Expression and localization of hepcidin in macrophages:
a role in host defense against tuberculosis
Fatoumata B. Sow,*,†William C. Florence,*,†Abhay R. Satoskar,*,†Larry S. Schlesinger,†,‡,§
Bruce S. Zwilling,*,†and William P. Lafuse†,‡,1
Departments of *Microbiology,‡Molecular Virology, Immunology and Medical Genetics, and§Internal Medicine,
Division of Infectious Diseases, and†Center for Microbial Interface Biology, The Ohio State University, Columbus,
produced by the liver in response to inflammatory
stimuli and iron overload. Hepcidin regulates iron
homeostasis by mediating the degradation of the
iron export protein ferroportin 1, thereby inhibit-
ing iron absorption from the small intestine and
release of iron from macrophages. Here, we exam-
ined the expression of hepcidin in macrophages
infected with the intracellular pathogens Mycobac-
terium avium and Mycobacterium tuberculosis.
Stimulation of the mouse RAW264.7 macrophage
cell line and mouse bone marrow-derived macro-
phages with mycobacteria and IFN-? synergisti-
cally induced high levels of hepcidin mRNA and
protein. Similar results were obtained using the
human THP-1 monocytic cell line. Stimulation of
macrophages with the inflammatory cytokines IL-6
and IL-? did not induce hepcidin mRNA expres-
sion. Iron loading inhibited hepcidin mRNA ex-
pression induced by IFN-? and M. avium, and iron
chelation increased hepcidin mRNA expression.
Intracellular protein levels and secretion of hepci-
din were determined by a competitive chemilumi-
nescence ELISA. Stimulation of RAW264.7 cells
with IFN-? and M. tuberculosis induced intracellu-
lar expression and secretion of hepcidin. Further-
more, confocal microscopy analyses showed that
hepcidin localized to the mycobacteria-containing
phagosomes. As hepcidin has been shown to pos-
sess direct antimicrobial activity, we investigated
its activity against M. tuberculosis. We found that
hepcidin inhibited M. tuberculosis growth in vitro
and caused structural damage to the mycobacteria.
In summary, our data show for the first time that
hepcidin localizes to the phagosome of infected,
IFN-?-activated cells and has antimycobacterial
activity. J. Leukoc. Biol. 82: 000–000; 2007.
Hepcidin is an antimicrobial peptide
Key Words: innate immunity ? cytokines ? antimicrobial peptides
Mycobacteria are engulfed by macrophages through phagocy-
tosis and reside within the phagosomes of these cells [1, 2].
Antimycobacterial activity is mediated primarily by CD4?and
CD8?T cells through the secretion of cytokines, which in-
crease the antimicrobial activity of macrophages [3, 4]. IFN-?
is the major regulator of the immune response to mycobacteria
[5, 6]. Activation of macrophages by IFN-? in mice induces the
expression of NO synthase 2 (NOS2), resulting in the produc-
tion of NO, which leads to killing of the intracellular myco-
bacteria . IFN-? also promotes the fusion of the mycobac-
teria-containing phagosome with late endosomal vesicles and
lysosomal vesicles [8, 9], resulting in the delivery of lysosomal
enzymes to the phagosome and the acidification of the phago-
Iron is essential for the growth and survival of mycobacteria,
but it is also involved in host macrophage defense by catalyzing
the production of toxic hydroxyl radicals via the Haber-Weiss/
Fenton reactions [10, 11]. During infection, a competition for
iron occurs between the host macrophage and the pathogen.
The macrophage attempts to suppress pathogen proliferation by
complex, iron-withholding mechanisms. Activation of macro-
phages by IFN-? results in a decrease in macrophage iron
content, ferritin levels, and expression of the transferrin recep-
tor [12–15]. In response, Mycobacterium tuberculosis, present
in activated macrophages, increases the expression of the
iron-binding siderophores, mycobactin and carboxymycobac-
tin, which are involved in binding and transporting iron [16–
The effectors of innate immunity also include antimicrobial
peptides , which are found in prokaryotes, plants, and
animals and have broad activities against bacteria and fungi.
Hepcidin, originally identified as a 25-amino acid antimicro-
bial peptide present in serum and urine [21, 22], is produced
from a propeptide precursor by liver hepatocytes during the
acute-phase response. Recent studies have shown that hepci-
din also acts as a negative regulator of iron absorption by the
duodenum [23, 24] and inhibits release of recycled iron by
macrophages . Studies by Nemeth et al.  have shown
that hepcidin binds to ferroportin 1, which is the sole iron
export protein in mammalian cells, and mediates its internal-
1Correspondence: Department of Molecular Virology, Immunology and
Medical Genetics, The Ohio State University, 333 W. 10th Ave., Columbus,
OH 43210, USA. E-mail: email@example.com
Received April 10, 2007; revised June 6, 2007; accepted June 7, 2007.
0741-5400/07/0082-0001 © Society for Leukocyte Biology
Journal of Leukocyte Biology
Volume 82, October 2007
Uncorrected Version. Published on July 3, 2007 as DOI:10.1189/jlb.0407216
Copyright 2007 by The Society for Leukocyte Biology.
ization and degradation, resulting in a decrease in iron release.
Thus, knockout of hepcidin gene expression in mice resulted in
iron overload resembling hemochromatosis , and overex-
pression of hepcidin in transgenic mice resulted in severe iron
deficiency anemia and death at birth .
