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

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.

1. 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.

2. 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).

3. 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.

4. 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 

1. 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.

2. 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.

3. 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.