Myelo-erythroid commitment after burn injury is under ß-adrenergic control via MafB regulation.

Severely injured burn patients receive multiple blood transfusions for anemia of critical illness despite the adverse consequences. One limiting factor to consider alternate treatment strategies is the lack of a reliable test platform to study molecular mechanisms of impaired erythropoiesis. This study illustrates how conditions resulting in a high catecholamine microenvironment such as burns can instigate myelo-erythroid reprioritization influenced by ß-adrenergic stimulation leading to anemia. In a mouse model of scald burn injury, we observed, along with a threefold increase in bone marrow LSK cells (linneg Sca1+cKit+), that the myeloid shift is accompanied with a significant reduction in megakaryocyte erythrocyte progenitors (MEPs). ß-Blocker administration (propranolol) for 6 days after burn, not only reduced the number of LSKs and MafB+ cells in multipotent progenitors, but also influenced myelo-erythroid bifurcation by increasing the MEPs and reducing the granulocyte monocyte progenitors in the bone marrow of burn mice. Furthermore, similar results were observed in burn patients' peripheral blood mononuclear cell-derived ex vivo culture system, demonstrating that commitment stage of erythropoiesis is impaired in burn patients and intervention with propranolol (nonselective ß1,2-adrenergic blocker) increases MEPs. Also, MafB+ cells that were significantly increased following standard burn care could be mitigated when propranolol was administered to burn patients, establishing the mechanistic regulation of erythroid commitment by myeloid regulatory transcription factor MafB. Overall, results demonstrate that ß-adrenergic blockers following burn injury can redirect the hematopoietic commitment toward erythroid lineage by lowering MafB expression in multipotent progenitors and be of potential therapeutic value to increase erythropoietin responsiveness in burn patients.

Burn injury dampens erythroid cell production through reprioritizing bone marrow hematopoietic response.

BACKGROUND: Anemia in burn patients is due to surgical blood loss and anemia of critical illness. Because the commitment paradigm of common bone marrow progenitors dictates the production of erythroid, myeloid, and lymphoid cells, we hypothesized that skewed bone marrow lineage commitment decreases red cell production and causes anemia after a burn injury. METHODS: After anesthesia, B(6)D(2)F(1) mice received a 15% total body surface area dorsal scald burn. The sham group did not receive scald burn. Femoral bone marrow was harvested on 2, 5, 7, 14, and 21 postburn days (PBD). Total bone marrow cells were labeled with specific antibodies to erythroid (CD71/Ter119), myeloid (CD11b), and lymphoid (CD19) lineages and analyzed by flow cytometry. To test whether erythropoietin (EPO) could increase red blood cell production, EPO was administered to sham and burn animals and their reticulocyte response was measured on PBD 2 and PBD 7. RESULTS: Burn injury reduced the erythroid cells of the bone marrow from 35% in sham to 17% by PBD 5 and remained at similar level until PBD 21. Myeloid cells, however, increased from 42% in sham to 60% on PBD 5 and 77% on PBD 21. Burn injury reduced reticulocyte counts on PBD 2 and PBD 7 indicating that the erythroid compartment is severely depleted. This depleted compartment, however, responded to EPO but was not sufficient to change red cell production. CONCLUSION: Burn injury skews the bone marrow hematopoietic commitment away from erythroid and toward myeloid cells. Shrinkage of the erythroid compartment contributes to resistance to EPO and the anemia of critical illness.

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.

An Assessment of Research Priorities to Dampen the Pendulum Swing of Burn Resuscitation.

On June 17 to 18, 2019, the American Burn Association, in conjunction with Underwriters Laboratories, convened a group of experts on burn resuscitation in Washington, DC. The goal of the meeting was to identify and discuss novel research and strategies to optimize the process of burn resuscitation. Patients who sustain a large thermal injury (involving >20% of the total body surface area [TBSA]) face a sequence of challenges, beginning with burn shock. Over the last century, research has helped elucidate much of the underlying pathophysiology of burn shock, which places multiple organ systems at risk of damage or dysfunction. These studies advanced the understanding of the need for fluids for resuscitation. The resultant practice of judicious and timely infusion of crystalloids has improved mortality after major thermal injury. However, much remains unclear about how to further improve and customize resuscitation practice to limit the morbidities associated with edema and volume overload. Herein, we review the history and pathophysiology of shock following thermal injury, and propose some of the priorities for resuscitation research. Recommendations include: studying the utility of alternative endpoints to resuscitation, reexamining plasma as a primary or adjunctive resuscitation fluid, and applying information about inflammation and endotheliopathy to target the underlying causes of burn shock. Undoubtedly, these future research efforts will require a concerted effort from the burn and research communities.

