PKM2 Is Required to Activate Myeloid Dendritic Cells from Patients 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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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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Study on the Pathways to Damage Hematopoiesis by CD8+ Effector T Cells of the Patients with Severe Aplastic Anemia

Blood ◽

10.1182/blood.v118.21.4371.4371 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4371-4371

Author(s):

Zonghong Shao ◽

Le Feng ◽

Rong Fu ◽

Jun Wang ◽

Chunyan Liu ◽

...

Keyword(s):

T Cells ◽

Aplastic Anemia ◽

Cd8 T Cells ◽

Bone Marrow Failure ◽

Granzyme B ◽

Effector T Cells ◽

Severe Aplastic Anemia ◽

Control Group ◽

Hla Dr ◽

Normal Controls

Abstract Abstract 4371 Objective To investigate the quantity and their pathways to damage hematopoietic cells of CD8+CD25+ and CD8+HLA-DR+ effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA) and explore the heterogeneous immunopathogenesis of SAA further. Methods The quantity of CD8+CD25+and CD8+HLA-DR+ cells in PB and the expressions of perforin, granzyme B, tumor necrosis factor-β(TNF -β) and FasL of 29 SAA (14 untreated and 15 recovered) patients and 12 normal controls were analyzed by flow cytometry. Results The ratio of CD8+CD25+T cells in CD8+ T cells was (3.67±2.58)% in untreated SAA patients, (5.19±4.29)% in recovered patients and (4.84±2.31)% in normal controls, and the ratios of CD8+CD25+T cells in CD3+ cells in three groups were (2.25±1.35)%, (2.98±1.35)% and (2.11±1.88)% respectively. There was no statistic difference among 3 groups(P>0.05). The ratio of CD8+HLA-DR+T cells in CD8+T cells was (39.30±8.13)% in untreated patients, which was significantly higher than that of recovered patients[(20.65±5.38%)] and controls [(18.34±6.68%)](P<0.001). There was no statistic difference between recovered patients and controls(P>0.05). CD8+HLA-DR+T cells in CD3+ cells was (27.81±7.10)% in untreated group, higher than that of recovered patients (12.02±3.03)% and controls(8.50±2.33)%(P<0.01). And the ratio in recovered group was higher than in control group(P<0.05). The expressions of perforin, granzyme B, TNF-β and FasL of CD8+HLA-DR+ T cells of untreated SAA patients were 8.51% A96.08% A72.11% and 94.25% respectively, higher than those of recovered patients(1.78% A85.20% A34.38% A51.20%)and controls(1.86% A82.09% A17.92% A32.91%). There was no statistic difference between recovered patients and controls(P>0.05). Conclusion There were elevated quantity of CD8+HLA-DR+ T cells and high expressions of perforin, granzyme B, TNF-β and FasL in SAA, which might contribute to the bone marrow failure of SAA. Disclosures: No relevant conflicts of interest to declare.

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

International Journal of Hematology ◽

10.1007/s12185-010-0761-z ◽

2011 ◽

Vol 93 (2) ◽

pp. 156-162 ◽

Cited By ~ 21

Author(s):

Shao Zonghong ◽

Tu Meifeng ◽

Wang Huaquan ◽

Xing Limin ◽

Wang Jun ◽

...

Keyword(s):

Dendritic Cells ◽

Aplastic Anemia ◽

Severe Aplastic Anemia ◽

Myeloid Dendritic Cells

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Recurrent 6pLOH Is the Most Common Somatic Lesion in Refractory Cytopenia of Childhood and Occurs Very Infrequently in Severe Aplastic Anemia

Blood ◽

10.1182/blood.v120.21.644.644 ◽

2012 ◽

Vol 120 (21) ◽

pp. 644-644 ◽

Cited By ~ 1

Author(s):

Marcin W Wlodarski ◽

Shinsuke Hirabayashi ◽

Brigitte Strahm ◽

Sandra Urbaniak ◽

Brigitte Schlegelberger ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Telomere Length ◽

