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RED CELLS, IRON, AND ERYTHROPOIESIS
Blunted hepcidin response to inflammation in the absence of Hfe and
transferrin receptor 2
Daniel F. Wallace,1 Cameron J. McDonald,1 Lesa Ostini,1 and V. Nathan Subramaniam1,2
1The Membrane Transport Laboratory, Queensland Institute of Medical Research, and 2Liver Research Centre, The School of Medicine,
The University of Queensland, Brisbane, Australia
The induction of the iron-regulatory peptide hepcidin by proinflammatory cytokines is thought to result in the withholding of iron from invading pathogens. Hfe
and transferrin receptor 2 (Tfr2) are involved in the homeostatic regulation of
hepcidin and their disruption causes hereditary hemochromatosis (HH). To determine whether either Hfe or Tfr2 is involved in the inflammatory pathway
regulating hepcidin, we analyzed the effect of inflammation in 3 mouse models of
HH. The inflammatory response and indicators of iron homeostasis were measured in wild-type, Hfeⴚ/ⴚ, Tfr2ⴚ/ⴚ, and
Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice injected with lipopolysaccharide (LPS). The administration of
LPS significantly reduced serum iron in
wild-type and Hfeⴚ/ⴚ mice, with smaller
reductions in Tfr2ⴚ/ⴚ and Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ
mice. Low basal levels of hepcidin in the
Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice were increased in response to LPS, but remained significantly
lower than in the other strains of mice.
These results suggest that despite the
absence of Hfe and Tfr2, hepcidin is responsive to inflammation; however, the
low basal expression and subsequent
low levels of circulating hepcidin are
insufficient to reduce serum iron effectively. This suggests that in HH, the ironwithholding response to invading pathogens may be inadequate, and this is
especially the case in the absence of
both Hfe and Tfr2. (Blood. 2011;117(10):
2960-2966)
Introduction
Iron is an important element in biologic processes because it is
required for the activity of many enzymes and proteins. Therefore,
its regulation is important and a deficiency or excess of iron can be
detrimental to health. Mammals have developed an intricate system
for the regulation of body iron levels. At the center of this is the
antimicrobial peptide and iron-regulatory hormone hepcidin. Hepcidin functions by binding to the cellular iron-exporter ferroportin,
causing its internalization and degradation.1 This results in reduced
release of iron from cells expressing ferroportin, such as duodenal
enterocytes and cells of the reticuloendothelial system. As a
consequence, intestinal iron absorption and iron recycling are
reduced and serum iron levels and transferrin saturation decrease.
The production of hepcidin is restricted mainly to the liver, where
its expression is regulated by several important molecules involved
in iron metabolism and hereditary hemochromatosis (HH).2 Hepcidin is up-regulated by increased iron stores, proinflammatory
cytokines, and bone-morphogenetic proteins (BMPs). The hemojuvelin (HJV) protein is a BMP coreceptor, and its cell-associated
form is required for the regulation of hepcidin through the
BMP-SMAD signaling pathway.3 Lack of functional HJV results in
very low hepcidin levels and early-onset juvenile hemochromatosis
(type 2A).4 Adult-onset forms of HH caused by mutations in HFE
and TFR2 are also characterized by low hepcidin relative to iron
stores. Both of these proteins have been implicated in the regulation of hepcidin, although their exact mechanisms of action remain
unresolved.5,6
We recently showed that double knockout of Hfe and Tfr2 in
mice results in more severe iron loading and lower hepcidin levels
than in the 2 single knockouts, with a phenotype similar to juvenile
hemochromatosis.7 This suggests that the loss of Hfe and Tfr2 has a
compounding effect on hepcidin regulation and that the 2 molecules can function independently to regulate hepcidin. The
pathways through which Hfe and Tfr2 regulate hepcidin are not
well defined, but there is some evidence that the Erk1/2 and/or
Smad pathways may be involved.7,8 Recently, new evidence for the
role of Hfe in the regulation of hepcidin through the Bmp/Smad
pathway has been presented, showing that Hfe⫺/⫺ mice have higher
hepatic Bmp6 expression but lower levels of phospho-Smad1/5/8
in relation to iron stores than wild-type mice.9 In addition, low
doses of Bmp6 were less effective at up-regulating hepcidin in
hepatocytes isolated from Hfe⫺/⫺ mice compared with wild-type
mice.9 A similar pattern of increased BMP6 expression concurrent
with low expression of BMP target genes and phospho-SMAD1/
5/8 relative to iron stores has also been observed in liver biopsies
from patients with HFE-HH.10 These studies suggest that there is an
impairment in Bmp-Smad signaling in the absence of Hfe, resulting
in a blunted hepcidin response. Treatment of Hfe⫺/⫺ mice with high
doses of Bmp6, however, can overcome this blunted hepcidin
response, increasing hepcidin and reducing serum iron levels, and
suggesting that Hfe is not necessary for BMP6-mediated induction
of hepcidin.11
Hepcidin also plays a role in the immune response to pathogens.