Expression of hepcidin mRNA is induced in human hepa-
tocytes by IL-6 and LPS [29, 30] and in mouse hepatocytes, by
IL-1 and IL-6 . In vivo, liver hepcidin mRNA is up-
regulated following injection of mice with LPS, turpentine, and
CFA [32–34]. Liver hepcidin mRNA expression is also in-
creased by iron overload  and decreased by anemia in-
duced by bleeding or hemolysis . A recent study 
reported that infection of mouse bone marrow-derived macro-
phages (BMDM) with the extracellular pathogens Pseudomonas
aeruginosa and group A Streptococcus induced hepcidin mRNA
expression in macrophages. However, no similar studies have
been done with intracellular pathogens of macrophages. The
previous study also did not determine if macrophage activation
by IFN-? regulates hepcidin expression or the localization of
hepcidin within the macrophage. We investigated the expres-
sion of hepcidin mRNA and protein in Mycobacterium avium-
and M. tuberculosis-infected macrophages. We demonstrate
that mycobacteria infection and IFN-? stimulation synergisti-
cally induce high levels of hepcidin mRNA and protein in the
macrophage. We also show that hepcidin is present in the M.
tuberculosis-containing phagosomes and that at least in vitro,
hepcidin can inhibit M. tuberculosis growth. Thus, these stud-
ies indicate that hepcidin production by infected macrophages
is an IFN-?-induced host defense mechanism against infection
MATERIALS AND METHODS
M. avium strain Mac101 [American Type Culture Collection (ATCC) 70998]
and M. tuberculosis H37Rv strain (ATCC 27294) were obtained from ATCC
(Manassas, VA, USA). M. avium was cultured in Middlebrook 7H9 media
supplemented with oleic acid-albumin-dextrose-catalase (OADC; Difco, De-
troit, MI, USA) and stored in 1 ml aliquots in 10% glycerol at –70°C until used.
Lyophilized M. tuberculosis H37Rv was reconstituted in pyogen-free water
(Sigma Chemical Co., St. Louis, MO, USA), plated on 7H11 agar plates, and
incubated at 37°C in 5% CO2for 14 days, and bacterial suspensions were
generated as described . ?-Irradiated M. tuberculosis (Colorado State
University, Fort Collins, CO, USA; National Institutes of Health Contract
NIAID-N01-AI-40091) was resuspended in PBS, briefly sonicated, and cen-
trifuged at 800 rpm for 10 min to eliminate clumped bacteria. The protein
concentration in the supernatant was determined by the Bradford protein assay
(Bio-Rad, Hercules, CA, USA).
The RAW264.7 mouse macrophage cell line (TIB-71) was plated at 5 ? 106
cells per well in six-well culture plates containing DMEM supplemented with
10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA) and penicillin-
streptomycin. The macrophages were allowed to adhere for 4 h at 37°C in 5%
CO2in air. The nonadherent cells were washed away with DMEM without
antibiotics, and the macrophage monolayers were infected with M. avium strain
Mac101 or with live M. tuberculosis H37RV at a ratio of 10:1, bacterium:
macrophage. Macrophage monolayers were also stimulated with 200 U/ml
mouse IFN-? (Roche, Indianapolis, IN, USA), with and without mycobacteria
infection. RAW264.7 cells were also stimulated with IFN-? in combination
with 100 ?g/ml ?-irradiated M. tuberculosis H37Rv. In other experiments,
RAW264.7 cells were also stimulated with the proinflammatory cytokines IL-6,
IL-1?, and TNF-? at 10 ng/ml (Alpha Diagnostic, San Antonio, TX, USA) in
combination with M. avium infection. For iron-loading experiments,
RAW264.7 cells were pretreated for 1 h with Fe-nitrilotriacetate (FeNTA;
molar ratio 1:4), prepared from FeCl3and sodium NTA (Sigma Chemical Co.)
before stimulation with IFN-? and ?-irradiated M. tuberculosis. For iron
chelation experiments, the RAW264.7 cells were pretreated with the iron
chelator desferrioxamine (Sigma Chemical Co.) for 1 h prior to stimulation.
A RAW264.7 cell line, constitutively expressing Flag-tagged hepcidin
under control of the CMV promoter, was created by cloning full-length hep-
cidin cDNA into the pCMV-3Tag 8 expression vector (Stratagene, La Jolla, CA,
USA). The sequence of the hepcidin cDNA was confirmed by DNA sequencing.
RAW264.7 cells were transfected with the plasmid using Lipofectamine (In-
vitrogen, Carlsbad, CA, USA). Stable clones were obtained by hygromycin
selection and limiting dilution cloning. High expressing clones were identified
by confocal microscopy as described below using the M2 anti-Flag mAb (Sigma
Chemical Co.) and Alexa 488-coupled F(ab?)2goat anti-mouse IgG antibody
(Invitrogen) as the secondary antibody.
The human THP-1 monocytic cell line (ATCC TIB-202) was plated at 5 ?
106cells per well in six-well culture plates containing RPMI 1640, supple-
mented with 10% heat-inactivated FBS and penicillin-streptomycin at 37°C in
5% CO2for 2 h. The cells were then infected with live M. tuberculosis H37Rv
at a ratio of 20:1, bacterium:macrophage, and stimulated with 200 units/ml
human IFN-? (Roche).
Cells were isolated from the marrow of femurs and tibias of C57/BL6J mice.
The cells were plated in complete DMEM, supplemented with 10 ng/ml
GM-CSF (Peprotech, Rocky Hill, NJ, USA). After 3 and 5 days of culture, 50%
and 75% of the medium was removed and replaced with fresh medium,
supplemented with GM-CSF, respectively. Mature, adherent BMDM were
harvested after 7 days of culture and subjected to stimulation with M. avium,
IFN-?, or a combination of IFN-? and M. avium for 24 h.
RAW264.7 macrophages were lysed using Qiagen lysis buffer and 2-ME and
homogenized by passing the cell lysates through QiaShredders (Qiagen, Va-
lencia, CA, USA). The RNA was then isolated using the RNeasy mini kit. RNA
was isolated from THP-1 and BMDM using the High Pure RNA isolation kit
(Roche). Residual DNA was removed during RNA purification by on-column
DNase digestion in both procedures.
