1. by Rodrigo Abreu, Frederick Quinn, and Pramod K. Giri
Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells
by Rodrigo Abreu, Frederick Quinn, and Pramod K. Giri
BloodAdv
Volume 2(10):
May 22, 2018
© 2018 by The American Society of Hematology

2. Rodrigo Abreu et al. Blood Adv 2018;2:1089-1100
© 2018 by The American Society of Hematology

3. Iron increases TLR4-mediated hepcidin expression, whereas TLR2 activation does not induce hepcidin expression.
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 < All data were from 3 independent experiments. ns, not significant; UT, untreated; WT, wild type.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

4. TLR2 ligand inhibits ferroportin expression.
TLR2 ligand inhibits ferroportin expression. (A) THP-1 cells were stimulated with TLR2 or TLR4 ligand, and ferroportin expression was quantified by qRT-PCR. GAPDH expression was used as a control. (B) Surface ferroportin staining in THP-1 cells treated with TLR2 or TLR4 ligands was assessed by immunofluorescence microscopy (original magnification ×630). (C) MFI was assessed from 5 microscopic fields in panel B. (D) Surface ferroportin expression in THP-1 cells treated with TLR2 or TLR4 ligands compared with untreated cells was assessed by flow cytometry. All data were from 3 independent experiments. **P < .01, ***P < DAPI, 4′,6-diamidino-2-phenylindole; FPN, ferroportin.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

5. TLR2 induces intracellular iron sequestration through a hepcidin-independent mechanism.
TLR2 induces intracellular iron sequestration through a hepcidin-independent mechanism. (A) Ferritin mRNA transcription levels from PMA-differentiated THP-1 cells treated with TLR2 or TLR4 ligands for 24 hours quantified by qRT-PCR and normalized against untreated controls. GAPDH expression was used as a relative control. (B) THP-1 cells treated as in panel A and cell lysates were subjected to immunoblotting with the antiferritin (anti-FTH) antibody. (C) Densitometry analysis from western blot in panel B. (D) Prussian blue staining of PMA-differentiated THP-1 cells treated as described in panel A. Low magnification field (×10) is shown above, with high magnification (×40) shown below. All data were from 3 independent experiments. *P < .05.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

6. TLR2 inhibits ferroportin expression independent of hepcidin.
TLR2 inhibits ferroportin expression independent of hepcidin. Hepcidin gene silencing in THP-1 cells was achieved by lentiviral-based shRNA transduction. Scramble shRNA (Sh Scram) was used as a negative control. (A) Hepcidin expression in Sh Scram and hepcidin-specific shRNA (Sh HAMP) silenced THP-1 cells. (B) Surface ferroportin expression in Sh Scram and Sh HAMP THP-1 cells after Pam3CSK4 or LPS treatment for 24 hours. All data were from 3 independent experiments.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

7. TLR signaling induces hypoferremia through 2 independent pathways.
TLR signaling induces hypoferremia through 2 independent pathways. (A-B) Hepcidin expression in THP-1 cells treated with different TLR ligands and organized to show hepcidin noninducers (A) and hepcidin inducers (B). (C-D) Ferroportin (FPN) expression in THP-1 cells treated with TLR ligands and organized as in panels A and B. (E-F) Ferritin (FTH) expression in THP-1 cells treated with different TLR ligands and organized as in panels A and B. All data were from 3 independent experiments. *P < .05, **P < .01, ***P < .001.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

8. TLR-mediated hepcidin induction is independent of IL-6 signaling.
TLR-mediated hepcidin induction is independent of IL-6 signaling. (A-B) IL-8 expression in TLR-treated macrophages and organized to show hepcidin noninducers (A) and hepcidin inducers (B). (C-D) IL-6 expression in TLR-treated macrophages and organized as in panels A and B. (E-F) IL-6 secretion in TLR-treated macrophages and organized as in panels A and B. (G) IL-6 secretion in macrophages treated with Pam3CSK4 or LPS for 24 hours. (H) Hepcidin secretion in macrophages treated with Pam3CSK4 or LPS for 24 hours in presence of 1 μg/mL of IL-6 receptor–blocking antibody. All data were from 3 independent experiments. **P < .01, ***P < LOD, limit of detection.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

9. BCG induces iron sequestration through hepcidin induction and ferroportin downregulation.
BCG induces iron sequestration through hepcidin induction and ferroportin downregulation. (A-C) Transcriptional changes of hepcidin (A), ferroportin (B), and ferritin (C) expression in BCG-infected THP-1 cells differentiated with 50 nM of PMA for 24 hours. (D) Hepcidin mRNA levels of BCG-infected WT and ΔMyD88 THP-1 cells. (E) Hepcidin secretion in BCG- or L monocytogenes (L mono)–infected THP-1 cells differentiated as in panel A. (F) Ferroportin expression in L mono–infected THP-1 cells. All data were from 3 independent experiments. ***P < .001.
Rodrigo Abreu et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology