The 8–12 week-old male C57BL/6 mice were obtained from the Laboratory Animal Center of National Yang Ming Chiao Tung University (Taipei Yang Ming Campus). D-(+)-Glucosamine hydrochloride (purity 99.9%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). D-(+)-Glucosamine hydrochloride dissolved in phosphate-buffered saline (PBS) was administered via an intraperitoneal (i.p.) injection (30 mg/mouse/day) into the mice for 14 days according to a previous report (20) or implanted subcutaneously using Alzet model 1002 osmotic pumps (1 M, 0.25 μL/h) (DURECT Corp., Cupertino, CA, USA) to maintain a steady GlcN concentration. After 14-day of GlcN treatment, mice were sacrificed with carbon dioxide inhalation. Mice treated with PBS were used as control. Complete blood count analysis was performed on a XN-450 hematology analyser (Sysmex, Kobe, Hyogo, Japan). All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University (Taipei Yang Ming campus) (IACUC number: 1081005).

Bone marrow (BM) cells were carefully isolated from the tibia and femur. For spleen (SP) cell preparation, a small piece of excised SP was pressed through a strainer using the plunger end of a syringe. Subsequently, BM and SP cells were washed through the strainer and collected in PBS containing 2% fetal bovine serum (FBS), which was obtained from Gibco (Grand Island, NY, USA), for immediate use (21, 22).

For hematopoietic cell analysis, flow cytometric analyses were performed as previously described (23). Briefly, antibodies specific for the following surface antigens were purchased from BD Biosciences (Franklin Lakes, NJ, USA): B220 (RA3-6B2), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), CD34 (RAM34), CD41 (MWReg30), CD45 (30-F11), CD48 (HM48-1), CD150 (Q38-480), FcγRIII/FcγRII (2.4G2), Gr-1 (RB6-8C5), IgM (R6-60.2), IL-7Ra (B12-1), c-Kit (2B8), Ly-6C (AL-21), Ly-6G (1A8), Sca-1 (D7), streptavidin and Ter119 (TER-119). CD19 (1D3) and Thy1.2 (53-2.1) were purchased from eBioscience (San Diego, CA, USA) (The detail antibody panels for detecting different hematopoietic cells can be found in Supplementary Tables 1–4). For detection of reactive oxygen species, cells were stained with 5 μM 2′,7′-dichlorofluorescin diacetate (Merck, Darmstadt, Germany). Flow cytometry analyses were performed on a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) and results were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

For isolating Lin⁻c-Kit⁺Sca-1⁺ (LSK) cell population, which expresses Sca-1 and c-Kit but lacks the lineage (Lin) markers expressed on mature myeloid and lymphoid cells, myeloid progenitors (MP, Lin⁻c-Kit⁺Sca-1⁻) and granulocyte-macrophage progenitors (GMP, Lin⁻c-Kit⁺Sca-1⁻CD34⁺FcRII/III⁺), BM were extracted and stained with antibodies (Supplementary Table 2). The c-Kit⁺ cells were enriched firstly by using anti-allophycocyanin (APC) Magnetic Particles (E30-221), which was obtained from BD Bioscience. Then, LSK, MP, and GMP cells were sorted from the c-Kit⁺ cell fraction using BD FACS Melody cell sorter (BD Biosciences). The sorted cells were washed with cold PBS twice, and the RNA was extracted using RNeasy Plus Mini Kit (Qiagen).

For detecting phosphorylated proteins in CD11b⁺Gr-1⁺ cells, cell treatment and flow cytometric analysis were modified according to the previous report (24). BM cells were deprived of serum and cytokines at least 30 min. Then, cells were stimulated with GlcN, collected at the indicated time points, and fixed immediately with 4% paraformaldehyde (Merck, Darmstadt, Germany). The ice-cold 100% methanol (Merck) was used for permeabilization. Cells were blocked with mice Fc Blocker for 15 min, and stained with directly conjugated CD11b, Gr-1, ERK1/2-pT202/pY204 (20A) and Stat3-pY705 (4/P-STAT3) antibodies (BD Biosciences) (Supplementary Table 5) for 30 min. Flow cytometry analyses were performed on a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) and results were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

BM cells were resuspended in RPMI 1640 (Gibco) containing 10% FBS, 10 ng/mL interleukin (IL)-6, 10 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF), 10 ng/mL granulocyte (G)-CSF, 0.2 ng/mL transforming growth factor-β, and 50 μM 2-mercaptoethanol at 1 × 10⁶ cells/mL. Next, 0.5 mL resuspended cells were cultured in a 24-well plate. GlcN were added to the cells and incubated at 37°C and 5% CO₂ for 72 h. The cells were washed and stained with direct conjugated antibodies.