Copy Number ◽

Bone Marrow Failure ◽

Good Prognosis ◽

Severe Aplastic Anemia ◽

Diamond Blackfan Anemia ◽

Immune Mediated ◽

Somatic Aberrations

Abstract Abstract 644 Refractory cytopenia of the childhood (RCC) and severe aplastic anemia (SAA) are the most common causes of acquired hypoplastic bone marrow failure (BMF) in children. Although predisposing genetic factors and inciting immunological events had been implicated, little is known about the molecular origins of these conditions. Whole-genome scanning using single nucleotide polymorphism arrays (SNP-A) can complement standard cytogenetics in terms of the detection of submicrocopic aberrations and regions with copy number neutral loss of heterozygosity (CN-LOH). Here we present the results of an analysis of 181 children with bone marrow failure: MDS, n=106; SAA, n=41; and for hematological control, 34 patients with Diamond Blackfan anemia (DBA). To enrich for the myeloid lineage, we used granulocytes from bone marrow as source for DNA. We employed Affymetrix 6.0 arrays and used CNAG v3.3 and Genotyping Console v4.0 platforms for data analysis. To identify somatic aberrations that might have been missed by standard metaphase cytogenetics (MC), we initially looked at cases with abnormal MC, presenting as RCC or advanced MDS (n=25, median age 12.4 years). While SNP-A generally confirmed the cytogenetic lesions identified by MC, no novel recurrent somatic aberrations with pathogenic character were discovered. In the next step we focused on the analysis of RCC patients with normal karyotype (N=81, median age 10.2 years). In one case a small monosomy 7 clone was identified using SNP-A, that has been missed by standard MC. Most strikingly, 11 patients (14%) carried clones with a terminal CN-LOH of the short arm of chromosome 6 (6pLOH), with a length of 30–42Mb and various clonal size. The somatic myeloid origin was confirmed by the analysis of CD4+/CD8+ sorted T-cells in several cases. The 6pLOH lesion encompasses the HLA gene cluster leading to the loss of one HLA haplotype, has previously been reported in adults with SAA diagnosed in USA and Japan (Afable/Wlodarski, and Katagiri 2011), and was generally associated with a good prognosis. Regarding treatment modalities in RCC patients, the 6pLOH clone was overrepresented in the watch and wait (W&W) cohort: 10/55 (18%) of W&W cases carried this clone, as compared to 1/26 (4%) patient who received therapy within 6 months from diagnosis. Out of the 10 W&W patients with 6pLOH clone, only two required later therapeutic interventions (stem cell transplantation) due to progressive cytopenia at 10 and 11 months after diagnosis. The remaining 8/10 patients are still under W&W strategy with a median FUP of 6.6 (1.6.-12.2) years. In summary, all 11 RCC 6pLOH patients are alive with a median FUP of 6.6 years without disease progression. Interestingly, the 6pLOH clone survives over a long period of time, as confirmed in 2 patients in different bone marrow samples obtained 7 years apart. To answer the question if the persistence of the 6pLOH clones has an effect on telomere maintenance in the hematopoietic cell compartment, we measured telomere length using a quantitative PCR-based approach. All 11 RCC cases with 6pLOH studied had normal telomere length as compared to age-matched controls. Since the immune-mediated attack is the main operating mechanism of BMF in SAA, we next asked whether the 6pLOH clone can also arise in the bone marrow of children with SAA. We SNP-A genotyped a cohort of 41 SAA patients (median age 10.4 years) including 5 hepatitis-associated SAA (HSAA) cases. While no genomic copy number alterations were found, the somatic 6pLOH clone was discovered in only one HSAA patient, in whom the SAA developed 6 months after onset of hepatitis. When compared to the SAA patients, the 6pLOH clone is significantly more frequent in the RCC W&W cohort (P <0.04). Finally, to compare the results to a “non-immune-mediated” BMF, we analyzed 34 children with Diamond Blackfan anemia in whom no 6pLOH clone or other somatic defects were identified. In summary, 6pLOH is the most common somatic lesion found in the myeloid compartment in RCC patients and correlates with a very good prognosis. The absence of 6pLOH in children with SAA with no other associated pathologies supports the concept that RCC and SAA are two distinct entities in children. Disclosures: No relevant conflicts of interest to declare.

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Abnormal Levels of PKM2 in Mdcs from Patients with Severe Aplastic

Blood ◽

10.1182/blood.v126.23.4766.4766 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4766-4766

Author(s):

Zonghong Shao ◽

Mengying Zheng

Keyword(s):

Bone Marrow ◽

Pyruvate Kinase ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Myeloid Dendritic Cells ◽

Pyruvate Kinase M2 ◽

Rate Limiting Step ◽

Quantitative Changes ◽

Normal Controls

Abstract Severe aplastic anemia (SAA) is a hematologic disease characterized by pancytopenia with severe bone marrow failure. Our previous studies have demonstrated that activated myeloid dendritic cells (mDCs) increased in the bone marrow of SAA patients, which might promote Th0 cells to polarize to Th1 cells and cause the subsequent over-function of T lymphocytes and hematopoiesis failure in SAA. The other notable finding in our study is that abnormal expression of pyruvate kinase M2 (PKM2) in mDCs from SAA patients may be one of the reason for mDC hyperfunction. Human pyruvate kinase M2, which catalyzes the last but rate-limiting step of glycolysis, is exclusively expressed in actively proliferated cells ,especially in embryonic and adult dividing/tumor cells. It remains unclear how PKM2 may change in patients with SAA. This study aims at exploring the role of PKM2 in SAA. In this study, the quantitative changes of mDCs in peripheral blood of 15 SAA patients before immunosuppressive therapy (IST) °¢ 13 SAA patients after IST and 26 normal controls were determined by flow cytometry. Results showed that the levels of PKM2 in mDCs were significantly increased in SAA patients(59.1±15.8)%. After IST, the levels of PKM2 in mDCs decreased(42.6±22.1)% (P<0.05).In normal controls group , the level of PKM2 in mDCs was (32.7±20.2)%. These changes reveal that dysregulation of PKM2 expression and activation in mDCs from SAA patients may contribute to hyperfunction of mDCs, thus leading to the imbalance of downstream Th1/Th2 subsets which followed by hematopoiesis failure in SAA. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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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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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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