Burn patients experience erythropoietin resistant anemia in which early commitment and late maturation of erythroblasts are defective. We previously showed that propranolol (Prop) treatment restores erythroid committed progenitors, but terminal maturation remains impaired. Hemoglobinization and maturation occurs during terminal erythropoiesis and these processes are aided by an erythroblast intrinsic functional protein called alpha hemoglobin stabilizing protein (AHSP). We 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 seventeen 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 post burn), 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 to SBC. Furthermore, at late maturation, Prop had 50% more reticulocytes and 30% less PolyEs in CP versus BP compared to 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.

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

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 TNFa was also explored. Neutralization experiments were carried out by adding varying concentrations (25 ng-400 ng/mL) of Anti-TNFa Ab to either CPþTNFa 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 TNFa supplementation caused maximum diminution at erythroblast enucleation stage. Supplementation with anti-TNFa 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 TNFa impairs late-stage erythropoiesis by blocking enucleation, but neutralization of TNFa in BP only partially restored terminal enucleation indicating additional plasma factor(s) impair(s) late-stage RBC maturation in burn patients.

Introduction During anemia of critical illness such as burn injury, erythropoietin (Epo) resistance is accompanied by impaired bone marrow (BM) erythropoiesis. The non-selective β1, β2 -adrenergic receptor (AR) blocker propranolol was effective in mitigating MafB expression in multi potential progenitors and rescuing their erythrocyte commitment in burn patients. However, peripheral HGB levels did not reflect the effect of propranolol perhaps due to defective maturation of late erythroblasts, not regulated by propranolol. Subsequently, we reported that early commitment and late maturation stages are independently orchestrated via discrete β2- and β3-AR mechanisms respectively in the BM of mice with a parallel increase in blood HGB levels. Nonetheless, it is not clear whether commitment of progenitors or maturation of erythroblasts is essential to restore erythropoietic homeostasis after burn injury. In adult mice, the spleen serves as an extra medullary erythropoietic organ under hypoxia and also functions as a primary organ of erythropoietic homeostasis (Bozzini CE et al. Am J Physiol. 1970; 219(3):724). We examined in mice, erythropoietic responses to burn injury in two organs, the spleen and BM to understand the reciprocal relationship, if any, in erythropoiesis between the two adrenergically innervated organs in response to propranolol, and selective β2- and β3- AR blockers. As variation in the size of RBCs is an indication of ineffective maturation, we also evaluated coefficient of variation of RBC size (RDW) in peripheral blood from mice. To validate our animal studies, we also measured RBC parameters in human burn patients. Methods Animal study: B6D2F1 mice were randomized into 2 groups, 15% TBSA (total burn surface area) scald burn and sham burn. Burn mice were given β-AR blockers or saline either via Alzet pumps eight hours after injury or by daily injections. All treatments were terminated 24 hours before harvest. Animals were euthanized to obtain spleen, femurs and blood on post burn days (PBD) as specified in results. Single cell suspensions from spleen and BM were characterized for early erythroblasts (CD71+Ter119neg), orthochromatic erythroblasts (Ter119+CD71+Syto16+), reticulocytes (Ter119+CD71+Syto16neg), erythrocytes (Ter119+), and non-erythroid cells (Ter119neg CD71neg) by flow cytometry, and expressed as 103/106 total splenocytes or total BM cells. RDW was measured using HemaTrue analyzer. Human study: Retrospective analysis was done on 19 adult burn patients enrolled in IRB approved study. All patients received standard burn care (SBC) during the study period of seven weeks. As part of another clinical trial, 9/19 patients had received propranolol along with SBC (SBC+PR). Cohorts matched for age, gender and %TBSA. Results Animal study: Splenic erythropoiesis was increased after burn injury on PBD 3, 7, 14 and 21 as measured by erythrocyte and erythroblast production. Propranolol treatment for 14 days via Alzet pump alone or in combination with Epo was not sufficient to reduce splenic erythrocytes and erythroblasts that were elevated after burn injury. Between the selective blockers, only SR59320A (β3-AR) and not butoxamine (β2-AR) significantly reduced splenic erythropoiesis. This was accompanied by a decrease in splenomegaly and a reciprocal increase in BM erythropoiesis only with SR59230A treatment. RDW was significantly increased in blood after burn injury, while MCV remained within normal range. Interestingly, only SR59230A treatment and not propranolol or butoxamine reversed RDW to sham levels. Human study: Hemoglobin levels were higher at PBD 1-3 (due to hemo concentration) and decreased significantly in both groups through PBD 30-48. Mean RDW was normal at PBD 1-3 (Normal range =11.5% -14.5%) in all burn patients. Although RDW seemed to stabilize initially in SBC+PR treated patients at PBD 7-10, it increased to SBC levels by PBD 30-48. Mean corpuscular volume (MCV) remained within normal limits. Conclusion Effective maturation of late erythroblasts in BM is essential for the spleen to sense erythropoietic homeostasis, which is reestablished with β3-AR blockade after burn injury. Results emphasize only β3-AR antagonist restores RDW to sham levels and not β1/ β2-AR blocker. Increased RDW in burn patients corroborates with animal studies. Overall, this model system is suitable to study mechanisms of early and late erythropoiesis after burn injury. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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.