Originally isolated as a liver-expressed antimicrobial peptide with
activity against a range of bacteria and fungi,12,13 hepcidin is
induced by inflammatory stimuli and has been implicated in the
anemia of inflammation.14 The administration of inflammatory
agents such as lipopolysaccharide (LPS), turpentine, or Freund’s
complete adjuvant has been shown to increase hepatic Hamp1 (the
Submitted August 24, 2010; accepted December 21, 2010. Prepublished
online as Blood First Edition paper, January 18, 2011; DOI 10.1182/blood2010-08-303859.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2011 by The American Society of Hematology
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BLOOD, 10 MARCH 2011 䡠 VOLUME 117, NUMBER 10
gene encoding hepcidin) expression and reduce serum iron levels
in mice.14-16 The proinflammatory cytokine interleukin-6 (IL-6)
plays an important role in this inflammation-mediated regulation of
Hamp1 expression.15 The proinflammatory cytokines IL-1␣ and
IL-1␤ have also been shown to induce hepcidin.17 Hepcidin is
regulated by IL-6 through the gp130 receptor and the Stat3
signaling pathway.18 The role of Hfe and Tfr2 in the inflammationmediated induction of hepcidin has also been explored. It was
originally reported that Hfe⫺/⫺ mice were unable to mount an
appropriate hepcidin response to LPS, implicating Hfe in the
inflammation-mediated pathway regulating hepcidin.19 However,
2 subsequent studies indicated that Hfe was not required for
inflammatory-mediated induction of hepcidin, and that Hfe⫺/⫺
mice were able to increase hepcidin and reduce serum iron in
response to inflammation.16,20 One of these studies also suggested
that Tfr2 was not required for inflammation-mediated hepcidin
expression.20
We used Hfe⫺/⫺, Tfr2⫺/⫺, and double-knockout Hfe⫺/⫺/Tfr2⫺/⫺
mice on an identical genetic background to comprehensively and
comparatively explore the roles of Hfe and Tfr2 in the inflammatorymediated regulation of hepcidin and serum iron levels.
Hfe/Tfr2 REGULATION OF HEPCIDIN IN INFLAMMATION
2961
[TBS-T]) overnight at 4°C. Blots were incubated with the primary
antibodies anti-ferritin (1:2000; Sigma-Aldrich), anti–phospho-Stat3 (1:1000;
Cell Signaling Technology), anti-Stat3 (1:1000; Cell Signaling Technology), anti-ferroportin (1:2000; Alpha Diagnostics International), and anti–
glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:100 000; Millipore) in Western blocking buffer overnight. Blots were washed in TBS-T
and incubated with anti–rabbit or anti–mouse horseradish peroxidase
(1:10 000; Invitrogen) for 1 hour at room temperature. After washing in
TBS-T, blots were incubated with Immobilon Western Chemiluminescent
Horseradish Peroxidase Substrate (Millipore) and exposed to film. Bands
were quantitated by densitometry using the GeneGenius Imaging System
(Syngene). Blots probed with phospho-Stat3 were stripped by incubating
with stripping buffer (1% sodium dodecyl sulfate; 62.5mM Tris-HCl, pH
6.8; 0.7% 2-mercaptoethanol) at 50°C for 30 minutes. Blots were then
rinsed with 3 10-minute washes and reblocked with Western blocking
buffer at room temperature for 1 hour before incubation with antibodies to
total Stat3.
