Fig. 4

A specific subset of Cd163-cells does not express classical microglial markers. (a) Heatmap (a1) and gen-set-score (a2) of the expression of ten selected myeloid derived cell (MDC) specific genes (Cd163-cluster), quantified by Array Microfluidic Cards, in human hippocampus. (a1) Color key code represents Z-score distribution, from −1.5 (blue) to 1.5 (red). Subjects were ordered in an unsupervised manner using hierarchical clustering (Ward´s linkage method, Manhattan distance). At the top of the heatmap, a color bar represents each subject Braak stage (Braak 0, n = 8, Braak II, n = 15; Braak III-IV, n = 15, and Braak V–VI, n = 23). (a2) Braak dependent variations of the gene set score of Cd163-cluster. Note the clear up-regulation of these specific MDC genes mainly in Braak V–VI subjects. (b) Graphs represented active microglia (b1) and perivascular macrophages (b2). The expression of all different genes was assayed in parallel using fluidic cards. Data were shown as violin plots including individual samples. Significance, indicated in the figure, was tested using the Kruskal–Wallis test followed by the Dunn test. (c) Imaris-generated 3D surfaces images (c1) reveal different plaque-associated myeloid cells in the triple Cd163/Iba1/Abeta immunofluorescence (c2), showing the co-localization (yellow color, c3) between Iba1 and Cd163 in the nearness of two Ab plaques (white color). (d) Confocal microscopy of triple Iba1/Trem2/Cd163 immunofluorescence (d1), showing the corresponding Imaris-generated 3D reconstruction of the co-localization channel (yellow color) between the three markers analyzed (d2, d3). (e) Graphs showed the quantitative analysis of the different microglial/myeloid cells per Abeta plaque observed in c and d. Each point represented the proportion of the different cell population vs the total number of cells per plaque in AD cases (n = 3–7 BraakV–VI samples and 18–73 plaques for Iba1/Cd163/ or Trem2/Cd163 experiments, respectively). Scale bars: c1, c2 and c3, 30 μm; d1, d2 and d3, 10 μm