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. 2021 Dec 13;14(1):e14502. doi: 10.15252/emmm.202114502

Figure 7. Proteasome inhibitors are capable of reprogramming human M2 macrophage into M1‐like macrophage.

Figure 7

  • A
    Carfilzomib, Bortezomib, and MLN9708 promote the expression of M1 macrophage markers (Il‐1β, Il‐6, Tnfα) and reduce the expression of M2 macrophage markers (Il‐10, Tgfβ) in macrophages derived from PBMCs. After induced by IL‐4 (20 ng/ml) for 24 h to differentiate into mature M2 macrophages, PBMCs‐derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from cells 6 h after stimulation and the expression of mRNA was quantified through RT‐qPCR. Data from three experiments are presented as the mean ± SD. T‐test was used for statistical analysis of differences between groups. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t‐test).
  • B
    Carfilzomib, Bortezomib, and MLN9708 promote the secretion of inflammatory‐related cytokines in M2 macrophages derived from PBMCs. After induced by IL‐4 (20 ng/ml) for 24 h to differentiate into M2 macrophages, PBMCs‐derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM), or MLN9708 (500 nM). The secretion of IL‐1β, IL‐6, and TNFα was detected through ELISA experiments 24 h after stimulation. Data from three experiments are presented as the mean ± SD. T‐test was used for statistical analysis of differences between groups. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t‐test).
  • C–F
    Carfilzomib, Bortezomib, and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL‐4 (20 ng/ml) for 24 h to differentiate into mature M2 macrophages, PBMCs‐derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM), or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 h after stimulation. (D, F) Statistics represent the proportion of CD80‐ (D) or CD206‐ (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T‐test was used for statistical analysis of differences between groups. *P < 0.05, **P < 0.01 (Student’s t‐test).
  • G
    Carfilzomib, Bortezomib, and MLN9708 enhance the phagocytic ability of macrophages derived from PBMCs. After induced by IL‐4 (20 ng/ml) for 24 h to differentiate into mature M2 macrophages, PBMCs‐derived macrophages were stimulated with Carfilzomib (500 nM), Bortezomib (500 nM), or MLN9708 (500 nM) for 12 h. The indicated drugs‐induced macrophages were starved for 2 h and incubated with L1210‐GFP cells as targets in serum‐free medium for another 2 h. Macrophages were thoroughly washed and stained with fluorochrome‐conjugated anti‐CD11B antibody and analyzed through flow cytometry. Phagocytosis efficiency was determined as the percentage of GFP‐positive cells in CD11B+ population.
  • H
    Statistics represent the percentage of phagocytes and data are means ± SD of three independent experiments. *P < 0.05 (Student’s t‐test).