American Society of Hematology

Figure 2

$Figure 2. IL-6 and TGF-β1, but not BMPs, induce hepcidin mRNA in human PBMCs. (A-B) Time course of hepcidin induction by IL-6 and TGF-β1. qRT-PCR measurement of hepcidin mRNA induction in 5 × 106 PBMCs cultured for various times in the presence or absence of IL-6 (50 ng/mL) and TGF-β1 (10 ng/mL). Data are presented as (A) expression relative to GAPDH, and (B) fold-change in hepcidin expression relative to the untreated sample from the same timepoint. Datapoints represent means and ranges for PBMCs obtained from 3 donors, run in singlicate. (C-D) BMP6 and BMP9 do not induce hepcidin. Hepcidin mRNA expression in PBMCs cultured for 1 hour with BMP6, BMP9 (both 50 ng/mL), or TGF-β1 (10 ng/mL) in the presence or absence of IL-6 (50 ng/mL). Data are presented as (C) expression relative to GAPDH, and (D) fold-change in hepcidin expression relative to the untreated sample from the same individual. Dashed lines represent the mean values for PBMCs obtained from 4 donors, indicated by different symbols. (E, F) Small molecule and antibody-mediated inhibition of TGF-β1– and IL-6–mediated hepcidin induction. Hepcidin mRNA expression in PBMCs (N = 5 donors) cultured for 1 hour with combinations of IL-6 (50 ng/mL), TGF-β1 (10 ng/mL), the small molecule Alk5 (TGF-β receptor) inhibitor SD208 (1μM), and a mixture of neutralizing anti–IL-6 and anti–IL-6R antibodies (10 μg/mL each). Data are presented as (E) relative to GAPDH and (F) as fold-changes as in panels C, D. Data for anti–IL-6 treatment were not obtained for 2 PBMC preparations. (G-H) IL-6 treatment of human PBMCs renders them refractory to hepcidin induction by subsequent retreatment with IL-6, but not TGF-β1. Hepcidin mRNA expression in PBMCs (N = 3 donors) cultured for a total of 4 hours: at time zero, cells were treated with or without IL-6 (50 ng/mL); after 3 hours, they were treated with either IL-6 (50 ng/mL), TGF-β1 (10 ng/mL) or both. RNA was extracted after 4 hours. Data are presented as (G) relative to GAPDH and (H) fold-changes relative to the untreated sample from the same individual.$

IL-6 and TGF-β1, but not BMPs, induce hepcidin mRNA in human PBMCs. (A-B) Time course of hepcidin induction by IL-6 and TGF-β1. qRT-PCR measurement of hepcidin mRNA induction in 5 × 10⁶ PBMCs cultured for various times in the presence or absence of IL-6 (50 ng/mL) and TGF-β1 (10 ng/mL). Data are presented as (A) expression relative to GAPDH, and (B) fold-change in hepcidin expression relative to the untreated sample from the same timepoint. Datapoints represent means and ranges for PBMCs obtained from 3 donors, run in singlicate. (C-D) BMP6 and BMP9 do not induce hepcidin. Hepcidin mRNA expression in PBMCs cultured for 1 hour with BMP6, BMP9 (both 50 ng/mL), or TGF-β1 (10 ng/mL) in the presence or absence of IL-6 (50 ng/mL). Data are presented as (C) expression relative to GAPDH, and (D) fold-change in hepcidin expression relative to the untreated sample from the same individual. Dashed lines represent the mean values for PBMCs obtained from 4 donors, indicated by different symbols. (E, F) Small molecule and antibody-mediated inhibition of TGF-β1– and IL-6–mediated hepcidin induction. Hepcidin mRNA expression in PBMCs (N = 5 donors) cultured for 1 hour with combinations of IL-6 (50 ng/mL), TGF-β1 (10 ng/mL), the small molecule Alk5 (TGF-β receptor) inhibitor SD208 (1μM), and a mixture of neutralizing anti–IL-6 and anti–IL-6R antibodies (10 μg/mL each). Data are presented as (E) relative to GAPDH and (F) as fold-changes as in panels C, D. Data for anti–IL-6 treatment were not obtained for 2 PBMC preparations. (G-H) IL-6 treatment of human PBMCs renders them refractory to hepcidin induction by subsequent retreatment with IL-6, but not TGF-β1. Hepcidin mRNA expression in PBMCs (N = 3 donors) cultured for a total of 4 hours: at time zero, cells were treated with or without IL-6 (50 ng/mL); after 3 hours, they were treated with either IL-6 (50 ng/mL), TGF-β1 (10 ng/mL) or both. RNA was extracted after 4 hours. Data are presented as (G) relative to GAPDH and (H) fold-changes relative to the untreated sample from the same individual.

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