Fms-like tyrosine kinase-3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse

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1 Accepted Manuscript Fms-like tyrosine kinase-3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse Rebecca N. Jacobsen, Bianca Nowlan, Marion E. Brunck, Valerie Barbier, Ingrid G. Winkler, Jean-Pierre Levesque PII: DOI: S31-472X(15)741-9 Reference: EXPHEM /j.exphem To appear in: Experimental Hematology Received Date: 4 August 215 Revised Date: 15 October 215 Accepted Date: 5 November 215 Please cite this article as: Jacobsen RN, Nowlan B, Brunck ME, Barbier V, Winkler IG, Levesque J- P, Fms-like tyrosine kinase-3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse, Experimental Hematology (215), doi: 1.116/j.exphem This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Fms-like tyrosine kinase-3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse Rebecca N Jacobsen a,c, Bianca Nowlan a, Marion E Brunck a, Valerie Barbier b, Ingrid G Winkler b, Jean-Pierre Levesque a,c a Stem Cell Biology Group, b Stem Cells and Cancer Group, Mater Research Institute University of Queensland, Woolloongabba, Queensland, Australia; c School of Medicine, University of Queensland, Herston, Queensland, Australia. Corresponding authors: Jean-Pierre Levesque Mater Research Intitute The University of Queensland Translational Research Institute 37 Kent Street Woolloongabba, 412 Phone: Fax: jp.levesque@mater.uq.edu.au 1

3 Fms-like tyrosine kinase-3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse Rebecca N Jacobsen a,c, Bianca Nowlan a, Marion E Brunck a, Valerie Barbier b, Ingrid G Winkler b, Jean-Pierre Levesque a,c a Stem Cell Biology Group, b Stem Cells and Cancer Group, Mater Research Institute University of Queensland, Woolloongabba, Queensland, Australia; c School of Medicine, University of Queensland, Herston, Queensland, Australia. Corresponding authors: Jean-Pierre Levesque Mater Research Intitute The University of Queensland Translational Research Institute 37 Kent Street Woolloongabba, 412 Phone: Fax: jp.levesque@mater.uq.edu.au 1

4 ABSTRACT The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans and mice. We have recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. Herein we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3- L caused a whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However unlike G-CSF, this effect of Flt3-L on EI macrophages was indirect as Flt3 was not detected at the surface of EI macrophages or erythroid progenitors. 2

5 INTRODUCTION Erythropoiesis is a highly regulated process that takes place primarily in the bone marrow (BM) in adult mammals. This process is in part regulated by the erythroblastic island (EI) macrophage, a central macrophage surrounded by erythroblasts at varying stages of development [1, 2]. It has been proposed these macrophages play a role in iron transportation to erythroblasts [3, 4], secretion of erythroblastic survival cytokines [5, 6], synthesis of the heme, as well as phagocytosing and degrading the nuclei extruded by erythroblasts maturing into anucleated erythrocytes [7-9]. We have previously shown that mobilizing doses of recombinant human granulocyte colony-stimulating factor (G-CSF) inhibit red blood cell formation in the BM in vivo in mice by depleting erythroid island (EI) macrophages, defined phenotypically by positive expression of CD11b, F4/8, VCAM-1, CD169, ER-HR3 and Ly-6G cell surface antigens [1]. This depletion of EI macrophages was accompanied by an accumulation of pro-erythroblasts and a loss of all subsequent developmental red blood cell populations. Interestingly, an increase in splenic EI macrophage number and erythropoiesis was observed in these G-CSF-treated mice. Recently, it has been shown that the constitutive over expression of Flt3 ligand (Flt3-L) in transgenic mice leads to a decrease in the number of erythroid progenitors in the bone marrow and anemia (decreased hematocrit) [11]. Flt3 (CD135), the receptor for Flt3-L is expressed by a number of hematopoietic progenitor and stem cell (HSPC) populations [12-14]. Administration of Flt3-L for 5 1 days results in HSPC mobilization in primates [15], humans [16, 17] and mice [18-2]. In this report we investigated whether similar to G-CSF, the alternative HSPC mobilizing cytokine Flt3-L caused similar depletion of EI macrophages and inhibited red blood cell development in the bone marrow of treated mice. MATERIAL AND METHODS Mice and treatments All procedures had been approved by the Animal Experimentation Ethics Committee of the University of Queensland. C57BL/6 mice were purchased from the Animal Resource Centre (Perth, Australia). All experiments were performed on 6-8 week old mice. 3

