T4 Mechanistic Insights on Late Stage Erythropoiesis in Burn Patients Treated with Propranolol

Abstract

Burn patients suffer from Epo resistant anemia in which the Epo-independent stages of early commitment and late maturation of erythroblasts are defective. We have previously shown in mice and patients, that propranolol (prop) treatment after burn injury restored erythroid committed progenitors, while terminal maturation was still impaired. During terminal erythropoiesis, hemoglobinization is aided by alpha hemoglobin stabilizing protein (AHSP), which is an erythroblast intrinsic functional protein predominantly expressed in nucleated erythroblasts. Effective hemoglobinization results in maturation of polychromatic erythroblasts (PolyE) to reticulocytes. Here we demonstrate in PBMC derived ex vivo erythroblast cultures, AHSP is positively associated with late stage maturation in the presence of allogeneic control plasma (CP); and more so in combination with prop than with standard burn care (SBC) in burn patients. After IRB approval, blood samples were collected from 6 burn patients (age=39±16 yeas; %TBSA=32±7) at three time points on post burn days 2–4, 7–10 and 27–33. Control plasma was obtained from healthy volunteers. Fifteen PBMC samples were placed in growth factor cocktail (SCF, GMCSF, IL-3, Cyclosporine) for 0–5 days to nurture erythroid progenitors. On day 5, each sample was split into two and supplemented with either autologous burn plasma (BP) or CP along with SCF, EPO, and holotransferrin from days 5–15. We harvested cells on culture days 9, 11 and 15; subjected them to FACS and AMNIS to determine the expression (mean fluorescence intensity) and percentage of AHSP+ erythroblasts on days 9 and 11 and the percentage of Reticulocytes (Sytox- CD235a+ CD71+/- Syto16-) and PolyE (Sytox- CD235a+ CD71+/- Syto16+ SSChigh) on day 15. See figure. Overall, CP supplementation in PBMC cultures produced more reticulocytes with a proportional decrease in PolyE, indicating enhanced late stage maturation. Nonetheless, the decrease in PolyE and increase in reticulocytes are more striking in prop treated patients compared to SBC. This is consistent with about 20% increase in AHSP+ cells and AHSP expression in developing erythroblasts in prop compared to SBC. Together, CP imparts a positive influence on reticulocyte maturation by augmenting AHSP expression in PBMC derived erythroblasts. Plasma resuscitation in addition to prop treatment is a promising alternate strategy to reduce transfusions in burn patients.