The methylcellulose-based medium MethoCult™ M3434 was obtained from StemCell Technologies (Vancouver, Canada). Colony-forming assays were performed as previously described (22). To detect burst-forming unit-erythroids and other types of colonies, including oligopotential progenitor colony-forming unit (CFU)-granulocytes; erythrocytes, monocytes/macrophages, and megakaryocytes (GEMM); lineage-restricted progenitor CFU-granulocyte and monocytes/macrophages (GM); and precursors of granulocytes (G) and monocytes/macrophages (M), 2 × 10⁴ BM cells were plated in duplicate on MethoCult™ M3434 methylcellulose medium according to the manufacturer's protocol. Colonies were counted after 10 days of culture.

RNA isolation and first-strand cDNA synthesis were performed using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) and RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocols. The indicated transcripts in cDNA samples were quantified using Fast SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The reaction and signal detection were measured with a QuantStudio 3 Real-Time PCR System (Applied Biosystems). The primer sets used for gene detection are listed in Table 1. The gene expression level relative to that of GAPDH was calculated using the difference in threshold cycle method for reverse transcription-quantitative PCR analysis.

To analyze the immunosuppressive activity of MDSCs, the assay was performed according to the previous report (25). Murine CD11b⁺Gr1⁺ MDSCs were isolated from BM cells by using BD FACS Melody cell sorter. Murine splenocytes were incubated with 5 uM carboxylfluorescein succinimidyl ester (CFSE) (Biolegend, San Diego, CA, USA) for 5 min, and then washed with RPMI 1640 twice. The sorted CD11b⁺Gr1⁺ MDSCs were immediately co-cultured with CFSE-stained splenocytes at a density of 5 × 10⁵ cells per well at a ratio of 1:4 (MDSCs:T cells) in the presence of anti-CD3 (145-2C11) and anti-CD28 (37.51) antibodies, which were purchased from BD Biosciences and used for T cell activation. After 48 h, T-cell proliferation was evaluated by CFSE dilution by flow cytometry. The suppressive ability of MDSCs was expressed as percentage of CD8⁺ T cell proliferation (Supplementary Table 6).

To examine the influence of GlcN in ERK1/2 and STAT3 activation, the CD11b⁺ cells were isolated from BM cells by using anti-APC Magnetic Particles (see above). The sorted CD11b⁺ cells were incubated at 37°C in IMDM medium supplemented with 1% BSA for at least 30 min. Then, sorted CD11b⁺ cells (1.5 × 10⁶ cells/mL) were treated with 1 mM GlcN for 0, 5, 15, and 30 min. Cells were collected, and lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 0.1% SDS; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 10 μg/ml aprotinin; 10 μg/ml leupeptin; 10 μg/ml pepstatin; 1% sodium deoxycholate; 1 mM sodium fluoride; 1 mM sodium orthovanadate; 25 mM β-glycerophosphate). All reagents used for RIPA buffer preparation were purchased from Sigma-Aldrich. Western blot was performed with the following antibodies: Anti-phospho-STAT3 antibody (Tyr705) (M9C6), anti-STAT3 antibody (79D7), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (polyclonal) and anti-p44/42 MAPK antibody (polyclonal). All of these antibodies were purchased from Cell Signaling Technology. Detections were performed using Clarity Western ECL Substrate (Bio-Rad, USA), and digital images were captured by using Amersham Imager 680 (GE Healthcare Lifescience, Chicago, IL, USA). For detection of phosphorylated form and total proteins on the same membrane, the phosphorylated proteins were firstly detected, and then the membrane was stripped by using stripping reagent (Bionovas, Toronto, Ontario, Canada) before re-probing with another antibody.

Statistical analyses were performed using GraphPad Prism 6 software (GraphPad, Inc., La Jolla, CA, USA). For two groups, Student's t test was used; for multiple groups, one-way analysis of variance followed by Tukey multiple comparison test was used. Values are presented as the mean ± standard deviation. p < 0.05 was considered to indicate statistically significant results.