6 Mice were injected intra-peritoneally daily with 1µg recombinant human Flt3- L-human IgG1 fusion protein (BioXCell, West Lebanon, NH) for 6 consecutive days, and sacrificed 24 hours following the last injection. Tissue sampling Mice were anesthetized and 1mL blood collected into tubes containing heparin and EDTA by cardiac exsanguinations. Red cells were lysed from the blood as previously described in [21]. Spleens were harvested, weighed and dissociated in 1mL Iscove modified Dulbecco s medium (IMDM) with 1% FCS using a GentleMACS Dissociator tissue homogenizer with matching C tubes (Miltenyi Biotec) on spleen 3 setting, twice. Femurs were cleaned of any remaining muscles with a scalpel and paper towel, BM cells were flushed out using a 21-gauge needle and a 1mL syringe containing 1mL PBS with 2% FCS. BM leukocytes were dissociated by successive pipetting with the mounted syringe. Colony assays Colony assays were performed as described in [22] and counted after 7 days of culture. Flow Cytometry Bone marrow and splenic cells were pelleted at 37g for 5 minutes at 4 C and resuspended in CD16/32 hybridoma 2.4G2 supernatant. For red blood cell staining, cells were stained with Ter119-PECy7, CD44-PE, CD71-FITC, CD45-APCCy7, and 5µM Hoescht33342 as previously described [1]. When Flt3 expression was examined, CD135-PECF594 antibody was used in combination with anti-ter119- FITC, CD44-APC and CD45-APCCY7. For EI macrophages staining, cells were stained with CD11b-Brilliant Violet 65, anti-ly6g-apccy7, anti-f4/8-a647, anti-vcam-1-pacific Blue, CD169- FITC, Ter119-PECy7, anti-flt3-pe, biotinylated ER-HR3 and streptavidin-pecf594 and gated as previously described [1]. Matched isotype controls for VCAM-1-Pacific Blue, CD169-FITC, biotinylated ER-HR3 and Flt3-PE were used to set positive gates. For HSPC staining, cells were stained with FITC conjugated lineage antibodies (CD3ε, CD5, B22, Gr-1, CD11b and Ter119), anti-sca1-pecy7, anti-mouse c-kit- 4

7 APCCy7, CD48 Pacific Blue and anti-mouse Flt3-PE. A matched isotype control for Flt3-PE was used to set positive gates. For myeloid progenitor staining, cells were stained with FITC conjugated lineage antibodies (CD3ε, CD5, B22, Gr-1, CD11b and Ter119), anti-sca1-pecy7, anti-mouse c-kit-apccy7, CD34-e66, CD16/32-PE, biotinylated anti-mouse IL7 receptor α chain (CD127), streptavidin-bv65 and anti-mouse Flt3-PECF594. In all stains, 5µg/ml 7-amino actinomycin D was added 15 minutes before acquisition to exclude dead leukocytes. Data were acquired on a CyAn (Dako Cytomation) flow cytometer and analysed following compensation with single colour controls using FloJo Software (Tree Star). RNA extraction and quantitative reverse transcription polymerase chain reaction (qrt-pcr) Bone marrow EI macrophages and HSPCs (Lineage neg Kit + Sca-1 + ) were sorted directly into 1.5ml of Trizol LS (Life Technologies). RNA was extracted following manufacturer s instructions. Reverse transcription was performed using SensiFAST cdna synthesis kit (Bioline) as per manufacturer s instructions. qrt-pcr was performed using Taqman Universal PCR master mix (ABI, Life Technologies) as previously described [1]. Primers were Taqman Gene Expression Assays (ABI) for Flt3 (Mm43916_m1), β2-microglobulin (B2m Mm437762_m1) and β-actin (Actb Mm125647_m1). Statistical Analysis All data are presented as mean ± standard deviation (SD). Statistical differences were calculated using a Student s t-test. RESULTS AND DISCUSSION C57BL/6 mice were administered human Flt3-L or saline daily for 6 days and BM, blood and spleen were harvested to measure HSPC mobilization, changes in RBC development and EI macrophage populations. Flt3-L treatment caused significant increase in white blood cell counts, spleen weight and cellularity (Supplementary figure 1 online only, available at Colony assays and flow cytometry staining for HSPC cell populations on blood and spleen of treated mice 5