Total RNA (1 ?g) was reverse-transcribed using 100 ?M dNTPs, 15 units
avian myloblastosis virus RT, and 0.5 ?g oligo(dT)15primer in RT buffer for
1 h at 42°C (Promega, Madison, WI, USA). The expression of mouse hepcidin
1 and GAPDH mRNA was analyzed by real-time RT-PCR in the Roto-gene
2000 real-time cycler (Phenix Research Products, Candler, NC, USA) using
FastStart DNA SYBR Green I reaction mix (Roche). The primer sequences are
mouse GAPDH: GTGTGAACGGATTTGGCCGTATTGGGCG (sense) and
TCGCTCCTGGAAGATGGTGATGGGC (antisense); mouse ?-actin: TACAG-
CTTCACCACCACAGC (sense) and AAGGAAGGCTGGAAAAGAGC (anti-
sense); mouse hepcidin 1: GCAGAAGAGAAGGAAGAGAGACACC (sense)
and TGTAGAGAGGTCAGGATGTGGCTC (antisense); human GAPDH: GAA-
GGTGAAGGTCGGAGTC (sense) and GAAGATGGTGATGGGATTTC (anti-
sense); human hepcidin: GCACTGAGCTCCCAGATCTG (sense) and CTAC-
GTCTTGCAGCACATCC (antisense). The primers were designed using
MacVector primer software (Accelrys, San Diego, CA, USA). Specificity of
primers was confirmed by GenBank Blast searches. Mice express two dupli-
cated hepcidin genes, hepc1 and hepc2 . hepc2 has only 58% identity with
hepc1 and does not appear to be involved in regulating iron metabolism.
Primers were designed to amplify hepcidin 1 cDNA and not hepcidin 2 cDNA.
The amplification conditions were 95°C for 10 min followed by 40 cycles of
95°C for 15 s, 60°C for 5 s, and 72°C for 20 s. The relative expression of each
sample was calculated using mouse GAPDH as a reference and the ?Ct
method as described previously . GAPDH mRNA levels were used as the
reference mRNA in all experiments, except for experiments in which
RAW264.7 cells were loaded with iron and treated with the iron chelator
2Journal of Leukocyte Biology
Volume 82, October 2007
desferrioxamine. ?-Actin was used as the reference mRNA in these experi-
ments. Preliminary experiments showed that GAPDH mRNA levels increased
when macrophages were treated with desferrioxamine. ?-Actin mRNA levels
were not changed by iron-loading or iron chelation.
Hepcidin-competitive chemiluminescence ELISA
RAW264.7 cells in six-well culture plates were stimulated for 24 h with IFN-?
(200 units/ml), 100 ?g/ml ?-irradiated M. tuberculosis, and the combination of
IFN-? and ?-irradiated M. tuberculosis in serum-free DMEM. Hepcidin se-
creted into the culture media and intracellular hepcidin were measured by a
competitive chemiluminescence ELISA with biotinylated mouse hepcidin (Al-
pha Diagnostic). To measure cellular hepcidin expression, the RAW264.7
cells were harvested by scrapping and then pelleted by centrifugation. The
cells were lysed on ice for 10 min with 1% Triton X-100 in PBS with protease
inhibitor cocktail tablets (Roche), and the cell debris was removed by centrif-
ugation for 10 min at 14,000 rpm. Protein concentration of the lysate was
determined using the Bradford method (Bio-Rad). The ELISA was performed
by coating white ELISA plates overnight at 4°C with 100 ?l per well 1 ug/ml
affinity-purified rabbit anti-mouse hepcidin IgG (Alpha Diagnostic) in coating
buffer (Alpha Diagnostic). The plates were washed three times with washing
buffer (PBS, 0.050% Tween-20) and incubated with blocking solution, I-Block
(Applied Biosystems, Foster City, CA, USA), in PBS, 0.050% Tween-20, for
1 h at room temperature. Blocking solution was removed, and 50 ?l per well
samples and standards (0–10 pg/ml hepcidin) diluted in blocking buffer was
added to the plate. After 1 h incubation at room temperature, 50 ?l 5 ng/ml
biotinylated mouse hepcidin (Alpha Diagnostic) in blocking buffer was added
to each well, and the plate was incubated for an additional hour at room
temperature. The plate was washed three times with washing buffer. To detect
the bound, biotinylated hepcidin, 100 ul avidin/biotin-AP solution (ABC
Reagent, Vector Laboratories, Burlingame, CA, USA) was added to each well
and plate, incubated for 1 h at room temperature. The plate was washed four
times in washing buffer and once in 1? assay buffer. The substrate (CDP-Star
with Sapphire II enhancer, Applied Biosytems), 50 ?l/well, was added to the
plate and incubated for 10 min at room temperature. The plate was then read
using a microplate luminometer (Packard, Meriden, CT, USA). The concen-
tration of hepcidin in the samples was determined by regression analysis of the
RAW264.7 cells were plated on coverslips in 24-well plates at 5.0 ? 105
cells/well and stimulated with IFN-? and M. tuberculosis at 2:1, bacteria:
macrophage. The cells were fixed in 4% paraformaldehyde in PBS for 20 min,
washed twice with PBS, and then permeabilized with 0.1% Triton X-100 in
PBS for 10 min and washed twice with PBS. Monolayers were incubated in
blocking solution (1% BSA, 10% heat-inactivated goat serum, in PBS) for 3 h
at room temperature. To detect hepcidin, affinity-purified rabbit anti-mouse
hepcidin (20–25 hepc; Alpha Diagnostic), raised against the C terminus of the
mature, 25 amino acid hepcidin, was used. Residual M. tuberculosis-reactive
antibodies were removed by two rounds of absorption with ?-irradiated M.
tuberculosis for 1 h at 4°C prior to use. Antibodies were added at 1:200 in
blocking solution for 3 h at room temperature, followed by extensive washing
with 0.5% BSA in PBS and detection with Alexa 488-coupled F(ab?)2goat
anti-rabbit IgG antibody (Invitrogen). To detect mycobacteria in infected
RAW264.7 cells, coverslips were stained with auramine-rhodamine (Difco),
counterstained with 5% potassium permanganate as described by Ferguson et
al. , and followed by detection of hepcidin by immunofluorescence as
described above. The exclusion of primary antibody was used as a negative
control. A second negative control consisted of the hepcidin antibody preab-
sorbed with the mature, 25-amino acid peptide. Coverslips were removed from
24-well plates and mounted on slides with Prolong mounting medium (Invitro-
gen). Fluorescence was visualized by cross-sectional confocal microscopy
using a Zeiss LSM 510 confocal microcscope. The percentage of phagosomes
positive for hepcidin was determined by counting 25–50 phagosomes. Intensity
of the green immunofluorescence was measured from images using Sigma
ScanPro image analysis software (SPSS Science, Chicago, IL, USA).