IL-6 measurement
The concentration of IL-6 was measured in serum samples using an
enzyme-linked immunosorbent assay (ELISA; Life Research) according to
the manufacturer’s instructions.
Statistical analysis
Methods
Animals
Animal studies were approved by the Queensland Institute of Medical
Research Animal Ethics Committee. All animals had free access to water
and food under standard conditions and received humane care. The Hfe⫺/⫺,
Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice on a C57BL/6 background have been
described previously.7 Male mice were weaned at 21 days and maintained
on standard laboratory chow ad libitum (iron content, 120.1 mg/kg). All
mice were fasted overnight before being killed at 5 weeks and tissues
collected for analysis. Control mice were injected intraperitoneally with
saline 4 days before being killed at 5 weeks. LPS-treated mice were injected
intraperitoneally with 1␮g/g of LPS from Escherichia coli 055:B5 (SigmaAldrich) 6 hours before being killed at 5 weeks.
Measurement of iron indices
Serum iron and transferrin saturation were measured using an iron and
iron-binding–capacity kit (Sigma-Aldrich). Nonheme hepatic and splenic
iron concentrations were measured using the method of Torrance and
Bothwell.21
Real-time PCR analysis of mRNA transcripts
Primer pairs for detecting hepcidin mRNA transcripts and inflammatory
markers have been described previously.22,23 Primer sequences for detecting
IL-6 mRNA were forward 5⬘-ATGGATGCTACCAAACTGGAT-3⬘ and
reverse 5⬘-TGAAGGACTCTGGCTTTGTCT-3⬘. The quantitation of mRNA
transcripts in liver and spleen was determined by real-time polymerase
chain reaction (PCR) using LightCycler 480 SYBR Green Mix in a
LightCycler 480 (Roche Diagnostics), as described previously.23
Western blotting
Liver and spleen samples were homogenized in phosphatase inhibitor lysis
buffer (200mM Tris pH 8.0, 100mM NaCl, 1mM EDTA, 0.5% NP-40, 10%
glycerol, 1mM NaF, 1mM sodium orthovanadate, 1mM sodium pyrophosphate, 1:1000 protease inhibitor cocktail, 2mM phenylmethanesulfonyl
fluoride, and 10␮g/mL of DNase). Samples (25 ␮g) were electrophoresed
on 10% Tris-glycine sodium dodecyl sulfate–polyacrylamide gels, transferred onto Hybond-C⫹ membranes, and blocked in Western blocking
buffer (10% skim milk powder, 0.5% Tween 20 in Tris-buffered saline
Variables were compared between groups using 1-way ANOVA or 2-tailed
Student t test in Prism 5 software (GraphPad). A P value ⬍ .05 was
considered statistically significant. Graphs were prepared using Prism 5.
Results
Reduction in serum iron and transferrin saturation is blunted in
Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice exposed to LPS
The effect of inflammation on iron homeostasis was assessed in
5-week-old wild-type, Hfe⫺/⫺, Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice
by injection with LPS. The effect of LPS on serum iron and
transferrin saturation was measured. In all mice, there was a close
correlation between serum iron and transferrin saturation (Figure
1A-B). In wild-type mice, serum iron and transferrin saturation
were significantly reduced after administration of LPS. Similarly,
in the 3 knockout strains, the serum iron and transferrin saturation
were reduced in mice exposed to LPS (Figure 1A-B). However, the
magnitude of reduction varied between the strains. Hfe⫺/⫺ mice had
a large reduction in serum iron and transferrin saturation that
reached statistical significance, Tfr2⫺/⫺ mice had an intermediate
reduction that did not quite reach statistical significance, and
Hfe⫺/⫺/Tfr2⫺/⫺ mice had a small reduction that was not statistically
significant (Figure 1A-B).