8 confirmed that Flt3-L treatment caused mobilization of colony-forming units (CFU), Lin neg Sca1 + Kit + HSPC and Lin neg Sca1 + Kit + CD48 - CD15 + phenotypic HSC into blood and spleen (Supplementary figure 2). Femoral BM cell suspensions from Flt3-L treated mice were whitened compared to saline-treated control mice as previously reported for G-CSF treatment [1] (data not shown). We next examined the CD45 neg cell population stained with CD44 and Ter119 to delineate the different differentiation stages of erythroblasts, reticulocytes and erythrocytes as previously reported [23-25] (Fig 1A). A 3-fold decrease in the number of basophilic, polychromatic and orthochromatic erythroblasts as well as reticulocytes was observed in the BM of treated mice (Fig 1B), however the number of mature red blood cells was not significantly changed. The number of CD44 + Ter119 low pro-erythroblasts was unchanged by Flt3-L treatment (Fig 1B) unlike G-CSF treatment [1]. Macrophage populations in the BM were examined following Flt3-L treatment to assess changes to the EI macrophage population (Fig 1C). Phenotypic EI macrophages, defined as CD11b + F4/8 + VCAM-1 + CD169 + ER-HR3 + Ly6G + [1], were decreased 12-fold (Fig 1D) in the BM of Flt3L treated mice. No significant change to the CD11b + F4/8 + VCAM-1 + CD169 + ER-HR3 + Ly6G neg non-ei macrophage population was found. Therefore Flt3-L reduces medullary erythropoiesis and number of EI macrophages in a similar manner to the other HSC mobilizing agent G-CSF [1]. We next determined the effect of Flt3-L on splenic erythropoiesis. Flt3-L treatment increased the number of pro-erythroblasts (5 fold), orthochromatic erythroblasts (2.6 fold), reticulocytes (2.7 fold) and mature red blood cells (3 fold) in the spleen (Figs 1A and 1B). In conjunction with this increase in erythropoiesis, the splenic EI macrophage population was increased 1.7 fold, as well as non-ei macrophages (Ly6G neg ) (1.5 fold) (Figs 1C and 1D). Therefore, similar to mobilizing doses of G-CSF [1], Flt3-L exerted opposite effects on erythropoiesis and EI macrophages depending on the tissue considered, with a suppressive effect in the BM and a stimulatory effect in the spleen. As a result of this splenic compensation, Flt3-L treated mice were not anemic after a 6 day treatment (Supplementary figure 1B). In order to determine whether the suppressive effect of Flt3-L on EI macrophages was direct, we stained them for expression of Flt3, the only known receptor of Flt3-L. We could not detect Flt3 receptor at the surface of EI macrophages 6

