Myelodysplastic syndrome (MDS) is a heterogeneous group of malignant and clonogenic diseases that originate from hematopoietic stem cells. The main features are abnormal hematopoiesis (myeloid cell development abnormalities) and ineffective hematopoiesis (one line or multilineage). Approximately 30% of patients develop acute myeloid leukemia (AML) during the course of the disease. The pathogenesis of MDS is associated with genetic mutations, epigenetic changes, and bone marrow microenvironments [
The monocyte macrophage system is mainly composed of monocytes and macrophages. Its main function is to remove pathogens or waste materials from the blood and tissues, and it also plays a key role in the induction and regulation of the adaptive immune response [
Our previous studies showed that the number of monocytes in the peripheral blood of MDS patients increased, but the ability to differentiate into macrophages and the phagocytic function decreased [
In this study, we evaluated M1 and M2 macrophages from the bone marrow of MDS and the culture of M1 macrophages
The MDS group enrolled 38 newly diagnosed MDS patients in the Department of Hematology of General Hospital of Tianjin Medical University from June 2015 to June 2016, including 20 males and 18 females, with a median age of 58 (range, 21-79) years. According to the International Prognostic Score System (IPSS), the patients were divided into the lower-risk (LR) MDS group (15 cases) and the higher-risk (HR) MDS group (23 cases) (detail in Table
The characteristics of myelodysplastic syndrome patients.
Case | Sex | Age | Diagnosis | Cytogenetics | IPSS |
---|---|---|---|---|---|
1 | Male | 21 | RARS | 46,XY | Low |
2 | Male | 63 | RCMD | 46,XY | Low |
3 | Female | 38 | RAEB2 | 46,XY | Int-2 |
4 | Male | 46 | RCMD | 46,XY,-2,-12,+mar,19+,9P+ | Int-2 |
5 | Female | 57 | RAEB2 | 46,XX | Int-2 |
6 | Male | 58 | RAEB2 | 46,XY | Int-2 |
7 | Male | 59 | RAEB2 | 46,XY | Int-2 |
8 | Male | 59 | RAEB2 | 46,XY | Int-2 |
9 | Female | 59 | RAEB1 | 46,XY,13q+ | Int-2 |
10 | Male | 62 | RAEB2 | 46,XY | Int-2 |
11 | Female | 64 | RAEB2 | 46,XX | Int-2 |
12 | Male | 65 | RCMD | 46,XY,del17q31 | Int-2 |
13 | Male | 67 | RAEB2 | 46,XY | Int-2 |
14 | Female | 69 | RAEB2 | 46,XX | Int-2 |
15 | Female | 70 | RAEB2 | 46,XX | Int-2 |
16 | Male | 76 | RAEB2 | No result | Int-2 |
17 | Female | 79 | RAEB2 | 46,XX | Int-2 |
18 | Male | 42 | RARS | 46,XY,del20q11 | Int-1 |
19 | Female | 47 | RARS | 46,XX | Int-1 |
20 | Female | 49 | RARS | 46,XX | Int-1 |
21 | Male | 50 | RAEB1 | 46,XX | Int-1 |
22 | Male | 50 | RCMD | 47,XY,+8/46,XY | Int-1 |
23 | Female | 51 | RAEB1 | 46,XX | Int-1 |
24 | Male | 57 | RAEB1 | 46,XY | Int-1 |
25 | Male | 58 | RAEB1 | 46,XY | Int-1 |
26 | Female | 62 | 5q- | 5q- | Int-1 |
27 | Male | 62 | RA | 46,XY | Int-1 |
28 | Female | 64 | RAEB1 | 46,XX | Int-1 |
29 | Female | 74 | RARS | 46,XX | Int-1 |
30 | Female | 74 | RCMD | 46,XX | Int-1 |
31 | Male | 27 | RAEB2 | 3p+,-18,+mar | High |
32 | Female | 29 | RAEB2 | 20q-,5q-,7q- | High |
33 | Male | 30 | RAEB2 | 47,XY,+8/46,XY | High |
34 | Male | 60 | RAEB2 | 45,XY,-7 | High |
35 | Male | 68 | RAEB2 | 46,XY,+8/45,XY+8,-6,-7 | High |
36 | Female | 76 | RAEB2 | del5q33,del5q31,del7q311,del7q3 | High |
37 | Female | 77 | RAEB2 | 45,XX,-5,-2,45,XX,+mar,-5,3P- | High |
38 | Female | 79 | RAEB2 | 45,XX,-7 | High |
Bone marrow samples were obtained by standard bone marrow puncture using sterile heparin anticoagulant tubes. Bone marrow samples were filtered using flow cytometry tubes. CD14-FITC (Cat No.: 555397), CD68-PE (Cat No.: 565595), CD64-APC (Cat No.: 561189), CD40-PEcy7 (Cat No.: 561215), CD206-PE (Cat No.: 555954), CD163-PEcy7 (Cat No.: 556018), and isotype control antibodies (BD Biosciences, USA) were added to the tubes. The samples were then stained for 15 min in the dark at room temperature. After red blood cell lysis, the cells were washed with PBS. Finally, the cells were detected using a FACSCalibur flow cytometer (BD Biosciences, USA). Data analysis was performed using the Cell Quest software (Becton Dickinson, version 3.1).