Hepatic iron concentration (HIC) and hepatic ferritin protein
expression were also measured in all mice. As expected, all
3 knockout strains had significantly elevated HIC and hepatic
ferritin expression compared with wild-type mice (Figure 1B-C).
There were no significant changes in HIC or hepatic ferritin after
exposure to LPS in any of the mouse strains (Figure 1B-C).
The hepcidin response to LPS is inadequate in
Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice
To investigate the response to LPS in the mouse models of HH, we
analyzed the expression of hepatic hepcidin mRNA. In salineinjected mice, we found a significant increase in Hamp1 mRNA
expression in Hfe⫺/⫺ mice and a significant decrease in Hamp1
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BLOOD, 10 MARCH 2011 䡠 VOLUME 117, NUMBER 10
Figure 1. Iron indices in wild-type (WT), Hfeⴚ/ⴚ, Tfr2ⴚ/ⴚ, and Hfeⴚ/ⴚ/
Tfr2ⴚ/ⴚ mice treated with LPS. (A) Serum iron, (B) transferrin saturation,
(C) HIC, and (D) hepatic ferritin levels were measured in 5-week-old male
WT, Hfe⫺/⫺, Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice injected with saline (white
bars) or LPS (black bars) for 6 hours (n ⫽ 3-4 per group). Data are shown
as the mean; error bars indicate SEM. Statistical comparisons were
performed using the Student t test. Significant differences (P ⬍ .05) are
denoted between control and LPS treatments (*) and between strains
compared with WT (a), with Hfe⫺/⫺ (b), and with Tfr2⫺/⫺ (c).
mRNA expression in Hfe⫺/⫺/Tfr2⫺/⫺ mice compared with wildtype (Figure 2A). We previously observed a similar increase in
Hamp1 mRNA in 5-week-old Hfe⫺/⫺ mice on the C57BL6
background; however, when iron stores were taken into account,
these mice actually had a relative reduction in Hamp1 mRNA that
was probably insufficient to keep iron stores within normal limits.7
When iron stores were taken into account by calculating the ratio of
Hamp1 to HIC, we saw a decreasing gradient from wild-type to
Hfe⫺/⫺ to Tfr2⫺/⫺ to Hfe⫺/⫺/Tfr2⫺/⫺ mice (Figure 2B). LPS
administration resulted in an increase in Hamp1 expression in
wild-type mice that almost reached statistical significance
(P ⫽ .052). Increases in Hamp1 were also observed in Hfe⫺/⫺,
Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice after LPS administration, but the
increase was statistically significant only in the Hfe⫺/⫺/Tfr2⫺/⫺
mice (Figure 2A). The basal Hamp1 expression levels in the
saline-injected Hfe⫺/⫺/Tfr2⫺/⫺ mice were more than 10-fold lower
than in the other strains; therefore, although there was a 7-fold
increase in Hamp1 expression after LPS administration in the
Hfe⫺/⫺/Tfr2⫺/⫺ mice, the actual expression levels were still below
that seen in the wild-type saline-injected mice. When iron stores
were taken into account, the Hamp1/HIC ratio was increased after
LPS administration in all strains of mice, but this only reached
statistical significance in the wild-type mice (Figure 2B). Again,
there was a decreasing gradient in Hamp1/HIC values from
wild-type to Hfe⫺/⫺ to Tfr2⫺/⫺ to Hfe⫺/⫺/Tfr2⫺/⫺. This suggests that
although the ability to up-regulate Hamp1 in response to LPS was
retained in the Hfe⫺/⫺/Tfr2⫺/⫺ mice, the increased levels were
insufficient to reduce serum iron levels effectively. This was likely
Figure 2. Hepatic hepcidin (Hamp1) mRNA expression in wild-type
(WT), Hfeⴚ/ⴚ, Tfr2ⴚ/ⴚ, and Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice treated with LPS.
