Figure 1.

Mitapivat reduces transfusion burden in β-thalassemia mice exposed to chronic transfusion with associated repro-gramming of splenic macrophage phenotype. (A) Experimental study design to assess the effects of mitapivat on hematologic phenotype of β-thalassemia (β-thal) mice exposed to chronic transfusion. (B) Hemoglobin (Hb) changes over time in transfused (Tr.) β-thal (Hbb^th3/+) mice treated with either vehicle or mitapivat (50 mg/kg twice daily [BID]) shown as single animals (n=3 vehicle-treated mice; n=4 mitapivat-treated mice). Grey dotted line shows the transfusion threshold (10.5 g/dL). (C) Transfusion time intervals in β-thal (Hbb^th3/+) mice treated with either vehicle or mitapivat (50 mg/kg BID). Data are presented as means ± standard error of the mean (SEM) (n=3 vehicle-treated mice; n=4 mitapivat-treated mice); ^#P<0.05 compared to vehicle-treated transfused β-thal mice. (D) Iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in spleen from Hbb^th3/+ mice treated with either vehicle or transfusion plus vehicle or transfusion plus mitapivat. One representative image from 3 with similar results. Left panel: quantification of iron staining in spleen. Data are mean ± SEM (n=3). *P<0.05 compared with vehicle Hbb^th3/+ mice and ^#P<0.05 compared with vehicle-treated transfused Hbb^th3/+ mice. (E) Flow cytometric analysis (CD44⁺Ter119⁺ and cell size markers, see also the Online Supplementary Figure S2) of bone marrow and spleen from Hbb^th3/+ mice exposed to either vehicle or to chronic transfusion with and without mitapivat treatment (see also Matte et al.⁵). Data are mean ± SEM (n=3-4). *P<0.05 compared with vehicle Hbb^th3/+ mice and ^#P<0.05 compared with vehicle-treated transfused Hbb^th3/+ mice. (F) Maturation index as ratio between pop II (Baso E.) and pop IV (Ortho E.) in bone marrow and spleen from Hbb^th3/+ mice treated with either vehicle or exposed to chronic transfusion with or without mitapivat, analyzed by flow cytometry. Data are mean ± SEM (n=3-4). (G) Flow cytometric quantification of M1 (CD80) and M2 (CD206) expression on spleen macrophage cell surface from wild-type (WT) or Hbb^th3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Spleen macrophages (MΦ) were isolated with the GentleMACS cell dissociator (Miltenyi Biotech, Germany). MΦ were identified and gated as CD45+/F4/80+ cells. Anti-CD45 PE-Cy5.5, F4/80 PE, CD206 PerCP-Cy5.5 and CD80 were from BioLegend, USA. Data are mean ± SEM (n=3-4). MFI: mean fluorescence intensity; RBC: red blood cells.