Subtype-Specific Surface Proteins on Adipose Tissue Macrophages and Their Association to Obesity-Induced Insulin Resistance

Figures

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  Figure 1. Identification and characterization of macrophage populations in blood and adipose tissue of humans with obesity. (A) Flow cytometry gating scheme used to identify monocytes and macrophages. Arrows indicate the sequence of gating. (B) Representative flow cytometry plots showing CD206 and CD11c expression on monocytes (CD11c+CD206-), M1-(CD11c+CD206+) and M2-like (CD206+CD11c-) macrophages from PBMC, SAT, and VAT samples from one patient. (C) The total monocyte and macrophage population as a fraction of total CD45+ cells in PBMC (n=47), SAT (n=54), and VAT (n=54). (D) Monocytes, M1- and M2-like macrophages (MΦ) as a fraction of the total monocyte and macrophage population in PBMC (n=47), SAT (n=54), and VAT (n=54). (E) Representative stainings for the surface proteins shown in (F-L). (F-L) Scatter plots showing expression of surface proteins (F) CCR2, (G) CD44, (H) CD16, (I) CD14, (J) HLA-DR, (K) CD40, and (L) CD163 on monocytes (grey), M1 (red)-, and M2 (blue)-like macrophages (MΦ’s) from PBMC (n=47), SAT, and VAT (n=54). For (F, H, L) scatter plots showing percentage of cells expressing the proteins. For (G, I-K) scatter plots showing expression (mean fluorescence intensity,…

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  Figure 2. Surface proteome analysis of adipose tissue macrophages. (A) Experimental overview of the surface proteome screening using the LEGENDscreen™ kit. (B) Expression of surface proteins on blood monocytes (Blood-mono) and AT myeloid cells. Heatmap based on the percentage of cells expressing each surface protein, with a cut-off of minimum 20% expression by at least one of the populations. (C) Expression of surface proteins on AT M1- and M2-like macrophages and monocytes (mono). Heatmap based on the percentage of cells expressing each surface protein, with a cut-off of minimum 20% expression by at least one of the populations. Representative staining of a selection of surface proteins expressed on M1-like (dark green) or M2-like (light green) ATMs.

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  Figure 3. Verification of surface protein expression on M1- and M2-like adipose tissue macrophages from humans with obesity. (A) Scatter plots showing expression of surface proteins CCR2, CD85a, CD48, CD371, CD26, CD116, CD51, and integrin α9β1 on monocytes (grey), M1 (red)-, and M2 (blue)-like macrophages (MΦ’s) from PBMC (n=17), SAT (N=23), and VAT (n=23). (B) UMAP plots showing expression of the surface proteins analyzed in (A) with blue indicating low and red indicating high expression as well as UMAP plots colored according to cell origin (top) and tissue origin (bottom).

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  Figure 4. Insulin resistance, dyslipidemia, and inflammatory markers in SAT and VAT. (A) Pearson correlations between monocytes, M1-, and M2-like macrophages and M1/M2 ratio in SAT and VAT and biochemical parameters, corrected for BMI (n=80). Size and color intensities of circles indicate correlation coefficients. Red squares indicate significant (p<0.05) correlations. (B) Relative gene expression of CD68, MCP1, TREM1, TNF, IFNG, IL6, CD163, and TREM2 for n = 46 (39 matched) SAT and VAT samples. Results are presented as fold changes (2-ΔΔCT) of target gene and normalized to IPO8. Expression levels of the SAT samples were set to 100%. (C) Representative pictures showing from SAT and VAT samples stained with CD68 antibody identifying crown-like structures (CLS) defined as three or more macrophages surrounding an adipocyte. (D) Graph showing numbers of CLS in SAT and VAT for n = 46 (39 matched) SAT and VAT samples. (E) Graph showing median adipocyte size in µm2 in SAT and VAT for n = 53 SAT and VAT samples. Bars in (B) represent mean and error bars represent SD. For (D, E) line represents the mean and error bars represents SD. The Wilcoxon matched pairs signed rank test was used for…