To determine the effect of GlcN on hematopoietic reprogramming, GlcN was delivered into C57BL/6 mice using two different methods, i.p. injection according to a previous report (20) and osmotic pump release for maintaining a steady GlcN concentration (Figure 1A). Supplementation with GlcN did not change the body weight or fasting glucose level of the mice after i.p. injection (Figure 1B). The white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin level, and platelet count of the mice were not affected by treatment with GlcN by i.p. injection (Figure 1C) and pump release (Figure 1D). Notably, the frequency of CD11b⁺Gr1⁺ MDSCs in the peripheral blood (PB) were elevated after GlcN supplementation via i.p. injection, whereas that of CD11b⁺Gr1⁻ myeloid cells were not (Figure 1E). Similar results were observed in the pump release group (Figure 1F).

Next, polymorphonuclear and monocytic subpopulations of MDSCs (defined as PMN- and M-MDSCs, respectively) in the CD11b⁺ myeloid cell compartments were examined by flow cytometry (Figure 2A). Similar to the results for CD11b⁺Gr1⁺ MDSCs (Figures 1E,F), both PMN- and M-MDSCs were elevated in the PB of mice following i.p. injection of GlcN (Figure 2B). Consistent with the results for the i.p. injection method, GlcN delivery via pump release elevated PMN- and M-MDSCs in the PB (Figure 2E). However, 14-day i.p. GlcN treatment did not further increase PMN- and M-MDSCs in BM (Figure 2C) and SP (Figure 2D). The in vitro myeloid cell differentiation assay using murine BM cells supported that GlcN enhanced the production of CD11b⁺Gr1⁺ MDSCs (Figure 2F). Moreover, GlcN supplementation favored the production of PMN-MDSCs (Figure 2G) rather than of M-MDSCs in vitro in our differentiation model (Figure 2H). These results suggest that GlcN treatment interferes with myelopoiesis to promote MDSC production.

As MDSCs are a heterogeneous population of immature myeloid cells, we focused on whether GlcN supplementation affected HSPC compartment and myelopoiesis-related gene expression (Figure 3A). To determine the impact of GlcN supplementation on HSPCs, we examined different subpopulations of HSPCs isolated from the BM of injected mice. GlcN significantly elevated the population of hematopoietic stems cells (HSCs), which locate at the top of the hematopoietic hierarchy, as well as multipotent progenitor cells (MPPs) in BM (Figure 3C). The representative flow cytometric figures and gating strategies for HSC and MPP analysis were shown in Figure 3B. HSCs and MPPs are contained in the Lin⁻c-Kit⁺Sca-1⁺ (LSK) cell population, which expresses Sca-1 and c-Kit but lacks the lineage (Lin) markers expressed on mature myeloid and lymphoid cells. LSK cells and the following myeloid progenitor cells: total myeloid progenitors (MPs, Lin⁻c-Kit⁺Sca-1⁻), common myeloid progenitors (CMPs, Lin⁻c-Kit⁺Sca-1⁻CD34⁺FcRII/III⁻), granulocyte-macrophage progenitors (GMPs, Lin⁻c-Kit⁺Sca-1⁻CD34⁺FcRII/III⁺), and megakaryocyte-erythroid progenitors (MEP, Lin⁻c-Kit⁺Sca-1⁻CD34⁻FcRII/III⁻) in the BM were analyzed by flow cytometry (Figure 3D). Consistent with the results of HSCs and MPPs, GlcN significantly elevated the population of various types of hematopoietic progenitor cells (Figure 3E).

Previous studies indicated glucose transporters facilitate GlcN transport (3, 26). In hematopoietic system, differently expressed Glut1 and 4 affect glucose uptake during erythropoiesis (27), and disruption of Glut1 in HSCs interferes myelopoiesis (28). Thus, LSKs, MPs and GMPs in BM were isolated from the pump release groups, and the expression of Glut1, 2, 3, and 4 were analyzed (Figure 4A). Our results indicated that GlcN treatment significantly increase Glut1 expression in LSKs (Figure 4B). Because GlcN treatment expanded MDSCs in the PB, the critical transcription factors and cytokines involved in myelopoiesis (Figure 3A) were analyzed. GlcN treatment significantly suppressed the expression of these transcription factors, including PU.1, GATA1, C/EBPα, C/EBPβ, IRF8, Rb, c-Jun, as well as cytokines, including IL-6, GM-CSF, and G-CSF in BM cells (Figure 5). These results suggest that HSPCs might uptake GlcN through Glut1, and then GlcN weakens the differentiation function of HSPCs.

