Figure 5
IL-6, but not OSM, directly acts on macrophages to potentiate their alternatively activated phenotype. Bone-marrow derived macrophages were cultured from naïve C57BL/6 for either 18 or 30 hours with recombinant OSM, IL-6, IL-4/IL-13, alone or in combination as indicated. AMs were isolated from naïve mice by adherence. BMDMs were either lysed and processed for (A) arginase activity assay, or analysed by flow cytometry to show (B) percentage of arg1+CD206+ macrophages, (C–D) fold change of percentage of IL-4Rα+ cells from the arg1+CD206+ population relative to controls. At least 100,000 events were captured per condition, and repeated twice. (E) Western blot analysis of cell lysates probed for arginase-1, pSTAT6, pSTAT3, and Actin. (F) Densitometry of pSTAT6 (corrected to STAT6), (G) arginase-1 (corrected to actin) and (H) pSTAT3 (corrected to STAT3) represented as a fold-change relative to control. (I) Normalized mRNA counts of Osmrβ and Il6rα from control (unstimulated) BMDMs and AMs. Flow cytometry and western blot results are from experiments completed in duplicates. The appropriate Western blot area depicting the antibody band was cropped and enclosed by black boxes, as indicated above. All samples were derived at the same time and processed in parallel. For Nanostring gene expression, lower than 5 counts was considered not detected “ND”. Bar graphs represent mean ± SEM from 2-3 replicates per group (showing one of two representative experiments), *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.05; ##P < 0.01; ###P < 0.001; *represent a difference between any sample relative to the control (control vs. IL-4/IL-13, IL-6 vs. IL-4/IL-13 + IL-6); #represent a difference between the indicated groups. Significance was established using GraphPad, Prism 7.0 with One-way ANOVA and non-parametric independent Student’s t-test.
