Panel A shows a flow cytometry plot with gates displaying CD169 on the y-axis and CD45 on the x-axis, with a subplot showing F4/80 on the y-axis and CD64 on the x-axis. Panel B presents flow cytometry plots of CD169 expression in different cell populations. Each plot has CD169 on the y-axis and MHCII on the x-axis. A bar graph shows the percentages of CD169+ cells in each population, with p-values calculated using one-way ANOVA. Panel C displays a flow cytometry plot. The plot has MHCII on the x-axis and CD169 on the y-axis. Panel D shows representative flow cytometry plots of various markers in different macrophage populations. Panel E presents immunostaining images of IBA1, CD169, and DAPI in mouse normal back skin, with a bar graph showing the ratio of CD169 positive cells in the dermis, dermal white adipose tissue (DWAT), and adventitia. Panel F illustrates the experimental scheme for CD169 positive macrophage depletion by anti-CSF1R mAb, with bar graphs showing the counts of different macrophage populations and Langerhans cells, and p-values calculated using Student's t-test.
Identification of CSF1R-dependent CD169+ macrophages in the normal skin. (A) Representative flow cytometry plots with gates showing CD169+ macrophages in the mouse normal back skin (representative of five mice). (B) Representative flow cytometry plots of CD169 expression in skin macrophages (Mϕ), LCs, DCs, and monocytes (representative of five mice). The graph indicated the percentages of CD169+ cells in each cell population. P value was calculated using one-way ANOVA. (C) Three macrophage populations based on MHCII and CD169 expression. (D) Representative flow cytometry plots of CD64, F4/80, CD11c, MHCII, CCR1, CX3CR1, LYZM, MERTK, Ly6C, LYVE1, and CD206 and positivity of DAPI and FVD in CD169+ Mϕ (magenta), CD169− MHCII+ Mϕ (blue), and CD169− MHCII− Mϕ (orange) (representative of at least three mice). (E) Immunostaining of IBA1 (red), CD169 (green), and DAPI (blue) in mouse normal back skin. The ratio of CD169+ cells in the dermis, DWAT, and adventitia was analyzed using Imaris (n = 3). Scale bar = 50 μm. P value was calculated using one-way ANOVA. (F) Experimental scheme for CD169+ macrophage depletion by anti-CSF1R mAb. 200 μg isotype mAb or anti-CSF1R mAb was subcutaneously injected into the mouse back skin on days −3 and −1, and then each skin macrophage population and LC were analyzed on day 0 using flow cytometry (n = 5 for each group, data represent two independent experiments). Each circle represents one mouse. P value was calculated using Student’s t test.