Glucosamine Interferes With Myelopoiesis and Enhances the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells

Abstract

Glucosamine (GlcN) is the most widely consumed dietary supplement and exhibits anti-inflammatory effects. However, the influence of GlcN on immune cell generation and function is largely unclear. In this study, GlcN was delivered into mice to examine its biological function in hematopoiesis. We found that GlcN promoted the production of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs), both in vivo and in vitro. Additionally, GlcN upregulated the expression of glucose transporter 1 in hematopoietic stem and progenitor cells (HSPCs), influenced HSPC functions, and downregulated key genes involved in myelopoiesis. Furthermore, GlcN increased the expression of arginase 1 and inducible nitric oxide synthase to produce high levels of reactive oxygen species, which was regulated by the STAT3 and ERK1/2 pathways, to increase the immunosuppressive ability of MDSCs. We revealed a novel role for GlcN in myelopoiesis and MDSC activity involving a potential link between GlcN and immune system, as well as the new therapeutic benefit.

Keywords: d-Glucosamine hydrochloride (PubChem CID: 91431); hematopoietic stem cell (HSC); immunosuppression; myeloid-derived suppressive cell; myelopoiesis.

Figure 1

GlcN expands the myeloid compartment of mice. (A) Schematic of GlcN supplementation methods to mice. (B) Body weight and fasting glucose level of GlcN-supplemented mice after 14-day intraperitoneal (i.p.) injection. (C) WBC count, RBC count, hemoglobin level, and platelet count following i.p. treatment. (D) WBC count, RBC count, hemoglobin level, and platelet count following 14-day pump release. (E) Myeloid compartment of peripheral blood from mice given i.p. injection. (F) Myeloid compartment of peripheral blood from osmotic pump-treated mice. The results are presented as the mean ± standard deviation ^*P < 0.05.

Figure 2

GlcN supplementation expands the compartment of MDSCs. (A) Schematic of PMN-MDSC and M-MDSC flow cytometry gating. PMN-MDSC and M-MDSC levels in (B) peripheral blood (PB), (C) bone marrow (BM), (D) spleen (SP) following i.p. injection. (E) PMN-MDSC and M-MDSC levels in the PB following pump release. Wild-type BM cells were stimulated with IL-6, GM-CSF, G-CSF, and transforming growth factor-β (TGF-β) to induce MDSC expansion in vitro. (F) Expanded CD11b⁺Gr1⁺ MDSC frequency. (G) Expanded PMN-MDSC frequency. (H) Expanded M-MDSC frequency. The quantified results are presented as the mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

GlcN increases hematopoietic stem and progenitor cell compartment. (A) Schematic of hematopoietic hierarchy and critical transcriptional factors and cytokines in myelopoiesis. (B) Schematics of identifying hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs). (C) The frequency of HSCs and MPPs in BM after i.p. injection. (D) Schematics of identifying Lin⁻c-Kit⁺Sca-1⁺ cells (LSKs), 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⁻). (E) The frequency of LSKs, MPs, CMPs, GMPs and MEPs in BM after i.p. injection. The quantified results are presented as the mean ± standard deviation. *P < 0.05; **P < 0.01.

Figure 4

The influence of GlcN treatment in the expression of glucose transporters of hematopoietic stem and progenitor cells. (A) The schematic of glucose transporter expression analysis in sorted LSKs, MPs, and GMPs. (B) Relative mRNA expression of Glut1, 2, 3 and 4 in LSKs, MPs, and GMPs were determined by quantitative reverse transcription-PCR. GlcN treatment significantly increase Glut1 expression in LSKs. The quantified results are presented as the mean ± standard deviation. ^**P < 0.01.

Figure 5

GlcN suppresses the expression of transcriptional factors and cytokines involved in myelopoiesis. Relative mRNA expression of PU.1, GATA1, C/EBPα, C/EBPβ, IRF8, Rb, c-Jun, IL-6, GM-CSF, and G-CSF in the BM cells of GlcN-treated mice were determined by quantitative reverse transcription-PCR. The quantified results are presented as the mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

GlcN treatment weakens hematopoietic stem and progenitor cell differentiation. BM cells from i.p.-injected mice were extracted and directly cultured in methylcellulose-based medium M3434. Colonies were counted after 10 days of culture. Quantification of CFU-GEMM, CFU-GM, CFU-G, CFU-M, BFU-E and CFU-E. Quantified results are presented as the mean ± standard deviation. *P < 0.05; **P < 0.01.

Figure 7

GlcN treatment enhances the immunosuppressive ability of MDSCs. Mice were treated with GlcN by i.p. injection for 14 days. Arg-1, iNOS, and COX2 expression levels in (A) total BM cells and (B) BM CD11b⁺Gr1⁺ MDSCs. ROS levels in (C) PMN-MDSCs and (D) M-MDSCs isolated from BM, SP, and PB. (E) T cell immunosuppressive ability of BM CD11b⁺Gr1⁺ MDSCs. (F) Quantification of PD-L1 expression levels in BM cells. MFI: Mean fluorescence intensity. Quantified results are presented as the mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8

GlcN stimulates the activation of STAT3 and ERK1/2 in MDSCs. The level and mean fluorescence intensity (MFI) of (A) phosphorylated STAT3 (pSTAT3) and (B) phosphorylated ERK1/2 (pERK1/2) in BM CD11b⁺Gr1⁺, CD11b⁺Gr1⁻, and CD11b⁻Gr1⁻ cells were shown. (C) The levels of phospho-STAT3, STAT3, phospho-ERK1/2 and ERK1/2 in GlcN-treated CD11b⁺ cells were detected by Western analysis. (D) JAK2 inhibitor AG490 and (E) MEK inhibitor PD98059 were used to suppress GlcN-induced phosphorylation of STAT3 and ERK1/2 proteins, respectively, in BM CD11b⁺Gr1⁺ cells. (F) Inhibition of either STAT3 or ERK1/2 decreased GlcN-promoted ROS production in CD11b⁺Gr1⁺ MDSCs. Quantified results are presented as the mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 9

Schematic illustration showing the influence of GlcN in myelopoiesis and MDSC functions. GlcN interferes myelopoiesis by downregulating the gene expression of critical transcription factors and cytokines involved in HSPC differentiation and myelopoiesis to produce more immature myeloid cells, MDSCs. In addition, GlcN increases STAT3 and ERK1/2 phosphorylation to enhance the MDSC immunosuppressive ability.