Iron increases TLR4-mediated hepcidin expression, whereas TLR2 activation does not induce hepcidin expression. (A) THP-1 human monocytic cells were differentiated with 50 nM of PMA and rested overnight in C-RPMI or C-RPMI supplemented with 100 μM of ferric ammonium citrate (FeAC) and then stimulated for 24 hours with 500 ng/mL of ultrapure LPS. (B) Differentiated THP-1 cells were treated as in panel A and stained for intracellular hepcidin using human antihepcidin antibody (mAb2.7) and analyzed by flow cytometry. (C) Differentiated THP-1 cells were treated with TLR2 ligand (Pam3CSK4) or TLR4 ligand (ultrapure LPS) for 24 hours, and hepcidin expression was quantified by quantitative reverse transcription PCR (qRT-PCR). (D-E) Macrophages treated as in panel C for 24 hours were stained for intracellular hepcidin using human antihepcidin antibody (mAb2.7) and analyzed by flow cytometry. Hepcidin expression was quantified by MFI. (E) Summary data from 3 independent experiments as represented in panel D. (F) Hepcidin expression in MyD88-deficient THP-1 (THP-1 ΔMyD88) cells treated with LPS as in panel A was measured by qRT-PCR. Hepcidin expression levels were analyzed by qRT-PCR, and GAPDH expression was used as a control. *P < .05, **P < .01, ***P < .001. All data were from 3 independent experiments. ns, not significant; UT, untreated; WT, wild type.