Real-time PCR was used to quantitate hepatic Hamp1 mRNA transcript
levels in 5-week-old male WT, Hfe⫺/⫺, Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice
injected with saline (white bars) or LPS (black bars) for 6 hours
(n ⫽ 3-4 per group). (A) Hamp1 mRNA transcript levels are shown
normalized to ␤-actin. (B) Hamp1 mRNA transcript levels normalized to
␤-actin are shown relative to iron stores as a ratio to HIC. Data are shown
as the mean; error bars indicate SEM. Statistical comparisons were
performed using the Student t test. Significant differences (P ⬍ .05) are
denoted between control and LPS treatments (*) and between strains
compared with WT (a), with Hfe⫺/⫺ (b), and with Tfr2⫺/⫺ (c).
because of the low basal levels of Hamp1 in relation to iron stores.
This was also the case, but to a lesser extent, in Tfr2⫺/⫺ mice, and
although the Hfe⫺/⫺ mice had a robust response to LPS in terms of
serum iron reduction, the hepcidin response was smaller compared
with wild-type mice.
Splenic ferroportin protein is unaffected by LPS in
Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice
Hepcidin indirectly controls serum iron levels by affecting the
cell-surface expression of the iron-exporter ferroportin by inducing
its internalization and degradation. We investigated the mechanism
underlying the LPS-mediated reduction in serum iron by analyzing
the levels of ferroportin protein in the spleen. The spleen was
chosen because it has a high proportion of macrophage cells and
high expression of ferroportin. The expression of ferroportin
protein was analyzed by Western blotting and the levels quantitated
(Figure 3A-B). There was a statistically significant reduction in
ferroportin protein after LPS administration in the spleens of
wild-type and Hfe⫺/⫺ mice, but no significant reduction in Tfr2⫺/⫺
and Hfe⫺/⫺/Tfr2⫺/⫺ mice (Figure 3A-B). The decrease in spleen
ferroportin protein in wild-type and Hfe⫺/⫺ mice exposed to LPS is
consistent with the observed reduction in serum iron and the
increase in hepatic Hamp1 expression. The splenic iron concentration was also determined, and although an increase was observed in
all strains, there were no significant changes after LPS administration (Figure 3C). This may be because of the relatively short time
course of the experiment (6 hours).
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BLOOD, 10 MARCH 2011 䡠 VOLUME 117, NUMBER 10
Hfe/Tfr2 REGULATION OF HEPCIDIN IN INFLAMMATION
2963
Figure 3. Splenic ferroportin expression and splenic iron concentration in WT, Hfeⴚ/ⴚ, Tfr2ⴚ/ⴚ, and Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice treated with LPS. (A) Western blotting was
used to determine the expression of ferroportin in the spleens of 5-week-old male WT, Hfe⫺/⫺, Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice injected with saline or LPS for 6 hours. The blots
in panel A were quantitated and results are shown relative to Gapdh for saline-injected (white bars) and LPS-injected (black bars) mice (B). Splenic iron concentration was
determined in all mice (C). Data are shown as the means; error bars indicate SEM. Statistical comparisons were performed using the Student t test. Significant differences
(P ⬍ .05) are denoted between control and LPS treatments (*) and between strains compared with WT (a), with Hfe⫺/⫺ (b), and with Tfr2⫺/⫺ (c).
