Clec4f⁺Marco^high macrophages are important for erythrocyte clearance. (A) Comparisons of the expression levels of proinflammatory and anti-inflammatory markers in 7 distinct macrophage subsets identified by scRNA-seq of liver cells from TNF-challenged 2-month-old A/A (n = 2) and S/S mice (n = 2). Among them, the Clec4f⁺ Kupffer cells expressed higher levels of Mrc1 and Marco than of other groups, identifying them as restorative macrophages. (B) Representative transmission electron microscopic images showing increased erythrophagocytosis in hepatic macrophages of S/S mice than those in A/A mice. There were dramatically increased interactions between erythrocytes (red) and macrophages (blue) and phagocytosis of erythrocytes by macrophages in the liver of S/S mice. (C) 3D rendering of confocal images showing the clearance of erythrocytes by macrophages in the liver of A/A and S/S mice. Side views are shown on the left and bottom. The sections were stained with primary antibodies to MARCO (a scavenger receptor), macrophages (F4/80), and erythrocytes (Ter119). There was a dramatic increase of Marco^high macrophages in the liver of S/S mice and these Marco^high macrophages showed increased clearance of erythrocytes in the liver of S/S mice. DAPI cell nuclear staining (n = 3 mice per group). (D) Quantification of MARCO fluorescence intensity (left) and the colocalization of MARCO⁺ macrophages with erythrocytes (right). Data represent mean ± standard deviation. ∗∗∗P < .001; ∗∗∗∗P < .0001, 2-tailed, unpaired Student t test.