Macrophage Depletion Leads to Modification of Thalassemic Phenotype.

Abstract 2023

Poster Board I-1045

Macrophages represent an important link between erythropoiesis and iron metabolism. They support maturation and differentiation of erythroblasts and clear senescent red cells from circulation, thus recycling iron within the hematopoietic system. However, little is known about the role of macrophages in conditions of ineffective erythropoiesis like beta-thalassemia intermedia (β-TI) or sickle cell anemia (SCD). Our goal was to study the role of macrophages under such conditions, where there is a large expansion of the erythron, the lifespan of the red cells is decreased, and both iron absorption and organ iron content are increased.

Clodronate liposomes have been shown to efficiently eliminate macrophages from spleen, liver and bone marrow when administered by intravenous injection. Therefore, we utilized this technique to eliminate macrophages from the spleen, liver and bone marrow of β-TI and SCD mice. Three different treatment schedules were used; a) acute high dose; b) medium term split dose; c) chronic low dose. Animals were analyzed for erythropoietic and pathological parameters.

All treatments effectively depleted splenic and bone marrow macrophages as shown by flow citometric and immunohistochemical analyses. At doses that led to only a small decrease of hemoglobin (Hb) levels in normal mice, clodronate treatment induced high level of mortality in β-TI and SCD mice (70% and 75%, respectively). In contrast, administration of clodronate to mice affected by beta-thalassemia major, mice that do not make any adult or fetal Hb/mature red cells, caused no mortality suggesting that circulating abnormal red cells might be responsible for the mortality observed in β-TI and SCD mice. Pathological analysis of β-TI mice revealed the formation of thrombi in the lungs and heart to be the likely cause of death. Analysis of SCD mice is in progress. β-TI and SCD have been described as hypercoagulable states with a high risk of thrombosis. Damaged red cell membranes and ROS have been implicated as factors that increase the risk of thrombosis in β-TI and SCD patients. We hypothesize that clodronate treatment enhances this process. To test this hypothesis, we administered an anti-coagulant (heparin) and an anti-oxidant (NAC) to β-TI mice. Both these treatments resulted in increased survival from 31% to 65% compared to clodronate alone. Analysis of SCD animals is in progress. β-TI mice that survived clodronate treatment exhibited a milder thalassemic phenotype, characterized by increased Hb (∼1.5g/dL), HCT (∼7%) and RBC count (∼2e6 cells/ul), partially associated with a decreased RBC clearance. Ineffective erythropoiesis and splenomegaly were also ameliorated (∼40% decrease in spleen size). Animals depleted of macrophages also showed a marked decrease in serum iron parameters, but no difference in their organ iron concentrations. Analysis of hepcidin expression is in progress. To further corroborate these observations, β-TI mice are being crossed with mice expressing the human diphtheria toxin (CD11b-DTR) which allows for conditional depletion of macrophages.

Our data suggests that macrophages play a major role in conditions of abnormal red cell production and ineffective erythropoiesis. In the former case, they have an important protective function related to the high risk of thrombosis associated with β-TI and SCD. In the latter, macrophages may act as negative modulators of erythropoietic development. The amelioration of splenomegaly and ineffective erythropoiesis observed suggests that erythroid cells might proliferate less and/or differentiate more in the absence of macrophages. One hypothesis is that macrophages sense erythroid maturation and/or differentiation and act as modulators of these processes to ensure the release of high quality red cells into the circulation. Second, macrophages, under conditions of iron overload, might produce inflammatory cytokines that further impair erythropoiesis in thalassemia. Analysis of RNA extracted from macrophages and of inflammatory cytokines in β-TI mice before and after clodronate treatment is in progress.