Beta3-Adrenergic Blockade Restores Erythropoietic Homeostasis in Anemia of Critical Illness
Authors: Muthumalaiappan Kuzhali , Hasan Shirin , Kini Ameet , Saini Pravesh , Walczak Julia , Baldea Anthony J 
Published: October 2019

Abstract
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.
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Disclosures
No relevant conflicts of interest to declare.