Macrophages were defined as CD14+CD68+ cells. M1 macrophages were defined as CD64+CD40+ macrophages. M2 macrophages were defined as CD206+CD163+ macrophages (detail in Supplemental Figure
Peripheral blood mononuclear cells (PBMCs) were separated from fresh heparinized blood samples (5 mL) using Ficoll Solution (Suolaibao, China). The PBMCs were seeded at 3 million cells/mL in sterile RPMI 1640 (Invitrogen, CA, USA) and cultured for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) (Huabei Pharmacy, Shijiazhuang, China), interferon-gamma (Sigma, USA), and lipopolysaccharide (Sigma, USA). On day 7, macrophages were collected from the bottom of the culture dishes.
Total RNA from macrophages was extracted using TRIzol (Takara Bio, CA, USA), and cDNA was generated using a reverse transcriptase kit (Takara Bio, CA, USA). Gene expression was quantified by qPCR (SYBR® Premix Ex Taq II, Takara Bio, China). The primer sequences were as follows: IL-1
The results were analyzed using the GraphPad Prism 8.0 program (GraphPad Software, Inc., San Diego, CA). Data with normal distribution were presented as
The proportion of bone marrow monocytes was
The quantity of monocytes in bone marrow of patients with MDS. (a) Representative dot plots from flow cytometric (FACS) analyses showing the CD14+ cell frequency among bone marrow mononuclear cells obtained from healthy controls. (b) Representative dot plots from FACS analyses showing the CD14+ cell frequency among bone marrow mononuclear cells obtained from MDS patients. (c) The proportion of CD14+ cells from bone marrow of MDS patients and controls. (d) The median fluorescence intensity (MFI) of CD14+ cells from bone marrow of MDS patients and controls.
The median fluorescence intensity (MFI) of CD14+ cells from the bone marrow in the control group, LR-MDS group, and HR-MDS group was
The proportion of M1 macrophages in the bone marrow monocytes was
The proportion of M2 macrophages in the bone marrow monocyte was
The ratio of M1 to M2 macrophages was
The percentage of macrophages in bone marrow of patients with MDS. (a) Representative dot plots from flow cytometric (FACS) analyses showing the macrophage (CD14+CD68+ cells) frequency among bone marrow mononuclear cells. (b) Representative dot plots from FACS analyses showing the M1 macrophage (CD64+CD40+ macrophages) frequency among bone marrow mononuclear cells. (c) Representative dot plots from FACS analyses showing the M2 macrophage (CD206+CD163+ macrophages) frequency among bone marrow mononuclear cells. (d) The frequency of M1 macrophages from bone marrow of MDS patients and controls. (e) The frequency of M2 macrophages from bone marrow of MDS patients and controls. (f) The ratio of M1/M2 macrophages from bone marrow of MDS patients and controls.
The level of IL-1
The level of TNF-alpha mRNA was
The expression of IL-1
Bone marrow macrophages play an important role in maintaining the homeostasis of the hematopoietic stem cell niche. Removing macrophages can release hematopoietic stem cells into the peripheral blood [
Tissue macrophages and inflammatory macrophages are derived from monocytes in the peripheral blood or from the embryonic origin of tissue macrophages, which have strong plasticity [
In this study, we compared the proportion of macrophages, the ratio of M1 to M2, and the expression of macrophage surface molecules between patients with MDS and the control group. We found that the ratio of M2 macrophages to monocytes was higher in patients with MDS. The ratio of M1 to M2 macrophages was lower in the MDS group. There was no significant difference in the proportion of M1 macrophages between MDS patients and the control group. The results showed that with the development of MDS, the macrophages in the bone marrow further polarized to the M2 subtype and not to the M1 subtype, and the antitumor effect of macrophages was insufficient.
In this study, we found that the expression of IL-1
The increase in M2 polarization in the bone marrow of patients with MDS is beneficial for the proliferation of MDS clonal cells. Repolarization of M2 cells to the M1 phenotype is a method of cancer immunotherapy, which can effectively restore the response of the innate and adaptive immune systems, leading to tumor regression [
Our study has some limitations, such as whether the induced M1 macrophages express the surface markers of M1 cells, such as iNOS and STAT-1, and the levels of TNF-
In conclusion, we found that the polarization of bone marrow macrophages in patients with MDS was abnormal, M1 macrophages were relatively reduced, and IL-1
The data used to support the findings of this study are included within the article.
All authors declare no conflict of interest.
Gaochao Zhang, Liyan Yang, and Yu Han contributed equally to this work. G.Z., L.Y., and Y.H. performed research and analyzed the data; H.W. designed studies, ensured the correct analysis of the data, and drafted the manuscript; H.N., L.Y., L.X., and Z.S. assisted in research design, oversaw data collection, and contributed to the writing of the manuscript. All authors carefully revised the manuscript and finally approved the manuscript.
This project is partly supported by the National Natural Science Foundation of China (No. 81170472) and Key Technology Research and Development Program of Tianjin China (18ZXDBSY00140).
Supplemental Figure 1: the gating strategy for macrophages. (A) Bone marrow mononucleated cells were gated with SSC and FSC. (B) Monocytes were gated with CD14. (C) Macrophages were defined with CD14+CD68+ cells. (D) M1 macrophages were defined with CD14+CD68+CD40+CD64+ cells. (E) M2 macrophages were defined with CD14+CD68+CD163+CD206+ cells.