Fig. 3: Multiplexed flow cytometry reveals the regulatory action of GSDME on infiltration and status of macrophages/monocytes in the vascular wall of AAA.

A Representative experimental design of multiplexed flow cytometry in CD45⁺ immune cells isolated from AAA lesion vasculature. B High-dimensional analysis of multiplexed flow cytometry using an unbiased nonlinear dimensionality reduction algorithm (t-distributed stochastic neighbor embedding, t-SNE) to identify clustering of subpopulations in CD45+ cells isolated from AAA lesion vasculature. Live CD45⁺ leukocyte cells were gated with macrophages/monocytes-related markers (CD11b, Ly6G, F4/80, TIM4, MHCII, and Ly6C). The multiplexed flow cytometry results were concatenated, transformed, and plotted in 2D t-SNE plots using R software. The subgroups of macrophages/monocytes are indicated in the image. C Comparison of t-SNE dimensionality reduction and embedding of CD45⁺ cells pooled from vasculatures of WT (red) and KO (blue) mice with AAA. D Heatmap showing the differences in t-SNE dimensionality reduction and embedding of CD45⁺ cells from vasculatures of WT and KO mice with AAA. Four different regions (R1 to R4) are indicated by dotted lines. Their properties are indicated in (B). E Representative flow cytometry plots and quantification analyses with manual gating on the proportions of neutrophils, macrophages, and monocytes within CD45⁺ cells from vasculatures of WT and KO mice with AAA. F Representative flow cytometry plots and quantification analyses on the subclusters of macrophages by markers of TIM4 and MHCII. The TIM4⁺ macrophages, MHCII⁺ macrophages, and double-negative macrophages (DN-macrophages) were gated and calculated. G Representative flow cytometry plots and quantification analyses on the subclusters of monocytes by markers of Ly6C. The Ly6C^hi monocytes, Ly6C^int monocytes, and Ly6C^lo monocytes were gated and calculated. H Representative immunofluorescence staining and quantitative analysis of CD68 and F4/80 in lesion vasculature of WT and KO mice with the P-AAA model. Data are presented as mean ± SEM. N = 6 biological replicates. Unpaired Student’s t-tests were used. P-AAA refers to PPE + BAPN-induced AAA.