Adipose Tissue-Derived Mesenchymal Stromal Cells from Ex-Morbidly Obese Individuals Instruct Macrophages towards a M2-Like Profile In Vitro

Figures

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  Fig. 1. Characterization and quantification of macrophages in the subcutaneous white adipose tissue of overweight (OW) and ex-morbidly obese (Ex-Ob) subjects. Hematoxylin-Eosin staining of OW (A) and Ex-Ob (B) adipose tissue. Immunohistochemistry for CD68 (D, E), FXIII-A (G, H), and CD206 (I, J) in OW and Ex-Ob. (C) Number of adipocytes per microscopic field of view. Bars express mean±SEM (p=0,0846); (F) Percentage of CD68+ (n=9 for OW and n=15 for Ex-Ob, *p<0.0001) and FXIII-A+ (n=9 for OW and n=17 for Ex-Ob, p=0,3738) macrophages in relation to the number of adipocytes. (K) Correlation analysis between the number of CD68+ macrophages and the BMI of Ex-Ob patients, immediately before the plastic surgery (Spearman’s correlation=0.4545 and linear correlation=0.1336). Immunofluo-rescence for CD68 (green, L, P, O, S) and FXIII-A (red, M, Q, O, S) in OW (L∼O) and Ex-Ob (P∼S) samples. Dapi (blue, N, R, O, S) stains nuclei. Merged sections (O, S). Scale bars=25 μm.

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  Fig. 2. Immunophenotype of ADSCs isolated from Ex-Ob and OW patients. FACS analysis of the hematopoietic markers CD45 (A) and CD14 (B) in cultured ADSCs from Ex-Ob patients (solid red lines). (C∼L) Histograms showing the expression of CD34 (C, D), CD31 (E, F), CD105 (G, H), CD44 (I, J) and CD90 (K, L) antigens in cultured ADSCs from Ex-Ob (solid red lines) and OW (solid blue lines) individuals. Solid gray lines in histograms show the negative control. Data are representative of n=3 Ex-Ob and n=3 OW subjects.

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  Fig. 3. Expression of CD16 in peripheral blood CD14++ monocytes following coculture with stromal cells. (A, B) Sorting strategy of peripheral blood monocytes showing selected region of FSC versus SSC. (A) combined with the region of CD14Hi, CD16− events (B). (C, D) post-sort analysis showing 96.4% pure CD14Hi, CD16− cells. (E) Experimental design of CD14Hi, CD16− selected peripheral blood monocytes co-cultured with BMSC and ADSCs. (F∼I) FACS analysis of CD14 and CD16 expressions by sorted monocytes after 48 h of culture in contact with OW-ADSCs (F), Ex-Ob-ADSCs (G), BMSCs (H), and plastic (control; No MSC) (I). Numbers represent the percentage of positive cells inside the region. (J) Percentage of CD16+ cells within the CD14+ cell population. Bars express mean±SEM (*p<0,05). Data of n=3 independent experiments.

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  Fig. 4. Expression of macrophage markers by unsorted peripheral blood monocytes cocultured with stromal cells and challenged with LPS. (A) Experimental design: Whole fraction, unsorted monocytes obtained from peripheral blood were maintained in culture for seven days, then co-cultured with OW-ADSCs, Ex-Ob ADSCs, and BMSCs for another four days, and finally treated with LPS for 18 hours. At the end of the experiment, cells were harvested for flow cytometry (B∼E) and the supernatants were harvested for ELISA (F∼I). (B∼E) Histograms showing the expression of CD14 (B), HLA-DR (C), CD206 (D), and CD86 (E) by macrophages in co-culture with Ex-Ob ADSCs (solid red line), OW ADSCs (solid black line), BMSCs (solid blue line), and alone (solid green line). Isotype controls are shown as solid gray line. Data are representative of n=4 independent experiments. (F∼I) Mean fluorescence intensity (MFI) of CD14 (F), HLA-DR (G), CD206 (H), and CD86 (I) expressions. Bars show mean±SEM of n=4 independent experiments. *p<0,05. MC: macrophage-only cultures.

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  Fig. 5. Secretion of cytokines by LPS-activated macrophages cultured for 4 days in the absence (MC, macrophage-only) or presence of stromal cells and stimulated with LPS. (A) Representative histogram of the FACS analysis of the intracellular TNF-α expression in CD14+ macrophages cultured in the absence (solid green line) or presence of Ex-Ob ADSCs (solid red line), OW ADSCs (solid black line), and BMSCs (solid blue line). Isotype control is represented by the solid gray line. Percentage of CD14+ TNF-α+ cells (B) and the MFI for TNF-α (C). Data show mean±SEM of n=3 independent experiments. (D∼G) Concentration of TNF-α (D), IL-10 (E), MIP-1α (F), and MIP-1β (G) in the supernatants of macrophages cultured alone (MC) or in the presence of Ex-Ob-(MC+Ex-Ob ADSC) and OW-(MC+OW ADSC) derived ADSCs and BMSCs (MC+BMSC). Ex-Ob ADSC, OW ADSC, and BMSC represent the conditions where stromal cells were cultured alone. Data show mean±SEM of n=3 independent experiments. *p<0.05.