Figure 4

The immunomodulatory effect of mouse BMSCs on macrophages is dependent on hepcidin. IL-10 (A–D) and TNF-α (E–G) levels in supernatants of wild-type (WT) or Hamp^−/− BMSCs co-cultured with macrophage cell lines or peritoneal lavage cells after overnight incubation followed by 6 h of LPS stimulation. IL-10 ELISA measurements from SNs of co-cultures of different types of macrophages with WT and Hamp^−/− BMSCs. (A) Co-culture of RAW264.7 cells and (B) J774.2 cells with BMSCs derived from either WT or Hamp^−/− mice stimulated with 1 µg/mL LPS. (C) Co-culture of peritoneal lavage cells with BMSCs. (1 µg/mL LPS). (D) Co-culture using RAW264.7 macrophages, wild type BMSCs and anti-hepcidin antibodies (1 µg/mL LPS). (E) Co-culture of wild-type or Hamp^−/− BMSCs and J774.2 macrophages (1 µg/mL LPS) or peritoneal lavage cells. (F) Co-culture of wild-type or Hamp^−/− BMSCs and human THP-1 cells (1 µg/mL LPS). (G) TNF-α levels derived from peritoneal macrophages from zymosan (1 µg/mL)-induced peritonitis. (H) Peritoneal cells were stained with CD45, Gr-1 and Cd11b antibodies to detect polymorphonuclear cells (PMNs). As shown we observed a decrease (p < 0.05) in the percentage of PMNs in the live CD45 + population from mice treated with WT BMSCs (gray bar) compared to Hamp^−/− BMSC-treated animals (black bar) or vehicle (PBS—white bar) treated controls. PMF peritoneal macrophage. We found the same differences in the complete peritoneal lavage cell numbers between groups (n = 8 per group, pooled data). One-way ANOVA was used. *p < 0.05; **p < 0.01; ***p < 0.001; ns non-significant. 5–8 mice were used per group; the experiments were done at least twice.