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.

A Shift in Myeloid Cell Phenotype via Down Regulation of Siglec-1 in Island Macrophages of Bone Marrow Is Associated With Decreased Late Erythroblasts Seen in Anemia of Critical Illness.

Burn injury has been shown to significantly dampen erythropoiesis in both burn patients and in murine models. Our previous findings elucidated the erythropoietin independent defects in red cell development stages involving erythroid progenitor production and late stage erythroblast enucleation processes. We hypothesized that macrophages (MØ) in erythroblast islands (EBI) could be yet another roadblock impeding erythropoiesis following burn injury. Here we highlight that the methodology to study EBI can be achieved with single cell suspensions using a simple technique such as flow cytometry, as obtaining the central erythroblast island macrophages (EBIMØs) of interest is a delicate process. We elucidated the requisite of EBIMØ from the erythroblast as well as the MØ perspective. In addition to the primary erythropoiesis organ, the bone marrow (BM), spleens were also examined for extra-medullary erythropoiesis. Femurs and spleens were harvested from adult mice (B6D2F1) subjected to 15% total body surface area (TBSA) scald burn (B) or sham burn (S). Total bone marrow cells (TBM) and splenocytes were probed for total erythrons, early and late erythroblasts and EBIMØ by flow cytometry. There was only a marginal increase in the number of EBIMØ after burn, but, between the signatures of EBIMØ, Siglec-1 expression (MFI) was reduced by 40% in B with and a parallel 44% decrease in TBM erythrons in the BM. There were more (2.5-fold) EEBs and less LEBs (2.4-fold) per million TBM cells in B; with a corresponding decrease in Siglec-1 and Ly6G expressions in EBIMØ associated with EEB. Conversely, extra-medullary erythropoiesis was robust in spleens from B. Not only were the numbers of EBIMØs increased in B (p < 0.002), both EEBs and LEBs associated with EBIMØ were higher by 30 and 75%, respectively. Importantly, an increase in Siglec-1 and Vcam1 expressing F480+ splenic macrophages was observed after burn injury. Therefore, stagnant EEBs in the BM after burn injury could be due to low Siglec1 expressing EBIMØ, which perhaps impede their maturation into LEBs and reticulocytes. Repercussion of myeloid cell phenotype specific to BM after burn injury could plausibly account for a defective late stage RBC maturation resulting in anemia of critical illness. Summary Sentence: Characterization of erythroblast island macrophages (EBIMØ) in the bone marrow and spleen at different stages of erythropoiesis after burn injury.

Terminal Maturation of Orthochromatic Erythroblasts Is Impaired in Burn Patients.

Mechanisms of erythropoietin (Epo)-resistant anemia in burn patients are poorly understood. We have recently found that administering a nonselective beta 1,2-adrenergic blocker propranolol (PR) was effective in reversing myelo-erythroid commitment through MafB regulation and increase megakaryocyte erythrocyte progenitors in burn patients' peripheral blood mononuclear cell (PBMC)-derived ex vivo culture system. Having known that Epo-dependent proliferation of early erythroblasts is intact after burn injury, here we inquired whether or not Epo-independent maturation stage of erythropoiesis is affected by burn injury and the relative role of PR on late-stage erythropoiesis. While majority of erythropoiesis occurs in the bone marrow, maturation into reticulocytes is crucial for their release into sinusoids to occupy the peripheral circulation for which enucleation is vital. peripheral blood mononuclear cells (PBMCs) from burn patients were extended beyond commitment and proliferation stages to late maturation stage in ex vivo culture to understand the role of PR in burn patients. Burn impedes late maturation of orthochromatic erythroblasts into reticulocytes by restricting the enucleation step. Late-stage erythropoiesis is impaired in burn patients irrespective of PR treatment. Further, substituting the microenvironment with control plasma (homologous) in place of autologous plasma rescues the conversion of orthochromatic erythroblasts to reticulocytes. Results show promise in formulating interventions to regulate late-stage erythropoiesis, which can be used in combination with PR to reduce the number of transfusions.

