to cause stress erythropoiesis in the spleen [
10,
11,
63]. Our study demonstrates that the expression of TNF-α, and IL-1β on BM macrophages also increases upon GM-CSF treatment. TNF-α and IL-1β have been shown to inhibit the proliferation and differentiation of erythroid cells in BM [
64,
65] while also promoting the expansion and differentiation of stress erythroid progenitors (SEPs) in the spleen in the mouse model of AI [
10,
11,
63]. However, although our data indicates that GM-CSF single injection leads to inhibition of BM steady-state erythropoiesis, it does not induce stress erythropoiesis in the spleen. Overall, these data provide new insights into the complexities of BM and spleen erythroid cells and the ways in which the unique BM and spleen microenvironment affect their maturation.
In summary, we have identified previously unknown roles of proinflammatory cytokine GM-CSF in erythropoiesis. Based on our findings, we propose that GM-CSF significantly impairs erythropoiesis by affecting the functioning of EBI macrophages. This conclusion is supported by several lines of evidence. (1) GM-CSF impairs human and mouse EBI formation by decreasing the expression of adhesion molecule CD163. (2) GM-CSF reduces mouse BM erythroblast numbers by decreasing the numbers of EBI macrophages and EBI formation. (3) GM-CSF impairs phagocytosis of senescent RBCs by decreasing the engulfment-related expression of Mertk, Axl and Timd4. Although we cannot exclude the effect of GM-CSF on erythropoiesis through influencing on other myeloid cells, our findings nonetheless provide new insights into GM-CSF’s role in impairing terminal erythroid differentiation at least in part by affecting EBI macrophages, and our findings can help better understanding of elevated GM-CSF levels of inflammatory diseases. Targeting GM-CSF or GM-CSF inhibitors might be a novel option for treating AI. This conclusion must be confirmed in mice in which the GM-CSFR gene is specifically depleted by EBI macrophages.
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