Fig. 6: Reduced erythrophagocytosis by Muc2^−/− macrophages.

a, b Erythrocytes from Muc2^−/− and Muc2^+/+ littermates were isolated, labeled with a lipophilic dye PHK26 ex vivo and injected back into congenic recipients. Blood was drawn from PHK26⁺ erythrocytes recipient animals at the indicated time points and RBCs were stained with the erythrocyte marker Ter119 to identify PKH26⁺ RBCs. a Representative dot plots (one of three experiments) showing remaining labeled RBCs in the circulation after 16 h (n = 6) and b quantification showing percentage of PKH26⁺TER119⁺CD45⁻ cells remaining in the circulation at indicated time points (n = 6). Data are representative from three independent experiments. c Distribution of PKH26⁺CD45⁺ leukocytes in the spleen after 16 h post labeled RBCs challenge showing uptake of erythrocytes by these cells. Cells were gated as CD45⁺CD3⁻CD19⁻NK1.1-leukocytes in the spleen. Data represents three independent experiments, means ± SEM (n = 5 per group). d Representative data (one of three experiments) on distribution of PKH26⁺F4/80⁺ macrophages in spleen 16 h post labeled RBCs injection in mice as visualized by a Quorum WaveFx spinning disk confocal upright microscope. Labeled RBCs from one genotype were injected in the same genotype of mice (homologous group) can be seen in circulation (arrow, upper left panel) in Muc2^+/+ spleen as compared to Muc2^−/− animals where they seem to be out of the circulation. Quantitative analysis of percentage of PKH26⁺F4/80⁺ cells in spleen suggests inefficient erythrophagocytosis in Muc2^−/− spleen. Means ± SEM (n = 5), paired Student’s t test. e Quantitative analysis of RBCs aggregates in the spleen suggests significantly higher proportion of senescent RBCs were not phagocytosed in Muc2^−/− littermates (n = 5–8). f Representative images showing presence of macrophages in spleen after heterologous transfer (RBCs from Muc2^+/+ were transferred into Muc2^−/− littermates and vice versa) with labeled RBCs. Quantitative analysis of percentage of PKH26⁺F4/80⁺ cells show inefficient clearance of Muc2^+/+ RBCs in Muc2^−/− spleen (n = 5–8). g Quantitative analysis of PKH26⁺ RBCs uptake by splenic macrophages in bone-marrow chimeras. Data represents two independent experiments. Means ± SEM (n = 3), paired Student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001.