Panel A shows flow-cytometry gating plots distinguishing infiltrating and resident hepatic macrophage populations from mouse liver samples. Panel B presents scatter plots comparing phagocytic receptor expression in infiltrating and resident macrophages from Mdr2-/- mice and control counterpart (Mdr2+/+). Panel C shows t-SNE maps visualizing macrophage subclusters and receptor-expression patterns in healthy and diseased livers. Panel D presents scatter plots quantifying Annexin-V-positive neutrophils, T cells, and parenchymal cells in Mdr2-/- livers over time. Panel E shows flow-cytometry gating plots and bar graphs identifying phagocytic-receptor-enriched and receptor-deficient hepatic macrophage populations in Mdr2-/- livers. Panel F presents bar graphs quantifying conjugated and unconjugated bile-acid accumulation within distinct Mdr2-/- hepatic macrophage subpopulations. Panel G shows a bar graph comparing Lxra messenger-RNA expression between hepatic Mdr2-/- macrophage subpopulations with differing efferocytic profiles. Panel H presents scatter plots comparing CCR2, CX3CR1, MHC-II, and CD206 expression across hepatic macrophage subsets in Mdr2-/- livers.
Hepatic Mφ in Mdr2 −/− mice show an altered efferocytic signature and accumulation of bile acids. (A) Flow cytograms representing the gating strategy adopted for analysis of hepatic Mφ. Dead cells and doublets were excluded. Mφ were gated as CD45+Ly6G−CD11cdim/loCD11b+F4/80+ and further divided into infiltrating (Ly6C+CCR2+CX3CR1+) and resident (Ly6C−CCR2−CX3CR1−CLEC4F+) Mφ. (B) Pooled data reporting frequency of infiltrating and resident Mφ expressing the receptors CD36, MERTK, TIM4, and CD64 isolated from the liver of Mdr2+/+ (clear dots) and Mdr2−/− (filled dots) mice at 12 wk of age. n = 11–12 mice/time point. Mean ± SEM. Mann–Whitney U test. (C) t-SNE maps, concatenated from 11 to 12 mice/condition, showing the expression of the receptors CD36, MERTK, TIM4, and CD64 in infiltrating and resident Mφ within the Ly6C+ and Ly6C− clusters in 12-wk-old Mdr2+/+ and Mdr2−/− mice. (D) Percentage of Annexin V+ cells within neutrophils (CD45+Ly6G+), T cells (CD45+CD3+), and parenchymal cells (CD45−) isolated from the liver of Mdr2−/− mice at 8, 12, and 35 wk of age and analyzed by flow cytometry. n = 3–4 mice/time point. (E) Flow cytograms depicting gating strategy for FACS-sorting of hepatic Mφ (CD45+Ly6G− CD11b+F4/80+) isolated from 8- to 12-wk-old Mdr2−/− mice, based on the expression of the efferocytic receptors MERTK and CD36 (as Phag. R–deficient Mφ or Phag. R–enriched Mφ). A bar graph reporting their absolute number per liver is also shown. (F) Quantification of conjugated bile acids (CBAs), unconjugated bile acids (UBAs), and the sum of all analyzed bile acids are classified based on bile acid classes. Data are presented as fold change relative to Phag. R–deficient Mφ. Four independent experiments were performed. Each dot corresponds to a pool of 3–6 mice. n = 4–7; mean ± SEM, Mann–Whitney U test. (G)Lxra mRNA amount in Phag. R–enriched Mφ or Phag. R–deficient Mφ FACS-sorted as reported in E from 8- to 12-wk-old Mdr2−/− mice, detected by qPCR. Four independent experiments were performed. n = 6–13 liver pooled/experiment; Mean ± SEM. Mann–Whitney U test. (H) Frequencies of Phag. R–enriched Mφ or Phag. R–deficient Mφ expressing CCR2, CX3CR1, MHC-II, and CD206 (out of CD45+Ly6G−CD11c−CD11b+F4/80+ cells), retrieved from the liver of Mdr2−/− mice. n = 12 mice; mean ± SEM. Mann–Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.