Circulating myeloid dendritic cells are increased in individuals with severe aplastic anemia

Severe aplastic anemia (SAA) is an autoimmune disease in which bone marrow failure is mediated by activated myeloid dendritic cells (mDCs) and T lymphocytes. Recent research has identified a strong immunomodulatory effect of pyruvate kinase M2 (PKM2) on dendritic cells in immune-mediated diseases. In this study, we aimed to explore the role of PKM2 in the activation of mDCs in SAA. We observed conspicuously higher levels of PKM2 in mDCs from SAA patients compared to normal controls at both the gene and protein levels. Concurrently, we unexpectedly discovered that after the mDC-specific downregulation of PKM2, mDCs from patients with SAA exhibited weakened phagocytic activity and significantly decreased and shortened dendrites relative to their counterparts from normal controls. The expression levels of the costimulatory molecules CD86 and CD80 were also reduced on mDCs. Our results also suggested that PKM2 knockdown in mDCs reduced the abilities of these cells to promote the activation of CD8+ T cells (CTLs), leading to the decreased secretion of cytotoxic factors by the latter cell type. These findings demonstrate that mDC activation requires an elevated intrinsic PKM2 level and that PKM2 improves the immune status of patients with SAA by enhancing the functions of mDCs and, consequently, CTLs.

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Effects of Shikonin on the Functions of Myeloid Dendritic Cells in a Mouse Model of Severe Aplastic Anemia

Mediators of Inflammation ◽

10.1155/2020/9025705 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Mengying Zheng ◽

Bingnan Liu ◽

Yuanyuan Shao ◽

Luogang Hua ◽

Rong Fu ◽

...

Keyword(s):

Dendritic Cells ◽

Mouse Model ◽

Cyclosporin A ◽

Aplastic Anemia ◽

Survival Rates ◽

Severe Aplastic Anemia ◽

High Survival ◽

Myeloid Dendritic Cells ◽

Cd8 Cells

This study is aimed at investigating the effects of shikonin, a pyruvate kinase M2 (PKM2) inhibitor, on the functions of myeloid dendritic cells (mDCs) in a mouse model of severe aplastic anemia (AA) generated by total body irradiation and lymphocyte infusion. Flow cytometry and qPCR were used to determine the proportions of PKM2+ mDCs and other immune indicators in the AA mice. Glucose consumption level, pyruvate generation level, and ATP content were used to determine the level of glycolytic metabolism in the mDCs. The survival rates of AA mice were evaluated after the administration of shikonin or the immunosuppressive agent cyclosporin A. The AA mice displayed pancytopenia, decreased CD4+/CD8+ cell ratio, increased perforin and granzyme levels in CD8+ cells, increased costimulatory CD80 and CD86 expressions, and inadequate regulatory T cell number. In vivo animal experiments showed that the shikonin-mediated inhibition of the PKM2 expression in mice was associated with high survival rates. In addition, the administration of cyclosporin A or shikonin decreased the expression of cytotoxic molecules and costimulatory CD80 and CD86 on CD8+ cells. Taken together, the results of this study indicated that shikonin could inhibit the activation and proliferation of mDCs as well as the activation of downstream cytotoxic T cells by reducing the PKM2 level in mDCs.

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Upregulated Expression of Profilin1 on Dendritic Cells in Patients With Severe Aplastic Anemia

Frontiers in Immunology ◽

10.3389/fimmu.2021.631954 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hong Yu ◽

Yang Zhao ◽

Xiaofeng Pan ◽

Chunyan Liu ◽

Rong Fu

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Cell Movement ◽

Severe Aplastic Anemia ◽

Myeloid Dendritic Cells ◽

Life Threatening ◽

Movement Ability ◽

Antigen Presenting

Severe aplastic anemia (SAA) is a life-threatening form of bone marrow failure that is associated with very high mortality. Dendritic cells (DCs) are antigen presenting cells (APCs) with powerful movement ability, which is an important factor affecting immune function. The expression of profilin1 (Pfn1) plays an important role in the regulation of cell movement ability. We detected the expression of Pfn1 mRNA in the bone marrow (BM) myeloid dendritic cells (mDCs) from patients with SAA using RT-PCR. Next, we examined Pfn1 expression on mDCs using flow cytometry (FCM). We also assessed the relationship between Pfn1 expression and cytokine levels. Our data showed increased Pfn1 mRNA expression in patients with SAA. The expression of Pfn1 in BM mDCs increased in SAA patients. The expression of Pfn1 on mDCs and cytokines (TNF-α and IFN-γ) were positively correlated in the serum of untreated patients with SAA. Taken together, we found that the expression of Pfn1 on mDCs of SAA patients increased, which may affect the function of mDCs. Profilin 1 may be involved in the immunopathogenesis of SAA.