The inflammatory response to LPS was similar in all mice
To determine whether the differences in hepcidin response to LPS
in the knockout mice was because of an impaired inflammatory
response, the levels of the inflammatory cytokine IL-6 and other
markers of the inflammatory response were measured. The expression of IL-6 mRNA in the spleen was measured by real-time PCR
and serum concentrations of IL-6 were measured by ELISA. The
levels of IL-6 were elevated in the groups treated with LPS, and
there were no significant differences between genotype groups
(Figure 4A-B). To assess the inflammatory response in the liver, the
mRNA expression of 2 inflammatory markers was measured by
real time PCR. Orosomucoid (Orm), also known as ␣-1-acid
glycoprotein (Agp) is an acute phase plasma glycoprotein expressed predominantly in the liver. Serum amyloid A (Saa) is also
an acute-phase plasma apolipoprotein that is produced in the liver
Figure 4. Expression of IL-6 and hepatic inflammatory markers in
wild-type (WT), Hfeⴚ/ⴚ, Tfr2ⴚ/ⴚ, and Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ mice treated with
LPS. Real-time PCR was used to quantitate mRNA transcript levels in
5-week-old male WT, Hfe⫺/⫺, Tfr2⫺/⫺, and Hfe⫺/⫺/Tfr2⫺/⫺ mice injected
with saline (white bars) or LPS (black bars) for 6 hours (n ⫽ 3-4 per group).
In the spleen, IL-6 mRNA transcripts levels were measured relative to Hprt
(A). An ELISA was used to determine IL-6 concentrations in the serum of
all mice (B). In the liver, Orm (C) and Saa (D) mRNA transcript levels were
measured relative to ␤-actin. Data are shown as the means; error bars
indicate SEM. Statistical comparisons were performed using the Student
t test. Significant differences (P ⬍ .05) are denoted between control and
LPS treatments (*) and between strains compared with WT (a), with Hfe⫺/⫺
(b), and with Tfr2⫺/⫺ (c).
during inflammation. The expression of these 2 inflammatory
markers was increased markedly in mice treated with LPS (Figure
4C-D). There did not appear to be any consistent differences in the
induction of these 2 inflammatory markers between the wild-type
and knockout strains of mice. This suggests that the inflammatory
response in the liver is similar in all strains of mice. The induction
of hepcidin by the inflammatory cytokine IL-6 involves the
activation of the Stat3 transcription factor through gp130.18 Stat3
also plays an important role in the cytokine-mediated induction of
Saa.24 Western blots were used to determine the levels of phosphorylated and total Stat3 in mouse livers (Figure 5A). Phospho-Stat3
was undetectable in control saline-injected mice. LPS administration led to a massive increase in the levels of phospho-Stat3 in
wild-type and all 3 knockout strains (Figure 5). Compared with the
levels of total Stat3, there was a significant decline in the ratio of
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Figure 5. Expression of activated Stat3 in the livers
of wild-type (WT), Hfeⴚ/ⴚ, Tfr2ⴚ/ⴚ and Hfeⴚ/ⴚ/Tfr2ⴚ/ⴚ
mice treated with LPS. (A) Western blotting was used
to determine the expression of phosphorylated Stat3 in
the livers of 5-week-old male WT, Hfe⫺/⫺, Tfr2⫺/⫺, and
Hfe⫺/⫺/Tfr2⫺/⫺ mice injected with saline or LPS for
6 hours. The blots in panel A were quantitated using
SynGene GeneTools, and the results are shown as
ratios of phospho-Stat3/total-Stat3 (B), phospho-Stat3/
Gapdh (C), and total-Stat3/Gapdh (D) for saline-injected
(white bars) and LPS-injected (black bars) mice. Data
are shown as the means; error bars indicate SEM.
Statistical comparisons were performed using the Student t test. Significant differences (P ⬍ .05) are denoted
between control and LPS treatments (*) and between
strains compared with WT (a), with Hfe⫺/⫺ (b), and with
Tfr2⫺/⫺ (c).
phospho-Stat3 in the 3 knockout strains compared with wild-type
mice (Figure 5B). However, the amount of phospho-Stat3 (relative
to Gapdh) was actually similar across all mouse strains, with the
decreasing ratio to total-Stat3 resulting from increased levels of
total-Stat3 (relative to Gapdh) in the knockout strains (Figure
5C-D). The reason for the increase in total-Stat3 in the knockout
mice is unknown, and the effect of this, if any, on signaling is
unclear. Overall, our results suggest that the Hfe⫺/⫺, Tfr2⫺/⫺, and
Hfe⫺/⫺/Tfr2⫺/⫺ mice have a normal cytokine-mediated response to
inflammation through the Stat3 pathway.
