48 Burn Injury Impedes Late Erythropoiesis via Decreased Alpha Hemoglobin Stabilizing Protein and siglec1

Abstract

Abstract Introduction Transfusions are of no benefit to burn patients with chronic anemia. We are elucidating the mechanisms of erythropoietin resistant anemia in burn injury using a mouse model in addition to a contrived system with human PBMCs. We have found that Alpha Hemoglobin Stabilizing Protein (AHSP), an erythroblast specific protein essential to effective hemoglobinization, is decreased in PBMC derived erythroblasts (EB) from burn patients (male + female). Hemoglobinization begins at the polychromatic EB (PolyE) stage and intensifies at the orthochromatic EB (OrthoE) stage, culminating in ejection of the nucleus and the cell becoming a reticulocyte (Reti). EB needs the support of a central island macrophage (EBI MØ) until they progress to maturation. We have seen in bone marrow from mice (male) following burn injury that Siglec1, an essential adhesion molecule is decreased in EBI MØ. It is these MØ that provide iron for the assembly of the hemoglobin unit in late erythropoiesis. EB maturation defects seen in burn injury could be due to the intrinsic changes in EB phenotype or the phenotype of supporting EBI MØ or a combination of the two. Our goal was to investigate in the animal model using male and female mice, how myeloid/erythroid intrinsic changes after burn impact the progression of late erythropoiesis from an EB and EBI MØ perspectives. Methods With IACUC approval, 12 male and female mice were anesthetized and subjected to sham burn or scald burn (15% TBSA) upon dorsal immersion in a 100oC water bath for 8 seconds. Animals were sacrificed on post burn day 7 (PBD 7). Total bone marrow cells (TBM) were harvested from bilateral femurs. Cells were characterized using CD71, Ter119, and Syto16 antibodies. EBI MØ were identified using a combination of F4/80, ERHR3, Vcam1, Ly6G and Siglec 1 antibodies. Geometric mean of fluorescence intensity (MFI) and cell counts were obtained by flow cytometric analysis and presented as x103 per million TBM cells. Peripheral blood was harvested via cardiac puncture and hemoglobin concentrations (g/dL) were obtained using a hematology analyzer. Results Among the erythroblasts, EEBs were 2.4 fold higher with a 2.4 fold lower AHSP expression in PBD 7 mice compared to sham as shown in left, top and bottom figures. Similarly, more EEBs were associated with EBIMØ with a lower Siglec1 expression as shown in right, top and bottom figures. Blood Hbg concentrations were lower in burn (sham=15.5±0.7 vs PBD 7= 13.1±0.5; p< 0.01). Conclusions The combined influence of Siglec1 and AHSP expression interferes with effective hemoglobinization impeding late maturation of erythroblasts following burn injury. Whether there is a synergistic action between the two deficiencies is being studied. Applicability of Research to Practice Underpinning the mechanism of erythropoiesis will lead to better options to ineffective transfusions in burn patients with anemia.