To determine the localization of hepcidin to the phagosome in RAW264.7
cells constitutively expressing hepcidin, the RAW-264.7-hepcidin-Flag cell
line was plated onto coverslips and infected with live M. tuberculosis for 2 h.
The coverslips were then processed for confocal microscopy as described
above. M. tuberculosis was detected by auramine-rhodamine staining and
Flag-tagged hepcidin with the M2 anti-Flag mAb (1:500; Sigma Chemical Co.)
and Alexa 488-coupled F(ab?)2goat anti-mouse IgG antibody (Invitrogen) as
the secondary antibody.
Antimicrobial assay for hepcidin
The 25-amino acid form of hepcidin (Alpha Diagnostics) was tested for
antimicrobial activity against M. tuberculosis by incubating various concentra-
tions of this peptide (20, 50, 100, and 200 ?g/ml) with 1 ? 104M. tuberculosis
H37Rv at 37°C in 100 ?l 7H9 broth. At 6 and 72 h, viable bacteria were
assessed by culturing serial dilutions onto Middle-brook 7H11 agar plates
(Becton Dickinson, San Jose, CA, USA), supplemented with OADC (Becton
Dickinson). Colonies were counted after 21 days at 37°C.
Transmission electron microscopy (TEM)
M. tuberculosis H37Rv bacteria were plated at 1 ? 108in 100 ?l 7H9 broth,
with or without hepcidin (200 ug/ml), in 24-well culture plates. After 24 h, the
cell suspensions were centrifuged at 10,000 g for 15 min, washed with PBS,
and resuspended in fixative (3% glutaraldehyde and 4% paraformaldehyde in
0.1 M cacodylate buffer at pH 7.2) overnight at 4°C. The next day, the cells
were washed three times in sodium cacodylate buffer, postfixed in 1% osmium
tetroxide, and en bloc-stained in 1% uranyl acetate for 90 min. The cells were
then dehydrated in gradient ethanol (45 min at 50%, 45 min at 70%, 50 min
at 80%, 1 h at 95%, and 90 min with three changes at 100%), followed by
treatment with propylene oxide and covering with polybed and propylene oxide
(2:1) overnight. The samples were then embedded in polybed resin for 20 h at
60°C, followed by thin sectioning at 70 nm using a Leica EM UC6 ultramic-
rotome and staining in 2% uranyl acetate and Reynold’s lead citrate. The
samples were then observed and photographed in a FEI Technai Spirit TEM at
Results were analyzed by one-way ANOVA with Tukey’s test and t-test using
SigmaSTAT (SPSS Science).
Induction of macrophage hepcidin mRNA
expression by mycobacteria and IFN-?
We examined the mRNA expression of hepcidin in macro-
phages infected with M. avium and M. tuberculosis. Figure 1
shows that IFN-? and M. avium strain Mac101 synergistically
induced production of hepcidin mRNA in RAW264.7 macro-
phages (Fig. 1A). The increase in hepcidin mRNA expression
was apparent at 12 h after stimulation with IFN-? and M.
avium. A 100-fold increase in hepcidin mRNA was observed at
24 h. Infection with M. avium alone induced low levels of
hepcidin mRNA (threefold increase at 24 h), and treatment
with IFN-? alone did not induce any significant changes in the
expression of hepcidin mRNA. The synergistic effect observed
was dependent on the IFN-? concentration and the M. avium
dose used. Maximal effect was reached at a dose of 200
units/ml (Fig. 1B) and a M. avium ratio of 20:1 (Fig. 1C).
Heat-killed M. avium was as effective as live bacteria in
inducing hepcidin mRNA expression, and phagocytosis of la-
tex beads had no effect (data not shown).
The results in Figure 1D show that IFN-? and M. avium also
synergistically produced hepcidin mRNA in mouse BMDM.
The combination of IFN-? and M. avium induced a 40-fold
increase in hepcidin mRNA.
Sow et al.
Hepcidin expression in mycobacteria-infected macrophages3
Live M. tuberculosis H37Rv and IFN-? also synergistically
induced hepcidin mRNA expression in RAW264.7 macro-
phages (Fig. 2 A and B). The results in Figure 2A show the
level of hepcidin mRNA expression after 24 h, and those in
Figure 2B show the effect of increasing doses of M. tuberculo-
sis. Overall, M. tuberculosis induced high levels of hepcidin
mRNA expression. M. tuberculosis alone induced a hepcidin
mRNA level, which was 25–50 times higher than control cells.
The combination of IFN-? and M. tuberculosis increased the
levels to 250–500 times greater than control cells. Synergy was
also observed in RAW264.7 cells stimulated with IFN-? and
?-irradiated M. tuberculosis (Fig. 2, C and D). We also
observed similar synergy in the induction of hepcidin by
?-irradiated M. tuberculosis and IFN-? using the mouse
macrophage cell line J774A.1 and mouse BMDM (not
shown). To determine if M. tuberculosis and IFN-? induce
hepcidin mRNA in human cells, we examined the expres-
sion of hepcidin mRNA in the human monocytic cell line
THP1. As shown in Figure 2E, live M. tuberculosis and
IFN-? induced hepcidin mRNA in THP1 cells.
Hepcidin mRNA in macrophages is not induced
by proinflammatory cytokines or iron overload
As hepcidin mRNA expression is induced in human hepatocytes
by IL-6 and LPS [29, 30] and in mouse hepatocytes by IL-1 and
IL-6 , we determined if these proinflammatory cytokines in-
duce hepcidin mRNA in mouse macrophages. The results in
Figure 3 show that IL-1? and IL-6 do not induce hepcidin
mRNA in RAW264.7 macrophages. Hepcidin mRNA was also
not induced by TNF-?. IL-1? and IL-6 did significantly increase
hepcidin mRNA induced by M. avium. However, the effect of
these cytokines on hepcidin mRNA in M. avium-infected macro-
phages was much less than observed with IFN-? (Fig. 1).