Discrete ß-adrenergic mechanisms regulate early and late erythropoiesis in erythropoietin-resistant anemia.

BACKGROUND: Anemia of critical illness is resistant to exogenous erythropoietin. Packed red blood cells transfusions is the only treatment option, and despite related cost and morbidity, there is a need for alternate strategies. Erythrocyte development can be divided into erythropoietin-dependent and erythropoietin-independent stages. We have shown previously that erythropoietin-dependent development is intact in burn patients and the erythropoietin-independent early commitment stage, which is regulated by ß1/ß2-adrenergic mechanisms, is compromised. Utilizing the scald burn injury model, we studied erythropoietin-independent late maturation stages and the effect of ß1/ß2, ß-2, or ß-3 blockade in burn mediated erythropoietin-resistant anemia. METHODS: Burn mice were randomized to receive daily injections of propranolol (nonselective ß1/ß2 antagonist), nadolol (long-acting ß1/ß2 antagonist), butoxamine (selective ß2 antagonist), or SR59230A (selective ß3 antagonist) for 6 days after burn. Total bone marrow cells were characterized as nonerythroid cells, early and late erythroblasts, nucleated orthochromatic erythroblasts and enucleated reticulocyte subsets using CD71, Ter119, and Syto-16 by flow cytometry. Multipotential progenitors were probed for MafB expressing cells. RESULTS: Although propranolol improved early and late erythroblasts, only butoxamine and selective ß3-antagonist administrations were positively reflected in the peripheral blood hemoglobin and red blood cells count. While burn impeded early commitment and late maturation stages, ß1/ß2 antagonism increased the early erythroblasts through commitment stages via ß2 specific MafB regulation. ß3 antagonism was more effective in improving overall red blood cells through late maturation stages. CONCLUSION: The study unfolds novel ß2 and ß3 adrenergic mechanisms orchestrating erythropoietin resistant anemia after burn, which impedes both the early commitment stage and the late maturation stages, respectively.

Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness.

Patients who survive initial burn injury are susceptible to nosocomial infections. Anemia of critical illness is a compounding factor in burn patients that necessitates repeated transfusions, which further increase their susceptibility to infections and sepsis. Robust host response is dependent on an adequate number and function of monocytes/macrophages and dendritic cells. In addition to impaired RBC production, burn patients are prone to depletion of dendritic cells and an increase in deactivated monocytes. In steady-state hematopoiesis, RBCs, macrophages, and dendritic cells are all generated from a common myeloid progenitor within the bone marrow. We hypothesized in a mouse model of burn injury that an increase in myeloid-specific transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homolog B at the common myeloid progenitor stage steers their lineage potential away from the megakaryocyte erythrocyte progenitor production and drives the terminal fate of common myeloid progenitors to form macrophages vs. dendritic cells, with the consequences being anemia, monocytosis, and dendritic cell deficits. Results indicate that, even though burn injury stimulated bone marrow hematopoiesis by increasing multipotential stem cell production (LinnegSca1poscKitpos), the bone marrow commitment is shifted away from the megakaryocyte erythrocyte progenitor and toward granulocyte monocyte progenitors with corresponding alterations in peripheral blood components, such as hemoglobin, hematocrit, RBCs, monocytes, and granulocytes. Furthermore, burn-induced V-maf musculoaponeurotic fibrosarcoma oncogene homolog B in common myeloid progenitors acts as a transcriptional activator of M-CSFR and a repressor of transferrin receptors, promoting macrophages and inhibiting erythroid differentiations while dictating a plasmacytoid dendritic cell phenotype. Results from small interfering RNA and gain-of-function (gfp-globin transcription factor 1 retrovirus) studies indicate that targeted interventions to restore V-maf musculoaponeurotic fibrosarcoma oncogene homolog B/globin transcription factor 1 balance can mitigate both immune imbalance and anemia of critical illness.

Dendritic cell depletion in burn patients is regulated by MafB expression.

Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23­68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.