Figure 4: BIO induces anti-inflammatory M2 macrophage polarization in differentiating monocytes.

(A) Schematic of the co-culture system used to analyze the influence of rat cardiac fibroblasts on human THP-1 monocyte differentiation into macrophages. (B) Co-culture of cardiac fibroblasts with THP-1 monocytes for 72 hours induced macrophage differentiation. Treatment with 200 nM PMA was used as a positive control for inducing macrophage differentiation. n = 4, *p < 0.05 vs. monocultures, t-test. (C) Addition of 5 μM BIO or 10 ng/mL IL-4 to the co-cultures induced IL-10 expression. n = 4, *p < 0.05, t-test. (D) Treatment of co-cultures with BIO induced the expression of IL-10 mRNA. n = 4, *p < 0.05 compared to untreated co-cultures, t-test. (E) Treatment of co-cultures with BIO or IL-4 increased Arg activity. Treatment with LiCl did not increase Arg activity. n = 4, *p < 0.05, t-test. (F) Schematic of the ex vivo system used to analyze the effect of BIO treatment on macrophage differentiation in mouse bone marrow-derived monocytes (BMDMs). The images of the mouse and bone are used under the Creative Commons Licenses (CC BY-SA 2.0 FR and CC BY-SA 3.0; bone image citation: “Blausen gallery 2014”. Wikiversity Journal of Medicine. DOI: 10.15347/wjm/2014.010. ISSN 20018762; mouse image citations: David Liao and User:Rama). (G) Conditioned media (CM) from rat cardiac fibroblasts can induce macrophage differentiation in BMDMs after for 72 h. n = 4, *p < 0.05, t-test. (H) Differentiating BMDMs treated with cardiac fibroblast CM and BIO showed increased expression of IL-10 mRNA. Treatment with LiCl did not increase IL-10, whereas treatment with IL-4 or 10 ng/mL LPS increased IL-10 expression. Densitometry analysis indicated that treatment with BIO or IL-4 induced IL-10 expression to a greater degree than BMDMs treated with LPS or CM alone. n = 4, *p < 0.05 vs. BMDMs treated with cardiac fibroblast CM, ¶p < 0.05 vs. BMDMs treated with CM and LPS, t-test. (I) Real-time PCR analysis indicated that treatment of BMDMs with BIO increased the expression of IL-10. As a positive control for M2 macrophage polarization, BMDMs were treated with 50 ng/mL TGF-β and 10 ng/mL IL-4 for 72 hours. n = 4, *p < 0.05 vs. BMDM treated with LiCl and LPS; ¶p < 0.05 vs. BMDMs differentiated in the presence of LPS, t-test. (J) Treatment of differentiation BMDMs with BIO or IL-4 increased activity of the M2 macrophage marker, arginase-1. Moreover, BIO treatment increased Arg activity in the presence of LPS. n = 4, *p < 0.05, t-test.