Figure 3
Figure 3. Hepcidin induction in human PBMCs by bacterial and fungal-related TLR agonists. (A-B) Hepcidin and IL-6 induction. qRT-PCR measurement of (A) hepcidin and (B) IL-6 mRNA in PBMCs cultured for 3 hours in the presence of various TLR agonists derived from bacteria or fungi, or their mimics: the TLR2 agonist Heat-killed L monocytogenes (HKLM, 7-8 × 107 cells/mL); the TLR1/2 agonist Pam3CSK4, a synthetic bacterial lipopeptide mimic (0.5 μg/mL); the TLR4 agonist lipopolysaccharide (LPS) derived from E coli K12 (1 μg/mL); the TLR5 agonist S typhimurium flagellin, a flagellar motor protein (0.5 μg/mL); and the TLR6/2 agonist FSL1, a synthetic Mycoplasma lipoprotein mimic (0.5 μg/mL). Muramyl dipeptide (MDP), a bioactive motif found within bacterial peptidoglycan and agonist for the cytoplasmically-expressed NLR NOD2 was also tested, separated from TLR agonists by a vertical dotted line. Data are presented as fold-changes in expression relative to untreated samples from the same individual (N = 9 PBMC donors, each symbol represents one PBMC preparation; not every individual was tested with every TLR agonist; datapoints represent, in the majority of cases, mean values of biologic duplicates; dashed line represents the mean for each dataset). *P < .05, **P < .01, Wilcoxon matched pairs signed-rank test, comparing with untreated samples: note that to account for variability in baseline hepcidin levels between individuals, statistics were generated based on the data presented in supplemental Figure 3A-B (see supplemental Methods). (C-D) Time course of hepcidin and IL-6 induction by Flagellin and LPS. Fold-changes in (C) hepcidin and (D) IL-6 expression (relative to untreated samples from the same timepoint and individual) in PBMCs (N = 3 donors) treated with LPS (1μg/ml) Flagellin (0.5 μg/ml) or IL-6 (50 ng/mL) for 1, 3, 6, or 20 hours. Mean and range are shown. (E-F) Hepcidin and IL-6 induction in PBMCs by titrations of S. typhimurium and B subtilis flagellins. (E) Hepcidin and (F) IL-6 mRNA expression relative to GAPDH in PBMCs (N = 1 donor) cultured for 3 hours with increasing concentrations of flagellins derived from S typhimurium (purple line) and B subtilis (red line). (G) Inhibition of flagellin-mediated hepcidin induction in PBMCs. Fold-changes in hepcidin mRNA expression (relative to untreated samples from the same individual; N = 5 donors) in PBMCs cultured for 3 hours in the presence or absence of: flagellin (0.5 μg/mL); the Alk5/TGF-β pathway inhibitor, SD208 (1μM); and a mix of neutralizing anti–IL-6 and anti–IL-6R antibodies (10 μg/mL each, added 15 minutes before addition of flagellin/SD208 to the cultures). P < .001, Friedman test; **P < .01 indicates Dunn multiple comparison posttest.

Hepcidin induction in human PBMCs by bacterial and fungal-related TLR agonists. (A-B) Hepcidin and IL-6 induction. qRT-PCR measurement of (A) hepcidin and (B) IL-6 mRNA in PBMCs cultured for 3 hours in the presence of various TLR agonists derived from bacteria or fungi, or their mimics: the TLR2 agonist Heat-killed L monocytogenes (HKLM, 7-8 × 107 cells/mL); the TLR1/2 agonist Pam3CSK4, a synthetic bacterial lipopeptide mimic (0.5 μg/mL); the TLR4 agonist lipopolysaccharide (LPS) derived from E coli K12 (1 μg/mL); the TLR5 agonist S typhimurium flagellin, a flagellar motor protein (0.5 μg/mL); and the TLR6/2 agonist FSL1, a synthetic Mycoplasma lipoprotein mimic (0.5 μg/mL). Muramyl dipeptide (MDP), a bioactive motif found within bacterial peptidoglycan and agonist for the cytoplasmically-expressed NLR NOD2 was also tested, separated from TLR agonists by a vertical dotted line. Data are presented as fold-changes in expression relative to untreated samples from the same individual (N = 9 PBMC donors, each symbol represents one PBMC preparation; not every individual was tested with every TLR agonist; datapoints represent, in the majority of cases, mean values of biologic duplicates; dashed line represents the mean for each dataset). *P < .05, **P < .01, Wilcoxon matched pairs signed-rank test, comparing with untreated samples: note that to account for variability in baseline hepcidin levels between individuals, statistics were generated based on the data presented in supplemental Figure 3A-B (see supplemental Methods). (C-D) Time course of hepcidin and IL-6 induction by Flagellin and LPS. Fold-changes in (C) hepcidin and (D) IL-6 expression (relative to untreated samples from the same timepoint and individual) in PBMCs (N = 3 donors) treated with LPS (1μg/ml) Flagellin (0.5 μg/ml) or IL-6 (50 ng/mL) for 1, 3, 6, or 20 hours. Mean and range are shown. (E-F) Hepcidin and IL-6 induction in PBMCs by titrations of S. typhimurium and B subtilis flagellins. (E) Hepcidin and (F) IL-6 mRNA expression relative to GAPDH in PBMCs (N = 1 donor) cultured for 3 hours with increasing concentrations of flagellins derived from S typhimurium (purple line) and B subtilis (red line). (G) Inhibition of flagellin-mediated hepcidin induction in PBMCs. Fold-changes in hepcidin mRNA expression (relative to untreated samples from the same individual; N = 5 donors) in PBMCs cultured for 3 hours in the presence or absence of: flagellin (0.5 μg/mL); the Alk5/TGF-β pathway inhibitor, SD208 (1μM); and a mix of neutralizing anti–IL-6 and anti–IL-6R antibodies (10 μg/mL each, added 15 minutes before addition of flagellin/SD208 to the cultures). P < .001, Friedman test; **P < .01 indicates Dunn multiple comparison posttest.

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