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Differential expression of the proteome of myeloid dendritic cells in severe aplastic anemia

Cellular Immunology ◽

10.1016/j.cellimm.2013.09.007 ◽

2013 ◽

Vol 285 (1-2) ◽

pp. 141-148 ◽

Cited By ~ 9

Author(s):

Chunyan Liu ◽

Weiwei Sheng ◽

Rong Fu ◽

Huaquan Wang ◽

Lijuan Li ◽

...

Keyword(s):

Dendritic Cells ◽

Aplastic Anemia ◽

Differential Expression ◽

Severe Aplastic Anemia ◽

Myeloid Dendritic Cells

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Changes of Subsets of DC 1 in the Bone Marrow of Severe Aplastic Anemia Patients.

Blood ◽

10.1182/blood.v104.11.3712.3712 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3712-3712

Author(s):

Guangsheng He ◽

Zhonghong Shao ◽

De Pei Wu ◽

Xiao Ma ◽

Aining Sun

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

T Lymphocytes ◽

Aplastic Anemia ◽

Severe Aplastic Anemia ◽

Stable Form ◽

Th1 Cells ◽

Th1 Cell ◽

Cd8 Cells ◽

Normal Controls

Abstract Objective To measure the changes of subsets of dendritic cells 1 (DC1) in the bone marrow of severe aplastic anemia (SAA) patients and evaluate the relationships between the CD11c+CD83+cells and Th1 cells, CD3+CD8+ cells or hematopoietic function. Methods By FACS, the quantity and ratio of CD11c+CD1a+ cells, CD11c+CD83+ cells, Th1 cells, and CD3+CD8+ cells in the bone marrow of SAA patients and normal controls were detected respectively. The relationships between CD3+CD8+ cells and Ret or ANC, between Th1 cells and CD3+CD8+ cells, Ret or ANC, between CD11c+CD83+ cells, and Th1 cells, CD3+CD8+ cells, Ret or ANC were evaluated. Results In normal control’s bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells, CD11c+CD83+ cells and ratio of CD11c+CD83+ /CD11c+CD1a+ was 0.42±0.30%, 0.38±0.29%, 0.37±0.32% and 1.07±0.10 respectively. In the untreated SAA patient’s bone marrow, they were 4.87±0.54%, 1.73±0.24%, 3.38±0.56% and 2.21±0.32 respectively, and increased markedly(p<0.01). In recovering SAA patient’s bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells and CD11c+CD83+ cells decreased significantly[0.53±0.22%, 0.61±0.23%, 0.65±0.22%, respectively (p<0.01)]. The ratio of CD11c+CD83+/CD11c+ CD1a+ of recovering SAA patients was 1.37±0.25 which was similar to that of normal controls (p>0.05). The percentages of CD3+CD8+ cells of untreated SAA patients was 32.32±10.22%, and that of recovering SAA patients decreased to 13.67%±5.24 significantly (p<0.01). The percentages of CD3+CD8+ cells of SAA patients were correlated to their Ret and ANC (P<0.05) negatively. Their Th1 cell percentages were correlated to their CD3+CD8+ cells positively (P<0.01), but to their Ret and ANC negatively(p<0.01). SAA patient’s CD11c+CD83+ cell percentages were correlated to their Th1 cell and CD3+CD8+ cells positively (P<0.01, P<0.05), but to their Ret and ANC negatively(p<0.01). Conclusion Both immature DC1 and activated DC1 increased in the bone marrow of SAA patients, and the balance of subsets of DC1 shifted from stable form to active one, which might promote Th0 cells to polarize to Th1 cells, then cause the over-function of T lymphocytes and hematopoietic failure in SAA.

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In Vitro Study of the Function of Dendritic Cells on Stimulating T Lymphocytes Proliferation in Severe Aplastic Anemia

Blood ◽

10.1182/blood.v112.11.4919.4919 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4919-4919

Author(s):

Zonghong Shao ◽

Jun Wang ◽

Rong Fu ◽

Lijuan Li ◽

Hui Liu ◽

...