We observed that the addition of iron in RAW264.7 macrophages
inhibited the induction of hepcidin mRNA by M. tuberculosis ?
IFN-?, and iron chelation increased hepcidin mRNA expression
(Fig. 4). These effects of iron and iron chelation occurred only in M.
effect on hepcidin mRNA expression (not shown).
Fig. 1. IFN-? and M. avium synergistically induce hepcidin mRNA expression. (A) Time-course experiment in which RAW264.7 macrophages were stimulated with
IFN-? (200 U/ml), M. avium infection (10:1 ratio), and IFN-? ? M. avium. (B) IFN-? dose-response experiment. RAW264.7 macrophages were stimulated with M. avium
(10:1) and the indicated doses of IFN-? for 24 h. (C) M. avium dose experiment. RAW264.7 macrophages were stimulated with IFN-? and M. avium for 24 h. (D) BMDM
were isolated from C57BL/6J mice as described in Materials and Methods and stimulated with IFN-? (200 U/ml), M. avium infection (30:1 ratio), and IFN-? ? M. avium
and expressed as fold induction relative to untreated RAW264.7 cells. The data represent the mean ? SD of three separate experiments.
4 Journal of Leukocyte Biology
Volume 82, October 2007
Detection of hepcidin protein in activated
macrophages by a competitive ELISA
We determined if IFN-? and mycobacteria induced the ap-
pearance of hepcidin protein in macrophages using a compet-
itive chemiluminescence ELISA. The antibody used to detect
hepcidin is an affinity-purified rabbit anti-mouse hepcidin
produced by immunization with a 13-amino acid peptide lo-
cated in the C terminus of the mature, 25-amino acid form of
mouse hepcidin. In the competitive ELISA, the antibody was
coated onto the wells of a 96-well plate, and hepcidin levels
were determined by competition of standards and samples with
biotinylated hepcidin; hepcidin protein expression was in-
creased in cell lysates of RAW264.7 cells stimulated with
?-irradiated M. tuberculosis (Fig. 5A). The highest level of
hepcidin protein expression (?160 pg hepcidin/mg protein)
was observed in RAW264.7 cells stimulated with ?-irradiated
M. tuberculosis and IFN-?. Stimulation with ?-irradiated M.
tuberculosis and IFN-? also resulted in the secretion of up to 50
pg/ml hepcidin by the macrophage (Fig. 5B).
Hepcidin is present in mycobacteria-containing
The expression of hepcidin was examined in RAW264.7 cells
infected with M. tuberculosis by cross-sectional, confocal mi-
croscopy (Fig. 6). As CFA was used in the production of the
antihepcidin rabbit antibody (Alpha Diagnostic, personal com-
munication), the antibody was absorbed with ?-irradiated M.
tuberculosis to remove any M. tuberculosis-reactive antibodies,
which may have remained after affinity purification. The ab-
sorbed antibody was negative against M. tuberculosis by West-
ern blot analysis with M. tuberculosis SDS lysates and negative
by immunofluorescence of M. tuberculosis adhered to cover-
slips (data not shown). Hepcidin was not detected in untreated
Fig. 2. IFN-? and M. tuberculosis synergistically in-
duce hepcidin mRNA expression. (A) RAW264.7 mac-
rophages were infected with M. tuberculosis (M.tb; 10:1)
and stimulated with IFN-? (200 U/ml) for 24 h. (B) M.
tuberculosis dose response. RAW264.7 macrophages
were infected with M. tuberculosis and stimulated with
IFN-? for 24 h. (C) Time-course experiment using
?-irradiated M. tuberculosis. RAW264.7 macrophages
were stimulated with ?-irradiated (irra.) M. tuberculosis
(100 ?g protein/ml) and IFN-? (200 U/ml). (D) Dose
response using ?-irradiated M. tuberculosis at the indi-
cated protein concentrations. RAW264.7 macrophages
were stimulated with ?-irradiated M. tuberculosis and
IFN-? for 24 h. (E) Induction of hepcidin mRNA in human THP1 cells, which were infected with M. tuberculosis (20:1) and 200 units/ml human IFN-? for 24 h.
(A–D) Total RNA was extracted, and hepcidin mRNA was detected by real-time RT-PCR using primers specific for mouse hepcidin and GAPDH. Mean ? SD of
three separate experiments. (E) Primers specific for human hepcidin and GAPDH were used. The hepcidin mRNA expression levels were normalized to GAPDH
mRNA levels and expressed as fold induction relative to untreated cells.
Fig. 3. Induction of hepcidin in macrophages by cytokines. RAW264.7
macrophages were stimulated with 10 ng/ml IL-1?, IL-6, TNF-?, and M. avium
infection (10:1 ratio) and a combination of cytokines and M. avium for 24 h.
Total RNA was extracted, and hepcidin mRNA was detected by real-time
RT-PCR. The hepcidin mRNA expression levels were normalized to GAPDH
mRNA levels and expressed as fold-induction relative to untreated RAW264.7
cells. The data represent the mean ? SD of three separate experiments. *,
IL-1? and IL-6 significantly increased hepcidin mRNA levels induced by M.
avium; P ? 0.05 by ANOVA when compared with M. avium.
Sow et al.
Hepcidin expression in mycobacteria-infected macrophages5
cells (Fig. 6A) or in cells treated with IFN-? alone (Fig. 6B).