Discussion
The regulation of iron homeostasis is essential for maintaining
body iron levels within safe limits and is also important in the
immune response to pathogens. Hepcidin, the master regulator of
iron homeostasis, is implicated in both of these processes. The
important role of the HH-associated proteins Hfe and Tfr2 in
regulating hepcidin, and ultimately iron absorption and body iron
stores, has been shown by us and others.5,7,22,25-27 Hepcidin is also
up-regulated by proinflammatory cytokines during inflammation,
and this aspect of hepcidin regulation has implicated it in the
anemia of chronic disease. Hepcidin causes the sequestration of
iron within cells, reducing serum iron levels and essentially
withholding iron from potential invading pathogens. The roles of
Hfe and Tfr2 in the immune response to pathogens are less clear.
Studies in Hfe⫺/⫺ mice16 and hepatocytes deficient in Hfe or Tfr220
suggest that these genes are not involved in the inflammatory
pathway regulating hepcidin, because hepcidin was up-regulated
after exposure to an inflammatory stimulus or to IL-6 in a pattern
similar to that of wild-type controls. In Hfe⫺/⫺ mice, this was
accompanied by a significant reduction in serum transferrin
saturation.16
In this study, we have shown that the iron-withholding response
after an inflammatory stimulus is blunted in mice deficient in both
Hfe and Tfr2, and to a lesser extent in mice deficient in Tfr2 alone.
The inflammatory response in the knockout mice appears to be
intact, as evidenced by the up-regulation of IL-6 mRNA in the
spleen and increased levels of IL-6 in the serum of mice exposed to
LPS. Inflammatory markers (Orm and Saa) in the livers of the
knockout mice also suggest that the response to inflammation in the
liver is not abrogated. There was also robust activation of Stat3 in
the livers of all mice treated with LPS. Although absolute levels of
phospho-Stat3 were similar between the strains, the levels of
total-Stat3 were increased in the knockout stains of mice. The
reasons for the increase in total-Stat3 are unclear, and whether it
had an impact on Stat3 activation and the inflammatory response in
the knockout mice is uncertain. The response of Saa, a gene known
to be regulated by Stat3, to LPS suggests that Stat3 activity is not
impaired in the knockout mice. Our observation of a normal
inflammatory response to LPS in our mouse models of HH
contrasts with another study that observed an attenuated inflammatory response in Hfe⫺/⫺ mice. In that study, Hfe⫺/⫺ mice infected
orally with Salmonella typhimurium showed reduced macrophage
expression of tumor necrosis factor-alpha and IL-6.28 In agreement
with our results, however, another study that infected mice
intraperitoneally with S typhimurium observed no differences between the expression of inflammatory cytokines in macrophages
from wild-type and Hfe⫺/⫺ mice.29
The blunting of the iron-withholding response in the Hfe⫺/⫺/
Tfr2⫺/⫺ mice appears to be due to inadequate levels of the
iron-regulatory hormone hepcidin. Whereas mice deficient in both
Hfe and Tfr2 are capable of up-regulating the expression of Hamp1
in response to an inflammatory stimulus, the absolute levels,
although elevated from baseline, remain low. This low level of
circulating hepcidin is probably insufficient to adequately downregulate the cell-surface expression of ferroportin and to reduce
iron release from cells. Indeed, we have shown that the levels of
ferroportin protein in the spleen are significantly reduced after
exposure to LPS in wild-type and Hfe⫺/⫺ mice, but not in Tfr2⫺/⫺
or Hfe⫺/⫺/Tfr2⫺/⫺ mice. The higher basal Hamp1 expression in the
Tfr2⫺/⫺ mice compared with Hfe⫺/⫺/Tfr2⫺/⫺ mice would allow
greater hepcidin expression during inflammation and a larger
decrease in serum iron, but would still be inadequate to reduce
serum iron down to wild-type levels. The even higher basal Hamp1
levels in Hfe⫺/⫺ mice would allow a more marked decrease in
serum iron levels, close to that observed in wild-type mice and
similar to that observed in other studies.16 However, the Hfe⫺/⫺
mice exposed to LPS did have lower Hamp1 in relation to iron
stores compared with wild-type, and this may suggest a slight
deficiency in the iron-withholding response to pathogens in
Hfe⫺/⫺ mice.
