Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

Figures

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  Figure 1. Cell viability by MTT assay on RAW 264.7 cells. Cells were exposed for 24 h to H2O2 or Triton (0.05 and 0.1 mM), and to L2 or BNP (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2 and 100 ng/mL). n = 8–9/condition. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding untreated control group.

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  Figure 2. Effects of L2 on LPS-induced inflammatory cytokines by ELISA assay. Levels of (a) TNF-α, (b) IL-6 and (c) IL-10, and (d) TNF-α/IL-10 ratio after L2 treatment of LPS-activated RAW 264.7 cells. After LPS (1 µg/mL) priming for 24 h, cells were treated with different concentrations of L2 (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 ng/mL) for 24 h to determine TNF-α and IL-6, and for 48 h to assess IL-10 levels. Natriuretic peptide receptor antagonist isatin (0.1 mM) was added 20 min before L2 treatment. n = 3–6/condition. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS untreated group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. corresponding control L2 untreated group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. corresponding LPS + L2 treated group.

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  Figure 3. Effects of BNP on LPS-induced inflammatory cytokines by ELISA assay. Levels of (a) TNF-α, (b) IL-6 and (c) IL-10, and (d) TNF-α/IL-10 ratio after BNP treatment of LPS-activated RAW 264.7 cells. After LPS (1 µg/mL) priming for 24 h, cells were treated with different concentrations of BNP (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 ng/mL) for 24 h to determine TNF-α and IL-6, and for 48 h to assess IL-10 levels. Natriuretic peptide receptor antagonist isatin (0.1 mM) was added 20 min before BNP treatment. n = 3–6/condition. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS untreated group; $ p < 0.05, $$ p < 0.01 vs. corresponding control BNP untreated group; # p < 0.05, ### p < 0.001 vs. corresponding LPS + BNP treated group.

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  Figure 4. Effect of L2 and BNP on macrophage polarization in LPS-activated RAW264.7 cells. (a) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68+/CD206− cells, Q1 population) and M2-like macrophages (CD68+/CD206+/MRC-1, Q2 population) in control and treated cells. (b,c) Effect of L2 on M1 and M2 macrophage subtype expression. (d,e) Effect of BNP on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 or BNP (0, 0.2, 0.4 and 0.8 ng/mL) and isatin (0.1 mM), added 20 min earlier. (f) M2/M1 ratio in LPS-activated RAW264.7 cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68+/CD206ࢤ cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$ p < 0.01,…

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  Figure 5. Effect of L2 on macrophage polarization in LPS-activated RAW264.7 cells after interleukin-10 inhibition. (a,b) Effect of interlekin-10 (IL-10) on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS for 24 h followed by 48 h treatment with or without exogenous IL-10 (0, 10 and 20 ng/mL). (c,d) Effect of L2 on M1 and M2 macrophage subtype expression after IL-10 inhibition. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 (0, 0.4 or 0.8 ng/mL) and IL-10 inhibitor at 10 µg/mL, added 20 min earlier. (e) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68+/CD206− cells, Q1 population) and M2-like macrophages (CD68+/CD206+/MRC-1, Q2 population) in control and treated cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68+/CD206− cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro…

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  Figure 6. Protocol to investigate the effect of L2 on macrophage polarization in vitro on lipopolysaccharide (LPS)-activated RAW264.7 macrophages and to explore the associated underlying mechanisms.