Plasma TNFα and Unknown Factor/s Potentially Impede Erythroblast Enucleation Obstructing Terminal Maturation of Red Blood Cells in Burn Patients.

INTRODUCTION: In this study, using burn patient's peripheral blood mononuclear cells (PBMCs), we have shown that the Epo independent stage of terminal enucleation to reticulocyte formation is impeded in the presence of autologous plasma (BP). Furthermore, substitution with allogeneic control plasma (CP) from the healthy individual in place of BP rectified this enucleation defect. The exclusive role of burn microenvironment in late-stage erythropoiesis defect was further demarcated through control healthy human bone marrow cells cultured in the presence of CP, BP, and cytokines. METHODS: PBMCs and human bone marrow (huBM) were differentiated ex vivo to enucleated reticulocytes in the presence of required growth factors and 5% CP or BP. Effect of systemic mediators in burn microenvironment like IL-6, IL-15, and TNFα was also explored. Neutralization experiments were carried out by adding varying concentrations (25 ng-400 ng/mL) of Anti-TNFα Ab to either CP+TNFα or BP. RESULTS: Reticulocyte proportion and maturation index were significantly improved upon substituting BP with CP during differentiation of burn PBMCs. In the huBM ex vivo culture, addition of IL-6 and IL-15 to CP inhibited the proliferation stages of erythropoiesis, whereas TNFα supplementation caused maximum diminution at erythroblast enucleation stage. Supplementation with anti-TNFα in the BP showed significant but partial restoration in the enucleation process, revealing the possibility of other crucial microenvironmental factors that could impact RBC production in burn patients. CONCLUSION: Exogenous TNFα impairs late-stage erythropoiesis by blocking enucleation, but neutralization of TNFα in BP only partially restored terminal enucleation indicating additional plasma factor(s) impair(s) late-stage RBC maturation in burn patients.

Transient Improvement in Erythropoiesis Is Achieved Via the Chaperone AHSP With Early Administration of Propranolol in Burn Patients.

Burn patients experience erythropoietin resistant anemia in which early commitment and late maturation of erythroblasts are defective. The authors previously showed that propranolol (Prop) treatment restores erythroid committed progenitors, but terminal maturation remains impaired. Hemoglobinization and maturation occur during terminal erythropoiesis and these processes are aided by an erythroblast intrinsic functional protein called alpha-hemoglobin stabilizing protein (AHSP). The authors evaluated the role of AHSP in PBMC- (peripheral blood mono nuclear cell) derived erythroblasts and the implications of Prop in burn patients. Blood samples were collected at three time points from 17 patients receiving standard burn care (SBC) or Prop. Five healthy volunteers provided control plasma (CP). PBMCs were placed in biphasic cultures with 5% autologous plasma (BP) or CP. Erythroblasts were harvested during mid and late maturation stages; the percentage of AHSP+ erythroblasts, AHSP expression, and relative distribution of reticulocytes and polychromatophilic erythroblasts (PolyE) were determined by cytometry. During the second time point (7-10 days postburn), Prop cohort required 35% less transfusions. At mid maturation, PBMCs from Prop-treated patients cultured in BP had 33% more AHSP+ erythroblasts and 40% more AHSP expression compared with SBC. Furthermore, at late maturation, Prop had 50% more reticulocytes and 30% less PolyEs in CP vs BP compared with SBC (11% and 6%, respectively). AHSP is positively associated with late-stage maturation of PBMC-derived erythroblasts in the presence of CP. Albeit transiently, this is more pronounced in Prop than SBC. Early administration of propranolol in burn patients supports erythropoiesis via the chaperone AHSP.

Stage Specific Expression Pattern of Alpha-Hemoglobin-Stabilizing-Protein (AHSP) Portrayed in Erythroblast Chronology.

During erythropoiesis, the molecular chaperone alpha-hemoglobin-stabilizing protein (AHSP) sequesters free alpha-hemoglobin (αHb) and prevents precipitation of excess αHb. While AHSP is linked to hereditary anemia, the pattern of expression during specific erythroblast stages is poorly understood. We investigated gene and protein expressions of AHSP throughout progressive maturation stages of erythroblasts in biphasic cultures of blood and bone marrow samples from healthy donors. Differentiating erythroblasts were periodically subjected to flow cytometry, Amnis imaging and RT-qPCR analyses. We made parallel in vivo validations from naive murine bone marrow cells. Percentages of AHSP+ erythroblasts, protein expressions and AHSP gene expressions are negligible on culture day 6 (CFU-Es) and progressively increases from culture days 8-12 (peaks on day 12) and declines on day 14. Notably, sub-cellular location of AHSP is both in the cytoplasm and nucleus in the early erythroblasts while in the late stages of maturation AHSP is found predominantly in the nucleus, being expelled with it during enucleation. As both human bone marrow and peripheral blood mononuclear cells (PBMC) derived erythroblasts demonstrated similar expression patterns, sampling of erythroblasts from day 11 cultures could portray erythroblast chronology and provide optimum representative stage specific expression patterns. PBMCs may be suitable for comparison studies of AHSP expression in pathologic erythropoiesi.