The low basal hepcidin expression in HH and Hfe⫺/⫺ mice is
likely because of deficiencies in the BMP-SMAD signaling pathway, as has been suggested by several recent studies.9-11 Our
demonstration of an attenuated hepcidin response to inflammation
in Hfe⫺/⫺/Tfr2⫺/⫺ mice suggests that a functional BMP-SMAD
pathway is also required for an effective iron-withholding response
during inflammation. This has implications for patients with HH
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 10 MARCH 2011 䡠 VOLUME 117, NUMBER 10
Hfe/Tfr2 REGULATION OF HEPCIDIN IN INFLAMMATION
because it may lead to a poorer protective response to invading
pathogens. It has been documented that HH patients are more
susceptible to infection with the pathogenic gram-negative bacteria
Vibrio vulnificus, which has been attributed to the higher availability of iron in the blood of patients with HH.30,31 Patients with iron
overload are also more susceptible to other aggressive bacteria that
require iron for growth and enhanced virulence, such as Listeria
monocytogenes, Klebsiella sp, and Yersinia sp32 Our results suggest that a slightly blunted hepcidin response to inflammation in
HH patients infected with V vulnificus or other iron-requiring
bacteria may also contribute to the greater susceptibility to
infection. In contrast, Hfe⫺/⫺ mice may be protected against
infection with bacteria whose pathogenicity depends on their
ability to invade the host’s macrophages (such as S typhimurium).29
The relative iron deficiency of Hfe⫺/⫺ macrophages and the high
expression of ferroportin has been proposed to partially underlie
the protection against S typhimurium and other intramacrophage
pathogens in HH.33-36 Other mechanisms for the protection in
Hfe⫺/⫺ mice have been proposed, such as the up-regulation of
lipocalin 2, a molecule capable of capturing iron-laden bacterial
siderophores.29 In another study, an attenuated immune response
observed in Hfe⫺/⫺ macrophages suggested that the absence of Hfe
does not protect against these bacteria.28
In conclusion, we have shown that in the absence of Hfe and
Tfr2, the low basal expression of hepcidin contributes to an
inadequate iron-withholding response during inflammation. This is
the case to a lesser extent in the absence of Tfr2 and possibly Hfe
alone. This has implications for patients with HH and may partly
2965
explain their greater susceptibility to infection with some ironrequiring pathogens.
Acknowledgments
We thank Emily Crampton for assistance with the animal
experiments.
This work was supported in part by a program grant from the
National Health and Medical Research Council (NHMRC) of
Australia (no. 339400 to V.N.S.) and by an NHMRC R. D. Wright
Career Development Award (no. 443026 to D.F.W.).
Authorship
Contribution: D.F.W. designed and performed the research, analyzed the data, and wrote the paper; C.J.M. and L.O. performed the
research and analyzed the data; and V.N.S. designed the research,
analyzed the data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Assoc Prof V. Nathan Subramaniam, Membrane Transport Laboratory, The Queensland Institute of Medical
Research, 300 Herston Rd, Herston, Brisbane, QLD 4006, Australia; e-mail: [email protected].
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2011 117: 2960-2966
doi:10.1182/blood-2010-08-303859 originally published
online January 18, 2011
Blunted hepcidin response to inflammation in the absence of Hfe and
transferrin receptor 2
Daniel F. Wallace, Cameron J. McDonald, Lesa Ostini and V. Nathan Subramaniam
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