FIGURE 6.

Differential contribution of IL-10, HO-1, and IL-4 to flAcrp-mediated M2 polarization in RAW 264.7 macrophages. A, RAW264.7 macrophages were treated with gAcrp or flAcrp for 18 h, and IL-4 mRNA accumulation was measured by qRT-PCR, and IL-4 protein expression was assessed by Western blot (inset). Kupffer cells isolated from chow-fed control rats were cultured with flAcrp for 18 h, and accumulation of IL-4 protein in the media was assessed by Western blot (inset). flAcrp increased IL-4 protein by 2.0 ± 0.4-fold (n = 5) in Kupffer cells and 1.6 ± 0.2-fold (n = 7) in RAW264.7 macrophages when compared with untreated cells. B–D, RAW264.7 cells were transfected or not with either 100 nm of IL-10, IL-4, HO-1. or scrambled siRNA and then cultured with or without 45 nm flAcrp for 18 h. Arg-1, Mgl2, and IL-4Rα mRNA were measured by qRT-PCR and normalized to 18 S mRNA. n = 4. Values from flAcrp-treated cells with different superscripts (a–c) are significantly different from each other, p < 0.05. E, Kupffer cells were treated with gAcrp or flAcrp for 60 min, and phosphorylation of STAT6 and STAT3 was assessed by Western blot. Expression of STAT6, STAT3, and Hsc70 was measured as loading controls. F, RAW264.7 macrophages were transfected with siRNA against IL-10 or IL-4 and then stimulated with gAcrp or flAcrp for 60 min. Phosphorylation of STAT6 and STAT3 was assessed by Western blot. Expression of Hsc70 was measured as a loading control. E and F, images are representative of at least four independent experiments.