Keyword(s):

Growth Rate ◽

Dendritic Cells ◽

T Lymphocytes ◽

Aplastic Anemia ◽

Mononuclear Cells ◽

Statistical Significance ◽

In Vitro Study ◽

Severe Aplastic Anemia ◽

Normal Controls

Abstract Objective To investigate the function of dendritic cells (DCs) in severe aplastic anemia (SAA). Methods Fifteen untreated patients, 10 recovered patients with SAA and 12 normal controls were enrolled in this study. Their mDCs were induced from their bone marrow mononuclear cells with rhIL-4, rhGM-CSF and rhTNF in vitro. Then mDCs were co-cultured with allogenic lymphocytes (mixture lymphocyte reaction, MLR). The growth rate of lymphocyte was measured by MTT colorimetry and the concentrations of IL-12 and IFNγ in MLR supernatant were measured by ELISA. The correlation between the growth rate and the concentration of IL-12 or IFNγ was analyzed. Results When mDCs and lymphocytes were co-cultured at the ratio of 1:50, the growth rate of lymphocytes stimulated with the mDCs from untreated cases was (322.13±171.07)%, which was higher than those stimulated with the mDCs from recovered and controls [(180.90±79.12)%, (192.25±91.93)%] (P<0.05). There was no statistical significance between the growth rates of lymphocytes stimulated with the mDCs from the recovered and controls (P>0.05). The mDCs of 8 recovered cases and 4 controls were co-cultured with allogenic lymphocytes at the ratio of 1:100, 1:50, 1:20 and 1:10, but no statistical significance was found between two groups at each ratio (P>0.05). Cross MLR showed only untreated cases versus normal had statistical significance (P<0.05). The concentrations of IL-12 and IFNγ in MLR supernatant of untreated cases were higher than those of the recovered or controls (P<0.05), but there was no statistical significance between the recovered and controls (P>0.05). The concentration of IL-12 in MLR supernatant correlated positively with the growth rate of lymphocyte (r = 0.529,P=0.001), which was also positively correlated with the concentration of IFNγ (r = 0.381,P=0.024). Conclusion The function of mDCs to stimulate T lymphocytes proliferation by secretingt IL-12 in SAA was enhanced, which might play an important role in the immunopathogenesis of SAA.

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The Role of PKM2 in the Activation of Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia

Blood ◽

10.1182/blood.v128.22.1497.1497 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1497-1497

Author(s):

Zonghong Shao ◽

Chunyan Liu ◽

Mengying Zheng ◽

Huaquan Wang ◽

Rong Fu

Keyword(s):

Flow Cytometry ◽

Dendritic Cells ◽

Aplastic Anemia ◽

Normal Control ◽

Control Group ◽

Sirna Transfection ◽

Myeloid Dendritic Cells ◽

Ifn Γ ◽

Protein Components

Abstract Severe aplastic anemia (SAA) is a hematologic disease characterized by severe bone marrow failure. The abnormal activation of myeloid dendritic cells (mDCs) in SAA might accelerate the polarization of Th0 cells to Th1 cells ,thus causing the over-function of T lymphocytes and apoptosis of hematopoietic cells. Hence, mDCs played an important role in the primary stage of the immune response. Here, we analyze the protein components of mDC from SAA as well as normal control to explore the possible reason that activate mDC. The protein components of mDCs was detected by proteomics technology. Furtherly we applied both realtime PCR and western blot from a perspective of mRNA and protein to validate our discovery. Results revealed that expression of pyruvate kinase M2(PKM2) were enhanced in mDCs from SAA patients at an early stage of the onset. Concurrently, the level of PKM2 in mDCs of SAA patients was conspicuously higher than that in normal control not only at a protein level but also at the gene level. To clarify the role of PKM2 in the activation of mDCs, we investigate the effect of targeted PKM2 siRNA -knockout in mDCs at the cellular level. We measured the levels of costimulatory molecules CD80 and CD86 on mDCs by flow cytometry and identified significantly higher expression of CD86 in the control group relative to the transfected group. To quantify antigen uptake and processing capacities of mDCs, cells were treated with carboxylate-modified yellow-green (YG) microspheres and then analyzed by flow cytometry. The percentage of phagocytosis (PP) was determined by the following formula: PP = M1 + M2 + M3 + M4, where M1, M2, M3, and M4 correspond to the number of cells with one, two, three and four microspheres. Phagocytosis index(PI)=(intracellular cpms/ intracellular + extracellular cpms)x100%.After PKM2 siRNA transfection, we observed lower phagocytic ability of mDCs. Additionally, the level of PP of mDCs from control group was higher than that of PKM2 siRNA -knockout group. Through scanning electron microscopy (SEM), mDCs before siRNA transfection from SAA patients presented longer and more branched protrusions than those seen in PKM2 siRNA transfected mDCs. Next, the effects of PKM2 on cytotoxic T cells(CTL) activation by mDCs from SAA patients were also determined. mDCs before and after PKM2 siRNA transfection were co cultured with CTL cells for 72 hours. After cell co culture, the mRNA expressions of perforin were identified significantly higher in the control group relative to the transfected group. Culture supernatant were collected for quantification of IFN-γ and IL-4 by commercial ELISA kits. The levels of IFN-γ and IL-4 in contral group were much higher than that of transfected group .Additionally, flow cytometry experiments assessing annexin V/propidium iodide staining showed that high PKM2 expression in mDCs was significantly associated with reduced apoptosis rate of CTLs. Therefore, the enhanced function of mDCs from SAA could activate the downstream CTL function and then enhance the killing effect to target cells. Our findings demonstrated that elevated levels of PKM2 in mDCs play an important role in the activation of mDCs and thereby affecting the immune status of SAA by enhancing the function of mDCs and downstream CTLs. Disclosures No relevant conflicts of interest to declare.