To further examine whether GlcN affected the proliferation and differentiation capacity of HSPCs, BM cells isolated from control or GlcN-treated mice were subjected to colony-forming unit (CFU) analysis. Consistent with the transcription factor expression, the ability of HSPCs to form CFU-granulocytes, erythrocytes, monocytes/macrophages, and megakaryocytes (GEMM); CFU-granulocytes and monocytes/macrophages (GM); and CFU-granulocytes (G) were weakened. However, the CFU-monocytes/macrophages (M) were not affected. Interestingly, the earlier erythroid progenitors, burst-forming unit erythroid (BFU-E) cells, were decreased, whereas more mature erythroid progenitor cells, named as CFU-erythrocytes (E), were not affected in GlcN-supplemented mice (Figure 6).

To study the impact of GlcN on MDSC functions, genes associated with MDSC functions were examined. Cyclooxygenase 2 (COX2) is an important upstream regulator of Arg-1 and iNOS in MDSCs (29). In vivo GlcN treatment increased the expression of Arg-1, iNOS, and COX2 in BM cells (Figure 7A), as well as in CD11b⁺Gr1⁺ MDSCs isolated from the BM (Figure 7B). Since the increase of Arg-1 might contribute to ROS accumulation in cells, ROS levels in PMN- and M-MDSCs, which were isolated from BM, SP and PB in GlcN treated mice, were analyzed. In vivo GlcN treatment elevated ROS levels in both PMN-MDSC and M-MDSCs (Figures 7C,D).

To further examine the impact of GlcN on the immunosuppressive ability of MDSCs, MDSC-mediated suppression in CD8⁺ T cell proliferation was assessed. After 48 h of co-culture of CD11b⁺Gr1⁺ MDSCs and splenocytes, GlcN significantly enhanced the suppressive ability of MDSCs in a dose-dependent manner (Figure 7E). Previous results indicated that the expression of programmed death-ligand 1 (PD-L1) on MDSCs is involved in MDSC-mediated T-cell suppression (30). However, GlcN treatment did not influence PD-L1 expression (Figure 7F). These results indicate that GlcN enhanced the immunosuppressive ability of MDSCs through ROS production, but did not affect PD-L1 expression.

STAT3 and ERK1/2 signaling pathways are involved in the MDSC-mediated suppressive function (31, 32). To determine whether GlcN treatment influenced these two signaling pathways to modulate MDSC functions, the phosphorylation of STAT3 and ERK1/2 in MDSCs was analyzed. BM cells were stimulated with GlcN (1 mM) for 5, 10, and 30 min, and the phosphorylation of STAT3 (Figure 8A) and ERK1/2 (Figure 8B) was examined by flow cytometry. Phosphorylation of STAT3 reached a peak after 5 min of GlcN stimulation in CD11b⁺Gr1⁺ MDSCs and CD11b⁺Gr1⁻ myeloid cells, but not in non-myeloid cells (CD11b⁻Gr1⁻ cells) (Figure 8A). GlcN stimulation also increased the phosphorylation of ERK1/2 in CD11b⁺Gr1⁺ MDSCs, CD11b⁺Gr1⁻ myeloid cells, and CD11b⁻Gr1⁻ non-myeloid cells (Figure 8B). Furthermore, Western analysis was used to confirmed the results from flow cytometry. The GlcN treatment increased the phosphorylation of STAT3 and ERK1/2 in sorted CD11b⁺ cells (Figure 8C).

The AG490 (JAK2 inhibitor) and PD98059 (MEK inhibitor) have been extensively used to inhibit the JAK2–STAT3 and MEK–ERK1/2 pathways, respectively. Inhibition of these pathways reversed GlcN-mediated activation of STAT3 and ERK1/2, respectively (Figures 8D,E). Furthermore, inhibition of either the JAK2/STAT3 or MEK/ERK1/2 pathway downregulated GlcN-promoted ROS production in CD11b⁺Gr1⁺ MDSCs (Figure 8F). Our results clearly demonstrated that GlcN activates the STAT3 and ERK1/2 pathway to enhance the immunosuppressive ability of MDSCs.