9 (Fig 1E) or at the surface of any other F4/8 + macrophage in the BM (data not shown). Absence of Flt3 protein expression on EI macrophages was confirmed at the RNA level, by the very low level of Flt3 mrna detected by qrt-pcr in sorted EI macrophages whilst it was readily detected in sorted Lin neg Kit + Sca1 + HSPC (Fig 1F). We further delineated Flt3 expression during myeloid differentiation by flow cytometry on BM cells from untreated mice (Supplementary figure 3). As expected, 37±4 % of Lin - Kit + Sca1 + HSPCs were positive for cell surface Flt3 as previously reported [12, 26]. Flt3 was also expressed on 12±1% of common myeloid progenitors (Lin - IL7Rα - Kit + Sca1 - CD34 + CD16/32 - ) but was absent from the surface of granulocyte-monocyte progenitors (Lin - IL7Rα - Kit + Sca1 - CD34 + CD16/32 - ). Therefore the suppressive effect of Flt3-L on EI macrophages is likely to be indirect. Finally we assessed expression of Flt3 on BM erythroid cells by flow cytometry. 3.5±.5% megakaryocyte-erythrocyte progenitors (Lin - IL7Rα - Kit + Sca1 - CD34 - CD16/32 - ) expressed Flt3 (Supplementary figure 3). However none of the Ter119 + erythroid populations (from pro-erythroblast to reticulocytes) expressed surface Flt3 at the cell surface (Supplementary figure 4). Therefore the suppressive effect of Flt3-L treatment on medullary erythropoiesis is unlikely to be due to a a direct effect of Flt3-L on erythroblasts. In conclusion, mobilizing doses of Flt3-L have very similar effects on medullary and splenic erythropoiesis to those reported for G-CSF with suppression of medullary EI macrophages and erythropoiesis compensated by increased erythropoiesis and EI macrophages in the spleen. This may explain why transgenic mice overexpressing Flt3-L become anemic over time [11]. However, there are some important differences between medullar anemia caused by G-CSF and Flt3-L. Indeed, unlike G-CSF [1] the Flt3-L-induced suppression of medullary erythropoiesis was not accompanied by accumulation of pro-erythroblasts (Fig 1B). In addition, while the effect mediated by G-CSF may be direct since EI macrophages express its cognate receptor Csf3r [1], the effect of Flt3-L is indirect as EI macrophages (Fig. 1E) and granulocyte-monocyte progenitors (Supplementary Figure 3) do not express Flt3. As pro-erythroblasts, erythroblasts or reticulocytes do not express Flt3 either (Supplementary Figure 4), the suppressive effect of Flt3-L on erythropoiesis is likely to be mediated indirectly via suppression of EI macrophages. The small subset of megakaryocyte-erythrocyte progenitors that was positive for Flt3 is unlikely to be responsible for erythropoiesis suppression as the number of pro-erythroblasts in the BM was not reduced by Flt3-L 7

10 treatment. As Flt3-L stimulates dendritic cell development [17, 27], the effect of Flt3- L on EI macrophages may be indirectly mediated by other Flt3 + cells such as dendritic cells or other lympho-myeloid progenitors. Further mechanistic studies to understand how Flt3-L suppresses medullary EI macrophages and erythropoiesis are warranted. As Flt3-L is being contemplated as an alternative mobilizing agent for HSCs and dendritic cells [15, 17], its potential effect on erythropoiesis is of relevance. Finally, in light of these results it would also be interesting to examine the effect on erythropoiesis of other HSPC mobilizing cytokines used in the clinic such as Kit ligand / stem cell factor [28-3]. Acknowledgements This work was supported by Project Grant from the National Health and Medical Research Council of Australia (to JPL, ARP and IGW). RNJ was supported by an Australian Post-graduate Award from the Commonwealth of Australia, JPL and IGW are supported by a Senior Research Fellowship #14491 and a Career Development Fellowship # respectively from the National Health and Medical Research Council. Conflict of interest disclosure The authors have no conflict of interest to declare. 8

11 REFERENCES 1 Bessis M. L'ilot erythroblastique. Unite fonctionelle de la moelle osseuse. Rev Hematol. 1958;13: Chasis JA, Mohandas N. Erythroblastic islands: niches for erythropoiesis. Blood. 28;112: Bessis MC, Breton-Gorius J. Iron Metabolism in the Bone Marrow as Seen by Electron Microscopy: A Critical Review. Blood. 1962;19: Leimberg MJ, Prus E, Konijn AM, Fibach E. Macrophages function as a ferritin iron source for cultured human erythroid precursors. J Cell Biochem. 28;13: Manwani D, Bieker JJ. The Erythroblastic Island. In: James JB, ed. Current Topics in Developmental Biology: Academic Press; 28. p Sawada K, Krantz SB, Dessypris EN, Koury ST, Sawyer ST. Human colonyforming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development. J Clin Invest. 1989;83: Skutelsky E, Danon D. On the expulsion of the erythroid nucleus and its phagocytosis. Anat Rec. 1972;173: Soni S, Bala S, Gwynn B, Sahr KE, Peters LL, Hanspal M. Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion. J Biol Chem. 26;281: Jacobsen RN, Perkins AC, Levesque JP. Macrophages and regulation of erythropoiesis. Curr Opin Hematol. 215;22: Jacobsen RN, Forristal CE, Raggatt LJ, et al. Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/8+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse. Exp Hematol. 214;42: e Tsapogas P, Swee LK, Nusser A, et al. In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development. Haematologica. 214;99: Adolfsson J, Mansson R, Buza-Vidas N, et al. Identification of Flt3+ lymphomyeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell. 25;121: Boyer Scott W, Schroeder Aaron V, Smith-Berdan S, Forsberg EC. All Hematopoietic Cells Develop from Hematopoietic Stem Cells through Flk2/Flt3- Positive Progenitor Cells. Cell Stem Cell. 211;9: Beaudin AE, Boyer SW, Forsberg EC. Flk2/Flt3 promotes both myeloid and lymphoid development by expanding non-self-renewing multipotent hematopoietic progenitor cells. Exp Hematol. 214;42: e Papayannopoulou T, Nakamoto B, Andrews RG, Lyman SD, Lee MY. In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor. Blood. 1997;9:

12 16 Lebsack ME, McKenna HJ, Hoek J, Feng A, Maraskovsky E, Hayes FA. Flt-3 ligand (Mobist) administered in combination with GMCSF or G-CSF to healthy volunteers. J Clin Oncol. 1998;17:78 (abstract). 17 Anandasabapathy N, Breton G, Hurley A, et al. Efficacy and safety of CDX-31, recombinant human Flt3L, at expanding dendritic cells and hematopoietic stem cells in healthy human volunteers. Bone Marrow Transplant. 215;5: Molineux G, McCrea C, Yan XQ, Kerzic P, McNiece I. Flt-3 ligand synergizes with granulocyte colony-stimulating factor to increase neutrophil numbers and to mobilize peripheral blood stem cells with long-term repopulating potential. Blood. 1997;89: Sudo Y, Shimazaki C, Ashihara E, et al. Synergistic Effect of FLT-3 Ligand on the Granulocyte Colony-Stimulating Factor Induced Mobilization of Hematopoietic Stem Cells and Progenitor Cells Into Blood in Mice. Blood. 1997;89: de Kruijf E-JFM, Hagoort H, Velders GA, Fibbe WE, van Pel M. Hematopoietic stem and progenitor cells are differentially mobilized depending on the duration of Flt3-ligand administration. Haematologica. 21;95: Levesque JP, Hendy J, Winkler IG, Takamatsu Y, Simmons PJ. Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-kit receptor (CD117) from the surface of hematopoietic progenitor cells. Exp Hematol. 23;31: Winkler IG, Sims NA, Pettit AR, et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 21;116: Kingsley PD, Greenfest-Allen E, Frame JM, et al. Ontogeny of erythroid gene expression. Blood. 213;121:e5-e Chen K, Liu J, Heck S, Chasis JA, An X, Mohandas N. Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis. Proc Nat Acad Sci USA. 29;16: Fraser ST, Midwinter RG, Coupland LA, et al. Heme oxygenase-1 deficiency alters erythroblastic island formation, steady-state erythropoiesis and red blood cell lifespan in mice. Haematologica. 215;1: Adolfsson J, Borge OJ, Bryder D, et al. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity. 21;15: Ding Y, Wilkinson A, Idris A, et al. FLT3-Ligand Treatment of Humanized Mice Results in the Generation of Large Numbers of CD141+ and CD1c+ Dendritic Cells In Vivo. The Journal of Immunology. 214;192: Briddell R, Hartley C, Smith K, McNiece I. Recombinant rat stem cell factor synergizes with recombinant human granulocyte colony-stimulating factor in vivo in mice to mobilize peripheral blood progenitor cells that have enhanced repopulating potential. Blood. 1993;82: Roberts MM, Swart BW, Simmons PJ, Basser RL, Begley CG, To LB. Prolonged release and c-kit expression of haemopoietic precursor cells mobilized by stem 1