Figure 6C shows the results when macrophages were stimu-
lated with live M. tuberculosis alone. In Figure 6D, the mac-
rophages were stimulated with M. tuberculosis and IFN-?. M.
tuberculosis was detected by staining the coverslips with aura-
mine-rhodamine  prior to detection of hepcidin by immu-
nofluorescence. Hepcidin was highly localized to the phago-
some. In the RAW264.7 cells stimulated with M. tuberculosis
and IFN-?, punctate staining was also observed outside of the
phagosome (Fig. 6D). The intensity of green immunofluores-
cence in the phagosome was significantly higher in cells stim-
ulated with M. tuberculosis and IFN-? (Fig. 6D) compared with
cells stimulated with M. tuberculosis alone (Fig. 6C). The
fluorescence intensity of phagosomes in macrophages stimu-
lated with M. tuberculosis ? IFN-? was 3.47 ? 0.33 ? 104
green fluorescence intensity units/phagosome compared with
1.44 ? 0.12 ? 104green fluorescence intensity units/phago-
some for phagosomes in macrophages infected with M. tuber-
culosis alone (P?0.001). Pretreatment of the hepcidin antibody
with the 25-amino acid hepcidin peptide blocked the fluores-
cence detection of hepcidin (Fig. 6E). There was also no green
fluorescence detected in control experiments in which
RAW264.7 cells treated with M. tuberculosis ? IFN-? were
incubated with the secondary antibody only (not shown).
Localization of hepcidin to M. tuberculosis phagosomes was
also examined in time-course experiments. RAW264.7 were
infected with M. tuberculosis and IFN-? for 0, 4, 8, 16, and
24 h, and hepcidin expression was examined by confocal
microscopy (Fig. 7A). Hepcidin begins to localize to the
phagosome by 4 h (Fig. 7B). Approximately 80% of the phago-
somes were positive for hepcidin by 8 h after infection. Hep-
cidin expression was quantified by measuring the green immu-
nofluorescence intensity of hepcidin-positive phagosomes (Fig.
7C). The intensity increased with infection. Peak levels were
obtained at 16 and 24 h, consistent with the increase in
hepcidin mRNA levels at these time-points.
To establish further that hepcidin localizes to the phagosome
following infection with M. tuberculosis, hepcidin epitope
tagged with Flag at the C terminus was stably expressed in
RAW264.7 cells. Expression of the hepcidin in the transfected
cells was examined by confocal microscopy using mouse anti-
Flag mAb. The cells constitutively express high levels of
hepcidin-Flag in intracellular vesicles (Fig. 8A). After infec-
tion with M. tuberculosis, the hepcidin-Flag localized to the
phagosome (Fig. 8B). Thus, our studies indicate that hepcidin
redistributes to the phagosome during infection.
Hepcidin has direct, antibacterial activity against
As hepcidin has antibacterial activity against a number of
microorganisms [21, 22], we determined if hepcidin has anti-
Fig. 4. Addition of iron to IFN-? ? M. tuberculosis-stimulated macrophages
inhibits hepcidin mRNA expression, and iron chelation increases expression.
RAW264.7 macrophages were incubated with FeNTA and the iron chelator
desferrioxamine for 1 h prior to stimulation with IFN-? (200 U/ml) and
?-irradiated M. tuberculosis (100 ?g/ml) for 24 h. Total RNA was extracted,
and hepcidin mRNA was detected by real-time RT-PCR. The hepcidin mRNA
expression levels were normalized to ?-actin mRNA levels and expressed as
fold induction relative to untreated RAW264.7 cells. The data represent the
mean ? SD of three separate experiments. Addition of iron decreased hepcidin
mRNA expression significantly; P ? 0.001 by ANOVA. Iron chelation in-
creased hepcidin mRNA expression significantly; P ? 0.001 by ANOVA.
Fig. 5. Effect of M. tuberculosis and IFN-? on hepcidin protein expression by
RAW264.7 cells, which in serum-free media, were stimulated for 24 h with
IFN-? (200 units/ml) and ?-irradiated M. tuberculosis (100 ?g/ml). Hepcidin
was detected in cell lysates (A) and culture media (B) by a competitive
chemiluminescent ELISA. Results are the mean ? SD of two experiments.
Hepcidin levels in cell lysates were increased significantly in RAW264.7 cells
stimulated with M. tuberculosis (*, P?0.001, t-test) and the combination of M.
tuberculosis and IFN-? (*, P?0.001, t-test) compared with control cells.
Hepcidin, secreted into the culture media, was increased significantly by
treatment with M. tuberculosis and IFN-? (*, P?0.05, t-test) compared with
6Journal of Leukocyte Biology
Volume 82, October 2007
bacterial against M. tuberculosis in vitro. The 25-amino acid
hepcidin peptide was incubated with M. tuberculosis in 7H9
broth. At 6 and 72 h, the effect of hepcidin on M. tuberculosis
viability was determined by CFU. As shown in Table 1,
treatment of M. tuberculosis with hepcidin for 6 h reduced
CFUs, and CFUs of M. tuberculosis were reduced by 50%
compared with control, untreated M. tuberculosis at the highest
concentration of hepcidin used. The effect of hepcidin on M.
Fig. 6. Localization of hepcidin to the M. tuberculosis phagosome. RAW264.7 cells were infected with M. tuberculosis and stimulated with 200 units/ml IFN-?
for 24 h. Representative confocal microscopy images of control, untreated RAW264.7 cells (A), RAW264.7 cells stimulated with IFN-? alone (B), RAW264.7 cells
infected with M. tuberculosis alone (C), and RAW264.7 cells infected with M. tuberculosis and stimulated with IFN-? (D). Hepcidin was detected in permeablized
cells with rabbit anti-mouse hepcidin antibody. M. tuberculosis was detected by staining with auramine-rhodamine prior to immunofluorescence detection. The
secondary antibody was Alexa 488-conjugated F(ab?)2goat anti-rabbit IgG. (E) Control experiment in which the rabbit anti-mouse hepcidin was absorbed with the
mature, 25-amino acid hepcidin peptide prior to fluorescence microscopy. Results are representative of three experiments.
Sow et al.