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Pyruvate Kinase M2 Regulates Hif-1alpha Activity in Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia

Blood ◽

10.1182/blood-2021-150219 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1109-1109

Author(s):

Xiaowei Liang ◽

Lijie Zeng ◽

Xiaoyu Zhao ◽

Chunyan Liu ◽

Zonghong Shao ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Aplastic Anemia ◽

Mrna Expression ◽

Immune Cells ◽

Expression Level ◽

Control Group ◽

Mrna Expression Level ◽

Myeloid Dendritic Cells ◽

Normal Controls

Abstract Hypoxia-inducible factor 1 (HIF-1) is a nuclear protein with transcriptional activity. HIF-1 can be activated by immune cells exposed to hypoxia and regulate glycolysis. In this regulation, pyruvate kinase M2 (PKM2), a key enzyme in the cell glycolytic pathway, is an important regulatory site. Our previous studies suggest that PKM expression is increased in myeloid dendritic cells (mDCs) in SAA patients. Therefore, we investigated the expression level of HIF-1α in mDCs and its interaction with PKM2 in SAA patients. The HIF-1α expression of mRNA and protein on mDCs in SAA untreated patients was significantly higher than that in SAA remission patients and normal controls. In the SAA patients, the HIF-1α expression on mDCs was positively correlated with the numbers of mDCs (p < 0.01), CD80+mDC/mDC (r = 0.689, p < 0.01), and CD86+mDC/mDC in PB (p < 0.05). In SAA patients, the HIF-1α expression on mDCs was negatively correlated with the CD4+T/CD8+T ratio in peripheral blood (PB) (p < 0.05), the percentage of granulocytoid and erythroid cells in bone marrow (p < 0.05), the WBC count in PB (p < 0.05), ANC in PB (p < 0.05), and the percentage of Rets in the PB (p < 0.05); was positively correlated with the percentage of lymphoid cells in bone marrow(p < 0.05); and was not statistically correlated with the megakaryocyte number in bone marrow, absolute PBL count, HGB in PB, absolute Ret count in PB, or PLT count in PB. In the correlation analysis we observed that there was a positively correlation between HIF-1α and PKM2 expression on mDCs in SAA patients (p < 0.001). To evaluate whether there is a mutual adjustment relationship between HIF-1α and PKM2, we successfully reduced PKM2 gene expression in this cell population via siRNA transfection. This process resulted in significantly lower levels of PKM2 protein expression relative to cells transfected with siControl which were evaluated by western blotting. We observed that the relative expression levels of HIF-1α mRNA in mDCs transfected with PKM2 siRNA was lower than siControl group（P< 0.01）. Conclusions In this study, we found that untreated patients with SAA had higher HIF-1α expression on mDCs compared with recovering SAA patients and normal controls. The expression of HIF-1α was correlated with the number and function in mDCs and the severity of pancytopenia of SAA. The results indicated that the mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The above experimental results confirmed that HIF-1α plays an important role in the abnormal immune response in patients with severe aplastic anemia, mainly by regulating the activity of PKM2 and thereby affecting the energy metabolism in immune cells. Disclosures No relevant conflicts of interest to declare.

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