13 cell factor and granulocyte colony stimulating factor. Br J Haematol. 1999;14: To LB, Bashford J, Durrant S, et al. Successful mobilization of peripheral blood stem cells after addition of ancestim (stem cell factor) in patients who had failed a prior mobilization with filgrastim (granulocyte colony-stimulating factor) alone or with chemotherapy plus filgrastim. Bone Marrow Transplant. 23;31:

14 FIGURE LEGENDS Figure 1. Flt3-L blocks medullary erythropoiesis and reduces the number of erythroid island macrophages in the bone marrow. A) Representative flow dot-plots showing alterations in erythroid subsets in the BM of mice following 6 days of treatment with either saline (Sal) or Flt3-L (Flt3L). The left dot-plots show Ter119 vs CD44 among gated CD45 neg cells. Proerythroblasts were gated as CD44 + Ter119 low (population I). Within the CD45 neg Ter cell population, basophilic erythroblasts (population II), polychromatic erythroblasts (population III), orthochromatic erythroblasts (population IV), reticulocytes (population V) and erythrocytes (population VI) were identified according to their forward scatter (FSC) and CD44 expression. B) Quantitation of erythroid populations in the femoral BM after 6 days of saline or Flt-3 ligand treatment. C) Representative flow dot-plots showing changes in myeloid cell populations in the BM gated by expression of CD11b, F4/8, VCAM-1, CD169, ERHR3 and Ly6G antigens, after 6 days of treatment with either Saline or Flt3L. D) Quantification of macrophage populations in the BM of treated mice following 6 days of either Saline or Flt3L treatment. E) Expression of Flt-3 on BM HSPC, defined as Lin neg Kit + Sca-1 +, and EI macrophages (black line) with the isotype control shaded in gray. F) Quantification of Flt-3 mrna in HSPC and EI macrophages sorted from the BM of untreated mice. Data are mean ± SD. n = 4 mice per group. Differences were evaluated with a t-test *p.5; **p.1 Figure 2. Flt-3 ligand does not inhibit erythropoiesis in the spleen. A) Representative flow dot-plots showing changes to developing red blood cell populations in the spleen of mice following 6 days of treatment with either saline (Sal) or Flt3-L. The left dotplots show Ter119 vs CD44 among gated CD45 neg cells. Pro-erythroblasts were gated as CD44+ Ter-119low (population I). Within the CD45 neg Ter cell population, basophilic erythroblasts (population II), polychromatic erythroblasts (population III), orthochromatic erythroblasts (population IV), reticulocytes (population V) and erythrocytes (population VI) were identified according to their forward scatter (FCS) and CD44 expression. B) Quantitation of erythroid populations in the spleen after 6 days of saline or Flt-3 ligand treatment. C) Representative flow dot-plots showing changes in myeloid cell populations in the spleen gated by expression of CD11b, F4/8, VCAM1, CD169, ERHR3 and Ly6G antigens, after 6 days of treatment with 12

15 either Saline or Flt3L. D) Quantification of changes in macrophage populations in the spleen of treated mice following 6 days of either Saline or Flt3L treatment. Data are mean ± SD. n = 4 mice per group. Differences were evaluated with a t-test *p.5; **p.1 ***p.1. 13

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18 WBC 1 3 / µl Sal WBC * Flt-3L Spleen weight, mg RBC 1 6 / µl Sal RBC Spleen weight Sal *** Flt-3L Flt-3L HGb, g / dl Splenocytes 1 6 / spleen Sal HGb Flt-3L Spleen cellularity Sal *** Flt-3L PLT 1 3 / µl 1,2 1, Sal PLT * Flt-3L Supplementary Figure 1: Blood (A) and spleen (B) of mice treated with Flt3-L. A) Quantification of the number of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGb) and platelets (PLT) in peripheral blood of mice treated with saline (Sal) or Flt3-L. B) Effect of Fl3-L treatment on spleen weight and cellularity. Data represent mean ± SD of 4 mice per experimental group. * p <.5; **.1 < p <.1; *** p <.1.