Hepcidin expression in mycobacteria-infected macrophages7
tuberculosis was also observed after 72 h. M. tuberculosis CFUs
at 72 h of the hepcidin treatment were comparable with CFUs
at 6 h. In contrast, CFUs of the control cultures of M. tuber-
culosis at 72 h increased by 30% compared with the number of
CFUs at 6 h. To determine if hepcidin also damaged the
mycobacteria structurally, we examined M. tuberculosis by
TEM. We examined 481 control M. tuberculosis bacteria incu-
bated in media only and 416 M. tuberculosis bacteria incubated
with hepcidin for 24 h. In the hepcidin-treated M. tuberculosis,
39% of the mycobacteria visualized had lost their normal
architecture (disruption of membrane and loss of cytosol) com-
pared with only 7.9% in the control. Representative TEM
photographs are shown in Figure 9.
Hepcidin acts as a hormone to maintain iron homeostasis by
inhibiting intestinal iron absorption [22, 33] and the macro-
phage release of iron [25, 39]. Hepcidin is produced rapidly by
the liver in mice in response to inflammatory stimuli, LPS,
Freund adjuvant, and turpentine [29, 32, 33, 40]. Hepcidin is
also produced by the liver following in vivo iron loading, and
anemia and hypoxia lead to a decrease in production [30, 33].
Bacterial induction of hepcidin expression in mouse macro-
phages and neutrophils following infection with P. aeruginosa
and group A Streptococcus has been reported recently by Pey-
ssonnaux et al. . Other studies showed that LPS induced
hepcidin mRNA in mouse macrophages [41, 42]. However,
these studies focused on extracellular pathogens of macro-
phages and LPS and did not examine effects of IFN-? on
hepcidin production induced by bacterial infection. Here, we
report that hepcidin is also produced in mouse macrophages
infected with intracellular mycobacteria. Furthermore, we
found that high levels of hepcidin mRNA and protein are
induced in the RAW264.7 mouse macrophage cell line and
BMDM infected with M. avium or M. tuberculosis and stimu-
lated with IFN-?. Mycobacteria and IFN-? acted synergisti-
Fig. 7. Time course of the localization of hepcidin to the M. tuberculosis phagosome. RAW264.7 cells were infected with M. tuberculosis and stimulated with 200
units/ml IFN-? for 0, 4, 8, 16, and 24 h. (A) Representative confocal images. Hepcidin was detected with rabbit anti-mouse hepcidin antibody and Alexa
488-conjugated F(ab?)2goat anti-rabbit IgG secondary antibody. M. tuberculosis was detected by staining with auramine-rhodamine prior to immunofluorescence
detection. (B) Quantitative analysis of the percentage of M. tuberculosis phagosomes containing hepcidin. Results are the pooled data from two independent
experiments. (C) Quantitative analysis of the green fluorescence intensity of hepcidin localized to the M. tuberculosis phagosome. The increase in fluorescence
intensity with time was statistically significant by one-way ANOVA; P ? 0.01.
8 Journal of Leukocyte Biology
Volume 82, October 2007
cally to induce hepcidin mRNA expression. We also observed
that M. tuberculosis and IFN-? synergistically induced hepci-
din mRNA expression in the human monocytic THP1 cell line,
indicating that hepcidin mRNA can also be induced in human
cells. Heat-killed M. avium and ?-irradiated M. tuberculosis
were as effective as live mycobacteria in inducing expression.
However, phagocytosis of latex beads in combination with
IFN-? did not induce expression of hepcidin mRNA. Thus, our
data suggest that a component(s) of mycobacteria, rather than
just phagocytosis itself, induces hepcidin mRNA.
Hepcidin production in mouse hepatocytes is induced by
IL-1? and IL-6 . However, we found that IL-1? and IL-6
had no effect on hepcidin mRNA expression in RAW264.7
macrophages. We did observe an increase in hepcidin mRNA
when IL-1? or IL-6 was added to RAW264.7 cells with M.
avium infection. However, the effect of IL-1? and IL-6 was
much less than that observed with IFN-?. Conditioned media
from macrophages stimulated with LPS have been shown to
induce hepcidin expression in hepatocytes [29, 30]. However,
we have found that conditioned media from RAW264.7 mac-
rophages treated with M. avium and IFN-? did not induce
hepcidin mRNA in fresh RAW264.7 cells (data not shown).
This suggests that cytokines released from the activated mac-
rophages are not responsible for induction of hepcidin mRNA
expression in mouse macrophages. These results also suggest
that expression of hepcidin mRNA is differentially regulated in
macrophages and hepatocytes.
Expression of hepcidin mRNA is also differentially regu-
lated following iron loading. Previous studies have shown that
liver hepcidin mRNA is increased by iron loading . How-
ever, we have found that iron has an opposite effect on mac-
rophage hepcidin mRNA expression. Incubation of RAW264.7
cells with iron decreased hepcidin mRNA expression induced
Fig. 8. Hepcidin localizes to the M. tuberculosis phagosome in RAW264.7 cells, constitutively expressing high levels of Flag epitope-tagged hepcidin.
RAW-hepcidin-Flag cells were infected with live M. tuberculosis for 2 h. Hepcidin-Flag was detected with anti-Flag mAb and Alexa 488-conjugated F(ab?)2goat
anti-mouse IgG secondary antibody. M. tuberculosis was detected by staining with auramine-rhodamine prior to immunofluorescence detection. (A) Representative
confocal images of hepcidin-Flag expression in unstimulated cells. (B) Representative images of hepcidin-Flag in M. tuberculosis-infected cells. Results are
representative of two experiments.
TABLE 1.Hepcidin Antimicrobial Assaya
6 h72 h
0 95.5 ? 1.5
62.3 ? 10.1b
72.6 ? 1.5b
57.0 ? 4.6b
132.6 ? 19.1
86.0 ? 0.0b
84.0 ? 1.41b
66.3 ? 9.9b
62.3 ? 3.1b
aValues are CFU ? 103? SD pooled from three independent experiments.
bP ? 0.002 compared with control. NT, Not tested.
Sow et al.
