Hepcidin and IL-6 induction by viral-like TLR agonists and whole live Influenza A virus. (A-B) Hepcidin and IL-6 induction in PBMCs by viral-like TLR agonists. qRT-PCR measurement or fold-changes in (A) hepcidin and (B) IL-6 mRNA expression relative to untreated samples from the same individual in PBMCs (N = 5 donors, represented by different symbols) cultured for 3 hours in the presence of various viral-like TLR agonists: the TLR3 agonist poly(I:C), a synthetic mimic of viral double-stranded RNA (10 μg/mL); the TLR7 agonist R837 (Imiquimod, 1 μg/mL); the TLR8 agonist ssRNA40, representing viral single-stranded RNA (4 μg/mL); and the TLR9 agonist ODN2006, a synthetic unmethylated CpG-containing oligonucleotide (2μM [except individual denoted by black diamond, 1.25μM]). The dashed line represents the mean for each dataset; not every individual was tested with every TLR agonist. (C-D) Hepcidin and IL-6 induction by live Influenza A virus in PBMCs. Fold-changes in (C) hepcidin and (D) IL-6 mRNA expression in PBMCs (N = 10 donors) cultured for 3 hours in the presence of Influenza A virus (A/PR/8/34) in serum-free media (10 PFU per PBMC). ***P < .001, paired t test (C); **P < .01, Wilcoxon matched pairs test (D). (E) Effect of anti–IL-6/anti–IL-6R antibodies on Influenza A virus mediated hepcidin induction. Fold-changes in hepcidin mRNA expression in a subset of the PBMC samples from panel C also cultured with and without Influenza A virus in the presence of a mix of neutralizing anti–IL-6 and anti–IL6-R antibodies (10 μg/mL each, added 15 minutes before addition of virus to cultures). Values for Influenza A virus alone are those from panel C. P < .001, Friedman test; **P < .01, ***P < .001 indicate Dunn multiple comparison test. (F) Effect of the TGF-β pathway inhibitor SD208 on Influenza A virus mediated hepcidin induction. Fold-changes in hepcidin mRNA expression in a subset of the PBMC samples from panel C also cultured with and without Influenza A virus in the presence of SD208 (1 μM). Values for Influenza A virus alone are those from panel C. P < .001, 1-way repeated measures ANOVA; *P < .05 indicates significant Bonferroni posttest. (G-H) Hepcidin induction by live Influenza A virus in Hep3B and HepG2 cells. Fold-changes in hepcidin mRNA expression (relative to untreated cells) in (G) Hep3B cells and (H) HepG2 cells treated with Influenza A virus (A/PR/8/34; 100:1 PFU:Hep3B, and 10:1 PFU:HepG2) or recombinant human BMP6 (18nM) for 24 hours. Note that despite equivalent cell numbers, the quantities of GAPDH mRNA detected in infected cultures were ∼ 3-fold lower in infected cells than in untreated controls (data not shown), yet amounts of hepcidin mRNA were ∼ 30-fold greater, corresponding to the displayed ∼ 100-fold increases in infected cells. Data represent the mean + SD of single experiments carried out in biologic triplicate. **P < .01, t test (Gaussian distribution assumed).