19 A CFU/ml Blood CFU/Spleen B C No. of Colonies Cells/ml Blood Cells/Spleen 9, 8, 7, 6, 5, 4, 3, 2, 1, 16, 14, 12, 1, 8, 6, 4, 2, 45, 4, 35, 3, 25, 2, 15, 1, 5, Sal Flt-3L Lin neg Sca + Kit + Sal Flt-3L Lin neg Sca + Kit + Sal Flt-3L No. of Colonies Cells/ml Blood 25, *** *** Cells/Spleen 2, 15, 1, 5, Sal Flt-3L Lin neg Sca + Kit + Slam + CD48 neg 1,6 1,4 1,2 1, Sal Flt-3L Lin neg Sca + Kit + Slam + CD48 neg Sal Flt-3L Cells/ml Blood Cells/Spleen 8, 7, 6, 5, 4, 3, 2, 1, Lin neg Sca neg Kit + ** ** *** 18, 16, 14, 12, 1, 8, 6, 4, 2, Sal Flt-3L Lin neg Sca neg Kit + * * ** Sal Flt-3L Supplementary Figure 2: Flt-3 ligand induced mobilization in mice. A) Quantification of the number of colony forming units (CFU) per ml of blood and per spleen in mice after 6 days of treatment with either saline or Flt3 ligand. B) Change in the number of phenotypic HSPCs found in the blood and C) spleen of mice after 6 days of treatment with either saline or Flt3 ligand. Data represent mean ± SD of 4 mice per experimental group. * p <.5; **.1 < p <.1; *** p <.1.

20 A LKS- LKS+ 1 3 GMP IL7Rα % of Max % of Max B Lineage LKS Flt3 MEP Flt3 4.3 % of Max Kit CMP Sca C Flt3 Flt3+ cells, % of total % of Max CD16/ MEP GMP CD34.53 CMP Flt3 LKS+ CMP GMP MEP Supplementary Figure 3: Expression of Flt3 through myeloid differentiation stages in the adult mouse BM. A) Gating strategy to identify HSPCs (Lin - IL7Rα - Kit + Sca-1 + ) and myeloid progenitors included in the (Lin - IL7Rα - Kit + Sca-1 - gate. B) Overlay of cells stained anti-flt3 antibody (think line curve) and control stain without Flt3 antibody (grey shaded area) on LKS+ HSPCs, common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP). C) Proportion of Flt3 + cells within each indicated population. Data represent mean ± SD of 3 untreated mice.

21 A I CD CD CD V IV III II B % of Max % of Max C I Ter Flt3 IV Flt3 Flt3+ cells, % of total K 2K 3K II FSC Flt3 1K 2K 3K I II III IV V VI V Flt3 8.75e III VI VI FSC Flt Flt3 Supplementary Figure 4: Expression of Flt3 through erythroid differentiation stages in the adult mouse BM. A) Gating strategy to identify pro-erythroblasts were gated as (population I), basophilic erythroblasts (population II), polychromatic erythroblasts (population III), orthochromatic erythroblasts (population IV), reticulocytes (population V) and erythrocytes (population VI) according to decreasing forward scatter and CD44 expression within Ter119 + erythroid cells. B) Overlay showing staining with anti-flt3 antibody (line curve) and control stain without Flt3 antibody (grey shaded area) C) Proportion of Flt3 + cells within each indicated population. Data represent mean ± SD of 3 untreated mice.

22 Highlights: Daily administration of Flt3 ligand in mice depletes erythroblastic island macrophages and inhibits erythropoiesis in the bone marrow. Suppression of medullary erythropoiesis is compensated by increase in erythroblastic island macrophage numbers and erythropoiesis in the spleen. The suppressive effect of Flt3 ligand on medullary erythroblastic island macrophages and erythropoiesis is likely to be indirect as Flt3 receptor is not expressed by macrophages or erythroid progenitors.