Hepcidin expression in mycobacteria-infected macrophages9
by IFN-? and M. tuberculosis, and iron chelation increased
hepcidin mRNA expression. A similar effect of iron on mouse
macrophage inducible NOS promoter activity has been re-
ported . Thus, the effects of iron on hepcidin expression in
macrophages and hepatocytes may reflect differences in pro-
moter activity. The increased hepcidin mRNA in response to
iron chelation in macrophages is consistent with studies, which
show iron chelation inhibits M. tuberculosis growth .
The ELISA and immunofluorescence studies show that hep-
cidin protein is also induced by IFN-? and mycobacterial
infection. Intracellular hepcidin was detected by ELISA in
RAW264.7 cells treated with irradiated M. tuberculosis. The
highest levels of hepcidin protein were detected in RAW264.7
cells, which were also stimulated with IFN-?. Hepcidin is also
secreted by RAW264.7 cells stimulated with M. tuberculosis
and IFN-?. Hepcidin protein was also observed in RAW264.7
cells infected with live M. tuberculosis alone and in RAW264.7
cells treated with M. tuberculosis and IFN-?; the latter condi-
tion demonstrated greater fluorescence intensity. In these ex-
periments, we found hepcidin to highly localize to the phago-
some. Hepcidin was present in the phagosome beginning at 4 h
after infection. Fluorescence intensity increased with time after
infection and stimulation with IFN-?. This increase in hepci-
din protein paralleled the increase in hepcidin mRNA expres-
sion. To our knowledge, this is the first report that indicates
that hepcidin trafficks to the phagosome.
Hepcidin is synthesized as a propeptide precursor, which is
then processed into the 25-amino acid, mature form [22, 45].
The hepcidin antibody used in these studies was raised with a
peptide from the C terminus of the mature form of hepcidin.
Absorption with the 25 amino acid hepcidin peptide removed
the immunofluorescence activity of the antibody, indicating the
antibody is reactive with the mature hepcidin peptide. How-
ever, the antibody could also potentially react with the propep-
tide precursor. Our immunofluorescence microscopy studies
show a low level of punctate staining throughout the cell,
consistent with hepcidin being present in intracellular vesi-
cles. Thus, our studies suggest that hepcidin has two pathways
of trafficking within macrophages: fusion of intracellular vesi-
cles with phagosomes, resulting in localization of hepcidin
within the phagosome, and trafficking to the cell surface,
resulting in secretion. At present, we do not know at what step
in the trafficking the hepcidin propeptide precursor is pro-
cessed into the mature form.
In macrophages, mycobacteria acquire iron from extracellu-
lar sources, including iron bound to transferrin, lactoferrin, and
low molecular weight chelates, and from the intracellular labile
iron pool [46, 47]. IFN-? is critical in restricting the growth of
mycobacteria [5, 6]. One of the antimicrobial mechanisms of
IFN-? is its ability to decrease macrophage iron levels. De-
creased macrophage iron is thought to result from decreased
transferrin receptor and ferritin expression induced by IFN-?
[12–15, 46]. Our observations, that IFN-? regulates expression
of hepcidin, raise the question of whether this protein has a
role in the antimicrobial activity of the IFN-?-activated mac-
rophage. Hepcidin was identified, first as an antimicrobial
peptide [21, 22]. To determine if hepcidin has antibacterial
activity against M. tuberculosis, in this study, we incubated M.
tuberculosis with hepcidin in vitro. We observed growth inhi-
bition of M. tuberculosis at 6 and 72 h. Examination of M.
tuberculosis by electron microscopy showed that hepcidin
caused lysis of M. tuberculosis, suggesting that hepcidin acts by
causing loss of membrane integrity. This could occur by open-
ing pores in the cell membrane of M. tuberculosis through the
insertion of hepcidin into the cell membrane or by hepcidin
interacting with transport proteins present in the cell mem-
brane. The maximal growth inhibition hepcidin observed at
earlier times-points was no greater than 50%. This observation
suggests that M. tuberculosis can repair some of the damage
caused by hepcidin or is able to partially degrade or inactivate
hepcidin. The concentration of hepcidin required to maximally
inhibit M. tuberculsosis growth in the in vitro assay is also high
(100–200 ?g/ml). Whether this concentration is reached in the
Fig. 9. Hepcidin causes lysis of M. tuberculosis, which was incubated for 24 h
in 7H9 broth (Control M. tuberculosis) or 7H9 broth with 200 ?g/ml hepcidin
(Hepcidin treated M. tuberculosis). (A) Representative TEM photograph of
intact M. tuberculosis from the control culture. (B) Representative TEM pho-
tograph of M. tuberculosis from the hepcidin-treated culture. Present in B are
intact M. tuberculosis and M. tuberculosis, which have lost normal architecture
as a result of disruption of membrane and loss of cytosol.
10Journal of Leukocyte Biology
Volume 82, October 2007
phagosome is unknown. However, in the macrophage, hepcidin
may act in concert with other IFN-?-induced antimicrobial
mechanisms and thus, may be more effective at lower concen-
trations. Also, the conditions in the phagosome are likely to be
more optimal for killing M. tuberculosis than the in vitro
During infection, hepcidin is released into the blood by liver
hepatocytes and is believed to be responsible for the anemia
associated with chronic disease and inflammation [48, 49].
Tuberculosis is a chronic disease, and anemia is a common
complication of pulmonary tuberculosis . Dietary iron over-
load has been shown to support the growth of M. tuberculosis
and is a risk factor for active tuberculosis [51, 52]. These
studies would suggest that hepcidin production is likely to be
a factor in the anemia associated with tuberculosis. How much
hepcidin secreted by mycobacteria-infected macrophages con-
tributes to levels of hepcidin in the blood is not known. It is
more likely that the effect of hepcidin production by infected
macrophages would be local, mediating antimicrobial activity
and inhibiting iron recycling from dead cells by surrounding
This work was supported by Grant RO1DK-57667 from the
National Institutes of Health. We thank Dr. Georg Pongratz,
Dr. Travis McCarthy, Gail Alvarez, and Steve Oghumu for
technical help, Dr.Virginia Sanders for equipment use, and
Jane Dudek from Roche Applied Science for help with real-
time RT-PCR assays.
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12Journal of Leukocyte Biology
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